Is high heat primer necessary?

Molecular Biology: Which primer would anneal best at a relatively high temperature?

• Which primer (listed below) would anneal best at a relatively high temperature and WHY? Which primers (listed below) would be able to prime DNA synthesis at all at a relatively low temp and WHY? Primer A: CCAccCGTTA Primer B: CCAGTCGTcc Primer C: CCAGTCGTTA DS-DNA Target: 5'-ACGTTGCACCAGTCGTTATGACCAGGATAACGCATA-… Thank you for your help on this!!

• Answer:

  Specific target region: CCAGTCGTTA Primer A has 2 mismatches in the middle Primer B has 2 mismatches at the 3' end Primer C has no mismatches, so it would anneal best, at high temperatures. At high temperatures, molecules "vibrate" more, and hydrogen bonds become weaker. Only primers that have the best match, will be able to still stay annealed. Any mismatch will make the binding weaker, so at higher temperatures, the primers with mismatches will not be able to bind anymore. At relative low temperatures, more mismatches are allowed, and primers with some mismatches can now bind as well. So now primers A and B, both with 2 mismatches, might bind, and primer C will bind as well. However, primer B has 2 mismatches at the 3' end, where DNA extension will start, and a DNA polymerase might not recognize this as good priming. So at relative low temps, primers A and C only will allow priming.