Developments of New Methods of Genetic Engineering and Site-Directed Mutagenesis Using DNA Polymerase Chain Reaction.

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Polymerase chain reaction (PCR) with the extremely thermostable DNA polymerase is useful for specific amplification in not less than 10^5-fold of a very small amount of any genes. The present research utilizing PCR aimed at establishing novel methods of molecular cloning without constructing genomic or cDNA libraries, genetic manipulation by introducing unique restriction sites, and site-directed mutagenesis changing randomly or rapidly into all kinds of amino acid residues. The following results were obtained in this research performed from 1990 to 1992.1) By using PCR, a new method of site-directed mutagenesis has been developed for replacing the active-site lysyl residue of microbial aspartate transaminase by 20 amino acid residues in a single round of cloning procedure.2) A system for the efficient expression in Escherichia coli of higher plant UDP-glucose pyrophosphorylase cDNA has been constructed by introducing new restriction enzyme sites with the use of PCR.3) PCR-based random mutation has been applied to the substrate-binding site of chicken muscle adenylate kinase, and a new method of rapid screening of mutant genes has been devised.4) Several DNA fragments from cDNAs coding for two higher plant alpha-glucan phosphorylase isozymes have been amplified by PCR with synthetic oligonucleotides having various restriction enzyme sites as primers, and chimeric enzymes composed of the two phosphorylase isozymes and the rabbit muscle enzyme have been constructed. One of the chimeric enzyme has been found to show extraordinarily high affinities for various glucan substrates.