Shotgun Hairpin-Bisulfite PCR Reveals Genome Methylation and Sequence Variation

描述（由申请方提供）：DNA甲基化和单核苷酸多态性（SNP）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，将准确地揭示表观遗传DNA甲基化模式和遗传信息的等位基因的个别细胞。我们以前已经获得了遗传和表观遗传信息的启动子的个别等位基因的一小部分基因座。我们已经证明，鸟枪发夹-亚硫酸氢盐PCR是可行的人类LINE-1（L1）基因座，并建议将这种方法扩展到启动子区含有CpG岛。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。所述方法包括（i）发夹-亚硫酸氢盐PCR，以确定单个DNA分子的双链DNA甲基化模式和SNP;（ii）发夹接头与L1的CpG岛的鸟枪连接，以捕获许多L1基因座的广泛代表性;（iii）单个基因组DNA片段的批量标记和条形码化，以鉴定每个测序的分子;（iv）群体表观遗传建模，以定量和统计地分析位点特异性甲基化模式。需要资金来筛选正常人类白细胞中约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一个更系统的表征结合表观遗传和遗传变异的正常和病变细胞。

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.