Author: Lovejoy Katherine
Institution: Chemistry and Integrated Science
Date: June 2002
Only a small amount of the human genome was sequenced between 1970 and 1990. Suddenly, in June 2000, all 3 million base pairs of the human genome were known, 5 years ahead of schedule and at a tiny fraction of the projected cost. Was it wizardry? Or technicians working serious overtime hours? Neither - an innovative team of analytic chemists was behind this acceleration. Their novel technique for separating and identifying DNA bases not only revolutionized the sequencing of DNA, but may eventually lead to "while-you-wait" disease diagnosis for throat and urinary infections, soil fertility profiling, and improved quality control measures for food supplements and antibiotics.
A method known as capillary electrophoresis powered the sequencing of the human genome. Capillary electrophoresis technology is based on the technique of separation by electrophoresis, which has long been used to separate proteins and DNA. The original method is based on the principle that an ion, a charged particle, will feel a force when placed in an electric field. Separation occurs because the negative ions move toward the positive plate and positive ions move toward the negative plate. Additionally, the lighter the molecule, or the more compact its shape, the greater the distance it travels in a given amount of time. As a result, an undifferentiated sample can be separated such that the lightest molecules are closest to the charged poles and the heaviest ones are closest to the source of the charge. DNA is negatively charged in aqueous solutions, and will move from the anode to the cathode when separated by electrophoresis.
At first, geneticists attempted to sort the tangled human genome using gel electrophoresis, a time-consuming, labor intensive, and expensive method. This method, also known as the Sanger method, uses radioactively labeled dideoxynucleotides (ddNTPs) to produce fragments terminated at each of the four bases, in separate reactions for each base. A limit on the maximum voltage that can be applied is a major problem with this technique. With a higher voltage, the sample can be separated more quickly, but higher voltage also means an increase in the heat produced. As the aqueous medium dissipates the electrical energy, the temperature of the sample also increases. At high voltages, heat-sensitive compounds are damaged. Therefore, the separation time associated with gel electrophoresis is very high. Additionally, samples have to be loaded in each well using micropipettes, a requirement that adds to the tedium of this technique.
The Entrance of Analytical Chemistry
Analytical chemists, accustomed to completely-automated, instrument-based methods, reviewed the process of using gel electrophoresis to separate DNA fragments and improved it, simultaneously advancing the fields of chromatography and genomics. Capillary electrophoresis on the micrometer scale was first described in 1979, when James Jorgenson of the University of North Carolina began work to increase the voltage that could be applied in electrophoresis experiments. In order to protect the sample from the heat generated by the increased voltage, the researchers reduced the size of the electrophoresis sample container from several inches to a few tens of micrometers. With the smaller container, they experienced a gain in the ability of the aqueous matrix to dissipate heat and could apply a much higher voltage to the sample, thus decreasing the time needed to sort the sample.
Developing an automated detection system to analyze separated DNA was another piece of the capillary electrophoresis puzzle. Fluorescence detection, a very precise and accurate method for detecting fluorescent particles, was chosen as a likely method. The difficulty was that there was no known method to attach fluorescent labels to DNA. Lloyd Smith of the University of Wisconsin devised a technique involving the use of four different fluorescent dyes: One each for adenine, guanine, cytosine, and thymine. Because each base was labeled with a different dye, researchers could run the entire sequencing reaction in one lane on a gel, rather than keeping the different bases in separate lanes. Laser light was then focused on a small spot of the capillary tube to excite the bases as they came through the tube (Figure 1).
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Additional advances in the capillary electrophoresis technique included the development of arrays of capillary tubes, which was critical to achieving high throughput, and the detection of such arrays using a charge-coupled device (CCD) camera. By the end of the Human Genome Project, commercial automated sequencing instruments had been produced that could run up to 96 capillaries simultaneously (Figure 2). Now, instead of a lab full of technicians tediously pipetting samples one at a time, the human genome could be analyzed by a tireless army of robots and tiny capillary systems. Long DNA sequences could be churned out in a matter of minutes, instead of months.
Other Uses of Capillary Zone Electrophoresis
Capillary Electrophoresis on Living Organisms
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Since the sequencing of the human genome, capillary electrophoresis techniques continue to improve and carry out new functions. A group at Iowa State University led by Dan Armstrong has shown that capillary electrophoresis can be performed not only on individual molecules, but also on intact living cells, including yeast and bacteria. This high-efficiency microbial separation (HEMS) begins with an undifferentiated aqueous sample of live bacteria and yields a separated sample of still-living bacteria. So far, the technique has been used to determine the percentage of living and dead cells in a given sample. The team has discovered that a large percentage of microorganisms in many commercial food supplements, such as microbial preparations of Lactobacillus acidophilus for the treatment of lactose intolerance, are in fact dead or nonviable. HEMS could also be adapted to monitor antibiotics fermentation, identify microbes in soil or urine samples, or facilitate automatic quality control analysis.
Adaptations for Neutral Molecules
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Micellar electrokinetic capillary chromatography (MECC) is a newly-developed technique that allows separation of neutral compounds, not just negatively- or positively-charged molecules. The separation involves the addition of a surfactant, usually sodium dodecyl sulfate, to the sample. The surfactant molecule is composed of head groups that are tolerant of water and tails that are intolerant of water. Above a certain concentration, the molecules form spherical micelles in which the head groups face outward and are in contact with the water. The tails are hidden on the inside of the micelle and are protected from the water. Because these micelles move at a speed different than that of the bulk flow of the buffer, any neutral analytes that interact with these micelles are slowed in comparison to the surrounding buffer. The selectivity of MECC is controlled not only by changing the surfactant, but also by adding modifiers, such as organic solvents, to the buffer.
Lab-on-a-Chip Devices
Researchers are also interested in building miniature devices with the ability to perform chemical and biochemical experiments that previously could only be done at the bench top on a large scale. Researchers hope that capillary electrophoresis will follow the model of the evolution of microelectronics and move from instruments taking up entire rooms to hand-held models that would reduce the time and cost of acquiring information. One such micro-scale device, developed by Stephen Quake of the California Institute of Technology, is a microfabricated fluorescence-activated cell sorter. Current eukaryotic cell sorting technology, although very efficient, involves the use of complex and costly machines. The work of the Quake group involved the use of a microfabricated fluidic device for cell sorting. The cells were electrokinetically directed into one of two output channels by selecting which output channel was connected to the driving voltage. The microfabricated chip was produced at a negligible cost and the overall cost of the smaller, more efficient machine was only $15,000 - far less than earlier instruments costing about $250,000 (Figure 3). In the future, researchers envision a "plug and play" interface where a generic controller device allows the insertion of a variety of microfluidic chips, each of which would be designed to perform different assays employing a form of the capillary electrophoresis technology developed for the human genome project.
However bright the predictions for the future of capillary electrophoresis are, it is important to reflect on all that has already been done with this extraordinary technology. The development of new quality control methods and analytical techniques, and, most importantly, the sequencing of the human genome, have paved the way for scientists who wish to miniaturize capillary electrophoresis technology for industrial use. The application of chemical ideas to a biological problem has demonstrated the importance of communication between biologists and chemists and should encourage even greater collaborations between scientists of all fields.
Suggested Reading
Armstrong, D. W., et al. (2001) Rapid CE Microbial Assays for Consumer Products that Contain Active Bacteria. FEMS Microbiol. Lett., 194: 33-37.
Fu, A. Y. et. al. (1999) A Microfabricated Fluorescence-Activated Cell Sorter. Nature Biotechnology. 17, 11: 1109-1111. http://www.nature.com/cgi-taf/DynaPage.taf?file=/nbt/journal/v17/n11/full/nbt1199_1109.html
Ramsey, J. M. (1999) The Burgeoning Power of the Shrinking Laboratory. Nature Biotechnology. 17, 11: 1061-1062. http://www.nature.com/cgi-taf/DynaPage.taf?file=/nbt/journal/v17/n11/full/nbt1199_1061.html
Wilkinson, Deborah. (2000) Capillary Action. The Scientist. 14, 10: 21.
Zubritsky, E. (2002) How Analytical Chemists Saved the Human Genome Project. Analytical Chemistry. 74, 1: 23A-26A.