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PubMedDate not supplied

Mitochondrial DNA analysis of Sporothrix schenckii in Costa Rica.

Mitochondrial DNA (mtDNA) types using restriction patterns with HaeIII were investigated in 21 clinical isolates of Sporothrix schenckii in Costa Rica. Nineteen isolates were found to be a new mtDNA type (Type 14), while the remaining two were Type 3 and Type 6. Based on the phylogeny of 14 mtDNA types constructed by estimating sequence divergences of mtDNA, S. schenckii were clustered into two main groups: Group A and Group B, with Type 14 and Type 3 included in Group A, and Type 6 in Group B. The results suggest that isolates of Group A may exist mainly in Central America including Costa Rica.

Costa Rica↗
PubMed1997

DNA analysis using an electrospray scanning mobility particle sizer.

A scanning mobility particle sizer (SMPS) allows size separation of gas phase particles according to their electrophoretic mobilities. The addition of an electrospray source (ES) recently allowed extension of SMPS analysis to the macromolecular range. We demonstrate here the application of ES-SMPS to nucleic acids analysis. Single- and double-stranded DNA molecules ranging from 6.1 kDa (single-stranded DNA 20 nucleotides in length) to 300 kDa (500 base-pair double-stranded DNA) were separated and detected by ES-SMPS at the picomole to femtomole levels. The measured electrophoretic mobility diameters were found to correlate with the analytes' molecular weights, while the peak areas could yield quantitative information. No fragmentation of DNA was observed under the conditions employed. Different apparent densities were observed for single-stranded and double-stranded DNAs, showing a different behavior for each type of biomolecule. The total analysis time was about 3 min/spectrum. Further optimization of ES-SMPS is expected to make it a fast and sensitive technique for biopolymer characterization.

DNA↗
PubMed2000

Effects of individual dental factors on genomic DNA analysis.

The use in forensic medicine of methods pertaining to molecular biology has made it possible to identify human remains through the analysis of polymorphic profiles of human DNA. Voluntary, accidental, or natural postmortem degradation, as well as environmental conditions, influences the preservation state of the corpse, making it sometimes difficult to obtain biologic material suitable for genetic analysis (e.g., hair, soft and/or hard tissue). According to their anatomic/morphologic characteristics, dental formations are particularly resistant to external insults and are thus suitable for this kind of research. The purpose of this work, conducted on nonselected dental findings (presenting intrinsic characteristics similar to those usually found in forensic cases) that were homogeneous with regard to environmental factors, was to determine an operative protocol that will enable combination of the maximum availability of genomic DNA with the preservation of the morphologic characteristics of the tooth for classic anthropologic evaluations.

Adult↗
PubMed1998

DNA analysis of a grandfather-father-son relationship from 300-year-old remains of the Date clan in Japan.

The grandfather-father-son relationship of the first three lords of the Date clan in Japan was ascertained by HLA-DNA sequencing analysis. From their hairs and dried lung tissue found in ca. 300-year-old remains, DNA was extracted with usual phenol-chloroform method followed by purification with cetyltrimethylammonium bromide (CTAB). Two HLA class II genes, HLA-DQA1 and -DPB1, were amplified by seminested, or dual/triple PCR. The PCR products were cloned and analyzed by automated sequencing. Since the three lords shared a haplotype of DQA1*0301-DPB1*0402, we concluded that there is no inconsistency in their lineage. This is the first case of biological evidence for a historical Samurai family relationship in the 17-18th centuries.

Base Sequence↗
PubMed1989

Flow cytometric analysis DNA content in esophageal carcinoma. Correlation with histologic and clinical features.

Cell nuclear DNA content was determined by flow cytometric analysis in 36 patients with esophageal carcinoma. Aneuploid DNA content was observed in 25 cases (69.4%). The relationships among DNA content, histopathologic features, and clinical characteristics were investigated. The patients with aneuploid DNA content had a high frequency of local lymph node metastasis, invasion to surrounding tissues, poor histologic differentiation, and advanced tumor stages as compared with those patients with diploid DNA content. In contrast to this, there was no correlation in patients' sex, age, and the sites of the disease.

Adult↗
PubMed1990

[Cytofluorometric nuclear DNA analysis and immunohistochemical study on the proliferative activity of the trophoblasts in human early gestation].

Changes in proliferative activity in trophoblastic cells were evaluated in various different sites of implantation. DNA content analysis by cytofluorometry and immunohistochemical study using a monoclonal antibody Ki-67 was performed in 5 cases with a gestation period of 8-9 weeks. The measurement of proliferative activity of trophoblastic cells was analysed in four different sites of implantation: (1) the top of the anchoring villi, (2) the cell column, (3) the intermediate trophoblasts in the decidua basalis and myometrium, and (4) the free villi. A large number of Langhans cells on the top of the anchoring villi (L-cells) were mononuclear and tetraploid. It was demonstrated that the L-cell group had 2C, 4C and 8C peaks and also had a 4C mode. S-phase fraction was also found in the L-cell group. The so-called intermediate trophoblasts (IMT) in the cell column, decidua basalis and myometrium had 2C and 4C peaks and no S-phase fraction. Ki-67 positive cells existed only in the L-cell group. The findings are as follows: 1. The L-cell group had a higher proliferative activity than the other trophoblasts. It was found to have a mononuclear tetraploidy pattern. 2. It is suspected that the cells having 4C DNA content in the IMT originated in the L-cell group. 3. In conclusion, L-cells may differentiate to syncytial trophoblasts and IMT.

Adult↗
PubMed2006

Comparative phylogeography of two bitterlings, Tanakia lanceolata and T. limbata (Teleostei, Cyprinidae), in Kyushu and adjacent districts of western Japan, based on mitochondrial DNA analysis.

To understand the geographical patterns of genetic variation in freshwater fishes in western Japan, the genetic structures of populations of Tanakia lanceolata and T. limbata (Teleostei, Cyprinidae) in this area were investigated using mitochondrial DNA (mtDNA) cytb sequences. Neighbor-joining trees of mtDNA haplotypes revealed four and three genetically divergent groups in T. lanceolata and T. limbata, respectively. Each group was restricted to one or the other of the geographical regions in the area studied. The patterns of geographical divergence in the two species showed some similarities, which seem to reflect common historical events experienced by freshwater fishes distributed in western Japan. On the other hand, dissimilarities were also found in the patterns, indicating that species-specific historical processes also occurred. Within one region, T. lanceolata was less differentiated than T. limbata, suggesting a difference in the dispersal abilities of the two bitterlings. In addition, several individuals in Kunichika River on Shikoku island were morphologically identified as T. lanceolata but had mtDNA haplotypes of T. limbata. We tentatively suggest that these individuals are hybrids of the two species, but further studies employing nuclear markers are necessary to validate this hypothesis.

Animals↗
PubMed1999

[DNA analysis for paternity testing and forensic identification].

Non coding DNA of human genome contains an important part of tandem repetitive DNA which last years has been extensively applied to solve paternity disputes and individual identification cases. A class of this DNA are minisatellites and microsatellites loci. Such loci are highly polimorphics because they have a multiple of different alleles to make them ideals genetic markers for these purposes. This article is a review about these loci, methods of their analysis and practical aspects about their applications to determine biological paternity or individual identification in the forensic genetic field are considered.

DNA↗
PubMed2003

Cationic comb-type copolymers for DNA analysis.

Genetic diagnoses, such as single nucleotide polymorphism (SNP) typing, allow elucidation of gene-based physiological differences, such as susceptibility to diseases and response to drugs, among individuals. Many detection technologies, including allele-specific hybridization, allele-specific primer extension and oligonucleotide ligation, are being used to discriminate SNP alleles. These methods still have many unsolved practical issues. In general they require adequate and specific hybridizations of primer or probe DNAs with target DNAs. This frequently needs optimization of the probe/primer structures and operating conditions. In nature, highly homology-sensitive hybridization is assisted by a nucleic acid chaperone that reduces the energy barrier associated with breakage and reassociation of nucleic base pairs. Here we report a simple, quick, precise but enzyme-free method for SNP analysis. The method uses cationic comb-type copolymers (CCCs) producing high nucleic acid chaperone activities. A single-base mismatch in 20-mer DNA can be detected within a few minutes at ambient temperatures (25-37 degrees C). Even without careful optimization processes, the method has the sensitivity to detect the mismatches causing subtle changes (Delta T(m) equals approximately 1 degree C) in duplex thermal stability. CCCs may have various bioanalytical applications where precise hybridization of nucleic acids is needed.

Aryl Hydrocarbon Hydroxylases↗
PubMed2001

Microfabricated capillary array electrophoresis DNA analysis systems.

Microfabricated "laboratory-on-a-chip" systems are revolutionizing all aspects of genetic analysis. The development of capillary array electrophoresis (CAE) microchannel plate devices makes possible the performance of 96 or more high-speed separations in parallel on a single wafer-scale device. The fluorescently labeled DNA samples are detected within the microchannels with a novel four-color rotary confocal fluorescence scanner. The capabilities of this system for genotyping are demonstrated through multiplex separations of short tandem repeat and hereditary haemochromatosis allele-specific amplicons. Furthermore, with newly developed folded channel designs that maintain high resolution, these CAE microplate systems are used to perform 96 high-quality DNA sequencing separations in parallel to approximately 500 bases per capillary in less than 30 min. These densely packed microfabricated device technologies will facilitate the even more rapid collection of vast amounts of genetic data in the future.

Base Sequence↗
PubMed1992

Cytometric DNA analysis in the management of cancer. Clinical and laboratory considerations.

Clinically, cytometric DNA (ploidy and proliferative fraction) analysis is primarily used in the determination of tumor prognosis. This determination has a clinical impact primarily in low stage tumors where therapeutic options are available, rather than in higher stage tumors that will be treated the same independent of their ploidy or proliferative fraction. In general, the best available data indicate that for most tumors aneuploidy is an indicator of more aggressiveness. Ploidy analysis is independent of other prognostic indicators in many tumors and is the most significant prognostic indicator in some. More recent data suggest that proliferative fraction, the percentage of tumor cells in the S-phase of the cell cycle, may be as important an indicator of tumor prognosis as simple presence of aneuploidy. However, the poor interlaboratory reproducibility of S-phase measurements as currently performed limits the general applicability of this measurement in the management of patients.

Cell Division↗
PubMed2002

Diversity of Streptococcus mutans bacteriocins as confirmed by DNA analysis using specific molecular probes.

Mutacin-producing strains have been classified into 24 groups (designated by letters A to X) by similarity in activity spectra and cross-immunity. Similarity in primary structure among these groups can be revealed using DNA hybridization. The amino acid sequences of four mutacins (B-Ny266, 1140/mutacin III and mutacin II) were used to design two DNA probes in order to detect similar genes among groups of Streptococcus mutans strains demonstrating inhibitory activity. In addition to the appropriate parent strain, each probe hybridized with the total DNA from only two out of the 24 mutacin group type strains. Thus, the remaining 18 groups of strains produce mutacins that differ from the mutacins sequenced to date. In order to explore the similarity between genes coding for mutacins B-Ny266 and JH1140, the group B specific probe was utilized to detect a DNA fragment of 1.9 kb in the genome of S. mutans strain Ny266. The sequence of the cloned fragment codes for three open reading frames (lanA, lanA' and lanB) similar to those of strains JH1140 and UA787. The gene lanA' is strongly similar to the structural gene lanA (67%), but only one RNA transcript of about 300 bases was detected by Northern hybridization using the lanA-lanA' probe. Transcription of lanA alone was verified by RT-PCR.

Amino Acid Sequence↗
PubMed2006

Pseudo-outbreak of Mycobacterium gordonae: usefulness of randomly amplified polymorphic DNA analysis to assess the clonality of the isolates.

Mycobacterium gordonae was detected in 18 of 21 clinical samples processed during the same day from patients with clinical suspicion of tuberculosis. Randomly amplified polymorphic DNA (RAPD) analysis revealed that all the isolates generated an identical pattern with each of the five primers used, and that these patterns were different from those of epidemiologically non-related isolates of M. gordonae. M. gordonae was not detected in the distilled water used for the procedures, and following replacement of the commercial products and sterilisation of home-made reagents, no more isolates belonging to the same clone of M. gordonae were detected.

Disease Outbreaks↗
PubMed2000

Randomly amplified polymorphic DNA analysis of Erysipelothrix spp.

The usefulness of randomly amplified polymorphic DNA method (RAPD) to identify each species of genus Erysipelothrix and for epidemiological analysis of this genus was studied. Eighty-one strains and 18 random primers were tested. Among the tested primers, the primers NK51 (GGTGGTGGTATC) and NK6 (CCCGCGCCCC) produced noticeable results. The primer NK51 revealed four species-specific RAPD patterns. Of the 66 strains of E. rhusiopathiae, 64 had the same unique band of 884 bp. Of the 12 strains of E. tonsillarum, 11 produced a 1,265-bp band. In addition, two strains, previously thought to be E. rhusiopathiae, produced the 1,265-bp band, suggesting that they had been misclassified. One strain of E. tonsillarum produced the 884-bp band, suggesting that it too was E. rhusiopathiae. The E. rhusiopathiae strain of serovar 13 produced a 650-bp band, and the strain of serovar 18 produced a clear 420-bp band as well as three weak bands of 1,265, 918, and 444 bp. The primer NK6 revealed 14 RAPD patterns that were not serovar specific. However, different patterns were produced among strains of the same serovar showing that the RAPD method is able to identify the genetic variations of strains of this genus and can rapidly and easily differentiate strains of the same serovar. Based on these results, we concluded that the RAPD method with primers NK51 and NK6 is a rapid and reliable method to identify the species of this genus; we also concluded that this method might be a useful tool for the epidemiological analysis of the Erysipelothrix species.

Erysipelothrix↗
PubMed1999

Specificity and sensitivity of random amplified polymorphic DNA analysis for the identification of single larvae of Trichinella after experimental infection of pigs.

Single muscle larvae (ML) of Trichinella belonging to 5 species and 2 genotypes collected from 126 experimentally infected pigs and preserved in 75% ethyl alcohol at room temperature were identified by random amplified polymorphic DNA (RAPD) analysis to evaluate the specificity and sensitivity of this assay. A double-blind protocol was used to ensure that the interpretation of electrophoretic patterns after RAPD amplification was not influenced by an expected result. RAPD analysis of one larva from each sample showed a sensitivity of 100% when only samples with undamaged DNA were considered, and the specificity resulted in an 88% match with the species or genotype that had been used to infect pigs, whereas the identification reached 100% specificity when an additional one to four ML were examined.

Animals↗
PubMed1994

Sex identification of old skeletal remains by DNA analysis: the report of three cases.

We analyzed the DNA in the remains of three skeletons affected by severe post-mortem modification to identify the sex of the cases. We analyzed DNA extracted from bone, bone marrow and a tooth by Southern blot hybridization with Y chromosome specific probe and PCR to amplify the sex chromosome specific sequence. The first case had been preserved underground in a deep grave for two months, the second case was found in a forest after more than five years, and the last case had been kept in a styrofoam box for few years. The detective sensitivity of Southern hybridization improved to nearly the same level as PCR with the aid of computed image processing. Although PCR was of obvious benefit when analyzing small amounts of biological evidence, amplification was impossible in case the target sequence became lost or changed. As a result, we identify the sexes of the three cases by Southern blot hybridization or PCR using bone, bone marrow and tooth DNA. The old skeletal remains were suitable for sex identification in spite of severe post-mortem change, due to preservation in a condition protected from external influences.

Adult↗
PubMed2003

Genomic DNA analysis of genes encoding (hemi-)cellulolytic enzymes of the anaerobic fungus Piromyces sp. E2.

Anaerobic fungi contain more than one copy of genes encoding (hemi-)cellulases in their genome. The arrangement of these genes on the chromosomes was not known. A genomic DNA (gDNA) library of Piromyces sp. E2 was screened with different probes specific for (hemi-)cellulolytic enzymes. This screening resulted in three gDNA clones with genes encoding glycoside hydrolase enzymes of families 1 (beta-glucosidase), 6 (exoglucanase) and 26 (mannanase). Each clone contained two or more genes of the same family. Comparison of the gene copies on a clone revealed that they were highly homologous, and in addition, 54-75% of the substitutions was synonymous. One of the mannanase genes contained an intron. PCR with selected primers resulted in a gDNA clone with a new representative (cel9B) of glycoside hydrolase family 9 (endoglucanase). Comparison with cel9A revealed that cel9B had 67% homology on the nucleotide level. Furthermore, three introns were present. All results of this paper taken together provided evidence for duplications of (hemi-)cellulolytic genes, which resulted in clusters of almost identical genes arranged head-to-tail on the genome. In contrast to other eukaryotes, this phenomenon appears frequently in anaerobic fungi.

3' Flanking Region↗