优化重叠延伸PCR条件构建BTRCP-CypA融合基因

Optimization of Construction of Fusion Gene-BTRCP-CypA by Using SOE-PCR Technique

目的:通过对重叠延伸PCR技术的改进,建立一种更有效的构建融合基因的方法。方法:优化SOE-PCR体系的模板浓度、中间引物互补长度、起始模板的纯度及循环数等条件。结果:中间引物的互补序列长度一般应在大约25bp时扩增效果最好;降低第一轮PCR循环数至5个后作为模板进行SOE-PCR。成功构建了BTRCP-CypA融合基因,获得了特异性强和保真度高的连接产物。结论:改进后的SOE-PCR提高了特异性,为广泛进行基因特别是高GC含量或复杂模板基因片段的连接操作奠定了基础。

Objective： To discuss the SOE - PCR technique in long piece or multiple pieces gene infusion operation. Method ： The concen- tration of template, intermediate primers complementary length, initial template purity and cycle number, etc. of the SOE -PCR system were discussed. Result：The length of complementary sequences of primers could be about 25bp was the best in the experiment. The PCR prod- ucts of the first PCR for 5 cycle were used as templates for SOE - PCR. The fusion gene - BTRCP - CypA was successfully constructed with strong specificity and high - fidelity. Conclusion ： The modified SOE - PCR improves the specificity and fidelity, and may be meanful for chimerical genes operations especially for gene with GC - rich or complex structure.