The microarray screen performed by Ooi et al. . The S. cerevisiae-Escherichia coli shuttle plasmids pRS416 are linearized with the restriction endonuclease EcoRI. The mutant pools are transformed in parallel with circular and linearized pRS416 plasmids and the transformants selected by plating on synthetic complete medium without uracil (SC-URA). Mutants with wild-type NHEJ activity (Mutant 1, blue) will be able to repair the linearized plasmid and grow to levels similar to those of the same Mutant 1 transformed with circular plasmid. In contrast, mutants deficient in NHEJ (Mutant 2, pink) will die on SC-URA plates because they fail to recircularize the plasmid. As each deletion is flanked with UPTAG and DOWNTAG as well as universal priming sites (gray boxes), PCR can be used to amplify the UPTAGs (or DOWNTAGs) using labeled primers to the universal sites; Cy5-(red) labeled primers were used for DNA prepared from circular pRS416 transformants, whereas for linearized pRS416 transformants Cy3-(green) labeled primers were used. The fluorescently labeled PCR products were used as probes for hybridization to a DNA microarray bearing each tag in triplicate; if both are present (Mutant 1) the array appears yellow, but mutants deficient in NHEJ (Mutant 2) would be under-represented after selection on SC-URA plates and therefore the Cy3 signal will be reduced and it will appear as red.
Jazayeri and Jackson Genome Biology 2002 3:reviews1009.1 doi:10.1186/gb-2002-3-4-reviews1009