Position of the fluorescent label is a crucial factor determining signal intensity in microarray hybridizations.

A key issue in applications of short oligonucleotide-based microarrays is how to design specific probes with high sensitivity. Some details of the factors affecting microarray hybridization remain unclear, hampering a reliable quantification of target nucleic acids. We have evaluated the effect of the position of the fluorescent label [position of label (POL)] relative to the probe-target duplex on the signal output of oligonucleotide microarrays. End-labelled single-stranded DNA targets of different lengths were used for hybridization with perfect-match oligonucleotide probe sets targeting different positions of the same molecule. Hybridization results illustrated that probes targeting the labelled terminus of the target showed significantly higher signals than probes targeting other regions. This effect was independent of the target gene, the fluorophore and the slide surface chemistry. Comparison of microarray signal patterns of fluorescently end-labelled, fluorescently internally random-labelled and radioactively end-labelled target-DNAs with the same set of oligonucleotide probes identified POL as a critical factor affecting signal intensity rather than binding efficiency. Our observations define a novel determinant for large differences of signal intensities. Application of the POL effect may contribute to better probe design and data interpretation in microarray applications.