The research performed in Phase I of this project will demonstrate the feasibility of performing a kinetic DNA hybridization assay on a fiber optic sensor (FOS) with fluorescence detection. The FOS is mounted within a temperature controlled reaction cell. DNA sequences will be amplified using the polymerase chain reaction (PCR) and hybridized to specific probes immobilized to fiber optic sensors. The hybridization event will be monitored by measuring the fluorescence emission of labeled nucleic acids (dNTPs) that are used in PCR. Phase II will incorporate an internal standard (IS) sequence which is coamplified along with the target sequence. The IS hybridizes to a different probe on the same FOS. Using fluorescence energy transfer and a two-color fluorescence detector, IS allows normalization of the amplification event. The prototype instrument will be self-contained, requiring only the application of sample to a reaction cell that contains the FOS and the expendable biochemical reagents. This system will allow quantitative PCR for viruses and other infectious agents, determination of metastasis, mutational analysis and other genetic determinations. The FOS will be reusable and readily calibrated, and probes that are developed as a model system will allow for quality control of manufacturing.
This integrated nucleic acid amplification and detection system provides features not found in available methods, including in situ amplification, real-time monitoring of hybridization in a homogeneous assay format, quantification by means of an internal standard, re-use of probes, ease of quality control, and instrument portability. The system can be adapted to a variety of amplification formats, and has the potential for use in genetics research laboratories and for clinical determinations in hospital labs.
