GENOME AMPLIFICATION TOLERANT TO SAMPLE DEGRADATION

描述（由申请人提供）：从已知临床结果的存档的福尔马林固定石蜡包埋（FFPE）组织样本中进行基因组、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，从而改善癌症诊断、预后和治疗。然而，在这些样本中对均匀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差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），一种基于等温滚动圈扩增的新方法，克服了局限性，并有望提供从部分降解的FFPE样本中获得一分钟活检和基因分型或微阵列筛选之间所需的联系。RCA-RCA使全基因组/mRNA扩增可以调整到FFPE样品降解的程度，因为这是通过实时PCR评估的。因此，RCA-RCA能够从退化的样品中检索尽可能多的信息。在修订后的申请中，除了采纳研究组的建议外，还包括对RCA-RCA的进一步改进，即mRCA-RCA。mRCA-RCA扩增DNA，同时在全基因组基础上保留表观遗传修饰（“全甲基组扩增”），从而允许在新鲜或FFPE样品中高度扩展甲基化检测。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/甲基组（DNA）或转录组（RNA）。随后，将建立标准并验证扩增材料作为最常用分子分析（突变/SNP检测、微卫星不稳定性/LOH、阵列- cgh、表达谱分析和甲基化检测）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具有已知结果的存档组织的微小活检，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.