For my research work on bacterial fingerprinting, I have to run DGGE analysis. I have to design my primers and for DGGE I need to add gc-clamp to primers. Some articles says gc-clamp to be added to forward primers while few have added to reverse. Most have added to 5'end while rare have 3' end.
What will better option for adding gc-clamp? Can same gc-clamp be added to both the primers? If yes wont same clamp will anneal?
The purpose of a gc clamp in DGGE is to stop the entire amplimer from melting completely. It creates a region that will not melt and changes the melting characteristics of the region beside it so that melting is partial and the changes in sequence in the adjacent region have the chance to melt at different denaturant or temperature conditions.
You could add the clamp to either end of the amplimer and it will work but if one end of your amplimer is already GC rich thenit might be better to make the clamp at that end so changes in the AT rich end melt easily and separate well from changes in the high GC end making the result easier to read. Adding clamps at both ends is possible and will create a different pattern of melting as both ends will stay double stranded and you will just pick up bubbles in the middle melting domain and the differently melted sequences may be much closer together. If you want to label both ends it is probably better to have both F and R primers labelled and also unlabelled and run F lab-R unlab and r lab -F unlabelled separately to create melting domains at both ends of the amplimer. GC clamps are quite long and will need purification so on cost grounds you might be better off running just one clamped primer or even looking into psoralen clamping at one end
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