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. ? ? ?
