How to know if observed methylation is true or a consequence of incomplete or fa - (Jan/09/2013 )
Hello, I've been doing some BIS modification using the Genetic Signature MethylEasy Kit. I got bands of the correct size with my modified primers and immediately ligated the products and procede with cloning in pGEMTeasty. My product is 1626bp in length. I've sent 10 clones for sequencing and the sequences that I have received show most of the cytosine residues to be methylated excepted those in the first ~250bp which are non-methylated and have been converted to Ts. Is this possible or is it more likely that the treatment has failed?
Thanks in advance!
I can't really imagine bisulfite treatment that would ommit only some part of sequence. It either works on all or not.
Amplifying 1.6 kb product from bisulfite treated DNA is very difficult if not impossible due to DNA degradation during treatment. I doubt the product is from treated DNA template.
I'd suggest that your redesign your primers to allow for a product size not greater than 500 bp.
Your concern is right that incomplete conversion can give your false positive results. To control for this, you can include known unmethylated DNA as a negative control.