PCR - What is it about?

What is PCR? 

PCR is the abbreviation of polymerase chain reaction. This term recently got popularity after the COVID-19 pandemic. Also offered job opportunities for many people. That is one amazing fact about PCR! 

Well, it is one of the major milestones and a scientific discovery which redefined the molecular biology. This allows rapid and cheap amplification of the desired DNA sequence in a sample. This was developed by Kary Mullis in 1983 and he was awarded Nobel prize this invention in 1993.

How does it work? 

 5 major ingredients are required to set up a PCR. These are, 

1. DNA template to be amplified.
2. Primers – 2 types forward and reverse primer. Short stretches of DNA to start the PCR reaction. 
3. DNA nucleotide bases (dNTPs) - deoxy nucleotide triphosphates such dATPs, dCTPs, dGTPs and dCTPs. A, T, G and C are the building blocks for DNA and are required to synthesize new strand. 
4. DNA polymerase enzyme - They duplicate the DNA strand by adding nucleotides at 3'-OH end. Taq polymerase enzyme extracted from the bacterium Thermus aquaticus is used as it is thermostable. 
5. Buffer – Provide the right conditions for the PCR reaction (also act as a suitable environment for Taq polymerase) such as pH, ions etc.  

Steps involved: 

• Denaturation – the hydrogen bonds between the two DNA strands get break at temperatures >90⁰C. So, the double stranded DNA converts to single stranded DNA. The denaturation temperature varies for each DNA sequence based on the A-T and G-C pairs. 
• Annealing – The temperature cooled down to 40-60⁰C, the bonding between primer and desired DNA sequence occurs in this temperature. The precise temperature is to be optimized for each primer and PCR reaction.  The primer has free 3'-OH end where the DNA polymerase can act. That is the next step. 
• Extension – This is the DNA synthesizing step. The temperature is raised up to around 72⁰C, which is the optimum temperature for the DNA polymerase enzyme, which add nucleotides at 3' end and the DNA synthesis (new strand) proceeds from 5'end to 3'end.  

Thus, after repeating these 3 steps for several times (approximately 30-40 cycles) results in million copies of DNA. The number of double stranded DNA is doubled in each cycle. After the 'n' number of cycles, 2ⁿ copies of DNA is produced. E.g.: after 5 cycles, you have 32 copies of DNA! 

                                                Three cycles in PCR, 5'-3' denotes primer 

Where it all takes place? 

Thermocycler (DNA amplifier) is used to amplify the desired DNA sequence. These are the simple devices which provide the precise control of temperature and similar rates of cooling and heating for PCR tubes in different parts of the heating blocks. The tubes with PCR reaction mixture can be placed in the holes of the heating block. 

Important points to set up a PCR 

• As the PCR technique deals with very minute amounts of DNA, even small traces of contaminants can severely interfere the entire process. So, good cleanliness should be maintained while setting up a PCR.  
• Even the aerosols from the pipetting can cause cross contamination, so good pipetting technique is needed.  
•  Proper labelling of tubes and quality control procedures are required. 
• PCR set up should be done in a separate sterile hood or flow cabinet. 

Detection 

PCR products are analyzed by agarose gel electrophoresis. The PCR products are identified by ethidium bromide or non-toxic dyes such as SYBR green. The band intensity is used to identify the desired PCR product with respect to the DNA ladder (fragments of DNA of known base pair). 

Applications of PCR

Area where PCR used	Application
Molecular Diagnostics	Genetic testing, detection of oncogenic mutations, detection of infectious diseases
Forensic Science	Scenes of crime
Agriculture	Food pathogen detection, plant genotyping
Sequence analysis	DNA sequencing
Protein Engineering	Production of novel proteins

References

•  W. (2010). Principles and Techniques of Biochemistry and Molecular Biology (7^th ed.). Cambridge India 
•  P. Arora, M. (2005). Genetic Engineering (1st ed.). Himalaya Publishing House. 
• What is PCR (polymerase chain reaction)? (2021, July 21). Yourgenome. https://www.yourgenome.org/facts/what-is-pcr-polymerase-chain-reaction 

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