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METHODS AND TECHNOLOGIES

Polymerase chain reaction

POLYMERASE CHAIN REACTION

The polymerase chain reaction (PCR) is a method for the rapid amplification of specific DNA segments.

For PCR, a sequence-specific, complementary oligonucleotide pair (sense and antisense primers), thermostable DNA polymerase, and a mix of the four nucleotides guanine (G), adenine (A), thymine (T), and cytosine (C) are required. The primers are 16–24 base pairs long and are designed to flank the target sequence. Primer selection is a critical step, as it is essential to avoid the amplification of nonspecific products, especially pseudogenes. Pseudogenes are inactive genes with sequences very similar to the target genes, which can lead to false results. Therefore, primer design ensures that the primer sequence is specific to the desired PCR product and does not target regions with frequent polymorphisms, which could prevent binding to the DNA template (risk of allele dropout).

PCR is performed in a specialized device (thermocycler) capable of rapidly changing the temperature of the reaction block. Different temperatures are necessary to denature the DNA (formation of single strands at 95°C), allow primer annealing (50–65°C), and ensure the elongation of primers along the DNA template into new daughter strands (optimum temperature for DNA polymerase at 72°C). This process (denaturation, annealing, strand elongation) is referred to as a PCR cycle and is repeated 30–40 times. Ideally, the DNA segment between the primers doubles with each cycle, and the entire reaction typically takes 1–2 hours.

PCR products can be used for various purposes. Most commonly, they are utilized in analytical techniques such as sequencing, MLPA, real-time PCR, RFLP, fragment length analysis, etc.

PCR is also an inexpensive method for directly detecting specific genotypes. For example, known deletions or duplications of a gene can be identified using (long-range) PCR (e.g., CYP2D6 amplification/deletion, alpha-globin gene deletions), or point mutations can be analyzed using sequence-specific primers.

The main advantages of PCR include its robustness, specificity, and sensitivity. Even the smallest amounts of DNA can be detected and used for diagnostic purposes. However, this also poses the risk of contamination. Therefore, it is crucial to ensure that DNA samples are not contaminated with DNA from other sources. To safeguard against this, contamination controls are included in every assay.

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