You want to create a point mutation in you PCR product by modifying one nucleoti

Q: You want to create a point mutation in you PCR product by modifying one nucleoti

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.

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.

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You want to create a point mutation in you PCR product by modifying one nucleoti

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