一种改进后的重叠延伸PCR法对被孢霉重组载体的构建  

DNA Molecule Construction by a Modified Overlap Extension PCR

作　　者：孟祥亮[1] 王宁新[1] 牛丽华[1] 李培光[1] 李洋[1] 黄大卫[1,2] 

机构地区：[1]山东农业大学植物保护学院,泰安271018 [2]中国科学院动物研究所,北京100101

出　　处：《生物技术通报》2013年第9期62-67,共6页Biotechnology Bulletin

基　　金：国家自然科学基金项目(NSFC 31101634)

摘　　要：为了提高DNA大片段的拼接效率,通过引入逐次退火的PCR的方法,改良了传统的重叠延伸PCR方法。逐次退火PCR法,一方面延长了重叠区的PCR引物长度;另一方面把原来在1个循环中1个退火温度改成若干个,逐次降低退火温度,适用于Tm值相差比较大的引物;相邻的退火温度之间相差3-6℃。结果显示,通过此种方法成功拼接了ω3(2)和HCT两个大片段;PCR产物电泳条带单一,克隆测序证实序列完全正确,可以直接应用于后续试验。这种改进后的方法可以有效减少非特异性扩增,提高灵敏度,把这种方法称之为逐次退火重叠延伸PCR。In order to assemble large fragments of DNA sequence efficiently,the study modified a advanced method,successive anneal overlap extension PCR,base on the traditional overlap extension PCR.The method of successive anneal overlap extension PCR lengthens the primer sequences corresponding to overlapping region between two fragments and uses successive annealing ranging from 3 to 6℃ in each single PCR cycles of the second PCR process.In this study,we assembled two large DNA fragments,ω3（2）and HCT,using this advanced method.A single band was obtained with the PCR product and the sequence was proved to be right after sequencing repectively,which would contribute to next experiments.This modified method has higher specificity and sensitivity,and is called successive anneal overlap extension PCR.

关 键 词：逐次退火 重叠区域PCR 突变 拼接 

分 类 号：Q503[生物学—生物化学]