Automated fragment length analysis of fluorescently-labeled 16S rDNA after digestion with 4-base cutting restriction enzymes

1998 ◽  
Vol 32 (1) ◽  
pp. 55-63 ◽  
Author(s):  
R Pukall ◽  
E Brambilla ◽  
E Stackebrandt
Keyword(s):  
16S Rdna ◽  
Fragment Length ◽  
Restriction Enzymes ◽  
Length Analysis

scholarly journals First Report of an Aster Yellows Phytoplasma Associated with Cabbage in Southern Texas

Plant Disease ◽  
2001 ◽  
Vol 85 (4) ◽  
pp. 447-447 ◽  
Author(s):  
I.-M. Lee ◽  
R. A. Dane ◽  
M. C. Black ◽  
Noel Troxclair
Keyword(s):  
16S Rdna ◽  
Nested Pcr ◽  
Restriction Enzymes ◽  
Diagnostic Procedures ◽  
Acid Extraction ◽  
Insect Vector ◽  
Aster Yellows ◽  
First Report ◽  
Pcr Products ◽  
Aster Yellows Phytoplasma

In early spring 2000 carrot crops in southwestern Texas were severely infected by an outbreak of phyllody associated with aster yellows phytoplasma. Cabbage crops that had been planted adjacent to these carrot fields began to display previously unobserved symptoms characteristic of phytoplasma infection. Symptoms included purple discoloration in leaf veins and at the outer edges of leaves on cabbage heads. Proliferation of sprouts also occurred at the base of the stem and between leaf layers of some plants, and sprouts sometimes continued to proliferate on extended stems. About 5% of cabbage plants in the field exhibited these symptoms. Two symptomless and four symptomatic cabbage heads were collected in early April from one cabbage field. Veinal tissues were stripped from each sample and used for total nucleic acid extraction. To obtain specific and sufficient amount of PCR products for analysis, nested PCR was performed by using primer pairs (first with P1/P7 followed by R16F2n/R16R2) (1,2) universal for phytoplasma detection. A specific 16S rDNA fragment (about 1.2 kb) was strongly amplified from the four symptomatic but not from the two asymptomatic samples. The nested PCR products obtained from the four symptomatic samples were then analyzed by restriction fragment length polymorphism (RFLP) using the restriction enzymes MseI, HhaI, and HpaII, and the RFLP patterns were compared to the published patterns of known phytoplasmas (1). The resulting RFLP patterns were identical to those of a phytoplasma belonging to subgroup B of the aster yellows phytoplasma group (16SrI). These RFLP patterns were also evident in putative restriction sites observed in a 1.5 kbp nucleotide sequence of the 16S rDNA. This is the first report of aster yellows phytoplasma associated disease symptoms in cabbage in Texas. The occurrence of cabbage proliferation coincided with the presence of high populations of the insect vector, aster leafhopper. References: (1) I.-M. Lee et al. Int. J. Syst. Bacteriol. 48:1153, 1998. (2) B. Schneider et al. 1995. Molecular and Diagnostic Procedures in Mycoplasmology, Vol. I. Academic Press, San Diego, CA.

Analysis of the Bacterial Diversity Existing on Animal Hide and Wool: Development of a Preliminary PCR-Restriction Fragment Length Polymorphism Fingerprint Database for Identifying Isolates

2012 ◽  
Vol 95 (6) ◽  
pp. 1750-1754 ◽  
Author(s):  
Chen Yu ◽  
Gao Hongwei ◽  
Zhang Yanming ◽  
Deng Mingjun ◽  
Wu Zhenxing ◽  
...  
Keyword(s):  
Restriction Fragment Length Polymorphism ◽  
16S Rdna ◽  
Restriction Fragment ◽  
Length Polymorphism ◽  
Fragment Length ◽  
Fragment Length Polymorphism ◽  
Nutrient Broth ◽  
Universal Primers ◽  
Comamonas Testosteroni ◽  
Restriction Fragment Length

Abstract Twenty-one bacterial strains were isolated from imported cattle hide and rabbit wool using two types of media, nutrient broth, and nutrient broth with serum. The bacteria identified were Brevibacillus laterosporus, Leclercia adecarboxylata, Peptococcus niger, Bacillus circulans, Raoultella ornithinolytica, Bacillus subtilis, Bacillus cereus, Bacillus thermobacillus, Bacillus choshinensis, Bacillus sphaericus, Acinetobacter haemolyticus, Sphingomonas paucimobilis, Bacillus thuringiensis, Staphylococcus intermedius, Mycobacteria, Moraxella, Klebsiella pneumoniae, Ralstonia pickettii, Staphylococcus chromogenes, Comamonas testosteroni, and Cupriavidus pauculus. The 16s rDNA gene of each bacterium was amplified using the universal primers 27f and 1492r. The amplicons were digested with AvaI, BamHI, BgII, DraI, EcoRI, EcoRV, HindIII, HinfI, HpaI, PstI, SmaI, TaqI, XbaI, XmaI, AluI, XhoI, and PvuI individually. A specific fingerprint from the PCR-restriction fragment length polymorphism method based on 16s rDNA was obtained for each bacterium. The results showed that the method developed was useful not only for bacterial identification but also for the etiological investigation of pathogens in imported animal hair and wool.

scholarly journals Identification of Pneumococcal Serotypes by PCR–Restriction Fragment Length Polymorphism

Diagnostics ◽  
2019 ◽  
Vol 9 (4) ◽  
pp. 196 ◽  
Author(s):  
García-Suárez ◽  
González-Rodríguez ◽  
Cima-Cabal ◽  
Yuste ◽  
Vazquez ◽  
...  
Keyword(s):  
Restriction Fragment Length Polymorphism ◽  
Restriction Fragment ◽  
Length Polymorphism ◽  
Dna Sequences ◽  
Fragment Length ◽  
Fragment Length Polymorphism ◽  
Rflp Analysis ◽  
Restriction Enzymes ◽  
Pcr Rflp ◽  
Restriction Fragment Length

Streptococcus pneumoniae shows more than 90 capsular serotypes that can be distinguished by their reactivity against antisera. The main objective of this work was the development of a molecular method for serotyping without the use of antisera. A computer program containing an algorithm was used to search in a database for potentially useful enzymes for Restriction Fragment Length Polymorphism-RFLP typing, in order to maximize the discrimination between different serotypes. DNA sequences of 90 serotypes for the region between dexB and aliA genes were compiled, and a computer screening of restriction enzymes was performed. The wzg–wzh–wzd–wze region and Sse9I restriction predicted unique PCR-RFLP patterns for 39 serotypes and eight serogroups. A second restriction enzyme resolved fragment specific patterns for 25 serotypes. The method was tested with 98 serotype-unknown clinical isolates. PCR-RFLP analysis deduced correct serotypes that were confirmed by Quellung reaction for 78.5% of the isolates.

Phylogenetic relationships of the pigeonpea (Cajanus cajan) based on nuclear restriction fragment length polymorphisms

Genome ◽  
1993 ◽  
Vol 36 (2) ◽  
pp. 216-223 ◽  
Author(s):  
Ram G. Nadimpalli ◽  
R. L. Jarret ◽  
Sharad C. Phatak ◽  
Gary Kochert
Keyword(s):  
Restriction Fragment ◽  
Fragment Length ◽  
Phylogenetic Relationships ◽  
Seed Protein ◽  
Restriction Enzymes ◽  
Restriction Fragment Length Polymorphisms ◽  
Length Polymorphisms ◽  
Genomic Probes ◽  
Clustering Patterns ◽  
Restriction Fragment Length

Nuclear restriction fragment length polymorphisms (RFLPs) were used to determine phylogenetic relationships in the genus Cajanus using 15 random genomic probes and six restriction enzymes. Twenty-four accessions representing 12 species of four genera (Cajanus, Dunbaria, Eriosema, and Rhynchosia) were examined to determine phylogenetic relationships in the genus Cajanus. Eriosema parviflorum was selected as the out-group. Sufficient RFLP polymorphisms were detected among species to resolve in-group taxa into distinct clusters. Topologies of trees from parsimony and similarity matrix analyses were similar but not identical, and clustering patterns agreed broadly with published phylogenies based on seed protein data and, to a lesser extent, data from cytology and breeding experiments. Accessions of cultivated C. cajan shared more DNA fragments with C. scarabaeoides than with C. cajanifolia. Inconsistencies in taxonomic relationships based on data from morphology, cytology, crossability, and RFLPs are discussed.Key words: pigeonpea, systematics, taxonomy, evolution, germplasm.

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