83 research outputs found
Nonequilibrium effects in DNA microarrays: a multiplatform study
It has recently been shown that in some DNA microarrays the time needed to
reach thermal equilibrium may largely exceed the typical experimental time,
which is about 15h in standard protocols (Hooyberghs et al. Phys. Rev. E 81,
012901 (2010)). In this paper we discuss how this breakdown of thermodynamic
equilibrium could be detected in microarray experiments without resorting to
real time hybridization data, which are difficult to implement in standard
experimental conditions. The method is based on the analysis of the
distribution of fluorescence intensities I from different spots for probes
carrying base mismatches. In thermal equilibrium and at sufficiently low
concentrations, log I is expected to be linearly related to the hybridization
free energy with a slope equal to , where is
the experimental temperature and R is the gas constant. The breakdown of
equilibrium results in the deviation from this law. A model for hybridization
kinetics explaining the observed experimental behavior is discussed, the
so-called 3-state model. It predicts that deviations from equilibrium yield a
proportionality of to . Here, is an
effective temperature, higher than the experimental one. This behavior is
indeed observed in some experiments on Agilent arrays. We analyze experimental
data from two other microarray platforms and discuss, on the basis of the
results, the attainment of equilibrium in these cases. Interestingly, the same
3-state model predicts a (dynamical) saturation of the signal at values below
the expected one at equilibrium.Comment: 27 pages, 9 figures, 1 tabl
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
Arrested spinodal decomposition in polymer brush collapsing in poor solvent
We study the Brownian dynamics of flexible and semiflexible polymer chains
densely grafted on a flat substrate, upon rapid quenching of the system when
the quality of solvent becomes poor and chains attempt collapse into a globular
state. The collapse process of such a polymer brush differs from individual
chains, both in its kinetics and its structural morphology. We find that the
resulting collapsed brush does not form a homogeneous dense layer, in spite of
all chain monomers equally attracting each other via a model Lennard-Jones
potential. Instead, a very distinct inhomogeneous density distribution in the
plane forms, with a characteristic length scale dependent on the quenching
depth (or equivalently, the strength of monomer attraction) and the geometric
parameters of the brush. This structure is identical to the
spinodal-decomposition structure, however, due to the grafting constraint we
find no subsequent coarsening: the established random bundling with
characteristic periodicity remains as the apparently equilibrium structure. We
compare this finding with a recent field-theoretical model of bundling in a
semiflexible polymer brush.This work was funded by the Osk. Huttunen Foundation (Finland) and the Cambridge Theory of Condensed Matter Grant from EPSRC. Simulations were performed using the Darwin supercomputer of the University of Cambridge High Performance Computing Service provided by Dell Inc. using Strategic Research Infrastructure funding from the Higher Education Funding Council for England.This is the accepted manuscript. The final version is available at http://pubs.acs.org/doi/abs/10.1021/ma501985r
Immobilization of nucleic acids at solid surfaces: effect of oligonucleotide length on layer assembly.
This report investigates the effect of DNA length and the presence of an anchoring group on the assembly of presynthesized oligonucleotides at a gold surface. The work seeks to advance fundamental insight into issues that impact the structure and behavior of surface-immobilized DNA layers, as in, for instance, DNA microarray and biosensor devices. The present study contrasts immobilization of single-stranded DNA (ssDNA) containing a terminal, 5' hexanethiol anchoring group with that of unfunctionalized oligonucleotides for lengths from 8 to 48 bases. Qualitatively, the results indicate that the thiol anchoring group strongly enhances oligonucleotide immobilization, but that the enhancement is reduced for longer strand lengths. Interestingly, examination of the probe coverage as a function of strand length suggests that adsorbed thiol-ssDNA oligonucleotides shorter than 24 bases tend to organize in end-tethered, highly extended configurations for which the long-term surface coverage is largely independent of oligonucleotide length. For strands longer than 24 bases, the surface coverage begins to decrease notably with probe length. The decrease is consistent with a less ordered arrangement of the DNA chains, presumably reflecting increasingly polymeric behavior
Controlled and Efficient Hybridization Achieved with DNA Probes Immobilized Solely through Preferential DNA-Substrate Interactions
Reliable microspotting methodology for peptide-nucleic acid layers with high hybridization efficiency on gold SPR imaging chips
Drop drying on surfaces determines chemical reactivity - the specific case of immobilization of oligonucleotides on microarrays
BACKGROUND: Drop drying is a key factor in a wide range of technical applications, including spotted microarrays. The applied nL liquid volume provides specific reaction conditions for the immobilization of probe molecules to a chemically modified surface. RESULTS: We investigated the influence of nL and μL liquid drop volumes on the process of probe immobilization and compare the results obtained to the situation in liquid solution. In our data, we observe a strong relationship between drop drying effects on immobilization and surface chemistry. In this work, we present results on the immobilization of dye labeled 20mer oligonucleotides with and without an activating 5'-aminoheptyl linker onto a 2D epoxysilane and a 3D NHS activated hydrogel surface. CONCLUSIONS: Our experiments identified two basic processes determining immobilization. First, the rate of drop drying that depends on the drop volume and the ambient relative humidity. Oligonucleotides in a dried spot react unspecifically with the surface and long reaction times are needed. 3D hydrogel surfaces allow for immobilization in a liquid environment under diffusive conditions. Here, oligonucleotide immobilization is much faster and a specific reaction with the reactive linker group is observed. Second, the effect of increasing probe concentration as a result of drop drying. On a 3D hydrogel, the increasing concentration of probe molecules in nL spotting volumes accelerates immobilization dramatically. In case of μL volumes, immobilization depends on whether the drop is allowed to dry completely. At non-drying conditions, very limited immobilization is observed due to the low oligonucleotide concentration used in microarray spotting solutions. The results of our study provide a general guideline for microarray assay development. They allow for the initial definition and further optimization of reaction conditions for the immobilization of oligonucleotides and other probe molecule classes to different surfaces in dependence of the applied spotting and reaction volume
On the Nature of DNA Self-Assembled Monolayers on Au: Measuring Surface Heterogeneity with Electrochemical in Situ Fluorescence Microscopy
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