We endeavor to advance the use of molecular diagnostic methods for the analysis of urine and other body fluids. Our research has focused on developing robust techniques for identifying DNA sequence changes in order to perform rapid screening for somatic mutations. Techniques were developed to amplify disease-related DNA sequences in human genomic DNA with the polymerase chain reaction (PCR), and to detect mutations by single-stranded conformational polymorphism (SSCP) analysis. In order to be compatible with clinical laboratory testing, our PCR-SSCP methods are non-isotopic and partially automated. We have succeeded in screening for mutations in the human p53 tumor-suppressor gene, the K-ras oncogene, as well as in the thyroid hormone receptor-beta gene. Using DNA from cell lines, tumors, and blood, the rate of mutation detection by our SSCP method was compared to that obtained by the Sanger sequencing method. In addition, we recently began using DNA hybridization strategies to detect K-ras mutations. Since our objective is to detect mutations in DNA samples obtained from heterogeneous cell populations, we are developing enrichment procedures which should significantly improve our ability to find mutations in body fluids. Our immediate goal is to refine existing technology to perform high- throughput mutation detection with the quality and sensitivity requisite for clinical testing. Subsequently, we wish to test our methods with clinical samples in order to validate molecular screening for early-stage tumors.
