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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. Template and four primers (FT, FS, RT, RS) were mixed and the entire plasmid template was amplified. Four PCR products were created (Products 1–4). The 5'-adapter sequences were incorporated at the opposite terminal ends of the Products 1 and 2. These are productive as they contribute to a hybrid that can form a non-covalent closed circle. Products 3 and 4 are not productive in this regard. The PCR fragments were then subjected to denaturation and re-annealing. Productive hybrids form when a strand from Product 1 forms a hybrid with a strand from Product 2 and creates a double-stranded fragment with complementary overhangs that can circularize. Fourteen non-clonable hybrids also form (data not shown).

(B) Sequence deletion. As for sequence insertion, inverse PCR was performed on the circular plasmid template. To simplify the figure, only the initial primer binding sites and the final deleted product are shown. The region to be deleted is denoted by a gray rectangle. The 5' portion of the tailed forward primer (FT) contains sequence complementary to DNA sequence (hash-lined square) adjacent to the region to be deleted. The reverse tailed primer (RT) contains a 5' tail complementary to the same region adjacent to the deletion. The short gene-specific primers are located immediately 3' to the deletion (FS) and 5' to the adjacent region (RS). Four PCR products were created in the amplification step, two of which were productive products. Following denaturing and re-annealing, 16 possible hybrids form, two of which were productive hybrids. The final product with the gray rectangle deleted is obtained after transformation.

(C) Sequence exchange (mutagenesis).Inverse PCR was performed on circular plasmid template. To simplify the figure, only the initial primer binding sites are shown. The region undergoing mutagenesis is represented by a hash-lined rectangle with the positions of mutation indicated by an asterisk. The 5' portion of each tailed primer (FT and RT) carries the mutation(s) to be made on the target sequence. The resulting linear PCR products carry the designed mutations on the adapters. PCR, hybridization and transformation were performed as described for sequence insertion.

for more info read the full article http://ukpmc.ac.uk/picrender.cgi?artid=227979&blobtype=pdf