You want to create a point mutation in you PCR product by modifying one nucleoti
ID: 141075 • Letter: Y
Question
You want to create a point mutation in you PCR product by modifying one nucleotide in Primer 1. This means that the primer/template binding at one nucleotide will not be complementary but binding of the other 5 nucleotides will still occur in a complementary manner. You will now need to (increase/decrease) the annealing temperature of your reaction. Which of the following best explains why?
A. Increase, because this is now a less favorable interaction
B. Increase, because you need to make sure that the stands are capable of denaturing
C. Decrease, but only if the nucleotide change is from an G or C to a A or T
D. Decrease, because fewer hydrogen bonds are going to form between the primer/template
Explanation / Answer
Answer is d
Because if temperature is too high, primers will not bind to the template. Use a temperature that is 5 °C lower than the Tm of the primer. And if the no. Of nucleotide pair decrease there will be less hydrogen bonding that may result in instability at high temp.