Bioinformatics Preprint 04-003
[Full text not available since the prospective publisher explicitly prohibits preprints]
The sensitivity of microarray oligonucleotide probes - variability and the effect of base composition
Hans Binder, Toralf Kirsten, Markus Loeffler, Peter F. Stadler
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The optimisation of both probe design and analysis algorithms for microarray experiments requires improved understanding and predictability of oligonucleotide hybridisation behaviour. We present here a physico-chemical theory of GeneChip© probe sensitivities in terms of normalised intensities. Our concept divides the probe intensity into an averaged intensity value which serves as a relative measure of the RNA-target concentration and into the sensitivity of each probe, which characterises its ability to detect a certain amount of RNA in microarray hybridisation experiments. The sensitivity decomposes into additive terms owing to specific and non-specific hybridisation, saturation, the heterogeneous distribution of labels and intramolecular folding of target and probe. We analysed microarray intensity data taken from Affymetrix GeneChips© within the light of the theoretical predictions. The effect of saturation and non-specific hybridisation are studied and correlated with the variability of probe sensitivity. The statistical error and the heterogeneous distribution of labels only slightly contribute to the observed variability of probe sensitivities. Instead, the observed heterogeneity is mainly caused by variations of the probe affinity for target binding owing to differences between the probes on the molecular level. The sensitivity values are therefore analysed in terms of simple molecular characteristics, which consider the base composition and sequence of the probes. We found that the mean sensitivity, averaged over all probes of a chip containing a certain number of bases of one type, strongly increases with increasing number of C residues per oligo whereas A nucleotides show the opposite tendency. These trends are asymmetrical with respect to the number of G and T, which have a much weaker and partly even the opposite effect in probes of intermediate and high sensitivity. Probes of weak sensitivity can be identified by their G/C (or T/A) content. The middle base systematically affects the relation between the sensitivities of perfect match (PM) and mismatch (MM) probes. MM probes with a self complementary (SC) base pair in the middle of their sequence are on the average more sensitive than the respective PM probes if their middle base is a purine. For pyrimidines this relation reverses. This purine-pyrimidine asymmetry is related to the effect of labelling. The presented results provide the starting point for the development of new sequence-related models of probe sensitivities with the potency to improve existing algorithms of gene expression analysis.
hybridisation theory, DNA/RNA duplex formation, binding affinity, folding and saturation, gene expression analysis, chip design
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