Schematic representation of MGK. (a) Mixed library is grown in a reference and selective condition, and genomic DNA is isolated from each population. (b) Using genomic DNA as template, single-stranded DNA flanks are generated by linear extension of outward-facing insertion cassette-specific biotinylated primers (blue arrows). (c) The biotinylated flanks are separated from the template using streptavidin-coated magnetic beads, and polyadenylated at the 3'-ends using terminal deoxynucleotidyl transferase in the presence of dATP. (d) Microarray targets are PCR-amplified using an oligo d(T) primer (red arrows) and a nested Kmr-specific primer (black arrows). Amino-allyl dUTP is incorporated during this step. (e) Fluorescent dyes are conjugated to microarray targets. (f) Differentially labeled targets are mixed and hybridized to a custom DNA microarray. Kmr, kanamycin resistance; MGK, Monitoring of Gene Knockouts; PCR, polymerase chain reaction.