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Sample records for dna quantification methods

  1. Comparison of five DNA quantification methods

    DEFF Research Database (Denmark)

    Nielsen, Karsten; Mogensen, Helle Smidt; Hedman, Johannes

    2008-01-01

    Six commercial preparations of human genomic DNA were quantified using five quantification methods: UV spectrometry, SYBR-Green dye staining, slot blot hybridization with the probe D17Z1, Quantifiler Human DNA Quantification kit and RB1 rt-PCR. All methods measured higher DNA concentrations than...... Quantification kit in two experiments. The measured DNA concentrations with Quantifiler were 125 and 160% higher than expected based on the manufacturers' information. When the Quantifiler human DNA standard (Raji cell line) was replaced by the commercial human DNA preparation G147A (Promega) to generate the DNA...... standard curve in the Quantifiler Human DNA Quantification kit, the DNA quantification results of the human DNA preparations were 31% higher than expected based on the manufacturers' information. The results indicate a calibration problem with the Quantifiler human DNA standard for its use...

  2. Comparison of DNA Quantification Methods for Next Generation Sequencing.

    Science.gov (United States)

    Robin, Jérôme D; Ludlow, Andrew T; LaRanger, Ryan; Wright, Woodring E; Shay, Jerry W

    2016-04-06

    Next Generation Sequencing (NGS) is a powerful tool that depends on loading a precise amount of DNA onto a flowcell. NGS strategies have expanded our ability to investigate genomic phenomena by referencing mutations in cancer and diseases through large-scale genotyping, developing methods to map rare chromatin interactions (4C; 5C and Hi-C) and identifying chromatin features associated with regulatory elements (ChIP-seq, Bis-Seq, ChiA-PET). While many methods are available for DNA library quantification, there is no unambiguous gold standard. Most techniques use PCR to amplify DNA libraries to obtain sufficient quantities for optical density measurement. However, increased PCR cycles can distort the library's heterogeneity and prevent the detection of rare variants. In this analysis, we compared new digital PCR technologies (droplet digital PCR; ddPCR, ddPCR-Tail) with standard methods for the titration of NGS libraries. DdPCR-Tail is comparable to qPCR and fluorometry (QuBit) and allows sensitive quantification by analysis of barcode repartition after sequencing of multiplexed samples. This study provides a direct comparison between quantification methods throughout a complete sequencing experiment and provides the impetus to use ddPCR-based quantification for improvement of NGS quality.

  3. Comparison of Suitability of the Most Common Ancient DNA Quantification Methods.

    Science.gov (United States)

    Brzobohatá, Kristýna; Drozdová, Eva; Smutný, Jiří; Zeman, Tomáš; Beňuš, Radoslav

    2017-04-01

    Ancient DNA (aDNA) extracted from historical bones is damaged and fragmented into short segments, present in low quantity, and usually copurified with microbial DNA. A wide range of DNA quantification methods are available. The aim of this study was to compare the five most common DNA quantification methods for aDNA. Quantification methods were tested on DNA extracted from skeletal material originating from an early medieval burial site. The tested methods included ultraviolet (UV) absorbance, real-time quantitative polymerase chain reaction (qPCR) based on SYBR ® green detection, real-time qPCR based on a forensic kit, quantification via fluorescent dyes bonded to DNA, and fragmentary analysis. Differences between groups were tested using a paired t-test. Methods that measure total DNA present in the sample (NanoDrop ™ UV spectrophotometer and Qubit ® fluorometer) showed the highest concentrations. Methods based on real-time qPCR underestimated the quantity of aDNA. The most accurate method of aDNA quantification was fragmentary analysis, which also allows DNA quantification of the desired length and is not affected by PCR inhibitors. Methods based on the quantification of the total amount of DNA in samples are unsuitable for ancient samples as they overestimate the amount of DNA presumably due to the presence of microbial DNA. Real-time qPCR methods give undervalued results due to DNA damage and the presence of PCR inhibitors. DNA quantification methods based on fragment analysis show not only the quantity of DNA but also fragment length.

  4. Quantification of total phosphorothioate in bacterial DNA by a bromoimane-based fluorescent method.

    Science.gov (United States)

    Xiao, Lu; Xiang, Yu

    2016-06-01

    The discovery of phosphorothioate (PT) modifications in bacterial DNA has challenged our understanding of conserved phosphodiester backbone structure of cellular DNA. This exclusive DNA modification in bacteria is not found in animal cells yet, and its biological function in bacteria is still poorly understood. Quantitative information about the bacterial PT modifications is thus important for the investigation of their possible biological functions. In this study, we have developed a simple fluorescence method for selective quantification of total PTs in bacterial DNA, based on fluorescent labeling of PTs and subsequent release of the labeled fluorophores for absolute quantification. The method was highly selective to PTs and not interfered by the presence of reactive small molecules or proteins. The quantification of PTs in an E. coli DNA sample was successfully achieved using our method and gave a result of about 455 PTs per million DNA nucleotides, while almost no detectable PTs were found in a mammalian calf thymus DNA. With this new method, the content of phosphorothioate in bacterial DNA could be successfully quantified, serving as a simple method suitable for routine use in biological phosphorothioate related studies. Copyright © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. Critical points of DNA quantification by real-time PCR – effects of DNA extraction method and sample matrix on quantification of genetically modified organisms

    Directory of Open Access Journals (Sweden)

    Žel Jana

    2006-08-01

    Full Text Available Abstract Background Real-time PCR is the technique of choice for nucleic acid quantification. In the field of detection of genetically modified organisms (GMOs quantification of biotech products may be required to fulfil legislative requirements. However, successful quantification depends crucially on the quality of the sample DNA analyzed. Methods for GMO detection are generally validated on certified reference materials that are in the form of powdered grain material, while detection in routine laboratories must be performed on a wide variety of sample matrixes. Due to food processing, the DNA in sample matrixes can be present in low amounts and also degraded. In addition, molecules of plant origin or from other sources that affect PCR amplification of samples will influence the reliability of the quantification. Further, the wide variety of sample matrixes presents a challenge for detection laboratories. The extraction method must ensure high yield and quality of the DNA obtained and must be carefully selected, since even components of DNA extraction solutions can influence PCR reactions. GMO quantification is based on a standard curve, therefore similarity of PCR efficiency for the sample and standard reference material is a prerequisite for exact quantification. Little information on the performance of real-time PCR on samples of different matrixes is available. Results Five commonly used DNA extraction techniques were compared and their suitability for quantitative analysis was assessed. The effect of sample matrix on nucleic acid quantification was assessed by comparing 4 maize and 4 soybean matrixes. In addition 205 maize and soybean samples from routine analysis were analyzed for PCR efficiency to assess variability of PCR performance within each sample matrix. Together with the amount of DNA needed for reliable quantification, PCR efficiency is the crucial parameter determining the reliability of quantitative results, therefore it was

  6. Critical points of DNA quantification by real-time PCR--effects of DNA extraction method and sample matrix on quantification of genetically modified organisms.

    Science.gov (United States)

    Cankar, Katarina; Stebih, Dejan; Dreo, Tanja; Zel, Jana; Gruden, Kristina

    2006-08-14

    Real-time PCR is the technique of choice for nucleic acid quantification. In the field of detection of genetically modified organisms (GMOs) quantification of biotech products may be required to fulfil legislative requirements. However, successful quantification depends crucially on the quality of the sample DNA analyzed. Methods for GMO detection are generally validated on certified reference materials that are in the form of powdered grain material, while detection in routine laboratories must be performed on a wide variety of sample matrixes. Due to food processing, the DNA in sample matrixes can be present in low amounts and also degraded. In addition, molecules of plant origin or from other sources that affect PCR amplification of samples will influence the reliability of the quantification. Further, the wide variety of sample matrixes presents a challenge for detection laboratories. The extraction method must ensure high yield and quality of the DNA obtained and must be carefully selected, since even components of DNA extraction solutions can influence PCR reactions. GMO quantification is based on a standard curve, therefore similarity of PCR efficiency for the sample and standard reference material is a prerequisite for exact quantification. Little information on the performance of real-time PCR on samples of different matrixes is available. Five commonly used DNA extraction techniques were compared and their suitability for quantitative analysis was assessed. The effect of sample matrix on nucleic acid quantification was assessed by comparing 4 maize and 4 soybean matrixes. In addition 205 maize and soybean samples from routine analysis were analyzed for PCR efficiency to assess variability of PCR performance within each sample matrix. Together with the amount of DNA needed for reliable quantification, PCR efficiency is the crucial parameter determining the reliability of quantitative results, therefore it was chosen as the primary criterion by which to

  7. Critical points of DNA quantification by real-time PCR – effects of DNA extraction method and sample matrix on quantification of genetically modified organisms

    Science.gov (United States)

    Cankar, Katarina; Štebih, Dejan; Dreo, Tanja; Žel, Jana; Gruden, Kristina

    2006-01-01

    Background Real-time PCR is the technique of choice for nucleic acid quantification. In the field of detection of genetically modified organisms (GMOs) quantification of biotech products may be required to fulfil legislative requirements. However, successful quantification depends crucially on the quality of the sample DNA analyzed. Methods for GMO detection are generally validated on certified reference materials that are in the form of powdered grain material, while detection in routine laboratories must be performed on a wide variety of sample matrixes. Due to food processing, the DNA in sample matrixes can be present in low amounts and also degraded. In addition, molecules of plant origin or from other sources that affect PCR amplification of samples will influence the reliability of the quantification. Further, the wide variety of sample matrixes presents a challenge for detection laboratories. The extraction method must ensure high yield and quality of the DNA obtained and must be carefully selected, since even components of DNA extraction solutions can influence PCR reactions. GMO quantification is based on a standard curve, therefore similarity of PCR efficiency for the sample and standard reference material is a prerequisite for exact quantification. Little information on the performance of real-time PCR on samples of different matrixes is available. Results Five commonly used DNA extraction techniques were compared and their suitability for quantitative analysis was assessed. The effect of sample matrix on nucleic acid quantification was assessed by comparing 4 maize and 4 soybean matrixes. In addition 205 maize and soybean samples from routine analysis were analyzed for PCR efficiency to assess variability of PCR performance within each sample matrix. Together with the amount of DNA needed for reliable quantification, PCR efficiency is the crucial parameter determining the reliability of quantitative results, therefore it was chosen as the primary

  8. Comparison of methods for quantification of global DNA methylation in human cells and tissues.

    Directory of Open Access Journals (Sweden)

    Sofia Lisanti

    Full Text Available DNA methylation is a key epigenetic modification which, in mammals, occurs mainly at CpG dinucleotides. Most of the CpG methylation in the genome is found in repetitive regions, rich in dormant transposons and endogenous retroviruses. Global DNA hypomethylation, which is a common feature of several conditions such as ageing and cancer, can cause the undesirable activation of dormant repeat elements and lead to altered expression of associated genes. DNA hypomethylation can cause genomic instability and may contribute to mutations and chromosomal recombinations. Various approaches for quantification of global DNA methylation are widely used. Several of these approaches measure a surrogate for total genomic methyl cytosine and there is uncertainty about the comparability of these methods. Here we have applied 3 different approaches (luminometric methylation assay, pyrosequencing of the methylation status of the Alu repeat element and of the LINE1 repeat element for estimating global DNA methylation in the same human cell and tissue samples and have compared these estimates with the "gold standard" of methyl cytosine quantification by HPLC. Next to HPLC, the LINE1 approach shows the smallest variation between samples, followed by Alu. Pearson correlations and Bland-Altman analyses confirmed that global DNA methylation estimates obtained via the LINE1 approach corresponded best with HPLC-based measurements. Although, we did not find compelling evidence that the gold standard measurement by HPLC could be substituted with confidence by any of the surrogate assays for detecting global DNA methylation investigated here, the LINE1 assay seems likely to be an acceptable surrogate in many cases.

  9. Multiplex cDNA quantification method that facilitates the standardization of gene expression data

    Science.gov (United States)

    Gotoh, Osamu; Murakami, Yasufumi; Suyama, Akira

    2011-01-01

    Microarray-based gene expression measurement is one of the major methods for transcriptome analysis. However, current microarray data are substantially affected by microarray platforms and RNA references because of the microarray method can provide merely the relative amounts of gene expression levels. Therefore, valid comparisons of the microarray data require standardized platforms, internal and/or external controls and complicated normalizations. These requirements impose limitations on the extensive comparison of gene expression data. Here, we report an effective approach to removing the unfavorable limitations by measuring the absolute amounts of gene expression levels on common DNA microarrays. We have developed a multiplex cDNA quantification method called GEP-DEAN (Gene expression profiling by DCN-encoding-based analysis). The method was validated by using chemically synthesized DNA strands of known quantities and cDNA samples prepared from mouse liver, demonstrating that the absolute amounts of cDNA strands were successfully measured with a sensitivity of 18 zmol in a highly multiplexed manner in 7 h. PMID:21415008

  10. Radiation dose determines the method for quantification of DNA double strand breaks

    International Nuclear Information System (INIS)

    Bulat, Tanja; Keta, Olitija; Korićanac, Lela; Žakula, Jelena; Petrović, Ivan; Ristić-Fira, Aleksandra; Todorović, Danijela

    2016-01-01

    Ionizing radiation induces DNA double strand breaks (DSBs) that trigger phosphorylation of the histone protein H2AX (γH2AX). Immunofluorescent staining visualizes formation of γH2AX foci, allowing their quantification. This method, as opposed to Western blot assay and Flow cytometry, provides more accurate analysis, by showing exact position and intensity of fluorescent signal in each single cell. In practice there are problems in quantification of γH2AX. This paper is based on two issues: the determination of which technique should be applied concerning the radiation dose, and how to analyze fluorescent microscopy images obtained by different microscopes. HTB140 melanoma cells were exposed to γ-rays, in the dose range from 1 to 16 Gy. Radiation effects on the DNA level were analyzed at different time intervals after irradiation by Western blot analysis and immunofluorescence microscopy. Immunochemically stained cells were visualized with two types of microscopes: AxioVision (Zeiss, Germany) microscope, comprising an ApoTome software, and AxioImagerA1 microscope (Zeiss, Germany). Obtained results show that the level of γH2AX is time and dose dependent. Immunofluorescence microscopy provided better detection of DSBs for lower irradiation doses, while Western blot analysis was more reliable for higher irradiation doses. AxioVision microscope containing ApoTome software was more suitable for the detection of γH2AX foci. (author)

  11. Radiation dose determines the method for quantification of DNA double strand breaks

    Energy Technology Data Exchange (ETDEWEB)

    Bulat, Tanja; Keta, Olitija; Korićanac, Lela; Žakula, Jelena; Petrović, Ivan; Ristić-Fira, Aleksandra [University of Belgrade, Vinča Institute of Nuclear Sciences, Belgrade (Serbia); Todorović, Danijela, E-mail: dtodorovic@medf.kg.ac.rs [University of Kragujevac, Faculty of Medical Sciences, Kragujevac (Serbia)

    2016-03-15

    Ionizing radiation induces DNA double strand breaks (DSBs) that trigger phosphorylation of the histone protein H2AX (γH2AX). Immunofluorescent staining visualizes formation of γH2AX foci, allowing their quantification. This method, as opposed to Western blot assay and Flow cytometry, provides more accurate analysis, by showing exact position and intensity of fluorescent signal in each single cell. In practice there are problems in quantification of γH2AX. This paper is based on two issues: the determination of which technique should be applied concerning the radiation dose, and how to analyze fluorescent microscopy images obtained by different microscopes. HTB140 melanoma cells were exposed to γ-rays, in the dose range from 1 to 16 Gy. Radiation effects on the DNA level were analyzed at different time intervals after irradiation by Western blot analysis and immunofluorescence microscopy. Immunochemically stained cells were visualized with two types of microscopes: AxioVision (Zeiss, Germany) microscope, comprising an ApoTome software, and AxioImagerA1 microscope (Zeiss, Germany). Obtained results show that the level of γH2AX is time and dose dependent. Immunofluorescence microscopy provided better detection of DSBs for lower irradiation doses, while Western blot analysis was more reliable for higher irradiation doses. AxioVision microscope containing ApoTome software was more suitable for the detection of γH2AX foci. (author)

  12. Quantification of viral DNA during HIV-1 infection: A review of relevant clinical uses and laboratory methods.

    Science.gov (United States)

    Alidjinou, E K; Bocket, L; Hober, D

    2015-02-01

    Effective antiretroviral therapy usually leads to undetectable HIV-1 RNA in the plasma. However, the virus persists in some cells of infected patients as various DNA forms, both integrated and unintegrated. This reservoir represents the greatest challenge to the complete cure of HIV-1 infection and its characteristics highly impact the course of the disease. The quantification of HIV-1 DNA in blood samples constitutes currently the most practical approach to measure this residual infection. Real-time quantitative PCR (qPCR) is the most common method used for HIV-DNA quantification and many strategies have been developed to measure the different forms of HIV-1 DNA. In the literature, several "in-house" PCR methods have been used and there is a need for standardization to have comparable results. In addition, qPCR is limited for the precise quantification of low levels by background noise. Among new assays in development, digital PCR was shown to allow an accurate quantification of HIV-1 DNA. Total HIV-1 DNA is most commonly measured in clinical routine. The absolute quantification of proviruses and unintegrated forms is more often used for research purposes. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

  13. A direct qPCR method for residual DNA quantification in monoclonal antibody drugs produced in CHO cells.

    Science.gov (United States)

    Hussain, Musaddeq

    2015-11-10

    Chinese hamster ovary (CHO) cells are the host cell of choice for manufacturing of monoclonal antibody (mAb) drugs in the biopharmaceutical industry. Host cell DNA is an impurity of such manufacturing process and must be controlled and monitored in order to ensure drug purity and safety. A conventional method for quantification of host residual DNA in drug requires extraction of DNA from the mAb drug substance with subsequent quantification of the extracted DNA using real-time PCR (qPCR). Here we report a method where the DNA extraction step is eliminated prior to qPCR. In this method, which we have named 'direct resDNA qPCR', the mAb drug substance is digested with a protease called KAPA in a 96-well PCR plate, the protease in the digest is then denatured at high temperature, qPCR reagents are added to the resultant reaction wells in the plate along with standards and controls in other wells of the same plate, and the plate subjected to qPCR for analysis of residual host DNA in the samples. This direct resDNA qPCR method for CHO is sensitive to 5.0fg of DNA with high precision and accuracy and has a wide linear range of determination. The method has been successfully tested with four mAbs drug, two IgG1 and two IgG4. Both the purified drug substance as well as a number of process intermediate samples, e.g., bioreactor harvest, Protein A column eluate and ion-exchange column eluates were tested. This method simplifies the residual DNA quantification protocol, reduces time of analysis and leads to increased assay sensitivity and development of automated high-throughput methods. Copyright © 2015 Elsevier B.V. All rights reserved.

  14. Accurate and precise DNA quantification in the presence of different amplification efficiencies using an improved Cy0 method.

    Science.gov (United States)

    Guescini, Michele; Sisti, Davide; Rocchi, Marco B L; Panebianco, Renato; Tibollo, Pasquale; Stocchi, Vilberto

    2013-01-01

    Quantitative real-time PCR represents a highly sensitive and powerful technology for the quantification of DNA. Although real-time PCR is well accepted as the gold standard in nucleic acid quantification, there is a largely unexplored area of experimental conditions that limit the application of the Ct method. As an alternative, our research team has recently proposed the Cy0 method, which can compensate for small amplification variations among the samples being compared. However, when there is a marked decrease in amplification efficiency, the Cy0 is impaired, hence determining reaction efficiency is essential to achieve a reliable quantification. The proposed improvement in Cy0 is based on the use of the kinetic parameters calculated in the curve inflection point to compensate for efficiency variations. Three experimental models were used: inhibition of primer extension, non-optimal primer annealing and a very small biological sample. In all these models, the improved Cy0 method increased quantification accuracy up to about 500% without affecting precision. Furthermore, the stability of this procedure was enhanced integrating it with the SOD method. In short, the improved Cy0 method represents a simple yet powerful approach for reliable DNA quantification even in the presence of marked efficiency variations.

  15. Formic acid hydrolysis/liquid chromatography isotope dilution mass spectrometry: An accurate method for large DNA quantification.

    Science.gov (United States)

    Shibayama, Sachie; Fujii, Shin-Ichiro; Inagaki, Kazumi; Yamazaki, Taichi; Takatsu, Akiko

    2016-10-14

    Liquid chromatography-isotope dilution mass spectrometry (LC-IDMS) with formic acid hydrolysis was established for the accurate quantification of λDNA. The over-decomposition of nucleobases in formic acid hydrolysis was restricted by optimizing the reaction temperature and the reaction time, and accurately corrected by using deoxynucleotides (dNMPs) and isotope-labeled dNMPs as the calibrator and the internal standard, respectively. The present method could quantify λDNA with an expanded uncertainty of 4.6% using 10fmol of λDNA. The analytical results obtained with the present method were validated by comparing with the results of phosphate-base quantification by inductively coupled plasma-mass spectrometry (ICP-MS). The results showed good agreement with each other. We conclude that the formic acid hydrolysis/LC-IDMS method can quantify λDNA accurately and is promising as the primary method for the certification of DNA as reference material. Copyright © 2016 Elsevier B.V. All rights reserved.

  16. Improved Methods of Carnivore Faecal Sample Preservation, DNA Extraction and Quantification for Accurate Genotyping of Wild Tigers

    Science.gov (United States)

    Harika, Katakam; Mahla, Ranjeet Singh; Shivaji, Sisinthy

    2012-01-01

    Background Non-invasively collected samples allow a variety of genetic studies on endangered and elusive species. However due to low amplification success and high genotyping error rates fewer samples can be identified up to the individual level. Number of PCRs needed to obtain reliable genotypes also noticeably increase. Methods We developed a quantitative PCR assay to measure and grade amplifiable nuclear DNA in feline faecal extracts. We determined DNA degradation in experimentally aged faecal samples and tested a suite of pre-PCR protocols to considerably improve DNA retrieval. Results Average DNA concentrations of Grade I, II and III extracts were 982pg/µl, 9.5pg/µl and 0.4pg/µl respectively. Nearly 10% of extracts had no amplifiable DNA. Microsatellite PCR success and allelic dropout rates were 92% and 1.5% in Grade I, 79% and 5% in Grade II, and 54% and 16% in Grade III respectively. Our results on experimentally aged faecal samples showed that ageing has a significant effect on quantity and quality of amplifiable DNA (pDNA degradation occurs within 3 days of exposure to direct sunlight. DNA concentrations of Day 1 samples stored by ethanol and silica methods for a month varied significantly from fresh Day 1 extracts (p0.05). DNA concentrations of fresh tiger and leopard faecal extracts without addition of carrier RNA were 816.5pg/µl (±115.5) and 690.1pg/µl (±207.1), while concentrations with addition of carrier RNA were 49414.5pg/µl (±9370.6) and 20982.7pg/µl (±6835.8) respectively. Conclusions Our results indicate that carnivore faecal samples should be collected as freshly as possible, are better preserved by two-step method and should be extracted with addition of carrier RNA. We recommend quantification of template DNA as this facilitates several downstream protocols. PMID:23071624

  17. Simultaneous Assessment of Cardiomyocyte DNA Synthesis and Ploidy: A Method to Assist Quantification of Cardiomyocyte Regeneration and Turnover.

    Science.gov (United States)

    Richardson, Gavin D

    2016-05-23

    Although it is accepted that the heart has a limited potential to regenerate cardiomyocytes following injury and that low levels of cardiomyocyte turnover occur during normal ageing, quantification of these events remains challenging. This is in part due to the rarity of the process and the fact that multiple cellular sources contribute to myocardial maintenance. Furthermore, DNA duplication within cardiomyocytes often leads to a polyploid cardiomyocyte and only rarely leads to new cardiomyocytes by cellular division. In order to accurately quantify cardiomyocyte turnover discrimination between these processes is essential. The protocol described here employs long term nucleoside labeling in order to label all nuclei which have arisen as a result of DNA replication and cardiomyocyte nuclei identified by utilizing nuclei isolation and subsequent PCM1 immunolabeling. Together this allows the accurate and sensitive identification of the nucleoside labeling of the cardiomyocyte nuclei population. Furthermore, 4',6-diamidino-2-phenylindole labeling and analysis of nuclei ploidy, enables the discrimination of neo-cardiomyocyte nuclei from nuclei which have incorporated nucleoside during polyploidization. Although this method cannot control for cardiomyocyte binucleation, it allows a rapid and robust quantification of neo-cardiomyocyte nuclei while accounting for polyploidization. This method has a number of downstream applications including assessing the potential therapeutics to enhance cardiomyocyte regeneration or investigating the effects of cardiac disease on cardiomyocyte turnover and ploidy. This technique is also compatible with additional downstream immunohistological techniques, allowing quantification of nucleoside incorporation in all cardiac cell types.

  18. Hepatitis B virus DNA quantification with the three-in-one (3io) method allows accurate single-step differentiation of total HBV DNA and cccDNA in biopsy-size liver samples.

    Science.gov (United States)

    Taranta, Andrzej; Tien Sy, Bui; Zacher, Behrend Johan; Rogalska-Taranta, Magdalena; Manns, Michael Peter; Bock, Claus Thomas; Wursthorn, Karsten

    2014-08-01

    Hepatitis B virus (HBV) replicates via reverse transcription converting its partially double stranded genome into the covalently closed circular DNA (cccDNA). The long-lasting cccDNA serves as a replication intermediate in the nuclei of hepatocytes. It is an excellent, though evasive, parameter for monitoring the course of liver disease and treatment efficiency. To develop and test a new approach for HBV DNA quantification in serum and small-size liver samples. The p3io plasmid contains an HBV fragment and human β-actin gene (hACTB) as a standard. Respective TaqMan probes were labeled with different fluorescent dyes. A triplex real-time PCR for simultaneous quantification of total HBV DNA, cccDNA and hACTB could be established. Three-in-one method allows simultaneous analysis of 3 targets with a lower limit of quantification of 48 copies per 20 μl PCR reaction and a wide range of linearity (R(2)>0.99, pDNA samples from HBV infected patients. Total HBV DNA and cccDNA could be quantified in 32 and 22 of 33 FFPE preserved liver specimens, respectively. Total HBV DNA concentrations quantified by the 3io method remained comparable with Cobas TaqMan HBV Test v2.0. The three-in-one protocol allows the single step quantification of viral DNA in samples from different sources. Therefore lower sample input, faster data acquisition, a lowered error and significantly lower costs are the advantages of the method. Copyright © 2014 Elsevier B.V. All rights reserved.

  19. Advances in forensic DNA quantification: a review.

    Science.gov (United States)

    Lee, Steven B; McCord, Bruce; Buel, Eric

    2014-11-01

    This review focuses upon a critical step in forensic biology: detection and quantification of human DNA from biological samples. Determination of the quantity and quality of human DNA extracted from biological evidence is important for several reasons. Firstly, depending on the source and extraction method, the quality (purity and length), and quantity of the resultant DNA extract can vary greatly. This affects the downstream method as the quantity of input DNA and its relative length can determine which genotyping procedure to use-standard short-tandem repeat (STR) typing, mini-STR typing or mitochondrial DNA sequencing. Secondly, because it is important in forensic analysis to preserve as much of the evidence as possible for retesting, it is important to determine the total DNA amount available prior to utilizing any destructive analytical method. Lastly, results from initial quantitative and qualitative evaluations permit a more informed interpretation of downstream analytical results. Newer quantitative techniques involving real-time PCR can reveal the presence of degraded DNA and PCR inhibitors, that provide potential reasons for poor genotyping results and may indicate methods to use for downstream typing success. In general, the more information available, the easier it is to interpret and process the sample resulting in a higher likelihood of successful DNA typing. The history of the development of quantitative methods has involved two main goals-improving precision of the analysis and increasing the information content of the result. This review covers advances in forensic DNA quantification methods and recent developments in RNA quantification. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  20. LORD-Q: a long-run real-time PCR-based DNA-damage quantification method for nuclear and mitochondrial genome analysis

    Science.gov (United States)

    Lehle, Simon; Hildebrand, Dominic G.; Merz, Britta; Malak, Peter N.; Becker, Michael S.; Schmezer, Peter; Essmann, Frank; Schulze-Osthoff, Klaus; Rothfuss, Oliver

    2014-01-01

    DNA damage is tightly associated with various biological and pathological processes, such as aging and tumorigenesis. Although detection of DNA damage is attracting increasing attention, only a limited number of methods are available to quantify DNA lesions, and these techniques are tedious or only detect global DNA damage. In this study, we present a high-sensitivity long-run real-time PCR technique for DNA-damage quantification (LORD-Q) in both the mitochondrial and nuclear genome. While most conventional methods are of low-sensitivity or restricted to abundant mitochondrial DNA samples, we established a protocol that enables the accurate sequence-specific quantification of DNA damage in >3-kb probes for any mitochondrial or nuclear DNA sequence. In order to validate the sensitivity of this method, we compared LORD-Q with a previously published qPCR-based method and the standard single-cell gel electrophoresis assay, demonstrating a superior performance of LORD-Q. Exemplarily, we monitored induction of DNA damage and repair processes in human induced pluripotent stem cells and isogenic fibroblasts. Our results suggest that LORD-Q provides a sequence-specific and precise method to quantify DNA damage, thereby allowing the high-throughput assessment of DNA repair, genotoxicity screening and various other processes for a wide range of life science applications. PMID:24371283

  1. Influence of DNA extraction methods, PCR inhibitors and quantification methods on real-time PCR assay of biotechnology-derived traits.

    Science.gov (United States)

    Demeke, Tigst; Jenkins, G Ronald

    2010-03-01

    Biotechnology-derived varieties of canola, cotton, corn and soybean are being grown in the USA, Canada and other predominantly grain exporting countries. Although the amount of farmland devoted to production of biotechnology-derived crops continues to increase, lingering concerns that unintended consequences may occur provide the EU and most grain-importing countries with justification to regulate these crops. Legislation in the EU requires traceability of grains/oilseeds, food and feed products, and labelling, when a threshold level of 0.9% w/w of genetically engineered trait is demonstrated to be present in an analytical sample. The GE content is routinely determined by quantitative PCR (qPCR) and plant genomic DNA provides the template for the initial steps in this process. A plethora of DNA extraction methods exist for qPCR applications. Implementing standardized methods for detection of genetically engineered traits is necessary to facilitate grain marketing. The International Organization for Standardization draft standard 21571 identifies detergent-based methods and commercially available kits that are widely used for DNA extraction, but also indicates that adaptations may be necessary depending upon the sample matrix. This review assesses advantages and disadvantages of various commercially available DNA extraction kits, as well as modifications to published cetyltrimethylammonium bromide methods. Inhibitors are a major obstacle for efficient amplification in qPCR. The types of PCR inhibitors and techniques to minimize inhibition are discussed. Finally, accurate quantification of DNA for applications in qPCR is not trivial. Many confounders contribute to differences in analytical measurements when a particular DNA quantification method is applied and different methods do not always provide concordant results on the same DNA sample. How these differences impact measurement uncertainty in qPCR is considered.

  2. Analytical Validation of Quantitative Real-Time PCR Methods for Quantification of Trypanosoma cruzi DNA in Blood Samples from Chagas Disease Patients.

    Science.gov (United States)

    Ramírez, Juan Carlos; Cura, Carolina Inés; da Cruz Moreira, Otacilio; Lages-Silva, Eliane; Juiz, Natalia; Velázquez, Elsa; Ramírez, Juan David; Alberti, Anahí; Pavia, Paula; Flores-Chávez, María Delmans; Muñoz-Calderón, Arturo; Pérez-Morales, Deyanira; Santalla, José; Marcos da Matta Guedes, Paulo; Peneau, Julie; Marcet, Paula; Padilla, Carlos; Cruz-Robles, David; Valencia, Edward; Crisante, Gladys Elena; Greif, Gonzalo; Zulantay, Inés; Costales, Jaime Alfredo; Alvarez-Martínez, Miriam; Martínez, Norma Edith; Villarroel, Rodrigo; Villarroel, Sandro; Sánchez, Zunilda; Bisio, Margarita; Parrado, Rudy; Maria da Cunha Galvão, Lúcia; Jácome da Câmara, Antonia Cláudia; Espinoza, Bertha; Alarcón de Noya, Belkisyole; Puerta, Concepción; Riarte, Adelina; Diosque, Patricio; Sosa-Estani, Sergio; Guhl, Felipe; Ribeiro, Isabela; Aznar, Christine; Britto, Constança; Yadón, Zaida Estela; Schijman, Alejandro G

    2015-09-01

    An international study was performed by 26 experienced PCR laboratories from 14 countries to assess the performance of duplex quantitative real-time PCR (qPCR) strategies on the basis of TaqMan probes for detection and quantification of parasitic loads in peripheral blood samples from Chagas disease patients. Two methods were studied: Satellite DNA (SatDNA) qPCR and kinetoplastid DNA (kDNA) qPCR. Both methods included an internal amplification control. Reportable range, analytical sensitivity, limits of detection and quantification, and precision were estimated according to international guidelines. In addition, inclusivity and exclusivity were estimated with DNA from stocks representing the different Trypanosoma cruzi discrete typing units and Trypanosoma rangeli and Leishmania spp. Both methods were challenged against 156 blood samples provided by the participant laboratories, including samples from acute and chronic patients with varied clinical findings, infected by oral route or vectorial transmission. kDNA qPCR showed better analytical sensitivity than SatDNA qPCR with limits of detection of 0.23 and 0.70 parasite equivalents/mL, respectively. Analyses of clinical samples revealed a high concordance in terms of sensitivity and parasitic loads determined by both SatDNA and kDNA qPCRs. This effort is a major step toward international validation of qPCR methods for the quantification of T. cruzi DNA in human blood samples, aiming to provide an accurate surrogate biomarker for diagnosis and treatment monitoring for patients with Chagas disease. Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

  3. A flexible fluorescence correlation spectroscopy based method for quantification of the DNA double labeling efficiency with precision control

    International Nuclear Information System (INIS)

    Hou, Sen; Tabaka, Marcin; Sun, Lili; Trochimczyk, Piotr; Kaminski, Tomasz S; Kalwarczyk, Tomasz; Zhang, Xuzhu; Holyst, Robert

    2014-01-01

    We developed a laser-based method to quantify the double labeling efficiency of double-stranded DNA (dsDNA) in a fluorescent dsDNA pool with fluorescence correlation spectroscopy (FCS). Though, for quantitative biochemistry, accurate measurement of this parameter is of critical importance, before our work it was almost impossible to quantify what percentage of DNA is doubly labeled with the same dye. The dsDNA is produced by annealing complementary single-stranded DNA (ssDNA) labeled with the same dye at 5′ end. Due to imperfect ssDNA labeling, the resulting dsDNA is a mixture of doubly labeled dsDNA, singly labeled dsDNA and unlabeled dsDNA. Our method allows the percentage of doubly labeled dsDNA in the total fluorescent dsDNA pool to be measured. In this method, we excite the imperfectly labeled dsDNA sample in a focal volume of <1 fL with a laser beam and correlate the fluctuations of the fluorescence signal to get the FCS autocorrelation curves; we express the amplitudes of the autocorrelation function as a function of the DNA labeling efficiency; we perform a comparative analysis of a dsDNA sample and a reference dsDNA sample, which is prepared by increasing the total dsDNA concentration c (c > 1) times by adding unlabeled ssDNA during the annealing process. The method is flexible in that it allows for the selection of the reference sample and the c value can be adjusted as needed for a specific study. We express the precision of the method as a function of the ssDNA labeling efficiency or the dsDNA double labeling efficiency. The measurement precision can be controlled by changing the c value. (letter)

  4. Method evaluation of Fusarium DNA extraction from mycelia and wheat for down-stream real-time PCR quantification and correlation to mycotoxin levels.

    Science.gov (United States)

    Fredlund, Elisabeth; Gidlund, Ann; Olsen, Monica; Börjesson, Thomas; Spliid, Niels Henrik Hytte; Simonsson, Magnus

    2008-04-01

    Identification of Fusarium species by traditional methods requires specific skill and experience and there is an increased interest for new molecular methods for identification and quantification of Fusarium from food and feed samples. Real-time PCR with probe technology (Taqman) can be used for the identification and quantification of several species of Fusarium from cereal grain samples. There are several critical steps that need to be considered when establishing a real-time PCR-based method for DNA quantification, including extraction of DNA from the samples. In this study, several DNA extraction methods were evaluated, including the DNeasy Plant Mini Spin Columns (Qiagen), the Bio robot EZ1 (Qiagen) with the DNeasy Blood and Tissue Kit (Qiagen), and the Fast-DNA Spin Kit for Soil (Qbiogene). Parameters such as DNA quality and stability, PCR inhibitors, and PCR efficiency were investigated. Our results showed that all methods gave good PCR efficiency (above 90%) and DNA stability whereas the DNeasy Plant Mini Spin Columns in combination with sonication gave the best results with respect to Fusarium DNA yield. The modified DNeasy Plant Mini Spin protocol was used to analyse 31 wheat samples for the presence of F. graminearum and F. culmorum. The DNA level of F. graminearum could be correlated to the level of DON (r(2) = 0.9) and ZEN (r(2) = 0.6) whereas no correlation was found between F. culmorum and DON/ZEA. This shows that F. graminearum and not F. culmorum, was the main producer of DON in Swedish wheat during 2006.

  5. [DNA quantification of blood samples pre-treated with pyramidon].

    Science.gov (United States)

    Zhu, Chuan-Hong; Zheng, Dao-Li; Ni, Rao-Zhi; Wang, Hai-Sheng; Ning, Ping; Fang, Hui; Liu, Yan

    2014-06-01

    To study DNA quantification and STR typing of samples pre-treated with pyramidon. The blood samples of ten unrelated individuals were anticoagulated in EDTA. The blood stains were made on the filter paper. The experimental groups were divided into six groups in accordance with the storage time, 30 min, 1 h, 3 h, 6 h, 12 h and 24h after pre-treated with pyramidon. DNA was extracted by three methods: magnetic bead-based extraction, QIAcube DNA purification method and Chelex-100 method. The quantification of DNA was made by fluorescent quantitative PCR. STR typing was detected by PCR-STR fluorescent technology. In the same DNA extraction method, the sample DNA decreased gradually with times after pre-treatment with pyramidon. In the same storage time, the DNA quantification in different extraction methods had significant differences. Sixteen loci DNA typing were detected in 90.56% of samples. Pyramidon pre-treatment could cause DNA degradation, but effective STR typing can be achieved within 24 h. The magnetic bead-based extraction is the best method for STR profiling and DNA extraction.

  6. Real-Time PCR Quantification of Chloroplast DNA Supports DNA Barcoding of Plant Species.

    Science.gov (United States)

    Kikkawa, Hitomi S; Tsuge, Kouichiro; Sugita, Ritsuko

    2016-03-01

    Species identification from extracted DNA is sometimes needed for botanical samples. DNA quantification is required for an accurate and effective examination. If a quantitative assay provides unreliable estimates, a higher quantity of DNA than the estimated amount may be used in additional analyses to avoid failure to analyze samples from which extracting DNA is difficult. Compared with conventional methods, real-time quantitative PCR (qPCR) requires a low amount of DNA and enables quantification of dilute DNA solutions accurately. The aim of this study was to develop a qPCR assay for quantification of chloroplast DNA from taxonomically diverse plant species. An absolute quantification method was developed using primers targeting the ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) gene using SYBR Green I-based qPCR. The calibration curve was generated using the PCR amplicon as the template. DNA extracts from representatives of 13 plant families common in Japan. This demonstrates that qPCR analysis is an effective method for quantification of DNA from plant samples. The results of qPCR assist in the decision-making will determine the success or failure of DNA analysis, indicating the possibility of optimization of the procedure for downstream reactions.

  7. Nuclear and mitochondrial DNA quantification of various forensic materials.

    Science.gov (United States)

    Andréasson, H; Nilsson, M; Budowle, B; Lundberg, H; Allen, M

    2006-12-01

    Due to the different types and quality of forensic evidence materials, their DNA content can vary substantially, and particularly low quantities can impact the results in an identification analysis. In this study, the quantity of mitochondrial and nuclear DNA was determined in a variety of materials using a previously described real-time PCR method. DNA quantification in the roots and distal sections of plucked and shed head hairs revealed large variations in DNA content particularly between the root and the shaft of plucked hairs. Also large intra- and inter-individual variations were found among hairs. In addition, DNA content was estimated in samples collected from fingerprints and accessories. The quantification of DNA on various items also displayed large variations, with some materials containing large amounts of nuclear DNA while no detectable nuclear DNA and only limited amounts of mitochondrial DNA were seen in others. Using this sensitive real-time PCR quantification assay, a better understanding was obtained regarding DNA content and variation in commonly analysed forensic evidence materials and this may guide the forensic scientist as to the best molecular biology approach for analysing various forensic evidence materials.

  8. Image cytometry: nuclear and chromosomal DNA quantification.

    Science.gov (United States)

    Carvalho, Carlos Roberto; Clarindo, Wellington Ronildo; Abreu, Isabella Santiago

    2011-01-01

    Image cytometry (ICM) associates microscopy, digital image and software technologies, and has been particularly useful in spatial and densitometric cytological analyses, such as DNA ploidy and DNA content measurements. Basically, ICM integrates methodologies of optical microscopy calibration, standard density filters, digital CCD camera, and image analysis softwares for quantitative applications. Apart from all system calibration and setup, cytological protocols must provide good slide preparations for efficient and reliable ICM analysis. In this chapter, procedures for ICM applications employed in our laboratory are described. Protocols shown here for human DNA ploidy determination and quantification of nuclear and chromosomal DNA content in plants could be used as described, or adapted for other studies.

  9. Pitfalls of DNA Quantification Using DNA-Binding Fluorescent Dyes and Suggested Solutions.

    Science.gov (United States)

    Nakayama, Yuki; Yamaguchi, Hiromi; Einaga, Naoki; Esumi, Mariko

    2016-01-01

    The Qubit fluorometer is a DNA quantification device based on the fluorescence intensity of fluorescent dye binding to double-stranded DNA (dsDNA). Qubit is generally considered useful for checking DNA quality before next-generation sequencing because it measures intact dsDNA. To examine the most accurate and suitable methods for quantifying DNA for quality assessment, we compared three quantification methods: NanoDrop, which measures UV absorbance; Qubit; and quantitative PCR (qPCR), which measures the abundance of a target gene. For the comparison, we used three types of DNA: 1) DNA extracted from fresh frozen liver tissues (Frozen-DNA); 2) DNA extracted from formalin-fixed, paraffin-embedded liver tissues comparable to those used for Frozen-DNA (FFPE-DNA); and 3) DNA extracted from the remaining fractions after RNA extraction with Trizol reagent (Trizol-DNA). These DNAs were serially diluted with distilled water and measured using three quantification methods. For Frozen-DNA, the Qubit values were not proportional to the dilution ratio, in contrast with the NanoDrop and qPCR values. This non-proportional decrease in Qubit values was dependent on a lower salt concentration, and over 1 mM NaCl in the DNA solution was required for the Qubit measurement. For FFPE-DNA, the Qubit values were proportional to the dilution ratio and were lower than the NanoDrop values. However, electrophoresis revealed that qPCR reflected the degree of DNA fragmentation more accurately than Qubit. Thus, qPCR is superior to Qubit for checking the quality of FFPE-DNA. For Trizol-DNA, the Qubit values were proportional to the dilution ratio and were consistently lower than the NanoDrop values, similar to FFPE-DNA. However, the qPCR values were higher than the NanoDrop values. Electrophoresis with SYBR Green I and single-stranded DNA (ssDNA) quantification demonstrated that Trizol-DNA consisted mostly of non-fragmented ssDNA. Therefore, Qubit is not always the most accurate method for

  10. A novel automatic quantification method for high-content screening analysis of DNA double strand-break response.

    Science.gov (United States)

    Feng, Jingwen; Lin, Jie; Zhang, Pengquan; Yang, Songnan; Sa, Yu; Feng, Yuanming

    2017-08-29

    High-content screening is commonly used in studies of the DNA damage response. The double-strand break (DSB) is one of the most harmful types of DNA damage lesions. The conventional method used to quantify DSBs is γH2AX foci counting, which requires manual adjustment and preset parameters and is usually regarded as imprecise, time-consuming, poorly reproducible, and inaccurate. Therefore, a robust automatic alternative method is highly desired. In this manuscript, we present a new method for quantifying DSBs which involves automatic image cropping, automatic foci-segmentation and fluorescent intensity measurement. Furthermore, an additional function was added for standardizing the measurement of DSB response inhibition based on co-localization analysis. We tested the method with a well-known inhibitor of DSB response. The new method requires only one preset parameter, which effectively minimizes operator-dependent variations. Compared with conventional methods, the new method detected a higher percentage difference of foci formation between different cells, which can improve measurement accuracy. The effects of the inhibitor on DSB response were successfully quantified with the new method (p = 0.000). The advantages of this method in terms of reliability, automation and simplicity show its potential in quantitative fluorescence imaging studies and high-content screening for compounds and factors involved in DSB response.

  11. Comparison of three DNA extraction methods for the detection and quantification of GMO in Ecuadorian manufactured food.

    Science.gov (United States)

    Pacheco Coello, Ricardo; Pestana Justo, Jorge; Factos Mendoza, Andrés; Santos Ordoñez, Efrén

    2017-12-20

    In Ecuador, food products need to be labeled if exceeded 0.9% of transgenic content in whole products. For the detection of genetically modified organisms (GMOs), three DNA extraction methods were tested in 35 food products commercialized in Ecuador. Samples with positive amplification of endogenous genes were screened for the presence of the Cauliflower mosaic virus 35S-promoter (P35S) and the nopaline synthase-terminator (Tnos). TaqMan™ probes were used for determination of transgenic content of the GTS 40-3-2 and MON810 events through quantitative PCR (qPCR). Twenty-six processed food samples were positive for the P35S alone and eight samples for the Tnos and P35S. Absolute qPCR results indicated that eleven samples were positive for GTS 40-3-2 specific event and two for MON810 specific event. A total of nine samples for events GTS 40-3-2 and MON810 exceeded the umbral allowed of transgenic content in the whole food product with the specific events. Different food products may require different DNA extraction protocols for GMO detection through PCR. Among the three methods tested, the DNeasy mericon food kit DNA extraction method obtained higher proportion of amplified endogenous genes through PCR. Finally, event-specific GMOs were detected in food products in Ecuador.

  12. A fully validated bioanalytical method using an UHPLC-MS/MS system for quantification of DNA and RNA oxidative stress biomarkers.

    Science.gov (United States)

    Cervinkova, Barbora; Krcmova, Lenka Kujovska; Sestakova, Veronika; Solichova, Dagmar; Solich, Petr

    2017-05-01

    A new, rapid and effective ultra-high-performance liquid chromatography method with mass spectrometry detection is described for the separation and quantification of 8-hydroxy-2-deoxyguanosine, 8-hydroxyguanosine and creatinine in human urine. The present study uses an isotope-labelled internal standard ([ 15 N] 5 -8-hydroxy-2-deoxyguanosine), a BIO core-shell stationary phase and an isocratic elution of methanol and water. Sample preparation of human urine was performed by solid-phase extraction (SPE) on Oasis HLB cartridges with methanol/water 50:50 (v/v) elution. Extraction recoveries ranged from 98.1% to 109.2%. Biological extracts showed high short-term stability. Several aspects of this procedure make it suitable for both clinical and research purposes: a short elution time of less than 3.2 min, an intra-day precision of 2.5-8.9%, an inter-day precision of 3.4-8.7% and low limits of quantification (27.7 nM for 8-hydroxyguanosine, 6.0 nM for 8-hydroxy-2-deoxyguanosine). Finally, simultaneous analysis of DNA and RNA oxidative stress biomarkers is a useful tool for monitoring disease progression in neurodegenerative disorders and cancer. Graphical abstract UHPLC-MS/MS analysis of DNA and RNA oxidative stress biomarkers.

  13. Quantification of cellular uptake of DNA nanostructures by qPCR

    DEFF Research Database (Denmark)

    Okholm, Anders Hauge; Nielsen, Jesper Sejrup; Vinther, Mathias

    2014-01-01

    interactions and structural and functional features of the DNA delivery device must be thoroughly investigated. Here, we present a rapid and robust method for the precise quantification of the component materials of DNA origami structures capable of entering cells in vitro. The quantification is performed...

  14. Toward metrological traceability for DNA fragment ratios in GM quantification. 1. Effect of DNA extraction methods on the quantitative determination of Bt176 corn by real-time PCR.

    Science.gov (United States)

    Corbisier, Philippe; Broothaerts, Wim; Gioria, Sabrina; Schimmel, Heinz; Burns, Malcolm; Baoutina, Anna; Emslie, Kerry R; Furui, Satoshi; Kurosawa, Yasunori; Holden, Marcia J; Kim, Hyong-Ha; Lee, Yun-Mi; Kawaharasaki, Mamoru; Sin, Della; Wang, Jing

    2007-05-02

    An international CCQM-P60 pilot study involving eight national metrological institutes was organized to investigate if the quantification of genetically modified (GM) corn powder by real-time PCR was affected by the DNA extraction method applied. Four commonly used extraction methods were compared for the extraction of DNA from a GM Bt176 corn powder. The CTAB-based method yielded the highest DNA template quantity and quality. A difference in the 260 nm/230 nm absorbance ratio was observed among the different extraction methods. Real-time amplification of sequences specific for endogenous genes zein and hmg as well as transgenic sequences within the cryIA(b) gene and a fragment covering the junction between the transformed DNA and the plant genome were used to determine the GM percentage. The detection of the transgenic gene was affected by the quantity and quality of template used for the PCR reaction. The Bt176 percentages measured on diluted or purified templates were statistically different depending on the extraction method applied.

  15. Direct qPCR quantification using the Quantifiler(®) Trio DNA quantification kit.

    Science.gov (United States)

    Liu, Jason Yingjie

    2014-11-01

    The effectiveness of a direct quantification assay is essential to the adoption of the combined direct quantification/direct STR workflow. In this paper, the feasibility of using the Quantifiler(®) Trio DNA quantification kit for the direct quantification of forensic casework samples was investigated. Both low-level touch DNA samples and blood samples were collected on PE swabs and quantified directly. The increased sensitivity of the Quantifiler(®) Trio kit enabled the detection of less than 10pg of DNA in unprocessed touch samples and also minimizes the stochastic effect experienced by different targets in the same sample. The DNA quantity information obtained from a direct quantification assay using the Quantifiler(®) Trio kit can also be used to accurately estimate the optimal input DNA quantity for a direct STR amplification reaction. The correlation between the direct quantification results (Quantifiler(®) Trio kit) and the direct STR results (GlobalFiler™ PCR amplification kit(*)) for low-level touch DNA samples indicates that direct quantification using the Quantifiler(®) Trio DNA quantification kit is more reliable than the Quantifiler(®) Duo DNA quantification kit for predicting the STR results of unprocessed touch DNA samples containing less than 10pg of DNA. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  16. Evaluation of a Rapid One-step Real-time PCR Method as a High-throughput Screening for Quantification of Hepatitis B Virus DNA in a Resource-limited Setting.

    Science.gov (United States)

    Rashed-Ul Islam, S M; Jahan, Munira; Tabassum, Shahina

    2015-01-01

    Virological monitoring is the best predictor for the management of chronic hepatitis B virus (HBV) infections. Consequently, it is important to use the most efficient, rapid and cost-effective testing systems for HBV DNA quantification. The present study compared the performance characteristics of a one-step HBV polymerase chain reaction (PCR) vs the two-step HBV PCR method for quantification of HBV DNA from clinical samples. A total of 100 samples consisting of 85 randomly selected samples from patients with chronic hepatitis B (CHB) and 15 samples from apparently healthy individuals were enrolled in this study. Of the 85 CHB clinical samples tested, HBV DNA was detected from 81% samples by one-step PCR method with median HBV DNA viral load (VL) of 7.50 × 10 3 lU/ml. In contrast, 72% samples were detected by the two-step PCR system with median HBV DNA of 3.71 × 10 3 lU/ml. The one-step method showed strong linear correlation with two-step PCR method (r = 0.89; p Tabassum S. Evaluation of a Rapid One-step Real-time PCR Method as a High-throughput Screening for Quantification of Hepatitis B Virus DNA in a Resource-limited Setting. Euroasian J Hepato-Gastroenterol 2015;5(1):11-15.

  17. qPCR-based mitochondrial DNA quantification: Influence of template DNA fragmentation on accuracy

    International Nuclear Information System (INIS)

    Jackson, Christopher B.; Gallati, Sabina; Schaller, André

    2012-01-01

    Highlights: ► Serial qPCR accurately determines fragmentation state of any given DNA sample. ► Serial qPCR demonstrates different preservation of the nuclear and mitochondrial genome. ► Serial qPCR provides a diagnostic tool to validate the integrity of bioptic material. ► Serial qPCR excludes degradation-induced erroneous quantification. -- Abstract: Real-time PCR (qPCR) is the method of choice for quantification of mitochondrial DNA (mtDNA) by relative comparison of a nuclear to a mitochondrial locus. Quantitative abnormal mtDNA content is indicative of mitochondrial disorders and mostly confines in a tissue-specific manner. Thus handling of degradation-prone bioptic material is inevitable. We established a serial qPCR assay based on increasing amplicon size to measure degradation status of any DNA sample. Using this approach we can exclude erroneous mtDNA quantification due to degraded samples (e.g. long post-exicision time, autolytic processus, freeze–thaw cycles) and ensure abnormal DNA content measurements (e.g. depletion) in non-degraded patient material. By preparation of degraded DNA under controlled conditions using sonification and DNaseI digestion we show that erroneous quantification is due to the different preservation qualities of the nuclear and the mitochondrial genome. This disparate degradation of the two genomes results in over- or underestimation of mtDNA copy number in degraded samples. Moreover, as analysis of defined archival tissue would allow to precise the molecular pathomechanism of mitochondrial disorders presenting with abnormal mtDNA content, we compared fresh frozen (FF) with formalin-fixed paraffin-embedded (FFPE) skeletal muscle tissue of the same sample. By extrapolation of measured decay constants for nuclear DNA (λ nDNA ) and mtDNA (λ mtDNA ) we present an approach to possibly correct measurements in degraded samples in the future. To our knowledge this is the first time different degradation impact of the two

  18. qPCR-based mitochondrial DNA quantification: Influence of template DNA fragmentation on accuracy

    Energy Technology Data Exchange (ETDEWEB)

    Jackson, Christopher B., E-mail: Christopher.jackson@insel.ch [Division of Human Genetics, Departements of Pediatrics and Clinical Research, Inselspital, University of Berne, Freiburgstrasse, CH-3010 Berne (Switzerland); Gallati, Sabina, E-mail: sabina.gallati@insel.ch [Division of Human Genetics, Departements of Pediatrics and Clinical Research, Inselspital, University of Berne, Freiburgstrasse, CH-3010 Berne (Switzerland); Schaller, Andre, E-mail: andre.schaller@insel.ch [Division of Human Genetics, Departements of Pediatrics and Clinical Research, Inselspital, University of Berne, Freiburgstrasse, CH-3010 Berne (Switzerland)

    2012-07-06

    Highlights: Black-Right-Pointing-Pointer Serial qPCR accurately determines fragmentation state of any given DNA sample. Black-Right-Pointing-Pointer Serial qPCR demonstrates different preservation of the nuclear and mitochondrial genome. Black-Right-Pointing-Pointer Serial qPCR provides a diagnostic tool to validate the integrity of bioptic material. Black-Right-Pointing-Pointer Serial qPCR excludes degradation-induced erroneous quantification. -- Abstract: Real-time PCR (qPCR) is the method of choice for quantification of mitochondrial DNA (mtDNA) by relative comparison of a nuclear to a mitochondrial locus. Quantitative abnormal mtDNA content is indicative of mitochondrial disorders and mostly confines in a tissue-specific manner. Thus handling of degradation-prone bioptic material is inevitable. We established a serial qPCR assay based on increasing amplicon size to measure degradation status of any DNA sample. Using this approach we can exclude erroneous mtDNA quantification due to degraded samples (e.g. long post-exicision time, autolytic processus, freeze-thaw cycles) and ensure abnormal DNA content measurements (e.g. depletion) in non-degraded patient material. By preparation of degraded DNA under controlled conditions using sonification and DNaseI digestion we show that erroneous quantification is due to the different preservation qualities of the nuclear and the mitochondrial genome. This disparate degradation of the two genomes results in over- or underestimation of mtDNA copy number in degraded samples. Moreover, as analysis of defined archival tissue would allow to precise the molecular pathomechanism of mitochondrial disorders presenting with abnormal mtDNA content, we compared fresh frozen (FF) with formalin-fixed paraffin-embedded (FFPE) skeletal muscle tissue of the same sample. By extrapolation of measured decay constants for nuclear DNA ({lambda}{sub nDNA}) and mtDNA ({lambda}{sub mtDNA}) we present an approach to possibly correct measurements in

  19. Accurate quantification of supercoiled DNA by digital PCR

    Science.gov (United States)

    Dong, Lianhua; Yoo, Hee-Bong; Wang, Jing; Park, Sang-Ryoul

    2016-01-01

    Digital PCR (dPCR) as an enumeration-based quantification method is capable of quantifying the DNA copy number without the help of standards. However, it can generate false results when the PCR conditions are not optimized. A recent international comparison (CCQM P154) showed that most laboratories significantly underestimated the concentration of supercoiled plasmid DNA by dPCR. Mostly, supercoiled DNAs are linearized before dPCR to avoid such underestimations. The present study was conducted to overcome this problem. In the bilateral comparison, the National Institute of Metrology, China (NIM) optimized and applied dPCR for supercoiled DNA determination, whereas Korea Research Institute of Standards and Science (KRISS) prepared the unknown samples and quantified them by flow cytometry. In this study, several factors like selection of the PCR master mix, the fluorescent label, and the position of the primers were evaluated for quantifying supercoiled DNA by dPCR. This work confirmed that a 16S PCR master mix avoided poor amplification of the supercoiled DNA, whereas HEX labels on dPCR probe resulted in robust amplification curves. Optimizing the dPCR assay based on these two observations resulted in accurate quantification of supercoiled DNA without preanalytical linearization. This result was validated in close agreement (101~113%) with the result from flow cytometry. PMID:27063649

  20. Digital PCR for direct quantification of viruses without DNA extraction.

    Science.gov (United States)

    Pavšič, Jernej; Žel, Jana; Milavec, Mojca

    2016-01-01

    DNA extraction before amplification is considered an essential step for quantification of viral DNA using real-time PCR (qPCR). However, this can directly affect the final measurements due to variable DNA yields and removal of inhibitors, which leads to increased inter-laboratory variability of qPCR measurements and reduced agreement on viral loads. Digital PCR (dPCR) might be an advantageous methodology for the measurement of virus concentrations, as it does not depend on any calibration material and it has higher tolerance to inhibitors. DNA quantification without an extraction step (i.e. direct quantification) was performed here using dPCR and two different human cytomegalovirus whole-virus materials. Two dPCR platforms were used for this direct quantification of the viral DNA, and these were compared with quantification of the extracted viral DNA in terms of yield and variability. Direct quantification of both whole-virus materials present in simple matrices like cell lysate or Tris-HCl buffer provided repeatable measurements of virus concentrations that were probably in closer agreement with the actual viral load than when estimated through quantification of the extracted DNA. Direct dPCR quantification of other viruses, reference materials and clinically relevant matrices is now needed to show the full versatility of this very promising and cost-efficient development in virus quantification.

  1. Quantification of trace-level DNA by real-time whole genome amplification.

    Science.gov (United States)

    Kang, Min-Jung; Yu, Hannah; Kim, Sook-Kyung; Park, Sang-Ryoul; Yang, Inchul

    2011-01-01

    Quantification of trace amounts of DNA is a challenge in analytical applications where the concentration of a target DNA is very low or only limited amounts of samples are available for analysis. PCR-based methods including real-time PCR are highly sensitive and widely used for quantification of low-level DNA samples. However, ordinary PCR methods require at least one copy of a specific gene sequence for amplification and may not work for a sub-genomic amount of DNA. We suggest a real-time whole genome amplification method adopting the degenerate oligonucleotide primed PCR (DOP-PCR) for quantification of sub-genomic amounts of DNA. This approach enabled quantification of sub-picogram amounts of DNA independently of their sequences. When the method was applied to the human placental DNA of which amount was accurately determined by inductively coupled plasma-optical emission spectroscopy (ICP-OES), an accurate and stable quantification capability for DNA samples ranging from 80 fg to 8 ng was obtained. In blind tests of laboratory-prepared DNA samples, measurement accuracies of 7.4%, -2.1%, and -13.9% with analytical precisions around 15% were achieved for 400-pg, 4-pg, and 400-fg DNA samples, respectively. A similar quantification capability was also observed for other DNA species from calf, E. coli, and lambda phage. Therefore, when provided with an appropriate standard DNA, the suggested real-time DOP-PCR method can be used as a universal method for quantification of trace amounts of DNA.

  2. DNA degradation in genetically modified rice with Cry1Ab by food processing methods: implications for the quantification of genetically modified organisms.

    Science.gov (United States)

    Xing, Fuguo; Zhang, Wei; Selvaraj, Jonathan Nimal; Liu, Yang

    2015-05-01

    Food processing methods contribute to DNA degradation, thereby affecting genetically modified organism detection and quantification. This study evaluated the effect of food processing methods on the relative transgenic content of genetically modified rice with Cry1Ab. In steamed rice and rice noodles, the levels of Cry1Ab were ⩾ 100% and <83%, respectively. Frying and baking in rice crackers contributed to a reduction in Pubi and Cry1Ab, while microwaving caused a decrease in Pubi and an increase in Cry1Ab. The processing methods of sweet rice wine had the most severe degradation effects on Pubi and Cry1Ab. In steamed rice and rice noodles, Cry1Ab was the most stable, followed by SPS and Pubi. However, in rice crackers and sweet rice wine, SPS was the most stable, followed by Cry1Ab and Pubi. Therefore, Cry1Ab is a better representative of transgenic components than is Pubi because the levels of Cry1Ab were less affected compared to Pubi. Copyright © 2014 Elsevier Ltd. All rights reserved.

  3. Quantification and presence of human ancient DNA in burial place ...

    African Journals Online (AJOL)

    Quantification and presence of human ancient DNA in burial place remains of Turkey using real time polymerase chain reaction. ... A published real-time PCR assay, which allows for the combined analysis of nuclear or ancient DNA and mitochondrial DNA, was modified. This approach can be used for recovering DNA from ...

  4. Quantification bias caused by plasmid DNA conformation in quantitative real-time PCR assay.

    Science.gov (United States)

    Lin, Chih-Hui; Chen, Yu-Chieh; Pan, Tzu-Ming

    2011-01-01

    Quantitative real-time PCR (qPCR) is the gold standard for the quantification of specific nucleic acid sequences. However, a serious concern has been revealed in a recent report: supercoiled plasmid standards cause significant over-estimation in qPCR quantification. In this study, we investigated the effect of plasmid DNA conformation on the quantification of DNA and the efficiency of qPCR. Our results suggest that plasmid DNA conformation has significant impact on the accuracy of absolute quantification by qPCR. DNA standard curves shifted significantly among plasmid standards with different DNA conformations. Moreover, the choice of DNA measurement method and plasmid DNA conformation may also contribute to the measurement error of DNA standard curves. Due to the multiple effects of plasmid DNA conformation on the accuracy of qPCR, efforts should be made to assure the highest consistency of plasmid standards for qPCR. Thus, we suggest that the conformation, preparation, quantification, purification, handling, and storage of standard plasmid DNA should be described and defined in the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) to assure the reproducibility and accuracy of qPCR absolute quantification.

  5. Quantification of 5-methyl-2'-deoxycytidine in the DNA.

    Science.gov (United States)

    Giel-Pietraszuk, Małgorzata; Insińska-Rak, Małgorzata; Golczak, Anna; Sikorski, Marek; Barciszewska, Mirosława; Barciszewski, Jan

    2015-01-01

    Methylation at position 5 of cytosine (Cyt) at the CpG sequences leading to formation of 5-methyl-cytosine (m(5)Cyt) is an important element of epigenetic regulation of gene expression. Modification of the normal methylation pattern, unique to each organism, leads to the development of pathological processes and diseases, including cancer. Therefore, quantification of the DNA methylation and analysis of changes in the methylation pattern is very important from a practical point of view and can be used for diagnostic purposes, as well as monitoring of the treatment progress. In this paper we present a new method for quantification of 5-methyl-2'deoxycytidine (m(5)C) in the DNA. The technique is based on conversion of m(5)C into fluorescent 3,N(4)-etheno-5-methyl-2'deoxycytidine (εm(5)C) and its identification by reversed-phase high-performance liquid chromatography (RP-HPLC). The assay was used to evaluate m(5)C concentration in DNA of calf thymus and peripheral blood of cows bred under different conditions. This approach can be applied for measuring of 5-methylcytosine in cellular DNA from different cells and tissues.

  6. Interaction between insulin and calf thymus DNA, and quantification of insulin and calf thymus DNA by a resonance Rayleigh scattering method

    International Nuclear Information System (INIS)

    Kong, L.; Liu, Z.; Hu, X.; Liu, S.; Li, W.

    2012-01-01

    The interaction of insulin with calf thymus deoxyribonucleic acid (ctDNA) leads to a complex that displays remarkably enhanced resonance Rayleigh scattering (RRS). The complex and its formation were investigated by atomic force microscopy and by absorption, fluorescence and circular dichroism spectroscopies. We show that the Tyr B16, Tyr B26 and Phe B24 amino acids near the active center (Phe B25) were influenced by the interaction, whereas Tyr A14, Tyr A19 and Phe B1 (which are located far away from the active center) were less influenced. The interaction provide a way in the quantitation of both ctDNA and insulin with high sensitivity. When ctDNA is used as a probe to quantify insulin, the detection limit (3σ) is 6.0 ng mL -1 . If, inversely, insulin is used as a probe to quantify ctDNA, the detection limit (3σ) is 7.2 ng mL -1 . The analysis of synthetic DNA samples and an insulin infection sample provided satisfactory results. (author)

  7. Spectroscopic quantification of 5-hydroxymethylcytosine in genomic DNA.

    Science.gov (United States)

    Shahal, Tamar; Gilat, Noa; Michaeli, Yael; Redy-Keisar, Orit; Shabat, Doron; Ebenstein, Yuval

    2014-08-19

    5-Hydroxymethylcytosine (5hmC), a modified form of the DNA base cytosine, is an important epigenetic mark linked to regulation of gene expression in development, and tumorigenesis. We have developed a spectroscopic method for a global quantification of 5hmC in genomic DNA. The assay is performed within a multiwell plate, which allows simultaneous recording of up to 350 samples. Our quantification procedure of 5hmC is direct, simple, and rapid. It relies on a two-step protocol that consists of enzymatic glucosylation of 5hmC with an azide-modified glucose, followed by a "click reaction" with an alkyne-fluorescent tag. The fluorescence intensity recorded from the DNA sample is proportional to its 5hmC content and can be quantified by a simple plate reader measurement. This labeling technique is specific and highly sensitive, allowing detection of 5hmC down to 0.002% of the total nucleotides. Our results reveal significant variations in the 5hmC content obtained from different mouse tissues, in agreement with previously reported data.

  8. Mutant DNA quantification by digital PCR can be confounded by heating during DNA fragmentation.

    Science.gov (United States)

    Kang, Qing; Parkin, Brian; Giraldez, Maria D; Tewari, Muneesh

    2016-04-01

    Digital PCR (dPCR) is gaining popularity as a DNA mutation quantification method for clinical specimens. Fragmentation prior to dPCR is required for non-fragmented genomic DNA samples; however, the effect of fragmentation on DNA analysis has not been well-studied. Here we evaluated three fragmentation methods for their effects on dPCR point mutation assay performance. Wild-type (WT) human genomic DNA was fragmented by heating, restriction digestion, or acoustic shearing using a Covaris focused-ultrasonicator. dPCR was then used to determine the limit of blank (LoB) by quantifying observed WT and mutant allele counts of the proto-oncogenes KRAS and BRAF in the WT DNA sample. DNA fragmentation by heating to 95°C, while the simplest and least expensive method, produced a high background mutation frequency for certain KRAS mutations relative to the other methods. This was due to heat-induced mutations, specifically affecting dPCR assays designed to interrogate guanine to adenine (G>A) mutations. Moreover, heat-induced fragmentation overestimated gene copy number, potentially due to denaturation and partition of single-stranded DNA into different droplets. Covaris acoustic shearing and restriction enzyme digestion showed similar LoBs and gene copy number estimates to one another. It should be noted that moderate heating, commonly used in genomic DNA extraction protocols, did not significantly increase observed KRAS mutation counts.

  9. Comparison of Methods of Extracting Salmonella enterica Serovar Enteritidis DNA from Environmental Substrates and Quantification of Organisms by Using a General Internal Procedural Control

    NARCIS (Netherlands)

    Klerks, M.M.; Bruggen, van A.H.C.; Zijlstra, C.; Donnikov, M.; Vos, de R.

    2006-01-01

    This paper compares five commercially available DNA extraction methods with respect to DNA extraction efficiency of Salmonella enterica serovar Enteritidis from soil, manure, and compost and uses an Escherichia coli strain harboring a plasmid expressing green fluorescent protein as a general

  10. 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. Copyright © 2016 The Author(s). Published by Elsevier Ireland Ltd.. All rights reserved.

  11. Direct Quantification of Campylobacter jejuni in Chicken Fecal Samples Using Real-Time PCR: Evaluation of Six Rapid DNA Extraction Methods

    DEFF Research Database (Denmark)

    Garcia Clavero, Ana Belén; Kamara, Judy N.; Vigre, Håkan

    2013-01-01

    of this study, the Easy-DNA (Invitrogen) method generated lower Ct values, the best amplification efficiency (AE = 93.2 %) and good precision (R squared = 0.996). The method NucleoSpin® Tissue was able to detect samples spiked with the lowest Campylobacter concentration level (10 CFU/ml) but the amplification...... efficiency was not optimal (AE = 139.5 %). DNA extraction methods Easy-DNA Invitrogen, MiniMAG® and NucleoSpin® Tissue produced good real-time PCR reproducibility generating standard deviations from 0.3 to 0.8 between replicates....

  12. Comparison of DNA quantification methodology used in the DNA extraction protocol for the UK Biobank cohort.

    Science.gov (United States)

    Welsh, Samantha; Peakman, Tim; Sheard, Simon; Almond, Rachael

    2017-01-05

    UK Biobank is a large prospective cohort study in the UK established by the Medical Research Council (MRC) and the Wellcome Trust to enable approved researchers to investigate the role of genetic factors, environmental exposures and lifestyle in the causes of major diseases of late and middle age. A wide range of phenotypic data has been collected at recruitment and has recently been enhanced by the UK Biobank Genotyping Project. All UK Biobank participants (500,000) have been genotyped on either the UK Biobank Axiom® Array or the Affymetrix UK BiLEVE Axiom® Array and the workflow for preparing samples for genotyping is described. The genetic data is hoped to provide further insight into the genetics of disease. All data, including the genetic data, is available for access to approved researchers. Data for two methods of DNA quantification (ultraviolet-visible spectroscopy [UV/Vis]) measured on the Trinean DropSense™ 96 and PicoGreen®) were compared by two laboratories (UK Biobank and Affymetrix). The sample processing workflow established at UK Biobank, for genotyping on the custom Affymetrix Axiom® array, resulted in high quality DNA (average DNA concentration 38.13 ng/μL, average 260/280 absorbance 1.91). The DNA generated high quality genotype data (average call rate 99.48% and pass rate 99.45%). The DNA concentration measured on the Trinean DropSense™ 96 at UK Biobank correlated well with DNA concentration measured by PicoGreen® at Affymetrix (r = 0.85). The UK Biobank Genotyping Project demonstrated that the high throughput DNA extraction protocol described generates high quality DNA suitable for genotyping on the Affymetrix Axiom array. The correlation between DNA concentration derived from UV/Vis and PicoGreen® quantification methods suggests, in large-scale genetic studies involving two laboratories, it may be possible to remove the DNA quantification step in one laboratory without affecting downstream analyses. This would result in

  13. Digital PCR for direct quantification of viruses without DNA extraction

    OpenAIRE

    Pav?i?, Jernej; ?el, Jana; Milavec, Mojca

    2015-01-01

    DNA extraction before amplification is considered an essential step for quantification of viral DNA using real-time PCR (qPCR). However, this can directly affect the final measurements due to variable DNA yields and removal of inhibitors, which leads to increased inter-laboratory variability of qPCR measurements and reduced agreement on viral loads. Digital PCR (dPCR) might be an advantageous methodology for the measurement of virus concentrations, as it does not depend on any calibration mat...

  14. Molecular quantification of environmental DNA using microfluidics and digital PCR.

    Science.gov (United States)

    Hoshino, Tatsuhiko; Inagaki, Fumio

    2012-09-01

    Real-time PCR has been widely used to evaluate gene abundance in natural microbial habitats. However, PCR-inhibitory substances often reduce the efficiency of PCR, leading to the underestimation of target gene copy numbers. Digital PCR using microfluidics is a new approach that allows absolute quantification of DNA molecules. In this study, digital PCR was applied to environmental samples, and the effect of PCR inhibitors on DNA quantification was tested. In the control experiment using λ DNA and humic acids, underestimation of λ DNA at 1/4400 of the theoretical value was observed with 6.58 ng μL(-1) humic acids. In contrast, digital PCR provided accurate quantification data with a concentration of humic acids up to 9.34 ng μL(-1). The inhibitory effect of paddy field soil extract on quantification of the archaeal 16S rRNA gene was also tested. By diluting the DNA extract, quantified copy numbers from real-time PCR and digital PCR became similar, indicating that dilution was a useful way to remedy PCR inhibition. The dilution strategy was, however, not applicable to all natural environmental samples. For example, when marine subsurface sediment samples were tested the copy number of archaeal 16S rRNA genes was 1.04×10(3) copies/g-sediment by digital PCR, whereas real-time PCR only resulted in 4.64×10(2) copies/g-sediment, which was most likely due to an inhibitory effect. The data from this study demonstrated that inhibitory substances had little effect on DNA quantification using microfluidics and digital PCR, and showed the great advantages of digital PCR in accurate quantifications of DNA extracted from various microbial habitats. Copyright © 2012 Elsevier GmbH. All rights reserved.

  15. A Microneedle Functionalized with Polyethyleneimine and Nanotubes for Highly Sensitive, Label-Free Quantification of DNA

    OpenAIRE

    Saadat-Moghaddam, Darius; Kim, Jong-Hoon

    2017-01-01

    The accurate measure of DNA concentration is necessary for many DNA-based biological applications. However, the current methods are limited in terms of sensitivity, reproducibility, human error, and contamination. Here, we present a microneedle functionalized with polyethyleneimine (PEI) and single-walled carbon nanotubes (SWCNTs) for the highly sensitive quantification of DNA. The microneedle was fabricated using ultraviolet (UV) lithography and anisotropic etching, and then functionalized w...

  16. Quantification of cellular uptake of DNA nanostructures by qPCR.

    Science.gov (United States)

    Okholm, Anders Hauge; Nielsen, Jesper Sejrup; Vinther, Mathias; Sørensen, Rasmus Schøler; Schaffert, David; Kjems, Jørgen

    2014-05-15

    DNA nanostructures facilitating drug delivery are likely soon to be realized. In the past few decades programmed self-assembly of DNA building blocks have successfully been employed to construct sophisticated nanoscale objects. By conjugating functionalities to DNA, other molecules such as peptides, proteins and polymers can be precisely positioned on DNA nanostructures. This exceptional ability to produce modular nanoscale devices with tunable and controlled behavior has initiated an interest in employing DNA nanostructures for drug delivery. However, to obtain this the relationship between cellular interactions and structural and functional features of the DNA delivery device must be thoroughly investigated. Here, we present a rapid and robust method for the precise quantification of the component materials of DNA origami structures capable of entering cells in vitro. The quantification is performed by quantitative polymerase chain reaction, allowing a linear dynamic range of detection of five orders of magnitude. We demonstrate the use of this method for high-throughput screening, which could prove efficient to identify key features of DNA nanostructures enabling cell penetration. The method described here is suitable for quantification of in vitro uptake studies but should easily be extended to quantify DNA nanostructures in blood or tissue samples. Copyright © 2014 Elsevier Inc. All rights reserved.

  17. Raman spectroscopy for DNA quantification in cell nucleus.

    Science.gov (United States)

    Okotrub, K A; Surovtsev, N V; Semeshin, V F; Omelyanchuk, L V

    2015-01-01

    Here we demonstrate the feasibility of a novel approach to quantify DNA in cell nuclei. This approach is based on spectroscopy analysis of Raman light scattering, and avoids the problem of nonstoichiometric binding of dyes to DNA, as it directly measures the signal from DNA. Quantitative analysis of nuclear DNA contribution to Raman spectrum could be reliably performed using intensity of a phosphate mode at 1096 cm(-1) . When compared to the known DNA standards from cells of different animals, our results matched those values at error of 10%. We therefore suggest that this approach will be useful to expand the list of DNA standards, to properly adjust the duration of hydrolysis in Feulgen staining, to assay the applicability of fuchsines for DNA quantification, as well as to measure DNA content in cells with complex hydrolysis patterns, when Feulgen densitometry is inappropriate. © 2014 International Society for Advancement of Cytometry.

  18. Quantification of DNA in Neonatal Dried Blood Spots by Adenine Tandem Mass Spectrometry.

    Science.gov (United States)

    Durie, Danielle; Yeh, Ed; McIntosh, Nathan; Fisher, Lawrence; Bulman, Dennis E; Birnboim, H Chaim; Chakraborty, Pranesh; Al-Dirbashi, Osama Y

    2018-01-02

    Newborn screening programs have expanded to include molecular-based assays as first-tier tests and the success of these assays depends on the quality and yield of DNA extracted from neonatal dried blood spots (DBS). To meet high throughput and rapid turnaround time requirements, newborn screening laboratories adopted rapid DNA extraction methods that produce crude extracts. Quantification of DNA in neonatal DBS is not routinely performed due to technical challenges; however, this may enhance the performance of assays that are sensitive to amounts of input DNA. In this study, we developed a novel high throughput method to quantify total DNA in DBS. It is based on specific acid-catalyzed depurination of DNA followed by mass spectrometric quantification of adenine. The amount of adenine was used to calculate DNA quantity per 3.2 mm DBS. Reference intervals were established using archived, neonatal DBS (n = 501) and a median of 130.6 ng of DNA per DBS was obtained, which is in agreement with literature values. The intra- and interday variations were quantification were 12.5 and 37.8 nmol/L adenine, respectively. We demonstrated that DNA from neonatal DBS can be successfully quantified in high throughput settings using instruments currently deployed in NBS laboratories.

  19. Accurate quantification of mouse mitochondrial DNA without co-amplification of nuclear mitochondrial insertion sequences.

    Science.gov (United States)

    Malik, Afshan N; Czajka, Anna; Cunningham, Phil

    2016-07-01

    Mitochondria contain an extra-nuclear genome in the form of mitochondrial DNA (MtDNA), damage to which can lead to inflammation and bioenergetic deficit. Changes in MtDNA levels are increasingly used as a biomarker of mitochondrial dysfunction. We previously reported that in humans, fragments in the nuclear genome known as nuclear mitochondrial insertion sequences (NumtS) affect accurate quantification of MtDNA. In the current paper our aim was to determine whether mouse NumtS affect the quantification of MtDNA and to establish a method designed to avoid this. The existence of NumtS in the mouse genome was confirmed using blast N, unique MtDNA regions were identified using FASTA, and MtDNA primers which do not co-amplify NumtS were designed and tested. MtDNA copy numbers were determined in a range of mouse tissues as the ratio of the mitochondrial and nuclear genome using real time qPCR and absolute quantification. Approximately 95% of mouse MtDNA was duplicated in the nuclear genome as NumtS which were located in 15 out of 21 chromosomes. A unique region was identified and primers flanking this region were used. MtDNA levels differed significantly in mouse tissues being the highest in the heart, with levels in descending order (highest to lowest) in kidney, liver, blood, brain, islets and lung. The presence of NumtS in the nuclear genome of mouse could lead to erroneous data when studying MtDNA content or mutation. The unique primers described here will allow accurate quantification of MtDNA content in mouse models without co-amplification of NumtS. Copyright © 2016 Elsevier B.V. and Mitochondria Research Society. All rights reserved.

  20. Development of Quantification Method for Bioluminescence Imaging

    International Nuclear Information System (INIS)

    Kim, Hyeon Sik; Min, Jung Joon; Lee, Byeong Il; Choi, Eun Seo; Tak, Yoon O; Choi, Heung Kook; Lee, Ju Young

    2009-01-01

    Optical molecular luminescence imaging is widely used for detection and imaging of bio-photons emitted by luminescent luciferase activation. The measured photons in this method provide the degree of molecular alteration or cell numbers with the advantage of high signal-to-noise ratio. To extract useful information from the measured results, the analysis based on a proper quantification method is necessary. In this research, we propose a quantification method presenting linear response of measured light signal to measurement time. We detected the luminescence signal by using lab-made optical imaging equipment of animal light imaging system (ALIS) and different two kinds of light sources. One is three bacterial light-emitting sources containing different number of bacteria. The other is three different non-bacterial light sources emitting very weak light. By using the concept of the candela and the flux, we could derive simplified linear quantification formula. After experimentally measuring light intensity, the data was processed with the proposed quantification function. We could obtain linear response of photon counts to measurement time by applying the pre-determined quantification function. The ratio of the re-calculated photon counts and measurement time present a constant value although different light source was applied. The quantification function for linear response could be applicable to the standard quantification process. The proposed method could be used for the exact quantitative analysis in various light imaging equipment with presenting linear response behavior of constant light emitting sources to measurement time

  1. Urinary Cell-Free DNA Quantification as Non-Invasive Biomarker in Patients with Bladder Cancer.

    Science.gov (United States)

    Brisuda, Antonin; Pazourkova, Eva; Soukup, Viktor; Horinek, Ales; Hrbáček, Jan; Capoun, Otakar; Svobodova, Iveta; Pospisilova, Sarka; Korabecna, Marie; Mares, Jaroslav; Hanuš, Tomáš; Babjuk, Marek

    2016-01-01

    Concentration of urinary cell-free DNA (ucfDNA) belongs to potential bladder cancer markers, but the reported results are inconsistent due to the use of various non-standardised methodologies. The aim of the study was to standardise the methodology for ucfDNA quantification as a potential non-invasive tumour biomarker. In total, 66 patients and 34 controls were enrolled into the study. Volumes of each urine portion (V) were recorded and ucfDNA concentrations (c) were measured using real-time PCR. Total amounts (TA) of ucfDNA were calculated and compared between patients and controls. Diagnostic accuracy of the TA of ucfDNA was determined. The calculation of TA of ucfDNA in the second urine portion was the most appropriate approach to ucfDNA quantification, as there was logarithmic dependence between the volume and the concentration of a urine portion (p = 0.0001). Using this methodology, we were able to discriminate between bladder cancer patients and subjects without bladder tumours (p = 0.0002) with area under the ROC curve of 0.725. Positive and negative predictive value of the test was 90 and 45%, respectively. Quantification of ucf DNA according to our modified method could provide a potential non-invasive biomarker for diagnosis of patients with bladder cancer. © 2015 S. Karger AG, Basel.

  2. Accurate episomal HIV 2-LTR circles quantification using optimized DNA isolation and droplet digital PCR.

    Science.gov (United States)

    Malatinkova, Eva; Kiselinova, Maja; Bonczkowski, Pawel; Trypsteen, Wim; Messiaen, Peter; Vermeire, Jolien; Verhasselt, Bruno; Vervisch, Karen; Vandekerckhove, Linos; De Spiegelaere, Ward

    2014-01-01

    In HIV-infected patients on combination antiretroviral therapy (cART), the detection of episomal HIV 2-LTR circles is a potential marker for ongoing viral replication. Quantification of 2-LTR circles is based on quantitative PCR or more recently on digital PCR assessment, but is hampered due to its low abundance. Sample pre-PCR processing is a critical step for 2-LTR circles quantification, which has not yet been sufficiently evaluated in patient derived samples. We compared two sample processing procedures to more accurately quantify 2-LTR circles using droplet digital PCR (ddPCR). Episomal HIV 2-LTR circles were either isolated by genomic DNA isolation or by a modified plasmid DNA isolation, to separate the small episomal circular DNA from chromosomal DNA. This was performed in a dilution series of HIV-infected cells and HIV-1 infected patient derived samples (n=59). Samples for the plasmid DNA isolation method were spiked with an internal control plasmid. Genomic DNA isolation enables robust 2-LTR circles quantification. However, in the lower ranges of detection, PCR inhibition caused by high genomic DNA load substantially limits the amount of sample input and this impacts sensitivity and accuracy. Moreover, total genomic DNA isolation resulted in a lower recovery of 2-LTR templates per isolate, further reducing its sensitivity. The modified plasmid DNA isolation with a spiked reference for normalization was more accurate in these low ranges compared to genomic DNA isolation. A linear correlation of both methods was observed in the dilution series (R2=0.974) and in the patient derived samples with 2-LTR numbers above 10 copies per million peripheral blood mononuclear cells (PBMCs), (R2=0.671). Furthermore, Bland-Altman analysis revealed an average agreement between the methods within the 27 samples in which 2-LTR circles were detectable with both methods (bias: 0.3875±1.2657 log10). 2-LTR circles quantification in HIV-infected patients proved to be more

  3. DNA damage assessment by visualization and quantification of DNA damage response

    International Nuclear Information System (INIS)

    Matsuda, Shun; Matsuda, Tomonari; Ikura, Tsuyoshi

    2017-01-01

    DNA damage response (DDR) carries out signal transduction for DNA repair, activation of cell cycle checkpoint, and apoptosis to maintain genome integrity, in response to DNA damage. Many proteins and their post-translational modifications participate in the process. Especially, S139-phosphorylated histone H2AX (γH2AX), which is formed by DNA double-strand breaks (DSBs), is an important factor to bring and retain other DDR proteins to DSB sites, Thus, γH2AX is used as a good indicator of DSBs in clinical study and pharmacology for efficacy evaluation of chemotherapy and radiotherapy, detection of precancerous regions, and others. In regulatory science, γH2AX is also a useful biomarker of genotoxicity of chemicals, since a wide range of genotoxic chemicals induce γH2AX. However, conventional detection methods of γH2AX absolutely require anti-γH2AX antibody whose staining is burdensome and time-consuming, and some of these methods are not so superior in quantitativity. In this review, we introduce two new methods to overcome these limitations, involving an easy-to-use genotoxicity assay using DDR-visualizing cells and an absolute quantification method of γH2AX using liquid chromatography-tandem mass spectrometry (LC/MS/MS). (author)

  4. A nuclear DNA-based species determination and DNA quantification assay for common poultry species.

    Science.gov (United States)

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

    2014-12-01

    DNA testing for food authentication and quality control requires sensitive species-specific quantification of nuclear DNA from complex and unknown biological sources. We have developed a multiplex assay based on TaqMan® real-time quantitative PCR (qPCR) for species-specific detection and quantification of chicken (Gallus gallus), duck (Anas platyrhynchos), and turkey (Meleagris gallopavo) nuclear DNA. The multiplex assay is able to accurately detect very low quantities of species-specific DNA from single or multispecies sample mixtures; its minimum effective quantification range is 5 to 50 pg of starting DNA material. In addition to its use in food fraudulence cases, we have validated the assay using simulated forensic sample conditions to demonstrate its utility in forensic investigations. Despite treatment with potent inhibitors such as hematin and humic acid, and degradation of template DNA by DNase, the assay was still able to robustly detect and quantify DNA from each of the three poultry species in mixed samples. The efficient species determination and accurate DNA quantification will help reduce fraudulent food labeling and facilitate downstream DNA analysis for genetic identification and traceability.

  5. Inter-laboratory assessment of different digital PCR platforms for quantification of human cytomegalovirus DNA.

    Science.gov (United States)

    Pavšič, Jernej; Devonshire, Alison; Blejec, Andrej; Foy, Carole A; Van Heuverswyn, Fran; Jones, Gerwyn M; Schimmel, Heinz; Žel, Jana; Huggett, Jim F; Redshaw, Nicholas; Karczmarczyk, Maria; Mozioğlu, Erkan; Akyürek, Sema; Akgöz, Müslüm; Milavec, Mojca

    2017-04-01

    Quantitative PCR (qPCR) is an important tool in pathogen detection. However, the use of different qPCR components, calibration materials and DNA extraction methods reduces comparability between laboratories, which can result in false diagnosis and discrepancies in patient care. The wider establishment of a metrological framework for nucleic acid tests could improve the degree of standardisation of pathogen detection and the quantification methods applied in the clinical context. To achieve this, accurate methods need to be developed and implemented as reference measurement procedures, and to facilitate characterisation of suitable certified reference materials. Digital PCR (dPCR) has already been used for pathogen quantification by analysing nucleic acids. Although dPCR has the potential to provide robust and accurate quantification of nucleic acids, further assessment of its actual performance characteristics is needed before it can be implemented in a metrological framework, and to allow adequate estimation of measurement uncertainties. Here, four laboratories demonstrated reproducibility (expanded measurement uncertainties below 15%) of dPCR for quantification of DNA from human cytomegalovirus, with no calibration to a common reference material. Using whole-virus material and extracted DNA, an intermediate precision (coefficients of variation below 25%) between three consecutive experiments was noted. Furthermore, discrepancies in estimated mean DNA copy number concentrations between laboratories were less than twofold, with DNA extraction as the main source of variability. These data demonstrate that dPCR offers a repeatable and reproducible method for quantification of viral DNA, and due to its satisfactory performance should be considered as candidate for reference methods for implementation in a metrological framework.

  6. Effects of humic substances on fluorometric DNA quantification and DNA hybridization

    NARCIS (Netherlands)

    Bachoon, DS; Otero, E; Hodson, RE

    2001-01-01

    DNA extracts from sediment and water samples are often contaminated with coextracted humic-like impurities, Estuarine humic substances and vascular plant extract were used to evaluate the effect of the presence of such impurities on DNA hybridization and quantification. The presence of humic

  7. Reference Materials for Calibration of Analytical Biases in Quantification of DNA Methylation.

    Science.gov (United States)

    Yu, Hannah; Hahn, Yoonsoo; Yang, Inchul

    2015-01-01

    Most contemporary methods for the quantification of DNA methylation employ bisulfite conversion and PCR amplification. However, many reports have indicated that bisulfite-mediated PCR methodologies can result in inaccurate measurements of DNA methylation owing to amplification biases. To calibrate analytical biases in quantification of gene methylation, especially those that arise during PCR, we utilized reference materials that represent exact bisulfite-converted sequences with 0% and 100% methylation status of specific genes. After determining relative quantities using qPCR, pairs of plasmids were gravimetrically mixed to generate working standards with predefined DNA methylation levels at 10% intervals in terms of mole fractions. The working standards were used as controls to optimize the experimental conditions and also as calibration standards in melting-based and sequencing-based analyses of DNA methylation. Use of the reference materials enabled precise characterization and proper calibration of various biases during PCR and subsequent methylation measurement processes, resulting in accurate measurements.

  8. Rapid quantification of semen hepatitis B virus DNA by real-time polymerase chain reaction

    Science.gov (United States)

    Qian, Wei-Ping; Tan, Yue-Qiu; Chen, Ying; Peng, Ying; Li, Zhi; Lu, Guang-Xiu; Lin, Marie C.; Kung, Hsiang-Fu; He, Ming-Ling; Shing, Li-Ka

    2005-01-01

    AIM: To examine the sensitivity and accuracy of real-time polymerase chain reaction (PCR) for the quantification of hepatitis B virus (HBV) DNA in semen. METHODS: Hepatitis B viral DNA was isolated from HBV carriers’ semen and sera using phenol extraction method and QIAamp DNA blood mini kit (Qiagen, Germany). HBV DNA was detected by conventional PCR and quantified by TaqMan technology-based real-time PCR (quantitative polymerase chain reaction (qPCR)). The detection threshold was 200 copies of HBV DNA for conventional PCR and 10 copies of HBV DNA for real time PCR per reaction. RESULTS: Both methods of phenol extraction and QIAamp DNA blood mini kit were suitable for isolating HBV DNA from semen. The value of the detection thresholds was 500 copies of HBV DNA per mL in the semen. The viral loads were 7.5 × 107 and 1.67 × 107 copies of HBV DNA per mL in two HBV infected patients’ sera, while 2.14 × 105 and 3.02 × 105 copies of HBV DNA per mL in the semen. CONCLUSION: Real-time PCR is a more sensitive and accurate method to detect and quantify HBV DNA in the semen. PMID:16149152

  9. DNA imaging and quantification using chemi-luminescent probes

    International Nuclear Information System (INIS)

    Dorner, G.; Redjdal, N.; Laniece, P.; Siebert, R.; Tricoire, H.; Valentin, L.

    1999-01-01

    During this interdisciplinary study we have developed an ultra sensitive and reliable imaging system of DNA labelled by chemiluminescence. Based on a liquid nitrogen cooled CCD, the system achieves sensitivities down to 10 fg/mm 2 labelled DNA over a surface area of 25 x 25 cm 2 with a sub-millimeter resolution. Commercially available chemi-luminescent - and enhancer molecules are compared and their reaction conditions optimized for best signal-to-noise ratios. Double labelling was performed to verify quantification with radioactive probes. (authors)

  10. The practical analysis of food: the development of Sakalar quantification table of DNA (SQT-DNA).

    Science.gov (United States)

    Sakalar, Ergün

    2013-11-15

    Practical and highly sensitive Sakalar quantification table of DNA (SQT-DNA) has been developed for the detection% of species-specific DNA amount in food products. Cycle threshold (Ct) data were obtained from multiple curves of real-time qPCR. The statistical analysis was done to estimate the concentration of standard dilutions. Amplicon concentrations versus each Ct value were assessed by the predictions of targets at known concentrations. SQT-DNA was prepared by using the percentage versus each Ct values. The applicability of SQT-DNA to commercial foods was proved by using sausages containing varying ratios of beef, chicken, and soybean. The results showed that SQT-DNA can be used to directly quantify food DNA by a single PCR without the need to construct a standart curve in parallel with the samples every time the experiment is performed, and also quantification by SQT-DNA is as reliable as standard curve quantification for a wide range of DNA concentrations. Copyright © 2013 Elsevier Ltd. All rights reserved.

  11. Effects of humic acid on DNA quantification with Quantifiler® Human DNA Quantification kit and short tandem repeat amplification efficiency.

    Science.gov (United States)

    Seo, Seung Bum; Lee, Hye Young; Zhang, Ai Hua; Kim, Hye Yeon; Shin, Dong Hoon; Lee, Soong Deok

    2012-11-01

    Correct DNA quantification is an essential part to obtain reliable STR typing results. Forensic DNA analysts often use commercial kits for DNA quantification; among them, real-time-based DNA quantification kits are most frequently used. Incorrect DNA quantification due to the presence of PCR inhibitors may affect experiment results. In this study, we examined the alteration degree of DNA quantification results estimated in DNA samples containing a PCR inhibitor by using a Quantifiler® Human DNA Quantification kit. For experiments, we prepared approximately 0.25 ng/μl DNA samples containing various concentrations of humic acid (HA). The quantification results were 0.194-0.303 ng/μl at 0-1.6 ng/μl HA (final concentration in the Quantifiler reaction) and 0.003-0.168 ng/μl at 2.4-4.0 ng/μl HA. Most DNA quantity was undetermined when HA concentration was higher than 4.8 ng/μl HA. The C (T) values of an internal PCR control (IPC) were 28.0-31.0, 36.5-37.1, and undetermined at 0-1.6, 2.4, and 3.2 ng/μl HA. These results indicate that underestimated DNA quantification results may be obtained in the DNA sample with high C (T) values of IPC. Thus, researchers should carefully interpret the DNA quantification results. We additionally examined the effects of HA on the STR amplification by using an Identifiler® kit and a MiniFiler™ kit. Based on the results of this study, it is thought that a better understanding of various effects of HA would help researchers recognize and manipulate samples containing HA.

  12. Introduction to DNA methods

    International Nuclear Information System (INIS)

    Delincee, H.

    1991-01-01

    The purpose of this session is to discuss the various possibilities for detecting modifications in DNA after irradiation and whether these changes can be utilized as an indicator for the irradiation treatment of foods. The requirement to be fulfilled is that the method be able to distinguish irradiated food without the presence of a control sample, thus the measured response after irradiation must be large enough to supersede background levels from other treatments. Much work has been performed on the effects of radiation on DNA, particularly due to its importance in radiation biology. The main lesions of DNA as a result of irradiation are base damage, damage of the sugar moiety, single strand and double strand breaks. Crosslinking between bases also occurs, e.g. production of thymine dimers, or between DNA and protein. A valuable review on how to utilize these DNA changes for detection purposes has already appeared. Tables 1, 2 and 3 list the proposed methods of detecting changes in irradiated DNA, some identified products as examples for a possible irradiation indicator, in the case of immunoassay the substance used as antigen, and some selected literature references. In this short review, it is not intended to provide a complete literature survey

  13. The Method of Manufactured Universes for validating uncertainty quantification methods

    KAUST Repository

    Stripling, H.F.; Adams, M.L.; McClarren, R.G.; Mallick, B.K.

    2011-01-01

    The Method of Manufactured Universes is presented as a validation framework for uncertainty quantification (UQ) methodologies and as a tool for exploring the effects of statistical and modeling assumptions embedded in these methods. The framework

  14. Direct quantification of cell-free, circulating DNA from unpurified plasma.

    Science.gov (United States)

    Breitbach, Sarah; Tug, Suzan; Helmig, Susanne; Zahn, Daniela; Kubiak, Thomas; Michal, Matthias; Gori, Tommaso; Ehlert, Tobias; Beiter, Thomas; Simon, Perikles

    2014-01-01

    Cell-free DNA (cfDNA) in body tissues or fluids is extensively investigated in clinical medicine and other research fields. In this article we provide a direct quantitative real-time PCR (qPCR) as a sensitive tool for the measurement of cfDNA from plasma without previous DNA extraction, which is known to be accompanied by a reduction of DNA yield. The primer sets were designed to amplify a 90 and 222 bp multi-locus L1PA2 sequence. In the first module, cfDNA concentrations in unpurified plasma were compared to cfDNA concentrations in the eluate and the flow-through of the QIAamp DNA Blood Mini Kit and in the eluate of a phenol-chloroform isoamyl (PCI) based DNA extraction, to elucidate the DNA losses during extraction. The analyses revealed 2.79-fold higher cfDNA concentrations in unpurified plasma compared to the eluate of the QIAamp DNA Blood Mini Kit, while 36.7% of the total cfDNA were found in the flow-through. The PCI procedure only performed well on samples with high cfDNA concentrations, showing 87.4% of the concentrations measured in plasma. The DNA integrity strongly depended on the sample treatment. Further qualitative analyses indicated differing fractions of cfDNA fragment lengths in the eluate of both extraction methods. In the second module, cfDNA concentrations in the plasma of 74 coronary heart disease patients were compared to cfDNA concentrations of 74 healthy controls, using the direct L1PA2 qPCR for cfDNA quantification. The patient collective showed significantly higher cfDNA levels (mean (SD) 20.1 (23.8) ng/ml; range 5.1-183.0 ng/ml) compared to the healthy controls (9.7 (4.2) ng/ml; range 1.6-23.7 ng/ml). With our direct qPCR, we recommend a simple, economic and sensitive procedure for the quantification of cfDNA concentrations from plasma that might find broad applicability, if cfDNA became an established marker in the assessment of pathophysiological conditions.

  15. Uncertainty Quantification in Alchemical Free Energy Methods.

    Science.gov (United States)

    Bhati, Agastya P; Wan, Shunzhou; Hu, Yuan; Sherborne, Brad; Coveney, Peter V

    2018-05-02

    Alchemical free energy methods have gained much importance recently from several reports of improved ligand-protein binding affinity predictions based on their implementation using molecular dynamics simulations. A large number of variants of such methods implementing different accelerated sampling techniques and free energy estimators are available, each claimed to be better than the others in its own way. However, the key features of reproducibility and quantification of associated uncertainties in such methods have barely been discussed. Here, we apply a systematic protocol for uncertainty quantification to a number of popular alchemical free energy methods, covering both absolute and relative free energy predictions. We show that a reliable measure of error estimation is provided by ensemble simulation-an ensemble of independent MD simulations-which applies irrespective of the free energy method. The need to use ensemble methods is fundamental and holds regardless of the duration of time of the molecular dynamics simulations performed.

  16. In vivo quantification of DNA double strand breaks

    International Nuclear Information System (INIS)

    Simonsson, M.; Qvarnstroem, F.; Turesson, I.; Johansson, K.-A.; Nyman, J.; Hermansson, I.; Oden, A.; Book, M.

    2003-01-01

    DNA double strand breaks (DSBs) can be introduced in the genome by exposure to exogenous agents such as ionising radiation and radio-mimetic chemicals. The biological importance of these breaks is significant even at low numbers. Inaccurate repair or lack of repair of a single DSB has the potential to kill a cell or lead to tumourigenesis. Thus the induction and repair of DSBs are crucial events in the onset of malignancies. Following the induction of DSBs, the core histone H2AX is rapidly phosphorylated at residue serine 139. This phosphorylated form of H2AX is referred to as gH2AX. Histones wrapped in megabase regions flanking these breaks are involved in this process, which results in the formation of discrete nuclear foci. It has previously been shown that a single DSB is sufficient to produce a detectable focus. So far there has been a lack of methods capable of measuring the amount of DSBs at clinically relevant quantities. Such a method would embrace a wide field of applications. It could be applied as a biological dosimeter when studying carcinogenic effects and provide the basis for an assay predicting individual radiosensitivity. We describe a measurement procedure that detects and quantifies small amounts of DSBs in vivo. This is accomplished using immunofluorescence detection of the molecular marker gH2AX. The gH2AX foci are quantified in histological sections using basic digital image analysis methods as the main component. In a primary assessment of the procedure we analysed the in vivo dose response of prostate cancer patients in clinical practice undergoing radiotherapy. Epidermal nucleated cells in skin biopsies taken 30 minutes following the first single dose delivered show linear dose response for low doses ranging from 0 - 1.2 Gy. The described procedure for double strand break quantification can detect dose changes as low as 0.18 Gy

  17. New Detection Modality for Label-Free Quantification of DNA in Biological Samples via Superparamagnetic Bead Aggregation

    Science.gov (United States)

    Leslie, Daniel C.; Li, Jingyi; Strachan, Briony C.; Begley, Matthew R.; Finkler, David; Bazydlo, Lindsay L.; Barker, N. Scott; Haverstick, Doris; Utz, Marcel; Landers, James P.

    2012-01-01

    Combining DNA and superparamagnetic beads in a rotating magnetic field produces multiparticle aggregates that are visually striking, and enables label-free optical detection and quantification of DNA at levels in the picogram per microliter range. DNA in biological samples can be quantified directly by simple analysis of optical images of microfluidic wells placed on a magnetic stirrer without DNA purification. Aggregation results from DNA/bead interactions driven either by the presence of a chaotrope (a nonspecific trigger for aggregation) or by hybridization with oligonucleotides on functionalized beads (sequence-specific). This paper demonstrates quantification of DNA with sensitivity comparable to that of the best currently available fluorometric assays. The robustness and sensitivity of the method enable a wide range of applications, illustrated here by counting eukaryotic cells. Using widely available and inexpensive benchtop hardware, the approach provides a highly accessible low-tech microscale alternative to more expensive DNA detection and cell counting techniques. PMID:22423674

  18. Simultaneous quantification of ten constituents of Xanthoceras sorbifolia Bunge using UHPLC-MS methods and evaluation of their radical scavenging, DNA scission protective, and α-glucosidase inhibitory activities.

    Science.gov (United States)

    Zhang, Yu; Ma, Jian-Nan; Ma, Chun-Li; Qi, Zhi; Ma, Chao-Mei

    2015-11-01

    The present study was designed to investigate the bioactive constituents of Xanthoceras sorbifolia in terms of amounts and their antioxidant, DNA scission protection, and α-glucosidase inhibitory activities. Simultaneous quantification of 10 X. sorbifolia constituents was carried out by a newly established ultra-high performance liquid chromatography-quadrupole mass spectrometry method (UHPLC-MS). The antioxidant activities were evaluated by measuring DPPH radical scavenging and DNA scission protective activities. The α-glucosidase inhibitory activities were investigated by using an assay with α-glucosidase from Bacillus Stearothermophilus and disaccharidases from mouse intestine. We found that the wood of X. sorbifolia was rich in phenolic compounds with the contents of catechin, epicatechin, myricetin, and dihydromyricetin being 0.12-0.19, 1.94-2.16, 0.77-0.91, and 6.76-7.89 mg·g(-1), respectively. The four constituents strongly scavenged DPPH radicals (with EC50 being 4.2, 3.8 and 5.7 μg·mL(-1), respectively) and remarkably protected peroxyl radical-induced DNA strand scission (92.10%, 94.66%, 75.44% and 89.95% of protection, respectively, at a concentration of 10 μmol·L(-1)). A dimeric flavan 3-ol, epigallocatechin-(4β→8, 2β→O-7)-epicatechin potently inhibited α-glucosidase with an IC50 value being as low as 1.2 μg·mL(-1). The established UHPLC-MS method could serve as a quality control tool for X. sorbifolia. In conclusion, the high contents of antioxidant and α-glucosidase inhibitory constituents in X. sorbifolia support its use as complementation of other therapeutic agents for metabolic disorders, such as diabetes and hypertension. Copyright © 2015 China Pharmaceutical University. Published by Elsevier B.V. All rights reserved.

  19. Model Uncertainty Quantification Methods In Data Assimilation

    Science.gov (United States)

    Pathiraja, S. D.; Marshall, L. A.; Sharma, A.; Moradkhani, H.

    2017-12-01

    Data Assimilation involves utilising observations to improve model predictions in a seamless and statistically optimal fashion. Its applications are wide-ranging; from improving weather forecasts to tracking targets such as in the Apollo 11 mission. The use of Data Assimilation methods in high dimensional complex geophysical systems is an active area of research, where there exists many opportunities to enhance existing methodologies. One of the central challenges is in model uncertainty quantification; the outcome of any Data Assimilation study is strongly dependent on the uncertainties assigned to both observations and models. I focus on developing improved model uncertainty quantification methods that are applicable to challenging real world scenarios. These include developing methods for cases where the system states are only partially observed, where there is little prior knowledge of the model errors, and where the model error statistics are likely to be highly non-Gaussian.

  20. Digital quantification of rolling circle amplified single DNA molecules in a resistive pulse sensing nanopore.

    Science.gov (United States)

    Kühnemund, M; Nilsson, M

    2015-05-15

    Novel portable, sensitive and selective DNA sensor methods for bio-sensing applications are required that can rival conventionally used non-portable and expensive fluorescence-based sensors. In this paper, rolling circle amplification (RCA) products are detected in solution and on magnetic particles using a resistive pulse sensing (RPS) nanopore. Low amounts of DNA molecules are detected by padlock probes which are circularized in a strictly target dependent ligation reaction. The DNA-padlock probe-complex is captured on magnetic particles by sequence specific capture oligonucleotides and amplified by a short RCA. Subsequent RPS analysis is used to identify individual particles with single attached RCA products from blank particles. This proof of concept opens up for a novel non-fluorescent digital DNA quantification method that can have many applications in bio-sensing and diagnostic approaches. Copyright © 2014 Elsevier B.V. All rights reserved.

  1. Quantification of transplant-derived circulating cell-free DNA in absence of a donor genotype.

    Science.gov (United States)

    Sharon, Eilon; Shi, Hao; Kharbanda, Sandhya; Koh, Winston; Martin, Lance R; Khush, Kiran K; Valantine, Hannah; Pritchard, Jonathan K; De Vlaminck, Iwijn

    2017-08-01

    Quantification of cell-free DNA (cfDNA) in circulating blood derived from a transplanted organ is a powerful approach to monitoring post-transplant injury. Genome transplant dynamics (GTD) quantifies donor-derived cfDNA (dd-cfDNA) by taking advantage of single-nucleotide polymorphisms (SNPs) distributed across the genome to discriminate donor and recipient DNA molecules. In its current implementation, GTD requires genotyping of both the transplant recipient and donor. However, in practice, donor genotype information is often unavailable. Here, we address this issue by developing an algorithm that estimates dd-cfDNA levels in the absence of a donor genotype. Our algorithm predicts heart and lung allograft rejection with an accuracy that is similar to conventional GTD. We furthermore refined the algorithm to handle closely related recipients and donors, a scenario that is common in bone marrow and kidney transplantation. We show that it is possible to estimate dd-cfDNA in bone marrow transplant patients that are unrelated or that are siblings of the donors, using a hidden Markov model (HMM) of identity-by-descent (IBD) states along the genome. Last, we demonstrate that comparing dd-cfDNA to the proportion of donor DNA in white blood cells can differentiate between relapse and the onset of graft-versus-host disease (GVHD). These methods alleviate some of the barriers to the implementation of GTD, which will further widen its clinical application.

  2. Quantification of transplant-derived circulating cell-free DNA in absence of a donor genotype.

    Directory of Open Access Journals (Sweden)

    Eilon Sharon

    2017-08-01

    Full Text Available Quantification of cell-free DNA (cfDNA in circulating blood derived from a transplanted organ is a powerful approach to monitoring post-transplant injury. Genome transplant dynamics (GTD quantifies donor-derived cfDNA (dd-cfDNA by taking advantage of single-nucleotide polymorphisms (SNPs distributed across the genome to discriminate donor and recipient DNA molecules. In its current implementation, GTD requires genotyping of both the transplant recipient and donor. However, in practice, donor genotype information is often unavailable. Here, we address this issue by developing an algorithm that estimates dd-cfDNA levels in the absence of a donor genotype. Our algorithm predicts heart and lung allograft rejection with an accuracy that is similar to conventional GTD. We furthermore refined the algorithm to handle closely related recipients and donors, a scenario that is common in bone marrow and kidney transplantation. We show that it is possible to estimate dd-cfDNA in bone marrow transplant patients that are unrelated or that are siblings of the donors, using a hidden Markov model (HMM of identity-by-descent (IBD states along the genome. Last, we demonstrate that comparing dd-cfDNA to the proportion of donor DNA in white blood cells can differentiate between relapse and the onset of graft-versus-host disease (GVHD. These methods alleviate some of the barriers to the implementation of GTD, which will further widen its clinical application.

  3. HPCE quantification of 5-methyl-2'-deoxycytidine in genomic DNA: methodological optimization for chestnut and other woody species.

    Science.gov (United States)

    Hasbún, Rodrigo; Valledor, Luís; Rodríguez, José L; Santamaria, Estrella; Ríos, Darcy; Sanchez, Manuel; Cañal, María J; Rodríguez, Roberto

    2008-01-01

    Quantification of deoxynucleosides using micellar high-performance capillary electrophoresis (HPCE) is an efficient, fast and inexpensive evaluation method of genomic DNA methylation. This approach has been demonstrated to be more sensitive and specific than other methods for the quantification of DNA methylation content. However, effective detection and quantification of 5-methyl-2'-deoxycytidine depend of the sample characteristics. Previous works have revealed that in most woody species, the quality and quantity of RNA-free DNA extracted that is suitable for analysis by means of HPCE varies among species of the same gender, among tissues taken from the same tree, and vary in the same tissue depending on the different seasons of the year. The aim of this work is to establish a quantification method of genomic DNA methylation that lends itself to use in different Castanea sativa Mill. materials, and in other angiosperm and gymnosperm woody species. Using a DNA extraction kit based in silica membrane has increased the resolutive capacity of the method. Under these conditions, it can be analyzed different organs or tissues of angiosperms and gymnosperms, regardless of their state of development. We emphasized the importance of samples free of nucleosides, although, in the contrary case, the method ensures the effective separation of deoxynucleosides and identification of 5-methyl-2'-deoxycytidine.

  4. DNA quantification of basidiomycetous fungi during storage of logging residues

    Directory of Open Access Journals (Sweden)

    Isabella Børja

    2015-04-01

    Full Text Available The demand for bioenergy caused an increased use of logging residues, branches and treetops that were previously left on the ground after harvesting. Residues are stored outdoors in piles and it is unclear to what extent fungi transform this material. Our objective was to quantify the amount of wood degrading fungi during storage using quantitative real-time PCR (qPCR to detect basidiomycetous DNA in logging residues, a novel approach in this field. We found that the qPCR method was accurate in quantifying the fungal DNA during storage. As the moisture content of the piled logging residues decreased during the storage period, the fungal DNA content also decreased. Scots pine residues contained more fungal DNA than residues from Norway spruce. Loose piles had generally more fungal DNA than bundled ones.

  5. Multiplex electrochemical DNA platform for femtomolar-level quantification of genetically modified soybean.

    Science.gov (United States)

    Manzanares-Palenzuela, C Lorena; de-los-Santos-Álvarez, Noemí; Lobo-Castañón, María Jesús; López-Ruiz, Beatriz

    2015-06-15

    Current EU regulations on the mandatory labeling of genetically modified organisms (GMOs) with a minimum content of 0.9% would benefit from the availability of reliable and rapid methods to detect and quantify DNA sequences specific for GMOs. Different genosensors have been developed to this aim, mainly intended for GMO screening. A remaining challenge, however, is the development of genosensing platforms for GMO quantification, which should be expressed as the number of event-specific DNA sequences per taxon-specific sequences. Here we report a simple and sensitive multiplexed electrochemical approach for the quantification of Roundup-Ready Soybean (RRS). Two DNA sequences, taxon (lectin) and event-specific (RR), are targeted via hybridization onto magnetic beads. Both sequences are simultaneously detected by performing the immobilization, hybridization and labeling steps in a single tube and parallel electrochemical readout. Hybridization is performed in a sandwich format using signaling probes labeled with fluorescein isothiocyanate (FITC) or digoxigenin (Dig), followed by dual enzymatic labeling using Fab fragments of anti-Dig and anti-FITC conjugated to peroxidase or alkaline phosphatase, respectively. Electrochemical measurement of the enzyme activity is finally performed on screen-printed carbon electrodes. The assay gave a linear range of 2-250 pM for both targets, with LOD values of 650 fM (160 amol) and 190 fM (50 amol) for the event-specific and the taxon-specific targets, respectively. Results indicate that the method could be applied for GMO quantification below the European labeling threshold level (0.9%), offering a general approach for the rapid quantification of specific GMO events in foods. Copyright © 2015 Elsevier B.V. All rights reserved.

  6. Assessment of the real-time PCR and different digital PCR platforms for DNA quantification.

    Science.gov (United States)

    Pavšič, Jernej; Žel, Jana; Milavec, Mojca

    2016-01-01

    Digital PCR (dPCR) is beginning to supersede real-time PCR (qPCR) for quantification of nucleic acids in many different applications. Several analytical properties of the two most commonly used dPCR platforms, namely the QX100 system (Bio-Rad) and the 12.765 array of the Biomark system (Fluidigm), have already been evaluated and compared with those of qPCR. However, to the best of our knowledge, direct comparison between the three of these platforms using the same DNA material has not been done, and the 37 K array on the Biomark system has also not been evaluated in terms of linearity, analytical sensitivity and limit of quantification. Here, a first assessment of qPCR, the QX100 system and both arrays of the Biomark system was performed with plasmid and genomic DNA from human cytomegalovirus. With use of PCR components that alter the efficiency of qPCR, each dPCR platform demonstrated consistent copy-number estimations, which indicates the high resilience of dPCR. Two approaches, one considering the total reaction volume and the other considering the effective reaction size, were used to assess linearity, analytical sensitivity and variability. When the total reaction volume was considered, the best performance was observed with qPCR, followed by the QX100 system and the Biomark system. In contrast, when the effective reaction size was considered, all three platforms showed almost equal limits of detection and variability. Although dPCR might not always be more appropriate than qPCR for quantification of low copy numbers, dPCR is a suitable method for robust and reproducible quantification of viral DNA, and a promising technology for the higher-order reference measurement method.

  7. Evaluation of plasmid and genomic DNA calibrants used for the quantification of genetically modified organisms.

    Science.gov (United States)

    Caprioara-Buda, M; Meyer, W; Jeynov, B; Corbisier, P; Trapmann, S; Emons, H

    2012-07-01

    The reliable quantification of genetically modified organisms (GMOs) by real-time PCR requires, besides thoroughly validated quantitative detection methods, sustainable calibration systems. The latter establishes the anchor points for the measured value and the measurement unit, respectively. In this paper, the suitability of two types of DNA calibrants, i.e. plasmid DNA and genomic DNA extracted from plant leaves, for the certification of the GMO content in reference materials as copy number ratio between two targeted DNA sequences was investigated. The PCR efficiencies and coefficients of determination of the calibration curves as well as the measured copy number ratios for three powder certified reference materials (CRMs), namely ERM-BF415e (NK603 maize), ERM-BF425c (356043 soya), and ERM-BF427c (98140 maize), originally certified for their mass fraction of GMO, were compared for both types of calibrants. In all three systems investigated, the PCR efficiencies of plasmid DNA were slightly closer to the PCR efficiencies observed for the genomic DNA extracted from seed powders rather than those of the genomic DNA extracted from leaves. Although the mean DNA copy number ratios for each CRM overlapped within their uncertainties, the DNA copy number ratios were significantly different using the two types of calibrants. Based on these observations, both plasmid and leaf genomic DNA calibrants would be technically suitable as anchor points for the calibration of the real-time PCR methods applied in this study. However, the most suitable approach to establish a sustainable traceability chain is to fix a reference system based on plasmid DNA.

  8. Controls to validate plasma samples for cell free DNA quantification

    DEFF Research Database (Denmark)

    Pallisgaard, Niels; Spindler, Karen-Lise Garm; Andersen, Rikke Fredslund

    2015-01-01

    , are diverging due to methodological differences with lack of standardisation and definition of sensitivity. The new biological information has not yet come into routine use. The present study presents external standardisation by spiking with non-human DNA fragments to control for loss of DNA during sample...... preparation and measurement. It also suggests a method to control for admixture of DNA from normal lymphocytes by utilizing the unique immunoglobulin gene rearrangement in the B-cells. The results show that this approach improves the quality of the analysis and lowers the risk of falsely increased values...

  9. [Real-time quantification to analyze historical Colombian samples detecting a short fragment of hypervariable region II of mitochondrial DNA].

    Science.gov (United States)

    Pérez, Luz Adriana; Rodríguez, Freddy; Langebaek, Carl Henrik; Groot, Helena

    2016-09-01

    Unlike other molecular biology studies, the analysis of ancient DNA (aDNA) requires special infrastructure and methodological conditions to guarantee the quality of the results. One of the main authenticity criteria is DNA quantification, where quantitative real-time PCR is often used given its sensitivity and specificity. Nevertheless, the implementation of these conditions and methodologies to fulfill authenticity criteria imply higher costs. Objective: To develop a simple and less costly method for mitochondrial DNA quantification suitable for highly degraded samples. Materials and methods: The proposed method is based on the use of mini-primers for the specific amplification of short fragments of mitochondrial DNA. The subsequent purification of these amplified fragments allows a standard curve to be constructed with concentrations in accordance to the state of degradation of the samples. Results: The proposed method successfully detected DNA from ancient samples including bone remains and mummified tissue. DNA inhibitory substances were also detected. Conclusion: The proposed method represents a simpler and cost-effective way to detect low amounts of aDNA, and a tool to differentiate DNA-free samples from samples with inhibitory substances.

  10. Quantification of differential gene expression by multiplexed targeted resequencing of cDNA

    Science.gov (United States)

    Arts, Peer; van der Raadt, Jori; van Gestel, Sebastianus H.C.; Steehouwer, Marloes; Shendure, Jay; Hoischen, Alexander; Albers, Cornelis A.

    2017-01-01

    Whole-transcriptome or RNA sequencing (RNA-Seq) is a powerful and versatile tool for functional analysis of different types of RNA molecules, but sample reagent and sequencing cost can be prohibitive for hypothesis-driven studies where the aim is to quantify differential expression of a limited number of genes. Here we present an approach for quantification of differential mRNA expression by targeted resequencing of complementary DNA using single-molecule molecular inversion probes (cDNA-smMIPs) that enable highly multiplexed resequencing of cDNA target regions of ∼100 nucleotides and counting of individual molecules. We show that accurate estimates of differential expression can be obtained from molecule counts for hundreds of smMIPs per reaction and that smMIPs are also suitable for quantification of relative gene expression and allele-specific expression. Compared with low-coverage RNA-Seq and a hybridization-based targeted RNA-Seq method, cDNA-smMIPs are a cost-effective high-throughput tool for hypothesis-driven expression analysis in large numbers of genes (10 to 500) and samples (hundreds to thousands). PMID:28474677

  11. Quantification and Sequencing of Crossover Recombinant Molecules from Arabidopsis Pollen DNA.

    Science.gov (United States)

    Choi, Kyuha; Yelina, Nataliya E; Serra, Heïdi; Henderson, Ian R

    2017-01-01

    During meiosis, homologous chromosomes undergo recombination, which can result in formation of reciprocal crossover molecules. Crossover frequency is highly variable across the genome, typically occurring in narrow hotspots, which has a significant effect on patterns of genetic diversity. Here we describe methods to measure crossover frequency in plants at the hotspot scale (bp-kb), using allele-specific PCR amplification from genomic DNA extracted from the pollen of F 1 heterozygous plants. We describe (1) titration methods that allow amplification, quantification and sequencing of single crossover molecules, (2) quantitative PCR methods to more rapidly measure crossover frequency, and (3) application of high-throughput sequencing for study of crossover distributions within hotspots. We provide detailed descriptions of key steps including pollen DNA extraction, prior identification of hotspot locations, allele-specific oligonucleotide design, and sequence analysis approaches. Together, these methods allow the rate and recombination topology of plant hotspots to be robustly measured and compared between varied genetic backgrounds and environmental conditions.

  12. Single DNA imaging and length quantification through a mobile phone microscope

    Science.gov (United States)

    Wei, Qingshan; Luo, Wei; Chiang, Samuel; Kappel, Tara; Mejia, Crystal; Tseng, Derek; Chan, Raymond Yan L.; Yan, Eddie; Qi, Hangfei; Shabbir, Faizan; Ozkan, Haydar; Feng, Steve; Ozcan, Aydogan

    2016-03-01

    The development of sensitive optical microscopy methods for the detection of single DNA molecules has become an active research area which cultivates various promising applications including point-of-care (POC) genetic testing and diagnostics. Direct visualization of individual DNA molecules usually relies on sophisticated optical microscopes that are mostly available in well-equipped laboratories. For POC DNA testing/detection, there is an increasing need for the development of new single DNA imaging and sensing methods that are field-portable, cost-effective, and accessible for diagnostic applications in resource-limited or field-settings. For this aim, we developed a mobile-phone integrated fluorescence microscopy platform that allows imaging and sizing of single DNA molecules that are stretched on a chip. This handheld device contains an opto-mechanical attachment integrated onto a smartphone camera module, which creates a high signal-to-noise ratio dark-field imaging condition by using an oblique illumination/excitation configuration. Using this device, we demonstrated imaging of individual linearly stretched λ DNA molecules (48 kilobase-pair, kbp) over 2 mm2 field-of-view. We further developed a robust computational algorithm and a smartphone app that allowed the users to quickly quantify the length of each DNA fragment imaged using this mobile interface. The cellphone based device was tested by five different DNA samples (5, 10, 20, 40, and 48 kbp), and a sizing accuracy of <1 kbp was demonstrated for DNA strands longer than 10 kbp. This mobile DNA imaging and sizing platform can be very useful for various diagnostic applications including the detection of disease-specific genes and quantification of copy-number-variations at POC settings.

  13. Chromatin Immunoprecipitation (ChIP): Revisiting the Efficacy of Sample Preparation, Sonication, Quantification of Sheared DNA, and Analysis via PCR

    Science.gov (United States)

    Schoppee Bortz, Pamela D.; Wamhoff, Brian R.

    2011-01-01

    The “quantitative” ChIP, a tool commonly used to study protein-DNA interactions in cells and tissue, is a difficult assay often plagued with technical error. We present, herein, the process required to merge multiple protocols into a quick, reliable and easy method and an approach to accurately quantify ChIP DNA prior to performing PCR. We demonstrate that high intensity sonication for at least 30 min is required for full cellular disruption and maximum DNA recovery because ChIP lysis buffers fail to lyse formaldehyde-fixed cells. In addition, extracting ChIP DNA with chelex-100 yields samples that are too dilute for evaluation of shearing efficiency or quantification via nanospectrophotometry. However, DNA extracted from the Mock-ChIP supernatant via the phenol-chloroform-isoamyl alcohol (PCIA) method can be used to evaluate DNA shearing efficiency and used as the standard in a fluorescence-based microplate assay. This enabled accurate quantification of DNA in chelex-extracted ChIP samples and normalization to total DNA concentration prior to performing real-time PCR (rtPCR). Thus, a quick ChIP assay that can be completed in nine bench hours over two days has been validated along with a rapid, accurate and repeatable way to quantify ChIP DNA. The resulting rtPCR data more accurately depicts treatment effects on protein-DNA interactions of interest. PMID:22046253

  14. The value of fluorimetry (Qubit) and spectrophotometry (NanoDrop) in the quantification of cell-free DNA (cfDNA) in malignant melanoma and prostate cancer patients.

    Science.gov (United States)

    Ponti, Giovanni; Maccaferri, Monia; Manfredini, Marco; Kaleci, Shaniko; Mandrioli, Mauro; Pellacani, Giovanni; Ozben, Tomris; Depenni, Roberta; Bianchi, Giampaolo; Pirola, Giacomo Maria; Tomasi, Aldo

    2018-04-01

    Circulating cell-free tumor DNA (cfDNA) is of crucial interest in oncology. cfDNA constitutes a potential prognostic and therapeutic marker for different solid tumors and can be used in the diagnostic and therapeutic management of cancer patients for which nowadays there are no valid laboratory markers. In the present study, the quality and quantity of the cfDNA were assessed by different quantification procedures, in order to identify the potential applications of these techniques in the preliminary cfDNA quantification. Qubit with single (ss) and double strand (ds) DNA assay kits, NanoDrop and quantitative Real Time PCR (qPCR), were adopted to assess the cfDNA in the blood samples of 18 melanoma patients, 67 prostate cancer patients and 15 healthy controls. The quantification by NanoDrop (average value 8.48ng/μl, 95% confidence limit (CL)=7.23-9.73), Qubit ssDNA (average value 23.08ng/μl, CL=19.88-26.28), dsDNA (average value 4.32ng/μl, CL=3.52-5.12) assay kits and qPCR (average value 0.39ng/μl, CL=0.31-0.47) revealed differences among the four procedures. Qubit 2.0 ss-DNA kit gave higher cfDNA concentration values for all the samples analyzed. In detail, Qubit ssDNA assay revealed higher sensitivity in the quantification of small amounts of pure ss-DNA and ds-DNA, while NanoDrop allowed the assessment of the purity of cfDNA samples. The NanoDrop and Qubit 2.0 measurements were analyzed in order to define their correlation with qPCR cfDNA assessment, showing good correlation values with the qPCR that should be considered the "gold standard". In our proposal, the sequential combination of NanoDrop and Qubit ssDNA methods should be adopted for a cost-effective preliminary assessment of total circulating cfDNA in melanoma and prostate cancer patients, and only discordant values should undergo qPCR assessment. Copyright © 2018 Elsevier B.V. All rights reserved.

  15. High throughput multiplex real time PCR assay for the simultaneous quantification of DNA and RNA viruses infecting cassava plants

    OpenAIRE

    Otti, Gerald; Bouvaine, Sophie; Kimata, Bernadetha; Mkamillo, Geoffrey; Kumar, Lava; Tomlins, Keith; Maruthi, M.N.

    2016-01-01

    Aims: To develop a multiplex TaqMan-based real-time PCR assay (qPCR) for the simultaneous detection and quantification of both RNA and DNA viruses affecting cassava (Manihot esculenta) in eastern Africa.\\ud \\ud Methods and Results: The diagnostic assay was developed for two RNA viruses; Cassava brown streak virus (CBSV) and Uganda cassava brown streak virus (UCBSV) and two predominant DNA viruses; African cassava mosaic virus (ACMV) and East African cassava mosaic virus (EACMV), which cause t...

  16. Quantification Methods of Management Skills in Shipping

    Directory of Open Access Journals (Sweden)

    Riana Iren RADU

    2012-04-01

    Full Text Available Romania can not overcome the financial crisis without business growth, without finding opportunities for economic development and without attracting investment into the country. Successful managers find ways to overcome situations of uncertainty. The purpose of this paper is to determine the managerial skills developed by the Romanian fluvial shipping company NAVROM (hereinafter CNFR NAVROM SA, compared with ten other major competitors in the same domain, using financial information of these companies during the years 2005-2010. For carrying out the work it will be used quantification methods of managerial skills to CNFR NAVROM SA Galati, Romania, as example mentioning the analysis of financial performance management based on profitability ratios, net profit margin, suppliers management, turnover.

  17. Standardless quantification methods in electron probe microanalysis

    Energy Technology Data Exchange (ETDEWEB)

    Trincavelli, Jorge, E-mail: trincavelli@famaf.unc.edu.ar [Facultad de Matemática, Astronomía y Física, Universidad Nacional de Córdoba, Ciudad Universitaria, 5000 Córdoba (Argentina); Instituto de Física Enrique Gaviola, Consejo Nacional de Investigaciones Científicas y Técnicas de la República Argentina, Medina Allende s/n, Ciudad Universitaria, 5000 Córdoba (Argentina); Limandri, Silvina, E-mail: s.limandri@conicet.gov.ar [Facultad de Matemática, Astronomía y Física, Universidad Nacional de Córdoba, Ciudad Universitaria, 5000 Córdoba (Argentina); Instituto de Física Enrique Gaviola, Consejo Nacional de Investigaciones Científicas y Técnicas de la República Argentina, Medina Allende s/n, Ciudad Universitaria, 5000 Córdoba (Argentina); Bonetto, Rita, E-mail: bonetto@quimica.unlp.edu.ar [Centro de Investigación y Desarrollo en Ciencias Aplicadas Dr. Jorge Ronco, Consejo Nacional de Investigaciones Científicas y Técnicas de la República Argentina, Facultad de Ciencias Exactas, de la Universidad Nacional de La Plata, Calle 47 N° 257, 1900 La Plata (Argentina)

    2014-11-01

    The elemental composition of a solid sample can be determined by electron probe microanalysis with or without the use of standards. The standardless algorithms are quite faster than the methods that require standards; they are useful when a suitable set of standards is not available or for rough samples, and also they help to solve the problem of current variation, for example, in equipments with cold field emission gun. Due to significant advances in the accuracy achieved during the last years, product of the successive efforts made to improve the description of generation, absorption and detection of X-rays, the standardless methods have increasingly become an interesting option for the user. Nevertheless, up to now, algorithms that use standards are still more precise than standardless methods. It is important to remark, that care must be taken with results provided by standardless methods that normalize the calculated concentration values to 100%, unless an estimate of the errors is reported. In this work, a comprehensive discussion of the key features of the main standardless quantification methods, as well as the level of accuracy achieved by them is presented. - Highlights: • Standardless methods are a good alternative when no suitable standards are available. • Their accuracy reaches 10% for 95% of the analyses when traces are excluded. • Some of them are suitable for the analysis of rough samples.

  18. Genomic DNA-based absolute quantification of gene expression in Vitis.

    Science.gov (United States)

    Gambetta, Gregory A; McElrone, Andrew J; Matthews, Mark A

    2013-07-01

    Many studies in which gene expression is quantified by polymerase chain reaction represent the expression of a gene of interest (GOI) relative to that of a reference gene (RG). Relative expression is founded on the assumptions that RG expression is stable across samples, treatments, organs, etc., and that reaction efficiencies of the GOI and RG are equal; assumptions which are often faulty. The true variability in RG expression and actual reaction efficiencies are seldom determined experimentally. Here we present a rapid and robust method for absolute quantification of expression in Vitis where varying concentrations of genomic DNA were used to construct GOI standard curves. This methodology was utilized to absolutely quantify and determine the variability of the previously validated RG ubiquitin (VvUbi) across three test studies in three different tissues (roots, leaves and berries). In addition, in each study a GOI was absolutely quantified. Data sets resulting from relative and absolute methods of quantification were compared and the differences were striking. VvUbi expression was significantly different in magnitude between test studies and variable among individual samples. Absolute quantification consistently reduced the coefficients of variation of the GOIs by more than half, often resulting in differences in statistical significance and in some cases even changing the fundamental nature of the result. Utilizing genomic DNA-based absolute quantification is fast and efficient. Through eliminating error introduced by assuming RG stability and equal reaction efficiencies between the RG and GOI this methodology produces less variation, increased accuracy and greater statistical power. © 2012 Scandinavian Plant Physiology Society.

  19. Optimal purification and sensitive quantification of DNA from fecal samples

    DEFF Research Database (Denmark)

    Jensen, Annette Nygaard; Hoorfar, Jeffrey

    2002-01-01

    Application of reliable, rapid and sensitive methods to laboratory diagnosis of zoonotic infections continues to challenge microbiological laboratories. The recovery of DNA from a swine fecal sample and a bacterial culture extracted by a conventional phenol-chloroform extraction method was compared...... = 0.99 and R-2 = 1.00). In conclusion, silica-membrane, columns can provide a more convenient and less hazardous alternative to the conventional phenol-based method. The results have implication for further improvement of sensitive amplification methods for laboratory diagnosis....

  20. Highly sensitive polymerase chain reaction-free quantum dot-based quantification of forensic genomic DNA

    International Nuclear Information System (INIS)

    Tak, Yu Kyung; Kim, Won Young; Kim, Min Jung; Han, Eunyoung; Han, Myun Soo; Kim, Jong Jin; Kim, Wook; Lee, Jong Eun; Song, Joon Myong

    2012-01-01

    Highlights: ► Genomic DNA quantification were performed using a quantum dot-labeled Alu sequence. ► This probe provided PCR-free determination of human genomic DNA. ► Qdot-labeled Alu probe-hybridized genomic DNAs had a 2.5-femtogram detection limit. ► Qdot-labeled Alu sequence was used to assess DNA samples for human identification. - Abstract: Forensic DNA samples can degrade easily due to exposure to light and moisture at the crime scene. In addition, the amount of DNA acquired at a criminal site is inherently limited. This limited amount of human DNA has to be quantified accurately after the process of DNA extraction. The accurately quantified extracted genomic DNA is then used as a DNA template in polymerase chain reaction (PCR) amplification for short tandem repeat (STR) human identification. Accordingly, highly sensitive and human-specific quantification of forensic DNA samples is an essential issue in forensic study. In this work, a quantum dot (Qdot)-labeled Alu sequence was developed as a probe to simultaneously satisfy both the high sensitivity and human genome selectivity for quantification of forensic DNA samples. This probe provided PCR-free determination of human genomic DNA and had a 2.5-femtogram detection limit due to the strong emission and photostability of the Qdot. The Qdot-labeled Alu sequence has been used successfully to assess 18 different forensic DNA samples for STR human identification.

  1. Fragmentation of DNA affects the accuracy of the DNA quantitation by the commonly used methods

    Directory of Open Access Journals (Sweden)

    Sedlackova Tatiana

    2013-02-01

    Full Text Available Abstract Background Specific applications and modern technologies, like non-invasive prenatal testing, non-invasive cancer diagnostic and next generation sequencing, are currently in the focus of researchers worldwide. These have common characteristics in use of highly fragmented DNA molecules for analysis. Hence, for the performance of molecular methods, DNA concentration is a crucial parameter; we compared the influence of different levels of DNA fragmentation on the accuracy of DNA concentration measurements. Results In our comparison, the performance of the currently most commonly used methods for DNA concentration measurement (spectrophotometric, fluorometric and qPCR based were tested on artificially fragmented DNA samples. In our comparison, unfragmented and three specifically fragmented DNA samples were used. According to our results, the level of fragmentation did not influence the accuracy of spectrophotometric measurements of DNA concentration, while other methods, fluorometric as well as qPCR-based, were significantly influenced and a decrease in measured concentration was observed with more intensive DNA fragmentation. Conclusions Our study has confirmed that the level of fragmentation of DNA has significant impact on accuracy of DNA concentration measurement with two of three mostly used methods (PicoGreen and qPCR. Only spectrophotometric measurement was not influenced by the level of fragmentation, but sensitivity of this method was lowest among the three tested. Therefore if it is possible the DNA quantification should be performed with use of equally fragmented control DNA.

  2. Quantification of DNA cleavage specificity in Hi-C experiments.

    Science.gov (United States)

    Meluzzi, Dario; Arya, Gaurav

    2016-01-08

    Hi-C experiments produce large numbers of DNA sequence read pairs that are typically analyzed to deduce genomewide interactions between arbitrary loci. A key step in these experiments is the cleavage of cross-linked chromatin with a restriction endonuclease. Although this cleavage should happen specifically at the enzyme's recognition sequence, an unknown proportion of cleavage events may involve other sequences, owing to the enzyme's star activity or to random DNA breakage. A quantitative estimation of these non-specific cleavages may enable simulating realistic Hi-C read pairs for validation of downstream analyses, monitoring the reproducibility of experimental conditions and investigating biophysical properties that correlate with DNA cleavage patterns. Here we describe a computational method for analyzing Hi-C read pairs to estimate the fractions of cleavages at different possible targets. The method relies on expressing an observed local target distribution downstream of aligned reads as a linear combination of known conditional local target distributions. We validated this method using Hi-C read pairs obtained by computer simulation. Application of the method to experimental Hi-C datasets from murine cells revealed interesting similarities and differences in patterns of cleavage across the various experiments considered. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  3. Selective detection and quantification of modified DNA with solid-state nanopores.

    Science.gov (United States)

    Carlsen, Autumn T; Zahid, Osama K; Ruzicka, Jan A; Taylor, Ethan W; Hall, Adam R

    2014-10-08

    We demonstrate a solid-state nanopore assay for the unambiguous discrimination and quantification of modified DNA. Individual streptavidin proteins are employed as high-affinity tags for DNA containing a single biotin moiety. We establish that the rate of translocation events corresponds directly to relative concentration of protein-DNA complexes and use the selectivity of our approach to quantify modified oligonucleotides from among a background of unmodified DNA in solution.

  4. Quantification of integrated HIV DNA by repetitive-sampling Alu-HIV PCR on the basis of poisson statistics.

    Science.gov (United States)

    De Spiegelaere, Ward; Malatinkova, Eva; Lynch, Lindsay; Van Nieuwerburgh, Filip; Messiaen, Peter; O'Doherty, Una; Vandekerckhove, Linos

    2014-06-01

    Quantification of integrated proviral HIV DNA by repetitive-sampling Alu-HIV PCR is a candidate virological tool to monitor the HIV reservoir in patients. However, the experimental procedures and data analysis of the assay are complex and hinder its widespread use. Here, we provide an improved and simplified data analysis method by adopting binomial and Poisson statistics. A modified analysis method on the basis of Poisson statistics was used to analyze the binomial data of positive and negative reactions from a 42-replicate Alu-HIV PCR by use of dilutions of an integration standard and on samples of 57 HIV-infected patients. Results were compared with the quantitative output of the previously described Alu-HIV PCR method. Poisson-based quantification of the Alu-HIV PCR was linearly correlated with the standard dilution series, indicating that absolute quantification with the Poisson method is a valid alternative for data analysis of repetitive-sampling Alu-HIV PCR data. Quantitative outputs of patient samples assessed by the Poisson method correlated with the previously described Alu-HIV PCR analysis, indicating that this method is a valid alternative for quantifying integrated HIV DNA. Poisson-based analysis of the Alu-HIV PCR data enables absolute quantification without the need of a standard dilution curve. Implementation of the CI estimation permits improved qualitative analysis of the data and provides a statistical basis for the required minimal number of technical replicates. © 2014 The American Association for Clinical Chemistry.

  5. A Microneedle Functionalized with Polyethyleneimine and Nanotubes for Highly Sensitive, Label-Free Quantification of DNA.

    Science.gov (United States)

    Saadat-Moghaddam, Darius; Kim, Jong-Hoon

    2017-08-16

    The accurate measure of DNA concentration is necessary for many DNA-based biological applications. However, the current methods are limited in terms of sensitivity, reproducibility, human error, and contamination. Here, we present a microneedle functionalized with polyethyleneimine (PEI) and single-walled carbon nanotubes (SWCNTs) for the highly sensitive quantification of DNA. The microneedle was fabricated using ultraviolet (UV) lithography and anisotropic etching, and then functionalized with PEI and SWCNTs through a dip coating process. The electrical characteristics of the microneedle change with the accumulation of DNA on the surface. Current-voltage measurements in deionized water were conducted to study these changes in the electrical properties of the sensor. The sensitivity test found the signal to be discernable from the noise level down to 100 attomolar (aM), demonstrating higher sensitivity than currently available UV fluorescence and UV absorbance based methods. A microneedle without any surface modification only had a 100 femtomolar (fM) sensitivity. All measurement results were consistent with fluorescence microscopy.

  6. Combination of pentafluorophenylhydrazine derivatization and isotope dilution LC-MS/MS techniques for the quantification of apurinic/apyrimidinic sites in cellular DNA.

    Science.gov (United States)

    Li, Jie; Leung, Elvis M K; Choi, Martin M F; Chan, Wan

    2013-05-01

    Apurinic/apyrimidinic (AP) sites are common DNA lesions arising from spontaneous hydrolysis of the N-glycosidic bond and base-excision repair mechanisms of the modified bases. Due to the strong association of AP site formation with physically/chemically induced DNA damage, quantifying AP sites provides important information for risk assessment of exposure to genotoxins and oxidative stress. However, rigorous quantification of AP sites in DNA has been hampered by technical problems relating to the sensitivity and selectivity of existing analytical methods. We have developed a new isotope dilution liquid chromatography-coupled tandem mass spectrometry (LC-MS/MS) method for the rigorous quantification of AP sites in genomic DNA. The method entails enzymatic digestion of AP site-containing DNA by endo- and exonucleases, derivatization with pentafluorophenylhydrazine (PFPH), addition of an isotopically labeled PFPH derivative as internal standard, and quantification by LC-MS/MS. The combination of PFPH derivatization with LC-MS/MS analysis on a triple quadrupole mass spectrometer allows for sensitive and selective quantification of AP sites in DNA at a detection limit of 6.5 fmol, corresponding to 4 AP sites/10(9) nt in 5 μg of DNA, which is at least ten times more sensitive than existing analytical methods. The protocol was validated by AP site-containing oligonucleotides and applied in quantifying methyl methanesulfonate-induced formation of AP sites in cellular DNA.

  7. Detection and quantification of 4-ABP adducts in DNA from bladder cancer patients.

    Science.gov (United States)

    Zayas, Beatriz; Stillwell, Sara W; Wishnok, John S; Trudel, Laura J; Skipper, Paul; Yu, Mimi C; Tannenbaum, Steven R; Wogan, Gerald N

    2007-02-01

    We analyzed bladder DNA from 27 cancer patients for dG-C8-4-aminobiphenyl (dG-C8-ABP) adducts using the liquid chromatography tandem mass spectrometry method with a 700 attomol (1 adduct in 10(9) bases) detection limit. Hemoglobin (Hb) 4-aminobiphenyl (4-ABP) adduct levels were measured by gas chromatography-mass spectrometry. After isolation of dG-C8-ABP by immunoaffinity chromatography and further purification, deuterated (d9) dG-C8-ABP (MW=443 Da) was added to each sample. Structural evidence and adduct quantification were determined by selected reaction monitoring, based on the expected adduct ion [M+H+]+1, at m/z 435 with fragmentation to the product ion at m/z 319, and monitoring of the transition for the internal standard, m/z 444-->328. The method was validated by analysis of DNA (100 microg each) from calf thymus; livers from ABP-treated and untreated rats; human placentas; and TK6 lymphoblastoid cells. Adduct was detected at femtomol levels in DNA from livers of ABP-treated rats and calf thymus, but not in other controls. The method was applied to 41 DNA samples (200 microg each) from 27 human bladders; 28 from tumor and 14 from surrounding non-tumor tissue. Of 27 tissues analyzed, 44% (12) contained 5-80 dG-C8-ABP adducts per 10(9) bases; only 1 out of 27 (4%) contained adduct in both tumor and surrounding tissues. The Hb adduct was detected in samples from all patients, at levels of 12-1960 pg per gram Hb. There was no correlation between levels of DNA and Hb adducts. The presence of DNA adducts in 44% of the subjects and high levels of Hb adducts in these non-smokers indicate environmental sources of exposure to 4-ABP.

  8. The Qiagen Investigator® Quantiplex HYres as an alternative kit for DNA quantification.

    Science.gov (United States)

    Frégeau, Chantal J; Laurin, Nancy

    2015-05-01

    The Investigator® Quantiplex HYres kit was evaluated as a potential replacement for dual DNA quantification of casework samples. This kit was determined to be highly sensitive with a limit of quantification and limit of detection of 0.0049ng/μL and 0.0003ng/μL, respectively, for both human and male DNA, using full or half reaction volumes. It was also accurate in assessing the amount of male DNA present in 96 mock and actual casework male:female mixtures (various ratios) processed in this exercise. The close correlation between the male/human DNA ratios expressed in percentages derived from the Investigator® Quantiplex HYres quantification results and the male DNA proportion calculated in mixed AmpFlSTR® Profiler® Plus or AmpFlSTR® Identifiler® Plus profiles, using the Amelogenin Y peak and STR loci, allowed guidelines to be developed to facilitate decisions regarding when to submit samples to Y-STR rather than autosomal STR profiling. The internal control (IC) target was shown to be more sensitive to inhibitors compared to the human and male DNA targets included in the Investigator® Quantiplex HYres kit serving as a good quality assessor of DNA extracts. The new kit met our criteria of enhanced sensitivity, accuracy, consistency, reliability and robustness for casework DNA quantification. Crown Copyright © 2015. Published by Elsevier Ireland Ltd. All rights reserved.

  9. Accurate and sensitive quantification of protein-DNA binding affinity.

    Science.gov (United States)

    Rastogi, Chaitanya; Rube, H Tomas; Kribelbauer, Judith F; Crocker, Justin; Loker, Ryan E; Martini, Gabriella D; Laptenko, Oleg; Freed-Pastor, William A; Prives, Carol; Stern, David L; Mann, Richard S; Bussemaker, Harmen J

    2018-04-17

    Transcription factors (TFs) control gene expression by binding to genomic DNA in a sequence-specific manner. Mutations in TF binding sites are increasingly found to be associated with human disease, yet we currently lack robust methods to predict these sites. Here, we developed a versatile maximum likelihood framework named No Read Left Behind (NRLB) that infers a biophysical model of protein-DNA recognition across the full affinity range from a library of in vitro selected DNA binding sites. NRLB predicts human Max homodimer binding in near-perfect agreement with existing low-throughput measurements. It can capture the specificity of the p53 tetramer and distinguish multiple binding modes within a single sample. Additionally, we confirm that newly identified low-affinity enhancer binding sites are functional in vivo, and that their contribution to gene expression matches their predicted affinity. Our results establish a powerful paradigm for identifying protein binding sites and interpreting gene regulatory sequences in eukaryotic genomes. Copyright © 2018 the Author(s). Published by PNAS.

  10. Performance of the Real-Q EBV Quantification Kit for Epstein-Barr Virus DNA Quantification in Whole Blood.

    Science.gov (United States)

    Huh, Hee Jae; Park, Jong Eun; Kim, Ji Youn; Yun, Sun Ae; Lee, Myoung Keun; Lee, Nam Yong; Kim, Jong Won; Ki, Chang Seok

    2017-03-01

    There has been increasing interest in standardized and quantitative Epstein-Barr virus (EBV) DNA testing for the management of EBV disease. We evaluated the performance of the Real-Q EBV Quantification Kit (BioSewoom, Korea) in whole blood (WB). Nucleic acid extraction and real-time PCR were performed by using the MagNA Pure 96 (Roche Diagnostics, Germany) and 7500 Fast real-time PCR system (Applied Biosystems, USA), respectively. Assay sensitivity, linearity, and conversion factor were determined by using the World Health Organization international standard diluted in EBV-negative WB. We used 81 WB clinical specimens to compare performance of the Real-Q EBV Quantification Kit and artus EBV RG PCR Kit (Qiagen, Germany). The limit of detection (LOD) and limit of quantification (LOQ) for the Real-Q kit were 453 and 750 IU/mL, respectively. The conversion factor from EBV genomic copies to IU was 0.62. The linear range of the assay was from 750 to 10⁶ IU/mL. Viral load values measured with the Real-Q assay were on average 0.54 log₁₀ copies/mL higher than those measured with the artus assay. The Real-Q assay offered good analytical performance for EBV DNA quantification in WB.

  11. Accurate quantification of microRNA via single strand displacement reaction on DNA origami motif.

    Directory of Open Access Journals (Sweden)

    Jie Zhu

    Full Text Available DNA origami is an emerging technology that assembles hundreds of staple strands and one single-strand DNA into certain nanopattern. It has been widely used in various fields including detection of biological molecules such as DNA, RNA and proteins. MicroRNAs (miRNAs play important roles in post-transcriptional gene repression as well as many other biological processes such as cell growth and differentiation. Alterations of miRNAs' expression contribute to many human diseases. However, it is still a challenge to quantitatively detect miRNAs by origami technology. In this study, we developed a novel approach based on streptavidin and quantum dots binding complex (STV-QDs labeled single strand displacement reaction on DNA origami to quantitatively detect the concentration of miRNAs. We illustrated a linear relationship between the concentration of an exemplary miRNA as miRNA-133 and the STV-QDs hybridization efficiency; the results demonstrated that it is an accurate nano-scale miRNA quantifier motif. In addition, both symmetrical rectangular motif and asymmetrical China-map motif were tested. With significant linearity in both motifs, our experiments suggested that DNA Origami motif with arbitrary shape can be utilized in this method. Since this DNA origami-based method we developed owns the unique advantages of simple, time-and-material-saving, potentially multi-targets testing in one motif and relatively accurate for certain impurity samples as counted directly by atomic force microscopy rather than fluorescence signal detection, it may be widely used in quantification of miRNAs.

  12. Accurate Quantification of microRNA via Single Strand Displacement Reaction on DNA Origami Motif

    Science.gov (United States)

    Lou, Jingyu; Li, Weidong; Li, Sheng; Zhu, Hongxin; Yang, Lun; Zhang, Aiping; He, Lin; Li, Can

    2013-01-01

    DNA origami is an emerging technology that assembles hundreds of staple strands and one single-strand DNA into certain nanopattern. It has been widely used in various fields including detection of biological molecules such as DNA, RNA and proteins. MicroRNAs (miRNAs) play important roles in post-transcriptional gene repression as well as many other biological processes such as cell growth and differentiation. Alterations of miRNAs' expression contribute to many human diseases. However, it is still a challenge to quantitatively detect miRNAs by origami technology. In this study, we developed a novel approach based on streptavidin and quantum dots binding complex (STV-QDs) labeled single strand displacement reaction on DNA origami to quantitatively detect the concentration of miRNAs. We illustrated a linear relationship between the concentration of an exemplary miRNA as miRNA-133 and the STV-QDs hybridization efficiency; the results demonstrated that it is an accurate nano-scale miRNA quantifier motif. In addition, both symmetrical rectangular motif and asymmetrical China-map motif were tested. With significant linearity in both motifs, our experiments suggested that DNA Origami motif with arbitrary shape can be utilized in this method. Since this DNA origami-based method we developed owns the unique advantages of simple, time-and-material-saving, potentially multi-targets testing in one motif and relatively accurate for certain impurity samples as counted directly by atomic force microscopy rather than fluorescence signal detection, it may be widely used in quantification of miRNAs. PMID:23990889

  13. Accurate quantification of microRNA via single strand displacement reaction on DNA origami motif.

    Science.gov (United States)

    Zhu, Jie; Feng, Xiaolu; Lou, Jingyu; Li, Weidong; Li, Sheng; Zhu, Hongxin; Yang, Lun; Zhang, Aiping; He, Lin; Li, Can

    2013-01-01

    DNA origami is an emerging technology that assembles hundreds of staple strands and one single-strand DNA into certain nanopattern. It has been widely used in various fields including detection of biological molecules such as DNA, RNA and proteins. MicroRNAs (miRNAs) play important roles in post-transcriptional gene repression as well as many other biological processes such as cell growth and differentiation. Alterations of miRNAs' expression contribute to many human diseases. However, it is still a challenge to quantitatively detect miRNAs by origami technology. In this study, we developed a novel approach based on streptavidin and quantum dots binding complex (STV-QDs) labeled single strand displacement reaction on DNA origami to quantitatively detect the concentration of miRNAs. We illustrated a linear relationship between the concentration of an exemplary miRNA as miRNA-133 and the STV-QDs hybridization efficiency; the results demonstrated that it is an accurate nano-scale miRNA quantifier motif. In addition, both symmetrical rectangular motif and asymmetrical China-map motif were tested. With significant linearity in both motifs, our experiments suggested that DNA Origami motif with arbitrary shape can be utilized in this method. Since this DNA origami-based method we developed owns the unique advantages of simple, time-and-material-saving, potentially multi-targets testing in one motif and relatively accurate for certain impurity samples as counted directly by atomic force microscopy rather than fluorescence signal detection, it may be widely used in quantification of miRNAs.

  14. DNA imaging and quantification using chemi-luminescent probes; Imagerie et quantification d`ADN par chimiluminescence

    Energy Technology Data Exchange (ETDEWEB)

    Dorner, G; Redjdal, N; Laniece, P; Siebert, R; Tricoire, H; Valentin, L [Groupe I.P.B., Experimental Research Division, Inst. de Physique Nucleaire, Paris-11 Univ., 91 - Orsay (France)

    1999-11-01

    During this interdisciplinary study we have developed an ultra sensitive and reliable imaging system of DNA labelled by chemiluminescence. Based on a liquid nitrogen cooled CCD, the system achieves sensitivities down to 10 fg/mm{sup 2} labelled DNA over a surface area of 25 x 25 cm{sup 2} with a sub-millimeter resolution. Commercially available chemi-luminescent - and enhancer molecules are compared and their reaction conditions optimized for best signal-to-noise ratios. Double labelling was performed to verify quantification with radioactive probes. (authors) 1 fig.

  15. A performance study on three qPCR quantification kits and their compatibilities with the 6-dye DNA profiling systems.

    Science.gov (United States)

    Lin, Sze-Wah; Li, Christina; Ip, Stephen C Y

    2018-03-01

    DNA quantification plays an integral role in forensic DNA profiling. Not only does it estimate the total amount of amplifiable human autosomal and male DNA to ensure optimal amplification of target DNA for subsequent analysis, but also assesses the extraction efficiency and purity of the DNA extract. Latest DNA quantification systems even offer an estimate for the degree of DNA degradation in a sample. Here, we report the performance of three new generation qPCR kits, namely Investigator ® Quantiplex HYres Kit from QIAGEN, Quantifiler ® Trio DNA Quantification Kit from Applied Biosystems™, and PowerQuant ® System from Promega, and their compatibilities with three 6-dye DNA profiling systems. Our results have demonstrated that all three kits generate standard curves with satisfactory consistency and reproducibility, and are capable of screening out traces of male DNA in the presence of 30-fold excess of female DNA. They also exhibit a higher tolerance to PCR inhibition than Quantifiler ® Human DNA Quantification Kit from Applied Biosystems™ in autosomal DNA quantification. PowerQuant ® , as compared to Quantiplex HYres and Quantifiler ® Trio, shows a better precision for both autosomal and male DNA quantifications. Quantifiler ® Trio and PowerQuant ® in contrast to Quantiplex HYres offer better correlations with lower discrepancies between autosomal and male DNA quantification, and their additional degradation index features provide a detection platform for inhibited and/or degraded DNA template. Regarding the compatibility between these quantification and profiling systems: (1) both Quantifiler ® Trio and PowerQuant ® work well with GlobalFiler and Fusion 6C, allowing a fairly accurate prediction of their DNA typing results based on the quantification values; (2) Quantiplex HYres offers a fairly reliable IPC system for detecting any potential inhibitions on Investigator 24plex, whereas Quantifiler ® Trio and PowerQuant ® suit better for Global

  16. Quantification of eDNA shedding rates from invasive bighead carp Hypophthalmichthys nobilis and silver carp Hypophthalmichthys molitrix

    Science.gov (United States)

    Klymus, Katy E.; Richter, Catherine A.; Chapman, Duane C.; Paukert, Craig P.

    2015-01-01

    Wildlife managers can more easily mitigate the effects of invasive species if action takes place before a population becomes established. Such early detection requires sensitive survey tools that can detect low numbers of individuals. Due to their high sensitivity, environmental DNA (eDNA) surveys hold promise as an early detection method for aquatic invasive species. Quantification of eDNA amounts may also provide data on species abundance and timing of an organism’s presence, allowing managers to successfully combat the spread of ecologically damaging species. To better understand the link between eDNA and an organism’s presence, it is crucial to know how eDNA is shed into the environment. Our study used quantitative PCR (qPCR) and controlled laboratory experiments to measure the amount of eDNA that two species of invasive bigheaded carps (Hypophthalmichthys nobilis and Hypophthalmichthys molitrix) shed into the water. We first measured how much eDNA a single fish sheds and the variability of these measurements. Then, in a series of manipulative lab experiments, we studied how temperature, biomass (grams of fish), and diet affect the shedding rate of eDNA by these fish. We found that eDNA amounts exhibit a positive relationship with fish biomass, and that feeding could increase the amount of eDNA shed by ten-fold, whereas water temperature did not have an effect. Our results demonstrate that quantification of eDNA may be useful for predicting carp density, as well as densities of other rare or invasive species.

  17. Quantification of DNA damage by single-cell electrophoresis

    International Nuclear Information System (INIS)

    Ikushima, Takaji

    1990-01-01

    A simple technique of micro-agarose gel electrophoresis has been developed to quantify DNA damage in individual cells. Cells are embedded in agarose gel on microscope slides, lysed by detergents and then electrophoresed for a short time under neutral or alkaline condition. In irradiated cells, DNA migrates from the nucleus toward the anode, displaying commet-like pattern by staining with DNA-specific fluorescence dye. DNA damage is evaluated by measuring the distance of DNA migration. The technique was applied for measuring DNA damage in single cells exposed to 60 Co γ-rays, or to KUR radiation in the presence or absence of 10 B-enriched boric acid. The enhanced production of double-stranded DNA breaks by 10 B(n,α) 7 Li reaction was demonstrated here. The significant increase in the length of DNA migration was observed in single cells exposed to such a low dose as 20 cGy after alkaline micro electrophoresis. (author)

  18. Absolute and direct microRNA quantification using DNA-gold nanoparticle probes.

    Science.gov (United States)

    Degliangeli, Federica; Kshirsagar, Prakash; Brunetti, Virgilio; Pompa, Pier Paolo; Fiammengo, Roberto

    2014-02-12

    DNA-gold nanoparticle probes are implemented in a simple strategy for direct microRNA (miRNA) quantification. Fluorescently labeled DNA-probe strands are immobilized on PEGylated gold nanoparticles (AuNPs). In the presence of target miRNA, DNA-RNA heteroduplexes are formed and become substrate for the endonuclease DSN (duplex-specific nuclease). Enzymatic hydrolysis of the DNA strands yields a fluorescence signal due to diffusion of the fluorophores away from the gold surface. We show that the molecular design of our DNA-AuNP probes, with the DNA strands immobilized on top of the PEG-based passivation layer, results in nearly unaltered enzymatic activity toward immobilized heteroduplexes compared to substrates free in solution. The assay, developed in a real-time format, allows absolute quantification of as little as 0.2 fmol of miR-203. We also show the application of the assay for direct quantification of cancer-related miR-203 and miR-21 in samples of extracted total RNA from cell cultures. The possibility of direct and absolute quantification may significantly advance the use of microRNAs as biomarkers in the clinical praxis.

  19. The impact of targeting repetitive BamHI-W sequences on the sensitivity and precision of EBV DNA quantification.

    Directory of Open Access Journals (Sweden)

    Armen Sanosyan

    Full Text Available Viral load monitoring and early Epstein-Barr virus (EBV DNA detection are essential in routine laboratory testing, especially in preemptive management of Post-transplant Lymphoproliferative Disorder. Targeting the repetitive BamHI-W sequence was shown to increase the sensitivity of EBV DNA quantification, but the variability of BamHI-W reiterations was suggested to be a source of quantification bias. We aimed to assess the extent of variability associated with BamHI-W PCR and its impact on the sensitivity of EBV DNA quantification using the 1st WHO international standard, EBV strains and clinical samples.Repetitive BamHI-W- and LMP2 single- sequences were amplified by in-house qPCRs and BXLF-1 sequence by a commercial assay (EBV R-gene™, BioMerieux. Linearity and limits of detection of in-house methods were assessed. The impact of repeated versus single target sequences on EBV DNA quantification precision was tested on B95.8 and Raji cell lines, possessing 11 and 7 copies of the BamHI-W sequence, respectively, and on clinical samples.BamHI-W qPCR demonstrated a lower limit of detection compared to LMP2 qPCR (2.33 log10 versus 3.08 log10 IU/mL; P = 0.0002. BamHI-W qPCR underestimated the EBV DNA load on Raji strain which contained fewer BamHI-W copies than the WHO standard derived from the B95.8 EBV strain (mean bias: - 0.21 log10; 95% CI, -0.54 to 0.12. Comparison of BamHI-W qPCR versus LMP2 and BXLF-1 qPCR showed an acceptable variability between EBV DNA levels in clinical samples with the mean bias being within 0.5 log10 IU/mL EBV DNA, whereas a better quantitative concordance was observed between LMP2 and BXLF-1 assays.Targeting BamHI-W resulted to a higher sensitivity compared to LMP2 but the variable reiterations of BamHI-W segment are associated with higher quantification variability. BamHI-W can be considered for clinical and therapeutic monitoring to detect an early EBV DNA and a dynamic change in viral load.

  20. The impact of targeting repetitive BamHI-W sequences on the sensitivity and precision of EBV DNA quantification.

    Science.gov (United States)

    Sanosyan, Armen; Fayd'herbe de Maudave, Alexis; Bollore, Karine; Zimmermann, Valérie; Foulongne, Vincent; Van de Perre, Philippe; Tuaillon, Edouard

    2017-01-01

    Viral load monitoring and early Epstein-Barr virus (EBV) DNA detection are essential in routine laboratory testing, especially in preemptive management of Post-transplant Lymphoproliferative Disorder. Targeting the repetitive BamHI-W sequence was shown to increase the sensitivity of EBV DNA quantification, but the variability of BamHI-W reiterations was suggested to be a source of quantification bias. We aimed to assess the extent of variability associated with BamHI-W PCR and its impact on the sensitivity of EBV DNA quantification using the 1st WHO international standard, EBV strains and clinical samples. Repetitive BamHI-W- and LMP2 single- sequences were amplified by in-house qPCRs and BXLF-1 sequence by a commercial assay (EBV R-gene™, BioMerieux). Linearity and limits of detection of in-house methods were assessed. The impact of repeated versus single target sequences on EBV DNA quantification precision was tested on B95.8 and Raji cell lines, possessing 11 and 7 copies of the BamHI-W sequence, respectively, and on clinical samples. BamHI-W qPCR demonstrated a lower limit of detection compared to LMP2 qPCR (2.33 log10 versus 3.08 log10 IU/mL; P = 0.0002). BamHI-W qPCR underestimated the EBV DNA load on Raji strain which contained fewer BamHI-W copies than the WHO standard derived from the B95.8 EBV strain (mean bias: - 0.21 log10; 95% CI, -0.54 to 0.12). Comparison of BamHI-W qPCR versus LMP2 and BXLF-1 qPCR showed an acceptable variability between EBV DNA levels in clinical samples with the mean bias being within 0.5 log10 IU/mL EBV DNA, whereas a better quantitative concordance was observed between LMP2 and BXLF-1 assays. Targeting BamHI-W resulted to a higher sensitivity compared to LMP2 but the variable reiterations of BamHI-W segment are associated with higher quantification variability. BamHI-W can be considered for clinical and therapeutic monitoring to detect an early EBV DNA and a dynamic change in viral load.

  1. Application of Fuzzy Comprehensive Evaluation Method in Trust Quantification

    Directory of Open Access Journals (Sweden)

    Shunan Ma

    2011-10-01

    Full Text Available Trust can play an important role for the sharing of resources and information in open network environments. Trust quantification is thus an important issue in dynamic trust management. By considering the fuzziness and uncertainty of trust, in this paper, we propose a fuzzy comprehensive evaluation method to quantify trust along with a trust quantification algorithm. Simulation results show that the trust quantification algorithm that we propose can effectively quantify trust and the quantified value of an entity's trust is consistent with the behavior of the entity.

  2. Toward metrological traceability for DNA fragment ratios in GM quantification. 3. Suitability of DNA calibrants studied with a MON 810 corn model.

    Science.gov (United States)

    Charels, Diana; Broeders, Sylvia; Corbisier, Philippe; Trapmann, Stefanie; Schimmel, Heinz; Emons, Hendrik

    2007-05-02

    The quantification of GMOs by real-time PCR relies on an external calibrant. In this paper the suitability of two DNA calibrants, genomic DNA from plant leaves and plasmidic DNA, was investigated. The PCR efficiencies, the correlation coefficients of the calibration curves, and the ratios between PCR efficiencies of transgenic and endogenous sequences were compared for both calibrants using 59 data sets produced by 43 laboratories. There were no significant differences between plasmidic and genomic DNA except for the PCR efficiencies of the calibration curves for the transgene of the construct-specific real-time PCR method. In the GM system investigated, PCR efficiencies of plasmidic calibrants were slightly closer to the PCR efficiencies observed for the unknowns than those of the genomic DNA calibrant. Therefore, plasmidic DNA was the more suitable calibrant for the PCR measurements on genomic DNA extracted from MON 810 seeds. It is shown that plasmidic DNA is an appropriate choice for the calibration of measurements of MON 810 corn with respect to the DNA copy number ratio.

  3. Quantification of arbuscular mycorrhizal fungal DNA in roots: how important is material preservation?

    Science.gov (United States)

    Janoušková, Martina; Püschel, David; Hujslová, Martina; Slavíková, Renata; Jansa, Jan

    2015-04-01

    Monitoring populations of arbuscular mycorrhizal fungi (AMF) in roots is a pre-requisite for improving our understanding of AMF ecology and functioning of the symbiosis in natural conditions. Among other approaches, quantification of fungal DNA in plant tissues by quantitative real-time PCR is one of the advanced techniques with a great potential to process large numbers of samples and to deliver truly quantitative information. Its application potential would greatly increase if the samples could be preserved by drying, but little is currently known about the feasibility and reliability of fungal DNA quantification from dry plant material. We addressed this question by comparing quantification results based on dry root material to those obtained from deep-frozen roots of Medicago truncatula colonized with Rhizophagus sp. The fungal DNA was well conserved in the dry root samples with overall fungal DNA levels in the extracts comparable with those determined in extracts of frozen roots. There was, however, no correlation between the quantitative data sets obtained from the two types of material, and data from dry roots were more variable. Based on these results, we recommend dry material for qualitative screenings but advocate using frozen root materials if precise quantification of fungal DNA is required.

  4. Direct quantification of fungal DNA from soil substrate using real-time PCR.

    Science.gov (United States)

    Filion, Martin; St-Arnaud, Marc; Jabaji-Hare, Suha H

    2003-04-01

    Detection and quantification of genomic DNA from two ecologically different fungi, the plant pathogen Fusarium solani f. sp. phaseoli and the arbuscular mycorrhizal fungus Glomus intraradices, was achieved from soil substrate. Specific primers targeting a 362-bp fragment from the SSU rRNA gene region of G. intraradices and a 562-bp fragment from the F. solani f. sp. phaseoli translation elongation factor 1 alpha gene were used in real-time polymerase chain reaction (PCR) assays conjugated with the fluorescent SYBR(R) Green I dye. Standard curves showed a linear relation (r(2)=0.999) between log values of fungal genomic DNA of each species and real-time PCR threshold cycles and were quantitative over 4-5 orders of magnitude. Real-time PCR assays were applied to in vitro-produced fungal structures and sterile and non-sterile soil substrate seeded with known propagule numbers of either fungi. Detection and genomic DNA quantification was obtained from the different treatments, while no amplicon was detected from non-seeded non-sterile soil samples, confirming the absence of cross-reactivity with the soil microflora DNA. A significant correlation (Pgenomic DNA of F. solani f. sp. phaseoli or G. intraradices detected and the number of fungal propagules present in seeded soil substrate. The DNA extraction protocol and real-time PCR quantification assay can be performed in less than 2 h and is adaptable to detect and quantify genomic DNA from other soilborne fungi.

  5. Quantification and presence of human ancient DNA in burial place ...

    African Journals Online (AJOL)

    STORAGESEVER

    2009-10-19

    Oct 19, 2009 ... burial place remains of Turkey using real time ... DNA was isolaled from fossil bone tissue remains with Bio Robot EZ1 and ... the increase in the amount of DNA as it is amplified. The ... species or human blood in this work.

  6. How Severely Is DNA Quantification Hampered by RNA Co-extraction?

    Science.gov (United States)

    Sanchez, Ignacio; Remm, Matthieu; Frasquilho, Sonia; Betsou, Fay; Mathieson, William

    2015-10-01

    The optional RNase digest that is part of many DNA extraction protocols is often omitted, either because RNase is not provided in the kit or because users do not want to risk contaminating their laboratory. Consequently, co-eluting RNA can become a "contaminant" of unknown magnitude in a DNA extraction. We extracted DNA from liver, lung, kidney, and heart tissues and established that 28-52% of the "DNA" as assessed by spectrophotometry is actually RNA (depending on tissue type). Including an RNase digest in the extraction protocol reduced 260:280 purity ratios. Co-eluting RNA drives an overestimation of DNA yield when quantification is carried out using OD 260 nm spectrophotometry, or becomes an unquantified contaminant when spectrofluorometry is used for DNA quantification. This situation is potentially incompatible with the best practice guidelines for biobanks issued by organizations such as the International Society for Biological and Environmental Repositories, which state that biospecimens should be accurately characterized in terms of their identity, purity, concentration, and integrity. Consequently, we conclude that an RNase digest must be included in DNA extractions if pure DNA is required. We also discuss the implications of unquantified RNA contamination in DNA samples in the context of laboratory accreditation schemes.

  7. The Method of Manufactured Universes for validating uncertainty quantification methods

    KAUST Repository

    Stripling, H.F.

    2011-09-01

    The Method of Manufactured Universes is presented as a validation framework for uncertainty quantification (UQ) methodologies and as a tool for exploring the effects of statistical and modeling assumptions embedded in these methods. The framework calls for a manufactured reality from which experimental data are created (possibly with experimental error), an imperfect model (with uncertain inputs) from which simulation results are created (possibly with numerical error), the application of a system for quantifying uncertainties in model predictions, and an assessment of how accurately those uncertainties are quantified. The application presented in this paper manufactures a particle-transport universe, models it using diffusion theory with uncertain material parameters, and applies both Gaussian process and Bayesian MARS algorithms to make quantitative predictions about new experiments within the manufactured reality. The results of this preliminary study indicate that, even in a simple problem, the improper application of a specific UQ method or unrealized effects of a modeling assumption may produce inaccurate predictions. We conclude that the validation framework presented in this paper is a powerful and flexible tool for the investigation and understanding of UQ methodologies. © 2011 Elsevier Ltd. All rights reserved.

  8. Quantification of camel DNA from three camel types 2017

    OpenAIRE

    Alhaddad, Hasan

    2017-01-01

    This poster is a summary of comparisons of the quantity DNA extracted from blood, saliva, and hair and from three camel types Majaheem, Sufor, Wadh. The poster was presented at Kuwait University-research sector poster day 2017

  9. Development of salt-tolerance interface for an high performance liquid chromatography/inductively coupled plasma mass spectrometry system and its application to accurate quantification of DNA samples.

    Science.gov (United States)

    Takasaki, Yuka; Sakagawa, Shinnosuke; Inagaki, Kazumi; Fujii, Shin-Ichiro; Sabarudin, Akhmad; Umemura, Tomonari; Haraguchi, Hiroki

    2012-02-03

    Accurate quantification of DNA is highly important in various fields. Determination of phosphorus by ICP-MS is one of the most effective methods for accurate quantification of DNA due to the fixed stoichiometry of phosphate to this molecule. In this paper, a smart and reliable method for accurate quantification of DNA fragments and oligodeoxythymidilic acids by hyphenated HPLC/ICP-MS equipped with a highly efficient interface device is presented. The interface was constructed of a home-made capillary-attached micronebulizer and temperature-controllable cyclonic spray chamber (IsoMist). As a separation column for DNA samples, home-made methacrylate-based weak anion-exchange monolith was employed. Some parameters, which include composition of mobile phase, gradient program, inner and outer diameters of capillary, temperature of spray chamber etc., were optimized to find the best performance for separation and accurate quantification of DNA samples. The proposed system could achieve many advantages, such as total consumption for small amount sample analysis, salt-tolerance for hyphenated analysis, high accuracy and precision for quantitative analysis. Using this proposed system, the samples of 20 bp DNA ladder (20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 300, 400, 500 base pairs) and oligodeoxythymidilic acids (dT(12-18)) were rapidly separated and accurately quantified. Copyright © 2011 Elsevier B.V. All rights reserved.

  10. Quantification of residual host cell DNA in adenoviral vectors produced on PER.C6 cells

    NARCIS (Netherlands)

    Gijsbers, Linda; Koel, Björn; Weggeman, Miranda; Goudsmit, Jaap; Havenga, Menzo; Marzio, Giuseppe

    2005-01-01

    Recombinant adenoviral vectors for gene therapy and vaccination are routinely prepared on cultures of immortalized cells, allowing the production of vector batches of high titer and consistent quality. Quantification of residual DNA from the producing cell line is part of the purity tests for

  11. Accurate Digital Polymerase Chain Reaction Quantification of Challenging Samples Applying Inhibitor-Tolerant DNA Polymerases.

    Science.gov (United States)

    Sidstedt, Maja; Romsos, Erica L; Hedell, Ronny; Ansell, Ricky; Steffen, Carolyn R; Vallone, Peter M; Rådström, Peter; Hedman, Johannes

    2017-02-07

    Digital PCR (dPCR) enables absolute quantification of nucleic acids by partitioning of the sample into hundreds or thousands of minute reactions. By assuming a Poisson distribution for the number of DNA fragments present in each chamber, the DNA concentration is determined without the need for a standard curve. However, when analyzing nucleic acids from complex matrixes such as soil and blood, the dPCR quantification can be biased due to the presence of inhibitory compounds. In this study, we evaluated the impact of varying the DNA polymerase in chamber-based dPCR for both pure and impure samples using the common PCR inhibitor humic acid (HA) as a model. We compared the TaqMan Universal PCR Master Mix with two alternative DNA polymerases: ExTaq HS and Immolase. By using Bayesian modeling, we show that there is no difference among the tested DNA polymerases in terms of accuracy of absolute quantification for pure template samples, i.e., without HA present. For samples containing HA, there were great differences in performance: the TaqMan Universal PCR Master Mix failed to correctly quantify DNA with more than 13 pg/nL HA, whereas Immolase (1 U) could handle up to 375 pg/nL HA. Furthermore, we found that BSA had a moderate positive effect for the TaqMan Universal PCR Master Mix, enabling accurate quantification for 25 pg/nL HA. Increasing the amount of DNA polymerase from 1 to 5 U had a strong effect for ExTaq HS, elevating HA-tolerance four times. We also show that the average Cq values of positive reactions may be used as a measure of inhibition effects, e.g., to determine whether or not a dPCR quantification result is reliable. The statistical models developed to objectively analyze the data may also be applied in quality control. We conclude that the choice of DNA polymerase in dPCR is crucial for the accuracy of quantification when analyzing challenging samples.

  12. Absolute Quantification of the Host-To-Parasite DNA Ratio in Theileria parva-Infected Lymphocyte Cell Lines.

    Science.gov (United States)

    Gotia, Hanzel T; Munro, James B; Knowles, Donald P; Daubenberger, Claudia A; Bishop, Richard P; Silva, Joana C

    2016-01-01

    Theileria parva is a tick-transmitted intracellular apicomplexan pathogen of cattle in sub-Saharan Africa that causes East Coast fever (ECF). ECF is an acute fatal disease that kills over one million cattle annually, imposing a tremendous burden on African small-holder cattle farmers. The pathology and level of T. parva infections in its wildlife host, African buffalo (Syncerus caffer), and in cattle are distinct. We have developed an absolute quantification method based on quantitative PCR (qPCR) in which recombinant plasmids containing single copy genes specific to the parasite (apical membrane antigen 1 gene, ama1) or the host (hypoxanthine phosphoribosyltransferase 1, hprt1) are used as the quantification reference standards. Our study shows that T. parva and bovine cells are present in similar numbers in T. parva-infected lymphocyte cell lines and that consequently, due to its much smaller genome size, T. parva DNA comprises between 0.9% and 3% of the total DNA samples extracted from these lines. This absolute quantification assay of parasite and host genome copy number in a sample provides a simple and reliable method of assessing T. parva load in infected bovine lymphocytes, and is accurate over a wide range of host-to-parasite DNA ratios. Knowledge of the proportion of target DNA in a sample, as enabled by this method, is essential for efficient high-throughput genome sequencing applications for a variety of intracellular pathogens. This assay will also be very useful in future studies of interactions of distinct host-T. parva stocks and to fully characterize the dynamics of ECF infection in the field.

  13. Absolute quantification of olive oil DNA by droplet digital-PCR (ddPCR): Comparison of isolation and amplification methodologies.

    Science.gov (United States)

    Scollo, Francesco; Egea, Leticia A; Gentile, Alessandra; La Malfa, Stefano; Dorado, Gabriel; Hernandez, Pilar

    2016-12-15

    Olive oil is considered a premium product for its nutritional value and health benefits, and the ability to define its origin and varietal composition is a key step towards ensuring the traceability of the product. However, isolating the DNA from such a matrix is a difficult task. In this study, the quality and quantity of olive oil DNA, isolated using four different DNA isolation protocols, was evaluated using the qRT-PCR and ddPCR techniques. The results indicate that CTAB-based extraction methods were the best for unfiltered oil, while Nucleo Spin-based extraction protocols showed greater overall reproducibility. The use of both qRT-PCR and ddPCR led to the absolute quantification of the DNA copy number. The results clearly demonstrate the importance of the choice of DNA-isolation protocol, which should take into consideration the qualitative aspects of DNA and the evaluation of the amplified DNA copy number. Copyright © 2016 Elsevier Ltd. All rights reserved.

  14. eMethylsorb: electrochemical quantification of DNA methylation at CpG resolution using DNA-gold affinity interactions.

    Science.gov (United States)

    Sina, Abu Ali Ibn; Howell, Sidney; Carrascosa, Laura G; Rauf, Sakandar; Shiddiky, Muhammad J A; Trau, Matt

    2014-11-07

    We report a simple electrochemical method referred to as "eMethylsorb" for the detection of DNA methylation. The method relies on the base dependent affinity interaction of DNA with gold. The methylation status of DNA is quantified by monitoring the electrochemical current as a function of the relative adsorption level of bisulphite treated DNA samples onto a bare gold electrode. This method can successfully distinguish methylated and unmethylated epigenotypes at single CpG resolution.

  15. Quantification of Paratrichodorus allius in DNA extracted from soil using TaqMan probe and SYBR green real-time PCR assays

    Science.gov (United States)

    The ectoparasitic stubby root nematode Paratrichodorus allius transmits Tobacco rattle virus, which causes corky ringspot disease resulting in significant economic losses in the potato industry. This study developed a diagnostic method for direct quantification of P. allius from soil DNA using a Taq...

  16. Quantification of DNA by Agarose Gel Electrophoresis and Analysis of the Topoisomers of Plasmid and M13 DNA Following Treatment with a Restriction Endonuclease or DNA Topoisomerase I

    Science.gov (United States)

    Tweedie, John W.; Stowell, Kathryn M.

    2005-01-01

    A two-session laboratory exercise for advanced undergraduate students in biochemistry and molecular biology is described. The first session introduces students to DNA quantification by ultraviolet absorbance and agarose gel electrophoresis followed by ethidium bromide staining. The second session involves treatment of various topological forms of…

  17. Digital quantification of gene methylation in stool DNA by emulsion-PCR coupled with hydrogel immobilized bead-array.

    Science.gov (United States)

    Liu, Yunlong; Wu, Haiping; Zhou, Qiang; Song, Qinxin; Rui, Jianzhong; Zou, Bingjie; Zhou, Guohua

    2017-06-15

    Aberrations of gene methylation in stool DNA (sDNA) is an effective biomarker for non-invasive colorectal cancer diagnosis. However, it is challenging to accurately quantitate the gene methylation levels in sDNA due to the low abundance and degradation of sDNA. In this study, a digital quantification strategy was proposed by combining emulsion PCR (emPCR) with hydrogel immobilized bead-array. The assay includes following steps: bisulfite conversion of sDNA, pre-amplification by PCR with specific primers containing 5' universal sequences, emPCR of pre-amplicons with beaded primers to achieve single-molecular amplification and identification of hydrogel embedding beads coated with amplicons. The sensitivity and the specificity of the method are high enough to pick up 0.05% methylated targets from unmethylated DNA background. The successful detection of hypermethylated vimentin gene in clinical stool samples suggests that the proposed method should be a potential tool for non-invasive colorectal cancer screening. Copyright © 2016 Elsevier B.V. All rights reserved.

  18. Enhancement of Electroluminescence (EL) image measurements for failure quantification methods

    DEFF Research Database (Denmark)

    Parikh, Harsh; Spataru, Sergiu; Sera, Dezso

    2018-01-01

    Enhanced quality images are necessary for EL image analysis and failure quantification. A method is proposed which determines image quality in terms of more accurate failure detection of solar panels through electroluminescence (EL) imaging technique. The goal of the paper is to determine the most...

  19. Evaluation of assays for quantification of DNA in canine plasma as an indirect marker of NETosis.

    Science.gov (United States)

    Smith, Stephanie A; Lawson, Corinne M; McMichael, Maureen A; Jung, Katrina; O'Brien, Mauria; Achiel, Ron

    2017-06-01

    Neutrophil extracellular traps (NET), consisting of a filamentous DNA/chromatin-histone scaffold originating from neutrophils are part of the innate immune response, may be released under a variety of inflammatory conditions and are associated with an increased risk for thrombosis. The purpose of this study was to evaluate a SYTOX green fluorescence assay and a histone-DNA complex (hisDNA) ELISA for quantification of NET-related DNA in canine plasma. The influence of variations in blood sample handling on assay results was tested. Accuracy of the SYTOX green fluorescence and the hisDNA ELISA was evaluated with dilutional linearity using serial dilutions. Interference was assessed by addition of purified bilirubin or hemoglobin. Precision was determined by calculating the intra- and inter-assay CV. Preanalytic sample handling did not influence DNA measurements by either assay. Citrate and EDTA plasma samples were equivalent. For the DNA fluorescence assay, dilutional linearity was poor due to autofluorescence, which was corrected by addition of canine plasma to the diluent. The presence of bilirubin and hemoglobin also increased autofluorescence, and resulted in falsely low concentrations of DNA. On the hisDNA ELISA, pigmentemia had no effect. Both assays as modified in this study are suitable for measuring DNA in canine EDTA or citrate plasma. However, performance of the fluorescence assay was impacted by pigmentemia, and it was less sensitive than the ELISA in detecting the presence of nucleosome material in the plasma. © 2017 American Society for Veterinary Clinical Pathology.

  20. Quantification of the mitochondrial DNA common deletion in presbycusis.

    Science.gov (United States)

    Markaryan, Adam; Nelson, Erik G; Hinojosa, Raul

    2009-06-01

    This study was conducted to evaluate the association between the mitochondrial DNA (mtDNA) common deletion (CD) level in cochlear tissue and the severity of hearing loss in individuals with presbycusis. Nineteen individuals with presbycusis, ranging from 60 to 87 years of age, who met strict audiometric criteria were compared with four age frequency-matched normal hearing controls ranging from 51 to 76 years of age. Five additional normal hearing individuals, ranging from 9 to 50 years of age, were also studied. A duplex real time polymerase chain reaction assay was used to quantify the mtDNA in archival cochlear tissue samples. Linear regression models were used for comparison of the CD level between groups. The presbycusis group had a mean CD level of 32% with a standard deviation of 14%, and the normal hearing age matched control group had a mean CD level of 12% with a standard deviation of 2%. This difference in CD levels reached statistical significance (P = .011) and remained significant after adjusting for any differences in age between the two groups (age-adjusted P = .007). Furthermore, there was evidence for a significant association between the CD level and the severity of hearing loss based on audiometric thresholds at 8 kHz (r = 0.44, P = .034; age-adjusted partial correlation = 0.55, P = .007). For the first time, to our knowledge, these results demonstrate a relationship between quantitatively measured levels of the CD in human cochlear tissue and the severity of hearing loss in individuals with presbycusis. Laryngoscope, 2009.

  1. Imaging and high-sensitivity quantification of chemiluminescent labeled DNA-blots

    International Nuclear Information System (INIS)

    Dorner, G.

    1997-01-01

    The present thesis has for objective the development of both, methods of DNA labeling by chemiluminescence (via the catalytic activity of the enzyme alkaline phosphatase - AP) and an appropriate imaging system. Offering a competitive alternative to the detection of classical radio-labels in molecular-biological experiments of the blotting type, this technique should permit the realization of quantitative studies of gene expression at ultra-high sensitivity necessary in particular for differential-screening experiments. To reach our aim. we separated the project into three different parts. In a first step an imager based on a liquid-nitrogen-cooled CCD coupled to a standard optics (50 mm/fl.2) has been installed and characterized. This system offers a sensitive area of up to 625 cm 2 , a spatial resolution of 0.3-1 mm (depending on the field of view) and a sensitivity sufficient to detect 10 fg/mm 2 labeled DNA. In a second part, the chemiluminescent light-generation process in solution has been investigated to optimize the parameters temperature. pH and concentration of the substrate as well as the enzyme. The substrate offering the highest light yield (CDP-Star in addition with the enhancer EMERALD II) allows quantification of AP down to 10 -15 M within a dynamic range of 10 4 in solution. Finally. preparation, immobilization and detection of AP-labeled DNA probes (via a biotin-streptavidin-biotin-AP bridge) on nylon membranes has been optimized. A linear relation between the light intensities and the amount of DNA was observed in a range of 10 fg/mm 2 - 100 pg/mm 2 . Hybridization of the probes to bacterial cloned target-DNA has been addressed after examination of the best hybridization conditions. Our protocol includes the treatment of a proteinase, which resulted in a significantly lower background on the filter. The results of our investigations suggest that the main conditions for a reliable differential-screening experiment are fulfilled when using

  2. The diagnostic value of circulating cell free DNA quantification in non-small cell lung cancer: A systematic review with meta-analysis.

    Science.gov (United States)

    Jiang, Tao; Zhai, Changyun; Su, Chunxia; Ren, Shengxiang; Zhou, Caicun

    2016-10-01

    The aim of the current study was to assess the diagnostic value of circulating cell free DNA (cfDNA) quantification in discriminating non-small cell lung cancer (NSCLC) from healthy individuals. An electronic search was conducted on PubMed, EMBASE, Web of Science, and Cochrane Library. Eligible studies regarding to examine the diagnostic value of cfDNA in the detection of NSCLC were extracted and analyzed. We identified 15 eligible studies with a total of 2125 patients. The pooled results for quantification of cfDNA in lung cancer screening in the included studies were as follows: sensitivity, 81% (95% confidence interval (CI), 76%-84%); specificity, 85% (95% CI, 77%-91%); diagnostic odds ratio, 23.87 (95% CI, 13.37-42.61); and areas under the summary receiver operating characteristic curves were 0.89 (95% CI, 0.86-0.92). Subgroup analyses according to the time of sample collection, sample materials, test method, reference gene and cutoff value did not improve sensitivity, but specificity could be significantly improved when we only included the studies using cfDNA sample before surgery or antitumor treatment and real-time PCR to detect cfDNA and human β-actin as a reference gene. Quantification of cfDNA was a promising and effective biomarker for discriminating NSCLC from healthy individuals. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  3. Quantification of plasmid DNA reference materials for Shiga toxin-producing Escherichia coli based on UV, HR-ICP-MS and digital PCR.

    Science.gov (United States)

    Liang, Wen; Xu, Li; Sui, Zhiwei; Li, Yan; Li, Lanying; Wen, Yanli; Li, Chunhua; Ren, Shuzhen; Liu, Gang

    2016-01-01

    The accuracy and metrology traceability of DNA quantification is becoming a critical theme in many fields, including diagnosis, forensic analysis, microorganism detection etc. Thus the research of DNA reference materials (RMs) and consistency of DNA quantification methods has attracted considerable research interest. In this work, we developed 3 plasmid candidate RMs, containing 3 target genes of Escherichia coli O157:H7 (E. coli O157:H7) and other Shiga toxin-producing Escherichia coli (STEC): stx1, stx2, and fliC (h7) respectively. Comprehensive investigation of the plasmid RMs was performed for their sequence, purity, homogeneity and stability, and then the concentration was quantified by three different methods: ultraviolet spectrophotometer (UV), high resolution inductively coupled plasma mass spectrometry (HR-ICP-MS) and digital PCR. As a routinely applied method for DNA analysis, UV was utilized for the quantification (OD260) and purity analysis for the plasmids. HR-ICP-MS quantified the plasmid DNA through analysing the phosphorus in DNA molecules. Digital PCR distributed the DNA samples onto a microarray chip containing thousands of reaction chambers, and quantified the DNA copy numbers by analysing the number of positive signals without any calibration curves needed. Based on the high purification of the DNA reference materials and the optimization of dPCR analysis, we successfully achieved good consistency between UV, HR-ICP-MS and dPCR, with relative deviations lower than 10 %. We then performed the co-quantification of 3 DNA RMs with three different methods together, and the uncertainties of their concentration were evaluated. Finally, the certified values and expanded uncertainties for 3 DNA RMs (pFliC, pStx1 and pStx2) were (1.60 ± 0.10) × 10(10) copies/μL, (1.53 ± 0.10) × 10(10) copies/μL and (1.70 ± 0.11) × 10(10) copies/μL respectively.Graphical abstractWe developed 3 plasmid candidate RMs, containing 3 target genes of

  4. New LightCycler PCR for Rapid and Sensitive Quantification of Parvovirus B19 DNA Guides Therapeutic Decision-Making in Relapsing Infections

    Science.gov (United States)

    Harder, Timm C.; Hufnagel, Markus; Zahn, Katrin; Beutel, Karin; Schmitt, Heinz-Josef; Ullmann, Uwe; Rautenberg, Peter

    2001-01-01

    Detection of parvovirus B19 DNA offers diagnostic advantages over serology, particularly in persistent infections of immunocompromised patients. A rapid, novel method of B19 DNA detection and quantification is introduced. This method, a quantitative PCR assay, is based on real-time glass capillary thermocycling (LightCycler [LC]) and fluorescence resonance energy transfer (FRET). The PCR assay allowed quantification over a dynamic range of over 7 logs and could quantify as little as 250 B19 genome equivalents (geq) per ml as calculated for plasmid DNA (i.e., theoretically ≥5 geq per assay). Interrater agreement analysis demonstrated equivalence of LC-FRET PCR and conventional nested PCR in the diagnosis of an active B19 infection (kappa coefficient = 0.83). The benefit of the new method was demonstrated in an immunocompromised child with a relapsing infection, who required an attenuation of the immunosuppressive therapy in addition to repeated doses of immunoglobulin to eliminate the virus. PMID:11724854

  5. A probabilistic generative model for quantification of DNA modifications enables analysis of demethylation pathways.

    Science.gov (United States)

    Äijö, Tarmo; Huang, Yun; Mannerström, Henrik; Chavez, Lukas; Tsagaratou, Ageliki; Rao, Anjana; Lähdesmäki, Harri

    2016-03-14

    We present a generative model, Lux, to quantify DNA methylation modifications from any combination of bisulfite sequencing approaches, including reduced, oxidative, TET-assisted, chemical-modification assisted, and methylase-assisted bisulfite sequencing data. Lux models all cytosine modifications (C, 5mC, 5hmC, 5fC, and 5caC) simultaneously together with experimental parameters, including bisulfite conversion and oxidation efficiencies, as well as various chemical labeling and protection steps. We show that Lux improves the quantification and comparison of cytosine modification levels and that Lux can process any oxidized methylcytosine sequencing data sets to quantify all cytosine modifications. Analysis of targeted data from Tet2-knockdown embryonic stem cells and T cells during development demonstrates DNA modification quantification at unprecedented detail, quantifies active demethylation pathways and reveals 5hmC localization in putative regulatory regions.

  6. An Optimized Method for Quantification of Pathogenic Leptospira in Environmental Water Samples.

    Science.gov (United States)

    Riediger, Irina N; Hoffmaster, Alex R; Casanovas-Massana, Arnau; Biondo, Alexander W; Ko, Albert I; Stoddard, Robyn A

    2016-01-01

    Leptospirosis is a zoonotic disease usually acquired by contact with water contaminated with urine of infected animals. However, few molecular methods have been used to monitor or quantify pathogenic Leptospira in environmental water samples. Here we optimized a DNA extraction method for the quantification of leptospires using a previously described Taqman-based qPCR method targeting lipL32, a gene unique to and highly conserved in pathogenic Leptospira. QIAamp DNA mini, MO BIO PowerWater DNA and PowerSoil DNA Isolation kits were evaluated to extract DNA from sewage, pond, river and ultrapure water samples spiked with leptospires. Performance of each kit varied with sample type. Sample processing methods were further evaluated and optimized using the PowerSoil DNA kit due to its performance on turbid water samples and reproducibility. Centrifugation speeds, water volumes and use of Escherichia coli as a carrier were compared to improve DNA recovery. All matrices showed a strong linearity in a range of concentrations from 106 to 10° leptospires/mL and lower limits of detection ranging from Leptospira in environmental waters (river, pond and sewage) which consists of the concentration of 40 mL samples by centrifugation at 15,000×g for 20 minutes at 4°C, followed by DNA extraction with the PowerSoil DNA Isolation kit. Although the method described herein needs to be validated in environmental studies, it potentially provides the opportunity for effective, timely and sensitive assessment of environmental leptospiral burden.

  7. Correction of the lack of commutability between plasmid DNA and genomic DNA for quantification of genetically modified organisms using pBSTopas as a model.

    Science.gov (United States)

    Zhang, Li; Wu, Yuhua; Wu, Gang; Cao, Yinglong; Lu, Changming

    2014-10-01

    Plasmid calibrators are increasingly applied for polymerase chain reaction (PCR) analysis of genetically modified organisms (GMOs). To evaluate the commutability between plasmid DNA (pDNA) and genomic DNA (gDNA) as calibrators, a plasmid molecule, pBSTopas, was constructed, harboring a Topas 19/2 event-specific sequence and a partial sequence of the rapeseed reference gene CruA. Assays of the pDNA showed similar limits of detection (five copies for Topas 19/2 and CruA) and quantification (40 copies for Topas 19/2 and 20 for CruA) as those for the gDNA. Comparisons of plasmid and genomic standard curves indicated that the slopes, intercepts, and PCR efficiency for pBSTopas were significantly different from CRM Topas 19/2 gDNA for quantitative analysis of GMOs. Three correction methods were used to calibrate the quantitative analysis of control samples using pDNA as calibrators: model a, or coefficient value a (Cva); model b, or coefficient value b (Cvb); and the novel model c or coefficient formula (Cf). Cva and Cvb gave similar estimated values for the control samples, and the quantitative bias of the low concentration sample exceeded the acceptable range within ±25% in two of the four repeats. Using Cfs to normalize the Ct values of test samples, the estimated values were very close to the reference values (bias -13.27 to 13.05%). In the validation of control samples, model c was more appropriate than Cva or Cvb. The application of Cf allowed pBSTopas to substitute for Topas 19/2 gDNA as a calibrator to accurately quantify the GMO.

  8. CometQ: An automated tool for the detection and quantification of DNA damage using comet assay image analysis.

    Science.gov (United States)

    Ganapathy, Sreelatha; Muraleedharan, Aparna; Sathidevi, Puthumangalathu Savithri; Chand, Parkash; Rajkumar, Ravi Philip

    2016-09-01

    DNA damage analysis plays an important role in determining the approaches for treatment and prevention of various diseases like cancer, schizophrenia and other heritable diseases. Comet assay is a sensitive and versatile method for DNA damage analysis. The main objective of this work is to implement a fully automated tool for the detection and quantification of DNA damage by analysing comet assay images. The comet assay image analysis consists of four stages: (1) classifier (2) comet segmentation (3) comet partitioning and (4) comet quantification. Main features of the proposed software are the design and development of four comet segmentation methods, and the automatic routing of the input comet assay image to the most suitable one among these methods depending on the type of the image (silver stained or fluorescent stained) as well as the level of DNA damage (heavily damaged or lightly/moderately damaged). A classifier stage, based on support vector machine (SVM) is designed and implemented at the front end, to categorise the input image into one of the above four groups to ensure proper routing. Comet segmentation is followed by comet partitioning which is implemented using a novel technique coined as modified fuzzy clustering. Comet parameters are calculated in the comet quantification stage and are saved in an excel file. Our dataset consists of 600 silver stained images obtained from 40 Schizophrenia patients with different levels of severity, admitted to a tertiary hospital in South India and 56 fluorescent stained images obtained from different internet sources. The performance of "CometQ", the proposed standalone application for automated analysis of comet assay images, is evaluated by a clinical expert and is also compared with that of a most recent and related software-OpenComet. CometQ gave 90.26% positive predictive value (PPV) and 93.34% sensitivity which are much higher than those of OpenComet, especially in the case of silver stained images. The

  9. Electrical detection and quantification of single and mixed DNA nucleotides in suspension

    Science.gov (United States)

    Ahmad, Mahmoud Al; Panicker, Neena G.; Rizvi, Tahir A.; Mustafa, Farah

    2016-09-01

    High speed sequential identification of the building blocks of DNA, (deoxyribonucleotides or nucleotides for short) without labeling or processing in long reads of DNA is the need of the hour. This can be accomplished through exploiting their unique electrical properties. In this study, the four different types of nucleotides that constitute a DNA molecule were suspended in a buffer followed by performing several types of electrical measurements. These electrical parameters were then used to quantify the suspended DNA nucleotides. Thus, we present a purely electrical counting scheme based on the semiconductor theory that allows one to determine the number of nucleotides in a solution by measuring their capacitance-voltage dependency. The nucleotide count was observed to be similar to the multiplication of the corresponding dopant concentration and debye volume after de-embedding the buffer contribution. The presented approach allows for a fast and label-free quantification of single and mixed nucleotides in a solution.

  10. Real-time ligation chain reaction for DNA quantification and identification on the FO-SPR.

    Science.gov (United States)

    Knez, Karel; Spasic, Dragana; Delport, Filip; Lammertyn, Jeroen

    2015-05-15

    Different assays have been developed in the past years to meet point-of-care diagnostic tests requirements for fast and sensitive quantification and identification of targets. In this paper, we developed the ligation chain reaction (LCR) assay on the Fiber Optic Surface Plasmon Resonance (FO-SPR) platform, which enabled simultaneous quantification and cycle-to-cycle identification of DNA during amplification. The newly developed assay incorporated FO-SPR DNA melting assay, previously developed by our group. This required establishment of several assay parameters, including buffer ionic strength and thermal ramping speed as these parameters both influence the ligation enzyme performance and the hybridization yield of the gold nanoparticles (Au NPs) on the FO-SPR sensor. Quantification and identification of DNA targets was achieved over a wide concentration range with a calibration curve spanning 7 orders of magnitude and LOD of 13.75 fM. Moreover, the FO-SPR LCR assay could discriminate single nucleotide polymorphism (SNPs) without any post reaction analysis, featuring thus all the essential requirements of POC tests. Copyright © 2014 Elsevier B.V. All rights reserved.

  11. Quantification of 3-nitrobenzanthrone-DNA adducts using online column-switching HPLC-electrospray tandem mass spectrometry.

    Science.gov (United States)

    Gamboa da Costa, Gonçalo; Singh, Rajinder; Arlt, Volker M; Mirza, Amin; Richards, Meirion; Takamura-Enya, Takeji; Schmeiser, Heinz H; Farmer, Peter B; Phillips, David H

    2009-11-01

    The aromatic nitroketone 3-nitrobenzanthrone (3-nitro-7H-benz[de]anthracen-7-one; 3-NBA) is an extremely potent mutagen and a suspected human carcinogen detected in the exhaust of diesel engines and in airborne particulate matter. 3-NBA is metabolically activated via reduction of the nitro group to the hydroxylamine (N-OH-3-ABA) to form covalent DNA adducts. Thus far, the detection and quantification of covalent 3-NBA-DNA adducts has relied solely on (32)P-postlabeling methodologies. In order to expand the range of available techniques for the detection and improved quantification of 3-NBA-DNA adducts, we have developed a method based upon online column-switching HPLC coupled to electrospray tandem mass spectrometry, with isotopic dilution of (15)N-labeled internal standards. This methodology was applied to the determination of three 3-NBA-derived adducts: 2-(2'-deoxyguanosin-N(2)-yl)-3-aminobenzanthrone (dG-N(2)-3-ABA), N-(2'-deoxyguanosin-8-yl)-3-aminobenzanthrone (dG-C8-N-3-ABA) and 2-(2'-deoxyguanosine-8-yl)-3-aminobenzanthrone (dG-C8-C2-3-ABA). Dose-dependent increases were observed for all three adducts when salmon testis DNA was reacted with N-acetoxy-3-aminobenzanthrone (N-AcO-3-ABA). dG-C8-C2-3-ABA was detected at much lower levels (overall 1%) than the other two adducts. DNA samples isolated from tissues of rats treated either intratracheally with 3-NBA or intraperitoneally with N-OH-3-ABA were analyzed by mass spectrometry, and the results compared to those obtained by (32)P-postlabeling. The method required 50 microg of hydrolyzed animal DNA on column and the limit of detection was 2.0 fmol for each adduct. dG-C8-C2-3-ABA was not observed in any of the samples providing confirmation that it is not formed in vivo. Linear regression analysis of the levels of dG-N(2)-3-ABA and dG-C8-N-3-ABA in the rat DNA showed a reasonable correlation between the two methods (R(2) = 0.88 and 0.93, respectively). In summary, the mass spectrometric method is a faster, more

  12. Collaborative framework for PIV uncertainty quantification: comparative assessment of methods

    International Nuclear Information System (INIS)

    Sciacchitano, Andrea; Scarano, Fulvio; Neal, Douglas R; Smith, Barton L; Warner, Scott O; Vlachos, Pavlos P; Wieneke, Bernhard

    2015-01-01

    A posteriori uncertainty quantification of particle image velocimetry (PIV) data is essential to obtain accurate estimates of the uncertainty associated with a given experiment. This is particularly relevant when measurements are used to validate computational models or in design and decision processes. In spite of the importance of the subject, the first PIV uncertainty quantification (PIV-UQ) methods have been developed only in the last three years. The present work is a comparative assessment of four approaches recently proposed in the literature: the uncertainty surface method (Timmins et al 2012), the particle disparity approach (Sciacchitano et al 2013), the peak ratio criterion (Charonko and Vlachos 2013) and the correlation statistics method (Wieneke 2015). The analysis is based upon experiments conducted for this specific purpose, where several measurement techniques are employed simultaneously. The performances of the above approaches are surveyed across different measurement conditions and flow regimes. (paper)

  13. Correlation Coefficients Between Different Methods of Expressing Bacterial Quantification Using Real Time PCR

    Directory of Open Access Journals (Sweden)

    Bahman Navidshad

    2012-02-01

    Full Text Available The applications of conventional culture-dependent assays to quantify bacteria populations are limited by their dependence on the inconsistent success of the different culture-steps involved. In addition, some bacteria can be pathogenic or a source of endotoxins and pose a health risk to the researchers. Bacterial quantification based on the real-time PCR method can overcome the above-mentioned problems. However, the quantification of bacteria using this approach is commonly expressed as absolute quantities even though the composition of samples (like those of digesta can vary widely; thus, the final results may be affected if the samples are not properly homogenized, especially when multiple samples are to be pooled together before DNA extraction. The objective of this study was to determine the correlation coefficients between four different methods of expressing the output data of real-time PCR-based bacterial quantification. The four methods were: (i the common absolute method expressed as the cell number of specific bacteria per gram of digesta; (ii the Livak and Schmittgen, ΔΔCt method; (iii the Pfaffl equation; and (iv a simple relative method based on the ratio of cell number of specific bacteria to the total bacterial cells. Because of the effect on total bacteria population in the results obtained using ΔCt-based methods (ΔΔCt and Pfaffl, these methods lack the acceptable consistency to be used as valid and reliable methods in real-time PCR-based bacterial quantification studies. On the other hand, because of the variable compositions of digesta samples, a simple ratio of cell number of specific bacteria to the corresponding total bacterial cells of the same sample can be a more accurate method to quantify the population.

  14. Toponomics method for the automated quantification of membrane protein translocation.

    Science.gov (United States)

    Domanova, Olga; Borbe, Stefan; Mühlfeld, Stefanie; Becker, Martin; Kubitz, Ralf; Häussinger, Dieter; Berlage, Thomas

    2011-09-19

    Intra-cellular and inter-cellular protein translocation can be observed by microscopic imaging of tissue sections prepared immunohistochemically. A manual densitometric analysis is time-consuming, subjective and error-prone. An automated quantification is faster, more reproducible, and should yield results comparable to manual evaluation. The automated method presented here was developed on rat liver tissue sections to study the translocation of bile salt transport proteins in hepatocytes. For validation, the cholestatic liver state was compared to the normal biological state. An automated quantification method was developed to analyze the translocation of membrane proteins and evaluated in comparison to an established manual method. Firstly, regions of interest (membrane fragments) are identified in confocal microscopy images. Further, densitometric intensity profiles are extracted orthogonally to membrane fragments, following the direction from the plasma membrane to cytoplasm. Finally, several different quantitative descriptors were derived from the densitometric profiles and were compared regarding their statistical significance with respect to the transport protein distribution. Stable performance, robustness and reproducibility were tested using several independent experimental datasets. A fully automated workflow for the information extraction and statistical evaluation has been developed and produces robust results. New descriptors for the intensity distribution profiles were found to be more discriminative, i.e. more significant, than those used in previous research publications for the translocation quantification. The slow manual calculation can be substituted by the fast and unbiased automated method.

  15. Improved Method for PD-Quantification in Power Cables

    DEFF Research Database (Denmark)

    Holbøll, Joachim T.; Villefrance, Rasmus; Henriksen, Mogens

    1999-01-01

    n this paper, a method is described for improved quantification of partial discharges(PD) in power cables. The method is suitable for PD-detection and location systems in the MHz-range, where pulse attenuation and distortion along the cable cannot be neglected. The system transfer function...... was calculated and measured in order to form basis for magnitude calculation after each measurements. --- Limitations and capabilities of the method will be discussed and related to relevant field applications of high frequent PD-measurements. --- Methods for increased signal/noise ratio are easily implemented...

  16. New Highly Sensitive Real-Time PCR Assay for HIV-2 Group A and Group B DNA Quantification.

    Science.gov (United States)

    Bertine, Mélanie; Gueudin, Marie; Mélard, Adeline; Damond, Florence; Descamps, Diane; Matheron, Sophie; Collin, Fidéline; Rouzioux, Christine; Plantier, Jean-Christophe; Avettand-Fenoel, Véronique

    2017-09-01

    HIV-2 infection is characterized by a very low replication rate in most cases and low progression. This necessitates an approach to patient monitoring that differs from that for HIV-1 infection. Here, a new highly specific and sensitive method for HIV-2 DNA quantification was developed. The new test is based on quantitative real-time PCR targeting the long terminal repeat (LTR) and gag regions and using an internal control. Analytical performance was determined in three laboratories, and clinical performance was determined on blood samples from 63 patients infected with HIV-2 group A ( n = 35) or group B ( n = 28). The specificity was 100%. The 95% limit of detection was three copies/PCR and the limit of quantification was six copies/PCR. The within-run coefficients of variation were between 1.03% at 3.78 log 10 copies/PCR and 27.02% at 0.78 log 10 copies/PCR. The between-run coefficient of variation was 5.10%. Both manual and automated nucleic acid extraction methods were validated. HIV-2 DNA loads were detectable in blood cells from all 63 patients. When HIV-2 DNA was quantifiable, median loads were significantly higher in antiretroviral-treated than in naive patients and were similar for groups A and B. HIV-2 DNA load was correlated with HIV-2 RNA load ( r = 0.68; 95% confidence interval [CI], 0.4 to 0.8; P < 0.0001). Our data show that this new assay is highly sensitive and quantifies the two main HIV-2 groups, making it useful for the diagnosis of HIV-2 infection and for pathogenesis studies on HIV-2 reservoirs. Copyright © 2017 American Society for Microbiology.

  17. Developmental validation of the Quantifiler(®) HP and Trio Kits for human DNA quantification in forensic samples.

    Science.gov (United States)

    Holt, Allison; Wootton, Sharon Chao; Mulero, Julio J; Brzoska, Pius M; Langit, Emanuel; Green, Robert L

    2016-03-01

    The quantification of human genomic DNA is a necessary first step in the DNA casework sample analysis workflow. DNA quantification determines optimal sample input amounts for subsequent STR (short tandem repeat) genotyping procedures, as well as being a useful screening tool to identify samples most likely to provide probative genotypic evidence. To better mesh with the capabilities of newest-generation STR analysis assays, the Quantifiler(®) HP and Quantifiler(®) Trio DNA Quantification Kits were designed for greater detection sensitivity and more robust performance with samples that contain PCR inhibitors or degraded DNA. The new DNA quantification kits use multiplex TaqMan(®) assay-based fluorescent probe technology to simultaneously quantify up to three human genomic targets, allowing samples to be assessed for total human DNA, male contributor (i.e., Y-chromosome) DNA, as well as a determination of DNA degradation state. The Quantifiler HP and Trio Kits use multiple-copy loci to allow for significantly improved sensitivity compared to earlier-generation kits that employ single-copy target loci. The kits' improved performance provides better predictive ability for results with downstream, newest-generation STR assays, and their shortened time-to-result allows more efficient integration into the forensic casework analysis workflow. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  18. Biosensor for label-free DNA quantification based on functionalized LPGs.

    Science.gov (United States)

    Gonçalves, Helena M R; Moreira, Luis; Pereira, Leonor; Jorge, Pedro; Gouveia, Carlos; Martins-Lopes, Paula; Fernandes, José R A

    2016-10-15

    A label-free fiber optic biosensor based on a long period grating (LPG) and a basic optical interrogation scheme using off the shelf components is used for the detection of in-situ DNA hybridization. A new methodology is proposed for the determination of the spectral position of the LPG mode resonance. The experimental limit of detection obtained for the DNA was 62±2nM and the limit of quantification was 209±7nM. The sample specificity was experimentally demonstrated using DNA targets with different base mismatches relatively to the probe and was found that the system has a single base mismatch selectivity. Copyright © 2015 Elsevier B.V. All rights reserved.

  19. Quantification of damage in DNA recovered from highly degraded samples – a case study on DNA in faeces

    Directory of Open Access Journals (Sweden)

    Eveson J Paige

    2006-08-01

    Full Text Available Abstract Background Poorly preserved biological tissues have become an important source of DNA for a wide range of zoological studies. Measuring the quality of DNA obtained from these samples is often desired; however, there are no widely used techniques available for quantifying damage in highly degraded DNA samples. We present a general method that can be used to determine the frequency of polymerase blocking DNA damage in specific gene-regions in such samples. The approach uses quantitative PCR to measure the amount of DNA present at several fragment sizes within a sample. According to a model of random degradation the amount of available template will decline exponentially with increasing fragment size in damaged samples, and the frequency of DNA damage (λ can be estimated by determining the rate of decline. Results The method is illustrated through the analysis of DNA extracted from sea lion faecal samples. Faeces contain a complex mixture of DNA from several sources and different components are expected to be differentially degraded. We estimated the frequency of DNA damage in both predator and prey DNA within individual faecal samples. The distribution of fragment lengths for each target fit well with the assumption of a random degradation process and, in keeping with our expectations, the estimated frequency of damage was always less in predator DNA than in prey DNA within the same sample (mean λpredator = 0.0106 per nucleotide; mean λprey = 0.0176 per nucleotide. This study is the first to explicitly define the amount of template damage in any DNA extracted from faeces and the first to quantify the amount of predator and prey DNA present within individual faecal samples. Conclusion We present an approach for characterizing mixed, highly degraded PCR templates such as those often encountered in ecological studies using non-invasive samples as a source of DNA, wildlife forensics investigations and ancient DNA research. This method will

  20. Development of a method for detection and quantification of B. brongniartii and B. bassiana in soil

    Science.gov (United States)

    Canfora, L.; Malusà, E.; Tkaczuk, C.; Tartanus, M.; Łabanowska, B. H.; Pinzari, F.

    2016-03-01

    A culture independent method based on qPCR was developed for the detection and quantification of two fungal inoculants in soil. The aim was to adapt a genotyping approach based on SSR (Simple Sequence Repeat) marker to a discriminating tracing of two different species of bioinoculants in soil, after their in-field release. Two entomopathogenic fungi, Beauveria bassiana and B. brongniartii, were traced and quantified in soil samples obtained from field trials. These two fungal species were used as biological agents in Poland to control Melolontha melolontha (European cockchafer), whose larvae live in soil menacing horticultural crops. Specificity of SSR markers was verified using controls consisting of: i) soil samples containing fungal spores of B. bassiana and B. brongniartii in known dilutions; ii) the DNA of the fungal microorganisms; iii) soil samples singly inoculated with each fungus species. An initial evaluation of the protocol was performed with analyses of soil DNA and mycelial DNA. Further, the simultaneous detection and quantification of B. bassiana and B. brongniartii in soil was achieved in field samples after application of the bio-inoculants. The protocol can be considered as a relatively low cost solution for the detection, identification and traceability of fungal bio-inoculants in soil.

  1. 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

    2016-02-05

    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. Copyright © 2015 Elsevier B.V. All rights reserved.

  2. Comparison of quantification algorithms for circulating cell-free DNA methylation biomarkers in blood plasma from cancer patients.

    Science.gov (United States)

    de Vos, Luka; Gevensleben, Heidrun; Schröck, Andreas; Franzen, Alina; Kristiansen, Glen; Bootz, Friedrich; Dietrich, Dimo

    2017-01-01

    SHOX2 and SEPT9 methylation in circulating cell-free DNA (ccfDNA) in blood are established powerful and clinically valuable biomarkers for diagnosis, staging, prognosis, and monitoring of cancer patients. The aim of the present study was to evaluate different quantification algorithms (relative quantification, absolute quantification, quasi-digital PCR) with regard to their clinical performance. Methylation analyses were performed in a training cohort (141 patients with head and neck squamous cell carcinoma [HNSCC], 170 control cases) and a testing cohort (137 HNSCC cases, 102 controls). DNA was extracted from plasma samples, bisulfite-converted, and analyzed via quantitative real-time PCR. SHOX2 and SEPT9 methylations were assessed separately and as panel [mean SEPT9 / SHOX2 ] using the ΔCT method for absolute quantification and the ΔΔCT-method for relative quantification. Quasi-digital PCR was defined as the number of amplification-positive PCR replicates. The diagnostic (sensitivity, specificity, area under the curve (AUC) of the receiver operating characteristic (ROC)) and prognostic accuracy (hazard ratio (HR) from Cox regression) were evaluated. Sporadic methylation in control samples necessitated the introduction of cutoffs resulting in 61-63% sensitivity/90-92% specificity ( SEPT9 /training), 53-57% sensitivity/87-90% specificity ( SHOX2 /training), and 64-65% sensitivity/90-91% specificity (mean SEPT9 / SHOX2 /training). Results were confirmed in a testing cohort with 54-56% sensitivity/88-90% specificity ( SEPT9 /testing), 43-48% sensitivity/93-95% specificity ( SHOX2 /testing), and 49-58% sensitivity/88-94% specificity (mean SEPT9 / SHOX2 /testing). All algorithms showed comparable cutoff-independent diagnostic accuracy with largely overlapping 95% confidence intervals ( SEPT9 : AUC training  = 0.79-0.80; AUC testing  = 0.74-0.75; SHOX2 : AUC training  = 0.78-0.81, AUC testing  = 0.77-0.79; mean SEPT9 / SHOX2 : AUC training  = 0

  3. Direct detection and quantification of abasic sites for in vivo studies of DNA damage and repair

    International Nuclear Information System (INIS)

    Wang Yanming; Liu Lili; Wu Chunying; Bulgar, Alina; Somoza, Eduardo; Zhu Wenxia; Gerson, Stanton L.

    2009-01-01

    Use of chemotherapeutic agents to induce cytotoxic DNA damage and programmed cell death is a key strategy in cancer treatments. However, the efficacy of DNA-targeted agents such as temozolomide is often compromised by intrinsic cellular responses such as DNA base excision repair (BER). Previous studies have shown that BER pathway resulted in formation of abasic or apurinic/apyrimidinic (AP) sites, and blockage of AP sites led to a significant enhancement of drug sensitivity due to reduction of DNA base excision repair. Since a number of chemotherapeutic agents also induce formation of AP sites, monitoring of these sites as a clinical correlate of drug effect will provide a useful tool in the development of DNA-targeted chemotherapies aimed at blocking abasic sites from repair. Here we report an imaging technique based on positron emission tomography (PET) that allows for direct quantification of AP sites in vivo. For this purpose, positron-emitting carbon-11 has been incorporated into methoxyamine ([ 11 C]MX) that binds covalently to AP sites with high specificity. The binding specificity of [ 11 C]MX for AP sites was demonstrated by in vivo blocking experiments. Using [ 11 C]MX as a radiotracer, animal PET studies have been conducted in melanoma and glioma xenografts for quantification of AP sites. Following induction of AP sites by temozolomide, both tumor models showed significant increase of [ 11 C]MX uptake in tumor regions in terms of radioactivity concentration as a function of time, which correlates well with conventional aldehyde reactive probe (ARP)-based bioassays for AP sites.

  4. A practical method for accurate quantification of large fault trees

    International Nuclear Information System (INIS)

    Choi, Jong Soo; Cho, Nam Zin

    2007-01-01

    This paper describes a practical method to accurately quantify top event probability and importance measures from incomplete minimal cut sets (MCS) of a large fault tree. The MCS-based fault tree method is extensively used in probabilistic safety assessments. Several sources of uncertainties exist in MCS-based fault tree analysis. The paper is focused on quantification of the following two sources of uncertainties: (1) the truncation neglecting low-probability cut sets and (2) the approximation in quantifying MCSs. The method proposed in this paper is based on a Monte Carlo simulation technique to estimate probability of the discarded MCSs and the sum of disjoint products (SDP) approach complemented by the correction factor approach (CFA). The method provides capability to accurately quantify the two uncertainties and estimate the top event probability and importance measures of large coherent fault trees. The proposed fault tree quantification method has been implemented in the CUTREE code package and is tested on the two example fault trees

  5. The effects of metal ion PCR inhibitors on results obtained with the Quantifiler(®) Human DNA Quantification Kit.

    Science.gov (United States)

    Combs, Laura Gaydosh; Warren, Joseph E; Huynh, Vivian; Castaneda, Joanna; Golden, Teresa D; Roby, Rhonda K

    2015-11-01

    Forensic DNA samples may include the presence of PCR inhibitors, even after extraction and purification. Studies have demonstrated that metal ions, co-purified at specific concentrations, inhibit DNA amplifications. Metal ions are endogenous to sample types, such as bone, and can be introduced from environmental sources. In order to examine the effect of metal ions as PCR inhibitors during quantitative real-time PCR, 2800 M DNA was treated with 0.0025-18.750 mM concentrations of aluminum, calcium, copper, iron, nickel, and lead. DNA samples, both untreated and metal-treated, were quantified using the Quantifiler(®) Human DNA Quantification Kit. Quantification cycle (Cq) values for the Quantifiler(®) Human DNA and internal PCR control (IPC) assays were measured and the estimated concentrations of human DNA were obtained. Comparisons were conducted between metal-treated and control DNA samples to determine the accuracy of the quantification estimates and to test the efficacy of the IPC inhibition detection. This kit is most resistant to the presence of calcium as compared to all metals tested; the maximum concentration tested does not affect the amplification of the IPC or quantification of the sample. This kit is most sensitive to the presence of aluminum; concentrations greater than 0.0750 mM negatively affected the quantification, although the IPC assay accurately assessed the presence of PCR inhibition. The Quantifiler(®) Human DNA Quantification Kit accurately quantifies human DNA in the presence of 0.5000 mM copper, iron, nickel, and lead; however, the IPC does not indicate the presence of PCR inhibition at this concentration of these metals. Unexpectedly, estimates of DNA quantity in samples treated with 18.750 mM copper yielded values in excess of the actual concentration of DNA in the samples; fluorescence spectroscopy experiments indicated this increase was not a direct interaction between the copper metal and 6-FAM dye used to label the probe that

  6. Distortion of genetically modified organism quantification in processed foods: influence of particle size compositions and heat-induced DNA degradation.

    Science.gov (United States)

    Moreano, Francisco; Busch, Ulrich; Engel, Karl-Heinz

    2005-12-28

    Milling fractions from conventional and transgenic corn were prepared at laboratory scale and used to study the influence of sample composition and heat-induced DNA degradation on the relative quantification of genetically modified organisms (GMO) in food products. Particle size distributions of the obtained fractions (coarse grits, regular grits, meal, and flour) were characterized using a laser diffraction system. The application of two DNA isolation protocols revealed a strong correlation between the degree of comminution of the milling fractions and the DNA yield in the extracts. Mixtures of milling fractions from conventional and transgenic material (1%) were prepared and analyzed via real-time polymerase chain reaction. Accurate quantification of the adjusted GMO content was only possible in mixtures containing conventional and transgenic material in the form of analogous milling fractions, whereas mixtures of fractions exhibiting different particle size distributions delivered significantly over- and underestimated GMO contents depending on their compositions. The process of heat-induced nucleic acid degradation was followed by applying two established quantitative assays showing differences between the lengths of the recombinant and reference target sequences (A, deltal(A) = -25 bp; B, deltal(B) = +16 bp; values related to the amplicon length of the reference gene). Data obtained by the application of method A resulted in underestimated recoveries of GMO contents in the samples of heat-treated products, reflecting the favored degradation of the longer target sequence used for the detection of the transgene. In contrast, data yielded by the application of method B resulted in increasingly overestimated recoveries of GMO contents. The results show how commonly used food technological processes may lead to distortions in the results of quantitative GMO analyses.

  7. Comparison of the quantification of KRAS mutations by digital PCR and E-ice-COLD-PCR in circulating-cell-free DNA from metastatic colorectal cancer patients.

    Science.gov (United States)

    Sefrioui, David; Mauger, Florence; Leclere, Laurence; Beaussire, Ludivine; Di Fiore, Frédéric; Deleuze, Jean-François; Sarafan-Vasseur, Nasrin; Tost, Jörg

    2017-02-01

    Circulating cell-free DNA (ccfDNA) bears great promise as biomarker for personalized medicine, but ccfDNA is present only at low levels in the plasma or serum of cancer patients. E-ice-COLD-PCR is a recently developed enrichment method to detect and identify mutations present at low-abundance in clinical samples. However, recent studies have shown the importance to accurately quantify low-abundance mutations as clinically important decisions will depend on certain mutation thresholds. The possibility for an enrichment method to accurately quantify the mutation levels remains a point of concern and might limit its clinical applicability. In the present study, we compared the quantification of KRAS mutations in ccfDNA from metastatic colorectal cancer patients by E-ice-COLD-PCR with two digital PCR approaches. For the quantification of mutations by E-ice-COLD-PCR, cell lines with known mutations diluted into WT genomic DNA were used for calibration. E-ice-COLD-PCR and the two digital PCR approaches showed the same range of the mutation level and were concordant for mutation levels below the clinical relevant threshold. E-ice-COLD-PCR can accurately detect and quantify low-abundant mutations in ccfDNA and has a shorter time to results making it compatible with the requirements of analyses in a clinical setting without the loss of quantitative accuracy. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  8. Overview of hybrid subspace methods for uncertainty quantification, sensitivity analysis

    International Nuclear Information System (INIS)

    Abdel-Khalik, Hany S.; Bang, Youngsuk; Wang, Congjian

    2013-01-01

    Highlights: ► We overview the state-of-the-art in uncertainty quantification and sensitivity analysis. ► We overview new developments in above areas using hybrid methods. ► We give a tutorial introduction to above areas and the new developments. ► Hybrid methods address the explosion in dimensionality in nonlinear models. ► Representative numerical experiments are given. -- Abstract: The role of modeling and simulation has been heavily promoted in recent years to improve understanding of complex engineering systems. To realize the benefits of modeling and simulation, concerted efforts in the areas of uncertainty quantification and sensitivity analysis are required. The manuscript intends to serve as a pedagogical presentation of the material to young researchers and practitioners with little background on the subjects. We believe this is important as the role of these subjects is expected to be integral to the design, safety, and operation of existing as well as next generation reactors. In addition to covering the basics, an overview of the current state-of-the-art will be given with particular emphasis on the challenges pertaining to nuclear reactor modeling. The second objective will focus on presenting our own development of hybrid subspace methods intended to address the explosion in the computational overhead required when handling real-world complex engineering systems.

  9. Immunological detection and quantification of DNA components structurally modified by alkylating carcinogens, mutagens and chemotherapeutic agents

    International Nuclear Information System (INIS)

    Rajewsky, M.F.

    1983-01-01

    The detection and quantification of defined reaction products of chemical mutagens and carcinogens (and of many cancer chemotherapeutic agents) with DNA require highly sensitive analytical techniques. The exceptional capability of immunoglobulins to recognize subtle alterations of molecular structure (especially when monoclonal antibodies are used to maximize specificity), outstanding sensitivity of immunoanalysis by high-affinity antibodies, and the fact that radioactively-labelled agents are not required suggest the utility of a radioimmunoassay to recognize and quantitate alkylated DNA products. We have recently developed a set of high-affinity monoclonal antibodies (secreted by mouse x mouse as well as by rat x rat hybridomas; antibody affinity constants, 10 9 to > 10 10 lmol) specifically directed against several DNA alkylation products with possible relevance in relation to both mutagenesis and malignant transformation of mammalian cells. These alkylation products include 0 6 -N-butyldeoxyguanosine, and 0 4 -ethyldeoxythymidine. When used in a radioimmunassay, an antibody specific for 0 6 -ethyldeoxyguanosine, for example, will detect this product at an 0 6 -ethyldeoxyguanosine/deoxyguanosine molar ratio of approx. 3 x 10 -7 in a hydrolysate of 100 ug of DNA. The limit of detection can be lowered further if the respective alkyldeoxynucleosides are separated by HPLC from the DNA hydrolysate prior to the RIA. The anti-alkyldeoxynucleoside monoclonal antibodies can also be used to visualize, by immunostaining and fluorescence microscopy combined with electronic image intensification, specific alkylation products in the nuclear DNA of individual cells, and to localize structurally modified bases in double-stranded DNA molecules by transmission electron microscopy

  10. 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.

  11. Method for assessing damage to mitochondrial DNA caused by radiation and epichlorohydrin

    International Nuclear Information System (INIS)

    Singh, G.; Hauswirth, W.W.; Ross, W.E.; Neims, A.H.

    1985-01-01

    This paper describes a rapid and reliable method for quantification of damage to mitochondrial DNA (mtDNA), especially strand breaks. The degree of damage to mtDNA is assessed by the proportion of physical forms (i.e., supercoiled versus open-circular and linear forms) upon agarose gel electrophoresis, blotting, and visualization by hybridization with [ 32 P]mtDNA probes. The use of a radiolabeled probe is a crucial step in the procedure because it provides both a means to quantify by radioautography and to obtain the mtDNA specificity required to eliminate misinterpretation due to nuclear DNA contamination. To demonstrate the utility of this technique, X-irradiation and epichlorohydrin are shown to damage both isolated mtDNA and mtDNA in whole cells in a dose-dependent fashion

  12. Reliability and discriminatory power of methods for dental plaque quantification

    Directory of Open Access Journals (Sweden)

    Daniela Prócida Raggio

    2010-04-01

    Full Text Available OBJECTIVE: This in situ study evaluated the discriminatory power and reliability of methods of dental plaque quantification and the relationship between visual indices (VI and fluorescence camera (FC to detect plaque. MATERIAL AND METHODS: Six volunteers used palatal appliances with six bovine enamel blocks presenting different stages of plaque accumulation. The presence of plaque with and without disclosing was assessed using VI. Images were obtained with FC and digital camera in both conditions. The area covered by plaque was assessed. Examinations were done by two independent examiners. Data were analyzed by Kruskal-Wallis and Kappa tests to compare different conditions of samples and to assess the inter-examiner reproducibility. RESULTS: Some methods presented adequate reproducibility. The Turesky index and the assessment of area covered by disclosed plaque in the FC images presented the highest discriminatory powers. CONCLUSION: The Turesky index and images with FC with disclosing present good reliability and discriminatory power in quantifying dental plaque.

  13. Molecular Combing of DNA: Methods and Applications

    DEFF Research Database (Denmark)

    Nazari, Zeniab Esmail; Gurevich, Leonid

    2013-01-01

    studies to nanoelectronics. While molecular combing has been applied in a variety of DNA-related studies, no comprehensive review has been published on different combing methods proposed so far. In this review, the underlying mechanisms of molecular combing of DNA are described followed by discussion...

  14. Properties of targeted preamplification in DNA and cDNA quantification.

    Science.gov (United States)

    Andersson, Daniel; Akrap, Nina; Svec, David; Godfrey, Tony E; Kubista, Mikael; Landberg, Göran; Ståhlberg, Anders

    2015-01-01

    Quantification of small molecule numbers often requires preamplification to generate enough copies for accurate downstream enumerations. Here, we studied experimental parameters in targeted preamplification and their effects on downstream quantitative real-time PCR (qPCR). To evaluate different strategies, we monitored the preamplification reaction in real-time using SYBR Green detection chemistry followed by melting curve analysis. Furthermore, individual targets were evaluated by qPCR. The preamplification reaction performed best when a large number of primer pairs was included in the primer pool. In addition, preamplification efficiency, reproducibility and specificity were found to depend on the number of template molecules present, primer concentration, annealing time and annealing temperature. The amount of nonspecific PCR products could also be reduced about 1000-fold using bovine serum albumin, glycerol and formamide in the preamplification. On the basis of our findings, we provide recommendations how to perform robust and highly accurate targeted preamplification in combination with qPCR or next-generation sequencing.

  15. UV-Visible Spectroscopy-Based Quantification of Unlabeled DNA Bound to Gold Nanoparticles.

    Science.gov (United States)

    Baldock, Brandi L; Hutchison, James E

    2016-12-20

    DNA-functionalized gold nanoparticles have been increasingly applied as sensitive and selective analytical probes and biosensors. The DNA ligands bound to a nanoparticle dictate its reactivity, making it essential to know the type and number of DNA strands bound to the nanoparticle surface. Existing methods used to determine the number of DNA strands per gold nanoparticle (AuNP) require that the sequences be fluorophore-labeled, which may affect the DNA surface coverage and reactivity of the nanoparticle and/or require specialized equipment and other fluorophore-containing reagents. We report a UV-visible-based method to conveniently and inexpensively determine the number of DNA strands attached to AuNPs of different core sizes. When this method is used in tandem with a fluorescence dye assay, it is possible to determine the ratio of two unlabeled sequences of different lengths bound to AuNPs. Two sizes of citrate-stabilized AuNPs (5 and 12 nm) were functionalized with mixtures of short (5 base) and long (32 base) disulfide-terminated DNA sequences, and the ratios of sequences bound to the AuNPs were determined using the new method. The long DNA sequence was present as a lower proportion of the ligand shell than in the ligand exchange mixture, suggesting it had a lower propensity to bind the AuNPs than the short DNA sequence. The ratio of DNA sequences bound to the AuNPs was not the same for the large and small AuNPs, which suggests that the radius of curvature had a significant influence on the assembly of DNA strands onto the AuNPs.

  16. Influence of DNA isolation on Q-PCR-based quantification of methanogenic Archaea in biogas fermenters.

    Science.gov (United States)

    Bergmann, I; Mundt, K; Sontag, M; Baumstark, I; Nettmann, E; Klocke, M

    2010-03-01

    Quantitative real-time PCR (Q-PCR) is commonly applied for the detection of certain microorganisms in environmental samples. However, some environments, like biomass-degrading biogas fermenters, are enriched with PCR-interfering substances. To study the impact of the DNA extraction protocol on the results of Q-PCR-based analysis of the methane-producing archaeal community in biogas fermenters, nine different protocols with varying cell disruption and DNA purification approaches were tested. Differences in the quantities of the isolated DNA and the purity parameters were found, with the best cell lysis efficiencies being obtained by a combined lysozyme/SDS-based lysis. When DNA was purified by sephacryl columns, the amount of DNA decreased by one log cycle but PCR inhibitors were eliminated sufficiently. In the case of detection of methanogenic Archaea, the chosen DNA isolation protocol strongly influenced the Q-PCR-based determination of 16S rDNA copy numbers. For example, with protocols including mechanical cell disruption, the 16S rDNA of Methanobacteriales were predominantly amplified (81-90% of the total 16S rDNA copy numbers), followed by the 16S rDNA of Methanomicrobiales (9-18%). In contrast, when a lysozyme/SDS-based cell lysis was applied, the 16S rDNA copy numbers determined for these two orders were the opposite (Methanomicrobiales 82-95%, Methanobacteriales 4-18%). In extreme cases, the DNA isolation method led to discrimination of some groups of methanogens (e.g. members of the Methanosaetaceae). In conclusion, for extraction of high amounts of microbial DNA with high purity from samples of biogas plants, a combined lysozyme/SDS-based cell lysis followed by a purification step with sephacryl columns is recommended. Copyright 2010 Elsevier GmbH. All rights reserved.

  17. Usefulness of in-house real time PCR for HBV DNA quantification in serum and oral fluid samples.

    Science.gov (United States)

    Portilho, Moyra Machado; Mendonça, Ana Carolina da Fonseca; Bezerra, Cristianne Sousa; do Espirito-Santo, Márcia Paschoal; de Paula, Vanessa Salete; Nabuco, Leticia Cancella; Villela-Nogueira, Cristiane Alves; Lewis-Ximenez, Lia Laura; Lampe, Elisabeth; Villar, Livia Melo

    2018-06-01

    For quantification of hepatitis B virus DNA (HBV DNA), commercial assays are used with serum or plasma samples, but oral fluid samples could be an alternative for HBV diagnosis due to ease of collection. This study aims to develop in-house real time PCR using synthetic curve for HBV DNA quantification for serum and oral fluid samples. Samples were collected from 103 individuals (55 HBsAg reactive and HBV DNA reactive by commercial assay and 48 without HBV markers) and submitted to two in-house real time PCR assays for HBV pre-S/S region with different standard curves: qPCR plasmidial and qPCR synthetic. A total of 27 serum samples were HBV DNA positive by qPCR plasmidial and 40 with qPCR synthetic (72% and 85% of concordance, respectively). Quantitative PCR synthetic presented efficiency of 99% and sensitivity of 2log10 copies/mL. Among oral fluid samples, five and ten were detected using qPCR plasmidial and synthetic, respectively. This study demonstrated that qPCR synthetic using serum samples could be used as alternative for HBV DNA quantification due to its sensitivity. In addition, it was possible to quantify HBV DNA in oral fluid samples suggesting the potential of this specimen for molecular diagnosis of HBV. Copyright © 2018 Elsevier B.V. All rights reserved.

  18. Assessment of DNA degradation induced by thermal and UV radiation processing: implications for quantification of genetically modified organisms.

    Science.gov (United States)

    Ballari, Rajashekhar V; Martin, Asha

    2013-12-01

    DNA quality is an important parameter for the detection and quantification of genetically modified organisms (GMO's) using the polymerase chain reaction (PCR). Food processing leads to degradation of DNA, which may impair GMO detection and quantification. This study evaluated the effect of various processing treatments such as heating, baking, microwaving, autoclaving and ultraviolet (UV) irradiation on the relative transgenic content of MON 810 maize using pRSETMON-02, a dual target plasmid as a model system. Amongst all the processing treatments examined, autoclaving and UV irradiation resulted in the least recovery of the transgenic (CaMV 35S promoter) and taxon-specific (zein) target DNA sequences. Although a profound impact on DNA degradation was seen during the processing, DNA could still be reliably quantified by Real-time PCR. The measured mean DNA copy number ratios of the processed samples were in agreement with the expected values. Our study confirms the premise that the final analytical value assigned to a particular sample is independent of the degree of DNA degradation since the transgenic and the taxon-specific target sequences possessing approximately similar lengths degrade in parallel. The results of our study demonstrate that food processing does not alter the relative quantification of the transgenic content provided the quantitative assays target shorter amplicons and the difference in the amplicon size between the transgenic and taxon-specific genes is minimal. Copyright © 2013 Elsevier Ltd. All rights reserved.

  19. Robust sleep quality quantification method for a personal handheld device.

    Science.gov (United States)

    Shin, Hangsik; Choi, Byunghun; Kim, Doyoon; Cho, Jaegeol

    2014-06-01

    The purpose of this study was to develop and validate a novel method for sleep quality quantification using personal handheld devices. The proposed method used 3- or 6-axes signals, including acceleration and angular velocity, obtained from built-in sensors in a smartphone and applied a real-time wavelet denoising technique to minimize the nonstationary noise. Sleep or wake status was decided on each axis, and the totals were finally summed to calculate sleep efficiency (SE), regarded as sleep quality in general. The sleep experiment was carried out for performance evaluation of the proposed method, and 14 subjects participated. An experimental protocol was designed for comparative analysis. The activity during sleep was recorded not only by the proposed method but also by well-known commercial applications simultaneously; moreover, activity was recorded on different mattresses and locations to verify the reliability in practical use. Every calculated SE was compared with the SE of a clinically certified medical device, the Philips (Amsterdam, The Netherlands) Actiwatch. In these experiments, the proposed method proved its reliability in quantifying sleep quality. Compared with the Actiwatch, accuracy and average bias error of SE calculated by the proposed method were 96.50% and -1.91%, respectively. The proposed method was vastly superior to other comparative applications with at least 11.41% in average accuracy and at least 6.10% in average bias; average accuracy and average absolute bias error of comparative applications were 76.33% and 17.52%, respectively.

  20. Multiple tag labeling method for DNA sequencing

    Science.gov (United States)

    Mathies, R.A.; Huang, X.C.; Quesada, M.A.

    1995-07-25

    A DNA sequencing method is described which uses single lane or channel electrophoresis. Sequencing fragments are separated in the lane and detected using a laser-excited, confocal fluorescence scanner. Each set of DNA sequencing fragments is separated in the same lane and then distinguished using a binary coding scheme employing only two different fluorescent labels. Also described is a method of using radioisotope labels. 5 figs.

  1. Numerical Continuation Methods for Intrusive Uncertainty Quantification Studies

    Energy Technology Data Exchange (ETDEWEB)

    Safta, Cosmin [Sandia National Lab. (SNL-NM), Albuquerque, NM (United States); Najm, Habib N. [Sandia National Lab. (SNL-NM), Albuquerque, NM (United States); Phipps, Eric Todd [Sandia National Lab. (SNL-NM), Albuquerque, NM (United States)

    2014-09-01

    Rigorous modeling of engineering systems relies on efficient propagation of uncertainty from input parameters to model outputs. In recent years, there has been substantial development of probabilistic polynomial chaos (PC) Uncertainty Quantification (UQ) methods, enabling studies in expensive computational models. One approach, termed ”intrusive”, involving reformulation of the governing equations, has been found to have superior computational performance compared to non-intrusive sampling-based methods in relevant large-scale problems, particularly in the context of emerging architectures. However, the utility of intrusive methods has been severely limited due to detrimental numerical instabilities associated with strong nonlinear physics. Previous methods for stabilizing these constructions tend to add unacceptably high computational costs, particularly in problems with many uncertain parameters. In order to address these challenges, we propose to adapt and improve numerical continuation methods for the robust time integration of intrusive PC system dynamics. We propose adaptive methods, starting with a small uncertainty for which the model has stable behavior and gradually moving to larger uncertainty where the instabilities are rampant, in a manner that provides a suitable solution.

  2. High throughput DNA damage quantification of human tissue with home-based collection device

    Energy Technology Data Exchange (ETDEWEB)

    Costes, Sylvain V.; Tang, Jonathan; Yannone, Steven M.

    2018-04-03

    Kits, methods and systems for providing a service to provide a subject with information regarding the state of a subject's DNA damage. Collection, processing and analysis of samples are also described.

  3. High throughput DNA damage quantification of human tissue with home-based collection device

    Science.gov (United States)

    Costes, Sylvain V.; Tang, Jonathan; Yannone, Steven M.

    2018-04-03

    Kits, methods and systems for providing a service to provide a subject with information regarding the state of a subject's DNA damage. Collection, processing and analysis of samples are also described.

  4. Liquid chromatography-tandem mass spectrometry quantification of 6-thioguanine in DNA using endogenous guanine as internal standard

    DEFF Research Database (Denmark)

    Jacobsen, Jack Hummeland; Schmiegelow, Kjeld; Nersting, Jakob

    2012-01-01

    was estimated at 63% (RSD 26%), which is corrected for by the internal standard resulting in stable quantification. The TG levels found were above the LOQ in 18 out of 18 childhood leukemia patients on 6-mercaptopurine/methotrexate maintenance therapy (median 377, range 45-1190 fmol/μg DNA) with intra...

  5. Chip-Oriented Fluorimeter Design and Detection System Development for DNA Quantification in Nano-Liter Volumes

    Directory of Open Access Journals (Sweden)

    Da-Sheng Lee

    2009-12-01

    Full Text Available The chip-based polymerase chain reaction (PCR system has been developed in recent years to achieve DNA quantification. Using a microstructure and miniature chip, the volume consumption for a PCR can be reduced to a nano-liter. With high speed cycling and a low reaction volume, the time consumption of one PCR cycle performed on a chip can be reduced. However, most of the presented prototypes employ commercial fluorimeters which are not optimized for fluorescence detection of such a small quantity sample. This limits the performance of DNA quantification, especially low experiment reproducibility. This study discusses the concept of a chip-oriented fluorimeter design. Using the analytical model, the current study analyzes the sensitivity and dynamic range of the fluorimeter to fit the requirements for detecting fluorescence in nano-liter volumes. Through the optimized processes, a real-time PCR on a chip system with only one nano-liter volume test sample is as sensitive as the commercial real-time PCR machine using the sample with twenty micro-liter volumes. The signal to noise (S/N ratio of a chip system for DNA quantification with hepatitis B virus (HBV plasmid samples is 3 dB higher. DNA quantification by the miniature chip shows higher reproducibility compared to the commercial machine with respect to samples of initial concentrations from 103 to 105 copies per reaction.

  6. Quantification of DNA fragmentation in processed foods using real-time PCR.

    Science.gov (United States)

    Mano, Junichi; Nishitsuji, Yasuyuki; Kikuchi, Yosuke; Fukudome, Shin-Ichi; Hayashida, Takuya; Kawakami, Hiroyuki; Kurimoto, Youichi; Noguchi, Akio; Kondo, Kazunari; Teshima, Reiko; Takabatake, Reona; Kitta, Kazumi

    2017-07-01

    DNA analysis of processed foods is performed widely to detect various targets, such as genetically modified organisms (GMOs). Food processing often causes DNA fragmentation, which consequently affects the results of PCR analysis. In order to assess the effects of DNA fragmentation on the reliability of PCR analysis, we investigated a novel methodology to quantify the degree of DNA fragmentation. We designed four real-time PCR assays that amplified 18S ribosomal RNA gene sequences common to various plants at lengths of approximately 100, 200, 400, and 800 base pairs (bp). Then, we created an indicator value, "DNA fragmentation index (DFI)", which is calculated from the Cq values derived from the real-time PCR assays. Finally, we demonstrated the efficacy of this method for the quality control of GMO detection in processed foods by evaluating the relationship between the DFI and the limit of detection. Copyright © 2017 Elsevier Ltd. All rights reserved.

  7. A Real Time PCR Platform for the Simultaneous Quantification of Total and Extrachromosomal HIV DNA Forms in Blood of HIV-1 Infected Patients

    Science.gov (United States)

    Canovari, Benedetta; Scotti, Maddalena; Acetoso, Marcello; Valentini, Massimo; Petrelli, Enzo; Magnani, Mauro

    2014-01-01

    Background The quantitative measurement of various HIV-1 DNA forms including total, unintegrated and integrated provirus play an increasingly important role in HIV-1 infection monitoring and treatment-related research. We report the development and validation of a SYBR Green real time PCR (TotUFsys platform) for the simultaneous quantification of total and extrachromosomal HIV-1 DNA forms in patients. This innovative technique makes it possible to obtain both measurements in a single PCR run starting from frozen blood employing the same primers and standard curve. Moreover, due to identical amplification efficiency, it allows indirect estimation of integrated level. To specifically detect 2-LTR a qPCR method was also developed. Methodology/Findings Primers used for total HIV-1 DNA quantification spanning a highly conserved region were selected and found to detect all HIV-1 clades of group M and the unintegrated forms of the same. A total of 195 samples from HIV-1 patients in a wide range of clinical conditions were analyzed with a 100% success rate, even in patients with suppressed plasma viremia, regardless of CD4+ or therapy. No significant correlation was observed between the two current prognostic markers, CD4+ and plasma viremia, while a moderate or high inverse correlation was found between CD4+ and total HIV DNA, with strong values for unintegrated HIV DNA. Conclusions/Significance Taken together, the results support the use of HIV DNA as another tool, in addition to traditional assays, which can be used to estimate the state of viral infection, the risk of disease progression and to monitor the effects of ART. The TotUFsys platform allowed us to obtain a final result, expressed as the total and unintegrated HIV DNA copy number per microgram of DNA or 104 CD4+, for 12 patients within two working days. PMID:25364909

  8. Quantification methods of Black Carbon: Comparison of Rock-Eval analysis with traditional methods

    NARCIS (Netherlands)

    Poot, A.; Quik, J.T.K.; Veld, H.; Koelmans, A.A.

    2009-01-01

    Black Carbon (BC) quantification methods are reviewed, including new Rock-Eval 6 data on BC reference materials. BC has been reported to have major impacts on climate, human health and environmental quality. Especially for risk assessment of persistent organic pollutants (POPs) it is important to

  9. Real-Time PCR Quantification of Heteroplasmy in a Mouse Model with Mitochondrial DNA of C57BL/6 and NZB/BINJ Strains

    Science.gov (United States)

    Sangalli, Juliano Rodrigues; Rodrigues, Thiago Bittencourt; Smith, Lawrence Charles; Meirelles, Flávio Vieira; Chiaratti, Marcos Roberto

    2015-01-01

    Mouse models are widely employed to study mitochondrial inheritance, which have implications to several human diseases caused by mutations in the mitochondrial genome (mtDNA). These mouse models take advantage of polymorphisms between the mtDNA of the NZB/BINJ and the mtDNA of common inbred laboratory (i.e., C57BL/6) strains to generate mice with two mtDNA haplotypes (heteroplasmy). Based on PCR followed by restriction fragment length polymorphism (PCR-RFLP), these studies determine the level of heteroplasmy across generations and in different cell types aiming to understand the mechanisms underlying mitochondrial inheritance. However, PCR-RFLP is a time-consuming method of low sensitivity and accuracy that dependents on the use of restriction enzyme digestions. A more robust method to measure heteroplasmy has been provided by the use of real-time quantitative PCR (qPCR) based on allelic refractory mutation detection system (ARMS-qPCR). Herein, we report an ARMS-qPCR assay for quantification of heteroplasmy using heteroplasmic mice with mtDNA of NZB/BINJ and C57BL/6 origin. Heteroplasmy and mtDNA copy number were estimated in germline and somatic tissues, providing evidence of the reliability of the approach. Furthermore, it enabled single-step quantification of heteroplasmy, with sensitivity to detect as low as 0.1% of either NZB/BINJ or C57BL/6 mtDNA. These findings are relevant as the ARMS-qPCR assay reported here is fully compatible with similar heteroplasmic mouse models used to study mitochondrial inheritance in mammals. PMID:26274500

  10. Fluorescent strategy based on cationic conjugated polymer fluorescence resonance energy transfer for the quantification of 5-(hydroxymethyl)cytosine in genomic DNA.

    Science.gov (United States)

    Hong, Tingting; Wang, Tianlu; Guo, Pu; Xing, Xiwen; Ding, Fei; Chen, Yuqi; Wu, Jinjun; Ma, Jingwei; Wu, Fan; Zhou, Xiang

    2013-11-19

    DNA methylation is dynamically reprogrammed during early embryonic development in mammals. It can be explained partially by the discovery of 5-(hydroxymethyl)cytosine (5-hmC), 5-formylcytosine (5-fC), and 5-carboxylcytosine (5-caC), which are identified as key players involved in both active and passive demethylation pathways. As one of the ten-eleven translocation oxidation products, 5-hmC was found relatively abundant in neuron cells and embryonic stem cells. Herein we report a new method for 5-hmC quantification in genomic DNA based on CCP-FRET (cationic conjugated polymers act as the energy donor and induce fluorescence resonance energy transfer) assay combined with KRuO4 oxidation. 5-hmC in genomic DNA can be selectively transformed into 5-fC by the oxidation of KRuO4 and then labeled with hydroxylamine-BODIPY (BODIPY = 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene) fluorophore through the reaction between 5-fC and hydroxylamine-BODIPY. After the fluorescently labeled DNA was captured by CCP through electrostatic interactions, a significant FRET between CCP and hydroxylamine-BODIPY fluorophore was observed. This CCP-FRET-based assay benefits from light-harvesting, large Stokes shift, and optical signal amplification properties of the CCP. Furthermore, this CCP-FRET-based assay was quite successfully demonstrated for the 5-hmC quantification in three types of cells (mESc, HeLa, HEK 293T), providing a much more convenient choice for 5-hmC quantification in genomic DNA.

  11. A force-based, parallel assay for the quantification of protein-DNA interactions.

    Science.gov (United States)

    Limmer, Katja; Pippig, Diana A; Aschenbrenner, Daniela; Gaub, Hermann E

    2014-01-01

    Analysis of transcription factor binding to DNA sequences is of utmost importance to understand the intricate regulatory mechanisms that underlie gene expression. Several techniques exist that quantify DNA-protein affinity, but they are either very time-consuming or suffer from possible misinterpretation due to complicated algorithms or approximations like many high-throughput techniques. We present a more direct method to quantify DNA-protein interaction in a force-based assay. In contrast to single-molecule force spectroscopy, our technique, the Molecular Force Assay (MFA), parallelizes force measurements so that it can test one or multiple proteins against several DNA sequences in a single experiment. The interaction strength is quantified by comparison to the well-defined rupture stability of different DNA duplexes. As a proof-of-principle, we measured the interaction of the zinc finger construct Zif268/NRE against six different DNA constructs. We could show the specificity of our approach and quantify the strength of the protein-DNA interaction.

  12. A force-based, parallel assay for the quantification of protein-DNA interactions.

    Directory of Open Access Journals (Sweden)

    Katja Limmer

    Full Text Available Analysis of transcription factor binding to DNA sequences is of utmost importance to understand the intricate regulatory mechanisms that underlie gene expression. Several techniques exist that quantify DNA-protein affinity, but they are either very time-consuming or suffer from possible misinterpretation due to complicated algorithms or approximations like many high-throughput techniques. We present a more direct method to quantify DNA-protein interaction in a force-based assay. In contrast to single-molecule force spectroscopy, our technique, the Molecular Force Assay (MFA, parallelizes force measurements so that it can test one or multiple proteins against several DNA sequences in a single experiment. The interaction strength is quantified by comparison to the well-defined rupture stability of different DNA duplexes. As a proof-of-principle, we measured the interaction of the zinc finger construct Zif268/NRE against six different DNA constructs. We could show the specificity of our approach and quantify the strength of the protein-DNA interaction.

  13. Establishment of a novel two-probe real-time PCR for simultaneously quantification of hepatitis B virus DNA and distinguishing genotype B from non-B genotypes.

    Science.gov (United States)

    Wang, Wei; Liang, Hongpin; Zeng, Yongbin; Lin, Jinpiao; Liu, Can; Jiang, Ling; Yang, Bin; Ou, Qishui

    2014-11-01

    Establishment of a simple, rapid and economical method for quantification and genotyping of hepatitis B virus (HBV) is of great importance for clinical diagnosis and treatment of chronic hepatitis B patients. We hereby aim to develop a novel two-probe real-time PCR for simultaneous quantification of HBV viral concentration and distinguishing genotype B from non-B genotypes. Conserved primers and TaqMan probes for genotype B and non-B genotypes were designed. The linear range, detection sensitivity, specificity and repeatability of the method were assessed. 539 serum samples from HBV-infected patients were assayed, and the results were compared with commercial HBV quantification and HBV genotyping kits. The detection sensitivity of the two-probe real-time PCR was 500IU/ml; the linear range was 10(3)-10(9)IU/ml, and the intra-assay CVs and inter-assay CVs were between 0.84% and 2.80%. No cross-reaction was observed between genotypes B and non-B. Of the 539 detected samples, 509 samples were HBV DNA positive. The results showed that 54.0% (275/509) of the samples were genotype B, 39.5% (201/509) were genotype non-B and 6.5% (33/509) were mixed genotype. The coincidence rate between the method and a commercial HBV DNA genotyping kit was 95.9% (488/509, kappa=0.923, PDNA qPCR kit were achieved. A novel two-probe real-time PCR method for simultaneous quantification of HBV viral concentration and distinguishing genotype B from non-B genotypes was established. The assay was sensitive, specific and reproducible which can be applied to areas prevalent with HBV genotypes B and C, especially in China. Copyright © 2014 Elsevier B.V. All rights reserved.

  14. DNA adduct quantification in Eisenia fetida after subchronic exposures to creosote contaminated soils

    Energy Technology Data Exchange (ETDEWEB)

    Charrois, J.W.A.; McGill, W.B. [Alberta Univ., Dept. of Renewable Resources, Edmonton, AB (Canada)

    1999-07-01

    Within soil ecosystems contaminant toxicity can vary from acute and chronic, depending on the time of exposure. Due to the long times involved chronic toxicity is difficult to determine. DNA adducts fall into the category of biochemical markers that act as an early warning system in environmental monitoring. It has been proposed that they could be used as a sensitive method to determine environmental exposures to compounds such as polycyclic aromatic hydrocarbons (PAHs), which can occur, although not exclusively, in creosote. In this connection, Benzo[a]pyrene (BaP) is a PAH that can be transformed into an electrophilic metabolite, which ultimately results in DNA adduct formation. Use was made of a 32P postlabeling method to quantify the number of DNA adducts occurring in the earthworm Eisenia fetida after exposure to weathered creosote contaminated- and biotreated-soils with and without additions of extra BaP. DNA adducts can be measured in earthworms exposed to creosote contaminated- and biotreated-soils. E. fetida exposed to weathered creosote contaminated soils had significantly more DNA adducts than those exposed to a pristine control soil. Exposures to creosote contaminated soils with additional BaP (1000 mg/kg) or biotreatment did not yield statistically significant increases in DNA adducts compared to the pristine control. (Abstract only)

  15. Design and performance testing of a DNA extraction assay for sensitive and reliable quantification of acetic acid bacteria directly in red wine using real time PCR

    Directory of Open Access Journals (Sweden)

    Cédric eLONGIN

    2016-06-01

    Full Text Available Although strategies exist to prevent AAB contamination, the increased interest for wines with low sulfite addition leads to greater AAB spoilage. Hence there is a real need for a rapid, specific, sensitive and reliable method for detecting these spoilage bacteria. All these requirements are met by real time Polymerase Chain Reaction (or quantitative PCR; qPCR. Here, we compare existing methods of isolating DNA and their adaptation to a red wine matrix. Two different protocols for isolating DNA and three PCR mix compositions were tested to select the best method. The addition of insoluble polyvinylpolypyrrolidone (PVPP at 1% (v/v during DNA extraction using a protocol succeeded in eliminating PCR inhibitors from red wine. We developed a bacterial internal control which was efficient in avoiding false negative results due to decreases in the efficiency of DNA isolation and/or amplification. The specificity, linearity, repeatability and reproducibility of the method were evaluated. A standard curve was established for the enumeration of AAB inoculated into red wines. The limit of quantification in red wine was 3.7 log AAB/mL and about 2.8 log AAB/mL when the volume of the samples was increased from 1 mL to 10 mL. Thus the DNA extraction method developed in this paper allows sensitive and reliable AAB quantification without underestimation thanks to the presence of an internal control. Moreover, monitoring of both the AAB population and the amount of acetic acid in ethanol medium and red wine highlighted that a minimum about 6.0 log cells/mL of AAB is needed to significantly increase the production of acetic acid leading to spoilage.

  16. Mitochondrial DNA as a non-invasive biomarker: Accurate quantification using real time quantitative PCR without co-amplification of pseudogenes and dilution bias

    International Nuclear Information System (INIS)

    Malik, Afshan N.; Shahni, Rojeen; Rodriguez-de-Ledesma, Ana; Laftah, Abas; Cunningham, Phil

    2011-01-01

    Highlights: → Mitochondrial dysfunction is central to many diseases of oxidative stress. → 95% of the mitochondrial genome is duplicated in the nuclear genome. → Dilution of untreated genomic DNA leads to dilution bias. → Unique primers and template pretreatment are needed to accurately measure mitochondrial DNA content. -- Abstract: Circulating mitochondrial DNA (MtDNA) is a potential non-invasive biomarker of cellular mitochondrial dysfunction, the latter known to be central to a wide range of human diseases. Changes in MtDNA are usually determined by quantification of MtDNA relative to nuclear DNA (Mt/N) using real time quantitative PCR. We propose that the methodology for measuring Mt/N needs to be improved and we have identified that current methods have at least one of the following three problems: (1) As much of the mitochondrial genome is duplicated in the nuclear genome, many commonly used MtDNA primers co-amplify homologous pseudogenes found in the nuclear genome; (2) use of regions from genes such as β-actin and 18S rRNA which are repetitive and/or highly variable for qPCR of the nuclear genome leads to errors; and (3) the size difference of mitochondrial and nuclear genomes cause a 'dilution bias' when template DNA is diluted. We describe a PCR-based method using unique regions in the human mitochondrial genome not duplicated in the nuclear genome; unique single copy region in the nuclear genome and template treatment to remove dilution bias, to accurately quantify MtDNA from human samples.

  17. Quantification of meat proportions by measuring DNA contents in raw and boiled sausages using matrix-adapted calibrators and multiplex real-time PCR.

    Science.gov (United States)

    Köppel, René; Eugster, Albert; Ruf, Jürg; Rentsch, Jürg

    2012-01-01

    The quantification of meat proportions in raw and boiled sausage according to the recipe was evaluated using three different calibrators. To measure the DNA contents from beef, pork, sheep (mutton), and horse, a tetraplex real-time PCR method was applied. Nineteen laboratories analyzed four meat products each made of different proportions of beef, pork, sheep, and horse meat. Three kinds of calibrators were used: raw and boiled sausages of known proportions ranging from 1 to 55% of meat, and a dilution series of DNA from muscle tissue. In general, results generated using calibration sausages were more accurate than those resulting from the use of DNA from muscle tissue, and exhibited smaller measurement uncertainties. Although differences between uses of raw and boiled calibration sausages were small, the most precise and accurate results were obtained by calibration with fine-textured boiled reference sausages.

  18. High-throughput multiplex real-time PCR assay for the simultaneous quantification of DNA and RNA viruses infecting cassava plants.

    Science.gov (United States)

    Otti, G; Bouvaine, S; Kimata, B; Mkamillo, G; Kumar, P L; Tomlins, K; Maruthi, M N

    2016-05-01

    To develop a multiplex TaqMan-based real-time PCR assay (qPCR) for the simultaneous detection and quantification of both RNA and DNA viruses affecting cassava (Manihot esculenta) in eastern Africa. The diagnostic assay was developed for two RNA viruses; Cassava brown streak virus (CBSV) and Uganda cassava brown streak virus (UCBSV) and two predominant DNA viruses; African cassava mosaic virus (ACMV) and East African cassava mosaic virus (EACMV), which cause the economically important cassava brown streak disease (CBSD) and cassava mosaic disease (CMD) respectively. Our method, developed by analysing PCR products of viruses, was highly sensitive to detect target viruses from very low quantities of 4-10 femtograms. Multiplexing did not diminish sensitivity or accuracy compared to uniplex alternatives. The assay reliably detected and quantified four cassava viruses in field samples where CBSV and UCBSV synergy was observed in majority of mixed-infected varieties. We have developed a high-throughput qPCR diagnostic assay capable of specific and sensitive quantification of predominant DNA and RNA viruses of cassava in eastern Africa. The qPCR methods are a great improvement on the existing methods and can be used for monitoring virus spread as well as for accurate evaluation of the cassava varieties for virus resistance. © 2016 The Society for Applied Microbiology.

  19. Ultrasensitive Direct Quantification of Nucleobase Modifications in DNA by Surface-Enhanced Raman Scattering: The Case of Cytosine.

    Science.gov (United States)

    Morla-Folch, Judit; Xie, Hai-nan; Gisbert-Quilis, Patricia; Gómez-de Pedro, Sara; Pazos-Perez, Nicolas; Alvarez-Puebla, Ramon A; Guerrini, Luca

    2015-11-09

    Recognition of chemical modifications in canonical nucleobases of nucleic acids is of key importance since such modified variants act as different genetic encoders, introducing variability in the biological information contained in DNA. Herein, we demonstrate the feasibility of direct SERS in combination with chemometrics and microfluidics for the identification and relative quantification of 4 different cytosine modifications in both single- and double-stranded DNA. The minute amount of DNA required per measurement, in the sub-nanogram regime, removes the necessity of pre-amplification or enrichment steps (which are also potential sources of artificial DNA damages). These findings show great potentials for the development of fast, low-cost and high-throughput screening analytical devices capable of detecting known and unknown modifications in nucleic acids (DNA and RNA) opening new windows of activity in several fields such as biology, medicine and forensic sciences. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  20. Evaluating Digital PCR for the Quantification of Human Genomic DNA: Accessible Amplifiable Targets.

    Science.gov (United States)

    Kline, Margaret C; Romsos, Erica L; Duewer, David L

    2016-02-16

    Polymerase chain reaction (PCR) multiplexed assays perform best when the input quantity of template DNA is controlled to within about a factor of √2. To help ensure that PCR assays yield consistent results over time and place, results from methods used to determine DNA quantity need to be metrologically traceable to a common reference. Many DNA quantitation systems can be accurately calibrated with solutions of DNA in aqueous buffer. Since they do not require external calibration, end-point limiting dilution technologies, collectively termed "digital PCR (dPCR)", have been proposed as suitable for value assigning such DNA calibrants. The performance characteristics of several commercially available dPCR systems have recently been documented using plasmid, viral, or fragmented genomic DNA; dPCR performance with more complex materials, such as human genomic DNA, has been less studied. With the goal of providing a human genomic reference material traceably certified for mass concentration, we are investigating the measurement characteristics of several dPCR systems. We here report results of measurements from multiple PCR assays, on four human genomic DNAs treated with four endonuclease restriction enzymes using both chamber and droplet dPCR platforms. We conclude that dPCR does not estimate the absolute number of PCR targets in a given volume but rather the number of accessible and amplifiable targets. While enzymatic restriction of human genomic DNA increases accessibility for some assays, in well-optimized PCR assays it can reduce the number of amplifiable targets and increase assay variability relative to uncut sample.

  1. A simple method for normalization of DNA extraction to improve the quantitative detection of soil-borne plant pathogenic oomycetes by real-time PCR.

    Science.gov (United States)

    Li, M; Ishiguro, Y; Kageyama, K; Zhu, Z

    2015-08-01

    Most of the current research into the quantification of soil-borne pathogenic oomycetes lacks determination of DNA extraction efficiency, probably leading to an incorrect estimation of DNA quantity. In this study, we developed a convenient method by using a 100 bp artificially synthesized DNA sequence derived from the mitochondrion NADH dehydrogenase subunit 2 gene of Thunnus thynnus as a control to determine the DNA extraction efficiency. The control DNA was added to soils and then co-extracted along with soil genomic DNA. DNA extraction efficiency was determined by the control DNA. Two different DNA extraction methods were compared and evaluated using different types of soils, and the commercial kit was proved to give more consistent results. We used the control DNA combined with real-time PCR to quantify the oomycete DNAs from 12 naturally infested soils. Detectable target DNA concentrations were three to five times higher after normalization. Our tests also showed that the extraction efficiencies varied on a sample-to-sample basis and were simple and useful for the accurate quantification of soil-borne pathogenic oomycetes. Oomycetes include many important plant pathogens. Accurate quantification of these pathogens is essential in the management of diseases. This study reports an easy method utilizing an external DNA control for the normalization of DNA extraction by real-time PCR. By combining two different efficient soil DNA extraction methods, the developed quantification method dramatically improved the results. This study also proves that the developed normalization method is necessary and useful for the accurate quantification of soil-borne plant pathogenic oomycetes. © 2015 The Society for Applied Microbiology.

  2. Absolute quantification of DNA methylation using microfluidic chip-based digital PCR.

    Science.gov (United States)

    Wu, Zhenhua; Bai, Yanan; Cheng, Zule; Liu, Fangming; Wang, Ping; Yang, Dawei; Li, Gang; Jin, Qinghui; Mao, Hongju; Zhao, Jianlong

    2017-10-15

    Hypermethylation of CpG islands in the promoter region of many tumor suppressor genes downregulates their expression and in a result promotes tumorigenesis. Therefore, detection of DNA methylation status is a convenient diagnostic tool for cancer detection. Here, we reported a novel method for the integrative detection of methylation by the microfluidic chip-based digital PCR. This method relies on methylation-sensitive restriction enzyme HpaII, which cleaves the unmethylated DNA strands while keeping the methylated ones intact. After HpaII treatment, the DNA methylation level is determined quantitatively by the microfluidic chip-based digital PCR with the lower limit of detection equal to 0.52%. To validate the applicability of this method, promoter methylation of two tumor suppressor genes (PCDHGB6 and HOXA9) was tested in 10 samples of early stage lung adenocarcinoma and their adjacent non-tumorous tissues. The consistency was observed in the analysis of these samples using our method and a conventional bisulfite pyrosequencing. Combining high sensitivity and low cost, the microfluidic chip-based digital PCR method might provide a promising alternative for the detection of DNA methylation and early diagnosis of epigenetics-related diseases. Copyright © 2017 Elsevier B.V. All rights reserved.

  3. A SIMPLE METHOD FOR THE EXTRACTION AND QUANTIFICATION OF PHOTOPIGMENTS FROM SYMBIODINIUM SPP.

    Science.gov (United States)

    John E. Rogers and Dragoslav Marcovich. Submitted. Simple Method for the Extraction and Quantification of Photopigments from Symbiodinium spp.. Limnol. Oceanogr. Methods. 19 p. (ERL,GB 1192). We have developed a simple, mild extraction procedure using methanol which, when...

  4. An overview of quantification methods in energy-dispersive X-ray ...

    Indian Academy of Sciences (India)

    methods for thin samples, samples with intermediate thickness and thick ... algorithms and quantification methods based on scattered primary radiation. ... technique for in situ characterization of materials such as contaminated soil, archaeo-.

  5. Evaluation of two autoinducer-2 quantification methods for application in marine environments

    KAUST Repository

    Wang, Tian-Nyu; Kaksonen, Anna H.; Hong, Pei-Ying

    2018-01-01

    This study evaluated two methods, namely high performance liquid chromatography with fluorescence detection (HPLC-FLD) and Vibrio harveyi BB170 bioassay, for autoinducer-2 (AI-2) quantification in marine samples. Using both methods, the study also

  6. Droplet digital PCR for detection and quantification of circulating tumor DNA in plasma of head and neck cancer patients.

    Science.gov (United States)

    van Ginkel, Joost H; Huibers, Manon M H; van Es, Robert J J; de Bree, Remco; Willems, Stefan M

    2017-06-19

    During posttreatment surveillance of head and neck cancer patients, imaging is insufficiently accurate for the early detection of relapsing disease. Free circulating tumor DNA (ctDNA) may serve as a novel biomarker for monitoring tumor burden during posttreatment surveillance of these patients. In this exploratory study, we investigated whether low level ctDNA in plasma of head and neck cancer patients can be detected using Droplet Digital PCR (ddPCR). TP53 mutations were determined in surgically resected primary tumor samples from six patients with high stage (II-IV), moderate to poorly differentiated head and neck squamous cell carcinoma (HNSCC). Subsequently, mutation specific ddPCR assays were designed. Pretreatment plasma samples from these patients were examined on the presence of ctDNA by ddPCR using the mutation-specific assays. The ddPCR results were evaluated alongside clinicopathological data. In all cases, plasma samples were found positive for targeted TP53 mutations in varying degrees (absolute quantification of 2.2-422 mutational copies/ml plasma). Mutations were detected in wild-type TP53 background templates of 7667-156,667 copies/ml plasma, yielding fractional abundances of down to 0.01%. Our results show that detection of tumor specific TP53 mutations in low level ctDNA from HNSCC patients using ddPCR is technically feasible and provide ground for future research on ctDNA quantification for the use of diagnostic biomarkers in the posttreatment surveillance of HNSCC patients.

  7. A highly sensitive method for quantification of iohexol

    DEFF Research Database (Denmark)

    Schulz, A.; Boeringer, F.; Swifka, J.

    2014-01-01

    -chromatography-electrospray-massspectrometry (LC-ESI-MS) approach using the multiple reaction monitoring mode for iohexol quantification. In order to test whether a significantly decreased amount of iohexol is sufficient for reliable quantification, a LC-ESI-MS approach was assessed. We analyzed the kinetics of iohexol in rats after application...... of different amounts of iohexol (15 mg to 150 1.tg per rat). Blood sampling was conducted at four time points, at 15, 30, 60, and 90 min, after iohexol injection. The analyte (iohexol) and the internal standard (iotha(amic acid) were separated from serum proteins using a centrifugal filtration device...... with a cut-off of 3 kDa. The chromatographic separation was achieved on an analytical Zorbax SB C18 column. The detection and quantification were performed on a high capacity trap mass spectrometer using positive ion ESI in the multiple reaction monitoring (MRM) mode. Furthermore, using real-time polymerase...

  8. Simultaneous DNA-RNA Extraction from Coastal Sediments and Quantification of 16S rRNA Genes and Transcripts by Real-time PCR.

    Science.gov (United States)

    Tatti, Enrico; McKew, Boyd A; Whitby, Corrine; Smith, Cindy J

    2016-06-11

    Real Time Polymerase Chain Reaction also known as quantitative PCR (q-PCR) is a widely used tool in microbial ecology to quantify gene abundances of taxonomic and functional groups in environmental samples. Used in combination with a reverse transcriptase reaction (RT-q-PCR), it can also be employed to quantify gene transcripts. q-PCR makes use of highly sensitive fluorescent detection chemistries that allow quantification of PCR amplicons during the exponential phase of the reaction. Therefore, the biases associated with 'end-point' PCR detected in the plateau phase of the PCR reaction are avoided. A protocol to quantify bacterial 16S rRNA genes and transcripts from coastal sediments via real-time PCR is provided. First, a method for the co-extraction of DNA and RNA from coastal sediments, including the additional steps required for the preparation of DNA-free RNA, is outlined. Second, a step-by-step guide for the quantification of 16S rRNA genes and transcripts from the extracted nucleic acids via q-PCR and RT-q-PCR is outlined. This includes details for the construction of DNA and RNA standard curves. Key considerations for the use of RT-q-PCR assays in microbial ecology are included.

  9. Use of spontaneously mutated human DNA as competitive internal standard for nucleic acid quantification by reverse transcription-polymerase chain reaction (RT-PCR)

    International Nuclear Information System (INIS)

    Rudnicka, L.; Diaz, A.; Varga, J.; Jimenez, S.A.; Christiano, A.; Uitto, J.

    1995-01-01

    Quantification of gene expression is of increasing interest in many medical sciences. Methods based on reverse transcription-polymerase chain reactions (RT-PCRs) are timesaving and require only very small amounts of RNA. A limiting factor, however, is the significant fluctuation in the efficacy of reverse transcription as well in the polymerase chain reactions. Various external and internal standards have been suggested for correcting these fluctuations. We describe a novel way of creating an internal standard for assessing the expression of type VII collagen in human cells. The total RNA of a patient with hereditary 'epidermilysis bulosa dystrophica' associated with a homozygous T to A point mutation in type VII collagen gene was reverse transcribed and a 382bp fragment of type VII collagen cDNA containing the mutation was amplified. The mutated cDNA, unlike normal type VII collagen cDNA could be cleaved by 'Ear I' endonuclease into 244bp and 138bp fragments. Semiquantitative PCR was performed with the mutated cDNA as internal standard and the studied cDNA sample in the same tube in the presence of α 32 P-labelled dCTP. The reaction was followed by 'Ear I' digestion, electrophoresis on a polyacrylamide gel and exposure to a X-ray film. In conclusion, we describe a timesaving method for creating internal standards for semiquantitative RT-PCR. (author). 12 refs, 3 figs

  10. Methods for external event screening quantification: Risk Methods Integration and Evaluation Program (RMIEP) methods development

    International Nuclear Information System (INIS)

    Ravindra, M.K.; Banon, H.

    1992-07-01

    In this report, the scoping quantification procedures for external events in probabilistic risk assessments of nuclear power plants are described. External event analysis in a PRA has three important goals; (1) the analysis should be complete in that all events are considered; (2) by following some selected screening criteria, the more significant events are identified for detailed analysis; (3) the selected events are analyzed in depth by taking into account the unique features of the events: hazard, fragility of structures and equipment, external-event initiated accident sequences, etc. Based on the above goals, external event analysis may be considered as a three-stage process: Stage I: Identification and Initial Screening of External Events; Stage II: Bounding Analysis; Stage III: Detailed Risk Analysis. In the present report, first, a review of published PRAs is given to focus on the significance and treatment of external events in full-scope PRAs. Except for seismic, flooding, fire, and extreme wind events, the contributions of other external events to plant risk have been found to be negligible. Second, scoping methods for external events not covered in detail in the NRC's PRA Procedures Guide are provided. For this purpose, bounding analyses for transportation accidents, extreme winds and tornadoes, aircraft impacts, turbine missiles, and chemical release are described

  11. Quantification and genome-wide mapping of DNA double-strand breaks.

    Science.gov (United States)

    Grégoire, Marie-Chantal; Massonneau, Julien; Leduc, Frédéric; Arguin, Mélina; Brazeau, Marc-André; Boissonneault, Guylain

    2016-12-01

    DNA double-strand breaks (DSBs) represent a major threat to the genetic integrity of the cell. Knowing both their genome-wide distribution and number is important for a better assessment of genotoxicity at a molecular level. Available methods may have underestimated the extent of DSBs as they are based on markers specific to those undergoing active repair or may not be adapted for the large diversity of naturally occurring DNA ends. We have established conditions for an efficient first step of DNA nick and gap repair (NGR) allowing specific determination of DSBs by end labeling with terminal transferase. We used DNA extracted from HeLa cells harboring an I-SceI cassette to induce a targeted nick or DSB and demonstrated by immunocapture of 3'-OH that a prior step of NGR allows specific determination of loci-specific or genome wide DSBs. This method can be applied to the global determination of DSBs using radioactive end labeling and can find several applications aimed at understanding the distribution and kinetics of DSBs formation and repair. Copyright © 2016 Elsevier B.V. All rights reserved.

  12. Label-free DNA quantification via a 'pipette, aggregate and blot' (PAB) approach with magnetic silica particles on filter paper.

    Science.gov (United States)

    Li, Jingyi; Liu, Qian; Alsamarri, Hussein; Lounsbury, Jenny A; Haversitick, Doris M; Landers, James P

    2013-03-07

    Reliable measurement of DNA concentration is essential for a broad range of applications in biology and molecular biology, and for many of these, quantifying the nucleic acid content is inextricably linked to obtaining optimal results. In its most simplistic form, quantitative analysis of nucleic acids can be accomplished by UV-Vis absorbance and, in more sophisticated format, by fluorimetry. A recently reported new concept, the 'pinwheel assay', involves a label-free approach for quantifying DNA through aggregation of paramagnetic beads in a rotating magnetic field. Here, we describe a simplified version of that assay adapted for execution using only a pipet and filter paper. The 'pipette, aggregate, and blot' (PAB) approach allows DNA to induce bead aggregation in a pipette tip through exposure to a magnetic field, followed by dispensing (blotting) onto filter paper. The filter paper immortalises the extent of aggregation, and digital images of the immortalized bead conformation, acquired with either a document scanner or a cell phone camera, allows for DNA quantification using a noncomplex algorithm. Human genomic DNA samples extracted from blood are quantified with the PAB approach and the results utilized to define the volume of sample used in a PCR reaction that is sensitive to input mass of template DNA. Integrating the PAB assay with paper-based DNA extraction and detection modalities has the potential to yield 'DNA quant-on-paper' devices that may be useful for point-of-care testing.

  13. Effect of DNA extraction and sample preservation method on rumen bacterial population.

    Science.gov (United States)

    Fliegerova, Katerina; Tapio, Ilma; Bonin, Aurelie; Mrazek, Jakub; Callegari, Maria Luisa; Bani, Paolo; Bayat, Alireza; Vilkki, Johanna; Kopečný, Jan; Shingfield, Kevin J; Boyer, Frederic; Coissac, Eric; Taberlet, Pierre; Wallace, R John

    2014-10-01

    The comparison of the bacterial profile of intracellular (iDNA) and extracellular DNA (eDNA) isolated from cow rumen content stored under different conditions was conducted. The influence of rumen fluid treatment (cheesecloth squeezed, centrifuged, filtered), storage temperature (RT, -80 °C) and cryoprotectants (PBS-glycerol, ethanol) on quality and quantity parameters of extracted DNA was evaluated by bacterial DGGE analysis, real-time PCR quantification and metabarcoding approach using high-throughput sequencing. Samples clustered according to the type of extracted DNA due to considerable differences between iDNA and eDNA bacterial profiles, while storage temperature and cryoprotectants additives had little effect on sample clustering. The numbers of Firmicutes and Bacteroidetes were lower (P rumen fluid subjected to the eDNA isolation procedure considerably changed the ratio of molecular operational taxonomic units (MOTUs) of Bacteroidetes and Firmicutes. Intracellular DNA extraction using bead-beating method from cheesecloth sieved rumen content mixed with PBS-glycerol and stored at -80 °C was found as the optimal method to study ruminal bacterial profile. Copyright © 2013 Elsevier Ltd. All rights reserved.

  14. Quantification of Cell-Free HER-2 DNA in Plasma from Breast Cancer Patients

    DEFF Research Database (Denmark)

    Sørensen, Patricia Diana; Andersen, Rikke Fredslund; Pallisgaard, Niels

    2015-01-01

    The purpose of this study was to quantify the free-circulating plasma HER-2 DNA (cfHER-2 DNA) and to assess the ability of analysis to discriminate between patients with primary breast cancer and healthy controls in order to detect metastatic recurrence in comparison with serum HER-2 protein...... and also HER-2 gene amplification. The study population consisted of 100 patients with primary breast cancer and 50 healthy female donors. An additional 22 patients with metastases were subsequently included. cfHER-2 DNA was quantified with a quantitative PCR method together with a reference gene. RESULTS......: Using a cut-off of 2.5 for the ratio of the cfHER-2 DNA/reference gene, three (of 15) tissue HER-2-positive patients had a ratio >2.5 prior to the detection of metastatic disease. In the post-metastatic/pre-chemotherapy setting, 11 (of 23) tissue HER-2-positive patients with metastases had a ratio >2...

  15. Fluorescent Quantification of DNA Based on Core-Shell Fe3O4@SiO2@Au Nanocomposites and Multiplex Ligation-Dependent Probe Amplification.

    Science.gov (United States)

    Fan, Jing; Yang, Haowen; Liu, Ming; Wu, Dan; Jiang, Hongrong; Zeng, Xin; Elingarami, Sauli; Ll, Zhiyang; Li, Song; Liu, Hongna; He, Nongyue

    2015-02-01

    In this research, a novel method for relative fluorescent quantification of DNA based on Fe3O4@SiO2@Au gold-coated magnetic nanocomposites (GMNPs) and multiplex ligation- dependent probe amplification (MLPA) has been developed. With the help of self-assembly, seed-mediated growth and chemical reduction method, core-shell Fe3O4@SiO2@Au GMNPs were synthesized. Through modified streptavidin on the GMNPs surface, we obtained a bead chip which can capture the biotinylated probes. Then we designed MLPA probes which were tagged with biotin or Cy3 and target DNA on the basis of human APP gene sequence. The products from the thermostable DNA ligase induced ligation reactions and PCR amplifications were incubated with SA-GMNPs. After washing, magnetic separation, spotting, the fluorescent scanning results showed our method can be used for the relative quantitative analysis of the target DNA in the concentration range of 03004~0.5 µM.

  16. Methods for microbial DNA extraction from soil for PCR amplification

    Directory of Open Access Journals (Sweden)

    Yeates C

    1998-01-01

    Full Text Available Amplification of DNA from soil is often inhibited by co-purified contaminants. A rapid, inexpensive, large-scale DNA extraction method involving minimal purification has been developed that is applicable to various soil types (1. DNA is also suitable for PCR amplification using various DNA targets. DNA was extracted from 100g of soil using direct lysis with glass beads and SDS followed by potassium acetate precipitation, polyethylene glycol precipitation, phenol extraction and isopropanol precipitation. This method was compared to other DNA extraction methods with regard to DNA purity and size.

  17. Method for indirect quantification of CH4 production via H2O production using hydrogenotrophic methanogens

    Directory of Open Access Journals (Sweden)

    Ruth-Sophie eTaubner

    2016-04-01

    Full Text Available ydrogenotrophic methanogens are an intriguing group of microorganisms from the domain Archaea. They exhibit extraordinary ecological, biochemical, physiological characteristics colorbox{yellow}{and have a huge biotechnological potential}. Yet, the only possibility to assess the methane (CH$_4$ production potential of hydrogenotrophic methanogens is to apply gas chromatographic quantification of CH$_4$.In order to be able to effectively screen pure cultures of hydrogenotrophic methanogens regarding their CH$_4$ production potential we developed a novel method for indirect quantification of colorbox{yellow}{the} volumetric CH$_4$ production rate by measuring colorbox{yellow}{the} volumetric water production rate. This colorbox{yellow}{ } method was established in serum bottles for cultivation of methanogens in closed batch cultivation mode. Water production was colorbox{yellow}{estimated} by determining the difference in mass increase in an isobaric setting.This novel CH$_4$ quantification method is an accurate and precise analytical technique, colorbox{yellow}{which can be used} to rapidly screen pure cultures of methanogens regarding colorbox{yellow}{their} volumetric CH$_{4}$ evolution rate. colorbox{yellow}{It} is a cost effective alternative colorbox{yellow}{determining} CH$_4$ production of methanogens over CH$_4$ quantification by using gas chromatography, especially if colorbox{yellow}{ } applied as a high throughput quantification method. colorbox{yellow}{Eventually, the} method can be universally applied for quantification of CH$_4$ production from psychrophilic, thermophilic and hyperthermophilic hydrogenotrophic methanogens.

  18. Traditional Mold Analysis Compared to a DNA-based Method of Mold Analysis with Applications in Asthmatics' Homes

    Science.gov (United States)

    Traditional environmental mold analysis is based-on microscopic observations and counting of mold structures collected from the air on a sticky surface or culturing of molds on growth media for identification and quantification. A DNA-based method of mold analysis called mol...

  19. Nanobiosensor for Detection and Quantification of DNA Sequences in Degraded Mixed Meats

    Directory of Open Access Journals (Sweden)

    M. E. Ali

    2011-01-01

    Full Text Available A novel class of nanobiosensor was developed by integrating a 27-nucleotide AluI fragment of swine cytochrome b (cytb gene to a 3-nm diameter citrate-tannate coated gold nanoparticle (GNP. The biosensor detected 0.5% and 1% pork in raw and 2.5-h autoclaved pork-beef binary admixtures in a single step without any separation or washing. The hybridization kinetics of the hybrid sensor was studied with synthetic and AluI digested real pork targets from moderate to extreme target concentrations and a sigmoidal relationship was found. Using the kinetic curve, a convenient method for quantifying and counting target DNA copy number was developed. The accuracy of the method was over 90% and 80% for raw and autoclaved pork-beef binary admixtures in the range of 5–100% pork adulteration. The biosensor probe identified a target DNA sequence that was several-folds shorter than a typical PCR-template. This offered the detection and quantitation of potential targets in highly processed or degraded samples where PCR amplification was not possible due to template crisis. The assay was a viable alternative approach of qPCR for detecting, quantifying and counting copy number of shorter size DNA sequences to address a wide ranging biological problem in food industry, diagnostic laboratories and forensic medicine.

  20. Multiplex quantification of four DNA targets in one reaction with Bio-Rad droplet digital PCR system for GMO detection

    Science.gov (United States)

    Dobnik, David; Štebih, Dejan; Blejec, Andrej; Morisset, Dany; Žel, Jana

    2016-10-01

    The advantages of the digital PCR technology are already well documented until now. One way to achieve better cost efficiency of the technique is to use it in a multiplexing strategy. Droplet digital PCR platforms, which include two fluorescence filters, support at least duplex reactions and with some developments and optimization higher multiplexing is possible. The present study not only shows a development of multiplex assays in droplet digital PCR, but also presents a first thorough evaluation of several parameters in such multiplex digital PCR. Two 4-plex assays were developed for quantification of 8 different DNA targets (7 genetically modified maize events and maize endogene). Per assay, two of the targets were labelled with one fluorophore and two with another. As current analysis software does not support analysis of more than duplex, a new R- and Shiny-based web application analysis tool (http://bit.ly/ddPCRmulti) was developed that automates the analysis of 4-plex results. In conclusion, the two developed multiplex assays are suitable for quantification of GMO maize events and the same approach can be used in any other field with a need for accurate and reliable quantification of multiple DNA targets.

  1. Prospective comparison of liver stiffness measurements between two point wave elastography methods: Virtual ouch quantification and elastography point quantification

    Energy Technology Data Exchange (ETDEWEB)

    Yoo, Hyun Suk; Lee, Jeong Min; Yoon, Jeong Hee; Lee, Dong Ho; Chang, Won; Han, Joon Koo [Seoul National University Hospital, Seoul (Korea, Republic of)

    2016-09-15

    To prospectively compare technical success rate and reliable measurements of virtual touch quantification (VTQ) elastography and elastography point quantification (ElastPQ), and to correlate liver stiffness (LS) measurements obtained by the two elastography techniques. Our study included 85 patients, 80 of whom were previously diagnosed with chronic liver disease. The technical success rate and reliable measurements of the two kinds of point shear wave elastography (pSWE) techniques were compared by χ{sup 2} analysis. LS values measured using the two techniques were compared and correlated via Wilcoxon signed-rank test, Spearman correlation coefficient, and 95% Bland-Altman limit of agreement. The intraobserver reproducibility of ElastPQ was determined by 95% Bland-Altman limit of agreement and intraclass correlation coefficient (ICC). The two pSWE techniques showed similar technical success rate (98.8% for VTQ vs. 95.3% for ElastPQ, p = 0.823) and reliable LS measurements (95.3% for VTQ vs. 90.6% for ElastPQ, p = 0.509). The mean LS measurements obtained by VTQ (1.71 ± 0.47 m/s) and ElastPQ (1.66 ± 0.41 m/s) were not significantly different (p = 0.209). The LS measurements obtained by the two techniques showed strong correlation (r = 0.820); in addition, the 95% limit of agreement of the two methods was 27.5% of the mean. Finally, the ICC of repeat ElastPQ measurements was 0.991. Virtual touch quantification and ElastPQ showed similar technical success rate and reliable measurements, with strongly correlated LS measurements. However, the two methods are not interchangeable due to the large limit of agreement.

  2. Quantification of miRNAs by a simple and specific qPCR method

    DEFF Research Database (Denmark)

    Cirera Salicio, Susanna; Busk, Peter K.

    2014-01-01

    MicroRNAs (miRNAs) are powerful regulators of gene expression at posttranscriptional level and play important roles in many biological processes and in disease. The rapid pace of the emerging field of miRNAs has opened new avenues for development of techniques to quantitatively determine mi...... in miRNA quantification. Furthermore, the method is easy to perform with common laboratory reagents, which allows miRNA quantification at low cost....

  3. Comparison of Quantifiler(®) Trio and InnoQuant™ human DNA quantification kits for detection of DNA degradation in developed and aged fingerprints.

    Science.gov (United States)

    Goecker, Zachary C; Swiontek, Stephen E; Lakhtakia, Akhlesh; Roy, Reena

    2016-06-01

    The development techniques employed to visualize fingerprints collected from crime scenes as well as post-development ageing may result in the degradation of the DNA present in low quantities in such evidence samples. Amplification of the DNA samples with short tandem repeat (STR) amplification kits may result in partial DNA profiles. A comparative study of two commercially available quantification kits, Quantifiler(®) Trio and InnoQuant™, was performed on latent fingerprint samples that were either (i) developed using one of three different techniques and then aged in ambient conditions or (ii) undeveloped and then aged in ambient conditions. The three fingerprint development techniques used were: cyanoacrylate fuming, dusting with black powder, and the columnar-thin-film (CTF) technique. In order to determine the differences between the expected quantities and actual quantities of DNA, manually degraded samples generated by controlled exposure of DNA standards to ultraviolet radiation were also analyzed. A total of 144 fingerprint and 42 manually degraded DNA samples were processed in this study. The results indicate that the InnoQuant™ kit is capable of producing higher degradation ratios compared to the Quantifiler(®) Trio kit. This was an expected result since the degradation ratio is a relative value specific for a kit based on the length and extent of amplification of the two amplicons that vary from one kit to the other. Additionally, samples with lower concentrations of DNA yielded non-linear relationships of degradation ratio with the duration of aging, whereas samples with higher concentrations of DNA yielded quasi-linear relationships. None of the three development techniques produced a noticeably different degradation pattern when compared to undeveloped fingerprints, and therefore do not impede downstream DNA analysis. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  4. Sample preparation methods for quantitative detection of DNA by molecular assays and marine biosensors.

    Science.gov (United States)

    Cox, Annie M; Goodwin, Kelly D

    2013-08-15

    The need for quantitative molecular methods is growing in environmental, food, and medical fields but is hindered by low and variable DNA extraction and by co-extraction of PCR inhibitors. DNA extracts from Enterococcus faecium, seawater, and seawater spiked with E. faecium and Vibrio parahaemolyticus were tested by qPCR for target recovery and inhibition. Conventional and novel methods were tested, including Synchronous Coefficient of Drag Alteration (SCODA) and lysis and purification systems used on an automated genetic sensor (the Environmental Sample Processor, ESP). Variable qPCR target recovery and inhibition were measured, significantly affecting target quantification. An aggressive lysis method that utilized chemical, enzymatic, and mechanical disruption enhanced target recovery compared to commercial kit protocols. SCODA purification did not show marked improvement over commercial spin columns. Overall, data suggested a general need to improve sample preparation and to accurately assess and account for DNA recovery and inhibition in qPCR applications. Published by Elsevier Ltd.

  5. Chemically modified DNA : methods and applications

    NARCIS (Netherlands)

    Gjonaj, Lorina

    2013-01-01

    Over het toepassen van DNA in de chemie DNA is een fascinerend biopolymeer dat gemakkelijk gemanipuleerd kan worden. Doel van het onderzoek van Lorina Gjonaj was het ontwikkelen van nieuwe methodologie om DNA chemisch te modificeren. Verschillende functionaliteiten zijn aan DNA gekoppeld. Deze

  6. Quantification of Leishmania infantum DNA in females, eggs and larvae of Rhipicephalus sanguineus

    Directory of Open Access Journals (Sweden)

    Otranto Domenico

    2011-04-01

    Full Text Available Background Leishmania infantum is a widespread parasite that affects dogs and humans worldwide. It is transmitted primarily by phlebotomine sand flies, but recently there has been much discussion on the role of the brown dog tick, Rhipicephalus sanguineus, as a potential vector for this protozoan. Recent laboratory and field investigations have contributed to this hypothesis, but a proof of the vector capacity of R. sanguineus has yet to be provided. Following a recent study suggesting that L. infantum passes transovarially from the female tick to her progeny the current study provides new evidence of the transovarial transmission of L. infantum in R. sanguineus. Methods Engorged females of R. sanguineus were collected from the environment in a dog shelter of southern Italy, where canine leishmaniosis is endemic. In the laboratory, 97 females that successfully laid eggs, their eggs and the originated larvae were subjected to DNA extraction and then tested by a TaqMan-based real time PCR targeting a fragment of the kinetoplast DNA (kDNA of L. infantum. Results and conclusions L. infantum kDNA was detected in engorged females, their eggs and originating larvae, with a parasite load ranging from 1.8 × 10-4 to 10.0 × 100. Certainly, the current study provides further evidence on the passage of L. infantum from R. sanguineus females to their offspring. The observation of promastigote forms in larvae is necessary to definitively confirm this hypothesis, which would raise interesting questions about the possible role of ticks in the maintenance of L. infantum infection among dogs in certain areas.

  7. Method for priming and DNA sequencing

    Energy Technology Data Exchange (ETDEWEB)

    Mugasimangalam, R.C.; Ulanovsky, L.E.

    1997-12-01

    A method is presented for improving the priming specificity of an oligonucleotide primer that is non-unique in a nucleic acid template which includes selecting a continuous stretch of several nucleotides in the template DNA where one of the four bases does not occur in the stretch. This also includes bringing the template DNA in contract with a non-unique primer partially or fully complimentary to the sequence immediately upstream of the selected sequence stretch. This results in polymerase-mediated differential extension of the primer in the presence of a subset of deoxyribonucleotide triphosphates that does not contain the base complementary to the base absent in the selected sequence stretch. These reactions occur at a temperature sufficiently low for allowing the extension of the non-unique primer. The method causes polymerase-mediated extension reactions in the presence of all four natural deoxyribonucleotide triphosphates or modifications. At this high temperature discrimination occurs against priming sites of the non-unique primer where the differential extension has not made the primer sufficiently stable to prime. However, the primer extended at the selected stretch is sufficiently stable to prime.

  8. RNAontheBENCH: computational and empirical resources for benchmarking RNAseq quantification and differential expression methods

    KAUST Repository

    Germain, Pierre-Luc

    2016-06-20

    RNA sequencing (RNAseq) has become the method of choice for transcriptome analysis, yet no consensus exists as to the most appropriate pipeline for its analysis, with current benchmarks suffering important limitations. Here, we address these challenges through a rich benchmarking resource harnessing (i) two RNAseq datasets including ERCC ExFold spike-ins; (ii) Nanostring measurements of a panel of 150 genes on the same samples; (iii) a set of internal, genetically-determined controls; (iv) a reanalysis of the SEQC dataset; and (v) a focus on relative quantification (i.e. across-samples). We use this resource to compare different approaches to each step of RNAseq analysis, from alignment to differential expression testing. We show that methods providing the best absolute quantification do not necessarily provide good relative quantification across samples, that count-based methods are superior for gene-level relative quantification, and that the new generation of pseudo-alignment-based software performs as well as established methods, at a fraction of the computing time. We also assess the impact of library type and size on quantification and differential expression analysis. Finally, we have created a R package and a web platform to enable the simple and streamlined application of this resource to the benchmarking of future methods.

  9. RNAontheBENCH: computational and empirical resources for benchmarking RNAseq quantification and differential expression methods

    KAUST Repository

    Germain, Pierre-Luc; Vitriolo, Alessandro; Adamo, Antonio; Laise, Pasquale; Das, Vivek; Testa, Giuseppe

    2016-01-01

    RNA sequencing (RNAseq) has become the method of choice for transcriptome analysis, yet no consensus exists as to the most appropriate pipeline for its analysis, with current benchmarks suffering important limitations. Here, we address these challenges through a rich benchmarking resource harnessing (i) two RNAseq datasets including ERCC ExFold spike-ins; (ii) Nanostring measurements of a panel of 150 genes on the same samples; (iii) a set of internal, genetically-determined controls; (iv) a reanalysis of the SEQC dataset; and (v) a focus on relative quantification (i.e. across-samples). We use this resource to compare different approaches to each step of RNAseq analysis, from alignment to differential expression testing. We show that methods providing the best absolute quantification do not necessarily provide good relative quantification across samples, that count-based methods are superior for gene-level relative quantification, and that the new generation of pseudo-alignment-based software performs as well as established methods, at a fraction of the computing time. We also assess the impact of library type and size on quantification and differential expression analysis. Finally, we have created a R package and a web platform to enable the simple and streamlined application of this resource to the benchmarking of future methods.

  10. Impact of temperature and time storage on the microbial detection of oral samples by Checkerboard DNA-DNA hybridization method.

    Science.gov (United States)

    do Nascimento, Cássio; dos Santos, Janine Navarro; Pedrazzi, Vinícius; Pita, Murillo Sucena; Monesi, Nadia; Ribeiro, Ricardo Faria; de Albuquerque, Rubens Ferreira

    2014-01-01

    Molecular diagnosis methods have been largely used in epidemiological or clinical studies to detect and quantify microbial species that may colonize the oral cavity in healthy or disease. The preservation of genetic material from samples remains the major challenge to ensure the feasibility of these methodologies. Long-term storage may compromise the final result. The aim of this study was to evaluate the effect of temperature and time storage on the microbial detection of oral samples by Checkerboard DNA-DNA hybridization. Saliva and supragingival biofilm were taken from 10 healthy subjects, aliquoted (n=364) and processed according to proposed protocols: immediate processing and processed after 2 or 4 weeks, and 6 or 12 months of storage at 4°C, -20°C and -80°C. Either total or individual microbial counts were recorded in lower values for samples processed after 12 months of storage, irrespective of temperatures tested. Samples stored up to 6 months at cold temperatures showed similar counts to those immediately processed. The microbial incidence was also significantly reduced in samples stored during 12 months in all temperatures. Temperature and time of oral samples storage have relevant impact in the detection and quantification of bacterial and fungal species by Checkerboard DNA-DNA hybridization method. Samples should be processed immediately after collection or up to 6 months if conserved at cold temperatures to avoid false-negative results. Copyright © 2013 Elsevier Ltd. All rights reserved.

  11. Electrochemical Branched-DNA Assay for Polymerase Chain Reaction-Free Detection and Quantification of Oncogenes in Messenger RNA

    Energy Technology Data Exchange (ETDEWEB)

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

    2008-12-01

    We describe a novel electrochemical branched-DNA (bDNA) assay for polymerase chain reaction (PCR)-free detection and quantification of p185 BCR-ABL leukemia fusion transcript in the population of messenger RNA (mRNA) extracted from cell lines. The bDNA amplifier carrying high loading of alkaline phosphatase (ALP) tracers was used to amplify targets 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-napthyl-phosphate. The specificity and sensitivity of assay enabled direct detection of target transcript in as little as 4.6 ng mRNA 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 transcript in total mRNA population. The approach thus provides a simple, sensitive, accurate and quantitative tool alternate to the RQ-PCR for early disease diagnosis.

  12. Multiplex quantification of 16S rDNA of predominant bacteria group within human fecal samples by polymerase chain reaction--ligase detection reaction (PCR-LDR).

    Science.gov (United States)

    Li, Kai; Chen, Bei; Zhou, Yuxun; Huang, Rui; Liang, Yinming; Wang, Qinxi; Xiao, Zhenxian; Xiao, Junhua

    2009-03-01

    A new method, based on ligase detection reaction (LDR), was developed for quantitative detection of multiplex PCR amplicons of 16S rRNA genes present in complex mixtures (specifically feces). LDR has been widely used in single nucleotide polymorphism (SNP) assay but never applied for quantification of multiplex PCR products. This method employs one pair of DNA probes, one of which is labeled with fluorescence for signal capture, complementary to the target sequence. For multiple target sequence analysis, probes were modified with different lengths of polyT at the 5' end and 3' end. Using a DNA sequencer, these ligated probes were separated and identified by size and dye color. Then, relative abundance of target DNA were normalized and quantified based on the fluorescence intensities and exterior size standards. 16S rRNA gene of three preponderant bacteria groups in human feces: Clostridium coccoides, Bacteroides and related genera, and Clostridium leptum group, were amplified and cloned into plasmid DNA so as to make standard curves. After PCR-LDR analysis, a strong linear relationship was found between the florescence intensity and the diluted plasmid DNA concentrations. Furthermore, based on this method, 100 human fecal samples were quantified for the relative abundance of the three bacterial groups. Relative abundance of C. coccoides was significantly higher in elderly people in comparison with young adults, without gender differences. Relative abundance of Bacteroides and related genera and C. leptum group were significantly higher in young and middle aged than in the elderly. Regarding the whole set of sample, C. coccoides showed the highest relative abundance, followed by decreasing groups Bacteroides and related genera, and C. leptum. These results imply that PCR-LDR can be feasible and flexible applied to large scale epidemiological studies.

  13. DNA microsatellite region for a reliable quantification of soft wheat adulteration in durum wheat-based foodstuffs by real-time PCR.

    Science.gov (United States)

    Sonnante, Gabriella; Montemurro, Cinzia; Morgese, Anita; Sabetta, Wilma; Blanco, Antonio; Pasqualone, Antonella

    2009-11-11

    Italian industrial pasta and durum wheat typical breads must be prepared using exclusively durum wheat semolina. Previously, a microsatellite sequence specific of the wheat D-genome had been chosen for traceability of soft wheat in semolina and bread samples, using qualitative and quantitative Sybr green-based real-time experiments. In this work, we describe an improved method based on the same soft wheat genomic region by means of a quantitative real-time PCR using a dual-labeled probe. Standard curves based on dilutions of 100% soft wheat flour, pasta, or bread were constructed. Durum wheat semolina, pasta, and bread samples were prepared with increasing amounts of soft wheat to verify the accuracy of the method. Results show that reliable quantifications were obtained especially for the samples containing a lower amount of soft wheat DNA, fulfilling the need to verify labeling of pasta and typical durum wheat breads.

  14. Comparing transformation methods for DNA microarray data

    Directory of Open Access Journals (Sweden)

    Zwinderman Aeilko H

    2004-06-01

    Full Text Available Abstract Background When DNA microarray data are used for gene clustering, genotype/phenotype correlation studies, or tissue classification the signal intensities are usually transformed and normalized in several steps in order to improve comparability and signal/noise ratio. These steps may include subtraction of an estimated background signal, subtracting the reference signal, smoothing (to account for nonlinear measurement effects, and more. Different authors use different approaches, and it is generally not clear to users which method they should prefer. Results We used the ratio between biological variance and measurement variance (which is an F-like statistic as a quality measure for transformation methods, and we demonstrate a method for maximizing that variance ratio on real data. We explore a number of transformations issues, including Box-Cox transformation, baseline shift, partial subtraction of the log-reference signal and smoothing. It appears that the optimal choice of parameters for the transformation methods depends on the data. Further, the behavior of the variance ratio, under the null hypothesis of zero biological variance, appears to depend on the choice of parameters. Conclusions The use of replicates in microarray experiments is important. Adjustment for the null-hypothesis behavior of the variance ratio is critical to the selection of transformation method.

  15. Quantification of dsDNA using the Hitachi F-7000 Fluorescence Spectrophotometer and PicoGreen dye.

    Science.gov (United States)

    Moreno, Luis A; Cox, Kendra L

    2010-11-05

    Quantification of DNA, especially in small concentrations, is an important task with a wide range of biological applications including standard molecular biology assays such as synthesis and purification of DNA, diagnostic applications such as quantification of DNA amplification products, and detection of DNA molecules in drug preparations. During this video we will demonstrate the capability of the Hitachi F-7000 Fluorescence Spectrophotometer equipped with a Micro Plate Reader accessory to perform dsDNA quantification using Molecular Probes Quant-it PicoGreen dye reagent kit. The F-7000 Fluorescence Spectrophotometer offers high sensitivity and high speed measurements. It is a highly flexible system capable of measuring fluorescence, luminescence, and phosphorescence. Several measuring modes are available, including wavelength scan, time scan, photometry and 3-D scan measurement. The spectrophotometer has sensitivity in the range of 50 picomoles of fluorescein when using a 300 μL sample volume in the microplate, and is capable of measuring scan speeds of 60,000 nm/minute. It also has a wide dynamic range of up to 5 orders of magnitude which allows for the use of calibration curves over a wide range of concentrations. The optical system uses all reflective optics for maximum energy and sensitivity. The standard wavelength range is 200 to 750 nm, and can be extended to 900 nm when using one of the optional near infrared photomultipliers. The system allows optional temperature control for the plate reader from 5 to 60 degrees Celsius using an optional external temperature controlled liquid circulator. The microplate reader allows for the use of 96 well microplates, and the measuring speed for 96 wells is less than 60 seconds when using the kinetics mode. Software controls for the F-7000 and Microplate Reader are also highly flexible. Samples may be set in either column or row formats, and any combination of wells may be chosen for sample measurements. This allows

  16. A phase quantification method based on EBSD data for a continuously cooled microalloyed steel

    Energy Technology Data Exchange (ETDEWEB)

    Zhao, H.; Wynne, B.P.; Palmiere, E.J., E-mail: e.j.palmiere@sheffield.ac.uk

    2017-01-15

    Mechanical properties of steels depend on the phase constitutions of the final microstructures which can be related to the processing parameters. Therefore, accurate quantification of different phases is necessary to investigate the relationships between processing parameters, final microstructures and mechanical properties. Point counting on micrographs observed by optical or scanning electron microscopy is widely used as a phase quantification method, and different phases are discriminated according to their morphological characteristics. However, it is difficult to differentiate some of the phase constituents with similar morphology. Differently, for EBSD based phase quantification methods, besides morphological characteristics, other parameters derived from the orientation information can also be used for discrimination. In this research, a phase quantification method based on EBSD data in the unit of grains was proposed to identify and quantify the complex phase constitutions of a microalloyed steel subjected to accelerated coolings. Characteristics of polygonal ferrite/quasi-polygonal ferrite, acicular ferrite and bainitic ferrite on grain averaged misorientation angles, aspect ratios, high angle grain boundary fractions and grain sizes were analysed and used to develop the identification criteria for each phase. Comparing the results obtained by this EBSD based method and point counting, it was found that this EBSD based method can provide accurate and reliable phase quantification results for microstructures with relatively slow cooling rates. - Highlights: •A phase quantification method based on EBSD data in the unit of grains was proposed. •The critical grain area above which GAM angles are valid parameters was obtained. •Grain size and grain boundary misorientation were used to identify acicular ferrite. •High cooling rates deteriorate the accuracy of this EBSD based method.

  17. Direct liquid chromatography method for the simultaneous quantification of hydroxytyrosol and tyrosol in red wines.

    Science.gov (United States)

    Piñeiro, Zulema; Cantos-Villar, Emma; Palma, Miguel; Puertas, Belen

    2011-11-09

    A validated HPLC method with fluorescence detection for the simultaneous quantification of hydroxytyrosol and tyrosol in red wines is described. Detection conditions for both compounds were optimized (excitation at 279 and 278 and emission at 631 and 598 nm for hydroxytyrosol and tyrosol, respectively). The validation of the analytical method was based on selectivity, linearity, robustness, detection and quantification limits, repeatability, and recovery. The detection and quantification limits in red wines were set at 0.023 and 0.076 mg L(-1) for hydroxytyrosol and at 0.007 and 0.024 mg L(-1) for tyrosol determination, respectively. Precision values, both within-day and between-day (n = 5), remained below 3% for both compounds. In addition, a fractional factorial experimental design was developed to analyze the influence of six different conditions on analysis. The final optimized HPLC-fluorescence method allowed the analysis of 30 nonpretreated Spanish red wines to evaluate their hydroxytyrosol and tyrosol contents.

  18. Towards a new method for the quantification of metabolites in the biological sample

    International Nuclear Information System (INIS)

    Neugnot, B.

    2005-03-01

    The quantification of metabolites is a key step in drug development. The aim of this Ph.D. work was to study the feasibility of a new method for this quantification, in the biological sample, without the drawbacks (cost, time, ethics) of the classical quantification methods based on metabolites synthesis or administration to man of the radiolabelled drug. Our strategy consists in determining the response factor, in mass spectrometry, of the metabolites. This approach is based on tritium labelling of the metabolites, ex vivo, by isotopic exchange. The labelling step was studied with deuterium. Metabolites of a model drug, recovered from in vitro or urinary samples, were labelled by three ways (Crab tree's catalyst ID2, deuterated trifluoroacetic acid or rhodium chloride ID20). Then, the transposition to tritium labelling was studied and the first results are very promising for the ultimate validation of the method. (author)

  19. A method for simultaneous quantification of phospholipid species by routine 31P NMR

    DEFF Research Database (Denmark)

    Brinkmann-Trettenes, Ulla; Stein, Paul C.; Klösgen, Beate Maria

    2012-01-01

    We report a 31P NMR assay for quantification of aqueous phospholipid samples. Using a capillary with trimethylphosphate as internal standard, the limit of quantification is 1.30mM. Comparison of the 31P NMR quantification method in aqueous buffer and in organic solvent revealed that the two methods...... are equal within experimental error. Changing the pH of the buffer enables peak separation for different phospholipid species. This is an advantage compared to the commercial enzyme assay based on phospholipase D and choline oxidase. The reported method, using routine 31P NMR equipment, is suitable when...... fast results of a limited number of samples are requested. © 2012 Elsevier B.V.....

  20. An external standard method for quantification of human cytomegalovirus by PCR

    International Nuclear Information System (INIS)

    Rongsen, Shen; Liren, Ma; Fengqi, Zhou; Qingliang, Luo

    1997-01-01

    An external standard method for PCR quantification of HCMV was reported. [α- 32 P]dATP was used as a tracer. 32 P-labelled specific amplification product was separated by agarose gel electrophoresis. A gel piece containing the specific product band was excised and counted in a plastic scintillation counter. Distribution of [α- 32 P]dATP in the electrophoretic gel plate and effect of separation between the 32 P-labelled specific product and free [α- 32 P]dATP were observed. A standard curve for quantification of HCMV by PCR was established and detective results of quality control templets were presented. The external standard method and the electrophoresis separation effect were appraised. The results showed that the method could be used for relative quantification of HCMV. (author)

  1. Impact and quantification of the sources of error in DNA pooling designs.

    Science.gov (United States)

    Jawaid, A; Sham, P

    2009-01-01

    The analysis of genome wide variation offers the possibility of unravelling the genes involved in the pathogenesis of disease. Genome wide association studies are also particularly useful for identifying and validating targets for therapeutic intervention as well as for detecting markers for drug efficacy and side effects. The cost of such large-scale genetic association studies may be reduced substantially by the analysis of pooled DNA from multiple individuals. However, experimental errors inherent in pooling studies lead to a potential increase in the false positive rate and a loss in power compared to individual genotyping. Here we quantify various sources of experimental error using empirical data from typical pooling experiments and corresponding individual genotyping counts using two statistical methods. We provide analytical formulas for calculating these different errors in the absence of complete information, such as replicate pool formation, and for adjusting for the errors in the statistical analysis. We demonstrate that DNA pooling has the potential of estimating allele frequencies accurately, and adjusting the pooled allele frequency estimates for differential allelic amplification considerably improves accuracy. Estimates of the components of error show that differential allelic amplification is the most important contributor to the error variance in absolute allele frequency estimation, followed by allele frequency measurement and pool formation errors. Our results emphasise the importance of minimising experimental errors and obtaining correct error estimates in genetic association studies.

  2. An efficient method for DNA extraction from Cladosporioid fungi.

    Science.gov (United States)

    Moslem, M A; Bahkali, A H; Abd-Elsalam, K A; Wit, P J G M

    2010-11-23

    We developed an efficient method for DNA extraction from Cladosporioid fungi, which are important fungal plant pathogens. The cell wall of Cladosporioid fungi is often melanized, which makes it difficult to extract DNA from their cells. In order to overcome this we grew these fungi for three days on agar plates and extracted DNA from mycelium mats after manual or electric homogenization. High-quality DNA was isolated, with an A(260)/A(280) ratio ranging between 1.6 and 2.0. Isolated genomic DNA was efficiently digested with restriction enzymes and produced distinct banding patterns on agarose gels for the different Cladosporium species. Clear DNA fragments from the isolated DNA were amplified by PCR using small and large subunit rDNA primers, demonstrating that this method provides DNA of sufficiently high quality for molecular analyses.

  3. Quantification of Cooperativity in Heterodimer-DNA Binding Improves the Accuracy of Binding Specificity Models*

    Science.gov (United States)

    Isakova, Alina; Berset, Yves; Hatzimanikatis, Vassily; Deplancke, Bart

    2016-01-01

    Many transcription factors (TFs) have the ability to cooperate on DNA elements as heterodimers. Despite the significance of TF heterodimerization for gene regulation, a quantitative understanding of cooperativity between various TF dimer partners and its impact on heterodimer DNA binding specificity models is still lacking. Here, we used a novel integrative approach, combining microfluidics-steered measurements of dimer-DNA assembly with mechanistic modeling of the implicated protein-protein-DNA interactions to quantitatively interrogate the cooperative DNA binding behavior of the adipogenic peroxisome proliferator-activated receptor γ (PPARγ):retinoid X receptor α (RXRα) heterodimer. Using the high throughput MITOMI (mechanically induced trapping of molecular interactions) platform, we derived equilibrium DNA binding data for PPARγ, RXRα, as well as the PPARγ:RXRα heterodimer to more than 300 target DNA sites and variants thereof. We then quantified cooperativity underlying heterodimer-DNA binding and derived an integrative heterodimer DNA binding constant. Using this cooperativity-inclusive constant, we were able to build a heterodimer-DNA binding specificity model that has superior predictive power than the one based on a regular one-site equilibrium. Our data further revealed that individual nucleotide substitutions within the target site affect the extent of cooperativity in PPARγ:RXRα-DNA binding. Our study therefore emphasizes the importance of assessing cooperativity when generating DNA binding specificity models for heterodimers. PMID:26912662

  4. Quantification of Cooperativity in Heterodimer-DNA Binding Improves the Accuracy of Binding Specificity Models.

    Science.gov (United States)

    Isakova, Alina; Berset, Yves; Hatzimanikatis, Vassily; Deplancke, Bart

    2016-05-06

    Many transcription factors (TFs) have the ability to cooperate on DNA elements as heterodimers. Despite the significance of TF heterodimerization for gene regulation, a quantitative understanding of cooperativity between various TF dimer partners and its impact on heterodimer DNA binding specificity models is still lacking. Here, we used a novel integrative approach, combining microfluidics-steered measurements of dimer-DNA assembly with mechanistic modeling of the implicated protein-protein-DNA interactions to quantitatively interrogate the cooperative DNA binding behavior of the adipogenic peroxisome proliferator-activated receptor γ (PPARγ):retinoid X receptor α (RXRα) heterodimer. Using the high throughput MITOMI (mechanically induced trapping of molecular interactions) platform, we derived equilibrium DNA binding data for PPARγ, RXRα, as well as the PPARγ:RXRα heterodimer to more than 300 target DNA sites and variants thereof. We then quantified cooperativity underlying heterodimer-DNA binding and derived an integrative heterodimer DNA binding constant. Using this cooperativity-inclusive constant, we were able to build a heterodimer-DNA binding specificity model that has superior predictive power than the one based on a regular one-site equilibrium. Our data further revealed that individual nucleotide substitutions within the target site affect the extent of cooperativity in PPARγ:RXRα-DNA binding. Our study therefore emphasizes the importance of assessing cooperativity when generating DNA binding specificity models for heterodimers. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  5. Examination of packaging materials in bakery products : a validated method for detection and quantification

    NARCIS (Netherlands)

    Raamsdonk, van L.W.D.; Pinckaers, V.G.Z.; Vliege, J.J.M.; Egmond, van H.J.

    2012-01-01

    Methods for the detection and quantification of packaging materials are necessary for the control of the prohibition of these materials according to Regulation (EC)767/2009. A method has been developed and validated at RIKILT for bakery products, including sweet bread and raisin bread. This choice

  6. To beat or not to beat a tick: comparison of DNA extraction methods for ticks (Ixodes scapularis

    Directory of Open Access Journals (Sweden)

    Alyssa D. Ammazzalorso

    2015-08-01

    Full Text Available Background. Blacklegged ticks (Ixodes scapularis are important disease vectors in the United States, known to transmit a variety of pathogens to humans, including bacteria, protozoa, and viruses. Their importance as a disease vector necessitates reliable and comparable methods for extracting microbial DNA from ticks. Furthermore, to explore the population genetics or genomics of this tick, appropriate DNA extraction techniques are needed for both the vector and its microbes. Although a few studies have investigated different methods of DNA isolation from ticks, they are limited in the number and types of DNA extraction and lack species-specific quantification of DNA yield.Methods. Here we determined the most efficient and consistent method of DNA extraction from two different developmental stages of I. scapularis—nymph and adult—that are the most important for disease transmission. We used various methods of physical disruption of the hard, chitinous exoskeleton, as well as commercial and non-commercial DNA isolation kits. To gauge the effectiveness of these methods, we quantified the DNA yield and confirmed the DNA quality via PCR of both tick and microbial genetic material.Results. DNA extraction using the Thermo GeneJET Genomic DNA Purification Kit resulted in the highest DNA yields and the most consistent PCR amplification when combined with either cutting or bead beating with select matrices across life stages. DNA isolation methods using ammonium hydroxide as well as the MoBio PowerSoil kit also produced strong and successful PCR amplification, but only for females.Discussion. We contrasted a variety of readily available methods of DNA extraction from single individual blacklegged ticks and presented the results through a quantitative and qualitative assessment.

  7. Quantification of histoautoradiographic evidence of DNA repair synthesis in the liver

    International Nuclear Information System (INIS)

    Hochmann, J.; Stambergova, H.

    1988-01-01

    Histoautoradiography was used to detect dimethylnitrosamine-induced 3 H-thymidine incorporation in vivo into G phase hepatocytes. The description of the standard procedure for counting the grains and the mode of mathematical evaluation are presented. The results exhibited higher sensitivity than those in the investigation of the DNA repair synthesis by means of a scintillation counter using the method of detecting hydroxyurea-resistant incorporation of 3 H-thymidine. Thus, it was possible to simplify the investigation by lowering the number of evaluated cells. A suitable compromise between precision and laboriousness will probably be achieved by counting 20 hepatocytes per animal. In case of striking differences between the experimental and the control groups a qualitative conclusion may be drawn even without counting the grains. (author). 5 tabs., 10 refs

  8. Quantification of organellar DNA and RNA using real-time PCR.

    Science.gov (United States)

    Weihe, Andreas

    2014-01-01

    Quantitative (real-time) polymerase chain reaction (PCR) allows the measurement of relative organellar gene copy numbers as well as transcript abundance of individual mitochondrial or plastidial genes. Requiring only minute amounts of total DNA or RNA, the described method can replace traditional analyses like Southern or Northern hybridization which require large amounts of organellar nucleic acids and usually provide only semiquantitative data. Here we describe prerequisites, reaction conditions, and data analysis principles, which should be applicable for a wide range of plant species and experimental situations where comparative and precise determination of gene copy numbers or transcript abundance is requested. Sequences of amplification primers for qPCR of organellar genes from Arabidopsis are provided.

  9. Interpretation of biological and mechanical variations between the Lowry versus Bradford method for protein quantification

    OpenAIRE

    Tzong-Shi Lu; Szu-Yu Yiao; Kenneth Lim; Roderick V. Jensen; Li-Li Hsiao

    2010-01-01

    Background: The identification of differences in protein expression resulting from methodical variations is an essential component to the interpretation of true, biologically significant results. Aims: We used the Lowry and Bradford methods- two most commonly used methods for protein quantification, to assess whether differential protein expressions are a result of true biological or methodical variations. Material & Methods: Differential protein expression patterns was assessed by western bl...

  10. Forest Carbon Leakage Quantification Methods and Their Suitability for Assessing Leakage in REDD

    Directory of Open Access Journals (Sweden)

    Sabine Henders

    2012-01-01

    Full Text Available This paper assesses quantification methods for carbon leakage from forestry activities for their suitability in leakage accounting in a future Reducing Emissions from Deforestation and Forest Degradation (REDD mechanism. To that end, we first conducted a literature review to identify specific pre-requisites for leakage assessment in REDD. We then analyzed a total of 34 quantification methods for leakage emissions from the Clean Development Mechanism (CDM, the Verified Carbon Standard (VCS, the Climate Action Reserve (CAR, the CarbonFix Standard (CFS, and from scientific literature sources. We screened these methods for the leakage aspects they address in terms of leakage type, tools used for quantification and the geographical scale covered. Results show that leakage methods can be grouped into nine main methodological approaches, six of which could fulfill the recommended REDD leakage requirements if approaches for primary and secondary leakage are combined. The majority of methods assessed, address either primary or secondary leakage; the former mostly on a local or regional and the latter on national scale. The VCS is found to be the only carbon accounting standard at present to fulfill all leakage quantification requisites in REDD. However, a lack of accounting methods was identified for international leakage, which was addressed by only two methods, both from scientific literature.

  11. Pore REconstruction and Segmentation (PORES) method for improved porosity quantification of nanoporous materials

    Energy Technology Data Exchange (ETDEWEB)

    Van Eyndhoven, G., E-mail: geert.vaneyndhoven@uantwerpen.be [iMinds-Vision Lab, University of Antwerp, Universiteitsplein 1, B-2610 Wilrijk (Belgium); Kurttepeli, M. [EMAT, University of Antwerp, Groenenborgerlaan 171, B-2020 Antwerp (Belgium); Van Oers, C.J.; Cool, P. [Laboratory of Adsorption and Catalysis, University of Antwerp, Universiteitsplein 1, B-2610 Wilrijk (Belgium); Bals, S. [EMAT, University of Antwerp, Groenenborgerlaan 171, B-2020 Antwerp (Belgium); Batenburg, K.J. [iMinds-Vision Lab, University of Antwerp, Universiteitsplein 1, B-2610 Wilrijk (Belgium); Centrum Wiskunde and Informatica, Science Park 123, NL-1090 GB Amsterdam (Netherlands); Mathematical Institute, Universiteit Leiden, Niels Bohrweg 1, NL-2333 CA Leiden (Netherlands); Sijbers, J. [iMinds-Vision Lab, University of Antwerp, Universiteitsplein 1, B-2610 Wilrijk (Belgium)

    2015-01-15

    Electron tomography is currently a versatile tool to investigate the connection between the structure and properties of nanomaterials. However, a quantitative interpretation of electron tomography results is still far from straightforward. Especially accurate quantification of pore-space is hampered by artifacts introduced in all steps of the processing chain, i.e., acquisition, reconstruction, segmentation and quantification. Furthermore, most common approaches require subjective manual user input. In this paper, the PORES algorithm “POre REconstruction and Segmentation” is introduced; it is a tailor-made, integral approach, for the reconstruction, segmentation, and quantification of porous nanomaterials. The PORES processing chain starts by calculating a reconstruction with a nanoporous-specific reconstruction algorithm: the Simultaneous Update of Pore Pixels by iterative REconstruction and Simple Segmentation algorithm (SUPPRESS). It classifies the interior region to the pores during reconstruction, while reconstructing the remaining region by reducing the error with respect to the acquired electron microscopy data. The SUPPRESS reconstruction can be directly plugged into the remaining processing chain of the PORES algorithm, resulting in accurate individual pore quantification and full sample pore statistics. The proposed approach was extensively validated on both simulated and experimental data, indicating its ability to generate accurate statistics of nanoporous materials. - Highlights: • An electron tomography reconstruction/segmentation method for nanoporous materials. • The method exploits the porous nature of the scanned material. • Validated extensively on both simulation and real data experiments. • Results in increased image resolution and improved porosity quantification.

  12. QUANTIFICATION OF GLOMERULAR EPITHELIAL-CELL ADHESION BY USING ANTI-DNA ANTIBODIES IN ELISA

    NARCIS (Netherlands)

    COERS, W; SMEENK, RJT; SALANT, DJ; WEENING, JJ

    A sensitive and reproducible microassay is described for quantification of adhesion of cells to matrix-coated 96-wells plates under different experimental conditions. For this purpose glomerular visceral epithelial cells (GVEC) were used. Attached GVEC were fixed with methanol and incubated with a

  13. Quantification of transcription factor-DNA binding affinity in a living cell.

    Science.gov (United States)

    Belikov, Sergey; Berg, Otto G; Wrange, Örjan

    2016-04-20

    The apparent dissociation constant (Kd) for specific binding of glucocorticoid receptor (GR) and androgen receptor (AR) to DNA was determined in vivo in Xenopus oocytes. The total nuclear receptor concentration was quantified as specifically retained [(3)H]-hormone in manually isolated oocyte nuclei. DNA was introduced by nuclear microinjection of single stranded phagemid DNA, chromatin is then formed during second strand synthesis. The fraction of DNA sites occupied by the expressed receptor was determined by dimethylsulphate in vivo footprinting and used for calculation of the receptor-DNA binding affinity. The forkhead transcription factor FoxA1 enhanced the DNA binding by GR with an apparent Kd of ∼1 μM and dramatically stimulated DNA binding by AR with an apparent Kd of ∼0.13 μM at a composite androgen responsive DNA element containing one FoxA1 binding site and one palindromic hormone receptor binding site known to bind one receptor homodimer. FoxA1 exerted a weak constitutive- and strongly cooperative DNA binding together with AR but had a less prominent effect with GR, the difference reflecting the licensing function of FoxA1 at this androgen responsive DNA element. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  14. Novel Quantitative Real-Time LCR for the Sensitive Detection of SNP Frequencies in Pooled DNA: Method Development, Evaluation and Application

    Science.gov (United States)

    Psifidi, Androniki; Dovas, Chrysostomos; Banos, Georgios

    2011-01-01

    Background Single nucleotide polymorphisms (SNP) have proven to be powerful genetic markers for genetic applications in medicine, life science and agriculture. A variety of methods exist for SNP detection but few can quantify SNP frequencies when the mutated DNA molecules correspond to a small fraction of the wild-type DNA. Furthermore, there is no generally accepted gold standard for SNP quantification, and, in general, currently applied methods give inconsistent results in selected cohorts. In the present study we sought to develop a novel method for accurate detection and quantification of SNP in DNA pooled samples. Methods The development and evaluation of a novel Ligase Chain Reaction (LCR) protocol that uses a DNA-specific fluorescent dye to allow quantitative real-time analysis is described. Different reaction components and thermocycling parameters affecting the efficiency and specificity of LCR were examined. Several protocols, including gap-LCR modifications, were evaluated using plasmid standard and genomic DNA pools. A protocol of choice was identified and applied for the quantification of a polymorphism at codon 136 of the ovine PRNP gene that is associated with susceptibility to a transmissible spongiform encephalopathy in sheep. Conclusions The real-time LCR protocol developed in the present study showed high sensitivity, accuracy, reproducibility and a wide dynamic range of SNP quantification in different DNA pools. The limits of detection and quantification of SNP frequencies were 0.085% and 0.35%, respectively. Significance The proposed real-time LCR protocol is applicable when sensitive detection and accurate quantification of low copy number mutations in DNA pools is needed. Examples include oncogenes and tumour suppressor genes, infectious diseases, pathogenic bacteria, fungal species, viral mutants, drug resistance resulting from point mutations, and genetically modified organisms in food. PMID:21283808

  15. Use of DNA quantification to measure growth and autolysis of Lactococcus and Propionibacterium spp. in mixed populations.

    Science.gov (United States)

    Treimo, Janneke; Vegarud, Gerd; Langsrud, Thor; Rudi, Knut

    2006-09-01

    Autolysis is self-degradation of the bacterial cell wall that results in the release of enzymes and DNA. Autolysis of starter bacteria, such as lactococci and propionibacteria, is essential for cheese ripening, but our understanding of this important process is limited. This is mainly because the current tools for measuring autolysis cannot readily be used for analysis of bacteria in mixed populations. We have now addressed this problem by species-specific detection and quantification of free DNA released during autolysis. This was done by use of 16S rRNA gene single-nucleotide extension probes in combination with competitive PCR. We analyzed pure and mixed populations of Lactococcus lactis subsp. lactis and three different species of Propionibacterium. Results showed that L. lactis subsp. lactis INF L2 autolyzed first, followed by Propionibacterium acidipropionici ATCC 4965, Propionibacterium freudenreichii ISU P59, and then Propionibacterium jensenii INF P303. We also investigated the autolytic effect of rennet (commonly used in cheese production). We found that the effect was highly strain specific, with all the strains responding differently. Finally, autolysis of L. lactis subsp. lactis INF L2 and P. freudenreichii ISU P59 was analyzed in a liquid cheese model. Autolysis was detected later in this cheese model system than in broth media. A challenge with DNA, however, is DNA degradation. We addressed this challenge by using a DNA degradation marker. We obtained a good correlation between the degradation of the marker and the target in a model experiment. We conclude that our DNA approach will be a valuable tool for use in future analyses and for understanding autolysis in mixed bacterial populations.

  16. 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. © The Author 2014. Published by

  17. Methods to maximise recovery of environmental DNA from water samples.

    Directory of Open Access Journals (Sweden)

    Rheyda Hinlo

    Full Text Available The environmental DNA (eDNA method is a detection technique that is rapidly gaining credibility as a sensitive tool useful in the surveillance and monitoring of invasive and threatened species. Because eDNA analysis often deals with small quantities of short and degraded DNA fragments, methods that maximize eDNA recovery are required to increase detectability. In this study, we performed experiments at different stages of the eDNA analysis to show which combinations of methods give the best recovery rate for eDNA. Using Oriental weatherloach (Misgurnus anguillicaudatus as a study species, we show that various combinations of DNA capture, preservation and extraction methods can significantly affect DNA yield. Filtration using cellulose nitrate filter paper preserved in ethanol or stored in a -20°C freezer and extracted with the Qiagen DNeasy kit outperformed other combinations in terms of cost and efficiency of DNA recovery. Our results support the recommendation to filter water samples within 24hours but if this is not possible, our results suggest that refrigeration may be a better option than freezing for short-term storage (i.e., 3-5 days. This information is useful in designing eDNA detection of low-density invasive or threatened species, where small variations in DNA recovery can signify the difference between detection success or failure.

  18. Genomic DNA extraction method from pearl millet ( Pennisetum ...

    African Journals Online (AJOL)

    DNA extraction is difficult in a variety of plants because of the presence of metabolites that interfere with DNA isolation procedures and downstream applications such as DNA restriction, amplification, and cloning. Here we describe a modified procedure based on the hexadecyltrimethylammonium bromide (CTAB) method to ...

  19. Genomic DNA extraction method from Annona senegalensis Pers ...

    African Journals Online (AJOL)

    Extraction of DNA in many plants is difficult because of the presence of metabolites that interfere with DNA isolation procedures and downstream applications such as DNA restriction, replications, amplification, as well as cloning. Modified procedure based on the hexadecyltrimethyl ammoniumbromide (CTAB) method is ...

  20. Microbial diversity in fecal samples depends on DNA extraction method

    DEFF Research Database (Denmark)

    Mirsepasi, Hengameh; Persson, Søren; Struve, Carsten

    2014-01-01

    was to evaluate two different DNA extraction methods in order to choose the most efficient method for studying intestinal bacterial diversity using Denaturing Gradient Gel Electrophoresis (DGGE). FINDINGS: In this study, a semi-automatic DNA extraction system (easyMag®, BioMérieux, Marcy I'Etoile, France......BACKGROUND: There are challenges, when extracting bacterial DNA from specimens for molecular diagnostics, since fecal samples also contain DNA from human cells and many different substances derived from food, cell residues and medication that can inhibit downstream PCR. The purpose of the study...... by easyMag® from the same fecal samples. Furthermore, DNA extracts obtained using easyMag® seemed to contain inhibitory compounds, since in order to perform a successful PCR-analysis, the sample should be diluted at least 10 times. DGGE performed on PCR from DNA extracted by QIAamp DNA Stool Mini Kit DNA...

  1. Quantification of diatoms in biofilms: Standardisation of methods

    Digital Repository Service at National Institute of Oceanography (India)

    Patil, J.S.; Anil, A.C.

    of the difficulty in sampling and enumeration. Scraping or brushing are the traditional methods used for removal of diatoms from biofilms developed on solid substrata. The method of removal is the most critical step in enumerating the biofilm diatom community...

  2. Quantification of Epstein-Barr virus-DNA load in lung transplant recipients : A comparison of plasma versus whole blood

    NARCIS (Netherlands)

    Bakker, Nicolaas A.; Verschuuren, Erik A.; Veeger, Nic J.; van der Bij, Wim; van Imhoff, Gustaaf W.; Kallenberg, Cees G.; Hepkema, Bouke G.

    2008-01-01

    Background: Monitoring of the Epstein-Barr virus-DNA load is frequently used to identify patients at risk for post-transplant lymphoproliferative disease (PTLD). Epstein-Barr virus DNA can be measured in the plasma and whole blood serum compartments. Methods: We compared levels of Epstein-Barr virus

  3. Critical assessment of three high performance liquid chromatography analytical methods for food carotenoid quantification

    NARCIS (Netherlands)

    Dias, M.G.; Oliveira, L.; Camoes, M.F.G.F.C.; Nunes, B.; Versloot, P.; Hulshof, P.J.M.

    2010-01-01

    Three sets of extraction/saponification/HPLC conditions for food carotenoid quantification were technically and economically compared. Samples were analysed for carotenoids a-carotene, ß-carotene, ß-cryptoxanthin, lutein, lycopene, and zeaxanthin. All methods demonstrated good performance in the

  4. 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.

  5. Image correlation method for DNA sequence alignment.

    Science.gov (United States)

    Curilem Saldías, Millaray; Villarroel Sassarini, Felipe; Muñoz Poblete, Carlos; Vargas Vásquez, Asticio; Maureira Butler, Iván

    2012-01-01

    The complexity of searches and the volume of genomic data make sequence alignment one of bioinformatics most active research areas. New alignment approaches have incorporated digital signal processing techniques. Among these, correlation methods are highly sensitive. This paper proposes a novel sequence alignment method based on 2-dimensional images, where each nucleic acid base is represented as a fixed gray intensity pixel. Query and known database sequences are coded to their pixel representation and sequence alignment is handled as object recognition in a scene problem. Query and database become object and scene, respectively. An image correlation process is carried out in order to search for the best match between them. Given that this procedure can be implemented in an optical correlator, the correlation could eventually be accomplished at light speed. This paper shows an initial research stage where results were "digitally" obtained by simulating an optical correlation of DNA sequences represented as images. A total of 303 queries (variable lengths from 50 to 4500 base pairs) and 100 scenes represented by 100 x 100 images each (in total, one million base pair database) were considered for the image correlation analysis. The results showed that correlations reached very high sensitivity (99.01%), specificity (98.99%) and outperformed BLAST when mutation numbers increased. However, digital correlation processes were hundred times slower than BLAST. We are currently starting an initiative to evaluate the correlation speed process of a real experimental optical correlator. By doing this, we expect to fully exploit optical correlation light properties. As the optical correlator works jointly with the computer, digital algorithms should also be optimized. The results presented in this paper are encouraging and support the study of image correlation methods on sequence alignment.

  6. Quantification of DNA in simple eukaryotic cells using Fourier transform infrared spectroscopy.

    Science.gov (United States)

    Whelan, Donna R; Bambery, Keith R; Puskar, Ljiljana; McNaughton, Don; Wood, Bayden R

    2013-10-01

    A technique capable of detecting and monitoring nucleic acid concentration offers potential in diagnosing cancer and further developing an understanding of the biochemistry of disease. The application of Fourier transform infrared (FTIR) spectroscopy has previously been hindered by the supposed non-Beer-Lambert absorption behavior of DNA in intact cells making elucidation of the DNA bands difficult. We use known composition DNA/hemoglobin standards to successfully estimate the DNA content in avian erythrocyte nuclei (44.2%) and intact erythrocytes (12.8%). Furthermore we demonstrate that the absorption of cellular DNA does follow the Beer-Lambert Law and highlights the role of conformation and hydration in FTIR spectroscopy of biological samples. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  7. Performance Characteristics and Comparison of Abbott and artus Real-Time Systems for Hepatitis B Virus DNA Quantification

    Science.gov (United States)

    Ismail, Ashrafali M.; Sivakumar, Jayashree; Anantharam, Raghavendran; Dayalan, Sujitha; Samuel, Prasanna; Fletcher, Gnanadurai J.; Gnanamony, Manu; Abraham, Priya

    2011-01-01

    Virological monitoring of hepatitis B virus (HBV) DNA is critical to the management of HBV infection. With several HBV DNA quantification assays available, it is important to use the most efficient testing system for virological monitoring. In this study, we evaluated the performance characteristics and comparability of three HBV DNA quantification systems: Abbott HBV real-time PCR (Abbott PCR), artus HBV real-time PCR with QIAamp DNA blood kit purification (artus-DB), and artus HBV real-time PCR with the QIAamp DSP virus kit purification (artus-DSP). The lower limits of detection of these systems were established against the WHO international standards for HBV DNA and were found to be 1.43, 82, and 9 IU/ml, respectively. The intra-assay and interassay coefficients of variation of plasma samples (1 to 6 log10 IU/ml) ranged between 0.05 to 8.34% and 0.16 to 3.48% for the Abbott PCR, 1.53 to 26.85% and 0.50 to 12.89% for artus-DB, and 0.29 to 7.42% and 0.94 to 3.01% for artus-DSP, respectively. Ninety HBV clinical samples were used for comparison of assays, and paired quantitative results showed strong correlation by linear regression analysis (artus-DB with Abbott PCR, r = 0.95; Abbott PCR with artus-DSP, r = 0.97; and artus-DSP with artus-DB, r = 0.94). Bland-Altman analysis showed a good level of agreement for Abbott PCR and artus-DSP, with a mean difference of 0.10 log10 IU/ml and limits of agreement of −0.91 to 1.11 log10 IU/ml. No genotype-specific bias was seen in all three systems for HBV genotypes A, C, and D, which are predominant in this region. This finding illustrates that the Abbott real-time HBV and artus-DSP systems show more comparable performance than the artus-DB system, meeting the current guidelines for assays to be used in the management of hepatitis B. PMID:21795507

  8. Performance characteristics and comparison of Abbott and artus real-time systems for hepatitis B virus DNA quantification.

    Science.gov (United States)

    Ismail, Ashrafali M; Sivakumar, Jayashree; Anantharam, Raghavendran; Dayalan, Sujitha; Samuel, Prasanna; Fletcher, Gnanadurai J; Gnanamony, Manu; Abraham, Priya

    2011-09-01

    Virological monitoring of hepatitis B virus (HBV) DNA is critical to the management of HBV infection. With several HBV DNA quantification assays available, it is important to use the most efficient testing system for virological monitoring. In this study, we evaluated the performance characteristics and comparability of three HBV DNA quantification systems: Abbott HBV real-time PCR (Abbott PCR), artus HBV real-time PCR with QIAamp DNA blood kit purification (artus-DB), and artus HBV real-time PCR with the QIAamp DSP virus kit purification (artus-DSP). The lower limits of detection of these systems were established against the WHO international standards for HBV DNA and were found to be 1.43, 82, and 9 IU/ml, respectively. The intra-assay and interassay coefficients of variation of plasma samples (1 to 6 log(10) IU/ml) ranged between 0.05 to 8.34% and 0.16 to 3.48% for the Abbott PCR, 1.53 to 26.85% and 0.50 to 12.89% for artus-DB, and 0.29 to 7.42% and 0.94 to 3.01% for artus-DSP, respectively. Ninety HBV clinical samples were used for comparison of assays, and paired quantitative results showed strong correlation by linear regression analysis (artus-DB with Abbott PCR, r = 0.95; Abbott PCR with artus-DSP, r = 0.97; and artus-DSP with artus-DB, r = 0.94). Bland-Altman analysis showed a good level of agreement for Abbott PCR and artus-DSP, with a mean difference of 0.10 log(10) IU/ml and limits of agreement of -0.91 to 1.11 log(10) IU/ml. No genotype-specific bias was seen in all three systems for HBV genotypes A, C, and D, which are predominant in this region. This finding illustrates that the Abbott real-time HBV and artus-DSP systems show more comparable performance than the artus-DB system, meeting the current guidelines for assays to be used in the management of hepatitis B.

  9. Interpretation of biological and mechanical variations between the Lowry versus Bradford method for protein quantification.

    Science.gov (United States)

    Lu, Tzong-Shi; Yiao, Szu-Yu; Lim, Kenneth; Jensen, Roderick V; Hsiao, Li-Li

    2010-07-01

    The identification of differences in protein expression resulting from methodical variations is an essential component to the interpretation of true, biologically significant results. We used the Lowry and Bradford methods- two most commonly used methods for protein quantification, to assess whether differential protein expressions are a result of true biological or methodical variations. MATERIAL #ENTITYSTARTX00026; Differential protein expression patterns was assessed by western blot following protein quantification by the Lowry and Bradford methods. We have observed significant variations in protein concentrations following assessment with the Lowry versus Bradford methods, using identical samples. Greater variations in protein concentration readings were observed over time and in samples with higher concentrations, with the Bradford method. Identical samples quantified using both methods yielded significantly different expression patterns on Western blot. We show for the first time that methodical variations observed in these protein assay techniques, can potentially translate into differential protein expression patterns, that can be falsely taken to be biologically significant. Our study therefore highlights the pivotal need to carefully consider methodical approaches to protein quantification in techniques that report quantitative differences.

  10. Tissue-specific Calibration of Real-time PCR Facilitates Absolute Quantification of Plasmid DNA in Biodistribution Studies

    Directory of Open Access Journals (Sweden)

    Joan K Ho

    2016-01-01

    Full Text Available Analysis of the tissue distribution of plasmid DNA after administration of nonviral gene delivery systems is best accomplished using quantitative real-time polymerase chain reaction (qPCR, although published strategies do not allow determination of the absolute mass of plasmid delivered to different tissues. Generally, data is expressed as the mass of plasmid relative to the mass of genomic DNA (gDNA in the sample. This strategy is adequate for comparisons of efficiency of delivery to a single site but it does not allow direct comparison of delivery to multiple tissues, as the mass of gDNA extracted per unit mass of each tissue is different. We show here that by constructing qPCR standard curves for each tissue it is possible to determine the dose of intact plasmid remaining in each tissue, which is a more useful parameter when comparing the fates of different formulations of DNA. We exemplify the use of this tissue-specific qPCR method by comparing the delivery of naked DNA, cationic DNA complexes, and neutral PEGylated DNA complexes after intramuscular injection. Generally, larger masses of intact plasmid were present 24 hours after injection of DNA complexes, and neutral complexes resulted in delivery of a larger mass of intact plasmid to the spleen.

  11. Performance of spectral fitting methods for vegetation fluorescence quantification

    NARCIS (Netherlands)

    Meroni, M.; Busetto, D.; Colombo, R.; Guanter, L.; Moreno, J.; Verhoef, W.

    2010-01-01

    The Fraunhofer Line Discriminator (FLD) principle has long been considered as the reference method to quantify solar-induced chlorophyll fluorescence (F) from passive remote sensing measurements. Recently, alternative retrieval algorithms based on the spectral fitting of hyperspectral radiance

  12. Estimation and quantification of human DNA in dental calculus: A pilot study.

    Science.gov (United States)

    Singh, Udita; Goel, Saurabh

    2017-01-01

    Identification using DNA has proved its accuracy multiple times in the field of forensic investigations. Investigators usually rely on either teeth or bone as the DNA reservoirs. However, there are instances where the skeletal or dental remains are not available or not preserved properly. Moreover, due to religious beliefs, the family members of the dead do not allow the investigating team to damage the remains for the sole purpose of identification. To investigate the presence of human DNA in dental calculus and to quantify the amount, if present. This prospective single-blinded pilot study included twenty subjects selected from the patients visiting a dental college. The samples of dental calculus were collected from the thickest portion of calculus deposited on the lingual surfaces of mandibular incisors. These samples were decontaminated and subjected to gel electrophoresis for DNA extraction. DNA was found in 85% cases. The amount of DNA varied from 21 to 37 μg/ml of dental calculus. Dental calculus is a rich reservoir of human DNA.

  13. Quantification of Maternal Serum Cell-Free Fetal DNA in Early-Onset Preeclampsia

    Directory of Open Access Journals (Sweden)

    Mulan Ren

    2013-04-01

    Full Text Available The aim of this study was to determine whether the increased serum cell-free fetal DNA (cffDNA level of gravidas developed into early-onset preeclampsia (EOPE subsequently in the early second trimesters is related to prenatal screening markers. Serum was collected from 1011 gravidas. The level of cffDNA and prenatal screening markers were analyzed in 20 cases with EOPE and 20 controls. All fetuses were male. The maternal serum cffDNA level was assessed by amplification of the Y chromosome specific gene. Correlations between the variables were examined. (Logged cffDNA in EOPE (median, 3.08; interquartile range, 2.93–3.68 was higher than controls (median, 1.79; interquartile range, 1.46–2.53. The increased level of (logged cffDNA was correlated significantly with the increased human chorionic gonadotropin (HCG level (r = 0.628, p < 0.001. Significant reciprocal correlations between cffDNA and babies’ birth weight as well as gestation weeks at delivery were noted (r = −0.516, p = 0.001; r = −0.623, p < 0.001, respectively. The sensitivity and specificity of cffDNA to discriminate between the EOPE cases and the controls were 90% and 85%, respectively. CffDNA is a potential marker for EOPE, which had a significant reciprocal correlation with babies’ birth weight and gestation weeks at delivery. Moreover, it may help in indicating the underlying hypoxic condition in the placenta.

  14. Enzymatic quantification of strand breaks of DNA induced by vacuum-UV radiation

    International Nuclear Information System (INIS)

    Ito, Takashi

    1986-01-01

    Hind3 digested plasmid DNA dried on an aluminum plate was irradiated by vacuum-UV at 160 and 195 nm using a synchrotron irradiation system. A change induced in the DNA, presumably a single strand break, was quantified by the aid of the strand break-derived stimulation of poly(ADP-ribose) synthetase activity. The end group of strand breaks so induced was recognized by the enzyme as effectively as that by DNase 1 treatment, suggesting a nicking as the major lesion inflicted on the DNA. The fluence (UV) dependent stimulation of poly(ADP-ribose) synthetase activity was much higher upon 160 nm irradiation than upon 195 nm irradiation. (Auth.)

  15. Introduction to DNA methods for identification of irradiated foods

    International Nuclear Information System (INIS)

    Delincee, H.

    1996-01-01

    This brief introduction sets the scene with respect to the presentations in this ADMIT meeting dealing with DNA changes as a tool to detect the radiation processing of food. The choice to examine DNA seems obvious, since DNA is a sensitive cellular target to irradiation and the changes in DNA are responsible for many effects observed in irradiated foods, such as the inactivation of microorganisms, elimination of insects, inhibition of sprouting in bulbs and tubers and delay of ripening in several fruits. Therefore, these changes in DNA should be discernible in microbial or insect DNA or in the nucleic acids in the food itself. If DNA changes were specific to irradiation, a detection method could be designed which would have wide applicability, since most foods are derived from living organisms which all contain DNA. Such a method could almost be the universal method for detecting the radiation treatment of foods. Radiation-induced changes in DNA can be analysed by a variety of analytical techniques, which have mostly been employed on pure DNA or on DNA in living cells in radiation biology research. Whether or not some of these techniques can be utilised to detect irradiated food has recently been very briefly discussed. (author)

  16. Developing a method for quantification of Ascaris eggs on hands

    DEFF Research Database (Denmark)

    Jeandron, Aurelie; Ensink, Jeroen J. H.; Thamsborg, Stig Milan

    In transmission of soil transmitted helminths, especially with Ascaris and Trichuris infections, the importance of hands is unclear and very limited literature exists. This is partly because of the absence of a reliable method to quantify the number of helminth eggs on hands. The aim of this study...... was to develop a method to assess the number of Ascaris eggs on hands and determine the egg recovery rate of the method. Under laboratory conditions, hands were contaminated with app. 1000 Ascaris eggs, air dried and washed in a plastic bag retaining the washing water, in order to determine recovery rates...... of eggs for two different detergents (cationic [benzethonium chloride 0.1%], anionic [7X 1% - quadrafos, glycol ether, and dioctyl sulfoccinate sodium salt]) and de-ionized water used as control. The highest recovery rate (95.6%) was achieved with a hand rinse performed with 7X 1%. Washing hands...

  17. Gallic Acid: Review of the Methods of Determination and Quantification.

    Science.gov (United States)

    Fernandes, Felipe Hugo Alencar; Salgado, Hérida Regina Nunes

    2016-05-03

    Gallic acid (3,4,5 trihydroxybenzoic acid) is a secondary metabolite present in most plants. This metabolite is known to exhibit a range of bioactivities including antioxidant, antimicrobial, anti-inflammatory, and anticancer. There are various methods to analyze gallic acid including spectrometry, chromatography, and capillary electrophoresis, among others. They have been developed to identify and quantify this active ingredient in most biological matrices. The aim of this article is to review the available information on analytical methods for gallic acid, as well as presenting the advantages and limitations of each technique.

  18. Validated RP-HPLC Method for Quantification of Phenolic ...

    African Journals Online (AJOL)

    Purpose: To evaluate the total phenolic content and antioxidant potential of the methanol extracts of aerial parts and roots of Thymus sipyleus Boiss and also to determine some phenolic compounds using a newly developed and validated reversed phase high performance liquid chromatography (RP-HPLC) method.

  19. Development, optimization, and single laboratory validation of an event-specific real-time PCR method for the detection and quantification of Golden Rice 2 using a novel taxon-specific assay.

    Science.gov (United States)

    Jacchia, Sara; Nardini, Elena; Savini, Christian; Petrillo, Mauro; Angers-Loustau, Alexandre; Shim, Jung-Hyun; Trijatmiko, Kurniawan; Kreysa, Joachim; Mazzara, Marco

    2015-02-18

    In this study, we developed, optimized, and in-house validated a real-time PCR method for the event-specific detection and quantification of Golden Rice 2, a genetically modified rice with provitamin A in the grain. We optimized and evaluated the performance of the taxon (targeting rice Phospholipase D α2 gene)- and event (targeting the 3' insert-to-plant DNA junction)-specific assays that compose the method as independent modules, using haploid genome equivalents as unit of measurement. We verified the specificity of the two real-time PCR assays and determined their dynamic range, limit of quantification, limit of detection, and robustness. We also confirmed that the taxon-specific DNA sequence is present in single copy in the rice genome and verified its stability of amplification across 132 rice varieties. A relative quantification experiment evidenced the correct performance of the two assays when used in combination.

  20. Sample preparation methods for quantitative detection of DNA by molecular assays and marine biosensors

    International Nuclear Information System (INIS)

    Cox, Annie M.; Goodwin, Kelly D.

    2013-01-01

    Highlights: • DNA extraction methods affected measured qPCR target recovery. • Recovery and variability differed, sometimes by more than an order of magnitude. • SCODA did not offer significant improvement with PCR-inhibited seawater. • Aggressive lysis did appear to improve target recovery. • Reliable and affordable correction methods are needed for quantitative PCR. -- Abstract: The need for quantitative molecular methods is growing in environmental, food, and medical fields but is hindered by low and variable DNA extraction and by co-extraction of PCR inhibitors. DNA extracts from Enterococcus faecium, seawater, and seawater spiked with E. faecium and Vibrio parahaemolyticus were tested by qPCR for target recovery and inhibition. Conventional and novel methods were tested, including Synchronous Coefficient of Drag Alteration (SCODA) and lysis and purification systems used on an automated genetic sensor (the Environmental Sample Processor, ESP). Variable qPCR target recovery and inhibition were measured, significantly affecting target quantification. An aggressive lysis method that utilized chemical, enzymatic, and mechanical disruption enhanced target recovery compared to commercial kit protocols. SCODA purification did not show marked improvement over commercial spin columns. Overall, data suggested a general need to improve sample preparation and to accurately assess and account for DNA recovery and inhibition in qPCR applications

  1. An efficient method for DNA extraction from Cladosporioid fungi

    OpenAIRE

    Moslem, M.A.; Bahkali, A.H.; Abd-Elsalam, K.A.; Wit, de, P.J.G.M.

    2010-01-01

    We developed an efficient method for DNA extraction from Cladosporioid fungi, which are important fungal plant pathogens. The cell wall of Cladosporioid fungi is often melanized, which makes it difficult to extract DNA from their cells. In order to overcome this we grew these fungi for three days on agar plates and extracted DNA from mycelium mats after manual or electric homogenization. High-quality DNA was isolated, with an A260/A280 ratio ranging between 1.6 and 2.0. Isolated genomic DNA w...

  2. Quantification of Plasmodiophora brassicae Using a DNA-Based Soil Test Facilitates Sustainable Oilseed Rape Production

    OpenAIRE

    Ann-Charlotte Wallenhammar; Albin Gunnarson; Fredrik Hansson; Anders Jonsson

    2016-01-01

    Outbreaks of clubroot disease caused by the soil-borne obligate parasite Plasmodiophora brassicae are common in oilseed rape (OSR) in Sweden. A DNA-based soil testing service that identifies fields where P. brassicae poses a significant risk of clubroot infection is now commercially available. It was applied here in field surveys to monitor the prevalence of P. brassicae DNA in field soils intended for winter OSR production and winter OSR field experiments. In 2013 in Scania, prior to plantin...

  3. Direct infusion-SIM as fast and robust method for absolute protein quantification in complex samples

    Directory of Open Access Journals (Sweden)

    Christina Looße

    2015-06-01

    Full Text Available Relative and absolute quantification of proteins in biological and clinical samples are common approaches in proteomics. Until now, targeted protein quantification is mainly performed using a combination of HPLC-based peptide separation and selected reaction monitoring on triple quadrupole mass spectrometers. Here, we show for the first time the potential of absolute quantification using a direct infusion strategy combined with single ion monitoring (SIM on a Q Exactive mass spectrometer. By using complex membrane fractions of Escherichia coli, we absolutely quantified the recombinant expressed heterologous human cytochrome P450 monooxygenase 3A4 (CYP3A4 comparing direct infusion-SIM with conventional HPLC-SIM. Direct-infusion SIM revealed only 14.7% (±4.1 (s.e.m. deviation on average, compared to HPLC-SIM and a decreased processing and analysis time of 4.5 min (that could be further decreased to 30 s for a single sample in contrast to 65 min by the LC–MS method. Summarized, our simplified workflow using direct infusion-SIM provides a fast and robust method for quantification of proteins in complex protein mixtures.

  4. Identification of person and quantification of human DNA recovered from mosquitoes (Culicidae).

    Science.gov (United States)

    Curic, Goran; Hercog, Rajna; Vrselja, Zvonimir; Wagner, Jasenka

    2014-01-01

    Mosquitoes (Culicidae) are widespread insects and can be important in forensic context as a source of human DNA. In order to establish the quantity of human DNA in mosquitoes' gut after different post-feeding interval and for how long after taking a bloodmeal the human donor could be identified, 174 blood-engorged mosquitoes (subfamily Anophelinae and Culicinae) were captured, kept alive and sacrificed at 8h intervals. Human DNA was amplified using forensic PCR kits (Identifiler, MiniFiler, and Quantifiler). A full DNA profiles were obtained from all Culicinae mosquitoes (74/74) up to 48 h and profiling was successful up to 88 h after a bloodmeal. Duration of post-feeding interval had a significant negative effect on the possibility of obtaining a full profile (pfeeding interval. Culicinae mosquitoes are a suitable source of human DNA for forensic STR kits more than three days after a bloodmeal. Human DNA recovered from mosquito can be used for matching purposes and could be useful in revealing spatial and temporal relation of events that took place at the crime scene. Therefore, mosquitoes at the crime scene, dead or alive, could be a valuable piece of forensic evidence. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  5. Selection of Suitable DNA Extraction Methods for Genetically Modified Maize 3272, and Development and Evaluation of an Event-Specific Quantitative PCR Method for 3272.

    Science.gov (United States)

    Takabatake, Reona; Masubuchi, Tomoko; Futo, Satoshi; Minegishi, Yasutaka; Noguchi, Akio; Kondo, Kazunari; Teshima, Reiko; Kurashima, Takeyo; Mano, Junichi; Kitta, Kazumi

    2016-01-01

    A novel real-time PCR-based analytical method was developed for the event-specific quantification of a genetically modified (GM) maize, 3272. We first attempted to obtain genome DNA from this maize using a DNeasy Plant Maxi kit and a DNeasy Plant Mini kit, which have been widely utilized in our previous studies, but DNA extraction yields from 3272 were markedly lower than those from non-GM maize seeds. However, lowering of DNA extraction yields was not observed with GM quicker or Genomic-tip 20/G. We chose GM quicker for evaluation of the quantitative method. We prepared a standard plasmid for 3272 quantification. The conversion factor (Cf), which is required to calculate the amount of a genetically modified organism (GMO), was experimentally determined for two real-time PCR instruments, the Applied Biosystems 7900HT (the ABI 7900) and the Applied Biosystems 7500 (the ABI7500). The determined Cf values were 0.60 and 0.59 for the ABI 7900 and the ABI 7500, respectively. To evaluate the developed method, a blind test was conducted as part of an interlaboratory study. The trueness and precision were evaluated as the bias and reproducibility of the relative standard deviation (RSDr). The determined values were similar to those in our previous validation studies. The limit of quantitation for the method was estimated to be 0.5% or less, and we concluded that the developed method would be suitable and practical for detection and quantification of 3272.

  6. Non-linear methods for the quantification of cyclic motion

    OpenAIRE

    Quintana Duque, Juan Carlos

    2016-01-01

    Traditional methods of human motion analysis assume that fluctuations in cycles (e.g. gait motion) and repetitions (e.g. tennis shots) arise solely from noise. However, the fluctuations may have enough information to describe the properties of motion. Recently, the fluctuations in motion have been analysed based on the concepts of variability and stability, but they are not used uniformly. On the one hand, these concepts are often mixed in the existing literature, while on the other hand, the...

  7. A PRACTICAL METHOD FOR QUANTIFICATION OF PLEURAL EFFUSION BY USG

    OpenAIRE

    Swish Kumar; Dinesh Kumar; Suganita; Singh; Vijay Shankar; Rajeev; Ajay; Anjali

    2016-01-01

    OBJECTIVE The aim of this study is to find a correlation between pleural separation and amount of aspirated effusion. METHODS Total 20 adult patients with 25 effusions were taken into the study with chest x-ray showing homogeneous opacity in either one or both of the lung field, which was confirmed on USG. Only uncomplicated pleural effusion were taken into study. Effusion with septations or encysted effusion or pyothorax were excluded from the study. RESULTS...

  8. Rationalization of thermal injury quantification methods: application to skin burns.

    Science.gov (United States)

    Viglianti, Benjamin L; Dewhirst, Mark W; Abraham, John P; Gorman, John M; Sparrow, Eph M

    2014-08-01

    Classification of thermal injury is typically accomplished either through the use of an equivalent dosimetry method (equivalent minutes at 43 °C, CEM43 °C) or through a thermal-injury-damage metric (the Arrhenius method). For lower-temperature levels, the equivalent dosimetry approach is typically employed while higher-temperature applications are most often categorized by injury-damage calculations. The two methods derive from common thermodynamic/physical chemistry origins. To facilitate the development of the interrelationships between the two metrics, application is made to the case of skin burns. This thermal insult has been quantified by numerical simulation, and the extracted time-temperature results served for the evaluation of the respective characterizations. The simulations were performed for skin-surface exposure temperatures ranging from 60 to 90 °C, where each surface temperature was held constant for durations extending from 10 to 110 s. It was demonstrated that values of CEM43 at the basal layer of the skin were highly correlated with the depth of injury calculated from a thermal injury integral. Local values of CEM43 were connected to the local cell survival rate, and a correlating equation was developed relating CEM43 with the decrease in cell survival from 90% to 10%. Finally, it was shown that the cell survival/CEM43 relationship for the cases investigated here most closely aligns with isothermal exposure of tissue to temperatures of ~50 °C. Copyright © 2013 Elsevier Ltd and ISBI. All rights reserved.

  9. Alternative method for quantification of alfa-amylase activity.

    Science.gov (United States)

    Farias, D F; Carvalho, A F U; Oliveira, C C; Sousa, N M; Rocha-Bezerrra, L C B; Ferreira, P M P; Lima, G P G; Hissa, D C

    2010-05-01

    A modification of the sensitive agar diffusion method was developed for macro-scale determination of alfa-amylase. The proposed modifications lower costs with the utilisation of starch as substrate and agar as supporting medium. Thus, a standard curve was built using alfa-amylase solution from Aspergillus oryzae, with concentrations ranging from 2.4 to 7,500 U.mL-1. Clear radial diffusion zones were measured after 4 hours of incubation at 20 A degrees C. A linear relationship between the logarithm of enzyme activities and the area of clear zones was obtained. The method was validated by testing alpha-amylase from barley at the concentrations of 2.4; 60; 300 and 1,500 U.mL-1. The proposed method turned out to be simpler, faster, less expensive and able to determine on a macro-scale alpha-amylase over a wide range (2.4 to 7,500 U.mL-1) in scientific investigation as well as in teaching laboratory activities.

  10. Alternative method for quantification of alfa-amylase activity

    Directory of Open Access Journals (Sweden)

    DF. Farias

    Full Text Available A modification of the sensitive agar diffusion method was developed for macro-scale determination of alfa-amylase. The proposed modifications lower costs with the utilisation of starch as substrate and agar as supporting medium. Thus, a standard curve was built using alfa-amylase solution from Aspergillus oryzae, with concentrations ranging from 2.4 to 7,500 U.mL-1. Clear radial diffusion zones were measured after 4 hours of incubation at 20 °C. A linear relationship between the logarithm of enzyme activities and the area of clear zones was obtained. The method was validated by testing α-amylase from barley at the concentrations of 2.4; 60; 300 and 1,500 U.mL-1. The proposed method turned out to be simpler, faster, less expensive and able to determine on a macro-scale α-amylase over a wide range (2.4 to 7,500 U.mL-1 in scientific investigation as well as in teaching laboratory activities.

  11. Rapid and inexpensive method for isolating plasmid DNA

    International Nuclear Information System (INIS)

    Aljanabi, S. M.; Al-Awadi, S. J.; Al-Kazaz, A. A.; Baghdad Univ.

    1997-01-01

    A small-scale and economical method for isolating plasmid DNA from bacteria is described. The method provides DNA of suitable quality for most DNA manipulation techniques. This DNA can be used for restriction endonuclease digestion, southern blot hybridization, nick translation and end labeling of DNA probes, Polymerase Chain Reaction (PCR) -based techniques, transformation, DNA cycle-sequencing, and Chain-termination method for DNA sequencing. The entire procedure is adapted to 1.5 ml microfuge tubes and takes approximately 30 mins. The DNA isolated by this method has the same purity produced by CTAB and cesium chloride precipitation and purification procedures respectively. The two previous methods require many hours to obtain the final product and require the use of very expensive equipment as ultracentrifuge. This method is well suited for the isolation of plasmid DNA from a large number of bacterial samples and in a very short time and low cost in laboratories where chemicals, expensive equipment and finance are limited factors in conducting molecular research. (authors). 11refs. 11refs

  12. Development of an efficient fungal DNA extraction method to be used in random amplified polymorphic DNA-PCR analysis to differentiate cyclopiazonic acid mold producers.

    Science.gov (United States)

    Sánchez, Beatriz; Rodríguez, Mar; Casado, Eva M; Martín, Alberto; Córdoba, Juan J

    2008-12-01

    A variety of previously established mechanical and chemical treatments to achieve fungal cell lysis combined with a semiautomatic system operated by a vacuum pump were tested to obtain DNA extract to be directly used in randomly amplified polymorphic DNA (RAPD)-PCR to differentiate cyclopiazonic acid-producing and -nonproducing mold strains. A DNA extraction method that includes digestion with proteinase K and lyticase prior to using a mortar and pestle grinding and a semiautomatic vacuum system yielded DNA of high quality in all the fungal strains and species tested, at concentrations ranging from 17 to 89 ng/microl in 150 microl of the final DNA extract. Two microliters of DNA extracted with this method was directly used for RAPD-PCR using primer (GACA)4. Reproducible RAPD fingerprints showing high differences between producer and nonproducer strains were observed. These differences in the RAPD patterns did not differentiate all the strains tested in clusters by cyclopiazonic acid production but may be very useful to distinguish cyclopiazonic acid producer strains from nonproducer strains by a simple RAPD analysis. Thus, the DNA extracts obtained could be used directly without previous purification and quantification for RAPD analysis to differentiate cyclopiazonic acid producer from nonproducer mold strains. This combined analysis could be adaptable to other toxigenic fungal species to enable differentiation of toxigenic and non-toxigenic molds, a procedure of great interest in food safety.

  13. A simple and fast method for extraction and quantification of cryptophyte phycoerythrin

    OpenAIRE

    Thoisen, Christina; Hansen, Benni Winding; Nielsen, S?ren Laurentius

    2017-01-01

    The microalgal pigment phycoerythrin (PE) is of commercial interest as natural colorant in food and cosmetics, as well as fluoroprobes for laboratory analysis. Several methods for extraction and quantification of PE are available but they comprise typically various extraction buffers, repetitive freeze-thaw cycles and liquid nitrogen, making extraction procedures more complicated. A simple method for extraction of PE from cryptophytes is described using standard laboratory materials and equip...

  14. Standardless quantification approach of TXRF analysis using fundamental parameter method

    International Nuclear Information System (INIS)

    Szaloki, I.; Taniguchi, K.

    2000-01-01

    New standardless evaluation procedure based on the fundamental parameter method (FPM) has been developed for TXRF analysis. The theoretical calculation describes the relationship between characteristic intensities and the geometrical parameters of the excitation, detection system and the specimen parameters: size, thickness, angle of the excitation beam to the surface and the optical properties of the specimen holder. Most of the TXRF methods apply empirical calibration, which requires the application of special preparation technique. However, the characteristic lines of the specimen holder (Si Kα,β) present information from the local excitation and geometrical conditions on the substrate surface. On the basis of the theoretically calculation of the substrate characteristic intensity the excitation beam flux can be approximated. Taking into consideration the elements are in the specimen material a system of non-linear equation can be given involving the unknown concentration values and the geometrical and detection parameters. In order to solve this mathematical problem PASCAL software was written, which calculates the sample composition and the average sample thickness by gradient algorithm. Therefore, this quantitative estimation of the specimen composition requires neither external nor internal standard sample. For verification of the theoretical calculation and the numerical procedure, several experiments were carried out using mixed standard solution containing elements of K, Sc, V, Mn, Co and Cu in 0.1 - 10 ppm concentration range. (author)

  15. Quantification of emissions from knapsack sprayers: 'the weight method

    Science.gov (United States)

    Garcia-Santos, Glenda; Binder, Claudia R.

    2010-05-01

    Misuse of pesticides kill or seriously sicken thousands of people every year and poison the natural environment. Investigations of occupational and environmental risk have received considerable interest over the last decades. And yet, lack of staff and analytical equipments as well the costs of chemical analyses make difficult, if not impossible, the control of the pesticide contamination and residues in humans, air, water, and soils in developing countries. To assess emissions of pesticides (transport and deposition) during spray application and the risk for the human health and the environment, tracers can be useful tools. Uranine was used to quantify drift airborne and later deposition on the neighbouring field and clothes of the applicator after spraying with a knapsack sprayer in one of the biggest areas of potato production in Colombia. Keeping the same setup the amount of wet drift was measured by difference in the weight of high absorbent papers used to collect the tracer. Surprisingly this weight method (Weight-HAP) was able to explain 71% of the drift variance measured with the tracer. Therefore the weight method is presented as a suitable rapid low cost screening tool, complementary to toxicological tests, to assess air pollution, occupational and environmental exposure generated by the emissions from knapsack sprayers during pesticide application. This technique might be important in places were there is lack of analytical instruments.

  16. LC-MS-based quantification method for Achyranthes root saponins.

    Science.gov (United States)

    Kawahara, Yuki; Hoshino, Tatsuro; Morimoto, Hidetaka; Shinizu, Tomofumi; Narukawa, Yuji; Fuchino, Hiroyuki; Kawahara, Nobuo; Kiuchi, Fumiyuki

    2016-01-01

    A liquid chromatography mass spectrometry (LC-MS) method was developed for simultaneous quantitative analysis of Achyranthes root saponins: chikusetsusaponins IVa (1) and V (2), achyranthosides B (3), C (4), D (5), E (6), and G (7), sulfachyranthosides B (8) and D (9), and betavulgarosides II (10) and IV (11). Satisfactory separation of the saponins was achieved with the use of a volatile ion-pair reagent (dihexyl ammonium acetate) on a phenyl-hexylated silica gel column, and the amounts of saponins extracted under three different conditions were determined. When Achyranthes root was extracted with water at room temperature, achyranthosides B (3) and D (5) were the major saponins, and smaller amounts of other saponins (4, 6-11) were present. However, the amounts of chikusetsusaponins (1 and 2) were negligible. Under the condition to make a standard decoction of a Kampo formula, the major saponins were achyranthosides B (3), C (4), and D (5), and small amounts of chikusetsusaponins IVa (1) and V (2) appeared, whereas prolonged heating largely increased the amounts of chikusetsusaponins. This method can be used for quality control of Achyranthes root.

  17. Quantification of Circulating Free DNA as a Diagnostic Marker in Gall Bladder Cancer.

    Science.gov (United States)

    Kumari, Swati; Tewari, Shikha; Husain, Nuzhat; Agarwal, Akash; Pandey, Anshuman; Singhal, Ashish; Lohani, Mohtashim

    2017-01-01

    Gall bladder Carcinoma (GBC) is the fifth most common cancer of the digestive tract and frequently diagnosed in late stage of disease. Estimation of circulating free DNA (cfDNA) in serum has been applied as a "liquid biopsy" in several deep seated malignancies. Its value in diagnosis of gall bladder carcinoma has not been studied. The present study was designed to assess the role of cfDNA in the diagnosis of GBC and correlate levels with the TNM stage. Serum was collected from 34 patients with GBC and 39 age and sex matched controls including 22 cholecystitis and 17 healthy individuals. Serum cfDNA levels were measured through quantitative polymerase chain reaction (qPCR) by amplification of β-globin gene. Performance of the assay was calculated through the receiver operating characteristic (ROC) curve. The cfDNA level was significantly lower in healthy controls and cholecystitis (89.32 ± 59.76 ng/ml, 174.21 ± 99.93 ng/ml) compared to GBC (1245.91 ± 892.46 ng/ml, p = <0.001). The cfDNA level was significantly associated with TNM stage, lymph node involvement and jaundice (0.002, 0.027, and 0.041, respectively). Area under curve of ROC analysis for cancer group versus healthy and cholecystitis group was 1.00 and 0.983 with sensitivity of 100 %, 88.24 % and specificity of 100 % respectively. Quantitative analysis of cfDNA may distinguish cholecystitis and gall bladder carcinoma and may serve as new diagnostic, noninvasive marker adjunct to imaging for the diagnosis of GBC.

  18. [Doppler echocardiography of tricuspid insufficiency. Methods of quantification].

    Science.gov (United States)

    Loubeyre, C; Tribouilloy, C; Adam, M C; Mirode, A; Trojette, F; Lesbre, J P

    1994-01-01

    Evaluation of tricuspid incompetence has benefitted considerably from the development of Doppler ultrasound. In addition to direct analysis of the valves, which provides information about the mechanism involved, this method is able to provide an accurate evaluation, mainly through use of the Doppler mode. In addition to new criteria being evaluated (mainly the convergence zone of the regurgitant jet), some indices are recognised as good quantitative parameters: extension of the regurgitant jet into the right atrium, anterograde tricuspid flow, laminar nature of the regurgitant flow, analysis of the flow in the supra-hepatic veins, this is only semi-quantitative, since the calculation of the regurgitation fraction from the pulsed Doppler does not seem to be reliable; This accurate semi-quantitative evaluation is made possible by careful and consistent use of all the criteria available. The authors set out to discuss the value of the various evaluation criteria mentioned in the literature and try to define a practical approach.

  19. Validation of a spectrophotometric method for quantification of carboxyhemoglobin.

    Science.gov (United States)

    Luchini, Paulo D; Leyton, Jaime F; Strombech, Maria de Lourdes C; Ponce, Julio C; Jesus, Maria das Graças S; Leyton, Vilma

    2009-10-01

    The measurement of carboxyhemoglobin (COHb) levels in blood is a valuable procedure to confirm exposure to carbon monoxide (CO) either for forensic or occupational matters. A previously described method using spectrophotometric readings at 420 and 432 nm after reduction of oxyhemoglobin (O(2)Hb) and methemoglobin with sodium hydrosulfite solution leads to an exponential curve. This curve, used with pre-established factors, serves well for lower concentrations (1-7%) or for high concentrations (> 20%) but very rarely for both. The authors have observed that small variations on the previously described factors F1, F2, and F3, obtained from readings for 100% COHb and 100% O(2)Hb, turn into significant changes in COHb% results and propose that these factors should be determined every time COHb is measured by reading CO and O(2) saturated samples. This practice leads to an increase in accuracy and precision.

  20. Powder X-ray diffraction method for the quantification of cocrystals in the crystallization mixture.

    Science.gov (United States)

    Padrela, Luis; de Azevedo, Edmundo Gomes; Velaga, Sitaram P

    2012-08-01

    The solid state purity of cocrystals critically affects their performance. Thus, it is important to accurately quantify the purity of cocrystals in the final crystallization product. The aim of this study was to develop a powder X-ray diffraction (PXRD) quantification method for investigating the purity of cocrystals. The method developed was employed to study the formation of indomethacin-saccharin (IND-SAC) cocrystals by mechanochemical methods. Pure IND-SAC cocrystals were geometrically mixed with 1:1 w/w mixture of indomethacin/saccharin in various proportions. An accurately measured amount (550 mg) of the mixture was used for the PXRD measurements. The most intense, non-overlapping, characteristic diffraction peak of IND-SAC was used to construct the calibration curve in the range 0-100% (w/w). This calibration model was validated and used to monitor the formation of IND-SAC cocrystals by liquid-assisted grinding (LAG). The IND-SAC cocrystal calibration curve showed excellent linearity (R(2) = 0.9996) over the entire concentration range, displaying limit of detection (LOD) and limit of quantification (LOQ) values of 1.23% (w/w) and 3.74% (w/w), respectively. Validation results showed excellent correlations between actual and predicted concentrations of IND-SAC cocrystals (R(2) = 0.9981). The accuracy and reliability of the PXRD quantification method depend on the methods of sample preparation and handling. The crystallinity of the IND-SAC cocrystals was higher when larger amounts of methanol were used in the LAG method. The PXRD quantification method is suitable and reliable for verifying the purity of cocrystals in the final crystallization product.

  1. A simple and fast method for extraction and quantification of cryptophyte phycoerythrin.

    Science.gov (United States)

    Thoisen, Christina; Hansen, Benni Winding; Nielsen, Søren Laurentius

    2017-01-01

    The microalgal pigment phycoerythrin (PE) is of commercial interest as natural colorant in food and cosmetics, as well as fluoroprobes for laboratory analysis. Several methods for extraction and quantification of PE are available but they comprise typically various extraction buffers, repetitive freeze-thaw cycles and liquid nitrogen, making extraction procedures more complicated. A simple method for extraction of PE from cryptophytes is described using standard laboratory materials and equipment. The cryptophyte cells on the filters were disrupted at -80 °C and added phosphate buffer for extraction at 4 °C followed by absorbance measurement. The cryptophyte Rhodomonas salina was used as a model organism. •Simple method for extraction and quantification of phycoerythrin from cryptophytes.•Minimal usage of equipment and chemicals, and low labor costs.•Applicable for industrial and biological purposes.

  2. A simple and fast method for extraction and quantification of cryptophyte phycoerythrin

    DEFF Research Database (Denmark)

    Thoisen, Christina Vinum; Hansen, Benni Winding; Nielsen, Søren Laurentius

    2017-01-01

    The microalgal pigment phycoerythrin (PE) is of commercial interest as natural colorant in food and cosmetics, as well as fluoroprobes for laboratory analysis. Several methods for extraction and quantification of PE are available but they comprise typically various extraction buffers, repetitive...... freeze-thaw cycles and liquid nitrogen, making extraction procedures more complicated. A simple method for extraction of PE from cryptophytes is described using standard laboratory materials and equipment. Filters with the cryptophyte were frozen (−80 °C) and added phosphate buffer for extraction at 4 °C...... followed by absorbance measurement. The cryptophyte Rhodomonas salina was used as a model organism. •Simple method for extraction and quantification of phycoerythrin from cryptophytes. •Minimal usage of equipment and chemicals, and low labor costs. •Applicable for industrial and biological purposes....

  3. Quantification of cellular viability for the MTT method

    International Nuclear Information System (INIS)

    Altanes, M.

    2001-01-01

    In the last years, the scientists have been given to the task of finding new biomaterials whose biocompatibility with the human body allow them to substitute parts of the organism, as the bones and the junctures and also that they are little rejected. In the following work it was evaluated and quantified by the method of the MTT, a colorimetric, quick, simple and economic technical, the possible citohepatic effect of a watery extract of a biomaterials of polymeric origin (acrilamide and metacrilic acid) obtained for technical of gamma irradiation, consistent in culture medium 199 and veal serum, in cells Vero. In order to compare the answer of the analysis material, they were used the controls negative (polyethylene of high molecular weight), and positive (Sulphate of Streptomycin), just as they indicate it the established norms, being achieved the waited result of each one of them. In the observation made to the optic microscope, after 24 and 48 hours of contact between the cells and the extract of the analysis material, they were not perceived indicative of damage or cellular lysis, or morphologic changes in the structure of cells that it indicated us the possible cytotoxic effect of this biomaterials, therefore the qualitative analysis was not enough for the determination of the cytotoxic effect of the analysis sample

  4. Mathematical methods for quantification and comparison of dissolution testing data.

    Science.gov (United States)

    Vranić, Edina; Mehmedagić, Aida; Hadzović, Sabira

    2002-12-01

    In recent years, drug release/dissolution from solid dosage forms has been the subject of intense and profitable scientific developments. Whenever a new solid dosage form is developed or produced, it is necessary to ensure that drug dissolution occurs in an appropriate manner. The pharmaceutical industry and the registration authorities do focus, nowadays, on drug dissolution studies. The quantitative analysis of the values obtained in dissolution/release tests is easier when mathematical formulas that express the dissolution results as a function of some of the dosage forms characteristics are used. This work discusses the analysis of data obtained for dissolution profiles under different media pH conditions using mathematical methods of analysis described by Moore and Flanner. These authors have described difference factor (f1) and similarity factor (f2), which can be used to characterise drug dissolution/release profiles. In this work we have used these formulas for evaluation of dissolution profiles of the conventional tablets in different pH of dissolution medium (range of physiological variations).

  5. Quantification and persistence of recombinant DNA of Roundup Ready corn and soybean in rotation.

    Science.gov (United States)

    Lerat, Sylvain; Gulden, Robert H; Hart, Miranda M; Powell, Jeff R; England, Laura S; Pauls, K Peter; Swanton, Clarence J; Klironomos, John N; Trevors, Jack T

    2007-12-12

    The presence of the recombinant cp4 epsps gene from Roundup Ready (RR) corn and RR soybean was quantified using real-time PCR in soil samples from a field experiment growing RR and conventional corn and soybean in rotation. RR corn and RR soybean cp4 epsps persisted in soil for up to 1 year after seeding. The concentration of recombinant DNA in soil peaked in July and August in RR corn and RR soybean plots, respectively. A small fraction of soil samples from plots seeded with conventional crops contained recombinant DNA, suggesting transgene dispersal by means of natural process or agricultural practices. This research will aid in the understanding of the persistence of recombinant DNA in agricultural cropping systems.

  6. Quantification of immobilized Candida antarctica lipase B (CALB) using ICP-AES combined with Bradford method.

    Science.gov (United States)

    Nicolás, Paula; Lassalle, Verónica L; Ferreira, María L

    2017-02-01

    The aim of this manuscript was to study the application of a new method of protein quantification in Candida antarctica lipase B commercial solutions. Error sources associated to the traditional Bradford technique were demonstrated. Eight biocatalysts based on C. antarctica lipase B (CALB) immobilized onto magnetite nanoparticles were used. Magnetite nanoparticles were coated with chitosan (CHIT) and modified with glutaraldehyde (GLUT) and aminopropyltriethoxysilane (APTS). Later, CALB was adsorbed on the modified support. The proposed novel protein quantification method included the determination of sulfur (from protein in CALB solution) by means of Atomic Emission by Inductive Coupling Plasma (AE-ICP). Four different protocols were applied combining AE-ICP and classical Bradford assays, besides Carbon, Hydrogen and Nitrogen (CHN) analysis. The calculated error in protein content using the "classic" Bradford method with bovine serum albumin as standard ranged from 400 to 1200% when protein in CALB solution was quantified. These errors were calculated considering as "true protein content values" the results of the amount of immobilized protein obtained with the improved method. The optimum quantification procedure involved the combination of Bradford method, ICP and CHN analysis. Copyright © 2016 Elsevier Inc. All rights reserved.

  7. Quantification of Plasmodiophora brassicae Using a DNA-Based Soil Test Facilitates Sustainable Oilseed Rape Production.

    Science.gov (United States)

    Wallenhammar, Ann-Charlotte; Gunnarson, Albin; Hansson, Fredrik; Jonsson, Anders

    2016-04-22

    Outbreaks of clubroot disease caused by the soil-borne obligate parasite Plasmodiophora brassicae are common in oilseed rape (OSR) in Sweden. A DNA-based soil testing service that identifies fields where P. brassicae poses a significant risk of clubroot infection is now commercially available. It was applied here in field surveys to monitor the prevalence of P. brassicae DNA in field soils intended for winter OSR production and winter OSR field experiments. In 2013 in Scania, prior to planting, P. brassicae DNA was detected in 60% of 45 fields on 10 of 18 farms. In 2014, P. brassicae DNA was detected in 44% of 59 fields in 14 of 36 farms, in the main winter OSR producing region in southern Sweden. P. brassicae was present indicative of a risk for >10% yield loss with susceptible cultivars (>1300 DNA copies g soil(-1)) in 47% and 44% of fields in 2013 and 2014 respectively. Furthermore, P. brassicae DNA was indicative of sites at risk of complete crop failure if susceptible cultivars were grown (>50 000 copies g(-1) soil) in 14% and 8% of fields in 2013 and 2014, respectively. A survey of all fields at Lanna research station in western Sweden showed that P. brassicae was spread throughout the farm, as only three of the fields (20%) showed infection levels below the detection limit for P.brassicae DNA, while the level was >50,000 DNA copies g(-1) soil in 20% of the fields. Soil-borne spread is of critical importance and soil scraped off footwear showed levels of up to 682 million spores g(-1) soil. Soil testing is an important tool for determining the presence of P. brassicae and providing an indication of potential yield loss, e.g., in advisory work on planning for a sustainable OSR crop rotation. This soil test is gaining acceptance as a tool that increases the likelihood of success in precision agriculture and in applied research conducted in commercial oilseed fields and at research stations. The present application highlights the importance of prevention of

  8. Quantification of Plasmodiophora brassicae Using a DNA-Based Soil Test Facilitates Sustainable Oilseed Rape Production

    Directory of Open Access Journals (Sweden)

    Ann-Charlotte Wallenhammar

    2016-04-01

    Full Text Available Outbreaks of clubroot disease caused by the soil-borne obligate parasite Plasmodiophora brassicae are common in oilseed rape (OSR in Sweden. A DNA-based soil testing service that identifies fields where P. brassicae poses a significant risk of clubroot infection is now commercially available. It was applied here in field surveys to monitor the prevalence of P. brassicae DNA in field soils intended for winter OSR production and winter OSR field experiments. In 2013 in Scania, prior to planting, P. brassicae DNA was detected in 60% of 45 fields on 10 of 18 farms. In 2014, P. brassicae DNA was detected in 44% of 59 fields in 14 of 36 farms, in the main winter OSR producing region in southern Sweden. P. brassicae was present indicative of a risk for >10% yield loss with susceptible cultivars (>1300 DNA copies g soil−1 in 47% and 44% of fields in 2013 and 2014 respectively. Furthermore, P. brassicae DNA was indicative of sites at risk of complete crop failure if susceptible cultivars were grown (>50 000 copies g−1 soil in 14% and 8% of fields in 2013 and 2014, respectively. A survey of all fields at Lanna research station in western Sweden showed that P. brassicae was spread throughout the farm, as only three of the fields (20% showed infection levels below the detection limit for P.brassicae DNA, while the level was >50,000 DNA copies g−1 soil in 20% of the fields. Soil-borne spread is of critical importance and soil scraped off footwear showed levels of up to 682 million spores g−1 soil. Soil testing is an important tool for determining the presence of P. brassicae and providing an indication of potential yield loss, e.g., in advisory work on planning for a sustainable OSR crop rotation. This soil test is gaining acceptance as a tool that increases the likelihood of success in precision agriculture and in applied research conducted in commercial oilseed fields and at research stations. The present application highlights the importance of

  9. Comparison of quantification methods for the analysis of polychlorinated alkanes using electron capture negative ionization mass spectrometry.

    NARCIS (Netherlands)

    Rusina, T.; Korytar, P.; de Boer, J.

    2011-01-01

    Four quantification methods for short-chain chlorinated paraffins (SCCPs) or polychlorinated alkanes (PCAs) using gas chromatography electron capture negative ionisation low resolution mass spectrometry (GC-ECNI-LRMS) were investigated. The method based on visual comparison of congener group

  10. Comparison of quantification methods for the analysis of polychlorinated alkanes using electron capture negative ionisation mass spectrometry

    NARCIS (Netherlands)

    Rusina, T.; Korytar, P.; Boer, de J.

    2011-01-01

    Four quantification methods for short-chain chlorinated paraffins (SCCPs) or polychlorinated alkanes (PCAs) using gas chromatography electron capture negative ionisation low resolution mass spectrometry (GC-ECNI-LRMS) were investigated. The method based on visual comparison of congener group

  11. Highly efficient DNA extraction method from skeletal remains

    Directory of Open Access Journals (Sweden)

    Irena Zupanič Pajnič

    2011-03-01

    Full Text Available Background: This paper precisely describes the method of DNA extraction developed to acquire high quality DNA from the Second World War skeletal remains. The same method is also used for molecular genetic identification of unknown decomposed bodies in routine forensic casework where only bones and teeth are suitable for DNA typing. We analysed 109 bones and two teeth from WWII mass graves in Slovenia. Methods: We cleaned the bones and teeth, removed surface contaminants and ground the bones into powder, using liquid nitrogen . Prior to isolating the DNA in parallel using the BioRobot EZ1 (Qiagen, the powder was decalcified for three days. The nuclear DNA of the samples were quantified by real-time PCR method. We acquired autosomal genetic profiles and Y-chromosome haplotypes of the bones and teeth with PCR amplification of microsatellites, and mtDNA haplotypes 99. For the purpose of traceability in the event of contamination, we prepared elimination data bases including genetic profiles of the nuclear and mtDNA of all persons who have been in touch with the skeletal remains in any way. Results: We extracted up to 55 ng DNA/g of the teeth, up to 100 ng DNA/g of the femurs, up to 30 ng DNA/g of the tibias and up to 0.5 ng DNA/g of the humerus. The typing of autosomal and YSTR loci was successful in all of the teeth, in 98 % dekalof the femurs, and in 75 % to 81 % of the tibias and humerus. The typing of mtDNA was successful in all of the teeth, and in 96 % to 98 % of the bones. Conclusions: We managed to obtain nuclear DNA for successful STR typing from skeletal remains that were over 60 years old . The method of DNA extraction described here has proved to be highly efficient. We obtained 0.8 to 100 ng DNA/g of teeth or bones and complete genetic profiles of autosomal DNA, Y-STR haplotypes, and mtDNA haplotypes from only 0.5g bone and teeth samples.

  12. Comparing transformation methods for DNA microarray data

    NARCIS (Netherlands)

    Thygesen, Helene H.; Zwinderman, Aeilko H.

    2004-01-01

    Background: When DNA microarray data are used for gene clustering, genotype/phenotype correlation studies, or tissue classification the signal intensities are usually transformed and normalized in several steps in order to improve comparability and signal/noise ratio. These steps may include

  13. Quantification of bovine leukemia virus proviral DNA using a low-cost real-time polymerase chain reaction.

    Science.gov (United States)

    Petersen, M I; Alvarez, I; Trono, K G; Jaworski, J P

    2018-04-11

    The detection of bovine leukemia virus (BLV) proviral DNA is an important tool to address whether an animal is infected with BLV. Compared with serological assays, real-time PCR accounts for greater sensitivity and can serve as a confirmatory test for the clarification of inconclusive or discordant serological test results. However, the high cost related to real-time PCR assays has limited their systematic inclusion in BLV surveillance and eradication programs. The aim of the present study was to validate a low-cost quantitative real-time PCR. Interestingly, by using SYBR Green detection dye, we were able to reduce the cost of a single reaction by a factor of 5 compared with most common assays based on the use of fluorogenic probes (i.e., TaqMan technology). This approach allowed a highly sensitive and specific detection and quantification of BLV proviral DNA from purified peripheral blood leukocytes and a milk matrix. Due to its simplicity and low cost, our in-house BLV SYBR quantitative real-time PCR might be used either as a screening or as a confirmatory test in BLV control programs. Copyright © 2018 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  14. A Universal Fast Colorimetric Method for DNA Signal Detection with DNA Strand Displacement and Gold Nanoparticles

    Directory of Open Access Journals (Sweden)

    Xin Li

    2015-01-01

    Full Text Available DNA or gene signal detection is of great significance in many fields including medical examination, intracellular molecular monitoring, and gene disease signal diagnosis, but detection of DNA or gene signals in a low concentration with instant visual results remains a challenge. In this work, a universal fast and visual colorimetric detection method for DNA signals is proposed. Specifically, a DNA signal amplification “circuit” based on DNA strand displacement is firstly designed to amplify the target DNA signals, and then thiol modified hairpin DNA strands and gold nanoparticles are used to make signal detection results visualized in a colorimetric manner. If the target DNA signal exists, the gold nanoparticles aggregate and settle down with color changing from dark red to grey quickly; otherwise, the gold nanoparticles’ colloids remain stable in dark red. The proposed method provides a novel way to detect quickly DNA or gene signals in low concentrations with instant visual results. When applied in real-life, it may provide a universal colorimetric method for gene disease signal diagnosis.

  15. Development of real-time PCR method for the detection and the quantification of a new endogenous reference gene in sugar beet "Beta vulgaris L.": GMO application.

    Science.gov (United States)

    Chaouachi, Maher; Alaya, Akram; Ali, Imen Ben Haj; Hafsa, Ahmed Ben; Nabi, Nesrine; Bérard, Aurélie; Romaniuk, Marcel; Skhiri, Fethia; Saïd, Khaled

    2013-01-01

    KEY MESSAGE : Here, we describe a new developed quantitative real-time PCR method for the detection and quantification of a new specific endogenous reference gene used in GMO analysis. The key requirement of this study was the identification of a new reference gene used for the differentiation of the four genomic sections of the sugar beet (Beta vulgaris L.) (Beta, Corrollinae, Nanae and Procumbentes) suitable for quantification of genetically modified sugar beet. A specific qualitative polymerase chain reaction (PCR) assay was designed to detect the sugar beet amplifying a region of the adenylate transporter (ant) gene only from the species of the genomic section I of the genus Beta (cultivated and wild relatives) and showing negative PCR results for 7 species of the 3 other sections, 8 related species and 20 non-sugar beet plants. The sensitivity of the assay was 15 haploid genome copies (HGC). A quantitative real-time polymerase chain reaction (QRT-PCR) assay was also performed, having high linearity (R (2) > 0.994) over sugar beet standard concentrations ranging from 20,000 to 10 HGC of the sugar beet DNA per PCR. The QRT-PCR assay described in this study was specific and more sensitive for sugar beet quantification compared to the validated test previously reported in the European Reference Laboratory. This assay is suitable for GMO quantification in routine analysis from a wide variety of matrices.

  16. Impact of two different commercial DNA extraction methods on BK virus viral load

    Directory of Open Access Journals (Sweden)

    Massimiliano Bergallo

    2016-03-01

    Full Text Available Background and aim: BK virus, a member of human polyomavirus family, is a worldwide distributed virus characterized by a seroprevalence rate of 70-90% in adult population. Monitoring of viral replication is made by evaluation of BK DNA by quantitative polymerase chain reaction. Many different methods can be applied for extraction of nucleic acid from several specimens. The aim of this study was to assess the impact of two different DNA extraction procedure on BK viral load. Materials and methods: DNA extraction procedure including the Nuclisens easyMAG platform (bioMerieux, Marcy l’Etoile, France and manual QIAGEN extraction (QIAGEN Hilden, Germany. BK DNA quantification was performed by Real Time TaqMan PCR using a commercial kit. Result and discussion: The samples capacity, cost and time spent were compared for both systems. In conclusion our results demonstrate that automated nucleic acid extraction method using Nuclisense easyMAG was superior to manual protocol (QIAGEN Blood Mini kit, for the extraction of BK virus from serum and urine specimens.

  17. QUANTIFICATION AND BIOREMEDIATION OF ENVIRONMENTAL SAMPLES BY DEVELOPING A NOVEL AND EFFICIENT METHOD

    Directory of Open Access Journals (Sweden)

    Mohammad Osama

    2014-06-01

    Full Text Available Pleurotus ostreatus, a white rot fungus, is capable of bioremediating a wide range of organic contaminants including Polycyclic Aromatic Hydrocarbons (PAHs. Ergosterol is produced by living fungal biomass and used as a measure of fungal biomass. The first part of this work deals with the extraction and quantification of PAHs from contaminated sediments by Lipid Extraction Method (LEM. The second part consists of the development of a novel extraction method (Ergosterol Extraction Method (EEM, quantification and bioremediation. The novelty of this method is the simultaneously extraction and quantification of two different types of compounds, sterol (ergosterol and PAHs and is more efficient than LEM. EEM has been successful in extracting ergosterol from the fungus grown on barley in the concentrations of 17.5-39.94 µg g-1 ergosterol and the PAHs are much more quantified in numbers and amounts as compared to LEM. In addition, cholesterol usually found in animals, has also been detected in the fungus, P. ostreatus at easily detectable levels.

  18. Quantification of Leishmania infantum DNA in the bone marrow, lymph node and spleen of dogs.

    Science.gov (United States)

    Ramos, Rafael Antonio Nascimento; Ramos, Carlos Alberto do Nascimento; Santos, Edna Michelly de Sá; de Araújo, Flábio Ribeiro; de Carvalho, Gílcia Aparecida; Faustino, Maria Aparecida da Gloria; Alves, Leucio Câmara

    2013-01-01

    The aim of the present study was to quantify the parasite load of Leishmania infantum in dogs using real-time PCR (qPCR). Bone marrow, lymph node and spleen samples were taken from 24 dogs serologically positive for L. infantum that had been put down by the official epidemiological surveillance service. According to the clinical signs the dogs were classified as asymptomatic or symptomatic. After DNA extraction, the samples were subjected to qPCR to detect and quantify L. infantum DNA. Out of the 24 dogs, 12.5% (3/24) were classified as asymptomatic and 87.5% (21/24) as symptomatic. Real-time PCR detected L. infantum DNA in all the animals, in at least one biological sample. In particular, 100% of bone marrow and lymph node scored positive, whereas in spleen, the presence of DNA was detected in 95.9% (23/24). In addition, out of 24 animals, 15 were microscopically positive to amastigote forms of L. infantum in bone marrow. No statistical significant difference was found in the overall mean quantity of DNA among the different biological samples (P = 0.518). Considering each organ separately, there was 100% positivity in bone marrow and lymph nodes, while among the spleen samples, 95.9% (23/24) were positive. Regarding the different clinical groups, the overall mean parasite load varied significantly (P = 0.022). According to the results obtained, it was not possible determine which biological sample was most suitable tissue for the diagnosis, based only on the parasite load. Therefore, other characteristics such as convenience and easily of obtaining samples should be taken into consideration.

  19. Quantification of complex DNA damage by ionising radiation. An experimental and theoretical approach

    International Nuclear Information System (INIS)

    Fulford, J.

    2000-05-01

    Ionising radiation potentially produces a broad spectrum of damage in DNA including single and double strand breaks (ssb and dsb) and base damages. It has been hypothesised that sites of complex damage within cellular DNA have particular biological significance due to an associated decreased efficiency in repair. The aim of this study is to gain further understanding of the formation of complex DNA damage. Irradiations of plasmid DNA illustrate that an increase in ionising density of the radiation results in a decrease in ssb yields/Gy but an increase in dsb per ssb, indicative of an increase in the number of complex damage sites per simple isolated damage site. As the mechanism for damage formation shifts from purely indirect at low scavenging capacities to a significant proportion of direct at higher scavenging capacities the proportion of complex damage increases. Comparisons with the yields of ssb and dsb simulated by Monte-Carlo calculations for Al K USX and α-particles also indicate this correspondence. The ionisation density of low energy, secondary electrons produced by photons was assessed experimentally from the dependence of the yield of OH radicals escaping intra-track recombination on photon energy. As energy decreases the OH radical yield initially decreases reflecting an increased ionisation density. However, with further decrease in photon energy the yield of OH radicals increases in line with theoretical calculations. Base damage yields were determined for low and high ionising density radiation over a range of scavenging capacities. As scavenging capacity increases the base damage: ssb ratios increases implying a contribution from electrons to base damage. It is proposed that base damage contributes to DNA damage complexity. Complex damage analysis reveals that at cell mimetic scavenging capacities, 23% and 72% of ssb have an additional spatially close damage site following γ-ray and α-particle irradiation respectively. (author)

  20. A method for the 3-D quantification of bridging ligaments during crack propagation

    International Nuclear Information System (INIS)

    Babout, L.; Janaszewski, M.; Marrow, T.J.; Withers, P.J.

    2011-01-01

    This letter shows how a hole-closing algorithm can be used to identify and quantify crack-bridging ligaments from a sequence of X-ray tomography images of intergranular stress corrosion cracking. This allows automatic quantification of the evolution of bridging ligaments through the crack propagation sequence providing fracture mechanics insight previously unobtainable from fractography. The method may also be applied to other three-dimensional materials science problems, such as closing walls in foams.

  1. DNA extraction methods for panbacterial and panfungal PCR detection in intraocular fluids.

    Science.gov (United States)

    Mazoteras, Paloma; Bispo, Paulo José Martins; Höfling-Lima, Ana Luisa; Casaroli-Marano, Ricardo P

    2015-07-01

    Three different methods of DNA extraction from intraocular fluids were compared with subsequent detection for bacterial and fungal DNA by universal PCR amplification. Three DNA extraction methods, from aqueous and vitreous humors, were evaluated to compare their relative efficiency. Bacterial (Gram positive and negative) and fungal strains were used in this study: Escherichia coli, Staphylococcus epidermidis and Candida albicans. The quality, quantification, and detection limit for DNA extraction and PCR amplification were analyzed. Validation procedures for 13 aqueous humor and 14 vitreous samples, from 20 patients with clinically suspected endophthalmitis were carried out. The column-based extraction method was the most time-effective, achieving DNA detection limits ≥10(2) and 10(3 )CFU/100 µL for bacteria and fungi, respectively. PCR amplification detected 100 fg, 1 pg and 10 pg of genomic DNA of E. coli, S. epidermidis and C. albicans respectively. PCR detected 90.0% of the causative agents from 27 intraocular samples collected from 20 patients with clinically suspected endophthalmitis, while standard microbiological techniques could detect only 60.0%. The most frequently found organisms were Streptococcus spp. in 38.9% (n = 7) of patients and Staphylococcus spp. found in 22.2% (n = 4). The column-based extraction method for very small inocula in small volume samples (50-100 µL) of aqueous and/or vitreous humors allowed PCR amplification in all samples with sufficient quality for subsequent sequencing and identification of the microorganism in the majority of them.

  2. Droplet digital PCR (ddPCR) vs quantitative real-time PCR (qPCR) approach for detection and quantification of Merkel cell polyomavirus (MCPyV) DNA in formalin fixed paraffin embedded (FFPE) cutaneous biopsies.

    Science.gov (United States)

    Arvia, Rosaria; Sollai, Mauro; Pierucci, Federica; Urso, Carmelo; Massi, Daniela; Zakrzewska, Krystyna

    2017-08-01

    Merkel cell polyomavirus (MCPyV) is associated with Merkel cell carcinoma and high viral load in the skin was proposed as a risk factor for the occurrence of this tumour. MCPyV DNA was detected, with lower frequency, in different skin cancers but since the viral load was usually low, the real prevalence of viral DNA could be underestimated. To evaluate the performance of two assays (qPCR and ddPCR) for MCPyV detection and quantification in formalin fixed paraffin embedded (FFPE) tissue samples. Both assays were designed to simultaneous detection and quantification of both MCPyV as well as house-keeping DNA in clinical samples. The performance of MCPyV quantification was investigated using serial dilutions of cloned target DNA. We also evaluated the applicability of both tests for the analysis of 76 FFPE cutaneous biopsies. The two approaches resulted equivalent with regard to the reproducibility and repeatability and showed a high degree of linearity in the dynamic range tested in the present study. Moreover, qPCR was able to quantify ≥10 5 copies per reaction, while the upper limit of ddPCR was 10 4 copies. There was not significant difference between viral load measured by the two methods The detection limit of both tests was 0,15 copies per reaction, however, the number of positive samples obtained by ddPCR was higher than that obtained by qPCR (45% and 37% respectively). The ddPCR represents a better method for detection of MCPyV in FFPE biopsies, mostly these containing low copies number of viral genome. Copyright © 2017 Elsevier B.V. All rights reserved.

  3. A Rapid Protocol of Crude RNA/DNA Extraction for RT-qPCR Detection and Quantification of 'Candidatus Phytoplasma prunorum'.

    Science.gov (United States)

    Minguzzi, Stefano; Terlizzi, Federica; Lanzoni, Chiara; Poggi Pollini, Carlo; Ratti, Claudio

    2016-01-01

    Many efforts have been made to develop a rapid and sensitive method for phytoplasma and virus detection. Taking our cue from previous works, different rapid sample preparation methods have been tested and applied to Candidatus Phytoplasma prunorum ('Ca. P. prunorum') detection by RT-qPCR. A duplex RT-qPCR has been optimized using the crude sap as a template to simultaneously amplify a fragment of 16S rRNA of the pathogen and 18S rRNA of the host plant. The specific plant 18S rRNA internal control allows comparison and relative quantification of samples. A comparison between DNA and RNA contribution to qPCR detection is provided, showing higher contribution of the latter. The method presented here has been validated on more than a hundred samples of apricot, plum and peach trees. Since 2013, this method has been successfully applied to monitor 'Ca. P. prunorum' infections in field and nursery. A triplex RT-qPCR assay has also been optimized to simultaneously detect 'Ca. P. prunorum' and Plum pox virus (PPV) in Prunus.

  4. An ImageJ-based algorithm for a semi-automated method for microscopic image enhancement and DNA repair foci counting

    International Nuclear Information System (INIS)

    Klokov, D.; Suppiah, R.

    2015-01-01

    Proper evaluation of the health risks of low-dose ionizing radiation exposure heavily relies on the ability to accurately measure very low levels of DNA damage in cells. One of the most sensitive methods for measuring DNA damage levels is the quantification of DNA repair foci that consist of macromolecular aggregates of DNA repair proteins, such as γH2AX and 53BP1, forming around individual DNA double-strand breaks. They can be quantified using immunofluorescence microscopy and are widely used as markers of DNA double-strand breaks. However this quantification, if performed manually, may be very tedious and prone to inter-individual bias. Low-dose radiation studies are especially sensitive to this potential bias due to a very low magnitude of the effects anticipated. Therefore, we designed and validated an algorithm for the semi-automated processing of microscopic images and quantification of DNA repair foci. The algorithm uses ImageJ, a freely available image analysis software that is customizable to individual cellular properties or experimental conditions. We validated the algorithm using immunolabeled 53BP1 and γH2AX in normal human fibroblast AG01522 cells under both normal and irradiated conditions. This method is easy to learn, can be used by nontrained personnel, and can help avoiding discrepancies in inter-laboratory comparison studies examining the effects of low-dose radiation. (author)

  5. An ImageJ-based algorithm for a semi-automated method for microscopic image enhancement and DNA repair foci counting

    Energy Technology Data Exchange (ETDEWEB)

    Klokov, D., E-mail: dmitry.klokov@cnl.ca [Canadian Nuclear Laboratories, Chalk River, Ontario (Canada); Suppiah, R. [Queen' s Univ., Dept. of Biomedical and Molecular Sciences, Kingston, Ontario (Canada)

    2015-06-15

    Proper evaluation of the health risks of low-dose ionizing radiation exposure heavily relies on the ability to accurately measure very low levels of DNA damage in cells. One of the most sensitive methods for measuring DNA damage levels is the quantification of DNA repair foci that consist of macromolecular aggregates of DNA repair proteins, such as γH2AX and 53BP1, forming around individual DNA double-strand breaks. They can be quantified using immunofluorescence microscopy and are widely used as markers of DNA double-strand breaks. However this quantification, if performed manually, may be very tedious and prone to inter-individual bias. Low-dose radiation studies are especially sensitive to this potential bias due to a very low magnitude of the effects anticipated. Therefore, we designed and validated an algorithm for the semi-automated processing of microscopic images and quantification of DNA repair foci. The algorithm uses ImageJ, a freely available image analysis software that is customizable to individual cellular properties or experimental conditions. We validated the algorithm using immunolabeled 53BP1 and γH2AX in normal human fibroblast AG01522 cells under both normal and irradiated conditions. This method is easy to learn, can be used by nontrained personnel, and can help avoiding discrepancies in inter-laboratory comparison studies examining the effects of low-dose radiation. (author)

  6. Quantification of organic acids in beer by nuclear magnetic resonance (NMR)-based methods

    Energy Technology Data Exchange (ETDEWEB)

    Rodrigues, J.E.A. [CICECO-Department of Chemistry, University of Aveiro, Campus de Santiago, 3810-193 Aveiro (Portugal); Erny, G.L. [CESAM - Department of Chemistry, University of Aveiro, Campus de Santiago, 3810-193 Aveiro (Portugal); Barros, A.S. [QOPNAA-Department of Chemistry, University of Aveiro, Campus de Santiago, 3810-193 Aveiro (Portugal); Esteves, V.I. [CESAM - Department of Chemistry, University of Aveiro, Campus de Santiago, 3810-193 Aveiro (Portugal); Brandao, T.; Ferreira, A.A. [UNICER, Bebidas de Portugal, Leca do Balio, 4466-955 S. Mamede de Infesta (Portugal); Cabrita, E. [Department of Chemistry, New University of Lisbon, 2825-114 Caparica (Portugal); Gil, A.M., E-mail: agil@ua.pt [CICECO-Department of Chemistry, University of Aveiro, Campus de Santiago, 3810-193 Aveiro (Portugal)

    2010-08-03

    The organic acids present in beer provide important information on the product's quality and history, determining organoleptic properties and being useful indicators of fermentation performance. NMR spectroscopy may be used for rapid quantification of organic acids in beer and different NMR-based methodologies are hereby compared for the six main acids found in beer (acetic, citric, lactic, malic, pyruvic and succinic). The use of partial least squares (PLS) regression enables faster quantification, compared to traditional integration methods, and the performance of PLS models built using different reference methods (capillary electrophoresis (CE), both with direct and indirect UV detection, and enzymatic essays) was investigated. The best multivariate models were obtained using CE/indirect detection and enzymatic essays as reference and their response was compared with NMR integration, either using an internal reference or an electrical reference signal (Electronic REference To access In vivo Concentrations, ERETIC). NMR integration results generally agree with those obtained by PLS, with some overestimation for malic and pyruvic acids, probably due to peak overlap and subsequent integral errors, and an apparent relative underestimation for citric acid. Overall, these results make the PLS-NMR method an interesting choice for organic acid quantification in beer.

  7. Quantification of organic acids in beer by nuclear magnetic resonance (NMR)-based methods

    International Nuclear Information System (INIS)

    Rodrigues, J.E.A.; Erny, G.L.; Barros, A.S.; Esteves, V.I.; Brandao, T.; Ferreira, A.A.; Cabrita, E.; Gil, A.M.

    2010-01-01

    The organic acids present in beer provide important information on the product's quality and history, determining organoleptic properties and being useful indicators of fermentation performance. NMR spectroscopy may be used for rapid quantification of organic acids in beer and different NMR-based methodologies are hereby compared for the six main acids found in beer (acetic, citric, lactic, malic, pyruvic and succinic). The use of partial least squares (PLS) regression enables faster quantification, compared to traditional integration methods, and the performance of PLS models built using different reference methods (capillary electrophoresis (CE), both with direct and indirect UV detection, and enzymatic essays) was investigated. The best multivariate models were obtained using CE/indirect detection and enzymatic essays as reference and their response was compared with NMR integration, either using an internal reference or an electrical reference signal (Electronic REference To access In vivo Concentrations, ERETIC). NMR integration results generally agree with those obtained by PLS, with some overestimation for malic and pyruvic acids, probably due to peak overlap and subsequent integral errors, and an apparent relative underestimation for citric acid. Overall, these results make the PLS-NMR method an interesting choice for organic acid quantification in beer.

  8. Methods of introducing nucleic acids into cellular DNA

    Energy Technology Data Exchange (ETDEWEB)

    Lajoie, Marc J.; Gregg, Christopher J.; Mosberg, Joshua A.; Church, George M.

    2017-06-27

    A method of introducing a nucleic acid sequence into a cell is provided where the cell has impaired or inhibited or disrupted DnaG primase activity or impaired or inhibited or disrupted DnaB helicase activity, or larger or increased gaps or distance between Okazaki fragments or lowered or reduced frequency of Okazaki fragment initiation, or the cell has increased single stranded DNA (ssDNA) on the lagging strand of the replication fork including transforming the cell through recombination with a nucleic acid oligomer.

  9. Evaluation of DNA extraction methods for PCR-based detection of Listeria monocytogenes from vegetables.

    Science.gov (United States)

    Vojkovska, H; Kubikova, I; Kralik, P

    2015-03-01

    Epidemiological data indicate that raw vegetables are associated with outbreaks of Listeria monocytogenes. Therefore, there is a demand for the availability of rapid and sensitive methods, such as PCR assays, for the detection and accurate discrimination of L. monocytogenes. However, the efficiency of PCR methods can be negatively affected by inhibitory compounds commonly found in vegetable matrices that may cause false-negative results. Therefore, the sample processing and DNA isolation steps must be carefully evaluated prior to the introduction of such methods into routine practice. In this study, we compared the ability of three column-based and four magnetic bead-based commercial DNA isolation kits to extract DNA of the model micro-organism L. monocytogenes from raw vegetables. The DNA isolation efficiency of all isolation kits was determined using a triplex real-time qPCR assay designed to specifically detect L. monocytogenes. The kit with best performance, the PowerSoil(™) Microbial DNA Isolation Kit, is suitable for the extraction of amplifiable DNA from L. monocytogenes cells in vegetable with efficiencies ranging between 29.6 and 70.3%. Coupled with the triplex real-time qPCR assay, this DNA isolation kit is applicable to the samples with bacterial loads of 10(3) bacterial cells per gram of L. monocytogenes. Several recent outbreaks of Listeria monocytogenes have been associated with the consumption of fruits and vegetables. Real-time PCR assays allow fast detection and accurate quantification of microbes. However, the success of real-time PCR is dependent on the success with which template DNA can be extracted. The results of this study suggest that the PowerSoil(™) Microbial DNA Isolation Kit can be used for the extraction of amplifiable DNA from L. monocytogenes cells in vegetable with efficiencies ranging between 29.6 and 70.3%. This method is applicable to samples with bacterial loads of 10(3) bacterial cells per gram of L. monocytogenes. © 2014

  10. Performance verification and comparison of TianLong automatic hypersensitive hepatitis B virus DNA quantification system with Roche CAP/CTM system.

    Science.gov (United States)

    Li, Ming; Chen, Lin; Liu, Li-Ming; Li, Yong-Li; Li, Bo-An; Li, Bo; Mao, Yuan-Li; Xia, Li-Fang; Wang, Tong; Liu, Ya-Nan; Li, Zheng; Guo, Tong-Sheng

    2017-10-07

    To investigate and compare the analytical and clinical performance of TianLong automatic hypersensitive hepatitis B virus (HBV) DNA quantification system and Roche CAP/CTM system. Two hundred blood samples for HBV DNA testing, HBV-DNA negative samples and high-titer HBV-DNA mixture samples were collected and prepared. National standard materials for serum HBV and a worldwide HBV DNA panel were employed for performance verification. The analytical performance, such as limit of detection, limit of quantification, accuracy, precision, reproducibility, linearity, genotype coverage and cross-contamination, was determined using the TianLong automatic hypersensitive HBV DNA quantification system (TL system). Correlation and Bland-Altman plot analyses were carried out to compare the clinical performance of the TL system assay and the CAP/CTM system. The detection limit of the TL system was 10 IU/mL, and its limit of quantification was 30 IU/mL. The differences between the expected and tested concentrations of the national standards were less than ± 0.4 Log 10 IU/mL, which showed high accuracy of the system. Results of the precision, reproducibility and linearity tests showed that the multiple test coefficient of variation (CV) of the same sample was less than 5% for 10 2 -10 6 IU/mL; and for 30-10 8 IU/mL, the linear correlation coefficient r 2 = 0.99. The TL system detected HBV DNA (A-H) genotypes and there was no cross-contamination during the "checkerboard" test. When compared with the CAP/CTM assay, the two assays showed 100% consistency in both negative and positive sample results (15 negative samples and 185 positive samples). No statistical differences between the two assays in the HBV DNA quantification values were observed ( P > 0.05). Correlation analysis indicated a significant correlation between the two assays, r 2 = 0.9774. The Bland-Altman plot analysis showed that 98.9% of the positive data were within the 95% acceptable range, and the maximum difference

  11. Spectroscopic quantification of 5-hydroxymethylcytosine in genomic DNA using boric acid-functionalized nano-microsphere fluorescent probes.

    Science.gov (United States)

    Chen, Hua-Yan; Wei, Jing-Ru; Pan, Jiong-Xiu; Zhang, Wei; Dang, Fu-Quan; Zhang, Zhi-Qi; Zhang, Jing

    2017-05-15

    5-hydroxymethylcytosine (5hmC) is the sixth base of DNA. It is involved in active DNA demethylation and can be a marker of diseases such as cancer. In this study, we developed a simple and sensitive 2-(4-boronophenyl)quinoline-4-carboxylic acid modified poly (glycidyl methacrylate (PBAQA-PGMA) fluorescent probe to detect the 5hmC content of genomic DNA based on T4 β-glucosyltransferase-catalyzed glucosylation of 5hmC. The fluorescence-enhanced intensity recorded from the DNA sample was proportional to its 5-hydroxymethylcytosine content and could be quantified by fluorescence spectrophotometry. The developed probe showed good detection sensitivity and selectivity and a good linear relationship between the fluorescence intensity and the concentration of 5 hmC within a 0-100nM range. Compared with other fluorescence detection methods, this method not only could determine trace amounts of 5 hmC from genomic DNA but also could eliminate the interference of fluorescent dyes and the need for purification. It also could avoid multiple labeling. Because the PBAQA-PGMA probe could enrich the content of glycosyl-5-hydroxymethyl-2-deoxycytidine from a complex ground substance, it will broaden the linear detection range and improve sensitivity. The limit of detection was calculated to be 0.167nM after enrichment. Furthermore, the method was successfully used to detect 5-hydroxymethylcytosine from mouse tissues. Copyright © 2016 Elsevier B.V. All rights reserved.

  12. Comparison of manual and automated quantification methods of 123I-ADAM

    International Nuclear Information System (INIS)

    Kauppinen, T.; Keski-Rahkonen, A.; Sihvola, E.; Helsinki Univ. Central Hospital

    2005-01-01

    123 I-ADAM is a novel radioligand for imaging of the brain serotonin transporters (SERTs). Traditionally, the analysis of brain receptor studies has been based on observer-dependent manual region of interest definitions and visual interpretation. Our aim was to create a template for automated image registrations and volume of interest (VOI) quantification and to show that an automated quantification method of 123 I-ADAM is more repeatable than the manual method. Patients, methods: A template and a predefined VOI map was created from 123 I-ADAM scans done for healthy volunteers (n=15). Scans of another group of healthy persons (HS, n=12) and patients with bulimia nervosa (BN, n=10) were automatically fitted to the template and specific binding ratios (SBRs) were calculated by using the VOI map. Manual VOI definitions were done for the HS and BN groups by both one and two observers. The repeatability of the automated method was evaluated by using the BN group. Results: For the manual method, the interobserver coefficient of repeatability was 0.61 for the HS group and 1.00 for the BN group. The intra-observer coefficient of repeatability for the BN group was 0.70. For the automated method, the coefficient of repeatability was 0.13 for SBRs in midbrain. Conclusion: An automated quantification gives valuable information in addition to visual interpretation decreasing also the total image handling time and giving clear advantages for research work. An automated method for analysing 123 I-ADAM binding to the brain SERT gives repeatable results for fitting the studies to the template and for calculating SBRs, and could therefore replace manual methods. (orig.)

  13. Comparison of manual and automated quantification methods of {sup 123}I-ADAM

    Energy Technology Data Exchange (ETDEWEB)

    Kauppinen, T. [Helsinki Univ. Central Hospital (Finland). HUS Helsinki Medical Imaging Center; Helsinki Univ. Central Hospital (Finland). Division of Nuclear Medicine; Koskela, A.; Ahonen, A. [Helsinki Univ. Central Hospital (Finland). Division of Nuclear Medicine; Diemling, M. [Hermes Medical Solutions, Stockholm (Sweden); Keski-Rahkonen, A.; Sihvola, E. [Helsinki Univ. (Finland). Dept. of Public Health; Helsinki Univ. Central Hospital (Finland). Dept. of Psychiatry

    2005-07-01

    {sup 123}I-ADAM is a novel radioligand for imaging of the brain serotonin transporters (SERTs). Traditionally, the analysis of brain receptor studies has been based on observer-dependent manual region of interest definitions and visual interpretation. Our aim was to create a template for automated image registrations and volume of interest (VOI) quantification and to show that an automated quantification method of {sup 123}I-ADAM is more repeatable than the manual method. Patients, methods: A template and a predefined VOI map was created from {sup 123}I-ADAM scans done for healthy volunteers (n=15). Scans of another group of healthy persons (HS, n=12) and patients with bulimia nervosa (BN, n=10) were automatically fitted to the template and specific binding ratios (SBRs) were calculated by using the VOI map. Manual VOI definitions were done for the HS and BN groups by both one and two observers. The repeatability of the automated method was evaluated by using the BN group. Results: For the manual method, the interobserver coefficient of repeatability was 0.61 for the HS group and 1.00 for the BN group. The intra-observer coefficient of repeatability for the BN group was 0.70. For the automated method, the coefficient of repeatability was 0.13 for SBRs in midbrain. Conclusion: An automated quantification gives valuable information in addition to visual interpretation decreasing also the total image handling time and giving clear advantages for research work. An automated method for analysing {sup 123}I-ADAM binding to the brain SERT gives repeatable results for fitting the studies to the template and for calculating SBRs, and could therefore replace manual methods. (orig.)

  14. Quantification of massively parallel sequencing libraries - a comparative study of eight methods

    DEFF Research Database (Denmark)

    Hussing, Christian; Kampmann, Marie-Louise; Mogensen, Helle Smidt

    2018-01-01

    Quantification of massively parallel sequencing libraries is important for acquisition of monoclonal beads or clusters prior to clonal amplification and to avoid large variations in library coverage when multiple samples are included in one sequencing analysis. No gold standard for quantification...... estimates followed by Qubit and electrophoresis-based instruments (Bioanalyzer, TapeStation, GX Touch, and Fragment Analyzer), while SYBR Green and TaqMan based qPCR assays gave the lowest estimates. qPCR gave more accurate predictions of sequencing coverage than Qubit and TapeStation did. Costs, time......-consumption, workflow simplicity, and ability to quantify multiple samples are discussed. Technical specifications, advantages, and disadvantages of the various methods are pointed out....

  15. A simple method to improve the quantification accuracy of energy-dispersive X-ray microanalysis

    International Nuclear Information System (INIS)

    Walther, T

    2008-01-01

    Energy-dispersive X-ray spectroscopy in a transmission electron microscope is a standard tool for chemical microanalysis and routinely provides qualitative information on the presence of all major elements above Z=5 (boron) in a sample. Spectrum quantification relies on suitable corrections for absorption and fluorescence, in particular for thick samples and soft X-rays. A brief presentation is given of an easy way to improve quantification accuracy by evaluating the intensity ratio of two measurements acquired at different detector take-off angles. As the take-off angle determines the effective sample thickness seen by the detector this method corresponds to taking two measurements from the same position at two different thicknesses, which allows to correct absorption and fluorescence more reliably. An analytical solution for determining the depth of a feature embedded in the specimen foil is also provided.

  16. DNA arrays : methods and protocols [Methods in molecular biology, v. 170

    National Research Council Canada - National Science Library

    Rampal, Jang B

    2001-01-01

    "In DNA Arrays: Methods and Protocols, Jang Rampal and a authoritative panel of researchers, engineers, and technologists explain in detail how to design and construct DNA microarrays, as well as how to...

  17. Real-time polymerase chain reaction assay for endogenous reference gene for specific detection and quantification of common wheat-derived DNA (Triticum aestivum L.).

    Science.gov (United States)

    Vautrin, Sonia; Zhang, David

    2007-01-01

    A species-specific endogenous reference gene system was developed for polymerase chain reaction (PCR)-based analysis in common wheat (Triticum aestivum L.) by targeting the ALMT1 gene, an aluminium-activated malate transporter. The primers and probe were elaborated for real-time PCR-based qualitative and quantitative assay. The size of amplified product is 95 base pairs. The specificity was assessed on 17 monocot and dicot plant species. The established real-time PCR assay amplified only T. aestivum-derived DNA; no amplification occurred on other phylogenetically related species, including durum wheat (T. durum). The robustness of the system was tested on the DNA of 15 common wheat cultivars using 20 000 genomic copies per PCR the mean cycle threshold (Ct) values of 24.02 +/- 0.251 were obtained. The absolute limits of detection and quantification of the real-time PCR assay were estimated to 2 and 20 haploid genome copies of common wheat, respectively. The linearity was experimentally validated on 2-fold serial dilutions of DNA from 650 to 20 000 haploid genome copies. All these results show that the real-time PCR assay developed on the ALMT1 gene is suitable to be used as an endogenous reference gene for PCR-based specific detection and quantification of T. aestivum-derived DNA in various applications, in particular for the detection and quantification of genetically modified materials in common wheat.

  18. DNA extraction method for PCR in mycorrhizal fungi.

    Science.gov (United States)

    Manian, S; Sreenivasaprasad, S; Mills, P R

    2001-10-01

    To develop a simple and rapid DNA extraction protocol for PCR in mycorrhizal fungi. The protocol combines the application of rapid freezing and boiling cycles and passage of the extracts through DNA purification columns. PCR amplifiable DNA was obtained from a number of endo- and ecto-mycorrhizal fungi using minute quantities of spores and mycelium, respectively. DNA extracted following the method, was used to successfully amplify regions of interest from high as well as low copy number genes. The amplicons were suitable for further downstream applications such as sequencing and PCR-RFLPs. The protocol described is simple, short and facilitates rapid isolation of PCR amplifiable genomic DNA from a large number of fungal isolates in a single day. The method requires only minute quantities of starting material and is suitable for mycorrhizal fungi as well as a range of other fungi.

  19. Quantification of radiation absorbed dose and DNA damages in subjects undergoing computer tomography imaging

    International Nuclear Information System (INIS)

    Kanagaraj, Karthik; Basheerudeen, Safa Abdul Syed; Tamizh Selvan, G.; Venkatachalam, Perumal; Jose, M.T.; Ozhimuthu, Annalakshmi; Panneer Selvam, S.; Pattan, Sudha

    2014-01-01

    X-rays are extensively used in medical field for imaging, diagnostic radiology and radiotherapy. Irrespective of the application, the procedures deliver a significant amount of dose to the subject, while undergoing the procedure, which vary from imaging (low dose in the order mGy) and therapy (high doses in the order of several Gy). Of the various imaging modalities, the computed tomography (CT) is commonly used to diagnose many health alignments, in all age groups. Though the personals involved in performing the procedures are monitored for the levels of exposure, it is uncommon to monitor the patient after the examination, as the benefits outweigh the risk. However an enhanced concern on the risk associated due to the exposure of low dose X-radiation in CT has been reported. Therefore, we aim to quantify the absorbed dose to the eye, thyroid and forehead using thermo luminescence dosimeter of Lithium Manganese Borate doped with Terbium (LMB:Tb) in subjects undergoing CT examination (n= 27), as a methodology to investigate the effects of low dose ionizing radiation. Further, the DNA damages was measured using chromosomal aberration (CA) and micronucleus (MN) assay, from the blood samples obtained from the study subjects before and after the procedures. The overall measured organ dose ranged between 1.92 and 520.14 mGy for eye, 0.84 and 210.33 mGy for forehead and 1.79-185 mGy for thyroid, with an average of 128.86 1 137.16, 78.25 1 69.02 and 48.86 1 63.60 respectively. The DNA damages measured using CA and MN assay, showed an extreme statistically significant (p<0.0001) increase in CA and significant increase (p<0.001) in MN frequency in post exposure when compared to that of unexposed control. The significance of the estimated dose and the DNA damages will be discussed. (author)

  20. SPECT quantification: a review of the different correction methods with compton scatter, attenuation and spatial deterioration effects

    International Nuclear Information System (INIS)

    Groiselle, C.; Rocchisani, J.M.; Moretti, J.L.; Dreuille, O. de; Gaillard, J.F.; Bendriem, B.

    1997-01-01

    SPECT quantification: a review of the different correction methods with Compton scatter attenuation and spatial deterioration effects. The improvement of gamma-cameras, acquisition and reconstruction software opens new perspectives in term of image quantification in nuclear medicine. In order to meet the challenge, numerous works have been undertaken in recent years to correct for the different physical phenomena that prevent an exact estimation of the radioactivity distribution. The main phenomena that have to betaken into account are scatter, attenuation and resolution. In this work, authors present the physical basis of each issue, its consequences on quantification and the main methods proposed to correct them. (authors)

  1. Comparison of Different Drying Methods for Recovery of Mushroom DNA.

    Science.gov (United States)

    Wang, Shouxian; Liu, Yu; Xu, Jianping

    2017-06-07

    Several methods have been reported for drying mushroom specimens for population genetic, taxonomic, and phylogenetic studies. However, most methods have not been directly compared for their effectiveness in preserving mushroom DNA. In this study, we compared silica gel drying at ambient temperature and oven drying at seven different temperatures. Two mushroom species representing two types of fruiting bodies were examined: the fleshy button mushroom Agaricus bisporus and the leathery shelf fungus Trametes versicolor. For each species dried with the eight methods, we assessed the mushroom water loss rate, the quality and quantity of extracted DNA, and the effectiveness of using the extracted DNA as a template for PCR amplification of two DNA fragments (ITS and a single copy gene). Dried specimens from all tested methods yielded sufficient DNA for PCR amplification of the two genes in both species. However, differences among the methods for the two species were found in: (i) the time required by different drying methods for the fresh mushroom tissue to reach a stable weight; and (ii) the relative quality and quantity of the extracted genomic DNA. Among these methods, oven drying at 70 °C for 3-4 h seemed the most efficient for preserving field mushroom samples for subsequent molecular work.

  2. Validation of an HPLC method for quantification of total quercetin in Calendula officinalis extracts

    International Nuclear Information System (INIS)

    Muñoz Muñoz, John Alexander; Morgan Machado, Jorge Enrique; Trujillo González, Mary

    2015-01-01

    Introduction: calendula officinalis extracts are used as natural raw material in a wide range of pharmaceutical and cosmetic preparations; however, there are no official methods for quality control of these extracts. Objective: to validate an HPLC-based analytical method for quantification total quercetin in glycolic and hydroalcoholic extracts of Calendula officinalis. Methods: to quantify total quercetin content in the matrices, it was necessary to hydrolyze flavonoid glycosides under optimal conditions. The chromatographic separation was performed on a C-18 SiliaChrom 4.6x150 mm 5 µm column, adapted to a SiliaChrom 5 um C-18 4.6x10 mm precolumn, with UV detection at 370 nm. The gradient elution was performed with a mobile phase consisting of methanol (MeOH) and phosphoric acid (H 3 PO 4 ) (0.08 % w/v). The quantification was performed through the external standard method and comparison with quercetin reference standard. Results: the studied method selectivity against extract components and degradation products under acid/basic hydrolysis, oxidation and light exposure conditions showed no signals that interfere with the quercetin quantification. It was statistically proved that the method is linear from 1.0 to 5.0 mg/mL. Intermediate precision expressed as a variation coefficient was 1.8 and 1.74 % and the recovery percentage was 102.15 and 101.32 %, for glycolic and hydroalcoholic extracts, respectively. Conclusions: the suggested methodology meets the quality parameters required for quantifying total quercetin, which makes it a useful tool for quality control of C. officinalis extracts. (author)

  3. Validation of methods for the detection and quantification of engineered nanoparticles in food

    DEFF Research Database (Denmark)

    Linsinger, T.P.J.; Chaudhry, Q.; Dehalu, V.

    2013-01-01

    the methods apply equally well to particles of different suppliers. In trueness testing, information whether the particle size distribution has changed during analysis is required. Results are largely expected to follow normal distributions due to the expected high number of particles. An approach...... approach for the validation of methods for detection and quantification of nanoparticles in food samples. It proposes validation of identity, selectivity, precision, working range, limit of detection and robustness, bearing in mind that each “result” must include information about the chemical identity...

  4. DNA-based methods of geochemical prospecting

    Energy Technology Data Exchange (ETDEWEB)

    Ashby, Matthew [Mill Valley, CA

    2011-12-06

    The present invention relates to methods for performing surveys of the genetic diversity of a population. The invention also relates to methods for performing genetic analyses of a population. The invention further relates to methods for the creation of databases comprising the survey information and the databases created by these methods. The invention also relates to methods for analyzing the information to correlate the presence of nucleic acid markers with desired parameters in a sample. These methods have application in the fields of geochemical exploration, agriculture, bioremediation, environmental analysis, clinical microbiology, forensic science and medicine.

  5. Improved LC-MS/MS method for the quantification of hepcidin-25 in clinical samples.

    Science.gov (United States)

    Abbas, Ioana M; Hoffmann, Holger; Montes-Bayón, María; Weller, Michael G

    2018-06-01

    Mass spectrometry-based methods play a crucial role in the quantification of the main iron metabolism regulator hepcidin by singling out the bioactive 25-residue peptide from the other naturally occurring N-truncated isoforms (hepcidin-20, -22, -24), which seem to be inactive in iron homeostasis. However, several difficulties arise in the MS analysis of hepcidin due to the "sticky" character of the peptide and the lack of suitable standards. Here, we propose the use of amino- and fluoro-silanized autosampler vials to reduce hepcidin interaction to laboratory glassware surfaces after testing several types of vials for the preparation of stock solutions and serum samples for isotope dilution liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS). Furthermore, we have investigated two sample preparation strategies and two chromatographic separation conditions with the aim of developing a LC-MS/MS method for the sensitive and reliable quantification of hepcidin-25 in serum samples. A chromatographic separation based on usual acidic mobile phases was compared with a novel approach involving the separation of hepcidin-25 with solvents at high pH containing 0.1% of ammonia. Both methods were applied to clinical samples in an intra-laboratory comparison of two LC-MS/MS methods using the same hepcidin-25 calibrators with good correlation of the results. Finally, we recommend a LC-MS/MS-based quantification method with a dynamic range of 0.5-40 μg/L for the assessment of hepcidin-25 in human serum that uses TFA-based mobile phases and silanized glass vials. Graphical abstract Structure of hepcidin-25 (Protein Data Bank, PDB ID 2KEF).

  6. TaqMan probe real-time polymerase chain reaction assay for the quantification of canine DNA in chicken nugget.

    Science.gov (United States)

    Rahman, Md Mahfujur; Hamid, Sharifah Bee Abd; Basirun, Wan Jefrey; Bhassu, Subha; Rashid, Nur Raifana Abdul; Mustafa, Shuhaimi; Mohd Desa, Mohd Nasir; Ali, Md Eaqub

    2016-01-01

    This paper describes a short-amplicon-based TaqMan probe quantitative real-time PCR (qPCR) assay for the quantitative detection of canine meat in chicken nuggets, which are very popular across the world, including Malaysia. The assay targeted a 100-bp fragment of canine cytb gene using a canine-specific primer and TaqMan probe. Specificity against 10 different animals and plants species demonstrated threshold cycles (Ct) of 16.13 ± 0.12 to 16.25 ± 0.23 for canine DNA and negative results for the others in a 40-cycle reaction. The assay was tested for the quantification of up to 0.01% canine meat in deliberately spiked chicken nuggets with 99.7% PCR efficiency and 0.995 correlation coefficient. The analysis of the actual and qPCR predicted values showed a high recovery rate (from 87% ± 28% to 112% ± 19%) with a linear regression close to unity (R(2) = 0.999). Finally, samples of three halal-branded commercial chicken nuggets collected from different Malaysian outlets were screened for canine meat, but no contamination was demonstrated.

  7. Localisation and quantification of alkali-labile sites in human spermatozoa by DNA breakage detection-fluorescence in situ hybridisation.

    Science.gov (United States)

    Cortés-Gutiérrez, E I; Dávila-Rodríguez, M I; Cerda-Flores, R M; Fernández, J L; López-Fernández, C; Aragón Tovar, A R; Gosálvez, J

    2015-03-01

    The localisation and quantification of constitutive alkali-labile sites (ALSs) were investigated using a protocol of DNA breakage detection plus fluorescence in situ hybridisation (DBD-FISH) and alkaline single-cell gel electrophoresis (SCGE or comet assay), in spermatozoa of infertile and fertile men. Semen samples from 10 normozoospermic patients undergoing infertility treatment and 10 fertile men were included in this study. ALSs were localised and quantified by DBD-FISH. The region most sensitive to alkali treatment in human spermatozoa was located in the basal region of the head. ALSs were more frequent in spermatozoa of infertile men than in those of fertile men. These results were confirmed by SCGE comet assays. In conclusion, the most intense localisation of hybridisation signals in human spermatozoa, representing the highest density of constitutive ALSs, was not randomly distributed and was predominantly located in the base of the head. Moreover, infertile men presented with an increase in ALS frequency. Further studies are necessary to determine the association between ALS, sperm chromatin organisation and infertility. © 2014 Blackwell Verlag GmbH.

  8. An ameliorative protocol for the quantification of purine 5',8-cyclo-2'-deoxynucleosides in oxidized DNA

    Science.gov (United States)

    Terzidis, Michael; Chatgilialoglu, Chryssostomos

    2015-07-01

    5',8-Cyclo-2'-deoxyadenosine (cdA) and 5',8-cyclo-2'-deoxyguanosine (cdG) are lesions resulting from hydroxyl radical (HO•) attack on the 5'H of the nucleoside sugar moiety and exist in both 5'R and 5'S diastereomeric forms. Increased levels of cdA and cdG are linked to Nucleotide Excision Repair mechanism deficiency and mutagenesis. Discrepancies in the damage measurements reported over recent years indicated the weakness of the actual protocols, in particular for ensuring the quantitative release of these lesions from the DNA sample and the appropriate method for their analysis. Herein we report the detailed revision leading to a cost-effective and efficient protocol for the DNA damage measurement, consisting of the nuclease benzonase and nuclease P1 enzymatic combination for DNA digestion followed by liquid chromatography isotope dilution tandem mass spectrometry analysis.

  9. A Simple and Effective Isocratic HPLC Method for Fast Identification and Quantification of Surfactin

    International Nuclear Information System (INIS)

    Muhammad Qadri Effendy Mubarak; Abdul Rahman Hassan; Aidil Abdul Hamid; Sahaid Khalil; Mohd Hafez Mohd Isa

    2015-01-01

    The aim of this study was to establish a simple, accurate and reproducible method for the identification and quantification of surfactin using high-performance liquid chromatography (HPLC). Previously reported method of identification and quantification of surfactin were time consuming and requires a large quantity of mobile phase. The new method was achieved by application of Chromolith® high performance RP-18 (100 x 4.6 mm, 5 μm) as the stationary phase and optimization of mobile phase ratio and flow rate. Mobile phase consisted of acetonitrile (ACN) and 3.8 mM trifluroacetic acid (TFA) solution of 80:20 ratio at flow rate of 2.2 mL/ min was obtained as the optimal conditions. Total elution time of the obtained surfactin peaks was four times quicker than various methods previously reported in the literature. The method described here allowed for fine separation of surfactin in standard sample (98 % purity) and surfactin in fermentation broth. (author)

  10. High-performance liquid chromatographic quantification of rifampicin in human plasma: method for Therapecutic drug monitoring

    International Nuclear Information System (INIS)

    Sameh, T.; Hanene, E.; Jebali, N.

    2013-01-01

    A high performance liquid chromatography (HPLC) method has been developed that allows quantification of Rifampicin in human plasma. The method is based on the precipitation of proteins in human plasma with methanol. Optimal assay conditions were found with a C18 column and a simple mobile phase consisting of 0.05 M dipotassic hydrogen phosphate buffer and acetonitrile (53/47, V/V) with 0.086 % diethylamin, pH = 4.46. The flow-rate was 0.6 ml /mm and the drug was monitored at 340 nm. Results from the HPLC analyses showed that the assay method is linear in the concentration range of 1-40 micro g/ml, (r2 >0.99). The limit of quantification and limit of detection of Rifampicin were 0.632 micro g/ml and 0.208 micro g/ml, respectively. Intraday and interday coefficient of variation and bias were below 10% for all samples, suggesting good precision and accuracy of the method. Recoveries were greater than 90% in a plasma sample volume of 100 micro l. The method is being successfully applied to therapeutic drug monitoring of Rifapicin in plasma samples of tuberculosis and staphylococcal infections patients. (author)

  11. Evaluation of two autoinducer-2 quantification methods for application in marine environments

    KAUST Repository

    Wang, Tian-Nyu

    2018-02-11

    This study evaluated two methods, namely high performance liquid chromatography with fluorescence detection (HPLC-FLD) and Vibrio harveyi BB170 bioassay, for autoinducer-2 (AI-2) quantification in marine samples. Using both methods, the study also investigated the stability of AI-2 in varying pH, temperature and media, as well as quantified the amount of AI-2 signals in marine samples.HPLC-FLD method showed a higher level of reproducibility and precision compared to V. harveyi BB170 bioassay. Alkaline pH > 8 and high temperature (> 37°C) increased the instability of AI-2. The AI-2 concentrations in seawater were low, ca. 3.2-27.6 pmol l-1 whereas 8- week old marine biofilm grew on an 18.8 cm2 substratum accumulated ca. 0.207 nmol of AI-2.Both methods have pros and cons for AI-2 quantification in marine samples. Regardless, both methods reported a ubiquitous presence of AI-2 in both planktonic and biomass fractions of seawater, as well as in marine biofilm.In this study, AI-2 signals were for the first time enumerated in marine samples to reveal the ubiquitous presence of AI-2 in this environment. The findings suggest a possible role of AI-2 in biofilm formation in marine environment, and the contribution of AI-2 in biofilm-associated problems such as biofouling and biocorrosion. This article is protected by copyright. All rights reserved.

  12. DNA Dynamics Studied Using the Homogeneous Balance Method

    International Nuclear Information System (INIS)

    Zayed, E. M. E.; Arnous, A. H.

    2012-01-01

    We employ the homogeneous balance method to construct the traveling waves of the nonlinear vibrational dynamics modeling of DNA. Some new explicit forms of traveling waves are given. It is shown that this method provides us with a powerful mathematical tool for solving nonlinear evolution equations in mathematical physics. Strengths and weaknesses of the proposed method are discussed. (general)

  13. Novel DNA sequence detection method based on fluorescence energy transfer

    International Nuclear Information System (INIS)

    Kobayashi, S.; Tamiya, E.; Karube, I.

    1987-01-01

    Recently the detection of specific DNA sequence, DNA analysis, has been becoming more important for diagnosis of viral genomes causing infections disease and human sequences related to inherited disorders. These methods typically involve electrophoresis, the immobilization of DNA on a solid support, hybridization to a complementary probe, the detection using labeled with /sup 32/P or nonisotopically with a biotin-avidin-enzyme system, and so on. These techniques are highly effective, but they are very time-consuming and expensive. A principle of fluorescene energy transfer is that the light energy from an excited donor (fluorophore) is transferred to an acceptor (fluorophore), if the acceptor exists in the vicinity of the donor and the excitation spectrum of donor overlaps the emission spectrum of acceptor. In this study, the fluorescence energy transfer was applied to the detection of specific DNA sequence using the hybridization method. The analyte, single-stranded DNA labeled with the donor fluorophore is hybridized to a probe DNA labeled with the acceptor. Because of the complementary DNA duplex formation, two fluorophores became to be closed to each other, and the fluorescence energy transfer was occurred

  14. Methods for the physical characterization and quantification of extracellular vesicles in biological samples.

    Science.gov (United States)

    Rupert, Déborah L M; Claudio, Virginia; Lässer, Cecilia; Bally, Marta

    2017-01-01

    Our body fluids contain a multitude of cell-derived vesicles, secreted by most cell types, commonly referred to as extracellular vesicles. They have attracted considerable attention for their function as intercellular communication vehicles in a broad range of physiological processes and pathological conditions. Extracellular vesicles and especially the smallest type, exosomes, have also generated a lot of excitement in view of their potential as disease biomarkers or as carriers for drug delivery. In this context, state-of-the-art techniques capable of comprehensively characterizing vesicles in biological fluids are urgently needed. This review presents the arsenal of techniques available for quantification and characterization of physical properties of extracellular vesicles, summarizes their working principles, discusses their advantages and limitations and further illustrates their implementation in extracellular vesicle research. The small size and physicochemical heterogeneity of extracellular vesicles make their physical characterization and quantification an extremely challenging task. Currently, structure, size, buoyant density, optical properties and zeta potential have most commonly been studied. The concentration of vesicles in suspension can be expressed in terms of biomolecular or particle content depending on the method at hand. In addition, common quantification methods may either provide a direct quantitative measurement of vesicle concentration or solely allow for relative comparison between samples. The combination of complementary methods capable of detecting, characterizing and quantifying extracellular vesicles at a single particle level promises to provide new exciting insights into their modes of action and to reveal the existence of vesicle subpopulations fulfilling key biological tasks. Copyright © 2016 Elsevier B.V. All rights reserved.

  15. Study on quantification method based on Monte Carlo sampling for multiunit probabilistic safety assessment models

    Energy Technology Data Exchange (ETDEWEB)

    Oh, Kye Min [KHNP Central Research Institute, Daejeon (Korea, Republic of); Han, Sang Hoon; Park, Jin Hee; Lim, Ho Gon; Yang, Joon Yang [Korea Atomic Energy Research Institute, Daejeon (Korea, Republic of); Heo, Gyun Young [Kyung Hee University, Yongin (Korea, Republic of)

    2017-06-15

    In Korea, many nuclear power plants operate at a single site based on geographical characteristics, but the population density near the sites is higher than that in other countries. Thus, multiunit accidents are a more important consideration than in other countries and should be addressed appropriately. Currently, there are many issues related to a multiunit probabilistic safety assessment (PSA). One of them is the quantification of a multiunit PSA model. A traditional PSA uses a Boolean manipulation of the fault tree in terms of the minimal cut set. However, such methods have some limitations when rare event approximations cannot be used effectively or a very small truncation limit should be applied to identify accident sequence combinations for a multiunit site. In particular, it is well known that seismic risk in terms of core damage frequency can be overestimated because there are many events that have a high failure probability. In this study, we propose a quantification method based on a Monte Carlo approach for a multiunit PSA model. This method can consider all possible accident sequence combinations in a multiunit site and calculate a more exact value for events that have a high failure probability. An example model for six identical units at a site was also developed and quantified to confirm the applicability of the proposed method.

  16. Identification and quantification of genetically modified Moonshade carnation lines using conventional and TaqMan real-time polymerase chain reaction methods.

    Science.gov (United States)

    Li, Peng; Jia, Junwei; Bai, Lan; Pan, Aihu; Tang, Xueming

    2013-07-01

    Genetically modified carnation (Dianthus caryophyllus L.) Moonshade was approved for planting and commercialization in several countries from 2004. Developing methods for analyzing Moonshade is necessary for implementing genetically modified organism labeling regulations. In this study, the 5'-transgene integration sequence was isolated using thermal asymmetric interlaced (TAIL)-PCR. Based upon the 5'-transgene integration sequence, conventional and TaqMan real-time PCR assays were established. The relative limit of detection for the conventional PCR assay was 0.05 % for Moonshade using 100 ng total carnation genomic DNA, corresponding to approximately 79 copies of the carnation haploid genome, and the limits of detection and quantification of the TaqMan real-time PCR assay were estimated to be 51 and 254 copies of haploid carnation genomic DNA, respectively. These results are useful for identifying and quantifying Moonshade and its derivatives.

  17. Method for construction of normalized cDNA libraries

    Science.gov (United States)

    Soares, Marcelo B.; Efstratiadis, Argiris

    1998-01-01

    This invention provides a method to normalize a directional cDNA library constructed in a vector that allows propagation in single-stranded circle form comprising: (a) propagating the directional cDNA library in single-stranded circles; (b) generating fragments complementary to the 3' noncoding sequence of the single-stranded circles in the library to produce partial duplexes; (c) purifying the partial duplexes; (d) melting and reassociating the purified partial duplexes to appropriate Cot; and (e) purifying the unassociated single-stranded circles, thereby generating a normalized cDNA library. This invention also provides normalized cDNA libraries generated by the above-described method and uses of the generated libraries.

  18. Development of DNA elution method to detect irradiated foodstuff

    International Nuclear Information System (INIS)

    Copin, M.P.; Bourgeois, C.M.

    1991-01-01

    The aim of the work is to develop a reliable method to detect whether a fresh and frozen foodstuff has been irradiated. The molecule of DNA is one of the targets of ionizing radiation. The induction of three major classes of lesion have been shown. Double strand breaks, single strand breaks and base damage. Among the different techniques used to observe and quantify the strand breaks, techniques of elution are very interesting. The method proposed consisted of a filtration of the DNA at the atmospheric pressure and in non denaturing conditions. The amount of DNA retained on the filter is measured after being suitably labelled by microfluorometry. A difference in the amount of DNA retained on a filter of 2 μm from a lysed muscular tissue sample between a frozen Norway lobster which has been irradiated and one which has not, is observed. 7 refs

  19. Application of DNA-based methods in forensic entomology.

    Science.gov (United States)

    Wells, Jeffrey D; Stevens, Jamie R

    2008-01-01

    A forensic entomological investigation can benefit from a variety of widely practiced molecular genotyping methods. The most commonly used is DNA-based specimen identification. Other applications include the identification of insect gut contents and the characterization of the population genetic structure of a forensically important insect species. The proper application of these procedures demands that the analyst be technically expert. However, one must also be aware of the extensive list of standards and expectations that many legal systems have developed for forensic DNA analysis. We summarize the DNA techniques that are currently used in, or have been proposed for, forensic entomology and review established genetic analyses from other scientific fields that address questions similar to those in forensic entomology. We describe how accepted standards for forensic DNA practice and method validation are likely to apply to insect evidence used in a death or other forensic entomological investigation.

  20. Quantification of Iodine-123-FP-CIT SPECT with a resolution-independent method

    International Nuclear Information System (INIS)

    Dobbeleir, A.A.; Ham, H.R.; Hambye, A.E.; Vervaet, A.M.

    2005-01-01

    Accurate quantification of small-sized objects by SPECT is hampered by the partial volume effect. The present work evaluates the magnitude of this phenomenon with Iodine- 123 in phantom studies, and presents a resolution- independent method to quantify striatal I-123 FP-CIT uptake in patients. At first five syringes with internal diameters varying between 9 and 29mm and an anthropomorphic striatal phantom were filled with known concentrations of Iodine-123 and imaged by SPECT using different collimators and radii of rotation. Data were processed with and without scatter correction. From the measured activities, calibration factors were calculated for each specific collimator. Then a resolution-independent method for FP-CIT quantification using large regions of interest was developed and validated in 34 human studies (controls and patients) acquired in 2 different hospitals, by comparing its results to those obtained by a semi- quantitative striatal-to-occipital analysis. Taking the injected activity and decay into account, the measured counts/volume could be converted into absolute tracer concentrations. For the fan-beam, high resolution and medium energy collimators, the measured maximum activity in comparison to the 29 mm-diameter syringe was respectively 38%, 16% and 9% for the 9 mm-diameter syringe and 82%, 80% and 30% for the 16 mm syringe, and not significantly modified after scatter correction. For the anthropomorphic phantom, the error in measurement in % of the true concentration ranged between 0.3-9.5% and was collimator dependent. Medium energy collimators yielded the most homogeneous results. In the human studies, inter- observer variability was 11.4% for the striatal-to-occipital ratio and 3.1% for the resolution-independent method, with correlation coefficients >0.8 between both. The resolution- independent method was 89%-sensitive and 100%-specific to separate the patients without and with abnormal FP-CIT uptake (accuracy: 94%). Also the

  1. Review of Polynomial Chaos-Based Methods for Uncertainty Quantification in Modern Integrated Circuits

    Directory of Open Access Journals (Sweden)

    Arun Kaintura

    2018-02-01

    Full Text Available Advances in manufacturing process technology are key ensembles for the production of integrated circuits in the sub-micrometer region. It is of paramount importance to assess the effects of tolerances in the manufacturing process on the performance of modern integrated circuits. The polynomial chaos expansion has emerged as a suitable alternative to standard Monte Carlo-based methods that are accurate, but computationally cumbersome. This paper provides an overview of the most recent developments and challenges in the application of polynomial chaos-based techniques for uncertainty quantification in integrated circuits, with particular focus on high-dimensional problems.

  2. Automated extraction of DNA from clothing

    DEFF Research Database (Denmark)

    Stangegaard, Michael; Hjort, Benjamin Benn; Nøhr Hansen, Thomas

    2011-01-01

    Presence of PCR inhibitors in extracted DNA may interfere with the subsequent quantification and short tandem repeat (STR) reactions used in forensic genetic DNA typing. We have compared three automated DNA extraction methods based on magnetic beads with a manual method with the aim of reducing...

  3. A validated Fourier transform infrared spectroscopy method for quantification of total lactones in Inula racemosa and Andrographis paniculata.

    Science.gov (United States)

    Shivali, Garg; Praful, Lahorkar; Vijay, Gadgil

    2012-01-01

    Fourier transform infrared (FT-IR) spectroscopy is a technique widely used for detection and quantification of various chemical moieties. This paper describes the use of the FT-IR spectroscopy technique for the quantification of total lactones present in Inula racemosa and Andrographis paniculata. To validate the FT-IR spectroscopy method for quantification of total lactones in I. racemosa and A. paniculata. Dried and powdered I. racemosa roots and A. paniculata plant were extracted with ethanol and dried to remove ethanol completely. The ethanol extract was analysed in a KBr pellet by FT-IR spectroscopy. The FT-IR spectroscopy method was validated and compared with a known spectrophotometric method for quantification of lactones in A. paniculata. By FT-IR spectroscopy, the amount of total lactones was found to be 2.12 ± 0.47% (n = 3) in I. racemosa and 8.65 ± 0.51% (n = 3) in A. paniculata. The method showed comparable results with a known spectrophotometric method used for quantification of such lactones: 8.42 ± 0.36% (n = 3) in A. paniculata. Limits of detection and quantification for isoallantolactone were 1 µg and 10 µg respectively; for andrographolide they were 1.5 µg and 15 µg respectively. Recoveries were over 98%, with good intra- and interday repeatability: RSD ≤ 2%. The FT-IR spectroscopy method proved linear, accurate, precise and specific, with low limits of detection and quantification, for estimation of total lactones, and is less tedious than the UV spectrophotometric method for the compounds tested. This validated FT-IR spectroscopy method is readily applicable for the quality control of I. racemosa and A. paniculata. Copyright © 2011 John Wiley & Sons, Ltd.

  4. A simple method of digitizing analog scintigrams for quantification and digital archiving

    International Nuclear Information System (INIS)

    Schramm, M.; Kaempfer, B.; Wolf, H.; Clausen, M.; Wendhausen, H.; Henze, E.

    1993-01-01

    This study was undertaken to evaluate a quick, reliable and cheap method of digitizing analog scintigrams. 40 whole-body bone scintigrams were obtained simultaneously in analog and genuine digital format. The analog scans on X-ray film were then digitized seecondarily by three different methods: 300 dpi flatbed scanning, high-resolution camera scanning and camcorder recording. A simple exposure approach using a light box, a cheap camcorder, a PC and image grabber hard- and software proved to be optimal. Visual interpretation showed no differences in clinical findings when comparing the analog images with their secondarily digitized counterparts. To test the possibility of quantification, 126 equivalent ROIs were drawn both in the genuine digital and the secondarily digitized images. Comparing the ROI count to whole-body count percentage of the corresponding ROIs showed the correlation to be linear. The evaluation of phantom studies showed the linear correlation to be true within a wide activity range. Thus, secondary digitalization of analog scintigrams is an easy, cheap and reliable method of archiving images and allows secondary digital quantification. (orig.) [de

  5. [A simple method of digitizing analog scintigrams for quantification and digital archiving].

    Science.gov (United States)

    Schramm, M; Kämpfer, B; Wolf, H; Clausen, M; Wendhausen, H; Henze, E

    1993-02-01

    This study was undertaken to evaluate a quick, reliable and cheap method of digitizing analog scintigrams. 40 whole-body bone scintigrams were obtained simultaneously in analog and genuine digital format. The analog scans on x-ray film were then digitized secondarily by three different methods: 300 dpi flat-bed scanning, high-resolution camera scanning and camcorder recording. A simple exposure approach using a light box, a cheap camcorder, a PC and image grabber hard- and software proved to be optimal. Visual interpretation showed no differences in clinical findings when comparing the analog images with their secondarily digitized counterparts. To test the possibility of quantification, 126 equivalent ROIs were drawn both in the genuine digital and the secondarily digitized images. Comparing the ROI count to whole-body count percentage of the corresponding ROIs showed the correlation to be linear. The evaluation of phantom studies showed the linear correlation to be true within a wide activity range. Thus, secondary digitalization of analog scintigrams is an easy, cheap and reliable method of archiving images and allows secondary digital quantification.

  6. Empirical quantification of lacustrine groundwater discharge - different methods and their limitations

    Science.gov (United States)

    Meinikmann, K.; Nützmann, G.; Lewandowski, J.

    2015-03-01

    Groundwater discharge into lakes (lacustrine groundwater discharge, LGD) can be an important driver of lake eutrophication. Its quantification is difficult for several reasons, and thus often neglected in water and nutrient budgets of lakes. In the present case several methods were applied to determine the expansion of the subsurface catchment, to reveal areas of main LGD and to identify the variability of LGD intensity. Size and shape of the subsurface catchment served as a prerequisite in order to calculate long-term groundwater recharge and thus the overall amount of LGD. Isotopic composition of near-shore groundwater was investigated to validate the quality of catchment delineation in near-shore areas. Heat as a natural tracer for groundwater-surface water interactions was used to find spatial variations of LGD intensity. Via an analytical solution of the heat transport equation, LGD rates were calculated from temperature profiles of the lake bed. The method has some uncertainties, as can be found from the results of two measurement campaigns in different years. The present study reveals that a combination of several different methods is required for a reliable identification and quantification of LGD and groundwater-borne nutrient loads.

  7. Simultaneous Quantification of Antidiabetic Agents in Human Plasma by a UPLC-QToF-MS Method.

    Directory of Open Access Journals (Sweden)

    Mariana Millan Fachi

    Full Text Available An ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry method for the simultaneous quantification of chlorpropamide, glibenclamide, gliclazide, glimepiride, metformin, nateglinide, pioglitazone, rosiglitazone, and vildagliptin in human plasma was developed and validated, using isoniazid and sulfaquinoxaline as internal standards. Following plasma protein precipitation using acetonitrile with 1% formic acid, chromatographic separation was performed on a cyano column using gradient elution with water and acetonitrile, both containing 0.1% formic acid. Detection was performed in a quadrupole time-of-flight analyzer, using electrospray ionization operated in the positive mode. Data from validation studies demonstrated that the new method is highly sensitive, selective, precise (RSD 0.99, free of matrix and has no residual effects. The developed method was successfully applied to volunteers' plasma samples. Hence, this method was demonstrated to be appropriate for clinical monitoring of antidiabetic agents.

  8. Method Development for Extraction and Quantification of Glycosides in Leaves of Stevia Rebaudiana

    International Nuclear Information System (INIS)

    Salmah Moosa; Hazlina Ahmad Hassali; Norazlina Noordin

    2015-01-01

    A solid-liquid extraction and an UHPLC method for determination of glycosides from the leave parts of Stevia rebaudiana were developed. Steviol glycosides found in the leaves of Stevia are natural sweetener and commercially sold as sugar substitutes. Extraction of the glycosides consisted of solvent extraction of leaf powder using various solvents followed by its concentration using rotary evaporator and analysis using Ultra High Performance Liquid Chromatography (UHPLC). Existing analytical methods are mainly focused on the quantification of either rebaudioside A or stevioside, whereas other glycosides, such as rebaudioside B and rebaudioside D present in the leaves also contribute to sweetness or its biological activity. Therefore, we developed an improved method by changing the UHPLC conditions to enable a rapid and reliable determination of four steviol glycosides rather than just two using an isocratic UHPLC method. (author)

  9. A simple method for DNA isolation from Xanthomonas spp.

    Directory of Open Access Journals (Sweden)

    Gomes Luiz Humberto

    2000-01-01

    Full Text Available A simple DNA isolation method was developed with routine chemicals that yields high quality and integrity preparations when compared to some of the most well known protocols. The method described does not require the use of lysing enzymes, water bath and the DNA was obtained within 40 minutes The amount of nucleic acid extracted (measured in terms of absorbancy at 260 nm from strains of Xanthomonas spp., Pseudomonas spp. and Erwinia spp. was two to five times higher than that of the most commonly used method.

  10. Prospects for DNA methods to measure human heritable mutation rates

    International Nuclear Information System (INIS)

    Mendelsohn, M.L.

    1985-01-01

    A workshop cosponsored by ICPEMC and the US Department of Energy was held in Alta, Utah, December 9-13, 1984 to examine the extent to which DNA-oriented methods might provide new approaches to the important but intractable problem of measuring mutation rates in control and exposed human populations. The workshop identified and analyzed six DNA methods for detection of human heritable mutation, including several created at the meeting, and concluded that none of the methods combine sufficient feasibility and efficiency to be recommended for general application. 8 refs

  11. Quantification of {sup 18}F-florbetapir PET: comparison of two analysis methods

    Energy Technology Data Exchange (ETDEWEB)

    Hutton, Chloe; Declerck, Jerome [Siemens Molecular Imaging, Oxford (United Kingdom); Mintun, Mark A.; Pontecorvo, Michael J.; Devous, Michael D.; Joshi, Abhinay D. [Avid Radiopharmaceuticals a wholly owned subsidiary of Eli Lilly and Company, Philadelphia, PA (United States); Collaboration: for the Alzheimer' s Disease Neuroimaging Initiative

    2015-04-01

    {sup 18}F-Florbetapir positron emission tomography (PET) can be used to image amyloid burden in the human brain. A previously developed research method has been shown to have a high test-retest reliability and good correlation between standardized uptake value ratio (SUVR) and amyloid burden at autopsy. The goal of this study was to determine how well SUVRs computed using the research method could be reproduced using an automatic quantification method, developed for clinical use. Two methods for the quantitative analysis of {sup 18}F-florbetapir PET were compared in a diverse clinical population of 604 subjects from the Alzheimer's Disease Neuroimaging Initiative (ADNI) and in a group of 74 younger healthy controls (YHC). Cortex to cerebellum SUVRs were calculated using the research method, which is based on SPM, yielding 'research SUVRs', and using syngo.PET Amyloid Plaque, yielding 'sPAP SUVRs'. Mean cortical SUVRs calculated using the two methods for the 678 subjects were correlated (r = 0.99). Linear regression of sPAP SUVRs on research SUVRs was used to convert the research method SUVR threshold for florbetapir positivity of 1.10 to a corresponding threshold of 1.12 for sPAP. Using the corresponding thresholds, categorization of SUVR values were in agreement between research and sPAP SUVRs for 96.3 % of the ADNI images. SUVRs for all YHC were below the corresponding thresholds. Automatic florbetapir PET quantification using sPAP yielded cortex to cerebellum SUVRs which were correlated and in good agreement with the well-established research method. The research SUVR threshold for florbetapir positivity was reliably converted to a corresponding threshold for sPAP SUVRs. (orig.)

  12. Quantification of 18F-florbetapir PET: comparison of two analysis methods

    International Nuclear Information System (INIS)

    Hutton, Chloe; Declerck, Jerome; Mintun, Mark A.; Pontecorvo, Michael J.; Devous, Michael D.; Joshi, Abhinay D.

    2015-01-01

    18 F-Florbetapir positron emission tomography (PET) can be used to image amyloid burden in the human brain. A previously developed research method has been shown to have a high test-retest reliability and good correlation between standardized uptake value ratio (SUVR) and amyloid burden at autopsy. The goal of this study was to determine how well SUVRs computed using the research method could be reproduced using an automatic quantification method, developed for clinical use. Two methods for the quantitative analysis of 18 F-florbetapir PET were compared in a diverse clinical population of 604 subjects from the Alzheimer's Disease Neuroimaging Initiative (ADNI) and in a group of 74 younger healthy controls (YHC). Cortex to cerebellum SUVRs were calculated using the research method, which is based on SPM, yielding 'research SUVRs', and using syngo.PET Amyloid Plaque, yielding 'sPAP SUVRs'. Mean cortical SUVRs calculated using the two methods for the 678 subjects were correlated (r = 0.99). Linear regression of sPAP SUVRs on research SUVRs was used to convert the research method SUVR threshold for florbetapir positivity of 1.10 to a corresponding threshold of 1.12 for sPAP. Using the corresponding thresholds, categorization of SUVR values were in agreement between research and sPAP SUVRs for 96.3 % of the ADNI images. SUVRs for all YHC were below the corresponding thresholds. Automatic florbetapir PET quantification using sPAP yielded cortex to cerebellum SUVRs which were correlated and in good agreement with the well-established research method. The research SUVR threshold for florbetapir positivity was reliably converted to a corresponding threshold for sPAP SUVRs. (orig.)

  13. Optimized methods for total nucleic acid extraction and quantification of the bat white-nose syndrome fungus, Pseudogymnoascus destructans, from swab and environmental samples.

    Science.gov (United States)

    Verant, Michelle L; Bohuski, Elizabeth A; Lorch, Jeffery M; Blehert, David S

    2016-03-01

    The continued spread of white-nose syndrome and its impacts on hibernating bat populations across North America has prompted nationwide surveillance efforts and the need for high-throughput, noninvasive diagnostic tools. Quantitative real-time polymerase chain reaction (qPCR) analysis has been increasingly used for detection of the causative fungus, Pseudogymnoascus destructans, in both bat- and environment-associated samples and provides a tool for quantification of fungal DNA useful for research and monitoring purposes. However, precise quantification of nucleic acid from P. destructans is dependent on effective and standardized methods for extracting nucleic acid from various relevant sample types. We describe optimized methodologies for extracting fungal nucleic acids from sediment, guano, and swab-based samples using commercial kits together with a combination of chemical, enzymatic, and mechanical modifications. Additionally, we define modifications to a previously published intergenic spacer-based qPCR test for P. destructans to refine quantification capabilities of this assay. © 2016 The Author(s).

  14. Optimized methods for total nucleic acid extraction and quantification of the bat white-nose syndrome fungus, Pseudogymnoascus destructans, from swab and environmental samples

    Science.gov (United States)

    Verant, Michelle; Bohuski, Elizabeth A.; Lorch, Jeffrey M.; Blehert, David

    2016-01-01

    The continued spread of white-nose syndrome and its impacts on hibernating bat populations across North America has prompted nationwide surveillance efforts and the need for high-throughput, noninvasive diagnostic tools. Quantitative real-time polymerase chain reaction (qPCR) analysis has been increasingly used for detection of the causative fungus, Pseudogymnoascus destructans, in both bat- and environment-associated samples and provides a tool for quantification of fungal DNA useful for research and monitoring purposes. However, precise quantification of nucleic acid fromP. destructans is dependent on effective and standardized methods for extracting nucleic acid from various relevant sample types. We describe optimized methodologies for extracting fungal nucleic acids from sediment, guano, and swab-based samples using commercial kits together with a combination of chemical, enzymatic, and mechanical modifications. Additionally, we define modifications to a previously published intergenic spacer–based qPCR test for P. destructans to refine quantification capabilities of this assay.

  15. Comparison of Six DNA Extraction Procedures and the Application of Plastid DNA Enrichment Methods in Selected Non-photosynthetic Plants

    Directory of Open Access Journals (Sweden)

    Shin-Yi Shyu

    2013-12-01

    Full Text Available Genomic DNA was isolated using three DNA extraction commercial kits and three CTAB-based methods for two non-photosynthetic plants, Balanophora japonica and Mitrastemon kanehirai. The quality of the isolated DNA was evaluated and subjected to following restriction enzyme digestions. All six procedures yielded DNA of sufficient quality for PCR, and the method described by Barnwell et al. (1998 performed well in isolating DNA from both species for restriction enzyme digestion. In addition, we succeeded to enrich plastid DNA content by using the methods depending on a high salt buffer to deplete nuclear material. The ‘high salt’ methods based on protocol presented by Milligan (1989 were able to increase plastid DNA effectively and significantly reduce nuclear DNA from M. kanehirai. The plastid DNA enrichment protocols are inexpensive and not time-consuming, and may be applicable to other non-photosynthetic plants.

  16. Emphysema quantification from CT scans using novel application of diaphragm curvature estimation: comparison with standard quantification methods and pulmonary function data

    Science.gov (United States)

    Keller, Brad M.; Reeves, Anthony P.; Yankelevitz, David F.; Henschke, Claudia I.; Barr, R. Graham

    2009-02-01

    Emphysema is a disease of the lungs that destroys the alveolar air sacs and induces long-term respiratory dysfunction. CT scans allow for the imaging of the anatomical basis of emphysema and quantification of the underlying disease state. Several measures have been introduced for the quantification emphysema directly from CT data; most,however, are based on the analysis of density information provided by the CT scans, which vary by scanner and can be hard to standardize across sites and time. Given that one of the anatomical variations associated with the progression of emphysema is the flatting of the diaphragm due to the loss of elasticity in the lung parenchyma, curvature analysis of the diaphragm would provide information about emphysema from CT. Therefore, we propose a new, non-density based measure of the curvature of the diaphragm that would allow for further quantification methods in a robust manner. To evaluate the new method, 24 whole-lung scans were analyzed using the ratios of the lung height and diaphragm width to diaphragm height as curvature estimates as well as using the emphysema index as comparison. Pearson correlation coefficients showed a strong trend of several of the proposed diaphragm curvature measures to have higher correlations, of up to r=0.57, with DLCO% and VA than did the emphysema index. Furthermore, we found emphysema index to have only a 0.27 correlation to the proposed measures, indicating that the proposed measures evaluate different aspects of the disease.

  17. A simple method for purification of herpesvirus DNA

    DEFF Research Database (Denmark)

    Christensen, Laurids Siig; Normann, Preben

    1992-01-01

    A rapid and reliable method for purification of herpesvirus DNA from cell cultures is described. The method is based on the isolation of virus particles and/or nucleocapsids by differential centrifugation and exploits the solubilizing and denaturing capabilities of cesium trifluoroacetate during...

  18. Sensitive quantification of apomorphine in human plasma using a LC-ESI-MS-MS method.

    Science.gov (United States)

    Abe, Emuri; Alvarez, Jean-Claude

    2006-06-01

    An analytical method based on liquid chromatography coupled with ion trap mass spectrometry (MS) detection with electrospray ionization interface has been developed for the identification and quantification of apomorphine in human plasma. Apomorphine was isolated from 0.5 mL of plasma using a liquid-liquid extraction with diethyl ether and boldine as internal standard, with satisfactory extraction recoveries. Analytes were separated on a 5-microm C18 Highpurity (Thermohypersil) column (150 mm x 2.1 mm I.D.) maintained at 30 degrees C, coupled to a precolumn (C18, 5-microm, 10 mm x 2.0 mm I.D., Thermo). The elution was achieved isocratically with a mobile phase of 2 mM NH4COOH buffer pH 3.8/acetonitrile (50/50, vol/vol) at a flow rate of 200 microL per minute. Data were collected either in full-scan MS mode at m/z 150 to 500 or in full-scan tandem mass spectrometry mode, selecting the [M+H]ion at m/z 268.0 for apomorphine and m/z 328.0 for boldine. The most intense daughter ion of apomorphine (m/z 237.1) and boldine (m/z 297.0) were used for quantification. Retention times were 2.03 and 2.11 minutes for boldine and apomorphine, respectively. Calibration curves were linear in the 0.025 to 20 ng/mL range. The limits of detection and quantification were 0.010 ng/mL and 0.025 ng/mL, respectively. Accuracy and precision of the assay were measured by analyzing 54 quality control samples for 3 days. At concentrations of 0.075, 1.5, and 15 ng/mL, intraday precisions were less than 10.1%, 5.3%, and 3.8%, and interday precisions were less than 4.8%, 6.6%, and 6.5%, respectively. Accuracies were in the 99.5 to 104.2% range. An example of a patient who was given 6 mg of apomorphine subcutaneously is shown, with concentrations of 14.1 ng/mL after 30 minutes and 0.20 ng/mL after 6 hours. The method described enables the unambiguous identification and quantification of apomorphine with very good sensitivity using only 0.5 mL of sample, and is very convenient for therapeutic drug

  19. A novel method of genomic DNA extraction for Cactaceae1

    Science.gov (United States)

    Fehlberg, Shannon D.; Allen, Jessica M.; Church, Kathleen

    2013-01-01

    • Premise of the study: Genetic studies of Cactaceae can at times be impeded by difficult sampling logistics and/or high mucilage content in tissues. Simplifying sampling and DNA isolation through the use of cactus spines has not previously been investigated. • Methods and Results: Several protocols for extracting DNA from spines were tested and modified to maximize yield, amplification, and sequencing. Sampling of and extraction from spines resulted in a simplified protocol overall and complete avoidance of mucilage as compared to typical tissue extractions. Sequences from one nuclear and three plastid regions were obtained across eight genera and 20 species of cacti using DNA extracted from spines. • Conclusions: Genomic DNA useful for amplification and sequencing can be obtained from cactus spines. The protocols described here are valuable for any cactus species, but are particularly useful for investigators interested in sampling living collections, extensive field sampling, and/or conservation genetic studies. PMID:25202521

  20. A RP-HPLC method for quantification of diclofenac sodium released from biological macromolecules.

    Science.gov (United States)

    Bhattacharya, Shiv Sankar; Banerjee, Subham; Ghosh, Ashoke Kumar; Chattopadhyay, Pronobesh; Verma, Anurag; Ghosh, Amitava

    2013-07-01

    Interpenetrating network (IPN) microbeads of sodium carboxymethyl locust bean gum (SCMLBG) and sodium carboxymethyl cellulose (SCMC) containing diclofenac sodium (DS), a nonsteroidal anti-inflammatory drug, were prepared by single water-in-water (w/w) emulsion gelation process using AlCl3 as cross-linking agent in a complete aqueous environment. Pharmacokinetic study of these IPN microbeads was then carried out by a simple and feasible high-performance liquid chromatographic method with UV detection which was developed and validated for the quantification of diclofenac sodium in rabbit plasma. The chromatographic separation was carried out in a Hypersil BDS, C18 column (250 mm × 4.6 mm; 5 m). The mobile phase was a mixture of acetonitrile and methanol (70:30, v/v) at a flow rate of 1.0 ml/min. The UV detection was set at 276 nm. The extraction recovery of diclofenac sodium in plasma of three quality control (QC) samples was ranged from 81.52% to 95.29%. The calibration curve was linear in the concentration range of 20-1000 ng/ml with the correlation coefficient (r(2)) above 0.9951. The method was specific and sensitive with the limit of quantification of 20 ng/ml. In stability tests, diclofenac sodium in rabbit plasma was stable during storage and assay procedure. Copyright © 2013. Published by Elsevier B.V.

  1. Methods for modeling and quantification in functional imaging by positron emissions tomography and magnetic resonance imaging

    International Nuclear Information System (INIS)

    Costes, Nicolas

    2017-01-01

    This report presents experiences and researches in the field of in vivo medical imaging by positron emission tomography (PET) and magnetic resonance imaging (MRI). In particular, advances in terms of reconstruction, quantification and modeling in PET are described. The validation of processing and analysis methods is supported by the creation of data by simulation of the imaging process in PET. The recent advances of combined PET/MRI clinical cameras, allowing simultaneous acquisition of molecular/metabolic PET information, and functional/structural MRI information opens the door to unique methodological innovations, exploiting spatial alignment and simultaneity of the PET and MRI signals. It will lead to an increase in accuracy and sensitivity in the measurement of biological phenomena. In this context, the developed projects address new methodological issues related to quantification, and to the respective contributions of MRI or PET information for a reciprocal improvement of the signals of the two modalities. They open perspectives for combined analysis of the two imaging techniques, allowing optimal use of synchronous, anatomical, molecular and functional information for brain imaging. These innovative concepts, as well as data correction and analysis methods, will be easily translated into other areas of investigation using combined PET/MRI. (author) [fr

  2. Methods for deep examination of DNA

    OpenAIRE

    Neiman, Mårten

    2013-01-01

    The development of sequencing technology has had a rapid pace during the last years and today, the sequencing instruments harbors enormous capacity. This thesis is about the development of methods to make the most out of this capacity and to use it for various applications. In paper I, a dual tagging system for sequencing large sample sets was developed. To proof the concept, 4,700 dogs were subjected to amplicon sequencing of the 2nd exon of the gene DLA-DRB1 using a 454...

  3. A machine learning approach for efficient uncertainty quantification using multiscale methods

    Science.gov (United States)

    Chan, Shing; Elsheikh, Ahmed H.

    2018-02-01

    Several multiscale methods account for sub-grid scale features using coarse scale basis functions. For example, in the Multiscale Finite Volume method the coarse scale basis functions are obtained by solving a set of local problems over dual-grid cells. We introduce a data-driven approach for the estimation of these coarse scale basis functions. Specifically, we employ a neural network predictor fitted using a set of solution samples from which it learns to generate subsequent basis functions at a lower computational cost than solving the local problems. The computational advantage of this approach is realized for uncertainty quantification tasks where a large number of realizations has to be evaluated. We attribute the ability to learn these basis functions to the modularity of the local problems and the redundancy of the permeability patches between samples. The proposed method is evaluated on elliptic problems yielding very promising results.

  4. Feasibility of the left ventricular volume measurement by acoustic quantification method. Comparison with ultrafast computed tomography

    International Nuclear Information System (INIS)

    Tomimoto, Shigehiro; Nakatani, Satoshi; Tanaka, Norio; Uematsu, Masaaki; Beppu, Shintaro; Nagata, Seiki; Hamada, Seiki; Takamiya, Makoto; Miyatake, Kunio

    1995-01-01

    Acoustic quantification (AQ: the real-time automated boundary detection system) allows instantaneous measurement of cardiac chamber volumes. The feasibility of this method was evaluated by comparing the left ventricular (LV) volumes obtained with AQ to those derived from ultrafast computed tomography (UFCT), which enables accurate measurements of LV volumes even in the presence of LV asynergy, in 23 patients (8 with ischemic heart disease, 5 with cardiomyopathy, 3 with valvular heart disease). Both LV end-diastolic and end-systolic volumes obtained with the AQ method were in good agreement with those obtained with UFCT (y=1.04χ-16.9, r=0.95; y=0.87χ+15.7, r=0.91; respectively). AQ was reliable even in the presence of LV asynergy. Interobserver variability for the AQ measurement was 10.2%. AQ provides a new, clinically useful method for real-time accurate estimation of the left ventricular volume. (author)

  5. Feasibility of the left ventricular volume measurement by acoustic quantification method. Comparison with ultrafast computed tomography

    Energy Technology Data Exchange (ETDEWEB)

    Tomimoto, Shigehiro; Nakatani, Satoshi; Tanaka, Norio; Uematsu, Masaaki; Beppu, Shintaro; Nagata, Seiki; Hamada, Seiki; Takamiya, Makoto; Miyatake, Kunio [National Cardiovascular Center, Suita, Osaka (Japan)

    1995-01-01

    Acoustic quantification (AQ: the real-time automated boundary detection system) allows instantaneous measurement of cardiac chamber volumes. The feasibility of this method was evaluated by comparing the left ventricular (LV) volumes obtained with AQ to those derived from ultrafast computed tomography (UFCT), which enables accurate measurements of LV volumes even in the presence of LV asynergy, in 23 patients (8 with ischemic heart disease, 5 with cardiomyopathy, 3 with valvular heart disease). Both LV end-diastolic and end-systolic volumes obtained with the AQ method were in good agreement with those obtained with UFCT (y=1.04{chi}-16.9, r=0.95; y=0.87{chi}+15.7, r=0.91; respectively). AQ was reliable even in the presence of LV asynergy. Interobserver variability for the AQ measurement was 10.2%. AQ provides a new, clinically useful method for real-time accurate estimation of the left ventricular volume. (author).

  6. Reliability Quantification Method for Safety Critical Software Based on a Finite Test Set

    International Nuclear Information System (INIS)

    Shin, Sung Min; Kim, Hee Eun; Kang, Hyun Gook; Lee, Seung Jun

    2014-01-01

    Software inside of digitalized system have very important role because it may cause irreversible consequence and affect the whole system as common cause failure. However, test-based reliability quantification method for some safety critical software has limitations caused by difficulties in developing input sets as a form of trajectory which is series of successive values of variables. To address these limitations, this study proposed another method which conduct the test using combination of single values of variables. To substitute the trajectory form of input using combination of variables, the possible range of each variable should be identified. For this purpose, assigned range of each variable, logical relations between variables, plant dynamics under certain situation, and characteristics of obtaining information of digital device are considered. A feasibility of the proposed method was confirmed through an application to the Reactor Protection System (RPS) software trip logic

  7. HUMAN ERROR QUANTIFICATION USING PERFORMANCE SHAPING FACTORS IN THE SPAR-H METHOD

    Energy Technology Data Exchange (ETDEWEB)

    Harold S. Blackman; David I. Gertman; Ronald L. Boring

    2008-09-01

    This paper describes a cognitively based human reliability analysis (HRA) quantification technique for estimating the human error probabilities (HEPs) associated with operator and crew actions at nuclear power plants. The method described here, Standardized Plant Analysis Risk-Human Reliability Analysis (SPAR-H) method, was developed to aid in characterizing and quantifying human performance at nuclear power plants. The intent was to develop a defensible method that would consider all factors that may influence performance. In the SPAR-H approach, calculation of HEP rates is especially straightforward, starting with pre-defined nominal error rates for cognitive vs. action-oriented tasks, and incorporating performance shaping factor multipliers upon those nominal error rates.

  8. Comparison of methods for the quantification of the different carbon fractions in atmospheric aerosol samples

    Science.gov (United States)

    Nunes, Teresa; Mirante, Fátima; Almeida, Elza; Pio, Casimiro

    2010-05-01

    Atmospheric carbon consists of: organic carbon (OC, including various organic compounds), elemental carbon (EC, or black carbon [BC]/soot, a non-volatile/light-absorbing carbon), and a small quantity of carbonate carbon. Thermal/optical methods (TOM) have been widely used for quantifying total carbon (TC), OC, and EC in ambient and source particulate samples. Unfortunately, the different thermal evolution protocols in use can result in a wide elemental carbon-to-total carbon variation. Temperature evolution in thermal carbon analysis is critical to the allocation of carbon fractions. Another critical point in OC and EC quantification by TOM is the interference of carbonate carbon (CC) that could be present in the particulate samples, mainly in the coarse fraction of atmospheric aerosol. One of the methods used to minimize this interference consists on the use of a sample pre-treatment with acid to eliminate CC prior to thermal analysis (Chow et al., 2001; Pio et al., 1994). In Europe, there is currently no standard procedure for determining the carbonaceous aerosol fraction, which implies that data from different laboratories at various sites are of unknown accuracy and cannot be considered comparable. In the framework of the EU-project EUSAAR, a comprehensive study has been carried out to identify the causes of differences in the EC measured using different thermal evolution protocols. From this study an optimised protocol, the EUSAAR-2 protocol, was defined (Cavali et al., 2009). During the last two decades thousands of aerosol samples have been taken over quartz filters at urban, industrial, rural and background sites, and also from plume forest fires and biomass burning in a domestic closed stove. These samples were analysed for OC and EC, by a TOM, similar to that in use in the IMPROVE network (Pio et al., 2007). More recently we reduced the number of steps in thermal evolution protocols, without significant repercussions in the OC/EC quantifications. In order

  9. Quantification of Pseudomonas fluorescens strains F113, CHA0 and Pf153 in the rhizosphere of maize by strain-specific real-time PCR unaffected by the variability of DNA extraction efficiency.

    Science.gov (United States)

    Von Felten, Andreas; Défago, Geneviève; Maurhofer, Monika

    2010-05-01

    Pseudomonas fluorescens strains F113 and CHA0 are well-known plant growth-promoting rhizobacteria (PGPR) often used as model strains in biocontrol experiments. To monitor their persistence in large scale field experiments, culture-independent methods are needed. In this study, a strain-specific real-time PCR quantification tool was developed based on sequence-characterized amplified regions (SCAR) for P. fluorescens strains F113, CHA0 and Pf153. Differences in DNA extraction efficiencies from rhizosphere samples were circumvented using plasmid APA9 as internal standard to normalize C(T) values after real-time amplification. The detection limits of the real-time PCR assays for all three strains were approximately 10 cells for genomic DNA and 10(4)cells/g rhizosphere for maize samples grown in different natural soils. Population sizes of the three strains in the rhizosphere of maize measured by the new real-time PCR approaches were similar to those measured by most probable number (MPN)-PCR. A persistence study of the three strains indicated that the strains persisted differently over a period of 5weeks. In conclusion the newly developed real-time PCR approach is a fast and resource efficient method for monitoring individual biocontrol strains in natural soil, which makes it an apt quantification tool for future large-scale field experiments. Copyright 2010 Elsevier B.V. All rights reserved.

  10. A Novel Computational Method for Detecting DNA Methylation Sites with DNA Sequence Information and Physicochemical Properties.

    Science.gov (United States)

    Pan, Gaofeng; Jiang, Limin; Tang, Jijun; Guo, Fei

    2018-02-08

    DNA methylation is an important biochemical process, and it has a close connection with many types of cancer. Research about DNA methylation can help us to understand the regulation mechanism and epigenetic reprogramming. Therefore, it becomes very important to recognize the methylation sites in the DNA sequence. In the past several decades, many computational methods-especially machine learning methods-have been developed since the high-throughout sequencing technology became widely used in research and industry. In order to accurately identify whether or not a nucleotide residue is methylated under the specific DNA sequence context, we propose a novel method that overcomes the shortcomings of previous methods for predicting methylation sites. We use k -gram, multivariate mutual information, discrete wavelet transform, and pseudo amino acid composition to extract features, and train a sparse Bayesian learning model to do DNA methylation prediction. Five criteria-area under the receiver operating characteristic curve (AUC), Matthew's correlation coefficient (MCC), accuracy (ACC), sensitivity (SN), and specificity-are used to evaluate the prediction results of our method. On the benchmark dataset, we could reach 0.8632 on AUC, 0.8017 on ACC, 0.5558 on MCC, and 0.7268 on SN. Additionally, the best results on two scBS-seq profiled mouse embryonic stem cells datasets were 0.8896 and 0.9511 by AUC, respectively. When compared with other outstanding methods, our method surpassed them on the accuracy of prediction. The improvement of AUC by our method compared to other methods was at least 0.0399 . For the convenience of other researchers, our code has been uploaded to a file hosting service, and can be downloaded from: https://figshare.com/s/0697b692d802861282d3.

  11. Application of radioanalytical methods in the quantification of solute transport in plants

    International Nuclear Information System (INIS)

    Hornik, M.

    2016-01-01

    The present habilitation thesis is elaborated as a compilation of published scientific papers supplemented with a commentary. The primary objective of the work was to bring the results and knowledge applicable to the further development of application possibilities of nuclear analytical chemistry, especially in the field of radioindication methods and application of positron emitters in connection with the positron emission tomography (PET) as well. In the work, these methods and techniques are developed mainly in the context of the solution of environmental issues related to the analysis and remediation of contaminated or degraded environment (water and soil), but also partially in the field of plant production or plant research. In terms of the achieved results and knowledge, the work is divided into three separated sections. The first part is dedicated to the application of radioindication methods, as well as others, non-radioanalytical methods and approaches in the characterization of plant biomass (biomass of terrestrial and aquatic mosses, and waste plant biomass) as alternative sorbents served to the separation and removal of (radio)toxic metals from contaminated or waste waters, as well as in the quantification and description of the sorption processes proceed under conditions of batch or continuous flow systems. The second part describes the results concerning on the quantification and visual description of the processes of (radio)toxic metals and microelements uptake and translocation in plant tissues using radioisotopes (β- and γ-emitters) of these metals and application of the methods of direct gamma spectrometry and autoradiography as well. The main aim of these experiments was to evaluate the possibilities of utilization of selected plant species in phytoremediation of contaminated soils and waters, as well as the possibilities affecting the effectiveness of uptake and translocation of these metals in the plant tissues mainly in dependence on their

  12. LuxGLM: a probabilistic covariate model for quantification of DNA methylation modifications with complex experimental designs.

    Science.gov (United States)

    Äijö, Tarmo; Yue, Xiaojing; Rao, Anjana; Lähdesmäki, Harri

    2016-09-01

    5-methylcytosine (5mC) is a widely studied epigenetic modification of DNA. The ten-eleven translocation (TET) dioxygenases oxidize 5mC into oxidized methylcytosines (oxi-mCs): 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). DNA methylation modifications have multiple functions. For example, 5mC is shown to be associated with diseases and oxi-mC species are reported to have a role in active DNA demethylation through 5mC oxidation and DNA repair, among others, but the detailed mechanisms are poorly understood. Bisulphite sequencing and its various derivatives can be used to gain information about all methylation modifications at single nucleotide resolution. Analysis of bisulphite based sequencing data is complicated due to the convoluted read-outs and experiment-specific variation in biochemistry. Moreover, statistical analysis is often complicated by various confounding effects. How to analyse 5mC and oxi-mC data sets with arbitrary and complex experimental designs is an open and important problem. We propose the first method to quantify oxi-mC species with arbitrary covariate structures from bisulphite based sequencing data. Our probabilistic modeling framework combines a previously proposed hierarchical generative model for oxi-mC-seq data and a general linear model component to account for confounding effects. We show that our method provides accurate methylation level estimates and accurate detection of differential methylation when compared with existing methods. Analysis of novel and published data gave insights into to the demethylation of the forkhead box P3 (Foxp3) locus during the induced T regulatory cell differentiation. We also demonstrate how our covariate model accurately predicts methylation levels of the Foxp3 locus. Collectively, LuxGLM method improves the analysis of DNA methylation modifications, particularly for oxi-mC species. An implementation of the proposed method is available under MIT license at https

  13. Abundance of four sulfur mustard-DNA adducts ex vivo and in vivo revealed by simultaneous quantification in stable isotope dilution-ultrahigh performance liquid chromatography-tandem mass spectrometry.

    Science.gov (United States)

    Yue, Lijun; Wei, Yuxia; Chen, Jia; Shi, Huiqin; Liu, Qin; Zhang, Yajiao; He, Jun; Guo, Lei; Zhang, Tingfen; Xie, Jianwei; Peng, Shuangqing

    2014-04-21

    Sulfur mustard (SM) is a highly reactive alkylating vesicant and causes blisters upon contact with skin, eyes, and respiratory organs. It covalently links with DNAs by forming four mono- or cross-link adducts. In this article, the reference standards of SM-DNA adducts and deuterated analogues were first synthesized with simplified procedures containing only one or two steps and using less toxic chemical 2-(2-chloroethylthio)ethanol or nontoxic chemical thiodiglycol as starting materials. A sensitive and high-throughput simultaneous quantification method of N(7)-[2-[(2-hydroxyethyl)thio]-ethyl]guanine (N(7)-HETEG), O(6)-[2-[(2-hydroxyethyl)thio]-ethyl]guanine (O(6)-HETEG), N(3)-[2-[(2-hydroxyethyl)thio]-ethyl]adenine (N(3)-HETEA), and bis[2-(guanin-7-yl)ethyl]sulfide (Bis-G) in the Sprague-Dawley rat derma samples was developed by stable isotope dilution-ultrahigh performance liquid chromatography-tandem mass spectrometry (ID-UPLC-MS/MS) with the aim of revealing the real metabolic behaviors of four adducts. The method was validated, the limit of detection (S/N ratio greater than 10) was 0.01, 0.002, 0.04, and 0.11 fmol on column for N(7)-HETEG, O(6)-HETEG, Bis-G, and N(3)-HETEA, respectively, and the lower limit of quantification (S/N ratio greater than 20) was 0.04, 0.01, 0.12, and 0.33 fmol on column for N(7)-HETEG, O(6)-HETEG, Bis-G, and N(3)-HETEA, respectively. The accuracy of this method was determined to be 76% to 129% (n = 3), and both the interday (n = 6) and intraday (n = 7) precisions were less than 10%. The method was further applied for the quantifications of four adducts in the derma of adult male Sprague-Dawley rats exposed to SM ex vivo and in vivo, and all adducts had time- and dose-effect relationships. To the best of our knowledge, this is the first time that the real presented status of four DNA adducts was simultaneously revealed by the MS-based method, in which Bis-G showed much higher abundance than the result previously reported and N(3

  14. Flow cytometry for intracellular SPION quantification: specificity and sensitivity in comparison with spectroscopic methods

    Directory of Open Access Journals (Sweden)

    Friedrich RP

    2015-06-01

    Full Text Available Ralf P Friedrich,1 Christina Janko,1 Marina Poettler,1 Philipp Tripal,1 Jan Zaloga,1 Iwona Cicha,1 Stephan Dürr,1,2 Johannes Nowak,3 Stefan Odenbach,3 Ioana Slabu,4 Maik Liebl,4 Lutz Trahms,4 Marcus Stapf,5 Ingrid Hilger,5 Stefan Lyer,1 Christoph Alexiou1 1Department of Otorhinolaryngology, Head and Neck Surgery, Section of Experimental Oncology and Nanomedicine, University hospital Erlangen, 2Department of Otorhinolaryngology, Head and Neck Surgery, Section of Phoniatrics and Pediatric Audiology, University hospital Erlangen, Erlangen, 3Technische Universität Dresden, Chair of Magnetofluiddynamics, Measuring and Automation Technology, Dresden, 4Physikalisch-Technische Bundesanstalt Berlin, Berlin, 5Department of Radiology, Division of Diagnostic and Interventional Radiology, Experimental Radiology, University hospital Jena, Jena, Germany Abstract: Due to their special physicochemical properties, iron nanoparticles offer new promising possibilities for biomedical applications. For bench to bedside translation of superparamagnetic iron oxide nanoparticles (SPIONs, safety issues have to be comprehensively clarified. To understand concentration-dependent nanoparticle-mediated toxicity, the exact quantification of intracellular SPIONs by reliable methods is of great importance. In the present study, we compared three different SPION quantification methods (ultraviolet spectrophotometry, magnetic particle spectroscopy, atomic adsorption spectroscopy and discussed the shortcomings and advantages of each method. Moreover, we used those results to evaluate the possibility to use flow cytometric technique to determine the cellular SPION content. For this purpose, we correlated the side scatter data received from flow cytometry with the actual cellular SPION amount. We showed that flow cytometry provides a rapid and reliable method to assess the cellular SPION content. Our data also demonstrate that internalization of iron oxide nanoparticles in human

  15. Automated quantification of renal interstitial fibrosis for computer-aided diagnosis: A comprehensive tissue structure segmentation method.

    Science.gov (United States)

    Tey, Wei Keat; Kuang, Ye Chow; Ooi, Melanie Po-Leen; Khoo, Joon Joon

    2018-03-01

    Interstitial fibrosis in renal biopsy samples is a scarring tissue structure that may be visually quantified by pathologists as an indicator to the presence and extent of chronic kidney disease. The standard method of quantification by visual evaluation presents reproducibility issues in the diagnoses. This study proposes an automated quantification system for measuring the amount of interstitial fibrosis in renal biopsy images as a consistent basis of comparison among pathologists. The system extracts and segments the renal tissue structures based on colour information and structural assumptions of the tissue structures. The regions in the biopsy representing the interstitial fibrosis are deduced through the elimination of non-interstitial fibrosis structures from the biopsy area and quantified as a percentage of the total area of the biopsy sample. A ground truth image dataset has been manually prepared by consulting an experienced pathologist for the validation of the segmentation algorithms. The results from experiments involving experienced pathologists have demonstrated a good correlation in quantification result between the automated system and the pathologists' visual evaluation. Experiments investigating the variability in pathologists also proved the automated quantification error rate to be on par with the average intra-observer variability in pathologists' quantification. Interstitial fibrosis in renal biopsy samples is a scarring tissue structure that may be visually quantified by pathologists as an indicator to the presence and extent of chronic kidney disease. The standard method of quantification by visual evaluation presents reproducibility issues in the diagnoses due to the uncertainties in human judgement. An automated quantification system for accurately measuring the amount of interstitial fibrosis in renal biopsy images is presented as a consistent basis of comparison among pathologists. The system identifies the renal tissue structures

  16. Recombinant methods for screening human DNA excision repair proficiency

    International Nuclear Information System (INIS)

    Athas, W.F.

    1988-01-01

    A method for measuring DNA excision repair in response to ultraviolet radiation (UV)-induced DNA damage has been developed, validated, and field-tested in cultured human lymphocytes. The methodology is amenable to population-based screening and should facilitate future epidemiologic studies seeking to investigate associations between excision repair proficiency and cancer susceptibility. The impetus for such endeavors derives from the belief that the high incidence of skin cancer in the genetic disorder xeroderma pigmentosum (XP) primarily is a result of the reduced capacity of patients cells to repair UV-induced DNA damage. For assay, UV-irradiated non-replicating recombinant plasmid DNA harboring a chloramphenicol acetyltransferase (CAT) indicator gene is introduced into lymphocytes using DEAE-dextran short-term transfection conditions. Exposure to UV induces transcriptionally-inactivating DNA photoproducts in the plasmid DNA which inactivate CAT gene expression. Excision repair of the damaged CAT gene is monitored indirectly as a function of reactivated CAT enzyme activity following a 40 hour repair/expression incubation period

  17. A Novel Computational Method for Detecting DNA Methylation Sites with DNA Sequence Information and Physicochemical Properties

    Directory of Open Access Journals (Sweden)

    Gaofeng Pan

    2018-02-01

    Full Text Available DNA methylation is an important biochemical process, and it has a close connection with many types of cancer. Research about DNA methylation can help us to understand the regulation mechanism and epigenetic reprogramming. Therefore, it becomes very important to recognize the methylation sites in the DNA sequence. In the past several decades, many computational methods—especially machine learning methods—have been developed since the high-throughout sequencing technology became widely used in research and industry. In order to accurately identify whether or not a nucleotide residue is methylated under the specific DNA sequence context, we propose a novel method that overcomes the shortcomings of previous methods for predicting methylation sites. We use k-gram, multivariate mutual information, discrete wavelet transform, and pseudo amino acid composition to extract features, and train a sparse Bayesian learning model to do DNA methylation prediction. Five criteria—area under the receiver operating characteristic curve (AUC, Matthew’s correlation coefficient (MCC, accuracy (ACC, sensitivity (SN, and specificity—are used to evaluate the prediction results of our method. On the benchmark dataset, we could reach 0.8632 on AUC, 0.8017 on ACC, 0.5558 on MCC, and 0.7268 on SN. Additionally, the best results on two scBS-seq profiled mouse embryonic stem cells datasets were 0.8896 and 0.9511 by AUC, respectively. When compared with other outstanding methods, our method surpassed them on the accuracy of prediction. The improvement of AUC by our method compared to other methods was at least 0.0399 . For the convenience of other researchers, our code has been uploaded to a file hosting service, and can be downloaded from: https://figshare.com/s/0697b692d802861282d3.

  18. A HPLC method for the quantification of butyramide and acetamide at ppb levels in hydrogeothermal waters

    Energy Technology Data Exchange (ETDEWEB)

    Gracy Elias; Earl D. Mattson; Jessica E. Little

    2012-01-01

    A quantitative analytical method to determine butyramide and acetamide concentrations at the low ppb levels in geothermal waters has been developed. The analytes are concentrated in a preparation step by evaporation and analyzed using HPLC-UV. Chromatographic separation is achieved isocratically with a RP C-18 column using a 30 mM phosphate buffer solution with 5 mM heptane sulfonic acid and methanol (98:2 ratio) as the mobile phase. Absorbance is measured at 200 nm. The limit of detection (LOD) for BA and AA were 2.0 {mu}g L{sup -1} and 2.5 {mu}g L{sup -1}, respectively. The limit of quantification (LOQ) for BA and AA were 5.7 {mu}g L{sup -1} and 7.7 {mu}g L{sup -1}, respectively, at the detection wavelength of 200 nm. Attaining these levels of quantification better allows these amides to be used as thermally reactive tracers in low-temperature hydrogeothermal systems.

  19. Rapid and simple colorimetric method for the quantification of AI-2 produced from Salmonella Typhimurium.

    Science.gov (United States)

    Wattanavanitchakorn, Siriluck; Prakitchaiwattana, Cheunjit; Thamyongkit, Patchanita

    2014-04-01

    The aim of this study was to evaluate the feasibility of Fe(III) ion reduction for the simple and rapid quantification of autoinducer-2 (AI-2) produced from bacteria using Salmonella Typhimurium as a model. Since the molecular structure of AI-2 is somewhat similar to ascorbic acid it was expected that AI-2 would also act as a reducing agent and reduce Fe(III) ions in the presence of 1,10-phenanthroline to form the colored [(o-phen)3 Fe(II)]SO4 ferroin complex that could be quantified colorimetrically. In support of this, colony rinses and cell free supernatants from cultures of all tested AI-2 producing strains, but not the AI-2 negative Sinorhizobium meliloti, formed a colored complex with a λmax of 510nm. The OD510 values of these culture supernatants or colony rinses were in broad agreement with the % activity observed in the same samples using the standard Vibrio harveyi bioluminescence assay for AI-2 detection, and with previously reported results. This methodology could potentially be developed as an alternative method for the simple and rapid quantification of AI-2 levels produced in bacterial cultures. Copyright © 2014 Elsevier B.V. All rights reserved.

  20. Quantification of Radiation-induced DNA Damage following intracellular Auger-Cascades

    DEFF Research Database (Denmark)

    Fredericia, Nina Pil Møntegaard

    2017-01-01

    Purpose: The aim my PhD study and the topic of this thesis is to investigate the radiotoxicity and the Relative Biological effectiveness (RBE) of intracellular Auger cascades. A special focus is kept on obtaining reliable absorbed dose calculations and using matched dose rate profiles for the Auger......-values (SC-values). The work can be divided into three steps; Examination of the bio-kinetics of the Auger emitter 131Cs used in the study, calculations of the SC-values and finally the measurement of the RBE of intracellular 131Cs decays, through ƴH2AX and clonogenic cell survival assay. Methods: A series....../(Bq*Sec)/pL for HeLa nuclei and from 7.45*10-4 to 7.63 *10-4 Gy/(Bq*Sec)/pL for V79 nuclei. The SC-values were shown to be were very robust and almost independent of cellular and nuclear size. A RBE value of 1 was obtained for HeLa cells using ƴH2AX assays. RBE values of 4.5 ± 0.5 and 3.8 ± 0.8 were obtained for He...

  1. Quantification of cell death in developing cerebellum by a 14C tracer method

    International Nuclear Information System (INIS)

    Griffin, W.S.; Woodward, D.J.; Chanda, R.

    1978-01-01

    To study the question of whether or not cell death contributes significantly to normal or stressed postnatal brain development in a way which is biochemically quantifiable, we carried out an experiment to assess the amount of cell death in developing cerebellum. By measuring the loss of DNA content and the loss of 14 C from labelled thymidine previously incorporated into the DNA fraction (DNAF) in X-irradiated neonatal animals, shown by histological methods to have cell death to the degree of degranulating the external granular layer (EGL), we showed that when cells die both label and DNA content are greatly decreased in the cerebellum. Experiments on both normal and malnourished animals showed that cell death does not contribute significantly to cerebellar development in either malnutrition-stressed or normal animals. Here, we present a biochemical tool for assessing cell death and evidence that cell death does not contribute significantly to cerebellar development

  2. Methods of direct (non-chromatographic) quantification of body metabolites utilizing chemical ionization mass spectrometry

    International Nuclear Information System (INIS)

    Mee, J.M.L.

    1978-01-01

    For quantitative determination of known metabolites from the biological sample by direct chemical ionization mass spectrometry (CI-MS), the method of internal standard using stable isotopically labelled analogs appears to be the method of choice. In the case where stable isotope ratio determinations could not be applied, and alternative quantification can be achieved using non-labelled external or internal standards and a calibration curve (sum of peak height per a given number of scans versus concentration). The technique of computer monitoring permits display and plotting of ion current profiles (TIC and SIC) or spectra per a given number of scans or a given range of mass per charge. Examples are given in areas of clinical application and the quantitative data show very good agreement with the conventional chromatographic measurements. (Auth.)

  3. ATPase activity measurement of DNA replicative helicase from Bacillus stearothermophilus by malachite green method.

    Science.gov (United States)

    Yang, Mu; Wang, Ganggang

    2016-09-15

    The DnaB helicase from Bacillus stearothermophilus (DnaBBst) was a model protein for studying the bacterial DNA replication. In this work, a non-radioactive method for measuring ATPase activity of DnaBBst helicase was described. The working parameters and conditions were optimized. Furthermore, this method was applied to investigate effects of DnaG primase, ssDNA and helicase loader protein (DnaI) on ATPase activity of DnaBBst. Our results showed this method was sensitive and efficient. Moreover, it is suitable for the investigation of functional interaction between DnaB and related factors. Copyright © 2016 Elsevier Inc. All rights reserved.

  4. Short overview of PSA quantification methods, pitfalls on the road from approximate to exact results

    International Nuclear Information System (INIS)

    Banov, Reni; Simic, Zdenko; Sterc, Davor

    2014-01-01

    Over time the Probabilistic Safety Assessment (PSA) models have become an invaluable companion in the identification and understanding of key nuclear power plant (NPP) vulnerabilities. PSA is an effective tool for this purpose as it assists plant management to target resources where the largest benefit for plant safety can be obtained. PSA has quickly become an established technique to numerically quantify risk measures in nuclear power plants. As complexity of PSA models increases, the computational approaches become more or less feasible. The various computational approaches can be basically classified in two major groups: approximate and exact (BDD based) methods. In recent time modern commercially available PSA tools started to provide both methods for PSA model quantification. Besides availability of both methods in proven PSA tools the usage must still be taken carefully since there are many pitfalls which can drive to wrong conclusions and prevent efficient usage of PSA tool. For example, typical pitfalls involve the usage of higher precision approximation methods and getting a less precise result, or mixing minimal cuts and prime implicants in the exact computation method. The exact methods are sensitive to selected computational paths in which case a simple human assisted rearrangement may help and even switch from computationally non-feasible to feasible methods. Further improvements to exact method are possible and desirable which opens space for a new research. In this paper we will show how these pitfalls may be detected and how carefully actions must be done especially when working with large PSA models. (authors)

  5. Surface Enhanced Raman Spectroscopy (SERS) methods for endpoint and real-time quantification of miRNA assays

    Science.gov (United States)

    Restaino, Stephen M.; White, Ian M.

    2017-03-01

    Surface Enhanced Raman spectroscopy (SERS) provides significant improvements over conventional methods for single and multianalyte quantification. Specifically, the spectroscopic fingerprint provided by Raman scattering allows for a direct multiplexing potential far beyond that of fluorescence and colorimetry. Additionally, SERS generates a comparatively low financial and spatial footprint compared with common fluorescence based systems. Despite the advantages of SERS, it has remained largely an academic pursuit. In the field of biosensing, techniques to apply SERS to molecular diagnostics are constantly under development but, most often, assay protocols are redesigned around the use of SERS as a quantification method and ultimately complicate existing protocols. Our group has sought to rethink common SERS methodologies in order to produce translational technologies capable of allowing SERS to compete in the evolving, yet often inflexible biosensing field. This work will discuss the development of two techniques for quantification of microRNA, a promising biomarker for homeostatic and disease conditions ranging from cancer to HIV. First, an inkjet-printed paper SERS sensor has been developed to allow on-demand production of a customizable and multiplexable single-step lateral flow assay for miRNA quantification. Second, as miRNA concentrations commonly exist in relatively low concentrations, amplification methods (e.g. PCR) are therefore required to facilitate quantification. This work presents a novel miRNA assay alongside a novel technique for quantification of nuclease driven nucleic acid amplification strategies that will allow SERS to be used directly with common amplification strategies for quantification of miRNA and other nucleic acid biomarkers.

  6. DNA based methods used for characterization and detection of food ...

    African Journals Online (AJOL)

    Detection of food borne pathogen is of outmost importance in the food industries and related agencies. For the last few decades conventional methods were used to detect food borne pathogens based on phenotypic characters. At the advent of complementary base pairing and amplification of DNA, the diagnosis of food ...

  7. A universal, rapid, and inexpensive method for genomic DNA ...

    Indian Academy of Sciences (India)

    MOHAMMED BAQUR SAHIB A. AL-SHUHAIB

    gels, containing 7% glycerol, and 1×TBE buffer. The gels were run under 200 .... Inc. Germany, GeneaidTM DNA Isolation Kit, Geneaid. Biotech., New Taipei City, .... C. L. and Arsenos G. 2015 Comparison of eleven methods for genomic DNA ...

  8. Track benchmarking method for uncertainty quantification of particle tracking velocimetry interpolations

    International Nuclear Information System (INIS)

    Schneiders, Jan F G; Sciacchitano, Andrea

    2017-01-01

    The track benchmarking method (TBM) is proposed for uncertainty quantification of particle tracking velocimetry (PTV) data mapped onto a regular grid. The method provides statistical uncertainty for a velocity time-series and can in addition be used to obtain instantaneous uncertainty at increased computational cost. Interpolation techniques are typically used to map velocity data from scattered PTV (e.g. tomographic PTV and Shake-the-Box) measurements onto a Cartesian grid. Recent examples of these techniques are the FlowFit and VIC+  methods. The TBM approach estimates the random uncertainty in dense velocity fields by performing the velocity interpolation using a subset of typically 95% of the particle tracks and by considering the remaining tracks as an independent benchmarking reference. In addition, also a bias introduced by the interpolation technique is identified. The numerical assessment shows that the approach is accurate when particle trajectories are measured over an extended number of snapshots, typically on the order of 10. When only short particle tracks are available, the TBM estimate overestimates the measurement error. A correction to TBM is proposed and assessed to compensate for this overestimation. The experimental assessment considers the case of a jet flow, processed both by tomographic PIV and by VIC+. The uncertainty obtained by TBM provides a quantitative evaluation of the measurement accuracy and precision and highlights the regions of high error by means of bias and random uncertainty maps. In this way, it is possible to quantify the uncertainty reduction achieved by advanced interpolation algorithms with respect to standard correlation-based tomographic PIV. The use of TBM for uncertainty quantification and comparison of different processing techniques is demonstrated. (paper)

  9. Leak Rate Quantification Method for Gas Pressure Seals with Controlled Pressure Differential

    Science.gov (United States)

    Daniels, Christopher C.; Braun, Minel J.; Oravec, Heather A.; Mather, Janice L.; Taylor, Shawn C.

    2015-01-01

    An enhancement to the pressure decay leak rate method with mass point analysis solved deficiencies in the standard method. By adding a control system, a constant gas pressure differential across the test article was maintained. As a result, the desired pressure condition was met at the onset of the test, and the mass leak rate and measurement uncertainty were computed in real-time. The data acquisition and control system were programmed to automatically stop when specified criteria were met. Typically, the test was stopped when a specified level of measurement uncertainty was attained. Using silicone O-ring test articles, the new method was compared with the standard method that permitted the downstream pressure to be non-constant atmospheric pressure. The two methods recorded comparable leak rates, but the new method recorded leak rates with significantly lower measurement uncertainty, statistical variance, and test duration. Utilizing this new method in leak rate quantification, projects will reduce cost and schedule, improve test results, and ease interpretation between data sets.

  10. DNA Checkerboard Method for Bacterial Pathogen Identification in Oral Diseases

    OpenAIRE

    Nascimento, Cássio do; Issa, João Paulo Mardegan; Watanabe, Evandro; Ito, Izabel Yoko

    2006-01-01

    This work aim to show by literature review the principal characteristics of the DNA checkerboard method for bacterial pathogens identification in oral diseases, showing the most varieties uses and applications of this technique Este trabajo tiene como objetivo, presentar en una revisión de la literatura, las principales características del método de chequeo del DNA para la identificación de bacterias patógenas en la cavidad oral, mostrando las diferentes utilizaciones y aplicaciones de est...

  11. A Method for Quantification of Epithelium Colonization Capacity by Pathogenic Bacteria

    DEFF Research Database (Denmark)

    Micha Pedersen, Rune; Grønnemose, Rasmus Birkholm; Stærk, Kristian

    2018-01-01

    a method in which epithelia/endothelia are simulated by flow chamber-grown human cell layers, and infection is induced by seeding of pathogenic bacteria on these surfaces under conditions that simulate the physiological microenvironment. Quantification of bacterial adhesion and colonization of the cell......Most bacterial infections initiate at the mucosal epithelium lining the gastrointestinal, respiratory, and urogenital tracts. At these sites, bacterial pathogens must adhere and increase in numbers to effectively breach the outer barrier and invade the host. If the bacterium succeeds in reaching...... the bloodstream, effective dissemination again requires that bacteria in the blood, reestablish contact to distant endothelium sites and form secondary site foci. The infectious potential of bacteria is therefore closely linked to their ability to adhere to, colonize, and invade epithelial and endothelial...

  12. Comparative analysis of experimental methods for quantification of small amounts of oil in water

    DEFF Research Database (Denmark)

    Katika, Konstantina; Ahkami, Mehrdad; Fosbøl, Philip Loldrup

    2016-01-01

    ) and the quantification of oil is then difficult. In this study, we compare four approaches to determine the volume of the collected oil fraction in core flooding effluents. The four methods are: Image analysis, UV/visible spectroscopy, liquid scintillation counting, and low-field nuclear magnetic resonance (NMR...... comparison to a pre-made standard curve. Image analysis, UV/visible spectroscopy, and liquid scintillation counting quantify only the oil fraction by comparing with a pre-made standard curve. The image analysis technique is reliable when more than 0.1 ml oil is present, whereas liquid scintillation counting...... performs well when less than 0.6 ml oil is present. Both UV/visible spectroscopy and NMR spectrometry produced high accuracy results in the entire studied range (0.006-1.1 ml). In terms of laboratory time, the liquid scintillation counting is the fastest and least user dependent, whereas the NMR...

  13. An HPLC-DAD method to quantification of main phenolic compounds from leaves of Cecropia species

    International Nuclear Information System (INIS)

    Costa, Geison M.; Ortmann, Caroline F.; Schenkel, Eloir P.; Reginatto, Flavio H.

    2011-01-01

    An efficient and reproducible HPLC-DAD method was developed and validated for the simultaneous quantification of major compounds (chlorogenic acid, isoorientin, orientin and isovitexin) present in the leaves of two Cecropia species, C. glaziovii and C. pachystachya. From the leaves of C. glaziovii and C. pachystachya were isolated the C-glycosylflavones isoorientin and isovitexin and identified on both species chlorogenic acid (3-O-caffeoylquinic acid) and the O-glycosylflavonol isoquercitrin. The C-glycosylflavone orientin was isolated only from C. pachystachya. Chlorogenic acid was the major compound in both species (11.1 mg g -1 of extract of C. glaziovii and 27.2 mg g -1 of extract of C. pachystachya) and for the flavonoids quantified, isovitexin was the main C-glycosylflavonoid for C. glaziovii (4.6 mg g -1 of extract) and isoorientin the main one for C. pachystachya (17.3 mg g -1 of extract). (author)

  14. Development of Uncertainty Quantification Method for MIR-PIV Measurement using BOS Technique

    International Nuclear Information System (INIS)

    Seong, Jee Hyun; Song, Min Seop; Kim, Eung Soo

    2014-01-01

    Matching Index of Refraction (MIR) is frequently used for obtaining high quality PIV measurement data. ven small distortion by unmatched refraction index of test section can result in uncertainty problems. In this context, it is desirable to construct new concept for checking errors of MIR and following uncertainty of PIV measurement. This paper proposes a couple of experimental concept and relative results. This study developed an MIR uncertainty quantification method for PIV measurement using SBOS technique. From the reference data of the BOS, the reliable SBOS experiment procedure was constructed. Then with the combination of SBOS technique with MIR-PIV technique, velocity vector and refraction displacement vector field was measured simultaneously. MIR errors are calculated through mathematical equation, in which PIV and SBOS data are put. These errors are also verified by another BOS experiment. Finally, with the applying of calculated MIR-PIV uncertainty, correct velocity vector field can be obtained regardless of MIR errors

  15. A Method for Quantification of Epithelium Colonization Capacity by Pathogenic Bacteria

    Directory of Open Access Journals (Sweden)

    Rune M. Pedersen

    2018-02-01

    Full Text Available Most bacterial infections initiate at the mucosal epithelium lining the gastrointestinal, respiratory, and urogenital tracts. At these sites, bacterial pathogens must adhere and increase in numbers to effectively breach the outer barrier and invade the host. If the bacterium succeeds in reaching the bloodstream, effective dissemination again requires that bacteria in the blood, reestablish contact to distant endothelium sites and form secondary site foci. The infectious potential of bacteria is therefore closely linked to their ability to adhere to, colonize, and invade epithelial and endothelial surfaces. Measurement of bacterial adhesion to epithelial cells is therefore standard procedure in studies of bacterial virulence. Traditionally, such measurements have been conducted with microtiter plate cell cultures to which bacteria are added, followed by washing procedures and final quantification of retained bacteria by agar plating. This approach is fast and straightforward, but yields only a rough estimate of the adhesive properties of the bacteria upon contact, and little information on the ability of the bacterium to colonize these surfaces under relevant physiological conditions. Here, we present a method in which epithelia/endothelia are simulated by flow chamber-grown human cell layers, and infection is induced by seeding of pathogenic bacteria on these surfaces under conditions that simulate the physiological microenvironment. Quantification of bacterial adhesion and colonization of the cell layers is then performed by in situ time-lapse fluorescence microscopy and automatic detection of bacterial surface coverage. The method is demonstrated in three different infection models, simulating Staphylococcus aureus endothelial infection and Escherichia coli intestinal- and uroepithelial infection. The approach yields valuable information on the fitness of the bacterium to successfully adhere to and colonize epithelial surfaces and can be used

  16. Diagnostic accuracy of detection and quantification of HBV-DNA and HCV-RNA using dried blood spot (DBS) samples - a systematic review and meta-analysis.

    Science.gov (United States)

    Lange, Berit; Roberts, Teri; Cohn, Jennifer; Greenman, Jamie; Camp, Johannes; Ishizaki, Azumi; Messac, Luke; Tuaillon, Edouard; van de Perre, Philippe; Pichler, Christine; Denkinger, Claudia M; Easterbrook, Philippa

    2017-11-01

    The detection and quantification of hepatitis B (HBV) DNA and hepatitis C (HCV) RNA in whole blood collected on dried blood spots (DBS) may facilitate access to diagnosis and treatment of HBV and HCV infection in resource-poor settings. We evaluated the diagnostic performance of DBS compared to venous blood samples for detection and quantification of HBV-DNA and HCV-RNA in two systematic reviews and meta-analyses on the diagnostic accuracy of HBV DNA and HCV RNA from DBS compared to venous blood samples. We searched MEDLINE, Embase, Global Health, Web of Science, LILAC and Cochrane library for studies that assessed diagnostic accuracy with DBS. Heterogeneity was assessed and where appropriate pooled estimates of sensitivity and specificity were generated using bivariate analyses with maximum likelihood estimates and 95% confidence intervals. We also conducted a narrative review on the impact of varying storage conditions or different cut-offs for detection from studies that undertook this in a subset of samples. The QUADAS-2 tool was used to assess risk of bias. In the quantitative synthesis for diagnostic accuracy of HBV-DNA using DBS, 521 citations were identified, and 12 studies met the inclusion criteria. Overall quality of studies was rated as low. The pooled estimate of sensitivity and specificity for HBV-DNA was 95% (95% CI: 83-99) and 99% (95% CI: 53-100), respectively. In the two studies that reported on cut-offs and limit of detection (LoD) - one reported a sensitivity of 98% for a cut-off of ≥2000 IU/ml and another reported a LoD of 914 IU/ml using a commercial assay. Varying storage conditions for individual samples did not result in a significant variation of results. In the synthesis for diagnostic accuracy of HCV-RNA using DBS, 15 studies met the inclusion criteria, and this included six additional studies to a previously published review. The pooled sensitivity and specificity was 98% (95% CI:95-99) and 98% (95% CI:95-99.0), respectively

  17. Comparison between PET template-based method and MRI-based method for cortical quantification of florbetapir (AV-45) uptake in vivo

    Energy Technology Data Exchange (ETDEWEB)

    Saint-Aubert, L.; Nemmi, F.; Peran, P. [Inserm, Imagerie Cerebrale et Handicaps neurologiques UMR 825, Centre Hospitalier Universitaire de Toulouse, Toulouse (France); Centre Hospitalier Universitaire de Toulouse, Universite de Toulouse, UPS, Imagerie Cerebrale et Handicaps Neurologiques UMR 825, Toulouse (France); Barbeau, E.J. [Universite de Toulouse, UPS, Centre de Recherche Cerveau et Cognition, France, CNRS, CerCo, Toulouse (France); Service de Neurologie, Pole Neurosciences, Centre Hospitalier Universitaire de Toulouse, Toulouse (France); Payoux, P. [Inserm, Imagerie Cerebrale et Handicaps neurologiques UMR 825, Centre Hospitalier Universitaire de Toulouse, Toulouse (France); Centre Hospitalier Universitaire de Toulouse, Universite de Toulouse, UPS, Imagerie Cerebrale et Handicaps Neurologiques UMR 825, Toulouse (France); Service de Medecine Nucleaire, Pole Imagerie, Centre Hospitalier Universitaire de Toulouse, Toulouse (France); Chollet, F.; Pariente, J. [Inserm, Imagerie Cerebrale et Handicaps neurologiques UMR 825, Centre Hospitalier Universitaire de Toulouse, Toulouse (France); Centre Hospitalier Universitaire de Toulouse, Universite de Toulouse, UPS, Imagerie Cerebrale et Handicaps Neurologiques UMR 825, Toulouse (France); Service de Neurologie, Pole Neurosciences, Centre Hospitalier Universitaire de Toulouse, Toulouse (France)

    2014-05-15

    Florbetapir (AV-45) has been shown to be a reliable tool for assessing in vivo amyloid load in patients with Alzheimer's disease from the early stages. However, nonspecific white matter binding has been reported in healthy subjects as well as in patients with Alzheimer's disease. To avoid this issue, cortical quantification might increase the reliability of AV-45 PET analyses. In this study, we compared two quantification methods for AV-45 binding, a classical method relying on PET template registration (route 1), and a MRI-based method (route 2) for cortical quantification. We recruited 22 patients at the prodromal stage of Alzheimer's disease and 17 matched controls. AV-45 binding was assessed using both methods, and target-to-cerebellum mean global standard uptake values (SUVr) were obtained for each of them, together with SUVr in specific regions of interest. Quantification using the two routes was compared between the clinical groups (intragroup comparison), and between groups for each route (intergroup comparison). Discriminant analysis was performed. In the intragroup comparison, differences in uptake values were observed between route 1 and route 2 in both groups. In the intergroup comparison, AV-45 uptake was higher in patients than controls in all regions of interest using both methods, but the effect size of this difference was larger using route 2. In the discriminant analysis, route 2 showed a higher specificity (94.1 % versus 70.6 %), despite a lower sensitivity (77.3 % versus 86.4 %), and D-prime values were higher for route 2. These findings suggest that, although both quantification methods enabled patients at early stages of Alzheimer's disease to be well discriminated from controls, PET template-based quantification seems adequate for clinical use, while the MRI-based cortical quantification method led to greater intergroup differences and may be more suitable for use in current clinical research. (orig.)

  18. Development of two dimensional electrophoresis method using single chain DNA

    International Nuclear Information System (INIS)

    Ikeda, Junichi; Hidaka, So

    1998-01-01

    By combining a separation method due to molecular weight and a method to distinguish difference of mono-bases, it was aimed to develop a two dimensional single chain DNA labeled with Radioisotope (RI). From electrophoretic pattern difference of parent and variant strands, it was investigated to isolate the root module implantation control gene. At first, a Single Strand Conformation Polymorphism (SSCP) method using concentration gradient gel was investigated. As a result, it was formed that intervals between double chain and single chain DNAs expanded, but intervals of both single chain DNAs did not expand. On next, combination of non-modified acrylic amide electrophoresis method and Denaturing Gradient-Gel Electrophoresis (DGGE) method was examined. As a result, hybrid DNA developed by two dimensional electrophoresis arranged on two lines. But, among them a band of DNA modified by high concentration of urea could not be found. Therefore, in this fiscal year's experiments, no preferable result could be obtained. By the used method, it was thought to be impossible to detect the differences. (G.K.)

  19. Quantification of organ motion based on an adaptive image-based scale invariant feature method

    Energy Technology Data Exchange (ETDEWEB)

    Paganelli, Chiara [Dipartimento di Elettronica, Informazione e Bioingegneria, Politecnico di Milano, piazza L. Da Vinci 32, Milano 20133 (Italy); Peroni, Marta [Dipartimento di Elettronica, Informazione e Bioingegneria, Politecnico di Milano, piazza L. Da Vinci 32, Milano 20133, Italy and Paul Scherrer Institut, Zentrum für Protonentherapie, WMSA/C15, CH-5232 Villigen PSI (Italy); Baroni, Guido; Riboldi, Marco [Dipartimento di Elettronica, Informazione e Bioingegneria, Politecnico di Milano, piazza L. Da Vinci 32, Milano 20133, Italy and Bioengineering Unit, Centro Nazionale di Adroterapia Oncologica, strada Campeggi 53, Pavia 27100 (Italy)

    2013-11-15

    Purpose: The availability of corresponding landmarks in IGRT image series allows quantifying the inter and intrafractional motion of internal organs. In this study, an approach for the automatic localization of anatomical landmarks is presented, with the aim of describing the nonrigid motion of anatomo-pathological structures in radiotherapy treatments according to local image contrast.Methods: An adaptive scale invariant feature transform (SIFT) was developed from the integration of a standard 3D SIFT approach with a local image-based contrast definition. The robustness and invariance of the proposed method to shape-preserving and deformable transforms were analyzed in a CT phantom study. The application of contrast transforms to the phantom images was also tested, in order to verify the variation of the local adaptive measure in relation to the modification of image contrast. The method was also applied to a lung 4D CT dataset, relying on manual feature identification by an expert user as ground truth. The 3D residual distance between matches obtained in adaptive-SIFT was then computed to verify the internal motion quantification with respect to the expert user. Extracted corresponding features in the lungs were used as regularization landmarks in a multistage deformable image registration (DIR) mapping the inhale vs exhale phase. The residual distances between the warped manual landmarks and their reference position in the inhale phase were evaluated, in order to provide a quantitative indication of the registration performed with the three different point sets.Results: The phantom study confirmed the method invariance and robustness properties to shape-preserving and deformable transforms, showing residual matching errors below the voxel dimension. The adapted SIFT algorithm on the 4D CT dataset provided automated and accurate motion detection of peak to peak breathing motion. The proposed method resulted in reduced residual errors with respect to standard SIFT

  20. Application and comparison of large-scale solution-based DNA capture-enrichment methods on ancient DNA

    DEFF Research Database (Denmark)

    Avila Arcos, Maria del Carmen; Cappellini, Enrico; Romero-Navarro, J. Alberto

    2011-01-01

    The development of second-generation sequencing technologies has greatly benefitted the field of ancient DNA (aDNA). Its application can be further exploited by the use of targeted capture-enrichment methods to overcome restrictions posed by low endogenous and contaminating DNA in ancient samples...

  1. Development and validation of a method for the quantification of fructooligosaccharides in a prebiotic ice cream

    Directory of Open Access Journals (Sweden)

    Claudia L. González-Aguirre

    2018-02-01

    Full Text Available Context: Fructooligosaccharides (FOS are known as oligofructanes, oligosaccharides or oligofructose, which fall within the concept of prebiotics. One of the methods most commonly used in the industry for quantification and quality control nutraceutical substances classification is the method of high performance liquid chromatography (HPLC. Aims: To develop a procedure for the determination of FOS by HPLC in raw materials and a prebiotic ice cream. Methods: For the chromatographic separation, an HPLC was used with a refractive index detector (IR. The separation was performed using two columns coupled Sugar-pak I™ using an isocratic procedure with water type 1 at 0.35 mL/min. Kestose (GF2, nistose (GF3 and fructofuranosylnystose (GF4 were used as standards. Robustness was assessed by applying the Youden and Steiner test. Results: Good linear correlations were obtained (y = 14191.4470 x + 285684.2, r2 = 0.9904 within the concentration range of 8.0-12.0 mg/mL. The FOS recoveries were 99.5% with the intra-day and inter-day relative standard deviation (RSD less than 0.8%. The robustness test showed that the temperature parameters of the column and flow velocity are critical factors in the method. Conclusions: This reliable, simple and cost-effective method could be applied to the routine monitoring of FOS (GF2, GF3, and GF4 in raw materials and prebiotic ice creams.

  2. Critical assessment of three high performance liquid chromatography analytical methods for food carotenoid quantification.

    Science.gov (United States)

    Dias, M Graça; Oliveira, Luísa; Camões, M Filomena G F C; Nunes, Baltazar; Versloot, Pieter; Hulshof, Paul J M

    2010-05-21

    Three sets of extraction/saponification/HPLC conditions for food carotenoid quantification were technically and economically compared. Samples were analysed for carotenoids alpha-carotene, beta-carotene, beta-cryptoxanthin, lutein, lycopene, and zeaxanthin. All methods demonstrated good performance in the analysis of a composite food standard reference material for the analytes they are applicable to. Methods using two serial connected C(18) columns and a mobile phase based on acetonitrile, achieved a better carotenoid separation than the method using a mobile phase based on methanol and one C(18)-column. Carotenoids from leafy green vegetable matrices appeared to be better extracted with a mixture of methanol and tetrahydrofuran than with tetrahydrofuran alone. Costs of carotenoid determination in foods were lower for the method with mobile phase based on methanol. However for some food matrices and in the case of E-Z isomer separations, this was not technically satisfactory. Food extraction with methanol and tetrahydrofuran with direct evaporation of these solvents, and saponification (when needed) using pyrogallol as antioxidant, combined with a HPLC system using a slight gradient mobile phase based on acetonitrile and a stationary phase composed by two serial connected C(18) columns was the most technically and economically favourable method. 2010. Published by Elsevier B.V.

  3. A method for uncertainty quantification in the life prediction of gas turbine components

    Energy Technology Data Exchange (ETDEWEB)

    Lodeby, K.; Isaksson, O.; Jaervstraat, N. [Volvo Aero Corporation, Trolhaettan (Sweden)

    1998-12-31

    A failure in an aircraft jet engine can have severe consequences which cannot be accepted and high requirements are therefore raised on engine reliability. Consequently, assessment of the reliability of life predictions used in design and maintenance are important. To assess the validity of the predicted life a method to quantify the contribution to the total uncertainty in the life prediction from different uncertainty sources is developed. The method is a structured approach for uncertainty quantification that uses a generic description of the life prediction process. It is based on an approximate error propagation theory combined with a unified treatment of random and systematic errors. The result is an approximate statistical distribution for the predicted life. The method is applied on life predictions for three different jet engine components. The total uncertainty became of reasonable order of magnitude and a good qualitative picture of the distribution of the uncertainty contribution from the different sources was obtained. The relative importance of the uncertainty sources differs between the three components. It is also highly dependent on the methods and assumptions used in the life prediction. Advantages and disadvantages of this method is discussed. (orig.) 11 refs.

  4. Comparative quantification of dietary supplemented neural creatine concentrations with (1)H-MRS peak fitting and basis spectrum methods.

    Science.gov (United States)

    Turner, Clare E; Russell, Bruce R; Gant, Nicholas

    2015-11-01

    Magnetic resonance spectroscopy (MRS) is an analytical procedure that can be used to non-invasively measure the concentration of a range of neural metabolites. Creatine is an important neurometabolite with dietary supplementation offering therapeutic potential for neurological disorders with dysfunctional energetic processes. Neural creatine concentrations can be probed using proton MRS and quantified using a range of software packages based on different analytical methods. This experiment examines the differences in quantification performance of two commonly used analysis packages following a creatine supplementation strategy with potential therapeutic application. Human participants followed a seven day dietary supplementation regime in a placebo-controlled, cross-over design interspersed with a five week wash-out period. Spectroscopy data were acquired the day immediately following supplementation and analyzed with two commonly-used software packages which employ vastly different quantification methods. Results demonstrate that neural creatine concentration was augmented following creatine supplementation when analyzed using the peak fitting method of quantification (105.9%±10.1). In contrast, no change in neural creatine levels were detected with supplementation when analysis was conducted using the basis spectrum method of quantification (102.6%±8.6). Results suggest that software packages that employ the peak fitting procedure for spectral quantification are possibly more sensitive to subtle changes in neural creatine concentrations. The relative simplicity of the spectroscopy sequence and the data analysis procedure suggest that peak fitting procedures may be the most effective means of metabolite quantification when detection of subtle alterations in neural metabolites is necessary. The straightforward technique can be used on a clinical magnetic resonance imaging system. Copyright © 2015 Elsevier Inc. All rights reserved.

  5. Standardization of a protocol to obtain genomic DNA for the quantification of 5mC in epicormics buds of Tectona grandis L.

    OpenAIRE

    Elisa Quiala; Luis Valledor; Rodrigo Hazbun; Raúl Barbón; Manuel de Feria; Maité Chávez

    2008-01-01

    The present investigation was carried out with the objective of defining an extraction and purification method that it provided deoxyribonucleic acid (DNA) appropriate to determine the percentage of 5mC in the genomic DNA of epicormics buds of Tectona grandis L. During the standardization of the protocol four methods were compared: 1 -classic based on saline shock solution with CTAB (hexadecil trimetil ammonium bromide), 2 - Kit of extraction of DNA plants DNeasy Plant Mini Kit (QIAGEN) accor...

  6. Quantitative Single-letter Sequencing: a method for simultaneously monitoring numerous known allelic variants in single DNA samples

    Directory of Open Access Journals (Sweden)

    Duborjal Hervé

    2008-02-01

    Full Text Available Abstract Background Pathogens such as fungi, bacteria and especially viruses, are highly variable even within an individual host, intensifying the difficulty of distinguishing and accurately quantifying numerous allelic variants co-existing in a single nucleic acid sample. The majority of currently available techniques are based on real-time PCR or primer extension and often require multiplexing adjustments that impose a practical limitation of the number of alleles that can be monitored simultaneously at a single locus. Results Here, we describe a novel method that allows the simultaneous quantification of numerous allelic variants in a single reaction tube and without multiplexing. Quantitative Single-letter Sequencing (QSS begins with a single PCR amplification step using a pair of primers flanking the polymorphic region of interest. Next, PCR products are submitted to single-letter sequencing with a fluorescently-labelled primer located upstream of the polymorphic region. The resulting monochromatic electropherogram shows numerous specific diagnostic peaks, attributable to specific variants, signifying their presence/absence in the DNA sample. Moreover, peak fluorescence can be quantified and used to estimate the frequency of the corresponding variant in the DNA population. Using engineered allelic markers in the genome of Cauliflower mosaic virus, we reliably monitored six different viral genotypes in DNA extracted from infected plants. Evaluation of the intrinsic variance of this method, as applied to both artificial plasmid DNA mixes and viral genome populations, demonstrates that QSS is a robust and reliable method of detection and quantification for variants with a relative frequency of between 0.05 and 1. Conclusion This simple method is easily transferable to many other biological systems and questions, including those involving high throughput analysis, and can be performed in any laboratory since it does not require specialized

  7. Quantification of methane emissions from 15 Danish landfills using the mobile tracer dispersion method

    Energy Technology Data Exchange (ETDEWEB)

    Mønster, Jacob [Department of Environmental Engineering, Technical University of Denmark, Miljøvej – Building 113, DK-2800 Lyngby (Denmark); Samuelsson, Jerker, E-mail: jerker.samuelsson@fluxsense.se [Chalmers University of Technology/FluxSense AB, SE-41296 Göteborg (Sweden); Kjeldsen, Peter [Department of Environmental Engineering, Technical University of Denmark, Miljøvej – Building 113, DK-2800 Lyngby (Denmark); Scheutz, Charlotte, E-mail: chas@env.dtu.dk [Department of Environmental Engineering, Technical University of Denmark, Miljøvej – Building 113, DK-2800 Lyngby (Denmark)

    2015-01-15

    Highlights: • Quantification of whole landfill site methane emission at 15 landfills. • Multiple on-site source identification and quantification. • Quantified methane emission from shredder waste and composting. • Large difference between measured and reported methane emissions. - Abstract: Whole-site methane emissions from 15 Danish landfills were assessed using a mobile tracer dispersion method with either Fourier transform infrared spectroscopy (FTIR), using nitrous oxide as a tracer gas, or cavity ring-down spectrometry (CRDS), using acetylene as a tracer gas. The landfills were chosen to represent the different stages of the lifetime of a landfill, including open, active, and closed covered landfills, as well as those with and without gas extraction for utilisation or flaring. Measurements also included landfills with biocover for oxidizing any fugitive methane. Methane emission rates ranged from 2.6 to 60.8 kg h{sup −1}, corresponding to 0.7–13.2 g m{sup −2} d{sup −1}, with the largest emission rates per area coming from landfills with malfunctioning gas extraction systems installed, and the smallest emission rates from landfills closed decades ago and landfills with an engineered biocover installed. Landfills with gas collection and recovery systems had a recovery efficiency of 41–81%. Landfills where shredder waste was deposited showed significant methane emissions, with the largest emission from newly deposited shredder waste. The average methane emission from the landfills was 154 tons y{sup −1}. This average was obtained from a few measurement campaigns conducted at each of the 15 landfills and extrapolating to annual emissions requires more measurements. Assuming that these landfills are representative of the average Danish landfill, the total emission from Danish landfills were calculated at 20,600 tons y{sup −1}, which is significantly lower than the 33,300 tons y{sup −1} estimated for the national greenhouse gas inventory for

  8. A method for the quantification of biased signalling at constitutively active receptors.

    Science.gov (United States)

    Hall, David A; Giraldo, Jesús

    2018-06-01

    Biased agonism, the ability of an agonist to differentially activate one of several signal transduction pathways when acting at a given receptor, is an increasingly recognized phenomenon at many receptors. The Black and Leff operational model lacks a way to describe constitutive receptor activity and hence inverse agonism. Thus, it is impossible to analyse the biased signalling of inverse agonists using this model. In this theoretical work, we develop and illustrate methods for the analysis of biased inverse agonism. Methods were derived for quantifying biased signalling in systems that demonstrate constitutive activity using the modified operational model proposed by Slack and Hall. The methods were illustrated using Monte Carlo simulations. The Monte Carlo simulations demonstrated that, with an appropriate experimental design, the model parameters are 'identifiable'. The method is consistent with methods based on the measurement of intrinsic relative activity (RA i ) (ΔΔlogR or ΔΔlog(τ/K a )) proposed by Ehlert and Kenakin and their co-workers but has some advantages. In particular, it allows the quantification of ligand bias independently of 'system bias' removing the requirement to normalize to a standard ligand. In systems with constitutive activity, the Slack and Hall model provides methods for quantifying the absolute bias of agonists and inverse agonists. This provides an alternative to methods based on RA i and is complementary to the ΔΔlog(τ/K a ) method of Kenakin et al. in systems where use of that method is inappropriate due to the presence of constitutive activity. © 2018 The British Pharmacological Society.

  9. Method validation using weighted linear regression models for quantification of UV filters in water samples.

    Science.gov (United States)

    da Silva, Claudia Pereira; Emídio, Elissandro Soares; de Marchi, Mary Rosa Rodrigues

    2015-01-01

    This paper describes the validation of a method consisting of solid-phase extraction followed by gas chromatography-tandem mass spectrometry for the analysis of the ultraviolet (UV) filters benzophenone-3, ethylhexyl salicylate, ethylhexyl methoxycinnamate and octocrylene. The method validation criteria included evaluation of selectivity, analytical curve, trueness, precision, limits of detection and limits of quantification. The non-weighted linear regression model has traditionally been used for calibration, but it is not necessarily the optimal model in all cases. Because the assumption of homoscedasticity was not met for the analytical data in this work, a weighted least squares linear regression was used for the calibration method. The evaluated analytical parameters were satisfactory for the analytes and showed recoveries at four fortification levels between 62% and 107%, with relative standard deviations less than 14%. The detection limits ranged from 7.6 to 24.1 ng L(-1). The proposed method was used to determine the amount of UV filters in water samples from water treatment plants in Araraquara and Jau in São Paulo, Brazil. Copyright © 2014 Elsevier B.V. All rights reserved.

  10. Quantification of self pollution from two diesel school buses using three independent methods

    Science.gov (United States)

    Sally Liu, L.-J.; Phuleria, Harish C.; Webber, Whitney; Davey, Mark; Lawson, Douglas R.; Ireson, Robert G.; Zielinska, Barbara; Ondov, John M.; Weaver, Christopher S.; Lapin, Charles A.; Easter, Michael; Hesterberg, Thomas W.; Larson, Timothy

    2010-09-01

    We monitored two Seattle school buses to quantify the buses' self pollution using the dual tracers (DT), lead vehicle (LV), and chemical mass balance (CMB) methods. Each bus drove along a residential route simulating stops, with windows closed or open. Particulate matter (PM) and its constituents were monitored in the bus and from a LV. We collected source samples from the tailpipe and crankcase emissions using an on-board dilution tunnel. Concentrations of PM 1, ultrafine particle counts, elemental and organic carbon (EC/OC) were higher on the bus than the LV. The DT method estimated that the tailpipe and the crankcase emissions contributed 1.1 and 6.8 μg m -3 of PM 2.5 inside the bus, respectively, with significantly higher crankcase self pollution (SP) when windows were closed. Approximately two-thirds of in-cabin PM 2.5 originated from background sources. Using the LV approach, SP estimates from the EC and the active personal DataRAM (pDR) measurements correlated well with the DT estimates for tailpipe and crankcase emissions, respectively, although both measurements need further calibration for accurate quantification. CMB results overestimated SP from the DT method but confirmed crankcase emissions as the major SP source. We confirmed buses' SP using three independent methods and quantified crankcase emissions as the dominant contributor.

  11. A fast isocratic liquid chromatography method for the quantification of xanthophylls and their stereoisomers.

    Science.gov (United States)

    Nimalaratne, Chamila; Lopes-Lutz, Daise; Schieber, Andreas; Wu, Jianping

    2015-12-01

    A fast isocratic liquid chromatography method was developed for the simultaneous quantification of eight xanthophylls (13-Z-lutein, 13'-Z-lutein, 13-Z-zeaxanthin, all-E-lutein, all-E-zeaxanthin, all-E-canthaxanthin, all-E-β-apo-8'-carotenoic acid ethyl ester and all-E-β-apo-8'-carotenal) within 12 min, compared to 90 min by the conventional high-performance liquid chromatography method. The separation was achieved on a YMC C30 reversed-phase column (100 mm x 2.0 mm; 3 μm) operated at 20°C using a methanol/tert-butyl methyl ether/water solvent system at a flow rate of 0.8 mL/min. The method was successfully applied to quantify lutein and zeaxanthin stereoisomers in egg yolk, raw and cooked spinach, and a dietary supplement. The method can be used for the rapid analysis of xanthophyll isomers in different food products and for quality control purposes. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. Development for 2D pattern quantification method on mask and wafer

    Science.gov (United States)

    Matsuoka, Ryoichi; Mito, Hiroaki; Toyoda, Yasutaka; Wang, Zhigang

    2010-03-01

    We have developed the effective method of mask and silicon 2-dimensional metrology. The aim of this method is evaluating the performance of the silicon corresponding to Hotspot on a mask. The method adopts a metrology management system based on DBM (Design Based Metrology). This is the high accurate contouring created by an edge detection algorithm used in mask CD-SEM and silicon CD-SEM. Currently, as semiconductor manufacture moves towards even smaller feature size, this necessitates more aggressive optical proximity correction (OPC) to drive the super-resolution technology (RET). In other words, there is a trade-off between highly precise RET and mask manufacture, and this has a big impact on the semiconductor market that centers on the mask business. 2-dimensional Shape quantification is important as optimal solution over these problems. Although 1-dimensional shape measurement has been performed by the conventional technique, 2-dimensional shape management is needed in the mass production line under the influence of RET. We developed the technique of analyzing distribution of shape edge performance as the shape management technique. On the other hand, there is roughness in the silicon shape made from a mass-production line. Moreover, there is variation in the silicon shape. For this reason, quantification of silicon shape is important, in order to estimate the performance of a pattern. In order to quantify, the same shape is equalized in two dimensions. And the method of evaluating based on the shape is popular. In this study, we conducted experiments for averaging method of the pattern (Measurement Based Contouring) as two-dimensional mask and silicon evaluation technique. That is, observation of the identical position of a mask and a silicon was considered. It is possible to analyze variability of the edge of the same position with high precision. The result proved its detection accuracy and reliability of variability on two-dimensional pattern (mask and

  13. Quantification of Parvovirus B19 DNA Using COBAS AmpliPrep Automated Sample Preparation and LightCycler Real-Time PCR

    Science.gov (United States)

    Schorling, Stefan; Schalasta, Gunnar; Enders, Gisela; Zauke, Michael

    2004-01-01

    The COBAS AmpliPrep instrument (Roche Diagnostics GmbH, D-68305 Mannheim, Germany) automates the entire sample preparation process of nucleic acid isolation from serum or plasma for polymerase chain reaction analysis. We report the analytical performance of the LightCycler Parvovirus B19 Quantification Kit (Roche Diagnostics) using nucleic acids isolated with the COBAS AmpliPrep instrument. Nucleic acids were extracted using the Total Nucleic Acid Isolation Kit (Roche Diagnostics) and amplified with the LightCycler Parvovirus B19 Quantification Kit. The kit combination processes 72 samples per 8-hour shift. The lower detection limit is 234 IU/ml at a 95% hit-rate, linear range approximately 104-1010 IU/ml, and overall precision 16 to 40%. Relative sensitivity and specificity in routine samples from pregnant women are 100% and 93%, respectively. Identification of a persistent parvovirus B19-infected individual by the polymerase chain reaction among 51 anti-parvovirus B19 IgM-negative samples underlines the importance of additional nucleic acid testing in pregnancy and its superiority to serology in identifying the risk of parvovirus B19 transmission via blood or blood products. Combination of the Total Nucleic Acid Isolation Kit on the COBAS AmpliPrep instrument with the LightCycler Parvovirus B19 Quantification Kit provides a reliable and time-saving tool for sensitive and accurate detection of parvovirus B19 DNA. PMID:14736825

  14. Optimized, fast through-put UHPLC-DAD based method for carotenoid quantification in spinach, serum, chylomicrons and faeces

    DEFF Research Database (Denmark)

    Eriksen, Jane Nygaard; Madsen, Pia Lisbeth; Dragsted, Lars Ove

    2017-01-01

    An improved UHPLC-DAD based method was developed and validated for quantification of major carotenoids present in spinach, serum, chylomicrons and faeces. Separation was achieved with gradient elution within 12.5 min for 6 dietary carotenoids and the internal standard, echinenone. The proposed me...

  15. Absolute quantification method and validation of airborne snow crab allergen tropomyosin using tandem mass spectrometry

    International Nuclear Information System (INIS)

    Rahman, Anas M. Abdel; Lopata, Andreas L.; Randell, Edward W.; Helleur, Robert J.

    2010-01-01

    Measuring the levels of the major airborne allergens of snow crab in the workplace is very important in studying the prevalence of crab asthma in workers. Previously, snow crab tropomyosin (SCTM) was identified as the major aeroallergen in crab plants and a unique signature peptide was identified for this protein. The present study advances our knowledge on aeroallergens by developing a method of quantification of airborne SCTM by using isotope dilution mass spectrometry. Liquid chromatography tandem mass spectrometry was developed for separation and analysis of the signature peptides. The tryptic digestion conditions were optimized to accomplish complete digestion. The validity of the method was studied using international conference on harmonization protocol, Where 2-9% for CV (precision) and 101-110% for accuracy, at three different levels of quality control. Recovery of the spiked protein from PTFE and TopTip filters was measured to be 99% and 96%, respectively. To further demonstrate the applicability and the validity of the method for real samples, 45 kg of whole snow crab were processed in an enclosed (simulated) crab processing line and air samples were collected. The levels of SCTM ranged between 0.36-3.92 μg m -3 and 1.70-2.31 μg m -3 for butchering and cooking stations, respectively.

  16. AO–MW–PLS method applied to rapid quantification of teicoplanin with near-infrared spectroscopy

    Directory of Open Access Journals (Sweden)

    Jiemei Chen

    2017-01-01

    Full Text Available Teicoplanin (TCP is an important lipoglycopeptide antibiotic produced by fermenting Actinoplanes teichomyceticus. The change in TCP concentration is important to measure in the fermentation process. In this study, a reagent-free and rapid quantification method for TCP in the TCP–Tris–HCl mixture samples was developed using near-infrared (NIR spectroscopy by focusing our attention on the fermentation process for TCP. The absorbance optimization (AO partial least squares (PLS was proposed and integrated with the moving window (MW PLS, which is called AO–MW–PLS method, to select appropriate wavebands. A model set that includes various wavebands that were equivalent to the optimal AO–MW–PLS waveband was proposed based on statistical considerations. The public region of all equivalent wavebands was just one of the equivalent wavebands. The obtained public regions were 1540–1868nm for TCP and 1114–1310nm for Tris. The root-mean-square error and correlation coefficient for leave-one-out cross validation were 0.046mg mL−1 and 0.9998mg mL−1 for TCP, and 0.235mg mL−1 and 0.9986mg mL−1 for Tris, respectively. All the models achieved highly accurate prediction effects, and the selected wavebands provided valuable references for designing specialized spectrometers. This study provided a valuable reference for further application of the proposed methods to TCP fermentation broth and to other spectroscopic analysis fields.

  17. Development and validation of an HPLC-FLD method for milbemectin quantification in dog plasma.

    Science.gov (United States)

    Xu, Qianqian; Xiang, Wensheng; Li, Jichang; Liu, Yong; Yu, Xiaolei; Zhang, Yaoteng; Qu, Mingli

    2010-07-15

    Milbemectin is a widely used veterinary antiparasitic agent. A high-performance liquid chromatography with fluorescent detection (HPLC-FLD) method is described for the determination of milbemectin in dog plasma. The derivative procedure included mixing 1-methylimizole [MI, MI-ACN (1:1, v/v), 100 microL], trifluoroacetic anhydride [TFAA, TFAA-ACN (1:2, v/v), 150 microL] with a subsequent incubation for 3s at the room temperature to obtain a fluorescent derivative, which is reproducible in different blood samples and the derivatives proved to be stable for at least 80 h at room temperature. HPLC method was developed on C18 column with FLD detection at an excitation wavelength of 365 nm and emission wavelength of 475 nm, with the mobile phase consisting of methanol and water in the ratio of 98:2 (v/v). The assay lower limit of quantification was 1 ng/mL. The calibration curve was linear over concentration range of 1-200 ng/mL. The intra- and inter-day accuracy was >94% and precision expressed as % coefficient of variation was <5%. This method is specific, simple, accurate, precise and easily adaptable to measure milbemycin in blood of other animals. Crown Copyright 2010. Published by Elsevier B.V. All rights reserved.

  18. Absolute quantification method and validation of airborne snow crab allergen tropomyosin using tandem mass spectrometry

    Energy Technology Data Exchange (ETDEWEB)

    Rahman, Anas M. Abdel, E-mail: anasar@mun.ca [Department of Chemistry, Memorial University of Newfoundland, St. John' s, Newfoundland A1B 3X7 (Canada); Lopata, Andreas L. [School of Applied Science, Marine Biomedical Sciences and Health Research Group, RMIT University, Bundoora, 3083 Victoria (Australia); Randell, Edward W. [Department of Laboratory Medicine, Memorial University of Newfoundland, Eastern Health, St. John' s, Newfoundland and Labrador A1B 3V6 (Canada); Helleur, Robert J. [Department of Chemistry, Memorial University of Newfoundland, St. John' s, Newfoundland A1B 3X7 (Canada)

    2010-11-29

    Measuring the levels of the major airborne allergens of snow crab in the workplace is very important in studying the prevalence of crab asthma in workers. Previously, snow crab tropomyosin (SCTM) was identified as the major aeroallergen in crab plants and a unique signature peptide was identified for this protein. The present study advances our knowledge on aeroallergens by developing a method of quantification of airborne SCTM by using isotope dilution mass spectrometry. Liquid chromatography tandem mass spectrometry was developed for separation and analysis of the signature peptides. The tryptic digestion conditions were optimized to accomplish complete digestion. The validity of the method was studied using international conference on harmonization protocol, Where 2-9% for CV (precision) and 101-110% for accuracy, at three different levels of quality control. Recovery of the spiked protein from PTFE and TopTip filters was measured to be 99% and 96%, respectively. To further demonstrate the applicability and the validity of the method for real samples, 45 kg of whole snow crab were processed in an enclosed (simulated) crab processing line and air samples were collected. The levels of SCTM ranged between 0.36-3.92 {mu}g m{sup -3} and 1.70-2.31 {mu}g m{sup -3} for butchering and cooking stations, respectively.

  19. Peritoneal Cell-free DNA: an innovative method for determining acute cell damage in peritoneal membrane and for monitoring the recovery process after peritonitis.

    Science.gov (United States)

    Virzì, Grazia Maria; Milan Manani, Sabrina; Brocca, Alessandra; Cantaluppi, Vincenzo; de Cal, Massimo; Pastori, Silvia; Tantillo, Ilaria; Zambon, Roberto; Crepaldi, Carlo; Ronco, Claudio

    2016-02-01

    Cell-free DNA (cfDNA) is present in the peritoneal effluent of stable peritoneal dialysis (PD) patients, but there are no data on cfDNA in PD patients with peritonitis. We investigated the variation of peritoneal cfDNA levels subsequent to peritonitis in PD patients. We enrolled 53 PD patients: 30 without any history of systemic inflammation or peritonitis in the last 3 months (group A) and 23 with acute peritonitis (group B). CfDNA was quantified in the peritoneal effluent. Peritoneal samples on days 1, 3, 10, 30 and until day 120 from the start of peritonitis were collected for white blood cells (WBC) count and cfDNA evaluation in group B. Quantitative analysis of cfDNA showed significantly higher levels in group B on day 1, 3, 10 and 30 compared with group A (p peritoneal cfDNA levels tended to progressively decline during follow-up of peritonitis. From this decreasing curve, we estimated that 49 days are necessary to reach the value of 51 genome equivalents (GE)/ml (75th percentile in controls) and 63 days to reach 31 GE/ml (median). Our results demonstrate that cfDNA increases in peritoneal effluent of PD patients with peritonitis and tends to progressively decline in step with peritonitis resolution and membrane repair process. Peritoneal cfDNA quantification could be an innovative method to determine acute damage and an inverse index of the repair process.

  20. A simple and efficient method for poly-3-hydroxybutyrate quantification in diazotrophic bacteria within 5 minutes using flow cytometry

    Directory of Open Access Journals (Sweden)

    L.P.S. Alves

    Full Text Available The conventional method for quantification of polyhydroxyalkanoates based on whole-cell methanolysis and gas chromatography (GC is laborious and time-consuming. In this work, a method based on flow cytometry of Nile red stained bacterial cells was established to quantify poly-3-hydroxybutyrate (PHB production by the diazotrophic and plant-associated bacteria, Herbaspirillum seropedicae and Azospirillum brasilense. The method consists of three steps: i cell permeabilization, ii Nile red staining, and iii analysis by flow cytometry. The method was optimized step-by-step and can be carried out in less than 5 min. The final results indicated a high correlation coefficient (R2=0.99 compared to a standard method based on methanolysis and GC. This method was successfully applied to the quantification of PHB in epiphytic bacteria isolated from rice roots.

  1. A simple and efficient method for poly-3-hydroxybutyrate quantification in diazotrophic bacteria within 5 minutes using flow cytometry.

    Science.gov (United States)

    Alves, L P S; Almeida, A T; Cruz, L M; Pedrosa, F O; de Souza, E M; Chubatsu, L S; Müller-Santos, M; Valdameri, G

    2017-01-16

    The conventional method for quantification of polyhydroxyalkanoates based on whole-cell methanolysis and gas chromatography (GC) is laborious and time-consuming. In this work, a method based on flow cytometry of Nile red stained bacterial cells was established to quantify poly-3-hydroxybutyrate (PHB) production by the diazotrophic and plant-associated bacteria, Herbaspirillum seropedicae and Azospirillum brasilense. The method consists of three steps: i) cell permeabilization, ii) Nile red staining, and iii) analysis by flow cytometry. The method was optimized step-by-step and can be carried out in less than 5 min. The final results indicated a high correlation coefficient (R2=0.99) compared to a standard method based on methanolysis and GC. This method was successfully applied to the quantification of PHB in epiphytic bacteria isolated from rice roots.

  2. A simple and efficient method for poly-3-hydroxybutyrate quantification in diazotrophic bacteria within 5 minutes using flow cytometry

    Science.gov (United States)

    Alves, L.P.S.; Almeida, A.T.; Cruz, L.M.; Pedrosa, F.O.; de Souza, E.M.; Chubatsu, L.S.; Müller-Santos, M.; Valdameri, G.

    2017-01-01

    The conventional method for quantification of polyhydroxyalkanoates based on whole-cell methanolysis and gas chromatography (GC) is laborious and time-consuming. In this work, a method based on flow cytometry of Nile red stained bacterial cells was established to quantify poly-3-hydroxybutyrate (PHB) production by the diazotrophic and plant-associated bacteria, Herbaspirillum seropedicae and Azospirillum brasilense. The method consists of three steps: i) cell permeabilization, ii) Nile red staining, and iii) analysis by flow cytometry. The method was optimized step-by-step and can be carried out in less than 5 min. The final results indicated a high correlation coefficient (R2=0.99) compared to a standard method based on methanolysis and GC. This method was successfully applied to the quantification of PHB in epiphytic bacteria isolated from rice roots. PMID:28099582

  3. Quantification of cell-free DNA in blood plasma and DNA damage degree in lymphocytes to evaluate dysregulation of apoptosis in schizophrenia patients.

    Science.gov (United States)

    Ershova, E S; Jestkova, E M; Chestkov, I V; Porokhovnik, L N; Izevskaya, V L; Kutsev, S I; Veiko, N N; Shmarina, G; Dolgikh, O; Kostyuk, S V

    2017-04-01

    Oxidative DNA damage has been proposed as one of the causes of schizophrenia (SZ), and post mortem data indicate a dysregulation of apoptosis in SZ patients. To evaluate apoptosis in vivo we quantified the concentration of plasma cell-free DNA (cfDNA index, determined using fluorescence), the levels of 8-oxodG in cfDNA (immunoassay) and lymphocytes (FL1-8-oxodG index, flow cytometry) of male patients with acute psychotic disorders: paranoid SZ (total N = 58), schizophreniform (N = 11) and alcohol-induced (N = 14) psychotic disorder, and 30 healthy males. CfDNA in SZ (N = 58) does not change compared with controls. In SZ patients. Elevated levels of 8-oxodG were found in cfDNA (N = 58) and lymphocytes (n = 45). The main sources of cfDNA are dying cells with oxidized DNA. Thus, the cfDNA/FL1-8-oxodG ratio shows the level of apoptosis in damaged cells. Two subgroups were identified among the SZ patients (n = 45). For SZ-1 (31%) and SZ-2 (69%) median values of cfDNA/FL1-8-oxodG index are related as 1:6 (p DNA in the patient's body tissues and may be a contributing cause of acute psychotic disorder. Copyright © 2016 Elsevier Ltd. All rights reserved.

  4. Development of a reliable extraction and quantification method for glucosinolates in Moringa oleifera.

    Science.gov (United States)

    Förster, Nadja; Ulrichs, Christian; Schreiner, Monika; Müller, Carsten T; Mewis, Inga

    2015-01-01

    Glucosinolates are the characteristic secondary metabolites of plants in the order Brassicales. To date the common DIN extraction 'desulfo glucosinolates' method remains the common procedure for determination and quantification of glucosinolates. However, the desulfation step in the extraction of glucosinolates from Moringa oleifera leaves resulted in complete conversion and degradation of the naturally occurring glucosinolates in this plant. Therefore, a method for extraction of intact Moringa glucosinolates was developed and no conversion and degradation of the different rhamnopyranosyloxy-benzyl glucosinolates was found. Buffered eluents (0.1 M ammonium acetate) were necessary to stabilize 4-α-rhamnopyranosyloxy-benzyl glucosinolate (Rhamno-Benzyl-GS) and acetyl-4-α-rhamnopyranosyloxy-benzyl glucosinolate isomers (Ac-Isomers-GS) during HPLC analysis. Due to the instability of intact Moringa glucosinolates at room temperature and during the purification process of single glucosinolates, influences of different storage (room temperature, frozen, thawing and refreezing) and buffer conditions on glucosinolate conversion were analysed. Conversion and degradations processes were especially determined for the Ac-Isomers-GS III. Copyright © 2014 Elsevier Ltd. All rights reserved.

  5. Sensitive liquid chromatography-tandem mass spectrometry method for quantification of hydrochlorothiazide in human plasma.

    Science.gov (United States)

    Ramakrishna, N V S; Vishwottam, K N; Manoj, S; Koteshwara, M; Wishu, S; Varma, D P

    2005-12-01

    A simple, rapid, sensitive and specific liquid chromatography-tandem mass spectrometry method was developed and validated for quantification of hydrochlorothiazide (I), a common diuretic and anti-hypertensive agent. The analyte and internal standard, tamsulosin (II) were extracted by liquid-liquid extraction with diethyl ether-dichloromethane (70:30, v/v) using a Glas-Col Multi-Pulse Vortexer. The chromatographic separation was performed on a reversed-phase column (Waters symmetry C18) with a mobile phase of 10 mm ammonium acetate-methanol (15:85, v/v). The protonated analyte was quantitated in negative ionization by multiple reaction monitoring with a mass spectrometer. The mass transitions m/z 296.1 solidus in circle 205.0 and m/z 407.2 solidus in circle 184.9 were used to measure I and II, respectively. The assay exhibited a linear dynamic range of 0.5-200 ng/mL for hydrochlorothiazide in human plasma. The lower limit of quantitation was 500 pg/mL, with a relative standard deviation of less than 9%. Acceptable precision and accuracy were obtained for concentrations over the standard curve ranges. A run time of 2.5 min for each sample made it possible to analyze a throughput of more than 400 human plasma samples per day. The validated method has been successfully used to analyze human plasma samples for application in pharmacokinetic, bioavailability or bioequivalence studies. (c) 2005 John Wiley & Sons, Ltd.

  6. Uncertainty analysis methods for quantification of source terms using a large computer code

    International Nuclear Information System (INIS)

    Han, Seok Jung

    1997-02-01

    Quantification of uncertainties in the source term estimations by a large computer code, such as MELCOR and MAAP, is an essential process of the current probabilistic safety assessments (PSAs). The main objectives of the present study are (1) to investigate the applicability of a combined procedure of the response surface method (RSM) based on input determined from a statistical design and the Latin hypercube sampling (LHS) technique for the uncertainty analysis of CsI release fractions under a hypothetical severe accident sequence of a station blackout at Young-Gwang nuclear power plant using MAAP3.0B code as a benchmark problem; and (2) to propose a new measure of uncertainty importance based on the distributional sensitivity analysis. On the basis of the results obtained in the present work, the RSM is recommended to be used as a principal tool for an overall uncertainty analysis in source term quantifications, while using the LHS in the calculations of standardized regression coefficients (SRC) and standardized rank regression coefficients (SRRC) to determine the subset of the most important input parameters in the final screening step and to check the cumulative distribution functions (cdfs) obtained by RSM. Verification of the response surface model for its sufficient accuracy is a prerequisite for the reliability of the final results obtained by the combined procedure proposed in the present work. In the present study a new measure has been developed to utilize the metric distance obtained from cumulative distribution functions (cdfs). The measure has been evaluated for three different cases of distributions in order to assess the characteristics of the measure: The first case and the second are when the distribution is known as analytical distributions and the other case is when the distribution is unknown. The first case is given by symmetry analytical distributions. The second case consists of two asymmetry distributions of which the skewness is non zero

  7. A novel method for quantification of beam's-eye-view tumor tracking performance.

    Science.gov (United States)

    Hu, Yue-Houng; Myronakis, Marios; Rottmann, Joerg; Wang, Adam; Morf, Daniel; Shedlock, Daniel; Baturin, Paul; Star-Lack, Josh; Berbeco, Ross

    2017-11-01

    In-treatment imaging using an electronic portal imaging device (EPID) can be used to confirm patient and tumor positioning. Real-time tumor tracking performance using current digital megavolt (MV) imagers is hindered by poor image quality. Novel EPID designs may help to improve quantum noise response, while also preserving the high spatial resolution of the current clinical detector. Recently investigated EPID design improvements include but are not limited to multi-layer imager (MLI) architecture, thick crystalline and amorphous scintillators, and phosphor pixilation and focusing. The goal of the present study was to provide a method of quantitating improvement in tracking performance as well as to reveal the physical underpinnings of detector design that impact tracking quality. The study employs a generalizable ideal observer methodology for the quantification of tumor tracking performance. The analysis is applied to study both the effect of increasing scintillator thickness on a standard, single-layer imager (SLI) design as well as the effect of MLI architecture on tracking performance. The present study uses the ideal observer signal-to-noise ratio (d') as a surrogate for tracking performance. We employ functions which model clinically relevant tasks and generalized frequency-domain imaging metrics to connect image quality with tumor tracking. A detection task for relevant Cartesian shapes (i.e., spheres and cylinders) was used to quantitate trackability of cases employing fiducial markers. Automated lung tumor tracking algorithms often leverage the differences in benign and malignant lung tissue textures. These types of algorithms (e.g., soft-tissue localization - STiL) were simulated by designing a discrimination task, which quantifies the differentiation of tissue textures, measured experimentally and fit as a power-law in trend (with exponent β) using a cohort of MV images of patient lungs. The modeled MTF and NPS were used to investigate the effect of

  8. Validation of an HPLC-UV method for the identification and quantification of bioactive amines in chicken meat

    Directory of Open Access Journals (Sweden)

    D.C.S. Assis

    2016-06-01

    Full Text Available ABSTRACT A high-performance liquid chromatography with ultraviolet detection (HPLC-UV method was validated for the study of bioactive amines in chicken meat. A gradient elution system with an ultraviolet detector was used after extraction with trichloroacetic acid and pre-column derivatization with dansyl chloride. Putrescine, cadaverine, histamine, tyramine, spermidine, and spermine standards were used for the evaluation of the following performance parameters: selectivity, linearity, precision, recovery, limits of detection, limits of quantification and ruggedness. The results indicated excellent selectivity, separation of all amines, a coefficient of determination greater than 0.99 and recovery from 92.25 to 102.25% at the concentration of 47.2mg.kg-1, with a limit of detection at 0.3mg.kg-1 and a limit of quantification at 0.9mg.kg-1 for all amines, with the exception of histamine, which exhibited the limit of quantification, of 1mg.kg-1. In conclusion, the performance parameters demonstrated adequacy of the method for the detection and quantification of bioactive amines in chicken meat.

  9. Comparison of DNA preservation methods for environmental bacterial community samples.

    Science.gov (United States)

    Gray, Michael A; Pratte, Zoe A; Kellogg, Christina A

    2013-02-01

    Field collections of environmental samples, for example corals, for molecular microbial analyses present distinct challenges. The lack of laboratory facilities in remote locations is common, and preservation of microbial community DNA for later study is critical. A particular challenge is keeping samples frozen in transit. Five nucleic acid preservation methods that do not require cold storage were compared for effectiveness over time and ease of use. Mixed microbial communities of known composition were created and preserved by DNAgard(™), RNAlater(®), DMSO-EDTA-salt (DESS), FTA(®) cards, and FTA Elute(®) cards. Automated ribosomal intergenic spacer analysis and clone libraries were used to detect specific changes in the faux communities over weeks and months of storage. A previously known bias in FTA(®) cards that results in lower recovery of pure cultures of Gram-positive bacteria was also detected in mixed community samples. There appears to be a uniform bias across all five preservation methods against microorganisms with high G + C DNA. Overall, the liquid-based preservatives (DNAgard(™), RNAlater(®), and DESS) outperformed the card-based methods. No single liquid method clearly outperformed the others, leaving method choice to be based on experimental design, field facilities, shipping constraints, and allowable cost. © 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

  10. Quantification of methane and nitrous oxide emissions from various waste treatment facilities by tracer dilution method

    Science.gov (United States)

    Mønster, Jacob; Rella, Chris; Jacobson, Gloria; Kjeldsen, Peter; Scheutz, Charlotte

    2013-04-01

    Urban activities generate solid and liquid waste, and the handling and aftercare of the waste results in the emission of various compounds into the surrounding environment. Some of these compounds are emitted as gasses into the atmosphere, including methane and nitrous oxide. Methane and nitrous oxide are strong greenhouse gases and are considered to have 25 and 298 times the greenhouse gas potential of carbon dioxide on a hundred years term (Solomon et al. 2007). Global observations of both gasses have shown increasing concentrations that significantly contribute to the greenhouse gas effect. Methane and nitrous oxide are emitted from both natural and anthropogenic sources and inventories of source specific fugitive emissions from the anthropogenic sources of methane and nitrous oxide of are often estimated on the basis of modeling and mass balance. Though these methods are well-developed, actual measurements for quantification of the emissions is a very useful tool for verifying the modeling and mass balance as well as for validation initiatives done for lowering the emissions of methane and nitrous oxide. One approach to performing such measurements is the tracer dilution method (Galle et al. 2001, Scheutz et al. 2011), where the exact location of the source is located and a tracer gas is released at this source location at a known flow. The ratio of downwind concentrations of the tracer gas and the methane and nitrous oxide gives the emissions rates of the greenhouse gases. This tracer dilution method can be performed using both stationary and mobile measurements and in both cases, real-time measurements of both tracer and quantified gas are required, placing high demands on the analytical detection method. To perform the methane and nitrous oxide measurements, two robust instruments capable of real-time measurements were used, based on cavity ring-down spectroscopy and operating in the near-infrared spectral region. One instrument measured the methane and

  11. Quantification of circulating cell-free DNA in the serum of patients with obstructive sleep apnea-hypopnea syndrome.

    Science.gov (United States)

    Ye, Liang; Ma, Guan-Hua; Chen, Ling; Li, Min; Liu, Jia-Lin; Yang, Kun; Li, Qing-Yun; Li, Ning; Wan, Huan-Ying

    2010-12-01

    Serum cell-free DNA concentrations have been reported to increase in many acute diseases as well as in some chronic conditions such as cancer and autoimmune diseases. The aim of this study was to examine whether serum DNA concentrations were elevated in patients with obstructive sleep apnea-hypopnea syndrome (OSAHS). The effects of nasal continuous positive airway pressure (nCPAP) on serum DNA were also investigated. One hundred twenty-seven people diagnosed with OSAHS by polysomnography (PSG) were admitted into the OSAHS group, and 52 subjects without OSAHS were recruited for the control group. The OSAHS group was further divided into mild, moderate, and severe OSAHS subgroups based on their apnea-hypopnea index (AHI) during sleep. Ten patients with moderate and severe OSAHS were treated with nCPAP. Serum DNA, interleukin-6 (IL-6), and malonaldehyde (MDA) concentrations were measured and were found to be significantly higher in patients with moderate and severe OSAHS groups than those in the mild OSAHS and control groups (p DNA correlated positively with AHI, oxygen desaturation index (ODI), IL-6, and MDA, and negatively correlated with minimal oxygen saturation (miniSaO(2)) (all p DNA concentrations. After 6 months of nCPAP therapy, serum concentrations of DNA, IL-6, and MDA were significantly decreased (p DNA in patients with OSAHS was positively correlated with disease severity. Serum DNA may become an important parameter for monitoring the severity of OSAHS and effectiveness of therapy.

  12. Mathematical modelling of the automated FADU assay for the quantification of DNA strand breaks and their repair in human peripheral mononuclear blood cells

    International Nuclear Information System (INIS)

    Junk, Michael; Salzwedel, Judy; Sindlinger, Thilo; Bürkle, Alexander; Moreno-Villanueva, Maria

    2014-01-01

    Cells continuously undergo DNA damage from exogenous agents like irradiation or genotoxic chemicals or from endogenous radicals produced by normal cellular metabolic activities. DNA strand breaks are one of the most common genotoxic lesions and they can also arise as intermediates of DNA repair activity. Unrepaired DNA damage can lead to genomic instability, which can massively compromise the health status of organisms. Therefore it is important to measure and quantify DNA damage and its repair. We have previously published an automated method for measuring DNA strand breaks based on fluorimetric detection of alkaline DNA unwinding [1], and here we present a mathematical model of the FADU assay, which enables to an analytic expression for the relation between measured fluorescence and the number of strand breaks. Assessment of the formation and also the repair of DNA strand breaks is a crucial functional parameter to investigate genotoxicity in living cells. A reliable and convenient method to quantify DNA strand breakage is therefore of significant importance for a wide variety of scientific fields, e.g. toxicology, pharmacology, epidemiology and medical sciences

  13. An HPLC-DAD method to quantification of main phenolic compounds from leaves of Cecropia species

    Energy Technology Data Exchange (ETDEWEB)

    Costa, Geison M.; Ortmann, Caroline F.; Schenkel, Eloir P.; Reginatto, Flavio H., E-mail: freginatto@hotmail.co [Universidade Federal de Santa Catarina (UFSC), Florianopolis (Brazil). Centro de Ciencias da Saude. Dept. de Ciencias Farmaceuticas

    2011-07-01

    An efficient and reproducible HPLC-DAD method was developed and validated for the simultaneous quantification of major compounds (chlorogenic acid, isoorientin, orientin and isovitexin) present in the leaves of two Cecropia species, C. glaziovii and C. pachystachya. From the leaves of C. glaziovii and C. pachystachya were isolated the C-glycosylflavones isoorientin and isovitexin and identified on both species chlorogenic acid (3-O-caffeoylquinic acid) and the O-glycosylflavonol isoquercitrin. The C-glycosylflavone orientin was isolated only from C. pachystachya. Chlorogenic acid was the major compound in both species (11.1 mg g{sup -1} of extract of C. glaziovii and 27.2 mg g{sup -1} of extract of C. pachystachya) and for the flavonoids quantified, isovitexin was the main C-glycosylflavonoid for C. glaziovii (4.6 mg g{sup -1} of extract) and isoorientin the main one for C. pachystachya (17.3 mg g{sup -1} of extract). (author)

  14. Digital quantification of fibrosis in liver biopsy sections: description of a new method by Photoshop software.

    Science.gov (United States)

    Dahab, Gamal M; Kheriza, Mohamed M; El-Beltagi, Hussien M; Fouda, Abdel-Motaal M; El-Din, Osama A Sharaf

    2004-01-01

    The precise quantification of fibrous tissue in liver biopsy sections is extremely important in the classification, diagnosis and grading of chronic liver disease, as well as in evaluating the response to antifibrotic therapy. Because the recently described methods of digital image analysis of fibrosis in liver biopsy sections have major flaws, including the use of out-dated techniques in image processing, inadequate precision and inability to detect and quantify perisinusoidal fibrosis, we developed a new technique in computerized image analysis of liver biopsy sections based on Adobe Photoshop software. We prepared an experimental model of liver fibrosis involving treatment of rats with oral CCl4 for 6 weeks. After staining liver sections with Masson's trichrome, a series of computer operations were performed including (i) reconstitution of seamless widefield images from a number of acquired fields of liver sections; (ii) image size and solution adjustment; (iii) color correction; (iv) digital selection of a specified color range representing all fibrous tissue in the image and; (v) extraction and calculation. This technique is fully computerized with no manual interference at any step, and thus could be very reliable for objectively quantifying any pattern of fibrosis in liver biopsy sections and in assessing the response to antifibrotic therapy. It could also be a valuable tool in the precise assessment of antifibrotic therapy to other tissue regardless of the pattern of tissue or fibrosis.

  15. Imaging-based quantification of hepatic fat: methods and clinical applications.

    Science.gov (United States)

    Ma, Xiaozhou; Holalkere, Nagaraj-Setty; Kambadakone R, Avinash; Mino-Kenudson, Mari; Hahn, Peter F; Sahani, Dushyant V

    2009-01-01

    Fatty liver disease comprises a spectrum of conditions (simple hepatic steatosis, steatohepatitis with inflammatory changes, and end-stage liver disease with fibrosis and cirrhosis). Hepatic steatosis is often associated with diabetes and obesity and may be secondary to alcohol and drug use, toxins, viral infections, and metabolic diseases. Detection and quantification of liver fat have many clinical applications, and early recognition is crucial to institute appropriate management and prevent progression. Histopathologic analysis is the reference standard to detect and quantify fat in the liver, but results are vulnerable to sampling error. Moreover, it can cause morbidity and complications and cannot be repeated often enough to monitor treatment response. Imaging can be repeated regularly and allows assessment of the entire liver, thus avoiding sampling error. Selection of appropriate imaging methods demands understanding of their advantages and limitations and the suitable clinical setting. Ultrasonography is effective for detecting moderate or severe fatty infiltration but is limited by lack of interobserver reliability and intraobserver reproducibility. Computed tomography allows quantitative and qualitative evaluation and is generally highly accurate and reliable; however, the results may be confounded by hepatic parenchymal changes due to cirrhosis or depositional diseases. Magnetic resonance (MR) imaging with appropriate sequences (eg, chemical shift techniques) has similarly high sensitivity, and MR spectroscopy provides unique advantages for some applications. However, both are expensive and too complex to be used to monitor steatosis. (c) RSNA, 2009.

  16. Study on Meshfree Hermite Radial Point Interpolation Method for Flexural Wave Propagation Modeling and Damage Quantification

    Directory of Open Access Journals (Sweden)

    Hosein Ghaffarzadeh

    Full Text Available Abstract This paper investigates the numerical modeling of the flexural wave propagation in Euler-Bernoulli beams using the Hermite-type radial point interpolation method (HRPIM under the damage quantification approach. HRPIM employs radial basis functions (RBFs and their derivatives for shape function construction as a meshfree technique. The performance of Multiquadric(MQ RBF to the assessment of the reflection ratio was evaluated. HRPIM signals were compared with the theoretical and finite element responses. Results represent that MQ is a suitable RBF for HRPIM and wave propagation. However, the range of the proper shape parameters is notable. The number of field nodes is the main parameter for accurate wave propagation modeling using HRPIM. The size of support domain should be less thanan upper bound in order to prevent high error. With regard to the number of quadrature points, providing the minimum numbers of points are adequate for the stable solution, but the existence of more points in damage region does not leads to necessarily the accurate responses. It is concluded that the pure HRPIM, without any polynomial terms, is acceptable but considering a few terms will improve the accuracy; even though more terms make the problem unstable and inaccurate.