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At least 775 records · Page 43Linked to original sources
PubMed1998

Clonal nature of enterotoxigenic Escherichia coli serotype O6:H16 revealed by randomly amplified polymorphic DNA analysis.

The genetic relatedness among 29 enterotoxigenic Escherichia coli strains of serotype O6:H16 was investigated by randomly amplified polymorphic DNA (RAPD) analysis. The strains were isolated in different parts of the world, displayed CS1-CS3 or CS2-CS3 profiles, and expressed heat-labile toxin (LT) and heat-stable toxin; a single strain expressed only LT. Ten RAPD types were distinguished and showed significant similarity, having on average 82% of the amplified bands in common. These results indicated that, irrespective of the different geographical origin or virulence factors, these strains belonged to a widespread clonal group.

Bacterial Toxins↗
PubMed1995

Efficient discrimination of Mycobacterium tuberculosis strains by 16S-23S spacer region-based random amplified polymorphic DNA analysis.

Amplification of the region separating the genes coding for 16S and 23S rRNA was performed with 15 Mycobacterium tuberculosis isolates and the type strain, ATCC 27294. Reproducible amplification patterns were obtained. PCR products were then used as target DNA for random amplified polymorphic DNA (RAPD) analysis. The discriminatory power was higher than when whole genomic DNA was used as a RAPD template. 16S-23S spacer region-based RAPD analysis was a simple and efficient method of differentiation. Consequently, it may be a useful tool for epidemiologic studies of tuberculosis.

Bacteriological Techniques↗
PubMed1991

Semiautomation of preparation of fixed paraffin-embedded tissue for DNA analysis.

The usual manual preparation of single-cell suspensions from fixed paraffin-embedded tissue sections for flow cytometric (FCM) DNA ploidy analysis is a time-consuming, labor-intensive technique that requires 70 minutes to deparaffinize and rehydrate 50 microns sections as the initial step. Manual deparaffinization was compared with two semiautomated methods using an automatic slide stainer with either a 70-minute or 35-minute schedule. Samples from 6 normal tissues and 21 tumors (13 diploid and 8 aneuploid) were prepared using all three methods and analyzed by FCM. The mean cell counts in all samples were over 10(6). The DNA indices for the three samples prepared from a given tissue showed no significant differences. Using the 70-minute automation schedule, no aneuploid peaks were lost, and the ratio of G0G1 normal cells to aneuploid tumor cells was maintained. The automation of deparaffinization can thus provide a significant reduction in the labor need to produce single-cell suspensions for FCM; it can be especially helpful when handling large numbers of tumors. At the same time, the automated procedure decreases the exposure to hazardous chemicals and lowers the chance of losing tissue.

DNA↗
PubMed2006

Random amplified polymorphic DNA analysis of genetically modified organisms.

Randomly amplified polymorphic DNA (RAPD) was used to analyzed 78 samples comprises of certified reference materials (soya and maize powder), raw seeds (soybean and maize), processed food and animal feed. Combination assay of two arbitrary primers in the RAPD analysis enable to distinguish genetically modified organism (GMO) reference materials from the samples tested. Dendrogram analysis revealed 13 clusters at 45% similarity from the RAPD. RAPD analysis showed that the maize and soybean samples were clustered differently besides the GMO and non-GMO products.

Food Analysis↗
PubMed1997

Random amplified polymorphic DNA analysis and variation of essential oil components of Atractylodes plants.

Total DNAs were prepared from the leaves of Atractylodes lancea DE CANDOLLE, A. chinensis KOIDZUMI, A. lancea var. simplicifolia KITAMURA, A. japonica KOIDZUMI ex KITAMURA and A. ovata DE CANDOLLE. The DNAs were subjected to random amplified polymorphic DNA (RAPD) analysis. Some primers showed the definitive polymorphic DNA patterns in A. lancea, A. japonica and A. ovata. The RAPD of A. lancea var. simplicifolia and one of A. chinensis gave similar patterns to those of A. lancea, but one of the other A. chinensis gave a similar pattern to A. japonica. Furthermore, quantitative analysis of atractylon, hinesol, beta-eudesmol and atractylodin in the rhizomes was done using gas chromatography. Though atractylon was detected not only in A. japonica and A. ovata but also in some of A. lancea, their RAPD profiles revealed the presence of intraspecific variation with A. lancea.

Chromatography, Gas↗
PubMed1994

Genome DNA analysis and genotyping of clinical isolates of Helicobacter pylori.

The genotype of genome DNA from seventeen clinical isolates of Helicobacter pylori by pulsed-field gel electrophoresis (PFGE) and arbitrarily primed polymerase chain reaction (AP-PCR) was determined. Three restriction enzymes, Apa I, Kpn I and Not I, were found to produce distributions of DNA fragments which were useful for analysis of the chromosome-sized DNA from H. pylori NCTC11637T by PFGE. Many of the isolates of H. pylori could not be genotyped by PFGE after digestion reaction with Apa I, Kpn I, and Not I. When AP-PCR with a ten-nucleotide primer was performed using the chromosomal genomic DNA from the isolates as templates, fifteen distinctly different profiles were obtained from the seventeen isolates. Thus, genotyping of the isolates was possible where PFGE profiles had not previously been informative. The results suggest that AP-PCR is more suitable for genotyping of clinical isolates of H. pylori in Japan than PFGE.

DNA, Bacterial↗
PubMed1997

DNA analysis of the androgen receptor gene in two cases with complete androgen insensitivity syndrome.

OBJECTIVE: Androgen insensitivity syndrome is an X-linked disorder of sexual differentiation resulting from abnormalities of the androgen receptor gene. In this study, we analyzed the androgen receptor gene in 2 cases with complete androgen insensitivity syndrome (CAIS). METHODS: DNAs were isolated from patients with CAIS, and the androgen receptor gene was amplified by a polymerase chain reaction. Sequence analysis of the androgen receptor gene was performed. RESULTS: In Patient 1, one substitutional mutation [glutamine (CAA) to arginine (CGA) at position 194] was identified in exon A, and the premature termination of the androgen receptor gene was also demonstrated due to the deletion of one nucleotide at the codon in exon C (position 597). In Patient 2, one substitutional mutation [arginine (CGC) to cysteine (TGC) at position 855] in exon G was identified. This position was located in the hormone-binding domain and appeared to be a hot spot of mutations because the mutations at the same position have been reported before in several unrelated cases. CONCLUSION: The results of this study suggest that these abnormalities might be related to the pathogenesis of complete androgen insensitivity syndrome.

Adult↗
PubMed2000

Genome-directed primers for selective labeling of bacterial transcripts for DNA microarray analysis.

DNA microarrays have the ability to analyze the expression of thousands of the same set of genes under at least two different experimental conditions. However, DNA microarrays require substantial amounts of RNA to generate the probes, especially when bacterial RNA is used for hybridization (50 microg of bacterial total RNA contains approximately 2 microg of mRNA). We have developed a computer-based algorithm for prediction of the minimal number of primers to specifically anneal to all genes in a given genome. The algorithm predicts, for example, that 37 oligonucleotides should prime all genes in the Mycobacterium tuberculosis genome. We tested the usefulness of the genome-directed primers (GDPs) in comparison to random primers for gene expression profiling using DNA microarrays. Both types of primers were used to generate fluorescent-labeled probes and to hybridize to an array of 960 mycobacterial genes. Compared to random-primer probes, the GDP probes were more sensitive and more specific, especially when mammalian RNA samples were spiked with mycobacterial RNA. The GDPs were used for gene expression profiling of mycobacterial cultures grown to early log or stationary growth phases. This approach could be useful for accurate genome-wide expression analysis, especially for in vivo gene expression profiling, as well as directed amplification of sequenced genomes.

Algorithms↗
PubMed1999

Molecular typing of vibrio vulnificus isolated from clinical specimens by pulsed-field gel electrophoresis and random amplified polymorphic DNA analysis.

Primary V. vulnificus septicemia has been continuously reported in Korea. We analyzed the molecular diversity of V. vulnificus strains isolated from clinical specimens using pulsed-field gel electrophoresis (PFGE) and random amplified polymorphic DNA (RAPD) analysis. We analyzed a total of 23 V. vulnificus strains isolated from 22 patients between 1992 and 1997. RAPD analysis was performed using five random primers, and we obtained chromosomal DNA restriction patterns with NotI and SfiI by PFGE. Two isolates from one patient disclosed similarity value 1.0 by RAPD and had an indistinguishable pattern when analyzed with PFGE, which indicated that they were the same strains. The remaining 22 isolates from 22 patients could be separated into 5 different molecular types in RAPD analysis. The range of similarity values among the isolates was wide (0.29-0.81). Of 22 strains, four strains could not be typed in repeated trials by PFGE with NotI and SfiI. The PFGE patterns of 18 strains showed a remarkable polymorphism consisting 12-19 fragments (20-870 kb). These results show that V. vulnificus strains isolated from Korea are genetically diversified for the most part.

Adult↗
PubMed1988

Flow cytometry DNA analysis on tumor cell subpopulation of human tumor specimens by exclusion of lymphohemopoietic cells.

We describe a new flow cytometry procedure in which DNA analyses can be obtained selectively on pure, freshly obtained tumor cell subpopulations of human tumor specimens. This procedure is based on exclusion from analysis of the contaminating lymphohemopoietic cells mixed with tumor cells in tumor specimens. This exclusion is made possible by labeling all lymphohemopoietic cells with an antibody to HLe-1 (HLE), which is present on all lymphohemopoietic cells but on no other cells, and by gating against these labeled cells when analyzing for DNA. For the model system, a 1:1 mixture of normal human peripheral blood leukocytes and either of two human cancer cell lines, HEp-2 and MCF-7, normal leukocyte contamination can be reduced to 3.1% while retaining 94.7% of tumor cells for DNA analysis. Four examples of human tumor samples, two cases each of malignant effusions and lymph node metastases, were analyzed with this procedure. The results clearly indicate that this new method will improve ploidy analysis/aneuploidy detection and will make it possible to obtain more accurate cell-cycle analyses of tumor cells than have previously been possible. This new procedure will contribute to clinical and biological studies involving DNA of human tumors.

Adenocarcinoma↗
PubMedDate not supplied

Cytogenetic and corresponding flow cytometric DNA analysis of renal cell neoplasms.

Results of flow cytometric DNA content and cytogenetic analyses of six neoplasms representing the spectrum of the morphologic subtypes of renal cell neoplasms are compared. Flow cytometric determinations of DNA diploidy and near diploidy in two renal cortical neoplasms correlated with modal chromosome numbers; however, in three of four neoplasms with a highly aneuploid DNA content, the modal chromosome numbers indicated diploidy or near-diploidy. Our data suggest that the short-term culture conditions used in cytogenetic analysis may favor growth of cells with diploid or near-diploid DNA content. This may explain the discrepancy frequently observed between results obtained using flow cytometry and karyotyping after short-term culture. We also observed (1) abnormalities in the short arm of chromosome 3 in all three nonpapillary neoplasms, (both clear and granular cell types); (2) aberrations in chromosomes 3 and 17 in the two papillary tumors; and (3) normal chromosome 3 in the presence of various random karyotypic anomalies including telomeric associations and a pseudo-diploid modal chromosome number in the only oncocytic neoplasm studied.

Aneuploidy↗
PubMed1999

Genetic diversity among Escherichia coli O149:K91 strains isolated from pigs with diarrhoea determined by randomly amplified polymorphic DNA analysis.

The genetic relatedness among 72 Escherichia coli strains of serotype O149:K91 isolated from pigs with diarrhoea was investigated by randomly amplified polymorphic DNA (RAPD) analysis. Fimbrial and toxic virulence markers of the isolates were also tested. Amplification with primer 1254 resulted in three different RAPD types whereas primer 1290 generated one RAPD profile only. Based on the RAPD and fimbrial/toxin types the strains were classified into five distinct groups.

Animals↗
PubMedDate not supplied

Evaluation of early marrow response in childhood aneuploid acute lymphoblastic leukemia: flow cytometric DNA analysis versus standard morphology.

Despite improved survival rates for childhood acute lymphoblastic leukemia (ALL), the relapse rate remains at 20-30%. Early peripheral blood and bone marrow (BM) responses have been associated with more favorable outcomes; all current children's cancer group (CCG) protocols for ALL require BM evaluation at days 7 and 14 with subsequent therapy based on the results. Morphologic interpretation of aspirate smears during induction chemotherapy is challenging, as the samples are often hypocellular, excessively friable, and cytologically altered by drugs. We have shown discordancy of day 7 and 14 BM lymphoblast counts using morphologic and flow cytometric immunophenotypic analyses (FC). The aim of our study was to determine the utility, reliability, and cost effectiveness of lymphoblast enumeration using DNA content analysis by flow cytometry (DNA-FC) and to further demonstrate the subjectivity of morphologic review. All new cases of ALL had DNA-FC and FC analyses. The percentage of lymphoblasts as determined by both methods was compared for 82 aneuploid cases. Three pathologists independently reviewed aspirate smears from 39 bone marrow samples of aneuploid ALL that were obtained during early induction. These results were compared among themselves and with the results obtained by DNA-FC. We found excellent correlation between the percentage of lymphoblasts as determined by DNA-FC and FC (R2 = 0.97) over a range of 0 to 99%. Pathologic review agreed with the DNA-FC, on average, 68%. The sensitivity, specificity, and positive and negative predictive values of morphologic review, averaged 53, 84, 78, and 63%, respectively, when using DNA-FC as the "gold standard." All three pathologists achieved agreement of lymphoblast percentage by morphology in 72%. In our laboratory, the use of DNA-FC equates to one-sixth the time and one-half the price of FC per exam. We have shown a strong correlation between blast counts determined by DNA-FC and FC. DNA-FC is an objective, economical, and reliable method to assess early response in induction marrows from aneuploid ALL where morphology is often uninterpretable. This test is highly reproducible and available at most pediatric institutions. Prospective studies need to be employed to evaluate the effect of more definitive methods (DNA-FC and FC) of assessing the early response in bone marrows on prognosis.

Aneuploidy↗
PubMed2001

Automated ribotyping and random amplified polymorphic DNA analysis for molecular typing of Salmonella enteritidis and Salmonella typhimurium strains isolated in Italy.

AIMS: The ability of automated ribotyping and random amplified polymorphic DNA (RAPD) analysis to differentiate Salmonella enteritidis and Salmonella typhimurium isolates in relation to their origin was evaluated. METHODS AND RESULTS: The restriction enzymes EcoRI, PvuII and PstI, and the random primers OPB17 and P1254, were tested for ribotyping and RAPD analysis, respectively. Seventeen subtypes were identified among the isolates of the two pathogenic Salmonella serovars using the RiboPrinter, and 25 subtypes using RAPD. CONCLUSIONS: The greatest degree of genetic diversity was observed among Salm. typhimurium isolates using both automated ribotyping (Simpson's index of discrimination 0878) and RAPD (Simpson's index of discrimination 0886). SIGNIFICANCE AND IMPACT OF THE STUDY: According to the results of this research, automated ribotyping and RAPD are two useful genotyping techniques for identifying unique and common subtypes associated with a specific source and location, and provide powerful tools for epidemiological investigations.

Animals↗
PubMed2000

Molecular diversity of Renibacterium salmoninarum isolates determined by randomly amplified polymorphic DNA analysis.

The molecular diversity among 60 isolates of Renibacterium salmoninarum which differ in place and date of isolation was investigated by using randomly amplified polymorphic DNA (RAPD) analysis. Isolates were grouped into 21 banding patterns which did not reflect the biological source. Four 16S-23S rRNA intergenic spacer (ITS1) sequence variations and two alleles of an exact tandem repeat locus, ETR-A, were the bases for formation of distinct groups within the RAPD clusters. This study provides evidence that the most common ITS1 sequence variant, SV1, possesses two copies of a 51-bp repeat unit at ETR-A and has been widely dispersed among countries which are associated with mainstream intensive salmonid culture.

Animals↗
PubMed1997

Minisequencing: a specific tool for DNA analysis and diagnostics on oligonucleotide arrays.

We describe a method for multiplex detection of mutations in which the solid-phase minisequencing principle is applied to an oligonucleotide array format. The mutations are detected by extending immobilized primers that anneal to their template sequences immediately adjacent to the mutant nucleotide positions with single labeled dideoxynucleoside triphosphates using a DNA polymerase. The arrays were prepared by coupling one primer per mutation to be detected on a small glass area. Genomic fragments spanning nine disease mutations, which were selected as targets for the assay, were amplified in multiplex PCR reactions and used as templates for the minisequencing reactions on the primer array. The genotypes of homozygous and heterozygous genomic DNA samples were unequivocally defined at each analyzed nucleotide position by the highly specific primer extension reaction. In a comparison to hybridization with immobilized allele-specific probes in the same assay format, the power of discrimination between homozygous and heterozygous genotypes was one order of magnitude higher using the minisequencing method. Therefore, single-nucleotide primer extension is a promising principle for future high-throughput mutation detection and genotyping using high density DNA-chip technology.

DNA-Directed DNA Polymerase↗
PubMed2000

Identification of mutans and other oral streptococci by random amplified polymorphic DNA analysis.

The identification and classification of the non-haemolytic or viridans group of streptococci have long been recognised as difficult and unsatisfactory. Phenotypic and genotypic heterogeneity have resulted in ambiguous speciation, particularly with mutans streptococci and other oral streptococci. This study was done to determine whether random amplified polymorphic DNA (RAPD) analysis is useful to identify and even classify oral and other streptococci. DNA was prepared and purified from 25 strains of mutans streptococci including 11 reference strains of Streptococcus mutans, seven of S. sobrinus, three of S. rattus and one each of the four other species of the mutans group, together with 20 other reference species, mostly streptococci, and from 49 fresh isolates of mutans streptococci and of S. mutans from human saliva and dental plaque. DNA amplification was primed with each of three arbitrarily selected primers nine or 10 nucleotides in length. The amplified DNA fragments (amplicons) obtained were compared by agarose gel electrophoresis. Species- and strain-specific RAPD fingerprints were obtained not only from pure genomic DNA, but also from the supernates of crude cellular or colony extracts. Pending the analysis of numerous other strains, the data suggest that RAPD may be of value: (i) to distinguish the species S. mutans and S. sobrinus from each other and potentially from other species of oral streptococci, (ii) to differentiate and possibly classify oral streptococci and (iii) as a valuable tool in mutans streptococci epidemiology and transmission studies, by virtue of its rapidity, efficiency and reproducibility in generating genetic fingerprints of streptococcal isolates.

Adult↗