[Method for automated extraction and purification of nucleic acids and its implementation in microfluidic system].

A method and a microfluidic device for automated extraction and purification of nucleic acids from biological samples have been developed. The method involves disruption of bacterial cells and/or viral particles by combining enzymatic and chemical lysis procedures followed by solid-phase sorbent extraction and purification of nucleic acids. The procedure is carried out in an automated mode in a microfluidic module isolated from the outside environment, which minimizes contact of the researcher with potentially infectious samples and, consequently, decreases the risk of laboratory-acquired infections. The module includes reservoirs with lyophilized components for lysis and washing buffers; a microcolumn with a solid-phase sorbent; reservoirs containing water, ethanol, and water-ethanol buffer solutions for dissolving freeze-dried buffer components, rinsing the microcolumn, and eluting of nucleic acids; and microchannels and valves needed for directing fluids inside the module. The microfluidic module is placed into the control unit that delivers pressure, heats, mixes reagents, and flows solutions within the microfluidic module. The microfluidic system performs extraction and purification of nucleic acids with high efficiency in 40 min, and nucleic acids extracted can be directly used in PCR reaction and microarray assays.

Effects of external transport on discrimination between perfect and mismatch duplexes on gel-based oligonucleotide microchips.

Using hydrogel-based oligonucleotide microchips developed previously for the choice of drugs during leukemia treatment and the other diseases, it is shown that the acceleration of external transport by mixing buffer solution with peristaltic pump not only enhances the observable fluorescence signals, but also improves significantly the discrimination between perfect and mismatch duplexes at the intermediate stage of hybridization on the oligonucleotide microchips. The discrimination efficiency for a given hybridization time grows monotonously with the frequency of flow pulsations. The mixing with frequency 10 Hz accelerates the hybridization rate approximately thrice and improves the discrimination efficiency 1.5-2.5 times higher for overnight hybridization. To study these effects, we have developed the special peristaltic pump mixing solution in a hybridization chamber of 35 mul in volume (area approximately 1 x 1 cm(2) and height 0.3 mm). We present also the brief theoretical summary for the interpretation and assessment of the observed experimental features.