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A universal method for directional cloning of PCR products based on asymmetric PCR
Author(s) -
Wang BaoLi,
Jiao YanLi,
Li XiaoXia,
Zheng Fang,
Liang Hui,
Sun ZiYang,
Guo Gang
Publication year - 2009
Publication title -
biotechnology and applied biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.468
H-Index - 70
eISSN - 1470-8744
pISSN - 0885-4513
DOI - 10.1042/ba20070210
Subject(s) - restriction enzyme , insert (composites) , cloning (programming) , restriction site , molecular cloning , biology , computational biology , fragment (logic) , dna , cloning vector , genetics , restriction digest , polymerase chain reaction , microbiology and biotechnology , gene , computer science , algorithm , peptide sequence , materials science , composite material , programming language
We have developed a novel protocol for directional cloning of PCR products into any vector. The target sequence is amplified in two parallel asymmetric PCRs using specially but simply designed primers. Two single‐stranded products are produced and they are annealed to form a double‐stranded DNA fragment bearing overhangs at both ends that correspond to the restriction overhangs of certain restriction enzymes. The fragment can then be cloned into a certain vector previously treated with the corresponding enzymes without restriction of the inserted fragment. Compared with previously published protocols, the procedure described in this paper is highly efficient and it is independent of the restriction sites of the insert and is therefore applicable to molecular biology and biotechnology studies.