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
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
Small Business Information at Submission:
Research Institution Information:
390 WAKARA WAY SALT LAKE CITY, UT 84108-1214 SALT LAKE CITY, UT 84108
Number of Employees:
UNIVERSITY OF UTAH
UNIVERSITY OF UTAH
SALT LAKE CITY, UT 84132
Nonprofit college or university