Real Time PCR Annealing vs. Melting - Relation of anneal temp rxn to melt temp primer (Dec/01/2004 )
I am wondering if anyone can explain to me the relationship of annealing temp for reaction to the melting temp of primers.
Whenever I design primers for Real Time PCR (Using Bio Rad Light Cycler), I use the nearest neighbor calc for Melt Temp of primers (factors in the MG conc and salt etc). I use the Beacon Primer design software, and do separate checks of the sequence on mfold and repeatmasker (to cut out any repeatative sequences or non distinct DNA). Beacon then gives an estimated TA for the annealing temp of the reaction.
How can I make this calculation? Beacon has no reference for their TA output in the design program. I have read on several internet sites different advice (first I check to see if they are doing similar PCR to mine because it vaires so much depending on regular PCR or quantitative). Some sites say to use annealing temp 5-10 degrees LOWER than TM of primers, others say to use 5 degrees above.
Can someone explain to me what happens during the annealing step? I know the primer is "laying down" on the DNA strand to prepare for extension, so should this happen at a lower temp than the melting temp of the primer?
The melting point is the temperature when 50 % of the primer is hybridizated an 50 % is unbound. Therefore a primer binds at lower temperatures more effectively -perhaps under perfoming mismatches- than at higher temperatures. At high temperatures it will bind more specific but noct quantitative. I prefer using a higher TA because it is more specific. Further more you have to consider the probe and allow it some extra time to hybridizate before it is displaces by the polymerase. But it depends on the experiment if a higher or lower TA is useful or more efective.
Our lab has the same problem to u. We desigh primers in Primer Premier5.0. As soon as we get the primers, we will test which temp will be the best in a ladder PCR cycler. There is a experiential conclusion that the suitable temp will be 10 degree lower than the software estimated.