If you have a high fever, runny nose, and body aches, you might think you have the flu or a cold. But if you're a soldier out in the field in today's meaner world, you could be infected by a biological or chemical warfare agent—but not know for sure. By the time you find out that you've been hit by the real thing, it may be too late to save you and other exposed people.

To shorten the time in diagnosing whether a whole battalion has a stomach ache from food poisoning or has been exposed to an artificially induced toxin, MITRE is working with DNA microarrays to develop a rapid diagnosis system. Microarrays are hundreds or thousands of microscopic dots of gene samples on a single microscope slide. The gene samples, or sequences, are so small that you can't see them with the naked eye.

"This technology lets you take a snapshot of all the genes in your body at any given time," says Olivia Peters, a group leader at MITRE. "The test allows us to take a blood sample and tells us how every gene in your body is responding to a specific stimulus." (See box at end of story.)

For instance, the genes in your body change when you're tired, explains Peters. "Certain genes increase. If you're tired and you're trying to stay awake while driving, your body will give off signs that you're tired. Your heart beat will slow down, and genes will tell your heart to do that. But since you're driving, and forcing yourself to stay awake, the genes responsible for making your adrenaline will increase."

A Signature Narrows the Field

For the military, the idea is to use microarrays to find a signature for the body's response to biological warfare agents such as cyclosarin, an extremely toxic, colorless liquid. Low-level exposures to cyclosarin give symptoms of teary eyes, dimmed vision, runny nose, and tightness in the chest. The signature will help the military determine when soldiers have been exposed to cyclosarin and by how much.

To get a signature, a microarray of 40,000 standard genes is tested to see which genes are "informative" and express a negative or positive value. "For cyclosarin, we'll get expression levels on 8,800 genes," says Andrea Jensenius, a senior artificial intelligence engineer. "Not all of the genes will be affected by an agent or they'll look the same whether or not they've been exposed, so we need to find a smaller group of genes."

For cyclosarin, a software program called a classifier uses a special algorithm to narrow the number of genes down to less than 30. The purpose of reducing the number of features is both to improve classifier accuracy by removing redundant or noisy features, and to provide input for a quicker experimental protocol. Microarray tests in the laboratory can take 24 to 48 hours. Once the number of genes has been narrowed down to a smaller number that are informative, another test called polymerase chain reaction (PCR) can tell in five minutes whether or not you've been exposed to cyclosarin.

The classifier starts with baselines of healthy activity for the genes and then compares them to the same cells exposed to different agents, such as anthrax. "The classifiers are trained to differentiate between cells under normal and pathogenic conditions," Peters says. "We received much of our data from several collaborators, including Walter Reed Army Institute of Research Department of Pathology and the Edgewood Biological and Chemical Command, which provided experimental data for multiple chemical and biological warfare agents."

The gene analysis approaches include several popular statistical and machine-learning methods. "We have been successful in building classifiers for cyclosarin that show with 80 to 99 percent accuracy that a person has been exposed and at what level," she adds.

Now that the MITRE-sponsored research project has come to a close, Peters' group is transferring the research to the Air Force and Army. The Air Force has shown interest in exposure to environmental toxic substances such as jet fuels and heavy metals and the Army is interested in detection of exposure to biological and chemical agents.

How Does DNA Microarray Technology Work? Courtesy: National Human Genome Research Institute DNA microarrays are created by robotic machines that arrange minuscule amounts of hundreds or thousands of gene sequences on a single microscope slide. Researchers have an unlimited number of sequences that they can use for this purpose. When a gene is activated, cellular machinery begins to copy certain segments of that gene. The resulting product is known as messenger RNA (mRNA). The mRNA produced by the cell is complementary and will bind to the original portion of the DNA strand from which it was copied. To determine which genes are turned on and which are turned off in a given cell, messenger RNA molecules present in that cell are collected. Each mRNA molecule is labeled by attaching a fluorescent dye. Next, the researcher places the labeled mRNA onto a DNA microarray slide. The messenger RNA and its fluorescent tag that was present in the cell then bind to its complementary DNA on the microarray. A researcher must then use a special scanner to measure the fluorescent areas on the microarray. If a particular gene is very active, it produces many molecules of messenger RNA, which bind to the DNA on the microarray and generate a very bright fluorescent area. Genes that are somewhat active produce fewer mRNAs, which result in dimmer fluorescent spots. If there is no fluorescence, none of the messenger molecules are binding to the DNA, indicating that the gene is inactive. Researchers frequently use this technique to examine the activity of various genes at different times.

—by David A. Van Cleave

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