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The Cutting Edge |
In the late 1960s, a useful molecular tool came to the rescue of these frustrated researchers, thanks to a series of studies by Werner Arber, in Switzerland, and Hamilton Smith, at Johns Hopkins University. These investigators were studying what at first seemed to be an unrelated problem. They were interested in understanding how some bacteria resist invasion by viruses. When viral DNA enters these bacteria, it is cut into small pieces and inactivated by enzymes called endonucleases. Smith showed that one of these enzymes cut the DNA at a specific short DNA sequence. Smith's colleague Daniel Nathans recognized that this provided a means of cutting a large DNA molecule into well-defined smaller fragments, and he used the method to generate the first physical map of a chromosome, that of the small monkey virus SV40. The map allowed Nathans to determine the arrangement of the individual genes within the DNA that forms the viral chromosome. With clairvoyance, Nathans speculated that larger chromosomes might be studied similarly. This heralded the mapping of chromosomes, an activity that forms the basis for the assignment of a disease gene to a specific region on a particular human chromosome.
The DNA cutting enzyme that Smith isolated was the first of over 1,000 "restriction enzymes" that have been discovered in just a few decades. Restriction enzymes not only allow chromosome mapping, they also enable researchers to generate large amounts of any specific DNA sequence of interest. These enzymes usually do not cut straight across the two strands of DNA, but cut in a staggered fashion. Consequently, their cuts create short, single-stranded tails on the ends of each fragment, called sticky ends. The sticky ends can be joined to other DNA strands with the aid of another type of enzyme, called ligase. By 1973, researchers were using restriction enzymes to cut specific DNA sequences of interest and join them to the DNA of bacteria. The bacteria then generated copies of the selected DNA with their own DNA each time they divided. Because a single bacterium grows rapidly, producing more than 1 billion copies of itself in 15 hours, large quantities of a specific DNA sequence can be produced in this manner--called cloning. This DNA can either be used for further study or to make DNA probes.
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