Allelic expression monitoring by array-based genotyping

描述（由申请人提供）：本项目的目标是开发用于测量等位基因表达的定量eDNA基因分型平台。这将为更好地理解基因组的转录调控提供工具。这种调节的功能障碍导致许多疾病状态，包括癌症。特别是，癌症基因经常被遗传和表观遗传机制激活或失活，导致细胞生长不受控制。表观遗传机制导致异常转录控制包括CpG岛甲基化、印记丢失和染色质修饰。所提出的eDNA基因分型方法包括将cDNA与DNA寡核苷酸阵列直接杂交，然后通过基于阵列的等位基因特异性引物延伸反应进行基因分型。除了等位基因的表达，阵列的效用在标准的基因表达定量也将被证明。将探索cDNA的全基因组扩增作为提高低拷贝数转录物的基因分型准确性的手段。在第一阶段，我们将在Sentrix TM Array of Array TM平台上使用一个模型系统，该系统采用了数百个基因中的数百个eDNA SNP。在第二阶段，eDNA阵列将扩展到我们的Sentrix BeadChip平台，包括数千个基因的全基因组分析。为了能够容易地进行等位基因频率校准，还将探索eDNA阵列上gDNA的直接基因分型。这些努力的结果将导致匹配的正常/肿瘤组织的全基因组等位基因表达研究，以确定参与肿瘤发展的基因位点。

DESCRIPTION (provided by applicant): The goal of this project is to develop a quantitative eDNA genotyping platform for measuring allelic gene expression. This will provide a tool for better understanding transcriptional regulation of the genome. Dysfunction in this regulation leads to a number of disease states including cancer. In particular, cancer genes are often activated or inactivated by genetic and epigenetie mechanisms leading to uncontrolled cell growth. Epigenetie mechanisms leading to aberrant transcriptional control include methylation of CpG islands, loss of imprinting, and chromatin modification. The proposed approach to eDNA genotyping involves direct hybridization of cDNA to a DNA oligonucleotide array and subsequent genotyping by an array-based allele-specific primer extension reaction. In addition to allelic expression, the array's utility in standard gene expression quantitation will also be demonstrated. Whole genome amplification of cDNA will be explored as a means to improve genotyping accuracy for low copy number transcripts. In phase I, a model system employing several hundred eDNA SNPs across a few hundred genes will be used on our Sentrix TM Array of Array TM platform. In phase II, the eDNA array will be scaled to our Sentrix BeadChip platform encompassing whole genome analysis of thousands of genes. To enable easy allele frequency calibration, the direct genotyping of gDNA on the eDNA array will also be explored. The culmination of these efforts will result in the whole genome allelic expression study of matched normal/tumor tissues to identify gene loci involved in tumor development.