What factors contribute to PCR-introduced mutations?
What factors contribute to PCR-introduced mutations?
The following factors can contribute to PCR-introduced mutations:
-Unbalanced dNTP concentrations
Unequal amounts of the four dNTPs can increase base substitution by as high as eight-fold. Using equal concentrations of the four dNTPs is critical for reducing the error rate of the polymerase.
-High enzyme concentration
-Long incubation times
-Lack of 3'?5' exonuclease activity
-Magnesium concentration
Fidelity is highest when the concentration of Mg2+ is equimolar to the total concentration of the dNTPs. Fidelity decreases when the concentration of free divalent cations increases.
-pH of the reaction
Lowering the pH of the reaction by three units can increase base substitutions up to 60-fold. Low pH (<6.0) may lead to spontaneous purine loss.
-DNA damage
DNA damage can occur at high temperatures, possibly increasing the rate of mutation. One frequent mutation is deamination of cytosine to produce uracil.
-The presence of A stretches in primer sequences
-Overcycling
Error rate is increased when the DNA concentration is increased during the final PCR cycles. The total number of cycles should be kept to a minimum to produce the desired PCR product without errors.