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

聚合酶链式反应（PCR）使用极耐热的DNA聚合酶，可用于对极少量的任何基因进行不少于10^5倍的特异性扩增。目前的研究利用PCR旨在建立新的分子克隆方法，而无需构建基因组或cDNA文库，通过引入独特的限制性位点进行遗传操作，以及随机或快速改变各种氨基酸残基的定点突变。本研究于1990 ~ 1992年进行，获得以下结果：1）采用PCR技术，已经开发了一种新的定点诱变方法，用于在单轮克隆过程中将微生物天冬氨酸转氨酶的活性位点赖氨酰残基替换为20个氨基酸残基。2）在大肠杆菌中高效表达高等植物UDP-通过使用PCR引入新的限制性酶切位点构建了葡萄糖焦磷酸化酶cDNA。3）将基于PCR的随机突变应用于鸡肌肉腺苷酸激酶的底物结合位点，4）从编码两个高等植物α-谷氨酰胺合成酶基因的cDNA中提取了几个DNA片段，并对它们进行了初步的鉴定，为快速筛选突变基因提供了一种新方法。用具有各种限制酶位点的合成寡核苷酸作为引物，通过PCR扩增葡聚糖磷酸化酶同工酶，并构建了由两种磷酸化酶同工酶和兔肌肉酶组成的嵌合酶。已发现嵌合酶之一对各种葡聚糖底物显示出非常高的亲和力。

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.