Articles

Real-Time PCR

Gabriela Colichio

Developed in the late 1980s by Kary Mullis who won the Nobel Chemistry Award for his discovery, the PCR revolutionized the techniques of molecular genetics. It can be applied to genetic identification, forensic medicine, clinical analysis, disease diagnosis, industrial quality control, among others. This technique is used routinely in laboratories to make millions or billions of DNA copies that are of interest to the researcher.

What is PCR?

PCR technology is based on duplicating DNA in vitro that can be repeated several times, resulting in millions of copies with just one fragment of DNA. With this amount of DNA, it is possible to perform several analyzes. The PCR is a very sensitive, specific and fast method, compared to other tests, taking 2 to 3 hours to present the result. For this reason, they are widely used in clinical infectology for the detection of pathogens and bacterial and viral identification, such as COVID-19. In addition, it is used in diagnosis in oncologies and genetic diseases, identifying mutations and genetic predisposition for certain diseases.

How is conventional PCR?

Source image: https://profissaobiotec.com.br/

PCR components

  • DNA sample of interest: model DNA strand
  • Primers: a short sequence of nucleotides that provides a starting point for DNA synthesis by Taq Polymerase.
  • Taq Polymerase: protein capable of producing new DNA strands using an existing strand as a model.
  • Nucleotides: used by Taq Polimerase for the construction of the new tapes.
  • Mix Buffer: buffer that keeps the reaction components in an ideal state.

PCR process

The reaction is carried out in the thermal cycler, which is a device that allows to program the temperature and time.

  • Denaturation (95ºC): heats the reaction so that the DNA denatures, that is, the double strand of DNA separates, providing a single strand template.
  • Annealing (55ºC): the lower temperature causes the primers to bind to the DNA strand.
  • Extension (72ºC): with the starting point already identified, Taq polymerase binds the strand signaled by the primer. Then the extension of the new DNA fragment begins, forming a double strand of DNA again.

This process is repeated 25 – 35 times and usually occurs in 2 – 4 hours, depending on the length of the DNA region to be copied. The conventional PCR results are visualized in the end of the process. This process is quick because not only the original DNA is used as a template, but also the new DNAs, that serve as templates for the synthesis of more copies. So, this growth pattern is exponential, as shown in the figure below:

Real-time PCR

A real-time polymerase chain reaction (real-time PCR), also known as quantitative polymerase chain reaction (qPCR) is an evolution of the conventional PCR method. It is based on conventional PCR, but the result is visualized immediately. There are two common methods for the detection of amplified DNA:

  • Fluorescent dyes: binds to any double stranded DNA.
  • DNA probes: sequence-specific DNA that are labeled with a fluorescent reporter, which allows detection only after hybridization of the probe to its complementary sequence.

Therefore, in qPCR, the DNA amplification is monitored at each cycle of PCR. There are numerous applications for quantitative polymerase chain, and It is commonly used for both diagnostic and basic research.

References:

[1] DEEPAK, S. A. et al. Real-time PCR: revolutionizing detection and expression analysis of genes. Current genomics, v. 8, n. 4, p. 234-251, 2007.

[2] KUBISTA, Mikael et al. The real-time polymerase chain reaction. Molecular aspects of medicine, v. 27, n. 2-3, p. 95-125, 2006.

[3] RAMAKERS, Christian et al. Assumption-free analysis of quantitative real-time polymerase chain reaction (PCR) data. Neuroscience letters, v. 339, n. 1, p. 62-66, 2003.

[4] WILHELM, Jochen; PINGOUD, Alfred. Real‐time polymerase chain reaction. Chembiochem, v. 4, n. 11, p. 1120-1128, 2003.

Leave a message