Real Time PCR Annealing vs. Melting
Posted 01 December 2004 - 10:03 AM
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?
Posted 01 December 2004 - 10:27 AM
Posted 01 December 2004 - 04:55 PM