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At least 199 records · Page 11Linked to original sources
PubMed2004

High-resolution genotyping of Listeria monocytogenes by fluorescent amplified fragment length polymorphism analysis compared to pulsed-field gel electrophoresis, random amplified polymorphic DNA analysis, ribotyping, and PCR-restriction fragment length polymorphism analysis.

The purpose of this study was to evaluate fluorescent amplified fragment length polymorphism (AFLP) analysis for the inter- and intraspecies differentiation of a collection of 96 strains of Listeria monocytogenes and 10 non-L. monocytogenes strains representing six other Listeria species of different origin. The AFLP technique was compared with three other molecular typing methods--ribotyping, random amplified polymorphic DNA analysis (RAPD), and pulsed-field gel electrophoresis (PFGE)--in terms of discriminatory ability. PCR-restriction fragment length polymorphism was included for virulence gene allele characterization. The 96 L. monocytogenes strains were divided into two major clusters by AFLP fingerprinting at a similarity level of 82% in concordance with the results of PFGE, RAPD, and ribotyping. One main cluster consisted of all of the 24 L. monocytogenes hly allele 1 strains, while another main cluster consisted of all of the 72 L. monocytogenes hly allele 2 strains. This indicates the existence of two distinct phylogenetic divisions. Isolates of the remaining Listeria species were not included in the clusters. AFLP, PFGE, and RAPD typing were highly discriminatory methods, with discrimination (D) indices of 0.974, 0.969, and 0.954, respectively, whereas ribotyping had a lower D index of 0.874. AFLP, PFGE, and RAPD typing showed some level of agreement in terms of strain grouping and differentiation. However, all three methods subdivided types of strains grouped by the other methods. Isolates with identical DNA profiles were distributed across the spectrum of origin. It was not possible to associate certain types with specific food sectors or clinical cases, which is indicative of the spread of L. monocytogenes clones across species. Overall, AFLP fingerprinting was suitable for the high-resolution genotyping of L. monocytogenes and had an equally high or higher differentiation power compared to PFGE or RAPD typing.

Cluster Analysis↗
PubMed1993

[Fluorocytometric DNA analysis of intestinal metaplasia associated with gastric carcinoma].

Histopathologic studies and fluorocytometric DNA analysis were carried out in 39 patients with intestinal metaplasia (IM) of the mucosa associated with gastric carcinoma, in order to determine possible relationships. According to the histological degree of IM, 39 cases were divided into 16 (41%) with slight-to-moderate, and 23 (59%) with severe IM. The 39 tumors were histologically divided into 20 (51%) differentiated tumors and 19 (49%) undifferentiated tumors. The DNA patterns of tumors revealed 17 (43%) to be euploidy and 22 (57%) to be aneuploidy. The DNA content of the metaplastic epithelial cells was measured, and the percentage of the cells in S and G2 + M phases of the cell cycle was taken as the index of proliferative activity. DNA analysis of the non-metaplastic epithelium was performed in 10 as a control group. While the histological differentiation of the tumors and the degree of IM were not related to the proliferative activity of IM, the IM around the aneuploid tumors did have higher proliferative activity (14.01 +/- 6.16%) than that around euploid tumors (11.08 +/- 7.13%) or of normal mucosa (9.95 +/- 9.92%). This approach will help to determine cell kinetics of IM around the gastric tumor. Our study indicated a relationship between DNA ploidy and the proliferative activity of IM around gastric cancer.

Cell Cycle↗
PubMed1994

Inter-institutional reproducibility of flow cytometric DNA-analysis in breast carcinomas.

In order to study interinstitutional reproducibility of flow cytometric DNA-analysis (DNA-FCM), frozen pieces from 30 consecutive breast carcinomas were analysed by 5 laboratories. Different instruments, preparation and DNA staining methods were used. A concordance in DNA-ploidy status was obtained in 26 of the 30 tumours. The discrepancy can mainly be explained by intratumoural DNA-heterogeneity since a complete agreement in ploidy status was obtained when four of the laboratories analysed the same cell suspension, where solid bits showed differing results. The sampling method seems therefore to be a crucial step for the results and needs further studies. As far as the estimation of S-phase fraction was concerned, one laboratory obtained significantly higher values compared to the other four. The correlation between the other four laboratories varied between r = 0.66-0.92.

Academies and Institutes↗
PubMed1984

Cytophotometric DNA analysis of mucosal and submucosal carcinoma of the esophagus.

Cell nuclear deoxyribonucleic acid (DNA) content was microspectrophotometrically determined in 35 cases of mucosal and submucosal carcinoma of the esophagus. DNA distribution pattern was classified into types I, II, III, and IV, according to the degree of dispersion on the DNA histogram, in the order of wider distribution. Patients with types I and II (relatively regular in DNA distribution) had an uneventful postoperative course and no recurrence, whereas 3 of 15 (20%), and 5 of 9 (55.6%) with type III and type IV, respectively (widely scattered DNA distribution), died following a recurrence. Cytophotometric DNA analysis of the cancer cells reflected well the outcome in patients with esophageal carcinoma. These results suggest the potential usefulness of cytophotometric DNA analysis for assessing the prognosis, even in the early stage of esophageal carcinoma.

Adult↗
PubMed1994

First trimester prenatal diagnosis of Menkes disease by DNA analysis.

Menkes disease is an X linked recessive disorder of copper metabolism characterised by neurological symptoms and connective tissue manifestations. The defective gene in Menkes disease has recently been isolated and the gene product is predicted to be a copper transporting ATPase. The diagnosis of Menkes disease has hitherto been performed by biochemical analysis, based on intracellular accumulation of copper. Cloning the gene opened up the possibility of establishing precise and reliable carrier and prenatal diagnosis by defining the molecular defect. In this report we describe the partial deletion of the Menkes gene in a patient who had inherited the mutation from his phenotypically normal mother. This information enabled us to perform prenatal diagnosis by direct mutation analysis of the mother's sixth pregnancy and we detected the same deletion, indicating that the male fetus was affected. This first prenatal diagnosis of Menkes disease by direct mutation analysis shows some advantages of DNA analysis compared to biochemical diagnosis.

Abortion, Induced↗
PubMed1991

Genetic markers in human bone: I. Deoxyribonucleic acid (DNA) analysis.

Deoxyribonucleic acid (DNA) was isolated from a number of spongy and compact human bone tissue specimens, and the yield was estimated on a "per milligram of starting tissue" basis. DNA was, in addition, isolated from a number of corresponding blood and bone tissue specimens. Spectrophotofluorometry and ethidium bromide visualization on minigels were used to estimate the quantity and degree of degradation of DNA. The DNA from several blood-bone pairs is shown to give concordant restriction fragment length polymorphism (RFLP) typing results by two different typing protocols with five different single-locus probes. DNA from several additional blood-bone pairs is shown to give concordant results for human leucocyte antigen (HLA)-DQ alpha phenotypes following polymerase chain reaction (PCR) amplification and hybridization to specific allele-specific oligonucleotide (ASO) probes, and for the variable numbers of tandem repeats (VNTR) length polymorphisms 3' to the human apolipoprotein B (APOB) gene following PCR amplification with specific primers and analysis of the products by electrophoresis and ethidium bromide visualization.

Base Sequence↗
PubMed2002

Traces of early Eurasians in the Mansi of northwest Siberia revealed by mitochondrial DNA analysis.

The mitochondrial DNA (mtDNA) of 98 Mansi, an ancient group (formerly known as "Vogul") of Uralic-speaking fishers and hunters on the eastern slope of the northern Ural Mountains, were analyzed for sequence variants by restriction fragment--length polymorphism analysis, control-region sequencing, and sequencing of additional informative sites in the coding region. Although 63.3% of the mtDNA detected in the Mansi falls into western Eurasian lineages (e.g., haplogroups UK, TJ, and HV), the remaining 36.7% encompass a subset of eastern Eurasian lineages (e.g., haplogroups A, C, D, F, G, and M). Among the western Eurasian lineages, subhaplogroup U4 was found at a remarkable frequency of 16.3%, along with lineages U5, U7, and J2. This suggests that the aboriginal populations residing immediately to the east of the Ural Mountains may encompass remnants of the early Upper Paleolithic expansion from the Middle East/southeastern Europe. The added presence of eastern Eurasian mtDNA lineages in the Mansi introduces the possibilities that proto-Eurasians encompassed a range of macrohaplogroup M and N lineages that subsequently became geographically distributed and that the Paleolithic expansion may have reached this part of Siberia before it split into western and eastern human groups.

Asia↗
PubMed2002

A simple DNA disc chip in a microarray design based on modified comparative genomic hybridization for sperm DNA analysis.

OBJECTIVE: A DNA disc chip assay, based on comparative genomic hybridization, was designed to measure changes in sperm DNA intensities. The objective was to analyze the DNA integrity of hyperactive sperm cells after mild heat treatment. DESIGN: The assay based on a multiple cell comet assay was used to analyze changes in genomic DNA. Washed sperm DNA were tested on the assay and images stored in a microarray design. SETTING: Clinical and academic research environment. PATIENT(S): Frozen-thawed washed sperm from different donors (n = 7). INTERVENTION(S): Discarded sperm leftover from trial washes carried out at 37 degrees and 40 degrees C were frozen and processed for the DNA disc chip assay. MAIN OUTCOME MEASURE(S): Fluorescent intensities of DNA disc chips and sperm variables. RESULT(S): Heat treatment resulted in more than eightfold increase in sperm hyperactive motility with little degradation in DNA integrity. Sperm with low hyperactivation was associated with alterations in DNA after heat treatment. CONCLUSION(S): The DNA disc chip assay was simple, inexpensive, and permitted assisted reproduction technologies laboratories to use comparative genomic hybridization for cytogenotoxicity testing. However, the assay required manual processing, a fluorescent microscope, and computer. The data showed an association between sperm hyperactivation and DNA integrity suggesting that the hyperactivation marker may be used for selecting quality sperm for intracytoplasmic sperm injection. More studies are needed to examine temperature effects on ejaculated human sperm.

DNA↗
PubMed1998

Geographic discrimination of Paracoccidioides brasiliensis strains by randomly amplified polymorphic DNA analysis.

Randomly amplified polymorphic DNA (RAPD) analysis of 33 Paracoccidioides brasiliensis strains from Argentina, Brazil, Colombia, Peru, and Venezuela produced reproducible amplification products which were sufficiently polymorphic to allow differentiation of the strains. Types generated with five primers (OPG 03, OPG 05, OPG 14, OPG 16, and OPG 18) resulted in a high discriminatory index (0.956). The discriminatory index was slightly reduced (0.940) when only two primers (OPG 3 and OPG 14) were used. A dendrogram based on these results showed a high degree of similarity among the strains, and genetic differences were expressed in clusters related to geographical regions but not to pathological features of the disease. With a few exceptions, strains were sorted into five groups by geographical origin as follows: group I, Venezuelan strains; group II, Brazilian strains; group III, Peruvian strains; group IV, Colombian strains; and group V, Argentinian strains. The group containing the most disparate strains was group V (discriminatory index, 0.633); the discriminatory index for the other four groups was 0.824. The use of primer OPG 18 by itself was sufficient to discriminate species specificity, and the use of primer OPG 14 by itself was sufficient to discriminate among the geographical locations of the strains in the sample. This method may be helpful for epidemiological studies of P. brasiliensis.

DNA, Fungal↗
PubMed1998

Genetic structure in wild populations of black tiger shrimp (Penaeus monodon) using randomly amplified polymorphic DNA analysis.

Randomly amplified polymorphic DNA (RAPD) analysis was used to examine genetic variation in wild black tiger shrimp, Penaeus monodon. Specimens were collected from five geographically separated locations (Satun-Trang, Phangnga, and Medan in the Andaman Sea and Chumphon and Trad in the Gulf of Thailand). A total of 100 P. monodon individuals were investigated using seven arbitrarily selected primers. Fifty-eight (72.5%) of eighty reproducible RAPD fragments ranging in size from 200 to 2200 bp were polymorphic. The percentages of polymorphic bands of the five geographic populations investigated varied from 51.5 to 57.7%. The genetic distance between populations and UPGMA dendrograms indicated that the Medan population was genetically different from Thai P. monodon (Dij = 14.976%). Within Thailand, the Satun-Trang P. monodon was separated from the remaining geographic populations with a genetic distance of 2.632%. RAPD analysis in the present study yielded a total of 252 genotypes. A Monte Carlo analysis illustrated geographic heterogeneity in genotype frequencies within this species, suggesting that genetic population structure does exist in this taxon (P < 0.001 for all primers). Signficant differences in genotype frequencies between Thai and Indonesian (Medan) P. monodon were observed (P < 0.0001). Within Thailand, the Andaman Sea P. monodon was significantly different from that of the Gulf of Thailand (P values between 0.0000 and 0.0387), indicating population differentiation between P. monodon from these two main fishery regions of Thailand.

Journal Article↗
PubMed1998

Cytoplasmic diversity in leaf beet cultivars as revealed by mitochondrial DNA analysis.

Mitochondrial (mt) DNA restriction fragment length polymorphisms are convenient markers for identifying cytoplasmic variation among plant cultivars. In an attempt to detect new cytoplasmic genotypes useful for sugarbeet breeding, we have compared the hybridization patterns of mtDNA from three groups of cultivated beets, viz. leaf beet, garden beet, and fodder beet. Utilized as probes were the two sugarbeet mtDNA clones that were capable of distinguishing normal fertile and different sources of male-sterile cytoplasms from one another. The analysis allowed the identification of four chondriome types among 14 leaf beet cultivars examined. Two out of the four chondriome types were found to be different from the previously described fertile or male-sterile chondriome type. Our results thus indicate that leaf beet cultivars and landraces make up the primary cytoplasmic gene pool of the sugarbeet.

Base Sequence↗
PubMed1995

Flow cytometric immunofluorescence and DNA analysis: using a 1.5 mW helium-neon laser (544 nm).

We evaluate a 1.5 mW HeNe laser (544 nm) for use on an EPICS Elite with a 76 microns Sortsense flow cell. The two applications chosen were immunofluorescence and DNA analysis. We measured the fluorescence threshhold of phycoerytherin calibration beads to be approximately 336 MESF. Cell analysis with a HeNe laser and Argon laser correlated well for the CD4PE, CD56PE, CD19PE conjugates, with correlation coefficients of 0.98, 0.99, 0.94, respectively. The % positive and mean channel fluorescence were comparable to the results obtained with a 15 mW Argon laser. In addition, a three-color configuration yielded excellent results. Cell analysis of CD4PE, CD3ECD and CD19Cy-Chrome with the HeNe laser and Argon laser correlated well with correlation coefficients of 0.96, 0.95, and 0.92, respectively. The histograms showed good separation between the negative cells, the dimly staining cells and the brightly staining cells. Propidium Iodide was chosen for DNA analysis. Full CV values for whole blood DNA fluorescence using the green laser were good at 2.6%. These data indicate the low power 544 nm laser is sufficient to do immunophenotyping and DNA analysis. Results may be explained by higher quantum efficiency and lower background fluorescence. The wavelength of the 544 nm laser is much closer to the excitation peaks of PI, PE, and the tandem dyes ECD and Cy-Chrome. Also, the Raman scattering of water for the 544 nm laser has a longer wavelength maximum than the emission peaks of PI, PE, and ECD. The major advantages of this laser for the research laboratory are small size, no cooling fan, low power requirements and low cost.

Adult↗
PubMed1990

DNA analysis of multiple synchronous renal cell carcinomas.

The authors used retrospective quantitative DNA analysis to study interrelationships between multiple synchronous renal cell carcinomas in seven patients. DNA content was determined by image analysis on Feulgen-stained nuclear smears prepared from multiple paraffin blocks from each tumor. Tumors were unilateral in four cases and bilateral in three. Ten tumors had homogeneous, and four heterogeneous DNA stemlines. Intertumoral heterogeneity in four cases suggested multifocal origin. Identical DNA aneuploid indices in bilateral tumors in one case suggested metastasis from a solitary origin. Abnormal DNA content and heterogeneous populations began to appear in the size range 2.0 to 5.0 cm. All tumors over 5.0 cm contained nondiploid populations. Although the interrelationships between these multiple synchronous neoplasms are not entirely clear, the DNA analysis suggests that the occurrence of nondiploid stemlines and heterogeneous DNA content may parallel both tumor growth and more aggressive behavior.

Adult↗
PubMed2000

An autopsy case of Klinefelter's syndrome suspected and its DNA analysis.

UNLABELLED: We experienced an autopsy case, small testes and tall stature, which suggested Klinefelter's syndrome. DNA analysis was performed to confirm the genetic abnormality. CASE HISTORY: A 28-year-old man who was single and lived with his parents. He suddenly lost his consciousness in a sitting room and died. Autopsy findings: He was 176 cm in height and 57 kg in weight. The post-mortem hypostasis was red-purple on his back, and rigor mortis was strong in each joint of the whole body. The heart weighted 340 g, in which dark red fluidal blood (300 ml) without coagulation was contained. The testes were smaller than normal adult male (left and right testes with epididymides weighted 8.1 g and 6.0 g, respectively). As a results of pathological examination, clumped Leydig cells, sclerotic and hyalined tubules were observed. Some germ cells with spermatozoid were also present. DNA Analysis: Generally, Klinefelter's syndrome is determined by karyotype analysis and/or the detection of sex chromatin. However, in this case, karyotype analysis and the detection of sex chromatin could not be demonstrated, because the blood which was collected in the autopsy became too old. Therefore, we tried sex determination and STR analysis (HPRT, HUMARA and DXS 1470) using DNA extracted from stored blood materials. Consequently, in the sex determination, no different situation was found in the X- and Y-specific bands from normal male's and as results of STR analyses, we could not corroborate the Klinefelter's syndrome.

Adult↗
PubMed2000

Use of an automated DNA analysis system (DARAS) for sequence-specific recognition of Neisseria meningitidis DNA.

OBJECTIVES: To combine use of the polymerase chain reaction (PCR) for rapid diagnosis of meningococcal meningitis with a novel automated detection system for sequence-specific recognition of PCR products. METHODS: DNA was extracted from cerebrospinal fluid (CSF) by a quick boil-lysis method, followed by PCR with primers specific for Neisseria meningitidis. Sequence-specific recognition of N. meningitidis DNA was performed with an automated DNA analysis system (DARAS) and the data were compared with results following agarose gel electrophoresis or conventional microbiological culture. RESULTS: The DARAS system had a sensitive detection limit of 102 meningococci/mL with spiked samples, compared with a detection limit of 104 meningococci/mL following agarose gel electrophoresis. When the system was used to examine 74 CSF samples, the 19 CSF samples positive for N. meningitidis by conventional microbiological methods were also all positive in the DARAS system and the 55 samples negative by DARAS for meningococci were also negative by conventional microbiological methods. CONCLUSION: The sensitivity and specificity of the DARAS system makes it a useful tool for the diagnosis of meningococcal meningitis. The system is user-friendly, requires minimal hands-on time and generates data in an informative numerical format.

Colony Count, Microbial↗
PubMed1985

Hemophilia A. Detection of molecular defects and of carriers by DNA analysis.

To understand the molecular basis of hemophilia A and to provide heterozygote detection and prenatal diagnosis by DNA analysis, we used cloned factor VIII:C DNA fragments to study 10 affected families. In four of these families, inhibitors of factor VIII:C had developed in affected persons. In one such family a deletion of approximately 80 kb within the factor VIII:C gene was identified. Carriers of the deletion were identified through detection of an abnormal DNA fragment located at the deletion end points. In another family a single nucleotide change in the coding region of the factor VIII:C gene produced a nonsense codon leading to premature termination of factor VIII:C synthesis. Carrier detection was performed in eight female members of this four-generation family. In a third family a small change in the size of a restriction-endonuclease fragment correlated with the presence of the mutant gene, and in the other seven families the molecular defect has not yet been identified. In addition, we used two common polymorphic sites in the factor VIII:C gene to differentiate the normal from the defective gene in four of six obligate female carriers from families with patients in whom inhibitors did not develop. Carrier detection was possible in other members of these families. These data suggest that DNA analysis of the factor VIII:C gene provides an accurate method of carrier detection and, potentially, of prenatal diagnosis in at least 50 per cent of the pedigrees affected by hemophilia A.

Chromosome Deletion↗
PubMed1996

Analysis of Vibrio cholerae O139 Bengal isolated from different geographical areas using macrorestriction DNA analysis.

Vibrio cholerae O139 isolated from different countries, as well as from different locations within a country, were examined using macrorestriction DNA analysis to determine the clonality of the O139 strains. NotI digests of genomic DNA of representative strains from Nepal, India, Bangladesh, China, Thailand, and Malaysia revealed very similar but not identical patterns. Examinations of the banding patterns generated by pulsed-field gel electrophoresis of strains isolated within countries revealed complete homogeneity. These results further reiterate the spread of an identical clone of V. cholerae O139 although it appears that genetic polymorphism among the O139 strains is becoming apparent.

Asia↗