Open AccessSite-directed, Ligase-Independent Mutagenesis (SLIM): a single-tube methodology approaching 100% efficiency in 4 h
Author(s) -
Joyce Chiu
Publication year - 2004
Publication title -
nucleic acids research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 9.008
H-Index - 537
eISSN - 1362-4954
pISSN - 0305-1048
DOI - 10.1093/nar/gnh172
Subject(s) - biology , ligase chain reaction , mutagenesis , inverse polymerase chain reaction , genetics , heteroduplex , dna ligase , plasmid , directed evolution , site directed mutagenesis , computational biology , overlap extension polymerase chain reaction , microbiology and biotechnology , gene , applications of pcr , directed mutagenesis , protein engineering , bacteriophage , polymerase chain reaction , mutation , multiplex polymerase chain reaction , biochemistry , mutant , enzyme , escherichia coli
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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