Southern blot v. bisulfite sequencing - which is better to detect methylation? (Sep/10/2005 )

NEED HELP!!!

My genomic DNA Southern blot indicates that a certain CpG in my gene of interest is methylated. On the other hand, results from bisulfite sequencing show that this CpG is not methylated. I was wondering which result I should trust and why I would have two different results.

For my Southern blot, I have been digesting 10ug of genomic DNA with the appropriate restriction enzymes.

For bisulfite sequencing, using the TOPO TA cloning kit (with TOP10F' competent cells), I cloned my PCR product into the vector. Then, I picked and sequenced 10 clones. Only one of the ten clones demonstrated methylation.

Any suggestions would be GREATLY appreciated.

Thanks!

hmmmm sounds odd.

I can think of a possilbe reason for your discrepancy. The restriction digest maybe incomplete giving rise to it being methylated.

Bisulfite sequencing is by far, more sensitive than southerns. A southern would give an indication of how methylated a region is while bisulfite tells you the methylation status of every CpG within that region.

It is possible that of the ten clones you have sequenced, you just happened to pick 9 that were unmethylated. If you sequence more you may get a better picture.

good luck

nick