Adsorption models of hybridization and post-hybridization behaviour on oligonucleotide microarrays

Burden, Conrad J.; Pittelkow, Yvonne; Wilson, Susan R.

doi:10.1088/0953-8984/18/23/024

Cited by 41 publications

(88 citation statements)

References 28 publications

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“…We assume in agreement with previous studies [8,9] that the dissociation rate is related to the stability of the respective duplex in terms its free energy of probe/target hybridization, which in turn depends on the equilibrium binding constant introduced in Eq. (1), ΔG P,h = -RT·lnK P,h (R is the gas constant), i.e.…”

Section: Resultssupporting

confidence: 72%

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Washing scaling of GeneChip microarray expression

2010

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BackgroundPost-hybridization washing is an essential part of microarray experiments. Both the quality of the experimental washing protocol and adequate consideration of washing in intensity calibration ultimately affect the quality of the expression estimates extracted from the microarray intensities.ResultsWe conducted experiments on GeneChip microarrays with altered protocols for washing, scanning and staining to study the probe-level intensity changes as a function of the number of washing cycles. For calibration and analysis of the intensity data we make use of the 'hook' method which allows intensity contributions due to non-specific and specific hybridization of perfect match (PM) and mismatch (MM) probes to be disentangled in a sequence specific manner. On average, washing according to the standard protocol removes about 90% of the non-specific background and about 30-50% and less than 10% of the specific targets from the MM and PM, respectively. Analysis of the washing kinetics shows that the signal-to-noise ratio doubles roughly every ten stringent washing cycles. Washing can be characterized by time-dependent rate constants which reflect the heterogeneous character of target binding to microarray probes. We propose an empirical washing function which estimates the survival of probe bound targets. It depends on the intensity contribution due to specific and non-specific hybridization per probe which can be estimated for each probe using existing methods. The washing function allows probe intensities to be calibrated for the effect of washing. On a relative scale, proper calibration for washing markedly increases expression measures, especially in the limit of small and large values.ConclusionsWashing is among the factors which potentially distort expression measures. The proposed first-order correction method allows direct implementation in existing calibration algorithms for microarray data. We provide an experimental 'washing data set' which might be used by the community for developing amendments of the washing correction.

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Section: Resultssupporting

confidence: 72%

“…Furthermore, theoretical approaches have been published which explicitly take into account washing and introduced 'washing terms' to improve the agreement with experimental spiked-in data [6-9]. Spike-in experiments typically provide the intensities of a set of selected probes as a function of their target concentration.…”

Section: Introductionmentioning

confidence: 99%

Washing scaling of GeneChip microarray expression

2010

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“…According to the simple Langmuir model, signal response to concentration should saturate at the same level for all probes. However, as shown in Figure 1, it is apparent that amongst different probes, there is up to a 30-fold difference in saturation levels and variation in these levels was found to correlate with probe sequences (7,8). …”

Section: Introductionmentioning

confidence: 97%

Explaining differences in saturation levels for Affymetrix GeneChip® arrays

Skvortsov

Abdueva

Curtis

et al. 2007

Nucleic Acids Research

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The experimental spike-in studies of microarray hybridization conducted by Affymetrix demonstrate a nonlinear response of fluorescence intensity signal to target concentration. Several theoretical models have been put forward to explain these data. It was shown that the Langmuir adsorption isotherm recapitulates a general trend of signal response to concentration. However, this model fails to explain some key properties of the observed signal. In particular, according to the simple Langmuir isotherm, all probes should saturate at the same intensity level. However, this effect was not observed in the publicly available Affymetrix spike-in data sets. On the contrary, it was found that the saturation intensities vary greatly and can be predicted based on the probe sequence composition. In our experimental study, we attempt to account for the unexplained variation in the observed probe intensities using customized fluidics scripts. We explore experimentally the effect of the stringent wash, target concentration and hybridization time on the final microarray signal. The washing effect is assessed by scanning chips both prior to and after the stringent wash. Selective labeling of both specific and non-specific targets allows the visualization and investigation of the washing effect for both specific and non-specific signal components. We propose a new qualitative model of the probe-target hybridization mechanism that is in agreement with observed hybridization and washing properties of short oligonucleotide microarrays. This study demonstrates that desorption of incompletely bound targets during the washing cycle contributes to the observed difference in saturation levels.

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“…As summarized in several recent reviews (5)(6)(7)(8), physical studies under simplified experimental conditions have begun to unravel the rich phenomenology that occurs in diagnostic assays. Applications typically operate away from equilibrium and are faced with a highly diverse pool of target sequences competing for the probe sites.…”

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confidence: 99%

DNA surface hybridization regimes

Gong

Levicky²

2008

Proc. Natl. Acad. Sci. U.S.A.

224

282

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Surface hybridization reactions, in which sequence-specific recognition occurs between immobilized and solution nucleic acids, are routinely carried out to quantify and interpret genomic information. Although hybridization is fairly well understood in bulk solution, the greater complexity of an interfacial environment presents new challenges to a fundamental understanding, and hence application, of these assays. At a surface, molecular interactions are amplified by the two-dimensional nature of the immobilized layer, which focuses the nucleic acid charge and concentration to levels not encountered in solution, and which impacts the hybridization behavior in unique ways. This study finds that, at low ionic strengths, an electrostatic balance between the concentration of immobilized oligonucleotide charge and solution ionic strength governs the onset of hybridization. As ionic strength increases, the importance of electrostatics diminishes and the hybridization behavior becomes more complex. Suppression of hybridization affinity constants relative to solution values, and their weakened dependence on the concentration of DNA counterions, indicate that the immobilized strands form complexes that compete with hybridization to analyte strands. Moreover, an unusual regime is observed in which the surface coverage of immobilized oligonucleotides does not significantly influence the hybridization behavior, despite physical closeness and hence compulsory interactions between sites. These results are interpreted and summarized in a diagram of hybridization regimes that maps specific behaviors to experimental ranges of ionic strength and probe coverage.biosensor ͉ ferrocene ͉ ionic strength ͉ microarray ͉ probe coverage S olid-phase or ''surface'' hybridization is the foundation of modern microarray and biosensing technologies widely used in applied genomics for genotyping, drug discovery, gene expression profiling, and related applications based on measurement of genomic information (1, 2). These tools function through detection of interactions between nucleic acids immobilized on a solid support, or ''probes,'' with analyte ''target'' nucleic acids present in solution. Binding, or hybridization, between probes and targets to form an immobilized duplex takes place based on the degree of complementarity between the probe and target base sequences. The tremendous growth in applications of surface hybridization has been mirrorred by increased emphasis on experimental and fundamental aspects of the assays that directly impact measurement and interpretation of data (3, 4).As summarized in several recent reviews (5-8), physical studies under simplified experimental conditions have begun to unravel the rich phenomenology that occurs in diagnostic assays. Applications typically operate away from equilibrium and are faced with a highly diverse pool of target sequences competing for the probe sites. A variety of aids to enhance hybridization performance is used, including surfactants and blocking agents to control nonspecific adsorp...

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Adsorption models of hybridization and post-hybridization behaviour on oligonucleotide microarrays

Cited by 41 publications

References 28 publications

Washing scaling of GeneChip microarray expression

Washing scaling of GeneChip microarray expression

Explaining differences in saturation levels for Affymetrix GeneChip® arrays

DNA surface hybridization regimes

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