Efficient strategy for introducing large and multiple changes in plasmid DNA

Fanli Zeng,Pan Li,Yanan Meng,Shixin Duan,Yibin Lin,Zhimin Hao,Jingao Dong,Suhua Zhang

doi:10.1038/s41598-018-20169-8

Abstract

While the QuikChange site-directed mutagenesis method and its later modifications are extremely useful and simple, they suffer from several drawbacks. Here, we propose a new method, named LFEAP mutagenesis (Ligation of Fragment Ends After PCR) for creating various mutations in plasmid by leveraging three existing concepts: inverse PCR, single primer PCR, and sticky-end assembly. The first inverse PCR on the target plasmid yielded linearized DNA fragments with mutagenic ends, and a second single primer PCR resulted in complementary single-stranded DNA fragments with the addition of overhangs at the 5′ end of each strand. The resulting single strands were then annealed to produce double-stranded DNA with free 5′ single-stranded DNA tails. These products with compatible sticky ends were efficiently assembled into a circular, mutagenized plasmid. With this strategy, multiple simultaneous changes (up to 15) and mutations in large plasmids (up to 50 kb) were achieved with high efficiency and fidelity. LFEAP mutagenesis is a versatile method that offers significant advantages for introducing large and multiple changes in plasmid DNA.

Highlights

Polymerase chain reaction (PCR)-based site-directed mutagenesis is an invaluable technique for altering genes and the structure and activity of individual proteins in a systematic way, opening up opportunities for investigating the structure-function relationships of protein, enzyme specificity and selectivity, or protein engineering[1,2,3]
Forward primer 2 (Fw2) and reverse primer 2 (Rv2) were designed to have additional overhang sequence at the 5′ ends that were incorporated into the second-round PCR products
The resulting PCR products contained an overhang at the 5′ terminus. (iii) After treating with polynucleotide kinase (PNK) for 5′ phosphorylation, the two complementary single-stranded DNAs generated in the second-round PCR were annealed to form double-stranded DNA with 5′ protruding ends. (iv) The double-stranded DNAs with sticky ends were joined using DNA ligase. (v) These ligated products were transformed into competent E. coli cells, and the presence of modifications was confirmed by DNA sequencing

Summary

Introduction

Polymerase chain reaction (PCR)-based site-directed mutagenesis is an invaluable technique for altering genes and the structure and activity of individual proteins in a systematic way, opening up opportunities for investigating the structure-function relationships of protein, enzyme specificity and selectivity, or protein engineering[1,2,3]. Stratagene’s QuikChange site-directed mutagenesis kit is extremely useful and simple, and probably one of the most favored[4] It requires a high-fidelity DNA polymerase that minimizes unwanted mutations, such as KOD. The originally developed QuikChange method requires the altered nucleotides to be introduced in the middle of both primers, limiting the introduction of multiple mutations[4] as well as large changes[9] To circumvent these limitations, many modified versions of the QuikChange site-directed mutagenesis method have been developed[4,10,11,12]. Many modified versions of the QuikChange site-directed mutagenesis method have been developed[4,10,11,12] These methods use partially overlapping primers to reduce the formation of primer dimers and improve PCR amplification efficiency.

Objectives

Methods

Results

Conclusion