GENOME AMPLIFICATION TOLERANT TO SAMPLE DEGRADATION

基因组扩增能够耐受样本降解

• 批准号: 6961398
• 负责人: G. Mike Makrigiorgos
• 金额: $ 14.62万
• 依托单位: DANA-FARBER CANCER INST
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-09-01 至 2007-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6961398
• 关键词: biotechnology; biotransformation; cell line; cell morphology; data collection methodology /evaluation; evaluation /testing; genetic screening; human tissue; methylation; neoplasm /cancer diagnosis; neoplasm /cancer genetics; nucleic acid amplification techniques; polymerase chain reaction; sample collection; technology /technique development; tissue /cell preparation; tissue resource /registry

DESCRIPTION (provided by applicant): Genomic, epigenetic and gene expression analysis from archived formalin-fixed paraffin embedded (FFPE) tissue samples with known clinical outcomes provides a unique opportunity for extraction of genetic information leading to improved cancer diagnosis, prognosis and therapy. However, extensive genotyping or microarray profiling on homogeneous cell populations within these samples often requires whole genome/mRNA amplification prior to screening. Major hurdles to this process are the introduction of amplification bias and the inhibitory effects of formalin fixation on DNA/RNA amplification. We have developed (RCA-RCA), a novel method based on isothermal rolling-circle amplification, that overcomes the limitations and promises to provide the needed link between obtaining a minute biopsy from partially degraded, FFPE samples and genotyping or micro-array screening. RCA-RCA enables whole genome/mRNA amplification that can be adjusted to the degree of FFPE sample degradation, as this is assessed via real time PCR. Thereby RCA-RCA enables retrieval of the maximum possible amount of information from the degraded sample. In the revised application, apart from adopting the Study Section's recommendations, a further enhancement of RCA-RCA is included, mRCA-RCA. mRCA-RCA amplifies DNA while retaining epigenetic modifications on a genome-wide basis ('whole methylome amplification'), thereby allowing highly expanded detection of methylation in fresh or FFPE samples. The R21 phase will examine the maximum capabilities of the technology and establish criteria for adjusting RCA-RCA to conform to the condition of the specific FFPE sample. The R33 phase will develop the technology for obtaining minute cancer biopsies from FFPE samples, assessing sample quality, and amplifying the whole genome/methylome (DNA) or transcriptome (RNA) without introducing amplification bias. Subsequently it will establish criteria and will validate the utility of the amplified material as input for the most frequently used molecular assays (mutation/SNP detection, microsatellite instability/LOH, array-CGH, expression profiling and methylation detection). By removing problems associated with sample degradation and biases associated with amplification this project will enable application of the newest technologies to the analysis of minute biopsies from archived tissue with known outcomes, thereby accelerating the process of candidate gene discovery.

描述(申请人提供)：对已知临床结果的福尔马林固定石蜡包埋(FFPE)存档组织样本的基因组、表观遗传学和基因表达分析为提取遗传信息提供了独特的机会，有助于改善癌症诊断、预后和治疗。然而，对这些样本中的同质细胞群体进行广泛的基因分型或微阵列分析通常需要在筛查之前进行全基因组/信使核糖核酸扩增。这一过程的主要障碍是引入了扩增偏差和福尔马林固定对DNA/RNA扩增的抑制作用。我们开发了一种基于等温滚环扩增的新方法(RCA-RCA)，它克服了这些限制，有望在从部分降解的FFPE样本中获得微小活检与基因分型或微阵列筛选之间提供所需的联系。RCA-RCA能够实现全基因组/信使核糖核酸的扩增，可以根据FFPE样本的降解程度进行调整，因为这是通过实时聚合酶链式反应进行评估的。因此，RCA-RCA能够从退化的样本中检索最大可能的信息量。在修订后的申请书中，除了采纳研究科的建议外，还包括了对区域协调-区域协调机制的进一步改进，即区域协调机制--区域协调机制。MRCA-RCA在保持全基因组基础上的表观遗传修饰的同时放大DNA(‘全甲基组扩增’)，从而允许在新鲜或FFPE样本中高度扩展的甲基化检测。R21阶段将审查技术的最大能力，并建立调整RCA-RCA的标准，以符合特定FFPE样品的条件。R33阶段将开发从FFPE样本中获取微小癌症活检组织、评估样本质量以及在不引入扩增偏差的情况下放大整个基因组/甲基组(DNA)或转录组(RNA)的技术。随后，它将建立标准，并将验证扩增材料作为最常用的分子分析(突变/SNP检测、微卫星不稳定性/杂合性缺失、阵列-CGH、表达谱分析和甲基化检测)的实用性。通过消除与样品降解相关的问题和与扩增相关的偏差，该项目将使最新技术能够应用于从已知结果的存档组织中进行微小活检的分析，从而加快候选基因发现的进程。