MOLECULAR BIOLOGY

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Core A is the molecular biology component of the Program. It will have four important functions: 1) Test all biopsies and eyebrow hair samples for the presence of beta HPV DMA. 2) Test sera for the presence of antibodies to beta HPVs. 3) Develop and maintain computerized databases that will be used to track tissue samples and mice, to generate work lists and to link histology and laboratory test results with individual mice or specimen. 4) Archive plasmids, cell lines and retroviruses. In addition this Core has a developmental component to establish a multiplex, high-throughput assay to detect HPV DMAs. For the detection or beta HPV DMAwe will be amplifying beta HPV DMA using a set of primers from a conserved region in the E1 open reading frame. The amplified DMAwill be cloned and sequenced (10 clones per sample). Blast searches will be use to identify the HPVs as known or novel HPVs. For the detection of antibodies to HPVs, we will adopt a method that has recently been established in our laboratory that has the ability to test for antibodies to numerous alpha, beta, gamma and mu HPV types simultaneously, and to do so in a highly sensitive and specific fashion. This method uses Luminex technology in which GST- LI proteins are coupled to colored polystyrene beads. The beads are mixed together, along with serum and a standard detection reagent, and the Luminex reads the amount of antibody bound to each bead. The greatest strengths of this Core are the laboratory personnel, the outside experts that have agreed to consult with us on the development and implementation of testing protocols, and the excellent facilities available at the Fred Hutchinson Cancer Center. All of Core A personnel have strong track records and extensive experience in the work required for this Core.