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

利用 DNA 聚合酶链式反应进行基因工程和定点诱变的新方法的发展。

基本信息

批准号：
02556013
负责人：
TANIZAWA Katsuyuki
金额：
$ 3.39万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Developmental Scientific Research (B)
财政年份：
1990
资助国家：
日本
起止时间：
1990 至 1992
项目状态：
已结题

项目摘要

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.

使用极其耐热的 DNA 聚合酶的聚合酶链式反应 (PCR) 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。目前利用PCR的研究旨在建立新的分子克隆方法，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诱变。 1990年至1992年进行的这项研究获得了以下结果。1)通过使用PCR，开发了一种新的定点诱变方法，可以在单轮中用20个氨基酸残基取代微生物天冬氨酸转氨酶的活性位点赖氨酰残基2)构建了高等植物UDP-葡萄糖焦磷酸化酶cDNA在大肠杆菌中高效表达的系统通过PCR引入新的限制性酶切位点。3）基于PCR的随机突变已应用于鸡肌肉腺苷酸激酶的底物结合位点，并设计了一种快速筛选突变基因的新方法。4）用具有各种限制性酶位点的合成寡核苷酸作为引物，以及由这两种磷酸化酶同工酶和兔组成的嵌合酶，通过PCR扩增了来自编码两种高等植物α-葡聚糖磷酸化酶同工酶的cDNA的几个DNA片段。肌肉酶已经构建。已发现其中一种嵌合酶对各种葡聚糖底物表现出极高的亲和力。