A note on the use of a plasmid as a DNA probe in the detection of a Lactobacillus fermentum strain in porcine stomach contents.

A plasmid (about 50 kb) was used as a DNA probe to enumerate, by colony hybridization, a strain of Lactobacillus fermentum in the stomach contents of eight piglets. The population sizes obtained by colony hybridization were in agreement with estimated levels calculated on the basis of plasmid profiling of colonies isolated at random from the total lactobacillus population.     

In situ RNA hybridization using Technovit resin in Arabidopsis thaliana

A protocol is described for RNA in situ hybridization using thin sections prepared by Technovit resin. Technovit is a widely used resin for histological examinations. Since it does not require time-consuming processes such as removal of the resin and can be performed without high temperature treatment, a high resolution of sections could be possible compared to other resins and paraffin. Thin sections (approximately 4 m) were made from inflorescences of Arabidopsis thaliana embedded in Technovit 8100 resin, and in situ hybridization was performed using the protocol described in this article. Hybridization signals were observed using LEAFY and other genes as probes, showing that this resin can be used for in situ analysis. In our experiments, the most important factor for a successful in situ hybridization pattern was to optimize the RNase A concentration after hybridization. We routinely used RNase A at a concentration of 2–5 ng/ml, a concentration much lower than that used for paraffin embedding method. Thus, the use of the Technovit resin for plant tissue embedding results in a faster protocol and greater quality than allowed by paraffin sections.     

FISH probes for the detection of the parasitic dinoflagellate Amoebophrya sp. infecting the dinoflagellate Akashiwo sanguinea in Chesapeake Bay

A comparison of the small subunit rRNA sequences of a Chesapeake Bay strain of the dinoflagellate Akashiwo sanguinea and the dinoflagellate Amoebophrya sp. parasitizing it revealed several potential target sites that could be used to detect the parasite through in situ hybridization. The fluorescence of probed cells under various conditions of hybridization was measured by using a spot meter on a Nikon UFX-II camera attachment so that the effect of various hybridization parameters on probe binding could be determined. Probes directed against both the junction between helices 8 and 11 and helix 46 could detect the parasite, although the helix 8/11 probe produced a stronger signal under the conditions tested. The fluorescence of the probed cells increased with increasing hybridization time up to approximately twelve hours. The background fluorescence was lower at the wavelengths used to detect Texas Red than at those used to detect fluorescein, so probed cells were more distinct when Texas Red was used as the label. Cells stored in cold paraformaldehyde for a year still bound the probes. Young stages of the parasite could be seen more readily after in situ hybridization than after protargol impregnation.     

A note on the use of a plasmid as a DNA probe in the detection of a Lactobacillus fermentum strain in porcine stomach contents

A plasmid (about 50 kb) was used as a DNA probe to enumerate, by colony hybridization, a strain of Lactobacillus fermentum in the stomach contents of eight piglets. The population sizes obtained by colony hybridization were in agreement with estimated levels calculated on the basis of plasmid profiling of colonies isolated at random from the total lactobacillus population.     

The use of microwave irradiation as a pretreatment toin situ hybridization for the detection of measles virus and chicken anaemia virus in formalin-fixed paraffin-embedded tissue

Summary Microwave irradiation was investigated as a pretreatment toin situ hybridization on formalin-fixed, paraffin-embedded tissue. Two probe/tissue systems were used: a single-stranded RNA probe for the detection of measles virus nucleocapsid genome in subacute sclerosing panencephalitis brain tissue, and a double stranded DNA probe for chicken anaemia virus in thymus of chicken infected with the virus. Microwaving, when used as sole pretreatment, was not as effective as the more traditional enzyme pretreatments forin situ hybridization. However, when used in combination with existing pretreatments, a significant increase was found in hybridization signal in both brain and thymus tissue. This was emphasized when combination enzyme/microwave pretreatments were used prior to detection of measles virus byin situ hybridization in a series of five archival subacute sclerosing panencephalitis cases. The use of microwave irradiation would be recommended as a means of supplementingin situ hybridization methods, especially when using long-term formalin fixed paraffin-embedded tissue.     

Rapid subchromosomal localization of cosmids by nonradioactive in situ hybridization

A rapid method for localizing large numbers of complete cosmids by nonradioactive in situ hybridization is described. The cosmids are nick translated in the presence of biotin-16-dUTP, incubated with an excess of sonicated human DNA, and used as a probe for in situ hybridization. Sites of hybridization are detected by successive treatments with FITC-labeled avidin and biotinylated anti-avidin antibody. Fifty-two cosmids were localized on chromosome 16 in 5 d relative to translocation breakpoints contained in two cell lines. Rapid identification of chromosome 16 was achieved by cohybridization with a chromosome 16-specific centromeric repeat probe.     

Quantitation of in situ hybridization of ribosomal ribonucleic acids to human diploid cells

The hybridization of 5S and 28S ribosomal RNAs to human fibroblast and leukocyte cells was used as a model system to quantitate the technique of in situ hybridization for human diploid cell types. Quantitation consisted of counting (scoring) the number of grains formed over both interphase nuclei and metaphase chromosomes on slides after various hybridization procedures. The average number of grains/nucleus per slide was then used to determine hybridization percentages. As with nitrocellulose filter hybridizations the kinetics of in situ hybridizations can be fit with a single first-order rate constant. However, the in situ hybridization rate was approximately 10 times slower than the corresponding filter hybridization rate. The efficiency of in situ hybridization was found to range between 5 and 15% for both leukocyte and fibroblast cell types and for both metaphase and interphase nuclei. Determination of the parameters of the in situ hybridization reaction of ribosomal RNAs to diploid chromosomes define the experimental conditions needed for the localization of single copy genes to diploid chromosomes.     

Dissociation analysis in polymerase chain reaction and 1X SSC buffer as a prerequisite for selection of 13mer microarray probe sets with uniform hybridization behavior

Homogeneous probe hybridization is a prerequisite for the robust design of microarrays. Elaborate algorithms were developed to select for probe sets with uniform melting temperatures (Tm). However, at least short oligonucleotides (<20 bp) show large variation in the on-chip hybridization efficiency even if designed with state-of-the-art algorithms. This variation can be explained by steric effects and interferences on the solid surface as well as by chemical conditions that may deviate from conditions used to develop the algorithms. We designed 412 random 13mer duplexes to study the differences between the Tm of nearest-neighbor algorithms and the Tm values measured by dissociation analysis in polymerase chain reaction (PCR) and 1X SSC buffer. We tested the effects of theoretical vs empirical Tm values on the hybridization variation of 40 duplexes on-chip. Although the empirical approach resulted in a slightly better prediction of hybridization efficiency, less than one-fifth (17%) of the observed variation could be explained by the factor Tm alone. We conclude that state-of-the-art algorithms can be used for a first selection of short oligonucleotide probes, but that it is then necessary to perform an on-chip selection to obtain a probe set with a uniform hybridization behavior.     

Detection and enumeration of virulent Yersinia enterocolitica in food by DNA colony hybridization.

A portion of a 44-megadalton plasmid found in Yersinia enterocolitica 8081 was used as a genetic probe to differentiate virulent and nonvirulent strains of the species. A DNA colony hybridization technique was employed. Three BamHI restriction endonuclease fragments labeled with 32P by nick translation were hybridized to lysed colonies of pure cultures, mixtures of virulent and nonvirulent cells, and portions of a food sample artificially contaminated with virulent Y. enterocolitica. The results of the colony hybridization test for virulence were the same as those obtained by the autoagglutination and suckling mouse tests. DNA colony hybridization detects pathogenic Y. enterocolitica in foods without the need for enrichment or pure cultures.     

Detection and enumeration of virulent Yersinia enterocolitica in food by DNA colony hybridization

A portion of a 44-megadalton plasmid found in Yersinia enterocolitica 8081 was used as a genetic probe to differentiate virulent and nonvirulent strains of the species. A DNA colony hybridization technique was employed. Three BamHI restriction endonuclease fragments labeled with 32P by nick translation were hybridized to lysed colonies of pure cultures, mixtures of virulent and nonvirulent cells, and portions of a food sample artificially contaminated with virulent Y. enterocolitica. The results of the colony hybridization test for virulence were the same as those obtained by the autoagglutination and suckling mouse tests. DNA colony hybridization detects pathogenic Y. enterocolitica in foods without the need for enrichment or pure cultures.     

Fluorescence in situ hybridization to interphase cell nuclei in suspension allows flow cytometric analysis of chromosome content and microscopic analysis of nuclear organization

Summary Fluorescence hybridization to interphase nuclei in liquid suspension allows quantification of chromosome-specific DNA sequences using flow cytometry and the analysis of the three-dimensional positions of these sequences in the nucleus using fluorescence microscopy. The three-dimensional structure of nuclei is substantially intact after fluorescence hybridization in suspension, permitting the study of nuclear organization by optical sectioning. Images of the distribution of probe and total DNA fluroescence within a nucleus are collected at several focal planes by quantitative fluorescence microscopy and image processing. These images can be used to reconstruct the three-dimensional organization of the target sequences in the nucleus. We demonstrate here the simultaneous localization of two human chromosomes in an interphase nucleus using two probe labeling schemes (AAF and biotin). Alternatively, dual-beam flow cytometry is used to quantify the amount of bound probe and total DNA content. We demonstrate that the intensity of probe-linked fluorescence following hybridization is proportional to the amount of target DNA over a 100-fold range in target content. This was shown using four human/hamster somatic cell hybrids carrying different numbers of human chromosomes and diploid and tetraploid human cell lines hybridized with human genomic DNA. We also show that populations of male, female, and XYY nuclei can be discriminated by measuring their fluores-cence intensity following hybridization with a Y-chromosome-specific repetitive probe. The delay in the increase in Y-specific fluorescence until the end of S-phase is consistent with the results recorded in previous studies indicating that these sequences are among the last to replicate in the genome. A chromosome-17-specific repetitive probe is used to demonstrate that target sequences as small as one megabase (Mb) can be detected using fluorescence hybridization and flow cytometry.     

Detection of DNA sequences in Plasmodium berghei by means of in situ hybridization

Summary A non-radioactive in situ hybridization technique, used to map unique DNA sequences to plant chromosomes, has been adapted for the localization of specific DNA sequences in nuclei of Plasmodium berghei. After hybridization using probes labeled with biotin-11-dUTP, the formed DNA/DNA hybrids were detected by fluorescence microscopy using a specific double-layer antibody technique. Besides its high resolution, this procedure is characterized by a high sensitivity, allowing the detection of a unique sequence as small as 2.5 kb.     

Genetic polymorphism in Xanthomonas albilineans strains originating from 11 geographical locations, revealed by two DNA probes

Two DNA fragments from Xanthomonas albilineans were used as probes to study the molecular diversity among strains of this pathogen. Two serologically distinct groups, serovars I and II, could be differentiated by hybridization to the probes. These probes, designated 830 and 838, were cloned after subtractive DNA hybridization of common sequences of Xanthomonas campestris pv. vasculorum from a serovar I strain of X. albilineans. They did not hybridize to the DNA of several other xanthomonads or to sugarcane DNA under the conditions of hybridization used. Faint bands were observed upon hybridization of probe 830 with one strain of X. campestris pv. phaseoli. The same banding patterns were obtained with a strain of X. albilineans from Burkina Faso and the serovar II strains of Mauritius. The serovar I strains from Mauritius and two other strains each from Reunion and South Africa had similar pattern.     

Detection of DNA sequences in Plasmodium berghei by means of in situ hybridization

A non-radioactive in situ hybridization technique, used to map unique DNA sequences to plant chromosomes, has been adapted for the localization of specific DNA sequences in nuclei of Plasmodium berghei. After hybridization using probes labeled with biotin-11-dUTP, the formed DNA/DNA hybrids were detected by fluorescence microscopy using a specific double-layer antibody technique. Besides its high resolution, this procedure is characterized by a high sensitivity, allowing the detection of a unique sequence as small as 2.5 kb.     

A novel method for determining linkage between DNA sequences: hybridization to paired probe arrays

Cooperative hybridization has been used to establish physical linkage between two loci on a DNA strand. Linkage was detected by hybridization to a new type of high-density oligonucleotide array. Each synthesis location on the array contains a mixture of two different probe sequences. Each of the two probes can hybridize independently to a different target sequence, but if the two target sequences are physically linked there is a cooperative increase in hybridization yield. The ability to create and control non-linear effects raises a host of possibilities for applications of oligonucleotide array hybridization. The method has been used to assign linkage in 50:50 mixtures of DNA containing single nucleotide polymorphisms (SNPs) separated by 17, 693, 1350 and 2038 bp and to reconstruct haplotypes. Other potential uses include increasing the specificity of hybridization in mutation detection and gene expression monitoring applications, determining SNP haplotypes, characterizing repetitive sequences, such as short tandem repeats, and aiding contig assembly in sequen-cing by hybridization.     

Detection of hepatopancreatic parvovirus in Thai shrimp Penaeus monodon by in situ hybridization,dot blot hybridization and PCR amplification

Hepatopancreatic parvovirus (HPV) infects the hepatopancreas in penaeid shrimp and retards their growth. The DNA sequence of HPV from Thai shrimp Penaeus monodon (HPVmon) differs from HPV of Penaeus chinensis (HPVchin) by approximately 30%. In spite of this difference, commercial PCR primers (DiagXotics) developed from HPVchin to yield a 350 bp PCR product do give a 732 bp product with HPVmon DNA template. On the other hand, the sensitivity of HPVmon detection with these primers and with hybridization probes designed for HPVchin is significantly lower than it is with HPVchin. To improve sensitivity for HPVmon detection, we used the sequence of the 732 bp HPVmon PCR amplicon described above to develop specific PCR primers (H441F and H441R) and hybridization probe. The primers could detect as little as 1 fg of purified HPVmon DNA while the 441 bp digoxygenin-labeled PCR product gave strong, specific reactions with in situ hybridization and with hybridization blots. In contrast, negative results were obtained using DNA from all other pathogens tested and from DNA of P. monodon. Supernatant solution from boiled, fresh shrimp fecal and postlarval samples homogenized in 0.025% NaOH/0.0125% SDS could be used to detect as little as 0.1 pg HPVmon DNA by the PCR reaction. By dot blot hybridization, a visible signal was obtained with purified HPVmon DNA at 0.01 pg, but detection in spiked feces and postlarval samples was only 1 and 0.1 pg, respectively.     

Accurate and high resolution in situ hybridization analysis of gene expression in secondary stem tissues

Accurate in situ hybridization analysis in secondary stem tissues of plants has been hindered by specific characteristics of these tissues. First, secondary cell walls non-specifically bind probes used for in situ hybridization thus preventing gene expression analysis in the lignified regions of the stem, such as the xylem. Second, the mRNA in the cambial meristem and its recent derivatives are prone to inadequate fixation when conventional techniques are used. Here we describe an in situ hybridization technique which uses fast freezing and freeze substitution to cryoimmobilize the mRNA followed by embedding in a methacrylate resin for high-resolution analysis of gene expression. By using a transgenic poplar line harbouring rolC:uidA, rolC:iaaM, the gene expression pattern could be compared with histochemical GUS staining. This in situ hybridization technique results in superior preservation of cellular contents, retention of mRNA in all cell types in the poplar stem, a significant reduction of non-specific binding to secondary cell walls and a resolution not previously possible in secondary tissues. This technique will be particularly valuable for the expression analysis of genes involved in xylogenesis and wood formation.     

Application of cloned satellite DNA sequences to molecular-cytogenetic analysis of constitutive heterochromatin heteromorphisms in man

Summary The cloned alpha-satellite DNA sequences were used to evaluate the specificity and possible variability of repetitive DNA in constitutive heterochromatin of human chromosomes. Five probes with high specificity to individual chromosomes (chromosomes 3, 11, 17, 18, and X) were in situ hybridized to metaphase chromosomes of different individuals. The stable position of alpha-satellite DNA sequences in heterochromatic regions of particular chromosomes was found. Therefore, the chromosome-specific alpha-satellite DNA sequences may be used as molecular markers for heterochromatic regions of certain human chromosomes. The homologous chromosomes of many individuals were characterized by cytologically visible heteromorphisms of hybridization intensity with chromosome-specific alpha-satellite DNA sequences. A special analysis of hybridization between homologues with morphological differences provided the evidence for a high resolution power of the in situ hybridization technique for evaluation of chromosome heteromorphisms. The approaches for detection of heteromorphisms in cases without morphological differences between homologues are discussed. The results obtained indicate that constitutive heterochromatin of human chromosomes has a variable amount of alphasatellite DNA sequences. In situ hybridization of cloned satellite DNA sequences may be used as a new general approach to analysis of chromosome heteromorphisms in man.     

Molecular cytogenetic research on the polymorphism of segments of the constitutive heterochromatin in human chromosomes

Cloned alpha-satellite DNA sequences were used to evaluate the specificity and possible variability of repetitive DNA in constitutive heterochromatin of human chromosomes. Five probes of high specificity to individual chromosomes (chromosomes 3, 11, 17, 18 and X) were hybridized in situ to metaphase chromosomes of different individuals. The stable position of alpha-satellite DNA sequences in definite heterochromatic regions of particular chromosomes was found. Therefore, the chromosome-specific alpha-satellite DNA sequences may be used as molecular markers for heterochromatic regions of certain human chromosomes. The significant interindividual differences in relative copy number of alpha-satellite DNA have been detected. The homologous chromosomes of many individuals were characterized by cytologically visible heteromorphisms, as shown by intensity of hybridization with chromosome-specific alpha-satellite DNA sequences. A special analysis of hybridization between homologues with morphological differences gives evidence for a high resolution power of in situ hybridization technique for evaluation of chromosome heteromorphisms. The approaches for detection of heteromorphisms in cases without morphological differences between homologues are discussed. The results obtained indicate that constitutive heterochromatin of human chromosomes is variable for amount of alpha-satellite DNA sequences. In situ hybridization of cloned satellite DNA sequences may be used as novel general approach to analysis of chromosome heteromorphisms in man.