Towards fluorescence anisotropy detection for real-time PCR assays

Real-time PCR methods play a valuable role in the characterization of mutations as well as gene expression studies. Existing techniques such as TaqMAN/sup /spl trade// and Molecular Beacons/sup /spl trade// employ specially designed oligonucleotides whose fluorescent resonance energy transfer (FRET) is affected by the reaction progression. Such methods have proven repeatable and accurate, however the challenges in design and cost of these reagents are limiting for high throughput applications. Other less expensive techniques, such as the use of intercalating probes, lack the specificity to allow simultaneous detection of multiple alleles. To overcome these limitations, we are developing an alternative real-time PCR detection technique based on fluorescence anisotropy (FA) that relies only on fluorescently labeled forward primers. Previous work on FA and PCR has highlighted several barriers to their combination. Here we analyze these challenges and study the specifications for instrumentation to overcome them. We report the construction of an apparatus testing these specifications in which very low noise FA measurements have been made throughout the progress of the PCR procedure. This work demonstrates the feasibility of FA detection for reduced cost analysis of real-time PCR assays for high throughput applications.