GENOME AMPLIFICATION TOLERANT TO SAMPLE DEGRADATION

描述（由申请人提供）：对具有已知临床结果的福尔马林固定石蜡包埋（FFPE）存档组织样本进行基因组、表观遗传和基因表达分析，为提取遗传信息提供了独特的机会，从而改善癌症诊断、预后和治疗。然而，对这些样品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选之前进行全基因组/mRNA扩增。这一过程的主要障碍是扩增偏倚的引入和福尔马林固定对DNA/RNA扩增的抑制作用。我们已经开发了（RCA-RCA），一种基于等温滚环扩增的新方法，该方法克服了局限性，并有望提供从部分降解的FFPE样品获得微小活检与基因分型或微阵列筛选之间的必要联系。RCA-RCA能够实现全基因组/mRNA扩增，其可以根据FFPE样品降解的程度进行调整，因为这是通过真实的时间PCR进行评估的。因此，RCA-RCA能够从劣化样品中检索最大可能量的信息。在修订后的应用程序中，除了采纳研究组的建议外，还包括RCA-RCA的进一步增强，即mRCA-RCA。mRCA-RCA扩增DNA，同时保留全基因组基础上的表观遗传修饰（“全甲基化组扩增”），从而允许在新鲜或FFPE样品中高度扩增甲基化检测。R21阶段将检查该技术的最大能力，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样本中获得微小癌症活检的技术，评估样本质量，并在不引入扩增偏倚的情况下扩增全基因组/甲基化组（DNA）或转录组（RNA）。随后，它将建立标准，并将验证扩增材料作为最常用的分子测定（突变/SNP检测、微卫星不稳定性/洛、阵列CGH、表达谱和甲基化检测）的输入的效用。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分析来自存档组织的具有已知结果的微小活检，从而加速候选基因发现的过程。

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.