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FIGURE 9-21 Photolithography. This technique for preparing a DNA microarray makes use of nucleotide precursors that are activated by light, joining one nucleotide to the next in a photoreaction (as opposed to the chemical process illustrated in Fig. 8-38). A computer is programmed with the oligonucleotide sequences to be synthesized at each point on a solid surface. The surface is washed successively with solutions containing one type of activated nucleotide (A*, G*, etc.). As in the chemical synthesis of DNA, the activated nucleotides are blocked so that only one can be added to a chain in each cycle. A screen covering the surface is opened over the areas programmed to receive a particular nucleotide, and a flash of light joins the nucleotide to the polymers in the uncovered areas. This continues until the required sequences are built up on each spot on the surface. Many polymers with the same sequence are generated on each spot, not just the single polymer shown. Also, the surfaces have thousands of spots with different sequences (see Fig. 9-22); this array shows just four spots, to illustrate the strategy.

or cell culture to identify the genes being expressed in those cells.

A microarray can answer such questions as which genes are expressed at a given stage in the development of an organism. The total complement of mRNA is isolated from cells at two different stages of development and converted to cDNA, using reverse transcriptase and fluorescently labeled deoxynucleotides. The fluorescent cDNAs are then mixed and used as probes, each hybridizing to complementary sequences on the microar-ray. In Figure 9-22, for example, the labeled nucleotides used to make the cDNA for each sample fluoresce in two different colors. The cDNA from the two samples is mixed and used to probe the microarray. Spots that fluoresce green represent mRNAs more abundant at the single-cell stage; those that fluoresce red represent sequences more abundant later in development. The mRNAs that are equally abundant at both stages of development fluoresce yellow. By using a mixture of two samples to measure relative rather than absolute abundance of sequences, the method corrects for variations in the amounts of DNA originally deposited in each spot on the grid and other possible inconsistencies among spots in the microarray. The spots that fluoresce provide a snapshot of all the genes being expressed in the cells at the moment they were harvested—gene expression examined on a genome-wide scale. For a gene of unknown function, the time and circumstances of its expression can provide important clues about its role in the cell.

An example of this technique is illustrated in Figure 9-23, showing the dramatic results this technique can produce. Segments from each of the more than 6,000 genes in the completely sequenced yeast genome were separately amplified by PCR, and each segment was deposited in a defined pattern to create the illustrated microarray. In a sense, this array provides a snapshot of the entire yeast genome.

Protein Chips Proteins, too, can be immobilized on a solid surface and used to help define the presence or absence of other proteins in a sample. For example, researchers prepare an array of antibodies to particular proteins by immobilizing them as individual spots on a solid surface. A sample of proteins is added, and if the protein that binds any of the antibodies is present in the sample, it can be detected by a solid-state form of the ELISA assay (see Fig. 5-28). Many other types and applications of protein chips are being developed.

Detection of Protein-Protein Interactions Helps to Define Cellular and Molecular Function

A key to defining the function of any protein is to determine what it binds to. In the case of protein-protein interactions, the association of a protein of unknown function with one whose function is known can provide a useful and compelling "guilt by association." The techniques used in this effort are quite varied.

Comparisons of Genome Composition Although not evidence of direct association, the mere presence of combinations of genes in particular genomes can hint at

Isolate mRNAs from cells at two stages of development; each mRNA sample represents all the genes expressed in the cells at that stage.

o mRNA

mRNA

Convert mRNAs to cDNAs by reverse transcriptase, using fluorescently labeled deoxyribonucleotide triphosphates.

reverse transcriptase

cDNA

Add the cDNAs to a microarray; fluorescent cDNAs anneal to complementary sequences on the microarray.

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