NOVEL ULTRASENSITIVE COLORIMETRIC ASSAY FOR DNA

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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.