Automated One-Double-Z Pair BaseScope™ for CircRNA In Situ Hybridization.

Circular RNAs (circRNAs) are single-stranded RNA, typically exons, connected head to tail by back-splicing. The functions of circRNAs include binding of microRNA, regulation of transcription, regulation of alternative splicing, and modulation of immune response. As for other RNA transcripts their levels vary during development and may also become deregulated during disease progression. Different from linear RNAs, the circRNAs are not susceptible to traditional exonuclease activity and therefore more stable in tissues and blood. This makes the circRNAs an attractive new group of potential biomarkers. Specific detection of circRNAs in situ is challenged by the need to discriminate bona fide circRNAs from the linear precursor forms and splice variants that contain largely overlapping sequences. Knowing the sequence around the splice junction site makes the branched DNA probe technology, BaseScope, suitable for selective detection of unique circRNAs. Here, we present the automated application of BaseScope with a one-double-Z pair probe set designed for the junction of circHIPK3.

Anti-counterfeiting DNA molecular tagging of pharmaceutical excipients: An evaluation of lactose containing tablets.

The licensed pharmaceutical industry and regulators use many approaches to control counterfeiting, but it remains a very difficult task to differentiate between counterfeit and real products. Moreover, there is a lack of techniques available for providing a batch specific molecular bar code for tablets that has the required traceability, specificity and sensitivity to be fit for purpose. The aim of this study was to evaluate DNA molecular tags as a potential anti-counterfeiting technology in tablets. Lactose tablets (400 mg) were used as a model to investigate incorporation DNA molecular tag into a solid dosage form: DNA authentication was carried out on an Applied DNA SigNify® qPCR instrument. Tablet batches were subjected to accelerated stability conditions (40 °C and 75% RH) for up to 6 months. All batches passed the monograph specifications of the British Pharmacopoeia (hardness, friability and mass uniformity) throughout the storage period. In all of recovery plots, the number of cycles required for DNA detection (Cq values) increased as a function of storage time, which indicated a reduction in tag levels, but it should be noted for all storage experiments the tag was clearly detected. It would appear that DNA molecular tags could feasibly be applied within the pharmaceutical development cycle when a new solid dosage form is brought to the market so as to mitigate the risk and dangers of counterfeiting.

Fluorescence In Situ Hybridization and Rehybridization Using Bacterial Artificial Chromosome Probes.

Fluorescence in situ hybridization (FISH) method enables in situ genetic analysis of both metaphase and interphase cells from different types of material, including cell lines, cell smears, and fresh and paraffin-embedded tissue. Despite the growing number of commercially available FISH probes, still for large number of gene loci or chromosomal regions commercial probes are not available. Here we describe a simple method for generating FISH probes using bacterial artificial chromosomes (BAC). Due to genome-wide coverage of BAC clones, there are almost unlimited possibilities for the analysis of any genomic regions using BAC FISH probes.

Electrophoretic mobility shift assay for the analysis of interactions of jasmonic acid-responsive transcription factors with DNA.

The electrophoretic mobility shift assay based on nondenaturing polyacrylamide gel electrophoresis is a simple, rapid, and sensitive method for the study of the interaction of transcription factors with DNA in vitro. It relies on a change in the electrophoretic mobility of a DNA fragment when bound to an interacting protein. The assay can be used to test DNA binding of either purified or recombinant proteins or uncharacterized binding activities present in crude protein extracts from plant cells or nuclei. It allows the determination of the abundance, affinity, association rate constants, dissociation rate constants, and binding specificity of DNA-binding proteins.

Transcript localization in Dictyostelium discoideum cells by RNA FISH.

Biogenesis of ribosomal RNA (rRNA) takes place preliminary in the nucleolus of eukaryotic cells, the site of rDNA transcription. Several processing steps of rRNA molecules have been implied to take place in the cytoplasm. To follow these processing events we have adapted protocols for fluorescence in situ hybridization (FISH) for use in Dictyostelium discoideum. We describe methods for the generation of suitable fluorescently labeled probes and the fixation of cells, by which we have localized different precursor and mature rRNA molecules to the nucleolus or the cytoplasm, respectively.

RNA imaging: tracking in real-time RNA transport in neurons using molecular beacons and confocal microscopy.

RNAs are present within eukaryotic cells and are involved in several biological processes. RNA transport within cell compartments is important for proper cell function. To understand in depth the cellular processes in which RNA is involved requires a method that reveals RNA localization in real time in a sub-cellular context in living cells. In this protocol we describe a method for imaging RNA in living cells and in particular in neuronal cultures based on cell microinjection of molecular beacons in conjunction with confocal microscopy. This methodology overcomes some of the main obstacles for imaging RNA in live cells since microinjection allows the delivery of the probe to a desired cellular compartment and MBs bind with high specificity to its target RNA without inhibiting its function. The proper design of the MBs is essential to obtain RNA-MB association at the temperature of the cell cytosol. MBs design with other purposes in mind (such as PCR experiments) have a design that facilitates association to its target at high temperatures, rendering them unsuitable for live cell imaging. Using the methodology described in this chapter allows the study of RNA transport to different regions of neurons and may be combined with the tagging of proteins of interest to measure co-transport of the protein and the RNA to different cellular regions.

Fluorescence in situ hybridization techniques for cytogenetic and genomic analyses.

Fluorescent in situ hybridization (FISH) is a powerful method to visualize DNA sequences in the context of the whole chromosome. Yet despite the value of FISH analysis for cytogenetic studies, there are surprisingly few labs that are able to adapt the technique for their experiments in chromosomal and genome biology. Here we present a comprehensive FISH protocol acquired from over 20 years of collective experience using different plant species. Our description uses rice as a model for performing a complete FISH procedure, but the protocol can be readily adapted for other plant species. We have provided more specialized instruction beyond routine FISH, which includes the preparation of meiotic and mitotic samples suitable for FISH analysis, procedures for direct and indirect labeling of DNA probes, and techniques for increasing signal strength using layers of antibodies.

A rapid, cost-effective method of assembly and purification of synthetic DNA probes >100 bp.

Here we introduce a rapid, cost-effective method of generating molecular DNA probes in just under 15 minutes without the need for expensive, time-consuming gel-extraction steps. As an example, we enzymatically concatenated six variable strands (50 bp) with a common strand sequence (51 bp) in a single pool using Fast-Link DNA ligase to produce 101 bp targets (10 min). Unincorporated species were then filtered out by passing the crude reaction through a size-exclusion column (<5 min). We then compared full-length product yield of crude and purified samples using HPLC analysis; the results of which clearly show our method yields three-quarters that of the crude sample (50% higher than by gel-extraction). And while we substantially reduced the amount of unligated product with our filtration process, higher purity and yield, with an increase in number of stands per reaction (>12) could be achieved with further optimization. Moreover, for large-scale assays, we envision this method to be fully automated with the use of robotics such as the Biomek FX; here, potentially thousands of samples could be pooled, ligated and purified in either a 96, 384 or 1536-well platform in just minutes.

Bacteriemias por Escherichia coli productor de betalactamasas de espectro extendido (BLEE): significación clínica y perspectivas actuales / Bacteraemia due to Escherichia coli producing extended-spectrum beta-lactamases (ESBL): clinical relevance and today’s’ insights

La resistencia bacteriana es un problema antiguo pero de gran actualidad, ya que en un marco de “austeridad” en cuanto al número de nuevas moléculas de antibiótico disponibles en el mercado, la presencia de microorganismos multirresistentes es cada vez más frecuente. E. coli es el microorganismo más frecuentemente implicado en bacteriemias nosocomiales y comunitarias, y el aislamiento de cepas productoras de BLEE se sitúa en torno al 10% en nuestro país. Las infecciones por E. coli con BLEE han experimentado importantes cambios epidemiológicos en los últimos tiempos y actualmente la atención se centra en el aumento de infecciones y colonizaciones en pacientes procedentes de la comunidad, sobre todo en relación con instituciones sanitarias, y la mayor incidencia de las CTX-M frente a otros tipos de BLEE. El papel de estas enzimas como factor de virulencia que aumente por sí mismo la mortalidad en los pacientes con bacteriemia por E. coli no queda claro. La principal repercusión clínica de las BLEE parece ser la mayor frecuencia con la que estos pacientes con infecciones graves reciben un tratamiento empírico inadecuado, de ahí la importancia de identificar qué factores predicen la presencia de una cepa con BLEE para poder ofrecer un tratamiento adecuado lo antes posible. En cuanto a las medidas de control de la diseminación de BLEE, la eficacia del aislamiento de contacto y la actuación frente a pacientes colonizados por E. coli con BLEE no están claras, pero es incuestionable la necesidad de implementar un uso correcto y responsable de los antibióticos para evitar la expansión de cepas resistentes(AU)

Antibiotic resistance is an old problem with new face as the rate of infections due to multidrug resistant bacteria is higher everyday and the number of new antibiotics to overwhelm the problem is becoming smaller. E. coli is the most frequent agent causing nosocomial or community-acquired bacteraemia being in our country 10% of them extended-spectrum betalactamases (ESBL) producing E. coli isolates. Nowadays the number of community- acquired or health-related infections caused by these ESBL producing E. coli is increasing. CTX-M has also become the most frequent ESBL compared to other enzymes. The role of these enzymes as a virulence factor increasing mortality in patients with bacteraemia due to E. coli is not well defined. The relevance of ESBL- E. coli seems to be related with the higher frequency of inadequate treatment and therefore the importance of identifying factors or features that might predict that the patient’s infection is due to one of these isolates. In terms of prevention and control of infection measures, the role of patient’s isolation is not clear but a proper prescription of antibiotics and antibiotic control policies are probably important to reduce the problem(AU)

Combining ligation reaction and capillary gel electrophoresis to obtain reliable long DNA probes.

New DNA amplification methods are continuously developed for sensitive detection and quantification of specific DNA target sequences for, e.g. clinical, environmental or food applications. These new applications often require the use of long DNA oligonucleotides as probes for target sequences hybridization. Depending on the molecular technique, the length of DNA probes ranges from 40 to 450 nucleotides, solid-phase chemical synthesis being the strategy generally used for their production. However, the fidelity of chemical synthesis of DNA decreases for larger DNA probes. Defects in the oligonucleotide sequence result in the loss of hybridization efficiency, affecting the sensitivity and selectivity of the amplification method. In this work, an enzymatic procedure has been developed as an alternative to solid-phase chemical synthesis for the production of long oligonucleotides. The enzymatic procedure for probe production was based on ligation of short DNA sequences. Long DNA probes were obtained from smaller oligonucleotides together with a short sequence that acts as bridge stabilizing the molecular complex for DNA ligation. The ligation reactions were monitored by capillary gel electrophoresis with laser-induced fluorescence detection (CGE-LIF) using a bare fused-silica capillary. The capillary gel electrophoresis-LIF method demonstrated to be very useful and informative for the characterization of the ligation reaction, providing important information about the nature of some impurities, as well as for the fine optimization of the ligation conditions (i.e. ligation cycles, oligonucleotide and enzyme concentration). As a result, the yield and quality of the ligation product were highly improved. The in-lab prepared DNA probes were used in a novel multiplex ligation-dependent genome amplification (MLGA) method for the detection of genetically modified maize in samples. The great possibilities of the whole approach were demonstrated by the specific and sensitive detection of transgenic maize at percentages lower than 1%.

Visualizing mRNAs in fixed and living yeast cells.

Localization of messenger RNA (mRNA) is a process used by eukaryotes to control the spatio-temporal expression of proteins involved in cellular motility, asymmetric cell division, or polarized cell growth. A better understanding of this process relies on methods to detect specifically the position of an mRNA in fixed or living cells. This chapter presents methods to visualize mRNA in both fixed and living yeast Saccharomyces cerevisiae. In fixed cells, position of mRNAs can be assessed by using Fluorescent In Situ Hybridization (FISH) that consists of the hybridization of fluorescent probes that target a specific transcript in situ. In living cells, dynamics of mRNAs can be monitored using a bipartite system composed of MS2 stem-loops inserted in the mRNA of interest. These stem-loops are recognized specifically by the MS2 RNA-binding protein, fused to a fluorescent protein. In vivo association between the reporter (fluorescent MS2 protein) and the MS2-tagged mRNA reconstitutes active fluorescent ribonucleoparticles that can be followed by live cell imaging. Detailed protocols for the realization of these methods are provided and several technical considerations are discussed. Together, these methods provide very robust tools to determine the intracellular position and dynamics of your mRNA of interest in yeast.

Fluorescence in situ hybridization analysis of formalin fixed paraffin embedded tissues, including tissue microarrays.

Formalin fixed paraffin embedded (FFPE) material is frequently the most convenient readily available source of diseased tissue, including tumors. Multiple cores of FFPE material are being used increasingly to construct tissue microarrays (TMAs) that enable simultaneous analyses of many archival samples. Fluorescence in situ hybridization (FISH) is an important approach to analyze FFPE material for specific genetic aberrations that may be associated with tumor types or subtypes, cellular morphology, and disease prognosis. Annealing, or hybridization of labeled nucleic acid sequences, or probes, to detect and locate one or more complementary nucleic acid sequences within fixed tissue sections allows the detection of structural (translocation/inversion) and numerical (deletion/gain) aberrations and their localization within tissues. The robust protocols described include probe preparation, hybridization, and detection and take 2-3 days to complete. A protocol is also described for the stripping of probes for repeat FISH in order to maximize the use of scarce tissue resources.

Electrophoretic mobility shift assays for the analysis of DNA-protein interactions.

Electromobility shift assay is a simple, efficient, and rapid method for the study of specific DNA-protein interactions. It relies on the reduction in the electrophoretic mobility conferred to a DNA fragment by an interacting protein. The technique is suitable to qualitative, quantitative, and kinetic analyses. It can also be used to analyze conformational changes.

A real-time Taqman method for hepatitis C virus genotyping and methods for further subtyping of isolates.

The HCV genome is highly heterogeneous; more and more genotypes, each with several distinct subtypes, are being identified around the world. Knowledge of genotype is important for planning of treatment regimes, whereas subtype identification is useful in epidemiological studies and outbreak investigation. We describe HCV genotyping and subtyping assays, based on real-time PCR, that are sensitive, specific, and reliable. These assays provide fast, accurate, and convenient methods for HCV genotyping/subtyping to support clinical practice.

An efficient chemical method to generate repetitive sequences depleted DNA probes.

We describe an efficient method to remove repetitive sequences from DNA of microdissected whole chromosomes, chromosome arms, locus specific probes, and specific bands. The chemical approach described removes human repetitive DNA sequences from a source DNA, eliminating the need for blocking such sequences when the source DNA used as a probe is hybridized with a target DNA. It employs subtracting hybridized biotin-labeled repetitive-sequence DNA complex with phenol and chloroform after incubation of hybridized products with avidin. The method produces unique products that are formed after such repetitive sequences have been removed from the DNA. We have applied our newly designed subtraction strategy to microdissected chromosomes in generating whole chromosome painting (e.g., chromosome 4), chromosome arm (e.g., 1q and 15q), and band (e.g., 3q26) specific probes, and we have demonstrated its utility using flow-sorted chromosome. FISH analyses using these probes show consistent strong signals with no cross-hybridization, and optimal hybridization results can be obtained relatively quickly.

Identification of an anonymous RFLP DNA probe through multiple arbitrary amplicon profiling and its use for strain differentiation in a field isolate of cellulose-degrading Aspergillus niger.

The genome of Aspergillus niger (MPS-002) was subjected to RAPD fingerprinting using none different random oligonucleotide primers. A 0.7 Kb PCR amplicon, generated by primer-3 could be used as a RFLP probe to differentiate A. niger (ATCC 16880) from A. niger (MPS-002). The probe revealed DNA polymorphism internal to two different EcoRI recognition sequences spaced apart at a distance of 0.4 Kb within a 4.0 Kb EcoRI fragment of the genome of both the strains. Localized genome mapping analysis further revealed that the 0.7 Kb RFLP probe was positioned at a distance of 2.7 Kb and 0.6 Kb from the two ends of a 4.0 Kb EcoRI fragment, respectively within the genome of the two strains of A. niger.

Methods of detection of Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis and Tannerella forsythensis in periodontal microbiology, with special emphasis on advanced molecular techniques: a review.

BACKGROUND: Certain specific bacterial species from the subgingival biofilm have demonstrated aetiological relevance in the initiation and progression of periodontitis. Among all the bacteria studied, three have shown the highest association with destructive periodontal diseases: Actinobacillus actinomycetemcomitans (Aa), Porphyromonas gingivalis (Pg) and Tannerella forsythensis (Tf). Therefore, the relevance of having accurate microbiological diagnostic techniques for their identification and quantification is clearly justified. AIM: To evaluate critically all scientific information on the currently available microbial diagnostic techniques aimed for the identification and quantification of Aa, Pg and Tf. SUMMARY: Bacterial culturing has been the reference diagnostic technique for many years and, in fact, most of our current knowledge on periodontal microbiology derives from cultural data. However, the advent of new microbial diagnostics, mostly based on immune and molecular technologies, has not only highlighted some of the shortcomings of cultural techniques but has also allowed their introduction as easy and available adjunct diagnostic tools to be used in clinical research and practice. These technologies, mostly polymerase chain reaction (PCR), represent a field of continuous development; however, we still lack the ideal diagnostic to study the subgingival microflora. Qualitative PCR is still hampered by the limited information provided. Quantitative PCR is still in development; however, the promising early results reported are still hampered by the high cost and the equipment necessary for the processing. CONCLUSION: Quantitative PCR technology may have a major role in the near future as an adjunctive diagnostic tool in both epidemiological and clinical studies in periodontology. However, culture techniques still hold some inherent capabilities, which makes this diagnostic tool the current reference standard in periodontal microbiology.

End labeling procedures: an overview.

There are two ways to label a DNA molecular; by the ends or all along the molecule. End labeling can be performed at the 3'- or 5'-end. Labeling at the 3' end is performed by filling 3'-end recessed ends with a mixture or labeled and unlabeled dNTPs using Klenow or T4 DNA polymerases. Both reactions are template dependent. Terminal deoxynucleotide transferase incorporates dNTPs at the 3' end of any kind of DNA molecule or RNA. Labels incorporated at the 3'-end of the DNA molecule prevent any further extension or ligation to any other molecule, but this can be overcome by labeling the 5'-end of the desired DNA molecule. 5'-end labeling is performed by enzymatic methods (T4 polynucleotide kinase exchange and forward reactions), by chemical modification of sensitized oligonucleotides with phosphoroamidite, or by combined methods. Probe cleanup is recommended when high background problems occur, but caution should be taken not to damage the attached probe with harsh chemicals or by light exposure.

Evaluation of dual-labeled fluorescent DNA probe purity versus performance in real-time PCR.

Real-time PCR technology using dual-labeled fluorescent oligonucleotide probes allows for sensitive, specific, and quantitative determination of mRNA or DNA targets. Historically, dual-labeled probes have been the most expensive reagent in real-time PCR because of the postsynthesis high-performance liquid chromatography (HPLC) and/or gel purification steps required due to limitations in traditional synthesis chemistry. The recent availability of quencher reagents that allow the 3' quencher incorporation as part of the on-machine synthesis has presented the possibility that probes, when carefully synthesized, may be used without extensive postsynthesis purification. This would substantially reduce cost, making the synthesis of dual-labeled fluorescent probes affordable to any DNA synthesis laboratory. The Nucleic Acids Research Group (NARG) of the Association of Biomolecular Resource Facilities (ABRF) (Santa Fe, NM, USA) tested the hypothesis that now any DNA synthesis laboratory is capable of making quality dual-labeled fluorescent probes suitable for real-time PCRs without the need for postsynthesis purification. Members of the DNA synthesis community synthesized dual-labeled human beta-actin probes and submitted them for quality and functional analysis. We found that probes that were at least 20% pure had the same efficiency as those near 100% purity, but the sensitivity of the assay was reduced as the level of purity decreased.

Distribution analysis of IS231-like sequences among Bacillus thuringiensis serovars inferred from restriction fragment length polymorphisms.

The distribution of IS231 has been analyzed in Bacillus thuringiensis serovars. A 723-bp HaeII conserved fragment from IS231M has been used as a probe against EcoRI-digested B. thuringiensis total DNA to yield serovar-specific hybridization profiles. The approach was useful at revealing the extent of distribution of IS231-like sequences between and within strains. Of the 88 B. thuringiensis strains tested, 70 showed hybridization banding patterns that comprised between one and 20 distinct bands. These 70 B. thuringiensis strains were grouped based on banding pattern similarities. Interestingly, intraserovar strains did not necessarily cluster together.