Advancing forensic DNA profiling through microchip technology

The virtues of microfabrication, chemistry and biology are exploited to develop an integrated microchip DNA typing system for fast, high-performance and automated forensic human identification. My early focus was on the development of a 96-lane microfabricated capillary array electrophoresis (muCAE) system for forensic short tandem repeat (STR) analysis, and the demonstration that it has twice the speed and six times the throughput of conventional CE. I validated this instrument and process for forensic laboratory use, leading to its adoption by the Virginia Department of Forensic Science.Next, I exploited fluorescent energy-transfer (ET)-dye cassette labeling technology to develop an ET version of the PowerPlex 16 STR typing kit with superior spectral properties. The ET 16-plex that I developed exhibited 2--9 fold higher allele fluorescence intensities compared to single-dye labeled primers, allowing me to reduce the input DNA and PCR cycles while producing complete STR profiles. Furthermore, this approach produces additional STR allele calls from problematic casework samples previously analyzed by the Palm Beach County Sheriff's Office. This demonstration facilitated the adoption of the ET-cassette technology by Promega Corp. for future high-sensitivity multiplex kit development.Finally, I developed an universal sample cleanup and concentration method and integrated it inline with a micro-CE (muCE) separation channel for STR analysis. A small streptavidin-acrylamide gel plug was precisely defined in a microchannel preceding the separation region by photopolymerization and then used to capture biotinylated STR products. This inline cleanup, concentration and injection method eliminates the electrokinetic injection bias in high-salt PCR samples and overcomes the inefficient sample use that plagues traditional cross injection. The capture-muCE process requires only 40 minutes and produced 10--14 fold higher allele fluorescent signals than cross injection. This enhanced sensitivity enabled the complete typing of STR samples at half the current profiling limit and the recovery of additional alleles from severely degraded DNA samples. This research has built the technical foundation for a fully integrated DNA profiling microdevice that can perform fast, highly sensitive, low-volume forensic STR analyses.