Sequence of pillars: Produces fastest ever separation of DNA and RNA
- Published: Apr 17, 2017
- Author: Jon Evans
- Channels: Electrophoresis
Across the channels
Using nanopillars in a nanoslit, a team of Japanese scientists has achieved the fastest ever separation of nucleic acids by microchip electrophoresis, separating micro-RNA from DNA in just 20 microseconds.
The team, led by Noritada Kaji at Nagoya University, employed photolithography to etch a cross-shaped microchannel system into a quartz wafer, and then fabricated the nanoslit and nanopillars in the right-hand microchannel. The 100μm-long nanoslit stretched across the 25μm-wide microchannel and sat 2μm above the channel floor, such that the nanoslit was only 100nm high. It was filled with a square array of nanopillars, each 100nm tall, 250nm wide and separated from each other by 750nm.
The idea is to introduce a mixed sample of nucleic acids into the top of the cross-shaped channel system, and drive them down the channel by electrophoresis. Once the nucleic acids reach the cross section, an electric current is applied across the channel system to drive the nucleic acids into the right-hand microchannel containing the nanoslit and array of nanopillars. This first requires the nucleic acids to find their way into the narrow opening of the nanoslit located 2μm above the channel floor and then negotiate their way through the array of nanopillars, both of which act to separate the nucleic acids based on length.
Skirting around nanopillars
The longer strands of nucleic acids have more chance of finding their way into the nanoslit than shorter strands, as some section of them will generally be near the opening. Once in the nanoslit, the close confines and electric current force the longer strands to adopt a long, straight configuration that isn’t hindered too much by the nanopillars. Whereas the smaller strands adopt a curled up configuration that results in them repeatedly wrapping themselves around the nanopillars. As such, driven by the electric current, longer strands enter and then travel through the right-hand microchannel faster than shorter strands.
In designing this novel microchip electrophoresis device, the main aim of Kaji and his colleagues was to separate nucleic acids as quickly as possible. The obvious way to do this is by applying as strong an electric current as possible, which meant making the device as robust as possible. Thus, they replaced the gel usually used in microchip electrophoresis, which can be degraded by strong electric currents, and fabricated the device in quartz, which is able to withstand strong currents. They also employed both a nanoslit and nanopillars to double the separation mechanism.
They were able to achieve a kind of virtuous circle with this approach. As the speed of the separation increased, they were able to apply even higher currents to increase the speed further, because they were applying the high currents for ever shorter periods of time and thus limiting any damaging effects. In this way, they found they could separate a mixture of micro-RNA, which is a short form of RNA involved in regulating gene expression, and DNA in just 20 microseconds. They were also able to separate a more complex mixture of micro-RNA, normal RNA and DNA in 100 microseconds.
Kaji and his colleagues think that such super-speedy separation should help with next-generation DNA and RNA sequencing. This is especially the case for nanopore sequencing, in which the sequence of bases is determined by alterations in the current as a strand of RNA or DNA passes through a tiny nanopore.
Unlike existing sequencing techniques, nanopore sequencing can work with individual strands, and thus requires mixtures of strands to be separated first. And because it can sequence strands at a rate of one base per millisecond, nanopore sequencing requires an equally first separation method, which Kaji and his colleagues have now delivered. They think their novel device could be particularly appropriate for sequencing micro-RNA, which previous research has indicated could act as effective cancer biomarkers.
Scientific Reports, 2017, 7, 43877: "A millisecond micro-RNA separation technique by a hybrid structure of nanopillars and nanoslits"
Article by Jon Evans
The views represented in this article are solely those of the author and do not necessarily represent those of John Wiley and Sons, Ltd.
Follow us on Twitter!