BI 5 PHN TCH TRNH T CHUI ADN V THIT K MI CHO PCR Ngi hng dn: TS.

V Vn Ton Hc vin: L Th Tuyt Mai Lp : Sinh hc thc nghim K14 5.1. Bi cnh v l thuyt 5.1.1. D on gen Cc phng php my tnh hin nay nh v gen trong DNA c da trn nhng c tnh thng k ca chui nucleotide, chng hn nh s dng codon hoc s thch v tr. Nhng phng php ny i hi mt b o to c xc nh, m ch c th thu c t trc xc nh cc gen m ha protein. V vy, cc cng c d on gen hin nay c ti u ha cho mt s lng hn ch ca cc sinh vt. Mt danh sch cc phn mm d on gene c sn ti http://igs-server.cnrsmrs.fr/igs/banbury/programs.html. Vic u i v v tr c th hin bi mi tng quan gia nucleotide lin k trong chui DNA nhim sc th. N c ngha l nu chng ta c mt nucleotide ca loi A bn trong mt gen, th chng ta s tm thy mt nucleotide ca B nhp vo cc v tr tip theo vi mt tn s c th khc bit vi tn s ca B nucleotide trong mt vng khng m ha. M hnh ny xc sut c th c m t bi mt chui Markov. Mt trong nhng cng c c GeneMark (http://opal.biology.gatech.edu/GeneMark/genemark_prok_gms_plus.cgi). Chng trnh GeneMark c truy cp vo cc tim nng m ha protein ca mt chui DNA (trong mt ca s trt) bng cch s dng cc m hnh Markov m ha v vng khng m ha. Cch tip cn ny l nhy cm vi cc bin th ca m ha tim nng ca a phng, v cc th GeneMark hin th chi tit ca vic phn phi tim nng m ha cng mt chui. Chng trnh GeneMark.hmm xc nh phn tch ti a kh nng ca cc chui protein m ha ton b vo gen (vi intron c th) v khu vc intergenic (http://opal.biology.gatech.edu/GeneMark/). *i vi phn tch ADN t cc sinh vt nhn s v sinh vt nhn chun thp, GeneMark v GeneMark.hmm c bao gm trong mt cng c d on kt hp (http://opal.biology.gatech.edu/GeneMark/gmhmm2_prok.cgi). *Khi mt gen gi nh c xc nh, cng c dch thut cho php ly c chui axit amin t chui DNA bng cch dch trong ba khung c c hai hng (5 ` 3` v 3 ` 5`). Ph bin nht l cc cng c dch thut EXPASY (http://www.expasy.ch/tools/dna.html). 5.1. Bi cnh v l thuyt 5.1.2. Thit k Primer

PCR (Polymerase Chain Reaction) by gi l mt trong nhng k thut ph bin nht c s dng trong nghin cu sinh hc v y sinh. C ch ca PCR l khuch i mt on DNA da trn mt cp mi, polymerase ADN v mt s ha cht cn thit. PCR l tit kim thi gian v nhy cm, tuy nhin, sai tn hiu ca phn ng l mt vn nghim trng Cc nguyn nhn ph bin ny l nhim, t dimerization v khng chnh xc DNA mu ca mi. Thit k mi l mt bc quan trng trong chin lc PCR. V mt l thuyt, cc tiu ch cho vic la chn mt cp mi l n gin nhng rt nghim ngt. Mt s tnh nng ca mi nh chiu di mi, nhit nng chy, nhit gn mi ... nn c thc hin m bo rng phn ng PCR l thnh cng v cc sn phm khuch i c sn xut. Hng dn tnh ton kim tra cc tnh nng cho trnh t nucleotide nhiu s la chn tt nht l rt nhiu thi gian. Ngy nay, nhiu chng trnh my tnh c xy dng bi cc nh nghin cu tin sinh hc gii quyt vn ny. Phn mm thng mi, thng c c lp cc chng trnh, c bn vi gi cao, v cc dch v da trn web l min ph nhng cht lng ca cc c tho lun Thng thng, mt phn mm thit k mi c th tnh ton nhit nng chy, cc xu hng hnh thnh vng kp tc, dimer ca mi, v cng l d-dimer ca mi pha trc v ngc li. Mt mi nn vt qua cc yu cu ny: 1. Bm mt cch chnh xc vi mu DNA. 2. Primer chiu di phi trong phm vi 18-30 nucleotide. 3. % GC nn trong khong t 50 - 60% 4. thit b u cui kt thc 3 `nn bm cht vo cc mu DNA 5. Nhit nng chy trong khong 48 - 65oC * 6. Trnh hnh thnh vng kp tc, dimer t v d dimer-** 7. S khc bit trong nhit nng chy ca mi pha trc v ngc li th khng cao hn 5oC Nh vy l mt s phng php tnh ton nhit nng chy ca mi: 1. 2AT 4 phng php GC: Tm = 2 * (A + T) + 4 * (G + C) 2. GC% cc phng php: Tm = 64,9 41 * (GC-16,4%) / chiu di

3. Hng xm gn nht-cc phng php: Tm = DH / (DS + R * ln (C / 4)) 16,6 * log ([K +] / (1 0,7 [K +]))- 273,15 (DH, DS l entanpy v Entropy ca trnh t nucleotide tng ng) ** Vng Hairpin 3`-GGGAAA |||| TATCTAGGACCTTA T-dimer 3`-GGGAAAATTCCAGGATCTAT |||||||| TATCTAGGACCTTAAAAGGG-3 ` D-dimer 5`-TATCTAGGACCTTAAAAGGG ||||| 3`-CATGGAAACTAGGGAC

Trong phn ny, hc vin s c hun luyn bit cch s dng FastPCR - mt phn mm min ph - thit k mi cho phn ng PCR. tm cc khung c m c th c trong mt trnh t DNA, chng ta s dng mt chng trnh c tn l ORF finder ca NCBI. Chng trnh ny s tm kim nhng khung c m c th c ca trnh t nhp vo v trnh t b sung ca n. Sau a ra bn khung c m vi cc trnh t dch m thnh trnh t amino acid. Bc 1: T google chng ta nhp vo t kha orf finder Bc 2: M trang ORF finder t trang ch NCBI bng cch nhn vo dng ORF finder. Nhp trnh t cc nucleotid bn trn vo or sequence in FASTA format Bc 3: Nhp trnh t DNA vo hp trnh t (sequence in FASTA format) hoc m s trnh t vo hp GI or ACESSSION (nu mun dng ton b trnh t trong c s d liu). La v tr dch m (From: To: ) v kiu m di truyn. Nhn nt OrfFind thc hin chng trnh. (Xem hnh trn) i kt qu xut hin sau vi pht (hoc c th lu hn). Kt qu c su khung dch m xut hin. Cc khung c m (nu c) s l nhng thanh c mu sm hn. La chn gii hn cch th hin bng tr s trong mc Redraw (50, 100, 300).

Kt qu th hin c dng tng t: Nhn ln trnh t khung c m s thy hin ln trnh t DNA v trnh t dch m amino acid tng t kt qu bn di. Nhn ln trnh t khung c m s thy hin ln trnh t DNA v trnh t dch m amino acid tng t kt qu bn di. 5.2. Thit k mi Thit k mi cho gen cytochrome P450 (GenBank nhp s: 13699817) khuch ai mt san phm DNA: khoang 500bp Khi ng chng trnh vi cc thng s mc inh, ban nhn c bao nhiu cp mi? M ca s " PCR primers or Probes design options ", trong the " General primer design options ", hy nh du " Show all possible primers " ng lai. Khi ng lai chng trnh, ban nhn c bao nhiu cp mi? Hy c gng tng s lng ti a oan mi c th n 3000. Chay chng trnh v chon cp mi cua ban. Xc inh vi tr cp mi cua ban, san phm PCR cua ban tn bao lu. Xc nh nhit nng chay, GC% v nhit u cua phan ng PCR vi mi cua ban c thit k. XIN CHN THANH CAM N QUY THY C VA CAC BN!