St. Louis, Aug. 16, 1999 -- Like a temperamental copy machine, the most commonly used enzyme in DNA sequencing has a few annoying quirks. It generates pages with blank spots, for example. But new X-ray images of the enzyme at work have suggested a way to fix these problems. The strategy works in the lab and is being tested by several companies in the United States.
"We expect this will make the enzyme an even better sequencing tool for the Human Genome Project," says Gabriel Waksman, Ph.D., an associate professor of biochemistry and molecular biophysics at Washington University School of Medicine in St. Louis, one of the world's major sequencing sites. Waksman and colleagues report their findings about the enzyme, Taq DNA polymerase, in the Aug. 17 issue of Proceedings of the National Academy of Sciences.
The Sanger method is the most common method for determining the order of genetic letters, called nucleotides, in DNA. It uses Taq to copy segments of DNA into chains of different lengths. Because Taq comes from a bacterium, Thermus aquaticus, that flourishes in hot springs, it can withstand the temperature changes necessary for copying.
Four genetic letters -- A, T, C and G -- make up the DNA code, and all of these nucleotides are included in the reaction mixture when Taq is put to work. But because a nucleotide-like compound called a dideoxynucleotide triphosphate -- ddC, ddG, ddA or ddT -- also is included, Taq stops copying when it incorporates that compound, like a train that halts on the track when it hits a rock. Therefore, the length of a new DNA chain indicates where that particular nucleotide occurred. After the copies are separated by size and reacted with fluorescent chemicals, DNA sequencing machines can determine which nucleotide terminates each chain. Because A is complementary to T and C to G, this reveals the order of the nucleotides in the original piece of DNA.
Waksman and colleagues obtained X-ray images of Taq at wor
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Contact: Linda Sage
sage@medicine.wustl.edu
314-286-0119
Washington University School of Medicine
17-Aug-1999