This is more of an biological protocol question. My problem at hand is the following one: Firstly, I need monitor DNA duplex formation by primer extension on a microarray and I am kind of limited to the use of Cy3 dye for this (but I'm open for suggestions). Secondly, I need to monitor the hybridization of another molecule to the duplex DNA. Here, I intent to use Cy5 dye attached to the molecule in question. The problem is that both dyes could come into close contact, which may result in a quenching bias... something I would rather avoid.
Does anybody have any experience about how this bias manifests or which dyes may be more appropriate for the task at hand, i.e. don't interact with each other?
To circumvent the bias I could think of removing the Cy3-label after the duplex formation has been confirmed. Is there any experience about how to remove a dye from nucleotides by e.g. photobleaching? I would prefer to physically remove the dye from the nucleotides without, of course, destroying the DNA duplex. However, depleting the emission capacity of the Cy3 label might also be an option.
while this forum so far attracted mainly informatics related expertise let's hope that we can enlist a few wetlab experts as well