Thermal Cycler: Real Time PCR

September 8, 2021by Kalstein

The thermal cycler is a device that allows the polymerase chain reaction (PCR) efficiently and quickly; by means of the automatic and cyclical realization of the temperature changes that are required for the amplification of a deoxyribonucleic acid (DNA) chain; from a thermostable enzyme.

Thermal cyclers are used for qualitative amplifications or to quantify the amount of amplified DNA. The results of these DNA amplification techniques have a great impact on our society; especially in this period of pandemic where they have become one of the main diagnostic tests, which is why it must be ensured that the thermal cyclers are accurate; accurate and consistent, in order to obtain reliable results.

What is the polymerase chain reaction or PCR?

The polymerase chain reaction, called PCR for short by its acronym in English (polymerase chain reaction), is a technique that allows the exponential amplification of a DNA fragment of interest. That is, from a low number of copies of this fragment, after the PCR reaction, millions of copies are obtained that are already easily detectable in the laboratory. PCR was invented by Kary Mullis in 1986 and earned him the Nobel Prize in 1993.

What is real-time PCR?

Real-time PCR is a modality of end-point PCR, where the accumulation of amplified DNA is detected and quantified as the reaction progresses, that is to say: “In real time” this is achieved by incorporating a fluorescent molecule that is associated with the Amplified DNA, where the increase in this fluorescence is proportional to the increase in the amount of amplified DNA molecules in the reaction.

Real-time PCR protocols can be designed to obtain quantitative results as well as demonstrate the presence or absence of a DNA or RNA fragment or quantitative results by calculating the number of DNA copies, which when compared to a standard curve, establishes the amount of microorganisms present in a given sample or to determine the number of molecules of an RNA to designate its expression, for example.

Real-time PCR is a technique that combines amplification and detection in one step, by correlating the PCR product of each of the cycles with a fluorescence intensity signal. They are composed of a thermal cycler coupled to an optical system, which monitors the signal of the fluorophores used to detect the amplified product. Because the fluorescence of these increases as the product is amplified, the amplification and detection processes are combined in a single step.

This technique has important characteristics such as high specificity, a wide detection range and speed in the visualization of the product. Real-time PCR systems include a thermal cycler and a unit capable of detecting fluorescent signals to monitor the progress of the amplification reaction, as well as software for data analysis. The fluorophores used to monitor DNA amplification during real-time PCR can be DNA affinity (SYBR Green) or DNA fragment specific probes (Taqman probes).

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