LI NI U

Sau s kin trng i - thng 6 nm 2000 khi trnh t phc tho b gen ngi c cng b
, tt c mi ngi trn hnh tinh u ch i mt ngy no con ngi c th tr c cc
bnh nan y v ngy n . Hin nay ngi ta c th chn on v iu tr c cc bnh
bng cng ngh DNA . S xut hin ca u d DNA cng vi PCR gii quyt c nhiu
vn bc thit ca Y hc , chng hn nh thay v phi ch i hng tun l c mt xt
nghim v vi khun lao th nay cng vic ch mt vi gi thm ch c th sm hn na .
iu ny gip ch cho cc bnh nhn c iu tr sm hn v trnh c s ly lan trong
cng ng . Vi phng php cha bnh bng gen hay gi vn tt l gen tr liu cha
c nhiu bnh di truyn nh bnh x nang , bnh au c Duchenne , bnh
Huntington , Hi chng gy nhim sc th X , U nguyn bo vng mc , bnh
Alzheimer , bnh x cng teo c ct bn , bnh tiu ng , bnh ung th v.v..
Trong tng lai cc bnh nh Cholesterol cao c tnh cht gia nh, cc bnh v gan
, bnh Lesh- Nyhan , bnh Gaucher v bnh a chy mu cng s c x l bng
gen tr liu .
Mi y nhm nghin cu ti Trng i hc California , Los Angles s dng liposom
c bao bi polyethylen glycol (PEG) a vo tn cc t bo no . Vic chuyn gen vo
no l mt thnh cng rt c ngha , n to tim nng cho vic tr cc bnh Parkinson
(New Scientist . Com-March 20, 2003)
iu m mi ngi ang ch i nht c l l cc bnh ung th v cn bnh th k HIVAIDS . Cho ti th im ny nhiu bnh ung th c tr bng gen tr liu v em li
nhng kt qu kh quan . Vi bnh AIDS , ngi ta cng thu c nhng kt qu rt khch l .
Chng nhng th cc th h vac xin AIDS ang c th nghim , chc chng ta ai cng ch
i ci giy pht m vac xin AIDS s thnh cng nh cc loi vac xin Vim gan B , vac
xin Cm hay mt loi vac xin hin i no ang c lu hnh .
Cun sch ny dnh cho cc bc s lm sng , nhng ngi ang ngy m theo di cc cn
bnh tng bnh nhn , lc no h cng ch i mt phng php hu hiu nht cu sng
ngi bnh . Tuy vy , sch cng gip ch cho tt c nhng ai quan tm ti Gen tr liu
mt phng php cha bnh mi hin ai v y tim nng . Sch cng gii thiu
cc dc phm c bo ch theo kiu Cng ngh DNA . Phn t Antisene l mt ch
phm thuc hon ton mi cha ng nhiu tim nng ng c quan tm . y khng ch
n thun l vn khoa hc m cn l mt lnh vc kinh t hp dn m cc nh hoch nh
chnh sch cn quan tm v li nhun thu c t cng ngh gen l khng l
.
Sch cng cp ti vic lm trong sch mi trng , ph v chu k dch bnh ca cc cn
trng v cc tc nhn gy bnh nguy him khc v.v..
Phn cui ca cun sch chng ti c cp ti mt s lnh vc mi trong cng ngh DNA
nh Tin -Sinh hc . S xut hin ca tin sinh hc lm chng ta an tm rng , mc d ta cn
ngho , trang thit b thiu thn , nhng chng ta vn c th bt tay ngay vo lnh vc gen tr
liu cha tr bnh cho cc bnh nhn . iu tht d hiu , v thi i ngy nay tt c
mi cng vic u mang tnh cht ton cu , cng ngh DNA ni chung hay Gen tr liu ni
ring cng khng ngoi quy lut y .
Vi lng mong mi chuyn ti cc thng tin cp nht nht trong lnh vc gen tr liu ti tt c
cc bn c , mc d c gng rt nhiu nhng chc chn khng trnh khi cc thiu st ,
mong bn c lng th .
Nhn y , chng ti xin chan thnh cm n Gio s o nh c , nguyn Ph Vin
Trng Vin Y hc lm sng nhit i c bn tho v ng gp nhiu kin b ch hon
thin cun sch . Chng ti cng chn thnh cm n Nh xut bn Y hc to mi iu
kin thun li cun sch sm ti tay c gi .
Tc gi xin chn thnh cm n nhng kin ng gp xy dng cun sch ngy cng c
hon chnh hn .
H ni thng 7 nm 2005.
Tc gi

Mc lc
Li ni u
Mc lc
Chng I : Nhng khi nim c bn v gen tr liu
1.1.S lc v gen tr liu
1.2.Khi nim v php cha bnh bng gen
1.3.C ch ca gen tr liu
1.4.Cc loi gen tr liu
1.5. Nhng bnh c th s dng c gen tr liu
1.5.1.iu tr cc bnh di truyn
1.5.1.1.Cc bnh di truyn gy nn bi cc locut gen n
1.5.1.2.Cc bnh do ri lon di truyn cc locut a gen
1.5.2. iu tr cc bnh do nhim trng
Chng II : Nguyn l c bn ca gen tr liu
2.1.Nguyn l ca gen tr liu
2.1.1.Cc t bo ch
2.1.1.1.Nhim sc th ca vi khun
2.1.1.2.Virut
2.1.2.Cc Enzym gii hn
2.1.3. Ligaza
2.1.4.Plasmid
2.2. Cc th nghim v DNA ti t hp
2.3. Nhng bn ci v tnh an ton trong gen tr liu
2.4. Tng lai ca cng ngh DNA ti t hp
2.5. Nhng tin b ng k trong GTL hin nay
Chng III : cc phng php ca cng ngh DNA
3.1. Thu thp gen
3.2. S la chn cc Vec t
3.3. S la chn cc t bo ch
3.4. S biu hin ca gen
3.5. Tp hp cc sn phm ca gen
3.6. Th vin gen
3.6.1.Thit lp mt th vin gen
3.6.2.Sng lc th vin gen
3.6.3. Th vin cDNA
Chng IV : Phn tch v chn on bng DNA
4.1. M u
4.2. Phng php phn tch DNA
4.2.1. u d DNA
4.2.2.Phn ng tng hp chui (PCR)
4.2.3. Khuch i tn hiu
4.2.4. Phn tch RFLP
4.3. Chn on cc bnh truyn nhim
4.3.1. Chn on Hi chng thiu ht min dch mc phi
4.3.2. Chn on bnh Lao
4.3.3. Chn on bnh Lyme
4.3.4. Chn on bnh u nh v cc bnh khc
4.4. Pht hin cc bnh do di truyn
4.4.1. Bnh x nang
4.4.2.Bnh au c Duchenne
4.4.3.Bnh Huntington
4.4.4. Hi chng gy nhim sc th X
4.4.5. U nguyn bo vng mc
4.4.6. Bnh Alzheimer
4.4.7. Bnh s cng teo c ct bn
4.4.8. Bnh tiu ng
4.4.9. Bnh ung th
4.4.10. Ngn hng gen
4.5. ng dng gen tr liu trong lm sng
4.5.1. iu tr bnh thiu ht min dich t hp trm trng (SCID)

4.5.2. Gen tr liu trong chng ung th

4.5.3. Nhng n lc hin ti v tng lai
4.5.3.1.Thay th gen khim khuyt trong bnh x nang
4.5.3.2. Bnh Cholesterol cao c tnh cht gia nh
4.5.3.3. GTL vi cc t bo gan
4.5.3.4. GTL trong Hi chng thiu ht min dch mc phi
4.5.3.6. Bnh Lesh-Nyhan
4.5.3.7. Bnh Gaucher v bnh a chy mu
4.6. Bo him an ton trong gen tril liu
Chng V : Cc Dc phm ch to theo cng ngh DNA
5.1. m u
5.2.Thay th cc protein ngi
5.2.1. Insulin
5.2.2. Hormon sinh trng ca ngi
5.2.3. Yu t VIII
5.3. Tr liu trn ngi
5.3.1. Cht hot ho Plasminogen ca m
5.3.2. Interferon
5.3.3. Cc phn t Antisene
5.4.Vac xin
5.4.1. Vac xin vim gan B
5.4.2. Vac xin AIDS
5.4.3. Cc loi vac xin khc
5.5. ng vt chuyn gen v nhng ng dng thc tin ca VCG
5.5.1. Nhng khi nim v ng vt chuyn gen v nhng ng dng ca chng
5.5.2. a DNA vo cc t bo ng vt c v
5.5.3. To cc ng vt theo mun
5.5.3.1. Chut mang h thng min dch ca ngi
5.5.3.2. Chut mang ung th
5.5.3.3. Cc ng vt chuyn gen khc
5.5.3.4. Cc cht phn ng sinh hc t ng vt
5.5.3.5. Hemoglobin ngi t ln
5.5.3.6. Cc sn phm khc ca ng vt chuyn gen
5.5.4.cc cht thay th mi trng
Chng VI : D n b gen ngi
6.1 ngha Y sinh hc
6.2. D n b gen ngi c nhiu mc ch
6.3.Trnh t phc tho ca b gen ngi c thng bo thng 6 nm 2000.
6.4.Hai nhm s dng cc phng php khc nhau
6.5. Vic xc nh trnh t b gen ngi em li ch li cho cc pht hin mi
6.6. Hu ht b gen ngi c xc nh trnh t
6.7. xc nh c rng b gen ngi m cho khong 30.000-40.000 protein
6.8. Ch c 1,1% n 1,5% b gen ngi dng m cho protein
6.9. Cu trc ca cc nhim sc th ngi thay i rt ln
6.10. Cc gen ca ngi hot ng nhiu hn cc gen ca cc t chc n gin hn .
6.11. H protein ngi phc tp hn ng vt c xng sng
6.12. Cc trnh t lp chim trn 50% b gen ngi
6.13. Nhng pht hin khc ng quan tm
6.14 .Lp k hoch cho nhng cng vic tip theo v b gen ngi v cc t chc khc
6.15 Nhng lin quan ti h protein , cng ngh sinh hc v tin sinh hc
6.16. Nhng lin quan ti Y hc
Gii thch mt s t chuyn mn
Ph lc
Tin-Sinh hc : Khi nim v ng dng
Mu d Axit Nucleic
Nhng sch tham kho chnh

Chng I
Nhng khi nim c bn v Gen tr liu
(phng php cha bnh bng gen) .
1.1.Lc s v gen tr liu:
Ci mc lch s l ngy 14 thng 9 nm 1990 khi cc nh khoa hc vin sc kho quc gia
Hoa k trnh by phng php gen tr liu (GTL) trn mt c b 4 tui tn l Ashanti Desiva
.Khi mi sinh ra em mc mt bnh di truyn him gp - bnh thiu ht min dch t hp
trm trng (severe combined immune deficiency SCID) ,v em b tn thng h min dch
ngay t phi thai . Tr em mc chng bnh ny thng pht trin rm r cc bnh nhim trng
v him khi tn ti ti tui trng thnh . Mt bnh thng thng nh bnh thu u cng c
th e do tnh mng , v th Ashanti phi giam mnh trong mt phng kn , trnh tip sc vi
mi ngi tr nhng ngi trong gia nh . Em c gi trong mi Trng v trng v phi
dng mt lng ln khng sinh chng tri li cc bnh thng thng .

Hnh 1.1. .nh 2 bnh nhn u tin c iu tr bnh bng gen . C gi pha bn tri l
Cynthia cn pha bn phi l Ashanti. C hai c gi ny u c cuc sng bnh thng do
nhn c cc t bo bin i gen y lui bnh do thiu ht ADA vo cui 1990 v u
1991 . Tm nh nh ny xut hin trn tp ch Time vo gia 1993.
Trong tin trnh gen tr liu , cc bc s phi rt ht bch cu ra khi c th Ashanti , ri cc
t bo ny pht trin phng th nghim v a cc gen b mt vo . Cc t bo c sa
i di truyn s c thm nhp qua mch mu bnh nhn . iu tht vui mng l cc test
phng th nghim (labor) cho thy h thng min dch ca Ashanti sau khi c tr liu kh
hn ln , em khng cn b cm cm thng xuyn na v c php ti Trng ri c tim
chng c vacxin ho g na .
Sau Ashanti l Cynthia 9 tui cng mc bnh suy gim min dch t hp trm trng cng c
cu cha bng GTL va cui 1990 u 1991.
Tuy nhin, vi GTL th khng th ch x l mt ln v cc bch cu dc x l di truyn ch
c th hot ng c trong vng vi thng nn liu trnh phi lp i lp li nhiu ln .
Cng cn phi nhn mnh rng ,tuy t c mt s thnh tu trong GTL, nhng nhng g
m ta thu c vn cn rt b nh so vi s lc quan trong mt cu chuyn di.
Nm 1999 vp phi mt kh khn v cng ln vi s c Jesse Gelsinger 18 tui b cht khi
tham gia GTL v bnh di truyn do thiu ht enzym Ornithin Transcarboxylaza (OTCD) .Jesse
b cht sau 4 ngy tr liu v c s p ng min dch mnh lit vi vt mang Adenovirut dn
n c th b hu hoi nghim trng .
Mt s kin trng i na l thng 1 nm 2003 , FDA ra lnh tm dng tt c cc GTL c s
dng vec t Retrovirut trong cc t bo ngun ca mu. S d c s c ny l do sau khi mt
em b ngi Php khi x l bng GTL li pht bnh ging nh bnh bch cu (Leukemia) .
iu ng ni l c 2 em b u pht bnh ging nhau khi dng GTL iu tr thnh cng bnh

thiu ht min dch t hp nghim trng SCID hay cn gi l hi chng Bubble Baby vo
thng 8 nm 2003.
Cng v l do ny nn U ban c vn sinh hc ca FDA cng bn lun v kh nng c th nh
lng s gen tr liu c s dng Retrovirut m bo s an ton cho GTL.
Con ng i ti s ph chun thc hin mt qui trnh GTL gp bit bao kh khn tr ngi
v cn rt nhiu vn cn tranh ci. Kha cnh Sinh hc ca GTL rt phc tp v nhiu k
thut cn phi pht trin thm cng nh cc bnh cn phi c hiu y hn na trc khi
mun ng dng php tr bnh bng gen .
Vic tranh ci cng khai xung quanh vn liu c nn s dng nhng cht liu (material) do
cng ngh gen cho con ngi hay khng th qu l qu phc tp .Tham gia tranh lun v vn
ny c cc chuyn gia nhiu lnh vc khc nhau nh Sinh hc , Qun l nh nc, Y hc ,
Trit hc , Chnh tr v Tn gio v.v.. Mi lnh vc nhn nhn mt khc v th c rt nhiu vn
cn phi tranh lun .
Mc d gp nhiu tr ngi , nhng GTL vn c nhng bc tin ng ghi nhn . Ch tnh n
cui 1994 c hn 500 bnh nhn c iu tr bng GTL vi nhiu loi bnh khc nhau ,
trong s ngi b ung th c iu tr vi liu php ny chim ti 69% ( nm 2001) , bnh
HIV-AIDS 11,8%.
Cho ti nay s bnh c iu tr bng GTL ngy cng nhiu , ngoi cc bnh ung th, SCID,
hng lot bnh khc cng s dng GTL nh x nang, thiu mu do hng cu hnh li lim,
bnh lo ho sm , bnh mu kh ng, bnh thiu ht trm trng enzym OTG ( do mt gen
trn NST gii tnh X ), bnh Huntington ( cha bng antisens RNA) (New scientist .Commarch 13.2003) . K thut to ra cc Liposom c kch thc siu nh (khong 25 nm) d dng
mang cc gen qua l mng nhn , hoc s dng vec t Liposom c bao bi mt lp v
polyethylen glycol PEG a cc gen vo t bo no cha cc bnh Parkinson.v.v..(New
Scientist .Com May 12-2002)
1.2.Khi nim v php cha bnh bng gen:
Gen l n v c s ca thng tin di truyn .Thut ng gen dng nh mt on ca thng tin di
truyn c phin m sang mt RNA n l v tip thng tin t phn t ny c dch m
sang mt protein nht nh .
Gen nm trn cc nhim sc th (NST) (v tr trn NST ni mt gen c th nh v gi l locut
ca gen ) . cc sinh vt lng bi , cc NST sp xp thnh cc cp tng ng ti cc v tr
tng ng tn ti cc dng khc nhau ca cng mt gen gi l ALEN.
Cu trc ca mt gen bao gm 3 vng chnh : Vng iu khin , vng mang m di truyn v
vng kt thc . Vng iu khin c mt s trnh t c hiu iu khin hot ng ca gen.
Vng mang m cha cc thng tin d truyn , c phin m sang RNA thng tin (mRNA) .
Gen cu trc c th c dch m to nn cc sn phm l protein . Vng kt thc mang cc
trnh t phn bit gia cc gen v cc trnh t kt thc qu trnh phin m . Cu trc ca gen
cn bao gm mt s cu trc c th nm trc,sau hoc trong gen nh cc trnh t iu ho,
vng tng cng , vng bt hot (silencer) , vng m (spacer).
Sn phm ca gen l protein mang nhng chc nng quan trng nht ca s sng v to nn cu
trc ca cc t bo . Khi gen b bin i th cc protein c m ho bi cc gen khng c
kh nng thc hin nhng chc nng thng thng ca chng - l nguyn nhn ca cc bnh
di truyn .
Php cha bnh bng gen hay cn gi l gen tr liu gen liu php v.v.. l mt k thut nhm
chun xc li cc gen b khim khuyt (l nguyn nhn pht sinh ra bnh ). Cc nh nghin cu
a ra mt s phng php chun xc li cc gen li nh sau :
*a mt gen hot ng bnh thng vo mt v tr khng c hiu trongb gen (genome)
thay th cc gen khng cn chc nng . y l cch ph bin nht .
*Mt gen d thng c th c i bng mt gen bnh thng thng qua ti t hp tng ng
.
*Mt gen d thng c th c sa cha thng qua t bin chn lc ngc (selective reserse
mutation) chuyn gen tr li chc nng bnh thng ca n .
*iu ho mt gen c bit no b bin i (mc ng, m gen).
* a mt gen bnh thng vo t bo hot ng ng thi vi cc gen gy bnh hn ch tc
ng ca gen gy bnh hoc h tr cho cc gen b h hng ,
*a mt gen bt hot vo t bo thay th cho mt gen bnh thng no nhm hn ch cc
sn phm khng cn thit ca gen lnh nhm to mt trng thi mi cho t bo.
1.3. C ch ca Gen tr liu:
Trong hu ht cc nghin cu v gen tr liu , mt gen bnh thng uc a vo b
gen(genome) thay th mt gen khng bnh thng-gen gy bnh .Phn t chuyn ch
Carrier c gi l Vec t lm nhim v chuyn gen tr liu ti cc t bo ch ca bnh nhn
. Hin nay, Vec t thng thng l cc Virut c bin i gen mang DNA ca ngi kho
mnh . Virut pht trin dn dn v cc gen ca chng c chuyn ti cc t bo ngi

nhim bnh . Ngi ta c gng to ra cc li th ca kh nng ny v vn dng genome virut

loi b cc gen gy bnh v a cc gen tr liu vo c th ngi bnh .
Cc t bo ch ca bnh nhn (chng hn nh cc t bo gan hoc phi ) s b nhim vi cc
vec t virut . Cc vec t sau li bc dcc vt liu di truyn ca n c cha cc gen tr liu
vo cc t bo ch. S sinh si ny n ca cc protein c chc nngc to ra t cc gen tr
liu s hon tr trng thi bnh thng ca t bo ch .
1.4.Cc loi gen tr liu :
V mt l thuyt ca GTL , ngi ta phn bit gia GTL i vi t bo Soma v GTL
i vi cc t bo mm (germ) lm nhim v sinh sn .
D nhin ch c cc t bo mm th mi c th mang cc gen truyn t th h ny sang th h
khc
Xung quanh vn ny c nhiu kin khc nhau:
Mt s ngi phn i bt k mt thao tc no ca GTL cho d l c nh tt (VII , Rifkin
1983) .
Mt s khc th ng TLG c dng cho cc t bo soma nhng li do d i vi cc t bo
mm v cha nhn trc c cc hu qu i vi cc th h tng lai .
Cn mt s khc li cho rng an ton cng nh s iu ho trong GTL cng cn phi un
nn thng xuyn theo nm thng v phi c s chp thun ca lun l .
Liu php gen Soma (Somatic Gene Therapy) l phng php iu tr thay hoc sa cha
cc gen hng, gen gy bnh ca cc t bo soma trong c th bnh nhn.
Liu php gen soma c th s dng mt s loi t bo nh lympho(lymphocyte),nguyn bo
si(Fibroblast),t bo gc (stem cells), t bo mu(hematocyte), t bo biu b(keratinocyte)...
Liu php ny c p dng vi mt s ln bnh nhn mc cc bnh him ngho nh ung
th ,thiu ht min dch t hp trm trng (SCID) , thiu mu do hng cu hnh li lim, x
nang v.v..
Liu php gen t bo mm (Germline Gene Therapy) l phng php iu tr , sa cha
hay thay th cc gen hng cho giao t (tinh trng hoc t bo trng) a cc t bo mm tr li
trng thi sinh l bnh thng . TLG t bo mm c th theo 2 cch :
a.iu tr cc phi giai on u (pre embryo) c cc khuyt tt di truyn nghim trng.
b.iu tr cc t bo mm (tinh trng hay t bo trng ) ca nhng ngi c khuyt tt v mt
di truyn m nhng khuyt tt ny c th truyn li cho cc th h sau . Cch tip cn ny i
hi s thnh tho v k thut . Tuy nhin, vn ny cn nhiu tranh ci ch yu v l do o
c v c s lin quan ti vic nhn bn ngi .
1.5.Nhng bnh c th s dng c GTL.
Hin nay nhng bnh no c th ng dng c phng php cha bnh bng gen ? y l mt
cu hi m nhiu ngi quan tm . Chng ta nh li rng cui nm 1993 GTL c s dng
cho cc bnh thiu ht min dch t hp nghim trng ,bnutng Cholesterol c tnh cht gia
nh,bnh nang x v bnh Gaucher. Cho ti nay phn ln cc protocol c p dng cho cc
bnh ung th v mt s t hng ti bnh AIDS . Cng c mt s bnh c bn lun s
dng GTL nh bnh Parkinson v bnh Alzheimer, bnh vim khp , cc bnh v tim (VII,
Wolff 1993).
D n b gen ngi ang n lc xc nh v tr ca tt c cc gen trong b gen ngi , sau
tip tc xc nh cc gen gy bnh di truyn . Cng trong d n ny Eve Nichols a ra cc
tiu chun la chn cc bnh s dng GTL nh sau :
1.Cc bnh nng ang e do s sng m khng c cch cu cha
2.Phi xc nh r rng cc t chc, m v cc t bo b nhm bnh .
3.Bn sao bnh thng ca cc gen khuyt tt phi c phn lp v tch dng .
4.Cc gen bnh thng c th c a vo tng b phn nh ca t bo m b bnh. Hoc khi
a mt gen vo mt m ch nh tu xng chng hn th n s lm thay i qu trnh din
tin cc m b bnh .
5. Gen c th c chuyn ti mt v tr thch hp khi n s ch o vic tng hp mt lng
protein c chc nng bnh thng to nn mt s khc bit c bit.
6.Cc k thut c p dng phi c xc nhn l an ton ( III,Nichols 1988,p.18)
Cho ti nay GTL c th c ng dng trong c c bnh c th nh sau :
1.5.1.iu tr cc bnh di truyn ( inherited disorders) .
1.5.1.1.Vi cc bnh di truyn gy nn bi cc locut gen n (monogenic ).
Nhng bnh ny thng gy t vong giai on sm v li cc di chng cho cc th h sau.
Mc di truyn l 100% . Mt s bnh in hnh l :
-Thiu ht min dch t hp trm trng (SCID)
-Thiu mu do hng cu hnh li lim (Sickle cell anemia)
-Bnh a chy mu (Hemophilia)
7

-Bnh x nang (cystic fibrosis) v vim phi cp.

-Bnh Parkinson
-Bnh Gaucher.
-Bnh ri lon mn tnh chc nng t chc ht (chronic granulomatoses disease)
1.5.1.2. Cc bnh do ri lon di truyn cc locut a gen (polygenic)
y l nhm bnh gy bi nhiu gen thuc cc locut khc nhau . Tu theo mc h hng
hoc mt chc nng gen ca mt hay nhiu gen (alelle) m mc biu hin bnh c khc
nhau . Nhng bnh ny ch di truyn li cho th h sau di 100%.C th k qua cc bnh sau
y:
-Bnh tim bm sinh (Congenital Heart disease)
-Ung th
-Bnh tiu ng (Diabetes)
-Bnh nghin ru (Alcoholism)
-Bnh tm thn phn lit (Schizophrenia)
-Bnh c hnh vi phm ti (Criminal behavior)
1.5.2.iu tr cc bnh do nhim trng :
GTL t hiu qu cao trong vic iu tr cc bnh nhim trng do vi khun v virut nh ung
th gan, lao , HIV-AIDS ,vim gan B ... Trong tng lai kh nng GTL s iu tr c nhiu
bnh truyn nhim vi hiu qu v cht lng cao , cu sng c nhiu ngi bnh .
Php cha bnh bng gen l mt phng php mi ,c hiu qu cao nhng hin nay vn b hn
ch v n qu phc tp, tn km v bt buc i hi phng tin k thut hin i. Chnh v vy
cho ti nay phng php ny ch mi gii hn trong cc bnh him ngho v ch c cc nc
pht trin vi tim lc kinh t cao mi c iu kin p dng. D nhin cn nhiu vn khc
na nh h s an ton v nhng hu qu ngoi mun ca gen tr liu ...cng lm cho ngi ta
thn trng hn khi s dng k thut ny .
Vi cc nc ang pht trin th gen tr liu mi ch gii hn nhng thc nghim, cha c
chin lc pht trim rm r .Nc ta cng vy, liu php gen cn rt xa l vi cc thy thuc ,
chng ta cha sn sng tip nhn mt v kh mi hin i ,hiu qu vt tri hn hn cc
phng php khc . n lc chng ta phi t ra mt chin lc pht trin c nh hng c
th v gen tr liu , v n c nhiu li th vt tri v chc chn s mang li hnh phc cho tt
c mi ngi .

Chng II
Nguyn l c bn ca Gen tr liu
2.1. Nguyn l ca gen tr liu
GTL l a mt gen mi (cn gi l gen liu php) vo trong t bo ngi lm cho n gn ng
vo v tr cn sa cha ca b gen v hot ng mt cch bnh thng trong t bo .Cc gen tr
liu ny to c cc sn phm c kh nng hn ch hoc km hm s tc ng ca cc gen
hng , do lm gim cc biu hin ca bnh .y chnh l k thut di truyn (genetic
engineering) hay cn gi l thao tc gen (gene manipulation) hay tch dng gen (gene cloning),
cng ngh DNA ti t hp (recombitant DNA technology) , sa i di truyn (genetic
modification) hoc cn gi l di truyn hc mi (new genetics).
Mc d nhiu k thut a dng v phc tp c s dng nhng cc nguyn l ca k thut di
truyn kh n gin . C s ca cng ngh ny l thng tin di truyn m ho trong DNA tn ti
dng cc gen . Cc thng tin ny c th sa i theo nhiu cch khc nhau t ti nhng
mc ch nht nh trong nghin cu c bn cng nh ng dng trong y sinh hc . im mu
cht ca k thut di truyn l tch chit mt on DNA ring bit t h gen ( l bn cht
ca tch dng gen ). Vi GTL qu trnh ny bao gm 4 bc sau y :
1.Tch dng gen tr liu (to ra cc on DNA).
2. Chn vec t chuyn gen ph hp vi gen tr liu v ni chng li vi nhau .
3. To cc vec t ti t hp v a cc vec t mang gen tr liu vo t bo ch v nhn ln .
4. Chn lc cc trnh t (sequence) quan tm ,theo di s hot ng v nhng biu hin ca
gen tr liu.
Chng ta hy lt qua i nt v
c s sinh hc ca cng ngh DNA .
2.1.1.Cc t bo ch (organnisms) :
Cng ngh DNA mi to dng c kh nng nghin cu trn vi khun v virut (Hnh 2.1.)

Hnh 2.1.Vi khun phc v con ngi. nh hin vi in t mt mu Pseudomonas. Anad

Chakrabarty s dng Pseudomonas bin i gen sn xut ra cc enzym phn hu du v
lm sch mi trng.
Nhng sinh vt ny c th nui cy c mt cch d dng v vic kho st Ho sinh cng
c thc hin mt cch d dng trong ng nghim.Vi khun c dng ph thng nht trong
9

cng ngh DNA l Escherichia Coli.Vic s dung rng ri u d vi khun l mt bin c lch
s .Nhng cng trnh nghin cu trn nhng sinh vt ny qua hng my thp k cho php
cc nh sinh hc hiu r hn v mt Ho sinh, hnh thi, sinh l,v di truyn hc ca chng hn
bt c sinh vt no khc (k c con ngi) v chng d dng c nui cy v khng phi l
yu t gy bnh cho con ngi . E. Coli tr thnh con nga th cho cc th nghim trong
cng ngh DNA .
2.1.1.1.Nhim sc th ca vi khun c bit thch hp cho cc th nghim DNA bi v
n ch thun c DNA , cn cc t bo nhn chun nh cc t bo ng vt th phc tp hn v
NST c cha nhiu protein gn cht vo DNA . Hn na , NST ca vi khun l hon ton n l
nn n c th t biu hin c m khng b ln t bi NST th hai . Ngc li NST ca cc t
bo nhn chun li l cp i v thng mt trong s l tri . Thm na l NST ca vi
khun thng nm t do trong t bo cht v th nghin cu n d dng hn . Trong khi
NST ca cc t bo nhn chun th li c mng nhn bao quanh nhn nn cng vic kho st
gp nhiu kh khn hn . Mt l do khc na l m di truyn ca cc sinh vt ny l rt ph
thng nn vi khun c th biu hin DNA ngoi lai t bt k c th no d di n my nu
nh gn c vo DNA ca vi khun . Trn thc t, mt gen ngoi lai khi gn vo NST
ca vi khun th n s c sao chp v phin m chnh xc ging y nh DNA vi khun m.
2.1.1.2.Virut : Cc virut thng c dng trong cc th nghim cng ngh DNA . Nhng
ht siu vi ny bao gm t hoc nhiu on RNA hoc DNA c bao trong mt ci o protein
v tu thuc vo tng loi virut m c loi cn c c mt lp lipid bao quanh. Virut sao chp
mt cch n c trong cc t bo sng. chng lt b phn v protein ca mnh v s
dng c my phn t ca t bo to nn cc virut mi .Virut DNA hay RNA hot ng nh
mt gen v ch o s tng hp cc ht virut mi . Khi cc virut RNA hay DNA tc ng
nh mt RNA thng tin cung cp cc m (Codon) cho cc protein enzym v cc thnh phn
cu trc ca virut .
i khi virut khng t sao chp tc khc m n li gn vo NST ca t bo ch v tr thnh
mt b phn ca b gen t bo . DNA ca Herpesvirus chng hn , n c th hi nhp vo b
gen ca t bo thn kinh v li nhiu nm gy nn s xm nhim tun hon herpes .Cng
tng t nh vy, virut gy hi chng thiu ht min dich trn ngi HIV chng lt b o trn
t bo ri RNA ca chng hot ng nh mt ci khun tng hp DNA. DNA ny t ci vo
genome ca t bo ch. Nh vy, t bo tr thnh vt mang virut DNA v b bnh HIV.
Kh nng virut ci c vo b gen ca t bo ch lm cc nh cng ngh DNA ch , h
nhn nhn n nh l mt cch mang gen vo trong t bo .
Nhng nghin cu nhng nm 1950 cng chng minh rng ti t hp gen cng c th xy ra
gia cc vi khun v virut.
Cc th nghim ca Griffith vi vi khun vo nm 1928 lm r s ti t hp gen v dn ti
s pht hin ca Avery v DNA l mt phn t c lin quan trong ti t hp. Cng trong
nhng nm 1950 , cc nh vi khun hc pht hin ra cc vt cht di truyn t cc mnh v vi
khun c th a uc vo cc vi khun sng , l mt hin tng t nhin . Hin tng ny
chnh l s bin np (transforrmation). Mc d s bin np ch xy ra vi t l t hn 1% trong
mt qun th vi khun , nhng iu ny dn n nhng thay i su sc trong di truyn hc .
Trong s bin np, mt s vi khun cho (donnor) b v ra v DNA ca chng c l ra thnh
cc mnh . Khi c mt vi khun nhn (recipient) th mt on DNA chui kp cha khong 1020 gen c th i qua vch v mng t bo ca chng . Mt enzym lm ho tan mt chui ca
DNA v chui cn li th i ch cho on DNA chui n trong NST vi khun nhn . Cc gen
ngoi lai ny sau t biu l trong qu trnh tng hp protein hon tt s bin np.
Di cc iu kin t nhin, s bin np xy ra nhng c th c DNA tng t .Mt trong s
cc h qu c th l lm tng tnh gy bnh cho c th nhn (trong th nghim Griffiths
pneumococci) . Mt h qu khc c th l pht trin tnh khng thuc . iu , c th gii
thch c mt s vi khun khng thuc trong nhng nm gn y nh th no .

10

Hnh 2.2. Cu trc ca cc Virut (a) Virut thc vt (virut khm c ci ). (b) Virut khm
thuc l .(c) Bacteriophage .
Tip tc nhng nghin cu trong nhng nm 1950 ngi ta ch r rng vi khun cng c
th c ti t hp bi qu trnh lin hp (conjugation) .

11

Hnh 2.3, C ch trong mt Retrovirut nh HIV c lin quan vi t bo vt ch . Acid

Nucleic ca Retrovirut l RNA.Enzymphin m ngc tng hp mt phn t DNA chui kp
thng (ds DNA), sau hp nht vi vo b gen t bo ch nh mt Provirut. nhng thi
im sau (gi, ngy , tun hoc di hn na) RNA polymeraza t vt ch s sao chp
provirut thnh dng genome RNA ca Retrovirut .Khi ng gi vi protein th genome ny to
nn mt b cc Virut mi ra khi t bo ny gy nhim cc t bo mi v tip tc chu
trnh.
S pht hin ca Joshua Lederberg, Francois Jacob v Elie Wollman c nh hng
su sc ti vic nghin cu . Trong khi lin hp 2 vi khun , mt cho v mt nhn tin li gn
nhau v ni vi nhau bi mt cu bo cht (cytoplasmic) . Sau DNA chui n t vi khun
cho i qua cu bo cht ti vi khun nhn .

Hnh 2.4. Bin np trong t bo vi khun .(a) Mt on DNA gii phng t mt t bo cho
cht .(b)on DNA i vo cc t bo nhn ang sng. (c)Mt chui b ho tan v (d) chui
cn li th mt on ca chui trong NST t bo nhn v bin np t bo ny. Bin np c
nghin cu su t nhng nm 1950 v c chng minh nhng iu xy ra trong phng th
nghim vo nhng nm 1970.
y n c th hp nht vi NST vi khun nhn (rt him) , hoc c th tn ti t bo cht
di dng mt vng DNA di ng t do (xem phn Plasmid).
Nhng gen mi thu nhn t vi khun cho sau ny t n biu hin (Hnh 2.5.)

12

Hnh 2.5. S hp nht trong vi khun. Hai t bo Escherichia Coli ni vi nhau qua cu tng
bo .Nhng t bo pha bn phi c lng trn b mt l cc t bo cho . Cc t bo pha
bn tri l cc t nhn .S hp nht to thun li cho vic vn chuyn cc cht liu di truyn t
t bo ny sang t bo khc . l mt phng php m vi khun thu c cc gen cho php
n khng li nhiu loi thuc .
S lin hp nh m t xy ra gia cc t bo t cc loi khc nhau ca vi khun , chng
hn nh gia Salmonella v Shigella .(ngc li, s bin np ch xy ra gia cc t bo c
b gen ging nhau). Nh vy, s lin hp c th coi nh vic a mt gen vo cc vi khun
khng c quan h di truyn v coi nh l s ti t hp ca cc mu phn k (divergent)
rng.
Dng th ba ca ti t hp c pht hin vo nhng nm 1950 l ca Joshua Ledeberg
v Norton Zinder. Nhng nh nghin cu ny pht hin ra rng virut c th truyn DNA
cho cc t bo vi khun v ti t hp c hiu qu cc t bo ny . Qu trnh ny gi l s
chuyn np (transduction).
Trong chuyn np , mt virut l bacteriophage (n gin gi l phage) chui vo mt t bo vi
khun v trao i DNA ca n vi NST vi khun .Ri cc virut ny c th c tho g khi
NST vi khun , nh vy n c th mang theo mt on nh DNA ca vi khun . Khi virut sao
chp n t ti bn DNA ca chnh n cng nh DNA vi khun v ng gi DNA vo trong cc
phage mi .V khi virut chui no trong mt vi khun mi n s t gn vo NST vi khun v
mang theo DNA ca vi khun
c gn t trc . Nh vy vi khun mi ny c ti t hp , hay cn gi l s chuyn
np (transduction).
Chuyn np l mt s kin t xy ra cc t bo vi khun , kh tm c cc v d . Tuy nhin,
tim nng ca chuyn np th rt to ln bi v nhiu virut c th trao i trong cc t bo vt
ch khi phage chuyn np .Chng hn nh Bacillus diphteria l nhng bn cng cho
bacteriophage m ho cho vic to ra cc c t ca chnh sinh vt ny . Salmonnella gy
nhim trng thc phm cng c hiu l vt vn chuyn bacteriophage . i vi ngi th cc
herpesvirus c th tn ti trong cc t bo thn kinh nh cp trn .Virut gy thu
u, virut gy nhim mononucleosis v HIV l cc virut c nghin cu k lng , n c
th ci a xit nucleic ca chng vo trong cc t bo ca ngi.

13

Hnh 2.6. nh qua hin vi in t ca mt Bacteriophage v vi khun ch ca n- E.Coli

.Nhiu Bacteriophage c th gn vo b mt t bo vi khun . Trong hnh phage ang sao chp
trong vi khun v c th nhn thy nhiu th trong t bo ch .
Qu trnh bin np, lin hp, v chuyn np cho php vi khun thu c nhng mu DNA mi
v duy tr c cc c tnh di truyn .
Trong nhng nm 1950 , cc nh sinh hc phn t ln u tin s dng t ti t hprecombined ch cc vi khun bin i gen v c DNA mi .Dn dn, t recombinant
DNA tr thnh thut ng Ho sinh . Cho ti nay, DNA ti t hp dng ch cc phn t
DNA c cha cc thnh phn DNA t nhin cng nh mt s DNA ngoi lai gn vnh vin vo
phn t gc . Nhng thc nghim vi vi khun trong nhng nm 1950 chng minh rng ti
t hp l mt kh nng xy ra trong t nhin . Cc nh khoa hc bt u t ra vn l c th
thc hin s ti t hp tng t trong phng th nghim khng ?
2.1.2.Cc enzym gii hn:
Cng trnh c quan tm ln trong nhng nm 1950 v DNA l cng trnh ca Salvador
Luria cng cng tc cung cp bng chng l E.Coli c th khng li s ph hu bi
bacteriophage . Tc l n c thhn ch s sao chp bi virut . Nm 1962, Werner Arber
v nhm cng tc chng minh rng c mt h thng enzym hn ch s sao chp ca virut do
n ct b DNA ca phage trc khi n ti t bo cht ca vi khun . H phn lp c
enzym t E.Coli v t tn l endonucleaza. Enzym ny ct DNA ca virut nhng khng ct
DNA ca vt ch vi khun bi v n c s ci bin trc tin DNA vi khun bng cch
gn thm cc nhm metyl vo DNA vi khun . V endonucleazahn ch s sao chp ca virut
(v bi v n lm khi ng v trhn ch s sao chp trn phn t DNA) nn dn dn c
gi l enzym ct gii hn (restriction enzyme).
Nhm nghin cu ca Arber pht hin ra rng enzym ct gii hn ca h t c gi tr thc
tin trong thao tc DNA v n ct DNA nhiu v tr . V th cho ti nm 1970 Hamilton
Smith v cng tc li phn lp c mt enzym gii hn mi t vi khun Haemophilus
influenzae . Enzym ny ct phn t DNA nhng im c th on trc c v v tr tc
ng ca n chnh xc hn enzym ca Arber. Khi s dng enzym ny Daniel Nathan v
nhm nghin cu ca ng phn lp c DNA t mt virut trn kh gi l Simian virut 40
(hay SV40) v chng minh rng enzym ca vi khun ny c th dng ct DNA virut mt
vi v tr no (Hnh 2.7.).

14

Hnh 2.7.Cc Enzym gii hn . (a) Werner Arber , Hamilton Smith v Daniel Nathans nhn
gii Nobel nm 1978 v Sinh l hc v y hc vi cng trnh v cc enzym gii hn . (b) Hot
tnh ca cc enzym gii hn (hoc Endonucleaza) . Enzym gii hn ct 2 chui ca phn t
DNA to thnh 2 mnh . (c)V tr nhn dng ca mt vi enzym gii hn. Mi tn ch chc
nng ca nhng enzym gii hn khc nhau . Lu rng v tr nhn dng c bit trnh t
nucleotit ca 2 chui DNA chy theo hng i nghch nhau . S i xng ny gi l i xng
quay 2 chiu .Cng lu rng 2 enzym HaeIII v Smal to ra cc mnh vi cc u khng sc
(blunt) trong khi enzym EIoRIv HindIII to nn cc u ung a . Nhng u ung a
ny cn cho k thut gn gen . Do nhng cng trnh ny m Arber,Smith v Nathan c
nhn gii thng Nobel v sinh l hc v y hc nm 1978.
Nh cc thnh tu nghin cu trong nhiu nm , cc nh cng ngh DNA thy rng h c th
s dng cc enzym gii hn ct nh phn t DNA theo mun bt k t ngun gc no .
Tht vy, enzym ct gii hn t vi khun c th dng ct mt phn t DNA im X , bt
chp n l DNA ca thc vt, ng vt hay ngi. Chng hn nh i vi mt s cc virut th
mt enzym c th ct c DNA ca cc virut khc nhau rt nhiu v tr .Thc t l c trn
1.200 enzym ct gii hn c phn lp v lm tinh khit t vi khun .Enzym thuc nhm

15

ny tc ng vo nhiu trnh t nucleotit khc nhau : c khong 75 enzym tr thnh thng

phm .
Cc enzym ct gii hn gi tn theo quy c quc t nh sau: Ch u tin vit ch in hoa ch
tn chi hoc loi vi khun m t cc enzym gii hn c phn lp (ch nghing)
Bng.2.1.

Hai ch tip theo vit ch thng ch ging vi khun (cng ch nghing) , tip theo l l mt
ch hoa ch chng vi khun v cui cng l s La m ch th t enzym gii hn c tm ra .
Chng hn nh cc enzym di y :
EcoRI t Escherichia (E)
co(co)
chng RY13(R)
Endonucleaza th nht (I)
BamHI t Bacillus(B)
amyloliquifaciens(am)
chng H(H)
endonucleaza th nht (I)
HindIII t Haemophilus (H)
influenzae(in)
chng Rd(d)
endonucleaza th III (III)
Vi mt c ch cha c xc nh , enzym gii hn qut trn phn t DNA v dng li khi n
nhn dng c mt trnh t ca 4 hay 6 nucleotit . Trnh t nhn dng (recognition sequence)
nh tn gi ca n l ni m phn t DNA b phn ct .Trnh t nhn dng biu l s i xng
2 vng , iu c ngha l cc nucleotit ui trnh t ny l b cu cho cc nucleotit mt
u khc (H.4.8c).
Thc cht 2 chui ca xon kp li c cng mt trnh t nucleotit chy theo hng i nghch
dc theo chiu di ca trnh t . Nhiu enzym gii hn ch ct cc v tr phn ui , nhng

16

mt s enzym gii hn khc li ct c nhng v tr cui ca nhng chui n , tc l khng

c nh (h 4.8c).
S sp xp c quyn nucleotit trn 2 chui c 2 tc dng:
1. V trnh t nhn dng l ging nhau trn c 2 chui (mc d chy tri chiu nhau), nn
cc enzym gii hn nhn dng v ct c c 2 chui ca DNA v th n ct c c 2 chui
xon kp .
2. Nhng v tr m 2 chui c ct thng khng phi l i nghch trc tip v th n c
th ct c mt vi nucleotit cc u cui cng .
C s ca s x dch ny l s b cu ln nhau ca cc nucleotit .
Mt iu quan trng cn nh l bt k mt ct b no bi enzym gii hn u xy ra cng
mt v tr nhn dng bt k ngun gc ca DNA t u (Hnh 2.8.)

Hnh 2.8. Thit lp mt phn t DNA ti t hp i t 2 vng DNA xa l . Enym gii hn EcoRI
tch DNA c 2 vng bi v mc du chng khng c quan h vi nhau nhng nhng vng ny
vn c cc v tr nhn dng ging nhau . Cc vng by gi tr nn thng . Ti im ny cc
u c th ni vi nhau to nn phn t DNA n di . S gn kt ny c xc tc bi
DNA Ligaza . Nhng s lin kt nh th l c s cho s ti t hp gen tng hp .
Mt iu na cng rt quan trng l v tr nhn dng c t nht l mt trn bt k mt DNA
no cho bt k ngun DNA no cng c th s dng c . Mi mnh DNA c cc nucleotit
u a (dangling) gi l u dnh-stickly ends l kt qu ca s tc ng bi enzym.
V bi v cc u ca chui n l b cu cho nhau nn chng c th cp i vi nhau hoc vi
cc on DNA khc c u dnh b cu. c tnh th hai ny to cho enzym gii hn tr thnh
mt cng c v gi i vi cng ngh gen tc l d ngun gc DNA c th l ca vi khun,
chim, hoc ca cy mao lng v d di n my i na th th cc u dnh ca cc on
DNA ny vn l b cu.
2.1.3.Ligaza :
Cc on DNA b cu khng th t lin kt vi nhau . iu chc chn l lin kt hydro s
c hnh thnh gia cc baz b cu . Nhng nhng lin kt ny khng bn kt ni cc
ui ny vi nhau mt cch v tn c bit l trong cc iu kin nhit sinh l . thc
hin vic gn lu di cc u cui ca DNA th phi s dng mt enzym , l DNA ligaza.

17

Ln u tin ngi ta phn lp c mt DNA ligaza t bacteriophage T4, n c th gn cc

b khung ho hc ca cc chui DNA do hnh thnh mt lin kt ho hc gia nhm phot phat
5t do ca cc on nucleotit vi phn t deoxy ribozaca nucleotit k tip. chnh l lin
kt phot pho dieste (phosphodiesster bond)-mt lin kt ng ho tr bn vng hn lin kt
hydro gia cc baz ni t i nghch . Lin kt phot pho dieste tn ti tt c cc nucleotit to
nn cc phn t DNA mi (Hnh 2.9.)

Hnh 2.9. Cc lin kt dnh cc on DNA vi nhau trong phn t DNA ti t hp . Lin kt
hydro l lin kt yu gia cc baz b cu , cn lin kt bn vng c to bi Ligaza . Lin
kt ny ni cc b khung ca chui DNA .
DNA ligaza c phn lp ln u tin vo nm1960. N chng nhng c trong cc virut m
cn c c trong cc t bo nhn chun v E.Coli. N ni cc on DNA vi nhau v tham gia
cng cc enzym khc trong s tng hp DNA .
Enzym ny cng gip cho vic sa cha DNA do n gn li cc mnh v ca DNA, nh vy
m lm hi phc li cc vt thng ca t bo . Trong cng ngh DNA n ng vai tr l s
gn kt cui cng trong cc chui s kin dn n s hnh thnh mt phn t DNA ti t hp
(H4.11)
2.1.4.Plasmid
Nhng nm u ca nhng nm 1970, Paul Berg v cng tc ti Trng i hc Stanford
kho st kh nng s dng cc enym gii hn lm thay i cu trc ca DNA. Nhm nghin
cu ca Berg phn lp c NST ca E.Coli v ct nh phn t DNA bng enzym gii hn .
Tuy nhin , DNA c cc ui n gin nn rt kh gn vo cc DNA ngoi lai chng hn nh
virut (cng tng t nh vic gn mt vin gch vo cui mt bc tng) .
Mc du v, ngui ta thng li trong vic to ra phn t DNA ti t hp u tin. Sau
Berg c chia gii Nobel ho hc nm 1980 do nhng cng hin ca mnh .
Cng vo thi im c 2 s kin xy ra lm mt cuc cch mng trong cng ngh DNA
. S kin th nht l Herbert Boyer tch chit c enzym gi hn EcoRI ct b cc
ui nh kiu tho cc l mng . S kin th hai xy ra Labo Stanley Cohen thuc
Trng i hc Stanford . Cohen thu thp c cc d liu trn cc vng DNA nh b thy
t bo cht ca vi khun (khng thy cc sinh vt c cu trc phc tp) . Cc vng DNA
chnh l Plasmid . Plasmid nm ngoi NST vi khun v chc nng ca n cha c hiu r
y . Plasmid cha t th mt t m nhiu th hng hng trm gen (vi khun c khong vi
ngn gen trong NST) n chim khong 20% thng tin di truyn ca c th . Mt iu r rng l
plasmid khng cn thit cho s sinh trng ca vi khun v khi loi b n i th cng chng
gy hi g cho vi khun .

18

Hnh 2.10. Hot tnh ca DNA Ligaza . DNA Ligaza gn cc on DNA vi nhau bng cch
lin kt gc phot phat v tr 5 ca on ny vi v tr 3 ca phn t Deoxyriboza tip theo.
chnh l lin kt photphodieste. Lin kt ny chc hn lin kt Hydro , n hnh thnh gia
cc nucleotit i du .
Nm 1972, Cohen thit lp c mt plasmid mi t mt plasmid c t trc E.Coli. Plasmid
mi ny c 3 c tnh quan trng :
1.N c mt v tr nhn dng n l ch EcoRI phn ct , v th m xc nh c ni m
plasmid s m ra .
2.C mt trnh t nucleotit gi l im gc sao chp (origin of replication) , trnh t y thc
y s sao chp ca plasmid trong c th tc ch .
3.Cha mt gen khng tetraxyclin .
V vy cc vi khun cha plasmid ny th khng li tetraxyclin , cn nhng vi khun khc
khng c plasmid s cht khi c mt tetraxyclin .Cohen t tn plasmid l pSC101
(SCtc l Stankey Cohen).
Mt c trng quan trng khc ca plasmid Cohen l d dng ci vo t bo tc ch .

Cohen pht hin ra rng c th ci cc plasmid vo cc vi khun mi bngo cch treo n

trong dung dch Canxi clorua , sau li nhanh chng lm nng vi khun ti 42 C . Vi cch
x l nh vy th vch v mng sinh cht ca vi khun s m ra , cho php plasmid i qua
vo t bo cht ca vi khun . Khi bn trong vi khun th mt plasmid n t n nhn ln
to nn hng t plasmid mi . Nu plasmid cha mt gen ngoi lai th gen ny c sao chp
cng vi nhng phn cn li ca phn t . V cc vi khun c cha cc plasmid cng c nhn
ln thng c 20 pht mt ln v mi mt vi khun mi li c vi plasmid mi . D nhin l
trc vi khun sn sinh hng triu th h con chu ri . Mt qun th nh th xut pht t
mt t bo cha m c gi l mt dng (clone). Tt c cc t bo trong cng mt dng u c
cc plasmid ng nht . Cng tng t nh vy, hin nay c hng triu bn sao ca cng mt
gen ngoi lai. V th gen cng phi tch dng .
Khng phi tng tng nhiu cng hiu c rng plasmid l cc vt mang hay l cc vec t
i vi gen ngi trong cc th nghim cng ngh DNA.
Giai on hin ti l tp hp cc th nghim DNA ti t hp lm thay i tin trnh lch s
Ho sinh .
2.2. cc th nghim v DNA ti t hp
Theo mt s s gia th nguyn l ca cng ngh DNA ging nh chic bnh sanwich b bin
Waikiki .Nm 1972, xy ra s kin nh sau : khi Herbert Boyer pht biu trong mt hi ngh

19

khoa hc Hawai v enzym gii hn EcoRI th Stanley Cohen cng tham gia hi ngh nhng
vi cng v l thnh gi . Sau khi kt thc din thuyt Cohen mi Boyer n tra v bc bch
rng h c th cng tc vi nhau trong hng lot cc th nghim . Sau 2 nh nghin cu
ngi li vi nhau v xem xt cc th nghim a cng ngh DNA vo mt k nguyn mi .
Cohen ch o thnh cng cc th nghim vi plasmid , nhng ng li gp kh khn khi ct m
rng cc plasmid v enzym EcoRI ca Boyer dng nh l mt gii php l tng v th Cohen
ng cng hp tc nghin cu . H dng plasmid ca Cohen v enzym ca Boyer thc hin
ti t hp DNA plasmid .
Trc tin, h s tin hnh t hp 2 plasmid to nn mt plasmid n.Nu thnh cng h s
a DNA ngoi lai vo plasmid to nn mt phn t DNA ti t hp . Boyer ng v bt
u vo cng vic .
Cc th nghim ca Boyer v Cohen c tin hnh vo nm 1973, sau 20 nm Watson v
Crick cng b trn bo v lch s cu trc DNA . Plasmid m Boyer v Cohen s dng l
pSC101 (Hnh 2.11.).

Hnh 2.11. nh hin vi in t ca Plasmid vi khun . Lu rng DNA dng vng khp kn .
Mt s vng li ni lng , trong khi c cc vng li son cht .
Trong cc th nghim u tin, Boyer v Cohen t hp thnh cng pSC101 vi pSC102 v
tch dng vi cc t bo E.Coli. Cch tip cn ca h tht l th v . Plasmid pSC101 c mt
gen khng cht khng sinh kanamyxin . Khi cc plasmid ti t hp hp nht c vi vi
khun th vi khun ny bc l kh nng di truyn mi tc l hn ch c tc dng ca c
tetraxyclin v kanamyxin.
Ri ti l th nghim th hai vi DNA ngoi lai (Hnh 2.12)

20

Hnh 2.12. Cc th nghim ca Boyer-Cohen nm 1973 .(a) Plasmid pSC101 c m ra bng

enzymr gii hn EcoRI im ch mi tn .Plasmid c cha gen khng tetracyclin c m hnh
l tet .(b)DNA t cc Xenopus Laevi cng c x l vi EcoRI v thu c cc on DNA
ngoi lai .Lu rng EcoRI tc ng nhng v tr nhn dng ging nhau vec t Plasmid v
DNA cho .(c)Cc on DNA cho gn vi plasmid m v xy ra s b cu cc baz . (d)
Khi thm DNA Ligaza , plasmid s ng li v hnh thnh phn t DNA ti t hp. (e) Plasmid
c a vo cc t bo E.Coli nhy cm vi tetracyclin (tets) bng cch x l t bo vi hp
cht can xi . Cc t bo E.Coli bin np . Cc plasmid s nhn ln trong t bo v m cho
cc protein chuyn ho bi DNA ca cc .(f) Khi vi khun c nui cy trong mi Trng c
tetracyclin th cc t bo c cha plasmid ti t hp s pht trin v to cc khun lc . iu
xy ra bi v n c gen khng li tetracyclin .Vi khun c cha plasmid bnh thng khng
c gen khng tetracyclin s khng to c khun lc trong mi trng .
Boyer v Cohen thu thp mt gen t cc t bo cc Chu Phi Xenopus Laevis .Gen ny m ho
cho mt on protein dng tng hp RNA riboxom . H s dng EcoRI ct DNA ca cc
, sau cng dng enzym ny m plasmid pSC101. S d dng EcoRI cho c hai bi v cc
ui ca chng u tng t nhau. Cc on DNA ca cc c trn vi DNA plasmid v s
cp i gia cc baz b cu xy ra trn c 2 on DNA . Thm DNA ligaza vo th cc
plasmid c ti sinh tun hon tr trng hp by gi plasmid li cha cc DNA c ci
vo t cc .Cc nh nghin cu gi plasmid ti t hp ny l Chimera.
Tip theo , ngi ta xem xt kh nng m ho protein ca cc DNA ngoi lai . Bng cch s
dng cc k thut tiu chun Cohen a plasmid vo trong cc t bo E.Coli v t vi khun
vo mi trng pht trin .Vi khun v plasmid ti t hp ca chng c sao chp tng
pht v trc cc t bo E.Coli to ra thm mt protein cho RNA riboxom m thng
thng protein ny ch c to nn t cc t bo ca cc . y E.Coli tng hp nn
protein ca n v tng hp thm c protein ca c th khc (X .laevis) .
Cc phng tin truyn thng quc gia c ng cho nhng thnh cng ca Boyer v
Cohen.Thc cht hai nh khoa hc ny ph v hng ro ngn cch gia cc mu sinh hc
tch bit v m ra mt k nguyn mi hin i ca cng ngh DNA . V vy mt quan st
21

vin ghi ch nh sau Cng ngh sinh hc trc Cohen ( BBC Before Boyer-Cohen) v
nay l sau Cohen ( ABC tc l After Boyer-Cohen)
Cc nh sinh hc nhanh chng xem xt cc mi lin quan ca cng ngh DNA ti t hp v
khn trng tin hnh cc th nghim thao tc gen .V ch trong my tun cc nh khoa hc
pht hin ra s chuyn gen v c gng vt qua cc phn cch v mu .Trong mt th nghim
ngi ta chuyn gen ca Staphylococcus Aureus cho E.Coli. Cc nh khoa hc khc li c
nh phn lp cc gen ngi v a vo plasmid ca vi khun . Cn s khc th i su vo cc
mi lin quan cht ch gia cc th nghim trong cng ngh DNA . Chng hn nh ngi ta suy
lun rng cc gen c th c a vo cc t bo sng gii to s thiu ht di truyn cng
nh c th to c mt lng ln protein dc phm him .
Ngi ta c vng c c nhng ngun nng lng sinh hc r tin hay tr em sinh ra khng
b khuyt tt. Dng nh khng c gii hn trong nn cng ngh mi ny .
V phng din k thut Trong Gen tr liu ngi ta s dng 2 phng php chuyn gen :
Phng php chuyn gen ngoi c th (ex vivo ) v phng php chuyn gen trc tip vo c
th (in vivo) .
Mt s k thut thng dng trong gen tr liu l : K thut vi tim , K thut in xung , k
thut bn gen , k thut liposom , k thut vin gen v.v.. Nhng vn ny chng ta s bn
lun sau .
2.3.Nhng bn c i v tnh an ton trong gen tr liu.
Mc d st sng vi cng ngh DNA nhng nhiu nh khoa hc cnh bo v nhng hu qu
nguy him ca cng ngh ny gi cn phi thn trng hn vi cc xu hng nghin cu .
Chng hn nh c s bo ng khi nhm nghin cu ca Paul Berg d nh a mt gen t
virut gy ung th vo E.Coli.Cc ng nghip ca ng vch ra rnh mch rng cc t bo
E.Coli ti t hp t phng th nghim khi vo c th ngi ( chng vn sng mt cch
bnh thng) th n s biu l cc gen ung th . Berg phi xem xt nhng li phn i v
hu b th nghim ny .
Nhng nhng vn v tnh an ton vn lun c t ra . Ch mi gn y thi cc nh sinh
hc phn t li bt u tho lun v cc vn lin quan n cng ngh DNA trong mt hi
ngh c t chc di s bo tr ca vin hn lm khoa hc quc gia .Ti nm 1974 , mt s
kin cha h thy xy ra , l mt bc th c c ch k ca Paul Berg v 9 nh khoa hc
khc xut hin ng thi trn 3 tp ch khoa hc uy tn nht Th gii vit bng ting Anh l
Science, Nature v Proceedings of the National Academy of Science. Bc th ch r cc
nguy hi tim tng ca cng ngh DNA ti t hp . Bc th c on vit:
Nhng tin b mi y trong k thut phn lp v ghp ni cc on DNA ti nay cho
php kin to cc phn t DNA c hot tnh sinh hc in vitro.
Mc d nhng th nghim ny c v to thun li cho cc gii php v cc vn thc tin v
l thuyt quan trng trong sinh hc nhng vic to nn cc dng DNA mi m bn cht sinh
hc ca cc phn t ca chng khng th d on c mt cch y
C mt vn lin quan rt nghim tc l mt s trong cc phn t DNA ny c th c
chng minh l c nguy hi v mt sinh hc

22

Hnh 2.13. Cng ngh DNA vi vic tch dng gen ngi v cc sn phm protein sn xut vi
s lng ln c m ho bi gen ny.
Bc th tip tc hi kin cc nh sinh hc phn t trn ton Th gii liu c t nguyn tr
honmt s th nghim v DNA cho ti khi c mt hi ngh quc t nhm hp tho thun
v cc nguy hi c th v cc bin php bo v cn thit hay khng?
Cc nh khoa hc cng nhc nh cn phi thn trng vi cc th nghim c lin quan ti vic
a mt gen khng cht khng sinh vo plasmid v gn mt gen ung th vo bt k mt phn t
cht mang DNA no .
y l ln u tin cc nh khoa hc thuc nhiu lnh vc khc nhau i hn ch cc nghin
cu mc d chng c bng chng no chng minh n l nguy him .
Mc d qu trnh ti t hp khng vi phm lut php , nhng cc nh khoa hc ton Th gii
vn phi ng k cc d nh trong cc th nghim khoa hc ca mnh.Thng 2-1975 , mt
nhm gm 139 nh nghin cu t 17 quc gia hp mt trong 4 ngy Asilomar , mt
trung tm v hi ngh Pacific Grove , California nhm a ra nhng hng dn v cc khuyn
co cho vic ch o cc th nghim trong cng ngh DNA . Mt trong s nhng iu trc
tin nhng c to phi lm l cam oan vi cng chng rng vi khun dng cho cc th nghim
DNA ti t hp l nhng ni c bit v gii tr qun b nn chng khng cn tn ti
ngoi phng th nghim . Cc nh khoa hc cng vt ln vi tnh trng kh x nht l thiu
cc bng chng hoc ng h hoc phn i v bn cht gy nguy hi ca cc cng trnh v
DNA .
Cui cng , cc nh nghin cu chn bin php nhc nh v hoch nh chnh sch cho cc
cng trnh ca h . Cc th nghim DNA ti t hp phi lun xem xt ti cc ri ro cc mc
thp, trung bnh v cao v quyt nh rng bt k mt th nghim no m c ri ro (nh Berg

23

 nu) u phi c ch o mt cch cht ch cho ti khi c mt phng php tt hn .

Chin lc ny bao hm c trong lnh vc vt l cng nh sinh hc v u tin nhiu cho vic
pht trin cc chng vi khun khng c kh nng sng bn ngoi phng th nghim .
Khuyn co Asilomar dn ti vic hnh thnh mt U ban c vn v DNA ti t hp ti vin
sc kho quc gia Hoa k(NIH). U ban ny s a ra cc hng dn y song song vi cc
khuyn co Asilomar.Nm thng tri qua , mi iu c sng t trc cng chng l cc
nh nghin cu v DNA khng xa ri cc o lut x hi.
2.4.Tng lai ca cng ngh DNA ti t hp
Nhng th nghim c thc hin nm 1972 v nhng nm sau tc ng su sc ti cch
suy ngh ca cc nh di truyn hc . Trc nhng thnh cng v cc th nghim tch gen , cc
nh di truyn hc suy lun qua cc nghin cu ca mnh rng tt c nhng iu m h hiu v
c im v s biu hin ca gen cho ti gi h u c th thao tc c . Cng ngh DNA
cho php cc nh di truyn hc hiu c cu trc , chc nng cng nh s iu ho gen v bt
u i su vo di truyn Ho sinh cc bnh lo khoa .
i i vi nhng thng li trong cng ngh DNA th ng thi cng xut hin thm cc nguyn
l ca cng ngh sinh hc . Cng ngh sinh hc l mt nn cng nghip rng ln v hon ton
mi trong sinh hc phn t c th tham gia gii quyt cc vn v nhim, cc ch phm
thc phm , nng lng v s tng hp cc loi dc phm mi . Cc nh Ho sinh sm ti
vi khun , coi l cc nh my ho hc tng lai v suy lun rng c th lp chng trnh cho
DNA tng hp mt s lng khng hn ch cc cht c ngha i vi cng nghip , kinh t
v y hc.
Sau nhng nm 1970, mt s ha hn thnh hin thc, nhiu nh my bt u p dng k
thut DNA sn xut nhiu sn phm hu ch. Chng hn nh nm 1980, mt cng ty thu
c insulin t vi khun ti t hp vi nhng gen ca cc t bo tu ngi . Mt cng ty khc
cng s dng cc t bo vi khun to ra interferon mt cht c ch virut m thng
thng ch c to ra t cc t bo ca ngi . Nhiu cng ty khc cng ti t hp DNA
trong vi khun cho vi khun ny c th to nn cc cht nh hormon sinh trng ca ngi ,
hay mt vacxin cho mt bnh no ca ng vt hoc cc enzym ho tan trong du
Trong nhng nm 1980, nhiu sn phm khc ca cng ngh DNA c d bo l hin thc
hoc l c kh nng tr thnh hin thc . Cc vi khun ti t hp c dng trong vic loi thi
cc cht c hi , ho tan cc cht kh tan trong nc nh tc chng hn . Vi khun ti t hp
tng hp c cc enzym ho tan cc mu ng ca ngi l Urokinaza v hormon ca thn
l erythropoietin . Hng trm cng ty trn Th gii ng dng cng ngh DNA trong cng
nghip .
Nhiu nh khoa hc pht biu mt cch lc quan v mt tng lai trong s th tinh s l
li thi , thc vt c th s dng cc c t vi khun xua ui cn trng v hoa mu c th
c trng cy m khng b ph hi ca sng gi .
Ti nhng nm 1990, cc nh nghin cu i qu xa so vi vic ci mt DNA ngoi lai vo
cc t bo vi khun v cng ngh DNA pht trin trong rt nhiu lnh vc . Chng hn nh
in du vn DNA l mt dng mi ca php y c cc h thng php lut chp thun .
Trong gen tr liu con ngi c truyn vo cc gen ngoi lai cha cc bnh .Hay ng
vt bin i gen c c h thng min dch ca ngi .Ri d n tm hiu trnh t Baz cc
gen n ca ngi
Hn bt k mt k thut no khc , cng ngh DNA s cho nhn loi c hi lm ch c
nhng phn t to nn chnh h. Cc nh khoa hc thch th khi con ngi cng hiu r hn
na v c th mnh cng nh cc ng tc nh b, bi li , chy nhy hay bay ln v.v..
Kh thy nhng vn v s tn ti ca con ngi m li khng ng ti cng ngh DNA cng
nh cc vn chnh tr ni ln i hi phi c cc gii php c tnh ton cu.Chng hn nh
liu cng ngh DNA c cho php gen can thip vo vic ci bin ging ni hay khng?
Hay cc vn thuc o c , nhng bn khon v tinh thn s cn cn tng bc ny sinh
trn con ng i ti ca D truyn hc .
Nhng c mt iu m ai cng phi tha nhn l cng ngh DNA i vo cuc sng con
ngi v tr thnh mt trong nhng ng dng c tnh thuyt phc nht .
2.5.Nhng tin b ng k trong gtl hin nay :
*Nhm nghin cu Trng i hc California , Los Angeles s dng Liposome c bao
polyme Polyethylen glycol (PEG) a vo no . Vic chuyn gen vo cc t bo no l mt
thnh cng rt c ngha bi v cc vec t virut l qu ln vt qua c hng ro gia no
v mu . Thnh cng ny to tim nng cho vic tr cc bnh Parkinson .(20/3/2003 New
Scientist .Com March 20, 2003 ).
*S can thip ca RNA hay cc gen ln c th l mt phng php mi iu tr bnh
Huntington. ( New Scientist . Com March 13, 2003).
24

*Phong php TLG theo cch sa cha nhng sai st trong RNA thng tin ca cc gen khim
khuyt . K thut ny l tim nng iu tr cc bnh v mu nh bnh thiu mu vng bin ,
bnh x nang ( mt bnh di truyn tc ng ti cc tuyn ngoi tit ) v mt s bnh ung th .
( New Scientist ,Com october 11, 2002.)
*TLG dng trong iu tr bnh X-SCID . Tuy nhin , vic ny dng li khi mt bnh nhn
Php b mc bnh bch cu (Leukemia) . (New Scientist .Com October 3, 2003).
*Cc nh nghin cu Trng H Case Western v Copernicus Therapeutics c th to c
cc liposome nh ti 25 nanomet , n c th mang DNA tr liu qua cc l nhn t bo (New
Scientist . Com May 12, 2002)
*Cc t bo hnh li lim iu tr thnh cng trn chut ( The Science March 18, 2002).

Chng III
Cc phng php ca cng ngh DNA
3.1.Thu thp gen
Cc t bo ca c th sng bao gm hng nghn gen hoc nhiu hn na.C th chng ta c ti
100.000 gen trong b gen (genome ).Nu ch cn nghin cu mt gen n th nhim v ca
nh Ho sinh cng rt ln bi v mt gen cng c ti hng nghn milimet chiu di DNA .
Tuy nhin, cng vic ny tr nn tht n gin nh s dng mt enzym ct gii hn(restriction
enzyme) , cc enzyme ny c th ct cc phn t DNA cc v tr c hiu , iu cho php
cc nh sinh Ho tch ring c cc on DNA.Vic s dng nhiu enzym gii hn lm
tng kh nng nghin cu cc gen nm trn nhng on DNA ny .
Tuy nhin nh s dng cc phn t RNA thng tin (mRNA) nn cng khng cn thit phi tm
bng c mt gen c hiu trong cc on DNA . Nh ta bit , biu hin gen thng qua s
tng hp protein trong t bo lin quan ti s m ho ca cc phn t mRNA . Khi ta mun
tch mt gen tng hp protein X th ta phi tm kim cc t bo hot ho to nn protein X
tch ra cc phn t mRNA t t bo cht ca t bo .Khi xc nh c chnh xc cc t bo
cn nghin cu , lc ny ta ph v t bo , cc thnh phn ca t bo c x l bng hng lot
cc phng php l ho hc v thu c cc cht nh protein, cc cht bo, carbohydrat v acid
nucleic . Cc phn t mRNA c thu thp nh li th ca cc ui poly-A (poly A tails),
phn RNA c ti 150-200 nucleotit cha adenin (v th mi c tn l poly A) . Phn ln mRNA
u gn vi cc ht xenluloza m trn b mt ca chng c nhiu mnh acid nucleic cha
thimin poly T. Cc mnh v cn li s c ra loi i . V by gi c th thu thp cc
phn t mRNA t cc ht xenluloza c c mRNA c c .
Sau khi c c mRNA tinh khit th nhng thng tin Ho sinh m n cha ng c th
chuyn ngc li trong DNA . thc hin c bc ny , cc trnh t ca cc baz ni t
trong mRNA c s dng nh mt ci khun tng hp cc trnh t baz b cu trong DNA
. Mt enzym gi l Enzym phin m ngc (Reverse transcriptase) gip cho vic tng hp ny
. Chng ta nh li rng vo nhng nm 1970 Howard Temin v David Baltimore pht
hin ra v enzym phin m ngc , ngi ta s dng RNA nh mt ci khun tng hp mt
phn t DNA b cu . Khi s dng enzym phin m ngc to nn cc phn t DNA th i
hi phi c mt cht mi (Primer)-hay l cn s c mt ca cc trnh t nucleotit khi u .

25

Cht mi ny bao gm cc chui nucleotit ca Thimin (polyT), cc chui ny gn vi ui poly

A ca mRNA v hot ng nh mt v tr khi u cho s tng hp DNA. Sau reverse
transcriptase dch chuyn dc theo cc phn t mRNA m ho cho mt phn t DNA b cu
vi mRNA ( complementary DNA= cDNA) .Phn cui cDNA c mt nucleotit vng xon
ngn c a vo nh mt enzym , l do ti sao vn cha r .Nu chui n cDNA l ph hp
vi mong mun th vng xon nucleotit ny s c loi b bi enzym nucleaza v cDNA c
th tch khi cc khun mRNA dng tinh khit.

Hnh 3.1. Phng php chung to DNA b cu (cDNA) (a) cc t bo tng hp protein nh
cc t bo tu c gi cn thn v mRNA c tch chit ra t nhng t bo ny , (b)mRNA
c x l vi enzym Transcriptaza ngc . Enzym ny s dng nhng m baz ni t trong
RNA tng hp chui b cu ca DNA . cDNA sau li dng tng hp mt chui DNA
b cu vi chui th nht .
to c mt gen lng vo t bo , ngi ta phi to ra cc phn t DNA chui kp .
hon tt qu trnh ny , vng xon nucleotit phi c t ng ch , khi phi dng enzym
DNA Polymeraza . Enzym ny s dng nucleotit vng nh mt cht mi v di chuyn xung
phn t DNA . S dng phn t ny nh mt ci khun tng hp phn t DNA b cu . Lc
ny vng xon c loi i nh nucleaza v kt qu l c mt phn t cDNA xon kp
tng t nh gen m ho nguyn gc phn t mRNA .
Tuy nhin , phn t cDNA khng c intron (trong gen nguyn gc th vn c) m n ch cha
cc exon ( Hnh 3.2)

26

Hnh 3.2. S hnh thnh mt phn t cDNA chui kp (bc 1)RNA chn c phn lp vi
ui poly-A trong phng th nghim . Transcriptaza ngc v Deoxynucleotit c s dng
tng hp DNA b cu (cDNA) trong c ui poly-T b cu .(Bc 2) phn t RNA c
phn hu bi kim , cn cDNA c cc vng hnh ci kp tc u 3 .(Bc3) cDNA va
ng vai tr l khun va l primer cho s tng hp mt chui cDNA b cu .(Bc4) vng
hnh cp tc vn gn vi nhn to mt cDNA chui kp ph hp ci vo mt vec t. Cc
cDNA ch c cc Exon.
cho cc cDNA c chc nng trong cc t bo nhn , cc phn t ny phi c kh nng t
nhn i. Di cc iu kin bnh thng , cc on DNA ngu nhin khng ti bn c
trong t bo sng .L do l enzym DNA polymeraza ch c khi ng nu c mt mt trnh
t c bit ca cc baz l cc gc sao chp ( origin of replication) . M cc origin of
replication li c cung cp bi cc DNA cht mang (carrier) , ni m cc cDNA gn vo .
Cc cht mang (carrier) thng l cc vec t.
3.2.S la chn cc vec t.
i vi cc th nghim cng ngh DNA th cc vec t thng c dng l cc plasmid.
Plasmid l mt DNA dng vng khp kn nm ngoi NST . ng quan tm l cc plasmid ca
vi khun (H nh 3.3.) , cn vi cc t bo nhn chun nh cc t bo ng vt v thc vt th
khng c plasmid .

27

Hnh 3.3. nh.hin vi in t hin r plasmid nh l mt phn t DNA iu ho . Trong

hnh Plasmid l nhng vng nh .Phn t DNA c phn lp t lc lp ca cy xanh c cu
trc vng ln gia hnh.
Chc nng ca plasmid cha c hiu y . Tuy nhin cc nh khoa hc xc nhn rng
plasmid khng cn cho s tn ti ca t bo . Vo nhng nm 1970, cc nh khoa hc Stanley
Cohen v cc cng tc pht hin rng plasmid c th c m ra v gn c vo cc on
DNA to nn plasmid ti t hp hay cn gi l chimera . Cc chimera cha cc origin of
replication i hi bi DNA polymeraza.Qu trnh gn thm mt on cDNA vo plasmid phi
s dng rt nhiu enzym gii hn . Cc enzym gii hn qet trn cc xon kp ca plasmid cho
ti khi nhn ra c trnh t baz c hiu . Sau plasmid c m ra dng ngon ngeo ,
loi i 4 baz trn mi si b ct . 4 baz ny gn lng lo vi cc baz b cu trn phn t
cDNA c m .Tip theo, enzym ligaza gn b khung pht pht , ng ca plasmid v
cDNA. Ngi ta ly DNA ligaza t bacteriophage T4 to ra lin kt bn vng v lm n
nh chimera a vo cc t bo tip nhn mi .
Plasmid cng nh cng c li cho cng ngh DNA v plasmid nh t b h hi hn cc plasmid
ln khi tri qua qu trnh phn lp.Hn na, mt plasmid nh c th gn vi t bo vt ch hu
hiu hn. Gii hn v kch c plasmid phi tnh sao cho cc phn t cDNA khng qu ln c
th c ci vo .
Theo chc nng cc gen trn plasmid ngi ta chia ra nhiu loi plasmid nh plasmid gii tnh
(F) , plasmid khng khng sinh (R) , plasmid col (c gen m ho Colicin)... Plasmid pht hin
u tin E.Coli c k hiu l ColE1.T cc plasmid t nhin phn lp c ngi ta to
nn nhiu th h plasmid nhn to khc nhau vi nhiu c im qu thun li cho vic tch
dng .
Plasmid th h th nht l nhng plasmid u tin c s dng tch dng vec t pSC101
(Stanley&Cohen ,1973),ColE1(Hershfield,1974) .
Plasmid th h th hai c to ra bng cch tp hp cc c tnh qu ca nhiu plasmid t
nhin hoc gn thm cc gen ch th c mt plasmid mi . Tiu biu cho plasmid th h
ny l pBK322 (Bolivar v cng tc ,1977). Plasmid pBK322 c kh nng ti bn cao , cho
php gn cc on DNA c ti 6kb vn hot ng bnh thng .
Plasmid th h th ba l nhng plasmid mnh ,kch thc rt nh v mt polylinker
(polycloning site ) rt c a chung trong cng ngh gen .Polylinker l mt on
polynucleotit tng hp mang mt chui cc v tr nhn dng duy nht ca nhiu loi RE
(restiction Enzyme)
Nhm cc plasmid pUC
in hnh l pUC18 c ci bin t pBK322 , kch thc khong
2.686bp mang gen ApR v mt phn gen lacZ , xen gia gen lacZ l polylinker. cc plasmid
nhm ny ch khc nhau v di ca polylinker.Plasmid nhm pUC c kch thc nh , vng
polylinker cho php gn bt k mt trnh t DNA l no .
Nhm plasmid pSP v Gemini c kch thc khong 3.000bp, mang cc gen ApR v
polylinker , khng mang gen lacZ. Vec t pSP mang promoter c trng cho RNA hai bn
vng polylinker (pSP64,pSP56,Gemini...) u im ni bt ca plasmid nhm ny l cho php

28

phin m cc on DNA ngn trong vec t to nn nhiu RNA, cc RNA ny c dng lm

mu d hay nghin cu cu trc , chc nng ca RNA.

Hnh 3.4. Tng hp v s dng Cosmid ,(a) DNA thu nhn t Bacteriophage Lamda ,(b) DNA
m ra v cc on cDNA c ci vo nh enzym ,(c) to thnh Cosmid,(d) Cc m di truyn
cha cosmid cho yu t ny i vo vi khun v nh vy l ho vi vi khun ,(e) y ngi
thng ip hnh thnh mt th ging plasmid c mang cDNA.
Mt vec t khc cng hay dng l cc Cosmid . Cosmid l mt on DNA c to ra bng
cch ci mt phn t cDNA vo gia cc trnh t Cos cui cc phn t DNA . Phn t DNA
dng to Cosmid c ly t virut, thng l bacteriophage lamda , n sao chp c trong
vi khun . Cc virut nh bacteriophage c cha cc m di truyn cho cc yu t cho php thm
nhp t bo khi sao chp . Khi bn trong t bo ch , DNA dng thng ca virut gi thng
ip ti cc vng ging plasmid do gn vo cc trnh t Cos ca n . S dng Cosmid cng
ging nh su ch vo kim ri kt li cc u lng lo to nn mt Plasmid . Cc Cosmid c
kh nng vn chuyn cc on DNA ln vo bn trongbt bo v chng c kh nng thm nhp
vo t bo mt cch d dng
Vec t th 3 l mt Virut c thit k li mang cc mu DNA ngoi lai nh
cDNA.Trong nhng nm gn y cc virut c RNA nh Retrovirut c dng chuyn
ch cc gen ti cc t bo ca ngi. Virut c th c thit lp cho vic vn chuyn m di
truyn cu cc on vi khun s dng n nh cc tc nhn gy min dch v thng gi l
vacxin vec t vi khun .N i din cho s tip cn mi vi vic tim chng cho mt qun th
dn c rng ln v cng l nguyn nhn lc quan v cc vac xin tng lai .
Mt virut c s dng rng ri vi t cch l mt vec t l Bacteriophage hay n gin
gi l Phage .Mc d khng cn thit xy ra , nhng b gen ca phage thng t sao chp
trong t bo cht ca vi khun . Nhiu dng ca phage b gen ca n li gn vi cc NST ca
Vi khun v nm lu di . V vy b gen ca phage c th dng chuyn cDNAti mt
NST vi khun . Mt qu trnh tng t nh vycng xy ra cc virut ca mt loi thc vt
hay cc virut ca ng vt (tc l retrovirut) vi cc t bo vt ch . Hn na , c mt im
gc cho s sao chp (origin of replication) th mt vec t thng ch c mt v tr n , ni dnh
cho enzym gii hn c bit , mc d enzym c th ct vec t rt nhiu im . Vec t ny cn
c mt b phn ging nh maker gene (du chun gen) gip cho vec t c th nh v c
trong cc t bo. Gen khng khng sinh l mt du chun thch hp bi v cc t bo c du
chun vn cn tn ti khi x l vi khng sinh cn nhng t bo khng c du chun th s b
cht. Gi thit l ci c mt gen th maker gene phi hot ng .
Mt yu t khc lm tng gi tr ca vec t ny l tnh thch nghi ca n vi cc t bo vt ch .
Tnh n nh ca vec t c th t c bng cch khi mun a mt gen vo th phi kim
sot cht ch thi im a gen vo sao cho ng thi k phin m , chng hn nh trong thi
k t bo sinh trng nhanh .Tiu im kim sot s biu hin gen trong cng ngh DNA l s
hot ng v tr promoter mt trnh t baz to nn s hnh thnh RNA thng tin .
Tnh n nh cng t c bng cch s dng NST thay v cc vec t plasmid .
gim bt kh nng mt ton b plasmid , cc nh cng ngh DNA a vo cc t bo
mt lng ln plasmid gi l high copy number-nhiu bn sao v c s c mt cc t bo c
cha plasmid trong lc phn chia t bo .

29

Hnh 3.5. .Bacteriophage Lamda Phage i vo cc t bo vi khun , li DNA ca n gn

vi NST ca vi khun . Khi t hp vi DNA ngoi lai , phage ng vai tr nh mt vec t cho
DNA .
Mt cch khc duy tr plasmid lm cho cc t bo ph thuc plasmid phi sng tip tc .
Chng hn nh mt gen cho mt enzym cn thit no c th c a vo plasmid cng vi
mt gen khc. Plasmid s ci vo t bo cc gen ca enzym cn thit . tn ti, cc t bo
phi c plasmid (v gen to ra cc enzym) . V th ch c cc t bo c plasmid th mi tn
ti c khi phn chia t bo .Cng c th l cn phi hp nht mt gen khng khng sinh vo
plasmid . V nh vy th ch cc t bo c cha plasmid khng li khng sinh mi tn ti cn
cc t bo khc th s b cht .
3.3.La chn cc t bo ch
Bn cht ca vec t cng quan trng nh bn cht ca cc t bo hay c quan vt ch . Mt i
hi khc l cc t bo vt ch phi thch hp c vi vic nui cy trong phng th nghim v
c kh nng hp nht vi cc vt cht di truyn ca vec t . V cc nh Ho sinh cng phi c
kh nng kim sot c s biu hin gen trong cc t bo ch v thu thp c cc sn phm
ca gen .
Mt trong s cc phng tin vn ti ln nht i vi s biu hin protein l vi khun
Escherichia Coli (Hnh 3.7a).
Nh cp cc chng trc, vi khun c s dng rng ri ngay t trong cc th
nghim u tin ca cng ngh DNA v ngi ta qu hiu v vi khun .i vi virut th
ngi ta cng s dng cc t liu nghin cu t nhng nm u 1950 . Tnh di truyn ca
chng c xc lp trong cc th nghim v bin np (transformation) v cng hp

30

(conjugation) trong nhng nm 1960 v n c dng gii m qu trnh tng hp protein

trong nhng nm 1960 v 1970.
Hn na , cc t bo E.Coli c s dng rng ri cn do chng c tc ti bn c bit cao
.Trong iu kin l tng, c 20 pht vi khun ny li nhn i mt ln . Khi vi khun ti sinh
th plasmid v cc gen c ci vo cng c ti sinh .V trong nhiu gi th s c mt qun
th c ti hng triu cc con chu ca vi khun cng nh hng triu bn sao ca cc plasmid
bin i gen .Nhng qn th nh vy gi l colony(khun lc) v gi l dng (clone) i vi
plasmid v gen . T lng ca cng ngh DNA gi l gen c tch dng(gene have been
cloned).Vic tch dng gen cc t bo ng vt th chm hn nhiu bi b cc t bo ny
nhn ln vi tc chm so vi cc t bo E.Coli.
Mc d E.Coli l con nga th (Workhorse) ca di truyn phn t , nhng nhng vi khun ny
cng c nhng im bt li .Chng hn nh mt s chng E.Coli cng gy bnh tiu chy tr
em v cc khch du lch (travelers diarrhea).
Hn na thnh t bo ca cc t bo E.Coli cn c c cc ni c t m cc ni c t ny li
c hi cho con ngi. Cc ni c t cng kh loi ra khi cc ch phm thuc .
E.Coli cng ch to ra c mt lng tng i t protein , y l im yu lm gim gi tr
ca n trong cng ngh DNA .
Mt sinh vt khc c bin i , l vi khun Bacillus Subtilis (Hnh 3.7b) . Sinh vt hnh
chic gy ny khng gy bnh . Nm 1958 n c bin np trong cc th nghim Griffith v t
sau di truyn vi khun c nghin cu thu o hn .
Chng B.Subtilis sn xut protein mt cch tch cc v chng ny c dng trong cng
nghip sn xt cc khng sinh , cc cht dit cn trng v cc enzym cng nghip . Plasmid
ca B.Subtilis v s tn cng ca virut s c cp k trong cc chng sau.
Trong nhng trng hp xc nh , ngi ta mun s dng cc t chc nhn chun tc l cc
t bo c nhn , cc c quan t v phc tp hn cc vi khun nhn trn . Sinh vt thch hp
trong trng hp ny l nm mem Saccharomycess Cerevisiae (Hnh 3.7c). Nm men khng
gy bnh , c thm d k cng v mt di truyn v c s dng mt cch thng qui
trong qu trnh ln men v lm bnh m .
S dng cc t chc nhn chan th tt hn cho vic sn xut cc protein cho ngi bi v cc
protein kh phc tp li c sn xut bi cc t chc phc tp hn . Mt vi sinh vt nhn
chun khc l nm cng c s dng trong cng ngh DNA , chng ta s xem xt sau.
Trong mt s cc th nghim ca cng ngh DNA li i hi cc t bo ng vt c v
(VCV) . Khi s dng cc t bo ng vt c v s c mt li th l trnh c cc c t nh
khi s dng vi khun . Cc nh Ho sinh cng pht hin rng mt s protein l qu ln v qu
phc tp nu phi tng hp bng vi khun (chng hn nh cc cht hot ho plasminogen ca
m) . V cc protein phc tp c xu hng cun li khng ng quy cch trong vi khun v s
khim khuyt ny c th dn ti s to thnh cc sn phm khng c hot tnh . Vi khun cn
c th thiu h enzym c lin quan ti s ci bin protein ti dng cui cng ca n nh vic
gn thm phn t cachohydrat chng hn .

31

Hnh 3.6. Cc t bo ch l vi sinh vt dng cho cng ngh DNA ,(a) Escherichia Coli ,(b)
Bacillus subtilis , (c) Saccharomyces cerevisiae .
Mt bt li khi lm vic vi cc t bo VCV l thao tc rt kh khn v t tin so vi vic
s dng cc t bo vi khun . Vic nui cy cc t bo VCV cng rt phc tp v vic ci cc
32

phn t vec t vo cc t bo VCV l cc k phc tp. Tuy nhin, cc nh cng ngh DNA
phn chia ra nhiu ng hng hon tt vic tng hp protein trong cc t bo VCV bng
cch s dng cc trnh t promoter, nhng vec t mi ,v c nhng phng php mi bi
xut protein .
Khi vec t c chun b ri th vic ci n vo cc t bo hay t chc tip nhn tng i
d dng . Plasmid v Cosmid s thm nhp vo cc t bo khi lm nng ln hay lm lnh i xen
k cng vi s c mt ca can xi clorua v virut xm nhp vo t bo trong lc cc qu trnh
sao chp ca chng vn din ra mt cch bnh thng .
Mt phng php khc dng ci l n sinh hc biological bullistic , cc
phng php vi tim (microsyring injection) s c bn sau.
3.4.S biu hin ca gen
Khi chun b c vec t v ci c vo cc t bo hay t chc ca vt ch th cc nh cng
ngh DNA phi thc ti vic xem xt v mt Ho sinh v n cn gp phn vo s thnh , bi
ca c qu trnh.
Nhng bc ny cng phi thn trng nh cc bc trc bi v cc qu trnh sinh ho cn
lin quan ti s biu hin gen bn trong t bo .
Mi bc nh th ph thuc vo s nh v chin lc (strategic location), tc l phi t chnh
xc gen cn ci vo ng v tr trn plasmid (H5.9) . Khi enzym RNA polymeraza phin m
DNA thnh mRNA th cng l lc bt u qu trnh nh v mt v tr nhn dng trn phn t
DNA .V tr ny gi l v tr promoter (promoter site), l mt trnh t baz ni ln rng
enzym bt u tng hp mRNA .Enzym ny sau di chuyn dc theo phn t DNA v
tng hp mRNA cho ti khi n m kt thc. c s biu hin gen th v tr promoter phi
c t ng du (spot) trn vec t c lin quan vi gen.

Hnh 3.7. Vai tr ca Vec t chng hn nh plasmid m ho Insulin. Plasmid c cc v tr

khi u s hnh thnh mRNA (promoter), n cho php gn vi Riboxom v kt thc s to
thnh mRNA (terminator) . Trong cc t bo nhn chun th mRMA m u c ci bin
bng cch loi i nhng intron to thnh phn t mRNA cui cng .
V tr kt thc (termination site) cng c tm quan trng nh v tr promoter. y l m ca cc
baz tn hiu kt thc qu trnh phin m . iu ny tht l hu ch v t mt tn hiu hu
hiu ngay sau gen ngn nga vic c ln c sang cc gen bn cnh .Gen mong mun s
c t tht chnh xc gia v tr promoter v v tr kt thc m bo cho s phin m hp
l .
Hn na, i vi cc v tr promoter v v tr kt thc th cc vec t phi cha mt trnh t baz
cho v tr gn riboxom (ribosomal binding site). V tr ny l cn thit bi v bi v phn t
mRNA c m ho bi gen phi c gn vi riboxom v lm c iu th cn phi c
mt trnh t baz b cu vi n trn riboxom .Nu vic gn khng thnh cng th pha dch m
tng hp protein s khng xy ra v gen khng c biu hin.
Mt vn khc cn phi xem xt i vi s biu hin gen c lin quan ti s tng hp cc
phn t mRNA. vi khun ton b trnh t DNA c phin m thnh phn t mRNA .Nhng

33

i vi cc t bo ng vt nhn chun th khng phi nh vy. Trong cc t bo ny cn c

mt s vng m gi l intron , cc intron s c loi khi phn t mRNA m u
(preliminary mRNA) trc khi tr thnh mRNA cui cng .Vng m cn li l cc exons (v
chng c biu hin expressing). V vy cc mRNA c to ra bi cc t bo nhn chun
trn thc t l t tp hp ca cc exons .Cc nh cng ngh DNA s cn phi gii quyt vn
ny bi v vi khun khng c enzym loi cc intron khi cc phn t mRNA . V th, tht l
kh khn khi s dng vi khun biu hin cc gen i t cc t bo ca ngi .
Cch mu tr khc phc tnh trng kh s ny l s dung DNAb cu (complementary DNA
= cDNA) i t s phin m ngc ca mRNA . cDNA c tng hp t s phin m ngc th
khng c intron m ch c cc exons .Cc cDNA c tng hp nh th c th d dng ci
c vo cc vi khun .
Mt cch khc lin quan ti vn intron-exons l tng hp mt gen nhn to (artificial gene)
vi cc khi kin trc nucleotit . Phng php ny i t cc tiu chun Ho sinh c in tc l
s dng cc trnh t amino axit ca protein nh mt c s suy ra trnh t baz cho s tng
hp DNA. iu ny i hi phi bit c ton b trnh t amino axit ca protein . Tc l cc
m di truyn ca cc amino axit s c hin r v sau th gn cc nucleotit c trng li vi
nhau .C mt s gen c tng hp theo cch ny . Ngi ta gi n l oligonucleotit (oligo
theo gc ch Hy lp c ngha l t) tc l mun cp ti cc gen c kch thc nh . Cc
oligonucleotit c th ci c vo cc vec t v dng biu hin protein, nhng phng php
ny rt kh .Mt trong nhng thch thc khc l phi tng hp c cc v tr gn promoter,
termination v riboxom .
Cc oligonucleotit c s dng nh l c s ca cc phn t antisen .
Tuy nhin , vic sn xut cc protein him khi ch dng giai on gn cc amino axit li vi
nhau m cn phi tri qua cc bc ci bin tip theo, l vn m cc nh cng ngh DNA
ang lu tm . Trng hp Insulin chng hn th mt t hp gm 35 amino axit phi c ct
b t cc phn t Insulin sm (early insulin) trc khi gi li cc amino axit to nn 2 chui
dnh vi nhau .
Vi khun th khng c kh nng ci bin ny nn cc nh cng ngh DNA phi bin i vi
khun chng to nn 2 chui tch bit , sau 2 chui ny s c gn li vi nhau bng
cc qu trnh Ho sinh tch bit ngoi vi khun .
Mt ci bin khc c th xy ra l cc chui protein c thm vo mt phn t cacbohydrat
hnh thnh nn phn t glycoprotein.
Hai dng Interferon hp cht khng virut u l glycoprotein . Vi khun khng c kh nng
gn thm cacbohydrat vo protein nh kh nng ca cc gen ngi nn vi khun khng c
dng sn xut interferon .
gii quyt khc mc ny cc nh cng ngh DNA li phi s dng ti cc t bo ng vt.
Trong cng ngh DNA ngi ta lun mong mun lm tng sn lng sn phm (Hnh
3.9..).Bc ny i hi phi tng mc biu hin gen . thc hin iu phi c nhiu
gii php .Chng hn nh c th tng s lng plasmid trong vi khun ( cng nhiu plasmid th
cng c nhiu mRNA v nhiu protein) .Cng c th lp chin cng bng cch nng cao hiu
lc ca cc tn hiu ho hc t v tr promoter khi u s biu hin gen , hoc tng lc gn
mRNA vi riboxom bng cch can thip vo gen ph trch v tr gn vi riboxom .Cc nh
cng ngh DNA kho st tt c cc phng php ny .
Mt phng php khc lm tng s biu hin gen l dng cc m li th nht cho mt protein
c bit mt t chc c bit .iu hin nhin l c c nhiu m cho mt amino axit , iu
quan trng l phi bit c m no l thch hp vi t chc ny .D nhin s c mt t m s
khng thch hp .
Tuy nhin, cc t bo sn xut ra protein c th cho ra cc sn phm hot ho t bo t tin
(chng hn nh trong s phn chia t bo) v nhng t bo sn xut ny c th gp bt li khi
c mt cc t bo bnh thng.
Cc nh cng ngh DNA v th vn ang ch i mt qun th t bo s c thit lp trc khi
c nh mun m rng ti a s biu hin gen v chc chn rng vn cha c s tranh ua t
bo trong k nguyn ny .
Gen promoter c th c kim sot bng cch cm s biu hin ca gen cu trc cho ti
khi n ri ng thi im gen c biu hin .
3.5.Tp hp cc sn phm ca gen
S khch l ca Ho sinh i vi s biu hin gen lm cc sn phm gen c tch t cng
nhiu . Nhim v lc ny l phi tp hp li cc sn phm gen. Qu trnh ny c th l phc tp
hn ta tng bi v mt sn phm gen thng l ngoi lai i vi t bo sn xut ra n. Chng
hn nh Insulin ngi ,n khng phi l mt sn phm protein thng thng ca vi khun .Tht
vy, vi mt protein ngoi lai nh vy th vi khun c enzym proteaza, enzym ny nhn dng v
ct bt mt phn phn t protein .Chnh v hot tnh ny ca enzym m n lm hn ch rt ln
kh nng sn xut insulin.
34

 gim bt s hu hoi tim tng cc sn phm gen cc nh cng ngh DNA s dng cc
chng vi khun khng c enzym proteaza. Hin ngi ta thng dng Escherichia Coli. Tt
nhin n l mt chng tt nht bi v n thiu hon ton enzym proteaza . Nhng nhng vi
khun ny c th b cht nu thiu s bo v nghim ngt .Mt phng php mi gii quyt
c vn ny l lm mt cu ch gia cc sn phm gen v cc protein nguyn bn khc
ca t bo .Phng php ny c s dng sn xut insulin v hormon sinh trng ca
ngi . Phng php mi nht l s dng cc t chc m cc protein ngoi lai c tch t
li mt cch tng th trong t bo cht v nh vy trnh c tc ng ca proteaza. Vic tch
chit cc protein ny cng l mt vn ,bi v cc phng php tch chit c th lm gy cc
phn t protein .
Mt vn cng cn c gii qut l s mt mt protein trong lc s biu hin gen vn
ang tip tc xy ra .Cc sn phm gen c th c tch t bn trong t bo cht ca t bo
hoc c th c a ra mi Trng bn ngoi t bo .Vic cho ra cc sn phm gen c li th
l c th lm tng sn lng sn phm ,d dng phc hi v c th lm tinh khit c .V vy
cn phi n lc tm kim cc phng php hu hiu trong vic xut khucc sn phm .C rt
nhiu cng trnh nhm vo Bacillus Subtilis , mt vi khun thng xut khu mt lng
ln protein . Cc nh cng ngh DNA khn kho a vo Bacillus subtilis cc trnh t tn
hiu kch thch s tit (secretion) m vn thng thy cc mu Bacillus khc .Cc vi
khun ny sau mc vo cc sn phm gen mt peptid tn hiu kch thch s xut khu
(Hnh 3.8.)

Hnh 3.8. S dng trnh t tn hiu v peptid tn hiu .(a)Vec t dng trong tng hp protein c
cha mt trnh t tn hiu m cho mt peptid tn hiu , n s gn vi cc sn phm , (b) peptid
tn hiu khch l s xut khu cc sn phm gen ra mi trng bn ngoi , (c) Khi xut
khu peptid ny s c loi i v sn phm gen c hi phc .
Peptid tn hiu ny s c loi i khi cc sn phm gen c lm tinh khit.
Nh vic s dng cc trnh t v cc peptid tn hiu nn nng cao c kh nng thu lm
cc sn phm gen khi t cc t bo trong cc iu kin sn xut protein. Qu trnh ny thch
hp vi vic sn xut Insulin v cc nghin cu xa hn na .
3.6.Th vin gen
Cc nh cng ngh DNA s phi i mt vi mt nhim v trng i l phn lp cc gen t cc
t bo gc phc v cho th nghim tng thi im .iu c ngha l cc gen phi c
gi an ton trong nhng kho cha sau khi chng c phn lp t DNA ca t bo v c lm
tinh khit. Cc kho cha ny c gi l th vin gen (gene libraries). Mi th vin l mt tp
hp ca cc t bo sng.
tin hnh thnh lp mt th vin gen ngi ta phi thu thp c mt khi lng ln t bo
c cha cc gen mong mun. Chng hn nh cc t bo tu ngi c cc gen ca Insulin .Vn
l ch cc t bo tu phi c khong 100.000 gen (tnh theo ton b t bo) v trong s
100.000 gen c mt th c khong 5% -10% thuc DNA ca t bo ,s cn li khong 90-95%
l cc gen lp khng c chc nng . V th gen ca Insulin phi c phn lp t mt khi
lng rt ln DNA .
By gi chng ta hay xem vn ny c hon thin nh th no ?.
3.6.1.Thit lp mt th vin.

35

 thit lp mt dng t bo c cc gen insulin cc nh cng ngh DNA phi bt u bng

vic thu lm cc gen ny . lm c iu , ngi ta phi s dng mt dy cc enzym
gii hn ct cc DNA t bo tu thnh nhng on nh . Mi on nh ny thng nh hn
mt gen c trung bnh v cha khong 4000 baz ni t , khong 4 kilobaz (kb).Mt NST c
trung bnh ca ngi c khong 100.000 kb , v th nn mi NST phi c ct thnh 25.000
on , mi on di 4 kb.V ngi c 23 NST khc nhau cho mi t bo nn tng s ca cc
on 4kb s l 575.000 on. Trong s 575.000 on ny c gen Inssulin .
By gi mt t chc vt ch s la chn cc on ny, chng hn nh Escherichia Coli hay
Bacillus mt vi khun ng rut cua ngi . Plasmid ca E.Coli s c phn lp v cc
on DNA t cc t bo tu s c ci vo cc plasmid (H 5.12). Cc plasmid ti t hp (bao
gm tt c cc on) sau li c ci vo cc t bo E.Coli v sau c kch thch cho n
nhn ln. Ni mt cch tng qut l cc t bo E.Coli by gi tr thnh cc kho cha tt c
DNA ca cc t bo ty ngi . Tp hp cc t bo ny chnh l mt th vin .V nh
vy, cc t bo s c khong 575.000 cun sch trong mt th vin . Loi th vin gen ny gi
l genomic library (Th vin B gen) .
Nhng cun sch no ca th vin ny c c cha cc gen sn xut insulin? tr li cu
hi ny ta phi sng lc trong th vin. S sng lc s xc nh c gen ch c hiu m
trong trng hp ny l Inssulin . Qu trnh ny s nhn din ra cc trnh t c bit ca cc vt
liu di truyn bn trong cc t bo bin np .
3.6.2.Sng lc th vin gen
Vic sng lc mt qun th t bo trong mt th vin gen tc l xem xt ti cc vt liu v
thng tin di truyn c th ca n. Chng hn nh , khi sng lc cc t bo sn xut Insulin
chng hn th ngi ta phi s dng cc phn t mRNA c hiu. Phn t mRNA ny c cc
trnh t baz b cu vi cc trnh t baz ca gen Insulin. Khi n hot ng nh mt dng u
dprobe gen , mRNA s gn c hiu vi gen Insulin . Nu u d c gn mt ng v
phng x th s thy c s tch t phng x cao ni ang xy ra phn ng .

36

Hnh 3.9. Thit lp mt th vin gen ,(a) DNA c phn lp t cc t bo ca m (chng hn

nh cc t bo tu),plasmid ly t cc t bo ch (chng hn nh vi khun) ,(b)s dng mt
enzym gii hn c bit phn gii DNA t bo v mt enzym gii hn tng t cng c
dng m cc plasmid . Kt qu ca s phn gii l m c plasmid v cc on DNA c
nhn ln theo mu t 1 n 9 ,(c)cc on DNA gn vi plasmid m gn li vi nhau
to nn plsssmid ti t hp . Lu rng cc plasmid m thu nhn c nhiu on DNA ,
(d)plasmid ti t hp c ci vo vi khun mi, chng s nm v nhn ln khi c s
nhn ln ca t bo. Qun th t bo vi khun ny l th vin gen .Cc t bo khc nhau trong
qun th c cha cc on DNA khc nhau . Khng phi t c cc t bo u cha cng mt
on DNA v mt s t bo c c cc bn sao c nhn ln ca plasmid ti t hp do s sao
chp ca plasmid . Th vin ny c th c sng lc theo chng trnh m t hnh tip theo.
tin hnh sng lc, cc t bo E.Coli bin np s c nui cy trong mt a Petri phng
th nghim c cha gel nui dng . Trong vng 1-2 ngy c th nhn thy cc khun lc trn
b mt gel. L tng l cc khun lc ch cha cc t bo c ci ch vi DNA ca ngi thi.
37

Tuy nhin, cc t bo cng c th c c cc plasmid bnh thng (khng c cc gen ca ngi)

hoc cng c th c cc plasmid ti t hp.V vy, mi khun lc li phi em kim tra xem n
c ci gen Insulin cha ?

Hnh 3.10. Dng k thut bc bn sao sng lc cc khun lc vi khun . Cc khun lc ca

vi khun c nui cy trn mt a Petri c cha gel nui dng , y l a gc . (a) Dng
mt mnh vi v trng t ln trn cc khun lc th mt s vi khun t mi khun lc c
chuyn sang tm vi . Sau tm vi ny c a vo mt a Petri mi c gel nui dng .
cc gel nui dng ny th cc khun lc s xut hin v vy m to ra c mt bn sao ca
mt a gc . (b) K thut ny c th c dng sng lc cc vi khun khng ampicillin (c
cha gen khng ampicillin) ang pht trin trn mt a c cha vi khun khng tetracyclin .
V bc Bn sao cha ampicillin nn ch cc vi khun khng ampicillin mi mc c . i
chiu vi cc a gc s bit c vi khun no c gen khng c tetracyclin v ampicillin .
Mun c cc bn sao vi sinh ca mt a dng cho cc cng vic sau ny , cc nh Ho sinh
s dng mt k thut c gi l bc cc bn sao(-replica plating)(Hnh 3.10)
.K thut ny c Joshua v Esther Lederberg pht trin t nm 1952. K thut ny l
p mt mnh gc v trng vo gel sau li nhn n vo mt a Petri mi c gel nui dng
thit lp mt a lm vic(-working) . Bng cch lm nh vy tc l sao c mt a
gc . By gi c th phn tch cc mu ca cc khun lc khc nhau t cc a lm vic tm
ra gen ch .
C nhiu kiu phn tch. i vi cc gen Insulin th s dng u d mRNA gn phng x .
Trc tin, ly mt mu vi khun t mt khun lc nghi ng ( c gi an ton) v DNA ca n
c ct thnh tng on v c phn lp trn giy lc nitroxenluloza c bit.Dng phng
php in di (electrophoresis) tch chit. Sau dng cc u d xem n c phn ng
vi mt on DNA bt k no khng (t cc t bo ny).Cng ging nh vic ly tay phi m
tay tri , y cc u d c trn cng vi cc phn t DNA tm ra phn b cu ca n
.Nu nh hot tnh phng x khng c tch t th cc nh k thut c th xc nh c rng
khng c phn ng xy ra v khng c gen Insulin.Cn nu hot tnh phng x li tch t mt
u th cc DNA ny c xc nh nh l mt cun sch trong th vin ( library
book)trong cha ng cc thng tin di truyn (Hnh3.11)

38

Hnh 3.11. Dng u d gen nh v gen Insulin . Saukhi mt bn sao ca gen nguyn gc
c hon tt (Hnh trc) , DNA t khun lc vi khun thu nhn c s cho bit nhng t
bo ny c cha cc gen Insulin mong mun hay cha . Khi n gn vi gen kia th tn hiu
phng x s pht ra ch rng xy ra s lin kt .Trong hnh ny th u d gn vi
on #5 , l gen Insulin.
thi im ny , khun lc cha kho (key) s nh v cc a lm vic.Vi phng php
kim tra kinh in , khun lc trong cc a gc cng c th c xc nh xem c cc gen
Insulin hay khng.
Cc nh cng ngh DNA mun lm vic vi cc gen Insulin c tch chit t cc t bo hn
l vi cc t bo tu nguyn gc .
3.6.3.Th vin cDNA
Trc khi lu li khi nim th vin gen , cn phi lu rng vn cn tn ti mt dng th hai
ca th vin , dng ny bao gm cc phn t DNA b cu (complementary DNA=cDNA) .Th
vin cDNA ca cc t chc (chng hn nh E.Coli), n mang cc on DNA c chc nng t
bo c hiu.(ngc li , th vin genomic th cha cc gen m ho cho cc chc nng chung
ca t bo ). Cc gen c hiu th nm cc th vin cDNA bi v th vin cDNA c tng
hp t qu trnh phin m ngc (transcription) i t cc thng tin c hiu trong mRNA .
sng lc th vin cDNA ngi ta phi s dng mt u d c bit . u d ny c chun
b t cc plasmid c phn lp cng vi cDNA ca n ci t cc t chc vt mang (carrier
organisms).
Cc cDNA plssmid sau c s l ho hc sao cho t 2 chui n c chuyn thnh dng
chui kp .Cc chui ny c gn vo giy lc nitro xenluloza . y c s pha trn cc phn
t mRNA vi giy lc .Cc phn t mRNA ny l b cu cho rt nhiu trnh t gen , v bt k
mt phn t mRNA no l b cu i vi DNA th chng s gn c. Cn cc phn t mRNA
khc s c loa khi giy lc .
Nhng phn t cn nm giy lc l mt qun th tng t cc phn t mRNA gn vi cc
phn t DNA chui n . T hp ny sau c ly ra khi giy v tch ra c RNA. Tip
theo, mRNA li c gn vo mt h thng t bo t do cng cc cht liu cn thit cho s
tng hp protein.V th cc protein c to ra l xc nh .
Ngc li,t cc protein s cho cc thng tin v cc m di truyn. V lm nh vy th cc t
bo cht mang (carrier cells) c xc nh bi v n c cha cDNA i vi mt protein
c bit .
V cDNA c xc nh theo phng php ny nn n c th dng nh mt u d gen
sng lc trnh t DNA hay RNA cho mt qun th t bo .Chng hn nh , cc sn phm c
tch chit t mt qun th t bo m ngi ta cha bt c th cho gn trn giy trn giy lc c
cDNA bit . Nu xy ra s gn b cu th cDNA trong th vin ca mt qun th t bo
cha bit s c xc nh . Cn nu khng xy ra phn ng th phi s dng cc u d khc
cho n khi xy ra s gn kt .
Mt k thut hin i l replica plating technique (k thut bc cc bn sao) c dng
sng lc mt th vin genomic hay cDNA .Trong qa trnh ny, cc khun lc ca cc t chc
vt mang (carrier organisms) s c nui cy trn mt gel a petri. Nhng phi thay vi v

39

trng bng giy lc xenluloza thu lm cc bn sao ca khun lc .Giy nitro xenluloza
c p vo b mt cc khun
lc c c cc mu sao , sau giy c lm t bi dung
dch NaCl nhit cao (65oC ) gii phng DNA khi t bo v tch cc chui ra ( tc lm
bin tnh) . By gi th cc u d gen c hot tnh phng x c a vo xem ni no xy
ra s gn b cu . to iu kin cho vic gn kt th phi iu kin thch hp (thng 24
g ) cho phn ng xy ra .
Sau thi k , cc t giy c ra loi b cc cht phng x v cc u d d tha . Sau
giy lc li c kp gia cc t phim x quang . Hot tnh phng x dng ht beta s c
gii phng khi cc u d phng x vo cc phim X quang v hin ln phim ny . Sau khi
ra phim , vng c hot tnh phng x s c cc vt sm. Bng cch so snh v tr cc khun lc
ca n trn cc a chun (original), cc nh cng ngh DNA c th xc nh c cc khun
lc c cha DNA.
Mt phng php khc dng sng lc th vin gen l vic s dng cc li th ca thiu ht
dinh dng .Chng hn nh mt vi khun t bin ch c kh nng pht trin trong mi
trng khi n c cung cp a xit amin Histidin (Cc vi khun t bin khng c kh nng t
tng hp Histidin).
Mt phng php khc cng c s dng l cc gen khng thuc.
Phng php cui cng l lm ng kh t bo . c duy tr theo kiu ny cc t bo cm
thy d chu v cc thng tin di truyn d dng c khi phc.

40

41

Hnh 3.12..Phng php sng lc mt th vin DNA , (a) cc t chc mang vt ch c cha
cc gen mong mun c nui cy trn gel nui dng. y n s hnh thnh cc khun lc
.(b) Dng mt tp giy lc nitroxenluloza t ln trn c c mt bn sao cc khun lc,
(c) bc giy lc nitroxenluloza ra khi mt gen ly cc khun lc vi khun ,(d) Giy c
x l ngt t bo v tch ra cc chui DNA . Sau t vo mt ti cht do bt kn vi
dung dch c cha u d gen c cht phng x .u d ny c hiu vi gen mong mun v s
phn ng vi DNA b cu trong thi k .(e) giy c p vi phim x quang. Nu phng x
pht ra do c s tng tc u d-gen th n s hot ho phim X quang, (f) vng ti xut hin
nhng ni c s tng tc xy ra .(g) Phim X quang c so snh vi a gc xc nh xem
cc khun lc no cha DNA pht ra phng x. Nhng khun lc ny s c vi khun c cc
gen mong mun.
Nh cp trn , c nhiu vn ny sinh trong cng ngh DNA c gii quyt nh
cc gii php thch hp . Cc nh cng ngh DNA thao tc gen gn ging nh cc chi
Ho sinh v thng li trong vic thu c cc gen theo s t cc. S pht trin thnh cng
cc phng php Ho sinh dn ti hng lot thng li trong vic s dng mt cch thc tin
cng ngh DNA ci thin cht lng cuc sng con ngi .

Chng IV
Phn tch v chn on bng DNA
4.1.M u.
T nhng nm 1950 , cc nh khoa hc pht hin ra nhiu k thut mi dng phan tch v
lm cc test trong y hc . Chng hn nh trc kia ngi ta tm thy cc khng th lin quan ti
mt bnh no mt bnh nhn th ngy nay h c th xc nh c t chc (vt) gy bnh
bng cc phng php m cc th h trc y khng th tng tng c . Trong nhiu
Trng hp , khi cn chn on mt bnh c th ch tp trung vo chnh t chc ny thi ch
khng cn phi xem xt cc bng chng khc.
Trung tm ca k thut mi ny l phn t DNA .Ngy nay ngi ta c th ti to li DNA
trong mt ng nghim , hay tch n ra thnh tng mnh , xc nh thnh phn, thay i cu
42

trc , chuyn i cc mnh v lp bn gen ca n . Nhng nguyn l thu lm c t

nhng th nghim c p dng xc nh cc bnh truyn nhim , tm du vt cc ti
phm, sng lc ung th ngi ln v cc bnh di truyn phi , m bo sc kho cho cng
ng bng vic xc nh cc yu t gy bnh trong mi trng .
Nhiu k thut c m ra trong c phn ng tng hp chui (polymerase chain
Reaction- PCR). Trc nhng nm 1985 him khi c nghe thut ng PCR;Vi nhng tiu
chun hin nay , mt phng th nghim DNA m khng c mt my PCR th cng ging nh
mt vn phng khng c my photocopy . Thc t , chc nng ca my PCR ging nh l mt
my photocopy. Nu ta ly mt mnh DNA ri n t ng sao chp th s c hng triu bn
c sao ch trong khong 3 gi ng h . Mt yu t cn thit khc trong vic phn tch DNA
l mt on DNA gi l u d DNA (hay cn gi l u d gen) .c pht trin t nhng
nm 1970 , u d DNA sn lng c mt on b cu ca n trong c mt m ly cc
tn hiu v vt liu t bo m c cc on DNA nh v .
Vic theo di mt gen hay tp hp cc gen l mt nhim v v cng to ln v khi ngi ta
phi xem xt ton b b gen ngi . Chng ta hy tng tng rng cc chui b cu ca DNA
nh l mt con ng cao tc 2 chiu . Nu chng ta ni ui vi ui ca 46 nhim sc th
vi nhau th con ng cao tc ny s c chiu di bao quanh c 3 triu vng tri t
(khong 65 t dm) . Vy m mt u d DNA c th pht hin hiu qu trn chiu di ti vi
ngn dm .
Chng ta thy c nhng thnh qu m PCR v u d DNA thu c trong phn tch v
chn on bnh. Trong nhiu trng hp , cc k thut ny l b sung cho cc phng php
phn tch truyn thng . Trong nhng Trng hp khc th n li c dngnh cc phng
php thay th . Nhng trong mt s chn on c th c thc hin m khng cn ti cc k
thut c lin quan ti DNA.
4.2.Phng php phn tch DNA.
Nm 1961 Sol Spiegelman v Edward Hall pht hin ra rng DNA chui n c th
gn vi mt chui b cu ca RNA to nn mt phn t DNA-RNA chui kp . 20 nm sau,
cc nh cng ngh DNA p dng nguyn l ny cho phn t DNA-DNA (tt hn phn t
DNA-RNA) v tm ra kh nng s dng chui DNA nhn dng mt phn t DNA b cu
gia mt hn hp cc phn t DNA khc . N cng ging nh mt chic cha kho tm ng
kho .Dn dn h a vo c mt yu t quan trng trong phn tch cc baz ca DNA
, l mt chui DNA gi l u d DNA .
4.2.1.u d (Mu d) DNA.
u d DNA l mt phn t DNA chui n tng i nh , n c th nhn dng v gn c
vo mt phn t b cu ca DNA hoc mt on ca mt phn t DNA ln (hnh 7.1) .Bi v
Baz ca DNA lun cp i A vi T v G vi C nn mt u d DNA c th tng tc cao c
bit vi cc trnh t ca a xit nucleic trong cc phn t DNA ch . Ging nh tay tri tm tay
phi , phn t DNA u d trn ln vo gia cc chui DNA cho n khi nh v c mt
chui hoc mt phn ca chui m n b cu , ri n gn vo chui hoc mt phn ca
chui .

43

Hnh 4.1. Hot ng ca mt u d gen : u d gen l mt on DNA chui n . Khi gn

vi mt phn t DNA c mt v tr b cu th u d gen s tm ra c v tr gn . Nu c
mt phn t phng x hoc mt nguyn t c gn vo u d ny th hot tnh phng x s
tch t v tr gn v tn hiu ny ni rng phn ng xy ra . Lu trong hnh v ch r cc
baz ca mt u d b cu cc baz ca on DNA nh th no .
S hot ng ca u d gn cht vi Sinh ho hc DNA . Trong nhng iu kin xc nh ca
phng th nghim phn t DNA chui kp c th c lm rui thng ra v khng gn vi
nhau na . Cc iu kin l :Lm nng ln ti trn 90 C hoc a DNA ti pH cao hn 10,5
hoc a thm vo mt hp cht hu c nh ur hoc formaldehyt. Khi lin kt Hydro gia
cc cp baz b bn mt v s b cu c thc hin . Qu trnh ny gi l s bin tnh .
Tuy nhin , s bin tnh c th c hi nguyn . Nu trong nhng iu kin c bit v nng
mui, nhit v pH ca phng th nghim 2 phn t DNA chui n s gn vi nhau
to thnh dng cp i nguyn bn . Qu trnh ny gi l s lai (Hybridization) hay l s
hon nguyn(renaturation) , l trung tm ca vic s dng d DNA .Di nhng
iu kin c kim sot cht ch s hnh thnh cp i DNA bn vng khi s b cu cc cp
baz hon ho dc theo ton b chiu di ca chui DNA . V th u d DNA s to c mt
sn phm n nh ch trn chui vi cc chui DNA b cu ca n .
Khi s dng vi mc ch lm test th u d DNA thng c gn vo mt cht pht hin ,
chng hn nh mt cht ng v phng x . Khi u d gn vo DNA b cu ca n th cc
ng v phng x cng gn dc theo v s tch t ca cc hot tnh phng x cho bit rng s
gn kt c thc hin . Tuy nhin, trc khi a u d vo th DNA ch c phn lp
v ct nh ra tng mnh . u d du chun s sn tm on b cu ca n trong cc phn t
DNA chui n .
Khi ng dng trong cng ngh hin i , u d DNA c th pht trin pht hin bt k mt
trnh t a xit nucleic no . Thm ch cng c nhng Trng hp khng cn phi bit trnh t ca
cc baz trong DNA ch . Chng hn nh DNA trong mt t bo c th c khch l hnh
thnh phn t RNA thng tin b cu (mRNA) . Nhng phn t mRNA ny sau c phn
lp v cho gn vi enzym Transcriptaza ngc cng vi cc nucleotit tng ng v cc vt liu
khc .Transcriptaza ngc c dng phn lp mRNA vi t cch nh l mt ci khun n
s tng hp mt phn t b cu ca DNA . DNA mi ny cng ng nht vi cc phn chc
nng ca DNA (exon) trong vic tng hp protein mt thi im nht nh .
Phn t DNA mi ny cn c dng nh mt u d pht hin DNA ca nhn (khi cn
phi nh loi) .
Mt u d c th c chiu di ch di 10 baz hoc c khi ln ti hn 10.000 baz.
R rng l, u d phi c kh nng gn (hoc lai) vi DNA ch . Nhng iu quan trng l
u d s khng lai vi cc phn t a xit nucleic khc cng c mt . V phn t u d
ch (probe target molecule)(hoc phn t lai) nu c hnh thnh th s gn kt ny phi
n nh .

44

Trong nhng nm 1980, iu lc quan ca vic s dng u d l gii quyt c vn

lun lun thiu DNA trong mu lm test . Tuy nhin, nu thiu DNA ch th cc tn hiu
phng x gn vi u d s khng hot ng tt . V th iu quan trng l phi khuch i
DNA ch . K thut khuch i DNA c gi l phn ng tng hp chui (polymerase chain
Reaction) (PCR).
4.2.2.Phn ng tng hp chui -(PCR).
PCR c Cetus Corporation pht trin nm 1984, n cho php cc nh cng ngh DNA ti to
ra hng t bn sao ca mt chui n DNA ch trong vng my gi ng h (s phn chia ca
t bo ung th cng phi mt hng thng mi t ti con s ny). nhn mt phn t DNA
vi PCR i hi phi c 4 vt liu sau : 1.Phn t DNA ch (c th c khuch i); 2.Cc
chui ngn DNA mi primer bit , n xc nh on phi sao v to c s cho vic bt u
qu trnh sao chp ; 3.DNA polumeraza , mt enzym hng dn s s sao chp DNA trong t
bo sng nhng khng khi ng c qu trnh sao chp; 4. Mt hn hp cc nucleotit - l
cc khi vt liu xy dng nn axit nucleic t m to nn cc DNA mi .

Hnh 4.2. Phn tch bng PCR : (a) DNA ly t mt t bo t vo mt ng nghim c cc vt

liu thch ng . (b) Enzym DNA polymeraza nhn i hng triu ln DNA ch . (c) Vi s
nhn ln cc bn sao DNA , u d DNA ca mt trnh t baz c bit d dng nh v v tr
gn b cu ca n .
cc khi vt liu ny gn kt vi nhau , PCR phi tin hnh theo 3 bc , tt c u t ng
trn my PCR . Bc th nht : DNA ch c lm nng ln b gy cc lin kt gia 2
chui v lm phn t rui thng ra . Ti bc th hai : Gim nhit cho cht mi gn vo
DNA ch . Nh vy n xc nh c vng phi sao chp . Cht mi l tn hiu bt u hay
l dng li qu trnh sao chp .
Bc th 3 :DNA polymeraza xc tc s hnh thnh cc phn t DNAchui n khi c cc
chui DNA ch nh l cc khun . S tng hp c bt u m du chun bi DNA mi.
Khi enzym di chuyn dc theo mi chui n s c cc trnh t ca cc baz , ri s dng n
nh mt ci khun gn vo mt chui b cu v to nn chui nucleotit mi (phn ng
chui). DNA polymeraza c s dng y l mt enzym chu nhit c bit c tn l Tag
Polymeraza . Enzym ny ly t mt vi khun chu nhit Thermus aquaticus , ln u tin c
Thomas Brock phn lp trong nhng nm 1980 .
S dung np nhit ca Tag polymeraza rt quan trng i vi qu trnh bi v enzym ny s
khng phi thay th sau mi bc c lm nng ln .
Kt thc bc 3 c 4 chui DNA (ban u ch c 2 chui) .

45

Khi qu trnh tip tc , PCR s lp li 30-60 ln . Mi chu k u bt u vi vic lm nng

hn hp (bc 1). Mi chu k mt khong 1-2 pht v mi on DNA mi s nh mt ci
khun cho cc bn sao . V th s bn sao ca DNA tng ln khng ngng . Vi 2 chui DNA
gc s c hng triu , hng t cc bn sao khc .
Tuy nhin PCR cng cn tn ti nhiu vn . Trc tin l vn dnh bn . Nu DNA c
dnh cc cht bn th nhng cht bn ny cng c khuch i cng vi cc DNA ch .
loi tr kh nng ny , cn phi ht sc thn trng khi chun b mu .Thng phi chun b
mu cc phng th nghim mnh , trang b t tin . Mt vn na l PCR khng nh lng
c . PCR ch c th xc nh xem mt on DNA c hiu no c mt hay khng m
thi ch khng nh lng c s chui l bao nhiu . V v PCR cn qu mi nn cng cn
phi c thi gian tiu chun ho . Mc du c mt vi tr ngi nh vy nhng PCR c s
dng kt ni vi u d , l nim hy vng trong tng lai . Tht vy, nm 1989 theo tnh
ton d bo cc nh cng ngh DNA thu c vi trm t Dola mi nm vi cng ngh ny .
4.2.3.Khuch i tn hiu.
Phn ng tng hp chui hot ng trn nguyn tc khuch i ch ; tc l DNA ch c
khuch i trc khi thm u d vo .Mt nguyn l thay th l khuch i tn hiu . Theo
nguyn l ny cho mt lng nh nht DNA gn vi u d , sau tn hiu c khuch i
ch rng phn ng hon tt .
c c s khuch i th phi c 2 s tip cn chung . Th nht l phi lm gim n
nn bng cch loi i cc DNA d tha . iu c thc hin bng cch tch phc hp u
d ch (target probe Complex) khi hn hp DNA c cc tp cht . thc hin vic tch
chit , cc chui nucleotit thymidin (Poly T) c gn vi cc ht t tnh . Cc chui b cu
ca Adenozin nucleotit (poly A) sau li c gn vo u d DNA . Sau khi u d DNA
c c hi gn vi DNA ch ca n th thm cc ht Poly-T vo . By gi th baz adenin ca
phc hp u d s gn vo baz thymin ca cc ht ny .Cc ht sau li c tch khi
hn hp c mang cc phc hp u d -ch .Nh vy n c c ng li v tch ra khi
DNA tp cht . iu c ngha l tn hiu (u d DNA phng x) c khuch i .
Vn th hai l khuch i vt pht hin (detector) c gn vi tn hiu , tc l khuch i
phn t DNA u d . thc hin c mc ch ny phi s dng mt u d th hai cho
DNA ch . u d ny c thit lp t mt RNA c mt cu trc bc 3 c quyn . u d
ny hot ng vi Q-beta replicaza, mt enzym xc tc s sao chp RNA . u d RNA gn
vi DNA ch cng vi DNA u d . Khi lin kt 3 gm RNA-DNA-DNA c tch ra khi
hn hp th thm Q-beta replicaza vo . Enzym ny s sao chp u d RNA . Nh vy
khuch i c vt pht hin (RNA) cng nh tn hiu(u d) v mt lng ln RNA c th
c pht hin bng cc k thut tiu chun .

46

Hnh 4.3. Chi tit mt phn ng tng hp chui (PCR) . (a) Dng nhit tch phn t DNA
chui kp {(ds) DNA }. Trong chu k 1. Mt hn hp nucleotit , enzym Polymeraza v mt
on mi DNA c thm vo .(b) polymeraza ni rng on mi v to nn 2 phn t DNA
chui kp {(ds) DNA }. (c) Qu trnh c lp li v cui chu k 2 s c 4 phn t DNA . Lp
li chu k 3 s to c tt c 8 phn t DNA . Cc chui c tng ln dn cc chu k tip
theo .
H thng u d RNA c li th v n rt nhanh , mi vng sao chp RNA ch mt 15 n 20
giy .K thut ny cng c dng nh lng DNA ch v cc nh nghin cu chp
nhn n pht hin RNA .

47

D nhin vi bt k mt k thut mi no cng c nhng vn cn phi gii quyt trc khi

n tr thnh tr ct trong cc Labo chn on .
4.2.4.Phn tch RFLP.
Cng c th khng cn thit phi xc nh mt gen c bit nu n l s bin i thch ng .
S bin i nh vy gi l tnh a hnh theo chiu di on gii hn (restriction fragment length
polymorphism) hay l RFLP c l rif lip . RFLP l mt on DNA dng lm du
chun mt gen c hiu . RFLP hin nhin khng c chc nng trong sinh l hc t bo . Nhiu
RFLP nh v mt cch ngu nhin khp trn cc NST ca ngi nh mt dng rc di truyn
.N c tc dng nh mt du chun gen khi mt gen c bit cha c tch dng hoc c kh
khn khi lm vic vi n (Hnh 4.4.)

Hnh 4.4. Pht hin mt RFLP .C th th nht c 3 v tr nhy cm vi HindII cho nn s to

c 2 on . on th nht 2 kilobaz (Kb) cn on th hai 4 Kb . C th th hai thiu mt
v tr nhy cm (H ) c th do t bin di truyn . V th enzym gii hn HindIII c thm
vo n ch ct phn t DNA 2 v tr v to nn mt on 6 Kb. Lu rng u d DNA i
vi on DNA ny nm gia v tr H v H c cc baz b cu cho c on nn n s phn
ng vi cc on 2 Kb,4 Kb, v 6 Kb .V th u d c th c dng pht hin tt c 3
on .Cc on l cc RFLP.
RFLP c th s dng nh mt du chun n phi cch xa gen t hn 5x106 cp baz (hoc 5
Centimorgans). Nu RFLP v gen trong khong cch ny th chc chn l chng gn vi
nhau v truyn li tt c cho cc th h sau .Cc nh cng ngh thng k vi 95% tin cy
rng nu mt c th c RFLP th ngi tra s tm ra c gen .
tm hiu bn cht ca RFLP chng ta phi hiu rng l on DNA c bit c th c
kch thc thay i . on ny c gi l a hnh (dch theo vn hc tc l nhiu th loi
(many forms) . Chng ta hy xem xt v d sau y :on DNA ko di t im A n F ,
trong cc ch kp ch cc im ct ca enzym gii hn .
-A- BB -CC DD EE FBy gi ta thm vo mt hn hp cc enzym gii hn (b,c,d v e)n s ct phn t DNA cc
ch kp .Kt qu l c 5 on c kch c th nh nhau :
-A-B

B-C

C-D

D-E

E-F

Qua nhng khong thi gian rt di , phn t DNAc s thay i , s thay i t ng trong cp
baz m n khng chu tc ng bi cc qu trnh Ho sinh hay sinh l hc m bi s tc ng

48

ca enzym .V th s bin i trong mt cp baz c th xy ra cc on ca DNA v to

c cc phn t sau :
-A-BB - CC DX EF FNu by gi chng ta x l phn t ny vi hn hp enzym gii hn (b,c,d,e) kt qu s cho cc
on sau :
-A B

BC

C-DX E
3

E- F
4

Cn phi lu rng kt qu tc ng ca enzym ch cho ra 4 on , bi v enzym dkhng c

hot tnh v tr DX(v t nguyn gc DD i thnh DE).
Cn mt ngha thc tin na l on { C-DX-E} L ngn hn 3 on kia .Nh vy cc on
DNA c kch c khc nhau , chnh s khc nhau v kch c ny to nn s a hnh . Thut ng
RFLP l i t Restriction Enzyme cn FL tc l fragment length.
s dng RFLP trong phn tch DNA , cc nh cng ngh DNA nm ly li th l h
bit trc chiu di cc cp baz ca mt RFLP c hiu .
Ngi ta ly cc t bo t mt ngi v phn lp DNA , ri x l n vi hn hp enzym gii
hn . Sau a hn hp ny vo trong my in di , khi cc polymorphism s c tch ra
theo kch c .
Qu trnh tch chit ny s cho ra mt mu cc bng ging nh m (Code) cc Bar trong mt
siu th .Khi phn tch mu , cc bng s phn nh c hay khng c RFLP c hiu , v n ph
thuc vo tnh di truyn ca c th . Nu s dng RFLP du chun th chc chn ti 95% l c
mt gen ny .
Trong thc t , mt NST c bit v cc gen ca n c th truyn t th h ny sang th h khc
cng vi RFLP du chun .
Khi phn tch mt gen gy bnh , mu RFLP ca mt c th s c so snh vi mu RFLP ca
nhng ngi bnh thng cng nh ca nhng ngi b tc ng bi gen ny .Nhng s so snh
nh vy cho php xc nh c c th c hay khng c RFLP v gen gy bnh . Cc thng
tin ny cng to c cc quyt nh quan trng vi cc bnh di truyn .
4.3.Chn on cc bnh truyn nhim.
Cc test chn on da trn c s u d v PCR u xut pht t cc thc tin trong qu kh
.Vi k nguyn ng i , cc thuc th cho cc test c 2 dng chung : Cc thuc th sinh ho
dng pht hin cc enzym c hiu v khng th dng pht hin protein .S hin din ca
u d DNA l mt lp hon ton mi ca cc thuc th chn on bi v n chng pht hin
enzym cng nh protein m li l trnh t gen .V s dng mt cch hiu qu u d DNA
th cn phi c mt lng ln DNA mi lm test v PCR p ng cyu cu ny .Cng
ngh khuch i v pht hin ca DNA ngy nay cho php lm c mt s test chn on cc
bnh truyn nhim v n khng ngng pht trin .
4.3.1.Chn on Hi chng thiu ht min dch mc phi .
S dng u d DNA v PCR c th pht hin c virut gy thiu ht min dch trn ngi
(Human immunodeficiency Virut)-(HIV) . Virut ny gy nn hi chng thiu ht min dch
mc phi (AIDS) . T nm 1985 tr li y FDA mi ch cp php cho test pht hin HIV da
trn c s khng th. Test hot ng da trn s phng on khi mt ngi phi nhim HIV th
khng th do c th to ra c th pht hin c trong phng th nghim .Nhng diu bt tin
l phi mt vi tun c th mi to ra lng khng th lm c mt test dng tnh .
Tm thi th test vn l m tnh mc d HIV c mt trong c th (Test m tnh gi -false
negative Test ) .
Vi vic a vo mt u d DNA v PCR n to kh nng cho test trc tip hn i v s
hin din ca HIV .Trong chu k xm nhim ca n , HIV tn ti nh mt on DNA ni vo
NST ca cc t bo ch . Cc t bo thuc h min dch ca mt bnh nhn c bit nh cc
Lympho T . thc hin mt test chn on trc tip ngi ta phi thu thp cc lympho T
t bnh nhn v ph v mt cch an ton DNA . Sau khuch i bi PCR , t c th thu
c hng na miligam DNA dng cho test .(Khi lng DNA khng c khuch i ch
c khong 1 triu sn phm cui cng) .By gi th cho u d vo . Trnh t baz ca u d
s b cu vi trnh t baz ca DNA virut . Nu c mt DNA virut th u d s nh v c v
gn vo n . S gn kt ny c bo bi tn hiu tch t hot tnh phng x . Kt qu l mt
test dng tnh .

49

Bi v cc k thut chn on HIV mi hn , xc nh tt hn so vi vic dng khng th virut

nn cc thy thuc c th tin tng hn v trng thi sc kho ca bnh nhn. Cc thuc c
k ra xc thc hn v cc gii php nhm trnh s lan truyn HIV s c th hin sm hn .
K thut ny cng gip cho y t cng ng xc nh c tnh hnh dch t ca AIDS d dng
hn bi v h c th hiu r hn ai c Virut m vn cha xut hin khng th . Chn on
HIV bng u d cn c th pht hin chnh xc hn s xm nhim HIV lc s sinh . Phng
php truyn thng l Test s sinh xem c khng th HIV khng .iu rc ri l ch khng
th ny c th t mu ca m truyn qua nhau thai v nh vy r rng l m c th b nhim
HIV nhiu hn l tr s sinh .Bng cch s dng test chn on trc tip DNA gn vi HIV,
ngi ta c th pht hin c HIV trong cc t bo ca tr s sinh v vic chn on sm hn
rt nhiu .
H qu ca s tip cn ny l cc nghin cu t nm 1992. Khi cc nh nghin cu pht
hin trong mt ngy sinh c 23 tr em dng tnh vi HIV khi c test vi u d DNA v
c 19 em l m tnh .
Ngy nay test u d DNA lun c cc thy thuc i hi . Tuy nhin test ny cn t v
cha c ph chun bi FDA v khi lng DNA dng cho test . V s ph chun ny c
thc hin vo nhng nm 1990 v khi n s tr thnh mt phng tin chnh xc nh
HIV .
4.3.2.Chn on Bnh lao .
Cc Test chn on bnh lao hin nay cn phi vi tun mi hon tt bi v vi khun lao
Mycobacterium Tubercolosis nhn ln rt chm , ti sinh mt t bo phi mt 24 gi .Trong
khi ch i chn on , cc thy thuc phi quyt nh iu tr da trn cc ngun thng tin rt
hn ch . iu dn n vic lm mt hiu lc ca thuc k n v sc kho ca bnh nhn
ti t hn . Hn na , bnh nhn ny c th truyn bnh ca mnh sang ngi khc khi ang
ch i kt qu xt nghim .
Xut pht t con om m , cc nh nghin cu pht trin mt Test chn on hon ton
mi v giu tng tng i vi bnh lao , test ny cng gip cho vic pht hin cc chng c
bit ca M. Tuberculosis khng thuc .C s ca s tip cn ny l enzym Luciferaza ca
om m . Enzym ny s pht ra tia sng trong vi khun lao ang sng .

Hnh 4.5. Pht hin vi khun lao Mycobacterium tubercolosis . (a) Bacteriophage (phage) c
hiu cho M. tuberculosis c mang gen sn xut ra luciferaza c a vo mi trng nui
cy cng vi vi khun . Nu vi khun l M. tuberculosis th phage s gn vo thnh t bo v
b gen (genome) ca phage vo vi khun . (b) B gen ca Phage c gen Luciferaza c ci

50

vo NST ca vi khun . (c) Vi khun bt u tng hp luciferaza . (d) Khi thm luciferin v
ATP vo mi Trng th enzym ny s phn gii luciferin .(e) Kt qu ca s phn gii l pht
ra mt chp sng iu ch rng vi khun chnh l M. tuberculosis. Nu l vi khun khc
th s khng c chp sng pht ra .
Qu trnh ny xy ra nh sau : Mt Bacteriophage c hiu cho M.Tubercolosis c mang gen
Luciferaza bi cng ngh di truyn . (Phage i khi cng c hiu l Mycophage cng nh
Luciferase reporter phage). Sau mu phage c nui cy cng vi mt vi khun cha bit .
Nu mi Trng c M. Tuberculosis th phage s thm nhp vo vi khun v ci vo NST ca vi
khun cng vi gen luciferaza .Vi khun ny ngay lp tc bt tay vo vic tng hp
luciferaza . By gi th thm vo mi trng nui cy mt hp cht gn vi Luciferaza l
Luciferin cng vi phn t ATP nng lng cao . Nu c mt Luciferaza th enzym ny s
phn gii luciferin v kt qu l c mt chp sng pht ra .
Dng mt cng c chuyn dng (luminometer) pht hin tia sng s cho ngay kt qu trong
mi trng nui cy c vi khun lao hay khng . Kt qu ny gip cho cc thy thuc hon tt
vic chn on .
xc nh nhy cm hay khng thuc th quy trnh cng tng t nh trn ch tr vic a
thm thuc vo mi Trng nui cy . Nu vi khun nhy cm vi thuc th n s b cht v ni
mt cch vn v l n ht sng (light go out) . Cn nu chng khng thuc th n vn tip
tc sng v sn xut ra Luciferaza .
Ma xun 1993, cc nh khoa hc New York k Albert Einstein College of Medicine and the
University of Pittsburgh thng bo pht trin c test ny trn tp ch Nature . Khi t
successful Test c ng ti trn cc trang nht ca cc tp ch th vic chn on bnh lao
tr nn ht sc sng sa .
4.3.3.Chn on bnh Lyme.
Bnh Lyme l mt v d khc ca bnh gy ra bi vi khun c th pht hin c bng u d
v k thut PCR . l mt bnh ca m lin kt gy ra bi xon khun Borrelia burgdorferi.
Hng nm M c ti 10.000 ngi trong 45 bang mc bnh st, da ni ban v cc triu
chng khc lin quan. Nhng ngi ny khng c iu tr ngay bng khng sinh mt cch
t v din tin ca bnh l lm ui sc , i khi gy vim khp nng v cc di chng thn kinh
.
Xon khun rt kh nhn thy di knh hin vi quang hc v gn nh khng c kh nng nui
cy trong phng th nghim , B. burgdorferi cng khng phi l ngoi l (Hnh 4.6.) .

Hnh 4.6. Borrelia burgdorferi qua hin vi in t qut , l xon khun gy bnh Lyme .
Cng ngh DNA ngy nay c th pht hin nhanh vi khun ny .
Nm 1975 khi ln u tin bnh ny c xc nh , vic chn on ch da trn cc cc triu
chng , cc quan st dch t (tc l nu b ve bt cn) v cc test khng th . Phi ni rng
chng c phng php no l hon ton tin cy v khi cha c chn on th vic iu tr s
b chm tr . Tuy nhin , cc nh khoa hc phng th nghim thuc federal Rocky Mountain

51

 Montana thnh cng trong vic s dng PCR khuch i DNA xon khun , h dng
u d DNA pht hin t nht l 5 xon khun trong mt mu mu ca mt bnh nhn . y
l mt test bt thng bi s xon khun trong mu thng rt t .
Thm mt gi tr na ca test l n pht hin c c nhng chng ch khc nhau cht t ca B.
burgdorferi c gy phn ng dng tnh . iu ny rt quan trng bi v khng cn thit phi
dng test khng th xc nh tt c cc chng . Hn na , test ny cn cn c th phn bit
c B. burgdorferi t cc mu Borrelia gy cm ng cc triu chng ca bnh Lyme .
Cng bng vic xc nh xon khun trc tip (hn l gin tip qua khng th) , cc nh nghin
cu s c kh nng nghin cu s pht trin bnh l hc ca bnh . Chng hn nh iu quan
trng l phi hiu c liu xon khun thc s c mt giai on mun ca bnh khi c
cc bin chng hay l cc bin chng l do cc on xon khun cn nm li sau khi n
c loi tr ?
4.3.4.Chn on bnh u nh v cc bnh khc .
Mt Test c FDA ph chun l test u d DNA dng xc nh virut gy u nh ngi
(human papilloma virut HPV) Virut ny gy nn bnh mn cm sinh dc (genital warts)
ViraPap kit l mt test dng u d DNA tm HPV trong cc mu ca m ly t c t cung ph
n .Cng ging nh test AIDS , mt u d DNA c mt trnh t baz b cu vi mt trnh t
baz ca DNA c mu m . (S khc bit hin nhin l DNA t virut gy u nh trn ngi
ch khng phi l virut AIDS) . Bi v mt s HPV cng lin quan ti ung th c t cung , v
th test ny c cc thy thuc cng nhn l c th s dng xc nh virut v lm gim
bt kh nng gy ung th .
Nhng nm gn y pht trin nhiu thuc khng virut mi (chng hn nh acyclovir ,
ganciclovir) lm tng nhu cu v cc test mi cho cc bnh gy bi virut . S d nh vy v
vic iu tr c th c bt u sm nu xc nh c bnh . cc thp k trc cc test
cng c lm phn no vi nhng ngi mc cc bnh do virut . Test HIV v HPV l nhng
test trc nht trong s cc test m chng ta mong i . Cc test u d DNA i vi vim gan
B , virut herpes simplex v cc test cho cc bnh gy bi virut tuyn nc bt cng trong
giai on thc nghim v chng s c a vo s dng trong lm sng nhng nm ti . Test
vim gan B nay tr thnh thng phm c s dng trn khp hnh tinh .
Mt test da trn c s phn tch bng u d DNA cng c dng cho cc bnh vim quanh
rng . Bnh vim quanh rng (periodontal disease) l mt bnh nhim trng gy thoi ho li
dn n rng rng . Mt cng ty cng ngh sinh hc pht trin mt u d DNA pht
hin 3 mu vi khun thng lin quan vi bnh ny . S pht hin hiu qu l cha kho chn
on bnh sm , nng cao hiu qu iu tr v phng nga tt hn vic rng rng .
4.4.Pht hin cc bnh do di truyn .
Cc test da trn c s DNA cng c gi tr trong vic pht hin s tim tng cu cc bnh di
truyn . Cc bnh di truyn thng khng th chn on ngay c cho n tn khi xut hin
y cc triu chng ca n .Tuy nhin, cc test DNA li c kh nng xc nh c s di
truyn ca nhng bnh ny .Nhng ngi mc cc bnh di truyn s c t vn v cc vn
trong tng lai cng nh c khuyn bo lm sao hn ch s di truyn ca gen ny . ng
thi cng xy dng cc phng php cha bnh bng gen .
Trc ht chng ta hy cp ti mt s bnh di truyn m u d DNA c s dng mt
cch hu hiu trong nhng nm qua .
4.4.1.Bnh x nang (Cystic Fibrosis).
X nang l mt bnh c th dng u d DNA xc nh gen gy bnh .Ngi mc bnh x
nang sn xut ra cht nhy dnh bt thng lm chn ng h hp . ( gip tr khc ra cc
cht nhy ,cha m phi v vo lng n nhiu ln).
Hng nm Hoa k c trn 30.000 Trng hp tr em b bnh x ho . Trong nhng iu kin
bnh thng mt protein (c m bi mt gen ) lm nhim v iu ho hp thu nc , gi cho
cc cht nhy chy t do dc theo b mt ca ng h hp . Nu mt protein khim khuyt
(do mt gen khim khuyt) c tng hp th nc s ra khi b mt ng h hp v lm cho
cht nhy ny cng dy ln .Mt kho st ti M cho thy c 12 triu ngi mang 2 bn sao
gen khim khuyt dn n bnh x nang .
Nm 1989 bnh x nang c Francis Collin Trng i hc Michigan v Lap chee
Tsui vin nhi Toronto xc nh gen gy bnh x ho nm NST s 7 ca ngi . Sau t
lu , u d DNA c pht trin gip cho vic nh v gen ny .Vic tng hp mt u d
DNA l bc quan trng u tin cho php cc nh nghin cu to ra c cc bn sao bnh
thng v bt bnh thng ca gen .
Nm 1990, cc nh nghin cu a cc gen CF bnh thng vo vo cc t bo b bnh ca
cc bnh nhn x nang trong cc ng nghim trong phng th nghim .H tht s tho mn khi
thy cc t bo ny hot ng ging nh cc t bo bnh thng .Chng hn nh mt c trng
52

ca x nang l clo-rua khng c vn chuyn qua cc knh ca mng t bo . Nhng trong

cc th nghim labo th cc knh m ra v clo-rua i ra i vo mt cch t do trong t bo .
Khi gen CF c xc nh , cc nh t vn c th ngi ta hiu rng con chu h cng s
mang cc gen CF trong cc bn sao . (Hnh 4.7.)

Hnh 4.7. Di truyn gen gy bnh x nang .Bnh x nang theo di truyn , tc l bnh c
pht trin khi c c mt 2 gen di truyn . Trong hnh , c b v m u mang gen di truyn (c)
i vi bnh x nang nhng h u khng b bnh bi v mi ngi u c mt gen tri (C)
trong iu kin bnh thng .Con c di truyn theo tng cp , mt bn sao t b v mt bn
sao t m . V th c 4 kh nng xy ra : 3 con bnh thng bi v chng c mt gen tri (2 con
c mang gen CF) cn mt con di truyn c 2 gen gy bnh v v th bnh pht trin .
Trong nhng thng trc khi sinh cn phi lm test trn cc m thai thu c bng th thut
chc mng i qua bng . Cc t bo ca thai c phn lp t cc mu ca dch mng i bao
thai . Trong Trng hp l lng nhung mng m th mu ly t mng m (lp ph ny
ging nh mng m bao quanh thai) .Cc t bo t mng m cho cc thng tin tng t nh
cc t bo t dch mng i v v mng m c hnh thnh trc mng i nn s pht hin gen
CF bnh l c th c thc hin sm hn .
Mt bc tin mi v bn cht c thng bo nm 1992. Thay v ly cc t bo ca thai
lm Test xc nh gen CF, cc nh khoa hc thnh cng lm test ny trn phi giai on
8 t bo .Trong nghin cu ny cc phi c th tinh in vitro trong phng th nghim . V b
m c mang cc gen CF nn s c 1 trong 4 c hi thai mc bnh . Cc nh nghin cu
ly cc t bo t mi phi v xc nh bng u d DNA xem chng c hai , mt hay khng c
bn sao no ca gen CF. Nhng phi c 1 hoc khng c bn sao no s c cy tr li t
cung ca m . Chn thng sau , tht vui mng khi mt a tr ra i hon ton kho mnh .
4.4.2.Bnh au c Duchenne.
S khim khuyt gen cng lin quan ti bnh au c Duchenne (tn ca Guillaume
Duchenne, nh thn kinh hc ngi Php m t bnh ny nm 1968). Nhng ngi mc
bnh ny th c ca h thiu mt protein lm cng c Dystrophin (c v nh va xy dng)
. Thiu dystrophin th c thm nhp vi cc m khc , v b gin ra . Nm 1986 cc nh nghin
cu Trng i hc Harvard xc nh c gen m cho dystrophin . y l mt trong
nhng gen ln nht c xc nh trong b gen ngi .
Bnh au c Duchenne l mt bnh c bit , pht trin ngm ngm tr em . R rng l
nhng a tr ny sinh ra vn pht trin mt cch bnh thng cho ti khong 3 tui . Sau

53

dn dn chn ca chng tr nn khng vng vng v khng cn lc v khng kim sot c c

. n 10 tui th phi gi trn gh c bnh xe y v thng cht la tui ln 10 .
Trong y hc hin i , ngi ta dng u d DNA xc nh mt on DNA nm gn gen
khim khuyt gy ra bnh au c . on ny chnh l RFLP . Nh cp trn , RFLP l
mt on du chun ca DNA vi nhiu dng , mi dng c mt chiu di khc nhau . Nu c
mt RFLP th chc chn ti 95% l c gen gy bnh au c Duchenne.
ng dng phn tch RFLP trong bnh Duchenne ngi ta phi thu thp DNA t nhiu ngi
c quan h h hng gm cha m, ng b , c d .v.v..Mu RFLP t nhng ngi ny c s
dng thit lp mu cho ton th gia nh . RFLP ca mi c nhn s c so snh vi cc
mu ca nhng ngi b nh hng v khng b nh hng bi gen gy bnh xem n ph
hp vi mu no nht .
Lu quan trng rng khng c mt test chung no cho RFLP v RFLP gia cc gia nh l
khc nhau . RFLP cng c s dng nh cc du chun xc nh cc bnh di truyn .
4.4.3.Bnh Huntington.
Cc u d DNA vi cc du chun RFLP cng c s dng pht hin bnh Huntington
.Chng ta u bit bnh ny git cht ca s nhc folk (dn gian ) Woody Guthrie nm 1967
.Huntington l mt bnh c t khi mang thai v khng cha tr c , n hu hoi cc t bo
no . Tin trin ca bnh l lm hng h thng thn kinh v thng dn n s p ph ,di
chuyn un o , in v cht .(tn c ca bnh ny l Huntingtons cholereia , cholereia theo
gc hy lp tc l s nhy ma tc l s di chuyn un o). Bnh ny c mang tn George
Huntington, bc s ngi M nghin cu bnh ny hi u th k 20.
Ngy nay chng ta bit rng bnh Huntington do mt gen khim khuyt nm st nh NST s 4
. Gen ny c phn bit bi s ln lp li ca cp 3 CAG m cho glutamin. phin bn bnh
thng ca gen ny th cp 3 CAG lp li t 11-34 ln , cn bnh nhn Huntington th lp li
ti 42-66 ln. (Cng ging nh hi chng gy nhim sc th X mt bnh vn ng kh khn
do tu sng tiu no s c cp di y).
Tuy nhin mc ca s lp li c lin quan ti tui ca bnh nhn .Nhng bnh nhn tr th
cp 3 ny lp li nhiu hn . Bnh xut hin do mt protein b bin i cu hnh hoc b khim
khuyt , protein ny lm cht cc t bo trong hch c bn (basal ganglia)- mt vng quan
trng nht ca no i vi chc nng vn ng . Khi dng u d DNA xc nh gen gy
bnh Huntington ln u tin James Gusella (Hnh 4.8.) nh v c mt RFLP gip
ranh va nm 1984.
Gusella v ng nghip dng mt u d DNA c thit k l G8 dng tm cc RFLP
nghi ng . Nhng h li khng bit chc chn l RFLP c gn vi G 8 ng hay khng . Tht
may l h c mt gia nh rt ln nghin cu mt gia nh sng gn h Maracaibo
Venezuela . Gia nh ny b suy thoi bi c mt ph n b tc ng bi bnh Huntington ,
ngi ny di c t chu u ti y t nm 1800. Cng trnh ca nh tm l hc lm sng
Nancy Wexler v Gusella cng ng nghip c th ch r ni ging ca gia nh ny qua 7
th h v s dng u d G8 h pht hin rng RFLP c mt nhng ni xy ra bnh . Mt
iu r rng l c s tng quan cht ch gia RFLP v bnh Huntington .
Khi tm kim RFLP du chun vi bnh Huntington , nhm ca Gusella t k hoch s tin
hnh xc nh gen Huntington trong 10 nm . Cc nh nghin cu chun b cc t bo lai
gia ngi v chut trong mi t bo ch c mt t nhim sc th ca ngi . Sau h phn
lp DNA t cc NST bit trong nhng t bo ny v gn n vi u d DNA G8 xem s
gn kt c xy ra khng .Kt qu nghin cu ca h ch r rng DNA t cc t bo c cha
NST s 4 ca ngi gn vi u d G8.V th h kt lun NST s 4 c mang gen gy bnh
Huntington.
Nm 1993, nhm ca Gusella hi tho quc t vi 6 n v nghin cu thng bo rng h
nh v c gen Huntington l nm cnh nh ca NST s 4 .
Cho ti 1996 vn cha c Test trc tip cho gen Huntington , do RFLP du chun vn tip
tc c s dng tm kim gen khim khuyt qua cc th h lin tip (test chn on chnh
gen th vn cha c th, mt phn bi gen ny qu phc tp). Vic bit c mt ngi no
c gen du chun rt quan trng bi v bnh Huntington thng chng c triu chng g cho
ti tui trung nin , v cho ti thi im rt c th gen ny c truyn li cho th h sau
ri .Tuy nhin , c trng ny gy bi mt gen tri , cho nn ch c mt mt gen l bnh c th
c biu hin .
Hng nm M c ti 20.000 ngi mc bnh ny . i vi Nancy Wexler , cng vic
Venezuela vn tip tc . Hng nm c u tr li h Maracaibo v ly c nhiu d liu v thu
c nhiu mu mu b sung thm cc cc s liu vo biu s gia tng ca bnh trong
i gia nh ny .Hn 12.000 ngi trong i gia nh hin nay c lm test v biu v
ni ging i gia nh ny che lp c 2 pha hnh lang bn ngoi ca Trng i hc Colombia
. i vi Nancy Wexler , cng vic ny cn c s quan tm c nhn bi v bn thn c cng
mang trong mnh mt gen gy bnh Huntington .
54

4.4.4.Hi chng gy nhim sc th X .

Mt bnh di truyn na cng s dng u d DNA , l hi chng gy nhim sc th X. Du
hiu in hnh ca hi chng ny l c s chm tr v tinh thn (mental retardation). Mt kho
st cho thy hng nm Hoa k c 2000 ph n v 1000 thanh nin mc hi chng ny . Hi
chng ny c gi nh vy bi v nhim sc th X ca t bo (NST gii tnh c nam v n)
c mt m nh, ti NST c th c cm ng mnh m di cc iu kin th nghim .
Nm 1991, cc nh nghin cu nh v c gen bnh l nm cnh m ny . Khi h tch
dng gen bnh l v kim tra trnh t baz ca n h pht hin c mt on di trong
c 3 baz ni t c lp li nhiu ln .Cc gen bnh thng th on lp li ngn cn nhng
ngi c hi chng gy nhim sc th X th c mt on cc di c lp li (ging nh mt
n accordion ang m ) . V gen bnh thng hot ng trn no nn nu c mt gen bnh
hot ng th c th gy nn s chm tr tinh thn . Tuy nhin , v c ch th vn cha r .
Cho ti nm 1991 pht hin hi chng gy nhim sc th X ngi ta nui cy cc t bo
bch cu ca mt ngi bnh trong phng th nghim v kim tra bng hin vi xem c phi
nhim sc th X b gy hay khng . V cng nm mt cng ty cng ngh sinh hc thng bo
ch to c test u d DNA pht hin gen khim khuyt ny . Thng bo c a ra
sau mt thng pht hin c gen ny . u d ny bt li cc nucleotit lp li v xc nh
c cc t bo s pht trin hi chng gy nhim sc th X .
4.4.5.U nguyn bo vng mc .
U nguyn bo vng mc l mt bnh ung th him gp mt , hng nm c khong 200 tr em
Hoa k b tc ng bi bnh ny . Mc du thng phi phu thut loi b nhn cu , nhng
mt s phng php km hiu qu hn cng c ng dng trong cha tr nh tia x, phu
thut la ze , lm ng lnh v.v.. nu c pht hin sm .
Trong nhng nm 1970 , cc nh nghin cu quan st thy rng con ca nhng bnh nhn mc
bnh u nguyn bo vng mc cng pht trin bnh u nguyn bo vng mc .V r rng l
khuynh hng dn ti ung th ny c th c di truyn .
Ti nhng nm 1980 , nhng cuc kho st c s dng u d DNA nh v c on
DNA b mt (on DNA b t bin) vng q14 ca NST s 13 nhng bnh nhn u nguyn
bo vng mc (Hnh 4.8.) . Sau ngi ta thy bnh c lin quan ti s khim khuyt ca
NST .

55

Hnh 4.8. S pht trin ca u nguyn bo vng mc . (a) ngi bnh thng on q14 ca
NST s 13 c chc nng . (b) khi mt NST b t bin vng q14 th vn l ngi bnh thng
nhng c mang gen u nguyn bo vng mc . (c) Khi c 2 NST u b t bin th bnh mi
pht trin .
Cui cng ti nm 1986 , cc nh nghin cu Massachusetts Eye and Ear Infirmary Boston
phn lp c gen iu khin bnh u nguyn bo vng mc . Mt iu r rng l gen ny
cm ng ung th khng phi bng s c mt ca n (nh thy cc gen gy ung th khc)
m bi s vng mt hay mt chc nng ca n (do mt on b mt do t bin ).Trong Trng
hp ny , gen bnh thng chnh l gen khng ung th .
S khm ph ra gen gy ung th nguyn bo vng mc l khuynh hng ln trong khoa hc bi
v n quan tm ti cc gen ung th do b thoi ho . ( Chng ta bit r gen gy ung th i t
cc gen tin ung th sau mi chuyn thnh gen ung th ). Cc gen ung th thoi ho l cc
gen c ng li vnh vin . Tuy nhin , mun xy ra ung th th phi c 2 bn sao . Khi c
t bin hay c s sai st ngu nhin khi sao chp th s to nn mt gen th hai gy ung th .
Vic sn lng cc gen gy ung th thoi ho khc vn ang c tip tc .
4.4.6.Bnh Alzheimer.
Alzheimer l bnh thoi ho no . Hng nm c ti 2,5 triu ngi M b tc ng bi bnh
ny .N l nguyn nhn gy cht ng hng th 4 i vi nhng ngi ga M .Tn ca bnh
c ly theo tn ca nh thn kinh hc ngi c Alois Alzheimer nhng nm u 1990.
l mt bnh i cng vi vic mt chc nng tr tu (lon tr) , mt tr nh v mt ht cc
kh nng ni chung . Bnh nhn thng khng ni c , i li kh khn hoc khng hng
c ti nhng iu tht yu nht ca h .
Qua nhiu nm , cc nh khoa hc khng gii thch c nguyn nhn ca bnh Alzheimer .
Ti nm 1987 , cc nh nghin cu mt vi vin xc nh c mt gen c hiu cm ng
cc m no c trng cho bnh ny . Hu nh ng thi , cc nhm khoa hc khc cng thng
bo rng nh vic s dng u d DNA h nh v c mt du chun di truyn (genetic

56

marker) ca bnh ny trn NST s 21 ca ngi .Mc du cc pht hin ny khng gi c

rng tt c cc trng hp Alzheimer u c lin quan ti di truyn , nhng h cng ch r c t
nht l mt dng Alzheimer gia nh (familial Alzheimers disease) (FAD) l c th c di
truyn .
Gen FAD p ng cho vic sn xut mt protein c tn l Amyloid . Amyloid l thnh phn
chnh ca cc mng cht ca cc si thn kinh ( cht) , n s kp cht no ca bnh nhn .
Cho ti thi im ny Amyliod vn cha phi l cha kho cho s hiu bit v Alzheimer . Rt
c th l gen Amyloid cng vi cc yu t mi trng khc s lm cho bnh biu hin . Nhiu
nh thn kinh hc th li cho rng Alzheimer c lin quan ti mt virut no .
4.4.7. Bnh s cng teo c ct bn (Amyotrophic lateral sclerosis ).
S cng teo c ct bn (ALS) l mt bnh thoi ho neuron vn ng ca no v dy xon
(spiral cord) . ALS tc ng khong 5000 ngi M hng nm v thng c gi l bnh Lou
Gehrig . l mt ngi M u tin New York b bnh ny.
.
Do b hu hoi neuron vn ng nn dn n c b yu , v ngi bnh phi b ri cht . Hi
chng ny thng xy ra la tui t 35-70 tui v thng cht sau 3-5 nm .
C khong 5%-10% ALS l di truyn li cho th h sau , cn nhng trng hp khc th ri
rc .
Mu di truyn ny thu ht cc nh di truyn phn t vi bnh ALS t nhng nm 1980 v
cc im l 1993 , h pht hin ra gen ca dng di truyn ny l ALS gia nh (familial
ALS) .
Trong cc iu kin bnh thng , gen bnh thng gi l sod1 , m cho enzym superoxit
dismutaza. Enzym ny gip t bo loi tr cc phn t c hot tnh o xy ho cao v c c tnh
l cc gc t do . (Cc gc t do nh hng n bnh Parkinson, Alzheimer v cc qu
trnh lin quan ti tui tc ). Thiu superoxit dismutaza th lng gc t do tng cao v neuron
b hu hoi do nhim c , dn dn dn ti ALS .
Mt nhm nghin cu v gen c thnh lp nm 1993 gm 31 nh khoa hc t 4 quc gia ,
ng u l Robert Brown v Robert Horvitz . Nhm nghin cu trc tin tp trung vo
gen superoxit dismutaza NST s 21 , sau pht hin ra gen ny bi t bin nhng bnh
nhn t 13 gia nh c cc thnh vin mc bnh ALS . H thy t bin xy ra 11 v tr khc
nhau . Mi t bin u dn n vic loi i mt amino axit phn cha kho ca enzym (trung
tm hot ng) .DNA ca nhng ngi bnh thng th khng b t bin .
Da trn cc kt qu nghin cu cho thy ALS c lin quan ti s khim khuyt trong hot
ng ca enzym superoxit dismutaza v dn ti s tch t cc gc t do .
Chng ta cng bit c rt nhiu loa thuc nh vitamin C, E c kh nng loi tr cc gc t do
v cng tht d hiu khi n c dng lm gim s tc ng ca ALS .
4.4.9.Bnh tiu ng.
Nhng ngi mc bnh tiu ng khng s dng c Glucoza trong qu trnh chuyn ho v
cng kh thu nhn nng lng ho hc m n c . Di cc iu kin bnh thng , tu sn
sinh ra hormon Insulin thc y chuyn ho Glucoza . bnh tiu ng typ I (pht sinh khi
cn tr) th chc nng ca nhng t bo sn xut ra insulin ca tu l khng thch ng , n
khng to c lng Insulin p li vi s trao i nng lng (s tiu hao nng lng
). Trong Trng hp ny khi tim insulin c sn xut bng cng ngh di truyn th bnh s
thuyn gim .Cn trong tiu ng typ II (pht sinh khi trng thnh) th c th mt kh nng
s dng insulin ngay c khi cc t bo tu sn xut lng insulin .
Tiu ng typ II t nguy him nhng li tc ng ti 10 triu ngi M hng nm . C th iu
tr bnh ny bng cch kim sot trng lng v ch n . Mc d nguyn nhn pht sinh
bnh th vn cha c xc nh , nhng theo thuyt ang thnh hnh th khng nh l do cc
t bo ca c th khng phn ng vi insulin bi v thiu v tr receptor cn thit trn b mt t
bo ca chng . V insulin thng thc y glucoza i vo cc t bo ca c th , do nu
thiu cc v tr receptor ny th chc nng ca insulin khng cn na .
Mt dng khng ph bin tiu ng typ II l tiu ng li pht sinh nhng ngi tr
(maturity onset diabetes of the young ) (MODY).
Tiu ng typ II thng pht trin sau tui 25 n 30 , cn MODY li pht sinh ngay khi cn
l thanh nin hay di 20 tui .
Nm 1992 , cc nh nghin cu Trng i hc Chicago ng u l Graem Bell thng bo
rng MODY c th pht trin c l do s khim khuyt mt gen c lin quan ti enzym
Glucokinaza. Cc nh nghin cu theo ui mt dy cc nghin cu kt hp cng vi cc
nh khoa hc Php . H xc nh c mt gen c hiu trn NST s 7 . l gen p ng
cho vic sn xut Glucokinaza. R rng cc t bo tu i hi enzym ny phi pht hin c
nng glucoza trong mu .
Nhm ca Bell ch r rng c mt gen khim khuyt lm cho bnh nhn MODY sn xut
khng Glucokinaza v thiu enzym ny th vic tit insulin ca tu b ph v . (y l mt
57

quan im mi v s pht trin bnh tiu ng v cho rng n khng ph thuc vo v tr cc

receptor hoc do thiu insulin ). Trong nhng nghin cu k tip cc nh nghin cu cng xc
nh c s khim khuyt cng mt gen trong mt gia nh b tiu ng typ II dng ph
thng hn .
Khi trnh din nhng kt qu ca mnh , nhm ca Bell s dng PCR khuch i nhng
phn m cho gen glucokinaza trong cc mu DNA ly t nhng ngi bnh thng v nhng
ngi b bnh tiu ng . Sau h sng lc cc cht liu di truyn bng u d DNA pht
hin cc t bin . Cc nh khoa hc pht hin rng c s khc nhau v mt baz ni t n
DNA nhng ngi b tiu ng . S pht hin ny gi li hnh nh v bnh thiu mu do cc
t bo hnh li lim c cp trn . V tht d hiu , sa cha nhng khim khuyt
ny th ch c th nh ti gen tr liu .
Nm 1993 cc nh khoa hc quc t xc nhn nhng kt qu ny v con s t bin ln
ti vi t .
4.4.10.Pht hin bnh ung th .
R rng l cc dng xc nh ca bnh tiu ng c lin quan n mt yu t di truyn v
cng r rng l s pht trin ca ung th l kt qu tc ng ca cc yu t ngu nhin cng
cc thm ho ca mi trng , tc l n cng l mt sn phm ca di truyn .Chng hn nh
cc nh ung th hc tnh ton c khong 10% ngi b u hc sc t th ngay t bm sinh
nhy cm vi ung th .Vic pht hin cc gen c hiu hin nhin l c s lin quan gia vic
pht hin ra nhng ngi d mc ung th v vic pht trin cc cch iu tr mi . Cc nh khoa
hc tm thy mt vi gen c th nhy cm vi u hc sc t nm NST s 1 v s 9.
Cc gen lin quan ti mt loi ung th khc cng c pht hin . Gen c tn l p53
(bi v protein c gen ny m ho c TLPT l 53 kilodalton) . Mt cch hin nhin , l
mt gen kim ch khi u (tumor suppressing gene) .Gen ny ln u tin c tch dng vo
nm 1986 , p53 cho mt hnh nh chung cho tt c cc khi u v ung th , c th v gen ny
nh mt ngi gc ca cho DNA ca t bo . Khi c s hu hoi trong DNA ca t bo (tc l
s hu hoi dn n hnh thnh khi u) th p53 m cho mt protein gn dc theo DNA b hu
hoi ny v c ch s sao chp .
V hot ng theo kiu nh th nn p53 cho php t bo c thi gian sa cha DNA ca n ,
nh vy trnh c vic truyn cc gen gy ung th , cc gen li ti cc t bo con chu trong
phn bo c t (mitosis). Nh vy p53 hot ng nh mt gen c ch khi u nhm trnh cho t
bo khng b ung th ho .
Vai tr ca p53 li cng nh gi cao bi v n phc hi li cc phin bn b bin i trong
cc t bo ung th ca cc bnh nhn ung th kt trng ,ung th v , ung th no, xng , phi ,
da , bng quang v ung th c t cung .
u d DNA v PCR l nhng k thut chnh dng xc nh gen ny. Nm 1992, cc nh
nghin cu Trng i hc John Hopkins phn lp c 4 protein c m bi p53 v c
nh rng chc nng c ch ph thuc vo hn mt protein n (tc ph thuc vo nhiu
protein n).
Vn v ngun gc ca p53 cng c nhiu nh nghin cu qua tm . Nguyn nhn ch yu
vn l cc c t ca mi trng lm thay i gen p53 to nn cc dng t bin v mt
bc i sai v sinh ho trong s sao chp DNA cng c th l mt nghuyn nnhn . Hn na ,
nhng t bin ny li c nhiu kh nng di truyn li v th n ng h cho quan im cho
rng c nhng dng ung th c di truyn trong cc gia nh . Ngi ta c th chng li c
s sao chp qu mc ny , tuy nhin hin tng ung th qu tht qu phc tp nn con ng i
ti trong vic chn on v iu tr ung th vn l vn rt nan gii .
4.4.8.Ngn hng gen
u d DNA c kh nng vi nhiu mc ch nh ta thy , nhng n cha c kh nng xc
nh c v s cc bnh nh ung th v , phi hay cc dng ung th thng thng khc . Tin
liu mt ngy no u d DNA s c kh nng mi mi ngi ti ch lu tr DNA ca h l ngn hng gen .
Mun thit lp mt ngn hng gen th phi c mt vin sn sng tch chit , lm ng kh ,v
tp hp DNA ca tng ngi . San francisco c t nht mt bnh vin tham gia chng trnh
ny . Tuy nhin ,
phi lo kinh ph ly mu t mi c nhn v DNA ca bch cu s c lu trong kho . Khi
u d pht hin mt bnh no pht trin th DNA c lm tan v test cc gen gy bnh
.Theo cc nh nghin cu , ngn hng gen thch hp cho nhng ngi mun bit liu h c
mang mt gen nguy him no khng nh ung th v chng hn .Mc du s co rn ca bnh tt
cn ph thuc vo s tng tc phc tp gia gen v cc yu t mi trng , nhng mt ngi
ph n khi bit rng mnh c mang du chun di truyn v ung th v th c th lm gim
thiu nhng ru ro bng cch thay i l sng chng hn . kt lun xem cc du chun gen
c mt hay khng th phi phn tch DNA t nhng ngi h hng t nht t 2 th h , bao gm
58

c mt s ngi mc bnh . Ngn hng gen m bo rng c th thu thp c DNA t nhng
ngi h hng khi h vn ang cn sng .D nhin khi bit c mt d chun ca mt bnh
no th ngi ta khng khi bn khon , nhng i vi nhiu ngi th s thiu hiu bit vn
nhiu hn l s s hi .
4.5. NG DNG GEN TR LIU TRONG LM SNG.
4.5.1. iu tr bnh thiu ht min dch t hp trm trng (SCID).
Hng nm ti Hoa k c khong 40 tr em mc bnh Thiu ht min dch t hp trm trng
(SCID) . Mt na s bnh nhn THMDTHTT c nghin cu thy c mt gen khim khuyt
trong cc t bo , v th n khng m c cho mt enzym c hiu .Nu thiu gen bnh thng
th enzym khng c tng hp . Enzym l Adenozin Desaminaza (ADA). Enzym ny
gip cho vic phn gia cc sn phm axit nucleic ca t bo . Nu khng c ADA trong
lympho T th mt enzym Kinaza s chuyn i mt sn phm chuyn ho thnh c t , v
c t ny s ph hu lympho T . Lympho T l mt t bo rt cn thit ca h min dch , n
chng nhng tham gia trc tip trong cc p ng min dch m cn kim sot s hot ng ca
lympho B loi t bo sn sinh khng th . V th thiu ht ADA s lm cho c th mt c ch
bo v ca c 2 loi t bo lympho . D nhin khi mt kh nng bo v th bnh nhn khng c
kh nng chng li s xm nhim ca cc bnh .Cht v cc bnh truyn nhim l hu qu ca
bnh SCID.
Ma h 1990 , cc nh nghin cu thuc vin quc gia y t Hoa k (National Institute of Health
NIH) nhn th ng dng GTL i vi bnh thiu ht ADA . (Gen ADA nm trn NST s
20 ca ngi v c 32.000 cp baz , 12 exon) . ng u nhm nghin cu l R.Michael
Blaese , W. French Anderson v Kenneth Culver .Cc nh nghin cu a gen ADA
vo cc t bo lympho ca cc bnh nhn SCID c chn lc . thc hin vic cc
nh nghin cu phi rt ht cc t bo lympho ra khi bnh nhn v phi by cc t bo c
hng t retrovirut c mang cc gen sn xut ADA .Sau khi cc gen ADA c a vo NST
ca t bo lympho th cc t bo ny s c a tr li cho bnh nhn . Nu mi vic xung
x , cc gen ADA s m cho enzym ADA v loi tr s thiu ht enzym ny . Sau vi tun li
lp li qui trnh ny bi v cc t bo lympho ch tn ti trong c th vi thng .
c ph chun thc nghim ADA cc nh khoa hc phi xin kin ca nhiu c quan
chc nng , trong c U ban c vn DNA ti t hp ca vin NIH (NIH recombinant DNA
advisory Commitee-RAC) , kin ca Human Gene Theory Subcommitee of RAC (tiu ban l
thuyt GTL ca RAC) , th 3 l s ph chun ca gim c NIH v th 4 l kin ph chun
ca FDA . Cng vic ny phi mt 3,5 nm mi hon tt . Cui cng , vo 14-9-1990 cc t bo
bin i gen u tin c a vo mch mu ca mt c b 4 tui l Ashanti .
Ma xun 1991, Blaese thng bo vi u ban GTL rng cc t bo lympho c thit lp li
(c cng ngh ho) sng c trong h tun hon ca c gi ny . Hn na li c bng
chng l cc t bo ny tn ti cn tt hn c cc t bo lympho ca c ta .Tuy nhin, lng
ADA c to ra bi cc t bo lympho bin i gen phi tng dn dn theo thi gian v phi
t ti 18% mc bnh thng . Thm vo ngi ta cng thy kh nng sn xut ra khng th
ca c gi ny cng tng ln ng k . Trong mt cuc tr chuyn vi cha ca c gi ng
m t gen ADA c ci vo nh l mt gen ci (laughing gene) v n l mt a tr hnh
phc hn nhng ngi khc ng ni , Bi v chu c kho hn .
Cng vo thi im mt c gi th hai 9 tui l Cynthia cng c tr liu theo cch ny .
Liu trnh bt u t 30-1-1991 .
Vi bt k mt phng php iu tr no cng vy u c cc mt vi kin tri ngc , vi
GTL trn bnh nhn thiu ht ADA cng vy .Chng hn nh ngi ta i hi cn phi ban
hnh nhng quy nh y v tnh an ton bi v cc bnh nhn SCID rt d b tn thng vi
cc bnh truyn nhim . Hn na , tnh trng cuc sng cng b e do nu virut tr gip (vec
t) li b nhim bn trong khi chun b vec t . V nu khng duy tr c mi trng v trng
trong lc ang sn xut v qun l cc vec t th cng c th dn n b xm nhim .
Cng c nhng kin i nghch cho rng vic tin hnh thay th phi c tin hnh nh th
no cho t mo him hn . Chng hn nh cy thnh cng tu xng th phi cho n
ngmdn vo bnh nhn nhng t bo bnh thng c mang gen ADA c chc nng . Mt
vn cn phi lu i vi quy trnh ny l tu xng ca ngi nhn v ngi cho phi rt
ph hp vi nhau (thng l anh ch em rut).
Mt vn khc l s loi thi t chc ghp ca bnh nhn .Nhng ngi xng ra GTL th
tranh lun rng c ch loi thi c th khng xy ra i vi GTL bi v n c truyn li chnh
nhng t bo ca c th mnh .
Mt phng php khc ca GTL l thay th enzym .Nhng nm u 1990 , FDA ph chun
cho php s dng ADA tinh khit gn vi cc phn t ha hc polyetylen glycol (polyethylene
glycol PEG). Cc phn t PEG c tc dng ko di s hot ng ca ADA trong c th . Kt
59

qu l nhng phn t PEG-ADA c th tim c cho nhng bnh nhn SCID gim nh s
thiu ht ADA .
Nhng kho st nh gi thuc hin nay tiu tn khong 50.000 USD hng nm , l mt
hnh nh r nt chng li vic dng thuc thay v s dng GTL.
Thng 9-1991 l thi im ngt ngo c bit cho 2 c b .C hai c c tip nhn cc t bo
bin i gen u c n trng , mt ti nh tr cn mt th vo lp 5 . C hai c gi u
biu l l h thng min dch du c phc hi r rt .Mt t bo ng trn trang nht vi
tiu tr li Trng hc vi nhng gen mi . iu ng vui l hin nay c hai c gi ny
u sng mt cch bnh thng nh bao nhiu ngi kho mnh khc .
4.5.2. Gen tr liu trong chng ung th .
Cng trong bui gp mt ta ni ph chun cho cc th nghim ADA , U ban c vn DNA ti
t hp ca NIH cng ph chun mt th nghim th 2 trong GTL .Th nghim ny cp ti
vic rt cc t bo lympho ca bnh nhn ung th ra v ci vo cc t bo gen khng ung th
. Cc t bo c thit k li ny sau li c a tr li cho bnh nhn vi hy vng l tc
nhn khng ung th c th git mt cch chn lc cc t bo ung th . Nhm khoa hc ny
c dn u bi Steven Rosenberg thuc NIH cng cc ng nghip, W. French
Anderson (ngi tham gia trong th nghim ADA) .Trong quy trnh th nghim ny , cc
nh khoa hc s dng mt dng lympho l lympho thm nhp khi u (tumor infiltrating
lymphocytes TIL) . l nhng t bo c bit ca h min dch . Di cc iu kin bnh
thng , TIL c kch thch bi cc yu t ho hc ca cc t bo ung th ri i vo cc khi u
rn chc v ph hu cc t bo khi u .
Mt v kh ph tr quan trng l cc yu t hoi t khi u ( Tumor necrosis factor TNF )
c gn vo cc tc nhn khng ung th . TNF l mt sn phm protein ca i thc bo - cc
t bo ca m ging nh amip s tham gia vo vic ph hu cc t bo ung th .Protein TNF
c th c sn xut bi cng ngh di truyn v c th c dng nng cao kh nng khng
ung th ca cc lympho b khi u thm nhp .
Rosenberg cng ng nghip d nh s iu tr cho nhng bnh nhn u hc sc t tin trin
(malignant melanoma) dng ung th da nguy him thng khng p ng vi bt c cch
iu tr no .
Trong gen tr liu , cc nh khoa hc ly mt mnh m ung th ca bnh nhn . H cng
phn lp TIL t mu ca bnh nhn . Cc TIL ny sau c nui cy cng vi cc t bo t
m b khi u kch thch TIL v nng cao tnh la chn ca cc t bo u hc sc t ca bnh
nhn . Ri thm c mt hn hp gi l Interleukin-2 . Interleukin-2 l mt protein ca t bo
lympho , n c tc dng thc y nhanh s nhn ln ca TIL .By gi n cng on ci gen
.Cc nh nghin cu trn TIL c kch thch vi mt s vec t retrovirut c mang gen
TNF (yu t hoi t khi u) .Cc vec t ny xm nhp vo t bo cht ca TIL v mang gen
TNF vo trong NST . Cc TIL cng ngh ho sau c nui cy mt thi gian to nn
mt qun th ca lympho thm nhp khi u sn xut TNF (TNF producing tumor
infiltrating lymphocytes ).
Ri tip tc kin nhn i vo cc bc tip theo ca th nghim . Cc TIL to TNF c cng
ngh ho s ngm dn vo h tun hon ca bnh nhn . Vi kh nng c tng cng n s
ph hu cc t bo ung th v c kh nng la chn vi cc t bo u hc sc t , TIL s tn
cng cc t bo ung th v chuyn giao tc nhn khng ung th TNF ca chng . Nhng b
phn kho mnh ca c th c th c phng trnh bi v TIL kch thch mt cch chn lc
vic tm kim cc t bo u hc sc t .
Nm 1995 nhm nghin cu ca Rosenberg thnh cng trong vic cho thm cc t bo
cng ngh ho vo hn chc bnh nhn b u hc sc t .Thng li ny iu kin tr li
cu hi liu TIL c thm vo nh v s lng v tr b u cha hoc l vn s
lng TNF cn trong tr liu cho bnh nhn . tr li cu hi ny v nhng vn khc ,
nhm ca Rosenberg lm th nghim vi cc dng vec t mi vi mc ch lm tng kh
nng ca TIL i vi s nh v .
Mt kiu GTL chng ung th km ph thng hn cng c ph chun vo nm 1993 i vi
cc th nghim trn lm sng .Cng trnh ny c thc hin bi trung tm y hc Methodist
thnh ph Iowa vi ngi ng u l Michael blaese v Kenneth Culver. H a vo
cc t bo u no mt gen m cho enzym Thymidin kinaza . Enzym ny c mt s vi sinh vt
s dng trong tng hp DNA khi phn chia t bo v n c th ngng hot ng bi thuc
Ganciclovir. iu hy vng l cc t bo ung th s gn vi cc gen ny v tr nn ph thuc
vo s hot ng ca thymidin Kinaza khi phn bo v s nhy cm vi ganciclovir ri cht .
Gen Thymidin Kinaza thng c gi l gen t st . Ganciclovir cng c dng tiu
dit cc khi u .

60

Hnh 4.9. Ganciclovirlm gin on s phn chia t bo nh th no . (a) Enzym Thymidin

Kinaza( trn cng) tc ng bng cch gn gc photphat vi nucleozit to thnh nucleotit
.Thuc Ganciclovir c cu trc tng t mt nucleozit , v th Thymidin kinaza kt hp nhm
vi Ganciclovir to thnh mt nucleotit gi . (b) Trong khi phn chia t bo th mt enzym
khc l DNA polymeraza( di cng) s gn vi nucleotit gi ny pht trin thnh mt phn
t DNA khi tng hp DNA . Tuy nhin, cc nucleotit ny li thiu im gn cho cc nucleotit
tip theo nn vic ko di phn t DNA b dng li t ngt . Bi v kt thc s tng hp
DNA nn cc t bo khng phn chia c thnh 2 t bo mi . Trong GTL, khi ci mt gen
Thymidin kinaza vo mt t bo s lm cho t bo nhy cm vi s tc ng ca ganciclovir .
Ganciclovir sau s c ch s phn chia t bo v git cht t bo ny . V th m Thymidin
kinaza c gi l gen t st .
Vec t s dng trong cc th nghim ny l cc retrovirut c thit k li vi gen sn xut
ra Thymidin kinaza ly t Virut herpes Simplex. Cc t bo no bnh thng th khng phn
chia nhng cc t bo u th phn chia mnh lit v cc retrovirut ci mt cch chn lc gen
Thymidin kinaza vo cc t bo u ca no .(Virut khi thm nhp vo cc t bo bnh thng th
khng c nhn ln). cung cp s lng virut , cc nh nghin cu phi tim vo
bnh nhn cc t bo da b nhim virut ly t mt con chut .Nhng t bo b nhim phng
thch virut ra ngoi c th vi mt tc u u .V th chc chn l virut c kh nng nhn
ln cc t bo khi u , chnh v th m c hi b nhim cc t bo khi u tng ln . Nhng t

61

bo chut s cht sau vi ngy do c ch loi thi bnh thng ca c th , cn cc t bo b

nhim khi u th li bt u sn xut ra thymidin kinaza . Khi c tim Ganciclovir th cc t
bo ny v khi u no s b ph hu .
Cc th nghim vn cn ang tip tc .
4.5.3.Nhng n lc hin ti v tng lai.
Ngay khi cc th nghim u tin v GTL c thc hin th cc nh khoa hc cng thc ho
cc test sp ti vi s tip cn mi trong y hc .
4.5.3.1.Mt trong s cc d kin l thay th gen khim khuyt trong bnh x
nang . Vn chnh cc bnh nhn x nang l cc ion clo rua c cng dy ln trong cc t
bo cc t chc v cc nang ca c th .Cc ion bnh thng vn i ra khi t bo qua mt
protein hnh ng gi l knh - l mt protein iu ho vn chuyn mng cc nang (cystic
transmembrane conductance regulator-CTCR ) . nhng ngi b x nang , protein CTCR
khng c to ra v khim khuyt gen v th cc ion b tch t trong cc t bo v rt nc
trong cc t bo , khi cc cht nhy b ng li trn cc ng dn ca c th , c bit l
cc ng dn kh . iu kin ny to thun li cho vi khun thm nhp (Hnh 4.10)

Hnh 4.10. Cc lng nhung ging nh tc mc ra t cc t bo ng h hp. Bnh thng th

nhng lng ny dng by bi v cc vi sinh vt , nhng trong bnh x nang th do d tha
cc cht nhy nn n dnh vo nhau v mt chc nng by bi v vi sinh vt .Do lm cho
ng h hp d b nhim trng
im ni bt nhng bnh nhn x nang l s b cht do nhim trng phi v ng h hp
trc tui 30 .
Vic pht hin ra gen khim khuyt trong cc bnh nhn x nang l mt thnh tu ng ghi
nh ca nm 1989. Trong nm c 2 nhm nghin cu d nh tr liu thay th gen khim
khuyt ny .
Ti Trng i hc Iowa , cc nh khoa hc s dng thnh cng vec t Virut ci vo
phin bn gen bnh thng ca cc t bo phi ca mt bnh nhn , c nui cy trong
phng th nghim . Cc t bo ny sau sn xut ra protein CTCR n nh l cc knh
cc ion i qua mng t bo v kch thch s loi thi cc ion clo rua d tha .
Nhm th hai lm vic ti Trng i hc Michigan cng thu c nhng kt qu tng t
nhng vi cc t bo tu. (trong cc t bo tu , nu thiu protein ny th dn ti vic gii
phng d lng enzym).
iu lc qua i vi vic iu tr x nang vi GTL cng tng ln bi nm 1991 c thng bo
v mt virut lnh tnh thng thng gim hot lc c th dng chuyn nhng gen bnh
thng vo trong cc t bo ch c nui trong phng th nghim.Nm cc nh nghin

62

cu ti vin huyt hc, tim ,phi quc gia Hoa k m t rng cc gen m cho cc enzym ca
ngi c th c gn vi virut ng h hp Adenovirut (Hnh 4.11.)

Hnh 4.11. nh qua hin vi in t ca Adenovirut (x300.000) , mt dng virut ng h hp

c dng nh mt vec t trong GTL .
B li vic khng c kh nng sao chp , cc adenovirut ny l mt vec t thch hp bi v n
xm nhim cc t bo ca ng h hp (mc du n khng phi l Retrovirrus).C th l cc
Adenovirut gim hot lc ny c th vn chuyn cc gen bnh thng vo cc t bo ng h
hp ca cc bnh nhn x nang thay th cho cc gen khim khuyt .
Nhng tin b trong nghin cu cng tng thm nhiu t nm 1993. Trc ht l nhm cc nh
sinh hc phn t ca Anh quc thng bo rng h sa cha c s khim khuyt gen x
nang chut . Sau n thng 4-1993, U ban c vn DNA ti t hp ca NIH ph chun
3 th nghim GTL v x nang c lin quan ti vec t adenovirut.T hp nghin cu ny c
iu hnh bi 3 nhm , mi nhm nghin cu chn 10 bnh nhn tui trn 21 cc bnh vin
Michigan , Lowa v Maryland . Hai nhm xt Adenovirut vo trong mi sau a vo
phi ca bnh nhn .
Cn nhm th 3 th ch xt vo mi bnh nhn thi . Mc tiu ban u ca test l xc nh xem
liu phng php tr liu c kh nng lm cho bnh nhn to ra c cc protein phng
chng bnh x nang hay khng .Cng cn phi lu rng Adenovirut khng hp nht vi cc
t bo nh Retrovirut v th bt k kt qu dng tnh no ca test cng l do c truyn li .
Cui 1994 ngi ta ci c gen ny vo cc t bo c mi cng nh phi ca bnh nhn
x nang .
4.5.3.2.Mt d kin khc ca GTL l hng ti cc bnh di truyn him gp ,
l bnh Cholesterol cao c tnh cht gia nh (familia hypercholesterol).
Nhng bnh nhn ny khng c kh nng loi b cholesterol trong mu . Ci ngun ca vn
l do mt gen khim khuyt m bnh thng n m cho cc receptor cholesterol. Cc Receptor
cholesterol l cc phn t protein b mt cc t bo gan n by cholesterol vo mu v loi
n ra khi h tun hon .Nu thiu cc receptor ny th cholesterol vn nm li trong mu v
tch t li lm tc nghn ng mch v tnh mch. Kt qu l lm tng huyt p v cc bnh tim
ngay c khi cn rt tr .
Nm 1993 cc nh khoa hc Trng i hc Michigan thng bo rng h thnh cng
trong vic thay th gen khim khuyt dn n tng cholesterol c tnh cht gia nh ny . Cc
nh khoa hc ly mt mu cc t bo gan ca mt ph n ri cho cc t bo ny nhim vi
virut vec t c mang gen ca receptor bnh thng v cho thm nhp cc t bo bin i ny

63

vo bnh nhn. Kt qu l mc cholesterol ca ngi ph n ny gim xung ng k ti mc

lm cho U ban c vn ti t hp DNA cho php m rng th nghim thm 5 bnh nhn na .

4.5.3.3.Mt hng khc ca GTL c lin quan ti cc t bo gan cng c d

nh bi cc nh nghin cu Trng i hc Baylor. D kin ca h l phn lp cc t bo
gan t nhng ngi tnh nguyn bnh thng ri nui cy nhng t bo ny trong phng th
nghim v nh du chng vi mt gen khng khng sinh . (Gen khng khng sinh thng
dng lm du chun bi v cc t bo tip nhn gen ny c th c test d dng v sc
khng i vi tc ng ca thuc) . Tip theo l cc t bo c nh du s c cy vo
mt ngi tip nhn tnh nguyn .Gen nh du ny s cho php cc nh nghin cu xc nh
cc t bo c cy tn ti tt nh th no trong c th ngi nhn .iu quan trng l phi
nh gi c s sng st ca cc t bo gan c nui cy bi v trong tng lai cy ghp
t bo c th thay th cho vic phi ghp ton b gan . Cng tht d hiu l quy trnh cy ghp
t bo c th c chp thun vi nhng ngi c cc bnh chuyn ho lin quan ti gan .
Trong nhng Trng hp nh vy , cc t bo c phn lp c th c th c bin i gen
to nn protein v nu thiu cc protein ny th s sinh ra cc bnh v gan .
4.5.3.4.Gen tr liu cng c th lm gim nh cc hiu ng ca hi chng thiu
ht min dch mc phi (Acquired immune deficiency Syndrrome)-AIDS).
Nm 1993 U ban c vn DNA ti t hp (RAC) ph chun cc test lm sng tr liu vi
vic chuyn gen mi vo cc bnh nhn nhim HIV .Test ny c lin quan ti mt ni t bin
ca HIVc cha cc gen khim khuyt rev v env . Ni ny c cc nh nghin cu
Trng i hc California San Diego to ra . Ni HIV t bin ny khng sao chp c bi
l cc gen rev v env b khim khuyt nn khng m c cho protein iu ho v protein
v bc ngoi ca virut , m c 2 protein ny u cn cho s sao chp ca virut . s dng
cc virut t bin nh l cc thit b tr liu , cc nh nghin cu tch cc Lympho T ra khi
bnh nhn nhim HIV v ci cc virut t bin vo nhng t bo ny . Sau ngi ta nui cy
c mt s lng ln cc t bo ny , ri sau li tim tr li cho bnh nhn . Nhng
lympho T c cha cc virut ny khng sn sinh c virut mi , ngc li n kch thch c
th sn sinh ra cc t bo Lympho killer CD8 . Nhng t bo ny c to ra phn ng c
hiu vi cc t bo b nhim HIV v trong cc test phng th nghim thy chng c kh nng
ph hu cc t bo ny . Hy vng iu cng xy ra trong c th .
Mt dng khc ca GTL i vi cc bnh nhn nhim HIV cng c RAC ph chun . Trong
test ny , cc gen m cho cc protein HIV c gn vo DNA ca virut chut nhng v hi vi
ngi . Cc virut c cng ngh ho ny c tim vo nhng ngi nhim HIV nhng
khng c cc triu chng biu hin bnh .Cc nh nghin cu tin tng rng cc gen HIV s
kch thch cc t bo bnh thng ca c th sn xut ra cc protein ca HIV . Cc protein ny
s li kch thch h min dch tit ra cc khng th HIV . Cc khng th ny c th phng trnh
s lan rng HIV trong c th bnh nhn v bit trc c s ph hu ca cc lympho- T , l
c trng thng thng ca cc Trng hp AIDS tin trin .
Mt kiu khc ca tr liu khng AIDS c lin quan ti mt qu trnh thc nghim ti phng th
nghim , nhng cha c thc hin trn lm sng . Qu trnh ny c ch ch ly cc t bo b
nhim HIV to ra cc khng th khng HIV . Cc nh cng ngh DNA vin ung th Dana
Father , Boston tng hp c gen ca khng th F105 . Khng th ny phn ng v gy
bt hot gp 120-mt glycoprotein c v HIV cn cho vic gn HIV vo cc t bo ch . Cc
nh khoa hc tinh ch gen ny sao cho cc phn t khng th c to ra li ni cht
(ER) ca t bo v mt thnh phn ca mng gp120 cng c tng hp .V h cho thm vo
mt on gen cho protein khng th neo vo li ni cht v khng ri khi t bo (ging
kiu hot ng ca khng th ). c thit lp nh vy nn cc gen ny c gn vo mt
plasmid chun v ri ci vo cc t bo nhim HIV t cc ng vt th nghim labo . Tht s
thnh cng , khng th F105 c to ra v tnh trng d thng l cc t bo b nhim to
ra c khng th chng li rt nhiu tc nhn gy nhim cho n . S tng hp gp120 gim gh
gm v s tng hp cc ht HIV cng chm i dng k . iu ng ni l cc t bo ny chng
h c hiu ng gy c no c .

64

4.5.3.5.Khng dng li , trong cc th nghim vi cc t bo phng th

nghim , cc nh nghin cu Trng i hc North Carolina thnh cng
trong vic thay th mt gen bnh thng thay cho gen gy thiu mu do t bo
hnh li lim .Gen c ci m cho mt globin bnh thng mt thnh phn ca
hemoglobin , gen ny b khim khuyt nhng bnh nhn thiu mu do t bo hnh li lim
.Tuy nhin nhng tin b t c trong vic iu tr bnh thiu mu do t bo hnh li lim
vn cn rt chm so vi s mong i . Hn na vic tng hp mt protein khn yu nh th s
i hi phi c s iu ho chnh xc s hot ng ca cc gen v s kim sot iu ho ny
hin nay l ngoi kh nng ca cc nh Ho sinh .
4.5.3.6.Mt ng c vin khc ca GTL l bnh Lesch Nyhan . Hng nm cn
bnh him ny tc ng c tnh 2000 ngi M . Bnh ny gy bi s khim khuyt mt gen
lm cho c th khng tng hp c mt enzym c tn l hypoxanthin-guanin-photphoribozyl
transferaza (HPRT). Thiu HPRT th s chuyn ho Guanin v Hypoxanthin b nh tr v to
nn axit uric . Kt qu l gy nn bnh gout trm trng v hu hoi thn , hay nhng a tr b
bnh b lit no, tinh thn chm pht trin cng nh c cc hnh vi k qui nh thc dc khng
kim sot c dn n p ph , n ni sng sng , gm vo mi v cc ngn tay , p u
vo tng v.v..
Nm 1984 , cc nh khoa hc Trng i hc California San Diego s dng vec t
retrovirut ci cc gen sn xut HPRT vo trong cc t bo ngi c phn lp .Trong cc
th nghim ca h cc gen lm tng mt cch ng k mc enzym trong t bo . Tuy nhin ,
cho ti thi im ngi ta vn cha c thc nghim no cy gen vo c th ngi .Mt
vn ln m cc nh khoa hc v no phi quan tm l h cha xc nh c liu enzym ny
c gii to c nhng hnh vi bt thng v s lit no hay khng v h cng khng bit chc
chn lng enzym then cht trong c th l bao nhiu .
4.5.3.7 Mt ng c vin khc ca GTL l bnh Gaucher v bnh a chy mu
(hemophilia) . Bnh Gaucher l mt bnh di truyn ca h thn kinh trung ng i cng vi
gan lch to , mn cc xng di v vng da ngi ln . Nm 1993 , RAC ph chun mt test
lm sng a vo cc t bo bnh nhn mt gen m cho enzym glucocerebrosidaza (v enzym
ny b mt hoc khim khuyt). Vic to ra enzym v s hot ng ca enzym ny c th
lm gim nh triu chng ca bnh .
Test ny hin vn ang cn tip tc .
Trong bnh a chy mu Hemophilia B th bnh nhn khng sn xut c mt protein lm
ng mu l factor IX (yu t IX) , gy nn chy mu qu mc nh trong hemophilia A . Nm
1993, cc nh nghin cu ca Trng i hc Baylor thng bo h ci thnh cng cc gen
ca factor IX vo trong cc t bo gan ca ch .
Mt Retrovirut c dng a cc gen ny vo trong cc t bo ; cc nh nghin cu thy
retrovirut c th lm cho gen ci trc tip vo cc ng vt sng . hon tt vic h
gii phu ly mt phn gan ca mt con ch , do d n kch thch cc t bo cn li phn chia
nhanh chng (nh khi hi phc sau khi b bnh hoc b chn thng ) . Cc nh cng ngh
DNA sau tim cc retrovirut cng ngh ho qua tnh mch ca chnh ca gan dn trc
tip vo gan . Khi cc t bo gan c nhn ln n s gn vi cc virut v hon tt vic ci gen
.
Theo nhiu nh gi th cc bnh di truyn gy nn bi s khim khguyt mt gen n ln ti
con s 2000 . Cc bnh di truyn lm cho mt s ln ngi khng c hng s ci xin cng
nh mt s ln tr em b cht v chm pht trin tinh thn . Hn na h cn l mt gnh nng
v kinh t cho x hi v l hi chung bo ng i vi cuc sng con ngi . Gen tr liu t ra
l mt phng php kh hu hiu i vi vic cu cha cc bnh di truyn .
4.6. bo him an ton trong gen tr liu .
Vn bo him an ton trong GTL kh phc tp , y chng ti ch trnh by mt cch vn
tt nhng quy nh tm thi Hoa k mt quc gia i hng u trong GTL v cng l quc
gia c nhng quy ch rt cht ch i vi mt phng thc iu tr mi y tim nng nhng
cng khng t ri ro ny .
lm lng xung s s hi i vi GTL v m bo c s bo v c bit ca x hi , hin
nay c mt h thng xt duyt nhiu tng trong thao tc i vi cc ngh ca cc nh
khoa hc . H thng ny cng to nhng thi c thun li cho u vo trong cng ng .
theo ui cc th nghim khoa hc , cc nh khoa hc phi chng minh c rng cc gen

65

c ci vo khng gy tc hi trong cc m. (chng hn nh h phi ch r c

rng cc gen ca vec t virut s khng hot ho cc gen ca ngi m n cn phi nm yn ).
H cng phi chng minh v an ton ca cc vec t virut trn ngi , bi v cng d hiu l
mt vec t c th c ci vo mt im trn NST , n c th lm t bin mt t bo do
lm sai trnh t gen . V h cng phi chng minh c rng cc virut tr gip (helper) s
phi c loi i ton b khi hn hp virut bi v cc virut gip l xa l vi cc
m ca ngi nn n c th gy bnh .
Cn mt vn na l vic ci cc gen vo cc t bo cc m c bit . n thi im
1996 , vic ci cc t bo tu xng vn c u tin nht bi v cc nh khoa hc tng lm
cc cng vic loi b ri cy tu xng t trn 20 nm nay .
Mt tr ngi ln tip theo l vic ci cc gen vo trong cc t bo khng phi l iu
d dng nh cc t bo no b chng hn .Thc vy,vic nhn ln mt gen n b
khim khuyt s nh hng ti cc t bo ca no b .
T 1984, h thng xt duyt nhiu tng ca GTL v cc th nghim c nhng
tin trin ng k. Gim th hip hi cng ngh DNA hin nay yu cu bt k mt nghin
cu no c lin quan ti ch th con ngi u phi c ph chun ca hi ng xt duyt
quc gia nhng ngi kho st trong bao hm c nhng vn thuc lun l v an ton sinh
hc .
Nhng d kin v GTL trn ngi c th phi c ph chun bi U ban c
vn DNA ti t hp ca NIH (RAC). RAC s thnh lp mt nhm cng tc c bit v
cc d kin GTL trn ngi . Mt tiu ban v GTL trn ngi c thnh lp ,
l mt nhm gm 4 nh khoa hc lm vic trong cc labo , 3 nh khoa hc lm sng , 3 chuyn
gia v lun l v 2 chuyn gia v chnh sch cng ng .
T 1994, RAC ph chun vic khng cn phi trnh tt c cc trng hp GTL m ch
cn ph chun cho cc th nghim khc thng (c bit).
Tiu ban GTL trn ngi ca RAC chun b mt ti liu tm ra nhng im cn xem xt
(points to consider) cho cc nh khoa hc lp ra k hoch trnh nhng ngh v cc nghin
cu GTL trn ngi .
Cc nh khoa hc phi cung cp chi tit cc thng tin v b nh m h mun
iu tr , nhng phng php tr liu gn nht v c gi c lin quan ti qu
trnh tr liu bng gen .
Cc c tnh ca cc vt liu di truyn d nh s c ci vo chng hn nh
nhng bng chng v tinh khit , cc kt qu trong labo trong nui cy t
bo v trong ng vt , cc tc dng ph c th trn bnh nhn v cc hiu
ng s c gim i ti a nh th no ;s la chn cc ti ;nhng bo
v ring cho bnh nhn nh th no?. Cc vn x hi v lun l nh tnh
cng khai v nhng ngn tr s s hi ca cng ng cng c cp .
V quyn hn ca RAC cng c quy nh r nm 1993 khi gim c NIH Bernadine Healy
hp vi u ban trong mt phin bt thng yu cu tng tc quy trnh ph chun GTL cho
lm t thin (compassionate use) i vi nhng ngi m yu liu mng mt phen .
Sau nhiu tranh lun , U ban ny lm cho NIH chnh thc chp thun ph chun v nh
gi nhng ngh GTL vi cc trng hp c bit . NIH cng chnh thc khin trch v vic
tham kho cc kin ca cc thnh vin RAC nu h thy cn phi c kin cho nhng trng
hp c bit.
Ny sinh nhng k hoch cho GTL cng l nhng ti c FDA quan tm c bit .C quan
ny tham gia vic xt duyt bi v quyn hn ca n l xem xt tt c cc nghin cu c lin
quan n vic s dng thuc trn ngi . FDA cng rt quan tm ti tnh an ton ca cc cht
liu dng trong thc nghim , h lun xem xt mt cch t m cc cht liu xem n c ng tin
cy c lp li trong cc th nghim tip theo hay khng .
Trong thm tm cc nh klhoa hc th cng nhiu lp xt duyt th s ph chun cng chm tr.
gi c lng tin ca cng ng , cc nh khoa hc v nhng ngi c trch nhim phi c
gng xc nh c a ch nhiu vn khoa hc rm ri v tham gia vo mt s vn thuc
lun l ni ln khi ng dng GTL. Cng chnh v th m phi mt 3,5 nm mi ph chun c
th nghim ADA nh ta cp phn trn .R rng l GTL ang c theo ui nhng
phi ht sc thn trng.

5.1.M u :

Chng V
Cc dc phm ch to theo cng ngh DNA .

66

Trong nhng nm 1940,vic pht trin v s dng khng sinh nh du mt bc chuyn

tip trong y hc .Trc cc thy thuc cng gip c cht t cho nhng bnh nhn b
tiu chy , thng hn , bch hu v cc bnh truyn nhim khc . Tuy nhin, khi a khng
sinh vo h c c mt cch iu tr mnh v lm lui bnh mt cch ng k . Khng sinh
lm thay i tn gc r trong y hc v vch ra mt hng mi trong iu tr m tc dng
ca n vn cn ko di cho ti tn hm nay .
T nhng nm u 1980, mt thay i tn gc khc cng xy ra trong vic iu tr cc bnh
tiu ng v bnh a chy mu . Bnh tiu ng l do tu khng sn xut c insulin , cn
bnh a chy mu th lin quan ti vic gan khng c kh nng sn xut ra mt yu t cn thit
cho s ng mu . C Insulin v yu t ng mu u l protein v c hai u c th c thay
th bng nhng ngun khc .Chng hn nh , Insulin c th thu nhn t tu ng vt v yu t
ng mu th i t huyt tng ca ngi cho. Nhng iu ng tic l vic vic thu nhn
protein nh th th rt dt v c th dnh cc cht bn c ngun gc t vi sinh vt v th n
c th gy phn ng d ng cho bnh nhn.
S tin cng ca cng ngh DNA : Trong hn 20 nm qua , cc nh khoa hc ng
dng cng ngh si ng ny pht trin nhng cch iu tr an ton hn i vi bnh tiu
ng v bnh a chy mu . Chng hn nh by gi c th thu nhn c insulin ngi t vi
khun c cng ngh ho (Hnh 5.1)

Hnh 5.1.Tinh th Insulin tinh khit c sn xut t vi khun . Cc tinh th Insulin ngi ny
c tng hp bi mt vi khun ng rut E.Coli bng cng ngh Ho sinh bng vic mang
gen v biu hin gen to Insulin .
v kh thi vic phn lp yu t ng mu t cc t bo ng vt c nui cy .
Chng ta s xem xt c 2 thnh tu ny phn sau .
Mt ci tin khc trong y hc l nng cao kh nng khng bnh ca c th . Chng hn
nh , cc t bo ca c th nh ta bit l n c th tng hp c cc cht khng virut nh
interferon . Cc thy thuc nhn thy rng cung cp interferon cho c th l mt cch hiu qu
iu tr cc bnh mn rp (herpes simplex), bnh si , thu u v vim tu xm .
Cc nh cng ngh DNA hin nay c th sn xut c Insulin vi s lng ln v c th thy
trc mt ngy no Interferon c s dng cha cc bnh do virut cng chng khc no
ta dng penicillin tr vi khun vy .
Mt cch mng trong y hc cng cn phi k n l vic thit k cc loi thuc . Cc thy
thuc c s dng cc sn phm ho hc ca cng ngh DNA lm bt hot mt phn t
n c lin quan ti bnh . Nhng loi thuc nh th gi l cc phn t Antisene , n phn
ng vi cc phn t mRNA , bi vy lm gin on qu trnh tng hp protein .
i vi vic phng bnh , cc nh cng ngh DNA mang ti mt lp vac xin hon ton mi
. Cc vac xin mi hn s kch thch h min dch hiu qu hn vac xin c v t tc dng ph
hn .Vi 2 li th nh vy cc vac xin mi s phng chng tt bnh tt trong tng lai.
Vic lm hi phc v thay th cc protein b mt nh insulin v cc yu t ng mu kch
thch c th thc hin c cc qu trnh sinh l bnh thng ca n . C th lm tng cng c
ch bo v ca c th vi interferon hoc nng cao kh nng khng li cc xm nhim mt cch
t nhin . V nh vic s dng cc thuc antisene lm bt hot mt phn t c hiu

67

khch l cc thy thuc hng vo cc cht c lin quan ti mt bnh xc nh .Trong tt c cc

trng hp nh vy , cc sn phm dc hot ng theo cch hp l v chng ta d dng hiu
c iu . Cng ngh DNA v th mang ti y hc mt cch tip cn tn tin tr bnh
chnh xc hn cc phng php khc k nguyn trc . Trong k nguyn liu lng v
cch pha ch thuc ca cc thy thuc cng cha c bnh , nhng h li chng hiu ti sao
li lm nh vy . By gi th khc , cc thy thuc chng nhng hiu k cng cc cng vic ca
mnh ang lm m cn d kin c nhng kh nng m h s lm c trong tng lai .
5.2.thay th cc protein ca ngi .
Mt t bo ca ngi c khong mt trm ngn gen , phn ln l m cho cc protein cn
thit ca c th . iu thng xy ra l mt gen b khim khuyt lm cho protein c tng hp
ra khng ng hoc hon ton khng c tng hp .Thiu mt protein c th khng ng lu
lm , nhng i khi s thiu ht ny li gy nn bnh . S thiu ht ny thng l di truyn v
th ni chung bnh nhng bnh ny lin quan n di truyn .
Hin nay c th iu tr c cc bnh c lin quan n di truyn nu xc nh c protein b
thiu ht .Trong phn ny chng ta s bn lun v mt s trng hp trong cc protein thiu
ht c pht hin v cng ngh DNA tng hp v hon tr cc protein b mt nh th
no .
5.2.1. INSULIN .
Mt v d v bnh c lin quan n di truyn l bnh tiu ng . Dng ph bin nht ca bnh
ny l do cc t bo Beta ca tu khng sn xut lng hormon insulin (Hnh 5.2)

Hnh 5.2.Phn t Insulin . Insulin l mt protein gm 2 chui polypeptid A v B .Chui A c 21

amino axit . Chui B c 31 amino axit .Cc chui ni vi nhau nhng im c bit . Cc
lin kt c hnh thnh gia cc chui polypeptid l cc lin kt Disunfit (-S-S-) .
Insulin c Frederick Banting v Howard Best xc nh trong mt dy cc th nghim
kinh in thc hin vo nm 1921 . Hormon ny c phn phi bi mch mu i ti tt c
mi t bo ca c th .
Insulin c tm quan trng ng k i vi c th bi v n to iu kin thun li cho s hp
th glucoza ca t bo s dng trong trao i nng lng .Thiu insulin th glucoza vn nm
li trong mch mu v sn phm ATP y nng lng b gim xung nghim trng .
Cc bnh nhn tiu ng rt yu v rt mt mi bi v t bo khng ly c nng lng t
glucoza . Thn s loi glucoza d tha ra khi mch mu v thi vo trong nc tiu
.(mellitusc ngha l ngt , tc l glucoza c trong nc tiu ). ho long glucoza th thn
cng phi thi ra mt lng ln nc mt cch bt thng . (Thut ng diabetes i t ch
siphon tc l mt th tch ln nc b dn ra khi c th).
Tiu ng s dn ti s h hi ca mt v m cng nh cc bnh v thn , thn kinh b hu
hoi v cc bnh thuc h tun hon trong c c hoi t v t qu . Mt nguy hi na ca
tiu ng l hn m tiu ng , s mt thc do mt cn bng pH do tng phn gii cht bo
ca c th .
Khong 60 triu ngi trn ton Th gii b bnh tiu ng . H phi thng xuyn tim
insulin trnh nhng hu qu nghim trng ca bnh .
Insulin l mt protein gm 51 amino axit , gm 2 chui ni vi nhau . Chui A c 31 amino
axit , cn chui B c 20 amino axit . Nh cc th nghim ca Banting v Best nn insulin c
th c thu nhn t tu ca ln v b t cc l st sinh .
Insulin ng vt cng tng t nh insulin ca ngi , n ch khc 1 hoc 3 amino axit (nh
insulin ca ln v tru b). S khc bit ny lm cho mt s ngi b tiu ng d ng vi cc
insulin ng vt . Mt vn khc l tnh d tip cn ch cn 7 n 10 pound m tu ln l
lng insulin iu tr cho mt bnh nhn trong mt nm .
Cui nhng nm 1970 , cc nh Ho sinh thy c dp may ng dng nguyn l ca cng
ngh DNA sn xut insulin. H ly mt on DNA t th vin gen ri gn gen cu trc ca
insulin ny vo plasmid ca vi khun E.Coli v ci v tr promoter cnh gen cu trc (Hnh 5.3)

68

Hnh 5.3. Sn xut Insulin E.Coli . (a) nh hin vi qut ca cc t bo E.Coli thu nhn c
khi chng ang sn xut Insulin . Nhng ch phnh trn r nt c th thy thnh t bo , c
th l hnh nh gii phng sn phm ca gen .
Hnh b sung ch cc t bo E.Coli bnh thng khng c cc ch phnh . (b)nh qua hin vi
in t vi lt ct siu mng ca cc t bo E. Coli khi ang sn xut Insulin . Nhng th ni
ln r rt (mi tn) c l lin quan ti s sn xut mnh m protein ang tip tc trong cc t
bo ny .
V tr promoter c s dng ly t Lac operon . Thay v tng hp enzym promoting tiu
ho lactoza th v tr promoter la khi u cho vic sn xut insulin ca ngi . V gen insulin
l gen ca ngi nn insulin c tng hp chc chn l insulin ngi .
Thng 7-1980 c 17 ngi tnh nguyn chp nhn tim insulin cng ngh gen bnh vin Guy
London .C l h l nhng ngi u tin nhn c cc ch phm dc sn xut theo cng
ngh DNA . Insulin ny hot ng ging nh cc insulin i t ng vt theo cch truyn thng
v nhng th nghim ny xc minh li tuyn b ca cc nh Ho sinh rng cng ngh DNA
c th c dng to ra cc cht c gi tr trong y hc cng nh thng mi .
Ngay t nhng nm sau , vic sn xut tng nhanh . Cc nh nghin cu c s sa i
plasmid vi khun bng cch thm mt on ngn DNA vo gia v tr promoter v gen cu trc
. on mi ny hng dn t bo vi khun gn vo mt peptid tn hiu gm 24 amino axit
mt u ca phn t insulin .Peptid tn hiu s kch thch vi khun tit ra insulin vo mi trng
xung quanh khi n c sn sinh , v vy gii quyt c mt tr ngi l phi thu lm insulin
t bn trong t bo . Peptid ny s c tch khi phn t insulin khi n i qua mng t bo .
Nh th ngha l vi khun li tip tc tng hp insulin mt cch cng nghip : Tc l a thc
phm vo vo mt u ca thng ln men v insulin c rt ra mt u khc .
Vic sn xut insulin cng tng nng xut bi v qu trnh to ra 2 chui ca insulin l tch bit
nhau . Trong c th ngi mt phn t ln l proinsulin c tng hp u tin .Phn t ny
gm c mt trnh t tn hiu v 3 khu amino axit . Trong tng hp insulin t nhin th trc
tin trnh t tn hiu s c loi b v to nn proinsulin. Sau mt on gm 33 amino axit

69

c ct b bi b my Golgi ca t bo v gi li 2 chui (A v B) ni vi nhau hnh thnh

nn phn t insulin .Vic tng hp proinsulin trn vi khun rt kh khn v cc nh cng ngh
DNA thy rng h c th tng bc chng trnh ho cho vi khun tng chui tch bit (A
v B) (Hnh 5.4)

Hnh 5.4. C ch tng hp Insulin . Gen chui A ca Insulin c ci vo plasmid ca mt t

bo E.Coli c nui cy cng vi mt gen cm ng (i) , mt v tr promoter (P) v mt gen
operator(O). Mt t bo nui cy khc c thit k vi gen chui B ca insulin .Operon c
s dng l Lac operon , v th lactoza c dng trong mi trng nui cy nh l mt cht
cm ng . Cc t bo c nui cy s tng hp cc chui A v B mt cch bit lp , sau
chng c phn lp ra . Khi ni chui A vi B s c mt phn t Insulin ca ngi .
Khi cc chui c tng hp th ngi ta chit xt chng t mi trng nui cy ri lm
tinh khit v ni bng phng php ho hc vi cc lin kt disunfit . Kt qu l phn t insulin
c tng hp .
Nm 1986, cng ty Eli Lilly c ph chun nhn hiu Humulin , l insulin cng
ngh DNA u tin c php s dng rng ri .
thi im , vic s dng thnh cng cng ngh DNA chng minh rng khng cn lo
lng vi vic vi sinh vt c th sn ra cc protein l dng cho ngi na .
Tuy nhin nhng nm tip theo cng c nhng kin tranh ci bi v cc nh khoa hc cng
pht hin ra phng php lm thay i cu trc ho hc ca insulin ln lm cho n ging
nh insulin ca ngi . V th hin nay insulin ngi c th c sn xut theo 2 cch : Cng
ngh DNA v bng cch bin i ho hc . Vy insulin ca ln c bin i hay insulin i t
plasmid s l insulin c la chn , iu vn cn cha r .

5.2.2. Hormon sinh trng ca ngi .

Ln (Dwarfism) l mt bnh ri lon chuyn ho khc c th iu tr bng cch hon tr mt
protein m n c to ra mc thp trong c th . Bnh ln xy ra khi tuyn Yn , mt tuyn
hnh qu u y no khng tit lng hormon sinh trng (Human growth hormone
HGH). Hormon ny l mt protein kch thch s sinh trng ca ton b c th bng cch lm

70

tng s tip nhn amino axit ca t bo v kch thch tng hp protein . HGH cng thc y
vic s dng cc cht bo lm nhin liu cho c th .
Khi tuyn Yn sn xut khng HGH ngay t thu nh th c th pht trin chm , khun
mt mm mm , gi l tr d mbng v cao khong 4 feet . Mc d tr no bnh thng
nhng nhng ngi b bnh ny c thn hnh khng cn xng v rt ln .
Nu hin tng ln c chn on sm khi xng cn ang pht trin th c th tim c
HGH. Vic iu tr ny rt tn km . Tuy nhin nu c t 8 xc cht th s HGH c th
dng iu tr trong mt nm cho mt ngi (bnh ny phi iu tr t 8 n 10 nm ) . iu
nguy him l no ca nhng xc cht c th b nhim bnh . V th nm 1985 vic s dng cc
m ca xc cht b hn ch Hoa k v Anh bi v c kh nng ly truyn hi chng
Creutzfeldt Jacob (CJ) . Bnh no ny gy nn bi mt virut hoc mt tc nhn tng t virut
. Bnh nhn b run , chn ng , sa st tr tu v teo c . Tht vy nm 1993 ta Php c 24
ca hi chng CJ c xc nh nhng ngi c tim HGH t cc m ca cc xc cht .
Cc nh cng ngh DNA ngy nay c th sn xut c hormon sinh trng qua c ch ca
cng ngh di truyn . Qu trnh ny cng tng t nh vi Insulin , tc l : Gen HGH c gn
vo plasmid vi khun sau a vo vi khun . Cc sn phm gen ca HGH trong trng hp
ny l mi trng ngoi t bo .
to c mt lng thch hp HGH , i hi cc nh cng ngh DNA phi thay i k thut
c bn i vi mt s vn Ho sinh .
Phn t HGH t nhin bao gm 191 amino axit . Khi tng hp HGH t nhin , mt phn t
trung gian c thm 26 amino axit c m bi mt peptid tn hiu . Chui amino axit ny dn
dn s c ct i khi chng c tit ra . tng hp gen HGH , phn t mRNA m cho
HGH c s dng nh mt m hnh tng hp phn t DNA b cu (cDNA). Vn ny
xy ra khi cDNA c ci vo E.Coli .Vi khun ny p ng mt cch tht thng bi v n
khng hiu ngha ca peptid tn hiu ny v n khng loi c peptid tn hiu khi phn
t HGH trung gian .Cc nh cng ngh DNA cng kh c kh nng loi i mt cch d dng
peptid tn hiu ny trong vi khun .
gii quyt vn ny cc nh cng ngh DNA lm vic vi phn t cDNA ct i
peptid tn hiu trn HGH . Nhng ng tic l khng c enzym gii hn bit no c th thc
hin c vic . Vn ny c gii quyt bng vic dng enzym gii hn EcolRI
loi i trnh t baz ca peptid tn hiu 26 amino axit) khi phn t cDNA v c trnh t ca
24 amino axit (tng cng l 50 amino axit). Sau cc trnh t ca 24 amino axit c tng
hp v thay th trn phn t cDNA lp cng thc ca gen HGH y (Hnh 5.5) . Khi
thiu trnh t peptid tn hiu ny th c th ci gen ny vo cc t bo E.Coli sn xut HGH .

71

Hnh 5.5. Gen sn xut HGH . Phn t cDNA c s dng nh gen hormon sinh trng ca
ngi (HGH) gm c mt trnh t tn hiu m n khng c phin trong vi khun cng nh
khng c loi i mt cch thch ng bng bin php ho hc . (a) gii quyt vn ny
ngi ta s dng enzym gii hn EcolRI loi i gen trnh t tn hiu cng vi gen cho 24
amino axit trong phn t HGH chn .(b) Sau gen ca trnh t 24 amino axit c tng hp
mt cch tch bit v gn vo phn t cDNA cn li bi Ligaza. (c) Do cu trc nh vy nn
vec t biu hin (Plasmid) khng c trnh t tn hiu lm gin on qu trnh tng hp
protein v . (d) Vec t c th c ci vo mt vi khun nh E.Coli chng hn .
Cc th nghim v hormon sinh trng ca ngi c thc hin t 1985. Nhn hiu
Protropin ca cng ty Genentech l mt hormon tng hp lm tng 3 ln tc tng
trng ca nhng tr em thiu ht hormon nm th nht v tng 2 ln nm th 2 v th 3
trong liu trnh tr liu . u tin , hormon ny ch c dng cho nhng tr em t chiu cao
rt thp ch vo klhong 3 % theo gii tnh v la tui .Ngy nay ngi ta m t l nhng
a tr rt thp. t c chiu cao bnh thing phi tim thuc hng ngy trong nhiu
nm , vi gi khong 20.000 USD mi nm . V iu khng vui cho cc nhng a tr c thn
hnh cc k thp na l vin cnh thm c vi inches vi gi tin phi tr nh vy th qu l
qu t .

72

Cng d hiu l HGH cng c tc dng dng tnh v cc tr em c mc hormon bnh thng
nhng nhng li b ri xung nc cht ca ng cong chiu cao chun . Nm 1990, NIH
u cho mt cng trnh nghin cu ko di 10 nm vi 80 tr em lnla tui ln 10 . Mt na
trong s c tim hormon cn mt na th dng thuc Placebo . Tuy nhin, lut s dng
thuc nu r nhng nghin cu v tm vc thp b c th l mt bt li x hi . Tuy nhin
vn c nhng vn tri ngc cho rng nhng ngi c tm vc thp cng c th c iu tr
nh mt bnh trong y hc thng thng . Nhng vn ny vn cn ang tip tc nghin cu .
5.2.3.Yu t VIII.
Hoa k , bnh di truyn c lin quan ti s ng mu ph bin nht l bnh a chy mu
hemophilia A . Bnh ny tc ng n khong 10.000 nam gii Hoa k v do nguyn nhn
khim khuyt di truyn i vi qu trnh ng mu . S khim khuyt ny lm cho c th
khng c kh nng sn xut mt yu t quan trng trong qu trnh ng mu , l yu t
VIII.
Ri cng ngh DNA bt u c ng dng khi m yu t VIII c thu thp t cc n v
mu ngi ton phn (Cn khong 800 pint (mi pint 0,5 lit) l c yu t ng mu dng
cho mt bnh nhn trong mt nm ) . Nhng ng tic l , trong cc mu mu c th dnh c
cc tc nhn gy bnh nht l t nhng nm 1980 khi c thm ho HIV . Trong nhng nm
c hng ngn ngi bnh a chy mu khi c truyn thuc (yu t ng mu) b nhim
HIV v pht trin thnh AIDS .
Sau nhng nm 1980 , cc nh cng ngh DNA nghin cu tng hp c yu t VIII bng
cng ngh di truyn . H bit rng gen ca yu t VIII trn ngi nm nhim sc th X ,
c khong 186.000 cp baz trong 26 exon xen vi nhiu intron (Hnh 5.6)

Hnh 5.6. Phc hp gen ca yu t VIII . Gen ny gm 186 cp baz (186 kb) t chc trong 26
exon . Trong hnh thy cc exon rt di . Xen ln cc exon l intron (t th 200 m nhiu th
ti 32.000 cp baz ) . Protein Yu t VIII cui cng c m bi gen ln ti 2323 amino axit
(Insulin ch c 51 amino axit).
Sau h phn lp ton b mRNA (c m bi DNA ) v tch ring mRNA chn (ch cha
exon khng c intron). mRNA chn ny c 9000 baz v m cho yu t VIII-mt protein c
2332 amino axit . Mt phn t b cu (cDNA) s c tng hp t mRNA ny , sau c
ci vo cc t bo thn ca chut bch, n m cho protein yu t VIII. Vic s dng cc t
bo ng vt c v l rt cn thit bi v yu t VIII rt ln v phc tp , n c t nht 25 v tr
c gn cc phn t carbohydrat .
Nm 1992, hai phin bn ca yu t VIII c sn xut t 2 cng ty dc phm v c 2 u
c cp giy php Recombinate mt sn phm ca vin di truyn Cambridge,
Massachusette v Kogenate -c sn xut ti phng th nghim Miles.
5.3.TR liu trn ngi
Ha hn ca cng ngh sinh hc cn c m rng sang lnh vc iu tr bnh . Cc nh
nghin cu v cc thy thuc ang pht trin nhng thuc mi s dng cng ngh DNA
cha bnh . Hai v d v cc thuc ang si ng l yu t plasminogen ca m (tisue
plasminogen factor TPA) v Interferon . C hai thuc ny u c cung cp rt t . Ngy
nay n c sn xut vi s lng ln dng trong iu tr . Hn na , mt lp hp cht
hon ton mi gi l cc phn t Antisene cng c pht trin rt mnh . Trong phn ny
chng ta s tm hiu v qu trnh sn xut cng nh cch s dng nhng loi thuc ny .
5.3.1.Cht hot ho plasminogen ca m (tisue Plasminogen Activator
TPA).
Trong nhiu nm qua , bnh nghn mch vnh c iu tr bng Streptokinaza v cc
enzym khc ph hu cc mu ng trong mch mu . Mt loi thuc mi c thm vo
trong danh mc thuc iu tr l mt protein c tn l cht hot ho Plasminogen ca m
(TPA) . TPA l mt proteaza c trong cc t bo ng vt c v . N t gn vo cc cc mu
ng v kch thch cc thnh phn khc ca mu phn gii cc mu ng m khng lm
gim kh nng ng mu nhng ni khc trong c th . Mt enzym khc cng lin quan ti
s ng mu l Urokinaza , enzym ny lm gim kh nng ng mu khp c th nn gy h
qu l gy cha mu trong . TPA tc ng trong mch mu , trong khi Urokinaza v

73

Strreptokinaza li tc ng trc tip vo cc tc nghn ; TPA tc ng nhanh hn urokinaza

v strreptokinaza .
TPA l thuc u tin c sn xut bng cc t bo ng vt nui cy . Trong nhng nm
1980 , cc nh cng ngh DNA tng hp c phn t DNA b cu cho TPA v gn n
bng phng php Ho sinh vo mt plasmid tng hp . tng hp TPA , ngi ta s dng
mt Plasmid c cha cc trnh t gen ca TPA (phn t cDNA) , mt trnh t tn hiu v cc v
tr promoter v kt thc . Sau a plasmid ny vo cc t bo ng vt , cc t bo ny s
tit ra mt lng ln TPA (Hnh 5.7) .

Hnh 5.7.Sn xut cht hot ho plasminogen ca m (TPA) trong cc t bo ng vt . (a)

Plasmid tng hp c a vo cc t bo ng vt . (b) Thm Methotrexate chn ra nhng
t bo c mang plasmid v c kh nng sn xut TPA cao . (c) Cc t bo c chuyn vo
bnh ln men cng nghip sn xut TPA cc t bo s tit TPA vo mi trng xung
quanh .
Khi nui cy trong mt bnh ln men ln , cc t bo ny s cung cp TPA cho nhu cu dc
phm .
Nm 1987 , cng ty cng ngh sinh hc Genentech nhn c giy php ca FDA cho nhn
hiu ca cht hot ho plasminogen ca m l Activase . Tc ng vi t cch l tc nhn lm
tan khi huyt , TPA bc l hot tnh ca mt proteaza do n phn gii mt enzym tin thn l
plasminogen chuyn thnh plasmin . Bn thn plasmin cng l mt proteaza , n phn gii
fibrin mt protein hnh thnh trong cc mu ng . Nhng bnh nhn c nhng du hiu
sm ca bnh tim v v t qu s c iu tr vi TPA . Nhiu thy thuc khuyn khch dng
ch phm ny . Nhng cn tn ti nhng vn khc l n vn khng vt tri hn hn
streptokinaza v urokinaza . Tuy nhin TPA lm gim cc tc dng ph so vi cc thuc
chng ng khc , l iu ng c trn trng .
Nm 1992, Genentech thc s thu li t cc sn phm TPA . Nm cng ty ny thu c
trn 230 t USD khi bn sn phm ny cho ton Th gii . Tht vy , nhiu cng ty thu c
nhng thnh qu trong cng ngh DNA (bng 5.1)
Bng 5.1 Cc sn phm bn ca Dc phm c sn xut theo cng ngh di
truyn nm 1992 .
sn phm

cng ty sn xut

74

Bn M

Bn trn Th gi

Erythropoietin
Vac xin vim gan B

Amgen ;vin di truyn

Biogen

600
260

(T USD)
1125
724

Insulin ngi

Genentech

245

625

Hormon sinh trng

ca ngi

Genentech, Biotechnology
General

270

575

Interferon Alpha

Genentech, Biogen
Wellcome .

135

565

Yu t kch thch
Amgen
khun lc bch cu ht.

295

544

Yu t hot ho
plasminogen m .

Genentech

180

230

Yu t kch thch
khun lc i thc
bo bch cu ht .

Immunex, genetic
institute .

50

70

Interferon Gamma

Genentech, Biogen

15

25

Interleukin -2

Immunex, Chiron

20

2055

4503

Tng cng

Nm c 27 sn phm sinh hc ca cng ngh DNA c bn Hoa k v 270 thuc iu tr

khc cng ang c th nghim trn lm sng . Erythropoeitin mt hormon kch thch
sn sinh hng cu thu c 1,1 t USD khi bn sn phm ny cho ton Th gii v dn u
v cc li nhun t sinh hc . Mt tc nhn tr liu khc l Interferon cng ng v tr hng
u trong cc sn phm c bn trn th Trng . Chng ta s bn lun v hot tnh v cch s
dng ca n sau y .
5.3.2.interferon .
Interferon l mt cht khng virut ln u tin c Alick Isaacs v Jean Lindenmann
xc nh nm 1957 . Interferon khng phi l mt n cht m l mt nhm gm trn 20 hp
cht c cu trc v chc nng tch bit c thit k vi 3 di nhm l Alpha, Beta v Gamma
Interferon . Cc interferon u l protein , nhiu Interferon l Glycoprotein , tc l chng c cc
phn t Carbohydrat gn vo cc amino axit nhng im khc nhau dc theo chui .
Interferon c to ra bi cc t bo khc nhau ca c th sau khi chng c kch thch bi
Virut . Interferon phn ng khng c hiu vi cc virut k c virut kch thch to ra
interferon bo v t bo . Hn na , nhiu interferon cn tiu dit c cc t bo ung th .
iu ng tic l interferon ca ngi th ch c to ra bi cc t bo ca ngi th mi c
tc dng cho ngi , cn cc interferon ca ch hay ca cc ng vt khc th khng c tc
dng .
Interferon c t bo to ra sau khi virut gii phng axit nucleic ca n vo trong tng bo
ca t bo .Interferon c tit ra t t bo v chng gn vo cc v tr receptor trn cc t bo
nm lin k .Khi i vo c t bo chng s sn xut ra cc protein bo v (h6.8) lm c
ch s sao chp ca virut bng nhng cch m cc nh virut cng cha hiu c y (mc
du c nhng bng chng l c t nht mt protein bo v trnh cho s xm nhp ca virut v
mt protein khc gn vo mRNA m bi virut ).

75

(T US

Interferon c chiu di lch s c kim chng . Cc nh Ho sinh rt quan tm ti s hot

ng ca n ngay t nhng nm 1960 , nhng h li khng thu thp lng intrferon
nghin cu v th vic nghin cu tr nn cc k kh khn (Cn trn 90.000 pint mu (45.000
lit) mi thu c 1 gam Interferon) . Tuy nhin , mt t ph ln xy ra nm 1980 khi cc nh
cng ngh sinh hc Thu s v Nht bn gii c m di truyn ca interferon alpha v h
ni mt on DNA thch hp vo trong cc plasmid ca E.Coli.
Cc interferon sn xut t vi khun lm gim bt triu chng cho nhng ngi mc cc bnh
gan , lm hn ch s ly lan ca cc Herpes Zoster (Bnh Zona) v lm nh li mt s khi u .

Hnh 5.8. C ch tc ng ca Interferon . Khi mt virut xm nhp t bo ch th axit nucleic

ca n s kch thch DNA ca t bo sn xut ra Interferon . Sau interferon i ra khi t
bo vo mt t bo lin k . y n kch thch DNA ca t bo m cho mt dy cc protein
khng virut . Nhng protein ny xut hin ngn nga s sao chp v th m to ra hiu ng
bo v trn t bo .
Nm 1984, hng cng ngh sinh hc Thu s bt u tip th Interferon . Nm 1986 , FDA
ph chun cho php s dng interferon alpha iu tr mt th bnh bch cu (Leukemia) ,
nm 1988 FDA ph chun dng cha cc mn ng sinh dc (genital Warts) . Nm 1996
interferon c ph chun cha cc sac cm Kaposi (mt dng ung th mu v da cc
bnh nhn AIDS) cng nh bnh u hc sc t , a u tu v mt s ung th thn .
Mt Inerferon khc l interferon beta -1b cng c cp php nm 1993 dng cha
bnh a s cng .
Nhn nhn Interferon l s rc sng hay l ti tm iu ph thuc vo vin cnh ca n
.Thm na l interferon c th s dng trong lm sng cha nhiu bnh do virut v cc bnh
ung th . Cc nh khoa hc ch r rng Interferon c th c s dng nh cc loi thuc xt
ngn chn cc bnh cm cm thng thng . V nhng bt li th interferon cn kh t
v vi khun khng th tng hp c mt glycoprotein ln nh interferon . khc phc tnh
trng ny cc nh khoa hc cm ng vi khun tng hp ra mt interferon lai (hybrid
interferon) , iu c ngha l interferon c tng hp theo m di truyn ca cc interferon
khc nhau c ni li vi nhau .
5.3.3. Cc phn t antisense.
u th k 20 , mt nh sinh ho c gii thng Nobel ng c ch l Paul Ehrlich , ng
a vo y hc mt khi nim vin n ma thut (magic bullets). Ehrlich mng tng
ra mt th thuc c kh nng tn cng cc vi sinh vt m khng gy ra mt tc dng ph no
trong c th . Dn dn , Ehrlich nh v c mt tc nhn ho tr liu i vi bnh giang
mai , v ng t mt vin tng cho ho tr liu hin i .
Cc nh nghin cu DNA ngy nay ang tm kim cc dng khc ca vin n ma lc ny -
l mt phn t nh hng vo mt ch trong t bo ch khng phi l vo ton b t bo . D
nhin ci ch khng c cc t bo kho mnh v n cng khng cn thit cho cc chc
76

nng ca t bo bnh thng . Hn na , n l mt phn t ho hc ch c sn xut ra khi t

bo b xm nhim . C th l vin n ma lc loi thi phn t ch v hon tr s kho
mnh cho t bo .
Chng hn nh chng ta hy xem xt ci g s xy ra khi mt ngi b nhim HIV hay AIDS ?
Trong trng hp ny th HIV nm trong nhn ca t bo b nhim nh mt phn t DNA .
y HIV m cho vic sn sinh cc virut mi . N c s dng nh mt ci khun tng hp
phn t mRNA .Ting lng trong Ho sinh tc l phn t mRNA ny mang thng tin c
ngha , tc l thng tin ny c th c dng tng hp mt enzym cho vic sn xut cc
virut AIDS mi .
lm ngng vic sn xut cc ht HIV mi cc nh Ho sinh thit k mt phn t mRNA
tng hp Phn t mRNA tng hp ny c mt trnh t cc baz ni t b cu vi cc phn t cn
pht hin (sense) .Phn t mRNA tng hp ny c gi l phn t antisense. Khi dng trong
tr liu cc antisense i vo cc t bo b xm nhim v gn c hiu vi phn t mRNA b cu
ca n , ging nh ly tay tri nm ly tay phi . Khi phn t mRNA bt hot th s tng hp
ca virut c kt thc v s xm nhim gim i .
Nm 1992 , ln u tin phn t Antisense c dng khng li HIV , bi v cc test vi cc
t bo b xm nhim trong phng th nghim cho kt qu lm gim mt cch ng k virut
trong t bo ( Hnh 5.9).

Hnh 5.9. Phn t Antisense dng lm gin on s sao chp ca virut trong cc t bo
nhim HIV .(a) Trong mt t bo b xm nhim th HIV nm trong t bo dng mt phn t
DNA gi l provirut . Provirut s dng mRNA m cho protein tng hp virut mi v s xm
nhim tip tc .(b) Phn t Antisense c mt trnh t baz b cu vi mRNA . Khi n vo
trong t bo th phn t Antisense hp nht vi mRNA (phn t cn pht hin) v lm bt hot
n . V khng c mRNA nn khng tng hp c protein v s khng tng hp c cc ht
virut mi . V l thuyt th s xm nhim virut s kt thc .
Mt ng dng thc tin khc ca antisense RNA cng c cc nh nghin cu Anh thng bo
nm 1988. Khi nghin cu to ra cc ging c chua tt hn cc nh nghin cu thnh cng
trong vic s dng antisense RNA kho s hot ng ca cc gen lm nu v lm thi c
chua chn ; c ch c vic lm nu th c chua v nho s c vn chuyn bng tu bin i
bn cc nc xa xi .
Cc phn t antisense cng c s dng lm nh ch s hot ng ca cc tc nhn gy
cm ng ung th Oncogenes . Chng hn nh nm 1989 , cc nh nghin cu Trng i
hc George town s dng thnh cng antisense RNA kho s biu hin ca mt
oncogene l raf . Gen ny c cc t bo ung th thanh qun c nui cy trong phng th
nghim . DNA ca antisense RNA c ci vo cc t bo ny sao cho cc t bo c th t sn

77

xut ra phnt antisense . Cc nh ung th hc ang hy vng c th dng s hot ng ca gen

cm ng bnh bch cu trong cc t bo tu xng bng cch ci DNA m cho antisense .
Trng hp tt nht l c th a cc t bo c x l vo tu xng ca bnh nhn tr
li cc chc nng bnh thng ca t bo . iu ny cng rt thch hp cho cc bnh nhn ung
th phi .
Trong nghin cu antisense , cc nh nghin cu ch trng vo mt thc t l ch c mt
chui DNA m cho mRNA cn chui kia th vn nm yn .Chui nm yn ny l phn t
antisense t nhin (natural antisense molecule) . Chnh v th cc nh khoa hc bt u vi mt
bn sao DNA chui kp ca mt gen gc , to nn mt dng c vai tr lm o ngc v hot
ho c chui nm yn . Nh vy chui antisense c chuyn thnh mt chui c ngha .
H hon tt vic chuyn i ngc ny bng cch ni v tr promoter vo cnh trnh t m
h mun t bo biu hin v bng cch chuyn cc ca b xng deoxyriboza-photphat .Chui
ny ang c theo hng ngc li by gi c promoter nn li c theo hng thun chiu .
Khi th chui hot tnh s l antisense v tr nn yn lng .Phn t c m bi chui DNA
hot ho mi l antisense RNA .
Ngi u tin ci DNA m cho antisense RNA l Harold Weinstraub trung tm ung
th Fred Hutchinson , Seatle . Gia nhng nm 1980 , Weinstraub nghin cu s hot ng
ca thymidin kinaza , mt enzym c s dng trong tng hp DNA . Nhm ca Weinstraub
ci c mt on DNA vo trong cc t bo ang tng hp DNA nhc nh cc t bo
sn xut antisense khng li mRNA m cho Thymidin kinaza. Khi antisense RNA lm tc
nghn mRNA bnh thng th s tng hp thymidin kinaza b dng li v s tng hp DNA
cng b dng li mt cch bt ng .
Weinsraub l mt trong nhng ngi u tin chng minh rng cc t bo ny c th cng hin
mt chng trnh Ho sinh cho vin n ma thut .
Cc nh Ho sinh cng c th tng hp c nhng on nh antisense DNA ng nht tm
hiu trnh t ca DNA . Nhng on ngn ny c mt chui n DNA tng hp , chng khng
i vo nhn v tc ng nh l mt DNA bnh thng .Tuy nhin , chng li i vo t bo cht ,
chng cng to c antisense RNA , tc l chng nhn dng c v gn vo cc trnh
t ch trn cc phn t DNA .
Nhng on DNA nh ny l cc oligonucleotit hay n gin gi l oligo . Cc oligo c
khong 15 cp baz ngn i c vo cc t bo . y chng gn vi trnh t mRNA
b cu ca chng to thnh phn t lai mRNA:DNA . Sau enzym Ribonucleaza tn cng
v phn hu phn t lai ny .
Cc nh nghin cu s dng oligo khng li cc virut AIDS , herpes Simplex v Influenza
v khng li trypanosoma brucei , protozoa gy bnh ng chu Phi (Hnh 5..10)

Hnh 5.10 Hai nh ca Trypanosoma brucei , mt protozoa gy nn bnh bun ng . Cc phn

t Antisense s dng c mt s kt qu l ph v oc nhng t chc ny trong c th . Hnh
th nht l T.brucei trn knh hin vi quang hc ; hnh th hai trn hin vi in t qut ca
T.brucei . Trong c 2 hnh cc i tng u bao quanh cc t bo hng cu .
H cng thit k oligo i vo tn nhn t bo v chng t p vo nhng phn ch c hiu
trn cc vng xon ca cc xon kp DNA . Khi gn vi DNA th n tr thnh mt oligo cp 3
(triplex) ngn nga kh nng c ch tng gp mRNA ca gen . Bng cch ny gen gy bnh

78

c th c dp tt . Cc th nghim oligo trn bnh nhn bnh bch cu v virut gy khi u

cng ang bt u .
iu lc quan ca phn t antisense l tt c cc mi lin quan c cp trc khi cc
phn t ny c s dng trong thc tin . Tuy nhin cc phn t c sn xut theo kiu ny
th gi thnh kh t v li phi s dng vi liu lng ln th mi c tc dng .
Nhng phn t ny c th i vo trong cc t bo (chng phi ho tan trong lipit ca mng t
bo ) v phi ho tan trong t bo cht (phi ho tan trong nc ) v phi khng li c s
hot ng ca cc enzym ca t bo v phi c kh nng gn vo cc phn t mRNA ch ca
chng .
Ngoi nhng mi lin quan ny , cn rt nhiu vn khc na : Chng hn nh phn t
antisense ch hng vo mRNA ca ngi , vy lm th no th test trn ng vt . Hay
lm sao cc nh nghin cu c th xc nh c rng oligo khng t ho nhp vi DNA bnh
thng ? V liu oligo c tn cng h min dich ca bnh nhn hay khng? V chng c th
c hng dn ti cc ch t bo c hiu ca chng nh th no ? Cn FDA li quan tm
ti iu antisense l mt sinh vt hay l mt th thuc ?
Cc phn t antisense i din cho mt ng dng hp dn ca cng ngh DNA v di truyn
phn t (mc d n l di truyn c in) . t nht l c hng chc cng ty cng ngh sinh
hc hin nay ang nghin cu v cc ng dng ca chng v cc nh cng ngh sinh hc ra
mt tp ch chuyn ngnh c tn l Antisense Rearch and development .
chnh xc ca cc phn t antisense lm chng ta hy vng rng chng khng gy ra cc tc
dng ph v antisense l mt tm gng khch l tr tng tng ca cc nh nh thc hnh
v nhiu vn qu gi trong y hc v trong cng ngh .
5.4.Vac xin
Truyn thng k la rng nhng thy thuc Trung hoa bit chng u ngay t u th k XI .
H co vt thng ca nhng ngi b u ma ri thi th bt vo mi ca nhng ngi
kho mnh . Kt qu l nhng ngi ny t b u ma khi dch bng pht .
Mt phng php khc cng c hiu qu nhng khoa hc hn l tim chng . Phng php ny
c pht trin t nhng nm 1700 bi mt thy thuc ngi Anh tn l Jenner. Jenner lu
thy rng nhng ngi lm trang tri v nhng ngi vt sa , tc l gn nhng con b th b
mt bnh u ma th nh gi l u b , hay l bnh u b . H cho rng nu ai t gy
cho mnh bnh u b th h s khng b bnh u ma nng . Jenner theo ui vic tim
chng nhng ngi tnh nguyn vi cc cht liu ly t cc vt thng u b . Sau my
tun ng th nghim tim chng vi cc cht liu ca u ma .
Tht l tuyt vi l nhng ngi c tim chng th khng b u ma na .
Nm 1798 Jenner xut bn mt cun sch nh lch s ni chi tit nhng li ch ca vic tim
chng u b cho ngi . Nhng thy thuc c ting xc nhn nhng khuyn co ca Jenner
v ch trong vi nm phng php ca Jenner c ng dng trn khp Th gii . Ch ring
Anh c trn 10.000 ngi c tim chng u nhng nm 1800. Chng bao lu dch u
ma lng xung . Tht vy , bnh u ma khng cn tm thy bt c u trn Th gii t
thng 10 nm 1977.
Cch y hn 100 nm cc nh khoa hc hiu c c s min dch ca bnh u ma :
l cc virut u b kch thch h min dch ca c th to ra cc phn t protein gi l khng
th . Cc khng th lm trung ho virut u b cng nh cc virut u ma nguy him cht
ngi .
Cc vac xin hin i cng hot ng theo kiu y . Chng hun luyn cho h min dch kh
nng nhn dng cc t chc ngoi lai v p ng bng khng th v cc bin php bo v khc
gi an ton cho c th .Cc vac xin hin i c tinh ch tt hn cc vac xin ca Jenner .
Chng bao gm 3 dng vt liu : Vi khun cht hoc bt hot ; hoc l cc on hay cc phn
t i t mt vi khun .Trong ba loi vac xin th loi th 3 l an ton nht bi v n khng
cha vi khun hoc virut c th gy c bnh cho c th . Nhng phn t vi khun hoc cc
on virut c s dng lm vac xin u l cc mnh di n v (subunit) . Nhng di n v
ny tc ng nh cc khng nguyn , tc l chng kch thch h min dch c p ng c hiu
. Mc du cc khng nguyn ch l cc phn t ho hc , nhng chng vn c th gi c mt
p ng khng th mnh chng li nhng t chc sng lt vo trong c th .
Bnh vim phi do vi khun l mt bnh c khng li bng cc vac xin di n v
(subunit) . Mt vac xin c bo ch t cc polysacarit v ca streptococcus pneumoniae ( mt
nguyn nhn chnh gy vim phi) c cp php nm 1983 .
iu ng tic l khng phi tt c cc vc xin di n v u thng li nh nhau . Chng hn
nh trong nhng nm u v gia 1980 , khi vc xin vim gan B oc s dng l loi vc xin
c sn xut t cc mnh virut thu nhn t mu ngi , m nh vy th c kh nng l trong
mu s c HIV v nhng ngi c tim vac xin ny s b nhim HIV . Loi vac xin ny ngy
nay khng cn dng na , thay vo l mt loi vac xin c sn xut bng cng ngh DNA .

79

Loi vac xin ny ang c s dng mt cch rng ri v chc chn n s cn c s dng
trong tng lai .
5.4.1.Vac xin vim gan B
Vac xin vim gan B th h mi c sn xut bng cng ngh DNA . Thnh phn chnh ca vac
xin ny l protein b mt ca virut vim gan B l HBsAg (khng nguyn b mt vim gan
B) . Protein ny l mt mnh virut thu nhn trc t mu ngi v c s dng lm vac xin .
Cc nh cng ngh DNA xc nh c gen ca HBsAg v dng cc t bo nm men to
ra cc bn sao ca khng nguyn dng trong vac xin di n v .Nm men c s dng
sn xut ra vac xin l Sacharomyces cereviciae mt loi nm men v hi dng trong ln men
v lm bnh m .
to cc di n v , gen HBsAg c ci vo mt vec t biu hin (plasmid) t mt t bo
nm men , cc nh Sinh ho s dng v tr promoter t gen ca nm men m cho enzym
alcohol dehydrogenaza; trn vec t plasmid cn c mt trnh t kt thc t gen nm men v
mt nguyn mu bn sao ca vi khun . Thm vo , vec t ny cn c c cc du chun
(markers) khng khng sinh v mt gen ch pht trin c khi c mt mt amino axit l xin .
Plasmid c cu trc nh th s c ci vo cc t bo nm men . Cc t bo nm men ti t
hp ny s c chn ra bng cch nui cy trong mi trng khng c l xin . Sau cc t
bo s pht trin dy c trong cc bnh ln men cng nghip . Mt trong s cc protein m
chng sn xut ra c xc nh l HBsAg . Di n v ny hin nay c th c tch chit
ra v s dng lm vac xin .
Nm 1987 , vac xin vim gan B tng hp u tin c php s dng trong cng ng .
Nhn hiu Recombivax v Engerix B l 2 sn phm ca 2 cng ty sn xut vac xin .
l cc vac xin an ton bi n khng cha ton b virut hoc virut c kh hot tnh hoc
lm gim hiu lc. Vac xin ny cng khng c kh nng gy phn ng d ng vi cc thnh
phn ca m sng (bi v n l vac xin ch cha cc virut c nui cy ) v n cng khng
b nhim cc cht bn t cc thnh phn ca nm men hoc ln cc protein khng mong mun .
Ngay t nhng nm u tin s dng , vc xin ny tr thnh mt v kh c chp thun
gii hn s lan trn ca virut vim gan B . N c dng tim chng cho nhng ngi lm
cng vic bo v sc kho nh cc bc s , cc phu thut vin , y t , bc s rng ming , cc k
thut vin trong phng th nghim. N cng c khuyn co cho bt k ai c tip sc vi mu
v cc cht tit ca c th , chng hn nh cc k thut vin lm nhim v cp cu hay nhng
ngi lm cng vic tang l , lnh cu ho , cng an v.v..
Cc bc s nhi khoa cng khuyn nn dng vac xin cho c tr s sinh , hin nay a vo
chng trnh tim chng m rng cho tr s sinh Vit nam .
Nhng vn cn tn ta mt vn l liu c m bo chc chn rng vac xin ny n vi tt
c nhng ngi cn phi gip khng ?
Vac xin ti t hp phi c tim 3 ln trong thi hn 6 thng . Nhng c nhiu ngi khng
c tim liu . Vn a c chua vim gan B vo s dng , cc nh khoa hc cng ty
cng ngh sinh hc Texas thnh cng trong vic ci gen vim gan B vo trong cc t bo ca
cy c chua . Nhng cy c cng ngh ho nh th s sn xut ra cc khng nguyn vim
gan B cng vi cc khng nguyn ca chnh chng . Cc nh cng ngh ca cng ty nhn
thy trc mt ngy no vic tim chng vim gan B s n gin nh khi chng ta n c
chua trong mt ba n tra .
5.4.2.Vac xin AIDS.
Nhng kinh nghim thu c i vi vac xin vim gan B khch l cc nh khoa hc s dng
cng ngh DNA sn xut cc loi vac xin khc . u tin nht trong s l vac xin AIDS .
Mt s vac xin AIDS hin nay ang pht trin hoc ang trong giai on th nghim . Trong
vac xin ny c cc di n v , c bit l cc glycoprotein gn vi HIV .
C 2 glycoprotein l gp 120 v gp 41 v HIV. Trc ht ta thy gp 120 c mt nhng mm
nhn trong lp v . Th nh l gp 41 nm di gp 120 . Khi HIV gn vo t bo lympho trong
c th th phn t gp 120 gn vi v tr receptor . Sau phn t gp 41gn vo mng t bo ch
v gip cho virut i vo t bo cht .
Cc nh sn xut vac xin nhn nhn qu trnh ny nh l mt con ng rng mnh mng trong
vic c ch HIV . H cng xc nh c gen ca phn t gp 120 v gp 41 v ci c
nhng gen ny vo cc t bo E.Coli . Vi khun ny sn xut ra mt lng ln gp 120 v gp
41 s dng nh mt vac xin . Khi tim vo nhng ngi tnh nguyn , cc glycoprotein s
kch thch to khng th gn vo cc glycoprotein tng ng . Kt qu l trung ho cc v tr
gn gp 120 v gp 41 v trnh cho HIV gn vo cc lympho-T ca tc ch (Hnh .5.11)

80

Hnh 5.11. Vac xin khng HIV hot ng nh th no ? (a) Khi HIV gn vi t bo ch ca n
th phn t gp 120 trong lp v ca virut gn vi v tr receptor CD4 trn b mt t bo ch .
(b) Vic gn khi u ny vn khng che khut phn t gp 41 cng trn v virut . Khi gp41
gn vo b mt t bo ch n m ng tin vo cho virut . (c) Mt vac xin khng HIV c th
cha cc phn t gp 120 v gp 41.Phn t gp 120 s kch thch h min dch ca c th to
ra cc khng th khng gp 120, trnh to lin hp gp20-CD4 .(d) Phn t gp 41 cng kch
thch h min dch to ra cc khng th khng gp41. Cc khng th ny trnh cho s lin
hp Virut t bo .Nu phng chng c s lin hp ny th s xm nhim s khng xy ra
na .
Trc khi vac xin AIDS tr thnh hin thc th c v s cc vn thit thc cn phi gii
quyt .Chng hn nh cc phn t gp120 v gp 41 l nhng cht kch thch rt km i vi h
min dch v th cn phi nh v c mt phn t cht mang (carrier) thch hp . Ri cn vn
test , bi v m hnh test trn ng vt ni chung l khng thch hp i vi cc th nghim
vc xin . Hn na HIV vn cn sng trong cc t bo ch di dng mt phn t DNA m cc
khng th th li khng vo c bn trong cc t bo ch . Cn mt vn na l HIV cng
c t bin do n cng lm gim tc dng ca vac xin . Cui cng l s thch thc v liu
lng HIV c th khng c qun l bi nhng ngi tim chng mt cch mo mui vac xin
ny . Tuy nhin, ch ring ti Hoa k hin nay c ti 20 tri ngi cn vac xin AIDS v th
vic pht trin vac xin ny vn l mt u tin hng u . n thi im ang vit cun sch ny
th c nhiu vac xin AIDS ang c th nghim trn ngi v chng ta hy vng chng s
sm c s dng mt cch hiu qu v an ton nh vac xin vim gan B .
5.4.3.Cc loi vac xin khc .
Cng ngh DNA cng c s dng trong nghin cu vac xin Influenza v herpes simplex. Mt
dng vac xin mi ang c nghin cu i vi nhng bnh ny . Vac xin ny bao gm vac xin
u b ( c Jenner s dng) c ni vi b gen (genome) ca mt s virut .Virut u b
l mt vt mang tt bi v chng khng nguy him v cc tnh cht Ho sinh c bit r .
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Hn na, virut ny khng tim tng trong cc t bo ca c th (cng nh cc virut khc nh

herpesvirut) v thao tc tng i d bi v chng c kch thc ln . V virut u b c th
iu tit c cc on DNA ngoi lai trong b gen ca n v khi gii phng vo trong tng
bo t bo ch th b gen biu hin thm c phn n mang theo . Virut ny nui cy c tng
i d dng v lm gim c gi thnh ch to . Hn na n c th c lm ng kh v gi
c nhiu nm , v th cho php vn chuyn i cc ni m khng cn phi gi iu kin lnh
m vn khng b mt hiu lc .
Li th quan trng ca vic s dng vac xin u b ti t hp l n kch thch khng rt cao
. Chng hn nh vac xin di n v ch kch thch cc lympho B to khng th . Th vac
xin virut ton phn kch thch c lympho B ln lympho T. Cc lympho T tn cng v
ph hu cc t bo nhim virut .Tht vy khi s dng vac xin cha virut ton phn th mc bo
v ca n rt cao v tnh min dch c ko di hn .
Virut u b ln u tin c s dng nh l mt cht mang c thng bo nm 1986 bi
cc nh virut hc Bernard Moss , Dennis Panicali v Enzo Paoletti. T mt quy
trnh gm 2 bc cng ngh ho cc vac xin di n v vi b gen u b . Bc th
nht l tp hp vec t ci plasmid . Plasmid ny c cha cc gen ngoi lai (chng hn nh
herpesvirut ,virut thu u), mt promoter u b t nhin khc hng plasmid vo mt
vng c hiu trong b gen u b . Plasmid ti t hp ny sau c khuch i (nhn ln )
trong cc t bo E.Coli ri c phn lp v lm tinh khit . Cc plasmid ny l cc vec t ci
plasmid .
Ti bc th 2 , cc nh virut hc ly cc t bo ng vt sng cho kch thch nhim vi cc
virut u b v cc vec t ci plasmid . Khi virut nhn ln th cc vec t ci plasmid t hp nht
mt im chuyn bit cho cc gen u b t nhin trn vec t (Hnh 5.12)

Hnh 5.12. Sn xut vec t u b s dng trong vac xin . (a) virut u b ti c x l
loi i DNA v cho thm vo cc gen vim gan B (HEP) .(b) Sau cc gen t virut herpes
Simplex(HER) v (c) v t virut Influenza (INF) c gn vo . Cc on DNA u b sau
c phn dng trong cc t bo E.Coli tng thm s lng v sn xut cc vec t ci
plasmid . (d) Cc plasmid c a vo cc t bo ch ng thi vi cc virut u b , Virut
u b ny cm ng DNA cu t bo m cho cc virut du b mi , (e) trong khi tng hp
Virut , plasmid gn vi cc DNA u b bnh thng sn xut ra cc virut u b ti t hp
. (f) Cc virut c gii phng ra t cc t bo c s dng nh cc vac xin .

82

S hp nht ny xy ra vng gen m cho enzym Thymidin kinaza (TK) lm cho gen ny s b
mt i v virut ti t hp ny s khng sn xut c Thymidin kinaza . Lm mt kh nng ny
c 2 tc dng ln : Th nht , v khng c enzym nn virut ny xm nhim t hn cc virut u
b bnh thng . Th 2 , c th nui c cc t bo virut ti t hp c chn ra t cc qun
th khi thm acyclovir vo . Acyclovir l mt thuc khng virut , n tiu dit cc t bo b
nhim cc virut biu hin thymidin kinaza .Thuc ny v hi vi cc t bo nhim cc virut m
khng tng hp enzym ny .
T cui nhng nm 1980 , cc vac xin kiu u b c sn xut khng li bnh vim
gan B , herpes Simplex , Influenza , bnh st rt cng nh khng li cc bnh di ng vt v
cc bnh mn nc ming . Cho ti thi im 1996 cha c loi vac xin no c cp giy
php ca FDA bi v n cn lin qua ti vn an ton . (Tuy nhin , cc vac xin da trn c s
virut i vi bnh thng hn th c cp php t nm 1990).
Cc tranh lun cho rng virut ti t hp c th pht tn vo ngi v gy nn nhng hu qu
bt ng hoc nhng bin chng e do ti cuc sng nh bnh vim no chng hn .
Nhng ngi xng ra loi vac xin ny ch r rng h sng lc cn thn v cc vn
tim n ca vac xin cng nh lm hn ch ti mc cao nht i vi c 2 vn lin quan .Mt
vn tim n na l vac xin ny c th khng c hiu qu i vi nhng ngi tim chng
vac xin u b phng trnh bnh u ma . Tuy nhin , tr em by gi khng cn dng vac
xin ny na nn iu ny khng cn lin quan g na trong tng lai .
tip tc cc vn lin quan ti virut u b , mt cng trnh nghin cu rng ln c
thc hin vi vic s dng virut Fowlpox nh mt cht mang .
Mi lin quan gn gi vi cc virut u b l virut Fowlpox ch nhn ln chim . Cc th
nghim ca Enzo Paoletti nm 1992 xc nh rng virut Fowlpox vn cn hot tnh
chuyn cc gen di n v thnh tc nhn gy bnh di .
Mt cht mang khc c th l mt vi khun , cc nh cng ngh DNA hin nay ang nghin
cu s dng BCG mang cc gen k sinh trng . BCG tc l bacille Calmette Guerin , l
mt chng Bacillus lnh tnh trn ng vt c dng nhiu ni trn Th gii (ngoi tr
Hoa k) gy min dch khng bnh lao . Cc nh cng ngh DNA thit k li Bacillus
ny bng cch thm mt gen t k sinh trng protozoa Leishmania tropia . K sinh trng ny
gy bnh Leishmaniasis , mt bnh mu tc ng n quan i trong cuc chin vng vnh Ba
t . BCG cng ngh ho biu hin gen k sinh trng trn b mt ca n v cm ng p ng
cc lympho T . Cc test trn ng vt ang c thc hin vi loi vac xin ny .Mt loi vac
xin ton phn khc c chun b bng cch lm thay i b gen ca t chc . lm tng kh
nng ng ch ngi ta thy rng trong cc nghin cu v vac xin trc cc nh khoa
hc chun b c mt chng gy bnh yu bng cch ko di qu trnh nui cy c th l
sau nhiu tun , nhiu thng hay nhiu nm mt t chc th s lm cho t chc y mt kh nng
gy bnh nhng vn gi c kh nng tc ng nh mt tc nhn gy min dch . Ngy nay
cc nh cng ngh DNA c th ct b cc gen ca mt t chc m cho cc protein cm ng
bnh .
Vac xin bnh t l mt v d v k thut theo kiu nh th thu c thng li . Bnh t l
mt bnh gy ra bi Vibrio Cholerae , n c trong nc v thc phm b nhim bn . Khi vo
trong ng tiu ho ca ngi , V.Cholerae sn xut ra mt protein c t lm cm ng cc t
bo thnh ng tiu ho , gii phng mt lng ln natri, bicacbonat ,v cc ion khc . Nc
cun i cng cc ion v bnh nhn sm b tiu chy t vi lng nc b mt t 8 n 10
Quart (n v o lng ca Anh bng 1,14 lit) trong vng vi gi v tnh mng bnh nhn lun
b e do .
gy min dch chng li bnh t , ngi ta vn dng vibrio Cholerae cht . Nm 1992 , cc
nh nghin cu Trng i hc Maryland xc nh c gen ca protein c t ny , sau
loi b nhng gen trong V. Cholerae ti phng th nghim .(Hnh 5.13)

83

Hnh5.13. nh qua knh hin vi in t qut ca Vibrrio Cholerae . Vi khun ny c chun

b nh mt vac xin bng cch loi b khi nhim sc th mt gen c tim n gy bnh .
Cc vi khun V. Cholerae b yu i v thng dng lm vac xin t .
Mt vac xin t mi hin ang trong giai on th nghim v trin vng s c mt vac xin khc
c kt qu tt ra i .
Nhng th nghim theo cch lm tn ph cc tc nhn gy bnh ton cu nh bnh st
rt , bnh Leishmanniasis , bnh gy bun ng v bnh sn mng (Schitosomiasis).
Mt tip cn mi i vi nghin cu vac xin bo trc rng s c nhng th h vac xin
tng lai c sn xut theo cng ngh DNA . Chc Edward Jenner cng t ho vi nhng
thnh qu t nhng nh khoa hc ni tip theo con ng ca ng .
5.5. NG VT CHUYN GEN V NHNG NG DNG THC TIN CA VCG.
K thut di truyn nh hng rt ln n vic nghin cu cu trc v biu hin ca gen trong
cc t bo ng vt . Trong vic sn xut protein ti t hp ngun gc ng vt bc cao thng
a li nhng kt qu tt nht khi s dng cc t bo ng vt nui cy , v nhiu khi chng l
vt ch duy nht m bo cho s biu hin bnh thng ca cc gen ng vt .
ng vt chuyn gen c s dng trong y hc v thu c nhng kt qu ng khch l .
D nhin cng khng trnh khi nhng hiu qu khng mong mun . Vic sn xut Insulin ,
cc cht hot ho plasminogen , cc yu t ng mu v.v.. u c s ng gp ca ng vt
chuyn gen . Di y chng ta hy xem xt chi tit i cht v ng vt chuyn gen cng nh
nhng ng dng c bn ca n trong y hc .
5.5.1. Nhng khi nim c bn v ng vt chuyn gen v nhng ng
dng ca chng.
Ngy nay, cc nh cng ngh sinh hc to c mt nhm ln cc ng vt c nhng
kh nng k diu tng chng nh ch l cc gic m trong khoa hc , chng hn
nh ln c th tng hp c hemoglobin ngi , b tng hp c cc protein gn st
ca ngi , d c th sn xut ra cc loi thuc cha bnh x nang, hay cc ng vt
khng li cc bnh khng c kh nng cu cha . chnh l cc ng vt chuyn
gen . Nhng con vt ny c cy thm mt hoc nhiu gen trong t bo . Mc d phc
tp v k thut , nhng quy trnh to dng mt ng vt chun gen th tng i n
gan . Cc gen ngoi lai , chng hn nh gen Hemoglobin ngi s c tim vo nhn
ca t bo trng th tinh , sau c nui cy v sng lc . Sau phi c a
vo cc b m thay cho n pht trn v sinh . D nhin l rt him trng hp
84

m tt c cc mnh ghp u ph hp nh kiu xp hnh ,v ch khi mi c c mt

ng vt chuyn gen.Trong nhng nm 1970, cc nh cng ngh DNA mit mi
nghin cu v lnh vc ny , bt u vi vi khun ri nm, protozoa ti thc vt . H
ph v hng ro ngn cch ca con ngi vi cc gen tr liu v thng li trong vic
thu c cc ng vt chuyn gen . Tt c cc c vng ca con ngi dn dn c p
ng.
5.5.2.a DNA vo cc t bo ng vt c v.
to ra mt ng vt chuyn gen ngi ta phi s dng cc phng php mi a
DNA vo trong cc t bo ng vt. Cho ti gia nhng nm 1970, mi ch c mt cch
l lai cc t bo soma . Trong k thut ny , 2 t bo khng c quan h h hng (mt t
bo cha DNA thc nghim) ho vo nhau v cc t bo ny c kch thch cho pht
trin v nhn ln . Nhng ng tic l khi hai t bo ho nhn vo nhau th thng loi
mt i mt s cht liu ca nhim sc th , trong c c DNA thc nghim. V nh
vy l qu trnh hp nht khng thnh cng .
Trong nhng nm gia 1970 c mt phng php mi s dng can xi phot phat , phng
php ny c cc nh nghin cu thuc Trng i hc Leiden H lan pht trin. Cc
nh nghin cu trn cc on DNA ngoi lai vi can xi phot phat to ra ta DNACan xi . Khi ta ny c trn vi cc t bo ng vt nui cy th cc t bo ny s
thm vo mnh hn rt nhiu so vi khi khng dng can xi . S kin ny c kh nng
dn ti s hnh thnh endocytosis trong t bo . Endocytosis l mt qu trnh trong t
bo hnh thnh mt lp mng bao quanh cc chn gi v y cc nang vo trong bo
tng ca n. Nhng c mt kh khn l khng c cch no xc nh xem t bo
c gn ln cc ht DNA- Can xi cha v bt k mt t bo no bin np th cng b
mt i cng vi m t bo khng bin np .
Ngy nay phng php vi tim c s dng rng ri , n rt n gin v thao tc
trc tip (H11.1) . Ngi ta ly mt pipet thu tinh mnh a vo ngn la lm cho u
mt ca n b nu chy. Mt cch nhanh chng , cc nh k thut to ra c mt u
mt siu mnh , ng knh vo khong t mt na n vi micromet (1% ng knh
ca si tc). Micropipet ny n gin l mt xy lanh c u cc mnh . N c a vo
mt my vi tim v c ht y vi cc on DNA hoc vec t c cha gen ci. Cc
nh th nghim kim sot micropipet bng cch nhn qua th knh ca mt knh hin vi v
dng pipet kp t bo cho ti khi thc hin c vic tim .

Hnh 5.14 :Thc hin vi tim . T bo trung tm c gi bi mt vt pha phi .

Mt microsyring c DNA ci vo t bo nm pha tri . S hp nht ca DNA vi
b gen ca t bo ng vt c th mc cao hoc thp tu thuc vo cc iu kin thc
nghim.

85

Thc hin vi tim rt mt thi gian v tn km ng thi cng lao ng li cao. Tuy
nhin, n li c nhiu li th bi v DNA t b mt hn so vi phng php dng can xi
v phng php ny khng o hi mt cch tuyt i v du chun gen ch r t bo
no a c DNA vo . Tuy nhin, t l hp nht DNA vi nhn t bo ng vt c
th tng i cao, tu thuc vo iu kin th nghim .Nh cp , phng php hp
nht DNA tr thnh mt cng c thch hp i vi chuyn gen ng vt.
i vi cc phng php i hi c du chun th ngi ta thng dng cc t bo ng
vt c gen m ho cho enzym Thymidin kinaza(TK). Chc nng ca enzym TK trong
cc t bo ng vt l rt ht cc nucleotit thymidin t cc DNA s dng t trc v
hp nht chng thnh cc phn t DNA mi . Thng thng th cc t bo ng vt
khng cn enzym TK bi v chng c cch thay th hon thnh mc ch .V vy
khi dng cc cht c ch (inhibitor) , cc nh cng ngh DNA c th git cht cc t bo
c s dng enzym TK v cho thy s chuyn ho thymidin bng cc phng php thay
th .Nh vy nhng t bo cn sng st sau qu trnh chn lc ny c cha cc phn t
thymidin l do chng t tng hp c ch khng phi a t bn ngoi vo.
tng hp thymidin , cc t bo phi c cc sn phm th t mi trng s ti . Tuy
nhin, nu Hypoxanthin v aminopterin c mt trong mi trng th chng s c ch
tng hp thymidin v t bo khng pht trin c . Vy bt buc trong trng hp
khng c thymidin trong mi trng th cc t bo s phi s dng cc cht trn.Tuy
nhin iu li khng xy ra , bi v cc t bo thiu enzym TK th li cn b sung
thm thymidin. V vy trong phng th nghim mt mi trng bao gm hypoxanthin
(H), aminopterin(A) v Thymin(T) gi l mi Trng HAT th cc t bo khng pht
trin c (Hnh 5.15).

86

Hnh 5.15: Dng du chun TK xc nh xem gen c ci cha. (a) Cc t bo

ng vt dng enzym Thymidin Kinaza (TK) thu nhn thymidin t mi trng sinh
trng v to nn DNA cho cc t bo mi . Nhng t bo ny s b cht do km hm s
hot ng ca TK . (a)Nhng t bo cn sng st l do s thu nhn thymidin bng cch
s dng cc gen X , Y v Z m cho thymidin trong t bo cht . Nhng t bo ny s
dng ci gen . (b) Cc t bo ny c a vo mi trng c cha hypoxanthin (H)
v aminopterin (A) , nhng cht ny c ch s tng hp thymidin do s can thip vo
hot ng ca cc gen X , Y v Z . V khi thiu thymidin th t bo s cht k c khi c
thymidin trong mi trng sinh trng . (c) Gen TK c gn vo mt on DNA ci
vo t bo. Nu ci thnh cng th gen TK s c chc nng .V mc d thiu H v A
trong mi trng sinh trng , nhng thymidin vn thu nhn c to thnh DNA cho
s sn xut t bo mi . Nhng t bo ny s pht trin v cc nh cng ngh c th m
bo rng gen mong mun c ci vo t bo .
Hin nay, cc nh cng ngh DNA ci mt gen ca enzym TK vo mt on DNA ri
li ci vo cc t bo ng vt .Nu cc on DNA v gen TK ca n ci c trong
t bo th gen TK ny s m ho cho enzym TK . Khi t bo c th s dng enzym
ny trong mi trng HAT thu nhn thymidin t mi trng v to thnh DNA mi
cho cc qu trnh ca t bo v lm cho n pht trin .

87

Nh vy, theo suy din th nu t bo pht trin tc l n tip nhn c gen TK .

Ngc li nu t bo b cht tc l cc t bo khng c kh nng tip nhn gen TK v
khng to ra c enzym TK v chng khng sng c trong mi Trng HAT.
Nm 1977, Michael Wiger v ng nghip Trng i hc Columbia s dng
thnh cng du chun TK chng minh s hp nht ca cc gen virut vi cc t bo
ng vt. Khi s dng can xi phot phat nh mt cht mang ngi ta ly cc gen t
virut Herpes Simplex ri gn chng vo gen TK . Qua ta DNA-Canxi thy r l cc
t bo ly t mt con chut b bin np. Nhng th nghim ny chng t cc du
chun TK s dng c hiu lc cc t bo ng vt .
Mt du chun khc cng sm c pht trin l du chun Alu (Alu marker). y l
trnh t lp ca cc cp baz , n c tn nh vy v enzym gii hn Alu1 iu khin du
chun ny .C khong 300.000 bn sao cc trnh t Alu trong b gen ngi . S bn sao
cc ng vt khc th c s thay i . Bng vic s dng cc u d c cht phng x
tm c s cc trnh t lp hin din v cc nh cng ngh DNA c th xc nh
c dng DNA no c mt . Chng hn nh mun xem xt s c mt ca DNA ngi
trong b gen ca ng vt th ngi ta tm kim du chun Alu ngi v xc nh c bao
nhiu bn sao tn ti trong cc t bo thc nghim ny .

Hnh 5.16: Nhng ng vt chuyn gen u tin . y l nhng con chut cng la ,
chut bn phi l bnh thng cn chut bn tri c gen ca hormon sinh trng c
ci vo trong t bo v th nn kch thc hai con chut khc hn nhau.

88

Phng php hp nht DNA v vic s dng cc du chun chn lc l rt cn thit i

vi cc th nghim cng ngh sinh hc nhm pht trin cc ng vt chuyn gen .
5.5.3..To cc ng vt theo mun.
Nm 1983, 2 con chut b nh dng nh ging nhau c sinh ra t mt chut m .C
hai u c mu xm, mt en v chng c th thay i c cc biu hin ny .Nhng
con vt ny khc nhau v mt di truyn . Mt con pht trin vi kch thc bnh thng
cn con kia th tr nn to ln cc i . Con th hai ny pht trin vi b gen c
bin i , l nhng con vt bin i gen u tin (H11.3).
Ralph Brinster Trng i hc Pensylvania v Richard Palmiter thuc Trng
i hc Washington l nhng ngi c nhng th nghim tin phong v cc ng vt
bin i gen . Brinster v Palmiter phn lp v tch dng gen m ho cho hormon sinh
trng ca chut ri gn n vo mt promoter. H dng promoter ca gen
metallothionein (MT) (mt gen m ho protein gn kim loi). Promoter ny ng mch
cho gen hormon sinh trng khi promoter MT c cm ng bi mt kim loi trong mi
trng , chng hn nh kim loi cadimi . S d nh vy bi v to hormon trong mt
m c hiu th u phi b sung thm cadimi-mt cht cm ng (kim loi) dng kch
thch promoter (MT) nhm thc y gen ngoi lai hot ng (gen hormon).
Khi s dng k thut nh th ny , Brinster v Palmiter chun b mt plasmid
chimeric c cha gen hormon sinh trng v promoter MT. Sau h a plssmid ny
vao mt tin nhn (pronuclei) ca trng chut c th tinh . Sau th tinh 12 gi , c
th thu thp cc t bo trng th tinh t vi t cung ca chut ci trc lc c s
phn chia t bo . Plasmid c th c tim vo tin nhn vi phng php vi tim.
Khi c tim plasmid , nhng trng th tinh c tch ra v a vo ng
Fallopian ca mt chut ci thay (surrogate). kch thch s mang thai mt cch t
nhin chut ci c giao phi vi mt chut c ct b ng dn tinh. Sau khi ,
nhng con chut con c lm test xem c mt gen hormon sinh trng hay cha .
Khi cc gen hp nht th thm Cadimi vo mi trng cm ng promoter MT v
hormon sinh trng c tng hp . iu l th vi cc nh nghin cu l h c c
mt con chut con pht trin vi kch thc gp i kch thc ca con chut bnh
thng.Tc l con chut con ny t kch c ca mt con chut ln .
Tuy nhin , thng li bc u ny li b lng xung khi nhn thy rng s ln ln v
kch c vn cn cha , bi v i vi chut chuyn gen cn v s cc vn v sinh
l hc cn phi gii quyt nh mt kh nng sinh sn hay b bnh cc c quan, hoc
vn thc n i vi ng vt chuyn gen cng cn c s chuyn i
5.5.3.1.Chut mang h thng min dch ca ngi
Nhng th nghim ca Brinster v Palmiter c v cc nh khoa hc khc theo ui
cc th nghim v ng vt chuyn gen . Mt trong s cc kt qu t c l to c
ging chut mang h thng min dch ca ngi (Human Mouse).ng vt
ny khng phi l sn phm ca vi tim gen , ni mt cch chnh xc l khng phi l
sn phm ca cng ngh DNA. y l mt phi c cc t bo ngi c pht trin cng
vi cc m ca chut v nhng t bo ngi s tr thnh mt b phn ca c th chut.
Cc th nghim v chut mang h min dch ngi c thc hin vo nm 1988 do cc
nh nghin cu thuc Trng i hc Stanford thc hin . Dn u l Joseph M.McCun,
nhm nghin cu ny la chn nhng con chut thiu ht min dch t hp nghim
trng (serve combined immunodeficiency SCID) . ng vt ny khng pht trin cc
t bo ca h thng min dch (na n nh bnh thiu ht min dch ADA).

89

Hnh 5.17. Chut c h thng min dch ca ngi .(a) Cc nh nghin cu a t m

tuyn c ngi vo di mng ca thn chut th n khng c kh nng to nn h
min dch. Tim m hch lympho vo di mng ca thn i ngc . (b) Cc t bo
h min dch (t bo lympho T) c tim qua ui tun hon trong chut . Nhng t
bo ny pht hin cch m tuyn c tin gn ti thn . (c) Cc t bo h min dch
chn (lympho T chn) xut hin t m tuyn c v i vo tun hon ri ti m hch
lympho . Nhng t bo ny c nhn ln trong m hch lympho v tr thnh cc hnh
tinh ca h min dch .
Cc nh nghin cu ly tuyn c , cc hch lympho v cc m gan t phi ngi b
sy thai a vo mng ta nh nang (capsulelike) bao quanh thn chut .Mt tun sau
ngi ta cy cc t bo ca h thng min dch cha chn t mt phi ngi vo chut ,
hy vng rng cc t bo ny s chn trong c th chut . Hai tun sau khi cy ghp ,
chut biu l cc c tnh p ng min dch vi c lympho T v lympho B . Nh th tc
l chut c c mt h thng min dch ca ngi . Chut c cha h thng min
dch ca ngi l mt ti sn v gi i vi nhiu th loi nghin cu , c bit i vi
cc nghin cu v AIDS. Cc nh nghin cu v AIDS b hn ch nghim trng bi
thiu cc mu ng vt tt lm cc test trong phng th nghim. Chng hn nh chng
90

c ng vt no tr tinh tinh mi c kh nng tip xc vi AIDS. Khi s dng mt ng

vt c cc t bo ca ngi s cho php cc nh nghin cu hiu c cc vac xin thc
nghim c p ng hay khng . Hn na, cc cng ty Dc c th quan st c hiu
ng ca cc thuc mi trc kh nng gy nhim ca HIV vi cc t bo ca h thng
min dch.
5.5.3.2.Chut mang ung th (Oncomouse)
Mt ng vt khc c loan tin vo nm 1988 l chut mang ung th . Con chut ny
c Philip Leder thuc Trng i hc Havard to nn . Leder to ra mt con
chut nhy cm cao vi ung th v .Vi mt con chut nh th n cho php cc nh
nghin cu test chnh xc hn , hiu qu hn cc tc nhn gy ung th (Carcinogens)
cng nh phng php iu tr bnh ung th hn l vi nhng con chut bnh thng .
Hn na , qua chut b gy ung th s lm tng s hiu bit v tc ng ca cc gen gy
ung th th nh th no .

Hnh5.18: ng vt u tin c nhn bng sng ch , l chut mang ung th .Bng

sng ch ca Trng i hc Havard v ng vt chuyn gen nm 1988..
Nhm ca Leder nghin cu gen gy ung th c tn l c-myc .Trong nhng nghin cu
trc, cc nh nghin cu ghi ch rng gen ny c lin quan n mt dng u bch
huyt (lymphomas) tr em.Tuy nhin, vi s c mt ca virut gy khi u trn v chut
(mouse mammary tumor-MMT) th gen ny b bin i cm ng ung th v trn cc
ng vt .
Vi s hiu bit ny , cc nh nghin cu tng hp c mt plasmid chimeric c
cha c virut MMT.Ngi ta tim chimera vo tin nhn ca cc t bo trng chut
th tinh v cy cc t bo ny vo cc chut m mang thai h . Nhng chut ny khng
biu hin b ung th v lc cn non , nhng khi trng thnh th gen MMT s hot ng
do n ph v cc hot ng bnh thng ca gen c-myc. Bnh ung th v i theo v
truyn li cho ti cc i con chu.
Ngy 12-4-1988 i vo lch s khi c quan v cc bng sng ch Hoa k cng
nhn bng sng ch cho Havard v chut chuyn gen nhy cm vi ung th (H11.5).
Nhng con chut ny v cc th h con chu ca chng chnh thc tr thnh Number
4.736.866 - l nhng ng vt u tin c nhn bng sng ch (sng ch u tin
i vi t chc chuyn gen c tha nhn nm 1980 l vi khun).

91

Bng sng ch th hai vi ng vt cng l ca Leder v nhm nghin cu thuc Trng

i hc Havard ca ng . Nm 1991, cc nh nghin cu pht trin mt con chut
ung th tuyn tin lit khi n pht trin . Tuyn tin lit l mt tuyn bao quanh
ng tit niu ca nam gii (con c) rt d b ung th v thng phi phu thut ct
b . chut th bnh thng n khng xy ra tr phi n b cm ng cc tc nhn mi
trng.
Nhm nghin cu ca Leder pht trin mt mu chut mi cng ging nh phng
php x l vi nhng con chut trc . H ghp gen gy ung th int-2 vo mt
promoter virrut v ci Chimera vo cc trng th tinh . Kt qu l nhng con chut
ny c gi l chut tin lit tuyn (prostate mice), biu l r rng tuyn tin lit phnh
i ng nh on t trc.
5.5.3.3.Cc ng vt chuyn gen khc.
Mt ng vt khc c to ra theo mun l chut mc bnh Alzheimer .
Postmortem phn tch no ca nhng ngi b bnh Alzheimer thy c nhng
tm t bo thn kinh b cht mc vo cc si ca mt protein gi l Amyloid .Vi nm
qua , cc nh nghin cu lm tinh khit c amyloid v xc nh c trnh t amino
axit ca chng . Ngay sau mt protein tin thn c xc nh v trnh t gen ca
n cng c xc nh sau ny .
Qua tt c cc th nghim , cu hi v mi lin quan ca amyloid trong bnh alzheimer
vn cha c gii p . N l nguyn nhn ca bnh hay ch l cc mnh v cn
li bi bnh ny? Cc th nghim c bo co nm 1991 lm sng t vn
ny.Nhng nh cng ngh DNA nm y c dn u bi Jon Gordon bnh vin
Mount Sinai New york bin i gen mt con chut n c du hiu v triu chng
ca bnh Alzheimer. Nhm ca Gordon a cc gen tin thn amyloid vo cc t bo
trng th tinh ca chut v quan st thy rng khi nhng con chut ny trng thnh
tng hp c Amyloid ca ngi.
Nh m amyloid c trong no ca ng vt m ta c c cu hnh c bit ca no
vi cc tm amyloid trn bnh nhn Alzheimer . M hnh ny sao chp li cc qu
trnh ca bnh mt cch chnh xc v lm cho chng ta hiu mt cch thc t v cn
bnh ny .
Mt trong s nhng mt ch phm nng bng nht trong di truyn phn t ng thi l
chut o vn (knockout mouse). l mt con chut m cc gen cho mt t chc n l
hay mt h thng cc c quan b loi b . Chut knockout to nn mt dng mi
ca cc th nghim cy ghp , cc th nghim m thi loi khng cn l vn nan gii
na . Thc vy, v cc ng vt th nghim khng c h min dch nn n khng loi
thi bt k mt m no c cy ghp. iu ny rt quan trng bi v chut b SCID
nguyn bn vn cn biu hin l t mt vi hot tnh ca h min dch , cng l
nguyn nhn gy nguy him cho cc th nghim ang din ra .
Li tr v cctin tc t 1992 ca Ralph Brinster v Richard Palmiter cng ng
nghip, thi im ny ngi ta s dng chut knockout c mt gen c th hu hoi
gan ca ng vt. Cc nghin cu pht trin mt gen t st (suicide gene), gen ny
c th git mt cch chn lc cc t bo gan chut . Theo sau s cht ca cc t bo gan
chut, mt mu cc t bo gan ngi c cy vo thay th cho cc t bo gan chut .
Bnh thng th h min dch s loi thi nhng t bo ny , nhng v s dng cc t bo
chut knockout nn s loi thi c loi tr bi v chut khng c h thng min dch .
V nh vy c th thay th hon ton c mt c quan nh vo cng ngh DNA.
5.5.3.4.Cc cht phn ng sinh hc t ng vt (Animal Bioreactor)
Mc du cc ng vt chuyn gen c quan tm trong cc phng th nghim v
em li nhng li ch ln lao trong y hc , nhng cng ngh Ho sinh DNA cng cn
phi c p dng vi cc ng vt trong cc trang tri v n cng em li nhng li ch
khng nh .
Tuy nhin, khi lm vic vi cc vt nui ln th kh khn hn v nhng ng vt ny
khng nhiu nh cc ng vt nh hn trong phng th nghim . Hn na vic thao
92

tc cy ghp phi vi cc b m mang thai h cng kh khn hn bi v kch c ca

nhng ng vt ny ln hn . Trng ca nhiu ng vt nui li c t bo cht m c ,
do khng th nhn thy nhn ca chng nu nh khng c cc k thut c bit tr
gip , chng hn nh cn phi c mt giai on ly tm ngn tch t bo cht khi
nhn t bo .
Tuy vy, bt k mt protein no c to ra bi con ngi th cng thc hin c trong
nhng ng vt khc min l cc gen ca ng vt c chng trnh ho d n di
n my.
Tim nng ny lm cho ng vt c kh nng tr thnh mt nh my sn xut cc cht
phn ng sinh hc. Thc cht l ng vt s tr thnh mt nh my sn xut thc nh
cc bo thng ghi lPharm animal (ng vt dc phm) .
Qu trnh to ra mt ng vt chuyn gen bt u bng vic ghp ni 2 gen t 2 dng
tch bit . Mt gen m cho cc protein theo mong mun cn gen kia th phn chiu li
cc t chc hay cc tuyn ca ng vt ni m proteinc to ra .Chng hn nh nu
mt protein c tch ra t sa th tuyn v l ni sn sinh ra cc sn phm ny . Hn
na gen th hai ny li ng vai tr k ton sinh ho khc i v cc sn phm
protein .
Cng vic tip theo l vi tim DNA lai vo cc t bo trng th tinh hoc vo trong
cc phi.Ngi ta thy khong 5%-10% cc trng hp DNA c ci mt cch ngu
nhin trong b gen ca phi. Sau tt c cc trng c chuyn ti cc con m v ri
cc con vt con s c sinh ra , chng s c kim tra xem c cc gen lai cha.Trong trng hp ca cc protein to sa th mun ghp thnh cng cc gen cn phi
thi gian lu hn bi v cn phi test li tht chnh xc v bn thn con ci l ni m
trc y n sn xut c sa , vn y l phi xc nh v so snh v cht
lng cng nh s lng ca sa to ra .V nu con chu ca chng l con c th qu
trnh ny li cng phi lu hn na .
Tuy nhin, ngi ta xc nh c ng vt nn tng (founder animal), n c th
c dng xy dng mt n m khng cn phi dng ti phng php vi tim . Cc
th h con chu ca n hot nh nh l cc cht phn ng sinh hc i t ng vt .
5.5.3.5.Hemoglobin ngi t ln
Mt v d v Bioreactor l ln chun gen , n tng hp c hemoglobin ngi .ng
vt ny l mt ct mc v hiu qu trong vic tm kim cc cht thay th mu . N c
dng thay th cho tt c cc nhm mu v gi c nhiu thng (cc loi mu ca ngi
cho ch gi c vi tun ). Hn na cht thay th mu c th khng c cc tc nhn gy
bnh . Nm 1991, cc nh nghin cu cng ty Cng ngh sinh hc New Jersy thng
bo pht trin c 3 con ln chuyn gen. H tim 2 gen m ho Hemoglobin vo
nhng phi gi sau cy vo ln m th . Trong th nghim ny ch c khong
5% s phi c tim l thng li v ch c 3 con ln cn sng c cho ti khi . Cc
nh nghien cu pht hin rng c khong 15% hng cu ln c cha Hemoglobin ngi
(85% c hemoglobin ln bnh thng).
tch chit c hemoglobin ngi tinh khit , cc nh nghin cu thu thp mu
ln v a n qua h thng tch chit v lm tinh khit ri phn bit u l hemoglobin
ngi v u l hemoglobin ln da trn s khc nhau v tch in ca cc phn t .
Hemoglobin ngi sau c tch chit bng cch cho hp ph vo mt cht liu tng
hp c bit . Sau li c ly ra khi cht tng hp ny v c lm tinh khit . Kh
nng ca cc hemoglobin cng ngh gen c th gn v vn chuyn oxy tng t nh
hemoglobin thc th ca ngi .
Khi dng t do trong mu , hemoglobin ln s b ph v trong vng vi gi n vi
ngy , iu cn ph thuc nhiu yu t .Tuy nhin, hemoglobin ny c th c n
nh do cc lin kt cho gia cc protein phn t v hemoglobin trn ny c th thc
hin c nhim v ging nh hemoblobin gn kt trong hng cu .

93

Hemoglobin trn c l khng c dng trong trng hp b mt nhiu mu qu , cn

khi ch mt 5-6 n v mu th vn dng c (khi phu thut rng). Hemoglobin ny
c th gip cho bnh nhn vt qua thi k nguy kch .
Li th chnh ca hemoglobin i t ng vt l n khng c cc yu t gy bnh cho
ngi .Vn loi thi cc cht nhim bn nng ln t nhng nm 1980 do s xut hin
ca HIV trong cc ngn hng mu.Chng ta cn nh s kin nm 1985 khi pht hin
nhiu ngi truyn mu b nhim virut v ngay c nhng nm gn y vo u nhng
nm 1990 cc mu mu c truyn pht hin thy virut vim gan C (virut vim gan B b
gim thiu v c cc test sng lc cc phng th nghim t vi nm trc) .Tuy
nhin , s dng hemoglobin trn c th cn thit cho vic xc nh cc type mu ca
ngi nhn v ngi cho bi v n khng c khng nguyn b mt ca hng cu . im
bt li l n c th c cha cc cht gy bn gy ra bi virut ng vt hay cc mnh v
ca t bo . Nhng th ny c th gy cm ng cc phn ng d ng .
5.5.3.6.Cc sn phm khc ca ng vt chuyn gen.
S pht trn thnh cng v ln chuyn gen lm ngi ta cng ch i v nhng ci c
to ra khi a mt gen vo c th ng vt .
Mt ng vt khc c nghin cu l b sn xut c lactoferrin ngi trong
sa ca n. Lactoferrin l mt protein gn st chnh ca sa gi chc nng lm cho tr
thng minh .Protein ny cng c c tnh khng khun mnh bi v n loi mt st trong
mi trng ( vi khun li s dng st ny sinh trng) .V lactoferrin lm tng s gn
kt st trong c th nn n cng c dng gii to s thiu mu do thiu st .
Nm 1993, mt cng ty cng ngh sinh hc ca H lan (gi chnh xc l Gen Pharm
International) to c mt s b sa chuyn gen (transgenic dairy calves) c gen
lactoferrin , cc nh nghin cu ny cng c nhng bc tin v cng ngh ci tin
cch chit sut v tinh ch protein .
V mi con b c th sn xut hng ngn lit sa mi nm nhiu hn rt nhiu d v
cu nn n l ng vt sn xut lactoferrin ng c ch .
Mt sn phm khc mt trang tri Scotland l cu chuyn gen , n sn xut c
protein alpha -1-antitrypsin. y l mt protein gi cho s n hi ca mng t bo
v v th m thc y mng cho cc cht bay hi (ga) , cc cht dinh dng v c nhng
cht thi i qua mng .
Cc nh nghin cu Scotland bo co nm 1992 l cu chuyn gen c th tit c 35
gam alpha-1-antitrypsin trong mt lit sa, cao hn 5 ln liu lng cn iu tr cho mt
bnh nhn mt nm.
Vi mc ch iu tr bnh x nang , mt cng ty cng ngh sinh hc Massachusett
s dng nhng con d chuyn gen sn xut ra mt protein iu ho dn in
vn chuyn mng ca nang (cystic transmembrane conductance Regulator protein)
(CTCR) . Protein CTCR l mt cht cn thit cho mng t bo vn chuyn cc ion.
Nhng bnh nhn b x nang thiu protein ny . Liu lng s dng protein ny cng d
hiu l lng cn s thay th cc CTCR b mt v gip gii quyt cc triu chng
v x nang .
Cht hot ho plasminogen ca m(Tissue plasminogen acitvator-TPA) hin nay
c sn xut mt cch cng nghip vi cc vi khun bin i gen (H11.8). TPA l mt
enzym rt quan trng n lm tan cc cc mu ng , dng lamg gim nh bnh nhi
mu c tim . Nm 1991, cc nh nghin cu Massachusett a cc gen TPA vo gia
cc gen i t cc t bo tuyn sa ca d v vi tim vo cc trng th tinh cc gen
c ghp ni . Trong s 29 hu th t 200 trng th c 2 con c gen TPA v khi 1 trong
s 2 hu th c gen ny chn th n con v 1 trong 5 hu th ca n di truyn c gen
TPA .
Cho ti nm 1994 c t nht 2 cng ty u chu thit lp cc trang tri cc ng vt
ln sn xut cc protein dn xut ng vt . Nhng nh my dc phm chuyn gen
nm ti Hoa k khi mt cng ty cng ngh sinh hc thng bo k hoch m rng
94

hng ngn con d bin i gen trn mt trang tri Massachusett. Nhng con d ny sn
xut ra cc protein trong sa ca chng iu tr v chn on bnh cho con ngi .
Cc sn phm ny tr thnh thng phm t nhiu nm nay.
5.5.4.Cc cht thay th mi trng.
Vic ph v cc chu k sinh bnh trong t nhin vn c cc nh cng ngh DNA xem
xt t m .Chng hn nh mt con c sn chuyn gen tm thng cng c th ngt c
chu k sng ca cc k sinh trng gy bnh sn mng (schistosomiasis) .Cc nh nghin
cu Php ngy nay c nh pht trin mt loi c sn chuyn gen khng li s xm
nhp v pht trin ca cc Schistosoma . Khi th vo mi trng nhng con c sn
chuyn gen ny n c th bao quanh cc con c sn t nhin v v th m ph v c
chu k ca giun k sinh ny .
Mi nm c chng khong 100 triu ngi trn ton Th gii mc cc bnh st, cm
lnh, vim ng rut v tiu chy gy ra bi Schistosomiasis .
Mt kch bn tng t c p dng cho mui chuyn gen , c bit l ging
Anopheles. Chng truyn k sinh trng protozoa gy bnh st rt . Cc nh nghin cu
xc nh c mt gen c nng lc vec t (vec t competence gene) , gen ny lm cho
mui anopheles c kh nng u li v truyn k sinh trng st rt. C l nn bin i
nhng gen c th to c cc cn trng t bin th vo vi mt slng ln ,
nhng con mui ny c th lm long bt hoc chn vi qun th mui t nhin v
cng nh c sn , n ph hu c mt chui truyn bnh .

95

Hnh 5.19. Mui Anopheles truyn plasmodium gy bnh st rt . Nhng con mui bin
i gen vi cc vec t thiu nng s c thay th cc mui t nhin ph v chu k
sng ca k sinh trng st rt .

Trung tm ca s quan tm trong cng ngh snh hc cn trng l hiu c nguyn l

ca vec t thiu nng (incompetence vec t) , iu c ngha l ti sao mt vec t
khng li c mt k sinh trng v lm th no m vec t ny c th lm cho c th
96

sinh vt khng mc bnh ? iu c th l do yu t gy bnh khng t thit lp

c trong cc m ca cn trng v vng c ch min dch hot ng trong rut cn
trng .Mt kh nng khc l k sinh trng khng pht trin ng mc bi v dinh dng
ca mi trng khng tt .Cng cn kh nng khc na l k sinh trng khng di
chuyn c ti tuyn nc bt ca cn trng ri pht tn i. Cc nh nghin cu
Notre Dame pht hin rng mui c vec t thiu nng c nhng l nh trong tuyn nc bt ca chng v th n sng lc virut ra mt cch hiu qu trc khi mui chch
nc bt ca n vo vt ch l ngi .Vy s khc bit ny do 1 gen hay 2 gen? V vy
cn phi tm hiu c ch ca vic to ra mui chuyn gen i khng .(H11,10)
c sn chuyn gen v mui chuyn gen l i din cho s tip cn ng n trong vic
can thip vo bnh tt . M hnh truyn thng c cp l cu cha ngi bnh, lm
vac xin phng bnh, cc cht dit cn trng v s thay i mi trng ngn nga s
truyn bnh. Nh chng ta thy, s tip cn ca di truyn i vi vic phng nga
bnh l thut ng s thay th (replacement) m trong lch s y hc him khi c s tip
cn nh th .

Chng VI
D n b gen ngi -nh cao ca Di truyn hc hin i
6.1. nghi y sinh hc .
Nhng thng tin v vic xc nh cc trnh t b gen ngi v cc t chc khc s lm thay i
ln trong sinh hc v y hc . Chng hn nh vi vic i chiu vi b gen ngi , ang bng
n nhng thng tin mi v ngun gc s sng ca chng ta , v cc gen gy bnh hay vic
chn on v nhng kh nng tin ti tr liu . Nhng tin b t c trong cc lnh vc nh
h gen , Proteomics (h protein) , tin sinh hc (bioinformatics) , h gen cc ch phm thuc
(pharmacogenomics) cng tng ln rt nhanh .
97

Trong chng ny chng ti gii thiu mt cch vn tt nhng pht hin chnh ca d n b
gen ngi ( The Human genome Project) (HGP) v nhng mi lin quan ca chng vi sinh , y
hc .
6.2.D n b gen ngi c nhiu mc ch .
D n b gen ngi bt u t nm 1990 , l mt n lc quc t m mc tiu chnh l xc
nh trnh t ton b b gen ngi v b gen ca mt vi t chc khc c cc c s v
nghin cu di truyn ( nh Escherichia Coli , nm men (Sacharomyces cerevisae) , rui dm
(Drosophila melanogaster) , giun a (Caenorhabditis elegans) v mt loi chut nh thng
gp (Mus Musculus).
Cho ti nay nhng mc tiu chnh ca d n c hon tt . Ti hoa k , trung tm quc gia
nghin cu b gen ngi (the National Center for Human Genome Rearch) (NCHGR) c
thnh lp nm 1989, khi u c hng dn bi Jame D. Watson v sau ny l Francis
Collins . NCHGR gi vai tr hng u trong vic nh hng cc n lc quc gia i vi d
n b gen ngi . Nm 1997 c quan ny tr thnh vin nghin cu quc gia b gen ngi (the
National Human Genome Rearch Institute NHGRI ), trong c s cng tc quc t ca cc
nhm t Hoa k , Anh , Nht bn , Php , c v Trung quc v n tr thnh mt tp on
quc t xc nh trnh t b gen ngi (The International Human Genome Sequencing
Consortium) (IHGSC ) . Lc u c quan ny ch t ra mt s mc tiu ngn hn nh to ra
bn b gen ngi vi cc du chun (marker ) 2-5 xng ti morgan (cM) ring r ri xy
dng nn mt bn vt l ca tt c 24 NST (22 nhim sc th thng v 2 NST gii tnh l X
v Y ) vi cc du chun c khong 100.000 cp baz (bp) . ( Hnh .1 ) Tm tt nhng im
khc bit gia bn di truyn , bn di truyn t bo v bn vt l ca mt
NST . Nhng mc tiu ny v nhng mc tiu ban u thu c thnh qu vt tri vo
nhng nm gia 1990 . Nm 1998 mt mc tiu mi ca HGP quc gia c thng bo .
l vic mong mun hon tt ton b trnh t vo cui 2003 hoc sm hn . Nhng mc tiu
c bit khc c lin quan ti cng ngh xc nh trnh t , so snh cc h gen , tin sinh hc ,
cc vn lin quan ti lun l v cc vn khc .

Hnh 6.1 Cc phng php chnh xc nh v phn lp cc gen bnh thng v gen gy
bnh .Vi bn di truyn , v tr ca mt vi du chun di truyn (marker) gi thuyt oc
ch r cng vi cc khong cch di truyn tnh bng xng ti morgan . Vng trn ch v tr ca
tm ng (centromere) . Vi bn di truyn t bo nhng v tr gn chnh xc ca cc du
chun c ch r cng vi cc khong cch vt l tng i tnh bng cp megabaz .
Nhng v d v bn gii hn , im tip cn (contig) v bn STS cng u c ch r .
Ma thu 1998 , c khong 6 % trnh t b gen ngi c hon tt v s t ra c s cho
nhng cng vic tng lai . Mt tin trin na l c mt nhm th 2 , l mt cng ty t nhn
Celera Genomics ng u l Craig Venter thng bo s nhn trch nhim xc nh trnh
t b gen ngi . Nm 1995 Venter v ng nghip xut bn cng trnh v trnh t ton b
b gen ca Haemophilus influenza v Mycoplasma genitalium , l cc mu u tin c
xc nh trnh t gen .Mt yu t quan trng mang li thng li cho nhng ngi lm cng vic
98

ny l s dng mt loi sng bn tc l sonicating DNA , xc nh trnh t cc on v ni

cc trnh t trn c s gi ln nhau . i vi vic so snh th c rt nhiu cch tip cn c s
dng nhng thi im khc nhau khi nghin cu cc gen bnh thng v cc gen gy bnh
(c ghi trong bng 6.1)
Bng 6.1 Nhng phng php chnh dng xc nh v phn lp cc gen
bnh thng v gen gy b
nh .
Quy trnh
Pht hin cc bnh lin
quan ti di truyn t
bo c bit .

Ghi ch
Nh trng hp t nh bng Xp 21.2 c lin quan n
gen gy bnh au c Deuchenne.

Nhng nghin cu tng

qut v NST .

Xc nh ni ging ca cc gia nh ln .Cc gen tri d

nhn dng hn cc gen lit .

Dng u d xc
nh cc locus du chun

u d c th xc nh c hng ngn STS , RFLP , SNP

khp trn NST . N thm d c 2 pha v phc ho r
rng .
Hin nay l phng php nhanh nht nh v mt gen hay
mt on DNA phn min NST ngi v xy dng bn
vt l.
Cho php chuyn mt gen ti mt NST c hiu nhng
khng ti cc phn min .

Lp bn lai bc x
(radiation hybrid mapping)
S dng cc t bo lai
sinh dng ca ngi
hay loi gm nhm.
Hunh quang trong lai
situ.

Cho php nh v mt gen ca mt bng NST .

Dng in di gel xung

in (PFGE) tch
chit cc on DNA ln.

Cho php phn lp cc on DNA di thu nhn t vic s

dng Endonucleaza gii hn ct rt hn ch DNA.

NST i do

Phn dng cc on lp DNA ; l quy trnh trong

phng th nghim thng ch khong 100-200 kb.
Ct DNA thnh nhng mnh tng i ln v quay vng ,
n c th c loi i nhanh chng hn v vi cc on
DNA di hn so vi NST i do .
Cho php phn lp cc on c chiu di thay i .

NST nhy
Phn dng qua YAC,
BAC, Cosmid , Phage,
v plasmid .
Pht hin s biu hin
ca mRNA trong cc
m bi cch thm
Northern khi dng mt
hay nhiu on ca gen
nh l mt u d .

mRNA c th biu hin trong cc m b tc ng .

PCR

S dng khuch i cc on gen v cc ng dng khc

Xc nh trnh t DNA.

Thit lp bn vt l gii php cao nht . Xc nh khung

c m .To thun li cho cc phng tin c th xc
nh c hng triu cp baz mi ngy .
So snh DNA v cc trnh t protein thu nhn t gen cha
bit vi trnh t bit trong d liu c th lm d dng vic
nh loi .

C s d liu

Ghi ch: STS : Sequence tagged site .

RFLP : Restriction fragment linked polymorphism .
SNP : Single nucleotite polymorphism.
YAC : Yeast artifical chromosome .
BAC : Bacterial artificial chromosome .

99

PCR : polymerase chain Reaction .

6.3.Trnh t phc tho ca B gen ngi c thng bo thng 6
nm 2000
Thng 6 -2000 , ngi ng u IHGSC v cng ty t nhn Celera Genomics thng bo
hon tt bn tho trnh t b gen ngi vi khong 90 % tng s . Nhng pht hin chnh ca 2
nhm c xut bn tch bit 2 t bo vo thng 1-2001 , IHGSC bo Nature v Celera
th Science . Bn tho c tp on ny xut bn l sn phm nghin cu t nht l 10 nm
ca 20 trung tm ti 6 quc gia . Xut bn phm ca Celera v cc trung tm hp tc khc l
sn phm ca mt cng trnh nghin cu vi thi gian t nht l 3 nm hoc ngn hn i cht
. Cn mt phn d liu l ca IHGSC . Thnh tu ca s kt hp ny thu ht s ch ca
cng chng v n cung cp cho chng ta mt th vin v s sng , mt bng lit k cc thi
k ca s sng , cn Holy Grail th cung cp cho chng ta nhng pht hin v di truyn con
ngi .
6.4.Hai nhm s dng cc phng php khc nhau .
Di y l tm tt nhng pht hin ln c ghi trong 2 bn tho v nhng ghi ch v cc ng
dng ca chng . Nhng s khc bit khng c nu ra y bi v nhng phn thng nht
chim t l qu ln . Tuy nhin cng cn phi tm tt nhng phng php khc nhau m 2
nhm s dng .V c bn th IHGSC trc ht dng bn sau ri mi n trnh t . S
d nh vy mt phn l do vic xc nh trnh t l mt qu trnh tin hnh chm chp khi m
d n chung bt u .V th chin lc ca ca tp on l phi vt thi gian bi t
c nhng tin b trong vic xc nh trnh t v cc k thut khc . Cch tip cn tng th l
i chiu trong khi xc nh trnh t cc mnh nh(shotgun) c th bc (hierarchical shotgun
sequencing) , bao hm vic ph v ra tng mnh ton b b gen thnh nhng on xp x 100200 kb v ci chng vo NST nhn to ca vi khun (BAC) . Nhng BAC ny sau c nh
v trn NST ngi bng cch tm cc trnh t du chun nh l cc v tr bt u cc trnh t
(sequence tagged sites -STS ) , nhng v tr ny th c xc nh . Cc STS u ngn
(thng nh hn 500 bp) , l locus h gen c quyn cho cc th nghim PCR . Cc dng
BAC sau c lm v thnh nhng mnh nh (shotgunting) . Sau mi mnh nh u
c xc nh trnh t v s dng nhng gii thut my tnh nhn dng cc thng tin trnh
t thch ng t cc on gi nhau thnh nhng mnh c cc trnh t y .
Celera th s dng phng php Whole genome shotgun (bn ton b b gen) . Cc on
shotgun c gn vi nhau bi nhng gii thut thnh nhng b khung ln v dng STS sa
cha cc v tr trn khung ny trong b gen . Mt khung bao gm mt dy cc contig nm
pha bn phi nhng khng cn thit phi ni vo cc trnh t lin tip .
Contig l cc trnh t k nhau ca DNA to bi s ghp li cc on trnh t gi nhau ca mt
NST t nhin hay mt BAC . C th tm kim c my xc nh trnh t vi s lng ln
(high-throughput sequenator ), cc chng trnh my tnh mnh (powerful computer programs)
, yu t cnh tranh v cc yu t khc c tnh ton lm nhanh cc tin b Tt c
c cc nhm thc hin t 1998 tr v trc .
6.5.Vic xc nh trnh t b gen ngi em li ch li cho cc
pht hin mi .
Chng ta hy trch dn mt vi pht hin chnh trong d n b gen ngi bng di y :
Bng 6.2. Nhng pht hin chnh trong bn tho th v b gen ngi .
*Trn 90 % B gen c xc nh trnh t , cn nhng ch trng ln v nh vn phi tip
tc b sung lm y .
*Xc nh c s gen m cho protein l 30.000 n 40.000 .
*Ch c 1,1 n 1,5 % b gen l m cho cc protein .
*C nhng thay i ln v hnh nh NST ngi (tc l s gen theo tng Mb , mt SNP ,
lng GC , s yu t c th c vn chuyn v cc o CpG , tc ti t hp .
*Cc gen ca ngi thc hin nhiu cng vic hn l gen ca giun a hay rui dm .
*H protein (proteome)) ngi phc tp hn so vi cc ng vt c xng sng khc .
*Cc trnh t lp chim khong 50% b gen .
*Khong 100 vng m ho c sao chp v c loi i bng nhng ht vn chuyn c c
s l RNA .
*Khong 200 gen c th truyn t vi khun sang.
*Trn 3 tiu SNP c xc nh .

100

6.6.Hu ht b gen ngi oc xc nh trnh t .

Trn 90 % b gen ngi c xc nh trnh t vo thng 7 -2000 , b xa b gen ln nht
c xc nh trnh t vi kch c khong 3,2 gigabaz (Gb) .Di b gen ngi th b gen ca
rui dm l ln nht (khong 180 Mb) . Vn cn nhng ch trng ln v nh v cht lng
ca mt s d liu v trnh t s phi s l li v mt s pht hin c l l cha c tht s
chnh xc .
6.7. xc nh c rng b gen ngi m cho khong 30.000 -40.000
protein.
iu ngc nhin nht l so vi nhng kt qu thu c t trc th r rng l s gen m cho
protein l thp , ch nm gia khong 30.000 n 40.000 .Theo nhng s liu mi th th con s
ny cao hn gp khong 2 ln i vi giun a (19.099) v gp 3 ln i vi rui dm (13.061)
. Nhng hnh nh ny gi rng phi c nhng gii thch khc chnh xc hn v s gen ca
ngi v ngi c t chc phc tp hn so vi 2 t chc n gin hn .
6.8.Ch c 1,1 n 1,5 % b gen ngi dng m cho protein .
Phn tch cc s liu thy rng ch c 1,1 -1,5 % b gen l cc exon . Khong 24 % l cc
intron v khong 75% cc trnh t nm trung gian (gen trung gian intergenic) . So snh cc
s liu trn giun a v rui dm cho thy kch thc ca exon c 3 mu tng i hng
nh (i vi ngi l 145 bp) . Tuy nhin , kch thc ca intron ngi c th bin i nhiu
hn ( khong trn 3300bp) . Kt qu cho thy c s thay i ln trong kch thc ca gen .
6.9.Cu trc ca cc nhim sc th ngi thay i rt ln .
C nhiu c tnh khc nhau gia cc NST ca ngi , chng hn nh s megabaz , mt
ca cc a hnh nucleotit n (SNP) , lng GC , s cc yu t vn chuyn v cc o CpG v
tc ti t hp . Ta hy ly mt v d : NST s 19 cha s gen nhiu nht (23 gen/ megabaz
) , trong khi NST s 13 v nhim sc th Y th ch tha tht (5 gen/ megabaz) . Ti thi
im ny vn cha gii thch c nhng s thay i ny .
6.10.Cc gen ca ngi hot ng nhiu hn cc gen ca cc t chc
n gin hn .
S ni thay th l kh ph bin ngi , chim t nht l 35% cc gen ca chng . Cc s liu
ch ra rng con s trung bnh ca cc bn sao khc bit /gen ca NST s 22 v 19 l 2,6 v 3,2 .
Nhng c tnh ny l cao hn giun trn , n ch c 12,2% gen l c ni thay th v ch c 1,34
thay i ni /gen .
6.11.H Protein (Proteome ) ngi phc tp hn ng vt c xng
sng .
C tng i t cc Domain protein mi xut hin cc ng vt c xng sng .Tuy nhin ,
ngi s cu trc Domain phn bit (khong 1800) ca protein gp 1,8 ln giun a v rui
dm . Khong 90 h protein c bit ca ng vt c xng sng c nhiu trong h min dch
v h thn kinh .
Nhng kt qu trong 2 bn tho rt giu thng tin v cc h v lp protein . V d trong bng
54-3 lit k cc lp chnh ca protein c m bi cc gen ca ngi . V lp ln nht l
lp cha r unknown . Vic xc nh cc protein cha r ny l mt tiu im ln ca
nhiu phng th nghim .
Bng 6.3. Nhng lp chnh ca protein c m bi cc gen ca ngi .
Lp protein
Cha r
Enzym axit nucleic
Cc yu t phin m
Cc Receptor
Hydrolaza
Cc phn t iu ho chn lc (tc l protein G,
cc cht iu ho chu k t bo .
Cc tin gen gy ung th
Cc ptrotein cu trc b khung t bo
Kinaza

S %
12.809 (41 %)
2.308 (7,5 %)
1.850 (6%)
1.534 (5%)
1.227 (4,0 %)
988 (3,2 %)
902 (2,8%)
876 (2,8%)
868 (2,8%)

6.12.Cc trnh t lp chim trn 50% b gen ngi .

Cc trnh t lp li tnh c t nht l chim mt na b gen .Chng ri v 5 lp sau y :
101

1.Cc lp ri rc (transposon derived repeats) (interspersed repeats) .

2. X l cc gen gi.
3.Lp cc trnh t n .
4.Sao chp tng on , to nn cc bn sao 10-300kb t mt vng ca b gen thnh mt vng
khc .
5.Cc khi trnh t lp ni tip nhau c on gia (centromere ) v on cui (telomere) v
cc vng khc ca NST .
Nhng thng tin chnh v trnh t ca hu ht cc lp trn c gi tr trong vic tm hiu kin
trc v s pht trin ca b gen ngi . C 2 im cn quan tm l nguyn t nhm c coi l
thnh vin ni bt nht (chim khong 10% tng s b gen) ca cc yu t ri rc ngn (short
interspersed elements SINE) , c th c cc vng giu GC . S trng i cc on ngi
thy ph bin hn giun a v rui dm . iu c th l nhng cu trc ny c lin quan ti
s tro exon v lm tng s bin i protein ngi .
6.13.Nhng pht hin khc ng quan tm .
Ba im chnh trong bng 54-2 tm tt cc vn ny . i gm cc ni dung sau : C
khong 100 vng m ho c xc nh l c s sao chp v c loi i bi cc ht chuyn c
c s l RNA (retrotransposons ). iu c th l mt s gen c th chp nhn nhng vai tr
mi theo tin trnh ca thi gian . Mt pht hin ng ngc nhin l trn 200 gen c th c
ngun gc t vi khun truyn ti . Khng thy cc gen ny cc t bo nhn chun m li
khng phi l ng vt c v . Trn 3 triu SNP c xc nh . iu ny chng minh v s
v gi ca cc kha cnh bn gen .
Cng cn phi nhn mnh rng nhng pht hin c lit k y ch l mt s nh trong ni
dung c trong cc bn tho . Bn c c th bn lun cc vn mt cch y trn cc bn
gc ca cc bo co (phn i chiu) .
6.14. Lp k hoch cho nhng cng vic tip theo v b gen ngi v
cc t chc khc .
IHGSC ch r rng s xc nh y cc trnh t vo nm 2003 . Nhim v ny c lin
quan ti vic lp y ch trng v xc nh cc gen mi cng nh v tr v chc nng ca chng
. S phi xc nh cc vng iu ho v cc trnh t ca cc b gen ln khc ( nh chut nh
Rattus norvegicus , mt loi chut ca Na uy ; Danio rerio mt loi c vn ; Fugu rubripes
mt loi c nc h (the tiger puffer fish) v mt hay nhiu ng vt linh chng ) . Thc vy
, bn tho b gen c nc h c xut bn nm 2002 . Cc SNP s c xc nh b sung ;
mt Catalog y ca cc phng n hy vng s c gi tr trong vic lp bn gen vi cc
chng phc tp v cho cc ng dng khc . Cng vi cc vn trn , cc c s d liu s c
thm dn cc thng tin mi v nhng c s d liu mi s c thit lp phc v cho cc
mc ch c bit . S tin hnh nhng nghin cu v h gen chc nng (functional genomics)
(tc l nghin cu b gen xc nh chc nng ca tt c cc gen v cc sn phm ca chng
).
6.15.Nhng lin quan ti h protein , cng ngh Sinh hc v tin sinh
hc .
Nhiu lnh vc s b nh hng bi nhng hiu bit v b gen ngi . Sau y ch nu mt vi
v d ngn gn :
Proteomics (H protein) theo ngha rng l nghin cu tt c cc protein c m trong mt
c th (tc l Proteome), bao gm cu trc , chc nng , s tng tc , v nhng thay i ca
chng . Cn theo ngha hp tc l s nh loi v s nghin cu cc protein gn vi cc hot
ng ca t bo , nhng khng cn thit phi l ton b Proteome . i vi con ngi th nhiu
protein c th s c xc nh cng cc c tnh ca chng . S tng tc v hm lng ca
chng s c xc nh c 2 trng thi : Sinh l v bnh l . Cc thng tin thu c s c
a vo cc d liu c s thch hp . Cc k thut nh in di 2 chiu , khi ph v cc
khng th s l trung tm m rng lnh vc ang pht trin nhanh chng ny . Trn ht l
h protein s to thn li ln cho chng ta hiu c bn v protein cng nh nui dng Cng
ngh sinh hc khi cc protein mi c dng chn on , tr liu v cc ng dng khc cng
nh cc bin php nhm pht trin sn xut kinh t . Cng i hi cc chuyn gia trong lnh
vc Tin sinh hc , bi v n s s ho rt nhanh cho cng vic qun l v s dng mt s
khng l cc d liu t cc nghin cu v h gen v h protein .
6.16.Nhng lin quan ti Y hc .
Trn thc t l mi lnh vc ca y hc u chu tc ng bi cc thng tin v b gen ngi , c
bit vic theo di cc gen gy bnh s c thun li rt nhiu . Nh cp trn , bn
SNP s gip ch rt nhiu trong vic xc nh cc gen lin quan ti cc bnh phc tp . u d
cho bt k mt gen no cng s c p dng nu cn thit , trc tin l vic ci tin cc test

102

chn on cc bnh c lin quan ti gen v cc gen lin quan trc tip n cn nguyn ca cc
bnh c bit . Trong lnh vc Dc phm (pharmacogenomics) cng ang c m rng v c
kh nng trong tng lai thuc s c bo ch iu tit nhng bin i trong cc
enzym v cc protein khc c lin quan ti hot ng v chuyn ho ca thuc
trong cc c th . Nghin cu v gen cn lin quan ti cc hnh vi c th hiu bit su sa
hn na v cc cn nguyn v kh nng iu tr cc bnh tm thn .
Nhiu vn thuc lun l , s ring t v vic s dng thng tin h gen vi mc ch thng
mi cng phi c cp .
Mt im na cng quan trng l mang nhng li ch v y hc v kinh t cho nhng ngi
thuc Th gii th ba trong cc lnh vc dch v y t , chn on v iu tr bnh .

gii thch mt s t chuyn mn .

A
activator protein Mt protein phn ng vi v tr iu ho trn phn t DNA v khch ln
s biu hin gen .
adenosine
mt nucleotit c cha riboza hoc deoxyriboza, phot phat v adenin .
adenovirus mt virut thng thng bao gm mt b gen DNA v mt capsid icosahedral ;
s dng nh mt vec t trong gen tr liu .
agarose mt cht liu gelatin dng trong k thut in di .
AIDS mt bnh do virut c c tnh l lm mt cc t bo Lympho T ca h min dch v
thng i km vi cc bnh do vi sinh vt gy nn cc nhim trng c hi gy t vong .
amino acid mt hp cht ho hc c cha t nht mt nhm amin v mt nhm axit hu c ;
n l cc n v cu thnh ca protein .
anticodon mt trnh t gm 3 baz ca phn t RNA b cu m trn phn t mRNA .
antisene molecule phn t mRNA phn ng v lm trung ho phn t mRNA dng
tng hp mt protein c bit ; phn t antisene c tc dng lm ngng s sn xut protein .
autosome 44 nhim sc th ca b gen ngi khng lin quan ti vic xc nh gii tnh (
nhim sc th thng ).
B
Bacillus thuringiensis vi khun hnh que m cc tinh th c t ca n tc ng nh mt
cht dit cn trng dng chng li mt s loi thuc chn khp ; ngun gen cm ng
khng li loi chn khp trong thc vt .
bacterriophage mt virut sao chp bn trong vi khun hoc hp nht vi cc cht liu di
truyn ca n vi NST ca vi khun ; s dng nh mt vec t trong cng ngh DNA .
beta-galactosidase mt enzym tiu ho c m bi mt operon c trong t bo vi khun
.
biolistic mt xy lanh c mt ng bng ny lon mang cc ht hnh cu dng bn gen vo
cc t bo thc vt .
biotechnology
nguyn l sinh hc trong cc qu trnh da trn c s Ho sinh nh
cng ngh DNA c dng cho cc mc ch thc tin .
bovine growth hormone (BGH) mt hormon c sn xut bng cng ngh di truyn ;
BGH kch thch tng sn xut sa ca tru b sa ; cng c gi l somatotropin .
C
cape mt nucleotit cha 7-metyl guanozin c thm vo u dn ca phn t mRNA trong
khi tng hp phn t ny .
cDNA phn t DNA c tng hp bi enzym phin m ngc (transcriptaza) trn RNA ;
trnh t baz ni t trong DNA b cu vi trnh t baz trong RNA .
cDNA library mt khi t bo c a vo cc plasmid ca chng mt b gen c bit ,
bng cch c th thu nhn gen tng i d dng .
centiMorgan (cM) mt n v ca di truyn hc phn t tng ng vi mt triu cp
baz trong mt phn t DNA .
chimera
mt plasmid hay mt vec t khc c cha DNA khng thng thy trong plasmid .

103

chimeric plasmid mt plasmid c cng ngh ho c mang mt hay nhiu gen

ngoi lai .
Chromosome Nhim sc th .
chromosome walking
mt phng php xc nh trnh t baz trong mt NST c
phn tch bng cch tri di ra ri chn ly mt mu mt thi im v xc nh trnh t baz
ca mu .
clone nhm ca cc t chc , hay t bo , phn t hay cc i tng khc - tt c u i t
mt c th n ; ng ngha vi tp on hay khun lc .
codon mt trnh t gm 3 baz trn phn t mRNA, hot ng cng vi anticodon trn phn
t tRNA nh r ni mt amino axit c t vo trong mt protein .
complementary thnh phn i nghch ca mt cp baz c nhn dng (b tr , b
sung) ; c bit l Adenin b cu vi thymin , Xytosin b cu vi Guanin.
Conjugation
qu trnh ti t hp trong mt vi khun sng nhn c cc on DNA t
mt vi khun sng khc v biu l protein c m bi DNA nhn c .
contig map bn NST trong cc on DNA ni vi nhau to nn mt phn t DNA k
nhau (k tip nhau) .
cosmid
mt on thng DNA m phn ui ca n c th an vo nhau to nn mt
vng ; cosmid c th s dng nh mt vec t trong cng ngh sinh hc .
cytogenetic map
bn NST trong cc gen hot tnh p ng vi mt thuc
nhum ha hc v biu l nh cc bng trn NST .
D
dieoxynucleotide mt nucleotit thiu 2 nguyn t oxy trong phn cacbohydrat .
DNA
axit deoxyribonucleic mt hp cht hu c ca gen.
DNA fingeprinting mt k thut tng hp DNA trong cc trnh t nht nh trong
DNA ca mt mu t bo xc nh v so snh vi trnh t baz DNA ca mt mu t bo
th hai xem chng ng nht mc thit lp tin tng cha .
DNA ligase
mt enzym ni cc on DNA vi nhau bng lin kt gia nhm phot phat
ca mt nucleotit vi phn t deoxyriboza ca mt nucleotit tip theo .
DNA probe mt phn t DNA tng hp chui n , nh gn vi chui DNA b cu ch
trong mt hn hp cc cht sinh hc .
double helix phn t chui kp b nt ra hnh thnh mt xon ; dng DNA trong NST .
Drosophila melanogaster tn khoa hc ca mt loi rui c s dng trong cng ngh
sinh hc .
E
EcolRI mt enzym gii hn t mt chng R ca vi khun Escherichia Coli.
electrophoresis
mt k thut Ho sinh trong cc phn t c dch chuyn theo kch
thc v cc c tnh ho hc ca chng trong Trng in t .
endonuclease mt enzym t bo phn ct phn t DNA .
enzyme mt protein xc tc mt phn ng ho hc ca s chuyn ho m n vn cn gi
nguyn sau phn ng .
eschrichia Coli mt chng vi khun thng thy trong ng tiu ho ca ngi c dng
trong cng ngh DNA nh mt t chc nhn trnh t gen .
eucaryote
mt t chc m cc t bo ca n c nhn chun (nh nm , protozoa , thc
vt , ng vt ).
eucaryotic thuc v mt t chc phc tp m cc t bo ca n c nhn v cc c quan t ,
nhn ln bng gin phn (mitose) v c cc c tnh phn bit vi cc bo procaryote n
gan hn . Cc t bo thc vt , ng vt v ngi l nhn chun
exon
phn mRNA c hiu cho mt trnh t ca mt amino axit ; exon vn c gi li
trong phn t mRNA cui cng v biu hin trnh t amino axit ca protein .
F
forensic

lm theo lut php .

G
gene
mt on DNA cung cp thng tin ho hc cho vic tng hp protein trong t bo ;
tp hp cc gen v xen vo cc trnh t DNA hp thnh mt NST .
gene bank ni cha cc kho t bo ca c th sau ny c th tin hnh phn tch di truyn
.
gene library mt dy cc t bo hoc cc c th n bo thng cha cc gen t cc t bo
ngoi lai ; cc gen ly t cc t bo ca th vin gen c dng trong cc th nghim cng
ngh DNA .

104

gene linkage map bn NST trong cc gen hot ng c nh v bng cch nh v

kt hp cht ch vi cc gen du chun .
gene probe phn t DNA hoc RNA kt hp vi on DNA hoc RNA b cu khi a vo
mt khi ln cc phn t axit nucleic .
genetic code
trnh t baz ni t trong phn t DNA chuyn ho cho mt trnh t ca mt
amino axit trong mt protein .
genome
ton b cc thnh phn DNA nhn ca tt c cc gen ca mt t bo hay mt c
th n bo
genomic library
mt tp hp ca cc t bo hp nht trong cc plasmid ca n tt c
cc gen ca mt t bo khc . Bng ch ny , vic thu nhn gen tng i d dng .
glycoprotein mt protein c cha mt hoc nhiu phn t cacbohydrat gn vi mt hoc
nhiu amino axit trong mt protein .
glycosylation qu trnh sinh ho hc trong cc phn t cacbohydrat c gn vo amino
axit ca mt protein nhng im khc nhau .
Golgi apparatus mt dy cc mng dt mng trong t bo nhn chun cng vi nhng th
x l protein trc khi s tng hp hon tt ; cng cn gi l th Golgi .
gp 120 mt glycptein nh v lp v ca virut HIV ; mt cht cn thit gn HIV vo t
bo ch trong qu trnh sao chp ; c th tng hp c bng cng ngh DNA s dng
trong vic sn xut vac xin .
H
helix
ging nh mt cun dy ni vi my in thoi (xon) , dng DNA trong NST .
hemophilia B mt dng a chy mu trong bnh nhan khng to c cc mu ng do
thiu mt yu t cn thit cho s ng mu , yu t ng mu tng hp c th lm nh bt triu
chng ny .
hepatitis B
mt bnh vim gan do virut truyn qua mu ; vac xin hepatitis B c
sn xut bng cng ngh DNA .
human genome
cha khong 100.000 gen trong mt t bo ngi .
human mouse
chut c thay i v gii phu c cha cc t bo chc nng ca
h min dch .
Huntingtons diseases
mt bnh di truyn c trng bi s h hng tin trin h
thn kinh cng vi s p ph .
hypercholesterolemia
mt bnh di truyn i km vi vic tng mc cholesterol trong
mu v vng mt receptor cholesterol .
I
ice minus bacterium
mt vi khun bin i gen to ra cc protein cm ng s hnh
thnh cc tinh th nhit thp hn bnh thng .
insulin hormon tuyn tu bao gm 51 amino axit , 2 chui ni vi nhau . Insulin lm thun
li cho vic chuyn glucoza vo mu . Insulin cng c sn xut bng vi khun bin i gen .
interferon
cc protein c c sn xut bi cc t bo ca ngi trong p ng vi
virut . Interferon kch thch s tng hp cc cht nhm chng li s thm nhp ca virut vo
trong t bo ; n cng c tng hp bng cc t bo vi khun bin i gen .
intron
phn xen vo phn t mRNA u tin , sau n c loi i trc khi to thnh
phn t mRNA cui cng .
K
kilobase
mt n v gm 1000 baz ni t trong mt phn t DNA hay RNA .
knockout mouse
chut bin i di truyn lm thiu cc gen cho c c quan hay c
h thng c quan .

L
lactoferrin
mt protein c trong sa c kh nng gn cc phn t st ; lactoferrin c
sn xut bng cc t bo ng vt chuyn gen .
M
mammalian ng vt c xng sng , mu nng c tuyn v , c lng tc ,chng hn nh
loi gm nhm , ng vt nui , ng vt bc cao v ngi .
mega YAC mt loi nm men ln t gp c NST nhn to .
messenger RNA (mRNA)
phn t RNA c tng hp vi cc baz ni t m b cu vi
DNA ; mRNA mang cc thng ip ti t bo cht ca t bo tng hp protein .

105

microinjection
mt k thut Ho sinh trong mt xy lanh nh c s dng thm
nhp vo trong t bo v y cc on DNA vo nhn ca t bo .
mus musculus
mt loi chut thng dng trong cng ngh DNA .
mutation thay i c tnh ca mt t chc do thay i cc gen trong t chc .
N
nanometer
n v o lng tng ng vi mt phn t met .
nucleic acid
mt hp cht hu c cu thnh t cc nucleotit ni vi nhau bi cc lin kt
phot phat .
nucleotide mt n v cu thnh ca axit nucleic .
O
oligonucleotide
cc on tng hp nh ca RNA hoc DNA ; mt dng ca gen tng
hp ; cng c gi l oligo ; cng l cc on nh DNA hot ng nh cc phn t antisene.
ongogenes
gen gy khi u hay ung th .
oncomouse chut bin i gen c kh nng b ung th v cao .
operator
mt dy cc baz ni t trong DNA ca mt operon protein c ch hay hot
ho c th iu chnh s biu hin ca gen .
operon phc hp cc gen cu trc v gen iu ho trong NST .
origin of replication mt trnh t baz ni t trn phn t DNA lm tn hiu cho im bt
u ca s sao chp ca phn t DNA .
P
peptide mt protein nh .
phage
vit tt ca bacteriophage ; mt virut sao chp trong vi khun hoc hp nht axit
nucleic ca n vi b gen vi khun ; c dng nh mt vec t .
pharm animal ng vt chuyn gen sn xut ra mt sn phm thuc xc nh .
phosphate group mt phn t gm phot pho , oxy , v nguyn t hydro i t axit phot pho
ric n c mt c trong RNA v DNA .
phosphodiester bond lin kt ho hc hnh thnh gia 5 phot phat t do ca mt nucleotit
v deoxyriboza ca nucleotit th hai .
physical map bn NST trong v tr ca gen hot ng c xc nh v bit s
baz gia cc gen hot ho .
plasmid mt vng ng cht ca DNA cha khong trn mi gen v cc bn sao nhn ln
trong tbo cht ca vi khun ; n c th l mt vec t ca gen trong cng ngh DNA .
poly A tail
mt trnh t ca nucleotit cha adenin cui phn t mRNA khng r v
chc nng nhng c th c dng trong cng ngh DNA xc nh phn t mRNA .
polygalacturonate mt enzym c trong thc vt c th tiu ho c pectin .
polymer mt phn t ho hc bao gm cc n v lp i lp li ca mt cht c bit .
polymerase chain reaction mt qu trnh sinh ho trong mt on DNA c nhn ln
hng triu ln nh s hot ng ca enzym polymeraza .
primer DNA mt on DNA khi u tng hp DNA , chng hn nh trong phn ng
chui PCR .
procaryote mt t chc m cc t bo ca n c nhn ri rc (chng hn nh vi khun ) .
prokaryotic thuc v mt t chc n gin m cc t bo ca n thiu nhn hoc cc bo
quan , nhn ln bng cch sinh sn n gin , n c cc c im khc bit hn so vi cc t
bo nhn chun . Cc t bo vi khun l nhn ri rc .
promoter site
mt trnh t ca nucleotit khi u cho s phin m ca mt m di truyn
trong DNA thnh mRNA .
protease mt enzym tiu ho protein ; vi khun , proteaza thng tiu ho cc protein
ngoi lai .
pseudomonas syringae
mt protein u my vi khun gy cm ng hnh thnh cc
tinh th mt cc t nhin . vi khun c th c bin i gen cm ng hnh thnh cc
tinh th nhit thp hn .
purine mt baz ni t . Vd nh adenin v guanin .
Q
Q-beta replicase mt enzym xc tc tng hp RNA s dng RNA nh mt ci khun ;
dng khuch i tn hiu trong PCR .
R
RAC U ban c vn ti t hp DNA ; mt nhm chuyn vin trong vin y hc quc gia Hoa
k NIH . Nhm ny c quyn ph chun hoc bi min cc d kin thc nghim c lin qua ti
gen tr liu .
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radioactive c th pht ra bc x nng lng c th pht hin c .

recognition sequence mt trnh t baz trn DNA c nhn dng ho hc bng mt
enzym gii hn .
regulatory site mt trnh t baz trn phn t DNA s biu hin gen c th c kim
sot bng phn ng vi cc protein kim ch hoc hot ho .
repressor protein mt protein phn ng vi v tr iu ho trn phn t DNA v kim ch
s biu hin ca gen bng cch c ch s phin m .
resolution gii php .
restriction enzyme mt enzym c th ct DNA mt v tr gii hn .
restriction fragment length polymorphism (RFLP) a hnh di cc on gii hn .
retrovirus mt virut thm nhp vo cc t bo ch v s dng enzym phin m ngc m
cho phn t DNA b cu cho RNA retrovirut ; n c dng nh mt vec t DNA .
reverse transcriptase mt enzym s dng cc trnh t baz trong phn t DNA nh mt m
hnh tng hp mt phn t DNA b cu .
RFLP (restriction fragment length polymorphism) mt on DNA phn lp bi cc enzym
gii hn v c chiu di rt thay i ; RFLP c dng trong chn on v in du DNA nhng
cha r chc nng ca chng .
ribose phn t 5 cac bon v l mt trong cc thnh phn ca RNA ; cng tng t nh
deoxyriboza ch khc l c thm vo mt nguyn t oxy .
ribosome binding site
mt dy cc baz ni t trong DNA ca mt operon m baz
trong mRNA l c hiu cho php mRNA gn vo riboxom .
RNA polymerase mt enzym c chc nng trong phin m v tng hp phn t RNA vi
cc baz b cu vi n trong DNA .
S
sequence map bn NST trong bn cht v v tr ca mi baz nit r sequence tag
site (STS) mt on DNA c gn vo mt v tr chn lc trn phn t DNA nh du v
tr u tin phn tch .
signal peptide mt protein nh to thun li cho vic vn chuyn mt protein c sn
xut ra khi t bo i vo mi Trng ngoi t bo .
Southern blotting mt k thut trong phng th nghim trong cc phn t c tch
chit bng in di sau thm khi gel nh loi .
sticky end
mt chui n nucleotit ni rng phn ui ca mt on DNA (u
dnh).
structural genes mt nhm gen trong operon m cho mt protein v s biu hin ca n
c kim sot bi mt s gen iu ho .
subunits cc phn t vi khun hoc cc on virut c th s dng lm vac xin ; n c sn
xut bng cng ngh DNA .
T
tag polymerase mt enzym chu nhit i t vi khun Thermus aquaticus ; enzym ny l mt
DNA polymerza c s dng trong PCR .
telomere
mt on DNA u NST ca nm men bo v NST khi b phn gii bi
enzym nucleaza .
termination site
mt dy cc nucleotit lm tn hiu kt thc s phin m t DNA
thnh mRNA .
thymidine mt nucleotit c cha deoxyriboza , phot phat v thymin .
thymidine kinase (TK) mt enzym c chc nng trong tng hp DNA bng cch gii
phng thymin t DNA s dng trc tng hp DNA mi ; n c s dng nh mt du
chun xc nh chc chn cho vic ci gen .
tissue plasminogen activator (TPA) mt protein tc ng nh mt proteaza v phn
gii cc cc mu ng . TPA cng c th sn xut bng cng ngh DNA .
transcription mt qu trnh m enzym tng hp phn t RNA khi s dng mt chui DNA
nh mt ci khun ; cc baz RNA b cu vi cc baz DNA .
transduction qu trnh ti t hp gen trong virut ca vi khun nhn c cc cc on
DNA ca vi khun khi sao chp virut v chuyn nhng on DNA ny vo mt vi khun sng
khc DNA c biu hin .
transfer RNA (tRNA) mt phn t RNA gn vo mt amino axit c hiu v vn chuyn
chng ti riboxom , xy ra s tng hp protein ; c nhiu tRNA cho mt amino axit .
transformation qu trnh ti t hp gen trong vi khun nhn c cc on DNA t mi
Trng bn ngoi v biu hin cc protein c m bi cc gen c trong cc on .
translation mt qu trnh sinh ho c lin quan ti cc enzym, riboxom, v cc thnh phn
khc trong phn t mRNA cung cp mt baz ni t m cho s thay th ca cc amino axit
trong s tng hp protein .
107

tumor-infiltrating lymphocyte (TIL) cc t bo lympho ca h min dch tn cng v

phn hu cc t bo khi u .
tumor necrosis factor (TNF) mt protein c to ra bi i thc bo ca ngi n kch
thch s ph v cc t bo khi u ; TNF c th c to ra bng cng ngh DNA .
U
ultramicroscopic

di kh nng quan st ca hin vi quang hc .

V
variable number tandem repeat ( xem VNTR) = on lp di .
vector thc cht ca vic vn chuyn DNA ngoi lai vo trong mt t bo ca mt t chc,
DNA c biu hin .; mt plasmid , cosmid hay mt virut c th nh l mt vec t .
vector incompetence khi mt t chc khng c kh nng t thit lp trong vt ch (bt lc
).
virus
mt on axit nucleic c bao quanh bi mt lp protein bao ngoi , trong mt s
Trng hp c c mt lp v ; c kh nng sao chp trong cc t bo vt ch v gy bnh trong
mt s Trng hp ; mt khc , axit nucleic ca virut c th hp nht vi NST ca t bo ; virut
thng c s dng nh cc vec t .
VNTR (variable number tandem repeat) mt on DNA c cha mt s c
bit cc trnh t baz ni t lp i lp li . VNTR c dng trong k thut in vn tay DNA
.(on lp di)
Y
yeast artificial chromosome (YAC) mt on DNA t mt t bo nm men v c
dng nh phn t c s gn vo cc on DNA khc .

PH LC
Tin sinh hc : khi nim v ng dng .
108

Nhng khi nim c bn v tin sinh hc (bioinformatics) .

Tin sinh hc (bioinrmatics) l s hi t ca ba lnh vc cng ngh hng u l tin hc
cng ngh thng tin v cng ngh sinh hc .Tin sinh hc l mt cng c mi y nhanh tc
nghin cu v ng dng ca cng ngh sinh hc .
Tin sinh hc l khoa hc bao gm vic xy dng , qun l v lu gi ngun d liu thng tin
ton cu , trn c s xy dng v hon thin cc chng trnh x l d liu ng dng lm
cng c h tr hiu qu cho vic nghin cu khm ph bn cht sinh hc ca gii t nhin
phc v nhng li ch ca con ngi .
Nhim v chnh ca tin sinh hc bao gm :
* Xy dng , b sung , t chc v khai thc c s d liu a dng v ton din trn quy m ton
cu lin quan n sinh hc v cc ngnh khoa hc khc .
* Xy dng v pht trin cc chng trnh x l d liu ng dng di dng cc chng trnh
x l d liu c lp c tch hp trong cc thit b phn tch hin i nhm cung cp cho cc
nh sinh hc phng tin xy dng phng n nghin cu hay phn tch x l kt qu vi s
tham gia t vn ca cc chuyn gia trn ton cu .
*o to v cp nht thng xuyn cho cc nh sinh hc k nng t duy v nng lc khai thc
hai ni dung trn vo hot ng khoa hc v cng ngh nhm to bc chuyn bin t ph
trong cch tip cn v nghin cu th gii sng ,to ra mt cuc cch mng thc s trong hot
ng sng to ca con ngi .
C th ni rng , ngy nay cc nh khoa hc bt k mt quc gia no cng u c c hi
ho nhp mt cch bnh ng trong nghin cu sinh hc mang li nhng li ch cho c nhn
loi . iu ny c ngha l mt nc ngho , vi trang thit b thng thng cng c th thc
hin c nhng chng trnh nghin cu phc tp thm ch cc k phc tp nh s h tr quc
t trn mng internet .
C s d liu cng ngh sinh hc .
c im ca d liu cng ngh sinh hc .
Ngun c s d liu sinh hc c truyn ti trn mng rt a dng v phong ph v chng loi
cng nh khi lng thng tin , vi tc ngy cng gia tng theo thi gian . V ni dung , c
s d liu tri rng trn tt c cc mt t cc thng tin chung v tim lc khoa hc v cng
ngh ca cc c quan n cc thng tin v cc cng trnh khoa hc cng b , cc tp ch
chuyn ngnh v.v..c im chung nht ca cc d liu ny l c biu din di dng s hay
k t trong cc tp d liu n l hay di dng cc chng trnh thut ton hon chnh rt
thun li ct gi hay trao i .Ngun tin ny c th chia thnh 2 mng ln l d liu s b v
d liu th cp .
*D liu s cp bao gm cc d liu thu c qua phn tch trc tip bng cc phng tin
tng ng (c s d liu phn tch cu trc DNA , cu trc enzym , amino axit v cc cht
khc )
*D liu th cp gm cc d liu v cc thng tin thu c trn c s phn tch , khi qutho ,
h thng ho hay thng tin m phng cho tng i tng hay nhm i tng sinh hc trong
th gii t nhin . Mng d liu ny bao gm c mng thng tin m qua nh sinh hc c th
khai thc cho vic nh hng , hoch nh k hoch v t chc thc nghim khoa hc tip
theo sao cho hiu qu hn . Hoc trn c s pht trin nm bt c quy lut vn ng ca t
nhin kt hp vi nn tng logic ca th gi sng c th thit knhngn phm hon ton
mi , thm ch cha tng xut hin trong t nhin .
Mt s c s d liu sinh hc ln trn Th gii .
A.D liu thng tin thng thng (sch ,tp ch ,ti liu thng tindng s ho ) :
-Cc cng trnh khoa hc cng b : PUBMED -).
-Cc d liu v Y Dc (http://www.embase.com).
-C s d liu nng nghip (http://www.nalusda.gov/general info/agricola/agricola.html) .
-C s d liu v c sinh hc v ng vt hoang d (http://www.biosis/org).
-C s d liu v bnh hc trong nng nghip (http://www.cabi.org).
B . D liu v phn loi hc (http:ww.ncbi.nlm.nih.gov/taxonomy).
C. D liu v cu trc v c tnh ca nucleotit v b gen (genome) .C th truy cp vo mt
trong 3 a ch sau : http://www.ncbi.nlm.nih.gov/Genbank/index.html.
109

http://www.ebi,ac .uk/embl/databases.
v http://www.ddbj.nig.ac.jp.
D . D liu b gen ngi c th truy cp vo cc a ch sau :OMIM:
http://www3.ncbi.nlm.nih.gov/Omim.
GDB: http://www.gdb.org.
C s d liu v vi khun E.Coli
:
http;//www.susi.bio.unigiessen.de/ecdc/ecdc.html.

http://cgsc.biology.yale.edu/top.html

C s d liu v nm men :http://www.mips.biochem.mpg.de/proj/yeast v

http://genomewww.stanford.edu/Saccharomyces.
E. D liu v cu trc v c tnh chui amino axit v protein :
-Protein Information Resourse PIR (http://www.nbrf.georetow.edu)
-SWISS-PROT (http:// expasy.ch) hoc (http://www.ebi.ac.uk/swissprot).
-trEMBL (http://www.ebi.ac.uk/trEMBL)
-PROSITE (http://www.expasy.ch/prosite).
-PRINT (http://www.bioinf.man.ac.uk/bsm/dbbrowser/PRINTS/PRNT.html).
F.D liu v proteomic (http://www.genom.ad.jp/kegg)
hoc (http://wit.mcs.ant.gov/WIT2) hoc (http://www..ncbi.nlm.nih.gov/COG).
G.D liu v cc enzym v cc con ng trao i cht :
ENZYM databases (http://www.expasy.ch/enzyme).
-c tnh enzym BRENDA (http://www..brenda.uni-koeln.de/brenda).
-Enzym v phn ng enzym (http://www.genome.ad.jp/dbget/ligand.html).
Gii thiu mt vi trung tm d liu ln nht Th gii :
TRUNG TM THNG TIN QUC GIA V CNG NGH SINH HC HOA K

PubMed

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111

National Center for Biotechnology Informatics (NCBI) c thnh lp nm

1988 l mt trong
cc c s d liu sinh hc ln nht Th gii . NCBI qun l khong 25.106 nhm d liu khc
nhau bao gm cc thng tin v cc cng trnh cng b n cu trc DNA , amino axit cng
nh cu trc gen ca cc loi v.v..
Mt s mng d liu ln ca trung tm ny l :
PubMed : Cng b cc kt qu nghin cu ca tt c cc tc gi . Gn y NCBI cn c
PubMed Central cung cp thm c nhng cng trnh khoa hc nm trong k hoch sp
pht hnh gii thiu trc .
GenBank l mng c s d liu v cu trc DNA v amino axit . C s d liu GenBank cng
l sn phm quc t gia 3 trung tm d liu gen ln nht Th gii l GenBank ca NBCI (Hoa
k) , DNA Data Bank (ca Nht bn) v European Molecular Biology Laboratory nucleotide
database (EMBL).
entrez System nhm kt ni cc lien thng gia cc mng d liu gip cho vic truy cp
nhanh v y cc thng tin tm kim . Tc l Entrez khng phi l mt c s d liu m l
ch l cng c gip cho ngi khai thc d dng tip cn cc thng tin lin quan t nhiu mng
d liu khc nhau .
c s d liu embl.
Phng th nghim Sinh hc phn t Chu u (European Molecular Laboratory
EMBL) c thnh lp t nm 1974 , l h thng lin kt cc phng th nghim sinh hc ca
17 nc Chu u v Israel . Trong tp trung vo 5 trung tm ln Heidelberg v
Hambur (c) , Grenoble(Php) , Hinston (Anh) v Monterotondo (Italia) . Vin tin
Sinh hc u chu (European Bioinformatics Institute , trc thuc EMBL ) c thnh lp vo
nm 1994 tr thnh mt trong 3 ngn hng d liu sinh hc ln nht Th gii .
EMBL Outstation - The European Bioinformatics Institute
EMBL Nucleotide Sequence Database
Release Notes
Release 64 Sep 2000
EMBL Outstation
European Bioinformatics Institute
Wellcome Trust Genome Campus
Hinxton
Cambridge CB10 1SD
United Kingdom
Telephone: +44-1223-494400
Telefax : +44-1223-494468
Electronic mail: datalib@ebi.ac.uk
URL: http://www.ebi.ac.uk

CONTENTS
* 1 RELEASE 64
o 1.1 Nine Billion Nucleotides
o 1.2 Draft Human Genome
112

o 1.2.1 Base Quality Values

o 1.2.2 ENSEMBL automatic annotation
o 1.3 Genomes Web Server
o 1.4 Cross-Reference Information
o 1.5 Database Files
o 1.5.1 EST Database Files
o 1.5.2 GSS Database Files
o 1.5.3 HUM Database Files
o 1.5.4 HTG Database Files
o 1.6 Sequence Retrieval System (SRS6)
o 1.7 EMBL Database FAQ
o 1.8 Disclaimer
* 2 FORTHCOMING CHANGES
o 2.1 Genome Representation
o 2.2 New HTC (High Throughput cDNA) division
o 2.3 EMBL Cumulative Update File
o 2.4 Splitting HTG and GSS division files
o 2.5 Next version of SRS indices
* 3 SEQUENCE SUBMISSION SYSTEMS
o 3.1 Checking Sequence Data For Vector Contamination
o 3.2 WebIn - WWW Sequence Submission System
o 3.3 Bulk Submissions
o 3.4 SEQUIN - Stand-alone Submission Program
o 3.5 Sequence Alignment Submissions
o 3.6 Further Submission Information
o 3.6.1 Annotation Guides
* 4 CITING THE EMBL NUCLEOTIDE SEQUENCE DATABASE
* 5 EBI NETWORK SERVICES
o 5.1 Electronic Mail Server
o 5.2 Anonymous FTP Server
o 5.3 World Wide Web (WWW) Server
o 5.4 Sequence Similarity Search Servers
* 6 DISTRIBUTION FILES
o 6.1 Release 64 Files
o 6.2 SRS Indices
* APPENDIX A DATABASE GROWTH TABLE
1 RELEASE 64
The EMBL Nucleotide Sequence Database was frozen to make Release 64 on
02-Sep-2000. The release contains 8,344,436 sequence entries comprising
9,650,223,037 nucleotides. This represents an increase of about 16% over
Release 63. A breakdown of Release 64 by division is shown below:
Division
Entries Nucleotides
----------------- ------------ --------------ESTs
5,565,880 2,194,418,599
Fungi
41,017 75,333,934
GSSs
1,717,212 950,099,606
HTG
77,671 4,263,600,014
Human
119,154 965,113,287
Invertebrates
54,900 329,846,226
Other Mammals
27,021 25,376,675
Organelles
72,962 61,665,029
Patents
207,677
67,411,887
Bacteriophage
1,595
4,385,850
113

Plants
68,956 221,131,770
Prokaryotes
86,977 218,928,626
Rodents
55,263 92,528,729
STSs
116,671 51,039,988
Synthetic
3,838
9,763,762
Unclassified
1,174
1,869,994
Viruses
102,523 90,011,114
Other Vertebrates
23,945 27,697,947
------------ --------------Total
8,344,436 9,650,223,037
1.1 Nine Billion Nucleotides
On 07-JUL-2000 the number of nucleotides in the EMBL Database has passed
the
9,000,000,000 mark. Over the last 12 months (compare Oct 1, 1999: 3.6
Gigabases)
the database size has increased by more than 160%.
EMBL database statistics are available at URL:
http://www3.ebi.ac.uk/Services/DBStats/
1.2 Draft Human Genome and HTG division
The completion of the human draft genome sequence has been announced on
26-June-2000. The draft sequence data is available from the EMBL Database
HTG and HUM divisions.
The total size of the euchromatic portion of the genome is estimated to be 3.2
Gbases. The fact that the total score (FIN + UNFIN) exceeds the size of the
genome is due to redundancy, the general assumption is that about 30% - 40%
of
the bases are redundant.
Below are the database statistics for finished and unfinished human sequence
in EMBL database from September 19, 2000.
YEAR
FIN_TOTAL UNFIN_TOTAL FIN + UNFIN
------ --------- ----------- ----------9/2000 910 Mb 3505 Mb 4415 Mb
See also the Genome Monitoring Table for further detailed information
available from the EBI at URL
http://www.ebi.ac.uk/Databases/Genome_MOT/genome_mot.html
1.2.1 Base quality values
Quality scores from draft HTG data are available on the EBI FTP server. The
gzip'ed files in the directory contain base quality values for unfinished human
sequences from Japanese, US and European sequencing centres. The FastAtype
headers contain the EMBL accession number/version of the corresponding
database
entries.
Example:
>AL009030.9 Phrap Quality (Length:229022, Min: 3, Max: 99)
In order to keep the size of the files within reasonable limits for handling
purposes, files which in uncompressed form are bigger than 1 Gb, are split
into smaller files.

114

Directory: ftp://ftp.ebi.ac.uk/pub/databases/embl/quality_scores
Current Files: /htg_sanger1.qscore.gz - /htg_sanger3.qscore.gz
/htg_genoscope1.qscore.gz
/htg_mpimg1.qscore.gz
/htg_gbf1.qscore.gz
/htg_japan1.qscore.gz
/htg_us1.qscore.gz - /htg_us9.qscore.gz
Quality score files are updated on a daily basis.
1.2.2 Ensembl automatic annotation
Ensembl provides automatic annotation to the human draft genome data
including
information on confirmed peptides, confirmed cDNAs and also predicted
peptides.
Additionally, repeat prediction along with integration of map information and
SNPs are available.
Updated human genome resources spanning the entire working draft are now
available. Ensembl has released its automatic annotation for a June 15th
"frozen" data set at http://freeze.ensembl.org. This URL will now be the stable
location for all subsequent "frozen" dataset updates.
The Ensembl web site is available at URL http://www.ensembl.org/
Ensembl is a joint project between the Sanger Centre and EMBL-EBI.
1.3 Genome WEB Server
Access to completed genomes
The first completed genomes from viruses, phages and organelles were
deposited
into the EMBL Database in the early 1980's. Since then, molecular biology's
shift to obtain the complete sequences of as many genomes as possible
combined
with major developments in sequencing technology resulted in hundreds of
complete genome sequences being added to the database, including Archaea,
Eubacteria and Eukaryota. Recent additions include Buchnera sp. APS
(acc# BA000003) and Pseudomonas aeruginosa (acc# AE004091).
EBI's Genome Web Server provides easy access to completed genome
sequences and
is available at URL: http://www.ebi.ac.uk/genomes/
Genome Monitoring Table
The Genome MOT presents the status of a number of large eukaryotic genome
sequencing projects. The tables are updated daily and also provide access to
EMBL database entries. The Genome MOTis available at URL:
http://www.ebi.ac.uk/Databases/Genome_MOT/genome_mot.html
1.4 Cross-Reference Information
Links to a growing list of external databases have been expanded allowing
integration with specialised data collections, such as protein databases,
species-specific databases, taxonomy databases etc. The WWW-based
sequence
retrieval system (SRS) enable users to easily navigate between cross-referenced
database entries.
EMBL links to other databases:
Database Nr of links
---------- ----------RZPD
2002574
TrEMBL
338688
115

Demeter
175252
SWISS-PROT 143124
MaizeDB
65929
FLYBASE
40968
IMGT/LIGM
37286
MENDEL
21033
GDB
8430
MGD
7998
TRANSFAC
6620
SGD
6029
EPD
3094
IMGT/HLA
2628
---------------------Total
2859653
A list of URLs which conform with current DR line references is available:
Demeter http://ars-genome.cornell.edu
EPD
http://www.epd.isb-sib.ch
FLYBASE http://www.fruitfly.org
GDB
http://www.gdb.org
IMGT/HLA http://www.ebi.ac.uk/imgt/hla
IMGT/LIGM http://imgt.cines.fr:8104
MGD
http://www.informatics.jax.org
MaizeDB http://www.agron.missouri.edu
MENDEL http://mbclserver.rutgers.edu/CPGN
RZPD
http://www.rzpd.de
SGD
http://genome-www.stanford.edu
SWISS-PROT http://www.expasy.ch
TRANSFAC http://transfac.gbf.de/TRANSFAC
TrEMBL http://www.ebi.ac.uk/swissprot/Information/information.html
1.5 Database Files
In order to keep the size of the data files within reasonable limits for
handling purposes, additional division files will be added in subsequent
releases as appropriate.
1.5.1 EST Database Files
EST files are now split according to taxonomic subdivisions following the
model
of the taxonomic split of all other EMBL database divisions, e.g. Release 64
includes files
est_fun.dat
Fungi ESTs
est_hum1.dat - est_hum23.dat Human ESTs
est_inv1.dat - est_inv4.dat
Invertebrate ESTs
est_mam1.dat - est_mam2.dat
Mammal ESTs
est_pln1.dat - est_pln8.dat
Plant ESTs
est_pro.dat
Prokaryote ESTs
est_rod1.dat - est_rod19.dat Rodent ESTs
est_vrt1.dat - est_vrt2.dat
Vertebrate ESTs
This should reduce significantly the volume of data users have to parse in
order to extract ESTs for specific groups of organisms.
1.5.2 GSS Database Files
The GSS division has been split into 18 files (gss1.dat-gss18.dat).

116

1.5.3 HUM Database Files

The HUM division has been split into 6 files (hum1.dat-hum6.dat).
1.5.4 HTG Database Files
The HTG division has been split into 11 files (htgo.dat and htg1.dat-htg10.dat).
htgo.dat includes all HTGS_PHASE0 entries. These typically consist of one-tofew
pass reads of a single clone, have not been assembled into contigs and are
unoriented, unordered, unannotated and contain gaps with runs of 'N's
separating
the reads. Low-pass sequence sampling is useful for identifying clones that
may
be gene-rich. Phase0 sequences are used to check whether another center is
already sequencing this clone. If not, it will be sequenced through phase 1 and
phase 2. When records are updated, the accession numbers will be preserved.
Files htg1-htg10 include all other HTG entries (HTGS_PHASE1 HTGS_PHASE2)
1.6 Sequence Retrieval System (SRS6)
As announced earlier EBI's SRS6 server is available at URL
http://srs.ebi.ac.uk/ now maps to http://srs6.ebi.ac.uk/.
All external services are available from the Tools button on EBI's Web pages.
If you have any comments and/or suggestions please send these to:
support@ebi.ac.uk
1.7 EMBL Database FAQ
An EMBL Database FAQ has been created and is available from the EBI at
URL
http://www.ebi.ac.uk/embl/Documentation/FAQ/
This document includes information on:
General questions about EMBL and other databases
Submission procedure
Updating database entries
WEBIN-specific questions
Navigation guide
1.8 Disclaimer
No guarantee is given as to the completeness and accuracy of the database
entries, in particular the conformity of sequence data in the database with
the journal publication where the sequence is also disclosed.
2 FORTHCOMING CHANGES
2.1 Genome Representation
At the May 2000 Collaborative Meeting it was confirmed by the sequence
database
117

collaboration DDBJ/EMBL/GenBank to go ahead to transform the currently

existing
experimental FTP directory representing genome data into a database division
CON (Constructed Sequences) to represent complete genomes and other long
sequences constructed from segment entries. The CON division entries will
contain construct information (accession numbers and sequence locations)
involved in building the genomes. CON entries and according information will
be included into the daily data exchange mechanism between the collaborating
databases.
The CON entry file includes construct information and all accession numbers
relevant to the genome. Additionally, the complete entry in EMBL format
(DNA and features) plus the complete DNA sequence in Fasta format is
provided.
These entries will be linked, searchable and retrievable through SRS and
available for BLAST and FASTA homology searching.
For an example representation, see the bacterial genome of Pseudomonas
aeruginosa (AE004091) in
ftp://ftp.ebi.ac.uk/pub/databases/embl/genomes/Bacteria/paeruginosa/
AE004091.con
AE004091.embl
AE004091.embl.Z
AE004091.fasta
AE004091.fasta.Z
2.2 New HTC (High Throughput cDNA) division
At the May 2000 collaborative meeting DDBJ/EMBL/GenBank agreed to create
a new
database division HTC to represent unfinished High Throughput cDNA
sequences.
HTC sequences may include 5'UTR and 3'UTR regions and (part of a) codin
region. Upon finishing of these sequences, they will be moved to the
corresponding taxonomic division. HTC sequence entries will include the
keyword
'HTC'. The keyword will be removed once the entry has been included in the
taxonomic division.
2.3 EMBL cumulative update file
We intend to discontinue the provision of the single cumulative update file.
Several sites have reported problems handling our EMBL cumulative update
file
when it grows beyond 2GB (uncompressed), because of file systems that do not
support files > 2Gb. Instead of the cumulative.dat.gz file, we will continue to
make available on our FTP server a set of smaller data files, that contain
together the same data as the full cumulative update file, named cum_*.dat.gz
For further details please check the README file in directory
ftp://ftp.ebi.ac.uk/pub/databases/embl/new/
2.4 Splitting HTG and GSS division files
We plan to split HTG and GSS division files according to taxonomic
subdivisions
following the model of the taxonomic split of all other EMBL database
divisions.
This should reduce significantly the volume of data users have to parse in order
to extract HTGs and GSSs for specific groups of organisms. Files will be named
118

accordingly e.g.
HTGS_PHASE0 sequences will be included in files htgo_hum.dat, htgo_inv.dat
htgo_rod.dat etc, while htgo.dat will include all remaining HTGS_PHASE0
entries.
HTGS_PHASE1 - HTGS_PHASE2 sequences will be included in files
htg_hum.dat,
htg_inv.dat, htg_rod.dat etc while htg.dat will include all remaining HTG
entries.
GSS sequences will be included in files gss_fun.dat, gss_hum.dat etc, while
gss.dat will include all remaining GSS entries.
2.5 Next version of SRS indices
Please note that the next version of SRS indices will be for version 607x and
not 606.
3 SEQUENCE SUBMISSION SYSTEMS
3.1 Checking Sequence Data For Vector Contamination
We urge submitters to remove vector contamination from sequence data before
submitting to the database. To assist submitters the EBI is providing a Vector
Screening Service using the latest implementation of the BLAST algorithm and
a
special sequence databank known as EMVEC. EMVEC is an extraction of
sequences
from the SYNthetic division of EMBL containing more than 2000 sequences
commonly used in cloning and sequencing experiments. EMVEC is by no
means a
complete vector databank but EBI believes it is representative of the kind of
material used in modern sequencing and should be useful to submitters. The
databank will be updated with each release of EMBL and made publicly
available
on the EBI's ftp server for those who wish to have it.
The interactive WWW service can be found at:
http://www.ebi.ac.uk/embl/Submission/webin.html
http://www.ebi.ac.uk/blastall/vectors.html
The results will list sequences producing significant alignments and associated
information like vector name, score, alignment etc
3.2 WebIn - WWW Sequence Submission System
WebIn is the preferred WWW Sequence Submission System for submitting
nucleotide
sequence data and associated biological information to the EMBL Nucleotide
Sequence Database at the European Bioinformatics Institute(EBI). To access
WebIn
at the EBI please use the following URL:
http://www.ebi.ac.uk/embl/Submission/webin.html
Database entries submitted to the EMBL Nucleotide Sequence Database at the
EBI
will be exchanged and shared among the International Collaboration of
Nucleotide
Sequence Databases (DDBJ/EMBL/GenBank).
WebIn guides the user through a sequence of WWW forms allowing the
submission
of sequence data and descriptive information in an interactive and easy way.
119

All the information required to create a database entry will be collected

during this process:
1 Submitter Information
2 Release Date Information
3 Sequence Data, Description and Source Information
4 Reference Citation Information
5 Feature Information (e.g. coding regions, regulators,
signals etc.)
EBI staff will process data submissions within 2 working days and send the
database accession number(s) assigned to your data to your e-mail address.
3.3 Bulk Submissions
With the aim to make bulk sequence submission less time consuming for the
submitters, a new web-based submission system can now be accessed from the
WebIn page. Authors planning to submit a large number of similar sequences
(i.e.,>25) are presented with an option for "Bulk WebIn Submission". When
choosing thebulk path, submitters carry on the usual WebIn submission
procedure
untilhaving finished a first and single representative sequence. During the
submission process database staff will interactively assist in making the
submission of this specific data as convenient as possible, thus saving the
author the time and effort required to complete numerous submission events
individually.
Alternatively, authors planning to submit very large numbers of similar
sequences should contact the database before submitting the data. Database
staff will create series of templates and communicate these to the author for
completion with just the information unique to each sequence required.
Please contact database staff if you require further information.
e-mail: datasubs@ebi.ac.uk
Tel: +44-1223-494499
Fax: +44-1223-494472
3.4 SEQUIN - Stand-alone Submission Program
Sequin is the multi-platform (Mac/PC/Unix) stand-alone software tool
developed
by the NCBI for submitting entries to the EMBL, GenBank, or DDBJ sequence
databases. The Sequin program, along with detailed downloading and
installation
instructions plus general information are available from the EBI via WWW and
anonymous FTP.
http://www3.ebi.ac.uk/Services/Sequin/
ftp://ftp.ebi.ac.uk/pub/software/sequin/
3.5 Sequence Alignment Submissions
The EBI accepts submissions of alignment data (e.g. from phylogenetic and
population analysis etc) of both nucleotide or amino-acid sequences, database
staff assigns an alignment number (e.g. ds38200), which is then communicated
to
the submitter. We suggest that this number is quoted in the resulting
publication.
Alignment data and associated information are made available via EBI's
network
servers (see below).
120

ALIGNMENT FORMATS:
As well as your alignment data we require information describing your
alignment
(see table below) Please provide information for all fields.
Description Field
TITLE:
SUBMITTER:

Information required
Title of alignment
Name, Affiliation, Phone, Fax, Email

RELEASE DATE:
Public Immediately / if Confidential please
provide hold date
CITATION:

If known please provide complete Author list,

Title, Journal, Year of publication, Page
numbers

ALIGNMENT METHOD: Method of alignment and format submitted,

parameters of alignment sequences used (if
appropriate)
DESCRIPTION OF
SYMBOLS:

e.g. Gaps indicated by a dash '-'

DESCRIPTION OF
Describe sequences aligned, including accession
ALIGNMENT:
numbers (if known) and abbreviation of clones or
taxon used in alignment file. If your alignment
contains sequences derived from multiple
taxoonomic sources, please provide the full name
of each organism
FILE FORMAT:
We suggest submission in STANDARD ALIGNMENT FORMATS eg.
(NEXUS, PHYLIP,
CLUSTALW etc) or Sequin output.
A sample alignment in NEXUS format can be viewed at
ftp://ftp.ebi.ac.uk/pub/databases/embl/align/ds32096.dat
NOTE 1: Alignments can be created within Sequin or imported into Sequin
from
files in a standard alignment format like NEXUS or PHYLIP.
NOTE 2: If reporting new primary sequence data, we suggest that you submit
the complete individual sequence files (e.g. via Sequin or WebIn), in order to
include the sequence data as individual entries in the EMBL database. If gaps
have been introduced for the alignment, please leave them out when sending
the
individual sequence files.
SENDING ALIGNMENT DATA to the EMBL Nucleotide Sequence Database
Sequence alignment data can be sent to the Nucleotide Sequence Database by
Electronic mail to datasubs@ebi.ac.uk
ACCESSING ALIGNMENT DATA
Alignment data and additional information are available via the EBI servers:
EBI WWW server:
http://www.ebi.ac.uk/embl/Submission/alignment.html
121

ftp://ftp.ebi.ac.uk/pub/databases/embl/align/
EBI FTP server: by anonymous FTP from FTP.EBI.AC.UK in directory
pub/databases/embl/align
EBI File server: by sending an e-mail message to netserv@ebi.ac.uk
including the line HELP ALIGN or GET ALIGN:DS8200.DAT

3.6 Further Submission Information

3.6.1 Annotation Guides
To help and guide submitters in annotating their sequences, two online guides
are available via hyperlinks from within WebIn:
EMBL Annotation Examples (http://www3.ebi.ac.uk/Services/Standards/web/)
and
EMBL Features and Qualifiers (http://www3.ebi.ac.uk/Services/WebFeat/). The
annotation examples consist of a list of EMBL approved feature table
annotations for common biological sequences. The EMBL Features and
Qualifiers
is a complete list of feature table key and qualifier definitions providing
detailed descriptions, mandatory and optional qualifiers and usage examples.
For further information on submission of sequence data to the EMBL
Nucleotide
Sequence Database please access:
http://www.ebi.ac.uk/embl/Submission/
or contact database staff at:
EMBL Nucleotide Sequence Submissions
e-mail: datasubs@ebi.ac.uk
telephone: +44-1223-494499
telefax: +44-1223-494472
4 CITING THE EMBL NUCLEOTIDE SEQUENCE DATABASE
We encourage authors to include a reference to the EMBL Database in
publications related to their research.
When citing data in the EMBL Database, we suggest to give the according
primary accession and the publication in which the sequence first appeared.
For unpublished data, we suggest to contact the original submitters for
recent publication information or revisions of the data.
We suggest to also provide a reference for the EMBL Database itself. Our
recent publication describing the EMBL database should be cited:
Baker W., van den Broek, A., Camon E., Hingamp P., Sterk P., Stoesser G.,
and Tuli M.A.. 'The EMBL Nucleotide Sequence Database',
Nucl. Acids Res., 28 (1), 19-23 (2000).
Example: The numbers in parentheses refer to the REFERENCE in the EMBL
database entry, and to the EMBL citation above.
"Sequence entry X56734 (1) has been retrieved from the EMBL Database (2)
and showed significant sequence similarity to ..."
(1) Oxtoby, E., et al., Plant Mol. Biol. 17:209-219(1991).
(2) Baker, W., et al., Nucl. Acids Res. 28:19-23(2000)
122

5 EBI NETWORK SERVICES

5.1 Electronic Mail Server
Computer users with access to Internet (directly or via a gateway) can obtain
copies of database entries, documentation or the data submission form, by
sending commands to a file server running at EBI. New and updated EMBL
nucleotide sequence entries are made available on the server on a daily basis.
To use this facility, send file server commands (as electronic mail) to the
address netserv@ebi.ac.uk. Each line of the mail message should consist of a
single file server request.
The most important file server request, to get started, is:
HELP
If the file server receives this command, it will return a helpfile to the
sender, explaining in some detail how to use the facility. For example, to
request a copy of the nucleotide sequence with accession number X55652, use
the command:
GET NUC:X55652
The file server offers various other services, (eg., access to nucleotide and
protein sequence data, protein structure data, software), details of which are
provided in the HELP file.
5.2 Anonymous FTP Server
An alternative method of accessing the EBI archives is to use the Internet
File transfer protocol (ftp). Researchers with direct access to the Internet
can use the FTP program on their local machine to connect to the host
FTP.EBI.AC.UK and enter the username "anonymous" and their email address
as
password.
The directory pub/help contains detailed information about the data available
from the EBI anonymous FTP server which includes the complete EMBL
Nucleotide
Sequence Database releases as well as daily and weekly updates and a
cumulative
update file (in UNIX-compressed format)in the following directories:
EMBL quarterly release: pub/databases/embl/release
EMBL updates: pub/databases/embl/new
5.3 World Wide Web (WWW) Server
The EBI operates a WWW server with URL http://www.ebi.ac.uk/ which gives
access
to information about the EBI and it's products and services. Nucleotide
sequences can be retrieved by a simple query by accession number, or more
complex queries can be contructed using an SRS WWW databank browser.
Nucleotide
sequences can also be submitted to the database using the interactive
submission
system WebIn at URL:
http://www.ebi.ac.uk/embl/Submission/webin.html

123

5.4 Sequence Similarity Search Servers

The EBI offers two network servers for sequence similarity searches via
electronic mail or interactive WWW forms:
FASTA based on W. Pearson's FASTA algorithm. Allows local similarity
searches of protein and nucleotide sequence databases.
Send "help" to Fasta@EBI.AC.UK or use
URL http://www.ebi.ac.uk/fasta3/
BLAST based on the NCBI and WU-Blast software Send "help" to
Blast@EBI.AC.UK or use URL http://www.ebi.ac.uk/blast2/
BLITZ allows very fast searches of protein sequence databases for
local similarities using an exhaustive Smith-Waterman matching
algorithm. Compugen's BIC_SW software is running on a
Biocellerator (BIC-2) Send "help" to Blitz@EBI.AC.UK or
use URL http://www.ebi.ac.uk/bic_sw/
6 DISTRIBUTION FILES
6.1 Release 64 Files
The release contains the files shown below, in the order listed. File sizes are
given as numbers of records.
File Number File Name
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34

Description

Number of Records

DELETEAC.TXT Deleted accession numbers

44649
FTABLE.TXT Feature Table Documentation
465
RELNOTES.TXT Release Notes (this document)
915
SUBFORM.TXT Data Submission Form
418
SUBINFO.TXT Data Submission Documentation
333
UPDATE.TXT Data Update Form
107
USRMAN.TXT User Manual
1469
ACNUMBER.NDX Accession Number Index
8372365
CITATION.NDX Citation Index
1872434
DIVISION.NDX Division Index
23
KEYWORD.NDX Keyword Index
3109242
SHORTDIR.NDX Short Directory Index
21428207
SPECIES.NDX Species Index
2888410
EST_FUN.DAT EST Sequences
3491596
EST_HUM1.DAT EST Sequences
7242162
EST_HUM2.DAT EST Sequences
7383411
EST_HUM3.DAT EST Sequences
7092087
EST_HUM4.DAT EST Sequences
6958043
EST_HUM5.DAT EST Sequences
7086795
EST_HUM6.DAT EST Sequences
7098043
EST_HUM7.DAT EST Sequences
7136249
EST_HUM8.DAT EST Sequences
7031857
EST_HUM9.DAT EST Sequences
7156374
EST_HUM10.DAT EST Sequences
6859020
EST_HUM11.DAT EST Sequences
6661083
EST_HUM12.DAT EST Sequences
6431484
EST_HUM13.DAT EST Sequences
6811351
EST_HUM14.DAT EST Sequences
6856402
EST_HUM15.DAT EST Sequences
7036586
EST_HUM16.DAT EST Sequences
7306475
EST_HUM17.DAT EST Sequences
7263236
EST_HUM18.DAT EST Sequences
7357458
EST_HUM19.DAT EST Sequences
7444208
EST_HUM20.DAT EST Sequences
7476190
124

35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98

EST_HUM21.DAT EST Sequences

6699624
EST_HUM22.DAT EST Sequences
6963358
EST_HUM23.DAT EST Sequences
4588499
EST_INV1.DAT EST Sequences
6431773
EST_INV2.DAT EST Sequences
6042873
EST_INV3.DAT EST Sequences
6293598
EST_INV4.DAT EST Sequences
4046341
EST_MAM1.DAT EST Sequences
6114230
EST_MAM2.DAT EST Sequences
2356039
EST_PLN1.DAT EST Sequences
6750911
EST_PLN2.DAT EST Sequences
6219344
EST_PLN3.DAT EST Sequences
5830564
EST_PLN4.DAT EST Sequences
7215994
EST_PLN5.DAT EST Sequences
7046836
EST_PLN6.DAT EST Sequences
6762278
EST_PLN7.DAT EST Sequences
6720107
EST_PLN8.DAT EST Sequences
6029205
EST_PRO.DAT EST Sequences
38548
EST_ROD1.DAT EST Sequences
7331559
EST_ROD2.DAT EST Sequences
7567611
EST_ROD3.DAT EST Sequences
7220551
EST_ROD4.DAT EST Sequences
7549688
EST_ROD5.DAT EST Sequences
6811012
EST_ROD6.DAT EST Sequences
7086810
EST_ROD7.DAT EST Sequences
9771985
EST_ROD8.DAT EST Sequences
9130283
EST_ROD9.DAT EST Sequences
7665029
EST_ROD10.DAT EST Sequences
9177208
EST_ROD11.DAT EST Sequences
9743196
EST_ROD12.DAT EST Sequences
9700691
EST_ROD13.DAT EST Sequences
9653685
EST_ROD14.DAT EST Sequences
9473210
EST_ROD15.DAT EST Sequences
9015774
EST_ROD16.DAT EST Sequences
6666497
EST_ROD17.DAT EST Sequences
7649778
EST_ROD18.DAT EST Sequences
7420422
EST_ROD19.DAT EST Sequences
738690
EST_VRT1.DAT EST Sequences
7641169
EST_VRT2.DAT EST Sequences
2254064
FUN.DAT
Fungi Sequences
3736027
GSS1.DAT Genome Survey Sequences
6116578
GSS2.DAT Genome Survey Sequences
6118824
GSS3.DAT Genome Survey Sequences
6268149
GSS4.DAT Genome Survey Sequences
6628318
GSS5.DAT Genome Survey Sequences
6554451
GSS6.DAT Genome Survey Sequences
6616068
GSS7.DAT Genome Survey Sequences
6639716
GSS8.DAT Genome Survey Sequences
6644800
GSS9.DAT Genome Survey Sequences
6958158
GSS10.DAT Genome Survey Sequences
6788195
GSS11.DAT Genome Survey Sequences
7155659
GSS12.DAT Genome Survey Sequences
6988978
GSS13.DAT Genome Survey Sequences
6978243
GSS14.DAT Genome Survey Sequences
6402203
GSS15.DAT Genome Survey Sequences
6646868
GSS16.DAT Genome Survey Sequences
7448747
GSS17.DAT Genome Survey Sequences
6669805
GSS18.DAT Genome Survey Sequences
1027489
HTG1.DAT High Throughput Genome Sequences
7854248
HTG2.DAT High Throughput Genome Sequences
5995734
HTG3.DAT High Throughput Genome Sequences
4210260
HTG4.DAT High Throughput Genome Sequences
4724917
HTG5.DAT High Throughput Genome Sequences
8718298
HTG6.DAT High Throughput Genome Sequences
8721834
125

99
100
101
102
103
104
105
106
107
108
109
110
111
112
113
114
115
116
117
118
119
120
121
122
123

HTG7.DAT High Throughput Genome Sequences

8979368
HTG8.DAT High Throughput Genome Sequences
8137472
HTG9.DAT High Throughput Genome Sequences
7846179
HTG10.DAT High Throughput Genome Sequences
4273070
HTGO. DAT High Throughput Genome Sequences
8701440
HUM1.DAT Human Sequences
9494007
HUM2.DAT Human Sequences
5320579
HUM3.DAT Human Sequences
3561983
HUM4.DAT Human Sequences
2858503
HUM5.DAT Human Sequences
2298449
HUM6.DAT Human Sequences
1644433
INV.DAT
Invertebrate Sequences
9495348
MAM.DAT
Other Mammal Sequences
1908267
ORG.DAT
Organelle Sequences
5140625
PATENT.DAT Patent Sequences
8110279
PHG.DAT
Bacteriophage Sequences
217840
PLN.DAT
Plant Sequences
8269953
PRO1.DAT Prokaryote Sequences
6104496
PRO2.DAT Prokaryote Sequences
4233076
ROD.DAT
Rodent Sequences
4755562
STS.DAT
STS Sequences
7970081
SYN.DAT
Synthetic Sequences
394629
UNC.DAT
Unclassified Sequences
106371
VRL.DAT
Viral Sequences
7545287
VRT.DAT
Other Vertebrate Sequences
1787491

6.2 SRS Indices

SRS indices can be found on the FTP server in the srs directory
ftp://ftp.ebi.ac.uk/pub/databases/embl/release/srs/.
See README file for details.
Please note that the next version of SRS indices will be for version 607x
and not 606.
APPENDIX A
DATABASE GROWTH TABLE
The following table shows the growth of the EMBL Nucleotide Sequence
Database at each release.
Release Month

Entries

Nucleotides

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18

568
811
1481
1698
2378
4835
5789
6395
7630
8817
11621
12706
14397
15344
17961
19592
20695
22938

585433
1114447
1654863
2147205
2874493
4567592
5622638
6353040
7813214
9766948
12189783
13638061
16023478
17272160
20318442
22625941
24211054
27249830

06/1982
04/1983
12/1983
08/1984
04/1985
08/1985
12/1985
04/1986
09/1986
12/1986
04/1987
07/1987
10/1987
01/1988
05/1988
08/1988
11/1988
02/1989

126

19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64

05/1989
08/1989
11/1989
02/1990
05/1990
08/1990
11/1990
02/1991
05/1991
09/1991
12/1991
03/1992
06/1992
09/1992
12/1992
03/1993
06/1993
09/1993
12/1993
03/1994
06/1994
09/1994
12/1994
03/1995
06/1995
09/1995
12/1995
03/1996
06/1996
09/1996
12/1996
03/1997
06/1997
10/1997
12/1997
03/1998
06/1998
09/1998
12/1998
03/1999
06/1999
09/1999
12/1999
03/2000
06/2000
09/2000

24365
26223
28679
31508
34902
37784
41580
43745
46871
54558
57655
63378
72481
79377
89100
99591
108973
127933
146576
167777
182615
209352
230950
303206
420111
506190
622566
701246
827174
928067
1047263
1187455
1432941
1787004
1917868
2125225
2330040
2689618
3046471
3272064
3952878
4719266
5303436
5865742
6760113
8344436

29066676
31240948
34748087
38165786
42923803
47354438
52900354
55859549
59915244
70448052
75400487
83574342
94390065
101292310
111413979
121420828
131880111
145401156
158171400
177550115
192195819
211017104
226259607
262559786
315840053
363273777
427620278
473691480
550739395
608931850
696183789
789755858
931351601
1181167498
1281391651
1427634373
1607673907
1904091473
2164718256
2355200790
2924568545
3543553093
4508169737
6120908677
8255674441
9650223037.

C s d liu CIB-ddBJ .
center for Information Biology and DNA Data Bank of Japan
(CIB-DDBJ) l c s d liu ca trung tm thng tin Sinh hc , vin Di truyn
Quc gia Nht bn .

127

CIB DDBJ l c s d liu cng ngh Sinh hc quan trng v l c s d liu DNA duy nht
Nht bn . Bn cnh CIB-DDBJ , vin Di truyn Quc gia Nht bn cn qun l nhiu mng
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using gene therapy?

Gene therapy links

What is gene therapy?

Genes, which are carried on chromosomes, are the basic
physical and functional units of heredity. Genes are
specific sequences of bases that encode instructions on
how to make proteins. Although genes get a lot of
attention, its the proteins that perform most life functions
and even make up the majority of cellular structures.
When genes are altered so that the encoded proteins are
unable to carry out their normal functions, genetic
disorders can result.

Gene therapy is a technique for correcting defective genes

Medicine
& responsible for disease development. Researchers may use
the New Genetics one of several approaches for correcting faulty genes:
Home
A normal gene may be inserted into a nonspecific
Gene
Testing
location within the genome to replace a
nonfunctional gene. This approach is most
Gene
Therapy
common.
Pharmacogenomics
An abnormal gene could be swapped for a normal
Disease
gene through homologous recombination.
Information
Genetic
The abnormal gene could be repaired through
Counseling
selective reverse mutation, which returns the gene
Ethical,
Legal,
to its normal function.
Social
Issues
Home
The regulation (the degree to which a gene is
turned on or off) of a particular gene could be
Privacy
altered.
Legislation
Gene
Testing
How does gene therapy work?
Patenting
Forensics
In most gene therapy studies, a "normal" gene is inserted
into the genome to replace an "abnormal," disease-causing
Genetically
Modified
Food gene. A carrier molecule called a vector must be used to
deliver the therapeutic gene to the patient's target cells.
Behavioral
Currently, the most common vector is a virus that has
Genetics
been genetically altered to carry normal human DNA.
Minorities, Race, Viruses have evolved a way of encapsulating and
delivering their genes to human cells in a pathogenic
Genetics
manner. Scientists have tried to take advantage of this
Genetics
in capability and manipulate the virus genome to remove
Courtroom
disease-causing genes and insert therapeutic genes.
Education
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Target cells such as the patient's liver or lung cells are

infected with the viral vector. The vector then unloads its
genetic material containing the therapeutic human gene
into the target cell. The generation of a functional protein

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product from the therapeutic gene restores the target cell

to a normal state. See a diagram depicting this process.
Some of the different types of viruses used as gene
therapy vectors:

Retroviruses - A class of viruses that can create

double-stranded DNA copies of their RNA
genomes. These copies of its genome can be
integrated into the chromosomes of host cells.
Human immunodeficiency virus (HIV) is a
retrovirus.

Adenoviruses - A class of viruses with doublestranded DNA genomes that cause respiratory,
intestinal, and eye infections in humans. The virus
that causes the common cold is an adenovirus.

Adeno-associated viruses - A class of small,

single-stranded DNA viruses that can insert their
genetic material at a specific site on chromosome
19.

Herpes simplex viruses - A class of doublestranded DNA viruses that infect a particular cell
type, neurons. Herpes simplex virus type 1 is a
common human pathogen that causes cold sores.

Besides virus-mediated gene-delivery systems, there are

several nonviral options for gene delivery. The simplest
method is the direct introduction of therapeutic DNA into
target cells. This approach is limited in its application
because it can be used only with certain tissues and
requires large amounts of DNA.
Another nonviral approach involves the creation of an
artificial lipid sphere with an aqueous core. This liposome,
which carries the therapeutic DNA, is capable of passing
the DNA through the target cell's membrane.
Therapeutic DNA also can get inside target cells by
chemically linking the DNA to a molecule that will bind
to special cell receptors. Once bound to these receptors,
the therapeutic DNA constructs are engulfed by the cell
membrane and passed into the interior of the target cell.
This delivery system tends to be less effective than other
options.

Researchers also are experimenting with introducing a

47th (artificial human) chromosome into target cells. This
chromosome would exist autonomously alongside the
standard 46 --not affecting their workings or causing any
Search This mutations. It would be a large vector capable of carrying
Site
substantial amounts of genetic code, and scientists
anticipate that, because of its construction and autonomy,
the body's immune systems would not attack it. A
problem with this potential method is the difficulty in
delivering such a large molecule to the nucleus of a target
cell.
Contact
Us
Privacy Statement What is the current status of gene therapy research?

130

Site Stats
Credits

and The Food and Drug Administration (FDA) has not yet
approved any human gene therapy product for sale.
Current gene therapy is experimental and has not proven
very successful in clinical trials. Little progress has been
made since the first gene therapy clinical trial began in
1990. In 1999, gene therapy suffered a major setback with
the death of 18-year-old Jesse Gelsinger. Jesse was
participating in a gene therapy trial for ornithine
transcarboxylase deficiency (OTCD). He died from
multiple organ failures 4 days after starting the treatment.
His death is believed to have been triggered by a severe
immune response to the adenovirus carrier.
Another major blow came in January 2003, when the
FDA placed a temporary halt on all gene therapy trials
using retroviral vectors in blood stem cells. FDA took this
action after it learned that a second child treated in a
French gene therapy trial had developed a leukemia-like
condition. Both this child and another who had developed
a similar condition in August 2002 had been successfully
treated by gene therapy for X-linked severe combined
immunodeficiency disease (X-SCID), also known as
"bubble baby syndrome."
FDA's Biological Response Modifiers Advisory
Committee (BRMAC) met at the end of February 2003 to
discuss possible measures that could allow a number of
retroviral gene therapy trials for treatment of lifethreatening diseases to proceed with appropriate
safeguards. FDA has yet to make a decision based on the
discussions and advice of the BRMAC meeting.
What factors have kept gene therapy from becoming an
effective treatment for genetic disease?

Short-lived nature of gene therapy - Before gene

therapy can become a permanent cure for any
condition, the therapeutic DNA introduced into
target cells must remain functional and the cells
containing the therapeutic DNA must be longlived and stable. Problems with integrating
therapeutic DNA into the genome and the rapidly
dividing nature of many cells prevent gene therapy
from achieving any long-term benefits. Patients
will have to undergo multiple rounds of gene
therapy.

Immune response - Anytime a foreign object is

introduced into human tissues, the immune system
is designed to attack the invader. The risk of
stimulating the immune system in a way that
reduces gene therapy effectiveness is always a
potential risk. Furthermore, the immune system's
enhanced response to invaders it has seen before
makes it difficult for gene therapy to be repeated
in patients.

Problems with viral vectors - Viruses, while the

carrier of choice in most gene therapy studies,
present a variety of potential problems to the
patient --toxicity, immune and inflammatory

131

responses, and gene control and targeting issues.

In addition, there is always the fear that the viral
vector, once inside the patient, may recover its
ability to cause disease.

Multigene disorders - Conditions or disorders that

arise from mutations in a single gene are the best
candidates for gene therapy. Unfortunately, some
the most commonly occurring disorders, such as
heart disease, high blood pressure, Alzheimer's
disease, arthritis, and diabetes, are caused by the
combined effects of variations in many genes.
Multigene or multifactorial disorders such as these
would be especially difficult to treat effectively
using gene therapy. For more information on
different types of genetic disease, see Genetic
Disease Information.

What are some recent developments in gene therapy

research?

University of California, Los Angeles, research

team gets genes into the brain using liposomes
coated in a polymer call polyethylene glycol
(PEG). The transfer of genes into the brain is a
significant achievement because viral vectors are
too big to get across the "blood-brain barrier." This
method has potential for treating Parkinson's
disease. See Undercover genes slip into the brain
at NewScientist.com (March 20, 2003).

RNA interference or gene silencing may be a new

way to treat Huntington's. Short pieces of doublestranded RNA (short, interfering RNAs or
siRNAs) are used by cells to degrade RNA of a
particular sequence. If a siRNA is designed to
match the RNA copied from a faulty gene, then
the abnormal protein product of that gene will not
be produced. See Gene therapy may switch off
Huntington's at NewScientist.com (March 13,
2003).

New gene therapy approach repairs errors in

messenger RNA derived from defective genes.
Technique has potential to treat the blood disorder
thalassaemia, cystic fibrosis, and some cancers.
See Subtle gene therapy tackles blood disorder at
NewScientist.com (October 11, 2002).

Gene therapy for treating children with X-SCID

(sever combined immunodeficiency) or the
"bubble boy" disease is stopped in France when
the treatment causes leukemia in one of the
patients. See 'Miracle' gene therapy trial halted at
NewScientist.com (October 3, 2002).

Researchers at Case Western Reserve University

and Copernicus Therapeutics are able to create
tiny liposomes 25 nanometers across that can carry

132

therapeutic DNA through pores in the nuclear

membrane. See DNA nanoballs boost gene therapy
at NewScientist.com (May 12, 2002).

Sickle cell is successfully treated in mice. See

Murine Gene Therapy Corrects Symptoms of
Sickle Cell Disease from March 18, 2002, issue of
The Scientist.

What are some of the ethical considerations for using

gene therapy?
--Some Questions to Consider...

What is normal and what is a disability or

disorder, and who decides?

Are disabilities diseases? Do they need to be cured

or prevented?

Does searching for a cure demean the lives of

individuals presently affected by disabilities?

Is somatic gene therapy (which is done in the adult

cells of persons known to have the disease) more
or less ethical than germline gene therapy (which
is done in egg and sperm cells and prevents the
trait from being passed on to further generations)?
In cases of somatic gene therapy, the procedure
may have to be repeated in future generations.

Preliminary attempts at gene therapy are

exorbitantly expensive. Who will have access to
these therapies? Who will pay for their use?

Gene Therapy Links

General Information

MEDLINEplus: Genes and Gene Therapy - Access

news, information from the National Institutes of
Health, clinical trials information, research, and
more.

Recombinant DNA and Gene Transfer - National

Institutes of Health Guidelines

Questions and Answers about Gene Therapy - A

fact sheet from the National Cancer Institute.

Introduction to Gene Therapy - An overview by

Access Excellence.

A Gene Therapy Primer - Introduction to gene

therapy from the bio.com.

Gene Therapy and Your Child - From KidsHealth

for Parents.

133

Pioneering gene treatment gives frail toddler a new

lease of life

Gene Transfer - An overview of gene therapy

science issues, ethical concerns, and regulation
and policy from the Genetics & Public Policy
Center.

Cures - An introduction to gene therapy provided

by discoveryhealth.com.

Delivering the Goods - An article describing the

different types of gene therapy approaches. From
October 2, 2000, issue of The Scientist.

How to Turn on a Gene - An article from Wired

Magazine.

How Viruses Are Used in Gene Therapy - From

The DNA Files, a series of radio programs from
SoundVision Productions.

Human Gene Therapy: Present and Future - A

Human Genome News article.

Gene Therapy - A NewsHour with Jim Lehrer

transcript covering the death of gene therapy
patient, Jesse Gelsinger (February 2, 2000).

Animations from the Tokyo Medical University

Department of Paediatrics Genetics Study Group
o

Animations of Induction of Genes (Gene

Therapy)

Animations of Problems in Gene Therapy

FDA Information

FDA Advisory Committee Discusses Steps for

Potentially Continuing Certain Gene Therapy
Trials That Were Recently Placed on Hold 2/28/2003

FDA Places Temporary Halt On Gene Therapy

Trials Using Retroviral Vectors In Blood Stem
Cells - 1/14/2003

New Initiatives to Protect Participants in Gene

Therapy Trials - 3/7/2000

Human Gene Therapy and The Role of the Food

and Drug Administration - An overview from the
Center for Biologics Evaluations and Research of
the U.S. Food and Drug Administration.

Human Gene Therapy Harsh Lessons, High Hopes

- An article published in the September-October
2000 issue of FDA Consumer magazine.

The Last Word: Researchers React to Gene

Therapy's Pitfalls and Promises - An article
published in the September-October 2000 issue of

134

FDA Consumer magazine.

Fundamentals of Gene Therapy - Diagrams and

basic description of gene therapy from the FDA.

Gene Therapy Ethics

Ethical Issues in Human Gene Therapy - A Human

Genome News article.

Special Report: Ethics of Genetics - From

Guardian Unlimited.

Ethical Issues in Human Gene Therapy - A Human

Genome News article.

Gene Therapy Clinical Trials

University of Pittsburgh Molecular Medicine

Institute - Contains information about ongoing and
completed clinical trials.

Gene therapy studies in ClinicalTrials.gov - The

U.S. National Institutes of Health resource for
public access to information on clinical research
studies.

Gene Therapy Clinical Trials - Access to a

worldwide database of gene therapy clinical trials
at this Web site from the publishers of The Journal
of Gene Medicine. To search the database, click
on "Interactive Database" at the top of the page.
Access to charts, statistics, and abstracts from
clinical trials results also provided.

Professional Associations

American Society of Gene Therapy

Australasian Gene Therapy Society (AGTS)

European Society of Gene Therapy (ESGT)

Gene
Therapy
Journals
(Scientific, peer-reviewed publications targeted to
clinicians and researchers. Access to full-text articles in
these journals typically requires a subscription.)

Cancer Gene Therapy - From the publishers of

Nature.

Current Gene Therapy - From Bentham Science

Publishers.

Gene Therapy - From the publishers of Nature.

Human Gene Therapy - Journal published by Mary

Anne Liebert, Inc.

The Journal of Gene Medicine - Official journal of

the European Society of Gene Therapy (ESGT),
Japan Society of Gene Therapy (JSGT), and the
Australasian Gene Therapy Society (AGTS).

Molecular Therapy - A monthly journal published

135

by the American Society of Gene Therapy

(ASGT).
Other Publications

Vector - Magazine of the Gene Therapy Center at

the University of Alabama at Birmingham. Issues
available for download as PDF.

Send the url of this page to a friend

To read pdf files, download the free Acrobat Reader software.
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Lai phn t axit nucleic c dng chn on cc trng hp nhim khun m nhng
phng php kinh in khng th pht hin c gen gy bnh .
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mi , l u d axit nucleic .
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c xc nh th cDNA c th s dng sng lc th vin gen v tch chnh on trnh t
gen .Tc l cDNA c th to ra t qun th v em s dng , khng cn tch dng cc cDNA .
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biu hingen ny (mu d tr) . Bng cch lai ghp cc dng c th c xc nh thng qua
cc kiu lai ca chng vi cc mu d cng v tr .
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cc mu d d tng ng hoc xc nh cc dng khc c cha cc phn b sung ca gen
nghin cu .
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gip cho vic xc nh cc on trnh t gi ln nhau v sau nu ni cc mu li s c cc
mch DNA di c xc nh .

136

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protein c m bi gen ch . Dng m di truyn xc nh trnh t gen tng ng v to ra
oligonucleotit.
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hiu v vy cc gen cha tch dng vn c th xc nh trnh t thng qua trnh t ca cc on
peptit v c th thit k c cc mu d tng ng .
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ton chnh xc trnh t gen. Nhng khng phi l kh khn ln v ta c th s dng cc mu
d hn hp bao gm tt c cc trnh t c th c .
Mu d axit nucleic ang tr thnh mt cng c hu hiu trong nhiu lnh vc nghin cu v
ng dng cng ngh sinh hc . Mt ng dng thit thc l cho php chn on cc trng hp
nhim khun m khng pht hin c khng nguyn . V d , i vi cc axit nucleic ca gen
gy bnh nm ngoi NST hoc loi ho nhp , khng c biu hin r qua kiu hnh th
phng php lai phn t vn cho php chn on c .
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ni ring .

nhng ti liu tham kho chnh

1. Nguyn Vn Cch . Tin-Sinh hc . Nxb . KHKT, HN ,2005.
2. L nh Lng . Nguyn l k thut Di truyn . Nxb . KHKT, HN , 2001.
3. Phan C Nhn .Di truyn hc ng vt .Nxb. KHKT, HN, 2001.
4. Khut Hu Thanh . Liu php gen . Nxb. KHKT , HN , 2005 .
5. Bruce A . Chabner (Editor) ; Dan L . Longo (Editor) . Cancer Chemotherapy and Biotherapy
: Principles and Practice .
Lippincott Williams Wilkins Publisher , 1996.
6. I.Edward Alcamo . DNA Technology
the awasome skill . Wm. C. Brown Publisher.
Dubuque , IA Bogota Boston Buenos Aires Caracas Chicago Guilford , CT London
Madrid Mexico City Sedney Toronto , 1996.
7. Jackque B. Wallach . Interpretation of Diagnostic Test 7th Edition .
Lippincott Williams & Wilkin Publisher , 2000.
8. J. Caroline . Suicide gene therapy . Springer , 2004.
*David C. Dale (Editor) . Infectious Diseases : The Clinicans Guide to Diagnosis , Treatment
and Prevention . Webmd Scientific American Medicine by WebMD Professional Publishing ,
2003.
9. K Fran , Md . Austen (Editor) ; michael M., Md . frank (Editor) ; Havey I., Md .Canter
(Editor) ; John
P.,Md . Atkinson (Editor) ; Max Samter (Editor) . Samters Immunologic
Diseases 6th Edition .
Lippincott Williams & Wilkins Publishers , 2001.
10. Robert K . Murray ; Daryl K. Granner ; Peter A. Mayer ; Victor W. Rodwell . Harpers
Illustracted Biochemistry .
137

Lange Medical Book / Mc Graw Hill .

Medical Publisher Division
New York Chicago San Francisco London Madrid
San Juan Singapore Sedney Toronto , 2003.

Mexico City

Milan New Delhi

11. Gerhard Krauss . Biochemistry of Signal Transduction and Regulation .

Second Edition . Wiley VCH Verlag GmbH, 2001.
12. Robert L. Souhan (Editor) ; Ian Tannock (Editor) ; Petter Hohenberen (Editor) ; JeanClaude Horiot (Editor) . Oxford Textbook of Oncology .
Oxford Press , 2002.
13. Lehninger . Principle of Biochemistry .
Fourth Edition . 2005. Acrobat_60.
14. Mathews , Van holde , Ahern . Biochemistry.
Third Editon . Web site , 2004.
15.Robert M. Watcher (Editor) ; lee Golman (Editor) ; Harry Hollander (Editor) . Hospital
Medicine .
Lippincott Williams & Wilkin , 2000.
16. Bernard N. Fields ; David M. Knipe ; Teter M. Howley . Fundamental Virology .
Lippincott Williams & Wilkin .
Philadelphia Baltimore New York London Boenos Aires Hong kong Sydney
Tokyo , 1996.
17. Voet Pratt . Biochemistry . Web site , 2005.
18. Walter R. Wilson ; W. Laurence ; MD Drew ; Nancy K., Phd hery ; Merle A., MD Sande ;
David A., Md Relman ; James M.., MD Steckelberg ; Julie Louise ; MD Gerberding . Current
Diagnosis & Treatment Infectious Diseases 1th Editon .
Mc Graw-hill/ Appleton & Lange , 2001.
19. Prein Seth . Adenovirus : Basic Biology to Gene Therapy .
R . G . Lands Company , Austin , Texas USA , 1999 .
Mt s trang Web
http://www.nature.com/gt/journal/v12/n14/3302503a.html.
http://www.medicalnewstoday.com/medicalnews.php?newid=27707.
http://www.ncbi.nlm.nih.gov.
www.ebi.ac.uk/databases.
www.nig.ac.jp/section/service.html.
www.expassy.org.
www.atcc.org.
www.dsmz.de

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