Getting kids to line up single-file can be a challenge, but lining up single molecules for analyses can be just as frustrating. Now, researchers at Cornell report that they’ve done just that—they can line up single DNA molecules in a tiny channel in a nanofluidic device, sort them according to methylation state, then recover them for later experiments.
In a proof-of-principle study, the researchers combined nanofluidics and sorting technology into one device that analyzes single methylated DNA molecules in real time. Basically, fluorescently labeled methylated and unmethylated DNAs are added in the input section, and the molecules line up in single-file in a nanofluidic channel. They get analyzed, and then methylated molecules are automatically sorted into one channel, while the unmethylated molecules go off into a different channel. And there’s enough sample left over to do other analysis with it (unlike what often happens with bisulfite conversion).
They demonstrated the device by analyzing a mixture of methylated and unmethylated linearized plasmids (just 11 femtograms of DNA). The DNAs were labeled red, while MBD1 (which binds to methylcytosines) was labeled with a green fluorescent marker. The device sorted methylated DNAs bound to MBD1 into one channel and unmethylated DNAs that didn’t bind to MBD1 into another channel.
The scientists report up to 98% sorting accuracy, and they could do qPCR on the recovered material. The enrichment was similar to that reported for other fluorescence-activated sorting methods that only sort much larger particles, such as cells. These results suggest that the new device could be the first part of a multi-step analysis of epigenetic modifications on DNAs.
Line up now for your glimpse of the details at PNAS, May 2012.