Hello everbody,
recently, I plan to check the influence of DNA methylation and methylation levels on protein activities in vitro (a protein with both methyl-DNA and unmethyl-DNA binding properties), So I need first try to amplify a specific fragment with different methylation levels.
for an example, 50% methylation levels, each DNA molecule is methylated, and 50% cytosine is replaced by methyl-cytosine occasionally, rather than 50% DNA molecules are methylated, 50% unmethylated (which I can acquire by mix unmethylated and methylated DNA)
As I consumed, If I mix different ratio of dCTP:dmCTP for PCR mixtures (50%), during PCR, 1/2 of cytosine (C) position will be replaced by methyl-cytosine (mC) accasionally. the final products will contain 50% mC.
after, I need to check if the DNA contains different levels mC. the slot blot is used, and anti-5mC antibody is applied to detect the methylation levels.
the unthylated PCR product (no dmCTP) show no signal, while the 100% methylated DNA (no dCTP) show strong signal. but the PCR products amplified with different dCTP:dmCTP ratio show similiar signals (10%, 25%, 50%, 75%) to 100% methylated DNA.
could I conclude that all PCR products, even the one with only 10% dmCTP added, are 100% methylated, all C is replaced by mC.
if not, which methods should be applied to detect the methylation levels.
Hi,
I don't think this approach is a good idea as for methylation only CpGs are modified, so the methylation of all does not mimic this behavior completely. Would SSS methylase based methylation be an option? For detection of methylation I would go with either Sanger or Pyro Seq
Sven
You need to check the sensitivity of your antibody assay. Since you may be experiencing some sort of signal saturation and signals from 10% methylation and 100% look the same. An antibody titration might come in handy, but considering the type of assay it may not work.
To detect CpG site-specific DNA methylation, qPCR (e.g. methylight) is a good option. If you want to analyze the methylation status of several CpG sites at once (methylation "level" within a group of possibly methylated Cs), bisulfite sequencing (sanger sequencing variant) is the way to go. This will give you a better idea of the methylation status. Be sure to include your methylation controls.
The ratio of dC versus dmC incorporated will depend on the preference of the polymerase used to synthesize the DNA. If you wish to assess this ratio reliably, you should hydrolyze the DNA to single nucleotides (e.g. with a mixture of DNase and snake venom exonuclease) and then quantify the ratio dC:dmC by some chromatographic technique (thin layer, paper, HPLC etc.), using appropriate dC and dmC standards.
- Badges
- Science method