FAST. Faster. Fastest. In the commercial arena, getting big jobs done fast requires automation, For the Human Genome Project at Lawrence Livermore National Laboratory, the key to uncovering thousands of yet-to-be-identified human genes is to automate and speed up the specialized biotechnical equipment that prepares and sequences DNA samples.
The point of all this urgency is the gold mine of information contained within the structure of the genes themselves. Genes and the proteins they produce hold the key to unlocking the mysteries of genetic diseases. Once the genetic code for a disease is understood, researchers can begin developing gene and drug therapies for that particular disease. The ultimate goal of the worldwide Human Genome Project is to find all the genes in the DNA sequence, develop tools for using this information in the study of human biology and medicine, and improve human health.
Sequencing involves determining the exact order of the four individual chemical building blocks, or bases, that form DNA. The total DNA in a single human cell has approximately 3 billion pairs of the chemical building blocks adenine, thymine, guanine, and cystosine. (For more information on the Laboratory's other work in DNA sequencing, see S&TR, November 1996.)
For a multidisciplinary team of engineers, chemists, computer scientists, and biologists at Livermore, Joe Balch is project leader for developing a next-generation instrument for sequencing DNA. When this high-throughput DNA sequencer is built and its operating conditions are optimized, it will ultimately read nearly 600,000 bases per eight-hour shift, about 12 times faster than current instruments, which manage at most 48,000 bases per shift.
"There is a worldwide push in the field to 'pick up the speed' with which DNA is sequenced," said Balch. "The current strategy is to do it with existing technology and just turn the crank a lot of times with more people. It's very people-intensive. The next-generation sequencing machine we are developing will allow us to leave the old technology behind and take the next step in automation." Livermore expertise in microfabrication, bioinformatics, and biochemistry makes this move possible.
Faster sequencing will also provide other Livermore programs with faster access to information in nonproliferation projects to detect biological signatures of collected samples and in bioremediation projects to optimize micro-organism action.

Sequencing: How It Works
When biological researchers want to sequence a section of DNA, they clone fragments of that section and then run four nearly identical reactions on those fragments. In these reactions, the four bases are chemically labeled with four different fluorescent dyes.
The sequencing machines currently used at the Laboratory are based on a gel-electrophoresis system, which works like this. The DNA samples are loaded by hand into a 200- to 400-micrometer-thick polyacrylamide gel, rather like thin Jello, which is sandwiched between two large glass plates, 48 centimeters long by 25 centimeters wide. The plates can hold 36 samples at a time. An electric current is then applied to the gel, and, because the DNA itself has a negative charge, the fragments migrate in 36 columns or "lanes" from the top to the bottom of the plate. The DNA fragments move at different rates depending on their size: smaller ones move faster than larger ones. As the fragments migrate past a certain point in the gel, a laser beam scans back and forth across the plate, exciting the dyes on the DNA bases. As the fragments pass the laser, the bases are separated from smallest to largest. The fluorescent signals generated by the laser are detected by photomultiplier tubes (or other detectors), and a computer captures, stores, and processes them (Figure 1 above).
When cleaning, loading, and running times are all taken into account, it takes between five to seven hours to complete a run. Each sample contains about 500 bases, which means each run of 36 samples yields no more than 18,000 bases. According to Balch, conventional technology is expanding the number of lanes to 64, which will increase the yield to about 32,000. But to increase those numbers significantly, say, by an order of magnitude, requires applying some new technologies.
There are a number of ways to increase this yield, explained Balch, who is also the former head of the Laboratory's Microtechnology Center. (For an overview of the Center, see pp. 11-17 of this issue). "You can increase the number of lanes on a single run. You can increase the speed at which you do a run--in other words, apply more electric field to the fragments. You can also look for ways to cut down on the loading and cleanup times, which often take a couple of hours. But to do all of these things, you need to move outside the current technologies and look for different ways to get the job done." That is what they are doing for their new sequencing machine (Figure 2).






Increase the Lanes
In the current system, although the samples travel in "lanes," no physical barriers divide one lane from the next. "And even though you have an electric field pulling the fragments to the bottom, they still wander a bit," said Balch. "Right now, we correct the wandering with software, with what is called 'lane tracking.' But if we start packing more lanes in, there's a problem with the columns blurring into each other."
So Balch and his team are taking a different tack: fabricating small, exact lanes, or microchannels, in large glass plates through which the gel medium flows. This effort got its start in 1993 with seed money from the Lawrence Livermore's Laboratory Directed Research and Development Program. In 1994, the Laboratory entered a year-and-a-half agreement with Perkin Elmer's Applied Biosystems Division to further develop this technology and some of the others needed for the new system. This effort is now being supported by grants from the National Institutes of Health and the DOE Human Genome Project.
Last year, Steve Swierkowski and Courtney Davidson of the Microtechnology Center successfully demonstrated the fabrication of a 96-lane array on a piece of glass 7.5 centimeters wide and 55 centimeters long--in other words, twice the lanes that current technology can offer in less than a third of the space. Ultimately, the team will be producing plates with 384 channels.
"These high-density microchannel glass plates are the really novel piece of our instrument," noted Balch. "The fabrication process involves three steps, each of which we need to continue to build and improve upon." (See Figure 2a.)
The first is the photolithography step, where the pattern of the channels is defined on a photomask plate. The second involves using that plate to chemically etch that very small pattern on the glass to very exact specifications. The final step is bonding the top piece of glass to the etched glass plate. Figure 2c shows the current prototype instrument developed by Davidson, Larry Brewer, Joe Kimbrough and Ron Pastrone.

Increase the Speed
Another way to speed up the process is to increase the electric field. The velocity of the DNA increases proportionally. In the current system, however, just increasing the field leads to other problems.
A higher electric field increases the power dissipation, which increases the temperature in the sieving media, explained Balch. And when the gel heats up--and the DNA samples in it--thermal diffusion causes the fluorescent bands to spread out. The bands run into each other and can no longer be identified as individual and distinct bands. This problem can be significantly reduced by using a very thin gel (about 50 micrometers thick) or other sieving media in place of the polyacrylamide gel now being used. The thinner gel means that the temperature gradient across the width of the gel is smaller, and the thermal diffusion of the DNA fragment bands is less.
With this thin sieving media, the instrument can run with an electrical field three to four times higher than that used on the conventional instrument. Thus, the speed of the run increases by the same factor.

Decrease the Clean-Up Time
Another improvement involves using small syringe pumps to inject thin sieving media into the microchannels of the new instrument when a run begins and then automatically pumping it out when a run is complete. This procedure will significantly speed up the overall time it takes to complete a run.
"With the polyacrylamide gel now in use, you have to go through a lengthy preparation at the start of a run to make a fresh gel. Then at the end, you have to take the plates apart, remove the old gel, and clean the plates for the next run. With this new media, we just pump it in and out through channels and capillaries without removing the microchannel plate from the instrument," said Balch. "We figure that when the system is up and running, one run will take two to three hours from start to finish, compared with four to five hours using the polyacrylamide gel."

Putting It All Together
Because the new system has different performance specifications than the old, simply loading the new glass plates with the new medium into the old machine is not the only change.
For instance, because the DNA fragments are moving at a higher velocity when they come to the laser, the laser has to scan across the plates faster. In addition, given the 96 lanes now and the 384 to come in the future, Balch and his team are exploring several concepts for automatic sample loading.
Other systems being developed include the laser-induced fluorescent detection system, the fluidic and pumping system for the polymer medium, a temperature control system, and analysis software. "These improvements plus the microchannel plates themselves add up to seven major parts of the high-throughput DNA sequencer that we must eventually meld together," said Balch. "The final production system is still down the road. When it's ready, we plan to make it available to others within the Department of Energy and the human genome community."

-- Ann Parker

Key Words: DNA sequencing, Human Genome Project, microchannel, polyacrylamide gel.

For further information contact Joe Balch (510) 422-8643 (balch1@llnl.gov).

The Human Genome Center's Internet home page is at http://www.llnl.gov/bbrp/genome/genome.html.

The Department of Energy's "Primer on Molecular Genetics" is located at http://www.gdb.org/Dan/DOE/intro.html.


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