Individual identification by DNA analysis: points to consider. Ad Hoc Committee on individual identification by DNA analysis, The American Society of Human Genetics.
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DNA↗
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Optimization of three- and four-color multiparameter DNA analysis in lymphoma specimens.
BACKGROUND: Simultaneous analysis of DNA and immunophenotype of lymphoma cells by flow cytometry allows the calculation of the proliferative activity and aneuploidy in even a small lymphoma population. Unfavorable DNA binding characteristics or spectral features of DNA dyes impair the accuracy of multiparameter DNA analysis and limit their clinical application. We describe here a reliable and reproducible application of both three- and four-color multiparameter DNA analysis. METHODS: After immunostaining of fresh samples of peripheral blood, bone marrow and single cell suspensions of lymph nodes from healthy and lymphoma patients, a methanol fixation for TO-PRO-3 and DRAQ5 staining was tested. RESULTS: The red-excitable TO-PRO-3 on a FACSCalibur is limited to two-color antigen staining including fluorescein-isothiocyanate and phycoerythrin-labeled monoclonal antibodies due to its broad excitation spectrum. Although DRAQ5 is only applicable to flow cytometers equipped with a single argon laser emitting 488-nm light, its emission spectrum can be easily separated from the FITC, PE, and PE/Texas-Red emissions. DRAQ5 showed almost identical stoichiometric DNA binding characteristics as propidium iodide. Coefficient of variation produced by DRAQ5 staining is in the range of 3.5 and is adequate for detecting aneuploid amd near-diploid cells. CONCLUSIONS: These advantageous features of DRAQ5 make it a reliable candidate for multiparameter clinical studies.
Anthraquinones↗
[Treatment of hepatocellular carcinoma from the aspect of nuclear DNA analysis].
Flow cytometric DNA analysis was done on 302 resected hepatocellular carcinoma and 30 specimens by fine needle aspiration biopsy. Patients with DNA aneuploid tumors had a significant worse prognosis than those with DNA diploid tumors in subdivided groups by curability (p less than 0.01). The recurrence after hepatectomy occurred frequently in DNA aneuploid or non-curative resected group. In a cox multivariate analysis, nuclear DNA content provided significant prognostic value (p = 0.008) as did vascular invasion and intrahepatic metastasis. For the multiple tumors of hepatocellular carcinoma, it is helpful to get the DNA ploidy pattern to diagnose tumors with intrahepatic metastasis or multicentric origin. Nuclear DNA analysis by fine needle aspiration biopsy was useful to determine the treatment method of hepatocellular carcinoma.
Carcinoma, Hepatocellular↗
Prenatal diagnosis of cystic fibrosis: microvillar enzymes and DNA analysis compared.
We have established reference ranges for three microvillar intestinal enzymes--alkaline phosphatase (EC 3.1.3.1), gamma-glutamyltransferase (EC 2.3.2.1), and leucine aminopeptidase (EC 3.4.1.1)--measured in amniotic fluid in a reference population of 1875 women presenting for routine amniocentesis. These data were derived for use in prenatal diagnostic studies in a population at risk (1:4) for cystic fibrosis. False-positive or indeterminate results were noted for fewer than 3.5% of all low-risk cases for each enzyme evaluated. Total alkaline phosphatase and its isoenzymes and leucine amino-peptidase and gamma-glutamyltransferase were measured in amniotic fluid sampled between the 15th and 19th weeks of gestation. Restriction fragment length polymorphism analysis of DNA was also performed when possible. In 52 cases examined for cystic fibrosis thus far, 46 were diagnosed on the basis of DNA analysis and (or) by sweat testing; for the other six cases, only abnormal enzyme results were obtained before termination of pregnancy. Predictions based on microvillar enzyme results were falsely negative in three cases. In only one case was there a discrepancy between enzyme results and DNA analysis. Diagnostic accuracy was highest during the 17th and 18th week of gestation. Preliminary results suggest the false-negative rate of this diagnostic strategy may be greater than or equal to 10%.
Alkaline Phosphatase↗
[Flow cytometric DNA analysis and cytology in diagnosis and prognosis of bladder tumors: preliminary results of a comparative study of bladder lavage].
OBJECTIVE: To compare flow cytometric data (ploidy and proliferative activity or percentage SG2M-phase cells) to cytologic and histologic data of the bladder carcinomas. MATERIALS AND METHODS: Cytologic and flow cytometric analysis of DNA content were performed on 48 bladder washings: 28 bladder washings from patients being followed for urothelial carcinomas and 20 control washings from individuals undergoing cytoscopy for other reasons. RESULTS: Cytological sensitivity and specificity of bladder washing were 75% and 91% respectively. Specificity was increased to 94% using flow cytometric DNA analysis whereas sensibility was moderately decreased to 68%. Combination of flow cytometry and cytology increased the diagnostic yield to 100%. The study of the patient group showed an increased abnormalities (aneuploidy and/or proliferate activity SG2M > 10%) according to the tumor grading and tumor staging. A cytometric test was positive in 80% for G3 tumours and in 68% for G2 tumours. The staging tumor was positive in 46%, 89% and 100% of the pTa-pT1, pT2 and pT4 tumours respectively. Otherwise the comparison of control group with patients showed a statistical correlation between cytometric test, staging tumour and tumoral grading as showed in the following groups: control/G1-G2 (p < 0.05), control/G3 (p < 0.001), control/pTa-pT (p < 0.05), control/pT2-pT4 (p < 0.001). CONCLUSION: We confirmed through this study the interest of the flow cytometric DNA analysis in the diagnosis and prognosis of bladder carcinomas, and we showed the importance of the histogram classification in order to facilitate their interpretation and to avoid the trap of false aneuploidy.
Carcinoma↗
Hemoglobin Pasadena: identification of the gene mutant by DNA analysis using synthetic DNA probes.
Hemoglobin Pasadena [beta 75(E19)Leu----Arg] was found in a boy who had an acute episode of anemia and rapid splenic enlargement. His father was the only other member of a large family with this hemoglobinopathy. We have used gene mapping techniques for direct identification of the beta-globin gene mutation. To correlate the DNA findings with the structural identification of this variant, we have also performed globin chain separation and analysis of the tryptic peptides using high performance liquid chromatography and secondary ion mass spectral analysis.
Adult↗
Preoperative detection of a highly malignant type of early gastric carcinoma by cytophotometric DNA analysis.
A cytophotometric DNA analysis was made of gastroendoscopically biopsied materials of 43 patients with early gastric carcinoma and the data were compared with the DNA patterns of the resected specimens of these patients. The DNA patterns could be classified into two types, Low Ploidy and High ploidy, according to the degree of dispersion of the DNA value. The Ploidy to Ploidy coincidence of the DNA patterns between biopsied material and the resected specimen in the same lesion was noted in 93 per cent (39/42). The DNA patterns of the resected specimens were consistent in the majority of cases, regardless of the areas examined, center or periphery, and mucosa or submucosa of the same lesion. This study provides evidence that a highly malignant type of early gastric carcinoma, Pen A type, could be differentiated in gastroendoscopically biopsied materials by a cytophotometric DNA analysis.
Biopsy↗
Differentiation of growth patterns of early gastric carcinoma determined by cytophotometric DNA analysis.
Cell nuclear DNA content was microspectrophotometrically measured in 60 cases of early and six of advanced gastric carcinoma. DNA distribution pattern was classified into Types I, II and III, according to the degree of dispersion on the DNA histogram. Five of 6 cases of the advanced carcinomas were Type III. In early carcinoma, the majority of Super (superficially spreading) type were Type II, while all of Pen A (expansively penetrating growth) type were Type III. In Pen B (infiltratively penetrating growth) type, six of ten cases were Type II and the other four were Type III. Thus, there was a distinct difference in the DNA pattern between Super and Pen Types, i.e., the DNA analysis reflected these growth types. Since about 20% of those with small mucosal carcinomas, not classifiable into the Super or the Pen types presented Type III, the cytophotometric DNA analysis should be useful in the detection of Pen A like carcinoma, in its early stage. The development from early to advanced carcinoma was discussed in terms of the similarity of the DNA histogram patterns.
Bone Neoplasms↗
High-throughput DNA analysis by microchip electrophoresis.
DNA analysis plays a great role in genetic and medical research, and clinical diagnosis of inherited diseases and particular cancers. Development of new methods for high throughput DNA analysis is necessitated with incoming of post human genome era. A new powerful analytical technology, called microchip capillary electrophoresis (MCE), can be integrated with some experimental units and is characterized by high-speed, small sample and reagent requirements and high-throughput. This new technology, which has been applied successfully to the separation of DNA fragments, analysis of polymerase chain reaction (PCR) products, DNA sequencing, and mutation detection, for example, will become an attractive alternative to conventional methods such as slab gel electrophoresis, Southern blotting and Northern blotting for DNA analysis. This review is focused on some basic issues about DNA analysis by MCE, such as fabrication methods for microchips, detection system and separation schemes, and several key applications are summarized.
DNA↗
Pitfalls in the analysis of mitochondrial DNA from ancient specimens and the consequences for forensic DNA analysis: the historical case of the putative heart of Louis XVII.
Amplification of mtDNA D-loop fragments with a length of 200 bp or more from ancient and even from fairly recent biological samples, can lead to erroneous results. This was clearly illustrated in our investigation of the putative heart of Louis XVII. By selecting different sets of primers which amplified shorter fragments of mtDNA (length 109 bp-201 bp), authentic polymorphisms could be visualised which remained undetected with the more classical primers for fragment sizes > 210 bp. Here we have extended those findings to other biological materials. A competitive PCR assay for quantitation of the amount of mtDNA for different fragment lengths, using a 10 bp deletion construct, was applied to ancient material and on a set of hairs of various ages of sampling (1966 up to the present). The results showed that DNA degradation started a few years after sampling. In the DNA extracts of the older hair shafts (1983-1995), the proportion of the number of short fragments to the number of long fragments is on average 4 in contrast to the most recent hair shafts. The numbers of amplifiable mtDNA copies for the hairs from 1975 and older were too small to show a clear difference. Use of long PCR fragments in such cases can yield misleading results. Use of short PCR fragments for the analysis of mtDNA from shed hair, in combination with a competitive PCR assay to determine the state of degradation, should improve the reliability of forensic mtDNA analysis considerably.
Base Sequence↗
RAPD (random amplified polymorphic DNA) analysis of Giardia DNA and correlation with isoenzyme data.
Fourteen Giardia duodenalis isolates were examined using the RAPD (random amplified polymorphic deoxyribonucleic acid) technique. Simple reproducible polymorphisms were generated using 3 different RAPD primers. The results generated by each primer were very similar and were significantly correlated with each other. These data were then compared to existing isoenzyme electrophoresis data on the same isolates. The RAPD data divided the isolates into 10 groupings or rapdemes while the isoenzyme data divided them into 10 similar zymodemes. Both methods grouped 4 isolates (BAH42, BAH44c9, BAH12c9 and BAH39c7), which comprised a phenotypically heterogeneous assemblage with respect to growth rate and metabolism, into similar groupings. The 2 methods were significantly correlated (P < 0.001). It will therefore be possible to use RAPD for the characterization of isolates of Giardia, and other parasites such as Cryptosporidium, which are refractory to cultivation in vitro.
Animals↗
Human mitochondrial DNA: analysis of 7S DNA from the origin of replication.
Heat-treated samples of human mitochondrial DNA (mtDNA) exhibited a set of three low molecular weight DNA bands in addition to the major mtDNA band when electrophoresed in polyacrylamide gels. These DNA components were seen only after heat treatment or after relaxation of the mtDNA with a restriction endonuclease. The three components were single stranded and had sizes of 550, 585, and 629 nucleotides, close to the size (600 nucleotides) estimated from contour length measurements for the 7S DNA from the D loop of human mtDNA. Hybridization of the components with restriction endonuclease fragments of known position in the mtDNA confirmed this identification. Digestion of each 7S DNA component with the restriction endonuclease Hae III produced three fragments, two of which were identical in size among the components and the third of which varied. This third fragment, shown to be from the 5' end of each component, differed in length by approximately 35 nucleotides among the components. These results suggest that human 7S mtDNA synthesis is terminated at a distinct position and that it is either initiated at one of three possible sites in the same mtDNA or that the mtDNA population consists of three subpopulations, each differing from the others by the presence or absence of a nucleotide sequence immediately adjacent to the origin of replication.
DNA Restriction Enzymes↗
Interaction of several Ni(III) complexes of peptides with DNA; analysis by DNA-fiber EPR.
EPR spectra of Ni(III) complexes of GGH, GHG, and GHK were obtained by in-situ oxidation of the Ni(II) complexes in DNA-pellet and on DNA-fibers. A species with an apical coordination of nitrogenous base of DNA was detected for Ni(III) GGH. Both GHG and GHK complexes showed the EPR signals of Ni(III) species when the ligand to metal ion ratio was 2:1. The complexes bind as Ni(III)(N6), NI(III)(N5), and Ni(III)(N4) species on DNA.
DNA↗
Interaction of RNA polymerase from Escherichia coli with DNA. Analysis of T7 DNA early-promoter sites.
A method was devised for directing RNA polymerase on a single promoter site on T7 DNA. Initiation complexes were formed on each of the three main promoter sites using one dinucleotide plus one nucleoside triphosphate. The ternary initiation complexes are resistant to rifampicin action, to inhibition by (rI)n at 0 degrees C and are stable at high salt concentrations. A minimum of a trinucleotide is required to form a stable ternary complex. To determine which promoter site was selected by RNA polymerase during initiation, the (rI)n-resistant RNA was digested by RNAse III to generate three characteristic initiator RNA fragments, resolved by gel electrophoresis. The three major promoter sites could be selected individually by using different primer and substrate combinations ApC plus ATP selected promoter A3, CpG plus CTP selected A2 and CpC plus ATP specified preferentially A1. A number of primer-substrate combinations specified each site at low salt concentration but the substrate requirement became very stringent at high salt concentration, suggesting that the postulated local opening of the promoter site could be more or less extensive, depending on the ionic strength. The minimum opening observed at high salt concentration corresponded to the insertion of a leader trinucleotide sequence. The promoter region melted by RNA polymerase at low salt concentration was (G plus C)-rich and corresponded to about 9 to 11 base pairs. Sequences of the melting recognition regions were tentatively inferred from the results.
Coliphages↗
Value of DNA analysis with multiple DNA probes for the detection of hemophilia A carriers.
Detection of hemophilia carriers is an important issue and should be addressed with great care. The allelic frequencies of three intragenic probes (Bcl I for probe p114.12, Xba I for probe p482.6, and Bgl I for probe C) and one linked probe (Bgl II for probe DX 13) are reported, together with their diagnostic yield singly and in combination. In this series, 725 individuals (405 females) in 156 families were analyzed for restriction fragment-length polymorphisms. A total of 255 females (63%) were found to be informative for their carrier state with one or more probes. The most informative intragenic probe was p482.6 (useful in 49% of informative females). The most informative probe was DX 13 (useful in 59% of informative females), but this is a linked probe that carries a 5% risk of cross-over. By the use of probes p114.12, p482.6, and DX 13, almost 98% of all the informative females could be detected. In about 71% of families with a family history and a known carrier, prenatal diagnosis was feasible.
Alleles↗
Specific labelling of replicating SPP1 DNA: analysis of viral DNA synthesis and identification of phage DNA-genes.
Specific labelling of replicating bacteriophage SPP1 DNA can be achieved by infection at nonpermissive temperature of a B. subtilis strain carrying the initation mutation dnaB ts134. Under these conditions host DNA synthesis is reduced by 90 to 95%. This technique was used to identify cistrons of SPP1 involved in phage DNA synthesis and to define intermediates in SPP1 replication.
Bacillus subtilis↗
DNA analysis on microfabricated electrophoretic devices with bubble cells.
Microfluidic devices with bubble cells have been fabricated on poly(methyl methacrylate) (PMMA) plates and have been employed for the analysis of DNA using polyethylene oxide (PEO) solutions. First, the separation channel was fabricated using a wire-imprinting method. Then, wires with greater sizes or a razor blade glued in a polycarbonate plate was used to fabricate bubble cells, with sizes of 190-650 microm. The improvements in resolution and sensitivity have been achieved for large DNA (> 603 base pair, bp) using such devices, which depend on the geometry of the bubble cell. The main contributor for optimal resolution is mainly due to DNA migration at lower electric field strengths inside the bubble cell. On the other hand, slight losses of resolution for small DNA fragments have been found mainly due to diffusion, supported by the loss of resolution when separating two small solutes. With a bubble cell of 75 microm (width) x 500 microm (depth), the sensitivity improvement up to 17-fold has been achieved for the 271 bp fragment in the separation of PhiX-174/HaeIII DNA restriction fragments. We have also found that a microfluidic device with a bubble cell of 360 microm x 360 microm is appropriate for DNA analysis. Such a device has been used for separating DNA ranging from 8 to 2176 bp and polymerase chain reaction (PCR) products amplified after 30 cycles, with rapidity and improvements in the sensitivity as well as resolution.
Base Sequence↗
Flow cytometric DNA analysis of submucosal carcinoma of the esophagus.
Carcinoma of the esophagus confined to the submucosa presents clinicopathologic features different from those of either carcinoma limited to the mucosa or more advanced lesions. The present study was designed to find out whether or not analysis of DNA content would yield prognostic information allowing the clinical outcome of carcinoma of the esophagus confined to the submucosa to be predicted. Cell nuclear DNA content was determined by flow cytometry in paraffin-embedded blocks from 55 patients with carcinoma of the esophagus confined to the submucosa. This allowed classification of DNA content into diploid and aneuploid patterns based on combined DNA histogram and cytogram data. The aneuploid pattern included typical, multiple clone and small clone aneuploid subtypes, as classified by our new criteria. A DNA aneuploid pattern was identified in 26 cases (47.3%) and found to correlate significantly with lymph node metastasis (P < 0.01). The incidence of distant metastasis was significantly higher in the aneuploid (4/26) than in the diploid (0/29) group (P < 0.05). The mortality from cancer within two years of surgery was significantly higher in the aneuploid (4/26) than in the diploid (0/29) group (P < 0.05). In conclusion, flow-cytometric DNA analysis is a valuable method for predicting malignant potential in carcinoma of the esophagus confined to the submucosa.
Adult↗