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PubMed1993

Flow cytometric DNA analysis does not predict the radiochemoresponsiveness of esophageal cancer.

The relationship between the DNA pattern and the responsiveness to chemotherapy or chemoradiotherapy has been evaluated in 30 patients with squamous cell carcinoma of the esophagus. In 24 patients polychemotherapy with cisplatin (100 mg/m2 on day 1) and 5-fluorouracil (1,000 mg/m2/24 h, continuous infusion of 120 h) every 3 weeks, was performed. Six other patients received chemoradiotherapy with cisplatin 80 mg/m2 on day 1 and 18.5 Gy (split course). Before treatment, at least three endoscopic biopsies were taken from each tumor and frozen at -85 degrees C. Five patients were excluded from the evaluation, three because of interrupted treatment and two due to unsuitable biopsy material obtained endoscopically. The response rate to the cytoreductive treatment was 40% (10/25). DNA content was analyzed with flow cytometry. Out of 25 evaluable patients, a diploid and aneuploid tumor was present in 8 (32.0%) and 17 (68.0%) patients, respectively. According to the DNA pattern, a major response was observed in 4 of 8 patients with a diploid tumor and in 6 of 17 patients with an aneuploid tumor (P = 0.5). No relationship between the percentage of cells in the S-phase and the response to the cytoreductive treatment was evident. Although a slightly higher percentage of major responses was found in euploid tumors, there is no evidence that flow-cytometric DNA analysis can be helpful in the selection of patients for chemotherapy or chemoradiotherapy.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed1982

Clonal evolution demonstrated by flow cytometric DNA analysis of a human colonic carcinoma grown in nude mice.

A spontaneous change in DNA content of a human colonic carcinoma grown in nude mice was observed fortuitously. The tumor initially had a G1 cell DNA content of 1.3 times that of normal cells. Flow cytometric DNA analysis showed in transplant generation 56 the appearance of a new subpopulation which in three passages completely overgrew the original population. The DNA content of the new subpopulation was twice that of the original population. The observation supports the hypothesis of clonal evolution of tumor cell populations. The growth rates of the tumor before and after the change showed no significant difference (p greater than 0.05). Cell kinetic factors, therefore, offer no obvious explanation of how the overgrowth took place. It is not known whether the original population disappeared completely or survived as a small population below the detection limit. The heterogeneity created by clonal evolution of a tumor would be less pronounced if old subpopulations often become extinct as new ones emerge. Heterogeneity of human tumors is of clinical importance because the individual subpopulations may have different sensitivity patterns to antineoplastic drugs.

Animals↗
PubMed1986

Programming for the DNA analysis of FCM data on an IBM microcomputer.

The analysis of data generated on a flow cytometer (FCM) is often performed on a computer obtained especially for dedicated use with the flow cytometer. This computer component can be expensive and also presents the FCM user with the added burden of mastering specialized programming language or of accepting the secret analytical processes of protected proprietary program routines. We believe that the evolution of more accurate and efficient FCM analyses that have the power to consider complex signal distributions can be assisted by the availability of analysis programs written in languages common to many users. DNA analysis routines written for a relatively inexpensive microcomputer (IBM PC/XT) in Basic and Pascal are described here. The routines can automatically process multiple FCM data files and can provide high-resolution graphic hardcopy. A foreground/background utilization is also described that allows the computer to be available for other uses in the laboratory.

Animals↗
PubMed1984

Identification of Campylobacter coli isolates from animals and humans by bacterial restriction endonuclease DNA analysis.

Ninety-nine Campylobacter coli isolates were examined by bacterial restriction endonuclease DNA analysis (BRENDA) with HindIII. Isolates from poultry from the same environment had identical patterns, patterns of isolates carried by suckling piglets were generally the same as those of isolates recovered from their dams, and one human patient yielded the same BRENDA type when sampled 6 weeks later. The 14 human isolates examined produced 11 distinct BRENDA types. Forty-three C. coli isolates from pigs were represented by 20 BRENDA types. Ten C. coli isolates from the feces of gulls yielded five different BRENDA types. Thirty-two C. coli isolates from live chickens and processed chicken yielded five different BRENDA types. Three human isolates had identical DNA patterns; two were from brothers living in the same house, and the third was from a human with no apparent relationship to the brothers. Another human isolate was identical to a poultry isolate. None of the pig strains had DNA patterns resembling those of human strains, nor were the DNA patterns like those of any strains recovered from poultry or gulls. Four C. coli isolates were subcultured onto agar 23 times over a period of 45 days, and their BRENDA patterns were preserved. BRENDA shows great promise for use in epidemiological studies of C. coli.

Animals↗
PubMed2004

Circle-to-circle amplification for precise and sensitive DNA analysis.

We present a tightly controlled process for strand-specific amplification of circularized DNA molecules. Tandem repeated complements of DNA circles are generated by rolling-circle replication, and converted to monomer circles of opposite polarity to that of the starting material. These circles are then subjected to one more round of rolling-circle replication and circularization, and the process can be further repeated. The method can be directed to produce single-stranded circular or linear monomers, or linear concatemers of the desired polarity. The reaction is not product inhibited, and can yield approximately 100-fold higher concentrations of monomer products than PCR. Each generation of the amplification process proceeds in a linear fashion, ensuring precise quantification. The procedure is suitable for parallel amplification of large numbers of DNA circles, because the few cycles and the robust reaction mechanism preserves the proportion of amplified molecules. We demonstrate the utility of the method for multiplexed genotyping of polymorphic loci and for quantitative DNA analysis.

Base Sequence↗
PubMed1995

The value of cytometric DNA analysis as a prognostic tool in neuroendocrine neoplastic diseases.

In several traditionally non-endocrine, common, human, neoplastic diseases, it has become well established during the last few years, that cytometric analyses of the DNA distribution pattern of the nuclei of tumour cells can be an excellent supplement to the conventional prognostic tools, (such as clinical staging and histopathologic malignancy assessments). When analogous studies of the value of DNA analysis by means of flow cytometry and/or image cytometry are made in neuroendocrine (NE) neoplastic diseases, the ensuing results often become rather disappointing. Thus, clear-cut aneuploid DNA histograms can be found in the neoplastic cell nuclei of clinically and histopathologically completely benign NE adenomas (and even hyperplastic nodules). In contrast, highly aggressive NE carcinomas not seldom reveal themselves to be composed of tumour cells with nuclei, displaying an euploid, i.e. normal, DNA pattern. Statements of this kind have been based on the results of comprehensive investigations in several laboratories, analysing such NE tumours as insulomas/insular carcinomas, bronchial/gastrointestinal carcinoids, phaeochromocytomas, paragangliomas, neuroblastomas, adenomas of the anterior pituitary gland, parathyroid adenomas, medullary carcinoma of the thyroid and Merkel-cell tumours of the skin. Thus, the prognostic value of the cytometric DNA ploidy pattern of the nuclei of neoplastic parenchymal cells is definitely lower in NE tumours than in most of the traditionally non-endocrine carcinomas and sarcomas. Data from published and unpublished series of these kinds of NE tumours, and those of prostatic and breast carcinomas with NE differentiation, are given. By means of a new, consecutive double staining technique, it was shown that in idiopathic nesidioblastosis, the hyperinsulinism is caused by beta cells with a nuclear DNA ploidy pattern of euploid type. By the same technique, it can be shown that in the pathogenesis of the hypergastrinaemia-induced ECL-cell carcinoids of the stomach, a switch from an euploid to an aneuploid nuclear DNA distribution pattern occurs in the ECL-cells when they pass from a state of hyperplasia to that of a genuine neoplasia. In neuroblastomas, a triploid (i.e. aneuploid) DNA pattern is part of an algorithm capable of predicting a 96% survival rate, whereas a diploid/tetraploid (i.e. euploid) DNA pattern predicts a 0% survival.

DNA, Neoplasm↗
PubMed1999

Mutation identification DNA analysis system (MIDAS) for detection of known mutations.

We introduce a novel experimental strategy for DNA mutation detection named the Mismatch Identification DNA Analysis System (MIDAS) [1, 2], which has an associated isothermal probe amplification step to increase target DNA detection sensitivity to attomole levels. MIDAS exploits DNA glycosylases to remove the sugar moiety on one strand (the probe strand) at a DNA base pair mismatch. The resulting apyrimidinic/ apurinic (AP) site is cleaved by AP endonucleases/lyases either associated with the DNA glycosylase or externally added to the reaction mixture. MIDAS utilizes 32p- or FITC-labeled oligonucleotides as mutation probes. Generally between 20-50 nucleotides in length, the probe hybridizes to the target sequence at the reaction temperature. Mismatch repair enzymes (MREs) then cut the probe at the point of mismatch. Once the probe is cleaved, the fragments become thermally unstable and fall off the target, thereby allowing another full-length probe to hybridize. This oscillating process amplifies the signal (cleaved probe). Cleavage products can be detected by electrophoretic separation followed by autoradiography, or by laser-induced fluorescence-capillary electrophoresis (LIF-CE) of fluorophore-labeled probes in two minutes using a novel CE matrix. In the present experiments, we employed the mesophilic Escherichia coli enzyme deoxyinosine 3'-endonuclease (Endo V), and a novel thermostable T/G DNA glycosylase, TDG mismatch repair enzyme (TDG-MRE). MIDAS differentiated between a clinical sample BRCA 1 wild-type sequence and a BRCA1 185delAG mutation without the need for polymerase chain reaction (PCR). The combination of MIDAS with LIF-CE should make detection of known point mutations, deletions, and insertions a rapid and cost-effective technique well suited for automation.

BRCA1 Protein↗
PubMed1992

Diagnostic approaches to renal genetic disorders using DNA analysis.

Recent developments in molecular genetic technology have made it possible to diagnose many genetic disorders affecting the kidney before they are clinically manifest. For a disorder to be diagnosed by DNA analysis, either the causative gene must be known and cloned, or a closely linked DNA segment must have been identified. If one of these criteria is met, the disorder may be diagnosed either by direct detection of a mutation, if it is known, or indirectly by linkage analysis of the region using closely linked genetic markers. The methodology currently employed for direct detection of mutation includes the Southern blot, which will detect large structural alterations of genes or mutations altering a restriction recognition site, or the use of allele-specific oligonucleotides, which will detect specific point mutations. Linkage analysis is performed on DNA from multiple family members of the person at risk. Polymorphic markers are "tracked" in the family to determine the allele segregating with the disease gene. These methods are now routinely applied to the diagnosis of mendelian disorders affecting the kidney. It is anticipated that progress over the next decade will extend these applications to detection of the genetic component(s) contributing to multifactorial conditions.

Blotting, Southern↗
PubMed1990

Bone metastases of medulloblastoma in childhood; correlation with flow cytometric DNA analysis.

Thirty children with medulloblastoma who were treated between 1980 and 1986 are presented. Flow cytometric DNA analysis using formalin-fixed and paraffin-embedded tissues showed that 15 were DNA aneuploid, 13 diploid and 2 tetraploid. After homogeneous treatment with a combination of radical resection and radiation therapy, patients with DNA aneuploid medulloblastoma showed significantly better outcome (p = 0.003). Among these 30 patients, 6 (20%) had bone metastases of medulloblastoma during a minimum of 2-year follow-up period. DNA ploidy of these patients was diploid in 5 and aneuploid in 1. Incidence of bone metastases appears to correlate with DNA ploidy (p = 0.046). At the time of discovery of bone metastases, 4 had no demonstrable recurrence in the central nervous system (CNS). Radionuclide bone scan was more sensitive in disclosing these lesions, while only one patient had positive results of bone marrow aspirates/biopsy. Three patients were treated with chemotherapy, two had a combination of regional irradiation and chemotherapy and one refused treatment. All treated bone metastases in 5 patients responded clinically and radiographically, but only one patient is alive 66 months after therapy. Four patients died due to either CNS recurrence of septic complication of chemotherapy. Early detection of bone metastases by use of frequent bone scans is important. Patients with diploid medulloblastoma should be treated with adjuvant chemotherapy in order to prevent recurrences and metastases.

Adolescent↗
PubMed1992

Evolutionary history of the honey bee Apis mellifera inferred from mitochondrial DNA analysis.

Variability of mitochondrial DNA (mtDNA) of the honey bee Apis mellifera L. has been investigated by restriction and sequence analyses on a sample of 68 colonies from ten different subspecies. The 19 mtDNA types detected are clustered in three major phylogenetic lineages. These clades correspond well to three groups of populations with distinct geographical distributions: branch A for African subspecies (intermissa, monticola, scutellata, andansonii and capensis), branch C for North Mediterranean subspecies (caucasica, carnica and ligustica) and branch M for the West European populations (mellifera subspecies). These results partially confirm previous hypotheses based on morphometrical and allozymic studies, the main difference concerning North African populations, now assigned to branch A instead of branch M. The pattern of spatial structuring suggests the Middle East as the centre of dispersion of the species, in accordance with the geographic areas of the other species of the same genus. Based on a conservative 2% divergence rate per Myr, the separation of the three branches has been dated at about 1 Myr BP.

Animals↗
PubMed1992

Nonfamilial and unusual cases of Leber's hereditary optic neuropathy identified by mitochondrial DNA analysis.

Peripheral blood mitochondrial DNA (mtDNA) samples from 11 patients with acute optic neuritis or insidious optic atrophy were examined for the mutation at nt 11778 and nt 3460 in polymerase chain reaction products. The mtDNA mutation at nt 11778 was evident in 8 cases, which led to a definite diagnosis of Leber's hereditary optic neuropathy (LHON); 4 of the cases were familial and the remaining 4 cases were nonfamilial. None of the 11 patients showed the nt 3460 mutation. The symptoms and signs were variable among the LHON cases with nt 11778 mutation. Assessment of mtDNA provides a useful diagnostic aid for clinically undefined, seemingly nonfamilial or atypical cases of Leber's hereditary optic neuropathy, particularly in bilateral, insidious optic nerve disease in early childhood.

Acute Disease↗
PubMed2004

Bone metastases: prognostic applications of cytometric DNA analysis.

OBJECTIVE: To examine DNA parameters as prognostic factors for developing metastases. STUDY DESIGN: Image cytometry was used to determine DNA content of 21 tumors and 28 metastases. DNA ploidy status, 2c deviation index (2cDI) and DNA malignancy grade (DNA-MG) (based on the variation of nuclear DNA content of tumor cells around the normal DNA [2c] peak) were examined for their prognostic value. RESULTS: Twenty of 21 tumors showed aneuploid content, and 1 tumor showed diploid DNA content. Twenty-one bone metastases showed aneuploid cells. In 6 cases both euploid and aneuploid cells were detected. In 1 metastasis only euploid cells were present. DNA-MG was increased in bone metastases (mean, 2.4) as compared to the corresponding primary tumor (mean, 2.2) in most of the cases. The mean value of the 2cDI was 30.07 in primary tumors and 42.5 in metastases. Twelve bone metastases had a higher 5cEE than did the primary tumor. CONCLUSION: Diploid and aneuploid cells were able to leave a tumor and establish metastases. DNA-MG and 2cDI were increased in metastases in comparison with the primary tumor, but even tumors with lower DNA-MG had metastatic potential.

Adult↗
PubMed1997

[Genetic divergence of Cordyceps sinensis as estimated by random amplified polymorphic DNA analysis].

Random amplified polymorphic DNA(RAPD) has been used in analyzing 13 winter worm, summer grasses (Cordyceps sinensis [Berk.], Sacc.) from 5 areas in Qingzang plateau. For 19 arbitrary primers, each individual showed about 65 RAPD markers. The genetic distances among those 13 winter worm, summer grasses were correlated to their geographic distances. Molecular phylogenetic tree constructed by UPGMA and NJ suggested that 13 winter worm, summer grasses could be divided into 3 clusters: east cluster, north cluster and middle cluster, in consistence with their origins and genetic divergence among different clusters. Furthermore, our results implied that the RAPD markers of winter worm, summer grasses showed a geographic specificity and could be a better genetic marker in studying genetic diversity and phylogeny of winter worm, summer grasses.

Genetic Variation↗
PubMedDate not supplied

[Measuring precision in determining the length of restriction fragments within the scope of DNA analysis].

Because of the measurement error that is known from the length determination of restriction fragments during DNA analysis, it is necessary to determine the precision by which identical DNA can be evaluated. Besides the collection of frequency data from a certain population this is one of the basic prerequisites to calculate the likelihood ratio of an established polymorphism. By multiple measurement of restriction fragments from different persons indicated by the probes MS1, MS31, MS43A, g3 and YNH24 on different blots the within-laboratory variation was determined in the complete separation area from 1 to 19 kb. The values were compared to procedures used by other laboratories.

Blood Group Antigens↗
PubMed1990

[Prenatal diagnosis of hemophilia A by DNA analysis].

Prenatal diagnosis of hemophilia A and B was carried out in our hospital by DNA analysis in early and late villi and amniotic cells. Fifteen women with family history of hemophilia were studied by different sets of DNA probes and restrictive enzymes from Nov. 1986 to March 1989. Two of the 15 women were proved not carriers by blood sample assaying. Two did not show sites of polymorphism and another was not reported owing too small a DNA sample of amniotic cells. Among other 10 cases, 4 were proven normal male fetus, 1 proven affected male fetus was induced, and 5 were proven female fetus with 2 carriers.

Amniotic Fluid↗
PubMed1996

Tissue disaggregation for flow cytometric DNA analysis: comparison of fine-needle aspiration and an automated mechanical procedure.

Flow cytometric DNA analysis was performed on unfixed frozen samples from 56 breast cancer patients. From each patient, two samples were analyzed. The only difference in the handling of the paired samples was the mechanical disaggregation of one sample by fine-needle aspiration compared to an automated mechanical disaggregation method (Medimachine) of the other sample. With the two methods for tissue disaggregation, the same resolution of the DNA histograms was obtained, indicated by median coefficients of variation (CV) of 1.5% for the DNA diploid G1 peaks. Also, the frequencies of DNA diploid and aneuploid cases as well as the fractions of DNA aneuploid cells were comparable. This indicates that the two methods did not differ in ability to detect DNA aneuploid tumor clones. Automated mechanical disaggregation resulted in DNA histograms with significantly less debris and with lower S-phase fractions. In practice, the procedure of automated mechanical disaggregation was rapid, easy, and safer because of minimal handling of the unfixed tissue compared to the fine-needle aspiration.

Automation↗
PubMed1982

The prognostic value of DNA analysis in primary carcinoma in situ of the urinary bladder.

20 patients with primary carcinoma in situ of the urinary bladder were studied by flow cytofluorometric DNA analysis (FCM). Aneuploidy, i.e. abnormal DNA pattern, was found in all cases. As a rule the aneuploid cell populations were found in the tri-tetraploid region, corresponding to 70--92 chromosomes in the tumour cell nuclei. In seven cases more than one aneuploid cell line was found. All these seven cases showed progression in the tumour disease within 2 years. By FCM it was also possible to determine the proliferation rate of the tumours and the relative number of aneuploid cells in each tumour cell sample. In progressive cases high values of these two variables were found.

Aged↗
PubMed1987

Restriction endonuclease DNA analysis of Clostridium difficile.

HindIII restriction enzyme digests of genomic DNA from nine distinct strains of Clostridium difficile were undertaken, and the results were related to those of a previously established typing method based on [35S]methionine-labeled protein profiles. Each of the typed strains identified by its protein profile could also be distinguished by its unique DNA digestion pattern. Analysis of strains isolated from 10 patients during a hospital outbreak of antibiotic-associated colitis revealed identical DNA profiles, confirming a single strain as the source of cross-infection. Characterization of isolates from worldwide sources revealed similar digestion patterns within the same strain type. Restriction endonuclease DNA analysis provides a sensitive and useful technique for studying the epidemiology of C. difficile.

Clostridium↗