The power of the PCR reaction lies in its ability to amplify vanishingly small amounts of DNA to produce quantities sufficient for measurement. Unfortunately, amplification typically produces errors that become greater with increasing number of cycles so that accuracy is lost before accurate measurement is possible. The proposed solution is to reduce the need for amplification by employing more sensitive detection. SeaLite Sciences has developed a flash type bioluminescent tag, AquaLite. The great sensitivity of AquaLite detection has made it possible, in preliminary studies, to develop highly accurate, rapid and inexpensive quantitative PCR and RT-PCR assays. This project will marshal the expertise of the developers of AquaLite with investigators with many years experience in PCR and RT-PCR reactions to perfect these assays to the point that they can be automated and used with the ease and precision of contemporary immunoassays. The specific aims are first, to develop an assay that can quantitatively measure specific DNA in 4 hours with a coefficient of variations less than 15%. And second, to develop an RT-PCR assay for quantitative analysis of cytokine message from peripheral blood cells of needle biopsy tissue. The project will proceed via a series of steps to optimize PCR reaction, determine optimal means of capturing DNA on micrometer plates, optimize hybridization conditions, optimize probe detection and optimize efficiency of the bioluminescent reaction. The second aim will be approached by evaluating methods for extraction of RNA from tissue, standardization of RNA concentration necessary to detect PCR products, optimization of bioluminescent detection and identification of ideal RT-PCR controls. In Phase II, these methods will be used to develop commercial assays for cytokines and other analytes.
