DNA Probe Arrays Research Articles

Underwater application of quantitative PCR on an ocean mooring.

The Environmental Sample Processor (ESP) is a device that allows for the underwater, autonomous application of DNA and protein probe array technologies as a means to remotely identify and quantify, in situ, marine microorganisms and substances they produce. Here, we added functionality to the ESP through the development and incorporation of a module capable of solid-phase nucleic acid extraction and quantitative PCR (qPCR). Samples collected by the instrument were homogenized in a chaotropic buffer compatible with direct detection of ribosomal RNA (rRNA) and nucleic acid purification. From a single sample, both an rRNA community profile and select gene abundances were ascertained. To illustrate this functionality, we focused on bacterioplankton commonly found along the central coast of California and that are known to vary in accordance with different oceanic conditions. DNA probe arrays targeting rRNA revealed the presence of 16S rRNA indicative of marine crenarchaea, SAR11 and marine cyanobacteria; in parallel, qPCR was used to detect 16S rRNA genes from the former two groups and the large subunit RuBisCo gene (rbcL) from Synecchococcus. The PCR-enabled ESP was deployed on a coastal mooring in Monterey Bay for 28 days during the spring-summer upwelling season. The distributions of the targeted bacterioplankon groups were as expected, with the exception of an increase in abundance of marine crenarchaea in anomalous nitrate-rich, low-salinity waters. The unexpected co-occurrence demonstrated the utility of the ESP in detecting novel events relative to previously described distributions of particular bacterioplankton groups. The ESP can easily be configured to detect and enumerate genes and gene products from a wide range of organisms. This study demonstrated for the first time that gene abundances could be assessed autonomously, underwater in near real-time and referenced against prevailing chemical, physical and bulk biological conditions.

Analytical Ancestry: Evolution of the Array in Analysis

Macro-, micro-, and nanosized arrays of test sites at various densities have emerged as important types of analytical devices in response to the need for high volume parallel analysis in both the research and the clinical laboratory. This review explores the diversity of arrays of reaction vessels and arrays of reagents and of samples, with an emphasis on the earliest descriptions of the different variations. The scope of such arrays includes linear and 2-dimensional arrays of reaction vessels (e.g., microwell strips, microplates); linear and 2-dimensional arrays of reagents arrayed on pillars and posts; beads in wells; and reagents randomly arrayed (or dis-ordered) for use in next-generation sequencing. Micro- and nanofabrication technologies have been applied to the miniaturization of arrays to increase array density (e.g., DNA probe arrays) and produce arrays of analytical structures (e.g., cantilevers, nanoelectrospray nozzles). Arrays are now firmly established in many types of analytical devices, and this analytical format has gained widespread acceptance owing to the advantages of high-throughput automation and multiplex analysis. Ongoing "big biology" genomic and proteomic studies will ensure the continued dominance of array-based methods into the foreseeable future.

What is DNA array?

DNA probe arrays - DNA arrays or DNA chips for short have recently emerged as one of the core genome technologies. They provide a cost-effective method for obtaining fast and accurate results in a wide range of genomic analyses, including gene expression monitoring, mutation detection, and single nucleotide polymorphism analysis (see [1] for a survey). The number of applications is growing at a high rate already covering a diversity of fields ranging from health care to environmental sciences and law enforcement.

Rapid discrimination of single‐nucleotide mismatches based on reciprocating flow on a compact disc microfluidic device

In this study, a compact disc (CD) microfluidic device based on reciprocating flow is developed for the rapid discrimination of single-nucleotide mismatch (SNM). This device consists of a PDMS CD slab containing eight SNM assay units and a glass substrate immobilized with DNA probe array. The rapid SNM assay is realized by two-step controllable reciprocating flow in the microchannel, resulting from centrifugal force and capillary action as well. A perfect-match probe and three mismatched probes containing a SNM at various positions were designed to investigate the ability of this device to discriminate SNM. Based on the difference between the dissociation kinetics of perfect-match and mismatched duplexes, this CD device was able to discriminate SNM within 400 s. Moreover, only sub-microliter volume DNA samples (350 nL) and washing buffers (350 nL) were required per assay unit. The results indicate that this device has the potential to be used for fast, accurate gene diagnostics, especially for the detection of SNPs.

Remote Detection of Marine Microbes, Small Invertebrates, Harmful Algae, and Biotoxins using the Environmental Sample Processor (ESP)

The advent of ocean observatories is creating unique opportunities for deploying novel sensor systems. We are exploring that potential through the development and application of the Environmental Sample Processor (ESP). ESP is an electromechanical/fluidic system designed to collect discrete water samples, concentrate microorganisms, and automate application of molecular probe technologies. Development and application of ESP grew from extensive partnerships galvanized by the National Oceanographic Partnership Program. Near-real-time observations are currently achieved using low-density DNA probe and protein arrays. Filter-based sandwich hybridization methodology enables direct detection of ribosomal RNA sequences diagnostic for groups of bacteria and archaea, as well as a variety of invertebrates and harmful algal species. An antibody-based technique is used for detecting domoic acid, an algal biotoxin. To date, ESP has been deployed in ocean waters from the near surface to 1000 m. Shallow-water deployments demonstrated application of all four types of assays in single deployments lasting up to 30 days and provided the first remote detection of such phylogenetically diverse organisms and metabolites on one platform. Deep-water applications focused on detection of invertebrates associated with whale falls, using remotely operated vehicle-based operations lasting several days. Current work emphasizes incorporating a four-channel, real-time polymerase chain reaction module, extending operations to 4000-m water depth, and increasing deployment duration.

Remote, subsurface detection of the algal toxin domoic acid onboard the Environmental Sample Processor: Assay development and field trials

The ability to detect harmful algal bloom (HAB) species and their toxins in real- or near real-time is a critical need for researchers studying HAB/toxin dynamics, as well as for coastal resource managers charged with monitoring bloom populations in order to mitigate their wide ranging impacts. The Environmental Sample Processor (ESP), a robotic electromechanical/fluidic system, was developed for the autonomous, subsurface application of molecular diagnostic tests and has successfully detected several HAB species using DNA probe arrays during field deployments. Since toxin production and thus the potential for public health and ecosystem effects varies considerably in natural phytoplankton populations, the concurrent detection of HAB species and their toxins onboard the ESP is essential. We describe herein the development of methods for extracting the algal toxin domoic acid (DA) from Pseudo-nitzschia cells (extraction efficiency >90%) and testing of samples using a competitive ELISA onboard the ESP. The assay detection limit is in the low ng/mL range (in extract), which corresponds to low ng/L levels of DA in seawater for a 0.5 L sample volume acquired by the ESP. We also report the first in situ detection of both a HAB organism (i.e., Pseudo-nitzschia) and its toxin, domoic acid, via the sequential (within 2–3 h) conduct of species- and toxin-specific assays during ESP deployments in Monterey Bay, CA, USA. Efforts are now underway to further refine the assay and conduct additional calibration exercises with the aim of obtaining more reliable, accurate estimates of bloom toxicity and thus their potential impacts.

Rapid nanoliter DNA hybridization based on reciprocating flow on a compact disk microfluidic device

We present a compact disk (CD) microfluidic device capable of generating the reciprocating flow of DNA samples within the microchannels and demonstrate its application in rapid DNA hybridization assay with nanoliter-volume samples. This device consists of a polydimethylsiloxane (PDMS) CD slab containing twelve DNA hybridization functional units and a glass substrate with immobilized DNA probe array. A reciprocating flow is produced with simple rotation-pause operation of the CD device. When spinning the CD device, centrifugal force drives the sample solution to flow through the hybridization channel into the temporary collection reservoir. When stopping the rotation of the CD device, capillary action pulls the sample solution to reversely flow back into the hybridization channel because of hydrophilic surface of the hybridization channel. We used Dengue virus gene sequence (18mer) as a model to demonstrate that the reciprocating flow of DNA samples significantly enhanced the mass transfer rate in the hybridization reaction, reducing the hybridization time to 90 s. Moreover, only nanoliter-volume DNA samples were required per assay unit. We also compared the performance between the reciprocating-flow hybridization and the flow-through hybridization using the same sample concentrations. A fluorescence intensity was observed to increase up to threefold in the reciprocating-flow hybridization compared to the flow-through hybridization with the same hybridization time (90 s) and sample volumes (350 nL). This CD microfluidic device has the potential for automated, rapid and multiple DNA-based diagnostics.

Field applications of the second‐generation Environmental Sample Processor (ESP) for remote detection of harmful algae: 2006‐2007

We assess the application of the second‐generation Environmental Sample Processor (ESP) for the detection of harmful algal bloom (HAB) species in field and laboratory settings using two molecular probe techniques: a sandwich hybridization assay (SHA) and fluorescent in situ hybridization (FISH). During spring 2006, the first time this new instrument was deployed, the ESP successfully automated application of DNA probe arrays for various HAB species and other planktonic taxa, but non‐specific background binding on the SHA probe array support made results interpretation problematic. Following 2006, the DNA array support membrane that we were using was replaced with a different membrane, and the SHA chemistry was adjusted. The sensitivity and dynamic range of these modifications were assessed using 96‐well plate and ESP array SHA formats for several HAB species found commonly in Monterey Bay over a range of concentrations; responses were significantly correlated (p < 0.01). Modified arrays were deployed in 2007. Compared to 2006, probe arrays showed improved signal:noise, and remote detection of various HAB species was demonstrated. We confirmed that the ESP and affiliated assays can detect HAB populations at levels below those posing human health concerns, and results can be related to prevailing environmental conditions in near real‐time.

High-throughput SPR sensor for food safety

High-throughput surface plasmon resonance (SPR) biosensor for rapid and parallelized detection of nucleic acids identifying specific bacterial pathogens is reported. The biosensor consists of a high-performance SPR imaging sensor with polarization contrast and internal referencing (refractive index resolution 2 × 10 −7 RIU) and an array of DNA probes microspotted on the surface of the SPR sensor. It is demonstrated that short sequences of nucleic acids (20–23 bases) characteristic for bacterial pathogens such as Brucella abortus, Escherichia coli, and Staphylococcus aureus can be detected at 100 pM levels. Detection of specific DNA or RNA sequences can be performed in less than 15 min by the reported SPR sensor.

Photolithographic synthesis of high-density DNA probe arrays: Challenges and opportunities

The continual need for increased manufacturing capacity in the production of GeneChip™ DNA probe arrays, and the expanding use of these arrays into new areas of application such as molecular medicine, has stimulated the development of new chemistries and production methods with higher efficiency and resolution. For current production methods based on contact photolithography, modifications in substrate materials and photoactivated synthesis reagents have provided significant improvements in array performance and information content (≥4×106 sequences∕cm2). An alternative next-generation manufacturing process is also in development, which utilizes photoacid generating polymer films, and automated projection lithography systems. This process has the ability to fabricate arrays with 1 micron feature pitch and smaller, providing an unprecedented sequence density of 108∕cm2 and greater.

Microarray Analysis in the Archaeon Halobacterium salinarum Strain R1

BackgroundPhototrophy of the extremely halophilic archaeon Halobacterium salinarum was explored for decades. The research was mainly focused on the expression of bacteriorhodopsin and its functional properties. In contrast, less is known about genome wide transcriptional changes and their impact on the physiological adaptation to phototrophy. The tool of choice to record transcriptional profiles is the DNA microarray technique. However, the technique is still rarely used for transcriptome analysis in archaea.Methodology/Principal FindingsWe developed a whole-genome DNA microarray based on our sequence data of the Hbt. salinarum strain R1 genome. The potential of our tool is exemplified by the comparison of cells growing under aerobic and phototrophic conditions, respectively. We processed the raw fluorescence data by several stringent filtering steps and a subsequent MAANOVA analysis. The study revealed a lot of transcriptional differences between the two cell states. We found that the transcriptional changes were relatively weak, though significant. Finally, the DNA microarray data were independently verified by a real-time PCR analysis.Conclusion/SignificanceThis is the first DNA microarray analysis of Hbt. salinarum cells that were actually grown under phototrophic conditions. By comparing the transcriptomics data with current knowledge we could show that our DNA microarray tool is well applicable for transcriptome analysis in the extremely halophilic archaeon Hbt. salinarum. The reliability of our tool is based on both the high-quality array of DNA probes and the stringent data handling including MAANOVA analysis. Among the regulated genes more than 50% had unknown functions. This underlines the fact that haloarchaeal phototrophy is still far away from being completely understood. Hence, the data recorded in this study will be subject to future systems biology analysis.

Kinetics of Oligonucleotide Hybridization to DNA Probe Arrays on High-Capacity Porous Silica Substrates

We have investigated the kinetics of DNA hybridization to oligonucleotide arrays on high-capacity porous silica films that were deposited by two techniques. Films created by spin coating pure colloidal silica suspensions onto a substrate had pores of ∼23 nm, relatively low porosity (35%), and a surface area of 17 times flat glass (for a 0.3- μm film). In the second method, latex particles were codeposited with the silica by spin coating and then pyrolyzed, which resulted in larger pores (36 nm), higher porosity (65%), and higher surface area (26 times flat glass for a 0.3- μm film). As a result of these favorable properties, the templated silica hybridized more quickly and reached a higher adsorbed target density (11 vs. 8 times flat glass at 22°C) than the pure silica. Adsorption of DNA onto the high-capacity films is controlled by traditional adsorption and desorption coefficients, as well as by morphology factors and transient binding interactions between the target and the probes. To describe these effects, we have developed a model based on the analogy to diffusion of a reactant in a porous catalyst. Adsorption values ( k a, k d, and K) measured on planar arrays for the same probe/target system provide the parameters for the model and also provide an internally consistent comparison for the stability of the transient complexes. The interpretation of the model takes into account factors not previously considered for hybridization in three-dimensional films, including the potential effects of heterogeneous probe populations, partial probe/target complexes during diffusion, and non-1:1 binding structures. The transient complexes are much less stable than full duplexes (binding constants for full duplexes higher by three orders of magnitude or more), which may be a result of the unique probe density and distribution that is characteristic of the photolithographically patterned arrays. The behavior at 22°C is described well by the predictive equations for morphology, whereas the behavior at 45°C deviates from expectations and suggests that more complex phenomena may be occurring in that temperature regime.

Using Luminescent Nanoparticles as Staining Probes for Affymetrix GeneChips

Microarray technology provides efficient access to genetic information using miniaturized, high-density arrays of DNA probes. We investigated the application of luminescent nanoparticles as probes for Affymetrix GeneChips detection without the need for signal amplification. Our goal is to investigate the feasibility of using luminescent nanoparticles as probes in a commercial microarray system without changing its configurations. With the present imaging modality and existing optical excitation and detection systems of the Affymetrix GeneChips, our early results indicate that nanoparticles not only can be used for GeneChip labeling but also are superior to the traditional fluorescent protein streptavidin-phycoerythrin (SAPE). The advantage of the particles lies in a simplified staining procedure, higher photobleaching threshold, and enhanced luminescence signal. The nanoparticles can be used for detection of low-abundance targets without any amplification step. A concentration detection limit of 50 fM has been achieved. This work demonstrates the feasibility of using luminescent nanoparticles as probes for commercial microarray systems, making them less costly, more reproducible, and potentially quantitative.

Application of environmental sample processor (ESP) methodology for quantifying Pseudo‐nitzschia australis using ribosomal RNA‐targeted probes in sandwich and fluorescent in situ hybridization formats

In this contribution, we assess the application of methodology used in a novel in situ remote sampling platform, the environmental sample processor (ESP), for the identification and enumeration of a harmful algal species, the domoic acid‐producing diatom Pseudo‐nitzschia australis, using 2 molecular assays: a sandwich hybridization assay (SHA) and fluorescent in situ hybridization (FISH). Both the SHA and FISH assays were initially designed as laboratory‐based methods that are now emulated in the ESP. Response of the SHA to a range of concentrations of P. australis using the laboratory (96‐well plate) and ESP (DNA probe array) formats showed that the two were highly correlated. Enumeration of cells filtered and archived for FISH using a manifold designed for laboratory applications agreed well with counts of cells filtered and archived in the ESP at ≥2.5 × 104 cells L−1. At lower concentrations, it becomes statistically unlikely to derive a reliable abundance estimate, suggesting that FISH is better suited for qualitative analyses unless the target organism is very abundant. We also assessed the suitability of an oligonucleotide as synthetic target sequence to act as a SHA reagent quality control and internal standard for the plate assay. This was successful, but the probe and/or associated reagents were stable for only ~60 days. Our results show that the ESP is capable of detecting P. australis in near real‐time and also supports whole‐cell archival samples, making it a potentially useful tool for future research and monitoring initiatives.

European Multicenter Evaluation of High-Density DNA Probe Arrays for Detection of Hepatitis B Virus Resistance Mutations and Identification of Genotypes

Polymorphisms along the hepatitis B virus (HBV) genome have an impact on disease outcome, sensitivity to antiviral treatment, escape from vaccination, and laboratory diagnosis. We have designed a diagnostic tool based on duplex amplification of the whole HBV genome and a high-density DNA chip designed to detect 245 mutations, 20 deletions, and 2 insertions at 151 positions and to determine the genotype of the virus in serum. Assay performances were evaluated with 170 samples, characterized by determination of viral load and sequencing of the Pol, S, and precore genes and the basal core promoter. One hundred fifty-three samples (90%) could be amplified and analyzed by the chip. Only two samples with more than 10(3) genome copies/ml could not be analyzed. Genotype had no impact on analytical sensitivity. Reproducibility studies showed no difference between repeats for codon and genotype determination. Genotype determination by sequencing and the chip were concordant in 148 of 151 samples. Twelve thousand one hundred sixty-one codons were analyzed by both techniques. Only 89.4% could be determined by sequencing, and among the remaining 11,335 codons, 92.8% were identical by sequencing and the chip. Failures to identify an amino acid by the chip were mainly due to reduced hybridization efficiency attributed to unexpected polymorphisms. Optimization of the chip-based reagent for the analysis of the HBV genome is ongoing. This first evaluation showed that DNA chip technology can provide important information in relation to the clinical management of chronic hepatitis B.

Computer-Aided Optimization of DNA Array Design and Manufacturing

DNA probe arrays, or DNA chips, have emerged as a core genomic technology that enables cost-effective gene expression monitoring, mutation detection, single nucleotide polymorphism analysis, and other genomic analyses. DNA chips are manufactured through a highly scalable process called very large-scale immobilized polymer synthesis (VLSIPS) that combines photolithographic technologies adapted from the semiconductor industry with combinatorial chemistry. Commercially available DNA chips contain more than half a million probes and are expected to exceed 100 million probes in the next generation. This paper is one of the first attempts to apply very large scale integration (VLSI) computer-aided design methods to the physical design of DNA chips, where the main objective is to minimize total border cost (i.e., the number of nucleotide mismatches between adjacent sites). By exploiting analogies between manufacturing processes for DNA arrays and for VLSI chips, the authors demonstrate the potential for transfer of methodologies from the 40-year-old field of electronic design automation to the newer DNA array design field. The main contributions of this paper are the following. First, it proposes several partitioning-based algorithms for DNA probe placement that improve solution quality by over 4% compared to best previously known methods. Second, it gives a new design flow for DNA arrays, which enhances current methodologies by adding flow awareness to each optimization step and introducing feedback loops. Third, it proposes solution methods for new formulations integrating multiple design steps, including probe selection, placement, and embedding. Finally, it introduces new techniques to experimentally evaluate the scalability and suboptimality of existing and newly proposed probe placement algorithms. Interestingly, the authors find that DNA placement algorithms appear to have better suboptimality properties than those recently reported for VLSI placement algorithms [C.C. Chang et al., Optimality and scalability study of existing placement algorithms, Proc. Asia South-Pacific Design Automation Conf., Kitakyushu, Japan, p.621-7, Jan. 2003; J. Cong et al., Optimality, scalability and stability study of partitioning and placement algorithms, Proc. Int. Symp. Physical Design (ISPD), Monterey, CA, p.88-94, 2003]

DNA probe array for the simultaneous identification of herpesviruses, enteroviruses, and flaviviruses.

Viral infections of the central nervous system (CNS) are caused by a variety of viruses, namely, herpesviruses, enteroviruses, and flaviviruses. The similar clinical signs provoked by these viruses make the diagnosis difficult. We report on the simultaneous detection of these major CNS pathogens using amplification by PCR and detection of amplified products using DNA microarray technology. Consensus primers were used for the amplification of all members of each genus. Sequences specific for the identification of each virus species were selected from the sequence alignments of each target gene and were synthesized on a high-density microarray. The amplified products were pooled, labeled, and cleaved, followed by hybridization on a single array. This method was successfully used to identify herpesviruses, namely, herpes simplex virus type 1 (HSV-1), HSV-2, and cytomegalovirus; all serotypes of human enteroviruses; and five flaviviruses (West Nile virus, dengue viruses, and Langat virus). This approach, which used highly conserved consensus primers for amplification and specific sequences for identification, would be extremely useful for the detection of variants and would probably help solve some unexplained cases of encephalitis. The analytical sensitivity of the method was shown to be 500 genome equivalents ml(-1) for HSV-1, 0.3 50% tissue culture infectious doses (TCID50s) ml(-1) for the enterovirus coxsackievirus A9, and 200 TCID50s ml(-1) for West Nile virus. The clinical sensitivity of this method must now be evaluated.

High-density DNA probe arrays for identification of staphylococci to the species level

Rapid and accurate identification and speciation of staphylococci clinical isolates is important for predicting medical pathology. We evaluated the ability of a high-density DNA probe array based on 16S rDNA sequences to identify Staphylococcus species. Correct identification was observed for 185 out of the 201 strains (92%). Of the 33 tested species, the array was able to correctly identify 30 of them. The total time required for identification of 4 isolates was 5 h. Such a tool represents a powerful method for routine microbiological diagnostic as well as for epidemiological studies.

Photo-removable protecting groups for in situ DNA microarray synthesis.

High-density DNA probe arrays are finding widespread use in biomedical research and diagnostics because of their ability to simultaneously address large numbers of genes. GeneChip® probe arrays are manufactured by use of photolithography techniques (1)(2)(3)(4). At the base of the current Affymetrix process of synthesis of oligo-DNA probes is the use of nucleoside monomers protected with photo-removable groups (Fig. 1A⇓ ). Irradiation of the partially built oligomer with near-ultraviolet wavelengths (mainly 365 nm) deprotects the terminal hydroxyl group, and the use of appropriate masks allows for control of the sequence of the probe and the size of the features. The sensitivity of DNA to ultraviolet radiation limits the range of wavelengths usable for the photolitographic step to mainly the Hg atomic emission at 365 nm. Therefore, the choice of photo-removable hydroxyl protecting groups depends on the absorbance at this wavelength (4). Our ongoing investigations concerning the fabrication of DNA microarrays have several goals, including increasing the rate and the efficiency of photo-deprotection. The rate of deprotection, or photospeed (PS), is a function of the molar absorptivity of the chromophore at the wavelength of irradiation and the quantum yield of the desired photochemical product formation process. Increasing …

Detection of human immunodeficiency virus type 1 antiretroviral resistance mutations by high-density DNA probe arrays.

Genotypic resistance testing has become an important tool in the clinical management of patients infected with human immunodeficiency virus type 1 (HIV-1). Standard sequencing methodology and hybridization-based technology are the two principal methods used for HIV-1 genotyping. This report describes an evaluation of a new hybridization-based HIV-1 genotypic test of 99 clinical samples from patients infected mostly with HIV-1 subtype B and receiving treatment. This test combines RNA extraction with magnetic silica particles, amplification by nested reverse transcriptase PCR, and detection with high-density probe arrays designed to detect 204 antiretroviral resistance mutations simultaneously in Gag cleavage sites, protease, reverse transcriptase, integrase, and gp41. The nested reverse transcriptase PCR success rates at viral loads exceeding 1,000 copies/ml were 98% for the 2.1-kb amplicon that covers the Gag cleavage sites and the protease and reverse transcriptase genes, 92% for the gp41 amplicon, and 100% for the integrase amplicon. We analyzed 4,465 relevant codons with the HIV-1 DNA chip genotyping assay and the classic sequence-based method. Key resistance mutations in protease and reverse transcriptase were identified correctly 95 and 92% of the time, respectively. This test should be a valuable alternative to the standard sequence-based system for HIV-1 drug resistance monitoring and a useful diagnostic tool for simultaneous multiple genetic analyses.