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Specific and nonspecific hybridization of oligonucleotide probes on microarrays

Item Type:Article
Title:Specific and nonspecific hybridization of oligonucleotide probes on microarrays
Creators Name:Binder, H. and Preibisch, S.
Abstract:Gene expression analysis by means of microarrays is based on the sequence-specific binding of RNA to DNA oligonucleotide probes and its measurement using fluorescent labels. The binding of RNA fragments involving sequences other 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 article presents a molecular signature of specific and nonspecific 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 nonspecific 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 PM, namely a triplet-like (C > G approximately T > A > 0) and a duplet-like (C approximately T > 0 > G approximately A) pattern of the PM-MM log-intensity difference upon binding of specific and nonspecific RNA fragments, respectively. The systematic behavior of the intensity difference can be rationalized on the level of basepairings of DNA/RNA oligonucleotide duplexes in the middle of the probe sequence. Nonspecific binding is characterized by the reversal of the central Watson-Crick (WC) pairing for each PM/MM probe pair, whereas specific binding refers to the combination of a WC and a self-complementary (SC) pairing in PM and MM probes, respectively. The Gibbs free energy contribution of WC pairs to duplex stability is asymmetric for purines and pyrimidines of the PM and decreases according to C > G approximately T > A. SC pairings on the average only weakly contribute to duplex stability. The intensity of complementary MM introduces a systematic source of variation which decreases the precision of expression measures based on the MM intensities.
Keywords:Biophysics, DNA, DNA Probes, Drug Dose-Response Relationship, Gene Expression Regulation, Genetic Hybridization, Genetic Techniques, HeLa Cells, Nucleic Acid Hybridization, Oligonucleotide Array Sequence Analysis, Oligonucleotide Probes, Oligonucleotides, RNA, Statistical Models
Source:Biophysical Journal
Publisher:Biophysical Society
Page Range:337-352
Date:July 2005
Official Publication:https://doi.org/10.1529/biophysj.104.055343
PubMed:View item in PubMed

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