I have designed a real-time assay to amplify a target RNA isothermally (@41degrees throughout). My problem is that there is a lot of variation within the gene which leads to quite a lot of variation in the secondary structure of the amplification product and therefore decreased sensitivity of the assay. The assay does not contain a denaturation step (with the exception of an initial denaturation step before the addition of enzymes)so I need another way of melting the secondary structure of the amplification product to see if this will help to increase the sensitivity of the assay? A collegue has suggested DMSO...this has quite a significant effect on the Tm of the probe though (according to literature) and I have not seen it used as such for this sort of assay, mainly PCR. I am looking for other suggestions if anybody has some!
Thanks in advance =D
