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1、,第五章 目的基因與載體的,體外重組,基因工程的操作過程,切,接,轉(zhuǎn),增,檢,連接時(shí)考慮因素,實(shí)驗(yàn)步驟盡量簡(jiǎn)單易行,連接形成的節(jié)點(diǎn)序列應(yīng)能被重新切割,對(duì)轉(zhuǎn)錄、轉(zhuǎn)譯中密碼閱讀不發(fā)生干擾,粘性末端連接,平末端連接,末端延伸連接：,連接方式,同聚物加尾,加人工接頭,加,DNA,銜接物,粘性末端連接,單酶切產(chǎn)生的粘性末端的連接,（同種酶）,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,

2、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,粘性末端連接,單酶切產(chǎn)生的粘性末端的連接,（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,TG

3、ATCA,ACTAGT,5,BclI,T,GATCC,ACTAG,G,5,5,G,GATCA,CCTAG,T,粘性末端連接,雙酶切產(chǎn)生的粘性末端的連接,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,平末端連接,Hpa

4、I,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,平末端連接,-,不同粘性末端的平端連接,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

5、,C,5,CTGCAG,GACGTC,5,PstI,3,T4-DNA pol,切平,5,C,G,5,G,C,Klenow,補(bǔ)平,5,G,GATC,CCTAG,GATCC,5,CTAG,G,C,GATCC,G,CTAG,G,5,5,G,GATC,G,CCTAG,C,粘性末端的更換,BamHI,5,5,GGATCC,CCTAGG,T4-DNA ligase,Klenow,補(bǔ)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,CTGCA

6、G,GACGTC,PstI linker,末端轉(zhuǎn)移酶（,TDT,）：在沒有模板存在的條件下，能催化,dNTP,隨機(jī)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,）,末端延伸連接,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

7、,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,加熱,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,末端延伸連接,同聚物加尾 （,平齊末端）,末端延伸連接,同聚物加尾 （,3,突出末端）,CTGCA,3,G,G,3,A

8、CGTC,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,GT

9、AC,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,末端延伸連接,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,補(bǔ)平,Klenow,補(bǔ)平,5,G,GATC,CCTAG,GATCC,5,CTAG,G,5,G,AA

10、TT,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,

11、BamH I,EcoR I,BamH I,人工接頭：化學(xué)合成的兩條互補(bǔ)寡核苷酸連，上面,帶有一 至數(shù)個(gè)限制酶識(shí)別位點(diǎn),作用：加至待克隆的平末端,DNA,片段兩側(cè)，酶切后,產(chǎn)生粘性末端,末端延伸連接,人工接頭加尾,CC,GAATTC,GG,5,GG,CTTAAG,CC,5,EcoR I,末端延伸連接,人工接頭加尾,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,C

12、C,CC,G,AATTC,GG,CTTAA,G,退火,EcoR I,EcoR I,EcoR I,CC,GAATTC,GG,GG,CTTAAG,CC,DNA,銜接物：人工合成的雙鏈寡核苷酸片段，一頭為平末端,（與目的基因連接），另一頭為特定酶的切后粘,性末端（與載體粘端連接）,末端延伸連接,DNA,銜接物加尾,CC,G,5,GG,CTTAA,5,EcoR I,AATTC,GG,5,G,CC,5,EcoR I,重組率,重組率的定義：,重組率=含有外源,DNA,的重組分子數(shù)/載體分子總數(shù),（在常規(guī)實(shí)驗(yàn)條件下，重組率一般為,25-75%,）,重組率是衡量連接反應(yīng)效率的重要指標(biāo)，較高的重組率可以大大簡(jiǎn)化

13、,DNA,重組的后續(xù)操作。,重組率,提高重組率的方法：,提高外源,DNA,片段與載體的分子比,：,5,:,1,10,:,1,載體,DNA,分子在連接前先除去磷酸基團(tuán)：,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,堿性磷酸酶,5,GAATTC,CTTAAG

14、,5,重組率,提高重組率的方法：,人工構(gòu)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!,