methylated genomic DNA PCR - (Nov/17/2004 )
Recent, I am doing the Restriction Digestion/PCR to assay the genomic DNA methylation status. I isolated genomic DNA from testis and liver by using promega kit. Then I digested DNA by AvaII, HpaII and TaiI. All these enzymes are methylation sensetive. Before I digested the DNAs I did a PCR by using genomic DNA as template. I got very clear band in testis sample (the DNA is demethylation) but no band in the liver DNA sample ( the DNA is hypermethylation). After I digested the DNA by BamHI, and run a agarose gel. I saw both testis and liver DNA show a stronge smear around 20kb. Does any have experience on mehtylated genomic DNA PCR can give me any comments?
If I understand you correctly, you are trying to amplify POST-digestion with methylation-sensitive enzymes. Right? If this is the case, why do you not get a band in your genomic DNA (even if it is methylated) before digestion? If your primers are working, this DNA should amplify just fine. The PCR after digestion is the one that is your experimental variable. To get this to be cleaner and unsmeared, just purify (I use Qiagen columns) your restriction mixture and use the eluted DNA for PCR.
Also, try posting this in the methylation forum.