e166 Nucleic Acids Research, 2005, Vol. 33, No. 19 used (Figure 5). These minor variations might indeed be attrib- uted to secondary structure or steric effects. The obvious difference among hybrids with end-labelled targets is POL and ﬂuorescence. Since the observed strong effect cannot be attributed to differences in hybridization efﬁciency, it is most probably caused by increased ﬂuorescence signal intens- ity. The dramatic variation of signal output might be attributed to multiple factors, however the physical and chemical basis of the POL effect is not yet fully understood. The ﬂexibility of the labelled strand might increase with the distance to the double helix, therefore strands might fray with each other, leading to a decreased ﬂuorescence. However, a nucleotide–ﬂuorophore quenching effect may occur. It has been shown that ﬂuores- cence resonance energy transfer and contact-mediated quench- ing exist between ﬂuorophores and different nucleotides (20). Probe-target hybrids with distant label, having a higher ﬂexibility of the strand, might have a higher chance of ﬂuorophore–nucleotide interaction and thus quenching of the ﬂuorescent signal. Likewise, ﬂuorescence might increase in proximity to a DNA double helix. We obtained similar results with other PCR products of the nifH gene (genes from other microorganisms, truncated frag- ments), and with a second gene (nifD) that is phylogenetically, functionally and structurally different. This indicates that the effect is sequence independent and applicable to a wide range of targets. Moreover, our data suggest that the effect is largely independent from the chemical nature of the ﬂuorophore or the microarray slide surface. Therefore it should be applicable to a wide range of oligonucleotide microarrays using end-labelled targets. They are widely used techniques in the ﬁeld of SNP analysis, diagnostics, as well as microbial ecology and divers- ity studies. Moreover, the effect is not only observed for short oligonucleotide probes, but also for longer probes (50mers), albeit at a weaker level. There is always a combined set of factors inﬂuencing signal output in microarray experiments. However, the POL effect should be considered as a critical factor for microarray per- formance when probes of similar features are compared. The observed differences of probe efﬁciency towards the same target molecule indicate the importance of the POL effect for probe design. Oligonucleotide probe design taking into account the POL effect can minimize false negative results, and can improve the selection of probes for maximum signal intensities. The sensitivity of microarray applications could be increased if the probes were selected from the proper regions. Also data interpretation, i.e. the comparison of signal intens- ities obtained for a set of probes, is affected: consideration of the POL effect will improve quantitative evaluation of microarray results. In conclusion, our observations provide an important insight into factors affecting microarray perform- ance and deﬁne a novel determinant for large differences of signal intensities. ACKNOWLEDGEMENTS We would like to thank Andrea Krause for critical reading of the manuscript and Prof. F. Widdel, MPI Bremen, for the kind permission to use the radioactivity facilities. This work was linked to the CAG supported by the Land Bremen and the German Federal Ministry of Education and Research (BMBF) and was supported by a grant of the BMBF in the PAGE 8 OF 8 BIOLOG framework to B.R.-H. (grant no. 01LC0021). An application for a patent on the POL effect has been filed by the University Bremen. Funding to pay the Open Access publication charges for this article was provided by BMBF. 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