Development of Selectors for Cancer Mutation Analysis

癌症突变分析选择器的开发

基本信息

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
项目类别：
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

项目摘要

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）：该提案描述了一种用于实现癌症高通量突变分析的新型系统的开发。目前，聚合酶链式反应 (PCR) 是扩增基因组选定区域（例如基因外显子）以进行后续重测序的关键组成部分。 PCR 的一个主要限制是，当大量特异性引物对同时添加到反应中时，会出现扩增假象。我们针对这个问题开发了一种解决方案，能够对特定目标序列进行多重 PCR 扩增，而不会产生扩增产物。该过程基于称为选择器的寡核苷酸构建体。选择器识别确定的靶核酸序列，并且它们充当连接模板来指导这些靶标的环化。选择器包含通用引物对基序，允许使用通用 PCR 引物对在高度多重反应中扩增环化靶标。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 PieceMaker 的计算机程序，它可以为给定的选择器应用找到最佳的选择器探针序列。在此应用中，我们建议将选择器技术与大规模并行测序系统相结合，以实现大量癌症基因的测序。作为一个测试案例，我们的技术开发重点是开发一种选择器测定法，以在单个反应体积中并行测序十个癌症基因。我们将使用一组经过高度表征的 50 多个结直肠癌细胞系，其中许多已鉴定出诸如 TP53 基因等突变。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、插入和缺失突变的能力，并使用已知突变来确定总体敏感性和特异性。特定基因中遗传错误（也称为突变）的积累会导致癌症的行为。识别这些突变的技术有限，因此，仅在特定基因中识别出相对较少的突变。我们正在开发一种新技术，可以简化突变识别过程，从而可以并行分析大量基因。随着进一步的发展，这项技术将能够更大规模、更全面地识别癌症中的关键突变，并且这些突变的识别有可能成为重要的临床测试。