DNA Hybridization Method Research Articles

The distribution of P elements in D. melanogaster along the coast of China studied by DNA hybridization method.

The distribution of the P-M system in four local Drosophila melanogaster populations obtained in southern China has been examined by mating tests. There was no P strain. A HindIII fragment of the P element has been cloned, named as pPH 0.86, and used as probe in this study. The distribution of the P element itself in thirteen local D. melanogaster populations along the eastern coast of China has been studied by the method of DNA hybridization. It is found that all the populations obtained from the north of Shanghai carry P elements but few populations from the south of Shanghai carry P elements. In comparison with the mating test, the method of DNA hybridization is more direct and reliable. The origin and trends of distribution of P elements in China are discussed in this paper.

Restriction fragment length polymorphism and multiple copies of DNA sequences homologous with probes for P-fimbriae and hemolysin genes among uropathogenic Escherichia coli.

Hemolysin and P-fimbriae are two virulence traits frequently found together in uropathogenic Escherichia coli. Previous studies have discovered evidence both for linkage between the genes for these traits and for their duplication in the chromosomes of a limited number of strains. To test whether these observations are characteristic of uropathogenic Escherichia coli, the method of DNA hybridization to DNA restriction fragments separated by electrophoresis and transferred to nylon was used to determine copy number of genes for P-fimbriae (pap) among 51 E. coli strains isolated from symptomatic urinary tract infections. Twenty percent of the strains had more than one copy of pap homologous sequences. Fifteen strains, each representing a unique clone, were examined for the presence of sequences homologous with cloned hemolysin genes (hly). Samples of DNA from 14 of the 15 strains hybridized with hly probes. In eight strains the number of copies of pap equalled the number of copies of hly, including one strain with two apparent copies of each. Five strains appeared to have one more copy of pap than of hly, and one strain had an extra copy of hly.

Small-scale DNA preparation for rapid genetic identification of Campylobacter species without radioisotope.

A simplified and rapid genetic identification method for Campylobacter species without radioisotope was established. Three different amounts of DNA (200, 50, and 12.5 ng) extracted from each type strain of Campylobacter species with standard Marmur's procedure were spotted on a nitrocellulose filter. DNA obtained from one ml bacterial suspension at a concentration of McFarland standard turbidity No. 1 of Campylobacter fetus, C. jejuni, C. coli, and C. pylori isolates were sufficiently labeled with photo-biotin within 15 min and clearly hybridized with the type strain of the corresponding species within four to six hours. Hybridized spots were visualized with alkaline-phosphatase-conjugated streptavidin color-detection method. The reaction was usually stopped within 30 min. Atypical clinical isolates such as a nitrate-negative C. jejuni, two nalidixic acid-resistant C. jejuni, and two strains of C. fetus able to grow at 42 C, which were tentatively identified as such, were definitely identified by the simplified DNA hybridization method presented here. This method will be applicable routinely for the definite identification of atypical strains of Campylobacter species and other gram-negative bacteria difficult to identify biochemically.

Comparison of Cultural Methods and the DNA Hybridization Test for Detection of Salmonellae in Ground Beef

ABSTRACTA commercially available (Gene‐Trak®) DNA hybridization (DNAH) test was compared with conventional cultural methods for detection of salmonellae in naturally contaminated dairy cow ground beef. All subsamples which were Salmonella‐positive by cultural methods were also positive by DNAH. There was a small percentage (3.7%) of false‐positive subsamples by the DNAH method, but there were no false‐negative results. The DNAH test appears to be a reliable method for detection of salmonellae in ground beef.

Improved DNA hybridization method for detection of acyclovir-resistant herpes simplex virus

A simplified DNA hybridization method was developed to detect acyclovir-resistant isolates of herpes simplex virus. Herpes simplex virus-infected cell cultures in microtiter plates were treated with concentrations of acyclovir ranging from 8 to 0.015 micrograms/ml. At 48 h postinfection, infected cells were lysed by a one-step procedure and lysates were absorbed to membranes. Without further treatment, membranes were hybridized by using a herpes simplex virus-specific radioiodinated probe. The membranes were then washed and counted in a gamma counter. The elapsed time for assay performance was 4 h. Parallel plaque reduction assays were performed for comparison. The mean 50% inhibitory dose of in vivo- and in vitro-derived acyclovir-resistant, thymidine kinase-negative isolates was greater than 2 micrograms/ml by DNA hybridization. The 50% inhibitory dose of acyclovir-susceptible, thymidine kinase-positive isolates ranged from 0.01 to 1.1 micrograms/ml. This assay is simple and objective and should facilitate antiviral susceptibility testing in diagnostic laboratories.

RANDOM-FRAGMENT HYBRIDIZATION ANALYSIS OF EVOLUTION IN THE GENUS NEUROSPORA: THE STATUS OF FOUR-SPORED STRAINS.

A random-fragment hybridization method employing nuclear DNA has been developed to explore phylogenetic relationships in the genus Neurospora. Four cloned fragments and repetitive rDNA sequences were examined for restriction-fragment polymorphisms among 14 strains representing four species. The findings demonstrate that variation among randomly selected nuclear fragments can be employed to group related taxa with a higher degree of resolution than has been obtained with other DNA hybridization methods, isozyme electrophoresis, or restriction analysis of repetitive DNA. Based on our analysis of cloned fragments, we conclude that four-spored, secondarily-homothallic strains collected worldwide represent a monophyletic group. Trees constructed on the basis of restriction-fragment cataloging and coarse-structure restriction-site maps are for the most part consistent with the present mating-based species concept. We are encouraged that this method will provide an additional important experimental tool for evolutionary studies.

DNA Hybridization Assay for Detection of Salmonella in Foods: Collaborative Study

A collaborative study was performed in 11 laboratories to validate a DNA hybridization (DNAH) procedure for detection of Salmonella in foods. The DNAH procedure was compared to the standard culture method for detection of Salmonella in 6 foods: ground pepper, soy flour, dry whole egg, milk chocolate, nonfat dry milk, and raw deboned turkey. With the exception of turkey which was naturally contaminated, uninoculated and inoculated samples of each food group were analyzed. Results for the DNAH method were significantly better than for the standard culture method at the 5% probability level for the detection of Salmonella in turkey. There was no significant difference between the methods for the other 5 foods. The method has been adopted official first action.

Comparative Study of a DNA Hybridization Method and the Conventional Culture Procedure for Detection of Salmonella in Foods

ABSTRACTA commercial DNA hybridization assay (DNAH) for rapid detection of Salmonella in foods was compared to the conventional culture procedure. The DNAH method employed preenrichment, selective, and post‐enrichment steps (44°hr) prior to performing the assay (4hr). Confirmation of positive DNAH assays was accomplished using standard culture methods for isolation of Salmonella. More than 1,600 samples were tested, representing 23 food types and including naturally contaminated, artificially inoculated, and uninoculated foods. Based on the data generated, the DNAH method was as productive as the standard culture method for detection of Salmonella in all foods and was significantly better than the culture method for certain foods.

Immunologic selection of simian virus 40 (SV40) T-antigen-negative tumor cells which arise by excision of early SV40 DNA.

A clonal line of highly oncogenic spontaneously transformed mouse cells (104C) was transformed in tissue culture by simian virus 40 (SV40) and subsequently recloned (106CSC). This 106CSC cell line expressed T antigen and transplantation antigen but was about 100 times less tumorigenic than the 104C parent. When 10(5) 106CSC cells were injected into immunocompetent syngeneic mice, tumors were produced. From such tumors, cell lines were established in culture, all of which were consistently negative for T antigen. We found previously by solution DNA hybridization methods that the tumor cells were depleted in the early region of SV40 DNA which codes for the T antigen. We postulated that this loss occurs through a DNA rearrangement of unknown mechanism in one or a few 106CSC cells and that the tumors are then produced from such a cell or cells, whereas all the T-antigen-positive 106CSC cells are rejected by immunologic means. In this investigation we showed by the DNA transfer method using appropriately selected SV40 DNA probes that indeed the tumor cell clone (130CSCT) we selected to investigate came from one 106CSC cell in which the T-antigen-coding SV40 DNA sequences (but not all the early SV40 DNA sequences) were lost by an excision and recombination mechanism. We also showed that the 130CSCT cells, which are highly tumorigenic, could again be transformed by SV40 and that the resulting T-antigen-positive cloned derivative cells became much less tumorigenic (approximately 10(5)-fold), apparently again because of immunologic recognition and rejection. Indeed, when 10(7) T-antigen-positive cloned cells were injected, all the T-antigen-positive cells were rejected and the tumor was produced again from one or more T-antigen-negative cells. Thus, a one-step in vivo transplantation experiment allowed a selection (for tumorigenicity and against the SV40 T antigen) of a mutant mammalian cell with a DNA deletion at a definable site.

Direct detection of exogenous mouse mammary tumor virus sequences in lymphoid cells of BALB/cfC3H female mice.

The presence of exogenous mouse mammary tumor virus (MMTV) (C3H) DNA sequences in lymphoid tissue (spleen, bone marrow, and thymus) and nonlymphoid tissue (liver and kidney) of BALB/cfC3H female mice was directly assessed by DNA hybridization methods. Lymphoid tissues were found positive for integrated MMTV(C3H) sequences in females as young as 4 weeks. In most samples, the level of splenic MMTV(C3H) infection was low (2 to 5%). Infection remained throughout the life of the animal. The percentage of spleen samples found positive for exogenous viral infection was significantly higher in females bearing mammary tumors, whether virgin or multiparous. Liver and kidney DNAs were negative for exogenous MMTV sequences, suggesting tissue type selectivity in MMTV infection.

Demonstration of pseudorabies virus DNA in the mouse inner ear by an in situ nucleic acid hybridization technique in plastic embedded bony material.

This investigation is concerned with the possibility of identifying viral DNA using the in situ DNA hybridization method in methylmethacrylate-embedded material. As an experimental model we chose viral labyrinthitis produced by intranasal infection of the mouse with pseudorabies virus. Fixation and embedding methods specially adapted to this procedure and bony histology preparation technique (specimens by grinding or micromilling) made it possible to identify viral DNA directly morphologically and virologically in the inner ear. Quantitative microphotometric analyses of trans-sagittal sections of the entire skull after in situ DNA hybridization are presented and discussed here as an explicit method of investigating the path of distribution of viral DNA in the brain and the inner ear.

Use of a species-specific DNA hybridization probe for enumerating Bacteroides vulgatus in human feces.

pBV-1, a recombinant plasmid that contains a chromosomal DNA fragment from Bacteroides vulgatus, hybridized to DNA from B. vulgatus but not to DNA from other colonic Bacteroides species. This plasmid was used as a DNA probe to detect and enumerate B. vulgatus in pure culture, in mixed cultures, and in a bacterial fraction from human feces. Bacteria in a pure or mixed culture were lysed by heating the culture in NaOH. The DNA in the disrupted cell suspension was then trapped on nitrocellulose paper by vacuum filtration. If fecal samples were used instead of pure or mixed cultures, it was first necessary to partially purify the DNA by low-speed centrifugation (2,000 X g) and phenol-chloroform extraction before filtering. When 32P-labeled pBV-1 was incubated with filters containg B. vulgatus DNA, the amount of radioactivity that bound to the filters was proportional to the number of B. vulgatus filtered as long as the filtering capacity of the nitrocellulose was not exceeded. Using this procedure, we obtained a value for the concentration of B. vulgatus in human feces (2 X 10(10) to 3 X 10(10) per g of dry weight) that is similar to values obtained by other investigators using conventional bacteriological techniques (3 X 10(10) to 6 X 10(10) per g of dry weight). The advantage of the DNA hybridization method over conventional techniques is that it is not necessary to isolate pure cultures of bacteria from complex specimens such as feces. Furthermore, our method bypasses the cumbersome set of biochemical tests normally used to identify anaerobic bacteria. The major limitation of our method is its sensitivity.(ABSTRACT TRUNCATED AT 250 WORDS)

DNA hybridization technique to detect Shigella species and enteroinvasive escherichia coli.

A DNA hybridization method for the detection of Shigella species and enteroinvasive Escherichia coli is described. It is based upon the high degree of homology shared by the virulence plasmids present in all pathogenic strains. After 32P labeling, a 17-kilobase EcoRI restriction fragment of a virulence plasmid belonging to Shigella flexneri serotype 5 was used as a probe to detect Shigella species and enteroinvasive E. coli in isolated colonies. This method proved highly specific as well as sensitive and should be particularly useful for the characterization of atypical isolates and for large-scale epidemiological studies in endemic areas.

DNA: DNA hybridization method for the diagnosis of hepatitis b infection

Hepatitis B viral (HBV) DNA was detected in a hepatoma cell line which produces hepatitis B surface antigen (HBsAg) and in patients with acute hepatitis B. The serum of one patient with acute hepatitis B was found to be infectious when injected i.v. into a chimpanzee up to a dilution of 10 −8. Hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) were detectable in the same serum sample by radioimmunoassay up to a dilution of 10 −5 and of 10 −3, respectively. Using DNA: DNA hybridization on nitrocellulose membranes, HBV DNA sequences were detectable up to 10 −8 dilution corresponding to the infectivity level. Based on this finding, it appears that DNA: DNA hybridization is the most sensitive method for detecting hepatitis B virus (HBV) infection. In situations with low virus levels it may be the only indicator of the presence of infectious hepatitis B virus. The use of a tritium-labelled probe makes the method economical and adaptable to hospital laboratories.

DNA Hybridization Technique for the Detection of Neisseria gonorrhoeae in Men with Urethritis

A technique to detect Neisseria gonorrhoeae directly in clinical specimens was developed using a modified DNA-hybridization method. It uses the gonococcal cryptic plasmid as the radiolabeled probe, can detect as few as 100 colony-forming units of N gonorrhoeae or as little as 0.1 pg of purified gonococcal plasmid DNA, and is highly specific. This technique for differentiating between gonococcal and nongonococcal urethritis was evaluated in men with symptomatic urethritis in Seattle. Sixty-three (89%) of 71 who had cultures positive for N gonorrhoeae were also positive by DNA hybridization, and all 42 whose cultures were negative were also negative by DNA hybridization. Five of six isolates from patients who were positive by culture but negative by hybridization lacked the gonococcal cryptic plasmid and belonged to a unique auxo-type which requires proline, citrulline, and uracil for growth.

The isolation and characterization of the Escherichia coli DNA adenine methylase (dam) gene.

The E. coli dam (DNA adenine methylase) enzyme is known to methylate the sequence GATC. A general method for cloning sequence-specific DNA methylase genes was used to isolate the dam gene on a 1.14 kb fragment, inserted in the plasmid vector pBR322. Subsequent restriction mapping and subcloning experiments established a set of approximate boundaries of the gene. The nucleotide sequence of the dam gene was determined, and analysis of that sequence revealed a unique open reading frame which corresponded in length to that necessary to code for a protein the size of dam. Amino acid composition derived from this sequence corresponds closely to the amino acid composition of the purified dam protein. Enzymatic and DNA:DNA hybridization methods were used to investigate the possible presence of dam genes in a variety of prokaryotic organisms.

DNA HYBRIDIZATION METHOD FOR DETECTING ENTEROTOXIGENIC ESCHERICHIA COLI IN HUMAN ISOLATES AND ITS POSSIBLE APPLICATION TO FOOD SAMPLES1

A DNA hybridization assay was used to detect isolates of Escherichia coli that have the genetic potential to produce toxin. This method was compared with the traditional Y‐1 mouse adrenal cell method for detecting pathogenic E. coli isolated from humans. Results showed that the DNA hybridization method has the potential to detect foodborne bacterial pathogens. The advantages and disadvantages of this genetic method are compared with those of the traditional assays for pathogenicity.

Intragenomic DNA sequence homologies in the chicken and other members of the class Aves: DNA re-association under reduced stringency conditions.

We have investigated the intragenomic DNA sequence homologies of twelve species of birds representing five orders, and emphasizing Galliformes. This study differs in two important ways from the classical approaches taken in constructing and evaluating phylogenies based on DNA sequence similarities. Comparisons are made on the basis of sequence homologies within genomes of related birds, rather than between genomes. DNA is reassociated at 50 degrees C in 0.5M phosphate buffer; these conditions allow formation and detection of duplexes containing more mismatch than would normally be permitted using more stringent conditions, affording an opportunity to observe more ancient sequence homologies. Thermal stability profiles of DNA duplexes formed under these conditions are the basis of comparison; three general patterns were observed. This approach emphasizes differences in sequence composition between genomes while the more traditional method of intergenomic tracer DNA hybridization at higher stringency emphasizes sequence similarities. No correlation was found between taxonomic position and intragenomic sequence composition, either within or between lineages. The thermal stability profiles of DNA duplexes formed within avian genomes did not reflect the biological similarities inferred from morphology, karyotype, and studies of interspecific hybridization. While all of the differences observed could have occurred over geological time, it was surprising that the genomes of the domestic chicken and the Red Jungle Fowl (Gallus gallus) differ in their sequence compositions. It appears that amolification/reduction events and/or positional changes occur rather often during evolution of a lineage.