CORE--INSTRUMENTATION AND MUTATION DETECTION

核心——仪器仪表和突变检测

• 批准号: 6300473
• 负责人: FRANCIS BARANY
• 金额: $ 21.96万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-02-16 至 2002-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6300473
• 关键词: biomedical equipment resource; biomedical facility; diagnosis design /evaluation; gene mutation; ligase; neoplasm /cancer diagnosis; nucleic acid sequence; phosphoester ligase; polymerase chain reaction; restriction endonucleases

(Applicant's Description) Identifying multiple cancer-associated mutations will require high throughput detection methods. The goal of this core is to provide the instrumentation and mutation detection support to achieve large-scale detection and analysis of mutations. This core will have the following responsibilities: (1) Provide instrumentation for oligonucleotide synthesis and for analysis of mutations associated with cancer. Core A will provide the large numbers of oligonucleotides necessary to fully develop the PCR/LDR and PCR/RE/LDR methods described in Project 1. The products of these ligation detection reactions are currently separated and quantified on an ABI 373A DNA sequencer. In the future, LDR products may be simultaneously detected using addressable oligonucleotide or PNA zip-code arrays. (ii) Provide instrumentation for confirming the nature of cancer mutations and identification of additional thermophilic ligase genes by DNA sequencing. Automatic PCR-based sequencing technology will be applied both for assessing the reliability of PCR/LDR and determining the sequences of additional thermophilic ligase genes. (iii) Testing polymerase fidelity and the efficiency of nucleotide conversions using convertide oligonucleotides. The PCR/RE/LDR cancer detection method has the potential of detecting cancer mutations at a sensitivity of 1 in 100,000 to 1,000,000. The sensitivity of PCR/RE/LDR is dependent on the fidelity of polymerase extension from primers containing a 3' nucleotide analogue (Project 2). An assay has been developed to test both the relative efficiency and fidelity of different polymerases for each nucleotide conversion.

（申请人的描述） 突变将需要高通量检测方法。这个目标 核心是提供仪器和突变检测支持， 实现大规模的突变检测和分析。 该核心将承担以下职责： (1)提供 用于寡核苷酸合成和分析的仪器 与癌症相关的突变。 核心A将提供大量的 PCR/LDR和PCR/RE/LDR的充分开发所需的寡核苷酸 项目1中描述的方法。这些连接检测的产物 反应目前在ABI 373 A DNA上分离和定量 测序仪未来可能会同时检测LDR产品 使用可寻址的寡核苷酸或PNA邮政编码阵列。 (ii)提供 用于确认癌症突变性质的仪器， 通过DNA测序鉴定另外的嗜热连接酶基因。 基于PCR的自动测序技术将应用于 评估PCR/LDR的可靠性并确定 另外的嗜热连接酶基因。 (iii)测试聚合酶保真度 以及使用Convertide的核苷酸转化效率 寡核苷酸 PCR/RE/LDR癌症检测方法具有 以十万分之一的灵敏度检测癌症突变的潜力 到一百万PCR/RE/LDR的灵敏度取决于保真度 从含有3'核苷酸类似物的引物进行聚合酶延伸 （项目2）。已经开发了一种测定方法来测试相对 不同聚合酶对每种核苷酸的效率和保真度 转换.