Cu

trc c bn ca gen chuyn vo thc vt

Phng php sng lc PCR
Da vo promoter 35S Da vo terminator nos

Phng php Real-Time PCR So snh Ti liu tham kho

L vng iu khin s biu Terminator l vng ng vai tr du hin gen, ni nhn dng v hiu dng ko di ca RNA polymerase ca qu trnh phin m, II trong phin m im nhn dng v ngng hot ng ca RNA polymerase II.

P-35S; promoter FBP1; promoter SSU

terminator PinII; T-35S; terminator ORF25PolyA; terminator OCSt; T-nos

L phng php pht hin cc yu t di truyn thng thng i vi mt s sinh vt chuyn gen. Ch yu da vo cc cp mi c hiu cho vng promoter v terminator. Hu ht cc trnh t DNA chuyn vo h gen thc vt di s iu khin CaMV 35S-promoter v T-Nos terminator.

Nguyn tc
Vng promoter 35S c khuch i vi cp mi p35S-cf3 v p35S-cr4. Sn phm khuch i c pht hin v kt qu dng tnh s cho mt vch sng

c im ca cp mi p35S-cf3 v p35S-cr4

Nguyn tc
Vng terminator nos c khuch i vi cp mi HA-nos 118-f v HA-nos 118-r. Sn phm khuch i c pht hin v kt qu dng tnh s cho mt vch sng

c im ca cp mi HA-nos 118-f v HA-nos 118-r

Thnh phn phn ng PCR

Chng trnh PCR thch hp

Vt liu th nghim

- i chng dng: plasmid pBARGUS. - i chng m: nc ct siu sch. - Cc mu c kt qu tch chit DNA t cht lng tt.

Kt qu in di:

Ging 1: Thang chun 100bp SmartLadder SF Ging 2 11: mu t 1 10 theo th t bng kt qu. Ging 12: i chng dng (plasmid pBar Gus) Ging 13: i chng m (H2O)

Kt qu in di sng lc vi cp mi p35S-cf3 v p35S-cr4

Kt qu in di sng lc vi cp mi HA-nos 118-f v HA-nos 118-r

Nguyn tc: khc bit nhit nng chy sn phm khuch i.

Vi thuc nhum SYBR Green I,sn phm c kim tra bng phn tch Melt curve.

Phn tch Melt curve Thit lp trn my vi tnh cng vi chng trnh chy PCR v t ng thc hin ngay sau khi phn ng khuch i DNA hon tt. Sn phm khuch i c nung bin tnh t t. Tn hiu hunh quang thu nhn s c my tnh phn tch.

Chng trnh nhit phn tch Melt curve

- Kt qu hin th mc Data Analysis. - Phn tch kt qu trn mn hnh DNA Quantification - Phn tch nhit nng chy ca sn phm PCR:
Phn tch cc nh nhit nng chy trn

biu dF/dT so vi nhit (-dF/dT vs Temperature) Xc nh s hin din ca nh nhit nng chy 86,5oC tng ng vi on khuch i c hiu vng promoter 35S.

PCR chun Thi gian Thao tc c hi chnh xc ngoi nhim lu Nhiu,phc tp Cao Thp hn Cao

Real-time PCR Nhanh t,n gin hn Thp Cao khng

Kh nng nh lng Cng c

Khng
n gin,gi thp

C
Phc tp,gi cao

i vi ng vt chuyn gen, thng cc promoter s dng c ngun gc hoc t ng vt nh methallothionein (MT), thymidine kinase, -actin... hoc t virus ng vt nh Simian virus (SV40), Rous sarcoma virus (RSV)... Thng s dng promoter c ngun gc t virus ng vt c th c s dng sng lc s c mt ca DNA c ngun gc ng vt.

www.tailieu.vn http://DOCX.vn K thut gen nguyn l v ng dng. TG-PGS,TS Khut Hu Thanh.

L Th Kim Anh H Ngc Hong Kim Nguyn Th Kim TRang