To say researchers have been pretty amped about 5-Hydroxymethylcytosine (5-hmC) this last year would be an understatement. Is this 6th base for real, or is it some kind of intermediate of it’s more abundant cousin 5-mC? So far its low key presence has been detected in embryonic stem cells, brain, and other organs but there are still far more questions than answers for 5-hmC.
Like most new discoveries, there are going to be a lot of questions and speculation, but 5-hmC has maintained its aura of mystery in part because of the lack of tools available to analyze it. To date, only methods for obtaining a global snapshot or regional glimpse into 5-hmC’s presence have been commercialized.
Bisulfite conversion, the workhorse of most site-specific methylation analyses can’t tell the difference between 5-hmC and 5-mC, so researchers haven’t had the opportunity to effectively look at 5-hmC up close and personal, at the base level.
Until now. (Cue dramatic entry…)
A Much Needed Sugar High
Glucose, the simple sugar that keeps life moving, can be added to 5-hmC using a 5-hmC Glucosyltransferase from uridine diphosphoglucose (UDPG). The reaction takes longer to spell check than run.
Once glucosylated, 5-hmC sticks out of the crowd practically begging to be diced by glucosyl 5-hydroxymethylcytosine sensitive restriction enzymes. These GSREs (not to be confused with the lame grad school tests) slice and dice 5-mC and 5-hmC, but leave glucosylated 5-hmC intact, ultimately enabling site specific 5-hmC profiling.
A Blast from the Past
The whole 5-hmC Glucose, restriction digestion process has been around since Janis Joplin’s prime, but the clever folks at Zymo Research recently optimized the process for 5-hmC analysis. We caught up with James Yen, a Scientist with the company to hear about their approach.
“At the heart of the Quest 5-hmC Detection Kit is a unique 5-hmC Glucosyltransferase that specifically modifies 5-hydroxymethylcytosine residues in DNA with a sugar moiety. The reactions will be very familiar to anyone who sets up in vitro enzyme reactions (e.g. – DNA methylation or DNA endonuclease digestion). All reactions are carried out in an easy one tube format so there is no unnecessary sample processing in between steps. The whole process is done within a few hours and the location of 5-hmC can be analyzed by amplification methods like qPCR or NextGen sequencing methods.”
Next Up, Next Gen
Sequencing is on everybody’s short list today and since 5-hmC profiling is begging for it, researchers will be stoked to know that this approach can head right into their library construction weapon of choice. With this new approach, we’ll be waiting with keyboards primed to pass on the first publications with site-specific 5-hmC profiling data sets.
Major thanks to Dr. James Yen and the crew over at Zymo Research for busting out another great product and keeping us in the loop. Get the full scoop on Zymo’s Quest 5-hmC Detection Kit over at their site.