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PubMed1997

DNA analysis of squamous cell carcinoma in the oral cavity by image cytometry and its rating in the assessment of tumor prognosis.

Image cytometric measurements on the DNA content of squamous cell carcinoma of the oral cavity were carried out to evaluate its relationship with clinicopathological features. In the prospective study 52 patients with operable oral cavity carcinoma in stage T1-4 and N0-3 were studied. The tumors were classified according to the TNM classification (UICC 1987) and graded histopathologically. A positive correlation between tumor size and ploidy status was observed. There was a significant correlation between ploidy status and histologically confirmed cervical lymph node metastases (P < 0.03). A relationship between histopathological grading and ploidy status was not found. The cumulative 5-year survival rate was significantly higher (P < 0.03) among patients with diploid DNA findings than among those with nondiploid findings. When assessing the prognosis of squamous cell carcinoma, DNA cytometry analysis should be used as an additional method. Particularly the primarily existing, significantly higher N stage, a higher frequency of metastases, and a significantly lower survival rate with nondiploid tumors underline the importance of this examination method.

Adult↗
PubMed2004

Randomly amplified polymorphic DNA analysis of Trypanosoma rangeli and allied species from human, monkeys and other sylvatic mammals of the Brazilian Amazon disclosed a new group and a species-specific marker.

We characterized 14 trypanosome isolates from sylvatic mammals (9 from primates, 1 from sloth, 2 from anteaters and 2 from opossum) plus 2 human isolates of Brazilian Amazon. These isolates were proven to be Trypanosoma rangeli by detection of metacyclic trypomastigotes in the salivary glands of triatomines and by a specific PCR assay. Polymorphism determined by randomly amplified polymorphic DNA (RAPD) revealed that most (12) of the Brazilian T. rangeli isolates from the Amazon differed from those of other geographical regions, thus constituting a new group of T. rangeli. Four Brazilian isolates clustered together with a previously described group (A) that was described as being composed of isolates from Colombia and Venezuela. Isolates from Panama and El Salvador form another group. The isolate from Southern Brazil did not cluster to any of the above-mentioned groups. This is the first study that assesses the genetic relationship of a large number of isolates from wild mammals, especially from non-human primates. A randomly-amplified DNA fragment (Tra625) exclusive to T. rangeli was used to develop a PCR assay able to detect all T. rangeli groups.

Animals↗
PubMed1996

Investigation of an apparent cluster of Klebsiella pneumoniae bacteremias using random amplified polymorphic DNA analysis.

A cluster of bacteremia episodes with Klebsiella pneumoniae was noted in patients in a hematology-oncology ward during a 3-week period. Random amplified polymorphic DNA (RAPD) analysis, a novel technique for generating chromosomal fingerprints from bacterial isolates, was used as an aid to the epidemiological investigation of this cluster. For each of the two patients from whom multiple isolates had been obtained, identical RAPD patterns were observed in the serial isolates, even for a patient where the isolates had different biotypes. Isolates from different patients gave distinct patterns. Random amplified polymorphic DNA was found to be a useful typing technique for this cluster of K pneumonia bacteremias.

Bacteremia↗
PubMed1994

Identification of the remains of the Romanov family by DNA analysis.

Nine skeletons found in a shallow grave in Ekaterinburg, Russia, in July 1991, were tentatively identified by Russian forensic authorities as the remains of the last Tsar, Tsarina, three of their five children, the Royal Physician and three servants. We have performed DNA based sex testing and short tandem repeat (STR) analysis and confirm that a family group was present in the grave. Analysis of mitochondrial (mt) DNA reveals an exact sequence match between the putative Tsarina and the three children with a living maternal relative. Amplified mtDNA extracted from the remains of the putative Tsar has been cloned to demonstrate heteroplasmy at a single base within the mtDNA control region. One of these sequences matches two living maternal relatives of the Tsar. We conclude that the DNA evidence supports the hypothesis that the remains are those of the Romanov family.

Base Sequence↗
PubMed2000

Molecular cloning and DNA analysis of the orotidine-5'-phosphate decarboxylase gene from the yeast Saccharomyces exiguus Yp74L-3.

The orotidine-5'-phosphate decarboxylase gene of Saccharomyces exiguus Yp74L-3 was cloned as a DNA fragment complementing a ura4 mutation of this yeast. The coding region of the gene is 807 bp in length, and represents 68.7% similarity to the corresponding gene of S. cerevisiae (URA3). The cloned URA4 gene was shown to be located on the 790-kbp Chromosome (chr) VIII of S. exiguus Yp74L-3. The neighbor-joining phylogenetic tree based on the orotidine-5'-phosphate decarboxylase coding sequences indicates that S. exiguus Yp74L-3 is closely related to Kluyveromyces yeasts, as well as to a S. cerevisiae laboratory strain.

Chromosome Mapping↗
PubMed2005

Mitochondrial DNA analysis of horses recovered from a frozen tomb (Berel site, Kazakhstan, 3rd Century BC).

Sequence polymorphism of the mitochondrial DNA D-loop was used to determine the genetic diversity of horses recovered from a Scythian princely tomb dating from the beginning of the 3rd century BC. Eight haplotypes were found among the 13 ancient horse samples tested. Phylogenetical analysis showed that these ancient horse's sequences, along with two Yakut ones, were distributed throughout the tree defined by modern horses' sequences and are closely related to them. No clear geographical affiliation of the specimens studied was thus determined. Our work, among others, supports the very ancient origin of the matrilines in horses.

Animals↗
PubMed1994

Biomphalaria glabrata: extensive genetic variation in Brazilian isolates revealed by random amplified polymorphic DNA analysis.

To examine the extent of genetic variation in Biomphalaria glabrata, the intermediate host of Schistosoma mansoni, specimens from seven Brazilian isolates were studied. Regions of genomic DNA were amplified using short, arbitrarily selected oligonucleotide primers under low-stringency conditions (random amplified polymorphic DNAs--RAPDs) and analyzed by polyacrylamide gel electrophoresis followed by silver staining. The RAPD profiles of snails of the same isolate were relatively homogeneous, with the majority of bands being common to all individuals studied. In contrast, the profiles of snails from different isolates were quite distinct, with less than 10% of amplified DNA fragments being common to all of the specimens studied. This was found to be true irrespective of the primer, indicating that B. glabrata is remarkably genetically heterogeneous. The data are consistent with the view that genetic drift plays a major role in the genetic structure of populations of B. glabrata due to their ability to reconstitute populations from very small numbers of individuals by self-fertilization. The great variety of polymorphic genetic markers identified in this restricted survey indicates that RAPD analysis may make a major contribution to the study of the genetics of Biomphalaria.

Animals↗
PubMed1992

PCR amplification of tandemly repeated DNA: analysis of intra- and interchromosomal sequence variation and homologous unequal crossing-over in human alpha satellite DNA.

Tandemly repeated DNA can comprise several percent of total genomic DNA in complex organisms and, in some instances, may play a role in chromosome structure or function. Alpha satellite DNA is the major family of tandemly repeated DNA found at the centromeres of all human and primate chromosomes. Each centromere is characterized by a large contiguous array of up to several thousand kb which can contain several thousand highly homogeneous repeat units. By using a novel application of the polymerase chain reaction (repPCR), we are able to amplify a representative sampling of multiple repetitive units simultaneously, allowing rapid analysis of chromosomal subsets. Direct sequence analysis of repPCR amplified alpha satellite from chromosomes 17 and X reveals positions of sequence heterogeneity as two bands at a single nucleotide position on a sequencing ladder. The use of TdT in the sequencing reactions greatly reduces the background associated with polymerase pauses and stops, allowing visualization of heterogeneous bases found in as little as 10% of the repeat units. Confirmation of these heterogeneous positions was obtained by comparison to the sequence of multiple individual cloned copies obtained both by PCR and non-PCR based methods. PCR amplification of alpha satellite can also reveal multiple repeat units which differ in size. Analysis of repPCR products from chromosome 17 and X allows rapid determination of the molecular basis of these repeat unit length variants, which appear to be a result of unequal crossing-over. The application of repPCR to the study of tandemly repeated DNA should allow in-depth analysis of intra- and interchromosomal variation and unequal crossing-over, thus providing insight into the biology and genetics of these large families of DNA.

Base Sequence↗
PubMed1995

Subdivision of Burkholderia pseudomallei ribotypes into multiple types by random amplified polymorphic DNA analysis provides new insights into epidemiology.

Ribotyping has previously been used for epidemiological studies of Burkholderia pseudomallei (previously Pseudomonas pseudomallei). We show here that random amplified polymorphic DNA (RAPD) analysis allows subdivision of strains of the same ribotype. With five different primers, no two epidemiologically unrelated isolates of any single ribotype in this study of 102 isolates from humans, goats, cats, and soil had identical RAPD patterns. Conversely, RAPD analysis showed clonality for isolates from each of two animal outbreaks of melioidosis and from a nontropical focus of animal and human melioidosis spanning 25 years. Some soil isolates were identical to epidemiologically related animal and human isolates as determined by RAPD typing. There was no evidence that the clinical outcome of melioidosis was related to RAPD patterns.

Animals↗
PubMed2006

Evaluation of differential gene expression in susceptible and resistant clinical isolates of Klebsiella pneumoniae by DNA microarray analysis.

DNA microarray technology was used to evaluate differential gene expression in a susceptible Klebsiella pneumoniae isolate and a resistant clinical derivative. Nineteen genes were up-regulated in the resistant isolate when compared with the susceptible isolate. An ABC transporter-related gene, ycjV, was strongly over-expressed, suggesting the existence of a novel active efflux mechanism. Approximately half of the up-regulated genes coded for ribosomal proteins, or proteins involved in tRNA metabolism. Among 33 downregulated genes, almost one-third were related to nitrogen metabolism. A possible role of fitness in the development of antimicrobial resistance is suggested.

Anti-Bacterial Agents↗
PubMed1990

Kaposi's sarcoma in patients with acquired immune deficiency syndrome. A flow cytometric DNA analysis of 26 lesions in 21 patients.

The DNA content of formalin-fixed, paraffin-embedded tissue from 21 patients (26 lesions) with Kaposi's sarcoma associated with acquired immune deficiency syndrome (AIDS) was analyzed by flow cytometry. The lesions were classified histologically into the following three types according to the predominant pattern: granulation tissue-like (two lesions), angioma/angiosarcoma-like (ten lesions), and spindle cell (14 lesions). All lesions showed exclusively DNA diploidy, and the S- phase fractions were usually low, ranging from 2% to 13% (mean, 6.9%). The follow-up periods were from 2 to 60 months. Five patients died, with one of the deaths attributed to pulmonary failure resulting from diffuse alveolar damage and Kaposi's sarcoma; the other deaths were attributed to infections. These results indicate that Kaposi's sarcoma is not a high-grade neoplasm and, combined with previously reported evidence, may suggest that Kaposi's sarcoma associated with AIDS is a proliferative process rather than a malignant neoplasm.

Acquired Immunodeficiency Syndrome↗
PubMed1999

Rapid DNA extraction methods and new primers for randomly amplified polymorphic DNA analysis of Giardia duodenalis.

A randomly amplified polymorphic DNA (RAPD) procedure using simple genomic DNA preparation methods and newly designed primers was optimized for analyzing Giardia duodenalis strains. Genomic DNA was extracted from in vitro cultivated trophozoites by five freezing-thawing cycles or by sonic treatment. Compared to a conventional method involving proteinase K digestion and phenol extraction, both freezing-thawing and sonication were equally efficient, yet with the advantage of being much less time- and labor-intensive. Five of the 10 tested RAPD primers produced reproducible polymorphisms among five human origin G. duodenalis strains, and grouping of these strains based on RAPD profiles was in agreement among these primers. The consistent classification of two standard laboratory reference strains, Portland-1 and WB, in the same group confirmed previous results using other fingerprinting methods, indicating that the reported simple DNA extraction methods and the selected primers are useful in RAPD for molecular characterization of G. duodenalis strains.

Animals↗
PubMed1999

Purification of nuclear DNA from single hair shafts for DNA analysis in forensic sciences.

The typing of nuclear DNA from hair shafts has often been unsuccessful to date. We tried to type one of the nuclear DNA loci, HLA-DQA1, from hair shafts, using an efficient cetyl-trimethyl ammonium bromide (CTAB) precipitation for DNA purification and a sensitive semi-nested PCR. After thorough washing with ethanol and water, hair shafts were digested by proteinase K in the presence of dithiothreitol, followed by a purification step including CTAB-DNA precipitation. The specific region of HLA-DQA1 gene was amplified by the semi-nested PCR, and the amplified products were cloned and sequenced. The HLA-DQA1 genotype was determined by comparing the sequence to the known sequence of each allele. All genotypes of HLA-DQA1 were successfully typed with hair shafts from six known heterozygotes, although one of them showed the predominant appearance of one allele. For correct typing, a template DNA equivalent to a hair shaft of 5 or 10 cm in length was necessary. Without the CTAB-DNA precipitation step, DNA extract from such hair shafts inevitably contains enough melanin to inhibit PCR. The present results suggest that hair shafts can be used for the typing of nuclear DNA loci.

Journal Article↗
PubMed1999

Molecular epidemiology of Fonsecaea pedrosoi using mitochondrial DNA analysis.

Restriction fragment length polymorphism (RFLP) of mitochondrial DNA (mtDNA) from 49 clinical Fonsecaea pedrosoi isolates (18 isolates from Japan, 17 from Madagascar, 7 from Argentina, 5 from Venezuela, 1 from Costa Rica and 1 unknown) was studied. The 49 isolates were composed of 20 isolates of Type 1, 16 of Type 2, 12 of Type 4 and 1 of a new mtDNA type, Type 7, which was closely related to Type 2. On the bases of the results of 120 isolates of the present (49 isolates) and previous (71 isolates) studies, F. pedrosoi was classified into seven mtDNA types and according to the relationship between mtDNA types and geographic origins: in Japan and probably in China, Type 1 isolates; in Zaire and Madagascar, Type 2; in Central and South America, Type 4 and Type 1. These results indicated that the geographical origin of F. pedrosoi isolate could be roughly inferred from its mtDNA type.

Chromoblastomycosis↗
PubMed1991

Flow cytometric DNA analysis of poorly differentiated adenocarcinoma of the colorectum.

DNA ploidy patterns in 11 poorly differentiated adenocarcinomas of the colorectum were examined by flow cytometry using paraffin-embedded specimens. Measurements of DNA content were made of the superficial (Sup) half and deeper (Deep) half of the primary tumors in all cases, and of lymph node metastases in five cases. All the primary tumors showed invasion beyond the muscularis propria of the colorectum. Aneuploidy or polyploidy in either Sup or Deep of the primary tumor was found in six of the 11 (54.5%) tumors. Out of the six aneuploid tumors, five were in Dukes' stage C with distant metastases at the time of operation, and four died within one year of surgery. Conversely, out of five diploid tumors, none had distant metastases at the time of operation and two survived for longer than three years after surgery. The DNA ploidy pattern of Deep differed from that of Sup in four out of six aneuploid tumors, and two showed aneuploidy in Sup and diploidy in Deep. All the lymph node metastases in the five tumors had a diploid pattern, although three had aneuploid patterns in the primary tumors. The findings suggest the DNA ploidy pattern of a primary tumor to be correlated with the degree of metastasis at the time of operation or prognosis, but the population of tumor cells having different DNA contents may be apt to change between Sup and Deep in aneuploid tumor.

Adenocarcinoma↗
PubMed1990

Cytophotometric DNA analysis on canine stomach carcinogenesis induced by ENNG.

DNA content and nuclear area were measured by microspectrophotometry in gastric carcinogenesis of three adult wolfdogs induced by N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG). The mean values and standard deviations of DNA content and nuclear area in normal gastric mucosa were 10.03 +/- 2.30 AU and 28.76 +/- 5.85/microns2; those in atrophic gastritis were 12.04 +/- 3.34 AU and 28.69 +/- 8.02/microns2; in mild dysplasia 13.52 +/- 3.73 AU and 28.23 +/- 8.12/microns2; in moderate dysplasia 20.88 +/- 4.57 AU and 47.58 +/- 10.74/microns2; in severe dysplasia 24.01 +/- 4.48 AU and 56.64 +/- 12.53/microns2; in well-differentiated adenocarcinoma 33.07 +/- 9.38 AU and 72.99 +/- 15.57/microns. These figures were different (P less than 0.01). The nuclear area of gastric carcinoma increased with DNA content (r = 0.73, P less than 0.01). The distribution patterns of DNA content in the histogram showed that diploidy was decreased and polyploidy increased in cancer cells. These findings indicate that DNA ploidy patterns and nuclear area can be useful indices for differentiating carcinoma from precancerous lesions.

Animals↗
PubMed1993

Flow cytometric DNA analysis of 20 bronchopulmonary neuroendocrine tumours.

Deoxyribonucleic acid (DNA) content of bronchopulmonary neuroendocrine tumours was measured by flow cytometry in order to investigate correlations between ploidy, S-phase fraction (SPF) and two histological classifications (World Health Organization (WHO), Warren and Gould), and clinical staging. A paraffin-embedded technique was used on 20 surgical specimens. Cases comprised (according to the classification of Warren and Gould) 7 carcinoids, 3 well-differentiated neuroendocrine carcinomas (WDNC), 6 intermediate neuroendocrine carcinomas (INC), and 4 small cell neuroendocrine carcinomas (SCNC). DNA aneuploidy was demonstrated in 3 out of 7 of the carcinoids, 3 out of 9 of the WDNCs and INCs, and 2 out of 4 of the SCNCs. A variable SPF was found in each group, except for the SCNCs which showed a constantly high SPF. In our small series, no correlation was noted between high SPF or aneuploidy and metastases. In conclusion, we observed no diagnostic value of malignancy for DNA aneuploidy. The SCNC group appeared to be an homogeneous group according to the SPF compared to the small cell carcinoma (SCC) group of the WHO classification. This and the prognostic incidence of high SPF need to be further studied.

Adult↗