5 replies to this topic

[mexemi]

Is there a method to have an estimate (even qualitative) of overall DNA methylation in health and disease, even if the difference could be really small? Thanx

[kharald]

mexemi, on Oct 7 2004, 08:32 AM, said:

Is there a method to have an estimate (even qualitative) of overall DNA methylation in health and disease, even if the difference could be really small? Thanx

I think HPLC is a good method for detecting the amount of genomewhide methylcytosine content
Methylcytosines photometric maximum is at 283nm
you extrakt DNA then digest it with nuclease P1 and Alkaline Phosphatase
and inject it into a HPLC System RP18 collumn
7mdG& some others can be detected by electochemical detection & HPLC

[pcrman]

Apart from HPLC and other chromatography based methods, SssI methyltransferase assay is simple method for measuring global methylation or the content of 5 methyl-cytosines. Although not as sensitive as HPLC, people have used this assay to detect methylation and found that cancer tissues usually have lower level of global methylation compared to normal tissues.

[mexemi]

Hi there. Thank you all for your replies. You replied to my overall DNA methylation estimation problem. You talked about using an RP18 column for HPLC and digesting DNA with nuclease P1 and alkaline phosphatase. Could you give me a more detailed protocol such as quantities, incubation times etc? I have never done this before. There is a person that uses HPLC in my lab. So is nuclease P1 used to "cut" DNA into single nucleotides and then you can measure the absorbance of methylcyosines? Hope you can help me again. Thank you so much for your time
Regarding the other method SssI methyltransferase assay, do you have a detailed protocol? I have checked some literature but it seems to me you should have already an idea of which genomic region you want to study and not the WHOLE genome. Is this true? thanx again!

[spjgjsm]

I've got a similar question - i'd like to measure quantitatively global methylation. I have been told that the chloracetaldehyde reaction is the best method. Does anyone have any experience of using this, and could anyone point me in the direction of an online protocol??

Cheers

Jon

[Rovy]

mexemi, on Oct 7 2004, 08:32 AM, said:

Is there a method to have an estimate (even qualitative) of overall DNA methylation in health and disease, even if the difference could be really small? Thanx

,sorry to trouble you for the same question .I also want to know methods measuring overall methylation.Now I've used FACS with 5-m C antibody.However,I still need to do HPLC to quantitate the overall methylation.And do you know some other methods which can show redistribution of the whole methyaltion?
Best Wishes and forward to your reply!