Single-step nested PCR: how to investigate dynamics? - (Jun/14/2013 )

Hi,

I've designed a single-step nested PCR in which two sets of nested primers amplify an outer and an inner amplicon (see attachment). By using a lower inner primer concentration I obtain a large majority of outer amplicons.I'm an now interested in the dynamics of this PCR. I would like to see when the outer primers actually start taking over the amplification from the inner primers and see what influence primer concentration, Tm etc. have on this reaction.

I thought of using probes which are specific for the outer amplicon and one which is non-specific in order to calculate a ratio of each amplicon after each cycle. But designing a specific probe for the outer amplicon proves difficult. Does anybody have an idea how to solve this?

Regards,

Bram

Could you label the primers for the outer amplicon (and the inner ones with a different label) and use that to see?

Hi Bob, this would be possible if I would use capillary electrophoresis. But using fluorescent labels such as FAM or NED in QPCR on you primers does not work as they fluoresce without even being incorporated into the amplicon. I need to have some kind of fluorescently labeled hydrolysis probe with a quencher so I can measure its fluorescence when it gets incorporated. Regards,Bram