A Highly Efifcient In Vitro Site-directed Mutagenesis Protocol for Introducing Multiple-site Mutations into Target Genes
10.3779/j.issn.1009-3419.2014.06.06
- VernacularTitle:在基因序列中高效引入多位点突变的方法
- Author:
MENG FANRONG
1
;
CHEN CHEN
;
LI YONGWEN
;
WAN HAISU
;
ZHOU QINGHUA
Author Information
1. 300052天津，天津医科大学总医院，天津市肺癌研究所，天津市肺癌转移与肿瘤微环境实验室
- Keywords:
Multiple-site mutagenesis;
Mutagenic primers;
Type IIs restriction enzyme
- From:
Chinese Journal of Lung Cancer
2014;(6):469-473
- CountryChina
- Language:Chinese
-
Abstract:
Background and objective hTe methods for introducing point mutations into target genes are impor-tant for dissecting the relationship of gene structure and function. Up to date, there are numbers of protocols available for the purpose. However, many of them are suited for introducing single site mutation into the target gene. For introducing multiple-site mutations simultaneously into the target genes, it is required to further improve the related methods. Methods In this report, we describe an improvement on the type IIs restriction enzyme-dependent site-directed mutagenesis method and the improved protocol is highly effcient for multiple-site mutagenesis. In our method, a pair of mutagenic primers are synthesized for each desired site and each mutagenic primer contains a selected type IIs restriction site. hTe DNA fragments between two neighboring sites are ampliifed with PCR. All ampliifed fragments are then digested by the selected Type IIs restriction enzyme. hTe expected mutant is eventually generated by ligation of these digested DNA fragments. Results hTe improved protocol is very easy and can be achieved in just one day. As a proof of principle, we have introduced multiple-site, i.e., 3-site or 4-site mu-tations into the fusion gene of nm23 and EGFP (enhanced green lfuorecence protein). hTe mutagenic frequencies are almost reached 100%. Conclusion Our protocol provides a useful tool for gene function research.
