Cloning and Engineering of Nicking enzymes

切口酶的克隆和工程

• 批准号: 6737237
• 负责人: SHUANG-YONG XU
• 金额: $ 10.4万
• 依托单位: NEW ENGLAND BIOLABS, INC.
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-03-01 至 2004-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6737237
• 关键词: Chlorophyta; biotechnology; deoxyribonuclease I; endodeoxyribonuclease; endonuclease; gel mobility shift assay; genetic manipulation; genetic polymorphism; high throughput technology; methyltransferase; molecular cloning; polymerase chain reaction; recombinant DNA; technology /technique development

DESCRIPTION (provided by applicant): This proposal is aimed towards cloning, expression and engineering of DNA nicking enzymes. Two nicking enzymes CviNY2A and CviNYSI have been found in Chlorella-like algae virus strains NY2A and NYSI with three-nucleotide recognition sequence. The cloning and expression of these two enzymes will facilitate application of nicking enzymes in detection of genetic alterations and recombinant DNA technology. Sapl (GCTCTTC NIlN4) is one of the few type IIS endonucleases that have 7-bp recognition sequences. The second goal is to engineer the Sapl restriction endonuclease to isolate variants that nick DNA. There are only seven nicking enzymes are currently commercially available. Due to the difficulty in making large amount of lysates, CviNY2A and CviNYSI nicking enzymes are only available in small quantity and are often in short supply. Therefore CviNY2 and CviNYSI are in urgent need to be cloned and over-expressed to meet the demand in the research community. The following applications may employ the use of nicking enzymes: DNA amplification, recombinant DNA technology for gene assembly, genetic polymorphism detection, specific genome targeting, preparation of nicked duplex DNA for studying DNA mismatch excision repair. Four cloning strategies will be used to clone CviNY2A and CviNYSI: methylase selection of M.CviNY2A and M.CviNYSI and then cloning of the adjacent ORFs, "endo-blue" method for direct cloning of CviNY2A and CviNYSI nicking enzymes, using antibodies to N6A or C5 methylase modified DNA, PCR using degenerate PCR primers based on the conserved amino acid residues of N6A or C5 methylases. Four experimental strategies will be used to engineer Sapl endonuclease and isolate variants that nick DNA: genetic selection by transformation of I exonuclease treated Sapl nicked-circular DNA to enrich plasmids encoding Sapl nicking activity, molecular break lights generated by Sapl nicking enzyme using a high throughput screening system, DNA mobility shift assay, and in vivo SOS induction using a dinD: lacZ indicator strain.

描述（由申请人提供）：该提案旨在克隆、表达和改造DNA切口酶。在小球藻类藻病毒NY 2A和NYSI株中发现了两种切口酶CviNY 2A和CviNYSI，其识别序列均为三核苷酸。这两种酶的克隆和表达将有助于切口酶在基因突变检测和重组DNA技术中的应用。SapI（GCTCTTC NIIN 4）是具有7-bp识别序列的少数IIS型内切核酸酶之一。第二个目标是工程化SapI限制性内切核酸酶以分离使DNA产生切口的变体。目前只有七种切口酶是可商购的。由于制备大量裂解物的困难，CviNY 2A和CviNYSI切口酶仅以少量可用，并且通常供应短缺。因此，迫切需要克隆和过表达CviNY 2和CviNYSI以满足研究界的需求。以下应用可以使用切口酶：DNA扩增、用于基因组装的重组DNA技术、遗传多态性检测、特异性基因组靶向、用于研究DNA错配切除修复的切口双链DNA的制备。 将使用四种克隆策略来克隆CviNY 2A和CviNYSI：M.CviNY2A和M.CviNYSI的甲基化酶选择，然后克隆相邻的ORF，用于直接克隆CviNY 2A和CviNYSI切口酶的“内切蓝”方法，使用针对N6 A或C5甲基化酶修饰的DNA的抗体，使用基于N6 A或C5甲基化酶的保守氨基酸残基的简并PCR引物的PCR。将使用四种实验策略来工程化Sapl内切核酸酶并分离使DNA产生切口的变体：通过转化I外切核酸酶处理的Sapl切口环状DNA以富集编码Sapl切口活性的质粒进行遗传选择，使用高通量筛选系统通过Sapl切口酶产生分子断裂光，DNA迁移率变动测定，以及使用dinD：lacZ指示菌株进行体内SOS诱导。