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PCR as a method of diagnosing genetic diseases
Last reviewed: 04.07.2025
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PCR is a new achievement of molecular genetics, used for DNA amplification and allows rapid in vitro multiplication of a specific DNA region (i.e. any gene of interest) more than 200,000 times. To carry out the reaction, it is enough to have DNA material from one cell; the amount of DNA amplified by PCR is so great that this DNA can simply be stained (the use of radioactive probes after electrophoresis is not required). A prerequisite for carrying out PCR is knowledge of the nucleotide sequence of the amplified DNA region for the correct selection of artificially synthesized primers.
Currently, PCR is a single-tube process consisting of repeated cycles of amplification (reproduction, copying) of a specific DNA molecule sequence in order to obtain a sufficiently large number of copies that can be identified by electrophoresis. One of the key components of the reaction is "primers" - synthetic oligonucleotides consisting of 20-30 bases complementary to the "sites" (areas) of annealing (attachment) on the identified area of the matrix DNA.
PCR occurs automatically in a programmable thermostat - a thermal cycler (amplifier). The three-stage cycle, which results in exact copies of the identified section of matrix DNA, is repeated 30-50 times in accordance with the specified thermal cycler program. In the first cycle, oligoprimers hybridize with the original matrix DNA, and then (in subsequent cycles) with newly synthesized DNA molecules as they accumulate in the reaction mixture. In the latter case, DNA synthesis ends not as a result of a change in temperature, but upon reaching the DNA polymerase limit of the amplified section, which determines the size of the newly synthesized DNA section with an accuracy of one nucleotide.
Electrophoresis is used as a method for detecting the obtained DNA molecules, with the help of which the amplified material is separated according to the size of the amplicons (amplification products).
PCR can be used to directly examine the locations of suspected mutations or polymorphic sites, as well as to study the presence of any other specific DNA features.
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