PAGE 5 OF 8 Nucleic Acids Research, 2005, Vol. 33, No. 19 e166 Figure 3. (A) Hybridization of Cy5-labelled Z2 or Z3 sense strand ssDNA, respectively, with antisense reverse probes targeting different regions. Four spots are shown for the probes targeting 10 positions on Z2 sense strand (upper panel) and 4 positions on Z3 sense strand (lower panel). (B) Hybridization of a Cy5-labelled nifD sense strand ssDNA with antisense reverse probes targeting different regions. The hybridization images were adjusted for best viewing (quantitative conclusions drawn from the image may be misleading). The quantitative analysis below (arbitrary fluorescence units given with standard deviations) was calculated from 24 spots on three different slides. with perfect-match primers to nifH of strain BH72 instead of degenerate primers. This allowed the design of perfect-match probes spanning also the region of the PCR primer (A1–A14 in Figure 3). As expected from our POL hypothesis, probe A1 showed the highest signal, with a successive decrease of signal intensities with distance increase down to Probe A188. The difference in signal intensities of probe A1 and A64 was 15- fold, that of probe A1 and A188 was >355-fold (Figure 3), the latter can be regarded as a false negative signal. Thereafter (A188–A307) signals increased slightly. To exclude the possibility that the probes corresponding to the middle positions provided low signals because the decrease of signal intensities was dependent on the sequence, they were brought more closely towards the labelled end of the target by shortening the target. Target Z3 differed from Z1 by a truncation of 153 nt at the 50 end that was also Cy5- labelled. Probe A114 that gave almost negligible hybridization results with full-length target showed high signal intensities when it became positioned at the labelled end of the truncated target (Figure 3, dashed line), demonstrating that it can effect- ively hybridize with the target. In concordance with the pos- tulated position effect, more distantly hybridizing probes resulted in lower signals (5.2-, 2.5- or 1.8-fold lower for A188, A235 or A307, respectively, statistically signiﬁcant at P < 0.01 level). The position effect was not speciﬁc for the nifH gene of Azoarcus sp. BH72, since similar results were obtained for the Cy3-labelled Z1 fragment of nifH ampliﬁed from another spe- cies, A.indigens VB32T with perfect-match probes numbered according to the same criteria (VB-A34, VB-A164 and VB- A307) (94% identity of DNA sequence). The probe hybridiz- ing most closely to the labelled end showed the highest signal, 39.5 ± 5.2 or 13.8 ± 2.9-fold higher than VB-A164 or VB- A307, respectively. The differences were statistically signiﬁc- ant (P < 0.001). Also for a phylogenetically more divergent gene encoding the vanadium nitrogenase vnfH of A.vinelandii (84% DNA identity), results showed the same trend: vnf-A47 showed the highest signal intensity, vnf-A110 13.8 ± 2.6-fold, or vnf-A309 4.4 ± 0.98-fold lower signals, respectively. The differences were statistically signiﬁcant (P < 0.001). More- over, we used an entirely different gene, nifD, encoding a component of the dinitrogenase that is phylogenetically, func- tionally and structurally different from nifH encoding the dinitrogenase reductase. A fragment of 365 nt length (position