A Method for Introducing Mutations into Large Vectors
10.3779/j.issn.1009-3419.2014.07.12
- VernacularTitle:在长载体中引入定点突变的方法
- Author:
MENG FANRONG
1
;
CHEN CHEN
;
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:
Site-directed mutagenesis;
Large vector;
Type IIs endonuclease;
Bridge primer
- From:
Chinese Journal of Lung Cancer
2014;(7):563-568
- CountryChina
- Language:Chinese
-
Abstract:
Background and objective In vitro site-directed mutagenesis is a routine technique in molecular biol-ogy labs. However, although there are numbers of related methods available, most of these methods are not suitable for intro-ducing mutations into large vectors. Methods In this report, we describe a method which is highly effective for this purpose. Our method is based on the other site-directed method we recently reported. hTe basic protocol of our method is as follows:(1) Synthesize a pair of vector primers based on the sequences around the region to be mutated, each containing a suitable type IIs endonuclease restriction site;meanwhile, synthesize a pair of short complementary oligonucleotides which forms a mutagenic fragment atfer annealing;(2) Synthesize a pair of bridge primers which can speciifcally bind to a site in the vector sequence, each containing a suitable type IIs endonuclease restriction site;(3) Perform PCR reactions using these Vector primers and Bridge primers;(4) Digest the PCR products with the corresponding type IIs restriction enzyme;(5) Ligate the digested frag-ment with the mutagenic fragment to make the desired mutant. Results Using this protocol, we have introduced mutations into a vector larger than 9 kb. hTe results shows that the mutation rates are more that 90%. Conclusion Our method provides a useful tool for performing site-directed mutagenesis experiment in large vector.
