Arnaud Buhot

Sensitivity, Specificity, and the Hybridization Isotherms of DNA Chips

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.

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 c50 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.

Simple strong glass forming models: mean-field solution with activation

We introduce simple models, inspired by previous models for froths and covalent glasses, with trivial equilibrium properties but dynamical behaviour characteristic of strong glass forming systems. These models are also a generalization of backgammon or urn models to a non-constant number of particles, where entropic barriers are replaced by energy barriers, allowing for the existence of activated processes. We formulate a mean-field version of the models, which keeps most of the features of the finite dimensional ones, and solve analytically the out-of-equilibrium dynamics in the low temperature regime where activation plays an essential role.

Salt Concentration Effects on Equilibrium Melting Curves from DNA Microarrays

DNA microarrays find applications in an increasing number of domains where more quantitative results are required. DNA being a charged polymer, the repulsive interactions between the surface of the microarray and the targets in solution are increasing upon hybridization. Such electrostatic penalty is generally reduced by increasing the salt concentration. In this article, we present equilibrium-melting curves obtained from dedicated physicochemical experiments on DNA microarrays in order to get a better understanding of the electrostatic penalty incurred during the hybridization reaction at the surface. Various salt concentrations have been considered and deviations from the commonly used Langmuir adsorption model are experimentally quantified for the first time in agreement with theoretical predictions.

Physico-chemical foundations underpinning microarray and next-generation sequencing experiments

Hybridization of nucleic acids on solid surfaces is a key process involved in high-throughput technologies such as microarrays and, in some cases, next-generation sequencing (NGS). A physical understanding of the hybridization process helps to determine the accuracy of these technologies. The goal of a widespread research program is to develop reliable transformations between the raw signals reported by the technologies and individual molecular concentrations from an ensemble of nucleic acids. This research has inputs from many areas, from bioinformatics and biostatistics, to theoretical and experimental biochemistry and biophysics, to computer simulations. A group of leading researchers met in Ploen Germany in 2011 to discuss present knowledge and limitations of our physico-chemical understanding of high-throughput nucleic acid technologies. This meeting inspired us to write this summary, which provides an overview of the state-of-the-art approaches based on physico-chemical foundation to modeling of the nucleic acids hybridization process on solid surfaces. In addition, practical application of current knowledge is emphasized.

Point Mutation Detection by Surface Plasmon Resonance Imaging Coupled with a Temperature Scan Method in a Model System

The detection of point mutations in genes presents clear biological and medical interest. Various methods have been considered. In this paper, we take advantage of surface plasmon resonance imaging, a technique allowing detection of unlabeled DNA hybridization. Coupled with a temperature scan, this approach allows us to determine the presence of single-point mutations in oligonucleotide samples from the analysis of DNAs melting curves in either the homozygous or heterozygous case. Moreover, these experimental data are confirmed in good agreement with numerical calculations.

Numerical Solution of Hard-Core Mixtures

We study the equilibrium phase diagram of binary mixtures of hard spheres as well as of parallel hardcubes. A superior cluster algorithm allows us to establish and to access the demixed phase for bothsystems and to investigate the subtle interplay between short-range depletion and long-range demixing.[S0031-9007(98)05907-9]

Effects of formamide on the thermal stability of DNA duplexes on biochips

In molecular biology, formamide (FA) is a commonly used denaturing agent for DNA. Although its influence on DNA duplex stability in solution is well established, little is known about immobilized DNA on microarrays. We measured thermal denaturation curves for oligonucleotides immobilized by two standard protocols: thiol self-assembling and pyrrole electrospotting. A decrease of the DNA denaturation temperature with increasing FA fraction of the solvent was observed on sequences with mutations for both surface chemistries. The average dissociation temperature decrease was found to be -0.58+/-0.05 degrees C/% FA (v/v) independently of grafting chemistry and probe sequence.

Real time monitoring of thrombin interactions with its aptamers: Insights into the sandwich complex formation

Aptamers are raising an increasing interest for biosensor applications as replacements for antibodies due to their high stability and low cost. Thrombin, a key enzyme in the coagulation cascade, is an archetypical target against which two different aptamers, binding to two different exosites, have been selected. Recent studies dedicated to thrombin monitoring applications of biosensors have taken advantage of a potential sandwich-like structure between thrombin and these two aptamers for amplification purposes. However, in most cases, only end-point analysis was observed as a result of labeling requirements, thus preventing access to the kinetics of the complex formation. By using Surface Plasmon Resonance (SPR) imaging of aptamer-functionalized biosensors, we followed the binding of thrombin on the sensor and its interaction with a second reporter aptamer in real-time and in a label-free manner. Surprisingly, we showed that the injection of a second unlabeled-aptamer following the previous thrombin injection destabilized the thrombin-aptamer complex formed on the sensor surface, thus limiting any further amplification. However, the direct co-injection of thrombin, pre-complexed with a biotinylated aptamer bound to streptavidin efficiently increased the SPR signal by comparison to single thrombin detection. The various injection sequences performed may be rationalized considering a poor selectivity of one of the aptamers towards its exosite and a further negative allosteric effect upon sandwich complexation of the thrombin with its aptamers.

Effects of stacking on the configurations and elasticity of single-stranded nucleic acids

Stacking interactions in single-stranded nucleic acids give rise to configurations of an annealed rod-coil multiblock copolymer. Theoretical analysis identifies the following resulting signatures for long homopolynucleotides: a nonmonotonic dependence of size on temperature, the corresponding effects on cyclization and a plateau in the extension force law. Explicit numerical results for polydeoxyadenylate [poly(dA)] and polyriboadenylate [poly(rU)] are presented.