权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Shotgun Hairpin-Bisulfite PCR Reveals Genome Methylation and Sequence Variation

鸟枪发夹-亚硫酸氢盐 PCR 揭示基因组甲基化和序列变异

基本信息

批准号：
7491490
负责人：
CHARLES D LAIRD
金额：
$ 25.75万
依托单位：
UNIVERSITY OF WASHINGTON
依托单位国家：
美国
项目类别：
财政年份：
2006
资助国家：
美国
起止时间：
2006-09-30 至 2010-08-31
项目状态：
已结题

项目摘要

DESCRIPTION (provided by applicant): Both DNA methylation and single nucleotide polymorphisms (SNPs) are considered major contributors to human phenotypic variation. We propose a genomic approach that will accurately reveal both epigenetic DNA methylation patterns and genetic information for alleles of individual cells. We have previously obtained both genetic and epigenetic information for promoters of individual alleles for a small number of loci. We have demonstrated that shotgun hairpin-bisulfite PCR is feasible for human LINE-1 (L1) loci and propose to extend this approach to promoter regions containing CpG islands. Our "shotgun hairpin-bisulfite PCR" approach is made possible by combining experimental and analytical methods recently developed in the Pi's laboratory. The methods include (i) hairpin-bisulfite PCR to determine double-strand DNA methylation patterns and SNPs for individual DNA molecules; (ii) shotgun ligation of hairpin linkers to CpG-islands of L1s to capture a broad representation of many L1 loci; (iii) batch-stamping and barcoding of individual genomic DNA fragments to authenticate each sequenced molecule; (iv) population-epigenetic modeling to analyze site-specific methylation patterns quantitatively and statistically. Funds are requested to screen about 5000 gene promoters in normal human leukocytes, covering approximately 15-30% of human CpG islands. Our innovative strategy will initiate a more systematic characterization of combined epigenetic and genetic variations in normal and diseased cells.

描述(申请人提供)：DNA甲基化和单核苷酸多态(SNPs)都被认为是人类表型变异的主要贡献者。我们提出了一种基因组方法，它将准确地揭示表观遗传DNA甲基化模式和单个细胞等位基因的遗传信息。我们以前已经获得了少数基因座个体等位基因启动子的遗传和表观遗传学信息。我们已经证明了对人类LINE-1(L1)基因座进行鸟枪式发夹-亚硫酸氢盐聚合酶链式反应是可行的，并建议将该方法扩展到包含CpG岛的启动子区域。我们的“鸟枪式发夹-亚硫酸氢盐聚合酶链式反应”方法是通过结合实验和分析方法而实现的，这些方法是最近在PI的实验室开发的。这些方法包括(I)发夹-亚硫酸氢盐聚合酶链式反应，以确定单个DNA分子的双链DNA甲基化模式和SNPs；(Ii)将发夹连接到L1s的CpG岛上，以捕捉许多L1基因座的广泛代表性；(Iii)对单个基因组DNA片段进行批量标记和条形码编码，以验证每个测序的分子；(Iv)群体表观遗传学建模，以定量和统计地分析特定位点的甲基化模式。需要资金在正常人白细胞中筛选约5000个基因启动子，覆盖大约15%-30%的人类CpG岛。我们的创新战略将启动对正常和疾病细胞中表观遗传和遗传变异组合的更系统的表征。