TECHNOLOGY FOR DETECTION OF SINGLE BASE MUTATIONS

单碱基突变检测技术

• 批准号: 3493087
• 负责人: MARIANNA Mansfield GOLDRICK
• 金额: $ 4.86万
• 依托单位: AMBION, INC.
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-04-01 至 1992-12-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3493087
• 关键词: Escherichia coli; RNA; RNase protection assay; acidity /alkalinity; genetic disorder diagnosis; method development; molecular cloning; nucleic acid hybridization; nucleic acid probes; nucleobase; pancreatic ribonuclease; point mutation; polynucleotides; radiotracer

DESCRIPTION (Adapted from applicant's abstract): The goal of this project is to develop a ribonuclease protection/mismatch assay, which will allow the detection of essentially all single-base mutations. Such an assay would provide an inexpensive and widely applicable method for screening for genetic diseases, detecting mutations in oncogenes and tumor suppressor genes,typing viruses and for other research and diagnostic applications. Current ribonuclease protection assay procedures are able to detect only about two-thirds of single-base mutations. Preliminary results with RNase I from E.coli, which has recently been cloned and over expressed, indicate that it's capable of detecting significantly more single-base mutations than can the conventionally used RNases A and T1. RNase I is able to cleave after all four bases, unlike the combination of RNase A and T1 which are only able to cleave afterC, G and U residues. It will be determined how effective RNase I is at detecting all of the 12 possible single-base mismatches. In addition, reactionconditions for detection of single-base mismatches will be optimized, and, if necessary, an assay which will rapidly identify in crude cell homogenates novel ribonucleases with the ability to cleave at single-base mismatches refractory to cleavage with currently known ribonnucleases will be developed. This new technology will be tested using a series of p53 genes single-base mutations.

描述（改编自申请人的摘要）：本项目的目标 是开发一种核糖核酸酶保护/错配测定， 检测基本上所有的单碱基突变。 这样的测定 将提供一种廉价且广泛适用的筛选方法 对于遗传性疾病，检测癌基因和肿瘤中的突变 抑制基因、分型病毒和用于其他研究和诊断 应用. 目前的核糖核酸酶保护测定方法能够 只能检测到大约三分之二的单碱基突变。 初步 结果与RNase I从大肠杆菌，这是最近克隆， 过度表达，表明它能够检测到明显更多的 单碱基突变，而不是常规使用的RNA酶A和T1。 RNase I能够在所有四个碱基后切割，不像 RNase A和T1只能切割C、G和U残基。 它 将确定RNase I在检测所有12种 可能的单碱基错配 此外， 单碱基错配的检测将被优化，如果需要， 一种将在粗细胞匀浆中快速鉴定新的 具有单碱基错配切割能力的核糖核酸酶 用目前已知的核糖核酸酶难以切割的， 开发 这项新技术将使用一系列p53基因进行测试。 单碱基突变