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Sample records for dna assay system

  1. DNA-PK assay

    Science.gov (United States)

    Anderson, Carl W.; Connelly, Margery A.

    2004-10-12

    The present invention provides a method for detecting DNA-activated protein kinase (DNA-PK) activity in a biological sample. The method includes contacting a biological sample with a detectably-labeled phosphate donor and a synthetic peptide substrate defined by the following features to provide specific recognition and phosphorylation by DNA-PK: (1) a phosphate-accepting amino acid pair which may include serine-glutamine (Ser-Gln) (SQ), threonine-glutamine (Thr-Gln) (TQ), glutamine-serine (Gln-Ser) (QS), or glutamine-threonine (Gln-Thr) (QT); (2) enhancer amino acids which may include glutamic acid or glutamine immediately adjacent at the amino- or carboxyl- side of the amino acid pair and forming an amino acid pair-enhancer unit; (3) a first spacer sequence at the amino terminus of the amino acid pair-enhancer unit; (4) a second spacer sequence at the carboxyl terminus of the amino acid pair-enhancer unit, which spacer sequences may include any combination of amino acids that does not provide a phosphorylation site consensus sequence motif; and, (5) a tag moiety, which may be an amino acid sequence or another chemical entity that permits separating the synthetic peptide from the phosphate donor. A compostion and a kit for the detection of DNA-PK activity are also provided. Methods for detecting DNA, protein phosphatases and substances that alter the activity of DNA-PK are also provided. The present invention also provides a method of monitoring protein kinase and DNA-PK activity in living cells. -A composition and a kit for monitoring protein kinase activity in vitro and a composition and a kit for monitoring DNA-PK activities in living cells are also provided. A method for identifying agents that alter protein kinase activity in vitro and a method for identifying agents that alter DNA-PK activity in living cells are also provided.

  2. Branched DNA-based Alu quantitative assay for cell-free plasma DNA levels in patients with sepsis or systemic inflammatory response syndrome.

    Science.gov (United States)

    Hou, Yan-Qiang; Liang, Dong-Yu; Lou, Xiao-Li; Zhang, Mei; Zhang, Zhen-huan; Zhang, Lu-rong

    2016-02-01

    Cell-free circulating DNA (cf-DNA) can be detected by various of laboratory techniques. We described a branched DNA-based Alu assay for measuring cf-DNA in septic patients. Compared to healthy controls and systemic inflammatory response syndrome (SIRS) patients, serum cf-DNA levels were significantly higher in septic patients (1426.54 ± 863.79 vs 692.02 ± 703.06 and 69.66 ± 24.66 ng/mL). The areas under the receiver operating characteristic curve of cf-DNA for normal vs sepsis and SIRS vs sepsis were 0.955 (0.884-1.025), and 0.856 (0.749-0.929), respectively. There was a positive correlation between cf-DNA and interleukin 6 or procalcitonin or Acute Physiology and Chronic Health Evaluation II. The cf-DNA concentration was higher in intensive care unit nonsurviving patients compared to surviving patients (2183.33 ± 615.26 vs 972.46 ± 648.36 ng/mL; P DNA-based Alu assays are feasible and useful to quantify serum cf-DNA levels. Increased cf-DNA levels in septic patients might complement C-reactive protein and procalcitonin in a multiple marker format. Cell-free circulating DNA might be a new marker in discrimination of sepsis and SIRS.

  3. Standardization of anti-DNA antibody assays.

    Science.gov (United States)

    Pisetsky, David S

    2013-07-01

    Antibodies to DNA (anti-DNA) are the serological hallmark of systemic lupus erythematosus and represent important biomarkers for clinical and research purposes. These antibodies are part of a family of antibodies to nucleosomes and bind to conserved sites widely present on DNA. While the value of anti-DNA as a biomarker is well established, the assay for these antibodies has involved a variety of DNA sources and systems to detect DNA-anti-DNA interactions. The influence of these variations on antibody detection has complicated assay standardization. As an antigen, DNA has unique features since it is a highly charged polymer that has structural heterogeneity. This heterogeneity can affect antigenicity which can vary on the basis of DNA origin, size, conformation and mobility. In addition, as a polymer, DNA can promote patterns of antibody binding based on monogamous or bivalent interaction which require an extended polynucleotide structure. Understanding the nature of DNA as an antigen can facilitate interpretation of serological tests and underpin efforts at better standardization.

  4. Using Electrophoretic Mobility Shift Assays to Measure Equilibrium Dissociation Constants: GAL4-p53 Binding DNA as a Model System

    Science.gov (United States)

    Heffler, Michael A.; Walters, Ryan D.; Kugel, Jennifer F.

    2012-01-01

    An undergraduate biochemistry laboratory experiment is described that will teach students the practical and theoretical considerations for measuring the equilibrium dissociation constant (K[subscript D]) for a protein/DNA interaction using electrophoretic mobility shift assays (EMSAs). An EMSA monitors the migration of DNA through a native gel;…

  5. The effects of affinity-purified anti-DNA antibodies from patients with systemic lupus erythematosus on the fluorescent antinuclear antibody assay using HEp-2 cells.

    Science.gov (United States)

    Suzuki, Kimihiro; Kawamura, Masahide; Mineo, Midori; Shinohara, Tadashi; Kataharada, Koji; Okada, Makoto; Takada, Kunio; Miyawaki, Shoji; Ohsuzu, Fumitaka

    2002-01-01

    The aim of this study was to clarify the effects of anti-dsDNA antibodies on the titer and the nuclear staining pattern(s) in a fluorescent antinuclear antibody (FANA) assay using HEp-2 cells. Anti-dsDNA derived from 14 patients with systemic lupus erythematosus (SLE) was individually affinity-purified. The anti-dsDNA titer of the purified anti-dsDNA solution was measured by radioimmunoassay (RIA) or by enzyme-linked immunosorbent assay (ELISA). In the FANA assay, the anti-dsDNA solution was diluted in a stepwise manner and its titer was expressed by the endpoint dilution. The nuclear staining pattern in the anti-dsDNA solution was examined at the 1:5 and 1:20 dilutions and at the endpoint dilution. The anti-dsDNA titers of the affinity-purified anti-dsDNA solution were high enough (13 to 126 IU/ml) to be measured by RIA. However, the antinuclear antibody (ANA) titers of this solution were relatively low: 1:20 to 1:320. In the study of nuclear staining the peripheral pattern was observed in nine of the 14 cases at a 1:5 dilution. However, at the endpoint dilution, all cases exhibited the homogeneous pattern. These findings indicate that in the FANA assay using HEp-2 cells, 1) although serum samples show high anti-dsDNA titers by RIA or by ELISA, the antibodies' direct contribution to ANA titers is limited, and 2) when samples reveal a homogeneous staining pattern at the endpoint dilution, this suggests the presence of anti-dsDNA.

  6. Rover waste assay system

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    Akers, D.W.; Stoots, C.M.; Kraft, N.C.; Marts, D.J. [Idaho National Engineering Lab., Idaho Falls, ID (United States)

    1997-11-01

    The Rover Waste Assay System (RWAS) is a nondestructive assay system designed for the rapid assay of highly-enriched {sup 235}U contaminated piping, tank sections, and debris from the Rover nuclear rocket fuel processing facility at the Idaho Chemical Processing Plant. A scanning system translates a NaI(Tl) detector/collimator system over the structural components where both relative and calibrated measurements for {sup 137}Cs are made. Uranium-235 concentrations are in operation and is sufficiently automated that most functions are performed by the computer system. These functions include system calibration, problem identification, collimator control, data analysis, and reporting. Calibration of the system was done through a combination of measurements on calibration standards and benchmarked modeling. A description of the system is presented along with the methods and uncertainties associated with the calibration and analysis of the system for components from the Rover facility. 4 refs., 2 figs., 4 tabs.

  7. Auto-antibodies to double-stranded DNA as biomarker in systemic lupus erythematosus : comparison of different assays during quiescent and active disease

    NARCIS (Netherlands)

    de Leeuw, Karina; Bungener, Laura; Roozendaal, Caroline; Bootsma, Hendrika; Stegeman, Coen A

    2017-01-01

    OBJECTIVE: Auto-antibodies directed to dsDNA (anti-dsDNA) are used in diagnosis and follow-up for SLE. However, multiple assays are used. The objective of this study was to determine the best-performing assays, especially in prediction of exacerbations. METHODS: Seven assays were compared during LN

  8. Detection of Trichomonas vaginalis DNA by use of self-obtained vaginal swabs with the BD ProbeTec Qx assay on the BD Viper system.

    Science.gov (United States)

    Van Der Pol, Barbara; Williams, James A; Taylor, Stephanie N; Cammarata, Catherine L; Rivers, Charles A; Body, Barbara A; Nye, Melinda; Fuller, Deanna; Schwebke, Jane R; Barnes, Mathilda; Gaydos, Charlotte A

    2014-03-01

    Trichomonas vaginalis is the most prevalent nonviral sexually transmitted infection worldwide, and improved diagnostic methods are critical for controlling this pathogen. Diagnostic assays that can be used in conjunction with routine chlamydia/gonorrhea nucleic acid-based screening are likely to have the most impact on disease control. Here we describe the performance of the new BD T. vaginalis Qx (TVQ) amplified DNA assay, which can be performed on the automated BD Viper system. We focus on data from vaginal swab samples, since this is the specimen type routinely used for traditional trichomonas testing and the recommended specimen type for chlamydia/gonorrhea screening. Vaginal swabs were obtained from women attending sexually transmitted disease or family planning clinics at 7 sites. Patient-collected vaginal swabs were tested by the TVQ assay, and the Aptima T. vaginalis (ATV) assay was performed using clinician-collected vaginal swabs. Additional clinician-collected vaginal swabs were used for the wet mount and culture methods. Analyses included comparisons versus the patient infection status (PIS) defined by positive results with the wet mount method or culture, direct comparisons assessed with κ scores, and latent class analysis (LCA) as an unbiased estimator of test accuracy. Data from 838 women, 116 of whom were infected with T. vaginalis, were analyzed. The TVQ assay sensitivity and specificity estimates based on the PIS were 98.3% and 99.0%, respectively. The TVQ assay was similar to the ATV assay (κ=0.938) in direct analysis. LCA estimated the TVQ sensitivity and specificity as 98.3 and 99.6%, respectively. The TVQ assay performed well using self-collected vaginal swabs, the optimal sample type, as recommended by the CDC for chlamydia/gonorrhea screening among women.

  9. Directly Transforming PCR-Amplified DNA Fragments into Plant Cells Is a Versatile System That Facilitates the Transient Expression Assay

    Science.gov (United States)

    Lu, Yuming; Chen, Xi; Wu, Yuxuan; Wang, Yanping; He, Yuqing; Wu, Yan

    2013-01-01

    A circular plasmid containing a gene coding sequence has been broadly used for studying gene regulation in cells. However, to accommodate a quick screen plasmid construction and preparation can be time consuming. Here we report a PCR amplified dsDNA fragments (PCR-fragments) based transient expression system (PCR-TES) for suiting in the study of gene regulation in plant cells. Instead of transforming plasmids into plant cells, transient expression of PCR-fragments can be applicable. The transformation efficiency and expression property of PCR-fragments are comparable to transformation using plasmids. We analyzed the transformation efficiency in PCR-TES at transcription and protein levels. Our results indicate that the PCR-TES is as versatile as the conventional transformation system using plasmid DNA. Through reconstituting PYR1-mediated ABA signaling pathway in Arabidopsis mesophyll protoplasts, we were not only validating the practicality of PCR-TES but also screening potential candidates of CDPK family members which might be involved in the ABA signaling. Moreover, we determined that phosphorylation of ABF2 by CPK4 could be mediated by ABA-induced PYR1 and ABI1, demonstrating a crucial role of CDPKs in the ABA signaling. In summary, PCR-TES can be applicable to facilitate analyzing gene regulation and for the screen of putative regulatory molecules at the high throughput level in plant cells. PMID:23468926

  10. Directly transforming PCR-amplified DNA fragments into plant cells is a versatile system that facilitates the transient expression assay.

    Directory of Open Access Journals (Sweden)

    Yuming Lu

    Full Text Available A circular plasmid containing a gene coding sequence has been broadly used for studying gene regulation in cells. However, to accommodate a quick screen plasmid construction and preparation can be time consuming. Here we report a PCR amplified dsDNA fragments (PCR-fragments based transient expression system (PCR-TES for suiting in the study of gene regulation in plant cells. Instead of transforming plasmids into plant cells, transient expression of PCR-fragments can be applicable. The transformation efficiency and expression property of PCR-fragments are comparable to transformation using plasmids. We analyzed the transformation efficiency in PCR-TES at transcription and protein levels. Our results indicate that the PCR-TES is as versatile as the conventional transformation system using plasmid DNA. Through reconstituting PYR1-mediated ABA signaling pathway in Arabidopsis mesophyll protoplasts, we were not only validating the practicality of PCR-TES but also screening potential candidates of CDPK family members which might be involved in the ABA signaling. Moreover, we determined that phosphorylation of ABF2 by CPK4 could be mediated by ABA-induced PYR1 and ABI1, demonstrating a crucial role of CDPKs in the ABA signaling. In summary, PCR-TES can be applicable to facilitate analyzing gene regulation and for the screen of putative regulatory molecules at the high throughput level in plant cells.

  11. Alkaline Comet Assay for Assessing DNA Damage in Individual Cells.

    Science.gov (United States)

    Pu, Xinzhu; Wang, Zemin; Klaunig, James E

    2015-08-06

    Single-cell gel electrophoresis, commonly called a comet assay, is a simple and sensitive method for assessing DNA damage at the single-cell level. It is an important technique in genetic toxicological studies. The comet assay performed under alkaline conditions (pH >13) is considered the optimal version for identifying agents with genotoxic activity. The alkaline comet assay is capable of detecting DNA double-strand breaks, single-strand breaks, alkali-labile sites, DNA-DNA/DNA-protein cross-linking, and incomplete excision repair sites. The inclusion of digestion of lesion-specific DNA repair enzymes in the procedure allows the detection of various DNA base alterations, such as oxidative base damage. This unit describes alkaline comet assay procedures for assessing DNA strand breaks and oxidative base alterations. These methods can be applied in a variety of cells from in vitro and in vivo experiments, as well as human studies.

  12. Development of fluorescent methods for DNA methyltransferase assay

    Science.gov (United States)

    Li, Yueying; Zou, Xiaoran; Ma, Fei; Tang, Bo; Zhang, Chun-yang

    2017-03-01

    DNA methylation modified by DNA methyltransferase (MTase) plays an important role in regulating gene transcription, cell growth and proliferation. The aberrant DNA MTase activity may lead to a variety of human diseases including cancers. Therefore, accurate and sensitive detection of DNA MTase activity is crucial to biomedical research, clinical diagnostics and therapy. However, conventional DNA MTase assays often suffer from labor-intensive operations and time-consuming procedures. Alternatively, fluorescent methods have significant advantages of simplicity and high sensitivity, and have been widely applied for DNA MTase assay. In this review, we summarize the recent advances in the development of fluorescent methods for DNA MTase assay. These emerging methods include amplification-free and the amplification-assisted assays. Moreover, we discuss the challenges and future directions of this area.

  13. A rapid and efficient assay for extracting DNA from fungi

    Science.gov (United States)

    Griffin, Dale W.; Kellogg, C.A.; Peak, K.K.; Shinn, E.A.

    2002-01-01

    Aims: A method for the rapid extraction of fungal DNA from small quantities of tissue in a batch-processing format was investigated. Methods and Results: Tissue (DNA for PCR/ sequencing applications. Conclusions: The method allowed batch DNA extraction from multiple fungal isolates using a simple yet rapid and reliable assay. Significance and Impact of the Study: Use of this assay will allow researchers to obtain DNA from fungi quickly for use in molecular assays that previously required specialized instrumentation, was time-consuming or was not conducive to batch processing.

  14. An immunochemical assay to detect DNA damage in bovine sperm

    NARCIS (Netherlands)

    Schans, G.P. van der; Haring, R.; Dijk- Knijnenburg, H.C.M. van; Bruijnzeel, P.L.B.; Daas, N.H.G. den

    2000-01-01

    An immunochemical assay has been developed to detect oxidative damage in bovine sperm DNA. Sperm DNA contains a large amount of oxidative damage as a result of exposure to exogenous agents, but damage also can caused by normal metabolic processes and the absence of DNA repair in the later stages of

  15. Analysis of JC virus DNA replication using a quantitative and high-throughput assay.

    Science.gov (United States)

    Shin, Jong; Phelan, Paul J; Chhum, Panharith; Bashkenova, Nazym; Yim, Sung; Parker, Robert; Gagnon, David; Gjoerup, Ole; Archambault, Jacques; Bullock, Peter A

    2014-11-01

    Progressive Multifocal Leukoencephalopathy (PML) is caused by lytic replication of JC virus (JCV) in specific cells of the central nervous system. Like other polyomaviruses, JCV encodes a large T-antigen helicase needed for replication of the viral DNA. Here, we report the development of a luciferase-based, quantitative and high-throughput assay of JCV DNA replication in C33A cells, which, unlike the glial cell lines Hs 683 and U87, accumulate high levels of nuclear T-ag needed for robust replication. Using this assay, we investigated the requirement for different domains of T-ag, and for specific sequences within and flanking the viral origin, in JCV DNA replication. Beyond providing validation of the assay, these studies revealed an important stimulatory role of the transcription factor NF1 in JCV DNA replication. Finally, we show that the assay can be used for inhibitor testing, highlighting its value for the identification of antiviral drugs targeting JCV DNA replication.

  16. Analysis of JC virus DNA replication using a quantitative and high-throughput assay

    Energy Technology Data Exchange (ETDEWEB)

    Shin, Jong; Phelan, Paul J.; Chhum, Panharith; Bashkenova, Nazym; Yim, Sung; Parker, Robert [Department of Developmental, Molecular and Chemical Biology, Tufts University School of Medicine, Boston, MA 02111 (United States); Gagnon, David [Institut de Recherches Cliniques de Montreal (IRCM), 110 Pine Avenue West, Montreal, Quebec, Canada H2W 1R7 (Canada); Department of Biochemistry and Molecular Medicine, Université de Montréal, Montréal, Quebec (Canada); Gjoerup, Ole [Molecular Oncology Research Institute, Tufts Medical Center, Boston, MA 02111 (United States); Archambault, Jacques [Institut de Recherches Cliniques de Montreal (IRCM), 110 Pine Avenue West, Montreal, Quebec, Canada H2W 1R7 (Canada); Department of Biochemistry and Molecular Medicine, Université de Montréal, Montréal, Quebec (Canada); Bullock, Peter A., E-mail: Peter.Bullock@tufts.edu [Department of Developmental, Molecular and Chemical Biology, Tufts University School of Medicine, Boston, MA 02111 (United States)

    2014-11-15

    Progressive Multifocal Leukoencephalopathy (PML) is caused by lytic replication of JC virus (JCV) in specific cells of the central nervous system. Like other polyomaviruses, JCV encodes a large T-antigen helicase needed for replication of the viral DNA. Here, we report the development of a luciferase-based, quantitative and high-throughput assay of JCV DNA replication in C33A cells, which, unlike the glial cell lines Hs 683 and U87, accumulate high levels of nuclear T-ag needed for robust replication. Using this assay, we investigated the requirement for different domains of T-ag, and for specific sequences within and flanking the viral origin, in JCV DNA replication. Beyond providing validation of the assay, these studies revealed an important stimulatory role of the transcription factor NF1 in JCV DNA replication. Finally, we show that the assay can be used for inhibitor testing, highlighting its value for the identification of antiviral drugs targeting JCV DNA replication. - Highlights: • Development of a high-throughput screening assay for JCV DNA replication using C33A cells. • Evidence that T-ag fails to accumulate in the nuclei of established glioma cell lines. • Evidence that NF-1 directly promotes JCV DNA replication in C33A cells. • Proof-of-concept that the HTS assay can be used to identify pharmacological inhibitor of JCV DNA replication.

  17. Click-iT assay with improved DNA distribution histograms.

    Science.gov (United States)

    Hamelik, Ronald M; Krishan, Awtar

    2009-10-01

    The Click-iT Assay developed and commercialized by Invitrogen is based on incorporation of a new 5-bromo-2'-deoxyuridine analog, 5-ethynyl-2'-deoxyuridine (EdU) into newly synthesized DNA and its recognition by azide dyes via a copper mediated "click" reaction. This relatively convenient and useful procedure depends on fixation of cells with paraformaldehyde and staining of the DNA with 7-aminoactinomycin-D (7-AAD). Both of these procedures result in DNA histograms with broad coefficients of variation (CV's). In this report, we have shown that after EdU incorporation, nuclei isolated by lysis can be incubated with the Click-iT Assay and stained with propidium iodide for generation of DNA histograms with low CV's. This modified procedure results in better DNA histograms by replacing 7-AAD with propidium iodide and also saves processing time by eliminating the fixation and permeabilization steps.

  18. Photoelectrochemical competitive DNA hybridization assay using semiconductor quantum dot conjugated oligonucleotides.

    Science.gov (United States)

    Baş, Deniz; Boyaci, Ismail Hakki

    2011-05-01

    A competitive DNA hybridization assay based on the photoelectrochemistry of the semiconductor quantum dot-single stranded DNA conjugates (QD-ssDNA) was developed. Hybridization of QD-ssDNA with the capture probe DNA immobilized on the indium-tin oxide electrodes enables photocurrent generation when the electrochemical cell was illuminated with a light source. Upon the competition between QD-ssDNA and single-stranded target DNA, the photocurrent response decreased with the increase in the target DNA concentration. A linear relationship between the photocurrent and the target DNA concentration was obtained (R(2) = 0.991). The selectivity of system towards the target DNA was also demonstrated using non-complementary sample.

  19. High-throughput DNA droplet assays using picoliter reactor volumes.

    Science.gov (United States)

    Srisa-Art, Monpichar; deMello, Andrew J; Edel, Joshua B

    2007-09-01

    The online characterization and detection of individual droplets at high speeds, low analyte concentrations, and perfect detection efficiencies is a significant challenge underpinning the application of microfluidic droplet reactors to high-throughput chemistry and biology. Herein, we describe the integration of confocal fluorescence spectroscopy as a high-efficiency detection method for droplet-based microfluidics. Issues such as surface contamination, rapid mixing, and rapid detection, as well as low detections limits have been addressed with the approach described when compared to conventional laminar flow-based fluidics. Using such a system, droplet size, droplet shape, droplet formation frequencies, and droplet compositions can be measured accurately and precisely at kilohertz frequencies. Taking advantage of this approach, we demonstrate a high-throughput biological assay based on fluorescence resonance energy transfer (FRET). By attaching a FRET donor (Alexa Fluor 488) to streptavidin and labeling a FRET acceptor (Alexa Fluor 647) on one DNA strand and biotin on the complementary strand, donor and acceptor molecules are brought in proximity due to streptavidin-biotin binding, resulting in FRET. Fluorescence bursts of the donor and acceptor from each droplet can be monitored simultaneously using separate avalanche photodiode detectors operating in single photon counting mode. Binding assays were investigated and compared between fixed streptavidin and DNA concentrations. Binding curves fit perfectly to Hill-Waud models, and the binding ratio between streptavidin and biotin was evaluated and found to be in agreement with the biotin binding sites on streptavidin. FRET efficiency for this FRET pair was also investigated from the binding results. Efficiency results show that this detection system can precisely measure FRET even at low FRET efficiencies.

  20. A DNA immunoprecipitation assay used in quantitative detection of in vitro DNA-protein complex binding.

    Science.gov (United States)

    Kim, Min Young; Chae, Ji Hyung; Oh, Chang-Ho; Kim, Chul Geun

    2013-10-15

    To begin gene transcription, several transcription factors must bind to specific DNA sequences to form a complex via DNA-protein interactions. We established an in vitro method for specific and sensitive analyses of DNA-protein interactions based on a DNA immunoprecipitation (DIP) method. We verified the accuracy and efficiency of the DIP assay in quantitatively measuring DNA-protein binding using transcription factor CP2c as a model. With our DIP assay, we could detect specific interactions within a DNA-CP2c complex, with reproducible and quantitative binding values. In addition, we were able to effectively measure the changes in DNA-CP2c binding by the addition of a small molecule, FQI1 (factor quinolinone inhibitor 1), previously identified as a specific inhibitor of this binding. To identify a new regulator of DNA-CP2c binding, we analyzed several CP2c binding peptides and found that only one class of peptide severely inhibits DNA-CP2c binding. These data show that our DIP assay is very useful in quantitatively detecting the binding dynamics of DNA-protein complex. Because DNA-protein interaction is very dynamic in different cellular environments, our assay can be applied to the detection of active transcription factors, including promoter occupancy in normal and disease conditions. Moreover, it may be used to develop a targeted regulator of specific DNA-protein interaction.

  1. Forensically relevant SNaPshot(®) assays for human DNA SNP analysis: a review.

    Science.gov (United States)

    Mehta, Bhavik; Daniel, Runa; Phillips, Chris; McNevin, Dennis

    2017-01-01

    Short tandem repeats are the gold standard for human identification but are not informative for forensic DNA phenotyping (FDP). Single-nucleotide polymorphisms (SNPs) as genetic markers can be applied to both identification and FDP. The concept of DNA intelligence emerged with the potential for SNPs to infer biogeographical ancestry (BGA) and externally visible characteristics (EVCs), which together enable the FDP process. For more than a decade, the SNaPshot(®) technique has been utilised to analyse identity and FDP-associated SNPs in forensic DNA analysis. SNaPshot is a single-base extension (SBE) assay with capillary electrophoresis as its detection system. This multiplexing technique offers the advantage of easy integration into operational forensic laboratories without the requirement for any additional equipment. Further, the SNP panels from SNaPshot(®) assays can be incorporated into customised panels for massively parallel sequencing (MPS). Many SNaPshot(®) assays are available for identity, BGA and EVC profiling with examples including the well-known SNPforID 52-plex identity assay, the SNPforID 34-plex BGA assay and the HIrisPlex EVC assay. This review lists the major forensically relevant SNaPshot(®) assays for human DNA SNP analysis and can be used as a guide for selecting the appropriate assay for specific identity and FDP applications.

  2. Proofreading genotyping assays mediated by high fidelity exo+ DNA polymerases.

    Science.gov (United States)

    Zhang, Jia; Li, Kai; Pardinas, Jose R; Sommer, Steve S; Yao, Kai-Tai

    2005-02-01

    DNA polymerases with 3'-5' proofreading function mediate high fidelity DNA replication but their application for mutation detection was almost completely neglected before 1998. The obstacle facing the use of exo(+) polymerases for mutation detection could be overcome by primer-3'-termini modification, which has been tested using allele-specific primers with 3' labeling, 3' exonuclease-resistance and 3' dehydroxylation modifications. Accordingly, three new types of single nucleotide polymorphism (SNP) assays have been developed to carry out genome-wide genotyping making use of the fidelity advantage of exo(+) polymerases. Such SNP assays might also provide a novel approach for re-sequencing and de novo sequencing. These new mutation detection assays are widely adaptable to a variety of platforms, including real-time PCR, multi-well plate and microarray technologies. Application of exo(+) polymerases to genetic analysis could accelerate the pace of personalized medicine.

  3. Detection of radiation treatment of beans using DNA comet assay

    Science.gov (United States)

    Khan, Ashfaq A.; Khan, Hasan M.; Delincée, Henry

    2002-03-01

    A simple technique of microgel electrophoresis of single cells (DNA Comet Assay) enabled a quick detection of radiation treatment of several kinds of leguminous beans (azuki, black, black eye, mung, pinto, red kidney and white beans). Each variety was exposed to radiation doses of 0.5, 1 and 5kGy covering the permissible limits for insect disinfestation. The cells or nuclei from beans were extracted in cold PBS, embedded in agarose on microscope slides, lysed between 15 and 60min in 2.5% SDS and electrophoresis was carried out at a voltage of 2V/cm for 2-2.5min. After silver staining, the slides were evaluated through an ordinary transmission microscope. In irradiated samples, fragmented DNA stretched towards the anode and the damaged cells appeared as a comet. The density of DNA in the tails increased with increasing radiation dose. However, in non-irradiated samples, the large molecules of DNA remained relatively intact and there was only minor or no migration of DNA; the cells were round or had very short tails only. Hence, the DNA comet assay provides an inexpensive, rapid and relatively simple screening method for the detection of irradiated beans.

  4. Electrochemical DNA sandwich assay with a lipase label for attomole detection of DNA

    DEFF Research Database (Denmark)

    Ferapontova, Elena; Hansen, Majken Nørgaard; Saunders, Aaron Marc

    2010-01-01

    A fast and sensitive electrochemical lipase-based sandwich hybridization assay for detection of attomole levels of DNA has been developed. A combination of magnetic beads, used for pre-concentration and bioseparation of the analyte with a lipase catalyst label allowed detection of DNA with a limit...

  5. The comet assay: assessment of in vitro and in vivo DNA damage.

    Science.gov (United States)

    Bajpayee, Mahima; Kumar, Ashutosh; Dhawan, Alok

    2013-01-01

    Rapid industrialization and pursuance of a better life have led to an increase in the amount of chemicals in the environment, which are deleterious to human health. Pesticides, automobile exhausts, and new chemical entities all add to air pollution and have an adverse effect on all living organisms including humans. Sensitive test systems are thus required for accurate hazard identification and risk assessment. The Comet assay has been used widely as a simple, rapid, and sensitive tool for assessment of DNA damage in single cells from both in vitro and in vivo sources as well as in humans. Already, the in vivo comet assay has gained importance as the preferred test for assessing DNA damage in animals for some international regulatory guidelines. The advantages of the in vivo comet assay are its ability to detect DNA damage in any tissue, despite having non-proliferating cells, and its sensitivity to detect genotoxicity. The recommendations from the international workshops held for the comet assay have resulted in establishment of guidelines. The in vitro comet assay conducted in cultured cells and cell lines can be used for screening large number of compounds and at very low concentrations. The in vitro assay has also been automated to provide a high-throughput screening method for new chemical entities, as well as environmental samples. This chapter details the in vitro comet assay using the 96-well plate and in vivo comet assay in multiple organs of the mouse.

  6. A filter microplate assay for quantitative analysis of DNA binding proteins using fluorescent DNA.

    Science.gov (United States)

    Yang, William C; Swartz, James R

    2011-08-15

    We present a rapid method for quantifying the apparent DNA binding affinity and capacity of recombinant transcription factors (TFs). We capture His6-tagged TFs using nickel-nitrilotriacetic acid (Ni-NTA) agarose and incubate the immobilized TFs with fluorescently labeled cognate DNA probes. After washing, the strength of the fluorescence signal indicates the extent of DNA binding. The assay was validated using two pluripotency-regulating TFs: SOX2 and NANOG. Using competitive binding analysis with nonlabeled competitor DNA, we show that SOX2 and NANOG specifically bind to their consensus sequences. We also determined the apparent affinity of SOX2 and NANOG for their consensus sequences to be 54.2±9 and 44.0±6nM, respectively, in approximate agreement with literature values. Our assay does not require radioactivity, but radioactively labeling the TFs enables the measurement of absolute amounts of immobilized SOX2 and NANOG and, hence, a DNA-to-protein binding ratio. SOX2 possesses a 0.95 DNA-to-protein binding ratio, whereas NANOG possesses a 0.44 ratio, suggesting that most of the SOX2 and approximately half of the NANOG are competent for DNA binding. Alternatively, the NANOG dimer may be capable of binding only one DNA target. This flexible DNA binding assay enables the analysis of crude or purified samples with or without radioactivity.

  7. Detection of DNA cross-links in tumor cells with the ethidium bromide fluorescence assay

    NARCIS (Netherlands)

    de Jong, Steven; Zijlstra, J G; Timmer-Bosscha, H; Mulder, N H; de Vries, Liesbeth

    1986-01-01

    Until now the fluorescence assay with ethidium bromide has only been used on pure DNA. This assay depends on the difference in fluorescence between single- and double-stranded DNA (dsDNA). Cross-links in DNA are measured by the return of fluorescence of dsDNA after heat denaturation at pH 12. Under

  8. The low molecular weight DNA diffusion assay as an indicator of cytotoxicity for the in vitro comet assay.

    Science.gov (United States)

    Speit, Günter; Vesely, Alexandra; Schütz, Petra; Linsenmeyer, Regina; Bausinger, Julia

    2014-07-01

    The low molecular weight DNA diffusion assay (LMW assay) has been recommended as a measure for cytotoxicity for the in vivo comet assay. To better understand the relationship between effects in the LMW assay, DNA migration in the comet assay and effects in established cytotoxicity tests, we performed in vitro experiments with cultured human cell lines (TK6, A549) and comparatively investigated five test substances (methyl methanesulfonate, (±)-benzo[a]pyrene diol epoxide, sodium dodecyl sulphate, menthol and sodium arsenite). We measured DNA migration (tail intensity) in the comet assay and the frequency of 'hedgehogs' (cells with almost all DNA in the tail), DNA diffusion in the LMW assay, cell viability (trypan blue and fluorescein diacetate/ethidium bromide staining) and inhibition of proliferation (relative cell counts). Our in vitro experiments indicate that effects in the LMW assay occur independently from DNA effects in the comet assay and are not related to the occurrence of hedgehogs. Results from the LMW assay are in good agreement with results from viability assays and seem to allow discriminating genotoxic from non-genotoxic substances when appropriate preparation times are considered. Measurements of cytotoxicity by these methods only at an early preparation time after exposure to genotoxic substances may lead to erroneous results.

  9. Assessment of Human DNA Repair (NER) Capacity With DNA Repair Rate (DRR) by Comet Assay

    Institute of Scientific and Technical Information of China (English)

    WEI ZHENG; JI-LIANG HE; LI-FEN JIN; JIAN-LIN LOU; BAO-HONG WANG

    2005-01-01

    Objective Alkaline comet assay was used to evaluate DNA repair (nucleotide excision repair, NER) capacity of human fresh lymphocytes from 12 young healthy non-smokers (6 males and 6 females). Methods Lymphocytes were exposed to UV-C (254 nm) at the dose rate of 1.5 J/m2/sec. Novobiocin (NOV) and aphidicolin (APC), DNA repair inhibitors, were utilized to imitate the deficiency of DNA repair capacity at the incision and ligation steps of NER. Lymphocytes from each donor were divided into three grougs: UVC group, UVC plus NOV group, and UVC plus APC group. DNA single strand breaks were detected in UVC irradiated cells incubated for 0, 30, 60, 90, 120, 180, and 240 min after UVC irradiation. DNA repair rate (DRR) served as an indicator of DNA repair capacity. Results The results indicated that the maximum DNA damage (i.e. maximum tail length) in the UVC group mainly appeared at 90 min. The ranges of DRRs in the UVC group were 62.84%-98.71%. Average DRR value was 81.84%. The DRR difference between males and females was not significant (P<0.05). However, the average DRR value in the UVC plus NOV group and the UVC plus APC group was 52.98% and 39.57% respectively, which were significantly lower than that in the UVC group (P<0.01). Conclusion The comet assay is a rapid, simple and sensitive screening test to assess individual DNA repair (NER) capacity. It is suggested that the time to detect DNA single strand breaks in comet assay should include 0 (before UV irradiation), 90 and 240 min after exposure to 1.5 J·m-2 UVC at least. The DRR, as an indicator, can represent the individual DNA repair capacity in comet assay.

  10. Quantitative assay of photoinduced DNA strand breaks by real-time PCR.

    Science.gov (United States)

    Wiczk, Justyna; Westphal, Kinga; Rak, Janusz

    2016-09-05

    Real-time PCR (qPCR) - a modern methodology primarily used for studying gene expression has been employed for the quantitative assay of an important class of DNA damage - single strand breaks. These DNA lesions which may lead to highly cytotoxic double strand breaks were quantified in a model system where double stranded DNA was sensitized to UV photons by labeling with 5-bromo-2'-deoxyuridine. The amount of breaks formed due to irradiation with several doses of 320nm photons was assayed by two independent methods: LC-MS and qPCR. A very good agreement between the relative damage measured by the two completely different analytical tools proves the applicability of qPCR for the quantitative analysis of SSBs. Our results suggest that the popularity of the hitherto underestimated though accurate and site-specific technique of real-time PCR may increase in future DNA damage studies.

  11. Identification of irradiated refrigerated pork with the DNA comet assay

    Energy Technology Data Exchange (ETDEWEB)

    Araujo, M.M. E-mail: villavic@net.ipen.br; Marin-Huachaca, N.S.; Mancini-Filho, J. E-mail: jmancini@usp.br; Delincee, H.; Villavicencio, A.L.C.H. E-mail: henry.delincee@bfe.uni-karlsruhe.de

    2004-10-01

    Food irradiation can contribute to a safer and more plentiful food supply by inactivating pathogens, eradicating pests and by extending shelf-life. Particularly in the case of pork meat, this process could be a useful way to inactivate harmful parasites such as Trichinella and Taenia solium. Ionizing radiation causes damage to the DNA of the cells (e.g. strand breaks), which can be used to detect irradiated food. Microelectrophoresis of single cells ('Comet Assay') is a simple and rapid test for DNA damage and can be used over a wide dose range and for a variety of products. Refrigerated pork meat was irradiated with a {sup 60}Co source, Gammacell 220 (A.E.C.L.) installed in IPEN (Sao Paulo, Brazil). The doses given were 0, 1.5, 3.0 and 4.5 kGy for refrigerated samples. Immediately after irradiation the samples were returned to the refrigerator (6 deg. C). Samples were kept in the refrigerator after irradiation. Pork meat was analyzed 1, 8 and 10 days after irradiation using the DNA 'Comet Assay'. This method showed to be an inexpensive and rapid technique for qualitative detection of irradiation treatment.

  12. Identification of irradiated refrigerated pork with the DNA comet assay

    Science.gov (United States)

    Araújo, M. M.; Marin-Huachaca, N. S.; Mancini-Filho, J.; Delincée, H.; Villavicencio, A. L. C. H.

    2004-09-01

    Food irradiation can contribute to a safer and more plentiful food supply by inactivating pathogens, eradicating pests and by extending shelf-life. Particularly in the case of pork meat, this process could be a useful way to inactivate harmful parasites such as Trichinella and Taenia solium. Ionizing radiation causes damage to the DNA of the cells (e.g. strand breaks), which can be used to detect irradiated food. Microelectrophoresis of single cells (``Comet Assay'') is a simple and rapid test for DNA damage and can be used over a wide dose range and for a variety of products. Refrigerated pork meat was irradiated with a 60Co source, Gammacell 220 (A.E.C.L.) installed in IPEN (Sa~o Paulo, Brazil). The doses given were 0, 1.5, 3.0 and 4.5kGy for refrigerated samples. Immediately after irradiation the samples were returned to the refrigerator (6°C). Samples were kept in the refrigerator after irradiation. Pork meat was analyzed 1, 8 and 10 days after irradiation using the DNA ``Comet Assay''. This method showed to be an inexpensive and rapid technique for qualitative detection of irradiation treatment.

  13. Sperm DNA assays and their relationship to sperm motility and morphology in bulls (Bos Taurus).

    Science.gov (United States)

    Serafini, Rosanna; Romano, Juan E; Varner, Dickson D; Di Palo, Rossella; Love, Charles C

    2015-08-01

    The relationship among sperm DNA assays in bulls with different sperm motility and morphology measures has not been reported. The objectives of the present study were to (1) describe Comet assay measures and examine their repeatability (inter- and intra-assay); (2) compare sperm DNA quality assays (i.e., Sperm Chromatin Structure Assay-SCSA; alkaline and neutral Comet assays and Sperm Bos Halomax assay-SBH) in two groups of bulls selected on either greater and lesser sperm motility and morphology (greater compared with lesser); (3) determine the relationship among DNA assays and sperm motility and morphology values. Inter-assay repeatability was greater for the neutral Comet assay as compared to the alkaline Comet assay. Intra-assay repeatability was greater than inter-assay repeatability for both Comet assays. Comet assay dimension measures and percentage tail DNA were the most repeatable for both Comet assays. Among sperm DNA quality assays, only SCSA measures and neutral Comet assay Ghosts (% Ghosts), head diameter and area, and comet area were different between greater and lesser sperm quality groups (P<0.05). The SCSA measures were inversely correlated with neutral Comet head measures (diameter, area, and intensity) and positively with percentage Ghosts (P<0.05). The % Ghosts and COMP-αt were correlated with some measures of sperm morphology and sperm motility. The neutral Comet assay was more appropriate for sperm evaluation than the alkaline Comet assay for distinguishing among groups with different sperm quality.

  14. Environmental DNA (eDNA) metabarcoding assays to detect invasive invertebrate species in the Great Lakes.

    Science.gov (United States)

    Klymus, Katy E; Marshall, Nathaniel T; Stepien, Carol A

    2017-01-01

    Describing and monitoring biodiversity comprise integral parts of ecosystem management. Recent research coupling metabarcoding and environmental DNA (eDNA) demonstrate that these methods can serve as important tools for surveying biodiversity, while significantly decreasing the time, expense and resources spent on traditional survey methods. The literature emphasizes the importance of genetic marker development, as the markers dictate the applicability, sensitivity and resolution ability of an eDNA assay. The present study developed two metabarcoding eDNA assays using the mtDNA 16S RNA gene with Illumina MiSeq platform to detect invertebrate fauna in the Laurentian Great Lakes and surrounding waterways, with a focus for use on invasive bivalve and gastropod species monitoring. We employed careful primer design and in vitro testing with mock communities to assess ability of the markers to amplify and sequence targeted species DNA, while retaining rank abundance information. In our mock communities, read abundances reflected the initial input abundance, with regressions having significant slopes (p<0.05) and high coefficients of determination (R2) for all comparisons. Tests on field environmental samples revealed similar ability of our markers to measure relative abundance. Due to the limited reference sequence data available for these invertebrate species, care must be taken when analyzing results and identifying sequence reads to species level. These markers extend eDNA metabarcoding research for molluscs and appear relevant to other invertebrate taxa, such as rotifers and bryozoans. Furthermore, the sphaeriid mussel assay is group-specific, exclusively amplifying bivalves in the Sphaeridae family and providing species-level identification. Our assays provide useful tools for managers and conservation scientists, facilitating early detection of invasive species as well as improving resolution of mollusc diversity.

  15. Identification of irradiated refrigerated poultry with the DNA comet assay

    Energy Technology Data Exchange (ETDEWEB)

    Villavicencio, A.L.C.H. E-mail: villavic@net.ipen.br; Araujo, M.M.; Marin-Huachaca, N.S.; Mancini-Filho, J. E-mail: jmancini@usp.br; Delincee, H. E-mail: henry.delincee@bfe.uni-karlsruhe.de

    2004-10-01

    Food irradiation could make a significant contribution to the reduction of food-borne diseases caused by harmful bacteria such as Salmonella and parasites. In fact these organisms cause an increasing number of diseases and eventually deaths all over the world, also in industrialized countries. Radiation processing has the advantage that in addition to eliminating pathogens, thereby enhancing food safety, it also extends shelf life through destruction of spoilage organisms. The DNA molecule because of its big size is an easy target for ionizing radiation, therefore, changes in DNA offer potential to be used as a detection method for the irradiation treatment. In our study, poultry has been irradiated and changes in DNA analyzed by the Comet Assay. Samples were packed in plastic bags and irradiated. Doses were 0, 1.5, 3.0 and 4.5 kGy. Immediately after irradiation the samples were returned to the refrigerator (4 deg. C). Samples were analyzed 1 and 10 days after irradiation. This method proved to be an inexpensive and rapid screening technique for qualitative detection of irradiation treatment.

  16. Identification of irradiated refrigerated poultry with the DNA comet assay

    Science.gov (United States)

    Villavicencio, A. L. C. H.; Araújo, M. M.; Marin-Huachaca, N. S.; Mancini-Filho, J.; Delincée, H.

    2004-09-01

    Food irradiation could make a significant contribution to the reduction of food-borne diseases caused by harmful bacteria such as Salmonella and parasites. In fact these organisms cause an increasing number of diseases and eventually deaths all over the world, also in industrialized countries. Radiation processing has the advantage that in addition to eliminating pathogens, thereby enhancing food safety, it also extends shelf life through destruction of spoilage organisms. The DNA molecule because of its big size is an easy target for ionizing radiation, therefore, changes in DNA offer potential to be used as a detection method for the irradiation treatment. In our study, poultry has been irradiated and changes in DNA analyzed by the Comet Assay. Samples were packed in plastic bags and irradiated. Doses were 0, 1.5, 3.0 and 4.5kGy. Immediately after irradiation the samples were returned to the refrigerator (4°C). Samples were analyzed 1 and 10 days after irradiation. This method proved to be an inexpensive and rapid screening technique for qualitative detection of irradiation treatment.

  17. Detection of Irradiation Treatment of Foods Using DNA `Comet Assay'

    Science.gov (United States)

    Khan, Hasan M.; Delincée, Henry

    1998-06-01

    Microgel electrophoresis of single cells (DNA comet assay) has been investigated to detect irradiation treatment of some food samples. These samples of fresh and frozen rainbow trout, red lentil, gram and sliced almonds were irradiated to 1 or 2 kGy using 10 MeV electron beam from a linear accelerator. Rainbow trout samples yielded good results with samples irradiated to 1 or 2 kGy showing fragmentation of DNA and, therefore, longer comets with no intact cells. Unirradiated samples showed shorter comets with a significant number of intact cells. For rainbow trout stored in a freezer for 11 days the irradiated samples can still be discerned by electrophoresis from unirradiated samples, however, the unirradiated trouts also showed some longer comets besides some intact cells. Radiation treatment of red lentils can also be detected by this method, i.e. no intact cells in 1 or 2 kGy irradiated samples and shorter comets and some intact cells in unirradiated samples. However, the results for gram and sliced almond samples were not satisfactory since some intact DNA cells were observed in irradiated samples as well. Probably, incomplete lysis has led to these deviating results.

  18. Electrochemical chip-based genomagnetic assay for detection of high-risk human papillomavirus DNA.

    Science.gov (United States)

    Bartosik, Martin; Durikova, Helena; Vojtesek, Borivoj; Anton, Milan; Jandakova, Eva; Hrstka, Roman

    2016-09-15

    Cervical cancer, being the fourth leading cause of cancer death in women worldwide, predominantly originates from a persistent infection with a high-risk human papillomavirus (HPV). Detection of DNA sequences from these high-risk strains, mostly HPV-16 and HPV-18, represents promising strategy for early screening, which would help to identify women with higher risk of cervical cancer. In developing countries, inadequate screening options lead to disproportionately high mortality rates, making a fast and inexpensive detection schemes highly important. Electrochemical sensors and assays offer an alternative to current methods of detection. We developed an electrochemical-chip based assay, in which target HPV DNA is captured via magnetic bead-modified DNA probes, followed by an antidigoxigenin-peroxidase detection system at screen-printed carbon electrode chips, enabling parallel measurements of eight samples simultaneously. We show sensitive detection in attomoles of HPV DNA, selective discrimination between HPV-16 and HPV-18 and good reproducibility. Most importantly, we show application of the assay into both cancer cell lines and cervical smears from patients. The electrochemical results correlated well with standard methods, making this assay potentially applicable in clinical practice.

  19. Automated DNA Sequencing System

    Energy Technology Data Exchange (ETDEWEB)

    Armstrong, G.A.; Ekkebus, C.P.; Hauser, L.J.; Kress, R.L.; Mural, R.J.

    1999-04-25

    Oak Ridge National Laboratory (ORNL) is developing a core DNA sequencing facility to support biological research endeavors at ORNL and to conduct basic sequencing automation research. This facility is novel because its development is based on existing standard biology laboratory equipment; thus, the development process is of interest to the many small laboratories trying to use automation to control costs and increase throughput. Before automation, biology Laboratory personnel purified DNA, completed cycle sequencing, and prepared 96-well sample plates with commercially available hardware designed specifically for each step in the process. Following purification and thermal cycling, an automated sequencing machine was used for the sequencing. A technician handled all movement of the 96-well sample plates between machines. To automate the process, ORNL is adding a CRS Robotics A- 465 arm, ABI 377 sequencing machine, automated centrifuge, automated refrigerator, and possibly an automated SpeedVac. The entire system will be integrated with one central controller that will direct each machine and the robot. The goal of this system is to completely automate the sequencing procedure from bacterial cell samples through ready-to-be-sequenced DNA and ultimately to completed sequence. The system will be flexible and will accommodate different chemistries than existing automated sequencing lines. The system will be expanded in the future to include colony picking and/or actual sequencing. This discrete event, DNA sequencing system will demonstrate that smaller sequencing labs can achieve cost-effective the laboratory grow.

  20. The archaeal Ced system imports DNA

    Science.gov (United States)

    van Wolferen, Marleen; Wagner, Alexander; van der Does, Chris; Albers, Sonja-Verena

    2016-01-01

    The intercellular transfer of DNA is a phenomenon that occurs in all domains of life and is a major driving force of evolution. Upon UV-light treatment, cells of the crenarchaeal genus Sulfolobus express Ups pili, which initiate cell aggregate formation. Within these aggregates, chromosomal DNA, which is used for the repair of DNA double-strand breaks, is exchanged. Because so far no clear homologs of bacterial DNA transporters have been identified among the genomes of Archaea, the mechanisms of archaeal DNA transport have remained a puzzling and underinvestigated topic. Here we identify saci_0568 and saci_0748, two genes from Sulfolobus acidocaldarius that are highly induced upon UV treatment, encoding a transmembrane protein and a membrane-bound VirB4/HerA homolog, respectively. DNA transfer assays showed that both proteins are essential for DNA transfer between Sulfolobus cells and act downstream of the Ups pili system. Our results moreover revealed that the system is involved in the import of DNA rather than the export. We therefore propose that both Saci_0568 and Saci_0748 are part of a previously unidentified DNA importer. Given the fact that we found this transporter system to be widely spread among the Crenarchaeota, we propose to name it the Crenarchaeal system for exchange of DNA (Ced). In this study we have for the first time to our knowledge described an archaeal DNA transporter. PMID:26884154

  1. Development of a lipase-based optical assay for detection of DNA

    DEFF Research Database (Denmark)

    Pinijsuwan, Suttiporn; Shipovskov, Stepan; Surareungchai, Werasak

    2011-01-01

    A lipase-based assay for detection of specific DNA sequences has been developed. Lipase from Candida antarctica was conjugated to DNA and captured on magnetic beads in a sandwich assay, in which the binding was dependent on the presence of a specific target DNA. For amplification and to generate...

  2. Detection of Hepatitis B Virus DNA by Duplex Scorpion Primer-based PCR Assay

    Institute of Scientific and Technical Information of China (English)

    KONG De-Ming孔德明; SHEN Han-Xi沈含熙; MI Huai-Feng宓怀风

    2004-01-01

    The application of a new fiuorogenic probe-based PCR assay (PCR duplex scorpion primer assay) to the detection of Hepatitis B virus (HBV) DNA in human sera was described. Duplex scorpion primer is a modified variant of duplex Amplifluor, and the incorporation of a PCR stopper between probe and primer sequences improve the detection specificity and sensitivity. Combined with PCR amplification, this probe can give unambiguous positive results for the reactions initiated with more than 20 HBV molecules. In addition, the particular unimolecular probing mechanism of this probe makes the use of short target-specific probe sequence possible, which will render this probe applicable in some specific systems.

  3. Assessment and reduction of comet assay variation in relation to DNA damage: studies from the European Comet Assay Validation Group

    DEFF Research Database (Denmark)

    Møller, Peter; Möller, Lennart; Godschalk, Roger W L

    2010-01-01

    The alkaline single cell gel electrophoresis (comet) assay has become a widely used method for the detection of DNA damage and repair in cells and tissues. Still, it has been difficult to compare results from different investigators because of differences in assay conditions and because the data ...

  4. High accuracy genotyping directly from genomic DNA using a rolling circle amplification based assay

    Directory of Open Access Journals (Sweden)

    Du Yuefen

    2003-05-01

    Full Text Available Abstract Background Rolling circle amplification of ligated probes is a simple and sensitive means for genotyping directly from genomic DNA. SNPs and mutations are interrogated with open circle probes (OCP that can be circularized by DNA ligase when the probe matches the genotype. An amplified detection signal is generated by exponential rolling circle amplification (ERCA of the circularized probe. The low cost and scalability of ligation/ERCA genotyping makes it ideally suited for automated, high throughput methods. Results A retrospective study using human genomic DNA samples of known genotype was performed for four different clinically relevant mutations: Factor V Leiden, Factor II prothrombin, and two hemochromatosis mutations, C282Y and H63D. Greater than 99% accuracy was obtained genotyping genomic DNA samples from hundreds of different individuals. The combined process of ligation/ERCA was performed in a single tube and produced fluorescent signal directly from genomic DNA in less than an hour. In each assay, the probes for both normal and mutant alleles were combined in a single reaction. Multiple ERCA primers combined with a quenched-peptide nucleic acid (Q-PNA fluorescent detection system greatly accellerated the appearance of signal. Probes designed with hairpin structures reduced misamplification. Genotyping accuracy was identical from either purified genomic DNA or genomic DNA generated using whole genome amplification (WGA. Fluorescent signal output was measured in real time and as an end point. Conclusions Combining the optimal elements for ligation/ERCA genotyping has resulted in a highly accurate single tube assay for genotyping directly from genomic DNA samples. Accuracy exceeded 99 % for four probe sets targeting clinically relevant mutations. No genotypes were called incorrectly using either genomic DNA or whole genome amplified sample.

  5. Direct observation of enzymes replicating DNA using a single-molecule DNA stretching assay

    NARCIS (Netherlands)

    Kulczyk, A.W.; Tanner, N.A.; Loparo, J.J.; Richardson, C.C.; Oijen, A.M. van

    2010-01-01

    We describe a method for observing real time replication of individual DNA molecules mediated by proteins of the bacteriophage replication system. Linearized lambda DNA is modified to have a biotin on the end of one strand, and a digoxigenin moiety on the other end of the same strand. The biotinylat

  6. One-step assay for the quantification of T4 DNA ligase.

    Science.gov (United States)

    Franke, Steffi; Kreisig, Thomas; Buettner, Karin; Zuchner, Thole

    2015-02-01

    As one of the most commonly used enzyme in molecular biology, the T4 DNA ligase presents an important tool for the manipulation of DNA. T4 DNA ligase activity measurements are based on the use of radioactivity or rather labor-intense procedures including gel-based analysis. We therefore established a homogeneous T4 DNA ligase assay utilizing a specifically designed fluorescein- and dark quencher-labeled DNA molecule. Upon ligation of both DNA molecules, a quenching occurs and the fluorescence intensity decreases with increasing ligase concentrations. The assay allows a sensitive and precise quantification (CV, 4.6-5.5 %) of T4 DNA ligase activities and showed a high specificity when tested against other ligases of related and different species. Most importantly, this T4 DNA ligase assay requires only one working and incubation step before measurement can take place at room temperature and may therefore offer an interesting alternative to existing, more laborious ligase assays.

  7. Mobile non-destructive assay system

    Energy Technology Data Exchange (ETDEWEB)

    Colarusso, A.P.; Audas, J.H.; Bieri, J.M.; Herrera, G.C.; Hastings, R.D.; Horton, W.S.; Kuckertz, T.H.; Kunz, W.E.; Medvick, P.A.; Vogel, P.A.

    1987-07-01

    A mobile system for non-destructive assay (NDA), developed at the Los Alamos National Laboratory, provides accurate and sensitive measurements for transuranic (TRU) isotopes contained in 208-iota drums of miscellaneous nuclear wastes. The NDA unit consists of four major subsystems: an assay chamber, counting and digital electronics, data acquisition, and a neutron generator. It performs both active and passive neutron waste measurements. The former determines the amount of fissile isotopes at a sensitivity level of 1 mg plutonium. The latter determines spontaneous fission and ..cap alpha..,n) isotopes at a comparable level. A complete assay consists of sequential active and passive measurements. The assay measurement and other supporting data are incorporated in a commercial spreadsheet program (Lotus 1,2,3) for further analysis, which includes various matrix corrections and a determination of whether or not the drum exceeds the 100-nCi/g threshold for TRU wastes. Field tests have been performed on three separate occasions, accomplishing more than 1800 waste drum assays. These waste drum assays are discussed, especially those comparing passive and active neutron measurements with independent segmented gamma scan assays. Results obtained with a set of 15 drums containing plutonium prepared from standards and actual hot waste matrices are also reviewed.

  8. Ultrasensitive Detection of RNA and DNA Viruses Simultaneously Using Duplex UNDP-PCR Assay

    Science.gov (United States)

    Wang, Zengguo; Zhang, Xiujuan; Zhao, Xiaomin; Du, Qian; Chang, Lingling; Tong, Dewen

    2015-01-01

    Mixed infection of multiple viruses is common in modern intensive pig rearing. However, there are no methods available to detect DNA and RNA viruses in the same reaction system in preclinical level. In this study, we aimed to develop a duplex ultrasensitive nanoparticle DNA probe-based PCR assay (duplex UNDP-PCR) that was able to simultaneously detect DNA and RNA viruses in the same reaction system. PCV2 and TGEV are selected as representatives of the two different types of viruses. PCV2 DNA and TGEV RNA were simultaneously released from the serum sample by boiling with lysis buffer, then magnetic beads and gold nanoparticles coated with single and/or duplex specific probes for TGEV and PCV2 were added to form a sandwich-like complex with nucleic acids released from viruses. After magnetic separation, DNA barcodes specific for PCV2 and TGEV were eluted using DTT and characterized by specific PCR assay for specific DNA barcodes subsequently. The duplex UNDP-PCR showed similar sensitivity as that of single UNDP-PCR and was able to detect 20 copies each of PCV2 and TGEV in the serum, showing approximately 250-fold more sensitivity than conventional duplex PCR/RT-PCR assays. No cross-reaction was observed with other viruses. The positive detection rate of single MMPs- and duplex MMPs-based duplex UNDP-PCR was identical, with 29.6% for PCV2, 9.3% for TGEV and 3.7% for PCV2 and TGEV mixed infection. This duplex UNDP-PCR assay could detect TGEV (RNA virus) and PCV2 (DNA virus) from large-scale serum samples simultaneously without the need for DNA/RNA extraction, purification and reverse transcription of RNA, and showed a significantly increased positive detection rate for PCV2 (29%) and TGEV (11.7%) preclinical infection than conventional duplex PCR/RT-PCR. Therefore, the established duplex UNDP-PCR is a rapid and economical detection method, exhibiting high sensitivity, specificity and reproducibility. PMID:26544710

  9. Ultrasensitive Detection of RNA and DNA Viruses Simultaneously Using Duplex UNDP-PCR Assay.

    Directory of Open Access Journals (Sweden)

    Yong Huang

    Full Text Available Mixed infection of multiple viruses is common in modern intensive pig rearing. However, there are no methods available to detect DNA and RNA viruses in the same reaction system in preclinical level. In this study, we aimed to develop a duplex ultrasensitive nanoparticle DNA probe-based PCR assay (duplex UNDP-PCR that was able to simultaneously detect DNA and RNA viruses in the same reaction system. PCV2 and TGEV are selected as representatives of the two different types of viruses. PCV2 DNA and TGEV RNA were simultaneously released from the serum sample by boiling with lysis buffer, then magnetic beads and gold nanoparticles coated with single and/or duplex specific probes for TGEV and PCV2 were added to form a sandwich-like complex with nucleic acids released from viruses. After magnetic separation, DNA barcodes specific for PCV2 and TGEV were eluted using DTT and characterized by specific PCR assay for specific DNA barcodes subsequently. The duplex UNDP-PCR showed similar sensitivity as that of single UNDP-PCR and was able to detect 20 copies each of PCV2 and TGEV in the serum, showing approximately 250-fold more sensitivity than conventional duplex PCR/RT-PCR assays. No cross-reaction was observed with other viruses. The positive detection rate of single MMPs- and duplex MMPs-based duplex UNDP-PCR was identical, with 29.6% for PCV2, 9.3% for TGEV and 3.7% for PCV2 and TGEV mixed infection. This duplex UNDP-PCR assay could detect TGEV (RNA virus and PCV2 (DNA virus from large-scale serum samples simultaneously without the need for DNA/RNA extraction, purification and reverse transcription of RNA, and showed a significantly increased positive detection rate for PCV2 (29% and TGEV (11.7% preclinical infection than conventional duplex PCR/RT-PCR. Therefore, the established duplex UNDP-PCR is a rapid and economical detection method, exhibiting high sensitivity, specificity and reproducibility.

  10. Detection of human papillomavirus DNA by the hybrid capture assay

    Directory of Open Access Journals (Sweden)

    Carvalho Maria Odete O.

    2003-01-01

    Full Text Available Human Papillomavirus (HPV infection is the main cause of cervical cancers and cervical intraepithelial neoplasias (CIN worldwide. Consequently, it would be useful to evaluate HPV testing to screen for cervical cancer. Recently developed, the second-generation Hybrid Capture (HCA II test is a non-radioactive, relatively rapid, liquid hybridization assay designed to detect 18 HPV types, divided into high and low-risk groups. We evaluated 1055 women for HPV infection with the HCA II test. Five hundred and ten (48.3% of these women had HPV infection; 60 (11.8% had low cancer-risk HPV DNA; 269 (52.7% had high-risk HPV types and 181 (35.5% had both groups. Hence, 450 women (88.2% in this HPV-infected group had at least one high risk HPV type, and were therefore considered to be at high risk for cancer. Among the group with Papanicolaou (Pap test results, the overall prevalence of HPV DNA was 58.4%. Significant differences in HPV infection of the cervix were detected between Pap I (normal smears and Pap IV (carcinomas (p<0.0001. Values of HPV viral load obtained for Pap I and SILs were significantly different, with an upward trend (p<0.0001, suggesting a positive correlation between high viral load values and risk of SIL. Because of the high costs of the HCA II test, its use for routine cervical mass screening cannot be recommended in poor countries. Nevertheless, it is a useful tool when combined with cytology, diagnosing high-risk infections in apparently normal tissues. Use of this technique could help reduce the risk of cancer.

  11. Lens-free imaging of magnetic particles in DNA assays.

    Science.gov (United States)

    Colle, Frederik; Vercruysse, Dries; Peeters, Sara; Liu, Chengxun; Stakenborg, Tim; Lagae, Liesbet; Del-Favero, Jurgen

    2013-11-01

    We present a novel opto-magnetic system for the fast and sensitive detection of nucleic acids. The system is based on a lens-free imaging approach resulting in a compact and cheap optical readout of surface hybridized DNA fragments. In our system magnetic particles are attracted towards the detection surface thereby completing the labeling step in less than 1 min. An optimized surface functionalization combined with magnetic manipulation was used to remove all nonspecifically bound magnetic particles from the detection surface. A lens-free image of the specifically bound magnetic particles on the detection surface was recorded by a CMOS imager. This recorded interference pattern was reconstructed in software, to represent the particle image at the focal distance, using little computational power. As a result we were able to detect DNA concentrations down to 10 pM with single particle sensitivity. The possibility of integrated sample preparation by manipulation of magnetic particles, combined with the cheap and highly compact lens-free detection makes our system an ideal candidate for point-of-care diagnostic applications.

  12. Validation of a high-throughput in vitro alkaline elution/rat hepatocyte assay for DNA damage.

    Science.gov (United States)

    Gealy, Robert; Wright-Bourque, Jennifer L; Kraynak, Andrew R; McKelvey, Troy W; Barnum, John E; Storer, Richard D

    2007-04-20

    In vitro alkaline elution is a sensitive and specific short term assay which measures DNA strand breakage in a mammalian test system (primary rat hepatocytes). This lab has previously demonstrated the performance of the assay with known genotoxic and non-genotoxic compounds. The methodology employed has relatively low sample throughput and is labor-intensive, requiring a great deal of manual processing of samples in a format that is not amenable to automation. Here, we present an automated version of the assay. This high-throughput alkaline elution assay (HT-AE) was made possible through 3 key developments: (1) DNA quantitation using PicoGreen and OliGreen fluorescent DNA binding dyes; (2) design and implementation of a custom automation system; and (3) reducing the assay to a 96-well plate format. The assay can now be run with 5-50mg of test compound. HT-AE was validated in a similar manner as the original assay, including assessment of non-genotoxic and non-carcinogenic compounds and evaluation of cytotoxicity to avoid confounding effects of toxicity-associated DNA degradation. The validation test results from compounds of known genotoxic potential were used to set appropriate criteria to classify alkaline elution results for genotoxicity.

  13. Assessment of DNA interstrand crosslinks using the modified alkaline comet assay.

    Science.gov (United States)

    Wu, Jian Hong; Jones, Nigel J

    2012-01-01

    The single cell gel electrophoresis (SCGE) assay, more commonly known as the comet assay, due to the "comet-like" appearance of the cells, was originally developed as a technique to measure the presence of DNA single-strand breaks. The assay is performed on single cells embedded in agar and placed in an electrical field at alkaline pH, so that fragments of negatively charged single-stranded DNA move through the gel toward the positively charged anode. Undamaged DNA moves relatively slowly, forming the head of the comet, while DNA fragmented due to the presence of single-strand breaks, moves more quickly giving the appearance of the tail. The extent of DNA migration is a measure of the DNA damage present. Since it was first developed, the comet assay has been adapted for measuring other types of DNA damage. The neutral comet assay has been employed for DNA double-strand breaks, while techniques using DNA repair enzymes to cleave specific adducts, UvrABC for ultraviolet radiation induced adducts, for example, have also been described. Here, we describe a modified version of the comet assay for the measurement of interstrand crosslinks (ICLs). Interstrand crosslinking agents include the chemotherapeutic agents mitomycin C and cis-platin, psoralen plus UVA light (PUVA) used to treat hyperproliferative skin disorders and diepoxybutane, a metabolite of 1,3-butadiene used in industrial processes and an environmental pollutant. ICLs are a potent and cytotoxic form of DNA damage as they prevent DNA strand separation, thereby preventing DNA replication. Their removal requires several different DNA repair processes including translesion synthesis and homologous recombination. As ICLs prevent separation of the DNA strands, their presence results in less DNA migration in the comet assay. To successfully measure ICLs, it is necessary to incorporate a step that induces single-strand breaks (using a defined dose of ionizing radiation) that allows the crosslinked DNA to migrate.

  14. Endonuclease modified comet assay for oxidative DNA damage induced by detection of genetic toxiants

    Institute of Scientific and Technical Information of China (English)

    赵健

    2014-01-01

    Objective The aim of this study was to investigate the use of the lesion-specific endonucleases-modifiedcomet assay for analysis of DNA,oxidation in cell lines.Methods DNA breaks and oxidative damage were evaluated by normal alkaline and formamidopyrimidine-DNAglycosylase(FPG)modified comet assays.Cytotoxicity was assessed by MTT method.The human bronchial epi-

  15. Inter-laboratory variation in DNA damage using a standard comet assay protocol

    DEFF Research Database (Denmark)

    Forchhammer, Lykke; Ersson, Clara; Loft, Steffen

    2012-01-01

    There are substantial inter-laboratory variations in the levels of DNA damage measured by the comet assay. The aim of this study was to investigate whether adherence to a standard comet assay protocol would reduce inter-laboratory variation in reported values of DNA damage. Fourteen laboratories ...

  16. Estimates of DNA strand breakage in bottlenose dolphin (Tursiops truncatus leukocytes measured with the Comet and DNA diffusion assays

    Directory of Open Access Journals (Sweden)

    Adriana Díaz

    2009-01-01

    Full Text Available The analysis of DNA damage by mean of Comet or single cell gel electrophoresis (SCGE assay has been commonly used to assess genotoxic impact in aquatic animals being able to detect exposure to low concentrations of contaminants in a wide range of species. The aims of this work were 1 to evaluate the usefulness of the Comet to detect DNA strand breakage in dolphin leukocytes, 2 to use the DNA diffusion assay to determine the amount of DNA strand breakage associated with apoptosis or necrosis, and 3 to determine the proportion of DNA strand breakage that was unrelated to apoptosis and necrosis. Significant intra-individual variation was observed in all of the estimates of DNA damage. DNA strand breakage was overestimated because a considerable amount (~29% of the DNA damage was derived from apoptosis and necrosis. The remaining DNA damage in dolphin leukocytes was caused by factors unrelated to apoptosis and necrosis. These results indicate that the DNA diffusion assay is a complementary tool that can be used together with the Comet assay to assess DNA damage in bottlenose dolphins.

  17. Comet assay as an indirect measure of systemic oxidative stress.

    Science.gov (United States)

    Fang, Lei; Neutzner, Albert; Turtschi, Stephanie; Flammer, Josef; Mozaffarieh, Maneli

    2015-05-22

    Higher eukaryotic organisms cannot live without oxygen; yet, paradoxically, oxygen can be harmful to them. The oxygen molecule is chemically relatively inert because it has two unpaired electrons located in different pi * anti-bonding orbitals. These two electrons have parallel spins, meaning they rotate in the same direction about their own axes. This is why the oxygen molecule is not very reactive. Activation of oxygen may occur by two different mechanisms; either through reduction via one electron at a time (monovalent reduction), or through the absorption of sufficient energy to reverse the spin of one of the unpaired electrons. This results in the production of reactive oxidative species (ROS). There are a number of ways in which the human body eliminates ROS in its physiological state. If ROS production exceeds the repair capacity, oxidative stress results and damages different molecules. There are many different methods by which oxidative stress can be measured. This manuscript focuses on one of the methods named cell gel electrophoresis, also known as "comet assay" which allows measurement of DNA breaks. If all factors known to cause DNA damage, other than oxidative stress are kept constant, the amount of DNA damage measured by comet assay is a good parameter of oxidative stress. The principle is simple and relies upon the fact that DNA molecules are negatively charged. An intact DNA molecule has such a large size that it does not migrate during electrophoresis. DNA breaks, however, if present result in smaller fragments which move in the electrical field towards the anode. Smaller fragments migrate faster. As the fragments have different sizes the final result of the electrophoresis is not a distinct line but rather a continuum with the shape of a comet. The system allows a quantification of the resulting "comet" and thus of the DNA breaks in the cell.

  18. An ECVAG trial on assessment of oxidative damage to DNA measured by the comet assay

    DEFF Research Database (Denmark)

    Johansson, Clara; Møller, Peter; Forchhammer, Lykke;

    2010-01-01

    The increasing use of single cell gel electrophoresis (the comet assay) highlights its popularity as a method for detecting DNA damage, including the use of enzymes for assessment of oxidatively damaged DNA. However, comparison of DNA damage levels between laboratories can be difficult due...... assay end points to number of lesions/10(6) bp by calibration with ionizing radiation. The aim of this study was to investigate the inter-laboratory variation in assessment of oxidatively damaged DNA by the comet assay in terms of oxidized purines converted to strand breaks with formamidopyrimidine DNA...... to differences in assay protocols (e.g. lysis conditions, enzyme treatment, the duration of the alkaline treatment and electrophoresis) and in the end points used for reporting results (e.g. %DNA in tail, arbitrary units, tail moment and tail length). One way to facilitate comparisons is to convert primary comet...

  19. A multiplex calibrated real-time PCR assay for quantitation of DNA of EBV-1 and 2.

    Science.gov (United States)

    Gatto, Francesca; Cassina, Giulia; Broccolo, Francesco; Morreale, Giuseppe; Lanino, Edoardo; Di Marco, Eddi; Vardas, Efthiya; Bernasconi, Daniela; Buttò, Stefano; Principi, Nicola; Esposito, Susanna; Scarlatti, Gabriella; Lusso, Paolo; Malnati, Mauro S

    2011-12-01

    Accurate and highly sensitive tests for the diagnosis of active Epstein-Barr virus (EBV) infection are essential for the clinical management of individuals infected with EBV. A calibrated quantitative real-time PCR assay for the measurement of EBV DNA of both EBV-1 and 2 subtypes was developed, combining the detection of the EBV DNA and a synthetic DNA calibrator in a multiplex PCR format. The assay displays a wide dynamic range and a high degree of accuracy even in the presence of 1μg of human genomic DNA. This assay measures with the same efficiency EBV DNA from strains prevalent in different geographic areas. The clinical sensitivity and specificity of the system were evaluated by testing 181 peripheral blood mononuclear cell (PBMCs) and plasma specimens obtained from 21 patients subjected to bone marrow transplantation, 70 HIV-seropositive subjects and 23 healthy controls. Patients affected by EBV-associated post-transplant lymphoprolipherative disorders had the highest frequency of EBV detection and the highest viral load. Persons infected with HIV had higher levels of EBV DNA load in PBMCs and a higher frequency of EBV plasma viremia compared to healthy controls. In conclusion, this new assay provides a reliable high-throughput method for the quantitation of EBV DNA in clinical samples.

  20. A robust assay to measure DNA topology-dependent protein binding affinity.

    Science.gov (United States)

    Litwin, Tamara R; Solà, Maria; Holt, Ian J; Neuman, Keir C

    2015-04-20

    DNA structure and topology pervasively influence aspects of DNA metabolism including replication, transcription and segregation. However, the effects of DNA topology on DNA-protein interactions have not been systematically explored due to limitations of standard affinity assays. We developed a method to measure protein binding affinity dependence on the topology (topological linking number) of supercoiled DNA. A defined range of DNA topoisomers at equilibrium with a DNA binding protein is separated into free and protein-bound DNA populations using standard nitrocellulose filter binding techniques. Electrophoretic separation and quantification of bound and free topoisomers combined with a simple normalization procedure provide the relative affinity of the protein for the DNA as a function of linking number. Employing this assay we measured topology-dependent DNA binding of a helicase, a type IB topoisomerase, a type IIA topoisomerase, a non-specific mitochondrial DNA binding protein and a type II restriction endonuclease. Most of the proteins preferentially bind negatively supercoiled DNA but the details of the topology-dependent affinity differ among proteins in ways that expose differences in their interactions with DNA. The topology-dependent binding assay provides a robust and easily implemented method to probe topological influences on DNA-protein interactions for a wide range of DNA binding proteins.

  1. Test procedure for boxed waste assay system

    Energy Technology Data Exchange (ETDEWEB)

    Wachter, J. [Los Alamos National Lab., NM (United States)

    1994-12-07

    This document, prepared by Los Alamos National Laboratory`s NMT-4 group, details the test methodology and requirements for Acceptance/Qualification testing of a Boxed Waste Assay System (BWAS) designed and constructed by Pajarito Scientific Corporation. Testing of the BWAS at the Plutonium Facility (TA55) at Los Alamos National Laboratory will be performed to ascertain system adherence to procurement specification requirements. The test program shall include demonstration of conveyor handling capabilities, gamma ray energy analysis, and imaging passive/active neutron accuracy and sensitivity. Integral to these functions is the system`s embedded operating and data reduction software.

  2. DNA-repair measurements by use of the modified comet assay

    DEFF Research Database (Denmark)

    Godschalk, Roger W L; Ersson, Clara; Riso, Patrizia;

    2013-01-01

    The measurement of DNA-repair activity by extracts from cells or tissues by means of the single-cell gel electrophoresis (comet) assay has a high potential to become widely used in biomonitoring studies. We assessed the inter-laboratory variation in reported values of DNA-repair activity...... line as having the highest level of DNA-repair activity. The two laboratories that reported discordant results (with another cell line having the highest level of DNA-repair activity) were those that reported to have little experience with the modified comet assay to assess DNA repair. The laboratories...

  3. DETECTION OF LOW DOSE RADIATION INDUCED DNA DAMAGE USING TEMPERATURE DIFFERENNTIAL FLUORESENCE ASSAY

    Science.gov (United States)

    A rapid and sensitive fluorescence assay for radiation-induced DNA damage is reported. Changes in temperature-induced strand separation in both calf thymus DNA and plasmid DNA (puc 19 plasmid from Escherichia coli) were measured after exposure to low doses of radiation. Exposures...

  4. DETECTION OF LOW DOSE RADIATION INDUCED DNA DAMAGE USING TEMPERATURE DIFFERENTIAL FLUORESCENCE ASSAY

    Science.gov (United States)

    A rapid and sensitive fluorescence assay for radiation-induced DNA damage is reported. Changes in temperature-induced strand separation in both calf thymus DNA and plasmid DNA (puc 19 plasmid from Escherichia coli) were measured after exposure to low doses of radiation. Exposur...

  5. Hairpin DNA probe based surface plasmon resonance biosensor used for the activity assay of E. coli DNA ligase.

    Science.gov (United States)

    Luan, Qingfen; Xue, Ying; Yao, Xin; Lu, Wu

    2010-02-01

    Using hairpin DNA probe self-structure change during DNA ligation process, a sensitive, label-free and simple method of E. coli DNA ligase assay via a home-built high-resolution surface plasmon resonance (SPR) instrument was developed. The DNA ligation process was monitored in real-time and the effects of single-base mutation on the DNA ligation process were investigated. Then an assay of E. coli DNA ligase was completed with a lower detection limit (0.6 nM), wider concentration range and better reproducibility. Moreover, the influence of Quinacrine on the activity of E. coli DNA ligase was also studied, which demonstrated that our method was useful for drug screening.

  6. Persistence of DNA of Gaeumannomyces graminis var. tritici in soil as measured by a DNA-based assay.

    Science.gov (United States)

    Herdina; Neate, Stephen; Jabaji-Hare, Suha; Ophel-Keller, Kathy

    2004-02-01

    There are an increasing number of assays available for fungal plant pathogens based on DNA technology. We have developed such an assay for Gaeumannomyces graminis var. tritici (Ggt) in soil, using slot-blot hybridisation. To ensure the validity of DNA-based soil assays for the fungus, it is important to determine the stability of Ggt DNA in soil. This study was undertaken to quantify the DNA degradation of dead Ggt in soil using a DNA-based assay. Mycelia were killed using various treatments, then DNA was extracted and estimated by a slot-blot hybridisation technique using the specific Ggt DNA probe, pG158. Mycelia were also killed using a fungicide (triadimefon) at a concentration of 150-250 microg ml(-1). The amount of detectable DNA of Ggt, killed using triadimefon, declined by 82-93%. Inoculum in the form of diseased wheat roots, artificially inoculated ryegrass seed, particulate soil organic matter and whole soil was killed using heat-treatment. The amount of detectable DNA of Ggt declined markedly (90%) in both heat-treated roots and inoculated ryegrass seeds, and declined by 50% in both treated soil and soil organic matter. The rate of DNA degradation of Ggt in soil varied with the type of inoculum. The amount of detectable DNA of Ggt in dead mycelia declined by 99.8% after 4 days of incubation in soil. No DNA was detected after 8 days of incubation. In contrast, Ggt DNA in live mycelia declined by 70% after 8 days of incubation and declined to 10% of original DNA level after 32 days. In ground ryegrass seed inoculum, DNA in both killed and live Ggt declined by 50% after 8 days. In diseased roots, DNA from both live and killed Ggt did not appear to decline over 16 days. Estimates of the amount of Ggt in the soil using a DNA-based assay reflect both live and dead populations of the fungus. The rate of breakdown of DNA of the dead fungus is very high and the presence of dead fungi in roots probably a rare event so the DNA from dead fungus probably contributes

  7. Comet assay: a reliable tool for the assessment of DNA damage in different models.

    Science.gov (United States)

    Dhawan, Alok; Bajpayee, Mahima; Parmar, Devendra

    2009-02-01

    New chemicals are being added each year to the existing burden of toxic substances in the environment. This has led to increased pollution of ecosystems as well as deterioration of the air, water, and soil quality. Excessive agricultural and industrial activities adversely affect biodiversity, threatening the survival of species in a particular habitat as well as posing disease risks to humans. Some of the chemicals, e.g., pesticides and heavy metals, may be genotoxic to the sentinel species and/or to non-target species, causing deleterious effects in somatic or germ cells. Test systems which help in hazard prediction and risk assessment are important to assess the genotoxic potential of chemicals before their release into the environment or commercial use as well as DNA damage in flora and fauna affected by contaminated/polluted habitats. The Comet assay has been widely accepted as a simple, sensitive, and rapid tool for assessing DNA damage and repair in individual eukaryotic as well as some prokaryotic cells, and has increasingly found application in diverse fields ranging from genetic toxicology to human epidemiology. This review is an attempt to comprehensively encase the use of Comet assay in different models from bacteria to man, employing diverse cell types to assess the DNA-damaging potential of chemicals and/or environmental conditions. Sentinel species are the first to be affected by adverse changes in their environment. Determination of DNA damage using the Comet assay in these indicator organisms would thus provide information about the genotoxic potential of their habitat at an early stage. This would allow for intervention strategies to be implemented for prevention or reduction of deleterious health effects in the sentinel species as well as in humans.

  8. The alkaline comet assay: towards validation in biomonitoring of DNA damaging exposures.

    Science.gov (United States)

    Møller, Peter

    2006-04-01

    Generation of DNA damage is considered to be an important initial event in carcinogenesis. The single cell gel electrophoresis (comet) assay is a technically simple and fast method that detects genotoxicity in virtually any mammalian cell type without requirement for cell culture. This review discusses the strength of the comet assay in biomonitoring at its present state of validation. The simple version of the alkaline comet assay detects DNA migration caused by strand breaks, alkaline labile sites, and transient repair sites. By incubation with bacterial glycosylase/endonuclease enzymes, broad classes of oxidative DNA damage, alkylations, and ultraviolet light-induced photoproducts are detected as additional DNA migration. The most widely measured enzyme sensitive sites have been those detected by formamidopyrimidine DNA glycosylase (FPG) and endonuclease III (ENDOIII). Reports from biomonitoring studies show that the basal level of DNA damage in leukocytes is influenced be a variety of lifestyle and environmental exposures, including exercise, air pollution, sunlight, and diet. Although not all types of carcinogenic exposures should be expected to damage DNA in leukocytes, the comet assay is a valuable method for detection of genotoxic exposure in humans. However, the predictive value of the comet assay is unknown because it has not been investigated in prospective cohort studies. Also, it is important that the performance of the assay is investigated in multi-laboratory validation trials. As a tool in risk assessment the comet assay can be used in characterization of hazards.

  9. Nondestructive boxed transuranic (TRU) waste assay systems

    Science.gov (United States)

    Caldwell, John T.; Jones, Stephanie A.; Lucero, Randy F.

    1999-01-01

    A brief history of boxed waste assay systems (primarily those developed at Los Alamos National Laboratory) is presented. The characteristics and design process involved with current generation systems--as practiced by BII--are also discussed in some detail. Finally, a specific boxed waste assay system and acceptance test results are presented. This system was developed by BII and installed at the Waste Receiving and Packaging (WRAP) facility in Hanford, Washington in early 1997. The WRAP system combines imaging passive/active neutron (IPAN) techniques with gamma- ray energy analysis (GEA) to assay crates up to 2.5 m X 2.5 m X 6.5 m in size. (Systems that incorporate both these methodologies are usually denoted IPAN/GEA types.) Two separate gamma-ray measurements are accomplished utilizing 16 arrayed NaI detectors and a moveable HPGe detector, while 3He detectors acquire both active and passive neutron data. These neutron measurements use BII's proprietary imaging methodology. Acceptance testing of the system was conducted at Hanford in January 1998. The system's operating performance was evaluated based on accuracy and sensitivity requirements for three different matrix types. Test results indicate an average 13% active mode accuracy for 10 nCi/g loadings of Pu waste and 5% passive mode accuracy for 10 g loadings of Pu waste. Sensitivity testing demonstrated an active mode lower limit of detection of less than 5 nCi/g of 239Pu for the medium matrix and less than 20 pCi/g of fission and activation products at 3(sigma) above background.

  10. Lymphocyte DNA damage in Turkish asphalt workers detected by the comet assay.

    Science.gov (United States)

    Bacaksiz, Aysegul; Kayaalti, Zeliha; Soylemez, Esma; Tutkun, Engin; Soylemezoglu, Tulin

    2014-01-01

    Asphalt has a highly complex structure and it contains several organic compounds including polycyclic aromatic hydrocarbons and heterocyclic compounds. In this study, comet assay was used to detect the DNA damage in blood lymphocytes of 30 workers exposed to asphalt fumes and 30 nonexposed controls. This is the first report on Turkish asphalt workers' investigated DNA damage using the alkaline single cell gel electrophoresis (SCGE). The DNA damage was evaluated by the percentage of DNA in the comet tail (% tail DNA) for each cell. According to our results, workers exposed to asphalt fumes had higher DNA damage than the control group (p asphalt fumes caused a significant increase in DNA damage and the comet assay is a suitable method for determining DNA damage in asphalt workers.

  11. DNA strand breaks (comet assay) in blood lymphocytes from wild bottlenose dolphins.

    Science.gov (United States)

    Lee, Richard F; Bulski, Karrie; Adams, Jeffrey D; Peden-Adams, Margie; Bossart, Gregory D; King, Lydia; Fair, Patricia A

    2013-12-15

    The comet assay was carried out on blood lymphocytes from a large number of wild dolphins (71 from Indian River Lagoon, FL, USA; 51 from Charleston Harbor, SC, USA) and provides a baseline study of DNA strand breaks in wild dolphin populations. There were no significant differences in the comet assay (% DNA in tail) results between the different age and sex categories. Significant difference in DNA strand breaks were found between Charleston Harbor dolphins (median--17.4% DNA in tail) and Indian River Lagoon dolphins (median--14.0% DNA in tail). A strong correlation found between T-cell proliferation and DNA strand breaks in dolphin lymphocytes suggests that dolphins with a high numbers of DNA strand breaks have a decreased ability to respond to infection. Higher concentrations of genotoxic agents in Charleston Harbor compared with Indian River lagoon may have been one of the causes of higher DNA strand breaks in these dolphins.

  12. Development of a Radioactive Waste Assay System

    Energy Technology Data Exchange (ETDEWEB)

    Kang, Duck Won; Song, Myung Jae; Shin, Sang Woon; Sung, Kee Bang; Ko, Dae Hach [Korea Electric Power Research Institute, Taejon (Korea, Republic of); Kim, Kil Jeong; Park, Jong Mook; Jee, Kwang Yoong [Korea Atomic Energy Research Institute, Taejon (Korea, Republic of)

    1996-12-31

    Nuclear Act of Korea requires the manifest of low and intermediate level radioactive waste generated at nuclear power plants prior to disposal sites.Individual history records of the radioactive waste should be contained the information about the activity of nuclides in the drum, total activity, weight, the type of waste. A fully automated nuclide analysis assay system, non-destructive analysis and evaluation system of the radioactive waste, was developed through this research project. For the nuclides that could not be analysis directly by MCA, the activities of the representative {gamma}-emitters(Cs-137, Co-60) contained in the drum were measured by using that system. Then scaling factors were used to calculate the activities of {alpha}, {beta}-emitters. Furthermore, this system can automatically mark the analysis results onto the drum surface. An automated drum handling system developed through this research project can reduce the radiation exposure to workers. (author). 41 refs., figs.

  13. Measurement of DNA damage in individual cells using the Single Cell Gel Electrophoresis (Comet) assay.

    Science.gov (United States)

    Hartley, Janet M; Spanswick, Victoria J; Hartley, John A

    2011-01-01

    The Single Cell Gel Electrophoresis (Comet) assay is a simple, versatile and sensitive method for measuring DNA damage in individual cells, allowing the determination of heterogeneity of response within a cell population. The basic alkaline technique described is for the determination of DNA strand break damage and its repair at a single cell level. Specific modifications to the method use a lower pH ('neutral' assay), or allow the measurement of DNA interstrand cross-links. It can be further adapted to, for example, study specific DNA repair mechanisms, be combined with fluorescent in situ hybridisation, or incorporate lesion specific enzymes.

  14. A Structural Bisulfite Assay to Identify DNA Cruciforms.

    Science.gov (United States)

    Gentry, Matthew; Hennig, Lars

    2016-09-06

    In the half century since the discovery of the double-helix structure of DNA, it has become increasingly clear that DNA functionality is based on much more than its sequence in a double-helical structure. Further advances have highlighted the importance of additional aspects of DNA structure: its packaging in the higher order chromatin structure, positioning of nucleosomes along the DNA, and the occurrence of non-helical DNA structures. Of these, the latter has been problematic to prove empirically. Here, we describe a method that uses non-denaturing bisulfite sequencing on isolated Arabidopsis thaliana nuclei to determine the location of cytosines positioned outside the double helix as a result of non-B-form DNA structures. We couple this with computational methods and S1 nuclease digest to reliably identify stable, non-B-form, cruciform structures. This enables us to identify a palindrome in the promoter of FLOWERING LOCUS T that forms a stable non-B-form structure. The stronger conservation of the ability to form a non-helical secondary structure than of the sequence suggests that this structure is biologically relevant.

  15. A novel SERRS sandwich-hybridization assay to detect specific DNA target.

    Directory of Open Access Journals (Sweden)

    Cécile Feuillie

    Full Text Available In this study, we have applied Surface Enhanced Resonance Raman Scattering (SERRS technology to the specific detection of DNA. We present an innovative SERRS sandwich-hybridization assay that allows specific DNA detection without any enzymatic amplification, such as is the case with Polymerase Chain Reaction (PCR. In some substrates, such as ancient or processed remains, enzymatic amplification fails due to DNA alteration (degradation, chemical modification or to the presence of inhibitors. Consequently, the development of a non-enzymatic method, allowing specific DNA detection, could avoid long, expensive and inconclusive amplification trials. Here, we report the proof of concept of a SERRS sandwich-hybridization assay that leads to the detection of a specific chamois DNA. This SERRS assay reveals its potential as a non-enzymatic alternative technology to DNA amplification methods (particularly the PCR method with several applications for species detection. As the amount and type of damage highly depend on the preservation conditions, the present SERRS assay would enlarge the range of samples suitable for DNA analysis and ultimately would provide exciting new opportunities for the investigation of ancient DNA in the fields of evolutionary biology and molecular ecology, and of altered DNA in food frauds detection and forensics.

  16. A quantitative and high-throughput assay of human papillomavirus DNA replication.

    Science.gov (United States)

    Gagnon, David; Fradet-Turcotte, Amélie; Archambault, Jacques

    2015-01-01

    Replication of the human papillomavirus (HPV) double-stranded DNA genome is accomplished by the two viral proteins E1 and E2 in concert with host DNA replication factors. HPV DNA replication is an established model of eukaryotic DNA replication and a potential target for antiviral therapy. Assays to measure the transient replication of HPV DNA in transfected cells have been developed, which rely on a plasmid carrying the viral origin of DNA replication (ori) together with expression vectors for E1 and E2. Replication of the ori-plasmid is typically measured by Southern blotting or PCR analysis of newly replicated DNA (i.e., DpnI digested DNA) several days post-transfection. Although extremely valuable, these assays have been difficult to perform in a high-throughput and quantitative manner. Here, we describe a modified version of the transient DNA replication assay that circumvents these limitations by incorporating a firefly luciferase expression cassette in cis of the ori. Replication of this ori-plasmid by E1 and E2 results in increased levels of firefly luciferase activity that can be accurately quantified and normalized to those of Renilla luciferase expressed from a control plasmid, thus obviating the need for DNA extraction, digestion, and analysis. We provide a detailed protocol for performing the HPV type 31 DNA replication assay in a 96-well plate format suitable for small-molecule screening and EC50 determinations. The quantitative and high-throughput nature of the assay should greatly facilitate the study of HPV DNA replication and the identification of inhibitors thereof.

  17. Multiplex DNA assay based on nanoparticle probes by single particle inductively coupled plasma mass spectrometry.

    Science.gov (United States)

    Zhang, Shixi; Han, Guojun; Xing, Zhi; Zhang, Sichun; Zhang, Xinrong

    2014-04-01

    A multiplex DNA assay based on nanoparticle (NP) tags detection utilizing single particle mode inductively coupled plasma mass spectrometry (SP-ICP-MS) as ultrasensitive readout has been demonstrated in the article. Three DNA targets associated with clinical diseases (HIV, HAV, and HBV) down to 1 pM were detected by DNA probes labeled with AuNPs, AgNPs, and PtNPs via DNA sandwich assay. Single nucleotide polymorphisms in genes can also be effectively discriminated. Since our method is unaffected by the sample matrix, it is well-suited for diagnostic applications. Moreover, with the high sensitivity of SP-ICP-MS and the variety of NPs detectable by SP-ICP-MS, high-throughput DNA assay could be achieved without signal amplification or chain reaction amplification.

  18. Performance of MycAssay Aspergillus DNA real-time PCR assay compared with the galactomannan detection assay for the diagnosis of invasive aspergillosis from serum samples.

    Science.gov (United States)

    Danylo, Alexis; Courtemanche, Chantal; Pelletier, René; Boudreault, Alexandre A

    2014-08-01

    Invasive aspergillosis (IA) is a major problem in the immunocompromised population, and its diagnosis is difficult due to the low sensitivity of available tests. Detection of Aspergillus nucleic acid by polymerase chain reaction (PCR) in serum samples is a promising diagnostic tool; however, use of multiple "in-house" methods precludes standardization. The first commercial PCR assay, MycAssay Aspergillus (Myconostica, Ltd), became available recently, and its performance in the diagnosis of IA was evaluated and compared with the galactomannan (GM) assay. Serum samples obtained from patients with hematological cancer were tested retrospectively with MycAssay Aspergillus PCR. Per-episode and per-test analyses were undertaken with 146 sera from 35 hematological patients. Sixteen patients had proven or probable IA and 19 had possible or no IA. In per-episode analysis, MycAssay Aspergillus had a sensitivity of 43.8% (95% confidence interval [CI], 19.8%-70.1%) and a specificity of 63.2% (95% CI, 38.4%-83.7%) for IA diagnosis. In per-test analyses, MycAssay Aspergillus had a lower specificity than the GM assay (83.3% vs. 93.1%, P = 0.04). The addition of PCR to routine clinical practice would have permitted the diagnosis of one additional probable IA in our cohort. Use of PCR instead of GM assay would have delayed the diagnosis in two cases. Aspergillus DNA detection by PCR with serum specimens using MycAssay showed a lower specificity than the GM assay and was associated with a low sensitivity for IA diagnosis. More studies are needed to determine the exact role of MycAssay in IA diagnosis in patients with hematological malignancy.

  19. Mass Spectrometry Based Ultrasensitive DNA Methylation Profiling Using Target Fragmentation Assay.

    Science.gov (United States)

    Lin, Xiang-Cheng; Zhang, Ting; Liu, Lan; Tang, Hao; Yu, Ru-Qin; Jiang, Jian-Hui

    2016-01-19

    Efficient tools for profiling DNA methylation in specific genes are essential for epigenetics and clinical diagnostics. Current DNA methylation profiling techniques have been limited by inconvenient implementation, requirements of specific reagents, and inferior accuracy in quantifying methylation degree. We develop a novel mass spectrometry method, target fragmentation assay (TFA), which enable to profile methylation in specific sequences. This method combines selective capture of DNA target from restricted cleavage of genomic DNA using magnetic separation with MS detection of the nonenzymatic hydrolysates of target DNA. This method is shown to be highly sensitive with a detection limit as low as 0.056 amol, allowing direct profiling of methylation using genome DNA without preamplification. Moreover, this method offers a unique advantage in accurately determining DNA methylation level. The clinical applicability was demonstrated by DNA methylation analysis using prostate tissue samples, implying the potential of this method as a useful tool for DNA methylation profiling in early detection of related diseases.

  20. Antimutagenic activity of casein against MNNG in the E. coli DNA repair host-mediated assay.

    NARCIS (Netherlands)

    Boekel, van M.A.J.S.; Goeptar, A.R.; Alink, G.M.

    1997-01-01

    The effect of caseinate and soy protein in the diet on the mutagenicity induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) was assessed in-vivo and ex-vivo in the DNA-repair host-mediated assay and liquid suspension assay, respectively. Of the two proteins only casein showed a strong antimutagen

  1. Electrophoretic mobility shift assays for the analysis of DNA-protein interactions.

    Science.gov (United States)

    Gaudreault, Manon; Gingras, Marie-Eve; Lessard, Maryse; Leclerc, Steeve; Guérin, Sylvain L

    2009-01-01

    Electromobility shift assay is a simple, efficient, and rapid method for the study of specific DNA-protein interactions. It relies on the reduction in the electrophoretic mobility conferred to a DNA fragment by an interacting protein. The technique is suitable to qualitative, quantitative, and kinetic analyses. It can also be used to analyze conformational changes.

  2. Improved benzodiazepine radioreceptor assay using the MultiScreen (R) Assay System

    NARCIS (Netherlands)

    Janssen, MJ; Ensing, K; de Zeeuw, RA

    1999-01-01

    In this article, an improved benzodiazepine radioreceptor assay is described, which allows substantial reduction in assay time, The filtration in this method was performed by using the MultiScreen(R) Assay System. The latter consists of a 96-well plate with glass fibre filters sealed at the bottom,

  3. Increased knowledge of Francisella genus diversity highlights the benefits of optimised DNA-based assays.

    Science.gov (United States)

    Ahlinder, Jon; Öhrman, Caroline; Svensson, Kerstin; Lindgren, Petter; Johansson, Anders; Forsman, Mats; Larsson, Pär; Sjödin, Andreas

    2012-09-25

    Recent advances in sequencing technologies offer promising tools for generating large numbers of genomes, larger typing databases and improved mapping of environmental bacterial diversity. However, DNA-based methods for the detection of Francisella were developed with limited knowledge about genetic diversity. This, together with the high sequence identity between several Francisella species, means there is a high risk of false identification and detection of the highly virulent pathogen Francisella tularensis. Moreover, phylogenetic reconstructions using single or limited numbers of marker sequences often result in incorrect tree topologies and inferred evolutionary distances. The recent growth in publicly accessible whole-genome sequences now allows evaluation of published genetic markers to determine optimal combinations of markers that minimise both time and laboratory costs. In the present study, we evaluated 38 previously published DNA markers and the corresponding PCR primers against 42 genomes representing the currently known diversity of the genus Francisella. The results highlight that PCR assays for Francisella tularensis are often complicated by low specificity, resulting in a high probability of false positives. A method to select a set of one to seven markers for obtaining optimal phylogenetic resolution or diagnostic accuracy is presented. Current multiple-locus sequence-typing systems and detection assays of Francisella, could be improved by redesigning some of the primers and reselecting typing markers. The use of only a few optimally selected sequence-typing markers allows construction of phylogenetic topologies with almost the same accuracy as topologies based on whole-genome sequences.

  4. Assessment of gamma ray-induced DNA damage in Lasioderma serricorne using the comet assay

    Science.gov (United States)

    Kameya, Hiromi; Miyanoshita, Akihiro; Imamura, Taro; Todoriki, Setsuko

    2012-03-01

    We attempted a DNA comet assay under alkaline conditions to verify the irradiation treatment of pests. Lasioderma serricorne (Fabricius) were chosen as test insects and irradiated with gamma rays from a 60Co source at 1 kGy. We conducted the comet assay immediately after irradiation and over time for 7 day. Severe DNA fragmentation in L. serricorne cells was observed just after irradiation and the damage was repaired during the post-irradiation period in a time-dependent manner. The parameters of the comet image analysis were calculated, and the degree of DNA damage and repair were evaluated. Values for the Ratio (a percentage determined by fluorescence in the damaged area to overall luminance, including intact DNA and the damaged area of a comet image) of individual cells showed that no cells in the irradiated group were included in the Ratiocomet assay under alkaline conditions, combined with comet image analysis, can be used to identify irradiation history.

  5. Extraction, amplification and detection of DNA in microfluidic chip-based assays

    KAUST Repository

    Wu, Jinbo

    2013-12-20

    This review covers three aspects of PCR-based microfluidic chip assays: sample preparation, target amplification, and product detection. We also discuss the challenges related to the miniaturization and integration of each assay and make a comparison between conventional and microfluidic schemes. In order to accomplish these essential assays without human intervention between individual steps, the micro-components for fluid manipulation become critical. We therefore summarize and discuss components such as microvalves (for fluid regulation), pumps (for fluid driving) and mixers (for blending fluids). By combining the above assays and microcomponents, DNA testing of multi-step bio-reactions in microfluidic chips may be achieved with minimal external control. The combination of assay schemes with the use of micro-components also leads to rapid methods for DNA testing via multi-step bioreactions. Contains 259 references.

  6. Increased DNA damage in blood cells of rat treated with lead as assessed by comet assay

    Directory of Open Access Journals (Sweden)

    Mohammad Arif

    2008-06-01

    Full Text Available A growing body of evidence suggests that oxidative stress is the key player in the pathogenesis of lead-induced toxicity. The present study investigated lead induced oxidative DNA damage, if any in rat blood cells by alkaline comet assay. Lead was administered intraperitoneally to rats at doses of 25, 50 and 100 mg/kg body weight for 5 days consecutively. Blood collected on day six from sacrificed lead-treated rats was used to assess the extent of DNA damage by comet assay which entailed measurement of comet length, olive tail moment, tail DNA (% and tail length. The results showed that treatment with lead significantly increased DNA damage in a dose-dependent manner. Therefore, our data suggests that lead treatment is associated with oxidative stress-induced DNA damage in rat blood cells which could be used as an early bio-marker of lead-toxicity.

  7. Sensitive, simultaneous quantitation of two unlabeled DNA targets using a magnetic nanoparticle-enzyme sandwich assay.

    Science.gov (United States)

    Zhang, Yue; Pilapong, Chalermchai; Guo, Yuan; Ling, Zhenlian; Cespedes, Oscar; Quirke, Philip; Zhou, Dejian

    2013-10-01

    We report herein the development of a simple, sensitive colorimetric magnetic nanoparticle (MNP)-enzyme-based DNA sandwich assay that is suitable for simultaneous, label-free quantitation of two DNA targets down to 50 fM level. It can also effectively discriminate single-nucleotide polymorphisms (SNPs) in genes associated with human cancers (KRAS codon 12/13 SNPs). This assay uses a pair of specific DNA probes, one being covalently conjugated to an MNP for target capture and the other being linked to an enzyme for signal amplification, to sandwich a DNA target, allowing for convenient magnetic separation and subsequent efficient enzymatic signal amplification for high sensitivity. Careful optimization of the MNP surfaces and assay conditions greatly reduced the background, allowing for sensitive, specific detection of as little as 5 amol (50 fM in 100 μL) of target DNA. Moreover, this sensor is robust, it can effectively discriminate cancer-specific SNPs against the wild-type noncancer target, and it works efficiently in 10% human serum. Furthermore, this sensor can simultaneously quantitate two different DNA targets by using two pairs of unique capture- and signal-DNA probes specific for each target. This general, simple, and sensitive DNA sensor appears to be well-suited for a wide range of genetics-based biosensing and diagnostic applications.

  8. A high-throughput fluorescence resonance energy transfer-based assay for DNA ligase.

    Science.gov (United States)

    Shapiro, Adam B; Eakin, Ann E; Walkup, Grant K; Rivin, Olga

    2011-06-01

    DNA ligase is the enzyme that catalyzes the formation of the backbone phosphodiester bond between the 5'-PO(4) and 3'-OH of adjacent DNA nucleotides at single-stranded nicks. These nicks occur between Okazaki fragments during replication of the lagging strand of the DNA as well as during DNA repair and recombination. As essential enzymes for DNA replication, the NAD(+)-dependent DNA ligases of pathogenic bacteria are potential targets for the development of antibacterial drugs. For the purposes of drug discovery, a high-throughput assay for DNA ligase activity is invaluable. This article describes a straightforward, fluorescence resonance energy transfer-based DNA ligase assay that is well suited for high-throughput screening for DNA ligase inhibitors as well as for use in enzyme kinetics studies. Its use is demonstrated for measurement of the steady-state kinetic constants of Haemophilus influenzae NAD(+)-dependent DNA ligase and for measurement of the potency of an inhibitor of this enzyme.

  9. DNA Strand Breaks in Mitotic Germ Cells of Caenorhabditis elegans Evaluated by Comet Assay.

    Science.gov (United States)

    Park, Sojin; Choi, Seoyun; Ahn, Byungchan

    2016-03-01

    DNA damage responses are important for the maintenance of genome stability and the survival of organisms. Such responses are activated in the presence of DNA damage and lead to cell cycle arrest, apoptosis, and DNA repair. In Caenorhabditis elegans, double-strand breaks induced by DNA damaging agents have been detected indirectly by antibodies against DSB recognizing proteins. In this study we used a comet assay to detect DNA strand breaks and to measure the elimination of DNA strand breaks in mitotic germline nuclei of C. elegans. We found that C. elegans brc-1 mutants were more sensitive to ionizing radiation and camptothecin than the N2 wild-type strain and repaired DNA strand breaks less efficiently than N2. This study is the first demonstration of direct measurement of DNA strand breaks in mitotic germline nuclei of C. elegans. This newly developed assay can be applied to detect DNA strand breaks in different C. elegans mutants that are sensitive to DNA damaging agents.

  10. A Broadly Applicable Assay for Rapidly and Accurately Quantifying DNA Surface Coverage on Diverse Particles.

    Science.gov (United States)

    Yu, Haixiang; Xu, Xiaowen; Liang, Pingping; Loh, Kang Yong; Guntupalli, Bhargav; Roncancio, Daniel; Xiao, Yi

    2017-04-19

    DNA-modified particles are used extensively for applications in sensing, material science, and molecular biology. The performance of such DNA-modified particles is greatly dependent on the degree of surface coverage, but existing methods for quantitation can only be employed for certain particle compositions and/or conjugation chemistries. We have developed a simple and broadly applicable exonuclease III (Exo III) digestion assay based on the cleavage of phosphodiester bonds-a universal feature of DNA-modified particles-to accurately quantify DNA probe surface coverage on diverse, commonly used particles of different compositions, conjugation chemistries, and sizes. Our assay utilizes particle-conjugated, fluorophore-labeled probes that incorporate two abasic sites; these probes are hybridized to a complementary DNA (cDNA) strand, and quantitation is achieved via cleavage and digestion of surface-bound probe DNA via Exo III's apurinic endonucleolytic and exonucleolytic activities. The presence of the two abasic sites in the probe greatly speeds up the enzymatic reaction without altering the packing density of the probes on the particles. Probe digestion releases a signal-generating fluorophore and liberates the intact cDNA strand to start a new cycle of hybridization and digestion, until all fluorophore tags have been released. Since the molar ratio of fluorophore to immobilized DNA is 1:1, DNA surface coverage can be determined accurately based on the complete release of fluorophores. Our method delivers accurate, rapid, and reproducible quantitation of thiolated DNA on the surface of gold nanoparticles, and also performs equally well with other conjugation chemistries, substrates, and particle sizes, and thus offers a broadly useful assay for quantitation of DNA surface coverage.

  11. The synchronous active neutron detection assay system

    Energy Technology Data Exchange (ETDEWEB)

    Pickrell, M.M.; Kendall, P.K.

    1994-08-01

    We have begun to develop a novel technique for active neutron assay of fissile material in spent nuclear fuel. This approach will exploit a 14-MeV neutron generator developed by Schlumberger. The technique, termed synchronous active neutron detection (SAND), follows a method used routinely in other branches of physics to detect very small signals in presence of large backgrounds. Synchronous detection instruments are widely available commercially and are termed ``lock-in`` amplifiers. We have implemented a digital lock-in amplifier in conjunction with the Schlumberger neutron generator to explore the possibility of synchronous detection with active neutrons. The Schlumberger system can operate at up to a 50% duty factor, in effect, a square wave of neutron yield. Results are preliminary but promising. The system is capable of resolving the fissile material contained in a small fraction of the fuel rods in a cold fuel assembly; it also appears resilient to background neutron interference. The interrogating neutrons appear to be non-thermal and penetrating. Work remains to fully explore relevant physics and optimize instrument design.

  12. Optimization of Neutral Comet Assay for studying DNA double-strand breaks in pea and wheat

    Directory of Open Access Journals (Sweden)

    Ivelina Nikolova

    2013-01-01

    Full Text Available This study describes an adaptation of the Comet assay under neutral conditions for mono- and dicotyledonous plants pea (Pisum sativum L. and wheat (Triticum aestivum L.. Modifications concern lysis and electrophoresis steps, respectively. Electrophoresis was carried out varying the intensity of the electric field. A linear relationship between the percentages of DNA in the tail from control background with alteration of intensity was found. Trypan blue dye exclusion test was used in order to determine the intactness of nuclear membrane of the isolated nuclei from both plant model systems. Assessment was conducted on non-irradiated and irradiated nuclei on a monolayer with three doses of UVC. It was found that the share of intact nuclei (trypan blue negative ones is about 95% in controls. Gradual dose-related increase of damaged nuclei was observed in both species, reaching statistical significance only at the higher dose applied.

  13. Design and validation of DNA libraries for multiplexing proximity ligation assays.

    Science.gov (United States)

    Gobet, Nicolas; Ketterer, Simon; Meier, Matthias

    2014-01-01

    Here, we present an in silico, analytical procedure for designing and testing orthogonal DNA templates for multiplexing of the proximity ligation assay (PLA). PLA is a technology for the detection of protein interactions, post-translational modifications, and protein concentrations. To enable multiplexing of the PLA, the target information of antibodies was encoded within the DNA template of a PLA, where each template comprised four single-stranded DNA molecules. Our DNA design procedure followed the principles of minimizing the free energy of DNA cross-hybridization. To validate the functionality, orthogonality, and efficiency of the constructed template libraries, we developed a high-throughput solid-phase rolling-circle amplification assay and solid-phase PLA on a microfluidic platform. Upon integration on a microfluidic chip, 640 miniaturized pull-down assays for oligonucleotides or antibodies could be performed in parallel together with steps of DNA ligation, isothermal amplification, and detection under controlled microenvironments. From a large computed PLA template library, we randomly selected 10 template sets and tested all DNA combinations for cross-reactivity in the presence and absence of antibodies. By using the microfluidic chip application, we determined rapidly the false-positive rate of the design procedure, which was less than 1%. The combined theoretical and experimental procedure is applicable for high-throughput PLA studies on a microfluidic chip.

  14. Assessing the efficiency and significance of Methylated DNA Immunoprecipitation (MeDIP assays in using in vitro methylated genomic DNA

    Directory of Open Access Journals (Sweden)

    Jia Jinsong

    2010-09-01

    Full Text Available Abstract Background DNA methylation contributes to the regulation of gene expression during development and cellular differentiation. The recently developed Methylated DNA ImmunoPrecipitation (MeDIP assay allows a comprehensive analysis of this epigenetic mark at the genomic level in normal and disease-derived cells. However, estimating the efficiency of the MeDIP technique is difficult without previous knowledge of the methylation status of a given cell population. Attempts to circumvent this problem have involved the use of in vitro methylated DNA in parallel to the investigated samples. Taking advantage of this stratagem, we sought to improve the sensitivity of the approach and to assess potential biases resulting from DNA amplification and hybridization procedures using MeDIP samples. Findings We performed MeDIP assays using in vitro methylated DNA, with or without previous DNA amplification, and hybridization to a human promoter array. We observed that CpG content at gene promoters indeed correlates strongly with the MeDIP signal obtained using in vitro methylated DNA, even when lowering significantly the amount of starting material. In analyzing MeDIP products that were subjected to whole genome amplification (WGA, we also revealed a strong bias against CpG-rich promoters during this amplification procedure, which may potentially affect the significance of the resulting data. Conclusion We illustrate the use of in vitro methylated DNA to assess the efficiency and accuracy of MeDIP procedures. We report that efficient and reproducible genome-wide data can be obtained via MeDIP experiments using relatively low amount of starting genomic DNA; and emphasize for the precaution that must be taken in data analysis when an additional DNA amplification step is required.

  15. Application of single-cell gel electrophoresis (comet) assay for assessing levels of DNA damage in canine and feline leukocytes.

    Science.gov (United States)

    Heaton, Paul R; Ransley, Raymond; Charlton, Chris J; Mann, Sarah J; Stevenson, Joy; Smith, Brigitte H E; Rawlings, John M; Harper, E Jean

    2002-06-01

    Increasing evidence suggests involvement of free-radical species in the development of oxidative DNA damage, the consequences of which have been implicated in a number of degenerative disorders associated with the aging process. Here we report the application of a single-cell gel electrophoresis (comet) assay for assessing levels of DNA damage in canine and feline leukocytes. Leukocytes were collected from 24 healthy adult cats and dogs and subjected to DNA damage ex vivo by exposure to a range of hydrogen peroxide (H(2)O(2)) concentrations (0-250 micromol/L). The optimal concentration of H(2)O(2) to induce a significant increase in DNA damage was 100 micromol/L for both canine and feline leukocyte samples. Levels of DNA damage were assessed and quantified by visual and computer image analysis. The results obtained showed high correlations between visual scoring and computer image analysis for feline samples (percentage DNA in tail, R(2) > 0.99; tail moment, R(2) > 0.95; tail length, R(2) > 0.90) and canine samples (percentage DNA in tail, R(2) > 0.97; tail moment, R(2) > 0.95; tail length, R(2) > 0.91). In conclusion, this method provides a way of assessing levels of DNA damage utilizing visual and/or computer image analysis in the feline and canine systems. With the capacity of the comet assay to be able to measure end products of free-radical reactions, it is a useful tool for determining the optimal effects of dietary antioxidants on a reliable biomarker of oxidative stress such as cellular DNA status in cats and dogs.

  16. A Microfluidic Microbeads Fluorescence Assay with Quantum Dots-Bead-DNA Probe.

    Science.gov (United States)

    Ankireddy, S R; Kim, Jongsung

    2016-03-01

    A microfluidic bead-based nucleic acid sensor for the detection of tumor causing N-Ras genes using quantum dots has been developed. Presently, quantum dots-bead-DNA probe based hybridization detection methods are often called as 'bead based assays' and their success is substantially influenced by the dispensing and manipulation capability of the microfluidic technology. This study reports the detection of N-Ras cancer gene by fluorescence quenching of quantum dots immobilized on the surface of polystyrene beads. A microfluidic chip was constructed in which the quantum dots-bead-DNA probes were packed in the channel. The target DNA flowed across the beads and hybridized with immobilized probe sequences. The target DNA can be detected by the fluorescence quenching of the quantum dots due to their transfer of emission energy to intercalation dye after DNA hybridization. The mutated gene also induces fluorescence quenching but with less degree than the perfectly complementary target DNA.

  17. Real-time PCR assays for hepatitis B virus DNA quantification may require two different targets.

    Science.gov (United States)

    Liu, Chao; Chang, Le; Jia, Tingting; Guo, Fei; Zhang, Lu; Ji, Huimin; Zhao, Junpeng; Wang, Lunan

    2017-05-12

    Quantification Hepatitis B virus (HBV) DNA plays a critical role in the management of chronic HBV infections. However, HBV is a DNA virus with high levels of genetic variation, and drug-resistant mutations have emerged with the use of antiviral drugs. If a mutation caused a sequence mismatched in the primer or probe of a commercial DNA quantification kit, this would lead to an underestimation of the viral load of the sample. The aim of this study was to determine whether commercial kits, which use only one pair of primers and a single probe, accurately quantify the HBV DNA levels and to develop an improved duplex real-time PCR assay. We developed a new duplex real-time PCR assay that used two pairs of primers and two probes based on the conserved S and C regions of the HBV genome. We performed HBV DNA quantitative detection of HBV samples and compared the results of our duplex real-time PCR assays with the COBAS TaqMan HBV Test version 2 and Daan real-time PCR assays. The target region of the discordant sample was amplified, sequenced, and validated using plasmid. The results of the duplex real-time PCR were in good accordance with the commercial COBAS TaqMan HBV Test version 2 and Daan real-time PCR assays. We showed that two samples from Chinese HBV infections underestimated viral loads when quantified by the Roche kit because of a mismatch between the viral sequence and the reverse primer of the Roche kit. The HBV DNA levels of six samples were undervalued by duplex real-time PCR assays of the C region because of mutations in the primer of C region. We developed a new duplex real-time PCR assay, and the results of this assay were similar to the results of commercial kits. The HBV DNA level could be undervalued when using the COBAS TaqMan HBV Test version 2 for Chinese HBV infections owing to a mismatch with the primer/probe. A duplex real-time PCR assay based on the S and C regions could solve this problem to some extent.

  18. How to evaluate PCR assays for the detection of low-level DNA

    DEFF Research Database (Denmark)

    Banch-Clausen, Frederik; Urhammer, Emil; Rieneck, Klaus

    2015-01-01

    are needed and how many of these should be positive or what amount of template should be used? We developed a mathematical model to obtain a simple tool for quick PCR assay evaluation before laboratory optimization and validation procedures. The model was based on the Poisson distribution and the Binomial...... not significantly different from experimental data generated by testing of cell-free foetal DNA. Also, the simplified formula was applicable for fast and accurate assay evaluation. In conclusion, the model can be applied for evaluation of sensitivity of real-time PCR-based detection of low-level DNA, and may also...

  19. LAMP assay for rapid diagnosis of cow DNA in goat milk and meat samples

    Science.gov (United States)

    Deb, R.; Sengar, G. S.; Singh, U.; Kumar, S.; Raja, T. V.; Alex, R.; Alyethodi, R. R.; Prakash, B.

    2017-01-01

    Animal species detection is one of the crucial steps for consumer’s food analysis. In the present study we developed an in-house built loop-mediated isothermal amplification (LAMP) assay for rapid detection of adulterated cow DNA in goat milk/meat samples. The cow milk/tissue DNA in goat milk/meat samples were identified in the developed LAMP assay by either naked eye visualizing with SYBR Green I dyes or by detecting the typical ladder pattern on gel electrophoresis. This test can detect up to minimum 5% level of cow components admixed in goat milk/meat samples and can be completed within 1 h 40 min starting from DNA extraction from milk/meat samples and can be performed in a water bath. Developed LAMP methodology is simple; rapid and sensitive techniques that can detect adulterant like cow components in goat milk/meat are more accurate than other existing DNA based technologies. PMID:28775755

  20. Testing the feasibility of DNA typing for human identification by PCR and an oligonucleotide ligation assay

    Energy Technology Data Exchange (ETDEWEB)

    Delahunty, C.; Ankener, W.; Deng, Qiang [Univ. of Washington, Seattle, WA (United States)] [and others

    1996-06-01

    The use of DNA typing in human genome analysis is increasing and finding widespread application in the area of forensic and paternity testing. In this report, we explore the feasibility of typing single nucleotide polymorphisms (SNPs) by using a semiautomated method for analyzing human DNA samples. In this approach, PCR is used to amplify segments of human DNA containing a common SNP. Allelic nucleotides in the amplified product are then typed by a calorimetric implementation of the oligonucleotide ligation assay (OLA). The results of the combined assay, PCR/OLA, are read directly by a spectrophotometer; the absorbances are compiled and the genotypes are automatically determined. A panel of 20 markers has been developed for DNA typing and has been tested using a sample panel from the CEPH pedigrees (CEPH parents). The results of this typing, as well as the potential to apply this method to larger populations, are discussed. 62 refs., 2 figs., 4 tabs.

  1. A superstructure-based electrochemical assay for signal-amplified detection of DNA methyltransferase activity.

    Science.gov (United States)

    Zhang, Hui; Yang, Yin; Dong, Huilei; Cai, Chenxin

    2016-12-15

    DNA methyltransferase (MTase) activity is highly correlated with the occurrence and development of cancer. This work reports a superstructure-based electrochemical assay for signal-amplified detection of DNA MTase activity using M.SssI as an example. First, low-density coverage of DNA duplexes on the surface of the gold electrode was achieved by immobilized mercaptohexanol, followed by immobilization of DNA duplexes. The duplex can be cleaved by BstUI endonuclease in the absence of DNA superstructures. However, the cleavage is blocked after the DNA is methylated by M.SssI. The DNA superstructures are formed with the addition of helper DNA. By using an electroactive complex, RuHex, which can bind to DNA double strands, the activity of M.SssI can be quantitatively detected by differential pulse voltammetry. Due to the high site-specific cleavage by BstUI and signal amplification by the DNA superstructure, the biosensor can achieve ultrasensitive detection of DNA MTase activity down to 0.025U/mL. The method can be used for evaluation and screening of the inhibitors of MTase, and thus has potential in the discovery of methylation-related anticancer drugs.

  2. Evaluation of sperm DNA damage in bulls by TUNEL assay as a parameter of semen quality

    OpenAIRE

    TAKEDA, Kumiko; UCHIYAMA, Kyoko; KINUKAWA, Masashi; Tagami, Takahiro; Kaneda, Masahiro; Watanabe, Shinya

    2015-01-01

    Sperm DNA damage affects the conception rate resulting from human assisted reproduction technology. The objective of this study was to adapt the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay to provide a quality parameter for bull semen based on the detection of sperm DNA damage. Fresh semen was collected from two Japanese Black bulls (A, B) several times over the course of a year, and the percentage of TUNEL-positive spermatozoa (sperm TUNEL index) was d...

  3. Effects of seven chemicals on DNA damage in the rat urinary bladder: a comet assay study.

    Science.gov (United States)

    Wada, Kunio; Yoshida, Toshinori; Takahashi, Naofumi; Matsumoto, Kyomu

    2014-07-15

    The in vivo comet assay has been used for the evaluation of DNA damage and repair in various tissues of rodents. However, it can give false-positive results due to non-specific DNA damage associated with cell death. In this study, we examined whether the in vivo comet assay can distinguish between genotoxic and non-genotoxic DNA damage in urinary bladder cells, by using the following seven chemicals related to urinary bladder carcinogenesis in rodents: N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN), glycidol, 2,2-bis(bromomethyl)-1,3-propanediol (BMP), 2-nitroanisole (2-NA), benzyl isothiocyanate (BITC), uracil, and melamine. BBN, glycidol, BMP, and 2-NA are known to be Ames test-positive and they are expected to produce DNA damage in the absence of cytotoxicity. BITC, uracil, and melamine are Ames test-negative with metabolic activation but have the potential to induce non-specific DNA damage due to cytotoxicity. The test chemicals were administered orally to male Sprague-Dawley rats (five per group) for each of two consecutive days. Urinary bladders were sampled 3h after the second administration and urothelial cells were analyzed by the comet assay and subjected to histopathological examination to evaluate cytotoxicity. In the urinary bladders of rats treated with BBN, glycidol, and BMP, DNA damage was detected. In contrast, 2-NA induced neither DNA damage nor cytotoxicity. The non-genotoxic chemicals (BITC, uracil, and melamine) did not induce DNA damage in the urinary bladders under conditions where some histopathological changes were observed. The results indicate that the comet assay could distinguish between genotoxic and non-genotoxic chemicals and that no false-positive responses were obtained.

  4. A DNA Microarray-Based Assay to Detect Dual Infection with Two Dengue Virus Serotypes

    OpenAIRE

    Alvaro Díaz-Badillo; María de Lourdes Muñoz; Gerardo Perez-Ramirez; Victor Altuzar; Juan Burgueño; Mendoza-Alvarez, Julio G.; Martínez-Muñoz, Jorge P.; Alejandro Cisneros; Joel Navarrete-Espinosa; Feliciano Sanchez-Sinencio

    2014-01-01

    Here; we have described and tested a microarray based-method for the screening of dengue virus (DENV) serotypes. This DNA microarray assay is specific and sensitive and can detect dual infections with two dengue virus serotypes and single-serotype infections. Other methodologies may underestimate samples containing more than one serotype. This technology can be used to discriminate between the four DENV serotypes. Single-stranded DNA targets were covalently attached to glass slides and hybrid...

  5. Micropatterned comet assay enables high throughput and sensitive DNA damage quantification.

    Science.gov (United States)

    Ge, Jing; Chow, Danielle N; Fessler, Jessica L; Weingeist, David M; Wood, David K; Engelward, Bevin P

    2015-01-01

    The single cell gel electrophoresis assay, also known as the comet assay, is a versatile method for measuring many classes of DNA damage, including base damage, abasic sites, single strand breaks and double strand breaks. However, limited throughput and difficulties with reproducibility have limited its utility, particularly for clinical and epidemiological studies. To address these limitations, we created a microarray comet assay. The use of a micrometer scale array of cells increases the number of analysable comets per square centimetre and enables automated imaging and analysis. In addition, the platform is compatible with standard 24- and 96-well plate formats. Here, we have assessed the consistency and sensitivity of the microarray comet assay. We showed that the linear detection range for H2O2-induced DNA damage in human lymphoblastoid cells is between 30 and 100 μM, and that within this range, inter-sample coefficient of variance was between 5 and 10%. Importantly, only 20 comets were required to detect a statistically significant induction of DNA damage for doses within the linear range. We also evaluated sample-to-sample and experiment-to-experiment variation and found that for both conditions, the coefficient of variation was lower than what has been reported for the traditional comet assay. Finally, we also show that the assay can be performed using a 4× objective (rather than the standard 10× objective for the traditional assay). This adjustment combined with the microarray format makes it possible to capture more than 50 analysable comets in a single image, which can then be automatically analysed using in-house software. Overall, throughput is increased more than 100-fold compared to the traditional assay. Together, the results presented here demonstrate key advances in comet assay technology that improve the throughput, sensitivity, and robustness, thus enabling larger scale clinical and epidemiological studies.

  6. Detection of DNA damage induced by topoisomerase II inhibitors, gamma radiation and crosslinking agents using the comet assay.

    Science.gov (United States)

    Hazlehurst, Lori A

    2009-01-01

    The comet assay is a simple gel electrophoresis method for visualizing and quantifying DNA damage. The comet assay is sensitive and reproducible and can be used to detect single-strand DNA breaks, double-strand DNA breaks, protein-associated DNA strand breaks and DNA crosslinks. The comet assay uses fluorescent DNA-binding dyes to detect both damaged DNA that resides in the tail region and undamaged DNA that is retained in the head region following gel electrophoresis. This assay is a single cell-based assay and thus is highly adaptable for measuring DNA damage in clinical samples. Furthermore, unlike other assays the detection of DNA damage is not dependent on the random incorporation of radiolabeled nucleotides. Again this can be problematic with clinical samples as proliferation rates are often slow and culturing of primary patient specimens for 48 h required to randomly label DNA is often not possible. In this chapter we will outline the comet assay for the detection of DNA damage induced by topoisomerase II inhibitors, cross-linking agents and gamma radiation.

  7. Increased knowledge of Francisella genus diversity highlights the benefits of optimised DNA-based assays

    Directory of Open Access Journals (Sweden)

    Ahlinder Jon

    2012-09-01

    Full Text Available Abstract Background Recent advances in sequencing technologies offer promising tools for generating large numbers of genomes, larger typing databases and improved mapping of environmental bacterial diversity. However, DNA-based methods for the detection of Francisella were developed with limited knowledge about genetic diversity. This, together with the high sequence identity between several Francisella species, means there is a high risk of false identification and detection of the highly virulent pathogen Francisella tularensis. Moreover, phylogenetic reconstructions using single or limited numbers of marker sequences often result in incorrect tree topologies and inferred evolutionary distances. The recent growth in publicly accessible whole-genome sequences now allows evaluation of published genetic markers to determine optimal combinations of markers that minimise both time and laboratory costs. Results In the present study, we evaluated 38 previously published DNA markers and the corresponding PCR primers against 42 genomes representing the currently known diversity of the genus Francisella. The results highlight that PCR assays for Francisella tularensis are often complicated by low specificity, resulting in a high probability of false positives. A method to select a set of one to seven markers for obtaining optimal phylogenetic resolution or diagnostic accuracy is presented. Conclusions Current multiple-locus sequence-typing systems and detection assays of Francisella, could be improved by redesigning some of the primers and reselecting typing markers. The use of only a few optimally selected sequence-typing markers allows construction of phylogenetic topologies with almost the same accuracy as topologies based on whole-genome sequences.

  8. Variables Affecting Two Electron Transport System Assays

    OpenAIRE

    Burton, G. Allen; Lanza, Guy R.

    1986-01-01

    Several methodological variables were critical in two commonly used electron transport activity assays. The dehydrogenase assay based on triphenyl formazan production exhibited a nonlinear relationship between formazan production (dehydrogenase activity) and sediment dilution, and linear formazan production occurred for 1 h in sediment slurries. Activity decreased with increased time of sediment storage at 4°C. Extraction efficiencies of formazan from sediment varied with alcohol type; methan...

  9. Systems, devices, and methods for agglutination assays using sedimentation

    Science.gov (United States)

    Schaff, Ulrich Y.; Sommer, Gregory J.; Singh, Anup K.

    2016-01-26

    Embodiments of the present invention include methods for conducting agglutination assays using sedimentation. Aggregates may be exposed to sedimentation forces and travel through a density medium to a detection area. Microfluidic devices, such as microfluidic disks, are described for conducting the agglutination assays, as are systems for conducting the assays.

  10. Different sensitivities of cultured mammalian cells towards aphidicolin-enhanced DNA effects in the comet assay.

    Science.gov (United States)

    Speit, Günter; Schütz, Petra; Bausinger, Julia

    2016-06-01

    The comet assay in combination with the polymerase inhibitor aphidicolin (APC) has been used to measure DNA excision repair activity, DNA repair kinetics and individual DNA repair capacity. Since APC can enhance genotoxic effects of mutagens measured by the comet assay, this approach has been proposed for increasing the sensitivity of the comet assay in human biomonitoring. The APC-modified comet assay has mainly been performed with human blood and it was shown that it not only enhances the detection of DNA damage repaired by nucleotide excision repair (NER) but also damage typically repaired by base excision repair (BER). Recently, we reported that in contrast to blood leukocytes, A549 cells (a human lung adenocarcinoma cell line) seem to be insensitive towards the repair-inhibiting action of APC. To further elucidate the general usefulness of the APC-modified comet assay for studying repair in cultured mammalian cells, we comparatively investigated further cell lines (HeLa, TK6, V79). DNA damage was induced by BPDE (benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide) and MMS (methyl methanesulfonate) in the absence and presence of APC (3 or 15μM). APC was either added for 2h together with the mutagen or cells were pre-incubated for 30min with APC before the mutagen was added. The results indicate that the cell lines tested differ fundamentally with regard to their sensitivity and specificity towards the repair-inhibiting effect of APC. The actual cause for these differences is still unclear but potential molecular explanations are discussed. Irrespective of the underlying mechanism(s), our study revealed practical limitations of the use of the APC-modified comet assay.

  11. Inter-laboratory variation in DNA damage using a standard comet assay protocol.

    Science.gov (United States)

    Forchhammer, Lykke; Ersson, Clara; Loft, Steffen; Möller, Lennart; Godschalk, Roger W L; van Schooten, Frederik J; Jones, George D D; Higgins, Jennifer A; Cooke, Marcus; Mistry, Vilas; Karbaschi, Mahsa; Collins, Andrew R; Azqueta, Amaya; Phillips, David H; Sozeri, Osman; Routledge, Michael N; Nelson-Smith, Kirsty; Riso, Patrizia; Porrini, Marisa; Matullo, Giuseppe; Allione, Alessandra; Stępnik, Maciej; Steepnik, Maciej; Komorowska, Magdalena; Teixeira, João Paulo; Costa, Solange; Corcuera, Laura-Ana; López de Cerain, Adela; Laffon, Blanca; Valdiglesias, Vanessa; Møller, Peter

    2012-11-01

    There are substantial inter-laboratory variations in the levels of DNA damage measured by the comet assay. The aim of this study was to investigate whether adherence to a standard comet assay protocol would reduce inter-laboratory variation in reported values of DNA damage. Fourteen laboratories determined the baseline level of DNA strand breaks (SBs)/alkaline labile sites and formamidopyrimidine DNA glycosylase (FPG)-sensitive sites in coded samples of mononuclear blood cells (MNBCs) from healthy volunteers. There were technical problems in seven laboratories in adopting the standard protocol, which were not related to the level of experience. Therefore, the inter-laboratory variation in DNA damage was only analysed using the results from laboratories that had obtained complete data with the standard comet assay protocol. This analysis showed that the differences between reported levels of DNA SBs/alkaline labile sites in MNBCs were not reduced by applying the standard assay protocol as compared with the laboratory's own protocol. There was large inter-laboratory variation in FPG-sensitive sites by the laboratory-specific protocol and the variation was reduced when the samples were analysed by the standard protocol. The SBs and FPG-sensitive sites were measured in the same experiment, indicating that the large spread in the latter lesions was the main reason for the reduced inter-laboratory variation. However, it remains worrying that half of the participating laboratories obtained poor results using the standard procedure. This study indicates that future comet assay validation trials should take steps to evaluate the implementation of standard procedures in participating laboratories.

  12. Comet Assay: A Method to Evaluate Genotoxicity of Nano-Drug Delivery System

    Directory of Open Access Journals (Sweden)

    Morteza Eskandani

    2011-08-01

    Full Text Available Introduction: Drug delivery systems could induce cellular toxicity as side effect of nanomaterials. The mechanism of toxicity usually involves DNA damage. The comet assay or single cell gel electrophoresis (SCGE is a sensitive method for detecting strand damages in the DNA of a cell with applications in genotoxicity testing and molecular epidemiology as well as fundamental research in DNA damage and repair. Methods: In the current study, we reviewed recent drug delivery researches related to SCGE. Results: We found that one preference for choosing the assay is that comet images may result from apoptosis-mediated nuclear fragmentation. This method has been widely used over the last decade in several different areas. Overall cells, such as cultured cells are embedded in agarose on a microscope slide, lysed with detergent, and treated with high salt. Nucleoids are supercoiled DNA form. When the slide is faced to alkaline electrophoresis any breakages present in the DNA cause the supercoiling to relax locally and loops of DNA extend toward the anode as a ‘‘comet tail’’. Conclusion: This article provides a relatively comprehensive review upon potentiality of the comet assay for assessment of DNA damage and accordingly it can be used as an informative platform in genotoxicity studies of drug delivery systems.

  13. Inter- and intra-laboratory standardization of TUNEL assay for assessment of sperm DNA fragmentation.

    Science.gov (United States)

    Ribeiro, S; Sharma, R; Gupta, S; Cakar, Z; De Geyter, C; Agarwal, A

    2017-05-01

    One of the challenges with the sperm DNA fragmentation results is the inconsistency and the large variability in the results obtained by different techniques. The terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay quantifies the incorporation of fluoresceinated dUTP into single- and double-strand DNA breaks by labeling the 3'-OH terminal with TdT. The goal of this study was optimize the TUNEL protocol for assessment of sperm DNA fragmentation by standardization of the method and comparison of the data across two reference laboratories (i) at Basel, Switzerland and (ii) Cleveland Clinic, Ohio, USA. Semen samples from 31 subjects grouped into three cohorts. Sperm DNA fragmentation was data measured by two experienced operators at two different laboratories using identical semen samples, assay kit, protocol and acquisition settings using identical flow cytometers (BD Accuri C6). No significant differences were observed between the duplicates in any of the experiments performed. By including an additional washing step after fixation in paraformaldehyde, a high correlation was seen between the two laboratories (r = 0.94). A strong positive correlation was observed between the average sperm DNA fragmentation rates (r = 0.719). The mean sperm DNA fragmentation measured in each laboratory was similar. Both flow cytometers were identical in their settings and performance. This inter- and intra-laboratory study establishes that TUNEL is a reproducible assay when utilizing a standardized staining protocol and flow cytometer acquisition settings. Standardization and consensual guidelines for TUNEL validate the assay and establishes TUNEL as a robust test for measuring sperm DNA fragmentation especially in a multicenter setting. © 2017 American Society of Andrology and European Academy of Andrology.

  14. Evaluation of DNA damage induced by environmental exposure to mercury in Liza aurata using the comet assay.

    Science.gov (United States)

    Pereira, Carla Sofia Alves; Guilherme, Sofia Isabel Antunes Gomes; Barroso, Carlos Miguel Miguez; Verschaeve, Luc; Pacheco, Mário Guilherme Garcês; Mendo, Sónia Alexandra Leite Velho

    2010-01-01

    Mercury (Hg) is one of the major aquatic contaminants even though emissions have been reduced over the years. Despite the relative abundance of investigations carried out on Hg toxicity, there is a scarcity of studies on its DNA damaging effects in fish under realistic exposure conditions. This study assessed the Hg genotoxicity in Golden grey mullets (Liza aurata) at Laranjo basin, a particularly contaminated area of Ria de Aveiro (Portugal) well known for its Hg contamination gradient. (1) Fish were seasonally caught at Laranjo basin and at a reference site (S. Jacinto), and (2) animals from the reference site were transplanted and caged (at bottom and surface), for 3 days, in two different locations within Laranjo basin. Using the comet assay, blood was analyzed for genetic damage and apoptotic cell frequency. The seasonal survey showed greater DNA damage in the Hg-contaminated area for all sampling seasons excluding winter. The temporal variation pattern of DNA lesions was: summer approximately autumn > winter > spring. Fish caged at Laranjo also exhibited greater DNA damage than those caged at the reference site, highlighting the importance of gill uptake on the toxicity of this metal. No increased susceptibility to apoptosis was detected in either wild or caged fish, indicating that mercury damages DNA of blood cells by a nonapoptotic mechanism. Both L. aurata and the comet assay proved to be sensitive and suitable for genotoxicity biomonitoring in mercury-contaminated coastal systems.

  15. A Multiplexed, Probe-Based Quantitative PCR Assay for DNA of Phytophthora sojae

    Science.gov (United States)

    Phytophthora sojae (Kaufm. & Gerd.) causes seed rot, pre- and post-emergence damping off, and sometimes foliar blight in soybean (Glycine max). Crop loss may approach 100% with susceptible cultivars. We report here the development of a unique quantitative PCR assay specific to DNA of P. sojae, and a...

  16. High-throughput genotoxicity assay identifies antioxidants as inducers of DNA damage response and cell death

    Science.gov (United States)

    Human ATAD5 is an excellent biomarker for identifying genotoxic compounds because ATADS protein levels increase post-transcriptionally following exposure to a variety of DNA damaging agents. Here we report a novel quantitative high-throughput ATAD5-Iuciferase assay that can moni...

  17. Detection and quantification of DNA strand breaks using the ROPS (random oligonucleotide primed synthesis) assay.

    Science.gov (United States)

    Boyko, Alex; Kovalchuk, Igor

    2010-01-01

    DNA double strand breaks (DSBs) arise from spontaneous DNA damage due to metabolic activities or from direct and indirect damaging effects of stress. DSBs are also formed transiently during such processes as replication, transcription, and DNA repair. The level of DSBs positively correlates with the activities of homologous and nonhomologous DNA repair pathways, which in turn inversely correlate with methylation levels and chromatin structure. Thus, measurement of strand breaks can provide an informative picture of genome stability of a given cell. The use of random oligonucleotide-primed synthesis for the analysis of DSB levels is described. Applications of the assay for quantitative detection of 3'OH, 3'P, or DNA strand breaks at a cleavage site of the deoxyribose residue are discussed.

  18. DNA methylation profiling using the methylated-CpG island recovery assay (MIRA).

    Science.gov (United States)

    Rauch, Tibor A; Pfeifer, Gerd P

    2010-11-01

    The methylated-CpG island recovery assay (MIRA) exploits the intrinsic specificity and the high affinity of a methylated-CpG-binding protein complex (MBD2B and MBD3L1) to methylated CpG dinucleotides in genomic DNA. The MIRA approach works on double-stranded DNA and does not depend on the application of methylation-sensitive restriction enzymes. It can be performed on a few hundred nanograms of genomic DNA. Recently, the MIRA technique has been used to profile DNA methylation patterns at a resolution of 100 base pairs along the entire genome of normal human B-lymphocytes. The MIRA method is compatible with microarray and next generation DNA sequencing approaches. We describe the principles and details of this method applied for methylation profiling of genomes containing methylated CpG sequences.

  19. The alkaline comet assay as a method to investigate the DNA strand breaking effect of phenylpropanoids in mammalian cells

    Directory of Open Access Journals (Sweden)

    Sabrina Haupenthal

    2015-05-01

    Therefore, a modified alkaline comet assay to investigate DNA strand breaking effects of the asarone isomers in hamster lung fibroblasts (V79 cells and with human cytochrome P450 1A2 and human sulfotransferase 1C2 transfected V79 cells was used. Furthermore, the DNA repair enzyme formamidopyrimidine DNA glycosylase (FPG as a marker for oxidative DNA damage was also included in our test system. aA, bA as well as gA significantly induce DNA damage in V79 cells at concentrations > 10 µM. However, no enhanced DNA damage was observed after FPG-incubation of V79 cells. In contrast, in metabolic competent transfected V79 cells both propenylic asarone derivatives aA and bA significantly increase the amount of FPG-sensitive sites, whereas the allylic compound gA was not effective. This study assumes that an impact on the cellular redox status of the propenylic asarone isomers and their respective metabolites might contribute to their genotoxic properties. In conclusion, further in vitro studies on DNA damage and repair to elucidate the mechanism of genotoxicity and mutagenicity of these carcinogenic plant constituents are still under investigation.

  20. Bioluminescence regenerative cycle (BRC) system for nucleic acid quantification assays

    Science.gov (United States)

    Hassibi, Arjang; Lee, Thomas H.; Davis, Ronald W.; Pourmand, Nader

    2003-07-01

    A new label-free methodology for nucleic acid quantification has been developed where the number of pyrophosphate molecules (PPi) released during polymerization of the target nucleic acid is counted and correlated to DNA copy number. The technique uses the enzymatic complex of ATP-sulfurylase and firefly luciferase to generate photons from PPi. An enzymatic unity gain positive feedback is also implemented to regenerate the photon generation process and compensate any decay in light intensity by self regulation. Due to this positive feedback, the total number of photons generated by the bioluminescence regenerative cycle (BRC) can potentially be orders of magnitude higher than typical chemiluminescent processes. A system level kinetic model that incorporates the effects of contaminations and detector noise was used to show that the photon generation process is in fact steady and also proportional to the nucleic acid quantity. Here we show that BRC is capable of detecting quantities of DNA as low as 1 amol (10-18 mole) in 40μlit aqueous solutions, and this enzymatic assay has a controllable dynamic range of 5 orders of magnitude. The sensitivity of this technology, due to the excess number of photons generated by the regenerative cycle, is not constrained by detector performance, but rather by possible PPi or ATP (adenosine triphosphate) contamination, or background bioluminescence of the enzymatic complex.

  1. Detection of irradiated quail meat by using DNA comet assay and evaluation of comets by image analysis

    Science.gov (United States)

    Erel, Yakup; Yazici, Nizamettin; Özvatan, Sumer; Ercin, Demet; Cetinkaya, Nurcan

    2009-09-01

    A simple technique of microgel electrophoresis of single cells (DNA comet assay) was used to detect DNA comets in irradiated quail meat samples. Obtained DNA comets were evaluated by both photomicrographic and image analysis. Quail meat samples were exposed to radiation doses of 0.52, 1.05, 1.45, 2.00, 2.92 and 4.00 kGy in gamma cell (gammacell 60Co, dose rate 1.31 kGy/h) covering the permissible limits for enzymatic decay and stored at 2 °C. The cells isolated from muscle (chest, thorax) in cold PBS were analyzed using the DNA comet assay on 1, 2, 3, 4, 7, 8 and 11 day post irradiation. The cells were lysed between 2, 5 and 9 min in 2.5% SDS and electrophorosis was carried out at a voltage of 2 V/cm for 2 min. After propidium iodide staining, the slides were evaluated through a fluorescent microscope. In all irradiated samples, fragmented DNA stretched towards the anode and damaged cells appeared as a comet. All measurement data were analyzed using BS 200 ProP with software image analysis (BS 200 ProP, BAB Imaging System, Ankara, Turkey). The density of DNA in the tails increased with increasing radiation dose. However, in non-irradiated samples, the large molecules of DNA remained relatively intact and there was only minor or no migration of DNA; the cells were round or had very short tails only. The values of tail DNA%, tail length and tail moment were significantly different and identical between 0.9 and 4.0 kGy dose exposure, and also among storage times on day 1, 4 and 8. In conclusion, the DNA Comet Assay EN 13784 standard method may be used not only for screening method for detection of irradiated quail meat depending on storage time and condition but also for the quantification of applied dose if it is combined with image analysis. Image analysis may provide a powerful tool for the evaluation of head and tail of comet intensity related with applied doses.

  2. How to evaluate PCR assays for the detection of low-level DNA

    DEFF Research Database (Denmark)

    Banch-Clausen, Frederik; Urhammer, Emil; Rieneck, Klaus

    2015-01-01

    distribution describing parameters for singleplex real-time PCR-based detection of low-level DNA. The model was tested against experimental data of diluted cell-free foetal DNA. Also, the model was compared with a simplified formula to enable easy predictions. The model predicted outcomes that were......High sensitivity of PCR-based detection of very low copy number DNA targets is crucial. Much focus has been on design of PCR primers and optimization of the amplification conditions. Very important are also the criteria used for determining the outcome of a PCR assay, e.g. how many replicates...... are needed and how many of these should be positive or what amount of template should be used? We developed a mathematical model to obtain a simple tool for quick PCR assay evaluation before laboratory optimization and validation procedures. The model was based on the Poisson distribution and the Binomial...

  3. Environmental DNA assays for the sister taxa sauger (Sander canadensis) and walleye (Sander vitreus).

    Science.gov (United States)

    Dysthe, Joseph C; Carim, Kellie J; Ruggles, Michael; McKelvey, Kevin S; Young, Michael K; Schwartz, Michael K

    2017-01-01

    Sauger (Sander canadensis) and walleye (S. vitreus) are percid fishes that naturally co-occur throughout much of the eastern United States. The native range of sauger extends into the upper Missouri River drainage where walleye did not historically occur, but have been stocked as a sport fish. Sauger populations have been declining due to habitat loss, fragmentation, and competition with non-native species, such as walleye. To effectively manage sauger populations, it is necessary to identify areas where sauger occur, and particularly where they co-occur with walleye. We developed quantitative PCR assays that can detect sauger and walleye DNA in filtered water samples. Each assay efficiently detected low quantities of target DNA and failed to detect DNA of non-target species with which they commonly co-occur.

  4. Integrated microfluidic systems for DNA analysis.

    Science.gov (United States)

    Njoroge, Samuel K; Chen, Hui-Wen; Witek, Małgorzata A; Soper, Steven A

    2011-01-01

    The potential utility of genome-related research in terms of evolving basic discoveries in biology has generated widespread use of DNA diagnostics and DNA forensics and driven the accelerated development of fully integrated microfluidic systems for genome processing. To produce a microsystem with favorable performance characteristics for genetic-based analyses, several key operational elements must be strategically chosen, including device substrate material, temperature control, fluidic control, and reaction product readout. As a matter of definition, a microdevice is a chip that performs a single processing step, for example microchip electrophoresis. Several microdevices can be integrated to a single wafer, or combined on a control board as separate devices to form a microsystem. A microsystem is defined as a chip composed of at least two microdevices. Among the many documented analytical microdevices, those focused on the ability to perform the polymerase chain reaction (PCR) have been reported extensively due to the importance of this processing step in most genetic-based assays. Other microdevices that have been detailed in the literature include those for solid-phase extractions, microchip electrophoresis, and devices composed of DNA microarrays used for interrogating DNA primary structure. Great progress has also been made in the areas of chip fabrication, bonding and sealing to enclose fluidic networks, evaluation of different chip substrate materials, surface chemistries, and the architecture of reaction conduits for basic processing steps such as mixing. Other important elements that have been developed to realize functional systems include miniaturized readout formats comprising optical or electrochemical transduction and interconnect technologies. These discoveries have led to the development of fully autonomous and functional integrated systems for genome processing that can supply "sample in/answer out" capabilities. In this chapter, we focus on

  5. A highly scalable peptide-based assay system for proteomics.

    Directory of Open Access Journals (Sweden)

    Igor A Kozlov

    Full Text Available We report a scalable and cost-effective technology for generating and screening high-complexity customizable peptide sets. The peptides are made as peptide-cDNA fusions by in vitro transcription/translation from pools of DNA templates generated by microarray-based synthesis. This approach enables large custom sets of peptides to be designed in silico, manufactured cost-effectively in parallel, and assayed efficiently in a multiplexed fashion. The utility of our peptide-cDNA fusion pools was demonstrated in two activity-based assays designed to discover protease and kinase substrates. In the protease assay, cleaved peptide substrates were separated from uncleaved and identified by digital sequencing of their cognate cDNAs. We screened the 3,011 amino acid HCV proteome for susceptibility to cleavage by the HCV NS3/4A protease and identified all 3 known trans cleavage sites with high specificity. In the kinase assay, peptide substrates phosphorylated by tyrosine kinases were captured and identified by sequencing of their cDNAs. We screened a pool of 3,243 peptides against Abl kinase and showed that phosphorylation events detected were specific and consistent with the known substrate preferences of Abl kinase. Our approach is scalable and adaptable to other protein-based assays.

  6. Evaluation of the DNA damaging effects of amitraz on human lymphocytes in the Comet assay.

    Science.gov (United States)

    Radakovic, Milena; Stevanovic, Jevrosima; Djelic, Ninoslav; Lakic, Nada; Knezevic-Vukcevic, Jelena; Vukovic-Gacic, Branka; Stanimirovic, Zoran

    2013-03-01

    Amitraz is formamidine pesticide widely used as insecticide and acaricide. In veterinary medicine, amitraz has important uses against ticks, mites and lice on animals. Also, amitraz is used in apiculture to control Varroa destructor. It this study, the alkaline Comet assay was used to evaluate DNA damaging effects of amitraz in human lymphocytes. Isolated human lymphocytes were incubated with varying concentrations of amitraz (0.035, 0.35, 3.5, 35 and 350 mu g/mL). The Comet assay demonstrated that all concentrations of amitraz caused statistically significant increase in the level of DNA damage, thus indicating that amitraz possesses genotoxic potential. The concentration of amitraz that produced the highest DNA damage (3.5 mu g/mL) was chosen for further analysis with the antioxidant catalase. The obtained results showed that co-treatment with antioxidant catalase (100 IU/mL or 500 IU/mL) significantly reduced the level of DNA damage, indicating the possible involvement of reactive oxygen species in DNA damaging effects of amitraz. Flow cytometric analysis revealed increase of the apoptotic index following treatment with amitraz. However, co-treatment with catalase reduced the apoptotic index, while treatment with catalase alone reduced the percentage of apoptotoc cells even in comparison with the negative control. Therefore, catalase had protective effects against ROS-mediated DNA damage and apoptosis.

  7. Evaluation of the DNA damaging effects of amitraz on human lymphocytes in the Comet assay

    Indian Academy of Sciences (India)

    Milena Radakovic; Jevrosima Stevanovic; Ninoslav Djelic; Nada Lakic; Jelena Knezevic-Vukcevic; Branka Vukovic-Gacic; Zoran Stanimirovic

    2013-03-01

    Amitraz is formamidine pesticide widely used as insecticide and acaricide. In veterinary medicine, amitraz has important uses against ticks, mites and lice on animals. Also, amitraz is used in apiculture to control Varroa destructor. It this study, the alkaline Comet assay was used to evaluate DNA damaging effects of amitraz in human lymphocytes. Isolated human lymphocytes were incubated with varying concentrations of amitraz (0.035, 0.35, 3.5, 35 and 350 g/mL). The Comet assay demonstrated that all concentrations of amitraz caused statistically significant increase in the level of DNA damage, thus indicating that amitraz possesses genotoxic potential. The concentration of amitraz that produced the highest DNA damage (3.5 g/mL) was chosen for further analysis with the antioxidant catalase. The obtained results showed that co-treatment with antioxidant catalase (100 IU/mL or 500 IU/mL) significantly reduced the level of DNA damage, indicating the possible involvement of reactive oxygen species in DNA damaging effects of amitraz. Flow cytometric analysis revealed increase of the apoptotic index following treatment with amitraz. However, co-treatment with catalase reduced the apoptotic index, while treatment with catalase alone reduced the percentage of apoptotoc cells even in comparison with the negative control. Therefore, catalase had protective effects against ROS-mediated DNA damage and apoptosis.

  8. Microelectronic DNA assay for the detection of BRCA1 gene mutations

    Science.gov (United States)

    Chen, Hua; Han, Jie; Li, Jun; Meyyappan, Meyya

    2004-01-01

    Mutations in BRCA1 are characterized by predisposition to breast cancer, ovarian cancer and prostate cancer as well as colon cancer. Prognosis for this cancer survival depends upon the stage at which cancer is diagnosed. Reliable and rapid mutation detection is crucial for the early diagnosis and treatment. We developed an electronic assay for the detection of a representative single nucleotide polymorphism (SNP), deletion and insertion in BRCA1 gene by the microelectronics microarray instrumentation. The assay is rapid, and it takes 30 minutes for the immobilization of target DNA samples, hybridization, washing and readout. The assay is multiplexing since it is carried out at the same temperature and buffer conditions for each step. The assay is also highly specific, as the signal-to-noise ratio is much larger than recommended value (72.86 to 321.05 vs. 5) for homozygotes genotyping, and signal ratio close to the perfect value 1 for heterozygotes genotyping (1.04).

  9. Characterization of Sporothrix schenckii by random amplification of polymorphic DNA assay

    Institute of Scientific and Technical Information of China (English)

    刘晓明; 廉翠红; 金礼吉; 安利佳; 杨国玲; 林熙然

    2003-01-01

    Objectives To investigate the DNA polymorphism of Sporothrix schenckii (S.schenckii) and to find the relationship between DNA patterns and geographic areas and clinical manifestations. Method The total DNA was extracted with hexadecyltrimethyl-ammonium bromide. Random A mplification of Polymorphic DNA (RAPD) assay was used to study DNA typing of 24 strains of S.schenckii collected from different areas and isolated from di fferent clinical types. Results Of seven random primers used, three primers (OPAA11, OPD18 and OPB07) gave good reactions, the sequences of which were 5'-ACCCGACCTG-3', 5'-GAGAGCCAAC-3', 5 '-GGTGAC~GCAG-3' respectively. The RAPD patterns of the 24 isolates were not completely identical, showing certain degrees of hereditary variability. Differ ent isolates showed a common conserved DNA band with the same primer. Different clinical types showed different genotypes. Conclusion RAPD analysis is useful in DNA typing of S.schenckii, the DNA band type of which is related to geographic origin and Clinical manifestation.

  10. Detection of bovine herpesvirus 4 glycoprotein B and thymidine kinase DNA by PCR assays in bovine milk

    NARCIS (Netherlands)

    Wellenberg, G.J.; Verstraten, E.; Belak, S.; Verschuren, S.B.E.; Rijsewijk, F.A.M.; Peshev, R.; Oirschot, van J.T.

    2001-01-01

    A polymerase chain reaction (PCR) assay was developed to detect bovine herpesvirus 4 (BHV4) glycoprotein B (gB) DNA, and a nested-PCR assay was modified for the detection of BHV4 thymidine kinase (TK) DNA in bovine milk samples. To identify false-negative PCR results, internal control templates were

  11. Detection of bovine herpesvirus 4 glycoprotein B and thymidine kinase DNA by PCR assays in bovine milk

    NARCIS (Netherlands)

    Wellenberg, G.J.; Verstraten, E.; Belak, S.; Verschuren, S.B.E.; Rijsewijk, F.A.M.; Peshev, R.; Oirschot, van J.T.

    2001-01-01

    A polymerase chain reaction (PCR) assay was developed to detect bovine herpesvirus 4 (BHV4) glycoprotein B (gB) DNA, and a nested-PCR assay was modified for the detection of BHV4 thymidine kinase (TK) DNA in bovine milk samples. To identify false-negative PCR results, internal control templates were

  12. Specific detection of DNA and RNA targets using a novel isothermal nucleic acid amplification assay based on the formation of a three-way junction structure.

    Science.gov (United States)

    Wharam, S D; Marsh, P; Lloyd, J S; Ray, T D; Mock, G A; Assenberg, R; McPhee, J E; Brown, P; Weston, A; Cardy, D L

    2001-06-01

    The formation of DNA three-way junction (3WJ) structures has been utilised to develop a novel isothermal nucleic acid amplification assay (SMART) for the detection of specific DNA or RNA targets. The assay consists of two oligonucleotide probes that hybridise to a specific target sequence and, only then, to each other forming a 3WJ structure. One probe (template for the RNA signal) contains a non-functional single-stranded T7 RNA polymerase promoter sequence. This promoter sequence is made double-stranded (hence functional) by DNA polymerase, allowing T7 RNA polymerase to generate a target-dependent RNA signal which is measured by an enzyme-linked oligosorbent assay (ELOSA). The sequence of the RNA signal is always the same, regardless of the original target sequence. The SMART assay was successfully tested in model systems with several single-stranded synthetic targets, both DNA and RNA. The assay could also detect specific target sequences in both genomic DNA and total RNA from Escherichia coli. It was also possible to generate signal from E.coli samples without prior extraction of nucleic acid, showing that for some targets, sample purification may not be required. The assay is simple to perform and easily adaptable to different targets.

  13. Rapid detection of microbial DNA by a novel isothermal genome exponential amplification reaction (GEAR) assay.

    Science.gov (United States)

    Prithiviraj, Jothikumar; Hill, Vincent; Jothikumar, Narayanan

    2012-04-20

    In this study we report the development of a simple target-specific isothermal nucleic acid amplification technique, termed genome exponential amplification reaction (GEAR). Escherichia coli was selected as the microbial target to demonstrate the GEAR technique as a proof of concept. The GEAR technique uses a set of four primers; in the present study these primers targeted 5 regions on the 16S rRNA gene of E. coli. The outer forward and reverse Tab primer sequences are complementary to each other at their 5' end, whereas their 3' end sequences are complementary to their respective target nucleic acid sequences. The GEAR assay was performed at a constant temperature 60 °C and monitored continuously in a real-time PCR instrument in the presence of an intercalating dye (SYTO 9). The GEAR assay enabled amplification of as few as one colony forming units of E. coli per reaction within 30 min. We also evaluated the GEAR assay for rapid identification of bacterial colonies cultured on agar media directly in the reaction without DNA extraction. Cells from E. coli colonies were picked and added directly to GEAR assay mastermix without prior DNA extraction. DNA in the cells could be amplified, yielding positive results within 15 min.

  14. Direct identification of chlamydiae from clinical samples using a DNA microarray assay: a validation study.

    Science.gov (United States)

    Borel, Nicole; Kempf, Evelyne; Hotzel, Helmut; Schubert, Evelyn; Torgerson, Paul; Slickers, Peter; Ehricht, Ralf; Tasara, Taurai; Pospischil, Andreas; Sachse, Konrad

    2008-02-01

    While DNA microarrays have become a widely accepted tool for mRNA expression monitoring, their use in rapid diagnosis of bacterial and viral pathogens is only emerging. So far, insufficient sensitivity and high costs have been the major limiting factors preventing more widespread use of microarray platforms in direct testing of clinical samples. In the present study, a total of 339 samples, among them 293 clinical specimens from animals and humans, were examined by the ArrayTube (AT) DNA microarray assay to detect chlamydial DNA and identify the species of Chlamydia and Chlamydophila involved. Samples included nasal and conjunctival swabs, formalin-fixed, paraffin-embedded and fresh organ tissue, milk, feces and cell culture. Notably, the AT test was shown to detect mixed infections in clinical samples. The calculated median sensitivity of 0.81 over the entire panel of clinical samples was comparable to conventional 16S PCR, but slightly lower than real-time PCR and other PCR assays. However, when a panel of long-time stored swab samples was excluded from the calculation, the sensitivity was clearly higher (0.87) and equivalent to that of real-time PCR. Altogether, the data demonstrate the suitability of this DNA microarray assay for routine diagnosis.

  15. Evaluation of the neutral comet assay for detection of alpha-particle induced DNA-double-strand-breaks; Evaluation des Comet Assays bei neutralem pH zur Detektion von α-Partikel induzierten DNA-Doppelstrangbruechen

    Energy Technology Data Exchange (ETDEWEB)

    Hofbauer, Daniela

    2010-10-20

    Aim of this study was to differentiate DNA-double-strand-breaks from DNA-single-strand-breaks on a single cell level, using the comet assay after α- and γ-irradiation. Americium-241 was used as a alpha-irradiation-source, Caesium-137 was used for γ-irradiation. Because of technical problems with both the neutral and alkaline comet assay after irradiation of gastric cancer cells and human lymphocytes, no definite differentiation of DNA-damage was possible.

  16. Detection of DNA damage induced by heavy ion irradiation in the individual cells with comet assay

    Science.gov (United States)

    Wada, S.; Natsuhori, M.; Ito, N.; Funayama, T.; Kobayashi, Y.

    2003-05-01

    Investigating the biological effects of high-LET heavy ion irradiation at low fluence is important to evaluate the risk of charged particles. Especially it is important to detect radiation damage induced by the precise number of heavy ions in the individual cells. Thus we studied the relationship between the number of ions traversing the cell and DNA damage produced by the ion irradiation. We applied comet assay to measure the DNA damage in the individual cells. Cells attached on the ion track detector CR-39 were irradiated with ion beams at TIARA, JAERI-Takasaki. After irradiation, the cells were stained with ethidium bromide and the opposite side of the CR-39 was etched. We observed that the heavy ions with higher LET values induced the heavier DNA damage. The result indicated that the amount of DNA damage induced by one particle increased with the LET values of the heavy ions.

  17. Highly sensitive detection of human IgG using a novel bio-barcode assay combined with DNA chip technology

    Science.gov (United States)

    Liu, Zhenbao; Zhou, Bo; Wang, Haiqing; Lu, Feng; Liu, Tianjun; Song, Cunxian; Leng, Xigang

    2013-09-01

    A simple and ultrasensitive detection of human IgG based on signal amplification using a novel bio-barcode assay and DNA chip technology was developed. The sensing platform was a sandwich system made up of antibody-modified magnetic microparticles (Ab-MMPs)/human IgG/Cy3-labeled single-stranded DNA and antibody-modified gold nanoparticles (Cy3-ssDNA-Ab-AuNPs). The MMPs (2.5 μm in diameter) modified with mouse anti-human IgG monoclonal-antibodies could capture human IgG and further be separated and enriched via a magnetic field. The AuNPs (13 nm in diameter) conjugated with goat anti-human IgG polyclonal-antibodies and Cy3-ssDNA could further combine with the human IgG/Ab-MMP complex. The Cy3-ssDNA on AuNPs was then released by TCEP to hybridize with the DNA chip, thus generating a detectable signal by the fluorescence intensity of Cy3. In order to improve detection sensitivity, a three-level cascaded signal amplification was developed: (1) The MMP enrichment as the first-level; (2) Large quantities of Cy3-ssDNA on AuNPs as the second-level; (3) The Cy3-ssDNA conjugate with DNA chip as the third-level. The highly sensitive technique showed an increased response of the fluorescence intensity to the increased concentration of human IgG through a detection range from 1 pg mL-1 to 10 ng mL-1. This sensing technique could not only improve the detection sensitivity for the low concentration of human IgG but also present a robust and efficient signal amplification model. The detection method has good stability, specificity, and reproducibility and could be applied in the detection of human IgG in the real samples.

  18. Highly sensitive detection of human IgG using a novel bio-barcode assay combined with DNA chip technology

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Zhenbao [Central South University, School of Pharmaceutical Sciences (China); Zhou, Bo, E-mail: zhoubo1771@163.com [The Affiliated Zhongda Hospital of Southeast University, Department of Gerontology (China); Wang, Haiqing; Lu, Feng; Liu, Tianjun; Song, Cunxian; Leng, Xigang, E-mail: lengxigyky@163.com [Institute of Biomedical Engineering, Chinese Academy of Medical Sciences and Peking Union Medical College (China)

    2013-09-15

    A simple and ultrasensitive detection of human IgG based on signal amplification using a novel bio-barcode assay and DNA chip technology was developed. The sensing platform was a sandwich system made up of antibody-modified magnetic microparticles (Ab-MMPs)/human IgG/Cy3-labeled single-stranded DNA and antibody-modified gold nanoparticles (Cy3-ssDNA-Ab-AuNPs). The MMPs (2.5 {mu}m in diameter) modified with mouse anti-human IgG monoclonal-antibodies could capture human IgG and further be separated and enriched via a magnetic field. The AuNPs (13 nm in diameter) conjugated with goat anti-human IgG polyclonal-antibodies and Cy3-ssDNA could further combine with the human IgG/Ab-MMP complex. The Cy3-ssDNA on AuNPs was then released by TCEP to hybridize with the DNA chip, thus generating a detectable signal by the fluorescence intensity of Cy3. In order to improve detection sensitivity, a three-level cascaded signal amplification was developed: (1) The MMP enrichment as the first-level; (2) Large quantities of Cy3-ssDNA on AuNPs as the second-level; (3) The Cy3-ssDNA conjugate with DNA chip as the third-level. The highly sensitive technique showed an increased response of the fluorescence intensity to the increased concentration of human IgG through a detection range from 1 pg mL{sup -1} to 10 ng mL{sup -1}. This sensing technique could not only improve the detection sensitivity for the low concentration of human IgG but also present a robust and efficient signal amplification model. The detection method has good stability, specificity, and reproducibility and could be applied in the detection of human IgG in the real samples.

  19. Fast and automated DNA assays on a compact disc (CD)-based microfluidic platform

    Science.gov (United States)

    Jia, Guangyao

    Nucleic acid-based molecular diagnostics offers enormous potential for the rapid and accurate diagnosis of infectious diseases. However, most of the existing commercial tests are time-consuming and technically complicated, and are thus incompatible with the need for rapid identification of infectious agents. We have successfully developed a CD-based microfluidic platform for fast and automated DNA array hybridization and a low cost, disposable plastic microfluidic platform for polymerase chain reaction (PCR). These platforms have proved to be a promising approach to meet the requirements in terms of detection speed and operational convenience in diagnosis of infectious diseases. In the CD-based microfluidic platform for DNA hybridization, convection is introduced to the system to enhance mass transport so as to accelerate the hybridization rate since DNA hybridization is a diffusion limited reaction. Centrifugal force is utilized for sample propulsion and surface force is used for liquid gating. Standard microscope glass slides are used as the substrates for capture probes owing to their compatibility with commercially available instrumentation (e.g. laser scanners) for detection. Microfabricated polydimethylsiloxane (PDMS) structures are used to accomplish the fluidic functions required by the protocols for DNA hybridization. The assembly of the PDMS structure and the glass slide forms a flow-through hybridization unit that can be accommodated onto the CD platform for reagent manipulation. The above scheme has been validated with oligonucleotides as the targets using commercially available enzyme-labeled fluorescence (ELF 97) for detection of the hybridization events, and tested with amplicons of genomic staphylococcus DNA labeled with Cy dye. In both experiments, significantly higher fluorescence intensities were observed in the flow-through hybridization unit compared to the passive assays. The CD fluidic scheme was also adapted to the immobilization of

  20. Observation of DNA damage of human hepatoma cells irradiated by heavy ions using comet assay

    Institute of Scientific and Technical Information of China (English)

    Li-Mei Qiu; Wen-Jian Li; Xin-Yue Pang; Qing-Xiang Gao; Yan Feng; Li-Bin Zhou; Gao-Hua Zhang

    2003-01-01

    AIM: Now many countries have developed cancer therapy with heavy ions, especially in GSI (Gesellschaft fur Schwerionenforschung mbH, Darmstadt, Germany),remarkable results have obtained, but due to the complexity of particle track structure, the basic theory still needs further researching. In this paper, the genotoxic effects of heavy ions irradiation on SMMC-7721 cells were measured using the single cell gel electrophoresis (comet assay). The information about the DNA damage made by other radiations such as X-ray, γ-ray, UV and fast neutron irradiation is very plentiful, while little work have been done on the heavy ions so far. Hereby we tried to detect the reaction of liver cancer cells to heavy ion using comet assay, meanwhile to establish a database for clinic therapy of cancer with the heavy ions.METHODS: The human hepatoma cells were chosen as the test cell line irradiated by 80Mev/u 20Ne10+ on HIRFL (China), the radiation-doses were 0, 0.5, 1, 2, 4 and 8 Gy,and then comet assay was used immediately to detect the DNA damages, 100-150 cells per dose-sample (30-50 cells were randomly observed at constant depth of the gel). The tail length and the quantity of the cells with the tail were put down. EXCEL was used for statistical analysis.RESULTS: We obtained clear images by comet assay and found that SMMC-7721 cells were all damaged apparently from the dose 0.5Gy to 8Gy (t-test: P<0.001, vs control).The tail length and tail moment increased as the doses increased, and the number of cells with tails increased with increasing doses. When doses were higher than 2Gy, nearly 100 % cells were damaged. Furthermore, both tail length and tail moment, showed linear equation.CONCLUSION: From the clear comet assay images, our experiment proves comet assay can be used to measure DNA damages by heavy ions. Meanwhile DNA damages have a positive correlation with the dose changes of heavy ions and SMMC-7721 cells have a great radiosensitivity to 20Ne10+.Different reactions

  1. Demonstration of DSI-semen--A novel DNA methylation-based forensic semen identification assay.

    Science.gov (United States)

    Wasserstrom, Adam; Frumkin, Dan; Davidson, Ariane; Shpitzen, Moshe; Herman, Yael; Gafny, Ron

    2013-01-01

    Determining whether the source tissue of biological material is semen is important in confirming sexual assaults, which account for a considerable percentage of crime cases. The gold standard for confirming the presence of semen is microscopic identification of sperm cells, however, this method is labor intensive and operator-dependent. Protein-based immunologic assays, such as PSA, are highly sensitive and relatively fast, but suffer from low specificity in some situations. In addition, proteins are less stable than DNA under most environmental insults. Recently, forensic tissue identification advanced with the development of several approaches based on mRNA and miRNA for identification of various body fluids. Herein is described DNA source identifier (DSI)-semen, a DNA-based assay that determines whether the source tissue of a sample is semen based on detection of semen-specific methylation patterns in five genomic loci. The assay is comprised of a simple single tube biochemical procedure, similar to DNA profiling, followed by automatic software analysis, yielding the identification (semen/non-semen) accompanied by a statistical confidence level. Three additional internal control loci are used to ascertain the reliability of the results. The assay, which aims to replace microscopic examination, can easily be integrated by forensic laboratories and is automatable. The kit was tested on 135 samples of semen, saliva, venous blood, menstrual blood, urine, and vaginal swabs and the identification of semen vs. non-semen was correct in all cases. In order to test the assay's applicability in "real-life" situations, 33 actual casework samples from the forensic biological lab of the Israeli police were analyzed, and the results were compared with microscopic examination performed by Israeli police personnel. There was complete concordance between both analyses except for one sample, in which the assay identified semen whereas no sperm was seen in the microscope. This

  2. Assessment of DNA integrity (COMET assay) in sperm cells of boron-exposed workers.

    Science.gov (United States)

    Duydu, Yalçin; Başaran, Nurşen; Ustündağ, Aylin; Aydin, Sevtap; Undeğer, Ulkü; Ataman, Osman Yavuz; Aydos, Kaan; Düker, Yalçin; Ickstadt, Katja; Waltrup, Britta Schulze; Golka, Klaus; Bolt, Hermann M

    2012-01-01

    An extension of a male reproductive study conducted in a boric acid/borate production zone at Bandırma, Turkey, is presented. The relation between DNA-strand breaks (COMET assay, neutral and alkaline version) in sperm cells and previously described sperm quality parameters was investigated in boron-exposed males. A correlation between blood boron levels and mean DNA-strand breaks in sperm was weak, and DNA-strand breaks in sperm were statistically not different between control and exposed groups. Therefore, increasing boron exposures had no additional contribution in addition to already pre-existing DNA-strand breaks in the sperm cells. Weak but statistically significant correlations between DNA-strand breaks and motility/morphology parameters of sperm samples were observed in the neutral version of the COMET assay, while correlations between the same variables were statistically not significant in the alkaline version. A likely reason for these negative results, even in highly exposed humans, is that experimental exposures that had led to reproductive toxicity in animals were significantly higher than any boron exposures, which may be reached under realistic human conditions.

  3. Additional survey on genotoxicity of natural anthraquinones in the hepatocyte primary culture/DNA repair assay.

    Science.gov (United States)

    Mori, H; Yoshimi, N; Iwata, H; Tanaka, T; Kawai, K; Sankawa, U

    1988-08-01

    Genotoxicity of fungal anthraquinones of islandicin, iridoskyrin and (-) rubroskyrin, and a colorant of insect origin, cochineal and its component, carminic acid, an anthraquinone, was examined in the hepatocyte primary culture/DNA repair test. The results were compared with that of versicolorin A, an anthraquinone with bisfuran ring, which had been proved to be genotoxic on this assay. All of these anthraquinones, differently from versicolorin A did not show clear response of DNA repair. The results suggest that these agents are not genotoxic carcinogens.

  4. The Comet-FISH assay for the analysis of DNA damage and repair.

    Science.gov (United States)

    Spivak, Graciela

    2010-01-01

    In this chapter, I describe the alkaline single-cell gel electrophoresis (Comet assay) combined with fluorescence in situ hybridization (FISH) technology, used in our laboratory, to study the incidence and repair of lesions induced in human cells by ultraviolet light. The Comet-FISH method permits the simultaneous and comparative analysis of DNA damage and its repair throughout the genome and in defined chromosomal regions. This very sensitive approach can be applied to any lesion, such as those induced by chemical carcinogens and products of cellular metabolism that can be converted to DNA single- or double-strand breaks. The unique advantages and limitations of the method for particular applications are discussed.

  5. Direct LAMP Assay without Prior DNA Purification for Sex Determination of Papaya

    Directory of Open Access Journals (Sweden)

    Chi-Chu Tsai

    2016-09-01

    Full Text Available Papaya (Carica papaya L. is an economically important tropical fruit tree with hermaphrodite, male and female sex types. Hermaphroditic plants are the major type used for papaya production because their fruits have more commercial advantages than those of female plants. Sex determination of the seedlings, or during the early growth stages, is very important for the papaya seedling industry. Thus far, the only method for determining the sex type of a papaya at the seedling stage has been DNA analysis. In this study, a molecular technique—based on DNA analysis—was developed for detecting male-hermaphrodite-specific markers to examine the papaya’s sex type. This method is based on the loop-mediated isothermal amplification (LAMP and does not require prior DNA purification. The results show that the method is an easy, efficient, and inexpensive way to determine a papaya’s sex. This is the first report on the LAMP assay, using intact plant materials-without DNA purification-as samples for the analysis of sex determination of papaya. We found that using high-efficiency DNA polymerase was essential for successful DNA amplification, using trace intact plant material as a template DNA source.

  6. Direct LAMP Assay without Prior DNA Purification for Sex Determination of Papaya.

    Science.gov (United States)

    Tsai, Chi-Chu; Shih, Huei-Chuan; Ko, Ya-Zhu; Wang, Ren-Huang; Li, Shu-Ju; Chiang, Yu-Chung

    2016-09-24

    Papaya (Carica papaya L.) is an economically important tropical fruit tree with hermaphrodite, male and female sex types. Hermaphroditic plants are the major type used for papaya production because their fruits have more commercial advantages than those of female plants. Sex determination of the seedlings, or during the early growth stages, is very important for the papaya seedling industry. Thus far, the only method for determining the sex type of a papaya at the seedling stage has been DNA analysis. In this study, a molecular technique-based on DNA analysis-was developed for detecting male-hermaphrodite-specific markers to examine the papaya's sex type. This method is based on the loop-mediated isothermal amplification (LAMP) and does not require prior DNA purification. The results show that the method is an easy, efficient, and inexpensive way to determine a papaya's sex. This is the first report on the LAMP assay, using intact plant materials-without DNA purification-as samples for the analysis of sex determination of papaya. We found that using high-efficiency DNA polymerase was essential for successful DNA amplification, using trace intact plant material as a template DNA source.

  7. The next-generation Hybrid Capture High-Risk HPV DNA assay on a fully automated platform.

    Science.gov (United States)

    Eder, Paul S; Lou, Jianrong; Huff, John; Macioszek, Jerzy

    2009-07-01

    A next-generation diagnostic system has been developed at QIAGEN. The QIAensemble system consists of an analytical subsystem (JE2000) that utilizes a re-engineered Hybrid Capture chemistry (NextGen) to maintain the high level of clinical sensitivity established by the digene High-Risk HPV DNA Test (HC2), while creating improved analytical specificity as shown both in plasmid-based analyses and in processing of clinical specimens. Limit-of-detection and cross-reactivity experiments were performed using plasmid DNA constructs containing multiple high-risk (HR) and low-risk (LR) HPV types. Cervical specimens collected into a novel specimen collection medium, DCM, were used to measure stability of specimens, as well as analytical specificity. Signal carryover, instrument precision, and specimen reproducibility were measured on the prototype JE2000 system using the automated NextGen assay. The Limit of Detection (LOD) is HPV 16 plasmid in the automated assay. No cross-reactivity (signal above cutoff) was detected on the automated system from any of 13 LR types tested at 10(7) copies per assay. Within-plate, plate-to-plate, and day-to-day performance in the prototype system yielded a CV of 20%. No indication of target carryover was found when samples containing up to 10(9) copies/ml of HPV DNA type 16 were processed on the JE2000 instrument. In an agreement study with HC2, 1038 donor cervical specimens were tested in both the manual NextGen assay and HC2 to evaluate agreement between the two tests. After eliminating discrepant specimens that were adjudicated by HR-HPV genotyping, the adjudicated positive agreement was 98.5% (95% CI: 94.6, 99.6). The JE2000 prototype system automates NextGen assay processing, yielding accurate, reproducible, and highly specific results with both plasmid analytical model tests and cervical specimens collected in DCM. The final system will process more than 2000 specimens in an 8-hour shift, with fully continuous loading.

  8. Development of an Easy and High-Throughput Cell Assay System with a Culture Chip and an Assay Chip

    Science.gov (United States)

    Sugiura, Kanako; Kaji, Noritada; Okamoto, Yukihiro; Tokeshi, Manabu; Baba, Yoshinobu

    High throughput cell assay is significantly important in drug screening, assessment of toxicity etc. Cell assay with a microchip is one of the candidates for high throughput cell assay. However, reported cell assay system with the microchip requires expensive apparatus for refluxing medium and investigation of optimum experimental condition for steady data. For an inexpensive, easy and high throughput cell assay, we introduce a new cell assay system combined with a culture chip and an assay chip made of poly(dimethyl siloxane). Cell culture chips enabled cell to proliferate along the microchannel without refluxing medium and permitted to prepare cell patterning easily. Also, assay chips formed concentration gradient inside the chip and allowed the cell assay with different concentrations of drug at the same time. Thus, our developed cell assay system can overcome the problems of the present cell assay and would promote the drug discovery, assessment of toxicity etc.

  9. Optimal dose selection of N-methyl-N-nitrosourea for the rat comet assay to evaluate DNA damage in organs with different susceptibility to cytotoxicity.

    Science.gov (United States)

    Kitamoto, Sachiko; Matsuyama, Ryoko; Uematsu, Yasuaki; Ogata, Keiko; Ota, Mika; Yamada, Toru; Miyata, Kaori; Funabashi, Hitoshi; Saito, Koichi

    2015-07-01

    The in vivo rodent alkaline comet assay (comet assay) is a promising technique to evaluate DNA damage in vivo. However, there is no agreement on a method to evaluate DNA damage in organs where cytotoxicity is observed. As a part of the Japanese Center for the Validation of Alternative Methods (JaCVAM)-initiative international validation study of the comet assay, we examined DNA damage in the liver, stomach, and bone marrow of rats given three oral doses of N-methyl-N-nitrosourea (MNU) up to the maximum tolerated dose based on systemic toxicity. MNU significantly increased the % tail DNA in all the organs. Histopathological analysis showed no cytotoxic effect on the liver, indicating clearly that MNU has a genotoxic potential in the liver. In the stomach, however, the cytotoxic effects were very severe at systemically non-toxic doses. Low-dose MNU significantly increased the % tail DNA even at a non-cytotoxic dose, indicating that MNU has a genotoxic potential also in the stomach. Part of the DNA damage at cytotoxic doses was considered to be a secondary effect of severe cell damage. In the bone marrow, both the % tail DNA and incidence of micronucleated polychromatic erythrocytes significantly increased at non-hematotoxic doses, which were different from the non-cytotoxic doses for liver and stomach. These findings indicate that an optimal dose for detecting DNA damage may vary among organs and that careful attention is required to select an optimum dose for the comet assay based on systemic toxicity such as mortality and clinical observations. The present study shows that when serious cytotoxicity is suggested by increased % hedgehogs in the comet assay, histopathological examination should be included for the evaluation of a positive response.

  10. Development of two highly sensitive forensic sex determination assays based on human DYZ1 and Alu repetitive DNA elements.

    Science.gov (United States)

    Fazi, Amanda; Gobeski, Brianne; Foran, David

    2014-11-01

    Sex determination is a critical component of forensic identification, the standard genetic method for which is detection of the single copy amelogenin gene that has differing homologues on the X and Y chromosomes. However, this assay may not be sensitive enough when DNA samples are minute or highly compromised, thus other strategies for sex determination are needed. In the current research, two ultrasensitive sexing assays, based on real-time PCR and pyrosequencing, were developed targeting the highly repetitive elements DYZ1 on the Y chromosome and Alu on the autosomes. The DYZ1/Alu strategy was compared to amelogenin for overall sensitivity based on high molecular weight and degraded DNA, followed by assaying the sex of 34 touch DNA samples and DNA from 30 hair shafts. The real-time DYZ1/Alu assay proved to be approximately 1500 times more sensitive than its amelogenin counterpart based on high molecular weight DNA, and even more sensitive when sexing degraded DNA. The pyrosequencing DYZ1/Alu assay correctly sexed 26 of the touch DNAs, compared to six using amelogenin. Hair shaft DNAs showed equally improved sexing results using the DYZ1/Alu assays. Overall, both DYZ1/Alu assays were far more sensitive and accurate than was the amelogenin assay, and thus show great utility for sexing poor quality and low quantity DNA evidence.

  11. Quantification of applied dose in irradiated citrus fruits by DNA Comet Assay together with image analysis.

    Science.gov (United States)

    Cetinkaya, Nurcan; Ercin, Demet; Özvatan, Sümer; Erel, Yakup

    2016-02-01

    The experiments were conducted for quantification of applied dose for quarantine control in irradiated citrus fruits. Citrus fruits exposed to doses of 0.1 to 1.5 kGy and analyzed by DNA Comet Assay. Observed comets were evaluated by image analysis. The tail length, tail moment and tail DNA% of comets were used for the interpretation of comets. Irradiated citrus fruits showed the separated tails from the head of the comet by increasing applied doses from 0.1 to 1.5 kGy. The mean tail length and mean tail moment% levels of irradiated citrus fruits at all doses are significantly different (p Comet Assay may be a practical quarantine control method for irradiated citrus fruits since it has been possible to estimate the applied low doses as small as 0.1 kGy when it is combined with image analysis.

  12. Reproducibility of Digital PCR Assays for Circulating Tumor DNA Analysis in Advanced Breast Cancer

    Science.gov (United States)

    Hrebien, Sarah; O’Leary, Ben; Beaney, Matthew; Schiavon, Gaia; Fribbens, Charlotte; Bhambra, Amarjit; Johnson, Richard; Turner, Nicholas

    2016-01-01

    Circulating tumor DNA (ctDNA) analysis has the potential to allow non-invasive analysis of tumor mutations in advanced cancer. In this study we assessed the reproducibility of digital PCR (dPCR) assays of circulating tumor DNA in a cohort of patients with advanced breast cancer and assessed delayed plasma processing using cell free DNA preservative tubes. We recruited a cohort of 96 paired samples from 71 women with advanced breast cancer who had paired blood samples processed either immediately or delayed in preservative tubes with processing 48–72 hours after collection. Plasma DNA was analysed with multiplex digital PCR (mdPCR) assays for hotspot mutations in PIK3CA, ESR1 and ERBB2, and for AKT1 E17K. There was 94.8% (91/96) agreement in mutation calling between immediate and delayed processed tubes, kappa 0.88 95% CI 0.77–0.98). Discordance in mutation calling resulted from low allele frequency and likely stochastic effects. In concordant samples there was high correlation in mutant copies per ml plasma (r2 = 0.98; pprocessed tubes, although overall quantification of total cell free plasma DNA had similar prognostic effects in immediate (HR 3.6) and delayed (HR 3.0) tubes. There was moderate agreement in changes in allele fraction between sequential samples in quantitative mutation tracking (r = 0.84, p = 0.0002). Delayed processing of samples using preservative tubes allows for centralized ctDNA digital PCR mutation screening in advanced breast cancer. The potential of preservative tubes in quantitative mutation tracking requires further research. PMID:27760227

  13. Probing the functional impact of sequence variation on p53-DNA interactions using a novel microsphere assay for protein-DNA binding with human cell extracts.

    Directory of Open Access Journals (Sweden)

    Maher A Noureddine

    2009-05-01

    Full Text Available The p53 tumor suppressor regulates its target genes through sequence-specific binding to DNA response elements (REs. Although numerous p53 REs are established, the thousands more identified by bioinformatics are not easily subjected to comparative functional evaluation. To examine the relationship between RE sequence variation -- including polymorphisms -- and p53 binding, we have developed a multiplex format microsphere assay of protein-DNA binding (MAPD for p53 in nuclear extracts. Using MAPD we measured sequence-specific p53 binding of doxorubicin-activated or transiently expressed p53 to REs from established p53 target genes and p53 consensus REs. To assess the sensitivity and scalability of the assay, we tested 16 variants of the p21 target sequence and a 62-multiplex set of single nucleotide (nt variants of the p53 consensus sequence and found many changes in p53 binding that are not captured by current computational binding models. A group of eight single nucleotide polymorphisms (SNPs was examined and binding profiles closely matched transactivation capability tested in luciferase constructs. The in vitro binding characteristics of p53 in nuclear extracts recapitulated the cellular in vivo transactivation capabilities for eight well-established human REs measured by luciferase assay. Using a set of 26 bona fide REs, we observed distinct binding patterns characteristic of transiently expressed wild type and mutant p53s. This microsphere assay system utilizes biologically meaningful cell extracts in a multiplexed, quantitative, in vitro format that provides a powerful experimental tool for elucidating the functional impact of sequence polymorphism and protein variation on protein/DNA binding in transcriptional networks.

  14. DNA-labelled cytidine assay for the quantification of CAG repeats.

    Science.gov (United States)

    Pérez-Bello, Dannelys; Xu, Z H; Higginson-Clarke, David; Rojas, Ana María Riverón; Le, Weidong; Rodríguez-Tanty, Chryslaine

    2008-03-30

    The sequencing procedure has been used to determine the size of the CAG repeat expansion for the diagnosis of genetic disorders. Likewise, standard polymerase chain reaction (PCR) and gel electrophoresis techniques are applied for screening large number of patients. The trinucleotide repeats (TNR) region amplification by means of the PCR procedure was initially performed using 32-P end-labelled primers and currently carried out with fluorescently end-labelled primers. The goal to obtain reliable TNR quantification assays, at low cost and short assay times, represents a challenge for the molecular diagnosis aimed at massive screening of affected populations. In the current work, we obtained preliminary results of a new methodology for the detection and size estimation of CAG expanded alleles. The assay was based on an indirect enzyme linked immunosorbent assay (ELISA) for quantifying the amount of labelled cytidines in DNA molecules. The label, 6-(p-bromobenzamido)caproyl radical, was introduced by the transamination and acylation reactions. A group of model sequences containing different numbers of CAG repeats, as well as the ATXN3 (ataxin 3) gene (from subjects suffering type 3 spinocerebellar ataxia SCA3) were used for assay standardization. The assay is simple, inexpensive, and easy to perform and differentiates distinct degrees of CAG expansions.

  15. Evaluation of a fluorescent DNA hybridization assay for the detection of Neisseria gonorrhoeae.

    Science.gov (United States)

    Cano, R J; Palomares, J C; Torres, M J; Klem, R E

    1992-07-01

    This study evaluates a four-hour fluorescent DNA hybridization assay using both known bacterial isolates and clinical specimens. A biotinylated oligonucleotide probe from a sequence of the plasmid-encoded gene cppB was used. Hybrids were detected by addition of a streptavidin-alkaline phosphatase conjugate, followed by incubation for 30 min in a fluorescent substrate for alkaline phosphatase. The level of detection of the fluorescent assay was 0.1 pg of cryptic plasmid DNA or 200 cfu of the plasmid-containing strain NG 34/85 of Neisseria gonorrhoeae. A total of 119 reference strains of Neisseria gonorrhoeae and other related bacteria were tested for reactivity with the probe. All Neisseria gonorrhoeae strains, including eight plasmid-free strains, hybridized with the probe. Fluorescence ratios were 2.67 for plasmid-free strains and 3.85 for plasmid-containing strains. Of the heterologous microorganisms tested, only one of six strains of Neisseria cinerea gave a fluorescence ratio above the 2.0 cut-off value for positivity with the probe at a cell density of 1 x 10(4) cfu. The probe was also evaluated using clinical specimens from 100 patients attending a clinic for sexually transmitted diseases. The sensitivity of the assay was 100% while the specificity was 97.5%. Positive and negative predictive values were 91.2% and 100%, respectively. The fluorescent DNA hybridization assay for the detection of Neisseria gonorrhoeae described here thus appears to be a highly specific and sensitive assay.

  16. Significance of enzyme linked immunosorbent assay (ELISA) for antibodies to double stranded and single stranded DNA in patients with lupus nephritis: correlation with severity of renal histology.

    Science.gov (United States)

    Okamura, M; Kanayama, Y; Amastu, K; Negoro, N; Kohda, S; Takeda, T; Inoue, T

    1993-01-01

    The correlation between renal histology and class specific (IgG and IgM) antibodies to double stranded DNA (dsDNA) and single stranded DNA (ssDNA) was studied by enzyme linked immunosorbent assay (ELISA) in 40 untreated patients with systemic lupus erythematosus (SLE). The levels of IgG antibodies to dsDNA were significantly higher in patients with World Health Organisation class IV nephritis than in those with class I, class II, or class III nephritis. IgG antibodies to ssDNA were higher in patients with class IV than in those with class II nephritis. IgG antibodies to dsDNA showed a close correlation with the histological activity score and the amount of electron dense deposit. IgG antibodies to ssDNA showed only a weak correlation with the renal histological activity score. IgM antibodies to dsDNA and IgM antibodies to ssDNA were not correlated with renal histological features. Patients with moderate to severe nephritis had a lower ratio of IgM antibodies to dsDNA to IgG antibodies to dsDNA than those with mild nephritis. These results indicate that the measurement of IgG antibodies to dsDNA is predictive in evaluating renal histological activity in patients with SLE.

  17. Genotyping performance assessment of whole genome amplified DNA with respect to multiplexing level of assay and its period of storage.

    Directory of Open Access Journals (Sweden)

    Daniel W H Ho

    Full Text Available Whole genome amplification can faithfully amplify genomic DNA (gDNA with minimal bias and substantial genome coverage. Whole genome amplified DNA (wgaDNA has been tested to be workable for high-throughput genotyping arrays. However, issues about whether wgaDNA would decrease genotyping performance at increasing multiplexing levels and whether the storage period of wgaDNA would reduce genotyping performance have not been examined. Using the Sequenom MassARRAY iPLEX Gold assays, we investigated 174 single nucleotide polymorphisms for 3 groups of matched samples: group 1 of 20 gDNA samples, group 2 of 20 freshly prepared wgaDNA samples, and group 3 of 20 stored wgaDNA samples that had been kept frozen at -70°C for 18 months. MassARRAY is a medium-throughput genotyping platform with reaction chemistry different from those of high-throughput genotyping arrays. The results showed that genotyping performance (efficiency and accuracy of freshly prepared wgaDNA was similar to that of gDNA at various multiplexing levels (17-plex, 21-plex, 28-plex and 36-plex of the MassARRAY assays. However, compared with gDNA or freshly prepared wgaDNA, stored wgaDNA was found to give diminished genotyping performance (efficiency and accuracy due to potentially inferior quality. Consequently, no matter whether gDNA or wgaDNA was used, better genotyping efficiency would tend to have better genotyping accuracy.

  18. Capability of Vitamin E as a Radioprotector in Suppressing DNA Damage Determined with Comet Assay

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    Darlina Darlina

    2017-07-01

    Full Text Available Radiation has a potent to damage cells. Radiation may act directly or indirectly on deoxyribonucleic acid (DNA that results in the degeneration of tissues and necrotic, and thereby it needs a potent radioprotector to prevent these damages. Vitamin E is natural product known as an antioxidant which has potential as radioprotector. This research aimed to determine the capability of vitamin E with emphasized on the searching for its optimal concentration as radioprotector of DNA damage. This study used blood samples of healthy person irradiated with gamma rays at a dose of 6 Gy as the lethal dose to lymphocytes. The cocentrations of vitamin E from 0 to 0.8 mM was added into blood 15 minutes before irradiation. Isolation of lymphocytes was done using gradient centrifugation method. Evaluation on the capability of this compound in suppressing DNA damage was done by using alkaline Comet assay and data analysis was done using CaspLab program. The results show that addition of vitamin E could suppres these DNA damages and 0.8 mM of vitamin could reduce DNA damage up to 94.2%. We conclude that vitamin E effectively suppresed DNA damages induced by radiation. This information may benefit to the patient from negative impacts of radiotherapy.

  19. A bead-based multiplex assay for the detection of DNA viruses infecting laboratory rodents.

    Science.gov (United States)

    Höfler, Daniela; Nicklas, Werner; Mauter, Petra; Pawlita, Michael; Schmitt, Markus

    2014-01-01

    The Federation of European Laboratory Animal Science Association (FELASA) recommends screening of laboratory rodents and biological materials for a broad variety of bacterial agents, viruses, and parasites. Methods commonly used to date for pathogen detection are neither cost-effective nor time- and animal-efficient or uniform. However, an infection even if silent alters experimental results through changing the animals' physiology and increases inter-individual variability. As a consequence higher numbers of animals and experiments are needed for valid and significant results. We developed a novel high-throughput multiplex assay, called rodent DNA virus finder (rDVF) for the simultaneous identification of 24 DNA viruses infecting mice and rats. We detected all 24 DNA viruses with high specificity and reproducibility. Detection limits for the different DNA viruses varied between 10 and 1000 copies per PCR. The validation of rDVF was done with DNA isolated from homogenised organs amplified by pathogen specific primers in one multiplex PCR. The biotinylated amplicons were detected via hybridisation to specific oligonucleotide probes coupled to spectrally distinct sets of fluorescent Luminex beads. In conclusion, rDVF may have the potential to replace conventional testing and may simplify and improve routine detection of DNA viruses infecting rodents.

  20. A bead-based multiplex assay for the detection of DNA viruses infecting laboratory rodents.

    Directory of Open Access Journals (Sweden)

    Daniela Höfler

    Full Text Available The Federation of European Laboratory Animal Science Association (FELASA recommends screening of laboratory rodents and biological materials for a broad variety of bacterial agents, viruses, and parasites. Methods commonly used to date for pathogen detection are neither cost-effective nor time- and animal-efficient or uniform. However, an infection even if silent alters experimental results through changing the animals' physiology and increases inter-individual variability. As a consequence higher numbers of animals and experiments are needed for valid and significant results. We developed a novel high-throughput multiplex assay, called rodent DNA virus finder (rDVF for the simultaneous identification of 24 DNA viruses infecting mice and rats. We detected all 24 DNA viruses with high specificity and reproducibility. Detection limits for the different DNA viruses varied between 10 and 1000 copies per PCR. The validation of rDVF was done with DNA isolated from homogenised organs amplified by pathogen specific primers in one multiplex PCR. The biotinylated amplicons were detected via hybridisation to specific oligonucleotide probes coupled to spectrally distinct sets of fluorescent Luminex beads. In conclusion, rDVF may have the potential to replace conventional testing and may simplify and improve routine detection of DNA viruses infecting rodents.

  1. Detection of Nicotiana DNA in Tobacco Products Using a Novel Multiplex Real-Time PCR Assay.

    Science.gov (United States)

    Korchinski, Katie L; Land, Adrian D; Craft, David L; Brzezinski, Jennifer L

    2016-07-01

    Establishing that a product contains tobacco is a requirement for the U.S. Food and Drug Administration's regulation and/or prosecution of tobacco products. Therefore, a multiplex real-time PCR method was designed to determine if Nicotiana (tobacco) DNA is present in tobacco products. The PCR method simultaneously amplifies a 73 bp fragment of the cytochrome P450 monoxygenase CYP82E4 gene and 66 bp fragment in the nia-1 gene for nitrate reductase, which are detected using dual-labeled TaqMan probes. The assay is capable of detecting approximately 7.8 pg purified tobacco DNA, with a similar sensitivity for either gene target while incorporating an internal positive control (IPC). DNA was extracted from prepared tobacco products-including chewing tobacco, pipe tobacco, and snuff-or from the cut fill (no wrapper) of cigarettes and cigars. Of the 13 products analyzed, 12 were positive for both tobacco-specific markers and the IPC. DNA was also extracted from the fill of five varieties of herbal cigarettes, which were negative for both tobacco-specific gene targets and positive for the IPC. Our method expands on current assays by introducing a multiplex reaction, targeting two sequences in two different genes of interest, incorporating an IPC into the reaction, and lowering the LOD and LOQ while increasing the efficiency of the PCR.

  2. Analysis of DNA damage using the comet assay in infants fed cow's milk.

    Science.gov (United States)

    Dündaröz, Ruşen; Ulucan, Hakan; Aydin, Halil Ibrahim; Güngör, Tayfun; Baltaci, Volkan; Denli, Metin; Sanisoğlu, Yavuz

    2003-01-01

    It has been hypothesized that non-human milk feeding may increase the risk for cancer or for a specific cancer or group of cancers as well as the risk for diseases such as type-1 diabetes mellitus and Crohn's disease. Regarding DNA damage leading to cancer development in the absence of human milk protection, a comparison between infants fed human milk and cow's milk has been performed. Each group consisted of 35 infants, whose ages ranged from 9 to 12 months. The level of DNA damage in the peripheral blood lymphocytes of infants has been studied by the comet assay. A significant increase has been found in the number of limited DNA-damaged (p cow's milk. To our knowledge, this is the first study using the comet assay on infants not breast-fed. Supporting our previous SCE study, these results suggest that there is some level of DNA damage in the lymphocytes of infants not breast-fed and this may lead to malignancy in childhood or later in life.

  3. Human semen cryopreservation: a sperm DNA fragmentation study with alkaline and neutral Comet assay.

    Science.gov (United States)

    Ribas-Maynou, J; Fernández-Encinas, A; García-Peiró, A; Prada, E; Abad, C; Amengual, M J; Navarro, J; Benet, J

    2014-01-01

    Sperm cryopreservation is widely used for both research and reproduction purposes, but its effect on sperm DNA damage remains controversial. Sperm DNA fragmentation (SDF) has become an important biomarker to assess male infertility. In particular, the differentiation between single- and double-stranded DNA fragmentation (ssSDF and dsSDF) has clinical implications for male infertility where ssSDF is associated with reduced fertility, whereas dsSDF is associated with increased risk of miscarriage. In this study, semen samples from 30 human males have been analysed in both fresh and cryopreserved using the alkaline and neutral Comet assays. Results show an increase of about 10% of ssSDF, assessed by the alkaline Comet assay, regardless of the male fertility status. Neutral Comet analysis of dsSDF does not show any statistical increase when comparing fresh and cryopreserved samples in any of the patient groups. Results support previous reports that oxidative stress is the major effector in DNA damage during sample cryopreservation, as, on one hand, ssSDF has previously been related to oxidative damage and, on the other hand, we have not found any effect on dsSDF. Therefore, there might be a slight risk of decreased fertility after using a freezed sample, but no evidence for increased miscarriage risk from cryopreserved spermatozoa should be expected. © 2013 American Society of Andrology and European Academy of Andrology.

  4. Green synthesis of gold nanoparticles for staining human cervical cancer cells and DNA binding assay.

    Science.gov (United States)

    De, Swati; Kundu, Rikta; Ghorai, Atanu; Mandal, Ranju Prasad; Ghosh, Utpal

    2014-11-01

    Gold nanoparticles have been functionalized by non-ionic surfactants (polysorbates) used in pharmaceutical formulations. This results in the formation of more well-dispersed gold nanoparticles (GNPs) than the GNPs formed in neat water. The synthesized GNPs show good temporal stability. The synthesis conditions are mild and environmentally benign. The GNPs can bind to ct-DNA and displace bound dye molecules. The DNA-binding assay is significant as it preliminarily indicated that DNA-GNP conjugates can be formed. Such conjugates are extremely promising for applications in nanobiotechnology. The GNPs can also stain the human cervical cancer (HeLa) cells over a wide concentration range while remaining non-cytotoxic, thus providing a non invasive cell staining method. This result is very promising as we observe staining of HeLa cells at very low GNP concentrations (1 μM) while the cell viability is retained even at 10-fold higher GNP concentrations.

  5. Comparison of DNA Fragmentation Assay in Frozen-Thawed Cat Epididymal Sperm.

    Science.gov (United States)

    Kunkitti, P; Sjödahl, A; Bergqvist, A-S; Johannisson, A; Axnér, E

    2016-08-01

    DNA fragmentation of frozen-thawed feline epididymal sperm from corpus and cauda regions was evaluated by three different techniques. The DNA fragmentation index (DFI) was compared between techniques: the sperm chromatin structural assay (SCSA(®) ), acridine orange staining techniques (AOT) and the sperm chromatin dispersion (SCD). There were significant differences in DFI among the techniques (p < 0.05) with no correlations. Only DFI values obtained from SCD revealed a significantly higher DFI in corpus compared with cauda spermatozoa (p < 0.05). The discrepancy between techniques might be due to the sensitivity of each technique, differences in severity of DNA damaged that can be detected. The difference in DFI between epididymal regions from SCD technique might indicate different maturational stages of spermatozoa, with less chromatin condensation of spermatozoa in corpus compared with cauda epididymis. © 2016 Blackwell Verlag GmbH.

  6. DNA damage in grasshoppers' larvae--comet assay in environmental approach.

    Science.gov (United States)

    Augustyniak, Maria; Orzechowska, Helena; Kędziorski, Andrzej; Sawczyn, Tomasz; Doleżych, Bogdan

    2014-02-01

    The comet assay that provides a quantitative measure of the DNA-strand breaks may be used for assessing the 'genotoxic potential' of the environment. Young adults of Chorthippus brunneus (Orthoptera), collected at three sites in Southern Poland, differing in the level of pollution, particularly with heavy metals: Pilica (reference), Olkusz (moderately polluted) and Szopienice (heavily polluted) - were allowed to mate under laboratory conditions that were free from any pollution. Egg-pods were collected and, after diapause, brain cells from one-day old larvae were used for the comet assay. We compared the level of DNA damage in the larvae originating from these sites and also measured time-dependent DNA repair after single 10min. application of H2O2 (20μM final concentration). The DNA damage was relatively low in larval cells irrespectively of the site pollution their parents came from. However, measured comet parameters - tail DNA content (TDNA), tail length (TL), and olive tail moment (OTM) - were significantly higher in larvae originating from the Szopienice site than in those from the reference site. Incubation of cells with H2O2 resulted in significantly higher values of the comet parameters in the insects from all the study sites with the highest ones observed in the offspring of grasshoppers from Szopienice. Moreover, DNA repair, following the treatment, did not occur in the latter group. These data contribute to almost unexplored subject of genotoxic effects of environmental pollutants in insects. They are discussed in the light of the concept of adaptive strategies in energy allocation depending on the level of biotope pollution.

  7. Double-stranded DNA breaks hidden in the neutral Comet assay suggest a role of the sperm nuclear matrix in DNA integrity maintenance.

    Science.gov (United States)

    Ribas-Maynou, J; Gawecka, J E; Benet, J; Ward, W S

    2014-04-01

    We used a mouse model in which sperm DNA damage was induced to understand the relationship of double-stranded DNA (dsDNA) breaks to sperm chromatin structure and to the Comet assay. Sperm chromatin fragmentation (SCF) produces dsDNA breaks located on the matrix attachment regions, between protamine toroids. In this model, epididymal sperm induced to undergo SCF can religate dsDNA breaks while vas deferens sperm cannot. Here, we demonstrated that the conventional neutral Comet assay underestimates the epididymal SCF breaks because the broken DNA ends remain attached to the nuclear matrix, causing the DNA to remain associated with the dispersion halo, and the Comet tails to be weak. Therefore, we term these hidden dsDNA breaks. When the Comet assay was modified to include an additional incubation with sodium dodecyl sulfate (SDS) and dithiothreitol (DTT) after the conventional lysis, thereby solubilizing the nuclear matrix, the broken DNA was released from the matrix, which resulted in a reduction of the sperm head halo and an increase in the Comet tail length, exposing the hidden dsDNA breaks. Conversely, SCF-induced vas deferens sperm had small halos and long tails with the conventional neutral Comet assay, suggesting that the broken DNA ends were not tethered to the nuclear matrix. These results suggest that the attachment to the nuclear matrix is crucial for the religation of SCF-induced DNA breaks in sperm. Our data suggest that the neutral Comet assay identifies only dsDNA breaks that are released from the nuclear matrix and that the addition of an SDS treatment can reveal these hidden dsDNA breaks.

  8. Optimization and validation of DNA extraction and real-time PCR assay for the quantitative measurement of residual host cell DNA in biopharmaceutical products.

    Science.gov (United States)

    Hu, B; Sellers, J; Kupec, J; Ngo, W; Fenton, S; Yang, T-Y; Grebanier, A

    2014-01-01

    Host cell DNA contamination occurs during the production of biopharmaceuticals and must be controlled and monitored for the purity and safety of the drug products. A sodium iodide-based DNA extraction and a subsequent real time PCR assay were developed and validated for the quantitative measurement of residual host cell DNA impurity in monoclonal antibody therapeutic products. A sodium iodide-based commercial kit was optimized for the removal of interfering matrices. Several incubation steps from the kit protocol were combined and a neutralization buffer was introduced to protein digestion step to eliminate any precipitation from the detergent. The elimination of the two washing steps significantly reduced assay variability from loss of DNA pellets. The optimized DNA extraction procedure can recover DNA close to 100% for DNA concentrations from 10 to 100,000pg/mL. Of the published sequences of repetitive interspersed nuclear elements, we identified a nucleotide mismatch from the published CHO probe. Correction of this nucleotide increased DNA amplification by a thousand fold. The optimized assay was further validated for the quantitation of residual CHO DNA according to ICH guidelines with preset assay acceptance criteria. The method met all assay acceptance criteria and was found linear, accurate and precise for the quantitation of residual CHO in the linear range of 10-100,000pg DNA/mL. LOQ was measured at 10pg DNA/mL and LOD at 1pg DNA/mL. No matrix interference to our validated assay was detected from bioreactor harvest, Protein A eluate or eluate from ion exchange columns.

  9. Use of the Comet assay to investigate the role of superoxide in glutathione-induced DNA damage.

    Science.gov (United States)

    Thomas, S; Lowe, J E; Hadjivassiliou, V; Knowles, R G; Green, I C; Green, M H

    1998-02-04

    Although glutathione is an important scavenging molecule within the cell, it can also act as a pro-oxidant and at biological concentrations (1 mM) can induce DNA damage. We have used a sensitive cell-free Comet assay for DNA strand breakage to investigate this damage and to try to determine the active species involved. We show a substantial protection against glutathione-mediated DNA damage by superoxide dismutase (200 U/ml) and complete protection by combined superoxide dismutase and catalase. Damage is also prevented by EDTA but only at 100 mM and is not prevented by the chelating agent diethylenetriamine-pentaacetic acid (100 microM). Although superoxide is known to potentiate DNA damage by other reactive species, none of these indirect mechanisms seem to account for our results and it is possible that superoxide may damage DNA directly. Under the same experimental conditions, S-nitrosoglutathione requires ultraviolet A photolysis to cause DNA strand breakage and superoxide dismutase increases the level of this damage. When intact human lymphocytes are incubated with glutathione (1 mM) in phosphate buffer, DNA damage is also observed, but in this case it is completely preventable by catalase, with no protective effect of superoxide dismutase. Since cellular scavenging systems are not completely protective against reactive species formed from autooxidation of extracellular glutathione and since glutathione and oxygen are ubiquitously present within cells, our results imply that cells may have a mechanism of preventing autooxidation, rather than simply relying on scavenging the reactive species formula.

  10. Fluorescence-based electrophoretic mobility shift assay in the analysis of DNA-binding proteins.

    Science.gov (United States)

    Steiner, Sebastian; Pfannschmidt, Thomas

    2009-01-01

    Changes in gene expression mediated by DNA-binding protein factors are a crucial part of many signal transduction pathways. Generally, these regulatory proteins are low abundant and thus their purification and characterisation is labour- and time-intensive. Here we describe a workflow for purification, characterisation and identification of DNA-binding proteins. We show the use of a fluorescence-based electrophoretic mobility shift assay (fEMSA) and describe its advantages for a rapid and convenient screening for regulatory cis-elements. This involves a crude enrichment of nucleic acid binding proteins by heparin-Sepharose chromatography and the characterisation of fractions using overlapping fluorescence-labelled DNA probes spanning the promoter region of interest. The determined protein-binding sites can then be used for sequence-specific DNA-affinity chromatography to purify specifically interacting proteins. Finally, the DNA-binding complexes can be characterised and identified using two-dimensional EMSA, UV-cross-linking and mass spectrometry.

  11. Droplet centrifugation, droplet DNA extraction, and rapid droplet thermocycling for simpler and faster PCR assay using wire-guided manipulations

    Directory of Open Access Journals (Sweden)

    You David J

    2012-09-01

    Full Text Available Abstract A computer numerical control (CNC apparatus was used to perform droplet centrifugation, droplet DNA extraction, and rapid droplet thermocycling on a single superhydrophobic surface and a multi-chambered PCB heater. Droplets were manipulated using “wire-guided” method (a pipette tip was used in this study. This methodology can be easily adapted to existing commercial robotic pipetting system, while demonstrated added capabilities such as vibrational mixing, high-speed centrifuging of droplets, simple DNA extraction utilizing the hydrophobicity difference between the tip and the superhydrophobic surface, and rapid thermocycling with a moving droplet, all with wire-guided droplet manipulations on a superhydrophobic surface and a multi-chambered PCB heater (i.e., not on a 96-well plate. Serial dilutions were demonstrated for diluting sample matrix. Centrifuging was demonstrated by rotating a 10 μL droplet at 2300 round per minute, concentrating E. coli by more than 3-fold within 3 min. DNA extraction was demonstrated from E. coli sample utilizing the disposable pipette tip to cleverly attract the extracted DNA from the droplet residing on a superhydrophobic surface, which took less than 10 min. Following extraction, the 1500 bp sequence of Peptidase D from E. coli was amplified using rapid droplet thermocycling, which took 10 min for 30 cycles. The total assay time was 23 min, including droplet centrifugation, droplet DNA extraction and rapid droplet thermocycling. Evaporation from of 10 μL droplets was not significant during these procedures, since the longest time exposure to air and the vibrations was less than 5 min (during DNA extraction. The results of these sequentially executed processes were analyzed using gel electrophoresis. Thus, this work demonstrates the adaptability of the system to replace many common laboratory tasks on a single platform (through re-programmability, in rapid succession (using droplets

  12. Quantitative Fissile Assay In Used Fuel Using LSDS System

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    Lee YongDeok

    2017-01-01

    Full Text Available A quantitative assay of isotopic fissile materials (U235, Pu239, Pu241 was done at Korea Atomic Energy Research Institute (KAERI, using lead slowing down spectrometer (LSDS. The optimum design of LSDS was performed based on economics, easy maintenance and assay effectiveness. LSDS system consists of spectrometer, neutron source, detection and control. LSDS system induces fissile fission and fast neutrons are collected at fission chamber. The detected signal has a direct relation to the mass of existing fissile isotopes. Many current commercial assay technologies have a limitation in direct application on isotopic fissile assay of spent fuel, except chemical analysis. In the designed system, the fissile assay model was setup and the correction factor for self-shield was obtained. The isotopic fissile content assay was performed by changing the content of Pu239. Based on the fuel rod, the isotopic content was consistent with ~2% uncertainty for Pu239. By applying the covering (neutron absorber, the effective shielding was obtained and the activation was calculated on the target. From the assay evaluation, LSDS technique is very powerful and direct to analyze the isotopic fissile content. LSDS is applicable for nuclear fuel cycle and spent fuel management for safety and economics. Additionally, an accurate fissile content will contribute to the international transparency and credibility on spent fuel.

  13. Quantitative Fissile Assay In Used Fuel Using LSDS System

    Science.gov (United States)

    Lee, YongDeok; Jeon, Ju Young; Park, Chang-Je

    2017-09-01

    A quantitative assay of isotopic fissile materials (U235, Pu239, Pu241) was done at Korea Atomic Energy Research Institute (KAERI), using lead slowing down spectrometer (LSDS). The optimum design of LSDS was performed based on economics, easy maintenance and assay effectiveness. LSDS system consists of spectrometer, neutron source, detection and control. LSDS system induces fissile fission and fast neutrons are collected at fission chamber. The detected signal has a direct relation to the mass of existing fissile isotopes. Many current commercial assay technologies have a limitation in direct application on isotopic fissile assay of spent fuel, except chemical analysis. In the designed system, the fissile assay model was setup and the correction factor for self-shield was obtained. The isotopic fissile content assay was performed by changing the content of Pu239. Based on the fuel rod, the isotopic content was consistent with 2% uncertainty for Pu239. By applying the covering (neutron absorber), the effective shielding was obtained and the activation was calculated on the target. From the assay evaluation, LSDS technique is very powerful and direct to analyze the isotopic fissile content. LSDS is applicable for nuclear fuel cycle and spent fuel management for safety and economics. Additionally, an accurate fissile content will contribute to the international transparency and credibility on spent fuel.

  14. Single Cell Gel Electrophoresis Assay of Porcine Leydig Cell DNA Damage Induced by Zearalenone

    Institute of Scientific and Technical Information of China (English)

    Jianwei ZHEN; Qincl LIU; Jianhong GU; Yan YUAN; Xuezhong LIU; Handong WANG; Zongping LIU; Jianchun BIAN

    2012-01-01

    Abstract [Objective] This study aimed to investigate the effect of zearalenone (ZEN) on DNA damage of porcine leydig cells. [Method] Porcine leydig cells cultured in vitro were collected to determine the median lethal dose (LD~o) of ZEN with tetra- zolium-based colorimetric assay (MTT assay). Comet assay was carried out to de- tect the DNA damage of porcine leydig cells exposed to at 0 (negative group), 1, 5, 10, 20, 40 tJmol/L of ZEN. [Result] The percentage of cell tail was 16.67%, 34.00%, 40.67%. 52.00% and 64.67% under 0, 1, 5, 10 and 20 ~mo~/L o~ ZEN, respectively; the differences between the percentages of celt tail in various experimental groups had extremely significant statistical significance compared with the negative group (P〈0.01), showing a significant dose-effect relationship; Tail length in various groups was 57.60_+4.78, 57.75_+6.25, 78.97_+5.83, 100.50~6.94 and 146.83_+12.31 ~m, re- spectively; Tail DNA % in various groups was 21.29_+2.25%, 22.24_+2.43%, 31.21_+ 6.27%, 37.45_+4.33% and 60.68_+9.83%, respectively; Tail length and Tail DNA % in experimental groups with ZEN concentration above 5 ~mo~/L showed significant dif- ferences (P〈0.05) compared with the negative group, which showed an upward trend with the increase of ZEN concentration. [Conclusion] ZEN has genotoxic effect on porcine leydig cells, which can cause DNA damage, with a significant dose-effect relationship.

  15. Combining peptide and DNA for protein assay: CRIP1 detection for breast cancer staging.

    Science.gov (United States)

    Xie, Haona; Li, Hao; Huang, Yue; Wang, Xiaoying; Yin, Yongmei; Li, Genxi

    2014-01-01

    In this work, a novel method for a protein assay is proposed which uses the specific protein-binding peptide of the target protein and sequence-specific DNA to interact with the target as the capture and detection probe, respectively. Meanwhile, since the DNA sequence can be coupled with gold nanoparticles to amplify the signal readout, a sensitive and easily operated method for protein assay is developed. We have also employed a transcription factor named as cysteine-rich intestinal protein 1 (CRIP1), which has been identified as an ideal biomarker for staging of breast cancer, as the model protein for this study. With the proposed method, CRIP1 can be determined in a linear range from 1.25 to 10.13 ng/mL, with a detection limit of 1.25 ng/mL. Furthermore, the proposed method can be directly used to assay CRIP1 in tissue samples. Owing to its desirable sensitivity, excellent reproducibility, and high selectivity, the proposed method may hold great potential in clinical practice in the future.

  16. Microfludic device for creating ionic strength gradients over DNA microarrays for efficient DNA melting studies and assay development.

    Directory of Open Access Journals (Sweden)

    Jesper Petersen

    Full Text Available The development of DNA microarray assays is hampered by two important aspects: processing of the microarrays is done under a single stringency condition, and characteristics such as melting temperature are difficult to predict for immobilized probes. A technical solution to these limitations is to use a thermal gradient and information from melting curves, for instance to score genotypes. However, application of temperature gradients normally requires complicated equipment, and the size of the arrays that can be investigated is restricted due to heat dissipation. Here we present a simple microfluidic device that creates a gradient comprising zones of defined ionic strength over a glass slide, in which each zone corresponds to a subarray. Using this device, we demonstrated that ionic strength gradients function in a similar fashion as corresponding thermal gradients in assay development. More specifically, we noted that (i the two stringency modulators generated melting curves that could be compared, (ii both led to increased assay robustness, and (iii both were associated with difficulties in genotyping the same mutation. These findings demonstrate that ionic strength stringency buffers can be used instead of thermal gradients. Given the flexibility of design of ionic gradients, these can be created over all types of arrays, and encompass an attractive alternative to temperature gradients, avoiding curtailment of the size or spacing of subarrays on slides associated with temperature gradients.

  17. Disclosing bias in bisulfite assay: MethPrimers underestimate high DNA methylation.

    Directory of Open Access Journals (Sweden)

    Andrea Fuso

    Full Text Available Discordant results obtained in bisulfite assays using MethPrimers (PCR primers designed using MethPrimer software or assuming that non-CpGs cytosines are non methylated versus primers insensitive to cytosine methylation lead us to hypothesize a technical bias. We therefore used the two kinds of primers to study different experimental models and methylation statuses. We demonstrated that MethPrimers negatively select hypermethylated DNA sequences in the PCR step of the bisulfite assay, resulting in CpG methylation underestimation and non-CpG methylation masking, failing to evidence differential methylation statuses. We also describe the characteristics of "Methylation-Insensitive Primers" (MIPs, having degenerated bases (G/A to cope with the uncertain C/U conversion. As CpG and non-CpG DNA methylation patterns are largely variable depending on the species, developmental stage, tissue and cell type, a variable extent of the bias is expected. The more the methylome is methylated, the greater is the extent of the bias, with a prevalent effect of non-CpG methylation. These findings suggest a revision of several DNA methylation patterns so far documented and also point out the necessity of applying unbiased analyses to the increasing number of epigenomic studies.

  18. A signal amplification assay for HSV type 1 viral DNA detection using nanoparticles and direct acoustic profiling

    Directory of Open Access Journals (Sweden)

    Hammond Richard

    2010-02-01

    Full Text Available Abstract Background Nucleic acid based recognition of viral sequences can be used together with label-free biosensors to provide rapid, accurate confirmation of viral infection. To enhance detection sensitivity, gold nanoparticles can be employed with mass-sensitive acoustic biosensors (such as a quartz crystal microbalance by either hybridising nanoparticle-oligonucleotide conjugates to complimentary surface-immobilised ssDNA probes on the sensor, or by using biotin-tagged target oligonucleotides bound to avidin-modified nanoparticles on the sensor. We have evaluated and refined these signal amplification assays for the detection from specific DNA sequences of Herpes Simplex Virus (HSV type 1 and defined detection limits with a 16.5 MHz fundamental frequency thickness shear mode acoustic biosensor. Results In the study the performance of semi-homogeneous and homogeneous assay formats (suited to rapid, single step tests were evaluated utilising different diameter gold nanoparticles at varying DNA concentrations. Mathematical models were built to understand the effects of mass transport in the flow cell, the binding kinetics of targets to nanoparticles in solution, the packing geometries of targets on the nanoparticle, the packing of nanoparticles on the sensor surface and the effect of surface shear stiffness on the response of the acoustic sensor. This lead to the selection of optimised 15 nm nanoparticles that could be used with a 6 minute total assay time to achieve a limit of detection sensitivity of 5.2 × 10-12 M. Larger diameter nanoparticles gave poorer limits of detection than smaller particles. The limit of detection was three orders of magnitude lower than that observed using a hybridisation assay without nanoparticle signal amplification. Conclusions An analytical model was developed to determine optimal nanoparticle diameter, concentration and probe density, which allowed efficient and rapid optimisation of assay parameters

  19. The effect of inhibited replication on DNA migration in the comet assay in relation to cytotoxicity and clastogenicity.

    Science.gov (United States)

    Speit, Günter; Schütz, Petra

    2008-01-01

    The DNA-replication inhibitors aphidicolin (APC) and hydroxyurea (HU) were tested for their ability to induce effects on DNA in the in vitro alkaline comet assay with V79 cells. APC concentrations up to 15 microM and HU concentrations up to 500 microM did not significantly increase the extent of DNA migration after treatment during 4h. Treatment for 18 h, however, led to inconsistently significant increase in DNA migration. These increases in DNA migration were accompanied by severe cell-cycle disturbances, cytotoxic effects (reduced population doubling and reduced mitotic index) and increased frequencies of cells with chromosome aberrations. The results indicate that substances with such secondary effects on DNA (in contrast to agents that directly damage DNA) only induce effects in the comet assay after prolonged exposure, together with cytotoxic effects. We conclude that slight inhibition of DNA replication and cell-cycle delay per se do not cause significant effects in the in vitro comet assay under standard test conditions. Furthermore, the in vitro comet assay seems to be less sensitive towards this type of secondary DNA effects than the in vitro chromosome aberration test.

  20. Improved methods for capture, extraction, and quantitative assay of environmental DNA from Asian bigheaded carp (Hypophthalmichthys spp.).

    Science.gov (United States)

    Turner, Cameron R; Miller, Derryl J; Coyne, Kathryn J; Corush, Joel

    2014-01-01

    Indirect, non-invasive detection of rare aquatic macrofauna using aqueous environmental DNA (eDNA) is a relatively new approach to population and biodiversity monitoring. As such, the sensitivity of monitoring results to different methods of eDNA capture, extraction, and detection is being investigated in many ecosystems and species. One of the first and largest conservation programs with eDNA-based monitoring as a central instrument focuses on Asian bigheaded carp (Hypophthalmichthys spp.), an invasive fish spreading toward the Laurentian Great Lakes. However, the standard eDNA methods of this program have not advanced since their development in 2010. We developed new, quantitative, and more cost-effective methods and tested them against the standard protocols. In laboratory testing, our new quantitative PCR (qPCR) assay for bigheaded carp eDNA was one to two orders of magnitude more sensitive than the existing endpoint PCR assays. When applied to eDNA samples from an experimental pond containing bigheaded carp, the qPCR assay produced a detection probability of 94.8% compared to 4.2% for the endpoint PCR assays. Also, the eDNA capture and extraction method we adapted from aquatic microbiology yielded five times more bigheaded carp eDNA from the experimental pond than the standard method, at a per sample cost over forty times lower. Our new, more sensitive assay provides a quantitative tool for eDNA-based monitoring of bigheaded carp, and the higher-yielding eDNA capture and extraction method we describe can be used for eDNA-based monitoring of any aquatic species.

  1. Improved methods for capture, extraction, and quantitative assay of environmental DNA from Asian bigheaded carp (Hypophthalmichthys spp..

    Directory of Open Access Journals (Sweden)

    Cameron R Turner

    Full Text Available Indirect, non-invasive detection of rare aquatic macrofauna using aqueous environmental DNA (eDNA is a relatively new approach to population and biodiversity monitoring. As such, the sensitivity of monitoring results to different methods of eDNA capture, extraction, and detection is being investigated in many ecosystems and species. One of the first and largest conservation programs with eDNA-based monitoring as a central instrument focuses on Asian bigheaded carp (Hypophthalmichthys spp., an invasive fish spreading toward the Laurentian Great Lakes. However, the standard eDNA methods of this program have not advanced since their development in 2010. We developed new, quantitative, and more cost-effective methods and tested them against the standard protocols. In laboratory testing, our new quantitative PCR (qPCR assay for bigheaded carp eDNA was one to two orders of magnitude more sensitive than the existing endpoint PCR assays. When applied to eDNA samples from an experimental pond containing bigheaded carp, the qPCR assay produced a detection probability of 94.8% compared to 4.2% for the endpoint PCR assays. Also, the eDNA capture and extraction method we adapted from aquatic microbiology yielded five times more bigheaded carp eDNA from the experimental pond than the standard method, at a per sample cost over forty times lower. Our new, more sensitive assay provides a quantitative tool for eDNA-based monitoring of bigheaded carp, and the higher-yielding eDNA capture and extraction method we describe can be used for eDNA-based monitoring of any aquatic species.

  2. Improved Methods for Capture, Extraction, and Quantitative Assay of Environmental DNA from Asian Bigheaded Carp (Hypophthalmichthys spp.)

    Science.gov (United States)

    Turner, Cameron R.; Miller, Derryl J.; Coyne, Kathryn J.; Corush, Joel

    2014-01-01

    Indirect, non-invasive detection of rare aquatic macrofauna using aqueous environmental DNA (eDNA) is a relatively new approach to population and biodiversity monitoring. As such, the sensitivity of monitoring results to different methods of eDNA capture, extraction, and detection is being investigated in many ecosystems and species. One of the first and largest conservation programs with eDNA-based monitoring as a central instrument focuses on Asian bigheaded carp (Hypophthalmichthys spp.), an invasive fish spreading toward the Laurentian Great Lakes. However, the standard eDNA methods of this program have not advanced since their development in 2010. We developed new, quantitative, and more cost-effective methods and tested them against the standard protocols. In laboratory testing, our new quantitative PCR (qPCR) assay for bigheaded carp eDNA was one to two orders of magnitude more sensitive than the existing endpoint PCR assays. When applied to eDNA samples from an experimental pond containing bigheaded carp, the qPCR assay produced a detection probability of 94.8% compared to 4.2% for the endpoint PCR assays. Also, the eDNA capture and extraction method we adapted from aquatic microbiology yielded five times more bigheaded carp eDNA from the experimental pond than the standard method, at a per sample cost over forty times lower. Our new, more sensitive assay provides a quantitative tool for eDNA-based monitoring of bigheaded carp, and the higher-yielding eDNA capture and extraction method we describe can be used for eDNA-based monitoring of any aquatic species. PMID:25474207

  3. A DNA Microarray-Based Assay to Detect Dual Infection with Two Dengue Virus Serotypes

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    Alvaro Díaz-Badillo

    2014-04-01

    Full Text Available Here; we have described and tested a microarray based-method for the screening of dengue virus (DENV serotypes. This DNA microarray assay is specific and sensitive and can detect dual infections with two dengue virus serotypes and single-serotype infections. Other methodologies may underestimate samples containing more than one serotype. This technology can be used to discriminate between the four DENV serotypes. Single-stranded DNA targets were covalently attached to glass slides and hybridised with specific labelled probes. DENV isolates and dengue samples were used to evaluate microarray performance. Our results demonstrate that the probes hybridized specifically to DENV serotypes; with no detection of unspecific signals. This finding provides evidence that specific probes can effectively identify single and double infections in DENV samples.

  4. A DNA Microarray-Based Assay to Detect Dual Infection with Two Dengue Virus Serotypes

    Science.gov (United States)

    Díaz-Badillo, Alvaro; de Lourdes Muñoz, María; Perez-Ramirez, Gerardo; Altuzar, Victor; Burgueño, Juan; Mendoza-Alvarez, Julio G.; Martínez-Muñoz, Jorge P.; Cisneros, Alejandro; Navarrete-Espinosa, Joel; Sanchez-Sinencio, Feliciano

    2014-01-01

    Here; we have described and tested a microarray based-method for the screening of dengue virus (DENV) serotypes. This DNA microarray assay is specific and sensitive and can detect dual infections with two dengue virus serotypes and single-serotype infections. Other methodologies may underestimate samples containing more than one serotype. This technology can be used to discriminate between the four DENV serotypes. Single-stranded DNA targets were covalently attached to glass slides and hybridised with specific labelled probes. DENV isolates and dengue samples were used to evaluate microarray performance. Our results demonstrate that the probes hybridized specifically to DENV serotypes; with no detection of unspecific signals. This finding provides evidence that specific probes can effectively identify single and double infections in DENV samples. PMID:24776933

  5. Interpreting sperm DNA damage in a diverse range of mammalian sperm by means of the two-tailed comet assay

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    Elva I eCortes-Gutierrez

    2014-11-01

    Full Text Available Sperm DNA damage is associated with fertilization failure, impaired pre-and post- embryo implantation and poor pregnancy outcome. A series of methodologies to assess DNA damage in spermatozoa have been developed but most are unable to differentiate between single-stranded DNA breaks (SSBs and double-stranded DNA breaks (DSBs on the same sperm cell. The two-dimensional Two-Tailed Comet assay (TT-comet protocol highlighted in this review overcomes this limitation and emphasizes the importance in accounting for difference in sperm protamine composition at a species-specific level for the appropriate preparation of the assay. The TT-comet is a modification of the original comet assay that uses a two dimensional electrophoresis to allow for the simultaneous evaluation of relatively high numbers of DSBs and SSBs in mammalian spermatozoa. Here we have compiled a retrospective overview of how the TT-comet assay has been used to investigate the structure and function of sperm DNA across a diverse range of mammalian species (eutheria, metatheria and prototheria. When conducted as part of the TT-comet assay, we discuss how (a the alkaline comet single assay has been used to help understand the constitutive and transient changes in DNA structure associated with chromatin packing, (b the capacity of the TT-comet to differentiate between the presence of SSBs and DSBs (c and the possible implications of SSBs or DSBs for the assessment of infertility.

  6. DNA-lipid systems: A physical chemistry study

    Directory of Open Access Journals (Sweden)

    R. Dias

    2002-05-01

    Full Text Available It is well known that the interaction of polyelectrolytes with oppositely charged surfactants leads to an associative phase separation; however, the phase behavior of DNA and oppositely charged surfactants is more strongly associative than observed in other systems. A precipitate is formed with very low amounts of surfactant and DNA. DNA compaction is a general phenomenon in the presence of multivalent ions and positively charged surfaces; because of the high charge density there are strong attractive ion correlation effects. Techniques like phase diagram determinations, fluorescence microscopy, and ellipsometry were used to study these systems. The interaction between DNA and catanionic mixtures (i.e., mixtures of cationic and anionic surfactants was also investigated. We observed that DNA compacts and adsorbs onto the surface of positively charged vesicles, and that the addition of an anionic surfactant can release DNA back into solution from a compact globular complex between DNA and the cationic surfactant. Finally, DNA interactions with polycations, chitosans with different chain lengths, were studied by fluorescence microscopy, in vivo transfection assays and cryogenic transmission electron microscopy. The general conclusion is that a chitosan effective in promoting compaction is also efficient in transfection.

  7. A comprehensive assay for targeted multiplex amplification of human DNA sequences.

    Science.gov (United States)

    Krishnakumar, Sujatha; Zheng, Jianbiao; Wilhelmy, Julie; Faham, Malek; Mindrinos, Michael; Davis, Ronald

    2008-07-01

    We developed a robust and reproducible methodology to amplify human sequences in parallel for use in downstream multiplexed sequence analyses. We call the methodology SMART (Spacer Multiplex Amplification Reaction), and it is based, in part, on padlock probe technology. As a proof of principle, we used SMART technology to simultaneously amplify 485 human exons ranging from 100 to 500 bp from human genomic DNA. In multiple repetitions, >90% of the targets were successfully amplified with a high degree of uniformity, with 70% of targets falling within a 10-fold range and all products falling within a 100-fold range of each other in abundance. We used long padlock probes (LPPs) >300 bases in length for the assay, and the increased length of these probes allowed for the capture of human sequences up to 500 bp in length, which is optimal for capturing most human exons. To engineer the LPPs, we developed a method that generates ssDNA molecules with precise ends, using an appropriately designed dsDNA template. The template has appropriate restriction sites engineered into it that can be digested to generate nucleotide overhangs that are suitable for lambda exonuclease digestion, producing a single-stranded probe from dsDNA. The SMART technology is flexible and can be easily adapted to multiplex tens of thousands of target sequences in a single reaction.

  8. Development and assessment of a multiplex real-time PCR assay for quantification of human immunodeficiency virus type 1 DNA.

    Science.gov (United States)

    Beloukas, A; Paraskevis, D; Haida, C; Sypsa, V; Hatzakis, A

    2009-07-01

    Previous studies showed that high levels of human immunodeficiency virus type 1 (HIV-1) DNA are associated with a faster progression to AIDS, an increased risk of death, and a higher risk of HIV RNA rebound in patients on highly active antiretroviral therapy. Our objective was to develop and assess a highly sensitive real-time multiplex PCR assay for the quantification of HIV-1 DNA (RTMP-HIV) based on molecular beacons. HIV-1 DNA quantification was carried out by RTMP in a LightCycler 2.0 apparatus. HIV-1 DNA was quantified in parallel with CCR5 as a reference gene, and reported values are numbers of HIV-1 DNA copies/10(6) peripheral blood mononuclear cells (PBMCs). The clinical sensitivity of the assay was assessed for 115 newly diagnosed HIV-1-infected individuals. The analytical sensitivity was estimated to be 12.5 copies of HIV-1 DNA per 10(6) PBMCs, while the clinical sensitivity was 100%, with levels ranging from 1.23 to 4.25 log(10) HIV-1 DNA copies/10(6) PBMCs. In conclusion, we developed and assessed a new RTMP-HIV assay based on molecular beacons, using a LightCycler 2.0 instrument. This multiplex assay has comparable sensitivity, reproducibility, and accuracy to single real-time PCR assays.

  9. DNA damage and repair assessed by comet assay in workers exposed to lead in a battery recycling.

    Directory of Open Access Journals (Sweden)

    Mahara Valverde

    2015-05-01

    In addition to these findings, DNA damage determined by comet assay was sensible to reflect lead exposure levels related to specific activities inside this factory. Human biomonitoring studies through comet assay could be robust when additional biomarkers are determined at time.

  10. Microplate-based platform for combined chromatin and DNA methylation immunoprecipitation assays

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    Yu Jingjing

    2011-11-01

    Full Text Available Abstract Background The processes that compose expression of a given gene are far more complex than previously thought presenting unprecedented conceptual and mechanistic challenges that require development of new tools. Chromatin structure, which is regulated by DNA methylation and histone modification, is at the center of gene regulation. Immunoprecipitations of chromatin (ChIP and methylated DNA (MeDIP represent a major achievement in this area that allow researchers to probe chromatin modifications as well as specific protein-DNA interactions in vivo and to estimate the density of proteins at specific sites genome-wide. Although a critical component of chromatin structure, DNA methylation has often been studied independently of other chromatin events and transcription. Results To allow simultaneous measurements of DNA methylation with other genomic processes, we developed and validated a simple and easy-to-use high throughput microplate-based platform for analysis of DNA methylation. Compared to the traditional beads-based MeDIP the microplate MeDIP was more sensitive and had lower non-specific binding. We integrated the MeDIP method with a microplate ChIP assay which allows measurements of both DNA methylation and histone marks at the same time, Matrix ChIP-MeDIP platform. We illustrated several applications of this platform to relate DNA methylation, with chromatin and transcription events at selected genes in cultured cells, human cancer and in a model of diabetic kidney disease. Conclusion The high throughput capacity of Matrix ChIP-MeDIP to profile tens and potentially hundreds of different genomic events at the same time as DNA methylation represents a powerful platform to explore complex genomic mechanism at selected genes in cultured cells and in whole tissues. In this regard, Matrix ChIP-MeDIP should be useful to complement genome-wide studies where the rich chromatin and transcription database resources provide fruitful foundation

  11. DNA-repair measurements by use of the modified comet assay: an inter-laboratory comparison within the European Comet Assay Validation Group (ECVAG).

    Science.gov (United States)

    Godschalk, Roger W L; Ersson, Clara; Riso, Patrizia; Porrini, Marisa; Langie, Sabine A S; van Schooten, Frederik-Jan; Azqueta, Amaya; Collins, Andrew R; Jones, George D D; Kwok, Rachel W L; Phillips, David H; Sozeri, Osman; Allione, Alessandra; Matullo, Giuseppe; Möller, Lennart; Forchhammer, Lykke; Loft, Steffen; Møller, Peter

    2013-09-18

    The measurement of DNA-repair activity by extracts from cells or tissues by means of the single-cell gel electrophoresis (comet) assay has a high potential to become widely used in biomonitoring studies. We assessed the inter-laboratory variation in reported values of DNA-repair activity on substrate cells that had been incubated with Ro19-8022 plus light to generate oxidatively damaged DNA. Eight laboratories assessed the DNA-repair activity of three cell lines (i.e. one epithelial and two fibroblast cell lines), starting with cell pellets or with cell extracts provided by the coordinating laboratory. There was a large inter-laboratory variation, as evidenced by the range in the mean level of repair incisions between the laboratory with the lowest (0.002incisions/10(6)bp) and highest (0.988incisions/10(6)bp) incision activity. Nevertheless, six out of eight laboratories reported the same cell line as having the highest level of DNA-repair activity. The two laboratories that reported discordant results (with another cell line having the highest level of DNA-repair activity) were those that reported to have little experience with the modified comet assay to assess DNA repair. The laboratories were also less consistent in ordering the repair activity of the other two cell lines, probably because the DNA-repair activity by extracts from these cell lines were very similar (on average approximately 60-65% of the cell line with the highest repair capacity). A significant correlation was observed between the repair activity found in the provided and the self-made cell extracts (r=0.71, Pcomet assay for base-excision repair.

  12. Inter-laboratory comparison of the in vivo comet assay including three image analysis systems.

    Science.gov (United States)

    Plappert-Helbig, Ulla; Guérard, Melanie

    2015-12-01

    To compare the extent of potential inter-laboratory variability and the influence of different comet image analysis systems, in vivo comet experiments were conducted using the genotoxicants ethyl methanesulfonate and methyl methanesulfonate. Tissue samples from the same animals were processed and analyzed-including independent slide evaluation by image analysis-in two laboratories with extensive experience in performing the comet assay. The analysis revealed low inter-laboratory experimental variability. Neither the use of different image analysis systems, nor the staining procedure of DNA (propidium iodide vs. SYBR® Gold), considerably impacted the results or sensitivity of the assay. In addition, relatively high stability of the staining intensity of propidium iodide-stained slides was found in slides that were refrigerated for over 3 months. In conclusion, following a thoroughly defined protocol and standardized routine procedures ensures that the comet assay is robust and generates comparable results between different laboratories.

  13. DETECTION OF LOW DOSE RADIATION-AND CHEMICALLY-INDUCED DNA DAMAGE USING TEMPERATURE DIFFERENTIAL FLUORESCENCE ASSAYS

    Science.gov (United States)

    Rapid, sensitive and simple assays for radiation- and chemically-induced DNA damage can be of significant benefit to a number of fields including radiation biology, clinical research, and environmental monitoring. Although temperature-induced DNA strand separation has been use...

  14. Induction and repair of DNA damage measured by the comet assay in human T lymphocytes separated by immunomagnetic cell sorting.

    Science.gov (United States)

    Bausinger, Julia; Speit, Günter

    2014-11-01

    The comet assay is widely used in human biomonitoring to measure DNA damage in whole blood or isolated peripheral blood mononuclear cells (PBMC) as a marker of exposure to genotoxic agents. Cytogenetic assays with phytohemagglutinin (PHA)-stimulated cultured T lymphocytes are also frequently performed in human biomonitoring. Cytogenetic effects (micronuclei, chromosome aberrations, sister chromatid exchanges) may be induced in vivo but also occur ex vivo during the cultivation of lymphocytes as a consequence of DNA damage present in lymphocytes at the time of sampling. To better understand whether DNA damage measured by the comet assay in PBMC is representative for DNA damage in T cells, we comparatively investigated DNA damage and its repair in PBMC and T cells obtained by immunomagnetic cell sorting. PBMC cultures and T cell cultures were exposed to mutagens with different modes of genotoxic action and DNA damage was measured by the comet assay after the end of a 2h exposure and after 18h post-incubation. The mutagens tested were methyl methanesulfonate (MMS), (±)-anti-B[a]P-7,8-dihydrodiol-9,10-epoxide (BPDE), 4-nitroquinoline-1-oxide (4NQO), styrene oxide and potassium bromate. MMS and potassium bromate were also tested by the modified comet assay with formamido pyrimidine glycosylase (FPG) protein. The results indicate that the mutagens tested induce DNA damage in PBMC and T cells in the same range of concentrations and removal of induced DNA lesions occurs to a comparable extent. Based on these results, we conclude that the comet assay with PBMC is suited to predict DNA damage and its removal in T cells.

  15. Impact of simultaneous assay, the PCNA, cyclinD1, and DNA content with specimens before and after preoperative radiotherapy on prognosis of esophageal cancer-possible incorporation into clinical TNM staging system

    Institute of Scientific and Technical Information of China (English)

    Shu-Chai Zhu; Ren Li; Yu-Xiang Wang; Wei Feng; Juan Li; Rong Qiu

    2005-01-01

    AIM: The aim of the present study is to use immunohistochemical methods to investigate the clinical implications of tumor markers in esophageal squamous cell carcinoma and evaluate their impact on prognosis.METHODS: From November 1990 to December 1996, 47patients were treated with preoperative radiation followed by radical esophagectomy. All patients were confirmed pathologically as suffering from squamous cell carcinoma.Immunohistochemical stain was done for PCNA, cyclinD1 protein expression and DNA content analyzed by image cytometry. Kaplan-Meier method for single prognostic factor and log-rank test was used to test the significant difference. Cox stepwise regression model and prognosis index model were used for survival analysis with multiple prognostic factors.RESULTS: Radio-pathological change, T stage and N stage, as the traditional prognostic factors had statistical difference in 3-, 5- and 10-year survival rates. While, tumor cell proliferating marked PCNA, cyclinD1 and DNA content served as independent prognostic factors of esophageal carcinoma. There was definitely an identity between the single and multiple factor analyses. PI was more accurate to evaluate the prognosis of esophageal carcinoma.CONCLUSION: It is possible that tumor cell proliferating marked PCNA, cyclinD1 and DNA content would become the endpoints for evaluating the prognosis of esophageal carcinoma.

  16. Sensitive detection of point mutation by electrochemiluminescence and DNA ligase-based assay

    Science.gov (United States)

    Zhou, Huijuan; Wu, Baoyan

    2008-12-01

    The technology of single-base mutation detection plays an increasingly important role in diagnosis and prognosis of genetic-based diseases. Here we reported a new method for the analysis of point mutations in genomic DNA through the integration of allele-specific oligonucleotide ligation assay (OLA) with magnetic beads-based electrochemiluminescence (ECL) detection scheme. In this assay the tris(bipyridine) ruthenium (TBR) labeled probe and the biotinylated probe are designed to perfectly complementary to the mutant target, thus a ligation can be generated between those two probes by Taq DNA Ligase in the presence of mutant target. If there is an allele mismatch, the ligation does not take place. The ligation products are then captured onto streptavidin-coated paramagnetic beads, and detected by measuring the ECL signal of the TBR label. Results showed that the new method held a low detection limit down to 10 fmol and was successfully applied in the identification of point mutations from ASTC-α-1, PANC-1 and normal cell lines in codon 273 of TP53 oncogene. In summary, this method provides a sensitive, cost-effective and easy operation approach for point mutation detection.

  17. A novel mini-DNA barcoding assay to identify processed fins from internationally protected shark species.

    Science.gov (United States)

    Fields, Andrew T; Abercrombie, Debra L; Eng, Rowena; Feldheim, Kevin; Chapman, Demian D

    2015-01-01

    There is a growing need to identify shark products in trade, in part due to the recent listing of five commercially important species on the Appendices of the Convention on International Trade in Endangered Species (CITES; porbeagle, Lamna nasus, oceanic whitetip, Carcharhinus longimanus scalloped hammerhead, Sphyrna lewini, smooth hammerhead, S. zygaena and great hammerhead S. mokarran) in addition to three species listed in the early part of this century (whale, Rhincodon typus, basking, Cetorhinus maximus, and white, Carcharodon carcharias). Shark fins are traded internationally to supply the Asian dried seafood market, in which they are used to make the luxury dish shark fin soup. Shark fins usually enter international trade with their skin still intact and can be identified using morphological characters or standard DNA-barcoding approaches. Once they reach Asia and are traded in this region the skin is removed and they are treated with chemicals that eliminate many key diagnostic characters and degrade their DNA ("processed fins"). Here, we present a validated mini-barcode assay based on partial sequences of the cytochrome oxidase I gene that can reliably identify the processed fins of seven of the eight CITES listed shark species. We also demonstrate that the assay can even frequently identify the species or genus of origin of shark fin soup (31 out of 50 samples).

  18. A novel mini-DNA barcoding assay to identify processed fins from internationally protected shark species.

    Directory of Open Access Journals (Sweden)

    Andrew T Fields

    Full Text Available There is a growing need to identify shark products in trade, in part due to the recent listing of five commercially important species on the Appendices of the Convention on International Trade in Endangered Species (CITES; porbeagle, Lamna nasus, oceanic whitetip, Carcharhinus longimanus scalloped hammerhead, Sphyrna lewini, smooth hammerhead, S. zygaena and great hammerhead S. mokarran in addition to three species listed in the early part of this century (whale, Rhincodon typus, basking, Cetorhinus maximus, and white, Carcharodon carcharias. Shark fins are traded internationally to supply the Asian dried seafood market, in which they are used to make the luxury dish shark fin soup. Shark fins usually enter international trade with their skin still intact and can be identified using morphological characters or standard DNA-barcoding approaches. Once they reach Asia and are traded in this region the skin is removed and they are treated with chemicals that eliminate many key diagnostic characters and degrade their DNA ("processed fins". Here, we present a validated mini-barcode assay based on partial sequences of the cytochrome oxidase I gene that can reliably identify the processed fins of seven of the eight CITES listed shark species. We also demonstrate that the assay can even frequently identify the species or genus of origin of shark fin soup (31 out of 50 samples.

  19. A Novel Mini-DNA Barcoding Assay to Identify Processed Fins from Internationally Protected Shark Species

    Science.gov (United States)

    Fields, Andrew T.; Abercrombie, Debra L.; Eng, Rowena; Feldheim, Kevin; Chapman, Demian D.

    2015-01-01

    There is a growing need to identify shark products in trade, in part due to the recent listing of five commercially important species on the Appendices of the Convention on International Trade in Endangered Species (CITES; porbeagle, Lamna nasus, oceanic whitetip, Carcharhinus longimanus scalloped hammerhead, Sphyrna lewini, smooth hammerhead, S. zygaena and great hammerhead S. mokarran) in addition to three species listed in the early part of this century (whale, Rhincodon typus, basking, Cetorhinus maximus, and white, Carcharodon carcharias). Shark fins are traded internationally to supply the Asian dried seafood market, in which they are used to make the luxury dish shark fin soup. Shark fins usually enter international trade with their skin still intact and can be identified using morphological characters or standard DNA-barcoding approaches. Once they reach Asia and are traded in this region the skin is removed and they are treated with chemicals that eliminate many key diagnostic characters and degrade their DNA (“processed fins”). Here, we present a validated mini-barcode assay based on partial sequences of the cytochrome oxidase I gene that can reliably identify the processed fins of seven of the eight CITES listed shark species. We also demonstrate that the assay can even frequently identify the species or genus of origin of shark fin soup (31 out of 50 samples). PMID:25646789

  20. Study the effects of divalent metallic ions on the combination of DNA and histones with fluorescence anisotropy assays

    Institute of Scientific and Technical Information of China (English)

    LIU YuYing; WANG PengYe; DOU ShuoXing; XIE Ping; XI XuGuang

    2007-01-01

    The effects of divalent ions (Mn2+, Mg2+, Ca2+) on the interaction between DNA and histone are studied using a fluorescence anisotropy assay. Fluorescence anisotropies of DNA and DNA-histone in the presence of divalent ions (Mn2+, Mg2+, Ca2+) are measured. The results indicate that histone reduces the fluorescence anisotropy of lambda DNA while the divalent ions (Mn2+, Mg2+, Ca2+) significantly enhance the fluorescence anisotropy. Compared to the case of DNA incubated with histone alone, there are more histones binding to DNA when divalent ion, histone and DNA are incubated together. We also find that Mn2+ makes the DNA-histone complexes more condensed than the other ions do.

  1. Optimization of a DNA Nicking Assay to Evaluate Oenocarpus bataua and Camellia sinensis Antioxidant Capacity

    Directory of Open Access Journals (Sweden)

    Louis-Jérôme Leba

    2014-10-01

    Full Text Available This study was aimed at assessing the DNA damage protective activity of different types of extracts (aqueous, methanolic and acetonic using an in vitro DNA nicking assay. Several parameters were optimized using the pUC18 plasmid, especially FeSO4, EDTA, solvent concentrations and incubation time. Special attention has been paid to removing the protective and damaging effect of the solvent and FeSO4 respectively, as well as to identifying the relevant positive and negative controls. For each solvent, the optimal conditions were determined: (i for aqueous extracts, 0.33 mM of FeSO4 and 0.62 mM of EDTA were incubated for 20 min at 37 °C; (ii for acetone extracts, 1.16% solvent were incubated for 15 min at 37 °C with 1.3 mM of FeSO4 and 2.5 mM of EDTA and (iii for methanol extracts, 0.16% solvent, were incubated for 1.5 h at 37 °C with 0.33 mM of FeSO4 and 0.62 mM of EDTA. Using the optimized conditions, the DNA damage protective activity of aqueous, methanolic and acetonic extracts of an Amazonian palm berry (Oenocarpus bataua and green tea (Camellia sinensis was assessed. Aqueous and acetonic Oenocarpus bataua extracts were protective against DNA damage, whereas aqueous, methanolic and acetonic extracts of Camellia sinensis extracts induced DNA damage.

  2. Quantitative real-time PCR (qPCR) assay for human-dog-cat species identification and nuclear DNA quantification.

    Science.gov (United States)

    Kanthaswamy, S; Premasuthan, A; Ng, J; Satkoski, J; Goyal, V

    2012-03-01

    In the United States, human forensic evidence collected from crime scenes is usually comingled with biomaterial of canine and feline origins. Knowledge of the concentration of nuclear DNA extracted from a crime scene biological sample and the species from which the sample originated is essential for DNA profiling. The ability to accurately detect and quantify target DNA in mixed-species samples is crucial when target DNA may be overwhelmed by non-target DNA. We have designed and evaluated a species-specific (human, dog and cat) nuclear DNA identification assay based on the TaqMan(®) quantitative real-time PCR (qPCR) technology that can simultaneously detect and measure minute quantities of DNA specific to either humans, dogs and/or cats. The fluorogenic triplex assay employs primers and hydrolysis probes that target the human TH01 locus as well as the dog and cat Melanocortin 1 Receptor (MC1R) sequences in a species-specific manner. We also demonstrate that the assay is a highly sensitive, reliable and robust method for identifying and quantifying mixed-species templates of human-dog-cat origin with as little as 0.4 pg of human and cat nuclear DNA, respectively, and 4.0 pg of dog nuclear DNA.

  3. An in vitro DNA double-strand break repair assay based on end-joining of defined duplex oligonucleotides.

    Science.gov (United States)

    Datta, Kamal; Purkayastha, Shubhadeep; Neumann, Ronald D; Winters, Thomas A

    2012-01-01

    DNA double-strand breaks (DSBs) are caused by endogenous cellular processes such as oxidative metabolism, or by exogenous events like exposure to ionizing radiation or other genotoxic agents. Repair of these DSBs is essential for the maintenance of cellular genomic integrity. In human cells, and cells of other higher eukaryotes, DSBs are primarily repaired by the nonhomologous end-joining (NHEJ) DSB repair pathway. Most in vitro assays that have been designed to measure NHEJ activity employ linear plasmid DNA as end-joining substrates, and such assays have made significant contributions to our understanding of the biochemical mechanisms of NHEJ. Here we describe an in vitro end-joining assay employing linear oligonucleotides that has distinct advantages over plasmid-based assays for the study of structure-function relationships between the proteins of the NHEJ pathway and synthetic DNA end-joining substrates possessing predetermined DSB configurations and chemistries.

  4. Evaluation of the Comet Assay for Assessing the Dose-Response Relationship of DNA Damage Induced by Ionizing Radiation

    Directory of Open Access Journals (Sweden)

    Qiang Liu

    2013-11-01

    Full Text Available Dose- and time-response curves were combined to assess the potential of the comet assay in radiation biodosimetry. The neutral comet assay was used to detect DNA double-strand breaks in lymphocytes caused by γ-ray irradiation. A clear dose-response relationship with DNA double-strand breaks using the comet assay was found at different times after irradiation (p < 0.001. A time-response relationship was also found within 72 h after irradiation (p < 0.001. The curves for DNA double-strand breaks and DNA repair in vitro of human lymphocytes presented a nice model, and a smooth, three-dimensional plane model was obtained when the two curves were combined.

  5. Fabrication of uniform DNA-conjugated hydrogel microparticles via replica molding for facile nucleic acid hybridization assays.

    Science.gov (United States)

    Lewis, Christina L; Choi, Chang-Hyung; Lin, Yan; Lee, Chang-Soo; Yi, Hyunmin

    2010-07-01

    We identify and investigate several critical parameters in the fabrication of single-stranded DNA conjugated poly(ethylene glycol) (PEG) microparticles based on replica molding (RM) for highly uniform and robust nucleic acid hybridization assays. The effects of PEG-diacrylate, probe DNA, and photoinitiator concentrations on the overall fluorescence and target DNA penetration depth upon hybridization are examined. Fluorescence and confocal microscopy results illustrate high conjugation capacity of the probe and target DNA, femtomole sensitivity, and sequence specificity. Combined, these findings demonstrate a significant step toward simple, robust, and scalable procedures to manufacture highly uniform and high-capacity hybridization assay particles in a well-controlled manner by exploiting many advantages that the batch processing-based RM technique offers. We envision that the results presented here may be readily applied to rapid and high-throughput hybridization assays for a wide variety of applications in bioprocess monitoring, food safety, and biological threat detection.

  6. Sensitive detection of colorectal cancer in peripheral blood by septin 9 DNA methylation assay.

    Directory of Open Access Journals (Sweden)

    Robert Grützmann

    Full Text Available BACKGROUND: Colorectal cancer (CRC is the second leading cause of cancer deaths despite the fact that detection of this cancer in early stages results in over 90% survival rate. Currently less than 45% of at-risk individuals in the US are screened regularly, exposing a need for better screening tests. We performed two case-control studies to validate a blood-based test that identifies methylated DNA in plasma from all stages of CRC. METHODOLOGY/PRINCIPAL FINDINGS: Using a PCR assay for analysis of Septin 9 (SEPT9 hypermethylation in DNA extracted from plasma, clinical performance was optimized on 354 samples (252 CRC, 102 controls and validated in a blinded, independent study of 309 samples (126 CRC, 183 controls. 168 polyps and 411 additional disease controls were also evaluated. Based on the training study SEPT9-based classification detected 120/252 CRCs (48% and 7/102 controls (7%. In the test study 73/126 CRCs (58% and 18/183 control samples (10% were positive for SEPT9 validating the training set results. Inclusion of an additional measurement replicate increased the sensitivity of the assay in the testing set to 72% (90/125 CRCs detected while maintaining 90% specificity (19/183 for controls. Positive rates for plasmas from the other cancers (11/96 and non-cancerous conditions (41/315 were low. The rate of polyp detection (>1 cm was approximately 20%. CONCLUSIONS/SIGNIFICANCE: Analysis of SEPT9 DNA methylation in plasma represents a straightforward, minimally invasive method to detect all stages of CRC with potential to satisfy unmet needs for increased compliance in the screening population. Further clinical testing is warranted.

  7. Mutagenicity test system based on a reporter gene assay for short-term detection of mutagens (MutaGen assay).

    Science.gov (United States)

    Schmid, Claudia; Arndt, Christian; Reifferscheid, Georg

    2003-02-05

    The construction of a bacterial mutation assay system detecting reversions of base substitutions and frameshifts in tetracycline (tet) and ampicillin resistance genes located on low copy plasmids is described. Frameshift mutations were introduced into repetitive GC-sequences and G-repeats known to be mutagenic hot-spots. Base pair substitutions were inserted in or around the active site of the ampicillinase gene thus generating reversibility of the ampicilline sensitivity. The plasmids carry genes to enable sensitive, fast and specific detection of mutagens in bacteria. MucAB was cloned into the test plasmid to enhance error-prone DNA-repair. The conventional reversion principle has been combined with the luminometric measurement of an inducible reporter gene. The revertants are detected after induction of the beta-galactosidase-producing lacZ-gene either controlled by its natural lac-promotor or by the more stringently repressed (anhydrotetracyclin inducible) tetA promotor. The tester strains containing the tetA/lacZ reporter gene construct can grow in full medium over the complete assay. This test procedure enables screening for mutations within one working day. Incubation for 16 h reveals high sensitivity.

  8. Comparison of commercial and in-house Real-time PCR assays for quantification of Epstein-Barr virus (EBV DNA in plasma

    Directory of Open Access Journals (Sweden)

    Pollara Caterina

    2007-03-01

    Full Text Available Abstract Background Epstein-Barr virus (EBV DNA load monitoring is known to be useful for the diagnosis and monitoring of EBV-associated diseases. The aim of this study is to compare the performance of two real-time PCR assays for EBV DNA: a commercial kit as the Q-EBV Real-Time System (Q-EBV PCR, Amplimedical, Turin, Italy and an in-house assay (EBV RQ-PCR. Results The range of linearity and the degree of precision of the two assays were similar. The clinical sensitivity of Q-EBV PCR was higher for reference samples containing less than 1,000 EBV DNA copies/ml. The absolute quantitative results of the two methods were statistically correlated (R2 = 0.7789; p Conclusion Both the commercial and the in-house assay may be appropriate for clinical use, but common standards are advisable for comparable absolute values, as these would improve the clinical utility of EBV DNA load measurement.

  9. Immobilization of proteins onto microbeads using a DNA binding tag for enzymatic assays.

    Science.gov (United States)

    Kojima, Takaaki; Mizoguchi, Takuro; Ota, Eri; Hata, Jumpei; Homma, Keisuke; Zhu, Bo; Hitomi, Kiyotaka; Nakano, Hideo

    2016-02-01

    A novel DNA-binding protein tag, scCro-tag, which is a single-chain derivative of the bacteriophage lambda Cro repressor, has been developed to immobilize proteins of interest (POI) on a solid support through binding OR consensus DNA (ORC) that is tightly bound by the scCro protein. The scCro-tag successfully bound a transglutaminase 2 (TGase 2) substrate and manganese peroxidase (MnP) to microbeads via scaffolding DNA. The resulting protein-coated microbeads can be utilized for functional analysis of the enzymatic activity using flow cytometry. The quantity of bead-bound proteins can be enhanced by increasing the number of ORCs. In addition, proteins with the scCro-tag that were synthesized using a cell-free protein synthesis system were also immobilized onto the beads, thus indicating that this bead-based system would be applicable to high-throughput analysis of various enzymatic activities.

  10. Evaluation of the IrisPlex DNA-based eye color prediction assay in a United States population.

    Science.gov (United States)

    Dembinski, Gina M; Picard, Christine J

    2014-03-01

    DNA phenotyping is a rapidly developing area of research in forensic biology. Externally visible characteristics (EVCs) can be determined based on genotype data, specifically based on single nucleotide polymorphisms (SNPs). These SNPs are chosen based on their association with genes related to the phenotypic expression of interest, with known examples in eye, hair, and skin color traits. DNA phenotyping has forensic importance when unknown biological samples at a crime scene do not result in a criminal database hit; a phenotypic profile of the sample can therefore be used to develop investigational leads. IrisPlex, an eye color prediction assay, has previously shown high prediction rates for blue and brown eye color in a Dutch European population. The objective of this work was to evaluate its utility in a North American population. We evaluated six SNPs included in the IrisPlex assay in population sample collected from a USA college campus. We used a quantitative method of eye color classification based on (RGB) color components of digital photographs of the eye taken from each study volunteer so that each eye was placed in one of three eye color categories: brown, intermediate, or blue. Objective color classification was shown to correlate with basic human visual determination making it a feasible option for use in future prediction assay development. Using these samples and various models, the maximum prediction accuracies of the IrisPlex system after allele frequency adjustment was 58% and 95% brown and blue eye color predictions, respectively, and 11% for intermediate eye colors. Future developments should include incorporation of additional informative SNPs, specifically related to the intermediate eye color, and we recommend the use of a Bayesian approach as a prediction model as likelihood ratios can be determined for reporting purposes.

  11. Evaluation of the mitochondrial respiratory chain and oxidative phosphorylation system using polarography and spectrophotometric enzyme assays.

    Science.gov (United States)

    Barrientos, Antoni; Fontanesi, Flavia; Díaz, Francisca

    2009-10-01

    The oxidative phosphorylation (OXPHOS) system consists of five multimeric complexes embedded in the mitochondrial inner membrane. They work in concert to drive the aerobic synthesis of ATP. Mitochondrial and nuclear DNA mutations affecting the accumulation and function of these enzymes are the most common cause of mitochondrial diseases and have also been associated with neurodegeneration and aging. For this reason, several approaches for the assessment of the OXPHOS system enzymes have been developed. Based on the methods described elsewhere, the assays describe methods that form a biochemical characterization of the OXPHOS system in cells and mitochondria isolated from cultured cells or tissues.

  12. A Colorimetric Microplate Assay for DNA-Binding Activity of His-Tagged MutS Protein.

    Science.gov (United States)

    Banasik, Michał; Sachadyn, Paweł

    2016-09-01

    A simple microplate method was designed for rapid testing DNA-binding activity of proteins. The principle of the assay involves binding of tested DNA by his-tagged protein immobilized on a nickel-coated ELISA plate, following colorimetric detection of biotinylated DNA with avidin conjugated to horseradish peroxidase. The method was used to compare DNA mismatch binding activities of MutS proteins from three bacterial species. The assay required relatively low amounts of tested protein (approximately 0.5-10 pmol) and DNA (0.1-10 pmol) and a relatively short time of analysis (up to 60 min). The method is very simple to apply and convenient to test different buffer conditions of DNA-protein binding. Sensitive colorimetric detection enables naked eye observations and quantitation with an ELISA reader. The performance of the assay, which we believe is a distinguishing trait of the method, is based on two strong and specific molecular interactions: binding of a his-tagged protein to a nickel-coated microplate and binding of biotinylated DNA to avidin. In the reported experiments, the solution was used to optimize the conditions for DNA mismatch binding by MutS protein; however, the approach could be implemented to test nucleic acids interactions with any protein of interest.

  13. Detection of genotoxic carcinogens in the in vivo-in vitro hepatocyte DNA repair assay.

    Science.gov (United States)

    Mirsalis, J C; Tyson, C K; Butterworth, B E

    1982-01-01

    The in vivo-in vitro hepatocyte DNA repair assay has been shown to be useful in the evaluation of the carcinogenic potential of chemicals. The purpose of this study was to apply this assay to determining the genotoxicity of the compounds from a wide variety of structural classes. Male Fischer-334 rats were treated by gavage or ip injection with compounds dissolved in either corn oil, water, or dimethyl sulfoxide (DMSO). At selected times after treatment, hepatocytes were isolated by liver perfusion and cultured with 3H-thymidine, Unscheduled DNA synthesis (UDS) was measured by quantitative autoradiography as net grains/nucleus (NG); greater than or equal to 5 NG was considered positive. Water, corn oil, or DMSO controls produced -3 to -6 NG with less than or equal to 6% of the cells in repair. All genotoxic hepatocarcinogens tested produced strong positive responses of greater than 15 NG including dimethylnitrosamine, 2-acetylaminofluorene (2-AAF), azoxymethane, 1,2-dimethylhydrazine, benzidine, aflatoxin B1, 2,6-dinitrotoluene, and 2,4-diaminotoluene. The noncarcinogen, 2,6-diaminotoluene, was negative. The mutagen and rat brain carcinogen methyl methanesulfonate (MMS) and the rat pancreatic carcinogen azaserine were also positive. The carcinogens benzo(a)pyrene and 7,12-dimethylbenz(a)anthracene yielded from -2 to -4 NG. This negative response is consistent with their lack of carcinogenic activity in rat liver. MMS produced the greatest amount of UDS 2 hr after treatment whereas 2-AAF did not induce its maximum response until 12 hr post-treatment. The potent hepatotoxin carbon tetrachloride induced a 40-fold elevation in DNA replication 48 hr after a 400 mg/kg dose, but no UDS was observed at 2, 12, 24, or 48 hr post-treatment. The weak hepatocarcinogen safrole induced no UDS suggesting that it is either nongenotoxic or is metabolized to an active form at an extremely slow rate following a single administration. These results demonstrate that this assay is

  14. Comparison of kDNA PCR-hybridization assay with three PCR methods for canines visceral Leishmaniasis diagnosis

    Energy Technology Data Exchange (ETDEWEB)

    Pilatti, Marcia M.; Andrade, Antero S.R. [Centro de Desenvolvimento da Tecnologia Nuclear (CDTN/CNEN-MG), Belo Horizonte, MG (Brazil)], e-mail: marciapilatti@yahoo.com.br, e-mail: antero@cdtn.br; Ferreira, Sidney A. [Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, MG (Brazil). Dept. de Parasitologia], e-mail: saninoalmeida@gmail.com

    2009-07-01

    The sensitivity of the kDNA PCR-Hybridization assay, which uses radioactive DNA probes (labeled with {sup 32}P), was compared with three conventional PCR methods used for canine visceral leishmaniasis diagnosis. All PCR methods had two steps: a first amplification followed by hybridization or by a new amplification (nested or semi nested). Two methods (kDNA PCR-Hybridization and kDNA snPCR) used primers addressed to kinetoplast minicircles and the other two methods to the coding (LnPCR) and intergenic noncoding regions (ITS-1 nPCR) of the ribosomal rRNA genes. The comparison was accomplished in two groups of 23 infected dogs using samples collected by the conjunctival swab procedure. In the Group 1 the DNA was extracted from cotton swabs by phenol-chloroform and in Group 2 by boiling. The most efficient PCR methods in the Group 1 were those based on kDNA targets. The kDNA PCR-Hybridization was able to detect parasites in 22/23 dogs (95.6%) and in 40/46 samples (86.9%). The kDNA snPCR was positive for 21/23 dogs (91.3%) and for 40/46 samples (86.9%). The positivities of the kDNA based methods were significantly higher than the positivities verified for the methods based on ribosomal rRNA genes (p<0.05). In the Group 2 the kDNA PCR- Hybridization showed a better performance detecting parasites in 18/23 dogs (78.3%) and in 31/46 samples (67.4%), significantly higher than the other three methods (p<0.05). The higher sensitivity of the minicircle kDNA based assays reported by others was confirmed in this study and kDNA PCR-Hybridization showed the best sensitivity among the assays evaluated. (author)

  15. A system for performing high throughput assays of synaptic function.

    Directory of Open Access Journals (Sweden)

    Chris M Hempel

    Full Text Available Unbiased, high-throughput screening has proven invaluable for dissecting complex biological processes. Application of this general approach to synaptic function would have a major impact on neuroscience research and drug discovery. However, existing techniques for studying synaptic physiology are labor intensive and low-throughput. Here, we describe a new high-throughput technology for performing assays of synaptic function in primary neurons cultured in microtiter plates. We show that this system can perform 96 synaptic vesicle cycling assays in parallel with high sensitivity, precision, uniformity, and reproducibility and can detect modulators of presynaptic function. By screening libraries of pharmacologically defined compounds on rat forebrain cultures, we have used this system to identify novel effects of compounds on specific aspects of presynaptic function. As a system for unbiased compound as well as genomic screening, this technology has significant applications for basic neuroscience research and for the discovery of novel, mechanism-based treatments for central nervous system disorders.

  16. Adaptation of the neutral bacterial comet assay to assess antimicrobial-mediated DNA double-strand breaks in Escherichia coli

    Science.gov (United States)

    SOLANKY, DIPESH; HAYDEL, SHELLEY E.

    2012-01-01

    This study aimed to determine the mechanism of action of a natural antibacterial clay mineral mixture, designated CB, by investigating the induction of DNA double-strand breaks (DSBs) in Escherichia coli. To quantify DNA damage upon exposure to soluble antimicrobial compounds, we modified a bacterial neutral comet assay, which primarily associates the general length of an electrophoresed chromosome, or comet, with the degree of DSB-associated DNA damage. To appropriately account for antimicrobial-mediated strand fragmentation, suitable control reactions consisting of exposures to water, ethanol, kanamycin, and bleomycin were developed and optimized for the assay. Bacterial exposure to the CB clay resulted in significantly longer comet lengths, compared to water and kanamycin exposures, suggesting that the induction of DNA DSBs contributes to the killing activity of this antibacterial clay mineral mixture. The comet assay protocol described herein provides a general technique for evaluating soluble antimicrobial-derived DNA damage and for comparing DNA fragmentation between experimental and control assays. PMID:22940101

  17. Clinical performances of two real-time PCR assays and bDNA/TMA to early monitor treatment outcome in patients with chronic hepatitis C.

    Science.gov (United States)

    Martinot-Peignoux, Michelle; Khiri, Hacène; Leclere, Laurence; Maylin, Sarah; Marcellin, Patrick; Halfon, Philippe

    2009-11-01

    Early viral monitoring is essential for the management of treatment outcome in patients with chronic hepatitis C. A variety of commercially available assays are now available to quantify HCV-RNA in routine clinical practice. Compare the clinical results of 3 commercially available assays to evaluate the positive predictive value (PPV) and the negative predictive value (NPV) of rapid virological response (RVR) at week 4 and early virological response (EVR) at week 12. 287 patients treated with standard care regimen combination therapy were studied. HCV-RNA values measured at baseline, week 4, week 12 with VERSANT HCV 3.0 Assay (bDNA), and VERSANT HCV-RNA Qualitative Assay (TMA) (bDNA/TMA); COBAS Ampliprep/COBAS/TaqMan (CAP/CTM) and Abbott m2000sp extraction/m2000rt amplification system (ART). RVR was defined as undetectable serum HCV-RNA and EVR as a > OR =2 log decline in baseline viral load (BLV). Median (range) BVLs were: 5.585(2.585-6.816), 5.189(2.792-7.747) and 4.804(2.380-6.580) log(10)IU/ml, with bDNA/TMA, CAP/CTM and ART, respectively (pTMA, CAP/CTM and ART, respectively (p=0.317). EVR was observed in 76%, 73% and 67% of the patients and NPVs were 93%, 83% and 79% with bDNA/TMA, CAP/CTM and ART, respectively (p=0.09). Treatment monitoring should include both detection of serum HCV-RNA at week 4 to predict SVR and at week 12 to predict non-SVR. The value of all 3 assays was similar for evaluating RVR or EVR. Because of viral load discrepancies the same assay should be used throughout patient treatment follow-up.

  18. Detection of Cryptococcus neoformans DNA in Tissue Samples by Nested and Real-Time PCR Assays

    Science.gov (United States)

    Bialek, Ralf; Weiss, Michael; Bekure-Nemariam, Kubrom; Najvar, Laura K.; Alberdi, Maria B.; Graybill, John R.; Reischl, Udo

    2002-01-01

    Two PCR protocols targeting the 18S rRNA gene of Cryptococcus neoformans were established, compared, and evaluated in murine cryptococcal meningitis. One protocol was designed as a nested PCR to be performed in conventional block thermal cyclers. The other protocol was designed as a quantitative single-round PCR adapted to LightCycler technology. One hundred brain homogenates and dilutions originating from 20 ICR mice treated with different azoles were examined. A fungal burden of 3 × 101 to 2.9 × 104 CFU per mg of brain tissue was determined by quantitative culture. Specific PCR products were amplified by the conventional and the LightCycler methods in 86 and 87 samples, respectively, with products identified by DNA sequencing and real-time fluorescence detection. An analytical sensitivity of 1 CFU of C. neoformans per mg of brain tissue and less than 10 CFU per volume used for extraction was observed for both PCR protocols, while homogenates of 70 organs from mice infected with other fungi were PCR negative. Specificity testing was performed with genomic DNA from 31 hymenomycetous fungal species and from the ustilaginomycetous yeast Malassezia furfur, which are phylogenetically related to C. neoformans. Twenty-four strains, including species of human skin flora like M. furfur and Trichosporon spp., were PCR negative. Amplification was observed with Cryptococcus amylolentus, Filobasidiella depauperata, Cryptococcus laurentii, and five species unrelated to clinical specimens. LightCycler PCR products from F. depauperata and Trichosporon faecale could be clearly discriminated by melting curve analysis. The sensitive and specific nested PCR assay as well as the rapid and quantitative LightCycler PCR assay might be useful for the diagnosis and monitoring of human cryptococcal infections. PMID:11874894

  19. Evaluating genomic DNA extraction methods from human whole blood using endpoint and real-time PCR assays.

    Science.gov (United States)

    Koshy, Linda; Anju, A L; Harikrishnan, S; Kutty, V R; Jissa, V T; Kurikesu, Irin; Jayachandran, Parvathy; Jayakumaran Nair, A; Gangaprasad, A; Nair, G M; Sudhakaran, P R

    2017-02-01

    The extraction of genomic DNA is the crucial first step in large-scale epidemiological studies. Though there are many popular DNA isolation methods from human whole blood, only a few reports have compared their efficiencies using both end-point and real-time PCR assays. Genomic DNA was extracted from coronary artery disease patients using solution-based conventional protocols such as the phenol-chloroform/proteinase-K method and a non-phenolic non-enzymatic Rapid-Method, which were evaluated and compared vis-a-vis a commercially available silica column-based Blood DNA isolation kit. The appropriate method for efficiently extracting relatively pure DNA was assessed based on the total DNA yield, concentration, purity ratios (A260/A280 and A260/A230), spectral profile and agarose gel electrophoresis analysis. The quality of the isolated DNA was further analysed for PCR inhibition using a murine specific ATP1A3 qPCR assay and mtDNA/Y-chromosome ratio determination assay. The suitability of the extracted DNA for downstream applications such as end-point SNP genotyping, was tested using PCR-RFLP analysis of the AGTR1-1166A>C variant, a mirSNP having pharmacogenetic relevance in cardiovascular diseases. Compared to the traditional phenol-chloroform/proteinase-K method, our results indicated the Rapid-Method to be a more suitable protocol for genomic DNA extraction from human whole blood in terms of DNA quantity, quality, safety, processing time and cost. The Rapid-Method, which is based on a simple salting-out procedure, is not only safe and cost-effective, but also has the added advantage of being scaled up to process variable sample volumes, thus enabling it to be applied in large-scale epidemiological studies.

  20. Genotyping of Chlamydophila psittaci using a new DNA microarray assay based on sequence analysis of ompA genes

    Directory of Open Access Journals (Sweden)

    Schubert Evelyn

    2008-04-01

    Full Text Available Abstract Background The currently used genotyping system for the avian zoonotic pathogen Chlamydophila (C. psittaci has evolved from serology and is based on ompA sequence variations. It includes seven avian and two non-avian genotypes. Restriction enzyme cleavage of the amplified ompA gene and, less frequently, ompA sequencing are being used for examination, but, beside methodological limitations, an increasing number of recently tested strains could not be assigned to any established genotype. Results Comprehensive analysis of all available ompA gene sequences has revealed a remarkable genetic diversity within the species C. psittaci, which is only partially covered by the present genotyping scheme. We suggest adjustments and extensions to the present scheme, which include the introduction of subgroups to the more heterogeneous genotypes A, E/B and D, as well as six provisional genotypes representing so far untypable strains. The findings of sequence analysis have been incorporated in the design of a new DNA microarray. The ArrayTube™ microarray-based ompA genotyping assay has been shown to discriminate among established genotypes and identify so far untyped strains. Its high specificity, which allows detection of single-nucleotide polymorphisms, is due to the parallel approach consisting in the use of 35 hybridization probes derived from variable domains 2 and 4 of the ompA gene. Conclusion The traditional genotyping system does not adequately reflect the extent of intra-species heterogeneity in ompA sequences of C. psittaci. The newly developed DNA microarray-based assay represents a promising diagnostic tool for tracing epidemiological chains, exploring the dissemination of genotypes and identifying non-typical representatives of C. psittaci.

  1. DNA aptamer beacon assay for C-telopeptide and handheld fluorometer to monitor bone resorption.

    Science.gov (United States)

    Bruno, John Gordon; Carrillo, Maria P; Phillips, Taylor; Hanson, Douglas; Bohmann, Jonathan A

    2011-09-01

    A novel DNA aptamer beacon is described for quantification of a 26-amino acid C-telopeptide (CTx) of human type I bone collagen. One aptamer sequence and its reverse complement dominated the aptamer pool (31.6% of sequenced clones). Secondary structures of these aptamers were examined for potential binding pockets. Three-dimensional computer models which analyzed docking topologies and binding energies were in agreement with empirical fluorescence experiments used to select one candidate loop for beacon assay development. All loop structures from the aptamer finalists were end-labeled with TYE 665 and Iowa Black quencher for comparison of beacon fluorescence levels as a function of CTx concentration. The optimal beacon, designated CTx 2R-2h yielded a low ng/ml limit of detection using a commercially available handheld fluorometer. The CTx aptamer beacon bound full-length 26-amino acid CTx peptide, but not a shorter 8-amino acid segment of CTx peptide which is a common target for commercial CTx ELISA kits. The prototype assay was shown to detect CTx peptide from human urine after creatinine and urea were removed by size-exclusion chromatography to prevent nonspecific denaturing of the aptamer beacon. This work demonstrates the potential of aptamer beacons to be utilized for rapid and sensitive bone health monitoring in a handheld or point-of-care format.

  2. Transcriptional profiling of TLR-4/7/8-stimulated guinea pig splenocytes and whole blood by bDNA assay.

    Science.gov (United States)

    Ching, Lance K; Mompoint, Farah; Guderian, Jeffrey A; Picone, Alex; Orme, Ian M; Coler, Rhea N; Reed, Steven G; Baldwin, Susan L

    2011-10-28

    Toll-like receptor (TLR) agonists are currently being examined as adjuvants for vaccines, with several lead candidates now in licensed products or in late-stage clinical development. Guinea pigs are widely used for preclinical testing of drugs and vaccines; however, evaluation of TLR agonists in this model is hindered by the limited availability of immunological tools and reagents. In this study, we validated the use of a branched-chain DNA (bDNA) assay known as the QuantiGene Plex 2.0 Reagent System for measuring innate cytokine and chemokine mRNA levels following TLR stimulation of guinea pig cells. Gene expression for T-helper-1 (Th1) polarizing cytokines (TNF-α, IL-1β, IL-12) and chemokines (CXCL1, CCL2) was upregulated following ex vivo stimulation of guinea pig splenocytes and whole blood with TLR-4 or TLR-7/8 agonists. These data confirm the utility of the QuantiGene system both as an alternative to RT-PCR for measuring transcript levels and as a high-throughput screening tool for dissecting the immunological response to TLR stimulation in guinea pigs. Overall, the QuantiGene platform is reliable, reproducible, and sensitive. These agonists have the potential to be used as adjuvant components in vaccines against various pathogens. Copyright © 2011 Elsevier B.V. All rights reserved.

  3. Evaluation of sperm DNA damage in bulls by TUNEL assay as a parameter of semen quality.

    Science.gov (United States)

    Takeda, Kumiko; Uchiyama, Kyoko; Kinukawa, Masashi; Tagami, Takahiro; Kaneda, Masahiro; Watanabe, Shinya

    2015-01-01

    Sperm DNA damage affects the conception rate resulting from human assisted reproduction technology. The objective of this study was to adapt the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay to provide a quality parameter for bull semen based on the detection of sperm DNA damage. Fresh semen was collected from two Japanese Black bulls (A, B) several times over the course of a year, and the percentage of TUNEL-positive spermatozoa (sperm TUNEL index) was determined. Individual differences in semen were detected using the sperm TUNEL index in these bulls (P bulls with a conception rate lower than 10%) and Holstein (n = 34) bulls were analyzed. The average sperm TUNEL index and conception rate resulting from artificial insemination (AI) were 4.7% and 55.7% for Japanese Black, and 4.9% and 39.5% for Holstein, respectively. A weak negative correlation between sperm TUNEL index and conception rate was observed in Holstein bulls (P bulls with more than 10% sperm TUNEL index were studied, and these samples showed low sperm viability. However, semen resulting in a very low conception rate did not have a high sperm TUNEL index. Although it would be difficult to predict a low conception rate resulting from AI using the sperm TUNEL index alone, the index can be used as an additional parameter to provide a more comprehensive description of semen quality.

  4. DNA comet assay to identify different freezing temperatures of irradiated liver chicken

    Energy Technology Data Exchange (ETDEWEB)

    Duarte, Renato C.; Mozeika, Michel A.; Fanaro, Gustavo B.; Villavicencio, Anna L.C.H. [Instituto de Pesquisas Energeticas e Nucleares (IPEN/CNEN-SP), Sao Paulo, SP (Brazil)], e-mail: renatocduarte@yahoo.com.br; Marchioni, Eric [Universite de Strasbourg, Illkirch (France). Faculte de Pharmacie. Lab. de Chimie Analytique et Sciences de l' Aliment

    2009-07-01

    The cold chain is a succession of steps which maintain the food at low temperature. The thawed food never be frozen again and the best solution being to consume it quickly to avoid the microorganism growth which causes decay and nutrients damage. One of most important point is that freezing process, unlike irradiation, do not destroy microorganisms, only inactive them as long as they remain in a frozen state. The Comet Assay is an original test used to detect irradiated foods that's recognize the DNA damage and can then be used to control the overall degradation of the food and in a certain extend to evaluate the damage caused by irradiation, different forms of freeze and storage time on liver chicken cells. Different freezing temperatures were used, deep freeze -196 deg C and slow freeze -10 deg C. Samples were irradiated in a {sup 60}Co irradiator with 1.5, 3.0 and 4.5 kGy radiation doses. Fast freezing technique induces a low percent of DNA degradation comparing to slow freezing technique. This procedure could be a good choose to chicken freezing processing. (author)

  5. Variation in the measurement of DNA damage by comet assay measured by the ECVAG dagger inter-laboratory validation trial

    DEFF Research Database (Denmark)

    Forchhammer, Lykke; Johansson, Clara; Loft, Steffen;

    2010-01-01

    The comet assay has become a popular method for the assessment of DNA damage in biomonitoring studies and genetic toxicology. However, few studies have addressed the issue of the noted inter-laboratory variability of DNA damage measured by the comet assay. In this study, 12 laboratories analysed...... by the different laboratories as evidenced by an inter-laboratory coefficient of variation (CV) of 47%. Adjustment of the primary comet assay end points by a calibration curve prepared in each laboratory reduced the CV to 28%, a statistically significant reduction (P ... of the inter-laboratory variation originated from differences in image analysis, whereas the intra-laboratory variation was considerably smaller than the variation between laboratories. In summary, adjustment of primary comet assay results by reference standards reduces inter-laboratory variation in the level...

  6. Comparison of three human papillomavirus DNA assays and one mRNA assay in women with abnormal cytology

    DEFF Research Database (Denmark)

    Rebolj, Matejka; Lynge, Elsebeth; Ejegod, Ditte;

    2014-01-01

    OBJECTIVE: To compare the clinical characteristics of four human papillomavirus (HPV) assays: hybrid capture 2 (HC2), cobas, CLART, and APTIMA in Danish women with abnormal cytology. METHODS: SurePath samples from 367 consecutive women from Copenhagen, with atypical squamous cells of undetermined...

  7. Novel wide-range quantitative nested real-time PCR assay for Mycobacterium tuberculosis DNA: development and methodology.

    Science.gov (United States)

    Takahashi, Teruyuki; Tamura, Masato; Asami, Yukihiro; Kitamura, Eiko; Saito, Kosuke; Suzuki, Tsukasa; Takahashi, Sachiko Nonaka; Matsumoto, Koichi; Sawada, Shigemasa; Yokoyama, Eise; Takasu, Toshiaki

    2008-05-01

    Previously, we designed an internally controlled quantitative nested real-time (QNRT) PCR assay for Mycobacterium tuberculosis DNA in order to rapidly diagnose tuberculous meningitis. This technique combined the high sensitivity of nested PCR with the accurate quantification of real-time PCR. In this study, we attempted to improve the original QNRT-PCR assay and newly developed the wide-range QNRT-PCR (WR-QNRT-PCR) assay, which is more accurate and has a wider detection range. For use as an internal-control "calibrator" to measure the copy number of M. tuberculosis DNA, an original new-mutation plasmid (NM-plasmid) was developed. It had artificial random nucleotides in five regions annealing specific primers and probes. The NM-plasmid demonstrated statistically uniform amplifications (F = 1.086, P = 0.774) against a range (1 to 10(5)) of copy numbers of mimic M. tuberculosis DNA and was regarded as appropriate for use as a new internal control in the WR-QNRT-PSR assay. In addition, by the optimization of assay conditions in WR-QNRT-PCR, two-step amplification of target DNA was completely consistent with the standard curve of this assay. Due to the development of the NM-plasmid as the new internal control, significantly improved quantitative accuracy and a wider detection range were realized with the WR-QNRT-PCR assay. In the next study, we will try to use this novel assay method with actual clinical samples and examine its clinical usefulness.

  8. Further evaluation of an updated PCR assay for the detection of Schistosoma mansoni DNA in human stool samples

    Directory of Open Access Journals (Sweden)

    Luciana I Gomes

    2009-12-01

    Full Text Available A previously reported sensitive PCR assay for the detection of Schistosoma mansoni DNA was updated and evaluated. Changes in the DNA extraction method, including the use of a worldwide available commercial kit and the inclusion of additional quality control measures, increased the robustness of the test, as confirmed by the analysis of 67 faecal samples from an endemic area in Brazil. The PCR assay is at hand as a proven, reliable diagnostic test for the control of schistosomiasis in specific settings.

  9. RNA (E6 and E7) assays versus DNA (E6 and E7) assays for risk evaluation for women infected with human papillomavirus.

    Science.gov (United States)

    Cattani, Paola; Siddu, Alessia; D'Onghia, Sara; Marchetti, Simona; Santangelo, Rosaria; Vellone, Valerio G; Zannoni, Gian Franco; Fadda, Giovanni

    2009-07-01

    In the majority of cases, high-risk human papillomavirus (HR HPV) infections regress spontaneously, with only a small percentage progressing to high-grade lesions. Current screening methods are based on DNA detection. An alternative would be to monitor expression of the E6 and E7 viral oncogenes continuously expressed by malignant phenotypes. In the work reported in this paper, we compared the two methods for a group of women with high-risk HPV infections. Cervical specimens from 400 women, previously found to be HPV DNA positive, were analyzed for HPV DNA by a liquid hybridization assay and typed by multiplex PCR (for types 16, 18, 31, and 33). Identification of HR HPV E6 and E7 RNA transcripts was performed using real-time reverse transcription-PCR and nucleic acid sequence-based amplification assays. Results were compared with concurrent cytological data. HR HPVs were found in 61.2% of patients. The most common genotype was HPV type 16 (HPV-16) (47.1%), followed by HPV-18, HPV-31, and HPV-33. Nine percent of cases involved other genotypes. Among 223 HPV DNA-positive samples, only 118 were positive in the RNA test. Among HPV DNA-positive patients with normal cytology, we detected E6 and E7 RNA transcripts in two cases (18.2%). The rate of detection increased gradually with the grade of the observed lesions, rising from 20% for patients with atypical squamous cells of undetermined significance to 48.1% for women with low-grade squamous intraepithelial lesions and 86.3% for those with high-grade squamous intraepithelial lesions. These results suggest that testing for HPV E6 and E7 transcripts could be a useful tool for screening and patient management, providing more accurate predictions of risk than those obtained by DNA testing.

  10. A facile, branched DNA assay to quantitatively measure glucocorticoid receptor auto-regulation in T-cell acute lymphoblastic leukemia

    Institute of Scientific and Technical Information of China (English)

    Jason R. Schwartz; Purvaba J. Sarvaiya; Lily E. Leiva; Maria C. Velez; Tammuella C. Singleton; Lolie C. Yu; Wayne V. Vedeckis

    2012-01-01

    Glucocorticoid (GC) steroid hormones are used to treat acute lymphoblastic leukemia (ALL) because of their pro-apoptotic effects in hematopoietic cells.However,not all leukemia cells are sensitive to GC,and no assay to stratify patients is available.In the GC-sensitive T-cell ALL cell line CEM-C7,auto-up-regulation of RNA transcripts for the glucocorticoid receptor (GR) correlates with increased apoptotic response.This study aimed to determine if a facile assay of GR transcript levels might be promising for stratifying ALL patients into hormone-sensitive and hormone-resistant populations.The GR transcript profiles of various lymphoid cell lines and 4 bone marrow samples from patients with T-cell ALL were analyzed using both an optimized branched DNA (bDNA) assay and a real-time quantitative reverse transcription-polymerase chain reaction assay.There were significant correlations between both assay platforms when measuring total GR (exon 5/6) transcripts in various cell lines and patient samples,but not for a probe set that detects a specific,low abundance GR transcript (exon 1A3).Our results suggest that the bDNA platform is reproducible and precise when measuring total GR transcripts and,with further development,may ultimately offer a simple clinical assay to aid in the prediction of GC-sensitivity in ALL patients.

  11. Freeze-dried stallion spermatozoa: evaluation of two chelating agents and comparative analysis of three sperm DNA damage assays.

    Science.gov (United States)

    Olaciregui, M; Luño, V; Martí, J I; Aramayona, J; Gil, L

    2016-11-01

    During the freeze-drying procedure, sperm DNA might become damaged by both freezing and drying stresses. Sperm DNA status can be detected using well-established assays; however, most techniques are expensive and involve elaborate protocols and equipment. Indirect assessments can provide alternative strategies. The objective of this study was to compare a simple test of DNA status using Diff-Quik (DQ) with two established procedures: acridine orange test (AOT) and sperm chromatin dispersion (SCD) on freeze-dried (FD) stallion spermatozoa. Ejaculated spermatozoa from three stallions were freeze-dried in basic medium supplemented with two different chelating agents: EGTA or EDTA. After rehydration, the spermatozoa were subjected to DNA damage detection using a SCDt, AOT and DQ stain simultaneously. The results showed that the DNA damage levels in the EGTA group were significantly lower than those in the EDTA group. AOT detected a significantly higher proportion of spermatozoa with fragmented DNA than DQ and SCD. The results of the SCD test and DQ stain exhibited a significant positive correlation for DNA fragmentation (r = 0.528), whereas a negative correlation was observed between SCD, DQ and AOT (r = -0.134 and r = -0.332 respectively). The present study shows that both the SCD test and DQ assay are effective methods for detecting FD stallion sperm DNA fragmentation, whereas using of AOT is questionable.

  12. Pretreatment with mixed-function oxidase inducers increases the sensitivity of the hepatocyte/DNA repair assay

    Energy Technology Data Exchange (ETDEWEB)

    Shaddock, J.G.; Heflich, R.H.; McMillan, D.C.; Hinson, J.A.; Casciano, D.A. (National Center for Toxicological Research, Jefferson, AK (USA) Univ. of Arkansas for Medical Sciences, Little Rock (USA))

    1989-01-01

    A recent National Toxicology Program evaluation indicates that the rat hepatocyte/DNA repair assay has a high false-negative rate and that it is insensitive to some genotoxic hepatocarcinogens as well as other species and organ-specific carcinogens. In this study, the authors examined whether the sensitivity of the hepatocyte/DNA repair assay might be increased through animal pretreatment with various hepatic mixed-function oxidase inducers, i.e., Aroclor 1254, phenobarbital, and 3,3{prime},4,4{prime}-tetrachloroazobenzene (TCAB). The effects on unscheduled DNA synthesis (UDS), a measured of DNA damage and repair, were studied in cultures exposed to known and/or potential carcinogens that had been evaluated as negative or questionable or that produced conflicting results with hepatocytes isolated from uninduced animals. 4,4{prime}-Oxydianiline, 1-nitropy-rene, and TCAB produced concentration-dependent increases in UDS in hepatocytes from rats pretreated with Aroclor 1254. 4,4{prime}-Oxydianiline and TCAB also induced a dose-dependent increase in DNA repair in hepatocytes from rats pretreated with phenobarbital, whereas 1-nitropyrene was negative. These data indicate that the limited sensitivity to chemical carcinogens displayed by the hepatocyte/DNA repair assay may be increased by using hepatocytes isolated from animals exposed to hepatic mixed-function oxidase inducers.

  13. DNA-PKcs-OBA/Ku associate in the absence of DNA, as revealed by two-dimensional capillary gel electromobility shift assay.

    Science.gov (United States)

    Ruiz, Marcia T; Nichols, Amanda; Price, Gerald B; Zannis-Hadjopoulos, Maria

    2002-08-01

    Ors-binding activity (OBA) has been previously purified by its ability to specifically interact with A3/4, a 36-bp mammalian origin consensus sequence [1]. Peptide sequence analyses identified OBA as Ku86, the largest subunit of Ku antigen, a heterodimeric protein (Ku70/Ku86) involved in several autoimmune disorders [2-5]. The affinity-purified fraction containing OBA/Ku is also enriched for DNA-dependent protein kinase DNA-PKcs, the catalytic subunit of the DNA-PK holoenzyme, of which Ku antigen is the DNA-binding subunit [6-8]. Glycerol-gradient sedimentation analyses have demonstrated the presence of OBA/Ku in a high-molecular-weight complex. In order to investigate whether OBA/Ku and DNA-PKcs are associated in this fraction, we have used a modification of the two-dimensional gel electrophoresis technique originally described [9]. Electromobility shift assays were developed in native capillary gels, which were subsequently used for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in the second dimension. The gels were then processed for Western blotting using the Ku70, Ku86 and DNA-PKcs antibodies. This approach has revealed the association of OBA/Ku and DNA-PKcs to give rise to the DNA-PK holoenzyme irrespective of the presence, or the absence of DNA. Altogether, we have proven the utility of this technique for the study of protein-protein and protein-DNA interactions.

  14. Detection of DNA damage by Escherichia coli UvrB-binding competition assay is limited by the stability of the UvrB-DNA complex.

    Science.gov (United States)

    Routledge, M N; Allan, J M; Garner, R C

    1997-07-01

    To investigate the use of UvrB-binding to detect DNA damage, mobility shift gel electrophoresis was used to detect binding of UvrB protein to a 136 bp DNA fragment that was randomly adducted with aflatoxin B1 8,9-epoxide and end-labelled with 32P. After polyacrylamide gel electrophoresis, the shifted band that contained DNA bound by UvrB was quantified as a percentage of total radioactive substrate DNA. This method was applied to analyse plasmid DNA that was adducted with various DNA modifying agents in vitro. These adducts competed for UvrB-binding to the labelled substrate. By competing for UvrB-binding with 10 ng of plasmid DNA that was adducted with known levels of aflatoxin B1, 2-amino-3-methylimidazo[4,5-f]quinoline, or benzo[a]pyrene diol epoxide, UvrB competition could be quantified for DNA adducted with between one adduct in 10(2) and one adduct in 10(5) normal nucleotides. However, plasmid DNA exposed to N-methyl-N-nitrosourea or methylene blue + visible light, did not compete for UvrB-binding, even though the presence of UvrABC sensitive sites were confirmed on this DNA by a UvrABC incision assay. Mono-adducted 96-bp DNA substrates, which contained an internal 32P-label and either a single apurinic site, aflatoxin B1-guanine adduct, O6-methylguanine, 8-oxo-deoxyguanosine or non-adducted guanine, were also used as substrates for UvrA- and UvrB-binding to examine the stability of UvrB-DNA complexes with specific adducts. Under similar conditions used for the competition assay, significant UvrB-binding was seen only for the aflatoxin adducted substrate. These results suggest that stability of UvrB-binding varies greatly between bulky and non-bulky adducts. It was also found that rat liver DNA from untreated rats inhibited UvrB-binding to the substrate DNA in the competition assay, to a degree that was equivalent to competition with plasmid adducted at one adduct in 10(3) normal nucleotides.

  15. Application of the comet assay for investigation of oxidative DNA damage in equine peripheral blood mononuclear cells.

    Science.gov (United States)

    Marlin, David J; Johnson, Lucy; Kingston, Demelza A; Smith, Nicola C; Deaton, Chris M; Mann, Sarah; Heaton, Paul; Van Vugt, Fenneke; Saunders, Kelly; Kydd, Julia; Harris, Pat A

    2004-08-01

    Oxidative stress occurs when antioxidant defense mechanisms are overwhelmed by free radicals and may lead to DNA damage, which has been implicated in processes such as aging and diseases such as cancer. The two main techniques presently used to quantify DNA damage are measurement of 8-hydroxydeoxyguanosine and the Comet assay (also known as single-cell gel electrophoresis). The aim of this study was to apply the comet assay to equine peripheral blood mononuclear cells (PBMCs) and identify two conditions in which we hypothesized that oxidative DNA damage would be increased in PBMCs: aging and equine recurrent airway obstruction (RAO, a condition similar to human asthma). The images obtained were similar to those previously published for humans, cats, and dogs. The optimum concentration of H(2)O(2) to estimate susceptibility to exogenous damage was 50 microM. Mean intraassay coefficients of variation were 4.7 and 9.7% for endogenous and exogenous tail-DNA quantities, respectively, and 7.3 and 8.3%, respectively, for interassay coefficients. There was no significant difference in either endogenous or exogenous percentages of tail DNA for samples collected from six ponies on three consecutive days. There was no significant difference in endogenous, exogenous, or exogenous (corrected for endogenous) oxidative DNA damage between mature and aged ponies. However, young pony foals had significantly less endogenous DNA damage than mature or aged ponies (P < 0.05). RAO-affected horses without airway inflammation (i.e., in clinical remission) had significantly greater endogenous damage compared with non-RAO-affected control animals (P = 0.009). There was a significant correlation between endogenous percentage of tail DNA in PBMCs and red blood cell hemolysate glutathione concentration (r = 0.720; P < 0.001). In conclusion, the comet assay appears to be suitable for investigating DNA damage in equine PBMCs.

  16. Relation between DNA damage measured by comet assay and OGG1 Ser326Cys polymorphism in antineoplastic drugs biomonitoring

    Directory of Open Access Journals (Sweden)

    Carina Ladeira

    2015-09-01

    Full Text Available Antineoplastic drugs are hazardous chemical agents used mostly in the treatment of patients with cancer, however health professionals that handle and administer these drugs can become exposed and develop DNA damage. Comet assay is a standard method for assessing DNA damage in human biomonitoring and, combined with formamidopyrimidine DNA glycosylase (FPG enzyme, it specifically detects DNA oxidative damage.The aim of this study was to investigate genotoxic effects in workers occupationally exposed to cytostatics (n = 46, as compared to a control group with no exposure (n = 46 at two Portuguese hospitals, by means of the alkaline comet assay. The potential of the OGG1 Ser326Cys polymorphism as a susceptibility biomarker was also investigated. Exposure was evaluated by investigating the contamination of surfaces and genotoxic assessment was done by alkaline comet assay in peripheral blood lymphocytes. OGG1 Ser326Cys (rs1052133 polymorphism was studied by Real Time PCR.As for exposure assessment, there were 121 (37% positive samples out of a total of 327 samples analysed from both hospitals. No statistically significant differences (Mann-Whitney test, p > 0.05 were found between subjects with and without exposure, regarding DNA damage and oxidative DNA damage, nevertheless the exposed group exhibited higher values. Moreover, there was no consistent trend regarding the variation of both biomarkers as assessed by comet assay with OGG1 polymorphism.Our study was not statistically significant regarding occupational exposure to antineoplastic drugs and genetic damage assessed by comet assay. However, health professionals should be monitored for risk behaviour, in order to ensure that safety measures are applied and protection devices are used correctly.

  17. Ultrasensitive electrochemical DNA assay based on counting of single magnetic nanobeads by a combination of DNA amplification and enzyme amplification.

    Science.gov (United States)

    Zhang, Xiaoli; Li, Linlin; Li, Lu; Chen, Jia; Zou, Guizheng; Si, Zhikun; Jin, Wenrui

    2009-03-01

    An ultrasensitive electrochemical method for determination of DNA is developed based on counting of single magnetic nanobeads (MNBs) corresponding to single DNA sequences combined with a double amplification (DNA amplification and enzyme amplification). In this method, target DNA (t-DNA) is captured on a streptavidin-coated substrate via biotinylated capture DNA. Then, MNBs functionalized with first-probe DNAs (p1-DNA-MNBs) are conjugated to t-DNA sequences with a ratio of 1:1. Subsequently, the p1-DNA-MNBs are released from the substrate via dehybridization. The released p1-DNA-MNBs are labeled with alkaline phosphatase (AP) using biotinylated second-probe DNAs (p2-DNAs) and streptavidin-AP conjugates. The resultant AP-p2-DNA-p1-DNA-MNBs with enzyme substrate disodium phenyl phosphate (DPP) are continuously introduced through a capillary as the microsampler and microreactor at 40 degrees C. AP on the AP-p2-DNA-p1-DNA-MNBs converts a huge number of DPP into its product phenol, and phenol zones are produced around each moving AP-p2-DNA-p1-DNA-MNB. The phenol zones are continuously delivered to the capillary outlet and detected by a carbon fiber disk bundle electrode at 1.05 V. An elution curve with peaks is obtained. Each peak is corresponding to a phenol zone relative to single t-DNA sequence. The peaks on the elution curve are counted for quantification. The number of the peaks is proportional to the concentration of t-DNA in a range of 5.0 x 10(-16) to 1.0 x 10(-13) mol/L.

  18. Shortening of alkaline DNA unwinding time does not interfere with detecting DNA damage to mouse and human spermatozoa in the comet assay

    Institute of Scientific and Technical Information of China (English)

    Hirokazu Kusakabe; Hiroyuki Tateno

    2011-01-01

    The comet assay was performed on mouse and human spermatozoa to examine the effect of alkaline DNA unwinding time.The spermatozoa were treated in vitrowith the DNA-damaging agents,methyl methanesulfonate(MMS)or hydrogen peroxide(H2O2),and then embedded in agarose gel on glass slides.The slides were immersed in alkaline solution(>pH 1.3)for 1,5,10 and 20 min,and then subjected to the electrophoresis under neutral conditions.In mouse spermatozoa,comet tails seen in solvent controls became brighter and longer as the alkaline DNA unwinding time increased.However,in the MMS-treated mouse spermatozoa,a smaller difference in the damage from that in the solvent control was seen with time within a dose.DNA damage induced by H2O2 could also be detected accurately after alkali treatment for 1-20 min.In human spermatozoa,DNA damage induced by MMS and H2O2 could be detected in a dose-dependent manner after alkali treatment for 1 min.The ability of the comet assay to detect DNA damage was not adversely affected by the short period(1 min)of the alkaline DNA unwinding time.

  19. A Functional Henipavirus Envelope Glycoprotein Pseudotyped Lentivirus Assay System

    Directory of Open Access Journals (Sweden)

    Broder Christopher C

    2010-11-01

    Full Text Available Abstract Background Hendra virus (HeV and Nipah virus (NiV are newly emerged zoonotic paramyxoviruses discovered during outbreaks in Queensland, Australia in 1994 and peninsular Malaysia in 1998/9 respectively and classified within the new Henipavirus genus. Both viruses can infect a broad range of mammalian species causing severe and often-lethal disease in humans and animals, and repeated outbreaks continue to occur. Extensive laboratory studies on the host cell infection stage of HeV and NiV and the roles of their envelope glycoproteins have been hampered by their highly pathogenic nature and restriction to biosafety level-4 (BSL-4 containment. To circumvent this problem, we have developed a henipavirus envelope glycoprotein pseudotyped lentivirus assay system using either a luciferase gene or green fluorescent protein (GFP gene encoding human immunodeficiency virus type-1 (HIV-1 genome in conjunction with the HeV and NiV fusion (F and attachment (G glycoproteins. Results Functional retrovirus particles pseudotyped with henipavirus F and G glycoproteins displayed proper target cell tropism and entry and infection was dependent on the presence of the HeV and NiV receptors ephrinB2 or B3 on target cells. The functional specificity of the assay was confirmed by the lack of reporter-gene signals when particles bearing either only the F or only G glycoprotein were prepared and assayed. Virus entry could be specifically blocked when infection was carried out in the presence of a fusion inhibiting C-terminal heptad (HR-2 peptide, a well-characterized, cross-reactive, neutralizing human mAb specific for the henipavirus G glycoprotein, and soluble ephrinB2 and B3 receptors. In addition, the utility of the assay was also demonstrated by an examination of the influence of the cytoplasmic tail of F in its fusion activity and incorporation into pseudotyped virus particles by generating and testing a panel of truncation mutants of NiV and HeV F

  20. Nested real-time quantitative polymerase chain reaction assay for detection of hepatitis B virus covalently closed circular DNA

    Institute of Scientific and Technical Information of China (English)

    XU Chun-hai; LI Zhao-shen; DAI Jun-ying; ZHU Hai-yang; YU Jian-wu; L(U) Shu-Ian

    2011-01-01

    peripheral blood mononuclear cells; marrow mononuclear cells Background Successful treatment of hepatitis B can be achieved only if the template for hepatitis B virus (HBV) DNA replication, the covalently closed circular HBV DNA (cccDNA) can be completely cleared. To date, detecting cccDNA remains clinically challenging. The purpose of this study was to develop a nested real-time quantitative polymerase chain reaction (PCR) assay for detecting HBV cccDNA in peripheral blood mononuclear cells (PBMCs) and bone marrow mononuclear cells (MMNCs).Methods Based on the structural differences between HBV cccDNA and HBV relaxed circular DNA (rcDNA), two pairs of primers were synthesized as well as a downstream TaqMan probe. Blood and bone marrow samples were collected from hepatitis B patients and healthy controls. To remove rcDNA, samples were incubated with mung bean nuclease and the resultant purified HBV cccDNA was then amplified by nested real-time fluorescence quantitative PCR. The cccDNA levels were calculated using a positive standard.Results The nested real-time fluorescence quantitative PCR method for HBV cccDNA was successful, with a linear range of 3.0x102 copies/ml to 3.9x108 copies/ml. Of the 25 PBMC samples and 7 MMNC samples obtained from chronic hepatitis B or liver cirrhosis patients, 3 MMNC samples and 9 PBMC samples were positive for HBV cccDNA, while all of the 21 PBMC samples from healthy controls were negative.Conclusion The nested real-time fluorescence quantitative PCR may be used as an important tool for detecting cccDNA in hepatitis B patients.

  1. Human cell chips: adapting DNA microarray spotting technology to cell-based imaging assays.

    Science.gov (United States)

    Hart, Traver; Zhao, Alice; Garg, Ankit; Bolusani, Swetha; Marcotte, Edward M

    2009-10-28

    Here we describe human spotted cell chips, a technology for determining cellular state across arrays of cells subjected to chemical or genetic perturbation. Cells are grown and treated under standard tissue culture conditions before being fixed and printed onto replicate glass slides, effectively decoupling the experimental conditions from the assay technique. Each slide is then probed using immunofluorescence or other optical reporter and assayed by automated microscopy. We show potential applications of the cell chip by assaying HeLa and A549 samples for changes in target protein abundance (of the dsRNA-activated protein kinase PKR), subcellular localization (nuclear translocation of NFkappaB) and activation state (phosphorylation of STAT1 and of the p38 and JNK stress kinases) in response to treatment by several chemical effectors (anisomycin, TNFalpha, and interferon), and we demonstrate scalability by printing a chip with approximately 4,700 discrete samples of HeLa cells. Coupling this technology to high-throughput methods for culturing and treating cell lines could enable researchers to examine the impact of exogenous effectors on the same population of experimentally treated cells across multiple reporter targets potentially representing a variety of molecular systems, thus producing a highly multiplexed dataset with minimized experimental variance and at reduced reagent cost compared to alternative techniques. The ability to prepare and store chips also allows researchers to follow up on observations gleaned from initial screens with maximal repeatability.

  2. Human cell chips: adapting DNA microarray spotting technology to cell-based imaging assays.

    Directory of Open Access Journals (Sweden)

    Traver Hart

    Full Text Available Here we describe human spotted cell chips, a technology for determining cellular state across arrays of cells subjected to chemical or genetic perturbation. Cells are grown and treated under standard tissue culture conditions before being fixed and printed onto replicate glass slides, effectively decoupling the experimental conditions from the assay technique. Each slide is then probed using immunofluorescence or other optical reporter and assayed by automated microscopy. We show potential applications of the cell chip by assaying HeLa and A549 samples for changes in target protein abundance (of the dsRNA-activated protein kinase PKR, subcellular localization (nuclear translocation of NFkappaB and activation state (phosphorylation of STAT1 and of the p38 and JNK stress kinases in response to treatment by several chemical effectors (anisomycin, TNFalpha, and interferon, and we demonstrate scalability by printing a chip with approximately 4,700 discrete samples of HeLa cells. Coupling this technology to high-throughput methods for culturing and treating cell lines could enable researchers to examine the impact of exogenous effectors on the same population of experimentally treated cells across multiple reporter targets potentially representing a variety of molecular systems, thus producing a highly multiplexed dataset with minimized experimental variance and at reduced reagent cost compared to alternative techniques. The ability to prepare and store chips also allows researchers to follow up on observations gleaned from initial screens with maximal repeatability.

  3. Label-free detection of kanamycin based on a G-quadruplex DNA aptamer-based fluorescent intercalator displacement assay

    Science.gov (United States)

    Xing, Yun-Peng; Liu, Chun; Zhou, Xiao-Hong; Shi, Han-Chang

    2015-01-01

    This work was the first to report that the kanamycin-binding DNA aptamer (5'-TGG GGG TTG AGG CTA AGC CGA-3') can form stable parallel G-quadruplex DNA (G4-DNA) structures by themselves and that this phenomenon can be verified by nondenaturing polyacrylamide gel electrophoresis and circular dichroism spectroscopy. Based on these findings, we developed a novel label-free strategy for kanamycin detection based on the G4-DNA aptamer-based fluorescent intercalator displacement assay with thiazole orange (TO) as the fluorescence probe. In the proposed strategy, TO became strongly fluorescent upon binding to kanamycin-binding G4-DNA. However, the addition of kanamycin caused the displacement of TO from the G4-DNA-TO conjugate, thereby resulting in decreased fluorescent signal, which was inversely related to the kanamycin concentration. The detection limit of the proposed assay decreased to 59 nM with a linear working range of 0.1 μM to 20 μM for kanamycin. The cross-reactivity against six other antibiotics was negligible compared with the response to kanamycin. A satisfactory recovery of kanamycin in milk samples ranged from 80.1% to 98.0%, confirming the potential of this bioassay in the measurement of kanamycin in various applications. Our results also served as a good reference for developing similar fluorescent G4-DNA-based bioassays in the future.

  4. DNA-Based Enzyme Reactors and Systems

    Directory of Open Access Journals (Sweden)

    Veikko Linko

    2016-07-01

    Full Text Available During recent years, the possibility to create custom biocompatible nanoshapes using DNA as a building material has rapidly emerged. Further, these rationally designed DNA structures could be exploited in positioning pivotal molecules, such as enzymes, with nanometer-level precision. This feature could be used in the fabrication of artificial biochemical machinery that is able to mimic the complex reactions found in living cells. Currently, DNA-enzyme hybrids can be used to control (multi-enzyme cascade reactions and to regulate the enzyme functions and the reaction pathways. Moreover, sophisticated DNA structures can be utilized in encapsulating active enzymes and delivering the molecular cargo into cells. In this review, we focus on the latest enzyme systems based on novel DNA nanostructures: enzyme reactors, regulatory devices and carriers that can find uses in various biotechnological and nanomedical applications.

  5. DNA damage by ochratoxin A in rat kidney assessed by the alkaline comet assay

    Directory of Open Access Journals (Sweden)

    D. Zeljezic

    2006-12-01

    Full Text Available There are few studies of ochratoxin A (OTA genotoxicity in experimental animals and the results obtained with cell cultures are inconsistent, although the carcinogenic potential of OTA for the kidney of experimental animals has been well established. We studied the genotoxic potential of OTA in the kidney of adult female Wistar rats (5 in each group treated intraperitoneally with OTA (0.5 mg kg body weight-1 day-1 for 7, 14, and 21 days measuring DNA mobility on agarose gel stained with ethidium-bromide using standard alkaline single-cell gel electrophoresis (comet assay. Negative control animals were treated with solvent (Tris buffer, 1.0 mg/kg and positive control animals were treated with methyl methanesulfonate (40 mg/kg according to the same schedule. OTA concentrations in plasma and kidney homogenates in 7-, 14-, and 21-day treated animals were 4.86 ± 0.53, 7.52 ± 3.32, 7.85 ± 2.24 µg/mL, and 0.87 ± 0.09, 0.99 ± 0.06, 1.09 ± 0.15 µg/g, respectively. In all OTA-treated groups, the tail length, tail intensity, and tail moment in kidney tissue were significantly higher than in controls (P < 0.05. The tail length and tail moment were higher after 14 days than after 7 days of treatment (P < 0.05, and still higher after 21 days (P < 0.05. The highest tail intensity was observed in animals treated for 21 days, and it differed significantly from animals treated for 7 and 14 days (P < 0.05. OTA concentrations in plasma and kidney tissue increased steadily and OTA concentration in kidney tissue strongly correlated with tail intensity and tail moment values. These results confirm the genotoxic potential of OTA, and show that the severity of DNA lesions in kidney correlates with OTA concentration.

  6. DNA damage detected by the alkaline comet assay in the liver of mice after oral administration of tetrachloroethylene

    DEFF Research Database (Denmark)

    Cederberg, H.; Henriksson, J.; Binderup, Mona-Lise

    2010-01-01

    Induction of DNA damage in the liver and kidney of male CD1 mice was studied by means of the alkaline Comet assay after oral administration of tetrachloroethylene at the doses of 1000 and 2000 mg/kg/day. A statistically significant dose-related increase in tail intensity was established...

  7. Measurement of oxidatively-induced clustered DNA lesions using a novel adaptation of single cell gel electrophoresis (comet assay).

    Science.gov (United States)

    Georgakilas, Alexandros G; Holt, Stewart M; Hair, Jessica M; Loftin, Charles W

    2010-12-01

    The two basic groups of complex DNA damage are double-strand breaks (DSBs) and non-DSB oxidatively-induced clustered DNA lesions (OCDLs). The single-cell gel electrophoresis (SCGE) or comet assay has been widely used for the detection of low levels of various types of DNA lesions including single-strand breaks (SSBs), DSBs, and oxidized bases per individual cell. There are limited data on the use of the comet assay for the detection of non-DSB clustered DNA lesions using different repair enzymes as enzymatic probes. This unit discusses a novel adaptation of the comet assay used to measure these unique types of lesions. Until now OCDL yields have been measured using primarily pulsed-field agarose gel electrophoresis. The advantages offered by the current approach are: (1) measurement of OCDL levels per individual cell; (2) use of a small number of cells (∼10,000) and relatively low doses of ionizing radiation (1 to 2 Gy) or low levels of oxidative stress, which are not compatible with standard agarose gel electrophoresis; and finally, (3) the assay is fast and allows direct comparison with pulsed-field gel electrophoresis results.

  8. Application of a novel microtitre plate-based assay for the discovery of new inhibitors of DNA gyrase and DNA topoisomerase VI.

    Science.gov (United States)

    Taylor, James A; Mitchenall, Lesley A; Rejzek, Martin; Field, Robert A; Maxwell, Anthony

    2013-01-01

    DNA topoisomerases are highly exploited targets for antimicrobial drugs. The spread of antibiotic resistance represents a significant threat to public health and necessitates the discovery of inhibitors that target topoisomerases in novel ways. However, the traditional assays for topoisomerase activity are not suitable for the high-throughput approaches necessary for drug discovery. In this study we validate a novel assay for screening topoisomerase inhibitors. A library of 960 compounds was screened against Escherichia coli DNA gyrase and archaeal Methanosarcina mazei DNA topoisomerase VI. Several novel inhibitors were identified for both enzymes, and subsequently characterised in vitro and in vivo. Inhibitors from the M. mazei topoisomerase VI screen were tested for their ability to inhibit Arabidopsis topoisomerase VI in planta. The data from this work present new options for antibiotic drug discovery and provide insight into the mechanism of topoisomerase VI.

  9. Molecular biological identification of Babesia, Theileria, and Anaplasma species in cattle in Egypt using PCR assays, gene sequence analysis and a novel DNA microarray.

    Science.gov (United States)

    El-Ashker, Maged; Hotzel, Helmut; Gwida, Mayada; El-Beskawy, Mohamed; Silaghi, Cornelia; Tomaso, Herbert

    2015-01-30

    In this preliminary study, a novel DNA microarray system was tested for the diagnosis of bovine piroplasmosis and anaplasmosis in comparison with microscopy and PCR assay results. In the Dakahlia Governorate, Egypt, 164 cattle were investigated for the presence of piroplasms and Anaplasma species. All investigated cattle were clinically examined. Blood samples were screened for the presence of blood parasites using microscopy and PCR assays. Seventy-one animals were acutely ill, whereas 93 were apparently healthy. In acutely ill cattle, Babesia/Theileria species (n=11) and Anaplasma marginale (n=10) were detected. Mixed infections with Babesia/Theileria spp. and A. marginale were present in two further cases. A. marginale infections were also detected in apparently healthy subjects (n=23). The results of PCR assays were confirmed by DNA sequencing. All samples that were positive by PCR for Babesia/Theileria spp. gave also positive results in the microarray analysis. The microarray chips identified Babesia bovis (n=12) and Babesia bigemina (n=2). Cattle with babesiosis were likely to have hemoglobinuria and nervous signs when compared to those with anaplasmosis that frequently had bloody feces. We conclude that clinical examination in combination with microscopy are still very useful in diagnosing acute cases of babesiosis and anaplasmosis, but a combination of molecular biological diagnostic assays will detect even asymptomatic carriers. In perspective, parallel detection of Babesia/Theileria spp. and A. marginale infections using a single microarray system will be a valuable improvement.

  10. Comparison of two molecular assays for detection of cytomegalovirus DNA in whole blood and plasma samples from transplant recipients.

    Science.gov (United States)

    Costa, Cristina; Sidoti, Francesca; Mantovani, Samantha; Gregori, Gabriella; Proietti, Alex; Ghisetti, Valeria; Cavallo, Rossana

    2016-07-01

    In immunosuppressed patients, pre-emptive therapy and a strict follow-up of CMV infection are the standard of care for the prevention of CMV disease. Several real-time PCR assays for CMV DNA quantification on whole blood (WB) and plasma (PL) are commercially available. This study compared and correlated CMV viral loads obtained by the Cobas AmpliPrep/Cobas TaqMan (CAP/CTM) platform on plasma specimens with those obtained on corresponding whole blood specimens by the real-time PCR assay (ELITe MGB-CMV) in 185 sequential samples from 41 immunosuppressed patients. Correlation between the two assays was good. Kinetics of CMV DNA within the same patient was similar, but PL viral load was constantly 1 log lower than WB. In patients under antiviral therapy, low level of CMV DNA persisted in WB, while it was absent in PL. The good correlation between CMV DNA detected on both PL and WB supports the reliability of the two matrices for viral monitoring and the therapeutic management of CMV infection. Nevertheless, due to significant quantification differences between PL and WB CMV DNA, the same biological specimen should be used for a sequential and reliable follow-up of patients at high risk of CMV infection.

  11. Detection limits of quantitative and digital PCR assays and their influence in presence-absence surveys of environmental DNA

    Science.gov (United States)

    Hunter, Margaret; Dorazio, Robert M.; Butterfield, John S.; Meigs-Friend, Gaia; Nico, Leo; Ferrante, Jason

    2017-01-01

    A set of universal guidelines is needed to determine the limit of detection (LOD) in PCR-based analyses of low concentration DNA. In particular, environmental DNA (eDNA) studies require sensitive and reliable methods to detect rare and cryptic species through shed genetic material in environmental samples. Current strategies for assessing detection limits of eDNA are either too stringent or subjective, possibly resulting in biased estimates of species’ presence. Here, a conservative LOD analysis grounded in analytical chemistry is proposed to correct for overestimated DNA concentrations predominantly caused by the concentration plateau, a nonlinear relationship between expected and measured DNA concentrations. We have used statistical criteria to establish formal mathematical models for both quantitative and droplet digital PCR. To assess the method, a new Grass Carp (Ctenopharyngodon idella) TaqMan assay was developed and tested on both PCR platforms using eDNA in water samples. The LOD adjustment reduced Grass Carp occupancy and detection estimates while increasing uncertainty – indicating that caution needs to be applied to eDNA data without LOD correction. Compared to quantitative PCR, digital PCR had higher occurrence estimates due to increased sensitivity and dilution of inhibitors at low concentrations. Without accurate LOD correction, species occurrence and detection probabilities based on eDNA estimates are prone to a source of bias that cannot be reduced by an increase in sample size or PCR replicates. Other applications also could benefit from a standardized LOD such as GMO food analysis, and forensic and clinical diagnostics.

  12. Variation in assessment of oxidatively damaged DNA in mononuclear blood cells by the comet assay with visual scoring

    DEFF Research Database (Denmark)

    Forchhammer, Lykke; Bräuner, Elvira Vaclavik; Folkmann, Janne Kjaersgaard;

    2008-01-01

    The comet assay is popular for assessments of genotoxicity, but the comparison of results between studies is challenging because of differences in experimental procedures and reports of DNA damage in different units. We investigated the variation of DNA damage in mononuclear blood cells (MNBCs......) measured by the comet assay with focus on the variation related to alkaline unwinding and electrophoresis time, number of cells scored, as well as the putative benefits of transforming the primary end points to common units by the use of reference standards and calibration curves. Eight experienced......-response relationships of cells exposed to gamma-radiation and it was possible to reduce the variation in oxidized purines in MNBCs from humans by adjusting the level of lesions with protocol-specific calibration curves. However, there was a difference in the level of DNA damage measured by different investigators...

  13. A sensitive high performance liquid chromatography assay for the quantification of doxorubicin associated with DNA in tumor and tissues.

    Science.gov (United States)

    Lucas, Andrew T; O'Neal, Sara K; Santos, Charlene M; White, Taylor F; Zamboni, William C

    2016-02-01

    Doxorubicin, a widely used anticancer agent, exhibits antitumor activity against a wide variety of malignancies. The drug exerts its cytotoxic effects by binding to and intercalating within the DNA of tumor and tissue cells. However, current assays are unable to accurately determine the concentration of the intracellular active form of doxorubicin. Thus, the development of a sample processing method and a high-performance liquid chromatography (HPLC) methodology was performed in order to quantify doxorubicin that is associated with DNA in tumors and tissues, which provided an intracellular cytotoxic measure of doxorubicin exposure after administration of small molecule and nanoparticle formulations of doxorubicin. The assay uses daunorubicin as an internal standard; liquid-liquid phase extraction to isolate drug associated with DNA; a Shimadzu HPLC with fluorescence detection equipped with a Phenomenex Luna C18 (2μm, 2.0×100mm) analytical column and a gradient mobile phase of 0.1% formic acid in water or acetonitrile for separation and quantification. The assay has a lower limit of detection (LLOQ) of 10ng/mL and is shown to be linear up to 3000ng/mL. The intra- and inter-day precision of the assay expressed as a coefficient of variation (CV%) ranged from 4.01 to 8.81%. Furthermore, the suitability of this assay for measuring doxorubicin associated with DNA in vivo was demonstrated by using it to quantify the doxorubicin concentration within tumor samples from SKOV3 and HEC1A mice obtained 72h after administration of PEGylated liposomal doxorubicin (Doxil(®); PLD) at 6mg/kg IV x 1. This HPLC assay allows for sensitive intracellular quantification of doxorubicin and will be an important tool for future studies evaluating intracellular pharmacokinetics of doxorubicin and various nanoparticle formulations of doxorubicin.

  14. Pu-238 assay performance with the Canberra IQ3 system

    Energy Technology Data Exchange (ETDEWEB)

    Booth, L.; Gillespie, B.; Seaman, G.

    1997-11-01

    Canberra Industries has recently completed a demonstration project at the Westinghouse Savannah River Site (WSRC) to characterize 55-gallon drums containing Pu-238 contaminated waste. The goal of this project was to detect and quantify Pu-238 contaminated waste. The goal of this project was to detect and quantify Pu-238 waste to detection limits of less than 50 nCi/g using gamma assay techniques. This would permit reclassification of these drums from transuranic (TRU) waste to low-level waste (LLW). The instrument used for this assay was a Canberra IQ3 high sensitivity gamma assay system, mounted in a trailer. The results of the measurements demonstrate achievement of detection levels as low as 1 nCi/g for low density waste drums, and good correlation with known concentrations in several test drums. In addition, the data demonstrates significant advantages for using large area low-energy germanium detectors for achieving the lowest possible MDAs for gamma rays in the 80-250 keV range. 1 fig., 2 tabs.

  15. Multiplex time-reducing quantitative polymerase chain reaction assay for determination of telomere length in blood and tissue DNA.

    Science.gov (United States)

    Jiao, Jingjing; Kang, Jing X; Tan, Rui; Wang, Jingdong; Zhang, Yu

    2012-04-01

    In this paper we describe a multiplex time-reducing quantitative polymerase chain reaction (qPCR) method for determination of telomere length. This multiplex qPCR assay enables two pairs of primers to simultaneously amplify telomere and single copy gene (albumin) templates, thus reducing analysis time and labor compared with the previously established singleplex assay. The chemical composition of the master mix and primers for the telomere and albumin were systematically optimized. The thermal cycling program was designed to ensure complete separation of the melting processes of the telomere and albumin. Semi-log standard curves of DNA concentration versus cycle threshold (C (t)) were established, with a linear relationship over an 81-fold DNA concentration range. The well-performed intra-assay (RSD range 2.4-4.7%) and inter-assay (RSD range: 3.1-5.0%) reproducibility were demonstrated to ensure measurement stability. Using wild-type, Lewis lung carcinoma and H22 liver carcinoma C57BL/6 mouse models, significantly different telomere lengths among different DNA samples were not observed in wild-type mice. However, the relative telomere lengths of the tumor DNA in the two strains of tumor-bearing mice were significantly shorter than the lengths in the surrounding non-tumor DNA of tumor-bearing mice and the tissue DNA of wild-type mice. These results suggest that the shortening of telomere lengths may be regarded as an important indicator for cancer control and prevention. Quantification of telomere lengths was further confirmed by the traditional Southern blotting method. This method could be successfully used to reduce the time needed for rapid, precise measurement of telomere lengths in biological samples.

  16. Cy5 labeled single-stranded DNA-polydopamine nanoparticle conjugate-based FRET assay for reactive oxygen species detection

    Directory of Open Access Journals (Sweden)

    Lina Ma

    2015-03-01

    Full Text Available This work reports on a simple and feasible fluorescence resonance energy transfer (FRET assay for detecting reactive oxygen species (ROS both in solution and living cell using polydopamine nanoparticle (PDA NP as energy acceptor and Cy5 labeled single-stranded DNA (Cy5-ssDNA as energy donor. The Cy5-ssDNA and PDA NPs form self-assembled conjugates (Cy5-ssDNA-PDA NP conjugates via π-stacking interactions. In the presence of ROS, the PDA NP adsorbed Cy5-ssDNAs can be effectively cleaved, resulting in the release of Cy5 molecules into solution and recovery of fluorescence emission of Cy5. In order to obtain ROS solution, the glucose oxidase-catalyzed oxidation reaction of glucose with O2 is employed to generate hydrogen peroxide for Fenton-like reaction. The formation of ROS in Fenton-like reaction can be detected as low as glucose oxidase-catalyzed oxidation of 100 pM glucose by the Cy5-ssDNA-PDA NP conjugate-based FRET assay. The recovery ratio of Cy5 fluorescence intensity is increased linearly with logarithm of glucose concentration from 100 pM to 1 μM, demonstrating that the FRET assay has wide dynamic range. In particular, intracellular ROS has been successfully detected in chemical stimulated HepG-2 cells by the Cy5-ssDNA-PDA NP conjugate-based FRET assay with a fluorescence microscopy, indicating that this approach has great potential to monitor ROS in living cells.

  17. Rapid assessment of the effect of ciprofloxacin on chromosomal DNA from Escherichia coli using an in situ DNA fragmentation assay

    Directory of Open Access Journals (Sweden)

    Gosalvez Jaime

    2009-04-01

    Full Text Available Abstract Background Fluoroquinolones are extensively used antibiotics that induce DNA double-strand breaks (DSBs by trapping DNA gyrase and topoisomerase IV on DNA. This effect is usually evaluated using biochemical or molecular procedures, but these are not effective at the single-cell level. We assessed ciprofloxacin (CIP-induced chromosomal DNA breakage in single-cell Escherichia coli by direct visualization of the DNA fragments that diffused from the nucleoid obtained after bacterial lysis in an agarose microgel on a slide. Results Exposing the E. coli strain TG1 to CIP starting at a minimum inhibitory concentration (MIC of 0.012 μg/ml and at increasing doses for 40 min increased the DNA fragmentation progressively. DNA damage started to be detectable at the MIC dose. At a dose of 1 μg/ml of CIP, DNA damage was visualized clearly immediately after processing, and the DNA fragmentation increased progressively with the antibiotic incubation time. The level of DNA damage was much higher when the bacteria were taken from liquid LB broth than from solid LB agar. CIP treatment produced a progressively slower rate of DNA damage in bacteria in the stationary phase than in the exponentially growing phase. Removing the antibiotic after the 40 min incubation resulted in progressive DSB repair activity with time. The magnitude of DNA repair was inversely related to CIP dose and was noticeable after incubation with CIP at 0.1 μg/ml but scarce after 10 μg/ml. The repair activity was not strictly related to viability. Four E. coli strains with identified mechanisms of reduced sensitivity to CIP were assessed using this procedure and produced DNA fragmentation levels that were inversely related to MIC dose, except those with very high MIC dose. Conclusion This procedure for determining DNA fragmentation is a simple and rapid test for studying and evaluating the effect of quinolones.

  18. Protective effect of dietary curcumin in Anabas testudineus (Bloch) with a special note on DNA fragmentation assay on hepatocytes and micronucleus assay on erythrocytes in vivo.

    Science.gov (United States)

    Manju, Maniyan; Vijayasree, Appiyathu Saraswathy; Akbarsha, Mohammad Abdulkader; Oommen, Oommen Vilaverthottathil

    2013-10-01

    The present study was conducted to evaluate the safety of long-term dietary curcumin at doses 0.5 and 1% in Anabas testudineus employing hematological and cytological techniques. The fish were fed with curcumin-supplemented feed for 6 months. Fine blood smears were prepared and subjected to three different staining techniques. The erythrocyte micronucleus frequency (MN) and the cytometric measurements of erythrocytes were determined. Blood from the control and treated fish was subjected to the assessment of several hematological parameters. Also, DNA fragmentation assay on hepatocytes was conducted. The results showed that hemoglobin content, RBC count and hematocrit increased in the curcumin-fed fish compared to control, whereas WBC count, platelet count, mean corpuscular volume, mean corpuscular hemoglobin and mean corpuscular hemoglobin concentration were unaffected. WBC/RBC ratio was lower in the case of curcumin-treated fish. The cytometric measurements revealed no change in the erythrocytes and their nuclei after curcumin treatment. DNA fragmentation assay revealed intact DNA in curcumin-fed group, ruling out the possibility of curcumin-induced DNA damage. The positive control group showed a significant increase in MN frequency compared to negative control and curcumin-fed groups. In fact, the MN frequency decreased in 1% curcumin-fed group compared to the negative control and 0.5% curcumin groups. All these indicated a state of well-being of the curcumin-treated fish. Therefore, it is concluded that curcumin could be used as a safe feed ingredient to improve the growth of finfish in aquaculture.

  19. Hopping of a processivity factor on DNA revealed by single-molecule assays of diffusion

    NARCIS (Netherlands)

    Komazin-Meredith, Gloria; Mirchev, Rossen; Golan, David E.; Oijen, Antoine M. van; Coen, Donald M.; Richardson, Charles C.

    2008-01-01

    Many DNA-interacting proteins diffuse on DNA to perform their biochemical functions. Processivity factors diffuse on DNA to permit unimpeded elongation by their associated DNA polymerases, but little is known regarding their rates and mechanisms of diffusion. The processivity factor of herpes simple

  20. Design of a titering assay for lentiviral vectors utilizing direct extraction of DNA from transduced cells in microtiter plates

    Directory of Open Access Journals (Sweden)

    Michele E Murphy

    2016-01-01

    Full Text Available Using lentiviral vector products in clinical applications requires an accurate method for measuring transduction titer. For vectors lacking a marker gene, quantitative polymerase chain reaction is used to evaluate the number of vector DNA copies in transduced target cells, from which a transduction titer is calculated. Immune Design previously described an integration-deficient lentiviral vector pseudotyped with a modified Sindbis virus envelope for use in cancer immunotherapy (VP02, of the ZVex platform. Standard protocols for titering integration-competent lentiviral vectors employ commercial spin columns to purify vector DNA from transduced cells, but such columns are not optimized for isolation of extrachromosomal (nonintegrated DNA. Here, we describe a 96-well transduction titer assay in which DNA extraction is performed in situ in the transduction plate, yielding quantitative recovery of extrachromosomal DNA. Vector titers measured by this method were higher than when commercial spin columns were used for DNA isolation. Evaluation of the method's specificity, linear range, and precision demonstrate that it is suitable for use as a lot release assay to support clinical trials with VP02. Finally, the method is compatible with titering both integrating and nonintegrating lentiviral vectors, suggesting that it may be used to evaluate the transduction titer for any lentiviral vector.

  1. Devices, systems, and methods for conducting sandwich assays using sedimentation

    Energy Technology Data Exchange (ETDEWEB)

    Schaff, Ulrich Y; Sommer, Gregory J; Singh, Anup K; Hatch, Anson V

    2015-02-03

    Embodiments of the present invention are directed toward devices, systems, and method for conducting sandwich assays using sedimentation. In one example, a method includes generating complexes on a plurality of beads in a fluid sample, individual ones of the complexes comprising a capture agent, a target analyte, and a labeling agent. The plurality of beads including the complexes may be transported through a density media, wherein the density media has a density lower than a density of the beads and higher than a density of the fluid sample, and wherein the transporting occurs, at least in part, by sedimentation. Signal may be detected from the labeling agents of the complexes.

  2. On the efficiency calibration of a drum waste assay system

    CERN Document Server

    Dinescu, L; Cazan, I L; Macrin, R; Caragheorgheopol, G; Rotarescu, G

    2002-01-01

    The efficiency calibration of a gamma spectroscopy waste assay system, constructed by IFIN-HH, was performed. The calibration technique was based on the assumption of a uniform distribution of the source activity in the drum and also a uniform sample matrix. A collimated detector (HPGe--20% relative efficiency) placed at 30 cm from the drum was used. The detection limit for sup 1 sup 3 sup 7 Cs and sup 6 sup 0 Co is approximately 45 Bq/kg for a sample of about 400 kg and a counting time of 10 min. A total measurement uncertainty of -70% to +40% was estimated.

  3. A sensitive colorimetric assay system for nucleic acid detection based on isothermal signal amplification technology.

    Science.gov (United States)

    Hu, Bo; Guo, Jing; Xu, Ying; Wei, Hua; Zhao, Guojie; Guan, Yifu

    2017-08-01

    Rapid and accurate detection of microRNAs in biological systems is of great importance. Here, we report the development of a visual colorimetric assay which possesses the high amplification capabilities and high selectivity of the rolling circle amplification (RCA) method and the simplicity and convenience of gold nanoparticles used as a signal indicator. The designed padlock probe recognizes the target miRNA and is circularized, and then acts as the template to extend the target miRNA into a long single-stranded nucleotide chain of many tandem repeats of nucleotide sequences. Next, the RCA product is hybridized with oligonucleotides tagged onto gold nanoparticles. This interaction leads to the aggregation of gold nanoparticles, and the color of the system changes from wine red to dark blue according to the abundance of miRNA. A linear correlation between fluorescence and target oligonucleotide content was obtained in the range 0.3-300 pM, along with a detection limit of 0.13 pM (n = 7) and a RSD of 3.9% (30 pM, n = 9). The present approach provides a simple, rapid, and accurate visual colorimetric assay that allows sensitive biodetection and bioanalysis of DNA and RNA nucleotides of interest in biologically important samples. Graphical abstract The colorimetric assay system for analyzing target oligonucleotides.

  4. Indicator-based and indicator-free magnetic assays connected with disposable electrochemical nucleic acid sensor system.

    Science.gov (United States)

    Karadeniz, Hakan; Erdem, Arzum; Kuralay, Filiz; Jelen, Frantisek

    2009-04-15

    An indicator-based and indicator-free magnetic assays connected with a disposable pencil graphite electrode (PGE) were successfully developed, and also compared for the electrochemical detection of DNA hybridization. The oxidation signals of echinomycin (ECHI) and electroactive DNA bases, guanine and adenine, respectively were monitored in the presence of DNA hybridization by using differential pulse voltammetry (DPV) technique. The biotinylated probe was immobilized onto the magnetic beads (magnetic particles, microspheres) and hybridization with its complementary target at the surface of particles within the medium was exhibited successfully using electrochemical sensor system. For the selectivity studies, the results represent that both indicator-based and indicator-free magnetic assays provide a better discrimination for DNA hybridization compared to duplex with one-base or more mismatches. The detection limits (S/N=3) of the magnetic assays based on indicator or indicator-free were found in nM concentration level of target using disposable sensor technology with good reproducibility. The characterization and advantages of both proposed magnetic assays connected with a disposable electrochemical sensor are also discussed and compared with those methods previously reported in the literature.

  5. A restriction enzyme-powered autonomous DNA walking machine: its application for a highly sensitive electrochemiluminescence assay of DNA

    Science.gov (United States)

    Chen, Ying; Xiang, Yun; Yuan, Ruo; Chai, Yaqin

    2014-12-01

    The construction of a restriction enzyme (Nt.AlwI)-powered DNA walking machine and its application for highly sensitive detection of DNA are described. DNA nanostructure tracks containing four overhang sequences with electrochemiluminescence (ECL) labels and complementary to the walker (target DNA) are self-assembled on the sensing electrode. The walker hybridizes with the complementary sequences on the tracks and forms specific recognition sites for Nt.AlwI, which cleaves the overhang sequences, releases the ECL labels and enables directional movement of the walker along the tracks. The formation of the nanostructure tracks and the Nt.AlwI-assisted cleavage of the overhang sequences in the presence of the walker are verified by using polyacrylamide gel electrophoresis analysis and cyclic voltammetry. The successive movement of the walker on the nanostructure tracks leads to continuous removal of massive ECL labels from the sensing electrode, which results in a significantly amplified suppression of the ECL emission for highly sensitive detection of sequence-specific DNA down to 0.19 pM. Results show that this DNA walking machine can also offer single-base mismatch discrimination capability. The successful application of the DNA walking machine for sequence-specific DNA detection can thus offer new opportunities for molecular machines in biosensing applications.The construction of a restriction enzyme (Nt.AlwI)-powered DNA walking machine and its application for highly sensitive detection of DNA are described. DNA nanostructure tracks containing four overhang sequences with electrochemiluminescence (ECL) labels and complementary to the walker (target DNA) are self-assembled on the sensing electrode. The walker hybridizes with the complementary sequences on the tracks and forms specific recognition sites for Nt.AlwI, which cleaves the overhang sequences, releases the ECL labels and enables directional movement of the walker along the tracks. The formation of the

  6. Preclinical detection of porcine circovirus type 2 infection using an ultrasensitive nanoparticle DNA probe-based PCR assay.

    Directory of Open Access Journals (Sweden)

    Yong Huang

    Full Text Available Porcine circovirus type 2 (PCV2 has emerged as one of the most important pathogens affecting swine production globally. Preclinical identification of PCV2 is very important for effective prophylaxis of PCV2-associated diseases. In this study, we developed an ultrasensitive nanoparticle DNA probe-based PCR assay (UNDP-PCR for PCV2 detection. Magnetic microparticles coated with PCV2 specific DNA probes were used to enrich PCV2 DNA from samples, then gold nanoparticles coated with PCV2 specific oligonucleotides were added to form a sandwich nucleic acid-complex. After the complex was formed, the oligonucleotides were released and characterized by PCR. This assay exhibited about 500-fold more sensitive than conventional PCR, with a detection limit of 2 copies of purified PCV2 genomic DNA and 10 viral copies of PCV2 in serum. The assay has a wide detection range for all of PCV2 genotypes with reliable reproducibility. No cross-reactivity was observed from the samples of other related viruses including porcine circovirus type 1, porcine parvovirus, porcine pseudorabies virus, porcine reproductive and respiratory syndrome virus and classical swine fever virus. The positive detection rate of PCV2 specific UNDP-PCR in 40 preclinical field samples was 27.5%, which appeared greater than that by conventional and real-time PCR and appeared application potency in evaluation of the viral loads levels of preclinical infection samples. The UNDP-PCR assay reported here can reliably rule out false negative results from antibody-based assays, provide a nucleic acid extraction free, specific, ultrasensitive, economic and rapid diagnosis method for preclinical PCV2 infection in field, which may help prevent large-scale outbreaks.

  7. Simultaneous detection of bovine and porcine DNA in pharmaceutical gelatin capsules by duplex PCR assay for Halal authentication.

    Science.gov (United States)

    Nikzad, Jafar; Shahhosseini, Soraya; Tabarzad, Maryam; Nafissi-Varcheh, Nastaran; Torshabi, Maryam

    2017-02-14

    In the pharmaceutical industry, hard- and soft-shelled capsules are typically made from gelatin, commonly derived from bovine and porcine sources. To ensure that pharmaceutical products comply with halal regulations in Muslim countries (no porcine products allowed), development of a valid, reliable, quick, and most importantly, cost-effective tests are of utmost importance. We developed a species-specific duplex polymerase chain reaction (PCR) assay targeting 149 bp porcine and 271 bp bovine mitochondrial DNA (mtDNA) to simultaneously detect both porcine and bovine DNA (in one reaction at the same time) in gelatin. Some additional simplex PCR tests (targeting 126 bp bovine and 212 bp porcine mtDNA) and real-time PCR using a commercially available kit (for identification of porcine DNA) were used to verify the selectivity and sensitivity of our duplex PCR. After optimization of DNA extraction and PCR methods, hard/soft pharmaceutical gelatin capsules (containing drug) were tested for the presence of porcine and/or bovine DNA. Duplex PCR detected the presence of as little as 0.1% porcine DNA, which was more accurate than the commercially available kit. Of all gelatin capsules tested (n = 24), 50% contained porcine DNA (pure porcine gelatin alone or in combination with bovine gelatin). Duplex PCR presents an easy-to-follow, quick, low-cost and reliable method to simultaneously detect porcine and bovine DNAs (>100 bp) in minute amounts in highly processed gelatin-containing pharmaceutical products (with a 0.1% sensitivity for porcine DNA) which may be used for halal authentication. Simultaneous detection of porcine and bovine DNA in gelatin capsules by duplex PCR.

  8. Analysis of In Vitro DNA Interactions of Brassinosteroid-Controlled Transcription Factors Using Electrophoretic Mobility Shift Assay.

    Science.gov (United States)

    Unterholzner, Simon J; Rozhon, Wilfried; Poppenberger, Brigitte

    2017-01-01

    Most signaling cascades ultimately lead to changes in gene expression by modulating the activity of transcription factors (TFs). The electrophoretic mobility shift assay (EMSA) is a simple but powerful in vitro method for investigation of specific protein-DNA interactions. It makes use of the fact that protein-DNA complexes have a lower electrophoretic mobility in gels than free DNA has. The application of labeled probes in combination with unlabeled competitors allows investigation of DNA-binding specificity and identification of binding motifs with single base-pair resolution. Here we describe the application of EMSAs for the study of interactions of the brassinosteroid-regulated TFs, BRASSINAZOLE-RESISTANT1, (BZR1), BRI1-ETHYL METHANESULFONATE-SUPPRESSOR1 (BES1)/BZR2, and CESTA with putative binding sites. The classical approach using radiolabeled probes, as well as the more recent application of fluorescent probes, is described and the advantages and disadvantages of both methods are discussed.

  9. Doping Level of Boron-Doped Diamond Electrodes Controls the Grafting Density of Functional Groups for DNA Assays.

    Science.gov (United States)

    Švorc, Ĺubomír; Jambrec, Daliborka; Vojs, Marian; Barwe, Stefan; Clausmeyer, Jan; Michniak, Pavol; Marton, Marián; Schuhmann, Wolfgang

    2015-09-02

    The impact of different doping levels of boron-doped diamond on the surface functionalization was investigated by means of electrochemical reduction of aryldiazonium salts. The grafting efficiency of 4-nitrophenyl groups increased with the boron levels (B/C ratio from 0 to 20,000 ppm). Controlled grafting of nitrophenyldiazonium was used to adjust the amount of immobilized single-stranded DNA strands at the surface and further on the hybridization yield in dependence on the boron doping level. The grafted nitro functions were electrochemically reduced to the amine moieties. Subsequent functionalization with a succinic acid introduced carboxyl groups for subsequent binding of an amino-terminated DNA probe. DNA hybridization significantly depends on the probe density which is in turn dependent on the boron doping level. The proposed approach opens new insights for the design and control of doped diamond surface functionalization for the construction of DNA hybridization assays.

  10. DNA damage detected by the alkaline comet assay in the liver of mice after oral administration of tetrachloroethylene.

    Science.gov (United States)

    Cederberg, Håkan; Henriksson, Jörgen; Binderup, Mona-Lise

    2010-03-01

    Induction of DNA damage in the liver and kidney of male CD1 mice was studied by means of the alkaline Comet assay after oral administration of tetrachloroethylene at the doses of 1000 and 2000 mg/kg/day. A statistically significant dose-related increase in tail intensity was established in hepatocytes, indicating that tetrachloroethylene induced DNA damage in the liver. No effect on DNA damage was observed in the kidney. The results are in agreement with carcinogenicity data in mice, in which tetrachloroethylene induced tumours in the liver but not in the kidney, and support that a genotoxic mode of action might be involved in liver carcinogenicity in mice. An alternative interpretation of the results conveyed by the Study director at the test facility, involving that tetrachloroethylene did not induce DNA damage in the liver and kidney of mice, is also presented and discussed.

  11. Rates of intercalator-driven platination of DNA determined by a restriction enzyme cleavage inhibition assay.

    Science.gov (United States)

    Choudhury, Jayati Roy; Rao, Lu; Bierbach, Ulrich

    2011-03-01

    A restriction enzyme cleavage inhibition assay was designed to determine the rates of DNA platination by four non-cross-linking platinum-acridine agents represented by the formula [Pt(am(2))LCl](NO(3))(2), where am is a diamine nonleaving group and L is an acridine derived from the intercalator 1-[2-(acridin-9-ylamino)ethyl]-1,3-dimethylthiourea (ACRAMTU). The formation of monofunctional adducts in the target sequence 5'-CGA was studied in a 40-base-pair probe containing the EcoRI restriction site GAATTC. The time dependence of endonuclease inhibition was quantitatively analyzed by polyacrylamide gel electrophoresis. The formation of monoadducts is approximately 3 times faster with double-stranded DNA than with simple nucleic acid fragments. Compound 1 (am(2) is ethane-1,2-diamine, L is ACRAMTU) reacts with a first-order rate constant of k (obs) = 1.4 ± 0.37 × 10(-4) s(-1) (t (1/2) = 83 ± 22 min). Replacement of the thiourea group in ACRAMTU with an amidine group (compound 2) accelerates the rate by fourfold (k (obs) = 5.7 ± 0.58 × 10(-4) s(-1), t (1/2) = 21 ± 2 min), and introduction of a propane-1,3-diamine nonleaving group results in a 1.5-fold enhancement in reactivity (compound 3, k (obs) = 2.1 ± 0.40 × 10(-4) s(-1), t (1/2) = 55 ± 10 min) compared with the prototype. Derivative 4, containing a 4,9-disubstituted acridine threading intercalator, was the least reactive compound in the series (k (obs) = 1.1 ± 0.40 × 10(-4) s(-1), t (1/2) = 104 ± 38 min). The data suggest a correlation may exist between the binding rates and the biological activity of the compounds. Potential pharmacological advantages of rapid formation of cytotoxic monofunctional adducts over the common purine-purine cross-links are discussed.

  12. Assessment of SYBR green I dye-based fluorescence assay for screening antimalarial activity of cationic peptides and DNA intercalating agents.

    Science.gov (United States)

    Bhatia, Rakesh; Gautam, Ankur; Gautam, Shailendra K; Mehta, Divya; Kumar, Vinod; Raghava, Gajendra P S; Varshney, Grish C

    2015-05-01

    The SYBR green I (SG) dye-based fluorescence assay for screening antimalarial compounds is based on direct quantitation of parasite DNA. We show that DNA-interacting cationic cell-penetrating peptides (CPPs) and intercalating agents compete with SG dye to bind to DNA. Therefore, readouts of this assay, unlike those of the [(3)H]hypoxanthine incorporation assay, for the antimalarial activity of the above DNA binding agents may be erroneous. In the case of CPPs, false readouts can be improved by the removal of excess peptides. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  13. Evaluation of γ-radiation-induced DNA damage in two species of bivalves and their relative sensitivity using comet assay.

    Science.gov (United States)

    Praveen Kumar, M K; Shyama, S K; Sonaye, B S; Naik, U Roshini; Kadam, S B; Bipin, P D; D'costa, A; Chaubey, R C

    2014-05-01

    Ionizing radiation is known to induce genetic damage in diverse groups of organisms. Under accidental situations, large quantities of radioactive elements get released into the environment and radiation emitted from these radionuclides may adversely affect both the man and the non-human biota. The present study is aimed (a) to know the genotoxic effect of gamma radiation on aquatic fauna employing two species of selected bivalves, (b) to evaluate the possible use of 'Comet assay' for detecting genetic damage in haemocytes of bivalves as a biomarker for environmental biomonitoring and also (c) to compare the relative sensitivity of two species of bivalves viz. Paphia malabarica and Meretrix casta to gamma radiation. The comet assays was optimized and validated using different concentrations (18, 32 and 56 mg/L) of ethyl methanesulfonate (EMS), a direct-acting reference genotoxic agent, to which the bivalves were exposed for various times (24, 48 and 72 h). Bivalves were irradiated (single acute exposure) with 5 different doses (viz. 2, 4, 6, 8 and 10 Gy) of gamma radiation and their genotoxic effects on the haemocytes were studied using the comet assay. Haemolymph was collected from the adductor muscle at 24, 48 and 72 h of both EMS-exposed and irradiated bivalves and comet assay was carried out using standard protocol. A significant increase in DNA damage was observed as indicated by an increase in % tail DNA damage at different concentrations of EMS and all the doses of gamma radiation as compared to controls in both bivalve species. This showed a dose-dependent increase of genetic damage induced in bivalves by EMS as well as gamma radiation. Further, the highest DNA damage was observed at 24h. The damage gradually decreased with time, i.e. was smaller at 48 and 72 h than at 24h post irradiation in both species of bivalves. This may indicate repair of the damaged DNA and/or loss of heavily damaged cells as the post irradiation time advanced. The present study

  14. Electrochemical branched-DNA assay for polymerase chain reaction-free detection and quantification of oncogenes in messenger RNA.

    Science.gov (United States)

    Lee, Ai-Cheng; Dai, Ziyu; Chen, Baowei; Wu, Hong; Wang, Jun; Zhang, Aiguo; Zhang, Lurong; Lim, Tit-Meng; Lin, Yuehe

    2008-12-15

    We describe a novel electrochemical branched-DNA (bDNA) assay for polymerase chain reaction (PCR)-free detection and quantification of p185 BCR-ABL leukemia fusion transcripts in the population of messenger ribonucleic acid (mRNA) extracted from cell lines. The bDNA amplifier carrying high loading of alkaline phosphatase (ALP) tracers was used to amplify the target signal. The targets were captured on microplate well surfaces through cooperative sandwich hybridization prior to the labeling of bDNA. The activity of captured ALP was monitored by square-wave voltammetric (SWV) analysis of the electroactive enzymatic product in the presence of 1-naphthyl phosphate. The voltammetric characteristics of substrate and enzymatic product as well as the parameters of SWV analysis were systematically optimized. A detection limit of 1 fM (1 x 10(-19) mol of target transcripts in 100 microL) and a 3-order-wide dynamic range of target concentration were achieved by the electrochemical bDNA assay. Such limit corresponded to approximately 17 fg of the p185 BCR-ABL fusion transcripts. The specificity and sensitivity of assay enabled direct detection of target transcripts in as little as 4.6 ng of mRNA population without PCR amplification. In combination with the use of a well-quantified standard, the electrochemical bDNA assay was capable of direct use for a PCR-free quantitative analysis of target transcripts in mRNA population. A mean transcript copy number of 62,900/ng of mRNA was determined, which was at least 50-fold higher than that of real-time quantitative PCR (qPCR). The finding was consistent with the underestimation of targets by qPCR reported earlier. In addition, the unique design based on bDNA technology increases the assay specificity as only the p185 BCR-ABL fusion transcripts will respond to the detection. The approach thus provides a simple, sensitive, accurate, and quantitative tool alternative to the qPCR for early disease diagnosis.

  15. Human DNA quantification and sample quality assessment: Developmental validation of the PowerQuant(®) system.

    Science.gov (United States)

    Ewing, Margaret M; Thompson, Jonelle M; McLaren, Robert S; Purpero, Vincent M; Thomas, Kelli J; Dobrowski, Patricia A; DeGroot, Gretchen A; Romsos, Erica L; Storts, Douglas R

    2016-07-01

    Quantification of the total amount of human DNA isolated from a forensic evidence item is crucial for DNA normalization prior to short tandem repeat (STR) DNA analysis and a federal quality assurance standard requirement. Previous commercial quantification methods determine the total human DNA and total human male DNA concentrations, but provide limited information about the condition of the DNA sample. The PowerQuant(®) System includes targets for quantification of total human and total human male DNA as well as targets for evaluating whether the human DNA is degraded and/or PCR inhibitors are present in the sample. A developmental validation of the PowerQuant(®) System was completed, following SWGDAM Validation Guidelines, to evaluate the assay's specificity, sensitivity, precision and accuracy, as well as the ability to detect degraded DNA or PCR inhibitors. In addition to the total human DNA and total human male DNA concentrations in a sample, data from the degradation target and internal PCR control (IPC) provide a forensic DNA analyst meaningful information about the quality of the isolated human DNA and the presence of PCR inhibitors in the sample that can be used to determine the most effective workflow and assist downstream interpretation.

  16. Highly selective and sensitive DNA assay based on electrocatalytic oxidation of ferrocene bearing zinc(II)-cyclen complexes with diethylamine.

    Science.gov (United States)

    Shiddiky, Muhammad J A; Torriero, Angel A J; Zeng, Zhanghua; Spiccia, Leone; Bond, Alan M

    2010-07-28

    A highly selective and sensitive electrochemical biosensor has been developed that detects DNA hybridization by employing the electrocatalytic activity of ferrocene (Fc) bearing cyclen complexes (cyclen = 1,4,7,10-tetraazacyclododecane, Fc[Zn(cyclen)H(2)O](2)(ClO(4))(4) (R1), Fc(cyclen)(2) (R2), Fc[Zn(cyclen)H(2)O](ClO(4))(2) (R3), and Fc(cyclen) (R4)). A sandwich-type approach, which involves hybridization of a target probe hybridized with the preimmobilized thiolated capture probe attached to a gold electrode, is employed to fabricate a DNA duplex layer. Electrochemical signals are generated by voltammetric interrogation of a Fc bearing Zn-cyclen complexes that selectively and quantitatively binds to the duplex layers through strong chelation between the cyclen complexes and particular nucleobases within the DNA sequence. Chelate formation between R1 or R3 and thymine bases leads to the perturbation of base-pair (A-T) stacking in the duplex structure, which greatly diminishes the yield of DNA-mediated charge transport and displays a marked selectivity to the presence of the target DNA sequence. Coupling the redox chemistry of the surface-bound Fc bearing Zn-cyclen complex and dimethylamine provides an electrocatalytic pathway that increases sensitivity of the assay and allows the 100 fM target DNA sequence to be detected. Excellent selectivity against even single-base sequence mismatches is achieved, and the DNA sensor is stable and reusable.

  17. A semi-quantitative assay of overall DNA methylation status using Methyl-CpG binding protein (MBD1

    Directory of Open Access Journals (Sweden)

    Zhang Chunxiao

    2012-05-01

    Full Text Available Abstract Background In mammals, DNA methylation at the 5-position of cytosine is the most essential epigenetic modification. Changes in the level of genome-wide DNA methylation (also known as overall DNA methylation are associated with alterations in gene expression, thereby contributing to the phenotypic and physiological diversity. Current technologies for detecting overall DNA methylation either suffer from low sensitivity or require sophisticated equipment. Studies on domestic animals are hampered by the lack of complete and annotated genomic information. Results Here we report a rapid slot blot method using methyl-CpG binding protein (MBD1 to exam the level of overall DNA methylation in pigs and chickens. Using this rapid approach, we determined the methylation status in various DNA samples of a Chinese indigenous (Erhualian and a Western (Large White breed of pigs. We also chose day 18 embryos (E18 and newly hatched chicks (D1 of a Chinese indigenous chicken breed (Wen’s yellow-feathered broiler chicken for genome-wide DNA methylation analysis. The results revealed tissue- and breed-specific differences, as well as age-dependent variations, in the level of overall DNA methylation. Conclusion The results showed that the slot blot assay is a sensitive, highly specific and convenient method for semi-quantitative estimation of overall DNA methylation with no species specificity. This method does not require sophisticated equipment, such as high performance liquid chromatography (HPLC, or expensive technologies like sequencing, thus providing a useful tool for overall DNA methylation studies on domestic animals.

  18. SWEPP Assay System Version 2.0 software design description

    Energy Technology Data Exchange (ETDEWEB)

    East, L.V.; Marwil, E.S.

    1996-08-01

    The Idaho National Engineering Laboratory (INEL) Stored Waste Examination Pilot Plant (SWEPP) operations staff use nondestructive analysis methods to characterize the radiological contents of contact-handled radioactive waste containers. Containers of waste from Rocky Flats Environmental Technology Site and other Department of Energy (DOE) sites are currently stored at SWEPP. Before these containers can be shipped to the Waste Isolation Pilot Plant (WIPP), SWEPP must verify compliance with storage, shipping, and disposal requirements. This program has been in operation since 1985 at the INEL Radioactive Waste Management Complex (RWMC). One part of the SWEPP program measures neutron emissions from the containers and estimates the mass of plutonium and other transuranic (TRU) isotopes present. A Passive/Active Neutron (PAN) assay system developed at the Los Alamos National Laboratory is used to perform these measurements. A computer program named NEUT2 was originally used to perform the data acquisition and reduction functions for the neutron measurements. This program was originally developed at Los Alamos and extensively modified by a commercial vendor of PAN systems and by personnel at the INEL. NEUT2 uses the analysis methodology outlined, but no formal documentation exists on the program itself. The SWEPP Assay System (SAS) computer program replaced the NEUT2 program in early 1994. The SAS software was developed using an `object model` approach and is documented in accordance with American National Standards Institute (ANSI) and Institute of Electrical and Electronic Engineers (IEEE) standards. The new program incorporates the basic analysis algorithms found in NEUT2. Additional functionality and improvements include a graphical user interface, the ability to change analysis parameters without program code modification, an `object model` design approach and other features for improved flexibility and maintainability.

  19. Confinement Vessel Assay System: Design and Implementation Report

    Energy Technology Data Exchange (ETDEWEB)

    Frame, Katherine C. [Los Alamos National Laboratory; Bourne, Mark M. [Los Alamos National Laboratory; Crooks, William J. [Los Alamos National Laboratory; Evans, Louise [Los Alamos National Laboratory; Mayo, Douglas R. [Los Alamos National Laboratory; Gomez, Cipriano D. [Retired CMR-OPS: OPERATIONS; Miko, David K. [Los Alamos National Laboratory; Salazar, William R. [Los Alamos National Laboratory; Stange, Sy [Los Alamos National Laboratory; Vigil, Georgiana M. [Los Alamos National Laboratory

    2012-07-18

    Los Alamos National Laboratory has a number of spherical confinement vessels remaining from tests involving nuclear materials. These vessels have an inner diameter of 6 feet with 1- to 2-inch thick steel walls. The goal of the Confinement Vessel Disposition (CVD) project is to remove debris and reduce contamination inside the vessels. We have developed a neutron assay system for the purposes of Materials Control and Accountability (MC&A) measurements of the vessel prior to and after cleanout. We present our approach to confronting the challenges in designing, building, and testing such a system. The system was designed to meet a set of functional and operational requirements. A Monte Carlo model was developed to aid in optimizing the detector design as well as to predict the systematic uncertainty associated with confinement vessel measurements. Initial testing was performed to optimize and determine various measurement parameters, and then the system was characterized using {sup 252}Cf placed a various locations throughout the measurement system. Measurements were also performed with a {sup 252}Cf source placed inside of small steel and HDPE shells to study the effect of moderation. These measurements compare favorably with their MCNPX model equivalent, making us confident that we can rely on the Monte Carlo simulation to predict the systematic uncertainty due to variations in response to material that may be localized at different points within a vessel.

  20. Differences in quantification of DNA double-strand breaks assessed by 53BP1/γH2AX focus formation assays and the comet assay in mammalian cells treated with irradiation and N-acetyl-L-cysteine.

    Science.gov (United States)

    Kurashige, Tomomi; Shimamura, Mika; Nagayama, Yuji

    2016-06-01

    The biological effect of ionizing radiation (IR) on genomic DNA is thought to be either direct or indirect; the latter is mediated by IR induction of free radicals and reactive oxygen species (ROS). This study was designed to evaluate the effect of N-acetyl-L-cysteine (NAC), a well-known ROS-scavenging antioxidant, on IR induction of genotoxicity, cytotoxicity and ROS production in mammalian cells, and aimed to clarify the conflicting data in previous publications. Although we clearly demonstrate the beneficial effect of NAC on IR-induced genotoxicity and cytotoxicity (determined using the micronucleus assay and cell viability/clonogenic assays), the data on NAC's effect on DNA double-strand break (DSB) formation were inconsistent in different assays. Specifically, mitigation of IR-induced DSBs by NAC was readily detected by the neutral comet assay, but not by the γH2AX or 53BP1 focus assays. NAC is a glutathione precursor and exerts its effect after conversion to glutathione, and presumably it has its own biological activity. Assuming that the focus assay reflects the biological responses to DSBs (detection and repair), while the comet assay reflects the physical status of genomic DNA, our results indicate that the comet assay could readily detect the antioxidant effect of NAC on DSB formation. However, NAC's biological effect might affect the detection of DSB repair by the focus assays. Our data illustrate that multiple parameters should be carefully used to analyze DNA damage when studying potential candidates for radioprotective compounds.

  1. DNA-based diagnosis of arylsulfatase A deficiencies as a supplement to enzyme assay: a case in point.

    Science.gov (United States)

    Coulter-Mackie, M B; Applegarth, D A; Toone, J; Vallance, H

    1997-02-01

    To identify the molecular basis of arylsulfatase A deficiency in a family at risk for metachromatic leukodystrophy (MLD) and determine the genetic risk in the offspring. Mutations in the arylsulfatase A gene were identified by PCR amplification and restriction enzyme digestion. Individuals had previously been tested for arylsulfatase A activity. Assays of arylsulfatase A activity had resulted in ambiguous results for MLD carrier identification. DNA analysis clearly identified two MLD mutations in the family, and an unsuspected arylsulfatase A pseudodeficiency. The DNA information immediately clarified the MLD risk for the family and confirmed that a newborn with low arylsulfatase A activity was unaffected. The overlap between activities for various combinations of MLD and pseudodeficiency alleles and the variability inherent in the assay of arylsulfatase A complicate the interpretation of activity levels in families at risk for MLD. Use of simple molecular biological tests for pseudodeficiency and the common MLD mutations in combination with the enzyme data can facilitate carrier identification and prenatal diagnosis.

  2. Genotoxicity of nano/microparticles in in vitro micronuclei, in vivo comet and mutation assay systems

    Directory of Open Access Journals (Sweden)

    Fukumori Nobutaka

    2009-09-01

    Full Text Available Abstract Background Recently, manufactured nano/microparticles such as fullerenes (C60, carbon black (CB and ceramic fiber are being widely used because of their desirable properties in industrial, medical and cosmetic fields. However, there are few data on these particles in mammalian mutagenesis and carcinogenesis. To examine genotoxic effects by C60, CB and kaolin, an in vitro micronuclei (MN test was conducted with human lung cancer cell line, A549 cells. In addition, DNA damage and mutations were analyzed by in vivo assay systems using male C57BL/6J or gpt delta transgenic mice which were intratracheally instilled with single or multiple doses of 0.2 mg per animal of particles. Results In in vitro genotoxic analysis, increased MN frequencies were observed in A549 cells treated with C60, CB and kaolin in a dose-dependent manner. These three nano/microparticles also induced DNA damage in the lungs of C57BL/6J mice measured by comet assay. Moreover, single or multiple instillations of C60 and kaolin, increased either or both of gpt and Spi- mutant frequencies in the lungs of gpt delta transgenic mice. Mutation spectra analysis showed transversions were predominant, and more than 60% of the base substitutions occurred at G:C base pairs in the gpt genes. The G:C to C:G transversion was commonly increased by these particle instillations. Conclusion Manufactured nano/microparticles, CB, C60 and kaolin, were shown to be genotoxic in in vitro and in vivo assay systems.

  3. DNA repair capacity of cultured human lymphocytes exposed to mutagens measured by the comet assay and array expression analysis.

    Science.gov (United States)

    Bausinger, Julia; Speit, Günter

    2015-11-01

    Repair of mutagen-induced DNA lesions during transportation, storage and cultivation of lymphocytes may have a significant impact on results obtained in human biomonitoring after occupational and environmental exposure of human populations to genotoxic chemicals. Using the comet assay in combination with the repair inhibitor aphidicolin and array gene expression analysis of 92 DNA repair genes, we investigated the repair of DNA lesions induced by methyl methanesulfonate (MMS) and benzo[a]pyrenediolepoxide (BPDE) in phytohaemagglutinin (PHA)-stimulated cultured human lymphocytes in the time segment before replication. The comet assay indicated fast repair of MMS-induced damage during the first hours of cultivation. In contrast, removal of BPDE-induced lesions was slower and significant amounts of damage seem to persist until S-phase. Gene expression analysis revealed that PHA stimulation had a clear effect on gene regulation in lymphocytes already during the first 18h of cultivation. Under the conditions of this study, genotoxic concentrations of MMS did not induce significant changes in gene expression. In contrast, exposure to BPDE led to altered expression of several genes in a time- and concentration-related manner. Of the significantly up-regulated genes, only two genes (XPA and XPC) were directly related to nucleotide excision repair. Our results suggest that PHA stimulation of human lymphocytes influences the expression of DNA repair genes in human lymphocytes. The effect of induced DNA damage on gene expression is comparatively low and depends on the mutagens used. PHA-stimulated lymphocytes repair induced DNA damage before they start to replicate but the repair activity during the first 18h of cultivation is not affected by changes in the expression of DNA repair genes during this period of time.

  4. A new assay format for NF-kappaB based on a DNA triple helix and a fluorescence resonance energy transfer.

    Science.gov (United States)

    Altevogt, Dominik; Hrenn, Andrea; Kern, Claudia; Clima, Lilia; Bannwarth, Willi; Merfort, Irmgard

    2009-10-07

    Herein we report a feasibility study for a new concept to detect DNA binding protein NF-kappaB based on a DNA triple helix formation in combination with a fluorescence resonance energy transfer (FRET). The new principle avoids expensive antibodies and radioactivity and might have implications for assays of other DNA binding proteins.

  5. Lack of DNA damage induced by fluoride on mouse lymphoma and human fibroblast cells by single cell gel (comet) assay

    OpenAIRE

    Ribeiro,Daniel Araki; Alves de Lima, Patrícia Lepage; Marques, Mariângela Esther Alencar; Salvadori,Daisy Maria Favero

    2006-01-01

    Fluoride has widely been used in Dentistry because it is a specific and effective caries prophylactic agent. However, excess fluoride may represent a hazard to human health, especially by causing injury on genetic apparatus. Genotoxicity tests constitute an important part of cancer research for risk assessment of potential carcinogens. In this study, the potential DNA damage associated with exposure to fluoride was assessed by the single cell gel (comet) assay in vitro. Mouse lymphoma and hum...

  6. SWEPP assay system version 2.0 software requirements specification

    Energy Technology Data Exchange (ETDEWEB)

    Matthews, S.D.; East, L.V.; Marwil, E.S.; Ferguson, J.J.

    1996-06-01

    The INEL Stored Waste Examination Pilot Plant (SWEPP) operations staff use nondestructive analysis methods to characterize the radiological contents of contact-handled radioactive waste containers. Containers of waste from Rocky Flats Environmental Technology Site and other DOE sites are currently stored at SWEPP. Before these containers can be shipped to WIPP, SWEPP must verify compliance with storage, shipping, and disposal requirements. One part of the SWEPP program measures neutron emissions from the containers and estimates the mass of Pu and other transuranic isotopes present. The code NEUT2 was originally used to perform data acquisition and reduction; the SWEPP Assay System (SAS) code replaced NEUT2 in early 1994. This document specifies the requirements for the SAS software as installed at INEL and was written to comply with RWMC (INEL Radioactive Waste Management Complex) quality requirements.

  7. Detection limits of quantitative and digital PCR assays and their influence in presence-absence surveys of environmental DNA.

    Science.gov (United States)

    Hunter, Margaret E; Dorazio, Robert M; Butterfield, John S S; Meigs-Friend, Gaia; Nico, Leo G; Ferrante, Jason A

    2017-03-01

    A set of universal guidelines is needed to determine the limit of detection (LOD) in PCR-based analyses of low-concentration DNA. In particular, environmental DNA (eDNA) studies require sensitive and reliable methods to detect rare and cryptic species through shed genetic material in environmental samples. Current strategies for assessing detection limits of eDNA are either too stringent or subjective, possibly resulting in biased estimates of species' presence. Here, a conservative LOD analysis grounded in analytical chemistry is proposed to correct for overestimated DNA concentrations predominantly caused by the concentration plateau, a nonlinear relationship between expected and measured DNA concentrations. We have used statistical criteria to establish formal mathematical models for both quantitative and droplet digital PCR. To assess the method, a new Grass Carp (Ctenopharyngodon idella) TaqMan assay was developed and tested on both PCR platforms using eDNA in water samples. The LOD adjustment reduced Grass Carp occupancy and detection estimates while increasing uncertainty-indicating that caution needs to be applied to eDNA data without LOD correction. Compared to quantitative PCR, digital PCR had higher occurrence estimates due to increased sensitivity and dilution of inhibitors at low concentrations. Without accurate LOD correction, species occurrence and detection probabilities based on eDNA estimates are prone to a source of bias that cannot be reduced by an increase in sample size or PCR replicates. Other applications also could benefit from a standardized LOD such as GMO food analysis and forensic and clinical diagnostics. Published 2016. This article is a U.S. Government work and is in the public domain in the USA.

  8. A T7 Endonuclease I Assay to Detect Talen-Mediated Targeted Mutation of HBV cccDNA.

    Science.gov (United States)

    Bloom, Kristie; Ely, Abdullah; Arbuthnot, Patrick

    2017-01-01

    Gene editing using designer nucleases is now widely used in many fields of molecular biology. The technology is being developed for the treatment of viral infections such as persistant hepatitis B virus (HBV). The replication intermediate of HBV comprising covalently closed circular DNA (cccDNA) is stable and resistant to available licensed antiviral agents. Advancing gene editing as a means of introducing targeted mutations into cccDNA thus potentially offers the means to cure infection by the virus. Essentially, targeted mutations are initiated by intracellular DNA cleavage, then error-prone nonhomologous end joining results in insertions and deletions (indels) at intended sites. Characterization of these mutations is crucial to confirm activity of potentially therapeutic nucleases. A convenient tool for evaluation of the efficiency of target cleavage is the single strand-specific endonuclease, T7EI. Assays employing this enzyme entail initial amplification of DNA encompassing the targeted region. Thereafter the amplicons are denatured and reannealed to allow hybridization between indel-containing and wild-type sequences. Heteroduplexes that contain mismatched regions are susceptible to action by T7EI and cleavage of the hybrid amplicons may be used as an indicator of efficiency of designer nucleases. The protocol described here provides a method of isolating cccDNA from transfected HepG2.2.15 cells and evaluation of the efficiency of mutation by a transcription activator-like effector nuclease that targets the surface open reading frame of HBV.

  9. Detection of Interaction of Binding Affinity of Aromatic Hydrocarbon Receptor to the Specific DNA by Exonuclease Protection Mediated PCR Assay

    Institute of Scientific and Technical Information of China (English)

    SUN Xi; XU Shunqing

    2005-01-01

    A novel exonuclease protection mediated PCR assay (EPM-PCR) to detect the interaction of protein and DNA at a dioxin-responsive enhancer (DRE) upstream of the CYP1A1 gene in rat hepatic cytosol was established. A double-stranded DNA fragment containing two binding sites was designed and incubated with the aryl hydrocarbon receptor (AhR) transformed by 2,3,7,8-tet rachlorodibenzo p dioxin (TCDD) to generate TCDD: AhR: DNA complex which could protect receptor-binding DNA against exonuclease Ⅲ (Exo Ⅲ) digestion. With ExoⅢ treatment, free DNAs were digested and receptor-bound DNAs remained that could be amplified by PCR. By agarose gel electrophoreses a clear band (285bp) was detected using TCDD-treated sample, while nothing with control samples. To detect transformed AhR-DRE complex, 2 fmol DNAs and 3 ug cytosol proteins were found to be sufficient in the experiment. Compared with gel retardation assay, this new method is more sensitive for monitoring the Ah receptor-enhancer interaction without radioactive pollution.

  10. Evaluation of DNA Recombinant Methodologies for the Diagnosis of Plasmodium falciparum and their Comparison with the Microscopy Assay

    Directory of Open Access Journals (Sweden)

    L Urdaneta

    1998-09-01

    Full Text Available Since 1984, DNA tests based on the highly repeated subtelomeric sequences of Plasmodium falciparum (rep 20 have been frequently used in malaria diagnosis. Rep 20 is very specific for this parasite, and is made of 21 bp units, organized in repeated blocks with direct and inverted orientation. Based in this particular organization, we selected a unique consensus oligonucleotide (pf-21 to drive a PCR reaction coupled to hybridization to non-radioactive labeled probes. The pf-21 unique oligo PCR (pf-21-I assay produced DNA amplification fingerprints when was applied on purified P. falciparum DNA samples (Brazil and Colombia, as well as in patient's blood samples from a large area of Venezuela. The performance of the Pf-21-I assay was compared against Giemsa stained thick blood smears from samples collected at a malaria endemic area of the Bolívar State, Venezuela, at the field station of Malariología in Tumeremo. Coupled to non-radioactive hybridization the pf-21-I performed better than the traditional microscopic method with a r=1.7:1. In the case of mixed infections the r value of P. falciparum detection increased to 2.5:1. The increased diagnostic sensitivity of the test produced with this homologous oligonucleotide could provide an alternative to the epidemiological diagnosis of P. falciparum being currently used in Venezuela endemic areas, where low parasitemia levels and asymptomatic malaria are frequent. In addition, the DNA fingerprint could be tested in molecular population studies

  11. The synchronous active neutron detection system for spent fuel assay

    Energy Technology Data Exchange (ETDEWEB)

    Pickrell, M.M.; Kendall, P.K.

    1994-10-01

    The authors have begun to develop a novel technique for active neutron assay of fissile material in spent nuclear fuel. This approach will exploit the unique operating features of a 14-MeV neutron generator developed by Schlumberger. This generator and a novel detection system will be applied to the direct measurement of the fissile material content in spent fuel in place of the indirect measures used at present. The technique they are investigating is termed synchronous active neutron detection (SAND). It closely follows a method that has been used routinely in other branches of physics to detect very small signals in the presence of large backgrounds. Synchronous detection instruments are widely available commercially and are termed {open_quotes}lock-in{close_quotes} amplifiers. The authors have implemented a digital lock-in amplifier in conjunction with the Schlumberger neutron generator to explore the possibility of synchronous detection with active neutrons. This approach is possible because the Schlumberger system can operate at up to a 50% duty factor, in effect, a square wave of neutron yield. The results to date are preliminary but quite promising. The system is capable of resolving the fissile material contained in a small fraction of the fuel rods in a cold fuel assembly. It also appears to be quite resilient to background neutron interference. The interrogating neutrons appear to be nonthermal and penetrating. Although a significant amount of work remains to fully explore the relevant physics and optimize the instrument design, the underlying concept appears sound.

  12. Scalable DNA-Based Magnetic Nanoparticle Agglutination Assay for Bacterial Detection in Patient Samples

    DEFF Research Database (Denmark)

    Mezger, Anja; Fock, Jeppe; Antunes, Paula Soares Martins

    2015-01-01

    We demonstrate a nanoparticle-based assay for the detection of bacteria causing urinary tract infections in patient samples with a total assay time of 4 h. This time is significantly shorter than the current gold standard, plate culture, which can take several days depending on the pathogen...

  13. A single molecule assay for measuring site-specific DNA cleavage.

    Science.gov (United States)

    Gambino, Stefano; Mousley, Briana; Cathcart, Lindsay; Winship, Janelle; Loparo, Joseph J; Price, Allen C

    2016-02-15

    Sequence-specific DNA cleavage is a key step in a number of genomic transactions. Here, we report a single-molecule technique that allows the simultaneous measurement of hundreds of DNAs, thereby collecting significant statistics in a single experiment. Microbeads are tethered with single DNA molecules in a microfluidic channel. After the DNA cleavage reaction is initiated, the time of cleavage of each DNA is recorded using video microscopy. We demonstrate the utility of our method by measuring the cleavage kinetics of NdeI, a type II restriction endonuclease.

  14. Comet assay measures of DNA damage as biomarkers of irinotecan response in colorectal cancer in vitro and in vivo.

    Science.gov (United States)

    Wood, Joanna P; Smith, Andrew J O; Bowman, Karen J; Thomas, Anne L; Jones, George D D

    2015-09-01

    The use of irinotecan to treat metastatic colorectal cancer (CRC) is limited by unpredictable response and variable toxicity; however, no reliable clinical biomarkers are available. Here, we report a study to ascertain whether irinotecan-induced DNA damage measures are suitable/superior biomarkers of irinotecan effect. CRC-cell lines (HCT-116 and HT-29) were treated in vitro with irinotecan and peripheral blood lymphocytes (PBL) were isolated from patients before and after receiving irinotecan-based chemotherapy. Levels of in vitro-, in vivo-, and ex vivo-induced DNA damage were measured using the Comet assay; correlations between damage levels with in vitro cell survival and follow-up clinical data were investigated. Irinotecan-induced DNA damage was detectable in both CRC cell-lines in vitro, with higher levels of immediate and residual damage noted for the more sensitive HT-29 cells. DNA damage was not detected in vivo, but was measurable in PBLs upon mitogenic stimulation prior to ex vivo SN-38 treatment. Results showed that, following corrections for experimental error, those patients whose PBLs demonstrated higher levels of DNA damage following 10 h of SN-38 exposure ex vivo had significantly longer times to progression than those with lower damage levels (median 291 vs. 173 days, P = 0.014). To conclude, higher levels of irinotecan-induced initial and residual damage correlated with greater cell kill in vitro and a better clinical response. Consequently, DNA damage measures may represent superior biomarkers of irinotecan effect compared to the more often-studied genetic assays for differential drug metabolism.

  15. Sensitive non-isotopic DNA hybridisation assay or immediate-early antigen detection for rapid identification of human cytomegalovirus in urine.

    Science.gov (United States)

    Kimpton, C P; Morris, D J; Corbitt, G

    1991-04-01

    A sensitive non-radioactive DNA hybridisation assay employing digoxigenin-labelled probes was compared with immediate-early antigen detection and conventional virus isolation for the identification of human cytomegalovirus (HCMV) in 249 urine samples. Of 44 specimens yielding HCMV by virus isolation, more were positive by DNA hybridisation (32; 73%) than by immediate-early antigen detection (25; 52%) (P = 0.05). The specificity of the hybridisation assay in 45 apparently falsely positive specimens was supported by detection of HCMV DNA in 40 of these specimens using the polymerase chain reaction. Many urine specimens may thus contain large amounts of non-viable virus or free viral DNA. Evaluation of various protocols for the extraction and denaturation of virus DNA prior to hybridisation showed that proteinase K digestion with phenol/chloroform extraction was the most sensitive and reliable procedure. We conclude that the non-radioactive DNA hybridisation assay described is a potentially valuable routine diagnostic test.

  16. Challenge assay: A functional biomarker for exposure-induced DNA repair deficiency and for risk of cancer.

    Science.gov (United States)

    Au, William W; Giri, Ashok K; Ruchirawat, Mathuros

    2010-01-01

    A variety of biomarkers have been used to monitor exposed populations to determine potential health hazards from their exposure to environmental toxic agents. However, the majority of these biomarkers have been focused onto the identification of biological damage from the exposure. Therefore, there is a need to develop functional biomarkers that can identify exposure-induced functional deficiencies. More importantly, these deficiencies should be positioned along pathways that are responsible for the development of specific diseases. One of such pathways belongs to the extensive and complex DNA-repair machinery. The machinery thus becomes a large target for damage from environmental toxic agents. The hypothesis is that damage to any component of a repair pathway will interfere with the pathway-specific repair activities. Therefore, when cells from exposed populations are challenged with a DNA-damaging agent in vitro, the in vivo exposure-induced repair deficiency will be dramatically amplified and the deficiency will be detectable in a challenge assay as increased chromosome aberrations, micronuclei or un-repaired DNA strand breaks. The challenge assay has been used in different laboratories to show that a variety of exposed populations (with exposure to air pollutants, arsenic, benzene, butadiene, cigarette smoke, incense smoke, lead, mercury, pesticides, uranium or xylene but not to low concentrations of air pollutants or butadiene) expressed abnormal challenge response. The predicted health consequences of some of these studies have also been validated. Therefore, the challenge assay is a useful functional biomarker for population studies. Details of the challenge assay and its application will be presented in this review.

  17. DNA damage in barn swallows (Hirundo rustica) from the Chernobyl region detected by use of the comet assay.

    Science.gov (United States)

    Bonisoli-Alquati, Andrea; Voris, Andrew; Mousseau, Timothy A; Møller, Anders Pape; Saino, Nicola; Wyatt, Michael D

    2010-04-01

    We investigated levels of DNA damage in blood cells of barn swallows (Hirundo rustica) inhabiting the Chernobyl region to evaluate whether chronic exposure to low-level radioactive contamination continues to induce genetic damage in free-living populations of animals. Blood samples were obtained from barn swallows collected at sites with different background levels of radiation, including a relatively uncontaminated area. The extent of DNA damage was evaluated using the alkaline (pH=12.1) comet assay, a robust and sensitive electrophoresis-based technique widely employed in research ranging from biomonitoring to clinical studies. We found that levels of DNA damage, as indexed by the extent of DNA migration, were increased in barn swallows living in areas surrounding Chernobyl when compared to swallows sampled at low-level sites. The results we obtained are consistent with previous findings on this same species, which showed that swallows breeding in areas heavily contaminated with radionuclides have increased mutation rates, higher oxidative stress and incidence of morphological aberrations and tumors. Overall, these results indicate that chronic exposure to radioactive contaminants, even 20years after the accident at the Chernobyl nuclear power plant, continues to induce DNA damage in cells of free-living animals.

  18. Resistance to UV-B induced DNA damage in extreme-tolerant cryptoendolithic Antarctic fungi: detection by PCR assays.

    Science.gov (United States)

    Selbmann, Laura; Isola, Daniela; Zucconi, Laura; Onofri, Silvano

    2011-10-01

    Cryptoendolithic Antarctic black fungi are adapted to the harshest terrestrial conditions as in the ice-free area of the McMurdo Dry Valleys. Recently, surviving space simulated conditions proves their bewildering extremotolerance. In order to investigate the potential DNA damage and their response after UV-B exposition, two strains of Antarctic cryptoendolithic black fungi, Cryomyces antarcticus CCFEE 534 and Cryomyces minteri CCFEE 5187, were irradiated at different UV-B doses. Since conventional methods cannot be applied to these organisms, the effect on the genome was assessed by RAPD and rDNA amplification PCR based assays; the results were compared with the responses of Saccharomyces pastorianus DBVPG 6283 treated with the same conditions. Results showed that template activity was drastically inhibited in S. pastorianus after irradiation. Dramatic changes in the RAPD profiles showed after 30 min of exposure while the rDNA amplification of SSU, LSU, and ITS portions failed after 30, 60, and 90 min of exposure respectively. No alteration was detected in the templates of the Antarctic strains where both RAPD profiles and rDNA PCR amplifications were unaffected even after 240 min of exposure. The electroferograms of the rDNA portions of Cryomyces strains were perfectly readable and conserved whilst the analyses revealed a marked alteration in S. pastorianus confirming the high resistance of the Antarctic strains to UV-B exposure.

  19. Evaluation of γ-radiation-induced DNA damage in two species of bivalves and their relative sensitivity using comet assay

    Energy Technology Data Exchange (ETDEWEB)

    Praveen Kumar, M.K., E-mail: here.praveen@gmail.com [Department of Zoology, Goa University, Goa 403206 (India); Shyama, S.K., E-mail: skshyama@gmail.com [Department of Zoology, Goa University, Goa 403206 (India); Sonaye, B.S. [Department of Radiation Oncology, Goa Medical College, Goa (India); Naik, U Roshini; Kadam, S.B.; Bipin, P.D.; D’costa, A. [Department of Zoology, Goa University, Goa 403206 (India); Chaubey, R.C. [Radiation Biology and Health Science Division, Bhabha Atomic Research Centre, Mumbai (India)

    2014-05-01

    Highlights: • Possible genotoxic effect of accidental exposure of aquatic fauna to γ radiation. • Relative sensitivity of bivalves to γ radiation is also analyzed using comet assay. • γ radiation induced significant genetic damage in both the species of bivalves. • P. malabarica and M. casta exhibited a similar level of sensitivity to γ radiation. • Comet assay may be used as a biomarker for the environmental biomonitoring. - Abstract: Ionizing radiation is known to induce genetic damage in diverse groups of organisms. Under accidental situations, large quantities of radioactive elements get released into the environment and radiation emitted from these radionuclides may adversely affect both the man and the non-human biota. The present study is aimed (a) to know the genotoxic effect of gamma radiation on aquatic fauna employing two species of selected bivalves, (b) to evaluate the possible use of ‘Comet assay’ for detecting genetic damage in haemocytes of bivalves as a biomarker for environmental biomonitoring and also (c) to compare the relative sensitivity of two species of bivalves viz. Paphia malabarica and Meretrix casta to gamma radiation. The comet assays was optimized and validated using different concentrations (18, 32 and 56 mg/L) of ethyl methanesulfonate (EMS), a direct-acting reference genotoxic agent, to which the bivalves were exposed for various times (24, 48 and 72 h). Bivalves were irradiated (single acute exposure) with 5 different doses (viz. 2, 4, 6, 8 and 10 Gy) of gamma radiation and their genotoxic effects on the haemocytes were studied using the comet assay. Haemolymph was collected from the adductor muscle at 24, 48 and 72 h of both EMS-exposed and irradiated bivalves and comet assay was carried out using standard protocol. A significant increase in DNA damage was observed as indicated by an increase in % tail DNA damage at different concentrations of EMS and all the doses of gamma radiation as compared to controls in

  20. Diagnosis of amebic liver abscess and amebic colitis by detection of Entamoeba histolytica DNA in blood, urine, and saliva by a real-time PCR assay.

    Science.gov (United States)

    Haque, Rashidul; Kabir, Mamun; Noor, Zannatun; Rahman, S M Mazidur; Mondal, Dinesh; Alam, Faisal; Rahman, Intekhab; Al Mahmood, Abdullh; Ahmed, Nooruddin; Petri, William A

    2010-08-01

    The noninvasive diagnosis of amebic liver abscess is challenging, as most patients at the time of diagnosis do not have a concurrent intestinal infection with Entamoeba histolytica. Fecal testing for E. histolytica parasite antigen or DNA is negative in most patients. A real-time PCR assay was evaluated for detection of E. histolytica DNA in blood, urine, and saliva samples from amebic liver abscess as well as amebic colitis patients in Bangladesh. A total of 98 amebic liver abscess and 28 amebic colitis patients and 43 control subjects were examined. The real-time PCR assay detected E. histolytica DNA in 49%, 77%, and 69% of blood, urine, and saliva specimens from the amebic liver abscess patients. For amebic colitis the sensitivity of the real-time PCR assay for detection of E. histolytica DNA in blood, urine, and saliva was 36%, 61%, and 64%, respectively. All blood, urine, and saliva samples from control subjects were negative by the real-time PCR assay for E. histolytica DNA. When the real-time PCR assay results of the urine and saliva specimens were taken together (positive either in urine or saliva), the real-time PCR assay was 97% and 89% sensitive for detection of E. histolytica DNA in liver abscess and intestinal infection, respectively. We conclude that the detection of E. histolytica DNA in saliva and urine could be used as a diagnostic tool for amebic liver abscess.

  1. Human Cell Chips: Adapting DNA Microarray Spotting Technology to Cell-Based Imaging Assays

    OpenAIRE

    Traver Hart; Alice Zhao; Ankit Garg; Swetha Bolusani; Marcotte, Edward M.

    2009-01-01

    Here we describe human spotted cell chips, a technology for determining cellular state across arrays of cells subjected to chemical or genetic perturbation. Cells are grown and treated under standard tissue culture conditions before being fixed and printed onto replicate glass slides, effectively decoupling the experimental conditions from the assay technique. Each slide is then probed using immunofluorescence or other optical reporter and assayed by automated microscopy. We show potential ap...

  2. Evaluation of DNA Damage in Common Carp (Cyprinus carpio L. by Comet Assay for Determination of Possible Pollution in Lake Mogan (Ankara

    Directory of Open Access Journals (Sweden)

    İsmet Çok

    2011-01-01

    Full Text Available Contamination of the aquatic environment with various concentrations of pollutants results in unexpected threats to humans and wildlife. The consequences of exposure and metabolism of pollutants/xenobiotics, especially carcinogens and mutagens, can be suitably assessed by investigating severe events, such as DNA damage; for example, DNA adducts and DNA strand breaks. One of the commonly used techniques to detect DNA damage in aquatic organisms is single-cell gel electrophoresis (comet assay. This study was carried out using Cyprinus carpio in order to identify the possible pollution in Lake Mogan, near Ankara, Turkey, where the city's sewer system and pesticides used in agriculture are believed to be the common causes of pollution. From the comet assay, the tail length (μm, tail intensity (%, and tail moment values of fish caught from Lake Mogan were found to be 31.10 ± 10.39, 7.77 ± 4.51, 1.50 ± 1.48, respectively, whereas for clean reference sites they were found to be 22.80 ± 1.08, 3.47 ± 1.59, 0.40 ± 0.51, respectively. The values are statistically different from each other (p < 0.0001, p < 0.0001, and p < 0.0013, respectively. These results indicate that Lake Mogan may be polluted with substances that have genotoxic effects and constitute an early warning for the lake system. Further detailed research is needed to establish the source of the pollution and the chemicals responsible.

  3. HPV Genotyping from the high risk mRNA Aptima assay- a direct approach using DNA from Aptima sample tubes.

    Science.gov (United States)

    Lillsunde Larsson, G; Kaliff, M; Bergengren, L; Karlsson, M G; Helenius, G

    2016-09-01

    The underlying cause of cervical cancer is an infection with the human papilloma virus (HPV) and HPV testing can be used for cervical cancer screening. The Aptima HPV assay from Hologic is an mRNA HPV test used to identify clinically relevant infections but the method does not discriminate between the different high risk genotypes. The aim of the current study was to evaluate if analyzed Aptima sample transfer tubes could be used as a source for HPV genotyping, using sample DNA. Study samples (n=108); were HPV-tested with mRNA Aptima assay and in parallel DNA was extracted and genotyped with Anyplex II HPV28. Analyzed mRNA Aptima tubes were thereafter used as source for a second DNA extraction and genotyping. Using mRNA Aptima result as reference, 90% of the samples (35/39) were high risk positive with the Anyplex II HPV28. Cohen's kappa 0.78 (95% CI: 0.66-0.90), sensitivity 0.90 (95% CI: 0.76-0.97) and specificity 0.90 (95% CI: 0.80-0.96). Two discordant samples carried low-risk genotypes (HPV 82 and HPV 44) and two were negative. DNA-genotyping results, in parallel to and after mRNA testing, were compared and differed significantly (McNemar test: P=0.021) possibly due to sample extraction volume difference. Cohen's kappa 0.81 (95% CI: 0.70-0.92), sensitivity 0.85 (95% CI: 0.74-0.93) and specificity 0.98 (95% CI: 0.88-1.00). In conclusion, analyzed mRNA Aptima sample tubes could be used as a source for DNA HPV genotyping. The sample volume used for extraction needs to be further explored.

  4. The effects on DNA migration of altering parameters in the comet assay protocol such as agarose density, electrophoresis conditions and durations of the enzyme or the alkaline treatments.

    Science.gov (United States)

    Ersson, Clara; Möller, Lennart

    2011-11-01

    The single cell gel electrophoresis (comet assay) is a popular method for measuring DNA migration as an estimate of DNA damage. No standardised comet assay protocol exists, which make comparisons between studies complicated. In a previous inter-laboratory validation study of the comet assay, we identified important parameters in the protocol that might affect DNA migration. The aim of this study was to assess how different comet assay protocols affect DNA migration. The results in this study suggest that (i) there is a significant linear dose-response relationship between the agarose gel's density and DNA migration and that damaged cells are more sensitive to the agarose gel's density; (ii) incubation with formamidopyrimidine DNA glycosylase for 10 min is inadequate, whereas 30 min is sufficient; (iii) the typically used 20 min of alkaline treatment might be to short when analysing samples that contain particular alkali-labile sites (ALS) and (iv) the duration of electrophoresis as well as the strength of the electric field applied affects the DNA migration. By using protocol-specific calibration curves, it is possible to reduce the variation in DNA migration caused by differences in comet assay protocols. This does, however, not completely remove the impact of the durations of alkaline treatment and electrophoresis when analysing cells containing ALS that are relatively resistant to high alkaline treatment.

  5. Development and utility of an internal threshold control (ITC) real-time PCR assay for exogenous DNA detection.

    Science.gov (United States)

    Ni, Weiyi; Le Guiner, Caroline; Moullier, Philippe; Snyder, Richard O

    2012-01-01

    Sensitive and specific tests for detecting exogenous DNA molecules are useful for infectious disease diagnosis, gene therapy clinical trial safety, and gene doping surveillance. Taqman real-time PCR using specific sequence probes provides an effective approach to accurately and quantitatively detect exogenous DNA. However, one of the major challenges in these analyses is to eliminate false positive signals caused by either non-targeted exogenous or endogenous DNA sequences, or false negative signals caused by impurities that inhibit PCR. Although multiplex Taqman PCR assays have been applied to address these problems by adding extra primer-probe sets targeted to endogenous DNA sequences, the differences between targets can lead to different detection efficiencies. To avoid these complications, a Taqman PCR-based approach that incorporates an internal threshold control (ITC) has been developed. In this single reaction format, the target sequence and ITC template are co-amplified by the same primers, but are detected by different probes each with a unique fluorescent dye. Sample DNA, a prescribed number of ITC template molecules set near the limit of sensitivity, a single pair of primers, target probe and ITC probe are added to one reaction. Fluorescence emission signals are obtained simultaneously to determine the cycle thresholds (Ct) for amplification of the target and ITC sequences. The comparison of the target Ct with the ITC Ct indicates if a sample is a true positive for the target (i.e. Ct less than or equal to the ITC Ct) or negative (i.e. Ct greater than the ITC Ct). The utility of this approach was demonstrated in a nonhuman primate model of rAAV vector mediated gene doping in vivo and in human genomic DNA spiked with plasmid DNA.

  6. Detection of ESKAPE Bacterial Pathogens at the Point of Care Using Isothermal DNA-Based Assays in a Portable Degas-Actuated Microfluidic Diagnostic Assay Platform.

    Science.gov (United States)

    Renner, Lars D; Zan, Jindong; Hu, Linda I; Martinez, Manuel; Resto, Pedro J; Siegel, Adam C; Torres, Clint; Hall, Sara B; Slezak, Tom R; Nguyen, Tuan H; Weibel, Douglas B

    2017-02-15

    An estimated 1.5 billion microbial infections occur globally each year and result in ∼4.6 million deaths. A technology gap associated with commercially available diagnostic tests in remote and underdeveloped regions prevents timely pathogen identification for effective antibiotic chemotherapies for infected patients. The result is a trial-and-error approach that is limited in effectiveness, increases risk for patients while contributing to antimicrobial drug resistance, and reduces the lifetime of antibiotics. This paper addresses this important diagnostic technology gap by describing a low-cost, portable, rapid, and easy-to-use microfluidic cartridge-based system for detecting the ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.) bacterial pathogens that are most commonly associated with antibiotic resistance. The point-of-care molecular diagnostic system consists of a vacuum-degassed microfluidic cartridge preloaded with lyophilized recombinase polymerase amplification (RPA) assays and a small portable battery-powered electronic incubator/reader. The isothermal RPA assays detect the targeted ESKAPE pathogens with high sensitivity (e.g., a limit of detection of ∼10 nucleic acid molecules) that is comparable to that of current PCR-based assays, and they offer advantages in power consumption, engineering, and robustness, which are three critical elements required for the point-of-care setting.

  7. Loop-mediated isothermal amplification assay for the detection of Ehrlichia canis DNA in blood samples from dogs

    Directory of Open Access Journals (Sweden)

    SA Faggion

    2013-01-01

    Full Text Available The rickettsial bacterium Ehrlichia canis is the etiological agent of canine monocytic ehrlichiosis, one of the most important canine tick-borne diseases in the world. In this study, a loop-mediated isothermal amplification (LAMP assay was developed for detection of E. canis DNA using LAMP primers targeting the groESL operon. Reactions were performed at 60°C for 60 min and the results were visualized by gel electrophoresis. Successful amplification was obtained using plasmid DNA containing a fragment of the groESL operon and DNA extracted from blood samples that tested positive for E. canis by real-time PCR. The specificity of amplification was confirmed by EcoRI restriction of internal sites in the LAMP primers and no cross-reactivity with blood samples positive for Babesia spp., another common tick-borne pathogen, was observed. The high cost of nucleic acid tests (NAT is one of the disadvantages for their large-scale use as routine diagnostic tests. The E. canis LAMP assay developed here is an interesting alternative to PCR since it does not require a thermocycler, thus reducing costs for the veterinary clinical laboratory.

  8. The potential value of the neutral comet assay and the expression of genes associated with DNA damage in assessing the radiosensitivity of tumor cells.

    Science.gov (United States)

    Jayakumar, Sundarraj; Bhilwade, Hari N; Pandey, Badri N; Sandur, Santosh K; Chaubey, Ramesh C

    2012-10-09

    The assessment of tumor radiosensitivity would be particularly useful in optimizing the radiation dose during radiotherapy. Therefore, the degree of correlation between radiation-induced DNA damage, as measured by the alkaline and the neutral comet assays, and the clonogenic survival of different human tumor cells was studied. Further, tumor radiosensitivity was compared with the expression of genes associated with the cellular response to radiation damage. Five different human tumor cell lines were chosen and the radiosensitivity of these cells was established by clonogenic assay. Alkaline and neutral comet assays were performed in γ-irradiated cells (2-8Gy; either acute or fractionated). Quantitative PCR was performed to evaluate the expression of DNA damage response genes in control and irradiated cells. The relative radiosensitivity of the cell lines assessed by the extent of DNA damage (neutral comet assay) immediately after irradiation (4Gy or 6Gy) was in agreement with radiosensitivity pattern obtained by the clonogenic assay. The survival fraction of irradiated cells showed a better correlation with the magnitude of DNA damage measured by the neutral comet assay (r=-0.9; Pcomet assay (r=-0.73; Pcomet assay was better than alkaline comet assay for assessment of radiosensitivities of tumor cells after acute or fractionated doses of irradiation.

  9. Development of DNA-Free Sediment for Ecological Assays with Genomic Endpoints (NAC SETAC)

    Science.gov (United States)

    Recent advances in genomics are currently being exploited to discern ecological changes that have conventionally been measured using laborious counting techniques. For example, next generation sequencing technologies can be used to create DNA libraries from benthic community ass...

  10. Development of DNA-Free Sediment for Ecological Assays with Genomic Endpoints

    Science.gov (United States)

    Recent advances in genomics are currently being exploited to discern ecological changes that have conventionally been measured using laborious counting techniques. For example, next generation sequencing technologies can be used to create DNA libraries from benthic community ass...

  11. Correlation between the genotoxicity endpoints measured by two different genotoxicity assays: comet assay and CBMN assay

    OpenAIRE

    Carina Ladeira; Susana Viegas; Manuel C. Gomes

    2015-01-01

    The cytokinesis-block micronucleus cytome (CBMN) assay is a comprehensive system for measuring DNA damage; cytostasis and cytotoxicity-DNA damage events are scored specifically in once-divided binucleated cells. The endpoints possible to be measured are micronuclei (MN), a biomarker of chromosome breakage and/or whole chromosome loss, nucleoplasmic bridges (NPB), a biomarker of DNA misrepair and/or telomere end-fusions, and nuclear buds (NBUD), a biomarker of elimination of amplified DNA and/...

  12. Microfludic device for creating ionic strength gradients over DNA microarrays for efficient DNA melting studies and assay development

    DEFF Research Database (Denmark)

    Petersen, Jesper; Poulsen, Lena; Birgens, Henrik

    2009-01-01

    is to use a thermal gradient and information from melting curves, for instance to score genotypes. However, application of temperature gradients normally requires complicated equipment, and the size of the arrays that can be investigated is restricted due to heat dissipation. Here we present a simple...... microfluidic device that creates a gradient comprising zones of defined ionic strength over a glass slide, in which each zone corresponds to a subarray. Using this device, we demonstrated that ionic strength gradients function in a similar fashion as corresponding thermal gradients in assay development. More...

  13. Quantitative data analysis methods for bead-based DNA hybridization assays using generic flow cytometry platforms.

    Science.gov (United States)

    Corrie, S R; Lawrie, G A; Battersby, B J; Ford, K; Rühmann, A; Koehler, K; Sabath, D E; Trau, M

    2008-05-01

    Bead-based assays are in demand for rapid genomic and proteomic assays for both research and clinical purposes. Standard quantitative procedures addressing raw data quality and analysis are required to ensure the data are consistent and reproducible across laboratories independent of flow platform. Quantitative procedures have been introduced spanning raw histogram analysis through to absolute target quantitation. These included models developed to estimate the absolute number of sample molecules bound per bead (Langmuir isotherm), relative quantitative comparisons (two-sided t-tests), and statistical analyses investigating the quality of raw fluorescence data. The absolute target quantitation method revealed a concentration range (below probe saturation) of Cy5-labeled synthetic cytokeratin 19 (K19) RNA of c.a. 1 x 10(4) to 500 x 10(4) molecules/bead, with a binding constant of c.a. 1.6 nM. Raw hybridization frequency histograms were observed to be highly reproducible across 10 triplex assay replicates and only three assay replicates were required to distinguish overlapping peaks representing small sequence mismatches. This study provides a quantitative scheme for determining the absolute target concentration in nucleic acid hybridization reactions and the equilibrium binding constants for individual probe/target pairs. It is envisaged that such studies will form the basis of standard analytical procedures for bead-based cytometry assays to ensure reproducibility in inter- and intra-platform comparisons of data between laboratories. (c) 2008 International Society for Advancement of Cytometry.

  14. A laboratory information management system for DNA barcoding workflows

    NARCIS (Netherlands)

    Vu, D.; Eberhardt, U.; Szöke, S.; Groenewald, M.; Robert, V.

    2012-01-01

    This paper presents a laboratory information management system for DNA sequences (LIMS) created and based on the needs of a DNA barcoding project at the CBS-KNAW Fungal Biodiversity Centre (Utrecht, the Netherlands). DNA barcoding is a global initiative for species identification through simple DNA

  15. Antibiotic microbial assay using kinetic-reading microplate system

    OpenAIRE

    Felipe Rebello Lourenço; Terezinha de Jesus Andreoli Pinto

    2011-01-01

    The aim of this study was to determine the optimal experimental conditions to develop a methodology for microbiological assay of apramycin employing microplate and kinetic reading mode, and to validate the developed method, through evaluation of parameters of selectivity, linearity, linear range, limits of detection and quantification, accuracy and precision. The turbidimetric assay principle is simple: the test solution is added to a suspension of test microorganism in culture media, the mix...

  16. Detection of beer spoilage bacteria Pectinatus and Megasphaera with acridinium ester labelled DNA probes using a hybridisation protection assay.

    Science.gov (United States)

    Paradh, A D; Hill, A E; Mitchell, W J

    2014-01-01

    DNA probes specific for rRNA of selected target species were utilised for the detection of beer spoilage bacteria of the genera Pectinatus and Megasphaera using a hybridisation protection assay (HPA). All the probes were modified during synthesis by addition of an amino linker arm at the 5' end or were internally modified by inserting an amine modified thymidine base. Synthesised probes then were labelled with acridinium ester (AE) and purified using reverse phase HPLC. The internally AE labelled probes were able to detect target RNA within the range of 0.016-0.0032pmol. All the designed probes showed high specificity towards target RNA and could detect bacterial contamination within the range of ca. 5×10(2)1×10(3) CFU using the HPA. The developed assay was also compatible with MRS, NBB and SMMP beer enrichment media, routinely used in brewing laboratories.

  17. Development of a nested PCR assay to detect equine infectious anemia proviral DNA from peripheral blood of naturally infected horses.

    Science.gov (United States)

    Dong, Jian-Bao; Zhu, Wei; Cook, Frank R; Goto, Yoshitaka; Horii, Yoichiro; Haga, Takeshi

    2012-11-01

    Equine infectious anemia (EIA) has posed a major challenge and caused significant losses to the equine industry worldwide. PCR detection methods have considerable potential as an adjunct to conventional serological diagnostic techniques. However, most published PCR methods, including that recommended by the OIE, were designed using laboratory-adapted virus strains and do not function with field isolates of EIA virus (EIAV). In the present study, a nested PCR assay for detection of EIAV proviral DNA in peripheral blood cells of naturally infected horses was developed. Primer sets were designed based on conserved 5' regions of the viral genome extending from the LTR to the tat gene. Preliminary studies demonstrated that the method has a detection limit of 10 genomic copies and, when applied to a naturally EIAV-infected feral horse population, shows 100 % correlation with conventional serological diagnostic techniques. This assay provides a powerful new tool in the control of EIAV.

  18. Development of a novel in vitro assay for the evaluation of integron DNA integrase activity

    Directory of Open Access Journals (Sweden)

    Fatemeh Tohidi

    2016-05-01

    Full Text Available Integrons play an important role in multidrug resistance. The integron platform codes for integrase (intI that is required for gene cassette integration through site-specific recombination. The recombination crossover occurs between the G and TT nucleotides in non-palindromic attI and palindromic attC sites. The aim of this study was to establish an efficient in vitro assay for integrase purification and activity detection. To this end, the intI gene was cloned into the pET-22b plasmid. Then, the resulting recombinant plasmid was transformed into Escherichia coli Origami™ strain. The recombinant protein expression was confirmed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE and western blot assays. The recombinant intI protein was purified by nickel–nitrilotriacetic acid (Ni–NTA affinity chromatography, and its activity was measured by a newly introduced assay. Briefly, specific primers for each side of attI and attC were used, thereby, a polymerase chain reaction would be performed, if a fused plasmid containing both attI and attC sites was created upon recombination. SDS-PAGE and western blotting confirmed the presence of a 38-kDa recombinant protein. Optimum conditions were established for the measurement of the integrase activity and a new model assay was conducted to analyse the recombination activity in vitro. Although the electrophoretic mobility shift assay is an efficient and reliable method, the newly introduced assay provided new or enhanced capability to determine the integrase activity, suggesting that there is no need for expensive and advanced equipment.

  19. Optimized modification of gold nanoparticles with a self-assembled monolayer for suppression of nonspecific binding in DNA assays

    Science.gov (United States)

    Esashika, Keiko; Saiki, Toshiharu

    2016-10-01

    Homogeneous DNA assays using gold nanoparticles (AuNPs) require the reduction of nonspecific binding between AuNPs to improve sensitivity in detecting the target molecule. In this study, we employed alkanethiol self-assembled monolayers (SAMs) for modifying the AuNP surface to attain both good dispersability and high hybridization efficiency. The alkanethiol SAMs enhance the repulsive interaction between AuNPs, reducing nonspecific binding and promoting the extension of surface-immobilized ssDNA into the solvent, thus enhancing the hybridization process. Introduction of oligoethylene glycol into the alkanethiol prevented nonspecific binding caused by the entanglement of alkane chains. Finally, the conditions were optimized by controlling the surface charge density through the introduction of a COOH group at the alkanethiol terminus, resulting in the complete blocking of nonspecific binding and the maintenance of high hybridization efficiency.

  20. Nucleobase-Based Barbiturates: Their Protective Effect against DNA Damage Induced by Bleomycin-Iron, Antioxidant, and Lymphocyte Transformation Assay

    Directory of Open Access Journals (Sweden)

    Bhaveshkumar D. Dhorajiya

    2014-01-01

    Full Text Available A number of nucleobase-based barbiturates have been synthesized by combination of nucleic acid bases and heterocyclic amines and barbituric acid derivatives through green and efficient multicomponent route and one pot reaction. This approach was accomplished efficiently using aqueous medium to give the corresponding products in high yield. The newly synthesized compounds were characterized by spectral analysis (FT-IR, 1H NMR, 13C NMR, HMBC, and UV spectroscopy and elemental analysis. Representative of all synthesized compounds was tested and evaluated for antioxidant, bleomycin-dependent DNA damage, and Lymphocyte Transformation studies. Compounds TBC > TBA > TBG showed highest lymphocyte transformation assay, TBC > TBA > BG showed inhibitory antioxidant activity using ABTS methods, and TBC > BPA > BAMT > TBA > 1, 3-TBA manifested the best protective effect against DNA damage induced by bleomycin.

  1. Detection of mitochondrial DNA with the compact bead array sensor system (cBASS)

    Science.gov (United States)

    Mulvaney, Shawn P.; Ibe, Carol N.; Caldwell, Jane M.; Levine, Jay F.; Whitman, Lloyd J.; Tamanaha, Cy R.

    2009-02-01

    Enteric pathogens are a significant contaminant in surface waters used for recreation, fish and shellfish harvesting, crop irrigation, and human consumption. The need for water monitoring becomes more pronounced when industrial, agricultural, and residential lands are found in close proximity. Fecal contamination is particularly problematic and identification of the pollution source essential to remediation efforts. Standard monitoring for fecal contamination relies on indicator organisms, but the technique is too broad to identify the source of contamination. Instead, real-time PCR of mitochondrial DNA (mtDNA) is an emerging method for identification of the contamination source. Presented herein, we evaluate an alternative technology, the compact Bead Array Sensor System (cBASS®) and its assay approach Fluidic Force Discrimination (FFD), for the detection of mtDNA. Previously, we achieved multiplexed, attomolar detection of toxins and femtomolar detection of nucleic acids in minutes with FFD assays. More importantly, FFD assays are compatible with a variety of complex matrices and therefore potentially applicable for samples where the matrix would interfere with PCR amplification. We have designed a triplex assay for the NADH gene found in human, swine, and bovine mtDNA and demonstrated the specific detection of human mtDNA spiked into a waste water sample.

  2. Measurement of DNA base and nucleotide excision repair activities in mammalian cells and tissues using the comet assay--a methodological overview.

    Science.gov (United States)

    Azqueta, Amaya; Langie, Sabine A S; Slyskova, Jana; Collins, Andrew R

    2013-11-01

    There is an increasing demand for phenotyping assays in the field of human functional genetics. DNA repair activity is representative of this functional approach, being seen as a valuable biomarker related to cancer risk. Repair activity is evaluated by incubating a cell extract with a DNA substrate containing lesions specific for the DNA repair pathway of interest. Enzymic incision at the lesion sites can be measured by means of the comet assay (single cell gel electrophoresis). The assay is particularly applicable for evaluation of base and nucleotide excision repair pathways (BER and NER). Substrate DNA containing oxidised purines gives a measure of BER, while UV-induced photolesions are the substrate for NER. While applications of comet-based DNA repair assays continue to increase, there are no commonly accepted standard protocols, which complicates inter-laboratory comparisons of results. Here we provide a comprehensive summary of protocols for the comet-based BER- and NER-specific in vitro DNA repair assays that can be applied to a wide spectrum of biological material--cultured cell lines, blood cells, animal tissue samples and human biopsies. Our intention is to provide a detailed and user-friendly account of the assays, including practical tips and recommendations to help in setting them up. By proposing standard protocols, we hope to facilitate comparison of results obtained in different laboratories.

  3. A facile, sensitive, and highly specific trinitrophenol assay based on target-induced synergetic effects of acid induction and electron transfer towards DNA-templated copper nanoclusters.

    Science.gov (United States)

    Li, Haiyin; Chang, Jiafu; Hou, Ting; Ge, Lei; Li, Feng

    2016-11-01

    Reliable, selective and sensitive approaches for trinitrophenol (TNP) detection are highly desirable with respect to national security and environmental protection. Herein, a simple and novel fluorescent strategy for highly sensitive and specific TNP assay has been successfully developed, which is based on the quenching of the fluorescent poly(thymine)-templated copper nanoclusters (DNA-CuNCs), through the synergetic effects of acid induction and electron transfer. Upon the addition of TNP, donor-acceptor complexes between the electron-deficient nitro-groups in TNP and the electron-donating DNA templates are formed, resulting in the close proximity between TNP and CuNCs. Moreover, the acidity of TNP contributes to the pH decrease of the system. These factors combine to dramatically quench the fluorescence of DNA-CuNCs, providing a "signal-off" strategy for TNP sensing. The as-proposed strategy demonstrates high sensitivity for TNP assay, and a detection limit of 0.03μM is obtained, which is lower than those reported by using organic fluorescent materials. More significantly, this approach shows outstanding selectivity over a number of TNP analogues, such as 2,4,6-trinitrotoluene (TNT), 2,4-dinitrotoluene (DNT), 2,4-dinitrophenol (DNP), 3-nitrophenol (NP), nitrobenzene (NB), phenol (BP), and toluene (BT). Compared with previous studies, this method does not need complex DNA sequence design, fluorescent dye labeling, or sophisticated organic reactions, rendering the strategy with additional advantages of simplicity and cost-effectiveness. In addition, the as-proposed strategy has been adopted for the detection of TNP in natural water samples, indicating its great potential to be applied in the fields of public safety and environmental monitoring.

  4. Development of a robust DNA quality and quantity assessment qPCR assay for targeted next-generation sequencing library preparation.

    Science.gov (United States)

    Dang, Jennifer; Mendez, Pedro; Lee, Sharon; Kim, James W; Yoon, Jun-Hee; Kim, Thomas W; Sailey, Charles J; Jablons, David M; Kim, Il-Jin

    2016-10-01

    Next-generation sequencing (NGS) is becoming a standard for genetic analyses of clinical samples. DNAs retrieved from formalin-fixed, paraffin-embedded (FFPE) tissue specimens are commonly degraded, and specimens such as core biopsies are sometimes too small to obtain enough DNA for NGS applications. Thus, it is important to measure both the DNA quantity and quality accurately from clinical samples. However, there is no standard method for DNA quantity and quality analyses for NGS library preparation. We tested four different methods (PicoGreen, Qubit® fluorometry, TaqMan and SYBR-Green-based qPCR assay) and compared each to RNase P TaqMan as a reference control. We found that SYBR-Green-based qPCR assay provides a consistent and accurate DNA quantification while keeping its cost relatively low and the throughput high. We designed a dual-probe SYBR-Green qPCR assay for DNA quantity and quality assessment for targeted NGS library preparation. This assay provides a Dscore (degradation score) of the interrogated DNA by analyzing two different sizes of amplicons. We show an example of a clinical sample with a very high Dscore (high degradation). With a regular DNA quantification, without considering the degradation status, no correct NGS libraries were obtained. However, after optimizing the library condition by considering its poor DNA quality, a reasonably good library and sequencing results were obtained. In summary, we developed and presented a new DNA quantity and quality analysis qPCR assay for the targeted NGS library preparation. This assay may be mostly efficient for the clinical samples with high degradation and poor DNA quality.

  5. Evaluation of DNA damage in agricultural workers exposed to pesticides using single cell gel electrophoresis (comet) assay.

    Science.gov (United States)

    Kaur, Raminderjeet; Kaur, Satbir; Lata, Mukesh

    2011-09-01

    Pesticides are used in agriculture to protect crops, but they pose a potential risk to farmers and environment. The aim of the present study is to investigate the relation between the occupational exposure to various pesticides and the presence of DNA damage. Blood samples of 210 exposed workers (after a day of intense spraying) and 50 control subjects belonging to various districts of Punjab (India) were evaluated using Comet assay. Sixty workers who showed DNA damage were selected for follow up at 5-6 months after the first sampling during a low or null spraying period. Significant differences were found in DNA damage between freshly exposed workers and controls and freshly exposed and followed up cases. There was significant increase in the comet parameters viz. mean comet tail length and frequency of cells showing migration in exposed workers as compared to controls (72.22 ± 20.76 vs. 46.92 ± 8.17, Pdamaged cells as compared to freshly exposed workers of first sampling (Ppesticide exposure, age, smoking, drinking and dietary habits etc which were expected to modulate the damage, were instead found to have no significant effect on DNA fragmentation. The evidence of a genetic hazard related to exposure resulting from the intensive use of pesticides stresses the need for educational programs for agricultural workers to reduce the use of chemicals in agriculture.

  6. Detection of xenobiotic-induced DNA damage by the comet assay applied to human and rat precision-cut liver slices

    NARCIS (Netherlands)

    Plazar, Janja; Hrejac, Irena; Pirih, Primoz; Filipic, Metka; Groothuis, Geny M. M.

    2007-01-01

    The comet assay is a simple and sensitive method for measuring DNA damage at the level of individual cells and is extensively used in genotoxicity studies. It is commonly applied to cultured cells. The aim of this study was to apply the comet assay for use in fresh liver tissue, where metabolic acti

  7. A defined system for in vitro lambda DNA packaging.

    Science.gov (United States)

    Hwang, Y; Feiss, M

    1995-08-20

    We constructed a defined in vitro system for packaging lambda DNA which is composed of purified proheads, the noncapsid proteins terminase and gpFI, and the Escherichia coli DNA binding/bending protein IHF. The defined packaging system: (i) is free from endogenous ATP, DNAs, and DNases and (ii) packages 30% of the input mature lambda DNA efficiently. In this defined packaging system, IHF and gpFI make modest contributions to packaging efficiency. The defined packaging reactions showed that DNA packaging gave a linear response to the concentration of mature lambda DNA and terminase. DNA packaging showed a sigmoidal relationship with respect to the concentration of ATP and proheads.

  8. Electrophoretic Mobility Shift Assay of DNA and CRISPR-Cas Ribonucleoprotein Complexes

    NARCIS (Netherlands)

    Künne, T.A.; Westra, E.R.; Brouns, S.J.J.

    2015-01-01

    The Electrophoretic Mobility Shift Assay is a straightforward and inexpensive method for the determination and quantification of protein-nucleic acid interactions. It relies on the different mobility of free and protein-bound nucleic acid in a gel matrix during electrophoresis. Nucleic acid affiniti

  9. Improvement in fetal DNA extraction from maternal plasma. Evaluation of the NucliSens Magnetic Extraction system and the QIAamp DSP Virus Kit in comparison with the QIAamp DNA Blood Mini Kit

    DEFF Research Database (Denmark)

    Clausen, Frederik Banch; Krog, Grethe Risum; Rieneck, Klaus

    2007-01-01

    Prenatal diagnostic assays have been developed using free fetal DNA circulating in the maternal blood of pregnant women. Efficient DNA extraction is crucial for a robust analysis. To improve fetal DNA yield, we tested two manual extraction methods--the NucliSens Magnetic Extraction (NMAG) system...... and the QIAamp DSP Virus Kit (QDSP)--against our current standard method, the widely used QIAamp DNA Blood Mini Kit (QDNA)....

  10. Combination of DNA ligase reaction and gold nanoparticle-quenched fluorescent oligonucleotides: a simple and efficient approach for fluorescent assaying of single-nucleotide polymorphisms.

    Science.gov (United States)

    Wang, Hao; Li, Jishan; Wang, Yongxiang; Jin, Jiangyu; Yang, Ronghua; Wang, Kemin; Tan, Weihong

    2010-09-15

    A new fluorescent sensing approach for detection of single-nucleotide polymorphisms (SNPs) is proposed based on the ligase reaction and gold nanoparticle (AuNPs)-quenched fluorescent oligonucleotides. The design exploits the strong fluorescence quenching of AuNPs for organic dyes and the difference in noncovalent interactions of the nanoparticles with single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA), where ssDNA can be adsorbed onto the surface of AuNPs while dsDNA cannot be. In the assay, two half primer DNA probes, one being labeled with a dye and the other being phosphorylated, were first incubated with a target DNA template. In the presence of DNA ligase, the two captured ssDNAs are linked for the perfectly matched DNA target to form a stable duplex, but the duplex could not be formed by the single-base mismatched DNA template. After addition of AuNPs, the fluorescence of dye-tagged DNA probe will be efficiently quenched unless the perfectly matched DNA target is present. To demonstrate the feasibility of this design, the performance of SNP detection using two different DNA ligases, T4 DNA ligase and Escherichia coli DNA ligase, were investigated. In the case of T4 DNA ligase, the signal enhancement of the dye-tagged DNA for perfectly matched DNA target is 4.6-fold higher than that for the single-base mismatched DNA. While in the presence of E. coli DNA ligase, the value raises to be 30.2, suggesting excellent capability for SNP discrimination.

  11. PCR real time assays for the early detection of BKV-DNA in immunocompromised patients.

    Science.gov (United States)

    Marinelli, Katia; Bagnarelli, Patrizia; Gaffi, Gianni; Trappolini, Silvia; Leoni, Pietro; Paggi, Alessandra Mataloni; Della Vittoria, Agnese; Scalise, Giorgio; Varaldo, Pietro Emanuele; Menzo, Stefano

    2007-07-01

    Testing for viral BKV-DNA in urine is a non-invasive early detection and monitoring tool in the diagnostic of BKV-related pathologies: quantitative analysis by Real-Time PCR can provide useful information in addition to cytologic analysis, although our study suggests that high BKV viruria is not necessarily associated with kidney or bladder damage.

  12. Persistence of Free Plasmid DNA in Soil Monitored by Various Methods, Including a Transformation Assay

    Science.gov (United States)

    Romanowski, Gerd; Lorenz, Michael G.; Sayler, Gary; Wackernagel, Wilfried

    1992-01-01

    The persistence and stability of free plasmid pUC8-ISP DNA introduced into 10-g samples of various soils and kept at 23°C were monitored over a period of 60 days. The soils were sampled at a plant science farm and included a loamy sand soil (no. 1), a clay soil (no. 2), and a silty clay soil (no. 3). Four different methods allowed monitoring of (i) the production of acid-soluble radioactive material from [3H]thymidine-labeled plasmid DNA, (ii) the decrease of hybridizing nucleotide sequences in slot blot analysis, (iii) the loss of plasmid integrity measured by Southern hybridization, and (iv) the decay of the biological activity as determined by transformation of Ca2+-treated Escherichia coli cells with the DNA extracted from soil. Acid-soluble material was not produced within the first 24 h but then increased to 45% (soil no. 1), 27% (soil no. 2), and 77% (soil no. 3) until the end of incubation. A quite parallel loss of material giving a slot blot hybridization signal was observed. Southern hybridization indicated that after 1 h in the soils, plasmid DNA was mostly in the form of circular and full-length linear molecules but that, depending on the soil type, after 2 to 5 days full-length plasmid molecules were hardly detectable. The transforming activity of plasmid DNA reextracted from the soils followed inactivation curves over 2 to 4 orders of magnitude and dropped below the detection limit after 10 days. The inactivation was slower in soil no. 2 (28.2-h half-life time of the transforming activity of a plasmid molecule) than in soils no. 3 (15.1 h) and no. 1 (9.1 h). The studies provide data on the persistence of free DNA molecules in natural bacterial soil habitats. The data suggest that plasmid DNA may persist long enough to be available for uptake by competent recipient cells in situ. Images PMID:16348772

  13. Microbead agglutination based assays

    KAUST Repository

    Kodzius, Rimantas

    2013-01-21

    We report a simple and rapid room temperature assay for point-of-care (POC) testing that is based on specific agglutination. Agglutination tests are based on aggregation of microbeads in the presence of a specific analyte thus enabling the macroscopic observation. Such tests are most often used to explore antibody-antigen reactions. Agglutination has been used for protein assays using a biotin/streptavidin system as well as a hybridization based assay. The agglutination systems are prone to selftermination of the linking analyte, prone to active site saturation and loss of agglomeration at high analyte concentrations. We investigated the molecular target/ligand interaction, explaining the common agglutination problems related to analyte self-termination, linkage of the analyte to the same bead instead of different microbeads. We classified the agglutination process into three kinds of assays: a two- component assay, a three-component assay and a stepped three- component assay. Although we compared these three kinds of assays for recognizing DNA and protein molecules, the assay can be used for virtually any molecule, including ions and metabolites. In total, the optimized assay permits detecting analytes with high sensitivity in a short time, 5 min, at room temperature. Such a system is appropriate for POC testing.

  14. A PCR-based assay for discriminating Cervus and Rangifer (Cervidae) antlers with mitochondrial DNA polymorphisms.

    Science.gov (United States)

    Kim, Young Hwa; Kim, Eung Soo; Ko, Byong Seob; Oh, Seung-Eun; Ryuk, Jin-Ah; Chae, Seong Wook; Lee, Hye Won; Choi, Go Ya; Seo, Doo Won; Lee, Mi Young

    2012-07-01

    This study describes a method for discriminating Rangifer antlers from true Cervus antlers using agarose gel electrophoresis, capillary electrophoresis, quantitative real-time PCR, and allelic discrimination. Specific primers labeled with fluorescent tags were designed to amplify fragments from the mitochondrial D-loop genes for various Cervus subspecies and Rangifer tarandus differentially. A 466-bp fragment that was observed for both Cervus and Rangifer antlers served as a positive control, while a 270-bp fragment was specifically amplified only from Rangifer antlers. Allelic discrimination was used to differentiate between Cervus and Rangifer antlers, based on the amplification of specific alleles for both types of antlers. These PCR-based assays can be used for forensic and quantitative analyses of Cervus and Rangifer antlers in a single step, without having to obtain any sequence information. In addition, multiple PCR-based assays are more accurate and reproducible than a single assay for species-specific analysis and are especially useful in this study for the identification of original Cervus deer products from fraudulent Rangifer antlers.

  15. DNA Profiling of Convicted Offender Samples for the Combined DNA Index System

    Science.gov (United States)

    Millard, Julie T

    2011-01-01

    The cornerstone of forensic chemistry is that a perpetrator inevitably leaves trace evidence at a crime scene. One important type of evidence is DNA, which has been instrumental in both the implication and exoneration of thousands of suspects in a wide range of crimes. The Combined DNA Index System (CODIS), a network of DNA databases, provides…

  16. DNA Profiling of Convicted Offender Samples for the Combined DNA Index System

    Science.gov (United States)

    Millard, Julie T

    2011-01-01

    The cornerstone of forensic chemistry is that a perpetrator inevitably leaves trace evidence at a crime scene. One important type of evidence is DNA, which has been instrumental in both the implication and exoneration of thousands of suspects in a wide range of crimes. The Combined DNA Index System (CODIS), a network of DNA databases, provides…

  17. Development of a new ultra sensitive real-time PCR assay (ultra sensitive RTQ-PCR for the quantification of HBV-DNA

    Directory of Open Access Journals (Sweden)

    Varaklioti Agoritsa

    2010-03-01

    Full Text Available Abstract Background Improved sensitivity of HBV-DNA tests is of critical importance for the management of HBV infection. Our aim was to develop and assess a new ultra sensitive in-house real-time PCR assay for HBV-DNA quantification (ultra sensitive RTQ-PCR. Results Previously used HBV-DNA standards were calibrated against the WHO 1st International Standard for HBV-DNA (OptiQuant® HBV-DNA Quantification Panel, Accrometrix Europe B.V.. The 95% and 50% HBV-DNA detection end-point of the assay were 22.2 and 8.4 IU/mL. According to the calibration results, 1 IU/mL equals 2.8 copies/mL. Importantly the clinical performance of the ultra sensitive real-time PCR was tested similar (67% to the Procleix Ultrio discriminatory HBV test (dHBV (70% in low-titer samples from patients with occult Hepatitis B. Finally, in the comparison of ultra sensitive RTQ-PCR with the commercially available COBAS TaqMan HBV Test, the in-house assay identified 94.7% of the 94 specimens as positive versus 90.4% identified by TaqMan, while the quantitative results that were positive by both assay were strongly correlated (r = 0.979. Conclusions We report a new ultra sensitive real time PCR molecular beacon based assay with remarkable analytical and clinical sensitivity, calibrated against the WHO 1st International standard.

  18. DNA-conjugated gold nanoparticles based colorimetric assay to assess helicase activity: a novel route to screen potential helicase inhibitors

    Science.gov (United States)

    Deka, Jashmini; Mojumdar, Aditya; Parisse, Pietro; Onesti, Silvia; Casalis, Loredana

    2017-03-01

    Helicase are essential enzymes which are widespread in all life-forms. Due to their central role in nucleic acid metabolism, they are emerging as important targets for anti-viral, antibacterial and anti-cancer drugs. The development of easy, cheap, fast and robust biochemical assays to measure helicase activity, overcoming the limitations of the current methods, is a pre-requisite for the discovery of helicase inhibitors through high-throughput screenings. We have developed a method which exploits the optical properties of DNA-conjugated gold nanoparticles (AuNP) and meets the required criteria. The method was tested with the catalytic domain of the human RecQ4 helicase and compared with a conventional FRET-based assay. The AuNP-based assay produced similar results but is simpler, more robust and cheaper than FRET. Therefore, our nanotechnology-based platform shows the potential to provide a useful alternative to the existing conventional methods for following helicase activity and to screen small-molecule libraries as potential helicase inhibitors.

  19. DNA-conjugated gold nanoparticles based colorimetric assay to assess helicase activity: a novel route to screen potential helicase inhibitors

    Science.gov (United States)

    Deka, Jashmini; Mojumdar, Aditya; Parisse, Pietro; Onesti, Silvia; Casalis, Loredana

    2017-01-01

    Helicase are essential enzymes which are widespread in all life-forms. Due to their central role in nucleic acid metabolism, they are emerging as important targets for anti-viral, antibacterial and anti-cancer drugs. The development of easy, cheap, fast and robust biochemical assays to measure helicase activity, overcoming the limitations of the current methods, is a pre-requisite for the discovery of helicase inhibitors through high-throughput screenings. We have developed a method which exploits the optical properties of DNA-conjugated gold nanoparticles (AuNP) and meets the required criteria. The method was tested with the catalytic domain of the human RecQ4 helicase and compared with a conventional FRET-based assay. The AuNP-based assay produced similar results but is simpler, more robust and cheaper than FRET. Therefore, our nanotechnology-based platform shows the potential to provide a useful alternative to the existing conventional methods for following helicase activity and to screen small-molecule libraries as potential helicase inhibitors. PMID:28287182

  20. A competition assay for DNA binding using the fluorescent probe ANS.

    Science.gov (United States)

    Taylor, Ian A; Kneale, G Geoff

    2009-01-01

    Fluorescence spectroscopy is a technique frequently employed to study protein-nucleic acid interactions. Often, the intrinsic fluorescence emission spectrum of tryptophan residues in a nucleic-acid-binding protein is strongly perturbed upon interaction with a target DNA or RNA. These spectral changes can then be exploited in order to construct binding isotherms and the extract equilibrium association constant together with the stoichiometry of an interaction. However, when a protein contains many tryptophan residues that are not located in the proximity of the nucleic-acid-binding site, changes in the fluorescence emission spectrum may not be apparent or the magnitude too small to be useful. Here, we make use of an extrinsic fluorescence probe, the environmentally sensitive fluorophore 1-anilinonaphthalene-8-sulphonic acid (1,8-ANS). Displacement by DNA of 1,8-ANS molecules from the nucleic-acid-binding site of the Type I modification methylase EcoR124I results in red shifting and an intensity decrease of the 1,8-ANS fluorescence emission spectrum. These spectral changes have been used to investigate the interaction of EcoR124I with DNA target recognition sequences.

  1. Direct assay of radiation-induced DNA base lesions in mammalian cells

    Energy Technology Data Exchange (ETDEWEB)

    1992-01-01

    Adenine (Ade), 2'-deoxyadenosine (dAdo), 5'-deoxyadenosine monophosphate (dAUT), single stranded poly adenylic acid [poly (dA)], double stranded deoxyadenylic-thymidylic acid [ds poly (dA-T)] and salmon testis DNA were irradiated with 500 Gy under oxic and anoxic conditions. The major damage products were analyzed by BPLC with optical detection and quantitated in terms of the percentage of the adenosine in each model compound found as a specific damage product. Outside of the Ade free base, 8-OH-dAdo was the major oxic damage product from each model compound. The type and quantity of the major damage products depended on the sequence and conformation of the model compounds under anoxic conditions. When dAdo and dAMP were irradiated under anoxic conditions, the major damage product was either the R or S isomer of 8,5'cdAdo and little Ade or [alpha]-dAdo was observed. However, when poly(dA), poly(dA-dT), and salmon testis DNA were [gamma]-irradiated under nitrogen, the major deoxyadenosine damage product was identified as the [alpha]-anomer of deoxyadenosine. No [alpha]-deoxyadenosine was detected after irradiation under oxic conditions. The presence of nucleotides with the [alpha]-configuration at the anomeric carbon atom in the DNA chain may have a significant effect on its tertiary structure and possibly modify its biological activity.

  2. The New Aptima HBV Quant Real-Time TMA Assay Accurately Quantifies Hepatitis B Virus DNA from Genotypes A to F.

    Science.gov (United States)

    Chevaliez, Stéphane; Dauvillier, Claude; Dubernet, Fabienne; Poveda, Jean-Dominique; Laperche, Syria; Hézode, Christophe; Pawlotsky, Jean-Michel

    2017-04-01

    Sensitive and accurate hepatitis B virus (HBV) DNA detection and quantification are essential to diagnose HBV infection, establish the prognosis of HBV-related liver disease, and guide the decision to treat and monitor the virological response to antiviral treatment and the emergence of resistance. Currently available HBV DNA platforms and assays are generally designed for batching multiple specimens within an individual run and require at least one full day of work to complete the analyses. The aim of this study was to evaluate the ability of the newly developed, fully automated, one-step Aptima HBV Quant assay to accurately detect and quantify HBV DNA in a large series of patients infected with different HBV genotypes. The limit of detection of the assay was estimated to be 4.5 IU/ml. The specificity of the assay was 100%. Intra-assay and interassay coefficients of variation ranged from 0.29% to 5.07% and 4.90% to 6.85%, respectively. HBV DNA levels from patients infected with HBV genotypes A to F measured with the Aptima HBV Quant assay strongly correlated with those measured by two commercial real-time PCR comparators (Cobas AmpliPrep/Cobas TaqMan HBV test, version 2.0, and Abbott RealTime HBV test). In conclusion, the Aptima HBV Quant assay is sensitive, specific, and reproducible and accurately quantifies HBV DNA in plasma samples from patients with chronic HBV infections of all genotypes, including patients on antiviral treatment with nucleoside or nucleotide analogues. The Aptima HBV Quant assay can thus confidently be used to detect and quantify HBV DNA in both clinical trials with new anti-HBV drugs and clinical practice. Copyright © 2017 American Society for Microbiology.

  3. Rapid assessment of repair of ultraviolet DNA damage with a modified host-cell reactivation assay using a luciferase reporter gene and correlation with polymorphisms of DNA repair genes in normal human lymphocytes

    Energy Technology Data Exchange (ETDEWEB)

    Qiao Yawei; Spitz, Margaret R.; Guo Zhaozheng; Hadeyati, Mohammad; Grossman, Lawrence; Kraemer, Kenneth H.; Wei Qingyi

    2002-11-30

    As DNA repair plays an important role in genetic susceptibility to cancer, assessment of the DNA repair phenotype is critical for molecular epidemiological studies of cancer. In this report, we compared use of the luciferase (luc) reporter gene in a host-cell reactivation (HCR) (LUC) assay of repair of ultraviolet (UV) damage to DNA to use of the chloramphenicol (cat) gene-based HCR (CAT) assay we used previously for case-control studies. We performed both the assays on cryopreserved lymphocytes from 102 healthy non-Hispanic white subjects. There was a close correlation between DNA repair capacity (DRC) as measured by the LUC and CAT assays. Although these two assays had similar variation, the LUC assay was faster and more sensitive. We also analyzed the relationship between DRC and the subjects' previously determined genotypes for four polymorphisms of two nucleotide-excision repair (NER) genes (in intron 9 of xeroderma pigmentosum (XP) C and exons 6, 10 and 23 of XPD) and one polymorphism of a base-excision repair gene in exon 10 of X-ray complementing group 1 (XRCC1). The DRC was significantly lower in subjects homozygous for one or more polymorphisms of the two NER genes than in subjects with other genotypes (P=0.010). In contrast, the polymorphic XRCC1 allele had no significant effect on DRC. These results suggest that the post-UV LUC assay measures NER phenotype and that polymorphisms of XPC and XPD genes modulate DRC. For population studies of the DNA repair phenotype, many samples need to be evaluated, and so the LUC assay has several advantages over the CAT assay: the LUC assay was more sensitive, had less variation, was not radioactive, was easier to perform, and required fewer cryopreserved cells. These features make the LUC-based HCR assay suitable for molecular epidemiological studies.

  4. Alkaline, Endo III and FPG modified comet assay as biomarkers for the detection of oxidative DNA damage in rats with experimentally induced diabetes.

    Science.gov (United States)

    Kushwaha, S; Vikram, A; Trivedi, P P; Jena, G B

    2011-12-24

    Increased production of reactive oxygen species under diabetic condition underlines the higher oxidatively damaged DNA in different tissues. However, it is practically difficult to assess the oxidatively damaged DNA in different internal organs. Therefore, the present study was aimed to evaluate the extent of oxidative stress-induced DNA damage in different organs with the progression of diabetes. Diabetic and control Sprague Dawley rats were sacrificed in time-dependent manner and the lung, liver, heart, aorta, kidney, pancreas and peripheral blood lymphocytes (PBL) were analyzed for both alkaline and modified comet assay with endonuclease-III (Endo III) and formamidopyrimidine-DNA glycosylase (FPG) (hereafter called modified comet assay) for the detection of oxidative DNA damage. The statistically significant increase in olive tail moment (OTM) was found in all the tested tissues. The extent of DNA damage was increased with the progression of diabetes as revealed by the parameter of OTM in alkaline and modified comet assay. Further, the positive correlations were observed between OTM of the lung, liver, heart, aorta, kidney and pancreas with PBL of diabetic rat in the alkaline and modified comet assay. Moreover, significant increase in the 8-oxodG positive nuclei in the lung, liver, heart, aorta, kidney and pancreas was observed in 4th and 8th week diabetic rat as compared to control. Results of the present study clearly indicated the suitability of alkaline and modified comet assay for the detection of multi-organ oxidative DNA damage in streptozotocin (STZ)-induced diabetic rat and showed that damaged DNA of PBL can be used as a suitable biomarker to assess the internal organs response to DNA damage in diabetes.

  5. Development and evaluation of a loop-mediated isothermal amplification assay for detection of Ehrlichia canis DNA in naturally infected dogs using the p30 gene.

    Science.gov (United States)

    Pinhanelli, V C; Costa, P N M; Silva, G; Aguiar, D M; Silva, C M L; Fachin, A L; Marins, M

    2015-12-22

    Canine monocytic ehrlichiosis (CME) is a common tick-borne disease caused by the rickettsial bacterium Ehrlichia canis (Rickettsiales: Anaplasmataceae). In view of the different stages and variable clinical signs of CME, which can overlap with those of other infections, a conclusive diagnosis can more readily be obtained by combining clinical and hematological evaluations with molecular diagnostic methods. In this study, a loop-mediated isothermal amplification (LAMP) assay targeting the p30 gene of E. canis was developed. The assay was developed using DNA extracted from E. canis-infected cultures of the macrophage cell line DH82 and samples from dogs testing positive for E. canis DNA by PCR. The LAMP assay was compared to a p30-based PCR assay, using DNA extracted from EDTA-anticoagulated blood samples of 137 dogs from an endemic region in Brazil. The LAMP assay was sensitive enough to detect a single copy of the target gene, and identified 74 (54.0%) E. canis DNA-positive samples, while the p30 PCR assay detected 50 positive samples (36.5%) among the field samples. Agreement between the two assays was observed in 42 positive and 55 negative samples. However, 32 positive samples that were not detected by the PCR assay were identified by the LAMP assay, while eight samples identified as E. canis-positive by PCR showed negative results in LAMP. The developed E. canis LAMP assay showed the potential to maximize the use of nucleic acid tests in a veterinary clinical laboratory, and to improve the diagnosis of CME.

  6. Optimization of loop-mediated isothermal amplification (LAMP) assays for the detection of Leishmania DNA in human blood samples.

    Science.gov (United States)

    Abbasi, Ibrahim; Kirstein, Oscar D; Hailu, Asrat; Warburg, Alon

    2016-10-01

    Visceral leishmaniasis (VL), one of the most important neglected tropical diseases, is caused by Leishmania donovani eukaryotic protozoan parasite of the genus Leishmania, the disease is prevalent mainly in the Indian sub-continent, East Africa and Brazil. VL can be diagnosed by PCR amplifying ITS1 and/or kDNA genes. The current study involved the optimization of Loop-mediated isothermal amplification (LAMP) for the detection of Leishmania DNA in human blood or tissue samples. Three LAMP systems were developed; in two of those the primers were designed based on shared regions of the ITS1 gene among different Leishmania species, while the primers for the third LAMP system were derived from a newly identified repeated region in the Leishmania genome. The LAMP tests were shown to be sufficiently sensitive to detect 0.1pg of DNA from most Leishmania species. The green nucleic acid stain SYTO16, was used here for the first time to allow real-time monitoring of LAMP amplification. The advantage of real time-LAMP using SYTO 16 over end-point LAMP product detection is discussed. The efficacy of the real time-LAMP tests for detecting Leishmania DNA in dried blood samples from volunteers living in endemic areas, was compared with that of qRT-kDNA PCR.

  7. Assessment of electron beam-induced DNA damage in larvae of chestnut weevil, Curculio sikkimensis (Heller) (Coleoptera: Curculionidae) using comet assay

    Energy Technology Data Exchange (ETDEWEB)

    Todoriki, Setsuko [Radiation and Information Technology Laboratory, National Food Research Institute, Tsukuba, Ibaraki 305-8642 (Japan)]. E-mail: setsuko@nfri.affrc.go.jp; Hasan, Mahbub [Laboratory for Stored Product Protection, Department of Zoology, Rajshahi University, Rajshahi 6205 (Bangladesh); Miyanoshita, Akihiro [Radiation and Information Technology Laboratory, National Food Research Institute, Tsukuba, Ibaraki 305-8642 (Japan); Imamura, Taro [Radiation and Information Technology Laboratory, National Food Research Institute, Tsukuba, Ibaraki 305-8642 (Japan); Hayashi, Toru [Radiation and Information Technology Laboratory, National Food Research Institute, Tsukuba, Ibaraki 305-8642 (Japan)

    2006-02-15

    Effect of electron beam treatment on DNA damage in mature larvae of chestnut weevil Curculio sikkimensis (Heller) was assessed using single-cell gel electrophoresis (DNA comet assay). Electrons at acceleration voltages of 0 (control), 300, 750, 1000, and 1500 kV at radiation doses of 1 and 4 kGy were used. Electron beam-treated chestnut larvae showed typical DNA fragmentation, compared with cells from non-treated ones which showed a more intact DNA. Investigations using the comet assay showed that the parameters including tail length, tail moment, olive tail moment as well as the quota of DNA damage at both the doses were significantly larger than the control batch larvae. Thus, this technique could contribute to analytical identification of an effective disinfestation and quarantine treatment.

  8. Assessment of electron beam-induced DNA damage in larvae of chestnut weevil, Curculio sikkimensis (Heller) (Coleoptera: Curculionidae) using comet assay

    Science.gov (United States)

    Todoriki, Setsuko; Hasan, Mahbub; Miyanoshita, Akihiro; Imamura, Taro; Hayashi, Toru

    2006-02-01

    Effect of electron beam treatment on DNA damage in mature larvae of chestnut weevil Curculio sikkimensis (Heller) was assessed using single-cell gel electrophoresis (DNA comet assay). Electrons at acceleration voltages of 0 (control), 300, 750, 1000, and 1500 kV at radiation doses of 1 and 4 kGy were used. Electron beam-treated chestnut larvae showed typical DNA fragmentation, compared with cells from non-treated ones which showed a more intact DNA. Investigations using the comet assay showed that the parameters including tail length, tail moment, olive tail moment as well as the quota of DNA damage at both the doses were significantly larger than the control batch larvae. Thus, this technique could contribute to analytical identification of an effective disinfestation and quarantine treatment.

  9. Isolation and characterization of species-specific DNA probes from Taenia solium and Taenia saginata and their use in an egg detection assay.

    Science.gov (United States)

    Chapman, A; Vallejo, V; Mossie, K G; Ortiz, D; Agabian, N; Flisser, A

    1995-05-01

    Cysticercosis results from ingestion of the eggs of the tapeworm Taenia solium. Reduction of the incidence of human and swine cysticercosis requires identification and treatment of individuals who carry the adult tapeworm. T. solium and Taenia saginata eggs cannot be differentiated on the basis of morphology; thus, in order to improve existing methods for the diagnosis of taeniasis, we have developed highly sensitive, species-specific DNA probes which differentiate T. solium and T. saginata. Recombinant clones containing repetitive DNA sequences which hybridize specifically with genomic DNAs from either species were isolated and characterized. T. solium-specific DNA sequences contained complete and truncated forms of a tandemly repeated 158-bp DNA sequence. An unrelated T. saginata DNA sequence was also characterized and shown to encode a portion of the mitochondrial cytochrome c oxidase I gene. T. solium- and T. saginata-specific DNA probes did not hybridize in dot blot assays either with genomic DNA from the platyhelminths Taenia hydatigena, Taenia pisiformis, Taenia taeniaeformis, Echinococcus granulosus, and Schistosoma mansoni or with genomic DNA from other eukaryotes, including Saccharomyces cerevisiae, Candida albicans, Cryptosporidium parvum, Entamoeba histolytica, Trypanosoma gambiense, Trypanosoma brucei, and Giardia lamblia, Caenorhabditis elegans, and human DNA. By using these T. solium and T. saginata DNA probes, a rapid, highly sensitive and specific dot blot assay for the detection of T. solium eggs was developed.

  10. A Fluid Membrane-Based Soluble Ligand Display System for Live CellAssays

    Energy Technology Data Exchange (ETDEWEB)

    Nam, Jwa-Min; Nair, Pradeep N.; Neve, Richard M.; Gray, Joe W.; Groves, Jay T.

    2005-10-14

    Cell communication modulates numerous biological processes including proliferation, apoptosis, motility, invasion and differentiation. Correspondingly, there has been significant interest in the development of surface display strategies for the presentation of signaling molecules to living cells. This effort has primarily focused on naturally surface-bound ligands, such as extracellular matrix components and cell membranes. Soluble ligands (e.g. growth factors and cytokines) play an important role in intercellular communications, and their display in a surface-bound format would be of great utility in the design of array-based live cell assays. Recently, several cell microarray systems that display cDNA, RNAi, or small molecules in a surface array format were proven to be useful in accelerating high-throughput functional genetic studies and screening therapeutic agents. These surface display methods provide a flexible platform for the systematic, combinatorial investigation of genes and small molecules affecting cellular processes and phenotypes of interest. In an analogous sense, it would be an important advance if one could display soluble signaling ligands in a surface assay format that allows for systematic, patterned presentation of soluble ligands to live cells. Such a technique would make it possible to examine cellular phenotypes of interest in a parallel format with soluble signaling ligands as one of the display parameters. Herein we report a ligand-modified fluid supported lipid bilayer (SLB) assay system that can be used to functionally display soluble ligands to cells in situ (Figure 1A). By displaying soluble ligands on a SLB surface, both solution behavior (the ability to become locally enriched by reaction-diffusion processes) and solid behavior (the ability to control the spatial location of the ligands in an open system) could be combined. The method reported herein benefits from the naturally fluid state of the supported membrane, which allows

  11. Evaluation of DNA damage in agricultural workers exposed to pesticides using single cell gel electrophoresis (comet assay

    Directory of Open Access Journals (Sweden)

    Raminderjeet Kaur

    2011-01-01

    Full Text Available Background : Pesticides are used in agriculture to protect crops, but they pose a potential risk to farmers and environment. The aim of the present study is to investigate the relation between the occupational exposure to various pesticides and the presence of DNA damage. Materials and Methods : Blood samples of 210 exposed workers (after a day of intense spraying and 50 control subjects belonging to various districts of Punjab (India were evaluated using Comet assay. Sixty workers who showed DNA damage were selected for follow up at 5-6 months after the first sampling during a low or null spraying period. Results : Significant differences were found in DNA damage between freshly exposed workers and controls and freshly exposed and followed up cases. There was significant increase in the comet parameters viz. mean comet tail length and frequency of cells showing migration in exposed workers as compared to controls (72.22 ± 20.76 vs. 46.92 ± 8.17, P<0.001; 31.79 vs. 5.77, P<0.001. In the second samples, followed up cases showed significant decrease in frequency of damaged cells as compared to freshly exposed workers of first sampling (P<0.05. The confounding factors such as variable duration of pesticide exposure, age, smoking, drinking and dietary habits etc which were expected to modulate the damage, were instead found to have no significant effect on DNA fragmentation. Conclusion : The evidence of a genetic hazard related to exposure resulting from the intensive use of pesticides stresses the need for educational programs for agricultural workers to reduce the use of chemicals in agriculture.

  12. Reactive oxygen species generation by copper(II) oxide nanoparticles determined by DNA damage assays and EPR spectroscopy.

    Science.gov (United States)

    Angelé-Martínez, Carlos; Nguyen, Khanh Van T; Ameer, Fathima S; Anker, Jeffrey N; Brumaghim, Julia L

    2017-03-01

    Copper(II) oxide nanoparticles ((NP)CuO) have many industrial applications, but are highly cytotoxic because they generate reactive oxygen species (ROS). It is unknown whether the damaging ROS are generated primarily from copper leached from the nanoparticles, or whether the nanoparticle surface plays a significant role. To address this question, we separated nanoparticles from the supernatant containing dissolved copper, and measured their ability to damage plasmid DNA with addition of hydrogen peroxide, ascorbate, or both. While DNA damage from the supernatant (measured using an electrophoresis assay) can be explained solely by dissolved copper ions, damage by the nanoparticles in the presence of ascorbate is an order of magnitude higher than can be explained by dissolved copper and must, therefore, depend primarily upon the nanoparticle surface. DNA damage is time-dependent, with shorter incubation times resulting in higher EC50 values. Hydroxyl radical ((•)OH) is the main ROS generated by (NP)CuO/hydrogen peroxide as determined by EPR measurements; (NP)CuO/hydrogen peroxide/ascorbate conditions generate ascorbyl, hydroxyl, and superoxide radicals. Thus, (NP)CuO generate ROS through several mechanisms, likely including Fenton-like and Haber-Weiss reactions from the surface or dissolved copper ions. The same radical species were observed when (NP)CuO suspensions were replaced with the supernatant containing leached copper, washed (NP)CuO, or dissolved copper solutions. Overall, (NP)CuO generate significantly more ROS and DNA damage in the presence of ascorbate than can be explained simply from dissolved copper, and the (NP)CuO surface must play a large role.

  13. A high-throughput assay for the comprehensive profiling of DNA ligase fidelity.

    Science.gov (United States)

    Lohman, Gregory J S; Bauer, Robert J; Nichols, Nicole M; Mazzola, Laurie; Bybee, Joanna; Rivizzigno, Danielle; Cantin, Elizabeth; Evans, Thomas C

    2016-01-29

    DNA ligases have broad application in molecular biology, from traditional cloning methods to modern synthetic biology and molecular diagnostics protocols. Ligation-based detection of polynucleotide sequences can be achieved by the ligation of probe oligonucleotides when annealed to a complementary target sequence. In order to achieve a high sensitivity and low background, the ligase must efficiently join correctly base-paired substrates, while discriminating against the ligation of substrates containing even one mismatched base pair. In the current study, we report the use of capillary electrophoresis to rapidly generate mismatch fidelity profiles that interrogate all 256 possible base-pair combinations at a ligation junction in a single experiment. Rapid screening of ligase fidelity in a 96-well plate format has allowed the study of ligase fidelity in unprecedented depth. As an example of this new method, herein we report the ligation fidelity of Thermus thermophilus DNA ligase at a range of temperatures, buffer pH and monovalent cation strength. This screen allows the selection of reaction conditions that maximize fidelity without sacrificing activity, while generating a profile of specific mismatches that ligate detectably under each set of conditions.

  14. A novel microRNA assay with optical detection and enzyme-free DNA circuits

    Science.gov (United States)

    Liao, Yuhui; Zhou, Xiaoming

    2014-09-01

    MicroRNAs (miRNAs) participate in the significant processes of life course, can be used as quantificational biomarkers for cellular level researches and related diseases. Conventional methods of miRNAs' quantitative detection are obsessed with low sensitivity, time and labour consuming. Otherwise, the emerging miRNAs detection approaches are mostly exposed to the expensive equipment demands and the professional operation, remains at the stage of laboratory and concept demonstration phase. Herein, we designed a novel miRNAs detection platform that based on enzyme-free DNA circuits and electrochemical luminescence (ECL). MicroRNA21 was chosen as the ideal analyte of this platform. The whole process consists of enzyme-free DNA circuits and ECL signal giving-out steps, achieves advantages of operating in constant temperature condition, without the participation of the enzyme, preferable sensitivity and specificity. Through this approach, the sensitivity achieved at 10pM. It is indicated that this miRNAs detection platform possesses potentials to be an innovation of miRNA detection technologies in routine tests. Benefits of the high penetration of ECL in well-equipped medical establishment, this approach could greatly lessen the obstacles in process of popularization and possess excellent prospects of practical application.

  15. Induction and repair of DNA cross-links induced by sulfur mustard in the A-549 cell line followed by a comet assay.

    Science.gov (United States)

    Jost, Petr; Svobodova, Hana; Stetina, Rudolf

    2015-07-25

    Sulfur mustard is a highly toxic chemical warfare agent with devastating impact on intoxicated tissues. DNA cross-links are probably the most toxic DNA lesions induced in the cell by sulfur mustard. The comet assay is a very sensitive method for measuring DNA damage. In the present study using the A-549 lung cell line, the comet assay protocol was optimized for indirect detection of DNA cross-links induced by sulfur mustard. The method is based on the additional treatment of the assayed cells containing cross-links with the chemical mutagen, styrene oxide. Alkali-labile adducts of styrene oxide cause DNA breaks leading to the formation of comets. A significant dose-dependent reduction of DNA migration of the comet's tail was found after exposing cells to sulfur mustard, indicative of the amount of sulfur mustard induced cross-links. The remarkable decrease of % tail DNA could be observed as early as 5min following exposure to sulfur mustard and the maximal effect was found after 30min, when DNA migration was reduced to the minimum. Sulfur mustard preincubated in culture medium without cells lost its ability to induce cross-links and had a half-life of about 15min. Pre-incubation longer than 30min does not lead to a significant increase in cross-links when applied to cells. However, the amount of cross-links is decreased during further incubation due to repair. The current modification of the comet assay provides a useful tool for detecting DNA cross-links induced by sulfur mustard and could be used for detection of other DNA cross-linking agents such as chemotherapeutic drugs.

  16. Quantitation of HTLV-I proviral load by a real-time PCR assay using SYBR Green: comparison of two methods for DNA isolation.

    Science.gov (United States)

    Altamirano, Natalia Andrea; Rocco, Carlos; Aulicino, Paula; Sen, Luisa; Mangano, Andrea

    2010-12-01

    A real-time quantitative PCR (qPCR) assay using SYBR Green was developed to determine HTLV-I proviral load (pVL) in peripheral blood mononuclear cells (PBMCs), and its performance was evaluated with samples processed as cell lysates and DNA isolated by salting out. Primers targeting the pol region were standardized against the MT2 cell line and HTLV-I copy number was normalized to the amount of cellular DNA by quantitation of the albumin gene. The sensitivity, specificity and reproducibility of the qPCR were assessed in the two methods used for DNA processing. The assay had a limit of detection of 400 HTLV-I copies/10(6) PBMCs for both methods, with a broad range of quantitation (2.6log(10) to >5log(10)), and without cross-reactivity with HTLV-II or with HIV-1. The inter- and intra-assay coefficients of variation were less than 2.4%. HTLV-I pVL quantitation in seven blood donor samples processed as either cell lysates or isolated DNA by salting out showed a strong linear correlation and no difference in the calculated pVL (Fisher's exact test, p>0.05). The assay was found to be a low cost, robust and reproducible assay for quantifying HTLV-I pVL in samples processed as cell lysates or as isolated DNA.

  17. A Bioluminescence Assay System for Imaging Metal Cationic Activities in Urban Aerosols.

    Science.gov (United States)

    Kim, Sung-Bae; Naganawa, Ryuichi; Murata, Shingo; Nakayama, Takayoshi; Miller, Simon; Senda, Toshiya

    2016-01-01

    A bioluminescence-based assay system was fabricated for an efficient determination of the activities of air pollutants. The following four components were integrated into this assay system: (1) an 8-channel assay platform uniquely designed for simultaneously sensing multiple optical samples, (2) single-chain probes illuminating toxic chemicals or heavy metal cations from air pollutants, (3) a microfluidic system for circulating medium mimicking the human body, and (4) the software manimulating the above system. In the protocol, we briefly introduce how to integrate the components into the system and the application to the illumination of the metal cationic activities in air pollutants.

  18. Performance validation of commercially available mobile waste-assay systems: Preliminary report

    Energy Technology Data Exchange (ETDEWEB)

    Schanfein, M.; Bonner, C.; Maez, R. [Los Alamos National Lab., NM (United States)] [and others

    1997-11-01

    Prior to disposal, nuclear waste must be accurately characterized to identify and quantify the radioactive content to reduce the radioactive hazard to the public. Validation of the waste-assay systems` performance is critical for establishing the credibility of the assay results for storage and disposal purposes. Canberra Nuclear has evaluated regulations worldwide and identified standard, modular, neutron- and gamma-waste-assay systems that can be used to characterize a large portion of existing and newly generated transuranic (TRU) and low-level waste. Before making claims of guaranteeing any system`s performance for specific waste types, the standardized systems` performance be evaluated. 7 figs., 11 tabs.

  19. Upscaled CTAB-based DNA extraction and real-time PCR assays for Fusarium culmorum and F. graminearum DNA in plant material with reduced sampling error.

    Science.gov (United States)

    Brandfass, Christoph; Karlovsky, Petr

    2008-11-01

    Fusarium graminearum Schwabe (Gibberella zeae Schwein. Petch.) and F. culmorum W.G. Smith are major mycotoxin producers in small-grain cereals afflicted with Fusarium head blight (FHB). Real-time PCR (qPCR) is the method of choice for species-specific, quantitative estimation of fungal biomass in plant tissue. We demonstrated that increasing the amount of plant material used for DNA extraction to 0.5-1.0 g considerably reduced sampling error and improved the reproducibility of DNA yield. The costs of DNA extraction at different scales and with different methods (commercial kits versus cetyltrimethylammonium bromide-based protocol) and qPCR systems (doubly labeled hybridization probes versus SYBR Green) were compared. A cost-effective protocol for the quantification of F. graminearum and F. culmorum DNA in wheat grain and maize stalk debris based on DNA extraction from 0.5-1.0 g material and real-time PCR with SYBR Green fluorescence detection was developed.

  20. H2O2-mediated DNA damage and repair in the brain cells in the aging rats detected by comet assay

    Institute of Scientific and Technical Information of China (English)

    Suming ZhangM.D., Ph.D; Zongchao Han, M.D.; Siyu Fang, M.D.; Ruan Yang, M.D; Wei Wang, M.D., Ph. D

    2000-01-01

    Objective: To identify the relation between DNA damage susceptibility/ DNA repair capability and aging process after insults, an observation of H2O2_induced DNA damage and the kinetics of DNA repair in senescent murine brain cells with the alkaline single cell gel electrophoresis (SCGE/Comet assay) was made. Methods: The dissociated brain cells harvested in the area of the cerebral cortex, hippocampus, basal gang]ion from 3-month (n=10), 8-month (n=8) and 26-month (n=5) old rats were respectively treated with H2O2 in gradient doses for 10 min, or without H2O2 as controls. The cells embedded in agarose were lysed, helix-untied, electrophoresed, stained with a fluorescence DNA binding stain, viewed under a fluorescence microscope. Individual image was optically recorded. The frequency of the tailed cells and the grade of tails wereused to analyze single strand breaks of DNA and injury intensity. Results: By the cell and DNA image like comets, a linear increase was noticed in vulnerability of DNA both to H2O2 doses and to the age. Regarding the damaged region of the brain, the cortex cells were more vulnerable to the insult than the hippocampus/basal ganglionic cells. Whatever aging or not the cells were, the maximum of ratio of DNA repair was only within 1 hour during the incubation for 0.5-4 hours after the insults. Furthermore, the more aging, the less ratio of DNA repair of sick cells. Conclusion: The DNA damagesusceptibility and the DNA repair capability of individual cells, whatever its age is, can be detected by this brain cell injury model. Comet assay is a sensitive way to find out DNA damage and repair of the cells. It should be more difficult for the cells to cope with an acute and excessive than with a persistent, chronic and mild DNA damage which is more related to an accumulating injury, the aging.

  1. Force-activated substrates for high-precision, high-throughput optical trapping assays of ssDNA motor proteins (Conference Presentation)

    Science.gov (United States)

    Okoniewski, Stephen; Perkins, Thomas T.

    2016-09-01

    Optical-trapping-based assays can measure individual proteins bind to and move along DNA with sub-nm resolution, and have yielded insight into a broad array of protein-DNA interactions. Unfortunately, collecting large numbers of high-resolution traces remains an ongoing challenge. Studying helicase motion along DNA exemplifies this challenge. One major difficulty is that helicase binding often requires a single stranded (ss)-double stranded (ds) DNA junction flanked by ssDNA with a minimum size and orientation. Historically, creating such DNA substrates is inefficient. More problematic is that data throughput is low in standard surface-based assays since all substrates are unwound upon introduction of ATP. The net result is 2-4 high-resolution traces on a good day. To improve throughput, we sought to turn-on or activate a substrate for a helicase one molecule at a time and thereby sequentially study many molecules on an individual microscope slide. As a first step towards this goal, we engineered a dsDNA that contains two site-specific nicks along the same strand of the dsDNA but no ssDNA. Upon overstretching the DNA (F = 65 pN), the strand between the two nicks was mechanically dissociated. We demonstrated this with two different substrates: one yielding an internal ssDNA region of 1100 nt and the other yielding a 20-bp long hairpin flanked by 30 nt of ssDNA. Unwinding a hairpin yields a 3-fold larger signal while the 30-nt ssDNA serves as the binding site for the helicase. We expect that these force-activated substrates to significantly accelerate high-resolution optical-trapping studies of DNA helicases.

  2. A DNA-Mediated Homogeneous Binding Assay for Proteins and Small Molecules

    DEFF Research Database (Denmark)

    Zhang, Zhao; Hejesen, Christian; Kjelstrup, Michael Brøndum

    2014-01-01

    Optical detection of molecular targets typically requires immobilization, separation, or chemical or enzymatic processing. An important exception is aptamers that allow optical detection in solution based on conformational changes. This method, however, requires the laborious selection of aptamers...... with high target specificity and affinity, and the ability to undergo the required conformational changes. Here we report on an alternative generic scheme for detecting small molecules and proteins in solution based on a shift in the equilibrium of DNA-based strand displacement competition reaction....... The shift occurs upon binding of a protein, for example, an antibody to its target. We demonstrate nanomolar detection of small molecules such as biotin, digoxigenin, vitamin D, and folate, in buffer and in plasma. The method is flexible, and we also show nanomolar detection of the respective antibodies...

  3. Use of a multi-thermal washer for DNA microarrays simplifies probe design and gives robust genotyping assays

    DEFF Research Database (Denmark)

    Petersen, J.; Poulsen, Lena; Petronis, S.

    2008-01-01

    DNA microarrays are generally operated at a single condition, which severely limits the freedom of designing probes for allele-specific hybridization assays. Here, we demonstrate a fluidic device for multi-stringency posthybridization washing of microarrays on microscope slides. This device...... is called a multi-thermal array washer (MTAW), and it has eight individually controlled heating zones, each of which corresponds to the location of a subarray on a slide. Allele-specific oligonucleotide probes for nine mutations in the beta-globin gene were spotted in eight identical subarrays at positions...... corresponding to the temperature zones of the MTAW. After hybridization with amplified patient material, the slides were mounted in the MTAW, and each subarray was exposed to different temperatures ranging from 22 to 40 degrees C. When processed in the MTAW, probes selected without considering melting...

  4. An Improved Multiplex Real-Time SYBR Green PCR Assay for Analysis of 24 Target Genes from 16 Bacterial Species in Fecal DNA Samples from Patients with Foodborne Illnesses.

    Science.gov (United States)

    Kawase, Jun; Etoh, Yoshiki; Ikeda, Tetsuya; Yamaguchi, Keiji; Watahiki, Masanori; Shima, Tomoko; Kameyama, Mitsuhiro; Horikawa, Kazumi; Fukushima, Hiroshi; Goto, Ryoichi; Shirabe, Komei

    2016-05-20

    Here, we developed a new version of our original screening system (Rapid Foodborne Bacterial Screening 24; RFBS24), which can simultaneously detect 24 genes of foodborne pathogens in fecal DNA samples. This new version (RFBS24 ver. 5) detected all known stx2 subtypes, enterotoxigenic Escherichia coli (STh genotype), and Vibrio parahaemolyticus (trh2), which were not detected by the original RFBS24 assay. The detection limits of RFBS24 ver. 5 were approximately 5.6 × 10(-2)-5.6 × 10(-5) (ng DNA)/reaction, significantly lower (10- to 100-fold) than those of the original RFBS24 for the 22 target genes analyzed here. We also tested the new assay on fecal DNA samples from patients infected with Salmonella, Campylobacter, or enterohemorrhagic E. coli. The number of bacterial target genes detected by RFBS24 ver. 5 was greater than that detected by RFBS24. RFBS24 ver. 5 combined with an Ultra Clean Fecal DNA Isolation Kit showed adequate performance (sensitivity and specificity 89% and 100%, respectively, for Salmonella spp. and 100% and 83%, respectively, for Campylobacter jejuni) in terms of rapid detection of a causative pathogen during foodborne-illness outbreaks. Thus, RFBS24 ver. 5 is more useful than the previous assay system for detection of foodborne pathogens and offers quick simultaneous analysis of many targets and thus facilitates rapid dissemination of information to public health officials.

  5. Effect of varying the exposure and /sup 3/H-thymidine labeling period upon the outcome of the primary hepatocyte DNA repair assay

    Energy Technology Data Exchange (ETDEWEB)

    Barfknecht, T.R.; Mecca, D.J.; Naismith, R.W.

    1988-06-01

    The results presented in this report demonstrate that an 18-20 hour exposure//sup 3/H-thymidine DNA labeling period is superior to a 4 hour incubation interval for general genotoxicity screening studies in the rat primary hepatocyte DNA repair assay. When DNA damaging agents which give rise to bulky-type DNA base adducts such as 2-acetylaminofluorene, aflatoxin B1 and benzidine were evaluated, little or no difference was observed between the 4 hour or an 18-20-hour exposure/labeling period. Similar results were also noted for the DNA ethylating agent diethylnitrosamine. However, when DNA damaging chemicals which produce a broader spectrum of DNA lesions were studied, differences in the amount of DNA repair as determined by autoradiographic analysis did occur. Methyl methanesulfonate and dimethylnitrosamine induced repairable DNA damage that was detected at lower dose levels with the 18-20 hour exposure/labeling period. Similar results were also observed for the DNA cross-linking agents, mitomycin C and nitrogen mustard. Ethyl methanesulfonate produced only a marginal amount of DNA repair in primary hepatocytes up to a dose level of 10(-3) M during the 4 hour incubation period, whereas a substantial amount of DNA repair was detectable at a dose level of 2.5 X 10(-4) M when the 18-20 hour exposure/labeling period was employed. The DNA alkylating agent 4-nitroquinoline-1-oxide, which creates DNA base adducts that are slowly removed from mammalian cell DNA, induced no detectable DNA repair in hepatocytes up to a toxic dose level of 2 X 10(-5) M with the 4 hour exposure period, whereas a marked DNA repair response was observed at 10(-5) M when the 18-20 hour exposure/labeling period was used.

  6. Assessment of DNA damage in underground coal miners using the cytokinesis-block micronucleus assay in peripheral blood lymphocytes.

    Science.gov (United States)

    Sinitsky, Maxim Yu; Minina, Varvara I; Gafarov, Nikolay I; Asanov, Maxim A; Larionov, Aleksey V; Ponasenko, Anastasia V; Volobaev, Valentin P; Druzhinin, Vladimir G

    2016-11-01

    Coal miners are exposed to coal dust, containing mineral particles, inorganic compounds and polycyclic aromatic hydrocarbons, and to ionizing radiation. These factors can induce oxidative stress and promote inflammation that leads to DNA damage. The aim of this investigation is to analyse the degree of DNA damage in miners working in underground coal mines in Kemerovo Region (Russian Federation) using the cytokinesis-block micronucleus assay (CBMN) in peripheral blood lymphocytes. The exposed group included 143 coal miners (mean age = 50.11±7.36 years; mean length of service in coal mining conditions = 23.26±9.66 years). As a control group, we have used venous blood extracted from 127 healthy non-exposed men. The mean age in this group was 47.67±8.45 years. We have discovered that coal miners are characterized by a significant increase in the frequency of binucleated lymphocytes with micronuclei (MN), nucleoplasmic bridges (NPBs) and protrusions (NBUDs) compared to non-exposed donors. In addition, we report, for the first time, a reduction of cell proliferation in a cohort of coal miners. These data are evidence of the genotoxic and cytostatic effects of occupational harmful factors of the coal mining industry. No correlation between the level of chromosome damage and age, smoking status or length of service in coal mining conditions were discovered. We suggest that the CBMN assay would be useful in biomonitoring studies to monitor hygiene and prevention strategies in occupational settings in coal mining countries. © The Author 2016. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  7. An improved method for the isolation of rat alveolar type II lung cells: Use in the Comet assay to determine DNA damage induced by cigarette smoke.

    Science.gov (United States)

    Dalrymple, Annette; Ordoñez, Patricia; Thorne, David; Dillon, Debbie; Meredith, Clive

    2015-06-01

    Smoking is a cause of serious diseases, including lung cancer, emphysema, chronic bronchitis and heart disease. DNA damage is thought to be one of the mechanisms by which cigarette smoke (CS) initiates disease in the lung. Indeed, CS induced DNA damage can be measured in vitro and in vivo. The potential of the Comet assay to measure DNA damage in isolated rat lung alveolar type II epithelial cells (AEC II) was explored as a means to include a genotoxicity end-point in rodent sub-chronic inhalation studies. In this study, published AEC II isolation methods were improved to yield viable cells suitable for use in the Comet assay. The improved method reduced the level of basal DNA damage and DNA repair in isolated AEC II. CS induced DNA damage could also be quantified in isolated cells following a single or 5 days CS exposure. In conclusion, the Comet assay has the potential to determine CS or other aerosol induced DNA damage in AEC II isolated from rodents used in sub-chronic inhalation studies.

  8. A New Lab Developed Real Time PCR Assay for Direct Detection of C. Difficle from Stool Sample without DNA Extraction.

    Science.gov (United States)

    Li, Brandon

    2016-09-01

    Clostridium difficile is a major cause of nosocomial antibiotic-associated infectious diarrhea and pseudomembranous colitis. Detection of C. difficile by anaerobic bacterial culture and/or cytotoxicity assays has been largely replaced by rapid enzyme immunoassays (EIA). However, due to the lack of sensitivity of stool EIA, we developed a multiplex real-time PCR assay targeting the C. difficile toxin genes tcdB. stool samples from hospitalized pediatric patients suspected of having C. difficile-associated disease were prospectively collected. Three testing modalities were evaluated, including enriched culture, cepheid Xpert and real-time Pcr (tcdB) on stool samples performed with tcdB gene-specific primers and hydrolysis probes. A total of 150 de-identified clinical specimen were analyzed. The sensitivities of stool real-time Pcr were 95% against cepheid Xpert C. difficile and 93% against enriched culture respectively, with a specificity of 97% and 94%. The lower limit of detection of the stool real-time PCR was 0.5 cFU/ml of per reaction for tcdB. Direct detection of C. difficile toxin genes in stool samples by real-time Pcr showed performance comparable to enriched culture. Real-time PCR of DNA from stool samples is a rapid and cost-effective diagnostic modality for patients that should facilitate appropriate patient management.

  9. IN-VIVO EVALUATION OF HEXAVALENT CHROMIUM INDUCED DNA DAMAGE BY ALKALINE COMET ASSAY AND OXIDATIVE STRESS IN CATLA CATLA

    Directory of Open Access Journals (Sweden)

    Kantha Deivi Arunachalam

    2013-01-01

    Full Text Available In the present study, the acute toxicity of Chromium in fingerlings of Catla catla, an Indian major carp, was evaluated with renewal bioassay method. In vivo studies were designed to assess the extent of Micronucleus Assay, Comet Assay under the exposure of common heavy-metal compounds, namely, Chromium Nitrate, using Catla catla (2n = 20, as a test model. The laboratory acclimatized fishes were divided into four groups. Group I served as positive control and the other three as exposed groups for three different time durations of 7, 14 and 21 days and were subjected to uninterrupted sub lethal concentrations (50% of 96 h LC50. The experiments were planned in such a way that fish from all the groups were sacrificed on the same day. The frequencies of micronuclei and bi-nuclei were evaluated comparatively in peripheral erythrocytes. As a result, it was observed that, the fishes and different tissues showed differential sensitivity to the heavy-metal treatment. A significant increase in the frequencies of micronucleated and binucleated cells and percentage increase in DNA tail (pCatla catla during sub lethal toxicity study was also calculated.

  10. Bacterial Bioburden Decrease in Orthokeratology Lens Storage Cases After Forewarning: Assessment by the DNA Dot Hybridization Assay.

    Science.gov (United States)

    Fang, Po-Chiung; Lo, Jung; Chang, Tsung C; Chien, Chun-Chih; Hsiao, Chang-Chun; Tseng, Shin-Ling; Lai, Yu-Hsuan; Kuo, Ming-Tse

    2017-05-01

    The aim of this study was to measure the changes in the bacterial bioburden in orthokeratology (OK) lens storage cases using the DNA dot hybridization assay (DHA) after forewarning patients about their bacterial contamination severity. Thirty-one OK lens wearers were prospectively enrolled in this study. Dot hybridization assay was used for serial measurements of bacterial bioburden in OK storage cases after lenses had been soaked for approximately 6 hr. After the first assessment, the lens wearers were informed of the extent of case contamination and the possible risk of microbial keratitis (MK), and best practices for lens care and lens case hygiene were reviewed and reinforced. A second assessment by the same DHA method was performed after approximately 6 months. Two universal bacterial probes confirmed a significant decrease in bacterial bioburden at the second assessment (P<0.01 and P<0.001). Genus-specific probes showed significant reductions in Acinetobacter and Klebsiella (P=0.02 and P=0.01), but not in Pseudomonas (P=0.42). Making OK lens wearers aware of the bacterial bioburden in their lens cases resulted in improved quality of case care and reduced bioburden. Our results suggest that a strategy of bioburden assessment with forewarning could be a useful method to decrease the incidence of OK-related MK.

  11. A New Lab Developed Real Time PCR Assay for Direct Detection of C. Difficle from Stool Sample without DNA Extraction

    Science.gov (United States)

    Li, Brandon

    2016-01-01

    Clostridium difficile is a major cause of nosocomial antibiotic-associated infectious diarrhea and pseudomembranous colitis. Detection of C. difficile by anaerobic bacterial culture and/or cytotoxicity assays has been largely replaced by rapid enzyme immunoassays (EIA). However, due to the lack of sensitivity of stool EIA, we developed a multiplex real-time PCR assay targeting the C. difficile toxin genes tcdB. stool samples from hospitalized pediatric patients suspected of having C. difficile-associated disease were prospectively collected. Three testing modalities were evaluated, including enriched culture, cepheid Xpert and real-time Pcr (tcdB) on stool samples performed with tcdB gene-specific primers and hydrolysis probes. A total of 150 de-identified clinical specimen were analyzed. The sensitivities of stool real-time Pcr were 95% against cepheid Xpert C. difficile and 93% against enriched culture respectively, with a specificity of 97% and 94%. The lower limit of detection of the stool real-time PCR was 0.5 cFU/ml of per reaction for tcdB. Direct detection of C. difficile toxin genes in stool samples by real-time Pcr showed performance comparable to enriched culture. Real-time PCR of DNA from stool samples is a rapid and cost-effective diagnostic modality for patients that should facilitate appropriate patient management. PMID:27829823

  12. DNA conformational behavior and compaction in biomimetic systems: Toward better understanding of DNA packaging in cell.

    Science.gov (United States)

    Zinchenko, Anatoly

    2016-06-01

    In a living cell, long genomic DNA is strongly compacted and exists in the environment characterized by a dense macromolecular crowding, high concentrations of mono- and divalent cations, and confinement of ca. 10μm size surrounded by a phospholipid membrane. Experimental modelling of such complex biological system is challenging but important to understand spatiotemporal dynamics and functions of the DNA in cell. The accumulated knowledge about DNA condensation/compaction in conditions resembling those in the real cell can be eventually used to design and construct partly functional "artificial cells" having potential applications in drug delivery systems, gene therapy, and production of synthetic cells. In this review, I would like to overview the past progress in our understanding of the DNA conformational behavior and, in particular, DNA condensation/compaction phenomenon and its relation to the DNA biological activity. This understanding was gained by designing relevant experimental models mimicking DNA behavior in the environment of living cell. Starting with a brief summary of classic experimental systems to study DNA condensation/compaction, in later parts, I highlight recent experimental methodologies to address the effects of macromolecular crowding and nanoscale and microscale confinements on DNA conformation dynamics. All the studies are discussed in the light of their relevance to DNA behavior in living cells, and future prospects of the field are outlined.

  13. DNA packaging induced by micellar aggregates: a novel in vitro DNA condensation system.

    Science.gov (United States)

    Ghirlando, R; Wachtel, E J; Arad, T; Minsky, A

    1992-08-11

    Evidence for a conceptually novel DNA packaging process is presented. X-ray scattering, electron microscopy, and circular dichroism measurements indicate that in the presence of positively charged micellar aggregates and flexible anionic polymers, such as negatively charged polypeptides or single-stranded RNA species, a complex is formed in which DNA molecules are partially embedded within a micellar scaffold and partially condensed into highly packed chiral structures. Based on studies of micelle-DNA and micelle-flexible anionic polymer systems, as well as on the known effects of a high charge density upon the micellar organization, a DNA packaging model is proposed. According to this model, the DNA induces the elongation of the micelles into rodlike aggregates, forming a closely packed matrix in which the DNA molecules are immobilized. In contrast, the flexible anionic polymers stabilize clusters of spherical micelles which are proposed to effect a capping of the rodlike micelles, thus arresting their elongation and creating surfactant-free segments of the DNA that are able to converge and collapse. Thus, unlike other in vitro DNA packaging systems, in which condensation follows encounters between charge-neutralized DNA molecules, a prepackaging phase where the DNA is immobilized within a matrix is proposed in this case. Cellular and nuclear membranes have been implicated in DNA packaging processes in vivo, and negatively charged polyelectrolytes were shown to be involved in the processes. These observations, combined with the basic tenets of the DNA condensation system described here, allow for the progression to the study of more elaborate model systems and thus might lead to insights into the nature and roles of the intricate in vivo DNA-membrane complexes.

  14. Use of the comet assay to measure DNA damage in cells exposed to photosensitizers and gamma radiation

    Science.gov (United States)

    Pouget, J.-P.; Ravanat, J.-L.; Douki, T.; Richard, M.-J.; Cadet, J.

    1999-01-01

    We used the comet assay associated with DNA-glycosylases to estimate DNA damage in cells exposed to gamma irradiation or photosensitized either with methylene blue or orange acridine. A calibration performed using irradiation allowed the measurement of the steady-state level and the yield of 8-oxodGuo as well as strand breaks and alkali-labile sites. Nous avons utilisé la méthode des comètes associée à des ADN-glycosylases, pour estimer les dommages de l'ADN dans des cellules après l'exposition à un rayonnement gamma ou après photosensibilisation par le bleu de méthylène ou l'acridine orange. Une calibration de la méthode des comètes a permis de mesurer le niveau basal et les taux de formation de 8-oxodGuo ainsi que le nombre de cassures de brins et de sites alcali labiles.

  15. The validation of forensic DNA extraction systems to utilize soil contaminated biological evidence.

    Science.gov (United States)

    Kasu, Mohaimin; Shires, Karen

    2015-07-01

    The production of full DNA profiles from biological evidence found in soil has a high failure rate due largely to the inhibitory substance humic acid (HA). Abundant in various natural soils, HA co-extracts with DNA during extraction and inhibits DNA profiling by binding to the molecular components of the genotyping assay. To successfully utilize traces of soil contaminated evidence, such as that found at many murder and rape crime scenes in South Africa, a reliable HA removal extraction system would often be selected based on previous validation studies. However, for many standard forensic DNA extraction systems, peer-reviewed publications detailing the efficacy on soil evidence is either lacking or is incomplete. Consequently, these sample types are often not collected or fail to yield suitable DNA material due to the use of unsuitable methodology. The aim of this study was to validate the common forensic DNA collection and extraction systems used in South Africa, namely DNA IQ, FTA elute and Nucleosave for processing blood and saliva contaminated with HA. A forensic appropriate volume of biological evidence was spiked with HA (0, 0.5, 1.5 and 2.5 mg/ml) and processed through each extraction protocol for the evaluation of HA removal using QPCR and STR-genotyping. The DNA IQ magnetic bead system effectively removed HA from highly contaminated blood and saliva, and generated consistently acceptable STR profiles from both artificially spiked samples and crude soil samples. This system is highly recommended for use on soil-contaminated evidence over the cellulose card-based systems currently being preferentially used for DNA sample collection. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  16. Identification of irradiated wheat by germination test, DNA comet assay and electron spin resonance

    Science.gov (United States)

    Barros, Adilson C.; Freund, Maria Teresa L.; Villavicencio, Ana Lúcia C. H.; Delincée, Henry; Arthur, Valter

    2002-03-01

    In several countries, there has been an increase in the use of radiation for food processing thus improving the quality and sanitary conditions, inhibiting pathogenic microorganisms, delaying the natural aging process and so extending product lifetime. The need to develop analytical methods to detect these irradiated products is also increasing. The goal of this research was to identify wheat irradiated using different radiation doses. Seeds were irradiated with a gamma 60Co source (Gammacell 220 GC) in the Centro de Energia Nuclear na Agricultura and the Instituto de Pesquisas Energéticas e Nucleares. Dose rate used were 1.6 and 5.8kGy/h. Applied doses were 0.0, 0.10, 0.25, 0.50, 0.75, 1.0, and 2.0kGy. After irradiation, seeds were analysed over a 6 month period. Three different detection methods were employed to determine how irradiation had modified the samples. Screening methods consisted of a germination test measuring the inhibition of shooting and rooting and analysis of DNA fragmentation. The method of electron spin resonance spectroscopy allowed a better dosimetric evaluation. These techniques make the identification of irradiated wheat with different doses possible.

  17. Development of an internal amplification control system for a real-time PCR assay for detection of Neisseria meningitidis in CSF and EDTA blood.

    Science.gov (United States)

    McIver, Christopher J; Bell, Sydney M; Er, Noel

    2014-06-01

    The aim of this study was to assemble and assess a non-competitive internal amplification control (IAC) system targeting the Escherichia coli alanine racemase (alr) gene to include in a real-time polymerase chain reaction (PCR) assay for Neisseria meningitidis. Primers and hybridisation probes specific for the IAC were designed and assessed for specificity. Amplification efficiency and limit of detection for the assembled assay was extrapolated using standard curves constructed with serial dilutions of N. meningitidis in saline, pooled cerebrospinal fluid (CSF) and EDTA blood. The 95% confidence limits (CI) were calculated for IAC crossing-points recorded for assays for N. meningitidis ctrA in saline (negative blank), and N. meningitides-negative samples of CSF and EDTA blood. These limits served as a reference range against which the IAC crossing-points recorded for prospective assays are compared to detect sample inhibition. This system was used in testing consecutive EDTA blood samples from two cases of meningococcal disease. The IAC system is specific for Escherichia coli and Shigella species. The amplification efficiency of the assembled assay for N. meningitidis and ability to detect low target DNA levels was not compromised with the inclusion of the IAC system. The IAC crossing-points varied in clinical samples of CSF and EDTA blood. The elucidated reference range for EDTA blood was used to detect sample inhibition in one of the two clinical cases investigated.The IAC system monitors the performance of all processes in the assembled assay for N. meningitidis. Measuring IAC crossing-points serves as an indicator of sample stability and inhibitory properties when testing single or multiple samples from the same patient. Specificity for E. coli and Shigella species enables inclusion in assays of different targets within the same laboratory. Reporting PCR assay results in the context of the IAC crossing-points and reference ranges validates against sample

  18. MSP-HTPrimer: a high-throughput primer design tool to improve assay design for DNA methylation analysis in epigenetics.

    Science.gov (United States)

    Pandey, Ram Vinay; Pulverer, Walter; Kallmeyer, Rainer; Beikircher, Gabriel; Pabinger, Stephan; Kriegner, Albert; Weinhäusel, Andreas

    2016-01-01

    Bisulfite (BS) conversion-based and methylation-sensitive restriction enzyme (MSRE)-based PCR methods have been the most commonly used techniques for locus-specific DNA methylation analysis. However, both methods have advantages and limitations. Thus, an integrated approach would be extremely useful to quantify the DNA methylation status successfully with great sensitivity and specificity. Designing specific and optimized primers for target regions is the most critical and challenging step in obtaining the adequate DNA methylation results using PCR-based methods. Currently, no integrated, optimized, and high-throughput methylation-specific primer design software methods are available for both BS- and MSRE-based methods. Therefore an integrated, powerful, and easy-to-use methylation-specific primer design pipeline with great accuracy and success rate will be very useful. We have developed a new web-based pipeline, called MSP-HTPrimer, to design primers pairs for MSP, BSP, pyrosequencing, COBRA, and MSRE assays on both genomic strands. First, our pipeline converts all target sequences into bisulfite-treated templates for both forward and reverse strand and designs all possible primer pairs, followed by filtering for single nucleotide polymorphisms (SNPs) and known repeat regions. Next, each primer pairs are annotated with the upstream and downstream RefSeq genes, CpG island, and cut sites (for COBRA and MSRE). Finally, MSP-HTPrimer selects specific primers from both strands based on custom and user-defined hierarchical selection criteria. MSP-HTPrimer produces a primer pair summary output table in TXT and HTML format for display and UCSC custom tracks for resulting primer pairs in GTF format. MSP-HTPrimer is an integrated, web-based, and high-throughput pipeline and has no limitation on the number and size of target sequences and designs MSP, BSP, pyrosequencing, COBRA, and MSRE assays. It is the only pipeline, which automatically designs primers on both genomic

  19. Evaluation of a novel real-time fluorescent polymerase chain reaction assay for high-risk human papilloma virus DNA genotypes in cytological cervical screening

    OpenAIRE

    Cheng, Jiaoying; BIAN, MEILU; CONG, XIAO; SUN, AIPING; Li, Min; Ma, Li; Chen, Ying; Liu,Jun

    2012-01-01

    It has been confirmed that detection of high-risk human papillomavirus (HR HPV) DNA is useful in cervical cancer (CC) screening. Recently, a new real-time fluorescent polymerase chain reaction (PCR) assay was developed to detect HR HPV. This assay can synchronize nucleic acid amplification and testing using specific primers for 13 types of HR HPV genomes, combined with specific TaqMan fluorescent marker probe techniques through the fluorescence automatic PCR instrument. Furthermore, it uses T...

  20. Simultaneous detection and subtyping of porcine endogenous retroviruses proviral DNA using the dual priming oligonucleotide system.

    Science.gov (United States)

    Moon, Hyoung Joon; Park, Seong Jun; Kim, Hye Kwon; Ann, Soo Kyung; Rho, Semi; Keum, Hyun Ok; Park, Bong Kyun

    2010-09-01

    The purpose of this study was to develop a multiplex PCR that can detect porcine endogenous retrovirus (PERV) proviral genes (pol, envA, envB, envC) and porcine mitochondrial DNA, using a dual priming oligonucleotide (DPO) system. The primer specifically detected the PERV proviral genes pol, envA, envB, envC, and porcine mitochondrial DNA only in samples of pig origin. The sensitivity of the primer was demonstrated by simultaneous amplification of all 5 target genes in as little as 10 pg of pig DNA containing PERV proviral genes and mitochondrial DNA. The multiplex PCR, when applied to field samples, simultaneously and successfully amplified PERV proviral genes from liver, blood and hair root samples. Thus, the multiplex PCR developed in the current study using DPO-based primers is a rapid, sensitive and specific assay for the detection and subtyping of PERV proviral genes.

  1. Antibiotic microbial assay using kinetic-reading microplate system

    Directory of Open Access Journals (Sweden)

    Felipe Rebello Lourenço

    2011-09-01

    Full Text Available The aim of this study was to determine the optimal experimental conditions to develop a methodology for microbiological assay of apramycin employing microplate and kinetic reading mode, and to validate the developed method, through evaluation of parameters of selectivity, linearity, linear range, limits of detection and quantification, accuracy and precision. The turbidimetric assay principle is simple: the test solution is added to a suspension of test microorganism in culture media, the mixture is incubated under appropriate conditions and the microbial growth is measured by photometric reading. Microplate with kinetic reading mode employed in antibiotic assay is of considerable interest since it allows reduction of material and analysis time and enables a large number of samples to be analyzed simultaneously, with automated reading and calculating. Established conditions considered the standard-curve of apramycin at concentrations from 5.0 to 35.0 μg mL-1, and tryptic soy broth inoculated with 5% Escherichia coli (ATCC 8739 suspension. Satisfactory results were obtained with 2 hours of incubation. The developed method showed appropriate selectivity, linearity in the range from 5.0 to 35.0 μg mL-1, limits of detection and quantification of 0.1 and 0.4 μg mL-1, respectively, as well as satisfactory accuracy (recuperation = 98.5% and precision (RSD = 6.0%. Microplate assay combined the characteristics of microbiological (evaluation of antibiotic activity against sensitive test microorganism and physico-chemical (operationally straightforward and faster results assays.O objetivo deste trabalho é determinar as condições experimentais ideais para o desenvolvimento de metodologia para a dosagem microbiológica de apramicina empregando microplacas e modo de leitura cinético e validar o método desenvolvido, através da avaliação dos parâmetros de especificidade e seletividade, linearidade, faixa ou intervalo linear, limite de detecção e

  2. Random, double- and single-strand DNA breaks can be differentiated in the method of Comet assay by the shape of the comet image.

    Science.gov (United States)

    Georgieva, Milena; Zagorchev, Plamen; Miloshev, George

    2015-10-01

    Comet assay is an invaluable tool in DNA research. It is widely used to detect DNA damage as an indicator of exposure to genotoxic stress. A canonical set of parameters and specialized software programs exist for Comet assay data quantification and analysis. None of them so far has proven its potential to employ a computer-based algorithm for assessment of the shape of the comet as an indicator of the exact mechanism by which the studied genotoxins cut in the molecule of DNA. Here, we present 14 unique measurements of the comet image based on the comet morphology. Their mathematical derivation and statistical analysis allowed precise description of the shape of the comet image which in turn discriminated the cause of genotoxic stress. This algorithm led to the development of the "CometShape" software which allowed easy discrimination among different genotoxins depending on the type of DNA damage they induce.

  3. Measurement of X-ray-induced DNA double-strand breaks at various stages of the cell cycle using the total fluorescence as a comet assay parameter

    Energy Technology Data Exchange (ETDEWEB)

    Attia, Atef M.M. [Department of Biochemistry, Biophysical laboratory, National Research Center, Dokki, Cairo (Egypt); Nabil, Ghada M., E-mail: gmnabilnooh@hotmail.com [Department of Biochemistry, Biophysical laboratory, National Research Center, Dokki, Cairo (Egypt); Frankenberg, Dieter; Frankenberg-Schwager, M. [Abteilung Klinische Strahlenbiologie und Klinische Strahlenphysik, Zentrum, Radiologie, Georg-August-Universitaet Goettingen, Von-Siebold-Str.3 (Germany)

    2011-11-15

    The aim of the study was to develop a protocol for both estimating cell cycle position and the level of ionizing radiation-induced DNA dsb using the neutral comet assay. Using DNA histograms, cell cycle positions were determined for human dermal fibroblasts. The tail intensity was used to estimate the level of DNA damage induced by X-rays, at different positions of the cell cycle. The results of tail intensity versus DNA content bivariate analysis of exponentially growing cells showed a remarkable decrease in tail intensity with transition of cells from G1 to S-phase and increases slightly with transition to G2/M phase. This effect is observed at all doses including unirradiated cells, indicating that the effect is not caused by X-rays and the comet assay based on the current tail parameters is not relevant to measure DNA damage at various stages of the cell cycle. The results of dose response curves showed a linear decrease in the comet fluorescence with the X-ray dose. This observation provides a basis for estimating the fraction of damaged DNA, based on the fluorescence decrement induced by ionizing radiation. The results of this new approach showed a linear increase in DNA damage with dose, at various stages of the cell cycle, with rates, which vary in the following order G0>G2/M>S/G1 cells. These results suggest that G0 and G2/M cells are the most sensitive to X-rays among all phases of the cell cycle and suggest synchronization of cells at these phases to increase the cellular radiosensitivity during radiotherapy. - Display Omitted Highlights: > Increase in DNA damage with dose. > Introduction of a new technique for measuring DNA damage using a new approach of the neutral comet assay. > Estimation of DNA damage in mammalian cells.

  4. A continuous-flow ATP amplification system for increasing the sensitivity of quantitative bioluminescence assay

    OpenAIRE

    Satoh, Tetsuya; Shinoda, Yasuharu; Alexandrov, Maxym; Kuroda, Akio; Murakami, Yuji

    2008-01-01

    We constructed a novel ATP amplification reactor using a continuous-flow system, and this allowed us to increase the sensitivity of quantitative bioluminescence assay by controlling the number of ATP amplification cycles. We previously developed a bioluminescence assay coupled with ATP amplification using a batch system. However, it was difficult to control the number of amplification cycles. In this study, ATP amplification was performed using a continuous-flow system, and significant linear...

  5. Simultaneous Detection of CDC Category "A" DNA and RNA Bioterrorism Agents by Use of Multiplex PCR & RT-PCR Enzyme Hybridization Assays

    Directory of Open Access Journals (Sweden)

    Kelly J. Henrickson

    2009-10-01

    Full Text Available Assays to simultaneously detect multiple potential agents of bioterrorism are limited. Two multiplex PCR and RT-PCR enzyme hybridization assays (mPCR-EHA, mRT-PCR-EHA were developed to simultaneously detect many of the CDC category “A” bioterrorism agents. The “Bio T” DNA assay was developed to detect: Variola major (VM, Bacillus anthracis (BA, Yersinia pestis (YP, Francisella tularensis (FT and Varicella zoster virus (VZV. The “Bio T” RNA assay (mRT-PCR-EHA was developed to detect: Ebola virus (Ebola, Lassa fever virus (Lassa, Rift Valley fever (RVF, Hantavirus Sin Nombre species (HSN and dengue virus (serotypes 1-4. Sensitivity and specificity of the 2 assays were tested by using genomic DNA, recombinant plasmid positive controls, RNA transcripts controls, surrogate (spiked clinical samples and common respiratory pathogens. The analytical sensitivity (limit of detection (LOD of the DNA asssay for genomic DNA was 1×100~1×102 copies/mL for BA, FT and YP. The LOD for VZV whole organism was 1×10-2 TCID50/mL. The LOD for recombinant controls ranged from 1×102~1×103copies/mL for BA, FT, YP and VM. The RNA assay demonstrated LOD for RNA transcript controls of 1×104~1×106 copies/mL without extraction and 1×105~1×106 copies/mL with extraction for Ebola, RVF, Lassa and HSN. The LOD for dengue whole organisms was ~1×10-4 dilution for dengue 1 and 2, 1×104 LD50/mL and 1×102 LD50/mL for dengue 3 and 4. The LOD without extraction for recombinant plasmid DNA controls was ~1×103 copies/mL (1.5 input copies/reaction for Ebola, RVF, Lassa and HSN. No cross-reactivity of primers and probes used in both assays was detected with common respiratory pathogens or between targeted analytes. Clinical sensitivity was estimated using 264 surrogate clinical samples tested with the BioT DNA assay and 549 samples tested with the BioT RNA assay. The clinical specificity is 99.6% and 99.8% for BioT DNA assay and BioT RNA assay, respectively. The

  6. Genetic diversity in Silene sennenii Pau (Caryophyllaceae assayed through DNA-based techniques

    Directory of Open Access Journals (Sweden)

    Blanché, C.

    2012-12-01

    Full Text Available Silene sennenii is a narrow endemic species from the NE of the Iberian Peninsula. It is considered as EN (“Endangered” according to the IUCN criteria and is under legal protection in Catalonia. In the present work we report an assay using three different approaches for surveying the genetic diversity in this rare, endangered campion: analysis of chloroplast haplotypes, AFLPs and transferability of microsatellites previously developed for Silene latifolia. None of the nine chloroplast regions sequenced showed any variability. Five out of the 21 microsatellite loci tested were polymorphic although more loci are required in order to perform a robust population genetics study. Regarding the AFLP analysis, five out of the 26 pairs of primers tested exhibited moderate levels of variability and therefore they could be useful for further investigating the genetic structure of S. sennenii. Although preliminary, our results based on three different genetic markers are in agreement with the low values of genetic variation already reported for this species employing allozymes.Silene sennenii es una especie endémica, circunscrita a un área extremadamente reducida al NE de la Península Ibérica. Está catalogada como EN («En Peligro» según criterios UICN y se encuentra legalmente protegida en Cataluña. En el presente trabajo se expone el ensayo de tres aproximaciones diferentes al estudio de su diversidad genética: análisis de haplotipos cloroplásticos, AFLPs y transferibilidad de microsatélites diseñados previamente para Silene latifolia. Ninguna de las nueve regiones cloroplásticas secuenciadas ha presentado variabilidad. Se han obtenido cinco loci microsatélites polimórficos de los 21 ensayados, cantidad insuficiente para llevar a cabo un estudio robusto sobre genética poblacional. En cuanto a AFLPs, cinco combinaciones de cebadores de las 26 probadas han mostrado niveles moderados de variabilidad siendo así útiles para posteriores

  7. Advanced Assay Systems for Radionuclide Contamination in Soils

    Energy Technology Data Exchange (ETDEWEB)

    J. R. Giles; L. G. Roybal; M. V. Carpenter; C. P. Oertel; J. A. Roach

    2008-02-01

    Through the support of the Department of Energy (DOE) Office of Environmental Management (EM) Technical Assistance Program, the Idaho National Laboratory (INL) has developed and deployed a suite of systems that rapidly scan, characterize, and analyze surface soil contamination. The INL systems integrate detector systems with data acquisition and synthesis software and with global positioning technology to provide a real-time, user-friendly field deployable turn-key system. INL real-time systems are designed to characterize surface soil contamination using methodologies set forth in the Multi-Agency Radiation Surveys and Site Investigation Manual (MARSSIM). MARSSIM provides guidance for planning, implementing, and evaluating environmental and facility radiological surveys conducted to demonstrate compliance with a dose or risk-based regulation and provides real-time information that is immediately available to field technicians and project management personnel. This paper discusses the history of the development of these systems and describes some of the more recent examples and their applications.

  8. The use of comet assay to assess DNA integrity of boar spermatozoa following liquid preservation at 5 degrees C and 16 degrees C.

    Directory of Open Access Journals (Sweden)

    J Strzezek

    2004-03-01

    Full Text Available The comet assay, under neutral conditions, allows the assessment of DNA integrity influenced by sperm ageing, which is manifested in DNA double-strand breaks. Here, we attempted to use a modified neutral comet assay test (single-cell gel electrophoresis, to our knowledge for the first time, to assess DNA integrity of boar spermatozoa during liquid storage for 96 h at 5 degrees C and 16 degrees C. In this comet assay protocol we used 2% beta-mercaptoethanol prior to the lysis procedure, to aid in removing nuclear proteins. Ejaculates from 3 boars (designated A, C and G were diluted with a standard semen extender, Kortowo-3 (K-3, which was supplemented with lipoprotein fractions extracted from hen egg yolk (LPFh or ostrich egg yolk (LPFo. Irrespective of the extender type, the percentage of comet-detected spermatozoa with damaged DNA increased gradually during prolonged storage at 5 degrees C and 16 degrees C. Spermatozoa stored in K-3 extender exhibited elevated levels of DNA damage at both storage temperatures. Significant differences in DNA damage among the boars were more pronounced during storage in LPF-based extenders at 5 degrees C: spermatozoa of boars A and G were less susceptible to DNA damage. The percent of tail DNA in comets was lower in LPF-based extenders, and there were individual variations among the boars. We observed that changes in DNA integrity were dependent on the extender type and storage temperature. A higher level of DNA instability was observed in K-3 extended semen compared with K-3/LPFh or K-3/LPFo extended semen during storage at 5 degrees C. No significant difference in the level of DNA damage between K-3/LPFh and K-3/LPFo was observed. It seems that a long-term storage can affect genomic integrity of boar spermatozoa. The modified neutral comet assay can be used to detect low levels of DNA damage in boar spermatozoa during liquid preservation. Therefore, screening for sperm DNA damage may be used as an additional

  9. Application of Comet assay to assess the effects of white bean meal on DNA of human lymphocytes

    Directory of Open Access Journals (Sweden)

    Luciana Lopes Silva Pereira

    2012-03-01

    Full Text Available This study was conducted to evaluate the potential induction of genotoxic effects of white bean flour using the Comet assay. The test was conducted with human lymphocytes present in whole blood immediately after collection, by incubation with white bean flour in three concentrations (3.92, 9.52 and 18.18 mg/mL at 37 ºC for 4 h followed by preparation of slides. Samples were considered positive (above 20% damage when the damage observed to cellular DNA was higher than the negative control. No genotoxic potential was found at the doses tested. However, it would be premature to suggest absence of risk to human health of DNA damage since the exposure of cells to the extract was restricted to four hours rather than a whole cell cycle. Additionally, further information on toxicology should be obtained in future studies.Este estudo foi realizado para avaliar o potencial de indução de efeitos genotóxicos da farinha de feijão branco utilizando o teste do Cometa. O ensaio foi realizado com linfócitos humanos presentes no sangue imediatamente após a coleta, por incubação com farinha de feijão branco em três concentrações (3,92, 9,52 e 18,18 mg/mL a 37 ºC por 4 h, seguida de preparação das lâminas. As amostras foram consideradas positivas (acima de 20% de dano, quando os danos observados no DNA celular foram maiores do que o controle negativo. Verificou-se que as doses testadas não mostraram potencial genotóxico. No entanto, seria prematuro fazer recomendações sobre o padrão de riscos para a saúde humana resultantes de danos ao DNA já que exposição das células ao extrato foi restrito ao período de quatro horas e não durante um ciclo celular completo. Além disso, outras informações sobre a toxicologia devem ser obtidas no futuro.

  10. Urolithins display both antioxidant and pro-oxidant activities depending on assay system and conditions.

    Science.gov (United States)

    Kallio, Tuija; Kallio, Johanna; Jaakkola, Mari; Mäki, Marianne; Kilpeläinen, Pekka; Virtanen, Vesa

    2013-11-13

    The biological effects of polyphenolic ellagitannins are mediated by their intestinal metabolites, urolithins. This study investigated redox properties of urolithins A and B using ORAC assay, three cell-based assays, copper-initiated pro-oxidant activity (CIPA) assay, and cyclic voltammetry. Urolithins were strong antioxidants in the ORAC assay, but mostly pro-oxidants in cell-based assays, although urolithin A was an antioxidant in cell culture medium. Parent compound ellagic acid was a strong extracellular antioxidant, but showed no response in the intracellular assay. The CIPA assay confirmed the pro-oxidant activity of ellagitannin metabolites. In the cell proliferation assay, urolithins but not ellagic acid decreased growth and metabolism of HepG2 liver cells. In cyclic voltammetry, the oxidation of urolithin A was partly reversible, but that of urolithin B was irreversible. These results illustrate how strongly measured redox properties depend on the employed assay system and conditions and emphasize the importance of studying pro-oxidant and antioxidant activities in parallel.

  11. Lab-on-a-chip-based PCR-RFLP assay for the confirmed detection of short-length feline DNA in food.

    Science.gov (United States)

    Ali, Md Eaqub; Al Amin, Md; Hamid, Sharifah Bee Abd; Hossain, M A Motalib; Mustafa, Shuhaimi

    2015-01-01

    Wider availability but lack of legal market trades has given feline meat a high potential for use as an adulterant in common meat and meat products. However, mixing of feline meat or its derivatives in food is a sensitive issue, since it is a taboo in most countries and prohibited in certain religions such as Islam and Judaism. Cat meat also has potential for contamination with of severe acute respiratory syndrome, anthrax and hepatitis, and its consumption might lead to an allergic reaction. We developed a very short-amplicon-length (69 bp) PCR assay, authenticated the amplified PCR products by AluI-restriction digestion followed by its separation and detection on a lab-on-a-chip-based automated electrophoretic system, and proved its superiority over the existing long-amplicon-based assays. Although it has been assumed that longer DNA targets are susceptible to breakdown under compromised states, scientific evidence for this hypothesis has been rarely documented. Strong evidence showed that shorter targets are more stable than the longer ones. We confirmed feline-specificity by cross-challenging the primers against 10 different species of terrestrial, aquatic and plant origins in the presence of a 141-bp site of an 18S rRNA gene as a universal eukaryotic control. RFLP analysis separated 43- and 26-bp fragments of AluI-digest in both the gel-image and electropherograms, confirming the original products. The tested detection limit was 0.01% (w/w) feline meat in binary and ternary admixed as well as meatball matrices. Shorter target, better stability and higher sensitivity mean such an assay would be valid for feline identification even in degraded specimens.

  12. INEL test plan for evaluating waste assay systems

    Energy Technology Data Exchange (ETDEWEB)

    Mandler, J.W.; Becker, G.K.; Harker, Y.D.; Menkhaus, D.E.; Clements, T.L. Jr.

    1996-09-01

    A test bed is being established at the Idaho National Engineering Laboratory (INEL) Radioactive Waste Management Complex (RWMC). These tests are currently focused on mobile or portable radioassay systems. Prior to disposal of TRU waste at the Waste Isolation Pilot Plant (WIPP), radioassay measurements must meet the quality assurance objectives of the TRU Waste Characterization Quality Assurance Program Plan. This test plan provides technology holders with the opportunity to assess radioassay system performance through a three-tiered test program that consists of: (a) evaluations using non-interfering matrices, (b) surrogate drums with contents that resemble the attributes of INEL-specific waste forms, and (c) real waste tests. Qualified sources containing a known mixture and range of radionuclides will be used for the non-interfering and surrogate waste tests. The results of these tests will provide technology holders with information concerning radioassay system performance and provide the INEL with data useful for making decisions concerning alternative or improved radioassay systems that could support disposal of waste at WIPP.

  13. Monitoring of DNA breakage in embryonic stages of the African catfish Clarias gariepinus (Burchell, 1822) after exposure to lead nitrate using alkaline comet assay

    NARCIS (Netherlands)

    Osman, A.G.M.; Mekkawy, Imam A.; Verreth, J.A.J.; Wuertz, S.; Kloas, W.; Kirschbaum, Frank

    2008-01-01

    Increasing lead contamination in Egyptian ecosystems and high lead concentrations in food items have raised concern for human health and stimulated studies on monitoring ecotoxicological impact of lead-caused genotoxicity. In this work, the alkaline comet assay was modified for monitoring DNA strand

  14. DNA Repair Systems: Guardians of the Genome

    Indian Academy of Sciences (India)

    2016-10-01

    The 2015 Nobel Prize in Chemistry was awarded jointly to Tomas Lindahl, Paul Modrich and Aziz Sancar to honour their accomplishments in the field of DNA repair. Ever since the discovery of DNA structure and their importance in the storage of genetic information, questions about their stability became pertinent. A molecule which is crucial for the development and propagation of an organism must be closely monitored so that the genetic information is not corrupted. Thanks to the pioneering research work of Lindahl, Sancar, Modrich and their colleagues, we now have an holistic awareness of how DNA damage occurs and how the damage is rectified in bacteria as well as in higher organisms including human beings. A comprehensive understanding of DNA repair has proven crucial in the fight against cancer and other debilitating diseases.

  15. A method for the determination of bacterial spore DNA content based on isotopic labelling, spore germination and diphenylamine assay; ploidy of spores of several Bacillus species.

    Science.gov (United States)

    Hauser, P M; Karamata, D

    1992-01-01

    A reliable method for measuring the spore DNA content, based on radioactive DNA labelling, spore germination in absence of DNA replication and diphenylamine assay, was developed. The accuracy of the method, within 10-15%, is adequate for determining the number of chromosomes per spore, provided that the genome size is known. B subtilis spores were shown to be invariably monogenomic, while those of larger bacilli Bacillus megaterium, Bacillus cereus and Bacillus thuringiensis, often, if not invariably, contain two genomes. Attempts to modify the spore DNA content of B subtilis by altering the richness of the sporulation medium, the sporulation conditions (liquid or solid medium), or by mutation, were apparently unsuccessful. An increase of spore size with medium richness, not accompanied by an increase in DNA content, was observed. The implication of the apparently species-specific spore ploidy and the influence of the sporulation conditions on spore size and shape are discussed.

  16. Influence of molecular weight of DNA on the determination of anti-DNA antibodies in systemic lupus erythematosus (SLE) sera by radioimmunoassay

    Energy Technology Data Exchange (ETDEWEB)

    Geisert, M.; Heicke, B.; Metzmann, E.; Zahn, R.K.

    1975-04-01

    Using a radioimmunoassay (RIA) based on the Farr technique with radioactively labeled /sup 3/H-DNA for quantitative measurements of anti-DNA antibodies in sera of patients with systemic lupus erythematosus (SLE), the influence of molecular weight of DNA (ranging from 0.1 x 10/sup 6/ to 22.0 x 10/sup 6/ daltons) on binding and precipitation in this system has been investigated. Comparing our results with mathematical models it follows that one antibody molecule is fixed on the average to a statistical DNA segment of 2 x 10/sup 6/ to 4 x 10/sup 6/ daltons. Furthermore binding capacity of the DNA was found to be independent of the molecular weight, as demonstrated in a double label experiment using /sup 14/C and /sup 3/H-labeled DNA of different size. However, the amount of radioactivity precipitated was found to depend on the molecular weight of the labeled DNA following a non-linear function. It was calculated that a minimal ratio of fixed antibody molecules per a certain size of DNA was necessary for precipitation. The mathematical treatment of the observed non-linear precipitation dependence will be discussed using various statistical models. The results indicate that the quantitative measurements of anti-DNA antibodies with the Farr technique e.g., for diagnosis and control of SLE in clinical immunology is highly dependent on the molecular weight of the labeled DNA used in the assay system and reliable results are only obtained with DNA of a sufficiently high molecular weight. (auth)

  17. Cytomegalovirus DNA quantification using an automated platform for nucleic acid extraction and real-time PCR assay setup.

    Science.gov (United States)

    Forman, Michael; Wilson, Andy; Valsamakis, Alexandra

    2011-07-01

    Analytical performance characteristics of the QIAsymphony RGQ system with artus cytomegalovirus (CMV) reagents were determined. Measurable range spanned 2.0 to ≥ 7.0 log(10) copies/ml. The detection limit was 23 copies/ml. Intrarun and interrun coefficients of variation were ≤ 2.1% at 3.0 and 5.0 log(10) copies/ml. In clinical specimens, RGQ values were ~0.2 log(10) copies/ml higher than those in an assay using a BioRobot M48 extraction/manual reaction setup/7500 Real-Time PCR instrument. No cross-contamination was observed.

  18. A sandwich-hybridization assay for simultaneous determination of HIV and tuberculosis DNA targets based on signal amplification by quantum dots-PowerVision™ polymer coding nanotracers.

    Science.gov (United States)

    Yan, Zhongdan; Gan, Ning; Zhang, Huairong; Wang, De; Qiao, Li; Cao, Yuting; Li, Tianhua; Hu, Futao

    2015-09-15

    A novel sandwich-hybridization assay for simultaneous electrochemical detection of multiple DNA targets related to human immune deficiency virus (HIV) and tuberculosis (TB) was developed based on the different quantum dots-PowerVision(TM) polymer nanotracers. The polymer nanotracers were respectively fabricated by immobilizing SH-labeled oligonucleotides (s-HIV or s-TB), which can partially hybrid with virus DNA (HIV or TB), on gold nanoparticles (Au NPs) and then modified with PowerVision(TM) (PV) polymer-encapsulated quantum dots (CdS or PbS) as signal tags. PV is a dendrimer enzyme linked polymer, which can immobilize abundant QDs to amplify the stripping voltammetry signals from the metal ions (Pb or Cd). The capture probes were prepared through the immobilization of SH-labeled oligonucleotides, which can complementary with HIV and TB DNA, on the magnetic Fe3O4@Au (GMPs) beads. After sandwich-hybridization, the polymer nanotracers together with HIV and TB DNA targets were simultaneously introduced onto the surface of GMPs. Then the two encoding metal ions (Cd(2+) and Pb(2+)) were used to differentiate two viruses DNA due to the different subsequent anodic stripping voltammetric peaks at -0.84 V (Cd) and -0.61 V (Pb). Because of the excellent signal amplification of the polymer nanotracers and the great specificity of DNA targets, this assay could detect targets DNA as low as 0.2 femtomolar and exhibited excellent selectivity with the dynamitic range from 0.5 fM to 500 pM. Those results demonstrated that this electrochemical coding assay has great potential in applications for screening more viruses DNA while changing the probes.

  19. [Method validation according to ISO 15189 and SH GTA 04: application for the extraction of DNA and its quantitative evaluation by a spectrophotometric assay].

    Science.gov (United States)

    Harlé, Alexandre; Lion, Maëva; Husson, Marie; Dubois, Cindy; Merlin, Jean-Louis

    2013-01-01

    According to the French legislation on medical biology (January 16th, 2010), all biological laboratories must be accredited according to ISO 15189 for at least 50% of their activities before the end of 2016. The extraction of DNA from a sample of interest, whether solid or liquid is one of the critical steps in molecular biology and specifically in somatic or constitutional genetic. The extracted DNA must meet a number of criteria such quality and also be in sufficient concentration to allow molecular biology assays such as the detection of somatic mutations. This paper describes the validation of the extraction and purification of DNA using chromatographic column extraction and quantitative determination by spectrophotometric assay, according to ISO 15189 and the accreditation technical guide in Human Health SH-GTA-04.

  20. Medically Relevant Assays with a Simple Smartphone and Tablet Based Fluorescence Detection System

    Directory of Open Access Journals (Sweden)

    Piotr Wargocki

    2015-05-01

    Full Text Available Cell phones and smart phones can be reconfigured as biomedical sensor devices but this requires specialized add-ons. In this paper we present a simple cell phone-based portable bioassay platform, which can be used with fluorescent assays in solution. The system consists of a tablet, a polarizer, a smart phone (camera and a box that provides dark readout conditions. The assay in a well plate is placed on the tablet screen acting as an excitation source. A polarizer on top of the well plate separates excitation light from assay fluorescence emission enabling assay readout with a smartphone camera. The assay result is obtained by analysing the intensity of image pixels in an appropriate colour channel. With this device we carried out two assays, for collagenase and trypsin using fluorescein as the detected fluorophore. The results of collagenase assay with the lowest measured concentration of 3.75 µg/mL and 0.938 µg in total in the sample were comparable to those obtained by a microplate reader. The lowest measured amount of trypsin was 930 pg, which is comparable to the low detection limit of 400 pg for this assay obtained in a microplate reader. The device is sensitive enough to be used in point-of-care medical diagnostics of clinically relevant conditions, including arthritis, cystic fibrosis and acute pancreatitis.

  1. A One-Step PCR-Based Assay to Evaluate the Efficiency and Precision of Genomic DNA-Editing Tools.

    Science.gov (United States)

    Germini, Diego; Bou Saada, Yara; Tsfasman, Tatiana; Osina, Kristina; Robin, Chloé; Lomov, Nikolay; Rubtsov, Mikhail; Sjakste, Nikolajs; Lipinski, Mar; Vassetzky, Yegor

    2017-06-16

    Despite rapid progress, many problems and limitations persist and limit the applicability of gene-editing techniques. Making use of meganucleases, TALENs, or CRISPR/Cas9-based tools requires an initial step of pre-screening to determine the efficiency and specificity of the designed tools. This step remains time consuming and material consuming. Here we propose a simple, cheap, reliable, time-saving, and highly sensitive method to evaluate a given gene-editing tool based on its capacity to induce chromosomal translocations when combined with a reference engineered nuclease. In the proposed technique, designated engineered nuclease-induced translocations (ENIT), a plasmid coding for the DNA-editing tool to be tested is co-transfected into carefully chosen target cells along with that for an engineered nuclease of known specificity and efficiency. If the new enzyme efficiently cuts within the desired region, then specific chromosomal translocations will be generated between the two targeted genomic regions and be readily detectable by a one-step PCR or qPCR assay. The PCR product thus obtained can be directly sequenced, thereby determining the exact position of the double-strand breaks induced by the gene-editing tools. As a proof of concept, ENIT was successfully tested in different cell types and with different meganucleases, TALENs, and CRISPR/Cas9-based editing tools.

  2. Evaluation and improvement of real-time PCR assays targeting lytA, ply, and psaA genes for detection of pneumococcal DNA.

    Science.gov (United States)

    Carvalho, Maria da Gloria S; Tondella, Maria Lucia; McCaustland, Karen; Weidlich, Luciana; McGee, Lesley; Mayer, Leonard W; Steigerwalt, Arnold; Whaley, Melissa; Facklam, Richard R; Fields, Barry; Carlone, George; Ades, Edwin W; Dagan, Ron; Sampson, Jacquelyn S

    2007-08-01

    The accurate diagnosis of pneumococcal disease has frequently been hampered not only by the difficulties in obtaining isolates of the organism from patient specimens but also by the misidentification of pneumococcus-like viridans group streptococci (P-LVS) as Streptococcus pneumoniae. This is especially critical when the specimen comes from the respiratory tract. In this study, three novel real-time PCR assays designed for the detection of specific sequence regions of the lytA, ply, and psaA genes were developed (lytA-CDC, ply-CDC, and psaA, respectively). These assays showed high sensitivity (<10 copies for lytA-CDC and ply-CDC and an approximately twofold less sensitivity for psaA). Two additional real-time PCR assays for lytA and ply described previously for pneumococcal DNA detection were also evaluated. A panel of isolates consisting of 67 S. pneumoniae isolates (44 different serotypes and 3 nonencapsulated S. pneumoniae isolates from conjunctivitis outbreaks) and 104 nonpneumococcal isolates was used. The 67 S. pneumoniae isolates were reactive in all five assays. The new real-time detection assays targeting the lytA and psaA genes were the most specific for the detection of isolates confirmed to be S. pneumoniae, with lytA-CDC showing the greatest specificity. Both ply PCRs were positive for all isolates of S. pseudopneumoniae, along with 13 other isolates of other P-LVS isolates confirmed to be non-S. pneumoniae by DNA-DNA reassociation. Thus, the use of the ply gene for the detection of pneumococci can lead to false-positive reactions in the presence of P-LVS. The five assays were applied to 15 culture-positive cerebrospinal fluid specimens with 100% sensitivity; and serum and ear fluid specimens were also evaluated. Both the lytA-CDC and psaA assays, particularly the lytA-CDC assay, have improved specificities compared with those of currently available assays and should therefore be considered the assays of choice for the detection of pneumococcal DNA

  3. Identification of the major capsid protein of erythrocytic necrosis virus (ENV) and development of quantitative real-time PCR assays for quantification of ENV DNA

    Science.gov (United States)

    Purcell, Maureen K.; Pearman-Gillman, Schuyler; Thompson, Rachel L.; Gregg, Jacob L.; Hart, Lucas M.; Winton, James R.; Emmenegger, Eveline J.; Hershberger, Paul K.

    2016-01-01

    Viral erythrocytic necrosis (VEN) is a disease of marine and anadromous fish that is caused by the erythrocytic necrosis virus (ENV), which was recently identified as a novel member of family Iridoviridae by next-generation sequencing. Phylogenetic analysis of the ENV DNA polymerase grouped ENV with other erythrocytic iridoviruses from snakes and lizards. In the present study, we identified the gene encoding the ENV major capsid protein (MCP) and developed a quantitative real-time PCR (qPCR) assay targeting this gene. Phylogenetic analysis of the MCP gene sequence supported the conclusion that ENV does not group with any of the currently described iridovirus genera. Because there is no information regarding genetic variation of the MCP gene across the reported host and geographic range for ENV, we also developed a second qPCR assay for a more conserved ATPase-like gene region. The MCP and ATPase qPCR assays demonstrated good analytical and diagnostic sensitivity and specificity based on samples from laboratory challenges of Pacific herring Clupea pallasii. The qPCR assays had similar diagnostic sensitivity and specificity as light microscopy of stained blood smears for the presence of intraerythrocytic inclusion bodies. However, the qPCR assays may detect viral DNA early in infection prior to the formation of inclusion bodies. Both qPCR assays appear suitable for viral surveillance or as a confirmatory test for ENV in Pacific herring from the Salish Sea.

  4. Identification of the major capsid protein of erythrocytic necrosis virus (ENV) and development of quantitative real-time PCR assays for quantification of ENV DNA.

    Science.gov (United States)

    Purcell, Maureen K; Pearman-Gillman, Schuyler; Thompson, Rachel L; Gregg, Jacob L; Hart, Lucas M; Winton, James R; Emmenegger, Eveline J; Hershberger, Paul K

    2016-07-01

    Viral erythrocytic necrosis (VEN) is a disease of marine and anadromous fish that is caused by the erythrocytic necrosis virus (ENV), which was recently identified as a novel member of family Iridoviridae by next-generation sequencing. Phylogenetic analysis of the ENV DNA polymerase grouped ENV with other erythrocytic iridoviruses from snakes and lizards. In the present study, we identified the gene encoding the ENV major capsid protein (MCP) and developed a quantitative real-time PCR (qPCR) assay targeting this gene. Phylogenetic analysis of the MCP gene sequence supported the conclusion that ENV does not group with any of the currently described iridovirus genera. Because there is no information regarding genetic variation of the MCP gene across the reported host and geographic range for ENV, we also developed a second qPCR assay for a more conserved ATPase-like gene region. The MCP and ATPase qPCR assays demonstrated good analytical and diagnostic sensitivity and specificity based on samples from laboratory challenges of Pacific herring Clupea pallasii The qPCR assays had similar diagnostic sensitivity and specificity as light microscopy of stained blood smears for the presence of intraerythrocytic inclusion bodies. However, the qPCR assays may detect viral DNA early in infection prior to the formation of inclusion bodies. Both qPCR assays appear suitable for viral surveillance or as a confirmatory test for ENV in Pacific herring from the Salish Sea.

  5. MGB probe assay for rapid detection of mtDNA11778 mutation in the Chinese LHON patients by real-time PCR

    Institute of Scientific and Technical Information of China (English)

    Jian-yong WANG; Yang-shun GU; Jing WANG; Yi TONG; Ying WANG; Jun-bing SHAO; Ming QI

    2008-01-01

    Objective:Leber's hereditary optic neuropathY (LHON)is a maternally inherited degeneration of the optic nerve caused by point mutations of mitochondrial DNA(mtDNA).Many unsolved questions regarding the penetrance and pathophysiological mechanism of LHON demand efficient and reliable mutation testing.This study aims to develop a minor groove binder(MGB) probe assay for rapid detection of mtDNA11778 mutation and heteroplasmy in Chinese LHON patients by real-time polymerase chain reaction(PCR).Methods:Forty-eight patients suspected of having LHON and their maternal relatives underwent a molecular genetic evaluation,with 20 normal individuals as a control group at the same time.A real-time PCR involving two MGB probes was used to detect the mtDNA 11778 mutation and heteroplasmy.A linear standard curve was obtained by pUCmLHONG and pUCmLHONA clones.Results:All 48 LHON patients and their matemal relatives were positive for mtDNA 11778 mutation in our assay,27 heteroplasmic and 21 homoplasmic.Eighteen cases did not show an occurrence of the disease,while 9 developed the disease among the 27 heteroplasmic mutation cases.Eleven did not show an occurrence of the disease,while 10 cases developed the disease among 21 homoplasmic mutation cases.There was a significant difierence in the incidence between the heteroplasmic and the homoplasmic mutation types.The time needed for running a real-time PCR assay was only 80 min.Conclusion:This real-time PCR assay is a rapid,reliable method for mtDNA mutation detection as well as heteroplasmy quantification.Detecting this ratio is very important for predicting phenotypic expression of unaffected carriers.

  6. Establishing a Gene Trap System Mediated by T-DNA(GUS) in Rice

    Institute of Scientific and Technical Information of China (English)

    Shi-Yan Chen; Ai-Min Wang; Wei Li; Zong-Yang Wang; Xiu-Ling Cai

    2008-01-01

    Two plasmids, p13GUS and p13GUS2, were constructed to create a gene trap system containing the promoterless β-glucuronidase (GUS) reporter gene in the T-DNA region. Transformation of these two plasmids into the rice variety Zhonghua 11 (Oryza sativa ssp. japonica cv.), mediated by Agrobacterium tumefaciens, resulted in 942 independent transgenic lines. Histochemical GUS assays revealed that 31 To plants had various patterns of the reporter gene expression, including expression in only one tissue, and simultaneously in two or more tissues. Hygromycin-resistant (hygr) homozygotes were screened and the copy number of the T-DNA inserts was determined in the GUS-positivs transgenic plants. The flanking sequences of the T-DNA were isolated by inverse-polymerase chain reaction and the insert positions on the rice genome of T-DNA were determined by a basic local alignment search tool in the GUS-positive transgenic plants transformed with plasmid p13GUS. Moreover, calii induced from the seeds of the T1 generation of 911 GUS-negative transgenic lines were subjected to stress and hormone treatments. Histochemical GUS assays were carried out on the calli before and after treatment. The results revealed that calli from 21 lines displayed differential GUS expression after treatment. All of these data demonstrated that this trap system is suitable for identifying rice genes, including those that are sensitive to induction.

  7. Evaluation of a novel real-time fluorescent polymerase chain reaction assay for high-risk human papilloma virus DNA genotypes in cytological cervical screening.

    Science.gov (United States)

    Cheng, Jiaoying; Bian, Meilu; Cong, Xiao; Sun, Aiping; Li, Min; Ma, Li; Chen, Ying; Liu, Jun

    2013-03-01

    It has been confirmed that detection of high-risk human papillomavirus (HR HPV) DNA is useful in cervical cancer (CC) screening. Recently, a new real-time fluorescent polymerase chain reaction (PCR) assay was developed to detect HR HPV. This assay can synchronize nucleic acid amplification and testing using specific primers for 13 types of HR HPV genomes, combined with specific TaqMan fluorescent marker probe techniques through the fluorescence automatic PCR instrument. Furthermore, it uses TaqGold™ DNA polymerase, which minimizes the amount of non-specific amplification and increases the sensitivity of the assay. The aim of this study was to evaluate the analytical and clinical performance of the real-time fluorescent PCR assay in CC screening, compared to the Qiagen Hybrid Capture(®) II High-Risk HPV DNA test(®) (HC II). In total, 1,252 cervical specimens were collected from women between 19 and 71 years of age. The specimens were examined with three different assays, real-time fluorescent PCR assay and HC II for HR HPV detection combined with liquid-based cytology. Women with cytological abnormalities or HR HPV-positive results underwent colposcopy and cervical biopsy. This study demonstrated good overall agreement between HC II and real-time fluorescent PCR assay (overall agreement, 92.25%; Cohen's κ=0.814). For the detection of high-grade cervical intraepithelial neoplasias (CIN) and CC, the sensitivity of HC II and real-time fluorescent PCR was 94.48 and 92.82%, respectively, and the negative predictive value was 98.85 and 98.54%, respectively. High HR HPV infection rate of the high-grade CIN and CC group was detected (PHPV detection and could be used in CC screening in clinic.

  8. Nanoparticle-based assays in automated flow systems: A review

    Energy Technology Data Exchange (ETDEWEB)

    Passos, Marieta L.C. [LAQV, REQUIMTE, Departamento de Ciências Químicas, Faculdade de Farmácia, Universidade do Porto, Rua Jorge Viterbo Ferreira, n° 228, 4050-313 Porto (Portugal); Pinto, Paula C.A.G., E-mail: ppinto@ff.up.pt [LAQV, REQUIMTE, Departamento de Ciências Químicas, Faculdade de Farmácia, Universidade do Porto, Rua Jorge Viterbo Ferreira, n° 228, 4050-313 Porto (Portugal); Santos, João L.M., E-mail: joaolms@ff.up.pt [LAQV, REQUIMTE, Departamento de Ciências Químicas, Faculdade de Farmácia, Universidade do Porto, Rua Jorge Viterbo Ferreira, n° 228, 4050-313 Porto (Portugal); Saraiva, M. Lúcia M.F.S., E-mail: lsaraiva@ff.up.pt [LAQV, REQUIMTE, Departamento de Ciências Químicas, Faculdade de Farmácia, Universidade do Porto, Rua Jorge Viterbo Ferreira, n° 228, 4050-313 Porto (Portugal); Araujo, André R.T.S. [LAQV, REQUIMTE, Departamento de Ciências Químicas, Faculdade de Farmácia, Universidade do Porto, Rua Jorge Viterbo Ferreira, n° 228, 4050-313 Porto (Portugal); Unidade de Investigação para o Desenvolvimento do Interior, Instituto Politécnico da Guarda, Av. Dr. Francisco de Sá Carneiro, n° 50, 6300-559 Guarda (Portugal)

    2015-08-19

    Nanoparticles (NPs) exhibit a number of distinctive and entrancing properties that explain their ever increasing application in analytical chemistry, mainly as chemosensors, signaling tags, catalysts, analytical signal enhancers, reactive species generators, analyte recognition and scavenging/separation entities. The prospect of associating NPs with automated flow-based analytical is undoubtedly a challenging perspective as it would permit confined, cost-effective and reliable analysis, within a shorter timeframe, while exploiting the features of NPs. This article aims at examining state-of-the-art on continuous flow analysis and microfluidic approaches involving NPs such as noble metals (gold and silver), magnetic materials, carbon, silica or quantum dots. Emphasis is devoted to NP format, main practical achievements and fields of application. In this context, the functionalization of NPs with distinct chemical species and ligands is debated in what concerns the motivations and strengths of developed approaches. The utilization of NPs to improve detector's performance in electrochemical application is out of the scope of this review. The works discussed in this review were published in the period of time comprised between the years 2000 and 2013. - Highlights: • The state of the art of flowing stream systems comprising NPs was reviewed. • The use of different types of nanoparticles in each flow technique is discussed. • The most expressive and profitable applications are summarized. • The main conclusions and future perspectives were compiled in the final section.

  9. DNA strand displacement system running logic programs.

    Science.gov (United States)

    Rodríguez-Patón, Alfonso; Sainz de Murieta, Iñaki; Sosík, Petr

    2014-01-01

    The paper presents a DNA-based computing model which is enzyme-free and autonomous, not requiring a human intervention during the computation. The model is able to perform iterated resolution steps with logical formulae in conjunctive normal form. The implementation is based on the technique of DNA strand displacement, with each clause encoded in a separate DNA molecule. Propositions are encoded assigning a strand to each proposition p, and its complementary strand to the proposition ¬p; clauses are encoded comprising different propositions in the same strand. The model allows to run logic programs composed of Horn clauses by cascading resolution steps. The potential of the model is demonstrated also by its theoretical capability of solving SAT. The resulting SAT algorithm has a linear time complexity in the number of resolution steps, whereas its spatial complexity is exponential in the number of variables of the formula. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  10. Targeting DNA-repair systems brings hopes to cancer patients

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    @@ CAS researchers have recently raised a hypothesis to circumvent tumor resistance to radio- and chemo-therapy and to enhance the efficacy of DNAdamaging agents by interfering with DNA repair. "There are emerging anticancer therapeutic opportunities in targeting DNA-repair systems," they asserted.

  11. DNA

    Science.gov (United States)

    Stent, Gunther S.

    1970-01-01

    This history for molecular genetics and its explanation of DNA begins with an analysis of the Golden Jubilee essay papers, 1955. The paper ends stating that the higher nervous system is the one major frontier of biological inquiry which still offers some romance of research. (Author/VW)

  12. Evaluation of the Punch-it™ NA-Sample kit for detecting microbial DNA in blood culture bottles using PCR-reverse blot hybridization assay.

    Science.gov (United States)

    Kim, Jungho; Wang, Hye-Young; Kim, Seoyong; Park, Soon Deok; Yu, Kwangmin; Kim, Hyo Youl; Uh, Young; Lee, Hyeyoung

    2016-09-01

    DNA extraction efficiency affects the success of PCR-based method applications. The Punch-it™ NA-Sample kit for extracting DNA by using paper chromatography is technically easy to use and requires just two reagents and only 10min to complete. The Punch-it™ NA-Sample kit could be offered as a rapid, accurate, and convenient method for extracting bacterial and fungal DNA from blood culture bottles. We compared the efficiencies of the commercial kit (Punch-it™ NA-Sample kit) and an in-house conventional boiling method with Chelex-100 resin for DNA extraction from blood culture bottles. The efficiency of the two DNA extraction methods was assessed by PCR-reverse blot hybridization assay (PCR-REBA, REBA Sepsis-ID) for detecting Gram positive (GP) bacteria, Gram negative (GN) bacteria, and Candida species with 196 positive and 200 negative blood culture bottles. The detection limits of the two DNA extraction methods were 10(3)CFU/mL for GP bacteria, 10(3)CFU/mL for GN bacteria, and 10(4)CFU/mL for Candida. The sensitivity and specificity of the Punch-it™ NA-Sample kit by REBA Sepsis-ID were 95.4% (187/196) and 100% (200/200), respectively. The overall agreement of the two DNA extraction methods was 98.9% (392/396). Three of four samples showing discrepant results between the two extraction methods were more accurately matched up with the Punch-it™ NA-Sample kit based on conventional culture methods. The results indicated that the Punch-it™ NA-Sample kit extracted bacterial and fungal DNA in blood culture bottles and allowed extracted DNA to be used in molecular assay. Copyright © 2016. Published by Elsevier B.V.

  13. Development of an Automated DNA Detection System Using an Electrochemical DNA Chip Technology

    Science.gov (United States)

    Hongo, Sadato; Okada, Jun; Hashimoto, Koji; Tsuji, Koichi; Nikaido, Masaru; Gemma, Nobuhiro

    A new compact automated DNA detection system Genelyzer™ has been developed. After injecting a sample solution into a cassette with a built-in electrochemical DNA chip, processes from hybridization reaction to detection and analysis are all operated fully automatically. In order to detect a sample DNA, electrical currents from electrodes due to an oxidization reaction of electrochemically active intercalator molecules bound to hybridized DNAs are detected. The intercalator is supplied as a reagent solution by a fluid supply unit of the system. The feasibility test proved that the simultaneous typing of six single nucleotide polymorphisms (SNPs) associated with a rheumatoid arthritis (RA) was carried out within two hours and that all the results were consistent with those by conventional typing methods. It is expected that this system opens a new way to a DNA testing such as a test for infectious diseases, a personalized medicine, a food inspection, a forensic application and any other applications.

  14. Development of a Portable DNA Sensor System

    Science.gov (United States)

    2008-12-01

    75 80 85 90 0.00 -0.10 -0.20 -0.30 -0.40 Potential (V) A C C ur re nt (n A ) The cyclic voltammetry curves resulting from the preliminary...and eDNA microfluidic chip. This chip uses external fluid reservoirs, heaters and pumps. 2.5 Circuitry and software The analog potentiostat ...electronics and potentiostat PCB assemblies (left). Concept for future, integrated eDNA biosensor. 2.5.3 Multi-Channel Potentiostat The

  15. DNA – A General Energy System Simulation Tool

    DEFF Research Database (Denmark)

    Elmegaard, Brian; Houbak, Niels

    2005-01-01

    operation. The program decides at runtime to apply the DAE solver if the system contains differential equations. This makes it easy to extend an existing steady state model to simulate dynamic operation of the plant. The use of the program is illustrated by examples of gas turbine models. The paper also......The paper reviews the development of the energy system simulation tool DNA (Dynamic Network Analysis). DNA has been developed since 1989 to be able to handle models of any kind of energy system based on the control volume approach, usually systems of lumped parameter components. DNA has proven...... to be a useful tool in the analysis and optimization of several types of thermal systems: Steam turbines, gas turbines, fuels cells, gasification, refrigeration and heat pumps for both conventional fossil fuels and different types of biomass. DNA is applicable for models of both steady state and dynamic...

  16. Enzyme-regulated activation of DNAzyme: a novel strategy for a label-free colorimetric DNA ligase assay and ligase-based biosensing.

    Science.gov (United States)

    He, Kaiyu; Li, Wang; Nie, Zhou; Huang, Yan; Liu, Zhuoliang; Nie, Lihua; Yao, Shouzhuo

    2012-03-26

    The DNA nick repair catalyzed by DNA ligase is significant for fundamental life processes, such as the replication, repair, and recombination of nucleic acids. Here, we have employed ligase to regulate DNAzyme activity and developed a homogeneous, colorimetric, label-free and DNAzyme-based strategy to detect DNA ligase activity. This novel strategy relies on the ligation-trigged activation or production of horseradish peroxidase mimicking DNAzyme that catalyzes the generation of a color change signal; this results in a colorimetric assay of DNA ligase activity. Using T4 DNA ligase as a model, we have proposed two approaches to demonstrate the validity of the DNAzyme strategy. The first approach utilizes an allosteric hairpin-DNAzyme probe specifically responsive to DNA ligation; this approach has a wide detection range from 0.2 to 40 U mL(-1) and a detection limit of 0.2 U mL(-1). Furthermore, the approach was adapted to probe nucleic acid phosphorylation and single nucleotide mismatch. The second approach employs a "split DNA machine" to produce numerous DNAzymes after being reassembled by DNA ligase; this greatly enhances the detection sensitivity by a signal amplification cascade to achieve a detection limit of 0.01 U mL(-1).

  17. Enzymic colorimetry-based DNA chip: a rapid and accurate assay for detecting mutations for clarithromycin resistance in the 23S rRNA gene of Helicobacter pylori.

    Science.gov (United States)

    Xuan, Shi-Hai; Zhou, Yu-Gui; Shao, Bo; Cui, Ya-Lin; Li, Jian; Yin, Hong-Bo; Song, Xiao-Ping; Cong, Hui; Jing, Feng-Xiang; Jin, Qing-Hui; Wang, Hui-Min; Zhou, Jie

    2009-11-01

    Macrolide drugs, such as clarithromycin (CAM), are a key component of many combination therapies used to eradicate Helicobacter pylori. However, resistance to CAM is increasing in H. pylori and is becoming a serious problem in H. pylori eradication therapy. CAM resistance in H. pylori is mostly due to point mutations (A2142G/C, A2143G) in the peptidyltransferase-encoding region of the 23S rRNA gene. In this study an enzymic colorimetry-based DNA chip was developed to analyse single-nucleotide polymorphisms of the 23S rRNA gene to determine the prevalence of mutations in CAM-related resistance in H. pylori-positive patients. The results of the colorimetric DNA chip were confirmed by direct DNA sequencing. In 63 samples, the incidence of the A2143G mutation was 17.46 % (11/63). The results of the colorimetric DNA chip were concordant with DNA sequencing in 96.83 % of results (61/63). The colorimetric DNA chip could detect wild-type and mutant signals at every site, even at a DNA concentration of 1.53 x 10(2) copies microl(-1). Thus, the colorimetric DNA chip is a reliable assay for rapid and accurate detection of mutations in the 23S rRNA gene of H. pylori that lead to CAM-related resistance, directly from gastric tissues.

  18. QUANTITATION OF DNA TOPOISOMERASE-II-ALPHA MESSENGER-RIBONUCLEIC-ACID LEVELS IN A SMALL-CELL LUNG-CANCER CELL-LINE AND 2 DRUG-RESISTANT SUBLINES USING A POLYMERASE CHAIN REACTION-AIDED TRANSCRIPT TITRATION ASSAY

    NARCIS (Netherlands)

    WITHOFF, S; SMIT, EF; MEERSMA, GJ; van den Berg, Anke; TIMMERBOSSCHA, H; KOK, K; POSTMUS, PE; MULDER, NH; DEVRIES, EGE; BUYS, CHCM

    1994-01-01

    BACKGROUND: We have modified a polymerase chain reaction (PCR)-aided transcript titration assay (1) in order to allow quantitation of low amounts of DNA topoisomerase II alpha mRNA in small RNA samples. EXPERIMENTAL DESIGN: The titration assay was used to quantitate the amount of DNA topoisomerase I

  19. Clinical performance of human papillomavirus E6, E7 mRNA flow cytometric assay compared to human papillomavirus DNA typing.

    Science.gov (United States)

    Kottaridi, Christine; Tsiodras, Sotirios; Spathis, Aris; Chranioti, Aikaterini; Pappas, Asimakis; Kassanos, Dimitrios; Panayiotides, Ioannis; Karakitsos, Petros

    2011-12-01

    To use flow cytometry to screen cervical samples for the overexpression of human papillomavirus (HPV) E6 and E7 mRNA and compare the performance of this assay with an HPV DNA array for the detection of high-grade cervical lesions. Cervical samples were analyzed for HPV DNA by clinical arrays, and the overexpression of E6 and E7 viral oncogenes was monitored using an HPV mRNA detection kit that quantifies the intracellular HPV E6 and E7 mRNA on a cell-by-cell basis. HPV positivity increased with severity of histologic lesions. On the basis of histology-confirmed CIN 2+ cases the specificity of HPV assay was 73.9% (95% CI 66.07, 80.88), whereas it was 39.3% (95% CI 31.85, 47.1) for the DNA assay. The HPV assay provides an early predictor of persistent HPV infection and may improve cervical cancer screening by increasing the specificity of detecting high-grade lesions.

  20. Detection of Morganella morganii, a prolific histamine former, by the polymerase chain reaction assay with 16S rDNA-targeted primers.

    Science.gov (United States)

    Kim, Shin-Hee; An, Haejung; Field, Katharine G; Wei, Cheng-I; Velazquez, Jorge Barros; Ben-Gigirey, Begoña; Morrissey, Michael T; Price, Robert J; Pitta, Thomas P

    2003-08-01

    A polymerase chain reaction (PCR) assay for the rapid and sensitive detection of the most prolific histamine former, Morganella morganii, was developed. 16S rDNA targeted PCR primers were designed, and the primer specificity and sensitivity of the PCR assay were evaluated. The 16S rDNA sequence (1,503 bp) for M. morganii showed 95% identity to those for enteric bacteria, i.e., Enterobacter spp., Klebsiella spp., Citrobacter spp., Hafnia alvei, Proteus spp., and Providencia spp. The unique primers for M. morganii were designed on the basis of the variable regions in the 16S rDNA sequence. The primers showed positive reactions with all M. morganii strains tested. However, PCR amplification was not detected when the primers were tested with other enteric or marine bacteria. When the sensitivity of the assay was evaluated, M. morganii was detected at levels ranging from 10(6) to 10(8) CFU/ml in albacore homogenate after the PCR amplification. The sensitivity of the assay was greatly improved with the enrichment of samples, and 9 CFU of M. morganii per ml of albacore homogenate was detected after 6 h of enrichment at 37 degrees C.

  1. Continuous-flow ATP amplification system for increasing the sensitivity of quantitative bioluminescence assay.

    Science.gov (United States)

    Satoh, Tetsuya; Shinoda, Yasuharu; Alexandrov, Maxym; Kuroda, Akio; Murakami, Yuji

    2008-08-01

    We constructed a novel ATP amplification reactor using a continuous-flow system, and this allowed us to increase the sensitivity of a quantitative bioluminescence assay by controlling the number of ATP amplification cycles. We previously developed a bioluminescence assay coupled with ATP amplification using a batch system. However, it was difficult to control the number of amplification cycles. In this study, ATP amplification was performed using a continuous-flow system, and significant linear correlations between amplified luminescence and initial ATP concentration were observed. When performing four cycles of continuous-flow ATP amplification, the gradient of amplification was 1.87(N). Whereas the lower quantifiable level was 500 pM without amplification, values as low as 50 pM ATP could be measured after amplification. The sensitivity thus increased 10-fold, with further improvements expected with additional amplification cycles. The continuous-flow system thus effectively increased the sensitivity of the quantitative bioluminescence assay.

  2. Total Measurement Uncertainty for the Plutonium Finishing Plant (PFP) Segmented Gamma Scan Assay System

    CERN Document Server

    Fazzari, D M

    2001-01-01

    This report presents the results of an evaluation of the Total Measurement Uncertainty (TMU) for the Canberra manufactured Segmented Gamma Scanner Assay System (SGSAS) as employed at the Hanford Plutonium Finishing Plant (PFP). In this document, TMU embodies the combined uncertainties due to all of the individual random and systematic sources of measurement uncertainty. It includes uncertainties arising from corrections and factors applied to the analysis of transuranic waste to compensate for inhomogeneities and interferences from the waste matrix and radioactive components. These include uncertainty components for any assumptions contained in the calibration of the system or computation of the data. Uncertainties are propagated at 1 sigma. The final total measurement uncertainty value is reported at the 95% confidence level. The SGSAS is a gamma assay system that is used to assay plutonium and uranium waste. The SGSAS system can be used in a stand-alone mode to perform the NDA characterization of a containe...

  3. Development and validation of a D-loop mtDNA SNP assay for the screening of specimens in forensic casework.

    Science.gov (United States)

    Chemale, Gustavo; Paneto, Greiciane Gaburro; Menezes, Meiga Aurea Mendes; de Freitas, Jorge Marcelo; Jacques, Guilherme Silveira; Cicarelli, Regina Maria Barretto; Fagundes, Paulo Roberto

    2013-05-01

    Mitochondrial DNA (mtDNA) analysis is usually a last resort in routine forensic DNA casework. However, it has become a powerful tool for the analysis of highly degraded samples or samples containing too little or no nuclear DNA, such as old bones and hair shafts. The gold standard methodology still constitutes the direct sequencing of polymerase chain reaction (PCR) products or cloned amplicons from the HVS-1 and HVS-2 (hypervariable segment) control region segments. Identifications using mtDNA are time consuming, expensive and can be very complex, depending on the amount and nature of the material being tested. The main goal of this work is to develop a less labour-intensive and less expensive screening method for mtDNA analysis, in order to aid in the exclusion of non-matching samples and as a presumptive test prior to final confirmatory DNA sequencing. We have selected 14 highly discriminatory single nucleotide polymorphisms (SNPs) based on simulations performed by Salas and Amigo (2010) to be typed using SNaPShot(TM) (Applied Biosystems, Foster City, CA, USA). The assay was validated by typing more than 100 HVS-1/HVS-2 sequenced samples. No differences were observed between the SNP typing and DNA sequencing when results were compared, with the exception of allelic dropouts observed in a few haplotypes. Haplotype diversity simulations were performed using 172 mtDNA sequences representative of the Brazilian population and a score of 0.9794 was obtained when the 14 SNPs were used, showing that the theoretical prediction approach for the selection of highly discriminatory SNPs suggested by Salas and Amigo (2010) was confirmed in the population studied. As the main goal of the work is to develop a screening assay to skip the sequencing of all samples in a particular case, a pair-wise comparison of the sequences was done using the selected SNPs. When both HVS-1/HVS-2 SNPs were used for simulations, at least two differences were observed in 93.2% of the comparisons

  4. Assessment of DNA damage of Lewis lung carcinoma cells irradiated by carbon ions and X-rays using alkaline comet assay

    Science.gov (United States)

    Li, Ping; Zhou, Li-Bin; Jin, Xiao-Dong; He, Jing; Dai, Zhong-Ying; Zhou, Guang-Ming; Gao, Qing-Xiang; Li, Sha; Li, Qiang

    2008-01-01

    DNA damage and cell reproductive death determined by alkaline comet and clonogenic survival assays were examined in Lewis lung carcinoma cells after exposure to 89.63 MeV/u carbon ion and 6 MV X-ray irradiations, respectively. Based on the survival data, Lewis lung carcinoma cells were verified to be more radiosensitive to the carbon ion beam than to the X-ray irradiation. The relative biological effectiveness (RBE) value, which was up to 1.77 at 10% survival level, showed that the DNA damage induced by the high-LET carbon ion beam was more remarkable than that induced by the low-LET X-ray irradiation. The dose response curves of “Tail DNA (%)” (TD) and “Olive tail moment” (OTM) for the carbon ion irradiation showed saturation beyond about 8 Gy. This behavior was not found in the X-ray curves. Additionally, the carbon ion beam produced a lower survival fraction at 2 Gy (SF2) value and a higher initial Olive tail moment 2 Gy (OTM2) than those for the X-ray irradiation. These results suggest that carbon ion beams having high-LET values produced more severe cell reproductive death and DNA damage in Lewis lung carcinoma cells in comparison with X-rays and comet assay might be an effective predictive test even combining with clonogenic assay to assess cellular radiosensitivity.

  5. A Comparison of Fresh Frozen vs. Formalin-Fixed, Paraffin-Embedded Specimens of Canine Mammary Tumors via Branched-DNA Assay

    Directory of Open Access Journals (Sweden)

    Florenza Lüder Ripoli

    2016-05-01

    Full Text Available Mammary neoplasms are the tumors most affecting female dogs and women. Formalin-fixed, paraffin-embedded (FFPE tissues are an invaluable source of archived biological material. Fresh frozen (FF tissue is considered ideal for gene expression analysis. However, strategies based on FFPE material offer several advantages. Branched-DNA assays permit a reliable and fast workflow when analyzing gene expression. The aim of this study was to assess the comparability of the branched-DNA assay when analyzing certain gene expression patterns between FF and FFPE samples in canine mammary tumors. RNA was isolated from 109 FFPE samples and from 93 FF samples of different canine mammary tissues. Sixteen (16 target genes (Tp53; Myc; HMGA1; Pik3ca; Mcl1; MAPK3; FOXO3; PTEN; GATA4; PFDN5; HMGB1; MAPK1; BRCA2; BRCA1; HMGA2; and Her2 were analyzed via branched-DNA assay (b-DNA. ACTB, GAPDH, and HPRT1 were used as data normalizers. Overall, the relative gene expression of the two different origins of samples showed an agreement of 63%. Still, care should be taken, as FFPE specimens showed lower expression of the analyzed targets when compared to FF samples. The fact that the gene expression in FFPE proved to be lower than in FF specimens is likely to have been caused by the effect of storage time. ACTB had the best performance as a data normalizer.

  6. DNA Nanocarriers for Systemic Administration: Characterization and In Vivo Bioimaging in Healthy Mice

    OpenAIRE

    David, Stéphanie; Passirani,Catherine; Carmoy, Nathalie; Morille, Marie; Mevel, Mathieu; Chatin, Benoit; Benoit, Jean-Pierre; Montier, Tristan; Pitard, Bruno

    2013-01-01

    International audience; We hereby present different DNA nanocarriers consisting of new multimodular systems (MMS), containing the cationic lipid dioleylaminesuccinylparomomycin (DNA MMS DOSP), or bis (guanidinium)-tren-cholesterol (DNA MMS BGTC), and DNA lipid nanocapsules (DNA LNCs). Active targeting of the asialoglycoprotein receptor (ASGP-R) using galactose as a ligand for DNA MMS (GAL DNA MMS) and passive targeting using a polyethylene glycol coating for DNA LNCs (PEG DNA LNCs) should imp...

  7. Multiplex quantification of Escherichia coli, Salmonella typhi and Vibrio cholera with three DNA targets in single reaction assay.

    Science.gov (United States)

    Jangampalli Adi, Pradeepkiran; Naidu, Jagadish R; Matcha, Bhaskar

    2017-09-01

    Escherichia coli (E. coli), Salmonella typhi and Vibrio cholera harmful pathogens, which causes various diseases in humans. Rapid diagnosis of bacterial infection is an important for patient management and appropriate therapy during the early phase of the bacterial infected diseases. Among the existing techniques for identifying pathogens were less sensitive and time-consuming processes. In the present study total, 48 clinical 31 blood and 17 urine samples of patients suspected with the infections were collected from SVRR Hospital and used to detect the pathogens. Multiplex polymerase chain reaction (PCR) assay was set to design for the identification of Escherichia coli, Salmonella typhi and Vibrio cholera from the different clinical samples. Rapid diagnosis of Escherichia coli (E. coli), Salmonella and Vibrio cholera pathogens can be done with simultaneously in a single multiplex PCR assay by using specific primers with adjusted PCR conditions. Through this approach, the results represented with out of 31 blood samples 1-15 shows the positive with E. coli and remaining 14 only 11 were correlated with multiplex results of Vibrio cholera, remaining the urine samples all are positive with 17 samples correlate with the Salmonella typhi. Through the high specificity benefits of excellent sensitivity, with high resolution and reproducibility. This method of results proved and illustrates the best potential system for diagnosing the infectious disease with modern trendy. Copyright © 2017 Elsevier Ltd. All rights reserved.

  8. Fluorescence bio-barcode DNA assay based on gold and magnetic nanoparticles for detection of Exotoxin A gene sequence.

    Science.gov (United States)

    Amini, Bahram; Kamali, Mehdi; Salouti, Mojtaba; Yaghmaei, Parichehreh

    2017-06-15

    Bio-barcode DNA based on gold nanoparticle (bDNA-GNPs) as a new generation of biosensor based detection tools, holds promise for biological science studies. They are of enormous importance in the emergence of rapid and sensitive procedures for detecting toxins of microorganisms. Exotoxin A (ETA) is the most toxic virulence factor of Pseudomonas aeruginosa. ETA has ADP-ribosylation activity and decisively affects the protein synthesis of the host cells. In the present study, we developed a fluorescence bio-barcode technology to trace P. aeruginosa ETA. The GNPs were coated with the first target-specific DNA probe 1 (1pDNA) and bio-barcode DNA, which acted as a signal reporter. The magnetic nanoparticles (MNPs) were coated with the second target-specific DNA probe 2 (2pDNA) that was able to recognize the other end of the target DNA. After binding the nanoparticles with the target DNA, the following sandwich structure was formed: MNP 2pDNA/tDNA/1pDNA-GNP-bDNA. After isolating the sandwiches by a magnetic field, the DNAs of the probes which have been hybridized to their complementary DNA, GNPs and MNPs, via the hydrogen, electrostatic and covalently bonds, were released from the sandwiches after dissolving in dithiothreitol solution (DTT 0.8M). This bio-barcode DNA with known DNA sequence was then detected by fluorescence spectrophotometry. The findings showed that the new method has the advantages of fast, high sensitivity (the detection limit was 1.2ng/ml), good selectivity, and wide linear range of 5-200ng/ml. The regression analysis also showed that there was a good linear relationship (∆F=0.57 [target DNA]+21.31, R(2)=0.9984) between the fluorescent intensity and the target DNA concentration in the samples.

  9. Ready to use dry-reagent PCR assays for the four common bacterial pathogens using switchable lanthanide luminescence probe system.

    Science.gov (United States)

    Lehmusvuori, A; Soikkeli, M; Tuunainen, E; Seppä, T; Spangar, A; Rantakokko-Jalava, K; von Lode, P; Karhunen, U; Soukka, T; Wittfooth, S

    2015-11-01

    Ready to use dry-reagent PCR assays for Escherichia coli, Staphylococcus aureus, Streptococcus pneumoniae, Pseudomonas spp. and for broad-range bacteria detection were developed. The assays were based on novel switchable lanthanide probes that provide sensitive target DNA detection with exceptionally high signal-to-background ratio, thus enabling clear discrimination between positive and negative results. For example, sensitivity of three S. aureus and two S. pneumonia bacteria (colony forming units) per PCR assay was measured with fluorescence signal more than 30 times over the background signal level. The rapid and easy-to-use assays are suitable for routine clinical diagnostics without molecular biology expertise and facilities.

  10. The comet assay as a rapid test in biomonitoring occupational exposure to DNA-damaging agents and effect of confounding factors.

    Science.gov (United States)

    Møller, P; Knudsen, L E; Loft, S; Wallin, H

    2000-10-01

    Within the last decade, the comet assay has been used with increasing popularity to investigate the level of DNA damage in terms of strand breaks and alkaline labile sites in biomonitoring studies. The assay is easily performed on WBCs and has been included in a wide range of biomonitoring studies of occupational exposures encompassing styrene, vinyl chloride, 1,3-butadiene, pesticides, hair dyes, antineoplastic agents, organic solvents, sewage and waste materials, wood dust, and ionizing radiation. Eleven of the occupational studies were positive, whereas seven were negative. Notably, the negative studies appeared to have less power than the positive studies. Also, there were poor dose-response relationships in many of the biomonitoring studies. Many factors have been reported to produce effects by the comet assay, e.g., age, air pollution exposure, diet, exercise, gender, infection, residential radon exposure, smoking, and season. Until now, the use of the comet assay has been hampered by the uncertainty of the influence of confounding factors. We argue that none of the confounding factors are unequivocally positive in the majority of the studies. We recommend that age, gender, and smoking status be used as criteria for the selection of populations and that data on exercise, diet, and recent infections be registered before blood sampling. Samples from exposed and unexposed populations should be collected at the same time to avoid seasonal variation. In general, the comet assay is considered a suitable and fast test for DNA-damaging potential in biomonitoring studies.

  11. Development and Evaluation of Loop-Mediated Isothermal Amplification Assay for Rapid Detection of Tylenchulus semipenetrans Using DNA Extracted from Soil

    Science.gov (United States)

    Song, Zhi-Qiang; Cheng, Ju-E; Cheng, Fei-Xue; Zhang, De-Yong; Liu, Yong

    2017-01-01

    Tylenchulus semipenetrans is an important and widespread plant-parasitic nematode of citrus worldwide and can cause citrus slow decline disease leading to significant reduction in tree growth and yield. Rapid and accurate detection of T. semipenetrans in soil is important for the disease forecasting and management. In this study, a loop-mediated isothermal amplification (LAMP) assay was developed to detect T. semipenetrans using DNA extracted from soil. A set of five primers was designed from the internal transcribed spacer region (ITS1) of rDNA, and was highly specific to T. semipenetrans. The LAMP reaction was performed at 63°C for 60 min. The LAMP product was visualized directly in one reaction tube by adding SYBR Green I. The detection limit of the LAMP assay was 10−2 J2/0.5 g of soil, which was 10 times more sensitive than conventional PCR (10−1 J2/0.5 g of soil). Examination of 24 field soil samples revealed that the LAMP assay was applicable to a range of soils infested naturally with T. semipenetrans, and the total assay time was less than 2.5 h. These results indicated that the developed LAMP assay is a simple, rapid, sensitive, specific and accurate technique for detection of T. semipenetrans in field soil, and contributes to the effective management of citrus slow decline disease. PMID:28381965

  12. A Real-Time PCR Assay Based on 5.8S rRNA Gene (5.8S rDNA) for Rapid Detection of Candida from Whole Blood Samples.

    Science.gov (United States)

    Guo, Yi; Yang, Jing-Xian; Liang, Guo-Wei

    2016-06-01

    The prevalence of Candida in bloodstream infections (BSIs) has increased. To date, the identification of Candida in BSIs still mainly relies on blood culture and serological tests, but they have various limitations. Therefore, a real-time PCR assay for the detection of Candida from whole blood is presented. The unique primers/probe system was designed on 5.8S rRNA gene (5.8S rDNA) of Candida genus. The analytical sensitivity was determined by numbers of positive PCRs in 12 repetitions. At the concentration of 10(1) CFU/ml blood, positive PCR rates of 100 % were obtained for C. albicans, C. parapsilosis, C. tropicalis, and C. krusei. The detection rate for C. glabrata was 75 % at 10(1) CFU/ml blood. The reaction specificity was 100 % when evaluating the assay using DNA samples from clinical isolates and human blood. The maximum CVs of intra-assay and inter-assay for the detection limit were 1.22 and 2.22 %, respectively. To assess the clinical applicability, 328 blood samples from 82 patients were prospectively tested and real-time PCR results were compared with results from blood culture. Diagnostic sensitivity of the PCR was 100 % using as gold standard blood culture, and specificity was 98.4 %. Our data suggest that the developed assay can be used in clinical laboratories as an accurate and rapid screening test for the Candida from whole blood. Although further evaluation is warranted, our assay holds promise for earlier diagnosis of candidemia.

  13. Recombinase polymerase and enzyme-linked immunosorbent assay as a DNA amplification-detection strategy for food analysis

    Energy Technology Data Exchange (ETDEWEB)

    Santiago-Felipe, S.; Tortajada-Genaro, L.A.; Puchades, R.; Maquieira, A., E-mail: amaquieira@qim.upv.es

    2014-02-06

    Graphical abstract: -- Highlights: •Recombinase polymerase amplification is a powerful DNA method operating at 40 °C. •The combination RPA–ELISA gives excellent performances for high-throughput analysis. •Screening of food safety threats has been done using standard laboratory equipment. •Allergens, GMOs, bacteria, and fungi have been successfully determined. -- Abstract: Polymerase chain reaction in conjunction with enzyme-linked immunosorbent assay (PCR–ELISA) is a well-established technique that provides a suitable rapid, sensitive, and selective method for a broad range of applications. However, the need for precise rapid temperature cycling of PCR is an important drawback that can be overcome by employing isothermal amplification reactions such as recombinase polymerase amplification (RPA). The RPA–ELISA combination is proposed for amplification at a low, constant temperature (40 °C) in a short time (40 min), for the hybridisation of labelled products to specific 5′-biotinylated probes/streptavidin in coated microtiter plates at room temperature, and for detection by colorimetric immunoassay. RPA–ELISA was applied to screen common safety threats in foodstuffs, such as allergens (hazelnut, peanut, soybean, tomato, and maize), genetically modified organisms (P35S and TNOS), pathogenic bacteria (Salmonella sp. and Cronobacter sp.), and fungi (Fusarium sp.). Satisfactory sensitivity and reproducibility results were achieved for all the targets. The RPA–ELISA technique does away with thermocycling and provides a suitable sensitive, specific, and cost-effective method for routine applications, and proves particularly useful for resource-limited settings.

  14. Monitoring of DNA breakage in embryonic stages of the African catfish Clarias gariepinus (Burchell, 1822) after exposure to lead nitrate using alkaline comet assay.

    Science.gov (United States)

    Osman, Alaa G M; Mekkawy, Imam A; Verreth, Johan; Wuertz, Sven; Kloas, Werner; Kirschbaum, Frank

    2008-12-01

    Increasing lead contamination in Egyptian ecosystems and high lead concentrations in food items have raised concern for human health and stimulated studies on monitoring ecotoxicological impact of lead-caused genotoxicity. In this work, the alkaline comet assay was modified for monitoring DNA strand breakage in sensitive early life stages of the African catfish Clarias gariepinus. Following exposure to 100, 300, and 500 microg/L lead nitrate, DNA strand breakage was quantified in embryos at 30, 48, 96, 144, and 168 h post-fertilization (PFS). For quantitative analysis, four commonly used parameters (tail % DNA, %TDNA; head % DNA, %HDNA; tail length, TL; tail moment, TM) were analyzed in 96 nuclei (in triplicates) at each sampling point. The parameter %TDNA revealed highest resolution and lowest variation. A strong correlation between lead concentration, time of exposure, and DNA strand breakage was observed. Here, genotoxicity detected by comet assay preceded the manifested malformations assessed with conventional histology. Qualitative evaluation was carried out using five categories are as follows: undamaged (%TDNA 75%) nuclei confirming a dose and time-dependent shift towards increased frequencies of highly and extremely damaged nuclei. A protective capacity provided by a hardened chorion is a an interesting finding in this study as DNA damage in the prehatching stages 30 h-PFS and 48 h-PFS was low in all treatments (qualitative and quantitative analyses). These results clearly show that the comet assay is a sensitive tool for the detection of genotoxicity in vulnerable early life stages of the African catfish and is a method more sensitive than histological parameters for monitoring genotoxic effects.

  15. Gene polymorphisms against DNA damage induced by hydrogen peroxide in leukocytes of healthy humans through comet assay: a quasi-experimental study

    Directory of Open Access Journals (Sweden)

    Klautau-Guimarães Maria N

    2010-05-01

    Full Text Available Abstract Background Normal cellular metabolism is well established as the source of endogenous reactive oxygen species which account for the background levels of oxidative DNA damage detected in normal tissue. Hydrogen peroxide imposes an oxidative stress condition on cells that can result in DNA damage, leading to mutagenesis and cell death. Several potentially significant genetic variants related to oxidative stress have already been identified, and angiotensin I-converting enzyme (ACE inhibitors have been reported as possible antioxidant agents that can reduce vascular oxidative stress in cardiovascular events. Methods We investigate the influences of haptoglobin, manganese superoxide dismutase (MnSOD Val9Ala, catalase (CAT -21A/T, glutathione peroxidase 1 (GPx-1 Pro198Leu, ACE (I/D and gluthatione S-transferases GSTM1 and GSTT1 gene polymorphisms against DNA damage and oxidative stress. These were induced by exposing leukocytes from peripheral blood of healthy humans (N = 135 to hydrogen peroxide (H2O2, and the effects were tested by comet assay. Blood samples were submitted to genotyping and comet assay (before and after treatment with H2O2 at 250 μM and 1 mM. Results After treatment with H2O2 at 250 μM, the GPx-1 polymorphism significantly influenced results of comet assay and a possible association of the Pro/Leu genotype with higher DNA damage was found. The highest or lowest DNA damage also depended on interaction between GPX-1/ACE and Hp/GSTM1T1 polymorphisms when hydrogen peroxide treatment increased oxidative stress. Conclusions The GPx-1 polymorphism and the interactions between GPX-1/ACE and Hp/GSTM1T1 can be determining factors for DNA oxidation provoked by hydrogen peroxide, and thus for higher susceptibility to or protection against oxidative stress suffered by healthy individuals.

  16. A simple and efficient method for DNA extraction from skin and paraffin-embedded tissues applicable to T-cell clonality assays.

    Science.gov (United States)

    Sidorova, Julia V; Biderman, Bella V; Nikulina, Elena E; Sudarikov, Andrey B

    2012-01-01

    PCR-based clonality assay of rearranged T-cell receptor genes gamma and beta (TCRG and TCRB) in a number of cases could be essential to discriminate between cutaneous T-cell lymphomas and reactive lymphoproliferative lesions in the skin. However, extraction of good-quality DNA from skin specimens (especially formalin-fixed paraffin-embedded) remains a challenge. Common procedures, being labour-intensive and time-consuming and requiring toxic solvents such as phenol and chloroform, still may end up with DNA sample of insufficient quality. We herewith present a simple and efficient method for DNA isolation based on ammonia extraction of tissue, followed by neutralization and simultaneous salting out of proteins with acetic acid. We have analysed 30 samples - 24 fresh (16 skin, two spleen and six lymph node) and six paraffin-embedded. Standard procedure (proteinase K digestion, followed by phenol/chloroform extraction) has been carried out simultaneously. We observed good PCR signal for TCRG rearrangements in 30 samples processed with the new protocol and only in 20 extracted with proteinase K/phenol/chloroform. For TCRB, the success rate was 29 of 30 with the new protocol, compared to 11 of 30 with conventional protocol. The proposed method of DNA extraction should improve the value of T-cell clonality assay, because insufficient DNA quality and quantity may bias analysis towards monoclonality and therefore cause false-positive results.

  17. Development of a nested PCR assay for the detection of Fusarium solani DNA and its evaluation in the diagnosis of invasive fusariosis using an experimental mouse model.

    Science.gov (United States)

    Ahmad, Suhail; Khan, Zia U; Theyyathel, Ajmal M

    2010-01-01

    Fusarium infections are increasingly being encountered in immunocompromised patients. Fusarium solani accounts for nearly half of these infections. A specific nested PCR (nPCR) assay has been developed by using DNA isolated from several Fusarium species and other common fungi. Furthermore, DNA samples isolated from bronchoalveolar lavage (BAL) and serum samples from mice infected intravenously with F. solani conidia and sacrificed on every third day post infection were used for the evaluation of the established nPCR protocol. The lung homogenate, BAL and blood from infected animals were also cultured. The nPCR assay was specific for F. solani and detected 450 fg of DNA corresponding roughly to 11 F. solani cells. Cultures of lung homogenate of infected animals up to day 16 yielded F. solani with decreasing fungal load and were negative thereafter. The nPCR positivity in BAL was 100% concordant with lung tissue culture results. Although detection of F. solani DNA in serum was less sensitive than in BAL, it remained positive for longer duration. Our data from an experimental mouse model show that detection of DNA in BAL and to a lesser extent in serum by nPCR offers a sensitive and specific diagnostic approach to invasive F. solani infection.

  18. Comparison of the artus Epstein-Barr virus (EBV) PCR kit and the Abbott RealTime EBV assay for measuring plasma EBV DNA loads in allogeneic stem cell transplant recipients.

    Science.gov (United States)

    Vinuesa, Víctor; Solano, Carlos; Giménez, Estela; Navarro, David

    2017-02-24

    The ability of the artus Epstein-Barr virus (EBV) PCR kit and the Abbott RealTime EBV PCR assay to detect and quantify plasma EBV DNAemia was compared. The agreement between these assays was 95.8%. The EBV DNA loads measured by the two assays significantly correlated (P=< 0.0001).

  19. Comparison of post-thaw DNA integrity of boar spermatozoa assessed with the neutral comet assay and Sperm-Sus Halomax test kit.

    Science.gov (United States)

    Fraser, L; Parda, A; Filipowicz, K; Strzeżek, J

    2010-10-01

    In this study, we tested the hypothesis whether the neutral Comet assay (NCA) and the Sperm-Sus-Halomax (SSH) test kit could provide similar measurements of post-thaw DNA fragmentation of boar spermatozoa. Whole ejaculates or sperm-rich fractions of boar semen were frozen in an extender containing lactose, lipoprotein fractions isolated from ostrich egg yolk (LPFo), glycerol (lactose-LPFo-G) or in a standard boar semen extender (K3), without the addition of cryoprotective substances. In all boars, both the NCA and SSH test showed similar levels of post-thaw sperm DNA fragmentation in samples of the same ejaculates, regardless of the ejaculate collection procedure and extender. Yet, the levels of post-thaw sperm DNA damage, detected by the NCA and SSH test, were more accentuated in spermatozoa frozen in the absence of cryoprotective substances. Both the NCA and SSH detected variations among individual boars in terms of post-thaw sperm DNA fragmentation. Agreement between the measurements of the NCA and SSH was confirmed by scatter plots of differences, suggesting that the DNA integrity tests could detect the same sperm populations, which were susceptible to cryo-induced DNA damage. The findings of this study indicate that the NCA and the SSH test are effective in detecting similar levels of sperm DNA fragmentation and reinforce their importance in the assessment of frozen-thawed boar semen quality.

  20. An eDNA assay for river otter detection: A tool for surveying a semi-aquatic mammal

    Science.gov (United States)

    Ticha M. Padgett-Stewart; Taylor M. Wilcox; Kellie J. Carim; Kevin S. McKelvey; Michael K. Young; Michael K. Schwartz

    2015-01-01

    Environmental DNA (eDNA) is an effective tool for the detection of elusive or low-density aquatic organisms. However, it has infrequently been applied to mammalian species. North American river otters (Lontra canadensis) are both broad ranging and semi-aquatic, making them an ideal candidate for examining the uses of eDNA for detection of mammals. We developed...

  1. A robotics platform for automated batch fabrication of high density, microfluidics-based DNA microarrays, with applications to single cell, multiplex assays of secreted proteins.

    Science.gov (United States)

    Ahmad, Habib; Sutherland, Alex; Shin, Young Shik; Hwang, Kiwook; Qin, Lidong; Krom, Russell-John; Heath, James R

    2011-09-01

    Microfluidics flow-patterning has been utilized for the construction of chip-scale miniaturized DNA and protein barcode arrays. Such arrays have been used for specific clinical and fundamental investigations in which many proteins are assayed from single cells or other small sample sizes. However, flow-patterned arrays are hand-prepared, and so are impractical for broad applications. We describe an integrated robotics/microfluidics platform for the automated preparation of such arrays, and we apply it to the batch fabrication of up to eighteen chips of flow-patterned DNA barcodes. The resulting substrates are comparable in quality with hand-made arrays and exhibit excellent substrate-to-substrate consistency. We demonstrate the utility and reproducibility of robotics-patterned barcodes by utilizing two flow-patterned chips for highly parallel assays of a panel of secreted proteins from single macrophage cells.

  2. Neospora caninum DNA detection by TaqMan real-time PCR assay in experimentally infected pregnant heifers.

    Science.gov (United States)

    Pereira, Gabriel Ribas; Vogel, Fernanda Silveira Flores; Bohrer, Rodrigo Camponogara; da Nóbrega, Janduí Escarião; Ilha, Gustavo Freitas; da Rosa, Paulo Roberto Antunes; Glanzner, Werner Giehl; Camillo, Giovana; Braunig, Patricia; de Oliveira, João Francisco Coelho; Gonçalves, Paulo Bayard Dias

    2014-01-31

    Neosporosis has been considered the main cause of abortion between the first and the second trimester of pregnancy in cattle. Therefore, the objective of this study was to identify the presence of Neospora caninum DNA obtained from experimental models based on the evaluation of different areas of the fetal nervous system and organs from heifers previously inoculated with NC-1 after or before insemination. This study was performed with Hereford × Nelore (n=29) heifers and all animals were considered free of diseases at the beginning of the experiment. All animals were bred by fixed-time artificial insemination (TAI) and allocated as follows: (a) seronegative heifers subjected to TAI (TAI, n=9), (b) heifers infected with N. caninun 60 days prior to TAI (NC-1+TAI, n=9), and (c) heifers submitted to TAI and infected with N. caninum 60 days later (TAI+NC-1, n=11). The pregnancy was confirmed by transrectal ultrasonography 35 days after TAI and evaluated every 30 days until the end of gestation. Fetuses were collected surgically at 170 days of gestation, and immediately necropsied to remove tissues aseptically. Samples of the central nervous system (CNS), heart, kidney, lung, liver, skeletal muscle and caruncle were collected for DNA extraction. Days of gestation at abortion and interval from abortion to first insemination were examined by Student's t-test. At 35 days of gestation the pregnancy rates in the group NC-1+TAI (4/9, 44.4%) was lower than in the control group (8/9, 88.8%, P<0.05). At 60 days, the pregnancy rates in the NC-1+TAI group (0/4, 0%) was lower compared to TAI+NC-1 (5/7, 71.4%) and control (6/8, 75.0%) groups (P<0.05). Animals from the group NC-1+TAI were re-inseminated 60 days after the first TAI. After pregnancy losses throughout the study, 5 animals (TAI), 3 animals (NC-1+TAI) and 5 animals (TAI+NC-1) maintained pregnancy until 170 days of gestation. TaqMan RT-PCR demonstrated the presence of N. caninum DNA in the medulla and right posterior

  3. Measurement of X-ray-induced DNA double-strand breaks at various stages of the cell cycle using the total fluorescence as a comet assay parameter

    Science.gov (United States)

    Attia, Atef M. M.; Nabil, Ghada M.; Frankenberg, Dieter; Frankenberg-Schwager, M.

    2011-11-01

    The aim of the study was to develop a protocol for both estimating cell cycle position and the level of ionizing radiation-induced DNA dsb using the neutral comet assay. Using DNA histograms, cell cycle positions were determined for human dermal fibroblasts. The tail intensity was used to estimate the level of DNA damage induced by X-rays, at different positions of the cell cycle. The results of tail intensity versus DNA content bivariate analysis of exponentially growing cells showed a remarkable decrease in tail intensity with transition of cells from G1 to S-phase and increases slightly with transition to G2/M phase. This effect is observed at all doses including unirradiated cells, indicating that the effect is not caused by X-rays and the comet assay based on the current tail parameters is not relevant to measure DNA damage at various stages of the cell cycle. The results of dose response curves showed a linear decrease in the comet fluorescence with the X-ray dose. This observation provides a basis for estimating the fraction of damaged DNA, based on the fluorescence decrement induced by ionizing radiation. The results of this new approach showed a linear increase in DNA damage with dose, at various stages of the cell cycle, with rates, which vary in the following order G0>G2/M>S/G1 cells.These results suggest that G0 and G2/M cells are the most sensitive to X-rays among all phases of the cell cycle and suggest synchronization of cells at these phases to increase the cellular radiosensitivity during radiotherapy.

  4. Functional activity of plasmid DNA after entry into the atmosphere of earth investigated by a new biomarker stability assay for ballistic spaceflight experiments.

    Directory of Open Access Journals (Sweden)

    Cora S Thiel

    Full Text Available Sounding rockets represent an excellent platform for testing the influence of space conditions during the passage of Earth's atmosphere and re-entry on biological, physical and chemical experiments for astrobiological purposes. We designed a robust functionality biomarker assay to analyze the biological effects of suborbital spaceflights prevailing during ballistic rocket flights. During the TEXUS-49 rocket mission in March 2011, artificial plasmid DNA carrying a fluorescent marker (enhanced green fluorescent protein: EGFP and an antibiotic resistance cassette (kanamycin/neomycin was attached on different positions of rocket exterior; (i circular every 90 degree on the outer surface concentrical of the payload, (ii in the grooves of screw heads located in between the surface application sites, and (iii on the surface of the bottom side of the payload. Temperature measurements showed two major peaks at 118 and 130 °C during the 780 seconds lasting flight on the inside of the recovery module, while outer gas temperatures of more than 1000 °C were estimated on the sample application locations. Directly after retrieval and return transport of the payload, the plasmid DNA samples were recovered. Subsequent analyses showed that DNA could be recovered from all application sites with a maximum of 53% in the grooves of the screw heads. We could further show that up to 35% of DNA retained its full biological function, i.e., mediating antibiotic resistance in bacteria and fluorescent marker expression in eukaryotic cells. These experiments show that our plasmid DNA biomarker assay is suitable to characterize the environmental conditions affecting DNA during an atmospheric transit and the re-entry and constitute the first report of the stability of DNA during hypervelocity atmospheric transit indicating that sounding rocket flights can be used to model the high-speed atmospheric entry of organics-laden artificial meteorites.

  5. Gelatin-based nanoparticles as DNA delivery systems: Synthesis, physicochemical and biocompatible characterization.

    Science.gov (United States)

    Morán, M C; Rosell, N; Ruano, G; Busquets, M A; Vinardell, M P

    2015-10-01

    The rapidly rising demand for therapeutic grade DNA molecules requires associated improvements in encapsulation and delivery technologies. One of the challenges for the efficient intracellular delivery of therapeutic biomolecules after their cell internalization by endocytosis is to manipulate the non-productive trafficking from endosomes to lysosomes, where degradation may occur. The combination of the endosomal acidity with the endosomolytic capability of the nanocarrier can increase the intracellular delivery of many drugs, genes and proteins, which, therefore, might enhance their therapeutic efficacy. Among the suitable compounds, the gelification properties of gelatin as well as the strong dependence of gelatin ionization with pH makes this compound an interesting candidate to be used to the effective intracellular delivery of active biomacromolecules. In the present work, gelatin (either high or low gel strength) and protamine sulfate has been selected to form particles by interaction of oppositely charged compounds. Particles in the absence of DNA (binary system) and in the presence of DNA (ternary system) have been prepared. The physicochemical characterization (particle size, polydispersity index and degree of DNA entrapment) have been evaluated. Cytotoxicity experiments have shown that the isolated systems and the resulting gelatin-based nanoparticles are essentially non-toxic. The pH-dependent hemolysis assay and the response of the nanoparticles co-incubated in buffers at defined pHs that mimic extracellular, early endosomal and late endo-lysosomal environments demonstrated that the nanoparticles tend to destabilize and DNA can be successfully released. It was found that, in addition to the imposed compositions, the gel strength of gelatin is a controlling parameter of the final properties of these nanoparticles. The results indicate that these gelatin-based nanoparticles have excellent properties as highly potent and non-toxic intracellular delivery

  6. Applicability of the comet assay in evaluation of DNA damage in healthcare providers' working with antineoplastic drugs: a systematic review and meta-analysis.

    Science.gov (United States)

    Zare Sakhvidi, Mohammad Javad; Hajaghazadeh, Mohammad; Mostaghaci, Mehrdad; Mehrparvar, Amir Houshang; Zare Sakhvidi, Fariba; Naghshineh, Elham

    2016-01-01

    Unintended occupational exposure to antineoplastic drugs (ANDs) may occur in medical personnel. Some ANDs are known human carcinogens and exposure can be monitored by genotoxic biomarkers. To evaluate the obstacles to obtaining conclusive results from a comet assay test to determine DNA damage among AND exposed healthcare workers. We systematically reviewed studies that used alkaline comet assay to determine the magnitude and significance of DNA damage among health care workers with potential AND exposure. Fifteen studies were eligible for review and 14 studies were used in the meta-analysis. Under random effect assumption, the estimated standardized mean difference (SMD) in the DNA damage of health care workers was 1.93 (95% CI: 1.15-2.71, p < 0.0001). The resulting SMD was reduced to 1.756 (95% CI: 0.992-2.52, p < 0.0001) when the analysis only included nurses. In subgroup analyses based on gender and smoking, heterogeneity was observed. Only for studies reporting comet moment, I2 test results, as a measure of heterogeneity, dropped to zero. Heterogeneity analysis showed that date of study publication was a possible source of heterogeneity (B = -0.14; p < 0.0001). A mixture of personal parameters, comet assay methodological variables, and exposure characteristics may be responsible for heterogenic data from comet assay studies and interfere with obtaining conclusive results. Lack of quantitative environmental exposure measures and variation in comet assay protocols across studies are important obstacles in generalization of results.

  7. Assessment of DNA damage and its modulation by dietary and genetic factors in smokers using the Comet assay: a biomarker model.

    Science.gov (United States)

    Glei, M; Habermann, N; Osswald, K; Seidel, C; Persin, C; Jahreis, G; Pool-Zobel, B L

    2005-01-01

    Methods are needed to assess exposure to genotoxins in humans and to improve understanding of dietary cancer prevention. The Comet assay was used to detect smoking-related exposures and dietary modulations in target tissues. Buccal scrapings, blood and faeces were collected from 38 healthy male volunteers (smokers and non-smokers) during a dietary intervention study with bread supplemented with prebiotics+/-antioxidants. GSTM1-genotype was determined with PCR. Buccal and peripheral lymphocytes were analysed for DNA damage using the Comet assay. Genotoxicity of faecal water (FW) was assayed in human colon HT29 clone 19A cells. 'Tail intensity' (TI) was used as a quantitative indicator of DNA damage in the Comet assay. Intervention with bread reduced DNA damage in lymphocytes of smokers (8.3+/-1.7% TI versus 10.2+/-4.1% TI, n=19), but not of non-smokers (8.6+/-2.8% TI versus 8.3+/-2.7% TI, n=15). Faecal water genotoxicity was reduced only in non-smokers (9.4+/-2.9% TI versus 18.9+/-13.1% TI, n=15) but not in smokers (15.5+/-10.7% TI versus 20.4+/-14.1% TI, n=13). The Comet assay was efficient in the detection of both smoking-related exposure (buccal cells) and efficacy of dietary intervention (faecal samples). Smokers and non-smokers profited differently from the intervention with prebiotic bread+/-antioxidants. Stratification of data by genotype enhanced specificity/sensitivity of the intervention effects and contributed important information on the role of susceptibility.

  8. The complement system of the goat: Haemolytic assays and isolation of major proteins

    Directory of Open Access Journals (Sweden)

    Moreno-Indias Isabel

    2012-06-01

    Full Text Available Abstract Background The aim of the present study was to develop a haemolytic assay for the study of the complement system in dairy goats (Capra aegagrus hircus and to characterize the major goat complement system proteins. Results The commonly used sheep erythrocyte sensitized with rabbit antibodies were not sensitive to lysis by goat serum, but the combination of human red blood cells (RBC plus rabbit antibodies was the best option found for goat complement assay. A buffer based on HEPES instead of the classical veronal (barbitone was developed. Three proteins were isolated: factor H, C1q and C3 and these were compared with the corresponding human proteins. A novel affinity chromatography technique was developed for isolation of factor H. Conclusions Human RBC plus rabbit antibodies were a suitable option for haemolytic assays. The isolated proteins are similar to the human counterparts.

  9. DNA-enabled integrated molecular systems for computation and sensing.

    Science.gov (United States)

    LaBoda, Craig; Duschl, Heather; Dwyer, Chris L

    2014-06-17

    CONSPECTUS: Nucleic acids have become powerful building blocks for creating supramolecular nanostructures with a variety of new and interesting behaviors. The predictable and guided folding of DNA, inspired by nature, allows designs to manipulate molecular-scale processes unlike any other material system. Thus, DNA can be co-opted for engineered and purposeful ends. This Account details a small portion of what can be engineered using DNA within the context of computer architectures and systems. Over a decade of work at the intersection of DNA nanotechnology and computer system design has shown several key elements and properties of how to harness the massive parallelism created by DNA self-assembly. This work is presented, naturally, from the bottom-up beginning with early work on strand sequence design for deterministic, finite DNA nanostructure synthesis. The key features of DNA nanostructures are explored, including how the use of small DNA motifs assembled in a hierarchical manner enables full-addressability of the final nanostructure, an important property for building dense and complicated systems. A full computer system also requires devices that are compatible with DNA self-assembly and cooperate at a higher level as circuits patterned over many, many replicated units. Described here is some work in this area investigating nanowire and nanoparticle devices, as well as chromophore-based circuits called resonance energy transfer (RET) logic. The former is an example of a new way to bring traditional silicon transistor technology to the nanoscale, which is increasingly problematic with current fabrication methods. RET logic, on the other hand, introduces a framework for optical computing at the molecular level. This Account also highlights several architectural system studies that demonstrate that even with low-level devices that are inferior to their silicon counterparts and a substrate that harbors abundant defects, self-assembled systems can still

  10. Improved Activity Assay Method for Arginine Kinase Based on a Ternary Heteropolyacid System

    Institute of Scientific and Technical Information of China (English)

    陈宝玉; 郭勤; 郭智; 王希成

    2003-01-01

    This paper presents a new system for the activity assay of arginine kinase (AK), based on the spectrophotometric determination of an ascorbic acid-reduced blue ternary heteropolyacid composed of bismuth, molybdate and the released phosphate from N-phospho-L-arginine (PArg) formed in the forward catalysis reaction.The assay conditions, including the formulation of the phosphate determination reagent (PDR), the assay timing, and the linear activity range of the enzyme concentration, have been tested and optimized.For these conditions, the ternary heteropolyacid color is completely developed within 1 min and is stable for at least 15 min, with an absorbance maximum at 700 nm and a molar extinction coefficient of 15.97 (mmol/L)-1 · cm-1 for the phosphate.Standard curves for phosphate show a good linearity of 0.999.Compared with previous activity assay methods for AK, this system exhibits superior sensitivity, reproducibility, and adaptability to various conditions in enzymological studies.This method also reduces the assay time and avoids the use of some expensive instruments and reagents.

  11. Development of a PCR Assay to Detect Low Level Trypanosoma cruzi in Blood Specimens Collected with PAXgene Blood DNA Tubes for Clinical Trials Treating Chagas Disease.

    Science.gov (United States)

    Wei, Bo; Chen, Lei; Kibukawa, Miho; Kang, John; Waskin, Hetty; Marton, Matthew

    2016-12-01

    Chagas disease is caused by the parasitic infection of Trypanosoma cruzi (T. cruzi). The STOP CHAGAS clinical trial was initiated in 2011 to evaluate posaconazole in treating Chagas disease, with treatment success defined as negative qualitative PCR results of detecting the parasites in blood specimens collected post-treatment. PAXgene Blood DNA tubes were utilized as a simple procedure to collect and process blood specimens. However, the PAXgene blood specimens challenged published T. cruzi PCR methods, resulting in poor sensitivity and reproducibility. To accurately evaluate the treatment efficacy of the clinical study, we developed and validated a robust PCR assay for detecting low level T. cruzi in PAXgene blood specimens. The assay combines a new DNA extraction method with a custom designed qPCR assay, resulting in limit of detection of 0.005 and 0.01 fg/μl for K98 and CL Brener, two representative strains of two of T. cruzi's discrete typing units. Reliable qPCR standard curves were established for both strains to measure parasite loads, with amplification efficiency ≥ 90% and the lower limit of linearity ≥ 0.05 fg/μl. The assay successfully analyzed the samples collected from the STOP CHAGAS study and may prove useful for future global clinical trials evaluating new therapies for asymptomatic chronic Chagas disease.

  12. Development of a PCR Assay to Detect Low Level Trypanosoma cruzi in Blood Specimens Collected with PAXgene Blood DNA Tubes for Clinical Trials Treating Chagas Disease

    Science.gov (United States)

    Wei, Bo; Chen, Lei; Kibukawa, Miho; Kang, John; Waskin, Hetty; Marton, Matthew

    2016-01-01

    Chagas disease is caused by the parasitic infection of Trypanosoma cruzi (T. cruzi). The STOP CHAGAS clinical trial was initiated in 2011 to evaluate posaconazole in treating Chagas disease, with treatment success defined as negative qualitative PCR results of detecting the parasites in blood specimens collected post-treatment. PAXgene Blood DNA tubes were utilized as a simple procedure to collect and process blood specimens. However, the PAXgene blood specimens challenged published T. cruzi PCR methods, resulting in poor sensitivity and reproducibility. To accurately evaluate the treatment efficacy of the clinical study, we developed and validated a robust PCR assay for detecting low level T. cruzi in PAXgene blood specimens. The assay combines a new DNA extraction method with a custom designed qPCR assay, resulting in limit of detection of 0.005 and 0.01 fg/μl for K98 and CL Brener, two representative strains of two of T. cruzi’s discrete typing units. Reliable qPCR standard curves were established for both strains to measure parasite loads, with amplification efficiency ≥ 90% and the lower limit of linearity ≥ 0.05 fg/μl. The assay successfully analyzed the samples collected from the STOP CHAGAS study and may prove useful for future global clinical trials evaluating new therapies for asymptomatic chronic Chagas disease. PMID:27906977

  13. Concordance study between the ParaDNA® Intelligence Test, a rapid DNA profiling assay, and a conventional STR typing kit (AmpFlSTR® SGM Plus®).

    Science.gov (United States)

    Ball, G; Dawnay, N; Stafford-Allen, B; Panasiuk, M; Rendell, P; Blackman, S; Duxbury, N; Wells, S

    2015-05-01

    The ParaDNA® Intelligence Test enables STR profiling directly from human biological samples and evidence items collected from crime scene in 75min. Designed for non-expert use this system allows DNA information to be available to investigators before it would typically be available from a laboratory. The ParaDNA Intelligence Test system amplifies D3S1358, D8S119, D16S539, D18S1358 and TH01 STR loci and the gender typing locus amelogenin and detects the alleles present with HyBeacon® probes. Individual DNA samples from 381 UK Caucasian individuals were analysed using AmpFlSTR® SGM Plus® and the ParaDNA Intelligence Test with the derived STR profiles compared. Here we describe the high level of concordance demonstrated between the two systems and discuss this with reference to allele frequencies and the discriminatory power offered by the ParaDNA Intelligence Test.

  14. A laboratory information management system for DNA barcoding workflows.

    Science.gov (United States)

    Vu, Thuy Duong; Eberhardt, Ursula; Szöke, Szániszló; Groenewald, Marizeth; Robert, Vincent

    2012-07-01

    This paper presents a laboratory information management system for DNA sequences (LIMS) created and based on the needs of a DNA barcoding project at the CBS-KNAW Fungal Biodiversity Centre (Utrecht, the Netherlands). DNA barcoding is a global initiative for species identification through simple DNA sequence markers. We aim at generating barcode data for all strains (or specimens) included in the collection (currently ca. 80 k). The LIMS has been developed to better manage large amounts of sequence data and to keep track of the whole experimental procedure. The system has allowed us to classify strains more efficiently as the quality of sequence data has improved, and as a result, up-to-date taxonomic names have been given to strains and more accurate correlation analyses have been carried out.

  15. In vivo assessment of DNA damage and protective effects of extracts from Miconia species using the comet assay and micronucleus test.

    Science.gov (United States)

    Serpeloni, Juliana Mara; Bisarro dos Reis, Mariana; Rodrigues, Juliana; Campaner dos Santos, Lourdes; Vilegas, Wagner; Varanda, Eliana A; Dokkedal, Anne L; Cólus, Ilce Mara S

    2008-11-01

    The genus Miconia comprises approximately 1000 species belonging to the Melastomataceae family. Several crude plant extracts from Miconia and their isolated compounds have shown biological activities, such as analgesic and anti-neoplastic action; however, no studies concerning their effects on DNA are available. The present study aimed to evaluate, in vivo, the genotoxic and mutagenic effects of four species of plants from Miconia genus using the comet assay and micronucleus test. Their possible protective effects were also evaluated in experiments associating the plant extracts with cyclophosphamide (CPA). The methanolic extracts of Miconia albicans, Miconia cabucu, Miconia rubiginosa, Miconia stenostachya and the chloroformic extract of M. albicans were investigated. For genotoxic and mutagenic evaluations, three concentrations were tested, 200, 400 and 540 mg/kg body weight (bw), based on the solubility limit of the extract in distilled water. For the protective effects, only the highest dose was evaluated against 40 mg/kg bw of CPA. Blood was removed from mice tails pre- (T0) and post-treatment (T1-30 h) for the micronucleus test and 24 h post-treatment for the comet assay. The Student's t-test was used to compare data obtained at T0 and T1, the analysis of variance-Tukey test was used to compare between groups in the micronucleus test and the Kruskal-Wallis and Dunn's test were used to compare different groups in the comet assay. All the extracts induced alterations in DNA migration (comet assay); however, no mutagenic effect was observed in the micronucleus assay. All extracts showed a protective effect against CPA in both assays. Our study showed that the use of crude extracts could be more advantageous than the use of isolated compounds. The interaction between phytochemicals in the extracts showed efficacy in reducing mutagenicity and improving the protective effects.

  16. An investigation of the potential effect of sperm nuclear vacuoles in human spermatozoa on DNA fragmentation using a neutral and alkaline Comet assay.

    Science.gov (United States)

    Pastuszek, E; Kiewisz, J; Skowronska, P; Liss, J; Lukaszuk, M; Bruszczynska, A; Jakiel, G; Lukaszuk, K

    2017-03-01

    Presence of vacuoles and degree of sperm DNA damage are considered to be the basic factors used for the assessment of sperm fertilization capacity. We aimed to investigate the link between these two parameters. According to our knowledge, this is the first study where the Comet assay was used to assess the degree of DNA fragmentation of sperm categorized by Motile Sperm Organelle Morphology Examination (MSOME) Grades. Semen samples from 10 patients were assessed. Spermatozoa were graded into four MSOME groups according to the Vanderzwalmen's criteria. A total of 3930 motile spermatozoa were selected one-by-one using an inverted microscope and transferred onto two different slides. The degree of DNA fragmentation was analyzed by alkaline and neutral Comet assay. Results of the neutral Comet assay showed that Grade I spermatozoa (absence of vacuoles) presented significantly lower dsDNA fragmentation level (mean: 3.13 ± 1.17%) than Grade II (maximum of two small vacuoles; mean: 10.34 ± 2.65%), Grade III (more than two small vacuoles or at least one large vacuole; mean: 23.88 ± 8.37%), and Grade IV (large vacuoles associated with abnormal head shapes or other abnormalities; mean: 36.94 ± 7.78%; p fragmentation level (mean: 8.33 ± 3.62%) than Grade III (mean: 25.64 ± 9.15%) and Grade IV (mean: 40.10 ± 9.10%, p  0.05). Probably, the vacuoles may be responsible for double strand DNA breaks rather than single strand DNA breaks (only 2.39% spermatozoa in MSOME Grade II, 1.76% in III, and 3.16% in IV has single strand breaks). The results demonstrate that lower MSOME grading correlates with lower sperm DNA fragmentation. Therefore, the observation of sperm nuclear vacuoles using real-time optical microscopy without precise DNA fragmentation examination is not sufficient for optimal sperm selection for intracytoplasmic sperm injection. © 2017 American Society of Andrology and European Academy of Andrology.

  17. Evaluation of radio-induced DNA damage and their repair in human lymphocytes by comet assay or single cell gel electrophoresis; Avaliacao do dano radioinduzido no DNA e reparo em linfocitos humanos pelo metodo do cometa (single cell gel electrophoresis)