The new ultrasensitive flow cytometer is 100 times faster and 200,000 times more sensitive than conventional gel electrophoresis at analyzing DNA samples. And the process requires only minute quantities of DNA to obtain a reliable result.

The new flow cytometer has applications beyond defense and counter-bioterrorism, according to its developers. In the food industry, for example, it can be used to detect the presence of salmonella and other bacteria. Public health and medical diagnostic workers will be able to use the flow cytometer to analyze outbreaks of E.coli, staph and other infectious diseases. The instrument also will aid in studies of the human genome.

"If there should be an outbreak of a bacterial disease, we need to know immediately what the pathogen is to be able to rapidly initiate treatment," said James Jett of Los Alamos' Bioscience Division. "We wouldn't have days to figure out what we're up against. This flow cytometer may help us to determine the proper treatment much faster than we normally would."

Project leader Jett, Babs Marrone, Dick Keller and Tom Yoshida, all of the Bioscience Division, developed the new cytometer.

First developed at Los Alamos more than 30 years ago, flow cytometers use lasers to analyze, characterize and sort thousands of biological cells, chromosomes or molecules in minutes. Applications include analysis of white blood cells, DNA and RNA as well as biological functions.

Researchers currently use pulsed-field gel electrophoresis to separate large DNA fragments according to their size. Although sizes can be determined with 90 percent accuracy, this method requires relatively large amounts of DNA - roughly one-millionth of a gram - and 14 to 24 hours to obtain a fingerprint from a prepared sample, especially for fragments larger than 10,000 base pairs.

The new Los Alamos-developed flow cytometer determines the fingerprint of DNA fragments with 98 percent or better accuracy in less than seven minutes from a prepared sample, regardless of the length of the fragments. Less than two-trillionths of a gram of DNA is required to perform the analysis.

Sample preparation in both methods takes the same amount of time - several minutes for isolated small fragments or several hours for a whole bacterial genome digest. The big difference is the time it takes to obtain a result. Flow cytometry can deliver results in one clinical shift, which will be especially valuable to hospital workers, compared with 24 hours for gel electrophoresis.

Once the DNA is purified from the bacterial cells, researchers introduce an enzyme into the sample that chops up the pathogen's DNA into a defined set of fragments. The fragments then are stained with a fluorescent dye, the amount of which is directly proportional to the fragments' size. The larger the fragment, the more dye is bound to it.

The stained fragments are passed through the flow cytometer. As the laser strikes the fluorescent dye molecules that are bound to the fragment, a photon "burst" occurs. Because the number of photons in each burst is directly proportional to the fragment's size, the cytometer counts the photons in a burst to obtain an accurate fragment size measurement.

The resulting distribution of fragment sizes in the sample is its DNA fingerprint. The researchers then simply compare the fingerprint to those from a Los Alamos-developed database to identify the pathogen.

"The new flow cytometer also has the potential to identify specific strains of bacterial species," noted Jett. This is crucial in epidemic tracing and for forensics, as well as for responding to a bio-agent attack where being able to identify the specific strain of an organism aids in tracing its origin.

An earlier version of the flow cytometer received a 1997 R&D 100 Award from the Illinois-based R&D Magazine as one of the 100 most significant products, materials or processes with commercial promise for that year.

Researchers are developing a smaller, portable version of the tool and are seeking an industrial partner to manufacture it. A patent has been granted.

Los Alamos has been designated as the National Flow Cytometry Resource by the National Center for Research Resources, a branch of the National Institutes of Health.

Community
Email This Article
Comment On This Article

Related Links
Space

Memory Foam Mattress Review

SPACE MEDIA NETWORK PROMOTIONS Solar Energy Solutions Tempur-Pedic Mattress Comparison ... Newsletters :: SpaceDaily :: SpaceWar :: TerraDaily :: Energy Daily XML Feeds :: Space News :: Earth News :: War News :: Solar Energy News