Swapping of functional domains using a chimeric monster deletion mutagenesis strategy

摘要

Site-directed mutagenesis is an effective method to determine the function of amino acid and nucleotide residues within protein and RNA molecules. Systematic site-directed mutagenesis for scanning of larger regions has proven useful when only limited structural information is available (2). A variation of the site-directed mutagenesis approach is the swapping of entire domains between two related molecules to determine their unique functional differences. For domains that correspond up to several hundred base pairs, it can be difficult to introduce mutations over these,larger regions for the generation of chimeric molecules, as it requires de novo gene synthesis or multiple mutagenesis steps followed by sequence verification at each step. Common restriction enzyme cutting sites for swapping domains between two related genes are rarely available and always predetermined. A relatively time-consuming method for changing large DNA segments involves preparation of uracil-containing ssDNA templates and asymmetric PCRs (7). PCR of two overlapping fragments and extension after annealing at the overlap (3) is another widely used approach to generate hybrid genes, but it is critically dependent on primer design and annealing temperatures used in amplifications of the desired DNA fragments. Here, we describe a robust strategy for generating chimeric molecules effectively and precisely. As in PCR of overlapping fragments (3), this approach is effective in the absence of convenient restriction enzyme cutting sites.