HOMOGENEOUS MULTIPLEX PCR BY FLUORESCENCE AND TM

DESCRIPTION (applicant's abstract): Our long-term goal is to advance techniques and instrumentation for rapid, automated analysis of nucleic acids during amplification. Our approach is to add fluorescent probes before amplification and analyze the results in real-time, completing the entire process in 10-20 min. No additional sample processing, membranes, arrays, or gels are necessary. In Phase I, we demonstrated the feasibility of a new solution multiplexing technique that uses both probe melting temperature (Tm) and color. In Phase II, our specific aims are to: 1. Prototype a flexible multi-spectral rapid thermal cycler that can monitor any visible fluorescent dye or combination of fluorescent dyes. Design and produce a small, inexpensive, multi-spectral rapid thermal cycler for the commercial market. 2. Provide software to select hybridization probes for multiplex analysis at highly polymorphic loci based on probe Tm. 3. Demonstrate multiplex analysis with "virtual" 2-dimensional arrays of color and Tm. Instead of physical separation, probe color and Tm are used to identify products. Human HLA loci will serve as highly polymorphic targets. When both probe color and Tm are used to identify products, high-order multiplexing can be performed in solution. PROPOSED COMMERCIAL APPLICATION: We propose a small footprint, rapid, inexpensive fluorescent thermal cycler with continuously variable excitation and emission wavelengths. This will be the first real-time instrument compatible with all visible/near IR fluorescent probes and amplification techniques. Multiplexing by fluorescence and Tm greatly expands the power of real-time nucleic acid analysis.