CORE--INSTRUMENTATION AND MUTATION DETECTION

(申请人描述)识别多个与癌症相关的 突变将需要高通量的检测方法。这样做的目的是 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将提供大量数据 充分发展PCR/LDR和PCR/RE/LDR所需的一系列寡核苷酸 项目1中描述的方法。这些连接检测的产物 目前，反应是在ABI 373A DNA上分离和定量的 定序器。未来，LDR产品可能会被同时检测到 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和 DNA测序鉴定嗜热连接酶附加基因。 基于聚合酶链式反应的自动测序技术将应用于 评价PCR/LDR的可靠性并确定其序列 额外的嗜热连接酶基因。(Iii)测试聚合酶保真度 以及使用转化肽进行核苷酸转换的效率 寡核苷酸。PCR/RE/LDR癌症检测方法具有 检测癌症突变的可能性为1/100,000 到一百万。PCR/RE/LDR的灵敏度依赖于保真度 含有3‘核苷酸类似物的引物的聚合酶延伸 (项目2)。已经开发出一种检测方法来检测这两种亲属 不同聚合酶对每个核苷酸的效率和保真度 转换。

(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.