Differentiating between DNA’s celebrity cytosine bases can be harder than remembering all the ‘celebrities’ caught in hollywood’s latest photo leak. But have no fear, because oxidative bisulfite conversion (ox-BS) is here to save the day in a reduced representation way.
Normally, bisulfite conversion doesn’t produce a difference in readout between 5mC and 5hmC, but ox-BS overcomes this by treating the DNA with an agent that selectively oxidizes 5hmC into 5-formylcytosine (5fC), which after bisulfite conversion is read as uracil (just like unmethylated cytosine). The next step involves a normal bisulfite conversion that is then subtracted from the true 5mC signal from the oxidative run and is used to tune into the remaining signal that represents 5hmC.
By utilizing a clever ‘hacking’ of ox-BS and the 450k array, a clever team from University College London (UK) led by Stephan Beck shows that it’s now possible to use the ever so popular technological workhorse to discriminate between 5mC and 5hmC in your precious human samples. Here’s the specs:
- It works on samples with global 5hmC content ranging from high (brain) to low (blood).
- 5hmC makes an appearance at more than 30% of the CpGs on the array.
- The technical replicates reproduced quite nicely.
When it comes down to it, this new killer combo allows for a lower cost and higher throughput than NGS for interrogating the dynamic methylome. And with the results standing up to the test of validation via mass spec and pyrosequencing, why wouldn’t you give it a go?
Go check out the optimized protocol in Methods, September 2014