July 18, 2005
Restriction mapping is a widely used technique in molecular biology for identifying gene mutations. Now, a research team led by Robert Austin, Ph.D., of Princeton University has created a microfluidic device that can perform restriction mapping in less than a minute on single DNA molecules. The investigators believe that this technique, which is reported in the journal Proceedings of the National Academy of Science USA, can also be used to study how certain enzymes interact with DNA.
Restriction mapping uses so-called restriction enzymes that recognize and cut DNA at specific sequences, known as “restriction sites,” producing well-defined DNA fragments. Each of the many commercially available restriction enzymes are capable of cutting DNA at a specific sequence, or order, of nucleotide bases (A,T,G and C) within the millions of bases in a piece of DNA. Most often, each restriction enzyme will find several of its particular restriction sites within a piece of DNA and will produce a series of fragments that can be easily separated by their length. When a mutation occurs within a restriction site, the corresponding restriction enzyme no longer recognizes that site and the resulting pattern of fragments changes, producing one longer rather than two shorter fragments for each missing restriction site. Restriction enzymes require magnesium ions to function, which turns out to be a property that the Princeton team took advantage of in constructing their device.
The key to restriction mapping on a microfluidic device is the ability to first stretch a piece of DNA within a nanoscale channel so that restriction enzymes can then find their corresponding restriction sites and cut the DNA molecule. The investigators accomplish this feat by constructing parallel channels 100 to 200 nanometers in diameter on polished quartz wafers and connecting them with a series of smaller channels running perpendicular between the two larger channels. The ends of each of the two larger channels were connected to electrodes, producing a system that can move DNA into and out of one of the two larger channels and magnesium ions into and out of the other of the two larger channels.
Preparing a restriction map starts when both of the two larger channels are loaded with a restriction enzyme. The electrodes are powered so that DNA moves into one channel and magnesium ions stay in the other channel. Restriction enzymes will quickly bind to the DNA molecule so that by the time it is fully stretched within the channel, the enzymes are ready to do their cutting. At this point, the voltage applied to the electrodes is changed so that magnesium ions flow into the DNA-containing channel, which enables the restriction enzymes to slice DNA into its expected fragments. The lengths of these fragments are easily measured using an optical microscope as they exit the device. This work is detailed in a paper titled, “Restriction mapping in nanofluidic devices.” A reprint of this paper is available at no cost at the journal’s website.View paper.
Posted by s4175728 - Colby Hart