Stringent Hybridization Conditions Research Articles

Next-generation in situ hybridization chain reaction: higher gain, lower cost, greater durability.

Hybridization chain reaction (HCR) provides multiplexed, isothermal, enzyme-free, molecular signal amplification in diverse settings. Within intact vertebrate embryos, where signal-to-background is at a premium, HCR in situ amplification enables simultaneous mapping of multiple target mRNAs, addressing a longstanding challenge in the biological sciences. With this approach, RNA probes complementary to mRNA targets trigger chain reactions in which metastable fluorophore-labeled RNA hairpins self-assemble into tethered fluorescent amplification polymers. The properties of HCR lead to straightforward multiplexing, deep sample penetration, high signal-to-background, and sharp subcellular signal localization within fixed whole-mount zebrafish embryos, a standard model system for the study of vertebrate development. However, RNA reagents are expensive and vulnerable to enzymatic degradation. Moreover, the stringent hybridization conditions used to destabilize nonspecific hairpin binding also reduce the energetic driving force for HCR polymerization, creating a trade-off between minimization of background and maximization of signal. Here, we eliminate this trade-off by demonstrating that low background levels can be achieved using permissive in situ amplification conditions (0% formamide, room temperature) and engineer next-generation DNA HCR amplifiers that maximize the free energy benefit per polymerization step while preserving the kinetic trapping property that underlies conditional polymerization, dramatically increasing signal gain, reducing reagent cost, and improving reagent durability.

Natural infection of sweet cherry trees with Apple scar skin viroid.

Apple scar skin viroid (ASSVd) is a fruit-damaging pathogen that causes significant economic losses to pome fruit trees. In the context of a survey on fruit tree viroids in Greece, ASSVd was initially detected by RT-PCR in two sweet cherry trees of cv. Tragana Edessis in an apple orchard in Florina (Macedonia, Greece). This finding was confirmed by direct viroid sequencing of the amplified RT-PCR products. In order to verify this finding, we further examined four sweet cherry trees cvs Tragana Edessis and Biggareau Burlat, two sweet cherry trees of undetermined cultivar, and fifteen neighboring apple trees in the same orchard for possible infection with ASSVd. The viroid assay was done by tissue print hybridization using an ASSVd-specific DIG-labeled probe at stringent hybridization conditions and by RT-PCR using two different ASSVd-specific primer pairs. ASSVd was detected in the six sweet cherry trees, including symptomatic samples, but not in any of the 15 apple trees. Purified ASSVd-positive RT-PCR products from sweet cherries were sequenced either directly or after cloning into pGEM-T or pCR II plasmid vectors. Sixteen ASSVd sequences obtained from five trees were 327-340 nucleotide long and shared 96- 99% identity with ASSVd isolates from Indian apples. There was no cherry-specific nucleotide changes in the ASSVd sequences obtained. The viroid was graft transmitted successfully from cherry trees to cherry rootstocks and the newly developed rootstock leaves were ASSVd-positive by RT-PCR. To our knowledge, this is the first molecular and biological analyses of ASSVd infecting sweet cherry trees.

Specificity of randomly generated genomic DNA fragment probes on a DNA array

The use of randomly generated DNA fragment sequences as probes on DNA arrays offers a unique potential for exploring unsequenced microorganisms. In this study, the detection specificity was evaluated with respect to probe-target sequence similarity using genomic DNAs of four Pseudomonas strains. Genome fragments averaging 2000bp were found to be specific enough to discriminate 85-90% similarity under highly stringent hybridization conditions. Such stringent conditions compromised signal intensities; however, specific signals remained detectable at the highest stringency (at 75°C hybridization) with negligible false negatives. These results suggest that, without any probe design or selection, genomic fragments can provide a reasonable specificity for microbial diagnostics or species delineation by DNA-DNA similarities.

In situ DNA amplification with magnetic primers for the electrochemical detection of food pathogens

A sensitive and selective genomagnetic assay for the electrochemical detection of food pathogens based on in situ DNA amplification with magnetic primers has been designed. The performance of the genomagnetic assay was firstly demonstrated for a DNA synthetic target by its double-hybridization with both a digoxigenin probe and a biotinylated capture probe, and further binding to streptavidin-modified magnetic beads. The DNA sandwiched target bound on the magnetic beads is then separated by using a magneto electrode based on graphite–epoxy composite. The electrochemical detection is finally achieved by an enzyme marker, anti-digoxigenin horseradish peroxidase (HRP). The novel strategy was used for the rapid and sensitive detection of polymerase chain reaction (PCR) amplified samples. Promising resultants were also achieved for the DNA amplification directly performed on magnetic beads by using a novel magnetic primer, i.e., the up PCR primer bound to magnetic beads. Moreover, the magneto DNA biosensing assay was able to detect changes at single nucleotide polymorphism (SNP) level, when stringent hybridization conditions were used. The reliability of the assay was tested for Salmonella spp., the most important pathogen affecting food safety.

Monitoring synaptogenesis in the developing mouse cerebellum with an original oligonucleotide microarray

Use of DNA microarrays in neuroscience have been limited to rough screening purposes, mainly because the reliability and sensitivity of available arrays are not high enough. Because only a few hundred to one thousand genes are usually found to change expression levels in most experiments, we attempted to develop a more quantitative array by the following strategies: 1) limit the genes to those relevant to the experimental system, 2) design oligonucleotide probes of specified molecular properties so that more stringent hybridization conditions can be employed, 3) place six spots per probe on one slide and use multiple normalization genes, and 4) use a new type of gold-coated slide with higher S/N ratio. Genes involved in the construction and functioning of the synapse were selected from the literature as well as experimentally by comparing cerebella from hypothyroid and control mice at postnatal day 15 (P15). Although the number of genes covered was not yet large (172 genes), the custom array "Synaptoarray" thus constructed was capable of detecting +/-20% difference in gene expression ratios. Analysis of the postnatal development of the mouse cerebellum by using Synaptoarray demonstrated a general expression pattern with a peak at P7, followed by a decline at P15 and a partial recovery after P21. P10 clearly marked the end of the initial growth stage and a major transcriptional turning point in this system. This result suggests that such a custom array should be useful in monitoring perturbations to the normal developmental program.

Intercalating Nucleic Acids with Pyrene Nucleotide Analogues as Next‐Nearest Neighbors for Excimer Fluorescence Detection of Single‐Point Mutations under Nonstringent Hybridization Conditions

AbstractFluorescence and hybridization specificity is reported for intercalating nucleic acids (INAs), which are here oligodeoxynucleotides (ODNs) synthesized with insertions using (S)‐1‐[bis(4‐methoxyphenyl)(phenyl)methoxy]‐3‐[(pyren‐1‐yl)methoxy]propan‐2‐ol phosphoramidites. It is shown that an INA with two insertions placed as next‐nearest neighbors can be used for discrimination between nucleic acids and their single‐point mutants. Quenching of an excimer band at 480 nm is observed upon hybridizing to a complementary sequence, whereas the excimer band is present when the nucleobase pair between the two pyrene moieties is mismatched. It is the first example of a solution based on fluorescence detection of single‐point mutants that uses excimer formation and does not rely on stringent hybridization conditions. Furthermore, it is shown that INAs with pyrene insertions retain their sequence specificity in thermal melting.

Chemistry of Minor Groove Binder–Oligonucleotide Conjugates

Various types of minor groove binders have been attached to synthetic oligodeoxynucleotides, and the interactions of these conjugates (MB-ODNs) with DNA are reviewed here. MB-ODNs have enhanced DNA affinity and have improved the hybridization properties of sequence-specific DNA probes. Short MB-ODNs hybridize with ssDNA to give more stable DNA duplexes than unmodified ODNs with similar lengths. Mismatch discrimination of short MB-ODNs is enhanced in comparison to longer unmodified ODNs. The stronger binding of MB-ODNs allows for more stringent hybridization conditions to be used in DNA probe-based assays. MB-ODNs are especially useful in quantitative "real-time" PCR assays since they bind efficiently during the high-temperature primer extension cycle. The synthesis and biophysical chemistry of MB-ODN conjugates are reviewed here. Four published structural classes of MB-ODNs and their various dsDNA binding modes are discussed, and the well-characterized DPI3-type MB-ODNs and their interactions with ssDNA target strands are described in detail.

Rearrangement of the genes for the biosynthesis of benzoxazinones in the evolution of Triticeae species.

Gramineous plants, including the major agricultural crops wheat (Triticum aestivum L.), rye (Secale cereale L.) and maize (Zea mays L.), accumulate benzoxazinones (Bxs) as defensive compounds. Previously, we isolated cDNAs of the Bx biosynthetic genes in wheat, TaBx2- TaBx5, that encode the enzymes catalyzing the sequential hydroxylation of indole to Bxs. In this study we isolated a cDNA of TaBx1, which encodes the first enzyme of the Bx pathway of wheat. The level of identity (80%) in deduced amino-acid sequence between TaBx1 and the corresponding maize gene Bx1 was as high as those shown between TaBx2- TaBx5 and the corresponding maize genes Bx2- Bx5, respectively. Southern blot analysis using the TaBx1- TaBx5 cDNAs as probes was conducted with aneuploid lines of hexaploid wheat in order to determine sub-chromosomal locations of the five Bx biosynthetic genes in Triticeae species. In wheat, TaBx1 and TaBx2 co-existed in specific regions of chromosomes 4A, 4B and 4D, and TaBx3- TaBx5 were localized together in the distal regions of the short arms of chromosomes 5A, 5B and 5D. TaBx3 and TaBx5 were found to have duplicated loci in the long arm and the short arm of chromosome 5B, respectively. In rye, homoeoloci of TaBx1 and TaBx2 were located on chromosome 7R and those for TaBx3- TaBx5 were located on chromosome 5R. In barley, no Southern blot band was detected with any of the probes under the highly stringent hybridization conditions, suggesting that the non-Bx phenotype of barley is attributable to the loss of Bx biosynthetic genes.

An efficient in situ hybridization protocol for multiple tissue sections and probes on miniaturized slides.

To facilitate detection of gene activity in tissue sections we combined common protocols of in situ hybridization on tissue sections (TSISH) with the technique of whole-mount in situ hybridization (WMISH). Miniature glass slides for mounting tissue sections were cut from regular microscope slides and handled for in situ hybridization in laboratory-made 2-ml containers (baskets) similar to those originally used for WMISH on Drosophila embryos. A salient point of the method is the use of airtight reaction vessels placed in a dry thermostat for critical hybridization steps as this facilitates reproducible and stringent hybridization conditions which are difficult to achieve on tissue sections otherwise. The practicability of the method is illustrated on consecutive serial frozen sections of murine neonatal cerebellum hybridized for math1 and neuroD, two developmentally regulated genes with distinct expression patterns. For both genes excellent spatial resolution and a highly dynamic range of signal intensity was obtained. The approach enables simple processing of multiple probes, allows the efficient and economic use of small tissue samples and is amenable to automation.

Mutation detection by electrocatalysis at DNA-modified electrodes

Detection of mutations and damaged DNA bases is important for the early diagnosis of genetic disease. Here we describe an electrocatalytic method for the detection of single-base mismatches as well as DNA base lesions in fully hybridized duplexes, based on charge transport through DNA films. Gold electrodes modified with preassembled DNA duplexes are used to monitor the electrocatalytic signal of methylene blue, a redox-active DNA intercalator, coupled to [Fe(CN)6]3-. The presence of mismatched or damaged DNA bases substantially diminishes the electrocatalytic signal. Because this assay is not a measure of differential hybridization, all single-base mismatches, including thermodynamically stable GT and GA mismatches, can be detected without stringent hybridization conditions. Furthermore, many common DNA lesions and "hot spot" mutations in the human p53 genome can be distinguished from perfect duplexes. Finally, we have demonstrated the application of this technology in a chip-based format. This system provides a sensitive method for probing the integrity of DNA sequences and a completely new approach to single-base mismatch detection.

Specific detection of green sulfur bacteria by in situ hybridization with a fluorescently labeled oligonucleotide probe.

An oligodeoxynucleotide probe (GSB-532) specific for green sulfur bacteria was developed. Highly stringent hybridization conditions were established using whole cells of Chlorobium limicola DSM249 immobilized on glass slides. At a formamide concentration of 10%, the optimum specificity was reached at 47 degrees C. When a conventional fixation procedure was used, a conspicuous autofluorescence developed within the cells. This autofluorescence was due to the liberation of bacteriochlorophyll by the detergent Triton X-100 and a subsequent conversion to bacteriophenophytin and related compounds. The signal-to-noise ratio could be increased by a final dehydration of the samples with methanol. Finally, the method was adapted to the hybridization of natural samples collected on polycarbonate membrane filters. In situ hybridization of pure cultures, various enrichments, and natural samples from the chemocline of a freshwater lake confirmed that probe GSB-532 hybridized exclusively to cells of green sulfur bacteria. Our protocol allows the highly specific detection of green sulfur bacteria in water samples and a rapid screening of natural bacterial communities. Employing probe GSB-532, the phylogenetic affiliation of the epibionts in "Chlorochromatium aggregatum" and "Pelochromatium roseum" could be demonstrated for the first time.

A PCR screen identifies a novel, unconventional myosin heavy chain gene (MYO1) in Tetrahymena thermophila.

Degenerate primers for two regions of sequence homology in the myosin head domain were used in a polymerase chain reaction screen of Tetrahymena thermophila genomic DNA to amplify a 765 bp fragment that was cloned and sequenced. Based on the presence of conserved, myosin-specific sequences, the 765 bp PCR product was identified as a fragment of a myosin gene, the first to be discovered in ciliated protozoa and herein referred to as MYO1. An inverse polymerase chain reaction strategy was used to obtain additional sequence data that included the entire head domain of MYO1. Alignment of the predicted amino acid sequence of the MYO1 head domain with known myosin sequences identified the ATP-binding site, a phosphorylation site, and other myosin-specific consensus regions. In a northern blot analysis, a 765 bp MYO1-specific probe detected a 6.6 kb transcript under highly stringent hybridization conditions. Phylogenetic analysis revealed that the predicted protein encoded by MYO1 is not a member of any of the previously defined myosin classes and therefore represents a presumptive new myosin class.

Rapid method for visual identification of specific DNA sequences based on DNA-tagged liposomes

We describe a rapid method for visually determining specific DNA sequences at femtomole concentrations. Liposomes, encapsulating a red dye and labeled with oligonucleotide, were used in a capillary migration-sandwich hybridization assay. Capture probe was immobilized on nitrocellulose strips, and liposomes, migrating along each strip, formed a visually discernible band in the presence of target DNA. One femtomole of synthetic target sequence could be detected in < 10 min. Sufficiently stringent hybridization conditions can be used to allow the discrimination of a 10% mismatch sequence from perfectly complementary DNA. A 366-base PCR product was detected at 200 fmol.

Incidence of human papilloma virus in patients with invasive cervical carcinoma

The dot-blot hybridization of biotin-d UTP-labeled HPV-16 DNA with genomic DNA extracted from biopsies taken from patients with invasive carcinoma and abnormal cytology showed the presence of HPV-DNA in 88% and 80% cases under relaxed conditions and 40% and 20% cases under stringent conditions of hybridization, respectively. Southern blot revealed the HPV-DNA in randomly integrated form in two cases and in episomal form in the other two.

Expression profiling of mRNA obtained from single identified crustacean motor neurons: determination of specificity of hybridization.

The purpose of this study was to determine if the technique of expression profiling would allow us to determine the changes in the abundance of certain mRNAs in identifiable, single neurons as a result of heightened electrical activity. In doing so we developed an approach to test the specificity of hybridization in expression profiling. Messenger RNA from single identified crayfish motor neurons was amplified by ligation-mediated reverse transcription PCR and hybridized by dot-blotting to 45 target cDNAs from different species. As a test of specificity, the hybridization was repeated using unlabelled cDNAs, the dots were excised, and the hybridized nucleic acids were re-amplified, cloned, and sequenced to confirm their identity. By cloning and sequencing the re-amplified product for each cDNA examined, one can determine the degree of background hybridization as compared to homologous hybridization. False positive results were also observed when a species-specific cDNA and highly stringent hybridization conditions were used. Our results demonstrate that ligation-mediated PCR is a useful technique for checking the specificity of expression profiling. This approach can easily be adapted to any situation when confirmation of the specificity of nucleic acid hybridization is required. During this study, part of a novel crayfish neuronal actin cDNA was cloned and sequenced.

CDNA sequence and expression pattern of the putative pheromone carrier aphrodisin.

The cDNA sequence for aphrodisin, a lipocalin from hamster vaginal discharge which is involved in pheromonal activity, has been determined. Corresponding genomic clones were isolated and the promoter region was identified. Primer extension analysis revealed an adenosine residue as the main transcription initiation site, located 50 bp upstream of the translation start codon ATG, which is surrounded by a typical Kozak sequence. However, data from polymerase chain reaction analysis suggest the existence of at least one alternative transcription initiation site. The aphrodisin cDNA is 732 bp long and codes for the mature 151-aa aphrodisin and an additional N-terminal 16-aa secretory signal peptide. The 3' nontranslated region is 228 bp long. Among the known sequences, the aphrodisin cDNA shares the highest homology with the rat odorant-binding protein cDNA (45%), which verifies the protein data. Vaginal tissue and Bartholin's glands are the main aphrodisin gene-expressing tissues of the female hamster genital tract, as demonstrated by Northern blot analysis. Under less stringent hybridization conditions, RNA isolated from rat Bartholin's glands also showed a signal, indicating the occurrence of aphrodisin-related mRNA in this species.

Characterization of a cloned pR72H probe for Vibrio parahaemolyticus detection and development of a nonisotopic colony hybridization assay.

Vibrio parahaemolyticus is a halophilic bacterium often found in shellfish and is an important causative agent of food poisoning in Taiwan. A rapid and efficient detection method is required to identify this foodborne pathogen. A 0.76-Kb HindIII DNA fragment was cloned from the chromosomal DNA of V. parahaemolyticus strain no. 93, designated as pR72H fragment, was used as a polynucleotide probe. It was labeled with digoxigenin-11-dUTP (DIG) by the random primer-labeling method. The sensitivity and specificity of the digoxigenin-labeled 0.76-Kb DNA probe was determined by colony hybridization assay. Under stringent hybridization conditions, 122 of 124 isolates of V. parahaemolyticus showed positive hybridization reaction with DIG-0.76-Kb DNA probe; the negative strains were attributed to slow growth. The DIG-0.76-Kb probe did not hybridize with 86 isolates of other vibrios and a number of other enterics as well as nonenteric microorganisms. The sensitivity and specificity of this DIG probe are 98% and 100%, respectively. This nonisotopic colony hybridization assay can be very useful for routine monitoring of V. parahaemolyticus in the food industry, environmental analysis and clinical laboratories.

Prolactin Receptor mRNA Localization in the Hypothalamus by in situ Hybridization

Prolactin receptors may mediate the action of prolactin in the brain to influence behavior and neuroendocrine secretions. We recently demonstrated prolactin receptor gene expression in the anterior and medial basal hypothalamus and not in the cortex by the reverse transcription-polymerase chain reaction. In this paper, we localize the prolactin receptor gene expression to individual cells with in situ hybridization. Several steps in the in situ hybridization method were modified to increase sensitivity by using (i) probes complementary to the coding sequence of the extracellular binding domain common to both long and short prolactin receptor, (ii) more stringent hybridization and wash conditions to reduce background and (iii) higher specific activity, more complex and saturating amounts of probe. We detected prolactin receptor gene expression in cells of the periventricular area of the preoptic nucleus, medial preoptic nucleus, supraoptic nucleus, rostral arcuate nucleus and choroid plexus. Cortical brain tissue, which has been demonstrated previously by reverse-transcription polymerase chain reaction to be lacking in prolactin receptor mRNA, did not have any detectable signal for the receptor mRNA and was used as an indication of background levels of signal. The mean area of silver grains over labeled cells in periventricular area of the preoptic nucleus, medial preoptic nucleus, supraoptic nucleus, arcuate nucleus, lateral ventromedial nucleus was at least 10 times greater than the background in the cortex of the same brain section.

Cellular localization of putative odorant receptor mRNAs in olfactory and chemosensory neurons: a non radioactive in situ hybridization study

The precise cellular localization of mRNAs for putative odorant receptors was investigated in the mouse chemosensory system (olfactory epithelium, septal organ and vomeronasal organ). Four additional members of the odorant receptor family were cloned from mouse olfactory mucosa and in situ hybridization was performed with paraffin-embedded tissue using digoxigenin labelled, non-radioactive antisense RNA probes for these individual receptor genes. The results clearly demonstrated expression of odorant receptors within single individual receptor neurons and there was no receptor expression either in the basal cells (stem cells) or supporting cells (sustentacular cells). In contrast to the uniform expression of olfactory marker protein mRNA within the layer of mature neurons, odorant receptor expression was localized in scattered individual cells but with a bilateral symmetry. The number of positive cells was far less than the number detected with the olfactory marker protein probe. Interestingly, rostro-caudal and dorso-ventral sites of expression were specific to each receptor probe. Under the highly stringent hybridization and washing conditions used here, even mixed RNA probes prepared from 4 different odorant receptor genes were only expressed in a maximum of 20–60 neurons per section (i.e. less than 0.1% of the population of total receptor neurons) suggesting the size of odorant receptor superfamilies to be larger than previously estimated. Some chemoreceptor neurons in the septal organ and vomeronasal organ also expressed odorant receptor mRNAs suggesting that these two additional non-olfactory chemosensory systems share the same chemoreceptive pathway as the olfactory system.

Evidence for a Wolbachia symbiont in Drosophila melanogaster.

The bacterial cell division gene, ftsZ, was used as a specific probe to show the presence of a symbiotic bacterium in two wild type strains of Drosophila melanogaster. Under stringent hybridization conditions we have shown that the bacterium is transferred to the progeny of these strains from infected mothers and can be eradicated by treatment with the antibiotic tetracycline. We have characterized this bacterium, by amplifying and sequencing its 16S rRNA gene, as being a member of the genus Wolbachia, an organism that is known to parasitize a range of insects including Drosophila simulans. In a series of reciprocal crosses no evidence was found that the symbiont causes cytoplasmic incompatibility (CI) which is known to occur in infected strains of D. simulans. The implications of these findings are discussed.