The choice of DNA's to be used in the spots on a microarray determines which genes can be detected in a comparative hybridization assay. For organisms whose genomes have been completely sequenced, including several bacteria and the the yeast Saccharomyces cerevisciae, it is possible to array genomic DNA from every known gene or suspected open reading frame (ORF) in the organism. Each gene or ORF is amplified from total genomic DNA by PCR, producing enough DNA to make unlimited numbers of arrays. The Pat Brown Lab at Stanford University has arrayed all known or suspected genes of S. cerevisciae (roughly 6100) on a single microarray.

Because the human genome has not been completely sequenced, we cannot yet produce a comprehensive array for all its genes. Moreover, the number of human genes has been estimated at somewhere between 10,000 and 100,000, so several arrays will probably be required to hold them all. Despite these limitations, several strategies can be used today to make arrays for studying human genes. We do know the location and sequence of quite a few human genes now, so the same method used to array yeast genes will produce at least a partial human genome array. There are two other ways to produce arrayable DNA even for unknown genes: amplify clone inserts from human cDNA libraries, or synthesize oligonucleotides directly from known expressed sequence information such as EST's. While neither of these methods will produce DNA's for every human gene, both can yield enough different expressed sequences to make substantial arrays. Both types of DNA have been used before in array-like applications: cDNA libraries were used for comparative hybridization before the advent of fluorescent microarrays, while oligonucleotide arrays are available commercially today from Affymetrix Corporation for rapid resequencing of a few genes important to AIDS and some cancers.

5. Scanning the Hybridized Array

Once the cDNA probes have been hybridized to the array and any loose probe has been washed off, the array must be scanned to determine how much of each probe is bound to each spot. The probes are tagged with fluorescent reporter molecules which emit detectable light when stimulated by a laser. The emitted light is captured by a detector, either a charge-coupled device (CCD) or a confocal microscope, which records its intensity. Spots with more bound probe will have more reporters and will therefore fluoresce more intensely.

Each of the two fluorescent reporters (fluors) used has a characteristic excitation wavelength; only light of this wavelength will cause the molecule to fluoresce. The emitted light has a characteristic emission wavelength which is different from the excitation wavelength. The detector for the emitted fluorescence from the array is sensitive to the emission wavelength but filters out the excitation wavelength; in this way, the fluorescent light of interest can be separated from the laser light scattered off the slide.

A good pair of fluors for a comparative hybridization experiment should have very different emission or excitation wavelengths. If the emission wavelengths are different, light emitted from the two fluors can be selectively filtered to measure the amount emitted by each fluor separately. If the excitation wavelengths are different, the two fluors can be stimulated and scanned one at a time. If one of these conditions is not met, the scanned intensities can be contaminated by crosstalk between the two fluorescent channels.

Although the purpose of the scanner is to pick up light emitted by probe cDNA's bound to their