|
|
Single-Primer PCR for Site-Directed Mutagenesis |
PENG Xiang-lei,WANG Ye,WANG Li-nan,SU Yan-bin,FU Yuan-hui,ZHENG Yan-peng,HE Jin-sheng() |
College of Life Sciences & Bioengineering, Beijing Jiaotong University, Beijing 100044, China |
|
|
Abstract Objectives: To introduce site-directed mutagenesis into the pcDNA3.1(+)-F plasmid containing respiratory syncytial virus F gene coding sequence by single circular PCR using single primer. Methods: First, three single-stranded primers with mutagenesis N70Q, I431N or Q270T were designed according to the template pcDNA3.1(+)-F plasmid, respectively. Second, once-single PCR with the double-stranded DNA template and each of the three single primers was performed. Next, the PCR products were treated with endonuclease Dpn I to eliminate the methylated template DNA and then transformed into E. coli DH5α. Finally, plasmids were extracted from the culture of the selected positive clones and sent for sequencing after digestion identification. Results: The results of enzymatic digestion and sequencing analysis were as expected. Three types of site-directed mutagenesis were successfully introduced, namely ‘_X_’, ‘X_X’, ‘XXX’ when ‘X’ means mutated nucleotide and ‘_’ means the opposite. Conclusions: Single-primer PCR is a simple, quick and effective innovation to introduce site-directed mutagenesis, which solved the problems of multiple PCRs, complicated procedures and low effectiveness in previous methods.
|
Received: 17 May 2020
Published: 10 September 2020
|
|
Corresponding Authors:
Jin-sheng HE
E-mail: jshhe@bjtu.edu.cn
|
|
|
[1] |
Zeng F L, Zhang S H, Hao Z M, et al. Efficient strategy for introducing large and multiple changes in plasmid DNA. Scientific Reports, 2018,8(1):1714.
doi: 10.1038/s41598-018-20169-8
pmid: 29379085
|
|
|
[2] |
Liu H, Naismith J H. An efficient one-step site-directed deletion, insertion, single and multiple-site plasmid mutagenesis protocol. BMC Biotechnology, 2008,8:91.
doi: 10.1186/1472-6750-8-91
pmid: 19055817
|
|
|
[3] |
Edelheit O, Hanukoglu A, Hanukoglu I. Simple and efficient site-directed mutagenesis using two single-primer reactions in parallel to generate mutants for protein structure-function studies. BMC Biotechnology, 2009,9:61.
doi: 10.1186/1472-6750-9-61
pmid: 19566935
|
|
|
[4] |
张敏, 辛绍杰, 段学章, 等. 单引物二次PCR法对重组质粒中HBV前C-C基因进行点突变的研究. 中华检验医学杂志, 2007,30(4):444-446.
|
|
|
[4] |
Zhang M, Xin S J, Duan X Z, et al. Establishment of a new “twice single primer PCR method” and application for site-directed mutation in HBV pre-core region. Chin J Lab Med, 2007,30(4):444-446.
|
|
|
[5] |
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. 中国生物工程杂志, 2015,35(5):61-65.
doi: 10.13523/j.cb.20150509
|
|
|
[5] |
Gao R P, Cheng L B, Li Z Q. A simple and rapid single primer PCR method for site-directed mutagenesis. China Biotechnology, 2015,35(5):61-65.
doi: 10.13523/j.cb.20150509
|
|
|
[6] |
Zhuo J, Ma B, Xu J, et al. Reconstruction of a hybrid nucleoside antibiotic gene cluster based on scarless modification of large DNA fragments. Sci China Life Sci, 2017,60:968-979.
doi: 10.1007/s11427-017-9119-1
pmid: 28840532
|
|
|
[7] |
Zeng F L, Zang J P, Zhang S H, et al. AFEAP cloning: a precise and efficient method for large DNA sequence assembly. BMC Biotechnology, 2017,17(1):81.
doi: 10.1186/s12896-017-0394-x
pmid: 29137618
|
|
|
[8] |
Zou X H, Bi Z X, Guo X J, et al. DNA assembly technique simplifies the construction of infectious clone of fowl adenovirus.. J Virol Methods 2018,257:85-92.
doi: 10.1016/j.jviromet.2018.04.001
pmid: 29703616
|
|
|
|
Viewed |
|
|
|
Full text
|
|
|
|
|
Abstract
|
|
|
|
|
Cited |
|
|
|
|
|
Shared |
|
|
|
|
|
Discussed |
|
|
|
|