e166 Nucleic Acids Research, 2005, Vol. 33, No. 19 2109–2473 nt on AF200742) was ampliﬁed, end-labelled with Cy5, and the ssDNA hybridized as in the previous experi- ments. Hybridization to the ﬁve oligonucleotide probes spe- ciﬁcally binding to this fragment showed a sharp decline of ﬂuorescence with increasing distance to the label (Figure 3B): probe nifD-A1 showed a 342-fold higher signal than probe nifD-A185 located in a middle position. Thus, a large POL effect was also observed for a non-homologous gene. Impact of the fluorophore, slide surface and other factors on the position effect In order to test whether the nature of the ﬂuorescent label had an impact on the position effect, the target sense strand was labelled with Cy5 instead of Cy3 (Figure 3). For the six probes tested in previous experiments (A20–A307) and a target of similar length labelled with Cy5, similar results were obtained (Figure 3, Z2). The impact of the slide surface of the microarray on the position effect was tested for ﬁve types of commercial slides with different chemically modiﬁed surfaces: aldehyde, amine (QMT Amino Slides, Pan Amine slides) or ‘aldehyde plus’ surfaces. With respect to the immobilization step, the isothiocyanate-functionalized surface in our lab has the advantages of simplicity and homogeneous distributions over the other ﬁve types of slides, since it does not require ﬁxation steps, such as baking at elevated temperature or irra- diation with UV light. The formation of strong ionic interac- tions or a varying number of covalent bonds between the surface and the DNA oligonucleotides are avoided. A20– A307 and S20–S307 were used as probes; Cy3-labelled Z1 sense and antisense ssDNAs were used as targets. With respect to the signal intensities obtained from hybridization experi- ments, the slides with aldehyde, amine or ‘aldehyde plus’ surface from Genetix and the slides with amine surface from MWG Biotech were found to yield low hybridization signals. However, the isothiocyanate-functionalized surface and the QMT Amino Slides revealed 10-fold higher ﬂuor- escence signals and more homogenous distributions. With respect to the POL effect, all ﬁve kinds of commercial slides showed similar signal variation patterns as the self-made slides, indicating that the observed phenomenon could not be attributed to the differences between microarray surfaces. To assess the effect of possible steric hindrance of the non- hybridized part of the target, the same target was used for hybridization; however, we reversed the orientation of the probes on the microarray by attaching them at their 30 end (Figure 4). The position effect was also apparent, probe A20 or A20-30 providing higher signals compared with A307, irrespective of the orientation of the bound target. The 30-immobilized probe A307-30, however, showed a false neg- ative result (Figure 4), which is probably caused by a com- bination of position effect, steric hindrance of hybridization and quenching of ﬂuorescence close to the slide surface. Impact of internally, randomly labelled ssDNA or radioactively labelled ssDNA on signal intensity The data suggested that the relative position of the ﬂuorescent end-label of the target is crucial for the observed effect. There- fore randomly labelled target was tested, which should dimin- ish the effect. After strand separation of the Z2 target ampliﬁed PAGE 6 OF 8 Figure 4. Hybridization of Cy3-labelled Z1 sense strand ssDNA with two pairs of antisense reverse probes with different immobilizing orientations. Schematic representation of different hybrids on the surface is shown along with four spots of hybridization image. The label located at the 50 end of the strand is shown as a star, poly(A) spacer of the probe as a dashed line. 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 16 spots on two different slides. with non-ﬂuorescent primers, the sense strand ssDNA was labelled internally and randomly with Alexa Fluor 647 included in the ULYSIS nucleic acid labelling kit, and then used as target to hybridize with the same set of 10 antisense reverse probes spotted on the slide as in Figure 4. In compar- ison with the microarray results of end-labelled targets, the variation of signal intensities of different probes was strongly reduced (Figure 5A). The ratio between the highest signal (A8) and the lowest signal (A188) was only 4.7-fold (P < 0.001, ANOVA, also for probes A1–A20 in comparison with probe A64 and higher), suggesting that the stronger effects observed for end-labelled probes were indeed related to the position of the ﬂuorescence label but not to the differences in hybridiza- tion efﬁciencies. If the effect was related to ﬂuorescence, a strong POL effect would not be expected for a radioactively labelled target. Therefore, the same set of probes was hybridized to the same type of probe end-labelled with the isotope [33P] (Figure 5B). Here, no sharp decrease in signal intensity was observed. Except for probes A14 or A20, respectively, versus A307, the signals were not signiﬁcantly different (P > 0.05, ANOVA). Impact of oligonucleotide probe length on the POL effect In many applications, e.g. in microbial ecology, longer oligo- nucleotide probes in the range of 50–70 nt are immobilized to the slide surface. Therefore we analysed the effect of probe length (50mers) on the signal intensity in hybridizations with randomly labelled (Alexa Fluor 647) or 50 end Cy5-labelled target fragment Z2 of nifH. The signals for the different probes close to the label (A1–50) or in the middle position (A174–50) were not signiﬁcantly different for the randomly labelled target: A1–50, 29 750 ± 1073; A19–68, 27 811 ± 3060; A174–223, 30 982 ± 956. In contrast, there was an enhance- ment of signal intensity when the end-label was close to the probe binding site, however, the factor was only 3.8: A1–50, 18 918 ± 712; A19–68, 14 575 ± 366; A174–223, 4975 ± 313.