The timetable for completing the Human Genome Project, the effort now underway to decipher the human genetic code, could be moved up considerably by a new technique for automated "DNA sequencing" -- determining the order of the nucleotide bases that are the "letters" of the code. Developed by scientists in LBL's Structural Biology Division (SBD), this new technique has the potential to be 50 to 100 times faster than the method most widely used today.
SBD's Richard Mathies, who is also a professor of chemistry at UC Berkeley (UCB), and Xiaohua Huang, a Department of Energy Human Genome Distinguished Postdoctoral Fellow, led the development of the new technique which is called "capillary array electrophoresis." This technique combines the use of gel-filled capillary tubes with a unique laser scanning system to sequence each of the four different types of bases -- adenine, cytosine, guanine, and thymine -- in a sample of DNA.
This work is part of a joint project by Mathies and SBD's Alex Glazer to develop new, highly sensitive detection methods and reagents for DNA mapping and sequencing. Working with them on this project, in addition to Huang, are Mark Quesada and Scott Benson, both postdoctoral fellows, plus UCB graduate students Hays Rye, Steve Clark, and Yimen Wang. The work was funded in part through LBL's Human Genome Center.
The Human Genome Project will require scientists to map the location of some 100,000 genes arranged along the 23 pairs of chromosomes that make up the full complement of human DNA. They will then have to sequence the 3 billion base pairs contained in these chromosomes in order to read the genome's message. With current technology, this is expected to take about 10 years and cost several billion dollars. Consequently, from the project's inception, the search has been on for ways of speeding up the work.
The standard DNA sequencing technique is the Sanger method, named for its developer, Frederick Sanger, who shared the 1980 Nobel Prize in Chemistry. This method begins with the use of special enzymes to synthesize fragments of DNA that terminate when a selected base appears in the stretch of DNA being sequenced. These fragments are then sorted according to size by placing them in a slab of polymeric gel and applying an electric field -- a technique called electrophoresis. Because of DNA's negative charge, the fragments move across the gel toward the positive electrode. The shorter the fragment, the faster it moves. Typically, each of the terminating bases within the collection of fragments is tagged with a radioactive probe for identification.
Although sensitive enough to sort and distinguish one type of base pair from another, the biggest drawback to the Sanger method is that it takes hours for DNA fragments to transverse a slab gel. Capillary array electrophoresis cuts this time down by replacing the slab with hundreds of tiny gel-filled capillaries, about 100 microns (four thousandths of an inch) in internal diameter, that can be bundled into a single array for automated detection. This permits the safe application of a much higher electrical field to make all of the fragments separate faster.
One of the major new developments behind the capillary array electrophoresis technique of Mathies and his colleagues is the use of laser-excited, confocal- fluorescence detection system that makes possible the analysis of more than one capillary at a time. In most fluorescence detection systems for capillary electrophoresis, the incident laser beam and emitted fluorescence are perpendicular to one another, making scans of more than one capillary difficult. By focusing the laser beam on a sample and gathering the emitted fluorescence through the same lens of a confocal microscope, Mathies and his colleagues set up a linear configuration that is ideal for scanning multiple capillaries.
"Using confocal excitation and detection, the depth of field of our optical system is sufficiently small that only the interior of each capillary is probed," says Mathies. "This gives us high spatial resolution and very efficient fluorescence collection and at the same time excellent rejection of scattered and stray light."
In the system developed by Mathies and his colleagues, an array of capillaries is placed in a holder and mounted onto a computer-controlled translation stage which brings each capillary into the path of the laser beam. The resulting fluorescence from the DNA sequencing fragments, which are labeled with special highly fluorescent dyes, is spectrally filtered and detected with a photomultiplier. Mathies, Huang, and Quesda reported the use of a 25-capillary array to sequence DNA at the rate of 10,000 base pairs an hour in a paper recently published in Analytical Chemistry. According to Mathies, this is about 10 times faster than current automated DNA sequencing methods.
"There is no reason we can't ultimately get to 50 to 100 kilobases per hour using 100-200 capillary arrays," says Mathies. "Once this performance level is achieved, we can talk seriously about sequencing the human genome."
Mathies and his group have also developed a new method for coding dye-labeled DNA fragments. In addition to needing a dye that is highly fluorescent, it is also critical that there be no electrophoretic mobility differences between sequencing fragments labeled with different colored dyes. Mathies and his colleagues made this requirement easier to meet by devising a unique binary coding protocol for labeling DNA fragments. This protocol lets them use only two different fluorescently labeled primers and two colors (red and green) to separate and distinguish all four sets of sequencing fragments in a single capillary tube.
Says Mathies, "With this new technology, sequencing itself is no longer the main limitation on how fast you can read a piece of DNA. The bottlenecks now are how fast you can prepare the sequencing samples and analyze the data."