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Results show hybridization efficiencies of 98% and 75% for the 12-mer and 88-mer colloidal gold particles, respectively. The enzymatic extension of the primers are attached to the particles. Data indicate that the reaction efficiency is dependent on the linker length and primer coverage.

Oligonucleotide microarrays are used to analyze target sequences on the basis of differences in hybridization stability between matched and mismatched probe-target duplexes. DNA microarray manufacture via photolithographic synthesis generates a minority of full-length oligonucleotide probes along with a series of 5'-truncated contaminants. In a model experiment, we now investigate the effect of truncated oligonucleotides on the ability to distinguish target sequence variants that differ in a single nucleotide position. A series of oligonucleotides, mixed in proportions simulating stepwise synthetic yields of between 82 and 100%, were bound to a solid support and allowed to hybridize to a target molecule. The extent of hybridization was monitored over a range of temperatures via the fluorescence of a double-strand-specific dye. The discriminatory power of pure oligonucleotide probes was found to be significantly greater than that of a population of truncated probes, but only over a limited temperature interval. We conclude that at optimal temperatures greater oligonucleotide quality can improve the performance of oligonucleotide hybridization microarrays.

A molecular beacon, an oligonucleotide probe with inherent signal transduction mechanisms, is an optimal tool for visualizing real-time mRNA hybridization in single living cells. Each molecular beacon (MB) consists of a single-stranded DNA molecule in a stem-loop conformation with a fluorophore linked to the 5' end and a quencher at the 3' end. In this study, we demonstrate real-time monitoring of mRNA*DNA hybridization inside living cells using molecular beacons. A MB specific for [beta]-actin mRNA has been designed and synthesized. After micro-injection into the cytoplasm of single living kangaroo rat kidney cells (PtK2 cells), the MB hybridizes with [beta]-actin mRNA as shown by fluorescence measurements over time. Hybridization dynamics have been followed. Strict control experiments have been carried out to confirm that the fluorescence signal increase is indeed due to the hybridization of mRNA inside single living cells. Variation in the MB/mRNA hybridization fluorescent signal has been observed for different PtK2 cells, which indicates the amount of mRNA in different cells is different. We have also monitored the [beta]-1 andrenergic receptor mRNA inside the PtK2 cells. These studies demonstrate the feasibility of using MBs and the ultrasensitivity achieved in our fluorescence imaging system for real-time detection of mRNA hybridization and for the visualization of oligo-nucleotide/mRNA interactions inside single living cells.

Closely related Prochlorococcus genotypes have been shown to have remarkably different depth distsributions in the Red Sea and the North Atlantic. An in situ hybridization method was used for identification of marine cyanobacteria using horseradish-peroxidase-labelled 16S rRNA targeted oligonucleotide probes in combination with tyramid signal amplification (TSA).

Repeated ruminal dosing of Ruminococcus spp. has been found not to result in persistence in studies related to fiber digestibility and whether it is the cellulolytic bacteria or the nature of plant cell walls that are the limiting factor. Changes in other microbial populations can be measured with unique quantitative 16S-rRna-based probes. Higher level probes were used to explore the effects of dosing on Fibrobacter, Ruminococcus, and eukaryotes.

Results point out that the tertiary interactions are localized to a G.U base-pair of oligonucleotides occurring at the intron's splice junction. Data reveal that oligonucleotides with nuclease stable backbones retain binding and reactivity properties.

Results demonstrate that the contaminating humic substances and extraneous DNA, in the RNA extracts from environmental samples, interfere in the rRNA-targeted oligonucleotide probe hybridization assays. Data suggest that the contaminants either saturate the membrane surface or reduce rRNA target accessibility.

Diversity and distribution of cyanobacteria in planktonic communities of lakes are analyzed by Oligonucleotide probes of 16S rRNA gene of the cyanobacteria. Distribution profiles from genetic analysis correlated with those of hydrobiological and hydrochemical analysis qualitatively but relatively less at quantitative levels.

Bacterial groups in human adult fecal flora have been quantified using oligonucleotide probe hybridization. The probe is designated S-G-Clept-1240-a-A-18. Clostridium leptum subgroup and Clostridium coccoides group-specific probes made up 16% +/- 7% and 14% +/- 6% respectively. Bifidobacterium and Lactobacillus groups accounted for less than 2%.

Precursor 16S rRNAs of Acinetobacter spp. in activated sludge waste-water treatment systems have been monitored and it appears they undergo a change in growth activity in wastewater treatment systems. Acinetobacter calcoaceticus grown in LB medium had a more substantial and faster increase in precursor 16S rRNA levels vs the increase in total 16S rRNA levels in exponential growth. Precursor 16S rRNa levels of Acinetobacter spp. increased transiently when activated sludge was mixed with influent wastewater in full- and lab-scale wastewater treatment systems.

Analysis of p53-regulated (tumor suppressor gene-regulated) gene expression patterns has been carried out using oligonucleotide arrays. The oligonucleotide microarrays were used to measure mRNA levels from a large number of genes regulated by the p53 transcription factor. It was found that the nature to the p53 response in different mRNA species depends on the cell type, levels of p53 protein in a cell, and the type of inducing agent or event.

Marine planktonic archaea and bacteria have been looked at and counted using polyribonucleotide probes and fluorescent in situ hybridization (FISH). FISH is greatly enhanced in sensitivity and applicability with seawater samples and is useful to depths of 3,400 m. It appears that archaea make up a large fraction of the total picoplankton biomass in coastal waters and vary by type in the different parts of the water column with maximum density of 10 to the fifth/ml. Data show the great majority of planktonic prokaryotes have significant numbers of ribosomes so they can easily be detected with polyribonucleotide probes. Most planktonic cells seen with 4',6-diamidino-2-phenylindole (DAPI) stain are not lysed cells or ghosts.

Oligonucleotide arrays can provide a broad picture of the state of the cell, by monitoring the expression level of thousands of genes at the same time. It is of interest to develop techniques for extracting useful information from the resulting data sets. Here we report the application of a two-way clustering method for analyzing a data set consisting of the expression patterns of different cell types. Gene expression in 40 tumor and 22 normal colon tissue samples was analyzed with an Affymetrix oligonucleotide array complementary to more than 6,500 human genes. An efficient two-way clustering algorithm was applied to both the genes and the tissues, revealing broad coherent patterns that suggest a high degree of organization underlying gene expression in these tissues. Coregulated families of genes clustered together, as demonstrated for the ribosomal proteins. Clustering also separated cancerous from noncancerous tissue and cell lines from in vivo tissues on the basis of subtle distributed patterns of genes even when expression of individual genes varied only slightly between the tissues. Two-way clustering thus may be of use both in classifying genes into functional groups and in classifying tissues based on gene expression.

The in situ community structure of Prochlorococcus populations in the eastern North Atlantic Ocean was examined by analysis of Prochlorococcus 16S rDNA sequences via cloning and sequencing, hybridization to specific oligonucleotide probes and denaturing gradient gel electrophoresis. Observations of a partitioning of Prochlorococcus genotypes in a stratified water column provide a genetic basis for the dim and bright Prochlorococcus populations observed in flow cytometric signatures in several oceanic provinces.

Anoxic sediments from Rotsee, Switzerland, were analyzed for the presence and diversity of methanogens by using molecular tools and for methanogenic activity by using radiotracer techniques, in addition to the measurement of chemical profiles. Discriminative oligonucleotide probes were used for the analysis of indigenous methanogens in a core of the anoxic sediment, in addition to domain-specific probes against members of the domains Bacteria and Archaea. Significant hydrogenotrophic methanogenesis was found in the organically enriched upper 2 centimeters of the sediment.

A method that is capable of counting ribosome-active bacteria in soil and can categorize soil bacteria by size was developed. This method uses a fluorescence in situ hybridization technique that is predicated on binding of a rhodamine-labelled oligonucleotide probe to 16S ribosomal RNA. Use of the proposed procedure and fluorescence microscopy led to the identification of active soil bacteria according to their size groups. This method will be useful for phylogenetic probes and for automatic soil bacteria counting.

Cyanobacterial cells have been identified by using a whole-cell hybridization method that uses horseradish peroxidase (HRP)-labeled, rRNA-targeted oligonucleotides. The technique was used because fluorescent monolabeled oligonucleotides fail to overstain the autofluorescence of the target cells. The genotypic method has adequate sensitivity to detect cyanobacterial cells in the deeper layers of microbial mats. Its cell fixation, specificity and sensitivity were evaluated by using four oligonucleotides to detect four groups of bacteria.

A ribosomal RNA fluorescent in situ hybridization approach using 2-hydroxy-3-naphthoic acid 2'-phenylanilide phosphate and Fast Red TR was developed to study planktonic bacterial cells in river water. The bacterial community in two sites with different organic carbon pollution level was investigated using oligonucleotide probes specific for the domain bacteria and five bacterial groups. Results showed that while the probes were able to identify 50% of the total cells at the eutrophic sites, only 26-38% of the cells were identified at the oligotrophic site.

Research was conducted to investigate the predation of attached Pseudomonas putida mt2 by the small ciliate Tetrahymena sp. under lotic and lentic conditions. Data were compared to grazing rates in batch systems containing suspended prey with densities of 2 x 10(super 8) bacteria per ml of pore space and 2 x10(super 8) bacteria per ml of suspension. Results indicate that the correlation between the retardation of transport and grazing activity is caused by the interaction between attached bacteria and active predators.

The composition of the cricket hindgut microbial community was examined with the use of fluorescently labeled rRNA-targeted probes. With the aid of phylogenetic probes with varying levels of specificity, the microbial community structures of different cricket species were compared and the impact of diet differences on the various microbial communities residing in the hindgut of Acheta domesticus was examined. The results are discussed.

A rapid oligonucleotide probe design strategy based on subtractive hybridization was devised to eliminate the need to sequence many similar clones of dominant members of a community. The method yields probes for 16S rRNA or rRNA genes of individual members of microbial communities specific within the context of those communities. Results revealed that only those clones with 100% sequence identity with the probe fragment were detected. Probes also were inclined to correspond to more variable regions of the 16S rRNA if the target sequences of other clones in the library.

A novel analytical method using PNA probes detected by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOFMS) was applied to type sequence polymorphisms within the human leukocyte antigen (HLA), DQA locus. Streptavidin-coated magnetic beads were used to immobilize biotinylated DNA. PNA probes representing possible alleles were then prepared for the immobilized DNA hybridization. The nonspecific PNA probes were removed with stringent washes. The PNA/DNA/beads conjugate was analyzed by MALDI-TOFMS. The genotype of the DNA was determined by the detected molecular masses of the released PNA probes. Reproducible and accurate genotyping was achieved by this analytical method.

Oligonucleotide probes directed against the ribosomal DNA in the 16S subunit were designed to survey bacterioplankton in marine and estuarine environments in the southeastern coast of the US. Results reveal the predominance of a cluster of marine bacteria in the alpha-3-subclass of Class Proteobacteria. The other applications of the generated ribosomal DNA probes are discussed.

The physiological role of Fos protein expressed in the nucleus tractus solitarii (NTS) in central cardiovascular regulation was assessed through bilateral microinjection of antisense oligonucleotide into the caudal NTS of adult male rats. Results have shown that microinjection of oligonucleotide enhanced the spontaneous baroreceptor reflex response. The presence of expressed Fos protein at the NTS was critical to beat-to-beat baroreflex regulation of systemic arterial pressure.

A new technique is described for the rapid detection of specific nucleic acid sequences in unamplified DNA samples. The method consists of using two nucleic acid probes complementary to different sites on a target DNA sequence. The two probes are each labeled with different fluorescent dyes. When mixed with a sample containing the target DNA, the two probes hybridize to their respective binding sites on the same target DNA molecule. The sample is then analyzed by a laser-based ultrasensitive fluorescence system capable of detecting single fluorescent molecules at two different wavelength channels simultaneously. Since the probes are bound to the same target DNA molecule, their signals appear simultaneously. Thus, coincident detection of both dyes provides the necessary specificity to detect an unamplified, single-copy target DNA molecule in a homogeneous assay. If the target is not present, only uncorrelated events originating from free probes will be observed at either channel. Phage [Lambda] DNA in a background of salmon genomic DNA was detected as a two-dye coincident signal at a relative concentration of one [Lambda] molecule per salmon genome. In a control sample, cleavage of the [Lambda] DNA between the two probe binding sites eliminated the coincident signals. In a second experiment, a single-copy transgene was detected in maize. Detection parameters and possible future applications to genetic analysis are discussed.

The molecular interactions of the prochiral oxygens of internucleotide phosphates in the trp repressor-operator complex were analyzed by utilizing chiral methylphosphonate derivatives. The pair of analogue DNA derivatives containing the trp operator sequence and a methylphosphonate linkage exhibited variable binding affinities that can be utilized for the characterization of prochiral phosphate oxygens. Analysis of the trp-repressor complex indicated the presence of a six internucleotide phosphonates that mediated interactions with the trp repressor.

A series of fluorescent oligonucleotide probes that can hybridize with unique sequences of the small subunit (SSU) ribosomal RNA (rRNA) of Paraphysomonas vestita were prepared for bacterial identification. The fluorescent oligonucleotide probes specifically bonded to Paraphysomonas vestita and imparted a strong fluorescence to the nanoflagellate. Furthermore, SSU rRNA gene of the nanoflagellate exhibited numerous sequence differences compared to other Paraphysomonas species.

A polymerase chain reaction (PCR)-based method was utilized to isolate genes that encode response regulators in 12 species of Gram-positive and Gram-negative bacteria. The PCR-based method was characterized by the construction of degenerate oligonucleotide primers that exhibited similar amino acid sequences to the response regulators of the bacterial two-component system. Furthermore, the strategy allowed the amplification of Gram-positive and Gram-negative internal receiver domains including a novel response regulator gene.

Monoclonal antibodies (MAbs) that reacted with Listeria monocytogenes serovar 4 and cluster IIB were utilized to identify the genomic regions responsible for the expression of surface antigens in the pathogenic microbe. The deoxyribonucleic acid (DNA) probe 1 from the two novel genomic regions of the microbe was specific for serotypes 4b, 4d and 4e strains. Furthermore, three Listeria innocua strains that were positive with MAbs c74.22 and c74.33 reacted with the DNA probes due to the presence of similar surface antigenic composition from both strains.

The Pseudomonas syringae pathovars pv. glycinea, pv. phaseolicola and pv. pisi produce significant amounts of ethylene in vitro and in planta. The strains show growth associated production of ethylene. The bean strains of P. syringae pv. phaseolicola and 17 other pathovars fail to produce ethylene. Two 22-bp oligonucleotide primers from the ethylene-forming enzyme gene of P. syringae pv. phaseolicola amplify a specific 0.99-kb fragment in all the strains, except the pv. pisi strain.

A non-culture approach for the specific detection of pathogenic fungal species, Candida albicans and C. tropicalis, was investigated. The method used in situ hybridization with fluorescently labelled 020 oligonucleotide probes which binds to the 18S rRNA of C. albicans and C. tropicalis. RNA was extracted from 47 fungal strains and hybridized with the probes at 43 degrees Centigrade. C. albicans and C. tropicalis reacted strongly with fluorescently labelled probe 020. Adherence assay using human umbilical vein endothelial cells showed discrimination between C. albicans and C. krusei, and only C. albicans cells were fluorescent. Germ tubes and hyphae from C. albicans were also fluorescently labelled after in situ hybridization.

Combination of PCR amplification and oligonucleotide probe hybridization (assay A) helps in better evaluation of the Cryptosporidium parvum in raw milk than assay B, which is based on the Laxer et. al. primer set. Assay A is effective in the detection of all bovine Cryptosporidium isolates except C. muris. Assay B amplifies the Eimeria acervulina DNA and detects the Giardia intestinalis DNA. The sensitivity in 20 ml of milk is 1 to 10 oocysts. There is a lack of reproducibility of when only few oocysts are present in the milk.

Oligonucleotide probe survey of ammonia-oxidizing nitrifiers in seawater aquarium biofilters demonstrates the need for different bacteria for ammonia-oxidation in freshwater aquaria and seawater aquaria. Analysis of nitrifying bacterial phylotypes associated with the water samples reveals high levels of ammonia-oxidizing bacteria, Nitrosomonas europaea, in seawater aquaria. However, the chemolithotrophic ammonia oxidation in the freshwater aquaria shows no sign of Nitrosomonas. Both the aquaria are characterized by the absence of rRNA from nitrite-oxidizing proteobacteria.

The degenerate oligonucleotide primers and PCR enable identification of the bacterial genes encoding sigma(super 54)-dependent transcription activators. The primers are specific for the catalytic region of the activators which contain a nucleotide binding site and two conserved sites. The primers amplify DNA containing the catalytic domain and null mutants are formed by homologous recombination. The probes identify DNA fragments in Rhizobium meliloti, Myxococcus xanthus and Bacillus subtilis. No DNA is identified from Synechococcus sp. strain PCC 7002 or Saccharomyces cerevisiae.

The bacterial population is different in the snow, slush and water of a high mountain lake but consists of mainly bacteria from the Proteobacteria beta subclass. In situ hybridization of bacteria with fluorescent labeled rRNA oligonucleotide probes shows high secondary production and level of bacteria reducing 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyltetrazolium chloride. The association between bacterial production, population composition and activity is different in the pelagic and slush layers.

Phylogenetic hierarchical 16S rRNA oligonucleotide probes specific for ammonia- and nitrite-oxidizing bacteria are suitable for the identification of bacteria in environmental samples by slot blot, whole-cell or in situ hybridization. The probes are specific for Proteobacteria beta subclass, Nitrosomonas, Nitrosovibrio-Nitrosolobus-Nitrosospira and Nitrobacter species. In situ hybridization results show that biofilm and nitrifying activated sludge samples contain mainly Nitrosomonas bacteria. Nitrosomonas bacteria are found near and sometimes associated with Nitrobacter bacteria.

Members of the domain Bacteria constitute 60% or more of the microbial cells colonizing limnetic macroscopic organic aggregates. The aggregates were collected in situ or formed of natural lake water in rolling cylinders under simulated in situ conditions and analyzed using rRNA-directed fluorescent oligonucleotide probes. Staining studies with 4',6-diamidino-2-phenylindole indicated that the beta-subclass proteobacteria were more abundant than the alpha and gamma-subclasses. The beta-subclass proteobacteria increased with time during time course experiments with aggregates made in the laboratory.

A combination of cytochemical methods and in situ rRNA probe hybridization signals are efficient in colocalization of phenotypic and genotypic characteristics of individual bacterial cells. The technique allows the assignment of biochemical properties such as substrate-enhanced tetrazolium salt reduction and cytochrome oxidase reaction to cells detected from rRNA probes. A study of digitized images allows the determination of the relationship between rRNA signal strength and the rate of reduction of the tetrazolium salt.

Oligonucleotide probes specific for the 16S rRNA of members of the cytophaga-flavobacter-bacteroides phylum accurately identify the bacteria in activated sludge and human feces. The probes are specific for the conserved 319th to 336th amino acids of the 16S rRNA. The application of the probes shows that activated sludge flocs contain many flavobacteria-cytophaga bacteria. The probes identify bacteria belonging to the Bacteroides and the Prevotella family in human feces even in the absence of anaerobic cultivation.

A fluorescent in situ hybridization technique using rRNA-targeted oligonucleotide probes is useful in the enumeration of the eukaryotic protists in environmental water samples and cultures. The counts of protists by this method are similar to those determined by staining methods using acridine orange and 4',-6-diamidino-2-phenylindole. However, the oligonucleotide probes method gives a higher total nanoplankton count. This is probably due to the inability of the staining method to detect small nanoplanktonic cells.

A method using many oligonucleotide probes in a single slot blot hybridization is applied for the identification of bacterial species and the study of bacterial colonies. Probes of varying lengths are labeled with 32P and hybridized on a membrane containing genes coding for 16S rRNAs from several bacterial species. The bound probes are separated after hybridization and the genes are identified. This technique is economical and less time-consuming because it uses less reagents and exposure to radioactivity.

Hybridization of the 16S ribosomal RNA genes of the Nitrosospira bacteria in various samples, using polymerase chain reaction amplification probes, shows the ubiquity of nitrosospiras in nature. The probes fail to directly detect the Nitrosomonas genus 16S rRNA. Incubation of the samples for two weeks enables the identification of Nitrosospira and Nitrosomonas. Nitrosospira bacteria are more commonly spread than the Nitrosomonas bacteria. The probes were prepared from the 16S rRNA oligonucelotides of ammonia-oxidizing bacteria.

Examination of the composition of dissolved DNA in marine environments by differential centrifugation and probing with 16S rRNA-targeted kingdom oligonucleotide probes reveals that 50% of the filterable DNA is free DNA, while the remaining portion contains vital particles and colloids. Viral abundance tests reveal that viral particles accounts for 17 to 30% of the remaining portion. Dissolved DNA undergoes hybridization with universal, eubacterial, and eucaryotic probes, while purified standard viral DNAs hybridize only with the universal probe.

A PCR-based assay helps identify Bacillus cereus in foods. Experimental studies help design three oligonucleotide primers within the sequence for cereolysin AB, a cytolytic determinant encoding hemolytic and lecithin-hydrolyzing activities of B. cereus. Examination of the specificity of these primers using a combined PCR-hybridization assay reveals the significant specificity of these primers for isolates of the B. cereus group. This combined assay helps detect B. cereus in artificially contaminated milk and the detection limit is 1 CFU ml of milk.

16S rRNA-targeted oligonucleotide hybridization probes help examine the bacterial interactions of ruminal microorganisms in defined mixed cultures with specific substrates. Ruminococcus probes help determine the populations of Ruminococcus albur and Ruminococcus flavefaciens in in vitro competition studies with Fibrobacter succinogens. Ruminal bacterium produces a bacteriocin-like substance and this production mechanism helps compete for nutrients.

16S rRNA-targeted oligonucleotide probes help examine microbial interactions between ruminal fibrolytic bacteria during fermentation of cellulose and alkaline hydrogen peroxide-treated wheat straw in mono-, di- and tricultures. In diculture Ruminococcus albur 8 inhibits the growth of R. flavefaciens FD-1 but there is no competition between R. albus 8 and Fibrobacter succinogenes S85. In tricultures with cellulose as carbon source R. albur 8 growth dominates initially while F. succinogenes S85 growth is dominant during the later stages. In alkaline hydrogen peroxide-treated wheat straw, the growth of F. succinogenes 85 dominates.

Studies using nucleotide sequences of synthetic oligonucleotide probes reveal that the major NAD(P)H-flavin oxidoreductase (flavin reductase) serves as a catalyst in the supply of reduced flavin mononucleotide in bioluminescence reaction. Flavin reductase exhibits low level of nitro reductase activity and is homologous to oxygen-insensitive NAD(P)H nitro reductases of E. coli and S. typhimurium, and together they form a novel protein family.