High-throughput human DNA purification on a centrifugal microfluidic device for rapid forensic sex-typing.

We introduce a novel centrifugal disc for purifying nucleic acid (NA) in a high-throughput manner to perform the human sex-typing of forensic samples. The centrifugal disc was designed with double-sided etched channels to fabricate 30 extraction units, which was capable of purifying 30 forensic samples in a single run. In order to introduce the washing solution (70% ethanol) and the elution buffer for the 30 extraction units in an automatic manner, we designed the aliquoting chambers that were connected with a zigzag delivery channel. The super-hydrophobic zigzag-shaped aliquot structure plays a crucial role in automatically dividing the washing solution and the elution buffer into 30 aliquots with one injection shot. The Whatman glass filter paper was used as an NA extraction matrix and sophisticated passive valves were equipped to avoid the overflowing of these buffers to the neighboring chamber during the injection. To operate the disc, we developed a portable workstation that consists of a buffer storage system, a buffer injection system, and a spinning unit. The entire process was automatically operated by the in-house portable workstation. Genomic DNA extraction using thirty forensic samples was completed in 10 min. Using the purified genomic DNA, we performed a loop-mediated isothermal amplification (LAMP) reaction for sex-typing by targeting the human alphoid repeat sequence of the Y-chromosome and the human 18S rRNA. The combination of the high-throughput centrifugal disc for NA extraction and the LAMP reaction enables us to complete the genetic sex-typing in 30 min.