-
Applied and Environmental Microbiology Dec 1993We evaluated the restriction fragment length polymorphism of genomic DNA among 53 strains of the phytopathogenic bacterium Pseudomonas syringae pv. syringae. Twenty-nine...
DNA sequence variation and phylogenetic relationships among strains of Pseudomonas syringae pv. syringae inferred from restriction site maps and restriction fragment length polymorphism.
We evaluated the restriction fragment length polymorphism of genomic DNA among 53 strains of the phytopathogenic bacterium Pseudomonas syringae pv. syringae. Twenty-nine strains were isolated from beans, and the rest were isolated from 11 other hosts. Southern blots of DNA digested with EcoRI or HindIII were hybridized to two random probes from a cosmid library of P. syringae pv. syringae and a hrp (hypersensitive reaction and pathogenicity) cluster cloned from P. syringae pv. syringae. The size of hybridizing fragments was determined, and a similarity matrix was constructed by comparing strains on a pairwise basis for the presence or absence of fragments. The proportion of shared fragments was then used to estimate sequence divergence. Dendrograms were produced by using the unweighted pair group method with averages and the neighbor-joining method. For the hrp region, BamHI, EcoRI, EcoRV, and HindIII restriction sites were mapped for six representative bean strains and used to construct EcoRI and HindIII restriction maps for all 30 strains pathogenic on beans. Restriction mapping revealed the presence of a 3-kb insertion in nine bean strains and a probable second insertion or deletion event on the left-hand side of the hrp cluster that biased estimates of nucleotide sequence divergence from fragment comparisons. This demonstrated that the determination of phylogenetic relationships among bacteria by using restriction fragment length polymorphism data requires mapping restriction sites to remove the effect of insertion or deletion events on the analysis.(ABSTRACT TRUNCATED AT 250 WORDS)
Topics: DNA, Bacterial; Genetic Linkage; Genetic Variation; Phylogeny; Polymorphism, Restriction Fragment Length; Pseudomonas; Restriction Mapping
PubMed: 7904440
DOI: 10.1128/aem.59.12.4180-4188.1993 -
Clinical and Diagnostic Laboratory... Mar 2001We developed and evaluated a PCR-based-restriction endonuclease analysis method to detect and analyze the tonB gene of Haemophilus influenzae and Haemophilus...
PCR-based detection, restriction endonuclease analysis, and transcription of tonB in Haemophilus influenzae and Haemophilus parainfluenzae isolates obtained from children undergoing tonsillectomy and adenoidectomy.
We developed and evaluated a PCR-based-restriction endonuclease analysis method to detect and analyze the tonB gene of Haemophilus influenzae and Haemophilus parainfluenzae from pediatric patients undergoing tonsillectomy and adenoidectomy. Multiple sites from the same patient, including the surface of adenoids and tonsils, as well as the core of tonsils, were cultured on chocolate agar and identified using standard procedures and the API NH Kit. A total of 55 H. influenzae isolates were recovered from different sites of 20 patients, and 32 H. parainfluenzae isolates were recovered from various sites of 12 patients. DNA was extracted from American Type Culture Collection strains and test isolates by the PureGene kit. Two primers, G1 (21-mer) and G2 (23-mer), were designed by us to amplify by PCR the tonB gene that consists of an 813-bp fragment. A nested PCR using primers T1 (23-mer) and T2 (24-mer) that flank an internal sequence to the gene of the order of 257 bp and restriction endonuclease digestion using XhoI and BglII were done to detect whether heterogeneity within the gene exists between the two species. Reverse transcription-PCR (RT-PCR) was finally done to detect transcription of the gene in both species. Our data have shown that the tonB gene was detected in both species. It is known to encode a virulent protein, TonB, in H. influenzae; however, demonstration of its presence in H. parainfluenzae is novel. Nested-PCR and restriction endonuclease analysis have shown that the tonB gene is apparently structurally the same in both species, with possible differences that may exist in certain H. parainfluenzae isolates. RT-PCR done on selected numbers of H. influenzae and H. parainfluenzae have shown that the tonB gene was transcribed in both species. This shows that the TonB protein, if expressed, may play a different role in the virulence in H. parainfluenzae since it is not needed for heme or heme complexes uptake as with H. influenzae.
Topics: Adenoidectomy; Bacterial Proteins; Child; Haemophilus Infections; Haemophilus influenzae; Humans; Membrane Proteins; Restriction Mapping; Reverse Transcriptase Polymerase Chain Reaction; Tonsillectomy; Tonsillitis; Transcription, Genetic
PubMed: 11238199
DOI: 10.1128/CDLI.8.2.221-224.2001 -
Journal of Bacteriology Mar 1991The genome of Myxococcus xanthus, which is 9,454 kbp, is one of the largest bacterial genomes. The organization of the DNA and the distribution of genes encoding social...
The genome of Myxococcus xanthus, which is 9,454 kbp, is one of the largest bacterial genomes. The organization of the DNA and the distribution of genes encoding social and developmental behaviors were examined by using pulsed field gel electrophoresis. Intact genomic DNA was digested with AseI into 16 restriction fragments, which were separated by contour-clamped homogeneous electric field electrophoresis, purified, and radiolabeled. Each AseI fragment was hybridized to SpeI-digested DNA and to an M. xanthus genomic library contained in yeast artificial chromosomes. Some SpeI restriction fragments and yeast artificial chromosome clones contained AseI sites and hybridized with two different AseI restriction fragments, providing evidence for the juxtaposition of these AseI restriction fragments in the chromosome. The deduced AseI physical map is circular, suggesting that this bacterium contains a single, circular chromosome. Transposable elements shown by transduction to be in or near genes of interest were located on specific AseI restriction fragments by restriction analysis and Southern hybridization. Most AseI restriction fragments contained genes involved in social and developmental behaviors.
Topics: Blotting, Southern; DNA Transposable Elements; DNA, Bacterial; Genes, Bacterial; Genetic Linkage; Myxococcales; Restriction Mapping
PubMed: 1848221
DOI: 10.1128/jb.173.6.2109-2115.1991 -
Bioinformatics (Oxford, England) Apr 2016The Optical Mapping System discovers structural variants and potentiates sequence assembly of genomes via scaffolding and comparisons that globally validate or correct...
MOTIVATION
The Optical Mapping System discovers structural variants and potentiates sequence assembly of genomes via scaffolding and comparisons that globally validate or correct sequence assemblies. Despite its utility, there are few publicly available tools for aligning optical mapping datasets.
RESULTS
Here we present software, named 'Maligner', for the alignment of both single molecule restriction maps (Rmaps) and in silico restriction maps of sequence contigs to a reference. Maligner provides two modes of alignment: an efficient, sensitive dynamic programming implementation that scales to large eukaryotic genomes, and a faster indexed based implementation for finding alignments with unmatched sites in the reference but not the query. We compare our software to other publicly available tools on Rmap datasets and show that Maligner finds more correct alignments in comparable runtime. Lastly, we introduce the M-Score statistic for normalizing alignment scores across restriction maps and demonstrate its utility for selecting high quality alignments.
AVAILABILITY AND IMPLEMENTATION
The Maligner software is written in C ++ and is available at https://github.com/LeeMendelowitz/maligner under the GNU General Public License.
CONTACT
Topics: Algorithms; Computer Simulation; Genome; Restriction Mapping; Sequence Alignment; Sequence Analysis, DNA; Software
PubMed: 26637292
DOI: 10.1093/bioinformatics/btv711 -
Eastern Mediterranean Health Journal =... Nov 1999It has been 20 years since DNA analysis was first used in the detection of sickle-cell anaemia. Here, techniques for detecting human mutations are reviewed. We describe... (Review)
Review
It has been 20 years since DNA analysis was first used in the detection of sickle-cell anaemia. Here, techniques for detecting human mutations are reviewed. We describe direct detection of mutations using restriction enzyme analysis and polymerase chain reaction amplification to detect gene deletions, rearrangements and point mutations. Indirect detection of mutations include the use of DNA polymorphisms in linkage analysis.
Topics: Chromosome Mapping; DNA Mutational Analysis; Gene Deletion; Gene Rearrangement; Point Mutation; Polymerase Chain Reaction; Polymorphism, Genetic; Restriction Mapping
PubMed: 11924102
DOI: No ID Found -
Briefings in Functional Genomics Dec 2010Next-generation sequencing technologies are making a substantial impact on many areas of biology, including the analysis of genetic diversity in populations. However,... (Comparative Study)
Comparative Study
Next-generation sequencing technologies are making a substantial impact on many areas of biology, including the analysis of genetic diversity in populations. However, genome-scale population genetic studies have been accessible only to well-funded model systems. Restriction-site associated DNA sequencing, a method that samples at reduced complexity across target genomes, promises to deliver high resolution population genomic data-thousands of sequenced markers across many individuals-for any organism at reasonable costs. It has found application in wild populations and non-traditional study species, and promises to become an important technology for ecological population genomics.
Topics: Animals; Base Sequence; Genetic Variation; Genetics, Population; Genome; Humans; Phylogeny; Polymorphism, Single Nucleotide; Restriction Mapping; Sequence Analysis, DNA
PubMed: 21266344
DOI: 10.1093/bfgp/elq031 -
GigaScience Dec 2016The pangolin is a Pholidota mammal with large keratin scales protecting its skin. Two pangolin species ( Manis pentadactyla and Manis javanica ) have been recorded as... (Comparative Study)
Comparative Study
BACKGROUND
The pangolin is a Pholidota mammal with large keratin scales protecting its skin. Two pangolin species ( Manis pentadactyla and Manis javanica ) have been recorded as critically endangered on the International Union for Conservation of Nature Red List of Threatened Species. Optical mapping constructs high-resolution restriction maps from single DNA molecules for genome analysis at the megabase scale and to assist genome assembly. Here, we constructed restriction maps of M. pentadactyla and M. javanica using optical mapping to assist with genome assembly and analysis of these species.
FINDINGS
Genomic DNA was nicked with Nt.BspQI and then labeled using fluorescently labeled bases that were detected by the Irys optical mapping system. In total, 3,313,734 DNA molecules (517.847 Gb) for M. pentadactyla and 3,439,885 DNA molecules (504.743 Gb) for M. javanica were obtained, which corresponded to approximately 178X and 177X genome coverage, respectively. Qualified molecules (≥150 kb with a label density of >6 sites per 100 kb) were analyzed using the de novo assembly program embedded in the IrysView pipeline. We obtained two maps that were 2.91 Gb and 2.85 Gb in size with N50s of 1.88 Mb and 1.97 Mb, respectively.
CONCLUSIONS
Optical mapping reveals large-scale structural information that is especially important for non-model genomes that lack a good reference. The approach has the potential to guide de novo assembly of genomes sequenced using next-generation sequencing. Our data provide a resource for Manidae genome analysis and references for de novo assembly. This note also provides new insights into Manidae evolutionary analysis at the genome structure level.
Topics: Animals; Endangered Species; Genome; Humans; Mammals; Optical Restriction Mapping
PubMed: 28369358
DOI: 10.1093/gigascience/giw001 -
Proceedings of the National Academy of... Jul 2005We have performed restriction mapping of DNA molecules using restriction endonucleases in nanochannels with diameters of 100-200 nm. The location of the restriction...
We have performed restriction mapping of DNA molecules using restriction endonucleases in nanochannels with diameters of 100-200 nm. The location of the restriction reaction within the device is controlled by electrophoresis and diffusion of Mg2+ and EDTA. We have successfully used the restriction enzymes SmaI, SacI, and PacI, and have been able to measure the positions of restriction sites with a precision of approximately 1.5 kbp in 1 min using single DNA molecules.
Topics: DNA; DNA Restriction Enzymes; Magnesium; Microscopy; Nanostructures; Nanotechnology; Restriction Mapping
PubMed: 16000405
DOI: 10.1073/pnas.0503809102 -
Journal of Bacteriology May 1997PacI and SpeI restriction maps were obtained for the two chromosomes of each of the six species of the genus Brucella: B. melitensis, B. abortus, B. suis, B. canis, B.... (Comparative Study)
Comparative Study
PacI and SpeI restriction maps were obtained for the two chromosomes of each of the six species of the genus Brucella: B. melitensis, B. abortus, B. suis, B. canis, B. ovis, and B. neotomae. Three complementary techniques were used: hybridization with the two replicons as probes, cross-hybridization of restriction fragments, and a new mapping method. For each type strain, a unique I-SceI site was introduced in each of the two replicons, and the location of SpeI sites was determined by linearization at the unique site, partial digestion, and end labeling of the fragments. The restriction and genetic maps of the six species were highly conserved. However, numerous small insertions or deletions, ranging from 1 to 34 kb, were observed by comparison with the map of the reference strain of the genus, B. melitensis 16M. A 21-kb Spel fragment specific to B. ovis was found in the small chromosome of this species. A 640-kb inversion was demonstrated in the B. abortus small chromosome. All of these data allowed the construction of a phylogenetic tree, which reflects the traditional phenetic classification of the genus.
Topics: Brucella; Deoxyribonucleases, Type II Site-Specific; Electrophoresis, Gel, Pulsed-Field; Genome, Bacterial; Phylogeny; Polymorphism, Restriction Fragment Length; Replicon; Restriction Mapping; Saccharomyces cerevisiae Proteins
PubMed: 9150220
DOI: 10.1128/jb.179.10.3244-3249.1997 -
Bioinformatics (Oxford, England) Nov 2012The R/Bioconductor package HiTC facilitates the exploration of high-throughput 3C-based data. It allows users to import and export 'C' data, to transform, normalize,...
SUMMARY
The R/Bioconductor package HiTC facilitates the exploration of high-throughput 3C-based data. It allows users to import and export 'C' data, to transform, normalize, annotate and visualize interaction maps. The package operates within the Bioconductor framework and thus offers new opportunities for future development in this field.
AVAILABILITY AND IMPLEMENTATION
The R package HiTC is available from the Bioconductor website. A detailed vignette provides additional documentation and help for using the package.
Topics: Animals; Chromosome Mapping; Chromosomes, Human, Pair 14; Data Display; Humans; Mice; Molecular Conformation; Restriction Mapping; Software
PubMed: 22923296
DOI: 10.1093/bioinformatics/bts521