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
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
Decrease the Clean-Up Time
Putting It All Together
-- Ann Parker
Key Words: DNA sequencing, Human Genome Project, microchannel, polyacrylamide gel.
For further information contact Joe Balch (510) 422-8643 (firstname.lastname@example.org).
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.