United S ta tes Pa tent [ 1 9 ]

M u l l is et a 1 .
[ 5 4 ] PROCES S FOR AM PLIFYING, DETECTING,
AND/OR-CLONING NUCLEIC ACID
S EQUENCES
[ 7 5 ] Inv ento r s : K a r y B . M u l l is , K ens ing to n; H enr y
A. Er l ic h , Oa k l a nd; No r ma n
Ar nh eim, Wo o dl a nd H il l s ; Gl enn T.
H o r n, Emer y v il l e; Ra nda l l K . S a ik i,
Ric h mo nd; S tep h en J . S c h a r f ,
B er k el ey , a l l o f Ca l if .
[ 7 3 ] As s ig nee: Cetu s Co r p o r a tio n, Emer y v il l e, Ca l if .
[ * ] No tic e: Th e p o r tio n o f th e ter m o f th is p a tent
s u b s eq u ent to J u l . 2 8 , 2 0 0 4 h a s b een
dis c l a imed.
[ 2 1 ] Ap p l . No . : 8 2 8 ,1 4 4
[ 2 2 ] Fil ed: Feb . 7 , 1 9 8 6
Rel a ted US . Ap p l ic a tio n Da ta
[ 6 0 ] Co ntinu a tio n-imp a r t o f S er . No . 8 2 4 ,0 4 4 , J a n. 3 0 , 1 9 8 6 ,
a b a ndo ned, wh ic h is a div is io n o f S er . No . 7 9 1 ,3 0 8 ,
Oc t. 2 5 , 1 9 8 5 , wh ic h is a c o ntinu a tio n-in-p a r t o f S er .
No . 7 1 6 ,9 7 5 , M a r . 2 8 , 1 9 8 5 , a b a ndo ned.
[ 5 1 ] Int. Cl . 4 . . . . . . . . . . . . . . . . . . . . . . . . C1 2 Q 1 /6 8 ; C1 2 P 1 9 /3 4 ;
C1 2 N 1 /0 0 ; C1 2 N 1 5 /0 0 ; GOl N 3 3 /4 8 ; G0 1 N
3 3 /0 0 ; G0 1 N 3 3 /5 6 6 ; GOIN 3 3 /5 6 4 ; CO7 H
2 1 /0 2 ; CO7 H 2 1 /0 4
[ 5 2 ] US . Cl . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 3 5 /6 ; 4 3 5 /9 1 ;
4 3 5 /1 7 2 . 3 ; 4 3 5 /3 1 7 ; 4 3 6 /6 3 ; 4 3 6 /9 4 ; 4 3 6 /5 0 1 ;
4 3 6 /5 0 8 ; 5 3 6 /2 7 ; 5 3 6 /2 8 ; 5 3 6 /2 9 ; 9 3 5 /1 7 ;
9 3 5 /1 8 ; 9 3 5 /7 6 ; 9 3 5 /7 7 ; 9 3 5 /7 8
[ 5 8 ] Fiel d o f S ea r c h . . . . . . . . . . . . . . . . . . . . . . 4 3 5 /9 1 , 1 7 2 . 3 , 3 1 7 ,
4 3 5 /6 ; 5 3 6 /2 7 , 2 8 , 2 9 ; 9 3 5 /1 7 , 1 8 , 7 8 , 7 7 , 7 6 ;
4 3 6 /6 3 , 9 4 , 5 0 1 , 5 0 8
[ 5 6 ] Ref er enc es Cited
U. S . PATENT DOCUM ENTS v
4 ,3 9 5 ,4 8 6 7 /1 9 8 3 Wil s o n et a 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 3 5 /6
OTH ER PUB LICATIONS
Go r s k i et a l , M o l ec u l a r o r g a niz a tio n o f th e H LA-S B
Reg io n o f th e H u ma n M a j o r H is to c o mp a tib il ity Co m
4 ,6 8 3 ,1 9 5
* J u l . 2 8 , 1 9 8 7
[ 1 1 ] Pa tent Nu mb er :
[ 4 5 ] Da te o f Pa tent:
p l ex a nd Ev idenc e f o r Two S B B eta -c h a in Genes ,
Pr o c . Na tl . Ac a d. S c i. US A 8 1 : 3 9 3 4 ( 1 9 8 4 ) .
B o s s et a 1 , Cl o ning a nd S eq u enc e Ana l y s is o f th e
H u ma n M a j o r H is to c o mp a tib il ity Co mp l ex Gene DC
3 b eta , Pr o c . Na t. Ac a d. S c i. US A 8 1 : 5 1 9 9 ( 1 9 8 4 ) .
Ok a da et a l , Gene Or g a niz a tio n o f DC a nd DX S u b r e
g io ns o f th e H u ma n M a j o r H is to c o mp a tib il ity Co m
p l ex , Pr o c . Na tl . Ac a d. S c i. US A 8 2 : 3 4 1 0 ( 1 9 8 5 ) .
S a l s er , Cl o ning c DNA s eq u enc es : A g ener a l Tec h
niq u e f o r Pr o p a g a ting Eu k a r y o tic Gene S eq u enc es in
B a c ter ia l Cel l s , in Genetic Eng ineer ing , 1 9 7 8 , Ch a r
r a b a r ty ( ed. ) , CRC Pr es s , Inc . B o c a Ra to n, Fl a . , p p .
5 3 -8 1 .
Ga u b a tz et a 1 , S tr a teg ies f o r Co ns tr u c ting Co mp l e
menta r y DNA f o r Cl o ning , J . Th eo r . B io l . 9 5 : 6 7 9
( 1 9 8 2 ) .
Ro s s i et a l . , J . B io l . Ch em, 2 5 7 , 9 2 2 6 -9 2 2 9 ( 1 9 8 2 ) .
Pr ima r y Ex a miner -J a mes M a r tinel l
Atto r ney , Ag ent, o r Fir m-J a net E. H a s a k ; Al b er t P.
H a l l u in
[ 5 7 ] AB S TRACT
Th e p r es ent inv entio n is dir ec ted to a p r o c es s f o r a mp l i
f y ing a nd detec ting a ny ta r g et nu c l eic a c id s eq u enc e
c o nta ined in a nu c l eic a c id o r mix tu r e th er eo f . Th e
p r o c es s c o mp r is es tr ea ting s ep a r a te c o mp l ementa r y
s tr a nds o f th e nu c l eic a c id with a mo l a r ex c es s o f two
o l ig o nu c l eo tide p r imer s , ex tending th e p r imer s to f o r m
c o mp l ementa r y p r imer ex tens io n p r o du c ts wh ic h a c t a s
temp l a tes f o r s y nth es iz ing th e des ir ed nu c l eic a c id s e
q u enc e, a nd detec ting th e s eq u enc e s o a mp l i? ed. Th e
s tep s o f th e r ea c tio n ma y b e c a r r ied o u t s tep wis e o r
s imu l ta neo u s l y a nd c a n b e r ep ea ted a s o f ten a s des ir ed.
In a dditio n, a s p ec i? c nu c l eic a c id s eq u enc e ma y b e
c l o ned into a v ec to r b y u s ing p r imer s to a mp l if y th e
s eq u enc e, wh ic h c o nta in r es tr ic tio n s ites o n th eir no n
c o mp l ementa r y ends , a nd a nu c l eic a c id f r a g ment ma y
b e p r ep a r ed f r o m a n ex is ting s h o r ter f r a g ment u s ing th e
a mp l i? c a tio n p r o c es s .
2 6 Cl a ims , 1 2 Dr a wing Fig u r es
US . Pa tent J u l . 2 8 , 1 9 8 7 S h eet 1 0 f 1 2 4 ,6 8 3 ,1 9 5
FIG. 1
DQu b Ie-S tr a nded 9 4 -b p S eq u enc e
TTTGC TTCTGACACA ACTGTGTTCA CTAGCAACCT ->'
AAACG AAGACTGTGT TGACACAAGT GATCGTTGGA
CAAACAGACA J
GTTTGTCTGT
CTGCCGTTAC
GACGGCAATG
Nc o I H inf I M s tl I
V V V
CCATGG TGCA CC TGAC TCCT GAGGAGAAGT >
GGTACCACGT GGACTGAGGA CICCTCTTCA
A1 1 e] ic b a s e
p a ir DNA
p o l y mo r p h is m
TGCCC TGTG '
ACGGGACAC
US . Pa tent J u l . 2 8 , 1 9 8 7 S h eet2 0 f 1 2 4 ,6 8 3 ,1 9 5
b p
1 8 8
1 8 1
4 1
FIGZ
US . Pa tent J u l . 2 8 , 1 9 8 7 S h eet3 o f 1 2 4 ,6 8 3 ,1 9 5
8 1 8 8 1 1
1 4 1
7 8
6 2
4 8
4 5
FIG. 3
US . Pa tent J u l . 2 8 , 1 9 8 7 S h eet4 0 f 1 2 4 ,6 8 3 ,1 9 5
D
F u mh z u . u wna u ez o m
---->
NOUL ( Ua GQOFUFPUOTE.
. o
U. S . Pa tent J u l . 2 8 , 1 9 8 7 S h eet6 0 f 1 2 4 ,6 8 3 ,1 9 5
. . GOPOOFFU< < OPOOa EGOOO UFOFUUUOFU ( PFQUUOPUP
U
. . . .
. . . . .
POFUUUOPU ( FPOUUOFUP POPu u UOPu ( PFOQUOh UF
. . . . . . . . . . . . . .
. . . . _. . . . . . _. . .
. . _. . . . . _. . _ . . . . . . . .
. _. . . . . . . . . .
OFFUPUUFUC
. inILu z Z Z u l
o c h h o u h k u h OOFFOH Z EOF OOk POUb QOF
>
_. . . . .
" mu QGE
. . . . . .
. . . . .
T2 1 2 5 3 5 :
Oh h h " ELL.
Z
US . Pa tent J u l . 2 8 , 1 9 8 7 S h eet 7 o f 1 2 4 ,6 8 3 ,1 9 5
7 8
6 2
4 8
4 5
FIG. 5
US . Pa tent J u l . 2 8 , 1 9 8 7 S h eet8 0 f 1 2 4 ,6 8 3 ,1 9 5
F1 6 5
B A CATGGTGCACCTGACTCCTGAGGAGAAGTCTGCCGTTACTGCCCTGTGGGGCAAGGTGAA
GTACCACGTGGACTGAGGACTCCTCTTCAGACGGCAATGACGGGACACCCCGTTCCACTT
B S CATGGTGCACCTGACTCCTGTGGAGAAGTCTGCCGTTACTGCCCTGTGGGGCAAGGTGAA
GTACCACGTGGACTGAGGACACCTCTTCAGACGGCAATGACGGGACACCCCGTTCCACTT
* M a r k s th e mu ta tio n ( A to T) in th e s ic k l e c e] 1 g ene wh ic h dis r u p ts
th e DdeI s ite
US . Pa tent J u l . 2 8 , 1 9 8 7 S h eet9 0 f 1 2 4 ,6 8 3 ,1 9 5
GACTCCTGAC
. TGAGGACTC B A
dena tu r e
a nnea ] to p r o b e
* --GACTCCTGAG p r o b e
CTGAGGACTC
dig es t with DdeI
( s -mer ) * --GACTCC TGAG
CTGAGGACT C
dig es t with H inf I
* GACTCC
TGAG
CTGAGGACT C
* 1 's l a b el
U. S . Pa tent J u l . 2 8 , 1 9 8 7
S h eet 1 0 o f 1 2 4 ,6 8 3 ,1 9 5
GACTCCTGTC
* is 1 a b e1
CTGAGGACAC a 5
dena tu r e
a nnea l to p r o b e
A
* --GACTCCTG G---p r o b e
CTGAGGAC C
A
d1 g es t with DdeI
A
* GAC TCC TG G
' CTGAGGAC C
A
dig es t with H i nf I
A
( 3 -mer ) * -G ACTCCTG G----
CTGA GGAC C
A
U. S . Pa tent J u l . 2 8 , 1 9 8 7 S h eet 1 1 0 f 1 2 4 ,6 8 3 ,1 9 5
AB CD
8 -M ER
3 -M ER
U S . Pa tent 3 1 mm 1 %? S h em 1 1 2 @ f 1 1 2 4 9 8 3 J 9 5
PO
1 . . 1
4 ,6 8 3 ,1 9 5
1
PROCES S FOR AM PLIFYING, DETECTING,
AND/OR-CLONING NUCLEIC ACID S EQUENCES
B ACK GROUND OF TH E INVENTION
Th is a p p l ic a tio n is a c o ntinu a tio n-in-p a r t a p p l ic a tio n
o f c o p ending U. S . S er . No . 8 2 4 ,0 4 4 ? l ed J a n. 3 0 , 1 9 8 6 ,
no w a b a ndo ned, wh ic h is a div is io na l a p p l ic a tio n o f
c o p ending U. S . S er . No . 7 9 1 ,3 0 8 ? l ed Oc t. 2 5 , 1 9 8 5 ,
wh ic h is a c o ntinu a tio n-in-p a r t a p p l ic a tio n o f c o p ending
U. S . a p p l ic a tio n S er . No . 7 1 6 ,9 7 5 ? l ed M a r . 2 8 , 1 9 8 5 ,
no w a b a ndo ned.
FIELD OF TH E INVENTION
Th e p r es ent inv entio n r el a tes to a p r o c es s f o r a mp l i
f y ing ex is ting nu c l eic a c id s eq u enc es if th ey a r e p r es ent
in a tes t s a mp l e a nd detec ting th em if p r es ent b y u s ing a
p r o b e. M o r e s p ec i? c a l l y , it r el a tes to a p r o c es s f o r p r o
du c ing a ny p a r tic u l a r nu c l eic a c id s eq u enc e f r o m a
g iv en s eq u enc e o f DNA o r RNA in a mo u nts wh ic h a r e
l a r g e c o mp a r ed to th e a mo u nt initia l l y p r es ent s o a s to
f a c il ita te detec tio n o f th e s eq u enc es . Th e DNA o r RNA
ma y b e s ing l e- o r do u b l e-s tr a nded, a nd ma y b e a r el a
tiv el y p u r e s p ec ies o r a c o mp o nent o f a mix tu r e o f nu
c l eic a c ids . Th e p r o c es s o f th e inv entio n u til iz es a r ep et
itiv e r ea c tio n to a c c o mp l is h th e a mp l i? c a tio n o f th e
des ir ed nu c l eic a c id s eq u enc e.
DES CRIPTION OF RELATED DIS CLOS URES
Fo r dia g no s tic a p p l ic a tio ns in p a r tic u l a r , th e ta r g et
nu c l eic a c id s eq u enc e ma y b e o nl y a s ma l l p o r tio n o f th e
DNA o r RNA in q u es tio n, s o th a t it ma y b e dif ? c u l t to
detec t its p r es enc e u s ing no nis o to p ic a l l y l a b el ed o r
end-l a b el ed o l ig o nu c l eo tide p r o b es . M u c h ef f o r t is
b eing ex p ended in inc r ea s ing th e s ens itiv ity o f th e p r o b e
detec tio n s y s tems , b u t l ittl e r es ea r c h h a s b een c o n
du c ted o n a mp l if y ing th e ta r g et s eq u enc e s o th a t it is
p r es ent in q u a ntities s u f ? c ient to b e r ea dil y detec ta b l e
u s ing c u r r entl y a v a il a b l e meth o ds .
S ev er a l meth o ds h a v e b een des c r ib ed in th e l iter a tu r e
f o r th e s y nth es is o f nu c l eic a c ids de no v o o r f r o m a n
ex is ting s eq u enc e. Th es e meth o ds a r e c a p a b l e o f p r o
du c ing l a r g e a mo u nts o f a g iv en nu c l eic a c id o f c o m
p l etel y s p ec i? ed s eq u enc e.
One k no wn meth o d f o r s y nth es iz ing nu c l eic a c ids de
no v o inv o l v es th e o r g a nic s y nth es is o f a nu c l eic a c id
f r o m nu c l eo s ide der iv a tiv es . Th is s y nth es is ma y b e p er
f o r med in s o l u tio n o r o n a s o l id s u p p o r t. One ty p e o f
o r g a nic s y nth es is is th e p h o s p h o tr ies ter meth o d, wh ic h
h a s b een u til iz ed to p r ep a r e g ene f r a g ments o r s h o r t
g enes . In th e p h o s p h o tr ies ter meth o d, o l ig o nu c l eo tides
a r e p r ep a r ed wh ic h c a n th en b e j o ined to g eth er to f o r m
l o ng er nu c l eic a c ids . Fo r a des c r ip tio n o f th is meth o d,
s ee Na r a ng , S . A. , et a l . , M eth . Enz y mo l , 6 8 , 9 0 ( 1 9 7 9 )
a nd U. S . Pa t. No . 4 ,3 5 6 ,2 7 0 . Th e p a tent des c r ib es th e
s y nth es is a nd c l o ning o f th e s o ma to s ta tin g ene.
A s ec o nd ty p e o f o r g a nic s y nth es is is th e p h o s p h o di
es ter meth o d, wh ic h h a s b een u til iz ed to p r ep a r e a
tRNA g ene. S ee B r o wn, E. L. , et a l . , M eth . Enz y mo L,
6 8 , 1 0 9 ( 1 9 7 9 ) f o r a des c r ip tio n o f th is meth o d. As in th e
p h o s p h o tr ies ter meth o d, th e p h o s p h o dies ter meth o d
inv o l v es s y nth es is o f o l ig o nu c l eo tides wh ic h a r e s u b s e
q u entl y j o ined to g eth er to f o r m th e des ir ed nu c l eic
a c id.
Al th o u g h th e a b o v e p r o c es s es f o r de no v o s y nth es is
ma y b e u til iz ed to s y nth es iz e l o ng s tr a nds o f nu c l eic
a c id, th ey a r e no t v er y p r a c tic a l to u s e f o r th e s y nth es is
o f l a r g e a mo u nts o f a nu c l eic a c id. B o th p r o c es s es a r e
4 0
4 5
6 5
2
l a b o r io u s a nd time-c o ns u ming , r eq u ir e ex p ens iv e eq u ip
ment a nd r ea g ents , a nd h a v e a l o w o v er a l l ef ? c ienc y .
Th e l o w o v er a l l ef ? c ienc y ma y b e c a u s ed b y th e inef ?
c ienc ies o f th e s y nth es is o f th e o l ig o nu c l eo tides a nd o f
th e j o ining r ea c tio ns . In th e s y nth es is o f a l o ng nu c l eic
a c id, o r ev en in th e s y nth es is o f a l a r g e a mo u nt o f a
s h o r ter nu c l eic a c id, ma ny o l ig o nu c l eo tides wo u l d need
to b e s y nth es iz ed a nd ma ny j o ining r ea c tio ns wo u l d b e
r eq u ir ed. Co ns eq u entl y , th es e meth o ds wo u l d no t b e
p r a c tic a l f o r s y nth es iz ing l a r g e a mo u nts o f a ny des ir ed
nu c l eic a c id.
M eth o ds a l s o ex is t f o r p r o du c ing nu c l eic a c ids in
l a r g e a mo u nts f r o m s ma l l a mo u nts o f th e initia l ex is ting
nu c l eic a c id. Th es e meth o ds inv o l v e th e c l o ning o f a
nu c l eic a c id in th e a p p r o p r ia te h o s t s y s tem, wh er e th e
des ir ed nu c l eic a c id is ins er ted into a n a p p r o p r ia te v ec
to r wh ic h is u s ed to tr a ns f o r m th e h o s t. Wh en th e h o s t
is c u l tu r ed th e v ec to r is r ep l ic a ted, a nd h enc e mo r e
c o p ies o f th e des ir ed nu c l eic a c id a r e p r o du c ed. Fo r a
b r ief des c r ip tio n o f s u b c l o ning nu c l eic a c id f r a g ments ,
s ee M a nia tis , T. , et a l . , M o l ec u l a r Cl o ning : A La b o r a to r y
M a nu a l , Co l d S p r ing H a r b o r La b o r a to r y , p p . 3 9 0 -4 0 1
( 1 9 8 2 ) . S ee a l s o th e tec h niq u es des c r ib ed in U. S . Pa t.
No s . 4 ,4 1 6 ,9 8 8 a nd 4 ,4 0 3 ,0 3 6 .
A th ir d meth o d f o r s y nth es iz ing nu c l eic a c ids , de
s c r ib ed in U. S . Pa t. No . 4 ,2 9 3 ,6 5 2 , is a h y b r id o f th e
a b o v e-des c r ib ed o r g a nic s y nth es is a nd mo l ec u l a r c l o n
ing meth o ds . In th is p r o c es s , th e a p p r o p r ia te nu mb er o f
o l ig o nu c l eo tides to ma k e u p th e des ir ed nu c l eic a c id
s eq u enc e is o r g a nic a l l y s y nth es iz ed a nd ins er ted s eq u en
tia l l y into a v ec to r wh ic h is a mp l i? ed b y g r o wth p r io r
to ea c h s u c c eeding ins er tio n.
Th e p r es ent inv entio n b ea r s s o me s imil a r ity to th e
mo l ec u l a r c l o ning meth o d; h o wev er , it do es no t inv o l v e
th e p r o p a g a tio n o f a ny o r g a nis m a nd th er eb y a v o ids th e
p o s s ib l e h a z a r ds o r inc o nv enienc e wh ic h th is enta il s .
Th e p r es ent inv entio n a l s o do es no t r eq u ir e s y nth es is o f
nu c l eic a c id s eq u enc es u nr el a ted to th e des ir ed s e
q u enc e, a nd th er eb y th e p r es ent inv entio n o b v ia tes th e
need f o r ex tens iv e p u r i? c a tio n o f th e p r o du c t f r o m a
c o mp l ic a ted b io l o g ic a l mix tu r e.
S UM M ARY OF TH E INVENTION
Th e p r es ent inv entio n r es ides in a p r o c es s f o r a mp l i
f y ing o ne o r mo r e s p ec i? c nu c l eic a c id s eq u enc es p r es
ent in a nu c l eic a c id o r mix tu r e th er eo f u s ing p r imer s
a nd a g ents f o r p o l y mer iz a tio n a nd th en detec ting th e
a mp l i? ed s eq u enc e. Th e ex tens io n p r o du c t o f o ne
p r imer wh en h y b r idiz ed to th e o th er b ec o mes a tem
p l a te f o r th e p r o du c tio n o f th e des ir ed s p ec i? c nu c l eic
a c id s eq u enc e, a nd v ic e v er s a , a nd th e p r o c es s is r e
p ea ted a s o f ten a s is nec es s a r y to p r o du c e th e des ir ed
a mo u nt o f th e s eq u enc e. Th is meth o d is ex p ec ted to b e
mo r e ef ? c ient th a n th e meth o ds des c r ib ed a b o v e f o r
p r o du c ing l a r g e a mo u nts o f nu c l eic a c id f r o m a ta r g et
s eq u enc e a nd to p r o du c e s u c h nu c l eic a c id in a c o mp a r
a tiv el y s h o r t p er io d o f time. Th e p r es ent meth o d is
es p ec ia l l y u s ef u l f o r a mp l if y ing r a r e s p ec ies o f nu c l eic
a c id p r es ent in a mix tu r e o f nu c l eic a c ids f o r ef f ec tiv e
detec tio n o f s u c h s p ec ies .
M o r e s p ec i? c a l l y , th e p r es ent inv entio n p r o v ides a
p r o c es s f o r detec ting th e p r es enc e o r a b s enc e o f a t l ea s t
o ne s p ec i? c nu c l eic a c id s eq u enc e in a s a mp l e c o nta in
ing a nu c l eic a c id o r mix tu r e o f nu c l eic a c ids , o r dis tin
g u is h ing b etween two dif f er ent f o r ms o f s eq u enc es in
s a id s a mp l e, wh er ein th e s a mp l e is s u s p ec ted o f c o nta in
4 ,6 8 3 ,1 9 5
3
ing s a id s eq u enc e o r s eq u enc es , wh ic h p r o c es s c o m
p r is es :
( a ) tr ea ting th e s a mp l e with o ne o l ig o nu c l eo tide
p r imer f o r ea c h s tr a nd o f ea c h dif f er ent s p ec i? c s e
q u enc e s u s p ec ted o f b eing p r es ent in th e s a mp l e, u nder
h y b r idiz ing c o nditio ns s u c h th a t f o r ea c h s tr a nd o f ea c h
dif f er ent s eq u enc e to b e detec ted a n ex tens io n p r o du c t
o f ea c h p r imer is s y nth es iz ed wh ic h is c o mp l ementa r y
to ea c h nu c l eic a c id s tr a nd, wh er ein s a id p r imer o r
p r imer s a r e s el ec ted s o a s to b e s u b s ta ntia l l y c o mp l e
menta r y to ea c h s tr a nd o f ea c h s p ec i? c s eq u enc e s u c h
th a t th e ex tens io n p r o du c t s y nth es iz ed f r o m o ne p r imer ,
wh en it is s ep a r a ted f r o m its c o mp l ement, c a n s er v e a s
a temp l a te f o r s y nth es is o f th e ex tens io n p r o du c t o f th e
o th er p r imer ;
( b ) tr ea ting th e s a mp l e u nder dena tu r ing c o nditio ns to
s ep a r a te th e p r imer ex tens io n p r o du c ts f r o m th eir tem
p l a tes if th e s eq u enc e o r s eq u enc es to b e detec ted a r e
p r es ent;
( c ) tr ea ting th e s a mp l e with o l ig o nu c l eo tides p r imer s
s u c h th a t a p r imer ex tens io n p r o du c t is s y nth es iz ed
u s ing ea c h o f th e s ing l e s tr a nds p r o du c ed in s tep ( b ) a s
a temp l a te, r es u l ting in a mp l i? c a tio n o f th e s p ec i? c
nu c l eic a c id s eq u enc e o r s eq u enc es if p r es ent;
( d) a dding to th e p r o du c t o f s tep ( c ) a l a b el ed p r o b e
c a p a b l e o f h y b r idiz ing to s a id s eq u enc e b eing detec ted
o r a mu ta tio n th er eo f ; a nd
( e) deter mining wh eth er s a id h y b r idiz a tio n h a s o c
c u r r ed.
Th e s tep s ( a ) ( c ) ma y b e c o ndu c ted s eq u entia l l y o r
s imu l ta neo u s l y . In a dditio n, s tep s ( b ) a nd ( c ) ma y b e
r ep ea ted u ntil th e des ir ed l ev el o f s eq u enc e a mp l i? c a
tio n is o b ta ined.
In o th er emb o diments th e inv entio n r el a tes to dia g
no s tic k its f o r th e detec tio n o f a t l ea s t o ne s p ec i? c nu
c l eic a c id s eq u enc e in a s a mp l e c o nta ining o ne o r mo r e
nu c l eic a c ids a t l ea s t o ne o f wh ic h nu c l eic a c id is s u s
p ec ted o f c o nta ining s a id s eq u enc e, wh ic h k it c o m
p r is es , in p a c k a g ed f o r m, a mu l tic o nta iner u nit h a v ing
( a ) o ne c o nta iner f o r ea c h o l ig o nu c l eo tide p r imer f o r
ea c h s tr a nd o f ea c h dif f er ent s eq u enc e to b e detec ted,
wh ic h p r imer o r p r imer s a r e s u b s ta ntia l l y c o mp l emen
ta r y to ea c h s tr a nd o f ea c h s p ec i? c nu c l eic a c id s e
q u enc e s u c h th a t a n ex tens io n p r o du c t s y nth es iz ed f r o m
o ne p r imer , wh en it is s ep a r a ted f r o m its c o mp l ement,
c a n s er v e a s a temp l a te f o r th e s y nth es is o f th e ex tens io n
p r o du c t o f th e o th er p r imer ;
( b ) a c o nta iner c o nta ining a n a g ent f o r p o l y mer iz a
tio n;
( 0 ) a c o nta iner f o r ea c h o f f o u r dif f er ent nu c l eo s ide
tr ip h o s p h a tes ;
( d) a c o nta iner c o nta ining a p r o b e c a p a b l e o f detec t
ing th e p r es enc e o f s a id s eq u enc e in s a id s a mp l e; a nd
( e) a c o nta iner c o nta ining mea ns f o r detec ting h y
b r ids o f s a id p r o b e a nd s a id s eq u enc e.
In y et a no th er emb o diment, th e inv entio n r el a tes to a
p r o c es s f o r c l o ning into a v ec to r a s p ec i? c nu c l eic a c id
s eq u enc e c o nta ined in a nu c l eic a c id o r a mix tu r e o f
nu c l eic a c ids , wh ic h p r o c es s c o mp r is es :
( a ) tr ea ting th e nu c l eic a c id( s ) with o ne o l ig o nu c l eo
tide p r imer f o r ea c h s tr a nd o f ea c h dif f er ent s p ec i? c
s eq u enc e b eing a mp l i? ed, u nder c o nditio ns s u c h th a t
f o r ea c h s tr a nd o f ea c h dif f er ent s eq u enc e b eing a mp l i
? ed a n ex tens io n p r o du c t o f ea c h p r imer is s y nth es iz ed
wh ic h is c o mp l ementa r y to ea c h nu c l eic a c id s tr a nd,
wh er ein s a id p r imer o r p r imer s a r e s el ec ted s o a s to b e
s u b s ta ntia l l y c o mp l ementa r y to ea c h s tr a nd o f ea c h
s p ec i? c s eq u enc e s u c h th a t th e ex tens io n p r o du c t s y n
4
th es iz ed f r o m o ne p r imer , wh en it is s ep a r a ted f r o m its
c o mp l ement, c a n s er v e a s a temp l a te f o r s y nth es is o f th e
ex tens io n p r o du c t o f th e o th er p r imer , a nd wh er ein s a id
p r imer o r p r imer s ea c h c o nta in a r es tr ic tio n s ite o n its 5
end wh ic h is th e s a me a s o r dif f er ent f r o m th e r es tr ic
_ tio n s ite( s ) o n th e o th er p r imer ( s ) ;
0
2 0
2 5
4 0
5 0
5 5
6 0
( b ) s ep a r a ting th e p r imer ex tens io n p r o du c ts f r o m th e
temp l a tes o n wh ic h th ey wer e s y nth es iz ed to p r o du c e
s ing l e-s tr a nded mo l ec u l es ;
( 0 ) tr ea ting th es ing l e-s tr a nded mo l ec u l es g ener a ted
f r o m s tep ( b ) with o l ig o nu c l eo tide p r imer s s u c h th a t a
p r imer ex tens io n p r o du c t is s y nth es iz ed u s ing ea c h o f
th e s ing l e s tr a nds p r o du c ed in s tep ( b ) a s a temp l a te,
wh er ein dep ending o n th e p a r tic u l a r s eq u enc e b eing
a mp l i? ed, s tep s ( a ) a nd ( c ) a r e c a r r ied o u t in th e p r es
enc e o f f r o m 0 u p to a n ef f ec tiv e a mo u nt o f dimeth y l
s u l f o x ide o r a t a temp er a tu r e o f u p to a b o u t 4 5 C. ;
( d) a dding to th e p r o du c t o f s tep ( c ) a r es tr ic tio n
enz y me f o r ea c h o f s a id r es tr ic tio n s ites to o b ta in
c l ea v ed p r o du c ts in a r es tr ic tio n dig es t; a nd
( e) l ig a ting th e c l ea v ed p r o du c t( s ) into o ne o r mo r e
c l o ning v ec to r s .
In y et a no th er emb o diment, th e inv entio n h er ein
r el a tes to a p r o c es s f o r s y nth es iz ing a nu c l eic a c id f r a g
ment f r o m a n ex is ting nu c l eic a c id f r a g ment h a v ing
f ewer nu c l eo tides th a n th e f r a g ment b eing s y nth es iz ed
a nd two o l ig o nu c l eo tide p r imer s , wh er ein th e nu c l eic
a c id b eing s y nth es iz ed is c o mp r is ed o f a l ef t s eg ment, a
c o r e s eg ment a nd a r ig h t s eg ment, a nd wh er ein th e c o r e
s eg ment r ep r es ents a t l ea s t s u b s ta ntia l l y th e nu c l eo tide
s eq u enc e o f s a id ex is ting nu c l eic a c id f r a g ment, a nd th e
r ig h t a nd l ef t s eg ments r ep r es ent th e s eq u enc e nu c l eo
tide p r es ent in th e 5 ends o f th e two p r imer s , th e 3 ' ends
o f wh ic h a r e c o mp l ementa r y o r s u b s ta ntia l l y c o mp l e
menta r y to th e 3 ' ends o f th e s ing l e s tr a nds p r o du c ed b y
s ep a r a ting th e s tr a nds o f s a id ex is ting nu c l eic a c id f r a g
ment, wh ic h p r o c es s c o mp r is es :
( a ) tr ea ting th e s tr a nds o f s a id ex is ting f r a g ment with
two o l ig o nu c l eo tide p r imer s u nder c o nditio n s u c h th a t
a n ex tens io n p r o du c t o f ea c h p r imer is s y nth es iz ed
wh ic h is c o mp l ementa r y to ea c h nu c l eic a c id s tr a nd,
wh er ein s a id p r imer s a r e s el ec ted s o a s to b e s u b s ta n
tia l l y c o mp l ementa r y to th e 3 end o f ea c h s tr a nd o f s a id
ex is ting f r a g ment s u c h th a t th e ex tens io n p r o du c t s y n
th es iz ed f r o m o ne p r imer , wh en it is s ep a r a ted f r o m its
c o mp l ement, c a n s er v e a s a temp l a te f o r s y nth es is o f th e
ex tens io n p r o du c t o f th e o th er p r imer , a nd wh er ein
ea c h p r imer c o nta ins , a t its 5 end, a s eq u enc e o f nu c l eo
tides wh ic h a r e no t c o mp l ementa r y to s a id ex is ting
f r a g ment a nd wh ic h c o r r es p o nd to th e two ends o f th e
nu c l eic a c id f r a g ment b eing s y nth es iz ed;
( b ) s ep a r a ting th e p r imer ex tens io n p r o du c ts f r o m th e
temp l a tes o n wh ic h th ey wer e s y nth es iz ed to p r o du c e
s ing l e-s tr a nded mo l ec u l es ;
( c ) tr ea ting th e s ing l e-s tr a nded mo l ec u l es g ener a ted
f r o m s tep ( b ) with th e p r imer s o f s tep ( a ) u nder c o ndi
tio ns s u c h th a t a p r imer ex tens io n p r o du c t is s y nth es iz ed
u s ing ea c h o f th e s ing l e s tr a nds p r o du c ed in s tep ( b ) a s
a temp l a te s o a s to p r o du c e two inter media te do u b l e
s tr a nded nu c l eic a c id mo l ec u l es , into ea c h o f wh ic h h a s
b een inc o r p o r a ted th e nu c l eo tide s eq u enc e p r es ent in
th e 5 ' end o f o ne o f th e o l ig o nu c l eo tide p r imer s , a nd
two f u l l -l eng th do u b l e-s tr a nded nu c l eic a c id mo l ec u l es ,
into ea c h o f wh ic h h a s b een inc o r p o r a ted th e nu c l eo tide
s eq u enc e p r es ent in th e 5 ends o f b o th o f th e o l ig o nu
c l eo tide p r imer s ;
4 ,6 8 3 ,1 9 5
5
( d) r ep ea ting s tep s ( b ) a nd ( c ) f o r a s u f ? c ient nu mb er
o f times to p r o du c e th e f u l l -l eng th do u b l e-s tr a nded
mo l ec u l e in a n ef f ec tiv e a mo u nt;
( e) tr ea ting th e s tr a nds o f th e p r o du c t o f s tep ( d) with
two p r imer s s o a s to l eng th en th e p r o du c t o f s tep ( d) o n
b o th ends ; a nd
( D r ep ea ting s tep s ( a ) -( d) u s ing th e p r o du c t o f s tep ( d)
a s th e c o r e f r a g ment a nd two o l ig o nu c l eo tide p r imer s
wh ic h a r e c o mp l ementa r y o r s u b s ta ntia l l y c o mp l emen
ta r y to th e 3 ends o f th e s ing l e s tr a nds p r o du c ed b y
s ep a r a ting th e s tr a nds o f th e p r o du c t o f s tep ( d) .
Th e c o r e f r a g ment ma y b e o b ta ined b y th e s tep s
c o mp r is ing :
( a ) r ea c ting two o l ig o nu c l eo tides , ea c h o f wh ic h c o n
ta in a t th eir 3 ' ends a nu c l eo tide s eq u enc e wh ic h is
c o mp l ementa r y to th e o th er o l ig o nu c l eo tide a t its 3
end, a nd wh ic h a r e no n-c o mp l ementa r y to ea c h o th er a t
th eir 5 ' ends , with a n a g ent f o r p o l y mer iz a tio n a nd f o u r
nu c l eo s ide tr ip h o s p h a tes u nder c o nditio ns s u c h th a t a n
ex tens io n p r o du c t o f ea c h o l ig o nu c l eo tide is s y nth e
s iz ed wh ic h is c o mp l ementa r y to ea c h nu c l eic a c id
s tr a nd;
( b ) s ep a r a ting th e ex tens io n p r o du c ts f r o m th e tem
p l a tes o n wh ic h th ey wer e s y nth es iz ed to p r o du c e s in
g l e-s tr a nded mo l ec u l es ; a nd
( c ) tr ea ting th e s ing l e-s tr a nded mo l ec u l es g ener a ted
f r o m s tep ( b ) with th e o l ig o nu c l eo tides o f s tep ( a ) u nder
c o nditio ns s u c h th a t a p r imer ex tens io n p r o du c t is s y n
th es iz ed u s ing ea c h o f th e s ing l e s tr a nds p r o du c ed in
s tep ( b ) a s a temp l a te, r es u l ting in a mp l i? c a tio n o f th e
c o r e f r a g ment.
B RIEF DES CRIPTION OF TH E DRAWINGS
FIG. 1 il l u s tr a tes a 9 4 b a s e p a ir l eng th s eq u enc e o f
h u ma n ,B -g l o b in des ir ed to b e a mp l i? ed. Th e s ing l e b a s e
p a ir c h a ng e wh ic h is a s s o c ia ted with s ic k l e c el l a nemia
is dep ic ted b enea th th e 9 4 -mer .
FIG. 2 il l u s tr a tes a p h o to g r a p h o f a n eth idiu m b r o
mide-s ta ined p o l y a c r y l a mide g el demo ns tr a ting a mp l i?
c a tio n o f th e 9 4 -mer c o nta ined in h u ma n wil d-ty p e
DNA a nd in a p l a s mid c o nta ining a 1 . 9 k b B a mI-II f r a g
ment o f th e no r ma l B -g l o b in g ene ( p B R3 2 8 : I-Ib A) .
FIG. 3 il l u s tr a tes a p h o to g r a p h o f a n eth idiu m b r o
mide-s ta ined p o l y a c r y l a mide g el demo ns tr a ting a mp l i?
c a tio n o f a ny o f th e s p ec i? c ta r g et 9 4 -mer s eq u enc e
p r es ent in p B R3 2 8 : H b A, a p l a s mid c o nta ining a 1 . 9 k b
B a mH I f r a g ment o f th e s ic k l e c el l a l l el e o f B -g l o b in
( p B R3 2 8 z H b S ) , p B R3 2 8 z H b A wh er e th e s eq u enc e to b e
a mp l i? ed is c l ea v ed with M s tII, a nd p B R3 2 8 z H b S
wh er e th e s eq u enc e to b e a mp l i? ed h a s b een tr ea ted b u t
no t c l ea v ed with M s tII.
FIG. 4 il l u s tr a tes in deta il th e s tep s a nd p r o du c ts o f
th e p o l y mer a s e c h a in r ea c tio n f o r a mp l i? c a tio n o f th e
des ir ed 9 4 -mer s eq u enc e o f h u ma n ,B -g l o b in f o r th r ee
c y c l es u s ing two o l ig o nu c l eo tides p r imer s .
FIG. 5 r ep r es ents a p h o to g r a p h o f a n eth idiu m b r o
mide-s ta ined p o l y a c r y l a mide g el demo ns tr a ting a mp l i?
c a tio n a f ter f o u r c y c l es o f a 2 4 0 -mer s eq u enc e in
p B R3 2 8 z H b A, wh er e th e a l iq u o ts a r e dig es ted with
Nc o I ( La ne 3 ) , M s tII ( La ne 4 ) o r H inf l ( La ne 5 ) . La ne
1 is th e mo l ec u l a r weig h t s ta nda r d a nd La ne 2 c o nta ins
th e inta c t 2 4 0 -b p p r o du c t.
FIG. 6 il l u s tr a tes th e s eq u enc e o f th e no r ma l ( 1 3 " ) a nd
s ic k l e c el l ( B 5 ) B -g l o b in g enes in th e r eg io n o f th e Ddel
a nd H inf l r es tr ic tio n s ites , wh er e th e s ing l e l ines f o r B A
ma r k th e p o s itio n o f th e DdeI s ite ( CTGAG) a nd th e
do u b l e b a r s f o r B A a nd B S ma r k th e p o s itio n o f th e H inf l
s ite ( GACTC) .
2 5
3 0
3 5
4 5
5 0
5 5
6 0
6 5
6
FIG. 7 il l u s tr a tes th e r es u l ts o f s eq u entia l dig es tio n o f
no r ma l B -g l o b in u s ing a 4 0 -mer p r o b e a nd Ddel f o l
l o wed b y I-Iinf I r es tr ic tio n enz y mes .
FIG. 8 il l u s tr a tes th e r es u l ts o f s eq u entia l dig es tio n o f
s ic k l e ,B -g l o b in u s ing th e s a me 4 0 -mer p r o b e a s in FIG.
7 a nd Ddel f o l l o wed b y H inf l r es tr ic tio n enz y mes .
FIG. 9 il l u s tr a tes a p h o to g r a p h o f a n eth idiu m b r o
mide-s ta ined p o l y a c r y l a mide g el demo ns tr a ting th e u s e
o f th e s a me 4 0 -mer p r o b e a s in FIG. 7 to s p ec i? c a l l y
c h a r a c ter iz e th e b eta -g l o b in a l l el es p r es ent in s a mp l es o f
wh o l e h u ma n DNA wh ic h h a v e b een s u b j ec ted to a m
p l i? c a tio n, h y b r idiz a tio n with th e p r o b e, a nd s eq u entia l
dig es tio n with Ddel a nd H inf l .
FIG. 1 0 il l u s tr a tes a p h o to g r a p h o f a 6 % Nu S iev e
a g a r o s e g el v is u a l iz ed u s ing eth idiu m b r o mide a nd UV
l ig h t. Th is p h o to g r a p h demo ns tr a tes a mp l i? c a tio n o f a
s u b -f r a g ment o f a l l O-b p a mp l i? c a tio n p r o du c t wh ic h
s u b -f r a g ment is a n inner nes ted s et with in th e 1 1 0 -b p
f r a g ment.
DETAILED DES CRIPTION OF TH E
PREFERRED EM B ODIM ENTS
Th e ter m o l ig o nu c l eo tide a s u s ed h er ein in r ef er
r ing to p r imer s , p r o b es , o l ig o mer f r a g ments to b e de
tec ted, o l ig o mer c o ntr o l s a nd u nl a b el ed b l o c k ing o l ig o
mer s is de? ned a s a mo l ec u l e c o mp r is ed o f two o r mo r e
deo x y r ib o nu c l eo tides o r r ib o nu c l eo tides , p r ef er a b l y
mo r e th a n th r ee. Its ex a c t s iz e wil l dep end o n ma ny
f a c to r s , wh ic h in tu r n dep end o n th e u l tima te f u nc tio n
o r u s e o f th e o l ig o nu c l eo tide.
Th e ter m p r imer a s u s ed h er ein r ef er s to a n o l ig o
nu c l eo tide wh eth er o c c u r r ing na tu r a l l y a s in-a p u r i? ed
r es tr ic tio n dig es t o r p r o du c ed s y nth etic a l l y , wh ic h is
c a p a b l e o f a c ting a s a p o int o f initia tio n o f s y nth es is
wh en p l a c ed u nder c o nditio ns in wh ic h s y nth es is o f a
p r imer ex tens io n p r o du c t wh ic h is c o mp l ementa r y to a
nu c l eic a c id s tr a nd is indu c ed, i. e. , in th e p r es enc e o f
nu c l eo tides a nd a n a g ent f o r p o l y mer iz a tio n s u c h a s
DNA p o l y mer a s e a nd a t a s u ita b l e temp er a tu r e a nd p H .
Th e p r imer is p r ef er a b l y s ing l e s tr a nded f o r ma x imu m
ef ? c ienc y in a mp l i? c a tio n, b u t ma y a l ter na tiv el y b e
do u b l e s tr a nded. If do u b l e s tr a nded, th e p r imer is ? r s t
tr ea ted to s ep a r a te its s tr a nds b ef o r e b eing u s ed to p r e
p a r e ex tens io n p r o du c ts . Pr ef er a b l y , th e p r imer is a n
o l ig o deo x y r ib o nu c l eo tide. Th e p r imer mu s t b e s u f ?
c ientl y l o ng to p r ime th e s y nth es is o f ex tens io n p r o du c ts
in th e p r es enc e o f th e a g ent f o r p o l y mer iz a tio n. Th e
ex a c t l eng th s o f th e p r imer s wil l dep end o n ma ny f a c
to r s , inc l u ding temp er a tu r e a nd s o u r c e o f p r imer . Fo r
ex a mp l e, dep ending o n th e c o mp l ex ity o f th e ta r g et
s eq u enc e, th e o l ig o nu c l eo tide p r imer ty p ic a l l y c o nta ins
1 5 -2 5 o r mo r e nu c l eo tides , a l th o u g h it ma y c o nta in
f ewer nu c l eo tides . S h o r t p r imer mo l ec u l es g ener a l l y
r eq u ir e c o o l er temp er a tu r es to f o r m s u f ? c ientl y s ta b l e
h y b r id c o mp l ex es with temp l a te.
Th e p r imer s h er ein a r e s el ec ted to b e s u b s ta ntia l l y
c o mp l ementa r y to th e dif f er ent s tr a nds o f ea c h s p ec i? c
s eq u enc e to b e a mp l i? ed. Th is mea ns th a t th e p r imer s
mu s t b e s u f ? c ientl y c o mp l ementa r y to h y b r idiz e with
th eir r es p ec tiv e s tr a nds . Th er ef o r e, th e p r imer s eq u enc e
need no t r ef l ec t th e ex a c t s eq u enc e o f th e temp l a te. Fo r
ex a mp l e, a no n-c o mp l ementa r y nu c l eo tide f r a g ment
ma y b e a tta c h ed to th e 5 end o f th e p r imer , with th e
r ema inder o f th e p r imer s eq u enc e b eing c o mp l ementa r y
to th e s tr a nd. Al ter na tiv el y , no n-c o mp l ementa r y b a s es
o r l o ng er s eq u enc es c a n b e inter s p er s ed into th e p r imer ,
p r o v ided th a t th e p r imer s eq u enc e h a s s u f f ic ient c o m
p l ementa r ity with th e s eq u enc e o f th e s tr a nd to b e a m
4 ,6 8 3 ,1 9 5
7
p l i? ed to h y b r idiz e th er ewith a nd th er eb y f o r m a tem
p l a te f o r s y nth es is o f th e ex tens io n p r o du c t o f th e o th er
p r imer .
As u s ed h er ein, th e ter ms " r es tr ic tio n endo nu c l ea s es "
a nd " r es tr ic tio n enz y mes r ef er to b a c ter ia l enz y mes
ea c h o f wh ic h c u t do u b l e-s tr a nded DNA a t o r nea r a
s p ec i? c nu c l eo tide s eq u enc e.
As u s ed h er ein, th e ter m DNA p o l y mo r p h is m" r e
f er s to th e c o nditio n in wh ic h two o r mo r e dif f er ent
nu c l eo tide s eq u enc es c o ex is t in th e s a me inter b r eeding
p o p u l a tio n in a DNA s eq u enc e.
Th e ter m " r es tr ic tio n f r a g ment l eng th p o l y mo r
p h is m" ( RFLP ) r ef er s to th e dif f er enc es in DNA
nu c l eo tide s eq u enc es th a t a r e r a ndo ml y dis tr ib u ted
th r o u g h o u t th e entir e h u ma n g eno me a nd th a t p r o du c e
dif f er ent r es tr ic tio n endo nu c l ea s e p a tter ns .
Th e p r es ent inv entio n is dir ec ted to a p r o c es s f o r
a mp l if y ing a ny o ne o r mo r e des ir ed s p ec i? c nu c l eic
a c id s eq u enc es s u s p ec ted o f b eing in a nu c l eic a c id.
B ec a u s e l a r g e a mo u nts o f a s p ec i? c s eq u enc e ma y b e
p r o du c ed b y th is p r o c es s , th e p r es ent inv entio n ma y b e
u s ed f o r imp r o v ing th e ef f ic ienc y o f c l o ning DNA o r
mes s eng er RNA a nd f o r a mp l if y ing a ta r g et s eq u enc e to
f a c il ita te detec tio n th er eo f .
In g ener a l , th e p r es ent p r o c es s inv o l v es a c h a in r ea c
tio n f o r p r o du c ing , in ex p o nentia l q u a ntities r el a tiv e to
th e nu mb er o f r ea c tio n s tep s inv o l v ed, a t l ea s t o ne s p e
' c i? c nu c l eic a c id s eq u enc e g iv en ( a ) th a t th e ends o f th e
r eq u ir ed s eq u enc e a r e k no wn in s u f ? c ient deta il th a t
o l ig o nu c l eo tides c a n b e s y nth es iz ed wh ic h wil l h y b r id
iz e to th em, a nd ( b ) th a t a s ma l l a mo u nt o f th e s eq u enc e
is a v a il a b l e to initia te th e c h a in r ea c tio n. Th e p r o du c t o f
th e c h a in r ea c tio n wil l b e a dis c r ete nu c l eic a c id du p l ex
with ter mini c o r r es p o nding to th e ends o f th e s p ec i? c
p r imer s emp l o y ed.
Any s o u r c e o f nu c l eic a c id, in p u r i? ed o r no np u r i? ed
f o r m, c a n b e u til iz ed a s th e s ta r ting nu c l eic a c id o r
a c ids , p r o v ided it is s u s p ec ted o f c o nta ining th e s p ec i? c
nu c l eic a c id s eq u enc e des ir ed. Th u s , th e p r o c es s ma y
emp l o y , f o r ex a mp l e, DNA o r RNA, inc l u ding mes s en
. g er RNA, wh ic h DNA o r RNA ma y b e s ing l e s tr a nded
o r do u b l e s tr a nded. In a dditio n, a DNA-RNA h y b r id
wh ic h c o nta ins o ne s tr a nd o f ea c h ma y b e u til iz ed. A
mix tu r e o f a ny o f th es e nu c l eic a c ids ma y a l s o b e em
p l o y ed, o r th e nu c l eic a c ids p r o du c ed f r o m a p r ev io u s
a mp l i? c a tio n r ea c tio n h er ein u s ing th e s a me o r dif f er ent
p r imer s ma y b e s o u til iz ed. Th e s p ec i? c nu c l eic a c id
s eq u enc e to b e a mp l i? ed ma y b e o nl y a f r a c tio n o f a
l a r g er mo l ec u l e o r c a n b e p r es ent initia l l y a s a dis c r ete
mo l ec u l e, s o th a t th e s p ec i? c s eq u enc e c o ns titu tes th e
entir e nu c l eic a c id. It is no t nec es s a r y th a t th e s eq u enc e
to b e a mp l i? ed b e p r es ent initia l l y in a p u r e f o r m; it ma y
b e a mino r f r a c tio n o f a c o mp l ex mix tu r e, s u c h a s a
p o r tio n o f th e B -g l o b in g ene c o nta ined in wh o l e h u ma n
DNA o r a p o r tio n o f nu c l eic a c id s eq u enc e du e to a
p a r tic u l a r mic r o o r g a nis m wh ic h o r g a nis m mig h t c o ns ti
tu te o nl y a v er y mino r f r a c tio n o f a p a r tic u l a r b io l o g ic a l
s a mp l e. Th e s ta r ting nu c l eic a c id ma y c o nta in mo r e
th a n o ne des ir ed s p ec i? c nu c l eic a c id s eq u enc e wh ic h
ma y b e th e s a me o r dif f er ent. Th er ef o r e, th e p r es ent
p r o c es s is u s ef u l no t o nl y f o r p r o du c ing l a r g e a mo u nts
o f o ne s p ec i? c nu c l eic a c id s eq u enc e, b u t a l s o f o r a mp l i
f y ing s imu l ta neo u s l y mo r e th a n o ne dif f er ent s p ec i? c
nu c l eic a c id s eq u enc e l o c a ted o n th e s a me o r dif f er ent
nu c l eic a c id mo l ec u l es . _
Th e nu c l eic a c id o r a c ids ma y b e o b ta ined f r o m a ny
s o u r c e, f o r ex a mp l e, f r o m p l a s mids s u c h a s p B R3 2 2 ,
f r o m c l o ned DNA o r RNA, o r f r o m na tu r a l DNA o r
0
5
2 5
3 0
3 5
4 5
5 5
6 0
6 5
8
RNA f r o m a ny s o u r c e, inc l u ding b a c ter ia , y ea s t, v i
r u s es , a nd h ig h er o r g a nis ms s u c h a s p l a nts o r a nima l s .
DNA o r RNA ma y b e ex tr a c ted f r o m b l o o d, tis s u e
ma ter ia l s u c h a s c h o r io nic v il l i o r a mnio tic c el l s b y a
v a r iety o f tec h niq u es s u c h a s th a t des c r ib ed b y M a nia tis
et a l . , M o l ec u l a r Cl o ning : A La b o r a to r y M a nu a l , ( New
Yo r k : Co l d S p r ing H a r b o r La b o r a to r y , 1 9 8 2 ) , p p
2 8 0 -2 8 1 .
Any s p ec i? c nu c l eic a c id s eq u enc e c a n b e p r o du c ed
b y th e p r es ent p r o c es s . It is o nl y nec es s a r y th a t a s u f ?
c ient nu mb er o f b a s es a t b o th ends o f th e s eq u enc e b e
k no wn in s u f ? c ient deta il s o th a t two o l ig o nu c l eo tide
p r imer s c a n b e p r ep a r ed wh ic h wil l h y b r idiz e to dif f er
ent s tr a nds o f th e des ir ed s eq u enc e a nd a t r el a tiv e p o s i
tio ns a l o ng th e s eq u enc e s u c h th a t a n ex tens io n p r o du c t
s y nth es iz ed f r o m o ne p r imer , wh en it is s ep a r a ted f r o m
its temp l a te ( c o mp l ement) , c a n s er v e a s a temp l a te f o r
ex tens io n o f th e o th er p r imer into a nu c l eic a c id o f
de? ned l eng th . Th e g r ea ter th e k no wl edg e a b o u t th e
b a s es a t b o th ends o f th e s eq u enc e, th e g r ea ter c a n b e
th e s p ec i? c ity o f th e p r imer s f o r th e ta r g et nu c l eic a c id
s eq u enc e, a nd th u s th e g r ea ter th e ef ? c ienc y o f th e
p r o c es s . It wil l b e u nder s to o d th a t th e wo r d p r imer a s
u s ed h er eina f ter ma y r ef er to mo r e th a n o ne p r imer ,
p a r tic u l a r l y in th e c a s e wh er e th er e is s o me a mb ig u ity in
th e inf o r ma tio n r eg a r ding th e ter mina l s eq u enc e( s ) o f
th e f r a g ment to b e a mp l i? ed. Fo r ins ta nc e, in th e c a s e
wh er e a nu c l eic a c id s eq u enc e is inf er r ed f r o m p r o tein
s eq u enc e inf o r ma tio n a c o l l ec tio n o f p r imer s c o nta ining
s eq u enc es r ep r es enting a l l p o s s ib l e c o do n v a r ia tio ns
b a s ed o n deg ener a c y o f th e g enetic c o de wil l b e u s ed
f o r ea c h s tr a nd. One p r imer f r o m th is c o l l ec tio n wil l b e
h o mo l o g o u s with th e end o f th e des ir ed s eq u enc e to b e
a mp l i? ed.
Th e o l ig o nu c l eo tide p r imer s ma y b e p r ep a r ed u s ing
a ny s u ita b l e meth o d, s u c h a s , f o r ex a mp l e, th e p h o s p h o
tr ies ter a nd p h o s p h o dies ter meth o ds des c r ib ed a b o v e,
o r a u to ma ted emb o diments th er eo f . In o ne s u c h a u to
ma ted emb o diment dieth y l p h o s p h o r a midites a r e u s ed a s
s ta r ting ma ter ia l s a nd ma y b e s y nth es iz ed a s des c r ib ed
b y B ea u c a g e et a l . , Tetr a h edr o n Letter s ( 1 9 8 1 ) ,
2 2 : 1 8 5 9 -1 8 6 2 . One meth o d f o r s y nth es iz ing o l ig o nu c l e
o tides o n a mo di? ed s o l id s u p p o r t is des c r ib ed in US .
Pa t. No . 4 ,4 5 8 ,0 6 6 . It is a l s o p o s s ib l e to u s e a p r imer
wh ic h h a s b een is o l a ted f r o m a b io l o g ic a l s o u r c e ( s u c h
a s a r es tr ic tio n endo nu c l ea s e dig es t) .
Th e s p ec i? c nu c l eic a c id s eq u enc e is p r o du c ed b y
u s ing th e nu c l eic a c id c o nta ining th a t s eq u enc e a s a
temp l a te. If th e nu c l eic a c id c o nta ins two s tr a nds , it is
nec es s a r y to s ep a r a te th e s tr a nds o f th e nu c l eic a c id
b ef o r e it c a n b e u s ed a s th e temp l a te, eith er a s a s ep a r a te
s tep o r s imu l ta neo u s l y with th e s y nth es is o f th e p r imer
ex tens io n p r o du c ts . Th is s tr a nd s ep a r a tio n c a n b e a c
c o mp l is h ed b y a ny s u ita b l e dena tu r ing meth o d inc l u d
ing p h y s ic a l , c h emic a l o r enz y ma tic mea ns . One p h y s i
c a l meth o d o f s ep a r a ting th e s tr a nds o f th e nu c l eic a c id
inv o l v es h ea ting th e nu c l eic a c id u ntil it is c o mp l etel y
( >9 9 %) dena tu r ed. Ty p ic a l h ea t dena tu r a tio n ma y
inv o l v e temp er a tu r e r a ng ing f r o m a b o u t 8 0 to 1 0 5 C.
f o r times r a ng ing f r o m a b o u t 1 to 1 0 minu tes . S tr a nd
s ep a r a tio n ma y a l s o b e indu c ed b y a n enz y me f r o m th e
c l a s s o f enz y mes k no wn a s h el ic a s es o r th e enz y me
Rec A, wh ic h h a s h el ic a s e a c tiv ity a nd in th e p r es enc e
o f r ib o ATP is k no wn to dena tu r e DNA. Th e r ea c tio n
c o nditio ns s u ita b l e f o r s ep a r a ting th e s tr a nds o f nu c l eic
a c ids with h el ic a s es a r e des c r ib ed b y Co l d S p r ing H a r
b o r S y mp o s ia o n Qu a ntita tiv e B io l o g y , Vo l . XLIII
DNA: Rep l ic a tio n a nd Rec o mb ina tio n ( New Yo r k :
4 ,6 8 3 ,1 9 5
9
Co l d S p r ing H a r b o r La b o r a to r y , 1 9 7 8 ) , B . K u h n et 2 1 1 . ,
DNA l -l el ic a s es " , p p . 6 3 -6 7 . a nd tec h niq u es f o r u s ing
Rec A a r e r ev iewed in C. Ra dding , Ann. Rev . Genetic s .
1 6 : 4 0 5 -3 7 ( 1 9 8 2 ) .
If th e o r ig ina l nu c l eic a c id c o nta ining th e s eq u enc e to
b e a mp l i? ed is s ing l e s tr a nded, its c o mp l ement is s y n
th es iz ed b y a dding o ne o r two o l ig o nu c l eo tide p r imer s
th er eto . If a n a p p r o p r ia te s ing l e p r imer is a dded. a
p r imer ex tens io n p r o du c t is s y nth es iz ed in th e p r es enc e
o f th e p r imer , a n a g ent f o r p o l y mer iz a tio n a nd th e f o u r
nu c l eo tides des c r ib ed b el o w. Th e p r o du c t wil l b e p a r
tia l l y c o mp l ementa r y to th e s ing l e-s tr a nded nu c l eic a c id
a nd wil l h y b r idiz e with th e nu c l eic a c id s tr a nd to f o r m
a du p l ex o f u neq u a l l eng th s tr a nds th a t ma y th en b e
s ep a r a ted into s ing l e s tr a nds a s des c r ib ed a b o v e to p r o
du c e two s ing l e s ep a r a ted c o mp l ementa r y s tr a nds . Al
ter na tiv el y , two a p p r o p r ia te p r imer s ma y b e a dded to
th e s ing l e-s tr a nded nu c l eic a c id a nd th e r ea c tio n c a r r ied
o u t.
If th e o r ig ina l nu c l eic a c id c o ns titu tes th e s eq u enc e to
b e a mp l i? ed, th e p r imer ex tens io n p r o du c t( s ) p r o du c ed
wil l b e c o mp l etel y c o mp l ementa r y to th e s tr a nds o f th e
o r ig ina l nu c l eic a c id a nd wil l h y b r idiz e th er ewith to
f o r m a du p l ex o f eq u a l l eng th s tr a nds to b e s ep a r a ted
into s ing l e-s tr a nded mo l ec u l es .
Wh en th e c o mp l ementa r y s tr a nds o f th e nu c l eic a c id
o r a c ids a r e s ep a r a ted, wh eth er th e nu c l eic a c id wa s
o r ig ina l l y do u b l e o r s ing l e s tr a nded, th e s tr a nds a r e
r ea dy to b e u s ed a s a temp l a te f o r th e s y nth es is o f a ddi
tio na l nu c l eic a c id s tr a nds . Th is s y nth es is c a n b e p er
f o r med u s ing a ny s u ita b l e meth o d. Gener a l l y it o c c u r s
in a b u f f er ed a q u eo u s s o l u tio n, p r ef er a b l y a t a p H o f
7 -9 , mo s t p r ef er a b l y a b o u t 8 . Pr ef er a b l y , a mo l a r ex c es s
( f o r c l o ned nu c l eic a c id, u s u a l l y a b o u t 1 0 0 0 : 1 p r imer :
temp l a te, a nd f o r g eno mic nu c l eic a c id, u s u a l l y a b o u t
1 0 6 : l p r imer z temp l a te) o f th e two o l ig o nu c l eo tide p r im
er s is a dded to th e b u f f er c o nta ining th e s ep a r a ted tem
p l a te s tr a nds . It is u nder s to o d, h o wev er , th a t th e
a mo u nt o f c o mp l ementa r y s tr a nd ma y no t b e k no wn if
th e p r o c es s h er ein is u s ed f o r dia g no s tic a p p l ic a tio ns , s o
th a t th e a mo u nt o f p r imer r el a tiv e to th e a mo u nt o f
c o mp l ementa r y s tr a nd c a nno t b e deter mined with c er
ta inty . As a p r a c tic a l ma tter , h o wev er , th e a mo u nt o f
p r imer a dded wil l g ener a l l y b e in mo l a r ex c es s o v er th e
a mo u nt o f c o mp l ementa r y s tr a nd ( temp l a te) wh en th e
s eq u enc e to b e a mp l i? ed is c o nta ined in a mix tu r e o f
c o mp l ic a ted l o ng -c h a in nu c l eic a c id s tr a nds . A l a r g e
mo l a r ex c es s is p r ef er r ed to imp r o v e th e ef ? c ienc y o f
th e p r o c es s .
Th e deo x y r ib o nu c l eo s ide tr ip h o s p h a tes dATP,
dCTP, dGTP a nd TTP a r e a l s o a dded to th e s y nth es is
mix tu r e in a deq u a te a mo u nts a nd th e r es u l ting s o l u tio n
is h ea ted to a b o u t 9 0 -1 0 0 C. f o r f r o m a b o u t 1 to 1 0
minu tes , p r ef er a b l y f r o m 1 to 4 minu tes . Af ter th is h ea t
ing p er io d th e s o l u tio n is a l l o wed to c o o l to f r o m
2 0 -4 0 C. , wh ic h is p r ef er a b l e f o r th e p r imer h y b r idiz a
tio n. To th e c o o l ed mix tu r e is a dded a n a g ent f o r p o l y
mer iz a tio n, a nd th e r ea c tio n is a l l o wed to o c c u r u nder
c o nditio ns k no wn in th e a r t. Th is s y nth es is r ea c tio n
ma y o c c u r a t f r o m r o o m temp er a tu r e u p to a temp er a
tu r e a b o v e wh ic h th e a g ent f o r p o l y mer iz a tio n no
l o ng er f u nc tio ns ef ? c ientl y . Th u s , f o r ex a mp l e, if DNA
p o l y mer a s e is u s ed a s th e a g ent f o r p o l y mer iz a tio n, th e
temp er a tu r e is g ener a l l y no g r ea ter th a n a b o u t 4 5 C.
Pr ef er a b l y a n a mo u nt o f dimeth y l s u l f o x ide ( DM S O) is
p r es ent wh ic h is ef f ec tiv e in detec tio n o f th e s ig na l o r
th e temp er a tu r e is 3 5 4 0 C. M o s t p r ef er a b l y , 5 -1 0 %
b y v o l u me DM S O is p r es ent a nd th e temp er a tu r e is
5
2 5
4 0
4 5
5 0
6 5
1 0
3 5 -4 0 C. Fo r c er ta in a p p l ic a tio ns , wh er e th e s eq u en
c es to b e a mp l i? ed a r e o v er 1 1 0 b a s e p a ir f r a g ments ,
s u c h a s th e H LA DQ-a o r -B g enes , a n ef f ec tiv e a mo u nt
( e. g . , 1 0 % b y v o l u me) o f DM S O is a dded to th e a mp l i?
c a tio n mix tu r e, a nd th e r ea c tio n is c a r r ied a t 3 5 ~ 4 0 C. ,
to o b ta in detec ta b l e r es u l ts o r to ena b l e c l o ning .
Th e a g ent f o r p o l y mer iz a tio n ma y b e a ny c o mp o u nd
o r s y s tem wh ic h wil l f u nc tio n to a c c o mp l is h th e s y nth e
s is o f p r imer ex tens io n p r o du c ts , inc l u ding enz y mes .
S u ita b l e enz y mes f o r th is p u r p o s e inc l u de, f o r ex a mp l e,
E. c o l i DNA p o l y mer a s e I, K l eno w f r a g ment o f E. 0 0 1 1 '
DNA p o l y mer a s e I, T4 DNA p o l y mer a s e, o th er a v a il
a b l e DNA p o l y mer a s es , r ev er s e tr a ns c r ip ta s e, a nd o th er
enz y mes , inc l u ding h ea ts ta b l e enz y mes , wh ic h wil l f a
c il ita te c o mb ina tio n o f th e nu c l eo tides in th e p r o p er
ma nner to f o r m th e p r imer ex tens io n p r o du c ts wh ic h
a r e c o mp l ementa r y to ea c h nu c l eic a c id s tr a nd. Gener
a l l y , th e s y nth es is wil l b e initia ted a t th e 3 ' end o f ea c h
p r imer a nd p r o c eed in th e 5 ' dir ec tio n a l o ng th e tem
p l a te s tr a nd, u ntil s y nth es is ter mina tes , p r o du c ing mo l e
c u l es o f dif f er ent l eng th s . Th er e ma y b e a g ents , h o w
ev er , wh ic h initia te s y nth es is a t th e 5 end a nd p r o c eed
in th e o th er dir ec tio n, u s ing th e s a me p r o c es s a s de
s c r ib ed a b o v e.
Th e newl y s y nth es iz ed s tr a nd a nd its c o mp l ementa r y
nu c l eic a c id s tr a nd f o r m a do u b l e-s tr a nded mo l ec u l e
wh ic h is u s ed in th e s u c c eeding s tep s o f th e p r o c es s . In
th e nex t s tep , th e s tr a nds o f th e do u b l e-s tr a nded mo l e
c u l e a r e s ep a r a ted u s ing a ny o f th e p r o c edu r es de
s c r ib ed a b o v e to p r o v ide s ing l e-s tr a nded mo l ec u l es .
New nu c l eic a c id is s y nth es iz ed o n th e s ing l e
s tr a nded mo l ec u l es . Additio na l indu c ing a g ent, nu c l eo
tides a nd p r imer s ma y b e a dded if nec es s a r y f o r th e
r ea c tio n to p r o c eed u nder th e c o nditio ns p r es c r ib ed
a b o v e. Ag a in, th e s y nth es is wil l b e initia ted a t o ne end
o f th e o l ig o nu c l eo tide p r imer s a nd wil l p r o c eed a l o ng
th e s ing l e s tr a nds o f th e temp l a te to p r o du c e a dditio na l
nu c l eic a c id. Af ter th is s tep , h a l f o f th e ex tens io n p r o d
u c t wil l c o ns is t o f th e s p ec i? c nu c l eic a c id s eq u enc e
b o u nded b y th e two p r imer s .
Th e s tep s o f s tr a nd s ep a r a tio n a nd ex tens io n p r o du c t
s y nth es is c a n b e r ep ea ted a s o f ten a s needed to p r o du c e
th e des ir ed q u a ntity o f th e s p ec i? c nu c l eic a c id s e
q u enc e. As wil l b e des c r ib ed in f u r th er deta il b el o w, th e
a mo u nt o f th e s p ec i? c nu c l eic a c id s eq u enc e p r o du c ed
wil l a c c u mu l a te in a n ex p o nentia l f a s h io n.
Wh en it is des ir ed to p r o du c e mo r e th a n o ne s p ec i? c
nu c l eic a c id s eq u enc e f r o m th e ? r s t nu c l eic a c id o r
mix tu r e o f nu c l eic a c ids , th e a p p r o p r ia te nu mb er o f
dif f er ent o l ig o nu c l eo tide p r imer s a r e u til iz ed. Fo r ex
a mp l e, if two dif f er ent s p ec i? c nu c l eic a c id s eq u enc es
a r e to b e p r o du c ed, f o u r p r imer s a r e u til iz ed. Two o f
th e p r imer s a r e s p ec i? c f o r o ne o f th e s p ec i? c nu c l eic
a c id s eq u enc es a nd th e o th er two p r imer s a r e s p ec i? c
f o r th e s ec o nd s p ec i? c nu c l eic a c id s eq u enc e. In th is
ma nner , ea c h o f th e two dif f er ent s p ec i? c s eq u enc es c a n
b e p r o du c ed ex p o nentia l l y b y th e p r es ent p r o c es s .
Th e p r es ent inv entio n c a n b e p ef o r med in a s tep -wis e
f a s h io n wh er e a f ter ea c h s tep new r ea g ents a r e a dded,
o r s imu l ta neo u s l y , wh er e a l l r ea g ents a r e a dded a t th e
initia l s tep , o r p a r tia l l y s tep -wis e a nd p a r tia l l y s imu l ta
neo u s , wh er e f r es h r ea g ent is a dded a f ter a g iv en nu m
b er o f s tep s . If a meth o d o f s tr a nd s ep a r a tio n, s u c h a s
h ea t, is emp l o y ed wh ic h wil l ina c tiv a te th e a g ent f o r
p o l y mer iz a tio n, a s in th e c a s e o f a h ea t-l a b il e enz y me,
th en it is nec es s a r y to r ep l enis h th e a g ent f o r p o l y mer i
z a tio n a f ter ev er y s tr a nd s ep a r a tio n s tep . Th e s imu l ta
neo u s meth o d ma y b e u til iz ed wh en a nu mb er o f p u r i
4 ,6 8 3 ,1 9 5
1 1
? ed c o mp o nents , inc l u ding a n enz y ma tic mea ns s u c h a s
h el ic a s e, is u s ed f o r th e s tr a nd s ep a r a tio n s tep . In th e
s imu l ta neo u s p r o c edu r e, th e r ea c tio n mix tu r e ma y c o n
ta in, in a dditio n to th e nu c l eic a c id s tr a nd( s ) c o nta ining
th e des ir ed s eq u enc e, th e s tr a nd-s ep a r a ting enz y me
( e. g . , h el ic a s e) , a n a p p r o p r ia te ener g y s o u r c e f o r th e
s tr a nd-s ep a r a ting enz y me, s u c h a s r ATP, th e f o u r nu
c l eo tides . th e o l ig o nu c l eo tide p r imer s in mo l a r ex c es s ,
a nd th e indu c ing a g ent, e. g . , K l eno w f r a g ment o f E. c o l i
DNA p o l y mer a s e I. If h ea t is u s ed f o r dena tu r a tio n in a
s imu l ta neo u s p r o c es s , a h ea t-s ta b l e indu c ing a g ent s u c h
a s a th er mo s ta b l e p o l y mer a s e ma y b e emp l o y ed wh ic h
wil l o p er a te a t a n el ev a ted temp er a tu r e, p r ef er a b l y
6 5 9 0 C. dep ending o n th e indu c ing a g ent, a t wh ic h
temp er a tu r e th e nu c l eic a c id wil l c o ns is t o f s ing l e a nd
do u b l e s tr a nds in eq u il ib r iu m. Fo r s ma l l er l eng th s o f
nu c l eic a c id, l o wer temp er a tu r es o f a b o u t 5 0 C. ma y b e
emp l o y ed. Th e u p p er temp er a tu r e wil l dep end o n th e
temp er a tu r e a t wh ic h th e enz y me wil l deg r a de o r th e
temp er a tu r e a b o v e wh ic h a n ins u f ? c ient l ev el o f p r imer
h y b r idiz a tio n wil l o c c u r . S u c h a h ea t-s ta b l e enz y me is
des c r ib ed, e. g . , b y A. S . K a l edin et a l . , B io k h iml 'y a , 4 5 ,
6 4 4 -6 5 1 ( 1 9 8 0 ) . Ea c h s tep o f th e p r o c es s wil l o c c u r
s eq u entia l l y no twith s ta nding th e initia l p r es enc e o f a l l
th e r ea g ents . Additio na l ma ter ia l s ma y b e a dded a s
nec es s a r y . Af ter th e a p p r o p r ia te l eng th o f time h a s
p a s s ed to p r o du c e th e des ir ed a mo u nt o f th e s p ec i? c
nu c l eic a c id s eq u enc e, th e r ea c tio n ma y b e h a l ted b y
ina c tiv a ting th e enz y mes in a ny k no wn ma nner o r s ep a
r a ting th e c o mp o nents o f th e r ea c tio n.
Th e p r o c es s o f th e p r es ent inv entio n ma y b e c o n
du c ted c o ntinu o u s l y . In o ne emb o diment o f a n a u to
ma ted p r o c es s , th e r ea c tio n ma y b e c y c l ed th r o u g h a
dena tu r ing r eg io n, a r ea g ent a dditio n r eg io n, a nd a
3 0
r ea c tio n r eg io n. In a no th er emb o diment, th e enz y me 3 5
u s ed f o r th e s y nth es is o f p r imer ex tens io n p r o du c ts c a n
1 2
th e des ir ed s eq u enc e [ 5 ] c o mp r is ed o f c o mp l ementa r y
s tr a nds [ 5 + ] a nd [ 5 -] is u til iz ed a s th e nu c l eic a c id.
Du r ing th e ? r s t a nd ea c h s u b s eq u ent r ea c tio n c y c l e
ex tens io n o f ea c h o l ig o nu c l eo tide p r imer o n th e o r ig ina l
temp l a te wil l p r o du c e o ne new s s DNA mo l ec u l e p r o d
u c t o f inde? nite l eng th wh ic h ter mina tes with o nl y o ne
o f th e p r imer s . Th es e p r o du c ts , h er ea f ter r ef er r ed to a s
" l o ng p r o du c ts ," wil l a c c u mu l a te in a l inea r f a s h io n;
th a t is , th e a mo u nt p r es ent a f ter a ny nu mb er o f c y c l es
wil l b e p r o p o r tio na l to th e nu mb er o f c y c l es .
Th e l o ng p r o du c ts th u s p r o du c ed wil l a c t a s tem
p l a tes f o r o ne o r th e o th er o f th e o l ig o nu c l eo tide p r im
er s du r ing s u b s eq u ent c y c l es a nd wil l p r o du c e mo l e
c u l es o f th e des ir ed s eq u enc e [ 5 + ] o r [ S ] . Th es e mo l e
c u l es wil l a l s o f u nc tio n a s temp l a tes f o r o ne o r th e o th er
o f th e o l ig o nu c l eo tide p r imer s , p r o du c ing f u r th er [ 5 + ]
a nd [ S r ] , a nd th u s a c h a in r ea c tio n c a n b e s u s ta ined
wh ic h wil l r es u l t in th e a c c u mu l a tio n o f [ S ] a t a n ex p o
nentia l r a te r el a tiv e to th e nu mb er o f c y c l es .
B y -p r o du c ts f o r med b y o l ig o nu c l eo tide h y b r idiz a
tio ns o th er th a n th o s e intended a r e no t s el f -c a ta l y tic
( ex c ep t in r a r e ins ta nc es ) a nd th u s a c c u mu l a te a t a l inea r
r a te.
Th e s p ec i? c s eq u enc e to b e a mp l i? ed, [ S ] , c a n b e
dep ic ted dia g r a mma tic a l l y a s :
[ 5 + ] 5 AAAAAAAAAAXXXXXXXXXXCCCCCCCCCC 3 '
[ 5 -] 5 'TTTTTTTTTTYYYYYYYYYYGGGGGGGGGG 5
Th e a p p r o p r ia te o l ig o nu c l eo tide p r imer s wo u l d b e: .
Pr imer t: GGGGGGGGGG
Pr imer 2 : AAAAAAAAAA
s o th a t if DNA c o nta ining [ S ]
. . . z z z z z z z z z z z z z z z z AAAAAAAAAAXXXXXXXXXXCCCCCCCCCCz z z z z z z z z z z z z z z z . . .
. . . z z z z z z z z z z z z z z z z TTTTTTTTTTYYYYYYYYYYGGGGGGGGGGz z z z z z z z z z z z z z z z . . .
b e immo b il iz ed in a c o l u mn. Th e o th er r ea c tio n c o mp o
nents c a n b e c o ntinu o u s l y c ir c u l a ted b y a p u mp th r o u g h
th e c o l u mn a nd a h ea ting c o il in s er ies ; th u s th e nu c l eic
a c ids p r o du c ed c a n b e r ep ea tedl y dena tu r ed with o u t
ina c tiv a ting th e enz y me.
4 5
is s ep a r a ted into s ing l e s tr a nds a nd its s ing l e s tr a nds a r e
h y b r idiz ed to Pr imer s l a nd 2 , th e f o l l o wing ex tens io n
r ea c tio ns c a n b e c a ta l y z ed b y DNA p o l y mer a s e in th e
p r es enc e o f th e f o u r deo x y r ib o nu c l eo s ide tr ip h o s p h a tes :
ex tends -GGGGGGGGGG Pr imer l
. . . z z z z z z z z z z z z z z z z AAAAAAAAAAXXXXXXXXXX C CCC C C CC C C z z z z z z z z z z z z z z z z . . .
o r ig ina l temp l a te s tr a nd +
o r ig ina l temp l a te s tr a nd
. . . z z z z z z z z z z z z z z z z TTTTTTTTTTYYYYYYYYYYGGGGGGGGGGz z z z z z z z z z z z z z z z . . .
Pr imer 2 AAAAAAAAAA->ex tends
5 ,
Th e p r es ent inv entio n is demo ns tr a ted dia g r a mma ti
c a l l y b el o w wh er e do u b l e-s tr a nded DNA c o nta ining
3 ,
On dena tu r a tio n o f th e two du p l ex es f o r med, th e p r o d
u c ts a r e:
5 ,
. . . z z z z z z z z z z z z z z z z TTTTTTTTTTYYYYYYYYYYGGGGGGGGGG
newl y s y nth es iz ed l o ng p r o du c t 1
S i 3 ,
. . . z z z z z z z z z z z z z z z z AAAAAAAAAAXXXXXXXXXX C C CC CCCC C Cz z z z z z z z z z z z z z z z . . .
o r ig ina l temp l a te s tr a nd+
4 ,6 8 3 ,1 9 5
. 1 3 1 4
-c o ntinu ed
3 ' 5 ,
. . . z z z z z z z z z z z z z z z z TTTTTTTTTTYYYYYYYYYYGGGGGGGGGGl z z z z z z z z z z z z z u . . .
o r ig ina l temp l a te s tr a nd "
3 ,
5 ,
AAAAAAAAAAXXXXXXXXXX CCCCCCC CCCz z z z z z z z z z z z z z z y , . . .
newl y s y nth es iz ed l o ng p r o du c t 3
If th es e f o u r s tr a nds a r e a l l o wed to r eh y b r idiz e with h u ma n g eno mic DNA, p r ef er a b l y th e nu mb er o f c y c l es
Pr imer s 1 a nd 2 in th e nex t c y c l e, a g ent f o r p o l y mer iz a - 1 0 is f r o m a b o u t 1 0 3 0 . u
tio n wil l c a ta l y z e th e f o l l o wing r ea c tio ns : Th e a mo u nt o f o r ig ina l nu c l eic a c id r ema ins c o ns ta nt
Pr imer Z 5 ' AAAAAAAAAA --9 ex tends to h er e
3 . . . z z z z z z z z z z z z z z z z z z TTTTTTTTTTYYYYYYYYYYGGGGGGGGGG 5
newl y s y nth es iz ed l o ng p r o du c t 1
ex tends GGGGGGGGGG 5 ' Pr imer l
5 ' . . . z z z z z z z z z z z z z z AAAAAAAAAAXXXXXXXXXXCCCCCCCCCCz z z z z z z z z z z z z z . . . 3 '
o r ig ina l temp l a te s tr a nd+
Pr imer 2 5 ' AAAAAAAAAA \ex tends
3 ' . . . z z z z z z z z z z z z z z z z z z TTTTTTTTTTYYYYYYYYYYGGGGGGGGGGz z z z z z z z z z . . . 5 '
o r ig ina l temp l a te s tr a nd
ex tends to h er e --_GGGGGGGGGG 5 Pr imer l
5 ' AAAAAAAAAAXXXXXXXXXXCCCCCCCCCCz z z z z z z z z z z z z z z z . . . 3 '
newl y s y nth es iz ed l o ng p r o du c t 2
If th e s tr a nds o f th e a b o v e f o u r du p l ex es a r e s ep a r a ted, in th e entir e p r o c es s , b ec a u s e it it no t r ep l ic a ted. Th e
th e f o l l o wing s tr a nds a r e f o u nd: a mo u nt o f th e l o ng p r o du c ts inc r ea s es l inea r l y b ec a u s e
5 ' AAAAAAAAAAXXXXXXXXXXC CCC C CCC C C 3 '
newl y s y nth es iz ed [ S + ]
3 ' l . . z z z z z z z z z z z z z z z z z z z TTTTTTTTT'IYYYYYYYYYYGGGGGGGGGG 5
? r s t c y c l e s y nth es iz ed l o ng p r o du c t 1
3 . . . z z z z z z z z z z z z z z z z z z z TTTTTTTTTTYYYYYYYYYYGGGGGGGGGG 5 '
newl y s y nth es iz ed l o ng p r o du c t 1
5 ' . . . z z z z z z z z z z z z z z z z z z z AAAAAAAAAAXXXXXXXXXXCCCCCCCCCCz z z z z z z z z . . . 3 '
o r ig ina l temp l a te s tr a nd+
5 AAAAAAAAAAXXXXXXXXXXCCCCCCCCCCz z z z z z z z z z z z z z z z . . . 3 '
newl y s y nth es iz ed l o ng p r o du c t 2
3 ' . . . z z z z z z z z z z z z z z z z z z z TTTTTTTTTTYYYYYYYYYYGGGGGGGGGGz z z z z z z z z z z z z z z z . . . 5
o r ig ina l temp l a te s tr a nd
3 ' TTTTTTTTTTYYYYYYYYYYGGGGGGGGGG 5 '
newl y s y nth es iz ed [ 8 -]
5 AAAAAAAAAAXXXXXXXXXXCCCCCCCCCCz z z z z z z z z z z z z z z z . . . 3
? r s t c y c l e s y nth es iz ed l o ng p r o du c t 2
th ey a r e p r o du c ed o nl y f r o m th e o r ig ina l nu c l eic a c id.
Th e a mo u nt o f th e s p ec i? c s eq u enc e inc r ea s es ex p o nen
tia l l y . Th u s , th e s p ec i? c s eq u enc e wil l b ec o me th e p r e
do mina nt s p ec ies . Th is is il l u s tr a ted in th e f o l l o wing
ta b l e, wh ic h indic a tes th e r el a tiv e a mo u nts o f th e s p e
c ies th eo r etic a l l y p r es ent a f ter n c y c l es , a s s u ming 1 0 0 %
ef ? c ienc y a t ea c h c y c l e:
It is s een th a t ea c h s tr a nd wh ic h ter mina tes with th e
o l ig o nu c l eo tide s eq u enc e o f o ne p r imer a nd th e c o mp l e
menta r y s eq u enc e o f th e o th er is th e s p ec i? c nu c l eic
a c id s eq u enc e [ S ] th a t is des ir ed to b e p r o du c ed. 6 0
Th e s tep s o f th is p r o c es s c a n b e r ep ea ted inde? nitel y ,
b eing l imited o nl y b y th e a mo u nt o f Pr imer s 1 a nd 2 , th e
a g ent f o r p o l y mer iz a tio n a nd nu c l eo tides p r es ent. Fo r
detec tio n, th e nu mb er o f c y c l es u s ed is th a t r eq u ir ed to Nu mb er o f Do u b l e S tr a nds
p r o du c e a detec ta b l e s ig na l , a n a mo u nt wh ic h w1 l l de- 6 5 Af ter 0 to n Cy c l es
p end, e. g . , o n th e na tu r e o f th e s a mp l e. Fo r ex a mp l e, if Lo ng S p ec i? c
th e s a mp l e is p u r e o r dil u ted, f ewer c y c l es ma y b e r e- Cy c l e Nu mb er Temp l a te Pmdu c ts S eq u enc e [ 5 ]
q u ir ed th a n if it is a c o mp l ex mix tu r e. If th e s a mp l e is 0 1 - _