Tn bi ging: PHNG PHP PCR

Gio vin : L QUC HI

1.Khi qut chung v PCR 1.1 Khi nim

- PCR (Polymerase chain reactions) l mt phng php to
dng in vitro nhm khuych i (to ra nhiu bn sao) mt on DNA trong ng nghim m khng cn s dng cc sinh vt sng..

PCR c pht minh nm 1985 Gii Nobel Ha hc 1993

Kary Mullis

1.Khi qut chung v PCR 1.2. ng dng

- Tch dng gene. - Xc nh huyt thng, truy tm du vt ti phm - Chun on nhng bnh - Xc nh cc on trnh t cn nghin cu - Pht hin t bin - Nghin cu qu trnh tin ho phn t - Phc hi cc gen tn ti hng triu nm - Chn ging vt nui, cy trng - Xc nh cc loi mi, cc loi c hu - L k thut nn cho cc k thut khc nh: RAPD, SSR

1.Khi qut chung v PCR 1.2. ng dng

To dng gen s dng Plasmid

2. Nguyn tc v quy trnh PCR 2.1 Nguyn tc PCR

- Tng hp DNA ngoi c th cng tun th nhng nguyn tc c bn ca sao chp DNA trong c th nhng c s khc bit: + Dng nhit cao tho xon thay cho enzim helicase. + Kt hp vi enzim DNA polymerase chu nhit tng hp DNA mi trong mi trng thch hp. + H thng iu nhit thch hp cng vi cc on mi c thit k chuyn bit, ch ng.

2. Nguyn tc v quy trnh PCR 2.2 Quy trnh PCR

Bc 1 30s-1pht, 94-960 Bc 2 30s-1pht, 40-700 Bc 3 30s1pht, 720

94-96

94-96

72

40-70o

2. Nguyn tc v quy trnh PCR 2.2 Quy trnh PCR

Cc bc ca 1 chu k ca PCR Nhit Cc din bin Thi gian

Bc 1: Bin tnh

94-96 C

Mu DNA v mi c phn tch hon ton v 30s-1 pht ch cn dng si n. Mi bt cp vi khun 30s-1 pht theo chiu 5/-3/ DNA polymerase gn tip vo on mi. N bt u bm vo v hot ng dc theo si DNA, tng hp chui DNA mi ging chui DNA gc. Ko di 30s n nhiu pht

Bc 2: Gn mi Bc 3:

40-70 C

Tng hp hay ko di

720 C

2. Nguyn tc v quy trnh PCR 2.2 Quy trnh PCR

Lng bn sao tng theo cp s nhn 2 4 8 16 32 64 128 256 512 1028

3. Cc yu cu trong PCR 3.1 Thnh phn mi trng * Nc - Nc s dng cho phn ng PCR phi tht tinh khit, khng cha ion no, khng cha DNAase, RNAase, enzim ct gii hn

4.1Thnh phn phn ng Buffer

n nh pH

DNA khun Mi

Xui v Ngc tng hp DNA Taq polymerase Tng hot lc ca men

Nucleotides

Men polymerase

MgCl2

Cn nhit chnh xc

My PCR

T ng iu chnh nhit theo chu k

3. Cc yu cu trong PCR 3.1 Thnh phn mi trng - L mu DNA m chng ta s khuch i. C th l DNA kp, n, hoc RNA c tch chit t cc i tng nghin cu. DNA khun c nguyn vn cao, tht tinh sch trnh ln tp cht.

3. Cc yu cu trong PCR 3.1 Thnh phn mi trng

Mi PCR
- Mi l nhng on DNA si n ngn v cn thit cho vic xc tin phn ng dy chuyn tng hp DNA. Chng nhn ra phn DNA cn c nhn ln, bt cp b sung vi mt u ca DNA mu v to ra v tr bt u ti bn. Cc mi ny c chiu ngc nhau, bao gm mt mi xui v mt mi ngc. Ti sao cn c mi PCR Polymerase ch c th bt u qu trnh tng hp t si i Bt u ti ni mi gn vo Mi PCR l g? Cc on DNA ngn, c tng hp nhn to 18-28 Nu

- Trong phng php PCR, on mi c hai vai tr chnh :

+ Khi ng men polymerase v men polymerase ch c th bt u tng hp si b sung cho chui DNA ch mt khi n nhn dng c u 3 (l u m n xc tc cho mt dNTP c gn vo) ang tnh trng si i + Quyt nh nn tnh c hiu ca sn phm PCR, v nu on mi c chn cng c hiu cho chui ch, ngha l ch c th bt cp trn chui ch m khng th bt cp c trn cc chui DNA khc ngoi chui ch, th sn phm PCR cng c hiu

Thermus aquaticus

Vi khun Thermus aquaticus c pht hin ln u tin mt con sui nc nng khu vc Great Fountain ca the Lower Geyser Basin ti cng vin quc gia Yellowstone. Taq polymerase c tch chit t vi khun ny Taq polymerase cn 30-60s tng hp 1 kilo base. V vy thi gian ko di ph thuc vo kch thc sn phm

Hot ng ca men DNA polymerase lun theo hng 5 3

5 3 5

5 3

3 5

iu kin PCR chun

Hu ht DNA c th c khuch i di iu kin chun

Thnh phn cho phn ng 100 L DNA khun 103 104 phn t Mi 0,2 uM mi mi MgCl2 1.5 mM dNTP 50 M mi loi Taq DNA polymerase 2 units Chng trnh PCR 30-40 chu k: Bin tnh 94C, 30 giy Mi bt cp 55C, 30 giy Ko di 72C, 30 giy Chu k cui: ko di ln cui 72C, 5 pht

Kinh nghim PCR

Khng bao gi pha 1 phn ng PCR duy nht
Nn chun b hn hp ch (master mix)
Buffer, mi, MgCl2, nc, dNTPs, Taq

Khi tnh th tch master mix, nn tr hao thm 1 mu v hao ht trong qu trnh ht bng pipet

Chng m
Khng c DNA khun
Kim tra s nhim

Chng dng
kim tra hot ng ca mix pha

in i: http://207.207.4.198/pub/flash/4/4.html

Cc vn thng gp khi lm PCR

Nhim Khng c sn phm hoc nng sn phm yu Mi t bt cp

Sn phm ph

Vn t nht NHIM

S sao chp qu mc DNA khun Qu nhy Mt lng rt nh cht nhim c th gy ra nhiu trc trc

Th phm chnh Sn phm PCR Cc on DNA ngn hon ton tng ng Nng qu cao C th ln t khun DNA mi

Gii php cho vn nhim

Dng ha cht tit trng Tch khu vc tin PCR v hu PCR Lun dng chng m Chia ha cht thnh nhiu ng nh C th b i khi c trc trc Dng u cn lc Ht cn thn bng pipet

Sn phm khng c hocDNA khng tt yu Mu

Qun mt thnh phn no Kim tra li s th nghim Lp li th nghim Nng sai DNA khun Mi Taq MgCl2 Sai mi Kim tra trnh t Thay mi khc Dng chng dng

Kim tra mu trn gel agarose Xem s tch on Cht c ch PCR B sung qu nhiu iu kin phn ng sai Gim tnh chnh xc Gim nhit bt cp Tng lng MgCl2 Nhum khng tt Kim tra hnh nh Dng thang chun

Mi t bt cp

Mi t bt cp vi nhau Sn phm nh 50-100 bp Thng do vn DNA khun Mi lun c gn vo trnh t no Gii php Chng dng Thit k li mi Dng Hot Start

Dimer

Hairpin loops

Sn phm khng c hiu

Cch pht hin in di trn gel agarose Kch thc sn phm sai Lun dng thang chun Bit kch thc Gii php Tng chnh xc Tng nhit bt cp Gim lng MgCl2 Thay i chng trnh Thi gian ko di Dng nhiu mi khc nhau Gim s chu k

u khuyt im ca PCR:

u im: Hiu qu cao: hot ng trn mt lng DNA cc nh Tnh c hiu cao Nhiu mi hot ng trn phm vi i tng rng (thc vt, ng vt, nm mc) n gin ha qu trnh gii trnh t DNA Khuyt im: Kh khn v tn km khi to mi PCR Gp trc trc khi phn ng tht bi