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Polymerase Chain Reaction

Medically Reviewed by Dr. S. Kevin Fernando, MBBS on Apr 19, 2016


What is a PCR?

PCR or Polymerase Chain Reaction is a revolutionary method that is used to amplify specific segments of DNA or RNA. This method was developed by Kary Mullis in the 1980s. Kary Mullis received the Nobel Prize in Chemistry in 1993, for his work on PCR.


PCR aids in increasing copies of genetic material that can be used for further analysis. This procedure allows genetic analysis from small samples of blood and tissue.

PCR amplification of DNA is used routinely in laboratories across the world, as it is fast and inexpensive.

How Does PCR Amplification Occur?

PCR amplification occurs through a series of steps using primers. Primers are laboratory synthesized nucleotide sequences that bind to the complementary strands of DNA. They are the starting points for synthesis and elongation. Primers are designed to include the regions of DNA that need to be amplified by the PCR technique.

Denaturation of DNA:

The sample DNA is heated to 94-960C for a few minutes to denature the DNA and to separate the two strands of DNA.

Anneal Primers:

The temperature is then cooled down instantly to 50-650C to facilitate the attachment of the DNA primers to the complementary strands of DNA.

Once the DNA primers are attached to the complementary strands of DNA from the sample, DNA strand elongation is ready to begin.

Strand Elongation:

In this stage, there is strand elongation which occurs at a temperature of 720C. The enzyme Taq polymerase mediates this process by attaching nucleotides to the DNA primers based on the nucleotides present in the DNA strand of the sample.

On completing this step, an identical copy of the original DNA strand will be obtained.

The steps from DNA denaturation till strand elongation are repeated to get multiple copies of the original DNA strand. The process repeats several times till a billion copies are generated within a few hours.

DNA Copies in Each PCR Cycle

Cycles in a PCRNo of targeted DNA copies
12
24
38
416
532
664
7128
8256
9512
101024
1132,768
121,048,576

What are Added to the Reaction Mixture to be Loaded into a PCR for DNA Amplification?

The following are added to the PCR reaction mixture for DNA amplification:

What are the Advances That Have Allowed DNA Amplification to be Automated Using a PCR Machine?

A. DNA polymerases that are thermostable: In Vivo enzymes are used to separate the complementary strands of DNA that are to be amplified. In a PCR, heat is used to denature complementary strands, Taq polymerase was isolated from Thermus aquaticus, an organism that can withstand high temperatures. This allowed DNA amplification using a PCR machine.

B. Altering temperature bath: Thermal cyclers that alternate between varying temperatures allow automation of DNA amplification.


What is the Plateau Effect in a PCR?

During the last stages of a PCR, when the concentration of the product is between 0.3nM to 1.0nM, there is an arrest in the exponential rate of DNA amplification. This could be due to:

What are the Different Types of PCR?

The different types of PCR are:

This may be carried out manually or by placing wax in between the reaction mixture and the specific component. As the temperature increases, the wax melts and the component is mixed with the reaction mixture.


What is the Difference Between DNA Replication In Vivo and Strand Amplification in a PCR?

DNA replication in vivo or within the living system is different from strand amplification in a PCR in some ways.

What are the Uses of Amplifying DNA Using a PCR?

The uses of DNA amplified by PCR are:

Limitations of PCR

Some of the limitations of PCR in gene synthesis are as follows:

References:

  1. Polymerase Chain Reaction (PCR) - (http://www.ncbi.nlm.nih.gov/probe/docs/techpcr/)
  2. Polymerase Chain Reaction (Interactive) - (https://www.dnalc.org/resources/animations/pcr.html)
  3. Polymerase Chain Reaction (PCR) Fact Sheet - (https://www.genome.gov/10000207)
  4. Principles and applications of polymerase chain reaction in medical diagnostic fields: a review - (http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3768498/)

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