DEVELOPMENT OF A HOMOGENEOUS GENOTYPING METHOD

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Single nucleotide substitutions are the most frequent form of polymorphism and disease-causing mutations in the human. An efficient method to identify and type these mutations will have many applications in both clinical diagnostics and genetics such as: prenatal diagnosis of genetic diseases, HLA-typing prior to transplantation, and forensics. The ability to type large numbers of genetic markers in large populations will accelerate the discovery of genes important in complex human diseases such as adult-onset diabetes, coronary artery disease, and cancer. This proposal aims to develop a high-throughput, homogeneous genotyping method based on minisequencing and fluorescence energy transfer. Fluorescent resonance energy transfer (FRET) is a physical phenomenon that provides a unique way to monitor the distance between two fluorescent dyes. Using FRET as a reporting system, one can monitor the incorporation of a specific dye-labeled nucleotide in the minisequencing genotyping reaction without having to separate or purify the DNA primer extension products. We also plan to couple PCR and minisequencing to further simplify the protocol. We will explore treatment of PCR products with shrimp alkaline phosphatase and exonuclease I as a way to inactivate excess PCR primers and dNTPs prior to the minisequencing reaction. If successful, we will have developed a one-vessel, homogeneous genotyping assay amenable to full automation, thereby opening up the way to truly large-scale population studies using genetic markers.