Written by Prerak Juthani (PEACE Advisor)
a) The polymerase denatures.
b) The primers don’t bind to their specific sequences.
c) The primers were manufactured incorrectly and lack the 2’ hydroxyl the polymerase needs to elongate.
d) The primers were manufactured incorrectly and lack the 3’ hydroxyl the polymerase needs to elongate.
e) None of the above
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In this case, the answer choice A is incorrect because the entire point of having the taq polymerase is so that it won't denature under the temperature-dependent conditions of PCR (in fact, any other polymerase would readily denature under PCR conditions, but taq is the one exception). Thus, the polymerase denaturing is not a valid explanation for why the PCR would stop working (making D incorrect)
B is incorrect because if the sequences of the primers are correct, then there is nothing that will stop it from binding - just because of the natural purine- pyrimidine binding (and the respective amount of hydrogen bindings).
C is incorrect because, as we remember from chemistry mechanisms, it is the 3' OH that does the attacking the phosphate - not the 2'. Thus, it is not the 2' hydroxyl that is needed to elongate; rather, it is the 3' hydroxyl, which makes D the correct answer.
D is also logical because if the primer were lacking a 3' hydroxyl, then it has no "nucleophile" to commence the elongation reaction, and thus, primers that have this defect would simply just prevent the PCR from running.