Research Project
6335572
DESCRIPTION (Applicant's Abstract): 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.
