分子生物学-10-4-基因克隆技术知识.pptVIP

SMARTTM 5’-RACE的原理是先是利用mRNA的3’末端的poly(A)尾巴作为一个引物结合位点，以Oligo(dT)30MN作为锁定引物。在反转录酶MMLV作用下合成标准第一链cDNA。 利用该反转录酶的末端转移酶活性，在第一链的5’末端自动加上3-5个dC，退火后dC与含有Oliogo(dG)通用接头引物配对后，第一链转换为模板。接头上带有通用引物UPM(universal primer,UPM)的结合位点，UPM作为上游引物，用一个基因特异引物2(genespecific primer,GSP2)作为下游引物，扩增出第二链 补充： SMART RACE 5’ First-strand synthesis is primed using a modified oligo (dT) primer. After reverse transcriptase reaches the end of the mRNA template, it adds several dC residues. The SMART II A Oligonucleotide anneals to the tail of the cDNA and serves as an extended template for PowerScript RT polymerase. Mechanism of SMART? cDNA synthesis （了解） 补充： End-to-End PCRand Ligation of 5- and 3-RACE Fragments End-to-End PCR If you know the extreme sequences of the 5- and 3-ends of your target cDNA, you can use the sequences to design 5 and 3 primers for the generation of your target full-length cDNA. The extreme sequences of the 5- and 3-ends can be obtained from the 5- and 3-RACE products of your gene that is generated by this kit as described in the Sections 5 and 6. If you know the sequence at the 5 and 3 ends of a target cDNA, you can synthesize 5 and 3 primers and perform PCR using them. Ligation of 5- and 3-RACE Fragments  If you know a unique restriction enzyme site in your target cDNA, you can use the restriction site to generate the full-length target cDNA. First, you can generate 5- and 3-RACE products of your target cDNA that should have the overlapping region between them. The unique restriction site should be present within the overlapping region. Second, the 5- and 3-RACE fragments are digested by the unique restriction enzyme and the digested 5- and 3-RACE fragments are ligated to generate full-length cDNA RACE （了解） 补充： TaKaRa 5‘ -Full RACE 技术 TaKaRa 5’-Full RACE Kit应用了“去帽法”原理 ：用烟草酸焦磷酸酶( Tobacco Acid Pyrophosphatase，TAP) 去掉mRNA的5‘ 帽子结构，加上一个 Adaptor，然后使用5 Adaptor上的引物与已知序列部分的引物GSP进行RT-PCR反应，高特异性地扩增cDNA 5 末端的全长序列。 RACE （了解） 补充： TaKaRa 5’-Full RACE Alkaline Phosphatase (CIAP) 对Total