PCR footprinting strategy used to confirm or reject putative essential genes. a) Diagrammatic representation of the strategy. PCR is performed using a gene specific primer 50–300 bases upstream of the start codon, and a primer corresponding to the transposon sequence. b) Agarose gel showing sample results. M – size markers, lane 1 – SAOUHSC_00862 (non-essential), lane 2 – SAOUHSC_01672 (essential). Each band represents a PCR product of a different size, corresponding to a transposon insertion in a different position. The white boxes represent the product size range expected for transposon insertions within the gene. For essential genes this region does not contain bands, since cells with insertions in this region are not viable.