A method for generating sticky-end PCR products which facilitates unidirectional cloning and the one-step assembly of complex DNA constructs.
We have developed and tested a method for the restriction enzyme-independent generation of sticky-end PCR products. The method is suitable for use with a proof-reading polymerase such as pfu, or any other heat-stable polymerase which produces a blunt-end product. The technique can be used to achieve unidirectional cloning of PCR products with an efficiency greater than 90%. Because the sequences of the sticky ends are defined by the user and potentially can be of any length, the method can also be exploited for the one-step construction of recombinant plasmids from multiple functional cassettes, without the use of restriction enzymes.
Walker
Andrew
A
Geneservice Ltd., 2 Cambridge Science Park, Milton Road, Cambridge CB4 0FE, UK.
Taylor
James
J
Rowe
Duncan
D
Summers
David
D
D003001:Cloning, Molecular; D004247:DNA / Q000737:chemistry*; D017931:DNA Primers / Q000737:chemistry*; D004274:DNA, Recombinant / Q000235:genetics; D004926:Escherichia coli / Q000378:metabolism; D005821:Genetic Techniques; D005822:Genetic Vectors; D006801:Humans; D008957:Models, Genetic; D008967:Molecular Biology; D010957:Plasmids / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods*; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics
D017931:DNA Primers; D004274:DNA, Recombinant; D014409:Tumor Necrosis Factor-alpha; D004247:DNA