Restriction site tiling analysis identifies polymorphisms and genotypes individuals by hybridization to a custom microarray. Fifty base pair tiles (white circles) are designed to be centered on restriction enzyme cut sites. DNA from an individual is extracted and randomly sheared by sonication. The sample is then divided in half: one part is treated with the restriction enzyme and labeled with green fluorescent dye (Cy3), the other part is treated as a control (without restriction enzyme) and labeled with red fluorescent dye (Cy5). The two parts are mixed and hybridized to the array. This DNA processing and hybridization result in different fluorescent signals reflecting the three possible genotypes for a polymorphic locus: when an individual is homozygous for the cut site (blue triangle) the digested DNA is cut and does not hybridize to the tile, resulting in a high red-to-green ratio (log2 Cy5/Cy3, left panel); however, if an individual is homozygous for a mutation in the cut site (yellow star) then the DNA remains intact and hybridizes to the tile, resulting in high green signal intensity or a low red-to-green ratio (right panel). Heterozygous individuals yield an intermediate red-to-green ratio. Polymorphic loci are identified based on the bi- or trimodal distribution of log ratios across sampled individuals. Individuals can be genotyped based on their log ratio.