Figure 1. Two main types of DNA arrays. (a) High-density oligonucleotide array synthesized using photolithographic methods. (b) cDNA array made by mechanically depositing pre-made PCR products. Images are shown following hybridization of labeled samples and fluorescence detection. In the case of photolithographically synthesized arrays, approximately 10 million copies of each selected oligonucleotide (usually 25 nucleotides in length) are synthesized base by base in a highly parallel, combinatorial synthesis strategy. For robotic deposition, approximately 1 ng of material is deposited at a center-to-center spot spacing of 100–300 m. (c) Different methods for preparing labeled material for measurements of gene expression (mRNA abundance) levels. RNA can be labeled directly using chemical or enzymatic methods, DNA can be end-labeled using terminal transferase and biotinylated nucleotides, and labeled nucleotides can be incorporated into cDNA during or after reverse transcription of polyadenylated RNA. In the protocol used most frequently for oligonucleotide arrays, cDNA is generated from cellular mRNA using an oligodeoxythymidine (dT) primer that carries a T7 promoter at its 5¢ end. The double-stranded cDNA intermediate serves as template for a reverse transcription reaction in which labeled nucleotides are incorporated into cRNA. The advantage of this approach is that the original, cellular mRNA is effectively amplified (typically by a factor of 50–200) in a linear, unbiased and reproducible fashion. Commonly used labeling groups include the fluorophores fluorescein, Cy3 (or Cy5 and other cyanine dyes), or nonfluorescent biotin, which is subsequently made fluorescent by staining with a streptavidin–phycoerythrin conjugate. (d) Two-color hybridization strategy often used with cDNA microarrays. cDNA from two different conditions is labeled with two different fluorescent dyes (e.g. Cy3 and Cy5), and the two samples are cohybridized to an array. After washing, the array is scanned at two different wavelengths to quantify the relative transcript abundance for each condition. (cDNA array image courtesy of J. DeRisi and P. O. Brown. Figure reprinted from Lockhart DJ and Winzeler EA (2000) Genomics, gene expression and DNA arrays. Nature 405: 827–836, with permission from Nature, ©2000, Macmillan Magazines Limited.)