Quantitative PCR

With the development of thermal cyclers incorporating fluorescent detection, PCR has a new, innovative application. In routine PCR, the critical result is the final quantity of amplicon generated after the process. Real-time or Quantitative PCR and RT-PCR use the linearity of DNA amplification to determine absolute or relative amounts of a known sequence in a sample. By using a fluorescent reporter in the reaction, it is possible to measure DNA generation.

In quantitative PCR, DNA amplification is monitored at each cycle of PCR. When the DNA is in the log linear phase of amplification, the amount of fluorescence increases above the background. The point at which the fluorescence becomes measurable is called the Threshold cycle (C_T) or crossing point. By using multiple dilutions of a known amount of standard DNA, a standard curve can be generated of log concentration against C_T. The amount of DNA or cDNA in an unknown sample can then be calculated from its C_T value.

Real time PCR also lends itself to relative studies. A reaction may be performed using primers unique to each region to be amplified and tagged with different fluorescent dyes. Several commercially available quantitative thermal cyclers include multiple detection channels. In this multiplex system, the amount of target DNA/cDNA can be compared to the amount of a housekeeping sequence e.g. GAPDH or β-actin.

Two types of detection chemistries are used for quantitative PCR. The first uses an intercalating dye that incorporates into double-stranded DNA. Of these fluorescent dyes, SYBR^® Green I dye is the most common one used. This detection method is suitable when a single amplicon is being studied, as the dye will intercalate into any double-stranded DNA generated.

The second detection method uses a primer or oligonucleotide specific to the target of interest, as in TaqMan^® probes, Molecular Beacons™, or Scorpion primers. The oligonucleotide is labeled with a fluorescent dye and quencher. The oligonucleotide itself has no significant fluorescence, but fluoresces either when annealed to the template (as in molecular beacons) or when the dye is clipped from the oligo during extension (as in TaqMan probes). Multiplex PCR is possible by using dyes with different fluorescent emissions for each primer.

Sigma has developed products for use with real-time instruments that use plates and tubes as well as those that use capillary reaction vessels.

For multiplex applications, Sigma-Genosys offers custom dual-labeled probes with dyes for quantitative PCR and is a licensed manufacturer of Molecular Beacons.