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。

• 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
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-02556013/
• 关键词: polymerase chain reaction (PCR); genetic engineering; site-directed mutagenesis; cDNA; random primer; restriction enzyme sites; ホスホリラーゼ; 基質特異性; 制限酵素; DNAポリメラ-ゼ反復反応(PCR); アデニル酸キナ-ゼ; UDP-グルコ-スピロホスホリラ-ゼ; ランダムプライマ-; アスパラギン酸トランスアミナ-ゼ; polymerase chain reaction (PCR); genetic engineering; site-directed mutagenesis; cDNA; random primer; restriction enzyme sites; ホスホリラーゼ; 基質特異性; 制限酵素; DNAポリメラ-ゼ反復反応(PCR); アデニル酸キナ-ゼ; UDP-グルコ-スピロホスホリラ-ゼ; ランダムプライマ-; アスパラギン酸トランスアミナ-ゼ

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.

聚合酶链反应（PCR）与极其热稳定的DNA聚合酶可用于特异性扩增，在不少于非常少量的任何基因的10^5倍中。本利用PCR的研究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位点来进行基因操作，以及定向定向的诱变随机或快速地变成各种氨基酸残基。 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已经通过使用PCR引入新的限制酶位点来构建。3）基于PCR的随机突变已应用于鸡肉肌肉腺苷酸激酶的底物结合位点，并且已经设计了一种新的突变基因筛选方法。具有各种限制酶位点作为引物的合成寡核苷酸，以及由两个磷酸化酶同工酶组成和兔肌肉酶组成的嵌合酶。已经发现，一种嵌合酶对各种葡萄糖底物显示出极高的亲和力。