PAGE 7 OF 8 Figure 5. Hybridization of alternatively labelled Z2 sense strand ssDNA with antisense reverse probes targeting different regions. (A) Random fluorescent labelling with Alexa Fluor 647. (B) Radioactive end-labelling with [33P]. Four spots are shown for the probes targeting 10 positions on Z2 sense strand. The hybridization images were adjusted for best viewing (quantitative conclu- sions drawn from the image may be misleading). The quantitative analysis below (arbitrary fluorescence units given with standard deviations) was calcu- lated from 24 spots on three different slides (A) or 16 spots from two different slides (B). This showed that in microarrays utilizing longer oligonuc- leotides, the POL effect is still occurring, albeit at a weaker level. DISCUSSION The results of our study indicated that the signal intensity in oligonucleotide-based microarray experiments is inﬂuenced by the relative position of the nucleic acid hybrid towards the ﬂuorescence label of the target: when oligonucleotides hybridize to the region closer to the end that is labelled, the ﬂuorescence signal is stronger. We term this effect as ‘position effect’ or ‘POL effect’. The following lines of evidence sug- gest that this is a signiﬁcant, previously unknown factor in microarray evaluation. The probes targeting different regions of our test targets shared similar characteristics; moreover, probes from six posi- tions were also applied as reverse complementary probes for comparison of the impact of complementary strands (Figure 1). Theoretically the complimentary probe pairs should have Nucleic Acids Research, 2005, Vol. 33, No. 19 e166 formed the same hybrids with their corresponding targets and thus given similar results in microarrays hybridizations. However, it was found in repeated experiments that probes targeting the regions close to the labelled ends generated higher signals than other probes, regardless of the target sequences (Figures 1 and 2). In most of the previous studies, signal variations were attrib- uted to the effect of secondary structure and steric hindrance, co-acting with certain extent. Secondary structure contributes to the low binding efﬁciency more predominantly, mainly owing to the complex secondary structure of long targets used in those experiments. In our study, several aspects suggest that the effect of secondary structure was not the main cause of the large variation between signal intensities: (i) ssDNA was routinely denatured before hybridization, and the hybridization was performed at high stringency (50% formamide) preventing the ssDNA from forming complex structures; (ii) base pair forming in different target regions calculated in silico (13) could not explain the observed hybrid- ization phenomenon, moreover reverse complementary hybridization experiments did not result in identical signal strengths (see above); (iii) DG and DS of all formed hybrids were estimated in the same range (16,17) (data not shown); and (iv) radioactively labelled target that is expected to have the same binding and secondary structure features did not show a signiﬁcant POL effect (Figure 5B). The target used in these experiments was relatively short (maximum 362 nt), therefore the chance of formation of complex secondary struc- tures was alleviated. Utilization of long target molecules could be a reason why the POL effect was not observed in previous oligonucleotide microarray applications. Long target sequences in comparison with short ones are more probably to fold on themselves as a result of intra-molecular Watson– Crick base pairing, which hides parts of the target from the oligonucleotide probes (18) and obscures quantitative results. The large variation of signal intensities observed is also unlikely to be the effect of steric hindrances. It has been observed in previous studies that immobilized probes could form hybrids more easily when they are away from the support surface (4). Although it was shown that the optimal spacer length should be at least 40 atoms (19), several studies have illustrated that spacer with 5, 6 or 12 atoms in length could affect the signal intensity signiﬁcantly as well, especially one using a similar test system as this study (18). The extension of spacers from A6 to A12 in our study did not have a signiﬁcant impact on the position effect (data not shown). Also a steric hindrance of hybridization owing to long overhanging ends of non-hybridized target DNA close to the array surface cannot explain the POL effect: comparison of different hybrids formed by the target with the same oligonucleotide having different immobilization directions and thus different steric hindrance (Figure 4) resulted in equally high signals as long as the ﬂuorescence label was located close to the hybrid. However, probes resulting in sterically favourable hybrids showed very low signal intensities when the label was distant to the hybrid (see e.g. Figure 2, S20 versus A20). Both signal-suppressing parameters are more or less correl- ated with the binding efﬁciency. However, the hybridization results of randomly, internally labelled target and of radioact- ively labelled target clearly showed that there was only a weak or no difference of binding efﬁciency for the different probes