Question 14 continued. d. On a different day, you realize you forgot to add one

Question

Question 14 continued. d. On a different day, you realize you forgot to add one of the primers to the tube. You stop the PCR after cycle 13, add the second primer and then let the PCR finish (25 cycles total) What is the expected result compared to "normal" PCR? (Put an X' in the blank for the BEST answer) There will be amplification but the fragment yield will be about half the amount. There will be amplification but the fragment yield will be dramatically lower. There will be amplification but the fragment size will be shorter than expected. There will not be amplification because both primers are needed at the beginning. e. Assuming that the primers you designed are 30 nucleotides long, how many nucleotides will be added by Taq to complete the desired fragment? f. During PCR, what takes the place of topoisomerase (required for DNA replication in vivo)?

Explanation / Answer

There will be amplification but the fragment yield will be dramatically lower.

In the first 13 cycles of PCR, the template is amplified only from one end. Longer fragments are produced during these cycles.

After 13 cycles, when second primer is added, it would bind to the amplified DNA since it contains the complementary sequence. The next 12 cycles would produce the desired fragment from third cycle onwards. Therefore, the yield would be very much lower than half the original. (the amplification results in exponential increase in yield)

e. the length of the original sequence and the positions where the primers bind are required.

f. topoisomerase overwinds or underwinds the DNA.

In PCR, temperature does that. No enzymes are added to PCR mixture except Taq polymerase.

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