Specific and non specific hybridization of oligonucleotide probes on microarrays

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  • 25 October 2004
Abstract
Gene expression analysis by means of microarrays is based on the sequence specific binding of mRNA to DNA oligonucleotide probes and its measurement using fluorescent labels. The binding of RNA fragments involving other sequences than the intended target is problematic because it adds a "chemical background" to the signal, which is not related to the expression degree of the target gene. The paper presents a molecular signature of specific and non specific hybridization with potential consequences for gene expression analysis. We analyzed the signal intensities of perfect match (PM) and mismatch (MM) probes of GeneChip microarrays to specify the effect of specific and non specific hybridization. We found that these events give rise to different relations between the PM and MM intensities as function of the middle base of the PMs, namely a triplet- (C>G=T>A>0) and a duplet-like (C=T>0>G=A) pattern of the PM-MM log-intensity difference upon binding of specific and non specific RNA fragments, respectively. The systematic intensity differences can be rationalized on the level of base pairings of DNA/RNA oligonucleotide duplexes in the middle of the probe sequence. Hybridization on GeneChip microarrays is compatible with conventional hybridization theory, which assumes a considerably reduced binding strength of non-WC mismatches compared with WC pairings.

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