PCR英文课件.ppt

单击此处编辑母版标题样式,单击此处编辑母版文本样式,第二级,第三级,第四级,第五级,*,High-Fidelity PCR,1,Review,The most important source of PCR product contamination is the generation of(_)that is associated with the post-PCR analysis.,aerosols of PCR,amplicons,2,PCR,管,移液器,手套,离心机,高压锅,超静台,冰箱,超纯水机,烘箱,3,以前,PCR,产生的,扩增子的回馈污染,，是主要的污染源。,控制污染,实验室应有紫外灯；在超静工作台里配,PCR,反应液；操作中总是带手套；使用正确放置的移液器和枪头、试剂等用品,.,4,污染源,污染可来自几个不同的污染源,:,(1),质粒的扩增与纯化,(2),模板基因组,DNA,的反复分离,(3),以前扩增的分子,(PCR,片段,),（产物污染）交差污染（如气溶胶从一个阳性标本扩散到原本阴性的标本）,(4),试剂污染（贮存液或工作液）,5,如何避免污染？,PCR,活动,:,前,PCR,(,样本制备和,PCR,的准备,),后,PCR,(PCR,进行与,PCR,产物分析,),避免方法,：将,DNA,样本制备,PCR,反应液的配制以及,PCR,过程和,PCR,产物的分析都分别指定在实验室的不同区域里完成。,PCR,活动的时空隔离,6,PCR,实验室的装备,为保证前,-PCR,活动和 后,-PCR,活动的隔离，每个区域都应该有专门的装备，包括移液器及枪头、试剂、移液器架等。,7,1,、,PCR,实验室区域隔离图,Fig.2,8,样本制备,实验台,超静工作台,冰箱,离心机,造水机,正压,前,PCR,区,高压锅,烘箱,9,2,、使用带过滤屏障的枪头,3,、实验室的日常安排,4,、实验室的环境处理,5,、,酶,核酸酶,DNase,I;UDG,尿嘧啶,-DNA-,糖基化酶,6,、试剂灭菌,7,、,配制最大反应液,8,、,使用阳性和阴性对照,10,Contamination Control Program,The essential parts of this contamination control program include,space and time separation,of,pre-and post-PCR activities,use of,physical aids,use of ultraviolet(,UV,)light,use of,aliquoted,PCR reagents,incorporation of,positive and negative control,。,11,7,、配制最大反应液,PCR,有几个平行反应，在一管里配制最大反应液，其中有,水,buffer,dNTPs,引物 和,Taq,DNA,聚合酶，,混匀后被等分到不同,PCR,管里,.MgCl2,和 模板,DNA,再分别加。,这可以减少移液误差和节省时间。,12,灭菌水,variable(34.75-35),10 PCR buffer 5l,（,1,）,2.5mM,dNTP,mix 4l(0.2mM),1.25g/l Primer I 1l(0.1-1M),1.25g/l Primer II 1l(0.1-1M),Taq,DNA Polymerase(5u/l)0.25l(1.25u/50l),25mM MgCl2 3l(1-4mM),*,Template DNA 1l(10pg-1g*),方法,1,）取出冻存试剂在冰盒里融开，稍离心集合液体。,2,）取,PCR,灭菌管，冰上加反应液的各个组分。,3,）轻弹管底使液体混匀，稍离心，集合液体于管底。,4,）,PCR,仪设热盖，反应液不覆盖石蜡油；否则，加,1/2,体积的石蜡油。,PCR,。,13,PCR PROGRAME,预变性（,Pre-,denaturation,),1-3min 95C,变性,(,Denaturation,)92C 45s,退火,(,Annealing,)55C 45s,延伸,(,Elongation,)72C 1min,25-30 cycles,最后延伸,(,Elongation,),72C 5-15min,14,1 2 3 4 5,a,500bp,1000bp,2000bp,750bp,250bp,100bp,1:control;2:P1;3:P2;4:P1+P2,5,：,DL2000 Marker,15,2 3 4 5 6 M 8 9 10 11 12,1 2 3 4 5 M -,16,筛选目的片段测序,1#,MdL1,caaGCTTGCATGCCTGCAGGT,CGACGATT,CGAAGCAGTCAAACCCTCCTAAGATGAAGACATTCAGTGTTGCGGCTGCAGTAACCGCCGTGCTCACCTTTATTTGCCTTCAGGAGAGCCCTGCTGCCTCAGTCACCGAGGTGCAAGAGCTGGAGGAGCCAATGAGCAATGGCAGTCCAGTTGCTGTACATGAAGAGATGTTAGAGGAGTCCTGGAAGATGCTGTATAGCAACAGACACAAGCGCAGCCCCGCTGACTGTCGCTTTTGCTGCGGTTGCTGTCCTAACATGAGCGGATGTGGTGTCTGCTGCAGGTTCA,ATCTCTA,GAGGATCCCCGGGTACCGAGCTCGAATTCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCATGTCCTAACATGAGCGGATGTGGTGTCTGCTGCAGGT,288bp,17,18,3,5,5,3,Taq,Taq,Step 3,Extension,Taq,is,polymerase,.,Taq,synthesises DNA in the 5 to 3 direction,The temperature is increased to 72,for,the polymerase,to extend the primers into new complementary strands,which uses up,dNTPs,and required Mg+.,19,PCR fidelity,PCR fidelity,is largely determined by the intrinsic,error rate of DNA polymerases,under the reaction conditions employed.,PCR,的保真性主要由,PCR,反应条件下,DNA,聚合酶固有的错误率所决定。,20,DNA polymerase fidelity,Parameters contributing to,DNA polymerase fidelity,include the tendency of a polymerase to incorporate,掺入,incorrect nucleotides,the rate at which the enzyme can extend from,mispaired,3 primer termini,21,proofreading,and the presence of 3,5,exonuclease,activity,which can remove,mispaired,bases.,22,The importance of,proofreading,is evident in comparisons of base substitution error rates between non-proofreading,(10,-2,10,-6,),and proofreading,(10,-7,),.,23,DNA,polymerases,error rates,are influenced by PCR conditions and can be minimized by optimizing,pH,Mg+concentration,and nucleotide concentrations.,24,Taq,DNA polymerase,Taq,DNA polymerase is suitable for a number of PCR applications and is still considered by many to be the industry standard.,25,However,the performance of,Taq,is limited in more challenging applications,such as those requiring,high fidelity,synthesis of long,(2 kb,),amplicons,and/or amplification of,GC-rich,sequences.,Taq,DNA polymerase,lacks proofreading activity,and,as a result,exhibits relatively,poor fidelity,.,26,High-fidelity PCR enzymes,are valuable for minimizing the amplification errors in products that will be cloned,sequenced,and expressed.,High-fidelity PCR enzymes,27,Proofreading,Archaeal,DNA Polymerases,High-fidelity PCR enzymes,include proofreading,archaeal,古细菌,DNA polymerases and DNA polymerase blends,混合物,.,28,Proofreading,Archaeal,DNA Polymerases,Commercial proofreading DNA polymerases have been obtained from,Thermococcus,and,Pyrococcus,species of,hyperthermophilic,超嗜热,archaea,(,古生菌,)and are classified as Family B-type DNA polymerases.,29,Unlike,thermophilic,eubacterial,DNA polymerases(e.g.,Tag,),which may or may not possess 3,5,exonuclease,activity,all,archaeal,B-type DNA polymerases,possess,proofreading activity and,lack,an associated 5,3,exonuclease,activity.,Proofreading,Archaeal,DNA Polymerases,30,Most,archaeal,proofreading DNA polymerases,(,Pfu,Vent,Deep Vent,)exhibit limited,processivities,持续合成能力,in vitro(6(10 kb).,42,Therefore,PCR enzyme fidelity is of very importance when amplifying longer DNA sequences that will be cloned,sequenced,and expressed.,43,Optimizing PCR fidelity,PCR error rates can be minimized by employing the highest-fidelity PCR enzyme available for the desired application.,44,In addition to enzyme choice,researchers should also,conside,optimizing reaction conditions to reduce PCR mutation frequency further.For example,assuming a 1-kb fragment using,Tag,(ER,810-6 mutations per,bp,per doubling),a PCR generating 5g of,amplicon,from 5 pg of target DNA has undergone 20 target doublings and produced 1.6 mutations per 10,000 bases.,45,In comparison,a PCR generating 5g of,amplicon,from 75,ng,of target DNA has undergone only 6 target doublings and introduced 0.5 mutations per 10,000 bases.Therefore,researchers can minimize mutation frequency by,limiting,the number of target duplications,;for example,by,increasing,the amount of input,DNA template,or,reducing,the number of,PCR cycles,.,46,Additional reduction in mutation frequency may be achieved by optimizing buffer composition,nucleotide and polymerase concentration,and/or cycling conditions for a particular PCR enzyme.,47,For example,the error rate of,pfu,decreases from 2.610-6 to 1.110-6 as the,nucleotide concentration,is lowered from 1,mM,to 100 m,each.In,addition,using shorter synthesis times and lower enzyme concentrations is likely minimize polymerase extension from,mispaired,or misaligned primer termini.,48,In fact,many of the new high-fidelity enzyme formulations provide significantly improved yield and target-length capability compared to,Tag.,49,Reading material,P21-27,Writing,Characterize High-Fidelity DNA Polymerase.,50,