COLD-PCR (Dissociation Curve) via qPCR - (Mar/05/2014 )
Has anyone tinkered with the cycling conditions to determine the melting curve of low frequency SNP samples using COLD-PCR? I tried using the regular melting curve protocol that is preloaded on our 7500 System, but the results are not great. Is a 1% temperature increase for the ramp sufficient to obtain a good melting curve? Any advice would be appreciated.
Are you trying to do some sort of dHPLC equivalent? I would have thought a very slow increase would be better for this sort of discrimination, but haven't tried it all.
I want to generate a high resolution melting curve, without using the expensive dyes. I have read that using SYBR Green is sufficient for obtaining a good melting curve (almost equivalent to HRM dyes), but I am having a hard time getting good data. It seems that my curve shifts between experiments and when I try using the determined denaturation temps for sequencing, I still cannot see any indication of a SNP being present. The SNP should be present from a good read via exome sequencing. I am puzzled.
Ok, I won't be any further use there. I know that dHPLC can be used for this sort of thing, so I guess it is more accurate/reproducible than melting curves, but the equipment is quite expensive and not all that common.
It is definitely an interesting approach, but the cost would be to great for the reward at this phase. Thanks for the suggestion.