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Results for “Random Amplified Polymorphic DNA Technique”

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PubMed1993

Genetic relatedness among rabbiteye blueberry (Vaccinium ashei) cultivars determined by DNA amplification using single primers of arbitrary sequence.

Most improved cultivars of commercially important hexaploid rabbiteye blueberry were developed from only four native selections collected from the wild; thus, many cultivars are closely related by lineage. The consanguinity among major cultivars is a potential problem, as the rabbiteye blueberries are highly self-incompatible natural outcrossers with potential inbreeding depression. We investigated the extent of genetic relatedness among 15 improved cultivars and four wild selections by the technique of random amplified polymorphic DNA, also referred to as arbitrarily primed polymerase chain reaction. Single decanucleotides of arbitrary sequence revealed polymorphism among cultivars and wild selections. Genetic distances were estimated based on the amount of band sharing. Cluster analysis of genetic distance estimates tended to group siblings with each other and with one or both parents. The average genetic distance between improved cultivars decreased relative to the four wild parental selections, which might indicate progression towards inbreeding. The significance of increased genetic relatedness among the improved cultivars of rabbiteye blueberry and the application of molecular methods in breeding and commercial cultivation is discussed.

Base Sequence↗

PubMed2006

Isolation of Wuchereria bancrofti microfilariae from archived stained blood slides for use in genetic studies and amplification of parasite and endosymbiont genes.

Information on change in genetic diversity of Wuchereria bancrofti is important in view of the launching of the Global Lymphatic Filariasis Elimination Programme, as it may have important consequences on the control operations and on the potential resurgence after their withdrawal. Since attention was not paid to generate such information when the programme was launched, use of archived parasite material will provide an opportunity to derive this information in a prospective manner. In this paper a simple and effective technique is reported for isolation of microfilariae of W. bancrofti from dried and stained slides archived for several years and their utility in analysis of genetic structure and amplification of certain genes of the parasite is tested. The method was found to be efficient in purifying mf from the dried smears and the DNA of the parasite found to be useful in studying the genetic structure of Wuchereria bancrofti populations using random amplified polymorphic DNA (RAPD)-PCR and for amplifying genes of the parasite and its endosymbiont, Wolbachia sp.

Animals↗

PubMed2005

Bacillus axarquiensis sp. nov. and Bacillus malacitensis sp. nov., isolated from river-mouth sediments in southern Spain.

Two Gram-positive, rod-shaped, endospore-forming bacteria (strains CR-119(T) and CR-95(T)) were isolated from brackish sediments in the mouth of the river Velez in Malaga, southern Spain, and subjected to a polyphasic taxonomic study. Phenotypic tests showed that these strains were related to other Bacillus species at a similarity level of less than 87.6 %. Both strains are halotolerant, aerobic, chemoheterotrophic, motile with peritrichous flagella and biosurfactant producers. Their endospores are oval, subterminal and non-deforming structures. The predominant menaquinone in both strains is MK-7. The fatty-acid profiles of both strains contain large quantities of branched and saturated fatty acids. The major fatty acids (%) are 15 : 0 anteiso (32.4), 15 : 0 iso (16.8), 17 : 0 iso (13.4), 16 : 0 (11.5) and 17 : 0 anteiso (10.2) in strain CR-119(T) and 15 : 0 anteiso (37.5), 17 : 0 iso (16.0) and 17 : 0 anteiso (15.8) in strain CR-95(T). The G + C contents of strains CR-119(T) and CR-95(T) are 41.0 and 42.5 mol%, respectively. RAPD analysis confirmed the low degree of similarity between the two strains and also amongst other Bacillus species. 16S rRNA gene analysis of strain CR-119(T) showed the highest sequence similarity to be 97.4 %, with Bacillus mojavensis and Bacillus subtilis subsp. spizizenii. In the case of strain CR-95(T), the maximum similarity value was 99.5 %, with B. mojavensis. DNA-DNA hybridization of strains CR-119(T) and CR-95(T) with the above species produced values lower than 46.9 %. Therefore, on the basis of phenotypic characteristics, phylogenetic data and genomic distinctiveness, we conclude that these Bacillus strains merit classification as novel species, for which we propose the names Bacillus axarquiensis sp. nov. (type strain CR-119(T) = CECT 5688(T) = LMG 22476(T)) and Bacillus malacitensis sp. nov. (type strain CR-95(T) = CECT 5687(T) = LMG 22477(T)).

Aerobiosis↗

PubMed2005

Gene expression analysis using a modified HiCEP method applicable to prokaryotes: a study of the response of Rhodococcus to isoniazid and ethambutol.

We established a novel method to analyze the gene expression of prokaryotes by modifying and optimizing the HiCEP (high coverage gene expression analysis) method, which was originally developed for eukaryotic gene expression profiling. Following the removal of abundant rRNA, the mRNA of prokaryotic cells was enzymatically polyadenylated and subjected to HiCEP analysis. This method was highly reproducible due to selective PCR that was performed by using adaptor specific primers. We confirmed induction of tipA and induction or suppression of cspA, which are genes that are obtained from distinctive actinomycetes and responded to thiostrepton and temperature stress, respectively. Further, we applied this method to explore the gene expression profile of Rhodococcus erythropolis in response to drugs that inhibit cell wall synthetic pathways, and we were able to identify 35 upregulated genes. Among these genes, we confirmed the upregulation of 22 genes by using RT-PCR (reverse transcriptase-polymerase chain reaction). Some of these genes are involved in the synthesis of mycolic acid and arabinogalactan, suggesting a cell response to drug treatment by regulation of the genes involved in cell wall synthesis. This method could prove to be a useful technique for gene expression analysis of prokaryotes, particularly nonmodel strains with unknown genome sequences.

Antitubercular Agents↗

PubMed2001

Molecular taxonomy of the genus Chlorobium.

This review summarizes molecular studies on the taxonomy of the genus Chlorobium. Furthermore, we try to introduce a comprehensive view of how joining traditional phenotypic-based taxonomy with the current molecular approaches leads to a more natural classification. Moreover, we introduce the current insights that molecular techniques have brought to the detection and identification of nonisolated Chlorobium strains in their environments.

Chlorobi↗

PubMed2002

Molecular markers for sex determination in papaya ( Carica papaya L.).

We have developed molecular markers tightly linked to Sex1, the gene that determines plant sex in papaya ( Carica papaya L.). Three RAPD products have been cloned and a portion of their DNA sequenced. Based on these sequences SCAR primers were synthesized. SCAR T12 and SCAR W11 produce products in hermaphrodite and male plants and only rarely in females. SCAR T1 produces a product in all papayas regardless of plant sex. SCAR T12 and SCAR W11 showed no recombination in a population of 182 F2 plants from a 'SunUp' by 'Kapoho' cross. Based on these results a PCR-based technique for rapidly and accurately determining the sex of papaya plants was developed using either W11 or T12 to detect the hermaphrodite or male allele and T1, which amplifies a product regardless of sex type, as a positive control. The sexing technique, using SCAR T12 and SCAR T1 as a positive control, was used to correctly predict hermaphrodite papaya plants in a population of seedlings with an overall accuracy of 99.2%.

Carica↗

PubMed2006

[Study of Papaver somniferum cultivars identification by TD-RAPD technique].

OBJECTIVE: The feasibility of Papaver somniferum L. cultivars identification was explored by TD-RAPD technique. METHOD: Genomic DNA was extracted by improved CTAB method. One sample of species from Papaver somniferum L in xishuangbanna area. was studied by using TD-RAPD method. RESULT: We established an optimal method of extracting genomic DNA. Six primers were picked out from 10 primers. CONCLUSION: TD-RAPD could be applied to researches of molecular marker of Papaver somniferum L. TD-RAPD technique provide a method to constitute DNA database of Papaver somniferum L. and conclude the source of opium poppy.

DNA Primers↗

PubMed2004

Establishment of the amplified fragment length polymorphism (AFLP) technique for genotyping of pollen beetle (Meligethes aeneus)--a noxious insect pest on oilseed rape (Brassica napus).

In order to conduct studies concerning genetic variability of pollen beetles (Meligethes aeneus), a genotyping protocol was established. No genome information is available for pollen beetles so the amplified fragment length polymorphism (AFLP) technique was chosen since it does not depend on any prior sequence information of the samples and also is a sensitive and robust technique. However, several modifications were needed in order to adapt the method for analysis of pollen beetles. Basic modifications included (i) alterations of DNA purification, (ii) use of two six-cutter restriction enzymes, (iii) and modified PCR conditions. This protocol resulted in a favourable number of fragments of an appropriate size range for standard gel analysis by a DNA sequencer applicable to a single insect and even body parts enabling different assays to be conducted on a single specimen. Pollen beetles from different areas of Sweden were analysed to verify the reproducibility and efficacy of the protocol as well as for phenetic analysis. The high reproducibility of the modified AFLP protocol allows it to be used as a reliable tool for genotype analysis of pollen beetles.

Animals↗

PubMed2002

Discrimination of Arcobacter butzleri isolates by polymerase chain reaction-mediated DNA fingerprinting.

AIMS: The objective of this study was to subtype Arcobacter butzleri isolates using RAPD-PCR. METHODS AND RESULTS: Thirty-five A. butzleri isolates obtained from chicken carcasses were examined. PCR-mediated DNA fingerprinting technique with primers of the variable sequence motifs was used to detect polymorphism within the isolates. Eleven distinct DNA profiles were obtained as follows: Of the 35 strains, 10 as profile 4; seven as profile 1; five as profile 3; three as profiles 2 and 9; two as profile 10; one as profiles 5, 6, 7, 8 and 11. CONCLUSIONS: Chicken carcasses sold in markets were found to be contaminated with several different strains of A. butzleri. RAPD-PCR technique was found to be a useful technique for distinguishing A. butzleri isolates. SIGNIFICANCE AND IMPACT OF THE STUDY: The presence of several different A. butzleri strains on chicken carcasses may indicate multiple sources of contamination. The epidemiological role of A. butzleri in human and animal diseases should be investigated further.

Animals↗

PubMed2000

Simultaneous identification of two cyclohexanone oxidation genes from an environmental Brevibacterium isolate using mRNA differential display.

The technique of mRNA differential display was used to identify simultaneously two metabolic genes involved in the degradation of cyclohexanone in a new halotolerant Brevibacterium environmental isolate. In a strategy based only on the knowledge that cyclohexanone oxidation was inducible in this strain, the mRNA population of cells exposed to cyclohexanone was compared to that of control cells using reverse transcription-PCR reactions primed with a collection of 81 arbitrary oligonucleotides. Three DNA fragments encoding segments of flavin monooxygenases were isolated with this technique, leading to the identification of the genes of two distinct cyclohexanone monooxygenases, the enzymes responsible for the oxidation of cyclohexanone. Each monooxygenase was expressed in Escherichia coli and characterized. This work validates the application of mRNA differential display for the discovery of new microbial metabolic genes.

Amino Acid Sequence↗

PubMed2004

Molecular diversity of Moraxella bovis isolated from Brazil, Argentina and Uruguay over a period of three decades.

The molecular profile of 30 Moraxella bovis strains, recovered from outbreaks of infectious bovine keratoconjunctivitis in Argentina, Brazil and Uruguay between 1974 and 2001, was determined through randomly applied polymorphic DNA (RAPD) analysis. Molecular profiles of nine strains recovered after 1990 varied from those recovered before 1990. The profiles of 13 strains (48%) differed from those of three vaccinal strains extensively used since 1984 in Argentina and Uruguay. Eight Argentinean strains, one from Brazil and two from Uruguay had identical RAPD profiles. Strains belonging to different serogroups had identical RAPD profiles, demonstrating that this technique was not able to discriminate among strains with low cross-reactivity indices. RAPD may be helpful in the primary characterization of M. bovis strains, but it does not replace serological characterization.

Animals↗

PubMed2004

Evaluation of a RAPD-based typing scheme in a molecular epidemiology study of Vibrio cholerae O1, Brazil.

AIMS: To evaluate the utility of random amplification of polymorphic DNA (RAPD) technique for routine practice in public health laboratories for epidemiological studies of Vibrio cholerae O1 isolates. MATERIALS AND RESULTS: Seventy-nine strains were examined by PCR for the toxin genes (ctx A, zot and ace), virulence-associated genes (tcp A and tox T) and RAPD sequences. Except for one strain (no. 1123) from the Amazonas State, all the strains analysed carried the genes ctx A, zot, ace, tcp A and tox T. RAPD fingerprinting revealed variability but no correlation with serotype, biotype or geographical origin of the isolates was found. CONCLUSION: A standardized RAPD method does not enable the establishment of a pattern data bank for the identification of V. cholerae O1 strains. SIGNIFICANCE AND IMPACT OF THE STUDY: The simplicity and discriminative capacity of this technique make it useful for detecting genetic diversity among micro-organisms from a defined group or for outbreak investigation.

Brazil↗

PubMed2004

RACE using only a gene-specific primer: application of a template-switching model.

This article describes a simple method for accurate rapid amplification of complementary deoxyribonucleic acid (cDNA) ends (RACE), the distinctive feature being that only a gene-specific primer is used, without an anchor or adapter primer. Under these conditions, Thermus aquaticus (Taq) polymerase synthesizes cDNA ends exactly, so that amplified products obtain a characteristic structure: a terminal inverted repeat composed of a gene-specific primer and occasionally several nucleotides from its 3' flanking sequence. These structures suggest a hypothetical mechanism of cDNA end synthesis in which Taq DNA polymerase synthesizes a sequence complementary to the gene-specific primer at the 3' end of the daughter strand by switching the template to the 5' terminal region through circularization of the DNA. As a result, the targeted cDNA will be efficiently amplified with only a single gene-specific primer. This technique, which provides highly specific amplification of the 5' and 3' ends of a cDNA, is especially useful for isolation of cDNA when the corresponding messenger ribonucleic acid is scarce.

Animals↗

PubMed1999

Validation of binary typing for Staphylococcus aureus strains.

Most of the DNA-based methods for genetic typing of Staphylococcus aureus strains generate complex banding patterns. Therefore, we have developed a binary typing procedure involving strain-differentiating DNA probes which were generated on the basis of randomly amplified polymorphic DNA (RAPD) analysis. We present and validate the usefulness of 15 DNA probes, according to generally accepted performance criteria for molecular typing systems. RAPD analysis with multiple primers was performed on 376 S. aureus strains of which 97% were methicillin resistant (MRSA). Among the 1,128 RAPD patterns generated, 66 were selected which identified 124 unique DNA fragments. From these amplicons, only 12% turned out to be useful for isolate-specific binary typing. The nature of the RAPD-generated DNA fragments was investigated by partial DNA sequence analysis. Several homologies with known S. aureus sequences and with genes from other species were discovered; however, 87% of the probe sequences are of previously unknown origin. The locations of most of the DNA probes on the chromosome of S. aureus NCTC 8325 were determined by hybridization. Seven fragments were randomly dispersed along the genome, five were clustered within the 2500- to 2600-kb position of the genome, and the remaining four did not recognize complementary sequences in S. aureus NCTC 8325. A total of 103 S. aureus strains (69% MRSA) were used for the validation of the binary typing technique. The 15 DNA probes provided stable epidemiological markers, both in vitro (type consistency after serial passages on culture media) and in vivo (comparison of sequential isolates recovered from cases of persistent colonization). The discriminatory power of binary typing (D = 0.998) exceeded that of pulsed-field gel electrophoresis (D = 0.966) and RAPD analysis (D = 0.949). Reproducibility, measured by analyzing multiple strains belonging to a multitude of different epidemiological clusters, was comparable to that of other genotyping techniques used. Contribution of the DNA probes to the discriminatory power of the system was analyzed by comparison of dendrograms. This study demonstrates that binary typing is a robust tool for the genetic typing of S. aureus isolates.

Base Sequence↗

PubMed2007

Evidence for a pseudo-outbreak of Candida guilliermondii fungemia in a university hospital in Brazil.

Fungal infections due to Candida species represent an important cause of nosocomial bloodstream infections. We report a large pseudo-outbreak of Candida guilliermondii fungemia that occurred in a university hospital in Brazil. C. guilliermondii was identified in 64 (43%) of the 149 blood samples drawn between June 2003 and July 2004. The samples were from patients in different wards of the hospital but concentrated in pediatric units. None of the patients had clinical signs of fungemia, and observational analysis revealed errors in the collection of blood samples. During the investigation of the pseudo-outbreak, C. guilliermondii was isolated from environmental surfaces and from the skin and nails of members of the nursing team. Through a subtyping analysis it was found that some of the nonpatient isolates were highly related to the patient isolates, and all the patient isolates were highly related. This is consistent with the hypothesis that the pseudo-outbreak was from a limited number of common sources. The adoption of intervention measures was effective in resolving the outbreak, supporting the hypothesis that the outbreak was due to poor techniques of drawing blood samples for culture.

Blood↗

PubMedDate not supplied

Correlation among phenotypical and molecular techniques in comparing ascomycetous yeast type strains.

Different phenotypical or molecular techniques can be used to describe and classify microorganisms for taxonomic, phylogenetic or genetic purposes. In yeast taxonomy the official hierarchic classification, based on morphological and physiological characters, is used together with more convenient molecular techniques such as the DNA sequencing. The question on whether these procedures produce coherent classifications is critical both to interpret taxonomic data consistently and to outline species correctly. In this paper, a set of type strains from the major genera of the budding hemiascomycetes yeast is examined with a series of physiological and molecular techniques, widely employed in taxonomy, in order to compare the among-strains correlations obtained with different methods. Results showed that the level of correlation among different techniques is relatively low, showing that different classifications and species organization could be obtained with diverse approaches. This is particularly interesting, considering that the official description of the yeast species is based on characters different from those becoming increasingly popular in the routine identification.

Animals↗

PubMed2001

Diversity of Geotrichum candidum strains isolated from traditional cheesemaking fabrications in France.

The diversity of French fungus-ripened cheeses is due partly to the succession of fungi that colonize the cheese during ripening. Geotrichum candidum appears in the early stages of ripening on soft cheeses such as Camembert and semihard cheeses such as St. Nectaire and Reblochon. Its lipases and proteases promote flavor development, and its aminopeptidases reduce bitterness imparted by low-molecular-weight peptides in cheese. We assessed the genetic diversity of G. candidum strains by using random amplification of polymorphic DNA (RAPD)-PCR correlated with phenotypic tests for carbon assimilation and salt tolerance. Strains were isolated from milk, curd, and cheese collected in seven major cheesemaking regions of France. Sixty-four isolates were characterized. We found high genetic diversity of G. candidum even within the same cheesemaking regions. Strains did not group according to region. All of the strains from the Haute-Savoie were able to assimilate lactate as the sole source of carbon, while lactate assimilation varied among strains from the Auvergne. Strains varied in D-mannitol assimilation, and none used citrate as the sole source of carbon. Yeast-like colony morphology predominated in Reblochon, while all of the strains isolated from St. Nectaire were filamentous. The RAPD-PCR technique readily differentiated Geotrichum fragrans isolated from milk and curd in a St. Nectaire cheesemaking facility. This study reveals an enormous diversity of G. candidum that has been empirically selected through the centuries by the cheesemakers of France.

Animals↗

PubMed2000

[AFLP analysis of photoperiod-sensitive genic male sterile(PGMS) rice mutant lines].

The reaction conditions for rice AFLP assay were optimized. The relative efficiencies for polymorphism detection of RFLP, RAPD and AFLP were compared through the analysis between a pair of PGMS allelic mutant lines(NK58S and NK58F). Results indicated that the efficiency for polymorphism detection in rice is in the order of AFLP > RAPD > RFLP, and also indicated that AFLP is a powerful DNA molecular marker technique for polymorphism detection, especially in the cases of extremely low polymorphism, such as isogeneic lines and allelic mutant lines. The advantages and disadvantages of these three molecular marker systems were discussed. Using AFLP in conjunction with bulked segregating analysis, 5106 AFLP loci were screened and 9 of them showed polymorphism between NK58S and NK58F, 4 of the polymorphic AFLP products were cloned, Southern bloting analysis showed that two of them were single copy sequences while the other two were low copy sequences in rice genome.

Mutation↗