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1、重組DNA技術(shù)的操作過(guò)程ADNA的體外重組（切與接）3重組DNA技術(shù)的操作過(guò)程同種內(nèi)切酶生產(chǎn)的粘性末端的連接同尾酶生產(chǎn)的粘性末端的連接不同粘性末端的連接人工粘性末端的連接重組率基于同源重組的In-Fusion連接同種內(nèi)切酶生產(chǎn)的粘性末端的連接5'5'GAATTCCTTAAGGAATTCCTTAAGEcoRI5'GCTTAAAATTCG5'5'GCTTAA5'5'AATTCG5'退火GCTTAAAATTCG5'GCTTAA5'AATTCGGCTTAAAATTCG5'GCTTAA5'AATTCGT4-DNA
2、ligaseGAATTCCTTAAG5'5'GAATTCCTTAAGGAATTCCTTAAG5'5'GAATTCCTTAAG同尾酶生產(chǎn)的粘性末端的連接BamHI5'5'GGATCCCCTAGGGGATCCCCTAGG5'GCCTAGGATCCG5'5'TACTAG5'5'GATCAT5'退火GCCTAGGATCCG5'TACTAG5'GATCATT4-DNAligaseTGATCCACTAGG5'5'GGATCACCTAGT5'TGATCAACTAGT5'BclIGCCTAGGATCCG5'TACTAG
3、5'GATCATTGATCCACTAGG5'5'GGATCACCTAGT不同粘性末端的連接BamHI5'5'GGATCCCCTAGGGGATCCCCTAGG5'GCCTAGGATCCG5'5'CTGCAG5'GACGTC3'T4-DNAligaseCGATCCGCTAGG5'5'GGATCGCCTAGC5'CTGCAGGACGTC5'PstI3'T4-DNApol切平5'CG5'GCKlenow補(bǔ)平5'GGATCCCTAGGATCC5'CTAGGCGATCCGCTAGG5'5'GGATCGCCTAGC人
4、工粘性末端的連接5‘突出末端5'GCCTAGGATCCG5'5'GCTTAA5'5'5'AATTCGT4-DNAligase5'5'Klenow補(bǔ)平Klenow補(bǔ)平5'GGATCCCTAGGATCC5'CTAGG5'GAATTCTTAA5'5'5'AATTCTTAAGTdTTdTdGTPdCTPGAATTGGGGGGCTTAAAATTCGGGGGGTTAAGGGATCCCCCCCCCTAGGATCCCCCCCCCTAGGGAATTGGGGGGGATCCCTTAACCCCCCCTAGGGGATCCCCCC
5、CAATTCCCTAGGGGGGGTTAAG5'5'GAATTGGGGGGGATCCCTTAACCCCCCCTAGGGGATCCCCCCCAATTCCCTAGGGGGGGTTAAG退火B(yǎng)amHIBamHIEcoRIBamHI人工粘性末端的連接3‘突出末端CTGCA3'GG3'ACGTC5'GCATG3'C5'C3'GTACGT4-DNAligaseTdTTdTdCTPdGTPGCATGCCCCCC3'C退火PstIPstIC3'CCCCCCGTACGCTGCAGGGGGG3'GG3'GGGGGGACGT
6、C5'5'GCATGCCCCCCGCGGGGGGACGTCCTGCAGGGGGGCGCCCCCCGTACG5'5'GCATGCCCCCCGCGGGGGGACGTCCTGCAGGGGGGCGCCCCCCGTACG5'5'GCATGCCCCCCTGCAGCGTACGGGGGGACGTCCTGCAGGGGGGCATGCGACGTCCCCCCGTACG5'5'GCATGCCCCCCTGCAGCGTACGGGGGGACGTCCTGCAGGGGGGCATGCGACGTCCCCCCGTACGKlenowPstISph
7、I人工粘性末端的連接平頭末端C3'GG5'G3'C5'C3'GT4-DNAligaseTdTTdTdTTPdATPGTTTTTTTTTT3'C退火C3'TTTTTTTTTTGCAAAAAAAAAA3'GG3'AAAAAAAAAAC5'5'GTTTTTTTTTTGCAAAAAAAAAACCAAAAAAAAAACGTTTTTTTTTTGS1C3'5'5'GTTTTTTTTTTGCAAAAAAAAAACCAAAAAAAAAACGTTTTTTTTTTG加熱GTCATTTTTTTTTGAAAAAAAAACCAGT
8、AAAAAAAAACTTTTTTTTTGTGACCAGT基于同源重組的In-Fusion連接載體質(zhì)粒重組質(zhì)粒每條引物外側(cè)設(shè)計(jì)含有與載體相應(yīng)末端同源的15個(gè)堿基序列引物引物In-Fusion酶加入In-Fusion酶37℃15min+50℃15minPCR擴(kuò)增轉(zhuǎn)化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DNA重組
