Advancing Forensic DNA Profiling Through Microchip Technology

• NCJ Number: 309939
• Author(s): Hang Ieng Yeung
• Date Published: 2008
• Length: 143 pages
• Grant Number(s): 2007-DN-BX-0003
• Agencies: NIJ-Sponsored
• Publication Type: Thesis/Dissertation; Research (Applied/Empirical)
• Award: An Integrated Microfabricated Sample Cleanup and Electrophoresis Analyzer for High-Performance STR Typing

Annotation

This dissertation investigates advances in forensic DNA profiling through microchip technology.

Abstract

This research builds the technical foundation for a fully integrated DNA profiling microdevice that can perform fast, highly sensitive, low-volume forensic STR analyses. 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. Initial focus was on the development of a 96-lane microfabricated capillary array electrophoresis (μCAE) system for forensic short tandem repeat (STR) analysis, with twice the speed and six times the throughput of conventional CE. The researcher validated this instrument and process for forensic laboratory use. The researcher 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 that exhibited 2-9 fold higher allele fluorescence intensities compared to single-dye labeled primers, allowing reduction of the input DNA and PCR cycles while producing complete STR profiles. This approach produced additional STR allele calls from problematic casework samples. This demonstration facilitated the adoption of the ET-cassette technology by Promega Corp. for future high-sensitivity multiplex kit development. Finally, the researcher developed an universal sample cleanup and concentration method and integrated it inline with a micro-CE (μCE) 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-μCE 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.