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PubMed1989

Identification and classification of Campylobacter strains by using nonradioactive DNA probes.

Acetylaminofluorene-labeled genomic DNA probes were used for the identification and classification of Campylobacter strains. Relationships among 17 well-known strains of Campylobacter species and subspecies were studied by comparing acetylaminofluorene- or 32P-labeled probes. Results obtained with both methods were closely correlated and were in agreement with those already reported. In an identification experiment, hybridization with nonradioactive probes was performed on 60 strains isolated from stool samples after subculturing and quick DNA extraction; conventional biochemical tests were conducted in parallel. A good correlation was found between the results obtained by nonradioactive hybridization and by biochemical tests. Thus, the acetylaminofluorene-labeled genomic DNA probe method is an interesting alternative for laboratories without access to radioisotopes for the identification and classification of bacteria.

2-Acetylaminofluorene↗

PubMedDate not supplied

The use of C0t-1 probe DNA for the detection of low levels of DNA fragmentation.

In many apoptotic systems the final demise of the DNA results in the generation of multinucleosomal-sized DNA fragments, which appear as a DNA ladder after agarose electrophoresis. Extensive DNA fragmentation can be detected by ethidium bromide staining. Visualization of low levels of DNA fragmentation, however, requires both a more sensitive detection system, as well as a method of DNA extraction, that limits the extent of high molecular weight DNA shearing. We have found that the use of C0t-1 repetitive DNA as a probe for Southern analysis of DNA ladders is a sensitive method to detect low levels of DNA fragmentation. We have applied this methodology in the detection of DNA fragmentation in the normal developing C57BL/6 mouse retina at stages in which there is known DNA fragmentation, as well as in human Y-79 retinoblastoma cells grown in culture. We have found that in many instances in which there is no detectable DNA ladder with ethidium bromide staining a very definitive DNA ladder can be visualized via Southern blot analysis with a C0t-1 DNA probe.

Animals↗

PubMedDate not supplied

A Mycoplasma gallisepticum strain-specific DNA probe.

Total DNA from the vaccine F strain (K810) and the reference S6-strain of Mycoplasma gallisepticum (MG) was cloned in Escherichia coli using the plasmid pUC8. A 6-kilobase fragment, specific for the vaccine strain, was identified by colony dot and Southern hybridization analyses. When labeled and used as a probe, this fragment hybridized with the homologous and one other vaccine F-strain (F2F10), but it did not hybridize with other MG strains (Fg38, S6, A5969, V503) or with three other species of avian mycoplasmas.

Bacterial Vaccines↗

PubMed1996

Pyrene-labeled DNA probes for homogeneous detection of complementary DNA sequences: poly(C) model system.

DNA and RNA probes are important analytical reagents in molecular biology and in the detection of infectious and genetic diseases. However, the present polynucleotide probe technology is complex and labor-intensive. We have been investigating the possibility of using fluorescent-labeled DNA probes to develop assays which do not require the separation of free from hybridized probe (homogeneous assays). Such assays are possible if the fluorescence efficiency or fluorescence anisotropy of the fluorescent label changes upon hybridization of probe with target DNA. In this article we examine pyrene as a fluorescent label for DNA or RNA probes. Experiments were performed using a model system in which poly(C) and poly(I) are respectively the probe and target sequences. A small fraction of the nucleotide bases of poly(C) was randomly labeled with pyrene using the bisulfite-catalyzed diamine reaction. The results show that the uncorrected emission spectrum of pyrene-poly(C) decreases by a factor of 4 and shifts toward longer wavelengths upon hybridization with poly(I) at saturating concentrations. The average lifetime changes from 10.78 to 4 ns. These fluorescence changes occur in a wide range of chemical environments, including the high salt concentrations normally used to increase the velocity of the hybridization reaction in clinical assays. The pyrene label can thus be used to readily detect the amount of poly(I) in an unknown sample without having to separate free and bound labeled probe. To unravel the mechanism responsible for the observed changes in fluorescence intensity upon hybridization, we have performed polarized fluorescence intensity measurements and analyzed the results by approximate steady-state expressions that allow evaluation of the relative contributions of changes in lifetimes (fluorescence efficiency) and rotational motions to the changes in fluorescence intensity. The results indicate that the latter changes are due chiefly to changes in lifetime or fluorescence efficiency and that these changes seem to be due to the movement of the pyrene label to a more hydrophilic environment upon hybridization.

DNA Probes↗

PubMed1984

Sex chromosome positions in human interphase nuclei as studied by in situ hybridization with chromosome specific DNA probes.

Two cloned repetitive DNA probes, pXBR and CY1, which bind preferentially to specific regions of the human X and Y chromosome, respectively, were used to study the distribution of the sex chromosomes in human lymphocyte nuclei by in situ hybridization experiments. Our data indicate a large variability of the distances between the sex chromosomes in male and female interphase nuclei. However, the mean distance observed between the X and Y chromosome was significantly smaller than the mean distance observed between the two X-chromosomes. The distribution of distances determined experimentally is compared with three model distributions of distances, and the question of a non-random distribution of sex chromosomes is discussed. Mathematical details of these model distributions are provided in an Appendix to this paper. In the case of a human translocation chromosome (Xqter----Xp22.2::Yq11----Yqter) contained in the Chinese hamster X human hybrid cell line 445 X 393, the binding sites of pXBR and CY1 were found close to each other in most interphase nuclei. These data demonstrate the potential use of chromosome-specific repetitive DNA probes to study the problem of interphase chromosome topography.

Cell Nucleus↗

PubMed1988

Detection of toxigenic Escherichia coli using biotin-labelled DNA probes following enzymatic amplification of the heat labile toxin gene.

Several types of DNA probes labelled with biotin were compared for their sensitivity to detect the heat labile toxin (LT) gene in toxigenic Escherichia coli. In addition, a procedure was developed for enzymatically amplifying LT gene sequences in toxigenic E. coli. Probes were labelled with biotinylated nucleotides by either nick translation; 3' tailing; primer extension of probe DNA cloned into bacteriophage M13; sandwich hybridization; or oligolabelling of isolated DNA fragments. A single stranded probe consisting of a DNA fragment from the LT gene cloned into the bacteriophage M13mp18 and detected by hybridization to oligolabelled biotinylated M13mp18 RF DNA in a sandwich hybridization was able to detect as little as 10 pg of toxin gene DNA. Cloned LT gene DNA was serially diluted and amplified enzymatically using synthetic oligonucleotide primers. Amplified DNA was detected using biotin-labelled M13-based probes. As little as 1 fg of LT DNA could be amplified to detectable levels by this method. Experiments with LT+ bacteria resulted in the detection of as few as 1000 bacteria. The combination of enzymatic amplification coupled with M13-based DNA probes provides a highly sensitive tool for detecting pathogenic microorganisms.

Bacterial Toxins↗

PubMed2002

High-density microarray of small-subunit ribosomal DNA probes.

Ribosomal DNA sequence analysis, originally conceived as a way to provide a universal phylogeny for life forms, has proven useful in many areas of biological research. Some of the most promising applications of this approach are presently limited by the rate at which sequences can be analyzed. As a step toward overcoming this limitation, we have investigated the use of photolithography chip technology to perform sequence analyses on amplified small-subunit rRNA genes. The GeneChip (Affymetrix Corporation) contained 31,179 20-mer oligonucleotides that were complementary to a subalignment of sequences in the Ribosomal Database Project (RDP) (B. L. Maidak et al., Nucleic Acids Res. 29:173-174, 2001). The chip and standard Affymetrix software were able to correctly match small-subunit ribosomal DNA amplicons with the corresponding sequences in the RDP database for 15 of 17 bacterial species grown in pure culture. When bacteria collected from an air sample were tested, the method compared favorably with cloning and sequencing amplicons in determining the presence of phylogenetic groups. However, the method could not resolve the individual sequences comprising a complex mixed sample. Given these results and the potential for future enhancement of this technology, it may become widely useful.

Air Microbiology↗

PubMed1995

Prenatal diagnosis of partial trisomy through in situ hybridization on amniocytes with whole chromosome and centromere-specific DNA probes. A case report.

BACKGROUND: DNA probes specific for whole chromosomes or portions of chromosomes can provide important information to aid the clinician in managing pregnancy and the geneticist in relaying accurate recurrence risk information to the patient. CASE: In this case, sonography was ordered because of a low fundal height in a 29-year-old primigravida at 35 weeks' gestational age; it revealed major fetal anomalies. A small supernumerary marker was seen in some cultured amniocytes. Metaphase spreads were analyzed by means of fluorescence in situ hybridization using a centromere probe specific for chromosome 22 and a whole chromosome probe for the 11 chromosome. In situ hybridization showed that the marker chromosome was a derivative of chromosome 22 with 11q material attached near the centromere. The fetal karyotype was 47,XY,+der(22) t(11;22)(q23.3;q11.2)mat. The mother was later found to be a balanced translocation carrier. CONCLUSION: It was possible to offer rapid prenatal diagnosis for this family using interphase analysis with the 22 centromere probe. The patient had chorionic villus sampling, and DNA probes were used to analyze cells directly from the biopsy. The signal representing the supernumerary marker was not observed. Karyotype analysis later showed that the fetus was normal but a translocation carrier. This report illustrates that rapid in situ hybridization can provide important information in known cases of translocation carriers.

Adult↗

PubMed2004

Dna probes for identification of leptospires and disease diagnosis.

A newly identified 1 kb DNA fragment amplified by PCR using (AG)8T inter-simple sequence repeats (ISSR) primer and a 631 bp segment of 16S rRNA ribosomal gene amplified by PCR using reported primers were labeled with a alpha32P dCTP for use as DNA probes. These probes were hybridized with DNA extracted from 19 standard pathogenic serovars, 3 standard saprophytic serovars, 33 pathogenic isolates (12 from patients, 1 from a tapwater source, and 20 from rodents), and 22 saprophytic isolates from environmental sources. The pathogen-specific 16S rRNA DNA probe specifically hybridized all 33 standard pathogenic serovars, to 13 pathogenic isolates. Similarly, the saprophyte specific 1 kb ISSR DNA probe specifically hybridized the 3 standard saprophytic serovars and the 22 saprophytic Leptospira isolates. The sensitivity of the 1 kb labeled saprophytic Leptospira specific DNA probe was 1.95 ng, and for the 16S rRNA pathogen specific probe 3.90 ng. The 16S rRNA gene segment DNA probe could also identify the leptospiremic stage in mice or guinea pigs infected experimentally with the pathogenic serovars australis, autumnalis or icterohaemorrhagiae. DNA probes therefore, owing to their high specificity and sensitivity, appear useful for easy, rapid, and reliable differentiation of pathogenic Leptospira strains and also hold promise for direct identification of organisms in blood samples to diagnose leptopsirosis.

Animals↗

PubMed1992

Babesia bigemina: quantitation of infection in nymphal and adult Boophilus microplus using a DNA probe.

Candidates for a subunit vaccine against bovine babesiosis include surface proteins of infective forms found in the salivary glands of tick vectors. However, low numbers of infective forms are present within ticks and hinder analysis of this stage. To solve this problem, conditions which yield high numbers of infective forms were investigated with the use of a Babesia bigemina-specific DNA probe. DNA from progeny of female Boophilus microplus infected with B. bigemina was hybridized to probe DNA to detect and quantitate infection. There was no difference in the prevalence of infection in progeny of three strains of Bo. microplus. However, within a strain, prevalence could be increased to 30% by combining selection of progeny from heavily (3+) infected female ticks and selection of eggs laid 120 hr postengorgement. Quantitation of infective forms within pooled salivary gland preparations of 10 infected nymphal and adult Bo. microplus demonstrated that Day 9 and 10 nymphal ticks contained the highest numbers of parasites and represented approximately 10(6) infective forms. This number of infective forms is suitable for isolation and further characterization.

Animals↗

PubMed2003

Monitoring molecular beacon DNA probe hybridization at the single-molecule level.

We have monitored the reaction dynamics of the DNA hybridization process on a liquid/solid interface at the single-molecule level by using a hairpin-type molecular beacon DNA probe. Fluorescence images of single DNA probes were recorded by using total internal reflection fluorescence microscopy. The fluorescence signal of single DNA probes during the hybridization to individual complementary DNA probes was monitored over time. Among 400 molecular beacon DNA probes that we tracked, 349 molecular beacons (87.5 %) were hybridized quickly and showed an abrupt fluorescence increase, while 51 probes (12.5 %) reacted slowly, resulting in a gradual fluorescence increase. This ratio stayed about the same when varying the concentrations of cDNA in MB hybridization on the liquid/surface interface. Statistical data of the 51 single-molecule hybridization images showed that there was a multistep hybridization process. Our results also showed that photostability for the dye molecules associated with the double-stranded hybrids was better than that for those with the single-stranded molecular beacon DNA probes. Our results demonstrate the ability to obtain a better understanding of DNA hybridization processes using single-molecule techniques, which will improve biosensor and biochip development where surface-immobilized molecular beacon DNA probes provide unique advantages in signal transduction.

Biosensing Techniques↗

PubMed1989

In situ DNA-RNA hybridization using in vivo bromodeoxyuridine-labeled DNA probe.

An in vivo 5'-bromodeoxyuridine (BrdUrd) labeled DNA probe was used for in situ DNA-RNA hybridization. BrdUrd was incorporated into plasmid DNA by inoculating E. coli with Luria-Bertani (LB) culture medium containing 500 mg/L of BrdUrd. After purification of the plasmid DNA, specific probes of the defined DNA fragments, which contained the cloned insert and short stretches of the vector DNA, were generated by restriction endonuclease. The enzymatic digestion pattern of the BrdUrd-labeled plasmid DNA was the same as that of the non-labeled one. BrdUrd was incorporated in 15%-20% of the total DNA, that is, about 80% of the thymidine was replaced by BrdUrd. Picogram amounts of the BrdUrd-labeled DNA probe itself and the target DNA were detectable on nitrocellulose filters in dot-blot spot and hybridization experiments using a peroxidase/diaminobenzidine combination. The BrdUrd-labeled DNA probe was efficiently hybridized with both single stranded DNA on nitrocellulose filters and cellular mRNA in in situ hybridization experiments. Through the reaction with BrdUrd in single stranded tails, hybridized probes were clearly detectable with fluorescent microscopy using a FITC-conjugated monoclonal anti-BrdUrd antibody. The in vivo labeling method did not require nick translation steps or in vitro DNA polymerase reactions. Sensitive, stable and efficient DNA probes were easily obtainable with this method.

Bromodeoxyuridine↗

PubMed1994

A comparative study for detection of Chlamydia trachomatis and Neisseria gonorrhoeae with DNA probe (a note).

A newly developed DNA probe assay (Gen-Probe Pace 2 San Diego, USA) was compared with Chlamydia trachomatis direct immunofluorescence and Neisseria gonorrhoeae culture. Detection of C. trachomatis in cervical specimens from women and urethral specimens from men showed 23% positivity out of 313 cases. Out of the 69 positive cases 40 were positive with both examinations, in 29 cases only with DNA probe. Examinations of N. gonorrhoeae in 254 patients gave 98% positivity. Sensitivity of DNA probe assay was 100%, specificity was 97.8%. On the basis of preliminary data Gen-Probe is suitable for the detection of both causative agents.

Chlamydia Infections↗

PubMed1989

DNA probes for antimicrobial susceptibility testing.

As DNA probes are used more frequently in the clinical laboratory for the detection and identification of pathogens in clinical samples, a means of determining the antimicrobial susceptibility profile of those pathogens will be required. DNA probes directed to specific resistance determinants offer a solution to this problem. Methods of determining the susceptibility of viruses to antiviral agents can also be accomplished with DNA probes. This article explores the advantages and disadvantages of using hybridization methods for susceptibility testing of organisms contained in clinical samples.

DNA Probes↗

PubMed1992

[Diagnosis on Mycoplasma pneumoniae infections by DNA-probe assay in comparison with serological tests].

To evaluate the usefulness of DNA diagnosis as a diagnostic approach to Mycoplasma pneumoniae infections, we compared the DNA-probe assay with serological tests in 32 patients who were clinically suspected of having Mycoplasma pneumonia. The DNA-probe assay was carried out using the Gen-probe kit. Serological tests included complement fixation and passive hemagglutination tests. At first visit 10 patients were positive for the DNA-probe assay, while only 2 of them were positive for serological tests. The other 3 patients became positive for serological tests during the clinical course. In accordance with clinical improvement, these patients became negative for the DNA-probe assay. Three patients were negative for DNA-probe assay while they were positive for the serological tests. However, these patients had already received antibiotics; therefore, their conditions were considered to have been improved at the time of the study. The other 19 patients were negative for both the DNA-probe assay and serological tests. These patients might have suffered from respiratory tract infections of pathogenic organisms other than M. pneumoniae or the number of M. pneumoniae might have been too small to be detected by the DNA-probe assay. The results of the present work demonstrate that the DNA-probe assay was valuable in the diagnosis of mycoplasmal infections at the early stage, which indicates that DNA diagnosis provides useful information to determine the most appropriate therapeutic regimen.

Child↗

PubMed1989

The use of DNA probes in preimplantation and prenatal diagnosis.

DNA probes are now widely used for prenatal diagnosis, but the prospect of preimplantation diagnosis of genetic disorders requires the development of sensitive genetic tests that can be performed on small numbers of cells removed from a preimplantation-stage pre-embryo. The sensitivity of molecular tests can now be increased by specifically amplifying the target DNA with the polymerase chain reaction. In situ hybridisation with chromosome-specific DNA probes to repeated sequences also permits the detection of particular numerical chromosome aberrations or the distinction of male and female pre-embryos when only a few interphase nuclei are available. We have used in situ hybridisation to a Y chromosome-specific DNA probe to sex preimplantation-stage pre-embryos and to sex fetuses from samples of chorionic villus cells, amniotic fluid cells, and fetal blood. These two approaches (amplification of target DNA and in situ hybridisation) provide suitable tests for improving prenatal diagnosis particularly when few cells are available and they offer the possibility of tests suitable for preimplantation diagnosis.

Blastocyst↗

PubMed1995

Development and evaluation of PCR assay for detection of low levels of Cowdria ruminantium infection in Amblyomma ticks not detected by DNA probe.

The sensitivities of a PCR assay and a DNA probe assay were compared for the detection of Cowdria ruminantium in Amblyomma ticks that were fed on C. ruminantium-infected, clinically reacting, and recovered carrier animals. The PCR assay and DNA probe detected infection in 86.0 and 37.0%, respectively, of 100 ticks fed on a febrile animal. In 75 ticks fed on carrier animals, PCR and the DNA probe detected infection in 28.0 and 1.33% of ticks, respectively. This demonstrates that the DNA probe has poor sensitivity for the detection of low levels of infection in ticks and that PCR is necessary for this purpose. The PCR assay had a detection limit of between 1 and 10 C. ruminantium organisms and did not amplify DNA from Ehrlichia canis, which is phylogenetically closely related to C. ruminantium, Theileria parva, or uninfected Amblyomma hebraeum or A. variegatum. PCR detected infection in A. hebraeum and A. variegatum adult ticks infected with one of six geographically different C. ruminantium strains. Amplification was also possible from desiccated ticks and ticks fixed in 70% ethanol, 10% buffered formalin, or 2% glutaraldehyde. The PCR assay supersedes the DNA probe and older detection methods for the detection of C. ruminantium in ticks, particularly those fed on carrier animals, and is suitable for both prospective and retrospective studies which require accurate detection of C. ruminantium in individual ticks. Application of the PCR assay should significantly improve the understanding of heartwater epidemiology, particularly through the determination of field tick infection rates.

Animals↗

PubMed2002

Atomic force microscopy identification of transcription factor NFkappaB bound to streptavidin-pin-holding DNA probe.

A novel method for identifying DNA-binding proteins from image analysis using AFM was developed. Here, transcription factor NFkappaB, which a well-studied example of transcription activator proteins, was used as a target protein. 5'-biotinlynated double-stranded DNA probe was labeled site specifically through high affinity with streptavidin. When the biotinylated DNA fragments were incubated with the streptavidin at a 1:2 molar ratio of DNA:streptavidin, the overall efficiency of labeling was over 90%. The double-stranded DNA probes were immobilized on a mica surface by the adsorption of streptavidin that attached to the 5'-end of DNA and applied for selection of the target protein NFkappaB in solution and then AFM was used to image the DNA probe-NFkappaB complexes. The length of the distance between 5'-labeled streptavidin and NFkappaB bound on DNA probes from AFM images is 0.64, the normalized position of the NFkappaB binding site, and this result is in close agreement with the expected 299 and 167bp values.

Base Sequence↗