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2005
LSU-HHMI Summer Undergraduate Research Program |
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Jillian Bybee (Carroll College) and Steven Soper, Chemistry
Detection of Sporadic Genetic Mutations Using an EndoV/Ligase Scanning Assay
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. Our 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
of K-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 mismatches, as well as some correctly paired bases,
within the gene sequence. A highly specific ligase was used
to reseal miscleaves, which eliminates artifacts, 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. |
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