PCR and qPCR

The polymerase chain reaction (PCR) is a molecular biology method that enables the rapid synthesis of many copies of a specific DNA fragment. The method is used, among other things, to amplify and clone genes for expression studies, but also to detect bacteria and viruses. In this summary, we will give you a brief overview of the general and different variants of PCR.

Polymerase Chain Reaction (PCR)

The four main components of a PCR are:

• Primer
• DNA Template
• thermostable DNA Polymerase
• Nucleotides (dNTPs)

The primers (forward and reverse) are created complementary to the ends of the DNA fragment to be amplified, while the DNA template provides the basis for the reaction as it contains the DNA fragment to be amplified. The polymerase synthesizes copies of the DNA fragment using the primers as a starting point and the nucleotides. The amplified DNA sequence is also called an amplicon.

There are three standard phases in a PCR:

• Denaturation
• Annealing
• Extension

Denaturation takes place at 95°C and cleaves the hydrogen bonds between the bases of the DNA double strand, resulting in two DNA single strands (Fig. 1.1). The subsequent primer hybridization takes place at a lower temperature between 45 and 60°C. This depends on the length and GC content of the primers. The primers bind complementarily to the section to be amplified on the single-stranded DNA, resulting in a short section of double-stranded DNA (Fig. 1. 2). In the final phase, elongation, this section serves as the starting point for the synthesis of the complementary strands by the DNA polymerase. Elongation usually takes place at 72 °C and the DNA fragment to be amplified is synthesized in the 5'-3' direction (Fig. 1.3). This cycle is repeated approximately 20 to 50 times [1]. The PCR products of the first cycle are used as starting material for the second cycle (etc.), which leads to exponential amplification [2]. 

Figure 1: PCR procedure. DNA template with the sequence to be amplified (sequence of interest), dNTPs, primers and DNA polymerase are required. Denaturation (1) cleaves the dsDNA, whereupon the primers hybridize (2) and the polymerase synthesizes the complementary strand (3). The cycle is repeated 20-50 times.

Agarose gel electrophoresis is usually used to determine whether the desired DNA fragment has been amplified and to analyze the PCR products. This checks whether the synthesized DNA pieces have the correct (expected) base pair length. The PCR products are then purified and further processed (e.g. cloning, sequencing).

Quantitative Polymerase Chain Reaction (qPCR)

With qPCR, or real-time PCR, it is possible to monitor the progress of DNA amplification during the process. The quantitative determination of the starting amount is useful for analyzing gene expression and determining DNA concentrations.

The amplification procedure is the same as described above, but in qPCR fluorescent dyes or probes are integrated into the reaction (Fig. 2 and 3). In addition, a thermal cycler with an optical detection module is required to measure the generated fluorescence signal [3].

In dye-based qPCR, fluorescence is produced by the binding of a fluorophore to the small groove of the dsDNA that is created during elongation (Fig. 2.3). The amount of amplified DNA can be estimated from the fluorescence measured after each cycle. Although this quantification option is inexpensive, it is not specific for the target DNA sequence to be amplified. This can result in off-target intercalations that can be misinterpreted. Melting curve analysis can be used to determine the fragment lengths and thus also the specificity of the measurements [4].

Figure 2: Process of dye-based qPCR. After denaturation (1) and Primer annealing (2), the fluorescent dye intercalates in the dsDNA produced during elongation (3). 

Probe-based qPCR uses fluorescent probes to specifically detect the target DNA sequence. Probes conjugated with a fluorescent reporter dye and a fluorescence-inhibiting quencher bind to a DNA sequence within the section to be amplified (Fig. 3.2). During elongation, the DNA polymerase hydrolyzes the probe and the fluorescence reporter is cleaved off (Fig. 3.3). Due to the spatial distance to the quencher, a fluorescence signal can be measured which is proportional to the amount of PCR products generated [3]. 

Figure 3: Process of probe-based qPCR. After denaturation (1), both the primers and the probes with a fluorescence reporter and a quencher that inhibits fluorescence hybridize (2). During elongation (3), the reporter is cleaved from the probe, allowing the fluorescence signal to be detected.

Reverse Transcription Polymerase Chain Reaction (RT-PCR)

In RT-PCR, a reverse transcription reaction is used prior to PCR-based amplification to generate cDNA (complementary DNA) from mRNA. An RNA sequence serves as a template for the reverse transcriptase and the resulting single-stranded DNA in turn serves as a template for the PCR. Poly-dT primers, which bind to the poly-A tail of the mRNA, are often used for RT-PCR. The steps include heating the single-stranded RNA to resolve the secondary structure, followed by transcription of the RNA into cDNA by reverse transcriptase. The synthesized cDNA is amplified using the PCR method described above and finally the PCR products are analyzed [5]. 

RT-qPCR is a combination of the methods described, in which the RNA is transcribed into cDNA. During the subsequent cDNA amplification, dyes or probes are used to achieve a quantitative analysis in real time [6].

Have we triggered a chain reaction in you? Then read our blog article on qPCR and discover suitable products from our partner AAT Bioquest. 

Optimized qPCRs with AAT Bioquest

Also take a look at our partner NZYtech, which offers a wide range of products for qPCR.

Our new Molecular Biology Expert: NZYtech

Sources

[1] https://de.wikipedia.org/wiki/Polymerase-Kettenreaktion (06.03.2024)

[2] Canene-Adams, K. (2013) „General PCR“, in Methods in Enzymology. Elsevier, S. 291–298.

[3] https://www.aatbio.com/catalog/real-time-pcr-qpcr (06.03.2024)

[4] https://de.wikipedia.org/wiki/Real_Time_Quantitative_PCR (12.03.2024)

[5] Bachman, J. (2013) „Reverse-Transcription PCR (RT-PCR)“, in Laboratory Methods in Enzymology: RNA. Elsevier, S. 67–74.

[6] https://www.medicoswab.com/de/what-are-the-differences-between-pcr-qpcr-and-rt-pcr/ (06.03.2024)