BO CO THC HNH CNG NGH

BO CO THC HNH CNG NGH

BI 4: CNG NGH PCR

NHM 4a

1. Nguyn Hng Ti (nhm trng)

2. o Bt

3. V Khc Long

4. Lu Xun Ngc

5. Nguyn Khc Qun

6. Cao Vn Hip

I. L thuyt

1. K thut PCR l g?

PCR l ch vit tt ca cm tPolymerase Chain Reaction, l mt k thut trong sinh hc phn t nhm khuch i s lng mt on DNA trong ng nghim s dng my iu nhit tun hon.

2. Nguyn l ca PCR.

PCR da trn c s phn ng m rng primer (mi) nh enzyme Taq-Polymerase khuch i in vitro cc nucleic acid c trng. PCR cho php khuch i theo hm s m ln n hng triu ln cc on DNA c chiu di t 200 n 3000bp. on DNA c khuch i (DNA ch) c nhn din nh cp mi c trng (oligonucleotide) thng c chiu di khong 20-25 nucleotide.

3. Quy trnh thc hin PCR.

- Quy trnh thc hin pcr gm nhiu chu k lp i lp li(t 25-30 chu k) .Mi chu k c 3 bc chnh:

Bc 1: (Bin tnh) tho xon(hay l tch) 2 si ca DNA gc di nhit ~94

Bc 2: (Gn mi) h nhit c th gn mi vo si DNA n (45-60 ) . Cc mi nhn bit v gn vo DNA theo nguyn tc b sung.

Bc3: (Ko di chui) DNA polymerase gn vo u 3 t do ca mi v s dng dNTP tng hp cc mch mi theo chiu 5-3.Kt thc 1 chu k,t 1 phn t DNA gc s thu c 2 phn t DNA ging ht ban u (sau n chu k c 2 m n bn sao) Sau chu k cui,nhit c duy tr 72 th 5-10 sao cho tt c cc si n xon li,sau h xung 4 bo qun sn phm.

4. Cc thnh phn tham gia PCR.

Cc thnh phn tham gia bao gm: Nc kh ion v cng,ion Mg2+, Mi, dung dch m, DNA khun, Nucleotide ( dNTP), taq polymerase. Trong :

+DNA khun: Khun DNA c vai tr rt quan trng, nh hng r rt n hiu qu PCR. Phn ng PCR ti u vi cc on khun hon ton tinh sch, khi cc on khun khng sch cn ln protein, hiu qu PCR gim theo t l thun vi tinh sch ca DNA khun.

+Nucleotide c nhim v tho xon 2 si DNA.+Mi l nhng on ngn ,si DNA nhn to-khong 20-25 nucleotides,ph hp 1 cch chnh xc im bt u v kt thc ca DNA cn khuch i.n gn cht vi DNA mu nhng im khi u v kt thc,ni m DNA-polymerase ni v bt u qu trnh tng hp si DNA.+Taq- polymerase l mt loi enzyme DNA polymerase chu nhit (c vi khun chu c nhit cao Thermus aquaticus) c dng tng hp cc on DNA mi trong mi trng c 4 loi deoxyribonucleotide (dATP, dTTP, dCTP, dGTP, c gi chung l dNTP) v hai mi. L do ta phi dng Taq-polymerase l n c th gi nguyn hot tnh n 94 .nh vy c ngha l khng phi b sung enzyme trong qu trnh phn ng xy ra.5. My iu nhit tun hon (Thermocyler)My iu nhit tun hon l mt dng c th nghim thng c dng khuch i cc on DNA thng qua chui phn ng polymerase. Trong cc chu trnh nhit c th iu chnh c nhit to iu kin cho cc phn ng thng qua cc bc ci t trn my.

My tun hon nhit (Ngun internet)6. ng Dng

Pht hin Clostridium perfringens b bng PCR Gii thiu:

Clostridium perfringens l mt tc nhn gy bnh phn b rng c bit l gy ra nhiu bnh cho con ngi v ng vt. Clostridium perfringens sn xut nhiu loi c t alpha, beta,epsilon, v ion c coi l c t chnhv c s dng nhm vo 5 loi A, B, C, D, E. Trong c t alpha tham gia vo bnh v sinh bnh. Ngi ta s dng PCR bng cch s dng b mi pht hin s hin din ca gen m ha cht c alpha.

Vt liu v phng php+ Chun b mu:

1 n 5 khun lc C.perfringens ly t BHI blood agar cultures c li tm vng/pht trong 5 pht, v phn ta c ha trong 50 ul 10mM Tris-HCl,pH 9.0,50 mM KCl, 2% Triton X-100. Hn hp c vortex, un si trong 10 pht ly gii t bo, ly tm 13000 vng/pht trong 2 pht, v 5l dch ni c dng l khun.

+PCR:

Mi oligonucleotide Alpha toxin gen (cpa) c la chn t trnh t (19): 5 GCT AAT GTT ACT GCC GTT GACC 3 v 3 TCT GAT AAC TCG TGT AAG 5. Phn ng PCR c thc hin mt th tch cui cng 50l vi cc thuc th sau y: 10Mm Tris-HCl, pH 8.3, 50Mm KCl, 50 mM MgCl2, 200 dNTP M, 10 pmol ca mi mi, 2U taq DNA polymerase, v 5 l ca mi DNA Hn hp c trong my tun hon nhit. Mu bin tnh nhit 95oC trong 5 pht v sau thc hin 35 chu k gm 1 pht ti 94oC, 1 pht ti 53oC v 1 pht 72oC. Vi 1 chu trnh m rng hn na t 10 pht 72OC. C.perfringens type A ATCC 3624 c dng nh i chng dng v nc l i chng m. Kt qu:Tng cng 89 chng C.perfringens c nh type s dng PCR. Gen m ha alpha-toxin (cpa) c pht hin (loi A ATCC 3624) v trong tt c cc chng phn lp.

II. THC HNH

Tn bi th nghim: K thut PCR.

1. Mc ch:khuch i (to ra nhiu bn sao) mt on DNA m khng cn s dng cc sinh vt sng nh E. coli hay nm men.2. Dng c th nghim:

+My ly tm

My ly tm loi nh(PTN H Cng ngh- HQG HN)

+ng th nghim

ng nghim Eppenclofr (PTN H Cng ngh- HQG HN+ Pipet:

Pipet (0.5-10l) PTN H Cng ngh - HQG HN

Pipet (2-20l) PTN H Cng ngh - HQG HN+ My tun hon nhit

My tun hon nhit (Ngun internet)3. Ha Cht

+ H2O kh ion v cng 15.5l+ m (Buffer for Taq Polymerasa) 2.5l+ Nucleotide ( dNTP ) 0.5l+ Mi xui 1l + Mi ngc 1l+ DNA khun 1l+ Enzim taq polymerase 0.5l4. M t th nghim.

+ Ta cn s dng y 2 ng nghim Eppenclofr+ Vi ng nghim 1 ta cho ln lt theo th t cc ha cht trnh by trn ( S dng 2 loi pipet trong phn dng c th nghim ly c mt cch chnh xc nht). Nhng cha cho Enzim taq polymerase.+ Sau ta dng my li tm loi nh v thit lp tc quay 10000 vng/pht. Mc ch l cho tt c cc ha cht tp trung y ng nghim. Lu l khi s dng my ly tm ta phi t hai ng nghim i xng nhau.

My ly tm loi nh

+ Sau khi ly tm xong ta ly ng nghim ra v cho vo Enzim taq polymerase.

+ Vi ng nghim 2 ta lm tng t vi ng nghim 1. Nhng khng cho Enzim taq polymerase. Mc ch l dng lm mu i chng.+ Tip theo ta xp ng nghim cnh nhau v gn gia. Sau n nt Start v bt u thit lp nh sau:

Bc 1: (Bin tnh )

+ t nhit ban u l 94oC trong 2 pht (Mc ch l ch nhit trong my ln 94oC)

+ Ta thit lp my nhit 94oC trong 45s Trong giai on ny, di tc ng ca nhit cao, phn t DNA khun dng xon kp c tch thnh hai si n (single trands).

Bc 2: (Gn mi)+ t nhit 52oC trong 30s. Giai on ny cc mi gn vo cc v tr c trnh t tng ng DNA khun.Bc 3: (Ko di chui)

+ t nhit 72oC trong 1 pht 20s. y l khong nhit ti thch cho Taq-polymerase tin hnh tng hp DNA bt u t c v tr c mi theo chiu 5- 3. Cc mi c mt vi base gn vo khun mu c mi lin kt ion mnh hn cc lc ph v cc lin kt ny s khng b t gy.Lu : Thi gian bc ny ty thuc vo chiu di ca khun DNA.+ t nhit kt thc l 72oC trong 5pht. cc phn t DNA no cha c gn mi th s c thi gian hon thnh.+ Cui cng thit lp gi sn phm 4OC.

+ Ta s thit lp y l 35 chu k. TI LIU THAM KHO:[1] Kha lun tt nghip, L Th Thy Dung, Cc k thut PCR v ng dng,[2] http://en.wikipedia.org/wiki/Thermal_cycler[3] http://vi.wikipedia.org/wiki/PCR#Nh.E1.BB.AFng_.E1.BB.A9ng_d.E1.BB.A5ng_c.E1.BB.A7a_PCR