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A general one‐step method for the cloning of PCR products
Author(s) -
Bolchi Angelo,
Ottonello Simone,
Petrucco Stefania
Publication year - 2005
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/ba20050050
Subject(s) - cloning (programming) , multiple cloning site , amplicon , cloning vector , computational biology , molecular cloning , biology , computer science , genetics , polymerase chain reaction , vector (molecular biology) , recombinant dna , complementary dna , gene , programming language
A very fast, highly efficient, versatile and low‐cost cloning of PCR products is described. PCR amplicons, obtained with any set of primers, is directly integrated into circular plasmid vectors by means of a one‐step restriction–ligation procedure. When using proof‐reading DNA polymerases, 100% cloning efficiency is easily achieved, implying that direct cloning into ‘final‐use’ vectors (i.e. avoiding any intermediate cloning step) is a feasible task. Albeit with a lower efficiency, the same procedure is also suitable for the cloning of PCR products generated by ‘non‐proof‐reading’ DNA polymerases. Furthermore, with a simple modification of the vector polylinker site, the present method can be easily adapted to the directional cloning of open‐reading‐frame‐encoding amplicons. This one‐step procedure thus couples high efficiency with high reliability and versatility, and lends itself as the method of choice for routine cloning of PCR products.