Detection of Sporadic Genetic Mutations Using an EndoV/Ligase Scanning Assay

Abstract

The identification of sporadic point mutations in tumor-suppressor genes, which are responsible for regulating cell growth, has been linked to the early detection of cancer, as well as with identifying those individuals with a genetic predisposition to developing cancer. My research investigates the use of a single-step endonuclease V/ligase scanning assay in the detection of unknown point mutations in genomic DNA. Specifically, the research focuses on the K-Ras gene, which has been linked to several different types of cancer, including colorectal cancer. A universal PCR strategy using fluorescently labeled universal primers and unlabeled gene specific primers allowed for amplification ofK-Ras oncogene sequences. Amplification was followed by heteroduplex generation from the universal PCR products. This procedure resulted in products with one of two mismatches (A/C or G/T) that could be used for endonuclease treatment. Endonuclease V recognizes and cleaves base pair mismatches within the gene sequence, but it also cleaves some correctly paired bases. A highly specific ligase was used to reseal these miscleaves, thus increasing signal intensity. Capillary gel electrophoresis was employed to distinguish the single-stranded products of the EndoV/ligase assay based on fragment-size differences. The early detection and identification of mutations could lead to increased survival rates for individuals testing positive for those mutations. Further research should include the potential to transfer the assay to a microelectrophoretic device with possible clinical applications.