DNA Oligonucleotide Probes Research Articles

Dramatically improved RNA in situ hybridization signals using LNA-modified probes

In situ detection of RNA by hybridization with complementary probes is a powerful technique. Probe design is a critical parameter in successful target detection. We have evaluated the efficiency of fluorescent DNA oligonucleotides modified to contain locked nucleic acid (LNA) residues. This increases the thermal stability of hybrids formed with RNA. The LNA-based probes detect specific RNAs in fixed yeast cells with an efficiency far better than conventional DNA oligonucleotide probes of the same sequence. Using this probe design, we were also able to detect poly(A)(+) RNA accumulation within the nucleus/ nucleolus of wild-type cells. LNA-based probes should be readily applicable to a diverse array of cells and tissue samples.

Application trials of in situ hybridization at the electron microscopic level

In situ hybridization (ISH) is a morphology-oriented technique for demonstrating the presence of specific nucleic acid sequences at chromosomal, cytological and histological levels. It is, however, sometimes difficult to recognize specific cell identity, early phase mRNA expression and alternative splicing because of the limited resolution of the light microscope. To overcome this limitation, we developed an improved technique for ISH at the electron microscopic level, in which pre-embedding hybridization with a non-radioactively labeled probe was used, followed by post-embedding immunoglobulin gold colloid staining. By applying this technique, early phase bone morphogenetic protein-3 mRNA in the nuclei and cytoplasm was successfully demonstrated in a differentiating chondrocytic cell lineage. Moreover, with oligo-DNA probes specific for alternative spliced forms of parathyroid hormone-related protein mRNA, we demonstrated such forms in a hyperplastic parathyroid gland attributed to renal failure.

An update and optimisation of oligonucleotide probes targeting methanogenic Archaea for use in fluorescence in situ hybridisation (FISH)

Fluorescence in situ hybridisation (FISH) is a common and popular method used to investigate microbial populations in natural and engineered environments. DNA oligonucleotide probes require accurate determination of the optimal experimental conditions for their use in FISH. Oligonucleotides targeting the rRNA of methanogenic Archaea at various taxonomic levels have previously been published, although when applied in FISH, no optimisation data has been presented. In this study, 3000 Euryarchaeota 16S rRNA gene sequences were phylogenetically analysed and previously published oligonucleotides were evaluated for target group accuracy. Where necessary, modifications were introduced or new probes were designed. The updated set of probes was optimised for use in FISH for a more accurate detection of methanogenic Archaea.

An optimized isolation and labeling platform for accurate microRNA expression profiling

MicroRNAs (miRNAs) are small, noncoding RNAs that regulate gene expression in both plants and animals. miRNA genes have been implicated in a variety of important biological processes, including development, differentiation, apoptosis, fat metabolism, viral infection, and cancer. Similar to protein-coding messenger RNAs, miRNA expression varies between tissues and developmental states. To acquire a better understanding of global miRNA expression in tissues and cells, we have developed isolation, labeling, and array procedures to measure the relative abundance of all of the known human mature miRNAs. The method relies on rapid isolation of RNA species smaller than ~40 nucleotides (nt), direct and homogenous enzymatic labeling of the mature miRNAs with amine modified ribonucleotides, and hybridization to antisense DNA oligonucleotide probes. A thorough performance study showed that this miRNA microarray system can detect subfemtomole amounts of individual miRNAs from <1 mug of total RNA, with 98% correlation between independent replicates. The system has been applied to compare the global miRNA expression profiles in 26 different normal human tissues. This comprehensive analysis identified miRNAs that are preferentially expressed in one or a few related tissues and revealed that human adult tissues have unique miRNA profiles. This implicates miRNAs as important components of tissue development and differentiation. Taken together, these results emphasize the immense potential of microarrays for sensitive and high-throughput analysis of miRNA expression in normal and disease states.

DNA and protein microarray printing on silicon nitride waveguide surfaces

Sputtered silicon nitride optical waveguide surfaces were silanized and modified with a hetero-bifunctional crosslinker to facilitate thiol-reactive immobilization of contact-printed DNA probe oligonucleotides, streptavidin and murine anti-human interleukin-1 beta capture agents in microarray formats. X-ray photoelectron spectroscopy (XPS) was used to characterize each reaction sequence on the native silicon oxynitride surface. Thiol-terminated DNA probe oligonucleotides exhibited substantially higher surface printing immobilization and target hybridization efficiencies than non-thiolated DNA probe oligonucleotides: strong fluorescence signals from target DNA hybridization supported successful DNA oligonucleotide probe microarray fabrication and specific capture bioactivity. Analogously printed arrays of thiolated streptavidin and non-thiolated streptavidin did not exhibit noticeable differences in either surface immobilization or analyte capture assay signals. Non-thiolated anti-human interleukin-1 beta printed on modified silicon nitride surfaces reactive to thiol chemistry exhibited comparable performance for capturing human interleukin-1 beta analyte to commercial amine-reactive microarraying polymer surfaces in sandwich immunoassays, indicating substantial non-specific antibody-surface capture responsible for analyte capture signal.

Specific and Nonspecific Hybridization of Oligonucleotide Probes on Microarrays

Gene expression analysis by means of microarrays is based on the sequence-specific binding of RNA to DNA oligonucleotide probes and its measurement using fluorescent labels. The binding of RNA fragments involving sequences other than the intended target is problematic because it adds a chemical background to the signal, which is not related to the expression degree of the target gene. The article presents a molecular signature of specific and nonspecific hybridization with potential consequences for gene expression analysis. We analyzed the signal intensities of perfect match (PM) and mismatch (MM) probes of GeneChip microarrays to specify the effect of specific and nonspecific hybridization. We found that these events give rise to different relations between the PM and MM intensities as function of the middle base of the PM, namely a triplet-like ( C > G ≈ T > A > 0) and a duplet-like ( C ≈ T > 0 > G ≈ A) pattern of the PM-MM log-intensity difference upon binding of specific and nonspecific RNA fragments, respectively. The systematic behavior of the intensity difference can be rationalized on the level of basepairings of DNA/RNA oligonucleotide duplexes in the middle of the probe sequence. Nonspecific binding is characterized by the reversal of the central Watson-Crick (WC) pairing for each PM/MM probe pair, whereas specific binding refers to the combination of a WC and a self-complementary (SC) pairing in PM and MM probes, respectively. The Gibbs free energy contribution of WC pairs to duplex stability is asymmetric for purines and pyrimidines of the PM and decreases according to C > G ≈ T > A. SC pairings on the average only weakly contribute to duplex stability. The intensity of complementary MM introduces a systematic source of variation which decreases the precision of expression measures based on the MM intensities.

Detecting hybridization of DNA by highly sensitive evanescent field etched core fiber Bragg grating sensors

Highly sensitive fiber Bragg grating sensors were developed by etching away the cladding and part of the core of the fiber and detecting the change of Bragg wavelength due to the change of index of the surrounding medium. A sensitivity of 1394 nm/riu was achieved when the diameter of the grating core was 3.4 /spl mu/m and the index of the surrounding medium was close to the index of the core of the fiber. Assuming a detectable spectral resolution of 0.01 nm realized in the experiment, the sensor achieves a minimum detectable index resolution of 7.2/spl times/10/sup -6/. Higher sensitivity at lower surrounding index was achieved by using higher order modes excited in the Bragg grating region. The use of the fiber Bragg grating sensor was further investigated to detect hybridization of DNA. Single stranded DNA oligonucleotide probes of 20 bases were immobilized on the surface of the fiber grating using relatively common glutarahyldehyde chemistry. Hybridization of complimentary target single strand DNA oligonucleotide was monitored in situ and successfully detected. The demonstrated fiber Bragg grating sensors provide an elegant method to monitor biological changes in an in situ manner, and provide temporal information in a single experiment.

Advantages of peptide nucleic acid oligonucleotides for sensitive site directed 16S rRNA fluorescence in situ hybridization (FISH) detection of Campylobacter jejuni, Campylobacter coli and Campylobacter lari

Traditionally fluorescence in situ hybridization (FISH) has been performed with labeled DNA oligonucleotide probes. Here we present for the first time a high affinity peptide nucleic acid (PNA) oligonucleotide sequence for detecting thermotolerant Campylobacter spp. using FISH. Thermotolerant Campylobacter spp, including the species Campylobacter coli, Campylobacter jejuni and Campylobacter lari, are important food and water borne pathogens. The designed PNA probe (CJE195) bound with higher affinity to a previously reported low affinity site on the 16S rRNA than the corresponding DNA probe. PNA also overcame the problem of the lack of affinity due to the location of the binding site and the variation of the target sequence within species. The PNA probe specificity was tested with several bacterial species, including other Campylobacter spp. and their close relatives. All tested C. coli, C. jejuni and C. lari strains were hybridized successfully. Aging of the Campylobacter cultures caused the formation of coccoid forms, which did not hybridize as well as bacteria in the active growth phase, indicating that the probe could be used to assess the physiological status of targeted cells. The PNA FISH methodology detected C. coli by membrane filtration method from C. coli spiked drinking water samples.

Silicon nanowires for sequence-specific DNA sensing: device fabrication and simulation

Highly sensitive, sequence-specific and label-free DNA sensors were demonstrated by monitoring the electronic conductance of silicon nanowires (SiNWs) with chemically bonded single-stranded (ss) DNA or peptide nucleic acid (PNA) probe molecules. For a 12-mer oligonucleotide, tens of pM of target ss-DNA in solution was recognized when the complementary DNA oligonucleotide probe was attached to the SiNW surfaces. In contrast, ss-DNA samples of ×1000 concentration with a single-base mismatch produce only a weak signal due to nonspecific binding. In order to gain a physical understanding of the change in conductance of the SiNWs with the attachment of the DNA targets and the probes, process and device simulations of the two-dimensional cross sections of the SiNWs were performed. The simulations explained the width dependence of the SiNW conductance and provided understanding to improve the sensor performance.

An oligonucleotide microarray for microRNA expression analysis based on labeling RNA with quantum dot and nanogold probe

MicroRNAs (miRNAs) play important regulatory roles in animals and plants by targeting mRNAs for cleavage or translational repression. They have diverse expression patterns and might regulate various developmental and physiological processes. Profiling miRNA expression is very helpful for studying biological functions of miRNAs. We report a novel miRNA profiling microarray, in which miRNAs were directly labeled at the 3′ terminus with biotin and hybridized with complementary oligo-DNA probes immobilized on glass slides, and subsequently detected by measuring fluorescence of quantum dots labeled with streptavidin bound to miRNAs through streptavidin–biotin interaction. The detection limit of this microarray for miRNA was ∼0.4 fmol, and the detection dynamic range spanned about 2 orders of magnitude. We made a model microarray to profile 11 miRNAs from leaf and root of rice (Oryza sativa L. ssp. indica) seedlings. The analysis results of the miRNAs had a good reproducibility and were consistent with the northern blot result. To avoid using high-cost detection equipment, colorimetric detection, a method based on nanogold probe coupled with silver enhancement, was also successfully introduced into miRNA profiling microarray detection.

High Reproducibility using Sodium Hydroxide-Stripped Long Oligonucleotide DNA Microarrays

Recently, long oligonucleotide (60- to 70-mer) microarrays for two-color experiments have been developed and are gaining widespread use. In addition, when there is limited availability of mRNA from tissue sources, RNA amplification can and is being used to produce sufficient quantities of cRNA for microarray hybridization. Taking advantage of the selective degradation of RNA under alkaline conditions, we have developed a method to "strip" glass-based oligonucleotide microarrays that use fluorescent RNA in the hybridization, while leaving the DNA oligonucleotide probes intact and usable for a second experiment. Replicate microarray experiments conducted using stripped arrays showed high reproducibility, however, we found that arrays could only be stripped and reused once without compromising data quality. The intraclass correlation (ICC) between a virgin array and a stripped array hybridized with the same sample showed a range of 0.90-0.98, which is comparable to the ICC of two virgin arrays hybridized with the same sample. Using this method, once-stripped oligonucleotide microarrays are usable, reliable, and help to reduce costs.

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 …

LNA-modified oligonucleotides are highly efficient as FISH probes

Fluorescence in situ hybridization (FISH) is a highly useful technique with a wide range of applications including the delineation of complex karyotypes, prenatal diagnosis of aneuploidies, screening for diagnostic or prognostic markers in cancer cells, gene mapping and gene expression studies. However, it is still a fairly time-consuming method with limitations in both sensitivity and resolution. Locked Nucleic Acids (LNAs) constitute a novel class of RNA analogs that have an exceptionally high affinity towards complementary DNA and RNA. Substitution of DNA oligonucleotide probes with LNA has shown to significantly increase their thermal duplex stability as well as to improve the discrimination between perfectly matched and mismatched target nucleic acids. To exploit the improved hybridization properties of LNA oligonucleotides in FISH, we have designed several LNA substituted oligonucleotide probes specific to different human-specific repetitive elements, such as the classical satellite-2, telomere and alpha-satellite repeats. In the present study we show that LNA modified oligonucleotides are excellent probes in FISH, combining high binding affinity with short hybridization time.

Alteration of the selectivity of hybridization of immobilized oligonucleotide probes by co-immobilization with charged oligomers of ethylene glycol

A significant challenge exists in the creation of an environment for immobilized probe oligonucleotides that offer good structural regularity and reproducibility, where nearest neighbour interactions provide for control of selectivity, yet where the degree of hybridization does not alter nearest neighbour interactions. This new work explores whether a “matrix isolation” method will produce the desired environment for the probe molecules. The DNA oligonucleotide probes are polyelectrolytes with charged backbones and significant flexibility. It is possible to isolate the probe molecules by surrounding each, on average, with a sheath of immobilized oligomer that is not based on complementary nucleic acid, yet that is a polyelectrolyte in order to control the surface density and charge within the mixed film. Preliminary work investigates a mixture of dT 20 as the probe oligonucleotide, and a 20-mer oligomer primarily containing ethylene glycol phosphate, as a matrix isolation material in a 1:20 mole ratio, respectively. Melt temperature ( T m) measurements indicate that the thermodynamic stability of the probe molecules can be adjusted using the oligomer matrix to achieve lower T m values by up to 5 °C, with full retention of selectivity for discrimination of single base pair mismatches even under conditions where the probes at a surface are saturated with complementary target.

A single species, Micromonas pusilla (Prasinophyceae), dominates the eukaryotic picoplankton in the Western English Channel.

The class Prasinophyceae (Chlorophyta) contains several photosynthetic picoeukaryotic species described from cultured isolates. The ecology of these organisms and their contributions to the picoeukaryotic community in aquatic ecosystems have received little consideration. We have designed and tested eight new 18S ribosomal DNA oligonucleotide probes specific for different Prasinophyceae clades, genera, and species. Using fluorescent in situ hybridization associated with tyramide signal amplification, these probes, along with more general probes, have been applied to samples from a marine coastal site off Roscoff (France) collected every 2 weeks between July 2000 and September 2001. The abundance of eukaryotic picoplankton remained high (>10(3) cells ml(-1)) during the sampling period, with maxima in summer (up to 2 x 10(4) cells ml(-1)), and a single green algal species, Micromonas pusilla (Prasinophyceae), dominated the community all year round. Members of the order Prasinococcales and the species Bathycoccus prasinos (Mamiellales) displayed sporadic occurrences, while the abundances of all other Prasinophyceae groups targeted remained negligible.

Preferred orientation of DNA oligonucleotide probes on the (2×4) reconstructed surface of (001) GaAs

Determination of the orientation of the thiol-derivatized DNA oligonucleotide monolayer on arsenic terminated GaAs(001) has been performed using grazing incidence x-ray scattering. DNA oligonucleotides were observed to orient along the trenches of the 2X direction of the GaAs(001)-(2×4) reconstructed surface, at an angle of 33° with respect to the surface. The addition of mercaptohexanol spacer tended to partially align the DNA oligonucleotides at almost 48° with respect to GaAs (001) surface.

Enzymatic Synthesis of Redox‐Labeled RNA and Dual‐Potential Detection at DNA‐Modified Electrodes

Ferrocene and anthraquinone were used to label RNA (red, dark blue), which could then be detected at an electrode surface bearing complementary DNA oligonucleotide probes (light blue). This approach is an electrochemical analog of popular fluorescence methods for gene expression analysis. Supporting information for this article is available on the WWW under http://www.wiley-vch.de/contents/jc_2002/2004/z52977_s.pdf or from the author. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Chromogenic In Situ Hybridization for α6β4 Integrin in Breast Cancer : Correlation with Protein Expression

The alpha6beta4 integrin is the receptor for the basement membrane protein laminin-5. Recent studies suggest that alpha6beta4 integrin expression in invasive breast carcinomas may be a poor prognostic factor. Because we have not had reliable results with commercially available antibodies for the immunohistochemical detection of alpha6beta4 integrin in archival paraffin-embedded tissues, we designed a probe to detect beta4 integrin subunit mRNA in paraffin sections. In situ hybridization for beta4 mRNA was performed on paraffin-embedded tissue sections of 25 invasive breast carcinomas using a hyperbiotinylated oligonucleotide DNA probe. Immunohistochemical staining was performed on corresponding frozen tumor sections using two commercially available antibodies to the beta4 integrin subunit. All cases positive for beta4 protein by one or both antibodies were also positive for beta4 mRNA by in situ hybridization, but three cases with beta4 mRNA expression were negative by immunohistochemistry with both antibodies. These findings suggest that in situ hybridization appears to be a sensitive method for detecting beta4 integrin mRNA, but it appears to identify some cases that either lack beta4 protein or express variants not recognized with commercial antibodies directed to particular extracellular or cytoplasmic domains.

Determination of Cryptosporidium parvum oocyst viability by fluorescence in situ hybridization using a ribosomal RNA-directed probe.

Fluorescence in situ hybridization (FISH) has been proposed for species-specific detection, and viability determination of Cryptosporidium parvum oocysts. FISH-based viability determination depends on rRNA decay after loss of viability. We examined the effects of RNase(s) and RNase inhibitors on FISH of C. parvum. FISH was performed using a 5'-Texas red-labelled DNA oligonucleotide probe at 1 pM microl(-1). Intact and heat-permeabilized oocysts were treated with 1-100 microg ml(-1) RNase. FISH of intact oocysts appeared unaffected by exogenous RNase if this was neutralized before permeabilization. FISH fluorescence of heat-killed oocysts stored in phosphate-buffered saline at room temperature decayed by 1/2 after 55 h, but remained detectable after 6 days. Addition of vanadyl ribonucleoside complex (VRC) extended rRNA half-life of heat-permeabilized oocysts to 155 h. Extended rRNA half-life may result in viability overestimation using FISH. RNase pretreatment before FISH is recommended to destroy residual rRNA in recently killed oocysts. Incorporation of 1-10 mM l(-1) VRC before FISH permeabilization steps should neutralize RNase activity. Elimination of FISH fluorescence of nonviable C. parvum is desirable. Use of RNase and VRC is suggested to reduce numbers of false-positive 'viable' oocysts.

Efficient method for mapping and characterizing structures of deletion mutations in gpt delta mice using Southern blot analysis with oligo DNA probes.

Efficient method for mapping and characterizing structures of deletion mutations in gpt delta mice using Southern blot analysis with oligo DNA probes.