SBIR-STTR Award

The objective of Phase 1 is to develop a sensitive test and kit for detection of the Factor V Leiden mutation in human blood using a new nucleic acid ligation-based technology invented at Lumigen. The ligation technology is based on the discovery that a multitude of short oligonucleotides can be contiguously ligated to a template-bound anchor sequence performed under conditions in which the short oligonucleotides do not stably hybridize to the template. The absence of stable hybridization of the short oligonucleotides during the ligation confers extremely high replication fidelity. Incorporation of detectable labels on some or all of the short oligonucleotides permits the design of molecular assays with excellent specificity and reliability of detection. We will use a small hapten label to be detected with an enzyme-antibody conjugate and a chemiluminescent enzyme substrate. PROPOSED COMMERCIAL APPLICATION: The proposed test methods and kits should provide the basis for developing a set of detection methods and products for analysis of many different SNPs. The tests involve a new amplification process which should be more specific than existing amplification technology while achieving comparable speed and sensitivity. The tests should be capable of automation on a high throughput platform including chip formats. The unique properties of this methodology have resulted in the creation of a new nucleic acid amplification technology which is more specific than PCR and yields cleaner products. We will use this amplification technique to enhance the sensitivity of the method to detect the single-nucleotide polymorphism (SNP) associated with this mutation. The feasibility of developing a single test for discrimination of all three possible genotypes of this mutation will also be explored. This test will utilize one of two patented methods developed at Lumigen for detection of two enzyme labeled analytes using proprietary chemiluminescent substrates.

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.

Thesaurus Terms:
diagnosis design /evaluation, diagnostic test, genetic disorder diagnosis blood coagulation disorder, coagulation factor V, fluorescent dye /probe, gene mutation, method development, prothrombin fluorescence resonance energy transfer