Polymerase chain reaction (PCR)

Polymerase chain reaction (PCR) is the most effective method of molecular diagnostics, which is widespread in the field of in vitro diagnosis. In the EU countries, the share of PCR amounts up to 80% of laboratory analysis. PCR is an experimental method of molecular biology, which provides a significant increase (amplification) of target nucleotide acid (DNA/RNA) fragment even from a low copy or complex samples.

The amplification process consists of three steps:

1. Denaturation - is a transformation of double-stranded DNA into single-stranded driven by disruption of hydrogen bonds between complementary pairs of bases during incubation.

2. Annealing - is hybridization of primers, flanking the target fragment, to the single-stranded DNA-template.

3. Elongation – is a buildthrough of a new chain driven by enzyme possessing polymerase activity (Taq-polymerase).

These steps are completed at specific temperatures comprising one cycle of temperature program. Cycles are repeating several times providing the sufficient amount of specific PCR product. At each cycle, the number of synthesized copies of DNA fragments doubles. The process of accumulation of specific products of amplification goes exponentially, and in some point due to exhaustion of reaction resources, the acceleration reaches its maximum and reaction starts to deaccelerate. As soon as resources fall critically, reaction reaches the plateau and stops.

For convenience, the reaction mixture can include additional components so called DNA-probes, which are artificially synthesized complementary oligonucleotides of a small size (up to 30 nucleotides) bearing fluorescent tags and thus allowing to follow the reaction progress in real time. This creates a main advantage of fluorescent PCR over conventional approaches – the possibility of running quantitative assays. At present Real-Time PCR is the most efficient method for solving molecular genetic tasks in the field of diagnostics of infectious diseases, analysis of complex biological systems and genetic polymorphisms.

The presented technology is successfully used for qualitative and quantitative analysis, as well as foranalysis of expression of genes.