Evaluation of hybridization conditions for spotted oligonucleotide-based DNA microarrays

We compared different hybridization conditions of oligonucleotide-based DNA microarray to acquire optimized and reliable microarray data. Several parameters were evaluated at different hybridization conditions, including signal-to-background (S:B) ratios, signal dynamic range, usable spots, and reproducibility. Statistical analysis showed that better results were obtained when spotted, presynthesized long oligonucleotide arrays were blocked with succinic anhydride and hybridized at 42°C in the presence of 50% formamide.     

Innovative instrumentation for microarray scanning and analysis: application for characterization of oligonucleotide duplexes behavior.

Accuracy in microarray technology requires new approaches to microarray reader development. A microarray reader system (optical scanning array or OSA reader) based on automated microscopy with large field of view, high speed 3 axis scanning at multiple narrow-band spectra of excitation light has been developed. It allows fast capture of high-resolution, multi-fluorescence images and is characterized by a linear dynamic range and sensitivity comparable to commonly used photo-multiplier tube (PMT)-based laser scanner. Controlled by high performance software, the instrument can be used for scanning and quantitative analysis of any type of dry microarray. Studies implying temperature-controlled hybridization chamber containing a microarray can also be performed. This enables the registration of kinetics and melting curves. This feature is required in a wide range of on-chip chemical and enzymatic reactions including on-chip PCR amplification. We used the OSA reader for the characterization of hybridization and melting behaviour of oligonucleotide:oligonucleotide duplexes on three-dimensional Code Link slides.     

Kinetics of multiplex hybridization: mechanisms and implications

Quantitative analysis of DNA microarray data is complicated by uncertainties inherent to the experimental setup. Using computer simulations and real-time experimental results, we have previously demonstrated effects of multiplex reactions on a single sensing zone of an array, which may be a leading factor in erroneous interpretation of experimental data. We suggest here that a simplified three-component kinetic model may present a sufficient approximation to describe the general case of DNA sensing in a complex sample milieu. We show that, by analyzing the real-time hybridization kinetics of a nontarget species, we can perform quantitative analysis of unlabeled targets of interest within a broad dynamic range of concentrations.     

Conditions to ensure competitive hybridization in two-color microarray: a theoretical and experimental analysis

We derived a theoretical model that explains certain biases observed in the two-color microarray hybridization experiments reported in the literature. We show that true competition is achieved only when the hybridization kinetics of the two differentially labeled probes are the same. If the hybridization kinetics of the two differentially labeled probes is different, which can occur when the labeling and hybridization conditions for the two probes are dissimilar, then differential expression observed becomes a function of the amount of the target (i.e., DNA spotted on the slide). We use this model to validate the microarray methodology by determining the differential expression of four select Arabidopsis genes and two human genes (beta-actin and GAPDH) as a function of the amount of target arrayed. We show through both modeling and experiments that the rate constants for Cy5- and Cy3-labeled probes are the same under our exrimental conditions. Therefore, the target concentrations need not greatly exceed the probe concentration. It is obvious from the data presented that a simple treatment of an individual hybridization rate calculation does notfully describe what is occuring in today's complex, multispecies experiments. The method of validation is easily implemented to ensure data reliability by two-color microarray.     

The effects of mismatches on hybridization in DNA microarrays: determination of nearest neighbor parameters

Quantifying interactions in DNA microarrays is of central importance for a better understanding of their functioning. Hybridization thermodynamics for nucleic acid strands in aqueous solution can be described by the so-called nearest neighbor model, which estimates the hybridization free energy of a given sequence as a sum of dinucleotide terms. Compared with its solution counterparts, hybridization in DNA microarrays may be hindered due to the presence of a solid surface and of a high density of DNA strands. We present here a study aimed at the determination of hybridization free energies in DNA microarrays. Experiments are performed on custom Agilent slides. The solution contains a single oligonucleotide. The microarray contains spots with a perfect matching (PM) complementary sequence and other spots with one or two mismatches (MM) : in total 1006 different probe spots, each replicated 15 times per microarray. The free energy parameters are directly fitted from microarray data. The experiments demonstrate a clear correlation between hybridization free energies in the microarray and in solution. The experiments are fully consistent with the Langmuir model at low intensities, but show a clear deviation at intermediate (non-saturating) intensities. These results provide new interesting insights for the quantification of molecular interactions in DNA microarrays.     

Single-Stranded DNA Catalyzes Hybridization of PCR-Products to Microarray Capture Probes

Since its development, microarray technology has evolved to a standard method in the biotechnological and medical field with a broad range of applications. Nevertheless, the underlying mechanism of the hybridization process of PCR-products to microarray capture probes is still not completely understood, and several observed phenomena cannot be explained with current models. We investigated the influence of several parameters on the hybridization reaction and identified ssDNA to play a major role in the process. An increase of the ssDNA content in a hybridization reaction strongly enhanced resulting signal intensities. A strong influence could also be observed when unlabeled ssDNA was added to the hybridization reaction. A reduction of the ssDNA content resulted in a massive decrease of the hybridization efficiency. According to these data, we developed a novel model for the hybridization mechanism. This model is based on the assumption that single stranded DNA is necessary as catalyst to induce the hybridization of dsDNA. The developed hybridization model is capable of giving explanations for several yet unresolved questions regarding the functionality of microarrays. Our findings not only deepen the understanding of the hybridization process, but also have immediate practical use in data interpretation and the development of new microarrays.     

Non-linear analysis of GeneChip arrays

The application of microarray hybridization theory to Affymetrix GeneChip data has been a recent focus for data analysts. It has been shown that the hyperbolic Langmuir isotherm captures the shape of the signal response to concentration of Affymetrix GeneChips. We demonstrate that existing linear fit methods for extracting gene expression measures are not well adapted for the effect of saturation resulting from surface adsorption processes. In contrast to the most popular methods, we fit background and concentration parameters within a single global fitting routine instead of estimating the background before obtaining gene expression measures. We describe a non-linear multi-chip model of the perfect match signal that effectively allows for the separation of specific and non-specific components of the microarray signal and avoids saturation bias in the high-intensity range. Multimodel inference, incorporated within the fitting routine, allows a quantitative selection of the model that best describes the observed data. The performance of this method is evaluated on publicly available datasets, and comparisons to popular algorithms are presented.     

Theoretically modeling microarray with the chemical equilibrium and thermodynamics

To date, the idea that microarray may shed the light on cellular processes by identifying groups of genes that appear to be co-expressed seems to remain a dream. This is partly because that there are some blank (meaning the knowledge is unavailable) or even erroneous areas in the fundamental theory in this field. This paper attempts to present the digest of microarray hybridization system with chemical thermodynamics, theoretically clarifying some misunderstandings and looking for answers to some critical questions around this technology, such as the mechanisms and conditions of quantitative measuring by hybridization reaction, the reasons of inconsistency of the data and the analysis results and the solutions, how to analyze the data, etc. A theoretical model for the next generation of microarray is proposed. We believe that this model is universal, laying the foundation for microarray technology from array design through the data analysis.     

The Potential of Genomic Approaches to Rotifer Ecology

Rotifers are a key component of many freshwater ecosystems, but surveys of the composition of rotifer communities are limited by the labor-intensiveness of sample processing, particularly of non-planktonic taxa, and by the shortage of investigators qualified to identify a broad range of rotifer species. Additional problems are posed by species that must be identified from living specimens, and by members of cryptic species complexes. As DNA sequencing becomes easier and cheaper, it has become practical to obtain representative DNA sequences from identified rotifer species for use in genome-based surveys to determine which rotifers are present in a new sample, avoiding the difficulties of traditional surveys. Here we discuss two genome-based tools used in surveys of microbial communities: serial analysis of gene tags (SAGT) and microarray hybridization. SAGT is a method for inexpensively obtaining characteristic short DNA sequences from a sample that can both identify taxa for which the tag sequence is known and signal the presence of additional uncharacterized species. Microarray hybridization allows detection of DNA sequences in the sample that are identical or similar to sequences present on the microarray. We also report the construction and hybridization of a small microarray of rotifer sequences, demonstrating that this method can discriminate among bdelloid families, and is likely to make much finer discriminations if appropriate sequences are present on the microarray. These techniques are most powerful when combined with traditional systematics in collaborative efforts, which may be fostered through the data base of rotifer biology, WheelBase (http://jbpc.mbl.edu/wheelbase).     

Cooperativity of paired oligonucleotide probes for microarray hybridization assays

Synthetic DNA probes attached to microarrays usually range in length from 25 to 70 nucleotides. There is a compromise between short probes with lower sensitivity, which can be accurately synthesized in higher yields, and long probes with greater sensitivity but lower synthesis yields. Described here are microarrays printed with spots containing a mixture of two short probes, each designed to hybridize at noncontiguous sites in the same targeted sequence. We have shown that, for a printed microarray, mixed probe spots containing a pair of 30mers show significantly greater hybridization than spots containing a single 30mer and can approach the amount of hybridization to spots containing a 60mer or a 70mer. These spots with mixed oligonucleotide probes display cooperative hybridization signals greater than those that can be achieved by either probe alone. Both the higher synthesis yields of short probes and the greater sensitivity of long oligonucleotides can be utilized. This strategy provides new design options for microarray hybridization assays to detect RNA abundance, RNA splice variants, or sequence polymorphisms.     

GenXHC: a probabilistic generative model for cross-hybridization compensation in high-density genome-wide microarray data

MOTIVATION: Microarray designs containing millions to hundreds of millions of probes that tile entire genomes are currently being released. Within the next 2 months, our group will release a microarray data set containing over 12,000,000 microarray measurements taken from 37 mouse tissues. A problem that will become increasingly significant in the upcoming era of genome-wide exon-tiling microarray experiments is the removal of cross-hybridization noise. We present a probabilistic generative model for cross-hybridization in microarray data and a corresponding variational learning method for cross-hybridization compensation, GenXHC, that reduces cross-hybridization noise by taking into account multiple sources for each mRNA expression level measurement, as well as prior knowledge of hybridization similarities between the nucleotide sequences of microarray probes and their target cDNAs. RESULTS: The algorithm is applied to a subset of an exon-resolution genome-wide Agilent microarray data set for chromosome 16 of Mus musculus and is found to produce statistically significant reductions in cross-hybridization noise. The denoised data is found to produce enrichment in multiple gene ontology-biological process (GO-BP) functional groups. The algorithm is found to outperform robust multi-array analysis, another method for cross-hybridization compensation.     

Diagnostic application of padlock probes--multiplex detection of plant pathogens using universal microarrays

Padlock probes (PLPs) are long oligonucleotides, whose ends are complementary to adjacent target sequences. Upon hybridization to the target, the two ends are brought into contact, allowing PLP circularization by ligation. PLPs provide extremely specific target recognition, which is followed by universal amplification and microarray detection. Since target recognition is separated from downstream processing, PLPs enable the development of flexible and extendable diagnostic systems, targeting diverse organisms. To adapt padlock technology for diagnostic purposes, we optimized PLP design to ensure high specificity and eliminating ligation on non-target sequences under real-world assay conditions. We designed and tested 11 PLPs to target various plant pathogens at the genus, species and subspecies levels, and developed a prototype PLP-based plant health chip. Excellent specificity was demonstrated toward the target organisms. Assay background was determined for each hybridization using a no-target reference sample, which provided reliable and sensitive identification of positive samples. A sensitivity of 5 pg genomic DNA and a dynamic range of detection of 100 were observed. The developed multiplex diagnostic system was validated using genomic DNAs of characterized isolates and artificial mixtures thereof. The demonstrated system is adaptable to a wide variety of applications ranging from pest management to environmental microbiology.     

Phenotypic and molecular characterization of partial trisomy 2q resulting from insertion-duplication in chromosome 18q: a case report and review of literature

Trisomy 2q is a well-documented chromosomal anomaly with considerable variation in the phenotype depending upon the breakpoints and the co-existing chromosomal aberrations. The case of a dysmorphic male infant found to have trisomy of the 2q31.1-q37.3 segment, resulting from insertion-duplication of this segment in chromosome 18q23 is reported here. The rearrangement was resolved in detail by cytogenetic microarray and whole chromosome paint-based fluorescence in situ hybridization studies. There is some overlap of the phenotypic features in the reported patient with those described in previously reported cases with partial trisomy 2q. A detailed review of the available literature on 2q trisomy has also been presented and delineation of the phenotypic characteristics common to all patients with 2q trisomy has been attempted.     

Properties of hybridization isotherms upon binding of ligands on microchips

A simple thermodynamic model describing microarray oligo-target hybridization has been constructed. The relationship between the hybridization signal intensity and Gibbs free energy for oligo-target duplex formation has been considered. The behavior of this function, which we called energy hybridization isotherm, in response to target concentration change was modeled at different ratios of oligo/target concentrations. The results of modeling were compared with the relevant and currently available data from microarray adsorption experiments.