Secondary structure in real time - (May/14/2010 )
I'm running real time PCR with a hydrolysis probe. I checked the secondary structure of my amplicon at an annealing temperature of 60. There is a hairpin loop where my probe binds. The melting temperature of this structure is 64.9, so it will be intact when my primers anneal at 60. I've been getting a high amplification efficiency and I wasn't sure if this secondary structure was attributing to it. Should I redesign? My probe starts at 28 bp-54 bp on the image below. Any feedback would be great. Thanks.
the probe anneals at a higher temperature compared to the primers, approx. at 70°C during the temperature decrease from the 95°C step to the 60°C. so it is likely that probe binding is not affected by this structure which explains your high efficiency.