Site-directed, Ligase-Independent Mutagenesis (SLIM): a single-tube methodology approaching 100% efficiency in 4 h

Abstract

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%.

Figure 1

Representation of SLIM. The gene-specific portion of each primer is denoted by arrows. Forward and reverse primers are shown in gray and black, respectively. The hash line represents the 5′-adapter of the tailed primer. (A) Sequence insertion. Template and four primers (F_T, F_S, R_T, R_S) 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 (F_T) contains sequence complementary to DNA sequence (hash-lined square) adjacent to the region to be deleted. The reverse tailed primer (R_T) 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 (F_S) and 5′ to the adjacent region (R_S). 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 (F_T and R_T) 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.

Figure 2

SLIM pT7pol-thio PCR products. Lane 1, products from PCR amplification used to create an insertion of six histidine codons; lane 2, products from PCR amplification used to delete the N-terminal six histidine codons. Arrowhead indicates the position of migration of the 6.6 kb linear plasmid band in 1% agarose gel electrophoresis. Photograph of an ethidium bromide stained gel is shown.