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.
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.
|Rothschild, C B; Brewer, C S; Loggie, B et al. (1997) Detection of colorectal cancer K-ras mutations using a simplified oligonucleotide ligation assay. J Immunol Methods 206:11-9|