Biotechnology and Bioinformatics (1+1)

4.1.3. Reaction components

4.1.3.1. Primer s

• Primers should be at least 18-20 (17-30) nucleotide s in length and should have a G/C content between 40 to 60% (otherwise low melting temperature is needed).
• The specificity of the PCR depends upon the primers.
• The aim of good primer design is to maximize both the specificity and efficiency of the amplification reaction.

4.1.3.2. Buffer

pH range 8.3-8.8.

4.1.3.3. Mg++ concentration

• Mg++ concentration can severely affect the efficiency of PCR as a con sequence of its complexing with dNTPs and the Mg++ requirement of the enzyme.
• An excess of Mg++ results in increased non specific priming whereas too low Mg++ levels reduce product yield.
• Optimum Mg++ concentration should be attained empirically by titrating in 0.5 mM increment between 0.5 mM and 5 mM. Mg++ is essential for enzyme activity.

4.1.3.4. Template

The ideal template for a PCR is free from contaminants such as nucleases.

4.1.3.5. Polymerase

DNA polymerase, an enzyme, can lengthen a short strand of DNA, called an oligonucleotide primer, if the strand is bound to a longer "template" strand of DNA. The polymerase does this by adding the appropriate complementary nucleotide to the three prime end of the bound primer. Optimum enzyme concentration is 0.005 – 0.025 units/ m l. Higher concentration may cause an increase in non-specific product gene ration. The DNA polymerase has a 5¹→3¹ polymerase activity but lacks 3¹ → 5¹ exonuclease activity. The enzyme has a half life of up to 40 min at 95 ° C but is destroyed within a few minutes at 100 ° C.

4.1.3.6. Thermal cycling

When optimizing PCR it is important to ensure complete thermal equilibrium of the reaction mix. Reaction volume (including oil or wax layer) and tube wall thickness are critical variables to consider when setting up cycling profiles. Reactions are carried out in 0.5 ml or 0.2 ml reaction tubes.

4.1.3.7. Final volume of the reaction

PCR requires rapid changes of temperature, which are accomplished by the thermal cycler. As a general rule, reactions are usually between 20 and 100 m l. Large – volume samples will be inefficiently heated and cooled, while small – volume reaction render insufficient product for manipulation and analysis.