MSP - (Jun/20/2008 )

Hello

I have just started with MS PCR to identify methylation patterns of three genes. I have used methPRIMER to design my primers and their specificity was then assessed using methBLAST.
There is very good amplification with methylated primers in case of positive control (gDNA treated with SssI methylase) as well as samples. But, I am not able to standardise the PCR conditions using unmethylated primers for these genes. I have used annealing temp. gradient of 44-59˚C and magnesium chloride concentrations ranging from 1 to 4 mM. Bisulphite modified DNA from PBLs of healthy individuals is being used as template to optimise the PCR conditions. Agarose gel shows a smear and primer dimers. Following are the melting temperatures of the methylated and unmethyalted primers:

Gene 1
MF- (Tm 52.8)
MR- (Tm 60.3)
UF- (Tm 51.5)
UR- (Tm 57.1)

Gene 2
MF- (Tm 57.1)
MR- (Tm 57.6)
UF- (Tm 56.4)
UR- (Tm 56.4)

Gene 3
MF- (Tm 59.7)
MR- (Tm 57.9)
UF- (Tm 56.9)
UR- (Tm 55.9)

What type of template DNa can be used as control for unmethylated PCR except human sperm DNA and the one that can be generated using cloning in dam- bacteria?

Please suggest me how can I alleviate this problem. Thanking you in anticipation.

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You could use WGA DNA. The technique removes all methyl groups and you only need 10 ng to start off with.

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Thanks a lot for your suggestion but WGA DNA is not an affordable option as commercial kit is required. I will appreciate if you can suggest any cheaper option.
Is there any manual chemical method available for the removal of methyl groups?

Thanks

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Hi Scooby,

if WGA is not an option, you can PCR amplify the region of interest with normal primers and this would wipe the methylation from your region of interest, it's not the best control as you reduce the complexity of your DNA sample when compared to genomic DNA, but it's better than a genomic DNA sample for which the methylation status of your region is unknown.

good luck

Nick

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