Molecular Biology. Practical Guide - Velikov V.A. 2013

PCR

PCR, or Polymerase Chain Reaction, is the enzymatic in vitro copying of a specific DNA fragment using a DNA polymerase, essentially acting as selective DNA Amplification. The BOUNDARIES OF THE fragment are defined by The nucleotide sequences of primers, ensuring that only a targeted Gene (the DNA target) is copied, rather than the entire genome as in in vivo Replication. Kary Mullis (1985) is credited as the pioneer of this method.

PCR involves three cyclically repeating stages (see App. 15):

1) DNA Denaturation – unwinding of The Double Helix and Separation of the polynucleotide strands;

2) primer annealing – Hybridization of primers and single-stranded target DNA to form double-stranded primer-template complexes, which are necessary to initiate DNA Synthesis from monomer building blocks, namely deoxynucleoside triphosphates;

3) polymerization – extension (elongation) of complementary DNA strands by DNA polymerase in the 5’→3’ direction, starting from the 3’-OH ends of the attached primers. This represents METABOLISM/36.html">Template-Directed DNA Synthesis (derived from the German Matrize, from the Latin matrix meaning womb, source, or origin).

The repeated (cyclic) execution of these three stages leads to an exponential enrichment of the reaction mixture with target DNA molecules. This occurs because, in each subsequent cycle, the template pool comprises not only the initial DNA but also all newly synthesized DNA from previous cycles, which explains why the reaction is chain-based. Theoretically, $N$ cycles can yield $2^N$ target DNA molecules. The progression of the PCR—transitioning from stage to stage and cycle to cycle—is regulated by precise Temperature cycling.

DNA denaturation temperature. For any high-molecular-weight genomic DNA, this is typically set at 95°C. For amplification products, and especially for primer-template complexes, this temperature is lower.

Primer annealing temperature is the temperature at which the primer oligonucleotide binds to the single-stranded DNA template during the cooling phase that follows the denaturation step. Calculated individually for each specific primer, it generally ranges between 50–65°C and serves as a variable parameter.

Elongation temperature depends on the type of enzyme used. For the enzymatic activity of Taq DNA polymerase, isolated from the thermophilic bacterium Thermus aquaticus, the optimal temperature is 72°C.

Commercial suppliers offer ready-to-use PCR reagent kits in which all reaction components—except for the primers and template—are pre-mixed at optimal concentrations (see Practical 6.2) and aliquoted into thin-walled PCR tubes. These kits typically include a hot-start enzyme that remains inactive until heated. The researcher's task is simply to add the required amounts of DNA template and specific primers to the tube, place it into a thermal cycler, and program the appropriate temperature profile.