Search results for "Genomic DNA"
showing 10 items of 67 documents
Rapid identification of minor histocompatibility antigen HA-1 subtypes H and R using fluorescence-labeled oligonucleotides
2000
Acknowledgments: We thank Brigitte Schuch and Karola Schmidt for excellent technical assistance. This work was supported by a grant of the Deutsche Krebshilfe (Nr. 70-2427 and Nr. 70-2428). Abstract: Donor-recipient disparitiy of the minor histocompatibility antigen HA-1 is relevant for the development of graft-versus-host disease after HLA-matched sibling allogeneic bone marrow transplantation in HLA-A*0201-positive individuals. Two different alleles of HA-1 with a single amino acid polymorphism have been identified. Here we describe a time- and cost-efficient method for HA-1 typing of genomic DNA, using site-specific hybridization probes with the LightCycler. This method was compared with…
Comparative genetic diversity of the narG, nosZ, and 16S rRNA genes in fluorescent Pseudomonads
2003
ABSTRACT The diversity of the membrane-bound nitrate reductase ( narG ) and nitrous oxide reductase ( nosZ ) genes in fluorescent pseudomonads isolated from soil and rhizosphere environments was characterized together with that of the 16S rRNA gene by a PCR-restriction fragment length polymorphism assay. Fragments of 1,008 bp and 1,433 bp were amplified via PCR with primers specific for the narG and nosZ genes, respectively. The presence of the narG and nosZ genes in the bacterial strains was confirmed by hybridization of the genomic DNA and the PCR products with the corresponding probes. The ability of the strains to either reduce nitrate or totally dissimilate nitrogen was assessed. Overa…
Terricaulis silvestris gen. nov., sp. nov., a novel prosthecate, budding member of the family Caulobacteraceae isolated from forest soil
2020
The family Caulobacteraceae comprises prosthecate bacteria with a dimorphic cell cycle and also non-prosthecate bacteria. Cells of all described species divide by binary fission. Strain 0127_4T was isolated from forest soil in Baden Württemberg (Germany) and determined to be the first representative of the family Caulobacteraceae which divided by budding. Cells of strain 0127_4T were Gram-negative, rod-shaped, prosthecate, motile by means of a polar flagellum, non-spore-forming and non-capsulated. The strain formed small white colonies and grew aerobically and chemo-organotrophically utilizing organic acids, amino acids and proteinaceous substrates. 16S rRNA gene sequence analysis indicated…
Complete decontamination and regeneration of DNA purification silica colum
2008
Silica columns are among the most used DNA purification systems, allowing a good yield of high-quality nucleic acids without organic extractions. Silica column regeneration protocols reported up to now to remove DNA traces are time-consuming, and their effectiveness on genomic DNA has not been demonstrated. Here we report a very rapid regeneration procedure that ensures no DNA carryover, independent of its size, without impairing column efficiency. The method takes advantage of the improved DNA removal by low concentrations of Triton X-100.
A simple and rapid DNA extraction method from leaves of grapevine suitable for polymerase chain reaction analysis.
2012
The genomic grapevine (Vitis vinifera L.) DNA extraction is difficult because of secondary metabolites that interfere with DNA isolation procedures and subsequent applications. We developed a simple, rapid and efficient method for the extraction of genomic DNA from asymptomatic and pathogen-infected grape leaves. The protocol reported, based on a modified cetyl trimethylammonium bromide (CTAB) extraction procedure, allowed the rapid DNA extraction from little amounts of leaf material without employment of liquid nitrogen for initial tissue grinding. The protocol included polyvinylpyrrolidone (PVP) to bind phenolic compounds, β-mercaptoethanol to inhibit the oxidation of polyphenols, and a h…
Interrogation of genomes by molecular copy-number counting (MCC)
2006
Human cancers and some congenital traits are characterized by cytogenetic aberrations including translocations, amplifications, duplications or deletions that can involve gain or loss of genetic material. We have developed a simple method to precisely delineate such regions with known or cryptic genomic alterations. Molecular copy-number counting (MCC) uses PCR to interrogate miniscule amounts of genomic DNA and allows progressive delineation of DNA content to within a few hundred base pairs of a genomic alteration. As an example, we have located the junctions of a recurrent nonreciprocal translocation between chromosomes 3 and 5 in human renal cell carcinoma, facilitating cloning of the br…
Deinococcus radiodurans' SRA-HNH domain containing protein Shp (Dr1533) is involved in faithful genome inheritance maintenance following DNA damage
2018
WOS:000452343100012; International audience; Background: Deinococcus radiodurans R1 (DR) survives conditions of extreme desiccation, irradiation and exposure to genotoxic chemicals, due to efficient DNA breaks repair, also through Mn2+ protection of DNA repair enzymes. Methods: Possible annotated domains of the DR1533 locus protein (Shp) were searched by bioinformatic analysis. The gene was cloned and expressed as fusion protein. Band-shift assays of Shp or the SRA and HNH domains were performed on oligonucleotides, genomic DNA from E. coif and DR. slip knock-out mutant was generated by homologous recombination with a kanamycin resistance cassette. Results: DR1533 contains an N-terminal SRA…
Pulsed-field gel electrophoresis for the discrimination of Oenococcus oeni isolates from different wine-growing regions in Germany
2008
Reliable techniques are needed for the identification individual Oenococcus oeni strains with desirable flavor characteristics and to monitor the survival and contribution of inoculated and indigenous bacteria. Therefore, we investigated the suitability of pulsed-field gel electrophoresis (PFGE) for the discrimination of 65 O. oeni isolates from six different wine-producing regions in Germany. Among the restriction enzymes tested, genomic DNA digestions with Sfi I were most effective by displaying 56 (86%) different banding profiles. Our results underline the high capacity of PFGE for strain identification and differentiation. Cluster analysis of the DNA restriction patterns revealed no dis…
Rapid 96-well plates DNA extraction and sequencing procedures to identify genome-wide transposon insertion sites in a difficult to lyse bacterium: La…
2014
International audience; Random transposon mutagenesis followed by adequate screening methods is an unavoidable procedure to characterize genetics of bacterial adaptation to environmental changes. We have recently constructed a mutant library of Lactobacillus casei and we aimed to fully annotate it. However, we have observed that, for L. casei which is a difficult to lyse bacterium, methods used to identify the transposon insertion site in a few mutants (transposon rescue by restriction and recircularization or PCR-based methods) were not transposable for a larger number because they are too time-consuming and sometimes not reliable. Here, we describe a method for large-scale and reliable id…
Thalassobacter stenotrophicus Macián et al. 2005 is a later synonym of Jannaschia cystaugens Adachi et al. 2004, with emended description of the genu…
2005
The type strains of Jannaschia cystaugens (LMG 22015T) and Thalassobacter stenotrophicus (CECT 5294T) were analysed by means of genomic DNA–DNA hybridization, comparison of 16S rRNA gene sequences and phenotypic properties determined under the same methodological conditions. J. cystaugens LMG 22015T showed DNA–DNA relatedness levels of 72 % when hybridized with the genomic DNA of T. stenotrophicus CECT 5294T. Sequence comparisons revealed that the 16S rRNA genes of the two strains had a similarity of 99·8 %. The cellular fatty acid and polar lipid compositions of the two strains and their DNA mol% G+C contents were almost identical. Bacteriochlorophyll a (Bchl a) and polyhydroxybutyrate wer…