( Larissa Tan Marni Brisson R...)Real-Time PCR/Melt-Curve Analysis: SNP Detection With FRET, Rev A

Larissa Tan, Marni Brisson, Robert Park, and Keith Hamby,
Bio-Rad Laboratories, 2000 Alfred Nobel Drive, Hercules, CA 94547 USA

Introduction
The detection of fluorescence resonance energy transfer (FRET) through real-time polymerase chain reaction (PCR) and melt-curve analysis is a powerful tool for the identification of single nucleotide polymorphisms (SNPs). The frequency of SNPs in association with disease makes the identification of specific sequence differences important. Through the use of real-time PCR and melt-curve analysis, genotyping can be done more rapidly than by the conventional method of restriction enzyme digestion of PCR products.

Several probe designs can be utilized for SNP detection including molecular beacons, TaqMan minor groove binder probes, and single-labeled probes. The technique described below involves the use of a Cy5-labeled reverse primer that is incorporated into the amplified PCR product. A sequencespecific 6-FAM-labeled probe, with the polymorphic nucleotide placed in the middle, hybridizes next to the Cy5 fluorophore in the PCR product. FRET occurs from FAM to Cy5 with excitation of the FAM fluorophore, and the iCycler iQ system detects the increase in emission signal of the Cy5 fluorophore.

Following amplification with the Cy5-labeled primer in the presence of a hybridization probe to the wild-type sequence, a melt-curve analysis is performed. Cy5 fluorescence is continuously monitored as the temperature is slowly increased. The Cy5 fluorescence decreases slowly with increasing temperature until the melting temperature of the probe-template hybrid is reached, at which time there is a more rapid decrease in the Cy5 fluorescence.
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