Assembling long heteroduplexes by asymmetric polymerase chain reaction and annealing the resulting single-stranded DNAs

Mugui Wang; Chuchu Wei; Xiufen Ye; Jianping Liu; Cuicui Zhang; Hao Chen; Xiaobo Zhang; Jumin Tu

doi:10.1016/j.ab.2014.12.017

Assembling long heteroduplexes by asymmetric polymerase chain reaction and annealing the resulting single-stranded DNAs

Anal Biochem. 2015 Apr 15:475:29-31. doi: 10.1016/j.ab.2014.12.017. Epub 2015 Jan 6.

Authors

Mugui Wang¹, Chuchu Wei¹, Xiufen Ye¹, Jianping Liu¹, Cuicui Zhang¹, Hao Chen¹, Xiaobo Zhang², Jumin Tu³

Affiliations

¹ Institute of Crop Sciences, College of Agriculture and Biotechnology, Zhejiang University, Hangzhou 310058, China.
² Institute of Crop Sciences, College of Agriculture and Biotechnology, Zhejiang University, Hangzhou 310058, China. Electronic address: hello.xiaobo@163.com.
³ Institute of Crop Sciences, College of Agriculture and Biotechnology, Zhejiang University, Hangzhou 310058, China. Electronic address: jtu@zju.edu.cn.

PMID: 25575760
DOI: 10.1016/j.ab.2014.12.017

Abstract

We developed an effective protocol for generating high-purity heteroduplexes via annealing single-stranded DNAs (ssDNAs) derived from plasmid DNA by asymmetric polymerase chain reaction (A-PCR). With the addition of dimethyl sulfoxide, a one-step A-PCR procedure can generate ssDNAs stably at a range of reaction temperatures. Several annealing buffers can anneal two ssDNAs into heteroduplexes effectively. We further developed a simple strategy to create d(GATC) hemimethylated heteroduplexes by annealing fully methylated homoduplexes in the presence of excessive unmethylated ssDNAs. The constructed heteroduplexes have been well tested as substrates for mismatch repair in Escherichia coli and, thus, can be used in various biotechnology applications.

Keywords: Asymmetric PCR; DNA mismatch repair; Heteroduplex; Single-stranded DNA.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

DNA, Bacterial / chemistry*
DNA, Bacterial / genetics
DNA, Single-Stranded / chemistry*
DNA, Single-Stranded / genetics
Escherichia coli / chemistry*
Escherichia coli / genetics
Polymerase Chain Reaction / methods*

Substances

DNA, Bacterial
DNA, Single-Stranded