SBIR-STTR Award

The detection of specific DNA sequences for the diagnosis of genetic diseases, cancer, and infectious disease is becoming increasingly widespread. A number of innovative techniques have been employed, including the oligonucleotide ligation assay and reverse dot-blot hybridization. The end-point, and to a certain extent the effectiveness of these assays is determined by the sensitivity of the DNA detection techniques used. Progress has been significantly enhanced through the use of the polymerase chain reaction (PCR). However, implementation of PCR can be expensive, as well as equipment and expertise intensive. We have applied a technique with proven effectiveness in the field of immunoassays for the non-isotopic detection of DNA; initial data indicates an increase in sensitivity 10(2)-10(3) times higher than conventional techniques. The assay, which we have called EDNA-ELCA, uses the amplification of the coagulation cascade to produce a colorimetrically detectable end product. A DNA template is labelled with biotin and a haptene, and is then immobilised in a streptavidin coated microtiter plate. An antibody against the haptene is conjugated to a snake venom coagulation activator (RVVXa), and introduced into the microtiter well. The assay is then developed by the addition of coagulation factors, including an enzyme labelled fibrinogen. The deposition of enzyme labelled fibrin is indicative of a positive reaction. In this proposal we have chosen three specific applications to prove the utility of this method. They are detection of the deltaF508 mutation of cystic fibrosis, a codon 12 mutation in the first exon of K- ras for colon cancer, and the mecA gene in methicillin resistant Staphlococcus aureus.

Thesaurus Terms:
DNA, blood coagulation, colorimetry, method development, polymerase chain reaction binding protein, biotin, clotting factor, fibrinogen, gene mutation, reptile poison, thrombin human subject

We describe an ultrasensitive calorimetric assay for DNA, the EDNA-ELCA, which uses amplification of the common pathway of coagulation to produce a calorimetrically detectable product. The EDNA-ELCA enables detection of attomole amounts of DNA in a microtiter plate, with a sensitivity 200- 1000 time higher than conventional ELISA techniques. The assay has been applied to quantitative detection of PCR amplified DNA, and combined with the oligonucleotide ligation assay (OLA) for the detection of mutations associated with the genetic diseases sickle cell anemia and cystic fibrosis, and the detection of specific mutations in the kras oncogene associated with colorectal cancer. We have also applied the technique to DNA hybridization assays. We have modified the technique for presentation as a dipstick assay which provides a rapid, sensitive, calorimetric assay with definitive results without specialized equipment. In this proposal we will continue to develop the dipstick assay format by further simplification, stabilization and presentation. We will also use the technology for rapid and sensitive visualization of specific electroblotted DNA sequences, and continue development of the assay for microtiter plate based hybridization, with or without PCR amplification. We have chosen three applications which will demonstrate the broad utility of the assay. These are 1) detection of known mutations in colorectal cancer in tissue and stool samples, 2) quantitation and strain determination of hepatitis C virus in blood and 3) the detection of human papilloma virus strains related to cervical cancer in smears and biopsies. These applications will make use of the strengths of the technology i.e. high throughput, sensitivity and simplicity, while allowing the development of different aspects of the technique. PROPOSED COMMERCIAL APPLICATION: This assay system would allow routine screening for DNA sequences, gen mutations or viral sequences specific for certain diseases. The assay could be automated and/or simplified to an extent where it will be suitable for a high throughput clinical laboratory or an office setting. Implementation of these approaches may greatly increase the accuracy of early cancer detection techniques. The assay is also suitable for research or diagnostic purposes in specialty research laboratories.

Thesaurus Terms:
DNA, colorimetry, method development, nucleic acid sequence blood coagulation, cervix neoplasm, colorectal neoplasm, communicable disease diagnosis, cystic fibrosis, feces, gene mutation, hepatitis C virus, human papillomavirus, neoplasm /cancer genetics, oligonucleotide, oncogene, sickle cell anemia, virus genetics biopsy, gel electrophoresis, human subject, nucleic acid hybridization, polymerase chain reaction