Question

After you've identified five homologs of your favorite gene in your dog, you are eager to clone them. You download the dog genome sequence and design and order primers for all five homologs. After the first PCR reaction you realize that now your are having issues with non-specific amplification. Which of the following adjustments to the PCR reaction could you do to increase the specificity of the reaction? (choose all that apply) and why 2 sentence

Raise the temperature of the annealing step.

Lower the temperature of the annealing step.

Add beta-clamp to the PCR reaction.

Decrease the amount of magnesium in the PCR reaction.

Increase the amount of magnesium in the PCR reaction.

Answer 1

Raising the annealing temperature : During the annealing step in PCR, the annealing temperature needs to be low enough to allow forward and reverse primers to bind to the template, but it should not be so low that it will lead to the formation of undesired, non-specific bands in the PCR reaction.Hence the annealing temperature should be kept high in case of non specific amplication in PCR.

As a rule of thumb use an annealing temperature which is 5°C lower than the Tm of the primer.

Adding beta clamp which is the DNA sliding activity present in DNA polymerase Inside the cell can not only reduce background but will increase the processivity along with the proofreading activity.The tag polymerase which we use in PCR is thermostable but do lack this clamp function.Therefore some advanced version of tag Pol is used like Pfu Enzyme for PCR reactions for these benefits.

Magnesium is used as a co-factor for thermostable DNA tag polymerase. Excessive magnesium chloride concentrations can also stabilize the DNA and therefore can inhibit complete denaturation of the DNA during PCR reducing the product yield.Hence low magnesium ion will inhibit the formation of the non specific bands in the PCR reaction.