qPCR of BS DNA - (Apr/30/2010 )
I have been successfully doing MSP for my target gene. My amplicon products are 116bp for methylated and 96bp for unmethylated.
I have tried to use the same primer set for SYBRgreen based qPCR. The results are unexpected. I get limited amplification in the samples that contain DNA but I get a strong signal in the negative controls.
I wondered if anyone had any advice or experience with BS DNA using SYBRgreen in qPCR.
Are your negative controls without template? How many times have you done this qPCR?
Also, what does the melting curve look like for the negatives? It could be because of primmer dimmer..
Yes my negative controls are without template. I think the amplification that I am seeing is probably due to primer dimers, after looking at teh melt curves.
I was having a read of the datasheet for Fast SYBRgreen and it contains uracil-N-glycosylase (UDG) and i was wondering if this could be a problem with bisulphite treated DNA as all the thymines converted to uracil would be degraded. This may explain why I was getting no amplification in my samples containing bisulphite template.
If anyone has any experience in this it would be greatly appreciated.
UDG treatment can prevent the reamplification of carryover PCR products by removing any uracil incorporated into single- or double- stranded amplicons (from the manual)
Its probably degrading the template, try it with a different mastermix and do a gradient with different primer concentrations to get rid of the primmer dimmers ( how many cycles are you running?)
I've ordered a mastermix without UDG so I'll see if that helps. I'm doing 40 cycles at the moment and the amnplification has a Ct value of about 35 (primer dimers).
I'll try the primers at different concentrations.
That is probably what is happening because you have UDG in there.
Also the SYBR Green dye will change the PCR dynamics a little and would also require some optimisation (potentially tweaking the Mg concentration).
Using a robust mastermix that works for most things would help, such as fastStart from roche.