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

• 批准号: 7201948
• 负责人: CHARLES D LAIRD
• 金额: $ 25.81万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-09-30 至 2009-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7201948
• 关键词: CpG islands; DNA methylation; epigenetics; female; genetic mapping; genetic regulation; human genetic material tag; human population genetics; leukocytes; method development; nucleic acid probes; nucleic acid quantitation /detection; polymerase chain reaction; single nucleotide polymorphism; sulfites

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（由申请人提供）：DNA 甲基化和单核苷酸多态性 (SNP) 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，可以准确揭示表观遗传 DNA 甲基化模式和单个细胞等位基因的遗传信息。我们之前已经获得了少数基因座的单个等位基因启动子的遗传和表观遗传信息。我们已经证明鸟枪发夹-亚硫酸氢盐 PCR 对于人类 LINE-1 (L1) 基因座是可行的，并建议将此方法扩展到包含 CpG 岛的启动子区域。我们的“鸟枪发夹-亚硫酸氢盐 PCR”方法是通过结合 Pi 实验室最近开发的实验和分析方法而实现的。这些方法包括 (i) 发夹-亚硫酸氢盐 PCR 以确定双链 DNA 甲基化模式和单个 DNA 分子的 SNP； (ii) 将发夹接头与 L1 的 CpG 岛进行鸟枪式连接，以捕获许多 L1 位点的广泛代表性； (iii) 对单个基因组 DNA 片段进行批量标记和条形码，以验证每个已测序分子； (iv) 群体表观遗传模型，用于定量和统计分析位点特异性甲基化模式。需要资金在正常人类白细胞中筛选约5000个基因启动子，覆盖约15-30%的人类CpG岛。我们的创新策略将启动对正常和患病细胞的表观遗传和遗传变异组合的更系统的表征。