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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.
Attached File

-ColoSarah-

hi,

the probe anneals at a higher temperature compared to the primers, approx. at 70C during the temperature decrease from the 95C step to the 60C. so it is likely that probe binding is not affected by this structure which explains your high efficiency.

-tea-test-