e166 Nucleic Acids Research, 2005, Vol. 33, No. 19 sensitivity level of the photomultiplier for each slide so that absolute signal intensities (arbitrary units) from independent experiments can be compared. One-way Analysis of Variance (ANOVA) was calculated with Graphpad instat 3.01 (Graph- Pad Software, Inc., San Diego, CA). Alternatively, microar- rays with radioactively labelled targets were exposed for 1 h at room temperature to a phosphoimager screen and analysed by a Typhoon 8600 Variable Mode Imager (Molecular Dynamics Inc., Sunnyvale, CA) in combination with ImageQuant v.5.1. RESULTS The design of the test system During the development of a diagnostic microarray for envir- onmental monitoring of nitrogenase gene diversity (nifH- phylochip), optimization experiments suggested that the relative position of the ﬂuorescence label might have an impact on signal intensities. In order to test the effect of differ- ent oligonucleotide probe positions with respect to the ﬂuore- scence label of the target sequence, we focussed on the nifH gene from Azoarcus sp. strain BH72 encoding the iron protein of the nitrogenase complex of this bacterium. NifH fragments were either end-labelled by ﬂuorescence-labelled primers used for ampliﬁcation by PCR, or randomly labelled after ampli- ﬁcation. Oligonucleotides targeting different sequence regions of these fragments were used as probes for microarray hybrid- izations with single-stranded targets, binding either the sense or the antisense strand. The binding positions with respect to the target sequence are shown in Figure 1. Antisense probes binding the sense strand are designated as A, sense probes binding the antisense stand as S, the numbers referring to the relative nucleotide position of the probe with respect to the sense strand of target Z1. All oligonucleotides represented perfect-match probes. Probe thermodynamics (length, Tm, %GC, DG and secondary structure) were carefully controlled to fall in a range as narrow as possible (Table 1). Effect of the position of fluorescent label relative to the probe-target duplex for end-labelled ssDNA targets: impact of target and secondary structure Six pairs of complementary probes targeting different regions on the sense strand of Z1 were ﬁrst evaluated for variations of signal intensities. Cy3-labelled Z1 sense strand ssDNA was used as target for hybridization with antisense probes spotted on the slide. Surprisingly, A20 showed the highest signals despite an expected strong steric hindrance by the non- bound target fragment (Figure 2A). The signal intensity decreased with increasing distance of the labelled nucleotide from the duplex formed by hybridization, being 638-fold lower for oligonucleotide A188 in a middle position for which the lowest intensity was obtained (Figure 2A). The increase of signal intensity when oligonucleotides hybridize more closely to the end-labelled target is referred to as ‘POL effect’ or simply ‘position effect’. To analyse the impact of putative secondary structure formation of the target on the observed effect, the microarray hybridization strategy was reversed (Figure 2B). The antisense strand was used as 50 end-labelled target, and the reverse PAGE 4 OF 8 Figure 2. Hybridization of Cy3-labelled Z1 sense strand (A) or antisense strand (B) ssDNAs with antisense reverse or sense probes, respectively, spotted on slides. Schematic representation of three pairs of hybrids on the surface is shown for three positions A/S 20, 114 or 307, respectively. The label located at the 50 end of the target is shown as a star, poly(A) spacers of the probes as a dashed line. Four spots are shown for the probes targeting six positions on antisense or sense strand, which are in the same order as the probe names in the quantitative analysis below (arbitrary fluorescence units given with standard deviations). The hybridization images were adjusted for best viewing (quanti- tative conclusions drawn from the image may be misleading). Data are from 24 spots on three different slides. complement of the oligonucleotides was used as probes binding to the same regions of the target as in Figure 2A (e.g. sense probe S20 reverse complement of A20). Since the complementary DNA strands of the target DNA were expected to form similar secondary structures in the corres- ponding regions (not shown), hybridization results would be expected to be similar for sense and antisense probes in case the secondary structure was the major determinant of the expected effect. In contrast, again the probe hybridizing closest to the ﬂuorescent end of the target (S307, Figure 2B) provided the strongest signal but not the distant probe S20, with the lowest signals occurring for probes in middle posi- tions (for S188 in comparison with S307 112-fold lower). This strengthened the assumption that the relative position effect caused differences in signal intensities. To analyse the impact of a shorter distance between the end-label of the target and the hybridized region, a different target Z2 was utilized that differed from Z1 by ampliﬁcation