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COLD-PCR (Dissociation Curve) via qPCR

cold-pcr pcr qpcr realtime

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4 replies to this topic

#1 jerryshelly1

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Posted 05 March 2014 - 02:53 PM

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.



#2 bob1

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Posted 06 March 2014 - 02:38 PM

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.



#3 jerryshelly1

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Posted 07 March 2014 - 08:41 AM

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.



#4 bob1

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Posted 07 March 2014 - 11:37 PM

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.



#5 jerryshelly1

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Posted 09 March 2014 - 09:13 AM

It is definitely an interesting approach, but the cost would be to great for the reward at this phase. Thanks for the suggestion.







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