Development of Selectors for Cancer Mutation Analysis

癌症突变分析选择器的开发

• 批准号: 7280527
• 负责人: Ronald Wayne Davis
• 金额: $ 23.68万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-08-10 至 2009-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7280527
• 关键词: Atlases; Base Sequence; Behavior; Bioinformatics; Biological Assay; Cancer cell line; Cell Line; Clinical; Colorectal; Colorectal Cancer; DNA; DNA Resequencing; DNA Sequence; DNA amplification; Development; Diagnostic; Exons; Genes; Genetic; Genetic Research; Genome; Genomics; Goals; Human Genome; Large Intestine Carcinoma; Ligation; Malignant Epithelial Cell; Malignant Neoplasms; Methods; Molecular; Morphologic artifacts; Mutation; Mutation Analysis; Nucleic acid sequencing; Numbers; Oligonucleotides; Oncogenes; Polymerase Chain Reaction; Preparation; Procedures; Process; Reaction; Research Proposals; Running; Sampling; Scanning; Sensitivity and Specificity; Sequence Analysis; Solutions; System; TP53 gene; Technology; Testing; Time; base; cancer genome; computer program; cost; design; insertion/deletion mutation; new technology; next generation; novel; rapid technique; scale up; technology development

DESCRIPTION (provided by applicant): This proposal describes the development of a novel system for enabling high throughput mutation analysis of cancer. Currently, polymerase chain reaction (PCR) is a critical component for amplifying selected regions of the genome such as gene exons for subsequent resequencing. A major limitation of PCR is that amplification artifacts arise when large numbers of specific primer pairs are simultaneously added to a reaction. We have developed a solution to this problem that enables multiplex PCR amplification of specific target sequences without amplification artifacts. The procedure is based on oligonucleotide constructs, called selectors. The selectors identify defined target nucleic acid sequences, and they act as ligation templates to direct circularization of these targets. The selectors contain a general primer-pair motif that allows the circularized targets to be amplified in highly multiplexed reactions using a universal PCR primer pair. As part of the bioinformatics for choosing selector sequences, we developed a computer program called PieceMaker that finds the optimal sequence of selector probes for a given selector application. In this application, we propose to adapt selector technology with massively parallel sequencing systems to enable sequencing of large number of cancer genes. As a test case, our technology development is focused on developing a selector assay to sequence in parallel ten cancer genes in a single reaction volume. We will use a highly characterized group of over fifty colorectal carcinoma cell lines, many of them having identified mutations such as the TP53 gene. These cell lines will be used to validate the development of selector technology, optimize its ability to detect missense, insertion and deletion mutations and use known mutations to determine overall sensitivity and specificity. The accumulation of genetic errors, otherwise known as mutations, in specific genes contributes to the behavior of a cancer. Technologies to identify these mutations is limited and as a result, only a relatively few of these mutations have been identified in specific genes. We are developing a new technology that streamlines the process of identifying mutations such that large numbers of genes can be analyzed in parallel. With additional development, this technology will enable larger, more comprehensive identification of critical mutations in cancer and identification of these mutations have the potential to become important clinical tests.

描述（由申请人提供）：该提案描述了一种新型系统，以实现癌症的高吞吐突变分析。当前，聚合酶链反应（PCR）是扩增基因组选定区域（例如基因外显子）的关键成分。 PCR的一个主要局限性是，当大量特定底漆对同时添加到反应中时，就会出现扩增伪像。我们已经为这个问题开发了一个解决方案，该解决方案可以使特定目标序列的多重PCR扩增而无需放大伪像。该过程基于寡核苷酸构建体，称为选择器。选择器鉴定了定义的靶核酸序列，它们充当连接模板，直接指导这些靶标的圆形化。选择器包含一个通用的引物对基序，该基序允许使用通用PCR引物对在高度多重反应中放大圆形靶标。作为选择选择器序列的生物信息学的一部分，我们开发了一个名为“零件制造商”的计算机程序，该程序为给定的选择器应用程序找到了选择器探针的最佳序列。在此应用中，我们建议将选择器技术与大量平行的测序系统适应，以实现大量癌症基因的测序。作为测试案例，我们的技术开发集中在开发选择器测定中以在单个反应体积中的十个癌症基因中对顺序进行顺序。我们将使用一个高度特征的五十多个结直肠癌细胞系，其中许多已经鉴定出诸如TP53基因之类的突变。这些细胞系将用于验证选择器技术的开发，优化其检测错义，插入和删除突变的能力，并使用已知突变来确定整体敏感性和特异性。特定基因中遗传错误的积累，也称为突变，有助于癌症的行为。鉴定这些突变的技术是有限的，因此，在特定基因中仅鉴定出相对较少的这些突变。我们正在开发一种新技术，该技术简化了识别突变的过程，以便可以并行分析大量基因。随着额外的发展，这项技术将使对癌症关键突变的更大，更全面的识别和这些突变的鉴定有可能成为重要的临床测试。