Proceedings of the relevance of mass spectrometry to DNA sequence determination: Research needs for the Human Genome Program

Energy Technology Data Exchange (ETDEWEB)

Edmonds, C.G.; Smith, R.D. (Pacific Northwest Lab., Richland, WA (USA)); Smith, L.M. (Wisconsin Univ., Madison, WI (USA))

1990-11-01

A workshop was sponsored for the US Department of Energy (DOE), Office of Health and Environmental Research by Pacific Northwest Laboratory, April 4--5, 1990, in Seattle, Washington, to examine the potential role of mass spectrometry in the joint DOE/National Institutes of Health (NIH) Human Genome Program. The workshop was occasioned by recent developments in mass spectrometry that are providing new levels for selectivity, sensitivity, and, in particular, new methods of ionization appropriate for large biopolymers such as DNA. During discussions, three general mass spectrometric approaches to the determination of DNA sequence were considered: (1) the mass spectrometric detection of isotopic labels from DNA sequencing mixtures separated using gel electrophoresis, (2) the direct mass spectrometric analysis from direct ionization of unfractionated sequencing mixtures where the measured mass of the constituents functions to identify and order the base sequence (replacing separation by gel electrophoresis), and (3) an approach in which a single highly charged molecular ion of a large DNA segment produced is rapidly sequenced in an ion cyclotron resonance ion trap. The consensus of the workshop was that, on the basis of the new developments, mass spectrometry has the potential to provide the substantial increases in sequencing speed required for the Human Genome Program. 66 refs., 3 tabs.

The use of car-borne gamma-ray spectrometric survey in a basin

International Nuclear Information System (INIS)

Liu Tengyao; Lu Shili; Luo Zongquan.

1985-01-01

This paper describes the geological results in a basin in the Inner Mongolia which were obtained by using our newly-developed and assembled car-borne gamma-ray spectrometric system. Combined with the work of regional geology and remote sensing, five relatively favourable uranium zones were located within the working area. The field procedures of car-borne gamma-ray spectrometry for less rigid regions is discussed. The gamma-ray spectrometric profiling is mainly used for regional reconnaissance. In the case of enhanced anomalious radioactivity the profiling with varying directions is adopted and the data are plotted on scale 1:50000 topographic map. It is suggested that the car-borne gamma-ray spectrometric system can be calibrated both by the pads specified for portable spectrometers and by testing site when the calibration facility for the car-borne gamma-ray spectrometric system is not available. The effect of rainfall on car-borne gamma-ray spectrometric survey and the simplified field qualitative determination of U-Ra disequilibrium are also briefly discussed

eSensor: an electrochemical detection-based DNA microarray technology enabling sample-to-answer molecular diagnostics

Science.gov (United States)

Liu, Robin H.; Longiaru, Mathew

2009-05-01

DNA microarrays are becoming a widespread tool used in life science and drug screening due to its many benefits of miniaturization and integration. Microarrays permit a highly multiplexed DNA analysis. Recently, the development of new detection methods and simplified methodologies has rapidly expanded the use of microarray technologies from predominantly gene expression analysis into the arena of diagnostics. Osmetech's eSensor® is an electrochemical detection platform based on a low-to- medium density DNA hybridization array on a cost-effective printed circuit board substrate. eSensor® has been cleared by FDA for Warfarin sensitivity test and Cystic Fibrosis Carrier Detection. Other genetic-based diagnostic and infectious disease detection tests are under development. The eSensor® platform eliminates the need for an expensive laser-based optical system and fluorescent reagents. It allows one to perform hybridization and detection in a single and small instrument without any fluidic processing and handling. Furthermore, the eSensor® platform is readily adaptable to on-chip sample-to-answer genetic analyses using microfluidics technology. The eSensor® platform provides a cost-effective solution to direct sample-to-answer genetic analysis, and thus have a potential impact in the fields of point-of-care genetic analysis, environmental testing, and biological warfare agent detection.

Processing of gamma-ray spectrometric logs

International Nuclear Information System (INIS)

Umiastowski, K.; Dumesnil, P.

1984-10-01

CEA (Commissariat a l'Energie Atomique) has developped a gamma-ray spectrometric tool, containing an analog-to-digital converter. This new tool permits to perform very precise uranium logs (natural gamma-ray spectrometry), neutron activation logs and litho-density logs (gamma-gamma spectrometric logs). Specific processing methods were developped to treate the particular problems of down-hole gamma-ray spectrometry. Extraction of the characteristic gamma-ray peak, even if they are superposed on the background radiation of very high intensity, is possible. This processing methode enables also to obtain geological informations contained in the continuous background of the spectrum. Computer programs are written in high level language for SIRIUS (VICTOR) and APOLLO computers. Exemples of uranium and neutron activation logs treatment are presented [fr

A system design of gamma-ray spectrometric data processing for gold prospecting

International Nuclear Information System (INIS)

Yin Xueqin; Cheng Xuchu; Fang Lianxi; Huang Zuofeng.

1992-01-01

Based on different correlation between gamma-ray spectrometric data and gold grade in different types of gold deposits, it is necessary and possible to establish a mathematical model of gamma-ray spectrometric data processing for predicting specific gold deposits. A system design of gamma-ray spectrometric data processing for prospecting gold deposits has been worked out according to the mathematical method and procedure of data processing. The prediction effectiveness of commonly used multiple linear regression analysis is always not ideal but regression accuracy will be evidently increased after pre-processing of the calculated weight, deviation and favorability on gamma-ray spectrometric data. This system can establish more than ten models at the same time which enable users to have more choice. Tree structure and Chinese menu prompting are adopted in this system which can be utilized separately, sub-systems at different levels can be also individually operated. It can be transplanted to data processing of other similar geological deposit models (including non-gamma ray spectrometric data). The system is rapid, accurate, simple, convenient and flexible in use, more practical and easily popularized

Marine gamma spectrometric survey

International Nuclear Information System (INIS)

Kostoglodov, V.V.

1979-01-01

Presented are theoretical problems physical and geochemical prerequisites and possibilities of practical application of the method of continuous submarine gamma-spectrometric survey and radiometric survey destined for rapid study of the surface layer of marine sediments. Shown is high efficiency and advantages of this method in comparison with traditional and widely spread in marine geology methods of bottom sediments investigation

Analysis of GAA/TTC DNA triplexes using nuclear magnetic resonance and electrospray ionization mass spectrometry.

Science.gov (United States)

Mariappan, S V Santhana; Cheng, Xun; van Breemen, Richard B; Silks, Louis A; Gupta, Goutam

2004-11-15

The formation of a GAA/TTC DNA triplex has been implicated in Friedreich's ataxia. The destabilization of GAA/TTC DNA triplexes either by pH or by binding to appropriate ligands was analyzed by nuclear magnetic resonance (NMR) and positive-ion electrospray mass spectrometry. The triplexes and duplexes were identified by changes in the NMR chemical shifts of H8, H1, H4, 15N7, and 15N4. The lowest pH at which the duplex is detectable depends upon the overall stability and the relative number of Hoogsteen C composite function G to T composite function A basepairs. A melting pH (pHm) of 7.6 was observed for the destabilization of the (GAA)2T4(TTC)2T4(CTT)2 triplex to the corresponding Watson-Crick duplex and the T4(CTT)2 overhang. The mass spectrometric analyses of (TTC)6.(GAA)6 composite function(TTC)6 triplex detected ions due to both triplex and single-stranded oligonucleotides under acidic conditions. The triplex ions disappeared completely at alkaline pH. Duplex and single strands were detectable only at neutral and alkaline pH values. Mass spectrometric analyses also showed that minor groove-binding ligands berenil, netropsin, and distamycin and the intercalating ligand acridine orange destabilize the (TTC)6.(GAA)6 composite function (TTC)6 triplex. These NMR and mass spectrometric methods may function as screening assays for the discovery of agents that destabilize GAA/TTC triplexes and as general methods for the characterization of structure, dynamics, and stability of DNA and DNA-ligand complexes.

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.

Potentialities of aberrantly methylated circulating DNA for diagnostics and post-treatment follow-up of lung cancer patients.

Science.gov (United States)

Ponomaryova, Anastasia A; Rykova, Elena Yu; Cherdyntseva, Nadezda V; Skvortsova, Tatiana E; Dobrodeev, Alexey Yu; Zav'yalov, Alexander A; Bryzgalov, Leonid O; Tuzikov, Sergey A; Vlassov, Valentin V; Laktionov, Pavel P

2013-09-01

To date, aberrant DNA methylation has been shown to be one of the most common and early causes of malignant cell transformation and tumors of different localizations, including lung cancer. Cancer cell-specific methylated DNA has been found in the blood of cancer patients, indicating that cell-free DNA circulating in the blood (cirDNA) is a convenient tumor-associated DNA marker that can be used as a minimally invasive diagnostic test. In the current study, we investigated the methylation status in blood samples of 32 healthy donors and 60 lung cancer patients before and after treatment with neoadjuvant chemotherapy followed by total tumor resection. Using quantitative methylation-specific PCR, we found that the index of methylation (IM), calculated as IM = 100 × [copy number of methylated/(copy number of methylated + unmethylated gene)], for the RASSF1A and RARB2 genes in the cirDNA isolated from blood plasma and cell-surface-bound cirDNA was elevated 2- to 3-fold in lung cancer patients compared with healthy donors. Random forest classification tree model based on these variables combined (RARB2 and RASSF1A IM in both plasma and cell-surface-bound cirDNA) lead to NSCLC patients' and healthy subjects' differentiation with 87% sensitivity and 75% specificity. An association of increased IM values with an advanced stage of non-small-cell lung cancer was found for RARB2 but not for RASSF1A. Chemotherapy and total tumor resection resulted in a significant decrease in the IM for RARB2 and RASSF1A, in both cirDNA fractions, comparable to the IM level of healthy subjects. Importantly, a rise in the IM for RARB2 was detected in patients within the follow-up period, which manifested in disease relapse at 9 months, confirmed with instrumental and pathologic methods. Our data indicate that quantitative analysis of the methylation status of the RARB2 and RASSF1A tumor suppressor genes in both cirDNA fractions is a useful tool for lung cancer diagnostics, evaluation of cancer

Spectrometric techniques 2

CERN Document Server

Vanasse, George A

2013-01-01

Spectrometric Techniques, Volume II provides information pertinent to vacuum ultraviolet techniques to complete the demonstration of the diversity of methods available to the spectroscopist interested in the ultraviolet visible and infrared spectral regions. This book discusses the specific aspects of the technique of Fourier transform spectroscopy.Organized into five chapters, this volume begins with an overview of the large number of systematic effects in the recording of an interferogram. This text then examines the design approach for a Fourier transform spectrometer with focus on optics.

Nuclear/Nucleolar morphometry and DNA image cytometry as a combined diagnostic tool in pathology of prostatic carcinoma

Energy Technology Data Exchange (ETDEWEB)

Kavantzas, N.; Agapitos, E.; Lazaris, A. C.; Pavlopulos, P.M.; Sofikitis, N.; Davaris, P. [National University of Athens, Dept. of Pathology, Medical School, Athens (Greece)

2001-12-01

Paraffin tissue sections from 50 patients with prostate adenocarcinoma were used to study nuclear and nucleolar morphometric features by image analysis. The results were compared to DNA ploidy and Gleason grade. In the examined histological samples nuclear and nucleolar areas were positively interrelated. It was also noticed that the higher the percentage of nucleolated nuclei, the bigger the nuclear and nucleolar areas. The morphometric characteristics did not differ significantly among the four grades of the examined specimens. In well-differentiated carcinomas the DNA index was lower than in the rest at a statistically significant level. Hypodiploid carcinomas were found to possess significantly bigger nuclear areas than any other DNA index group. Morphonuclear evidence of anaplasia and DNA aneuploidy may be used as diagnostic tools in prostate cancer in addition to Gleason grade.

Nuclear/Nucleolar morphometry and DNA image cytometry as a combined diagnostic tool in pathology of prostatic carcinoma

International Nuclear Information System (INIS)

Kavantzas, N.; Agapitos, E.; Lazaris, A. C.; Pavlopulos, P.M.; Sofikitis, N.; Davaris, P.

2001-01-01

Paraffin tissue sections from 50 patients with prostate adenocarcinoma were used to study nuclear and nucleolar morphometric features by image analysis. The results were compared to DNA ploidy and Gleason grade. In the examined histological samples nuclear and nucleolar areas were positively interrelated. It was also noticed that the higher the percentage of nucleolated nuclei, the bigger the nuclear and nucleolar areas. The morphometric characteristics did not differ significantly among the four grades of the examined specimens. In well-differentiated carcinomas the DNA index was lower than in the rest at a statistically significant level. Hypodiploid carcinomas were found to possess significantly bigger nuclear areas than any other DNA index group. Morphonuclear evidence of anaplasia and DNA aneuploidy may be used as diagnostic tools in prostate cancer in addition to Gleason grade

Back to basics: an evaluation of NaOH and alternative rapid DNA extraction protocols for DNA barcoding, genotyping, and disease diagnostics from fungal and oomycete samples.

Science.gov (United States)

Osmundson, Todd W; Eyre, Catherine A; Hayden, Katherine M; Dhillon, Jaskirn; Garbelotto, Matteo M

2013-01-01

The ubiquity, high diversity and often-cryptic manifestations of fungi and oomycetes frequently necessitate molecular tools for detecting and identifying them in the environment. In applications including DNA barcoding, pathogen detection from plant samples, and genotyping for population genetics and epidemiology, rapid and dependable DNA extraction methods scalable from one to hundreds of samples are desirable. We evaluated several rapid extraction methods (NaOH, Rapid one-step extraction (ROSE), Chelex 100, proteinase K) for their ability to obtain DNA of quantity and quality suitable for the following applications: PCR amplification of the multicopy barcoding locus ITS1/5.8S/ITS2 from various fungal cultures and sporocarps; single-copy microsatellite amplification from cultures of the phytopathogenic oomycete Phytophthora ramorum; probe-based P. ramorum detection from leaves. Several methods were effective for most of the applications, with NaOH extraction favored in terms of success rate, cost, speed and simplicity. Frozen dilutions of ROSE and NaOH extracts maintained PCR viability for over 32 months. DNA from rapid extractions performed poorly compared to CTAB/phenol-chloroform extracts for TaqMan diagnostics from tanoak leaves, suggesting that incomplete removal of PCR inhibitors is an issue for sensitive diagnostic procedures, especially from plants with recalcitrant leaf chemistry. NaOH extracts exhibited lower yield and size than CTAB/phenol-chloroform extracts; however, NaOH extraction facilitated obtaining clean sequence data from sporocarps contaminated by other fungi, perhaps due to dilution resulting from low DNA yield. We conclude that conventional extractions are often unnecessary for routine DNA sequencing or genotyping of fungi and oomycetes, and recommend simpler strategies where source materials and intended applications warrant such use. © 2012 Blackwell Publishing Ltd.

Gas Chromatography-Mass Spectrometric Analysis and Insecticidal ...

African Journals Online (AJOL)

HP

Original Research Article. Gas Chromatography-Mass Spectrometric Analysis and ... into a natural fumigant/insecticide for the control of stored product insects. Keywords: Mallotus ..... stability as well as reduce cost. ACKNOWLEDGEMENT.

Gas Chromatography-Mass Spectrometric Analysis of Nematicidal ...

African Journals Online (AJOL)

Gas Chromatography-Mass Spectrometric Analysis of Nematicidal Essential Oil of Valeriana ... Tropical Journal of Pharmaceutical Research ... have a potential to be developed to natural nematicides for the control of cereal cyst nematodes.

Spectrometric control of radionuclides production parameters

International Nuclear Information System (INIS)

Zhuk, I.; Potarenko, A.; Yarochevich, O.; Hluboky, N.; Kerko, P.; Bogdanov, V.; Dyatel, N.

2006-01-01

Full text: A radioactive preparations and sources are widely used all over the world for scientific, industrial and medical purposes. These preparations in Belarus are planned to produce by the Joint Belarussian-Russian Closed Joint Stock Company 'Isotope technologies' (CJSC IT). The company was created in 1998 by two leading scientific centers-SSI 'Joint Institute of Power and Nuclear Research-Sosny' the National Academy of Sciences of Belarus and the State Center of Science of the Russian Federation 'Scientific research institute of nuclear reactors'. One of the mainstream directions in CJSC IT activities is production of radioactive preparations for the industrial and scientific application (such as 133 Ba, 109 Cd, 63 Ni, 60 Co) and for the medical purposes (such as 19 '2Ir, 60 Co). All radioactive preparations have a good export potential and adequate to modern technical and consumer requirements. X-γ spectrometric analysis of considered radioactive sources is one of the basic methods for quality control of radioactive sources. At present, we are developing x-γ spectrometric support of purification process from contaminating radionuclides of 109 Cd -γ preparation and 63 Ni - β preparation. Work on x-γ spectrometric quality control of 133 Ba preparation is carried out. The description of the used equipment is given. Techniques of contaminating radionuclides determination (contents ∼10 - '6 from activity of the basic radionuclide) are presented. Problems of the choice of geometry of measurements of sources with activity about 10 7 -10 9 Bq and possible sources of errors are discussed. (author)

CMOS Time-Resolved, Contact, and Multispectral Fluorescence Imaging for DNA Molecular Diagnostics

Directory of Open Access Journals (Sweden)

Nan Guo

2014-10-01

Full Text Available Instrumental limitations such as bulkiness and high cost prevent the fluorescence technique from becoming ubiquitous for point-of-care deoxyribonucleic acid (DNA detection and other in-field molecular diagnostics applications. The complimentary metal-oxide-semiconductor (CMOS technology, as benefited from process scaling, provides several advanced capabilities such as high integration density, high-resolution signal processing, and low power consumption, enabling sensitive, integrated, and low-cost fluorescence analytical platforms. In this paper, CMOS time-resolved, contact, and multispectral imaging are reviewed. Recently reported CMOS fluorescence analysis microsystem prototypes are surveyed to highlight the present state of the art.

Gas Chromatography-Mass Spectrometric Analysis and Insecticidal ...

African Journals Online (AJOL)

Tropical Journal of Pharmaceutical Research ... apelta aerial parts was analyzed by gas chromatography/mass spectrometric (GC/MS) to determine its composition. ... into a natural fumigant/insecticide for the control of stored product insects.

Molecular phylogenetic analysis of Enterobius vermicularis and development of an 18S ribosomal DNA-targeted diagnostic PCR.

Science.gov (United States)

Zelck, Ulrike E; Bialek, Ralf; Weiss, Michael

2011-04-01

We genetically characterized pinworms obtained from 37 children from different regions of Germany and established new species-specific molecular diagnostic tools. No ribosomal DNA diversity was found; the phylogenetic position of Enterobius vermicularis within the Oxyurida order and its close relationship to the Ascaridida and Spirurida orders was confirmed.

A potential new diagnostic tool to aid DNA analysis from heat compromised bone using colorimetry: A preliminary study.

Science.gov (United States)

Fredericks, Jamie D; Ringrose, Trevor J; Dicken, Anthony; Williams, Anna; Bennett, Phil

2015-03-01

Extracting viable DNA from many forensic sample types can be very challenging, as environmental conditions may be far from optimal with regard to DNA preservation. Consequently, skeletal tissue can often be an invaluable source of DNA. The bone matrix provides a hardened material that encapsulates DNA, acting as a barrier to environmental insults that would otherwise be detrimental to its integrity. However, like all forensic samples, DNA in bone can still become degraded in extreme conditions, such as intense heat. Extracting DNA from bone can be laborious and time-consuming. Thus, a lot of time and money can be wasted processing samples that do not ultimately yield viable DNA. We describe the use of colorimetry as a novel diagnostic tool that can assist DNA analysis from heat-treated bone. This study focuses on characterizing changes in the material and physical properties of heated bone, and their correlation with digitally measured color variation. The results demonstrate that the color of bone, which serves as an indicator of the chemical processes that have occurred, can be correlated with the success or failure of subsequent DNA amplification. Copyright © 2014 Forensic Science Society. Published by Elsevier Ireland Ltd. All rights reserved.

Liquid chromatographic-mass spectrometric method for ...

African Journals Online (AJOL)

Tropical Journal of Pharmaceutical Research ... Purpose: To develop and validate a simple, efficient and reliable Liquid chromatographic-mass spectrometric (LC-MS/MS) method for the quantitative determination of two dermatological drugs, ... By Country · List All Titles · Free To Read Titles This Journal is Open Access.

Mass Spectrometric Analysis for Nuclear Safeguards

International Nuclear Information System (INIS)

Boulyga, S.

2013-01-01

The release of man-made radionuclides into the environment results in contamination that carries specific isotopic signatures according to the release scenarios and the previous usage of materials and facilities. In order to trace the origin of such contamination and/or to assess the potential impact on the public and environmental health, it is necessary to determine the isotopic composition and activity concentrations of radionuclides in environmental samples in an accurate and timely fashion. Mass spectrometric techniques, such as thermal ionization mass spectrometry (TIMS), secondary ion mass spectrometry (SIMS), and inductively coupled plasma mass spectrometry (ICP-MS) belong to the most powerful methods for analysis of nuclear and related samples in nuclear safeguards, forensics, and environmental monitoring. This presentation will address the potential of mass spectrometric analysis of actinides at ultra-trace concentration levels, isotopic analysis of micro-samples, age determination of nuclear materials as well as identification and quantification of elemental and isotopic signatures of nuclear samples in general. (author)

Molecular Phylogenetic Analysis of Enterobius vermicularis and Development of an 18S Ribosomal DNA-Targeted Diagnostic PCR▿

Science.gov (United States)

Zelck, Ulrike E.; Bialek, Ralf; Weiß, Michael

2011-01-01

We genetically characterized pinworms obtained from 37 children from different regions of Germany and established new species-specific molecular diagnostic tools. No ribosomal DNA diversity was found; the phylogenetic position of Enterobius vermicularis within the Oxyurida order and its close relationship to the Ascaridida and Spirurida orders was confirmed. PMID:21248085

Emission spectrometric isotope analyzer

International Nuclear Information System (INIS)

Mauersberger, K.; Meier, G.; Nitschke, W.; Rose, W.; Schmidt, G.; Rahm, N.; Andrae, G.; Krieg, D.; Kuefner, W.; Tamme, G.; Wichlacz, D.

1982-01-01

An emission spectrometric isotope analyzer has been designed for determining relative abundances of stable isotopes in gaseous samples in discharge tubes, in liquid samples, and in flowing gaseous samples. It consists of a high-frequency generator, a device for defined positioning of discharge tubes, a grating monochromator with oscillating slit and signal converter, signal generator, window discriminator, AND connection, read-out display, oscillograph, gas dosing device and chemical conversion system with carrier gas source and vacuum pump

Car-borne multichannel gamma-ray spectrometric system model CZD-6

International Nuclear Information System (INIS)

Lu Shili; Zhai Yugui; Ma Yanfang; Jiao Cangwen; Zhang Biao

1998-01-01

The car-borne multichannel gamma-ray spectrometric system Mode CZD-6 is composed of a HDY-256 of portable multichannel gamma-ray spectrometer developed by the Beijing Research Institute of Uranium Geology and a large volume scintillation detector. the position for each measurement point is determined by the GPS instrument. Its latitude and longitude, as well as measured 256 channels of γ-spectrometric data are collected by a notebook computer, which can show the 256 channels of spectra for each point during measuring processes. The complete system can be loaded in a field car. This system has been used to environmental radioactive monitoring after calibration by airborne radiometric models in Shijiazhuang, Hebei province. A lot of data confirm that the system works stably and reliably, and is a fast and advanced approach for environmental γ-spectrometric monitoring. It can be used not only to determination of contents of natural radioactive elements in environments, but also to monitoring nuclear pollution and emergency treatment in nuclear accidents significantly

The usage and current approaches of cell free fetal DNA (cffDNA as a prenatal diagnostic method in fetal aneuploidy screening

Directory of Open Access Journals (Sweden)

Hülya Erbaba

2015-12-01

Full Text Available Prenatal diagnosis of invasive and noninvasive tests can be done in a way (NIPT, but because of the invasive methods have risks of infection and abortion, diagnosing non-invasive procedure increasing day by day. One of the widespread cell free fetal DNA in maternal blood test (cffDNA that is increasing in clinical use has been drawing attention. The incidence of aneuploidy chromosomal anomaly of the kind in which all live births; Trisomy 21 (Down Syndrome 1/800, trisomy 13 (Patau syndrome 1 /10,000, trisomy 18 (Edwards syndrome is a form of 1/6000. Because of the high mortality and morbidity, it is vital that congenital anomalies should be diagnosed in prenatal period. Aneuploidy testing for high-risk pregnant women after the 10th week of pregnancy in terms of the blood sample is taken and free fetal DNA in maternal plasma is based on the measurement of the relative amount. Knowledge of the current criteria for use by healthcare professionals in the field test will allow the exclusion of maternal and fetal risks. In this study, it is aimed to demonstrate current international approaches related to the positive and negative sides of non-invasive that is one of the prenatal diagnostic methods of cffDNA test. J Clin Exp Invest 2015; 6 (4: 414-417

Energy dispersive X-Ray fluorescence spectrometric study of ...

African Journals Online (AJOL)

Energy dispersive X-Ray fluorescence spectrometric study of compositional differences in trace elements in dried Moringa oleifera leaves grown in two different agro-ecological locations in Ebonyi State, Nigeria.

DNA Diagnostics: Optical or by Electronics?

KAUST Repository

Khan, Hadayat Ullah

2016-01-15

In this paper, we very briefly review DNA biosensors based on optical and electrical detection principles, referring mainly to our past work applying both techniques but here using nearly identical sensor chip surface architectures, i.e., capture probe layers that were prepared based on a pulsed plasma deposition protocol for maleic anhydride and subsequent wet-chemical attachment of the amine-functionalized peptide nucleic acid (PNA) probe oligonucleotides. 15 mer DNA target strands, labeled with Cy5-chromophores that were attached at the 5’ end were used for surface plasmon optical detection and the same target DNA but without label was used in OTFT sensor-based detection where the mere charge density of the bound (hybridized) DNA molecules modulate the source-drain current. The sensing mechanisms and the detection limits of the devices are described in some detail. Both techniques allow for the monitoring of surface hybridization reactions, and offer the capacity to quantitatively discriminate between targets with different degrees of mismatched sequences.

DNA barcodes and molecular diagnostics to distinguish an introduced and native Laricobius (Coleoptera: Derodontidae) species in eastern North America

Science.gov (United States)

G.A. Davis; N.P. Havill; Z.N. Adelman; A. Caccone; L.T. Kok; S.M. Salom

2011-01-01

Molecular diagnostics based on DNA barcodes can be powerful identification tools in the absence of distinctive morphological characters for distinguishing between closely related species. A specific example is distinguishing the endemic species Laricobius rubidus from Laricobius nigrinus, a biological control agent of hemlock...

Comparative Analytical Utility of DNA Derived from Alternative Human Specimens for Molecular Autopsy and Diagnostics

Science.gov (United States)

Klassen, Tara L.; von Rüden, Eva-Lotta; Drabek, Janice; Noebels, Jeffrey L.; Goldman, Alica M.

2013-01-01

Genetic testing and research have increased the demand for high-quality DNA that has traditionally been obtained by venipuncture. However, venous blood collection may prove difficult in special populations and when large-scale specimen collection or exchange is prerequisite for international collaborative investigations. Guthrie/FTA card–based blood spots, buccal scrapes, and finger nail clippings are DNA-containing specimens that are uniquely accessible and thus attractive as alternative tissue sources (ATS). The literature details a variety of protocols for extraction of nucleic acids from a singular ATS type, but their utility has not been systematically analyzed in comparison with conventional sources such as venous blood. Additionally, the efficacy of each protocol is often equated with the overall nucleic acid yield but not with the analytical performance of the DNA during mutation detection. Together with a critical in-depth literature review of published extraction methods, we developed and evaluated an all-inclusive approach for serial, systematic, and direct comparison of DNA utility from multiple biological samples. Our results point to the often underappreciated value of these alternative tissue sources and highlight ways to maximize the ATS-derived DNA for optimal quantity, quality, and utility as a function of extraction method. Our comparative analysis clarifies the value of ATS in genomic analysis projects for population-based screening, diagnostics, molecular autopsy, medico-legal investigations, or multi-organ surveys of suspected mosaicisms. PMID:22796560

Mass spectrometric researches in isotope cosmochemistry

International Nuclear Information System (INIS)

Gopalan, K.

1979-01-01

Recent advances in the understanding of solar system processes, past and present, based on mass spectrometric researches on meteorites and moon samples are reviewed. The topics include the following : (1) Duration of nebular condensation, (2) Terminal stages of nucleosynthesis, (3) Planetary formation and evolution, (4) Heterogeneities in the solar nebula and (5) Solar wind composition. (auth.)

Spectrometric assembly for portable installations

International Nuclear Information System (INIS)

Kluger, A.; Popescu, C.

1997-01-01

The components of the portable spectrometric assembly are: - the detecting probe with Na I(Tl) crystal and air-tight case of industrial type; - a microcomputer; - a unit of analogical processing of the signal from the detecting probe; - a single-channel analyzer with adjustable threshold; - commands and display module; - a source of high voltage; - an electrical supply battery. The device uses the method of gamma photons detection in energetic windows. Through theoretical and experimental studies carried out during the prototype development phase, the superiority of this method has been proved as compared with the installations which make use of the classical principle of photon integral detection. The achieved prototype has a basic program enabling the setting of all working parameters (measuring time, discriminating thresholds, discriminators operating conditions, etc.). Through the included interface RS232 it is possible to transmit the data to a more powerful computer in order to continually process the results. The spectrometric assembly, realized on the basis of micro-computers, can be used in a wide range of applications: measurement of thickness and erosion of walls and tubes, measurement of level in closed containers, of soil density, etc. The adjustment for specific application is performed only through a program modification. (authors)

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

MOLECULAR DIAGNOSTICS OF YERSINIA RUCKERI

Directory of Open Access Journals (Sweden)

Yu. Rud

2014-06-01

Full Text Available Purpose. The analysis of nucleotide sequences of the 16S rDNA gene of virulent strains of Yersinia ruckeri and to develop the method of molecular diagnostic of enteric redmouth disease. Methodology. By the method of CLUSTALW algorithm in MEGA software version 6.0 the nucleotide sequences of the 16S rDNA gene of virulent strains of Yersinia ruckeri were analysed. For development of molecular diagnostic of Y. ruckeri the method of polymerase chain reaction (PCR was used. Primer selection was carried out in software VectorNTI11 and on-line-service BLAST. The PCR products were investigated by the methods of sequencing and nucleotide analysis. Findings. Based on PCR assay the method of molecular diagnostic of enteric redmouth disease agent, bacterium Y. ruckeri was developed. It was shown that specific oligonucleotide primers generated PCR products in size of 600 base pairs. PCR products were investigated by the sequencing that showed right targeting of primers in reaction. Originality. Among high-conservative gene of 16S rDNA of Y. ruckeri the fragment of DNA was determined to which the specific primers for rapid diagnostic of virulent strains were selected. Practical Value. Rapid diagnostic of yersiniosis will allow to identify an agent of this infectious disease, bacterium Y. ruckeri, and to provide the prophylactic or medical measures in the fish farming of Ukraine.

DNA-based techniques for authentication of processed food and food supplements.

Science.gov (United States)

Lo, Yat-Tung; Shaw, Pang-Chui

2018-02-01

Authentication of food or food supplements with medicinal values is important to avoid adverse toxic effects, provide consumer rights, as well as for certification purpose. Compared to morphological and spectrometric techniques, molecular authentication is found to be accurate, sensitive and reliable. However, DNA degradation and inclusion of inhibitors may lead to failure in PCR amplification. This paper reviews on the existing DNA extraction and PCR protocols, and the use of small size DNA markers with sufficient discriminative power for molecular authentication. Various emerging new molecular techniques such as isothermal amplification for on-site diagnosis, next-generation sequencing for high-throughput species identification, high resolution melting analysis for quick species differentiation, DNA array techniques for rapid detection and quantitative determination in food products are also discussed. Copyright © 2017 Elsevier Ltd. All rights reserved.

The theoretical study of full spectrum analysis method for airborne gamma-ray spectrometric data

International Nuclear Information System (INIS)

Ni Weichong

2011-01-01

Spectra of airborne gamma-ray spectrometry was found to be the synthesis of spectral components of radioelement sources by analyzing the constitution of radioactive sources for airborne gamma-ray spectrometric survey and establishing the models of gamma-ray measurement. The mathematical equation for analysising airborne gamma-ray full spectrometric data can be expressed into matrix and related expansions were developed for the mineral resources exploration, environmental radiation measurement, nuclear emergency monitoring, and so on. Theoretical study showed that the atmospheric radon could be directly computed by airborne gamma-ray spectrometric data with full spectrum analysis without the use of the accessional upward-looking detectors. (authors)

A New FRET-Based Sensitive DNA Sensor for Medical Diagnostics using PNA Probe and Water-Soluble Blue Light Emitting Polymer

Directory of Open Access Journals (Sweden)

Nidhi Mathur

2008-01-01

Full Text Available A reliable, fast, and low-cost biosensor for medical diagnostics using DNA sequence detection has been developed and tested for the detection of the bacterium “Bacillus anthracis.” In this sensor, Poly [9,9-di (6,6′- N, N′ trimethylammonium hexylfluorenyl-2, 7-diyl-alt-co- (1,4-phenylene] dibromide salt (PFP has been taken as cationic conjugated polymer (CCP and PNA attached with fluorescein dye (PNAC∗ as a probe. The basic principle of this sensor is that when a PNAC∗ probe is hybridized with a single strand DNA (ssDNA having complementary sequence, Forster resonance energy transfer (FRET may take place from PFP to the PNAC∗/DNA complex. If the FRET is efficient, the photoluminescence from the PFP will be highly quenched and that from PNAC∗ will be enhanced. On the other hand, if the DNA sequence is noncomplementary to PNA, FRET will not occur.

Rapid colorimetric detection of p53 protein function using DNA-gold nanoconjugates with applications for drug discovery and cancer diagnostics.

Science.gov (United States)

Assah, Enock; Goh, Walter; Zheng, Xin Ting; Lim, Ting Xiang; Li, Jun; Lane, David; Ghadessy, Farid; Tan, Yen Nee

2018-05-05

The tumor suppressor protein p53 plays a central role in preventing cancer through interaction with DNA response elements (REs) to regulate target gene expression in cells. Due to its significance in cancer biology, relentless efforts have been directed toward understanding p53-DNA interactions for the development of cancer therapeutics and diagnostics. In this paper, we report a rapid, label-free and versatile colorimetric assay to detect wildtype p53 DNA-binding function in complex solutions. The assay design is based on a concept that alters interparticle-distances between RE-AuNPs from a crosslinking effect induced through tetramerization of wildtype p53 protein (p53-WT) upon binding to canonical DNA motifs modified on gold nanoparticles (RE-AuNPs). This leads to a visible solution color change from red to blue, which is quantifiable by the UV- visible absorption spectra with a detection limit of 5 nM. Contrastingly, no color change was observed for the binding-deficient p53 mutants and non-specific proteins due to their inability to crosslink RE-AuNPs. Based on this sensing principle, we further demonstrate its utility for fast detection of drug-induced DNA binding function to cancer-associated Y220C mutant p53 protein using well-established reactivating compounds. By exploiting the dominant-negative property of mutant p53 over p53-WT and interactions with RE-AuNPs, this assay is configurable to detect low numbers of mutant p53 expressing cells in miniscule sample fractions obtained from typical core needle biopsy-sized tissues without signal attrition, alluding to the potential for biopsy sampling in cancer diagnostics or for defining cancer margins. This nanogold enabled colorimetric assay provides a facile yet robust method for studying important parameters influencing p53-DNA interactions with great promises for clinically pertinent applications. Copyright © 2018 Elsevier B.V. All rights reserved.

2012 HIV Diagnostics Conference: the molecular diagnostics perspective.

Science.gov (United States)

Branson, Bernard M; Pandori, Mark

2013-04-01

2012 HIV Diagnostic Conference Atlanta, GA, USA, 12-14 December 2012. This report highlights the presentations and discussions from the 2012 National HIV Diagnostic Conference held in Atlanta (GA, USA), on 12-14 December 2012. Reflecting changes in the evolving field of HIV diagnostics, the conference provided a forum for evaluating developments in molecular diagnostics and their role in HIV diagnosis. In 2010, the HIV Diagnostics Conference concluded with the proposal of a new diagnostic algorithm which included nucleic acid testing to resolve discordant screening and supplemental antibody test results. The 2012 meeting, picking up where the 2010 meeting left off, focused on scientific presentations that assessed this new algorithm and the role played by RNA testing and new developments in molecular diagnostics, including detection of total and integrated HIV-1 DNA, detection and quantification of HIV-2 RNA, and rapid formats for detection of HIV-1 RNA.

GNU polyxmass: a software framework for mass spectrometric simulations of linear (bio-polymeric analytes

Directory of Open Access Journals (Sweden)

Rusconi Filippo

2006-04-01

Full Text Available Abstract Background Nowadays, a variety of (bio-polymers can be analyzed by mass spectrometry. The detailed interpretation of the spectra requires a huge number of "hypothesis cycles", comprising the following three actions 1 put forth a structural hypothesis, 2 test it, 3 (invalidate it. This time-consuming and painstaking data scrutiny is alleviated by using specialized software tools. However, all the software tools available to date are polymer chemistry-specific. This imposes a heavy overhead to researchers who do mass spectrometry on a variety of (bio-polymers, as each polymer type will require a different software tool to perform data simulations and analyses. We developed a software to address the lack of an integrated software framework able to deal with different polymer chemistries. Results The GNU polyxmass software framework performs common (bio-chemical simulations–along with simultaneous mass spectrometric calculations–for any kind of linear bio-polymeric analyte (DNA, RNA, saccharides or proteins. The framework is organized into three modules, all accessible from one single binary program. The modules let the user to 1 define brand new polymer chemistries, 2 perform quick mass calculations using a desktop calculator paradigm, 3 graphically edit polymer sequences and perform (bio-chemical/mass spectrometric simulations. Any aspect of the mass calculations, polymer chemistry reactions or graphical polymer sequence editing is configurable. Conclusion The scientist who uses mass spectrometry to characterize (bio-polymeric analytes of different chemistries is provided with a single software framework for his data prediction/analysis needs, whatever the polymer chemistry being involved.

ROLE OF DNA METHYLATION AS A DIAGNOSTIC BIOMARKER OF SPORADIC BREAST CANCER

Directory of Open Access Journals (Sweden)

Wirsma Arif Harahap

2017-02-01

all methylation profiles fit within the same molecular subtype. Specific gene methylation profiles are identified for basal-like, luminal A and HER2-overexpressing breast cancers. A number of studies have analyzed the methylation status of BRCA1, a key player in TNBC. One study demonstrated that BRCA1 promoter was methylated in TNBC.  It was discovered that the sensitivity of TNBC cell lines to PARP inhibitors was increased when BRCA1 was methylated. Concurrently, BRCA1 methylation quantity was higher in patients with complete response than in those who are non-responders of neoadjuvant chemotherapy. Epigenetics is now the cutting edge of cancer research.  Advances in this field will have major implications in diagnosis, prevention, treatment of cancer, and formulation of new epigenetically targeted cancer drugs.   Keywords: breast cancer, epigenetic, DNA methylation, BRCA1, diagnostic marker

Structure elucidation of metabolite x17299 by interpretation of mass spectrometric data.

Science.gov (United States)

Zhang, Qibo; Ford, Lisa A; Evans, Anne M; Toal, Douglas R

2017-01-01

A major bottleneck in metabolomic studies is metabolite identification from accurate mass spectrometric data. Metabolite x17299 was identified in plasma as an unknown in a metabolomic study using a compound-centric approach where the associated ion features of the compound were used to determine the true molecular mass. The aim of this work is to elucidate the chemical structure of x17299, a new compound by de novo interpretation of mass spectrometric data. An Orbitrap Elite mass spectrometer was used for acquisition of mass spectra up to MS 4 at high resolution. Synthetic standards of N,N,N -trimethyl-l-alanyl-l-proline betaine (l,l-TMAP), a diastereomer, and an enantiomer were chemically prepared. The planar structure of x17299 was successfully proposed by de novo mechanistic interpretation of mass spectrometric data without any laborious purification and nuclear magnetic resonance spectroscopic analysis. The proposed structure was verified by deuterium exchanged mass spectrometric analysis and confirmed by comparison to a synthetic standard. Relative configuration of x17299 was determined by direct chromatographic comparison to a pair of synthetic diastereomers. Absolute configuration was assigned after derivatization of x17299 with a chiral auxiliary group followed by its chromatographic comparison to a pair of synthetic standards. The chemical structure of metabolite x17299 was determined to be l,l-TMAP.

Polymerase study: Improved detection of Salmonella and Campylobacter through the optimized use of DNA polymerases in diagnostic real-time PCR

DEFF Research Database (Denmark)

Søndergaard, Mette Sofie Rousing; Löfström, Charlotta; Al-Habib, Zahra Fares Sayer

DNA extractions and intermediate or bad with the crude extractions, while TaKaRa ExTaq HS only performed well with the purest extractions of fecal samples and intermediate with semi-automated magnetic beads based extracted fecal samples. In conclusion, our data shows that exchanging the DNA polymerase......Diagnostic analyses of foodborne pathogens are increasingly based on molecular methods such as PCR, which can improve the sensitivity and reduce the analysis time. The core of PCR is the enzyme performing the reaction: the DNA polymerase. Changing the polymerase can influence the sensitivity...... commercially available polymerases and four master mixes in two validated PCR assays, for Campylobacter and Salmonella, respectively, to develop more sensitive, robust and cost effective assays. The polymerases were screened on purified DNA and the five best performing, for each PCR assay, were then applied...

Using rapid diagnostic tests as source of malaria parasite DNA for molecular analyses in the era of declining malaria prevalence

DEFF Research Database (Denmark)

Ishengoma, Deus S; Lwitiho, Sudi; Madebe, Rashid A

2011-01-01

was conducted to examine if sufficient DNA could be successfully extracted from malaria rapid diagnostic tests (RDTs), used and collected as part of routine case management services in health facilities, and thus forming the basis for molecular analyses, surveillance and quality control (QC) testing of RDTs....... continued molecular surveillance of malaria parasites is important to early identify emerging anti-malarial drug resistance, it is becoming increasingly difficult to obtain parasite samples from ongoing studies, such as routine drug efficacy trials. To explore other sources of parasite DNA, this study...

Electronic equipment for spectrometric data processing

International Nuclear Information System (INIS)

Antonov, L.J.; Trenev, A.M.; Todorova, E.I.; Dimitrov, V.D.

1978-01-01

Electronic equipment carrying out logical operations and a full set of the arithmetic operations was developed for spectrometric data processing. The flowsheet of the computing part of the device, made on the basis of a specialized integral circuit, is given. The device includes input registers, multiplexor, matrix commutator, arithmetic unit and indication unit. The equipment is rated to carry out calculations according to comparatively complex formulae in several seconds

Uncertainty assessment in gamma spectrometric measurements of plutonium isotope ratios and age

Energy Technology Data Exchange (ETDEWEB)

Ramebaeck, H., E-mail: henrik.ramebeck@foi.se [Swedish Defence Research Agency, FOI, Division of CBRN Defence and Security, SE-901 82 Umea (Sweden); Chalmers University of Technology, Department of Chemical and Biological Engineering, Nuclear Chemistry, SE-412 96 Goeteborg (Sweden); Nygren, U.; Tovedal, A. [Swedish Defence Research Agency, FOI, Division of CBRN Defence and Security, SE-901 82 Umea (Sweden); Ekberg, C.; Skarnemark, G. [Chalmers University of Technology, Department of Chemical and Biological Engineering, Nuclear Chemistry, SE-412 96 Goeteborg (Sweden)

2012-09-15

A method for the assessment of the combined uncertainty in gamma spectrometric measurements of plutonium composition and age was evaluated. Two materials were measured. Isotope dilution inductively coupled plasma sector field mass spectrometry (ID-ICP-SFMS) was used as a reference method for comparing the results obtained with the gamma spectrometric method for one of the materials. For this material (weapons grade plutonium) the measurement results were in agreement between the two methods for all measurands. Moreover, the combined uncertainty in all isotope ratios considered in this material (R{sub Pu238/Pu239}, R{sub Pu240/Pu239}, R{sub Pu241/Pu239}, and R{sub Am241/Pu241} for age determination) were limited by counting statistics. However, the combined uncertainty for the other material (fuel grade plutonium) were limited by the response fit, which shows that the uncertainty in the response function is important to include in the combined measurement uncertainty of gamma spectrometric measurements of plutonium.

Mass Spectrometric Identification and Differentiation of Botulinum Neurotoxins through Toxin Proteomics.

Science.gov (United States)

Kalb, Suzanne R; Barr, John R

2013-08-01

Botulinum neurotoxins (BoNTs) cause the disease botulism, which can be lethal if untreated. There are seven known serotypes of BoNT, A-G, defined by their response to antisera. Many serotypes are distinguished into differing subtypes based on amino acid sequence and immunogenic properties, and some subtypes are further differentiated into toxin variants. Toxin characterization is important as different types of BoNT can respond differently to medical countermeasures for botulism, and characterization of the toxin can aid in epidemiologic and forensic investigations. Proteomic techniques have been established to determine the serotype, subtype, or toxin variant of BoNT. These techniques involve digestion of the toxin into peptides, tandem mass spectrometric (MS/MS) analysis of the peptides, and database searching to identify the BoNT protein. These techniques demonstrate the capability to detect BoNT and its neurotoxin-associated proteins, and differentiate the toxin from other toxins which are up to 99.9% identical in some cases. This differentiation can be accomplished from toxins present in a complex matrix such as stool, food, or bacterial cultures and no DNA is required.

Double positivity for HPV-DNA/p16ink4a is the biomarker with strongest diagnostic accuracy and prognostic value for human papillomavirus related oropharyngeal cancer patients.

Science.gov (United States)

Mena, Marisa; Taberna, Miren; Tous, Sara; Marquez, Sandra; Clavero, Omar; Quiros, Beatriz; Lloveras, Belen; Alejo, Maria; Leon, Xavier; Quer, Miquel; Bagué, Silvia; Mesia, Ricard; Nogués, Julio; Gomà, Montserrat; Aguila, Anton; Bonfill, Teresa; Blazquez, Carmen; Guix, Marta; Hijano, Rafael; Torres, Montserrat; Holzinger, Dana; Pawlita, Michael; Pavon, Miguel Angel; Bravo, Ignacio G; de Sanjosé, Silvia; Bosch, Francesc Xavier; Alemany, Laia

2018-03-01

The etiologic role of human papillomaviruses (HPV) in oropharyngeal cancer (OPC) is well established. Nevertheless, information on survival differences by anatomic sub-site or treatment remains scarce, and it is still unclear the HPV-relatedness definition with best diagnostic accuracy and prognostic value. We conducted a retrospective cohort study of all patients diagnosed with a primary OPC in four Catalonian hospitals from 1990 to 2013. Formalin-fixed, paraffin-embedded cancer tissues were subjected to histopathological evaluation, DNA quality control, HPV-DNA detection, and p16 INK4a /pRb/p53/Cyclin-D1 immunohistochemistry. HPV-DNA positive and a random sample of HPV-DNA negative cases were subjected to HPV-E6*I mRNA detection. Demographic, tobacco/alcohol use, clinical and follow-up data were collected. Multivariate models were used to evaluate factors associated with HPV positivity as defined by four different HPV-relatedness definitions. Proportional-hazards models were used to compare the risk of death and recurrence among HPV-related and non-related OPC. 788 patients yielded a valid HPV-DNA result. The percentage of positive cases was 10.9%, 10.2%, 8.5% and 7.4% for p16 INK4a , HPV-DNA, HPV-DNA/HPV-E6*I mRNA, and HPV-DNA/p16 INK4a , respectively. Being non-smoker or non-drinker was consistently associated across HPV-relatedness definitions with HPV positivity. A suggestion of survival differences between anatomic sub-sites and treatments was observed. Double positivity for HPV-DNA/p16 INK4a showed strongest diagnostic accuracy and prognostic value. Double positivity for HPV-DNA/p16 INK4a , a test that can be easily implemented in the clinical practice, has optimal diagnostic accuracy and prognostic value. Our results have strong clinical implications for patients' classification and handling and also suggest that not all the HPV-related OPC behave similarly. Copyright © 2018 Elsevier Ltd. All rights reserved.

Study on a hidden protein-DNA binding in salmon sperm DNA sample by dynamic kinetic capillary isoelectric focusing

International Nuclear Information System (INIS)

Liang Liang; Dou Peng; Dong Mingming; Ke Xiaokang; Bian Ningsheng; Liu Zhen

2009-01-01

Nuclease P1 is an important enzyme that hydrolyzes RNA or single-stranded DNA into nucleotides, and complete digestion is an essential basis for assays based on this enzyme. To digest a doubled-stranded DNA, the enzyme is usually combined with heat denaturing, which breaks doubled-stranded DNA into single strands. This paper presents an un-expected phenomenon that nuclease P1, in combination with heat denaturing, fails to completely digest a DNA sample extracted from salmon sperm. Under the experimental conditions used, at which nuclease P1 can completely digest calf thymus DNA, the digestion yield of salmon sperm DNA was only 89.5%. Spectrometric measurement indicated that a total protein of 4.7% is present in the DNA sample. To explain the reason for this phenomenon, the dynamic kinetic capillary isoelectric focusing (DK-CIEF) approach proposed previously, which allows for the discrimination of different types of protein-DNA interactions and the measurement of the individual dissociation rate constants, was modified and applied to examine possible protein-DNA interactions involved. It was found that a non-specific DNA-protein binding occurs in the sample, the dissociation rate constant for which was measured to be 7.05 ± 0.83 x 10 -3 s -1 . The formation of DNA-protein complex was suggested to be the main reason for the incomplete digestion of the DNA sample. The modified DK-CIEF approach can be applied as general DNA samples, with the advantages of fast speed and low sample consumption.

Spectrometric determination of ammonium-nitrogen with quinol in ...

African Journals Online (AJOL)

Quinol is proposed as a reagent for the spectrometric determination of ammonium-nitrogen (NH4+-N) in aqueous medium. Quinol forms a pink complex with ammonium salt in aqueous medium. Hydrogen peroxide is needed for colour accentuation. The quinol/ammonium charge transfer complex has absorption maximum ...

Electronic DNA detection and diagnostics

NARCIS (Netherlands)

De, Arpita

2013-01-01

The Nanopill project is an ambitious undertaking with the objective to develop an early-warning cancer diagnostic pill that is ingested by the patient. The Nanopill collects intestinal fluid as the pill travels down the intestinal tract, and tests for the presence of a free floating hyper-methylated

Spectrometric methods for the determination of chlorine in crude oil and petroleum derivatives — A review

International Nuclear Information System (INIS)

Doyle, Adriana; Saavedra, Alvaro; Tristão, Maria Luiza B.; Mendes, Luiz A.N.; Aucélio, Ricardo Q.

2013-01-01

Chlorine determination in crude oil is made in order to guarantee that the oil does not contain levels of this element that might cause damages in the oil processing equipment. In petroleum products, the determination of chlorine is made, for instance, to evaluate if there are proper concentrations of organochloride compounds, which are used as additives. Such determinations are currently performed following official guidelines from the ASTM International and from the United States Environmental Protection Agency as well as protocols indicated by the Universal Oil Products. X-ray fluorescence spectroscopy plays an important role in many of these official methods. In contrast, other spectrometric methods based on optical and mass detection are plagued by limitations related to both the fundamental characteristics of non-metals and to the complex sample matrices, which reflects in the small number of articles devoted to these applications. In this review, the current status of the spectrometric methods, especially the role played by X-ray fluorescence spectrometry, is evaluated in terms of the determination of chlorine in crude oil and petroleum derivatives. Comparison of the performance of the methods, limitations and potential new approaches to ensure proper spectrometric determinations of chlorine is indicated. - Highlights: • Critical evaluation of spectrometric methods for chlorine in petroleum products. • Reviews on element determination in petroleum have not address the case of chlorine. • Peculiarities of the spectrometric determination of Cl in petroleum are discussed. • The spectrometric approaches are detailed and compared to the official methods. • New trends in chlorine determination in petroleum products are indicated

Spectrometric properties and radiation damage of BGO crystals

Science.gov (United States)

Kim, Gen C.; Gasanov, Eldar M.

1997-07-01

Spectrometric properties, such as light output, energy resolution BGO crystals before and after (superscript 60)Co gamma-ray (dose 10(superscript 4) - 10(superscript 6) R) and neutron irradiation (fluence 10(superscript 14) cm(superscript -2)) are investigated. Condition for degradation of spectrometric properties and their recovering after irradiation are studied. The energy spectrum of the photons emitted from BGO crystals irradiated with neutron fluence contains the long living background peak which is caused by self-irradiation with radioactive isotopes produced in the crystals. The defect production was studied in crystals under the high dose gamma-irradiation with (superscript 60)Co isotope. It was found that after doses above 10(superscript 8) R the color center at 365 nm and doses higher than 10(superscript 9) R a wide absorption band in the region of 300 - 350 nm occur. Comparison of these results with those of reactor irradiation has shown that under the high dose gamma-irradiation the structure defect production takes place.

Mass spectrometric detection of radiocarbon for dating applications

Energy Technology Data Exchange (ETDEWEB)

Synal, H.-A., E-mail: synal@phys.ethz.ch [ETH Zurich, Laboratory of Ion Beam Physics, Building HPK, 8093 Zurich (Switzerland); Schulze-Koenig, T.; Seiler, M.; Suter, M.; Wacker, L. [ETH Zurich, Laboratory of Ion Beam Physics, Building HPK, 8093 Zurich (Switzerland)

2013-01-15

Radiocarbon is still the most important nuclide measured by accelerator mass spectrometry (AMS). The related capabilities for dating and tracer studies are eminent not only in archaeology but also drive important applications in the earth and environmental sciences as well as in biomedical research. So far, standard mass spectrometric systems have not been capable of radiocarbon dating because of interfering molecular isobars which, however, can be completely eliminated in charge changing processes at high ion beam energies (MeV) [1,2]. Here, we present a novel type mass spectrometry system for radiocarbon analyses. Radiocarbon dating was performed using 45 keV {sup 14}C ions from the ion source and a molecule dissociation unit kept at ground potential. This proof-of-principle experiment demonstrates for the first time the feasibility of mass spectrometric radiocarbon dating without an accelerator. The results obtained will be the basis of an optimized design for a radiocarbon dating instrument comparable in size, complexity and cost to standard mass spectrometers.

Mass spectrometric detection of radiocarbon for dating applications

International Nuclear Information System (INIS)

Synal, H.-A.; Schulze-König, T.; Seiler, M.; Suter, M.; Wacker, L.

2013-01-01

Radiocarbon is still the most important nuclide measured by accelerator mass spectrometry (AMS). The related capabilities for dating and tracer studies are eminent not only in archaeology but also drive important applications in the earth and environmental sciences as well as in biomedical research. So far, standard mass spectrometric systems have not been capable of radiocarbon dating because of interfering molecular isobars which, however, can be completely eliminated in charge changing processes at high ion beam energies (MeV) [1,2]. Here, we present a novel type mass spectrometry system for radiocarbon analyses. Radiocarbon dating was performed using 45 keV 14 C ions from the ion source and a molecule dissociation unit kept at ground potential. This proof-of-principle experiment demonstrates for the first time the feasibility of mass spectrometric radiocarbon dating without an accelerator. The results obtained will be the basis of an optimized design for a radiocarbon dating instrument comparable in size, complexity and cost to standard mass spectrometers.

Mass spectrometric detection of radiocarbon for dating applications

Science.gov (United States)

Synal, H.-A.; Schulze-König, T.; Seiler, M.; Suter, M.; Wacker, L.

2013-01-01

Radiocarbon is still the most important nuclide measured by accelerator mass spectrometry (AMS). The related capabilities for dating and tracer studies are eminent not only in archaeology but also drive important applications in the earth and environmental sciences as well as in biomedical research. So far, standard mass spectrometric systems have not been capable of radiocarbon dating because of interfering molecular isobars which, however, can be completely eliminated in charge changing processes at high ion beam energies (MeV) [1,2]. Here, we present a novel type mass spectrometry system for radiocarbon analyses. Radiocarbon dating was performed using 45 keV 14C ions from the ion source and a molecule dissociation unit kept at ground potential. This proof-of-principle experiment demonstrates for the first time the feasibility of mass spectrometric radiocarbon dating without an accelerator. The results obtained will be the basis of an optimized design for a radiocarbon dating instrument comparable in size, complexity and cost to standard mass spectrometers.

The application study on the prospecting for gold by car-borne gamma-ray spectrometric method in northern Hebei and other areas

International Nuclear Information System (INIS)

Ma Zhongxiang; Liu Tengyao; Zhang Peng; Lu Shili; Zhai Yugui; Ma Yanfang

1995-01-01

The prospecting method for gold by car-borne gamma-ray spectrometric survey is a new geophysical and geochemical technique in the search for concealed gold deposits which can be used to effectively predict the regional gold metallogenetic potential area. The method of car-borne gamma-ray spectrometric survey was adopted and 63416 data at gamma-ray spectrometric measuring sites from rock masses (or strata) in northern Hebei and other areas (49100 km 2 ) are obtained. On the basis of the size of geological bodies, the sliding filtering technique was applied to process the gamma-ray spectrometric data from regional geological bodies and maps showing the parameter distribution characteristics were plotted (11 sheets). In accordance with the thorough on the distribution characteristics of gamma-ray spectrometric parameters that reflect the regional geological environment of gold metallogenesis, a set of gamma-ray spectrometric combination parameters in correlation with regional gold metallogenesis has been established and the characteristic information of gamma-ray spectrometric parameters has been extracted. Combined with geological information, the gold metallogenetic geological environment in the working area was studied, and two potential areas were predicted. After verification, gold mineralization was encountered in some areas. The study results show that the car-borne gamma-ray spectrometric method used for regional gold prospecting is a rapid, economical and effective one

Rapid diagnostic tests as a source of DNA for Plasmodium species-specific real-time PCR

Directory of Open Access Journals (Sweden)

Van Esbroeck Marjan

2011-03-01

Full Text Available Abstract Background This study describes the use of malaria rapid diagnostic tests (RDTs as a source of DNA for Plasmodium species-specific real-time PCR. Methods First, the best method to recover DNA from RDTs was investigated and then the applicability of this DNA extraction method was assessed on 12 different RDT brands. Finally, two RDT brands (OptiMAL Rapid Malaria Test and SDFK60 malaria Ag Plasmodium falciparum/Pan test were comprehensively evaluated on a panel of clinical samples submitted for routine malaria diagnosis at ITM. DNA amplification was done with the 18S rRNA real-time PCR targeting the four Plasmodium species. Results of PCR on RDT were compared to those obtained by PCR on whole blood samples. Results Best results were obtained by isolating DNA from the proximal part of the nitrocellulose component of the RDT strip with a simple DNA elution method. The PCR on RDT showed a detection limit of 0.02 asexual parasites/μl, which was identical to the same PCR on whole blood. For all 12 RDT brands tested, DNA was detected except for one brand when a low parasite density sample was applied. In RDTs with a plastic seal covering the nitrocellulose strip, DNA extraction was hampered. PCR analysis on clinical RDT samples demonstrated correct identification for single species infections for all RDT samples with asexual parasites of P. falciparum (n = 60, Plasmodium vivax (n = 10, Plasmodium ovale (n = 10 and Plasmodium malariae (n = 10. Samples with only gametocytes were detected in all OptiMAL and in 10 of the 11 SDFK60 tests. None of the negative samples (n = 20 gave a signal by PCR on RDT. With PCR on RDT, higher Ct-values were observed than with PCR on whole blood, with a mean difference of 2.68 for OptiMAL and 3.53 for SDFK60. Mixed infections were correctly identified with PCR on RDT in 4/5 OptiMAL tests and 2/5 SDFK60 tests. Conclusions RDTs are a reliable source of DNA for Plasmodium real-time PCR. This study demonstrates the

Analysis of oxidative DNA damage, Part II: Synthesis of the internal standard 8-[18O]hydroxy-2'-deoxyguanosine

NARCIS (Netherlands)

Hermanns RCA; Zomer G; Stavenuiter JFC; Westra G; Visser T; van de Werken G

1993-01-01

In the project 'Oxidative DNA Damage' the first aim is to develop a mass spectrometric method for the quantification of 8-hydroxy-2'- deoxyguanosine (oh8dG). The required precision of the method requires the application of a labeled analogue as an internal standard. This report

Review of procedures involving separation and preconcentration for the determination of cadmium using spectrometric techniques

International Nuclear Information System (INIS)

Ferreira, Sergio L.C.; Andrade, Jailson B. de; Korn, Maria das Gracas A.; Pereira, Madson de G.; Lemos, Valfredo A.; Santos, Walter N.L. dos; Rodrigues, Frederico de Medeiros; Souza, Anderson S.; Ferreira, Hadla S.; Silva, Erik G.P. da

2007-01-01

Spectrometric techniques for the analysis of trace cadmium have developed rapidly due to the increasing need for accurate measurements at extremely low levels of this element in diverse matrices. This review covers separation and preconcentration procedures, such as electrochemical deposition, precipitation, coprecipitation, solid phase extraction, liquid-liquid extraction (LLE) and cloud point extraction (CPE), and consider the features of the their application with several spectrometric techniques

Low-activity spectrometric gamma-ray logging technique for delineation of coal/rock interfaces in dry blast holes

International Nuclear Information System (INIS)

Asfahani, J.; Borsaru, M.

2007-01-01

A low-activity spectrometric gamma-ray logging technique is proposed in this paper as a sensitive tool for the delineation of coal/rock interfaces in dry blast holes. The advantages and superiority of this technique over traditional micro-density non-spectrometric gamma-ray tools are demonstrated

Novel atomic absorption spectrometric and rapid spectrophotometric methods for the quantitation of paracetamol in saliva: application to pharmacokinetic studies.

Science.gov (United States)

Issa, M M; Nejem, R M; El-Abadla, N S; Al-Kholy, M; Saleh, Akila A

2008-01-01

A novel atomic absorption spectrometric method and two highly sensitive spectrophotometric methods were developed for the determination of paracetamol. These techniques based on the oxidation of paracetamol by iron (III) (method I); oxidation of p-aminophenol after the hydrolysis of paracetamol (method II). Iron (II) then reacts with potassium ferricyanide to form Prussian blue color with a maximum absorbance at 700 nm. The atomic absorption method was accomplished by extracting the excess iron (III) in method II and aspirates the aqueous layer into air-acetylene flame to measure the absorbance of iron (II) at 302.1 nm. The reactions have been spectrometrically evaluated to attain optimum experimental conditions. Linear responses were exhibited over the ranges 1.0-10, 0.2-2.0 and 0.1-1.0 mug/ml for method I, method II and atomic absorption spectrometric method, respectively. A high sensitivity is recorded for the proposed methods I and II and atomic absorption spectrometric method value indicate: 0.05, 0.022 and 0.012 mug/ml, respectively. The limit of quantitation of paracetamol by method II and atomic absorption spectrometric method were 0.20 and 0.10 mug/ml. Method II and the atomic absorption spectrometric method were applied to demonstrate a pharmacokinetic study by means of salivary samples in normal volunteers who received 1.0 g paracetamol. Intra and inter-day precision did not exceed 6.9%.

Mass spectrometric investigation of vinyl-substituted organic boron compounds

International Nuclear Information System (INIS)

Tarielashvili, V.O.; Ordzhonikidze, K.G.; Parulava, L.P.; Vakhaniya, G.V.

1992-01-01

Mass spectrometric investigation of vinyl-substituted organic compounds was conducted. Ionization was performed by electron shock. Possibility of determining boron isotope content is all analyzed organic boron vinyl-substituted compounds by direct method is shown. This simplifies sufficiently and lowers the price of analysis, improves its accuracy and rapidity

DNA Diagnostics: Optical or by Electronics?

KAUST Repository

Khan, Hadayat Ullah; Knoll, Wolfgang

2016-01-01

In this paper, we very briefly review DNA biosensors based on optical and electrical detection principles, referring mainly to our past work applying both techniques but here using nearly identical sensor chip surface architectures, i.e., capture

Significance of Airborne Gamma-ray spectrometric data of Umm bisilla Area, central Eastern Desert, Egypt

Energy Technology Data Exchange (ETDEWEB)

Rabie, S I [Nuclear materials authority, Maadi, Cairo, (Egypt)

1995-10-01

Umm bisilla area, located in the Central Eastern Desert of Egypt, consists mainly of basement rocks. The present investigation of the airborne spectrometric data is to define the meaningful anomalies from the raw data by applying the significance factor techniques, by calculating the significant radioactive provinces. Determination of the gross structural pattern and broad variations in composition of the crystalline basement, to define the relationships between the tectonic features of the area as interpreted from aeromagnetic data, with the significant anomalies revealed from spectrometric data was carried out through the application of different magnetic techniques. Five significant uraniferous zones were detected associated with Umm Bisilla granite, amphibolite, and grey granite. The intersection of the structural lineaments interpreted from aeromagnetic data illustrated good correlation with the significant uranium anomalous zones interpreted from spectrometric data, and indicated that the concentration is structurally. 15 figs.

Measurements of iodine uptake in thyroid after diagnostic administration of 131I

International Nuclear Information System (INIS)

Osko, J.; Pliszczynski, T.

2003-01-01

The measurements performed up to now, showed that the spectrometric measurements can be useful in selection of the patients who need special consideration during the 131 I diagnostics and treatment. The next step of the work will include the measurements of the real activity of 131 I in thyroid gland, after the therapeutic administration of radioiodine. A special collimator was designed for this purpose and the thyroid counter was calibrated using a phantom with inserts simulating different shapes of pathologically changed thyroid glands. It can be expected that the improvement of accuracy of the diagnostic measurements and better control of real activity of 131 I in thyroid gland after the therapeutic administration will contribute to the process of optimisation of radiation doses to the patients and medical personnel. (authors)

The strategies of DNA immobilization and hybridization detection mechanism in the construction of electrochemical DNA sensor: A review

Directory of Open Access Journals (Sweden)

Jahwarhar Izuan Abdul Rashid

2017-11-01

Full Text Available In recent years, electrochemical deoxyribonucleic acid (DNA sensor has recently emerged as promising alternative clinical diagnostic devices especially for infectious disease by exploiting DNA recognition events and converting them into an electrochemical signal. This is because the existing DNA diagnostic method possesses certain drawbacks such as time-consuming, expensive, laborious, low selectivity and sensitivity. DNA immobilization strategies and mechanism of electrochemical detection are two the most important aspects that should be considered before developing highly selective and sensitive electrochemical DNA sensor. Here, we focus on some recent strategies for DNA probes immobilization on the surface of electrochemical transducer such as adsorption, covalent bonding and Avidin/Streptavidin-Biotin interaction on the electrode surface for specific interaction with its complementary DNA target. A numerous approach for DNA hybridization detection based electrochemical technique that frequently used including direct DNA electrochemical detection and label based electrochemical (redox-active indicator, enzyme label and nanoparticles were also discussed in aiming to provide general guide for the design of electrochemical DNA sensor. We also discussed the challenges and suggestions to improve the application of electrochemical DNA sensor at point-care setting. Keywords: Electrochemical DNA sensor, DNA immobilization, DNA hybridization, Electrochemical mechanism

Mirion--a software package for automatic processing of mass spectrometric images.

Science.gov (United States)

Paschke, C; Leisner, A; Hester, A; Maass, K; Guenther, S; Bouschen, W; Spengler, B

2013-08-01

Mass spectrometric imaging (MSI) techniques are of growing interest for the Life Sciences. In recent years, the development of new instruments employing ion sources that are tailored for spatial scanning allowed the acquisition of large data sets. A subsequent data processing, however, is still a bottleneck in the analytical process, as a manual data interpretation is impossible within a reasonable time frame. The transformation of mass spectrometric data into spatial distribution images of detected compounds turned out to be the most appropriate method to visualize the results of such scans, as humans are able to interpret images faster and easier than plain numbers. Image generation, thus, is a time-consuming and complex yet very efficient task. The free software package "Mirion," presented in this paper, allows the handling and analysis of data sets acquired by mass spectrometry imaging. Mirion can be used for image processing of MSI data obtained from many different sources, as it uses the HUPO-PSI-based standard data format imzML, which is implemented in the proprietary software of most of the mass spectrometer companies. Different graphical representations of the recorded data are available. Furthermore, automatic calculation and overlay of mass spectrometric images promotes direct comparison of different analytes for data evaluation. The program also includes tools for image processing and image analysis.

New insights into Trypanosoma cruzi evolution, genotyping and molecular diagnostics from satellite DNA sequence analysis.

Directory of Open Access Journals (Sweden)

Juan C Ramírez

2017-12-01

Full Text Available Trypanosoma cruzi has been subdivided into seven Discrete Typing Units (DTUs, TcI-TcVI and Tcbat. Two major evolutionary models have been proposed to explain the origin of hybrid lineages, but while it is widely accepted that TcV and TcVI are the result of genetic exchange between TcII and TcIII strains, the origin of TcIII and TcIV is still a matter of debate. T. cruzi satellite DNA (SatDNA, comprised of 195 bp units organized in tandem repeats, from both TcV and TcVI stocks were found to have SatDNA copies type TcI and TcII; whereas contradictory results were observed for TcIII stocks and no TcIV sequence has been analyzed yet. Herein, we have gone deeper into this matter analyzing 335 distinct SatDNA sequences from 19 T. cruzi stocks representative of DTUs TcI-TcVI for phylogenetic inference. Bayesian phylogenetic tree showed that all sequences were grouped in three major clusters, which corresponded to sequences from DTUs TcI/III, TcII and TcIV; whereas TcV and TcVI stocks had two sets of sequences distributed into TcI/III and TcII clusters. As expected, the lowest genetic distances were found between TcI and TcIII, and between TcV and TcVI sequences; whereas the highest ones were observed between TcII and TcI/III, and among TcIV sequences and those from the remaining DTUs. In addition, signature patterns associated to specific T. cruzi lineages were identified and new primers that improved SatDNA-based qPCR sensitivity were designed. Our findings support the theory that TcIII is not the result of a hybridization event between TcI and TcII, and that TcIV had an independent origin from the other DTUs, contributing to clarifying the evolutionary history of T. cruzi lineages. Moreover, this work opens the possibility of typing samples from Chagas disease patients with low parasitic loads and improving molecular diagnostic methods of T. cruzi infection based on SatDNA sequence amplification.

New insights into Trypanosoma cruzi evolution, genotyping and molecular diagnostics from satellite DNA sequence analysis.

Science.gov (United States)

Ramírez, Juan C; Torres, Carolina; Curto, María de Los A; Schijman, Alejandro G

2017-12-01

Trypanosoma cruzi has been subdivided into seven Discrete Typing Units (DTUs), TcI-TcVI and Tcbat. Two major evolutionary models have been proposed to explain the origin of hybrid lineages, but while it is widely accepted that TcV and TcVI are the result of genetic exchange between TcII and TcIII strains, the origin of TcIII and TcIV is still a matter of debate. T. cruzi satellite DNA (SatDNA), comprised of 195 bp units organized in tandem repeats, from both TcV and TcVI stocks were found to have SatDNA copies type TcI and TcII; whereas contradictory results were observed for TcIII stocks and no TcIV sequence has been analyzed yet. Herein, we have gone deeper into this matter analyzing 335 distinct SatDNA sequences from 19 T. cruzi stocks representative of DTUs TcI-TcVI for phylogenetic inference. Bayesian phylogenetic tree showed that all sequences were grouped in three major clusters, which corresponded to sequences from DTUs TcI/III, TcII and TcIV; whereas TcV and TcVI stocks had two sets of sequences distributed into TcI/III and TcII clusters. As expected, the lowest genetic distances were found between TcI and TcIII, and between TcV and TcVI sequences; whereas the highest ones were observed between TcII and TcI/III, and among TcIV sequences and those from the remaining DTUs. In addition, signature patterns associated to specific T. cruzi lineages were identified and new primers that improved SatDNA-based qPCR sensitivity were designed. Our findings support the theory that TcIII is not the result of a hybridization event between TcI and TcII, and that TcIV had an independent origin from the other DTUs, contributing to clarifying the evolutionary history of T. cruzi lineages. Moreover, this work opens the possibility of typing samples from Chagas disease patients with low parasitic loads and improving molecular diagnostic methods of T. cruzi infection based on SatDNA sequence amplification.

Fast infectious diseases diagnostics based on microfluidic biochip system

Directory of Open Access Journals (Sweden)

Qin Huang

2017-03-01

Full Text Available Molecular diagnostics is one of the most important tools currently in use for clinical pathogen detection due to its high sensitivity, specificity, and low consume of sample and reagent is keyword to low cost molecular diagnostics. In this paper, a sensitive DNA isothermal amplification method for fast clinical infectious diseases diagnostics at aM concentrations of DNA was developed using a polycarbonate (PC microfluidic chip. A portable confocal optical fluorescence detector was specifically developed for the microfluidic chip that was capable of highly sensitive real-time detection of amplified products for sequence-specific molecular identification near the optical diffraction limit with low background. The molecular diagnostics of Listeria monocytogenes with nucleic acid extracted from stool samples was performed at a minimum DNA template concentration of 3.65aM, and a detection limit of less than five copies of genomic DNA. Contrast to the general polymerase chain reaction (PCR at eppendorf (EP tube, the detection time in our developed method was reduced from 1.5h to 45min for multi-target parallel detection, the consume of sample and reagent was dropped from 25μL to 1.45μL. This novel microfluidic chip system and method can be used to develop a micro total analysis system as a clinically relevant pathogen molecular diagnostics method via the amplification of targets, with potential applications in biotechnology, medicine, and clinical molecular diagnostics.

Stability and linearity control of spectrometric channels of the Cherenkov counters using controllable units

International Nuclear Information System (INIS)

Kollar, D.; Kollarova, L.; Khorvat, P.

1976-01-01

A system is elaborated to control stability and linearity of the Cherenkov counter spectrometric channels in an experiment on a magnetic monopole search. Linearity of a light characteristic of a photoelectric multiplier is checked with the help of the calibrated light-strikings of light emitting diodes with flare intensity adjusted by controlling generator voltage across the mercury body. A program algorithm is presented for checking stability and linearity of the Cherenkov counter spectrometric channels which helps to consider the fatigue effects of the photoelectric multiplier resulting from the considerable loads

Selection and optimization of spectrometric amplifiers for gamma spectrometry: part II - linearity, live time correction factors and software

International Nuclear Information System (INIS)

Moraes, Marco Antonio Proenca Vieira de; Pugliesi, Reinaldo

1996-01-01

The objective of the present work was to establish simple criteria to choose the best combination of electronic modules to achieve an adequate high resolution gamma spectrometer. Linearity, live time correction factors and softwares of a gamma spectrometric system composed by a Hp Ge detector have been studied by using several kinds of spectrometric amplifiers: Canberra 2021, Canberra 2025, Ortec 673 and Tennelec 244 and the MCA cards Ortec and Nucleus. The results showed low values of integral non-linearity for all spectrometric amplifiers connected to the Ortec and Nucleus boards. The MCA card should be able to correct amplifier dead time for 17 kcps count rates. (author)

Spectrometric microbiological analyzer

Science.gov (United States)

Schlager, Kenneth J.; Meissner, Ken E.

1996-04-01

Currently, there are four general approaches to microbiological analysis, i.e., the detection, identification and quantification of micro-organisms: (1) Traditional culturing and staining procedures, metabolic fermentations and visual morphological characteristics; (2) Immunological approaches employing microbe-specific antibodies; (3) Biotechnical techniques employing DNA probes and related genetic engineering methods; and (4) Physical measurement techniques based on the biophysical properties of micro-organisms. This paper describes an instrumentation development in the fourth of the above categories, physical measurement, that uses a combination of fluorometric and light scatter spectra to detect and identify micro-organisms at the species level. A major advantage of this approach is the rapid turnaround possible in medical diagnostic or water testing applications. Fluorometric spectra serve to define the biochemical characteristics of the microbe, and light scatter spectra the size and shape morphology. Together, the two spectra define a 'fingerprint' for each species of microbe for detection, identification and quantification purposes. A prototype instrument has been developed and tested under NASA sponsorship based on fluorometric spectra alone. This instrument demonstrated identification and quantification capabilities at the species level. The paper reports on test results using this instrument, and the benefits of employing a combination of fluorometric and light scatter spectra.

Newborn Congenital Cytomegalovirus Screening Based on Clinical Manifestations and Evaluation of DNA-based Assays for In Vitro Diagnostics.

Science.gov (United States)

Fujii, Tomoyuki; Oka, Akira; Morioka, Ichiro; Moriuchi, Hiroyuki; Koyano, Shin; Yamada, Hideto; Saito, Shigeru; Sameshima, Hiroshi; Nagamatsu, Takeshi; Tsuchida, Shinya; Inoue, Naoki

2017-10-01

To establish a strategy for congenital cytomegalovirus (cCMV) screening and to establish confirmatory assays approved as in vitro diagnostics by the regulatory authorities, we evaluated the clinical risks and performance of diagnostic assays developed by commercial companies, since cCMV infection has significant clinical consequences. Newborns with clinical manifestations considered to be consequences of cCMV infection (n = 575) were screened for the presence of cytomegalovirus (CMV) DNA in urine specimens collected onto filter paper placed in their diapers using the polymerase chain reaction-based assay reported previously. Liquid urine specimens were obtained from all of 20 CMV-positive newborns and 107 of the CMV-negative newborns identified in the screening. We used these 127 specimens, as well as 12 from cCMV cases identified in a previous study and 41 from healthy newborns, to compare the performance of 2 commercial assays and 1 in-house assay. The risk-based screening allowed the identification of cCMV cases at least 10-fold more efficiently than our previous universal screening, although there appears to be a limit to the identification of asymptomatically infected newborns. Although CMV-specific IgM during pregnancy was found frequently in mothers of cCMV newborns, CMV-IgM alone is not an effective diagnostic marker. The urine-filter-based assay and the 3 diagnostic assays yielded identical results. Although risk-based and universal newborn screening strategies for cCMV infection each have their respective advantages and disadvantages, urine-filter-based assay followed by confirmatory in vitro diagnostics assays is able to identify cCMV cases efficiently.

Advanced microarray technologies for clinical diagnostics

NARCIS (Netherlands)

Pierik, Anke

2011-01-01

DNA microarrays become increasingly important in the field of clinical diagnostics. These microarrays, also called DNA chips, are small solid substrates, typically having a maximum surface area of a few cm2, onto which many spots are arrayed in a pre-determined pattern. Each of these spots contains

Preparation and spectrometric identification of amide derivatives with antimalaric

International Nuclear Information System (INIS)

Ferreira, E.J.

1987-01-01

The structures of polymeric compounds using spectrometric analysis of infrared, ultraviolet and protonic magnetic resonance and 13 C nuclear magnetic resonance are studied. Some structural models are used such as antimalaric-sulfones and pyrimidinic derivatives, and non-polymeric derivatives of active compounds. A comparative analysis on structures less complexes is shown. (M.J.C.). Spectrums 186 p [pt

System for Gamma an X rays fluorescence spectrometric

International Nuclear Information System (INIS)

Alonso Abad, D.; Arista Romeu, E.; Bolanos Perez, L. and others

1997-01-01

A system for spectrometry of gamma or fluorescence X rays is presented. It sis composed by a Si(Li) semiconductors detector, a charge sensitive preamplifier, a high voltage power supply, a spectrometric amplifier and a monolithic 1024 channels multichannel analyzers or an IBM compatible 4096 channels add - on- card multichannel analyzer. The system can be configured as a 1024 or 4096 channels gamma or fluorescent X rays spectrometer

DNA methylation profiles of the brain-derived neurotrophic factor (BDNF gene as a potent diagnostic biomarker in major depression.

Directory of Open Access Journals (Sweden)

Manabu Fuchikami

Full Text Available Major depression, because of its recurring and life-threatening nature, is one of the top 10 diseases for global disease burden. Major depression is still diagnosed on the basis of clinical symptoms in patients. The search for specific biological markers is of great importance to advance the method of diagnosis for depression. We examined the methylation profile of 2 CpG islands (I and IV at the promoters of the brain-derived neurotrophic factor (BDNF gene, which is well known to be involved in the pathophysiology of depression. We analyzed genomic DNA from peripheral blood of 20 Japanese patients with major depression and 18 healthy controls to identify an appropriate epigenetic biomarker to aid in the establishment of an objective system for the diagnosis of depression. Methylation rates at each CpG unit was measured using a MassArray® system (SEQUENOM, and 2-dimensional hierarchical clustering analyses were undertaken to determine the validity of these methylation profiles as a diagnostic biomarker. Analyses of the dendrogram from methylation profiles of CpG I, but not IV, demonstrated that classification of healthy controls and patients at the first branch completely matched the clinical diagnosis. Despite the small number of subjects, our results indicate that classification based on the DNA methylation profiles of CpG I of the BDNF gene may be a valuable diagnostic biomarker for major depression.

Diagnostic Devices for Isothermal Nucleic Acid Amplification

Directory of Open Access Journals (Sweden)

Chia-Chen Chang

2012-06-01

Full Text Available Since the development of the polymerase chain reaction (PCR technique, genomic information has been retrievable from lesser amounts of DNA than previously possible. PCR-based amplifications require high-precision instruments to perform temperature cycling reactions; further, they are cumbersome for routine clinical use. However, the use of isothermal approaches can eliminate many complications associated with thermocycling. The application of diagnostic devices for isothermal DNA amplification has recently been studied extensively. In this paper, we describe the basic concepts of several isothermal amplification approaches and review recent progress in diagnostic device development.

Diagnostic devices for isothermal nucleic acid amplification.

Science.gov (United States)

Chang, Chia-Chen; Chen, Chien-Cheng; Wei, Shih-Chung; Lu, Hui-Hsin; Liang, Yang-Hung; Lin, Chii-Wann

2012-01-01

Since the development of the polymerase chain reaction (PCR) technique, genomic information has been retrievable from lesser amounts of DNA than previously possible. PCR-based amplifications require high-precision instruments to perform temperature cycling reactions; further, they are cumbersome for routine clinical use. However, the use of isothermal approaches can eliminate many complications associated with thermocycling. The application of diagnostic devices for isothermal DNA amplification has recently been studied extensively. In this paper, we describe the basic concepts of several isothermal amplification approaches and review recent progress in diagnostic device development.

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.

Partial Purification of a Megadalton DNA Replication Complex by Free Flow Electrophoresis.

Directory of Open Access Journals (Sweden)

Caroline M Li

Full Text Available We describe a gentle and rapid method to purify the intact multiprotein DNA replication complex using free flow electrophoresis (FFE. In particular, we applied FFE to purify the human cell DNA synthesome, which is a multiprotein complex that is fully competent to carry-out all phases of the DNA replication process in vitro using a plasmid containing the simian virus 40 (SV40 origin of DNA replication and the viral large tumor antigen (T-antigen protein. The isolated native DNA synthesome can be of use in studying the mechanism by which mammalian DNA replication is carried-out and how anti-cancer drugs disrupt the DNA replication or repair process. Partially purified extracts from HeLa cells were fractionated in a native, liquid based separation by FFE. Dot blot analysis showed co-elution of many proteins identified as part of the DNA synthesome, including proliferating cell nuclear antigen (PCNA, DNA topoisomerase I (topo I, DNA polymerase δ (Pol δ, DNA polymerase ɛ (Pol ɛ, replication protein A (RPA and replication factor C (RFC. Previously identified DNA synthesome proteins co-eluted with T-antigen dependent and SV40 origin-specific DNA polymerase activity at the same FFE fractions. Native gels show a multiprotein PCNA containing complex migrating with an apparent relative mobility in the megadalton range. When PCNA containing bands were excised from the native gel, mass spectrometric sequencing analysis identified 23 known DNA synthesome associated proteins or protein subunits.

Partial Purification of a Megadalton DNA Replication Complex by Free Flow Electrophoresis.

Science.gov (United States)

Li, Caroline M; Miao, Yunan; Lingeman, Robert G; Hickey, Robert J; Malkas, Linda H

2016-01-01

We describe a gentle and rapid method to purify the intact multiprotein DNA replication complex using free flow electrophoresis (FFE). In particular, we applied FFE to purify the human cell DNA synthesome, which is a multiprotein complex that is fully competent to carry-out all phases of the DNA replication process in vitro using a plasmid containing the simian virus 40 (SV40) origin of DNA replication and the viral large tumor antigen (T-antigen) protein. The isolated native DNA synthesome can be of use in studying the mechanism by which mammalian DNA replication is carried-out and how anti-cancer drugs disrupt the DNA replication or repair process. Partially purified extracts from HeLa cells were fractionated in a native, liquid based separation by FFE. Dot blot analysis showed co-elution of many proteins identified as part of the DNA synthesome, including proliferating cell nuclear antigen (PCNA), DNA topoisomerase I (topo I), DNA polymerase δ (Pol δ), DNA polymerase ɛ (Pol ɛ), replication protein A (RPA) and replication factor C (RFC). Previously identified DNA synthesome proteins co-eluted with T-antigen dependent and SV40 origin-specific DNA polymerase activity at the same FFE fractions. Native gels show a multiprotein PCNA containing complex migrating with an apparent relative mobility in the megadalton range. When PCNA containing bands were excised from the native gel, mass spectrometric sequencing analysis identified 23 known DNA synthesome associated proteins or protein subunits.

Biotechnology in diagnostics

International Nuclear Information System (INIS)

Koprowski, H.; Ferrone, S.; Albertini, A.

1985-01-01

In recent years much progress has been made in the area of biotechnology. The cellular and molecular cloning methodology to develop monoclonal antibodies and DNA probes have been extensively utilized in basic and clinical research. These investigations have provided the necessary information to apply these reagents to diagnostic problems. The RIA 85 meeting focused on the application of monoclonal antibodies and DNA probes in laboratory medicine. The papers presented at this meeting clearly indicate that biotechnology has already had a significant impact on clinical medicine. (Auth.)

High-efficient method for spectrometric data real time processing with increased resolution of a measuring channel

International Nuclear Information System (INIS)

Ashkinaze, S.I.; Voronov, V.A.; Nechaev, Yu.I.

1988-01-01

Solution of reduction problem as a mean to increase spectrometric tract resolution when it is realized using the digit-by-digit modified method and special strategy, significantly reducing the time of processing, is considered. The results presented confirm that the complex measurement tract plus microcomputer is equivalent to the use of the tract with a higher resolution, and the use of the digit-by-digit modified method permits to process spectrometric information in real time scale

Realisation of a {beta} spectrometer solenoidal and a double {beta} spectrometer at coincidence; Realisation d'un spectrometre {beta} solenoidal et d'un double spectrometre {beta} a coincidence

Energy Technology Data Exchange (ETDEWEB)

Moreau, J [Commissariat a l' Energie Atomique, Saclay (France). Centre d' Etudes Nucleaires

1955-06-15

The two spectrometers have been achieved to tackle numerous problems of nuclear spectrometry. They possess different fields of application that complete themselves. The solenoidal spectrometer permits the determination of the energy limits of {beta} spectra and of their shape; it also permits the determination of the coefficients of internal conversion and reports {alpha}{sub K} / {alpha}{sub L} and it is especially efficient for the accurate energy levels of the {gamma} rays by photoelectric effect. The double coincidence spectrometer has been conceived to get a good efficiency in coincidence: indeed, the sum of the solid angles used for the {beta} and {gamma} emission is rather little lower to 4{pi} steradians. To get this efficiency, one should have sacrificed a little the resolution that is lower to the one obtained with the solenoidal spectrometer for a same brightness. Each of the elements of the double spectrometer can also be adapted to the study of angular correlations {beta}{gamma} and e{sup -}{gamma}. In this use, it is superior to the thin magnetic lens used up to here. The double spectrometer also permits the survey of the coincidences e{sup -}e{sup -}, e{sup -}{beta} of a equivalent way to a double lens; it can also be consider some adaptation for the survey of the angular correlations e{sup -}e{sup -}, e{sup -}{beta}. Finally, we applied the methods by simple spectrometry and by coincidence spectrometry, to the study of the radiances of the following radioelements: {sup 76}As (26 h), {sup 122}Sb (2,8 j), {sup 124}Sb (60 j), {sup 125}Sb (2,7 years). (M.B.) [French] Les deux spectrometres qui ont ete realises permettent d'aborder un grand nombre de problemes de spectrometrie nucleaire. Ils possedent des champs d'application tres differents qui se completent. Le spectrometre solenoidal permet la determination des energies limites des spectres {beta} et de leur forme; il permet aussi la determination des coefficients de conversion interne et des rapports

DNA Charge Transport: From Chemical Principles to the Cell

Science.gov (United States)

Arnold, Anna R.; Grodick, Michael A.; Barton, Jacqueline K.

2016-01-01

The DNA double helix has captured the imagination of many, bringing it to the forefront of biological research. DNA has unique features that extend our interest into areas of chemistry, physics, material science and engineering. Our laboratory has focused on studies of DNA charge transport (CT), wherein charges can efficiently travel long molecular distances through the DNA helix while maintaining an exquisite sensitivity to base pair π-stacking. Because DNA CT chemistry reports on the integrity of the DNA duplex, this property may be exploited to develop electrochemical devices to detect DNA lesions and DNA-binding proteins. Furthermore, studies now indicate that DNA CT may also be used in the cell by, for example, DNA repair proteins, as a cellular diagnostic, in order to scan the genome to localize efficiently to damage sites. In this review, we describe this evolution of DNA CT chemistry from the discovery of fundamental chemical principles to applications in diagnostic strategies and possible roles in biology. PMID:26933744

Mass spectrometric detection of radiocarbon for dating applications

OpenAIRE

Synal Hans-Arno

2013-01-01

Radiocarbon is still the most important nuclide measured by accelerator mass spectrometry (AMS). The related capabilities for dating and tracer studies are eminent not only in archaeology but also drive important applications in the earth and environmental sciences as well as in biomedical research. So far standard mass spectrometric systems have not been capable of radiocarbon dating because of interfering molecular isobars which however can be completely eliminated in charge changing proces...

[Determination of trace cobalt in human urine by graphite furnace atomic absorption spectrometr].

Science.gov (United States)

Zhong, L X; Ding, B M; Jiang, D; Liu, D Y; Yu, B; Zhu, B L; Ding, L

2016-05-20

To establish a method to determine cobalt in human urine by graphite furnace atomic absorption spectrometry. Urine with 2% nitric acid diluted two-fold, to quantify the curve, graphite furnace atomic absorption spectrometric detection. Co was linear within 2.5~40.0 ng/ml with r>0.999. Spike experiment showed that Co received good recovery rate, which was 90.8%~94.8%. Intra-assay precisions were 3.2%~5.1% for Co, inter-assay precisions were 4.4%~5.2% for Co. The method by using graphite furnace atomic absorption spectrometr to determine urine Co was fast, accurate and with low matrix effect. It could meet the requirement in GBZ/T 210.5-2008.

Multi-color fluorescent DNA analysis in an integrated optofluidic lab-on-a-chip

NARCIS (Netherlands)

Dongre, C.; van Weerd, J.; van Weeghel, R.; Martinez-Vazquez, R.; Osellame, R.; Cerullo, G.; Besselink, G.A.J.; van den Vlekkert, H.H.; Hoekstra, Hugo; Pollnau, Markus

Sorting and sizing of DNA molecules within the human genome project has enabled the genetic mapping of various illnesses. By employing tiny lab-on-a-chip devices for such DNA analysis, integrated DNA sequencing and genetic diagnostics have become feasible. However, such diagnostic chips typically

DNA nanotechnology

Science.gov (United States)

Seeman, Nadrian C.; Sleiman, Hanadi F.

2018-01-01

DNA is the molecule that stores and transmits genetic information in biological systems. The field of DNA nanotechnology takes this molecule out of its biological context and uses its information to assemble structural motifs and then to connect them together. This field has had a remarkable impact on nanoscience and nanotechnology, and has been revolutionary in our ability to control molecular self-assembly. In this Review, we summarize the approaches used to assemble DNA nanostructures and examine their emerging applications in areas such as biophysics, diagnostics, nanoparticle and protein assembly, biomolecule structure determination, drug delivery and synthetic biology. The introduction of orthogonal interactions into DNA nanostructures is discussed, and finally, a perspective on the future directions of this field is presented.

New FORTRAN computer programs to acquire and process isotopic mass-spectrometric data

International Nuclear Information System (INIS)

Smith, D.H.

1982-08-01

The computer programs described in New Computer Programs to Acquire and Process Isotopic Mass Spectrometric Data have been revised. This report describes in some detail the operation of these programs, which acquire and process isotopic mass spectrometric data. Both functional and overall design aspects are addressed. The three basic program units - file manipulation, data acquisition, and data processing - are discussed in turn. Step-by-step instructions are included where appropriate, and each subsection is described in enough detail to give a clear picture of its function. Organization of file structure, which is central to the entire concept, is extensively discussed with the help of numerous tables. Appendices contain flow charts and outline file structure to help a programmer unfamiliar with the programs to alter them with a minimum of lost time

Detecting an elusive invasive species: a diagnostic PCR to detect Burmese python in Florida waters and an assessment of persistence of environmental DNA.

Science.gov (United States)

Piaggio, Antoinette J; Engeman, Richard M; Hopken, Matthew W; Humphrey, John S; Keacher, Kandy L; Bruce, William E; Avery, Michael L

2014-03-01

Recent studies have demonstrated that detection of environmental DNA (eDNA) from aquatic vertebrates in water bodies is possible. The Burmese python, Python bivittatus, is a semi-aquatic, invasive species in Florida where its elusive nature and cryptic coloration make its detection difficult. Our goal was to develop a diagnostic PCR to detect P. bivittatus from water-borne eDNA, which could assist managers in monitoring this invasive species. First, we used captive P. bivittatus to determine whether reptilian DNA could be isolated and amplified from water samples. We also evaluated the efficacy of two DNA isolation methods and two DNA extraction kits commonly used in eDNA preparation. A fragment of the mitochondrial cytochrome b gene from P. bivittatus was detected in all water samples isolated with the sodium acetate precipitate and the QIAamp DNA Micro Kit. Next, we designed P. bivittatus-specific primers and assessed the degradation rate of eDNA in water. Our primers did not amplify DNA from closely related species, and we found that P. bivittatus DNA was consistently detectable up to 96 h. Finally, we sampled water from six field sites in south Florida. Samples from five sites, where P. bivittatus has been observed, tested positive for eDNA. The final site was negative and had no prior documented evidence of P. bivittatus. This study shows P. bivittatus eDNA can be isolated from water samples; thus, this method is a new and promising technique for the management of invasive reptiles. Published 2013. This article is a U.S. Government work and is in the public domain in the USA.

An Attempt to automate the lithological classification of rocks using geological, gamma-spectrometric and satellite image datasets

International Nuclear Information System (INIS)

Fouad, M. K.; Mielik, M. L.; Gharieb, A. N.

2004-01-01

The present study aims essentially at proving that the application of the integrated airborne gamma spectrometric and satellite image data is capable of refining the mapped surface geology, and identification of anomalous zones of radioelement content that could provide favorable exploration targets for radioactive mineralizations.The application of the appropriate statistical technique to correlate between satellite image data and gamma-spectrometric data is of great significance in this respect. Experience shows that Landsat T M data in 7 spectral bands are successfully used in such studies rather than MSS. Multivariate statistical analysis techniques are applied to airborne spectrometric and different spectral Landsat T M data. Reduction of the data from n-dimensionality, both qualitatively as color composite image, and quantitatively, as principal component analysis, is performed using some statistical control parameters. This technique shows distinct efficiency in defining areas where different lit ho facies occur. An area located at the north of the Eastern Desert of Egypt, north of Hurgada town, was chosen to test the proposed technique of integrated interpretation of data of different physical nature. The reduced data are represented and interpreted both qualitatively and quantitatively. The advantages and limitations of applying such technique to the different airborne spectrometric, and Landsat T M data are identified. (authors)

Mass Spectrometric Characteristics of Prenylated Indole Derivatives from Marine-Derived Penicillium sp. NH-SL.

Science.gov (United States)

Ding, Hui; Ding, Wanjing; Ma, Zhongjun

2017-03-22

Two prenylated indole alkaloids were isolated from the ethyl acetate extracts of a marine-derived fungus Penicillium sp. NH-SL and one of them exhibited potent cytotoxic activity against mouse hepa 1c1c7 cells. In order to detect other bioactive analogs, we used liquid chromatogram tandem mass spectrometry (LC-MS/MS) to analyze the mass spectrometric characteristics of the isolated compounds as well as the crude extracts. As a result, three other analogs were detected, and their structures were deduced according to the similar fragmentation patterns. This is the first systematic report on the mass spectrometric characteristics of prenylated indole derivatives.

Molecular characterization of the rDNA-ITS sequence and a PCR diagnostic technique for Pileolaria terebinthi, the cause of pistachio rust

Directory of Open Access Journals (Sweden)

Hossein ALAEI

2013-01-01

Full Text Available Eleven samples of the most important pistachio rust (caused by Pileolaria terebinthi (DC. Cast.,, which causes disease on Beneh (Pistacia atlantica Desf. subsp. mutica (Fisch. & Mey. Rech. F and Kasoor (Pistacia khinjuk Stocks., were collected from herbarium specimens and pistachio fields at the Pistachio Research Institute in Rafsanjan, Iran. The complete sequences of ribosomal DNA internal transcribed spacers ITS1 and ITS2 (rDNA ITS from the samples were determined and analysed. In general, very little rDNA ITS sequence variation was observed between rDNA ITS sequences of P. terebinthi samples. The length of the PCR fragments was 621 bp (for ITS1F-ITS4 and 1177 bp (for ITS1F-rust1, and consisted of 67 bp at the 3 ́ end of 18S rDNA, 93 bp of ITS1 region, 154 bp of 5.8S rDNA, 246 bp of the ITS2 region, 57 bp (for ITS1F-ITS4 and 613 bp (for ITS1F-rust1 at the 5 ́ end of the 28S rDNA. Restriction fragment length polymorphisms (RFLPs of the rDNA-ITS region were used to identify Pileolaria terebinthi. Three strong bands of 105, 134 and 381 bp and five bands of 105, 134, 200, 301 and 437 bp are observed for the fragment of “ITS1F-ITS4” and “ITS1F-rust1”, respectively. A PCR-RFLP diagnostic technique provided effective identification of the species by a unique pattern with the specific restriction enzyme XapI (ApoI.

Determination of plutonium-241 half-life by mass spectrometric measurement

International Nuclear Information System (INIS)

Hiyama, Takashi; Wada, Yukio; Onishi, Koichi

1982-01-01

Much data for Pu-241 half-life have been reported, but these values range from 13.8 years to 15.1 years depending on investigators. In order to define the half-life of Pu-241, the half-life was calculated by analyzing the mass spectrometry data obtained in the author's laboratory over the past six years on Plutonium Isotopic Standard Reference Materials prepared at the National Bureau of Standards (NBS). The sample used for this work consisted of SRM-947 and SRM-948 prepared at NBS. Before mass spectrometric analysis, the plutonium aliquot was separated from its Am-241 daughter by anion exchange chromatography, since Am-241 is not distinguished from Pu-241 in the mass spectrometer. 241 Pu/ 239 Pu and 241 Pu/ 240 Pu ratios were calculated from the values of mass spectrometric measurement. From the relation of log N to time, the half-life of Pu-241 was determined, based on the slope using a least squares fit. The half-life of Pu-241 was estimated to be 14.29+-0.15 years. (Yoshitake, I.)

Mass spectrometric protein characterization in proteome analysis using GELoader tip micro-columns packed with various chromatographic material

International Nuclear Information System (INIS)

Larsen, M.R.

2001-01-01

In the early 90'ies mass spectrometry (MS) was introduced as a tool for identifying proteins in protein sequence databases. Since then it has become an integrated tool in protein characterization and is today routinely used to identify proteins separated by gel electrophoresis. A two-tiered mass spectrometric protein identification strategy has recently been proposed. In the first strategy peptide mass maps obtained from the protein of interest are compared with theoretically derived peptide mass maps from proteins in protein sequence databases. If the protein cannot be identified by this strategy, tandem mass spectrometric sequencing is used to generate enough sequence data to identify the protein in protein sequence databases or expressed sequence tag (EST) databases. However, the above strategies primarily identify a protein relatively to the DNA sequence, in which no information about e.g. post-translational modifications (PTMs) is stored. PTMs are known to modify the function, location, solubility and activity of proteins in the cell, and they are therefore very important for understanding living cells. More than 200 different PTMs are known, of which glycosylation, phosphorylation and proteolytic processing are the most common ones. Mass spectrometric analysis of PTMs on gel-separated proteins requires a higher amount of protein than for identification only. In addition, higher sequence coverage from the peptide mass maps or pre-purification of the modified peptides prior to MS analysis, is necessary for detection of putative modified peptides. In this study a multi-tiered strategy, in which GELoader tip micro-columns packed with increasingly more hydrophobic chromatographic material are used in combination with mass spectrometry, is described. The ultimate aim was to gain increased sequence coverage from peptide mixtures derived from gel-separated proteins, in order to locate modified peptides. Graphite powder is described as an alternative to traditional

Novel primer specific false terminations during DNA sequencing reactions: danger of inaccuracy of mutation analysis in molecular diagnostics

Science.gov (United States)

Anwar, R; Booth, A; Churchill, A J; Markham, A F

1996-01-01

The determination of nucleotide sequence is fundamental to the identification and molecular analysis of genes. Direct sequencing of PCR products is now becoming a commonplace procedure for haplotype analysis, and for defining mutations and polymorphism within genes, particularly for diagnostic purposes. A previously unrecognised phenomenon, primer related variability, observed in sequence data generated using Taq cycle sequencing and T7 Sequenase sequencing, is reported. This suggests that caution is necessary when interpreting DNA sequence data. This is particularly important in situations where treatment may be dependent on the accuracy of the molecular diagnosis. Images PMID:16696096

"Off-on" electrochemical hairpin-DNA-based genosensor for cancer diagnostics.

Science.gov (United States)

Farjami, Elaheh; Clima, Lilia; Gothelf, Kurt; Ferapontova, Elena E

2011-03-01

A simple and robust "off-on" signaling genosensor platform with improved selectivity for single-nucleotide polymorphism (SNP) detection based on the electronic DNA hairpin molecular beacons has been developed. The DNA beacons were immobilized onto gold electrodes in their folded states through the alkanethiol linker at the 3'-end, while the 5'-end was labeled with a methylene blue (MB) redox probe. A typical "on-off" change of the electrochemical signal was observed upon hybridization of the 27-33 nucleotide (nt) long hairpin DNA to the target DNA, in agreement with all the hitherto published data. Truncation of the DNA hairpin beacons down to 20 nts provided improved genosensor selectivity for SNP and allowed switching of the electrochemical genosensor response from the on-off to the off-on mode. Switching was consistent with the variation in the mechanism of the electron transfer reaction between the electrode and the MB redox label, for the folded beacon being characteristic of the electrochemistry of adsorbed species, while for the "open" duplex structure being formally controlled by the diffusion of the redox label within the adsorbate layer. The relative current intensities of both processes were governed by the length of the formed DNA duplex, potential scan rate, and apparent diffusion coefficient of the redox species. The off-on genosensor design used for detection of a cancer biomarker TP53 gene sequence favored discrimination between the healthy and SNP-containing DNA sequences, which was particularly pronounced at short hybridization times.

Gamma spectrometric analyses of environmental samples at PINSTECH

International Nuclear Information System (INIS)

Faruq, M.U.; Parveen, N.; Ahmed, B.; Aziz, A.

1979-01-01

Gamma spectrometric analyses of air and other environmental samples from PINSTECH were carried out. Air particulate samples were analyzed by a Ge(Li) detector on a computer-based multichannel analyzer. Other environmental samples were analyzed by a Na(T1) scintillation detector spectrometer and a multichannel analyzer with manual analysis. Concentration of radionuclides in the media was determined and the sources of their production were identified. Age of the fall out was estimated from the ratios of the fission products. (authors)

Human Papillomavirus (HPV) Infection in Squamous Cell Carcinomas Arising From the Oropharynx: Detection of HPV DNA and p16 Immunohistochemistry as Diagnostic and Prognostic Indicators—A Pilot Study

Energy Technology Data Exchange (ETDEWEB)

Bussu, Francesco, E-mail: francesco.bussu.md@gmail.com [Institute of Otolaryngology, Università Cattolica del Sacro Cuore, Policlinico A. Gemelli, Roma (Italy); Sali, Michela [Institute of Microbiology, Università Cattolica del Sacro Cuore, Policlinico A. Gemelli, Roma (Italy); Gallus, Roberto [Institute of Otolaryngology, Università Cattolica del Sacro Cuore, Policlinico A. Gemelli, Roma (Italy); Petrone, Gianluigi; Zannoni, Gian Franco [Institute of Histopathology, Università Cattolica del Sacro Cuore, Policlinico A. Gemelli, Roma (Italy); Autorino, Rosa; Dinapoli, Nicola [Institute of Radiotherapy, Università Cattolica del Sacro Cuore, Policlinico A. Gemelli, Roma (Italy); Santangelo, Rosaria [Institute of Microbiology, Università Cattolica del Sacro Cuore, Policlinico A. Gemelli, Roma (Italy); Vellone, Valerio Gaetano [Institute of Histopathology, Università Cattolica del Sacro Cuore, Policlinico A. Gemelli, Roma (Italy); Graziani, Cristina [Institute of Otolaryngology, Università Cattolica del Sacro Cuore, Policlinico A. Gemelli, Roma (Italy); Miccichè, Francesco [Institute of Radiotherapy, Università Cattolica del Sacro Cuore, Policlinico A. Gemelli, Roma (Italy); Almadori, Giovanni; Galli, Jacopo [Institute of Otolaryngology, Università Cattolica del Sacro Cuore, Policlinico A. Gemelli, Roma (Italy); Delogu, Giovanni; Sanguinetti, Maurizio [Institute of Microbiology, Università Cattolica del Sacro Cuore, Policlinico A. Gemelli, Roma (Italy); Rindi, Guido [Institute of Histopathology, Università Cattolica del Sacro Cuore, Policlinico A. Gemelli, Roma (Italy); and others

2014-08-01

Purpose: Human papillomavirus (HPV) 16 infection is associated with oropharyngeal carcinogenesis and is likely the cause of the reported increase in disease incidence. We evaluated the prevalence of HPV infection and the reliability of different diagnostic tools using primary tumor samples from a cohort of 50 patients. Methods and Materials: Formalin-fixed paraffin-embedded (FFPE) tumor samples were collected from all 50 consecutive primary oropharyngeal SCC patients who were enrolled in the study; fresh tumor samples were available in 22 cases. NucliSENS EasyQ HPVv1 was used for RNA, and Digene Hybrid Capture-2(HC2) was used for DNA detection. p16 Expression was evaluated by immunohistochemistry in FPPE specimens. Results: Based on the DNA detection assay on FFPE samples, the frequency of high-risk HPV infection was 32%. The agreement rate between HPV RNA and HPV DNA detection in fresh samples was 100%. The agreement rate between p16 immunohistochemistry (IHC) and the detection of HPV DNA in the FFPE samples was fair but not excellent (κ = 0.618). HPV DNA detection was highly significant, as measured by disease-specific survival and determined using a Wilcoxon test (P=.001). p16 IHC also exhibited a prognostic value but with a lower statistical significance (P=.0475). The detection of HPV DNA, but not p16 IHC, was also significantly correlated with locoregional control (P=.0461). Conclusion: Diagnostic methods based on the detection of HPV nucleic acids appear to be more reliable and objective because they do not require reading by a trained histopathologist. Furthermore, the detection of HPV DNA exhibits an improved correlation with survival, and therefore appears definitely more reliable than p16 IHC for routine use in clinical practice.

Human Papillomavirus (HPV) Infection in Squamous Cell Carcinomas Arising From the Oropharynx: Detection of HPV DNA and p16 Immunohistochemistry as Diagnostic and Prognostic Indicators—A Pilot Study

International Nuclear Information System (INIS)

Bussu, Francesco; Sali, Michela; Gallus, Roberto; Petrone, Gianluigi; Zannoni, Gian Franco; Autorino, Rosa; Dinapoli, Nicola; Santangelo, Rosaria; Vellone, Valerio Gaetano; Graziani, Cristina; Miccichè, Francesco; Almadori, Giovanni; Galli, Jacopo; Delogu, Giovanni; Sanguinetti, Maurizio; Rindi, Guido

2014-01-01

Purpose: Human papillomavirus (HPV) 16 infection is associated with oropharyngeal carcinogenesis and is likely the cause of the reported increase in disease incidence. We evaluated the prevalence of HPV infection and the reliability of different diagnostic tools using primary tumor samples from a cohort of 50 patients. Methods and Materials: Formalin-fixed paraffin-embedded (FFPE) tumor samples were collected from all 50 consecutive primary oropharyngeal SCC patients who were enrolled in the study; fresh tumor samples were available in 22 cases. NucliSENS EasyQ HPVv1 was used for RNA, and Digene Hybrid Capture-2(HC2) was used for DNA detection. p16 Expression was evaluated by immunohistochemistry in FPPE specimens. Results: Based on the DNA detection assay on FFPE samples, the frequency of high-risk HPV infection was 32%. The agreement rate between HPV RNA and HPV DNA detection in fresh samples was 100%. The agreement rate between p16 immunohistochemistry (IHC) and the detection of HPV DNA in the FFPE samples was fair but not excellent (κ = 0.618). HPV DNA detection was highly significant, as measured by disease-specific survival and determined using a Wilcoxon test (P=.001). p16 IHC also exhibited a prognostic value but with a lower statistical significance (P=.0475). The detection of HPV DNA, but not p16 IHC, was also significantly correlated with locoregional control (P=.0461). Conclusion: Diagnostic methods based on the detection of HPV nucleic acids appear to be more reliable and objective because they do not require reading by a trained histopathologist. Furthermore, the detection of HPV DNA exhibits an improved correlation with survival, and therefore appears definitely more reliable than p16 IHC for routine use in clinical practice

Differential Immuno-Reactivity to Genomic DNA, RNA and Mitochondrial DNA is Associated with Auto-Immunity

Directory of Open Access Journals (Sweden)

Vilena V. Ivanova

2014-12-01

Full Text Available Background: Circulating auto-reactive antibodies are hallmark features of auto-immune diseases, however little is known with respect to the specificity of such bio-markers. In the present study, we investigated the specificity of anti-nucleic acid antibodies in the blood of subjects with systemic lupus erythematosus (SLE and healthy controls. Methods: Sera from 12 SLE cases and 8 controls were evaluated for immuno-reactivity to purified RNA, DNA and mitochondrial DNA (mtDNA by enzyme-linked immuno-sorbent assay (ELISA. Results: As expected, immuno-reactivity to total nucleic acids was significantly higher in subjects with SLE when compared to healthy controls, however a clear distinction was observed among the various nucleic acid sub-types, with sera from SLE subjects displaying the greatest immuno-reactivity to RNA followed by mtDNA and then total DNA. Conclusion: The identification of auto-reactive antibodies can serve as highly sensitive biomarkers, although their specificity may not always allow diagnostic certainty. The knowledge that auto-antibodies in subjects with SLE display differential immuno-reactivity may help to improve existing diagnostics and may lead to a better understanding of the pathogenesis of auto-immune disorders.

Mass spectrometric investigation of fluorated europium β-diketonates

International Nuclear Information System (INIS)

Khomenko, V.S.; Lozinskij, M.O.; Fialkov, Yu.A.; Rasshinina, T.A.; Suboch, V.P.; AN Ukrainskoj SSR, Kiev. Inst. Organicheskoj Khimii)

1983-01-01

Ternary complexes of europium with two organic lidands - fluorated β-diketone and organic base - in the aqueous phase at electron shock are investigated; regroupings that take place in them are established. Mass-spectrometric investigation has shown that complexes with β-diketones, containing in oxygen atom in a fluorated radical, eliminate stable molecules of difluorophosgen, CoF 2 . For the complexes studied under the above conditions the Co molecule elimination is also typical which is accompanied by the formation of ions with a metal-carbon bond

[Diagnostic significance of serum free DNA human telomerase reverse transcriptase quantitative determination on spinal cord injury].

Science.gov (United States)

Yang, M K; Tang, J; Xiang, Z; Zhang, X; Wang, J; Li, Z; Li, Y; Sheng, W B

2018-02-06

Objective: To investigate the relationship between the content of human telomerase reverse transcriptase (hTERT) and its clinical features in serum free DNA in patients with different degree of spinal cord injury. Methods: From December 2013 to December 2016, inpatients of the Central Hospital of Bazhong, Sichuan Province were enrolledand divided into the experimental group, the disease control group and the negative control group. For the experimental group: 46 patients with spinal cord injury were graded according to the criteria of the American Association of Spinal Cord Injury (ASIA), including 12 cases of grade A, 10 cases of grade B, 10 cases of grade C, 7 cases of grade D and 7 cases of grade E; for the disease control group: 15 patients with spinal fractures (without spinal cord injury) at the same period were included; and for the negative control group: 20 healthy adult volunteers aged 18-50 years were selected.Real-time fluorescence quantitative PCR and immunoblotting were performed to detect the content of hTERT in serum free DNA both in patients and healthy controls and to compare the difference between them. The results of the somatosensory evoked potential (SEP) of all patients were compared and analyzed.The receiver operating characteristic (ROC) curve was used to analyze the diagnostic value of hTERT content in serum free DNA in patients with spinal cord injury. Results: Comparison of serum free DNA hTERT content: in the experimental group, the serum free DNA hTERT content of grade A, B, C, D, E was (99.63±8.23), (76.24±4.37), (46.07±5.43), (16.30±0.95) and (15.74±1.12)μg/L, respectively.While it was (15.01±1.39)μg/L in the disease control group and (14.54±1.03)μg/L in the negative control group. The total difference was statistically significant between patients of each group and the control group ( F =857.917, P spinal cord injury has a certain guiding significance for the diagnosis of spinal cord injury and the degree of injury.

Saliva Preservative for Diagnostic Purposes

Science.gov (United States)

Pierson, Duane L.; Mehta, Satish K.

2012-01-01

Saliva is an important body fluid for diagnostic purposes. Glycoproteins, glucose, steroids, DNA, and other molecules of diagnostic value are found in saliva. It is easier to collect as compared to blood or urine. Unfortunately, saliva also contains large numbers of bacteria that can release enzymes, which can degrade proteins and nucleic acids. These degradative enzymes destroy or reduce saliva s diagnostic value. This innovation describes the formulation of a chemical preservative that prevents microbial growth and inactivates the degradative enzymes. This extends the time that saliva can be stored or transported without losing its diagnostic value. Multiple samples of saliva can be collected if needed without causing discomfort to the subject and it does not require any special facilities to handle after it is collected.

Mass spectrometric identification of isocyanate-induced modifications of keratins in human skin

NARCIS (Netherlands)

Hulst, A.G.; Verstappen, D.R.W.; Riet-van Oeveren, D. van der; Vermeulen, N.P.E.; Noort, D.

2015-01-01

In the current paper we show that exposure of human callus to isocyanates leads to covalent modifications within keratin proteins. Mass spectrometric analyses of pronase digests of keratin isolated from exposed callus show that both mono- and di-adducts (for di-isocyanates) are predominantly formed

DNA replication stress restricts ribosomal DNA copy number.

Science.gov (United States)

Salim, Devika; Bradford, William D; Freeland, Amy; Cady, Gillian; Wang, Jianmin; Pruitt, Steven C; Gerton, Jennifer L

2017-09-01

Ribosomal RNAs (rRNAs) in budding yeast are encoded by ~100-200 repeats of a 9.1kb sequence arranged in tandem on chromosome XII, the ribosomal DNA (rDNA) locus. Copy number of rDNA repeat units in eukaryotic cells is maintained far in excess of the requirement for ribosome biogenesis. Despite the importance of the repeats for both ribosomal and non-ribosomal functions, it is currently not known how "normal" copy number is determined or maintained. To identify essential genes involved in the maintenance of rDNA copy number, we developed a droplet digital PCR based assay to measure rDNA copy number in yeast and used it to screen a yeast conditional temperature-sensitive mutant collection of essential genes. Our screen revealed that low rDNA copy number is associated with compromised DNA replication. Further, subculturing yeast under two separate conditions of DNA replication stress selected for a contraction of the rDNA array independent of the replication fork blocking protein, Fob1. Interestingly, cells with a contracted array grew better than their counterparts with normal copy number under conditions of DNA replication stress. Our data indicate that DNA replication stresses select for a smaller rDNA array. We speculate that this liberates scarce replication factors for use by the rest of the genome, which in turn helps cells complete DNA replication and continue to propagate. Interestingly, tumors from mini chromosome maintenance 2 (MCM2)-deficient mice also show a loss of rDNA repeats. Our data suggest that a reduction in rDNA copy number may indicate a history of DNA replication stress, and that rDNA array size could serve as a diagnostic marker for replication stress. Taken together, these data begin to suggest the selective pressures that combine to yield a "normal" rDNA copy number.

DNA replication stress restricts ribosomal DNA copy number

Science.gov (United States)

Salim, Devika; Bradford, William D.; Freeland, Amy; Cady, Gillian; Wang, Jianmin

2017-01-01

Ribosomal RNAs (rRNAs) in budding yeast are encoded by ~100–200 repeats of a 9.1kb sequence arranged in tandem on chromosome XII, the ribosomal DNA (rDNA) locus. Copy number of rDNA repeat units in eukaryotic cells is maintained far in excess of the requirement for ribosome biogenesis. Despite the importance of the repeats for both ribosomal and non-ribosomal functions, it is currently not known how “normal” copy number is determined or maintained. To identify essential genes involved in the maintenance of rDNA copy number, we developed a droplet digital PCR based assay to measure rDNA copy number in yeast and used it to screen a yeast conditional temperature-sensitive mutant collection of essential genes. Our screen revealed that low rDNA copy number is associated with compromised DNA replication. Further, subculturing yeast under two separate conditions of DNA replication stress selected for a contraction of the rDNA array independent of the replication fork blocking protein, Fob1. Interestingly, cells with a contracted array grew better than their counterparts with normal copy number under conditions of DNA replication stress. Our data indicate that DNA replication stresses select for a smaller rDNA array. We speculate that this liberates scarce replication factors for use by the rest of the genome, which in turn helps cells complete DNA replication and continue to propagate. Interestingly, tumors from mini chromosome maintenance 2 (MCM2)-deficient mice also show a loss of rDNA repeats. Our data suggest that a reduction in rDNA copy number may indicate a history of DNA replication stress, and that rDNA array size could serve as a diagnostic marker for replication stress. Taken together, these data begin to suggest the selective pressures that combine to yield a “normal” rDNA copy number. PMID:28915237

DNA replication stress restricts ribosomal DNA copy number.

Directory of Open Access Journals (Sweden)

Devika Salim

2017-09-01

Full Text Available Ribosomal RNAs (rRNAs in budding yeast are encoded by ~100-200 repeats of a 9.1kb sequence arranged in tandem on chromosome XII, the ribosomal DNA (rDNA locus. Copy number of rDNA repeat units in eukaryotic cells is maintained far in excess of the requirement for ribosome biogenesis. Despite the importance of the repeats for both ribosomal and non-ribosomal functions, it is currently not known how "normal" copy number is determined or maintained. To identify essential genes involved in the maintenance of rDNA copy number, we developed a droplet digital PCR based assay to measure rDNA copy number in yeast and used it to screen a yeast conditional temperature-sensitive mutant collection of essential genes. Our screen revealed that low rDNA copy number is associated with compromised DNA replication. Further, subculturing yeast under two separate conditions of DNA replication stress selected for a contraction of the rDNA array independent of the replication fork blocking protein, Fob1. Interestingly, cells with a contracted array grew better than their counterparts with normal copy number under conditions of DNA replication stress. Our data indicate that DNA replication stresses select for a smaller rDNA array. We speculate that this liberates scarce replication factors for use by the rest of the genome, which in turn helps cells complete DNA replication and continue to propagate. Interestingly, tumors from mini chromosome maintenance 2 (MCM2-deficient mice also show a loss of rDNA repeats. Our data suggest that a reduction in rDNA copy number may indicate a history of DNA replication stress, and that rDNA array size could serve as a diagnostic marker for replication stress. Taken together, these data begin to suggest the selective pressures that combine to yield a "normal" rDNA copy number.

Gamma Spectrometric Determination of U, Th, K and Some Geochemical Applications

International Nuclear Information System (INIS)

Dodona, A.; Tashko, A.

2001-01-01

The application of 'in situ' gamma-spectrometric method (''infinite'' environment), made possible the simultanious determination of U, Th and K. 4 channel gamma-spectrometric analyser with NaI(TI) scintilation counter crystal detector (103 cm 3 φ=50x50mm) was used to determin U, Th(more than 1-2 ppm) and K (more than 1%) in laboratory conditions. The detector was inserted into a lead camera and calibrated for measurement geometry with vessel of ''Marineli'' type of a 17o cm 3 volume. The study of main factors, which influence in the gamma spectrometric measurements, (the technical, physical, geometrical and time parameters) has been carried out. International standards of U, Th, K and internal monitoring standard samples are used for the calibration. External analytical control has been realized by other radiometric and chemical methods. The detection limits ( 1 ppm Th, 2ppm U and 1% K) and the relative errors (17-20% for 1-10 ppm U, Th and 10-15% for more than 10 ppm U, Th and more than 1% K) guarantee a quantitative analysis that may be used successfully in the geochemical studies. Some geochemical applications, based on the content of Th, U and Th/U ratio in rocks samples that we have we have analyzed with this method, are shown in this paper. U, Th and their ratio are used as trace elements to indicate the differences between the acidic magmatic rocks of Albania (Th/U ratio=2-6 and>10). The bimodal character of Th/U scattering in ignimbrides and monzonites (Korabi zone) shows that in addition to the ''normal'' rocks, there are also some ones enriched with Th, So, the differential analysis of Th, U, and K may be used as geochemical exploration criteria for the radioactive and non-radioactive mineralization, such as REE (Rare Earth Elements), phospghorites, bauxites, placers etc. (authors)

Spectrometric aerial survey as a new tool for geological survey and mining prospecting

International Nuclear Information System (INIS)

Cambon, R.

1997-01-01

Airborne survey for radioactive minerals started around 1945. The limited sensitivity of the tools used, the difficulties found for the topographic and training effect corrections, made difficult the evaluation of the results. The technical progresses realized in the recent past years in electronic and computer sciences allowed to overcome these difficulties and gave to the method all its potentialities. With the aerial spectrometric survey, a new step was made, because this method can be used for other topics than radioactive prospection such as geological survey and mining prospection for metallic and industrial minerals. The spectrometric method is based on the possibility to measure photopeak energies (gamma radiation) emitted by radioactive minerals and discriminate between them those emitted by U238, TI 208 and K40 respectively daughter products of uranium, thorium and potassium. For airborne survey, one consider that measuring instruments will allow to pick-up 80% of the radioactive emission concerning the first 15 to 30 centimetres of ground (1 metre maximum). The use of this method for geological and mineral exploration is based on the assumption that different rock types or ore bearing rock types are composed of certain amounts of rock forming minerals which comprise specific quantities of radioactive elements such as potassium, uranium and thorium (cf: Gabelman 77). To be able to evaluate the results of the spectrometric survey it will be necessary to know roughly the behaviour of the different radioactive elements through a complete geological cycle. (author)

Spectrometric and Voltammetric Analysis of Urease – Nickel Nanoelectrode as an Electrochemical Sensor

Directory of Open Access Journals (Sweden)

Rene Kizek

2007-07-01

Full Text Available Urease is the enzyme catalyzing the hydrolysis of urea into carbon dioxide andammonia. This enzyme is substrate-specific, which means that the enzyme catalyzes thehydrolysis of urea only. This feature is a basic diagnostic criterion used in thedetermination of many bacteria species. Most of the methods utilized for detection ofurease are based on analysis of its enzyme activity – the hydrolysis of urea. The aim of thiswork was to detect urease indirectly by spectrometric method and directly by voltammetricmethods. As spectrometric method we used is called indophenol assay. The sensitivity ofdetection itself is not sufficient to analyse the samples without pre-concentration steps.Therefore we utilized adsorptive transfer stripping technique coupled with differential pulse voltammetry to detect urease. The influence of accumulation time, pH of supporting electrolyte and concentration of urease on the enzyme peak height was investigated. Under the optimized experimental conditions (0.2 M acetate buffer pH 4.6 and accumulation time of 120 s the detection limit of urease evaluated as 3 S/N was 200 ng/ml. The activity of urease enzyme depends on the presence of nickel. Thus the influence of nickel(II ions on electrochemical response of the enzyme was studied. Based on the results obtained the interaction of nickel(II ions and urease can be determined using electrochemical methods. Therefore we prepared Ni nanoelectrodes to measure urease. The Ni nanoelectrodes was analysed after the template dissolution by scanning electron microscopy. The results shown vertically aligned Ni nanopillars almost covered the electrode surface, whereas the defect places are minor and insignificant in comparison with total electrode surface. We were able to not only detect urease itself but also to distinguish its native and denatured form.

Gamma spectrometric system based on the personal computer Pravetz-83

International Nuclear Information System (INIS)

Yanakiev, K; Grigorov, T.; Vuchkov, M.

1985-01-01

A gamma spectrometric system based on a personal microcomputer Pravets-85 is described. The analog modules are NIM standard. ADC data are stored in the memory of the computer via a DMA channel and a real-time data processing is possible. The results from a series of tests indicate that the performance of the system is comparable with that of comercially avalable computerized spectrometers Ortec and Canberra

Scratching the surface of tomorrow's diagnostics: the Editor-in-Chief's opinion at the 15th year of Expert Review of Molecular Diagnostics.

Science.gov (United States)

Lorincz, Attila; Raison, Claire

2015-01-01

Interview with Attila Lorincz by Claire Raison (Commissioning Editor) To mark the beginning of the 15th year of Expert Review of Molecular Diagnostics, the journal's Editor-in-Chief shares his expert knowledge on translational diagnostics, his opinion on recent controversies and his predictions for molecular diagnostics in 2015 and beyond. Attila Lorincz received his doctorate from Trinity College, Dublin, Republic of Ireland, and went on to become a research fellow at the University of California, Santa Barbara, CA, USA. During Professor Lorincz's research on human papillomavirus (HPV), he found several important and novel carcinogenic HPV types and pioneered the use of HPV DNA testing for clinical diagnostics. In 1988, Professor Lorincz's team produced the first HPV test to be FDA-approved for patients and in 2003, for general population cervical precancer screening. Now Professor of Molecular Epidemiology at the Centre for Cancer Prevention, Queen Mary University of London, UK, he and his team are furthering translational research into DNA methylation assays for cancer risk prediction.

A measuring generator for testing spectrometric channels

International Nuclear Information System (INIS)

Dinh Sy Hien; Kalinkin, A.I.

1984-01-01

A measuring generator for testing and tuning an amplitude spectrometric channel is described. The device consists of a pseudo random pulse generator, constructed on shifters, a sawtooth wave generator and a shaper of stable amplitude pulses with exponential decay times. The device is made as CAMAC unit width modules and has the following specifications: average pulse repetition rate of pseudo random pulses is 3.1; 25; 50; 100; 200 kHz; peak amplitude of 2 Hz pulse repetition of saw tooth pulses is 6 V; peak amplitude of exponential shape pulses is 5 V. The block-diagram and basic circuits of the device are given

Gamma-spectrometric analysis of river sediments collected around phosphate fertilizer industries

International Nuclear Information System (INIS)

Gallardo, M.C.; Garcia-Leon, M.; Mundi, M.; Respaldiza, M.A.

1993-01-01

Gamma-ray spectrometric analysis has been carried out on sediments collected in an estuarine system formed by two major rivers in southern Spain. The results show clearly that important amounts of natural radioactivity are accumulating on the bed of both rivers. This radioactivity appears to originate from effluent from several phoshate fertilizer factories adjacent to the estuary. (author)

Standard test methods for chemical, mass spectrometric, and spectrochemical analysis of nuclear-grade boron carbide

CERN Document Server

American Society for Testing and Materials. Philadelphia

2004-01-01

1.1 These test methods cover procedures for the chemical, mass spectrometric, and spectrochemical analysis of nuclear-grade boron carbide powder and pellets to determine compliance with specifications. 1.2 The analytical procedures appear in the following order: Sections Total Carbon by Combustion and Gravimetry 7-17 Total Boron by Titrimetry 18-28 Isotopic Composition by Mass Spectrometry 29-38 Chloride and Fluoride Separation by Pyrohydrolysis 39-45 Chloride by Constant-Current Coulometry 46-54 Fluoride by Ion-Selective Electrode 55-63 Water by Constant-Voltage Coulometry 64-72 Impurities by Spectrochemical Analysis 73-81 Soluble Boron by Titrimetry 82-95 Soluble Carbon by a Manometric Measurement 96-105 Metallic Impurities by a Direct Reader Spectrometric Method 106-114

Mass spectrometric analysis of a UV-cross-linked protein-DNA complex: tryptophans 54 and 88 of E. coli SSB cross-link to DNA

DEFF Research Database (Denmark)

Steen, Hanno; Petersen, Jørgen; Mann, Matthias

2001-01-01

acid and peptide entities present in such heteroconjugates. Sample preparation of the peptide-nucleic acid heteroconjugates is, therefore, a crucial step in any mass spectrometry-based analytical procedure. This study demonstrates the performance of four different MS-based strategies to characterize E....... coli single-stranded DNA binding protein (SSB) that was UV-cross-linked to a 5-iodouracil containing DNA oligomer. Two methods were optimized to circumvent the need for standard liquid chromatography and gel electrophoresis, thereby dramatically increasing the overall sensitivity of the analysis...

Microbial food safety: Potential of DNA extraction methods for use in diagnostic metagenomics

DEFF Research Database (Denmark)

Josefsen, Mathilde Hasseldam; Andersen, Sandra Christine; Christensen, Julia

2015-01-01

) yielding protocols. The PowerLyzer PowerSoil DNA Isolation Kit performed significantly better than all other protocols tested. Selected protocols were modified, i.e., extended heating and homogenization, resulting in increased yields of total DNA. For QIAamp Fast DNA Stool Mini Kit (Qiagen) a 7-fold...... of the protocols to extract DNA was observed. The highest DNA yield was obtained with the PowerLyzer PowerSoil DNA Isolation Kit, whereas the FastDNA SPIN Kit for Feces (MP Biomedicals) resulted in the highest amount of PCR-amplifiable C. jejuni DNA....

The measurement of mass spectrometric peak height ratio of helium isotope in trace samples

International Nuclear Information System (INIS)

Sun Mingliang

1989-01-01

An experiment study on the measurement of mass spectrometric peak height ratio of helium isotope in the trace gaseous sample is discussed by using the gas purification line designed by the authors and model VG-5400 static-vacuum noble gas mass spectrometer imported and air helium as a standard. The results show that the amount of He and Ne in natural gas sample is 99% after purification. When the amount of He in Mass Spectrometer is more than 4 x 10 -7 cm 3 STP, it's sensitivity remains stable, about 10 -4 A/cm 3 STP He and the precision of 3 He/ 4 He ratio within the following 17 days is 1.32%. The 'ABA' pattern and experiment condition in the measurement of mass spectrometric peak height ratio of He isotope are presented

Multi-color fluorescent DNA analysis in an integrated optofluidic lab-on-a-chip

OpenAIRE

Dongre, C.; van Weerd, J.; van Weeghel, R.; Martinez-Vazquez, R.; Osellame, R.; Cerullo, G.; Besselink, G.A.J.; van den Vlekkert, H.H.; Hoekstra, Hugo; Pollnau, Markus

2010-01-01

Sorting and sizing of DNA molecules within the human genome project has enabled the genetic mapping of various illnesses. By employing tiny lab-on-a-chip devices for such DNA analysis, integrated DNA sequencing and genetic diagnostics have become feasible. However, such diagnostic chips typically lack integrated sensing capability. We address this issue by combining microfluidic capillary electrophoresis with laser-induced fluorescence detection resulting in optofluidic integration towards an...

Morphological and molecular diagnostics of Phytoseiulus persimilis and Phytoseiulus macropilis (Acari: Phytoseiidae).

Science.gov (United States)

Okassa, Mireille; Tixier, Marie-Stéphane; Kreiter, Serge

2010-11-01

This study focuses on the diagnostics of two natural enemy species, belonging to the genus Phytoseiulus in the family Phytoseiidae (sub-family Amblyseiinae): P. macropilis and P. persimilis. These two species are of primary importance in biological control all over the world. However, they are morphologically very similar and specific diagnostics is difficult. This study utilizes mitochondrial molecular markers (12S rRNA and Cytb mtDNA) to differentiate these two species. Morphological analyses showed significant differences between P. persimilis and P. macropilis for 17 morphological characters of the 32 considered. However, despite these significant differences, the ranges of all characters overlap. Only the serration of the macroseta on the basitarsus (StIV) allows the differentiation between P. persimilis and P. macropilis. Despite these small morphological differences, molecular results, for both mitochondrial DNA fragments considered (rRNA and Cytb mtDNA), showed a clear delineation between the specimens of P. macropilis and P. persimilis. This study emphasizes (i) that only one morphological character (serration of the seta StIV) clearly separates these two species, and (ii) the usefulness of an automatical molecular and simple diagnostic tool for accurate differentiation of the two species and ensure the morphological diagnostics. Further studies are proposed, including more DNA sequences especially for P. macropilis.

Radiochemical separation and alpha-spectrometric determination of Americium in different matrixes

International Nuclear Information System (INIS)

Radenkovic, M.; Joksic, J.; Paligoric, D.

2009-01-01

A method of separation and alpha-spectrometric determination of americium, developed in VINCA Institute of Nuclear Sciences is described in the paper. The procedure is convenient to be used for 241 Am determination in environmental matrixes as well as samples of human origin if a very small concentrations are expected, using 243 Am as a tracer for radiochemical yield recovery. (author) [sr

Sensitive emission spectrometric method for the analysis of airborne particulate matter

International Nuclear Information System (INIS)

Sugimae, A.

1975-01-01

A rapid and sensitive emission spectrometric method for the routine analysis of airborne particulate matter collected on the glass fiber filter is reported. The method is a powder--dc arc technique involving no chemical pre-enrichment procedures. The elements--Ag, BA: Be, Bi, Cd, Co, Cr, Cu, Fe, Ga, La, Mn, Ni, Pb, Sn, V, Y, Yb, and Zn--were determined. (U.S.)

A Comparative Study for Detection of EGFR Mutations in Plasma Cell-Free DNA in Korean Clinical Diagnostic Laboratories

Directory of Open Access Journals (Sweden)

Yoonjung Kim

2018-01-01

Full Text Available Liquid biopsies to genotype the epidermal growth factor receptor (EGFR for targeted therapy have been implemented in clinical decision-making in the field of lung cancer, but harmonization of detection methods is still scarce among clinical laboratories. We performed a pilot external quality assurance (EQA scheme to harmonize circulating tumor DNA testing among laboratories. For EQA, we created materials containing different levels of spiked cell-free DNA (cfDNA in normal plasma. The limit of detection (LOD of the cobas® EGFR Mutation Test v2 (Roche Molecular Systems was also evaluated. From November 2016 to June 2017, seven clinical diagnostic laboratories participated in the EQA program. The majority (98.94% of results obtained using the cobas assay and next-generation sequencing (NGS were acceptable. Quantitative results from the cobas assay were positively correlated with allele frequencies derived from digital droplet PCR measurements and showed good reproducibility among laboratories. The LOD of the cobas assay was 5~27 copies/mL for p.E746_A750del (exon 19 deletion, 35~70 copies/mL for p.L858R, 18~36 copies/mL for p.T790M, and 15~31 copies/mL for p.A767_V769dup (exon 20 insertion. Deep sequencing of materials (>100,000X depth of coverage resulted in detection of low-level targets present at frequencies of 0.06~0.13%. Our results indicate that the cobas assay is a reliable and rapid method for detecting EGFR mutations in plasma cfDNA. Careful interpretation is particularly important for p.T790M detection in the setting of relapse. Individual laboratories should optimize NGS performance to maximize clinical utility.

Thermogravimetric-quadrupole mass-spectrometric analysis of geochemical samples.

Science.gov (United States)

Gibson, E. K., Jr.; Johnson, S. M.

1972-01-01

Thermogravimetric-quadrupole mass-spectrometric-analysis techniques can be used to study a wide variety of problems involving decomposition processes and identification of released volatile components. A recording vacuum thermoanalyzer has been coupled with a quadrupole mass spectrometer. The rapid scan capabilities of the quadrupole mass spectrometer are used to identify the gaseous components released. The capability of the thermogravimetric-quadrupole mass spectrometer to provide analytical data for identification of the released volatile components, for determination of their sequence of release and for correlation of thermal-decomposition studies is illustrated by an analysis of the Orgueil carbonaceous chondrite.

A DNA aptamer recognising a malaria protein biomarker can function as part of a DNA origami assembly

Science.gov (United States)

Godonoga, Maia; Lin, Ting-Yu; Oshima, Azusa; Sumitomo, Koji; Tang, Marco S. L.; Cheung, Yee-Wai; Kinghorn, Andrew B.; Dirkzwager, Roderick M.; Zhou, Cunshan; Kuzuya, Akinori; Tanner, Julian A.; Heddle, Jonathan G.

2016-01-01

DNA aptamers have potential for disease diagnosis and as therapeutics, particularly when interfaced with programmable molecular technology. Here we have combined DNA aptamers specific for the malaria biomarker Plasmodium falciparum lactate dehydrogenase (PfLDH) with a DNA origami scaffold. Twelve aptamers that recognise PfLDH were integrated into a rectangular DNA origami and atomic force microscopy demonstrated that the incorporated aptamers preserve their ability to specifically bind target protein. Captured PfLDH retained enzymatic activity and protein-aptamer binding was observed dynamically using high-speed AFM. This work demonstrates the ability of DNA aptamers to recognise a malaria biomarker whilst being integrated within a supramolecular DNA scaffold, opening new possibilities for malaria diagnostic approaches based on DNA nanotechnology. PMID:26891622

Electrospray Ionization Mass Spectrometric Analysis of Highly Reactive Glycosyl Halides

Directory of Open Access Journals (Sweden)

Lajos Kovács

2012-07-01

Full Text Available Highly reactive glycosyl chlorides and bromides have been analysed by a routine mass spectrometric method using electrospray ionization and lithium salt adduct-forming agents in anhydrous acetonitrile solution, providing salient lithiated molecular ions [M+Li]⁺, [2M+Li]⁺ etc. The role of other adduct-forming salts has also been evaluated. The lithium salt method is useful for accurate mass determination of these highly sensitive compounds.

Photometric and emission-spectrometric determination of boron in steels

International Nuclear Information System (INIS)

Thierig, D.

1982-01-01

A method for the photometric determination of boron in unalloyed and alloyed steels is described, in which Curcumine is used as reagent. A separation of boron is not necessary. Limit of detection: 0.0003% B. The decomposition of boron nitride in the steel is achieved by heating the whole sample in fuming sulphuric acid/phosphoric acid. For the emission spectrometric investigation of solid steel samples and for the spectrochemical analysis of solutions with plasma excitation working parameters are given and possibilities of interferences are demonstrated. (orig.) [de

Mass spectrometric investigation of fluorated europium. beta. -diketonates. [Electrons

Energy Technology Data Exchange (ETDEWEB)

Khomenko, V.S.; Lozinskij, M.O.; Fialkov, Yu.A.; Rasshinina, T.A.; Suboch, V.P. (AN Belorusskoj SSR, Minsk. Inst. Fiziki; AN Ukrainskoj SSR, Kiev. Inst. Organicheskoj Khimii)

Ternary complexes of europium with two organic ligands - fluorated ..beta..-diketone and organic base - in the aqueous phase at electron shock are investigated; regroupings that take place in them are established. Mass-spectrometric investigation has shown that complexes with ..beta..-diketones, containing in oxygen atom in a fluorated radical, eliminate stable molecules of difluorophosgen, CoF/sub 2/. For the complexes studied under the above conditions the Co molecule elimination is also typical which is accompanied by the formation of ions with a metal-carbon bond.

Determination of uranium enrichment by using gamma-spectrometric methods

International Nuclear Information System (INIS)

Kutnyj, D.V.; Telegin, Yu.N.; Odejchuk, N.P.; Mikhailov, V.A.; Tovkanets, V.E.

2009-01-01

By using commercial analysis programs MGAU (LLNL, USA) and FRAM (LANL, USA) the summary error of gamma-spectrometric uranium enrichment measurements was investigated. Uranium samples with enrichments of 0,71; 4,46 and 20,1 % were measured. The coaxial high purity germanium detector (type GC) and the planar germanium detector (type LEGe) were used as gamma-radiation detectors. It was shown that experimental equipment and mathematical software available in NSC KIPT allow us to measure uranium enrichment by nondestructive method with accuracy of not worse than 2%.

Disparities in the diagnostic process of Duchenne and Becker muscular dystrophy.

Science.gov (United States)

Holtzer, Caleb; Meaney, F John; Andrews, Jennifer; Ciafaloni, Emma; Fox, Deborah J; James, Katherine A; Lu, Zhenqiang; Miller, Lisa; Pandya, Shree; Ouyang, Lijing; Cunniff, Christopher

2011-11-01

To determine whether sociodemographic factors are associated with delays at specific steps in the diagnostic process of Duchenne and Becker muscular dystrophy. We examined abstracted medical records for 540 males from population-based surveillance sites in Arizona, Colorado, Georgia, Iowa, and western New York. We used linear regressions to model the association of three sociodemographic characteristics with age at initial medical evaluation, first creatine kinase measurement, and earliest DNA analysis while controlling for changes in the diagnostic process over time. The analytical dataset included 375 males with information on family history of Duchenne and Becker muscular dystrophy, neighborhood poverty levels, and race/ethnicity. Black and Hispanic race/ethnicity predicted older ages at initial evaluation, creatine kinase measurement, and DNA testing (P Becker muscular dystrophy predicted younger ages at initial evaluation, creatine kinase measurement and DNA testing (P Becker muscular dystrophy are evident even after adjustment for family history of Duchenne and Becker muscular dystrophy and changes in the diagnostic process over time. Black and Hispanic children are initially evaluated at older ages than white children, and the gap widens at later steps in the diagnostic process.

The interlaboratory experiment IDA-72 on mass spectrometric isotope dilution analysis. Vol. 2

International Nuclear Information System (INIS)

Beyrich, W.; Drosselmeyer, E.

1975-07-01

Volume II of the report on the IDA-72 experiment contains papers written by different authors on a number of special topics connected with the preparation, performance and evaluation of the interlaboratory test. In detail the sampling procedures for active samples of the reprocessing plant and the preparation of inactive reference and spike solution from standard material are described as well as new methods of sample conditioning by evaporation. An extra chapter is devoted to the chemical sample treatment as a preparation for mass spectrometric analysis of the U and Pu content of the solutions. Special topics are also methods for mass discrimination corrections, α-spectrometer measurements as a supplement for the determination of Pu-238 and the comparison of concentration determinations by mass spectrometric isotope dilution analysis with those performed by X-ray fluorescence spectrometry. The last part of this volume contains papers connected with the computerized statistical evaluation of the high number of data. (orig.) [de

Mass spectrometric detection of siRNA in plasma samples for doping control purposes.

Science.gov (United States)

Kohler, Maxie; Thomas, Andreas; Walpurgis, Katja; Schänzer, Wilhelm; Thevis, Mario

2010-10-01

Small interfering ribonucleic acid (siRNA) molecules can effect the expression of any gene by inducing the degradation of mRNA. Therefore, these molecules can be of interest for illicit performance enhancement in sports by affecting different metabolic pathways. An example of an efficient performance-enhancing gene knockdown is the myostatin gene that regulates muscle growth. This study was carried out to provide a tool for the mass spectrometric detection of modified and unmodified siRNA from plasma samples. The oligonucleotides are purified by centrifugal filtration and the use of an miRNA purification kit, followed by flow-injection analysis using an Exactive mass spectrometer to yield the accurate masses of the sense and antisense strands. Although chromatography and sensitive mass spectrometric analysis of oligonucleotides are still challenging, a method was developed and validated that has adequate sensitivity (limit of detection 0.25-1 nmol mL(-1)) and performance (precision 11-21%, recovery 23-67%) for typical antisense oligonucleotides currently used in clinical studies.

Gamma-spectrometric examination of hot particles emitted during the Chernobyl accident

International Nuclear Information System (INIS)

Balashazy, I.; Szabadine-Szende, G.; Loerinc, M.; Zombori, P.

1987-05-01

Ge(Li) gamma-spectrometric examination of hot particles prepared from air filtered dust of Budapest air after the Chernobyl accident is presented. The method of separating hot particles is described and their concentration in the air is determined. The radioactive isotope composition of hot particles is discussed and compared with that of dust samples. Finally, the inhalation probability and radiation burden of hot particles are evaluated. (author)

DNA origami nanorobot fiber optic genosensor to TMV.

Science.gov (United States)

Torelli, Emanuela; Manzano, Marisa; Srivastava, Sachin K; Marks, Robert S

2018-01-15

In the quest of greater sensitivity and specificity of diagnostic systems, one continually searches for alternative DNA hybridization methods, enabling greater versatility and where possible field-enabled detection of target analytes. We present, herein, a hybrid molecular self-assembled scaffolded DNA origami entity, intimately immobilized via capture probes linked to aminopropyltriethoxysilane, onto a glass optical fiber end-face transducer, thus producing a novel biosensor. Immobilized DNA nanorobots with a switchable flap can then be actuated by a specific target DNA present in a sample, by exposing a hemin/G-quadruplex DNAzyme, which then catalyzes the generation of chemiluminescence, once the specific fiber probes are immersed in a luminol-based solution. Integrating organic nanorobots to inorganic fiber optics creates a hybrid system that we demonstrate as a proof-of-principle can be utilized in specific DNA sequence detection. This system has potential applications in a wide range of fields, including point-of-care diagnostics or cellular in vivo biosensing when using ultrathin fiber optic probes for research purposes. Copyright © 2017 Elsevier B.V. All rights reserved.

Near-Infrared Ag2S Quantum Dots-Based DNA Logic Gate Platform for miRNA Diagnostics.

Science.gov (United States)

Miao, Peng; Tang, Yuguo; Wang, Bidou; Meng, Fanyu

2016-08-02

Dysregulation of miRNA expression is correlated with the development and progression of many diseases. These miRNAs are regarded as promising biomarkers. However, it is challenging to measure these low abundant molecules without employing time-consuming radioactive labeling or complex amplification strategies. Here, we present a DNA logic gate platform for miRNA diagnostics with fluorescence outputs from near-infrared (NIR) Ag2S quantum dots (QDs). Carefully designed toehold exchange-mediated strand displacements with different miRNA inputs occur on a solid-state interface, which control QDs release from solid-state interface to solution, responding to multiplex information on initial miRNAs. Excellent fluorescence emission properties of NIR Ag2S QDs certify the great prospect for amplification-free and sensitive miRNA assay. We demonstrate the potential of this platform by achieving femtomolar level miRNA analysis and the versatility of a series of logic circuits computation.

Neutron activation and mass spectrometric measurement of /sup 129/I

International Nuclear Information System (INIS)

Strebin, R.S. Jr.; Brauer, F.P.; Kaye, J.H.; Rapids, M.S.; Stoffels, J.J.

1987-11-01

An integrated procedure has been developed for measurement of /sup 129/I by neutron activation analysis and mass spectrometry. An iodine isolation procedure previously used for neutron activation has been modified to provide separated iodine suitable for mass spectrometric measurement as well. Agreement between both methods has been achieved within error limits. The measurement limit by each method is about 10/sup 7/ atoms (2 fg) of /sup 129/I. 13 refs,. 4 figs., 1 tab

The fast gamma spectrometric method of the Am-241 determination in Chernobyl restricted zone soils

International Nuclear Information System (INIS)

Gleisberg, B.; Lukachina, V.V.; Kirsenko, V.N.; Tepikin, V.E.; Rajevsky, V.S.; Libman, V.A.; Stoljarevsky, I.P.; Isajev, A.G.

1997-01-01

The known methods of the 241 Am contents determination in environmental objects, as a rule, is based on ion-chromatographic or extraction separation techniques. This approach reflects widespread opinion, that only the α-spectrometric analysis termination is suitable to ensure necessary sensitivity of the overall method of 241 Am actively determination. Really, the minimal detectable activity for such methods is about 0.05 Bq/kg (considering that Am is usually concentrated during separation procedure). However, because of α-spectrometry does not permit to separate the α-peaks of the 241 Am, and 238 Pu, but also in view of high requests to the α-spectrometric specimen purity, the multistage and laborious chemical procedures to separate 241 Am from plutonium radionuclides and other elements (with a thorough control of each separation stage) are needed

Winnowing DNA for rare sequences: highly specific sequence and methylation based enrichment.

Directory of Open Access Journals (Sweden)

Jason D Thompson

Full Text Available Rare mutations in cell populations are known to be hallmarks of many diseases and cancers. Similarly, differential DNA methylation patterns arise in rare cell populations with diagnostic potential such as fetal cells circulating in maternal blood. Unfortunately, the frequency of alleles with diagnostic potential, relative to wild-type background sequence, is often well below the frequency of errors in currently available methods for sequence analysis, including very high throughput DNA sequencing. We demonstrate a DNA preparation and purification method that through non-linear electrophoretic separation in media containing oligonucleotide probes, achieves 10,000 fold enrichment of target DNA with single nucleotide specificity, and 100 fold enrichment of unmodified methylated DNA differing from the background by the methylation of a single cytosine residue.

Winnowing DNA for rare sequences: highly specific sequence and methylation based enrichment.

Science.gov (United States)

Thompson, Jason D; Shibahara, Gosuke; Rajan, Sweta; Pel, Joel; Marziali, Andre

2012-01-01

Rare mutations in cell populations are known to be hallmarks of many diseases and cancers. Similarly, differential DNA methylation patterns arise in rare cell populations with diagnostic potential such as fetal cells circulating in maternal blood. Unfortunately, the frequency of alleles with diagnostic potential, relative to wild-type background sequence, is often well below the frequency of errors in currently available methods for sequence analysis, including very high throughput DNA sequencing. We demonstrate a DNA preparation and purification method that through non-linear electrophoretic separation in media containing oligonucleotide probes, achieves 10,000 fold enrichment of target DNA with single nucleotide specificity, and 100 fold enrichment of unmodified methylated DNA differing from the background by the methylation of a single cytosine residue.

DNA microarray technology in nutraceutical and food safety.

Science.gov (United States)

Liu-Stratton, Yiwen; Roy, Sashwati; Sen, Chandan K

2004-04-15

The quality and quantity of diet is a key determinant of health and disease. Molecular diagnostics may play a key role in food safety related to genetically modified foods, food-borne pathogens and novel nutraceuticals. Functional outcomes in biology are determined, for the most part, by net balance between sets of genes related to the specific outcome in question. The DNA microarray technology offers a new dimension of strength in molecular diagnostics by permitting the simultaneous analysis of large sets of genes. Automation of assay and novel bioinformatics tools make DNA microarrays a robust technology for diagnostics. Since its development a few years ago, this technology has been used for the applications of toxicogenomics, pharmacogenomics, cell biology, and clinical investigations addressing the prevention and intervention of diseases. Optimization of this technology to specifically address food safety is a vast resource that remains to be mined. Efforts to develop diagnostic custom arrays and simplified bioinformatics tools for field use are warranted.

Mass-spectrometric investigation of rare earth acetylacetonates dipivaloylmethanates and their adducts

International Nuclear Information System (INIS)

Gavrishzhuk, E.M.; Dzyubenko, N.G.; Martynenko, L.I.

1985-01-01

Peculiarities of fragmentation of molecular ions of rare earth acetylacetonates and dipivaloylmethanates under mass-spectrometric investigation of these compounds as well as their adducts with o-phenanthroline, α,α'-dipyridyl, triphenylphosphine oxide are considered in the given review. Similar data for identical derivants of some transitions metals are presented. Data on potentials of appearance and dissociation energy of basic ions in mass-spectra of the studied β-diketonates are analyzed

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.

Electronic transport in methylated fragments of DNA

Energy Technology Data Exchange (ETDEWEB)

Almeida, M. L. de; Oliveira, J. I. N.; Lima Neto, J. X.; Gomes, C. E. M.; Fulco, U. L., E-mail: umbertofulco@gmail.com; Albuquerque, E. L. [Departamento de Biofísica e Farmacologia, Universidade Federal do Rio Grande do Norte, 59072-970 Natal-RN (Brazil); Freire, V. N. [Departamento de Física, Universidade Federal do Ceará, 60455-760 Fortaleza, CE (Brazil); Caetano, E. W. S. [Instituto Federal de Educação, Ciência e Tecnologia do Ceará, 60040-531 Fortaleza, CE (Brazil); Moura, F. A. B. F. de; Lyra, M. L. [Instituto de Física, Universidade Federal de Alagoas, 57072-900 Maceió-AL (Brazil)

2015-11-16

We investigate the electronic transport properties of methylated deoxyribonucleic-acid (DNA) strands, a biological system in which methyl groups are added to DNA (a major epigenetic modification in gene expression), sandwiched between two metallic platinum electrodes. Our theoretical simulations apply an effective Hamiltonian based on a tight-binding model to obtain current-voltage curves related to the non-methylated/methylated DNA strands. The results suggest potential applications in the development of novel biosensors for molecular diagnostics.

Electronic transport in methylated fragments of DNA

International Nuclear Information System (INIS)

Almeida, M. L. de; Oliveira, J. I. N.; Lima Neto, J. X.; Gomes, C. E. M.; Fulco, U. L.; Albuquerque, E. L.; Freire, V. N.; Caetano, E. W. S.; Moura, F. A. B. F. de; Lyra, M. L.

2015-01-01

We investigate the electronic transport properties of methylated deoxyribonucleic-acid (DNA) strands, a biological system in which methyl groups are added to DNA (a major epigenetic modification in gene expression), sandwiched between two metallic platinum electrodes. Our theoretical simulations apply an effective Hamiltonian based on a tight-binding model to obtain current-voltage curves related to the non-methylated/methylated DNA strands. The results suggest potential applications in the development of novel biosensors for molecular diagnostics

Spectrometric storage device in VME standard; Spektrometricheskoe zapominayushchee ustrojstvo v standarte VME

Energy Technology Data Exchange (ETDEWEB)

Rezaev, V E

1996-12-31

Paper describes module of histogram Memory Module designed to implement hardware way of accumulation of spectrometric data in systems based on VME standard equipment. Application of HMM module enables to increase throughput of the system for data acquisition and processing as in this case VME bus trunk is not occupied and may be used to control experiment course. 2 figs.

Isothermal multiple displacement amplification: a methodical approach enhancing molecular routine diagnostics of microcarcinomas and small biopsies.

Science.gov (United States)

Mairinger, Fabian D; Walter, Robert Fh; Vollbrecht, Claudia; Hager, Thomas; Worm, Karl; Ting, Saskia; Wohlschläger, Jeremias; Zarogoulidis, Paul; Zarogoulidis, Konstantinos; Schmid, Kurt W

2014-01-01

Isothermal multiple displacement amplification (IMDA) can be a powerful tool in molecular routine diagnostics for homogeneous and sequence-independent whole-genome amplification of notably small tumor samples, eg, microcarcinomas and biopsies containing a small amount of tumor. Currently, this method is not well established in pathology laboratories. We designed a study to confirm the feasibility and convenience of this method for routine diagnostics with formalin-fixed, paraffin-embedded samples prepared by laser-capture microdissection. A total of 250 μg DNA (concentration 5 μg/μL) was generated by amplification over a period of 8 hours with a material input of approximately 25 cells, approximately equivalent to 175 pg of genomic DNA. In the generated DNA, a representation of all chromosomes could be shown and the presence of elected genes relevant for diagnosis in clinical samples could be proven. Mutational analysis of clinical samples could be performed without any difficulty and showed concordance with earlier diagnostic findings. We established the feasibility and convenience of IMDA for routine diagnostics. We also showed that small amounts of DNA, which were not analyzable with current molecular methods, could be sufficient for a wide field of applications in molecular routine diagnostics when they are preamplified with IMDA.

Tumor‐associated DNA mutation detection in individuals undergoing colonoscopy

OpenAIRE

Fleshner, Phillip; Braunstein, Glenn D.; Ovsepyan, Gayane; Tonozzi, Theresa R.; Kammesheidt, Anja

2017-01-01

Abstract The majority of colorectal cancers (CRC) harbor somatic mutations and epigenetic modifications in the tumor tissue, and some of these mutations can be detected in plasma as circulating tumor DNA (ctDNA). Precancerous colorectal lesions also contain many of these same mutations. This study examined plasma for ctDNA from patients undergoing a screening or diagnostic colonoscopy to determine the sensitivity and specificity of the ctDNA panel for detecting CRC and precancerous lesions. T...

Mass spectrometric identification of isoforms of PR proteins in xylem sap of fungus-infected tomato

NARCIS (Netherlands)

Rep, Martijn; Dekker, Henk L.; Vossen, Jack H.; de Boer, Albert D.; Houterman, Petra M.; Speijer, Dave; Back, Jaap W.; de Koster, Chris G.; Cornelissen, Ben J. C.

2002-01-01

The protein content of tomato (Lycopersicon esculentum) xylem sap was found to change dramatically upon infection with the vascular wilt fungus Fusarium oxysporum. Peptide mass fingerprinting and mass spectrometric sequencing were used to identify the most abundant proteins appearing during

Moessbauer spectrometric data acquisition based on FPGA

International Nuclear Information System (INIS)

Zhang Yuan; Li Shimin; Chen Nan; Zhu Jingbo; Xia Yuanfu

2008-01-01

FPGA(Field Programmable Gate Array) is a programmable device with strong logical function and timing control ability. It is extremely potent in acquiring and processing timing signals. By replacing the traditional used SCM (Single-Chip Microcomputer) with FPGA, counting speed of Moessbauer spectrometric data acquisition can be improved markedly with significantly decreased size of the spectrometer. The counter, RAM and RS-232 communication of the module are developed on Altera Cyclone series chip EP1C6T144C8 with Quartus II. EP1C6T144C8 has 5980 logical units accompanied by 92160 bits of memory space. It is so powerful that all needs in data acquisition of the Moessbauer spectrometer can be perfectly satisfied while allowing modifications in functions and parameters. (authors)

Potential of DNA methylation in rectal cancer as diagnostic and prognostic biomarkers

OpenAIRE

Exner, Ruth; Pulverer, Walter; Diem, Martina; Spaller, Lisa; Woltering, Laura; Schreiber, Martin; Wolf, Brigitte; Sonntagbauer, Markus; Schr?der, Fabian; Stift, Judith; Wrba, Fritz; Bergmann, Michael; Weinh?usel, Andreas; Egger, Gerda

2015-01-01

Background: Aberrant DNA methylation is more prominent in proximal compared with distal colorectal cancers. Although a number of methylation markers were identified for colon cancer, yet few are available for rectal cancer. Methods: DNA methylation differences were assessed by a targeted DNA microarray for 360 marker candidates between 22 fresh frozen rectal tumour samples and 8 controls and validated by microfluidic high-throughput and methylation-sensitive qPCR in fresh frozen and formalin-...

Detection of Non-Amplified Genomic DNA

CERN Document Server

Corradini, Roberto

2012-01-01

This book offers a state-of-the-art overview on non amplified DNA detection methods and provides chemists, biochemists, biotechnologists and material scientists with an introduction to these methods. In fact all these fields have dedicated resources to the problem of nucleic acid detection, each contributing with their own specific methods and concepts. This book will explain the basic principles of the different non amplified DNA detection methods available, highlighting their respective advantages and limitations. The importance of non-amplified DNA sequencing technologies will be also discussed. Non-amplified DNA detection can be achieved by adopting different techniques. Such techniques have allowed the commercialization of innovative platforms for DNA detection that are expected to break into the DNA diagnostics market. The enhanced sensitivity required for the detection of non amplified genomic DNA has prompted new strategies that can achieve ultrasensitivity by combining specific materials with specifi...

Mass spectrometric methods for trace analysis of metals

International Nuclear Information System (INIS)

Bahr, U.; Schulten, H.R.

1981-01-01

A brief outline is given of the principles of mass spectrometry (MS) and the fundamentals of qualitative and quantitative mass spectrometric analysis emphasizing recent developments and results. Classical methods of the analysis of solids, i.e. spark-source MS and thermal ionization MS, as well as recent methods of metal analysis are described. Focal points in this survey of recently developed techniques include secondary ion MS, laser probe MS, plasma ion source MS, gas discharge MS and field desorption MS. Here, a more detailed description is given and the merits of these emerging methods are discussed more explicitly. In particular, the results of the field desorption techniques in elemental analyses are reviewed and critically evaluated

DNA probes for distinguishing Psychodopygus wellcomei from Psychodopygus complexus (Diptera: Psychodidae

Directory of Open Access Journals (Sweden)

P. D. Ready

1991-03-01

Full Text Available Genomic DNA fragments from males of Psychodopygus wellcomei were isolated and shown to be useful as sensitive diagnostic probles for positively separting individuals of this species from those of Ps. complexus. These two members of the Ps. squamiventris series are found sympatrically in foci of cutaneous leishmaniasis in the hill forests of southern Pará State. Of the two species, only Ps. welcomei is thought to be an important vector of Leishmania braziliensis sensu stricto, buth this is based on circumstantial evidence because of the difficulties of identifying female sandflies wothin the series. The diagnostic probes were isolated from a library of Ps. wellcomei built by ligationg short fragments of Sau 3A-resistricted, genomic DNA into the plasmid vector PUC 18. Differential screening of 1316 library clones with total genomic DNA of Ps. Wellcomei and Ps. complexus identified 5 recombinants, with cross-hybridizing inserts of repetitive DNA, that showed strong specificity for Ps. wellcomei. As little as 0.4% of the DNA extracted from an individual sandfly (=ca. 0.5 namograms was specifically detected. The diagnostic probes were used to identify as Ps. wellcomei a wild-caught female sandfly found infected with L. braziliensis s.s., providing only the second positive association between these two species.

A fluorescence sedimentation assay for dsDNA antibodies

DEFF Research Database (Denmark)

Duus, K; Draborg, A H; Güven, E

2017-01-01

The Farr assay is a radioimmunoassay (RIA) for dsDNA antibodies, based on antibody precipitation using ammonium sulphate and quantification using radio-labelled dsDNA. The RIA-Farr assay offers outstanding clinical specificity and sensitivity for systemic lupus erythematosus (SLE) compared to other...... on precipitation with polyethylene glycol (PEG) and fluorescence of EvaGreen intercalated in dsDNA as detection principle. As dsDNA antibodies are quantified using fluorescence, the disadvantages of working with radioactivity are eliminated. The Fluoro-Farr assay was developed and validated, and the diagnostic...

Improved molecular detection of Babesia infections in animals using a novel quantitative real-time PCR diagnostic assay targeting mitochondrial DNA.

Science.gov (United States)

Qurollo, Barbara A; Archer, Nikole R; Schreeg, Megan E; Marr, Henry S; Birkenheuer, Adam J; Haney, Kaitlin N; Thomas, Brittany S; Breitschwerdt, Edward B

2017-03-07

Babesiosis is a protozoal, tick transmitted disease found worldwide in humans, wildlife and domesticated animals. Commonly used approaches to diagnose babesiosis include microscopic examination of peripheral blood smears, detection of circulating antibodies and PCR. To screen and differentiate canine Babesia infections many PCR assays amplify the 18S rRNA gene. These sequences contain hypervariable regions flanked by highly conserved regions allowing for amplification of a broad-range of Babesia spp. However, differences in the 18S rRNA gene sequence of distantly related clades can make it difficult to design assays that will amplify all Babesia species while excluding the amplification of other eukaryotes. By targeting Babesia mitochondrial genome (mtDNA), we designed a novel three primer qPCR with greater sensitivity and broader screening capabilities to diagnose and differentiate Babesia spp. Using 13 Babesia mtDNA sequences, a region spanning two large subunit rRNA gene fragments (lsu5-lsu4) was aligned to design three primers for use in a qPCR assay (LSU qPCR) capable of amplifying a wide range of Babesia spp. Plasmid clones were generated and used as standards to determine efficiency, linear dynamic range and analytical sensitivity. Animals naturally infected with vector-borne pathogens were tested retrospectively and prospectively to determine relative clinical sensitivity and specificity by comparing the LSU qPCR to an established 18S rDNA qPCR. The LSU qPCR efficiencies ranged between 92 and 100% with the limit of detection at five copies/reaction. The assay did not amplify mammalian host or other vector-borne pathogen gDNA except Cytauxzoon felis (a feline protozoal pathogen). The LSU qPCR assay amplified 12 different Babesia. sp. and C. felis from 31/31 (100%) archived samples, whereas the 18S qPCR amplified only 26/31 (83.9%). By prospective analysis, 19/394 diagnostic accessions (4.8%) were LSU qPCR positive, compared to 11/394 (2.8%) 18S rDNA q

Mass spectrometric confirmation criterion for product-ion spectra generated in flow-injection analysis. Environmental application

NARCIS (Netherlands)

Geerdink, R.B.; Niessen, W.M.A.; Brinkman, U.A.T.

2001-01-01

The suitability of a confirmation criterion recently recommended in the Netherlands for gas chromatography with mass spectrometric detection (GC-MS), was evaluated for flow-injection analysis (FIA) with atmospheric pressure chemical ionisation MS-MS detection. The main feature of the criterion is

AU2EU : Privacy-preserving matching of DNA sequences

NARCIS (Netherlands)

Ignatenko, T.; Petkovic, M.; Naccache, D.; Sauveron, D.

2014-01-01

Advances in DNA sequencing create new opportunities for the use of DNA data in healthcare for diagnostic and treatment purposes, but also in many other health and well-being services. This brings new challenges with regard to the protection and use of this sensitive data. Thus, special technical

Circulating tumour DNA methylation markers for diagnosis and prognosis of hepatocellular carcinoma

Science.gov (United States)

Xu, Rui-Hua; Wei, Wei; Krawczyk, Michal; Wang, Wenqiu; Luo, Huiyan; Flagg, Ken; Yi, Shaohua; Shi, William; Quan, Qingli; Li, Kang; Zheng, Lianghong; Zhang, Heng; Caughey, Bennett A.; Zhao, Qi; Hou, Jiayi; Zhang, Runze; Xu, Yanxin; Cai, Huimin; Li, Gen; Hou, Rui; Zhong, Zheng; Lin, Danni; Fu, Xin; Zhu, Jie; Duan, Yaou; Yu, Meixing; Ying, Binwu; Zhang, Wengeng; Wang, Juan; Zhang, Edward; Zhang, Charlotte; Li, Oulan; Guo, Rongping; Carter, Hannah; Zhu, Jian-Kang; Hao, Xiaoke; Zhang, Kang

2017-11-01

An effective blood-based method for the diagnosis and prognosis of hepatocellular carcinoma (HCC) has not yet been developed. Circulating tumour DNA (ctDNA) carrying cancer-specific genetic and epigenetic aberrations may enable a noninvasive `liquid biopsy' for diagnosis and monitoring of cancer. Here, we identified an HCC-specific methylation marker panel by comparing HCC tissue and normal blood leukocytes and showed that methylation profiles of HCC tumour DNA and matched plasma ctDNA are highly correlated. Using cfDNA samples from a large cohort of 1,098 HCC patients and 835 normal controls, we constructed a diagnostic prediction model that showed high diagnostic specificity and sensitivity (P < 0.001) and was highly correlated with tumour burden, treatment response, and stage. Additionally, we constructed a prognostic prediction model that effectively predicted prognosis and survival (P < 0.001). Together, these findings demonstrate in a large clinical cohort the utility of ctDNA methylation markers in the diagnosis, surveillance, and prognosis of HCC.

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.

Retrospective detection of exposure to organophosphorus anti-cholinesterases: Mass spectrometric analysis of phosphylated human butyrylcholinesterase

NARCIS (Netherlands)

Fidder, A.; Hulst, A.G.; Noort, D.; Ruiter, R. de; Schans, M.J. van der; Benschop, H.P.; Langenberg, J.P.

2002-01-01

In this paper a novel and general procedure is presented for detection of organophosphate-inhibited human butyrylcholinesterase (HuBuChE), which is based on electrospray tandem mass spectrometric analysis of phosphylated nonapeptides obtained after pepsin digestion of the enzyme. The utility of this

Malignant Catarrhal Fever: Understanding Molecular Diagnostics in Context of Epidemiology

Directory of Open Access Journals (Sweden)

Hong Li

2011-10-01

Full Text Available Malignant catarrhal fever (MCF is a frequently fatal disease, primarily of ruminants, caused by a group of gammaherpesviruses. Due to complexities of pathogenesis and epidemiology in various species, which are either clinically-susceptible or reservoir hosts, veterinary clinicians face significant challenges in laboratory diagnostics. The recent development of specific assays for viral DNA and antibodies has expanded and improved the inventory of laboratory tests and opened new opportunities for use of MCF diagnostics. Issues related to understanding and implementing appropriate assays for specific diagnostic needs must be addressed in order to take advantage of molecular diagnostics in the laboratory.

Mass spectrometric study of Nd2S3 vaporization

International Nuclear Information System (INIS)

Fenochka, B.V.

1987-01-01

The authors conduct a mass-spectrometric study of neodymium(III) sulfide vaporization. The chemical composition of the samples was stoichiometric and the samples were vaporized from tantalum effusion cells. When the vapor over Nd 2 S 3 is ionized by electrons the mass spectra shows monovalent cations of Nd, S, NdS, and NdO. The enthalpy of vaporization if Nd atoms from Nd 2 S 3 at average experimental temperatures and the standard enthalpy of reaction is shown. Also presented is the enthalpy of vaporization of NdS molecules from Nd 2 S 3 at average experimental temperatures and the standard enthalpy of reaction

On the Origin of Microheterogeneity : A Mass Spectrometric Study of Dimethyl Sulfoxide-Water Binary Mixture

NARCIS (Netherlands)

Shin, Dong Nam; Wijnen, Jan W.; Engberts, Jan B.F.N.; Wakisaka, Akihiro

2001-01-01

We have studied the microscopic solvent structure of dimethyl sulfoxide-water mixtures and its influence on the solvation structure of solute from a clustering point of View, by means of a specially designed mass spectrometric system. It was observed that the propensity to the cluster formation is

DNA-based approaches to identify forest fungi in Pacific Islands: A pilot study

Science.gov (United States)

Anna E. Case; Sara M. Ashiglar; Phil G. Cannon; Ernesto P. Militante; Edwin R. Tadiosa; Mutya Quintos-Manalo; Nelson M. Pampolina; John W. Hanna; Fred E. Brooks; Amy L. Ross-Davis; Mee-Sook Kim; Ned B. Klopfenstein

2013-01-01

DNA-based diagnostics have been successfully used to characterize diverse forest fungi (e.g., Hoff et al. 2004, Kim et al. 2006, Glaeser & Lindner 2011). DNA sequencing of the internal transcribed spacer (ITS) and large subunit (LSU) regions of nuclear ribosomal DNA (rDNA) has proved especially useful (Sonnenberg et al. 2007, Seifert 2009, Schoch et al. 2012) for...

Validating DNA barcodes: A non-destructive extraction protocol enables simultaneous vouchering of DNA and morphological vouchers

Science.gov (United States)

Morphology-based keys support accurate identification of many taxa. However, identification can be difficult for taxa that are not well studied, very small, members of cryptic species complexes, or represented by immature stages. For such cases, DNA barcodes may provide diagnostic characters. Ecolog...

A Ribeiroia spp. (Class: Trematoda) - Specific PCR-based diagnostic

Science.gov (United States)

Reinitz, David M.; Yoshino, T.P.; Cole, Rebecca A.

2007-01-01

Increased reporting of amphibian malformations in North America has been noted with concern in light of reports that amphibian numbers and species are declining worldwide. Ribeiroia ondatrae has been shown to cause a variety of types of malformations in amphibians. However, little is known about the prevalence of R. ondatrae in North America. To aid in conducting field studies of Ribeiroia spp., we have developed a polymerase chain reaction (PCR)-based diagnostic. Herein, we describe the development of an accurate, rapid, simple, and cost-effective diagnostic for detection of Ribeiroia spp. infection in snails (Planorbella trivolvis). Candidate oligonucleotide primers for PCR were designed via DNA sequence analyses of multiple ribosomal internal transcribed spacer-2 regions from Ribeiroia spp. and Echinostoma spp. Comparison of consensus sequences determined from both genera identified areas of sequence potentially unique to Ribeiroia spp. The PCR reliably produced a diagnostic 290-base pair (bp) product in the presence of a wide concentration range of snail or frog DNA. Sensitivity was examined with DNA extracted from single R. ondatrae cercaria. The single-tube PCR could routinely detect less than 1 cercariae equivalent, because DNA isolated from a single cercaria could be diluted at least 1:50 and still yield a positive result via gel electrophoresis. An even more sensitive nested PCR also was developed that routinely detected 100 fg of the 290-bp fragment. The assay did not detect furcocercous cercariae of certain Schistosomatidae, Echinostoma sp., or Sphaeridiotrema globulus nor adults of Clinostomum sp. or Cyathocotyle bushiensis. Field testing of 137 P. trivolvis identified 3 positives with no overt environmental cross-reactivity, and results concurred with microscopic examinations in all cases. ?? American Society of Parasitologists 2007.

Protected DNA strand displacement for enhanced single nucleotide discrimination in double-stranded DNA.

Science.gov (United States)

Khodakov, Dmitriy A; Khodakova, Anastasia S; Huang, David M; Linacre, Adrian; Ellis, Amanda V

2015-03-04

Single nucleotide polymorphisms (SNPs) are a prime source of genetic diversity. Discriminating between different SNPs provides an enormous leap towards the better understanding of the uniqueness of biological systems. Here we report on a new approach for SNP discrimination using toehold-mediated DNA strand displacement. The distinctiveness of the approach is based on the combination of both 3- and 4-way branch migration mechanisms, which allows for reliable discrimination of SNPs within double-stranded DNA generated from real-life human mitochondrial DNA samples. Aside from the potential diagnostic value, the current study represents an additional way to control the strand displacement reaction rate without altering other reaction parameters and provides new insights into the influence of single nucleotide substitutions on 3- and 4-way branch migration efficiency and kinetics.

Aspects of optical fibers and spectrometric sensors in chemical process and industrial environments

International Nuclear Information System (INIS)

Boisde, G.; Perez, J.J.

1988-01-01

For on-line control, the two alternatives of automatic sample transfer and in situ remote analysis are discussed. New concepts are emerging from the possibilities offered by optical fibers. Absorption in the visible, UV and IR, fluorescence and Raman spectrometric techniques are examined. The state of the art of optodes and devices in chemical process control are given, with some examples of applications in nuclear plants

An improved method of DNA extraction from plants for pathogen ...

African Journals Online (AJOL)

Polymerase chain reaction (PCR)-based applications in plant molecular biology and molecular diagnostics for plant pathogens require good quality DNA for reliable and reproducible results. Leaf tissue is often the choice for DNA extraction, but the use of other sources such as tubers, stems, or seeds, is not uncommon.

In situ gamma-ray spectrometric analysis of radionuclide distributions at a commercial shallow land burial site

International Nuclear Information System (INIS)

Kirby, L.J.; Campbell, R.M.

1984-10-01

Gamma-ray spectrometric analysis conducted at the Maxey Flats, Kentucky (USA) shallow land burial site confirmed that the waste radionuclides have been retained largely within the restricted area of the burial site. Concentrations of 137 Cs and 60 Co were comparable with those originating from global fallout and lower than concentrations measured in several other areas having similar rainfall. In-situ spectrometric analyses, corroborated by soil sample and vegetation analyses, indicate that the site has influenced 60 Co levels slightly in the west drainage channel, but 137 Cs did not originate from the site. Concentrations of 60 Co, 90 Sr and 137 Cs determined in subsurface soils by well logging techniques confirmed that subsurface migration of waste-derived radionuclides to points outside the restricted area has not been a significant source of contamination of the environs adjacent to the site. 8 references, 8 figures

Challenges and recent advances in mass spectrometric imaging of neurotransmitters

Science.gov (United States)

Gemperline, Erin; Chen, Bingming; Li, Lingjun

2014-01-01

Mass spectrometric imaging (MSI) is a powerful tool that grants the ability to investigate a broad mass range of molecules, from small molecules to large proteins, by creating detailed distribution maps of selected compounds. To date, MSI has demonstrated its versatility in the study of neurotransmitters and neuropeptides of different classes toward investigation of neurobiological functions and diseases. These studies have provided significant insight in neurobiology over the years and current technical advances are facilitating further improvements in this field. neurotransmitters, focusing specifically on the challenges and recent Herein, we advances of MSI of neurotransmitters. PMID:24568355

DNA Nanobiosensors: An Outlook on Signal Readout Strategies

Directory of Open Access Journals (Sweden)

Arun Richard Chandrasekaran

2017-01-01

Full Text Available A suite of functionalities and structural versatility makes DNA an apt material for biosensing applications. DNA-based biosensors are cost-effective and sensitive and have the potential to be used as point-of-care diagnostic tools. Along with robustness and biocompatibility, these sensors also provide multiple readout strategies. Depending on the functionality of DNA-based biosensors, a variety of output strategies have been reported: fluorescence- and FRET-based readout, nanoparticle-based colorimetry, spectroscopy-based techniques, electrochemical signaling, gel electrophoresis, and atomic force microscopy.

DNA barcoding for biosecurity: case studies from the UK plant protection program.

Science.gov (United States)

Hodgetts, Jennifer; Ostojá-Starzewski, Jozef C; Prior, Thomas; Lawson, Rebecca; Hall, Jayne; Boonham, Neil

2016-11-01

Since its conception, DNA barcoding has seen a rapid uptake within the research community. Nevertheless, as with many new scientific tools, progression towards the point of routine deployment within diagnostic laboratories has been slow. In this paper, we discuss the application of DNA barcoding in the Defra plant health diagnostic laboratories, where DNA barcoding is used primarily for the identification of invertebrate pests. We present a series of case studies that demonstrate the successful application of DNA barcoding but also reveal some potential limitations to expanded use. The regulated plant pest, Bursephalenchus xylophilus, and one of its vectors, Monochamus alternatus, were found in dining chairs. Some traded wood products are potentially high risk, allowing the movement of longhorn beetles; Trichoferus campestris, Leptura quadrifasciata, and Trichoferus holosericeus were found in a wooden cutlery tray, a railway sleeper, and a dining chair, respectively. An outbreak of Meloidogyne fallax was identified in Allium ampeloprasum and in three weed species. Reference sequences for UK native psyllids were generated to enable the development of rapid diagnostics to be used for monitoring following the release of Aphalara itadori as a biological control agent for Fallopia japonica.

Mass Spectrometric Analysis of Synthetic Organic Pigments.

Science.gov (United States)

Sugaya, Naeko; Takahashi, Mitsuko; Sakurai, Katsumi; Tanaka, Nobuko; Okubo, Ichiro; Kawakami, Tsuyoshi

2018-04-18

Though synthetic organic colorants are used in various applications nowadays, there is the concern that impurities by-produced during the manufacturing and degradation products in some of these colorants are persistent organic pollutants and carcinogens. Thus, it is important to identify the synthetic organic colorants in various products, such as commercial paints, ink, cosmetics, food, textile, and plastics. Dyes, which are soluble in water and other solvents, could be analyzed by chromatographic methods. In contrast, it is difficult to analyze synthetic organic pigments by these methods because of their insolubility. This review is an overview of mass spectrometric analysis of synthetic organic pigments by various ionization methods. We highlight a recent study of textile samples by atmospheric pressure solid analysis probe MS. Furthermore, the mass spectral features of synthetic organic pigments and their separation from other components such as paint media and plasticizers are discussed.

Maintenance of stability in γ spectrometric system of low active and environmental samples - a practical experience

International Nuclear Information System (INIS)

Ravishankar, R.; Bandyopadhyay, T.; Sarkar, P.K.

2011-01-01

Particle Accelerators are becoming part of the society with more and more medical and Industrial types are added every year in addition to research type of accelerators. The outflow of materials to the public domain from such accelerator facilities need to checked carefully and must be released after ensuring the activities of such materials should not exceed the regulatory limits. Health Physics Unit, VECC is involved in analyzing food product samples, seized samples which are suspected to contain Uranium etc and other environmental samples in addition to analyzing radioactive materials evolved from Operational Health Physics work. Most of these analyses involve γ Spectrometric Systems of high efficiency and high resolution types. The efficacy of the analysis and results depends on various parameters of the spectrometric system. The electrical noise from the power supply system and other noises picked up, even in the range of a few milli volts range, have been found to affect the stability of the system. These effects may not be present initially during installation but may creep in due course due to various reasons including weather conditions, wear and tear etc. Unless these problems are attended in regular intervals, the stability of the spectrometric systems and hence the results of analysis of the low active and environmental samples, will not be satisfactory. The work describes the practical problems faced by Health Physics Unit, the methods employed in identifying the problems, the necessary remedial measures taken, the final outcome in the stability and the procedures framed in order to avoid in future. (author)

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.

Modern spectrometric methods for the analysis of labelled compounds

International Nuclear Information System (INIS)

Kaspersen, F.M.; Funke, C.W.; Wagenaars, G.N.; Jacobs, P.L.

1988-01-01

A proper analysis of chemical compounds should give information about the chemical identity (not only the structure but also enantiomeric form), the chemical purity and chemical composition (e.g. giving information about counter-ions, solvents of crystallization). For labelled compounds information is also needed about isotopic purity (defined as the % of isotope present in the compound), the position/distribution of the isotope in the molecule and degree of labelling/specific activity. In the past ten years the possibilities for spectrometric analyses of labelled compounds have increased enormously and this chapter will give an overview of these methods with the exception of (radio)chromatography that will be dealt with in another chapter. (author)

5th colloquium on atomic spectrometric trace analysis

International Nuclear Information System (INIS)

Welz, B.

1989-01-01

This book deals with apparatus, use-oriented and theoretical aspects of trace analysis and spectroscopy. General articles are concerned with the analysis of environmentally relevant samples; a comparison of modern spectroscopic techniques, the coupling of hydride production, chromatography and spectrometry; chemical modifiers for graphite tube furnace atomic absorption spectroscopy (AAS), and possible applications of flow injection to atomic spectrometric trace analysis - one of the outstanding subjects of the colloquium. About one quarter of the 85 contributions deals with new techniques including flow injection. Other priority subjects are the theory and application of graphite tube furnace AAS, and a comparison between different dissolution methods and direct solid analysis. Medicine and toxicology, analysis of biological materials and environmentally relevant samples are in the foreground of use-oriented papers. (orig./BBR) [de

Radiation induced DNA double-strand breaks in radiology; Strahleninduzierte DNA-Doppelstrangbrueche in der Radiologie

Energy Technology Data Exchange (ETDEWEB)

Kuefner, M.A. [Dornbirn Hospital (Austria). Dept. of Radiology; Brand, M.; Engert, C.; Uder, M. [Erlangen University Hospital (Germany). Dept. of Radiology; Schwab, S.A. [Radiologis, Oberasbach (Germany)

2015-10-15

Shortly after the discovery of X-rays, their damaging effect on biological tissues was observed. The determination of radiation exposure in diagnostic and interventional radiology is usually based on physical measurements or mathematical algorithms with standardized dose simulations. γ-H2AX immunofluorescence microscopy is a reliable and sensitive method for the quantification of radiation induced DNA double-strand breaks (DSB) in blood lymphocytes. The detectable amount of these DNA damages correlates well with the dose received. However, the biological radiation damage depends not only on dose but also on other individual factors like radiation sensitivity and DNA repair capacity. Iodinated contrast agents can enhance the x-ray induced DNA damage level. After their induction DSB are quickly repaired. A protective effect of antioxidants has been postulated in experimental studies. This review explains the principle of the γ-H2AX technique and provides an overview on studies evaluating DSB in radiologic examinations.

Importance of the efficiency of double-stranded DNA formation in cDNA synthesis for the imprecision of microarray expression analysis.

Science.gov (United States)

Thormar, Hans G; Gudmundsson, Bjarki; Eiriksdottir, Freyja; Kil, Siyoen; Gunnarsson, Gudmundur H; Magnusson, Magnus Karl; Hsu, Jason C; Jonsson, Jon J

2013-04-01

The causes of imprecision in microarray expression analysis are poorly understood, limiting the use of this technology in molecular diagnostics. Two-dimensional strandness-dependent electrophoresis (2D-SDE) separates nucleic acid molecules on the basis of length and strandness, i.e., double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), and RNA·DNA hybrids. We used 2D-SDE to measure the efficiency of cDNA synthesis and its importance for the imprecision of an in vitro transcription-based microarray expression analysis. The relative amount of double-stranded cDNA formed in replicate experiments that used the same RNA sample template was highly variable, ranging between 0% and 72% of the total DNA. Microarray experiments showed an inverse relationship between the difference between sample pairs in probe variance and the relative amount of dsDNA. Approximately 15% of probes showed between-sample variation (P cDNA synthesized can be an important component of the imprecision in T7 RNA polymerase-based microarray expression analysis. © 2013 American Association for Clinical Chemistry

Bamboo tea: reduction of taxonomic complexity and application of DNA diagnostics based on rbcL and matK sequence data

Science.gov (United States)

Häser, Annette

2016-01-01

Background Names used in ingredient lists of food products are trivial and in their nature rarely precise. The most recent scientific interpretation of the term bamboo (Bambusoideae, Poaceae) comprises over 1,600 distinct species. In the European Union only few of these exotic species are well known sources for food ingredients (i.e., bamboo sprouts) and are thus not considered novel foods, which would require safety assessments before marketing of corresponding products. In contrast, the use of bamboo leaves and their taxonomic origin is mostly unclear. However, products containing bamboo leaves are currently marketed. Methods We analysed bamboo species and tea products containing bamboo leaves using anatomical leaf characters and DNA sequence data. To reduce taxonomic complexity associated with the term bamboo, we used a phylogenetic framework to trace the origin of DNA from commercially available bamboo leaves within the bambusoid subfamily. For authentication purposes, we introduced a simple PCR based test distinguishing genuine bamboo from other leaf components and assessed the diagnostic potential of rbcL and matK to resolve taxonomic entities within the bamboo subfamily and tribes. Results Based on anatomical and DNA data we were able to trace the taxonomic origin of bamboo leaves used in products to the genera Phyllostachys and Pseudosasa from the temperate “woody” bamboo tribe (Arundinarieae). Currently available rbcL and matK sequence data allow the character based diagnosis of 80% of represented bamboo genera. We detected adulteration by carnation in four of eight tea products and, after adapting our objectives, could trace the taxonomic origin of the adulterant to Dianthus chinensis (Caryophyllaceae), a well known traditional Chinese medicine with counter indications for pregnant women. PMID:27957401

DNA-informed breeding of rosaceous crops: promises, progress and prospects

Science.gov (United States)

Peace, Cameron P

2017-01-01

Crops of the Rosaceae family provide valuable contributions to rural economies and human health and enjoyment. Sustained solutions to production challenges and market demands can be met with genetically improved new cultivars. Traditional rosaceous crop breeding is expensive and time-consuming and would benefit from improvements in efficiency and accuracy. Use of DNA information is becoming conventional in rosaceous crop breeding, contributing to many decisions and operations, but only after past decades of solved challenges and generation of sufficient resources. Successes in deployment of DNA-based knowledge and tools have arisen when the ‘chasm’ between genomics discoveries and practical application is bridged systematically. Key steps are establishing breeder desire for use of DNA information, adapting tools to local breeding utility, identifying efficient application schemes, accessing effective services in DNA-based diagnostics and gaining experience in integrating DNA information into breeding operations and decisions. DNA-informed germplasm characterization for revealing identity and relatedness has benefitted many programs and provides a compelling entry point to reaping benefits of genomics research. DNA-informed germplasm evaluation for predicting trait performance has enabled effective reallocation of breeding resources when applied in pioneering programs. DNA-based diagnostics is now expanding from specific loci to genome-wide considerations. Realizing the full potential of this expansion will require improved accuracy of predictions, multi-trait DNA profiling capabilities, streamlined breeding information management systems, strategies that overcome plant-based features that limit breeding progress and widespread training of current and future breeding personnel and allied scientists. PMID:28326185

DIAGNOSTIC PROBLEMS OF MUCOVISCIDOSIS AND WAYS OF SOLUTION IN RUSSIA

Directory of Open Access Journals (Sweden)

A. A. Baranov

2014-01-01

Full Text Available Mucoviscidosis is a monogenic autosomal recessive caused by the CFTR gene mutations and characterized by pronounced genetic heterogeneity and clinical polymorphism, which emphasizes the need in comprehensive diagnosis and molecular-genetic verification of the final diagnosis. Quality and duration of a mucoviscidosis patient depend on early diagnosis and timely adequate therapy. The article presents mucoviscidosis diagnostic methods and protocols and capabilities of the modern molecular-genetic pathological diagnosis; a review of DNA diagnostic sets has been performed. As sets of some of the genes typical to the Russian population have not been registered in the Russian Federation, mutations in 20% of the patients cannot be specified. In order to solve this problem it is necessary to develop DNA diagnostic sets specific for the Russian Federation, ensure genetic diagnosis of the disease, including sequencing methods at the expense of the federal budget, increase the amount of information and improve quality of teaching this discipline to  students of medial universities and at postgraduate courses for appropriate specialists. 

Diagnostic methods and techniques in cervical cancer prevention Part II: Molecular diagnostics of HPV infection

Directory of Open Access Journals (Sweden)

Adriana Vince,

2010-02-01

Full Text Available Clinical diagnostics of HPV infection is based on analytically andclinically validated assays for qualitative detection of HPV DNAfrom high risk genotypes. New generation of HPV DNA assayscombines qualitative detection of 12 high-risk HPV genotypeswith HPV-16 and HPV-18 genotyping. New generation of HPVmolecular assays designed to increase clinical specificity of moleculartesting is based on detection of mRNA for E6 and E7.

Comparison of accelerator mass spectrometric measurement with liquid scintillation counting measurement for the determination of 14C in environmental samples

International Nuclear Information System (INIS)

Yasuike, Kaeko; Yamada, Yoshimune; Amano, Hikaru

2010-01-01

The concentrations of organically-bound 14 C in tree-ring cellulose of a Japanese Black Pine grown in Shika-machi (37.0 deg. N, 136.8 deg. E) and those of a Japanese Cedar grown in Kanazawa (36.5 deg. N, 136.7 deg. E), Japan, were analyzed for the ring-years from 1989 to 1998 by the accelerator mass spectrometric measurement. The results were compared with those of the same samples analyzed by the liquid scintillation counting measurement to determine the reliability of liquid scintillation counting measurement. An important result of this study is that the sensitivity and reproducibility of accelerator mass spectrometric measurement was almost equal to that of liquid scintillation counting measurement.

Isothermal multiple displacement amplification: a methodical approach enhancing molecular routine diagnostics of microcarcinomas and small biopsies

Directory of Open Access Journals (Sweden)

Mairinger FD

2014-08-01

Full Text Available Fabian D Mairinger,1 Robert FH Walter,2 Claudia Vollbrecht,3 Thomas Hager,1 Karl Worm,1 Saskia Ting,1 Jeremias Wohlschläger,1 Paul Zarogoulidis,4 Konstantinos Zarogoulidis,4 Kurt W Schmid1 1Institute of Pathology, 2Ruhrlandklinik, West German Lung Center, University Hospital Essen, Essen, 3Institute of Pathology, University Hospital Cologne, Cologne, Germany; 4Pulmonary Department, Oncology Unit, G Papanikolaou General Hospital, Aristotle University of Thessaloniki, Thessaloniki, Greece Background and methods: Isothermal multiple displacement amplification (IMDA can be a powerful tool in molecular routine diagnostics for homogeneous and sequence-independent whole-genome amplification of notably small tumor samples, eg, microcarcinomas and biopsies containing a small amount of tumor. Currently, this method is not well established in pathology laboratories. We designed a study to confirm the feasibility and convenience of this method for routine diagnostics with formalin-fixed, paraffin-embedded samples prepared by laser-capture microdissection. Results: A total of 250 µg DNA (concentration 5 µg/µL was generated by amplification over a period of 8 hours with a material input of approximately 25 cells, approximately equivalent to 175 pg of genomic DNA. In the generated DNA, a representation of all chromosomes could be shown and the presence of elected genes relevant for diagnosis in clinical samples could be proven. Mutational analysis of clinical samples could be performed without any difficulty and showed concordance with earlier diagnostic findings. Conclusion: We established the feasibility and convenience of IMDA for routine diagnostics. We also showed that small amounts of DNA, which were not analyzable with current molecular methods, could be sufficient for a wide field of applications in molecular routine diagnostics when they are preamplified with IMDA. Keywords: isothermal multiple displacement amplification, isothermal, whole

[REAL TIME POLYMERASE CHAIN REACTION IN TULAREMIA LABORATORY DIAGNOSTICS].

Science.gov (United States)

Kormilitsyna, M I; Mescheryakova, I S; Mikhailova, T V; Dobrovolsky, A A

2015-01-01

Enhancement of tularemia laboratory diagnostics by F. tularensis DNA determination in blood sera of patients using real time polymerase chain reaction (RT-PCR). 39 blood sera of patients obtained during transmissive epidemic outbreak of tularemia in Khanty-Mansiysk in 2013 were studied in agglutination reaction, passive hemagglutination, RT-PCR. Specific primers and fluorescent probes were used: ISFTu2F/R+ISFTu2P, Tu14GF/R+tul4-PR2. Advantages of using RT-PCR for early diagnostics of tularemia, when specific antibodies are not detected using traditional immunologic methods, were established. Use of a combination of primers and ISFTu2F/R+ISFTu2P probe allowed to detect F. tularensis DNA in 100% of sera, whereas Tul4G F/R+tul4-PR2 combination--92% of sera. The data were obtained when DNA was isolated from sera using "Proba Rapid" express method. Clinical-epidemiologic diagnosis oftularemia was confirmed by both immune-serologic and RT-PCR methods when sera were studied 3-4 weeks after the onset of the disease. RT-PCR with ISFTu2F/R primers and fluorescent probe ISFTu2P, having high sensitivity and specificity, allows to determine F. tularensis DNA in blood sera of patients at both the early stage and 3-4 weeks after the onset of the disease.

The dilemma of diagnostic testing for Prader-Willi syndrome

Science.gov (United States)

Hung, Dorothy

2017-01-01

Although Prader-Willi syndrome (PWS) is a well-described clinical dysmorphic syndrome, DNA testing is required for a definitive diagnosis. A definitive diagnosis can be made in approximately 99% of cases using DNA testing; there are a number of DNA tests that can be used for this purpose, although there is no set standard algorithm of testing. The dilemma arises because of the complex genetic mechanisms at the basis of PWS, which need to be elucidated. To establish the molecular mechanism with a complete work up, involves at least 2 tests. Here we discuss the commonly used tests currently available and suggest a cost—effective approach to diagnostic testing. PMID:28164030

Construction of recombinant DNA clone for bovine viral diarrhea virus

International Nuclear Information System (INIS)

Yeo, S.G.; Cho, H.J.; Masri, S.A.

1992-01-01

Molecular cloning was carried out on the Danish strain of bovine viral diarrhea virus (BVDV) to construct strategy for the diagnostic tools and effective vaccine of BVD afterwards. A recombinant DNA clone (No. 29) was established successfully from cDNA for viral RNA tailed with adenine homopolymer at 3 -end. 32 P-labeled DNA probes of 300~1, 800bp fragments, originating from the clone 29, directed specific DNA-RNA hybridization results with BVDV RNA. Recombinant DNA of the clone 29 was about 5,200bp representing 41.6% of the full length of Danish strain's RNA, and restriction sites were recognized for EooR I, Sst I, Hind III and Pst I restriction enzymes in the DNA fragment

The AS-76 interlaboratory experiment on the alpha spectrometric determination of Pu-238. Pt. 1

International Nuclear Information System (INIS)

Beyrich, W.; Spannagel, G.

1979-12-01

In cooperation with 26 laboratories of 11 countries or international organizations, the Safeguards Project of the Karlsruhe Nuclear Research Center carried out the interlaboratory program AS-76. It focused on the alpha-spectrometric determination of the Pu-238 isotope. The performance of the program as well as the results obtained are described. (orig.) 891 HP/orig. 892 MKO [de

Effect of sample storage time on detection of hybridization signals in Checkerboard DNA-DNA hybridization.

Science.gov (United States)

do Nascimento, Cássio; Muller, Katia; Sato, Sandra; Albuquerque Junior, Rubens Ferreira

2012-04-01

Long-term sample storage can affect the intensity of the hybridization signals provided by molecular diagnostic methods that use chemiluminescent detection. The aim of this study was to evaluate the effect of different storage times on the hybridization signals of 13 bacterial species detected by the Checkerboard DNA-DNA hybridization method using whole-genomic DNA probes. Ninety-six subgingival biofilm samples were collected from 36 healthy subjects, and the intensity of hybridization signals was evaluated at 4 different time periods: (1) immediately after collecting (n = 24) and (2) after storage at -20 °C for 6 months (n = 24), (3) for 12 months (n = 24), and (4) for 24 months (n = 24). The intensity of hybridization signals obtained from groups 1 and 2 were significantly higher than in the other groups (p  0.05). The Checkerboard DNA-DNA hybridization method was suitable to detect hybridization signals from all groups evaluated, and the intensity of signals decreased significantly after long periods of sample storage.

DNA methylation changes measured in pre‐diagnostic peripheral blood samples are associated with smoking and lung cancer risk

Science.gov (United States)

Baglietto, Laura; Ponzi, Erica; Haycock, Philip; Hodge, Allison; Bianca Assumma, Manuela; Jung, Chol‐Hee; Chung, Jessica; Fasanelli, Francesca; Guida, Florence; Campanella, Gianluca; Chadeau‐Hyam, Marc; Grankvist, Kjell; Johansson, Mikael; Ala, Ugo; Provero, Paolo; Wong, Ee Ming; Joo, Jihoon; English, Dallas R.; Kazmi, Nabila; Lund, Eiliv; Faltus, Christian; Kaaks, Rudolf; Risch, Angela; Barrdahl, Myrto; Sandanger, Torkjel M.; Southey, Melissa C.; Giles, Graham G.; Johansson, Mattias; Vineis, Paolo; Polidoro, Silvia; Relton, Caroline L.

2016-01-01

DNA methylation changes are associated with cigarette smoking. We used the Illumina Infinium HumanMethylation450 array to determine whether methylation in DNA from pre‐diagnostic, peripheral blood samples is associated with lung cancer risk. We used a case‐control study nested within the EPIC‐Italy cohort and a study within the MCCS cohort as discovery sets (a total of 552 case‐control pairs). We validated the top signals in 429 case‐control pairs from another 3 studies. We identified six CpGs for which hypomethylation was associated with lung cancer risk: cg05575921 in the AHRR gene (p‐valuepooled = 4 × 10−17), cg03636183 in the F2RL3 gene (p‐valuepooled = 2 × 10 − 13), cg21566642 and cg05951221 in 2q37.1 (p‐valuepooled = 7 × 10−16 and 1 × 10−11 respectively), cg06126421 in 6p21.33 (p‐valuepooled = 2 × 10−15) and cg23387569 in 12q14.1 (p‐valuepooled = 5 × 10−7). For cg05951221 and cg23387569 the strength of association was virtually identical in never and current smokers. For all these CpGs except for cg23387569, the methylation levels were different across smoking categories in controls (p‐valuesheterogeneity ≤ 1.8 x10 − 7), were lowest for current smokers and increased with time since quitting for former smokers. We observed a gain in discrimination between cases and controls measured by the area under the ROC curve of at least 8% (p‐values ≥ 0.003) in former smokers by adding methylation at the 6 CpGs into risk prediction models including smoking status and number of pack‐years. Our findings provide convincing evidence that smoking and possibly other factors lead to DNA methylation changes measurable in peripheral blood that may improve prediction of lung cancer risk. PMID:27632354

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

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

Molecular diagnostics based on clustering dynamics of magnetic nanobeads

DEFF Research Database (Denmark)

Donolato, Marco; Bejhed, Rebecca S.; de la Torre, Teresa Zardán Gómez

2014-01-01

transmission modulation caused by the AC magnetic field-stimulated reversible formation and disruption of elongated MNB supra-structures during a cycle of the uniaxial applied magnetic field. As a specific clinically relevant diagnostic case, we detect DNA coils formed via padlock probe recognition...

Nanolock-Nanopore Facilitated Digital Diagnostics of Cancer Driver Mutation in Tumor Tissue.

Science.gov (United States)

Wang, Yong; Tian, Kai; Shi, Ruicheng; Gu, Amy; Pennella, Michael; Alberts, Lindsey; Gates, Kent S; Li, Guangfu; Fan, Hongxin; Wang, Michael X; Gu, Li-Qun

2017-07-28

Cancer driver mutations are clinically significant biomarkers. In precision medicine, accurate detection of these oncogenic changes in patients would enable early diagnostics of cancer, individually tailored targeted therapy, and precise monitoring of treatment response. Here we investigated a novel nanolock-nanopore method for single-molecule detection of a serine/threonine protein kinase gene BRAF V600E mutation in tumor tissues of thyroid cancer patients. The method lies in a noncovalent, mutation sequence-specific nanolock. We found that the nanolock formed on the mutant allele/probe duplex can separate the duplex dehybridization procedure into two sequential steps in the nanopore. Remarkably, this stepwise unzipping kinetics can produce a unique nanopore electric marker, with which a single DNA molecule of the cancer mutant allele can be unmistakably identified in various backgrounds of the normal wild-type allele. The single-molecule sensitivity for mutant allele enables both binary diagnostics and quantitative analysis of mutation occurrence. In the current configuration, the method can detect the BRAF V600E mutant DNA lower than 1% in the tumor tissues. The nanolock-nanopore method can be adapted to detect a broad spectrum of both transversion and transition DNA mutations, with applications from diagnostics to targeted therapy.

Nanotechnology based diagnostics for neurological disorders

Energy Technology Data Exchange (ETDEWEB)

Kurek, Nicholas S; Chandra, Sathees B., E-mail: schandra@roosevelt.edu [Department of Biological, Chemical and Physical Sciences, Roosevelt University, Chicago, IL (United States)

2012-07-01

Nanotechnology involves probing and manipulating matter at the molecular level. Nanotechnology based molecular diagnostics have the potential to alleviate the suffering caused by many diseases, including neurological disorders, due to the unique properties of nanomaterials. Most neurological illnesses are multifactorial conditions and many of these are also classified as neurobehavioral disorders. Alzheimer's disease, Parkinson's disease, Huntington disease, cerebral ischemia, epilepsy, schizophrenia and autism spectrum disorders like Rett syndrome are some examples of neurological disorders that could be better treated, diagnosed, prevented and possibly cured using nanotechnology. In order to improve the quality of life for disease afflicted people, a wide range of nanomaterials that include gold and silica nanoparticles, quantum dots and DNA along with countless other forms of nanotechnology have been investigated regarding their usefulness in advancing molecular diagnostics. Other small scaled materials like viruses and proteins also have potential for use as molecular diagnostic tools. Information obtained from nanotechnology based diagnostics can be stored and manipulated using bioinformatics software. More advanced nanotechnology based diagnostic procedures for the acquisition of even greater proteomic and genomic knowledge can then be developed along with better ways to fight various diseases. Nanotechnology also has numerous applications besides those related to biotechnology and medicine. In this article, we will discuss and analyze many novel nanotechnology based diagnostic techniques at our disposal today. (author)

Nanotechnology based diagnostics for neurological disorders

Energy Technology Data Exchange (ETDEWEB)

Kurek, Nicholas S.; Chandra, Sathees B., E-mail: schandra@roosevelt.edu [Department of Biological, Chemical and Physical Sciences, Roosevelt University, Chicago, IL (United States)

2012-07-01

Nanotechnology involves probing and manipulating matter at the molecular level. Nanotechnology based molecular diagnostics have the potential to alleviate the suffering caused by many diseases, including neurological disorders, due to the unique properties of nanomaterials. Most neurological illnesses are multifactorial conditions and many of these are also classified as neurobehavioral disorders. Alzheimer's disease, Parkinson's disease, Huntington disease, cerebral ischemia, epilepsy, schizophrenia and autism spectrum disorders like Rett syndrome are some examples of neurological disorders that could be better treated, diagnosed, prevented and possibly cured using nanotechnology. In order to improve the quality of life for disease afflicted people, a wide range of nanomaterials that include gold and silica nanoparticles, quantum dots and DNA along with countless other forms of nanotechnology have been investigated regarding their usefulness in advancing molecular diagnostics. Other small scaled materials like viruses and proteins also have potential for use as molecular diagnostic tools. Information obtained from nanotechnology based diagnostics can be stored and manipulated using bioinformatics software. More advanced nanotechnology based diagnostic procedures for the acquisition of even greater proteomic and genomic knowledge can then be developed along with better ways to fight various diseases. Nanotechnology also has numerous applications besides those related to biotechnology and medicine. In this article, we will discuss and analyze many novel nanotechnology based diagnostic techniques at our disposal today. (author)

Nanotechnology based diagnostics for neurological disorders

International Nuclear Information System (INIS)

Kurek, Nicholas S.; Chandra, Sathees B.

2012-01-01

Nanotechnology involves probing and manipulating matter at the molecular level. Nanotechnology based molecular diagnostics have the potential to alleviate the suffering caused by many diseases, including neurological disorders, due to the unique properties of nanomaterials. Most neurological illnesses are multifactorial conditions and many of these are also classified as neurobehavioral disorders. Alzheimer's disease, Parkinson's disease, Huntington disease, cerebral ischemia, epilepsy, schizophrenia and autism spectrum disorders like Rett syndrome are some examples of neurological disorders that could be better treated, diagnosed, prevented and possibly cured using nanotechnology. In order to improve the quality of life for disease afflicted people, a wide range of nanomaterials that include gold and silica nanoparticles, quantum dots and DNA along with countless other forms of nanotechnology have been investigated regarding their usefulness in advancing molecular diagnostics. Other small scaled materials like viruses and proteins also have potential for use as molecular diagnostic tools. Information obtained from nanotechnology based diagnostics can be stored and manipulated using bioinformatics software. More advanced nanotechnology based diagnostic procedures for the acquisition of even greater proteomic and genomic knowledge can then be developed along with better ways to fight various diseases. Nanotechnology also has numerous applications besides those related to biotechnology and medicine. In this article, we will discuss and analyze many novel nanotechnology based diagnostic techniques at our disposal today. (author)

Towards diagnostic metagenomics of Campylobacter in fecal samples

DEFF Research Database (Denmark)

Andersen, Sandra Christine; Kiil, Kristoffer; Harder, Christoffer Bugge

2017-01-01

The development of diagnostic metagenomics is driven by the need for universal, culture-independent methods for detection and characterization of pathogens to substitute the time-consuming, organism-specific, and often culture-based laboratory procedures for epidemiological source-tracing. Some...... of the challenges in diagnostic metagenomics are, that it requires a great next-generation sequencing depth and unautomated data analysis. DNA from human fecal samples spiked with 7.75 × 101-7.75 × 107 colony forming unit (CFU)/ml Campylobacter jejuni and chicken fecal samples spiked with 1 × 102-1 × 106 CFU...... Campylobacter in all the clinical samples. Sensitivity in diagnostic metagenomics is improving and has reached a clinically relevant level. There are still challenges to overcome before real-time diagnostic metagenomics can replace quantitative polymerase chain reaction (qPCR) or culture-based surveillance...

A liquid chromatography-mass spectrometric method for the determination of oak moss allergens atranol and chloroatranol in perfumes

DEFF Research Database (Denmark)

Bossi, Rossana; Rastogi, Suresh Chandra; Bernard, Guillaume

2004-01-01

This paper describes a validated liquid chromatographic-tandem mass spectrometric method for quantitative analysis of the potential oak moss allergens atranol and chloroatranol in perfumes and similar products. The method employs LC-MS-MS with electrospray ionization (ESI) in negative mode...

Rapid DNA extraction from dried blood spots on filter paper: potential applications in biobanking.

Science.gov (United States)

Choi, Eun-Hye; Lee, Sang Kwang; Ihm, Chunhwa; Sohn, Young-Hak

2014-12-01

Dried blood spot (DBS) technology is a microsampling alternative to traditional plasma or serum sampling for pharmaco- or toxicokinetic evaluation. DBS technology has been applied to diagnostic screening in drug discovery, nonclinical, and clinical settings. We have developed an improved elution protocol involving boiling of blood spots dried on Whatman filter paper. The purpose of this study was to compare the quality, purity, and quantity of DNA isolated from frozen blood samples and DBSs. We optimized a method for extraction and estimation of DNA from blood spots dried on filter paper (3-mm FTA card). A single DBS containing 40 μL blood was used. DNA was efficiently extracted in phosphate-buffered saline (PBS) or Tris-EDTA (TE) buffer by incubation at 37°C overnight. DNA was stable in DBSs that were stored at room temperature or frozen. The housekeeping genes GAPDH and beta-actin were used as positive standards for polymerase chain reaction (PCR) validation of general diagnostic screening. Our simple and convenient DBS storage and extraction methods are suitable for diagnostic screening by using very small volumes of blood collected on filter paper, and can be used in biobanks for blood sample storage.

Diagnostic values for the viral load in peripheral blood mononuclear cells of patients with chronic active Epstein-Barr virus disease.

Science.gov (United States)

Ito, Yoshinori; Suzuki, Michio; Kawada, Jun-ichi; Kimura, Hiroshi

2016-04-01

Chronic active Epstein-Barr virus disease (CAEBV) is a distinct EBV-associated lymphoproliferative disease with a poor prognosis. Although the viral load in blood samples has been widely used for diagnosing CAEBV, well-defined viral load thresholds to guide clinicians are currently lacking. The aim of the present study was to determine standardized diagnostic values for EBV load in blood samples of CAEBV patients using the World Health Organization international standard for reporting. Levels of EBV DNA in 103 peripheral blood mononuclear cells (PBMCs) and 95 plasma/serum samples from 107 cases with CAEBV were quantified and expressed in international units. Receiver operating characteristic curves were analyzed to assess the most appropriate cut-off values for levels of EBV DNA to distinguish CAEBV from EBV-associated infectious mononucleosis (IM) and controls with past EBV infection. Levels of EBV DNA in PBMCs were significantly higher in the CAEBV group (median, 10(4.2) IU/μgDNA) compared to the IM (median, 10(2.1) IU/μgDNA) and control groups. An inconsistent qualitative result was seen in 13 of 86 CAEBV patients; in these, EBV-DNA was positive in PBMCs, but negative in plasma. Diagnostic cut-off values for viral load in PBMCs from CAEBV patients, as compared to those of healthy controls and IM patients, were 10(2.0) IU/μgDNA and 10(3.2) IU/μgDNA, respectively. For diagnostic purposes, the viral load of PBMCs was better than of plasma/serum. A diagnostic cut-off EBV load for CAEBV may be useful for the management of CAEBV patients. Copyright © 2015 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

In situ monitoring of molecular changes during cell differentiation processes in marine macroalgae through mass spectrometric imaging.

Science.gov (United States)

Kessler, Ralf W; Crecelius, Anna C; Schubert, Ulrich S; Wichard, Thomas

2017-08-01

Matrix-assisted laser desorption/ionization mass spectrometric imaging (MALDI-MSI) was employed to discriminate between cell differentiation processes in macroalgae. One of the key developmental processes in the algal life cycle is the production of germ cells (gametes and zoids). The gametogenesis of the marine green macroalga Ulva mutabilis (Chlorophyta) was monitored by metabolomic snapshots of the surface, when blade cells differentiate synchronously into gametangia and giving rise to gametes. To establish MSI for macroalgae, dimethylsulfoniopropionate (DMSP), a known algal osmolyte, was determined. MSI of the surface of U. mutabilis followed by chemometric data analysis revealed dynamic metabolomic changes during cell differentiation. DMSP and a total of 55 specific molecular biomarkers, which could be assigned to important stages of the gametogenesis, were detected. Our research contributes to the understanding of molecular mechanisms underlying macroalgal cell differentiation. Graphical abstract Molecular changes during cell differentiation of the marine macroalga Ulva were visualized by matrix assisted laser desorption/ionization mass spectrometric imaging (MALDI-MSI).

DNA methylation signatures for prediction of biochemical recurrence after radical prostatectomy of clinically localized prostate cancer

DEFF Research Database (Denmark)

Haldrup, Christa; Mundbjerg, Kamilla; Vestergaard, Else Marie

2013-01-01

Purpose Diagnostic and prognostic tools for prostate cancer (PC) are suboptimal, causing overtreatment of indolent PC and risk of delayed treatment of aggressive PC. Here, we identify six novel candidate DNA methylation markers for PC with promising diagnostic and prognostic potential. Methods...... Microarray-based screening and bisulfite sequencing of 20 nonmalignant and 29 PC tissue specimens were used to identify new candidate DNA hypermethylation markers for PC. Diagnostic and prognostic potential was evaluated in 35 nonmalignant prostate tissue samples, 293 radical prostatectomy (RP) samples...... into low- and high-methylation subgroups, was trained in cohort 1 (HR, 1.91; 95% CI, 1.26 to 2.90) and validated in cohort 2 (HR, 2.33; 95% CI, 1.31 to 4.13). Conclusion We identified six novel candidate DNA methylation markers for PC. C1orf114 hypermethylation and a three-gene methylation signature were...

Whole Genome Sequencing Increases Molecular Diagnostic Yield Compared with Current Diagnostic Testing for Inherited Retinal Disease.

Science.gov (United States)

Ellingford, Jamie M; Barton, Stephanie; Bhaskar, Sanjeev; Williams, Simon G; Sergouniotis, Panagiotis I; O'Sullivan, James; Lamb, Janine A; Perveen, Rahat; Hall, Georgina; Newman, William G; Bishop, Paul N; Roberts, Stephen A; Leach, Rick; Tearle, Rick; Bayliss, Stuart; Ramsden, Simon C; Nemeth, Andrea H; Black, Graeme C M

2016-05-01

To compare the efficacy of whole genome sequencing (WGS) with targeted next-generation sequencing (NGS) in the diagnosis of inherited retinal disease (IRD). Case series. A total of 562 patients diagnosed with IRD. We performed a direct comparative analysis of current molecular diagnostics with WGS. We retrospectively reviewed the findings from a diagnostic NGS DNA test for 562 patients with IRD. A subset of 46 of 562 patients (encompassing potential clinical outcomes of diagnostic analysis) also underwent WGS, and we compared mutation detection rates and molecular diagnostic yields. In addition, we compared the sensitivity and specificity of the 2 techniques to identify known single nucleotide variants (SNVs) using 6 control samples with publically available genotype data. Diagnostic yield of genomic testing. Across known disease-causing genes, targeted NGS and WGS achieved similar levels of sensitivity and specificity for SNV detection. However, WGS also identified 14 clinically relevant genetic variants through WGS that had not been identified by NGS diagnostic testing for the 46 individuals with IRD. These variants included large deletions and variants in noncoding regions of the genome. Identification of these variants confirmed a molecular diagnosis of IRD for 11 of the 33 individuals referred for WGS who had not obtained a molecular diagnosis through targeted NGS testing. Weighted estimates, accounting for population structure, suggest that WGS methods could result in an overall 29% (95% confidence interval, 15-45) uplift in diagnostic yield. We show that WGS methods can detect disease-causing genetic variants missed by current NGS diagnostic methodologies for IRD and thereby demonstrate the clinical utility and additional value of WGS. Copyright © 2016 American Academy of Ophthalmology. Published by Elsevier Inc. All rights reserved.

DNA barcoding of recently diverged species: relative performance of matching methods.

Directory of Open Access Journals (Sweden)

Robin van Velzen

Full Text Available Recently diverged species are challenging for identification, yet they are frequently of special interest scientifically as well as from a regulatory perspective. DNA barcoding has proven instrumental in species identification, especially in insects and vertebrates, but for the identification of recently diverged species it has been reported to be problematic in some cases. Problems are mostly due to incomplete lineage sorting or simply lack of a 'barcode gap' and probably related to large effective population size and/or low mutation rate. Our objective was to compare six methods in their ability to correctly identify recently diverged species with DNA barcodes: neighbor joining and parsimony (both tree-based, nearest neighbor and BLAST (similarity-based, and the diagnostic methods DNA-BAR, and BLOG. We analyzed simulated data assuming three different effective population sizes as well as three selected empirical data sets from published studies. Results show, as expected, that success rates are significantly lower for recently diverged species (∼75% than for older species (∼97% (P<0.00001. Similarity-based and diagnostic methods significantly outperform tree-based methods, when applied to simulated DNA barcode data (P<0.00001. The diagnostic method BLOG had highest correct query identification rate based on simulated (86.2% as well as empirical data (93.1%, indicating that it is a consistently better method overall. Another advantage of BLOG is that it offers species-level information that can be used outside the realm of DNA barcoding, for instance in species description or molecular detection assays. Even though we can confirm that identification success based on DNA barcoding is generally high in our data, recently diverged species remain difficult to identify. Nevertheless, our results contribute to improved solutions for their accurate identification.

Hereditary neuropathies: systematization and diagnostics (clinical case of hereditary motor and sensor neuropathy of the IA type

Directory of Open Access Journals (Sweden)

Kolokolova A.M.

2016-09-01

Full Text Available Aim: to study the value of routine methods (clinical symptoms, electrophysiological findings and results of DNA analysis in diagnostics of hereditary motor sensory neuropathy type IA in outpatient clinics. Material and Methods. The review of foreign literature is represented. The phenotypic polymorphism, genetic heterogeneity and the difficulties of diagnostics are identified. A family with hereditary motor sensory neuropathy of lAtype is presented, which was diagnosed on the base of available methods in outpatient practice (clinical symptoms, genealogical method, electro-physiological findings and DNA analysis results. Results. Routine algorithm (consistent valuation of clinical symptoms, neurophysiologic findings and the results of DNA analysis helped to verify the diagnosis of hereditary motor sensory neuropathy of lAtype in outpatient practice after more than 20 years of the onset of the disease. Conclusion. The neurologists of outpatient clinics and other specialists must be informed about the availability of diagnostics of hereditary diseases of nervous system.

GENE-07. MOLECULAR NEUROPATHOLOGY 2.0 - INCREASING DIAGNOSTIC ACCURACY IN PEDIATRIC NEUROONCOLOGY

Science.gov (United States)

Sturm, Dominik; Jones, David T.W.; Capper, David; Sahm, Felix; von Deimling, Andreas; Rutkoswki, Stefan; Warmuth-Metz, Monika; Bison, Brigitte; Gessi, Marco; Pietsch, Torsten; Pfister, Stefan M.

2017-01-01

Abstract The classification of central nervous system (CNS) tumors into clinically and biologically distinct entities and subgroups is challenging. Children and adolescents can be affected by >100 histological variants with very variable outcomes, some of which are exceedingly rare. The current WHO classification has introduced a number of novel molecular markers to aid routine neuropathological diagnostics, and DNA methylation profiling is emerging as a powerful tool to distinguish CNS tumor classes. The Molecular Neuropathology 2.0 study aims to integrate genome wide (epi-)genetic diagnostics with reference neuropathological assessment for all newly-diagnosed pediatric brain tumors in Germany. To date, >350 patients have been enrolled. A molecular diagnosis is established by epigenetic tumor classification through DNA methylation profiling and targeted panel sequencing of >130 genes to detect diagnostically and/or therapeutically useful DNA mutations, structural alterations, and fusion events. Results are aligned with the reference neuropathological diagnosis, and discrepant findings are discussed in a multi-disciplinary tumor board including reference neuroradiological evaluation. Ten FFPE sections as input material are sufficient to establish a molecular diagnosis in >95% of tumors. Alignment with reference pathology results in four broad categories: a) concordant classification (~77%), b) discrepant classification resolvable by tumor board discussion and/or additional data (~5%), c) discrepant classification without currently available options to resolve (~8%), and d) cases currently unclassifiable by molecular diagnostics (~10%). Discrepancies are enriched in certain histopathological entities, such as histological high grade gliomas with a molecularly low grade profile. Gene panel sequencing reveals predisposing germline events in ~10% of patients. Genome wide (epi-)genetic analyses add a valuable layer of information to routine neuropathological

Sequential mass spectrometric analysis of uranium and plutonium employing resin bead technique

International Nuclear Information System (INIS)

Ramakumar, K.L.; Aggarwal, S.K.; Chitambar, S.A.; Jain, H.C.

1985-01-01

Sequential mass spectrometric analysis of uranium and plutonium employing anion exchange resin bead technique is reported using a high sensitive single stage magnetic analyser instrument, the routinely employed rhenium double filament assembly and 0.5M HNO 3 as a wetting agent for loading the resin beads. A precision of bettter than 0.3per cent (2sigma) is obtained on the isotopic ratio measurements. However, extreme care has to be exercised to carry the resin bead experiments under ultra clean conditions so as to avoid pick up of contamination. (author)

Multi-color fluorescent DNA analysis in an integrated optofluidic lab on a chip

NARCIS (Netherlands)

Dongre, C.

2010-01-01

Abstract: Sorting and sizing of DNA molecules within the human genome project has enabled the genetic mapping of various illnesses. Furthermore by employing tiny lab-on-a-chip device, integrated DNA sequencing and genetic diagnostics have become feasible. We present the combination of capillary

Gold-based optical biosensor for single-mismatched DNA detection using salt-induced hybridization

DEFF Research Database (Denmark)

Zhan, Zongrui; Ma, Xingyi; Cao, Cuong

2011-01-01

In this study, a gold nanoparticle (Au-NP)-based detection method for sensitive and specific DNA-based diagnostic applications is described. A sandwich format consisting of Au-NPs/DNA/PMP (Streptavidin-coated MagnetSphere Para-Magnetic Particles) was fabricated. PMPs captured and separated target...

Analysis of relationship between tumor markers and quantification of free DNA in serum of lung cancer patients

International Nuclear Information System (INIS)

Yang Shunfang; Zhang Peiling; Cao Jie; Zeng Jun; Dong Qianggang

2006-01-01

To evaluate the diagnostic value and relationship between five tumor markers (CA19- 9,CA125,CYFRA21-1 ,CEA,NSE) and free DNA in serum for lung cancer detection and try to find a new and more efficient tumor marker, the amounts of CA19-9, CA125, CYFRA21-1, CEA, NSE were determined by RIA and free DNA was determined by the use of quantitative real time PCR amplification of the human epidermal growth factor receptor (EGFR) in 52 lung cancer patients and 8 cases of benign pulmonary disease and 10 healthy controls. The resulls showed that average concentration of free DNA in serum of lung cancer patients, benign pulmo- nary disease and healthy controls was 107.6ng/mL, 76.86ng/mL and 18.8ng/mL, respective- ly. The diagnostic sensitivity, specificity and accuracy of free DNA for lung cancer were 71. 2%, 50% and 68.3%, same as the diagnostic value of combined detection of five tumor markers. The sensitivity, specificity and accuracy of the five tumor markers and free DNA combinend detection for lung cancer were 94.2%, 25% and 85%, respectively. The free DNA in the serum of lung cancer patients may be a new and better tumor marker. (authors)

Determination of trace quantities of uranium in rocks mass spectrometric isotope dilution technique

International Nuclear Information System (INIS)

Kakazu, Mauricio Hiromitu

1980-01-01

A detailed experimental investigation on the thermionic emission of uranium deposited on a single flat type rhenium filament has been carried out. The study was aimed at determining the influence of various forms of deposition on the emission sensitivity and thermal stability of U + , UO + and UO 2 + ions. Based on these investigations, a technique, involving an addition of a small quantity of colloidal suspension of graphite on top of the uranyl nitrate sample deposited, was chosen because of its higher, emission sensitivity for uranium metal ions. The experimental parameters of the technique were optimised and the technique was employed in the determination of trace quantities of uranium in rock samples using mass spectrometric isotope dilution method. For the mass spectrometric isotope dilution analysis National Bureau of Standards uranium isotopic standard NBS-U 970 was employed as a tracer, where as the mass discrimination effect in the uranium isotope analysis was corrected using the uranium isotopic standard NBS-U500. Uranium was determined in each of the seven granite samples from Wyoming, USA and two USGS standard rocks. The precision of the analysis was found to be ±1% . The uranium values obtained on the rock samples were compared with the analyses of other investigators. Influence of the sample splitting on the uranium analysis was discussed in the light of the analytical results obtained.(author)

A direct detection of Escherichia coli genomic DNA using gold nanoprobes

Directory of Open Access Journals (Sweden)

Padmavathy

2012-02-01

Full Text Available Abstract Background In situation like diagnosis of clinical and forensic samples there exists a need for highly sensitive, rapid and specific DNA detection methods. Though conventional DNA amplification using PCR can provide fast results, it is not widely practised in diagnostic laboratories partially because it requires skilled personnel and expensive equipment. To overcome these limitations nanoparticles have been explored as signalling probes for ultrasensitive DNA detection that can be used in field applications. Among the nanomaterials, gold nanoparticles (AuNPs have been extensively used mainly because of its optical property and ability to get functionalized with a variety of biomolecules. Results We report a protocol for the use of gold nanoparticles functionalized with single stranded oligonucleotide (AuNP- oligo probe as visual detection probes for rapid and specific detection of Escherichia coli. The AuNP- oligo probe on hybridization with target DNA containing complementary sequences remains red whereas test samples without complementary DNA sequences to the probe turns purple due to acid induced aggregation of AuNP- oligo probes. The color change of the solution is observed visually by naked eye demonstrating direct and rapid detection of the pathogenic Escherichia coli from its genomic DNA without the need for PCR amplification. The limit of detection was ~54 ng for unamplified genomic DNA. The method requires less than 30 minutes to complete after genomic DNA extraction. However, by using unamplified enzymatic digested genomic DNA, the detection limit of 11.4 ng was attained. Results of UV-Vis spectroscopic measurement and AFM imaging further support the hypothesis of aggregation based visual discrimination. To elucidate its utility in medical diagnostic, the assay was validated on clinical strains of pathogenic Escherichia coli obtained from local hospitals and spiked urine samples. It was found to be 100% sensitive and proves to

Single-cell manipulation and DNA delivery technology using atomic force microscopy and nanoneedle.

Science.gov (United States)

Han, Sung-Woong; Nakamura, Chikashi; Miyake, Jun; Chang, Sang-Mok; Adachi, Taiji

2014-01-01

The recent single-cell manipulation technology using atomic force microscopy (AFM) not only allows high-resolution visualization and probing of biomolecules and cells but also provides spatial and temporal access to the interior of living cells via the nanoneedle technology. Here we review the development and application of single-cell manipulations and the DNA delivery technology using a nanoneedle. We briefly describe various DNA delivery methods and discuss their advantages and disadvantages. Fabrication of the nanoneedle, visualization of nanoneedle insertion into living cells, DNA modification on the nanoneedle surface, and the invasiveness of nanoneedle insertion into living cells are described. Different methods of DNA delivery into a living cell, such as lipofection, microinjection, and nanoneedles, are then compared. Finally, single-cell diagnostics using the nanoneedle and the perspectives of the nanoneedle technology are outlined. The nanoneedle-based DNA delivery technology provides new opportunities for efficient and specific introduction of DNA and other biomolecules into precious living cells with a high spatial resolution within a desired time frame. This technology has the potential to be applied for many basic cellular studies and for clinical studies such as single-cell diagnostics.

Carbon nanotubes as solid-phase extraction sorbents prior to atomic spectrometric determination of metal species: A review

International Nuclear Information System (INIS)

Herrero Latorre, C.; Álvarez Méndez, J.; Barciela García, J.; García Martín, S.; Peña Crecente, R.M.

2012-01-01

Highlights: ► The use of CNTs as sorbent for metal species in solid phase extraction has been described. ► Physical and chemical strategies for functionalization of carbon nanotubes have been discussed. ► Published analytical methods concerning solid phase extraction and atomic spectrometric determination have been reviewed. - Abstract: New materials have significant impact on the development of new methods and instrumentation for chemical analysis. From the discovery of carbon nanotubes in 1991, single and multi-walled carbon nanotubes – due to their high adsorption and desorption capacities – have been employed as sorption substrates in solid-phase extraction for the preconcentration of metal species from diverse matrices. Looking for successive improvements in sensitivity and selectivity, in the past few years, carbon nanotubes have been utilized as sorbents for solid phase extraction in three different ways: like as-grown, oxidized and functionalized nanotubes. In the present paper, an overview of the recent trends in the use of carbon nanotubes for solid phase extraction of metal species in environmental, biological and food samples is presented. The determination procedures involved the adsorption of metals on the nanotube surface, their quantitative desorption and subsequent measurement by means of atomic spectrometric techniques such as flame atomic absorption spectrometry, electrothermal atomic absorption spectrometry or inductively coupled plasma atomic emission spectrometry/mass spectrometry, among others. Synthesis, purification and types of carbon nanotubes, as well as the diverse chemical and physical strategies for their functionalization are described. Based on 140 references, the performance and general properties of the applications of solid phase extraction based on carbon nanotubes for metal species atomic spectrometric determination are discussed.

Carbon nanotubes as solid-phase extraction sorbents prior to atomic spectrometric determination of metal species: A review

Energy Technology Data Exchange (ETDEWEB)

Herrero Latorre, C., E-mail: carlos.herrero@usc.es [Universidad de Santiago de Compostela, Dpto. Quimica Analitica, Nutricion y Bromatologia, Facultad de Ciencias, Alfonso X el Sabio s/n, 27002 Lugo (Spain); Alvarez Mendez, J.; Barciela Garcia, J.; Garcia Martin, S.; Pena Crecente, R.M. [Universidad de Santiago de Compostela, Dpto. Quimica Analitica, Nutricion y Bromatologia, Facultad de Ciencias, Alfonso X el Sabio s/n, 27002 Lugo (Spain)

2012-10-24

Highlights: Black-Right-Pointing-Pointer The use of CNTs as sorbent for metal species in solid phase extraction has been described. Black-Right-Pointing-Pointer Physical and chemical strategies for functionalization of carbon nanotubes have been discussed. Black-Right-Pointing-Pointer Published analytical methods concerning solid phase extraction and atomic spectrometric determination have been reviewed. - Abstract: New materials have significant impact on the development of new methods and instrumentation for chemical analysis. From the discovery of carbon nanotubes in 1991, single and multi-walled carbon nanotubes - due to their high adsorption and desorption capacities - have been employed as sorption substrates in solid-phase extraction for the preconcentration of metal species from diverse matrices. Looking for successive improvements in sensitivity and selectivity, in the past few years, carbon nanotubes have been utilized as sorbents for solid phase extraction in three different ways: like as-grown, oxidized and functionalized nanotubes. In the present paper, an overview of the recent trends in the use of carbon nanotubes for solid phase extraction of metal species in environmental, biological and food samples is presented. The determination procedures involved the adsorption of metals on the nanotube surface, their quantitative desorption and subsequent measurement by means of atomic spectrometric techniques such as flame atomic absorption spectrometry, electrothermal atomic absorption spectrometry or inductively coupled plasma atomic emission spectrometry/mass spectrometry, among others. Synthesis, purification and types of carbon nanotubes, as well as the diverse chemical and physical strategies for their functionalization are described. Based on 140 references, the performance and general properties of the applications of solid phase extraction based on carbon nanotubes for metal species atomic spectrometric determination are discussed.

Rapid and highly fieldable viral diagnostic

Energy Technology Data Exchange (ETDEWEB)

McKnight, Timothy E.

2016-12-20

The present invention relates to a rapid, highly fieldable, nearly reagentless diagnostic to identify active RNA viral replication in a live, infected cells, and more particularly in leukocytes and tissue samples (including biopsies and nasal swabs) using an array of a plurality of vertically-aligned nanostructures that impale the cells and introduce a DNA reporter construct that is expressed and amplified in the presence of active viral replication.

Biofunctionalization of ZnO nanowires for DNA sensory applications

Energy Technology Data Exchange (ETDEWEB)

Schroeder, Ulrich Christian; Gnauck, Martin; Ronning, Carsten [Institute of Solid State Physics, University of Jena, Max-Wien-Platz 1, D-07743 Jena (Germany); Moeller, Robert; Rudolph, Bettina; Fritzsche, Wolfgang [Institut fuer Photonische Technologien e.V., Albert-Einstein-Strasse 9, D-07745 Jena (Germany)

2011-07-01

In recent years, DNA detecting systems have received a growing interest due to promising fields of application like DNA diagnostics, gene analysis, virus detection or forensic applications. Nanowire-based DNA biosensor allows both miniaturization and easy continuous monitoring of a detection signal by electrical means. The label free detection scheme based on electrochemical changes of the surface potential during immobilization of specific DNA probes was heretofore mainly studied for silicon. In this work a surface decoration process with bifunctional molecules known as silanization was applied to VLS-grown ZnO nanowires which both feature a large sensitivity for surface modification, are biocompatible and easy to synthesize as well. Successfully bound DNA was proved by fluorescence microscopy. Dielectrophoresis (DEP) was chosen and optimized for quickly contacting the ZnO nanowires. Furthermore, electrical signal characterization was performed in preparation for DNA sensory applications.

Laser mass spectrometry for DNA fingerprinting for forensic applications

Energy Technology Data Exchange (ETDEWEB)

Chen, C.H.; Tang, K.; Taranenko, N.I.; Allman, S.L.; Chang, L.Y.

1994-12-31

The application of DNA fingerprinting has become very broad in forensic analysis, patient identification, diagnostic medicine, and wildlife poaching, since every individual`s DNA structure is identical within all tissues of their body. DNA fingerprinting was initiated by the use of restriction fragment length polymorphisms (RFLP). In 1987, Nakamura et al. found that a variable number of tandem repeats (VNTR) often occurred in the alleles. The probability of different individuals having the same number of tandem repeats in several different alleles is very low. Thus, the identification of VNTR from genomic DNA became a very reliable method for identification of individuals. DNA fingerprinting is a reliable tool for forensic analysis. In DNA fingerprinting, knowledge of the sequence of tandem repeats and restriction endonuclease sites can provide the basis for identification. The major steps for conventional DNA fingerprinting include (1) specimen processing (2) amplification of selected DNA segments by PCR, and (3) gel electrophoresis to do the final DNA analysis. In this work we propose to use laser desorption mass spectrometry for fast DNA fingerprinting. The process and advantages are discussed.

Determination of radium isotopes by BaSO4 coprecipitation for the preparation of alpha-spectrometric sources

International Nuclear Information System (INIS)

Lozano, J.C.; Fernandez, F.; Gomez, J.M.G.

1997-01-01

A coprecipitation procedure for the preparation of α-spectrometric sources for radium, using BaSO 4 as carrier, has been applied to the determination of alpha radium isotopes in water samples. The use of 133 Ba as a suitable tracer for radium determination and possible losses of radon isotopes from the sources are studied and discussed. (author)

Analytical Devices Based on Direct Synthesis of DNA on Paper.

Science.gov (United States)

Glavan, Ana C; Niu, Jia; Chen, Zhen; Güder, Firat; Cheng, Chao-Min; Liu, David; Whitesides, George M

2016-01-05

This paper addresses a growing need in clinical diagnostics for parallel, multiplex analysis of biomarkers from small biological samples. It describes a new procedure for assembling arrays of ssDNA and proteins on paper. This method starts with the synthesis of DNA oligonucleotides covalently linked to paper and proceeds to assemble microzones of DNA-conjugated paper into arrays capable of simultaneously capturing DNA, DNA-conjugated protein antigens, and DNA-conjugated antibodies. The synthesis of ssDNA oligonucleotides on paper is convenient and effective with 32% of the oligonucleotides cleaved and eluted from the paper substrate being full-length by HPLC for a 32-mer. These ssDNA arrays can be used to detect fluorophore-linked DNA oligonucleotides in solution, and as the basis for DNA-directed assembly of arrays of DNA-conjugated capture antibodies on paper, detect protein antigens by sandwich ELISAs. Paper-anchored ssDNA arrays with different sequences can be used to assemble paper-based devices capable of detecting DNA and antibodies in the same device and enable simple microfluidic paper-based devices.

The gas-chromatographic and gas-chromatographic-mass-spectrometric identification of halogen-containing organic compounds

Science.gov (United States)

Gidaspov, B. V.; Zenkevich, I. G.; Rodin, A. A.

1989-09-01

The problem of identifying halogen-containing organic compounds in their gas-chromatographic and gas-chromatographic-mass-spectrometric (GC-MS) determination in different materials has been examined. Particular attention has been paid not to the complete characterisation of methods for carrying out this analysis but to the most important problem of increasing the selectivity at the stages of sampling, separation, and interpretation of the gas-chromatographic and GC-MS information. The bibliography contains 292 references.

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...

Current status and future perspectives on molecular and serological methods in diagnostic mycology.

Science.gov (United States)

Lau, Anna; Chen, Sharon; Sleiman, Sue; Sorrell, Tania

2009-11-01

Invasive fungal infections are an important cause of infectious morbidity. Nonculture-based methods are increasingly used for rapid, accurate diagnosis to improve patient outcomes. New and existing DNA amplification platforms have high sensitivity and specificity for direct detection and identification of fungi in clinical specimens. Since laboratories are increasingly reliant on DNA sequencing for fungal identification, measures to improve sequence interpretation should support validation of reference isolates and quality control in public gene repositories. Novel technologies (e.g., isothermal and PNA FISH methods), platforms enabling high-throughput analyses (e.g., DNA microarrays and Luminex xMAP) and/or commercial PCR assays warrant further evaluation for routine diagnostic use. Notwithstanding the advantages of molecular tests, serological assays remain clinically useful for patient management. The serum Aspergillus galactomannan test has been incorporated into diagnostic algorithms of invasive aspergillosis. Both the galactomannan and the serum beta-D-glucan test have value for diagnosing infection and monitoring therapeutic response.

DNA Methylation Biomarkers: Cancer and Beyond

Directory of Open Access Journals (Sweden)

Thomas Mikeska

2014-09-01

Full Text Available Biomarkers are naturally-occurring characteristics by which a particular pathological process or disease can be identified or monitored. They can reflect past environmental exposures, predict disease onset or course, or determine a patient’s response to therapy. Epigenetic changes are such characteristics, with most epigenetic biomarkers discovered to date based on the epigenetic mark of DNA methylation. Many tissue types are suitable for the discovery of DNA methylation biomarkers including cell-based samples such as blood and tumor material and cell-free DNA samples such as plasma. DNA methylation biomarkers with diagnostic, prognostic and predictive power are already in clinical trials or in a clinical setting for cancer. Outside cancer, strong evidence that complex disease originates in early life is opening up exciting new avenues for the detection of DNA methylation biomarkers for adverse early life environment and for estimation of future disease risk. However, there are a number of limitations to overcome before such biomarkers reach the clinic. Nevertheless, DNA methylation biomarkers have great potential to contribute to personalized medicine throughout life. We review the current state of play for DNA methylation biomarkers, discuss the barriers that must be crossed on the way to implementation in a clinical setting, and predict their future use for human disease.

Epigenetic reprogramming of pericentromeric satellite DNA in premalignant and malignant lesions

DEFF Research Database (Denmark)

Brückmann, Nadine Heidi; Pedersen, Christina Bøg; Ditzel, Henrik Jørn

2018-01-01

on pericentromeric satellites in primary melanocytes. This suggests that polycomb bodies form in cancer cells with global DNA demethylation to control the stability of pericentromeric satellite DNA. These results reveal a novel epigenetic perturbation specific to premalignant and malignant cells thatmaybe used...... as an early diagnostic marker for detection of precancerous changes and a new therapeutic entry point. Implications: Pericentromeric satellite DNA is epigenetically reprogrammed into polycomb bodies as a premalignant event with implications for transcriptional activity and genomic stability. Mol Cancer Res...

In situ probing of cholesterol in astrocytes at the single-cell level using laser desorption ionization mass spectrometric imaging with colloidal silver.

Science.gov (United States)

Perdian, D C; Cha, Sangwon; Oh, Jisun; Sakaguchi, Donald S; Yeung, Edward S; Lee, Young Jin

2010-04-30

Mass spectrometric imaging has been utilized to localize individual astrocytes and to obtain cholesterol populations at the single-cell level in laser desorption ionization (LDI) with colloidal silver. The silver ion adduct of membrane-bound cholesterol was monitored to detect individual cells. Good correlation between mass spectrometric and optical images at different cell densities indicates the ability to perform single-cell studies of cholesterol abundance. The feasibility of quantification is confirmed by the agreement between the LDI-MS ion signals and the results from a traditional enzymatic fluorometric assay. We propose that this approach could be an effective tool to study chemical populations at the cellular level. Published in 2010 by John Wiley & Sons, Ltd.

Status of mass spectrometric radiocarbon detection at ETHZ

Energy Technology Data Exchange (ETDEWEB)

Seiler, Martin; Maxeiner, Sascha; Wacker, Lukas; Synal, Hans-Arno

2015-10-15

A prototype of a mass spectrometric radiocarbon detection instrument without accelerator stage was built for the first time and set into operation at ETH Zurich. The system is designed as an experimental platform to optimize performance of {sup 14}C detection at low ion energies and to study the most relevant processes that may limit system performance. The optimized stripper unit incorporates differential pumping to maintain a low gas outflow and a revised tube design to better match the phase space volume of the ion beam at low energies. The system is fully operational and has demonstrated true radiocarbon dating capabilities. The overall beam transmission through the stripper tube is about 40% for the 1{sup +} charge state. Radiocarbon analyses with an overall precision of 0.6% were obtained on a single sample under regular measurement conditions. By analyzing multiple targets of the same sample material an uncertainty level of 0.3% has been reached. The background level corresponds to a radiocarbon age of 40,000 years.

Identification and reproducibility of diagnostic DNA markers for tuber starch and yield optimization in a novel association mapping population of potato (Solanum tuberosum L.).

Science.gov (United States)

Schönhals, E M; Ortega, F; Barandalla, L; Aragones, A; Ruiz de Galarreta, J I; Liao, J-C; Sanetomo, R; Walkemeier, B; Tacke, E; Ritter, E; Gebhardt, C

2016-04-01

SNPs in candidate genes Pain - 1, InvCD141 (invertases), SSIV (starch synthase), StCDF1 (transcription factor), LapN (leucine aminopeptidase), and cytoplasm type are associated with potato tuber yield, starch content and/or starch yield. Tuber yield (TY), starch content (TSC), and starch yield (TSY) are complex characters of high importance for the potato crop in general and for industrial starch production in particular. DNA markers associated with superior alleles of genes that control the natural variation of TY, TSC, and TSY could increase precision and speed of breeding new cultivars optimized for potato starch production. Diagnostic DNA markers are identified by association mapping in populations of tetraploid potato varieties and advanced breeding clones. A novel association mapping population of 282 genotypes including varieties, breeding clones and Andean landraces was assembled and field evaluated in Northern Spain for TY, TSC, TSY, tuber number (TN) and tuber weight (TW). The landraces had lower mean values of TY, TW, TN, and TSY. The population was genotyped for 183 microsatellite alleles, 221 single nucleotide polymorphisms (SNPs) in fourteen candidate genes and eight known diagnostic markers for TSC and TSY. Association test statistics including kinship and population structure reproduced five known marker-trait associations of candidate genes and discovered new ones, particularly for tuber yield and starch yield. The inclusion of landraces increased the number of detected marker-trait associations. Integration of the present association mapping results with previous QTL linkage mapping studies for TY, TSC, TSY, TW, TN, and tuberization revealed some hot spots of QTL for these traits in the potato genome. The genomic positions of markers linked or associated with QTL for complex tuber traits suggest high multiplicity and genome wide distribution of the underlying genes.

800,000 year old mammoth DNA, modern elephant DNA or PCR artefact?

Science.gov (United States)

Binladen, Jonas; Gilbert, M Thomas P; Willerslev, Eske

2007-02-22

Poulakakis and colleagues (Poulakakis et al. 2006: Biol. Lett. 2, 451-454), report the recovery of 'authentic' mammoth DNA from an 800,000-year-old fragment of bone excavated on the island of Crete. In light of results from other ancient DNA studies that indicate how DNA survival is unlikely in samples, which are recovered from warm environments and are relatively old (e.g. more than 100,000 years), these findings come as a great surprise. Here, we show that problems exist with the methodological approaches used in the study. First, the nested PCR technique as reported is nonsensical--one of the second round 'nested' primers falls outside the amplicon of the first round PCR. More worryingly, the binding region of one of the first round primers (Elcytb320R) falls within the short 43 base pair reported mammoth sequence, specifically covering two of the three reportedly diagnostic Elephas polymorphisms. Finally, we demonstrate using a simple BLAST search in GenBank that the claimed 'uniquely derived character state' for mammoths is in fact also found within modern elephants.

Enzymatic production of 'monoclonal stoichiometric' single-stranded DNA oligonucleotides.

Science.gov (United States)

Ducani, Cosimo; Kaul, Corinna; Moche, Martin; Shih, William M; Högberg, Björn

2013-07-01

Single-stranded oligonucleotides are important as research tools, as diagnostic probes, in gene therapy and in DNA nanotechnology. Oligonucleotides are typically produced via solid-phase synthesis, using polymer chemistries that are limited relative to what biological systems produce. The number of errors in synthetic DNA increases with oligonucleotide length, and the resulting diversity of sequences can be a problem. Here we present the 'monoclonal stoichiometric' (MOSIC) method for enzyme-mediated production of DNA oligonucleotides. We amplified oligonucleotides from clonal templates derived from single bacterial colonies and then digested cutter hairpins in the products, which released pools of oligonucleotides with precisely controlled relative stoichiometric ratios. We prepared 14-378-nucleotide MOSIC oligonucleotides either by in vitro rolling-circle amplification or by amplification of phagemid DNA in Escherichia coli. Analyses of the formation of a DNA crystal and folding of DNA nanostructures confirmed the scalability, purity and stoichiometry of the produced oligonucleotides.

Validated methodology for quantifying infestation levels of dreissenid mussels in environmental DNA (eDNA) samples.

Science.gov (United States)

Peñarrubia, Luis; Alcaraz, Carles; Vaate, Abraham Bij de; Sanz, Nuria; Pla, Carles; Vidal, Oriol; Viñas, Jordi

2016-12-14

The zebra mussel (Dreissena polymorpha Pallas, 1771) and the quagga mussel (D. rostriformis Deshayes, 1838) are successful invasive bivalves with substantial ecological and economic impacts in freshwater systems once they become established. Since their eradication is extremely difficult, their detection at an early stage is crucial to prevent spread. In this study, we optimized and validated a qPCR detection method based on the histone H2B gene to quantify combined infestation levels of zebra and quagga mussels in environmental DNA samples. Our results show specific dreissenid DNA present in filtered water samples for which microscopic diagnostic identification for larvae failed. Monitoring a large number of locations for invasive dreissenid species based on a highly specific environmental DNA qPCR assay may prove to be an essential tool for management and control plans focused on prevention of establishment of dreissenid mussels in new locations.

Variants of sequence family B Thermococcus kodakaraensis DNA polymerase with increased mismatch extension selectivity.

Directory of Open Access Journals (Sweden)

Claudia Huber

Full Text Available Fidelity and selectivity of DNA polymerases are critical determinants for the biology of life, as well as important tools for biotechnological applications. DNA polymerases catalyze the formation of DNA strands by adding deoxynucleotides to a primer, which is complementarily bound to a template. To ensure the integrity of the genome, DNA polymerases select the correct nucleotide and further extend the nascent DNA strand. Thus, DNA polymerase fidelity is pivotal for ensuring that cells can replicate their genome with minimal error. DNA polymerases are, however, further optimized for more specific biotechnological or diagnostic applications. Here we report on the semi-rational design of mutant libraries derived by saturation mutagenesis at single sites of a 3'-5'-exonuclease deficient variant of Thermococcus kodakaraensis DNA polymerase (KOD pol and the discovery for variants with enhanced mismatch extension selectivity by screening. Sites of potential interest for saturation mutagenesis were selected by their proximity to primer or template strands. The resulting libraries were screened via quantitative real-time PCR. We identified three variants with single amino acid exchanges-R501C, R606Q, and R606W-which exhibited increased mismatch extension selectivity. These variants were further characterized towards their potential in mismatch discrimination. Additionally, the identified enzymes were also able to differentiate between cytosine and 5-methylcytosine. Our results demonstrate the potential in characterizing and developing DNA polymerases for specific PCR based applications in DNA biotechnology and diagnostics.

Standard test methods for chemical and mass spectrometric analysis of nuclear-grade gadolinium oxide (Gd2O3) powder

CERN Document Server

American Society for Testing and Materials. Philadelphia

2006-01-01

1.1 These test methods cover procedures for the chemical and mass spectrometric analysis of nuclear-grade gadolinium oxide powders to determine compliance with specifications. 1.2 The analytical procedures appear in the following order: Sections Carbon by Direct CombustionThermal Conductivity C1408 Test Method for Carbon (Total) in Uranium Oxide Powders and Pellets By Direct Combustion-Infrared Detection Method Total Chlorine and Fluorine by Pyrohydrolysis Ion Selective Electrode C1502 Test Method for Determination of Total Chlorine and Fluorine in Uranium Dioxide and Gadolinium Oxide Loss of Weight on Ignition 7-13 Sulfur by CombustionIodometric Titration Impurity Elements by a Spark-Source Mass Spectrographic C761 Test Methods for Chemical, Mass Spectrometric, Spectrochemical,Nuclear, and Radiochemical Analysis of Uranium Hexafluoride C1287 Test Method for Determination of Impurities In Uranium Dioxide By Inductively Coupled Plasma Mass Spectrometry Gadolinium Content in Gadolinium Oxid...

A gas/liquid chromatographic-mass spectrometric method for the rapid screening of 250 pesticides in aqueous matrices

Energy Technology Data Exchange (ETDEWEB)

Chandramouli, B.; Harvan, D.; Brittain, S.; Hass, R. [Eno River Labs, LLC. Durham, NC (United States)

2004-09-15

Pesticide residues in food present a potentially serious and significant cause for concern. Many pesticides have been associated with significant health effects to the nervous and endocrine systems and some have been deemed carcinogenic. There are many well-established techniques for pesticide analysis. However, commercial pesticide methods have traditionally only been available for specific pesticide families, such as chlorinated pesticides or herbicides, and at detection limits ranging from 0.05 ppb to 1 ppm in aqueous matrices. Techniques that can quickly screen for the presence/absence of pesticide residues in food matrices are critical in ensuring the safety of food and water. This paper outlines a combined Gas Chromatographic-High Resolution Mass Spectrometric (GC-HRMS) and Liquid Chromatographic Tandem Mass Spectrometric (LC-MS/MS) screening assay for 250 pesticides that was developed for use in water, and soda samples at screening levels ranging from 0.1-5 ppb. The pesticides selected have been identified by the European Union as being of concern and the target of possible legislation. The list encompasses a variety of pesticide classes and compound groupings.

Rapid Multiplex Small DNA Sequencing on the MinION Nanopore Sequencing Platform

Directory of Open Access Journals (Sweden)

Shan Wei

2018-05-01

Full Text Available Real-time sequencing of short DNA reads has a wide variety of clinical and research applications including screening for mutations, target sequences and aneuploidy. We recently demonstrated that MinION, a nanopore-based DNA sequencing device the size of a USB drive, could be used for short-read DNA sequencing. In this study, an ultra-rapid multiplex library preparation and sequencing method for the MinION is presented and applied to accurately test normal diploid and aneuploidy samples’ genomic DNA in under three hours, including library preparation and sequencing. This novel method shows great promise as a clinical diagnostic test for applications requiring rapid short-read DNA sequencing.

How mass spectrometric approaches applied to bacterial identification have revolutionized the study of human gut microbiota.

Science.gov (United States)

Grégory, Dubourg; Chaudet, Hervé; Lagier, Jean-Christophe; Raoult, Didier

2018-03-01

Describing the human hut gut microbiota is one the most exciting challenges of the 21 st century. Currently, high-throughput sequencing methods are considered as the gold standard for this purpose, however, they suffer from several drawbacks, including their inability to detect minority populations. The advent of mass-spectrometric (MS) approaches to identify cultured bacteria in clinical microbiology enabled the creation of the culturomics approach, which aims to establish a comprehensive repertoire of cultured prokaryotes from human specimens using extensive culture conditions. Areas covered: This review first underlines how mass spectrometric approaches have revolutionized clinical microbiology. It then highlights the contribution of MS-based methods to culturomics studies, paying particular attention to the extension of the human gut microbiota repertoire through the discovery of new bacterial species. Expert commentary: MS-based approaches have enabled cultivation methods to be resuscitated to study the human gut microbiota and thus to fill in the blanks left by high-throughput sequencing methods in terms of culturing minority populations. Continued efforts to recover new taxa using culture methods, combined with their rapid implementation in genomic databases, would allow for an exhaustive analysis of the gut microbiota through the use of a comprehensive approach.

Gas chromatography of organic microcontaminants using atomic emission and mass spectrometric detection combined in one instrument (GC-AED/MS)

NARCIS (Netherlands)

Mol, H.G.J.; Hankemeier, T.; Brinkman, U.A.T.

1999-01-01

This study describes the coupling of an atomic-emission detector and mass-spectrometric detector to a single gas chromatograph. Splitting of the column effluent enables simultaneous detection by atomic-emission detection (AED) and mass spectrometry (MS) and yields a powerful system for the target

The downfall of TBA-354 - a possible explanation for its neurotoxicity via mass spectrometric imaging.

Science.gov (United States)

Ntshangase, Sphamandla; Shobo, Adeola; Kruger, Hendrik G; Asperger, Arndt; Niemeyer, Dagmar; Arvidsson, Per I; Govender, Thavendran; Baijnath, Sooraj

2017-10-13

1. TBA-354 was a promising antitubercular compound with activity against both replicating and static Mycobacterium tuberculosis (M.tb), making it the focal point of many clinical trials conducted by the TB Alliance. However, findings from these trials have shown that TBA-354 results in mild signs of reversible neurotoxicity; this left the TB Alliance with no other choice but to stop the research. 2. In this study, mass spectrometric methods were used to evaluate the pharmacokinetics and spatial distribution of TBA-354 in the brain using a validated liquid chromatography tandem-mass spectrometry (LCMS/MS) and mass spectrometric imaging (MSI), respectively. Healthy female Sprague-Dawley rats received intraperitoneal (i.p.) doses of TBA-354 (20 mg/kg bw). 3. The concentrationtime profiles showed a gradual absorption and tissue penetration of TBA-354 reaching the C max at 6 h post dose, followed by a rapid elimination. MSI analysis showed a time-dependent drug distribution, with highest drug concentration mainly in the neocortical regions of the brain. 4. The distribution of TBA-354 provides a possible explanation for the motor dysfunction observed in clinical trials. These results prove the importance of MSI as a potential tool in preclinical evaluations of suspected neurotoxic compounds.

Development of an electrochemical DNA biosensor for detection of ...

Indian Academy of Sciences (India)

2.4 million of deaths.1,2 Southern hybridization tech- niques, radiographic .... Electrochemical DNA sensors can be greatly affected .... 3.5 Diagnostic performance of the biosensor ... Silva M M S, Cavalcanti I T, Barroso M F, Sales M G F.

Determination of isotope fractionation effect using a double spike (242Pu+240Pu) during the mass spectrometric analysis of plutonium

International Nuclear Information System (INIS)

Chitambar, S.A.; Parab, A.R.; Khodade, P.S.; Jain, H.C.

1986-01-01

Isotope fractionation effect during the mass spectrometric analysis of plutonium has been investigated using a double spike ( 242 Pu+ 240 Pu) and the determination of concentration of plutonium in dissolver solution of irradiated fuel is reported. (author). 6 refs., 2 tables

Bayesian and maximum entropy methods for fusion diagnostic measurements with compact neutron spectrometers

International Nuclear Information System (INIS)

Reginatto, Marcel; Zimbal, Andreas

2008-01-01

In applications of neutron spectrometry to fusion diagnostics, it is advantageous to use methods of data analysis which can extract information from the spectrum that is directly related to the parameters of interest that describe the plasma. We present here methods of data analysis which were developed with this goal in mind, and which were applied to spectrometric measurements made with an organic liquid scintillation detector (type NE213). In our approach, we combine Bayesian parameter estimation methods and unfolding methods based on the maximum entropy principle. This two-step method allows us to optimize the analysis of the data depending on the type of information that we want to extract from the measurements. To illustrate these methods, we analyze neutron measurements made at the PTB accelerator under controlled conditions, using accelerator-produced neutron beams. Although the methods have been chosen with a specific application in mind, they are general enough to be useful for many other types of measurements

Construction of a fuzzy and all Boolean logic gates based on DNA

DEFF Research Database (Denmark)

M. Zadegan, Reza; Jepsen, Mette D E; Hildebrandt, Lasse

2015-01-01

to the operation of the six Boolean logic gates AND, NAND, OR, NOR, XOR, and XNOR. The logic gate complex is shown to work also when implemented in a three-dimensional DNA origami box structure, where it controlled the position of the lid in a closed or open position. Implementation of multiple microRNA sensitive...... DNA locks on one DNA origami box structure enabled fuzzy logical operation that allows biosensing of complex molecular signals. Integrating logic gates with DNA origami systems opens a vast avenue to applications in the fields of nanomedicine for diagnostics and therapeutics....

Alkaline Comet Assay and Micronucleus Test Parameters in Children Exposed to Diagnostic X-Ray Examination

International Nuclear Information System (INIS)

Gajski, G.; Geric, M.; Garaj-Vrhovac, V.; Milkovic, Dj.; Beck, N.; Ranogajec-Komor, M.; Miljanic, S.; Knezevic, Z.

2011-01-01

Chest radiograms represent the basic radiological examination of thorax and are the most frequently performed radiological diagnostic procedure in the child population. Understanding the risks of low doses of radiation is an important aspect in the risk benefit analysis in paediatric populations. To provide the best care for the young patients the effects of radiation should be minimized thus chest X-rays must be performed by highest standards to ensure that the young patient has the lowest risk possible. Since children are the most sensitive to radiation, there is a need for follow up of the young populations that receive these X-ray diagnostic examinations. Follow up would be especially advisable for children that are at higher risk of radiation induced damage, for example children with a predisposition to DNA damage, or for children that are constantly exposed to numerous radiological examinations due to their illness. In that manner, present study was undertaken to evaluate application of different dosimetry systems in conjunction with alkaline comet assay and micronucleus test for the assessment of different types of DNA and chromosomal alterations in child population exposed to acute diagnostic X-rays examination. For that purpose doses were measured using thermoluminescence (TL) and radiophotoluminescent (RPL) dosimetry systems. The study demonstrated that immediately after exposure to diagnostic X-irradiation, mean percentage of DNA in tail of the comets, which is indirect measures of DNA damage, was significantly changed. The same was noticed for mean total number of micronuclei as well. It was shown that children with pulmonary diseases subjected to diagnostic procedure develop a significant increase in mean total number of each measured parameter which are the biomarkers of genetic damage for carcinogenesis, than prior to diagnostic procedure and that interindividual differences exist for each monitored child. Our results show that genetic damage arises

Non-invasive aneuploidy detection using free fetal DNA and RNA in maternal plasma: recent progress and future possibilities.

NARCIS (Netherlands)

Go, A.T.; Vugt, J.M.G. van; Oudejans, C.B.

2011-01-01

BACKGROUND: Cell-free fetal DNA (cff DNA) and RNA can be detected in maternal plasma and used for non-invasive prenatal diagnostics. Recent technical advances have led to a drastic change in the clinical applicability and potential uses of free fetal DNA and RNA. This review summarizes the latest

Next Generation DNA Sequencing and the Future of Genomic Medicine

OpenAIRE

Anderson, Matthew W.; Schrijver, Iris

2010-01-01

In the years since the first complete human genome sequence was reported, there has been a rapid development of technologies to facilitate high-throughput sequence analysis of DNA (termed “next-generation” sequencing). These novel approaches to DNA sequencing offer the promise of complete genomic analysis at a cost feasible for routine clinical diagnostics. However, the ability to more thoroughly interrogate genomic sequence raises a number of important issues with regard to result interpreta...

A diagnostic algorithm for metabolic myopathies.

Science.gov (United States)

Berardo, Andres; DiMauro, Salvatore; Hirano, Michio

2010-03-01

Metabolic myopathies comprise a clinically and etiologically diverse group of disorders caused by defects in cellular energy metabolism, including the breakdown of carbohydrates and fatty acids to generate adenosine triphosphate, predominantly through mitochondrial oxidative phosphorylation. Accordingly, the three main categories of metabolic myopathies are glycogen storage diseases, fatty acid oxidation defects, and mitochondrial disorders due to respiratory chain impairment. The wide clinical spectrum of metabolic myopathies ranges from severe infantile-onset multisystemic diseases to adult-onset isolated myopathies with exertional cramps. Diagnosing these diverse disorders often is challenging because clinical features such as recurrent myoglobinuria and exercise intolerance are common to all three types of metabolic myopathy. Nevertheless, distinct clinical manifestations are important to recognize as they can guide diagnostic testing and lead to the correct diagnosis. This article briefly reviews general clinical aspects of metabolic myopathies and highlights approaches to diagnosing the relatively more frequent subtypes (Fig. 1). Fig. 1 Clinical algorithm for patients with exercise intolerance in whom a metabolic myopathy is suspected. CK-creatine kinase; COX-cytochrome c oxidase; CPT-carnitine palmitoyl transferase; cyt b-cytochrome b; mtDNA-mitochondrial DNA; nDNA-nuclear DNA; PFK-phosphofructokinase; PGAM-phosphoglycerate mutase; PGK-phosphoglycerate kinase; PPL-myophosphorylase; RRF-ragged red fibers; TFP-trifunctional protein deficiency; VLCAD-very long-chain acyl-coenzyme A dehydrogenase.

Aggressive NK-cell leukemia: A rare entity with diagnostic and therapeutic challenge

Directory of Open Access Journals (Sweden)

Alia Nazarullah

2016-06-01

Full Text Available Aggressive natural killer cell leukemia (ANKL is a rare neoplasm of mature natural killer cells, with an extremely poor overall survival, which is almost always EBV related, with majority of cases reported in East Asia. Here we report the case of an ANKL presenting in a young Hispanic male with secondary hemophagocytosis. Aggressive clinical course, high EBV DNA levels and leukemic presentation, often with associated hemophagocytosis, should raise suspicion of an NK/T-cell neoplasm like ANKL. Due to significant diagnostic overlap with extranodal NK/T-cell lymphoma, nasal type (ENKL, accurate diagnostic classification is crucial due to differing treatment and prognosis. L-asparaginase including chemotherapy followed by allogeneic stem cell transplantation appears to slightly prolong overall survival, but relapse is almost inevitable. Clinical monitoring of EBV DNA levels shows good correlation with disease activity.

Potential of DNA methylation in rectal cancer as diagnostic and prognostic biomarkers

Science.gov (United States)

Exner, Ruth; Pulverer, Walter; Diem, Martina; Spaller, Lisa; Woltering, Laura; Schreiber, Martin; Wolf, Brigitte; Sonntagbauer, Markus; Schröder, Fabian; Stift, Judith; Wrba, Fritz; Bergmann, Michael; Weinhäusel, Andreas; Egger, Gerda

2015-01-01

Background: Aberrant DNA methylation is more prominent in proximal compared with distal colorectal cancers. Although a number of methylation markers were identified for colon cancer, yet few are available for rectal cancer. Methods: DNA methylation differences were assessed by a targeted DNA microarray for 360 marker candidates between 22 fresh frozen rectal tumour samples and 8 controls and validated by microfluidic high-throughput and methylation-sensitive qPCR in fresh frozen and formalin-fixed paraffin-embedded (FFPE) samples, respectively. The CpG island methylator phenotype (CIMP) was assessed by MethyLight in FFPE material from 78 patients with pT2 and pT3 rectal adenocarcinoma. Results: We identified and confirmed two novel three-gene signatures in fresh frozen samples that can distinguish tumours from adjacent tissue as well as from blood with a high sensitivity and specificity of up to 1 and an AUC of 1. In addition, methylation of individual CIMP markers was associated with specific clinical parameters such as tumour stage, therapy or patients' age. Methylation of CDKN2A was a negative prognostic factor for overall survival of patients. Conclusions: The newly defined methylation markers will be suitable for early disease detection and monitoring of rectal cancer. PMID:26335606

Assessing patterns of hybridization between North Atlantic eels using diagnostic single-nucleotide polymorphisms

DEFF Research Database (Denmark)

Pujolar, José Martin; Jacobsen, M.W.; Als, Thomas Damm

2014-01-01

The two North Atlantic eel species, the European eel (Anguilla anguilla) and the American eel (Anguilla rostrata), spawn in partial sympatry in the Sargasso Sea, providing ample opportunity to interbreed. In this study, we used a RAD (Restriction site Associated DNA) sequencing approach to identify...... species-specific diagnostic single-nucleotide polymorphisms (SNPs) and design a low-density array that combined with screening of a diagnostic mitochondrial DNA marker. Eels from Iceland (N=159) and from the neighboring Faroe Islands (N=29) were genotyped, along with 94 larvae (49 European and 45 American...... eel male crosses, backcrosses were also detected, including a first-generation backcross (F1 hybrid × pure European eel) and three individuals identified as second-generation backcrosses originating from American eel × F1 hybrid backcrosses interbreeding with pure European eels. In comparison...

Mass spectrometric identification of diagnostic markers for chronic prostatitis in seminal plasma by analysis of seminal plasma protein clinical samples.

Science.gov (United States)

Rokka, A; Mehik, A; Tonttila, P; Vaarala, M

2017-08-15

There are few specific diagnostic markers for chronic prostatitis. Therefore, we used mass spectrometry to evaluate differences in seminal plasma protein expression among patients with prostatitis and young and middle-aged healthy controls. We analysed pooled seminal plasma protein samples from four prostatitis patients (two pools), three young controls (one pool), and three middle-aged controls (one pool). The samples were analysed by liquid chromatography-tandem mass spectrometry. Of the 349 proteins identified, 16 were differentially expressed between the two control pools. Five proteins were up- or down-regulated in both of the prostatitis pools compared to middle-aged controls but not between young and middle-aged pools. Progestagen-associated endometrial protein (PAEP) was over-expressed in prostatitis samples compared to young and middle-aged controls. Our findings and those of previous studies indicate that PAEP is a potential seminal plasma marker for chronic prostatitis. In conclusion, we found age-related changes in seminal plasma protein expression. PAEP expression in seminal plasma should be investigated further to evaluate its potential as a diagnostic marker for chronic prostatitis.

Enzyme-Free Detection of Mutations in Cancer DNA Using Synthetic Oligonucleotide Probes and Fluorescence Microscopy

DEFF Research Database (Denmark)

Miotke, Laura; Maity, Arindam; Ji, Hanlee

2015-01-01

BACKGROUND: Rapid reliable diagnostics of DNA mutations are highly desirable in research and clinical assays. Current development in this field goes simultaneously in two directions: 1) high-throughput methods, and 2) portable assays. Non-enzymatic approaches are attractive for both types...... 1000-fold above the potential detection limit. CONCLUSION: Overall, the novel assay we describe could become a new approach to rapid, reliable and enzyme-free diagnostics of cancer or other associated DNA targets. Importantly, stoichiometry of wild type and mutant targets is conserved in our assay...... of methods since they would allow rapid and relatively inexpensive detection of nucleic acids. Modern fluorescence microscopy is having a huge impact on detection of biomolecules at previously unachievable resolution. However, no straightforward methods to detect DNA in a non-enzymatic way using fluorescence...

Speciation and detection of arsenic in aqueous samples: A review of recent progress in non-atomic spectrometric methods

Energy Technology Data Exchange (ETDEWEB)

Ma, Jian [State Key Laboratory of Marine Environmental Science, College of the Environment and Ecology, Xiamen University, Xiamen 361102 (China); Department of Chemistry and Biochemistry, University of Texas, 700 Planetarium Place, Arlington, TX 76019 (United States); Sengupta, Mrinal K. [Department of Chemistry and Biochemistry, University of Texas, 700 Planetarium Place, Arlington, TX 76019 (United States); Thermo Fisher Scientific, Dionex Products, 445 Lakeside Drive, Sunnyvale, CA, 94085 (United States); Yuan, Dongxing [State Key Laboratory of Marine Environmental Science, College of the Environment and Ecology, Xiamen University, Xiamen 361102 (China); Dasgupta, Purnendu K., E-mail: Dasgupta@uta.edu [Department of Chemistry and Biochemistry, University of Texas, 700 Planetarium Place, Arlington, TX 76019 (United States)

2014-06-01

Highlights: • Compilation of principal official documents and major review articles, including the toxicology and chemistry of As. • Review of non-atomic spectrometric methods for speciation and detection of arsenic in aqueous samples (2005–2013) of the performance of field-usable methods. - Abstract: Inorganic arsenic (As) displays extreme toxicity and is a class A human carcinogen. It is of interest to both analytical chemists and environmental scientists. Facile and sensitive determination of As and knowledge of the speciation of forms of As in aqueous samples are vitally important. Nearly every nation has relevant official regulations on permissible limits of drinking water As content. The size of the literature on As is therefore formidable. The heart of this review consists of two tables: one is a compilation of principal official documents and major review articles, including the toxicology and chemistry of As. This includes comprehensive official compendia on As speciation, sample treatment, recommended procedures for the determination of As in specific sample matrices with specific analytical instrument(s), procedures for multi-element (including As) speciation and analysis, and prior comprehensive reviews on arsenic analysis. The second table focuses on the recent literature (2005–2013, the coverage for 2013 is incomplete) on As measurement in aqueous matrices. Recent As speciation and analysis methods based on spectrometric and electrochemical methods, inductively coupled plasma-mass spectrometry, neutron activation analysis and biosensors are summarized. We have deliberately excluded atomic optical spectrometric techniques (atomic absorption, atomic fluorescence, inductively coupled plasma-optical emission spectrometry) not because they are not important (in fact the majority of arsenic determinations are possibly carried out by one of these techniques) but because these methods are sufficiently mature and little meaningful innovation has been

Speciation and detection of arsenic in aqueous samples: A review of recent progress in non-atomic spectrometric methods

International Nuclear Information System (INIS)

Ma, Jian; Sengupta, Mrinal K.; Yuan, Dongxing; Dasgupta, Purnendu K.

2014-01-01

Highlights: • Compilation of principal official documents and major review articles, including the toxicology and chemistry of As. • Review of non-atomic spectrometric methods for speciation and detection of arsenic in aqueous samples (2005–2013) of the performance of field-usable methods. - Abstract: Inorganic arsenic (As) displays extreme toxicity and is a class A human carcinogen. It is of interest to both analytical chemists and environmental scientists. Facile and sensitive determination of As and knowledge of the speciation of forms of As in aqueous samples are vitally important. Nearly every nation has relevant official regulations on permissible limits of drinking water As content. The size of the literature on As is therefore formidable. The heart of this review consists of two tables: one is a compilation of principal official documents and major review articles, including the toxicology and chemistry of As. This includes comprehensive official compendia on As speciation, sample treatment, recommended procedures for the determination of As in specific sample matrices with specific analytical instrument(s), procedures for multi-element (including As) speciation and analysis, and prior comprehensive reviews on arsenic analysis. The second table focuses on the recent literature (2005–2013, the coverage for 2013 is incomplete) on As measurement in aqueous matrices. Recent As speciation and analysis methods based on spectrometric and electrochemical methods, inductively coupled plasma-mass spectrometry, neutron activation analysis and biosensors are summarized. We have deliberately excluded atomic optical spectrometric techniques (atomic absorption, atomic fluorescence, inductively coupled plasma-optical emission spectrometry) not because they are not important (in fact the majority of arsenic determinations are possibly carried out by one of these techniques) but because these methods are sufficiently mature and little meaningful innovation has been

Comparison of first-tier cell-free DNA screening for common aneuploidies with conventional publically funded screening.

Science.gov (United States)

Langlois, Sylvie; Johnson, JoAnn; Audibert, François; Gekas, Jean; Forest, Jean-Claude; Caron, André; Harrington, Keli; Pastuck, Melanie; Meddour, Hasna; Tétu, Amélie; Little, Julian; Rousseau, François

2017-12-01

This study evaluates the impact of offering cell-free DNA (cfDNA) screening as a first-tier test for trisomies 21 and 18. This is a prospective study of pregnant women undergoing conventional prenatal screening who were offered cfDNA screening in the first trimester with clinical outcomes obtained on all pregnancies. A total of 1198 pregnant women were recruited. The detection rate of trisomy 21 with standard screening was 83% with a false positive rate (FPR) of 5.5% compared with 100% detection and 0% FPR for cfDNA screening. The FPR of cfDNA screening for trisomies 18 and 13 was 0.09% for each. Two percent of women underwent an invasive diagnostic procedure based on screening or ultrasound findings; without the cfDNA screening, it could have been as high as 6.8%. Amongst the 640 women with negative cfDNA results and a nuchal translucency (NT) ultrasound, only 3 had an NT greater or equal to 3.5 mm: one had a normal outcome and two lost their pregnancy before 20 weeks. cfDNA screening has the potential to be a highly effective first-tier screening approach leading to a significant reduction of invasive diagnostic procedures. For women with a negative cfDNA screening result, NT measurement has limited clinical utility. © 2017 John Wiley & Sons, Ltd.

The Development of a Post-Baccalaureate Certificate Program in Molecular Diagnostics

Science.gov (United States)

Williams, Gail S.; Brown, Judith D.; Keagle, Martha B.

2000-01-01

A post-baccalaureate certificate program in diagnostic molecular sciences was created in 1995 by the Diagnostic Genetic Sciences Program in the School of Allied Health at the University of Connecticut. The required on-campus lecture and laboratory courses include basic laboratory techniques, health care issues, cell biology, immunology, human genetics, research, management, and molecular diagnostic techniques and laboratory in molecular diagnostics. These courses precede a 6-month, full-time practicum at an affiliated full-service molecular laboratory. The practicum includes amplification and blotting methods, a research project, and a choice of specialized electives including DNA sequencing, mutagenesis, in situ hybridization methods, or molecular diagnostic applications in microbiology. Graduates of the program are immediately eligible to sit for the National Credentialing Agency examination in molecular biology to obtain the credential Clinical Laboratory Specialist in Molecular Biology (CLSp(MB). This description of the University of Connecticut program may assist other laboratory science programs in creating similar curricula. PMID:11232107

iMS2Flux – a high–throughput processing tool for stable isotope labeled mass spectrometric data used for metabolic flux analysis

Directory of Open Access Journals (Sweden)

Poskar C Hart

2012-11-01

Full Text Available Abstract Background Metabolic flux analysis has become an established method in systems biology and functional genomics. The most common approach for determining intracellular metabolic fluxes is to utilize mass spectrometry in combination with stable isotope labeling experiments. However, before the mass spectrometric data can be used it has to be corrected for biases caused by naturally occurring stable isotopes, by the analytical technique(s employed, or by the biological sample itself. Finally the MS data and the labeling information it contains have to be assembled into a data format usable by flux analysis software (of which several dedicated packages exist. Currently the processing of mass spectrometric data is time-consuming and error-prone requiring peak by peak cut-and-paste analysis and manual curation. In order to facilitate high-throughput metabolic flux analysis, the automation of multiple steps in the analytical workflow is necessary. Results Here we describe iMS2Flux, software developed to automate, standardize and connect the data flow between mass spectrometric measurements and flux analysis programs. This tool streamlines the transfer of data from extraction via correction tools to 13C-Flux software by processing MS data from stable isotope labeling experiments. It allows the correction of large and heterogeneous MS datasets for the presence of naturally occurring stable isotopes, initial biomass and several mass spectrometry effects. Before and after data correction, several checks can be performed to ensure accurate data. The corrected data may be returned in a variety of formats including those used by metabolic flux analysis software such as 13CFLUX, OpenFLUX and 13CFLUX2. Conclusion iMS2Flux is a versatile, easy to use tool for the automated processing of mass spectrometric data containing isotope labeling information. It represents the core framework for a standardized workflow and data processing. Due to its flexibility

Development of gallium arsenide gamma spectrometric detector

International Nuclear Information System (INIS)

Kobayashi, T.; Kuru, I.

1975-03-01

GaAs semiconductor material has been considered to be a suitable material for gamma-ray spectrometer operating at room temperature since it has a wid-band gap, larger than that of silicon and germanium. The basic objective of this work is to develop a GaAs gamma-ray spectrometric detector which could be used for gamma spectrometric measurement of uranium and plutonium in nuclear fuel safeguards. Liquid phase epitaxial techniques using iron (Fe) as dopant have been developed in making high purity GaAs crystals suitable for gamma-ray spectrometer operating at room temperature. Concentration of Fe in the epitaxial crystal was controlled by initial growth temperature. The best quality epitaxial crystal was obtained under the following conditions: starting temperature is about 800degC, the proportion of Fe to Ga solvent is 1 to 300. Carrier concentration of epitaxial crystals grown distributed in the ranges of 10 12 cm -3 to 10 14 cm -3 at room temperature. The thickness of the crystals ranged from 38 μm to 120 μm. Au-GaAs surface barrier detector was made of epitaxial crystal. Some of the detector were encapsulated in a can with a 50 μm Be window by welding a can to the detector holder. The detector with high energy resolution and good charge collecting characteristics was selected by alpha spectrometry at room temperature. Energy resolution of the detector for gamma-rays up to about 200 keV was very good at room temperature operation. The best energy resolutions taken with a GaAs detector were 3 keV (fwhm) and 3.8 keV for 241 Am 59.6 keV and 57 Co 122 keV, respectively, at room temperature. In order to study the applicability of the detector for nuclear safeguards, the measurements of 235 U gamma-ray spectrum have been carried out at room temperature. It was clarified that the gamma-ray spectrum of enriched U sample could be measured in high resolution with GaAs detector at room temperature, and that the content of 235 U in enriched U sources could be determined by

DNA Sensors for Malaria Diagnosis

DEFF Research Database (Denmark)

Hede, Marianne Smedegaard; Fjelstrup, Søren; Knudsen, Birgitta R.

2015-01-01

In the field of malaria diagnosis much effort is put into the development of faster and easier alternatives to the gold standard, blood smear microscopy. Nucleic acid amplification based techniques pose some of the most promising upcoming diagnostic tools due to their potential for high sensitivity......, robustness and user-friendliness. In the current review, we will discuss some of the different DNA-based sensor systems under development for the diagnosis of malaria....

Mitochondrial DNA Mutation Associated with Leber's Hereditary Optic Neuropathy

Science.gov (United States)

Wallace, Douglas C.; Singh, Gurparkash; Lott, Marie T.; Hodge, Judy A.; Schurr, Theodore G.; Lezza, Angela M. S.; Elsas, Louis J.; Nikoskelainen, Eeva K.

1988-12-01

Leber's hereditary optic neuropathy is a maternally inherited disease resulting in optic nerve degeneration and cardiac dysrhythmia. A mitochondrial DNA replacement mutation was identified that correlated with this disease in multiple families. This mutation converted a highly conserved arginine to a histidine at codon 340 in the NADH dehydrogenase subunit 4 gene and eliminated an Sfa NI site, thus providing a simple diagnostic test. This finding demonstrated that a nucleotide change in a mitochondrial DNA energy production gene can result in a neurological disease.

Ecological niche modelling and nDNA sequencing support a new, morphologically cryptic beetle species unveiled by DNA barcoding.

Science.gov (United States)

Hawlitschek, Oliver; Porch, Nick; Hendrich, Lars; Balke, Michael

2011-02-09

DNA sequencing techniques used to estimate biodiversity, such as DNA barcoding, may reveal cryptic species. However, disagreements between barcoding and morphological data have already led to controversy. Species delimitation should therefore not be based on mtDNA alone. Here, we explore the use of nDNA and bioclimatic modelling in a new species of aquatic beetle revealed by mtDNA sequence data. The aquatic beetle fauna of Australia is characterised by high degrees of endemism, including local radiations such as the genus Antiporus. Antiporus femoralis was previously considered to exist in two disjunct, but morphologically indistinguishable populations in south-western and south-eastern Australia. We constructed a phylogeny of Antiporus and detected a deep split between these populations. Diagnostic characters from the highly variable nuclear protein encoding arginine kinase gene confirmed the presence of two isolated populations. We then used ecological niche modelling to examine the climatic niche characteristics of the two populations. All results support the status of the two populations as distinct species. We describe the south-western species as Antiporus occidentalis sp.n. In addition to nDNA sequence data and extended use of mitochondrial sequences, ecological niche modelling has great potential for delineating morphologically cryptic species.

Comparative analysis of protocols for DNA extraction from soybean caterpillars.

Science.gov (United States)

Palma, J; Valmorbida, I; da Costa, I F D; Guedes, J V C

2016-04-07

Genomic DNA extraction is crucial for molecular research, including diagnostic and genome characterization of different organisms. The aim of this study was to comparatively analyze protocols of DNA extraction based on cell lysis by sarcosyl, cetyltrimethylammonium bromide, and sodium dodecyl sulfate, and to determine the most efficient method applicable to soybean caterpillars. DNA was extracted from specimens of Chrysodeixis includens and Spodoptera eridania using the aforementioned three methods. DNA quantification was performed using spectrophotometry and high molecular weight DNA ladders. The purity of the extracted DNA was determined by calculating the A260/A280 ratio. Cost and time for each DNA extraction method were estimated and analyzed statistically. The amount of DNA extracted by these three methods was sufficient for PCR amplification. The sarcosyl method yielded DNA of higher purity, because it generated a clearer pellet without viscosity, and yielded high quality amplification products of the COI gene I. The sarcosyl method showed lower cost per extraction and did not differ from the other methods with respect to preparation times. Cell lysis by sarcosyl represents the best method for DNA extraction in terms of yield, quality, and cost effectiveness.

Selective Covalent Conjugation of Phosphorothioate DNA Oligonucleotides with Streptavidin

Directory of Open Access Journals (Sweden)

Christof M. Niemeyer

2011-08-01

Full Text Available Protein-DNA conjugates have found numerous applications in the field of diagnostics and nanobiotechnology, however, their intrinsic susceptibility to DNA degradation by nucleases represents a major obstacle for many applications. We here report the selective covalent conjugation of the protein streptavidin (STV with phosphorothioate oligonucleotides (psDNA containing a terminal alkylthiolgroup as the chemically addressable linking unit, using a heterobifunctional NHS-/maleimide crosslinker. The psDNA-STV conjugates were synthesized in about 10% isolated yields. We demonstrate that the terminal alkylthiol group selectively reacts with the maleimide while the backbone sulfur atoms are not engaged in chemical conjugation. The novel psDNA-STV conjugates retain their binding capabilities for both biotinylated macromolecules and the complementary nucleic acid. Moreover, the psDNA-STV conjugate retained its binding capacity for complementary oligomers even after a nuclease digestion step, which effectively degrades deoxyribonucleotide oligomers and thus the binding capability of regular DNA-STV conjugates. The psDNA-STV therefore hold particular promise for applications e.g. in proteome research and novel biosensing devices, where interfering endogenous nucleic acids need to be removed from analytes by nuclease digestion.

Synthetic LNA/DNA nano-scaffolds for highly efficient diagnostics of nucleic acids and autoimmune antibodies

DEFF Research Database (Denmark)

Astakhova, Irina Kira

2014-01-01

) strands and a series of fluorescent azides. The multiply labeled fluorescent LNA/DNA probes prepared herein generally display high binding affinity to complementary DNA/RNA, high quantum yields and, hence, high fluorescence brightness values. With the novel fluorescent probes, specific sensing...

Evaluation of radioactive environmental hazards in Area-3, Northern Palmyrides, Central Syria using airborne spectrometric gamma technique

International Nuclear Information System (INIS)

Asfahani, J.; Aissa, M.; Al-Hent, R.

2016-01-01

Airborne spectrometric gamma data are used in this paper to estimate the degree of radioactive hazard on humanity in Area-3, Northern Palmyrides, Central Syria. Exposure Rate (ER), Absorbed Dose Rate (ADR), Annual Effective Dose Rate (AEDR), and Heat Production (HP) of the eleven radiometric units included in the established lithological scored map in the study area have been computed to evaluate the radiation background influence in humans. The results obtained indicate that a human body in Area-3 is subjected to radiation hazards in the acceptable limits for long duration exposure. The highest radiogenetic heat production values in Area-3 correspond to the phosphatic locations characterized by relatively high values of uranium and thorium. - Highlights: • Degree of radioactive hazard has been estimated by using airborne spectrometric gamma data. • ER, ADR, AEDR, and HP of the eleven radiometric units have been computed. • Comparison of AEDR of Area-3 with the AEDR of Area-1. • Human body in Area-3 is subjected to radiation hazards in the acceptable limits for long duration exposure. • The highest heat production in Area-3 correspond to the phosphatic locations.

Reply to "On Vaporization of liquid Pb-Li eutectic alloy from 1000 K to 1200 K- A high temperature mass spectrometric study"

Science.gov (United States)

Jain, Uttam; Mukherjee, Abhishek

2018-03-01

This communication is in response to a letter to editor commenting on the authors' earlier paper "Vaporization of liquid Pb-Li eutectic alloy from 1000 K to 1200 K - A high temperature mass spectrometric study".

DNA decontamination methods for internal quality management in clinical PCR laboratories.

Science.gov (United States)

Wu, Yingping; Wu, Jianyong; Zhang, Zhihui; Cheng, Chen

2018-03-01

The polymerase chain reaction (PCR) technique, one of the most commonly applied methods in diagnostic and molecular biology, has a frustrating downside: the occurrence of false-positive signals due to contamination. In previous research, various DNA decontamination methods have been developed to overcome this limitation. Unfortunately, the use of random or poorly focused sampling methods for monitoring air and/or object surfaces leads to the incomplete elimination during decontamination procedures. We herein attempted to develop a novel DNA decontamination method (environmental surveillance, including surface and air sampling) and quality management program for clinical molecular diagnostic laboratories (or clinical PCR laboratories). Here, we performed a step-by-step evaluation of current DNA decontamination methods and developed an effective procedure for assessing the presence of decontaminating DNA via PCR analysis. Performing targeted environmental surveillance by sampling, which reached optimal performance over 2 weeks, and the decontamination process had been verified as reliable. Additionally, the process was validated to not affect PCR amplification efficiency based on a comparative study. In this study, effective guidelines for DNA decontamination were developed. The method employed ensured that surface DNA contamination could be effectively identified and eliminated. Furthermore, our study highlighted the importance of overall quality assurance and good clinical laboratory practices for preventing contamination, which are key factors for compliance with regulatory or accreditation requirements. Taken together, we provided the evidence that the presented scheme ranged from troubleshooting to the elimination of surface contamination, could serve as critical foundation for developing regular environmental surveillance guidelines for PCR laboratories. © 2017 Wiley Periodicals, Inc.

Characterisation of radioactive contaminated materials by combined radiometric and spectrometric methods

International Nuclear Information System (INIS)

Dulama, C.; Toma, A.; Dobrin, R.; Ciocîrlan, C.; Stoica, S.; Valeca, M.; Popescu, I. I.

2013-01-01

In the present paper, a combined analytical methodology is described, for characterization of radioactive contaminated materials. The subject of testing activities was a set of solutions provided by the Cernavoda NPP, which are originating from processes of radiological survey of workplaces in the plant. In the introduction section, a theoretical approach was given to the origin and nature of main radionuclides occurring in the primary cooling system of the nuclear power plant, with the aim to establish selection criteria and performance requirements for the analytical methods to be used in the development of the characterization methodology. A combination of radiometric and spectrometric methods was selected, based on gross beta counting, high resolution gamma-ray spectrometry and liquid scintillation counting. (authors)

Liquid chromatography-tandem mass spectrometric assay for the tyrosine kinase inhibitor afatinib in mouse plasma using salting-out liquid-liquid extraction

NARCIS (Netherlands)

Sparidans, Rolf W; van Hoppe, Stephanie; Rood, Johannes J M; Schinkel, Alfred H; Schellens, Jan H M; Beijnen, Jos H

2016-01-01

A quantitative bioanalytical liquid chromatography-tandem mass spectrometric (LC-MS/MS) assay for afatinib, an irreversible inhibitor of the ErbB (erythroblastic leukemia viral oncogene homolog) tyrosine kinase family, was developed and validated. Plasma samples were pre-treated using salting-out

DNA origami applications in cancer therapy.

Science.gov (United States)

Udomprasert, Anuttara; Kangsamaksin, Thaned

2017-08-01

Due to the complexity and heterogeneity of cancer, the development of cancer diagnosis and therapy is still progressing, and a complete understanding of cancer biology remains elusive. Recently, cancer nanomedicine has gained much interest as a promising diagnostic and therapeutic strategy, as a wide range of nanomaterials possess unique physical properties that can render drug delivery systems safer and more effective. Also, targeted drug delivery and precision medicine have now become a new paradigm in cancer therapy. With nanocarriers, chemotherapeutic drugs could be directly delivered into target cancer cells, resulting in enhanced efficiency with fewer side-effects. DNA, a biomolecule with molecular self-assembly properties, has emerged as a versatile nanomaterial to construct multifunctional platforms; DNA nanostructures can be modified with functional groups to improve their utilities as biosensors or drug carriers. Such applications have become possible with the advent of the scaffolded DNA origami method. This breakthrough technique in structural DNA nanotechnology provides an easier and faster way to construct DNA nanostructures with various shapes. Several experiments proved that DNA origami nanostructures possess abilities to enhance efficacies of chemotherapy, reduce adverse side-effects, and even circumvent drug resistance. Here, we highlight the principles of the DNA origami technique and its applications in cancer therapeutics and discuss current challenges and opportunities to improve cancer detection and targeted drug delivery. © 2017 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

DNA nanomaterials for preclinical imaging and drug delivery.

Science.gov (United States)

Jiang, Dawei; England, Christopher G; Cai, Weibo

2016-10-10

Besides being the carrier of genetic information, DNA is also an excellent biological organizer to establish well-designed nanostructures in the fields of material engineering, nanotechnology, and biomedicine. DNA-based materials represent a diverse nanoscale system primarily due to their predictable base pairing and highly regulated conformations, which greatly facilitate the construction of DNA nanostructures with distinct shapes and sizes. Integrating the emerging advancements in bioconjugation techniques, DNA nanostructures can be readily functionalized with high precision for many purposes ranging from biosensors to imaging to drug delivery. Recent progress in the field of DNA nanotechnology has exhibited collective efforts to employ DNA nanostructures as smart imaging agents or delivery platforms within living organisms. Despite significant improvements in the development of DNA nanostructures, there is limited knowledge regarding the in vivo biological fate of these intriguing nanomaterials. In this review, we summarize the current strategies for designing and purifying highly-versatile DNA nanostructures for biological applications, including molecular imaging and drug delivery. Since DNA nanostructures may elicit an immune response in vivo, we also present a short discussion of their potential toxicities in biomedical applications. Lastly, we discuss future perspectives and potential challenges that may limit the effective preclinical and clinical employment of DNA nanostructures. Due to their unique properties, we predict that DNA nanomaterials will make excellent agents for effective diagnostic imaging and drug delivery, improving patient outcome in cancer and other related diseases in the near future. Copyright © 2016 Elsevier B.V. All rights reserved.

Functional proteomics with new mass spectrometric and bioinformatics tools

International Nuclear Information System (INIS)

Kesners, P.W.A.

2001-01-01

A comprehensive range of mass spectrometric tools is required to investigate todays life science applications and a strong focus is on addressing the needs of functional proteomics. Application examples are given showing the streamlined process of protein identification from low femtomole amounts of digests. Sample preparation is achieved with a convertible robot for automated 2D gel picking, and MALDI target dispensing. MALDI-TOF or ESI-MS subsequent to enzymatic digestion. A choice of mass spectrometers including Q-q-TOF with multipass capability, MALDI-MS/MS with unsegmented PSD, Ion Trap and FT-MS are discussed for their respective strengths and applications. Bioinformatics software that allows both database work and novel peptide mass spectra interpretation is reviewed. The automated database searching uses either entire digest LC-MS n ESI Ion Trap data or MALDI MS and MS/MS spectra. It is shown how post translational modifications are interactively uncovered and de-novo sequencing of peptides is facilitated

Mass spectrometric study of vaporization of cesium tellurate and tellurite

International Nuclear Information System (INIS)

Semenov, G.A.; Fokina, L.A.; Mouldagalieva, R.A.

1994-01-01

The process of vaporization of cesium tellurate and tellurite was studied by the Knudsen effusion method with a mass spectrometric analysis of the vapor composition. The thermal dissociation of Cs 2 TeO 4 to Cs 2 TeO 3 and the congruent vaporization of Cs 2 TeO 3 were established. Thermodynamic functions for gaseous Cs 2 TeO 3 have been calculated. The standard enthalpy of sublimation Δ s H (298.15)=268.1±13.0 kJ mol -1 was determined by the 2nd and 3rd laws of thermodynamics. The enthalpy of formation Δ f H (298.15)=-725.1±13.0 kJ mol -1 for gaseous Cs 2 TeO 3 and the enthalpy of atomization Δ at H (298.15)=1841.3±15.0 kJ mol -1 have been computed. ((orig.))

Evidence of authentic DNA from Danish Viking Age skeletons untouched by humans for 1,000 years

DEFF Research Database (Denmark)

Melchior, Linea; Kivisild, Toomas; Lynnerup, Niels

2008-01-01

-PCR work was carried out in a "clean- laboratory" dedicated solely to ancient DNA work. Mitochondrial DNA was extracted and overlapping fragments spanning the HVR-1 region as well as diagnostic sites in the coding region were PCR amplified, cloned and sequenced. Consistent results were obtained...

Terverticillate Penicillia studied by direct electrospray mass spectrometric profiling of crude extracts: I. Chemosystematics

DEFF Research Database (Denmark)

Smedsgaard, Jørn; Frisvad, Jens Christian

1997-01-01

) and Yeast Extract Sucrose agar (YES) directly into the electrospray source of the mass spectrometer. A data matrix was made from each substrate by transferring the complete centroid mass spectrum from 200 to 700 amu as 501 variables to individual columns. No attempt was made to identify ions in the mass......A chemosystematic study of 339 isolates from all known terverticillate Penicillium taxa was performed using electrospray mass spectrometric analysis of extractable metabolites. The mass profiles were made by injecting crude plug extracts made from cultures grown on Czapek Yeast Autolysate agar (CYA...

Certification of model spectrometric alpha sources (MSAS) and problems of the MSAS system improvement

International Nuclear Information System (INIS)

Belyatskij, A.F.; Gejdel'man, A.M.; Egorov, Yu.S.; Nedovesov, V.G.; Chechev, V.P.

1984-01-01

Results of certification of standard spectrometric alpha sources (SSAS) of industrial production are presented: methods for certification by main radiation physical parameters: proper halfwidth of α-lines, activity of radionuclides in the source, energies of α-particle emitting sources and relative intensity of different energy α-particle groups - are analysed. The advantage for the SSAS system improvement - a set of model measures for α-radiation, a collection of interconnected data units on physical, engineering and design characteristics of SSAS, methods for their obtaining and determination, on instruments used, is considered

Enzymatic Production of Monoclonal Stoichiometric Single-Stranded DNA Oligonucleotides

Science.gov (United States)

Ducani, Cosimo; Kaul, Corinna; Moche, Martin; Shih, William M.; Högberg, Björn

2013-01-01

Single-stranded oligonucleotides are important as research tools as probes for diagnostics and gene therapy. Today, production of oligonucleotides is done via solid-phase synthesis. However, the capabilities of current polymer chemistry are limited in comparison to what can be produced in biological systems. The errors in synthetic DNA increases with oligonucleotide length, and sequence diversity can often be a problem. Here, we present the Monoclonal Stoichiometric (MOSIC) method for enzymatic DNA oligonucleotide production. Using this method, we amplify oligonucleotides from clonal templates followed by digestion of a cutter-hairpin, resulting in pools of monoclonal oligonucleotides with precisely controlled relative stoichiometric ratios. We present data where MOSIC oligonucleotides, 14–378 nt long, were prepared either by in vitro rolling-circle amplification, or by amplification in Escherichia coli in the form of phagemid DNA. The formation of a DNA crystal and folding of DNA nanostructures confirmed the scalability, purity and stoichiometry of the produced oligonucleotides. PMID:23727986

Does more favourable handling of the cerebrospinal fluid increase the diagnostic sensitivity of Borrelia burgdorferi sensu lato-specific PCR in Lyme neuroborreliosis?

Science.gov (United States)

Forselv, Kristine J N; Lorentzen, Åslaug R; Ljøstad, Unn; Mygland, Åse; Eikeland, Randi; Kjelland, Vivian; Noraas, Sølvi; Quarsten, Hanne

2018-04-01

Tests for direct detection of Borrelia burgdorferi sensu lato (Bb) in Lyme neuroborreliosis (LNB) are needed. Detection of Bb DNA using PCR is promising, but clinical utility is hampered by low diagnostic sensitivity. We aimed to examine whether diagnostic sensitivity can be improved by the use of larger cerebrospinal fluid (CSF) volumes and faster handling of samples. Patients who underwent CSF examination for LNB were included. We collected two millilitres of CSF for PCR analysis, extracted DNA from the pellets within 24 h and analysed the eluate by two real-time PCR protocols (16S rRNA and OspA). Patients who fulfilled diagnostic criteria for LNB were classified as LNB cases and the rest as controls. Bb DNA in CSF was detected by PCR in seven of 28 adults with LNB. Two were Bb antibody negative. No Bb DNA was detected in CSF from 137 controls. Diagnostic sensitivity was 25% and specificity 100%. There was a non-significant trend towards larger CSF sample volume, faster handling of the sample, shorter duration of symptoms, and higher CSF cell count in the PCR-positive cases. We did not find that optimized handling of CSF increased diagnostic sensitivity of PCR in adults with LNB. However, our case series is small and we hypothesize that the importance of these factors will be clarified in further studies with larger case series and altered study design. PCR for diagnosis of LNB may be useful in cases without Bb antibodies due to short duration of symptoms.

The rapid evolution of molecular genetic diagnostics in neuromuscular diseases.

Science.gov (United States)

Volk, Alexander E; Kubisch, Christian

2017-10-01

The development of massively parallel sequencing (MPS) has revolutionized molecular genetic diagnostics in monogenic disorders. The present review gives a brief overview of different MPS-based approaches used in clinical diagnostics of neuromuscular disorders (NMDs) and highlights their advantages and limitations. MPS-based approaches like gene panel sequencing, (whole) exome sequencing, (whole) genome sequencing, and RNA sequencing have been used to identify the genetic cause in NMDs. Although gene panel sequencing has evolved as a standard test for heterogeneous diseases, it is still debated, mainly because of financial issues and unsolved problems of variant interpretation, whether genome sequencing (and to a lesser extent also exome sequencing) of single patients can already be regarded as routine diagnostics. However, it has been shown that the inclusion of parents and additional family members often leads to a substantial increase in the diagnostic yield in exome-wide/genome-wide MPS approaches. In addition, MPS-based RNA sequencing just enters the research and diagnostic scene. Next-generation sequencing increasingly enables the detection of the genetic cause in highly heterogeneous diseases like NMDs in an efficient and affordable way. Gene panel sequencing and family-based exome sequencing have been proven as potent and cost-efficient diagnostic tools. Although clinical validation and interpretation of genome sequencing is still challenging, diagnostic RNA sequencing represents a promising tool to bypass some hurdles of diagnostics using genomic DNA.

Status and application of α-spectrometric 230Th/234U dating of fossil corals in Ryukyus, Japan and the Philippines

International Nuclear Information System (INIS)

Inagaki, Miyuki; Omura, Akio; Sasaki, Keiichi

2007-01-01

High-precision α-spectrometric 230 Th/ 234 U dating was achieved by recent improvements of measurement system and chemical procedures and enabled critical evaluation of age reliability. We review the status and application of α-spectrometric 230 Th/ 234 U dating of Pleistocene and Holocene corals to reconstruct past sea level changes and tectonic movements in Ryukyus, southwestern Japan and the Philippines in the western rim of circum-Pacific island arcs. The highest terrace in Kikai Island was formed during MIS 5c not MIS 5e that previously reported. Coral reef sediments deposited not only during MIS 5e but also during glacial periods, e.g. MIS 6 and 2, have been found in the Ryukyus. Coral reef sediments formed during MIS 2 were found at ca. 120 m below present sea level off Irabu Island located at 25degN. In addition, it was clear that three terraces developed during MIS 5e, 5c and 5a at Pamilacan Island on the Philippines. (author)

Synthesis and detection of 3'-OH terminal biotin-labeled DNA probes

International Nuclear Information System (INIS)

Brakel, C.L.; Engelhardt, D.L.

1985-01-01

Nick translation has been used to prepare biotin-dUTP-containing DNA probes. These stable DNA probes have been identified, following hybridization to target DNA, by fluorescence using antibiotin antibodies or by enzyme reactions in which the enzyme has been linked to avidin or streptavidin. It is probable that this technology will be applicable to certain diagnostic determinations and that, with sufficient sensitivity, this technology might provide a system for obtaining rapid and specific diagnoses in situations presently requiring time-consuming growth assays. The sensitivity of this assay can be increased in two ways: (1) by increasing the amount of biotin contained in the DNA probes, and (2) by increasing the response to individual biotin molecules in the DNA probes. This report demonstrates that terminal deoxynucleotide transferase can be employed to increase the biotin content of DNA probes. We also introduce a new streptavidin-linked enzyme system that produces a greater response to biotinylated DNA probes than does streptavidin-linked horseradish peroxidase

APPLICATION OF THE SPECTROMETRIC METHOD FOR CALCULATING THE DOSE RATE FOR CREATING CALIBRATION HIGHLY SENSITIVE INSTRUMENTS BASED ON SCINTILLATION DETECTION UNITS

Directory of Open Access Journals (Sweden)

R. V. Lukashevich

2017-01-01

Full Text Available Devices based on scintillation detector are highly sensitive to photon radiation and are widely used to measure the environment dose rate. Modernization of the measuring path to minimize the error in measuring the response of the detector to gamma radiation has already reached its technological ceiling and does not give the proper effect. More promising for this purpose are new methods of processing the obtained spectrometric information. The purpose of this work is the development of highly sensitive instruments based on scintillation detection units using a spectrometric method for calculating dose rate.In this paper we consider the spectrometric method of dosimetry of gamma radiation based on the transformation of the measured instrumental spectrum. Using predetermined or measured functions of the detector response to the action of gamma radiation of a given energy and flux density, a certain function of the energy G(E is determined. Using this function as the core of the integral transformation from the field to dose characteristic, it is possible to obtain the dose value directly from the current instrumentation spectrum. Applying the function G(E to the energy distribution of the fluence of photon radiation in the environment, the total dose rate can be determined without information on the distribution of radioisotopes in the environment.To determine G(E by Monte-Carlo method instrumental response function of the scintillator detector to monoenergetic photon radiation sources as well as other characteristics are calculated. Then the whole full-scale energy range is divided into energy ranges for which the function G(E is calculated using a linear interpolation.Spectrometric method for dose calculation using the function G(E, which allows the use of scintillation detection units for a wide range of dosimetry applications is considered in the article. As well as describes the method of calculating this function by using Monte-Carlo methods

A method for exploring the distribution of radioelements at depth using gamma-ray spectrometric data

International Nuclear Information System (INIS)

Li Qingyang

1997-01-01

Based on the inherent relation between radioelements and terrestrial heat flow, theoretically shows the possibility of exploring the distribution of radioelements at depth using gamma-ray spectrometric data, and a data-processing and synthesizing method has been adopted to deduce the calculation formula. The practical application in the uranium mineralized area No. 2801 in Yunnan Province proves that this method is of practical value, and it has been successfully applied to the data processing and good results have been obtained

DNA detection of Schistosoma japonicum: diagnostic validity of a LAMP assay for low-intensity infection and effects of chemotherapy in humans.

Directory of Open Access Journals (Sweden)

Jing Xu

2015-04-01

Full Text Available Schistosomiasis has decreased significantly in prevalence and intensity of infection in China, thus more accurate and sensitive methods are desperately needed for the further control of schistosomiasis. The present work aimed to assess the utility of the loop-mediated isothermal amplification (LAMP for detection of light intensity infection or false-negative patients and patients post-treatment, targeting the highly repetitive retrotransposon SjR2 of Schistosoma japonicum.LAMP was first assessed in rabbits with low intensity infection (EPG<10. Then 110 patient sera from Hunan Province, China, and 47 sera after treatment by praziquantel were used to evaluate the diagnostic validity of LAMP. Meanwhile, 42 sera from healthy individuals in a non-endemic area, and 60 sera from "healthy" residents who were identified as being negative for feces examination and immuno-methods in an endemic area were also examined. The results showed that LAMP could detect S. japonicum DNA in sera from rabbits at 3rd day post-infection. Following administration of praziquantel, the S. japonicum DNA in rabbit sera became negative at 10 weeks post-treatment. Of 110 sera from patients, LAMP showed 95.5% sensitivity, and even for 41 patients with less than 10 EPG, the sensitivity of LAMP still reached to 95.1%. For 47 patients after treatment, the negative conversion rate of S. japonicum DNA in patient sera increased from 23.4%, 61.7% to 83.0% at 3 months, 6 months and 9 months post-treatment, respectively. No false-positive result was obtained for 42 human sera from non-endemic area, while for the 60 "healthy" individuals from endemic area, 10 (16.7% individuals were positive by LAMP, which suggested that these individuals might be false-negative patients.The present study demonstrated that the LAMP assay is sensitive, specific, and affordable, which would help reduce schistosomiasis transmission through targeted treatment of individuals, particularly for those with

Fusion of Taq DNA polymerase with single-stranded DNA binding-like protein of Nanoarchaeum equitans-Expression and characterization.

Directory of Open Access Journals (Sweden)

Marcin Olszewski

Full Text Available DNA polymerases are present in all organisms and are important enzymes that synthesise DNA molecules. They are used in various fields of science, predominantly as essential components for in vitro DNA syntheses, known as PCR. Modern diagnostics, molecular biology and genetic engineering need DNA polymerases which demonstrate improved performance. This study was aimed at obtaining a new NeqSSB-TaqS fusion DNA polymerase from the Taq DNA Stoffel domain and a single-stranded DNA binding-like protein of Nanoarchaeum equitans in order to significantly improve the properties of DNA polymerase. The DNA coding sequence of Taq Stoffel DNA polymerase and the nonspecific DNA-binding protein of Nanoarchaeum equitans (NeqSSB-like protein were fused. A novel recombinant gene was obtained which was cloned into the pET-30 Ek/LIC vector and introduced into E. coli for expression. The recombinant enzyme was purified and its enzymatic properties including DNA polymerase activity, PCR amplification rate, thermostability, processivity and resistance to inhibitors, were tested. The yield of the target protein reached approximately 18 mg/l after 24 h of the IPTG induction. The specific activity of the polymerase was 2200 U/mg. The recombinant NeqSSB-TaqS exhibited a much higher extension rate (1000 bp template in 20 s, processivity (19 nt, thermostability (half-life 35 min at 95°C and higher tolerance to PCR inhibitors (0.3-1.25% of whole blood, 0.84-13.5 μg of lactoferrin and 4.7-150 ng of heparin than Taq Stoffel DNA polymerase. Furthermore, our studies show that NeqSSB-TaqS DNA polymerase has a high level of flexibility in relation to Mg2+ ions (from 1 to 5 mM and KCl or (NH42SO4 salts (more than 60 mM and 40 mM, respectively. Using NeqSSB-TaqS DNA polymerase instead of the Taq DNA polymerase could be a better choice in many PCR applications.

Development of spectrometric probes for emergency preparedness. Status and way forward; Entwicklung von spektrometrierenden Sonden fuer den Notfallschutz. Stand und Perspektiven

Energy Technology Data Exchange (ETDEWEB)

Zaehringer, M.; Bleher, M. [Bundesamt fuer Strahlenschutz (BfS), Salzgitter (Germany); Dambacher, M. [X-Ray Imaging Europe, Freiburg (Germany); and others

2014-01-20

Spectrometric probes with medium energy resolution (NaI, LaBr{sub 3}, CZT) have the potential to enhance existing gamma dose rate monitoring networks as a early warning system by increasing the sensitivity and specificity. In an emergency situation nuclide specific information with sufficient spatial resolution can be made available to decision makers. An ongoing research project between BfS and the Freiburg Material Research Centre (FMF) aims at the development of semiconductor detectors based on Cadmium-Zink-Telluride which can be used for low cost ruggedized spectrometric systems suitable for deployment in an environmental network. The new probe contains also a newly developed versatile multi channel analyser. The system has a modular design and is based - where ever possible - on open source components. It is complemented by specific analysis software using established data exchange formats. This facilitates international cooperation and exploits synergies through distributed development of components.

A Novel Low Temperature PCR Assured High-Fidelity DNA Amplification

Directory of Open Access Journals (Sweden)

Shaoxia Zhou

2013-06-01

Full Text Available As previously reported, a novel low temperature (LoTemp polymerase chain reaction (PCR catalyzed by a moderately heat-resistant (MHR DNA polymerase with a chemical-assisted denaturation temperature set at 85 °C instead of the conventional 94–96 °C can achieve high-fidelity DNA amplification of a target DNA, even after up to 120 PCR thermal cycles. Furthermore, such accurate amplification is not achievable with conventional PCR. Now, using a well-recognized L1 gene segment of the human papillomavirus (HPV type 52 (HPV-52 as the template for experiments, we demonstrate that the LoTemp high-fidelity DNA amplification is attributed to an unusually high processivity and stability of the MHR DNA polymerase whose high fidelity in template-directed DNA synthesis is independent of non-existent 3'–5' exonuclease activity. Further studies and understanding of the characteristics of the LoTemp PCR technology may facilitate implementation of DNA sequencing-based diagnostics at the point of care in community hospital laboratories.

INTERLABORATORY STUDY OF A THERMOSPRAY-LIQUID CHROMATOGRAPHIC/MASS SPECTROMETRIC METHOD FOR SELECTED N-METHYL CARBAMATES, N-METHYL CARBAMOYLOXIMES, AND SUBSTITUTED UREA PESTICIDES

Science.gov (United States)

A thermospray-liquid chromatographic/mass spectrometric (TS-LC/MS) method was evaluated in an interlaboratory study for determining 3 N-methyl carbamates (bendiocarb, carbaryl, and carbofuran), 3-N-methyl carbamoyloximes (aldicarb, methomyl, and oxamyl), 2 substituted urea pestic...

DNA methylation-based classification of central nervous system tumours

DEFF Research Database (Denmark)

Capper, David; Jones, David T.W.; Sill, Martin

2018-01-01

Accurate pathological diagnosis is crucial for optimal management of patients with cancer. For the approximately 100 known tumour types of the central nervous system, standardization of the diagnostic process has been shown to be particularly challenging - with substantial inter-observer variabil......Accurate pathological diagnosis is crucial for optimal management of patients with cancer. For the approximately 100 known tumour types of the central nervous system, standardization of the diagnostic process has been shown to be particularly challenging - with substantial inter......-observer variability in the histopathological diagnosis of many tumour types. Here we present a comprehensive approach for the DNA methylation-based classification of central nervous system tumours across all entities and age groups, and demonstrate its application in a routine diagnostic setting. We show...

Quantitative Field Testing Rotylenchulus reniformis DNA from Metagenomic Samples Isolated Directly from Soil

Science.gov (United States)

Showmaker, Kurt; Lawrence, Gary W.; Lu, Shien; Balbalian, Clarissa; Klink, Vincent P.

2011-01-01

A quantitative PCR procedure targeting the β-tubulin gene determined the number of Rotylenchulus reniformis Linford & Oliveira 1940 in metagenomic DNA samples isolated from soil. Of note, this outcome was in the presence of other soil-dwelling plant parasitic nematodes including its sister genus Helicotylenchus Steiner, 1945. The methodology provides a framework for molecular diagnostics of nematodes from metagenomic DNA isolated directly from soil. PMID:22194958

Anti-double stranded DNA antibodies in systemic lupus erythematosus : Detection and clinical relevance of IgM-class antibodies

NARCIS (Netherlands)

Bootsma, H; Spronk, PE; Hummel, EJ; deBoer, G; terBorg, EJ; Limburg, PC; Kallenberg, CGM

1996-01-01

We determined the discriminative value of the Farr assay in comparison to ELISA and Crithidia luciliae immunofluorescence assay (IFT) for detecting anti-dsDNA antibodies as a diagnostic tool for systemic lupus erythematosus (SLE). Special attention was paid to the diagnostic significance of

Knudsen cell mass spectrometric study of the Cs2IOH(g) molecule thermodynamics

International Nuclear Information System (INIS)

Roki, F-Z.; Ohnet, M-N.; Fillet, S.; Chatillon, C.; Nuta, I.

2013-01-01

Highlights: • The pronounced ionic character leads to only dissociative ionization processes. • Ions formed are same as those coming from pure dimmers. • De-convolution of the ions origin needs accurate thermodynamic values for the pure gas phase. • Mass spectrometric interpretation has to be performed gradually and as a function of suitable condensed compositions. • Thermal functions have to be fully estimated. -- Abstract: The gas phase of the CsI + CsOH system is analyzed by high temperature Knudsen cell mass spectrometry in order to confirm the existence of the Cs 2 IOH(g) complex molecule. The mass spectrometric analysis is quite complex since such molecules undergo dissociative ionization into fragment ions that mix with the same ions from dimers of the pure compounds in the same vapor phase. Varying the chemical conditions for vaporization by using different CsI + CsOH mixture contents showed that the ionization of the Cs 2 IOH(g) molecule led to five different fragment ions, Cs 2 OH + , Cs 2 I + , Cs + , CsOH + and CsI + . This complex ionization pattern was studied in relation with previous assessed values for the vaporization of CsOH and CsI pure compounds in which monomer and dimer molecules are predominant. The equilibrium constant for the reaction CsI(g) + CsOH(g) = Cs 2 IOH(g) was determined and, after modeling the structure of the Cs 2 IOH molecule, the enthalpy of formation was determined using the third law of thermodynamics, as follows: Δ f H°(Cs 2 IOH, g, 298.15 K) = −578 ± 14.7 kJ · mole −1

Label-free detection of DNA hybridization using carbon nanotube network field-effect transistors

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Star, Alexander; Tu, Eugene; Niemann, Joseph; Gabriel, Jean-Christophe P.; Joiner, C. Steve; Valcke, Christian

2006-01-01

We report carbon nanotube network field-effect transistors (NTNFETs) that function as selective detectors of DNA immobilization and hybridization. NTNFETs with immobilized synthetic oligonucleotides have been shown to specifically recognize target DNA sequences, including H63D single-nucleotide polymorphism (SNP) discrimination in the HFE gene, responsible for hereditary hemochromatosis. The electronic responses of NTNFETs upon single-stranded DNA immobilization and subsequent DNA hybridization events were confirmed by using fluorescence-labeled oligonucleotides and then were further explored for label-free DNA detection at picomolar to micromolar concentrations. We have also observed a strong effect of DNA counterions on the electronic response, thus suggesting a charge-based mechanism of DNA detection using NTNFET devices. Implementation of label-free electronic detection assays using NTNFETs constitutes an important step toward low-cost, low-complexity, highly sensitive and accurate molecular diagnostics. hemochromatosis | SNP | biosensor

SMA Diagnosis: Detection of SMN1 Deletion with Real-Time mCOP-PCR System Using Fresh Blood DNA.

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Niba, Emma Tabe Eko; Ar Rochmah, Mawaddah; Harahap, Nur Imma Fatimah; Awano, Hiroyuki; Morioka, Ichiro; Iijima, Kazumoto; Saito, Toshio; Saito, Kayoko; Takeuchi, Atsuko; Lai, Poh San; Bouike, Yoshihiro; Nishio, Hisahide; Shinohara, Masakazu

2017-12-18

Spinal muscular atrophy (SMA) is one of the most common autosomal recessive disorders. The symptoms are caused by defects of lower motor neurons in the spinal cord. More than 95% of SMA patients are homozygous for survival motor neuron 1 (SMN1) deletion. We previously developed a screening system for SMN1 deletion based on a modified competitive oligonucleotide priming-PCR (mCOP-PCR) technique using dried blood spot (DBS) on filter paper. This system is convenient for mass screening in the large population and/or first-tier diagnostic method of the patients in the remote areas. However, this system was still time-consuming and effort-taking, because it required pre-amplification procedure to avoid non-specific amplification and gel-electrophoresis to detect the presence or absence of SMN1 deletion. When the fresh blood samples are used instead of DBS, or when the gel-electrophoresis is replaced by real-time PCR, we may have a simpler and more rapid diagnostic method for SMA. To establish a simpler and more rapid diagnostic method of SMN1 deletion using fresh blood DNA. DNA samples extracted from fresh blood and stored at 4 ℃ for 1 month. The samples were assayed using a real-time mCOP-PCR system without pre-amplification procedures. DNA samples had already been genotyped by PCR-restriction fragment length polymorphism (PCR-RFLP), showing the presence or absence of SMN1 exon 7. The DNA samples were directly subjected to the mCOP-PCR step. The amplification of mCOP-PCR was monitored in a real-time PCR apparatus. The genotyping results of the real-time mCOP-PCR system using fresh blood DNA were completely matched with those of PCR-RFLP. In this real-time mCOP-PCR system using fresh blood-DNA, it took only four hours from extraction of DNA to detection of the presence or absence of SMN1 deletion, while it took more than 12 hours in PCR-RFLP. Our real-time mCOP-PCR system using fresh blood DNA was rapid and accurate, suggesting it may be useful for the first

BLOG 2.0: a software system for character-based species classification with DNA Barcode sequences. What it does, how to use it

NARCIS (Netherlands)

Weitschek, E.; Velzen, van R.; Felici, G.; Bertolazzi, P.

2013-01-01

BLOG (Barcoding with LOGic) is a diagnostic and character-based DNA Barcode analysis method. Its aim is to classify specimens to species based on DNA Barcode sequences and on a supervised machine learning approach, using classification rules that compactly characterize species in terms of DNA

Multifunctional Nanomaterials Utilizing Hybridization Chain Reaction for Molecular Diagnostics and Bioanalytical Applications

Science.gov (United States)

Rana, Md. Muhit

DNA nanotechnology has shown great promise in molecular diagnostic, bioanalytical and biomedical applications. The great challenge of detecting target analytes, biomarkers and small molecules, in molecular diagnostics is low yield sensitivity. To address this challenge, different nanomaterials have been used for a long time and to date there is no such cost-effective bioanalytical technique which can detect these target biomarkers (DNA, RNA, circulating DNA/miRNA) or environmental heavy metal ions (Hg2+ and Ag+) in a cost-effective and efficient manner. Herein, we initially discuss two possible bioanalytical detection methods- a) colorimetric and b) fluorometric assays which are very popular nowadays due to their distinctive spectroscopic properties. Finally, we report the promising colorimetric assay using a novel DNA based amplification strategy know as hybridization chain reaction (HCR) for potential application in the visual detection of low copies of biomarkers (miRNAs as little as 20 femtomole in an RNA pool and cell extracts in seven different combinations and Ebola virus DNA as low as 400 attomoles in liquid biopsy mimics in sixteen different combinations), environmental and biological heavy metal ions (mercury and silver concentrations as low as 10 pM in water, soil and urine samples) and also successfully applied to a molecular logic gate operation to distinguish OR and AND logic gates. No results showed any false-positive or false-negative information. On the other hand, we also discuss the future possibilities of HCR amplification technology, which is very promising for fluorometric bioanalysis. The HCR based nanoprobe technology has numerous remarkable advantages over other methods. It is re-programmable, simple, inexpensive, easy to assemble and operate and can be performed with visual and spectroscopic read-outs upon recognition of the target analytes. This rapid, specific and sensitive approach for biomarkers and heavy metal ion detection generates

Recent advances in salivary cancer diagnostics enabled by biosensors and bioelectronics.

Science.gov (United States)

Mishra, Saswat; Saadat, Darius; Kwon, Ohjin; Lee, Yongkuk; Choi, Woon-Seop; Kim, Jong-Hoon; Yeo, Woon-Hong

2016-07-15

There is a high demand for a non-invasive, rapid, and highly accurate tool for disease diagnostics. Recently, saliva based diagnostics for the detection of specific biomarkers has drawn significant attention since the sample extraction is simple, cost-effective, and precise. Compared to blood, saliva contains a similar variety of DNA, RNA, proteins, metabolites, and microbiota that can be compiled into a multiplex of cancer detection markers. The salivary diagnostic method holds great potential for early-stage cancer diagnostics without any complicated and expensive procedures. Here, we review various cancer biomarkers in saliva and compare the biomarkers efficacy with traditional diagnostics and state-of-the-art bioelectronics. We summarize biomarkers in four major groups: genomics, transcriptomics, proteomics, and metabolomics/microbiota. Representative bioelectronic systems for each group are summarized based on various stages of a cancer. Systematic study of oxidative stress establishes the relationship between macromolecules and cancer biomarkers in saliva. We also introduce the most recent examples of salivary diagnostic electronics based on nanotechnologies that can offer rapid, yet highly accurate detection of biomarkers. A concluding section highlights areas of opportunity in the further development and applications of these technologies. Copyright © 2016 Elsevier B.V. All rights reserved.

Role of Brush Biopsy and DNA Cytometry for Prevention, Diagnosis, Therapy, and Followup Care of Oral Cancer

Directory of Open Access Journals (Sweden)

Alfred Böcking

2011-01-01

Full Text Available Late diagnosis resulting in late treatment and locoregional failure after surgery are the main causes of death in patients with oral squamous cell carcinomas (SCCs. Actually, exfoliative cytology is increasingly used for early detection of oral cancer and has been the subject of intense research over the last five years. Significant advances have been made both in relation to screening and evaluation of precursor lesions. As this noninvasive procedure is well tolerated by patients, more lesions may be screened and thus more oral cancers may be found in early, curable stages. Moreover, the additional use of DNA image cytometry is a reasonable tool for the assessment of the resection margins of SCC. DNA image cytometry could help to find the appropriate treatment option for the patients. Finally, diagnostic DNA image cytometry is an accurate method and has internationally been standardized. In conclusion, DNA image cytometry has increasing impact on the prevention, diagnostic, and therapeutical considerations in head and neck SCC.

A review on the mass spectrometric studies of americium: Present status and future perspective.

Science.gov (United States)

Aggarwal, Suresh Kumar

2018-01-01

The manuscript reviews the various mass spectrometric techniques for analysis and chemical studies of Americium. These methods include thermal ionization mass spectrometry (TIMS), and inductively coupled plasma source mass spectrometry (ICPMS) for the determination of Am isotope ratios and concentration in nuclear fuel samples of interest in nuclear technology, and in complex biological and environmental samples. Ultra-sensitive mass spectrometric techniques of resonance-ionization mass spectrometry (RIMS), and accelerator-based mass spectrometry (AMS) are also discussed. The novel applications of electrospray ionization mass spectrometry (ESIMS) to understand the solution chemistry of Am and other actinides are presented. These studies are important in view of the world-wide efforts to develop novel complexing agents to separate lanthanides and minor actinides (Am, Np, and Cm) for partitioning and transmutation of minor actinides from the point of view of nuclear waste management. These mass spectrometry experiments are also of great interest to examine the covalent character of actinides with increasing atomic number. Studies on gas-phase chemistry of Am and its oxides with Knudsen effusion mass spectrometry (KEMS), Fourier-transform ion cyclotron resonance mass spectrometry (FTICR-MS), and laser-based experiments with reflectron time-of-flight mass spectrometer (R-ToF) are highlighted. These studies are important to understand the fundamental chemistry of 5f electrons in actinides. Requirement of certified isotopic reference materials of Am to improve the accuracy of experimental nuclear data (e.g., the half-life of 243 Am) is emphasized. © 2016 Wiley Periodicals, Inc. Mass Spec Rev. © 2016 Wiley Periodicals, Inc.

Electronic Transport in Single-Stranded DNA Molecule Related to Huntington's Disease

Science.gov (United States)

Sarmento, R. G.; Silva, R. N. O.; Madeira, M. P.; Frazão, N. F.; Sousa, J. O.; Macedo-Filho, A.

2018-04-01

We report a numerical analysis of the electronic transport in single chain DNA molecule consisting of 182 nucleotides. The DNA chains studied were extracted from a segment of the human chromosome 4p16.3, which were modified by expansion of CAG (cytosine-adenine-guanine) triplet repeats to mimics Huntington's disease. The mutated DNA chains were connected between two platinum electrodes to analyze the relationship between charge propagation in the molecule and Huntington's disease. The computations were performed within a tight-binding model, together with a transfer matrix technique, to investigate the current-voltage (I-V) of 23 types of DNA sequence and compare them with the distributions of the related CAG repeat numbers with the disease. All DNA sequences studied have a characteristic behavior of a semiconductor. In addition, the results showed a direct correlation between the current-voltage curves and the distributions of the CAG repeat numbers, suggesting possible applications in the development of DNA-based biosensors for molecular diagnostics.

Distribution of Brugia malayi larvae and DNA in vector and non-vector mosquitoes: implications for molecular diagnostics

Directory of Open Access Journals (Sweden)

Christensen Bruce M

2009-11-01

Full Text Available Abstract Background The purpose of this study was to extend prior studies of molecular detection of Brugia malayi DNA in vector (Aedes aegypti- Liverpool and non-vector (Culex pipiens mosquitoes at different times after ingestion of infected blood. Results Parasite DNA was detected over a two week time course in 96% of pooled thoraces of vector mosquitoes. In contrast, parasite DNA was detected in only 24% of thorax pools from non-vectors; parasite DNA was detected in 56% of midgut pools and 47% of abdomen pools from non-vectors. Parasite DNA was detected in vectors in the head immediately after the blood meal and after 14 days. Parasite DNA was also detected in feces and excreta of the vector and non-vector mosquitoes which could potentially confound results obtained with field samples. However, co-housing experiments failed to demonstrate transfer of parasite DNA from infected to non-infected mosquitoes. Parasites were also visualized in mosquito tissues by immunohistololgy using an antibody to the recombinant filarial antigen Bm14. Parasite larvae were detected consistently after mf ingestion in Ae. aegypti- Liverpool. Infectious L3s were seen in the head, thorax and abdomen of vector mosquitoes 14 days after Mf ingestion. In contrast, parasites were only detected by histology shortly after the blood meal in Cx. pipiens, and these were not labeled by the antibody. Conclusion This study provides new information on the distribution of filarial parasites and parasite DNA in vector and non-vector mosquitoes. This information should be useful for those involved in designing and interpreting molecular xenomonitoring studies.

Evaluation of Sample Stability and Automated DNA Extraction for Fetal Sex Determination Using Cell-Free Fetal DNA in Maternal Plasma

Directory of Open Access Journals (Sweden)

Elena Ordoñez

2013-01-01

Full Text Available Objective. The detection of paternally inherited sequences in maternal plasma, such as the SRY gene for fetal sexing or RHD for fetal blood group genotyping, is becoming part of daily routine in diagnostic laboratories. Due to the low percentage of fetal DNA, it is crucial to ensure sample stability and the efficiency of DNA extraction. We evaluated blood stability at 4°C for at least 24 hours and automated DNA extraction, for fetal sex determination in maternal plasma. Methods. A total of 158 blood samples were collected, using EDTA-K tubes, from women in their 1st trimester of pregnancy. Samples were kept at 4°C for at least 24 hours before processing. An automated DNA extraction was evaluated, and its efficiency was compared with a standard manual procedure. The SRY marker was used to quantify cfDNA by real-time PCR. Results. Although lower cfDNA amounts were obtained by automated DNA extraction (mean 107,35 GE/mL versus 259,43 GE/mL, the SRY sequence was successfully detected in all 108 samples from pregnancies with male fetuses. Conclusion. We successfully evaluated the suitability of standard blood tubes for the collection of maternal blood and assessed samples to be suitable for analysis at least 24 hours later. This would allow shipping to a central reference laboratory almost from anywhere in Europe.

Determination of iodine to compliment mass spectrometric measurements

International Nuclear Information System (INIS)

Hohorst, F.A.

1994-11-01

The dose of iodine-129 to facility personnel and the general public as a result of past, present, and future activities at DOE sites is of continuing interest, WINCO received about 160 samples annually in a variety of natural matrices, including snow, milk, thyroid tissue, and sagebrush, in which iodine-129 is determined in order to evaluate this dose, Currently, total iodine and the isotopic ratio of iodine-127 to iodine-129 are determined by mass spectrometry. These two measurements determine the concentration of iodine-129 in each sample, These measurements require at least 16 h of mass spectrometer operator time for each sample. A variety of methods are available which concentrate and determine small quantities of iodine. Although useful, these approaches would increase both time and cost. The objective of this effort was to determine total iodine by an alternative method in order to decrease the load on mass spectrometry by 25 to 50%. The preparation of each sample for mass spectrometric analysis involves a common step--collection of iodide on an ion exchange bed. This was the focal point of the effort since the results would be applicable to all samples

Paenibacillus larvae 16S-23S rDNA intergenic transcribed spacer (ITS) regions: DNA fingerprinting and characterization.

Science.gov (United States)

Dingman, Douglas W

2012-07-01

Paenibacillus larvae is the causative agent of American foulbrood in honey bee (Apis mellifera) larvae. PCR amplification of the 16S-23S ribosomal DNA (rDNA) intergenic transcribed spacer (ITS) regions, and agarose gel electrophoresis of the amplified DNA, was performed using genomic DNA collected from 134 P. larvae strains isolated in Connecticut, six Northern Regional Research Laboratory stock strains, four strains isolated in Argentina, and one strain isolated in Chile. Following electrophoresis of amplified DNA, all isolates exhibited a common migratory profile (i.e., ITS-PCR fingerprint pattern) of six DNA bands. This profile represented a unique ITS-PCR DNA fingerprint that was useful as a fast, simple, and accurate procedure for identification of P. larvae. Digestion of ITS-PCR amplified DNA, using mung bean nuclease prior to electrophoresis, characterized only three of the six electrophoresis bands as homoduplex DNA and indicating three true ITS regions. These three ITS regions, DNA migratory band sizes of 915, 1010, and 1474 bp, signify a minimum of three types of rrn operons within P. larvae. DNA sequence analysis of ITS region DNA, using P. larvae NRRL B-3553, identified the 3' terminal nucleotides of the 16S rRNA gene, 5' terminal nucleotides of the 23S rRNA gene, and the complete DNA sequences of the 5S rRNA, tRNA(ala), and tRNA(ile) genes. Gene organization within the three rrn operon types was 16S-23S, 16S-tRNA(ala)-23S, and l6S-5S-tRNA(ile)-tRNA(ala)-23S and these operons were named rrnA, rrnF, and rrnG, respectively. The 23S rRNA gene was shown by I-CeuI digestion and pulsed-field gel electrophoresis of genomic DNA to be present as seven copies. This was suggestive of seven rrn operon copies within the P. larvae genome. Investigation of the 16S-23S rDNA regions of this bacterium has aided the development of a diagnostic procedure and has helped genomic mapping investigations via characterization of the ITS regions. Copyright © 2012 Elsevier Inc

High-Throughput Block Optical DNA Sequence Identification.

Science.gov (United States)

Sagar, Dodderi Manjunatha; Korshoj, Lee Erik; Hanson, Katrina Bethany; Chowdhury, Partha Pratim; Otoupal, Peter Britton; Chatterjee, Anushree; Nagpal, Prashant

2018-01-01

Optical techniques for molecular diagnostics or DNA sequencing generally rely on small molecule fluorescent labels, which utilize light with a wavelength of several hundred nanometers for detection. Developing a label-free optical DNA sequencing technique will require nanoscale focusing of light, a high-throughput and multiplexed identification method, and a data compression technique to rapidly identify sequences and analyze genomic heterogeneity for big datasets. Such a method should identify characteristic molecular vibrations using optical spectroscopy, especially in the "fingerprinting region" from ≈400-1400 cm -1 . Here, surface-enhanced Raman spectroscopy is used to demonstrate label-free identification of DNA nucleobases with multiplexed 3D plasmonic nanofocusing. While nanometer-scale mode volumes prevent identification of single nucleobases within a DNA sequence, the block optical technique can identify A, T, G, and C content in DNA k-mers. The content of each nucleotide in a DNA block can be a unique and high-throughput method for identifying sequences, genes, and other biomarkers as an alternative to single-letter sequencing. Additionally, coupling two complementary vibrational spectroscopy techniques (infrared and Raman) can improve block characterization. These results pave the way for developing a novel, high-throughput block optical sequencing method with lossy genomic data compression using k-mer identification from multiplexed optical data acquisition. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Stress, burnout and depression : A systematic review on DNA methylation mechanisms

NARCIS (Netherlands)

Bakusic, Jelena; Schaufeli, Wilmar; Claes, Stephan; Godderis, Lode

2017-01-01

Despite that burnout presents a serious burden for modern society, there are no diagnostic criteria. Additional difficulty is the differential diagnosis with depression. Consequently, there is a need to dispose of a burnout biomarker. Epigenetic studies suggest that DNA methylation is a possible

Levelling Airborne and Ground Gamma-Ray Spectrometric Data to Assist Uranium Exploration

Energy Technology Data Exchange (ETDEWEB)

Matolin, M., E-mail: matolin@natur.cuni.cz [Charles University, Prague (Czech Republic); Minty, B. [Geoscience Australia, Canberra (Australia)

2014-05-15

Geophysical methods can be used for mapping in both 2 and 3 dimensions, as well as the direct detection of ore bodies. The gamma-ray spectrometric method is an efficient method for the regional assessment of uranium potential and the detection of surface mineralization. However, the full potential of the method can only be realized when the data are adequately standardized. Examples of this standardization at both regional and local scales are dealt in this paper. At a regional scale, it is shown how the levelling of airborne gamma-ray spectrometry data over Australia increases the value of the resulting data, and on a local scale a geometrical correction for ground gamma-ray spectrometry in shallow holes that improves the accuracy of measurements is introduced. (author)

Radio spectrometric survey of un-surveyed areas in Syria

International Nuclear Information System (INIS)

Aissa, M.; Al-Hent, R; Jubeli, Y.

2002-11-01

The values and distribution of the radioelements e U, e Th, % K and Ur units in the surface geological formations of the west and south sectors of Syrian region, were estimated using carbone gamma ray spectrometric survey. The radiometric maps were prepared, as well as, many geological profiles, cross sections studied in different locations and geochemical samples were analyzed by laboratory gamma ray spectrometry and by x-ray diffractometry, the results of the all sets were compared. In general, the survey shows, low radioelement concentrations in the area, especially on basic rocks (Jabal Al-arab, Hawran) south Syria, and on ultra basic rocks (ophiolitic complex) north-west Syria, but there are some separate anomalous spots were connected with phosphate rocks, detected on cretaceous and Palaeogene age. Some times we noticed high radioelement concentrations haloes associated with fractured zones were already arise from secondary uranium mineralization, as a result of solutions movement through fissures in carbonatic and/or chalk like limestone rocks. finally, the obtained concentrations, represent a background values which has no significant importance for uranium exploration point of view. (author)

Comparison of the diagnostic value of cervical cytology and HPV HR DNA testing for the diagnosis of low-grade and high-grade squamous intraepithelial lesions across different age groups.

Science.gov (United States)

Paluszkiewicz, Aleksandra; Pruski, Dominik; Iwaniec, Kinga; Kędzia, Witold

2017-01-01

To assess the diagnostic value of cervical cytology and HPV HR DNA testing for the diagnosis of low grade and high-grade squamous intraepithelial lesions across different age groups. The study included 1103 patients, age 25-70 years. All patients underwent in-depth diagnostic tests following either an abnormal Pap test result or a clinically suspicious cervical lesion. In all women the following examinations were performed: a molecular test detecting 14 high-risk types of HPV, a colposcopy examination, as well as directed-biopsy of the cervix. The studied population was subdivided into four age groups. It was observed that the percentage of high grade squamous intraepithelial lesions (HSIL) and cancers increased with women's age. Sensitivity of both methods for detecting high-grade squamous intraepithelial lesions was highest for women aged 40-49 years. Sensitivity values of HPV testing was higher than that of cervical cytology among women under age 50. Specificity of HPV testing increased significantly with age of women and was several fold higher across all age groups than the specificity of cervical cytology.

Nano-biotechnology for biomedical and diagnostic research

CERN Document Server

Zahavy, Eran; Yitzhaki, Shmuel

2011-01-01

The title ""Nano Biotechnology for Biomedical and Diagnostics Research"" will address research aspects related to nanomaterial in imaging and biological research, nanomaterials as a biosensing tool, DNA nanotechnology, nanomaterials for drug delivery, medicinal and therapeutic application and cytotoxicity of nanomaterials. These topics will be covered by 16 different manuscripts. Amongst the authors that will contribute to the book are major scientific leaders such as S. Weiss - UCLA, I. Willner, and G. Golomb -- HUJI, S. Esener - UCSD, E.C. Simmel - Tech. Univ. Munchen, I. Medintz -- NRL, N.

MASS SPECTROMETRIC ANALYSIS FOR THE IDENTIFICATION OF THUNNUS GENUS FOUR SPECIES

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T. Pepe

2011-01-01

Full Text Available An accurate identification of similar fish species is necessary to prevent illegal substitution and is imposed by labeling regulations in UE countries (1. The genus Thunnus comprises many species of different quality and commercial value. The increasing trade of fish preparations of the species included in this genus and the consequent loss of the external anatomical and morphological features enables fraudulent substitutions. This study reports data relating to the proteomic analysis of four tuna species (T. thynnus, T. alalunga, T. albacares, T. obesus. Sarcoplasmic proteins were studied by mono and two dimensional electrophoresis. The most significant proteins for the characterization of the species were analyzed by mass spectrometric techniques. As reported in a previous study (2, an accurate identification of the species seems possible, owing to the polymorphism displayed by the species of the Thunnus genus.

Development of DNA probes for Candida albicans

International Nuclear Information System (INIS)

Cheung, L.L.; Hudson, J.B.

1988-01-01

An attempt was made to produce DNA probes that could be used as a rapid and efficient means of detecting candidiasis (invasive Candida infection) in immunocompromised patients. Whole DNA from Candida albicans was digested with restriction endonuclease, and the resulting fragments were randomly cloned into a plasmid vector. Several recombinant plasmids were evaluated for cross-hybridization to various other Candida species, other fungal DNAs, and to nonfungal DNAs. Cross reactions were observed between the probes and different yeasts, but none with unrelated DNAs. Some recombinants were genus-specific, and two of these were applied to the analysis of C. albicans growth curves. It became evident that, although both 32 P- and biotin-labelled probes could be made quite sensitive, a possible limitation in their diagnostic potential was the poor liberation of Candida DNA from cells. Thus, better methods of treatment of clinical specimens will be required before such probes will be useful in routine diagnosis

Development of DNA probes for Candida albicans

Energy Technology Data Exchange (ETDEWEB)

Cheung, L.L.; Hudson, J.B.

1988-07-01

An attempt was made to produce DNA probes that could be used as a rapid and efficient means of detecting candidiasis (invasive Candida infection) in immunocompromised patients. Whole DNA from Candida albicans was digested with restriction endonuclease, and the resulting fragments were randomly cloned into a plasmid vector. Several recombinant plasmids were evaluated for cross-hybridization to various other Candida species, other fungal DNAs, and to nonfungal DNAs. Cross reactions were observed between the probes and different yeasts, but none with unrelated DNAs. Some recombinants were genus-specific, and two of these were applied to the analysis of C. albicans growth curves. It became evident that, although both /sup 32/P- and biotin-labelled probes could be made quite sensitive, a possible limitation in their diagnostic potential was the poor liberation of Candida DNA from cells. Thus, better methods of treatment of clinical specimens will be required before such probes will be useful in routine diagnosis.

cDNA microarray screening in food safety

International Nuclear Information System (INIS)

Roy, Sashwati; Sen, Chandan K.

2006-01-01

The cDNA microarray technology and related bioinformatics tools presents a wide range of novel application opportunities. The technology may be productively applied to address food safety. In this mini-review article, we present an update highlighting the late breaking discoveries that demonstrate the vitality of cDNA microarray technology as a tool to analyze food safety with reference to microbial pathogens and genetically modified foods. In order to bring the microarray technology to mainstream food safety, it is important to develop robust user-friendly tools that may be applied in a field setting. In addition, there needs to be a standardized process for regulatory agencies to interpret and act upon microarray-based data. The cDNA microarray approach is an emergent technology in diagnostics. Its values lie in being able to provide complimentary molecular insight when employed in addition to traditional tests for food safety, as part of a more comprehensive battery of tests

Increased Protein Structural Resolution from Diethylpyrocarbonate-based Covalent Labeling and Mass Spectrometric Detection

Science.gov (United States)

Zhou, Yuping; Vachet, Richard W.

2012-04-01

Covalent labeling and mass spectrometry are seeing increased use together as a way to obtain insight into the 3-dimensional structure of proteins and protein complexes. Several amino acid specific (e.g., diethylpyrocarbonate) and non-specific (e.g., hydroxyl radicals) labeling reagents are available for this purpose. Diethylpyrocarbonate (DEPC) is a promising labeling reagent because it can potentially probe up to 30% of the residues in the average protein and gives only one reaction product, thereby facilitating mass spectrometric analysis. It was recently reported, though, that DEPC modifications are labile for some amino acids. Here, we show that label loss is more significant and widespread than previously thought, especially for Ser, Thr, Tyr, and His residues, when relatively long protein digestion times are used. Such label loss ultimately decreases the amount of protein structural information that is obtainable with this reagent. We find, however, that the number of DEPC modified residues and, thus, protein structural information, can be significantly increased by decreasing the time between the covalent labeling reaction and the mass spectrometric analysis. This is most effectively accomplished using short (e.g., 2 h) proteolytic digestions with enzymes such as immobilized chymotrypsin or Glu-C rather than using methods (e.g., microwave or ultrasonic irradiation) that accelerate proteolysis in other ways. Using short digestion times, we show that the percentage of solvent accessible residues that can be modified by DEPC increases from 44% to 67% for cytochrome c, 35% to 81% for myoglobin, and 76% to 95% for β-2-microglobulin. In effect, these increased numbers of modified residues improve the protein structural resolution available from this covalent labeling method. Compared with typical overnight digestion conditions, the short digestion times decrease the average distance between modified residues from 11 to 7 Å for myoglobin, 13 to 10 Å for

A stochastic DNA walker that traverses a microparticle surface

Science.gov (United States)

Jung, C.; Allen, P. B.; Ellington, A. D.

2016-02-01

Molecular machines have previously been designed that are propelled by DNAzymes, protein enzymes and strand displacement. These engineered machines typically move along precisely defined one- and two-dimensional tracks. Here, we report a DNA walker that uses hybridization to drive walking on DNA-coated microparticle surfaces. Through purely DNA:DNA hybridization reactions, the nanoscale movements of the walker can lead to the generation of a single-stranded product and the subsequent immobilization of fluorescent labels on the microparticle surface. This suggests that the system could be of use in analytical and diagnostic applications, similar to how strand exchange reactions in solution have been used for transducing and quantifying signals from isothermal molecular amplification assays. The walking behaviour is robust and the walker can take more than 30 continuous steps. The traversal of an unprogrammed, inhomogeneous surface is also due entirely to autonomous decisions made by the walker, behaviour analogous to amorphous chemical reaction network computations, which have been shown to lead to pattern formation.

Genetic diagnosis of familial hypercholesterolemia using a DNA-array based platform

NARCIS (Netherlands)

Alonso, Rodrigo; Defesche, Joep C.; Tejedor, Diego; Castillo, Sergio; Stef, Marianne; Mata, Nelva; Gomez-Enterria, Pilar; Martinez-Faedo, Ceferino; Forga, Lluis; Mata, Pedro

2009-01-01

The aim of this study was to validate the Lipochip genetic diagnostic platform by assessing effectiveness, sensitivity, specificity and costs for the identification of patients with familial hypercholesterolemia (FH) in Spain. This platform includes the use of a DNA micro array, the detection of

2DB: a Proteomics database for storage, analysis, presentation, and retrieval of information from mass spectrometric experiments.

Science.gov (United States)

Allmer, Jens; Kuhlgert, Sebastian; Hippler, Michael

2008-07-07

The amount of information stemming from proteomics experiments involving (multi dimensional) separation techniques, mass spectrometric analysis, and computational analysis is ever-increasing. Data from such an experimental workflow needs to be captured, related and analyzed. Biological experiments within this scope produce heterogenic data ranging from pictures of one or two-dimensional protein maps and spectra recorded by tandem mass spectrometry to text-based identifications made by algorithms which analyze these spectra. Additionally, peptide and corresponding protein information needs to be displayed. In order to handle the large amount of data from computational processing of mass spectrometric experiments, automatic import scripts are available and the necessity for manual input to the database has been minimized. Information is in a generic format which abstracts from specific software tools typically used in such an experimental workflow. The software is therefore capable of storing and cross analysing results from many algorithms. A novel feature and a focus of this database is to facilitate protein identification by using peptides identified from mass spectrometry and link this information directly to respective protein maps. Additionally, our application employs spectral counting for quantitative presentation of the data. All information can be linked to hot spots on images to place the results into an experimental context. A summary of identified proteins, containing all relevant information per hot spot, is automatically generated, usually upon either a change in the underlying protein models or due to newly imported identifications. The supporting information for this report can be accessed in multiple ways using the user interface provided by the application. We present a proteomics database which aims to greatly reduce evaluation time of results from mass spectrometric experiments and enhance result quality by allowing consistent data handling

2DB: a Proteomics database for storage, analysis, presentation, and retrieval of information from mass spectrometric experiments

Directory of Open Access Journals (Sweden)

Hippler Michael

2008-07-01

Full Text Available Abstract Background The amount of information stemming from proteomics experiments involving (multi dimensional separation techniques, mass spectrometric analysis, and computational analysis is ever-increasing. Data from such an experimental workflow needs to be captured, related and analyzed. Biological experiments within this scope produce heterogenic data ranging from pictures of one or two-dimensional protein maps and spectra recorded by tandem mass spectrometry to text-based identifications made by algorithms which analyze these spectra. Additionally, peptide and corresponding protein information needs to be displayed. Results In order to handle the large amount of data from computational processing of mass spectrometric experiments, automatic import scripts are available and the necessity for manual input to the database has been minimized. Information is in a generic format which abstracts from specific software tools typically used in such an experimental workflow. The software is therefore capable of storing and cross analysing results from many algorithms. A novel feature and a focus of this database is to facilitate protein identification by using peptides identified from mass spectrometry and link this information directly to respective protein maps. Additionally, our application employs spectral counting for quantitative presentation of the data. All information can be linked to hot spots on images to place the results into an experimental context. A summary of identified proteins, containing all relevant information per hot spot, is automatically generated, usually upon either a change in the underlying protein models or due to newly imported identifications. The supporting information for this report can be accessed in multiple ways using the user interface provided by the application. Conclusion We present a proteomics database which aims to greatly reduce evaluation time of results from mass spectrometric experiments and enhance

Depletion of Human DNA in Spiked Clinical Specimens for Improvement of Sensitivity of Pathogen Detection by Next-Generation Sequencing

OpenAIRE

Hasan, Mohammad R.; Rawat, Arun; Tang, Patrick; Jithesh, Puthen V.; Thomas, Eva; Tan, Rusung; Tilley, Peter

2016-01-01

Next-generation sequencing (NGS) technology has shown promise for the detection of human pathogens from clinical samples. However, one of the major obstacles to the use of NGS in diagnostic microbiology is the low ratio of pathogen DNA to human DNA in most clinical specimens. In this study, we aimed to develop a specimen-processing protocol to remove human DNA and enrich specimens for bacterial and viral DNA for shotgun metagenomic sequencing. Cerebrospinal fluid (CSF) and nasopharyngeal aspi...

Multi-color fluorescent DNA analysis in an integrated optofluidic lab on a chip

OpenAIRE

Dongre, C.

2010-01-01

Abstract: Sorting and sizing of DNA molecules within the human genome project has enabled the genetic mapping of various illnesses. Furthermore by employing tiny lab-on-a-chip device, integrated DNA sequencing and genetic diagnostics have become feasible. We present the combination of capillary electrophoresis with laser-induced fluorescence for optofluidic integration toward an on-chip bio-analysis tool. Integrated optical fluorescence excitation allows for a high spatial resolution (12 μm) ...

Digital diffraction analysis enables low-cost molecular diagnostics on a smartphone.

Science.gov (United States)

Im, Hyungsoon; Castro, Cesar M; Shao, Huilin; Liong, Monty; Song, Jun; Pathania, Divya; Fexon, Lioubov; Min, Changwook; Avila-Wallace, Maria; Zurkiya, Omar; Rho, Junsung; Magaoay, Brady; Tambouret, Rosemary H; Pivovarov, Misha; Weissleder, Ralph; Lee, Hakho

2015-05-05

The widespread distribution of smartphones, with their integrated sensors and communication capabilities, makes them an ideal platform for point-of-care (POC) diagnosis, especially in resource-limited settings. Molecular diagnostics, however, have been difficult to implement in smartphones. We herein report a diffraction-based approach that enables molecular and cellular diagnostics. The D3 (digital diffraction diagnosis) system uses microbeads to generate unique diffraction patterns which can be acquired by smartphones and processed by a remote server. We applied the D3 platform to screen for precancerous or cancerous cells in cervical specimens and to detect human papillomavirus (HPV) DNA. The D3 assay generated readouts within 45 min and showed excellent agreement with gold-standard pathology or HPV testing, respectively. This approach could have favorable global health applications where medical access is limited or when pathology bottlenecks challenge prompt diagnostic readouts.

High-Quality Exome Sequencing of Whole-Genome Amplified Neonatal Dried Blood Spot DNA

DEFF Research Database (Denmark)

Poulsen, Jesper Buchhave; Lescai, Francesco; Grove, Jakob

2016-01-01

Stored neonatal dried blood spot (DBS) samples from neonatal screening programmes are a valuable diagnostic and research resource. Combined with information from national health registries they can be used in population-based studies of genetic diseases. DNA extracted from neonatal DBSs can...... be amplified to obtain micrograms of an otherwise limited resource, referred to as whole-genome amplified DNA (wgaDNA). Here we investigate the robustness of exome sequencing of wgaDNA of neonatal DBS samples. We conducted three pilot studies of seven, eight and seven subjects, respectively. For each subject...... we analysed a neonatal DBS sample and corresponding adult whole-blood (WB) reference sample. Different DNA sample types were prepared for each of the subjects. Pilot 1: wgaDNA of 2x3.2mm neonatal DBSs (DBS_2x3.2) and raw DNA extract of the WB reference sample (WB_ref). Pilot 2: DBS_2x3.2, WB...

Distribution of Plasmodium species on the island of Grande Comore on the basis of DNA extracted from rapid diagnostic tests

Directory of Open Access Journals (Sweden)

Papa Mze Nasserdine

2016-01-01

Full Text Available In the Union of Comoros, interventions for combating malaria have contributed to a spectacular decrease in the prevalence of the disease. We studied the current distribution of Plasmodium species on the island of Grande Comore using nested PCR. The rapid diagnostic tests (RDTs currently used in the Comoros are able to identify Plasmodium falciparum but no other Plasmodium species. In this study, we tested 211 RDTs (158 positive and 53 negative. Among the 158 positive RDTs, 22 were positive for HRP2, 3 were positive only for pLDH, and 133 were positive for HRP2 and pLDH. DNA was extracted from a proximal part of the nitrocellulose membrane of RDTs. A total of 159 samples were positive by nested PCR. Of those, 156 (98.11% were positive for P. falciparum, 2 (1.25% were positive for P. vivaxI, and 1 (0.62% was positive for P. malariae. None of the samples were positive for P. ovale. Our results show that P. falciparum is still the most dominant species on the island of Grande Comore, but P. vivax and P. malariae are present at a low prevalence.

Compartmentalized self-replication (CSR) selection of Thermococcus litoralis Sh1B DNA polymerase for diminished uracil binding.

Science.gov (United States)

Tubeleviciute, Agne; Skirgaila, Remigijus

2010-08-01

The thermostable archaeal DNA polymerase Sh1B from Thermococcus litoralis has a typical uracil-binding pocket, which in nature plays an essential role in preventing the accumulation of mutations caused by cytosine deamination to uracil and subsequent G-C base pair transition to A-T during the genomic DNA replication. The uracil-binding pocket recognizes and binds uracil base in a template strand trapping the polymerase. Since DNA replication stops, the repair systems have a chance to correct the promutagenic event. Archaeal family B DNA polymerases are employed in various PCR applications. Contrary to nature, in PCR the uracil-binding property of archaeal polymerases is disadvantageous and results in decreased DNA amplification yields and lowered sensitivity. Furthermore, in diagnostics qPCR, RT-qPCR and end-point PCR are performed using dNTP mixtures, where dTTP is partially or fully replaced by dUTP. Uracil-DNA glycosylase treatment and subsequent heating of the samples is used to degrade the DNA containing uracil and prevent carryover contamination, which is the main concern in diagnostic laboratories. A thermostable archaeal DNA polymerase with the abolished uracil binding would be a highly desirable and commercially interesting product. An attempt to disable uracil binding in DNA polymerase Sh1B from T. litoralis by generating site-specific mutants did not yield satisfactory results. However, a combination of random mutagenesis of the whole polymerase gene and compartmentalized self-replication was successfully used to select variants of thermostable Sh1B polymerase capable of performing PCR with dUTP instead of dTTP.

Topic model-based mass spectrometric data analysis in cancer biomarker discovery studies.

Science.gov (United States)

Wang, Minkun; Tsai, Tsung-Heng; Di Poto, Cristina; Ferrarini, Alessia; Yu, Guoqiang; Ressom, Habtom W

2016-08-18

A fundamental challenge in quantitation of biomolecules for cancer biomarker discovery is owing to the heterogeneous nature of human biospecimens. Although this issue has been a subject of discussion in cancer genomic studies, it has not yet been rigorously investigated in mass spectrometry based proteomic and metabolomic studies. Purification of mass spectometric data is highly desired prior to subsequent analysis, e.g., quantitative comparison of the abundance of biomolecules in biological samples. We investigated topic models to computationally analyze mass spectrometric data considering both integrated peak intensities and scan-level features, i.e., extracted ion chromatograms (EICs). Probabilistic generative models enable flexible representation in data structure and infer sample-specific pure resources. Scan-level modeling helps alleviate information loss during data preprocessing. We evaluated the capability of the proposed models in capturing mixture proportions of contaminants and cancer profiles on LC-MS based serum proteomic and GC-MS based tissue metabolomic datasets acquired from patients with hepatocellular carcinoma (HCC) and liver cirrhosis as well as synthetic data we generated based on the serum proteomic data. The results we obtained by analysis of the synthetic data demonstrated that both intensity-level and scan-level purification models can accurately infer the mixture proportions and the underlying true cancerous sources with small average error ratios (data, we found more proteins and metabolites with significant changes between HCC cases and cirrhotic controls. Candidate biomarkers selected after purification yielded biologically meaningful pathway analysis results and improved disease discrimination power in terms of the area under ROC curve compared to the results found prior to purification. We investigated topic model-based inference methods to computationally address the heterogeneity issue in samples analyzed by LC/GC-MS. We observed

A spectrometric approach in radiography for detection of materials by their effective atomic number

CERN Document Server

Ryzhikov, V D; Onyshchenko, G M; Lecoq, P; Smith, C F

2009-01-01

In this paper we report a spectrometric approach to dual-energy digital radiography that has been developed and applied to identify specific organic substances and discern small differences in their effective atomic number. An experimental setup has been designed, and a theoretical description proposed based on the experimental results obtained. The proposed method is based on the application of special reference samples made of materials with different effective atomic number and thickness parameters known to affect X-ray attenuation in the low-energy range. The results obtained can be used in the development of a new generation of multi-energy customs or medical X-ray scanners.

Assessment of current mass spectrometric workflows for the quantification of low abundant proteins and phosphorylation sites

Directory of Open Access Journals (Sweden)

Manuel Bauer

2015-12-01

Full Text Available The data described here provide a systematic performance evaluation of popular data-dependent (DDA and independent (DIA mass spectrometric (MS workflows currently used in quantitative proteomics. We assessed the limits of identification, quantification and detection for each method by analyzing a dilution series of 20 unmodified and 10 phosphorylated synthetic heavy labeled reference peptides, respectively, covering six orders of magnitude in peptide concentration with and without a complex human cell digest background. We found that all methods performed very similarly in the absence of background proteins, however, when analyzing whole cell lysates, targeted methods were at least 5–10 times more sensitive than directed or DDA methods. In particular, higher stage fragmentation (MS3 of the neutral loss peak using a linear ion trap increased dynamic quantification range of some phosphopeptides up to 100-fold. We illustrate the power of this targeted MS3 approach for phosphopeptide monitoring by successfully quantifying 9 phosphorylation sites of the kinetochore and spindle assembly checkpoint component Mad1 over different cell cycle states from non-enriched pull-down samples. The data are associated to the research article ‘Evaluation of data-dependent and data-independent mass spectrometric workflows for sensitive quantification of proteins and phosphorylation sites׳ (Bauer et al., 2014 [1]. The mass spectrometry and the analysis dataset have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org via the PRIDE partner repository with the dataset identifier PXD000964.

Thermodynamic framework to assess low abundance DNA mutation detection by hybridization

Science.gov (United States)

Willems, Hanny; Jacobs, An; Hadiwikarta, Wahyu Wijaya; Venken, Tom; Valkenborg, Dirk; Van Roy, Nadine; Vandesompele, Jo; Hooyberghs, Jef

2017-01-01

The knowledge of genomic DNA variations in patient samples has a high and increasing value for human diagnostics in its broadest sense. Although many methods and sensors to detect or quantify these variations are available or under development, the number of underlying physico-chemical detection principles is limited. One of these principles is the hybridization of sample target DNA versus nucleic acid probes. We introduce a novel thermodynamics approach and develop a framework to exploit the specific detection capabilities of nucleic acid hybridization, using generic principles applicable to any platform. As a case study, we detect point mutations in the KRAS oncogene on a microarray platform. For the given platform and hybridization conditions, we demonstrate the multiplex detection capability of hybridization and assess the detection limit using thermodynamic considerations; DNA containing point mutations in a background of wild type sequences can be identified down to at least 1% relative concentration. In order to show the clinical relevance, the detection capabilities are confirmed on challenging formalin-fixed paraffin-embedded clinical tumor samples. This enzyme-free detection framework contains the accuracy and efficiency to screen for hundreds of mutations in a single run with many potential applications in molecular diagnostics and the field of personalised medicine. PMID:28542229

Anomaly disentanglement on the basis of automatic processing of the aerial gamma-spectrometric survey data

International Nuclear Information System (INIS)

Perlovskij, V.A.; Zaika, S.D.; Lomtadze, V.V.

1976-01-01

Automated processing airborne gamma spectrometric data has been introduced with the use of the BESM-4 computer. Discrimination of anomalies is effected using a variable interval 200-300 m within which maximum values in the general counting channel and their coordinates are found; reference points are detected too against which one can determine the excess and amplitude of the anomalies. The discriminated anomalies are fixed on printer and charts of anomalies are constructed. The charts and tables characterizing the anomaly help in the identification of its potentialities and hence underlie a decision to start ground works

Recent progress in the development of CsI(Tl) crystal-Si-photodiode spectrometric detection assemblies

International Nuclear Information System (INIS)

Semynozhenko, V.P.; Grinyov, B.V.; Nekrasov, V.V.; Borodenko, Yu.A.

2005-01-01

Highly sensitive spectrometric γ-detection assemblies are developed using a comprehensive approach (optimization of crystal growth conditions as well as of treatment and packing of scintillators, creation of low-noise charge-sensitive preamplifiers and shapers). The detection assemblies with CsI(Tl)≤100 cm 3 have a sensitivity of about 20 pulse/s(mcR/h), their energy resolution with respect to 137 Cs γ-line being ≤8.5%. The assemblies with lesser scintillator volumes (1-5 cm 3 ) provide a resolution lower than 6% with respect to 137 Cs and ≤40% with respect to 241 Am

Molecular diagnostic methods for invasive fungal disease: the horizon draws nearer?

Science.gov (United States)

Halliday, C L; Kidd, S E; Sorrell, T C; Chen, S C-A

2015-04-01

Rapid, accurate diagnostic laboratory tests are needed to improve clinical outcomes of invasive fungal disease (IFD). Traditional direct microscopy, culture and histological techniques constitute the 'gold standard' against which newer tests are judged. Molecular diagnostic methods, whether broad-range or fungal-specific, have great potential to enhance sensitivity and speed of IFD diagnosis, but have varying specificities. The use of PCR-based assays, DNA sequencing, and other molecular methods including those incorporating proteomic approaches such as matrix-assisted laser desorption ionisation-time of flight mass spectroscopy (MALDI-TOF MS) have shown promising results. These are used mainly to complement conventional methods since they require standardisation before widespread implementation can be recommended. None are incorporated into diagnostic criteria for defining IFD. Commercial assays may assist standardisation. This review provides an update of molecular-based diagnostic approaches applicable to biological specimens and fungal cultures in microbiology laboratories. We focus on the most common pathogens, Candida and Aspergillus, and the mucormycetes. The position of molecular-based approaches in the detection of azole and echinocandin antifungal resistance is also discussed.

Laser mass spectrometry for DNA fingerprinting for forensic applications

Science.gov (United States)

Chen, C. H. Winston; Tang, Kai; Taranenko, N. I.; Allman, S. L.; Ch'ang, L. Y.

1994-10-01

The application of DNA fingerprinting has become very broad in forensic analysis, patient identification, diagnostic medicine, and wildlife poaching, since every individual's DNA structure is identical within all tissues oftheir body. DNA fingerprinting was initiated by the use of restriction fragment length polymorphisms (RFLP). In 1987, Nakamura et aL2 found that a variable number of tandem repeats (VNTR) often occurred in the alleles. The probability of different individuals having the same number of tandem repeats in several different alleles is very low. Thus, the identification of VNTR from genomic DNA became a very reliable method for identification of individuals. Take the Huntington gene as an example, there are CAG trinucleotide repeats. For normal people, the number of CAG repeats is usually between 10 and 40. Since people have chromosomes in pairs, the possibility oftwo individuals having the same VNTR in the Huntington gene is less than one percent, ifwe assume equal distribution for various repeats. When several allels containing VNTR are analyzed for the number of repeats, the possibility of two individuals being exactly identical becomes very unlikely. Thus, DNA fingerprinting is a reliable tool for forensic analysis. In DNA fingerprinting, knowledge of the sequence of tandem repeats and restriction endornuclease sites can provide the basis for identification.

Interlaboratory Reproducibility of Droplet Digital Polymerase Chain Reaction Using a New DNA Reference Material Format.

Science.gov (United States)

Pinheiro, Leonardo B; O'Brien, Helen; Druce, Julian; Do, Hongdo; Kay, Pippa; Daniels, Marissa; You, Jingjing; Burke, Daniel; Griffiths, Kate; Emslie, Kerry R

2017-11-07

Use of droplet digital PCR technology (ddPCR) is expanding rapidly in the diversity of applications and number of users around the world. Access to relatively simple and affordable commercial ddPCR technology has attracted wide interest in use of this technology as a molecular diagnostic tool. For ddPCR to effectively transition to a molecular diagnostic setting requires processes for method validation and verification and demonstration of reproducible instrument performance. In this study, we describe the development and characterization of a DNA reference material (NMI NA008 High GC reference material) comprising a challenging methylated GC-rich DNA template under a novel 96-well microplate format. A scalable process using high precision acoustic dispensing technology was validated to produce the DNA reference material with a certified reference value expressed in amount of DNA molecules per well. An interlaboratory study, conducted using blinded NA008 High GC reference material to assess reproducibility among seven independent laboratories demonstrated less than 4.5% reproducibility relative standard deviation. With the exclusion of one laboratory, laboratories had appropriate technical competency, fully functional instrumentation, and suitable reagents to perform accurate ddPCR based DNA quantification measurements at the time of the study. The study results confirmed that NA008 High GC reference material is fit for the purpose of being used for quality control of ddPCR systems, consumables, instrumentation, and workflow.

A probe-based quantitative PCR assay for detecting Tetracapsuloides bryosalmonae in fish tissue and environmental DNA water samples

Science.gov (United States)

Hutchins, Patrick; Sepulveda, Adam; Martin, Renee; Hopper, Lacey

2017-01-01

A probe-based quantitative real-time PCR assay was developed to detect Tetracapsuloides bryosalmonae, which causes proliferative kidney disease in salmonid fish, in kidney tissue and environmental DNA (eDNA) water samples. The limits of detection and quantification were 7 and 100 DNA copies for calibration standards and T. bryosalmonae was reliably detected down to 100 copies in tissue and eDNA samples. The assay presented here is a highly sensitive and quantitative tool for detecting T. bryosalmonae with potential applications for tissue diagnostics and environmental detection.

Diagnostic reasoning strategies and diagnostic success.

Science.gov (United States)

Coderre, S; Mandin, H; Harasym, P H; Fick, G H

2003-08-01

Cognitive psychology research supports the notion that experts use mental frameworks or "schemes", both to organize knowledge in memory and to solve clinical problems. The central purpose of this study was to determine the relationship between problem-solving strategies and the likelihood of diagnostic success. Think-aloud protocols were collected to determine the diagnostic reasoning used by experts and non-experts when attempting to diagnose clinical presentations in gastroenterology. Using logistic regression analysis, the study found that there is a relationship between diagnostic reasoning strategy and the likelihood of diagnostic success. Compared to hypothetico-deductive reasoning, the odds of diagnostic success were significantly greater when subjects used the diagnostic strategies of pattern recognition and scheme-inductive reasoning. Two other factors emerged as independent determinants of diagnostic success: expertise and clinical presentation. Not surprisingly, experts outperformed novices, while the content area of the clinical cases in each of the four clinical presentations demonstrated varying degrees of difficulty and thus diagnostic success. These findings have significant implications for medical educators. It supports the introduction of "schemes" as a means of enhancing memory organization and improving diagnostic success.

Solar thermal polymerase chain reaction for smartphone-assisted molecular diagnostics

Science.gov (United States)

Jiang, Li; Mancuso, Matthew; Lu, Zhengda; Akar, Gunkut; Cesarman, Ethel; Erickson, David

2014-02-01

Nucleic acid-based diagnostic techniques such as polymerase chain reaction (PCR) are used extensively in medical diagnostics due to their high sensitivity, specificity and quantification capability. In settings with limited infrastructure and unreliable electricity, however, access to such devices is often limited due to the highly specialized and energy-intensive nature of the thermal cycling process required for nucleic acid amplification. Here we integrate solar heating with microfluidics to eliminate thermal cycling power requirements as well as create a simple device infrastructure for PCR. Tests are completed in less than 30 min, and power consumption is reduced to 80 mW, enabling a standard 5.5 Wh iPhone battery to provide 70 h of power to this system. Additionally, we demonstrate a complete sample-to-answer diagnostic strategy by analyzing human skin biopsies infected with Kaposi's Sarcoma herpesvirus (KSHV/HHV-8) through the combination of solar thermal PCR, HotSHOT DNA extraction and smartphone-based fluorescence detection. We believe that exploiting the ubiquity of solar thermal energy as demonstrated here could facilitate broad availability of nucleic acid-based diagnostics in resource-limited areas.

A superheterodyne spectrometer for electronic paramagnetic. Resonance; Spectrometre superheterodyne de resonance paramagnetique electronique

Energy Technology Data Exchange (ETDEWEB)

Laffon, J L [Commissariat a l' Energie Atomique, Grenoble (France). Centre d' Etudes Nucleaires

1963-12-15

After a few generalities about electron paramagnetic resonance, a consideration of different experimental techniques authorises the choice of a particular type of apparatus. An EPR superheterodyne spectrometer built in the laboratory and having a novel circuit is described in detail. With this apparatus, many experimental results have been obtained and some of these are described as example. (author) [French] Apres quelques generalites sur le phenomene de resonance paramagnetique electronique, une synthese des differentes techniques experimentales, permet de fixer le choix d'un type d'appareillage. Un spectrometre de RPE superheterodyne realise en laboratoire et comportant un circuit original est expose dans le detail. Cet appareil a permis de nombreux resultats experimentaux dont quelques-uns sont decrits a titre d'exemple. (auteur)

Revisited diagnostics of hereditary epidermolysis bullosa

Directory of Open Access Journals (Sweden)

V. I. Albanova

2014-01-01

Full Text Available Hereditary epidermolysis bullosa is a big group of hereditary diseases with the main manifestations in the form of blisters on the skin and mucous coat after slight mechanical injuries. It is not always possible to diagnose this disease based on the clinical picture. The article discusses current laboratory diagnostics methods for hereditary epidermolysis bullosa including immunofluorescence antigen mapping (IFM, transmission electron microscopy (TEM and genetic analysis (molecular or DNA diagnostics as well as their advantages and disadvantages. TEM determines the micro splitting level and nature of ultrafine changes in the area of the dermoepidermal junction; at the same time, such tests need special expensive equipment. Substantial experience is also needed to analyze the resulting submicroscopic images. IFM determines whether expression of the affected protein related to the disease development is reduced or absent; however, invalid (false positive or false negative results can be obtained in patients with the reduced expression of the affected protein. Genetic analysis plays a key role for prenatal diagnostics. Therefore, to make an exact diagnosis of hereditary epidermolysis bullosa, it is expedient to apply IFM, TEM and genetic analysis. The need to set an exact diagnosis of the disease is related to the fact that the promising treatment methods being currently developed are aimed at treating patients with certain forms of the disease.

Radiation risk of diagnostical procedures

International Nuclear Information System (INIS)

Pohlit, W.

1986-01-01

The environmental radiation burden of man in Germany is about 1 mGy (Milligray) per year. This is, of course, also valid for children. Due to diagnostical procedures this burden is increased to about 1.3 mGy. The question arises wether this can be neglected, or important consequences have to be drawn. To give a clear answer, the action of ionizing radiation in living cells and in organisms is explained in detail. Many of the radiation actions at the DNA can soon be repaired by the cell, if the radiation dose was small. Some damage, however will remain irreparable for the cell and consequently leads to cell death, to mutations or to cell transformation. The number of these lesion increases or decreases linearily with radiation dose. Therefore, it must be expected that the risk of tumour induction is increased to above the normal background even by the smallest doses. This small but not negligible risk has to be compared with other risks of civilization or with other medical risks. But also the benefit and the efficacy of diagnostic procedures have to be considered. (orig./HSCH) [de

[Diagnostic tools for canine parvovirus infection].

Science.gov (United States)

Proksch, A L; Hartmann, K

2015-01-01

Canine parvovirus (CPV) infection is one of the most important and common infectious diseases in dogs, in particular affecting young puppies when maternal antibodies have waned and vaccine-induced antibodies have not yet developed. The mortality rate remains high. Therefore, a rapid and safe diagnostic tool is essential to diagnose the disease to 1) provide intensive care treatment and 2) to identify virus-shedding animals and thus prevent virus spread. Whilst the detection of antibodies against CPV is considered unsuitable to diagnose the disease, there are several different methods to directly detect complete virus, virus antigen or DNA. Additionally, to test in commercial laboratories, rapid in-house tests based on ELISA are available worldwide. The specificity of the ELISA rapid in-house tests is reported to be excellent. However, results on sensitivity vary and high numbers of false-negative results are commonly reported, which potentially leads to misdiagnosis. Polymerase chain reaction (PCR) is a very sensitive and specific diagnostic tool. It also provides the opportunity to differentiate vaccine strains from natural infection when sequencing is performed after PCR.

Comparative Study of Seven Commercial Kits for Human DNA Extraction from Urine Samples Suitable for DNA Biomarker-Based Public Health Studies

Science.gov (United States)

El Bali, Latifa; Diman, Aurélie; Bernard, Alfred; Roosens, Nancy H. C.; De Keersmaecker, Sigrid C. J.

2014-01-01

Human genomic DNA extracted from urine could be an interesting tool for large-scale public health studies involving characterization of genetic variations or DNA biomarkers as a result of the simple and noninvasive collection method. These studies, involving many samples, require a rapid, easy, and standardized extraction protocol. Moreover, for practicability, there is a necessity to collect urine at a moment different from the first void and to store it appropriately until analysis. The present study compared seven commercial kits to select the most appropriate urinary human DNA extraction procedure for epidemiological studies. DNA yield has been determined using different quantification methods: two classical, i.e., NanoDrop and PicoGreen, and two species-specific real-time quantitative (q)PCR assays, as DNA extracted from urine contains, besides human, microbial DNA also, which largely contributes to the total DNA yield. In addition, the kits giving a good yield were also tested for the presence of PCR inhibitors. Further comparisons were performed regarding the sampling time and the storage conditions. Finally, as a proof-of-concept, an important gene related to smoking has been genotyped using the developed tools. We could select one well-performing kit for the human DNA extraction from urine suitable for molecular diagnostic real-time qPCR-based assays targeting genetic variations, applicable to large-scale studies. In addition, successful genotyping was possible using DNA extracted from urine stored at −20°C for several months, and an acceptable yield could also be obtained from urine collected at different moments during the day, which is particularly important for public health studies. PMID:25365790

Comparative study of seven commercial kits for human DNA extraction from urine samples suitable for DNA biomarker-based public health studies.

Science.gov (United States)

El Bali, Latifa; Diman, Aurélie; Bernard, Alfred; Roosens, Nancy H C; De Keersmaecker, Sigrid C J

2014-12-01

Human genomic DNA extracted from urine could be an interesting tool for large-scale public health studies involving characterization of genetic variations or DNA biomarkers as a result of the simple and noninvasive collection method. These studies, involving many samples, require a rapid, easy, and standardized extraction protocol. Moreover, for practicability, there is a necessity to collect urine at a moment different from the first void and to store it appropriately until analysis. The present study compared seven commercial kits to select the most appropriate urinary human DNA extraction procedure for epidemiological studies. DNA yield has been determined using different quantification methods: two classical, i.e., NanoDrop and PicoGreen, and two species-specific real-time quantitative (q)PCR assays, as DNA extracted from urine contains, besides human, microbial DNA also, which largely contributes to the total DNA yield. In addition, the kits giving a good yield were also tested for the presence of PCR inhibitors. Further comparisons were performed regarding the sampling time and the storage conditions. Finally, as a proof-of-concept, an important gene related to smoking has been genotyped using the developed tools. We could select one well-performing kit for the human DNA extraction from urine suitable for molecular diagnostic real-time qPCR-based assays targeting genetic variations, applicable to large-scale studies. In addition, successful genotyping was possible using DNA extracted from urine stored at -20°C for several months, and an acceptable yield could also be obtained from urine collected at different moments during the day, which is particularly important for public health studies.

Surface acoustic wave nebulization facilitating lipid mass spectrometric analysis.

Science.gov (United States)

Yoon, Sung Hwan; Huang, Yue; Edgar, J Scott; Ting, Ying S; Heron, Scott R; Kao, Yuchieh; Li, Yanyan; Masselon, Christophe D; Ernst, Robert K; Goodlett, David R

2012-08-07

Surface acoustic wave nebulization (SAWN) is a novel method to transfer nonvolatile analytes directly from the aqueous phase to the gas phase for mass spectrometric analysis. The lower ion energetics of SAWN and its planar nature make it appealing for analytically challenging lipid samples. This challenge is a result of their amphipathic nature, labile nature, and tendency to form aggregates, which readily precipitate clogging capillaries used for electrospray ionization (ESI). Here, we report the use of SAWN to characterize the complex glycolipid, lipid A, which serves as the membrane anchor component of lipopolysaccharide (LPS) and has a pronounced tendency to clog nano-ESI capillaries. We also show that unlike ESI SAWN is capable of ionizing labile phospholipids without fragmentation. Lastly, we compare the ease of use of SAWN to the more conventional infusion-based ESI methods and demonstrate the ability to generate higher order tandem mass spectral data of lipid A for automated structure assignment using our previously reported hierarchical tandem mass spectrometry (HiTMS) algorithm. The ease of generating SAWN-MS(n) data combined with HiTMS interpretation offers the potential for high throughput lipid A structure analysis.

Regular square planer bis-(4,4,4-trifluoro-1-(thiophen-2-yl)butane-1,3-dione)/copper(II) complex: Trans/cis-DFT isomerization, crystal structure, thermal, solvatochromism, hirshfeld surface and DNA-binding analysis

Science.gov (United States)

Hema, M. K.; Karthik, C. S.; Warad, Ismail; Lokanath, N. K.; Zarrouk, Abdelkader; Kumara, Karthik; Pampa, K. J.; Mallu, P.

2018-04-01

Trans-[Cu(O∩O)2] complex, O∩O = 4,4,4-trifluoro-1-(thiophen-2-yl)butane-1,3-dione was reported with high potential toward CT-DNA binder. The solved XRD-structure of complex indicated a perfect regular square-planer geometry around the Cu(II) center. The trans/cis-DFT-isomerization calculation supported the XRD seen in reflecting the trans-isomer as the kinetic-favor isomer. The desired complex structure was also characterized by conductivity measurement, CHN-elemental analyses, MS, EDX, SEM, UV-Vis., FT-IR, HAS and TG/DTG. The Solvatochromism behavior of the complex was evaluated using four different polar solvents. MPE and Hirshfeld surface analysis (HSA) come to an agreement that fluoride and thiophene protons atoms are with suitable electro-potential environment to form non-classical H-bonds of type CThsbnd H⋯F. The DNA-binding properties were investigated by viscosity tests and spectrometric titrations, the results revealed the complex as strong calf-thymus DNA binder. High intrinsic-binding constants value ∼1.8 × 105 was collected.

Novel Readout Method for Molecular Diagnostic Assays Based on Optical Measurements of Magnetic Nanobead Dynamics

DEFF Research Database (Denmark)

Donolato, Marco; Antunes, Paula Soares Martins; Bejhed, Rebecca S.

2015-01-01

behavior. We prove that the optical transmission spectra are highly sensitive to the formation of permanent MNB clusters and, thereby to the target analyte concentration. As a specific clinically relevant diagnostic case, we detect DNA coils formed via padlock probe recognition and isothermal rolling...

Diagnostic strategy for occult hepatitis B virus infection

Science.gov (United States)

Ocana, Sara; Casas, Maria Luisa; Buhigas, Ingrid; Lledo, Jose Luis

2011-01-01

In 2008, the European Association for the study of the liver (EASL) defined occult hepatitis B virus infection (OBI) as the “presence of hepatitis B virus (HBV) DNA in the liver (with detectable or undetectable HBV DNA in the serum) of individuals testing hepatitis B surface antigen (HBsAg) negative by currently available assays”. Several aspects of occult HBV infection are still poorly understood, including the definition itself and a standardized approach for laboratory-based detection, which is the purpose of this review. The clinical significance of OBI has not yet been established; however, in terms of public health, the clinical importance arises from the risk of HBV transmission. Consequently, it is important to detect high-risk groups for occult HBV infection to prevent transmission. The main issue is, perhaps, to identify the target population for screening OBI. Viremia is very low or undetectable in occult HBV infection, even when the most sensitive methods are used, and the detection of the viral DNA reservoir in hepatocytes would provide the best evaluation of occult HBV prevalence in a defined set of patients. However, this diagnostic approach is obviously unsuitable: blood detection of occult hepatitis B requires assays of the highest sensitivity and specificity with a lower limit of detection < 10 IU/mL for HBV DNA and < 0.1 ng/mL for HBsAg. PMID:21472120

The potential role for use of mitochondrial DNA copy number as predictive biomarker in presbycusis.

Science.gov (United States)

Falah, Masoumeh; Houshmand, Massoud; Najafi, Mohammad; Balali, Maryam; Mahmoudian, Saeid; Asghari, Alimohamad; Emamdjomeh, Hessamaldin; Farhadi, Mohammad

2016-01-01

Age-related hearing impairment, or presbycusis, is the most common communication disorder and neurodegenerative disease in the elderly. Its prevalence is expected to increase, due to the trend of growth of the elderly population. The current diagnostic test for detection of presbycusis is implemented after there has been a change in hearing sensitivity. Identification of a pre-diagnostic biomarker would raise the possibility of preserving hearing sensitivity before damage occurs. Mitochondrial dysfunction, including the production of reactive oxygen species and induction of expression of apoptotic genes, participates in the progression of presbycusis. Mitochondrial DNA sequence variation has a critical role in presbycusis. However, the nature of the relationship between mitochondrial DNA copy number, an important biomarker in many other diseases, and presbycusis is undetermined. Fifty-four subjects with presbycusis and 29 healthy controls were selected after ear, nose, throat examination and pure-tone audiometry. DNA was extracted from peripheral blood samples. The copy number of mitochondrial DNA relative to the nuclear genome was measured by quantitative real-time polymerase chain reaction. Subjects with presbycusis had a lower median mitochondrial DNA copy number than healthy subjects and the difference was statistically significant ( P =0.007). Mitochondrial DNA copy number was also significantly associated with degree of hearing impairment ( P =0.025) and audiogram configuration ( P =0.022). The findings of this study suggest that lower mitochondrial DNA copy number is responsible for presbycusis through alteration of mitochondrial function. Moreover, the significant association of mitochondrial DNA copy number in peripheral blood samples with the degree of hearing impairment and audiogram configuration has potential for use as a standard test for presbycusis, providing the possibility of the development of an easy-to-use biomarker for the early detection of

Microfluidic DNA microarrays in PMMA chips: streamlined fabrication via simultaneous DNA immobilization and bonding activation by brief UV exposure

DEFF Research Database (Denmark)

Sabourin, David; Petersen, J; Snakenborg, Detlef

2010-01-01

This report presents and describes a simple and scalable method for producing functional DNA microarrays within enclosed polymeric, PMMA, microfluidic devices. Brief (30 s) exposure to UV simultaneously immobilized poly(T)poly(C)-tagged DNA probes to the surface of unmodified PMMA and activated...... the surface for bonding below the glass transition temperature of the bulk PMMA. Functionality and validation of the enclosed PMMA microarrays was demonstrated as 18 patients were correctly genotyped for all eight mutation sites in the HBB gene interrogated. The fabrication process therefore produced probes...... with desired hybridization properties and sufficient bonding between PMMA layers to allow construction of microfluidic devices. The streamlined fabrication method is suited to the production of low-cost microfluidic microarray-based diagnostic devices and, as such, is equally applicable to the development...

The Contribution of the Activation Entropy to the Gas-Phase Stability of Modified Nucleic Acid Duplexes

Science.gov (United States)

Hari, Yvonne; Dugovič, Branislav; Istrate, Alena; Fignolé, Annabel; Leumann, Christian J.; Schürch, Stefan

2016-07-01

Tricyclo-DNA (tcDNA) is a sugar-modified analogue of DNA currently tested for the treatment of Duchenne muscular dystrophy in an antisense approach. Tandem mass spectrometry plays a key role in modern medical diagnostics and has become a widespread technique for the structure elucidation and quantification of antisense oligonucleotides. Herein, mechanistic aspects of the fragmentation of tcDNA are discussed, which lay the basis for reliable sequencing and quantification of the antisense oligonucleotide. Excellent selectivity of tcDNA for complementary RNA is demonstrated in direct competition experiments. Moreover, the kinetic stability and fragmentation pattern of matched and mismatched tcDNA heteroduplexes were investigated and compared with non-modified DNA and RNA duplexes. Although the separation of the constituting strands is the entropy-favored fragmentation pathway of all nucleic acid duplexes, it was found to be only a minor pathway of tcDNA duplexes. The modified hybrid duplexes preferentially undergo neutral base loss and backbone cleavage. This difference is due to the low activation entropy for the strand dissociation of modified duplexes that arises from the conformational constraint of the tc-sugar-moiety. The low activation entropy results in a relatively high free activation enthalpy for the dissociation comparable to the free activation enthalpy of the alternative reaction pathway, the release of a nucleobase. The gas-phase behavior of tcDNA duplexes illustrates the impact of the activation entropy on the fragmentation kinetics and suggests that tandem mass spectrometric experiments are not suited to determine the relative stability of different types of nucleic acid duplexes.

Quantitative PCR--new diagnostic tool for quantifying specific mRNA and DNA molecules

DEFF Research Database (Denmark)

Schlemmer, B O; Sorensen, B S; Overgaard, J

2004-01-01

of a subset of ligands from the EGF system is increased in bladder cancer. Furthermore, measurement of the mRNA concentration gives important information such as the expression of these ligands correlated to the survival of the patients. In addition to the alterations at the mRNA level, changes also can occur...... at the DNA level in the EGF system. Thus, it has been demonstrated that the number of genes coding for the human epidermal growth factor receptor 2 (HER2) is increased in a number of breast tumors. It is now possible to treat breast cancer patients with a humanized antibody reacting with HER2...... of mRNA or DNA in biological samples. In this study quantitative PCR was used to investigate the role of the EGF (epidermal growth factor) system in cancer both for measurements of mRNA concentrations and for measurements of the number of copies of specific genes. It is shown that the mRNA expression...

Plasmodium vivax molecular diagnostics in community surveys: pitfalls and solutions.

Science.gov (United States)

Gruenberg, Maria; Moniz, Clara Antunes; Hofmann, Natalie Ellen; Wampfler, Rahel; Koepfli, Cristian; Mueller, Ivo; Monteiro, Wuelton Marcelo; Lacerda, Marcus; de Melo, Gisely Cardoso; Kuehn, Andrea; Siqueira, Andre M; Felger, Ingrid

2018-01-30

A distinctive feature of Plasmodium vivax infections is the overall low parasite density in peripheral blood. Thus, identifying asymptomatic infected individuals in endemic communities requires diagnostic tests with high sensitivity. The detection limits of molecular diagnostic tests are primarily defined by the volume of blood analysed and by the copy number of the amplified molecular marker serving as the template for amplification. By using mitochondrial DNA as the multi-copy template, the detection limit can be improved more than tenfold, compared to standard 18S rRNA targets, thereby allowing detection of lower parasite densities. In a very low transmission area in Brazil, application of a mitochondrial DNA-based assay increased prevalence from 4.9 to 6.5%. The usefulness of molecular tests in malaria epidemiological studies is widely recognized, especially when precise prevalence rates are desired. Of concern, however, is the challenge of demonstrating test accuracy and quality control for samples with very low parasite densities. In this case, chance effects in template distribution around the detection limit constrain reproducibility. Rigorous assessment of false positive and false negative test results is, therefore, required to prevent over- or under-estimation of parasite prevalence in epidemiological studies or when monitoring interventions.

Anomalies from aerial spectrometric and total count radiometric surveys in the southeastern United States

International Nuclear Information System (INIS)

Lee, C.H.; Lawton, D.E.

1978-01-01

Aerial radiometric reconnaissance surveys are conducted because of their cost, time, and manpower savings compared to surface studies. Two types of aerial surveys are being flown in the southeastern United States: total count gamma-ray surveys for the Coastal Plains Regional Commission and the US Geological Survey, and differential gamma-ray spectrometric surveys for the US Department of Energy. Anomalous radioactivity detected during aerial surveys is related to higher concentrations of naturally occurring uranium, or to cultural activities, natural causes, or mapping errors which simulate real uranium anomalies. Each anomaly should be ground checked; however, several types of anomalies may be eliminated by evaluation of the aerial data in the office if field time is limited

Study of natural diamond detector spectrometric properties under neutron irradiation

CERN Document Server

Alekseyev, A B; Kaschuck, Y; Krasilnikov, A; Portnov, D; Tugarinov, S

2002-01-01

Natural diamond detector (NDD) performance was studied up to a neutron fluence of 10 sup 1 sup 5 neutron/cm sup 2. The variations of the NDD spectrometric response to incident alpha-particles from sup 2 sup 4 sup 1 Am source after exposure to fast neutron fluences up to 3x10 sup 1 sup 6 n/cm sup 2 were examined. No significant variations up to the level of 10 sup 1 sup 4 n/cm sup 2 were observed. Degradation of charge collection efficiency at higher fluences is reported. No remarkable increase of the NDD leakage current and count rate change had been observed up to a neutron fluence of 3x10 sup 1 sup 6 n/cm sup 2. The charge collection efficiency variations of neutron irradiated diamond spectrometer were studied ex situ under gamma-rays, beta-radiation and visible light excitation. Charge collection efficiency restoration up to 75% level and the NDD performance stabilization by extrinsic low-intensity visible light (550 nm

Preimplantation genetic diagnosis for mitochondrial DNA mutations: analysis of one blastomere suffices.

Science.gov (United States)

Sallevelt, Suzanne C E H; Dreesen, Joseph C F M; Coonen, Edith; Paulussen, Aimee D C; Hellebrekers, Debby M E I; de Die-Smulders, Christine E M; Smeets, Hubert J M; Lindsey, Patrick

2017-10-01

Preimplantation genetic diagnosis (PGD) is a reproductive strategy for mitochondrial DNA (mtDNA) mutation carriers, strongly reducing their risk of affected offspring. Embryos either without the mutation or with mutation load below the phenotypic threshold are transferred to the uterus. Because of incidental heteroplasmy deviations in single blastomere and the relatively limited data available, we so far preferred relying on two blastomeres rather than one. Considering the negative effect of a two-blastomere biopsy protocol compared with a single-blastomere biopsy protocol on live birth delivery rate, we re-evaluated the error rate in our current dataset. For the m.3243A>G mutation, sufficient embryos/blastomeres were available for a powerful analysis. The diagnostic error rate, defined as a potential false-negative result, based on a threshold of 15%, was determined in 294 single blastomeres analysed in 73 embryos of 9 female m.3243A>G mutation carriers. Only one out of 294 single blastomeres (0.34%) would have resulted in a false-negative diagnosis. False-positive diagnoses were not detected. Our findings support a single-blastomere biopsy PGD protocol for the m.3243A>G mutation as the diagnostic error rate is very low. As in the early preimplantation embryo no mtDNA replication seems to occur and the mtDNA is divided randomly among the daughter cells, we conclude this result to be independent of the specific mutation and therefore applicable to all mtDNA mutations. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2017. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

Digitally encoded DNA nanostructures for multiplexed, single-molecule protein sensing with nanopores

Science.gov (United States)

Bell, Nicholas A. W.; Keyser, Ulrich F.

2016-07-01

The simultaneous detection of a large number of different analytes is important in bionanotechnology research and in diagnostic applications. Nanopore sensing is an attractive method in this regard as the approach can be integrated into small, portable device architectures, and there is significant potential for detecting multiple sub-populations in a sample. Here, we show that highly multiplexed sensing of single molecules can be achieved with solid-state nanopores by using digitally encoded DNA nanostructures. Based on the principles of DNA origami, we designed a library of DNA nanostructures in which each member contains a unique barcode; each bit in the barcode is signalled by the presence or absence of multiple DNA dumbbell hairpins. We show that a 3-bit barcode can be assigned with 94% accuracy by electrophoretically driving the DNA structures through a solid-state nanopore. Select members of the library were then functionalized to detect a single, specific antibody through antigen presentation at designed positions on the DNA. This allows us to simultaneously detect four different antibodies of the same isotype at nanomolar concentration levels.

Surveillance of intestinal schistosomiasis during control: a comparison of four diagnostic tests across five Ugandan primary schools in the Lake Albert region.

Science.gov (United States)

Al-Shehri, Hajri; Koukounari, Artemis; Stanton, Michelle C; Adriko, Moses; Arinaitwe, Moses; Atuhaire, Aaron; Kabatereine, Narcis B; Stothard, J Russell

2018-03-21

Programmatic surveillance of intestinal schistosomiasis during control can typically use four diagnostic tests, either singularly or in combination, but these have yet to be cross-compared directly. Our study assembled a complete diagnostic dataset, inclusive of infection intensities, from 258 children from five Ugandan primary schools. The schools were purposely selected as typical of the endemic landscape near Lake Albert and reflective of high- and low-transmission settings. Overall prevalence was: 44.1% (95% CI 38.0-50.2) by microscopy of duplicate Kato-Katz smears from two consecutive stools, 56.9% (95% CI 50.8-63.0) by urine-circulating cathodic antigen (CCA) dipstick, 67.4% (95% CI 61.6-73.1) by DNA-TaqMan® and 75.1% (95% CI 69.8-80.4) by soluble egg antigen enzyme-linked immunosorbent assay (SEA-ELISA). A cross-comparison of diagnostic sensitivities, specificities, positive and negative predictive values was undertaken, inclusive of a latent class analysis (LCA) with a LCA-model estimate of prevalence by each school. The latter ranged from 9.6% to 100.0%, and prevalence by school for each diagnostic test followed a static ascending order or monotonic series of Kato-Katz, urine-CCA dipstick, DNA-TaqMan® and SEA-ELISA. We confirm that Kato-Katz remains a satisfactory diagnostic standalone in high-transmission settings but in low-transmission settings should be augmented or replaced by urine-CCA dipsticks. DNA-TaqMan® appears suitable in both endemic settings though is only implementable if resources permit. In low-transmission settings, SEA-ELISA remains the method of choice to evidence an absence infection. We discuss the pros and cons of each method concluding that future surveillance of intestinal schistosomiasis would benefit from a flexible, context-specific approach both in choice and application of each diagnostic method, rather than a single one-size fits all approach.

Sr isotope geochemistry of East Alpine mineral deposits and mass spectrometric analyses of fluid inclusions

International Nuclear Information System (INIS)

Grum, W.

1995-05-01

Strontium isotope geochemistry and deformational history of selected carbonate-hosted deposits from different tectonic positions in the Eastern Alps were studied. In this context an equipment has been constructed to analyze the composition of volatile components of fluid inclusions (FI). Based on the results of Sr-isotopic investigations two groups of deposits are discriminated: Deposits formed by formation waters and/or metamorphogenic fluids: Tux (magnesite, scheelite), Otterzug (barite), Lassing (magnesite), Rabenwald (talc), Laussa and Mooseck (fluorite). The mineralizing fluids are derived from different sedimentary rock series and therefore 87 Sr/ 86 Sr-ratios vary between 0.707 and 0.719. Deposits situated along fault zones: Lassing (talc), Gasteiner Tal (Silberpfennig area; gold), Schlaining (stibnite) and Waldenstein (specularite). Sr isotope ratios of the mineralizing fluids from Lassing, Waldenstein and Schlaining ranges from 0.7112 to 0.7127 and are therefore thought to have scavenged the East Alpine crystalline. The mineralizing solutions of the Gasteiner Tal deposit may either have equilibrated with low radiogenic sedimentary or with (ultra-)basic rocks. A cracking chamber has been constructed in order to open FI by cracking for mass spectrometric analysis of volatile components. Gases are analysed using a quadrupole mass spectrometer. The desorption of gas from metal and sample surfaces during cracking can be neglected. The amount of gas released from the mineral lattices was studied. With that fast method ore bearing from barren host rocks have been distinguished by different composition of the FI at the Brixlegg barite mineralization (Eastern Alps). Within the Galway fluorite deposit (Ireland) different fluids were involved and mass spectrometric analysis were carried out to characterize these different fluids and to identify their origin. (author)

Gamma-spectrometric surveys in differentiated granites. I: a review of the method and of the geochemical behavior of K, Th and U

International Nuclear Information System (INIS)

Ulbrich, Horstpeter Herberto Gustavo Jose; Ulbrich, Mabel Norma Costas; Guimaraes, Gilson Burigo

2009-01-01

This contribution is part of a research project on the Neo proterozoic Cunhaporanga Granitic Complex (CGC), cropping out in the Ponta Grossa Arch (Parana state, SE Brazil). An initial study used the gamma-spectrometric data of the Serra do Mar Sul Aero geophysical Project, performed during the 70's for CPRM. Later, terrestrial gamma-spectrometric surveys focused on the study of the differentiated Joaquim Murtinho Granite (JMG) in the NW corner of CGC, and the Serra do Carambei Granite, to the SW. In this paper, the results obtained for JMG are presented in two parts. The first deals with methodology and the presentation of several gamma-spectrometric 'color-scale' maps, indicating that results obtained in granites depend strongly on a climatic factor, given the mobility of K during weathering in subtropical climates with strong rainfalls, also favoring a greater mobility of U. Minerals that are U and Th hosts, documented in granites, are reviewed, together with the weathering processes that control the mobility of K, U and Th in soils. Strong K signals in granitic areas submitted to these climates document the presence of fresh rock and/or effects of hydrothermal alteration, while weak or nil signals are evidence of strong leaching of K during weathering. U and Th will be retained in the residual soils, in part leading to their selective enrichment, also coupled with soil migration to lower topographic levels by colluvial transport. The larger solubility of U (as uranyl ion) allows its liberation under oxidizing conditions, and its migration, limited by the possibility of absorption in newly formed mineral and organic soil phases. Th should be retained almost totally in resistant phases and, when liberated in solution, will mostly be fixed in organic and inorganic soil substances. (author)

Synthesis and DNA-interactions of new Co(III), Fe(II), Ni(II), Ru(II ...

Indian Academy of Sciences (India)

Administrator

phen) or a modified phen, are particularly attractive species for developing new diagnostic and therapeutic agents that can recognise and cleave DNA. The ligands or the metal in these complexes can be varied in an easily controlled manner ...

Identification of Diagnostic Mitochondrial DNA Single Nucleotide Polymorphisms Specific to Sumatran Orangutan (Pongo abelii Populations

Directory of Open Access Journals (Sweden)

Puji Rianti

2015-10-01

Full Text Available The hypervariable region I of mitochondrial DNA has frequently been used to distinguish among populations, in particular in species with strong female philopatry. In such cases, populations are expected to diverge rapidly for hypervariable region I markers because of the smaller effective population size and thus increased genetic drift. This rapid divergence leads to the accumulation of mutations exclusively found in one population, which may serve as diagnostic single nucleotide polymorphisms (SNPs. To date, diagnostic SNPs distinctive to Sumatran orangutan populations have not yet been described. However, given the continuously declining numbers of Sumatran orangutans, this information can be vital for effective conservation measures, especially regarding reintroductions of orangutans in rehabilitation centers. Phylogenetic analyses of 54 samples of Sumatran orangutans from nine sampling sites with good provenance, we found five major clades and a total of 20 haplotypes. We propose a total of 52 diagnostic SNPs that are specific to Sumatran orangutan populations. Data can be used to develop restriction fragment length polymorphism assays to carry out genetic assignments using basic laboratory equipment to assign Sumatran orangutan to their population of origin.

Stk1-mediated phosphorylation stimulates the DNA-binding properties of the Staphylococcus aureus SpoVG transcriptional factor.

Science.gov (United States)

Bischoff, Markus; Brelle, Solène; Minatelli, Sabrina; Molle, Virginie

2016-05-13

The stage V sporulation protein G (SpoVG) homolog of Staphylococcus aureus is a modulator of virulence factor synthesis and antibiotic resistance in this clinically important gram-positive pathogen. Here we demonstrate that SpoVG can be phosphorylated by the staphylococcal Ser/Thr protein kinase Stk1 and that phosphorylation positively affects its DNA-binding properties. Mass spectrometric analyses and site directed mutagenesis identified Thr4, Thr13, Thr24 and Ser41 as phospho-acceptors. Stk1-mediated phosphorylation markedly enhanced the DNA binding activity of SpoVG towards the promoter regions of target genes such as capA, lip, and nuc1. Similarly, trans-complementation of the S. aureus ΔyabJ-spoVG mutant SM148 with a SpoVG derivative that mimics constitutive phosphorylation, SpoVG_Asp, exhibited capA, lip, and nuc1 transcript levels that were comparable to the levels seen with the wild-type, whereas trans-complementation with a phosphoablative variant of SpoVG (SpoVG_Ala) produced transcript levels similar to the ones seen in SM148. Our data suggest that the expression/activity of this transcription factor is tightly controlled in S. aureus by transcriptional, post-transcriptional and post-translational mechanisms. Copyright © 2016 Elsevier Inc. All rights reserved.

Mass-Spectrometric Studies of Catalytic Chemical Vapor Deposition Processes of Organic Silicon Compounds Containing Nitrogen

Science.gov (United States)

Morimoto, Takashi; Ansari, S. G.; Yoneyama, Koji; Nakajima, Teppei; Masuda, Atsushi; Matsumura, Hideki; Nakamura, Megumi; Umemoto, Hironobu

2006-02-01

The mechanism of catalytic chemical vapor deposition (Cat-CVD) processes for hexamethyldisilazane (HMDS) and trisdimethylaminosilane (TDMAS), which are used as source gases to prepare SiNx or SiCxNy films, was studied using three different mass spectrometric techniques: ionization by Li+ ion attachment, vacuum-ultraviolet radiation and electron impact. The results for HMDS show that Si-N bonds dissociate selectively, although Si-C bonds are weaker, and (CH3)3SiNH should be one of the main precursors of deposited films. This decomposition mechanism did not change when NH3 was introduced, but the decomposition efficiency was slightly increased. Similar results were obtained for TDMAS.

Organisatsiooni DNA neli komponenti / Gary Neilson, Bruce A. Pasterneck, Decio Mendes ; tõlk. Kadre Vaik

Index Scriptorium Estoniae

Neilson, Gary

2004-01-01

Autorid selgitavad organisatsiooni nelja põhikomponendi - struktuuri, otsustusõiguse, motivaatorite ning informatsiooni omavahelisi seoseid. Diagramm: Kontrolli ulatuse võrdlus. Skeem: Liivakell-organisatsioon. Vt. samas: Quest Diagnostic DNA analüüs; "Me leidsime just elu saladuse!"

Mass Spectrometric C-14 Detection Techniques: Progress Report

Science.gov (United States)

Synal, H.

2013-12-01

Accelerator Mass Spectrometry (AMS) has been established as the best-suited radiocarbon detection technique. In the past years, significant progress with AMS instrumentation has been made resulting in a boom of new AMS facilities around the World. Today, carbon only AMS systems predominantly utilize 1+ charge state and molecule destruction in multiple ion gas collisions in stripper gas cell. This has made possible a significant simplification of the instruments, a reduction of ion energies and related to this less required space of the installations. However, state-of-the-art AMS instruments have still not reached a development stage where they can be regarded as table-top systems. In this respect, more development is needed to further advance the applicability of radiocarbon not only in the traditional fields of dating but also in biomedical research and new fields in Earth and environmental sciences. In a the proof-of-principle experiment the feasibility of radiocarbon detection over the entire range of dating applications was demonstrated using a pure mass spectrometer and ion energies below 50 keV. Now an experimental platform has been completed to test performance and to explore operation and measurement conditions of pure mass spectrometric radiocarbon detection. This contribution will overview the physical principles, which make this development possible and discuss key parameters of the instrumental design and performance of such an instrument.

Template-directed covalent conjugation of DNA to native antibodies, transferrin and other metal-binding proteins

Science.gov (United States)

Rosen, Christian B.; Kodal, Anne L. B.; Nielsen, Jesper S.; Schaffert, David H.; Scavenius, Carsten; Okholm, Anders H.; Voigt, Niels V.; Enghild, Jan J.; Kjems, Jørgen; Tørring, Thomas; Gothelf, Kurt V.

2014-09-01

DNA-protein conjugates are important in bioanalytical chemistry, molecular diagnostics and bionanotechnology, as the DNA provides a unique handle to identify, functionalize or otherwise manipulate proteins. To maintain protein activity, conjugation of a single DNA handle to a specific location on the protein is often needed. However, preparing such high-quality site-specific conjugates often requires genetically engineered proteins, which is a laborious and technically challenging approach. Here we demonstrate a simpler method to create site-selective DNA-protein conjugates. Using a guiding DNA strand modified with a metal-binding functionality, we directed a second DNA strand to the vicinity of a metal-binding site of His6-tagged or wild-type metal-binding proteins, such as serotransferrin, where it subsequently reacted with lysine residues at that site. This method, DNA-templated protein conjugation, facilitates the production of site-selective protein conjugates, and also conjugation to IgG1 antibodies via a histidine cluster in the constant domain.

Efficiency and attenuation correction factors determination in gamma spectrometric assay of bulk samples using self radiation

International Nuclear Information System (INIS)

Haddad, Kh.

2009-02-01

Gamma spectrometry forms the most important and capable tool for measuring radioactive materials. Determination of the efficiency and attenuation correction factors is the most tedious problem in the gamma spectrometric assay of bulk samples. A new experimental and easy method for these correction factors determination using self radiation was proposed in this work. An experimental study of the correlation between self attenuation correction factor and sample thickness and its practical application was also introduced. The work was performed on NORM and uranyl nitrate bulk sample. The results of proposed methods agreed with those of traditional ones.(author)

Correlation of DNA Ploidy with Progression of Cervical Cancer

International Nuclear Information System (INIS)

Singh, M.; Kalra, N.; Shukla, Y.; Mehrotra, S.; Singh, U.

2008-01-01

The majority of squamous cell carcinomas of cervix are preceded by visible changes in the cervix, most often detected by cervical smear. As cervical cancer is preceded by long precancerous stages, identification of the high-risk population through detection of DNA ploidy may be of importance in effective management of this disease. Here we attempted to correlate aneuploidy DNA patterns and their influence on biological behavior of flow-cytometry analysis of DNA ploidy which was carried out in cytologically diagnosed cases of mild (79), moderate (36), and severe (12) dysplasia, as well as “atypical squamous cells of unknown significance (ASCUS)” (57) along with controls (69), in order to understand its importance in malignant progression of disease. Cytologically diagnosed dysplasias, which were employed for DNA ploidy studies, 39 mild, 28 moderate, and 11 severe dysplasia cases were found to be aneuploidy. Out of the 69 control subjects, 6 cases showed aneuploidy pattern and the rest 63 subjects were diploid. An aneuploidy pattern was observed in 8 out of 57 cases of cytologically evaluated ASCUS. The results of the followup studies showed that aberrant DNA content reliably predicts the occurrence of squamous cell carcinoma in cervical smear. Flow cytometric analysis of DNA ploidy may provide a strategic diagnostic tool for early detection of carcinoma cervix. Therefore, it is a concept of an HPV screening with reflex cytology in combination with DNA flow cytometry to detect progressive lesions with the greatest possible sensitivity and specificity.

PCR performance of a thermostable heterodimeric archaeal DNA polymerase

Science.gov (United States)

Killelea, Tom; Ralec, Céline; Bossé, Audrey; Henneke, Ghislaine

2014-01-01

DNA polymerases are versatile tools used in numerous important molecular biological core technologies like the ubiquitous polymerase chain reaction (PCR), cDNA cloning, genome sequencing, and nucleic acid based diagnostics. Taking into account the multiple DNA amplification techniques in use, different DNA polymerases must be optimized for each type of application. One of the current tendencies is to reengineer or to discover new DNA polymerases with increased performance and broadened substrate spectra. At present, there is a great demand for such enzymes in applications, e.g., forensics or paleogenomics. Current major limitations hinge on the inability of conventional PCR enzymes, such as Taq, to amplify degraded or low amounts of template DNA. Besides, a wide range of PCR inhibitors can also impede reactions of nucleic acid amplification. Here we looked at the PCR performances of the proof-reading D-type DNA polymerase from P. abyssi, Pab-polD. Fragments, 3 kilobases in length, were specifically PCR-amplified in its optimized reaction buffer. Pab-polD showed not only a greater resistance to high denaturation temperatures than Taq during cycling, but also a superior tolerance to the presence of potential inhibitors. Proficient proof-reading Pab-polD enzyme could also extend a primer containing up to two mismatches at the 3' primer termini. Overall, we found valuable biochemical properties in Pab-polD compared to the conventional Taq, which makes the enzyme ideally suited for cutting-edge PCR-applications. PMID:24847315

PCR performance of a thermostable heterodimeric archaeal DNA polymerase

Directory of Open Access Journals (Sweden)

Tom eKillelea

2014-05-01

Full Text Available DNA polymerases are versatile tools used in numerous important molecular biological core technologies like the ubiquitous polymerase chain reaction (PCR, cDNA cloning, genome sequencing and nucleic acid based diagnostics. Taking into account the multiple DNA amplification techniques in use, different DNA polymerases must be optimized for each type of application. One of the current tendencies is to reengineer or to discover new DNA polymerases with increased performance and broadened substrate spectra. At present, there is a great demand for such enzymes in applications, e.g., forensics or paleogenomics. Current major limitations hinge on the inability of conventional PCR enzymes, such as Taq, to amplify degraded or low amounts of template DNA. Besides, a wide range of PCR inhibitors can also impede reactions of nucleic acid amplification. Here we looked at the PCR performances of the proof-reading D-type DNA polymerase from P. abyssi, Pab-polD. Fragments, 3 kilobases in length, were specifically PCR-amplified in its optimized reaction buffer. Pab-polD showed not only a greater resistance to high denaturation temperatures than Taq during cycling, but also a superior tolerance to the presence of potential inhibitors. Proficient proof-reading Pab-polD enzyme could also extend a primer containing up to two mismatches at the 3’ primer termini. Overall, we found valuable biochemical properties in Pab-polD compared to the conventional Taq, which makes the enzyme ideally suited for cutting-edge PCR-applications.

Comparison of different tandem mass spectrometric techniques (ESI-IT, ESI- and IP-MALDI-QRTOF and vMALDI-TOF/RTOF) for the analysis of crocins and picrocrocin from the stigmas of Crocus sativus L.

Science.gov (United States)

Koulakiotis, Nikolaos Stavros; Pittenauer, Ernst; Halabalaki, Maria; Tsarbopoulos, Anthony; Allmaier, Günter

2012-03-30

The expensive spice saffron originating from the stigmas of Crocus sativus L. and also applied in traditional Chinese medicine (TCM) constitutes a complex mixture of glycoconjugates varying not only in the aglycon structure, but also in glycosylation pattern. Therefore, various tandem mass spectrometric techniques were evaluated for their usefulness in structural elucidation. Three selected constituents of the stigmas of Crocus sativus L., trans- and cis-crocin-4 as well as picrocrocin, were isolated and purified by HPLC and finally analyzed by ESI-MS (ion trap, QqRTOF), IP-MALDI-MS (QqRTOF) and vMALDI-MS (TOF/RTOF) in combination with tandem mass spectrometry in collision energy regimes ranging from a few eV (LE) to 20 keV (HE) collisions for the first time. These data aid in structurally elucidating minor, unknown glycoconjugates originating from this plant-derived spice. LE-CID of isomeric crocins on either an ion trap with ESI or a QqRTOF-instrument with ESI or IP-MALDI as desorption/ionization technique only yielded a limited number of structurally diagnostic sodiated product ions related to the carbohydrate moiety as well as to the intact aglycon in contrast to true HE-CID. The low MW constituent picrocrocin did not yield useful LE-CID spectra, but showed a high number of structurally diagnostic product ions by HE-CID utilizing a vMALDI TOF/RTOF-instrument. The highest number of structurally diagnostic product ions allowing also determination of the carbohydrate linkage of the gentiobiose-moiety of isomeric crocins ((0,4)A(2), (3,5)A(2) product ions indicating a 1→6 carbohydrate linkage) was only achievable by HE-CID. Fragmentation of the aglycon was not observed by any collision energy regime applied. Copyright © 2012 John Wiley & Sons, Ltd.

A Double Slow Neutron Spectrometer; Spectrometre double pour neutrons lents; Dvojnoj spektrometr medlennykh nejtronov; Espectrometro doble para neutrones lentos

Energy Technology Data Exchange (ETDEWEB)

Bondarenko, I I; Liforov, V G; Nikolaev, M N; Orlov, V V; Parfenov, V A; Semenov, V A; Smirnov, V I; Turchin, V F [Fehi, Moscow, SSSR (Russian Federation)

1963-01-15

The neutron spectrometer described in the paper is intended for measurements of the angular and energy distribution of monochromatic slow neutrons, inelasticaily scattered by liquid and solid bodies. Experiments of this type permit detailed information to be obtained concerning the dynamics of the atoms in various aggregate states of a substance. The spectromeeter is based on the time-of-flight method. The pulse source of neutrons is the IBR (1) reactor. A mechanical interrupter, rotating synchronously with the disc of the IBR and having a prescribed phase shift, serves as the monochromator. A special phasing system ensures a phasee stability better than 0.5{sup o}. The neutrons scattered by the sample are recorded by a scintillation detector set at a given angle to the neutron beam. The resolving power of the spectrometer is - 15 {mu}s/m. The paper gives a detailed description of the construction of the spectroscope and its characteristics. (author) [French] Le spectrometre neutronique decrit dans le memoire est destine a mesurer la distribution en energie, la distribution angulaire et la diffusion inelastique des neutrons lents monochromatiques dans des liquides et des solides. Les experiences de ce genre permettent d'obtenir des renseignements detailles sur la dynamique des atomes dans de divers etats de la matiere. Le spectrometre est fonde sur la mesure du temps de vol. Comme source puisee de neutrons on a utilise le reacteur IBR-1. Le systeme monochromateur etait constitue par un interrupteur mecanique en rotation synchronisee avec celle du disque du reacteur IBR, avec un dephasage determine. Un dispositif special de dephasage assure la stabilite de la phase a 0,5{sup o} pres. La diffusion des neutrons par l'echantillon est enregistree a l'aide d'un detecteur a scintillations dispose sous un angle determine par rapport au faisceau de neutrons. Le pouvoir de resolution du spectrometre est d'environ 15 {mu}s/m. Les auteurs decrivent en detail la construction du

Direct colorimetric detection of unamplified pathogen DNA by dextrin-capped gold nanoparticles.

Science.gov (United States)

Baetsen-Young, Amy M; Vasher, Matthew; Matta, Leann L; Colgan, Phil; Alocilja, Evangelyn C; Day, Brad

2018-03-15

The interaction between gold nanoparticles (AuNPs) and nucleic acids has facilitated a variety of diagnostic applications, with further diversification of synthesis match bio-applications while reducing biotoxicity. However, DNA interactions with unique surface capping agents have not been fully defined. Using dextrin-capped AuNPs (d-AuNPs), we have developed a novel unamplified genomic DNA (gDNA) nanosensor, exploiting dispersion and aggregation characteristics of d-AuNPs, in the presence of gDNA, for sequence-specific detection. We demonstrate that d-AuNPs are stable in a five-fold greater salt concentration than citrate-capped AuNPs and the d-AuNPs were stabilized by single stranded DNA probe (ssDNAp). However, in the elevated salt concentrations of the DNA detection assay, the target reactions were surprisingly further stabilized by the formation of a ssDNAp-target gDNA complex. The results presented herein lead us to propose a mechanism whereby genomic ssDNA secondary structure formation during ssDNAp-to-target gDNA binding enables d-AuNP stabilization in elevated ionic environments. Using the assay described herein, we were successful in detecting as little as 2.94 fM of pathogen DNA, and using crude extractions of a pathogen matrix, as few as 18 spores/µL. Copyright © 2017 Elsevier B.V. All rights reserved.

Detection of Target ssDNA Using a Microfabricated Hall Magnetometer with Correlated Optical Readout

Directory of Open Access Journals (Sweden)

Steven M. Hira

2012-01-01

Full Text Available Sensing biological agents at the genomic level, while enhancing the response time for biodetection over commonly used, optics-based techniques such as nucleic acid microarrays or enzyme-linked immunosorbent assays (ELISAs, is an important criterion for new biosensors. Here, we describe the successful detection of a 35-base, single-strand nucleic acid target by Hall-based magnetic transduction as a mimic for pathogenic DNA target detection. The detection platform has low background, large signal amplification following target binding and can discriminate a single, 350 nm superparamagnetic bead labeled with DNA. Detection of the target sequence was demonstrated at 364 pM (<2 target DNA strands per bead target DNA in the presence of 36 μM nontarget (noncomplementary DNA (<10 ppm target DNA using optical microscopy detection on a GaAs Hall mimic. The use of Hall magnetometers as magnetic transduction biosensors holds promise for multiplexing applications that can greatly improve point-of-care (POC diagnostics and subsequent medical care.

Mass Spectrometric Calibration of Controlled Fluoroform Leak Rate Devices Technique and Uncertainty Analysis

CERN Document Server

Balsley, S D; Laduca, C A

2003-01-01

Controlled leak rate devices of fluoroform on the order of 10 sup - sup 8 atm centre dot cc sec sup - sup 1 at 25 C are used to calibrate QC-1 War Reserve neutron tube exhaust stations for leak detection sensitivity. Close-out calibration of these tritium-contaminated devices is provided by the Gas Dynamics and Mass Spectrometry Laboratory, Organization 14406, which is a tritium analytical facility. The mass spectrometric technique used for the measurement is discussed, as is the first principals calculation (pressure, volume, temperature and time). The uncertainty of the measurement is largely driven by contributing factors in the determination of P, V and T. The expanded uncertainty of the leak rate measurement is shown to be 4.42%, with a coverage factor of 3 (k=3).

Culture-Negative Endocarditis Diagnosed Using 16S DNA Polymerase Chain Reaction

Directory of Open Access Journals (Sweden)

Stephen Duffett

2012-01-01

Full Text Available 16S DNA polymerase chain reaction (PCR is a molecular amplification technique that can be used to identify bacterial pathogens in culture-negative endocarditis. Bacterial DNA can be isolated from surgically excised valve tissue or from blood collected in EDTA vials. Use of this technique is particularly helpful in identifying the bacterial pathogen in cases of culture-negative endocarditis. A case involving a 48-year-old man who presented with severe aortic regurgitation and a four-month prodrome of low-grade fever is reported. Blood and valve tissue cultures following valve replacement were negative. A valve tissue sample was sent for investigation with 16S DNA PCR, which successfully identified Streptococcus salivarius and was interpreted as the true diagnosis. A review of the literature suggests that 16S DNA PCR from valve tissue is a more sensitive diagnostic test than culture. It is also extremely specific, based on a sequence match of at least 500 base pairs.

Towards writing the encyclopaedia of life: an introduction to DNA barcoding

Science.gov (United States)

Savolainen, Vincent; Cowan, Robyn S; Vogler, Alfried P; Roderick, George K; Lane, Richard

2005-01-01

An international consortium of major natural history museums, herbaria and other organizations has launched an ambitious project, the ‘Barcode of Life Initiative’, to promote a process enabling the rapid and inexpensive identification of the estimated 10 million species on Earth. DNA barcoding is a diagnostic technique in which short DNA sequence(s) can be used for species identification. The first international scientific conference on Barcoding of Life was held at the Natural History Museum in London in February 2005, and here we review the scientific challenges discussed during this conference and in previous publications. Although still controversial, the scientific benefits of DNA barcoding include: (i) enabling species identification, including any life stage or fragment, (ii) facilitating species discoveries based on cluster analyses of gene sequences (e.g. cox1=CO1, in animals), (iii) promoting development of handheld DNA sequencing technology that can be applied in the field for biodiversity inventories and (iv) providing insight into the diversity of life. PMID:16214739

A rapid novel derivatization of amphetamine and methamphetamine using 2,2,2-trichloroethyl chloroformate for gas chromatography electron ionization and chemical ionization mass spectrometric analysis.

Science.gov (United States)

Dasgupta, A; Spies, J

1998-05-01

Amphetamine and methamphetamine are commonly abused central nervous system stimulants. We describe a rapid new derivatization of amphetamine and methamphetamine using 2,2,2-trichloroethyl chloroformate for gas chromatography-mass spectrometric analysis. Amphetamine and methamphetamine, along with N-propyl amphetamine (internal standard), were extracted from urine using 1-chlorobutane. The derivatization with 2,2,2-trichloroethyl chloroformate can be achieved at room temperature in 10 minutes. The electron ionization mass spectrum of amphetamine 2,2,2-trichloroethyl carbamate showed two weak molecular ions at m/z 309 and 311, but showed diagnostic strong peaks at m/z 218, 220, and 222. In contrast, chemical ionization of the mass spectrum of amphetamine 2,2,2-trichloroethyl carbamate showed strong (M + 1) ions at m/z 310 and 312 and other strong diagnostic peaks at m/z 274 and 276. The major advantages of this derivative are the presence of a diagnostic cluster of peaks due to the isotopic effect of three chlorine atoms (isotopes 35 and 37) in the derivatized molecule and the relative ease of its preparation. We also observed strong molecular ions for derivatized methamphetamine in the chemical ionization mass spectrum, but the molecular ions were very weak in the electron ionization mass spectrum. We used the scan mode of mass spectrometry in all analyses. When using a urine standard containing 1,000 ng/mL of amphetamine (a 7.4-micromol/L concentration) and methamphetamine (a 6.7-micromol/L concentration), the within-run precisions were 4.8% for amphetamine and 3.6% for methamphetamine. The corresponding between-run precisions were 5.3% for amphetamine and 6.7% for methamphetamine. The assay was linear for amphetamine and methamphetamine concentrations of 250 to 5,000 ng/mL (amphetamine, 1.9-37.0 micromol/L; methamphetamine, 1.7-33.6 micromol/L). The detection limit was 100 ng/mL (amphetamine, 0.74 micromol/L; methamphetamine, 0.67 micromol/L) using the scan mode

Radioimmunoassay of single-stranded DNA antibodies for control of diagnosis and therapy

Energy Technology Data Exchange (ETDEWEB)

Meffert, H; Boehm, F; Soennichsen, N; Gens, J [Humboldt-Universitaet, Berlin (German Democratic Republic). Bereich Medizin (Charite)

1980-10-01

Several years experience in quantitative determination of single-stranded DNA antibodies is reported and the normal range as well as the diagnostic hit rate of the method is outlined. In the controls the mean DNA attachment rate was 1.5% and the upper normal range limit was 12.8%, the risk of erroneous rejection being 1%. The DNA binding rate was greater than 12.8% in 74.7% of untreated patients suffering from lupus erythematodes visceralis, in 47.6% of patients with circumscribed sclerodermia, in 14.4% of patients with progressive sclerodermia, and in 10.3% of those suffering from lupus erythematodes chronicus. The findings emphasize the importance of regulatory mechanisms of the immune system to the process of autosensitization.

Radioimmunoassay of single-stranded DNA antibodies for control of diagnosis and therapy

International Nuclear Information System (INIS)

Meffert, H.; Boehm, F.; Soennichsen, N.; Gens, J.

1980-01-01

Several years experience in quantitative determination of single-stranded DNA antibodies is reported and the normal range as well as the diagnostic hit rate of the method is outlined. In the controls the mean DNA attachment rate was 1.5% and the upper normal range limit was 12.8%, the risk of erroneous rejection being 1%. The DNA binding rate was greater than 12.8% in 74.7% of untreated patients suffering from lupus erythematodes visceralis, in 47.6% of patients with circumscribed sclerodermia, in 14.4% of patients with progressive sclerodermia, and in 10.3% of those suffering from lupus erythematodes chronicus. The findings emphasize the importance of regulatory mechanisms of the immune system to the process of autosensitization

Are the classic diagnostic methods in mycology still state of the art?

Science.gov (United States)

Wiegand, Cornelia; Bauer, Andrea; Brasch, Jochen; Nenoff, Pietro; Schaller, Martin; Mayser, Peter; Hipler, Uta-Christina; Elsner, Peter

2016-05-01

The diagnostic workup of cutaneous fungal infections is traditionally based on microscopic KOH preparations as well as culturing of the causative organism from sample material. Another possible option is the detection of fungal elements by dermatohistology. If performed correctly, these methods are generally suitable for the diagnosis of mycoses. However, the advent of personalized medicine and the tasks arising therefrom require new procedures marked by simplicity, specificity, and swiftness. The additional use of DNA-based molecular techniques further enhances sensitivity and diagnostic specificity, and reduces the diagnostic interval to 24-48 hours, compared to weeks required for conventional mycological methods. Given the steady evolution in the field of personalized medicine, simple analytical PCR-based systems are conceivable, which allow for instant diagnosis of dermatophytes in the dermatology office (point-of-care tests). © 2016 Deutsche Dermatologische Gesellschaft (DDG). Published by John Wiley & Sons Ltd.

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

Diagnostic accuracy of organ electrodermal diagnostics | Szopinski ...

African Journals Online (AJOL)

Objective. To estimate the diagnostic accuracy as well as the scope of utilisation of a new bio-electronic method of organ diagnostics. Design. Double-blind comparative study of the diagnostic results obtained by means of organ electrodermal diagnostics (OED) and clinical diagnoses, as a criterion standard. Setting.

Optimized acoustic biochip integrated with microfluidics for biomarkers detection in molecular diagnostics.

Science.gov (United States)

Papadakis, G; Friedt, J M; Eck, M; Rabus, D; Jobst, G; Gizeli, E

2017-09-01

The development of integrated platforms incorporating an acoustic device as the detection element requires addressing simultaneously several challenges of technological and scientific nature. The present work was focused on the design of a microfluidic module, which, combined with a dual or array type Love wave acoustic chip could be applied to biomedical applications and molecular diagnostics. Based on a systematic study we optimized the mechanics of the flow cell attachment and the sealing material so that fluidic interfacing/encapsulation would impose minimal losses to the acoustic wave. We have also investigated combinations of operating frequencies with waveguide materials and thicknesses for maximum sensitivity during the detection of protein and DNA biomarkers. Within our investigations neutravidin was used as a model protein biomarker and unpurified PCR amplified Salmonella DNA as the model genetic target. Our results clearly indicate the need for experimental verification of the optimum engineering and analytical parameters, in order to develop commercially viable systems for integrated analysis. The good reproducibility of the signal together with the ability of the array biochip to detect multiple samples hold promise for the future use of the integrated system in a Lab-on-a-Chip platform for application to molecular diagnostics.

Amplifiable DNA from Gram-negative and Gram-positive bacteria by a low strength pulsed electric field method

Science.gov (United States)

Vitzthum, Frank; Geiger, Georg; Bisswanger, Hans; Elkine, Bentsian; Brunner, Herwig; Bernhagen, Jürgen

2000-01-01

An efficient electric field-based procedure for cell disruption and DNA isolation is described. Isoosmotic suspensions of Gram-negative and Gram-positive bacteria were treated with pulsed electric fields of Pulses had an exponential decay waveform with a time constant of 3.4 µs. DNA yield was linearly dependent on time or pulse number, with several thousand pulses needed. Electrochemical side-effects and electrophoresis were minimal. The lysates contained non-fragmented DNA which was readily amplifiable by PCR. As the method was not limited to samples of high specific resistance, it should be applicable to physiological fluids and be useful for genomic and DNA diagnostic applications. PMID:10734214

DNA methylation-based classification of central nervous system tumours.

Science.gov (United States)

Capper, David; Jones, David T W; Sill, Martin; Hovestadt, Volker; Schrimpf, Daniel; Sturm, Dominik; Koelsche, Christian; Sahm, Felix; Chavez, Lukas; Reuss, David E; Kratz, Annekathrin; Wefers, Annika K; Huang, Kristin; Pajtler, Kristian W; Schweizer, Leonille; Stichel, Damian; Olar, Adriana; Engel, Nils W; Lindenberg, Kerstin; Harter, Patrick N; Braczynski, Anne K; Plate, Karl H; Dohmen, Hildegard; Garvalov, Boyan K; Coras, Roland; Hölsken, Annett; Hewer, Ekkehard; Bewerunge-Hudler, Melanie; Schick, Matthias; Fischer, Roger; Beschorner, Rudi; Schittenhelm, Jens; Staszewski, Ori; Wani, Khalida; Varlet, Pascale; Pages, Melanie; Temming, Petra; Lohmann, Dietmar; Selt, Florian; Witt, Hendrik; Milde, Till; Witt, Olaf; Aronica, Eleonora; Giangaspero, Felice; Rushing, Elisabeth; Scheurlen, Wolfram; Geisenberger, Christoph; Rodriguez, Fausto J; Becker, Albert; Preusser, Matthias; Haberler, Christine; Bjerkvig, Rolf; Cryan, Jane; Farrell, Michael; Deckert, Martina; Hench, Jürgen; Frank, Stephan; Serrano, Jonathan; Kannan, Kasthuri; Tsirigos, Aristotelis; Brück, Wolfgang; Hofer, Silvia; Brehmer, Stefanie; Seiz-Rosenhagen, Marcel; Hänggi, Daniel; Hans, Volkmar; Rozsnoki, Stephanie; Hansford, Jordan R; Kohlhof, Patricia; Kristensen, Bjarne W; Lechner, Matt; Lopes, Beatriz; Mawrin, Christian; Ketter, Ralf; Kulozik, Andreas; Khatib, Ziad; Heppner, Frank; Koch, Arend; Jouvet, Anne; Keohane, Catherine; Mühleisen, Helmut; Mueller, Wolf; Pohl, Ute; Prinz, Marco; Benner, Axel; Zapatka, Marc; Gottardo, Nicholas G; Driever, Pablo Hernáiz; Kramm, Christof M; Müller, Hermann L; Rutkowski, Stefan; von Hoff, Katja; Frühwald, Michael C; Gnekow, Astrid; Fleischhack, Gudrun; Tippelt, Stephan; Calaminus, Gabriele; Monoranu, Camelia-Maria; Perry, Arie; Jones, Chris; Jacques, Thomas S; Radlwimmer, Bernhard; Gessi, Marco; Pietsch, Torsten; Schramm, Johannes; Schackert, Gabriele; Westphal, Manfred; Reifenberger, Guido; Wesseling, Pieter; Weller, Michael; Collins, Vincent Peter; Blümcke, Ingmar; Bendszus, Martin; Debus, Jürgen; Huang, Annie; Jabado, Nada; Northcott, Paul A; Paulus, Werner; Gajjar, Amar; Robinson, Giles W; Taylor, Michael D; Jaunmuktane, Zane; Ryzhova, Marina; Platten, Michael; Unterberg, Andreas; Wick, Wolfgang; Karajannis, Matthias A; Mittelbronn, Michel; Acker, Till; Hartmann, Christian; Aldape, Kenneth; Schüller, Ulrich; Buslei, Rolf; Lichter, Peter; Kool, Marcel; Herold-Mende, Christel; Ellison, David W; Hasselblatt, Martin; Snuderl, Matija; Brandner, Sebastian; Korshunov, Andrey; von Deimling, Andreas; Pfister, Stefan M

2018-03-22

Accurate pathological diagnosis is crucial for optimal management of patients with cancer. For the approximately 100 known tumour types of the central nervous system, standardization of the diagnostic process has been shown to be particularly challenging-with substantial inter-observer variability in the histopathological diagnosis of many tumour types. Here we present a comprehensive approach for the DNA methylation-based classification of central nervous system tumours across all entities and age groups, and demonstrate its application in a routine diagnostic setting. We show that the availability of this method may have a substantial impact on diagnostic precision compared to standard methods, resulting in a change of diagnosis in up to 12% of prospective cases. For broader accessibility, we have designed a free online classifier tool, the use of which does not require any additional onsite data processing. Our results provide a blueprint for the generation of machine-learning-based tumour classifiers across other cancer entities, with the potential to fundamentally transform tumour pathology.

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.

Antimitochondrial antibodies and other antibodies in primary biliary cirrhosis: diagnostic and prognostic value.

Science.gov (United States)

Muratori, Luigi; Granito, Alessandro; Muratori, Paolo; Pappas, Georgios; Bianchi, Francesco B

2008-05-01

Antimitochondrial antibodies (AMA) are the serologic cornerstone in the diagnosis of primary biliary cirrhosis (PBC), even if they are not detectable in a proportion of patients, notwithstanding the most sensitive and sophisticated technologies used. To fill in the serologic gap in AMA-negative PBC, there is sound evidence to consider antinuclear antibody (ANA) patterns, such as anti-multiple nuclear dots and anti-membranous/rim-like, as PBC-specific surrogate hallmarks of the disease, and their detection can be considered virtually diagnostic. Furthermore, particular ANA specificities, such as anti-gp210, anti-p62, anticentromere antibodies, and anti-dsDNA, may provide additional diagnostic and prognostic information.

A tuberculosis biomarker database: the key to novel TB diagnostics

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Seda Yerlikaya

2017-03-01

Full Text Available New diagnostic innovations for tuberculosis (TB, including point-of-care solutions, are critical to reach the goals of the End TB Strategy. However, despite decades of research, numerous reports on new biomarker candidates, and significant investment, no well-performing, simple and rapid TB diagnostic test is yet available on the market, and the search for accurate, non-DNA biomarkers remains a priority. To help overcome this ‘biomarker pipeline problem’, FIND and partners are working on the development of a well-curated and user-friendly TB biomarker database. The web-based database will enable the dynamic tracking of evidence surrounding biomarker candidates in relation to target product profiles (TPPs for needed TB diagnostics. It will be able to accommodate raw datasets and facilitate the verification of promising biomarker candidates and the identification of novel biomarker combinations. As such, the database will simplify data and knowledge sharing, empower collaboration, help in the coordination of efforts and allocation of resources, streamline the verification and validation of biomarker candidates, and ultimately lead to an accelerated translation into clinically useful tools.

Clinical experience of laboratory follow-up with noninvasive prenatal testing using cell-free DNA and positive microdeletion results in 349 cases.

Science.gov (United States)

Schwartz, S; Kohan, M; Pasion, R; Papenhausen, P R; Platt, L D

2018-02-01

Screening via noninvasive prenatal testing (NIPT) involving the analysis of cell-free DNA (cfDNA) from plasma has become readily available to screen for chromosomal and DNA aberrations through maternal blood. This report reviews a laboratory's experience with follow-up of positive NIPT screens for microdeletions. Patients that were screened positive by NIPT for a microdeletion involving 1p, 4p, 5p, 15q, or 22q who underwent diagnostic studies by either chorionic villus sampling or amniocentesis were evaluated. The overall positive predictive value for 349 patients was 9.2%. When a microdeletion was confirmed, 39.3% of the cases had additional abnormal microarray findings. Unrelated abnormal microarray findings were detected in 11.8% of the patients in whom the screen positive microdeletion was not confirmed. Stretches of homozygosity in the microdeletion were frequently associated with a false positive cfDNA microdeletion result. Overall, this report reveals that while cfDNA analysis will screen for microdeletions, the positive predictive value is low; in our series it is 9.2%. Therefore, the patient should be counseled accordingly. Confirmatory diagnostic microarray studies are imperative because of the high percentage of false positives and the frequent additional abnormalities not delineated by cfDNA analysis. © 2018 John Wiley & Sons, Ltd.

Mass spectrometric isotope dilution analyses of palladium, silver, cadmium and tellurium in carbonaceous chondrites

International Nuclear Information System (INIS)

Loss, R.D.; Rosman, K.J.R.; De Laeter, J.

1984-01-01

The mass spectrometric isotope dilution technique was used to measure the elemental abundances of Pd, Ag, Cd and Te in Orgueil (C1), Ivuna (C1), Murray (C2) and Allende (C3) chondrites. The Pd abundance of 554 ppb for the Cl chondrites is almost identical to the recommended value of Anders and Ebihara (1982); that for Cd (712 ppb) is approximately 5% higher, whereas that for Ag (198 ppb) is approximately 10% lower than the recommended values. A smooth distribution for the abundances of the odd-A nuclides between 65 128 Te and 130 Te to lie approximately 30% above the r-process peak at A = 130, whereas the new value fits smoothly into the general trend. (author)

Fragmentasi DNA Spermatozoa: Penyebab, Deteksi, dan Implikasinya pada Infertilitas Laki-Laki

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Silvia W. Lestari

2015-12-01

andrology laboratory. Besides having better diagnostic outcome,sperm DNA fragmentation test also give better infertility prognosis including the prediction ofassisted reproductive technology (ART program outcome. Keywords : male infertility, sperm DNA fragmentation, SCD method Normal 0 false false false IN X-NONE X-NONE

Improving the Conservation of Mediterranean Chondrichthyans: The ELASMOMED DNA Barcode Reference Library.

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Alessia Cariani

Full Text Available Cartilaginous fish are particularly vulnerable to anthropogenic stressors and environmental change because of their K-selected reproductive strategy. Accurate data from scientific surveys and landings are essential to assess conservation status and to develop robust protection and management plans. Currently available data are often incomplete or incorrect as a result of inaccurate species identifications, due to a high level of morphological stasis, especially among closely related taxa. Moreover, several diagnostic characters clearly visible in adult specimens are less evident in juveniles. Here we present results generated by the ELASMOMED Consortium, a regional network aiming to sample and DNA-barcode the Mediterranean Chondrichthyans with the ultimate goal to provide a comprehensive DNA barcode reference library. This library will support and improve the molecular taxonomy of this group and the effectiveness of management and conservation measures. We successfully barcoded 882 individuals belonging to 42 species (17 sharks, 24 batoids and one chimaera, including four endemic and several threatened ones. Morphological misidentifications were found across most orders, further confirming the need for a comprehensive DNA barcoding library as a valuable tool for the reliable identification of specimens in support of taxonomist who are reviewing current identification keys. Despite low intraspecific variation among their barcode sequences and reduced samples size, five species showed preliminary evidence of phylogeographic structure. Overall, the ELASMOMED initiative further emphasizes the key role accurate DNA barcoding libraries play in establishing reliable diagnostic species specific features in otherwise taxonomically problematic groups for biodiversity management and conservation actions.

[The development of reagents set in the format of DNA-chip for genetic typing of strains of Vibrio cholerae].

Science.gov (United States)

Pudova, E A; Markelov, M L; Dedkov, V G; Tchekanova, T A; Sadjin, A I; Kirdiyashkina, N P; Bekova, M V; Deviyatkin, A A

2014-05-01

The necessity of development of methods of genic diagnostic of cholera is conditioned by continuation of the Seventh pandemic of cholera, taxonomic variability of strains of Vibrio cholerae involved into pandemic and also permanent danger of delivery of disease to the territory of the Russian Federation. The methods of genic diagnostic of cholera make it possible in a comparatively short time to maximally minutely characterize strains isolated from patients or their environment. The article presents information about working out reagents set for genetic typing of agents of cholera using DNA-chip. The makeup of DNA-chip included oligonucleotide probes making possible to differentiate strains of V. cholerae on serogroups and biovars and to determine their pathogenicity. The single DNA-chip makes it possible to genetically type up to 12 samples concurrently. At that, duration of analysis without accounting stage of DNA separation makes up to 5 hours. In the progress of work, 23 cholera and non-cholera strains were analyzed. The full compliance of DNA-chip typing results to previously known characteristics of strains. Hence, there is a reason to consider availability of further development of reagents set and possibility of its further application in laboratories of regional level and reference centers.

A new ultrafast and high-throughput mass spectrometric approach for the therapeutic drug monitoring of the multi-targeted anti-folate pemetrexed in plasma from lung cancer patients

NARCIS (Netherlands)

R.J.W. Meesters (Roland); R. Cornelissen (Robin); R.J. van Klaveren (Rob); R. de Jonge (Robert); E. den Boer (Ethan); J. Lindemans (Jan); T.M. Luider (Theo)

2010-01-01

textabstractAn analytical assay has been developed and validated for ultrafast and high-throughput mass spectrometric determination of pemetrexed concentrations in plasma using matrix assisted laser desorption/ionization-triple quadrupole-tandem mass spectrometry. Patient plasma samples spiked with

Discovery and validation of DNA hypomethylation biomarkers for liver cancer using HRM-specific probes.

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Barbara Stefanska

Full Text Available Poor prognosis of hepatocellular carcinoma (HCC associated with late diagnosis necessitates the development of early diagnostic biomarkers. We have previously delineated the landscape of DNA methylation in HCC patients unraveling the importance of promoter hypomethylation in activation of cancer- and metastasis-driving genes. The purpose of the present study was to test the feasibility that genes that are hypomethylated in HCC could serve as candidate diagnostic markers. We use high resolution melting analysis (HRM as a simple translatable PCR-based method to define methylation states in clinical samples. We tested seven regions selected from the shortlist of genes hypomethylated in HCC and showed that HRM analysis of several of them distinguishes methylation states in liver cancer specimens from normal adjacent liver and chronic hepatitis in the Shanghai area. Such regions were identified within promoters of neuronal membrane glycoprotein M6-B (GPM6B and melanoma antigen family A12 (MAGEA12 genes. Differences in HRM in the immunoglobulin superfamily Fc receptor (FCRL1 separated invasive tumors from less invasive HCC. The identified biomarkers differentiated HCC from chronic hepatitis in another set of samples from Dhaka. Although the main thrust in DNA methylation diagnostics in cancer is on hypermethylated genes, our study for the first time illustrates the potential use of hypomethylated genes as markers for solid tumors. After further validation in a larger cohort, the identified DNA hypomethylated regions can become important candidate biomarkers for liver cancer diagnosis and prognosis, especially in populations with high risk for HCC development.

Oral bacterial DNA findings in pericardial fluid

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Anne-Mari Louhelainen

2014-11-01

Full Text Available Background: We recently reported that large amounts of oral bacterial DNA can be found in thrombus aspirates of myocardial infarction patients. Some case reports describe bacterial findings in pericardial fluid, mostly done with conventional culturing and a few with PCR; in purulent pericarditis, nevertheless, bacterial PCR has not been used as a diagnostic method before. Objective: To find out whether bacterial DNA can be measured in the pericardial fluid and if it correlates with pathologic–anatomic findings linked to cardiovascular diseases. Methods: Twenty-two pericardial aspirates were collected aseptically prior to forensic autopsy at Tampere University Hospital during 2009–2010. Of the autopsies, 10 (45.5% were free of coronary artery disease (CAD, 7 (31.8% had mild and 5 (22.7% had severe CAD. Bacterial DNA amounts were determined using real-time quantitative PCR with specific primers and probes for all bacterial strains associated with endodontic disease (Streptococcus mitis group, Streptococcus anginosus group, Staphylococcus aureus/Staphylococcus epidermidis, Prevotella intermedia, Parvimonas micra and periodontal disease (Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Treponema denticola, Fusobacterium nucleatus, and Dialister pneumosintes. Results: Of 22 cases, 14 (63.6% were positive for endodontic and 8 (36.4% for periodontal-disease-associated bacteria. Only one case was positive for bacterial culturing. There was a statistically significant association between the relative amount of bacterial DNA in the pericardial fluid and the severity of CAD (p=0.035. Conclusions: Oral bacterial DNA was detectable in pericardial fluid and an association between the severity of CAD and the total amount of bacterial DNA in pericardial fluid was found, suggesting that this kind of measurement might be useful for clinical purposes.

Newer diagnostic approaches to intestinal protozoa.

Science.gov (United States)

van Lieshout, Lisette; Verweij, Jaco J

2010-10-01

To update the reader on the latest developments in the laboratory diagnosis of intestinal protozoa. Correct identification of a diarrhoea causing pathogens is essential for the choice of treatment in an individual patient as well as to map the aetiology of diarrhoea in a variety of patient populations. Classical diagnosis of diarrhoea causing protozoa by microscopic examination of a stool sample lacks both sensitivity and specificity. Alternative diagnostic platforms are discussed. Recent literature on the diagnosis of intestinal protozoa has focused mainly on nucleic acid-based assays, in particular the specific detection of parasite DNA in stool samples using real-time PCR. In addition, the trend has been moving from single pathogen detection to a multiplex approach, allowing simultaneous identification of multiple parasites. Different combinations of targets can be used within a routine diagnostic setting, depending on the patient population, such as children, immunocompromised individuals and those who have been travelling to tropical regions. Large-scale monitoring and evaluation of control strategies become feasible due to automation and high-throughput facilities. Improved technology also has become available for differentiating protozoa subspecies, which facilitates outbreak investigations and extensive research in molecular epidemiology.

Molecular diagnostics for the sigatoka disease complex of banana.

Science.gov (United States)

Arzanlou, Mahdi; Abeln, Edwin C A; Kema, Gert H J; Waalwijk, Cees; Carlier, Jean; Vries, Ineke de; Guzmán, Mauricio; Crous, Pedro W

2007-09-01

ABSTRACT The Sigatoka disease complex of banana involves three related ascomycetous fungi, Mycosphaerella fijiensis, M. musicola, and M. eumusae. The exact distribution of these three species and their disease epidemiology remain unclear, because their symptoms and life cycles are rather similar. Disease diagnosis in the Mycosphaerella complex of banana is based on the presence of host symptoms and fungal fruiting structures, which hamper preventive management strategies. In the present study, we have developed rapid and robust species-specific molecular-based diagnostic tools for detection and quantification of M. fijiensis, M. musicola, and M. eumusae. Conventional species-specific polymerase chain reaction (PCR) primers were developed based on the actin gene that detected DNA at as little as 100, 1, and 10 pg/mul from M. fijiensis, M. musicola, and M. eumusae, respectively. Furthermore, TaqMan real-time quantitative PCR assays were developed based on the beta-tubulin gene and detected quantities of DNA as low as 1 pg/mul for each Mycosphaerella sp. from pure cultures and DNA at 1.6 pg/mul per milligram of dry leaf tissue for M. fijiensis that was validated using naturally infected banana leaves.

Validation of a stability-indicating spectrometric method for the determination of sulfacetamide sodium in pure form and ophthalmic preparations

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Sofia Ahmed

2017-01-01

Full Text Available Introduction: Sulfacetamide sodium is a widely used sulfonamide for ophthalmic infections. Objective: A number of analytical methods have been reported for the analysis of sulfacetamide but they lack the ability to determine both the active drug and its major degradation product, sulfanilamide, simultaneously in a sample. Materials and Methods: In the present study a simple, rapid and economical stability-indicating UV spectrometric method has been validated for the simultaneous assay of sulfacetamide sodium and sulfanilamide in pure form and in ophthalmic preparations. Results: The method has been found to be accurate (recovery 100.03 ±0.589% and precise (RSD 0.587% with detectable and quantifiable limits of 1.67×10–6 M (0.04 mg% and 5.07×10–6 M (0.13 mg%, respectively for the assay of pure sulfacetamide sodium. The method is also found to be accurate and precise to small changes in wavelength, pH and buffer concentration as well as to forced degradation. The study further includes the validation of the method for the assay of pure sulfanilamide in solution, which has been found to be accurate, precise and robust. Conclusion: The results indicate that the proposed two-component spectrometric method is stability-indicating and can be used for the simultaneous assay of both sulfacetamide sodium and sulfanilamide in synthetic mixtures and degraded solutions.

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.

Applications of Next-Generation Sequencing Technologies to Diagnostic Virology

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Giorgio Palù

2011-11-01

Full Text Available Novel DNA sequencing techniques, referred to as “next-generation” sequencing (NGS, provide high speed and throughput that can produce an enormous volume of sequences with many possible applications in research and diagnostic settings. In this article, we provide an overview of the many applications of NGS in diagnostic virology. NGS techniques have been used for high-throughput whole viral genome sequencing, such as sequencing of new influenza viruses, for detection of viral genome variability and evolution within the host, such as investigation of human immunodeficiency virus and human hepatitis C virus quasispecies, and monitoring of low-abundance antiviral drug-resistance mutations. NGS techniques have been applied to metagenomics-based strategies for the detection of unexpected disease-associated viruses and for the discovery of novel human viruses, including cancer-related viruses. Finally, the human virome in healthy and disease conditions has been described by NGS-based metagenomics.

Real-Time Polymerase Chain Reaction: Applications in Diagnostic Microbiology

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Kordo B. A. Saeed

2013-11-01

Full Text Available The polymerase chain reaction (PCR has revolutionized the detection of DNA and RNA. Real-Time PCR (RT-PCR is becoming the gold standard test for accurate, sensitive and fast diagnosis for a large range of infectious agents. Benefits of this procedure over conventional methods for measuring RNA include its sensitivity, high throughout and quantification. RT-PCR assays have advanced the diagnostic abilities of clinical laboratories particularly microbiology and infectious diseases. In this review we would like to briefly discuss RT-PCR in diagnostic microbiology laboratory, beginning with a general introduction to RT-PCR and its principles, setting up an RT PCR, including multiplex systems and the avoidance and remediation of contamination issues. A segment of the review would be devoted to the application of RT-PCR in clinical practice concentrating on its role in the diagnosis and treatment of infectious diseases.

DNA-enhanced peroxidase-like activity of layered double hydroxide nanosheets and applications in H2O2 and glucose sensing.

Science.gov (United States)

Chen, Lijian; Sun, Kaifang; Li, Peipei; Fan, Xianzhong; Sun, Jianchao; Ai, Shiyun

2013-11-21

LDH nanosheets were obtained via continuous impaction and exfoliation by herring sperm DNA molecules using a constant vibration method. DNA-LDH nanohybrids were composed by electrostatic forces and they exhibited DNA-enhanced peroxidase-like activity. The morphology and structure of DNA-LDH nanohybrids were analyzed by transmission electron microscopy (TEM), selected-area electron diffraction (SAED), X-ray diffraction (XRD), and atomic force microscopy (AFM) characterization. On the basis of the high catalytic activity of DNA/CuAl-LDH nanosheets, a rapid, sensitive, and convenient approach was developed for colorimetric detection of H2O2 and blood glucose. This method can be potentially applied in medical diagnostics and biotechnology fields.

Elevated levels of circulating cell-free DNA and neutrophil proteins are associated with neonatal sepsis and necrotizing enterocolitis in immature mice, pigs and infants

DEFF Research Database (Denmark)

Nguyen, Duc Ninh; Stensballe, Allan; Lai, Jacqueline C.Y.

2017-01-01

Preterm infants are highly susceptible to late-onset sepsis (LOS) and necrotizing enterocolitis (NEC), but disease pathogenesis and specific diagnostic markers are lacking. Circulating cell-free DNA (cfDNA) and immune cell-derived proteins are involved in multiple immune diseases in adults but ha...

Acceleration of incubation processes in DNA bio chips by magnetic particles

International Nuclear Information System (INIS)

Heer, Rudolf; Eggeling, Moritz; Schotter, Joerg; Noehammer, Christa; Pichler, Rudolf; Mansfeld, Markus; Brueckl, Hubert

2007-01-01

In classical DNA chip analysis, the target DNA moves by diffusion and Brownian motion only. We introduce a system for enhancing the signals and reducing the hybridization times of bio chips. It allows active agitation within the hybridization buffer by controlled movement of magnetic particles within the analyte solution. First results show that the system easily achieves specific fluorescent signals about four times higher than the ones obtained by a referencing standard procedure within the same hybridization time, while unspecific signals remain unchanged. The device can easily be applied to existing bio chip applications and allows universal operation in the field of molecular diagnostics

A Quantitative Tool for Producing DNA-Based Diagnostic Arrays

Energy Technology Data Exchange (ETDEWEB)

Tom J. Whitaker

2008-07-11

The purpose of this project was to develop a precise, quantitative method to analyze oligodeoxynucleotides (ODNs) on an array to enable a systematic approach to quality control issues affecting DNA microarrays. Two types of ODN's were tested; ODN's formed by photolithography and ODN's printed onto microarrays. Initial work in Phase I, performed in conjunction with Affymetrix, Inc. who has a patent on a photolithographic in situ technique for creating DNA arrays, was very promising but did seem to indicate that the atomization process was not complete. Soon after Phase II work was under way, Affymetrix had further developed fluorescent methods and indicated they were no longer interested in our resonance ionization technique. This was communicated to the program manager and it was decided that the project would continue and be focused on printed ODNs. The method being tested is called SIRIS, Sputter-Initiated Resonance Ionization Spectroscopy. SIRIS has been shown to be a highly sensitive, selective, and quantitative tool for atomic species. This project was aimed at determining if an ODN could be labeled in such a way that SIRIS could be used to measure the label and thus provide quantitative measurements of the ODN on an array. One of the largest problems in this study has been developing a method that allows us to know the amount of an ODN on a surface independent of the SIRIS measurement. Even though we could accurately determine the amount of ODN deposited on a surface, the amount that actually attached to the surface is very difficult to measure (hence the need for a quantitative tool). A double-labeling procedure was developed in which 33P and Pt were both used to label ODNs. The radioactive 33P could be measured by a proportional counter that maps the counts in one dimension. This gave a good measurement of the amount of ODN remaining on a surface after immobilization and washing. A second label, Pt, was attached to guanine nucleotides in the

Diagnostics

DEFF Research Database (Denmark)

Donné, A.J.H.; Costley, A.E.; Barnsley, R.

2007-01-01

of the measurements—time and spatial resolutions, etc—will in some cases be more stringent. Many of the measurements will be used in the real time control of the plasma driving a requirement for very high reliability in the systems (diagnostics) that provide the measurements. The implementation of diagnostic systems...... on ITER is a substantial challenge. Because of the harsh environment (high levels of neutron and gamma fluxes, neutron heating, particle bombardment) diagnostic system selection and design has to cope with a range of phenomena not previously encountered in diagnostic design. Extensive design and R......&D is needed to prepare the systems. In some cases the environmental difficulties are so severe that new diagnostic techniques are required. The starting point in the development of diagnostics for ITER is to define the measurement requirements and develop their justification. It is necessary to include all...

Does Extracellular DNA Production Vary in Staphylococcal Biofilms Isolated From Infected Implants versus Controls?

Science.gov (United States)

Zatorska, Beata; Groger, Marion; Moser, Doris; Diab-Elschahawi, Magda; Lusignani, Luigi Segagni; Presterl, Elisabeth

2017-08-01

amount of eDNA was greater in biofilms of S epidermidis than in biofilms of S aureus (S aureus mean ± SD: 1.35% ± 2.0%; S epidermidis mean ± SD: 6.42% ± 10.6%; p biofilm formation. The extraction method also showed that clinical isolates produced substantially greater amounts of eDNA than controls. S aureus and S epidermidis exhibit a differential production of DNA with time. Clinical isolates associated with implant infections produce greater amounts of eDNA than controls. Future research might focus on the diagnostic value of eDNA as a surrogate laboratory marker for biofilm formation in implant infections. eDNA should be considered as a potential future diagnostic tool or even a possible target to modify biofilms for successful treatment of biofilm-associated infections.

A Rapid and Low-Cost PCR Thermal Cycler for Infectious Disease Diagnostics.

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Kamfai Chan

Full Text Available The ability to make rapid diagnosis of infectious diseases broadly available in a portable, low-cost format would mark a great step forward in global health. Many molecular diagnostic assays are developed based on using thermal cyclers to carry out polymerase chain reaction (PCR and reverse-transcription PCR for DNA and RNA amplification and detection, respectively. Unfortunately, most commercial thermal cyclers are expensive and need continuous electrical power supply, so they are not suitable for uses in low-resource settings. We have previously reported a low-cost and simple approach to amplify DNA using vacuum insulated stainless steel thermoses food cans, which we have named it thermos thermal cycler or TTC. Here, we describe the use of an improved set up to enable the detection of viral RNA targets by reverse-transcription PCR (RT-PCR, thus expanding the TTC's ability to identify highly infectious, RNA virus-based diseases in low resource settings. The TTC was successful in demonstrating high-speed and sensitive detection of DNA or RNA targets of sexually transmitted diseases, HIV/AIDS, Ebola hemorrhagic fever, and dengue fever. Our innovative TTC costs less than $200 to build and has a capacity of at least eight tubes. In terms of speed, the TTC's performance exceeded that of commercial thermal cyclers tested. When coupled with low-cost endpoint detection technologies such as nucleic acid lateral-flow assay or a cell-phone-based fluorescence detector, the TTC will increase the availability of on-site molecular diagnostics in low-resource settings.

Organelle DNA haplotypes reflect crop-use characteristics and geographic origins of Cannabis sativa.

Science.gov (United States)

Gilmore, Simon; Peakall, Rod; Robertson, James

2007-10-25

Comparative sequencing of cannabis individuals across 12 chloroplast and mitochondrial DNA loci revealed 7 polymorphic sites, including 5 length variable regions and 2 single nucleotide polymorphisms. Simple PCR assays were developed to assay these polymorphisms, and organelle DNA haplotypes were obtained for 188 cannabis individuals from 76 separate populations, including drug-type, fibre-type and wild populations. The haplotype data were analysed using parsimony, UPGMA and neighbour joining methods. Three haplotype groups were recovered by each analysis method, and these groups are suggestive of the crop-use characteristics and geographical origin of the populations, although not strictly diagnostic. We discuss the relationship between our haplotype data and taxonomic opinions of cannabis, and the implications of organelle DNA haplotyping to forensic investigations of cannabis.

Diagnostic value of enzyme linked immuno-sorbent assay for cytomegalovirus disease.

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Priya K

2002-07-01

Full Text Available BACKGROUND: Since interpretation of results of enzyme linked immuno-sorbent assay (ELISA for diagnosis of Cytomegalovirus (CMV infection in India is difficult, its diagnostic value required evaluation. AIMS: To evaluate the diagnostic value of ELISA against polymerase chain reaction (PCR in CMV disease. SETTINGS AND DESIGN: Results of ELISA test for CMV antibodies in CMV-DNA PCR positive and negative patients and normal healthy blood donors were analysed. METHODS AND MATERIAL: Anti-CMV antibodies were assayed by ELISA on the sera of 26 CMV PCR positive and 21 PCR negative patients and 35 normal healthy blood donors. STATISTICAL ANALYSIS: Chi square and Fischer exact test were used for statistical analysis. RESULTS: Anti-CMV antibodies (IgG or IgG and IgM were present in 20 (76.9% of 26 PCR positive and 13 (61.9% of 21 PCR negative patients. ELISA was negative in six (23.1% of 26 PCR positive patients. Of the 28 paediatric patients, ELISA was positive in 14 (73.7% of 19 PCR positive and three (33.3% of nine PCR negative patients showing a statistically significant difference (Chi square test, P value 0.038. Among the 19 patients having complications after organ transplant, ELISA showed anti-CMV antibodies in six (85.7% of seven PCR positive and 11 (91.7% of 12 PCR negative patients showing no significant difference. CMV-DNA was not detected in the buffy coat of 35 sero-positive blood donors. CONCLUSION: ELISA has no diagnostic value in the detection of CMV activation although it may help in the differential diagnosis of CMV infection in the paediatric age group.

Sensitive molecular diagnostics using surface-enhanced resonance Raman scattering (SERRS)

Science.gov (United States)

Faulds, Karen; Graham, Duncan; McKenzie, Fiona; MacRae, Douglas; Ricketts, Alastair; Dougan, Jennifer

2009-02-01

Surface enhanced resonance Raman scattering (SERRS) is an analytical technique with several advantages over competitive techniques in terms of improved sensitivity and multiplexing. We have made great progress in the development of SERRS as a quantitative analytical method, in particular for the detection of DNA. SERRS is an extremely sensitive and selective technique which when applied to the detection of labelled DNA sequences allows detection limits to be obtained which rival, and in most cases, are better than fluorescence. Here the conditions are explored which will enable the successful detection of DNA using SERRS. The enhancing surface which is used is crucial and in this case suspensions of nanoparticles were used as they allow quantitative behaviour to be achieved and allow analogous systems to current fluorescence based systems to be made. The aggregation conditions required to obtain SERRS of DNA are crucial and herein we describe the use of spermine as an aggregating agent. The nature of the label which is used, be it fluorescent, positively or negatively charged also effects the SERRS response and these conditions are again explored here. We have clearly demonstrated the ability to identify the components of a mixture of 5 analytes in solution by using two different excitation wavelengths and also of a 6-plex using data analysis techniques. These conditions will allow the use of SERRS for the detection of target DNA in a meaningful diagnostic assay.

Molecular Diagnostics in Autosomal Dominant Polycystic Kidney Disease: Utility and Limitations

Science.gov (United States)

Zhao, Xiao; Paterson, Andrew D.; Zahirieh, Alireza; He, Ning; Wang, Kairong; Pei, York

2008-01-01

Background and objectives: Gene-based mutation screening is now available and has the potential to provide diagnostic confirmation or exclusion of autosomal dominant polycystic kidney disease. This study illustrates its utility and limitations in the clinical setting. Design, setting, participants, & measurements: Using a molecular diagnostic service, genomic DNA of one affected individual from each study family was screened for pathologic PKD1 and PKD2 mutations. Bidirectional sequencing was performed to identify sequence variants in all exons and splice junctions of both genes and to confirm the specific mutations in other family members. In two multiplex families, microsatellite markers were genotyped at both PDK1 and PKD2 loci, and pair-wise and multipoint linkage analysis was performed. Results: Three of five probands studied were referred for assessment of renal cystic disease without a family history of autosomal dominant polycystic kidney disease, and two others were younger at-risk members of families with autosomal dominant polycystic kidney disease being evaluated as living-related kidney donors. Gene-based mutation screening identified pathogenic mutations that provided confirmation or exclusion of disease in three probands, but in the other two, only unclassified variants were identified. In one proband in which mutation screening was indeterminate, DNA linkage studies provided strong evidence for disease exclusion. Conclusions: Gene-based mutation screening or DNA linkage analysis should be considered in individuals in whom the diagnosis of autosomal dominant polycystic kidney disease is uncertain because of a lack of family history or equivocal imaging results and in younger at-risk individuals who are being evaluated as living-related kidney donors. PMID:18077784

DNA typing from vaginal smear slides in suspected rape cases

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Dayse Aparecida da Silva

Full Text Available In an investigation of suspected rape, proof of sexual assault with penetration is required. In view of this, detailed descriptions of the genitalia, the thighs and pubic region are made within the forensic medical service. In addition, vaginal swabs are taken from the rape victim and some of the biological material collected is then transferred to glass slides. In this report, we describe two rape cases solved using DNA typing from cells recovered from vaginal smear slides. In 1999, two young women informed the Rio de Janeiro Police Department that they had been victims of sexual assaults. A suspect was arrested and the victims identified him as the offender. The suspect maintained that he was innocent. In order to elucidate these crimes, vaginal smear slides were sent to the DNA Diagnostic Laboratory for DNA analysis three months after the crimes, as unique forensic evidence. To get enough epithelial and sperm cells to perform DNA analysis, we used protocols modified from the previously standard protocols used for DNA extraction from biological material fixed on glass slides. The quantity of cells was sufficient to perform human DNA typing using nine short tandem repeat (STR loci. It was 3.3 billion times more probable that it was the examined suspect who had left sperm cells in the victims, rather than any other individual in the population of Rio de Janeiro.

A Chemical Eight Group Separation Method for Routine Use in Gamma Spectrometric Analysis. II. Detailed analytical schema

Energy Technology Data Exchange (ETDEWEB)

Samsahl, K

1961-06-15

A detailed ion-exchange procedure for the separation of chemical elements in eight groups suitable for subsequent gamma spectrometric analysis is described. The method has been in use for gamma spectrometry of some inorganic - but mostly organic - samples for one year. The separation time for inorganic samples, is usually about 1.5 hours and for organic samples as least 2 hours. One man can separate and count three samples per day. In comparative measurements of short-lived isotopes in biological material 10-12 elements can be analysed thus making possible 30 - 35 determinations per day for one man.

Development of a gas-ionization {beta}-spectrometer; Etude et realisation d'un spectrometre {beta} a ionisation gazeuse

Energy Technology Data Exchange (ETDEWEB)

Le Du, R [Commissariat a l' Energie Atomique, Bruyeres-le-Chatel (France). Centre d' Etudes

1968-05-15

We intend to develop in our laboratory a {beta}-spectrometer. This apparatus will have two main objectives: 1 - the determination of the nature and the degree of purity of certain {beta}-emitting radioactive substances; 2 - the study of an activity calibration process for {beta}-emitting radioactive sources. (author) [French] Nous nous proposons d'etudier et de realiser dans notre laboratoire un spectrometre {beta}. Cet appareil aura deux buts principaux: 1 - determiner la nature et le degre de purete de certains corps radioactifs emetteurs {beta}; 2 - permettre l'etude d'un procede d'etalonnage en activite de sources radioactives emetteurs {beta}. (auteur)

Application of diagnostic methods and molecular diagnosis of hemoglobin disorders in Khuzestan province of Iran

Science.gov (United States)

Fakher, Rahim; Bijan, Kaeikhaei; Taghi, Akbari Mohammad

2007-01-01

BACKGROUND: The hemoglobinopathies refer to a diverse group of inherited disorders characterized by a reduced synthesis of one or more globin chains (thalassemias) or the synthesis of structurally abnormal hemoglobin (Hb). The thalassemias often coexist with a variety of structural Hb variants giving rise to complex genotypes and an extremely wide spectrum of clinical and hematological phenotypes. Hematological and biochemical investigations and family studies provide essential clues to the different interactions and are fundamental to DNA diagnostics of the Hb disorders. Although DNA diagnostics have made a major impact on our understanding and detection of the hemoglobinopathies, DNA mutation testing should never be considered a shortcut or the test of first choice in the workup of a hemoglobinopathy. MATERIALS AND METHODS: A careful three-tier approach involving: (1) Full blood count (2) Special hematological tests, followed by (3) DNA mutation analysis, provides the most effective way in which to detect primary gene mutations as well as gene-gene interactions that can influence the overall phenotype. With the exception of a few rare deletions and rearrangements, the molecular lesions causing hemoglobinopathies are all identifiable by PCR-based techniques. Furthermore, each at-risk ethnic group has its own combination of common Hb variants and thalassemia mutations. In Iran, there are many different forms of α and β thalassemia. Increasingly, different Hb variants are being detected and their effects per se or in combination with the thalassemias, provide additional diagnostic challenges. RESULTS: We did step-by-step diagnosis workup in 800 patients with hemoglobinopathies who referred to Research center of Thalassemia and Hemoglobinopathies in Shafa Hospital of Ahwaz Joundishapour University of medical sciences, respectively. We detected 173 patients as iron deficiency anemia (IDA) and 627 individuals as thalassemic patients by use of different indices. We

Analysis of the relationships between oxidative stress, DNA damage and sperm vitality in a patient population: development of diagnostic criteria.

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Aitken, R John; De Iuliis, Geoffry N; Finnie, Jane M; Hedges, Andrew; McLachlan, Robert I

2010-10-01

DNA damage in human spermatozoa is known to be associated with a variety of adverse clinical outcomes affecting both reproductive efficiency and the health and wellbeing of the offspring. However, the origin of this damage, its biochemical nature and strategies for its amelioration, still await resolution. Using novel methods to simultaneously assess DNA fragmentation (modified TUNEL assay), DNA-base adduct formation (8-hydroxy-2'-deoxyguanosine [8OHdG]) and cell vitality, spermatozoa from a cohort of 50 assisted conception patients were examined and compared with a group of donors. Receiver operating characteristic (ROC) curve analysis was then used to examine the frequency distribution of the data and to determine optimized thresholds for identifying patients exhibiting abnormally high levels of DNA damage. 8OHdG formation and DNA fragmentation were highly correlated with each other and frequently associated with cell death. Percoll centrifugation improved sperm quality but, unexpectedly, increased 8OHdG formation in live cells, as did sperm fractionation using Puresperm gradients. ROC analysis indicated that the frequency distribution of 8OHdG and DNA fragmentation data were significantly different between patients and donors (P live cells. However, the development of novel methods and optimized thresholds for diagnosing oxidative DNA damage in human spermatozoa should assist in the clinical management of this pathology.

Atomic absorption spectrometric determination of copper, zinc, and lead in geological materials

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Sanzolone, R.F.; Chao, T.T.

1976-01-01

An atomic absorption spectrometric method is described for the determination of copper, zinc, and lead in geological materials. The sample is digested with HF-HCl-H2O2; the final solution for analysis is in 10 % (v/v) HCl. Copper and zinc are determined directly by aspirating the solution into an air-acetylene flame. A separate aliquot of the solution is used for determination of lead; lead is extracted into TOPO-MIBK from the acidic solution in the presence of iodide and ascorbic acid. For a 0.50-g sample, the limits of determination are 10-2000 p.p.m. for Cu and Zn, and 5-5000 p.p.m. for Pb. As much as 40 % Fe or Ca. and 10 % Al, Mg, or Mn in the sample do not interfere. The proposed method can be applied to the determination of copper, zinc, and lead in a wide range of geological materials including iron- and manganese-rich, calcareous and carbonate samples. ?? 1976.

High frequency of parvovirus B19 DNA in bone marrow samples from rheumatic patients

DEFF Research Database (Denmark)

Lundqvist, Anders; Isa, Adiba; Tolfvenstam, Thomas

2005-01-01

BACKGROUND: Human parvovirus B19 (B19) polymerase chain reaction (PCR) is now a routine analysis and serves as a diagnostic marker as well as a complement or alternative to B19 serology. The clinical significance of a positive B19 DNA finding is however dependent on the type of tissue or body fluid...... analysed and of the immune status of the patient. OBJECTIVES: To analyse the clinical significance of B19 DNA positivity in bone marrow samples from rheumatic patients. STUDY DESIGN: Parvovirus B19 DNA was analysed in paired bone marrow and serum samples by nested PCR technique. Serum was also analysed...... negative group. A high frequency of parvovirus B19 DNA was thus detected in bone marrow samples in rheumatic patients. The clinical data does not support a direct association between B19 PCR positivity and rheumatic disease manifestation. Therefore, the clinical significance of B19 DNA positivity in bone...

Mitochondrial mosaics in the liver of 3 infants with mtDNA defects

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Scalais Emmanuel

2009-06-01

Full Text Available Abstract Background In muscle cytochrome oxidase (COX negative fibers (mitochondrial mosaics have often been visualized. Methods COX activity staining of liver for light and electron microscopy, muscle stains, blue native gel electrophoresis and activity assays of respiratory chain proteins, their immunolocalisation, mitochondrial and nuclear DNA analysis. Results Three unrelated infants showed a mitochondrial mosaic in the liver after staining for COX activity, i.e. hepatocytes with strongly reactive mitochondria were found adjacent to cells with many negative, or barely reactive, mitochondria. Deficiency was most severe in the patient diagnosed with Pearson syndrome. Ragged-red fibers were absent in muscle biopsies of all patients. Enzyme biochemistry was not diagnostic in muscle, fibroblasts and lymphocytes. Blue native gel electrophoresis of liver tissue, but not of muscle, demonstrated a decreased activity of complex IV; in both muscle and liver subcomplexes of complex V were seen. Immunocytochemistry of complex IV confirmed the mosaic pattern in two livers, but not in fibroblasts. MRI of the brain revealed severe white matter cavitation in the Pearson case, but only slight cortical atrophy in the Alpers-Huttenlocher patient, and a normal image in the 3rd. MtDNA in leucocytes showed a common deletion in 50% of the mtDNA molecules of the Pearson patient. In the patient diagnosed with Alpers-Huttenlocher syndrome, mtDNA was depleted for 60% in muscle. In the 3rd patient muscular and hepatic mtDNA was depleted for more than 70%. Mutations in the nuclear encoded gene of POLG were subsequently found in both the 2nd and 3rd patients. Conclusion Histoenzymatic COX staining of a liver biopsy is fast and yields crucial data about the pathogenesis; it indicates whether mtDNA should be assayed. Each time a mitochondrial disorder is suspected and muscle data are non-diagnostic, a liver biopsy should be recommended. Mosaics are probably more frequent

Analyte-Triggered DNA-Probe Release from a Triplex Molecular Beacon for Nanopore Sensing.

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Guo, Bingyuan; Sheng, Yingying; Zhou, Ke; Liu, Quansheng; Liu, Lei; Wu, Hai-Chen

2018-03-26

A new nanopore sensing strategy based on triplex molecular beacon was developed for the detection of specific DNA or multivalent proteins. The sensor is composed of a triplex-forming molecular beacon and a stem-forming DNA component that is modified with a host-guest complex. Upon target DNA hybridizing with the molecular beacon loop or multivalent proteins binding to the recognition elements on the stem, the DNA probe is released and produces highly characteristic current signals when translocated through α-hemolysin. The frequency of current signatures can be used to quantify the concentrations of the target molecules. This sensing approach provides a simple, quick, and modular tool for the detection of specific macromolecules with high sensitivity and excellent selectivity. It may find useful applications in point-of-care diagnostics with a portable nanopore kit in the future. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Developing a new diagnostic algorithm for human papilloma virus associated oropharyngeal carcinoma: an investigation of HPV DNA assays.

Science.gov (United States)

Cohen, Natasha; Gupta, Michael; Doerwald-Munoz, Lilian; Jang, Dan; Young, James Edward Massey; Archibald, Stuart; Jackson, Bernard; Lee, Jenny; Chernesky, Max

2017-02-13

Human papilloma virus (HPV) has been implicated in the development of a large proportion of oropharyngeal squamous cell carcinoma (OPSCC). Current techniques used to diagnose HPV etiology require histopathologic analysis. We aim to investigate the diagnostic accuracy of a new application non-histopathologic diagnostic tests to help assist diagnosis of HPV-related oropharyngeal tumors. Patients with OPSCC with nodal metastasis were consecutively recruited from a multidisciplinary cancer clinic. Appropriate samples were collected and analyzed. The various tests examined included COBAS® 4800, Cervista® HR and Genotyping. These tests were compared to p16 staining, which was used as the diagnostic standard. StataIC 14.2 was used to perform analysis, including sensitivity, specificity and receiver operator characteristic [ROC] curves. The COBAS® FNA (area under ROC 0.863) and saliva (area under ROC 0.847) samples performed well in diagnosing HPV positive and negative tumors. Samples tested with Cervista® did not corroborate p16 status reliably. We were able to increase the diagnostic yield of the COBAS® FNA samples by applying the results of the saliva test to negative FNA samples which correctly identified 11 additional p16 positive tumors (area under ROC 0.915). Surrogate testing for HPV using alternate methods is feasible and closely predicts the results of standard diagnostic methods. In the future, these could minimize invasive procedures for diagnosing HPV-related oropharyngeal cancer, but also help to diagnose and treat patients with unknown primaries.

Environmental toxicants cause sperm DNA fragmentation as detected by the Sperm Chromatin Structure Assay (SCSA[reg])

International Nuclear Information System (INIS)

Evenson, Donald P.; Wixon, Regina

2005-01-01

Studies over the past two decades have clearly shown that reproductive toxicants cause sperm DNA fragmentation. This DNA fragmentation can usually be detected prior to observing alterations of metaphase chromosomes in embryos. Thus, Sperm Chromatin Structure Assay (SCSA)-detected DNA damage is viewed as the molecular precursor to later gross chromosome damage observed under the light microscope. SCSA measurements of animal or human sperm consist of first obtaining a fresh or flash frozen neat semen sample in LN2 or dry ice. Samples are then sent to a SCSA diagnostic laboratory where the samples are thawed, diluted to ∼1-2 x 106 sperm/ml, treated for 30 s with a pH 1.2 detergent buffer and then stained with acridine orange (AO). The low pH partially denatures DNA at the sites of DNA strand breaks and the AO-ssDNA fluoresces red while the AO-dsDNA fluoresces green. Flow cytometry measurements of 5000 sperm/sample provide statistically robust data on the ratio of red to green sperm, the extent of the DNA fragmentation and the standard deviations of measures. Numerous experiments on rodents treated with reproductive toxicants clearly showed that SCSA measures are highly dose responsive and have a very low CV. Different agents that act on germ cells at various stages of development usually showed sperm DNA fragmentation when that germ cell fraction arrived in the epididymis or ejaculate. Some of these treated samples were capable of successful in vitro fertilization but with frequent embryo failure. A 2-year longitudinal study of men living a valley town with a reported abnormal level of infertility and spontaneous miscarriages and also a seasonal atmospheric smog pollution, showed, for the first time, that SCSA measurements of human sperm DNA fragmentation were detectable and correlated with dosage of air pollution while the classical semen measures were not correlated. Also, young men spraying pesticides without protective gear are at an increased risk for elevated

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.

Sensitivitas dan Spesifisitas Nested Polymerase Chain Reaction untuk Mendeteksi DNA Coxiella burnetii (SENSITIVITY AND SPECIFICITY OF NESTED POLYMERASE CHAIN REACTION FOR DETECTION OF COXIELLA BURNETII DNA

Directory of Open Access Journals (Sweden)

Trioso Purnawarman

2014-04-01

Full Text Available Sensitivity and specificity of nested polymerase chain reaction (nested PCR to detect Coxiella burnetii(C. burnetii DNA were studied. The primer system which consists of external primers (OMP1 and OMP2and internal primers (OMP3 and OMP4, was designed from the nucleotide sequence of the com I geneencoding for 27 kDa outer membrane protein and used to specifically amplify a 501 bp and 438 bp fragment.This nested PCR assay was 50 fold more sensitive than that of using PCR external primer only. TheNested PCR has a detection limit as low as 300 pg/?l. Specificity studies showed that nested PCR onlydetected C. burnetii DNA and did not happened Brucella abortus, Escherichia coli, Pseudomonas aeruginosaand Campylobacter Jejuni DNA. Nested PCR has high senstively and specificaly diagnostic method of C.burnetii as agent of Q fever disease.

Magnetophoresis of flexible DNA-based dumbbell structures

Science.gov (United States)

Babić, B.; Ghai, R.; Dimitrov, K.

2008-02-01

Controlled movement and manipulation of magnetic micro- and nanostructures using magnetic forces can give rise to important applications in biomedecine, diagnostics, and immunology. We report controlled magnetophoresis and stretching, in aqueous solution, of a DNA-based dumbbell structure containing magnetic and diamagnetic microspheres. The velocity and stretching of the dumbbell were experimentally measured and correlated with a theoretical model based on the forces acting on individual magnetic beads or the entire dumbbell structures. The results show that precise and predictable manipulation of dumbbell structures is achievable and can potentially be applied to immunomagnetic cell separators.

Prostate-based biofluids for the detection of prostate cancer: A comparative study of the diagnostic performance of cell-sourced RNA biomarkers

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Matthew J. Roberts

2016-09-01

Conclusion: PEUW contains prostatic material, but has limited diagnostic accuracy when considering cell-derived DNA analysis. PCA3-based markers in ejaculate are comparable to serum PSA and digital rectal examination–urine markers.

Cell-free mitochondrial DNA copy number variation in head and neck squamous cell carcinoma: A study of non-invasive biomarker from Northeast India.

Science.gov (United States)

Kumar, Manish; Srivastava, Shilpee; Singh, Seram Anil; Das, Anup Kumar; Das, Ganesh Chandra; Dhar, Bishal; Ghosh, Sankar Kumar; Mondal, Rosy

2017-10-01

Head and neck squamous cell carcinoma is the most commonly diagnosed cancer worldwide. The lifestyle, food habits, and customary practices manifest the Northeast Indian population toward higher susceptibility to develop head and neck squamous cell carcinoma. Here, we have investigated the association of smoke and smokeless tobacco, and alcohol with copy number variation of cell-free mitochondrial DNA and cell-free nuclear DNA in cases and controls. Cell-free DNA from plasma was isolated from 50 head and neck squamous cell carcinoma cases and 50 controls with informed written consent using QIAamp Circulating Nucleic Acid Kit. Real-time polymerase chain reaction was done for copy number variation in cell-free mitochondrial DNA and cell-free nuclear DNA. Receiver operating characteristic curve analysis was performed to evaluate the diagnostic application between the two study groups using clinicopathological parameters. The levels of cell-free nuclear DNA and cell-free mitochondrial DNA of cases in association with smoke and smokeless tobacco, alcohol with smoking (p squamous cell carcinoma cases and controls, we distinguished cell-free mitochondrial DNA (cutoff: 19.84 raw Ct; sensitivity: 84%; specificity: 100%; p < 0.001) and cell-free nuclear DNA (cutoff: 463,282 genomic equivalent/mL; sensitivity: 53%; specificity: 87%; p < 0.001). The copy number variation in cases (cell-free nuclear DNA: 5451.66 genomic equivalent/mL and cell-free mitochondrial DNA: 29,103,476.15 genomic equivalent/mL) and controls (cell-free nuclear DNA: 1650.9 genomic equivalent/mL and cell-free mitochondrial DNA: 9,189,312.54 genomic equivalent/mL), respectively. Our result indicates that the cell-free mitochondrial DNA content is highly associated with smoke and smokeless tobacco, betel quid chewing, and alcohol which shows greater promises, holding the key characteristics of diagnostic biomarkers, that is, minimal invasiveness, high specificity, and sensitivity.

Electrochemical DNA biosensor based on the BDD nanograss array electrode.

Science.gov (United States)

Jin, Huali; Wei, Min; Wang, Jinshui

2013-04-10

The development of DNA biosensor has attracted considerable attention due to their potential applications, including gene analysis, clinical diagnostics, forensic study and more medical applications. Using electroactive daunomycin as an indicator, the hybridization detection was measured by differential pulse voltammetry in this study. Electrochemical DNA biosensor was developed based on the BDD film electrode (fBDD) and BDD nanograss array electrode (nBDD). In comparison with fBDD and AuNPs/CA/fBDD electrode, the lower semicircle diameter of electrochemical impedance spectroscopy obtained on nBDD and AuNPs/CA/nBDD electrode indicated that the presence of nanograss array improved the reactive site, reduced the interfacial resistance, and made the electron transfer easier. Using electroactive daunomycin as an indicator, the hybridization detection was measured by differential pulse voltammetry. The experimental results demonstrated that the prepared AuNPs/CA/nBDD electrode was suitable for DNA hybridization with favorable performance of faster response, higher sensitivity, lower detection limit and satisfactory selectivity, reproducibility and stability.

Biomolecule Sequencer: Next-Generation DNA Sequencing Technology for In-Flight Environmental Monitoring, Research, and Beyond

Science.gov (United States)

Smith, David J.; Burton, Aaron; Castro-Wallace, Sarah; John, Kristen; Stahl, Sarah E.; Dworkin, Jason Peter; Lupisella, Mark L.

2016-01-01

On the International Space Station (ISS), technologies capable of rapid microbial identification and disease diagnostics are not currently available. NASA still relies upon sample return for comprehensive, molecular-based sample characterization. Next-generation DNA sequencing is a powerful approach for identifying microorganisms in air, water, and surfaces onboard spacecraft. The Biomolecule Sequencer payload, manifested to SpaceX-9 and scheduled on the Increment 4748 research plan (June 2016), will assess the functionality of a commercially-available next-generation DNA sequencer in the microgravity environment of ISS. The MinION device from Oxford Nanopore Technologies (Oxford, UK) measures picoamp changes in electrical current dependent on nucleotide sequences of the DNA strand migrating through nanopores in the system. The hardware is exceptionally small (9.5 x 3.2 x 1.6 cm), lightweight (120 grams), and powered only by a USB connection. For the ISS technology demonstration, the Biomolecule Sequencer will be powered by a Microsoft Surface Pro3. Ground-prepared samples containing lambda bacteriophage, Escherichia coli, and mouse genomic DNA, will be launched and stored frozen on the ISS until experiment initiation. Immediately prior to sequencing, a crew member will collect and thaw frozen DNA samples, connect the sequencer to the Surface Pro3, inject thawed samples into a MinION flow cell, and initiate sequencing. At the completion of the sequencing run, data will be downlinked for ground analysis. Identical, synchronous ground controls will be used for data comparisons to determine sequencer functionality, run-time sequence, current dynamics, and overall accuracy. We will present our latest results from the ISS flight experiment the first time DNA has ever been sequenced in space and discuss the many potential applications of the Biomolecule Sequencer for environmental monitoring, medical diagnostics, higher fidelity and more adaptable Space Biology Human

Identification of organic sulfur compounds in coal bitumen obtained by different extraction techniques using comprehensive two-dimensional gas chromatography coupled to time-of-flight mass spectrometric detection

Energy Technology Data Exchange (ETDEWEB)

Machado, Maria Elisabete; Cappelli Fontanive, Fernando; Bastos Caramao, Elina; Alcaraz Zini, Claudia [Universidade Federal do Rio Grande do Sul, Instituto de Quimica, Porto Alegre, RS (Brazil); Oliveira, Jose Vladimir de [URI, Universidade Regional Integrada do Alto Uruguai e das Missoes, Erechim, RS (Brazil)

2011-11-15

The determination of organic sulfur compounds (OSC) in coal is of great interest. Technically and operationally these compounds are not easily removed and promote corrosion of equipment. Environmentally, the burning of sulfur compounds leads to the emission of SO{sub x} gases, which are major contributors to acid rain. Health-wise, it is well known that these compounds have mutagenic and carcinogenic properties. Bitumen can be extracted from coal by different techniques, and use of gas chromatography coupled to mass spectrometric detection enables identification of compounds present in coal extracts. The OSC from three different bitumens were tentatively identified by use of three different extraction techniques: accelerated solvent extraction (ASE), ultrasonic extraction (UE), and supercritical-fluid extraction (SFE). Results obtained from one-dimensional gas chromatography (1D GC) coupled to quadrupole mass spectrometric detection (GC-qMS) and from two-dimensional gas chromatography with time-of-flight mass spectrometric detection (GC x GC-TOFMS) were compared. By use of 2D GC, a greater number of OSC were found in ASE bitumen than in SFE and UE bitumens. No OSC were identified with 1D GC-qMS, although some benzothiophenes and dibenzothiophenes were detected by use of EIM and SIM modes. GC x GC-TOFMS applied to investigation of OSC in bitumens resulted in analytical improvement, as more OSC classes and compounds were identified (thiols, sulfides, thiophenes, naphthothiophenes, benzothiophenes, and benzonaphthothiophenes). The roof-tile effect was observed for OSC and PAH in all bitumens. Several co-elutions among analytes and with matrix interferents were solved by use of GC x GC. (orig.)

Long-term frozen storage of urine samples: a trouble to get PCR results in Schistosoma spp. DNA detection?

Science.gov (United States)

Fernández-Soto, Pedro; Velasco Tirado, Virginia; Carranza Rodríguez, Cristina; Pérez-Arellano, José Luis; Muro, Antonio

2013-01-01

Human schistosomiasis remains a serious worldwide public health problem. At present, a sensitive and specific assay for routine diagnosis of schistosome infection is not yet available. The potential for detecting schistosome-derived DNA by PCR-based methods in human clinical samples is currently being investigated as a diagnostic tool with potential application in routine schistosomiasis diagnosis. Collection of diagnostic samples such as stool or blood is usually difficult in some populations. However, urine is a biological sample that can be collected in a non-invasive method, easy to get from people of all ages and easy in management, but as a sample for PCR diagnosis is still not widely used. This could be due to the high variability in the reported efficiency of detection as a result of the high variation in urine samples' storage or conditions for handling and DNA preservation and extraction methods. We evaluate different commercial DNA extraction methods from a series of long-term frozen storage human urine samples from patients with parasitological confirmed schistosomiasis in order to assess the PCR effectiveness for Schistosoma spp. detection. Patients urine samples were frozen for 18 months up to 7 years until use. Results were compared with those obtained in PCR assays using fresh healthy human urine artificially contaminated with Schistosoma mansoni DNA and urine samples from mice experimentally infected with S. mansoni cercariae stored frozen for at least 12 months before use. PCR results in fresh human artificial urine samples using different DNA based extraction methods were much more effective than those obtained when long-term frozen human urine samples were used as the source of DNA template. Long-term frozen human urine samples are probably not a good source for DNA extraction for use as a template in PCR detection of Schistosoma spp., regardless of the DNA method of extraction used.

Label-free detection of DNA hybridization and single point mutations in a nano-gap biosensor

International Nuclear Information System (INIS)

Zaffino, R L; Mir, M; Samitier, J

2014-01-01

We describe a conductance-based biosensor that exploits DNA-mediated long-range electron transport for the label-free and direct electrical detection of DNA hybridization. This biosensor platform comprises an array of vertical nano-gap biosensors made of gold and fabricated through standard photolithography combined with focused ion beam lithography. The nano-gap walls are covalently modified with short, anti-symmetric thiolated DNA probes, which are terminated by 19 bases complementary to both the ends of a target DNA strand. The nano-gaps are separated by a distance of 50nm, which was adjusted to fit the length of the DNA target plus the DNA probes. The hybridization of the target DNA closes the gap circuit in a switch on/off fashion, in such a way that it is readily detected by an increase in the current after nano-gap closure. The nano-biosensor shows high specificity in the discrimination of base-pair mismatching and does not require signal indicators or enhancing molecules. The design of the biosensor platform is applicable for multiplexed detection in a straightforward manner. The platform is well-suited to mass production, point-of-care diagnostics, and wide-scale DNA analysis applications. (paper)

THE CURRENT METHODS FOR MOLECULAR DIAGNOSTICS OF FISH DISEASES (REVIEW

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O. Zaloilo

2016-06-01

Full Text Available Purpose. The methods of molecular diagnostic (MMD gradually become widespread in modern fish farming. MMD contain a wide variety of specific approaches, each of which has distinct limits of their possible applications and is characterized by individual peculiarities in practical performance. In addition to high sensitivity and the possibility of rapid diagnostics, the main advantage of molecular methods is to determine the uncultivated infectious agents. DNA amplification allows identifying pathogenic microorganisms at very small quantities even in the minimum sample volume. Molecular methods of diagnostic enable the determination of infection in latent or acute phases. These methods allow showing the differences between pathogens with similar antigenic structures. The current literature data on this subject usually show a methodology in the narrow context of the tasks or practical results obtained through such approaches. Thus, a synthesis of existing information on the mechanisms of action and the limits of the typical problems of basic methods of molecular diagnostics are an urgent task of fish breeding. In particular, the following description will more effectively choose one or several approaches to identify pathogens in fish. Findings. This paper reviews the basic molecular methods that are used in the world's aquaculture for diagnosis of various diseases in commercial fish species. Originality. This work is a generalization of data on the principles and mechanisms for the implementation of diagnostics based on modern molecular techniques. For each of the mentioned approaches, the most promising areas of application were shown. The information is provided in the form of a comparative analysis of each methodology, indicating positive and negative practical aspects. Practical value. The current review of modern methods of molecular diagnostic in aquaculture is focused on practical application. Generalizing and analytical information can be

Spectrometric gamma radiation of shale cores applied to sweet spot discrimination in Eastern Pomerania, Poland

Science.gov (United States)

Skupio, Rafal; de Alemar Barberes, Gabriel

2017-12-01

This paper describes the application and calculation of hydrocarbon anomalies in two different boreholes located in Eastern Pomerania (northern Poland). Spectrometric data from borehole geophysical probe (borehole 1) and portable gamma logger (borehole 2) were used to analyze shale formations. The results from borehole 1 presented a statistically significant, moderate correlation between calculated hydrocarbon anomalies and hydrocarbon saturation data obtained from well log interpretation. Borehole 2 has been analyzed focusing on the gamma radiation of the core samples, and the positive results of borehole 1. Hydrocarbon anomalies calculated from spectral gamma radiation are reliable indicators of sweet spots, based solely on a cursory evaluation of core measurements. These preliminary information acquired from gamma-ray measurements could help increase sampling precision of further geochemical analysis.

Mass spectrometric analysis of EPO IEF-PAGE interfering substances in nitrile examination gloves.

Science.gov (United States)

Reichel, Christian

2012-10-01

Direct detection of doping with recombinant erythropoietins (rhEPO) is accomplished by isoelectric focusing (IEF) or sodium dodecylsulfate (SDS) polyacrylamide gel electrophoresis (PAGE). In a recent publication, Lasne et al. (Electrophoresis 2011, 32, 1444) showed that improper use of nitrile examination gloves during sample collection, sample preparation, and IEF-PAGE may lead to distorted or absent EPO IEF-profiles. In order to clarify which substances are responsible for this observation, a mass spectrometric study on water extractable compounds found in nitrile gloves was performed. Several substance classes were shown to be present, among them polyethylene glycols (PEG), anionic and nonionic surfactants, as well as alcohol ethoxylates and plasticizers. It could be demonstrated that alkylbenzenesulfonates, the main category of detectable anionic detergents, and among them sodium dodecylbenzenesulfonate (SDBS) and its homologs, are the prime reason for the interference of nitrile gloves with EPO IEF-PAGE. Copyright © 2012 John Wiley & Sons, Ltd.

Mass spectrometric identification of proteins that interact through specific domains of the poly(A) binding protein.

Science.gov (United States)

Richardson, Roy; Denis, Clyde L; Zhang, Chongxu; Nielsen, Maria E O; Chiang, Yueh-Chin; Kierkegaard, Morten; Wang, Xin; Lee, Darren J; Andersen, Jens S; Yao, Gang

2012-09-01

Poly(A) binding protein (PAB1) is involved in a number of RNA metabolic functions in eukaryotic cells and correspondingly is suggested to associate with a number of proteins. We have used mass spectrometric analysis to identify 55 non-ribosomal proteins that specifically interact with PAB1 from Saccharomyces cerevisiae. Because many of these factors may associate only indirectly with PAB1 by being components of the PAB1-mRNP structure, we additionally conducted mass spectrometric analyses on seven metabolically defined PAB1 deletion derivatives to delimit the interactions between these proteins and PAB1. These latter analyses identified 13 proteins whose associations with PAB1 were reduced by deleting one or another of PAB1's defined domains. Included in this list of 13 proteins were the translation initiation factors eIF4G1 and eIF4G2, translation termination factor eRF3, and PBP2, all of whose previously known direct interactions with specific PAB1 domains were either confirmed, delimited, or extended. The remaining nine proteins that interacted through a specific PAB1 domain were CBF5, SLF1, UPF1, CBC1, SSD1, NOP77, yGR250c, NAB6, and GBP2. In further study, UPF1, involved in nonsense-mediated decay, was confirmed to interact with PAB1 through the RRM1 domain. We additionally established that while the RRM1 domain of PAB1 was required for UPF1-induced acceleration of deadenylation during nonsense-mediated decay, it was not required for the more critical step of acceleration of mRNA decapping. These results begin to identify the proteins most likely to interact with PAB1 and the domains of PAB1 through which these contacts are made.

Chimeric proteins for detection and quantitation of DNA mutations, DNA sequence variations, DNA damage and DNA mismatches

Science.gov (United States)

McCutchen-Maloney, Sandra L.

2002-01-01

Chimeric proteins having both DNA mutation binding activity and nuclease activity are synthesized by recombinant technology. The proteins are of the general formula A-L-B and B-L-A where A is a peptide having DNA mutation binding activity, L is a linker and B is a peptide having nuclease activity. The chimeric proteins are useful for detection and identification of DNA sequence variations including DNA mutations (including DNA damage and mismatches) by binding to the DNA mutation and cutting the DNA once the DNA mutation is detected.

Rapid detection of cancer related DNA nanoparticulate biomarkers and nanoparticles in whole blood

Science.gov (United States)

Heller, Michael J.; Krishnan, Raj; Sonnenberg, Avery

2010-08-01

The ability to rapidly detect cell free circulating (cfc) DNA, cfc-RNA, exosomes and other nanoparticulate disease biomarkers as well as drug delivery nanoparticles directly in blood is a major challenge for nanomedicine. We now show that microarray and new high voltage dielectrophoretic (DEP) devices can be used to rapidly isolate and detect cfc-DNA nanoparticulates and nanoparticles directly from whole blood and other high conductance samples (plasma, serum, urine, etc.). At DEP frequencies of 5kHz-10kHz both fluorescent-stained high molecular weight (hmw) DNA, cfc-DNA and fluorescent nanoparticles separate from the blood and become highly concentrated at specific DEP highfield regions over the microelectrodes, while blood cells move to the DEP low field-regions. The blood cells can then be removed by a simple fluidic wash while the DNA and nanoparticles remain highly concentrated. The hmw-DNA could be detected at a level of <260ng/ml and the nanoparticles at <9.5 x 109 particles/ml, detection levels that are well within the range for viable clinical diagnostics and drug nanoparticle monitoring. Disease specific cfc-DNA materials could also be detected directly in blood from patients with Chronic Lymphocytic Leukemia (CLL) and confirmed by PCR genotyping analysis.

NMR studies of DNA oligomers and their interactions with minor groove binding ligands

Energy Technology Data Exchange (ETDEWEB)

Fagan, Patricia A. [Univ. of California, Berkeley, CA (United States). Dept. of Chemistry

1996-05-01

The cationic peptide ligands distamycin and netropsin bind noncovalently to the minor groove of DNA. The binding site, orientation, stoichiometry, and qualitative affinity of distamycin binding to several short DNA oligomers were investigated by NMR spectroscopy. The oligomers studied contain A,T-rich or I,C-rich binding sites, where I = 2-desaminodeoxyguanosine. I•C base pairs are functional analogs of A•T base pairs in the minor groove. The different behaviors exhibited by distamycin and netropsin binding to various DNA sequences suggested that these ligands are sensitive probes of DNA structure. For sites of five or more base pairs, distamycin can form 1:1 or 2:1 ligand:DNA complexes. Cooperativity in distamycin binding is low in sites such as AAAAA which has narrow minor grooves, and is higher in sites with wider minor grooves such as ATATAT. The distamycin binding and base pair opening lifetimes of I,C-containing DNA oligomers suggest that the I,C minor groove is structurally different from the A,T minor groove. Molecules which direct chemistry to a specific DNA sequence could be used as antiviral compounds, diagnostic probes, or molecular biology tools. The author studied two ligands in which reactive groups were tethered to a distamycin to increase the sequence specificity of the reactive agent.

CSF studies facilitate DNA diagnosis in familial Alzheimer's disease due to a presenilin-1 mutation

NARCIS (Netherlands)

de Bot, Susanne T; Kremer, H P H; Dooijes, Dennis; Verbeek, Marcel M

2009-01-01

In sporadic Alzheimer's disease (AD), cerebrospinal fluid (CSF) analysis is becoming increasingly relevant to establish an early diagnosis. We present a case of familial AD due to a presenilin-1 mutation in which CSF studies suggested appropriate DNA diagnostics. A 38 year old Dutch man presented

High quality DNA from human papillomavirus (HPV for PCR/RFLPs

Directory of Open Access Journals (Sweden)

Denise Wanderlei-Silva

2005-01-01

Full Text Available The analysis of DNA in clinical samples for a secure diagnostic has become indispensable nowadays. Techniques approaching isolation of high molecular weigth DNA of HPV could lead to efficient amplification and early clinical diagnosis of the virus DNA by PCR (polymerase chain reaction. We describe a fast, non-toxical, efficient and cheap method for DNA isolation of human papilloma virus (HPV from cervical smears using guanidine (DNAzol solution. A 450 bp DNA band correponding to the late region (L1 of the virus genome was detected by PCR, showing that the DNAzol extraction soluction generated a good viral DNA yield. The electrophoretic pattern after digestion with restriction endonucleases (RFLPs/PCR revealed the predominance of HPV-16 and HPV-33 in the samples from the State of Alagoas, Brazil.A detecção de DNA em amostras clínicas visando um diagnóstico mais seguro vem se tornando uma prática comum em laboratórios de análise clínica. Metodologias que objetivem o isolamento de DNA de alto peso molecular de HPV podem levar a uma amplificação precisa e diagnose precoce do DNA do vírus por PCR (reação de polimerase em cadeia. Nós descrevemos um método para o isolamento do DNA do vírus do papiloma humano de amostras cervicais utilizando o detergente guanidina (solução DNAzol. O método foi rápido, não-tóxico e eficiente. Uma banda de DNA de 450 pb correspondente à região tardia (L1 do genoma viral foi detectada por PCR, mostrando que a extração com DNAzol gerou quantidade suficiente de DNA para análise. O padrão eletroforético, após digestão com endonucleases de restrição (RFLPs/PCR, revelou predominância de HPV 16 e HPV-33 nas amostras no Estado de Alagoas, Brasil.