Components of polymerase chain reaction

  • There are various components of PCR for conducting this process.
  • They are DNA template, primers, Taq polymerase, deoxyribonucleoside triphosphates, PCR buffer, dimethyl sulfoxide and thermocycler.

 

1. DNA template

  • It contains the region of the DNA fragment to be amplified by the PCR process.
  • Successful amplification of the region of interest is dependent upon the amount and quantity of the template DNA.

2. Primers

  • First we should know the nucleotide sequence of target DNA and also of short segment on each side of target DNA for primer construction.
  • Such sequence are called flanking sequences of interest.
  • Flanking sequences are then used to construct two single stranded oligonucleotides primers (25-35 bp long) complementary to the flanking sequences.
  • Such synthetic oligonucleotides function as primer in the PCR reactions and the 3’- OH end of each primer point towards target sequence.
  • In short, a primer is a short segment of nucleotides, which is complementary to a section of DNA, which is to be amplified in the PCR.
  • Primers are annealed to the denatured DNA template to provide an initiation site for elongation of the new DNA molecule.

3. Taq polymerase

  • Taq polymerase is a heat stable DNA polymerase from the thermophillic bacterium, Thermus acquaticus which adds the deoxynucleotides to the DNA template.
  • This Taq polymerase enzyme can act or function at 720
  • Heating and cooling steps should be carried out on the same mixture without adding new enzyme.
  • This allows the procedure to be automated.

4. Deoxyribonucleoside triphosphates (dNTPs)

  • The dNTPS (dATP, dGTP, dCTP, dUTP) provide both energy and nucloeotides for the synthesis of DNA.
  • It is important to add equal amount of PCR mix to prevent mismatches of the bases.

5. PCR buffer

  • PCR buffer contains Tris, MgCl2 and KCl with pH 8.3.
  • It keeps the PCR mix at proper pH so that PCR will take place.
  • Further magnesium is the co-factor for Taq polymerase and affects primer annealing, strand dissociation temperatures, specificity and primer dimmers.

6. Dimethyl sufoxide (DMSO)

  • If multiple primer sets are used, DMSO is also added to the reaction.
  • It helps to lower the melting and strand separation temperatures.
  • It also alleviates some of the de-purination effects at high temperatures, which can results in smears on the gel.
  • A heated lead is placed on the top of reaction tubes or a layer of oil is put on the surface of the reaction mixture to prevent evaporation of the reaction mixture.

7. Thermocycler

  • It is a machine that takes PCR mixtures through 20-50 cycles, producing large amounts of synthetic DNA for subsequent analysis.

Components of polymerase chain reaction

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