DNA microarrays for functional plant genomics

DNA microarray technology is a key element in today's functional genomics toolbox. The power of the method lies in miniaturization, automation and parallelism permitting large-scale and genome-wide acquisition of quantitative biological information from multiple samples. DNA microarrays are currently fabricated and assayed by two main approaches involving either in situ synthesis of oligonucleotides (`oligonucleotide microarrays') or deposition of pre-synthesized DNA fragments (`cDNA microarrays') on solid surfaces. To date, the main applications of microarrays are in comprehensive, simultaneous gene expression monitoring and in DNA variation analyses for the identification and genotyping of mutations and polymorphisms. Already at a relatively early stage of its application in plant science, microarrays are being utilized to examine a range of biological issues including the circadian clock, plant defence, environmental stress responses, fruit ripening, phytochrome A signalling, seed development and nitrate assimilation. Novel insights are obtained into the molecular mechanisms co-ordinating metabolic pathways, regulatory and signalling networks. Exciting new information will be gained in the years to come not only from genome-wide expression analyses on a few model plant species, but also from extensive studies of less thoroughly studied species on a more limited scale. The value of microarray technology to our understanding of living processes will depend both on the amount of data to be generated and on its clever exploration and integration with other biological knowledge arising from complementary functional genomics tools for `profiling' the genome, proteome, metabolome and phenome.     

Conjugated polyelectrolytes for label-free DNA microarrays

Classical strategies for gene microarrays require labeling of probes or target nucleic acids with signaling molecules, a process that is expensive, time consuming and not always reliable. Bazan and colleagues showed that a nucleic acid-binding cationic conjugated polyelectrolyte can be used in label-free DNA microarrays based on surfaces modified with neutral peptide nucleic acid (PNA) probes. This technique provides a simple and sensitive method for DNA detection without the need for covalent labeling of target DNA.     

Towards quality control for DNA microarrays.

We present a framework for detecting probes in oligonucleotide microarrays that may add significant error to measurements in hybridization experiments. Four types of so-called degenerate probe behavior are considered: secondary structure formation, self-dimerization, cross-hybridization, and dimerization. The framework uses a well-established model for computing the free energy of nucleic acid sequence hybridization and a novel method for the detection of patterns in hybridization experiment data. Our primary result is the identification of unique patterns in hybridization experiment data that are shown to correlate with each type of degenerate probe behavior. A support function for identifying degenerate probes from a large set of hybridization experiments is given and some preliminary experimental results are given for the Affymetrix HuGeneFL GeneChip. Finally, we show a strong relationship between the Affymetrix discrimination measure for a probe and the free-energy estimate from theoretical models of hybridization. In particular, probes on the HuGeneFL GeneChip with high free-energy estimates (weak hybridization) have almost always approximately zero discrimination. The framework can be applied to any Affymetrix oligonucleotide array, and the software is made freely available to the community.     

Coating of nitrocellulose for colorimetric DNA microarrays

We report on the modification of a nitrocellulose film with copoly(DMA-NAS-MAPS), a tercopolymer based on N,N-dimethylacrylamide (DMA), N-acryloyloxysuccinimide (NAS), and 3-(trimethoxysilyl)propyl-methacrylate (MAPS). The chains of this polymer, interacting with nitrocellulose fibers, introduce active ester functionalities that promote the covalent binding of short oligonucleotide fragments to the nitrocellulose thin film. Using colorimetric detection, naked eye visible DNA microarrays are developed for easy identification of foodborne pathogens. The fast and robust procedure of nitrocellulose functionalization opens the opportunity to implement this material in disposable analytical microdevices that do not require sophisticated readout systems.     

Missing value estimation methods for DNA microarrays

MOTIVATION: Gene expression microarray experiments can generate data sets with multiple missing expression values. Unfortunately, many algorithms for gene expression analysis require a complete matrix of gene array values as input. For example, methods such as hierarchical clustering and K-means clustering are not robust to missing data, and may lose effectiveness even with a few missing values. Methods for imputing missing data are needed, therefore, to minimize the effect of incomplete data sets on analyses, and to increase the range of data sets to which these algorithms can be applied. In this report, we investigate automated methods for estimating missing data. RESULTS: We present a comparative study of several methods for the estimation of missing values in gene microarray data. We implemented and evaluated three methods: a Singular Value Decomposition (SVD) based method (SVDimpute), weighted K-nearest neighbors (KNNimpute), and row average. We evaluated the methods using a variety of parameter settings and over different real data sets, and assessed the robustness of the imputation methods to the amount of missing data over the range of 1--20% missing values. We show that KNNimpute appears to provide a more robust and sensitive method for missing value estimation than SVDimpute, and both SVDimpute and KNNimpute surpass the commonly used row average method (as well as filling missing values with zeros). We report results of the comparative experiments and provide recommendations and tools for accurate estimation of missing microarray data under a variety of conditions.     

Surface modification for DNA and protein microarrays

Microarrays of biomolecules are emerging as powerful tools for genomics, proteomics, and clinical assays, since they make it possible to screen biologically important binding events in a parallel and high throughput fashion. Because the microarrays are fabricated on a solid support, coating of the surface and immobilization strategy of the biomolecules are major issues for successful microarray fabrication. This review deals with both DNA microarrays and protein microarrays, and focuses on the various modification approaches for the two-dimensional surface materials and three-dimensional ones. In addition, the immobilization strategies including adsorption, covalent attachment, physical entrapment, and affinity attachment of the biomolecules are summarized, and advantage and limitation of representative efforts are discussed.     

Innovative metal oxide-based substrates for DNA microarrays

In the present report, we propose a novel approach to synthesize DNA microarrays that employs immobilization of the nucleic acid molecules onto zinc and iron oxide surfaces through their phosphate backbone. Oxide films were prepared by the sol–gel technique and the resulting surfaces were characterized especially with respect to surface chemistry and morphological features by both X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM). ZnO films annealed at T ? 300 °C show the most promising surface features to be employed for DNA microarray preparation, i.e. high density of binding sites (hydroxyl groups), smooth and homogeneous surfaces, high optical transmittance in the visible spectral range suitable for detection using fluorescence, and easy handling during preparation procedures. The analysis of nucleic acid retention on the oxide layers was performed by the scanning of dye-labelled DNA previously printed on the substrate using the DNA microarray robotic arm. Clearly visible spots with regular shape were revealed above the background noise indicating that anchoring of the DNA on the treated surface is efficient and does not interfere with hybridization processes. The use of suitably engineered zinc oxide film makes the immobilization strategy ideal for facile, efficient, and cost-effective manufacturing of DNA microarrays.     

Ultrathin functional star PEG coatings for DNA microarrays

In this study, star PEG coatings on glass substrates have been used as support material for oligonucleotide microarrays. These coatings are prepared from solutions of six armed star shaped prepolymers that carry reactive isocyanate endgroups. As described earlier, such films prevent the adsorption of proteins and the adhesion of cells but can easily be functionalized for specific biological recognition. Here we used the high functionality of these coatings for the covalent immobilization of amino terminated 20mer oligonucleotides, both by microcontact printing and spotting techniques. The permanent immobilization of fluorescently labeled DNA as well as hybridization of 20mer oligonucleotides have been monitored by fluorescence microscopy. The hybridization efficiency as determined by fluorescence intensity varied from 30% to 80% depending on the way of layer preparation. The direct spotting without additional activation and blocking steps of the surface demonstrates the potential of star PEG coatings as ultrathin surface modification for microarrays.     

PNA microarrays for hybridisation of unlabelled DNA samples

Several strategies have been developed for the production of peptide nucleic acid (PNA) microarrays by parallel probe synthesis and selective coupling of full-length molecules. Such microarrays were used for direct detection of the hybridisation of unlabelled DNA by time-of-flight secondary ion mass spectrometry. PNAs were synthesised by an automated process on filter-bottom microtitre plates. The resulting molecules were released from the solid support and attached without any purification to microarray surfaces via the terminal amino group itself or via modifications, which had been chemically introduced during synthesis. Thus, only full-length PNA oligomers were attached whereas truncated molecules, produced during synthesis because of incomplete condensation reactions, did not bind. Different surface chemistries and fitting modifications of the PNA terminus were tested. For an examination of coupling selectivity, bound PNAs were cleaved off microarray surfaces and analysed by MALDI-TOF mass spectrometry. Additionally, hybridisation experiments were performed to compare the attachment chemistries, with fully acetylated PNAs spotted as controls. Upon hybridisation of unlabelled DNA to such microarrays, binding events could be detected by visualisation of phosphates, which are an integral part of nucleic acids but missing entirely in PNA probes. Overall best results in terms of selectivity and sensitivity were obtained with thiol-modified PNAs on maleimide surfaces.     

Improved background correction for spotted DNA microarrays.

Most microarray scanning software for glass spotted arrays provides estimates for the intensity for the "foreground" and "background" of two channels for every spot. The common approach in further analyzing such data is to first subtract the background from the foreground for each channel and to use the ratio of these two results as the estimate of the expression level. The resulting ratios are, after possible averaging over replicates, the usual inputs for further data analysis, such as clustering. If, with this background correction procedure, the foreground intensity was smaller than the background intensity for a channel, that spot (on that array) yields no usable data. In this paper it is argued that this preprocessing leads to estimates of the expression that have a much larger variance than needed when the expression levels are low.