A general one-step method for the cloning of PCR products.
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.
Dipartimento di Biochimica e Biologia Molecolare, Università degli Studi di Parma, Parco Area delle Scienze 23/A, Parma I-43100, Italy. angelo.bolchi@unipr.it
Angelo
A
Bolchi
Simone
S
Ottonello
Stefania
S
Petrucco
D003001:Cloning, Molecular; D004262:DNA Restriction Enzymes / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D005822:Genetic Vectors; D008957:Models, Genetic; D016133:Polymerase Chain Reaction; D015203:Reproducibility of Results
D004259:DNA-Directed DNA Polymerase; D004262:DNA Restriction Enzymes