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