DNA analysis using an integrated microchip for multiplex PCR amplification and electrophoresis for reference samples.

A system that automatically performs the PCR amplification and microchip electrophoretic (ME) separation for rapid forensic short tandem repeat (STR) forensic profiling in a single disposable plastic chip is demonstrated. The microchip subassays were optimized to deliver results comparable to conventional benchtop methods. The microchip process was accomplished in sub-90 min compared with >2.5 h for the conventional approach. An infrared laser with a noncontact temperature sensing system was optimized for a 45 min PCR compared with the conventional 90 min amplification time. The separation conditions were optimized using LPA-co-dihexylacrylamide block copolymers specifically designed for microchip separations to achieve accurate DNA size calling in an effective length of 7 cm in a plastic microchip. This effective separation length is less than half of other reports for integrated STR analysis and allows a compact, inexpensive microchip design. This separation quality was maintained when integrated with microchip PCR. Thirty samples were analyzed conventionally and then compared with data generated by the microfluidic chip system. The microfluidic system allele calling was 100% concordant with the conventional process. This study also investigated allelic ladder consistency over time. The PCR-ME genetic profiles were analyzed using binning palettes generated from two sets of allelic ladders run three and six months apart. Using these binning palettes, no allele calling errors were detected in the 30 samples demonstrating that a microfluidic platform can be highly consistent over long periods of time.

Solid phase extraction of DNA from biological samples in a post-based, high surface area poly(methyl methacrylate) (PMMA) microdevice.

This work describes the performance of poly(methyl methacrylate) (PMMA) microfluidic DNA purification devices with embedded microfabricated posts, functionalized with chitosan. PMMA is attractive as a substrate for creating high surface area (SA) posts for DNA capture because X-ray lithography can be exploited for extremely reproducible fabrication of high SA structures. However, this advantage is offset by the delicate nature of the posts when attempting bonding to create a closed system, and by the challenge of functionalizing the PMMA surface with a group that invokes DNA binding. Methods are described for covalent functionalization of the post surfaces with chitosan that binds DNA in a pH-dependent manner, as well as for bonding methods that avoid damaging the underlying post structure. A number of geometric posts designs are explored, with the goal of identifying post structures that provide the requisite surface area without a concurrent rise in fluidic resistance that promotes device failure. Initial proof-of-principle is shown by recovery of prepurified human genomic DNA (hgDNA), with real-world utility illustrated by purifying hgDNA from whole blood and demonstrating it to be PCR-amplifiable.

A valveless microfluidic device for integrated solid phase extraction and polymerase chain reaction for short tandem repeat (STR) analysis.

A valveless microdevice has been developed for the integration of solid phase extraction (SPE) and polymerase chain reaction (PCR) on a single chip for the short tandem repeat (STR) analysis of DNA from a biological sample. The device consists of two domains--a SPE domain filled with silica beads as a solid phase and a PCR domain with an ~500 nL reaction chamber. DNA from buccal swabs was purified and amplified using the integrated device and a full STR profile (16 loci) resulted. The 16 loci Identifiler® multiplex amplification was performed using a non-contact infrared (IR)-mediated PCR system built in-house, after syringe-driven SPE, providing an ~80-fold and 2.2-fold reduction in sample and reagent volumes consumed, respectively, as well as an ~5-fold reduction in the overall analysis time in comparison to conventional analysis. Results indicate that the SPE-PCR system can be used for many applications requiring genetic analysis, and the future addition of microchip electrophoresis (ME) to the system would allow for the complete processing of biological samples for forensic STR analysis on a single microdevice.

A modular microfluidic system for deoxyribonucleic acid identification by short tandem repeat analysis.

Microfluidic technology has been utilized in the development of a modular system for DNA identification through STR (short tandem repeat) analysis, reducing the total analysis time from the ∼6 h required with conventional approaches to less than 3h. Results demonstrate the utilization of microfluidic devices for the purification, amplification, separation and detection of 9 loci associated with a commercially-available miniSTR amplification kit commonly used in the forensic community. First, DNA from buccal swabs purified in a microdevice was proven amplifiable for the 9 miniSTR loci via infrared (IR)-mediated PCR (polymerase chain reaction) on a microdevice. Microchip electrophoresis (ME) was then demonstrated as an effective method for the separation and detection of the chip-purified and chip-amplified DNA with results equivalent to those obtained using conventional separation methods on an ABI 310 Genetic Analyzer. The 3-chip system presented here demonstrates development of a modular, microfluidic system for STR analysis, allowing for user-discretion as to how to proceed after each process during the analysis of forensic casework samples.

An integrated, valveless system for microfluidic purification and reverse transcription-PCR amplification of RNA for detection of infectious agents.

We describe the first miniaturized device capable of the front-end sample preparation essential for detecting RNA-based infectious agents. The microfluidic device integrates sample purification and reverse transcription PCR (RT-PCR) amplification for the identification and detection of influenza A. The device incorporates a chitosan-based RNA binding phase for the completely aqueous isolation of nucleic acids, avoiding the PCR inhibitory effects of guanidine and isopropanol used in silica-based extraction methods. The purified nucleic acids and the reagents needed for single-step RT-PCR amplification are fluidically mobilized simultaneously to a PCR chamber. Utilizing infrared (IR)-mediated heating allowed for a > 5-fold decrease in RT-PCR analysis time compared to a standard thermal cycling protocol used in a conventional thermal cycler. Influenza A virus [A/PR/8/34 (H1N1)] was used as a simulant in this study for virus-based infectious and biowarfare agents with RNA genomes, and was successfully detected in a mock nasal swab sample at clinically relevant concentrations. Following on-chip purification, a fragment specific to the influenza A nucleoprotein gene was first amplified via RT-PCR amplification using IR-mediated heating to achieve more rapid heating and cooling rates. This was initially accomplished on a two-chip system to optimize the SPE and RT-PCR, and then translated to an integrated SPE-RT-PCR device.

Dual-domain microchip-based process for volume reduction solid phase extraction of nucleic acids from dilute, large volume biological samples.

A microfluidic device was developed to carry out integrated volume reduction and purification of nucleic acids from dilute, large volume biological samples commonly encountered in forensic genetic analysis. The dual-phase device seamlessly integrates two orthogonal solid-phase extraction (SPE) processes, a silica solid phase using chaotrope-driven binding and an ion exchange phase using totally aqueous chemistry (chitosan phase), providing the unique capability of removing polymerase chain reaction (PCR) inhibitors used in silica-based extractions (guanidine and isopropanol). Nucleic acids from a large volume sample are shown to undergo a substantial volume reduction on the silica phase, followed by a more stringent extraction on the chitosan phase. The key to interfacing the two steps is mixing of the eluted nucleic acids from the first phase with loading buffer which is facilitated by flow-mediated mixing over a herringbone mixing region in the device. The complete aqueous chemistry associated with the second purification step yields a highly concentrated PCR-ready eluate of nucleic acids devoid of PCR inhibitors that are reagent-based (isopropanol) and sample-based (indigo dye), both of which are shown to be successfully removed using the dual-phase device but not by the traditional microfluidic SPE (muSPE). The utility of the device for purifying DNA was demonstrated with dilute whole blood, dilute semen, a semen stain, and a blood sample inhibited with indigo dye, with the resultant DNA from all shown to be PCR amplifiable. The same samples purified using muSPE were not all PCR amplifiable due to a smaller concentration of the DNA and the lack of PCR-compatible aqueous chemistry in the extraction method. The utility of the device for the purification of RNA was also demonstrated, by the extraction of RNA from a dilute semen sample, with the resulting RNA amplified using reverse transcription (RT)-PCR. The vrSPE-SPE device reliably yields a volume reduction for DNA and RNA purification on the order of 50- and 14-fold, respectively, both compatible with downstream PCR analysis. In addition, purification of all samples consumed less reagents (2.6-fold) than traditional purification methods, with the added advantage of being a "closed system" that eliminates sample transfer steps, thereby reducing the possible entrance points for contaminants.

Volume reduction solid phase extraction of DNA from dilute, large-volume biological samples.

Microdevices are often designed to process sample volumes on the order of tens of microliters and cannot typically accommodate larger volume samples without adversely affecting efficiency and greatly increasing analysis time. However, dilute, large-volume biological samples are frequently encountered, especially in forensic or clinical laboratories. A microdevice, capable of efficiently processing 0.5-1 mL samples has been developed for solid phase extraction (SPE) of DNA. SPE was carried out on a microdevice utilizing magnetic silica particles and an optimized volumetric flow rate and elution buffer, resulting in a 50-fold decrease in volume and a 15-fold increase in DNA concentration. Device characterization studies showed DNA extraction efficiencies comparable with previously reported silica-based purification methods, with robust performance demonstrated by the successful amplification of a fragment from the gelsolin gene extracted from dilute whole blood. In addition, the microchip-based method for SPE of large volume, dilute samples was also used to demonstrate the first successful on-chip purification of mitochondrial DNA (mtDNA) from both dilute whole blood and a degraded blood stain.