Types of PCR
Conventional PCR methods, like the process aforementioned, can only amplify DNA and requires agarose gel electrophoresis to determine PCR success from the end-point of the reaction. This process is very time-consuming and is hindered by various caveats, including low sensitivity, low resolution, poor precision, non-automation, post-PCR processing, and a short dynamic range. To address these concerns, several iterations of the PCR process have been developed, including quantitative PCR (qPCR) for monitoring DNA amplification in real-time and reverse-transcription PCR (RT-PCR) for the detection of RNA, a tool that has become instrumental in viral diagnostics.
Reverse-Transcription PCR
Reverse-transcription polymerase chain reaction (RT-PCR) is a highly sensitive technique for the detection and quantitation of mRNA expression levels. In RT-PCR, the RNA template is reverse transcribed into complementary DNA (cDNA), using reverse transcriptase. The cDNA is then used as a template for exponential amplification using standard PCR procedure (denaturation, annealing, and elongation). RT-PCR is used in various applications, including gene expression analysis, microarray validation, pathogen detection, and disease research.
Quantitative PCR
Quantitative or real-time PCR (qPCR) enables researchers to monitor the amplification of a DNA template in real-time and not at its end-point, as in conventional PCR. It does so using fluorescent reporter molecules which bind to and detect products generated during each cycle of the PCR process. As the reaction proceeds, fluorescence increases due to the accumulation of the PCR product with each amplification cycle. These fluorescent reporter molecules include dyes that bind to the double-stranded DNA (dsDNA), such as Helixyte™ Green (Cat No. 17591) and Q4ever™ Green (Cat No. 17608), or fluorescently labeled sequence-specific probes, such as TaqMan® probes, Molecular Beacons and Scorpion® probes.
Dye-Based qPCR
In qPCR, dsDNA binding dyes are frequently used as fluorescent reporters to measure gene expression. The fluorescence of the reporter dye increases as the product accumulates with each successive cycle of amplification. Recording the amount of fluorescence emission at each cycle makes it possible to monitor the PCR reaction during the exponential phase. Compared to microarrays, qPCR is more sensitive at detecting modest changes in expression levels, making it well-suited for investigating small subsets of genes. Although dsDNA-binding dyes provide the most convenient and cheapest option for qPCR, the principal drawback to intercalation-based detection of PCR product accumulation is that both specific and nonspecific products generate signals.
Figure 1. qPCR using Helixyte™ Green (Cat No. 17591). During the extension phase, DNA polymerase extends the sequence-specific primer by incorporating dNTPs complementary to the DNA template. As newly synthesized double-stranded DNA is produced, Helixyte™ Green will bind to the DNA complexes and fluoresce (figure made in BioRender).
Table 3. Double-stranded DNA-binding dyes for qPCR.