SYBRvsFRET Real-time or qPCR uses one of two reporter dye schemes. One is an intercalating dye that fluoresces more brightly when bound to double-stranded DNA. SYBR-green with a 200:1 fluorescent differential is the dye of choice. It is inexpensive and requires only that a primer set for the specific gene of interest be optimized. However, there are problems associated with the use of an intercalating dye. The dye will report all double-stranded DNA made in the PCR reaction, making the dye problematic for reactions with side products. The nonspecific nature of the dye means that only one target may be quantified per reaction. Alternatively, fluorescent resonance energy transfer (FRET) probes can be used. Probes, oligonucleotides labeled with a reporter dye on one end and a quencher on the other, are designed to hybridize to the DNA sequence amplified by the PCR primers. The quencher absorbs the energy released by the fluorochrome reporter until the DNA is cleaved by the 5'- nuclease activity of the polymerase. When the fluorochrome and quencher are physically separated during synthesis, light is emitted by the unquenched fluorochrome when exposed to light at the appropriate excitation wavelength. FRET probes have two advantages. They report the amount of the specific PCR product because they hybridize only with the amplified product. Multiplexing is possible using labeling probes with different fluorochromes in the same tube or well. However, FRET probes are difficult to design. The MB&P Core has Primer/Probe set design software, ABI’s Primer Express, to assist in the design. Although FRET probes have become cheaper over time and the MB&P Core can synthesize them readily; they can be expensive if the expression of multiple genes is to be quantitated.
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