Differentiating methylated and unmethylated DNA hexamer on HPLC will be hard. Best will be probably reverse-phase system.

What about enzymatic degradation of the unmethylated DNA?

I think PAGE is most suitable, PAGE can be used to differentiate full length product >= 80% from failure sequences based on size and conformation. More over you have very few sample, it is sufficient to run PAGE as HPLC. Thanks for the query

Poly acrylamide gel Eletrophorisis is most suitable method

In my opinion, HPLC is not so hard. Any C18 reverse column can do it. The problem is the amount that you need. Preparative HPLC is not as usual as analytical HPLC, and I do know the availabitity that your have to a preparative equipment (large colums and so on).PAGE is of course also useful.

I think you didn't understand, SRIVARDHAN REDDY Gayam wants to separate methylated and unmethylated DNA, for that the PAGE will be useless.

I see, sorry, I did not understand. In your first answer you distinguish HPLC from reverse-phase system and you discarded HPLC as hard. But you are right, PAGE is probably not good for separating 6-mer DNA primers. Too small. Reverse-phase HPLC will be the most valuable technique. Cheers

You can use acrylamide gel electrophoresis for purification

Francisco Solano - I meant it will be hard to separate the methylated and unmethylated DNA, but still seems like the only option.

Hi again Tomas,

Well, probably we are not helping a lot to to Shrivardan (in fact he dissapeared from our talk). Any, Selma Sinan thinks different. I had a two-minutes look to bibliography, and I think that HPLC with MS detection is perhaps the most appropriate method. See for instance:

A rapid, quantitative, non-radioactive bisulfite-SNuPE-IP RP HPLC assay for methylation analysis at specific CpG sites

O El-Maarri, U Herbiniaux, J Walter… - Nucleic acids …, 2002

If you have well configured HPLC- UV detector, Fraction collector you can do it easily.The Primers can be separated on a cation exchange column using high levels of ammonium acetate at pH 4.6, or also can be separated on C18 with hexanesulfonate at about 15% MeOH/water at pH 2.5

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Last post from Edward Miracco seems the best for me. High resolution cation exchange can in fact do the job with a greatest resolution than reverse phase HPLC. I usually do this with MonoQ 10/100 GL column in denaturating conditions at RT (10 mM NaOH for buffer A, 10 mM NaOH and 1 M NaCl in buffer B). You will have enough resolution to separate your methylated and unmethylated oligonucleotides. Then, you just have to neutralize the sample to pH7, desalt and check by MS. This works well for short oligonucleotides, up to about 20 bases (the shortest, the greatest the resolution).

Good luck

Dear Francisco Solano and Tomas Hlusca

So far I could not solve my problem and I am trying to figure it out. Indeed your discussions and suggestion helped me a lot to gain the knowledge. Thank you both for consideration of my problem.

Dear Thomas Manesh, Edward Miracco and Yann -Vai Le Bihan

Thank you for consideration

I will try to find out the suitable protocol suggesting here.