Checking status of Bisulphite Treated DNA - How do i know the treatment has worked? (Sep/20/2005 )

Hello!

I am stuck with methylation once more! I have a quick question - is there a simple way - an Restriction Enzyme or something that i can use to check if my bisulphite treatment has worked before i do the PCR - i am trying to optimise treatment times and need to check if its working before i do anything else down stream.

Help soonest please! as usual my boss wants results ASAP!

thank you!

I can't think of a way of doing this without performing a PCR....conventional bisulfite is such that you loose the majority of the starting DNA.

if there is a way I would like to know, but how I do it is outlined within a pinned post.

Nick

Hi Nick,

I am actually using your protocol as a basis for mine... thanks for that!!

someone i spoke to here suggested a enzyme that cuts at CpG islands - so i might try that. I'll keep u posted!

Yes, you can use restriction enzymes to check the completion of conversion. You still need PCR after or before the digestion. But I doubt you will get conclusive data about that because lack of proper control DNA and problem related to digestion itself such as incomplete digestion.

oh good luck with that janbrisbane, as pcrman says, you still require a PCR step whether it's before or after the restriction digestion.

who's group are you working for in brissy? I am in cold but bearable melbourne :-)

Nick

it worked!!! i am so excited after many many tries! well atleast i think it did - my digested, bisulphite treated DNA did not get amplified with general used primers but had amplification (though non specific) with methylation specific primers. I can conclude that it worked then right??

Nick, i am working at the Mater Childrens Hospital in Brisbane, where are u in melbourne?

hmmm I don't think it's conclusive a result because you got non-specific amplification with your methylation specific primers, are the general primers you used directed towards your gene of interest before bisulfite conversion?? it's certainly a step in the right direction if it is!

I am working at the Murdoch Childrens Research Institute in melbourne.

I just thought i'd share that i managed to clean up my PCR product and sequenced it and the bisulphite treatment has defiantely worked - now to reproduce it! thats another story.....


Nick, what are u working on??

that's awesome news, i have found that the methylation is quite reproducible in my system.

I work on human centromeres. and you Janbrisbane?

Nick