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PubMed2002

DNA sequence analysis of DNA gyrase and DNA topoisomerase IV quinolone resistance-determining regions of Salmonella enterica serovar Typhi and serovar Paratyphi A.

The mutations that are responsible for fluoroquinolone resistance in the gyrA, gyrB, parC, and parE genes of Salmonella enterica serovar Typhi and serovar Paratyphi A were investigated. The sequences of the quinolone resistance-determining region of the gyrA gene in clinical isolates which showed decreased susceptibilities to fluoroquinolones had a single mutation at either the Ser-83 or the Asp-87 codon, and no mutations were found in the gyrB, parC, and parE genes.

Anti-Infective Agents↗

PubMed1999

Photocleavable peptide-DNA conjugates: synthesis and applications to DNA analysis using MALDI-MS.

The synthesis and characterization of photocleavable peptide-DNA conjugates is described along with their use as photocleavable mass marker (PCMM) hybridization probes for the detection of target DNA sequences by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. Three photocleavable peptide-DNA conjugates were synthesized, purified, and characterized using HPLC and denaturing gel electrophoresis, as well as IR-MALDI and UV-MALDI. The hybridization properties of the conjugates were also studied by monitoring their thermal denaturation with absorption spectroscopy. No significant difference in the melting temperature ( T (m)) of the duplexes was observed between the unmodified duplex and the duplex in which one strand was modified with the photocleavable peptide moiety. These conjugates were evaluated as hybridization probes for the detection of immobilized synthetic target DNAs using MALDI-MS. In these experiments, the DNA portion of the conjugate acts as a hybridization probe, whereas the peptide is photoreleased during the ionization/desorption step of UV-MALDI and can serve as a marker (mass tag) to identify a unique target DNA sequence. The method should be applicable to a wide variety of assays requiring highly multiplexed DNA/RNA analysis, including gene expression monitoring, genetic profiling and the detection of pathogens.

Base Sequence↗

PubMed2004

[Single cell DNA analysis: possibilities, methods, implications in relation to stem cell therapy].

In the analysis of circulating tumor cells or in the preimplantation genetic diagnosis it is frequently necessary to examine one single cell. Some methods are appropriate to isolate single cells: Laser Assisted Microdissection, magnetic cell separation or FACS. The use of the whole genome amplification methods are needful, because the amount of the DNA extracted from the isolated cells is very low and inappropriate for additional examinations. With different molecular biological methods (e.g. sequencing, chip-technology) it is possible to determine genetic alterations in the analysed cells, and to return the modified cells with in vitro gene technological methods (viral vectors, non viral methods). Our aim is to summarize the methods and the possible technical problems developing during the process of the single cell molecular biological analysis.

DNA Mutational Analysis↗

PubMed1984

Flow-cytometric DNA analysis in primary breast carcinomas and clinicopathological correlations.

Flow-cytometric DNA analyses of single cell nuclei were performed on nuclear suspensions prepared from biopsies of primary breast carcinomas in 638 patients. Propidium-iodide-stained cell nuclei were analysed in an Ortho 50-H Cytofluorograph. The patients were staged by the TMN classification. Sixty percent of all the patients had aneuploid primary breast carcinomas; of size T1, T2, T3, and T4 tumors, 51%, 63%, 67%, and 75% were aneuploid, respectively. The proportions of aneuploid tumors in each of the tumor stages SI, SII, SIII, and SIV were 47%, 62%, 67%, and 69%, respectively. This trend to increasing aneuploidy proportions with more advanced disease was significant in contrast to the degree of aneuploidy found in relation to axillary nodal tumor involvement. Multiple aneuploid cell populations were found in 109 (17%) tumors. With a mean follow-up time of 16 months, 92 patients have relapsed out of 540 completely staged patients with unilateral breast cancer with no distant metastases at the time of initial treatment. When the influence of various treatments and tumor stage are not considered, the recurrence rate was twice as high among patients with aneuploid primary tumors than among patients with euploid tumors. The differences in relapsing rates among patients with euploid and aneuploid primary tumors decreased with more advanced disease. Out of 170 patients with T1 tumors, 17 relapsed and 16 of these were aneuploid. No such difference in relapse rate in relation to ploidy was, however, found in patients with more advanced primary disease.

Adult↗

PubMed1995

Mitochondrial DNA analysis of Exophiala spinifera.

Restriction fragment length polymorphism (RFLP) of mitochondrial DNA (mtDNA) was examined in 36 isolates of Exophiala spinifera (8 isolates from Brazil, 9 from China, 15 from Columbia, 1 from the United States and 2 from Venezuela). E. spinifera isolates displayed a high degree of mtDNA diversity in RFLP patterns and were clustered into six genetically heterogeneous groups (Group 1 through Group 6). Isolates of Group 2 including the type strain seemed to have a worldwide distribution. RFLP patterns of E. spinifera were also distinct from those of other dematiaceous fungi, such as E. jeanselmei, E. moniliae, E. dermatitidis, Fonsecaea pedrosoi, Cladosporium carrionii and Phialophora verrucosa. These results indicate that E. spinifera may be a taxonomic complex and that RFLP patterns will be useful in the identification, typing and epidemiology of the E. spinifera variants.

DNA, Fungal↗

PubMedDate not supplied

Random amplified polymorphic DNA analysis of culture collection strains of Candida species.

Random amplified polymorphic DNA (RAPD) profiles, obtained when screening culture collection strains of Candida spp., showed several species to be highly heterogeneous. The RAPD-defined groups were as follows: C. catenulata, two groups among five strains; Debaryomyces hansenii (anamorph C. famata), three groups among five strains; C. sake, three groups among three strains; C. intermedia, two groups among two strains; C. rugosa, two groups among three strains and C. parapsilosis, three groups among four strains. The five strains of Issatchenkia orientalis (anamorph C. krusei) belonged to a single RAPD-defined group. The two strains of the teleomorphic yeast Arxiozyma telluris had distinct RAPD profiles but neither resembled profiles of its anamorph, C. pintolopesii. The two varieties of C. pintolopesii had different RAPD profiles. Researchers are cautioned that organisms with the same name may not be closely related.

Animals↗

PubMed1991

[Flow cytometric DNA analysis and its relationship with pathology and prognosis in lung carcinoma].

DNA content of paraffin-embedded tumour material from 110 patients with lung carcinoma was measured by means of flow cytometry. Aneuploidy was found in 78% of the patients. The mean DNA ploid, the percentage of hyperdiploidy and the percentage of multiploidy in small cell carcinoma and large cell carcinoma were higher than those obtained in squamous cell carcinoma and adenocarcinoma. These values were also higher in grade III squamous cell carcinoma and adenocarcinoma as compared with grade I and II squamous cell carcinoma, and adenocarcinoma. They were also higher in lung carcinoma with the primary tumor size greater than or equal to 3 cm than that less than 3 cm. The five-year survival rate and mid mean survival time of patients with hyperdiploidy were significantly lower than those of patients with diploidy and hypodiploidy. Conclusively, DNA content is considered closely related to the degree of malignancy of lung carcinoma and may be adapted as a reliable criterion in estimating the prognosis of lung carcinoma.

Adenocarcinoma↗

PubMed1990

[Carrier detection and prenatal diagnosis of Duchenne's muscular dystrophy based on DNA analysis].

Seven families with histories of Duchenne's muscular dystrophy underwent DNA diagnosis. The daughters of those consulted were examined for the carriage in 4 families. Their carriage was rejected or confirmed. Prenatal diagnosis was made in 2 families. In another family an abortion preceded obtaining molecular-genetic evidence. Probes 754, p20, XJI.I and primers for amplification of the site pERI87-15 containing a polymorphic locus were employed. The genetic risk was assessed using the computer program GenRisk adjusted for family history and DNA test allowances.

Adult↗

PubMed1990

[Determination of paternity using DNA analysis of chorionic villi].

Using probes recognizing hypervariable regions in human DNA high precision paternity testing can be provided even in the first trimester of pregnancy. Paternity tests were performed in 14 families using DNA isolated from chorionic villi. The results indicate that by a single probe confirmation or exclusion of paternity from a DNA sample of chorionic villi the 1st trimester of pregnancy can be performed with a probability more than 99.99%.

Blotting, Southern↗

PubMed1988

[DNA analysis of early esophageal cancer and epithelium adjacent to the cancer].

DNA distribution of 4 early esophageal cancers and their epithelial adjacent to cancer was analysed using photocytometry technique. In the tissues around the cancer, DNA content of the normal squamous epithelium always showed diploid (2 c) or proliferating diploid (less than 5 c). DNA content of 3 cases of the dysplastic cells in three specimens was predominantly proliferating diploid, comprising 68%, 38.2% and 59.4% but the aneuploid (greater than 5 c), 8%, 2.9% and 21.8%, respectively. In 4 cases of carcinoma in situ, the aneuploid increased to 86.7%, 68.7%, 17.2% and 28.6%, markedly higher than the dysplastic cells. In 3 cases of early esophageal cancer, the aneuploid accounted for 58.8%, 51.1% and 13.6%, similar to carcinoma in situ. The results indicate that in the dysplastic cells, there is a relative high ratio of the aneuploid cells except the increased proliferating diploid. This phenomenon could explain the unstability for dysplasia and might be the earliest manifestation of malignant change of the epithelial cells.

Aneuploidy↗

PubMed1989

Genomic DNA analysis of the estrogen receptor gene in breast cancer.

A human estrogen receptor (ER) cDNA probe was used to examine genomic DNA extracted from 59 primary invasive breast cancers. The tumors were also studied histopathologically, and their ER status was assessed by hormone-binding assay and immunohistochemical analysis. Southern blots of genomic DNA samples digested with restriction endonucleases (BamHI, EcoRI, HindIII, PvuII, XbaI) revealed identical restriction fragments for each tumor, indicating preservation of gross ER gene integrity regardless of ER status, clinical stage, or histopathologic appearance. Digestion with PvuII identified a single, two-allele polymorphism with band(s) at 1.6 and/or 0.7 kb. The homozygous 1.6 kb pattern was present in 14 (24%) patients, the heterozygous 1.6/0.7 kb pattern in 29 (49%) patients and the homozygous 0.7 kb pattern in 16 (27%) patients. The PvuII restriction fragment length polymorphism (RFLP) within the ER gene showed no correlation with the results of the ER binding assay, the immunohistochemical analysis, clinical stage, or histopathologic appearance. A significant correlation was found between the genotypes and patient age at the time of tumor diagnosis. Tumors with the homozygous 1.6 kb and the heterozygous 1.6/0.7 kb patterns were observed in older women with mean ages of 64.6 and 64.4 years, respectively. In contrast, patients with tumors containing the homozygous 0.7 kb pattern were significantly younger, with a mean age of 50.4 years (p-value = 0.0024). The mechanism by which the homozygous 0.7 kb genotype is associated with breast cancer in the premenopausal age group is unknown.

Adult↗

PubMedDate not supplied

[A case of myotonic dystrophy proved by DNA analysis].

The authors describe a case of a 53 years old man with myotonic dystrophy. Based on characteristic clinical symptoms and EMG results the diagnosis was established and proved on DNA examination. Myotonic dystrophy gene analysis showed on 3' untranslated region one hundred and fifty CTG triplet repeats. The accessory examinations revealed: cataract arrhythmias, gallbladder stones, impotence, cerebral atrophy on neuroimaging (CT and MRI). Apathy, somnolence, concentration troubles were present. Pedigree of the presented patient and possibility of spontaneous mutation are discussed.

Brain↗

PubMed2001

Forensic mitochondrial DNA analysis: two years of commercial casework experience in the United States.

AIM: To describe mitochondrial DNA (mtDNA) forensic casework experience in a commercial laboratory in the United States. METHODS: Frequency statistics were kept for two years on all aspects of mtDNA forensic cases, including types of clientele, types of samples, levels of sample success and failure, site heteroplasmy, length heteroplasmy, contamination, rates of failures to exclude, and match statistics using a mtDNA sequence database. RESULTS: Low sample failure rate was observed, especially since an "ancient DNA" approach was used for samples with degraded DNA. Levels of contamination were low, and the observed site and length heteroplasmy did not confound the interpretation of results. The data collected from mtDNA haplotypes developed in casework showed extremely high diversity of haplotypes consistent with other formally developed databases. CONCLUSIONS: MtDNA forensic analysis in the private sector was successfully applied to many different types of samples overall, with minimal rates of complication due to sample handling challenges (degraded DNA, minimal samples, contamination) and sequence-specific phenomena (site and length heteroplasmy).

Blood Chemical Analysis↗

PubMed1992

Flow cytometry DNA analysis and prediction of loco-regional recurrences after mastectomy in breast cancer.

The study concerns whether DNA flow cytometry and estrogen receptor analysis might help predict which breast cancer patients, particularly node-positive ones, were at the greatest risk of developing loco-regional recurrence (LRR). Such patients would best benefit from postoperative radiotherapy following modified radical mastectomy and axillary lymph node dissection. After this type of surgery, 506 patients were followed up for a median time of nearly 5 years. Among the 235 patients given postoperative radiotherapy, the loco-regional control rate was 100% in N0 cases (n = 93), 94% in cases with 1-3 positive nodes (n = 90), 93% in cases with 4-9 positive nodes (n = 43), and 67% in cases with 10 or more positive nodes (n = 9). Among the 271 non-irradiated patients, the corresponding figures for loco-regional control were 91% in N0 cases (n = 141), 71% in cases with 1-3 positive nodes (n = 84), 65% in cases with 4-9 positive nodes (n = 31), and 67% in cases with 10 or more positive nodes (n = 15). Ploidy status, level of S-phase fraction, estrogen receptor content, and primary tumor size did not, in the present material, yield significant additional information with regard to the risk of LRR in the different nodal subgroups, a finding confirmed in multivariate analysis where the only significant predictor of LRR was the number of positive nodes (p = 0.01). Adjuvant tamoxifen treatment could not replace postoperative radiotherapy for achieving loco-regional tumor control, the overall rate of which was 81% among patients treated with tamoxifen only (n = 117), as compared with 98% among those also treated with radiotherapy (n = 54) (p = 0.003).

Breast Neoplasms↗

PubMedDate not supplied

Subclinical human papillomavirus infection of the male lower genital tract: colposcopy, histology and DNA analysis.

In 25 partners of women with genital human papillomavirus (HPV) infection or cervical intraepithelial neoplasia, colposcopic examination revealed the existence of subclinical HPV infection of the male lower genital tract in 22 cases. It manifested either as short papillae tipped with acetowhite changes, or flat acetowhite lesions on the foreskin, glans, periurethral region, scrotum, perineum and/or perianal region. Multiple lesions involving several anogenital areas were common. Some of these abnormalities were small and inconspicuous. Of these 22 cases, 17 had histological evidence of HPV infection. Although Southern blot hybridization detected HPV DNA in only one case, polymerase chain reaction (PCR) analysis revealed HPV DNA in 20 cases. There were 10 cases of HPV 16. Subclinical HPV disease is best identified by colposcopy and confirmed by PCR. In treating HPV disease, colposcopic recognition of subclinical HPV disease forms an essential part of the management plan.

Biopsy↗

PubMed1999

Diversity among wild type and vaccination strains of Trichophyton verrucosum investigated using random amplified polymorphic DNA analysis.

We initially tested 20 primers for their ability to amplify genomic DNA of Trichophyton verrucosum using RAPD. Six of these were selected for further study aimed at discrimination of wild type and vaccination strains of T. verrucosum. The results indicate that RAPD successfully distinguished all strains included in the study. In addition, results of corrected cluster analysis were consistent with the fact that the avirulent vaccination strains (T. verrucosum TV-M9 and T. verrucosum TV-M-130) were prepared by ultraviolet (UV) light induced mutagenesis of the standard wild type strain T. verrucosum Strádznice. No marker for a/virulence was detected. These outcomes suggest new possibilities for epidemiological analyses, for discrimination among different vaccination strains and studies of fungal population in vaccinated/infected hosts.

Animals↗

PubMed1993

Circulating Borrelia burgdorferi in patients with acute Lyme disease: results of blood cultures and serum DNA analysis.

To determine the usefulness of blood culture and polymerase chain reaction (PCR) analysis in detecting circulating Borrelia burgdorferi or its DNA, blood and serum from untreated patients with acute Lyme disease were examined. None of the cultures of blood or serum from the 7 patients tested demonstrated spirochetes. Similarly, all patient serum samples, assayed in two laboratories, were negative for B. burgdorferi DNA using PCR amplification. These results suggest that in patients with acute Lyme disease, spirochetes, spirochete DNA, or both circulate early, only intermittently, or at low levels and that neither culture nor PCR testing of blood or serum, as currently done, appears likely to prove generally useful in the diagnosis of Lyme disease.

Acute Disease↗

PubMed2003

Development of a quality, high throughput DNA analysis procedure for skeletal samples to assist with the identification of victims from the World Trade Center attacks.

The attacks on the World Trade Center (WTC) Towers on September 11, 2001, represented the single largest terrorist-related mass fatality incident in the history of the United States. More than 2,700 individuals of varied racial and ethnic background lost their lives that day. Through the efforts of thousands of citizens, including recovery workers, medical examiners, and forensic scientists, the identification of approximately 1,500 victims had been accomplished through June 2003 (the majority of these identifications were made within the first 8-12 months). The principal role of The Bode Technology Group (Bode) in this process was to develop a quality, high throughput DNA extraction and short tandem repeat (STR) analysis procedure for skeletal elements, and to provide STR profiles to the Office of the Chief Medical Examiner (OCME) in New York City to be used for identification of the victims. A high throughput process was developed to include electronic accessioning of samples, so that the numbering system of the OCME was maintained; rapid preparation and sampling of skeletal fragments to allow for the processing of more than 250 fragments per day; use of a 96-well format for sample extraction, DNA quantification, and STR analysis; and use of the Applied Biosystems 3100 and 3700 instrumentation to develop STR profiles. Given the highly degraded nature of the skeletal remains received by Bode, an advanced DNA extraction procedure was developed to increase the quantity of DNA recovery and reduce the co-purification of polymerase chain reaction (PCR) amplification inhibitors. In addition, two new STR multiplexes were developed specifically for this project, which reduced the amplicon size of the STR loci, and therefore, enhanced the ability to obtain results from the most challenged of samples. In all, the procedures developed allowed for the analysis of more than 1,000 skeletal samples each week. Approximately 13,000 skeletal fragments were analyzed at least once, for a total of more than 18,000 analyses, and greater than 8,000 of the skeletal samples produced STR results (65%). The percentage of successful results was low in relation to previous mass fatality incidents involving airline disasters. However, when this same process was applied to the analysis of skeletal remains from the American Airlines Flight 587 disaster that occurred on November 12, 2001, the success rate was in line with expected results (ie, greater than 92% of the skeletal remains produced results). This illustrated the quality aspects of the procedure and the degree of degradation that had occurred for the remains of the WTC victims. For future mass fatality incidents, the quality, high throughput procedures developed by Bode will allow for more rapid DNA analysis of victim remains, more rapid identification of victims, and thus more rapid return of remains to family members.

Bone and Bones↗