TECHNOLOGY FOR DETECTION OF SINGLE-BASE MUTATIONS

单碱基突变检测技术

• 批准号: 2097782
• 负责人: MARIANNA Mansfield GOLDRICK
• 金额: $ 18.78万
• 依托单位: AMBION, INC.
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-07-15 至 1995-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2097782
• 关键词: RNase protection assay; diagnosis design /evaluation; early diagnosis; genetic disorder diagnosis; genetic markers; human tissue; method development; neoplasm /cancer diagnosis; nucleobase; pancreatic ribonuclease; point mutation; virus classification

The goal of this project is to develop ribonuclease protection/mismatch assays which will reliably and easily allow the detection of essentially all point mutations. Such assays will provide inexpensive and widely applicable methods for detecting mutations in oncogenes and tumor suppressor genes, screening for genetic diseases, typing viruses and a variety of other applications. Current methods, including SSCP do not achieve 100% detection efficiency and have other shortcomings as well. During Phase I we significantly improved reaction conditions which allowed us to detect point mutations in the p53 tumor suppressor gene which were refractory to RNase cleavage under standard conditions. In addition we developed a PCR/in vitro transcription strategy which allowed us to increase the efficiency of mutation detection. During Phase II we propose to further improve reaction conditions, develop specific reagents for mutation detection in p53, APC, NFI, and other genes, to work out techniques which maximize the size of the region which can be screened in a single assay, and to adapt these assays to non-radioisotopic formats. Collectively these improvements will make RNase protection/mismatch assays the most attractive technique for screening for single base mutations.

本项目的目标是开发核糖核酸酶保护/错配 将可靠且容易地允许检测 基本上都是点突变 这样的测定将提供廉价的 和广泛适用的检测癌基因突变的方法， 肿瘤抑制基因，遗传病筛查，病毒分型 以及各种其他应用。 目前的方法，包括SSCP 不能达到100%的检测效率， 也 在第一阶段，我们显著改善了反应条件 这使我们能够检测p53肿瘤抑制基因的点突变， 这些基因在标准条件下对RNA酶切割是难治的。 此外，我们开发了一种PCR/体外转录策略， 使我们能够提高突变检测的效率。 期间 第二阶段我们建议进一步改善反应条件， p53、APC、NFI等突变检测的特异性试剂 基因，研究出最大化区域大小的技术， 其可以在单一测定中筛选，并且为了使这些测定适应于 非放射性同位素形式。 总的来说，这些改进将使 RNA酶保护/错配测定是最有吸引力的技术， 筛选单碱基突变。