Unmethylated primer control - (Jul/21/2005 )
Hello everyone, I was wondering if there is a positive control for unmethylated primers. My unmethylated primers do not always show up when running my gel even when my MSP show. I am trying to troubleshoot my results as I don't know if they are accurate. I believe chemicon might have converted genomic DNA but I was hoping for a "cheaper" option. Thanks a bunch.
you can create your own unmethylated control. you simply amplify your region of interest by PCR, clone it into a dam and dcm - bacteria and isolate the clone for bisulfite conversion.
convert it and now you have an umethylated control for your PCR.
USP primers amplifying may suggest inefficient bisulfite conversion or, if it has been fully converted, indicates your population exists as methylated and unmethylated states.
Thank you very much for the reply. I have a couple more questions if you don't mind.
I was just looking up the dam gene and to my limited knowledge I'm observing that they methylate adenine. Now my question is why clone in a bacteria with a methyltransferase when we're essentialy trying to rid our sequence of methylation.
My second question is I have a range of multiple cell lines and my MSP is showing that most of my cells are methylated with only one of my cell lines showing partial methylation. This is with the same set of primers modified for methylated and unmethylated samples. This may sound good as I'm glad my methylated primers are lighting up, however I'm a bit of a skeptic. What would you do to verify your results as soon as possible. I'm really anxious especially since it may take some time to prepare my USP controls. Heelllllppppppp!!!!!!! and thank you.
you want to clone into dam negative and dcm negative lines! hope this clears it up.
As for confirmation of your results, you could look for methylation sensitive restriction sites close to or within your region of interest (such as HpaII and MspI). You then digest your genomic DNA with these enzymes and perform a normal PCR on the region of interest. If the restriction enzymes cut your region of interest you will get no amplification, this is quite a robust assay. (HpaII only digests on unmethylated CCGG sites). It's quick and dirty, but does the trick!!
Thanks a lot