Analyzing DNA

Advances in Miniaturizing the Chemical Steps May Lead to a Small, Cheap, Fast ID Device

by Robert Sanders

Berkeley scientists have succeeded in miniaturizing one of the crucial steps in the DNA analysis or "fingerprinting" process to the size of a credit card.

Their success brings closer the time when DNA analysis, like that being used to identify blood stains in the murder trial of O.J. Simpson, can be performed cheaply, quickly, and reproducibly using portable hand-held devices.

Chemist Richard A. Mathies and graduate student Adam T. Woolley have constructed a miniaturized electrophoresis gel about an inch long and only two thousands of an inch (50 microns) wide--50 times smaller than so-called "slab" gels used today. Gels are used to separate fragments of DNA into a unique genetic pattern that can be used for identification.

Electrophoresis is one of the major bottlenecks in DNA analysis because, though it may take only a day for fragments to spread out on a typical "slab" gel and form separate bands, it takes weeks to detect the fragment pattern by radioactive methods.

The gel-on-a-chip can do the same analysis in two minutes, Mathies says.

"Ultimately, perhaps five years down the road, we hope to miniaturize all DNA analysis to the size of a chip," says Mathies, professor of chemistry and a member of the structural biology division at Lawrence Berkeley Laboratory.

"With miniaturization you make the analysis faster and cheaper, you can use smaller samples, and you reduce the amount of chemicals needed for the test."

The scientists reported their success in the Nov. 22 issue of the Proceedings of the National Academy of Sciences.

Several other groups have built miniature electrophoresis gels the size of an integrated circuit chip, Mathies says, but they have not yet shown they can separate DNA.

A separate group at the Lawrence Livermore National Laboratory reported last year miniaturizing another step in the DNA analysis process, the polymerase chain reaction. The integration of such PCR amplifiers with electrophoresis chips will be an important step forward, Mathies says.

DNA profiling involves extracting DNA from blood, semen or tissue, amplifying the DNA perhaps a million fold, cutting it into many pieces, tagging those pieces that contain a specific marker, then separating the pieces along the length of an electrophoresis gel. The number and type of markers present are characteristic of an individual and can be used in court for identification, for example in murder or rape trials.

Miniaturizing the entire process would make blood and semen analysis in criminal cases faster and cheaper.

"This gel-on-a-chip is the first step in building a microchemical analysis system, where you put the DNA on the chip, it's amplified, loaded into a capillary array, analyzed and detected," Mathies says. "This is chemistry on a micron scale."

Mathies and his colleagues have been trying to miniaturize many of the steps in DNA analysis, and several years ago developed a compact alternative to the slab gel. It is called capillary array electrophoresis, because electrophoresis takes place in an array of up to one hundred hollow capillaries, at a rate 10-50 times faster than electrophoresis along a slab gel.

This method is at the core of a fast DNA analysis machine to be marketed early next year by Molecular Dynamics, Inc., of Sunnyvale, Calif.

Last month Molecular Dynamics and its partner Affymetrix, Inc. of Santa Clara, Calif., received one of the largest technology grants ever by the US Department of Commerce, a five-year, $31.5 million grant to develop miniaturized DNA diagnostic systems. The grant is from the Advanced Technology Program of the National Institute of Standards and Technology.

Mathies will collaborate with this group as they attempt to bring miniaturized chemical labs to the market for applications in forensic science, environmental testing, agriculture, and biomedical research, as well as most clinical diagnostic fields.

The gel-on-a-chip is created by chemically etching microscopic channels into a glass plate and covering them with a second glass plate. When filled with a liquid similar to that in a slab gel, DNA fragments migrate down the channel at a rate determined by their size.

George Sensabaugh, an expert on forensic DNA analysis and a professor in the School of Public Health, has seen the gel-on-a-chip and is enthusiastic about its potential.

"The gel-on-a-chip is the next wave of miniaturization," Sensabaugh says. "The emerging technology of capillary array electrophoresis, however, is quite workable. When everything goes right we can do 500-700 analyses in a single day, as compared to 25-50 per week in forensic labs today."

Sensabaugh has been testing a capillary array device in collaboration with Mathies' group, and recently submitted an article to the journal Analytical Chemistry describing their success in using the machine to do genetic typing of blood samples.

The potential applications of fast, miniaturized diagnostic devices are myriad, whether they are the size of capillary arrays or a credit card. Sensabaugh says forensic labs today typically have the time and staff to handle only 20 percent of all cases that come their way, which means potentially useful evidence in cases without suspects may never get analyzed. A fast and less labor-intensive method like this is sorely needed, he says.

The new devices also should be useful in establishing DNA profile databases on registered sex offenders and convicted violent offenders, as mandated by many state legislatures, Sensabaugh says. The establishment of these databases will involve analysis of tens of thousands of blood samples, a task currently beyond the capacity of current technology.

In addition, DNA analysis could speed diagnosis of infectious diseases and tissue typing for matching organs for transplant.

Mathies' work was funded by the US Department of Energy.


Copyright 1994, The Regents of the University of California.
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