PubMed Health⌕ Search

SEARCH · PubMed Health

Results for “DNA probe”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 37 records · Page 2Linked to original sources
PubMed2006

Redox polymer and probe DNA tethered to gold electrodes for enzyme-amplified amperometric detection of DNA hybridization.

The detection of nucleic acids based upon recognition surfaces formed by co-immobilization of a redox polymer mediator and DNA probe sequences on gold electrodes is described. The recognition surface consists of a redox polymer, [Os(2,2'-bipyridine)2(polyvinylimidazole)(10)Cl](+/2+), and a model single DNA strand cross-linked and tethered to a gold electrode via an anchoring self-assembled monolayer (SAM) of cysteamine. Hybridization between the immobilized probe DNA of the recognition surface and a biotin-conjugated target DNA sequence (designed from the ssrA gene of Listeria monocytogenes), followed by addition of an enzyme (glucose oxidase)-avidin conjugate, results in electrical contact between the enzyme and the mediating redox polymer. In the presence of glucose, the current generated due to the catalytic oxidation of glucose to gluconolactone is measured, and a response is obtained that is binding-dependent. The tethering of the probe DNA and redox polymer to the SAM improves the stability of the surface to assay conditions of rigorous washing and high salt concentration (1 M). These conditions eliminate nonspecific interaction of both the target DNA and the enzyme-avidin conjugate with the recognition surfaces. The sensor response increases linearly with increasing concentration of target DNA in the range of 1 x 10(-9) to 2 x 10(-6) M. The detection limit is approximately 1.4 fmol, (corresponding to 0.2 nM of target DNA). Regeneration of the recognition surface is possible by treatment with 0.25 M NaOH solution. After rehybridization of the regenerated surface with the target DNA sequence, >95% of the current is recovered, indicating that the redox polymer and probe DNA are strongly bound to the surface. These results demonstrate the utility of the proposed approach.

Avidin↗
PubMed1992

Centromere detection in vinblastine- and radiation-induced micronuclei of cytokinesis-blocked mouse cells by using in situ hybridization with a mouse gamma (major) satellite DNA probe.

Non-isotopic in situ hybridization using a mouse gamma (major) satellite probe DNA was applied to detect centromeres in micronuclei, which were induced in vitro in mouse liver cells by ionizing radiation and by vinblastine sulfate. In a cytokinesis-blocked micronucleus assay a dose-dependent induction of micronuclei was found for both agents. After vinblastine exposure the observed micronuclei showed centromere-positive hybridization signals in an order of magnitude of 70-90%, but after radiation exposure the magnitude was only 10-20%. Since the in situ hybridization technique detects centromeric DNA directly, it can be used in a cytokinesis-blocked micronucleus assay for a rapid and reliable discrimination between aneuploidy-inducing and clastogenic agents.

Animals↗
PubMed1985

The use of plasmid DNA to probe DNA repair functions in the yeast Saccharomyces cerevisiae.

The survival of plasmid YRp12 treated in vitro with ultraviolet- or gamma-radiation, or with restriction endonucleases, has been used to investigate in vivo RAD gene activity in Saccharomyces cerevisiae. Yields of pyrimidine dimers or single and double strand breaks in plasmid DNA were assayed by physical methods. The biological effects of these damages were assayed by transformation of wild-type cells and rad mutants from each of the major groups of radiosensitive mutants. After UV-irradiation plasmid survival depended qualitatively on the same host functions that are needed for cellular survival. After gamma-irradiation no such correspondence was found. Apart from a RAD52-dependent stimulation of transformation efficiency at low doses, other host repair functions had little effect. Stimulation of transformation corresponded with the production of double- but not single-strand breaks in plasmid sequences homologous with the yeast genome and may be linked with a transient increase in mitotic stability. More generally these data also show that transformation events using the LiCl protocol may entail the uptake of a very low number of plasmid molecules per cell over a 10-fold range of DNA concentrations.

DNA Repair↗
PubMed1988

DNA probes for mycobacteria. I. Isolation of DNA probes for the identification of Mycobacterium tuberculosis complex and for mycobacteria other than tuberculosis (MOTT).

Traditional methods used in identifying mycobacteria such as acid-fast bacillus stains and culture are often time-consuming, insensitive and non-specific. The isolation of DNA probes, coupled to a non-radioactive, e.g. biotin-based detection system, have the potential to foster the development of clinical assays for Mycobacterium tuberculosis and mycobacteria other than tuberculosis (MOTT) that are rapid, sensitive and specific. To this end, we have isolated two different probes: one which is specific for the Mtb complex and one which recognizes all other potentially pathogenic mycobacteria. The use of these probes in combination should allow the detection and differentiation of M. tuberculosis from MOTT. To isolate the first probe, we prepared a library of M. tuberculosis DNA fragments in a lambda EMBL phage vector. Recombinant phage were screened by plaque-lift hybridization procedures using nick-translated mycobacterial genomic DNA to identify sequences specific to the Mtb complex. Inserts from candidate recombinant phage were purified, nick-translated and hybridized against a wide variety of filter-bound mycobacterial and non-mycobacterial DNAs. Two clones were identified which hybridized to the closely related M. tuberculosis, M. bovis and M. microti but not to other species of mycobacteria. The second probe was isolated by preparing a library of M. malmoense DNA fragments in lambda EMBL and screening by plaque-lift hybridization. One clone was identified which, in addition to recognizing members of the Mtb complex, also hybridized to M. intracellulare, M. malmoense, M. scrofulaceum, M. simiae, M. xenopi, M. avium, M. szulgai, M. kansasii and M. haemophilum. None of the three clones hybridized to DNA from non-mycobacterial species.

Animals↗
PubMed1996

Use of asymmetric PCR to generate long primers and single-stranded DNA for incorporating cross-linking analogs into specific sites in a DNA probe.

Photoactivatable DNA analogs have been incorporated enzymatically into DNA and used to map the locations of polypeptides in protein complexes bound to DNA. We have developed a procedure for generating long primers from short oligodeoxyribonucleotides (oligos) to incorporate DNA cross-linkers at specific sites within either strand of DNA probes of < or = 206 bp. Single-stranded DNA molecules of 52-206 nucleotides in length were generated by asymmetric polymerase chain reactions (aPCR), using an excess of one short sense-strand primer to be extended and a limiting amount of each short antisense primer that is complementary to and defines the 3' end of the long primer to be generated. The noncross-linking strand of the DNA probe was also generated by aPCR from the DNA sequence of interest. The long primers were annealed to the full-length noncross-linking DNA strand to form a partially double-stranded DNA. Cross-linking analogs and radioactive deoxyribonucleotides (dNTPs), followed by normal dNTPs, were enzymatically incorporated onto the long primers to form the double-stranded DNA cross-linking probes. This method is reproducible and avoids many of the difficulties encountered by other published methods.

Base Sequence↗
PubMed1990

Detection of methicillin resistance in staphylococci by using a DNA probe.

A DNA probe derived from the PBP 2a gene of the methicillin-resistant Staphylococcus aureus COL was compared with phenotypic microbiologic tests for its ability to identify methicillin-resistant and -susceptible staphylococci. Lysates were applied to nitrocellulose with a dot blot apparatus. Isolates tested were both S. aureus and coagulase-negative staphylococci that had been recovered from a variety of geographic and clinical sources. When compared with a spread plate phenotypic test, the DNA probe gave sensitivity, specificity, and predictive values for both positive and negative tests of 100% for 204 S. aureus isolates (103 positive, 101 negative) and 99, 95, 99, and 95%, respectively, for 249 coagulase-negative staphylococci (210 positive, 39 negative). The probe was more sensitive than broth microdilution and more specific than agar dilution in identifying methicillin-resistant and -susceptible coagulase-negative staphylococci; all tests were equally accurate in identifying the methicillin susceptibility of S. aureus. DNA probe analysis for determining the methicillin susceptibility of staphylococci was rapid, easily interpretable, and equally accurate with radioactive and nonradioactive probes, and it gave results equivalent to the most sensitive microbiologic test for all staphylococcus species studied.

Bacterial Proteins↗
PubMed1995

[Surgical treatment of infective endocarditis: application of DNA probe method].

DNA probe method is a new bacteriological method for diagnosis of bacteria. The authors tried to apply the method to diagnosis of bacteremia and treatment of infective endocarditis. We could diagnose the patient's illness as bacteremia with this method even when blood cultures are not positive. We suggest that cardiac surgery should be performed in case bacteria is detected repeatedly with DNA probe method. Therefore it is useful for decision whether cardiac surgery for patients with active infective endocarditis should be done or not.

Adolescent↗
PubMed1983

Rapid and sensitive colorimetric method for visualizing biotin-labeled DNA probes hybridized to DNA or RNA immobilized on nitrocellulose: Bio-blots.

Biotin-labelled DNA probes, prepared by nick-translation in the presence of biotinylated analogs of TTP, are hybridized to DNA or RNA immobilized on nitrocellulose filters. After removal of residual probe, the filters are incubated for 2--5 min with a preformed complex made with avidin-DH (or streptavidin) and biotinylated polymers of intestinal alkaline phosphatase. The filters are then incubated with a mixture of 5-bromo-4-chloro-3-indolyl phosphate and nitro blue tetrazolium, which results in the deposition of a purple precipitate at the sites of hybridization. This procedure will detect target sequences in the 1- to 10-pg range after enzyme incubation periods of 1 hr or less. The incubation period can be extended up to 24 hr, if required, to increase the color intensity of the hybridization signal. Furthermore, at high probe concentrations (250--7560 ng/ml), biotin-labeled DNA exhibits lower nonspecific binding to nitrocellulose than does radiolabeled DNA, so hybridization times required for the analysis of unique mammalian gene sequences can be decreased to 1--2 hr. This nonradiographic method of probe detection should be of general utility for genetic studies using Southern, RNA, or dot-blot hybridization protocols.

Alkaline Phosphatase↗
PubMed1994

Rapid detection of mycobacteria by combining a radiometric detection system with DNA probes.

Forty-four Mycobacterium-spp. were isolated in 33 patients from an infectious diseases ward. All patients were HIV-positive and most of them were drug-abusers. M. avium-intracellulare was the most common type of MOTT (Mycobacteria other than tuberculosis) detected and the only microorganism isolated in patients with mycobacteriaemia. The radiometric method performed by the Bactec system enhanced the isolation rate of mycobacteria, especially from the bloodstream. The Gen-probe DNA hybridization system proved to be rapid diagnostic tool for the identification of strains.

DNA Probes↗
PubMed2006

Effect of external fluctuations on the affinity-specificity negative correlation in DNA-probe interactions.

We show that the site-specific interaction of a probe DNA with the template DNA can be well modeled as an unbiased random jump process, where the probe molecule first nonspecifically binds to the template DNA and then searches for the specific site via unbiased random jump motion on the template DNA. By investigating the effects of increasing the jump size, and the fluctuations in the position of the specific site and the fluctuations in the specific site interval on the affinity-specificity negative correlation, we show that (1) increasing the jump size will in turn increase the affinity of the probe toward its target site on the template DNA, however, with a limiting value--the maximum affinity condition; (2) the degree of supercoiling or condensation of the template DNA as well as the electrostatic interactions between the probe and the template in turn control the jump size associated with the dynamics of the probe on the template DNA; (3) under a maximum specificity condition (therefore with minimum affinity), by introducing an external fluctuation in the relative position of the target site on the template DNA with respect to the probe, one can still improve the affinity rate; (4) on the other hand, one can improve the specificity of the probe toward the target site on the template DNA by introducing external fluctuations in the target-site interval. Finally, we propose the design strategies and optimum experimental conditions to simultaneously enhance the affinity as well as the specificity of probe toward its target site on the template DNA.

Binding Sites↗
PubMed1995

DNA probe assay based on exonuclease III digestion of probes hybridized on target DNA.

A quantitative DNA probe assay process has been developed that uses exonuclease III. The fluorophore-labeled DNA probe is hybridized with specific sequences of the target DNA and then enzymatically digested. As these probe hybridization and digestion cycle reactions are repeated at a fixed temperature, digested probes (shortened probes) accumulate in the reaction mixture in a manner similar to a DNA polymerase chain reaction. Investigation of the digestion characteristics of the DNA probe showed that a slight digestion of a free single-stranded probe produces a large background signal, which results in low detection sensitivity. The digestion of single-stranded DNA probes is caused by double-stranded formations in the molecules. This digestion decreases and the double-stranded-specific digestion increases with increasing reaction temperature. When the reaction occurs at 45 degrees C, the association rate of the enzyme on the double-stranded DNA is 700 times faster than that on single-stranded DNA. This enables selective digestion of double-stranded DNA. The detection limit is 9 x 10(-19) mol for a M13-phage DNA.

Animals↗
PubMed1987

Rapid detection of a specific trimethoprim resistance gene using a biotinylated DNA probe.

A DNA probe specific for the dihydrofolate reductase (DHFR) type I gene was labelled with biotin by the process of nick-translation and used to screen 83 independently-derived trimethoprim R plasmids from Enterobacteriaceae. Hybridization was detected using streptavidine and a biotin-conjugated alkaline phosphatase to generate an insoluble coloured precipitate following the addition of an appropriate dye. Sixty-eight plasmids (81.9%) hybridized with the probe for DHFR type I. The method could be adapted for use with any antibiotic resistance gene for which a suitable DNA probe is available and has none of the drawbacks associated with the use of radioactively-labelled DNA in hybridization techniques.

Alkaline Phosphatase↗
PubMed2002

[The in situ hybridization effort estimation between antisense RNA probe and random primed DNA probe].

OBJECTIVE: To estimate the effort between antisense RNA probe and random primed DNA probe. METHODS: The in situ hybridization was conducted on parafin section from wounding model of rat skin. RESULTS: Although both probes appeared positive staining, RNA probes was superior to DNA probes in terms of depth of staining and background. CONCLUSION: RNA probe showed more satisfactorily on ISH.

Animals↗
PubMed1994

Evidence that Escherichia coli isolated from the intestine of healthy pigs hybridize with LT-II, ST-Ib and SLT-II DNA probes.

A DNA probe specific for genes coding for the heat-labile toxin type II (LT-II), heat-stable toxin type Ib (ST-Ib) and Shiga-like toxin type II (SLT-II), were used to examine 118 fecal Escherichia coli strains isolated from healthy pigs. Fifty-six (47.4%) of the isolated were hybridized with the LT-II probe. Thirty-nine strains (33%) possessed ST-Ib genes and five of the 118 isolates (4.2%) showed homology with the SLT-II DNA probe. E. coli that possessed unusual toxin genes for pigs were isolated with a high frequency, which indicates the importance of toxigenic clones found in nature. Uncommon virulence factors should be examined in order to improve the efficiency of diagnosis and control procedures.

Aging↗
PubMed1987

First-trimester prenatal diagnosis for Huntington's disease with DNA probes.

Polymorphic DNA probes linked to the locus for Huntington disease (HD) were used for prenatal diagnosis of a 10-week fetus at 25% risk for the disease. The fetus proved to have a 48% risk of having inherited the HD mutation which was similar to that for the at-risk parent (50%). On this basis the parents elected to terminate the pregnancy. When appropriate family members are available and DNA studies are informative, prenatal diagnosis of HD with polymorphic DNA probes can determine the at-risk status of the fetus with 96% accuracy.

Adult↗
PubMed1991

Detection of Listeria monocytogenes by direct colony hybridization on hydrophobic grid-membrane filters by using a chromogen-labeled DNA probe.

A DNA probe specific for Listeria monocytogenes was isolated from a beta-hemolytic recombinant clone of an L. monocytogenes gene bank. It was labeled with horseradish peroxidase and used in a direct colony hybridization method on hydrophobic grid-membrane filters for the detection of the organism. Following color development of the chromogen, a commercial counter (HGMF Interpreter) was able to detect and count the organisms electronically. The method gave a positive reaction with 70 L. monocytogenes strains, while showing a negative reaction with 10 strains of other Listeria spp. and with 20 organisms of other genera.

Chromogenic Compounds↗
PubMed1991

Identification of mesophilic lactic acid bacteria by using polymerase chain reaction-amplified variable regions of 16S rRNA and specific DNA probes.

Specific DNA probes based on variable regions V1 and V3 of 16S rRNA of lactic acid bacteria were designed. These probes were used in hybridization experiments with variable regions amplified by using the polymerase chain reaction. In this way, a rapid and sensitive method was developed for the identification and classification of Lactococcus and Leuconostoc species.

Base Sequence↗