An evaluation of DNA extraction and amplification performed in microfluidic systems was carried out, with the aim of integrating the two processes in a single microfluidic device. This integrated device will then be incorporated upstream of capillary gel electrophoresis and fluorescence-based detection for development of a completely integrated genetic analysis system.DNA extraction was performed using a silica substrate with both hydrodynamic and electro-osmotic pumping (EOP), resulting in maximum DNA extraction efficiencies of 82% and 52% respectively under optimised conditions. While the DNA extraction efficiency was lower using EOP, this method eliminates the need for external pumps and ensures easier mechanical connection to the microfluidic device. The use of thermally activated silica monoliths as the solid-phase resulted in superior DNA extraction efficiencies compared to when photo-initiated monoliths and silica beads were used.DNA amplification of up to nine forensically relevant loci was successfully achieved on the microfluidic device in volumes as low as 1.1 microlitres using Peltier heating. A combination of silanisation and dynamic passivation was required to prevent PCR inhibition resulting from DNA polymerase adsorption. A custom-built microwave heating system was also evaluated, which was capable of heating and cooling rates of 65degC/second and 58degC/second, respectively.EOP was used in the generation of an integrated microfluidic device, for DNA extraction and amplification. The silica monolith used as the solid-phase for DNA extraction also acted as a pump for electrokinetic movement. All necessary reagents for carrying out both DNA extraction and amplification were encapsulated in agarose gel and pre-loaded onto the microfluidic device creating a self-contained, ready-to-use system. Following addition of the biological sample to the microfluidic device, all electrokinetic movement and thermal cycling was controlled using a custom-built operating system.
展开▼
