PCR products are always unmethylated, that's the simple reason why it is not possible to assess methylation of a CpG island by PCR without bisulfite treatment.

You could do Southern blots using metyhlation-specific restriction enzymes and appropriate hybridization probes.

Hi Daniel,

that approach will not work, as with a conventional PCR amplification methylated Cs (5mCs) will not be maintained, but just replaced by a normal C. Only the original template DNA will contain 5mC. That is why people use bisulfite conversion, where all unmethylated Cs are converted to U (after PCR T). Actually, doing bisulfite conversion and then PCR with deamination-specific primers for your region of interest is not that complicated and would be the most sensitive and accurate method. Why you do not want to use bisulfite?

Thanks very much.

A bisulfite free method is Methylscreen:

http://www.ncbi.nlm.nih.gov/pubmed/18072598

Methylscreen does not require bisulfite conversion, but instead relies on the use of methylation sensitive and methylation dependent DNA cleaving enzymes. (see attached paper and link). 

http://www.oriongenomics.com/technologies-clinical-assays.html 

Commercial Methylscreen assays are available for certain organisms:

http://www.sabiosciences.com/dna_methylation_qPCRprimer.php

Thanks Achim and  Sabine for your answers. To answer Achim's question "why you do not want to use bisulfite?": I am new to epigenetics and I am exploring DNA methylation assays that are cost effective and can be run in a modestly resourced lab in a LMIC.  

I have another question though. Can the data from sequencing bisulphite converted DNA be used for SNP calling as well? It will be great to get methylation data and SNP genotyping from one experiment.   

Hi Daniel, yes you can call methylation and SNPs from bisulfite sequencing. I've done so in some of my past work. It also opens up the interesting world of CpG SNPs. But also point raised by Sabine about the need for conversion before PCR is important to keep in mind, since the methylation will not be replicated. 

As for locus specific alternatives, there are a number of methods using restriction enzymes, where one pair cuts methylated DNA and the other doesn't. There are also antibodies specific to DNA methylation. 

Here are some resources:

1) http://epigenie.com/epigenetics-research-methods-and-technology/methylcytosine-5mc-analysis/enzymatic-dna-methylation-analysis/

2) http://epigenie.com/epigenetics-research-methods-and-technology/methylcytosine-5mc-analysis/dna-methylation-enrichment-methods/

Thanks Benjamin. This is refreshing news. Can please share with me some of your previous work (and that of others) that called methylation and SNPs from bisulphite sequencing.

Thanks to David and others for sharing resources.