Outline of a typical microarray experiment

In a two-color experiment (panel a in the figure) DNA from individuals of the same species or different tissue from a single individual (e.g., normal and diseased cells) is extracted and differentially labeled with compatible fluorophores (e.g., Cy3, Cy5). Equal amounts of labeled DNA is hybridized to the microarray, and the hybridization reaction is allowed to come to equilibrium over a period of >12 hours. At most probes, equal amounts of the two samples will hybridize (yellow features on the array), reflecting the fact that most loci in the two genomes are present in equal amounts (for example region 3). Regions that are deleted in the sample genome (region 1 of sample A) will result in probes with increased relative Cy3 signal (green features). Alternatively, amplified regions in the sample (region 2 of sample A) will result in features with an increased relative Cy5 signal (red features). Over the entire microarray, the signal ratios at each feature follow a Gaussian distribution, and candidate copy number variations are identified on the basis of deviation of a particular probe ratio, using statistical cut-offs. Although the diagram illustrates the protocol for array comparative genome hybridization, all array procedures, including SNP discovery and insertion site mapping, are carried out in this way. One-color experiments (panel b in the figure) are performed in a similar manner, except that the DNA is labeled with a single color and hybridized to a microarray without a reference sample. The difference between two-color and one-color experiments is that in the former case two samples are compared within an experiment, whereas in the latter case two separate experiments are required to compare the samples. For Affymetrix-manufactured microarrays, the method entails labeling DNA with biotin, then adding streptavidin conjugated to phycoerythrin after hybridization (represented by yellow circles). Rather than a ratio, an absolute value of hybridization is determi