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Bisulfite Modification Efficiency - (Aug/20/2007 )

Hi, i'm just wondering if someone could help me. I have just performed my bisulfite modification on my control samples (fully methylated control and fully unmethylated controls which we purchased from chemicon and I have also modified a number of tumour samples. I'm new to the field and this may seem like an obvious question, but how do i tell if i have 100% conversion of my DNA? Is it possible to use normal genomic primers for any region and if i get a band then the conversion hasn't worked?

Thank you very much, this forum has helped me a lot so far.

-imprinter-

QUOTE (imprinter @ Aug 20 2007, 09:29 AM)
Hi, i'm just wondering if someone could help me. I have just performed my bisulfite modification on my control samples (fully methylated control and fully unmethylated controls which we purchased from chemicon and I have also modified a number of tumour samples. I'm new to the field and this may seem like an obvious question, but how do i tell if i have 100% conversion of my DNA? Is it possible to use normal genomic primers for any region and if i get a band then the conversion hasn't worked?

Thank you very much, this forum has helped me a lot so far.



you measure by looking at a C that should be converted to T (ie: not part of a CpG) we routinely get 98% or better...

-beccaf22-

QUOTE (beccaf22 @ Aug 20 2007, 03:06 PM)
you measure by looking at a C that should be converted to T (ie: not part of a CpG) we routinely get 98% or better...

Thanks for getting back to me beccaf,
Just wondering if i could run something by you?.... the primers i used on my modified DNA are just regular genomic primers we use routinely. The product contains a number of C's that are not in a CpG island, therefore should be converted to T's as you mentioned......but I managed to get a product on a number of my samples.....including my methylated control. i would assume therefore that I have incomplete modification?

-imprinter-

Well, I guess you are introducing a bias towards unmodified DNA by using primers for unmodified DNA. Even if you have an efficency of 98% for the modification, you can still amplify the unmodified 2%... Try designing primers biasing for fully converted template by introducing A's or T's in your primers that correspond to non-CpG C's (and G's respectively) in the unmodified sequence.

K.

-kr├╝melmonster-

Hi, I had thought of the bias you mentioned, and that definitely would make sense. I'm going to have a go at designing primers for fully converted template as you suggested. Thanks very much, it's much clearer in my head now!

-imprinter-

glad I could help smile.gif

-kr├╝melmonster-