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

文献类型： 外文期刊

第一作者： Wang, Mugui

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

作者机构：

关键词： Heteroduplex;Single-stranded DNA;Asymmetric PCR;DNA mismatch repair

期刊名称：ANALYTICAL BIOCHEMISTRY （ 影响因子：3.365； 五年影响因子：3.049 ）

ISSN： 0003-2697

年卷期： 2015 年 475 卷

页码：

收录情况： SCI

摘要： 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. (C) 2015 Elsevier Inc. All rights reserved.

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