The genomics revolution that reached its climax in 2000 owes its very existence to two men. The first is Frederick Sanger who in 1977 developed the method for DNA sequencing that now bears his name. The second is Leroy Hood, who (with colleagues Michael Hunkapiller and Lloyd Smith) in 1986 took Sanger's method and made it better.
Sanger's enzymatic approach relies on specially modified reagents (2',3'-dideoxynucleotide triphosphates) whose incorporation into a growing DNA strand terminates the extension reaction (see related story, p. 44). The method calls for extending a primer-template pair in the presence of a radioactive marker and, in four parallel reactions, either dideoxy-A, dideoxy-C, dideoxy-G, or dideoxy-T. The resulting products can then be resolved on a high-resolution polyacry-lamide gel to produce a four-lane-wide "ladder" that reveals the template's sequence. Brilliantly inventive, the technique is also painfully laborious, producing a few hundred or perhaps a thousand bases at a time, which then have to be read by hand.
Hood's invention, the automated DNA sequencer, simplified the process first by replacing the radioactive marker with safer fluorescent ones. As each terminator was labeled a different color – red, green, yellow, or blue – scientists could combine the four reactions into one, increasing the per-gel throughput. Better still, the design used a laser to interrogate the samples in the gel and a computer to read the results. That first system, marketed by Applied Biosystems of Foster City, Calif., could produce a then-remarkable 4,800 bases of sequence per day. Suddenly, institutional core sequencing facilities were a practical possibility.
Today, some companies still market systems based on this design. But the polyacrylamide gel of Hood's first foray for the most part has been replaced by arrays of tiny capillaries, each of which acts as a "lane" from an old-style gel. Applied Biosystems' 3730xl DNA Analyzer, whose parts are shown here, is one such system. Able to run as many as 96 capillaries in parallel, a single instrument can churn out approximately two million bases per day.
Jeffrey M. Perkel
1 The polymer pump loads the capillaries with separation matrix.
2 The DNA samples are loaded into the array by a short burst of electrophoresis called "electrokinetic injection."
3 The capillary array is then immersed in running buffer.
4 DNA fragments migrate through the capillaries by size, smallest to largest.
5 As they reach the detection window, the laser beam excites the dye molecules, causing them to fluoresce. Light from all 96 capillaries is collected at once, spectrally separated, and focused onto a CCD camera.
6 Computer software interprets the data to produce a graph of intensity versus run time, or an "electropherogram."