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DNA Amplification (dna + amplification)
Selected AbstractsMolecular typing of meningococci: recommendations for target choice and nomenclatureFEMS MICROBIOLOGY REVIEWS, Issue 1 2007Keith A. Jolley Abstract The diversity and dynamics of Neisseria meningitidis populations generate a requirement for high resolution, comprehensive, and portable typing schemes for meningococcal disease surveillance. Molecular approaches, specifically DNA amplification and sequencing, are the methods of choice for various reasons, including: their generic nature and portability, comprehensive coverage, and ready implementation to culture negative clinical specimens. The following target genes are recommended: (1) the variable regions of the antigen-encoding genes porA and fetA and, if additional resolution is required, the porB gene for rapid investigation of disease outbreaks and investigating the distribution of antigenic variants; (2) the seven multilocus sequence typing loci,these data are essential for the most effective national, and international management of meningococcal disease, as well as being invaluable in studies of meningococcal population biology and evolution. These targets have been employed extensively in reference laboratories throughout the world and validated protocols have been published. It is further recommended that a modified nomenclature be adopted of the form: serogroup: PorA type: FetA type: sequence type (clonal complex), thus: B: P1.19,15: F5-1: ST-33 (cc32). [source] Unscheduled DNA replication origin activation at inserted HPV 18 sequences in a HPV -18/MYC ampliconGENES, CHROMOSOMES AND CANCER, Issue 8 2007Chiara Conti Oncogene amplification is a critical step leading to tumorigenesis, but the underlying mechanisms are still poorly understood. Despite data suggesting that DNA replication is a major source of genomic instability, little is known about replication origin usage and replication fork progression in rearranged regions. Using a single DNA molecule approach, we provide here the first study of replication kinetics on a previously characterized MYC/papillomavirus (HPV18) amplicon in a cervical cancer. Using this amplicon as a model, we investigated the role DNA replication control plays in generating amplifications in human cancers. The data reveal severely perturbed DNA replication kinetics in the amplified region when compared with other regions of the same genome. It was found that DNA replication is initiated from both genomic and viral sequences, resulting in a higher median frequency of origin firings. In addition, it was found that the higher initiation frequency was associated with an equivalent increase in the number of stalled replication forks. These observations raise the intriguing possibility that unscheduled replication origin activation at inserted HPV -18 viral DNA sequences triggers DNA amplification in this cancer cell line and the subsequent overexpression of the MYC oncogene. This article contains Supplementary Material available at http://www.interscience.wiley.com/jpages/1045-2257/suppmat. © 2007 Wiley-Liss, Inc. [source] Molecular cytogenetic characterization of non-Hodgkin lymphoma cell linesGENES, CHROMOSOMES AND CANCER, Issue 3 2002Sukvarsha Mehra Spectral karyotyping (SKY) and comparative genomic hybridization (CGH) have greatly enhanced the resolution of cytogenetic analysis, enabling the identification of novel regions of rearrangement and amplification in tumor cells. Here we report the analysis of 10 malignant non-Hodgkin lymphoma (NHL) cell lines derived at the Ontario Cancer Institute (OCI), Toronto, designated as OCI-Ly1, OCI-Ly2, OCI-Ly3, OCI-LY4, OCI-Ly7, OCI-Ly8, OCI-Ly12, OCI-Ly13.2, OCI-Ly17, and OCI-Ly18, by G-banding, SKY, and CGH, and we present their comprehensive cytogenetic profiles. In contrast to the 52 breakpoints identified by G-banding, SKY identified 87 breakpoints, which clustered at 1q21, 7p15, 8p11, 13q21, 13q32, 14q32, 17q11, and 18q21. G-banding identified 10 translocations, including the previously described recurring translocations, t(8;14)(q24;q32) and t(14;18)(q32;q21). In contrast, SKY identified 60 translocations, including five that were recurring, t(8;14)(q24;q32), t(14;18)(q32;q21), t(4;7)(p12;q22), t(11;18)(q22;q21), and t(3;18)(q21;p11). SKY also identified the source of all the marker chromosomes. In addition, 10 chromosomes that were classified as normal by G-banding were found by SKY to be rearranged. CGH identified seven sites of high-level DNA amplification, 1q31-32, 2p12-16, 8q24, 11q23-25, 13q21-22, 13q32-34, and 18q21-23; of these, 1q31-32, 11q23-25, 13q21-22, and 13q32-34 have previously not been described as amplified in NHL. This comprehensive cytogenetic characterization of 10 NHL cell lines identified novel sites of rearrangement and amplification; it also enhances their value in experimental studies aimed at gene discovery and gene function. © 2002 Wiley-Liss, Inc. [source] Multiple pathways in the FGF signaling network are frequently deregulated by gene amplification in oral dysplasiasINTERNATIONAL JOURNAL OF CANCER, Issue 9 2009Ivy F.L. Tsui Abstract Genetic alteration in oral premalignant lesions (OPLs), the precursors of oral squamous cell carcinomas (OSCCs), may represent key changes in disease initiation and development. We ask if DNA amplification occurs at this early stage of cancer development and which oncogenic pathways are disrupted in OPLs. Here, we evaluated 50 high-grade dysplasias and low-grade dysplasias that later progressed to cancer for gene dosage aberrations using tiling-path DNA microarrays. Early occurrences of DNA amplification and homozygous deletion were frequently detected, with 40% (20/50) of these early lesions exhibiting such features. Expression for 88 genes in 7 recurrent amplicons were evaluated in 5 independent head and neck cancer datasets, with 40 candidates found to be overexpressed relative to normal tissues. These genes were significantly enriched in the canonical ERK/MAPK, FGF, p53, PTEN and PI3K/AKT signaling pathways (p = 8.95 × 10,3 to 3.18 × 10,2). These identified pathways share interactions in one signaling network, and amplification-mediated deregulation of this network was found in 30.0% of these preinvasive lesions. No such alterations were found in 14 low-grade dysplasias that did not progress, whereas 43.5% (10/23) of OSCCs were found to have altered genes within the pathways with DNA amplification. Multitarget FISH showed that amplification of EGFR and CCND1 can coexist in single cells of an oral dysplasia, suggesting the dependence on multiple oncogenes for OPL progression. Taken together, these findings identify a critical biological network that is frequently disrupted in high-risk OPLs, with different specific genes disrupted in different individuals. © 2009 UICC [source] Relationships between the olive fly and bacteriaJOURNAL OF APPLIED ENTOMOLOGY, Issue 9-10 2008P. Sacchetti Abstract The relationship between the olive fly population and epiphytic bacteria of the olive tree was investigated by carrying out a 1-year survey in the field. The olive fly population affected the number of bacteria present on the olive surface. Scanning electron microscope observations demonstrated that bacteria may be ingested by the fly's mouth apparatus through the midline of the pseudotracheae. DNA amplification of the oesophageal bulb content using 16S bacteria universal primers and DNA sequencing evidenced that Candidatus Erwinia dacicola was the predominant species present. The role of bacteria in olive fly biology is discussed. [source] Die-off of Cryptosporidium parvum in soil and wastewater effluentsJOURNAL OF APPLIED MICROBIOLOGY, Issue 1 2007A.M. Nasser Abstract Aims:, To determine the effect of biotic and abiotic components of soil on the viability and infectivity of Cryptosporidium parvum, and evaluate the suitability of viability tests as a surrogate for oocyst infectivity under various environmental settings. Methods and Results:, The die-off of C. parvum in saturated and dry loamy soil was monitored over time by immunofluorescence assay (IFA) and PCR to estimate oocysts viability and by cell culture to estimate oocysts infectivity. Pseudomonas aeruginosa activity resulted in digestion of the outer layer of the oocysts, as demonstrated by loss of the ability to react in IFA. Whereas, P. aeruginosa activity did not affect the DNA amplification by PCR. A 1-log reduction in the oocysts infectivity was observed at 30 °C in distilled water and in saturated soil while oocysts viability was unchanged. Incubation for 10 days in dry loamy soil at 32 °C resulted in a 3-log10 reduction in their infectivity while no change of oocysts viability was recorded. Conclusions:, Under low temperature, C. parvum oocysts may retain their infectivity for a long time. Soil desiccation and high temperatures enhance the die-off rate of C. parvum. Significance and Impact of the Study:, Previous die-off studies of C. parvum used viability tests that do not necessarily reflect the oocyst infectivity. Under low temperatures, there was an agreement observed between viability and infectivity tests and oocysts retained their infectivity for a long time. Desiccation and high temperatures enhance the loss of infectivity of C. parvum. The presented die-off data have significant implications on the management of wastewater reuse in warm environments. [source] DNA amplification and expression of FADD in oral squamous cell carcinomaJOURNAL OF ORAL PATHOLOGY & MEDICINE, Issue 7 2010Chanwit Prapinjumrune J Oral Pathol Med (2010) 39: 525,532 Background:, The Fas-associated death domain-containing protein, FADD, is an adaptor for relaying apoptotic signals. However, recent studies have shown that FADD also plays an important role in the growth and regulation of the cell cycle. The purpose of this study was to elucidate the role of FADD in oral squamous cell carcinoma (SCC). Methods:, The DNA amplification of FADD from 30 samples of tongue SCC was analyzed using real-time PCR and the protein expression of FADD from 60 samples of tongue SCC was analyzed using immunohistochemistry. Results:, The DNA amplifications of FADD were observed in 13 cases (44.3%) and were significantly correlated with the histopathological differentiation grade of SCCs (P = 0.009). FADD expression levels compared with the matched adjacent epithelium increased significantly (P = 0.000). Additionally, the positive expressions of FADD were significantly correlated with lymph node metastasis of SCCs (P = 0.029) and the 5-year disease-specific survival rates (P = 0.049). A positive association between FADD expression level and the histopathological differentiation grade was found to be limited to T1 SCCs (P = 0.019). DNA amplification was moderately correlated (correlation coefficient = 0.406, P = 0.026) with expression of FADD in 30 samples of tongue SCC. Conclusion:, In tongue SCCs, the expression of FADD was higher when compared with that of adjacent areas, which might be determined via genomic amplification in 11q13.3. Thus, SCC cells with the expression of FADD are possibly more likely to become metastatic and to worsen survival rates. [source] Human papillomavirus frequency in oral epithelial lesionsJOURNAL OF ORAL PATHOLOGY & MEDICINE, Issue 5 2004Carmen Maria Lazzari Background:, Oral human papillomavirus (HPV) prevalence varies according to geographical occurrence, the type of lesion, and the method of diagnosis. The polymerase chain reaction method (PCR) appears to be more sensitive and can be easily applicable to epidemiologic studies. Objectives:, To determine the frequency of HPV and its genotypes in oral lesions among patients attending a reference clinic of a university hospital. Methods:, PCR was performed to identify HPV DNA from samples of oral epithelial lesions in 80 patients. For HPV DNA amplification, MY09/MY11 consensus primers were used and specific genotypes were identified through restriction fragment of length polymorphism (RFLP) pattern. Results:, HPV DNA was present in 11.3% of patients, and the identified genotypes were 6b, MM4 (W13B), and MM9 (PAP238A). Conclusions:, HPV DNA frequency in patients with oral epithelial lesions was 11.3%. The genotypes MM4 and MM9 are uncommon in oral lesions, and they are characterized as high-risk HPV types in those types of lesions. [source] Unsuitable distinction between viable and dead Staphylococcus aureus and Staphylococcus epidermidis by ethidium bromide monoazideLETTERS IN APPLIED MICROBIOLOGY, Issue 5 2009H. Kobayashi Abstract Aims:, The DNA-intercalating dye ethidium bromide monoazide (EMA) has recently been used as a DNA binding agent to differentiate viable and dead bacterial cells by selectively penetrating through the damaged membrane of dead cells and blocking the DNA amplification during the polymerase chain reaction (PCR). We optimized and tested the assay in vitro using Staphylococcus aureus and Staphylococcus epidermidis cultures to distinguish viable from dead bacteria, with the goal of reducing false positive PCR results. Methods and Results:, Viable and heat-inactivated bacteria were treated with EMA or left untreated before DNA extraction. A real-time PCR assay for the detection of the tuf gene in each DNA extract was used. Our results indicated that EMA influenced viable bacteria as well as dead bacteria, and the effect of EMA depended on the EMA concentration and bacterial number. Conclusions:, EMA is not a suitable indicator of bacterial viability, at least with respect to Staphylococcus species. Significance and Impact of the Study:, Determining the viability of pathogens has a major impact on interpreting the results of molecular tests for bacteria and subsequent clinical management of patients. To this end, several methods are being evaluated. One of these methods , intercalating DNA of dead bacteria by EMA , looked very promising, but our study found it unsatisfactory for S. aureus and coagulase-negative Staphylococci. [source] Cytomegalovirus infection in the brain of liver transplant recipients: Do pathologically occult infections matter clinically?LIVER TRANSPLANTATION, Issue 3 2003Raymund R. Razonable MD Cytomegalovirus (CMV) infection remains a highly prevalent systemic complication following orthotopic liver transplantation (LT), accounting for a significant increase in morbidity and affiliated costs. However, unlike other immunosuppressed groups of population, CMV infection of the central nervous system in LT is rarely diagnosed, either clinically or postmortem. Furthermore, in 20% of the LT patients who develop preterminal neurological complications, the etiology remains undetermined. With the hypothesis that at least some of these cases could be related to an occult CMV infection, we examined brain tissue from 83 unselected autopsies of LT patients by morphological, immunohistochemical (IHC), in situ hybridization (ISH), and nested polymerase chain reaction (nested PCR) techniques. Microglial nodules were observed in 17 brains of the LT group (20.4%) but in none of the 36 controls. Isolated positive cells by either IHC, ISH, or both techniques, were identified in 11 LT patients (13.2%) and in 2 controls (5.5%). CMV DNA amplification was obtained from paraffin embedded tissues in 41 of 81 LT cases (50.6%), and in 5 controls (13.8%) (P=0.00017). Viral inclusion bodies, inflammatory infiltrates, or necrotizing changes were not identified in any case. Our findings indicate an increased susceptibility of the brain of LT patients to occult infection by CMV and suggest that a latent or low-grade infection of the central nervous system could operate as a reservoir of the CMV and play a role in some of the unexplained neurological symptoms that appear in the postoperative period. [source] Development and Characterization of Microsatellite Markers from an Enriched Genomic Library of Cucumber (Cucumis sativus)PLANT BREEDING, Issue 1 2008N. Watcharawongpaiboon Abstract The development, characterization and application of cucumber (Cucumis sativus L.) microsatellite markers was accomplished using a library-enrichment procedure. Fifty-seven primer pairs flanking the microsatellite repeats were used for DNA amplification. Sixteen C. sativus accessions were assessed for polymorphisms using 45 primer pairs. The average number of alleles per locus was 3.6, and up to seven alleles were found at one locus. The maximum polymorphism information content value was 0.78 with an average of 0.47. The cucumber microsatellite makers could be useful for seed purity control in hybridity testing. Some of these cucumber markers were transferable to other cucurbit species (i.e. melon, watermelon, pumpkin and bitter gourd). [source] Technical note: PCR analysis of minimum target amount of ancient DNAAMERICAN JOURNAL OF PHYSICAL ANTHROPOLOGY, Issue 2 2010Daniela Woide Abstract The study of ancient DNA plays an important role in archaeological and palaeontological research as well as in pathology and forensics. Here, we present a new tool for ancient DNA analysis, which overcomes contamination problems, DNA degradation, and the negative effects of PCR inhibitors while reducing the amount of starting target material in the picogram range. Ancient bone samples from four Egyptian mummies were examined by combining laser microdissection, conventional DNA extraction, and low-volume PCR. Initially, several bone particles (osteons) in the micrometer range were extracted by laser microdissection. Subsequently, ancient DNA amplification was performed to verify our extraction method. Amelogenin and ,-actin gene specific fragments were amplified via low-volume PCR in a total reaction volume of 1 ,l. Results of microdissected mummy DNA samples were compared to mummy DNA, which was extracted using a standard DNA extraction method based on pulverization of bone material. Our results highlight the combination of laser microdissection and low-volume PCR as a promising new technique in ancient DNA analysis. Am J Phys Anthropol, 2010. © 2010 Wiley-Liss, Inc. [source] Technical note: Removal of metal ion inhibition encountered during DNA extraction and amplification of copper-preserved archaeological bone using size exclusion chromatographyAMERICAN JOURNAL OF PHYSICAL ANTHROPOLOGY, Issue 2 2009Carney D. Matheson Abstract A novel technique for the removal of metal ions inhibiting DNA extraction and PCR of archaeological bone extracts is presented using size exclusion chromatography. Two case studies, involving copper inhibition, demonstrate the effective removal of metal ion inhibition. Light microscopy, SEM, elemental analysis, and genetic analysis were used to demonstrate the effective removal of metal ions from samples that previously exhibited molecular inhibition. This research identifies that copper can cause inhibition of DNA polymerase during DNA amplification. The use of size exclusion chromatography as an additional purification step before DNA amplification from degraded bone samples successfully removes metal ions and other inhibitors, for the analysis of archaeological bone. The biochemistry of inhibition is explored through chemical and enzymatic extraction methodology on archaeological material. We demonstrate a simple purification technique that provides a high yield of purified DNA (>95%) that can be used to address most types of inhibition commonly associated with the analysis of degraded archaeological and forensic samples. We present a new opportunity for the molecular analysis of archaeological samples preserved in the presence of metal ions, such as copper, which have previously yielded no DNA results. Am J Phys Anthropol, 2009. © 2009 Wiley-Liss, Inc. [source] Biomarker Assays in Nipple Aspirate FluidTHE BREAST JOURNAL, Issue 6 2001Pamela Klein MD The noninvasive technique of nipple aspiration as a potential source of biomarkers of breast cancer risk was evaluated. The feasibility of performing mutagenesis assays, amplifying DNA, and performing protein electrophoresis on nipple aspirate fluid was explored. A tool was developed to measure the level of discomfort, if any, from this procedure. Twenty-five healthy women (20 premenopausal and 5 postmenopausal) were enrolled. Fluid was obtained using a modified breast pump. Premenopausal women were scheduled for four to six weekly aspirations, and postmenopausal women were scheduled for one to two weekly aspirations. Mutagenesis assays were performed using the Salmonella (Ames) assay. DNA amplification of several microsatellite regions was carried out using polymerase chain reaction. Protein was quantified, and two-dimensional protein electrophoresis was performed. Overall, fluid was obtained from 80% of the women, and the level of discomfort was minimal. Acid hydrolysis of one sample resulted in mutagenicity; all six nonhydrolyzed samples were not mutagenic. The ability to amplify DNA ranged from 34% to 96%, depending on length of the microsatellite region examined. The average protein concentration was 71 ,g/mL. Two-dimensional protein electrophoresis was successfully performed on samples from two subjects. Nipple aspiration is a simple technique and is easily learned and well tolerated, which yields a reagent useful for a variety of investigations. This technique may facilitate the identification and application of biomarkers for future breast cancer risk assessment and chemopreventive protocols. [source] Cellular and biochemical markers in semen indicating male accessory gland inflammationANDROLOGIA, Issue 5 2003W. Krause Summary. Leucocytospermia is considered to be a sign of male accessory gland inflammation. The leucocytes in semen are mainly polymorphonuclear neutrophilic granulocytes. Leucocytospermia is not associated with the presence of bacteria and antibiotic treatment does not significantly lower the extent of leucocytospermia. A higher frequency of elevated herpes simplex antibodies titres were found in men with leucocytospermia. The concentration of inflammatory cytokines, interleukin-6 and -8, is closely correlated with the number of leucocytes. Their determination does not provide additional information. Reactive oxygen species (ROS) are generated at least in part by seminal leucocytes in response to stimulating factors. Purified leucocytes produce high levels of ROS. The determination of ROS appears to represent a parameter of functional activity of leucocytes. The role of chlamydiae in male accessory gland infection is unclear. Their determination in semen by DNA amplification and by immunological tests does not provide reliable results. [source] The DNA puff 4C expresses a salivary secretion protein in Trichosia pubescens (Diptera; Sciaridae)ARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY (ELECTRONIC), Issue 2 2008Luiz Paulo Andrioli Abstract DNA puffs are genomic regions of polytene chromosomes that undergo developmentally controlled DNA amplification and transcription in salivary glands of sciarid flies. Here, we tested the hypothesis that DNA puff genes code for salivary proteins in Trichosia pubescens. To do that, we generated antibodies against saliva and immunoscreened a cDNA library made from salivary glands. We isolated clones corresponding to DNA puff regions, including clone D-50 that contained the entire coding sequence of the previously isolated C4B1 gene from puff 4C. Indeed, we showed that puff 4C is a DNA puff region detecting its local transcription and its extra rounds of DNA incorporation compared to neighboring regions. We further confirmed D-50 clone identity in Western blots reacted with the anti-saliva anitiserum. We detected a recombinant protein expressed by this clone that had the expected size for a full-length product of the gene. We end with a discussion of the relationship between DNA puff genes and their products. Arch. Insect Biochem. Physiol. 2007. © 2007 Wiley-Liss, Inc. [source] Incorporating Genotype Uncertainty into Mark,Recapture-Type Models For Estimating Abundance Using DNA SamplesBIOMETRICS, Issue 3 2009Janine A. Wright Summary Sampling DNA noninvasively has advantages for identifying animals for uses such as mark,recapture modeling that require unique identification of animals in samples. Although it is possible to generate large amounts of data from noninvasive sources of DNA, a challenge is overcoming genotyping errors that can lead to incorrect identification of individuals. A major source of error is allelic dropout, which is failure of DNA amplification at one or more loci. This has the effect of heterozygous individuals being scored as homozygotes at those loci as only one allele is detected. If errors go undetected and the genotypes are naively used in mark,recapture models, significant overestimates of population size can occur. To avoid this it is common to reject low-quality samples but this may lead to the elimination of large amounts of data. It is preferable to retain these low-quality samples as they still contain usable information in the form of partial genotypes. Rather than trying to minimize error or discarding error-prone samples we model dropout in our analysis. We describe a method based on data augmentation that allows us to model data from samples that include uncertain genotypes. Application is illustrated using data from the European badger (Meles meles). [source] Simply and reliably integrating micro heaters/sensors in a monolithic PCR-CE microfluidic genetic analysis systemELECTROPHORESIS, Issue 8 2009Runtao Zhong Abstract A novel fabrication process was presented to construct a monolithic integrated PCR-CE microfluidic DNA analysis system as a step toward building a total genetic analysis microsystem. Microfabricated Titanium/Platinum (Ti/Pt) heaters and resistance temperature detectors (RTDs) were integrated on the backside of a bonded glass chip to provide good thermal transfer and precise temperature detection for the drilled PCR-wells. This heater/RTD integration procedure was simple and reliable, and the resulting metal layer can be easily renewed when the Ti/Pt layer was damaged in later use or novel heater/RTD design was desired. A straightforward "RTD-calibration" method was employed to optimize the chip-based thermal cycling conditions. This method was convenient and rapid, comparing with a conventional RTD-calibration/temperature adjustment method. The highest ramping rates of 14°C/s for heating and 5°C/s for cooling in a 3-,L reaction volume allow 30 complete PCR cycles in about 33,min. After effectively passivating the PCR-well surface, successful ,-phage DNA amplifications were achieved using a two- or three-temperature cycling protocol. The functionality and performance of the integrated microsystem were demonstrated by successful amplification and subsequent on-line separation/sizing of ,-phage DNA. A rapid assay for Hepatitis B virus, one of the major human pathogens, was performed in less than 45,min, demonstrating that the developed PCR-CE microsystem was capable of performing automatic and high-speed genetic analysis. [source] Identification of DNA amplifications near the center of the Streptomyces coelicolor M145 chromosomeFEMS MICROBIOLOGY LETTERS, Issue 1 2000Matthias Redenbach Abstract Linear streptomycete chromosomes frequently undergo spontaneous gross DNA rearrangements at the terminal regions. Large DNA deletions of the chromosome ends are in many cases associated with tandemly reiterated DNA amplifications, found at the border of the deletable areas. In contrast to previous reports, we have discovered amplifications near the center of the Streptomyces coelicolor M145 chromosome. The detected amplified units of DNA are 19.9 kb and 16 kb in length and exist in copy numbers of 30 and 40, respectively. Both amplifications were located in the same region and share at least 3.6 kb. [source] Molecular-cytogenetic comparison of mucosa-associated marginal zone B-cell lymphoma and large B-cell lymphoma arising in the gastro-intestinal tractGENES, CHROMOSOMES AND CANCER, Issue 4 2001Thomas F.E. Barth Extranodal B-cell lymphoma of mucosa-associated lymphoid tissue (MALT) type may represent a model of lymphoma progression, because a small cell component frequently occurs in the large cell variants. We studied 52 extranodal B-cell lymphomas: 18 extranodal marginal zone B-cell lymphomas of MALT type (MZBL,MT), 7 MZBL,MT of the gastro-intestinal tract with a diffuse large B-cell component (giMZBLplusLBCL), and 27 diffuse large B-cell lymphomas of the gastro-intestinal tract without small cell component (giLBCL). Analytical techniques were comparative genomic hybridization (CGH) and fluorescence in situ hybridization (FISH). The translocation t(11;18) was found as the sole aberration in two MZBL,MT only. In contrast to this, t(11;18)-negative MZBL,MT were characterized by frequent gains on chromosome 3 and DNA amplifications on 2p13,p15. Furthermore, we found a clonal lymphoma progression from the small to the large cell component with accumulation of gains and losses of chromosomal material in the large cell component in giMZBLplusLBCL. Aberrations overlapping with MZBL,MT and giMZBLplusLBCL included losses on chromosome 13, amplifications of the REL proto-oncogene, or gains on chromosome 12. In addition, the large cell component revealed gains on 8q24, including amplifications of the MYC proto-oncogene, and losses on 2q. The giLBCL had frequent gains on chromosomes 12 and 9, as well as on 11q, and losses on 6q. We conclude that, based on the distinctive and partly overlapping patterns of genetic aberrations, MALT lymphomas can be divided into different genetic subgroups. © 2001 Wiley-Liss, Inc. [source] The Application of Ultraviolet Irradiation to Exogenous Sources of DNA in Plasticware and Water for the Amplification of Low Copy Number DNAJOURNAL OF FORENSIC SCIENCES, Issue 4 2006Jeannie Tamariz B.S. ABSTRACT: Using high sensitivity forensic STR polymerase chain reaction (PCR) typing procedures, we have found low concentrations of DNA contamination in plasticware and water assumed to be sterile, which is not detected by standard DNA procedures. One technique commonly used to eliminate the presence of DNA is ultraviolet (UV) irradiation; we optimized such a protocol used in the treatment of water, tubes, plates, and tips for low copy number DNA (LCN) amplification. UV light from a Stratalinker® 2400 was administered to 0.2, 1.5 mL tubes, and PCR plates contaminated with up to 500 pg of DNA. They were subsequently quantified with an ALU-based real-time PCR method using the Rotorgene 3000. Overall, there was a decrease in concentration of DNA recovered as the duration of treatment increased. Nonetheless, following 45 min of irradiating a PCR plate with 500 pg of DNA, nearly 6 pg were still detected. However, when the plate was raised within an inch of the UV source, less than 0.2 pg of DNA was detected. Additionally, lining the area around the samples with aluminum foil further reduced the amount of time necessary for irradiation, as only 30 min eliminated the presence DNA in the raised PCR plate. Similar experiments were conducted using tubes filled with a solution of DNA and water in equivalent concentrations for 50, 15, and 1.5 mL tubes with comparative results. It is plausible that the aluminum foil increased the amount of reflection in the area thereby enhancing penetration of UV rays through the walls of the plasticware. This protocol was tested for the possibility of inhibitors produced from irradiation of plastic tubes. As our protocols require less irradiation time than previous studies, PCR sensitivity was not affected. Moreover, the lifespan of the UV lamps was extended. Our findings demonstrate that this method is useful as an additional precautionary measure to prevent amplification of extraneous DNA from plasticware and water without compromising the sensitivity of LCN DNA amplifications. [source] Low Frequency of Chromosomal Imbalances in Anaplastic Ependymomas as Detected by Comparative Genomic HybridizationBRAIN PATHOLOGY, Issue 2 2001Stefanie Scheil We screened 26 ependymomas in 22 patients (7 WHO grade I, myxopapillary, myE; 6 WHO grade II, E; 13 WHO grade III, anaplastic, aE) using comparative genomic hybridization (CGH) and fluorescence in situ hybridization (FISH). 25 out of 26 tumors showed chromosomal imbalances on CGH analysis. The chromosomal region most frequently affected by losses of genomic material clustered on 13q (9/26). 6/7 myE showed a loss on 13q14-q31. Other chromosomes affected by genomic losses were 6q (5/26), 4q (5/26), 10 (5/26), and 2q (4/26). The most consistent chromosomal abnormality in ependymomas so far reported, is monosomy 22 or structural abnormality 22q, identified in approximately one third of Giemsabanded cases with abnormal karyotypes. Using FISH, loss or monosomy 22q was detected in small subpopulations of tumor cells in 36% of cases. The most frequent gains involved chromosome arms 17 (8/26), 9q (7/26), 20q (7/26), and 22q (6/26). Gains on 1q were found exclusively in pediatric ependymomas (5/10). Using FISH, MYCN proto-oncogene DNA amplifications mapped to 2p23-p24 were found in 2 spinal ependymomas of adults. On average, myE demonstrated 9.14, E 5.33, and aE 1.77 gains and/or losses on different chromosomes per tumor using CGH. Thus, and quite paradoxically, in ependymomas, a high frequency of imbalanced chromosomal regions as revealed by CGH does not indicate a high WHO grade of the tumor but is more frequent in grade I tumors. [source] | ||||||||||||||||