Methylation Analysis-New methods? - (Jan/19/2006 )
Our lab would like to start doing some methylation assays simmilar to that done in Waterland and Jirtle, Molecular and Cellular Biology, Aug. 2003, p 5293. We want to be able to detect the degree of methylation at each CpG site in addition to finding if there is any methylation there at all. In this reference, they use phosphorimaging and manual sequencing to detect the percent methylation. Does anyone know of a way to detect percent methylation at particular CpG sites without using phosphorimaging and manual sequencing? Or is there a company that will do this analysis for hire? Any and all idea are welcome.
The method you mentioned is new (few people use it) but outdated. You can just do automatic dye terminator cycle sequencing and then measure the peak height of Cs and Ts on the chromatogram if they overlap.
Thanks for your response. We plan on doing automatic dye terminator sequencing to detect presence of methylation, but we didn't really think there was an accurate way of measuring peaks. Do you know how accurate that is for detecting percent methylation? Can you get a reliable and repeatable answer by doing it that way?
The most acurate method is PCR cloning and sequencing. You need to screen at least 10 clones for each PCR reaction. The more clones you screen the more acurate.
Actually, methylation levels measured from peak height on direct sequencing is pretty consistent with result from cloning.
Worm21,
look up Sequenom, I think that is something you are after, high throughput methylation anaylsis that is site specific, and I think this will become a service , it is in Australia, and it's cheaper than direct sequencing.
Nick
Hello,
Pyrosequencing is a method which is able to accurately tell you what the percentage of methylation is at each particular CpG site. It is even possible to send your samples to a company to have them do the testing.
Have a look at http://www.biotagebio.com/DynPage.aspx?id=8068 for some more info.
David
I had Biotage did my samples. At least my positive control seems worked. (SssI treatment)
Thank you all for your helpful suggestions.