FEATURE ARTICLE
Gene Chips and Functional Genomics
A new technology will allow environmental health scientists to track the expression of thousands of genes in a single, fast and easy test
Hisham Hamadeh, Cynthia Afshari
Microarrays
Until recently, assessing RNA in cells was painstaking and time-consuming. In 1995, researchers including Patrick Brown and colleagues at Stanford University developed a new technology adapted from the microchips used by the computer industry. The resulting DNA microarrays—the so-called gene chips—are indeed potent investigative tools, where the status of thousands of genes from any biological origin can be monitored simultaneously for changes in levels of gene expression. The difference between the old and new methods is striking. Traditional assays measure RNA transcripts from one gene at a time over a three-day period. Gene chips can measure transcripts from thousands of genes in a single afternoon.
The theory behind the gene chip is fairly simple and exploits a basic fact of the chemistry of DNA and RNA. An RNA molecule can bind with its DNA template, but not with DNA templates whose sequences are very different from its own. A gene chip holds copies of most of the DNA templates contained within a particular cell. The subset of genes being expressed by that cell type at any given time is expressed as a group of RNA molecules, which serve as messages to the protein-manufacturing machinery. In the laboratory, those RNA messages are transcribed once more to form complementary DNA (cDNA) message molecules. A cDNA can also bind with its complementary DNA template. When the cDNAs are exposed to the DNA chip, the message molecules recognize and adhere to the spots on the chip corresponding to their DNA templates. These message molecules have been tagged with fluorescent dyes, so when scientists look at this chip, they can see the pattern of genes being expressed at any particular time. They can also compare a spot and note that a gene is not being expressed under one circumstance, but it is under another. That is, no message is being manufactured under, say, the normal situation, but it is manufactured when the cell is exposed to a toxic chemical.
A DNA chip is made using a glass microscope slide, 7.62 centimeters by 2.54 centimeters and about 1.2 millimeters thick. Samples of DNA, in the form of spots, are "printed" on the slide, using a procedure similar to the one used to print computer chips. The DNA spots adhere to the slide, each spot being a cloned DNA sequence that represents a gene. The DNA molecules that make up the spots include either fully sequenced genes of known function, or collections of partially sequenced, unknown genes.
Chip manufacturing—printing or spotting—is done with a machine called an arrayer. Most arrayers are still custom-built instruments featuring a high-speed robotic arm fitted with a number of pins. The arm is controlled by software that allows the user to place genes in select areas and configurations on the glass slide to generate a cDNA microarray chip. The pins resemble the tips of quill pens. By capillary action, each pin draws up a small amount of a solution containing the DNA for a single gene and deposits it in a precise location on a glass slide. Since the arm holds many such pins, many genes are deposited on the slide at a time. Computers keep track of the location of each gene on the gene chip.
The arrayer is housed in a clean chamber where temperature and humidity are monitored and maintained constant so as to produce consistent and evenly sized spots. In some configurations it is possible to print up to 50,000 genes on one chip, and efforts are underway to increase that number as demand grows. The spotted genes/DNA are linked to the surface of the glass slide by either covalent bonds or charge interactions.
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