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At least 145 records · Page 8Linked to original sources
PubMed1992

Synthesis of digoxigenin-labeled cDNA as a test for mRNA integrity.

The nucleotide analogue digoxigenin-11-dUTP is widely used as a nonradioactive marker in a broad range of techniques including dot blots, Southern and Northern blots, colony and plaque screenings and in situ hybridizations. In this report, we describe the incorporation of this molecule into a cDNA synthesized by reverse transcriptase as a novel application for digoxigenin. This reaction can be performed as a useful control to check the integrity of a bulk mRNA preparation in the construction of a cDNA library, since the ability of mRNA to direct the synthesis of long molecules of first-strand cDNA is a sign of its integrity.

DNA↗
PubMed1992

[DNA probe labeling with digoxigenin-dUTP and its application in gene diagnosis].

In this paper, DNA probe labeling by the randomly primed incorporation of digoxigenin-dUTP is reported. The sensitivity of color reaction and hybridization were 32 fg and 200 fg, respectively, and both were specific for the target. Single-copy and multi-copy gene fragments among 2 micrograms human genomic DNA were detected by beta IVS II, Fr 3-42 and 3'HVR labeled with digoxigenin-dUTP. The results were consistent with a radioactive control assay. This method has been successfully used in the gene diagnosis of adult polycystic kidney disease.

DNA Probes↗
PubMed1992

[Non-enzymatic marking of gene probes using digoxigenin for the diagnosis of sickle cell mutation and beta thalassemia].

A method is described in which allele-specific oligonucleotides (ASO) are labeled non-enzymatically with digoxigenin (DIG). The ASOs were synthesized in a DNA synthesizer. A primary C6-alkylamine was incorporated at the 5'end of the ASO. The derivative was coupled to digoxigenin-3-0-methylcarbonyl-epsilon-aminocaproic acid-N-hydroxy-succinimide ester. The DIG-ASO probes were isolated from the crude preparation and hybridized with amplified genomic DNA. The annealed probes were detected with an ELISA test. The DIG-ASO probes were successfully used for the identification of sickle cell disease genotypes and the most common beta-thalassemia mutations.

Adult↗
PubMed1992

[Hybridization using a digoxigenin-labeled probe for the detection of hepatitis delta virus].

BACKGROUND: The presence of hepatitis delta virus was investigated in liver biopsies by in situ cyto-hybridization with a probe labelled with digoxigenin. METHODS: The techniques developed included extraction of plasmid DNA by lysis by alkali, electroelution, electrophoresis in agarose gel and digoxigenin labelling, and the application in liver tissue. RESULTS: Viral RNA was detected in 6 of the 10 patients, and the reactivity was exclusively restricted to the nucleolus of the hepatocytes. CONCLUSION: This method reveals as a sensitive and quick diagnostic procedure, which allows to study the intensity of the infection as well as the serological state of the patient.

Acute Disease↗
PubMed1992

Fluorescent in situ hybridization en suspension (FISHES) using digoxigenin-labeled probes and flow cytometry.

Fluorescent in situ hybridization has become a major technique for visualizing genetic material in fixed cells. Currently, many systems utilize the hybridization of labeled molecular probes to cells that are attached to slides. We have developed a technique that allows for in situ hybridization to be performed using cells in suspension. By using digoxigenin-labeled DNA probes and a fluoresceinated antibody directed against the digoxigenin, we can measure the resulting signal on a flow cytometer and the cells can be attached to microscope slides for visual analysis.

Animals↗
PubMed1991

Gene rearrangements detected by nonradioactive digoxigenin-labeled DNA probes.

The configurations of immunoglobulin, T-cell receptor beta chain and bcl-2 genes were analyzed in lympho-proliferative disorders using nonradioactive digoxigenin-labeled probes. The studies demonstrated the reliability of digoxigenin-labeled probes for the detection of single-copy genes after Southern blotting of genomic DNA: 1 microgram and sometimes even 0.2 microgram of restriction endonuclease-digested DNA could be detected either by JH, C beta or bcl-2 probes. The intensity of the signal was consistently satisfactory, and there was no background problem. Control experiments showed neither false-positive nor false-negative results caused by the use of the nonradioactive detection system.

Blotting, Southern↗
PubMed1992

PCR amplification and simultaneous digoxigenin incorporation of long DNA probes for fluorescence in situ hybridization.

Nonradioactive in situ hybridization has found widespread applications in cytogenetics. Basic requirements are DNA probes in sufficient amounts and of high specificity as well as a labeling protocol of good reproducibility. The PCR has been of fundamental importance for the amplification of DNA sequences and thus for the production of DNA probes. Meanwhile, PCR protocols for amplification of DNA have reached a high degree of automation. So far, incorporation of labeled nucleotides into these DNA probes has normally been done by nick translation. Here we show that in using the PCR, amplification of a DNA probe larger than one kilobase accompanied by simultaneous incorporation of digoxigenin-11-dUTP can be performed for in situ hybridization experiments. As an example, the DNA probe pUC 1.77 specific for the subcentromeric region q12 of chromosome number 1 was used and hybridized against metaphase chromosomes from human lymphocytes. The labeled chromosome region was detected by anti-digoxigenin-fluorescein, Fab fragments. The experiments were evaluated by digital image analysis of microphotographs.

Chromosomes, Human, Pair 1↗
PubMed1990

[Use of digoxigenin-labeled probes for the detection of papillomaviruses by in situ hybridization].

A technique of detection of human papillomaviruses by in situ hybridization in paraffin-embedded formalin-fixed sections of genito-anal lesions is described. The probes are labeled by incorporation of digoxigenin-labeled nucleotides. The hybrids are revealed by immunohistochemistry with an anti-digoxigenin antibody coupled with alkaline phosphatase. This technique is sensitive, cheap and may be used in routine in laboratories of pathology.

DNA Probes↗
PubMed1990

[Comparative study with digoxigenin and biotin markers for the DNA probe identification of genes coding for type TEM beta-lactamases].

We have tested two non-isotopic labels: digoxigenin--11-dUTP and biotin-7-dATP for the detection of TEM-1 beta-lactamase gene with a TEM-1 probe by DNA:DNA hybridisation (using spot technique). The use of radioactive labels is inconvenient and not available to all the clinical bacteriological laboratories. The strains tested were: 16 on file (14 carriers of different types of beta-lactamase) and 19 clinical isolated strains. Previously, the plasmid beta-lactamase of these strains had been identified by analytical isoelectric focusing. Using the probe labeled with digoxigenin-11-dUTP, the correlation between results obtained by DNA:DNA hybridisation and those by analytical isoelectric focusing was excellent, with no false positives; with the biotin--7-dATP labeled probe, reading was difficult and some false positives were observed.

Biotin↗
PubMed1990

Digoxigenin-labeled probes can detect single-copy genes in human metaphase chromosomes.

A technique of in situ hybridization on metaphases of chromosomes by a digoxigenin-labeled probe is described. This technique was able to detect single DNA sequences of 2 and 7 kilobases. The results obtained were compared with those of a biotin streptavidin alkaline phosphatase-based detection system. The digoxigenin method was at least as efficient and sensitive as the biotin-streptavidin method.

Alkaline Phosphatase↗
PubMed1994

Detection of ptc in archival formalin-fixed, paraffin-embedded tissues. Comparison of radiolabeled DNA hybridization and direct incorporation of digoxigenin-11-dUTP into RT-PCR products.

Archival pathological specimens are a source of RNA and DNA for clinical surveillance or retrospective studies. We employed a modification of the acid guanidium thiocyanate-phenol-chloroform extraction method for the recovery of total RNA from formalin-fixed, paraffin-embedded neoplastic thyroid tissue. The extracted RNA was used for reverse transcription of ptc and subsequent amplification of the complementary DNA (cDNA) by the reverse transcription-polymerase chain reaction (RT-PCR). In lieu of 32P-labeled DNA for hybridization studies, we supplemented the nucleotide pool in the amplification reaction with a modified pyrimidine, digoxigenin-11-dUTP. Digoxigenin-11-dUTP was incorporated directly into the PCR product, eliminating the need for hybridization, posthybridization washes, and prolonged autoradiography. These products were resolved by electrophoresis on agarose gels, Southern blotted to nylon membranes, and rapidly detected by chemiluminescence. This nonradioisotopic method has expedited and reduced the cost for molecular investigations with archival pathological specimens by providing equal sensitivity to or greater sensitivity than that of DNA-labeled radionuclides without the associated biological hazards.

Carcinoma, Papillary↗
PubMed1993

Spectrophotometric quantitation of DNA on blots after ethanol-solubilization of the MTT-formazan from anti-digoxigenin-based detection of nucleic acids.

The tetrazolium salt, 3-(4,5-dimethyl-thiazolyl-2)-2,5-diphenyl tetrazolium bromide (MTT) has recently been established as a substitute for Nitro-blue tetrazolium (NBT) in stain mixtures using antibody-conjugated alkaline phosphatase for the location of proteins on Western blots (Heegaard, 1990). Experiments reported here show that MTT is as sensitive as NBT in digoxigenin-labeled probe localization (on nucleic acid blots) utilizing alkaline-phosphatase-labelled, anti-digoxigenin antibodies. Moreover, as the formazan from MTT is soluble in ethanol, it is shown that spectrophotometric quantitation can be used to estimate the amount of target DNA on dot and Southern blots. For dot blotting, pBR328 was used as the probe and pBR322 as target. For Southern blots, human rDNA was used as the probe and total genomic calf DNA as the target. Staining response was linear over at least six twofold DNA dilutions in both types of blot.

Antibodies↗
PubMed1994

Double-label in situ hybridization using biotin and digoxigenin-tagged RNA probes.

We describe a double-label in situ hybridization protocol based on the optimized synthesis of biotin-labeled RNA probes. Biotin-labeled probes are used in conjunction with digoxigenin-labeled probes to simultaneously visualize two different transcripts. One transcript is hybridized with a biotin-labeled RNA probe and visualized as a brown peroxidase reaction product, and the other transcript is hybridized with a digoxigenin-labeled RNA probe and visualized as a blue alkaline phosphatase reaction product. We present several examples in which this double-labeling method has proven useful in determining the spatial and temporal relationships between various transcripts expressed during Drosophila embryogenesis, indicating that this method should be of general use in establishing the relationship of two independent transcription patterns.

Biotin↗
PubMed1994

Rapid chemiluminescent detection of the mRNA for uncoupling protein in brown adipose tissue by northern hybridization with a 32-mer oligonucleotide end-labelled with digoxigenin.

Sequences for the uncoupling protein gene (or mRNA) expressed in brown adipose tissue have been re-examined for homology between species. A 32 bp region is identical in rats and cattle, this representing a 5 bp extension of the 27 bp conserved sequence previously described. In mice, rabbits, Syrian hamsters, and humans there is a single bp difference with the 32 bp conserved rat/cattle sequence. A 32-mer antisense oligonucleotide (3'-TAGTG-GAAGGGCGACCTGTGGCGGTTTCAGGC-5') targeted to the 32 bp rat/cattle sequence has been synthesized and end-labelled (3' and 5' ends) with digoxigenin, as a simple, non-radioactive probe for uncoupling protein mRNA. Hybridization of the oligonucleotide to the mRNA was readily detected on Northern blots with total RNA from brown adipose tissue of rats, mice, and newborn lambs, using a chemiluminescence substrate and following short exposure to film (1 h, or less). With longer exposure (overnight), uncoupling protein mRNA was detected in as little as 250 ng of total RNA from rat brown fat. The 32-mer digoxigenin-labelled oligonucleotide, coupled with a chemiluminescence substrate, provides a rapid, sensitive, non-radioactive procedure for detecting uncoupling protein mRNA on Northern blots, applicable across species.

Adipose Tissue, Brown↗
PubMed1993

In situ hybridization with digoxigenin labeled oligonucleotide probes: detection of CAMK-II gene expression in primary cultures of cerebral endothelial cells.

A better understanding of the regulation of gene expression under physiological and experimental conditions is one of the most important goals of today's neurobiology. In order to accomplish this task a number of in-situ hybridization methods have been elaborated. In the past few years the use of nonradioactive procedures have gained more and more space among these efforts. One of the most promising methods in this field is the application of digoxigenin-labeled probes, which have been used successfully in several laboratories. Here we present a rapid method providing good spatial resolution and low background labeling for the detection of messenger RNAs in various cell culture systems using digoxigenin-labeled probes. By using oligonucleotides complementary to the alpha and beta subunit of the calcium/calmodulin dependent protein kinase II (CAMK-II) we were able to demonstrate the presence of this enzyme in cultured cerebral endothelial cells, an enzyme that plays an important role in mediating the effects of extracellular signals.

Animals↗
PubMed1993

[Detection of hepatitis A virus by riboprobe labeled with digoxigenin: comparison of methods].

A riboprobe (RNA probe), corresponding to the 5' end of the HM175 hepatitis A virus (HAV) genome, was synthetized in vitro and was digoxigenin-labeled. Then the riboprobe was used to detect the CF53 HAV strain. Conditions of virus denaturation (with or without SDS and proteinase K, timing of assay) to release viral RNA were tested by dot-blot hybridization on a ten fold dilution of HAV suspension. Densitometric measures of dot-blot spots allowed to appreciate optimization of the method. Sensitivity of hybridization was compared with sensitivity of radioimmunoassay (RIA) and cell culture methods. Hybridization signals and scale of HAV suspension were consistent when 0.05% SDS, 0.17 micrograms/ml Proteinase K, 37 degrees C, 30 mn or 3 hours are used. 8.10(2) TCID50 HAV was detected by hybridization with digoxigenin-labeled RNA probes. Detection threshold was the same as radioimmunoassay and lower comparatively to cell culture.

Densitometry↗
PubMed1993

In situ hybridization at the ultrastructural level: localization of cytomegalovirus DNA using digoxigenin labelled probes.

We describe a procedure for detecting and localizing cytomegalovirus DNA sequences based on in situ hybridization at the ultrastructural level. A digoxigenin-labelled probe, identified with an anti-digoxigenin colloidal gold-labelled antiserum, was employed on infected cells embedded in a new acrylic resin (Bioacryl). The silver enhancement method on the same specimen was used to more easily reveal the reaction also at low magnification. The immunolocalization was characterized by high specificity with virtually no background staining and a good maintenance of submicroscopic cell features.

Acrylic Resins↗
PubMed1993

Detection of hepatitis B virus in plasma using flow cytometric analyses of polymerase chain reaction-amplified DNA incorporating digoxigenin-11-dUTP.

Blood donations are routinely screened by multiple serologic assays for antigens/antibodies associated with infection by blood-borne viruses, including hepatitis B virus (HBV), hepatitis C virus (HCV), human immunodeficiency viruses (HIV-1 and HIV-2), and human T-cell lymphotropic virus (HTLV-I and HTLV-II). A direct detection of these viruses would be more effective for the prevention of transfusion-transmitted infections than the indirect measurement of the variable host immune response to these agents. Because the polymerase chain reaction (PCR) for viral gene amplification offers the most sensitive and direct means of detecting viruses in blood, we have developed a nonisotopic PCR procedure for the detection of HBV, chosen as a prototype. The problems, common to previously described PCR methods, of nucleic acid extraction and inhibition of the PCR by plasma proteins were overcome by isolation of HBV from plasma by means of 450-microns polystyrene beads covalently coated with monoclonal antibody to the Pre-S1 region of the viral envelope protein. Detergent lysis and proteinase K digestion of the immunocaptured virions isolated from plasma released the HBV DNA. A modified PCR-amplification protocol, incorporating digoxigenin-labeled dUTP in the amplified gene products followed by hybridization with a specific biotinylated oligonucleotide probe bound to streptavidin-coated 2.8-microns magnetic beads, allowed flow cytometric analyses of HBV-specific PCR products by means of antibodies to digoxigenin labeled with fluorescein isothiocyanate. The endpoint serial dilutions of pedigreed human plasma samples containing chimpanzee infectious dose (CID50) of 10(7) for adw and CID50 of 10(7.5) for the ayw subtypes were compared in repeated testing of PCR products by our immunoreactive bead (PCR-IRB) assay. HBV DNA was consistently detected in a 5 x 10(-10) dilution of each sample. In testing 20 coded specimens of blood donors, with or without serologic markers of HBV infection, the PCR-IRB was specific and more sensitive than the PCR analyses by slot blot hybridization with radioactive probe. The PCR-IRB assay can be adapted for simultaneous detection of multiple blood-borne viruses by an automated flow cytometric analysis system.

Bacterial Proteins↗