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PubMed1997

Identification by DNA analysis of the victims of the August 1996 Spitsbergen civil aircraft disaster.

Disaster victim identification traditionally relies on the combined efforts of police, dentists and pathologists, comparing ante mortem (AM) information from the missing persons with post mortem (PM) data from the dead bodies. In Western countries, dental evidence has ordinarily played the major role. DNA analysis has been used successfully in a number of large accidents to associate body parts and for purposes of identification, by comparing victims' DNA profiles with those of relatives. However, DNA typing is still not generally regarded as an essential part of disaster victim identification. Facing the August 1996 Spitsbergen aircraft accident in which 141 Russians and Ukrainians died and anticipating scanty ante mortem dental data, it was decided to use DNA profile analysis as the primary identification method. Material collected at the scene from all body parts, and blood sample from relatives were analysed at eight polymorphic microsatellite and minisatellite loci, DNA profile comparisons enabled us to sort the 257 typed body parts into 141 individuals, as well as identifying the 139 victims for whom reference samples were available. Identification by DNA analysis was then followed by comparisons of traditional AM and PM data, and within day 20 of the accident the identities of all victims were confidently established. This investigation indicates that it might be feasible to replace traditional identification efforts with DNA typing.

Aircraft↗

PubMed2003

Hb Q-India: an uncommon variant diagnosed in three Punjabi patients with diabetes is identified by a novel DNA analysis test.

AIMS: An abnormality in the glycated haemoglobin peak (Hb A1c) on Diastat (Bio-Rad) cation exchange low pressure liquid chromatography (LPLC) was found in three Punjabi patients with diabetes. The aims of this study were to identify the variant by chromatography and electrophoresis and to determine whether a DNA analysis test could be designed for confirmation that could be generally applied for the identification of any unusual abnormal haemoglobin. METHODS: The presence of an Hb variant was confirmed by cellulose acetate electrophoresis at pH 8.6. The variant was characterised further by high performance liquid chromatography (HPLC; Bio-Rad Variant) and isolelectric focusing (IEF) electrophoresis. A novel DNA analysis test based on the amplification refractory mutation system (ARMS) and the polymerase chain reaction (PCR) was developed to confirm the presence of the mutation for the uncommon variant. RESULTS: Comparison of the HPLC retention time and IEF band position determined the presence of the variant Hb Q-India in all three cases. Hb Q-India is caused by the mutation GAC --> CAC at codon 64 of the alpha-1 globin gene and is clinically silent. ARMS-PCR specific primers were designed and used successfully to confirm the presence of the mutation for Hb Q-India. CONCLUSIONS: The results show that the ARMS-PCR technique, developed previously for the diagnosis of beta thalassaemia mutations, can also be adapted to provide a simple, rapid, and inexpensive approach for the identification of abnormal haemoglobins.

Adult↗

PubMedDate not supplied

[The use of DNA analysis for diagnostics of hereditary premature ovarian failure].

Methods of DNA-analysis of 769G --> A mutations in INHalpha1 gene and CGG-repeats polymorphism in FMRI gene have been developed for creating test-systems for genetically caused forms of premature ovarian failure (POF) diagnostics. The frequency of 769G --> A mutation among women population in Ukraine was established and, by preliminary calculations, makes up 2.8%. Results of analysis of CGG-repeats numbers in FMRI gene in the group of 215 women (oocyte donors) revealed five persons with CGG-repeats numbers, that exceeds the normal one (42 copies). Thus the frequency of persons with allels with high risk of premutation in FMRI gene is 2.3%. The results of our research confirm the actuality of genetic tests of mutations in INHalpha1 and FMR1 genes among the women of reproductive age with the purpose of POF prognosis and prevention the birth of children with fragile X syndrome.

DNA↗

PubMed1996

Flow cytometric DNA analysis of cirrhotic liver cells in patients with hepatocellular carcinoma can provide a new prognostic factor.

BACKGROUND: DNA flow cytometry of hepatocellular carcinoma (HCC) cells has been investigated in many studies, but, to the best of our knowledge, there are no data on DNA analysis of cirrhotic parenchyma around the HCC. In this study, cell kinetics and ploidy of parenchymal cells around HCC were performed to ascertain if this would predict the possibility of recurrence in the cirrhotic areas. METHODS: The DNA content of 93 cases of HCC and of cirrhotic liver around the tumor nodules was analyzed by flow cytometry. Ploidy and proliferative index of HCC and cirrhotic liver were compared with macroscopic, histologic, and clinical features of each case and linked with the behavior of these tumors. Survival curves were assessed according to the Kaplan-Meier method. A multivariate analysis based on Cox proportional hazards regression model was performed on cases of diploid cirrhosis cells in which the S-phase fraction was evaluable. RESULTS: The univariate analysis of survival suggested significant roles for age, number of intrahepatic nodules, Edmondson-Steiner's classification, portal invasion, vascular invasion, presence of necrosis, hepatitis B surface antigen, alpha-feto-protein, Child's score, ploidy, and S-phase fraction of HCC cells. The DNA analysis of the cirrhotic cells showed that polyploidy was dramatically reduced in patients with HCC, compared with normal hepatocytes, and aneuploid clones were present among diploid cells. High S-phase fraction of cirrhotic cells and Child-Pugh classification were the strongest independent parameters affecting the tumor behavior in this study. CONCLUSIONS: The results of this study suggest that S-phase fraction of cirrhotic liver parenchyma may be employed as a new parameter in the prognostic evaluation of HCC patients.

Adult↗

PubMed1997

[The fragile X chromosome syndrome: DNA analysis in families with familial occurrence of mental retardation].

The fragile X syndrome, the most common form of inherited mental retardation, is characterized by unique genetic mechanisms, which include amplification of a CGG repeat and abnormal DNA methylation. Direct DNA analysis of fragile X mutations has already shown its clear superiority for postnatal and prenatal diagnosis of the disorder and for carrier detection. In this, paper the authors report on the results of DNA analysis in families with familial mental retardation. They present the various alternatives (probe/enzymes combinations) for Southern blot based diagnosis and protocols which gave optimal results for detection of patients segregating for fragile X syndrome. Totally, 36 members from 10 families were analyzed by Southern blotting, including 18 mentally affected patients. No CGG expansion was detected in 9 clinically affected patients of 5 families. Expansion of the CGG repeats was found in 9 clinically and cytogenetically affected males, in 5 unaffected carriers of premutation, and in 1 carrier of full mutation in the remaining 5 families. Carriers represented mothers of the patients. These results correlated with cytogenetic and clinical expression of fragile X syndrome. The application of the method for diagnosis of the disease is discussed. (Tab. 2, Fig. 3, Ref. 21.)

Blotting, Southern↗

PubMed1990

Flow-cytometric DNA analysis of hematopoietic and lymphoid proliferations: a comparison of fresh, formalin-fixed and B5-fixed tissues.

Flow-cytometric DNA analysis of human tumors using paraffin-embedded tissue samples is becoming an increasingly popular method of determining ploidy and proliferative rate. Particularly with hematopoietic/lymphoid proliferations, little is known about how these data compare with data from fresh suspension studies, or how B5-fixed tissues compare with those fixed in formalin. For these reasons, flow-cytometric DNA ploidy and cell cycle analysis was performed on 16 hyperplastic tonsils and 28 lymphoid/hematopoietic neoplasms using fresh (or fresh-frozen) cell suspensions (FR), B5-fixed paraffin-embedded (B5), and formalin-fixed paraffin-embedded (FO) tissue. Ploidy analysis showed all tonsil specimens to be diploid regardless of preparative method; however, for the neoplastic cases the FO and B5 preparations agreed with the FR preparations in 50% and 61% of the cases, respectively. Complete agreement between all three tissue preparation methods was present in only 36% of the neoplastic cases. Percent S or S + G2M phase fractions showed relatively poor correlation between the three preparative methods, with the best correlations found between the FR and B5 samples (S phase, r = 0.44; S + G2M, r = 0.43). In conclusion, while potentially useful data can be obtained from flow-cytometric DNA analysis of fixed, paraffin-embedded lymphoid/hematopoietic tissues, the specific limitations of such analyses must be recognized. Ploidy and proliferative phase data from fixed tissue preparations are not equivalent to information obtained from fresh suspension studies. B5-fixed tissue provides an acceptable alternative to formalin-fixed tissue for such analyses.

Cell Division↗

PubMed1988

Restriction endonuclease DNA analysis of antigenic variants of leptospires selected by monoclonal antibodies.

The genome of antigenic variant CV (CT3)-1 derived from Leptospira interrogans serovar canicola was compared by cleavage with restriction endonucleases with the parent and serovar bafani, to which the variant was serologically most closely related. No differences were observed between the parent and variant in DNA restriction endonuclease patterns using eight restriction endonucleases. Serovar bafani was different in the patterns from the parent and antigenic variant CV (CT3)-1. The two antigenic variants derived from serovar hebdomadis, HV (H16)-1 and HV (H19)-1 which belonged serologically to serovars jules and hebdomadis, respectively, were compared by restriction endonuclease DNA analysis with the parent and serovar jules. No differences were observed between the parent and variants in DNA restriction endonuclease patterns using the same enzymes. But some differences were observed in DNA restriction endonuclease patterns between HV (H16)-1 and serovar jules. Thus, the antigenic variant selected from the parent by the anti-parent monoclonal antibody and serologically different from the parent, being identified either as a new serovar or as a known one, was found to be similar to the parent by the restriction endonuclease DNA analysis.

Antibodies, Monoclonal↗

PubMedDate not supplied

Prognostic significance of flow cytometric DNA analysis in patients with malignant pleural mesothelioma.

Malignant pleural mesothelioma (MPM) due to environmental exposure to asbestos and erionite is a relatively common cancer in Turkey. In this study, we investigated the value of flow cytometric (FCM) DNA analysis and other prognostic factors such as age and etiologic factor in the patients with MPM, treated with surgery+/-combination chemotherapy+/-radiotherapy. A total of 40 patients with a median age of 50 (range 30-68) were included in the study. Twenty-nine patients had asbestos exposure in etiology, while 11 had fibrous zeolite (erionite). Paraffin-embedded tumor specimens were studied by FCM for DNA analysis. Twelve patients (30%) had aneuploid tumors and 28 (70%) had diploid ones. Mean S-phase fraction (SPF; %) was 9.1+/-1.1 and proliferation index (PI, SPF+G2/M phase; %) was 11.3+/-0.9. While the median overall survival (OS) was 10+/-2 months (6-14; 95% CI), 1-year survival rate was 45.2%. Only PI was found to be statistically significant for OS in univariate analysis (P=0.013). PI was also found to be an independent prognostic factor for all patients (P=0.035). Aneuploidy was significantly higher in erionite group compared with asbestos group. Male predominance and poor survival were also prominent in erionite group, though not statistically significant. In conclusion, PI is an independent prognostic factor for patients with MPM and the biologic features of the disease may show differences with respect to different etiologies.

Adult↗

PubMed1994

Effect of low power microwave on the mouse genome: a direct DNA analysis.

The potential mutagenic effect of low power microwave at the DNA sequence level in the mouse genome was evaluated by direct DNA analysis. Animals were exposed to microwave at a power density of 1 mW/cm2 for 2 h/day at a frequency of 2.45 GHz over a period of 120, 150 and 200 days. HinfI digested DNA samples from testis and brain of control and exposed animals were hybridized with a synthetic oligo probe (OAT 36) comprising nine repeats of 5'-GACA-3'. As compared to control animals, band patterns in exposed animals were found to be distinctly altered in the range of 7-8 kb which was also substantiated by densitometric analysis. Though the mechanism of this rearrangement is not yet clear, the results obtained at the present dose are of significance. This dose, which has been set as the safe limit for general public exposure by the Non-Ionizing Radiation Committee of the International Radiation Protection Association, may imply a need for (re)evaluation of the mutagenic potential of microwaves at the prescribed safe limit for the personnel and people who are being exposed.

Animals↗

PubMed1994

Prenatal detection of Canavan disease (aspartoacylase deficiency) by DNA analysis.

Amniocentesis was performed in four pregnancies at risk for Canavan disease (CD). In all families both parents were of Ashkenazi-Jewish origin and harboured the C854 mutation in the cDNA of the aspartoacylase gene. Using DNA analysis of the amniotic cells, three fetuses were predicted to be non-affected and one fetus was predicted to be affected. The concentration of N-acetylaspartic acid (NAA) in the amniotic fluid was in agreement with these results. In urine samples of the three newborns predicted to be non-affected, the concentration of NAA was normal. Tissues of the aborted fetus were not available. We conclude that DNA analysis is probably a reliable method for prenatal diagnosis of CD.

Amidohydrolases↗

PubMed1980

Flow microfluorometric deoxyribonucleic acid (DNA) analysis supplementing routine histopathologic diagnosis of biopsy specimens.

Flow microfluorometric DNA analysis was applied on a routine basis additionally to the conventional pathologic diagnostics of biopsy samples. The evaluation of the DNA distributions for the determination of the fractions of cells in different stages of the cell cycle was complicated by varying portions of background debris, which was generally found to be present in suspensions of single dispersed cells prepared from solid tissues. By means of computerized subtraction of exponentially decreasing corrections, it was possible to clear the original flow microfluorometric distributions. The contribution of background debris was found to cause erroneous overestimation, particularly in the evaluation of low S-phase fractions. By means of the corrections, even samples containing more than 65 per cent nuclear fragments could be evaluated with sufficient accuracy. The correct evaluation of the S-phase fraction possibly could indicate the degree of differentiation and proliferative activity of tissues. Low S-phase fractions within the range of normal regeneration were found in benign alterations and well differentiated carcinomas. In contrast, S-phase fractions up to 40 per cent were present in poorly differentiated tumors with extended cellular atypia, largely varying nuclear size and staining properties. The combination of conventional pathologic diagnostics with quantitative determination of the fractions of cells within the stages of the cell cycle in biopsy samples on a routine basis could supplement the diagnoses by therapeutically relevant information.

Biopsy↗

PubMed2004

DNA analysis by MALDI-TOF mass spectrometry.

The last decade has seen an increased demand for high-throughput DNA analysis. This is mainly due to the human genome sequencing project that is now completed. Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry was pinpointed early on as a technology that could be of great use for sequence variation analysis in the post-genome sequencing era. Applications developed first on this platform were for SNP genotyping. Several strategies for allele-discrimination (hybridization, cleavage, ligation, and primer extension) were combined with MALDI-TOF mass spectrometric detection. Nowadays, in practice, only primer extension methods are applied for large-scale SNP genotyping studies with MALDI-TOF detection. Problems surrounding the integration of SNP genotyping by MALDI-TOF mass spectrometry at high throughput are largely mastered now. Mass spectrometry geared presentations at the HUGO Mutation Detection Meeting in Palm Cove, Australia almost exclusively focused on novel applications that go beyond standard SNP genotyping. These applications are more demanding in terms of chemistry and molecular biology. Molecular haplotyping, expression profiling, DNA methylation analysis, and mutation detection are now being demonstrated.

DNA Mutational Analysis↗

PubMed1991

Nuclear DNA analysis of the periampullary carcinoma using cytologic bile specimens.

DNA cytofluorometric analysis of carcinoma cells of the periampullary region was performed using cytologic bile specimens. The materials were obtained from three cases of obstructive jaundice through percutaneous transhepatic biliary drainage tubes. The DNA ploidy histograms of bile samples could be evaluated as well as those of paraffin-embedded tissue samples. DNA analysis using cytologic bile specimens may reveal biological behavior of carcinoma cells and provide useful information for the choice of therapeutic interventions.

Adenocarcinoma↗

PubMed1993

DNA analysis and clinical outcome in pediatric adrenal cortical tumors.

Adrenal cortical neoplasms are uncommon and it is often difficult to separate benign tumors from those that are malignant. Histology does not provide a reliable guide, particularly in pediatric cases. It has been suggested that analysis of nuclear DNA content may complement conventional histopathologic methods in the diagnosis of malignancy. Six previously reported adrenal cortical tumors from children aged 6 mths to 6 yrs were analysed using a CAS 200 image analysis system which provided DNA histograms on each of the cases. In 5 cases a significant proportion of the tumors showed aneuploidy and in only one case did most of the tumor cells show a diploid pattern. All 6 cases were treated with surgery alone with long term survival and follow up of 2-15 yrs except in one child with a histologically benign aneuploid tumor who died post-operatively from measles pneumonia. No correlation between histological appearance, outcome and ploidy was seen. The aneuploid tumors included those classified by current histological methods as benign (1), indeterminant (2) and malignant (2). The one diploid tumor seen was a large tumor of indeterminant malignancy that did not recur. Although the series is quite small these observations suggest that abnormalities of DNA content may not provide an objective measure of the malignant potential of adrenal cortical tumors in children.

Adrenal Cortex Neoplasms↗

PubMed2006

Identification of the DNA bases of a DNase I footprint by the use of dye primer sequencing on an automated capillary DNA analysis instrument.

We have adapted the techniques of DNA footprint analysis to an Applied Biosystems 3730 DNA Analyzer. The use of fluorescently labeled primers eliminates the need for radioactively labeled nucleotides, as well as slab gel electrophoresis, and takes advantage of commonly available automated fluorescent capillary electrophoresis instruments. With fluorescently labeled primers and dideoxynucleotide DNA sequencing, we have shown that the terminal base of each digested fragment may be accurately identified with a capillary-based instrument. Polymerase chain reaction (PCR) was performed with a 6FAM-labeled primer to amplify a typical target promoter region. This PCR product was then incubated with a transcriptional activator protein, or bovine serum albumin as a control, and then partially digested with DNase I. A clone of the promoter was sequenced with the Thermo Sequenase Dye Primer Manual Cycle Sequencing kit (USB) and the FAM-labeled primer. Through the use of Genemapper software, the Thermo sequenase and DNasei digestion products were accurately aligned, providing a ready means to assign correct nucleotides to each peak from the DNA footprint. This method was used to characterize the binding of two different transcriptional activator proteins to their respective promoter regions.

Base Sequence↗

PubMed1995

Randomly amplified polymorphic DNA analysis provides rapid differentiation of methicillin-resistant coagulase-negative staphylococcus bacteremia isolates in pediatric hospital.

Coagulase-negative staphylococci (CoNS) are now recognized as the most common cause of nosocomial bacteremia in pediatric patients. Randomly amplified polymorphic DNA analysis was used to study the relationships among 12 isolates of CoNS obtained from eight patients with catheter-related bacteremia in two distinct wards of our hospital and 6 epidemiologically unrelated strains. With this method, we were able to discriminate isolates that otherwise were indistinguishable by conventional criteria such as biochemical typing and antibiotic susceptibility patterns. Our results indicate that there were episodes of cross-infections among four patients in one ward but independent infectious episodes among four patients in the other ward. Randomly amplified polymorphic DNA analysis is a rapid method which seems particularly well suited to the epidemiological study of CoNS isolates.

Bacteremia↗

PubMed1985

Cytophotometric DNA analysis of rectal mucosa of patients with ulcerative colitis.

Cytophotometric DNA analysis of rectal mucosa was carried out in 11 patients with ulcerative colitis, in attempt to determine the proliferative activity of rectal mucosa. There was no significant difference in the mean DNA value, modal DNA value, total variation of DNA content and population of over 3c cells of rectal mucosa between controls and the patients. However, the differential determination of the proliferative activity in the upper, middle and lower thirds of the crypt disclosed that a population of over 3c cells in the upper and middle thirds of the crypt was significantly larger in the patients with longstanding disease than in the controls.

Adult↗

PubMed1983

Cytophotometric DNA analysis of early esophageal carcinoma.

In four patients with early esophageal carcinoma who underwent radical surgery, one died of cancer recurrence 13 months after surgery, two had an uneventful postoperative course after over five years and one succumbed to an incidental pneumonia 18 months postoperatively. Cytophotometric DNA analysis of the cancer cells reflected well the outcome of these patients. One recurrent patient had malignant pattern similar to that seen in usual advanced carcinoma of the esophagus, whereas the remaining three patient without recurrence throughout the postoperative course showed less malignant patterns. These result suggested the potential usefulness of cytophotometric DNA analysis in assessing the prognosis of early esophageal carcinoma.

Carcinoma↗