产物可直接进行“TA”克隆的高保真PCR法

A High-fidelity PCR Method with Its Product Used in “TA” Cloning Directly

[目的]介绍一种PCR产物直接进行“TA”克隆的高保真PCR法,减少高保真PCR产物“TA”克隆的中间步骤。[方法]Trizol法提取人肝癌细胞SMMC-7721总RNA,并将mRNA反转录成cDNA。以cDNA为模板,联合不同特性的Taq DNA聚合酶扩增PKC基因的蛋白编码区。PCR产物经纯化后进行“TA”克隆、细菌转化、筛选培养、质粒抽提、酶切鉴定和测序鉴定等。[结果]高保真DNA聚合酶扩增的PCR产物未发现DNA突变,但不能直接进行“TA”克隆。Taq DNA聚合酶扩增的产物可直接进行“TA”克隆,但其内部存在多个突变位点,保真性明显不足。联合使用高保真DNA聚合酶和Taq DNA聚合酶(双酶联用)扩增的PCR产物可直接进行“TA”克隆,且DNA测序未发现突变。[结论]双酶联用PCR可以获得高保真PCR产物,并直接进行“TA”克隆。

[Objective]To introduce a high-fidelity PCR method with its product used in“TA”cloning directly,which could simplify the“TA”cloning step.[Method]Total RNAs were extracted from human hepatocellular carcinoma cell line SMMC-7721 by Trizol reagent,and the mRNAs were reverse transcribed into cDNAs.Employing cDNAs as the templates,the cDNA region that codes protein of PKC was amplified by different types of DNA polymerases.The PCR products were subjected to a series of processes such as“TA”cloning,bacterial transformation,screening culture,plasmid extraction,restriction enzyme digestion and DNA sequencing.[Result]No DNA mutation was found in the product of PCR amplification with high fidelity DNA polymerase,but the product could not be linked directly to T vector.On the other hand,the product of PCR amplification with Taq DNA polymerase could be cloned into T vector directly,but it possessed multiple DNA point mutations and its fault was low fidelity.The product of PCR amplification with an alliance of high fidelity DNA polymerase and Taq DNA polymerase could be directly inserted into T vector without any point mutation.[Conclusion]Combined utilization of high fidelity DNA polymerase and Taq DNA polymerase in PCR can obtain high fidelity PCR product which is utilized in“TA”cloning directly.