Published online 7 December 2004
Nucleic Acids Research, Vol. 32 No. 21 © Oxford University Press 2004; all rights reserved
Site-directed, Ligase-Independent Mutagenesis (SLIM): a single-tube methodology approaching 100% efficiency in 4 h
1 School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, NSW 2052, Australia and 2 Nucleics Pty Ltd, Suite 145, National Innovation Centre, Australian Technology Park, Eveleigh, Sydney, NSW 1430, Australia
* To whom correspondence should be addressed. Tel: +61 2 9209 4034; Fax: +61 2 9209 4084; Email: daniel{at}nucleics.com
Received November 2, 2004; Revised and Accepted November 18, 2004
Site-directed, Ligase-Independent Mutagenesis (SLIM) is a novel PCR-mediated mutagenesis approach that can accommodate all three sequence modification types (insertion, deletion and substitution). The method utilizes an inverse PCR amplification of the template by two tailed long primers and two short primers in a single reaction with all steps carried out in one tube. The tailed primers are designed to contain the desired mutation on complementary overhangs at the terminus of PCR products. Upon post-amplification denaturation and re-annealing, heteroduplex formation between the mixed PCR products creates the desired clonable mutated plasmid. The technique is highly robust and suitable for applications in high-throughput gene engineering and library constructions. In this study, SLIM was employed to create sequence insertions, deletion and substitution within bacteriophage T7 gene 5. The overall efficiency for obtaining the desired product was >95%.
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