The primary goal of the contract is to provide development and application of molecular assays on human specimens gathered in epidemiologic studies. These studies address whether there are different patterns of DNA damage in tumors initiated by different carcinogens, and the role of gene-environment interaction in disease. Although no one study will involve all assays, the contractor will be working with a variety of molecular techniques. Basic techniques include the following: 1. Extract high molecular weight DNA from frozen tumor tissue and from frozen samples of peripheral white cells, and from microtome slices of fixed tissues, along with histologic analysis and microdissection of tumor samples. 2. PCR amplification of selected genes, e.g., p53. Ability to amplify DNA from microdissected fixed tissue samples is particularly important. 3. Single strand conformation polymorphism (SSCP) analysis of amplified DNA to screen for mutations. 4. Direct sequencing fo PCR amplified DNA with sensitivity adequate to identify mutants present in 1 of 10 cells as a standard minimum. 5. Assay for loss of hetrozygosity in tumor compared to normal tissue samples. 6. PCR-RFLP analysis of known allelic variants of genes.
