Unmehylated control DNA - (Sep/30/2008 )

Hi,

I am about to trial the Illumina Infinium Methylation arrays. I want to include a negative control sample (0% methylation) on the arrays and in the initial bisulfite conversion step. I have read that people use whole genome amplified DNA. How does this make DNA unmethylated? What do people generally use as a negative control? I am aware of commercial DNA that is unmethylated (CpGenome) but have people had success using other options as this is quite expensive. Any assistance you can give me would be greatly appreciated.

Thanks.

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Ok WGA will dilute the methyl groups as during amplification you will incorporate cytosine (not methyl cytosine) - so you may amplify your DNA from 10ng to 10ug, 10ng of potentially methylated DNA is still present, it is just in the vast majority which is fine for most applications

we have used WGA dna as an unmethylated control and by sequenom it looks 0% methylated. the problem is that the WGA procedure is biased, and some amplicons are hard to amplify after the WGA-bisulfite

as for the "unmethylated control" DNA from millipore, i wouldnt bother with it - they just give you 2 tubes of DNA (one from blood, one from a fetal cell line i think) and say that if your gene is methylated in one, try the other one. waste of money

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the initial DNA template you use (methylated) is not significant after amplifying, don't spend money (specially if it is too much) on things you don't really need. if you ever find a way to amplify methylated DNA (not the indirect way that is bisulfite conversion) then make sure to tell us about it!

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Thank you for that. Do you have a protocol for whole genome amplification for DNA for use as an unmethylated control?

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our lab just uses the WGA2 kit from sigma, it costs ~$5 per reaction

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