Biotin-labeled DNA Probes Research Articles

In Situ Hybridization for Ribosomal RNA Sequences: A Rapid Sensitive Method for Diagnosis of Infectious Pathogens in Anatomic Pathology Substrates.

Ribosomal RNA (rRNA) sequences are present in all prokaryotic and eukaryotic cells. These sequences are highly conserved among organisms and are know to be species specific. Sequence analysis is widely utilized in diagnostic microbiology in order to identify infectious agents. A rapid in situ hybridization assay for detection of rRNA sequences in formalin or Bouin's fixed paraffin embedded tissues was developed using biotin-labeled oligonucleotide DNA probes specific for a variety of rRNA sequences. This method was used effectively to identify human, Aspergillus species, Pneumocystis carinii, and Toxoplasma gondii rRNA sequences in a protocol requiring less than 30min to complete. Utilization of site specific oligonucleotide probes specific for a variety of rRNA sequences may aid in the diagnosis of several medically important bacterial, fungal, and protozoal pathogens.

Incidence of chromosome 3, 7, 10, 11, 17 and X disomy in mature human sperm nuclei as determined by nonradioactive in situ hybridization.

In situ hybridizations were performed on mature human sperm cells with biotin-labeled alpha-satellite DNA probes specific for chromosomes 3, 7, 10, 11, 17, and X in order to reveal the disomy rate for each of these chromosomes. A total of 76,253 sperm nuclei from seven healthy probands aged 23-57 years were analyzed. An average of 12,000 sperm nuclei (at least 1500 per donor) showing hybridization were scored with each probe. The disomy rate as indicated by two distinct hybridization signals turned out to be similar for all chromosomes, ranging from 0.31% to 0.34%. There were no significant interindividual differences and no age correlation in the frequency of disomic sperm cells between the donors.

Use of fluorescent substrate 4-MUP in the detection of biotin-labeled DNA probes.

To improve the sensitivity of detecting biotin-labeled DNA Probes, a new fluorescent substrate of alkaline phosphatase, 4-methylum belliferylphosphate (4-mup) was studied instead of conventional BCIP-NBT. The result of dot-blot hybridization demonstrates that this new substrate can be used for the colorimetric detection of biotin-labeled probes after hybridization to immobilized nucleic acids. The sensitivity is about one order of magnitude higher than that of BCIP-NBT system, and the time required for color development is very short, only about five min. It is suggested that the Bio-SA-Bio-AP-4-MUP colorimetric detection system can be widely used in gene diagnosis.

Quantitative Measurement of Nonisotopically Labeled Polymerase Chain Reaction Product

A novel approach for evaluation of polymerase chain reaction product was described. A biotin-labeled DNA (or RNA) probe is hybridized to the digoxigenin-labeled target PCR product. The hybrids are retained on an anti-biotin antibody-coated plate. An anti-digoxigenin Fab conjugated with alkaline phosphatase is used for generating either a fluorescence or a chromagenic substance. Both fluorogenic and chromagenic systems can be used for quantitative measurement of the PCR product/probe hybrids. The method is specific, fast, nonisotopic, sensitive, quantitative, and practical.

Chromosomal homologies between Drosophila lebanonensis and D. melanogaster determined by in situ hybridization.

Twelve biotin-labelled recombinant DNA probes were hybridized to polytene chromosomes of Drosophila melanogaster and D. lebanonensis. Probes were chosen in order to cover the whole chromosomal complement. Six probes correspond to known genes from D. melanogaster (RpII215, H3-H4, MHC, hsp28/23, hsp83, hsp70), four probes are clones isolated from a D. subobscura library (Xdh, lambda DsubS3, lambdaDsubG3, lambdaDsubG4) and the remaining two probes correspond to the Adh gene of D. lebanonensis and to one sequence (262), not yet characterized, from the same species. The chromosomal homologies obtained from the in situ hybridization results allow us to determine that Muller's C and D chromosomal elements are fused in the karyotype of D. lebanonensis and constitute the large metacentric chromosome. Single pericentric inversions in the E and B elements have generated the medium and small metacentric chromosomes, respectively. No great changes are detected in Muller's A element, which remains acrocentric. The changes detected in the karyotypic evolution of D. lebanonensis are frequently observed in Drosophila evolution, as deduced from chromosomal homologies of several Drosophila species. The results are also consistent with Muller's proposal that chromosomal elements have been conserved during the evolution of Drosophila.

Membrane-linked probes: 5′-(polysulfonylmethyloxyhexaglycol) oligonucleotides

A novel approach for the synthesis of functional, film-forming polymers which consists of the assembly of oligonucleotides anchored on a solid support with a soluble polymeric reagent is described. Phosphoramidites of hydroxymethyl-polysulfone and hexaglycoloxymethyl-polysulfone were synthesized and were linked by phosphate ester bonds to fragments of DNA anchored on controlled pore glass supports in the last step of an automatic synthesis of oligonucleotides. Microtiter plates were coated with the oligonucleotide-hexaethyloxymethyl-polysulfone and hybridization with a complementary biotin-labelled DNA probe was applied followed by avidine-peroxidase and detection by the usual procedure. Control experiments using a non-relevant probe for hybridization or using hybridization solutions with no oligonucleotide were also performed.

Polymerase Chain Reaction and a Biotin-Labeled DNA Probe for Detection of Infectious Bronchitis Virus in Chickens

Polymerase chain reaction (PCR) and a biotin-labeled DNA probe were used to amplify and detect the genome of infectious bronchitis virus (IBV) from tracheal swabs taken from chickens that were experimentally inoculated with the IBV Beaudette, Arkansas, and Gray strains. The viral genome was successfully detected by PCR and confirmed by dot-hybridization assay using a biotin-labeled DNA probe on days 1, 3, 9, and 14 after exposure. Direct electron microscopy (EM) analysis was used to compare the ability of the two tests to detect IBV from the same tracheal swab samples. The EM analysis did not detect IBV in four of eight necropsy groups that were positive using PCR and the biotin-labeled DNA probe. Although histopathological lesions were observed in the tracheas, no clinical signs or specific antibody response were observed in the birds. The virus was also detected in the allantoic fluid of embryonating chicken eggs that had been inoculated with field samples suspected to be IBV. The field samples were passed four to six times in embryonating eggs, and 10 of 17 samples were positive using PCR and the biotin-labeled probe.

Polyethylene tube: an alternative support for synthesis and purification of biotin-labeled single-stranded DNA probes.

Polyethylene tube: an alternative support for synthesis and purification of biotin-labeled single-stranded DNA probes.

Detection of Epstein-Barr virus genome in nasopharyngeal carcinoma by in situ DNA hybridization.

To elucidate the possible role of Epstein-Barr virus (EBV) in nasopharyngeal carcinoma (NPC), we investigated the EBV genome in NPC by in situ DNA hybridization using radioisotope- and and biotin-labeled EBV DNA probes. The EBV genome was detected in the tumor cells in all (100%) 60 cases, irrespective of histological type, but not in the lymphocytes. Silver grains, which reflected the copy number of the EBV genome, were more abundant in the nonkeratinizing, spindle cell, and undifferentiated carcinomas than in keratinizing squamous cell carcinoma. In the keratinizing carcinoma, which was poorly differentiated in this series, the EBV genome was usually detected in anaplastic tumor cells, and not in the keratinizing areas. The sensitivity of the radioisotopic technique was superior to that of the biotinylated probe method (100 vs. 81.7%, p < 0.0003). These results suggest that EBV is etiologically related to nasopharyngeal carcinogenesis and that the differentiation of tumor cells in vivo, and probably also in vitro, may become incompatible with EBV replication.

Enterohemorrhagic Escherichia coli associated with hemolytic-uremic syndrome in Chilean children.

A clinicoepidemiological study was undertaken to determine if enterohemorrhagic Escherichia coli (EHEC) was associated with hemolytic-uremic syndrome (HUS) in children in Santiago, Valdivia, and Temuco, Chile. Prospective surveillance detected 20 hospitalized cases of HUS in children less than 4 years of age in these cities from March 1988 to March 1989. Each HUS patient was matched (by sex and age) with two control children (hospitalized elective-surgery patients). To detect EHEC, DNA from stool culture isolates of E. coli was detected by hybridization with biotin-labelled DNA probes specific for the EHEC virulence plasmid, Shiga-like toxin I (SLT-I) or SLT-II. Stool cultures from 6 of 20 cases (30%) and from 2 of 38 controls (5.3%) yielded EHEC (P = 0.0158). EHEC isolates from all HUS cases hybridized with the EHEC plasmid probe and with probes for SLT-I or -II (or both). The serogroups of the isolates included O157, O26, and O111. EHEC causes HUS in Chile, and the biotinylated gene probes are practical diagnostic tools for epidemiologic studies.

Detection of cytomegalovirus-infected cells by flow cytometry and fluorescence in suspension hybridisation (FLASH) using DNA probes labeled with biotin by polymerase chain reaction.

A biotin-labeled DNA probe specific for the immediate early gene of the cytomegalovirus (CMV) strain AD 169 was generated with the polymerase chain reaction. Nucleic acid hybridisation was carried out with CMV-infected T-lymphoblastoid cells (MOLT-4) after 4 hr to 6 days of culture. Biotin molecules were made visible with streptavidin coupled with fluorescein. The fluorescence signal of the hybridised probe was measured by flow cytometry in the cell suspension. The number of CMV-positive cells was 7% at 4 hr, 8% after 28 hr, 18% after 2 days, 26% after 3 days, 91% after 4 days, 97% after 5 days, and 98% after 6 days. The first detection of CMV antigen (pp65) was possible with immunoenzymatic labeling by day 4, whereas CMV-DNA was detected by PCR after 4 hr. CMV-specific RNA could be detected in a similar way. The analysis of mononuclear peripheral blood leukocytes in a patient with active CMV infection showed 14.7% CMV DNA-positive cells at day 1 and 7% at day 8, as compared to 0.9% and 0.0% cells which were positive for CMV antigen (pp65) by immunoenzymatic labeling at day 1 and day 8, respectively. We conclude that flow cytometry and fluorescence in suspension hybridisation (FLASH) offers a new tool for analysing exactly and quantifying large numbers of cells for specific DNA or RNA and may be useful for other laboratory and clinical applications.

Biotin-labeled Genomic DNA Probe for the Detection of Theileria sergenti and its Nucleotide Sequence

Biotin-labeled Genomic DNA Probe for the Detection of <i> Theileria sergenti</i> and its Nucleotide Sequence

In situ hybridization analysis of cytomegalovirus and adenovirus DNA in immunoglobulin A nephropathy.

Viruses are thought to play a role in the occurrence of IgA nephropathy. In fact, cytomegalovirus (CMV) antigens have been detected in the glomerular mesangium. To clarify this relationship, we used in situ hybridization with biotin-labeled DNA probes of CMV and adenovirus (AV) in 40 patients with IgA nephropathy to determine the presence or absence of virus in affected renal tissue. Renal tissue DNA from these patients did not hybridize with the CMV or AV probes. Our results fail to support the theory that consecutive regional renal infections with CMV and/or AV are involved in the pathogenesis of IgA nephropathy.

Microwave irradiation-accelerated in situ hybridization technique for HIV detection

High frequency irradiation generated in a common household microwave oven was used to establish an in situ hybridization technique for rapid detection of HIV sequences in infected cells. A biotin-labeled DNA probe was subsequently detected either by an alkaline phosphatase-based colorimetric reaction or by fluorescence. When compared to standard hybridization procedures with radioactive or nonradioactive probes, microwave energymediated hybridization results in equal sensitivity and diminished background. The main advantage of this method, however, is the drastic reduction in time, allowing completion of the whole procedure, from sample preparation to hybrid signal visualization, within one hour. In addition to HIV detection, the approach described can be applied for the diagnosis of other viral infections and may stimulate the development of nucleic acid hybridization techniques based on microwave irradiation.

Detection of novel trimethoprim resistance determinants in the United Kingdom using biotin–labelled DNA probes

Two collections of trimethoprim R plasmids, isolated from strains of Escherichia coli during 1978-83 and 1987-8 respectively, were retrospectively screened with specific biotinylated DNA probes for the presence of genes encoding particular DHFR enzymes. The results confirmed that the type I DHFR gene was the predominant plasmid-encoded gene conferring trimethoprim resistance in strains of E. coli from the Nottingham area of the UK, but indicated that genes encoding the more recently recognized types of DHFR enzymes had appeared in the bacterial gene pool and could be recognized with increased frequency in the latter plasmid collection. This was particularly true of the type IIIa and type VII enzymes which together accounted for 27% of the trimethoprim R plasmids examined in 1987-8.

成人喉頭乳頭腫ウイルスのＤＮＡ診断

Tissues from patients with adult-onset type laryngeal papilloma were examined for the presence of human papillomavirus (HPV).The presence of HPV DNA was determined by in situ hybridization with biotin-labelled HPV DNA probes and by dot blot hybridization with 32P-labelled HPV DNA probes.Two cases of adult-onset type multiple laryngeal papilloma were positive for HPV-6, but a case of single laryngeal papilloma was negative for HPV.

Practical and economical method for using biotinylated DNA probes with bacterial colony blots to identify diarrhea-causing Escherichia coli

A simple and economical method was developed for using biotinylated DNA probes to hybridize with bacterial colonies belonging to the various categories of diarrhea-causing Escherichia coli. Simplification and cost containment were achieved by using Whatman no. 541 filter papers instead of nitrocellulose, by minimizing the concentration of proteinase K (an expensive but necessary reagent used to pretreat the colony blots prior to hybridization with biotin-labeled DNA probes) and by reusing hybridization solution containing labeled probe DNA. After exposing the colony blots to lysing solution and steam, followed by lysozyme (1.5 mg/ml), sucrose (25%), and proteinase K (10 micrograms/ml) treatments, biotinylated probes were used to detect enterotoxigenic, enteropathogenic, enterohemorrhagic, diffuse adherence, and enteroinvasive categories of diarrhea-causing E. coli with a high level of sensitivity and specificity. Three independent observers who were experienced in reading DNA blots recorded remarkably similar results, while less satisfactory results were obtained when the blots were read by an inexperienced observer. This technique will be useful in laboratories in which radioactive isotopes are unavailable or impractical and in which budgets are restricted.

Sensitivity of digoxigenin and biotin labelled probes for detection of human papillomavirus by in situ hybridisation.

The sensitivity of digoxigenin and biotin labelled DNA probes for the detection of human papillomavirus (HPV) by dot blotting and in situ hybridisation was compared in tissues from cervical, laryngeal, and anogenital neoplasia. Probes were either labelled with digoxigenin by the random primer technique and detected with anti-digoxigenin antibody, or labelled with biotin by nick translation and detected with streptavidin, both methods having a common final visualisation procedure using alkaline phosphatase. Digoxigenin labelled probes proved two to 10-fold more sensitive by quantitative dot blotting and four-fold more sensitive in detecting HPV 16 DNA in a series of 31 anal carcinomas, compared with biotinylated probes. The digoxigenin method also produced less non-specific background staining of tissue sections than biotin labelled probes. It is concluded that digoxigenin DNA labelling and detection provides a simple, reliable, and efficient alternative to the use of biotin or radioactive isotopes for the detection of HPV DNA by in situ hybridisation. Digoxigenin labelled probes also offer the possibility of double labelling in situ hybridisation procedures when used with biotin labelled probes to provide simultaneous identification of different DNA sequences.

Identification of translocations in pea by in situ hybridization with chromosome-specific DNA probes

Biotin-labelled DNA probes for tandemly repeated sequences have been used in in situ hybridization experiments as chromosome-specific markers in pea. Six of the seven chromosome pairs can be marked at single sites in this way. Translocations from a standard karyotype are revealed as chromosomes that have two hybridization sites, rather than one. By probing a tester set of reciprocal translocation (or interchange) lines, some of the markers can be assigned to chromosomes. The method is rapid and simple and, in the absence of well-resolved chromosome bands, provides a means for clarifying some of the problems in pea cytology.Key words: in situ hybridization, translocations, chromosome markers.

Microplate hybridization of amplified viral DNA segment

We have developed a simple hybridization method for a DNA segment which is amplified by the polymerase chain reaction: after heat denaturation, the amplified DNA segment with a length of more than 300 bases is adsorbed to microplate wells in the presence of 1.5 M NaCl or 0.5 M ammonium sulfate; the immobilized DNA is hybridized with a biotin-labeled DNA probe; then, the hybridization signal is detected by streptavidin-conjugated beta-galactosidase or peroxidase. This method has several advantages over the conventional dot blot hybridization method: (i) radioisotopes are not used, (ii) synthetic oligonucleotide for the probe is not needed, (iii) the time required for washing of the solid phase is greatly reduced, and (iv) the baking and prehybridization procedures are eliminated. By this method, we were able to detect viral genomes in vesicle specimens from patients infected with varicella-zoster virus.