一种改进后的重叠延伸PCR法对被孢霉重组载体的构建

生物技术通报 ›› 2013, Vol. 0 ›› Issue (9): 62-67.

一种改进后的重叠延伸PCR法对被孢霉重组载体的构建

孟祥亮¹, 王宁新¹, 牛丽华¹, 李培光¹, 李洋¹, 黄大卫^1,2

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

收稿日期:2013-04-25 修回日期:2013-09-05 出版日期:2013-09-05 发布日期:2013-09-06
作者简介:孟祥亮，男，硕士，研究方向：真菌改造；E-mail：mengxiangliangoo@126.com
基金资助:
国家自然科学基金项目（NSFC 31101634）

DNA Molecule Construction by a Modified Overlap Extension PCR

Meng Xiangliang¹,Wang Ningxin¹,Niu Lihua¹,Li Peiguang¹,Li Yang¹,Hung Dawei^1,2

1. College of Plant Protection，Shandong Agricultural University，Tai'an 271018；
2. Institute of Zoology，Chinese Academy of Sciences，Beijing 100101

Received:2013-04-25 Revised:2013-09-05 Published:2013-09-05 Online:2013-09-06

摘要/Abstract

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。

Abstract: 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.

孟祥亮, 王宁新, 牛丽华, 李培光, 李洋, 黄大卫. 一种改进后的重叠延伸PCR法对被孢霉重组载体的构建[J]. 生物技术通报, 2013, 0(9): 62-67.

Meng Xiangliang, Wang Ningxin, Niu Lihua, Li Peiguang, Li Yang, Hung Dawei. DNA Molecule Construction by a Modified Overlap Extension PCR[J]. Biotechnology Bulletin, 2013, 0(9): 62-67.

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