Advancing Forensic DNA Processing with Optical Detection and Microfluidic Technology 935 views

DNA forensic technology capabilities continue to increase with growing demands for automated, accurate, and rapid processing methods. Unfortunately, automation is commonly associated with complex and expensive instruments, and is not a viable solution for low-resource laboratories. In an effort to keep current DNA processing simple and cost-effective, this work utilizes microfluidic technology and optical detection for rapid screening and purification of forensically-relevant samples. Fast DNA screening methods are necessary to provide contextual clues for criminal cases, and determine which forensic samples are to be further processed. Two inexpensive optical methods are introduced for sequence-specific detection of nucleic acids using a single temperature amplification method known as loop-mediated isothermal amplification (LAMP). The first method exploits the high affinity between biotin-labeled LAMP amplicons and streptavidin beads for nucleic acid detection. When a target sequence is present, the biotin amplicons tether the streptavidin beads together, resulting in a sequence-specific bead aggregation response that is optically detectable down to single copies of DNA or RNA. A second optical detection method utilized LAMP with an embedded metal-indicator dye to colorimetrically detect fluid-specific mRNA markers for a panel of 5 body fluids. An optimized universal sample procedure allowed for the identification of any combination of the targeted body fluids in up to 23 samples simultaneously using a smart phone camera. DNA extraction is a critical step in DNA processing following DNA screening that is largely dominated by expensive biorobotic instruments. An affordable handheld centrifugal system and disposable polyethylene terephthalate (Pe) microdevices were developed for cost-effective sample lysis and DNA purification. Pe is amendable to simple and rapid fabrication of a four-layer extraction device with on-board passive valving capability for increased fluidic control. Using an optimized extraction procedure, DNA was purified from whole blood samples and buccal swab lysates, which yielded strong short tandem repeat (STR) profiles. A lysis domain, specifically for buccal swab cuttings, was integrated with the extraction process to provide sample-to-PCR ready DNA within 30 minutes. Overall this work provides development towards cost-effective and rapid DNA processing methods that are amendable to automation and beneficial to all forensic laboratories.

PHD (Doctor of Philosophy)

Microfluidics; Forensics; DNA

English

2016-07-30