GENOTYPING

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The purpose of Core D is act as a centralized facility to perform large scale and high throughput genotyping of genetic polymorphisms as well as routine DNA sequencing. The advantages for such a centralized facility include: 1) Conservation of resources; the core is more time and cost efficient than individual labs; 2) Avoid unnecessary duplication of effort: 3) Standardization of quality control; and 4) Facilitation of data sharing. In addition, the Core will also function as an educational resource to train members of the Program Project laboratories in proper genotyping methodologies and to provide advice for Project Leaders in the design and implementation of relevant experiments. The training will be valuable for Postdoctoral Fellows and Graduate Students and useful if the demands on the Core exceed its capacity at a particular time. The Genotyping Core will provide one ABI Prism 7900HT Genetic Analyzer with SDS 384-well Block, and a semi-automatic robotic system, Hydra II for high throughput SNP genotyping. We will use the 5' nuclease allelic discrimination assay for large-scale, high throughput genotyping of SNPs. All SNP assays will be tested for their accuracy and reproducibility. We have developed a strict protocol for evaluation of each SNP assay where the SNP of interest is identified by the Wave system or direct sequencing. SNPs chosen from published databases will be first confirmed by direct sequencing, followed by testing for the robustness and reproducibility of the assay. If the SNP passes these tests, it will be genotyoed in the rest of study samples. For further quality control, we include two negative controls (no DNA), two wells of allele 1 standard, and two wells of allele 2 standard. The Genotyping Core represents an integral part of the Program Project. Projects 1 and 2 will use the SNP detection service extensively. The other projects will make use of the DNA sequencing facility for analysis of plasmid probes and cDNA cloning (Project 3).