title: Assembling long heteroduplexes by asymmetric polymerase chain reaction and annealing the resulting single-stranded DNAs.
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.
authors.affiliation: Institute of Crop Sciences, College of Agriculture and Biotechnology, Zhejiang University, Hangzhou 310058, China.
authors.forename: Mugui
authors.initials: M
authors.lastname: Wang
authors.affiliation: Institute of Crop Sciences, College of Agriculture and Biotechnology, Zhejiang University, Hangzhou 310058, China.
authors.forename: Chuchu
authors.initials: C
authors.lastname: Wei
authors.affiliation: Institute of Crop Sciences, College of Agriculture and Biotechnology, Zhejiang University, Hangzhou 310058, China.
authors.forename: Xiufen
authors.initials: X
authors.lastname: Ye
authors.affiliation: Institute of Crop Sciences, College of Agriculture and Biotechnology, Zhejiang University, Hangzhou 310058, China.
authors.forename: Jianping
authors.initials: J
authors.lastname: Liu
authors.affiliation: Institute of Crop Sciences, College of Agriculture and Biotechnology, Zhejiang University, Hangzhou 310058, China.
authors.forename: Cuicui
authors.initials: C
authors.lastname: Zhang
authors.affiliation: Institute of Crop Sciences, College of Agriculture and Biotechnology, Zhejiang University, Hangzhou 310058, China.
authors.forename: Hao
authors.initials: H
authors.lastname: Chen
authors.affiliation: Institute of Crop Sciences, College of Agriculture and Biotechnology, Zhejiang University, Hangzhou 310058, China. Electronic address: hello.xiaobo@163.com.
authors.forename: Xiaobo
authors.initials: X
authors.lastname: Zhang
authors.affiliation: Institute of Crop Sciences, College of Agriculture and Biotechnology, Zhejiang University, Hangzhou 310058, China. Electronic address: jtu@zju.edu.cn.
authors.forename: Jumin
authors.initials: J
authors.lastname: Tu
mesh_terms: D004269:DNA, Bacterial / Q000737:chemistry* / Q000235:genetics; D004277:DNA, Single-Stranded / Q000737:chemistry* / Q000235:genetics; D004926:Escherichia coli / Q000737:chemistry* / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*
chemical_list: D004269:DNA, Bacterial; D004277:DNA, Single-Stranded
keywords: Asymmetric PCR; DNA mismatch repair; Heteroduplex; Single-stranded DNA