Quantitative PCR

qPCR; Quantitative Analysis of DNA

With the development of thermal cyclers incorporating fluorescent detection, the polymerase chain reaction assay (PCR)has new, innovative applications. In routine PCR, the critical result is the final quantity of amplicon generated from the assay. Quantitative PCR, or real-time PCR, (qPCR) and reverse transcription PCR (RT-PCR) use the linearity of DNA amplification to determine absolute or relative quantities of a known sequence in a sample. By using a fluorescent reporter in the reaction, it is possible to measure DNA generation in the qPCR assay.

In qPCR, 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 (CT) or crossing point. By using multiple dilutions of a known amount of standard DNA, a standard curve can be generated of log concentration against CT. The amount of DNA or cDNA in an unknown sample can then be calculated from its CT value.

Real-time PCR also lends itself to assays to determine relative quantities of DNA. A reaction may be performed using primers unique to each target 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 produced during amplification can be compared to the amount of a housekeeping sequence like GAPDH or ß-actin.

Two types of detection chemistry are used for real-time PCR analysis. The first uses an intercalating dye that incorporates into double-stranded DNA. Of these fluorescent dyes, SYBR® Green I dye is used most commonly. 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.

The quantification of DNA can be useful in research and Sigma has the reagents to meet your research needs. In addition, Sigma′s products can be used 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 qPCR and is a licensed manufacturer of Molecular Beacons.