SBIR-STTR Award

Genetic markers generated by DNA sequence polymorphism with restriction enzymes have aided in constructing a detailed genetic linkage map for the human. More than 400 polymorphic DNA markers for human chromosomes have been described. These include some human genetic disorders such as Duchenne muscular dystrophy, polycystic kidney disease, and cystic fibrosis. As additional genetic markers are generated by RFLP analysis, more human genetic characteristics will be identified and diagnosed for disorders. A novel method is proposed for efficient and non-isotopic RFLP analyses. Synthetic oligomeric DNA probes will be prepared and labeled with fluorophores that are unique to each probe. The labeled probes will be simultaneously hybridized to genomic DNA fragment generated with a restriction enzyme. The hybridized probes will serve as primers for the sequence to elongate DNA. The elongated DNA fragments will be separated by HPLC using a newly introduced Gen-Pak(TM) FAX column, and will be detected with a fluorescence detector. The data will be compared to the Southern blot technique. The proposed analytical method is novel, efficient, and safe for large numbers of routine RFLP analyses.

Thesaurus Terms:
Biology, polymorphism, chemistry, analytical methods, chemistry, analytical methods, spectrometry, fluorescence, genetic mapping, genetic markers, human, clinical, physical separation, chromatography, high performance liquid chromatography chemical synthesis, design and production, nucleic acids, dyes, fluorescent dyes and probes, genetic mapping, linkage, genetics, chromosome complement, genome, nucleic acids, dna, nucleic acids, nucleic acid probes, nucleotides, oligonucleotides, nucleic acid hybridization nucleases, endonucleases, nucleic acids synthesis, dna, nucleotidyltransferases, dna nucleotidyltransferases, physical separation, electrophoresis, gel, plants, crops and foods, grains, corn

RFLP analysis has been shown to have tremendous implications for both the diagnosis of genetic tendencies for human disease and for applications to plant the capacity of what is primarily a research technology. A replacement technology is desired that is faster, cheaper, and with a higher throughput but yet at the same time yields comparable information. NPI has developed a PCR-based approach which appears to satisfy many of these criteria. This process delivers information as to the DNA sequence variability between individuals at specific loci which can then be used analogously to RFLP results. Reduction to practice requires that a better understanding of amplification kinetics be developed, exact conditions for sequence discrimination be further refined, and implementation of a capture-signal strategy is essential to remove the necessity for radioactive labels and gel analysis. The resulting process should require less than a day for completion, be cheaper by ten-fold over conventional less analysis, and facilitate the analysis of several loci for many samples, all in a laboratory with personnel much less sophisticated than now required.

Thesaurus Terms:
Biomedical engineering, technology development, chemical synthesis, design and production, nucleic acids, polymerase chain reaction (pcr), genetics, plants, nucleic acids sequencing genetic mapping, genetic markers, genetics, chromosome complement, genome, genetics, population genetics, polymorphism, restriction fragment length polymorphism (rflp), nucleic acids, dna, veterinary science, animal breeding, genetic mapping