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

作　　者：孟祥平[1] 杨建英[1] 乔晓岚[1] 梁玲霞[2] 席守民[3] 

机构地区：[1]河南科技大学医学技术与工程学院 [2]河南科技大学第一附属医院 [3]河南科技大学药理学与医学分子生物学重点实验室,河南洛阳471003

出　　处：《生物技术》2013年第6期51-54,共4页Biotechnology

基　　金：国家自然科学基金项目("T-2毒素对小鼠睾丸间质细胞的毒性分子机制";No.31201119)资助

摘　　要：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.

关 键 词：重叠延伸PCR(SOE—PCR) 融合基因 高GC含量 弥散带 

分 类 号：Q784[生物学—分子生物学]