A comparison of non-radioisotopic hybridization assay methods using fluorescent, chemiluminescent and enzyme labeled synthetic oligodeoxyribonucleotide probes

Urdea, Mickey S.; Warner, Brian D.; Running, Joyce A.; Stempien, Michelle M.; Clyne, Jennifer; Horn, Thomas

doi:10.1093/nar/16.11.4937

Cited by 231 publications

(105 citation statements)

References 35 publications

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“…Development of automated DNA electrophoresis, hybridization and electronic detection of signal (Zapolski etal., 1987). In addition to these improvements, there have been great advances in the development of novel non-radioactive probes, such as those based on incorporation of digoxigenin-dUTP (Boehringer Mannheim, 1988b), bioluminescence (Hauber and Geiger, 1988) and chemiluminescence (Thorpe and Kricka, 1987;Urdea et al, 1988); new hybridization amplification methods; and ELISA-based solution hybridization techniques. These developments are discussed in more detail below.…”

Section: Nucleic Acid Hybridization Methodsmentioning

confidence: 99%

New Approaches to the Detection of Microbial Plant Pathogens

Chu

Waterhouse

Martín

et al. 1989

Biotechnology and Genetic Engineering Reviews

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Section: Nucleic Acid Hybridization Methodsmentioning

confidence: 99%

New Approaches to the Detection of Microbial Plant Pathogens

Chu

Waterhouse

Martín

et al. 1989

Biotechnology and Genetic Engineering Reviews

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“…Besides MED134, probe DOK196 targets 27 uncultured marine clones and 28 cultured strains (5 Dokdonia sp., 5 Krokinobacter sp., 1 Flexibacter sp., 2 isolates from marine sponges and 15 from coastal bacterioplankton). Probes were supplied by Thermo Electron Corporation with an aminolink (C6) at the 5 0 end, ligated with a horseradish peroxidase enzyme (Urdea et al, 1988), and then optimized for catalyzed reporter deposition (CARD)-FISH (Pernthaler et al, 2002) with bacterial cells (fixed and filtered as before) from the target culture. Filter pieces were permeabilized with lysozyme and achromopeptidase before hybridizing overnight at 35 1C in a buffer containing 30% formamide (which gave the best results after trying a range from 10% to 60%).…”

Section: Natural Microbial Assemblagesmentioning

confidence: 99%

Grazing rates and functional diversity of uncultured heterotrophic flagellates

et al. 2009

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Aquatic assemblages of heterotrophic protists are very diverse and formed primarily by organisms that remain uncultured. Thus, a critical issue is assigning a functional role to this unknown biota. Here we measured grazing rates of uncultured protists in natural assemblages (detected by fluorescent in situ hybridization (FISH)), and investigated their prey preference over several bacterial tracers in short-term ingestion experiments. These included fluorescently labeled bacteria (FLB) and two strains of the Roseobacter lineage and the family Flavobacteriaceae, of various cell sizes, which were offered alive and detected by catalyzed reporter deposition-FISH after the ingestion. We obtained grazing rates of the globally distributed and uncultured marine stramenopiles groups 4 and 1 (MAST-4 and MAST-1C) flagellates. Using FLB, the grazing rate of MAST-4 was somewhat lower than whole community rates, consistent with its small size. MAST-4 preferred live bacteria, and clearance rates with these tracers were up to 2 nl per predator per h. On the other hand, grazing rates of MAST-1C differed strongly depending on the tracer prey used, and these differences could not be explained by cell viability. Highest rates were obtained using FLB whereas the flavobacteria strain was hardly ingested. Possible explanations would be that the small flavobacteria cells were outside the effective size range of edible prey, or that MAST-1C selects against this particular strain. Our original dual FISH protocol applied to grazing experiments reveals important functional differences between distinct uncultured protists and offers the possibility to disentangle the complexity of microbial food webs.

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“…Southern blots were hybridized with oligonucleotides either labelled with peroxidase o r labelled with [y-32P]dATP. Peroxidase labelling reactions were performed as described by Urdea et al (1988).…”

Section: Pahmentioning

confidence: 99%

Differential detection of key enzymes of polyaromatic-hydrocarbon-degrading bacteria using PCR and gene probes

Moser²,

et al. 1999

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Bacteria with ability to degrade polyaromatic hydrocarbons (PAHs), isolated from wastewater and soil samples, were investigated for their taxonomic, physiological and genetic diversity. Eighteen isolates able to metabolize naphthalene or phenanthrene as sole carbon source were taxonomically affiliated to different subclasses of the Proteobacteria (Sphingomonas spp., Acidovorax spp., Comamonas spp. and Pseudomonas spp.) and to phyla of Gram-positive bacteria with low and high DNA G+C content (Paenibacillus sp. and Rhodococcus spp., respectively). Representatives of the genera Pseudomonas and Sphingomonas formed a remarkably high fraction of these isolates; 9 out of 18 strains belonged to these groups. Tests for enzyme activities showed that the majority of the isolates growing with PAHs as sole sources of carbon and energy had an active catechol2,3-dioxygenase (C230). C230 specific activities were very diverse, ranging from 0-1 to 650 mU (mg protein)-'. Pseudomonas and Sphingomonas strains showed considerably higher activities than the other isolates. All PAH degraders were examined for the presence of an initial PAH dioxygenase and C230, which catalyse key steps of PAH degradation, by PCR amplification of gene fragments and subsequent hybridization. PCR primers and internal oligonucleotide probes were developed for the specific detection of the genes of Pseudomonas and Sphingomonas strains. (1996). The aim of the present investigation was to isolate bacterial PAH degraders from aquatic a n d terrestrial sites, to determine their taxonomic affiliation and to characterize them physiologically a n d genetically with respect to their ability to degrade PAHs. al., 1996) are specific for most genuine pseudornonads, distinct P. putida strains, and the genera Comamonas and Acidovorax, respectively. Keywords METHODSFurther examination of the isolates was done by physiological tests as reported previously (Kampfer et al., 1991). Fatty acid methyl ester (FAME) patterns were used as a further marker for identification. Preparation of FAME extracts, gas chromatographic analysis and numerical identification by the MIDI system (Sherlock 2.11) were performed as described previously (Kampfer et al., 1996).Determination of naphthalene and phenanthrene mineralization potential. The potential of the isolated bacteria for mineralization of PAHs was examined by gas chromatographically quantifying remaining PAH and sirnultaneously monitoring bacterial growth in batch cultures.For each tested strain, five airtight flasks were each filled with 10 rnl basal medium containing either 005 ' / o naphthalene or phenanthrene and were inoculated with approximately 10' cells. Cell number was estimated by measuring the optical density at 600nm, Remaining PAH in the cultures was extracted by vigorously shaking (30 rnin) with 5 rnl n-hexane for gas chromatographic analysis. The gas chromatograph HP 6890 was equipped with a flame-ionization detector and a 25 m capillary column (Ultra 2, cross-linked 5 % phenyl methyl siloxan ; Hewlett Packard) ....

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A comparison of non-radioisotopic hybridization assay methods using fluorescent, chemiluminescent and enzyme labeled synthetic oligodeoxyribonucleotide probes

Cited by 231 publications

References 35 publications

New Approaches to the Detection of Microbial Plant Pathogens

New Approaches to the Detection of Microbial Plant Pathogens

Grazing rates and functional diversity of uncultured heterotrophic flagellates

Differential detection of key enzymes of polyaromatic-hydrocarbon-degrading bacteria using PCR and gene probes

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