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PubMed2003

AmpFlSTR profiler Plus short tandem repeat DNA analysis of casework samples, mixture samples, and nonhuman DNA samples amplified under reduced PCR volume conditions (25 microL).

As part of the validation of the AmpFlSTR Profiler Plus short tandem repeat (STR) system, under reduced polymerase chain reaction (PCR) volume conditions (i.e., 25 microL), a total of 275 casework samples were processed. Examples of profiles are presented along with amplification conditions to improve the odds of obtaining balanced and complete profiles for samples showing partial results or profiles with a descending slope. Data collected and used to develop our interpretation guidelines are included. From the mixture studies, full profiles were obtained for minor contributors, using 2 ng of DNA, with ratios of 10:1 or 1:20 and using 1 ng of DNA, with ratios of 10:1 and 1:8. The specificity of the Profiler Plus amplification reaction performed in 25 microL was examined and confirmed using a large spectrum of nonhuman DNAs. This report supports the use of the AmpFlSTR Profiler Plus STR system for casework DNA typing under reduced PCR volume conditions.

Alleles↗
PubMed1988

[DNA analysis in the assessment of leukemia].

In 5 cases of leukaemia and in two other cases where a diagnosis of leukaemia was considered, no definitive classification was made by morphology, cytochemistry, patterns of surface markers and/or cytogenetics. Therefore, genotyping was performed by DNA analysis of leukaemic cells. Somatic rearrangements in leukaemic cell DNA of immunoglobulin and T cell receptor genes, and of the breakpoint cluster region on chromosome 22 involved in the Philadelphia chromosome translocation, proved to be useful DNA markers to confirm or refute particular types of leukaemia.

Antigens, Differentiation↗
PubMed2003

Confirmation by DNA analysis that Contarinia maculipennis (Diptera: Cecidomyiidae) is a polyphagous pest of orchids and other unrelated cultivated plants.

The cytochrome oxidase subunit I gene in mitochondrial DNA of 53 larvae of Contarinia maculipennis Felt from flower buds of various host plants, collected from Hawaii, Japan and Thailand was analysed. Monophyly of the clade including C. maculipennis from Hawaii, Thailand and Japan was supported. There was no sequential variation within the specimens from Hawaii and Japan, which differed from one another by 6 bp (1.37%). Three haplotypes were recognized in specimens from Thailand but differences from Hawaiian and Japanese specimens were small. Overall, there were no differences in the 146 deduced amino acid residues. It is therefore concluded that C. maculipennis is a polyphagous species that can develop on plant hosts representing at least seven botanical families. This pest of Dendrobium flower buds in glasshouses is considered to have entered Hawaii, Florida and Japan from Southeast Asia, and was recently intercepted in the Netherlands. Infestations have established and spread in orchid glasshouses, causing concern about the possibility of more extensive damage to orchids and to crops, such as bitter gourd, grown in close proximity to orchid glasshouses in Japan. The potential usefulness of DNA analysis in determining host plant ranges of morphologically identical cecidomyiid species that are currently identified solely on differences of host plant is emphasized.

Animals↗
PubMed1990

Prenatal diagnosis of hemophilia A by DNA analysis of chorionic villi.

Hemophilia A is an X-linked recessive single gene disease. If a female is a carrier, her sons have a 50% risk of being affected. Five gravidas, each with a positive family history of hemophilia A, were collected for prenatal diagnosis using DNA analysis on chorionic villi. We used two closely linked extragenic DNA markers, St14 and DX13, and three restriction enzymes, TaqI, MspI, and BglII, to study four restriction fragment length polymorphisms in the Xq28 region. From family analysis, haplotypes were constructed to track the inheritance of X-chromosomes. In family 1, the male fetus inherited the X-chromosome from his normal grandfather, thus his probability of being affected was predicted to be less than 5%. In families 2 and 3, the gravidas inherited the maternal X-chromosome with a different haplotype from that of the affected brother or nephew, thus they both have a low probability (less than 5%) of being carriers. In family 4, the gravida and her male fetus inherited the same X-chromosome as her affected brother. With coagulation assay also showing a high probability of the gravida being a carrier, the fetus was at high risk for the disease. On pregnancy termination, fetal plasma analysis confirmed the diagnosis. In family 5, the gravida's mother had homozygous haplotypes, making the distinction between two X-chromosomes impossible. Coagulation assay classified the gravida as a probable carrier.(ABSTRACT TRUNCATED AT 250 WORDS)

Chorionic Villi Sampling↗
PubMed1992

[Comparison of DNA analysis with histomorphologic parameters in stomach cancer].

After curative resection of stomach carcinomas (adenocarcinomas: n = 58, signet ring cell carcinomas: n = 24, undifferentiated carcinomas: n = 21) the DNA content of the tumor cells was compared with the histomorphological parameters. There was a correlation between the DNA content and the histomorphological parameters. The DNA analysis had no additionally prognostic influence. In the multivariate regression analysis the prognosis depended on lymph node status (p = 0.0009), pT-stage (p = 0.02), tumor localization (p = 0.03) and the histological type (p = 0.05). The prognosis was independent of the DNA content. Furthermore, neither did the degree of differentiation, the operative procedure, the safety distance, the size of the tumor, the sex nor the age of the patient have any influence on the prognosis.

Adenocarcinoma↗
PubMed1993

Flow cytometric DNA analysis of fresh prostatic resections. Correlation with conventional prognostic parameters in patients with prostate cancer.

BACKGROUND: DNA ploidy analysis has been investigated as a prognostic indicator in prostate cancer. Most of the data is derived from retrospective studies using paraffin-embedded tissue. This method has drawbacks related to the quality of DNA histograms and uncontrolled data collection. METHODS: DNA ploidy analysis of freshly resected prostatic tissue was prospectively compared with conventional prognostic variables in 97 men treated with radical prostatectomy for localized prostate cancer. RESULTS: Regarding the patients, 31.9% were African American and 66% had pathologic Stages C or D1 disease. Only 9.6% of patients with Stages A2 and B had a prostate-specific antigen (PSA) value greater than 10 ng/ml, whereas 97% of patients with PSA values greater than 20 ng/ml had pathologic Stages C and D1. PSA levels correlated with Gleason score (P = < 0.05); 51% and 100% of patients with Gleason score 5-7 and 8-10, respectively, had PSA values greater than 10 ng/ml. Twenty-two patients (23%) had DNA aneuploid tumors. Comparisons of mechanical to enzymatic cell suspensions indicated that DNA aneuploidy was better preserved in mechanical cell preparations. DNA ploidy correlated with pathologic stage (P = < 0.05) and Gleason score (P = < 0.05). Fifteen of 79 patients (18.9%) with Gleason score 5-7 had DNA aneuploid tumors versus 71.4% of patients with Gleason score 8-10. PSA groups correlated with ploidy status (P = 0.01). Although the majority of patients (19 of 22) with DNA aneuploid tumors had elevated preoperative PSA levels, none had a PSA value greater than 50 ng/ml. CONCLUSIONS: DNA ploidy analysis correlated with established prognostic indicators in prostate cancer; however, its independent correlation with natural history and treatment outcome must be established for it to have an effect on therapeutic decisions.

Aged↗
PubMed1990

Detection of genomic alterations in carcinogen-induced mouse liver tumors by DNA fingerprint analysis.

DNA fingerprint analysis was used to study structural abnormalities in the genome of mouse liver tumor cells. Liver tumors were induced in three strains of mice, namely C57BL/6J, C3H/He and B6C3F1, by a single injection of 20 micrograms/g body wt. diethylnitrosamine on day 15 after birth. DNA from liver tumors was digested with Hinfl restriction enzyme and hybridized on Southern blots with wild-type bacteriophage M13 DNA as probe. The resulting fingerprints of tumor DNA were compared with those of DNA from normal liver tissue. Genomic aberrations were detected in two out of 68 tumors analyzed, one stemming from a C57BL/6J and the other from a C3H/He mouse.

Animals↗
PubMed1999

Inactivation of Mycobacterium tuberculosis for DNA typing analysis.

DNA fingerprinting analysis of Mycobacterium tuberculosis is used for epidemiological studies and the control of laboratory cross-contamination. Because standardized procedures are not entirely safe for mycobacteriology laboratory staff, the paper proposes a new technique for the processing of specimens. The technique ensures the inactivation of M. tuberculosis before DNA extraction without the loss of DNA integrity. The control of inactivated cultures should be rigorous and should involve the use of two different culture media incubated for at least 4 months.

Bacterial Typing Techniques↗
PubMed1988

Service experience using DNA analysis for genetic prediction in Duchenne muscular dystrophy.

In August 1985 we instituted a carrier and prenatal testing service for Duchenne muscular dystrophy (DMD) using direct DNA analysis. The experience over the first nine months is described. We have analysed samples for RFLPs from 154 people including 53 women at risk of being DMD carriers from 37 families. We used the probes pERT87.8 (BstXI and TaqI polymorphisms), 87-15 (TaqI polymorphism), and pXJ1.1 (TaqI polymorphism). Forty-one of the women have had their risks altered. We found one deletion (pERT87-8) out of 23 DNA samples analysed from affected boys. We used a recombination fraction of 0.05 in risk calculations but did not detect any known crossovers. In nine of the families there is only an isolated case of DMD. In families where we have not been able to alter the risk of the women being a carrier (for example, because all brothers are dead), we have offered prenatal exclusion and have carried out one first trimester prenatal diagnosis on this basis. Lowering the risk of an affected fetus to less than 2.5% appears to be a satisfactory situation for many (most) of the women involved and seems to justify the introduction of genetic prediction based on single intragenic probes despite the 5% recombination frequency.

DNA↗
PubMedDate not supplied

The value of measurement of ras oncogenes and nuclear DNA analysis in the diagnosis of Hürthle cell tumors of the thyroid.

Hürthle cell tumors (HCT) remain difficult to treat because some which appear non-malignant on light microscopy later metastasize. In order to improve diagnostic accuracy, the value of ras mutations and nuclear DNA analysis was determined in 65 patients with HCT. Rapid nuclear DNA cytometry (MicroTICAS system) was performed. Mutations of H-ras, K-ras, and N-ras genes were analyzed by oligonucleotide probe hybridizations to polymerase chain reaction (PCR) amplified DNA. HCT were classified by light microscopy as benign (n = 22), intermediate (n = 30), and malignant (n = 13). After a mean follow-up of 7 years, 1 (4.5%) of 22 benign tumors and 4 (13%) of 30 intermediate tumors had metastasized, leading to tumor death in 3 of these 5 patients. Six of the 13 cancers diagnosed by light microscopy also resulted in tumor-related deaths. Aneuploidy was found in 83% of all Hürthle cell cancers, including 3 (60%) of the 5 cancers not diagnosed microscopically. However, 49% of non-malignant HCT also demonstrated aneuploidy. A nuclear area of less than 55 square microns was found in 83% of all Hürthle cell cancers and in 100% of those cancers not diagnosed by light microscopy. However, 47% of non-malignant HCT also demonstrated a "small" nuclear area. Aneuploidy correctly identified 8 of 9 cancers that resulted in tumor death and each of 3 other tumors that developed metastases. However, 1 patient with a diploid tumor died of metastatic cancer. A nuclear area of less than 55 square microns identified each cancer that resulted in a tumor death.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1990

Flow and image cytometric DNA analysis in rhabdomyosarcoma.

Rhabdomyosarcoma is the most common malignant soft-tissue tumor in childhood, with an overall 3-year disease-free survival of 73%. DNA content is known to correlate with prognosis and therapy response in many cancers. To determine the role of DNA content in rhabdomyosarcoma, 23 tumor samples were studied retrospectively: 18 primary tumors and 5 post-chemotherapy recurrences or specimens obtained at second-look surgeries. The DNA analysis was performed on disaggregated paraffin-embedded tissue nuclei by flow and image cytometry and correlated with the histology and clinical history. Of the primary tumors 4 were diploid, 4 polyploid, and 10 aneuploid (9 with a single aneuploid G0G1 peak and 1 multiploid) by flow cytometry. The concordance rate between flow and image cytometry was 19 of 23 (83%); one case did not have flow cytometry available. Most embryonal rhabdomyosarcomas were aneuploid (10 of 12; 83%), and they had a high incidence of recurrence in Stages III and IV (4 of 12; 33%). Although aneuploidy in pediatric cancers may predict a therapeutic response and good prognosis, this was not supported by our findings in rhabdomyosarcoma. The tumor DNA content correlated with the clinical stage but not with the patient's clinical course or tumor histopathological type. DNA content did not appear to be as important a prognostic tool as tumor stage.

Adolescent↗
PubMed1986

Flow cytometric DNA analysis of neuroblastoma. Correlation with histology and clinical outcome.

Clinical and histologic features of 38 cases of neuroblastoma were compared with data obtained by flow cytometric DNA analysis. Favorable clinical outcome was associated with an aneuploid stem line (P less than 0.01) and a low percentage of tumor cells in the S, G2, and M phases of the cell cycle (P less than 0.005). These favorable cytometric features were also associated with a favorable clinical stage (1, 2, 4s), and histologic evidence of Schwann's cell and ganglion cell differentiation. Consideration of cytometric data improved the sensitivity, specificity, and predictive efficiency of a current system for histologic grading of neuroblastoma.

Child↗
PubMed1990

Cytophotometry in tumor pathology. A critical review of methods and applications, and some results of DNA analysis.

In tumor pathology the quantitation of cellular substances can be of diagnostic value. Microscope cytophotometry and digital image analysis and, on the other hand, flow cytometry are supplementary methods for measuring, each with a typical spectrum of application. The methods are predominantly used for DNA analysis: Static and image cytophotometry are applicable to cytologic and histologic slides preferably for identifying stem lines in tumors of heterogenous morphology and in merely circumscribed lesions (e.g., precancerous lesions). On the other hand, sampling errors due to preselection, and the often low number of cells actually measured, may preclude the possibility of exact cell cycle analysis. This is, in fact, an important additional option of flow cytometry resulting from the high resolution of DNA histograms, which is explained by the large number of cells that can be measured in a short period. Sampling errors in flow cytometry may result from the preparation of single cell suspensions which in certain tumor entities may suppress a varying amount of particularly fragile cells or nuclei. The prognostic significance of DNA ploidy, stem line heterogeneity and S-phase fraction is clearly described in quite a number of tumor entities. Independent of its prognostic value, the cytometric identification of stem lines might be particularly useful in the follow-up of tumor patients, where it may indicate the effectivity of systemic therapy. The development of therapeutic concepts is aptly supported by flow cytometric cell cycle analysis which helps to assess the in vitro effect of combined cytostatics on the proliferative process. Moreover, multiparameter analysis of biopsy samples may provide greater accuracy in characterising individual tumor stem lines and may furthermore help to develop improved protocols for the therapy of solid tumors.

Cytophotometry↗
PubMed2004

Micro- and nanofluidics for DNA analysis.

Miniaturization to the micrometer and nanometer scale opens up the possibility to probe biology on a length scale where fundamental biological processes take place, such as the epigenetic and genetic control of single cells. To study single cells the necessary devices need to be integrated on a single chip; and, to access the relevant length scales, the devices need to be designed with feature sizes of a few nanometers up to several micrometers. We will give a few examples from the literature and from our own research in the field of miniaturized chip-based devices for DNA analysis, including dielectrophoresis for purification of DNA, artificial gel structures for rapid DNA separation, and nanofluidic channels for direct visualization of single DNA molecules.

Cell Separation↗
PubMed1994

Evaluation of malignant grade of salivary gland tumors: studies by cytofluorometric nuclear DNA analysis, histochemistry for nucleolar organizer regions and immunohistochemistry for p53.

Investigations were carried out in 109 surgically resected salivary gland tumors, 46 adenomas and 63 carcinomas for nuclear DNA content by cytofluorometry, nucleolar organizer regions (AgNOR) by histochemistry and nuclear p53 accumulation by immunohistochemistry. The mean nuclear DNA content and the incidence of aneuploidy as well as the mean number of the AgNOR in carcinomas were significantly higher than those in adenomas. In the DNA histogram pattern, the aneuploidy frequently appeared in high grade carcinoma groups (adenocarcinoma, salivary duct carcinoma, carcinoma in pleomorphic adenoma and oncocytic carcinoma). Immunohistochemical nuclear p53 accumulation was observed in 15 tumors, of which ten showed DNA aneuploidy. In high grade carcinomas, DNA aneuploidy was closely related to p53 immunoreactivity. This study revealed that combined cytofluorometric nuclear DNA analysis, histochemical AgNOR count and immunoreactivity for p53 might be quite useful for evaluating the malignant grade of salivary gland tumors.

Adenocarcinoma↗
PubMedDate not supplied

Taxonomy of pasteurella anatipestifer. 1. DNA base composition and DNA-DNA hybridization analysis.

DNA was isolated from 15 strains of Pasteurella anatipestifer and from one strain each of Moraxella nonliquefaciens, M. bovis, Pasteurella multocida, P. haemolytica, P. gallinarum, P. pneumotropica, and P. ureae. The guanine-plus-cytosine contents of P. anatipestifer ranged from 32 to 35 mole %, whereas those of Moraxella and Pasteurella spp. were much higher, ranging from 40 to 45 mole %. DNA-DNA hybridization analysis revealed that homology of nine P. anatipestifer strains to strains ATCC 11845 and PA 15 was 52 to 100%, whereas homology of Moraxella and Pasteurella strains to these strains was only 3 to 17%. Similarly, homology of P. anatipestifer strains, Moraxella, and Pasteurella species other than P. multocida to P. multocida reference strain P-2192 was low. These results strongly suggest that P. anatipestifer is genetically unrelated to either Pasteurella or Moraxella.

Base Composition↗