Phase I of this proposal proved the feasibility of detection of the factor V Leiden mutation in human blood and correct determination of sample genotype as compared to a reference PCR method. The protocol used a new nucleic acid ligation-based technology, LMO amplification, invented at Lumigen. The LMO technology is based on the contiguous ligation of a set of short oligonucleotides to a template-bound primer under conditions in which the short oligonucleotides are not hybridized to the template. We demonstrated successful amplification, adequate sensitivity, and evaluated several different amplification designs. The prototype assay is as rapid as many existing techniques for detecting mutations. The Phase I results have laid the groundwork for the development and standardization of research kits for factor V using a microtiter plate format in Phase II. We will expand this work to develop a rapid test and kits for discrimination the three possible genotypes in a two-stage test. This test will utilize dual fluorescer energy transfer (FRET) assays. Detection will be based on energy transfer from a donor fluorophore-labeled oligomer to an acceptor fluorophore-labeled oligomer which become incorporated via the ligation-amplification process. Successful implementation of FRET-based detection will significantly shorten time to result. We will use the expertise gained in Phase I to develop two additional commonly used mutation tests related to clotting disorders, Factor II and MTHFR. As a final goal we will devise a multiplex test format for screening blood samples for each of these tests simultaneously. Use of different color fluorescers will distinguish the three mutations. ? ?
