Into the Darkness

PCR has fundamentally changed molecular biology, offering a rapid means to amplify minute quantities of DNA. Real-time quantitative PCR (Q-PCR) techniques are designed to facilitate accurate measurement of target sequences by correlating amplification with the fluorescence of specially labeled probes in the reaction. Q-PCR methodologies using fluorescence resonance energy transfer (FRET) employ an oligonucleotide probe designed to hybridize to the center of the desired PCR product. This probe is labeled at the 5' end with a fluorescent dye and at the 3' end with a fluorescence quencher. The proximity of the dye to the quencher decreases the fluorescence of the dye. When the polymerase encounters the probe, the 5' dye is released and separated from the quencher, leading to bright fluorescence. Presently available dye/quencher pairs suffer from a number of drawbacks, including inherent fluorescence of the quencher and poor spectral overlap between the fluorescent dye and quencher molecule, resulting in ...