| This Ph.D. dissertation reports the study on rapid and automated extraction of PCR (polymerase chain reaction)-ready genomic DNA on a centrifugal fluidic platform. The emphasis is on investigating novel microfluidic functions that provide crucial means for enabling required tasks for nucleic acid extraction. This work also evaluates the validity of a selected protocol that features mechanical cell disruption followed by silica-based DNA purification.; Reagentless mechanical cell lysis was demonstrated by periodically shaking a CD (compact disk) incorporating beads. Study on the interactions between beads and cells identified the following parameters; bead density, angular velocity, acceleration rate, and solid volume fraction, as having significant contribution to cell disruption. Lysis efficiency relative to a conventional lysis protocol was about 65%.; Secondly, rapid and automated genomic DNA preparation was presented on a multiplexed CD platform using hard-to-lyse bacterial spores. Three fluidic functions including keystone effect, wax valve and siphon were in play to perform cell lysis and debris clarification. Performance of the CD was evaluated using a real-time PCR assay to monitor efficiency of Bacillus globigii spore lysis. PCR analysis showed that 5 minute long CD run gave spore lysis efficiency similar to that obtained with a commercial DNA extraction kit.; Silica-based DNA purification was explored in a flow-through format. A passive flow switch, which relies on Coriolis force, was exploited to direct flow-through fractions to either one of two feeding reservoirs. Mechanical lysis of E. coli cells followed by DNA extraction was demonstrated. The extracted genomic and plasmid DNA was verified in agarose gel analysis.; This work will contribute towards CD based sample-to-answer nucleic acid analysis which will include cell lysis, DNA purification, DNA amplification, and DNA hybridization detection. |
