DNA Preparation and Amplification on Plastic Microfluidic Devices for Genetic Analyses

Lounsbury, Jenny Ann, Department of Chemistry, University of Virginia
Landers, James, Department of Chemistry, University of Virginia
Allen, Ralph, Department of Chemistry, University of Virginia
Columbus, Linda, Department of Chemistry, University of Virginia
Cafiso, David, Department of Chemistry, University of Virginia

The utilization of a microfluidics platform for DNA analysis would provide numerous advantages over conventional DNA analysis methods, including a completely closed system and reduction in analytical time. Before DNA can be analyzed, cells must first be eluted from the substrate (e.g., cotton swabs). A novel buffer is described, increasing sperm cell recovery from a cotton swab matrix to 85-90 %, an ~2-fold increase in recovery compared to a traditional differential extraction buffer, while comprehensively lysing epithelial cells, thereby increasing the probability of generating a full male DNA profile. In addition, to eliminate multiple sample handling steps and binding DNA to silica for purification (which leads to irreversible binding, thus, loss of DNA), an enzyme-based DNA preparation method is characterized, optimized and applied to various samples, including buccal swabs and blood stains. This method provides PCR-ready DNA in approximately 20 minutes, an ~4-fold reduction in preparation time compared to conventional DNA extraction. Furthermore, development of a novel PCR method for poly(methyl methacrylate) (PMMA) microdevices using an in-house IR-PCR system, is detailed. The composition of the PCR master mix was optimized, through the use of a two-polymerase mixture. The expansion of bubbles, which form during heating due to the laser ablated PMMA surface, was reduced, using a biocompatible adhesive and manifold. As a result, full short tandem repeat (STR) profiles were generated in under 35 minutes, an ~5-fold reduction in amplification time as compared to a thermocycler. The analysis of profile quality metrics, including peak height ratio and stutter percentages (defined by the governing body for forensic STR analysis), reveals that the quality of the profiles generated using this microchip PCR iii method are comparable to profiles generated using conventional amplification protocols. Finally, the integration of enzyme-based DNA preparation and microchip PCR is demonstrated. Beginning with either dried buccal swabs or whole blood samples, DNA preparation and amplification in this disposable, integrated microdevice yields PCR products in under 45 minutes, an ~6-fold reduction in analysis time compared to conventional methods. This device represents the closed, integrated, microfluidic system that has the potential to begin a shift in forensic and/or clinical sample processing.

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PHD (Doctor of Philosophy)
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