Steps and procedure of polymerase chain reaction (PCR)

  • Many cycles of DNA synthesis are carried out in PCR.
  • These cycles are staged by controlling the temperature that reaction takes place.
  • The various steps are:

1.Denaturation of DNA

  • The double stranded DNA should be heated to 940– 960C to break a part the hydrogen bonds forming single strands.
  • Denaturing is carried out for an extended time to ensure that both the template DNA and primers have completely separated and are now single strand only.
  • It takes about 1-2 minutes.

2.Annealing of primers

  • The separated DNA strands are cooled and allowed to anneal (hybridize) to two primers, one for each strand.
  • Primers can attach themselves to the single DNA strands, usually 50C below their melting temperature (450-600C).
  • A wrong temperature during the annealing step can result in primers not bonding to the template DNA at all, or binding at random.
  • The annealing time is 1-2 minutes.

3.Elongation or chain extension

  • Two new strands complementary to original DNA strands are synthesized in presence of DNA polymerase, dNTPs and primers.
  • Generally it is carried out at 72oC for 45 seconds.
  • The elongation starts at the 3’-OH end of annealed primer, making complementary copies of the target.
  • PCR products may be thousands of base pairs long.
  • At the completion of one cycle of replication, the reaction mixture is heated again to denature the DNA strands.
  • Each DNA strands binds a complementary primer and the cycle of strand separation is repeated.
  • Thus each newly synthesized polynucleotide can act as a template for the successive cycles.
  • This leads to an exponential increase in the amount of target DNA with each cycle, thus the name PCR.

 

Procedure of PCR

  • It involves preparation of sample, the PCR mix and the primers followed by detection and analysis of the reaction products.

1.Sample preparation

  • DNA sample should be such that it contains at least one intact DNA strand encompassing the region to be amplified.
  • On doing so any impurities present, are sufficiently diluted so as not to inhibit the polymerization step of the PCR amplification.
  • Usually 1:5 dilution of sample with water is sufficient to dilute out any impurities, which may result from the purifying protocol.
  • It is often best to use the fewest steps possible in DNA preparation in order to prevent accidental contamination with unwanted DNA.

2.PCR mix preparation

  • The PCR mix contains all of the components necessary to make new strands of DNA in the PCR process.
  • The PCR mix reagents include buffer, deoxyribonucleotides, primers, Taq polymerase and template DNA.

3.Cycle parameters

  • The annealing temperature must be calculated for each primer set.
  • The higher temperature minimizes non-specific primer annealing, increasing the amount of specific product produced and reducing the amount of ‘primer-dimer’
  • The annealing temperature is usually 5oC less than the melting temperature (Tm) of the primer.
  • This temperature should be checked by performing the reaction at several temperatures.

4.Order of premix preparation

  1. distilled water
  2. PCR buffer
  3. dNTPs
  4. Taq polymerase
  5. MgCl2
  6. DNA template
  7. Primer
  8. Paraffin oil
  9. PCR cycle

Detection and analysis of reaction products

  • The PCR product is a fragment or fragments of DNA of defined length.
  • The simplest way to check for the presence of these fragments is to load a sample taken from a reaction product.
  • Also the appropriate DNA molecular weight markers should be taken along with the sample into an agarose gel.
  • Staining with ethidium bromide facilitates the visualization of DNA bands on the gel under UV trans-illumination.
  • Identification is done by comparing with the bands with that of known markers.
  • The DNA replication that takes place in PCR, like in vivo is not 100% accurate.
  • A mistake is made occasionally.
  • If the amplification fragment is to be cloned, the resulting clones must be sequenced to ensure that they carry a wild type copy of the gene.
  • If the amplified fragment is to be used as a probe, a few mutant copies in a mixture that contains a large number of wild type copies will not present a problem.
  • Thus, depending upon the use of PCR fragment, these contaminating mutant copies of the fragment must be accounted for.

Steps and procedure of polymerase chain reaction (PCR)

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