scholarly journals A NotI Restriction site of Campylobacter coli Chromosomal DNA Detected by Double-Digestion Analysis with ApaI.

1991 ◽  
Vol 53 (3) ◽  
pp. 545-547
Author(s):  
Motoo MATSUDA ◽  
Kazumasa MATSUMOTO ◽  
Takatsugu YAMADA ◽  
Choji KANEUCHI
Keyword(s):  
Restriction Site ◽  
Campylobacter Coli ◽  
Chromosomal Dna ◽  
Noti Restriction Site ◽  
Double Digestion ◽  
Noti Restriction

scholarly journals Differentiation of Pathogenic BartonellaSpecies by Infrequent Restriction Site PCR

2000 ◽  
Vol 38 (8) ◽  
pp. 3010-3015 ◽  
Author(s):  
Scott A. Handley ◽  
Russell L. Regnery
Keyword(s):  
Capillary Electrophoresis ◽  
Restriction Endonuclease ◽  
Restriction Site ◽  
Polyacrylamide Gel Electrophoresis ◽  
Ease Of Use ◽  
Dna Templates ◽  
Pattern Complexity ◽  
Double Digestion ◽  
Pcr Method ◽  
High Level

Infrequent restriction site PCR (IRS-PCR) is a recently described DNA fingerprinting technique based on selective amplification of restriction endonuclease-cleaved fragments. Bartonellaisolates associated with human disease and related nonhuman isolates were analyzed by IRS-PCR genomic fingerprinting. Preparation of DNA templates began with double digestion using three different restriction endonuclease combinations. Combinations included the frequently cutting endonuclease HhaI in conjunction with an infrequently cutting endonuclease, EagI, SmaI, orXbaI. Digestion was followed by ligation of oligonucleotide adapters designed with ends complementary to the restriction endonuclease sites. Amplification of fragments flanked with anEagI, SmaI, or XbaI site in combination with an HhaI site produced a series of different-sized amplicons resolvable into patterns by polyacrylamide gel electrophoresis (PAGE). The pattern complexity was varied by the addition of selective nucleotides to the 3′ ends of theEagI-, SmaI-, or XbaI-specific primers. Amplicons were also generated with fluorescently labeled primers and were subsequently resolved and detected by capillary electrophoresis. Analysis by traditional slab PAGE and capillary electrophoresis provided suitable resolution of patterns produced with the enzyme combinations EagI-HhaI andSmaI-HhaI. However, the combination ofXbaI-HhaI produced too many fragments for sufficient resolution by traditional PAGE, thus requiring the better resolving properties of capillary electrophoresis. Due to the flexibility in modulating the pattern complexity and electrophoresis methods, these techniques allow for a high level of experimental optimization. The results provide evidence of the discriminatory power, ease of use, and flexibility of the IRS-PCR method as it applies to the identification of human-pathogenic Bartonella species.

scholarly journals High-Resolution Genotyping ofCampylobacter Strains Isolated from Poultry and Humans with Amplified Fragment Length Polymorphism Fingerprinting

1999 ◽  
Vol 65 (6) ◽  
pp. 2369-2375 ◽  
Author(s):  
Birgitta Duim ◽  
Trudy M. Wassenaar ◽  
Alan Rigter ◽  
Jaap Wagenaar
Keyword(s):  
Length Polymorphism ◽  
Fragment Length ◽  
Fragment Length Polymorphism ◽  
Amplified Fragment Length Polymorphism ◽  
Pcr Primers ◽  
Epidemiological Studies ◽  
Aflp Analysis ◽  
Campylobacter Coli ◽  
Chromosomal Dna ◽  
Typing Methods

ABSTRACT For epidemiological studies of Campylobacterinfections, molecular typing methods that can differentiate campylobacters at the strain level are needed. In this study we used a recently developed genotyping method, amplified fragment length polymorphism (AFLP), which is based on selective amplification of restriction fragments of chromosomal DNA, for genetic typing ofCampylobacter jejuni and Campylobacter colistrains derived from humans and poultry. We developed an automated AFLP fingerprinting method in which restriction endonucleasesHindIII and HhaI were used in combination with one set of selective PCR primers. This method resulted in evenly distributed band patterns for amplified fragments ranging from 50 to 500 bp long. The discriminatory power of AFLP was assessed with aC. jejuni strain, an isogenic flagellin mutant, and distinct C. jejuni strains having known pulsed-field gel electrophoresis and fla PCR-restriction fragment length polymorphism genotypes. Unrelated C. jejuni strains produced heterogeneous patterns, whereas genetically related strains produced similar AFLP patterns. Twenty-five Campylobacterstrains obtained from poultry farms in The Netherlands grouped in threeC. jejuni clusters that were separate from a C. coli cluster. The band patterns of 10 C. jejunistrains isolated from humans were heterogeneous, and most of these strains grouped with poultry strains. Our results show that AFLP analysis can distinguish genetically unrelated strains from genetically related strains of Campylobacter species. However, desirable genetically related strains can be differentiated by using other genotyping methods. We concluded that automated AFLP analysis is an attractive tool which can be used as a primary method for subtyping large numbers of Campylobacter strains and is extremely useful for epidemiological investigations.

Ribosomal RNA Gene Restriction Fragment Diversity amongst Penner Serotypes of Campylobacter jejuni and Campylobacter coli

1998 ◽  
Vol 53 (1-2) ◽  
pp. 65-68
Author(s):  
S. I. Smith ◽  
D. K. Olukoya ◽  
A. J. Fox ◽  
A. O. Coker
Keyword(s):  
Campylobacter Jejuni ◽  
Ribosomal Rna ◽  
Rrna Gene ◽  
Campylobacter Coli ◽  
Chromosomal Dna ◽  
Ribosomal Rna Gene ◽  
Restriction Endonuclease Digest ◽  
Gene Restriction ◽  
Rna Genes ◽  
The 16S Rrna Gene

AbstractDiversity based on ribosomal RNA gene-restriction endonuclease digest patterns was detected amongst forty-seven strains of Campylobacter made up of 38 strains of Campylobacter jejuni and 9 strains of Campylobacter coli. Restriction digests of chromosomal DNA prepared by treating with Hae III were probed with an oligonucleotide specific for Campylobacter 16S ribosomal RNA genes. Seventeen distinct hybridization patterns, each indicating the presence of 2 - 4 copies of the 16S rRNA gene are encoded in Campylobacter DNA. Differences in fragment patterns were observed not only between members of two species, but also between individual strains of the same species. Ribopattern fragments of 8.71, 7.56, 2.81 and 1.0 kb were characteristic of the majority of C. jejuni, whereas 7.59 and 4.68 kb fragments were commonly present in C. coli.In conclusion, Hae III ribotyping was even more discriminatory than the Penner serotyping of C. jejuni and C. coli, as strains of the same serotype were distinguished.

DNA hybridization analysis of thePseudomonas aeruginosaelastase gene [lasB) from different clinical isolates

1995 ◽  
Vol 41 (10) ◽  
pp. 910-917 ◽  
Author(s):  
Abdul N. Hamood ◽  
John Griswold
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Structural Gene ◽  
Dna Hybridization ◽  
Restriction Site ◽  
Clinical Isolates ◽  
Upstream Region ◽  
Gene Probe ◽  
Chromosomal Dna ◽  
Hybridization Pattern ◽  
Hybridization Band

Pseudomonas aeruginosa produces several extracellular virulence factors including elastase (which is encoded by lasB). Recently, we examined several clinical isolates of P. aeruginosa for the production of toxin A, elastase, exoenzyme S, and phospholipase C. Although the majority of the isolates produced a high level of elastase, a few isolates produced either very low or no detectable elastase. In this study, we tried to determine the presence of restriction site heterogeneity within lasB from these isolates and the possible correlation between such heterogeneity and the observed variation in elastase production. Chromosomal DNA from the isolates was digested with different restriction enzymes and examined by Southern blot hybridization experiments using two lasB probes. One lasB probe covers 636 bp of lasB structural gene while the other covers 240 bp of the lasB upstream region. Chromosomal DNA from P. aeruginosa PAO1 and PA103 was used as controls. Results indicate that chromosomal DNA from all isolates hybridized to both lasB probes. Depending on the restriction enzyme used for DNA digestion, lasB from 3 to 12% of the isolates showed different patterns of hybridization with the lasB structural gene probe. However, no difference in the hybridization pattern was seen with the lasB upstream probe. With the exception of one isolate, hybridization of genomic DNA from different isolates (with both probes) produced a single hybridization band. In that isolate, an additional hybridization band was detected. Immunoblotting experiments confirmed that elastase protein is not produced by 6 out of 67 isolates. However, lasB from four of these elastase-deficient strains showed no difference in the hybridization pattern with either lasB probe. These results suggest that (i) lasB is present as a single copy in all but one isolate; (ii) with the exception of one, the lasB upstream region from different P. aeruginosa isolates contains no restriction site polymorphism; (iii) the observed heterogeneity within lasB structural genes is limited; and (iv) variations in the hybridization patterns of lasB from different isolates do not correlate with the differences between these isolates in elastase production.Key words: Pseudomonas aeruginosa, clinical isolates, DNA hybridization, elastase, lasB.

scholarly journals Identification and Mapping of Eastern Filbert Blight Resistance Quantitative Trait Loci in European Hazelnut Using Double Digestion Restriction Site Associated DNA Sequencing

2019 ◽  
Vol 144 (5) ◽  
pp. 295-304 ◽  
Author(s):  
Josh A. Honig ◽  
Megan F. Muehlbauer ◽  
John M. Capik ◽  
Christine Kubik ◽  
Jennifer N. Vaiciunas ◽  
...  
Keyword(s):  
United States ◽  
Genetic Diversity ◽  
Quantitative Trait ◽  
Genetic Linkage ◽  
Genetic Linkage Map ◽  
Restriction Site ◽  
Eastern United States ◽  
Double Digestion ◽  
Disease Pressure ◽  
Resistance Source

European hazelnut (Corylus avellana L.) is an economically important edible nut producing species, which ranked sixth in world tree nut production in 2016. European hazelnut production in the United States is primarily limited to the Willamette Valley of Oregon, and currently nonexistent in the eastern United States because of the presence of a devastating endemic disease, eastern filbert blight (EFB) caused by Anisogramma anomala (Peck) E. Muller. The primary commercial means of control of EFB to date is through the development and planting of genetically resistant european hazelnut cultivars, with an R-gene introduced from the obsolete, late-shedding pollinizer ‘Gasaway’. Although the ‘Gasaway’ resistance source provides protection against EFB in the Pacific northwestern United States (PNW), recent reports have shown that it is not effective in parts of the eastern United States. This may be in part because the identification and selection of ‘Gasaway’ and ‘Gasaway’-derived cultivars occurred in an environment (PNW) with limited genetic diversity of A. anomala. The objectives of the current research were to develop a genetic linkage map using double digestion restriction site associated DNA sequencing (ddRADseq) and identify quantitative trait loci (QTL) markers associated with EFB resistance from the resistant selection Rutgers H3R07P25 from southern Russia. A mapping population composed of 119 seedling trees was evaluated in a geographic location (New Jersey) where the EFB fungus is endemic, exhibits high disease pressure, and has a high level of genetic diversity. The completed genetic linkage map included a total of 2217 markers and spanned a total genetic distance of 1383.4 cM, with an average marker spacing of 0.65 cM. A single QTL region associated with EFB resistance from H3R07P25 was located on european hazelnut linkage group (LG) 2 and was responsible for 72.8% of the phenotypic variation observed in the study. Based on its LG placement, origin, and disease response in the field, this resistance source is different from the ‘Gasaway’ source located on LG6. The current results, in combination with results from previous research, indicate that the H3R07P25 source is likely exhibiting resistance to a broader range of naturally occurring A. anomala isolates. As such, H3R07P25 will be important for the development of new european hazelnut germplasm that combines EFB resistance from multiple sources in a gene pyramiding approach. Identification of EFB resistance in high disease pressure environments representing a diversity of A. anomala populations is likely a requirement for identifying plants expressing durable EFB resistance, which is a precursor to the development of a commercially viable european hazelnut industry in the eastern United States.

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