- 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.
- 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.
- 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
- distilled water
- PCR buffer
- Taq polymerase
- DNA template
- Paraffin oil
- 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.