digoxygenin-labelled primer - (Jan/19/2007 )
Has anyone ever tried using a digoxygenin-labelled primer rather than incorporation? I'm concerned about signal strength.
With a labeled primer, you get a single DIG at the 5' end. By spiking the PCR reaction with DIG-11-dUTP, you can control the fraction of the T sites in your probe having a DIG label, so, in principle, it could be more sensitive. I have never had a sensitivity problem with PCR spiking. I add the DIG-11-dUTP to a PCR supermix rather than making up the complicated dNTP mixture they recommend, and it works fine. Test your probe sensitivity by serial dilution and spotting onto a membrane, then detecting with your detection system. I use the Roche CPSD system, but there may be better ones. When the probe detection is working, check the sensitivity and optimize the hybridization by spotting serial dilutions of your target on a membrane and detect these. Only when this works should you bother with doing Southern/Northern gels.
Thanks for your reply, phage434, but our task is not to hybridise. We are considering alternatives to ethidium bromide (or silver) staining for an SSCP analysis. Since digoxygenin is a rather large molecule, I have reservations about trying to incorporate it into our PCR products. It might have a negative effect on the stabiliity of the conformations of the folded single strands. Hence the question about labelling a primer. I have never worked with digoxygenin, so I have no experience with signal strength and detection. We want to avoid using radioactivity. Can a weaker signal be detected with longer exposures, as with radioactivity? So, does anyone think primer labelling would be feasible?
I see. Yes, you probably want to use a labeled primer, and I would anticipate that you would be able to see it fine. The luminescent detection systems are very sensitive, with the proper detector. You need a chilled CCD imager to take the long exposures (30 minutes - several hours) to achieve very high sensitivity.