Figure 1

In situ single stranded cDNA microarray production method (a) Strand-specific end modifications (amino-linkers) are incorporated into DNA by two parallel PCR reactions. After clean-up, the PCR product for each end-modified strand is printed separately onto the same microarray glass slide. The amino terminal groups are further coupled to the glass. The probes are digested with 5'-3' T7 gene 6 exonuclease and then immersed in boiling water. As a result of this treatment, only end-modifed strands remain attached to the surface. (b) Hybridization of labelled in vitro transcribed RNA from the β-lactamase gene with β-lactamase single-stranded sense and antisense DNA capture probes. Control spots containing ds-DNA probes or amino-modified PCR primers alone were also included on the array (not shown). Both probes and controls were spotted in 10 × 10 replicates. (c) Scatter plot showing the distribution of raw median pixel signal intensities from the hybridization performed in (b). Signals from all replicates are clearly discriminated according to the strand from which they originated (sense or antisense). Control spots for background intensities (printing buffer, PCR and amplification primers) demonstrate the specificity of the single stranded probes.