基因工程第8章PCR技术

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1、Chapter8PCRtechnologyPCRpolymerasechainreactionAnothercloningmethodSelectiveamplificationofaDNAsequence.Insomesituation,itessentiallyreplacestraditionalcloningmethodology.PCRprinciplePCR:AtechniquetoselectivelyamplifyaspecificregionofDNA.AmethodlikeDNAreplication

2、invivo.·template：ssDNAordsDNA·primers：oligo-nucleotides·substrates:dNTP·DNApolymerase:TaqpolyBasicprinciple：DNAthynthesingtemplate/primer/DNApolyApplication:themal-resistanceDNApolymeraseDNAamplificationPrincipledATPdGTPdCTPdTTPMg2+buffer5'3'3'5'templateprimerD

3、NApolymerasedenatureannealingextensiondsDNAssDAN92-96C37-72C50-58C72C3．ReactionPCRreactionmixture1．缓冲液buffer(1×)50Mm(大于500bp)(70-100Mm,小于500bp)KCl引物退火annealing10mMTris·HClpH8.3-9.01.5mMMgCl2(0.5-2.5)Storedin20℃2.dNTPFinalconcentration0.2mM(即饱和浓度)pH7.0(浓度大于4mM

4、可能抑制扩增反应)·4种dNTP应平衡，提高忠实性·使用时应考虑Mg2+,引物，产量之间的关系如序列分析和制备探针时，用20-40m3.Primers(正反向、同源互补、正反义)Each1m(1pmol/l)OD260dsDNA50g/mlssDNA40g/mloligo33g/mlPrimerdesign①≥16bp,frequently20-30bp②Tm=81.5+16.61og[J+]+0.41(G+C)%-(675/N)N:lengthofstrandJ:concentrationofmonoval

5、entionIfN=20G+C%=55%[J+]=60mmol/LthenTm=81.5-20.3+22.6-33.75=50.05Tm=(G+C)×4+(A+T)×2forshortprimersOperon公司http://www.operon.com③避免二级结构的形成二聚体二级结构④G/C和A/T分布尽量均匀,一般G+C%45-55%oligodT/oligodC除外，⑤其它，如5’末端，限制酶位点的长度⑥rondomprimers随机引物6nt10-12nt4.Templategorng102----1051

6、g人类单拷贝基因组DNA3×105targets10ngyeastDNA3×1051ngE.coliDNA3×1051ng1kbDNA9×1061%M13plaque1065.DNApolymerase1-2U早期使用klenow(1)TaqDNApolymerase,75℃,94kDaThermusaquticus嗜热水生菌，水生栖热菌5’--3’polymerationand5’---3’exonucleaseTaqhalf-time>2hr92.5℃40min95℃5min97.5℃(2)TthDNApolyme

7、raseThermusthermophilusHB8,94kDa,Mg2+:5’---3’合成DNAMn2+:以RNA为模板合成cDNA可解决RNA反转录过程中形成的二级结构·反转录:primerextension,cDNA合成(3)DNApolymerasewith3’--5’exonucleaseandthemal-resistanceA.VentDNApoly(NewEnglandBlabs)Thermococcuslitoralis,85kDaHalf-life:1.8hrat100℃(使用MgSO4)butTa

8、q5minB.TliDNApoly(Promega)85kDa来源同VentC．PwoPyrococcuswoesei(BM---Roche)90kDaHalf-life>2hrat100℃<5minforTaqD.PfuPyrococcusfuriosus激烈热球菌（stratagene）E.商用混合酶pfuIde