Hi,Nick.
I don`t know how to use methylation specific enzyme to assess the transfer rate of the C to T.Can you explain how to do it and its principle.
Thanks.

Leydig

QUOTE (mushroom @ Sep 17 2006, 01:44 PM)

I have no experience in this area yet. But I know that Sigma JumpStart, while very robust, generates high percentage of PCR mutation. Is this a possible problem?

Please explain?? I was unaware of the high mutation rate. it should be no higher than normal taq form other suppliers

basically you want to test the conversion by either the gain or loss of a restriction enzyme

MspI is the best example, if you have a MspI site within your region of interest after bisulfite conversion this would be lost because:

MspI Site : CCGG

After bisulfite and PCR it would be either : TCGG or TTGG depending on the methylation status of the CG in the middle of the site and you are testing conversion by the loss of this restriction site. because your PCR would contain a heterogeneous population of amplicons with variable methylation and that bisulfite conversion is never 100% you will get a subpopulation whereby it has not converted well and with MspI this would result in the amplicon being cut and on a gel it presents as two bands.

Good luck!

Nick