Determination of optimal M. tuberculosis target-specific oligonucleotide concentration. A: Serial dilutions of 5'-Cy5-labeled 70-mer oligonucleotides to M. tuberculosis sodA (column 1) or aceA (column 2) were generated in 15% DMSO/1.5 M betaine in the presence or absence of 0.5 μM 5'-fluorescein-labeled tracking oligonucleotide and spotted onto poly L-lysine coated glass slides. Slides were processed, blocked, and scanned for fluorescein fluorescence (panel I) or Cy5 fluorescence (panel II). A subset of arrays was also hybridized with Cy3-labeled sodA- and aceA- specific PCR products and scanned for Cy3 fluorescence (panel III). No loss of hybridization signal is observed following addition of tracking oligonucleotide. B: Fluorescein (red) or Cy5 (black) mean relative fluorescence intensities from printed oligonucleotide were plotted as a function of Cy5-labeled target-specific oligonucleotide concentration. Note that the fluorescein intensity derived from the tracking oligonucleotide (red line) is informative over approximately two orders of magnitude of M. tuberculosis-specific 70-mer concentration. Bars represent S.D. Data were generated from 100 dilution series over 25 arrays.