designing primer for methylation - (Apr/23/2006 )

Hello,

Just wanted to add my two cents. Our lab originally started methylation analysis trying to work out methods for melting curve analysis.

In theory melting curve analysis should easily be able discriminate methylated from unmethylated DNA. If you design primers to only amplify a few CpG sites (very difficult) using bisulfite sequencing primers (by which I mean no CpG sites recognized by the primer). You can then perform melting analysis and determine how many methylated sites you have with the lightcyler. Higher CG (methylated) would equate to higher melting temperatures. Many papers claim that you can discriminate a single methylated site by a temperature change of .25 degrees. By creating a standard curve of known methylation (induced by enzyme methylation) you can determine how many sites you have methylated in your sample.

One of the major issues we ran into is that the melting curve analysis is very sequence specific. Designing primers can be a major pain in the ass. The more sites you have the less information you can glean from the melting analysis. Also if the product you are trying to amplify has a lot of AT runs or TTTTTTTT sequences, which is common after bisulfite treatment. What ends up occuring is that the product will give a very odd melting pattern resulting in multiple peaks which are nearly impossible to analyze. We have since switched gears to methyl-light, and pyrosequencing and haven't looked back.

-CW