Скачать или смотреть Animated conventional PCR procedure — OPERON

Polymerase chain reaction (PCR) is a technique used in molecular biology to amplify multiple copies of a particular DNA/RNA sequence, up to hundreds or thousands of copies of the same sequence.

It is a simple, cost-effective and reliable technique that can be applied to different fields in modern biology and related sciences. Typically, the aim of PCR is to obtain enough copies of the target DNA/RNA region to be analysed or used at a later stage. For example, PCR-amplified DNA/RNA (amplicons) can be sent for sequencing, visualised by electrophoresis gels or hybridisation techniques, or cloned into a plasmid for further experiments.

Conventional PCR is performed in a thermal cycler, an apparatus capable of rapidly heating and cooling samples to take advantage of the physicochemical qualities of the nucleic acids and the enzymatic qualities of the DNA polymerase.

The PCR process usually consists of a series of temperature changes repeated 25-40 times, called cycles, each with a minimum of three steps: the first allows the separation of the double-stranded nucleic acids; the second allows the binding of the primers to the DNA strand; the third facilitates polymerisation by the DNA polymerase.

In the case of detection of amplified DNA using hybridisation techniques, a molecule (e.g. biotin) is incorporated during PCR that will aid in subsequent detection. The amplified DNA will hybridise, i.e. bind, to membrane-bound DNA probes, and this amplified DNA-biotin + probe complex will be detected using a peroxidase-streptavidin conjugate, which will bind specifically to biotin. Subsequently, a substrate will be added which will react with the peroxidase resulting in the appearance of blue bands in the area of the membrane where the DNA probes were attached. Thus, a banding pattern is obtained which can be interpreted with the help of a template or a scanner.

The PCR technique can be carried out using DNA from different sources such as tissues and micro-organisms, including peripheral blood, skin, hair, saliva and bacteria. Only trace amounts of DNA are needed to generate sufficient copies to be analysed using conventional laboratory methods. For this reason, the PCR technique is very sensitive, so it is extremely important to maintain good laboratory practices, especially regarding hygiene, order and safety in any of the phases of the technique (extraction of nucleic acids, preparation of the reactions and detection of the amplified material), in order to avoid possible contamination that may affect the test result.

The video shows how the PCR reaction is produced at a molecular level, followed by strip hybridisation, so that this technique can be understood in a simple way.

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