RESUMEN

This protocol describes a method to image individual mRNA molecules in situ. About 50 oligonucleotides complementary to different regions of a target mRNA species are used simultaneously. Each probe is labeled with a single fluorescent moiety. When these probes bind to their target, each mRNA molecule becomes so intensely fluorescent that it can be seen as a fine fluorescent spot. Several different mRNA species can be detected in multiplex imaging using differently colored probe sets for each species. An automated image-processing program is used to count the number of mRNA molecules of each species that are expressed in each cell, thus yielding single-cell gene expression profiles.

Asunto(s)

RESUMEN

BACKGROUND: The purpose of this study was to design and evaluate fluorescent in situ hybridization (FISH) probes for the single-cell detection and enumeration of lactic acid bacteria, in particular organisms belonging to the major phylogenetic groups and species of oral lactobacilli and to Abiotrophia/Granulicatella. RESULTS: As lactobacilli are known for notorious resistance to probe penetration, probe-specific assay protocols were experimentally developed to provide maximum cell wall permeability, probe accessibility, hybridization stringency, and fluorescence intensity. The new assays were then applied in a pilot study to three biofilm samples harvested from variably demineralized bovine enamel discs that had been carried in situ for 10 days by different volunteers. Best probe penetration and fluorescent labeling of reference strains were obtained after combined lysozyme and achromopeptidase treatment followed by exposure to lipase. Hybridization stringency had to be established strictly for each probe. Thereafter all probes showed the expected specificity with reference strains and labeled the anticipated morphotypes in dental plaques. Applied to in situ grown biofilms the set of probes detected only Lactobacillus fermentum and bacteria of the Lactobacillus casei group. The most cariogenic biofilm contained two orders of magnitude higher L. fermentum cell numbers than the other biofilms. Abiotrophia/Granulicatella and streptococci from the mitis group were found in all samples at high levels, whereas Streptococcus mutans was detected in only one sample in very low numbers. CONCLUSIONS: Application of these new group- and species-specific FISH probes to oral biofilm-forming lactic acid bacteria will allow a clearer understanding of the supragingival biome, its spatial architecture and of structure-function relationships implicated during plaque homeostasis and caries development. The probes should prove of value far beyond the field of oral microbiology, as many of them detect non-oral species and phylogenetic groups of importance in a variety of medical conditions and the food industry.

Asunto(s)

RESUMEN

Analysis of the use of real-time PCR with fluorescent registration of results for gene diagnosis of infectious diseases showed that the sensitivity and reliability of quantitative evaluation of DNA targets directly depended on the method of purification of oligonucleotide probes. Chromatographic behavior of synthetic probes carrying various fluorophores and fluorescence quenchers was analyzed. Approaches to optimization of purification methods are proposed enabling elimination of previously undetectable admixtures. The importance of these studies is explained by the need in extending the armory of methods for the development and production of diagnosticums for detection of infectious and hereditary diseases, identification of genetically modified organisms, and for a wide spectrum of research in molecular biology and medicine.

Asunto(s)

RESUMEN

Fluorescent nucleoside analogs provide a means to study DNA interactive systems through direct measurement of fluorescence properties. As integrated parts of DNA, these probes provide opportunities for monitoring subtle changes in DNA structure as it meets and reacts with other molecules. This protocol describes modifications to standard DNA synthesis to efficiently use smaller volumes of the probe phosphoramidite, purification of pteridine-containing sequences and a deprotection procedure specific for 6MI-containing sequences. Yields for probe incorporation in DNA synthesis are comparable to those for standard phosphoramidites. Examples of the fluorescence signals one can expect are described. Automated synthesis, which is dependent on the length of the sequence, takes about 4-5 h for a 20-mer. The deprotection of 6MI-containing sequences takes approximately 6-7 h before the standard ammonium hydroxide overnight incubation. Purification through polyacrylamide gels, electroelution and ethanol precipitation can be accomplished in 6-8 h.

Asunto(s)

RESUMEN

The photostability and narrow emission spectra of non-organic quantum dot fluorophores (QDs) make them desirable candidates for fluorescent in situ hybridization (FISH) to study the expression of specific mRNA transcripts. We developed a novel method for direct QD labeling of modified oligonucleotide probes through streptavidin and biotin interactions, as well as protocols for their use in multiple-label FISH. We validated this technique in mouse brainstem sections. The subcellular localization of the vesicular monoamine transporter (Vmat2) mRNA corresponds when using probes labeled with two different QDs in the same hybridization. We developed protocols for combined direct QD FISH and QD immunohistochemical labeling within the same neurons as well as for simultaneous study of the subcellular distribution of multiple mRNA targets. We demonstrated increased sensitivity of FISH using QDs in comparison with organic fluorophores. These techniques gave excellent histological results both for multiplex FISH and combined FISH and immunohistochemistry. This approach can facilitate the ultrasensitive simultaneous study of multiple mRNA and protein markers in tissue culture and histological section.

Asunto(s)

RESUMEN

We report here a new amino-modifier reagent, which enable high-throughput purification of amino-modified oligonucleotides. Either monomethoxytrityl (MMT) or trifluoroacetyl (TFA) group has been used as the protecting group for the primary amine when amino-terminal oligonucleotides are prepared. Generally, the removal of MMT requires the stringent acidic treatment after the oligonucleotide synthesis. We chemically synthesized a novel amino-modifier with the MMT protection. It was found that the new amino-modifier released MMT group under mild acidic condition, and then rapid purification of diverse amino-modified oligonucleotides could be achieved with cartridge column of reverse phase. Furthermore, we tried to construct the new detection system to study gene expression using the amino-modified oligonucleotides. The new amino-modifier will be useful for molecular biology by facilitating the construction of oligonucleotide library and improving the chemical reactivity.

Asunto(s)

RESUMEN

While molecular beacons are primarily known as biosensors for the detection of nucleic acids, it has proven possible to adapt other nucleic acid binding species (aptamers) to function in a manner similar to molecular beacons, yielding fluorescent signals only in the presence of a cognate ligand. Unfortunately, engineering aptamer beacons requires a detailed knowledge of aptamer sequence and structure. In order to develop a general method for the direct selection of aptamer beacons we have first developed a selection method for molecular beacons. A pool of random sequence DNA molecules were immobilized via a capture oligonucleotide on an affinity column, and those variants that could be released from the column by a target oligonucleotide were amplified. After nine rounds of selection and amplification the elution characteristics of the population were greatly improved. A fluorescent reporter in the selected beacons was located adjacent to a DABCYL moiety in the capture oligonucleotide; addition of the target oligonucleotide led to release of the capture oligonucleotide and up to a 17-fold increase in fluorescence. Signaling was specific for the target oligonucleotide, and occurred via a novel mechanism, relative to designed molecular beacons. When the target oligonucleotide is bound it can form a stacked helical junction with an intramolecular hairpin in the selected beacon; formation of the intramolecular hairpin in turn leads to release of the capture oligonucleotide. The ability to select molecular beacons may prove useful for identifying available sites on complex targets, such as mRNAs, while the method for selection can be easily generalized to other, non-nucleic acid target classes.

Asunto(s)

RESUMEN

A rapid method for the identification of lactic acid bacteria (LAB) from wine has been developed. This method is based on fluorescence in situ hybridisation (FISH), using fluorescent oligonucleotide probes, homologous to 16S rDNA of those species of LAB commonly found in wines. The protocol for the specific detection of these bacteria was established through the hybridisation of 36 reference strains. The specificity of the probes was evaluated by using pure cultures. Probes were used to identify species in different wines, making it evident that direct identification and quantification from natural samples without culturing is also possible. The results show that FISH is a promising technique for the rapid identification of LAB, allowing positive identification in a few hours (4-16 h).

Asunto(s)

RESUMEN

Regulation of gene expression through interaction of proteins with specific DNA sequences is a central issue in functional genomics. Capillary electrophoretic mobility shift assay is an efficient novel method for the investigation of sequence specific protein-DNA interactions, allowing rapid and sensitive quantification of the complex formation. In this paper, we present a pilot study on capillary zone electrophoretic mobility shift assay (CZEMSA) to investigate the interaction between the transcription factors of HeLa nuclear extract and Sp1-specific fluorescein-labeled oligonucleotide, using the unlabeled probe as competitor. The mobility shift assay was accomplished by CZE in coated capillaries without polymeric buffer additives. Specificity of the DNA protein complex formation was verified by competition experiments, as well as by supershift assay with an anti-Sp1 antibody. The applied electric field strength did not affect the stability of DNA-protein complex during the electrophoretic analysis, allowing rapid identification and quantification of the protein DNA interaction. A practical application to study the interaction between Oryza sativa MADS-box transcription factor 4 (OsMADS4) and its consensus sequence is also reported.

Asunto(s)

RESUMEN

Monofunctional, p-isothiocyanatophenyl-derivatives of platinum (II)-coproporphyrin-I (PtCP-NCS) were evaluated as phosphorescent labelling reagents for synthetic oligonucleotides containing a 3'- or 5'-amino modification. Synthesis and purification conditions were optimised to generate high yields and purity of PtCP-labelled oligonucleotide probes. Phosphorescent properties of the PtCP label have been shown to be largely unaffected by conjugation to oligonucleotides of various length, GC composition and label attachment site. 5'-PtCP-labelled oligonucleotides were shown to work efficiently as primers in a standard PCR. A dedicated 532 nm laser-based time-resolved fluorescence plate reader enabled highly sensitive detection of PtCP-labelled oligonucleotides and PCR products, both in solution and in agarose gels, with limits of detection in the order of 0.3 pM. A model system employing two complementary oligonucleotides labelled with PtCP and QSY 7 dye (dark quencher) showed strong (approximately 20-fold) and specific proximity quenching of PtCP label upon hybridisation in solution. The potential applications of PtCP-labelled probes in hybridisation assays were discussed.

Asunto(s)

RESUMEN

Here we describe a process for the generation of oligonucleotide libraries representative of a given nucleic acid. Starting from at random pool of DNA oligonucleotides, the technique selects only those that hybridize to the nucleic acid template. This selection yields a highly specific library that represents an oligonucleotide image of the chosen template. The novel quality of this approach is the generation of amplifiable oligonucleotide probes that are of unique length and are easily subjected to differential selection. Here we apply this technique to produce different genomic oligonucleotide libraries and show that these genomic oligonucleotide libraries do not cross-hybridize. Differential selection of these genomic oligonucleotide libraries produces oligonucleotides that can be used in the identification, characterzation, and isolation of nucleic acids.

Asunto(s)

RESUMEN

Novel fluorescent oligonucleotides that contain a 3' minor groove binding group (MGB) hybridize to single-stranded targets with increased sequence-specificity compared to ordinary DNA probes. This reduces non-specific probe hybridization and results in low background fluorescence during the 5' nuclease PCR assay (TaqMan, Applied Biosystems, Foster City, CA). We developed a method for closed-tube genotyping using two allele-specific MGB probes labeled with different fluorophores in one reaction. After PCR, tubes were transported to a fluorescence plate-reader for analysis of fluorescence. Common spreadsheet software was used for automated genotype assignment. As an example, DNA samples from 172 hemochromatosis patients were selected and tested for molecular defects in the HFE gene, i.e., mutations in codon 63 and 282. Tight genotype clusters were observed for both codons and results with MGB probes were identical to conventional genotyping (PCR + restriction-fragment-length-polymorphism). We show that this fast and easy method can be used for large-scale (high-throughput) genetic studies but also for routine molecular diagnostics without post-PCR manipulation of amplicons or the need for real-time quantitative PCR machines. Hum Mutat 19:554-559, 2002.

Asunto(s)

RESUMEN

A fluorescence assay has been developed to study the multiple linked equilibria which function in regulation of the Escherichia coli galactose operon. Fluorescein 5-isothiocyanate was attached to Amino-Modifier C6dT at different positions in an oligonucleotide containing the sequence for the OE site of the galactose operon. These fluorescently labeled oligonucleotides were used to study OEDNA-GalR-d-galactose interactions. The data were analyzed and fit to various models including the classical competitive binding model as well as models involving the formation of a ternary DNA-repressor-inducer complex. Examination of the reduced chi-square of the various fits and comparison of fitted parameters with those obtained in independent experiments were used to distinguish different models. Since the ternary complex is likely to exist under physiological conditions, our results suggest that obligatory dissociation of GalR from DNA may not be required for induction of the gal operon. Rather, induction may involve the formation of a ternary complex of OEDNA with GalR and D-galactose with a different conformation than the GalR-OEDNA binary repressor complex.

Asunto(s)

Asunto(s)

RESUMEN

Synthetic oligonucleotides have been deduced and synthesized based on the sequence of a salmonella-specific polynucleotide probe. Two oligonucleotide probes, ST4 and ST15rev hybridized to 93 and 92 strains respectively out of 93 strains of Salmonella analysed. ST4, however, cross hybridized to one of the 28 strains of 16 genera of Enterobacteriaceae tested. Based on sequence alignment, in 16 strains of Salmonella, of a 114 base pair region, a Salm. typhimurium specific oligonucleotide probe, ST22, was identified. In colony hybridization, this probe detected all 47 strains of Salm. typhimurium analysed without hybridization to 94 strains of other Salmonella serotypes and to 26 strains of non-Salmonella bacteria.

Asunto(s)

RESUMEN

We report the synthesis of a radioactive, photolabile oligodeoxyribonucleotide probe and its exploitation in identifying 50S ribosomal subunit components neighboring its target site in 23S rRNA. The probe is complementary to 23S rRNA nucleotides 2475-2483, a single-stranded sequence (the 2475 loop) near the peptidyltransferase center of Escherichia coli ribosomes. On photolysis in the presence of 50S subunits, it site-specifically incorporates into proteins L1, L13, L16, L32, and L33 and into 23S rRNA nucleotides G2470, A2471, and G2472. These results provide clear evidence that C2475 in 23S rRNA is within 21 A (the distance between C2475 and the photogenerated nitrene) of proteins L1, L13, L16, L32, and L33. The implications of these results for the evolving model of the internal structure of the 50S subunit are considered.

Asunto(s)

RESUMEN

A simple and rapid method for the analysis of genetic polymorphisms has been developed using allele-specific oligonucleotide arrays bound to glass supports. Allele-specific oligonucleotides are covalently immobilized on glass slides in arrays of 3 mm spots. Genomic DNA is amplified by PCR using one fluorescently tagged primer oligonucleotide and one biotinylated primer oligonucleotide. The two complementary DNA strands are separated, the fluorescently tagged strand is hybridized to the support-bound oligonucleotide array, and the hybridization pattern is detected by fluorescence scanning. Multiple polymorphisms present in the PCR product may be detected in parallel. The effect of spacer length, surface density and hybridization conditions were evaluated, as was the relative efficacy of hybridization with single or double-stranded PCR products. The utility of the method was demonstrated in the parallel analysis of 5 point mutations from exon 4 of the human tyrosinase gene.

Asunto(s)

Asunto(s)

RESUMEN

Synthetic deoxyoligonucleotides complementary to different regions of genome RNA of tick-borne encephalitis (TBE) Sofjin virus strain were used to differentiate TBE virus strains. Nine TBE strains isolated in different geographical areas from different sources and several viruses of the TBE subgroup were tested. The probes revealed genetic heterogeneity of TBE strains. The probes complementary to different regions of the genome had different specificity. The pattern of hybridization of TBE virus strains with a panel of 11 oligonucleotide probes correlated significantly with the source of the virus strain and to a smaller extent with the geographical isolation site.

Asunto(s)

RESUMEN

Escherichia coli virus T1 encodes a DNA (N6-adenine)-methyltransferase (M.T1) with the same sequence specificity as the E. coli DNA (N6-adenine)-methyltransferase (M.Eco dam). This enzyme was purified to homogeneity and a partial amino acid sequence determined. Oligonucleotides were constructed and used not only as probes to map the gene on the T1 genome, but also as primers in sequencing reactions to establish the nucleotide sequence of the M.T1 locus by primer extension. These data represent the first analysis of the genomic organization of bacterial virus T1 on a molecular level. Significant homology to E. coli consensus transcription and translation-initiation signals suggest that the gene for M.T1 is most probably under control of its own promoter. It may be transcribed as a polycistronic mRNA, together with a downstream open reading frame which codes for a polypeptide containing 83 amino acids (HP 83). Both the deduced primary and the secondary structure of the M.T1 were compared to those of other known DNA methyltransferases, especially those recognizing the sequence, GATC; there is little similarity of the T1 enzyme to the other members of this family.

Asunto(s)