Site-directed, Ligase-Independent Mutagenesis (SLIM) is a type of PCR-mediated mutagenesis that can accommodate different 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. SLIM ca be applied to create sequence insertions, deletion and substitution. The efficiency is >95%.




(A) Sequence insertion.
(B) Sequence deletion.
(C) Sequence exchange (mutagenesis).

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