N. Reginald Beer

On-chip single-copy real-time reverse-transcription PCR in isolated picoliter droplets.

The first lab-on-chip system for picoliter droplet generation and RNA isolation, followed by reverse transcription, and PCR amplification with real-time fluorescence detection in the trapped droplets has been developed. The system utilized a shearing T-junction in a fused-silica device to generate a stream of monodisperse picoliter-scale droplets that were isolated from the microfluidic channel walls and each other by the oil-phase carrier. An off-chip valving system stopped the droplets on-chip, allowing thermal cycling for reverse transcription and subsequent PCR amplification without droplet motion. This combination of the established real-time reverse transcription-PCR assay with digital microfluidics is ideal for isolating single-copy RNA and virions from a complex environment and will be useful in viral discovery and gene-profiling applications.

Imaging Modes for Ground Penetrating Radar and Their Relation to Detection Performance

The focus of this paper is an empirical study conducted to determine how imaging modes for ground penetrating radar (GPR) affect buried object detection performance. GPR data were collected repeatedly over lanes whose buried objects were mostly nonmetallic. This data were collected and processed with a GPR antenna array, system hardware, and processing software developed by the authors and their colleagues. The system enables GPR data to be collected, imaged, and processed in real-time on a moving vehicle. The images are focused by applying multistatic and synthetic aperture imaging techniques either separately or jointly to signal scans acquired by the GPR antenna array. An image-based detection statistic derived from the ratio of buried object energy in the foreground to energy of soil in the background is proposed. Detection-false alarm performance improved significantly when the detection algorithm was applied to focused multistatic synthetic aperture radar (SAR) images rather than to unfocused GPR signal scans.

Change Detection in Constellations of Buried Objects Extracted From Ground-Penetrating Radar Data

Detection of deliberately buried objects in ground-penetrating radar (GPR) data acquired along a path is a clutter-limited problem. Detection-false alarm rate performance can be improved by replacing the detection statistic with a change statistic that incorporates information from previous path traversals. A constellation matching approach is developed for buried-object change detection in GPR data. Network topologies of buried objects detected in GPR data from previous path traversals are maintained in a constellation database. Localized groups of buried objects newly detected on the latest path traversal are matched to the constellation. Buried objects from the latest path traversal whose locations or strengths cannot be reconciled with the constellation are identified as changes. The system has one component that generates constellation databases offline and another component suitable for change detection in real time. It can tolerate paths with significant translational misalignments. The system uses the following: 1) a customized translational relaxation algorithm for point pattern matching that incorporates detection strength and a probabilistic uncertainty model for buried-object location into the objective function and 2) a change statistic that accounts for the magnitude of change relative to predicted detection strength. A constellation database can typically be generated offline from a single path traversal roughly two orders of magnitude faster than the time typically required for a vehicle to travel the extent of the path. Database sizes are typically four to five orders of magnitude smaller than the data sets of GPR signal scans or focused 3-D GPR images that they were generated from. On bumpy dirt roads buried exclusively with nonmetallic objects at various depths, detection-false alarm rate performance is shown to be significantly better for our change statistics than for our detection statistics.

In vitro double transposition for DNA identification

We present a double transposition technique that inserts two different transposons into target DNA to act as priming sites for amplifying the region between the two transposons for sequencing applications. Unlike some current sequencing approaches, the genome of the unknown target remains intact in this method. The transposition reaction, DNA repair, and subsequent sequencing were performed entirely in vitro, without the need for transformation into bacteria, and resulted in sequence homology with the plasmid DNA target. This approach can reduce the time required for the assay by more than a day compared with standard techniques and reduces the number of required enzymatic steps. In addition, the in vitro method enables transposition to be carried out in automated microfluidic platforms without the need for significant sample manipulation. As a demonstration of incorporating transposition techniques into high-throughput technologies, single transposition reactions were carried out in picoliter-sized droplets generated on a microfluidic platform.