Question

How would the yield of a PCR reaction be affected by the following mistakes?

Explain your answer.

1. You forgot to add template DNA

2. you added TWICE as much template DNA as is called for by the intructions

3. You forgot to add Taq polymerase

4. You add HALF as much Taq polymerase as is called for the by instructions

Answer 1

1. If you forget to add template DNA, then the primers will hybridize to form primer-dimer products. It will give rise to non-specific amplified small fragments.

2. If you add TWICE as much template DNA as is called for by the instructions, then the DNA may bind to magnesium ions of the reaction mixture in order to stabilize its own structure. However, since the magnesium ions are essential for the functioning of Taq polymerase, it will cause hindrance to the reaction. Apart from this, contaminants present in the template DNA will inhibit the reaction. Higher concentration of DNA also decreases amplicon specificity.

3. Since taq polymerase is an enzyme that copies DNA, if you forget to add Taq polymerase in the PCR reaction mixture then the template DNA will not be replicated and the desired PCR product will not be formed. You may not observe the expected bands on the gel.

4. If you add HALF as much Taq polymerase as is called for the by instructions, then it may give rise to incomplete elongation of primer, because the reaction will run out of Taq polymerase enzyme due to its low quantity. During the elongation step of the PCR cycle it may give rise to premature termination of PCR product synthesis.