A wide variety of technical needs exist for surveillance, monitoring, identifying, or detecting pathogens with potential
use as biological terrorism or warfare agents. Because the needs vary greatly among diverse applications, tailored
systems are needed that meet performance, information, and cost requirements. A systems perspective allows developers
to identify chokepoints for each application, and focus R&D investments on the limiting factors. Surveillance and
detection systems are comprised of three primary components: information (markers), chemistries (assays), and
instrumentation for "readout". Careful consideration of these components within the context of each application will
allow for increases in efficiency and performance not generally realized when researchers focus on a single component
in isolation. In fact, many application requirements can be met with simple novel combinations of existing technologies,
without the need for huge investments in basic research. Here we discuss some of the key parameters for surveillance,
detection, and identification of biothreat agents, and provide examples of focused development that addresses key
bottlenecks, and greatly improve system performance.
We have developed a SNP scoring platform, yielding high throughput, inexpensive assays. The basic platform uses fluorescently labeled DNA fragments bound to microspheres, which are analyzed using flow cytometry. SNP scoring is performed using minisequencing primers and fluorescently labeled dideoxynucleotides. Furthermore, multiplexed microspheres make it possible to score hundreds of SNPs simultaneously. Multiplexing, coupled with high throughput rates allow inexpensive scoring of several million SNPs/day. GAMMArrays use universal tags that consist of computer designed, unique DNA tails. These are incorporated into each primer, and the reverse-component is attached to a discrete population of microspheres in a multiplexed set. This enables simultaneous minisequencing of many SNPs in solution, followed by capture onto the appropriate microsphere for multiplexed analysis by flow cytometry. We present results from multiplexed SNP analyses of bacterial pathogens, and human mtDNA variation. Analytes are performed on PCR amplicons, each containing numerous SNPs scored simultaneously. In addition, these assays easily integrate into conventional liquid handling automation, and require no unique instrumentation for setup and analysis. Very high signal-to-noise ratios, ease of setup, flexibility in format and scale, and low cost make these assays extremely versatile and valuable tools for a wide variety of SNP scoring applications.
Functional analysis of the human genome, including the quantification of differential gene expression and the identification of polymorphic sites and disease genes, is an important element of the Human Genome Project. Current methods of analysis are mainly gel-based assays that are not well- suited to rapid genome-scale analyses. To analyze DNA sequence on a large scale, robust and high throughput assays are needed. We are developing a suite of microsphere-based approaches employing fluorescence detection to screen and analyze genomic sequence. Our approaches include competitive DNA hybridization to measure DNA or RNA targets in unknown samples, and oligo ligation or extension assays to analyze single-nucleotide polymorphisms. Apart from the advantages of sensitivity, simplicity, and low sample consumption, these flow cytometric approaches have the potential for high throughput multiplexed analysis using multicolored microspheres and automated sample handling.
In June 1994, the U.S. Navy and U.S. Marine Corps restructured the F/A-18D (RC) ATARS program. McDonnell Douglas Aerospace was selected as the prime contractor responsible for overall program management and integration of the ATARS with the F/A-18D (RC) weapon system. Loral Fairchild Systems (LFS) was selected as the subcontractor responsible for ATARS subsystem development and integration. The first part of the restructured program was called Element One and was a ten month risk reduction program ending in April 1995. The primary objective of Element One was to resolve the critical ATARS anomalies found during previous flight testing. In order to resolve these anomalies, MDA and LFS concurred that additional flight test data was required. In November/December 1994, eight flights were flown with a high fidelity instrumentation system to gather the additional data required for evaluation of the anomalies. Using this data, along with previously collected data, design 'fixes' were implemented to correct several of the system anomalies. In April 1995 and May 1995, approximately four flight tests were conducted to evaluate the performance of these design 'fixes.' A period of two months for flight tests was planned for July-August 1995 to complete this evaluation. This presentation describes the testing and results through May 1995 of the Element One flight test program.