DIAGNOSING MUTATIONS W/ THERMOPHILIC DNA REPAIR ENZYMES

使用嗜热 DNA 修复酶诊断突变

基本信息

批准号：
6016540
负责人：
Jay George
金额：
$ 10万
依托单位：
TREVIGEN, INC.
依托单位国家：
美国
项目类别：
财政年份：
1999
资助国家：
美国
起止时间：
1999-08-01 至 2000-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/6016540
关键词：
DNA repair cell line colorectal neoplasms enzyme activity genetic disorder diagnosis molecular cloning phosphoester ligase point mutation protein purification technology /technique development thermophilic organism

项目摘要

DESCRIPTION: (Adapted from the investigator's abstract) Trevigen has developed a patented technology, the Mismatch Identification DNA Analysis System (MIDAS), for the detection of point mutations without PCR amplification. MIDAS employs DNA mismatch repair enzymes (MREs) to cleave an oligonucleotide probe at sites of mismatches when hybridized to mutated target DNA. The investigators propose to optimize MREs and increase their sensitivity for successful commercialization. Based upon the thermodynamics of enzyme kinetics, their approach is to search for MREs from thermophilic organisms. The investigators have partially purified a MRE from the extreme thermophile, Thermotoga maritima, that recognizes and cuts at all possible base mismatches, operates at high temperatures, survives up to 20s temperature cycles from 65C to 95C, and works well in MIDAS and in heteroduplex analysis for scanning for unknown mutations. Specific Aims 1 and 2 of this Phase 1 SBIR address the need to functionally purify, characterize, and, if necessary, clone this MRE. Specific Aim 3 examines the utility of this thermophilic MRE in the detection of known point mutations in the APC and catenin genes in human colorectal cancer cell lines. The investigators predict that successful optimization of these MREs will quickly translate into commercial products for the detection of known point mutations in a variety of disease-causing genes, without the need for PCR amplification, and for scanning for unknown mutations and single nucleotide polymorphisms by heteroduplex analysis. PROPOSED COMMERCIAL APPLICATION: NOT AVAILABLE

描述：（改编自研究者的摘要）Trevigen 开发了专利技术，错配识别 DNA 分析系统 (MIDAS)，用于无需 PCR 扩增即可检测点突变。 MIDAS 员工 DNA 错配修复酶 (MRE) 在位点切割寡核苷酸探针与突变的靶 DNA 杂交时的错配。调查人员建议优化 MRE 并提高其成功的敏感性商业化。根据酶动力学的热力学，它们方法是从嗜热生物中寻找 MRE。调查人员从极端嗜热菌 Thermotoga 中部分纯化了 MRE maritima 能够识别并消除所有可能的碱基不匹配，在高温下，可承受 65°C 至 95°C 长达 20 秒的温度循环，在 MIDAS 和扫描未知的异源双链分析中效果良好突变。第一阶段 SBIR 的具体目标 1 和 2 解决了以下需求：功能纯化、表征，并在必要时克隆该 MRE。具体的目标 3 检查这种嗜热 MRE 在检测已知物质中的效用人结直肠癌细胞APC和连环蛋白基因点突变线。研究人员预测，这些 MRE 的成功优化将迅速转化为商业产品，用于检测已知的多种致病基因的点突变，无需 PCR 扩增，以及扫描未知突变和单核苷酸通过异源双链分析进行多态性。拟议的商业应用：不可用