一种简便快速的单引物PCR定点突变方法

doi:10.13523/j.cb.20150509

中国生物工程杂志

2015, Vol. 35

Issue (5): 61-65 DOI: 10.13523/j.cb.20150509

技术与方法

一种简便快速的单引物PCR定点突变方法

高瑞平, 程隆斌, 李振秋

河北大学生命科学学院保定 071002

A Simple and Rapid Single Primer PCR Method for Site-directed Mutagenesis

GAO Rui-ping, CHENG Long-bin, LI Zhen-qiu

College of Life Sciences, Hebei University, Baoding 071002, China

全文: PDF(775 KB) HTML

摘要：

为了解决在一些特殊位点上利用QuickChange方法进行定点突变时会在突变位点处额外插入引物序列导致突变失败的问题,对QuickChange法进行了改良。改良方法为:合成在突变位点处点突变的一对反向互补引物,分别进行单引物PCR扩增,将两种扩增产物混合,变性复性后加入Dpn I进行酶切,酶切产物转化大肠杆菌DH5α,抗性筛选阳性克隆进行测序验证。利用此法成功突变紫穗槐二烯合酶(amorpha-4, 11-diene synthase,ADS)基因中多个利用常规方法突变均因引入额外引物而无法成功的特殊位点,证明此方法实践上可行,而且也可以避免插入额外引物序列,这也从侧面证明额外引物插入的原因是双引物同时反应。

关键词： 单引物; 定点突变; PCR

Abstract:

To solve the problem that additional primers could be inserted at the mutation site when use QuickChange site-directed mutagenesis methods to mutate some specific sites, QuickChange method was modifed as followed:A pair of complementary primers containing mutations site were synthesized, and single primer PCR amplification was done respectively. The two PCR products from single amplification were mixed and denatured and renatured followed by addition of restriction endonuclease Dpn I. The product was transformed into competent E.coli DH5α, and positive clones were screened for DNA sequencing. Several sites in amorpha-4,11-diene synthase(ADS) gene that could not be successful mutated using tranditional method had been successfully mutated with this method without any additional primers. So this novel method was practicable and could solve the problem of extra primers insertion in QuickChange site-directed mutagenesis. This also indirectly proved that additional primers insertion was caused by putting two primers in one reaction system.

Key words: Single primer Site-directed mutagenesis PCR

收稿日期: 2015-02-05 出版日期: 2015-05-25

ZTFLH:

Q785

基金资助:

国家自然科学基金资助项目(30900111)

通讯作者: 李振秋 E-mail: cell@hbu.edu.cn

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引用本文:

高瑞平, 程隆斌, 李振秋. 一种简便快速的单引物PCR定点突变方法[J]. 中国生物工程杂志, 2015, 35(5): 61-65.

GAO Rui-ping, CHENG Long-bin, LI Zhen-qiu. A Simple and Rapid Single Primer PCR Method for Site-directed Mutagenesis. China Biotechnology, 2015, 35(5): 61-65.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20150509 或 https://manu60.magtech.com.cn/biotech/CN/Y2015/V35/I5/61

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