Hi All,
I am doing drug discovery work related to DNA methylation level in cancer cells. I wonder if there are new & good assay that allow me to efficiently track DNA methylation status on a specific promoter?
I know that I can do bisulfite sequencing, but its not fast and I cannot track many compounds. I wonder if there are newer assays out there that provide high throughput capacity?
Thanks,
Lyra
Hi Lyra,
You could use methylation specific PCR (e.g MethylLight protocol), which would still require you to bisulfite convert your samples but would avoid the need for sequencing. In my experience bisulfite sequencing is worth the time it takes as it is very easily analysed and aligned between samples, however the PCR approach may suit your needs.
http://www.nature.com/nbt/journal/v34/n7/full/nbt.3605.html
1. bs sequencing
2. Sequenome mass spectrometry assays of bs DNA - very good and suited to medium through put.
3. methylation specific PCR
4. pyrosequencing
Hi All,
Thank you all for your suggestions!
Looking at just gene expression alone probably won't be specific enough for us because lower RNA/protein level can mean lots of stuff other than DNA methylation (protein degradation, simple transcription factor repression, histone modification....etc).
Methylation specific PCR, pyrosequencing and sequenome mass spectrometry assay sounds interesting. I will look into that.
I would imagine for pyrosequencing I need special machine, right? Anyone know how much the machine or service cost? Is there any company out there provide reasonable price for pyrosequencing?
Thank you again and wish you all have a great day!
Lyra
Hi Lyra,
It also depends on the medicine you want to screen and what the impact on DNA methylation is at the specfic promoter. Large differences could need less accurate methods like MS-PCR.
I found that methylation specific high resolution melting worked quite OK on a specific promoter. With this method you also need to bisulfite convert your DNA, this is followed by QPCR and metlingcurve analysis. Of course, given that you have a QPCR machine capable of doing high res melting. But this method is quantitative and can be used for many samples at the same time. original paper doi:10.4161/epi.7.1.18815 and where I tried it on a specific promoter: dx.doi.org/10.1021/es405524b
Another method is BisPCR2, but does require an expensive illumina kit and a miSeq sequencer. It is a bisulfite sequencing method capable of measuring up to 48 samples at the same time, measuring multiple loci. 10.1186/s13072-015-0020-x
Good luck!
Jorke
Hi Jorke,
Thank you so much! I like your suggestion of methylation specific high resolution melting. I will definitely try that out! We do have a qPCR machine that can do melting. I don't know if the resolution will be good enough but for sure worth a try.
Thank you & wish you have a great day :-)
Lyra
Have anyone heard of PacBio sequencing. Somebody suggest it for fast DNA methylation tracking. Can someone share your experience with this technique?
Hi Lyra,
As far as I have heard, it is possible, but the technology is not accurate enough yet. But time is move forward and am really curious about the progression regarding pacbio and iontorrent
1. https://dx.doi.org/10.1101%2Fgr.6417007
High-throughput methylation profiling by MCA coupled to CpG island microarray
2.Cancer Res. 1999 May 15;59(10):2307-12.
Identification of differentially methylated sequences in colorectal cancer by methylated CpG island amplification.
Hi Lyra,
I study DNA methylation in a filamentous fungus. We routinely use methylation-specific restriction digest to asses DNA methylation levels at a specific loci. First, we isolate genomic DNA. Then we set up two separate restriction digests: 1.with a methylation-insensitive enzyme (DpnII); 2. with a methylation-sensitive enzyme such as BfuCI or Sau3A1 (you can also find other methylation-sensitive enzymes but in this case DpnII and BfuCI/Sau3A1 cut the same sequence). Then we run the digested genomic DNA on an agarose gel, perform Southern blotting and probe for a region of interest with a radioactively-labeled DNA probe. DpnII is going to cut every site regardless of its methylation status, but BfuCI is only going to cut when a site is not methylated, resulting in products of higher molecular weight (see attached example).
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