Site-directed, Ligase-Independent Mutagenesis (SLIM) is a type of PCR-mediated mutagenesis that can accommodate different sequence modification types (insertion, deletion and substitution). Similar to inverse PCR mutagenesis, the desired mutations were introduced by PCR amplification of the entire vector with the target modifications incorporated into the oligonucleotide primers. However, to generate clonable circular DNA, SLIM utilizes a ligation independent approach based on the formation of complementary 5? and 3? single-stranded overhangs following heteroduplex formation. The desired heteroduplexes are generated by performing a single PCR reaction using four primers (two tailed and two non-tailed). The tailed primers are designed to contain the desired mutation on complementary overhangs at the terminus of PCR products: using this strategy, it is possible to replace one or more nucleotides simultaneouslyUpon post-amplification denaturation and re-annealing, heteroduplex formation between the mixed PCR products creates the desired clonable mutated plasmid. Upon transformation into E.coli, only the circular DNA molecules (containing the desired mutation) give rise to colonies on selective media.

The technique is highly robust and suitable for applications in high-throughput gene engineering and library constructions. SLIM can be applied to create sequence insertions, deletion and substitution.

The SLIM technique provides mutagenic efficiency of >95% in 4 h




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

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