microarrays are little more than glass microscope slides studded with a large number of DNA fragments, each containing a nucleotide sequence that serves as a probe for a specific gene. The most dense arrays may contain tens of thousands of these fragments in an area smaller than a postage stamp, allowing thousands of hyb reactions to be performed in parallel. Some microarrays are generated from large DNA fragments that have been generated by PCR and then spotted onto the slides by a robot. Others contain short oligonucleotides that are synthesized on the surface of the glass wafer with techniques similar to those that are used to etch circuits onto computer chips. In either case, the exact sequence—and position—of every probe on the chip is known. Thus any nucleotide fragment that hybridizes to a probe on the array can be identified as the product of a specific gene simply by detecting the position to which it is bound.
Using DNA microarrays to monitor the expression of thousands of genes simultaneously. To prepare the microarray, DNA fragments—each corresponding to a gene—are spotted onto a slide by a robot. Prepared arrays are also available commercially
To use a DNA microarray to monitor gene expression, mRNA from the cells being studied is first extracted and converted to cDNA (see Figure 8-34). The cDNA is then labeled with a fluorescent probe. The microarray is incubated with this labeled cDNA sample and hybridization is allowed to occur (see Figure 8-62). The array is then washed to remove cDNA that is not tightly bound, and the positions in the microarray to which labeled DNA fragments have bound are identified by an automated scanning-laser microscope. The array positions are then matched to the particular gene whose sample of DNA was spotted in this location.
So far, DNA microarrays have been used to examine everything from the change in gene expression that make strawberries ripen to the gene expression “signatures” of different types of human cancer cells. Arrays that contain probes representing all 6000 yeast genes have been used to monitor the changes that occur in gene expression as yeast shift from fermenting glucose to growing on ethanol; as they respond to a sudden shift to heat or cold; and as they proceed through different stages of the cell cycle. The first study showed that, as yeast use up the last glucose in their medium, their gene expression pattern changes markedly: nearly 900 genes are more actively transcribed, while another 1200 decrease in activity. About half of these genes have no known function, although this study suggests that they are somehow involved in the metabolic reprogramming that occurs when yeast cells shift from fermentation to respiration.