PCR Protocol | A Step by Step Procedure

The polymerase chain reaction, or PCR, was originally developed by Kary Mullis, who won the Nobel Prize for this in 1993.

The PCR is a laboratory technique that is used to generate large quantities of specified DNA. The polymerase chain reaction produces the selective amplification of a specific type of DNA- fragment for cloning.

The polymerase chain reaction is the cell-free amplification technique, which is used to synthesize multiple identical copies of any DNA of interest.

PCR Principle

The DNA of interest is denatured by the action of restriction endonuclease, which separates the two strands of the DNA. The strand is hybridized with a primer called renaturation. The complex of the resultant duplex is used further for DNA synthesis.

The three steps of denaturation, renaturation, and synthesis take place again and again to produce copies of target DNA.

The PCR Protocol / Technique of polymerase chain Reaction

The following are the essential requirements for PCR procedure like

Reagents:

  1. A target DNA.
  2. Two primers (synthetic oligonucleotides of 17-30 nucleotides that are complementary to the DNA )
  3. 4- deoxyribonucleotides (d-ATP, d-TTP, d-GTP, d-CTP )
  4. A DNA polymerase that can withstand the temperature up to 95 degrees Celsius.
  5. Reverse transcriptase enzyme
  6. 30 cycles of PCR are done to acquire the desired DNA sample.

Equipment:

pcr protocol
PCR cycler
  1. Pcr machines
  2. Pcr plates

Working on the mechanism of PCR :

The actual mechanism of PCR involves the following steps, which include the repeated cycles of amplification of target DNA.

  1. Denaturation :

This step is also called as the melting of the target DNA. In this step, the DNA of interest is subjected to a high-temperature range from 94 to 96 degrees Celsius, resulting in the separation of the two strands of the DNA.  Every single strand of the target DNA then acts as the template for DNA synthesis.

  1. Renaturation or annealing

As the temperature of the above mixture is slowly cooled to about 55 degrees Celsius, the primers base pairs with the complementary region flanking the DNA target strands. The two oligonucleotides primers anneal or hybridize to each of the single-stranded DNA. This step operates at a low-temperature range of 40 to 60 degrees Celsius, depending upon the length and sequence of the primers.  This process is called annealing or renaturation. The high concentration of primers ensures annealing between each DNA strand and the primers rather than the two strands of DNA.

  1. Synthesis or extension of polymer chains

The starting of the synthesis step occurs in the 3’-hydroxyl part of each primer. The primers are elongated with the addition of the complementary bands.  The synthesis process in PCR is quite comparable to the DNA replication of the leading strand.

The final step is the extension, wherein Taq DNA polymerase (thermophilic bacterium Thermes aquatics) synthesizes the DNA region between, using dNTPs (deoxynucleotides triphosphates) and magnesium ions.  The maximum temperature for this step is 72 degrees Celsius.

Like this, all the steps are repeated again and again to give multiple copies of the target DNA.

The PCR finds its applications in many areas which are as follows:

  1. PCR in clinical diagnosis.
  2. In the prenatal diagnosis of inherited disease.
  3. Diagnosis of retroviral diseases.
  4. Diagnosis of bacterial diseases.
  5. In sex determination of embryo.
  6. In DNA sequencing.
  7. In a comparative study of genomes.

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