BIOTECHNOLOGY - Inshyna N.M. - 2009

CHAPTER 1. GENETIC ENGINEERING

Polymerase chain reaction as a method of DNA amplification

The process of generating additional copies of DNA nucleotide sequences is called Amplification.

DNA amplification is carried out either through cloning or via the Polymerase Chain Reaction (PCR) method. PCR is an efficient method for obtaining large quantities of specific nucleotide sequences in vitro.

In 1985, K.B. Mullis published and patented the PCR method, and 7 years later he was awarded the Nobel Prize in Chemistry.

The most important CHARACTERISTICS OF THE PCR method are:

- reaction Specificity;

- accuracy of DNA Synthesis;

- efficiency.

Carrying out PCR requires the following components:

- 2 synthetic primers 15 - 30 bp in length;

- a DNA template ranging from 100 to 35,000 bp in length;

- thermostable Taq DNA polymerase;

- 4 deoxynucleotides: (dATP, dGTP, dCTP, dTTP.

PCR is a cyclic process. Each cycle consists of the following stages:

1) DNA Denaturation (t 95°С);

2) renaturation — annealing of primers to the DNA template (t 55°С);

3) DNA synthesis — Taq polymerase extends the complementary DNA strands (t 72 - 75°С).

The schematic diagram of the PCR process is shown in Fig. 1.2.

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Fig. 1.2. Schematic diagram of the PCR process

The first PCR cycle is preceded by incubation of the mixture at 92 — 96 °C for 0.5 — 10 min, which results in the inactivation of protein impurities. During short-term heating (for 1–2 min), DNA melting occurs, and the DNA strands separate from one another. Upon cooling to a Temperature of 37 - 65 °C, the primers bind to the complementary Regions of the DNA, and Taq polymerase synthesizes new DNA strands. To carry out DNA synthesis, the temperature is raised to 72°C. Upon completion of the DNA synthesis stage, the PCR cycle is repeated automatically. Each PCR cycle lasts 3 - 5 min, repeating for 30 rounds. Upon completion of each cycle, The amount of DNA doubles, reaching several micrograms in a reaction mixture with a volume of 25 - 50 µl. The standard sample volume for PCR is 20 - 100 µl. During PCR, the amount of DNA increases until it reaches 10¹², after which it drops sharply due to the depletion of reaction substrates and primers. Upon completion of the final PCR cycle, the samples are kept at 72°C for 5 — 10 min.

PCR utilizes Taq polymerase, which is resistant to high temperatures. This enzyme was isolated from the thermophilic Bacteria Thermus aquaticus, which inhabit hot springs. Thermostable Taq polymerase remains active at temperatures of 95 °C and above.

L.I. Patrushev was one of the first researchers in Russia to obtain a high-yield bacterial producer strain of thermostable Taq polymerase and to develop efficient Methods for its purification (yielding 2 million units of activity of the highly purified enzyme from 100 g of bacterial biomass).

The PCR method makes it possible to increase the number of DNA fragments a million-fold. Using PCR, DNA segments ranging from 0.1 to 5–7 kbp and longer can be amplified in vitro. From the genomic DNA of 1–2 somatic Cells, 10²–10⁵ DNA copies can be obtained. Thus, PCR serves as an alternative method for the molecular cloning of short DNA fragments.

Fields of application for PCR:

- Diagnostics of infectious diseases (detection of pathogenic microorganisms in biological material);

- generation of large quantities of specific DNA fragments (amplification);

- Gene synthesis;

- DNA Sequencing;

- detection of spontaneous Mutations in genes that lead to Hereditary diseases.

This method is essential for forensic medicine and the Prenatal Diagnosis of genetic abnormalities in utero. To detect gene defects, restriction analysis is performed: the DNA is cleaved by restriction Enzymes, and the resulting

fragments are separated using gel Electrophoresis, with the electrophoretic bands forming a distinct pattern. If mutations are present, the banding pattern will change.

In 1992, T. Sano first developed the immuno-PCR method, which is the most sensitive technique for detecting Proteins and other Antigens. The sensitivity of this method exceeds that of enzyme immunoassay by 1,000 times. DNA-antibody fragment conjugates are used as probes to detect antigens. If an antigen is present in the biological sample, it binds to the antibody, the signal is transmitted to the DNA, and PCR is carried out. Consequently, DNA synthesis indicates the presence of antigens.

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