Detection of K-ras Colorectal Point Mutations Using Endonuclease V/AK16DLigase and Ligase Detection Reaction Assays

Abstract

This research investigates the benefits of utilizing PCR/Ligase Detection Reaction multiplexing coupled with endonuclease V/AK16D ligase treatment in the detection of known and unknown point mutations within DNA. The research focused specifically on the K-ras gene, implicated in the development of certain cancers including colorectal cancer. Identification of point mutations within this gene could lead to early detection of cancer, as well as identify those with a predisposition to develop cancer. An LDR technique was utilized to identify specific, known mutations within the gene sequence, while the endonuclease/ligase treatment identified unknown mutations within the gene sequence. Capillary electrophoresis and slab gel electrophoresis were employed to distinguish the products of both EndoV/ligase and LDR, respectively, based on the fragments’ size differences. These methods have been found to be highly sensitive and have high throughput. Further research should explore the potential to transfer the gel electrophoresis separation to a microelectrophoretic device with the potential for clinical applications.