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PubMed2002

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↗
PubMed1994

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↗
PubMed2001

Relationships among DNA Index, S-Phase, and invasive behavior in anterior pituitary adenomas. A cytometric study of 61 cases with Feulgen-positive DNA analysis.

BACKGROUND: Pituitary adenomas are usually well differentiated neoplasms, although in about 1/3 of cases they invade the surrounding dura mater and bone, as confirmed by surgical findings, resulting in a long-term possibility of relapse. METHODS: To identify the cellular growth rate and to correlate it with surgical evidence of invasiveness, we performed the analysis of DNA with static cytometric quantitation on fresh surgical specimens, using a computer-assisted image processor. The DNA index and the percentage of cells in S-phase (%SPh) were obtained in 61 pituitary tumors consecutively operated on. In relation to surgically verified infiltration of dura and bone, we identified 39 noninvasive and 22 invasive adenomas. The cavernous sinus (CS) was infiltrated in 13 cases. On the basis of immunohistochemical staining and endocrine activity we recognized 27 nonsecreting and 34 secreting adenomas. RESULTS: The DNA content was aneuploid in 33 cases (11 nonfunctioning, 22 functioning; p = 0.05); there was no correlation with the invasive behavior of the adenomas. The DNA index ranged between 0.93 and 2.50 (median 1.13); the range of %SPh was 0-12.00% (median 2.54%). In invasive adenomas the mean DNA index was 1.33 (p not significant) and the mean %SPh was 4.03% (p = 0.05). In CS-infiltrating pituitary adenomas, the mean DNA index was 1.44 (p = 0.04) and the mean %SPh was 4.52% (p = 0.05). CONCLUSIONS: Our preliminary results seem to reveal a correlation between DNA index, %SPh, and invasive behavior of pituitary adenomas, encouraging the use of DNA analysis in the prognostic evaluation of these tumors.

Adenoma↗
PubMed1995

Germ line mutations of hMSH2 and hMLH1 genes in Japanese families with hereditary nonpolyposis colorectal cancer (HNPCC): usefulness of DNA analysis for screening and diagnosis of HNPCC patients.

Mutations in hMSH2 and hMLH1 genes were analyzed in patients from 11 Japanese families that had been diagnosed as carrying hereditary nonpolyposis colorectal cancer (HNPCC) by clinical examination. Germ line mutations of hMSH2 gene were identified in 5 independent families in which colorectal (87% of patients), endometrial (30%), ovarian (17%), gastric (14%), and other cancers existed. Five mutations detected between codons 136 and 811 included single-base substitutions (C-->T and T-->G), a T deletion, and an A insertion, all of which produced stop codons resulting in truncated proteins, and an A-->T substitution at splice donor site of exon 5 which resulted in deletion of this exon. Moreover, one HNPCC family was presumed to have germ line mutation of hMSH2 gene because a somatic mutation of hMSH2 gene was detected in a cancer from a patient in this family. In addition to these 11 families already diagnosed with HNPCC, 3 new families with germ line mutations of hMSH2 gene and hMLH1 gene were found through analysis of DNA from patients who had multiple cancers with alteration in microsatellite DNA. These mutations included an AG deletion at codons 877-878 of hMSH2 gene, an AAG deletion at codons 616-618 of hMLH1 gene, and a C-->T single-base substitution at codon 217 of hMLH1 gene. Seven of eight germ line mutations found in this study are new mutations that have not been reported previously. In families in which germ line mutations were identified presymptomatic examination was then carried out using polymerase chain reaction single-strand conformation polymorphism analysis of DNA from peripheral blood, and the result was the detection of family members predisposed to HNPCC who did not yet show signs of cancer. These results indicate the value of DNA analysis in the screening and diagnosis of HNPCC patients and families.

Adult↗
PubMed1996

Method to make paraffin-embedded breast and lymph tissue mimic fresh tissue in DNA analysis.

Determination of DNA ploidy has been found to be of diagnostic and prognostic value with regard to many solid tumors. Flow cytometric analysis of DNA ploidy is dependent on the binding of fluorescent dyes to DNA. Preserving cell morphology by fixing the tissue in formalin interferes with the binding of propidium iodide (PI) and other fluorescent dyes to DNA. This distortion of DNA content measurement can cause inaccuracies in DNA-ploidy determinations of formalin-fixed tissue specimens and precludes the use of appropriate DNA standards. Therefore, it has been impossible to determine accurately the DNA ploidy of formalin-fixed, paraffin-embedded (FFPE) tissues. Using formalin-fixed cells as a model for FFPE cells, we developed a thermal treatment method to reverse the effect of formalin on the binding of propidium iodide to DNA. Applying this approach to the preparation of FFPE lymph node and breast tissue for DNA analysis, we have developed a method that makes the binding of PI to the DNA of FFPE tissue mimic that of fresh tissue. Following dewaxing, rehydration, and trypsin treatment, the FFPE tissue, resuspended in PBS, was heated to 75 degrees C for 90 min to restore the PI binding to that of fresh cells. This method makes it possible to use fresh, DNA-diploid cells as an internal control and, thus, determine more accurately the DNA ploidy of tumors preserved in formalin and paraffin.

Aneuploidy↗
PubMed1990

A review of techniques and results obtained in one laboratory by an integrated system of methods designed for routine clinical flow cytometric DNA analysis.

Establishing flow cytometric DNA analysis as a clinical routine procedure requires adequate and proven guidelines, by which the data can be obtained and interpreted to directly influence management of the individual patient with a specific neoplasm. The present paper is intended as a contribution to such guidelines, of which only fragments are available today. We have previously described a system of methods, designed for routine flow cytometric DNA analysis. In the present status report our experience, based on approximately 18,000 samples (clinical and experimental) is summarised. Sample acquisition with fine-needle aspiration, storage at -80 degrees C, internal standardization by chicken (CRBC) and trout red blood cells (TRBC), staining with propidium iodide (PI), and analysis in the flow cytometer is recapitulated, with emphasis on previously unpublished aspects. The method of statistical analysis which has an integrating role is described in some detail. A lack of linearity between channel number and DNA content was determined experimentally, and the coefficient of variation (CV) was found to decrease with increasing channel number. The corrections in the algorithm of deconvolution made necessary by these findings are fundamental for estimating the end results. The zero point adjustment and procedures for changing from one batch of standards to another are described. A systematic approach to interpretation of DNA histograms is attempted and illustrated by data from clinical specimens of malignant lymphoma, breast cancer, small cell lung cancer, cancer of the oral cavity, and bladder cancer. Some problems are still unsolved and visual inspection is required to determine if the quality of the individual histogram is satisfactory. Inspection of the fluorescence/light scatter dot-plot provides additional information for the recognition of artifacts. The results stress that good quality DNA histograms with as small CVs as possible are important for interpretation of the data. It is essential that statistical methods are employed to extract the key end-point results. These are the number of subpopulations and their relative representation, and for each subpopulation the DNA index (DI) and the fractions of cells in the cell cycle phases. For the DNA data to have any rationally based impact on clinical decision making, it must be demonstrated that they have an independent prognostic value. Strategies for final evaluation are discussed. Multicenter trials on fresh material, to accrue quickly the number of patients necessary for firm conclusions, are suggested.

DNA↗
PubMed1992

Diffuse and localized tenosynovial giant cell tumor and pigmented villonodular synovitis: a clinicopathologic and flow cytometric DNA analysis.

The DNA content and proliferative indexes of seven cases of tenosynovial giant cell tumor of tendon sheath, diffuse type (TGCT-D); 11 cases of tenosynovial giant cell tumor of tendon sheath, localized type (TGCT-L); and seven cases of pigmented villonodular synovitis (PVNS) were analyzed by flow cytometry in an attempt to assess objectively their biologic differences. Three cases of TGCT-D manifested an aneuploid DNA content and four had a diploid DNA pattern. All cases of TGCT-L and PVNS showed a diploid DNA content. The proliferative indexes for TGCT-D were significantly higher than those found in the other two groups. There was no histopathologic feature that correlated with the aneuploid DNA pattern found in two of the three cases of TGCT-D. Only one of the three aneuploid DNA content TGCT-D cases displayed marked cellular pleomorphism with dense fibrous stroma; in that case there was recurrence 4 years after initial excision. Our data further support that TGCT-D, TGCT-L, and PVNS are histopathologically similar but clinically distinct lesions. The high proliferative indexes of TGCT-D may reflect a rapid, uncontrolled growth that may explain its aggressive biologic behavior. The presence of an aneuploid DNA pattern in some cases of TGCT-D in this study, coupled with the reported chromosomal abnormalities and occurrence of malignant transformation in these lesions, clearly supports their neoplastic nature.

Adult↗
PubMed1996

The Drosophila montium subgroup species. Phylogenetic relationships based on mitochondrial DNA analysis.

Mitochondrial DNA (mtDNA) restriction site maps for three Drosophila montium subgroup species of the melanogaster species group, inhabiting Indian and Afrotropical montium subgroup territories, were established. Taking into account previous mtDNA data concerning six oriental montium species, a phylogeny was established using distance-matrix and parsimony methods. Both genetic diversity and mtDNA size variations were found to be very narrow, suggesting close phylogenetic relationships among all montium species studied. The phylogenetic trees that were constructed revealed three main lineages for the montium subgroup species studied: one consisting of the Afrotropical species Drosophila seguyi, which is placed distantly from the other species, one comprising the north-oriental (Palearctic) species, and one comprising the southwestern (south-oriental, Australasian, Indian, and Afrotropical) species. The combination of the mtDNA data presented here with data from other species belonging to the melanogaster and obscura subgroups revealed two major clusters: melanogaster and obscura. The melanogaster cluster is further divided into two compact lineages, comprising the montium subgroup species and the melanogaster complex species; the species of the other complex of the melanogaster subgroup, yakuba, disperse among the obscura species. The above grouping is in agreement with the mtDNA size variations of the species. Overall, among all subgroups studied, the species of the montium subgroup seem to be the most closely related.

Animals↗
PubMed2001

Ancient DNA analysis reveals divergence of the cave bear, Ursus spelaeus, and brown bear, Ursus arctos, lineages.

The cave bear, Ursus spelaeus, represents one of the most frequently found paleontological remains from the Pleistocene in Europe. The species has always been confined to Europe and was contemporary with the brown bear, Ursus arctos. Relationships between the cave bear and the two lineages of brown bears defined in Europe, as well as the origins of the two species, remain controversial, mainly due to the wide morphological diversity of the fossil remains, which makes interpretation difficult [1, 2]. Sequence analysis of ancient DNA is a useful tool for resolving such problems because it provides an independent source of data [3]. We previously amplified a short DNA fragment of the mitochondrial DNA control region (mt control region) of a 40,000-year-old Ursus spelaeus sample [4]. In this paper, we describe the DNA analysis of two mtDNA regions, the control region and the cytochrome b gene. Control region sequences were obtained from ten samples of cave bears ranging from 130,000 to 20,000 years BP, and one particularly well-conserved sample gave a complete cyt b sequence. Our data demonstrate that cave bears split largely before the lineages of brown bears around 1.2 million years ago. Given its abundance, its wide distribution in space and time, and its large morphological diversity, the cave bear is a promising model for direct observation of the evolution of sequences throughout time, extinction periods, and the differentiation of populations shaped by climatic fluctuations during the Pleistocene.

Animals↗
PubMed2002

Differential chemosensitivity in human intracerebral gliomas measured by flow cytometric DNA analysis.

The present study was designed to investigate the chemosensitivity of human intracerebral gliomas using the flow cytometric (FCM) analysis of DNA integrity as an in vitro drug sensitivity testing. We also correlated the in vitro results and the clinical responses to chemotherapy. Forty-one fresh tumor specimens were obtained at surgery, and exposed to 30 currently-available anticancer agents. Drug-induced nuclear damage such as chromatin condensation or DNA degradation were assessed both by morphological observation and FCM analysis of DNA integrity. The FCM assay could be performed in all the cases (100% success rate). The chemosensitivities of anaplastic astrocytoma, oligodendroglial tumors, and medulloblastoma were generally higher than those of glioblastoma multiforme and ependymoma. The in vitro chemosensitivity was markedly heterogeneous among patients with the same histological tumor. Clinical sensitivity was predicted in 86% of all evaluable patients and clinical resistance in 81%; the overall accuracy of the FCM assay was 82%. The FCM analysis of DNA integrity proved to be feasible and sufficiently reliable as a routine clinical examination for the purpose of screening new chemotherapeutic agents and individualizing chemotherapy regimens for patients with intracerebral gliomas.

Antibiotics, Antineoplastic↗
PubMed1989

Carcinoma of the anal canal and flow cytometric DNA analysis.

Using flow cytometric DNA analysis of paraffin embedded tissue, DNA histograms were successfully obtained from the anal cancers of 117 patients. DNA diploid patterns were given by 82 cancers (70%) and DNA non-diploid patterns by 35 cancers (30%): 15 DNA aneuploid, 20 DNA tetraploid. Well differentiated squamous cell cancers were mainly DNA diploid, while a larger proportion of poorly differentiated and small cell cancers were DNA non-diploid. The large majority of stage A cancers were DNA diploid. A greater proportion of tumours that had invaded through the anal sphincter or had lymph node metastases or distant spread were DNA non-diploid. Prognosis was slightly poorer for patients with DNA non-diploid cancers when compared to patients with DNA diploid tumours (P = 0.08) and significantly poorer for individuals with DNA aneuploid anal cancers (P = 0.037). However, in a multivariate analysis model, the DNA ploidy pattern of an anal cancer was not of independent prognostic significance alongside tumour histology and tumour stage.

Aged↗
PubMed1994

[DNA analysis in classic phenylketonuria--screening for mutations and haplotype analysis in Slovak families].

Authors in this contribution present the results of screening for mutations in PAH gene responsible for classical phenylketonuria (PKU), and that of haplotype analysis, based on DNA analysis in 49 Caucasian families with at least one affected child from Slovak Republic. The clearly predominant PKU mutation in this population was the R408W with proportion of 45.9% among all PKU mutations. In addition four other mutations have been identified: IVS12nt1-10.2%, R158Q-7.1%, R261Q-7.1%, and R252W-2.0%. the overall proportion of identified PKU mutations equals 72.4%. Considering the fact, that these mutations are amenable to rapid and rather simple detection using PCR, the DNA analysis is recommended as a method of direct diagnosis in clinical practice as well as in prevention.

DNA↗
PubMed1986

Methylation blockage and other improvements to a comprehensive DNA analysis program.

A comprehensive DNA analysis computer program was described in the second special issue of Nucleic Acids Research on the applications of computers to research on nucleic acids by Stone and Potter (1). Criteria used in designing the program were user friendliness, ability to handle large DNA sequences, low storage requirement, migratability to other computers and comprehensive analysis capability. The program has been used extensively in an industrial-research environment. This paper talks about improvements to that program. These improvements include testing for methylation blockage of restriction enzyme recognition sites, homology analysis, RNA folding analysis, integration of a large DNA database (GenBank), a site specific mutagenesis analysis, a protein database and protein searching programs. The original design of the DNA analysis program using a command executive from which any analytical programs can be called, has proven to be extremely versatile in integrating both developed and outside programs to the file management system employed.

Base Sequence↗
PubMed1991

[Study of incidental carcinoma of the prostate. II. Nuclear DNA analysis].

The clinical significance of cytofluorometric nuclear DNA analysis, ploidy pattern and DNA content, was investigated in 47 incidental prostatic carcinomas, 24 stage A1 and 23 stage A2 cases, 9 clinically advanced cases and 25 BPHs. The results were compared to clinical stage and histological differentiation. The mean nuclear DNA content of stage A1 cancer, which was similar to BPH, differed from that of stage A2 cases. The latter was almost identical to that of advanced cases. In moderately and poorly differentiated carcinomas it was higher than that of well differentiated ones. A non-diploid pattern was distributed in 33% of stage A1, 78% of stage A2 and 89% of clinically advanced cases. It was detected in 42% of the well-, 77% of the moderately- and 100% of the poorly differentiated adenocarcinomas. The DNA analysis of incidental prostatic cancer thus correlated well to the clinical and pathological parameters. If limited to well differentiated carcinomas, however, 17% of the stage A1 cases showed an aneuploid, and 29% of the stage A2 cases, a diploid pattern. No diploid pattern was detected in clinically advanced cases. Although we have not been able to prove any difference in prognosis in the present cases, these findings suggest that the nuclear DNA analysis is another parameter in defining the prognosis of incidental prostatic carcinoma. Further follow-up of the patients and accumulation of the data are necessary to determine the clinical validity of this method.

Adenocarcinoma↗
PubMed1999

Virtual DNA analysis as a platform for interlaboratory data exchange of HLA DNA typing results.

In 1998, the German DNA Exchange offered the possibility to report typing data as virtual DNA. Participating labs have been equipped with software based on the principle of Virtual DNA Analysis (VDA). This approach allows the combination of sequence-specific oligonucleotide (SSO), sequence-specific primer (SSP) and sequence-based typing (SBT) results. The use of all types of test kits has been allowed without any limitations, as long as basic sequence information on SSOs or SSPs was available, at least the sequence and the position of the detected motif on the sample DNA. Typing raw data of the actual SSO-SSP and, if performed, SBT information was collected. Participating labs received 20 DNA samples to type. Fourteen labs returned data on 1,250 single-locus testings. Reported data consisted of 317 SBT data, 452 SSO kits and 1,795 SSP kits with 43,312 single SSO/ SSP reactivities. One hundred and twenty-six different typing kits (unique laboratory-specific kits, commercial kits from 7 companies) have been used. In 30 (2.4%) single-locus testings, at least one single SSO/SSP reactivity has been false-positive or -negative, thus not leading to a valid result on primary evaluation. Eight of these 30 cases were due to the presence of a new DRB1*14 allele in sample no. 2. Thirty-five tests (2.8%) showed wrong allele assignments. This first attempt to collect raw typing data instead of typing interpretation on a larger scale shows the advantages of Virtual DNA Analysis like interlaboratory data exchange without loss of information, transparency of typing interpretation and reinterpretation of typing data with an updated allele database. The VDA format is a useful tool for workshops and bone marrow donor registries.

Alleles↗
PubMed1993

[Individualized DNA analysis in insurance medicine clarifications].

DNA investigations for the clarification of problems in insurance medicine can be subdivided into 3 groups (1) Traffic accidents (2) identification (3) questionable fraud cases. The development in individualisation by DNA analysis has progressed from the initial restriction fragment length polymorphisms (RFLPs) to the PCR-based polymorphisms. PCR systems are characterised by a very high sensitivity of detection and a high degree of success with highly degraded biological material. The current possibilities for DNA analysis are described and exemplified with special reference to PCR-based polymorphisms.

Accidents, Traffic↗
PubMed1997

Restriction fragment length polymorphism analysis and random amplified polymorphic DNA analysis of Campylobacter jejuni strains isolated from patients with Guillain-Barré syndrome.

Campylobacter jejuni serotype O19 strains associated with the Guillain-Barré syndrome (GBS) and other strains were examined by restriction fragment length polymorphism (RFLP) analysis of polymerase chain reaction products of the flaA genes and by random amplified polymorphic DNA (RAPD) analysis. RFLP analysis showed that regardless of LIO serotype, geographic origins, or association with GBS, the O19 isolates shared an identical digestion pattern by each of four restriction endonucleases, DdeI, MboI, MseI, and AluI. In contrast, among C. jejuni O1 or O2 strains, RFLP patterns were different even among strains of the same LIO serotype. The results of the RAPD analysis were consistent with the flaA RFLP data. These data indicate that all of the O19 strains that were tested were closely related to one another whether they were or were not associated with GBS.

Animals↗
PubMed1988

DNA analysis of stimulated lymphocytes by automatic sampling for flow cytometry.

A microsample delivery system (MSDS) was tested for automatic flow cytometry (FCM) analysis of DNA synthesis in stimulated human peripheral blood lymphocytes (PBL) cultivated in wells of microtiter plates. After incubation, either for 1-3 days with phytohemagglutinin, concanavalin A, and pokeweed mitogen, or for 7 days with allogenic PBL, the cells, while in the wells, were washed in hypotonic Tris buffer and stained with ethidium bromide-RNAse solution. The results obtained from quintuplicate replicated wells, each of the five containing the same control or stimulated cultures, were reproducible in terms of the number of nuclei counted in each histogram of control, mitogen-stimulated PBL, and mixed lymphocyte cultures (MLC). Using a computer program that superimposes histograms and calculates their differences on the scale of fluorescence intensity, it was possible to quantify the intensity of the response to the mitogenic stimuli. This approach to the study of lymphocyte proliferation offers not only a simpler and faster analysis of DNA synthesis than the method of 3H-thymidine incorporation, but it also allows for the analysis of other FCM parameters, such as forward and 90 degrees light scatter and double fluorescence labelling of PBL nuclei.

Cell Cycle↗