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）：该项目的目标 是开发核糖核酸酶保护/不匹配分析，这将允许 基本上所有单基突变的检测。 这样的测定法 将提供廉价且广泛适用的筛查方法 对于遗传疾病，检测癌基因和肿瘤中的突变 抑制基因，打字病毒以及其他研究和诊断 申请。 当前的核糖核酸酶保护测定程序能够 仅检测大约三分之二的单基突变。 初步的 来自E.Coli的RNase I的结果，最近被克隆了， 过度表达，表明它能够明显地检测到 单基突变比常规使用的RNase A和T1可以。 RNase I能够在所有四个基地后都能分裂，这与组合不同 RNase A和T1只能裂解AFTERC，G和U残基。 它 将确定RNase I在检测所有12个 可能的单碱基不匹配。 另外，反应条件 将优化单基碱不匹配的检测，并在必要时进行 一种将在粗细细胞匀浆中迅速鉴定出新颖的测定 核糖核酸酶具有在单基碱不匹配下切割的能力 对当前已知的核糖核酸酶的裂解的难治性将是 发达。 这项新技术将使用一系列p53基因进行测试 单基突变。