Polymerase chain reaction, application of PCR in clinical diagnostics, RT-PCR and use of this technique.

Polymerase Chain reaction is method of amplifying specific sequences of DNA. It is a method that popular used in the field of medicine due to sensitivity, specificity and how quickly it yields result.

Mechanism of PCR

Three main steps of PCR:

• Denaturation: DNA is denatured at a temperature of 90 degrees celsius. The double stranded helical DNA is separated into two separated strands
  • This is performed at the temperature of 90 degree celsius
  • The separated stands of DNA serve as template for amplification.
  • This process is important to enable the next step in the process, the hybridization of the primers.
• Annealing: Primers and deoxynucleoside triphosphate are added in excess to the sample of DNA, where in they anneal to the complementary sequences on the strands DNA
  • This process is performed at temperatures that fall in the range between 50-70 degrees celsius
    • Primer: a short oligonucleomers that is complementary to the target sequence that needs to the amplified
      • These primer determine the location of the target sequences on the DNA strands as well the length of target DNA fragment.
    • To prevent the quantity of primer and deoxynucleoside limiting the process of PCR, they are added in excess.
• Elongation: With the action of the DNA polymerase, the target sequences are amplified.
  • DNA polymerase used in this process is derived from Thermophilic bacteria , organism able to sustain high temperatures
  • Thus the DNA polymerase extracted from them are able to withstand the temperatures used within this process

This three step process is repeated for 20-40 cycles and with each cycle the products exponentially rise. Thus the samples of DNA required are significantly low, another reason why this method is popular method within the field of medicine and outside.

Depending on the use of PCR, some may use the method Gel electrophoresis with ethimidium bromide. It serves to help visualization of DNA products to separate the DNA fragments through the gel.

Another method dependent on conventional PCR is the Sanger method. This sequence of methods could be used to identify chromosomal aberrations.

Application of PCR

• The use of PCR in the field of microbiology
  • to identify etiological agents behind pathological processes such as upper respiratory infections and involving other organs in the body.
    • In this scenario, this method is particularly indicated but not limited to viral agents. They can be sued bacterial, parasites, fungi for example.
  • This method in this field can also be used to identify genes of resistance in conjunction with other methods
• The use of PCR (in conjunction with Sanger sequencing) can be used in the field of clinical genetics
  • when trying to identify genetic cause of a syndrome where theres is a suspicion of genetic component
  • When trying to identify potential pathogenic alleles in carriers (to potential identify the mode of inheritance for example)
  • They can be used to identify the potential risks of developing of a certain type of neoplasm (with family history)

Reverse Transcriptase PCR: RT PCR

This method is variant of PCR. This method varies from the conventional PCR with the use of reverse transcriptase

• Reverse transcriptase is derived from the RNA retroviruses and this type of PCR is preferred due to RT ability to convert RNA to DNA.

Thus the uses of this method would be:

• Predominant method used in during COVID 19 pandemic for the SARS-COV-2.
• Identify genes of antibiotic resistance and asses the expression of those genes quantitatively.
• Identify genes of resistance against antivirals in viruses
• Identify drug resistance in cancers

References

Rodwell, V.W. et al. (2018) Harper’s Illustrated Biochemistry 31. 31st edn. New York: McGraw-Hill Medical. Pg 1071 - 1074

Polymerase chain reaction (PCR) (n.d) Khan Academy. Available at: https://www.khanacademy.org/science/ap-biology/gene-expression-and-regulation/biotechnology/a/polymerase-chain-reaction-pcr#:~:text=PCR%20is%20used%20in%20many%20research%20labs%2C%20and%20it%20also,the%20case%20of%20prenatal%20testing). (Accessed: 02 April 2025).

Khehra, N., Padda, I.S. and Swift., C.J. (2023) Polymerase chain reaction (PCR), StatPearls [Internet]. Available at: https://www.ncbi.nlm.nih.gov/books/NBK589663/ (Accessed: 02 April 2025).

PCR technique and its applications in biology and medicine (2023) MyBioSource Learning Center. Available at: https://www.mybiosource.com/learn/pcr-technique-and-its-applications-in-biology-and-medicine/ (Accessed: 02 April 2025).

Murray, P.R., Rosenthal, K.S. and Pfaller, M.A. (2016) Medical microbiology. 8th edition. Philadelphia, PA: Elsevier. Page 23 - 25