Overcoming PCR bias before bisulfite sequencing?! - (May/29/2007 )
I just started a project on epigenetics. I am trying to determine the DNA methylation status of several genes by bisulfite sequencing. Unfortunately there seems to be a bias in the PCR reaction. In my control group I expect 50% methylation (as the gene is paternally methylated). But according to the sequences I obtain, about 90% of my clones appear as completely unmethylated.
I know there are some posts that contain hints about overcoming the bias problem. I thought it might be helpful (not only for me!) to share experiences about bias and how you solved this problem in a seperate post.
Has anyone tried betaine in the PCR reaction??
Thanks in advance, Pandreas!!
that is a known problem in BSP. There are some threads about that in the forum (e.g. this one). Main possibility is using an increased annealing temperature. Others have successfully used pirmers containing one CpG-C to reduce the preferential binding to unmethylated templates. There are alos some reports on the use of MGB-elements.
Check out Biotechniques, there have been several reports on that problem in the last issues.
Just because a "gene is paternally (or maternally) methylated, doesn't mean that it is going to have 50% methylation along it's length. It's quite possible that this paternal methylation that regulates the gene is only a short stretch somewhere associated with the gene.
Also something to note, is that if you are sequencing 10 clones from each sample and 1 shows methylation and the other 9 don't (or the other way around) that is not stastically significantly different to 50% methylation. I know that most people only sequence 10 clones from each sample, but from my experience, and some stastical analysis, unless you are looking at a clear cut 100% methylation/unmethylated, I don't really think that is sufficient.
Just my 2c