Hi, depending on your purpose and resources, I highly recommend using the MspJI family of modification dependent restriction enzymes for genome wide detection of methylated sites. The enzymes create a double strand cleavage of the DNA 16 bases away from the modified cytosine, forming a 32 base fragment with the fully methylated cytosine in the middle, allowing direct sequencing of the excised fully modified site. The main advantage is that since you only need to sequence the methylated sites you reduce the required sequencing power. The method is described in this paper: http://www.pnas.org/content/108/27/11040.long , and if you are interested in using it, I will be happy to provide additional details.

Check out pyrosequencing which is widely used for methylation sequencing of entire genes

Couple more:

http://salsahpc.indiana.edu/tutorial/slides/0729/cancer-epigenomics-kim.pdf

http://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/Sequencing/Epigenetic-Sequencing.html

we use bisulfite sequencing to detect promoter/gene specific DNA methylation.

check bisulfate Method. easy and cheap...

As far as I know the best accurated method is the bisulfite method analysed with a mass spec (Sequenom)

Yes, the method is bisulfite sequencing. We use three sets of primers to cover 19 CpGs.

There are currently 3 main strategies to pre-treat DNA in order to examine DNA methylation:

- Bisulfite conversion

- Methylation Sensitive Restriction Enzymes

- Precipitaion of methylated DNA using antibodies of other high affinity methylation-binding proteins

If you are interested in genome-wide DNA methylation patterns, any of the above pre-treatments can be coupled with next generation sequencing. A review of genomic methods can be found here:

http://www.nature.com/nrg/journal/v11/n3/full/nrg2732.html

or:

http://genome.cshlp.org/content/19/6/959.long

However, the gold standard is bisulfite coupled with next generation sequencing.

A recent review in Nature Methods focuses on this method:

http://www.nature.com/nmeth/journal/v9/n2/full/nmeth.1828.html

For locus-specific DNA methylation examination I strongly recomend pyrosequencing of bisulfite treated DNA. Very quantitative and reliable under our hands, but like any method it also has disadvantages.

amplification of intermethylated sites (AIMS) should be your method of choice. Just google it or use pubmed to find relevant papers.

Be careful with pyrosequencing (or bisulfite sequencing) methylated and non-methylated DNA sequences are amplified differently (after or not bisulfite conversion). This could induces biases during PCR amplifications and the biases are sequences specific. Therefore, controls are necessary to be sure of the levels of methylation that are measured. For instance, you need to generate a mixture of the DNA sequence that you want to analyze with a methylation level of 50% and be sure that you are able to measure a methylation level of 50%... This is not straight forward... Furthermore, pyrosequencing is not really convenient to detect non CG methylation.

This kind of biases are not really known when using next generation sequencing technics...

bisulfite sequencing is ok...to detect promoter and gene specific DNA methylation

Thanks to each and everyone who has replied to my question! I received a lot of useful information and leads from you all here, and will utilize them to look deeper into this subject.

As Gidon has already explained, there are three main approaches for detecting DNA methylation:

- Methylation sensitive andonuclease-based

- Sodium bisulfite treatment

- Biological affinity and precipitation

However, there are several methods that can be used to asses single nucleotide resolution or genome wide DNA methylation for every profiling approach. So I really recomend to take your time to analyse and compare the available methods, so that you can use the best four your needs. I have an excelent document that briefly describes almost all the existing methods (specially for genome wide methods), and I would like to share it with all of you.

Hope this could help everyone.

Sequencing will allow you to only infer epigenetic modificdations in promoter regions but not gene bodies.

Hi, depending on your purpose and resources, I highly recommend using the MspJI family of modification dependent restriction enzymes for genome wide detection of methylated sites. The enzymes create a double strand cleavage of the DNA 16 bases away from the modified cytosine, forming a 32 base fragment with the fully methylated cytosine in the middle, allowing direct sequencing of the excised fully modified site. The main advantage is that since you only need to sequence the methylated sites you reduce the required sequencing power. The method is described in this paper: http://www.pnas.org/content/108/27/11040.long , and if you are interested in using it, I will be happy to provide additional details.