11,848 research outputs found
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
Real-time DNA microarray analysis
We present a quantification method for affinity-based
DNA microarrays which is based on the
real-time measurements of hybridization kinetics.
This method, i.e. real-time DNA microarrays,
enhances the detection dynamic range of conventional
systems by being impervious to probe
saturation in the capturing spots, washing
artifacts, microarray spot-to-spot variations, and
other signal amplitude-affecting non-idealities. We
demonstrate in both theory and practice that the
time-constant of target capturing in microarrays,
similar to all affinity-based biosensors, is inversely
proportional to the concentration of the target
analyte, which we subsequently use as the fundamental
parameter to estimate the concentration
of the analytes. Furthermore, to empirically
validate the capabilities of this method in practical
applications, we present a FRET-based assay which
enables the real-time detection in gene expression
DNA microarrays
Microarray-based ultra-high resolution discovery of genomic deletion mutations
BACKGROUND: Oligonucleotide microarray-based comparative genomic hybridization (CGH) offers an attractive possible route for the rapid and cost-effective genome-wide discovery of deletion mutations. CGH typically involves comparison of the hybridization intensities of genomic DNA samples with microarray chip representations of entire genomes, and has widespread potential application in experimental research and medical diagnostics. However, the power to detect small deletions is low. RESULTS: Here we use a graduated series of Arabidopsis thaliana genomic deletion mutations (of sizes ranging from 4 bp to ~5 kb) to optimize CGH-based genomic deletion detection. We show that the power to detect smaller deletions (4, 28 and 104 bp) depends upon oligonucleotide density (essentially the number of genome-representative oligonucleotides on the microarray chip), and determine the oligonucleotide spacings necessary to guarantee detection of deletions of specified size. CONCLUSIONS: Our findings will enhance a wide range of research and clinical applications, and in particular will aid in the discovery of genomic deletions in the absence of a priori knowledge of their existence
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Aptamers in oncology: a diagnostic perspective
Nucleic acid sequences can produce a wide variety of three-dimensional conformations. Some of these structural forms are able to interact with proteins and small molecules with high affinity and specificity. These sequences, comprising either double or single stranded oligonucleotides, are called 'aptamers' based on the Greek word aptus, which means 'to fit'. Using an efficient selection process, randomised oligonucleotide libraries can be rapidly screened for aptamers with the appropriate binding characteristics. This technology has spawned the development of a new class of oligonucleotide therapeutic products. However, while interest among pharmaceutical companies continues to grow with some candidates already in clinical trials and one in the market, there appears to be some reluctance to fully explore the diagnostic potential of this technology. This article will review aptamer developments in diagnostics, compare them with other oligonucleotide therapeutics and highlight both potentials and pitfalls of technological development in this area
Linear model for fast background subtraction in oligonucleotide microarrays
One important preprocessing step in the analysis of microarray data is
background subtraction. In high-density oligonucleotide arrays this is
recognized as a crucial step for the global performance of the data analysis
from raw intensities to expression values.
We propose here an algorithm for background estimation based on a model in
which the cost function is quadratic in a set of fitting parameters such that
minimization can be performed through linear algebra. The model incorporates
two effects: 1) Correlated intensities between neighboring features in the chip
and 2) sequence-dependent affinities for non-specific hybridization fitted by
an extended nearest-neighbor model.
The algorithm has been tested on 360 GeneChips from publicly available data
of recent expression experiments. The algorithm is fast and accurate. Strong
correlations between the fitted values for different experiments as well as
between the free-energy parameters and their counterparts in aqueous solution
indicate that the model captures a significant part of the underlying physical
chemistry.Comment: 21 pages, 5 figure
Specific discrimination of three pathogenic salmonella enterica subsp enterica serotypes using CarB-based oligonuceotide microarray
It is important to rapidly and selectively detect and analyze pathogenic Salmonella enterica subsp. enterica in contaminated food to reduce the morbidity and mortality of Salmonella infection and to guarantee food safety. In the present work, we developed an oligonucleotide microarray containing duplicate specific capture probes based on the carB gene, which encodes the carbamoyl phosphate synthetase large subunit, as a competent biomarker evaluated by genetic analysis to selectively and efficiently detect and discriminate three S. enterica subsp. enterica serotypes: Choleraesuis, Enteritidis, and Typhimurium. Using the developed microarray system, three serotype targets were successfully analyzed in a range as low as 1.6 to 3.1 nM and were specifically discriminated from each other without nonspecific signals. In addition, the constructed microarray did not have cross-reactivity with other common pathogenic bacteria and even enabled the clear discrimination of the target Salmonella serotype from a bacterial mixture. Therefore, these results demonstrated that our novel carB-based oligonucleotide microarray can be used as an effective and specific detection system for S. enterica subsp. enterica serotypes.open117Nsciescopu
Probing Hybridization parameters from microarray experiments: nearest neighbor model and beyond
In this article it is shown how optimized and dedicated microarray
experiments can be used to study the thermodynamics of DNA hybridization for a
large number of different conformations in a highly parallel fashion. In
particular, free energy penalties for mismatches are obtained in two
independent ways and are shown to be correlated with values from melting
experiments in solution reported in the literature. The additivity principle,
which is at the basis of the nearest-neighbor model, and according to which the
penalty for two isolated mismatches is equal to the sum of the independent
penalties, is thoroughly tested. Additivity is shown to break down for a
mismatch distance below 5 nt. The behavior of mismatches in the vicinity of the
helix edges, and the behavior of tandem mismatches are also investigated.
Finally, some thermodynamic outlying sequences are observed and highlighted.
These sequences contain combinations of GA mismatches. The analysis of the
microarray data reported in this article provides new insights on the DNA
hybridization parameters and can help to increase the accuracy of
hybridization-based technologies.Comment: 13 pages, 11 figures, 1 table, Supplementary Data available in
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