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,第五章 目的基因与载体的,体外重组,基因工程的操作过程,切,接,转,增,检,连接时考虑因素,实验步骤尽量简单易行,连接形成的节点序列应能被重新切割,对转录、转译中密码阅读不发生干扰,粘性末端连接,平末端连接,末端延伸连接:,连接方式,同聚物加尾,加人工接头,加,DNA,衔接物,粘性末端连接,单酶切产生的粘性末端的连接,(同种酶),5,5,GAATTC,CTTAAG,GAATTC,CTTAAG,EcoRI,5,G,CTTAA,AATTC,G,5,退火,G,CTTAA,AATTC,G,5,G,CTTAA,5,AATTC,G,T4-DNA ligase,G,AATTC,CTTAA,G,5,5,G,AATTC,CTTAA,G,G,AATTC,CTTAA,G,5,5,G,AATTC,CTTAA,G,5,G,CTTAA,5,5,AATTC,G,5,5,GAATTC,CTTAAG,5,EcoRI,粘性末端连接,单酶切产生的粘性末端的连接,(同尾酶),BamHI,5,5,GGATCC,CCTAGG,GGATCC,CCTAGG,5,G,CCTAG,GATCC,G,5,5,T,ACTAG,5,5,GATCA,T,5,退火,G,CCTAG,GATCC,G,5,T,ACTAG,5,GATCA,T,T4-DNA ligase,T,GATCC,ACTAG,G,5,5,G,GATCA,CCTAG,T,5,TGATCA,ACTAGT,5,BclI,T,GATCC,ACTAG,G,5,5,G,GATCA,CCTAG,T,粘性末端连接,双酶切产生的粘性末端的连接,5,5,GAATTC,CTTAAG,GGACTT,CCTGAA,EcoRI,5,G,CCTGA,AATTC,G,5,退火,G,CCTGA,AATTC,G,5,G,CTTAA,5,GACTT,A,T4-DNA ligase,G,AATTC,CTTAA,G,5,5,G,GACTT,CCTGA,A,5,G,CTTAA,5,5,GACTT,A,5,5,GAATTC,CTTAAG,EcoRI,GGACTT,CCTGAA,Bam,Bam,平末端连接,HpaI,5,5,GTTAAC,CAATTG,GTTAAC,CAATTG,5,GTT,CAA,AAC,TTG,5,5,CCC,GGG,5,GGG,CCC,3,T4-DNA ligase,CCC,AAC,GGG,TTG,5,5,GTT,GGG,CAA,CCC,5,CCCGGG,GGGCCC,5,StuI,3,平末端连接,-,不同粘性末端的平端连接,BamHI,5,5,GGATCC,CCTAGG,GGATCC,CCTAGG,5,G,CCTAG,GATCC,G,5,5,CTGCA,G,5,G,ACGTC,3,T4-DNA ligase,C,GATCC,G,CTAG,G,5,5,G,GATC,G,CCTAG,C,5,CTGCAG,GACGTC,5,PstI,3,T4-DNA pol,切平,5,C,G,5,G,C,Klenow,补平,5,G,GATC,CCTAG,GATCC,5,CTAG,G,C,GATCC,G,CTAG,G,5,5,G,GATC,G,CCTAG,C,粘性末端的更换,BamHI,5,5,GGATCC,CCTAGG,T4-DNA ligase,Klenow,补平,GATCC,5,G,5,G,CCTAG,5,5,5,G,GATC,CCTAG,5,GATCC,CTAG,G,5,5,G,GATC,CTGCAG,GATCC,CCTAG,GACGTC,CTAG,G,5,5,PstI,CTGCAG,GACGTC,PstI linker,末端转移酶(,TDT,):在没有模板存在的条件下,能催化,dNTP,随机加于,DNA,分子的,3-OH,端,5,p,3,HO,OH,3,p,5,TDT,Mg,2+,dATP,AAAAAAAAAAAA OH,3,5,p,3,HO,p,5,同聚物尾巴,Poly,(,dA,),末端延伸连接,同聚物加尾,C,3,G,G,5,G,3,C,5,C,3,G,T4-DNA ligase,TdT,TdT,dTTP,dATP,G,TTTTTTTTTT,3,C,退火,C,3,TTTTTTTTTT,G,C,AAAAAAAAAA,3,G,G,3,AAAAAAAAAA,C,5,5,G,TTTTTTTTTT,G,C,AAAAAAAAAA,C,C,AAAAAAAAAA,C,G,TTTTTTTTTT,G,S1,C,3,5,5,G,TTTTTTTTTT,G,C,AAAAAAAAAA,C,C,AAAAAAAAAA,C,G,TTTTTTTTTT,G,加热,G,T,C,A,T,T,T,T,T,T,T,T,T,G,A,A,AAAA,A,A,A,C,C,A,G,T,A,A,A,A,A,A,A,A,A,C,T,T,TTTT,T,T,T,G,T,G,A,C,C,A,G,T,末端延伸连接,同聚物加尾 (,平齐末端),末端延伸连接,同聚物加尾 (,3,突出末端),CTGCA,3,G,G,3,ACGTC,5,GCATG,3,C,5,C,3,GTACG,T4-DNA ligase,TdT,TdT,dCTP,dGTP,GCATG,CCCCCC,3,C,退火,Pst I,Pst I,C,3,CCCCCC,GTACG,CTGCA,GGGGGG,3,G,G,3,GGGGGG,ACGTC,5,5,GCATG,CCCCCC,G,C,GGGGGG,ACGTC,CTGCA,GGGGGG,C,G,CCCCCC,GTACG,5,5,GCATG,CCCCCC,G,C,GGGGGG,ACGTC,CTGCA,GGGGGG,C,G,CCCCCC,GTACG,5,5,GCATG,CCCCCC,TGCA,G,C,GTAC,GGGGGG,ACGTC,CTGCA,GGGGGG,CATG,C,G,ACGT,CCCCCC,GTACG,5,5,GCATG,CCCCCC,TGCA,G,C,GTAC,GGGGGG,ACGTC,CTGCA,GGGGGG,CATG,C,G,ACGT,CCCCCC,GTACG,Klenow,Pst I,Sph I,末端延伸连接,同聚物加尾 (,5,突出末端),5,G,CCTAG,GATCC,G,5,5,G,CTTAA,5,5,5,AATTC,G,T4-DNA ligase,5,5,Klenow,补平,Klenow,补平,5,G,GATC,CCTAG,GATCC,5,CTAG,G,5,G,AATT,CTTAA,5,5,5,AATTC,TTAA,G,TdT,TdT,dGTP,dCTP,G,AATT,GGGGGG,CTTAA,AATTC,GGGGGG,TTAA,G,G,GATC,CCCCCC,CCTAG,GATCC,CCCCCC,CTAG,G,G,AATT,GGGGGG,GATCC,CTTAA,CCCCCC,CTAG,G,G,GATC,CCCCCC,AATTC,CCTAG,GGGGGG,TTAA,G,5,5,G,AATT,GGGGGG,GATCC,CTTAA,CCCCCC,CTAG,G,G,GATC,CCCCCC,AATTC,CCTAG,GGGGGG,TTAA,G,退火,BamH I,BamH I,EcoR I,BamH I,人工接头:化学合成的两条互补寡核苷酸连,上面,带有一 至数个限制酶识别位点,作用:加至待克隆的平末端,DNA,片段两侧,酶切后,产生粘性末端,末端延伸连接,人工接头加尾,CC,GAATTC,GG,5,GG,CTTAAG,CC,5,EcoR I,末端延伸连接,人工接头加尾,5,5,G,CTTAA,5,5,5,AATTC,G,T4-DNA ligase,5,5,T4-DNA ligase,5,CC,GAATTC,GG,5,GG,CTTAAG,CC,EcoR I,CC,G,GG,CTTAA,AATTC,GG,G,CC,G,AATTC,GG,CTTAA,G,CC,CC,G,AATTC,GG,CTTAA,G,退火,EcoR I,EcoR I,EcoR I,CC,GAATTC,GG,GG,CTTAAG,CC,DNA,衔接物:人工合成的双链寡核苷酸片段,一头为平末端,(与目的基因连接),另一头为特定酶的切后粘,性末端(与载体粘端连接),末端延伸连接,DNA,衔接物加尾,CC,G,5,GG,CTTAA,5,EcoR I,AATTC,GG,5,G,CC,5,EcoR I,重组率,重组率的定义:,重组率=含有外源,DNA,的重组分子数/载体分子总数,(在常规实验条件下,重组率一般为,25-75%,),重组率是衡量连接反应效率的重要指标,较高的重组率可以大大简化,DNA,重组的后续操作。,重组率,提高重组率的方法:,提高外源,DNA,片段与载体的分子比,:,5,:,1,10,:,1,载体,DNA,分子在连接前先除去磷酸基团:,5,5,GAATTC,CTTAAG,GAATTC,CTTAAG,EcoRI,5,G,CTTAA,p,AATTC,G,5,p,5,G,CTTAA,OH,5,5,AATTC,G,5,HO,退火,5,G,-,A,-,A,-,T,-,T,-,C,C,-,T,-,T,-,A,-,A,G,5,G,A,-,A,-,T,-,T,-,C,C,-,T,-,T,-,A,-,A,-,G,OH,HO,OH,HO,碱性磷酸酶,5,GAATTC,CTTAAG,5,重组率,提高重组率的方法:,人工构建粘性末端:,CTGCA,3,G,G,3,ACGTC,5,GCATG,3,C,5,C,3,GTACG,T4-DNA ligase,TdT,TdT,dCTP,dGTP,GCATG,CCCCCC,3,C,退火,C,3,CCCCCC,GTACG,CTGCA,GGGGGG,3,G,G,3,GGGGGG,ACGTC,5,5,GCATG,CCCCCC,G,C,GGGGGG,ACGTC,CTGCA,GGGGGG,C,G,CCCCCC,GTACG,5,5,GCATG,CCCCCC,TGCA,G,C,GTAC,GGGGGG,ACGTC,CTGCA,GGGGGG,CATG,C,G,ACGT,CCCCCC,GTACG,Klenow,Thank You!,
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