Using PCR, a student wants to amplify a sequence of known DNA from a genomic DNA sample. The two primers are both designed to pair with sequences that have a 40% GC content. Using a particular set of denaturation temperatures, annealing temperatures, and extension temperatures, the experiment does not produce the expected single DNA fragment of a single known size, but instead a set of fragments of varied sizes. Which of the following could account for these results?
A)The annealing temperature was too low so that the primers can bind with multiple genomic sequences that contain some mismatched bases.
B)The denaturation temperature was too high so that the primers can bind with each other as well as with multiple genomic sites containing mismatches.
C)The extension temperature was too low so that many fragments were terminated before the full fragment was replicated.

Question

Quizplus · Accepted Answer

The fact that multiple fragments of varied sizes were produced suggests that multiple sites were being amplified, which is likely due to the primers binding to multiple genomic sequences containing mismatches. A low annealing temperature can promote non-specific binding and allow the primers to bind to other genomic sequences that may contain some mismatches. The denaturation and extension temperatures are less likely to be the cause of the issue as long as they are within the appropriate range for PCR amplification.

Using PCR, a Student Wants to Amplify a Sequence of Known