In a splicing by overlap extension PCR, two different DNA molecules (e.g. A and B) share an overlap region. During the annealing step, a single strand from the two different DNA molecules anneal with each other via the overlap region, and DNA polymerase extends to form a combined product (A + B). Why won't the two single strands of A and B re-anneal to its complementary strand (i.e. one strand of A anneals to the complementary strand of A) rather than the overlap region? In a typical PCR, primers are at a much higher concentration than the template DNA, so they are more likely to anneal. However, in my splicing by overlap extension PCR, I did not add any primers.
I think that the comparison with ordinary pcr is good. The first few cycles of both pcr and oepcr are very inefficient because the template self anneals at the higher temperature but in both cases some primer (product overlap) does anneal and extend. Then in the next cycle we have a small percentage of AB which now has perfect homology with products A and B which are in excess over the AB so both A' and b. anneal well and extend to give more AB quite efficiently so with each second round cycle the amount of AB increases
Paul Rutland Thank you so much for your explanation - it was very clear! Just one more thing to clarify: In that case, will the optimal annealing temperature change throughout the PCR? I'm assuming that at the start, the fragments anneal via the overlap region, so Tm will be lower. However, with more cycles, some of the combined A+B product will become the template, and individual A or B fragments will anneal fully (not just via the overlap region), and will likely have a higher Tm?
Claire Lim my feeling is that the answer to your question depends on what you want at the end .....lots of product or less product but possibly fewer mutations in the product. You have already run many pcr cycles to generate first round product. Annealing at low temperature will generate less product but fewer pcr induced mutations because the lower annealing route uses mainly lower cycle first round product but I think that setting the second annealing temperature high will generate more product so more opportunities for taq errors to get incorporated into the final product but I would be interested in seeing what other researchers think
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