The polymerase chain reaction (PCR) can be used for the selective amplification of a specific segment (target sequence or amplicon) of a DNA molecule. The DNA to be amplified can theoretically be present in a very small amount—even as a single molecule. The PCR reaction is carried out in vitro and, as such, it does not require a host organism. The size of the DNA region amplified during the PCR reaction typically falls within the range of 100 bp to 10 kbp.
PCR is based on the reaction scheme described as follows (see also Figure 10.11). First, a heat-induced denaturation of the target DNA sequence (template, panel a) is performed in order to separate the constituent complementary DNA strands. Then, short single-stranded DNA molecules (oligonucleotide primers) are added that are complementary to the flanking regions of the target sequence. Cooling of the sample allows annealing (panel b). Subsequently, the strand elongation activity of a DNA polymerase enzyme leads to the formation of new DNA strands (so-called primer extension products) starting from the 3’ end of the annealed primers (panel c). After repeated heat denaturation and cooling, the primers will be able to anneal both to the original template molecules and to the primer extension products (panel d). In the latter case, the length of the nascent DNA strand will be limited by the primer extension product now serving as a template strand. This way, the resulting “end-product” strands will comprise the DNA segment defined by the template and the flanking primers (panel e). In further denaturation–annealing–synthesis cycles, the end-product strands will serve as templates for the synthesis of additional end-product strands. Therefore, the amount of these molecules will grow exponentially with the number of reaction cycles (panel f). Thus, the final result of the reaction will be a large amount of end-product molecules comprising the sequence flanked by the predefined primers. This highlights one of the crucial advantages of the PCR technique: via the design of primers, we can fully control which segment of the template DNA will be amplified—with only a few practical limitations.
In order to successfully perform the reaction described above, the following solution components are necessary:
DNA molecules that serve as template for the reaction. The amount of the template can be very low—in principle, the reaction can start even from a single template molecule. Another advantage of PCR is that the selective amplification of the desired DNA segment can be accomplished even using a heterogeneous DNA sample as template.
A pair of oligonucleotides serving as primers. The 3’ ends of the oligonucleotides must be able to anneal to the corresponding strands of the template. A further advantage of PCR is that the 5’ end of the applied primers may contain segments that do not anneal to the original template. These regions of the primers may be specific engineered sequences or even contain labelling or other modifications, which will be present in the end product and thus facilitate its further analysis and/or processing. (For instance, recognition sites of restriction endonucleases can be incorporated in order to facilitate the subsequent cloning of the PCR product.)
The DNA polymerase enzyme catalysing DNA synthesis. As the heat-induced denaturation of the template is required during each cycle, heat stable polymerases are usually applied that originate from thermophilic organisms (e.g. Thermus aquaticus (Taq) or Pyrococcus furiosus (Pfu) DNA polymerase).
Deoxyribonucleoside triphosphate (dNTP) molecules that serve as building blocks for the DNA strands to be synthesised. These include dATP (deoxyadenosine triphosphate), dGTP (deoxyguanosine triphosphate), dTTP (deoxythymidine triphosphate), and dCTP (deoxycytidine triphosphate).
A buffer providing optimal reaction conditions for the activity of DNA polymerase. Among other components, PCR buffers contain bivalent cations (e.g. Mg2+ or Mn2+).
For an effective polymerase chain reaction, it is necessary to change the temperature of the solution rapidly, cyclically and in a wide range (see below). This can be achieved by using a programmable instrument containing a thermoblock equipped with a Peltier cell. To achieve effective heat exchange, PCR reactions are performed in thin-walled plastic tubes in small reaction volumes (typically, in the order of 10-200 μl). The caps of the PCR tubes are constantly held at high temperature by heating the lid of the thermoblock, in order to prevent condensation of the reaction mixture in the upper part of the tubes. In the absence of a heated lid, oil or wax can be layered on top of the aqueous PCR samples in order to prevent evaporation.
The programmed heat profile of a PCR reaction generally consists of the following steps:
Initial denaturation of the template, performed at high temperature (typically, around 95°C).
Denaturation: Heat-induced separation of the strands of double-stranded DNA molecules at high temperature (typically, around 95°C).
Annealing: Cooling of the reaction mixture to a temperature around 45-65°C in order to facilitate the annealing of the oligonucleotide primers to complementary stretches on template DNA molecules.
DNA synthesis: This step takes place at a temperature around the optimum of the heat-stable DNA polymerase (typically, 72°C), for a time period dictated by the length of the DNA segment to be amplified (typically, 1 minute per kilo-base pair).
Steps (b)-(d) are repeated typically 20-35 times, depending on the application.
Final DNA synthesis step: After completion of the cycles consisting of steps (b)-(d), this step is performed at a temperature identical to that during step (d) (72oC), in order to produce complementary strands for all remaining single-stranded DNA molecules.
PCR reactions are widely applied in diverse areas of biology and medical science. In the following, we list a few examples.
Molecular cloning, production of recombinant DNA constructs and hybridisation probes, mutagenesis, sequencing;
Investigation of gene function and expression;
Medical diagnostics: identification of genotypes, hereditary disorders, pathogenic agents;
Forensic investigations: identification of samples and individuals based on DNA fingerprints (unique individual DNA sequence patterns);
Evolutionary biology, molecular evolution, phylogenetic investigations, analysis of fossils.