Core--Molecular genetics

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This core unit provides crucial molecular resources for each of the research projects, including: preparation and cataloging of DNA sample preparation and storage, genotyping, physical mapping and genetic marker development, informatic support, DNA sequencing, and identification of polymorphisms at targeted loci as candidate functional changes. 1. Preparation and storage of genomic DNA (gDNA). Consistent and reliable DNA samples for genotyping (in analyses of linkage, association, and candidate functional sequence changes) are essential to Projects 1 and 2. We collect 50 ml of EDTA-anticoagulated whole blood from each individual in the clinic. We prepare gDNA from the white cells in small amounts and the remaining blood is stored frozen for future use. 2. DNA Sequencing. Strategy. Each of Projects 1-5 has suggested biological candidate genes as potential contributors to genetic variation in autonomic control of blood pressure. Testing these candidate genes requires linkage, association, and haplotype analysis of known markers and more particularly of single point and haplotype alleles enriched in hypertensive subjects and their first-degree relatives. 3. Genotyping. Strategy and Methods. As described under Sequencing above, we use a two-tiered strategy to maximize efficiency. (1) The first tier involves direct PCR-based resequencing of candidate loci to improve the density and appropriateness of polymorphism selection for larger-scale genotyping. (2) In the second tier of the strategy, derived marker combinations are selected and used to allow efficient discrimination of haplotypes. Obtained genotype data is subsequently analyzed for statistical significance and population parameters in the informatics core, Core B (Informatics and statistical genetics), including additional population-based controls for likely accuracy. 4. Allele-specific expression analysis. Strategy. To detect the effects of allelic variants define in this program on expression level, this core will provide expression analysis based on allelic detection from genotyped samples and heterologous promoter fusions.