DNA hybridization to mismatched templates: A chip study (original) (raw)
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Thermodynamics of RNA/DNA hybridization in high-density oligonucleotide microarrays
Physica A: Statistical Mechanics and its Applications, 2006
We analyze a series of publicly available controlled experiments (Latin square) on Affymetrix high-density oligonucleotide microarrays using a simple physical model of the hybridization process. We plot for each gene the signal intensity vs. the hybridization free energy of RNA/DNA duplexes in solution, for perfect matching and mismatching probes. Both values tend to align on a single master curve in good agreement with Langmuir adsorption theory, provided one takes into account the decrease of the effective target concentration due to target-target hybridization in solution. We give an example of a deviation from the expected thermodynamical behavior for the probe set 1091_at due to annotation problems, i.e., the surface-bound probe is not the exact complement of the target RNA sequence, because of errors present in public databases at the time when the array was designed. We show that the parametrization of the experimental data with RNA/DNA free energy improves the quality of the fits and enhances the stability of the fitting parameters compared to previous studies. r (E. Carlon). grown in situ using photolitographic techniques. As a single probe of just 25 nucleotides may not provide enough specificity for a reliable measurement of the gene expression level, a set of 10-16 probes (the probe set) complementary to different regions of the same target sequence are present in the chip. For each perfect matching (PM) probe there is a sequence differing by a single nucleotide. These are referred to as mismatching (MM) probes and are used to quantify the effects of cross-hybridization .
On the hybridization isotherms of DNA microarrays: the Langmuir model and its extensions
The design of DNA chip experiments utilizes hybridization isotherms relating the equilibrium hybridization at the surface to the composition of the solution. Within this family, the Langmuir isotherm is the simplest and the most frequently used. This tutorial review summarizes the domain of validity of the Langmuir isotherm and discusses the modifications necessary to allow for competitive hybridization in the bulk and at the surface, probe polydispersity and interactions between the probe sites. The equilibrium constant of hybridization at an impenetrable surface is described, as well as the relative merits of the melting temperature and c 50 as design parameters. The relevance to various experimental situations, including two-colour experiments, study of point mutations for cancer diagnostics, genotyping of pooled samples and aspects of Latin square experiments, is discussed.
An improved physico-chemical model of hybridization on high-density oligonucleotide microarrays
Bioinformatics, 2008
Motivation: High-density DNA microarrays provide useful tools to analyze gene expression comprehensively. However, it is still difficult to obtain accurate expression levels from the observed microarray data because the signal intensity is affected by complicated factors involving probe-target hybridization, such as non-linear behavior of hybridization, non-specific hybridization, and folding of probe and target oligonucleotides. Various methods for microarray data analysis have been proposed to address this problem. In our previous report, we presented a benchmark analysis of probe-target hybridization using artificially synthesized oligonucleotides as targets, in which the effect of non-specific hybridization was negligible. The results showed that the preceding models explained the behavior of probetarget hybridization only within a narrow range of target concentrations. More accurate models are required for quantitative expression analysis.
Sensitivity, Specificity, and the Hybridization Isotherms of DNA Chips
Biophysical Journal, 2004
Competitive hybridization, at the surface and in the bulk, lowers the sensitivity of DNA chips. Competitive surface hybridization occurs when different targets can hybridize with the same probe. Competitive bulk hybridization takes place when the targets can hybridize with free complementary chains in the solution. The effects of competitive hybridization on the thermodynamically attainable performance of DNA chips are quantified in terms of the hybridization isotherms of the spots. These relate the equilibrium degree of the hybridization to the bulk composition. The hybridization isotherm emerges as a Langmuir isotherm modified for electrostatic interactions within the probe layer. The sensitivity of the assay in equilibrium is directly related to the slope of the isotherm. A simpler description is possible, in terms of c 50 values specifying the bulk composition corresponding to 50% hybridization at the surface. The effects of competitive hybridization are important for the quantitative analysis of DNA chip results, especially when used to study point mutations.
Hybridization to surface-bound oligonucleotide probes: Influence of point defects
Microarray-based genotyping is based on the high discrimination capability of oligonucleotide probes. For detection of Single Nucleotide Polymorphisms (SNPs) single-base discrimination is required. We investigate how various point-mutations, comprising single base mismatches (MMs), insertions and deletions, affect hybridization of DNA-DNA oligonucleotide duplexes. Employing light-directed in situ synthesis we fabricate DNA microarrays with comprehensive sets of cognate point-mutated probes, allowing us to systematically investigate the influence of defect type, position and nearest neighbor effects. Defect position has been identified as the dominating influential factor. This positional effect which is almost identical for the different point-mutation types, is biased from the local sequence environment. The impact of the MM type is largely determined by the type of base pair (either AT or CG) affected by the mismatch. We observe that single base insertions next to like-bases resul...
BioTechniques, 2005
The hybridization behavior of small oligonucleotides arrayed on glass slides is currently unpredictable. In order to examine the hybridization efficiency of capture probes along target nucleic acid, 20-mer oligonucleotide probes were designed to hybridize at different distances from the 5' end of two overlapping 402- and 432-bp ermB products amplified from the target DNA. These probes were immobilized via their 5' end onto glass slides and hybridized with the two labeled products. Evaluation of the hybridization signal for each probe revealed an inverse correlation with the length of the 5' overhanging end of the captured strand and the hybridization signal intensity. Further experiments demonstrated that this phenomenon is dependent on the reassociation kinetics of the free overhanging tail of the captured DNA strand with its complementary strand. This study delineates key predictable parameters that govern the hybridization efficiency of short capture probes arrayed on glass slides. This should be most useful for designing arrays for detection of PCR products and nucleotide polymorphisms.
Nucleic Acids Research, 2006
A key issue in applications of short oligonucleotidebased 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 singlestranded 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.
Molecular Modeling of DNA for a Mechanistic Understanding of Hybridization
2013
DNA microarrays are a potentially disruptive technology in the medical field, but their use in such settings is limited by poor reliability. Microarrays work on the principle of hybridization and can only be as reliable as this process is robust, yet little is known at the molecular level about how the surface affects the hybridization process. This work uses advanced molecular simulation techniques and an experimentally-parameterized coarsegrain model to determine the mechanism by which hybridization occurs on surfaces and to identify key factors that influence the accuracy of DNA microarrays. Comparing behavior in the bulk and on the surface showed, contrary to previous assumptions, that hybridization on surfaces is more energetically favorable than in the bulk. The results also show that hybridization proceeds through a mechanism where the untethered (target) strand often flips orientation. For evenly-lengthed strands, the surface stabilizes hybridization (compared to the bulk system) by reducing the barriers involved in the flipping event. Additional factors were also investigated, including the effects of stretching or compressing the probe strand as a model system to test the hypothesis that improving surface hybridization will improve microarray performance. The results in this regard indicate that selectivity can be increased by reducing overall sensitivity by a small degree. Another factor that was investigated was the effect of unevenly-lengthed strands. It was found that, when unevenly-lengthed strands were hybridized on a surface, the surface may destabilize hybridization compared to the bulk, but the degree of destabilization is dependent on the location of the matching sequence. Taken as a whole, the results offer an unprecedented view into the hybridization process on surfaces and provide some insights as to the poor reproducibility exhibited by microarrays. Namely, the prediction methods that are currently used to design microarrays based on duplex stability in the bulk do a poor job of estimating the stability of those duplexes in a microarray environment.
Kinetics of oligonucleotide hybridization to photolithographically patterned DNA arrays
Analytical Biochemistry, 2006
The hybridization kinetics of oligonucleotide targets to oligonucleotide probe arrays synthesized using photolithographic fabrication methods developed by Affymetrix have been measured. Values for the fundamental adsorption parameters, k a , k d , and K, were determined at both room temperature and 45°C by monitoring the hybridization of fluorescently labeled targets to the array. The values for these parameters and the adsorbed target density (61 pmol/cm 2 at saturation) agree relatively well with published values for arrays fabricated by immobilizing intact probes. The isotherms can be fit well with the Sips model, a generalization of the Langmuir model that allows for multiple binding energies. However, binding to these arrays also displays certain characteristics that may result from the close spacing of probes on the array. At high target concentrations and 22°C, an ''overshoot'' is observed, wherein a large amount of target binds rapidly and then desorbs to a final plateau. This binding mode may be a result of the unique nature of photolithographically patterned arrays given that targets initially can bind in lower stability binding modes by partially adsorbing to a probe and its neighbor(s), a process that can be viewed as a form of competitive hybridization for overlapping sites on a given probe. Using the values measured for k a and k d , an analytical model that accounts for this behavior is proposed. Alternatively, at 45°C, the adsorption approaches an initial plateau, rather than an overshoot, and then undergoes a ''secondary rise'' to a final value. A potential explanation for this phenomenon that is compatible with the analysis at lower temperature is offered.