One-pot preparation of mRNA/cDNA display by a novel and versatile puromycin-linker DNA.

Science.gov (United States)

Mochizuki, Yuki; Biyani, Manish; Tsuji-Ueno, Sachika; Suzuki, Miho; Nishigaki, Koichi; Husimi, Yuzuru; Nemoto, Naoto

2011-09-12

A rapid, easy, and robust preparation method for mRNA/cDNA display using a newly designed puromycin-linker DNA is presented. The new linker is structurally simple, easy to synthesize, and cost-effective for use in "in vitro peptide and protein selection". An introduction of RNase T1 nuclease site to the new linker facilitates the easy recovery of mRNA/cDNA displayed protein by an improvement of the efficiency of ligating the linker to mRNAs and efficient release of mRNA/cDNA displayed protein from the solid-phase (magnetic bead). For application demonstration, affinity selections were successfully performed. Furthermore, we introduced a "one-pot" preparation protocol to perform mRNA display easy. Unlike conventional approaches that require tedious and downstream multistep process including purification, this protocol will make the mRNA/cDNA display methods more practical and convenient and also facilitate the development of next-generation, high-throughput mRNA/cDNA display systems amenable to automation.

Sequencing historical specimens: successful preparation of small specimens with low amounts of degraded DNA.

Science.gov (United States)

Sproul, John S; Maddison, David R

2017-11-01

Despite advances that allow DNA sequencing of old museum specimens, sequencing small-bodied, historical specimens can be challenging and unreliable as many contain only small amounts of fragmented DNA. Dependable methods to sequence such specimens are especially critical if the specimens are unique. We attempt to sequence small-bodied (3-6 mm) historical specimens (including nomenclatural types) of beetles that have been housed, dried, in museums for 58-159 years, and for which few or no suitable replacement specimens exist. To better understand ideal approaches of sample preparation and produce preparation guidelines, we compared different library preparation protocols using low amounts of input DNA (1-10 ng). We also explored low-cost optimizations designed to improve library preparation efficiency and sequencing success of historical specimens with minimal DNA, such as enzymatic repair of DNA. We report successful sample preparation and sequencing for all historical specimens despite our low-input DNA approach. We provide a list of guidelines related to DNA repair, bead handling, reducing adapter dimers and library amplification. We present these guidelines to facilitate more economical use of valuable DNA and enable more consistent results in projects that aim to sequence challenging, irreplaceable historical specimens. © 2017 John Wiley & Sons Ltd.

A microfluidic DNA library preparation platform for next-generation sequencing.

Science.gov (United States)

Kim, Hanyoup; Jebrail, Mais J; Sinha, Anupama; Bent, Zachary W; Solberg, Owen D; Williams, Kelly P; Langevin, Stanley A; Renzi, Ronald F; Van De Vreugde, James L; Meagher, Robert J; Schoeniger, Joseph S; Lane, Todd W; Branda, Steven S; Bartsch, Michael S; Patel, Kamlesh D

2013-01-01

Next-generation sequencing (NGS) is emerging as a powerful tool for elucidating genetic information for a wide range of applications. Unfortunately, the surging popularity of NGS has not yet been accompanied by an improvement in automated techniques for preparing formatted sequencing libraries. To address this challenge, we have developed a prototype microfluidic system for preparing sequencer-ready DNA libraries for analysis by Illumina sequencing. Our system combines droplet-based digital microfluidic (DMF) sample handling with peripheral modules to create a fully-integrated, sample-in library-out platform. In this report, we use our automated system to prepare NGS libraries from samples of human and bacterial genomic DNA. E. coli libraries prepared on-device from 5 ng of total DNA yielded excellent sequence coverage over the entire bacterial genome, with >99% alignment to the reference genome, even genome coverage, and good quality scores. Furthermore, we produced a de novo assembly on a previously unsequenced multi-drug resistant Klebsiella pneumoniae strain BAA-2146 (KpnNDM). The new method described here is fast, robust, scalable, and automated. Our device for library preparation will assist in the integration of NGS technology into a wide variety of laboratories, including small research laboratories and clinical laboratories.

A microfluidic DNA library preparation platform for next-generation sequencing.

Directory of Open Access Journals (Sweden)

Hanyoup Kim

Full Text Available Next-generation sequencing (NGS is emerging as a powerful tool for elucidating genetic information for a wide range of applications. Unfortunately, the surging popularity of NGS has not yet been accompanied by an improvement in automated techniques for preparing formatted sequencing libraries. To address this challenge, we have developed a prototype microfluidic system for preparing sequencer-ready DNA libraries for analysis by Illumina sequencing. Our system combines droplet-based digital microfluidic (DMF sample handling with peripheral modules to create a fully-integrated, sample-in library-out platform. In this report, we use our automated system to prepare NGS libraries from samples of human and bacterial genomic DNA. E. coli libraries prepared on-device from 5 ng of total DNA yielded excellent sequence coverage over the entire bacterial genome, with >99% alignment to the reference genome, even genome coverage, and good quality scores. Furthermore, we produced a de novo assembly on a previously unsequenced multi-drug resistant Klebsiella pneumoniae strain BAA-2146 (KpnNDM. The new method described here is fast, robust, scalable, and automated. Our device for library preparation will assist in the integration of NGS technology into a wide variety of laboratories, including small research laboratories and clinical laboratories.

General method of preparation of uniformly 13C, 15N-labeled DNA fragments for NMR analysis of DNA structures

International Nuclear Information System (INIS)

Rene, Brigitte; Masliah, Gregoire; Zargarian, Loussine; Mauffret, Olivier; Fermandjian, Serge

2006-01-01

Summary 13 C, 15 N labeling of biomolecules allows easier assignments of NMR resonances and provides a larger number of NMR parameters, which greatly improves the quality of DNA structures. However, there is no general DNA-labeling procedure, like those employed for proteins and RNAs. Here, we describe a general and widely applicable approach designed for preparation of isotopically labeled DNA fragments that can be used for NMR studies. The procedure is based on the PCR amplification of oligonucleotides in the presence of labeled deoxynucleotides triphosphates. It allows great flexibility thanks to insertion of a short DNA sequence (linker) between two repeats of DNA sequence to study. Size and sequence of the linker are designed as to create restriction sites at the junctions with DNA of interest. DNA duplex with desired sequence and size is released upon enzymatic digestion of the PCR product. The suitability of the procedure is validated through the preparation of two biological relevant DNA fragments

A simple non-enzymatic method for the preparation of white spot syndrome virus (WSSV) DNA from the haemolymph of Marsupenaeus japonicus using FTA matrix cards.

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Sudhakaran, R; Mekata, T; Kono, T; Supamattaya, K; Linh, N T H; Suzuki, Y; Sakai, M; Itami, T

2009-07-01

White spot syndrome virus (WSSV) is an important shrimp pathogen responsible for large economic losses for the shrimp culture industry worldwide. The nucleic acids of the virus must be adequately preserved and transported from the field to the laboratory before molecular diagnostic analysis is performed. Here, we developed a new method to isolate WSSV-DNA using Flinders Technology Associates filter paper (FTA matrix card; Whatman) without centrifugation or hazardous steps involved. FTA technology is a new method allowing the simple collection, shipment and archiving of nucleic acids from haemolymph samples providing DNA protection against nucleases, oxidation, UV damage, microbial and fungal attack. DNA samples prepared from 10-fold dilutions of moribund shrimp haemolymph using FTA matrix cards were analysed using semi-quantitative and quantitative polymerase chain reaction (PCR) and were compared with two commercially available DNA isolation methods, the blood GenomicPrep Mini Spin Kit (GE Healthcare) and the DNAzol (Invitrogen). Sequence analysis was performed for the DNA samples prepared using the various isolation procedures and no differences in the sequence among these methods were identified. Results based on the initial copy number of DNA prepared from the GenomicPrep Mini Spin Kit are a little more sensitive than the DNA prepared from FTA matrix cards, whereas the DNAzol method is not suitable for blood samples. Our data shows the efficiency of retention capacity of WSSV-DNA samples from impregnated FTA matrix cards. Matrix cards were easy to store and ship for long periods of time. They provide ease of handling and are a reliable alternative for sample collection and for molecular detection and characterization of WSSV isolates.

Two mini-preparation protocols to DNA extraction from plants with ...

African Journals Online (AJOL)

Were standardized two previously reported standard plant DNA extraction methods, but improved them on mini preparations to use the samples for population genetic analysis. The combination of CTAB lysis procedure-solvent extraction and DNA column purification (DNeasy plant mini kit modification) enables a faster and ...

Y-STR analysis on DNA mixture samples--results of a collaborative project of the ENFSI DNA Working Group

DEFF Research Database (Denmark)

Parson, Walther; Niederstätter, Harald; Lindinger, Alexandra

2008-01-01

The ENFSI (European Network of Forensic Science Institutes) DNA Working Group undertook a collaborative project on Y-STR typing of DNA mixture samples that were centrally prepared and thoroughly tested prior to the shipment. Four commercial Y-STR typing kits (Y-Filer, Applied Biosystems, Foster C...... a laboratory-specific optimization process is indicated to reach a comparable sensitivity for the analysis of minute amounts of DNA....

Preparation of DNA from cytological material: effects of fixation, staining, and mounting medium on DNA yield and quality.

Science.gov (United States)

Dejmek, Annika; Zendehrokh, Nooreldin; Tomaszewska, Malgorzata; Edsjö, Anders

2013-07-01

Personalized oncology requires molecular analysis of tumor cells. Several studies have demonstrated that cytological material is suitable for DNA analysis, but to the authors' knowledge there are no systematic studies comparing how the yield and quality of extracted DNA is affected by the various techniques used for the preparation of cytological material. DNA yield and quality were compared using cultured human lung cancer cells subjected to different preparation techniques used in routine cytology, including fixation, mounting medium, and staining. The results were compared with the outcome of epidermal growth factor receptor (EGFR) genotyping of 66 clinical cytological samples using the same DNA preparation protocol. All tested protocol combinations resulted in fragment lengths of at least 388 base pairs. The mounting agent EcoMount resulted in higher yields than traditional xylene-based medium. Spray and ethanol fixation resulted in both a higher yield and better DNA quality than air drying. In liquid-based cytology (LBC) methods, CytoLyt solution resulted in a 5-fold higher yield than CytoRich Red. Papanicolaou staining provided twice the yield of hematoxylin and eosin staining in both liquid-based preparations. Genotyping outcome and quality control values from the clinical EGFR genotyping demonstrated a sufficient amount and amplifiability of DNA in both spray-fixed and air-dried cytological samples. Reliable clinical genotyping can be performed using all tested methods. However, in the cell line experiments, spray- or ethanol-fixed, Papanicolaou-stained slides provided the best results in terms of yield and fragment length. In LBC, the DNA recovery efficiency of the preserving medium may differ considerably, which should be taken into consideration when introducing LBC. Cancer (Cancer Cytopathol) 2013;121:344-353. © 2013 American Cancer Society. © 2013 American Cancer Society.

Data of self-made Taq DNA polymerase prepared for screening purposes

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E.V. Konovalova

2017-04-01

Full Text Available DNA analysis is a key procedure in genetic engineering. Nowadays the analysis is often done by PCR with Taq DNA polymerase. Although the last enzyme price is quite low, demand for numerous analyses results in much money expenditure which are not affordable for many laboratories. In a meanwhile, many screening tasks do not require the highly purified enzyme. Taking into account the enzyme unique properties it makes possible to marginally simplify its production without resorting to costly or lengthy techniques such as column chromatography and/or dialysis. Here the data of routine usage of Taq DNA polymerase prepared according to the protocol developed in our laboratory is presented. The protocol takes only several hours to realize and does not need qualified personnel or expensive equipment. Yet it gives the enzyme preparation suitable for most screening purposes. The isolated Taq DNA polymerase stock can be stored as ammonium sulfate suspension in a refrigerator for prolonged period, not less than 6 months. The working enzyme solution is prepared from the stock suspension on demand, not more than once in a month and can be stored also in a refrigerator.

DNA barcoding of commercially important catfishes in the Philippines.

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Quilang, Jonas P; Yu, Shiny Cathlynne S

2015-06-01

Many species of catfish are important resources for human consumption, for sport fishing and for use in aquarium industry. In the Philippines, some species are cultivated and some are caught in the wild for food and a few introduced species have become invasive. In this study, DNA barcoding using the mitochondrial cytochrome c oxidase I (COI) gene was done on commercially and economically important Philippine catfishes. A total of 75 specimens belonging to 11 species and 5 families were DNA barcoded. The genetic distances were computed and Neighbor-Joining (NJ) trees were constructed based on the Kimura 2-Parameter (K2P) method. The average K2P distances within species, genus, family and order were 0.2, 8.2, 12.7 and 21.9%, respectively. COI sequences clustered according to their species designation for 7 of the 11 catfishes. DNA barcoding was not able to discriminate between Arius dispar and A. manillensis and between Pterygoplichthys disjunctivus and P. pardalis. The morphological characters that are used to distinguish between these species do not complement molecular identification through DNA barcoding. DNA barcoding also showed that Clarias batrachus from the Philippines is different from the species found in India and Thailand, which supports earlier suggestions based on morphology that those found in India should be designated as C. magur and those in mainland Southeast Asia as C. aff. batrachus "Indochina". This study has shown that DNA barcoding can be used for species delineation and for tagging some species for further taxonomic investigation, which has implications on proper management and conservation strategies.

The effects of commercial preparations of herbal supplements commonly used by women on the biotransformation of fluorogenic substrates by human cytochromes P450.

Science.gov (United States)

Ho, Shirley H Y; Singh, Mohini; Holloway, Alison C; Crankshaw, Denis J

2011-07-01

The study set out to determine the potential for commercially available preparations of black cohosh (Actaea racemosa), chaste tree berry (Vitex agnus-castus), crampbark (Viburnum opulus) and false unicorn (Chamaelirium luteum) to inhibit the major human drug metabolizing enzymes CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4 as well as CYP1A1 which activates some carcinogens. In vitro microplate-based assays using cDNA-expressed CYP450 isoforms and fluorogenic substrates were used. Components of the commercial herbal preparations interfered with the assays and limited the concentration ranges that could be tested. Nevertheless, the fluorogenic assays were robust, reproducible and easy to perform and thus are still useful for initial screening for potential herb-drug interactions. None of the preparations affected CYPs 1A1 or 2C9 at the concentrations tested but all preparations inhibited some of the enzymes with potencies around 1 μg/mL. The three most potent interactions were: chaste tree berry and CYP2C19 (IC₅₀) 0.22 μg/mL); chaste tree berry and CYP3A4 (IC₅₀) 0.3 μg/mL); black cohosh and CYP2C19 (IC₅₀) 0.37 μg/mL,). Thus, the study successfully identified the potential for the commercial herbal preparations to inhibit human drug metabolizing enzymes. Whether this potential translates into clinically significant herb-drug interactions can only be confirmed by appropriate in vivo studies. Copyright © 2011 John Wiley & Sons, Ltd.

OUTLINE FOR OCCUPATIONAL HOME ECONOMICS COURSE IN COMMERCIAL AND INSTITUTIONAL FOOD PREPARATION.

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Alabama State Dept. of Education, Montgomery. Home Economics Service.

THE EXPERIMENTAL OUTLINE IS FOR TEACHER USE IN PLANNING A TWO-SEMESTER COURSE TO PREPARE 11TH AND 12TH GRADE STUDENTS FOR ENTRY LEVEL COMMERCIAL FOOD PREPARATION JOBS SUCH AS FOOD SERVICE WORKERS, COOK HELPERS, CATERER HELPERS, SALAD MAKERS, BAKER HELPERS, SHORT ORDER COOKS, AND TRAY LINE WORKERS. IT WAS DEVELOPED BY VOCATIONAL HOME ECONOMICS…

DIFFERENCES IN CULTURAL YIELD OF MYCOBACTERIUM TUBERCULOSIS ON MEDIA PREPARED USING COMMERCIAL AND HOUSEHOLD EGGS.

Science.gov (United States)

Noori, Muhammad Yahya; AJi, Zaheer; Khan, Ghazala; Sharafat, Shaheen; Masroor, Muhammad

2015-01-01

Mycobacterial culture is considered as the gold standard for TB diagnosis. It is performed on egg-based media using commercially available eggs to grow Mycobacteria from clinical samples. These eggs are known to contain high concentration of antibiotics, including fluoroquinolones, given to chicken to prevent early mortality. This study was performed to compare Mycobacterial growth on media prepared from commercial and antibiotic free household eggs. Sputum samples from negative (No bacilli in 100 oil immersion field), scanty (1-9 AFB in 100 fields), 1+ (10-99 bacilli per field), 2+ (1-10 bacilli per field) and 3+ (>10 bacilli per field) were inoculated dually on Ogawa medium prepared from commercial and household eggs. Tubes were inspected every fourth day for the appearance of colonies till 60 days. Data tabulations and statistical analysis (F test for variation and unpaired Student's t test) were performed on Microsoft Excel. One microscopically negative sample showed growth on media prepared from household eggs, while all were negative on that prepared from commercial eggs. There were significant differences in time to culture positivity for samples graded 1+ (p = 0.02), 2+ (p = 0.002) and 3+ (p = 0.0003). Commercial eggs containing antibiotics can be a source of false negativity in cultures especially in microscopically negative samples. This can be of special concern in HIV patients who have high smear negativity. It is therefore important to either develop provision of antibiotic free eggs for media preparation or to develop and validate other laboratory investigations for smear negative TB patients.

Differences in cultural yield of mycobacterium tuberculosis on media prepared using commercial and household eggs

International Nuclear Information System (INIS)

Noori, M.Y.; Khan, G.

2015-01-01

Mycobacterial culture is considered as the gold standard for TB diagnosis. It is performed on egg-based media using commercially available eggs to grow Mycobacteria from clinical samples. These eggs are known to contain high concentration of antibiotics, including fluoroquinolones, given to chicken to prevent early mortality. This study was performed to compare Mycobacterial growth on media prepared from commercial and antibiotic free household eggs. Methods: Sputum samples from negative (No bacilli in 100 oil immersion field), scanty (1-9 AFB in 100 fields), 1+ (10-99 bacilli per field), 2+ (1-10 bacilli per field) and 3+ (>10 bacilli per field) were inoculated dually on Ogawa medium prepared from commercial and household eggs. Tubes were inspected every fourth day for the appearance of colonies till 60 days. Data tabulations and statistical analysis (F test for variation and unpaired Student's t test) were performed on Microsoft Excel. Results: One microscopically negative sample showed growth on media prepared from household eggs, while all were negative on that prepared from commercial eggs. There were significant differences in time to culture positivity for samples graded 1+ (p=0.02), 2+ (p=0.002) and 3+ (p=0.0003). Conclusion: Commercial eggs containing antibiotics can be a source of false negativity in cultures especially in microscopically negative samples. This can be of special concern in HIV patients who have high smear negativity. It is therefore important to either develop provision of antibiotic free eggs for media preparation or to develop and validate other laboratory investigations for smear negative TB patients. (author)

COMPARISON OF COMMERCIAL DNA KITS AND TRADITIONAL DNA EXTRACTION PROCEDURE IN PCR DETECTION OF PORK IN DRY/FERMENTED SAUSAGES

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Ivona Djurkin Kušec

2015-09-01

Full Text Available In the present study four commercially available DNA extraction kits (Wizard® Genomic DNA Purification Kit, High Pure PCR Template Kit, DNeasy mericon Food and GeneJET PCR Purification Kit, as well as standard phenol/chloroform isolation technique have been evaluated regarding their concentration, purity and suitability for amplification of porcine DNA in dry/fermented sausages. The isolates were assessed for quantity and quality using spectrophotometer (IMPLEN GmbH, Germany. To verify template usability and quality of isolated DNA, the polymerase chain reaction (PCR targeting at porcine cytochrome b by species specific primers was used. The comparison of extraction methods revealed satisfactory efficiency and purity of all extraction kits, while with standard phenol/chloroform isolation method high concentrations of DNA with low A260/280 were obtained. However, all the investigated techniques proved to be suitable for identification of porcine DNA in dry/fermented sausage. Thus, the standard phenol/chloroform DNA extraction method, as the cost-effective one, can be recommended as a good alternative to more expensive isolation kits when investigating the presence of pork DNA in dry/ fermented meat products.

Facile preparation of a DNA sensor for rapid herpes virus detection

Energy Technology Data Exchange (ETDEWEB)

Tam, Phuong Dinh, E-mail: tampd-hast@mail.hut.edu.vn [Hanoi Advanced School of Science and Technology, Hanoi University of Technology (Viet Nam); Tuan, Mai Anh, E-mail: tuanma-itims@mail.hut.edu.vn [International Training Institute for Materials Science, Hanoi University of Technology (Viet Nam); Huy, Tran Quang [National Institute of Hygiene and Epidemiology (NIHE), 01 Yersin, Hai Ba Trung District, Hanoi (Viet Nam); Le, Anh-Tuan [Hanoi Advanced School of Science and Technology, Hanoi University of Technology (Viet Nam); Hieu, Nguyen Van, E-mail: hieu@itims.edu.vn [International Training Institute for Materials Science, Hanoi University of Technology (Viet Nam)

2010-10-12

In this paper, a simple DNA sensor platform was developed for rapid herpes virus detection in real samples. The deoxyribonucleic acid (DNA) sequences of the herpes simplex virus (DNA probe) were directly immobilized on the surface of interdigitated electrodes by electrochemical polymerization along with pyrrole monomers. The potential was scanned from - 0.7 to + 0.6 V, and the scanning rate was 100 mV/s. Fourier transform infrared spectroscopy was employed to verify specific DNA sequence binding and the conducting polymer. The morphology of the conducting polymer doped with DNA strands was characterized using a field emission scanning electron microscope. As-obtained DNA sensor was used to detect the herpes virus DNA in the real samples. The results show that the current DNA sensors detected the lowest DNA concentration of 2 nM. This sensitivity appears to be better than that of the DNA sensors prepared by immobilization of the DNA probe on the 3-aminopropyl-triethoxy-silance (APTS) membrane.

Facile preparation of a DNA sensor for rapid herpes virus detection

International Nuclear Information System (INIS)

Tam, Phuong Dinh; Tuan, Mai Anh; Huy, Tran Quang; Le, Anh-Tuan; Hieu, Nguyen Van

2010-01-01

In this paper, a simple DNA sensor platform was developed for rapid herpes virus detection in real samples. The deoxyribonucleic acid (DNA) sequences of the herpes simplex virus (DNA probe) were directly immobilized on the surface of interdigitated electrodes by electrochemical polymerization along with pyrrole monomers. The potential was scanned from - 0.7 to + 0.6 V, and the scanning rate was 100 mV/s. Fourier transform infrared spectroscopy was employed to verify specific DNA sequence binding and the conducting polymer. The morphology of the conducting polymer doped with DNA strands was characterized using a field emission scanning electron microscope. As-obtained DNA sensor was used to detect the herpes virus DNA in the real samples. The results show that the current DNA sensors detected the lowest DNA concentration of 2 nM. This sensitivity appears to be better than that of the DNA sensors prepared by immobilization of the DNA probe on the 3-aminopropyl-triethoxy-silance (APTS) membrane.

Preparation of fluorescent-dye-labeled cDNA from RNA for microarray hybridization.

Science.gov (United States)

Ares, Manuel

2014-01-01

This protocol describes how to prepare fluorescently labeled cDNA for hybridization to microarrays. It consists of two steps: first, a mixture of anchored oligo(dT) and random hexamers is used to prime amine-modified cDNA synthesis by reverse transcriptase using a modified deoxynucleotide with a reactive amine group (aminoallyl-dUTP) and an RNA sample as a template. Second, the cDNA is purified and exchanged into bicarbonate buffer so that the amine groups in the cDNA react with the dye N-hydroxysuccinimide (NHS) esters, covalently joining the dye to the cDNA. The dye-coupled cDNA is purified again, and the amount of dye incorporated per microgram of cDNA is determined.

DNA Barcoding as a Reliable Method for the Authentication of Commercial Seafood Products

Directory of Open Access Journals (Sweden)

Silvia Nicolè

2012-01-01

Full Text Available Animal DNA barcoding allows researchers to identify different species by analyzing a short nucleotide sequence, typically the mitochondrial gene cox1. In this paper, we use DNA barcoding to genetically identify seafood samples that were purchased from various locations throughout Italy. We adopted a multi-locus approach to analyze the cob, 16S-rDNA and cox1 genes, and compared our sequences to reference sequences in the BOLD and GenBank online databases. Our method is a rapid and robust technique that can be used to genetically identify crustaceans, mollusks and fishes. This approach could be applied in the future for conservation, particularly for monitoring illegal trade of protected and endangered species. Additionally, this method could be used for authentication in order to detect mislabeling of commercially processed seafood.

DNA Modified with Metal Nanoparticles: Preparation and Characterization of Ordered Metal-DNA Nanostructures in a Solution and on a Substrate

Directory of Open Access Journals (Sweden)

Nina Kasyanenko

2016-01-01

Full Text Available DNA interaction with silver and aluminum nanoparticles in a solution has been investigated with the AFM, SEM, dynamic light scattering, viscometry, and spectral methods. The comparison of DNA interaction with nanoparticles synthesized by the reduction of Ag+ ions and with nanoparticles obtained by the electric discharge plasma method was done. DNA metallization in a solution and on n-silicon surface with metal nanoparticles or by the reduction of silver ions after their binding to DNA was executed and studied. It was shown that DNA strands with regular location of silver or aluminum nanoparticles can be prepared. The conditions for the formation of silver nanoparticles and silver nanoclusters on DNA were analyzed.

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

Science.gov (United States)

Sakalar, Ergün

2013-11-15

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

Chromosome-specific DNA Repeat Probes

Energy Technology Data Exchange (ETDEWEB)

Baumgartner, Adolf; Weier, Jingly Fung; Weier, Heinz-Ulrich G.

2006-03-16

In research as well as in clinical applications, fluorescence in situ hybridization (FISH) has gained increasing popularity as a highly sensitive technique to study cytogenetic changes. Today, hundreds of commercially available DNA probes serve the basic needs of the biomedical research community. Widespread applications, however, are often limited by the lack of appropriately labeled, specific nucleic acid probes. We describe two approaches for an expeditious preparation of chromosome-specific DNAs and the subsequent probe labeling with reporter molecules of choice. The described techniques allow the preparation of highly specific DNA repeat probes suitable for enumeration of chromosomes in interphase cell nuclei or tissue sections. In addition, there is no need for chromosome enrichment by flow cytometry and sorting or molecular cloning. Our PCR-based method uses either bacterial artificial chromosomes or human genomic DNA as templates with {alpha}-satellite-specific primers. Here we demonstrate the production of fluorochrome-labeled DNA repeat probes specific for human chromosomes 17 and 18 in just a few days without the need for highly specialized equipment and without the limitation to only a few fluorochrome labels.

Salt and fat contents in preparations at commercial restaurants in Goiânia-GO

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Camila Silva Kunert

2013-03-01

Full Text Available Objective: To evaluate the sodium and fat contents added to preparations of commercial restaurants in Goiânia-GO, Brazil. Methods: This was an observational, cross-sectional and descriptive study. It included ‘pay-per-weight’ restaurants with a medium standard menu and having as daily preparations white rice, beans and grilled chicken. Among the establishments with these characteristics, three agreed to participate. The production process of the above-mentioned preparations was accompanied for three non-consecutive days in each establishment. For quantification of sodium and fat added into the preparations, oil and salt were weighed, as well as the finished preparation; the weight of the standard portion and the yield of the preparation expressed in number of portions prepared were settled. From these data, the per capita amount of salt and oil added to cook one portion of each kind of preparation was calculated by dividing the total quantity of salt and oil by the number of prepared portions. Results: The levels of salt (3.0, 2.7, and 4.1 g – restaurant A, B and C, respectively and oil (17.0, 11.3, and 11.2 g – restaurant A, B and C, respectively added in the three preparations are superior to the recommendations. Conclusion: The sodium and fat contents in the analyzed restaurants are higher than it is recommended by the Food Guide for the Brazilian Population. It is essential that commercial restaurants become partners of public policies on health promotion, adopting good nutritional practices, by reducing the sodium and fat contents, to offer healthy meals daily.

Adsorption of β-glucosidases in two commercial preparations onto pretreated biomass and lignin

DEFF Research Database (Denmark)

Haven, Mai Østergaard; Jørgensen, Henning

2013-01-01

adsorbed strongly to lignin.The extent of adsorption of β-glucosidase from Cellic® CTec2 was affected by both type of biomass and pretreatment method. With approximately 65% of the β-glucosidases from Cellic® CTec2 adsorbed onto lignin from pretreated wheat straw, the activity of the β......Background: Enzyme recycling is a method to reduce the production costs for advanced bioethanol by lowering the overall use of enzymes. Commercial cellulase preparations consist of many different enzymes that are important for efficient and complete cellulose (and hemicellulose) hydrolysis...... commercial preparations (Novozym 188 and Cellic® CTec2) to substrates mimicking the components in pretreated wheat straw revealed that the Aspergillus niger β-glucosidase in Novozym 188 did not adsorb significantly to any of the components in pretreated wheat straw, whereas the β-glucosidase in Cellic® CTec2...

Diversity and dynamics of the DNA- and cDNA-derived compost fungal communities throughout the commercial cultivation process for Agaricus bisporus.

Science.gov (United States)

McGee, C F; Byrne, H; Irvine, A; Wilson, J

2017-01-01

Commercial cultivation of the button mushroom Agaricus bisporus is performed through the inoculation of a semipasteurized composted material. Pasteurization of the compost material prior to inoculation results in a substrate with a fungal community that becomes dominated by A. bisporus. However, little is known about the composition and activity in the wider fungal community beyond the presence of A. bisporus in compost throughout the mushroom cropping process. In this study, the fungal cropping compost community was characterized by sequencing nuc rDNA ITS1-5.8S-ITS2 amplified from extractable DNA and RNA. The fungal community generated from DNA extracts identified a diverse community containing 211 unique species, although only 51 were identified from cDNA. Agaricus bisporus was found to dominate in the DNA-derived fungal community for the duration of the cropping process. However, analysis of cDNA extracts found A. bisporus to dominate only up to the first crop flush, after which activity decreased sharply and a much broader fungal community became active. This study has highlighted the diverse fungal community that is present in mushroom compost during cropping.

Impact of two different commercial DNA extraction methods on BK virus viral load

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Massimiliano Bergallo

2016-03-01

Full Text Available Background and aim: BK virus, a member of human polyomavirus family, is a worldwide distributed virus characterized by a seroprevalence rate of 70-90% in adult population. Monitoring of viral replication is made by evaluation of BK DNA by quantitative polymerase chain reaction. Many different methods can be applied for extraction of nucleic acid from several specimens. The aim of this study was to assess the impact of two different DNA extraction procedure on BK viral load. Materials and methods: DNA extraction procedure including the Nuclisens easyMAG platform (bioMerieux, Marcy l’Etoile, France and manual QIAGEN extraction (QIAGEN Hilden, Germany. BK DNA quantification was performed by Real Time TaqMan PCR using a commercial kit. Result and discussion: The samples capacity, cost and time spent were compared for both systems. In conclusion our results demonstrate that automated nucleic acid extraction method using Nuclisense easyMAG was superior to manual protocol (QIAGEN Blood Mini kit, for the extraction of BK virus from serum and urine specimens.

Preparation and Characterization of Cellulose Nanofibers from Two Commercial Hardwood and Softwood Pulps

DEFF Research Database (Denmark)

Stelte, Wolfgang; Sanadi, Anand R.

2009-01-01

The aim of this work was to study the mechanical fibrillation process for the preparation of cellulose nanofibers from two commercial hard- and softwood cellulose pulps. The process consisted of initial refining and subsequent high-pressure homogenization. The progress in fibrillation was studied...

Comparative evaluation of three automated systems for DNA extraction in conjunction with three commercially available real-time PCR assays for quantitation of plasma Cytomegalovirus DNAemia in allogeneic stem cell transplant recipients.

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Bravo, Dayana; Clari, María Ángeles; Costa, Elisa; Muñoz-Cobo, Beatriz; Solano, Carlos; José Remigia, María; Navarro, David

2011-08-01

Limited data are available on the performance of different automated extraction platforms and commercially available quantitative real-time PCR (QRT-PCR) methods for the quantitation of cytomegalovirus (CMV) DNA in plasma. We compared the performance characteristics of the Abbott mSample preparation system DNA kit on the m24 SP instrument (Abbott), the High Pure viral nucleic acid kit on the COBAS AmpliPrep system (Roche), and the EZ1 Virus 2.0 kit on the BioRobot EZ1 extraction platform (Qiagen) coupled with the Abbott CMV PCR kit, the LightCycler CMV Quant kit (Roche), and the Q-CMV complete kit (Nanogen), for both plasma specimens from allogeneic stem cell transplant (Allo-SCT) recipients (n = 42) and the OptiQuant CMV DNA panel (AcroMetrix). The EZ1 system displayed the highest extraction efficiency over a wide range of CMV plasma DNA loads, followed by the m24 and the AmpliPrep methods. The Nanogen PCR assay yielded higher mean CMV plasma DNA values than the Abbott and the Roche PCR assays, regardless of the platform used for DNA extraction. Overall, the effects of the extraction method and the QRT-PCR used on CMV plasma DNA load measurements were less pronounced for specimens with high CMV DNA content (>10,000 copies/ml). The performance characteristics of the extraction methods and QRT-PCR assays evaluated herein for clinical samples were extensible at cell-based standards from AcroMetrix. In conclusion, different automated systems are not equally efficient for CMV DNA extraction from plasma specimens, and the plasma CMV DNA loads measured by commercially available QRT-PCRs can differ significantly. The above findings should be taken into consideration for the establishment of cutoff values for the initiation or cessation of preemptive antiviral therapies and for the interpretation of data from clinical studies in the Allo-SCT setting.

Comparison of direct boiling method with commercial kits for extracting fecal microbiome DNA by Illumina sequencing of 16S rRNA tags.

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Peng, Xin; Yu, Ke-Qiang; Deng, Guan-Hua; Jiang, Yun-Xia; Wang, Yu; Zhang, Guo-Xia; Zhou, Hong-Wei

2013-12-01

Low cost and high throughput capacity are major advantages of using next generation sequencing (NGS) techniques to determine metagenomic 16S rRNA tag sequences. These methods have significantly changed our view of microorganisms in the fields of human health and environmental science. However, DNA extraction using commercial kits has shortcomings of high cost and time constraint. In the present study, we evaluated the determination of fecal microbiomes using a direct boiling method compared with 5 different commercial extraction methods, e.g., Qiagen and MO BIO kits. Principal coordinate analysis (PCoA) using UniFrac distances and clustering showed that direct boiling of a wide range of feces concentrations gave a similar pattern of bacterial communities as those obtained from most of the commercial kits, with the exception of the MO BIO method. Fecal concentration by boiling method affected the estimation of α-diversity indices, otherwise results were generally comparable between boiling and commercial methods. The operational taxonomic units (OTUs) determined through direct boiling showed highly consistent frequencies with those determined through most of the commercial methods. Even those for the MO BIO kit were also obtained by the direct boiling method with high confidence. The present study suggested that direct boiling could be used to determine the fecal microbiome and using this method would significantly reduce the cost and improve the efficiency of the sample preparation for studying gut microbiome diversity. © 2013 Elsevier B.V. All rights reserved.

Methodology for sample preparation and size measurement of commercial ZnO nanoparticles

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Pei-Jia Lu

2018-04-01

Full Text Available This study discusses the strategies on sample preparation to acquire images with sufficient quality for size characterization by scanning electron microscope (SEM using two commercial ZnO nanoparticles of different surface properties as a demonstration. The central idea is that micrometer sized aggregates of ZnO in powdered forms need to firstly be broken down to nanosized particles through an appropriate process to generate nanoparticle dispersion before being deposited on a flat surface for SEM observation. Analytical tools such as contact angle, dynamic light scattering and zeta potential have been utilized to optimize the procedure for sample preparation and to check the quality of the results. Meanwhile, measurements of zeta potential values on flat surfaces also provide critical information and save lots of time and efforts in selection of suitable substrate for particles of different properties to be attracted and kept on the surface without further aggregation. This simple, low-cost methodology can be generally applied on size characterization of commercial ZnO nanoparticles with limited information from vendors. Keywords: Zinc oxide, Nanoparticles, Methodology

Epidemiologic Investigation of Riemerella anatipestifer in a Commercial Duck Company by Serotyping and DNA Fingerprinting

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A commercial duck company that raises approximately two million Pekin ducks per year experienced an outbreak of Riemerella anatipestifer(RA)on nine farms over a one year period. Due to concerns that the bacteria was being spread from farm to farm, an investigation using serotyping and DNA fingerprin...

Development of a Novel Self-Enclosed Sample Preparation Device for DNA/RNA Isolation in Space

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Zhang, Ye; Mehta, Satish K.; Pensinger, Stuart J.; Pickering, Karen D.

2011-01-01

Modern biology techniques present potentials for a wide range of molecular, cellular, and biochemistry applications in space, including detection of infectious pathogens and environmental contaminations, monitoring of drug-resistant microbial and dangerous mutations, identification of new phenotypes of microbial and new life species. However, one of the major technological blockades in enabling these technologies in space is a lack of devices for sample preparation in the space environment. To overcome such an obstacle, we constructed a prototype of a DNA/RNA isolation device based on our novel designs documented in the NASA New Technology Reporting System (MSC-24811-1/3-1). This device is self-enclosed and pipette free, purposely designed for use in the absence of gravity. Our design can also be modified easily for preparing samples in space for other applications, such as flowcytometry, immunostaining, cell separation, sample purification and separation according to its size and charges, sample chemical labeling, and sample purification. The prototype of our DNA/RNA isolation device was tested for efficiencies of DNA and RNA isolation from various cell types for PCR analysis. The purity and integrity of purified DNA and RNA were determined as well. Results showed that our developed DNA/RNA isolation device offers similar efficiency and quality in comparison to the samples prepared using the standard protocol in the laboratory.

CGH and SNP array using DNA extracted from fixed cytogenetic preparations and long-term refrigerated bone marrow specimens

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MacKinnon Ruth N

2012-02-01

Full Text Available Abstract Background The analysis of nucleic acids is limited by the availability of archival specimens and the quality and amount of the extracted material. Archived cytogenetic preparations are stored in many laboratories and are a potential source of total genomic DNA for array karyotyping and other applications. Array CGH using DNA from fixed cytogenetic preparations has been described, but it is not known whether it can be used for SNP arrays. Diagnostic bone marrow specimens taken during the assessment of hematological malignancies are also a potential source of DNA, but it is generally assumed that DNA must be extracted, or the specimen frozen, within a day or two of collection, to obtain DNA suitable for further analysis. We have assessed DNA extracted from these materials for both SNP array and array CGH. Results We show that both SNP array and array CGH can be performed on genomic DNA extracted from cytogenetic specimens stored in Carnoy's fixative, and from bone marrow which has been stored unfrozen, at 4°C, for at least 36 days. We describe a procedure for extracting a usable concentration of total genomic DNA from cytogenetic suspensions of low cellularity. Conclusions The ability to use these archival specimens for DNA-based analysis increases the potential for retrospective genetic analysis of clinical specimens. Fixed cytogenetic preparations and long-term refrigerated bone marrow both provide DNA suitable for array karyotyping, and may be suitable for a wider range of analytical procedures.

Standard assays do not predict the efficiency of commercial cellulase preparations towards plant materials

NARCIS (Netherlands)

Kabel, M.A.; Maarel, van der M.J.E.C.; Klip, G.; Voragen, A.G.J.; Schols, H.A.

2006-01-01

Commercial cellulase preparations are potentially effective for processing biomass feedstocks in order to obtain bioethanol. In plant cell walls, cellulose fibrils occur in close association with xylans (monocotyls) or xyloglucans (dicotyls). The enzymatic conversion of cellulose/xylans is a complex

Standard Assays Do Not Predict the Efficiency of Commercial Cellulase Preparations Towards Plant Materials

NARCIS (Netherlands)

Kabel, Mirjam A.; Maarel, Marc J.E.C. van der; Klip, Gert; Voragen, Alphons G.J.; Schols, Henk A.

2006-01-01

Commercial cellulase preparations are potentially effective for processing biomass feedstocks in order to obtain bioethanol. In plant cell walls, cellulose fibrils occur in close association with xylans (monocotyls) or xyloglucans (dicotyls). The enzymatic conversion of cellulose/xylans is a complex

Mineralization by mesenchymal stromal cells is variously modulated depending on commercial platelet lysate preparations.

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Boraldi, Federica; Burns, Jorge S; Bartolomeo, Angelica; Dominici, Massimo; Quaglino, Daniela

2018-03-01

Numerous cellular models have been developed to investigate calcification for regenerative medicine applications and for the identification of therapeutic targets in various complications associated with age-related diseases. However, results have often been contradictory due to specific culture conditions, cell type ontogeny and aging status. Human platelet lysate (hPL) has been recently investigated as valuable alternative to fetal bovine serum (FBS) in cell culture and bone regeneration. A parallel comparison of how all these multiple factors may converge to influence mineralization has yet to be reported. To compare mineralization of human mesenchymal cell types known to differ in extracellular matrix calcification potency, bone marrow-derived mesenchymal stromal cells and dermal fibroblasts from neonatal and adult donors, at both low and high passages, were investigated in an ex vivo experimental model by supplementing the osteogenic induction medium with FBS or with hPL. Four commercial hPL preparations were profiled by liquid chromatography/electrospray ionization quadrupole time-of-flight spectrometry, and mineralization was visualized by von Kossa staining and quantified by morphometric evaluations after 9, 14 and 21 days of culture. Data demonstrate that (i) commercial hPL preparations differ according to mass spectra profiles, (ii) hPL variously influences mineral deposition depending on cell line and possibly on platelet product preparation methods, (iii) donor age modifies mineral deposition in the presence of the same hPL and (iv) reduced in vitro proliferative capacity affects osteogenic induction and response to hPL. Despite the standardized procedures applied to obtain commercial hPL, this study highlights the divergent effects of different preparations and emphasizes the importance of cellular ontology, donor age and cell proliferative capacity to optimize the osteogenic induction capabilities of mesenchymal stromal cells and design more effective

Different Analytical Approaches in Assessing Antibacterial Activity and the Purity of Commercial Lysozyme Preparations for Dairy Application

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Luisa Pellegrino

2013-05-01

Full Text Available Hen egg-white lysozyme (LSZ is currently used in the food industry to limit the proliferation of lactic acid bacteria spoilage in the production of wine and beer, and to inhibit butyric acid fermentation in hard and extra hard cheeses (late blowing caused by the outgrowth of clostridial spores. The aim of this work was to evaluate how the enzyme activity in commercial preparations correlates to the enzyme concentration and can be affected by the presence of process-related impurities. Different analytical approaches, including turbidimetric assay, SDS-PAGE and HPLC were used to analyse 17 commercial preparations of LSZ marketed in different countries. The HPLC method adopted by ISO allowed the true LSZ concentration to be determined with accuracy. The turbidimetric assay was the most suitable method to evaluate LSZ activity, whereas SDS-PAGE allowed the presence of other egg proteins, which are potential allergens, to be detected. The analytical results showed that the purity of commercially available enzyme preparations can vary significantly, and evidenced the effectiveness of combining different analytical approaches in this type of control.

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

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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.

Comparison of two commercial DNA extraction kits for the analysis of nasopharyngeal bacterial communities

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Keith A. Crandall

2016-04-01

Full Text Available Characterization of microbial communities via next-generation sequencing (NGS requires an extraction ofmicrobial DNA. Methodological differences in DNA extraction protocols may bias results and complicate inter-study comparisons. Here we compare the effect of two commonly used commercial kits (Norgen and Qiagenfor the extraction of total DNA on estimatingnasopharyngeal microbiome diversity. The nasopharynxis a reservoir for pathogens associated with respiratory illnesses and a key player in understandingairway microbial dynamics. Total DNA from nasal washes corresponding to 30 asthmatic children was extracted using theQiagenQIAamp DNA and NorgenRNA/DNA Purification kits and analyzed via IlluminaMiSeq16S rRNA V4 ampliconsequencing. The Norgen samples included more sequence reads and OTUs per sample than the Qiagen samples, but OTU counts per sample varied proportionallybetween groups (r = 0.732.Microbial profiles varied slightly between sample pairs, but alpha- and beta-diversity indices (PCoAand clustering showed highsimilarity between Norgen and Qiagenmicrobiomes. Moreover, no significant differences in community structure (PERMANOVA and adonis tests and taxa proportions (Kruskal-Wallis test were observed betweenkits. Finally, aProcrustes analysis also showed low dissimilarity (M2 = 0.173; P< 0.001 between the PCoAs of the two DNA extraction kits. Contrary to what has been observed in previous studies comparing DNA extraction methods, our 16S NGS analysis of nasopharyngeal washes did not reveal significant differences in community composition or structure between kits. Our findingssuggest congruence between column-based chromatography kits and supportthe comparison of microbiomeprofilesacross nasopharyngeal metataxonomic studies.

Using a commercially available DNA extraction kit to obtain high quality human genomic DNA suitable for PCR and genotyping from 11-year-old saliva saturated cotton spit wads

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Hudziak James J

2008-12-01

Full Text Available Abstract Background We sought to describe the integrity of human genomic DNA extracted from saliva saturated cotton spit wads stored at -20°C for approximately 11 years. 783 spit wad samples were collected from an ADHD sample population (Vermont Family Study during 1996–2000. Human genomic DNA was extracted from the spit wads using a commercially available kit; QIAamp DNA Blood Midi Kit (Qiagen, Inc., Valencia, CA. with a few modifications. Results The resulting DNA yield was more than adequate for genetic analysis and ranged from approximately 1 μg to a total of 80 μg (mean 17.3 μgs ± 11.9 μgs. A260/A280 ratios for the human genomic DNA extracted from the spit wads was consistently within the generally acceptable values of 1.7–2.0, with the lowest purity being 1.70, and a mean value of 1.937 ± 0.226 for the 783 samples. The DNA also was suitable for PCR reactions as evidenced by the amplification of the serotonin-transporter-linked polymorphic region, 5HTTLPR. 5HTTLPR is a functional polymorphism in the promoter region of the serotonin transporter gene (HTT, SLC6A4, or SERT, consisting of two intensively studied alleles. 770 of the 783 samples (98.3% produced fragments after PCR of the expected size with primers specific for 5HTTLPR. Conclusion High quality and abundant genomic DNA can be successfully retrieved from saliva saturated cotton spit wads using the commercially available kit, QIAamp DNA Blood Midi Kit from Qiagen, Inc. Furthermore, the DNA can be extracted in less than 3 hours and multiple samples can be processed simultaneously thus reducing processing time.

Novel PVA-DNA nanoparticles prepared by ultra high pressure technology for gene delivery

International Nuclear Information System (INIS)

Kimura, Tsuyoshi; Okuno, Akira; Miyazaki, Kozo; Furuzono, Tsutomu; Ohya, Yuichi; Ouchi, Tatsuro; Mutsuo, Shingo; Yoshizawa, Hidekazu; Kitamura, Yoshiro; Fujisato, Toshiyta; Kishida, Akio

2004-01-01

Polyvinyl alcohol (PVA)-DNA nanoparticles have been developed by ultra high pressure (UHP) technology. Mixture solutions of DNA and PVA having various molecular weights (Mw) and degree of saponifications (DS) were treated under 10,000 atmospheres (981 MPa) condition at 40 deg. C for 10 min. Agarose gel electrophoresis and scanning electron microscope observation revealed that the PVA-DNA nanoparticles with average diameter of about 200 nm were formed. Using PVA of higher Mw and degree of saponifications, the amount of nanoparticles formed increased. The driving force of nanoparticle formation was the hydrogen bonding between DNA and PVA. In order to apply the PVA-DNA nanoparticles for gene delivery, the cytotoxicity and the cellular uptake of them were investigated using Raw264 cell lines. The cell viability was not influenced whether the presence of the PVA-DNA nanoparticles. Further, the nanoparticles internalized into cells were observed by fluorescent microscope. These results indicates that the PVA-DNA nanoparticles prepared by UHP technology showed be useful as drug carrier, especially for gene delivery

The effect of swim-up and gradient sperm preparation techniques on deoxyribonucleic acid (DNA) fragmentation in subfertile patients.

Science.gov (United States)

Oguz, Yuksel; Guler, Ismail; Erdem, Ahmet; Mutlu, Mehmet Firat; Gumuslu, Seyhan; Oktem, Mesut; Bozkurt, Nuray; Erdem, Mehmet

2018-03-23

To compare the effect of two different sperm preparation techniques, including swim-up and gradient methods on sperm deoxyribonucleic acid (DNA) fragmentation status of semen samples from unexplained and mild male factor subfertile patients undergoing intrauterine insemination (IUI). A prospective randomized study was conducted in 65 subfertile patients, including 34 unexplained and 31 male factor infertility to compare basal and post-procedure DNA fragmentation rates in swim-up and gradient techniques. Sperm DNA fragmentation rates were evaluated by a sperm chromatin dispersion (SCD) test in two portions of each sample of semen that was prepared with either swim-up or gradient techniques. Sperm motility and morphology were also assessed based on WHO 2010 criteria. Swim-up but not gradient method yielded a statistically significant reduction in the DNA fragmented sperm rate after preparation as compared to basal rates, in the semen samples of both unexplained (41.85 ± 22.04 vs. 28.58 ± 21.93, p gradient) and mild male factor (46.61 ± 19.38 vs. 30.32 ± 18.20, p gradient) subgroups. Swim-up method significantly reduces sperm DNA fragmentation rates and may have some prognostic value on intrauterine insemination in patients with decreased sperm DNA integrity.

A rapid and economic in-house DNA purification method using glass syringe filters.

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Yun-Cheol Kim

Full Text Available BACKGROUND: Purity, yield, speed and cost are important considerations in plasmid purification, but it is difficult to achieve all of these at the same time. Currently, there are many protocols and kits for DNA purification, however none maximize all four considerations. METHODOLOGY/PRINCIPAL FINDINGS: We now describe a fast, efficient and economic in-house protocol for plasmid preparation using glass syringe filters. Plasmid yield and quality as determined by enzyme digestion and transfection efficiency were equivalent to the expensive commercial kits. Importantly, the time required for purification was much less than that required using a commercial kit. CONCLUSIONS/SIGNIFICANCE: This method provides DNA yield and quality similar to that obtained with commercial kits, but is more rapid and less costly.

DNA barcoding reveals cryptic diversity within commercially exploited Indo-Malay Carangidae (Teleosteii: Perciformes.

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Tun Nurul Aimi Mat Jaafar

Full Text Available BACKGROUND: DNA barcodes, typically focusing on the cytochrome oxidase I gene (COI in many animals, have been used widely as a species-identification tool. The ability of DNA barcoding to distinguish species from a range of taxa and to reveal cryptic species has been well documented. Despite the wealth of DNA barcode data for fish from many temperate regions, there are relatively few available from the Southeast Asian region. Here, we target the marine fish Family Carangidae, one of the most commercially-important families from the Indo-Malay Archipelago (IMA, to produce an initial reference DNA barcode library. METHODOLOGY/PRINCIPAL FINDINGS: Here, a 652 bp region of COI was sequenced for 723 individuals from 36 putative species of Family Carangidae distributed within IMA waters. Within the newly-generated dataset, three described species exhibited conspecific divergences up to ten times greater (4.32-4.82% than mean estimates (0.24-0.39%, indicating a discrepancy with assigned morphological taxonomic identification, and the existence of cryptic species. Variability of the mitochondrial DNA COI region was compared within and among species to evaluate the COI region's suitability for species identification. The trend in range of mean K2P distances observed was generally in accordance with expectations based on taxonomic hierarchy: 0% to 4.82% between individuals within species, 0% to 16.4% between species within genera, and 8.64% to 25.39% between genera within families. The average Kimura 2-parameter (K2P distance between individuals, between species within genera, and between genera within family were 0.37%, 10.53% and 16.56%, respectively. All described species formed monophyletic clusters in the Neighbour-joining phylogenetic tree, although three species representing complexes of six potential cryptic species were detected in Indo-Malay Carangidae; Atule mate, Selar crumenophthalmus and Seriolina nigrofasciata. CONCLUSION/SIGNIFICANCE: This

Energy intake from commercially-prepared meals by food source in Korean adults: Analysis of the 2001 and 2011 Korea National Health and Nutrition Examination Surveys

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Choi, Injoo; Kim, Won Gyoung

2017-01-01

BACKGROUND/OBJECTIVES The commercial foodservice industry in Korea has shown rapid growth recently. This study examined Korean adults' consumption of commercially-prepared meals based on where the food was prepared. SUBJECTS/METHODS Data from a 24-hour dietary recall of the 2001 and 2011 Korea National Health and Nutrition Examination Surveys were analyzed. A total of 10,539 subjects (n = 6,152 in 2001; n = 4,387 in 2011) aged 19-64 years were included for analysis. Commercially-prepared meals were classified into four food source groups based on where the food was prepared: Korean restaurants, Chinese/Western/Japanese restaurants, fast-food restaurants, and retail stores. Subjects' energy intake, including the amount and proportion of calories, was examined for each food source. The analysis was also conducted by gender for age-stratified groups: 19-29, 30-49, and 50-64 years old. RESULTS Korean adults' energy intake from commercially-prepared meals increased in the amount of calories (551 kcal to 635 kcal, P food source of commercially-prepared meals was Korean restaurants in both years. The amount and proportion of calories from retail stores increased from 83 kcal to 143 kcal (P Korean adults consumed about one-fourth of their energy intake from commercially-prepared meals. In particular, males aged 30-49 years and females aged 19-29 years consumed more than one-third of their energy intake from commercially-prepared meals. Korean restaurants played a significant role in Korean adults' energy intake. Retail stores increased influence on Korean adults' energy intake. These results could be useful for developing health promotion policies and programs. PMID:28386389

Preparation of Phi29 DNA polymerase free of amplifiable DNA using ethidium monoazide, an ultraviolet-free light-emitting diode lamp and trehalose.

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Hirokazu Takahashi

Full Text Available We previously reported that multiply-primed rolling circle amplification (MRPCA using modified random RNA primers can amplify tiny amounts of circular DNA without producing any byproducts. However, contaminating DNA in recombinant Phi29 DNA polymerase adversely affects the outcome of MPRCA, especially for negative controls such as non-template controls. The amplified DNA in negative control casts doubt on the result of DNA amplification. Since Phi29 DNA polymerase has high affinity for both single-strand and double-stranded DNA, some amount of host DNA will always remain in the recombinant polymerase. Here we describe a procedure for preparing Phi29 DNA polymerase which is essentially free of amplifiable DNA. This procedure is realized by a combination of host DNA removal using appropriate salt concentrations, inactivation of amplifiable DNA using ethidium monoazide, and irradiation with visible light from a light-emitting diode lamp. Any remaining DNA, which likely exists as oligonucleotides captured by the Phi29 DNA polymerase, is degraded by the 3'-5' exonuclease activity of the polymerase itself in the presence of trehalose, used as an anti-aggregation reagent. Phi29 DNA polymerase purified by this procedure has little amplifiable DNA, resulting in reproducible amplification of at least ten copies of plasmid DNA without any byproducts and reducing reaction volume. This procedure could aid the amplification of tiny amounts DNA, thereby providing clear evidence of contamination from laboratory environments, tools and reagents.

Flavonoids, total phenolics and antioxidant capacity: comparison between commercial green tea preparations

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Débora Harumi Kodama

2010-12-01

Full Text Available The potential health benefits attributed to green tea and its catechins such as antioxidant effects, cancer chemoprevention, and weight loss have led to a huge increase of green tea products in the food market. The objectives of this work were to analyze and compare these products in terms of phenolic contents and in vitro antioxidant capacity including tea bags, dehydrated leaves, and ready-to-drink preparations after standardization of the infusion preparation procedure. Total phenolics content in 1 cup of the different teas varied from 90 to 341 mg of catechin equivalents, and the highest and the lowest values were both those of the ready-to-drink products. Infusions prepared from tea bags had contents varying from 96 to 201 mg.200 mL-1, and there were no significant differences among batches. The DPPH radical scavenging and the Oxygen Radical Absorbing Capacities (ORAC varied largely among the different tea preparations, from 23 to 131 mmoles of Trolox Equivalents (TE.200 mL-1 (DPPH, and from 1.2 to 5.1 mmoles of TE.200 mL-1 (ORAC, but again there were no differences among infusions or ready-to-drink commercial preparations. However, the antioxidant capacity of ready-to-drink products was partially due to the presence of other non-phenolic compounds such as ascorbic acid

Degradability of polylactide films by commercial microbiological preparations for household composters

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Morawska Magda

2017-09-01

Full Text Available Environmentally friendly polymers such as polylactide are increasingly becoming available for use in packaging applications. The main advantages of polylactide packaging are evident. Polylactide is based on renewable resources and can be degraded in compost or soil. The studies on degradability of polylactide (PLA films by commercial preparation of mixture of multi-active saprophytic soil microorganisms, bacteria, actinomycetes and fungi have been done. Unmodified PLA film, metalized co-extruded PLA film and modified by silicon oxide PLA film were incubated in the liquid nutritious medium (TSB prepared to support the growth of microorganisms. The degradability of polylactide films was examined by macro and microscopic observations of surface, changes of mass and crystallinity of polymer samples before and after incubation. The obtained results indicate that the degradation of polylactide was accelerated by the presence of a biological vaccine. It was found that PLA degradation in the inoculated TSB broth was a result of both: enzymatic and chemical hydrolysis.

Preparation of a Nanoscaled Poly(vinyl alcohol/Hydroxyapatite/DNA Complex Using High Hydrostatic Pressure Technology for In Vitro and In Vivo Gene Delivery

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Tsuyoshi Kimura

2011-01-01

Full Text Available Our previous research showed that poly(vinyl alcohol (PVA nanoparticles incorporating DNA with hydrogen bonds obtained by high hydrostatic pressurization are able to deliver DNA without any significant cytotoxicity. To enhance transfection efficiency of PVA/DNA nanoparticles, we describe a novel method to prepare PVA/DNA nanoparticles encapsulating nanoscaled hydroxyapatites (HAps prepared by high hydrostatic pressurization (980 MPa, which is designed to facilitate endosomal escape induced by dissolving HAps in an endosome. Scanning electron microscopic observation and dynamic light scattering measurement revealed that HAps were significantly encapsulated in PVA/HAp/DNA nanoparticles. The cytotoxicity, cellular uptake, and transgene expression of PVA/HAp/DNA nanoparticles were investigated using COS-7 cells. It was found that, in contrast to PVA/DNA nanoparticles, their internalization and transgene expression increased without cytotoxicity occurring. Furthermore, a similar level of transgene expression between plasmid DNA and PVA/HAp/DNA nanoparticles was achieved using in vivo hydrodynamic injection. Our results show a novel method of preparing PVA/DNA nanoparticles encapsulating HAp nano-crystals by using high hydrostatic pressure technology and the potential use of HAps as an enhancer of the transfection efficiency of PVA/DNA nanoparticles without significant cytotoxicity.

Preparation of Oil of Wintergreen from Commercial Aspirin Tablets: A Microscale Experiment Highlighting Acyl Substitutions

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Hartel, Aaron M.; Hanna, James M., Jr.

2009-01-01

A single-pot procedure for the preparation of methyl salicylate (oil of wintergreen) from commercial aspirin tablets has been developed. The synthesis proceeds via a tandem transesterification-Fischer esterification using acidic methanol and can be carried out using either conventional or microwave heating. The experiment helps demonstrate acyl…

Effects of commercial pectolytic and cellulolytic enzyme preparations on the apple cell wall.

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Dongowski, G; Sembries, S

2001-09-01

The action of three different commercial enzyme combinations on apple cell wall material has been examined in a model system under conditions of mash and pomace treatment by using an alcohol-insoluble substance prepared from apples. A part of the total dietary fiber, for example, galacturonan (pectin), appeared in the soluble fraction after enzymatic mash treatment. The soluble fraction increased intensely during pomace treatment. Furthermore, enzyme actions caused a change in the water-binding capacity of residues as well as changes in the monosaccharide composition and in the molecular weight distribution of saccharides in filtrates (soluble parts). The extent of decomposition of cell wall material and the increase of soluble oligomeric and/or polymeric dietary fiber components are caused by both the composition (pectinases, cellulases, and hemicellulases) and the activities of the enzyme preparations. The model experiments allow an insight into the reactions occurring during enzyme action on the plant cell wall, for example, during apple juice production using pectolytic and cellulolytic enzyme preparations.

[Preparation of the cDNA microarray on the differential expressed cDNA of senescence-accelerated mouse's hippocampus].

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Cheng, Xiao-Rui; Zhou, Wen-Xia; Zhang, Yong-Xiang

2006-05-01

Alzheimer' s disease (AD) is the most common form of dementia in the elderly. AD is an invariably fatal neurodegenerative disorder with no effective treatment. Senescence-accelerated mouse prone 8 (SAMP8) is a model for studying age-related cognitive impairments and also is a good model to study brain aging and one of mouse model of AD. The technique of cDNA microarray can monitor the expression levels of thousands of genes simultaneously and can be used to study AD with the character of multi-mechanism, multi-targets and multi-pathway. In order to disclose the mechanism of AD and find the drug targets of AD, cDNA microarray containing 3136 cDNAs amplified from the suppression subtracted cDNA library of hippocampus of SAMP8 and SAMR1 was prepared with 16 blocks and 14 x 14 pins, the housekeeping gene beta-actin and G3PDH as inner conference. The background of this microarray was low and unanimous, and dots divided evenly. The conditions of hybridization and washing were optimized during the hybridization of probe and target molecule. After the data of hybridization analysis, the differential expressed cDNAs were sequenced and analyzed by the bioinformatics, and some of genes were quantified by the real time RT-PCR and the reliability of this cDNA microarray were validated. This cDNA microarray may be the good means to select the differential expressed genes and disclose the molecular mechanism of SAMP8's brain aging and AD.

Preparation of Mica and Silicon Substrates for DNA Origami Analysis and Experimentation

Science.gov (United States)

Pillers, Michelle A.; Shute, Rebecca; Farchone, Adam; Linder, Keenan P.; Doerfler, Rose; Gavin, Corey; Goss, Valerie; Lieberman, Marya

2015-01-01

The designed nature and controlled, one-pot synthesis of DNA origami provides exciting opportunities in many fields, particularly nanoelectronics. Many of these applications require interaction with and adhesion of DNA nanostructures to a substrate. Due to its atomically flat and easily cleaned nature, mica has been the substrate of choice for DNA origami experiments. However, the practical applications of mica are relatively limited compared to those of semiconductor substrates. For this reason, a straightforward, stable, and repeatable process for DNA origami adhesion on derivatized silicon oxide is presented here. To promote the adhesion of DNA nanostructures to silicon oxide surface, a self-assembled monolayer of 3-aminopropyltriethoxysilane (APTES) is deposited from an aqueous solution that is compatible with many photoresists. The substrate must be cleaned of all organic and metal contaminants using Radio Corporation of America (RCA) cleaning processes and the native oxide layer must be etched to ensure a flat, functionalizable surface. Cleanrooms are equipped with facilities for silicon cleaning, however many components of DNA origami buffers and solutions are often not allowed in them due to contamination concerns. This manuscript describes the set-up and protocol for in-lab, small-scale silicon cleaning for researchers who do not have access to a cleanroom or would like to incorporate processes that could cause contamination of a cleanroom CMOS clean bench. Additionally, variables for regulating coverage are discussed and how to recognize and avoid common sample preparation problems is described. PMID:26274888

DNA Qualification Workflow for Next Generation Sequencing of Histopathological Samples

Science.gov (United States)

Simbolo, Michele; Gottardi, Marisa; Corbo, Vincenzo; Fassan, Matteo; Mafficini, Andrea; Malpeli, Giorgio; Lawlor, Rita T.; Scarpa, Aldo

2013-01-01

Histopathological samples are a treasure-trove of DNA for clinical research. However, the quality of DNA can vary depending on the source or extraction method applied. Thus a standardized and cost-effective workflow for the qualification of DNA preparations is essential to guarantee interlaboratory reproducible results. The qualification process consists of the quantification of double strand DNA (dsDNA) and the assessment of its suitability for downstream applications, such as high-throughput next-generation sequencing. We tested the two most frequently used instrumentations to define their role in this process: NanoDrop, based on UV spectroscopy, and Qubit 2.0, which uses fluorochromes specifically binding dsDNA. Quantitative PCR (qPCR) was used as the reference technique as it simultaneously assesses DNA concentration and suitability for PCR amplification. We used 17 genomic DNAs from 6 fresh-frozen (FF) tissues, 6 formalin-fixed paraffin-embedded (FFPE) tissues, 3 cell lines, and 2 commercial preparations. Intra- and inter-operator variability was negligible, and intra-methodology variability was minimal, while consistent inter-methodology divergences were observed. In fact, NanoDrop measured DNA concentrations higher than Qubit and its consistency with dsDNA quantification by qPCR was limited to high molecular weight DNA from FF samples and cell lines, where total DNA and dsDNA quantity virtually coincide. In partially degraded DNA from FFPE samples, only Qubit proved highly reproducible and consistent with qPCR measurements. Multiplex PCR amplifying 191 regions of 46 cancer-related genes was designated the downstream application, using 40 ng dsDNA from FFPE samples calculated by Qubit. All but one sample produced amplicon libraries suitable for next-generation sequencing. NanoDrop UV-spectrum verified contamination of the unsuccessful sample. In conclusion, as qPCR has high costs and is labor intensive, an alternative effective standard workflow for

DNA qualification workflow for next generation sequencing of histopathological samples.

Directory of Open Access Journals (Sweden)

Michele Simbolo

Full Text Available Histopathological samples are a treasure-trove of DNA for clinical research. However, the quality of DNA can vary depending on the source or extraction method applied. Thus a standardized and cost-effective workflow for the qualification of DNA preparations is essential to guarantee interlaboratory reproducible results. The qualification process consists of the quantification of double strand DNA (dsDNA and the assessment of its suitability for downstream applications, such as high-throughput next-generation sequencing. We tested the two most frequently used instrumentations to define their role in this process: NanoDrop, based on UV spectroscopy, and Qubit 2.0, which uses fluorochromes specifically binding dsDNA. Quantitative PCR (qPCR was used as the reference technique as it simultaneously assesses DNA concentration and suitability for PCR amplification. We used 17 genomic DNAs from 6 fresh-frozen (FF tissues, 6 formalin-fixed paraffin-embedded (FFPE tissues, 3 cell lines, and 2 commercial preparations. Intra- and inter-operator variability was negligible, and intra-methodology variability was minimal, while consistent inter-methodology divergences were observed. In fact, NanoDrop measured DNA concentrations higher than Qubit and its consistency with dsDNA quantification by qPCR was limited to high molecular weight DNA from FF samples and cell lines, where total DNA and dsDNA quantity virtually coincide. In partially degraded DNA from FFPE samples, only Qubit proved highly reproducible and consistent with qPCR measurements. Multiplex PCR amplifying 191 regions of 46 cancer-related genes was designated the downstream application, using 40 ng dsDNA from FFPE samples calculated by Qubit. All but one sample produced amplicon libraries suitable for next-generation sequencing. NanoDrop UV-spectrum verified contamination of the unsuccessful sample. In conclusion, as qPCR has high costs and is labor intensive, an alternative effective standard

Salt and fat contents in preparations at commercial restaurants in Goiânia-GO

Directory of Open Access Journals (Sweden)

Camila Silva Kunert

2013-08-01

Full Text Available Objective: To evaluate the sodium and fat contents added to preparations of commercialrestaurants in Goiânia-GO, Brazil. Methods: This was an observational, cross-sectionaland descriptive study. It included ‘pay-per-weight’ restaurants with a medium standardmenu and having as daily preparations white rice, beans and grilled chicken. Among theestablishments with these characteristics, three agreed to participate. The production processof the above-mentioned preparations was accompanied for three non-consecutive days ineach establishment. For quantification of sodium and fat added into the preparations, oiland salt were weighed, as well as the finished preparation; the weight of the standard portion and the yield of the preparation expressed in number of portions prepared were settled. From these data, the per capita amount of salt and oil added to cook one portion of each kind of preparation was calculated by dividing the total quantity of salt and oil by the number of prepared portions. Results: The levels of salt (3.0, 2.7, and 4.1 g – restaurant A, B and C,respectively and oil (17.0, 11.3, and 11.2 g – restaurant A, B and C, respectively added inthe three preparations are superior to the recommendations. Conclusion: The sodium andfat contents in the analyzed restaurants are higher than it is recommended by the Food Guidefor the Brazilian Population. It is essential that commercial restaurants become partners ofpublic policies on health promotion, adopting good nutritional practices, by reducing the sodium and fat contents, to offer healthy meals daily.

Diagnostic PCR: validation and sample preparation are two sides of the same coin

DEFF Research Database (Denmark)

Hoorfar, Jeffrey; Wolffs, Petra; Radstrøm, Peter

2004-01-01

Increased use of powerful PCR technology for the routine detection of pathogens has focused attention on the need for international validation and preparation of official non-commercial guidelines. Bacteria of epidemiological importance should be the prime focus, although a "validation...... of quantitative reference DNA material and reagents, production of stringent protocols and tools for thermal cycler performance testing, uncomplicated sample preparation techniques, and extensive ring trials for assessment of the efficacy of selected matrix/pathogen detection protocols....

Lead contamination in Eugenia dyeriana herbal preparations from different commercial sources in Malaysia.

Science.gov (United States)

Ang, H H

2008-06-01

The Drug Control Authority (DCA) of Malaysia implemented the phase three registration of traditional medicines on 1 January, 1992. A total of 100 products in various pharmaceutical dosage forms of a herbal preparation, containing Eugenia dyeriana, either single or combined preparations (more than one medicinal plant), were analyzed for the presence of lead contamination, using atomic absorption spectrophotometry. These samples were bought from different commercial sources in the Malaysian market, after performing a simple random sampling. Results showed that 22% of the above products failed to comply with the quality requirement for traditional medicines in Malaysia. Although this study showed that 78% of the products fully complied with the quality requirement for traditional medicines in Malaysia pertaining to lead, however, they cannot be assumed safe from lead contamination because of batch-to-batch inconsistency.

Comparison of commercially-available preservatives for maintaining the integrity of bacterial DNA in human milk.

Science.gov (United States)

Lackey, Kimberly A; Williams, Janet E; Price, William J; Carrothers, Janae M; Brooker, Sarah L; Shafii, Bahman; McGuire, Mark A; McGuire, Michelle K

2017-10-01

Inhibiting changes to bacteria in human milk between sample collection and analysis is necessary for unbiased characterization of the milk microbiome. Although cold storage is considered optimal, alternative preservation is sometimes necessary. The objective of this study was to compare the effectiveness of several commercially-available preservatives with regard to maintaining bacterial DNA in human milk for delayed microbiome analysis. Specifically, we compared Life Technologies' RNAlater® stabilization solution, Biomatrica's DNAgard® Saliva, Advanced Instruments' Broad Spectrum Microtabs II™, and Norgen Biotek Corporation's Milk DNA Preservation and Isolation Kit. Aliquots of 8 pools of human milk were treated with each preservative. DNA was extracted immediately and at 1, 2, 4, and 6wk, during which time milk was held at 37°C. The V1-V3 region of the bacterial 16S rRNA gene was amplified and sequenced. Changes in bacterial community structure and diversity over time were evaluated. Comparable to other studies, the most abundant genera were Streptococcus (33.3%), Staphylococcus (14.0%), Dyella (6.3%), Pseudomonas (3.0%), Veillonella (2.5%), Hafnia (2.0%), Prevotella (1.7%), Rhodococcus (1.6%), and Granulicatella (1.4%). Overall, use of Norgen's Milk DNA Preservation and Isolation Kit best maintained the consistency of the bacterial community structure. Total DNA, diversity, and evenness metrics were also highest in samples preserved with this method. When collecting human milk for bacterial community analysis in field conditions where cold storage is not available, our results suggest that Norgen's Milk DNA Preservation and Isolation Kit may be a useful method, at least for a period of 2weeks. Copyright © 2017 Elsevier B.V. All rights reserved.

Preparation of methacrylate-based anion-exchange monolithic microbore column for chromatographic separation of DNA fragments and oligonucleotides

Energy Technology Data Exchange (ETDEWEB)

Sabarudin, Akhmad, E-mail: sabarjpn@ub.ac.id [Division of Nano-materials Science, EcoTopia Science Institute, Nagoya University, Furu-Cho, Chikusa-Ku, Nagoya 464-8603 (Japan); Department of Chemistry, Faculty of Science, Brawijaya University, Jl Veteran Malang 65145 (Indonesia); Huang, Junchao; Shu, Shin; Sakagawa, Shinnosuke [Division of Nano-materials Science, EcoTopia Science Institute, Nagoya University, Furu-Cho, Chikusa-Ku, Nagoya 464-8603 (Japan); Umemura, Tomonari, E-mail: umemura@apchem.nagoya-u.ac.jp [Division of Nano-materials Science, EcoTopia Science Institute, Nagoya University, Furu-Cho, Chikusa-Ku, Nagoya 464-8603 (Japan)

2012-07-29

Highlights: Black-Right-Pointing-Pointer Microbore-scale (1 mm i.d.) anion-exchange monolithic column. Black-Right-Pointing-Pointer Potentially preparative applications. Black-Right-Pointing-Pointer Separation of oligodeoxythymidylic acids and DNA fragments. - Abstract: In this paper, we report on the preparation of a microbore-scale (1 mm i.d.) anion-exchange monolithic column suitable not only for analytical purposes but also for potentially preparative applications. In order to meet the conflicting requirements of high permeability and good mechanical strength, the following two-step procedure was applied. First, an epoxy-containing monolith was synthesized by in situ copolymerization of glycidyl methacrylate (GMA) and ethylene dimethacrylate (EDMA) within the confines of a silicosteel tubing of 1.02 mm i.d. and 1/16 Double-Prime o.d. in the presence of a ternary porogenic mixture of 1-propanol, 1,4-butanediol, and water. The monolithic matrix was subsequently converted into weak anion-exchanger via the ring-opening reaction of epoxy group with diethyl amine. The dynamic binding capacity was 21.4 mg mL{sup -1} for bovine serum albumin (BSA) at 10% breakthrough. The morphology and porous structure of this monolith were assessed by scanning electron microscope (SEM) and inverse size exclusion chromatography (ISEC). To optimize the separation efficiency, the effects of various chromatographic parameters upon the separation of DNA fragments were investigated. The resulting monolithic anion exchanger demonstrated good potential for the separation of both single- and double-stranded DNA molecules using a gradient elution with NaCl in Tris-HCl buffer (20 mM). Oligodeoxythymidylic acids (dT{sub 12}-dT{sub 18}) were successfully resolved at pH 8, while the fragments of 20 bp DNA ladder, 100 bp DNA ladder, and pBR322-HaeIII digest were efficiently separated at pH 9.

Evaluation of library preparation methods for Illumina next generation sequencing of small amounts of DNA from foodborne parasites.

Science.gov (United States)

Nascimento, Fernanda S; Wei-Pridgeon, Yuping; Arrowood, Michael J; Moss, Delynn; da Silva, Alexandre J; Talundzic, Eldin; Qvarnstrom, Yvonne

2016-11-01

Illumina library preparation methods for ultra-low input amounts were compared using genomic DNA from two foodborne parasites (Angiostrongylus cantonensis and Cyclospora cayetanensis) as examples. The Ovation Ultralow method resulted in libraries with the highest concentration and produced quality sequencing data, even when the input DNA was in the picogram range. Published by Elsevier B.V.

Processing of commercial peanut cake into food-grade meal and its utilization in preparation of cookies.

Science.gov (United States)

Tate, P V; Chavan, J K; Patil, P B; Kadam, S S

1990-04-01

The commercial cake produced during expeller pressing of peanuts was extracted with n-hexane, and 80% ethanol followed by sieving through 80 mesh, to remove residual oil, pigments, bitter taste and fibrous material. The processed meal exhibited comparable composition with defatted peanut flour prepared in the laboratory by solvent extraction of full-fat peanut meal. However, the processed cake meal exhibited low methionine content and in vitro protein digestibility as compared with defatted peanut flour. The processed cake meal can be blended with wheat flour to the extent of 10% (w/w) to prepare acceptable cookies with improved protein and mineral contents.

Preparation and Characterization of Super-paramagnetic Nano-beads for DNA Isolation

Institute of Scientific and Technical Information of China (English)

Xin XIE; Xu ZHANG; Bing Bin YU; wei Yang FE

2004-01-01

Unique coupling reagent, bis-(2-hydroxyethyl methacrylate) phosphate was used to prepare coated and functionalized superparamagnetic nanobeads, leading to a simple, effective method for coating the nanobeads. With this method, the thickness of the coating layer and the functional group contents on the nano-beads could be controlled by changing the quantity of the coated monomers. The nanobeads were characterized by means of transmission electron microscopy (TEM) and Fourier transformation infrared spectroscopy (FTIR). The carboxyl-modified magnetic nano-beads were employed to streamline the protocol of isolation of genomic DNA from the human whole blood.

A high throughput DNA extraction method with high yield and quality

Directory of Open Access Journals (Sweden)

Xin Zhanguo

2012-07-01

Full Text Available Abstract Background Preparation of large quantity and high quality genomic DNA from a large number of plant samples is a major bottleneck for most genetic and genomic analyses, such as, genetic mapping, TILLING (Targeting Induced Local Lesion IN Genome, and next-generation sequencing directly from sheared genomic DNA. A variety of DNA preparation methods and commercial kits are available. However, they are either low throughput, low yield, or costly. Here, we describe a method for high throughput genomic DNA isolation from sorghum [Sorghum bicolor (L. Moench] leaves and dry seeds with high yield, high quality, and affordable cost. Results We developed a high throughput DNA isolation method by combining a high yield CTAB extraction method with an improved cleanup procedure based on MagAttract kit. The method yielded large quantity and high quality DNA from both lyophilized sorghum leaves and dry seeds. The DNA yield was improved by nearly 30 fold with 4 times less consumption of MagAttract beads. The method can also be used in other plant species, including cotton leaves and pine needles. Conclusion A high throughput system for DNA extraction from sorghum leaves and seeds was developed and validated. The main advantages of the method are low cost, high yield, high quality, and high throughput. One person can process two 96-well plates in a working day at a cost of $0.10 per sample of magnetic beads plus other consumables that other methods will also need.

Comparison of six commercial DNA extraction kits for detection of Brucella neotomae in Mexican and Central American-style cheese and other milk products.

Science.gov (United States)

Lusk, Tina S; Strain, Errol; Kase, Julie A

2013-05-01

Raw or inadequately pasteurized milk from infected animals and cheese made with such milk are a frequent vehicle for human brucellosis infection. Also, biological terrorism is a concern with certain Brucella spp. Due to matrix-associated real-time polymerase chain reaction (qPCR) inhibitors, robust sample preparations are crucial. We compared six commercial nucleic acid extraction kits using nine Mexican and Central American-style soft cheeses or creams and three liquid milk products inoculated with Brucella neotomae, a surrogate for pathogenic Brucella spp. Kits were evaluated by purity and quantity of DNA as determined by qPCR Ct values, reproducibility across cheese and milk types, and cost. At 10(7) CFU/g in four different cheeses, Qiagen statistically outperformed all other kits. When two cheese styles were inoculated at dual levels, Qiagen and High Pure kit extracted samples at 1.5 × 10(5) CFU/g produced average Ct values of 34-39, while PrepSEQ and MagMAX kit extracted samples exhibited higher or no Ct values. High Pure and Qiagen kits excelled also with liquid milk products. Considering matrices, inoculation levels, and kits evaluated, High Pure and Qiagen products produced Brucella DNA of high quality and quantity indicated by the lowest Ct values and were the least expensive. Copyright © 2012 Elsevier Ltd. All rights reserved.

Characteristics of canine platelet-rich plasma prepared with five commercially available systems.

Science.gov (United States)

Franklin, Samuel P; Garner, Bridget C; Cook, James L

2015-09-01

To characterize platelet-rich plasma (PRP) products obtained from canine blood by use of a variety of commercially available devices. Blood samples from 15 dogs between 18 months and 9 years of age with no concurrent disease, except for osteoarthritis in some dogs. PRP products were produced from blood obtained from each of the 15 dogs by use of each of 5 commercially available PRP-concentrating systems. Complete blood counts were performed on each whole blood sample and PRP product. The degree of platelet, leukocyte, and erythrocyte concentration or reduction for PRP, compared with results for the whole blood sample, was quantified for each dog and summarized for each concentrating system. The various PRP-concentrating systems differed substantially in the amount of blood processed, method of PRP preparation, amount of PRP produced, and platelet, leukocyte, and erythrocyte concentrations or reductions for PRP relative to results for whole blood. The characteristics of PRP products differed considerably. Investigators evaluating the efficacy of PRPs need to specify the characteristics of the product they are assessing. Clinicians should be aware of the data (or lack of data) supporting use of a particular PRP for a specific medical condition.

[Comet assay of DNA fragmentation: modification of silver staining for obtaining permanent preparations].

Science.gov (United States)

Kamins'kyĭ, V O; Lutsyk, M D; Stoĭka, R S

2005-01-01

Modification of comet analysis is proposed for obtaining permanent preparations by DNA staining with silver compounds. The sensitivity of staining is similar to that observed at the treatment by ethidium bromide and other fluorochromes. The advantages of the method are stability of slides and possibility of their reinvestigation by light microscopy. The method does not need expensive fluorescent microscope and lacks contacting with carcinogenic compounds and UV light irradiation.

Facile and rapid DNA extraction and purification from food matrices using IFAST (immiscible filtration assisted by surface tension).

Science.gov (United States)

Strotman, Lindsay N; Lin, Guangyun; Berry, Scott M; Johnson, Eric A; Beebe, David J

2012-09-07

Extraction and purification of DNA is a prerequisite to detection and analytical techniques. While DNA sample preparation methods have improved over the last few decades, current methods are still time consuming and labor intensive. Here we demonstrate a technology termed IFAST (Immiscible Filtration Assisted by Surface Tension), that relies on immiscible phase filtration to reduce the time and effort required to purify DNA. IFAST replaces the multiple wash and centrifugation steps required by traditional DNA sample preparation methods with a single step. To operate, DNA from lysed cells is bound to paramagnetic particles (PMPs) and drawn through an immiscible fluid phase barrier (i.e. oil) by an external handheld magnet. Purified DNA is then eluted from the PMPs. Here, detection of Clostridium botulinum type A (BoNT/A) in food matrices (milk, orange juice), a bioterrorism concern, was used as a model system to establish IFAST's utility in detection assays. Data validated that the DNA purified by IFAST was functional as a qPCR template to amplify the bont/A gene. The sensitivity limit of IFAST was comparable to the commercially available Invitrogen ChargeSwitch® method. Notably, pathogen detection via IFAST required only 8.5 μL of sample and was accomplished in five-fold less time. The simplicity, rapidity and portability of IFAST offer significant advantages when compared to existing DNA sample preparation methods.

Food Consumption Patterns and Micronutrient Density of Complementary Foods Consumed by Infants Fed Commercially Prepared Baby Foods.

Science.gov (United States)

Reidy, Kathleen C; Bailey, Regan Lucas; Deming, Denise M; O'Neill, Lynda; Carr, B Thomas; Lesniauskas, Ruta; Johnson, Wendy

2018-03-01

Nutrition is critically important in the first 1000 days, and while most American babies are fed commercial baby foods, there is little or no evidence from nationally representative data to understand the implications of such consumption. We used 24-hour dietary recall data for 505 infants from The Feeding Infants and Toddlers Study to describe food consumption patterns and micronutrient density of complementary foods consumed by infants fed commercially prepared baby food fruit, vegetables, and dinners and compared with those eaten by nonconsumers of these products. Results show that consumers were significantly more likely to report eating all vegetables (excluding white potatoes, 71% vs 51%), deep yellow vegetables (42% vs 18%), and fruits (79% vs 65%) and were less likely to report eating white potatoes (10% vs 24%), dark green vegetables (4% vs 20%), and sweets (23% vs 47%) than were nonconsumers. Nutrient density of the complementary foods of consumers was greater for fiber, potassium, vitamin A, vitamin C, vitamin E, and magnesium, but lower in sodium and vitamin D. We conclude that infants fed commercially prepared baby foods were more likely to eat vegetables and fruits, and their diets were higher in several micronutrients. These findings provide important insights on complementary feeding and are useful to support the development of evidence-based infant-feeding guidelines.

Food Consumption Patterns and Micronutrient Density of Complementary Foods Consumed by Infants Fed Commercially Prepared Baby Foods

Science.gov (United States)

Reidy, Kathleen C.; Bailey, Regan Lucas; Deming, Denise M.; O’Neill, Lynda; Carr, B. Thomas; Lesniauskas, Ruta; Johnson, Wendy

2018-01-01

Nutrition is critically important in the first 1000 days, and while most American babies are fed commercial baby foods, there is little or no evidence from nationally representative data to understand the implications of such consumption. We used 24-hour dietary recall data for 505 infants from The Feeding Infants and Toddlers Study to describe food consumption patterns and micronutrient density of complementary foods consumed by infants fed commercially prepared baby food fruit, vegetables, and dinners and compared with those eaten by nonconsumers of these products. Results show that consumers were significantly more likely to report eating all vegetables (excluding white potatoes, 71% vs 51%), deep yellow vegetables (42% vs 18%), and fruits (79% vs 65%) and were less likely to report eating white potatoes (10% vs 24%), dark green vegetables (4% vs 20%), and sweets (23% vs 47%) than were nonconsumers. Nutrient density of the complementary foods of consumers was greater for fiber, potassium, vitamin A, vitamin C, vitamin E, and magnesium, but lower in sodium and vitamin D. We conclude that infants fed commercially prepared baby foods were more likely to eat vegetables and fruits, and their diets were higher in several micronutrients. These findings provide important insights on complementary feeding and are useful to support the development of evidence-based infant-feeding guidelines. PMID:29706668

Enzymes in Commercial Cellulase Preparations Bind Differently to Dioxane Extracted Lignins

Energy Technology Data Exchange (ETDEWEB)

Yarbrough, John M.; Mittal, Ashutosh; Katahira, Rui; Mansfield, Elisabeth; Taylor, Larry E.; Decker, Stephen R.; Himmel, Michael E.; Vinzant, Todd

2017-04-24

Commercial fungal cellulases used in biomass-to-biofuels processes can be grouped into three general classes: native, augmented, and engineered. To evaluate lignin binding affinities of different enzyme activities in various commercial cellulase formulations in order to determine if enzyme losses due to lignin binding can be modulated by using different enzymes of the same activity We used water:dioxane (1:9) to extract lignin from pretreated corn stover. Commercial cellulases were incubated with lignin and the unbound supernatants were evaluated for individual enzyme loss by SDS=PAGE and these were correlated with activity loss using various pNP-sugar substrates. Colorimetric assays for general glycosyl hydrolase activities showed distinct differences in enzyme binding to lignin for each enzyme activity. Native systems demonstrated low binding of endo- and exo-cellulases, high binding of xylanase, and moderate ..beta..-glucosidase binding. Engineered cellulase mixtures exhibited low binding of exo-cellulases, very strong binding of endocellulases and ..beta..- glucosidase, and mixed binding of xylanase activity. The augmented cellulase had low binding of exocellulase, high binding of endocellulase and xylanase, and moderate binding of ..beta..-glucosidase activities. Bound and unbound activities were correlated with general molecular weight ranges of proteins as measured by loss of proteins bands in bound fractions on SDS-PAGE gels. Lignin-bound high molecular weight bands correlated with binding of ..beta..-glucosidase activity. While ..beta..-glucosidases demonstrated high binding in many cases, they have been shown to remain active. Bound low molecular weight bands correlated with xylanase activity binding. Contrary to other literature, exocellulase activity did not show strong lignin binding. The variation in enzyme activity binding between the three classes of cellulases preparations indicate that it is certainly possible to alter the binding of specific

DNA Barcode Authentication and Library Development for the Wood of Six Commercial Pterocarpus Species: the Critical Role of Xylarium Specimens.

Science.gov (United States)

Jiao, Lichao; Yu, Min; Wiedenhoeft, Alex C; He, Tuo; Li, Jianing; Liu, Bo; Jiang, Xiaomei; Yin, Yafang

2018-01-31

DNA barcoding has been proposed as a useful tool for forensic wood identification and development of a reliable DNA reference library is an essential first step. Xylaria (wood collections) are potentially enormous data repositories if DNA information could be extracted from wood specimens. In this study, 31 xylarium wood specimens and 8 leaf specimens of six important commercial species of Pterocarpus were selected to investigate the reliability of DNA barcodes for authentication at the species level and to determine the feasibility of building wood DNA barcode reference libraries from xylarium specimens. Four DNA barcodes (ITS2, matK, ndhF-rpl32 and rbcL) and their combination were tested to evaluate their discrimination ability for Pterocarpus species with both TaxonDNA and tree-based analytical methods. The results indicated that the combination barcode of matK + ndhF-rpl32 + ITS2 yielded the best discrimination for the Pterocarpus species studied. The mini-barcode ndhF-rpl32 (167-173 bps) performed well distinguishing P. santalinus from its wood anatomically inseparable species P. tinctorius. Results from this study verified not only the feasibility of building DNA barcode libraries using xylarium wood specimens, but the importance of using wood rather than leaves as the source tissue, when wood is the botanical material to be identified.

Rapid methods for the extraction and archiving of molecular grade fungal genomic DNA.

Science.gov (United States)

Borman, Andrew M; Palmer, Michael; Johnson, Elizabeth M

2013-01-01

The rapid and inexpensive extraction of fungal genomic DNA that is of sufficient quality for molecular approaches is central to the molecular identification, epidemiological analysis, taxonomy, and strain typing of pathogenic fungi. Although many commercially available and in-house extraction procedures do eliminate the majority of contaminants that commonly inhibit molecular approaches, the inherent difficulties in breaking fungal cell walls lead to protocols that are labor intensive and that routinely take several hours to complete. Here we describe several methods that we have developed in our laboratory that allow the extremely rapid and inexpensive preparation of fungal genomic DNA.

Commercial Banking Industry Survey.

Science.gov (United States)

Bright Horizons Children's Centers, Cambridge, MA.

Work and family programs are becoming increasingly important in the commercial banking industry. The objective of this survey was to collect information and prepare a commercial banking industry profile on work and family programs. Fifty-nine top American commercial banks from the Fortune 500 list were invited to participate. Twenty-two…

Characterization of Satellite DNA Sequences from the Commercially Important Marine Rotifers Brachionus rotundiformis and Brachionus plicatilis.

Science.gov (United States)

Boehm; Gibson; Lubzens

2000-01-01

This study was initiated to search for species-specific and strain-specific satellite DNA sequences for which oligonucleotide primers could be designed to differentiate between various commercially important strains of the marine monogonont rotifers Brachionus rotundiformis and Brachionus plicatilis. Two unrelated, highly reiterated satellite sequences were cloned and characterized. The eight sequenced monomers from B. rotundiformis and six from B. plicatilis had low intrarepeat variability and were similar in their overall lengths, A + T compositions, and high degrees of repeated motif substructure. However, hybridizations to 19 representative strains, sequence characterizations, and GenBank searches indicated that these two satellites are morphotype-specific and population-specific, respectively, and share little homology to each other or to other characterized sequences in the database. Primer pairs designed for the B. rotundiformis satellite confirmed hybridization specificities on polymerase chain reaction and could serve as a useful molecular diagnostic tool to identify strains belonging to the SS morphotype, which are gaining widespread usage as first feeds for marine fish in commercial production.

Private Astronaut Training Prepares Commercial Crews of Tomorrow

Science.gov (United States)

2015-01-01

A new company that includes a handful of former NASA personnel is already taking applications for the first comprehensive commercial astronaut training approved by the Federal Aviation Administration. Waypoint 2 Space, located at Johnson Space Center, hopes to draw space tourists and enthusiasts and future commercial crewmembers with first-hand NASA know-how, as well as agency training technology.

Laboratory evaluation of commercial interferon preparations

African Journals Online (AJOL)

The antiviral, antiproliferative and natural killer-cell. (NKC) stimulatory activities of four commercial the- ... activity; (ii) inhibition ofcell proliferation; and (iii) stimulation of natural killer-cells (NKCs). ..... on serial dilution and then dropped substantially when a specific dilution was reached. It was thus possible to define a 'titre' as.

Regulating DNA Self-assembly by DNA-Surface Interactions.

Science.gov (United States)

Liu, Longfei; Li, Yulin; Wang, Yong; Zheng, Jianwei; Mao, Chengde

2017-12-14

DNA self-assembly provides a powerful approach for preparation of nanostructures. It is often studied in bulk solution and involves only DNA-DNA interactions. When confined to surfaces, DNA-surface interactions become an additional, important factor to DNA self-assembly. However, the way in which DNA-surface interactions influence DNA self-assembly is not well studied. In this study, we showed that weak DNA-DNA interactions could be stabilized by DNA-surface interactions to allow large DNA nanostructures to form. In addition, the assembly can be conducted isothermally at room temperature in as little as 5 seconds. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

The use of environmental DNA in invasive species surveillance of the Great Lakes commercial bait trade.

Science.gov (United States)

Nathan, Lucas R; Jerde, Christopher L; Budny, Michelle L; Mahon, Andrew R

2015-04-01

Over 180 non-native species have been introduced in the Laurentian Great Lakes region, many posing threats to native species and ecosystem functioning. One potential pathway for introductions is the commercial bait trade; unknowing or unconcerned anglers commonly release unused bait into aquatic systems. Previous surveillance efforts of this pathway relied on visual inspection of bait stocks in retail shops, which can be time and cost prohibitive and requires a trained individual that can rapidly and accurately identify cryptic species. Environmental DNA (eDNA) surveillance, a molecular tool that has been used for surveillance in aquatic environments, can be used to efficiently detect species at low abundances. We collected and analyzed 576 eDNA samples from 525 retail bait shops throughout the Laurentian Great Lake states. We used eDNA techniques to screen samples for multiple aquatic invasive species (AIS) that could be transported in the bait trade, including bighead (Hypophthalmichthys nobilis) and silver carp (H. molitrix), round goby (Neogobius melanostomus), tubenose goby (Proterorhinus marmoratus), Eurasian rudd (Scardinius erythrophthalmus), and goldfish (Carassius auratus). Twenty-seven samples were positive for at least one target species (4.7% of samples), and all target species were found at least once, except bighead carp. Despite current regulations, the bait trade remains a potential pathway for invasive species introductions in the Great Lakes region. Alterations to existing management strategies regarding the collection, transportation, and use of live bait are warranted, including new and updated regulations, to prevent future introductions of invasive species in the Great Lakes via the bait trade. © 2014 Society for Conservation Biology.

Formation of PAH-DNA adducts after in vivo and vitro exposure of rats and lung cells to different commercial carbon blacks.

Science.gov (United States)

Borm, Paul J A; Cakmak, Gonca; Jermann, Erich; Weishaupt, Christel; Kempers, Pascal; van Schooten, Frederik Jan; Oberdörster, Günter; Schins, Roel P F

2005-06-01

The current study was designed to test the possible release and bioavailability of polycyclic aromatic hydrocarbons (PAHs) from a set of commercial carbon blacks (CBs) as well as the ability of these PAHs to form bulky DNA adducts. In four commercial CBs (Printex 90, Sterling V, N330, Lampblack 101), leaching of PAH was examined through (1) release of parent PAHs in saline with or without surfactant, and (2) PAH adducts in lung epithelial cells (A549) or in rat lungs after exposure to two CBs (Printex 90, Sterling V) for 13 weeks (50 mg/m(3)). In vitro experiments were done with original and extracted particles, as well as organic extracts of CB in DMSO. As positive controls, B[a]P (0.03 microM) and a mixture of 16 PAHs (0.1 microM) were used. No leaching of PAHs was measured in saline or surfactant-containing saline. In vitro incubations with CB particles (30-300 microg/cm(2)) revealed no adduct spots except for Sterling V. However, the spot was not concentration dependent and remains unidentified. Lung DNA from rats after inhalation of Printex 90 or Sterling V showed no spots related to PAH-DNA adduct formation compared to sham-exposed rats. The results suggest that PAHs are very tightly bound to these CBs. Only using organic extracts or particles of low-surface Sterling V, with high PAH content, PAHs may become available to form PAH-DNA adducts. However, the in vitro conditions showing this effect will not be encountered in vivo and renders this mechanism in particle-induced lung cancer at in vivo exposures highly unlikely.

Formation of PAH-DNA adducts after in vivo and vitro exposure of rats and lung cells to different commercial carbon blacks

International Nuclear Information System (INIS)

Borm, Paul J.A.; Cakmak, Gonca; Jermann, Erich; Weishaupt, Christel; Kempers, Pascal; Schooten, Frederik Jan van; Oberdoerster, Guenter; Schins, Roel P.F.

2005-01-01

Objective: The current study was designed to test the possible release and bioavailability of polycyclic aromatic hydrocarbons (PAHs) from a set of commercial carbon blacks (CBs) as well as the ability of these PAHs to form bulky DNA adducts. Methods: In four commercial CBs (Printex 90, Sterling V, N330, Lampblack 101), leaching of PAH was examined through (1) release of parent PAHs in saline with or without surfactant, and (2) PAH adducts in lung epithelial cells (A549) or in rat lungs after exposure to two CBs (Printex 90, Sterling V) for 13 weeks (50 mg/m 3 ). In vitro experiments were done with original and extracted particles, as well as organic extracts of CB in DMSO. As positive controls, B[a]P (0.03 μM) and a mixture of 16 PAHs (0.1 μM) were used. Results: No leaching of PAHs was measured in saline or surfactant-containing saline. In vitro incubations with CB particles (30-300 μg/cm 2 ) revealed no adduct spots except for Sterling V. However, the spot was not concentration dependent and remains unidentified. Lung DNA from rats after inhalation of Printex 90 or Sterling V showed no spots related to PAH-DNA adduct formation compared to sham-exposed rats. Conclusion: The results suggest that PAHs are very tightly bound to these CBs. Only using organic extracts or particles of low-surface Sterling V, with high PAH content, PAHs may become available to form PAH-DNA adducts. However, the in vitro conditions showing this effect will not be encountered in vivo and renders this mechanism in particle-induced lung cancer at in vivo exposures highly unlikely

Preparation of Calcium Phosphate/pDNA Nanoparticles for Exogenous Gene Delivery by Co-Precipitation Method: Optimization of Formulation Variables Using Box-Behnken Design.

Science.gov (United States)

Li, Wenpan; Zhang, Xirui; Jing, Shasha; Xin, Xiu; Chen, Kang; Chen, Dawei; Hu, Haiyang

2017-08-01

This research focused on optimizing the preparations of pDNA-loaded calcium phosphate (CaP) nanoparticles by employing a 3-factor, 3-level Box-Behnken design. Results indicated that a Ca/P ratio of 189.56, pH of 7.82, and a stirring speed of 528.83 rpm were the optimum conditions for preparation of the nanoparticles. The size of the optimized CaP/pDNA nanoparticles was 61.3 ± 3.64 nm, with a polydispersity index of 0.341 and an encapsulation efficiency of up to 92.11%. The optimized CaP/pDNA nanoparticles had high transfection efficiency and demonstrated good biocompatibility in vitro. Therefore, the Box-Behnken design method was successful in providing desirable CaP nanoparticle pDNA delivery systems by optimizing the experimental factors. Copyright © 2017 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

Garlic, onions and cardiovascular risk factors. A review of the evidence from human experiments with emphasis on commercially available preparations

NARCIS (Netherlands)

Kleijnen, J.; Knipschild, P.; ter Riet, G.

1989-01-01

1. Claims for beneficial effects on cholesterol levels, fibrinolytic activity, and platelet aggregation are attributed both to fresh garlic and onions (or their extracts) and to commercially available preparations. 2. Regarding fresh garlic, the claims have been confirmed, but so far only at very

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

Science.gov (United States)

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

2012-11-01

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

DNA as a component of ER materials

International Nuclear Information System (INIS)

Minagawa, K; Aoki, Y; Berber, M R; Mori, T; Tanaka, M

2009-01-01

Deoxyribonucleic acid (DNA), which is known as a typical biopolymer, has been utilized for a few types of ER materials. Suspensions were prepared with the particles of DNA, DNA/lipid complexes, and LDH (layered double hydroxide)/DNA composites. The purified DNA showed larger ER effect than the others, but this particle tended to absorb water, which caused less stability. Preliminary experiments of preparing composite with LDH indicated that this inorganic material would be useful for hydrophobic modification of DNA particles, although further optimization of composite preparation is needed. In addition, the LDH/DNA suspensions showed interesting behaviours under some conditions, which indicated possibility for controlling ER property in a wide range.

DNA as a component of ER materials

Energy Technology Data Exchange (ETDEWEB)

Minagawa, K; Aoki, Y; Berber, M R [Institute of Technology and Science, University of Tokushima, Tokushima 770-8506 (Japan); Mori, T [Department of Applied Chemistry, Faculty of Engineering, Kyushu University, Fukuoka 819-0395 (Japan); Tanaka, M [Faculty of Pharmaceutical Science, Tokushima Bunri University, Tokushima 770-8514 (Japan)], E-mail: minagawa@chem.tokushima-u.ac.jp

2009-02-01

Deoxyribonucleic acid (DNA), which is known as a typical biopolymer, has been utilized for a few types of ER materials. Suspensions were prepared with the particles of DNA, DNA/lipid complexes, and LDH (layered double hydroxide)/DNA composites. The purified DNA showed larger ER effect than the others, but this particle tended to absorb water, which caused less stability. Preliminary experiments of preparing composite with LDH indicated that this inorganic material would be useful for hydrophobic modification of DNA particles, although further optimization of composite preparation is needed. In addition, the LDH/DNA suspensions showed interesting behaviours under some conditions, which indicated possibility for controlling ER property in a wide range.

Methane emissions abatement by multi-ion-exchanged zeolite A prepared from both commercial-grade zeolite and coal fly ash.

Science.gov (United States)

Hui, K S; Chao, C Y H

2008-10-01

The performance of multimetal-(Cu, Cr, Zn, Ni, and Co)-ion-exchanged zeolite A prepared from both a commercial-grade sample and one produced from coal fly ash in methane emissions abatement was evaluated in this study. The ion-exchange process was used to load the metal ions in zeolite A samples. The methane conversion efficiency by the samples was studied under various parameters including the amount of metal loading (7.3-19.4 wt%), reaction temperature (25-500 degrees C), space velocity (8400-41 900 h(-1)), and methane concentration (0.5-3.2 vol %). At 500 degrees C, the original commercial-grade zeolite A catalyzed 3% of the methane only, whereas the addition of different percentages of metals in the sample enhanced the methane conversion efficiency by 40-85%. Greater methane conversion was observed by increasing the percentage of metals added to the zeolite even though the BET surface area of the zeolite consequently decreased. Higher percentage methane conversion over the multi-ion-exchanged samples was observed at lower space velocities indicating the importance of the mass diffusion of reactants and products in the zeolite. Compared to the multi-ion-exchanged zeolite A prepared from the commercial-grade zeolite, the one produced from coal fly ash demonstrated similar performances in methane emissions abatement, showing the potential use of this low cost recycled material in gaseous pollutant treatment.

Comparison of DNA extraction kits for detection of Burkholderia pseudomallei in spiked human whole blood using real-time PCR.

Directory of Open Access Journals (Sweden)

Nicole L Podnecky

Full Text Available Burkholderia pseudomallei, the etiologic agent of melioidosis, is endemic in northern Australia and Southeast Asia and can cause severe septicemia that may lead to death in 20% to 50% of cases. Rapid detection of B. pseudomallei infection is crucial for timely treatment of septic patients. This study evaluated seven commercially available DNA extraction kits to determine the relative recovery of B. pseudomallei DNA from spiked EDTA-containing human whole blood. The evaluation included three manual kits: the QIAamp DNA Mini kit, the QIAamp DNA Blood Mini kit, and the High Pure PCR Template Preparation kit; and four automated systems: the MagNAPure LC using the DNA Isolation Kit I, the MagNAPure Compact using the Nucleic Acid Isolation Kit I, and the QIAcube using the QIAamp DNA Mini kit and the QIAamp DNA Blood Mini kit. Detection of B. pseudomallei DNA extracted by each kit was performed using the B. pseudomallei specific type III secretion real-time PCR (TTS1 assay. Crossing threshold (C T values were used to compare the limit of detection and reproducibility of each kit. This study also compared the DNA concentrations and DNA purity yielded for each kit. The following kits consistently yielded DNA that produced a detectable signal from blood spiked with 5.5×10(4 colony forming units per mL: the High Pure PCR Template Preparation, QIAamp DNA Mini, MagNA Pure Compact, and the QIAcube running the QIAamp DNA Mini and QIAamp DNA Blood Mini kits. The High Pure PCR Template Preparation kit yielded the lowest limit of detection with spiked blood, but when this kit was used with blood from patients with confirmed cases of melioidosis, the bacteria was not reliably detected indicating blood may not be an optimal specimen.

Immunogenicity of a DNA-launched replicon-based canine parvovirus DNA vaccine expressing VP2 antigen in dogs.

Science.gov (United States)

Dahiya, Shyam S; Saini, Mohini; Kumar, Pankaj; Gupta, Praveen K

2012-10-01

A replicon-based DNA vaccine encoding VP2 gene of canine parvovirus (CPV) was developed by cloning CPV-VP2 gene into a replicon-based DNA vaccine vector (pAlpha). The characteristics of a replicon-based DNA vaccine like, self-amplification of transcripts and induction of apoptosis were analyzed in transfected mammalian cells. When the pAlpha-CPV-VP2 was injected intradermal as DNA-launched replicon-based DNA vaccine in dogs, it induced CPV-specific humoral and cell mediated immune responses. The virus neutralization antibody and lymphocyte proliferative responses were higher than conventional CPV DNA vaccine and commercial CPV vaccine. These results indicated that DNA-launched replicon-based CPV DNA vaccine was effective in inducing both CPV-specific humoral and cellular immune responses and can be considered as effective alternative to conventional CPV DNA vaccine and commercial CPV vaccine. Crown Copyright © 2012. Published by Elsevier India Pvt Ltd. All rights reserved.

Purification of Single-Stranded cDNA Based on RNA Degradation Treatment and Adsorption Chromatography.

Science.gov (United States)

Trujillo-Esquivel, Elías; Franco, Bernardo; Flores-Martínez, Alberto; Ponce-Noyola, Patricia; Mora-Montes, Héctor M

2016-08-02

Analysis of gene expression is a common research tool to study networks controlling gene expression, the role of genes with unknown function, and environmentally induced responses of organisms. Most of the analytical tools used to analyze gene expression rely on accurate cDNA synthesis and quantification to obtain reproducible and quantifiable results. Thus far, most commercial kits for isolation and purification of cDNA target double-stranded molecules, which do not accurately represent the abundance of transcripts. In the present report, we provide a simple and fast method to purify single-stranded cDNA, exhibiting high purity and yield. This method is based on the treatment with RNase H and RNase A after cDNA synthesis, followed by separation in silica spin-columns and ethanol precipitation. In addition, our method avoids the use of DNase I to eliminate genomic DNA from RNA preparations, which improves cDNA yield. As a case report, our method proved to be useful in the purification of single-stranded cDNA from the pathogenic fungus Sporothrix schenckii.

Preparation and Characterization of Cationic PLA-PEG Nanoparticles for Delivery of Plasmid DNA

Directory of Open Access Journals (Sweden)

Zou Weiwei

2009-01-01

Full Text Available Abstract The purpose of the present work was to formulate and evaluate cationic poly(lactic acid-poly(ethylene glycol (PLA-PEG nanoparticles as novel non-viral gene delivery nano-device. Cationic PLA-PEG nanoparticles were prepared by nanoprecipitation method. The gene loaded nanoparticles were obtained by incubating the report gene pEGFP with cationic PLA-PEG nanoparticles. The physicochemical properties (e.g., morphology, particle size, surface charge, DNA binding efficiency and biological properties (e.g., integrity of the released DNA, protection from nuclease degradation, plasma stability, in vitro cytotoxicity, and in vitro transfection ability in Hela cells of the gene loaded PLA-PEG nanoparticles were evaluated, respectively. The obtained cationic PLA-PEG nanoparticles and gene loaded nanoparticles were both spherical in shape with average particle size of 89.7 and 128.9 nm, polydispersity index of 0.185 and 0.161, zeta potentials of +28.9 and +16.8 mV, respectively. The obtained cationic PLA-PEG nanoparticles with high binding efficiency (>95% could protect the loaded DNA from the degradation by nuclease and plasma. The nanoparticles displayed sustained-release properties in vitro and the released DNA maintained its structural and functional integrity. It also showed lower cytotoxicity than Lipofectamine 2000 and could successfully transfect gene into Hela cells even in presence of serum. It could be concluded that the established gene loaded cationic PLA-PEG nanoparticles with excellent properties were promising non-viral nano-device, which had potential to make cancer gene therapy achievable.

Rapid detection of Listeria monocytogenes in foods, by a combination of PCR and DNA probe.

Science.gov (United States)

Ingianni, A; Floris, M; Palomba, P; Madeddu, M A; Quartuccio, M; Pompei, R

2001-10-01

Listeria monocytogenes is a frequent contaminant of water and foods. Its rapid detection is needed before some foods can be prepared for marketing. In this work L. monocytogenes has been searched for in foods, by a combination of polymerase chain reaction (PCR) and a DNA probe. Both PCR and the probe were prepared for recognizing a specific region of the internalin gene, which is responsible for the production of one of the most important pathogenic factors of Listeria. The combined use of PCR and the DNA probe was used for the detection of L. monocytogenes in over 180 environmental and food samples. Several detection methods were compared in this study, namely conventional culture methods; direct PCR; PCR after an enrichment step; a DNA probe alone; a DNA probe after enrichment and another commercially available gene-probe. Finally PCR and the DNA probe were used in series on all the samples collected. When the DNA probe was associated with the PCR, specific and accurate detection of listeria in the samples could be obtained in about a working-day. The present molecular method showed some advantages in terms of rapidity and specificity in comparison to the other aforementioned tests. In addition, it resulted as being easy to handle, even for non-specialized personnel in small diagnostic microbiology laboratories. Copyright 2001 Academic Press.

High Throughput Sample Preparation and Analysis for DNA Sequencing, PCR and Combinatorial Screening of Catalysis Based on Capillary Array Technique

Energy Technology Data Exchange (ETDEWEB)

Zhang, Yonghua [Iowa State Univ., Ames, IA (United States)

2000-01-01

Sample preparation has been one of the major bottlenecks for many high throughput analyses. The purpose of this research was to develop new sample preparation and integration approach for DNA sequencing, PCR based DNA analysis and combinatorial screening of homogeneous catalysis based on multiplexed capillary electrophoresis with laser induced fluorescence or imaging UV absorption detection. The author first introduced a method to integrate the front-end tasks to DNA capillary-array sequencers. protocols for directly sequencing the plasmids from a single bacterial colony in fused-silica capillaries were developed. After the colony was picked, lysis was accomplished in situ in the plastic sample tube using either a thermocycler or heating block. Upon heating, the plasmids were released while chromsomal DNA and membrane proteins were denatured and precipitated to the bottom of the tube. After adding enzyme and Sanger reagents, the resulting solution was aspirated into the reaction capillaries by a syringe pump, and cycle sequencing was initiated. No deleterious effect upon the reaction efficiency, the on-line purification system, or the capillary electrophoresis separation was observed, even though the crude lysate was used as the template. Multiplexed on-line DNA sequencing data from 8 parallel channels allowed base calling up to 620 bp with an accuracy of 98%. The entire system can be automatically regenerated for repeated operation. For PCR based DNA analysis, they demonstrated that capillary electrophoresis with UV detection can be used for DNA analysis starting from clinical sample without purification. After PCR reaction using cheek cell, blood or HIV-1 gag DNA, the reaction mixtures was injected into the capillary either on-line or off-line by base stacking. The protocol was also applied to capillary array electrophoresis. The use of cheaper detection, and the elimination of purification of DNA sample before or after PCR reaction, will make this approach an

Protective action of DNA preparations on the survival of cells and yield of 8-azaguanine resistant mutations in X-irradiated cell culture of chinese hamsters

International Nuclear Information System (INIS)

Kuznetsova, N.N.; Feoktistova, T.P.

1976-01-01

A DNA preparation (molecular weight 19.6-21.0x1O 6 daltons) administered to cell culture of Chinese hamsters in concentrations of 100 to 122 μg/ml 60 minutes before and in the course of 3 days after X-irradiation (600 R) decreased the lethality of irradiated cells and reduced induction of 8-azaguanine resistant genic mutations. DNA preparations with the concentrations under study had no toxic action on cells and were not mutagenous

Lipid oxidation and volatile production in irradiated raw pork batters prepared with commercial soybean oil containing vitamin E

Energy Technology Data Exchange (ETDEWEB)

Jo, Cheorun; Ahn, D.U.; Byun, M.W. E-mail: mwbyun@kaeri.re.kr

2001-04-01

An emulsion-type raw pork batter was prepared using 10% (meat weight) of backfat or commercial soybean oil enriched with vitamin E to determine the effect of irradiation on lipid oxidation and volatile production during storage. Batters (approximately 100 g) were vacuum- or aerobically packaged and irradiated at 0, 2.5 or 4.5 kGy. Irradiation increased lipid oxidation of aerobically packaged raw pork batters prepared with both backfat and soybean oil. Lipid oxidation of vacuum-packaged pork batters was not influenced by irradiation except for the batter prepared with backfat at day 0. Aerobically packaged batters prepared with soybean oil had lower (P<0.05) TBARS than that with backfat, but vacuum-packaged ones were not different. The sum of volatile compounds with short retention time (<1.80) increased by irradiation, and with storage time except for aerobic packaging at day 7. The amount of total volatile compounds had an increasing trend until day 3, but not at day 7. Irradiation increased the production of total volatile compounds in the batters prepared with soybean oil and vacuum packaged, but irradiation effect on volatile production was not consistent with other treatments.

Lipid oxidation and volatile production in irradiated raw pork batters prepared with commercial soybean oil containing vitamin E

International Nuclear Information System (INIS)

Jo, Cheorun; Ahn, D.U.; Byun, M.W.

2001-01-01

An emulsion-type raw pork batter was prepared using 10% (meat weight) of backfat or commercial soybean oil enriched with vitamin E to determine the effect of irradiation on lipid oxidation and volatile production during storage. Batters (approximately 100 g) were vacuum- or aerobically packaged and irradiated at 0, 2.5 or 4.5 kGy. Irradiation increased lipid oxidation of aerobically packaged raw pork batters prepared with both backfat and soybean oil. Lipid oxidation of vacuum-packaged pork batters was not influenced by irradiation except for the batter prepared with backfat at day 0. Aerobically packaged batters prepared with soybean oil had lower (P<0.05) TBARS than that with backfat, but vacuum-packaged ones were not different. The sum of volatile compounds with short retention time (<1.80) increased by irradiation, and with storage time except for aerobic packaging at day 7. The amount of total volatile compounds had an increasing trend until day 3, but not at day 7. Irradiation increased the production of total volatile compounds in the batters prepared with soybean oil and vacuum packaged, but irradiation effect on volatile production was not consistent with other treatments

DNA polymerase preference determines PCR priming efficiency.

Science.gov (United States)

Pan, Wenjing; Byrne-Steele, Miranda; Wang, Chunlin; Lu, Stanley; Clemmons, Scott; Zahorchak, Robert J; Han, Jian

2014-01-30

Polymerase chain reaction (PCR) is one of the most important developments in modern biotechnology. However, PCR is known to introduce biases, especially during multiplex reactions. Recent studies have implicated the DNA polymerase as the primary source of bias, particularly initiation of polymerization on the template strand. In our study, amplification from a synthetic library containing a 12 nucleotide random portion was used to provide an in-depth characterization of DNA polymerase priming bias. The synthetic library was amplified with three commercially available DNA polymerases using an anchored primer with a random 3' hexamer end. After normalization, the next generation sequencing (NGS) results of the amplified libraries were directly compared to the unamplified synthetic library. Here, high throughput sequencing was used to systematically demonstrate and characterize DNA polymerase priming bias. We demonstrate that certain sequence motifs are preferred over others as primers where the six nucleotide sequences at the 3' end of the primer, as well as the sequences four base pairs downstream of the priming site, may influence priming efficiencies. DNA polymerases in the same family from two different commercial vendors prefer similar motifs, while another commercially available enzyme from a different DNA polymerase family prefers different motifs. Furthermore, the preferred priming motifs are GC-rich. The DNA polymerase preference for certain sequence motifs was verified by amplification from single-primer templates. We incorporated the observed DNA polymerase preference into a primer-design program that guides the placement of the primer to an optimal location on the template. DNA polymerase priming bias was characterized using a synthetic library amplification system and NGS. The characterization of DNA polymerase priming bias was then utilized to guide the primer-design process and demonstrate varying amplification efficiencies among three commercially

Molecular authentication of Pargo fillets Lutjanus purpureus (Perciformes: Lutjanidae by DNA barcoding reveals commercial fraud

Directory of Open Access Journals (Sweden)

Ivana Veneza

2018-03-01

Full Text Available ABSTRACT The Caribbean Red Snapper (Pargo Lutjanus purpureus is the most economically important snapper in Brazil, which is sold, among other forms, as frozen fillets. During the process of transformation into fillets there is the removal of the distinctive morphological traits, being able to favor the substitution by less valued species. In addition, there is no national legislation requiring the insertion of the specific name on the product label. However, according to a Normative Instruction (IN N ° 29/2015 MAPA that correlates the common and specific names of the products destined to the national trade, in Brazil only L. purpureus and L. campechanus can be denominated “Pargo”. Thus, the DNA barcode tool was used to identify the fillets sold in north of Brazil, labeled “Pargo”, with the aid of sequences from the public and control databases. The results showed that among 142 fillets examined, 78% was identified as L. purpureus and 22% as Rhomboplites aurorubens, a snapper with low commercial value in the country, revealing commercial fraud. The molecular identification method successfully used in this study to authenticate fillets snappers may also be used by surveillance authorities in the quality control of processed fish products, towards ensuring consumer rights.

Physical association of pyrimidine dimer DNA glycosylase and apurinic/apyrimidinic DNA endonuclease essential for repair of ultraviolet-damaged DNA

International Nuclear Information System (INIS)

Nakabeppu, Y.; Sekiguchi, M.

1981-01-01

T4 endonuclease, which is involved in repair of uv-damaged DNA, has been purified to apparent physical homogeneity. Incubation of uv-irradiated poly(dA).poly(dT) with the purified enzyme preparations resulted in production of alkali-labile apyrimidinic sites, followed by formation of nicks in the polymer. By performing a limited reaction with T4 endonuclease V at pH 8.5, irradiated polymer was converted to an intermediate form that carried a large number of alkali-labile sites but only a few nicks. The intermediate was used as substrate for the assay of apurinic/apyrimidinic DNA endonuclease activity. The two activities, a pyrimidine dimer DNA glycosylase and an apurinic/apyrimidinic DNA endonuclease, were copurified and found in enzyme preparations that contained only a 16,000-dalton polypeptide. These results strongly suggested that a DNA glycosylase specific for pyrimidine dimers and an apurinic/apyrimidinic DNA endonuclease reside in a single polypeptide chain coded by the denV gene of bacteriophage T4

Preparation and optical characterization of DNA-riboflavin thin films

Science.gov (United States)

Paulson, Bjorn; Shin, Inchul; Kong, Byungjoo; Sauer, Gregor; Dugasani, Sreekantha Reddy; Khazaeinezhad, Reza; Jung, Woohyun; Joo, Boram; Oh, Kyunghwan

2016-09-01

Thin films of DNA biopolymer thin film are fabricated by a drop casting process on glass and silicon substrates, as well as freestanding. The refractive index is measured by elliposmetry and in bulk DNA film the refractive index is shown to be increased in the 600 to 900 nm DNA transparency window by doping with riboflavin. Further analysis with FT-IR, Raman, and XRD are used to determine whether binding between riboflavin and DNA occurs.

Advancement in shampoo (a dermal care product): preparation methods, patents and commercial utility.

Science.gov (United States)

Deeksha; Malviya, Rishabha; Sharma, Pramod K

2014-01-01

Shampoo is a cleaning aid for hair and is the most evolving beauty products in the present scenario. Today's shampoo products are of great importance as they provide cleaning of hair with the benefits of conditioning, smoothing and good health of hair i.e. dandruff, dirt, grease and lice free hair. Various types of shampoos depending upon function, nature of ingredient, and their special effects are elaborated in this study. Generally shampoos are evaluated in terms of physical appearance, detergency, surface tension, foam quality, pH, viscosity, and percent of solid content, flow property, dirt dispersion, cleaning action, stability and wetting time. The attention should be paid at its patent portion which attracts towards itself as it provides wide knowledge related to shampoo. This article reviews the various aspects of shampoo in terms of preparation methods, various patents and commercial value.

New liquid chromatographic-chemometric approach for the determination of sunset yellow and tartrazine in commercial preparation.

Science.gov (United States)

Dinç, Erdal; Aktaş, A Hakan; Ustündağ, Ozgür

2005-01-01

A new liquid chromatographic (LC)-chemometric approach was developed for the determination of sunset yellow (SUN) and tartrazine (TAR) in commercial preparations. This approach uses LC and chemometric calibration methods, i.e., classical least-squares (CLS), principal component regression (PCR), and partial-least squares (PLS), simultaneously. The combined LC-chemometric approaches, denoted as LC-CLS, LC-PCR, and LC-PLS, are based on photodiode array (PDA) detection at multiple wavelengths. Optimum chromatographic separation of SUN and TAR with allura red as the internal standard (IS) was obtained by using a Waters Symmetry C18 column, 5 microm, 4.6 x 250 mm, and 0.2 M acetate buffer (pH 5)-acetonitrile-methano-bidistilled water (55 + 20 + 15 + 10, v/v) as the mobile phase at a flow rate of 1.9 mL/min. The LC data sets consisting of the ratios of analyte peak areas to the IS peak area were obtained by using PDA detection at 5 wavelengths (465, 470, 475, 480, and 485 nm). LC-chemometric calibrations for SUN and TAR were separately constructed by using the relationship between the peak-area ratio and the training sets for each colorant. LC-chemometric approaches were tested for different synthetic mixtures containing SUN and TAR in the presence of the IS. These LC-chemometric calibrations were applied to a commercial preparation of the 2 colorants. The experimental results of the LC-chemometric approaches were compared with those obtained by a developed classical LC method using single-wavelength detection.

Preparation of carboxyl group-modified palladium nanoparticles in an aqueous solution and their conjugation with DNA

Science.gov (United States)

Wang, Zhifei; Li, Hongying; Zhen, Shuang; He, Nongyue

2012-05-01

The use of nanomaterials in biomolecular labeling and their corresponding detection has been attracting much attention, recently. There are currently very few studies on palladium nanoparticles (Pd NPs) due to their lack of appropriate surface functionalities for conjugation with DNA. In this paper, we thus firstly present an approach to prepare carboxyl group-modified Pd NPs (with an average size of 6 nm) by the use of 11-mercaptoundecanoic acid (MUDA) as a stabilizer in the aqueous solution. The effect of the various reducing reaction conditions on the morphology of the Pd NPs was investigated. The particles were further characterized by TEM, UV-vis, FT-IR and XPS techniques. DNA was finally covalently conjugated to the surface of the Pd NPs through the activation of the carboxyl group, which was confirmed by agarose gel electrophoresis and fluorescence analysis. The resulting Pd NPs-DNA conjugates show high single base pair mismatch discrimination capabilities. This work therefore sets a good foundation for further applications of Pd NPs in bio-analytical research.

Principles of DNA architectonics: design of DNA-based nanoobjects

International Nuclear Information System (INIS)

Vinogradova, O A; Pyshnyi, D V

2012-01-01

The methods of preparation of monomeric DNA blocks that serve as key building units for the construction of complex DNA objects are described. Examples are given of the formation of DNA blocks based on native and modified oligonucleotide components using hydrogen bonding and nucleic acid-specific types of bonding and also some affinity interactions with RNA, proteins, ligands. The static discrete and periodic two- and three-dimensional DNA objects reported to date are described systematically. Methods used to prove the structures of DNA objects and the prospects for practical application of nanostructures based on DNA and its analogues in biology, medicine and biophysics are considered. The bibliography includes 195 references.

A real-time polymerase chain reaction method for the identification of four commercially important salmon and trout species.

Science.gov (United States)

Feng, Junli; Wu, Zhigang; Xie, Xiao; Dai, Zhiyuan; Liu, Shasha

2017-01-01

A duplex quantitative real-time PCR (qPCR) assay was developed for rapid and accurate identification of four commercially important salmon and trout species (Oncorhynchus keta, Oncorhynchus nerka, Oncorhynchus mykiss, and Salmo salar) commonly used for production process of fish in China. The assays targeting the mitochondrial control region (CR) and 16S rRNA gene were able to simultaneously discriminate four target species and the family Salmonidae from processed as well as fresh fish. The qPCR efficiency of each reaction was calculated according to the standard curve, and the method was validated by amplification DNA extracted from single or artificial mixtures prepared with the reference salmon and trout species. Testing of 11 commercial salmon and trout products by the established qPCR assay demonstrated that it was really a useful and academic technique to identify four commercially important salmon and trout species.

Surface-assisted DNA self-assembly: An enzyme-free strategy towards formation of branched DNA lattice

International Nuclear Information System (INIS)

Bhanjadeo, Madhabi M.; Nayak, Ashok K.; Subudhi, Umakanta

2017-01-01

DNA based self-assembled nanostructures and DNA origami has proven useful for organizing nanomaterials with firm precision. However, for advanced applications like nanoelectronics and photonics, large-scale organization of self-assembled branched DNA (bDNA) into periodic lattices is desired. In this communication for the first time we report a facile method of self-assembly of Y-shaped bDNA nanostructures on the cationic surface of Aluminum (Al) foil to prepare periodic two dimensional (2D) bDNA lattice. Particularly those Y-shaped bDNA structures having smaller overhangs and unable to self-assemble in solution, they are easily assembled on the surface of Al foil in the absence of ligase. Field emission scanning electron microscopy (FESEM) analysis shows homogenous distribution of two-dimensional bDNA lattices across the Al foil. When the assembled bDNA structures were recovered from the Al foil and electrophoresed in nPAGE only higher order polymeric bDNA structures were observed without a trace of monomeric structures which confirms the stability and high yield of the bDNA lattices. Therefore, this enzyme-free economic and efficient strategy for developing bDNA lattices can be utilized in assembling various nanomaterials for functional molecular components towards development of DNA based self-assembled nanodevices. - Highlights: • Al foil surface-assisted self-assembly of monomeric structures into larger branched DNA lattice. • FESEM study confirms the uniform distribution of two-dimensional bDNA lattice structures across the surface of Al foil. • Enzyme-free and economic strategy to prepare higher order structures from simpler DNA nanostructures have been confirmed by recovery assay. • Use of well proven sequences for the preparation of pure Y-shaped monomeric DNA nanostructure with high yield.

Racemic DNA crystallography.

Science.gov (United States)

Mandal, Pradeep K; Collie, Gavin W; Kauffmann, Brice; Huc, Ivan

2014-12-22

Racemates increase the chances of crystallization by allowing molecular contacts to be formed in a greater number of ways. With the advent of protein synthesis, the production of protein racemates and racemic-protein crystallography are now possible. Curiously, racemic DNA crystallography had not been investigated despite the commercial availability of L- and D-deoxyribo-oligonucleotides. Here, we report a study into racemic DNA crystallography showing the strong propensity of racemic DNA mixtures to form racemic crystals. We describe racemic crystal structures of various DNA sequences and folded conformations, including duplexes, quadruplexes, and a four-way junction, showing that the advantages of racemic crystallography should extend to DNA. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Fast and reliable DNA extraction protocol for identification of species in raw and processed meat products sold on the commercial market

Directory of Open Access Journals (Sweden)

Alvarado Pavel Espinoza

2017-08-01

Full Text Available In this work a protocol for the extraction of DNA from the meat of different animals (beef, pork, and horse was established. The protocol utilized TE lysis buffer with varying concentrations of phenol and chloroform as a base reagent. Reactions were carried out for verying time periods and under differing temperatures. All samples analyzed were obtained from commercial grade meat sourced from the local region. 12 samples were used for methodological optimization with 30 repetitions per sample. Once optimized, purity results for the three species were 1.7 with a concentration (determined spectrophotometrically at 260 nm of 100 μl/ml of DNA. The protocol was tested using 465 different meat samples from different animal species. All meat used was fresh and processed. Results showed a purity of 1.35 ± 0.076 and a DNA concentration of 70 ± 0.31 μl for a time duration of 1.5 hours. These results were tested by polymerase chain reaction (PCR as reported by several authors. The extracts were tested using different PCR reactions using specific primers for horses. Results suggest that there was 39 positive samples. The proposed methodology provides an efficient way to detect DNA concentration and purity, suitable for amplification with PCR.

Program Incubation and Commercialization Best Practices Report

Energy Technology Data Exchange (ETDEWEB)

Richardson, Shannon

2018-04-06

As a reminder, the primary task of the 4C Program is to increase the commercialization rate of cleantech companies in California. Commercialization, broadly defined, is the innovation continuum of developing and introducing a new product or service into the market. For measurability, the 4C Program defines commercialization as encompassing a startup’s: (a) preparation, (b) incubation, (c) commercial-scale pilot / demonstration, and (d) first customer.

Colorimetric DNA detection of transgenic plants using gold nanoparticles functionalized with L-shaped DNA probes

Science.gov (United States)

Nourisaeid, Elham; Mousavi, Amir; Arpanaei, Ayyoob

2016-01-01

In this study, a DNA colorimetric detection system based on gold nanoparticles functionalized with L-shaped DNA probes was prepared and evaluated. We investigated the hybridization efficiency of the L-shaped probes and studied the effect of nanoparticle size and the L-shaped DNA probe length on the performance of the as-prepared system. Probes were attached to the surface of gold nanoparticles using an adenine sequence. An optimal sequence of 35S rRNA gene promoter from the cauliflower mosaic virus, which is frequently used in the development of transgenic plants, and the two complementary ends of this gene were employed as model target strands and probe molecules, respectively. The spectrophotometric properties of the as-prepared systems indicated that the large NPs show better changes in the absorption spectrum and consequently present a better performance. The results of this study revealed that the probe/Au-NPs prepared using a vertical spacer containing 5 thymine oligonucleotides exhibited a stronger spectrophotometric response in comparison to that of larger probes. These results in general indicate the suitable performance of the L-shaped DNA probe-functionalized Au-NPs, and in particular emphasize the important role of the gold nanoparticle size and length of the DNA probes in enhancing the performance of such a system.

ITS and trnH-psbA as Efficient DNA Barcodes to Identify Threatened Commercial Woody Angiosperms from Southern Brazilian Atlantic Rainforests.

Science.gov (United States)

Bolson, Mônica; Smidt, Eric de Camargo; Brotto, Marcelo Leandro; Silva-Pereira, Viviane

2015-01-01

The Araucaria Forests in southern Brazil are part of the Atlantic Rainforest, a key hotspot for global biodiversity. This habitat has experienced extensive losses of vegetation cover due to commercial logging and the intense use of wood resources for construction and furniture manufacturing. The absence of precise taxonomic tools for identifying Araucaria Forest tree species motivated us to test the ability of DNA barcoding to distinguish species exploited for wood resources and its suitability for use as an alternative testing technique for the inspection of illegal timber shipments. We tested three cpDNA regions (matK, trnH-psbA, and rbcL) and nrITS according to criteria determined by The Consortium for the Barcode of Life (CBOL). The efficiency of each marker and selected marker combinations were evaluated for 30 commercially valuable woody species in multiple populations, with a special focus on Lauraceae species. Inter- and intraspecific distances, species discrimination rates, and ability to recover species-specific clusters were evaluated. Among the regions and different combinations, ITS was the most efficient for identifying species based on the 'best close match' test; similarly, the trnH-psbA + ITS combination also demonstrated satisfactory results. When combining trnH-psbA + ITS, Maximum Likelihood analysis demonstrated a more resolved topology for internal branches, with 91% of species-specific clusters. DNA barcoding was found to be a practical and rapid method for identifying major threatened woody angiosperms from Araucaria Forests such as Lauraceae species, presenting a high confidence for recognizing members of Ocotea. These molecular tools can assist in screening those botanical families that are most targeted by the timber industry in southern Brazil and detecting certain species protected by Brazilian legislation and could be a useful tool for monitoring wood exploitation.

ITS and trnH-psbA as Efficient DNA Barcodes to Identify Threatened Commercial Woody Angiosperms from Southern Brazilian Atlantic Rainforests.

Directory of Open Access Journals (Sweden)

Mônica Bolson

Full Text Available The Araucaria Forests in southern Brazil are part of the Atlantic Rainforest, a key hotspot for global biodiversity. This habitat has experienced extensive losses of vegetation cover due to commercial logging and the intense use of wood resources for construction and furniture manufacturing. The absence of precise taxonomic tools for identifying Araucaria Forest tree species motivated us to test the ability of DNA barcoding to distinguish species exploited for wood resources and its suitability for use as an alternative testing technique for the inspection of illegal timber shipments. We tested three cpDNA regions (matK, trnH-psbA, and rbcL and nrITS according to criteria determined by The Consortium for the Barcode of Life (CBOL. The efficiency of each marker and selected marker combinations were evaluated for 30 commercially valuable woody species in multiple populations, with a special focus on Lauraceae species. Inter- and intraspecific distances, species discrimination rates, and ability to recover species-specific clusters were evaluated. Among the regions and different combinations, ITS was the most efficient for identifying species based on the 'best close match' test; similarly, the trnH-psbA + ITS combination also demonstrated satisfactory results. When combining trnH-psbA + ITS, Maximum Likelihood analysis demonstrated a more resolved topology for internal branches, with 91% of species-specific clusters. DNA barcoding was found to be a practical and rapid method for identifying major threatened woody angiosperms from Araucaria Forests such as Lauraceae species, presenting a high confidence for recognizing members of Ocotea. These molecular tools can assist in screening those botanical families that are most targeted by the timber industry in southern Brazil and detecting certain species protected by Brazilian legislation and could be a useful tool for monitoring wood exploitation.

Explanatory chapter: how plasmid preparation kits work.

Science.gov (United States)

Koontz, Laura

2013-01-01

To isolate plasmid DNA from bacteria using a commercial plasmid miniprep kit (if interested, compare this protocol with Isolation of plasmid DNA from bacteria). Copyright © 2013 Elsevier Inc. All rights reserved.

Determination of adducts of polycyclic aromatic hydrocarbons to DNA

International Nuclear Information System (INIS)

Bean, R.M.; Chess, E.K.; Thomas, B.L.; Mann, D.B.; Dankovic, D.A.; Franz, J.A.; Springer, D.L.

1987-01-01

Adducts to deoxyribonucleic acid (DNA), formed from metabolites of polynuclear aromatic compounds, are relatively persistent and correlate with bioresponse (carcinogenicity). Therefore, qualitative and quantitative analysis of adducts in the DNA of individuals may provide valuable information as to recent exposure to carcinogenic hydrocarbons. Further, the ability to detect adducts in a large segment of a population may have significant epidemiological significance. The current thrust of the analytical development at PNL is to isolate the DNA, liberate the adducted hydrocarbon residue from the DNA with acid hydrolysis, and prepare derivatives of the hydrolyzed species that will enhance its detection, quantitation, and characterization using gas chromatography/mass spectrometry (GC/MS). They have initiated the development of the necessary techniques using benzo[a]pyrene (B[a]P). Samples of DNA adducts of radiolabeled B[a]P have been prepared for study by reacting DNA isolated from calf thymus with benzo[a]pyrene-7,8-diol-9,10-epoxide (the ultimate carcinogenic form of B[a]P). Other DNA/B[a]P samples have been prepared by painting the skin of mice with radiolabeled B[a]P. The ability to prepare research quantities of adducts using the hepatocyte preparation method reported by Dankovic et al is a significant development to their DNA adduct analysis program

Development of a DNA-liposome complex for gene delivery applications

Energy Technology Data Exchange (ETDEWEB)

Rasoulianboroujeni, M. [Marquette University School of Dentistry, Milwaukee, WI 53233 (United States); Kupgan, G. [Department of Chemical Engineering, Oklahoma State University, 423 Engineering North, Stillwater, OK 74078 (United States); Moghadam, F. [School of Biological and Health Systems Engineering, Arizona State University, Tempe, AZ (United States); Tahriri, M. [Marquette University School of Dentistry, Milwaukee, WI 53233 (United States); Boughdachi, A. [Polymer Engineering Department, Amirkabir University of Technology, Tehran (Iran, Islamic Republic of); Khoshkenar, P. [Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA 01605 (United States); Ambrose, J.J. [Biomedical Engineering Department, Louisiana Tech University, Ruston, LA 71272 (United States); Kiaie, N. [Tissue Engineering Department, Faculty of Biomedical Engineering, Amirkabir University of Technology, Tehran (Iran, Islamic Republic of); Vashaee, D. [Electrical and Computer Engineering Department, North Carolina State University, Raleigh, NC 27606 (United States); Ramsey, J.D. [Department of Chemical Engineering, Oklahoma State University, 423 Engineering North, Stillwater, OK 74078 (United States); Tayebi, L., E-mail: lobat.tayebi@marquette.edu [Marquette University School of Dentistry, Milwaukee, WI 53233 (United States)

2017-06-01

The association structures formed by cationic liposomes and DNA (Deoxyribonucleic acid)-liposome have been effectively utilized as gene carriers in transfection assays. In this research study, cationic liposomes were prepared using a modified lipid film hydration method consisting of a lyophilization step for gene delivery applications. The obtained results demonstrated that the mean particle size had no significant change while the polydispersity (PDI) increased after lyophilization. The mean particle size slightly reduced after lyophilization (520 ± 12 nm to 464 ± 25 nm) while the PDI increased after lyophilization (0.094 ± 0.017 to 0.220 ± 0.004). In addition. The mean particle size of vesicles increases when DNA is incorporated to the liposomes (673 ± 27 nm). According to the Scanning Electron Microscopy (SEM) and transmission electron microscopy (TEM) images, the spherical shape of liposomes confirmed their successful preservation and reconstitution from the powder. It was found that liposomal formulation has enhanced transfection considerably compared to the naked DNA as negative control. Finally, liposomal formulation in this research had a better function than Lipofectamine® 2000 as a commercialized product because the cellular activity (cellular protein) was higher in the prepared lipoplex than Lipofectamine® 2000. - Highlights: • Liposomal formulation in this research had a better function than Lipofectamine® 2000. • The average particle size had no significant change while the PDI increased after lyophilization. • LacZ expression of the developed cationic liposomes is approximately equal to the Lipofectamine® 2000.

How to open the treasure chest? Optimising DNA extraction from herbarium specimens.

Science.gov (United States)

Särkinen, Tiina; Staats, Martijn; Richardson, James E; Cowan, Robyn S; Bakker, Freek T

2012-01-01

Herbarium collections are potentially an enormous resource for DNA studies, but the use of herbarium specimens in molecular studies has thus far been slowed down by difficulty in obtaining amplifiable DNA. Here we compare a set of commercially available DNA extraction protocols and their performance in terms of DNA purity and yield, and PCR amplification success as measured by using three differentially sized markers, the rbcL barcoding marker (cpDNA), the LEAFY exon 3 (nrDNA), and the trnL((UAA)) P6 loop (cpDNA). Results reveal large differences between extraction methods, where DNA purity rather than yield is shown to be strongly correlated with PCR success. Amplicon size shows similarly strong correlation with PCR success, with the shortest fragment showing the highest success rate (78%, P6 loop, 10-143 base pairs (bp)) and the largest fragment the lowest success (10%, rbcL, 670 bp). The effect of specimen preparation method on PCR success was also tested. Results show that drying method strongly affects PCR success, especially the availability of fragments longer than 250 bp, where longer fragments are more available for PCR amplification in air dried material compared to alcohol dried specimens. Results from our study indicate that projects relying on poor-quality starting material such as herbarium or scat samples should focus on extracting pure DNA and aim to amplify short target regions (herbarium samples available into barcoding initiatives and other molecular studies.

Racemic DNA Crystallography

OpenAIRE

Mandal , Pradeep K.; Collie , Gavin W.; Kauffmann , Brice; Huc , Ivan

2014-01-01

International audience; Racemates increase the chances of crystallization by allowing molecular contacts to be formed in a greater number of ways. With the advent of protein synthesis, the production of protein racemates and racemic-protein crystallography are now possible. Curiously, racemic DNA crystallography had not been investigated despite the commercial availability of Land D-deoxyribo-oligonucleotides. Here, we report a study into racemic DNA crystallography showing the strong propens...

Rogue athletes and recombinant DNA technology: challenges for doping control.

Science.gov (United States)

Azzazy, Hassan M E; Mansour, Mai M H

2007-10-01

The quest for athletic excellence holds no limit for some athletes, and the advances in recombinant DNA technology have handed these athletes the ultimate doping weapons: recombinant proteins and gene doping. Some detection methods are now available for several recombinant proteins that are commercially available as pharmaceuticals and being abused by dopers. However, researchers are struggling to come up with efficient detection methods in preparation for the imminent threat of gene doping, expected in the 2008 Olympics. This Forum article presents the main detection strategies for recombinant proteins and the forthcoming detection strategies for gene doping as well as the prime analytical challenges facing them.

DNA dosimetry assessment for sunscreen genotoxic photoprotection.

Directory of Open Access Journals (Sweden)

André Passaglia Schuch

Full Text Available Due to the increase of solar ultraviolet radiation (UV incidence over the last few decades, the use of sunscreen has been widely adopted for skin protection. However, considering the high efficiency of sunlight-induced DNA lesions, it is critical to improve upon the current approaches that are used to evaluate protection factors. An alternative approach to evaluate the photoprotection provided by sunscreens against daily UV radiation-induced DNA damage is provided by the systematic use of a DNA dosimeter.The Sun Protection Factor for DNA (DNA-SPF is calculated by using specific DNA repair enzymes, and it is defined as the capacity for inhibiting the generation of cyclobutane pyrimidine dimers (CPD and oxidised DNA bases compared with unprotected control samples. Five different commercial brands of sunscreen were initially evaluated, and further studies extended the analysis to include 17 other products representing various formulations and Sun Protection Factors (SPF. Overall, all of the commercial brands of SPF 30 sunscreens provided sufficient protection against simulated sunlight genotoxicity. In addition, this DNA biosensor was useful for rapidly screening the biological protection properties of the various sunscreen formulations.The application of the DNA dosimeter is demonstrated as an alternative, complementary, and reliable method for the quantification of sunscreen photoprotection at the level of DNA damage.

DNA dosimetry assessment for sunscreen genotoxic photoprotection.

Science.gov (United States)

Schuch, André Passaglia; Lago, Juliana Carvalhães; Yagura, Teiti; Menck, Carlos Frederico Martins

2012-01-01

Due to the increase of solar ultraviolet radiation (UV) incidence over the last few decades, the use of sunscreen has been widely adopted for skin protection. However, considering the high efficiency of sunlight-induced DNA lesions, it is critical to improve upon the current approaches that are used to evaluate protection factors. An alternative approach to evaluate the photoprotection provided by sunscreens against daily UV radiation-induced DNA damage is provided by the systematic use of a DNA dosimeter. The Sun Protection Factor for DNA (DNA-SPF) is calculated by using specific DNA repair enzymes, and it is defined as the capacity for inhibiting the generation of cyclobutane pyrimidine dimers (CPD) and oxidised DNA bases compared with unprotected control samples. Five different commercial brands of sunscreen were initially evaluated, and further studies extended the analysis to include 17 other products representing various formulations and Sun Protection Factors (SPF). Overall, all of the commercial brands of SPF 30 sunscreens provided sufficient protection against simulated sunlight genotoxicity. In addition, this DNA biosensor was useful for rapidly screening the biological protection properties of the various sunscreen formulations. The application of the DNA dosimeter is demonstrated as an alternative, complementary, and reliable method for the quantification of sunscreen photoprotection at the level of DNA damage.

DNA damage in plant herbarium tissue.

Science.gov (United States)

Staats, Martijn; Cuenca, Argelia; Richardson, James E; Vrielink-van Ginkel, Ria; Petersen, Gitte; Seberg, Ole; Bakker, Freek T

2011-01-01

Dried plant herbarium specimens are potentially a valuable source of DNA. Efforts to obtain genetic information from this source are often hindered by an inability to obtain amplifiable DNA as herbarium DNA is typically highly degraded. DNA post-mortem damage may not only reduce the number of amplifiable template molecules, but may also lead to the generation of erroneous sequence information. A qualitative and quantitative assessment of DNA post-mortem damage is essential to determine the accuracy of molecular data from herbarium specimens. In this study we present an assessment of DNA damage as miscoding lesions in herbarium specimens using 454-sequencing of amplicons derived from plastid, mitochondrial, and nuclear DNA. In addition, we assess DNA degradation as a result of strand breaks and other types of polymerase non-bypassable damage by quantitative real-time PCR. Comparing four pairs of fresh and herbarium specimens of the same individuals we quantitatively assess post-mortem DNA damage, directly after specimen preparation, as well as after long-term herbarium storage. After specimen preparation we estimate the proportion of gene copy numbers of plastid, mitochondrial, and nuclear DNA to be 2.4-3.8% of fresh control DNA and 1.0-1.3% after long-term herbarium storage, indicating that nearly all DNA damage occurs on specimen preparation. In addition, there is no evidence of preferential degradation of organelle versus nuclear genomes. Increased levels of C→T/G→A transitions were observed in old herbarium plastid DNA, representing 21.8% of observed miscoding lesions. We interpret this type of post-mortem DNA damage-derived modification to have arisen from the hydrolytic deamination of cytosine during long-term herbarium storage. Our results suggest that reliable sequence data can be obtained from herbarium specimens.

Planning and Operation of Commercial Application Center

International Nuclear Information System (INIS)

Kim, Jun Yeon; Kim, Kye Ryung; Lee, Tae Joon; Lee, Jae Hyeong; Park, Je Won; Lee, Jae Sang

2003-06-01

The objectives of this R and D project are as follows : First, transferring developed technologies to outside companies and operating technology market to vitalize technology transactions, Second, developing commercial application projects to transfer technologies for commercial purposes and to solve interface problems in commercial applications, Third, enhancing commercial utilizations of developed accelerator and beam utilization technologies, Finally. preparing infra-structures for the development of over 30 venture- businesses based on achieved technologies through the Proton Engineering Frontier Project

Planning and Operation of Commercial Application Center

Energy Technology Data Exchange (ETDEWEB)

Kim, Jun Yeon; Kim, Kye Ryung; Lee, Tae Joon; Lee, Jae Hyeong; Park, Je Won; Lee, Jae Sang

2003-06-15

The objectives of this R and D project are as follows : First, transferring developed technologies to outside companies and operating technology market to vitalize technology transactions, Second, developing commercial application projects to transfer technologies for commercial purposes and to solve interface problems in commercial applications, Third, enhancing commercial utilizations of developed accelerator and beam utilization technologies, Finally. preparing infra-structures for the development of over 30 venture- businesses based on achieved technologies through the Proton Engineering Frontier Project.

How We Make DNA Origami.

Science.gov (United States)

Wagenbauer, Klaus F; Engelhardt, Floris A S; Stahl, Evi; Hechtl, Vera K; Stömmer, Pierre; Seebacher, Fabian; Meregalli, Letizia; Ketterer, Philip; Gerling, Thomas; Dietz, Hendrik

2017-10-05

DNA origami has attracted substantial attention since its invention ten years ago, due to the seemingly infinite possibilities that it affords for creating customized nanoscale objects. Although the basic concept of DNA origami is easy to understand, using custom DNA origami in practical applications requires detailed know-how for designing and producing the particles with sufficient quality and for preparing them at appropriate concentrations with the necessary degree of purity in custom environments. Such know-how is not readily available for newcomers to the field, thus slowing down the rate at which new applications outside the field of DNA nanotechnology may emerge. To foster faster progress, we share in this article the experience in making and preparing DNA origami that we have accumulated over recent years. We discuss design solutions for creating advanced structural motifs including corners and various types of hinges that expand the design space for the more rigid multilayer DNA origami and provide guidelines for preventing undesired aggregation and on how to induce specific oligomerization of multiple DNA origami building blocks. In addition, we provide detailed protocols and discuss the expected results for five key methods that allow efficient and damage-free preparation of DNA origami. These methods are agarose-gel purification, filtration through molecular cut-off membranes, PEG precipitation, size-exclusion chromatography, and ultracentrifugation-based sedimentation. The guide for creating advanced design motifs and the detailed protocols with their experimental characterization that we describe here should lower the barrier for researchers to accomplish the full DNA origami production workflow. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

DNA Dosimetry Assessment for Sunscreen Genotoxic Photoprotection

Science.gov (United States)

Schuch, André Passaglia; Lago, Juliana Carvalhães; Yagura, Teiti; Menck, Carlos Frederico Martins

2012-01-01

Background Due to the increase of solar ultraviolet radiation (UV) incidence over the last few decades, the use of sunscreen has been widely adopted for skin protection. However, considering the high efficiency of sunlight-induced DNA lesions, it is critical to improve upon the current approaches that are used to evaluate protection factors. An alternative approach to evaluate the photoprotection provided by sunscreens against daily UV radiation-induced DNA damage is provided by the systematic use of a DNA dosimeter. Methodology/Principal Findings The Sun Protection Factor for DNA (DNA-SPF) is calculated by using specific DNA repair enzymes, and it is defined as the capacity for inhibiting the generation of cyclobutane pyrimidine dimers (CPD) and oxidised DNA bases compared with unprotected control samples. Five different commercial brands of sunscreen were initially evaluated, and further studies extended the analysis to include 17 other products representing various formulations and Sun Protection Factors (SPF). Overall, all of the commercial brands of SPF 30 sunscreens provided sufficient protection against simulated sunlight genotoxicity. In addition, this DNA biosensor was useful for rapidly screening the biological protection properties of the various sunscreen formulations. Conclusions/Significance The application of the DNA dosimeter is demonstrated as an alternative, complementary, and reliable method for the quantification of sunscreen photoprotection at the level of DNA damage. PMID:22768281

The hospital preparation of radiopharmaceuticals

International Nuclear Information System (INIS)

The subject is covered in sections: introduction; preparation ((general - sterilization), production areas (laboratories), working methods for injections, working methods for oral preparations and iodination procedures); analytical testing (general, standards common to injections and oral preparations, standards for injections, standards for oral preparations); reliable methods of preparing sup(99m)Tc-radiopharmaceuticals and 51 Cr-red cells; commercial radiopharmaceutical kits. (U.K.)

PREPARING THE PUBLIC FOR COMMERCIALIZATION AND GUIDANCE OF STRUCTURAL MEDIA SPACE TOWARDS ITS FUSION WITH ADVERTISING SPACE

Directory of Open Access Journals (Sweden)

Marina Đukić

2015-07-01

Full Text Available Through genre structure analysis of the Television´s Zagreb First Channel schedule from the beginning of 1970´s till the end of the 1980´s accompanied by analysis of advertising in same period, the paper will examine the ways and intensity of commercialization entrance in Croatian media space dominated then by state media. Television schedule genre change and the broadcast of economic propaganda program will point out the different character of the television. It can be said that it will serve for preparing the public for commercialization entrance and guidance of structural media space towards its fusion with advertising one. The assumption is that in spite of the TV schedule change, which was in economic sense accompanied by economy reforms in order to establish market economy, the public wasn´t yet delivered to advertisers. One of the clarification lies in the role of the media, which then had revolutionary function with main purpose of not the voters’ generation but only to create patriots. The paper will reproduce a kind of public transformation genesis from latent status in state guided media system to same status of latent consumers in dual media model.

Real-time polymerase chain reaction detection of parvovirus B19 DNA in blood donations using a commercial and an in-house assay.

Science.gov (United States)

Koppelman, M H G M; van Swieten, P; Cuijpers, H T M

2011-06-01

European regulations require testing of manufacturing plasma for parvovirus B19 (B19) DNA to limit the load of this virus to a maximum acceptable level of 10 IU/µL. To meet this requirement, most manufacturers introduced a test algorithm to identify and eliminate high-load donations before making large manufacturing pools of plasma units. Sanquin screens all donations using a commercial assay from Roche and an in-house assay. Between 2006 and 2009, 6.2 million donations were screened using two different polymerase chain reaction (PCR) assays targeting B19 DNA. Donations with B19 DNA loads of greater than 1 × 10(6) IU/mL showing significant differences in viral load between the two assays were further analyzed by sequencing analysis. A total of 396 donations with B19 DNA loads of greater than 1 × 10(6) IU/mL were identified. Fifteen samples (3.8%) had discordant test results; 10 samples (2.5%) were underquantified by the Roche assay, two samples (0.5%) were underquantified by the in-house assay, and three samples (0.8%) were not detected by the Roche assay. Sequencing analysis revealed mismatches in primer and probe-binding regions. Phylogenetic analysis showed that 12 samples were B19 Genotype 1. The three samples not detected by the Roche assay were B19 Genotype 2. This study shows that 3.8% of the viremic B19 DNA-positive donations are not quantified correctly by the Roche or in-house B19 DNA assays. B19 Genotype 1 isolates showing incorrect test results are more common than B19 Genotype 2 or 3 isolates. Newly designed B19 PCR assays for blood screening should preferably have multiplexed formats targeting multiple regions of the B19 genome. © 2010 American Association of Blood Banks.

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

Science.gov (United States)

Jia, Guangyao

thiolated oligonucleotides on gold surfaces and up to a 2.5 fold increase was observed for the rate of adsorption compared to passive immobilization. In order to reduce the reaction time for DNA amplification, a miniaturized fluidic platform was developed for rapid polymerase chain reaction (PCR). Commercially available, adhesive-coated aluminum foils and polypropylene films were laminated to structured polycarbonate films forming micro reactors in a card format. Ice valves were employed to seal the reaction chambers during thermal cycling and a Peltier-based thermal cycler was configured for rapid thermal cycling and ice valve actuation. Numerical modeling was conducted to optimize the design of the PCR reactor and explore the thermal gradient in the reaction chamber in the direction of sample depth. The PCR reactor was experimentally characterized by using thin foil thermocouples and validated by a successful amplification of 10 genome copies of E. coli ATCC 35401 tuf gene in 27 minutes. In the future, we will integrate sample preparation, PCR amplification and DNA detection into a single, centrifugal microfluidic disc that is practically affordable for molecular diagnostics.

Efficient preparation of shuffled DNA libraries through recombination (Gateway) cloning.

Science.gov (United States)

Lehtonen, Soili I; Taskinen, Barbara; Ojala, Elina; Kukkurainen, Sampo; Rahikainen, Rolle; Riihimäki, Tiina A; Laitinen, Olli H; Kulomaa, Markku S; Hytönen, Vesa P

2015-01-01

Efficient and robust subcloning is essential for the construction of high-diversity DNA libraries in the field of directed evolution. We have developed a more efficient method for the subcloning of DNA-shuffled libraries by employing recombination cloning (Gateway). The Gateway cloning procedure was performed directly after the gene reassembly reaction, without additional purification and amplification steps, thus simplifying the conventional DNA shuffling protocols. Recombination-based cloning, directly from the heterologous reassembly reaction, conserved the high quality of the library and reduced the time required for the library construction. The described method is generally compatible for the construction of DNA-shuffled gene libraries. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Extraction of ultrashort DNA molecules from herbarium specimens.

Science.gov (United States)

Gutaker, Rafal M; Reiter, Ella; Furtwängler, Anja; Schuenemann, Verena J; Burbano, Hernán A

2017-02-01

DNA extracted from herbarium specimens is highly fragmented; therefore, it is crucial to use extraction protocols that retrieve short DNA molecules. Improvements in extraction and DNA library preparation protocols for animal remains have allowed efficient retrieval of molecules shorter than 50 bp. Here, we applied these improvements to DNA extraction protocols for herbarium specimens and evaluated extraction performance by shotgun sequencing, which allows an accurate estimation of the distribution of DNA fragment lengths. Extraction with N-phenacylthiazolium bromide (PTB) buffer decreased median fragment length by 35% when compared with cetyl-trimethyl ammonium bromide (CTAB); modifying the binding conditions of DNA to silica allowed for an additional decrease of 10%. We did not observe a further decrease in length for single-stranded DNA (ssDNA) versus double-stranded DNA (dsDNA) library preparation methods. Our protocol enables the retrieval of ultrashort molecules from herbarium specimens, which will help to unlock the genetic information stored in herbaria.

DNA barcoding commercially important aquatic invertebrates of Turkey.

Science.gov (United States)

Keskin, Emre; Atar, Hasan Hüseyin

2013-08-01

DNA barcoding was used in order to identify aquatic invertebrates sampled from fisheries bycatch and discards. A total of 440 unique cytochrome c oxidase sub unit I (COI) barcodes were generated for 22 species from three important phyla (Arthropoda, Cnidaria, and Mollusca). All the species were sequenced and submitted to GenBank and Barcode of Life Database (BOLD) databases using 654 bp-long fragment of mitochondrial COI gene. Two of them (Pontastacus leptodactylus and Rapana bezoar) were first records of the species for the BOLD database and six of them (Carcinus aestuarii, Loligo vulgaris, Melicertus kerathurus, Nephrops norvegicus, Scyllarides latus, and Scyllarus arctus) were first standard (>648 bp) COI barcode records for the GenBank database. COI barcodes were analyzed for nucleotide composition, nucleotide pair frequencies, and Kimura's two-parameter genetic distance. Mean genetic distance among species was found increasing at higher taxonomic levels. Neighbor-joining trees generated were congruent with morphometric-based taxonomic classification. Findings of this study clearly demonstrate that DNA barcodes could be used as an efficient molecular tool in identification of not only target species from fisheries but also bycatch and discard species, and so it could provide us leverage for a better understanding in monitoring and management of fisheries and biodiversity.

Automated methods for single-stranded DNA isolation and dideoxynucleotide DNA sequencing reactions on a robotic workstation

International Nuclear Information System (INIS)

Mardis, E.R.; Roe, B.A.

1989-01-01

Automated procedures have been developed for both the simultaneous isolation of 96 single-stranded M13 chimeric template DNAs in less than two hours, and for simultaneously pipetting 24 dideoxynucleotide sequencing reactions on a commercially available laboratory workstation. The DNA sequencing results obtained by either radiolabeled or fluorescent methods are consistent with the premise that automation of these portions of DNA sequencing projects will improve the reproducibility of the DNA isolation and the procedures for these normally labor-intensive steps provides an approach for rapid acquisition of large amounts of high quality, reproducible DNA sequence data

Hydrophobically modified chitosan/gold nanoparticles for DNA delivery

International Nuclear Information System (INIS)

Bhattarai, Shanta Raj; Remant Bahadur, K.C.; Aryal, Santosh; Bhattarai, Narayan; Kim, Sun Young; Yi, Ho Keun; Hwang, Pyoung Han; Kim, Hak Yong

2008-01-01

Present study dealt an application of modified chitosan gold nanoparticles (Nac-6-Au) for the immobilization of necked plasmid DNA. Gold nanoparticles stabilized with N-acylated chitosan were prepared by graft-onto approach. The stabilized gold nanoparticles were characterized by different physico-chemical techniques such as UV-vis, TEM, ELS and DLS. MTT assay was used for in vitro cytotoxicity of the nanoparticles into three different cell lines (NIH 3T3, CT-26 and MCF-7). The formulation of plasmid DNA with the nanoparticles corresponds to the complex forming capacity and in-vitro/in-vivo transfection efficiency was studied via gel electrophoresis and transfection methods, respectively. Results showed the modified chitosan gold nanoparticles were well-dispersed and spherical in shape with average size around 10∼12 nm in triple distilled water at pH 7.4, and showed relatively no cytotoxicity at low concentration. Addition of plasmid DNA on the aqueous solution of the nanoparticles markedly reduced surface potential (50.0∼66.6%) as well as resulted in a 13.33% increase in hydrodynamic diameters of the formulated nanoparticles. Transfection efficiency of Nac-6-Au/DNA was dependent on cell type, and higher β-galactosidase activity was observed on MCF-7 breast cancer cell. Typically, this activity was 5 times higher in 4.5 mg/ml nanoparticles concentration than that achieved by the nanoparticles of other concentrations (and/or control). However, this activity was lower in in-vitro and dramatically higher in in-vivo than that of commercially available transfection kit (Lipofectin (registered) ) and DNA. From these results, it can be expected to develop alternative new vectors for gene delivery

Using Real-Time PCR as a tool for monitoring the authenticity of commercial coffees.

Science.gov (United States)

Ferreira, Thiago; Farah, Adriana; Oliveira, Tatiane C; Lima, Ivanilda S; Vitório, Felipe; Oliveira, Edna M M

2016-05-15

Coffee is one of the main food products commercialized in the world. Its considerable market value among food products makes it susceptible to adulteration, especially with cereals. Therefore, the objective of this study was to develop a method based on Real-Time Polymerase Chain Reaction (PCR) for detection of cereals in commercial ground roast and soluble coffees. After comparison with standard curves obtained by serial dilution of DNA extracted from barley, corn and rice, the method was sensitive and specific to quantify down to 0.6 pg, 14 pg and 16 pg of barley, corn and rice DNA, respectively. To verify the applicability of the method, 30 commercial samples obtained in different countries were evaluated and those classified as gourmets or superior did not present the tested cereals DNA. However, barley was detected in various traditional (cheaper) samples from South America. In addition, corn and rice were also detected in different samples. Real-Time PCR showed to be suitable for detection of food adulterants in commercial ground roast and soluble coffees. Copyright © 2015 Elsevier Ltd. All rights reserved.

Transferrin-mediated PEGylated nanoparticles for delivery of DNA/PLL

International Nuclear Information System (INIS)

Gu Wangwen; Xu Zhenghong; Gao Yu; Chen Lingli; Li Yaping

2006-01-01

The purpose of this work was to determine the stability of pDNA/poly(L-lysine) complex (DNA/PLL) during microencapsulation, prepare transferrin (TF) conjugated PEGylated nanoparticles (TF-PEG-NP) loading DNA/PLL, and assess its physicochemical characteristics and in vitro transfection efficiency. The DNA/PLL was prepared by mixing plasmid DNA (pDNA) in deionized water with various amounts of PLL. PEGylated nanoparticles (PEG-NP) loading DNA/PLL were prepared by a water-oil-water double emulsion solvent evaporation technique. TF-PEG-NP was prepared by coupling TF with PEG-NP. The physicochemical characteristics of TF-PEG-NP and in vitro transfection efficiency on K562 cells were measured. The results showed that free pDNA reserved its double supercoiled form (dsDNA) for only on average 25.7% after sonification, but over 70% of dsDNA was reserved after pDNA was contracted with PLL. The particle size range of TF-PEG-NP loading DNA/PLL was 150-450 nm with entrapment efficiency over 70%. TF-PEG-NP exhibited the low burst effect (<10%) within 1 day. After the first phase, DNA/PLL displayed a sustained release. The amount of cumulated DNA/PLL release from TF-PEG-NP with 2% polymer over 7 days was 85.4% for DNA/PLL (1:0.3 mass ratio), 59.8% and 43.1% for DNA/PLL (1:0.6) and DNA/PLL (1:1.0), respectively. To TF-PEG-NP loading DNA/PLL without chloroquine, the percentage of EGFP expressing cells was 28.9% for complexes consisting of DNA/PLL (1:0.3), 38.5% and 39.7% for DNA/PLL (1:0.6) and DNA/PLL (1:1.0), respectively. In TF-PEG-NP loading DNA/PLL with chloroquine, more cells were transfected, the percentage of positive cells was 37.6% (DNA/PLL, 1:0.3), 47.1% (DNA/PLL, 1:0.6) and 45.8% (DNA/PLL, 1:1.0), which meant that the transfection efficiency of pDNA was increased by over 50 times when PLL and TF-PEG-NP were jointly used as a plasmid DNA carrier, in particular, the maximal percentage of positive cells (47.1%) from TF-PEG-NP loading DNA/PLL (1:0.6) was about 70 times the

Determination of melting curves of irradiated DNA preparations and of preparations isolated from irradiated calf lymph nodes

International Nuclear Information System (INIS)

Grabowska, B.

1977-01-01

Measurements of melting curves enabled to establish differences of melting temperature, hyperchromic effect and breadth of the helix - coil phase transition dependent on dose of the ionizing radiation applied and on kind of the irradiated object. Changes of the investigated parameters of DNA irradiated after isolation were detectably more pronounced that of DNA from irradiated lymph nodes. The obtained results suggest a protective role of tissue to the secondary structure of DNA. (author)

Towards a commercially potential process

DEFF Research Database (Denmark)

Panpipat, Worawan; Xu, Xuebing; Guo, Zheng

2012-01-01

In order to examine the industrial potential to indirectly isolate phytosterols from deodoriser distillates (DODs), enzymatic transesterification of an industrial rapeseed and soybean oil DOD mixture with bioethanol was investigated using commercial lipases and a few newly immobilised preparations...

A facile electrode preparation method for accurate electrochemical measurements of double-side-coated electrode from commercial Li-ion batteries

Science.gov (United States)

Zhou, Ge; Wang, Qiyu; Wang, Shuo; Ling, Shigang; Zheng, Jieyun; Yu, Xiqian; Li, Hong

2018-04-01

The post mortem electrochemical analysis, including charge-discharge and electrochemical impedance spectroscopy (EIS) measurements, are critical steps for revealing the failure mechanisms of commercial lithium-ion batteries (LIBs). These post measurements usually require the reassembling of coin-cell with electrode which is often double-side-coated in commercial LIBs. It is difficult to use such double-side-coated electrode to perform accurate electrochemical measurements because the back side of the electrode is coated with active materials, rather than single-side-coated electrode that is often used in coin-cell measurements. In this study, we report a facile tape-covering sample preparation method, which can effectively suppress the influence of back side of the double-side-coated electrodes on capacity and EIS measurements in coin-cells. By tape-covering the unwanted side, the areal capacity of the desired investigated side of the electrode has been accurately measured with an experimental error of about 0.5% at various current densities, and accurate EIS measurements and analysis have been conducted as well.

Identification of Species in Tripterygium (Celastraceae) Based on DNA Barcoding.

Science.gov (United States)

Zhang, Xiaomei; Li, Na; Yao, Yuanyuan; Liang, Xuming; Qu, Xianyou; Liu, Xiang; Zhu, Yingjie; Yang, Dajian; Sun, Wei

2016-11-01

Species of genus Tripterygium (Celastraceae) have attracted much attention owing to their excellent effect on treating autoimmune and inflammatory diseases. However, due to high market demand causing overexploitation, natural populations of genus Tripterygium have rapidly declined. Tripterygium medicinal materials are mainly collected from the wild, making the quality of medicinal materials unstable. Additionally, identification of herbal materials from Tripterygium species and their adulterants is difficult based on morphological characters. Therefore, an accurate, convenient, and stability method is urgently needed. In this wok, we developed a DNA barcoding technique to distinguish T. wilfordii HOOK. f., T. hypoglaucum (LÉVL.) HUTCH, and T. regelii SPRAGUE et TAKEDA and their adulterants based on four uniform and standard DNA regions (internal transcribed spacer 2 (ITS2), matK, rbcL, and psbA-trnH). DNA was extracted from 26 locations of fresh leaves. Phylogenetic tree was constructed with Neighbor-Joining (NJ) method, while barcoding gap was analyzed to assess identification efficiency. Compared with the other DNA barcodes applied individually or in combination, ITS2+psbA-trnH was demonstrated as the optimal barcode. T. hypoglaucum and T. wilfordii can be considered as conspecific, while T. regelii was recognized as a separate species. Furthermore, identification of commercial Tripterygium samples was conducted using BLAST against GenBank and Species Identification System for Traditional Chinese Medicine. Our results indicated that DNA barcoding is a convenient, effective, and stability method to identify and distinguish Tripterygium and its adulterants, and could be applied as the quality control for Tripterygium medicinal preparations and monitoring of the medicinal herb trade in markets.

Towards quantitative viromics for both double-stranded and single-stranded DNA viruses

Directory of Open Access Journals (Sweden)

Simon Roux

2016-12-01

Full Text Available Background Viruses strongly influence microbial population dynamics and ecosystem functions. However, our ability to quantitatively evaluate those viral impacts is limited to the few cultivated viruses and double-stranded DNA (dsDNA viral genomes captured in quantitative viral metagenomes (viromes. This leaves the ecology of non-dsDNA viruses nearly unknown, including single-stranded DNA (ssDNA viruses that have been frequently observed in viromes, but not quantified due to amplification biases in sequencing library preparations (Multiple Displacement Amplification, Linker Amplification or Tagmentation. Methods Here we designed mock viral communities including both ssDNA and dsDNA viruses to evaluate the capability of a sequencing library preparation approach including an Adaptase step prior to Linker Amplification for quantitative amplification of both dsDNA and ssDNA templates. We then surveyed aquatic samples to provide first estimates of the abundance of ssDNA viruses. Results Mock community experiments confirmed the biased nature of existing library preparation methods for ssDNA templates (either largely enriched or selected against and showed that the protocol using Adaptase plus Linker Amplification yielded viromes that were ±1.8-fold quantitative for ssDNA and dsDNA viruses. Application of this protocol to community virus DNA from three freshwater and three marine samples revealed that ssDNA viruses as a whole represent only a minor fraction (<5% of DNA virus communities, though individual ssDNA genomes, both eukaryote-infecting Circular Rep-Encoding Single-Stranded DNA (CRESS-DNA viruses and bacteriophages from the Microviridae family, can be among the most abundant viral genomes in a sample. Discussion Together these findings provide empirical data for a new virome library preparation protocol, and a first estimate of ssDNA virus abundance in aquatic systems.

Immunoassay of DNA damage

International Nuclear Information System (INIS)

Gasparro, F.P.; Santella, R.M.

1988-01-01

The direct photomodification of DNA by ultraviolet light or the photo-induced addition of exogenous compounds to DNA components results in alterations of DNA structure ranging from subtle to profound. There are two consequences of these conformational changes. First, cells in which the DNA has been damaged are capable of executing repair steps. Second, the DNA which is usually of very low immunogenicity now becomes highly antigenic. This latter property has allowed the production of a series of monoclonal antibodies that recognize photo-induced DNA damage. Monoclonal antibodies have been generated that recognize the 4',5'-monoadduct and the crosslink of 8-methoxypsoralen in DNA. In addition, another antibody has been prepared which recognizes the furan-side monoadduct of 6,4,4'-trimethylangelicin in DNA. These monoclonal antibodies have been characterized as to sensitivity and specificity using non-competitive and competitive enzyme-linked-immunosorbent assays (ELISA). (author)

Immunoassay of DNA damage

Energy Technology Data Exchange (ETDEWEB)

Gasparro, F P; Santella, R M

1988-09-01

The direct photomodification of DNA by ultraviolet light or the photo-induced addition of exogenous compounds to DNA components results in alterations of DNA structure ranging from subtle to profound. There are two consequences of these conformational changes. First, cells in which the DNA has been damaged are capable of executing repair steps. Second, the DNA which is usually of very low immunogenicity now becomes highly antigenic. This latter property has allowed the production of a series of monoclonal antibodies that recognize photo-induced DNA damage. Monoclonal antibodies have been generated that recognize the 4',5'-monoadduct and the crosslink of 8-methoxypsoralen in DNA. In addition, another antibody has been prepared which recognizes the furan-side monoadduct of 6,4,4'-trimethylangelicin in DNA. These monoclonal antibodies have been characterized as to sensitivity and specificity using non-competitive and competitive enzyme-linked-immunosorbent assays (ELISA).

Commercial and PET radioisotope manufacturing with a medical cyclotron

International Nuclear Information System (INIS)

Boothe, T.E.; McLeod, T.F.; Plitnikas, M.; Kinney, D.; Tavano, E.; Feijoo, Y.; Smith, P.; Szelecsenyi, F.

1993-01-01

Mount Sinai has extensive experience in producing radionuclides for commercial sales and for incorporation into radiopharmaceuticals, including PET. Currently, an attempt is being made to supply radiochemicals to radiopharmaceutical manufacturers outside the hospital, to prepare radiopharmaceuticals for in-house use, and to prepare PET radiopharmaceuticals, such as 2-[F-18] FDG, for outside sales. This use for both commercial and PET manufacturing is atypical for a hospital-based cyclotron. To accomplish PET radiopharmaceutical sales, the hospital operates a nuclear pharmacy. A review of operational details for the past several years shows a continuing dependence on commercial sales which is reflected in research and developmental aspects and in staffing. Developmental efforts have centered primarily on radionuclide production, target development, and radiochemical processing optimization. (orig.)

Commercial and PET radioisotope manufacturing with a medical cyclotron

Science.gov (United States)

Boothe, T. E.; McLeod, T. F.; Plitnikas, M.; Kinney, D.; Tavano, E.; Feijoo, Y.; Smith, P.; Szelecsényi, F.

1993-06-01

Mount Sinai has extensive experience in producing radionuclides for commercial sales and for incorporation into radiopharmaceuticals, including PET. Currently, an attempt is being made to supply radiochemicals to radiopharmaceutical manufacturers outside the hospital, to prepare radiopharmaceuticals for in-house use, and to prepare PET radiopharmaceuticals, such as 2-[F-18] FDG, for outside sales. This use for both commercial and PET manufacturing is atypical for a hospital-based cyclotron. To accomplish PET radiopharmaceutical sales, the hospital operates a nuclear pharmacy. A review of operational details for the past several years shows a continuing dependence on commercial sales which is reflected in research and developmental aspects and in staffing. Developmental efforts have centered primarily on radionuclide production, target development, and radiochemical processing optimization.

Synthesis and characterization of a lamellar hydroxyapatite/DNA nanohybrid

Energy Technology Data Exchange (ETDEWEB)

Zuo Guifu; Wan Yizao; Meng Xianguang [School of Materials Science and Engineering, Tianjin University, Tianjin 300072 (China); Zhao Qing [School of Agriculture and Bioengineering, Tianjin University, Tianjin 300072 (China); Ren Kaijing [Department of Joint Surgery, Tianjin Hospital, Tianjin 300211 (China); Jia Shiru [Key Laboratory of Industrial Microbiology, Ministry of Education, Tianjin University of Science and Technology, 29, 13th Street, TEDA, Tianjin 300457 (China); Wang Jiehua, E-mail: gfzuo@tju.edu.cn [School of Agriculture and Bioengineering, Tianjin University, Tianjin 300072 (China)

2011-04-15

Research highlights: {yields} A lamellar hydroxyapatite (HAp)/DNA nanohybrid was prepared as a novel gene delivering vector. {yields} Gel electrophoresis analysis confirmed that the lamellar HAp could protect DNA from degradation of DNase I. {yields} The protected DNA in the HAp/DNA nanohybrid could be recovered readily under acid conditions. - Abstract: Two-dimensional layered materials exhibit desired functionalities when being used as gene delivery materials. In this study, a novel gene delivering vector, lamellar hydroxyapatite (HAp)/DNA nanohybrid was prepared. The structure of HAp/DNA nanohybrid was investigated by X-ray diffraction (XRD), scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Fourier transform infrared (FT-IR) spectroscopy analysis revealed that ion-exchange occurred during the process. Gel electrophoresis analysis confirmed that the lamellar HAp could protect DNA from degradation of DNase I and the protected DNA could be recovered readily under acid conditions. Furthermore, the integrity of released DNA was confirmed by UV-vis spectra.

Synthesis and characterization of a lamellar hydroxyapatite/DNA nanohybrid

International Nuclear Information System (INIS)

Zuo Guifu; Wan Yizao; Meng Xianguang; Zhao Qing; Ren Kaijing; Jia Shiru; Wang Jiehua

2011-01-01

Research highlights: → A lamellar hydroxyapatite (HAp)/DNA nanohybrid was prepared as a novel gene delivering vector. → Gel electrophoresis analysis confirmed that the lamellar HAp could protect DNA from degradation of DNase I. → The protected DNA in the HAp/DNA nanohybrid could be recovered readily under acid conditions. - Abstract: Two-dimensional layered materials exhibit desired functionalities when being used as gene delivery materials. In this study, a novel gene delivering vector, lamellar hydroxyapatite (HAp)/DNA nanohybrid was prepared. The structure of HAp/DNA nanohybrid was investigated by X-ray diffraction (XRD), scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Fourier transform infrared (FT-IR) spectroscopy analysis revealed that ion-exchange occurred during the process. Gel electrophoresis analysis confirmed that the lamellar HAp could protect DNA from degradation of DNase I and the protected DNA could be recovered readily under acid conditions. Furthermore, the integrity of released DNA was confirmed by UV-vis spectra.

DNA-assisted synthesis of chitosan/ α -Fe 2 O 3 nanocomposites for ...

Indian Academy of Sciences (India)

The FDnanoparticles were prepared by co-precipitation method using DNA as the capping agent. The samples were characterizedby XRD, EDAX, SEM and TEM. The hematite nanoparticles that were prepared using DNA were compared withthe samples prepared using EDTA and CTAB as capping agents. The effect of ...

Fleet DNA Brings Fleet Data to Life, Informs R&D | News | NREL

Science.gov (United States)

Fleet DNA Brings Fleet Data to Life, Informs R&D Fleet DNA Brings Fleet Data to Life, Informs R De La Rosa, NREL 34672 The Fleet DNA clearinghouse of commercial vehicle operations data features Odyne-have tapped into Fleet DNA." The data-driven insight and decision-making capabilities

Low cost extraction and isothermal amplification of DNA for infectious diarrhea diagnosis.

Directory of Open Access Journals (Sweden)

Shichu Huang

Full Text Available In order to counter the common perception that molecular diagnostics are too complicated to work in low resource settings, we have performed a difficult sample preparation and DNA amplification protocol using instrumentation designed to be operated without wall or battery power. In this work we have combined a nearly electricity-free nucleic acid extraction process with an electricity-free isothermal amplification assay to detect the presence of Clostridium difficile (C. difficile DNA in the stool of infected patients. We used helicase-dependent isothermal amplification (HDA to amplify the DNA in a low-cost, thermoplastic reaction chip heated with a pair of commercially available toe warmers, while using a simple Styrofoam insulator. DNA was extracted from known positive and negative stool samples. The DNA extraction protocol utilized an air pressure driven solid phase extraction device run using a standard bicycle pump. The simple heater setup required no electricity or battery and was capable of maintaining the temperature at 65°C±2°C for 55 min, suitable for repeatable HDA amplification. Experiments were performed to explore the adaptability of the system for use in a range of ambient conditions. When compared to a traditional centrifuge extraction protocol and a laboratory thermocycler, this disposable, no power platform achieved approximately the same lower limit of detection (1.25×10(-2 pg of C. difficile DNA while requiring much less raw material and a fraction of the lab infrastructure and cost. This proof of concept study could greatly impact the accessibility of molecular assays for applications in global health.

Low Cost Extraction and Isothermal Amplification of DNA for Infectious Diarrhea Diagnosis

Science.gov (United States)

Huang, Shichu; Do, Jaephil; Mahalanabis, Madhumita; Fan, Andy; Zhao, Lei; Jepeal, Lisa; Singh, Satish K.; Klapperich, Catherine M.

2013-01-01

In order to counter the common perception that molecular diagnostics are too complicated to work in low resource settings, we have performed a difficult sample preparation and DNA amplification protocol using instrumentation designed to be operated without wall or battery power. In this work we have combined a nearly electricity-free nucleic acid extraction process with an electricity-free isothermal amplification assay to detect the presence of Clostridium difficile (C. difficile) DNA in the stool of infected patients. We used helicase-dependent isothermal amplification (HDA) to amplify the DNA in a low-cost, thermoplastic reaction chip heated with a pair of commercially available toe warmers, while using a simple Styrofoam insulator. DNA was extracted from known positive and negative stool samples. The DNA extraction protocol utilized an air pressure driven solid phase extraction device run using a standard bicycle pump. The simple heater setup required no electricity or battery and was capable of maintaining the temperature at 65°C±2°C for 55 min, suitable for repeatable HDA amplification. Experiments were performed to explore the adaptability of the system for use in a range of ambient conditions. When compared to a traditional centrifuge extraction protocol and a laboratory thermocycler, this disposable, no power platform achieved approximately the same lower limit of detection (1.25×10−2 pg of C. difficile DNA) while requiring much less raw material and a fraction of the lab infrastructure and cost. This proof of concept study could greatly impact the accessibility of molecular assays for applications in global health. PMID:23555883

A rapid and low-cost DNA extraction method for isolating ...

African Journals Online (AJOL)

user

2011-02-21

Feb 21, 2011 ... Available online at http://www.academicjournals.org/AJB ... α-casein, produces PCR ready DNA at a fraction of the cost of commercial DNA extraction kits. Key words: DNA .... This experiment was performed to evaluate the efficiency of the ..... Zoetendal EG, Ben-Amor K, Akkermans AD, Abee T, De Vos WM.

Isolation/separation of plasmid DNA using hemoglobin modified magnetic nanocomposites as solid-phase adsorbent.

Science.gov (United States)

Chen, Xu-Wei; Mao, Quan-Xing; Liu, Jia-Wei; Wang, Jian-Hua

2012-10-15

Hemoglobin (Hb) modified magnetic nanocomposites are prepared by immobilization of Hb onto the surface of amino-functionalized Fe(3)O(4)@SiO(2) magnetic nanoparticles via covalent bonding with glutaraldehyde as cross-linker. The obtained nanocomposites are characterized with FT-IR, SEM, XRD and surface charge analysis. A direct solid-phase extraction procedure for the isolation/separation of plasmid DNA using this nanocomposite as a novel adsorbent is thus developed. Some important experimental parameters governing the sorption efficiency, i.e., the pH of sample solution and the ionic strength, are investigated. The Hb modified magnetic nanocomposites provide a sorption capacity of 27.86 mg g(-1) for DNA. By using 2.0mg of the nanocomposites as sorption medium and a suitable acidity of pH 6.1, a sorption efficiency of 93% is achieved for 25 μg mL(-1) of DNA in 1.0 mL of sample solution. Afterwards, the absorbed DNA could be readily recovered by using 1.0 mL of Tris-HCl buffer (pH 8.9, 0.01 mol L(-1)), giving rise to a recovery of ca. 68.3%. The present solid-phased extraction protocol is applied for the isolation of plasmid DNA from Escherichia coli culture, resulting in comparable yield and purity of plasmid DNA with respect to those obtained by using commercial kits. Copyright © 2012 Elsevier B.V. All rights reserved.

Comparative assessment of plasmid DNA delivery by encapsulation ...

African Journals Online (AJOL)

Purpose: To compare the gene delivery effectiveness of plasmid DNA (pDNA) encapsulated within poly (D,L-lactide-co-glycolide) (PLGA) nanoparticles with that adsorbed on PLGA nanoparticles. Methods: PLGA nanoparticles were prepared using solvent-evaporation method. To encapsulate pDNA within the particles, ...

A convenient method of preparing gene vector for real time monitoring transfection process based on the quantum dots

International Nuclear Information System (INIS)

Zhang, Hai-Li; Zhang, Ming-Zhen; Li, Xiang-Yong; Wan, Min; Li, Yong-Qiang; Zhang, Rong-Ying; Zhao, Yuan-Di

2012-01-01

Highlights: ► An easy and direct way to prepare QDs–DNA complexes was developed. ► Surface charge of QDs was tuned with different ratio of amino and glycolate. ► Transfection efficiency was dependent on the surface zeta potentials of QDs. ► Cellular toxicity of this gene vectors is much lower than commercial liposome. ► Whole intracellular behavior of QDs–DNA complexes can be monitored in real time. -- Abstract: Nanoparticle carrier has been developed by combining water-soluble quantum dots and plasmid DNA expressed enhanced green fluorescent protein (EGFP) in a convenient and direct way. First the QDs with different surface charges were obtained by coating with amino and carboxyl terminals at different ratios. Then plasmid DNA was conjugated to QDs via electrostatic interaction. The resultant QDs–DNA complexes showed enhanced resistance to DNase I digestion. The following transfection experiments demonstrated that the transfection efficiency was dependent on the surface charges on QDs. The real time imaging of the transfection process showed that the nanoparticles experienced binding, penetrating the cell membrane and entering cytoplasm in the first 6 h of transfection. The green fluorescence of EGFP began to appear after 18 h transfection and plasmid DNA was fully expressed in the following 6 h. This new QDs–DNA platform showed great potential as new gene delivery carrier.

A convenient method of preparing gene vector for real time monitoring transfection process based on the quantum dots

Energy Technology Data Exchange (ETDEWEB)

Zhang, Hai-Li; Zhang, Ming-Zhen; Li, Xiang-Yong [Britton Chance Center for Biomedical Photonics, Wuhan National Laboratory for Optoelectronics, Huazhong University of Science and Technology, Department of Biomedical Engineering, Wuhan 430074 (China); Key Laboratory of Biomedical Photonics of Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology, Department of Biomedical Engineering, Wuhan 430074 (China); Wan, Min [Key Laboratory of Biomedical Photonics of Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology, Department of Biomedical Engineering, Wuhan 430074 (China); Li, Yong-Qiang [Britton Chance Center for Biomedical Photonics, Wuhan National Laboratory for Optoelectronics, Huazhong University of Science and Technology, Department of Biomedical Engineering, Wuhan 430074 (China); Key Laboratory of Biomedical Photonics of Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology, Department of Biomedical Engineering, Wuhan 430074 (China); Zhang, Rong-Ying [Key Laboratory of Biomedical Photonics of Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology, Department of Biomedical Engineering, Wuhan 430074 (China); Zhao, Yuan-Di, E-mail: zydi@mail.hust.edu.cn [Britton Chance Center for Biomedical Photonics, Wuhan National Laboratory for Optoelectronics, Huazhong University of Science and Technology, Department of Biomedical Engineering, Wuhan 430074 (China); Key Laboratory of Biomedical Photonics of Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology, Department of Biomedical Engineering, Wuhan 430074 (China)

2012-11-15

Highlights: ► An easy and direct way to prepare QDs–DNA complexes was developed. ► Surface charge of QDs was tuned with different ratio of amino and glycolate. ► Transfection efficiency was dependent on the surface zeta potentials of QDs. ► Cellular toxicity of this gene vectors is much lower than commercial liposome. ► Whole intracellular behavior of QDs–DNA complexes can be monitored in real time. -- Abstract: Nanoparticle carrier has been developed by combining water-soluble quantum dots and plasmid DNA expressed enhanced green fluorescent protein (EGFP) in a convenient and direct way. First the QDs with different surface charges were obtained by coating with amino and carboxyl terminals at different ratios. Then plasmid DNA was conjugated to QDs via electrostatic interaction. The resultant QDs–DNA complexes showed enhanced resistance to DNase I digestion. The following transfection experiments demonstrated that the transfection efficiency was dependent on the surface charges on QDs. The real time imaging of the transfection process showed that the nanoparticles experienced binding, penetrating the cell membrane and entering cytoplasm in the first 6 h of transfection. The green fluorescence of EGFP began to appear after 18 h transfection and plasmid DNA was fully expressed in the following 6 h. This new QDs–DNA platform showed great potential as new gene delivery carrier.

Sample preparation for avian and porcine influenza virus cDNA amplification simplified: Boilign vs. conventional RNA extraction

International Nuclear Information System (INIS)

Fereidouni, S.R.; Starick, E.; Ziller, M.; Harder, T.C.; Unger, H.; Hamilton, K.; Globig, A.

2016-01-01

Full text: RNA extraction and purification is a fundamental step that allows for highly sensitive amplification of specific RNA targets in PCR applications. However, commercial extraction kits that are broadly used because of their robustness and high yield of purified RNA are expensive and labor-intensive. In this study, boiling in distilled water or a commerical lysis buffer of different sample matrices containing avian or porcine influenza viruses was tested as an alternative. Real-time PCR (RTaPCR) for nucleoprotein gene fragment was used as read out. Results were compared with freshly extracted RNA by use of a commercial extraction kit. Different batches of virus containing material materials, including diluted virus positive allontoic fluid or cell culture supernatnat, and avian faecal, cloacal or oropharyngeal swab samples were used in this study. Simple boiling of samples without any additional purification steps can be used as an alternative RNA preparation method to detect influenza A virus nucleoprotein RNA in oropharyngeal swab samples, allantoic fluid or cell-culture supernatant. The boiling method is not applicable for sample matrices containing faecal material. (author)

Highly immunogenic prime–boost DNA vaccination protects chickens against challenge with homologous and heterologous H5N1 virus

Directory of Open Access Journals (Sweden)

Anna Stachyra

2014-01-01

Full Text Available Highly pathogenic avian influenza viruses (HPAIVs cause huge economic losses in the poultry industry because of high mortality rate in infected flocks and trade restrictions. Protective antibodies, directed mainly against hemagglutinin (HA, are the primary means of protection against influenza outbreaks. A recombinant DNA vaccine based on the sequence of H5 HA from the H5N1/A/swan/Poland/305-135V08/2006 strain of HPAIV was prepared. Sequence manipulation included deletion of the proteolytic cleavage site to improve protein stability, codon usage optimization to improve translation and stability of RNA in host cells, and cloning into a commercially available vector to enable expression in animal cells. Naked plasmid DNA was complexed with a liposomal carrier and the immunization followed the prime–boost strategy. The immunogenic potential of the DNA vaccine was first proved in broilers in near-to-field conditions resembling a commercial farm. Next, the protective activity of the vaccine was confirmed in SPF layer-type chickens. Experimental infections (challenge experiments indicated that 100% of vaccinated chickens were protected against H5N1 of the same clade and that 70% of them were protected against H5N1 influenza virus of a different clade. Moreover, the DNA vaccine significantly limited (or even eliminated transmission of the virus to contact control chickens. Two intramuscular doses of DNA vaccine encoding H5 HA induced a strong protective response in immunized chicken. The effective protection lasted for a minimum 8 weeks after the second dose of the vaccine and was not limited to the homologous H5N1 virus. In addition, the vaccine reduced shedding of the virus.

Commercial Supersonics Technology Project - Status of Airport Noise

Science.gov (United States)

Bridges, James

2016-01-01

The Commercial Supersonic Technology Project has been developing databases, computational tools, and system models to prepare for a level 1 milestone, the Low Noise Propulsion Tech Challenge, to be delivered Sept 2016. Steps taken to prepare for the final validation test are given, including system analysis, code validation, and risk reduction testing.

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...

Chemical modifications and reactions in DNA nanostructures

DEFF Research Database (Denmark)

Gothelf, Kurt Vesterager

2017-01-01

such as hydrocarbons or steroids have been introduced to change the surface properties of DNA origami structures, either to protect the DNA nanostructure or to dock it into membranes and other hydrophobic surfaces. DNA nanostructures have also been used to control covalent chemical reactions. This article provides......DNA nanotechnology has the power to form self-assembled and well-defined nanostructures, such as DNA origami, where the relative positions of each atom are known with subnanometer precision. Our ability to synthesize oligonucleotides with chemical modifications in almost any desired position...... provides rich opportunity to incorporate molecules, biomolecules, and a variety of nanomaterials in specific positions on DNA nanostructures. Several standard modifications for oligonucleotides are available commercially, such as dyes, biotin, and chemical handles, and such modified oligonucleotides can...

Recent advances in yeast molecular biology: recombinant DNA

International Nuclear Information System (INIS)

1982-09-01

Separate abstracts were prepared for the 25 papers presented at a workshop focusing on chromosomal structure, gene regulation, recombination, DNA repair, and cell type control, that have been obtained by experimental approaches incorporating the new technologies of yeast DNA transformation, molecular cloning, and DNA sequence analysis

DNA nanochannels [version 1; referees: 2 approved

Directory of Open Access Journals (Sweden)

Dianming Wang

2017-04-01

Full Text Available Transmembrane proteins are mostly nanochannels playing a highly important role in metabolism. Understanding their structures and functions is vital for revealing life processes. It is of fundamental interest to develop chemical devices to mimic biological channels. Structural DNA nanotechnology has been proven to be a promising method for the preparation of fine DNA nanochannels as a result of the excellent properties of DNA molecules. This review presents the development history and current situation of three different types of DNA nanochannel: tile-based nanotube, DNA origami nanochannel, and DNA bundle nanochannel.

Antibody recognition of Z-DNA

International Nuclear Information System (INIS)

Lafer, E.M.; Moeller, A.; Valle, R.P.C.; Nordheim, V.A.; Rich, A.; Stollar, B.D.; Massachusetts Inst. of Tech., Cambridge)

1983-01-01

To measure serological reactions under physiological ionic strength, we prepared a brominated (Bl) poly(dG-dC).poly(dG-dC), which forms a stable Z helix in solutions of low salt concentration. Mice and rabbits were immunized with this polymer complexed with the basic protein methylated bovine serum albumin (MBSA), and it was discovered that the Z-DNA helix is a strong immunogen. Various antibody populations were purified from the rabbit serum by quantitative immunoprecipitation. Spleen cells from the mice were used for the preparation of hybridoma cell lines secreting monoclonal antibodies. Anti-Z-DNA antibodies were also raised by immunizing animals with poly(dG-dm 5 C).poly(dG-dm 5 C) under conditions where it was reported to be in the left-handed Z conformation as well as unmodified poly(dG-dC).poly(dG-dC) that was in the right-handed B conformation: both were complexed with MBSA. Z-DNA reactive antibodies were found in both murine and human SLE. A Z-DNA-specific as well as a dDNA and Z-DNA cross-reactive antibody population were distinguished by affinity chromatography of the SLE sera. The specificities of the various anti-Z-DNA antibody populations were measured by direct-binding and competitive radioimmunoassays, using synthetic polymers of defined structure under various ionic strengths. These studies allow us to map the possible antigenic sites for these antibodies, which serve as a model for DNA-protein recognition. The findings also established the usefulness of the antibodies as biochemical probes for Z-DNA. 29 references, 6 figures, 1 table

New expanded bed adsorbents for the recovery of DNA

DEFF Research Database (Denmark)

Theodossiou, Irini; Olander, M. A.; Sondergaard, M.

2000-01-01

A 20-40 mum pellicular high density (similar to3.7 g cm(-3)) expanded bed material has been designed for the capture of DNA and other large macromolecules. Anion exchangers fashioned out of these supports exhibited dramatically enhanced DNA binding capacities over commercial anion exchange...

Comparison of Six DNA Extraction Procedures and the Application of Plastid DNA Enrichment Methods in Selected Non-photosynthetic Plants

Directory of Open Access Journals (Sweden)

Shin-Yi Shyu

2013-12-01

Full Text Available Genomic DNA was isolated using three DNA extraction commercial kits and three CTAB-based methods for two non-photosynthetic plants, Balanophora japonica and Mitrastemon kanehirai. The quality of the isolated DNA was evaluated and subjected to following restriction enzyme digestions. All six procedures yielded DNA of sufficient quality for PCR, and the method described by Barnwell et al. (1998 performed well in isolating DNA from both species for restriction enzyme digestion. In addition, we succeeded to enrich plastid DNA content by using the methods depending on a high salt buffer to deplete nuclear material. The ‘high salt’ methods based on protocol presented by Milligan (1989 were able to increase plastid DNA effectively and significantly reduce nuclear DNA from M. kanehirai. The plastid DNA enrichment protocols are inexpensive and not time-consuming, and may be applicable to other non-photosynthetic plants.

Preparation of genomic DNA from a single species of uncultured magnetotactic bacterium by multiple-displacement amplification.

Science.gov (United States)

Arakaki, Atsushi; Shibusawa, Mie; Hosokawa, Masahito; Matsunaga, Tadashi

2010-03-01

Magnetotactic bacteria comprise a phylogenetically diverse group that is capable of synthesizing intracellular magnetic particles. Although various morphotypes of magnetotactic bacteria have been observed in the environment, bacterial strains available in pure culture are currently limited to a few genera due to difficulties in their enrichment and cultivation. In order to obtain genetic information from uncultured magnetotactic bacteria, a genome preparation method that involves magnetic separation of cells, flow cytometry, and multiple displacement amplification (MDA) using phi29 polymerase was used in this study. The conditions for the MDA reaction using samples containing 1 to 100 cells were evaluated using a pure-culture magnetotactic bacterium, "Magnetospirillum magneticum AMB-1," whose complete genome sequence is available. Uniform gene amplification was confirmed by quantitative PCR (Q-PCR) when 100 cells were used as a template. This method was then applied for genome preparation of uncultured magnetotactic bacteria from complex bacterial communities in an aquatic environment. A sample containing 100 cells of the uncultured magnetotactic coccus was prepared by magnetic cell separation and flow cytometry and used as an MDA template. 16S rRNA sequence analysis of the MDA product from these 100 cells revealed that the amplified genomic DNA was from a single species of magnetotactic bacterium that was phylogenetically affiliated with magnetotactic cocci in the Alphaproteobacteria. The combined use of magnetic separation, flow cytometry, and MDA provides a new strategy to access individual genetic information from magnetotactic bacteria in environmental samples.

Preparation of Tc-99m-macroaggregated albumin from recombinant human albumin for lung perfusion imaging.

Science.gov (United States)

Hunt, A P; Frier, M; Johnson, R A; Berezenko, S; Perkins, A C

2006-01-01

Human serum albumin (HSA) extracted from pooled blood taken from human donors is used in the production of (99m)Tc-labelled macroaggregated albumin (MAA) for lung perfusion imaging. However, concerns for the safety of blood-derived products due to potential contamination by infective agents (e.g. new variant CJD), make alternative production methods necessary. Recombinant DNA technology is a promising method of albumin production avoiding problems associated with human-derived HSA. This paper presents results comparing MAA prepared from recombinant human albumin (rHA, Recombumin) (rMAA) with in-house produced HSA MAA (hMAA) and commercially available MAA (cMAA). (99m)Tc-MAA was prepared using previously published production methods by heating a mixture of albumin and stannous chloride in acetate buffer (pH 5.4) at 70 degrees C for 20 min. Parameters investigated include aggregate size, radiolabelling efficiency, radiochemical and aggregate stability at 4 degrees C and in vitro (in whole human blood) at 37 degrees C and biodistribution studies. Results showed that rMAA could be produced with similar morphology, labelling efficiency and stability to hMAA and cMAA. Our findings confirm that rHA shows significant potential as a direct replacement for HSA in commercially available MAA.

Mendel Meets CSI: Forensic Genotyping as a Method to Teach Genetics & DNA Science

Science.gov (United States)

Kurowski, Scotia; Reiss, Rebecca

2007-01-01

This article describes a forensic DNA science laboratory exercise for advanced high school and introductory college level biology courses. Students use a commercial genotyping kit and genetic analyzer or gene sequencer to analyze DNA recovered from a fictitious crime scene. DNA profiling and STR genotyping are outlined. DNA extraction, PCR, and…

DNA vaccines for aquacultured fish

DEFF Research Database (Denmark)

Lorenzen, Niels; LaPatra, S.E.

2005-01-01

of licensing and public acceptance of the technology. The potential benefits of DNA vaccines for farmed fish include improved animal welfare, reduced environmental impacts of aquaculture activities, increased food quality and quantity, and more sustainable production. Testing under commercial production......Deoxyribonucleic acid (DNA) vaccination is based on the administration of the gene encoding the vaccine antigen, rather than the antigen itself. Subsequent expression of the antigen by cells in the vaccinated hosts triggers the host immune system. Among the many experimental DNA vaccines tested...... in various animal species as well as in humans, the vaccines against rhabdovirus diseases in fish have given some of the most promising results. A single intramuscular (IM) injection of microgram amounts of DNA induces rapid and long-lasting protection in farmed salmonids against economically important...

The future of human DNA vaccines.

Science.gov (United States)

Li, Lei; Saade, Fadi; Petrovsky, Nikolai

2012-12-31

DNA vaccines have evolved greatly over the last 20 years since their invention, but have yet to become a competitive alternative to conventional protein or carbohydrate based human vaccines. Whilst safety concerns were an initial barrier, the Achilles heel of DNA vaccines remains their poor immunogenicity when compared to protein vaccines. A wide variety of strategies have been developed to optimize DNA vaccine immunogenicity, including codon optimization, genetic adjuvants, electroporation and sophisticated prime-boost regimens, with each of these methods having its advantages and limitations. Whilst each of these methods has contributed to incremental improvements in DNA vaccine efficacy, more is still needed if human DNA vaccines are to succeed commercially. This review foresees a final breakthrough in human DNA vaccines will come from application of the latest cutting-edge technologies, including "epigenetics" and "omics" approaches, alongside traditional techniques to improve immunogenicity such as adjuvants and electroporation, thereby overcoming the current limitations of DNA vaccines in humans. Copyright © 2012 Elsevier B.V. All rights reserved.

Preparation of Fe 3O 4/poly(styrene-butyl acrylate-[2-(methacryloxy)ethyl]trimethylammonium chloride) by emulsifier-free emulsion polymerization and its interaction with DNA

Science.gov (United States)

Li, Xiaolong; Liu, Guoqiang; Yan, Wei; Chu, Paul K.; Yeung, Kelvin W. K.; Wu, Shuilin; Yi, Changfeng; Xu, Zushun

2012-04-01

Cationic magnetic polymer particles Fe3O4/poly(styrene-butyl acrylate-[2-(methacryloxy)ethyl]trimethylammonium chloride), a type of potential gene carrier, were prepared by emulsifier-free emulsion polymerization with oleic acid modified magnetite Fe3O4, styrene, butyl acrylate and [2-(methacryloxy)ethyl]trimethylammonium chloride) (METAC). The morphology of the particles was characterized by transmission electron microscopy and the composites of particles were characterized by FT-IR spectroscopy, X-ray diffraction. These results showed that magnetic particles were well dispersed in polymers with the content of about 15%(wt/wt). The composites exhibited superparamagnetism and possessed a certain level of magnetic response. The interactions between the particles with calf-thymus DNA (ct DNA) were confirmed by zeta potential measurement, UV-vis spectroscopy and fluorescence spectroscopy. The DNA-binding capacity determined by the agarose gel electrophoresis showed good binding capacity of the emulsion to DNA. These results suggested the potential of the cationic magnetic polymer emulsion as gene target delivery carrier.

Stability of Mixed Preparations Consisting of Commercial Moisturizing Creams with an Ointment Base Investigated by Magnetic Resonance Imaging.

Science.gov (United States)

Onuki, Yoshinori; Funatani, Chiaki; Yamamoto, Yoshihisa; Fukami, Toshiro; Koide, Tatsuo; Hayashi, Yoshihiro; Takayama, Kozo

2017-01-01

A moisturizing cream mixed with a steroid ointment is frequently prescribed to patients suffering from atopic dermatitis. However, there is a concern that the mixing operation causes destabilization. The present study was performed to investigate the stability of such preparations closely using magnetic resonance imaging (MRI). As sample preparations, five commercial moisturizing creams that are popular in Japan were mixed with an ointment base, a white petrolatum, at a volume ratio of 1 : 1. The mixed preparations were stored at 60°C to accelerate the destabilization processes. Subsequently, the phase separations induced by the storage test were monitored using MRI. Using advanced MR technologies including spin-spin relaxation time (T 2 ) mapping and MR spectroscopy, we successfully characterized the phase-separation behavior of the test samples. For most samples, phase separations developed by the bleeding of liquid oil components. From a sample consisting of an oil-in-water-type cream, Urepearl Cream 10%, a distinct phase-separation mode was observed, which was initiated by the aqueous component separating from the bottom part of the sample. The resultant phase separation was the most distinct among the test samples. To investigate the phase separation quantitatively and objectively, we conducted a histogram analysis on the acquired T 2 maps. The water-in-oil type creams were found to be much more stable after mixing with ointment base than those of oil-in-water type creams. This finding strongly supported the validity of the mixing operation traditionally conducted in pharmacies.

Evaluation of a Solid Phase DNA Binding Matrix for Downstream PCR Analysis

National Research Council Canada - National Science Library

Bader, Douglas E; Fisher, Glen R; Stratilo, Chad W

2005-01-01

A commercially available solid-phase DNA binding matrix (FTA cards) was evaluated for its ability to capture and release DNA for downstream gene amplification and detection assays using polymerase chain reaction (PCR...

Komparasi Metode Isolasi DNA Patogen Antraknosa dan Bulai untuk Deteksi PCR

Directory of Open Access Journals (Sweden)

Ade Syahputra

2016-11-01

Full Text Available Polymerase chain reaction (PCR is an important tool for detection, identification and monitoring of quarantine pests in Indonesia. DNA isolation method from target organism is an important step to provide adequate DNA template for performing PCR. Objective of the research was to compare conventional, commercial kit, FTA-card and its modification methods of DNA isolation to be used in PCR detection for Colletotrichum acutatum and Peronosclerospora sorghi from chili and maize, respectively. DNA obtained from various isolation methods were measured using UV-vis nanodrop-spectrophotometry.  DNA amplification was performed using DNA concentration of 15 ng µL-1 from each isolation method with gradual primer concentrations of 0.4; 0.6; 0.8; and 1.0 mM. The highest concentration of DNA was achieved with conventional methods for C. acutatum from pure culture and P. sorghi from maize leaf. Best DNA purity was obtained from isolation method using commercial kit for C. acutatum from infected fruit (1.94 and from conventional method for C. acutatum from pure culture (1.91. The highest total yield of isolated DNA was achieved by modified FTA-card for C. acutatum from pure culture. In general DNA amplification using various primer concentration gave positive results although DNA bands intensity was varied from faint to very bright.  Furthermore PCR optimization using the best primer concentration from previous reaction showed that all DNA templates resulted in thick and bright DNA bands.

Comparison between Mt-DNA D-Loop and Cyt B primers for porcine DNA detection in meat products

Science.gov (United States)

Hamzah, Azhana; Mutalib, Sahilah Abd.; Babji, Abdul Salam

2013-11-01

This study was conducted to detect the presence of porcine DNA in meat products in the market using conventional polymerase chain reaction (PCR) and commercial PCR-southern hybridization analysis. Porcine DNA detection in meat products was tested due to some issues associated with the adulteration of food products in Malaysia. This is an important issue especially for Halal authentication which is required for some religious practices such as in Islam and Hinduisms. Many techniques have been developed for determining the Halal status of food products. In this paper, mt-DNA D-loop primer and cytochrome (cyt) b were used to detect the presence of porcine DNA in meat products. Positive and negative controls were always present for each batch of extraction. DNA of raw pork meat was used as a positive control while nucleus free water is used as negative control. A pair of oligonucleotide primer was used namely Pork1 and Pork2 which produced amplicon of 531 base pair (bp) in size. While, PCR-southern hybridization was conducted using primers readily supplied by commercial PCR-Southern hybridization and produced amplicon with 276 bp in size. In the present study, demonstrated that none of the samples were contaminated with porcine residuals but selected samples with pork meat were positive. The species-specific PCR amplification yielded excellent results for identification of pork derivatives in food products and it is a potentially reliable and suitable technique in routine food analysis for Halal certification.

Bacterial community analysis of activated sludge: an evaluation of four commonly used DNA extraction methods

NARCIS (Netherlands)

Vanysacker, L.; Declerck, S.A.J.; Hellemans, B.; De Meester, L.; Vankelecom, I.; Declerck, P.

2010-01-01

The effectiveness of three commercially available direct DNA isolation kits (Mobio, Fast, Qiagen) and one published direct DNA extraction protocol (Bead) for extracting bacterial DNA from different types of activated sludge was investigated and mutually compared. The DNA quantity and purity were

Tranformasi Fragmen Dna Kromosom Xanthomonas Campestris ke dalam Escherichia Coli

Directory of Open Access Journals (Sweden)

Wibowo Mangunwardoyo

2002-04-01

Full Text Available Research on DNA transformation of Xanthomonas campestris into Escherichia coli DH5αα using plasmid vector Escherichia coli (pUC19. was carried out. DNA chromosome was isolated using CTAB method, alkali lysis method was used to isolate DNA plasmid. Both of DNA plasmid and chromosome were digested using restriction enzyme EcoRI. Competent cell was prepared with CaCl2 and heat shock method for transformation procedure. The result revealed transformation obtain 5 white colonies, with transformation frequency was 1,22 x 10-8 colony/competent cell. Electrophoresis analysis showed the DNA fragment (insert in range 0.5 – 7,5 kb. Further research should be carried out to prepare the genomic library to obtain better result of transformant.

DNA excision repair in cell extracts from human cell lines exhibiting hypersensitivity to DNA-damaging agents

International Nuclear Information System (INIS)

Hansson, J.; Keyse, S.M.; Lindahl, T.; Wood, R.D.

1991-01-01

Whole cell extracts from human lymphoid cell lines can perform in vitro DNA repair synthesis in plasmids damaged by agents including UV or cis-diamminedichloroplatinum(II) (cis-DDP). Extracts from xeroderma pigmentosum (XP) cells are defective in repair synthesis. We have now studied in vitro DNA repair synthesis using extracts from lymphoblastoid cell lines representing four human hereditary syndromes with increased sensitivity to DNA-damaging agents. Extracts of cell lines from individuals with the sunlight-sensitive disorders dysplastic nevus syndrome or Cockayne's syndrome (complementation groups A and B) showed normal DNA repair synthesis in plasmids with UV photoproducts. This is consistent with in vivo measurements of the overall DNA repair capacity in such cell lines. A number of extracts were prepared from two cell lines representing the variant form of XP (XP-V). Half of the extracts prepared showed normal levels of in vitro DNA repair synthesis in plasmids containing UV lesions, but the remainder of the extracts from the same cell lines showed deficient repair synthesis, suggesting the possibility of an unusually labile excision repair protein in XP-V. Fanconi's anemia (FA) cells show cellular hypersensitivity to cross-linking agents including cis-DDP. Extracts from cell lines belonging to two different complementation groups of FA showed normal DNA repair synthesis in plasmids containing cis-DDP or UV adducts. Thus, there does not appear to be an overall excision repair defect in FA, but the data do not exclude a defect in the repair of interstrand DNA cross-links

A rapid and low-cost DNA extraction method for isolating ...

African Journals Online (AJOL)

The price of commercial DNA extraction methods makes the routine use of polymerase chain reaction amplification (PCR) based methods rather costly for scientists in developing countries. A guanidium thiocayante-based DNA extraction method was investigated in this study for the isolation of Escherichia coli (E. coli) DNA ...

Evaluation and Adaptation of a Laboratory-Based cDNA Library Preparation Protocol for Retrospective Sequencing of Archived MicroRNAs from up to 35-Year-Old Clinical FFPE Specimens.

Science.gov (United States)

Loudig, Olivier; Wang, Tao; Ye, Kenny; Lin, Juan; Wang, Yihong; Ramnauth, Andrew; Liu, Christina; Stark, Azadeh; Chitale, Dhananjay; Greenlee, Robert; Multerer, Deborah; Honda, Stacey; Daida, Yihe; Spencer Feigelson, Heather; Glass, Andrew; Couch, Fergus J; Rohan, Thomas; Ben-Dov, Iddo Z

2017-03-14

Formalin-fixed paraffin-embedded (FFPE) specimens, when used in conjunction with patient clinical data history, represent an invaluable resource for molecular studies of cancer. Even though nucleic acids extracted from archived FFPE tissues are degraded, their molecular analysis has become possible. In this study, we optimized a laboratory-based next-generation sequencing barcoded cDNA library preparation protocol for analysis of small RNAs recovered from archived FFPE tissues. Using matched fresh and FFPE specimens, we evaluated the robustness and reproducibility of our optimized approach, as well as its applicability to archived clinical specimens stored for up to 35 years. We then evaluated this cDNA library preparation protocol by performing a miRNA expression analysis of archived breast ductal carcinoma in situ (DCIS) specimens, selected for their relation to the risk of subsequent breast cancer development and obtained from six different institutions. Our analyses identified six miRNAs (miR-29a, miR-221, miR-375, miR-184, miR-363, miR-455-5p) differentially expressed between DCIS lesions from women who subsequently developed an invasive breast cancer (cases) and women who did not develop invasive breast cancer within the same time interval (control). Our thorough evaluation and application of this laboratory-based miRNA sequencing analysis indicates that the preparation of small RNA cDNA libraries can reliably be performed on older, archived, clinically-classified specimens.

UVB DNA dosimeters analyzed by polymerase chain reactors

International Nuclear Information System (INIS)

Yoshida, Hiroko; Regan, J.D.; Florida Inst. of Tech., Melbourne, FL

1997-01-01

Purified bacteriophage λ DNA was dried on a UV-transparent polymer film and served as a UVB dosimeter for personal and ecological applications. Bacteriophage λ DNA was chosen because it is commercially available and inexpensive, and its entire sequence is known. Each dosimeter contained two sets of DNA sandwiched between UV-transparent polymer films, one exposed to solar radiation (experimental) and another protected from UV radiation by black paper (control). The DNA dosimeter was then analyzed by a polymerase chain reaction (PCR) that amplifies a 500 base pair specific region of λ DNA. Photoinduced damage in DNA blocks polymerase from synthesizing a new strand; therefore, the amount of amplified product in UV-exposed DNA was reduced from that found in control DNA. The dried λ DNA dosimeter is compact, robust, safe and transportable, stable over long storage times and provides the total UVB dose integrated over the exposure time. (author)

Semi-commercialization of PVP-carrageenan hydrogel

International Nuclear Information System (INIS)

Relleve, Lorna S.; Abad, Lucille V.; Aranilla, Charito T.; Dela Rosa, A.M.

2008-01-01

The Philippine Nuclear Research Institute (PNRI) has developed the PVP-Carrageenan hydrogel wound dressing by radiation processing. The PVP-Carrageenan hydrogel has undergone clinical testing for burn and bedsores. It has already a pending patent application (No. 1-2000-02471) at the Philippine Patent Office. The techno-economic feasibility study has also been completed. In order to commercialize this product, a project on semi-commercialization in partnership with the investor was proposed to Technology Incubation for Commercialization (TECHNICOM), a technology transfer program of the Department of Science and Technology (DOST). TECHNICOM was established in 2003 under the National Science and Technology Plan (2002-2020) as a strategic technology transfer program. The program aims to identify key technological breakthroughs especially those generated by R and D institutes. It can intervene through the following: technology assessment/commercial prototype development; business plan/feasibility study preparation; intellectual property rights protection; technology valuation negotiation and licensing; semi-commercial production assistance and training/consultancy services. High technology applications with commercial potentials are given priority. Under semi-commercialization stage, government funds will be provided to match private sector investment in the commercial application of a particular technology innovation. This will lessen the risk of commercialization and ensure commitment from the investors. Commercial success in the shortest time is ensured since scientist can then work closely with the private sector at the production floor while testing the gaps in the technology. (author)

Bypass of a psoralen DNA interstrand cross-link by DNA polymerases beta, iota, and kappa in vitro

Science.gov (United States)

Smith, Leigh A.; Makarova, Alena V.; Samson, Laura; Thiesen, Katherine E.; Dhar, Alok; Bessho, Tadayoshi

2012-01-01

Repair of DNA inter-strand cross-links in mammalian cells involves several biochemically distinctive processes, including the release of one of the cross-linked strands and translesion DNA synthesis (TLS). In this report, we investigated in vitro TLS activity of psoralen DNA inter-strand cross-link by three DNA repair polymerases, DNA polymerase beta, kappa and iota. DNA polymerase beta is capable of bypassing a psoralen cross-link with a low efficiency. Cell extracts prepared from DNA polymerase beta knockout mouse embryonic fibroblast showed a reduced bypass activity of the psoralen cross-link and purified DNA polymerase beta restored the bypass activity. In addition, DNA polymerase iota mis-incorporated thymine across the psoralen cross-link and DNA polymerase kappa extended these mis-paired primer ends, suggesting that DNA polymerase iota may serve as an inserter and DNA polymerase kappa may play a role as an extender in the repair of psoralen DNA inter-strand cross-links. The results demonstrated here indicate that multiple DNA polymerases could participate in TLS steps in mammalian DNA inter-strand cross-link repair. PMID:23106263

Human mitochondrial DNA (mtDNA) types in Malaysia

International Nuclear Information System (INIS)

Lian, L.H.; Koh, C.L.; Lim, M.E.

2000-01-01

Each human cell contains hundreds of mitochondria and thousands of double-stranded circular mtDNA. The delineation of human mtDNA variation and genetics over the past decade has provided unique and often startling insights into human evolution, degenerative diseases, and aging. Each mtDNA of 16,569 base pairs, encodes 13 polypeptides essential to the enzymes of the mitochondrial energy generating pathway, plus the necessary tRNAs and rRNAs. The highly polymorphic noncoding D-(displacement) loop region, also called the control region, is approximately 1.2 kb long. It contains two well-characterized hypervariable (HV-) regions, HV1 and HV2. MtDNA identification is usually based on these sequence differences. According to the TWTGDAM (Technical Working Group for DNA Analysis Methods), the minimum requirement for a mtDNA database for HV1 is from positions 16024 to 16365 and for HV2, from positions 00073 to 00340. The targeted Malaysian population subgroups for this study were mainly the Malays, Chinese, Indians, and indigenous Ibans, Bidayuhs, Kadazan-Dusuns, and Bajaus. Research methodologies undertaken included DNA extraction of samples from unrelated individuals, amplification of the specific regions via the polymerase chain reaction (PCR), and preparation of template DNA for sequencing by using an automated DNA sequencer. Sufficient nucleotide sequence data were generated from the mtDNA analysis. When the sequences were analyzed, sequence variations were found to be caused by nucleotide substitutions, insertions, and deletions. Of the three causes of the sequence variations, nucleotide substitutions (86.1%) accounted for the vast majority of polymorphism. It is noted that transitions (83.5%) were predominant when compared to the significantly lower frequencies of transversions (2.6%). Insertions (0.9%) and deletions (13.0%) were rather rare and found only in HV2. The data generated will also form the basis of a Malaysian DNA sequence database of mtDNA D

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.

One-pot synthesis of strongly fluorescent DNA-CuInS2 quantum dots for label-free and ultrasensitive detection of anthrax lethal factor DNA

International Nuclear Information System (INIS)

Liu, Ziping; Su, Xingguang

2016-01-01

Herein, high quality DNA-CuInS 2 QDs are facilely synthesized through a one-pot hydrothermal method with fluorescence quantum yield as high as 23.4%, and the strongly fluorescent DNA-CuInS 2 QDs have been utilized as a novel fluorescent biosensor for label-free and ultrasensitive detection of anthrax lethal factor DNA. L-Cysteine (L-Cys) and a specific-sequence DNA are used as co-ligands to stabilize the CuInS 2 QDs. The specific-sequence DNA consists of two domains: phosphorothiolates domain (sulfur-containing variants of the usual phosphodiester backbone) controls the nanocrystal passivation and serves as a ligand, and the functional domain (non-phosphorothioates) controls the biorecognition. The as-prepared DNA-CuInS 2 QDs have high stability, good water-solubility and low toxicity. Under the optimized conditions, a linear correlation was established between the fluorescence intensity ratio I/I 0 (I 0 is the original fluorescence intensity of DNA-CuInS 2 QDs, and I is the fluorescence intensity of DNA-CuInS 2 QDs/GO with the addition of various concentrations of anthrax lethal factor DNA) and the concentration of anthrax lethal factor DNA in the range of 0.029–0.733 nmol L −1 with a detection limit of 0.013 nmol L −1 . The proposed method has been successfully applied to the determination of anthrax lethal factor DNA sequence in human serum samples with satisfactory results. Because of low toxicity and fine biocompatibility, DNA-CuInS 2 QDs also hold potential applications in bioimaging. - Highlights: • Strongly fluorescent DNA-QDs were successfully prepared by a one-pot hydrothermal method with quantum yield up to 23.4%. • A biosensor for label-free detection of anthrax lethal factor DNA was established based on the as-prepared DNA-QDs. • The DNA sensor took advantage of the feature that ssDNA binds to GO with significantly higher affinity than dsDNA. • Good sensitivity and selectivity were obtained. • This method was utilized to detect

Gibberellin-induced change in the structure of chromatin in wheat sprouts: decrease in the accessibility of DNA in preparations of soluble chromatin to the action of EcoRII methylase

International Nuclear Information System (INIS)

Noskov, V.A.; Kintsurashvili, L.N.; Smirnova, T.A.; Manamsh'yan, T.A.; Kir'yanov, G.I.; Vanyushin, B.F.

1986-01-01

A method has been perfected for producing soluble chromatin from whole wheat sprouts at low ionic strength. The chromatin preparations isolated possess a native structure: they have a nucleosome organization. Under identical conditions the soluble wheat chromatin undergoes more profound degradation by DNase I and staphylococcal nuclease than the chromatin from the rat liver. The DNA contained in the isolated chromatin is capable of accepting CHnumber groups from S-[methyl- 3 H]-adenosylmethionine during incubation with DNA methylase EcoRII; not all the CC A/T GG sequences in DNA are methylated in vivo. Chromatin from gibberellin A 3 -treated wheat sprout DNA accepts 40% fewer CH 3 groups than that from the control sprouts, which is probably due to the greater compactness of the chromatin. In the case of longer incubation, the level of methylation of the chromatin falls, which may be associated with the presence of DNA-demethylating activity

Time of flight Laue fiber diffraction studies of perdeuterated DNA

Energy Technology Data Exchange (ETDEWEB)

Forsyth, V.T.; Whalley, M.A.; Mahendrasingam, A.; Fuller, W. [Keele Univ. (United Kingdom)] [and others

1994-12-31

The diffractometer SXD at the Rutherford Appleton Laboratory ISIS pulsed neutron source has been used to record high resolution time-of-flight Laue fiber diffraction data from DNA. These experiments, which are the first of their kind, were undertaken using fibers of DNA in the A conformation and prepared using deuterated DNA in order to minimis incoherent background scattering. These studies complement previous experiments on instrument D19 at the Institute Laue Langevin using monochromatic neutrons. Sample preparation involved drawing large numbers of these deuterated DNA fibers and mounting them in a parallel array. The strategy of data collection is discussed in terms of camera design, sample environment and data collection. The methods used to correct the recorded time-of-flight data and map it into the final reciprocal space fiber diffraction dataset are also discussed. Difference Fourier maps showing the distribution of water around A-DNA calculated on the basis of these data are compared with results obtained using data recorded from hydrogenated A-DNA on D19. Since the methods used for sample preparation, data collection and data processing are fundamentally different for the monochromatic and Laue techniques, the results of these experiments also afford a valuable opportunity to independently test the data reduction and analysis techniques used in the two methods.

Perfusion chromatography separation of the tomato fruit-specific pectin methylesterase from a semipurified commercial enzyme preparation.

Science.gov (United States)

Savary, B J

2001-08-01

A rapid and simple method was developed, using perfusion chromatography media, to separate the fruit-specific pectin methylesterase (PME) isoform from the depolymerizing enzyme polygalacturonase (PG) and other contaminating pectinases present in a commercial tomato enzyme preparation. Pectinase activities were adsorbed onto a Poros HS (a strong cation exchanger) column in 20 M HEPES buffer at pH 7.5. The fruit-specific PME was eluted from the column with 80 mM NaCl, followed by a step to 300 mM NaCl to elute PG activity. Rechromatography of the PME activity peak with a linear gradient further resolved two PME isoenzymes and removed residual traces of PG activity. The PG activity peak was further treated with lectin affinity chromatography to provide purified PG enzyme, which was separated from a salt-dependent PME (tentatively identified as a "ubiquitous-type" isoform), and a pectin acetylesterase. The later enzyme has not been reported previously in tomato. This method provides monocomponent enzymes that will be useful for studying enzyme mechanisms and for modifying pectin structure and functional properties.

Lipopolysaccharide contamination in intradermal DNA vaccination : toxic impurity or adjuvant?

NARCIS (Netherlands)

Berg, J.H. van den; Quaak, S.G.L.; Beijnen, J.H.; Hennink, W.E.; Storm, G.; Schumacher, T.N.; Haanen, J.B.A.G.; Nuijen, B.

Purpose: Lipopolysaccharides (LPS) are known both as potential adjuvants for vaccines and as toxic impurity in pharmaceutical preparations. The aim of this study was to assess the role of LPS in intradermal DNA vaccination administered by DNA tattooing. Method: Micewere vaccinated with a model DNA

Comparative Analysis of the Genomic DNA Isolation Methods on Inula sp. (Asteraceae

Directory of Open Access Journals (Sweden)

Emre SEVİNDİK

2016-12-01

Full Text Available Simple, fast, low-cost and high throughput protocols are required for DNA isolation of plant species. In this study, phenol chloroform isoamyl alcohol and commercial (Sigma DNA isolation kit methods were applied on some Inula species that belong to Asteraceae family. Genomic DNA amounts, A260, A280, A260/A230 and purity degrees (A260/A280 that were obtained through both methods were measured through electrophoresis and spectrophotometer. Additionally, PCR amplification was realized by primer pairs specific to nrDNA ITS, cpDNA ndhF (972F-1603R and trnL-F regions. Results showed that maximum genomic DNA in nanograms obtained by phenol chloroform isoamyl alcohol method. The study also revealed that I. macrocephala had the maximum DNA and I. heterolepis had the minimum DNA amount. A260/A280 purity degrees showed that the highest and lowest purity in gDNAs obtained through phenol-choloform isoamyl alcohol method were in I.aucheriana and I. salicina, respectively. The highest and lowest purity degrees of gDNAs obtained through commercial kit was observed in I. fragilis and I. macrocephala samples, respectively. PCR amplification results showed that while band profiles of each three regions (ITS, trnL-F and ndhF did not yield positive results in PCR amplifications using phenol-choloform isoamyl alcohol method; PCR band profiles obtained through commercial kit yielded positive results. As a result, it is fair to say that the relation of genomic DNA with PCR was found to be more efficient although the maximum amount of genomic DNA was obtained through phenol chloroform isoamyl alcohol method.

Physical-chemical and biological characterization of different preparations of equine chorionic gonadotropin

OpenAIRE

Alvarez, Rafael Herrera; Natal, Fabio Luis Nogueira; Ribela, Maria Teresa Carvalho Pinto; de Almeida, Beatriz Elane; de Oliveira, Jo?o Ezequiel; Bartolini, Paolo

2016-01-01

Ovarian stimulation with commercial preparations of equine chorionic gonadotropin (eCG) produces extremely variable responses in domestic animals, ranging from excessive stimulation to practically no stimulation, when applied on the basis of their declared unitage. This study was conducted to analyze four commercial preparations from different manufacturers via reversed-phase HPLC (RP-HPLC) in comparison with a reference preparation and an official International Standard from the World Health...

One-pot synthesis of strongly fluorescent DNA-CuInS{sub 2} quantum dots for label-free and ultrasensitive detection of anthrax lethal factor DNA

Energy Technology Data Exchange (ETDEWEB)

Liu, Ziping; Su, Xingguang, E-mail: suxg@jlu.edu.cn

2016-10-26

Herein, high quality DNA-CuInS{sub 2} QDs are facilely synthesized through a one-pot hydrothermal method with fluorescence quantum yield as high as 23.4%, and the strongly fluorescent DNA-CuInS{sub 2} QDs have been utilized as a novel fluorescent biosensor for label-free and ultrasensitive detection of anthrax lethal factor DNA. L-Cysteine (L-Cys) and a specific-sequence DNA are used as co-ligands to stabilize the CuInS{sub 2} QDs. The specific-sequence DNA consists of two domains: phosphorothiolates domain (sulfur-containing variants of the usual phosphodiester backbone) controls the nanocrystal passivation and serves as a ligand, and the functional domain (non-phosphorothioates) controls the biorecognition. The as-prepared DNA-CuInS{sub 2} QDs have high stability, good water-solubility and low toxicity. Under the optimized conditions, a linear correlation was established between the fluorescence intensity ratio I/I{sub 0} (I{sub 0} is the original fluorescence intensity of DNA-CuInS{sub 2} QDs, and I is the fluorescence intensity of DNA-CuInS{sub 2} QDs/GO with the addition of various concentrations of anthrax lethal factor DNA) and the concentration of anthrax lethal factor DNA in the range of 0.029–0.733 nmol L{sup −1} with a detection limit of 0.013 nmol L{sup −1}. The proposed method has been successfully applied to the determination of anthrax lethal factor DNA sequence in human serum samples with satisfactory results. Because of low toxicity and fine biocompatibility, DNA-CuInS{sub 2} QDs also hold potential applications in bioimaging. - Highlights: • Strongly fluorescent DNA-QDs were successfully prepared by a one-pot hydrothermal method with quantum yield up to 23.4%. • A biosensor for label-free detection of anthrax lethal factor DNA was established based on the as-prepared DNA-QDs. • The DNA sensor took advantage of the feature that ssDNA binds to GO with significantly higher affinity than dsDNA. • Good sensitivity and selectivity were

[The effect of structure of benzimidazoles on the character of forming intramolecular cross-links in DNA and chromatin].

Science.gov (United States)

Mil', E M; Zhil'tsova, V M; Biniukov, V I; Zhizhina, G P; Stoliarova, L G; Kuznetsov, Iu P

1994-01-01

An investigation of a number of benzimidazole class preparations, being distinguished by a position of aminomethyl substitutes, has been carried out. It has been shown, that the non-substituted preparation BIO-10 does not form UV-cross-links in DNA and chromatine; BIO-40, having one substitute in the position 2, causes the formation of inter-molecular cross-links DNA-DNA. The preparation BIO-50, having 2 aminomethyl groups in the imidazole nucleus positions 2 and 6, forms cross-links DNA-DNA and DNA-protein in chromatine. The generation of radicals by the preparations BIO-10 and BIO-50 has been studied by the EPR-method by use of spin trap. It has been demonstrated, that BIO-10, unlike BIO-50, actively generates superoxide. A supposition has been made, that an UV-formation of superoxide-radical in the presence of BIO-10 might be a reason of DNA-macromolecule destruction.

Survey of current trends in DNA synthesis core facilities.

Science.gov (United States)

Hager, K M; Fox, J W; Gunthorpe, M; Lilley, K S; Yeung, A

1999-12-01

The Nucleic Acids Research Group of the Association of Biomolecular Resource Facilities (ABRF) last surveyed DNA synthesis core facilities in April 1995. Because of the introduction of new technologies and dramatic changes in the market, we sought to update survey information and to determine how academic facilities responded to the challenge presented by commercial counterparts. The online survey was opened in January 1999 by notifying members and subscribers to the ABRF electronic discussion group. The survey consisted of five parts: general facility information, oligonucleotide production profile, oligonucleotide charges, synthesis protocols, and trends in DNA synthesis (including individual comments). All submitted data were anonymously coded. Respondents from DNA synthesis facilities were primarily from the academic category and were established between 1984 and 1991. Typically, a facility provides additional services such as DNA sequencing and has upgraded to electronic ordering. There is stability in staffing profiles for these facilities in that the total number of employees is relatively unchanged, the tenure for staff averages 5.9 years, and experience is extensive. On average, academic facilities annually produce approximately 1/16 the number of oligonucleotides produced by the average commercial facilities, but all facilities report an increase in demand. Charges for standard oligonucleotides from academic facilities are relatively higher than from commercial companies; however, the opposite is true for modified phosphoramidites. Subsidized facilities charge less than nonsubsidized facilities. Synthesis protocols and reagents are standard across the categories. Most facilities offer typical modifications such as biotinylation. Despite the competition by large commercial facilities that have reduced costs dramatically, academic facilities remain a stable entity. Academic facilities enhance the quality of service by focusing on nonstandard

Recent advances in yeast molecular biology: recombinant DNA. [Lead abstract

Energy Technology Data Exchange (ETDEWEB)

1982-09-01

Separate abstracts were prepared for the 25 papers presented at a workshop focusing on chromosomal structure, gene regulation, recombination, DNA repair, and cell type control, that have been obtained by experimental approaches incorporating the new technologies of yeast DNA transformation, molecular cloning, and DNA sequence analysis. (KRM)

Studies on DNA repair in Bacillus subtilis

International Nuclear Information System (INIS)

Inoue, Tadashi; Kada, Tsuneo

1977-01-01

An enzyme which enhances the priming activity of γ-irradiated DNA for type I DNA polymerase (EC 2.7.7.7) was identified and partially purified from extracts of Bacillus subtilis cells. The enzyme preferentially degraded γ-irradiated DNA into acid-soluble materials. DNA preparations treated with heat, ultraviolet light, pancreatic DNAase (EC 3.1.4.5) or micrococcal DNAase (EC 3.1.4.7) were not susceptible to the enzyme. However, sonication rendered DNA susceptible to the enzyme to some extent. From these results, it is supposed that this enzyme may function by 'cleaning' damaged terminals produced by γ-irradiation to serve as effective primer of sites for repair synthesis by the type I DNA polymerase

A Fast, Sensitive and Label Free Electrochemical DNA Sensor

International Nuclear Information System (INIS)

Chen Yu; Elling; Lee Yokeling; Chong Serchoong

2006-01-01

A label free and sensitive DNA/RNA silicon based electrochemical microsensor array was developed by using thin film of the conducting polymer polypyrrole doped with an oligonucleotide probe. The electrochemical potential pulse amperometry technique was used for a biowarfare pathogen target DNA detection. The electrical potential assistanted DNA hybridisation method was applied. The sensor signal was increased by increasing the electrical potential assistanted DNA hybridisation time. It was possible to detect 0.34pmol and 0.072fmol of complementary oligonucleotide target in 0.1ml in seconds by using unpolished and polished gold electrode respectively. The probe preparation was also in seconds time, comparing indirect electrochemical DNA sensor, it has a fast sensor preparation as well as sensor response and label free advantages. The silicon microfabrication technique was used for this sensor array fabrication, which holds the potential to integrate with sensor electrical circuits. The conducting polymer polypyrrole was electrochemically deposited on each electrode respectively which has a possibility to dope the different DNA probe into the individual electrode to form a sensor array

Comparison of Three Different DNA Extraction Methods for Linguatula serrata as a Food Born Pathogen

Directory of Open Access Journals (Sweden)

Gilda ESLAMI

2017-06-01

Full Text Available Background: One of the most important items in molecular characterization of food-borne pathogens is high quality genomic DNA. In this study, we investigated three protocols and compared their simplicity, duration and costs for extracting genomic DNA from Linguatula serrata.Methods: The larvae were collected from the sheep’s visceral organs from the Yazd Slaughterhouse during May 2013. DNA extraction was done in three different methods, including commercial DNA extraction kit, Phenol Chloroform Isoamylalcohol (PCI, and salting out. Extracted DNA in each method was assessed for quantity and quality using spectrophotometery and agarose gel electrophoresis, respectively.Results: The less duration was regarding to commercial DNA extraction kit and then salting out protocol. The cost benefit one was salting out and then PCI method. The best quantity was regarding to PCI with 72.20±29.20 ng/μl, and purity of OD260/OD280 in 1.76±0.947. Agarose gel electrophoresis for assessing the quality found all the same.Conclusion: Salting out is introduced as the best method for DNA extraction from L. seratta as a food-borne pathogen with the least costand appropriate purity. Although, the best purity was regarding to PCI but PCI is not safe as salting out. In addition, the duration of salting out was less than PCI. The least duration was seen in commercial DNA extraction kit, but it is expensive and therefore is not recommended for developing countries where consumption of offal is common.

DNA barcode identification of commercial fish sold in Mexican markets.

Science.gov (United States)

Sarmiento-Camacho, Stephanie; Valdez-Moreno, Martha

2018-04-24

The substitution of high-value fish species for those of lower value is common practice. Although numerous studies have addressed this issue, few have been conducted in Mexico. In this study, we sought to identify fresh fillets of fish, sharks, and rays using DNA barcodes. We analyzed material from "La Viga" in Mexico City, and other markets located on the Gulf and Caribbean coasts of Mexico. From 134 samples, we obtained sequences from 129, identified to 9 orders, 28 families, 38 genera, and 44 species. The most common species were Seriola dumerili, Pangasianodon hypophthalmus, Carcharhinus falciformis, Carcharhinus brevipinna, and Hypanus americanus. Pangasianodon hypophthalmus was most commonly used as a substitute for higher-value species. The substitution rate was 18% of the total. A review of the conservation status of the specimens identified against the IUNC list enabled us to establish that some species marketed in Mexico are threatened: Makaira nigricans, Lachnolaimus maximus, Hyporthodus flavolimbatus, and Isurus oxyrinchus are classified as vulnerable; Lopholatilus chamaeleonticeps and Sphyrna lewini are endangered; and the status of Hyporthodus nigritus is critical. These results will demonstrate to the Mexican authorities that DNA barcoding is a reliable tool for species identification, even when morphological identification is difficult or impossible.

A high throughput system for the preparation of single stranded templates grown in microculture.

Science.gov (United States)

Kolner, D E; Guilfoyle, R A; Smith, L M

1994-01-01

A high throughput system for the preparation of single stranded M13 sequencing templates is described. Supernatants from clones grown in 48-well plates are treated with a chaotropic agent to dissociate the phage coat protein. Using a semi-automated cell harvester, the free nucleic acid is bound to a glass fiber filter in the presence of chaotrope and then washed with ethanol by aspiration. Individual glass fiber discs are punched out on the cell harvester and dried briefly. The DNA samples are then eluted in water by centrifugation. The processing time from 96 microcultures to sequence quality templates is approximately 1 hr. Assuming the ability to sequence 400 bases per clone, a 0.5 megabase per day genome sequencing facility will require 6250 purified templates a week. Toward accomplishing this goal we have developed a procedure which is a modification of a method that uses a chaotropic agent and glass fiber filter (Kristensen et al., 1987). By exploiting the ability of a cell harvester to uniformly aspirate and wash 96 samples, a rapid system for high quality template preparation has been developed. Other semi-automated systems for template preparation have been developed using commercially available robotic workstations like the Biomek (Mardis and Roe, 1989). Although minimal human intervention is required, processing time is at least twice as long. Custom systems based on paramagnetic beads (Hawkins et al., 1992) produce DNA in insufficient quantity for direct sequencing and therefore require cycle sequencing. These systems require custom programing, have a fairly high initial cost and have not proven to be as fast as the method reported here.

Effect of DNA extraction in the Rosa canina L. identification under different processing temperature

Directory of Open Access Journals (Sweden)

Jana Žiarovská

2017-01-01

Full Text Available Rosa canina, L. is widely used for medicinal purposes as well as in food industry where it is a valuable source, bioactive compounds and natural colorants. Actually, no specific method is recommended as suitable one for DNA extraction from rose hips. The aim of the study was to compare three commercial and three non-commercial methods to extract total genomic DNA from rose hips hyphanthium. Four methods are based on the precipitation in principle and two methods are based on resin-binding. Extracted DNA was proved for the effectivity in following PCR. In total, six different DNA isolations was performed for differently heat processes rose hips - fresh hyphanthium, 2-weeks frozen hyphanthium, dried hyphanthium (50 °C and boiled hyphanthium (100 °C. The amplification parameters of 500 bp chloroplast gene amplicon were evaluated. Obtained amounts of extracted DNA was very variable not only for every individual method used but for individual treatment of samples, too. In general, non-commercial method provided higher amount of extracted DNA, but the A260/280 ratio was lower. When regarding the processing treatment of the samples, high differences were found among the samples untreated by heat and those that were dried or boiled for three of the used extraction methods. All the samples were positive for amplification, but different amounts of amplified product were obtained. The comparison of data for concentrations of extracted DNA and concentrations of amplified product showed large differences when regarding the achieved purity of DNA in extraction.

Study of microtip-based extraction and purification of DNA from human samples for portable devices

Science.gov (United States)

Fotouhi, Gareth

DNA sample preparation is essential for genetic analysis. However, rapid and easy-to-use methods are a major challenge to obtaining genetic information. Furthermore, DNA sample preparation technology must follow the growing need for point-of-care (POC) diagnostics. The current use of centrifuges, large robots, and laboratory-intensive protocols has to be minimized to meet the global challenge of limited access healthcare by bringing the lab to patients through POC devices. To address these challenges, a novel extraction method of genomic DNA from human samples is presented by using heat-cured polyethyleneimine-coated microtips generating a high electric field. The microtip extraction method is based on recent work using an electric field and capillary action integrated into an automated device. The main challenges to the method are: (1) to obtain a stable microtip surface for the controlled capture and release of DNA and (2) to improve the recovery of DNA from samples with a high concentration of inhibitors, such as human samples. The present study addresses these challenges by investigating the heat curing of polyethyleneimine (PEI) coated on the surface of the microtip. Heat-cured PEI-coated microtips are shown to control the capture and release of DNA. Protocols are developed for the extraction and purification of DNA from human samples. Heat-cured PEI-coated microtip methods of DNA sample preparation are used to extract genomic DNA from human samples. It is discovered through experiment that heat curing of a PEI layer on a gold-coated surface below 150°C could inhibit the signal of polymerase chain reaction (PCR). Below 150°C, the PEI layer is not completely cured and dissolved off the gold-coated surface. Dissolved PEI binds with DNA to inhibit PCR. Heat curing of a PEI layer above 150°C on a gold-coated surface prevents inhibition to PCR and gel electrophoresis. In comparison to gold-coated microtips, the 225°C-cured PEI-coated microtips improve the

Plant DNA Detection from Grasshopper Guts: A Step-by-Step Protocol, from Tissue Preparation to Obtaining Plant DNA Sequences

Directory of Open Access Journals (Sweden)

Alina Avanesyan

2014-02-01

Full Text Available Premise of the study: A PCR-based method of identifying ingested plant DNA in gut contents of Melanoplus grasshoppers was developed. Although previous investigations have focused on a variety of insects, there are no protocols available for plant DNA detection developed for grasshoppers, agricultural pests that significantly influence plant community composition. Methods and Results: The developed protocol successfully used the noncoding region of the chloroplast trnL (UAA gene and was tested in several feeding experiments. Plant DNA was obtained at seven time points post-ingestion from whole guts and separate gut sections, and was detectable up to 12 h post-ingestion in nymphs and 22 h post-ingestion in adult grasshoppers. Conclusions: The proposed protocol is an effective, relatively quick, and low-cost method of detecting plant DNA from the grasshopper gut and its different sections. This has important applications, from exploring plant “movement” during food consumption, to detecting plant–insect interactions.

Identification of G-Protein-Coupled Receptors (GPCRs) in Pulmonary Artery Smooth Muscle Cells as Novel Therapeutic Targets

Science.gov (United States)

2017-10-01

who do not have pulmonary hypertension (PH) Completed 12/2015 lb. Prepare RNA and cDNA from PASMCs and use Taqman GPCR arrays to identify and quantify...expression of GPCRs with known physiologic agonists .Completed 5/2017 le. Prepare RNA and cDNA from commercially available coronary artery and...Isolate and culture PASMCs from 8 (4 male; 4 female) 3 -month old rats and mice, prepare RNA and cDNA and assess expression of GPCRs with known

Comparison of three methods for recovery of Brucella canis DNA from canine blood samples.

Science.gov (United States)

Batinga, Maria Cryskely A; Dos Santos, Jaíne C; Lima, Julia T R; Bigotto, Maria Fernanda D; Muner, Kerstin; Faita, Thalita; Soares, Rodrigo M; da Silva, David A V; Oliveira, Trícia M F S; Ferreira, Helena L; Diniz, Jaqueline A; Keid, Lara B

2017-12-01

Brucella canis, a gram-negative, facultative intracellular and zoonotic bacterium causes canine brucellosis. Direct methods are the most appropriate for the detection of canine brucellosis and bacterial isolation from blood samples has been employed as gold-standard method. However, due to the delay in obtaining results and the biological risk of the bacterial culturing, the polymerase chain reaction (PCR) has been successfully used as an alternative method for the diagnosis of the infection. Sample preparation is a key step for successful PCR and protocols that provide high DNA yield and purity are recommended to ensure high diagnostic sensitivity. The objective of this study was to evaluate the performance of PCR for the diagnosis of B. canis infection in 36 dogs by testing DNA of whole blood obtained through different extraction and purification protocols. Methods 1 and 2 were based on a commercial kit, using protocols recommended for DNA purification of whole blood and tissue samples, respectively. Method 3 was an in-house method based on enzymatic lysis and purification using organic solvents. The results of the PCR on samples obtained through three different DNA extraction protocols were compared to the blood culture. Of the 36 dogs, 13 (36.1%) were positive by blood culturing, while nine (25.0%), 14 (38.8%), and 15 (41.6%) were positive by PCR after DNA extraction using methods 1, 2 and 3, respectively. PCR performed on DNA purified by Method 2 was as efficient as blood culturing and PCR performed on DNA purified with in-house method, but had the advantage of being less laborious and, therefore, a suitable alternative for the direct B. canis detection in dogs. Copyright © 2017. Published by Elsevier B.V.

DNA recognition by synthetic constructs.

Science.gov (United States)

Pazos, Elena; Mosquera, Jesús; Vázquez, M Eugenio; Mascareñas, José L

2011-09-05

The interaction of transcription factors with specific DNA sites is key for the regulation of gene expression. Despite the availability of a large body of structural data on protein-DNA complexes, we are still far from fully understanding the molecular and biophysical bases underlying such interactions. Therefore, the development of non-natural agents that can reproduce the DNA-recognition properties of natural transcription factors remains a major and challenging goal in chemical biology. In this review we summarize the basics of double-stranded DNA recognition by transcription factors, and describe recent developments in the design and preparation of synthetic DNA binders. We mainly focus on synthetic peptides that have been designed by following the DNA interaction of natural proteins, and we discuss how the tools of organic synthesis can be used to make artificial constructs equipped with functionalities that introduce additional properties to the recognition process, such as sensing and controllability. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Open source and DIY hardware for DNA nanotechnology labs.

Science.gov (United States)

Damase, Tulsi R; Stephens, Daniel; Spencer, Adam; Allen, Peter B

A set of instruments and specialized equipment is necessary to equip a laboratory to work with DNA. Reducing the barrier to entry for DNA manipulation should enable and encourage new labs to enter the field. We present three examples of open source/DIY technology with significantly reduced costs relative to commercial equipment. This includes a gel scanner, a horizontal PAGE gel mold, and a homogenizer for generating DNA-coated particles. The overall cost savings obtained by using open source/DIY equipment was between 50 and 90%.

Evaluation of Taste Properties of Commercially Available Salts

OpenAIRE

ISHIKAWA, Kyoko; SUGIMOTO, Maho; KUMAGAI, Masanori; MATSUNAGA, Ryuji

2006-01-01

This study examined commercially available salts'taste properties. The salts were used in preparation of four dishes: asazuke of cucumber, asazuke of Chinese cabbage, clear soup, and green soybean rice. The respective tastes of the salts in those prepared foods differed from those of the salts alone. We evaluated the parameters: saltiness, mildness, unpleasantness, and palatability. Differences of the salt samples affected the perception of saltiness. Results of taste sensor analyses showed t...

Plasmid DNA loaded chitosan nanoparticles for nasal mucosal immunization against hepatitis B.

Science.gov (United States)

Khatri, Kapil; Goyal, Amit K; Gupta, Prem N; Mishra, Neeraj; Vyas, Suresh P

2008-04-16

This work investigates the preparation and in vivo efficacy of plasmid DNA loaded chitosan nanoparticles for nasal mucosal immunization against hepatitis B. Chitosan pDNA nanoparticles were prepared using a complex coacervation process. Prepared nanoparticles were characterized for size, shape, surface charge, plasmid loading and ability of nanoparticles to protect DNA against nuclease digestion and for their transfection efficacy. Nasal administration of nanoparticles resulted in serum anti-HBsAg titre that was less compared to that elicited by naked DNA and alum adsorbed HBsAg, but the mice were seroprotective within 2 weeks and the immunoglobulin level was above the clinically protective level. However, intramuscular administration of naked DNA and alum adsorbed HBsAg did not elicit sIgA titre in mucosal secretions that was induced by nasal immunization with chitosan nanoparticles. Similarly, cellular responses (cytokine levels) were poor in case of alum adsorbed HBsAg. Chitosan nanoparticles thus produced humoral (both systemic and mucosal) and cellular immune responses upon nasal administration. The study signifies the potential of chitosan nanoparticles as DNA vaccine carrier and adjuvant for effective immunization through non-invasive nasal route.

Preparation, Single-Molecule Manipulation, and Energy Transfer Investigation of a Polyfluorene-graft-DNA polymer.

Science.gov (United States)

Madsen, Mikael; Christensen, Rasmus S; Krissanaprasit, Abhichart; Bakke, Mette R; Riber, Camilla F; Nielsen, Karina S; Zelikin, Alexander N; Gothelf, Kurt V

2017-08-04

Conjugated polymers have been intensively studied due to their unique optical and electronic properties combined with their physical flexibility and scalable bottom up synthesis. Although the bulk qualities of conjugated polymers have been extensively utilized in research and industry, the ability to handle and manipulate conjugated polymers at the nanoscale lacks significantly behind. Here, the toolbox for controlled manipulation of conjugated polymers was expanded through the synthesis of a polyfluorene-DNA graft-type polymer (poly(F-DNA)). The polymer possesses the characteristics associated with the conjugated polyfluorene backbone, but the protruding single-stranded DNA provides the material with an exceptional addressability. This study demonstrates controlled single-molecule patterning of poly(F-DNA), as well as energy transfer between two different polymer-DNA conjugates. Finally, highly efficient DNA-directed quenching of polyfluorene fluorescence was shown. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

The structure of DNA by direct imaging

KAUST Repository

Marini, Monica

2015-08-28

The structure of DNA was determined in 1953 by x-ray fiber diffraction. Several attempts have been made to obtain a direct image of DNA with alternative techniques. The direct image is intended to allow a quantitative evaluation of all relevant characteristic lengths present in a molecule. A direct image of DNA, which is different from diffraction in the reciprocal space, is difficult to obtain for two main reasons: the intrinsic very low contrast of the elements that form the molecule and the difficulty of preparing the sample while preserving its pristine shape and size. We show that through a preparation procedure compatible with the DNA physiological conditions, a direct image of a single suspended DNA molecule can be obtained. In the image, all relevant lengths of A-form DNA are measurable. A high-resolution transmission electron microscope that operates at 80 keV with an ultimate resolution of 1.5 Å was used for this experiment. Direct imaging of a single molecule can be used as a method to address biological problems that require knowledge at the single-molecule level, given that the average information obtained by x-ray diffraction of crystals or fibers is not sufficient for detailed structure determination, or when crystals cannot be obtained from biological molecules or are not sufficient in understanding multiple protein configurations.

The structure of DNA by direct imaging

KAUST Repository

Marini, Monica; Falqui, Andrea; Moretti, Manola; Limongi, Tania; Allione, Marco; Genovese, Alessandro; Lopatin, Sergei; Tirinato, Luca; Das, Gobind; Torre, Bruno; Giugni, Andrea; Gentile, Francesco; Candeloro, Patrizio; Di Fabrizio, Enzo M.

2015-01-01

The structure of DNA was determined in 1953 by x-ray fiber diffraction. Several attempts have been made to obtain a direct image of DNA with alternative techniques. The direct image is intended to allow a quantitative evaluation of all relevant characteristic lengths present in a molecule. A direct image of DNA, which is different from diffraction in the reciprocal space, is difficult to obtain for two main reasons: the intrinsic very low contrast of the elements that form the molecule and the difficulty of preparing the sample while preserving its pristine shape and size. We show that through a preparation procedure compatible with the DNA physiological conditions, a direct image of a single suspended DNA molecule can be obtained. In the image, all relevant lengths of A-form DNA are measurable. A high-resolution transmission electron microscope that operates at 80 keV with an ultimate resolution of 1.5 Å was used for this experiment. Direct imaging of a single molecule can be used as a method to address biological problems that require knowledge at the single-molecule level, given that the average information obtained by x-ray diffraction of crystals or fibers is not sufficient for detailed structure determination, or when crystals cannot be obtained from biological molecules or are not sufficient in understanding multiple protein configurations.

Performance of 2 commercial real-time polymerase chain reaction assays for the detection of Aspergillus and Pneumocystis DNA in bronchoalveolar lavage fluid samples from critical care patients.

Science.gov (United States)

Orsi, Carlotta Francesca; Gennari, William; Venturelli, Claudia; La Regina, Annunziata; Pecorari, Monica; Righi, Elena; Machetti, Marco; Blasi, Elisabetta

2012-06-01

This article investigates the performance of 2 commercial real-time polymerase chain reaction (PCR) assays, MycAssay™ Aspergillus (Myc(Asp)Assay) and MycAssay™ Pneumocystis (Myc(PCP)Assay), on the ABI 7300 platform for the detection of Aspergillus (Asp) or Pneumocystis jirovecii (Pj) DNA in bronchoalveolar lavage (BAL) samples from 20 patients. Operationally, patients enrolled were clustered into 3 groups: invasive aspergillosis group (IA, 7 patients), Pj pneumonia group (PCP, 8 patients), and negative control group (5 patients). All the IA patients were Myc(Asp)Assay positive, whereas 12 non-IA patients returned negative PCR results. Furthermore, 7 of 8 PCP patients were Myc(PCP)Assay positive, while 9 non-PCP patients were PCR negative. In conclusion, these data provide an early indication of the effectiveness of both the Myc(Asp)Assay and Myc(PCP)Assay on the ABI 7300 platform for the detection of either Asp or Pj DNA in BAL from patients with deep fungal infections. Copyright © 2012 Elsevier Inc. All rights reserved.

DNA Cleavage Activity of Diazonium Salts: Chemical Nucleases

OpenAIRE

KIZIL, Murat

2014-01-01

4-Fenoldiazonium tetrafluoroborate and 4-benzoicaciddiazonium tetrafluoroborate was prepared and was shown to be an effective DNA cleavage agent in the presence of the 1-electron donor copper(II) chloride. Its mechanism involves the generation of the aryl radical cleaving DNA by hydrogen atom abstraction from deoxyribose sugar.

Preparation of Fe3O4/poly(styrene-butyl acrylate-[2-(methacryloxy)ethyl]trimethylammonium chloride) by emulsifier-free emulsion polymerization and its interaction with DNA

International Nuclear Information System (INIS)

Li Xiaolong; Liu Guoqiang; Yan Wei; Chu, Paul K.; Yeung, Kelvin W.K.; Wu Shuilin; Yi Changfeng; Xu Zushun

2012-01-01

Cationic magnetic polymer particles Fe 3 O 4 /poly(styrene-butyl acrylate-[2-(methacryloxy)ethyl]trimethylammonium chloride), a type of potential gene carrier, were prepared by emulsifier-free emulsion polymerization with oleic acid modified magnetite Fe 3 O 4 , styrene, butyl acrylate and [2-(methacryloxy)ethyl]trimethylammonium chloride) (METAC). The morphology of the particles was characterized by transmission electron microscopy and the composites of particles were characterized by FT-IR spectroscopy, X-ray diffraction. These results showed that magnetic particles were well dispersed in polymers with the content of about 15%(wt/wt). The composites exhibited superparamagnetism and possessed a certain level of magnetic response. The interactions between the particles with calf-thymus DNA (ct DNA) were confirmed by zeta potential measurement, UV–vis spectroscopy and fluorescence spectroscopy. The DNA-binding capacity determined by the agarose gel electrophoresis showed good binding capacity of the emulsion to DNA. These results suggested the potential of the cationic magnetic polymer emulsion as gene target delivery carrier. - Highlights: ► A new type of cationic magnetic polymer particles was synthesized by emulsifier-free emulsion polymerization. ► Structural, morphological, and magnetic properties of the composite were evaluated. ► The interaction between cationic magnetic polymer particles with DNA was confirmed by zeta potential measurements. ► UV–vis spectrophotometry, fluorescent spectroscopy and agarose gel electrophoresis. ► This process may have potential applications to gene carrier and DNA separation.

Validation of DNA probes for molecular cytogenetics by mapping onto immobilized circular DNA

Energy Technology Data Exchange (ETDEWEB)

Greulich-Bode, Karin; Wang, Mei; Rhein, Andreas; Weier, Jingly; Weier, Heinz-Ulli

2008-12-16

Fluorescence in situ hybridization (FISH) is a sensitive and rapid procedure to detect gene rearrangements in tumor cells using non-isotopically labeled DNA probes. Large insert recombinant DNA clones such as bacterial artificial chromosome (BAC) or P1/PAC clones have established themselves in recent years as preferred starting material for probe preparations due to their low rates of chimerism and ease of use. However, when developing probes for the quantitative analysis of rearrangements involving genomic intervals of less than 100kb, careful probe selection and characterization are of paramount importance. We describe a sensitive approach to quality control probe clones suspected of carrying deletions or for measuring clone overlap with near kilobase resolution. The method takes advantage of the fact that P1/PAC/BAC's can be isolated as circular DNA molecules, stretched out on glass slides and fine-mapped by multicolor hybridization with smaller probe molecules. Two examples demonstrate the application of this technique: mapping of a gene-specific {approx}6kb plasmid onto an unusually small, {approx}55kb circular P1 molecule and the determination of the extent of overlap between P1 molecules homologous to the human NF-?B2 locus. The relatively simple method presented here does not require specialized equipment and may thus find widespread applications in DNA probe preparation and characterization, the assembly of physical maps for model organisms or in studies on gene rearrangements.

Validation of DNA probes for molecular cytogenetics by mapping onto immobilized circular DNA

Energy Technology Data Exchange (ETDEWEB)

Greulich-Bode, Karin M.; Wang, Mei; Rhein, Andreas P.; Weier, Jingly F.; Weier, Heinz-Ulli G.

2008-12-04

Fluorescence in situ hybridization (FISH) is a sensitive and rapid procedure to detect gene rearrangements in tumor cells using non-isotopically labeled DNA probes. Large insert recombinant DNA clones such as bacterial artificial chromosome (BAC) or P1/PAC clones have established themselves in recent years as preferred starting material for probe preparations due to their low rates of chimerism and ease of use. However, when developing probes for the quantitative analysis of rearrangements involving genomic intervals of less than 100kb, careful probe selection and characterization are of paramount importance. We describe a sensitive approach to quality control probe clones suspected of carrying deletions or for measuring clone overlap with near kilobase resolution. The method takes advantage of the fact that P1/PAC/BAC's can be isolated as circular DNA molecules, stretched out on glass slides and fine-mapped by multicolor hybridization with smaller probe molecules. Two examples demonstrate the application of this technique: mapping of a gene-specific {approx}6kb plasmid onto an unusually small, {approx}55kb circular P1 molecule and the determination of the extent of overlap between P1 molecules homologous to the human NF-{kappa}B2 locus. The relatively simple method presented here does not require specialized equipment and may thus find widespread applications in DNA probe preparation and characterization, the assembly of physical maps for model organisms or in studies on gene rearrangements.

DNA Source Selection for Downstream Applications Based on DNA Quality Indicators Analysis

Science.gov (United States)

Lucena-Aguilar, Gema; Sánchez-López, Ana María; Barberán-Aceituno, Cristina; Carrillo-Ávila, José Antonio; López-Guerrero, José Antonio

2016-01-01

High-quality human DNA samples and associated information of individuals are necessary for biomedical research. Biobanks act as a support infrastructure for the scientific community by providing a large number of high-quality biological samples for specific downstream applications. For this purpose, biobank methods for sample preparation must ensure the usefulness and long-term functionality of the products obtained. Quality indicators are the tool to measure these parameters, the purity and integrity determination being those specifically used for DNA. This study analyzes the quality indicators in DNA samples derived from 118 frozen human tissues in optimal cutting temperature (OCT) reactive, 68 formalin-fixed paraffin-embedded (FFPE) tissues, 119 frozen blood samples, and 26 saliva samples. The results obtained for DNA quality are discussed in association with the usefulness for downstream applications and availability of the DNA source in the target study. In brief, if any material is valid, blood is the most approachable option of prospective collection of samples providing high-quality DNA. However, if diseased tissue is a requisite or samples are available, the recommended source of DNA would be frozen tissue. These conclusions will determine the best source of DNA, according to the planned downstream application. Furthermore our results support the conclusion that a complete procedure of DNA quantification and qualification is necessary to guarantee the appropriate management of the samples, avoiding low confidence results, high costs, and a waste of samples. PMID:27158753

Adapting RNAseq sample preparation for ISS

Data.gov (United States)

National Aeronautics and Space Administration — The primary innovation for this CIF will be the ability to accomplish library preparation of isolated RNA that will enable transcriptional (RNA instead of DNA)...

Hair Dye–DNA Interaction: Plausible Cause of Mutation

Directory of Open Access Journals (Sweden)

Swati Maiti

2015-09-01

Full Text Available Hair dye is one of the most popular cosmetic products which are used more widely and frequently to improve an individual’s appearance. Although the genotoxic effects of dye ingredients are widely reported, hair dye in its usable form is not reported extensively. In this contribution, we report the possible mode of interaction of hair dye with DNA which leads to genotoxicity. The effect of dye DNA interaction was studied on the most popular and globally used hair dye with Calf Thymus DNA and plasmid DNA. This interaction of dye DNA was studied by spectroscopic analyses and gel electrophoresis. The result had shown positive interaction of dye with DNA. Gel electrophoresis study confirms the binding of dye with DNA which results in linearization and fragmentation of the plasmid DNA. Dye–DNA interaction causes fragmentation and oxidation of DNA in absence of any catalyst, implies high toxicity of commercial hair dyes. Thus, it can be deduced from the present studies that hair dye in its usable form may lead to its penetration through skin affecting genomic DNA possesses genotoxic property and can be treated as one of the most common mutagen.

Direct-to-PCR tissue preservation for DNA profiling.

Science.gov (United States)

Sorensen, Amy; Berry, Clare; Bruce, David; Gahan, Michelle Elizabeth; Hughes-Stamm, Sheree; McNevin, Dennis

2016-05-01

Disaster victim identification (DVI) often occurs in remote locations with extremes of temperatures and humidities. Access to mortuary facilities and refrigeration are not always available. An effective and robust DNA sampling and preservation procedure would increase the probability of successful DNA profiling and allow faster repatriation of bodies and body parts. If the act of tissue preservation also released DNA into solution, ready for polymerase chain reaction (PCR), the DVI process could be further streamlined. In this study, we explored the possibility of obtaining DNA profiles without DNA extraction, by adding aliquots of preservative solutions surrounding fresh human muscle and decomposing human muscle and skin tissue samples directly to PCR. The preservatives consisted of two custom preparations and two proprietary solutions. The custom preparations were a salt-saturated solution of dimethyl sulfoxide (DMSO) with ethylenediaminetetraacetic (EDTA) and TENT buffer (Tris, EDTA, NaCl, Tween 20). The proprietary preservatives were DNAgard (Biomatrica(®)) and Tissue Stabilising Kit (DNA Genotek). We obtained full PowerPlex(®) 21 (Promega) and GlobalFiler(®) (Life Technologies) DNA profiles from fresh and decomposed tissue preserved at 35 °C for up to 28 days for all four preservatives. The preservative aliquots removed from the fresh muscle tissue samples had been stored at -80 °C for 4 years, indicating that long-term archival does not diminish the probability of successful DNA typing. Rather, storage at -80 °C seems to reduce PCR inhibition.

Cationic Polybutyl Cyanoacrylate Nanoparticles for DNA Delivery

Directory of Open Access Journals (Sweden)

Jinghua Duan

2009-01-01

Full Text Available To enhance the intracellular delivery potential of plasmid DNA using nonviral vectors, we used polybutyl cyanoacrylate (PBCA and chitosan to prepare PBCA nanoparticles (NPs by emulsion polymerization and prepared NP/DNA complexes through the complex coacervation of nanoparticles with the DNA. The object of our work is to evaluate the characterization and transfection efficiency of PBCA-NPs. The NPs have a zeta potential of 25.53 mV at pH 7.4 and size about 200 nm. Electrophoretic analysis suggested that the NPs with positive charges could protect the DNA from nuclease degradation and cell viability assay showed that the NPs exhibit a low cytotoxicity to human hepatocellular carcinoma (HepG2 cells. Qualitative and quantitative analysis of transfection in HepG2 cells by the nanoparticles carrying plasmid DNA encoding for enhanced green fluorescent protein (EGFP-N1 was done by digital fluorescence imaging microscopy system and fluorescence-activated cell sorting (FACS. Qualitative results showed highly efficient expression of GFP that remained stable for up to 96 hours. Quantitative results from FACS showed that PBCA-NPs were significantly more effective in transfecting HepG2 cells after 72 hours postincubation. The results of this study suggested that PBCA-NPs have favorable properties for nonviral delivery.

A duplex DNA-gold nanoparticle probe composed as a colorimetric biosensor for sequence-specific DNA-binding proteins.

Science.gov (United States)

Ahn, Junho; Choi, Yeonweon; Lee, Ae-Ree; Lee, Joon-Hwa; Jung, Jong Hwa

2016-03-21

Using duplex DNA-AuNP aggregates, a sequence-specific DNA-binding protein, SQUAMOSA Promoter-binding-Like protein 12 (SPL-12), was directly determined by SPL-12-duplex DNA interaction-based colorimetric actions of DNA-Au assemblies. In order to prepare duplex DNA-Au aggregates, thiol-modified DNA 1 and DNA 2 were attached onto the surface of AuNPs, respectively, by the salt-aging method and then the DNA-attached AuNPs were mixed. Duplex-DNA-Au aggregates having the average size of 160 nm diameter and the maximum absorption at 529 nm were able to recognize SPL-12 and reached the equivalent state by the addition of ∼30 equivalents of SPL-12 accompanying a color change from red to blue with a red shift of the maximum absorption at 570 nm. As a result, the aggregation size grew to about 247 nm. Also, at higher temperatures of the mixture of duplex-DNA-Au aggregate solution and SPL-12, the equivalent state was reached rapidly. On the contrary, in the control experiment using Bovine Serum Albumin (BSA), no absorption band shift of duplex-DNA-Au aggregates was observed.

Sperm DNA fragmentation, recurrent implantation failure and recurrent miscarriage

Directory of Open Access Journals (Sweden)

Carol Coughlan

2015-01-01

Full Text Available Evidence is increasing that the integrity of sperm DNA may also be related to implantation failure and recurrent miscarriage (RM. To investigate this, the sperm DNA fragmentation in partners of 35 women with recurrent implantation failure (RIF following in vitro fertilization, 16 women diagnosed with RM and seven recent fathers (control were examined. Sperm were examined pre- and post-density centrifugation by the sperm chromatin dispersion (SCD test and the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL assay. There were no significant differences in the age of either partner or sperm concentration, motility or morphology between three groups. Moreover, there were no obvious differences in sperm DNA fragmentation measured by either test. However, whilst on average sperm DNA fragmentation in all groups was statistically lower in prepared sperm when measured by the SCD test, this was not seen with the results from the TUNEL assay. These results do not support the hypothesis that sperm DNA fragmentation is an important cause of RIF or RM, or that sperm DNA integrity testing has value in such patients. It also highlights significant differences between test methodologies and sperm preparation methods in interpreting the data from sperm DNA fragmentation tests.

A new approach for cloning hLIF cDNA from genomic DNA isolated from the oral mucous membrane.

Science.gov (United States)

Cui, Y H; Zhu, G Q; Chen, Q J; Wang, Y F; Yang, M M; Song, Y X; Wang, J G; Cao, B Y

2011-11-25

Complementary DNA (cDNA) is valuable for investigating protein structure and function in the study of life science, but it is difficult to obtain by traditional reverse transcription. We employed a novel strategy to clone human leukemia inhibitory factor (hLIF) gene cDNA from genomic DNA, which was directly isolated from the mucous membrane of mouth. The hLIF sequence, which is 609 bp long and is composed of three exons, can be acquired within a few hours by amplifying each exon and splicing all of them using overlap-PCR. This new approach developed is simple, time- and cost-effective, without RNA preparation or cDNA synthesis, and is not limited to the specific tissues for a particular gene and the expression level of the gene.

Studying of the standardization principles of pharmacological activity of recombinant erythropoietin preparations

OpenAIRE

A. K. Yakovlev; L. A. Gayderova; N. A. Alpatova; T. N. Lobanova; E. L. Postnova; E. I. Yurchikova; T. A. Batuashvili; R. A. Volkova; V. N. Podkuiko; Yu. V. Olefir

2016-01-01

Analysis of the publications devoted to the structure, functions, mechanism of action of erythropoietin is given in the article. Erythropoietin preparations derived from recombinant DNA technology are a mixture of isoforms with different biological activity, which determine the biological properties pharmacological activity, pharmacokinetics, efficacy and safety of medicinal product. Erythropoietin preparations derived by using recombinant DNA technology are a mixture of isoforms with differe...

Locally prepared antibiotic sensitivity discs: a substitute for imported ...

African Journals Online (AJOL)

Zones of inhibition were compared with those obtained from commercial antibiotic discs. Results obtained showed that discs prepared locally from antibiotic tablets, performed comparably with commercially obtained discs. There was no significant statistical difference between the two tested discs. We therefore recommend ...

Preparation of patient-related allergens for hyposensitization. Qualitative aspects

DEFF Research Database (Denmark)

Poulsen, L K; Søndergaard, I; Weeke, B

1988-01-01

An affinity chromatography method for preparation of patient-related antigens from commercially available allergen extracts has been investigated. IgG1,2,4 from a patient previously hyposensitized with dog hair and dandruff allergen was bound to protein A-sepharose. Secondly, commercial allergen ...

Preparation and characterization of the polyclonal antibody against ...

African Journals Online (AJOL)

To prepare the polyclonal antibody against GAR domain, cDNA encoding 466 amino acids protein of GAR domain was amplified from MG-63 cell by RT-PCR. The amplified cDNA, that exhibited 99% identity to the published sequence, was cloned into prokaryotic expression vector pQE-80L for the expression of GAR ...

High-Quality and -Quantity DNA Extraction from Frozen Archival Blood Clots for Genotyping of Single-Nucleotide Polymorphisms

DEFF Research Database (Denmark)

Bank, Steffen; Nexø, Bjørn Andersen; Andersen, Vibeke

2013-01-01

the efficiency of commercial purification kits for extracting DNA from long-term frozen clotted blood. Methods: Serum tubes with clotted blood were stored at −20°C for 1 to 2.5 years before DNA extraction. DNA was extracted from 10 blood clot samples using PureGene (Qiagen) with and without glycogen, the QIAamp...... with a median of 0.65 μg (range 0.5–2.6 μg) pr 300 μL total blood. Conclusion: The yield obtained by the different commercial kits varied considerably. Our work demonstrates that high-quality and -quantity DNA can be extracted with the Maxwell 16 Blood purification kit (Promega) from cryopreserved blood clots...

Plant DNA detection from grasshopper guts: A step-by-step protocol, from tissue preparation to obtaining plant DNA sequences1

Science.gov (United States)

Avanesyan, Alina

2014-01-01

• Premise of the study: A PCR-based method of identifying ingested plant DNA in gut contents of Melanoplus grasshoppers was developed. Although previous investigations have focused on a variety of insects, there are no protocols available for plant DNA detection developed for grasshoppers, agricultural pests that significantly influence plant community composition. • Methods and Results: The developed protocol successfully used the noncoding region of the chloroplast trnL (UAA) gene and was tested in several feeding experiments. Plant DNA was obtained at seven time points post-ingestion from whole guts and separate gut sections, and was detectable up to 12 h post-ingestion in nymphs and 22 h post-ingestion in adult grasshoppers. • Conclusions: The proposed protocol is an effective, relatively quick, and low-cost method of detecting plant DNA from the grasshopper gut and its different sections. This has important applications, from exploring plant “movement” during food consumption, to detecting plant–insect interactions. PMID:25202604

Synthesis and characterization of erbium-doped SiO2-TiO2 thin films prepared by sol-gel and dip-coating techniques onto commercial glass substrates as a route for obtaining active GRadient-INdex materials

International Nuclear Information System (INIS)

Gómez-Varela, Ana I.; Castro, Yolanda; Durán, Alicia; De Beule, Pieter A.A.; Flores-Arias, María T.; Bao-Varela, Carmen

2015-01-01

In this work, SiO 2 -TiO 2 films doped with erbium were prepared by dip-coating sol-gel process onto commercial glass substrates. The surface morphology of the films was characterized using atomic force microscopy, while thickness, refractive index, extinction coefficient and porosity of the films were determined by ellipsometric measurements in a wavelength region of 400-1000 nm. Optical constants and porosity were found to vary with erbium concentration. The proof of principle presented in this paper is applicable to systems of different nature by tailoring the sol-gel precursors in such a way that active GRadient-INdex media described by a complex, parabolic-like refractive index distribution for beam shaping purposes is obtained. - Highlights: • Sol-gel route for preparation of active GRadient-INdex materials is proposed. • SiO 2 -TiO 2 films doped with erbium were prepared by dipping onto commercial glasses. • Morphological and optical characterization of the samples was performed. • Optical constants and porosity were found to vary with erbium concentration. • Refractive index diminishes with dopant content; the contrary occurs for porosity

Image cytometry: nuclear and chromosomal DNA quantification.

Science.gov (United States)

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

2011-01-01

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

Bifunctional Rhodium Intercalator Conjugates as Mismatch-Directing DNA Alkylating Agents

OpenAIRE

Schatzschneider, Ulrich; Barton, Jacqueline K.

2004-01-01

A conjugate of a DNA mismatch-specific rhodium intercalator, containing the bulky chrysenediimine ligand, and an aniline mustard has been prepared, and targeting of mismatches in DNA by this conjugate has been examined. The preferential alkylation of mismatched over fully matched DNA is found by a mobility shift assay at concentrations where untethered organic mustards show little reaction. The binding site of the Rh intercalator was determined by DNA photocleavage, and the position of covale...

An improved protocol and a new grinding device for extraction of genomic DNA from microorganisms by a two-step extraction procedure.

Science.gov (United States)

Zhang, S S; Chen, D; Lu, Q

2012-05-21

Current protocols to extract genomic DNA from microorganisms are still laborious, tedious and costly, especially for the species with thick cell walls. In order to improve the effectiveness of extracting DNA from microbial samples, a novel protocol, defined as two-step extraction method, along with an improved tissue-grinding device, was developed. The protocol included two steps, disruption of microbial cells or spores by grinding the sample together with silica sand in a new device and extraction of DNA with an effective buffer containing cell lysis chemicals. The device was prepared by using a commercial electric mini-grinder, adapted with a grinding stone, and a sample cup processed by lathing from a polytetrafluoroethylene rod. We tested the method with vegetative cells of four microbial species and two microbial spores that have thick cell walls and are therefore hard to process; these included Escherichia coli JM109, Bacillus subtilis WB600, Sacchromyces cerevisiae INVSc1, Trichoderma viride AS3.3711, and the spores of S. cerevisiae and T. viride, respectively, representing Gram-positive bacteria, Gram-negative bacteria, yeast, filamentous fungi. We found that this new method and device extracted usable quantities of genomic DNA from the samples. The DNA fragments that were extracted exceeded 23 kb. The target sequences up to about 5 kb were successfully and exclusively amplified by PCR using extracted DNA as the template. In addition, the DNA extraction was finalized within 1.5 h. Thus, we conclude that this two-step extraction method is an effective and improved protocol for extraction of genomic DNA from microbial samples.

Fecal specimens preparation methods for PCR diagnosis of human taeniosis

Directory of Open Access Journals (Sweden)

Nunes Cáris Maroni

2006-01-01

Full Text Available Sample preparation and DNA extraction protocols for DNA amplification by PCR, which can be applied in human fecal samples for taeniasis diagnosis, are described. DNA extracted from fecal specimens with phenol/chloroform/isoamilic alcohol and DNAzol® reagent had to be first purified to generate fragments of 170 pb and 600 pb by HDP2-PCR. This purification step was not necessary with the use of QIAmp DNA stool mini kit®. Best DNA extraction results were achieved after eggs disruption with glass beads, either with phenol/chloroform/isoamilic alcohol, DNAzol® reagent or QIAmp DNA stool mini kit®.

Genetic stock assessment of spawning arctic cisco (Coregonus autumnalis) populations by flow cytometric determination of DNA content.

Science.gov (United States)

Lockwood, S F; Bickham, J W

1991-01-01

Intraspecific variation in cellular DNA content was measured in five Coregonus autumnalis spawning populations from the Mackenzie River drainage, Canada, using flow cytometry. The rivers assayed were the Peel, Arctic Red, Mountain, Carcajou, and Liard rivers. DNA content was determined from whole blood preparations of fish from all rivers except the Carcajou, for which kidney tissue was used. DNA content measurements of kidney and blood preparations of the same fish from the Mountain River revealed statistically indistinguishable results. Mosaicism was found in blood preparations from the Peel, Arctic Red, Mountain, and Liard rivers, but was not observed in kidney tissue preparations from the Mountain or Carcajou rivers. The Liard River sample had significantly elevated mean DNA content relative to the other four samples; all other samples were statistically indistinguishable. Significant differences in mean DNA content among spawning stocks of a single species reinforces the need for adequate sample sizes of both individuals and populations when reporting "C" values for a particular species.

Preparation of DNA biosensor application from fuel oil waste by functionalization and characterization of MWCNT

Directory of Open Access Journals (Sweden)

Ahmed Mishaal Mohammed

2017-11-01

Full Text Available The potential of using a multi-wall carbon nanotube (MWCNT synthesized from a fuel oil waste of power plants has discovered for the first time for DNA biosensors application. The MWCNT surface morphologies were examined by field emission scanning electron microscopy (FE-SEM and atomic force microscopy (AFM. The thickness of the MWCNT was found 203nm and confirmed by FESEM. The electrochemical DNA biosensor was successfully developed using a MWCNT modified on SiO2 thin films. The capacitance measurements were performed to detect the sensitivity of DNA detection. The change in capacitance before and after immobilization of the DNA was measured in the frequency range of 1Hz to 1MHz. The results indicate that bare device exhibited the lowest capacitance value, which was 32.7μF. The capacitance value of the DNA immobilization increase to 52μF. The permittivity and conductivity also were examined to study the effect of the DNA immobilization toward the MWCNT modified surface. This present demonstrated that the MWCNT modified SiO2 a thin film was successfully fabricated for DNA biosensor detection. Keywords: Carbon nanotubes, Sensors, Thin films, Electrochemical DNA

New scale-down methodology from commercial to lab scale to optimize plant-derived soft gel capsule formulations on a commercial scale.

Science.gov (United States)

Oishi, Sana; Kimura, Shin-Ichiro; Noguchi, Shuji; Kondo, Mio; Kondo, Yosuke; Shimokawa, Yoshiyuki; Iwao, Yasunori; Itai, Shigeru

2018-01-15

A new scale-down methodology from commercial rotary die scale to laboratory scale was developed to optimize a plant-derived soft gel capsule formulation and eventually manufacture superior soft gel capsules on a commercial scale, in order to reduce the time and cost for formulation development. Animal-derived and plant-derived soft gel film sheets were prepared using an applicator on a laboratory scale and their physicochemical properties, such as tensile strength, Young's modulus, and adhesive strength, were evaluated. The tensile strength of the animal-derived and plant-derived soft gel film sheets was 11.7 MPa and 4.41 MPa, respectively. The Young's modulus of the animal-derived and plant-derived soft gel film sheets was 169 MPa and 17.8 MPa, respectively, and both sheets showed a similar adhesion strength of approximately 4.5-10 MPa. Using a D-optimal mixture design, plant-derived soft gel film sheets were prepared and optimized by varying their composition, including variations in the mass of κ-carrageenan, ι-carrageenan, oxidized starch and heat-treated starch. The physicochemical properties of the sheets were evaluated to determine the optimal formulation. Finally, plant-derived soft gel capsules were manufactured using the rotary die method and the prepared soft gel capsules showed equivalent or superior physical properties compared with pre-existing soft gel capsules. Therefore, we successfully developed a new scale-down methodology to optimize the formulation of plant-derived soft gel capsules on a commercial scale. Copyright © 2017 Elsevier B.V. All rights reserved.

Plasmid DNA Delivery: Nanotopography Matters.

Science.gov (United States)

Song, Hao; Yu, Meihua; Lu, Yao; Gu, Zhengying; Yang, Yannan; Zhang, Min; Fu, Jianye; Yu, Chengzhong

2017-12-20

Plasmid DNA molecules with unique loop structures have widespread bioapplications, in many cases relying heavily on delivery vehicles to introduce them into cells and achieve their functions. Herein, we demonstrate that control over delicate nanotopography of silica nanoparticles as plasmid DNA vectors has significant impact on the transfection efficacy. For silica nanoparticles with rambutan-, raspberry-, and flower-like morphologies composed of spike-, hemisphere-, and bowl-type subunit nanotopographies, respectively, the rambutan-like nanoparticles with spiky surfaces demonstrate the highest plasmid DNA binding capability and transfection efficacy of 88%, higher than those reported for silica-based nanovectors. Moreover, it is shown that the surface spikes of rambutan nanoparticles provide a continuous open space to bind DNA chains via multivalent interactions and protect the gene molecules sheltered in the spiky layer against nuclease degradation, exhibiting no significant transfection decay. This unique protection feature is in great contrast to a commercial transfection agent with similar transfection performance but poor protection capability against enzymatic cleavage. Our study provides new understandings in the rational design of nonviral vectors for efficient gene delivery.

Metallization of DNA on silicon surface

International Nuclear Information System (INIS)

Puchkova, Anastasiya Olegovna; Sokolov, Petr; Petrov, Yuri Vladimirovich; Kasyanenko, Nina Anatolievna

2011-01-01

New simple way for silver deoxyribonucleic acid (DNA)-based nanowires preparation on silicon surface was developed. The electrochemical reduction of silver ions fixed on DNA molecule provides the forming of tightly matched zonate silver clusters. Highly homogeneous metallic clusters have a size about 30 nm. So the thickness of nanowires does not exceed 30–50 nm. The surface of n-type silicon monocrystal is the most convenient substrate for this procedure. The comparative analysis of DNA metallization on of n-type silicon with a similar way for nanowires fabrication on p-type silicon, freshly cleaved mica, and glass surface shows the advantage of n-type silicon, which is not only the substrate for DNA fixation but also the source of electrons for silver reduction. Images of bound DNA molecules and fabricated nanowires have been obtained using an atomic force microscope and a scanning ion helium microscope. DNA interaction with silver ions in a solution was examined by the methods of ultraviolet spectroscopy and circular dichroism.

DNA replication and the repair of DNA strand breaks in nuclei of Physarum polycephalum. Terminal report, August 1, 1978-March 31, 1980

International Nuclear Information System (INIS)

Brewer, E.N.; Evans, T.E.

1980-01-01

Nuclei isolated from Physarum are able to replicate approximately 15% of the total genome in a manner which is qualitatively similar to the DNA replication process occurring in the intact organism. Such nuclei, however, are defective in the joining of Okazaki intermediates in vitro. Two DNA polymerase species, isolated from nuclei or intact plasmodia of this organism, can be separated by sucrose density gradient centrifugation. Total DNA polymerase activity is low in nuclei isolated during mitosis. A heat-stable glycoprotein material present in aqueous nuclear extracts stimulates DNA synthesis in well-washed nuclei. A sub-nuclear preparation active in DNA synthesis in vitro has been obtained from isolated nuclei of Physarum. Radiation-induced DNA double-strand breaks are rejoined in intact plasmodia and isolated nuclei of Physarum in a cell cycle-dependent manner. This phenomenon does not appear to be due to an intrinsic difference in nuclear DNA endonuclease activity at different times of the mitotic cycle. DNA strand breaks and repair induced by the carcinogen 4-nitroquinoline-1-oxide is similar in several respects to that resulting from exposure of the organism to ionizing radiation. Temperature sensitive strains of Physarum have been constructed and preliminary genetical and biochemical characterizations have been carried out. Two of the strains appear to be conditionally defective in DNA metabolism. An isogenic ploidal series of amoebae has been prepared and characterized as to uv and ionizing radiation sensitivity (in terms of cell survival). There is a direct relationship between ploidy and resistance to uv whereas ploidal change does not appear to affect the response to ionizing radiation

Preparation of a differentially expressed, full-length cDNA expression library by RecA-mediated triple-strand formation with subtractively enriched cDNA fragments

NARCIS (Netherlands)

Hakvoort, T. B.; Spijkers, J. A.; Vermeulen, J. L.; Lamers, W. H.

1996-01-01

We have developed a fast and general method to obtain an enriched, full-length cDNA expression library with subtractively enriched cDNA fragments. The procedure relies on RecA-mediated triple-helix formation of single-stranded cDNA fragments with a double-stranded cDNA plasmid library. The complexes

Yeast identification by sequencing, biochemical kits, MALDI-TOF MS and rep-PCR DNA fingerprinting.

Science.gov (United States)

Zhao, Ying; Tsang, Chi-Ching; Xiao, Meng; Chan, Jasper F W; Lau, Susanna K P; Kong, Fanrong; Xu, Yingchun; Woo, Patrick C Y

2017-12-08

No study has comprehensively evaluated the performance of 28S nrDNA and ITS sequencing, commercial biochemical test kits, MALDI-TOF MS platforms, and the emerging rep-PCR DNA fingerprinting technology using a cohort of yeast strains collected from a clinical microbiology laboratory. In this study, using 71 clinically important yeast isolates (excluding Candida albicans) collected from a single centre, we determined the concordance of 28S nrDNA and ITS sequencing and evaluated the performance of two commercial test kits, two MALDI-TOF MS platforms, and rep-PCR DNA fingerprinting. 28S nrDNA and ITS sequencing showed complete agreement on the identities of the 71 isolates. Using sequencing results as the standard, 78.9% and 71.8% isolates were correctly identified using the API 20C AUX and Vitek 2 YST ID Card systems, respectively; and 90.1% and 80.3% isolates were correctly identified using the Bruker and Vitek MALDI-TOF MS platforms, respectively. Of the 18 strains belonging to the Candida parapsilosis species complex tested by DiversiLab automated rep-PCR DNA fingerprinting, all were identified only as Candida parapsilosis with similarities ≥93.2%, indicating the misidentification of Candida metapsilosis and Candida orthopsilosis. However, hierarchical cluster analysis of the rep-PCR DNA fingerprints of these three species within this species complex formed three different discrete clusters, indicating that this technology can potentially differentiate the three species. To achieve higher accuracies of identification, the databases of commercial biochemical test kits, MALDI-TOF MS platforms, and DiversiLab automated rep-PCR DNA fingerprinting needs further enrichment, particularly for uncommonly encountered yeast species. © The Author 2017. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Carcinogen-induced damage to DNA

International Nuclear Information System (INIS)

Strauss, B.; Altamirano, M.; Bose, K.; Sklar, R.; Tatsumi, K.

1979-01-01

Human cells respond to carcinogen-induced damage in their DNA in at least two ways. The first response, excision repair, proceeds by at least three variations, depending on the nature of the damage. Nucleotide excision results in relatively large repair patches but few free DNA breaks, since the endonuclease step is limiting. Apurinic repair is characterized by the appearance of numerous breaks in the DNA and by short repair patches. The pathways behave as though they function independently. Lymphoic cells derived from a xeroderma pigmentosum complementation group C patient are deficient in their ability to perform nucleotide excision and also to excise 6 methoxyguanine adducts, but they are apurinic repair competent. Organisms may bypass damage in their DNA. Lymphoblastoid cells, including those derived from xeroderma pigmentosum treated with 3 H-anti-BPDE, can replicate their DNA at low doses of carcinogen. Unexcised 3 H is found in the light or parental strand of the resulting hybrid DNA when replication occurs in medium with BrdUrd. This observation indicates a bypass reaction occurring by a mechanism involving branch migration at DNA growing points. Branch migration in DNA preparations have been observed, but the evidence is that most occurs in BrdUrd-containing DNA during cell lysis. The measurement of the bifilarly substituted DNA resulting from branch migration is a convenient method of estimating the proportion of new synthesis remaining in the vicinity of the DNA growing point. Treatment with carcinogens or caffeine results in accumulation of DNA growing points accompanied by the synthesis of shortened pieces of daughter DNA

All-in-one nanowire-decorated multifunctional membrane for rapid cell lysis and direct DNA isolation.

KAUST Repository

So, Hongyun; Lee, Kunwoo; Murthy, Niren; Pisano, Albert P

2014-01-01

This paper describes a handheld device that uses an all-in-one membrane for continuous mechanical cell lysis and rapid DNA isolation without the assistance of power sources, lysis reagents, and routine centrifugation. This nanowire-decorated multifunctional membrane was fabricated to isolate DNA by selective adsorption to silica surface immediately after disruption of nucleus membranes by ultrasharp tips of nanowires for a rapid cell lysis, and it can be directly assembled with commercial syringe filter holders. The membrane was fabricated by photoelectrochemical etching to create microchannel arrays followed by hydrothermal synthesis of nanowires and deposition of silica. The proposed membrane successfully purifies high-quality DNA within 5 min, whereas a commercial purification kit needs more than an hour.

All-in-one nanowire-decorated multifunctional membrane for rapid cell lysis and direct DNA isolation.

KAUST Repository

So, Hongyun

2014-11-24

This paper describes a handheld device that uses an all-in-one membrane for continuous mechanical cell lysis and rapid DNA isolation without the assistance of power sources, lysis reagents, and routine centrifugation. This nanowire-decorated multifunctional membrane was fabricated to isolate DNA by selective adsorption to silica surface immediately after disruption of nucleus membranes by ultrasharp tips of nanowires for a rapid cell lysis, and it can be directly assembled with commercial syringe filter holders. The membrane was fabricated by photoelectrochemical etching to create microchannel arrays followed by hydrothermal synthesis of nanowires and deposition of silica. The proposed membrane successfully purifies high-quality DNA within 5 min, whereas a commercial purification kit needs more than an hour.

Characterization of polymer, DNA-based, and silk thin film resistivities and of DNA-based films prepared for enhanced electrical conductivity

Science.gov (United States)

Yaney, Perry P.; Ouchen, Fahima; Grote, James G.

2009-08-01

DC resistivity studies were carried out on biopolymer films of DNA-CTMA and silk fibroin, and on selected traditional polymer films, including PMMA and APC. Films of DNA-CTMA versus molecular weight and with conductive dopants PCBM, BAYTRON P and ammonium tetrachloroplatinate are reported. The films were spin coated on glass slides configured for measurements of volume dc resistance. The measurements used the alternating polarity method to record the applied voltage-dependent current independent of charging and background currents. The Arrhenius equation plus a constant was fitted to the conductivity versus temperature data of the polymers and the non-doped DNA-based biopolymers with activation energies ranging from 0.8 to 1.4 eV.

Evaluation of three Brettanomyces qPCR commercial kits: results from an interlaboratory study

Directory of Open Access Journals (Sweden)

Cédric Longin

2016-12-01

This study highlights that quantification of B. bruxellensis in red wine using commercial kits requires a high level of expertise in molecular biology. We recommend that all users use a microbiological internal control to validate DNA extraction yield.

Next generation sequencing of DNA-launched Chikungunya vaccine virus

Energy Technology Data Exchange (ETDEWEB)

Hidajat, Rachmat; Nickols, Brian [Medigen, Inc., 8420 Gas House Pike, Suite S, Frederick, MD 21701 (United States); Forrester, Naomi [Institute for Human Infections and Immunity, Sealy Center for Vaccine Development and Department of Pathology, University of Texas Medical Branch, GNL, 301 University Blvd., Galveston, TX 77555 (United States); Tretyakova, Irina [Medigen, Inc., 8420 Gas House Pike, Suite S, Frederick, MD 21701 (United States); Weaver, Scott [Institute for Human Infections and Immunity, Sealy Center for Vaccine Development and Department of Pathology, University of Texas Medical Branch, GNL, 301 University Blvd., Galveston, TX 77555 (United States); Pushko, Peter, E-mail: ppushko@medigen-usa.com [Medigen, Inc., 8420 Gas House Pike, Suite S, Frederick, MD 21701 (United States)

2016-03-15

Chikungunya virus (CHIKV) represents a pandemic threat with no approved vaccine available. Recently, we described a novel vaccination strategy based on iDNA® infectious clone designed to launch a live-attenuated CHIKV vaccine from plasmid DNA in vitro or in vivo. As a proof of concept, we prepared iDNA plasmid pCHIKV-7 encoding the full-length cDNA of the 181/25 vaccine. The DNA-launched CHIKV-7 virus was prepared and compared to the 181/25 virus. Illumina HiSeq2000 sequencing revealed that with the exception of the 3′ untranslated region, CHIKV-7 viral RNA consistently showed a lower frequency of single-nucleotide polymorphisms than the 181/25 RNA including at the E2-12 and E2-82 residues previously identified as attenuating mutations. In the CHIKV-7, frequencies of reversions at E2-12 and E2-82 were 0.064% and 0.086%, while in the 181/25, frequencies were 0.179% and 0.133%, respectively. We conclude that the DNA-launched virus has a reduced probability of reversion mutations, thereby enhancing vaccine safety. - Highlights: • Chikungunya virus (CHIKV) is an emerging pandemic threat. • In vivo DNA-launched attenuated CHIKV is a novel vaccine technology. • DNA-launched virus was sequenced using HiSeq2000 and compared to the 181/25 virus. • DNA-launched virus has lower frequency of SNPs at E2-12 and E2-82 attenuation loci.

Next generation sequencing of DNA-launched Chikungunya vaccine virus

International Nuclear Information System (INIS)

Hidajat, Rachmat; Nickols, Brian; Forrester, Naomi; Tretyakova, Irina; Weaver, Scott; Pushko, Peter

2016-01-01

Chikungunya virus (CHIKV) represents a pandemic threat with no approved vaccine available. Recently, we described a novel vaccination strategy based on iDNA® infectious clone designed to launch a live-attenuated CHIKV vaccine from plasmid DNA in vitro or in vivo. As a proof of concept, we prepared iDNA plasmid pCHIKV-7 encoding the full-length cDNA of the 181/25 vaccine. The DNA-launched CHIKV-7 virus was prepared and compared to the 181/25 virus. Illumina HiSeq2000 sequencing revealed that with the exception of the 3′ untranslated region, CHIKV-7 viral RNA consistently showed a lower frequency of single-nucleotide polymorphisms than the 181/25 RNA including at the E2-12 and E2-82 residues previously identified as attenuating mutations. In the CHIKV-7, frequencies of reversions at E2-12 and E2-82 were 0.064% and 0.086%, while in the 181/25, frequencies were 0.179% and 0.133%, respectively. We conclude that the DNA-launched virus has a reduced probability of reversion mutations, thereby enhancing vaccine safety. - Highlights: • Chikungunya virus (CHIKV) is an emerging pandemic threat. • In vivo DNA-launched attenuated CHIKV is a novel vaccine technology. • DNA-launched virus was sequenced using HiSeq2000 and compared to the 181/25 virus. • DNA-launched virus has lower frequency of SNPs at E2-12 and E2-82 attenuation loci.

Scutellarin-graft cationic β-cyclodextrin-polyrotaxane: Synthesis, characterization and DNA condensation

Energy Technology Data Exchange (ETDEWEB)

Qin, Qi; Ma, Xue; Liao, Xiali; Yang, Bo, E-mail: yangbo6910@sina.com

2017-02-01

As a prerequisite of gene delivery in living cells, DNA condensation has attracted more and more attention. In order to improve the efficiencies of polyamine-β-cyclodextrin-based cationic polyrotaxanes (PR-EDA and PR-DETA) as DNA condensation materials, we have designed and prepared two novel scutellarin-grafted cationic polyrotaxanes (PR-EDA-SCU and PR-DETA-SCU), in which scutellarins (SCU), the planar molecules, were conjugated on the cyclodextrin molecules of PR-EDA and PR-DETA. These materials were characterized by 1D and 2D NMR, XRD, TG and DSC. The electrophoresis assays showed that pDNA condensation efficiencies of PR-EDA and PR-DETA were better than that of PR-EDA and PR-DETA. The complexes of PR-EDA, PR-DETA, PR-EDA-SCU and PR-DETA-SCU with pDNA were further investigated by zeta potential and atomic force microscopy analysis. The results indicated that the planar structure of SCU played an important role in improvement of pDNA condensation efficiencies of PR-EDA-SCU and PR-DETA-SCU. The satisfactory pDNA condensation abilities of PR-EDA-SCU and PR-DETA-SCU could be helpful in designing non-viral gene delivery vectors to control gene expression and delivery. - Highlights: • Scutellarin-graft cationic cyclodextrin-polyrotaxans were prepared and characterized. • Scutellarin-graft cationic cyclodextrin-polyrotaxans was abbreviated as PR-(D)EDA-SCU. • The Scutellarin of PR-(D)EDA-SCU may be helpful to DNA condensation. • This study opens new avenues for preparing non-viral gene delivery vectors.

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

Preparation of function-enhanced vegetable oils

Directory of Open Access Journals (Sweden)

Hiroshi Maeda

2016-01-01

Full Text Available Background: Previously, we (HM found that most commercially available edible oils, which were processed by hexane extraction followed by a number of purification steps, were extremely low in anti-peroxy radical (ROO., or radical scavenging activity. This is a great contrast to the respective virgin oils as exemplified by extra-virgin olive oil or crude rape seed oil [1-4] (Figure 1. Therefore, such highly purified oils will became prooxidant and less desirable food components in terms of health oriented diet. Oxidized oils may eventually cause DNA cleavages, modification of proteins, RNA, and lipids, as well as cellular damage, or promote inflammation and carcinogenesis at later time [5-9]. These commercial oils of low antioxidant activity may be improved by adding functionally effective antioxidative components, by using dried vegetable-waste such as tomato-juice-waste-residues and wine-ferment-waste-residues. Their antioxiative components will be transferred into the functionally poor grade edible oils, and consequently, one can improve the quality of such functionally poor oils and thereby contributing human health [2,8,9]. The purpose of this paper is to report a practical procedure to fortify functionally low grade conventional edible oils to functionally enriched edible oils using dried vegetable-waste residues such as tomato juice waste, and wine-ferment-residues, or other vegetable-waste residues. Methods: (1 Preparation and measurements of lycopene and carotenoid enriched oils. To 5.0g or 1.0g of the dried residue of tomato juice waste, 100ml of commercial rape seed (canola oil was added respectively. Each mixture was incubated at room temperature in dark for several weeks. Amount of lycopene and carotenoids extracted into the oil was monitored by increase of absorption (400-550nm and fluorescence at 470nm of carotenoid. Grape-juice ferment (wine waste was similarly prepared after hot air drying, and immersed in canola oil. (2

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

Construction of a Liposome Dialyzer for preparation of high-value, small-volume liposome formulations

OpenAIRE

Adamala, Katarzyna; Engelhart, Aaron E.; Kamat, Neha P.; Jin, Lin; Szostak, Jack W.

2015-01-01

The liposome dialyzer is a small-volume equilibrium dialysis device, built from commercially available materials, that is designed for rapid exchange of small volumes of an extraliposomal reagent pool against a liposome preparation. The dialyzer is prepared by modification of commercially available dialysis cartridges and consists of a reactor with two 300 µL chambers and a 1.56 cm2 dialysis surface area. The dialyzer is prepared in three stages: 1) disassembly of dialysis cartridges to obtai...

Evaluation of advanced technologies for residential appliances and residential and commercial lighting

Energy Technology Data Exchange (ETDEWEB)

Turiel, I.; Atkinson, B.; Boghosian, S.; Chan, P.; Jennings, J.; Lutz, J.; McMahon, J.; Rosenquist, G.

1995-01-01

Section 127 of the Energy Policy Act requires that the Department of Energy (DOE) prepare a report to Congress on the potential for the development and commercialization of appliances that substantially exceed the present federal or state efficiency standards. Candidate high-efficiency appliances must meet several criteria including: the potential exists for substantial improvement (beyond the minimum established in law) of the appliance`s energy efficiency; electric, water, or gas utilities are prepared to support and promote the commercialization of such appliances; manufacturers are unlikely to undertake development and commercialization of such appliances on their own, or development and production would be substantially accelerated by support to manufacturers. This report describes options to improve the efficiency of residential appliances, including water heaters, clothes washers and dryers, refrigerator/freezers, dishwashers, space heating and cooling devices, as well as residential and commercial lighting products. Data from this report (particularly Appendix 1)were used to prepare the report to Congress mentioned previously. For the residential sector, national energy savings are calculated using the LBL Residential Energy Model. This model projects the number of households and appliance saturations over time. First, end-use consumption is calculated for a base case where models that only meet the standard replace existing models as these reach the end of their lifetime. Second, models with efficiencies equal to the technology under consideration replace existing models that reach the end of their lifetime. For the commercial sector, the COMMEND model was utilized to project national energy savings from new technologies. In this report, energy savings are shown for the period 1988 to 2015.

A plasma modified cellulose-chitosan porous membrane allows efficient DNA binding and provides antibacterial properties: A step towards developing a new DNA collecting card.

Science.gov (United States)

Chumwangwapee, Sasiwimon; Chingsungnoen, Artit; Siri, Sineenat

2016-11-01

In forensic DNA analyses, biological specimens are collected and stored for subsequent recovery and analysis of DNA. A cost-effective and efficient DNA recovery approach is therefore a need. This study aims to produce a plasma modified cellulose-chitosan membrane (pCE-CS) that efficiently binds and retains DNA as a potential DNA collecting card. The pCE-CS membrane was produced by a phase separation of ionic liquid dissolving CE and CS in water with subsequent surface-modification by a two-step exposure of argon plasma and nitrogen gas. Through plasma modification, the pCE-CS membrane demonstrated better DNA retention after a washing process and higher rate of DNA recovery as compared with the original CE-CS membrane and the commercial FTA card. In addition, the pCE-CS membrane exhibited anti-bacterial properties against both Escherichia coli and Staphylococcus aureus. The results of this work suggest a potential function of the pCE-CS membrane as a DNA collecting card with a high recovery rate of captured DNA. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

SALP, a new single-stranded DNA library preparation method especially useful for the high-throughput characterization of chromatin openness states.

Science.gov (United States)

Wu, Jian; Dai, Wei; Wu, Lin; Wang, Jinke

2018-02-13

Next-generation sequencing (NGS) is fundamental to the current biological and biomedical research. Construction of sequencing library is a key step of NGS. Therefore, various library construction methods have been explored. However, the current methods are still limited by some shortcomings. This study developed a new NGS library construction method, Single strand Adaptor Library Preparation (SALP), by using a novel single strand adaptor (SSA). SSA is a double-stranded oligonucleotide with a 3' overhang of 3 random nucleotides, which can be efficiently ligated to the 3' end of single strand DNA by T4 DNA ligase. SALP can be started with any denatured DNA fragments such as those sheared by Tn5 tagmentation, enzyme digestion and sonication. When started with Tn5-tagmented chromatin, SALP can overcome a key limitation of ATAC-seq and become a high-throughput NGS library construction method, SALP-seq, which can be used to comparatively characterize the chromatin openness state of multiple cells unbiasly. In this way, this study successfully characterized the comparative chromatin openness states of four different cell lines, including GM12878, HepG2, HeLa and 293T, with SALP-seq. Similarly, this study also successfully characterized the chromatin openness states of HepG2 cells with SALP-seq by using 10 5 to 500 cells. This study developed a new NGS library construction method, SALP, by using a novel kind of single strand adaptor (SSA), which should has wide applications in the future due to its unique performance.

DNA imaging and quantification using chemi-luminescent probes

International Nuclear Information System (INIS)

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

1999-01-01

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

Assessing the bias linked to DNA recovery from biofiltration woodchips for microbial community investigation by fingerprinting.

Science.gov (United States)

Cabrol, Léa; Malhautier, Luc; Poly, Franck; Lepeuple, Anne-Sophie; Fanlo, Jean-Louis

2010-01-01

In this study, we explored methodological aspects of nucleic acid recovery from microbial communities involved in a gas biofilter filled with pine bark woodchips. DNA was recovered indirectly in two steps, comparing different methods: cell dispersion (crushing, shaking, and sonication) and DNA extraction (three commercial kits and a laboratory protocol). The objectives were (a) to optimize cell desorption from the packing material and (b) to compare the 12 combinations of desorption and extraction methods, according to three relevant criteria: DNA yield, DNA purity, and community structure representation by denaturing gradient gel electrophoresis (DGGE). Cell dispersion was not influenced by the operational parameters tested for shaking and blending, while it increased with time for sonication. DNA extraction by the laboratory protocol provided the highest DNA yields, whereas the best DNA purity was obtained by a commercial kit designed for DNA extraction from soil. After successful PCR amplification, the 12 methods did not generate the same bias in microbial community representation. Eight combinations led to high diversity estimation, independently of the experimental procedure. Among them, six provided highly similar DGGE profiles. Two protocols generated a significantly dissimilar community profile, with less diversity. This study highlighted the crucial importance of DNA recovery bias evaluation.

Evaluation and optimisation of preparative semi-automated electrophoresis systems for Illumina library preparation.

Science.gov (United States)

Quail, Michael A; Gu, Yong; Swerdlow, Harold; Mayho, Matthew

2012-12-01

Size selection can be a critical step in preparation of next-generation sequencing libraries. Traditional methods employing gel electrophoresis lack reproducibility, are labour intensive, do not scale well and employ hazardous interchelating dyes. In a high-throughput setting, solid-phase reversible immobilisation beads are commonly used for size-selection, but result in quite a broad fragment size range. We have evaluated and optimised the use of two semi-automated preparative DNA electrophoresis systems, the Caliper Labchip XT and the Sage Science Pippin Prep, for size selection of Illumina sequencing libraries. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Sequence specificity of DNA cleavage by Micrococcus luteus γ endonuclease

International Nuclear Information System (INIS)

Hentosh, P.; Henner, W.D.; Reynolds, R.J.

1985-01-01

DNA fragments of defined sequence have been used to determine the sites of cleavage by γ-endonuclease activity in extracts prepared from Micrococcus luteus. End-labeled DNA restriction fragments of pBR322 DNA that had been irradiated under nitrogen in the presence of potassium iodide or t-butanol were treated with M. luteus γ endonuclease and analyzed on irradiated DNA preferentially at the positions of cytosines and thymines. DNA cleavage occurred immediately to the 3' side of pyrimidines in irradiated DNA and resulted in fragments that terminate in a 5'-phosphoryl group. These studies indicate that both altered cytosines and thymines may be important DNA lesions requiring repair after exposure to γ radiation

Determination of mammalian deoxyribonucleic acid (DNA) in commercial vegetarian and vegan diets for dogs and cats.

Science.gov (United States)

Kanakubo, K; Fascetti, A J; Larsen, J A

2017-02-01

The determination of undeclared ingredients in pet food using different analytical methods has been reported in recent years, raising concerns regarding adequate quality control, dietary efficacy and the potential for purposeful adulteration. The objective of this study was to determine the presence or absence of mammalian DNA using multiplex polymerase chain reaction (PCR) on diets marketed as vegetarian or vegan for dogs and cats. The diets were tested in duplicate; two samples were purchased approximately 3 to 4 months apart with different lot numbers. Multiplex PCR-targeted mitochondrial DNA with two species-specific primers was used to amplify and sequence two sections of the cytochrome b gene for each of the 11 mammalian species. Half of the diets assessed (7/14) were positive for one or more undeclared mammalian DNA source (bovine, porcine, or ovine), and the result was repeatable for one or more species in six diets. While most of the detected DNA was found at both time points, in some cases, the result was positive only at one time point, suggesting the presence may have been due to unintentional cross-contact with animal-sourced ingredients. DNA from feline, cervine, canine, caprine, equine, murine (mouse and rat) and leporine was not identified in any samples. However, evidence of mammalian DNA does not confirm adulteration by the manufacturer nor elucidate its clinical significance when consumed by animals that may benefit from a vegetarian or vegan diet. Journal of Animal Physiology and Animal Nutrition © 2016 Blackwell Verlag GmbH.

Quality Control of the Traditional Patent Medicine Yimu Wan Based on SMRT Sequencing and DNA Barcoding

Science.gov (United States)

Jia, Jing; Xu, Zhichao; Xin, Tianyi; Shi, Linchun; Song, Jingyuan

2017-01-01

Substandard traditional patent medicines may lead to global safety-related issues. Protecting consumers from the health risks associated with the integrity and authenticity of herbal preparations is of great concern. Of particular concern is quality control for traditional patent medicines. Here, we establish an effective approach for verifying the biological composition of traditional patent medicines based on single-molecule real-time (SMRT) sequencing and DNA barcoding. Yimu Wan (YMW), a classical herbal prescription recorded in the Chinese Pharmacopoeia, was chosen to test the method. Two reference YMW samples were used to establish a standard method for analysis, which was then applied to three different batches of commercial YMW samples. A total of 3703 and 4810 circular-consensus sequencing (CCS) reads from two reference and three commercial YMW samples were mapped to the ITS2 and psbA-trnH regions, respectively. Moreover, comparison of intraspecific genetic distances based on SMRT sequencing data with reference data from Sanger sequencing revealed an ITS2 and psbA-trnH intergenic spacer that exhibited high intraspecific divergence, with the sites of variation showing significant differences within species. Using the CCS strategy for SMRT sequencing analysis was adequate to guarantee the accuracy of identification. This study demonstrates the application of SMRT sequencing to detect the biological ingredients of herbal preparations. SMRT sequencing provides an affordable way to monitor the legality and safety of traditional patent medicines. PMID:28620408

Are commercial 99m Tc-DTPA preparations reliable for quantitation of renal function

International Nuclear Information System (INIS)

Jeghers, O.; Kinthaert, J.; Georges, B.; Van Gansbeke, B.; Piepsz, A.; Ham, H.R.

1983-01-01

Bad labeling and stability of 99m-Tc-DTPA preparations were found on several occasions, resulting in important artefacts in quantitative renal studies. Further studies are needed in order to understand the origin of these inconstant findings. Repeated quality control on Tc-99m-DTPA preparations are mandatory

DNA isolation by galactoacrylate-based nano-poly(HEMA-co-Gal-OPA) nanopolymers.

Science.gov (United States)

Türkcan Kayhan, Ceren; Zeynep Ural, Fulden; Koruyucu, Meryem; Gül Salman, Yeşim; Uygun, Murat; Aktaş Uygun, Deniz; Akgöl, Sinan; Denizli, Adil

2017-10-01

Isolation of DNA is one of the important processes for biotechnological applications such as investigation of DNA structures and functions, recombinant DNA preparations, identification of genetic factors and diagnosis and treatment of genetic disorders. The aim of this study was to synthesis and characterizes the galactoacrylate based nanopolymers with high surface area and to investigate the usability of these synthesized nanopolymers for DNA isolation studies. Nanopolymers were synthesized by the surfactant free emulsion polymerization technique by using the monomers of 2-hydroxyl ethylmethacrylate and 6-O-(2 ' -hydroxy-3 ' -acryloyloxypropyl)-1,2:3,4-di-O-isopropylidene-α-D-galactopyranose. Galactoacrylate origin of these newly synthesized nanopolymers increased the interaction between DNA and nanopolymers. Prepared nanopolymers were characterized by SEM, FT-IR and ZETA sizer analysis. Synthesized nanopolymers were spherical, and their average particle size was about 246.8 nm. Adsorption of DNA onto galactoacrylate based nanopolymers was investigated by using different pHs, temperatures, ionic strength, DNA concentrations and desorption studies and maximum DNA adsorption was found to be as 567.12 mg/g polymer at 25 °C, in pH 5.0 acetate buffer. Reusability was investigated for 5 successive reuse and DNA adsorption capacity decreased only about 10% at the end of the 5th reuse.

Solution-mediated cladding doping of commercial polymer optical fibers

Science.gov (United States)

Stajanca, Pavol; Topolniak, Ievgeniia; Pötschke, Samuel; Krebber, Katerina

2018-03-01

Solution doping of commercial polymethyl methacrylate (PMMA) polymer optical fibers (POFs) is presented as a novel approach for preparation of custom cladding-doped POFs (CD-POFs). The presented method is based on a solution-mediated diffusion of dopant molecules into the fiber cladding upon soaking of POFs in a methanol-dopant solution. The method was tested on three different commercial POFs using Rhodamine B as a fluorescent dopant. The dynamics of the diffusion process was studied in order to optimize the doping procedure in terms of selection of the most suitable POF, doping time and conditions. Using the optimized procedure, longer segment of fluorescent CD-POF was prepared and its performance was characterized. Fiber's potential for sensing and illumination applications was demonstrated and discussed. The proposed method represents a simple and cheap way for fabrication of custom, short to medium length CD-POFs with various dopants.

Evaluation of the efficiency and utility of recombinant enzyme-free seamless DNA cloning methods

Directory of Open Access Journals (Sweden)

Ken Motohashi

2017-03-01

Full Text Available Simple and low-cost recombinant enzyme-free seamless DNA cloning methods have recently become available. In vivo Escherichia coli cloning (iVEC can directly transform a mixture of insert and vector DNA fragments into E. coli, which are ligated by endogenous homologous recombination activity in the cells. Seamless ligation cloning extract (SLiCE cloning uses the endogenous recombination activity of E. coli cellular extracts in vitro to ligate insert and vector DNA fragments. An evaluation of the efficiency and utility of these methods is important in deciding the adoption of a seamless cloning method as a useful tool. In this study, both seamless cloning methods incorporated inserting DNA fragments into linearized DNA vectors through short (15–39 bp end homology regions. However, colony formation was 30–60-fold higher with SLiCE cloning in end homology regions between 15 and 29 bp than with the iVEC method using DH5α competent cells. E. coli AQ3625 strains, which harbor a sbcA gene mutation that activates the RecE homologous recombination pathway, can be used to efficiently ligate insert and vector DNA fragments with short-end homology regions in vivo. Using AQ3625 competent cells in the iVEC method improved the rate of colony formation, but the efficiency and accuracy of SLiCE cloning were still higher. In addition, the efficiency of seamless cloning methods depends on the intrinsic competency of E. coli cells. The competency of chemically competent AQ3625 cells was lower than that of competent DH5α cells, in all cases of chemically competent cell preparations using the three different methods. Moreover, SLiCE cloning permits the use of both homemade and commercially available competent cells because it can use general E. coli recA− strains such as DH5α as host cells for transformation. Therefore, between the two methods, SLiCE cloning provides both higher efficiency and better utility than the iVEC method for seamless DNA plasmid

Normalization with Corresponding Naïve Tissue Minimizes Bias Caused by Commercial Reverse Transcription Kits on Quantitative Real-Time PCR Results.

Directory of Open Access Journals (Sweden)

Andreas Garcia-Bardon

Full Text Available Real-time reverse transcription polymerase chain reaction (PCR is the gold standard for expression analysis. Designed to improve reproducibility and sensitivity, commercial kits are commonly used for the critical step of cDNA synthesis. The present study was designed to determine the impact of these kits. mRNA from mouse brains were pooled to create serial dilutions ranging from 0.0625 μg to 2 μg, which were transcribed into cDNA using four different commercial reverse-transcription kits. Next, we transcribed mRNA from brain tissue after acute brain injury and naïve mice into cDNA for qPCR. Depending on tested genes, some kits failed to show linear results in dilution series and revealed strong variations in cDNA yield. Absolute expression data in naïve and trauma settings varied substantially between these kits. Normalization with a housekeeping gene failed to reduce kit-dependent variations, whereas normalization eliminated differences when naïve samples from the same region were used. The study shows strong evidence that choice of commercial cDNA synthesis kit has a major impact on PCR results and, consequently, on comparability between studies. Additionally, it provides a solution to overcome this limitation by normalization with data from naïve samples. This simple step helps to compare mRNA expression data between different studies and groups.

DNA adducts in senescent cells

International Nuclear Information System (INIS)

Gaubatz, J.W.

1987-01-01

Perturbations in DNA repair and other metabolic processes during development and aging might affect the steady-state level of genomic damage. The persistence or accumulation of DNA lesions in postmitotic cells could have a significant impact on proper cellular function, interfering with gene regulation for example. To test the notion that DNA damage increases as a function of age in non-dividing cells, DNA was purified from heart tissue of C57BL/6Nia mice at different ages and analyzed by post labeling techniques to detect DNA adducts. In the present experiments, four-dimensional, thin-layer chromatography was used to isolate aromatic adducts that were labeled with carrier-free (γ- 32 P) ATP under DNA-P excess conditions. The complexity and frequency of aromatic adducts varied between DNA samples. Several adducts were present in all preparations and were clearly more abundant in nucleotide maps of mature and old heart DNA. However, a direct correlation with age was not observed. In contrast, experiments in which aromatic adducts were first isolated by phase-transfer to 1-butanol, then labeled with excess (γ- 32 P)ATP indicated that there was an age-related increase in these adducts. The results are consistent with their earlier studies that showed alkyl adducts increased during aging of mouse myocardium and suggest that a common repair pathway might be involved

Structural mediation on polycation nanoparticles by sulfadiazine to enhance DNA transfection efficiency and reduce toxicity.

Science.gov (United States)

Long, Xingwen; Zhang, Zhihui; Han, Shangcong; Tang, Minjie; Zhou, Junhui; Zhang, Jianhua; Xue, Zhenyi; Li, Yan; Zhang, Rongxin; Deng, Liandong; Dong, Anjie

2015-04-15

Reducing the toxicity while maintaining high transfection efficiency is an important issue for cationic polymers as gene carriers in clinical application. In this paper, a new zwitterionic copolymer, polycaprolactone-g-poly(dimethylaminoethyl methyacrylate-co-sulfadiazine methacrylate) (PC-SDZ) with unique pH-sensitivity, was designed and prepared. The incorporation of sulfadiazine into poly(dimethylaminoethyl methacrylate) (PDMAEMA) chains successfully mediates the surface properties including compacter shell structure, lower density of positive charges, stronger proton buffer capability, and enhanced hydrophobicity, which lead to reduction in toxicity and enhancements in stability, cellular uptake, endosome escape, and transfection efficiency for the PC-SDZ2 nanoparticles (NPs)/DNA complexes. Excellent transfection efficiency at the optimal N/P ratio of 10 was observed for PC-SDZ2 NPs/DNA complexes, which was higher than that of the commercial reagent-branched polyethylenimine (PEI). The cytotoxicity was evaluated by CCK8 measurement, and the results showed significant reduction in cytotoxicity even at high concentration of complexes after sulfadiazine modification. Therefore, this work may demonstrate a new way of structural mediation of cationic polymer carriers for gene delivery with high efficiency and low toxicity.

Characteristics of DNA-AuNP networks on cell membranes and real-time movies for viral infection.

Science.gov (United States)

Li, Chunmei; Zheng, Linling; Yang, Xiaoxi; Wan, Xiaoyan; Wu, Wenbi; Zhen, Shujun; Li, Yuanfang; Luo, Lingfei; Huang, Chengzhi

2016-03-01

This data article provides complementary data for the article entitled "DNA-AuNP networks on cell membranes as a protective barrier to inhibit viral attachment, entry and budding" Li et al. (2016) [1]. The experimental methods for the preparation and characterization of DNA-conjugated nanoparticle networks on cell membranes were described. Confocal fluorescence images, agarose gel electrophoresis images and hydrodynamic diameter of DNA-conjugated gold nanoparticle (DNA-AuNP) networks were presented. In addition, we have prepared QDs-labeled RSV (QDs-RSV) to real-time monitor the RSV infection on HEp-2 cells in the absence and presence of DNA-AuNP networks. Finally, the cell viability of HEp-2 cells coated by six types of DNA-nanoparticle networks was determined after RSV infection.

Radiation-induced dissociation of stable DNA-protein complexes in Erlich ascites carcinoma cells

International Nuclear Information System (INIS)

Juhasz, P.P.; Sirota, N.P.; Gaziev, A.I.

1982-01-01

DNA of Ehrlich ascites carcinoma cells prepared under conditions that were highly denaturing for proteins but not for DNA, contained a group of nonhistone residual proteins. The amount of these proteins increased during DNA replication. The DNA-protein complex observed was sensitive to proteolytic enzymes and/or SH-reagents. γ-irradiation cells with moderate doses leads to a decrease in the amount of DNA-protein complexes. High-dose gamma-irradiation produces enhanced linking of chromosomal proteins with DNA. (author)

Genetic Diversity in Commercial Rapeseed (Brassica napus L.) Varieties from Turkey as Revealed by RAPD

OpenAIRE

Özlem ÖZBEK; Betül Uçar GIDIK

2013-01-01

In cultivated commercial crop species, genetic diversity tends to decrease because of the extensive breeding processes. Therefore, germplasm of commercial crop species, such as Brassica napus L. should be evaluated and the genotypes, which have higher genetic diversity index, should be addressed as potential parental cross materials in breeding programs. In this study, the genetic diversity was analysed by using randomly amplified polymorphic DNA analysis (RAPD) technique in nine Turkish com...

Structure and hardness of a hard metal alloy prepared with a WC powder synthesized at low temperature

Energy Technology Data Exchange (ETDEWEB)

Costa, F.A. da [Programa de Pos-Graduacao em Ciencia e Engenharia de Materiais, UFRN, Campus Universitario, 59072-970 Natal, RN (Brazil)], E-mail: francineac@yahoo.com; Medeiros, F.F.P. de [Programa de Pos-Graduacao em Ciencia e Engenharia de Materiais, UFRN, Campus Universitario, 59072-970 Natal, RN (Brazil); Silva, A.G.P. da [Laboratorio de Materiais Avancados, UENF, 28015-620 Campos de Goytacazes, RJ (Brazil); Gomes, U.U. [Departamento de Fisica Teorica e Experimental, UFRN, Campus Universitario, 59072-970 Natal, RN (Brazil); Filgueira, M. [Laboratorio de Materiais Avancados, UENF, 28015-620 Campos de Goytacazes, RJ (Brazil); Souza, C.P. de [Laboratorio de Termodinamica e Reatores, UFRN, Campus Universitario, 59072-970 Natal, RN (Brazil)

2008-06-25

The structure and hardness of a WC-10 wt% Co alloy prepared with an experimental WC powder are compared with those of another alloy of the same composition produced under the same conditions and prepared with a commercial WC powder. The experimental WC powder was synthesized by a gas-solid reaction between APT and methane at low temperature and the commercial WC powder was conventionally produced by a solid-solid reaction between tungsten and carbon black. WC-10 wt% Co alloys with the two powders were prepared under the same conditions of milling and sintering. The structure of the sample prepared with the experimental WC powder is homogeneous and coarse grained. The structure of the sample prepared with the commercial powder is heterogeneous. Furthermore the size and shape of the WC grains are significantly different.

A cost-effective method for Illumina small RNA-Seq library preparation using T4 RNA ligase 1 adenylated adapters

Directory of Open Access Journals (Sweden)

Chen Yun-Ru

2012-09-01

Full Text Available Abstract Background Deep sequencing is a powerful tool for novel small RNA discovery. Illumina small RNA sequencing library preparation requires a pre-adenylated 3’ end adapter containing a 5’,5’-adenyl pyrophosphoryl moiety. In the absence of ATP, this adapter can be ligated to the 3’ hydroxyl group of small RNA, while RNA self-ligation and concatenation are repressed. Pre-adenylated adapters are one of the most essential and costly components required for library preparation, and few are commercially available. Results We demonstrate that DNA oligo with 5’ phosphate and 3’ amine groups can be enzymatically adenylated by T4 RNA ligase 1 to generate customized pre-adenylated adapters. We have constructed and sequenced a small RNA library for tomato (Solanum lycopersicum using the T4 RNA ligase 1 adenylated adapter. Conclusion We provide an efficient and low-cost method for small RNA sequencing library preparation, which takes two days to complete and costs around $20 per library. This protocol has been tested in several plant species for small RNA sequencing including sweet potato, pepper, watermelon, and cowpea, and could be readily applied to any RNA samples.

Release of 3-methyladenine from linker and core DNA of chromatin by a purified DNA glycosylase

International Nuclear Information System (INIS)

Heller, E.P.; Goldthwait, D.A.

1983-01-01

Oligonucleosomes were isolated from [ 14 C]thymidine-labeled HeLa cells by digestion of the nuclei with micrococcal nuclease and were then alkylated with [ 3 H]methylnitrosourea. Nucleosome core particles were also prepared by further digestion of the oligonucleosomes. The distribution of 3 H-labeled methyl groups in the linker versus the core DNA was established by a determination of 3 H: 14 C ratios in oligonucleosome and core DNA. The ratios in the core DNA of 145 and 165 base pair DNA fragments were 5.2 and 5.4, respectively, while the ratio in the oligonucleosomal DNA was 8.2. Assuming an equal mixture (as determined) of 145 and 165 base pair fragments of DNA in the 185 base pair repeat, the relative concentration of 3 H methyl groups in the linker versus the core DNA was 4.2. Thus, 45% of the 3 H methyl groups were in the linker DNA, and 55% were in the core DNA. Some shielding of the DNA was evident during alkylation. The concentrations of alkyl groups on the linker and core DNA were 67 and 12% of that found on free DNA alkylated under comparable conditions. No evidence for preferential shielding of the major or minor groove was observed. The purified 3-methyladenine DNA glycosylase I of Escherichia coli released approximately 37% of the 3-methyladenine from the linker DNA and 13% from the core DNA. The limited enzymatic removal of 3-methyladenine in vitro compared to the efficient removal in vivo suggests that conformational changes of the oligonucleosome and core structure must occur for total repair

In Situ Dark Adaptation Enhances the Efficiency of DNA Extraction from Mature Pin Oak (Quercus palustris Leaves, Facilitating the Identification of Partial Sequences of the 18S rRNA and Isoprene Synthase (IspS Genes

Directory of Open Access Journals (Sweden)

Csengele E. Barta

2017-10-01

Full Text Available Mature oak (Quercus spp. leaves, although abundantly available during the plants’ developmental cycle, are rarely exploited as viable sources of genomic DNA. These leaves are rich in metabolites difficult to remove during standard DNA purification, interfering with downstream molecular genetics applications. The current work assessed whether in situ dark adaptation, to deplete sugar reserves and inhibit secondary metabolite synthesis could compensate for the difficulties encountered when isolating DNA from mature leaves rich in secondary metabolites. We optimized a rapid, commercial kit based method to extract genomic DNA from dark- and light-adapted leaves. We demonstrated that in situ dark adaptation increases the yield and quality of genomic DNA obtained from mature oak leaves, yielding templates of sufficiently high quality for direct downstream applications, such as PCR amplification and gene identification. The quality of templates isolated from dark-adapted pin oak leaves particularly improved the amplification of larger fragments in our experiments. From DNA extracts prepared with our optimized method, we identified for the first time partial segments of the genes encoding 18S rRNA and isoprene synthase (IspS from pin oak (Quercus palustris, whose full genome has not yet been sequenced.

Alkaline Extraction of DNA from Pathogenic Fungi for PCR-RFLP Analysis

OpenAIRE

Matsumoto, Masaru; Mishima, Shinobu; Matsuyama, Nobuaki; 松元, 賢; 松山, 宣明

1997-01-01

For the preparation of DNA samples from fungal mycelia alkaline extraction method was applied and assessed its usefulness for PCR-RFLP analysis. Using alkaline treatment protocols, 18S ribosomal DNAs (rDNA) derived from fungal genomic DNA of Pyricularia oryzae, P. zingiberi, Rhizoctonia solani and R. oryzae were PCR-amplified and digested with Hha I, Msp I and Hae ill. RFLP analysis with HhaI showed the divergent polymorphism between genus Pyricularia and Rhizoctonia. The alkaline DNA extract...

Failure to detect circulating DNA-anti-DNA complexes by four radioimmunological methods in patients with systemic lupus erythematosus

International Nuclear Information System (INIS)

Izui, S.; Lambert, P.H.; Miescher, P.A.

1977-01-01

The presence of DNA-anti-DNA complexes in sera from patients with systemic lupus erythematosus (SLE) was investigated by two new radioimmunoassays (RIA) developed for this purpose and by measuring the CLq and DNA binding activity of serum before and after treatment with DNAse. Two direct RIA developed in this study were based on the reactivity of [ 3 H]actinomycin D ([ 3 H]ACT-D) or solid-phase methylated bovine serum albumin (mBSA) with DNA-anti-DNA complexes. DNA-anti-DNA complexes prepared in vitro could be efficiently detected at various antigen-antibody ratios by these two RIA. Increased levels of circulating immune complexes as indicated by the CLq binding test were found in 52% of SLE sera. However, the frequency of specific DNA-anti-DNA complexes detected in SLE sera was very low. Only 6% of sera exhibited an increased value deviating by more than three s.d. from the normal mean when tested with the [ 3 H]ACT-D binding RIA or the solid-phase mBSA RIA. On the other hand, there was no significant difference in the serum CLq or DNA binding activity after treatment with DNAse. These results suggest that DNA-anti-DNA complexes do not occur frequently in circulating blood and represent only a very small portion of the immune complexes detected in serum from patients with SLE. (author)

DNA-dependent protein kinase (DAN-PK), a key enzyme in the re-ligation of DNA double-strand breaks

International Nuclear Information System (INIS)

Hennequin, C.; Averbeck, D.

1999-01-01

Repair pathways of DNA are now defined and some important findings have been discovered in the last few years. DNA non-homologous end-joining (NEH) is a crucial process in the repair of radiation-induced double-strand breaks (DSBs). NHEj implies at least three steps: the DNA free-ends must get closer, preparation of the free-ends by exonucleases and then a transient hybridization in a region of DNA with weak homology. DNA-dependent protein kinase (DNA-PK) is the key enzyme in this process. DNA-PK is a nuclear serine/threonine kinase that comprises three components: a catalytic subunit (DNA-PK cs ) and two regulatory subunits, DNA-binding proteins, Ku80 and Ku70. The severe combined immuno-deficient (scid) mice are deficient in DNA-PK cs : this protein is involved both in DNA repair and in the V(D)J recombination of immunoglobulin and T-cell receptor genes. It is a protein-kinase of the P13-kinase family and which can phosphorylate Ku proteins, p53 and probably some other proteins still unknown. DNA-PK is an important actor of DSBs repair (induced by ionising radiations or by drugs like etoposide), but obviously it is not the only mechanism existing in the cell for this function. Some others, like homologous recombination, seem also to have a great importance for cell survival. (authors)

A bipedal DNA motor that travels back and forth between two DNA origami tiles.

Science.gov (United States)

Liber, Miran; Tomov, Toma E; Tsukanov, Roman; Berger, Yaron; Nir, Eyal

2015-02-04

In this work, the successful operation of a dynamic DNA device constructed from two DNA origami building blocks is reported. The device includes a bipedal walker that strides back and forth between the two origami tiles. Two different DNA origami tiles are first prepared separately; they are then joined together in a controlled manner by a set of DNA strands to form a stable track in high yield as confirmed by single-molecule fluorescence (SMF). Second, a bipedal DNA motor, initially attached to one of the two origami units and operated by sequential interaction with "fuel" and "antifuel" DNA strands, moves from one origami tile to another and then back again. The operational yield, measured by SMF, was similar to that of a motor operating on a similar track embedded in a single origami tile, confirming that the transfer across the junction from one tile to the other does not result in dissociation that is any more than that of steps on a single tile. These results demonstrate that moving parts can reliably travel from one origami unit to another, and it demonstrates the feasibility of dynamic DNA molecular machines that are made of more than a single origami building block. This study is a step toward the development of motors that can stride over micrometer distances. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Leishmania diagnostic and identification py using 32P labelled DNA probes

International Nuclear Information System (INIS)

Andrade, Antero Silva Ribeiro de; Melo, Maria Norma de

1999-10-01

P 32 labelled DNA probes are valious instruments for the parasitic diseases by using hybridization reaction. In this paper we describe the methodology and present the foundations for the radioactive probes production, based on the kinetoplast DNA (kDNA), for the Leishmania diagnostic an identification. We also describe the kDNA purification protocol from Leishmania reference cepa, the process of P 32 labelling of the kDNA by using the nick translation method, gathering, sample preparation and treatment, the optimum conditions for the hybridization reaction and the procedures for the autoradiography

Ultrathin Two-Dimensional Covalent Organic Framework Nanosheets: Preparation and Application in Highly Sensitive and Selective DNA Detection

KAUST Repository

Peng, Yongwu

2017-06-03

The ability to prepare ultrathin two-dimensional (2D) covalent organic framework (COF) nanosheets (NSs) in high yield is of great importance for the further exploration of their unique properties and potential applications. Herein, by elaborately designing and choosing two flexible molecules with C3v molecular symmetry as building units, a novel imine-linked COF, namely TPA-COF, with hexagonal layered structure and sheet-like morphology, is synthesized. Since the flexible building units are integrated into the COF skeletons, the interlayer stacking becomes weak, resulting in the easy exfoliation of TPA-COF into ultrathin 2D NSs. Impressively, for the first time, the detailed structural information, i.e. the pore channels and individual building units in the NSs, is clearly visualized by using the recently developed low-dose imaging technique of transmission electron microscopy (TEM). As a proof-of-concept application, the obtained ultrathin COF NSs are used as a novel fluorescence sensing platform for the highly sensitive and selective detection of DNA.

Ultrathin Two-Dimensional Covalent Organic Framework Nanosheets: Preparation and Application in Highly Sensitive and Selective DNA Detection

KAUST Repository

Peng, Yongwu; Huang, Ying; Zhu, Yihan; Chen, Bo; Wang, Liying; Lai, Zhuangchai; Zhang, Zhicheng; Zhao, Meiting; Tan, Chaoliang; Yang, Nailiang; Shao, Fangwei; Han, Yu; Zhang, Hua

2017-01-01

The ability to prepare ultrathin two-dimensional (2D) covalent organic framework (COF) nanosheets (NSs) in high yield is of great importance for the further exploration of their unique properties and potential applications. Herein, by elaborately designing and choosing two flexible molecules with C3v molecular symmetry as building units, a novel imine-linked COF, namely TPA-COF, with hexagonal layered structure and sheet-like morphology, is synthesized. Since the flexible building units are integrated into the COF skeletons, the interlayer stacking becomes weak, resulting in the easy exfoliation of TPA-COF into ultrathin 2D NSs. Impressively, for the first time, the detailed structural information, i.e. the pore channels and individual building units in the NSs, is clearly visualized by using the recently developed low-dose imaging technique of transmission electron microscopy (TEM). As a proof-of-concept application, the obtained ultrathin COF NSs are used as a novel fluorescence sensing platform for the highly sensitive and selective detection of DNA.

Ultrathin Two-Dimensional Covalent Organic Framework Nanosheets: Preparation and Application in Highly Sensitive and Selective DNA Detection.

Science.gov (United States)

Peng, Yongwu; Huang, Ying; Zhu, Yihan; Chen, Bo; Wang, Liying; Lai, Zhuangchai; Zhang, Zhicheng; Zhao, Meiting; Tan, Chaoliang; Yang, Nailiang; Shao, Fangwei; Han, Yu; Zhang, Hua

2017-06-28

The ability to prepare ultrathin two-dimensional (2D) covalent organic framework (COF) nanosheets (NSs) in high yield is of great importance for the further exploration of their unique properties and potential applications. Herein, by elaborately designing and choosing two flexible molecules with C 3v molecular symmetry as building units, a novel imine-linked COF, namely, TPA-COF, with a hexagonal layered structure and sheet-like morphology, is synthesized. Since the flexible building units are integrated into the COF skeletons, the interlayer stacking becomes weak, resulting in the easy exfoliation of TPA-COF into ultrathin 2D NSs. Impressively, for the first time, the detailed structural information, i.e., the pore channels and individual building units in the NSs, is clearly visualized by using the recently developed low-dose imaging technique of transmission electron microscopy (TEM). As a proof-of-concept application, the obtained ultrathin COF NSs are used as a novel fluorescence sensing platform for the highly sensitive and selective detection of DNA.

Microfluidic Devices for Forensic DNA Analysis: A Review.

Science.gov (United States)

Bruijns, Brigitte; van Asten, Arian; Tiggelaar, Roald; Gardeniers, Han

2016-08-05

Microfluidic devices may offer various advantages for forensic DNA analysis, such as reduced risk of contamination, shorter analysis time and direct application at the crime scene. Microfluidic chip technology has already proven to be functional and effective within medical applications, such as for point-of-care use. In the forensic field, one may expect microfluidic technology to become particularly relevant for the analysis of biological traces containing human DNA. This would require a number of consecutive steps, including sample work up, DNA amplification and detection, as well as secure storage of the sample. This article provides an extensive overview of microfluidic devices for cell lysis, DNA extraction and purification, DNA amplification and detection and analysis techniques for DNA. Topics to be discussed are polymerase chain reaction (PCR) on-chip, digital PCR (dPCR), isothermal amplification on-chip, chip materials, integrated devices and commercially available techniques. A critical overview of the opportunities and challenges of the use of chips is discussed, and developments made in forensic DNA analysis over the past 10-20 years with microfluidic systems are described. Areas in which further research is needed are indicated in a future outlook.

Stimulation of Escherichia coli DNA photoreactivating enzyme activity by adenosine 5'-triphosphate

International Nuclear Information System (INIS)

Koka, P.

1984-01-01

A purification procedure consisting of Biorex-70, single-stranded DNA-agarose, and ultraviolet (UV) light irradiated DNA-cellulose chromatography has been adopted for the Escherichia coli photoreactivating enzyme, to obtain enzyme preparations that are free of extraneous nucleic acid or nucleotides. The purification yields high specific activities (75 000 pmol h -1 mg -1 ) with a 50% recovery. Enzyme preparations have also been obtained from UV-irradiated DNA-cellulose by exposure to visible light. These enzyme preparations contain oligoribonucleotides, up to 26 nucleotides in length in relation to DNA size markers, but these are not essential for enzymatic activity. When the enzyme is preincubated with exogenous ATP a 10-fold stimulation in the enzyme activity has been observed. It has been determined by polyacrylamide gel electrophoresis and high-voltage diethylaminoethyl paper electrophoresis that the light-released enzyme samples from a preincubated and washed mixture of the enzyme, [γ- 32 P]ATP, and UV-irradiated DNA-cellulose contained exogenous [γ- 32 P], which eluted with the enzyme-containing fractions when subjected to Bio-Gel P-30 chromatography. GTP caused a slight enhancement of the enzyme activity while ADP strongly inhibited photoreactivation, at the same concentration and conditions. Higher (X5) concentrations of ADP and adenosine 5'-(β, γ-methylenetriphosphate) totally inhibited the enzyme activity. Dialysis of a photoreactivating enzyme preparation against a buffer solution containing 1 mM ATP caused a 9-fold stimulation of the enzyme activity. In addition, there is an apparent hydrolysis of ATP during photoreactivation as measured by the release of 32 P from [γ- 32 P]ATP

dna profiling of capsicum annuum with the help of molecular markers

African Journals Online (AJOL)

isha

2013-07-24

Jul 24, 2013 ... Since the commercial value of chilli pepper is based on pungency level, ... Randomly Amplified Polymorphic DNA, Dendrogram, Polymerase Chain ..... the Amazon department in Columbia, characterization by AFLPs of.

Azide photochemistry for facile modification of graphitic surfaces: preparation of DNA-coated carbon nanotubes for biosensing

International Nuclear Information System (INIS)

Moghaddam, Minoo J; Yang Wenrong; Bojarski, Barbara; Gengenbach, Thomas R; Gao Mei; Zareie, Hadi; McCall, Maxine J

2012-01-01

A facile, two-step method for chemically attaching single-stranded DNA to graphitic surfaces, represented here by carbon nanotubes, is reported. In the first step, an azide-containing compound, N-5-azido-nitrobenzoyloxy succinimide (ANB-NOS), is used to form photo-adducts on the graphitic surfaces in a solid-state photochemical reaction, resulting in active ester groups being oriented for the subsequent reactions. In the second step, pre-synthesized DNA strands bearing a terminal amine group are coupled in an aqueous solution with the active esters on the photo-adducts. The versatility of the method is demonstrated by attaching pre-synthesized DNA to surfaces of carbon nanotubes in two platforms—as vertically-aligned multi-walled carbon nanotubes on a solid support and as tangled single-walled carbon nanotubes in mats. The reaction products at various stages were characterized by x-ray photoelectron spectroscopy. Two different assays were used to check that the DNA strands attached to the carbon nanotubes were able to bind their partner strands with complementary base sequences. The first assay, using partner DNA strands tethered to gold nanoparticles, enabled the sites of DNA attachment to the carbon nanotubes to be identified in TEM images. The second assay, using radioactively labelled partner DNA strands, quantified the density of functional DNA strands attached to the carbon nanotubes. The diversity of potential applications for these DNA-modified carbon-nanotube platforms is exemplified here by the successful use of a DNA-modified single-walled carbon-nanotube mat as an electrode for the specific detection of metal ions. (paper)

DNA Minicircle Technology Improves Purity of Adeno-associated Viral Vector Preparations

Directory of Open Access Journals (Sweden)

Maria Schnödt

2016-01-01

Full Text Available Adeno-associated viral (AAV vectors are considered as one of the most promising delivery systems in human gene therapy. In addition, AAV vectors are frequently applied tools in preclinical and basic research. Despite this success, manufacturing pure AAV vector preparations remains a difficult task. While empty capsids can be removed from vector preparations owing to their lower density, state-of-the-art purification strategies as of yet failed to remove antibiotic resistance genes or other plasmid backbone sequences. Here, we report the development of minicircle (MC constructs to replace AAV vector and helper plasmids for production of both, single-stranded (ss and self-complementary (sc AAV vectors. As bacterial backbone sequences are removed during MC production, encapsidation of prokaryotic plasmid backbone sequences is avoided. This is of particular importance for scAAV vector preparations, which contained an unproportionally high amount of plasmid backbone sequences (up to 26.1% versus up to 2.9% (ssAAV. Replacing standard packaging plasmids by MC constructs not only allowed to reduce these contaminations below quantification limit, but in addition improved transduction efficiencies of scAAV preparations up to 30-fold. Thus, MC technology offers an easy to implement modification of standard AAV packaging protocols that significantly improves the quality of AAV vector preparations.

DENV gene of bacteriophage T4 codes for both pyrimidine dimer-DNA glycosylase and apyrimidinic endonuclease activities

International Nuclear Information System (INIS)

McMillan, S.; Edenberg, H.J.; Radany, E.H.; Friedberg, R.C.; Friedberg, E.C.

1981-01-01

Recent studies have shown that purified preparations of phage T4 UV DNA-incising activity (T4 UV endonuclease or endonuclease V of phase T4) contain a pyrimidine dimer-DNA glycosylase activity that catalyzes hydrolysis of the 5' glycosyl bond of dimerized pyrimidines in UV-irradiated DNA. Such enzyme preparations have also been shown to catalyze the hydrolysis of phosphodiester bonds in UV-irradiated DNA at a neutral pH, presumably reflecting the action of an apurinic/apyrimidinic endonuclease at the apyrimidinic sites created by the pyrimidine dimer-DNA glycosylase. In this study we found that preparations of T4 UV DNA-incising activity contained apurinic/apyrimidinic endonuclease activity that nicked depurinated form I simian virus 40 DNA. Apurinic/apyrimidinic endonuclease activity was also found in extracts of Escherichia coli infected with T4 denV + phage. Extracts of cells infected with T4 denV mutants contained significantly lower levels of apurinic/apyrimidinic endonuclease activity; these levels were no greater than the levels present in extracts of uninfected cells. Furthermore, the addition of DNA containing UV-irradiated DNA and T4 enzyme resulted in competition for pyrimidine dimer-DNA glycosylase activity against the UV-irradiated DNA. On the basis of these results, we concluded that apurinic/apyrimidinic endonuclease activity is encoded by the denV gene of phage T4, the same gene that codes for pyrimidine dimer-DNA glycosylase activity

Non liquid nitrogen-based-method for isolation of DNA from ...

African Journals Online (AJOL)

A simple, efficient, reliable and cost-effective method for isolation of total genomic DNA from fungi, suitable for polymerase chain reaction (PCR) amplification and other molecular applications was described. The main advantages of the method are: (1) does not require the use of liquid nitrogen for preparation of fungi DNA; ...

DNA recovery from wild chimpanzee tools.

Directory of Open Access Journals (Sweden)

Fiona A Stewart

Full Text Available Most of our knowledge of wild chimpanzee behaviour stems from fewer than 10 long-term field sites. This bias limits studies to a potentially unrepresentative set of communities known to show great behavioural diversity on small geographic scales. Here, we introduce a new genetic approach to bridge the gap between behavioural material evidence in unhabituated chimpanzees and genetic advances in the field of primatology. The use of DNA analyses has revolutionised archaeological and primatological fields, whereby extraction of DNA from non-invasively collected samples allows researchers to reconstruct behaviour without ever directly observing individuals. We used commercially available forensic DNA kits to show that termite-fishing by wild chimpanzees (Pan troglodytes schweinfurthii leaves behind detectable chimpanzee DNA evidence on tools. We then quantified the recovered DNA, compared the yield to that from faecal samples, and performed an initial assessment of mitochondrial and microsatellite markers to identify individuals. From 49 termite-fishing tools from the Issa Valley research site in western Tanzania, we recovered an average of 52 pg/μl chimpanzee DNA, compared to 376.2 pg/μl in faecal DNA extracts. Mitochondrial DNA haplotypes could be assigned to 41 of 49 tools (84%. Twenty-six tool DNA extracts yielded >25 pg/μl DNA and were selected for microsatellite analyses; genotypes were determined with confidence for 18 tools. These tools were used by a minimum of 11 individuals across the study period and termite mounds. These results demonstrate the utility of bio-molecular techniques and a primate archaeology approach in non-invasive monitoring and behavioural reconstruction of unhabituated primate populations.

High-throughput automated microfluidic sample preparation for accurate microbial genomics.

Science.gov (United States)

Kim, Soohong; De Jonghe, Joachim; Kulesa, Anthony B; Feldman, David; Vatanen, Tommi; Bhattacharyya, Roby P; Berdy, Brittany; Gomez, James; Nolan, Jill; Epstein, Slava; Blainey, Paul C

2017-01-27

Low-cost shotgun DNA sequencing is transforming the microbial sciences. Sequencing instruments are so effective that sample preparation is now the key limiting factor. Here, we introduce a microfluidic sample preparation platform that integrates the key steps in cells to sequence library sample preparation for up to 96 samples and reduces DNA input requirements 100-fold while maintaining or improving data quality. The general-purpose microarchitecture we demonstrate supports workflows with arbitrary numbers of reaction and clean-up or capture steps. By reducing the sample quantity requirements, we enabled low-input (∼10,000 cells) whole-genome shotgun (WGS) sequencing of Mycobacterium tuberculosis and soil micro-colonies with superior results. We also leveraged the enhanced throughput to sequence ∼400 clinical Pseudomonas aeruginosa libraries and demonstrate excellent single-nucleotide polymorphism detection performance that explained phenotypically observed antibiotic resistance. Fully-integrated lab-on-chip sample preparation overcomes technical barriers to enable broader deployment of genomics across many basic research and translational applications.

An investigation of the effects of Cinnamomum cassia bark extracts on oxidative DNA damage and possible cytotoxic and apoptotic activities in transformed/untransformed cell lines from Type 1 diabetic patients, in vitro.

Directory of Open Access Journals (Sweden)

Ferzan Lermioglu Erciyas

2015-05-01

Full Text Available It was shown that patients with Type 1 diabetes mellitus (T1DM had increased level of oxidative DNA damage and decreased efficacy of DNA repair. These changes were implicated in the increased cancer risk in patients with diabetes mellitus. Cinnamon bark extracts have diverse biological activities including antidiabetic and anti-tumor properties. Cinnamomum cassia (C. cassia is a common used cinnamon species present in commercial cinnamon preparations. We aimed to investigate the effects of cinnamon extracts prepared from C. cassia bark on endogenous and hydrogen peroxide (H2O2-induced oxidative DNA damage, as well as cytotoxic and apoptotic activities in this study. Type 1 diabetic (T1DM lymphocytes (GM02765, GM01838 and fibroblasts (GM01837 were obtained from NIGMS Human Genetic Cell Repository of Coriell Institute, New Jersey, USA. Cytotoxicity analysis were performed by using a tetrazolium salt, 4-[3-(4-iodophenyl 2-(4-nitrophenyl 2H-5-tetrazolio] 1,3-benzene disulfonate (WST-1. The effects of extracts on endogenous and H2O2-induced oxidative DNA damage were studied using the single cell gel electrophoresis (SCGE; Comet Assay, a technique allowing DNA damage in a single cell. Apoptotic activities of extracts were investigated by TUNEL and Annexin V/PI assays. using flow cytometry. IC50 and IC20 values of the extracts varied and the effects on endogenous and H2O2-induced DNA damage were different regarding cell lines and extracts. Although their protective effects at some doses against to H2O2-induced oxidative damage, our results suggested DNA damaging and apoptotic potential of cinnamon bark extracts on Type 1 diabetic cell lines, in vitro.

Immunoglobulin preparations for intravenous administration. A review of their biologic activities and comparison of various preparation methods

DEFF Research Database (Denmark)

Nielsen, H

1994-01-01

procedures are employed by different commercial suppliers of immunoglobulins, and from the literature it appears that various important biologic functions, e.g., opsonic activity, complement fixation, and Fc-receptor function, are subject to alterations during the preparation. The best preservation...

Bifunctional rhodium intercalator conjugates as mismatch-directing DNA alkylating agents.

Science.gov (United States)

Schatzschneider, Ulrich; Barton, Jacqueline K

2004-07-21

A conjugate of a DNA mismatch-specific rhodium intercalator, containing the bulky chrysenediimine ligand, and an aniline mustard has been prepared, and targeting of mismatches in DNA by this conjugate has been examined. The preferential alkylation of mismatched over fully matched DNA is found by a mobility shift assay at concentrations where untethered organic mustards show little reaction. The binding site of the Rh intercalator was determined by DNA photocleavage, and the position of covalent modification was established on the basis of the enhanced depurination associated with N-alkylation. The site-selective alkylation at mismatched DNA renders these conjugates useful tools for the covalent tagging of DNA base pair mismatches and new chemotherapeutic design.

Chemical composition and methane potential of commercial food wastes.

Science.gov (United States)

Lopez, Victoria M; De la Cruz, Florentino B; Barlaz, Morton A

2016-10-01

There is increasing interest in anaerobic digestion in the U.S. However, there is little information on the characterization of commercial food waste sources as well as the effect of waste particle size on methane yield. The objective of this research was to characterize four commercial food waste sources: (1) university dining hall waste, (2) waste resulting from prepared foods and leftover produce at a grocery store, (3) food waste from a hotel and convention center, and (4) food preparation waste from a restaurant. Each sample was tested in triplicate 8L batch anaerobic digesters after shredding and after shredding plus grinding. Average methane yields for the university dining, grocery store, hotel, and restaurant wastes were 363, 427, 492, and 403mL/dry g, respectively. Starch exhibited the most complete consumption and particle size did not significantly affect methane yields for any of the tested substrates. Lipids represented 59-70% of the methane potential of the fresh substrates. Copyright © 2016 Elsevier Ltd. All rights reserved.

Preparation of Fe{sub 3}O{sub 4}/poly(styrene-butyl acrylate-[2-(methacryloxy)ethyl]trimethylammonium chloride) by emulsifier-free emulsion polymerization and its interaction with DNA

Energy Technology Data Exchange (ETDEWEB)

Li Xiaolong; Liu Guoqiang; Yan Wei [Ministry-of-Education Key Laboratory for the Green Preparation and Application of Functional Materials, Hubei University, Wuhan (China); Chu, Paul K. [Department of Physics and Materials Science, City University of Hong Kong, Tat Chee Avenue, Kowloon (Hong Kong); Yeung, Kelvin W.K. [Division of Spine Surgery, Department of Orthopaedics and Traumatology, The University of Hong Kong, Pokfulam (Hong Kong); Wu Shuilin; Yi Changfeng [Ministry-of-Education Key Laboratory for the Green Preparation and Application of Functional Materials, Hubei University, Wuhan (China); Department of Physics and Materials Science, City University of Hong Kong, Tat Chee Avenue, Kowloon (Hong Kong); Division of Spine Surgery, Department of Orthopaedics and Traumatology, The University of Hong Kong, Pokfulam (Hong Kong); Xu Zushun, E-mail: zushun25@yahoo.com.cn [Ministry-of-Education Key Laboratory for the Green Preparation and Application of Functional Materials, Hubei University, Wuhan (China); Department of Physics and Materials Science, City University of Hong Kong, Tat Chee Avenue, Kowloon (Hong Kong); Division of Spine Surgery, Department of Orthopaedics and Traumatology, University of Hong Kong, Pokfulam (Hong Kong)

2012-04-15

Cationic magnetic polymer particles Fe{sub 3}O{sub 4}/poly(styrene-butyl acrylate-[2-(methacryloxy)ethyl]trimethylammonium chloride), a type of potential gene carrier, were prepared by emulsifier-free emulsion polymerization with oleic acid modified magnetite Fe{sub 3}O{sub 4}, styrene, butyl acrylate and [2-(methacryloxy)ethyl]trimethylammonium chloride) (METAC). The morphology of the particles was characterized by transmission electron microscopy and the composites of particles were characterized by FT-IR spectroscopy, X-ray diffraction. These results showed that magnetic particles were well dispersed in polymers with the content of about 15%(wt/wt). The composites exhibited superparamagnetism and possessed a certain level of magnetic response. The interactions between the particles with calf-thymus DNA (ct DNA) were confirmed by zeta potential measurement, UV-vis spectroscopy and fluorescence spectroscopy. The DNA-binding capacity determined by the agarose gel electrophoresis showed good binding capacity of the emulsion to DNA. These results suggested the potential of the cationic magnetic polymer emulsion as gene target delivery carrier. - Highlights: Black-Right-Pointing-Pointer A new type of cationic magnetic polymer particles was synthesized by emulsifier-free emulsion polymerization. Black-Right-Pointing-Pointer Structural, morphological, and magnetic properties of the composite were evaluated. Black-Right-Pointing-Pointer The interaction between cationic magnetic polymer particles with DNA was confirmed by zeta potential measurements. Black-Right-Pointing-Pointer UV-vis spectrophotometry, fluorescent spectroscopy and agarose gel electrophoresis. Black-Right-Pointing-Pointer This process may have potential applications to gene carrier and DNA separation.

Characterizing commercial pureed foods: sensory, nutritional, and textural analysis.

Science.gov (United States)

Ettinger, Laurel; Keller, Heather H; Duizer, Lisa M

2014-01-01

Dysphagia (swallowing impairment) is a common consequence of stroke and degenerative diseases such as Parkinson's and Alzheimer's. Limited research is available on pureed foods, specifically the qualities of commercial products. Because research has linked pureed foods, specifically in-house pureed products, to malnutrition due to inferior sensory and nutritional qualities, commercial purees also need to be investigated. Proprietary research on sensory attributes of commercial foods is available; however direct comparisons of commercial pureed foods have never been reported. Descriptive sensory analysis as well as nutritional and texture analysis of commercially pureed prepared products was performed using a trained descriptive analysis panel. The pureed foods tested included four brands of carrots, of turkey, and two of bread. Each commercial puree was analyzed for fat (Soxhlet), protein (Dumas), carbohydrate (proximate analysis), fiber (total fiber), and sodium content (Quantab titrator strips). The purees were also texturally compared with a line spread test and a back extrusion test. Differences were found in the purees for sensory attributes as well as nutritional and textural properties. Findings suggest that implementation of standards is required to reduce variability between products, specifically regarding the textural components of the products. This would ensure all commercial products available in Canada meet standards established as being considered safe for swallowing.

DNA fingerprinting of pearls to determine their origins.

Directory of Open Access Journals (Sweden)

Joana B Meyer

Full Text Available We report the first successful extraction of oyster DNA from a pearl and use it to identify the source oyster species for the three major pearl-producing oyster species Pinctada margaritifera, P. maxima and P. radiata. Both mitochondrial and nuclear gene fragments could be PCR-amplified and sequenced. A polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP assay in the internal transcribed spacer (ITS region was developed and used to identify 18 pearls of unknown origin. A micro-drilling technique was developed to obtain small amounts of DNA while maintaining the commercial value of the pearls. This DNA fingerprinting method could be used to document the source of historic pearls and will provide more transparency for traders and consumers within the pearl industry.

Low cost plastic scintillator by using commercial polystyrene

International Nuclear Information System (INIS)

Oktar, O.; Ari, G.; Guenduez, O.; Demirel, H.; Demirbas, A.

2009-01-01

Plastic scintillation detectors have been used in nuclear and high energy physics for many decades. Among their benefits are fast response, ease of manufacture and versatility. Their main drawbacks are radiation resistance and cost. Plastic Scintillators can be described as solid materials which contain organic fluorescent compounds dissolved within a polymer matri10. Transparent plastics commonly used for light scintillation are Polystyrene (or PS, poly-vinyl-benzene) and polyvinyl-toluene (or PVT, poly-methyl-styron). In this activity, preliminary studies for low cost plastic scintillator production by using commercial polystyrene pellets and extrusion method were aimed. For this purpose, PS blocks consist of commercial fluorescent dopant were prepared by an extruder in SANAEM. Molds suitable for extruder were designed and manufactured and optimum production parameters such as extrusion temperature profile, extrusion rate and pressure were obtained. Plastic blocks prepared were optically and mechanically tested and its response against various radioactive sources was measured.This study has shown that plastic scintillators imported can be produced in SANAEM domestically and be used for detection of radioactive materials within the country or border gates.

Cyclen-based double-tailed lipids for DNA delivery: Synthesis and the effect of linking group structures.

Science.gov (United States)

Zhang, Yi-Mei; Chang, De-Chun; Zhang, Ji; Liu, Yan-Hong; Yu, Xiao-Qi

2015-09-01

The gene transfection efficiency (TE) of cationic lipids is largely influenced by the lipid structure. Six novel 1, 4, 7, 10-tetraazacyclododecane (cyclen)-based cationic lipids L1-L6, which contain double oleyl as hydrophobic tails, were designed and synthesized. The difference between these lipids is their diverse backbone. Liposomes prepared by the lipids and DOPE showed good DNA affinity, and full DNA condensation could be achieved at N/P of 4 to form lipoplexes with proper size and zeta-potentials for gene transfection. Structure-activity relationship of these lipids as non-viral gene delivery vectors was investigated. It was found that minor backbone structural variations, including linking group and the structural symmetry would affect the TE. The diethylenetriamine derived lipid L4 containing amide linking bonds gave the best TE, which was several times higher than commercially available transfection reagent lipofectamine 2000. Besides, these lipids exhibited low cytotoxicity, suggesting their good biocompatibility. Results reveal that such type of cationic lipids might be promising non-viral gene vectors, and also afford us clues for the design of novel vectors with higher TE and biocompatibility. Copyright © 2015 Elsevier Ltd. All rights reserved.

Mechanisms for radiation damage in DNA

International Nuclear Information System (INIS)

Sevilla, M.D.

1993-12-01

In this project the author has proposed several mechanisms for radiation damage to DNA and its constituents, and has detailed a series of experiments utilizing electron spin resonance spectroscopy, HPLC, GC-mass spectroscopy and ab initio molecular orbital calculations to test the proposed mechanisms. In this years work he has completed several experiments on the role of hydration water on DNA radiation damage, continued the investigation of the localization of the initial charges and their reactions on DNA, investigated protonation reactions in DNA base anions, and employed ab initio molecular orbital theory to gain insight into the initial events of radiation damage to DNA. Ab initio calculations have provided an understanding of the energetics evolved in anion and cation formation, ion radical transfer in DNA as well as proton transfer with DNA base pair radical ions. This has been extended in this years work to a consideration of ionization energies of various components of the DNA deoxyribose backbone and resulting neutral sugar radicals. This information has aided the formation of new radiation models for the effect of radiation on DNA. During this fiscal year four articles have been published, four are in press, one is submitted and several more are in preparation. Four papers have been presented at scientific meetings. This years effort will include another review article on the open-quotes Electron Spin Resonance of Radiation Damage to DNAclose quotes

Radiobiology with DNA ligands

International Nuclear Information System (INIS)

Weinreich, R.; Argentini, M.; Guenther, I.; Koziorowski, J.; Larsson, B.; Nievergelt-Egido, M.C.; Salt, C.; Wyer, L.; Dos Santos, D.F.; Hansen, H.J.

1997-01-01

The paper deals with the following topics: labelling of DNA ligands and other tumour-affinic compounds with 4.15-d 124 I, radiotoxicity of Hoechst 33258 and 33342 and of iodinated Hoechst 33258 in cell cultures, preparation of 76 Br-, 123 I-, and 221 At-labelled 5-halo-2'-deoxyuridine, chemical syntheses of boron derivatives of Hoechst 33258.III., Gadolinium neutron capture therapy

CRADA Carbon Sequestration in Soils and Commercial Products

Energy Technology Data Exchange (ETDEWEB)

Jacobs, G.K.

2002-01-31

ORNL, through The Consortium for Research on Enhancing Carbon Sequestration in Terrestrial Ecosystems (CSiTE), collaborated with The Village Botanica, Inc. (VB) on a project investigating carbon sequestration in soils and commercial products from a new sustainable crop developed from perennial Hibiscus spp. Over 500 pre-treated samples were analyzed for soil carbon content. ORNL helped design a sampling scheme for soils during the planting phase of the project. Samples were collected and prepared by VB and analyzed for carbon content by ORNL. The project did not progress to a Phase II proposal because VB declined to prepare the required proposal.

Platelet-Rich Plasma Preparation Types Show Impact on Chondrogenic Differentiation, Migration, and Proliferation of Human Subchondral Mesenchymal Progenitor Cells.

Science.gov (United States)

Kreuz, Peter Cornelius; Krüger, Jan Philipp; Metzlaff, Sebastian; Freymann, Undine; Endres, Michaela; Pruss, Axel; Petersen, Wolf; Kaps, Christian

2015-10-01

To evaluate the chondrogenic potential of platelet concentrates on human subchondral mesenchymal progenitor cells (MPCs) as assessed by histomorphometric analysis of proteoglycans and type II collagen. Furthermore, the migratory and proliferative effect of platelet concentrates were assessed. Platelet-rich plasma (PRP) was prepared using preparation kits (Autologous Conditioned Plasma [ACP] Kit [Arthrex, Naples, FL]; Regen ACR-C Kit [Regen Lab, Le Mont-Sur-Lausanne, Switzerland]; and Dr.PRP Kit [Rmedica, Seoul, Republic of Korea]) by apheresis (PRP-A) and by centrifugation (PRP-C). In contrast to clinical application, freeze-and-thaw cycles were subsequently performed to activate platelets and to prevent medium coagulation by residual fibrinogen in vitro. MPCs were harvested from the cortico-spongious bone of femoral heads. Chondrogenic differentiation of MPCs was induced in high-density pellet cultures and evaluated by histochemical staining of typical cartilage matrix components. Migration of MPCs was assessed using a chemotaxis assay, and proliferation activity was measured by DNA content. MPCs cultured in the presence of 5% ACP, Regen, or Dr.PRP formed fibrous tissue, whereas MPCs stimulated with 5% PRP-A or PRP-C developed compact and dense cartilaginous tissue rich in type II collagen and proteoglycans. All platelet concentrates significantly (ACP, P = .00041; Regen, P = .00029; Dr.PRP, P = .00051; PRP-A, P platelet concentrates but one (Dr.PRP, P = .63) showed a proliferative effect on MPCs, as shown by significant increases (ACP, P = .027; Regen, P = .0029; PRP-A, P = .00021; and PRP-C, P = .00069) in DNA content. Platelet concentrates obtained by different preparation methods exhibit different potentials to stimulate chondrogenic differentiation, migration, and proliferation of MPCs. Platelet concentrates obtained by commercially available preparation kits failed to induce chondrogenic differentiation of MPCs, whereas highly standardized PRP

Isolation and analysis of high quality nuclear DNA with reduced organellar DNA for plant genome sequencing and resequencing

Directory of Open Access Journals (Sweden)

Zdepski Anna

2011-05-01

Full Text Available Abstract Background High throughput sequencing (HTS technologies have revolutionized the field of genomics by drastically reducing the cost of sequencing, making it feasible for individual labs to sequence or resequence plant genomes. Obtaining high quality, high molecular weight DNA from plants poses significant challenges due to the high copy number of chloroplast and mitochondrial DNA, as well as high levels of phenolic compounds and polysaccharides. Multiple methods have been used to isolate DNA from plants; the CTAB method is commonly used to isolate total cellular DNA from plants that contain nuclear DNA, as well as chloroplast and mitochondrial DNA. Alternatively, DNA can be isolated from nuclei to minimize chloroplast and mitochondrial DNA contamination. Results We describe optimized protocols for isolation of nuclear DNA from eight different plant species encompassing both monocot and eudicot species. These protocols use nuclei isolation to minimize chloroplast and mitochondrial DNA contamination. We also developed a protocol to determine the number of chloroplast and mitochondrial DNA copies relative to the nuclear DNA using quantitative real time PCR (qPCR. We compared DNA isolated from nuclei to total cellular DNA isolated with the CTAB method. As expected, DNA isolated from nuclei consistently yielded nuclear DNA with fewer chloroplast and mitochondrial DNA copies, as compared to the total cellular DNA prepared with the CTAB method. This protocol will allow for analysis of the quality and quantity of nuclear DNA before starting a plant whole genome sequencing or resequencing experiment. Conclusions Extracting high quality, high molecular weight nuclear DNA in plants has the potential to be a bottleneck in the era of whole genome sequencing and resequencing. The methods that are described here provide a framework for researchers to extract and quantify nuclear DNA in multiple types of plants.

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

Science.gov (United States)

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

2017-05-12

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

Effects of tritium on DNA, 3

International Nuclear Information System (INIS)

Yamamoto, Osamu; Fuji, Izumi

1984-01-01

Lambda DNA solution was prepared at a concentration of 125μg/ml of 10mM Tris-HCl-5mM NaCl-1mM Na 2 EDTA (pH 7.4). Solutions were irradiated with 60 Co gamma-rays and 3 H beta-rays ( 3 HHO addition), respectively. After irradiation of DNA solutions, single-strand breaks and double-strand breaks were detected by a vertical gel electrophoretic system (BRL Model V16). In both strand breaks higher oxygen enhancement ratios were obtained with 60 Co erradiation. (J.P.N.)

Two mini-preparation protocols to DNA extraction from plants with ...

African Journals Online (AJOL)

AJB SERVER

2006-10-16

Oct 16, 2006 ... samples to process and it is also a non expensive protocol. This method also ... because many of those chemicals inhibit PCR reactions. (Pandey et al., 1996) ... Spin at 15,000 rpm for 15 min and wash the DNA pellet with 1.2 ml ... Protocol: To 200 mg frozen and ground tissue plant material, add 900 µl of.

Repair of DNA in xeroderma pigmentosum conjunctiva

International Nuclear Information System (INIS)

Newsome, D.A.; Kraemer, K.H.; Robbins, J.H.

1975-01-01

Xeroderma pigmentosum (XP) is an autosomal recessive disease with tumor formation on sun-exposed areas of the skin and eyes. Cells from most XP patients are deficient in repairing DNA damaged by ultraviolet (uv) light as shown by a reduced rate of tritiated thymidine (3HTdR) incorporation during their DNA repair synthesis. We have studied such repair synthesis in conjunctival cells from an XP patient with a conjunctival epithelioma and from normal cadaver conjunctiva. Cultured conjunctival cells were irradiated with uv light and then incubated with 3HTdR. Autoradiograms were prepared and showed that uv radiation induced a considerably slower rate of DNA repair synthesis in the XP cells than in normal cells. Many of the ocular abnormalities of XP, including tumor formation, may be the result of this defective DNA repair process

Problems in radioimmunoassay of human lutropin with commercially available regents

International Nuclear Information System (INIS)

Hammond, J.E.; Phillips, J.C.; Straight, C.B.; Hammond, M.G.

1980-01-01

To evaluate five commercially available reagent sets supplied for the radioimmunoassay of lutropin, we determined whether there was parallelism between the curve given by dilutions of the standards supplied by the manufacturers, by dilutions of a serum pool, and by dilutions of a standard preparation from human pituitaries, LER-907. These studies demonstrated significant analytical problems with three of the five sets. We conclude that each user should carefully evaluate all commercially available radioimmunoassays for lutropin (and, by inference, for other peptide hormones) before use

Failure to detect circulating DNA-anti-DNA complexes by four radioimmunological methods in patients with systemic lupus erythematosus

Energy Technology Data Exchange (ETDEWEB)

Izui, S; Lambert, P H; Miescher, P A [Hopital Cantonal Geneve (Switzerland)

1977-12-01

The presence of The DNA-anti-DNA complexes in sera from patients with systemic lupus erythematosus (SLE) was investigated by two new radioimmunoassays (RIA) developed for this purpose and by measuring the CLq and DNA binding activity of serum before and after treatment with DNAse. Two direct RIA developed in this study were based on the reactivity of (/sup 3/H)actinomycin D ((/sup 3/H)ACT-D) or solid-phase methylated bovine serum albumin (mBSA) with DNA-anti-DNA complexes. DNA-anti-DNA complexes prepared in vitro could be efficiently detected at various antigen-antibody ratios by these two RIA. Increased levels of circulating immune complexes as indicated by the CLq binding test were found in 52% of the SLE sera. However, the frequency of specific DNA-anti-DNA complexes detected in the SLE sera was very low. Only 6% of the sera exhibited an increased value deviating by more than three s.d. from the normal mean when tested with the (/sup 3/H)ACT-D binding RIA or the solid-phase mBSA RIA. On the other hand, there was no significant difference in the serum CLq or DNA binding activity after treatment with DNAse. These results suggest that DNA-anti-DNA complexes do not occur frequently in circulating blood and represent only a very small portion of the immune complexes detected in serum from patients with SLE.

How commercial and non-commercial swine producers move pigs in Scotland: a detailed descriptive analysis.

Science.gov (United States)

Porphyre, Thibaud; Boden, Lisa A; Correia-Gomes, Carla; Auty, Harriet K; Gunn, George J; Woolhouse, Mark E J

2014-06-25

The impact of non-commercial producers on disease spread via livestock movement is related to their level of interaction with other commercial actors within the industry. Although understanding these relationships is crucial in order to identify likely routes of disease incursion and transmission prior to disease detection, there has been little research in this area due to the difficulties of capturing movements of small producers with sufficient resolution. Here, we used the Scottish Livestock Electronic Identification and Traceability (ScotEID) database to describe the movement patterns of different pig production systems which may affect the risk of disease spread within the swine industry. In particular, we focused on the role of small pig producers. Between January 2012 and May 2013, 23,169 batches of pigs were recorded moving animals between 2382 known unique premises. Although the majority of movements (61%) were to a slaughterhouse, the non-commercial and the commercial sectors of the Scottish swine industry coexist, with on- and off-movement of animals occurring relatively frequently. For instance, 13% and 4% of non-slaughter movements from professional producers were sent to a non-assured commercial producer or to a small producer, respectively; whereas 43% and 22% of movements from non-assured commercial farms were sent to a professional or a small producer, respectively. We further identified differences between producer types in several animal movement characteristics which are known to increase the risk of disease spread. Particularly, the distance travelled and the use of haulage were found to be significantly different between producers. These results showed that commercial producers are not isolated from the non-commercial sector of the Scottish swine industry and may frequently interact, either directly or indirectly. The observed patterns in the frequency of movements, the type of producers involved, the distance travelled and the use of haulage

Awaken to the World of Food Service; Commercial Cooking and Baking--Basic: 9193.01.

Science.gov (United States)

Dade County Public Schools, Miami, FL.

This course outline has been prepared as a guide for the tenth grade student in commercial cooking and baking or food management, production, and services. It provides basic experiences in the field of commercial food service, the hotel and restaurant industry and types of food service establishments. The course consists of 90 clock hours, covered…

Evaluation of Patients with an Apparent False Positive Stool DNA Test: The Role of Repeat Stool DNA Testing.

Science.gov (United States)

Cooper, Gregory S; Markowitz, Sanford D; Chen, Zhengyi; Tuck, Missy; Willis, Joseph E; Berger, Barry M; Brenner, Dean E; Li, Li

2018-03-07

There is uncertainty as to the appropriate follow-up of patients who test positive on multimarker stool DNA (sDNA) testing and have a colonoscopy without neoplasia. To determine the prevalence of missed colonic or occult upper gastrointestinal neoplasia in patients with an apparent false positive sDNA. We prospectively identified 30 patients who tested positive with a commercially available sDNA followed by colonoscopy without neoplastic lesions. Patients were invited to undergo repeat sDNA at 11-29 months after the initial test followed by repeat colonoscopy and upper endoscopy. We determined the presence of neoplastic lesions on repeat evaluation stratified by results of repeat sDNA. Twelve patients were restudied. Seven patients had a negative second sDNA test and a normal second colonoscopy and upper endoscopy. In contrast, 5 of 12 subjects had a persistently positive second sDNA test, and 3 had positive findings, including a 3-cm sessile transverse colon adenoma with high-grade dysplasia, a 2-cm right colon sessile serrated adenoma with dysplasia, and a nonadvanced colon adenoma (p = 0.045). These corresponded to a positive predictive value of 0.60 (95% CI 0.17-1.00) and a negative predictive value of 1.00 (95% CI 1.00-1.00) for the second sDNA test. In addition, the medical records of all 30 subjects with apparent false positive testing were reviewed and no documented cases of malignant tumors were recorded. Repeat positive sDNA testing may identify a subset of patients with missed or occult colorectal neoplasia after negative colonoscopy for an initially positive sDNA. High-quality colonoscopy with careful attention to the right colon in patients with positive sDNA is critically important and may avoid false negative colonoscopy.

Electronic imaging systems for quantitative electrophoresis of DNA

International Nuclear Information System (INIS)

Sutherland, J.C.

1989-01-01

Gel electrophoresis is one of the most powerful and widely used methods for the separation of DNA. During the last decade, instruments have been developed that accurately quantitate in digital form the distribution of materials in a gel or on a blot prepared from a gel. In this paper, I review the various physical properties that can be used to quantitate the distribution of DNA on gels or blots and the instrumentation that has been developed to perform these tasks. The emphasis here is on DNA, but much of what is said also applies to RNA, proteins and other molecules. 36 refs

The detection of HBV DNA with gold-coated iron oxide nanoparticle gene probes

International Nuclear Information System (INIS)

Xi Dong; Luo Xiaoping; Lu Qianghua; Yao Kailun; Liu Zuli; Ning Qin

2008-01-01

Gold-coated iron oxide nanoparticle Hepatitis B virus (HBV) DNA probes were prepared, and their application for HBV DNA measurement was studied. Gold-coated iron oxide nanoparticles were prepared by the citrate reduction of tetra-chloroauric acid in the presence of iron oxide nanoparticles which were added as seeds. With a fluorescence-based method, the maximal surface coverage of hexaethiol 30-mer oligonucleotides and the maximal percentage of hybridization strands on gold-coated iron oxide nanoparticles were (120 ± 8) oligonucleotides per nanoparticle, and (14 ± 2%), respectively, which were comparable with those of (132 ± 10) and (22 ± 3%) in Au nanoparticle groups. Large network aggregates were formed when gold-coated iron oxide nanoparticle HBV DNA gene probe was applied to detect HBV DNA molecules as evidenced by transmission electron microscopy and the high specificity was verified by blot hybridization. Our results further suggested that detecting DNA with iron oxide nanoparticles and magnetic separator was feasible and might be an alternative effective method

Aligned deposition and electrical measurements on single DNA molecules

International Nuclear Information System (INIS)

Eidelshtein, Gennady; Kotlyar, Alexander; Hashemi, Mohtadin; Gurevich, Leonid

2015-01-01

A reliable method of deposition of aligned individual dsDNA molecules on mica, silicon, and micro/nanofabricated circuits is presented. Complexes of biotinylated double stranded poly(dG)–poly(dC) DNA with avidin were prepared and deposited on mica and silicon surfaces in the absence of Mg 2+ ions. Due to its positive charge, the avidin attached to one end of the DNA anchors the complex to negatively charged substrates. Subsequent drying with a directional gas flow yields DNA molecules perfectly aligned on the surface. In the avidin–DNA complex only the avidin moiety is strongly and irreversibly bound to the surface, while the DNA counterpart interacts with the substrates much more weakly and can be lifted from the surface and realigned in any direction. Using this technique, avidin–DNA complexes were deposited across platinum electrodes on a silicon substrate. Electrical measurements on the deposited DNA molecules revealed linear IV-characteristics and exponential dependence on relative humidity. (paper)

Použití metody kvantifikace DNA jako screeningového nástroje pro efektivní genotypování vzorků ve forenzní DNA laboratoři.

OpenAIRE

Koljenšič, Ivana

2011-01-01

Quantification of human DNA in forensic samples is an important step during STR profiling because the STR genotyping is sensitive to the quantity of DNA used in the PCR reaction. This study focuses on the importance of quantification in the entire process of genetic analysis. Two real time PCR platforms (Roche LightCycler480 System and ABI 7900 RT PCR) were used to compare two commercial kits in terms of DNA quantification. It was found out that accuracy of absolute quantification values in c...

Synthesis and characterization of erbium-doped SiO{sub 2}-TiO{sub 2} thin films prepared by sol-gel and dip-coating techniques onto commercial glass substrates as a route for obtaining active GRadient-INdex materials

Energy Technology Data Exchange (ETDEWEB)

Gómez-Varela, Ana I. [Microoptics and GRIN Optics Group, Department of Applied Physics, Faculty of Optics and Optometry and Faculty of Physics, Universidade de Santiago de Compostela, Campus Vida s/n, Santiago de Compostela E-15782 (Spain); Castro, Yolanda, E-mail: castro@icv.csic.es [Instituto de Cerámica y Vidrio (CSIC), Kelsen 5, Campus de Cantoblanco, Madrid 28049 (Spain); Durán, Alicia [Instituto de Cerámica y Vidrio (CSIC), Kelsen 5, Campus de Cantoblanco, Madrid 28049 (Spain); De Beule, Pieter A.A. [Applied Nano-Optics Laboratory, International Iberian Nanotechnology Laboratory, Braga 4715-330 (Portugal); Flores-Arias, María T. [Microoptics and GRIN Optics Group, Department of Applied Physics, Faculty of Optics and Optometry and Faculty of Physics, Universidade de Santiago de Compostela, Campus Vida s/n, Santiago de Compostela E-15782 (Spain); Bao-Varela, Carmen, E-mail: carmen.bao@usc.es [Microoptics and GRIN Optics Group, Department of Applied Physics, Faculty of Optics and Optometry and Faculty of Physics, Universidade de Santiago de Compostela, Campus Vida s/n, Santiago de Compostela E-15782 (Spain)

2015-05-29

In this work, SiO{sub 2}-TiO{sub 2} films doped with erbium were prepared by dip-coating sol-gel process onto commercial glass substrates. The surface morphology of the films was characterized using atomic force microscopy, while thickness, refractive index, extinction coefficient and porosity of the films were determined by ellipsometric measurements in a wavelength region of 400-1000 nm. Optical constants and porosity were found to vary with erbium concentration. The proof of principle presented in this paper is applicable to systems of different nature by tailoring the sol-gel precursors in such a way that active GRadient-INdex media described by a complex, parabolic-like refractive index distribution for beam shaping purposes is obtained. - Highlights: • Sol-gel route for preparation of active GRadient-INdex materials is proposed. • SiO{sub 2}-TiO{sub 2} films doped with erbium were prepared by dipping onto commercial glasses. • Morphological and optical characterization of the samples was performed. • Optical constants and porosity were found to vary with erbium concentration. • Refractive index diminishes with dopant content; the contrary occurs for porosity.

Hydrolysis of solubilized hemicellulose derived from wet-oxidized wheat straw by a mixture of commercial fungal enzyme preparations

Energy Technology Data Exchange (ETDEWEB)

Skammelsen Schmidt, Anette; Thomsen, Alle Belinda; Woidemann, Anders [Risoe National Lab. (Denmark); Tenkanen, Maija [VTT Biotechnology and Food Research (Finland)

1998-04-01

The enzymatic hydrolysis of the solubilized hemicellulose fraction from wet-oxidized wheat straw was investigated for quantification purposes. An optimal hydrolysis depends on factors such as composition of the applied enzyme mixture and the hydrolysis conditions (enzyme loading, hydrolysis time, pH-value, and temperature). A concentrated enzyme mixture was used in this study prepared at VTT Biotechnology and Food Research, Finland, by mixing four commercial enzyme preparations. No distinctive pH-value and temperature optima were identified after a prolonged incubation of 24 hours. By reducing the hydrolysis time to 2 hours a temperature optimum was found at 50 deg. C, where a pH-value higher than 5.2 resulted in reduced activity. An enzyme-substrate-volume-ratio of 0.042, a pH-value of 5.0, and a temperature of 50 deg. C were chosen as the best hydrolysis conditions due to an improved monosaccharide yield. The hydrolysis time was chosen to be 24 hours to ensure equilibrium and total quantification. Even under the best hydrolysis conditions, the overall sugar yield from the enzymatic hydrolysis was only 85% of that of the optimal acid hydrolysis. The glucose yield were approximately the same for the two types of hydrolyses, probably due to the high cellulase activity in the VTT-enzyme mixture. For xylose and arabinose the enzymatic hydrolysis yielded only 80% of that of the acid hydrolysis. As the pentoses existed mainly as complex polymers their degradation required many different enzymes, some of which might be missing from the VTT-enzyme mixture. Furthermore, the removal of side-choins from the xylan backbone during the wet-oxidation pretreatment process might enable the hemicellulosic polymers to interact and precipitate, hence, reducing the enzymatic digestibility of the hemicellulose. (au) 8 tabs., 10 ills., 65 refs.

Final Report: The DNA Files: Unraveling the mysteries of genetics, January 1, 1998-March 31, 1999

Energy Technology Data Exchange (ETDEWEB)

Scott, Bari

1999-05-01

The DNA Files is an award-winning radio documentary series on genetics created by SoundVision Productions. The DNA Files was hosted by John Hockenberry and was presented in documentary and discussion format. The programs covered a range of topics from prenatal and predictive gene testing, gene therapy, and commercialization of genetic information to new evolutionary genetic evidence, transgenic vegetables and use of DNA in forensics.

Preparation of alumina microspheres

International Nuclear Information System (INIS)

Santos, W.R. dos; Abrao, A.

1980-01-01

Inorganic exchangers are widely used for adsorption and column partition chromatography. The main difficulty of using commercial alumina (in powder) for column chromatography is related to its packing, and the operations through the column become diffcult and time-consuming; also it turns to be virtually impossible to use large dimension columns. In order to eliminate these problems, a process for the preparation of alumina micro-spheres was developed as an adaptation of a similar process used to prepare nuclear fuel microspheres (UO 2 , ThO 2 ). The flowsheet of this process is presented together with the analytical results of sphericity after calcination, granulometry, density and characterization by X-ray diffractometry. Solubility tests showed that the so-prepared microspheres are well resistant to strong acids and bases; retention tests showed their efficiency, mainly to copper. (C.L.B.) [pt

Simplified preparation of coniferyl and sinapyl alcohols.

Science.gov (United States)

Kim, Hoon; Ralph, John

2005-05-04

Coniferyl and sinapyl alcohols were prepared from commercially available coniferaldehyde and sinapaldehyde using borohydride exchange resin in methanol. This reduction is highly regioselective and exceptionally simple, making these valuable monolignols readily available to researchers lacking synthetic chemistry expertise.

Regulation of DNA replication in irradiated cells by trans-acting factors

International Nuclear Information System (INIS)

Wang, Y.; Huq, M.S.; Cheng, X.; Iliakis, G.

1995-01-01

We compared DNA replication activity in cytoplasmic extracts prepared from irradiated and nonirradiated HeLa cells using a simian virus 40 (SV40)-based in vitro replication assay. The assay measures semi-conservative DNA replication in a plasmid carrying the SV40 origin of replication and requires SV40 T antigen as the sole noncellular protein. The plasmid DNA used in the replication reaction is never exposed to radiation. We find that replication of plasmid DNA is significantly reduced when cytoplasmic extracts from irradiated cells are used. Since plasmid replication proceeds to completion in extracts from irradiated cells, the observed reduction in the overall replication activity is probably due to a reduction in the efficiency of initiation events. The degree of inhibition of DNA replication after exposure to 10, 30 and 50 Gy X rays as measured in vitro using this assay is similar to that measured in intact cells immediately before processing for extract preparation. These observations are compatible with the induction or activation by ionizing radiation of a factor(s) that inhibits in trans DNA replication. The results contribute to our understanding of the mechanism(s) developed by the cells to regulate DNA replication when exposed to clastogenic agents. Such processes may be of significance in the restoration of DNA integrity, and may define yet another checkpoint operating during S at the level of clusters of replicons. 26 refs., 4 figs

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

Directory of Open Access Journals (Sweden)

Da-Sheng Lee

2009-12-01

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

Ligation bias in illumina next-generation DNA libraries: implications for sequencing ancient genomes.

Directory of Open Access Journals (Sweden)

Andaine Seguin-Orlando

Full Text Available Ancient DNA extracts consist of a mixture of endogenous molecules and contaminant DNA templates, often originating from environmental microbes. These two populations of templates exhibit different chemical characteristics, with the former showing depurination and cytosine deamination by-products, resulting from post-mortem DNA damage. Such chemical modifications can interfere with the molecular tools used for building second-generation DNA libraries, and limit our ability to fully characterize the true complexity of ancient DNA extracts. In this study, we first use fresh DNA extracts to demonstrate that library preparation based on adapter ligation at AT-overhangs are biased against DNA templates starting with thymine residues, contrarily to blunt-end adapter ligation. We observe the same bias on fresh DNA extracts sheared on Bioruptor, Covaris and nebulizers. This contradicts previous reports suggesting that this bias could originate from the methods used for shearing DNA. This also suggests that AT-overhang adapter ligation efficiency is affected in a sequence-dependent manner and results in an uneven representation of different genomic contexts. We then show how this bias could affect the base composition of ancient DNA libraries prepared following AT-overhang ligation, mainly by limiting the ability to ligate DNA templates starting with thymines and therefore deaminated cytosines. This results in particular nucleotide misincorporation damage patterns, deviating from the signature generally expected for authenticating ancient sequence data. Consequently, we show that models adequate for estimating post-mortem DNA damage levels must be robust to the molecular tools used for building ancient DNA libraries.

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.

Pressure-driven one-step solid phase-based on-chip sample preparation on a microfabricated plastic device and integration with flow-through polymerase chain reaction (PCR).

Science.gov (United States)

Tran, Hong Hanh; Trinh, Kieu The Loan; Lee, Nae Yoon

2013-10-01

In this study, we fabricate a monolithic poly(methylmethacrylate) (PMMA) microdevice on which solid phase-based DNA preparation and flow-through polymerase chain reaction (PCR) units were functionally integrated for one-step sample preparation and amplification operated by pressure. Chelex resin, which is used as a solid support for DNA preparation, can capture denatured proteins but releases DNA, and the purified DNA can then be used as a template in a subsequent amplification process. Using the PMMA microdevices, DNA was successfully purified from both Escherichia coli and human hair sample, and the plasmid vector inserted in E. coli and the D1S80 locus in human genomic DNA were successfully amplified from on-chip purified E. coli and human hair samples. Furthermore, the integration potential of the proposed sample preparation and flow-through PCR units was successfully demonstrate on a monolithic PMMA microdevice with a seamless flow, which could pave the way for a pressure-driven, simple one-step sample preparation and amplification with greatly decreased manufacture cost and enhanced device disposability. Copyright © 2013 Elsevier B.V. All rights reserved.

Protein unfolding allows use of commercial antibodies in an apolipoprotein M sandwich ELISA

DEFF Research Database (Denmark)

Bosteen, Markus Høybye; Dahlbäck, Björn; Nielsen, Lars Bo

2015-01-01

that specifically recognizes human apoM in plasma using commercially available reagents. Commercial apoM antibodies were screened for compatibility in a sandwich ELISA-based assay. One optimal pair of antibodies was chosen, and sample preparation, buffers, and incubation times were optimized to generate a simple...... and reproducible method. Validation and comparison to a previously described ELISA for apoM confirmed that the assay displays a high degree of sensitivity, specificity, and precision. Our results show that commercially available antibodies can be used to accurately measure human plasma apoM. This method can...

MOLECULAR CLONING OF OVINE cDNA LEPTIN GENE

Directory of Open Access Journals (Sweden)

CLAUDIA TEREZIA SOCOL

2008-05-01

Full Text Available An efficient bacterial transformation system suitable for cloning the coding sequence of the ovine leptin gene in E. coli DH5α host cells using the pGEMT easy vector it is described in this paper. The necessity of producing leptin is based on the fact that the role of this molecule in the animal and human organism is still unknown, leptin not existing as commercial product on the Romanian market. The results obtained in the bacterial transformation, cloning, recombinant clones selection, control of the insertion experiments and DNA computational analysis represent the first steps in further genetic engineering experiments such as production of DNA libraries, DNA sequencing, protein expression, etc., for a further contribution in elucidating the role of leptin in the animal and human organism.

Human DNA Extraction by Two Extraction Methods for Forensic Typification from Human Feces on FTA Paper

Directory of Open Access Journals (Sweden)

Shirleny Monserrat Sandoval-Arias

2014-11-01

Full Text Available The identification of suspects in criminal investigations has been facilitated since DNA test are executed on different samples. The application of this technology for forensic typification from human fecal samples still presents complications therefore this research evaluated two DNA extraction protocols with modifications to determine that of major efficiency. Organic extractions and extractions using the commercial kit “IQTM DNA Casework Sample Kit for Maxwell ® 16” on FTA portions of 4cm2 and 1cm2 impregnated with feces from the same individual were done to accomplish the objective. In all the assays the results were useful, however; the best forensic typification (by the electropherogram characteristics was obtained by using the commercial kit in an area of 1 cm2 of FTA paper impregnated in a 1:4 dilution.

Commercialization plan laser-based decoating systems

International Nuclear Information System (INIS)

Freiwald, J.; Freiwald, D.A.

1998-01-01

F2 Associates Inc. (F2) is a small, high-technology firm focused on developing and commercializing environmentally friendly laser ablation systems for industrial-rate removal of surface coatings from metals, concrete, and delicate substrates such as composites. F2 has a contract with the US Department of Energy Federal Energy Technology Center (FETC) to develop and test a laser-based technology for removing contaminated paint and other contaminants from concrete and metal surfaces. Task 4.1 in Phase 2 of the Statement of Work for this DOE contract requires that F2 ''document its plans for commercializing and marketing the stationary laser ablation system. This document shall include a discussion of prospects for commercial customers and partners and may require periodic update to reflect changing strategy. This document shall be submitted to the DOE for review.'' This report is being prepared and submitted in fulfillment of that requirement. This report describes the laser-based technology for cleaning and coatings removal, the types of laser-based systems that have been developed by F2 based on this technology, and the various markets that are emerging for this technology. F2's commercialization and marketing plans are described, including how F2's organization is structured to meet the needs of technology commercialization, F2's strategy and marketing approach, and the necessary steps to receive certification for removing paint from aircraft and DOE certification for D and D applications. The future use of the equipment built for the DOE contract is also discussed

75 FR 12981 - Eligibility for Commercial Flats Failing Deflection

Science.gov (United States)

2010-03-18

... applicable only to automation flats, for all commercial flat-size mail except saturation and high-density...[supreg] pricing. If automation prices are denied, pieces that are prepared to be part of full-service IMb... compliance, but at additional cost; and quarter-folding would not work well with any inserts. Also, there was...

DNA migration mechanism analyses for applications in capillary and microchip electrophoresis

Science.gov (United States)

Forster, Ryan E.; Hert, Daniel G.; Chiesl, Thomas N.; Fredlake, Christopher P.; Barron, Annelise E.

2009-01-01

In 2009, electrophoretically driven DNA separations in slab gels and capillaries have the sepia tones of an old-fashioned technology in the eyes of many, even while they remain ubiquitously used, fill a unique niche, and arguably have yet to reach their full potential. For comic relief, what is old becomes new again: agarose slab gel separations are used to prepare DNA samples for “next-gen” sequencing platforms (e.g., the Illumina and 454 machines)—dsDNA molecules within a certain size range are “cut out” of a gel and recovered for subsequent “massively parallel” pyrosequencing. In this review, we give a Barron lab perspective on how our comprehension of DNA migration mechanisms in electrophoresis has evolved, since the first reports of DNA separations by CE (∼1989) until now, 20 years later. Fused silica capillaries, and borosilicate glass and plastic microchips, quietly offer increasing capacities for fast (and even “ultra-fast”), efficient DNA separations. While the channel-by-channel scaling of both old and new electrophoresis platforms provides key flexibility, it requires each unique DNA sample to be prepared in its own micro- or nanovolume. This Achille's heel of electrophoresis technologies left an opening through which pooled-sample, next-gen DNA sequencing technologies rushed. We shall see, over time, whether sharpening understanding of transitions in DNA migration modes in crosslinked gels, nanogel solutions, and uncrosslinked polymer solutions will allow electrophoretic DNA analysis technologies to flower again. Microchannel electrophoresis, after a quiet period of metamorphosis, may emerge sleeker and more powerful, to claim its own important niche applications. PMID:19582705

Preparation, characterization and application of some anti- corrosive molybdate pigments

International Nuclear Information System (INIS)

Abd El-Ghaffar, M.A.; El-Sawy, S.M.; Ahmed, N.M.

2005-01-01

Some molybdate pigments of single and mixed metal ions, namely, zinc, calcium and zinc-calcium molybdates were prepared, characterized and evaluated according to international standard methods. The evaluated pigments were incorporated in some paint formulations. The physicomechanical, chemical and corrosion protective properties of the paint films were measured; this was done in comparison with a commercial imported molybdate pigment. It was found that, the prepared pigments under investigation are fine white crystalline powders of suitable pigment properties. They can be successfully used as environmentally acceptable anti corrosive pigments. They can replace satisfactorily the similar commercial imported pigment and possess adequate or superior properties against corrosion

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.

Two efficient methods for isolation of high-quality genomic DNA from entomopathogenic fungi.

Science.gov (United States)

Serna-Domínguez, María G; Andrade-Michel, Gilda Y; Arredondo-Bernal, Hugo C; Gallou, Adrien

2018-03-27

Conventional and commercial methods for isolation of nucleic acids are available for fungal samples including entomopathogenic fungi (EPF). However, there is not a unique optimal method for all organisms. The cell wall structure and the wide range of secondary metabolites of EPF can broadly interfere with the efficiency of the DNA extraction protocol. This study compares three commercial protocols: DNeasy® Plant Mini Kit (Qiagen), Wizard® Genomic DNA Purification Kit (Promega), and Axygen™ Multisource Genomic DNA Miniprep Kit (Axygen) and three conventional methods based on different buffers: SDS, CTAB/PVPP, and CTAB/β-mercaptoethanol versus three cell lysis procedures: liquid nitrogen homogenization and two bead-beating materials (i.e., tungsten-carbide and stainless-steel) for four representative species of EPF (i.e., Beauveria bassiana, Hirsutella citriformis, Isaria javanica, and Metarhizium anisopliae). Liquid nitrogen homogenization combined with DNeasy® Plant Mini Kit (i.e., QN) or SDS buffer (i.e., SN) significantly improved the yield with a good purity (~1.8) and high integrity (>20,000 bp) of genomic DNA in contrast with other methods, also, these results were better when compared with the two bead-beating materials. The purified DNA was evaluated by PCR-based techniques: amplification of translation elongation factor 1-α (TEF) and two highly sensitive molecular markers (i.e., ISSR and AFLP) with reliable and reproducible results. Despite a variation in yield, purity, and integrity of extracted DNA across the four species of EPF with the different DNA extraction methods, the SN and QN protocols maintained a high-quality of DNA which is required for downstream molecular applications. Copyright © 2018 Elsevier B.V. All rights reserved.

Environmental DNA from seawater samples correlate with trawl catches of Subarctic, deepwater fishes

DEFF Research Database (Denmark)

Thomsen, Philip Francis; Møller, Peter Rask; Sigsgaard, Eva Egelyng

2016-01-01

such as bottom trawling, and on official reporting of global catches, which can be unreliable. Thus, there is need for alternative and non-invasive techniques for qualitative and quantitative oceanic fish surveys. Here we report environmental DNA (eDNA) metabarcoding of seawater samples from continental slope...... depths in Southwest Greenland. We collected seawater samples at depths of 188-918 m and compared seawater eDNA to catch data from trawling. We used Illumina sequencing of PCR products to demonstrate that eDNA reads show equivalence to fishing catch data obtained from trawling. Twenty-six families were...... found with both trawling and eDNA, while three families were found only with eDNA and two families were found only with trawling. Key commercial fish species for Greenland were the most abundant species in both eDNA reads and biomass catch, and interpolation of eDNA abundances between sampling sites...

Abnormal recovery of DNA replication in ultraviolet-irradiated cell cultures of Drosophila melanogaster which are defective in DNA repair

International Nuclear Information System (INIS)

Brown, T.C.; Boyd, J.B.

1981-01-01

Cell cultures prepared from embryos of a control stock of Drosophila melanogaster respond to ultraviolet light with a decline and subsequent recovery both of thymidine incorporation and in the ability to synthesize nascent DNA in long segments. Recovery of one or both capacities is absent or diminished in irradiated cells from ten nonallelic mutants that are defective in DNA repair and from four of five nonallelic mutagen-sensitive mutants that exhibit normal repair capabilities. Recovery of thymidine incorporation is not observed in nine of ten DNA repair-defective mutants. On the other hand, partial or complete recovery of incorporation is observed in all but one repair-proficient mutagen-sensitive mutant. (orig./AJ) [de

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

Directory of Open Access Journals (Sweden)

Alyssa D. Ammazzalorso

2015-08-01

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

Optimizing Frozen Sample Preparation for Laser Microdissection: Assessment of CryoJane Tape-Transfer System®.

Directory of Open Access Journals (Sweden)

Yelena G Golubeva

Full Text Available Laser microdissection is an invaluable tool in medical research that facilitates collecting specific cell populations for molecular analysis. Diversity of research targets (e.g., cancerous and precancerous lesions in clinical and animal research, cell pellets, rodent embryos, etc. and varied scientific objectives, however, present challenges toward establishing standard laser microdissection protocols. Sample preparation is crucial for quality RNA, DNA and protein retrieval, where it often determines the feasibility of a laser microdissection project. The majority of microdissection studies in clinical and animal model research are conducted on frozen tissues containing native nucleic acids, unmodified by fixation. However, the variable morphological quality of frozen sections from tissues containing fat, collagen or delicate cell structures can limit or prevent successful harvest of the desired cell population via laser dissection. The CryoJane Tape-Transfer System®, a commercial device that improves cryosectioning outcomes on glass slides has been reported superior for slide preparation and isolation of high quality osteocyte RNA (frozen bone during laser dissection. Considering the reported advantages of CryoJane for laser dissection on glass slides, we asked whether the system could also work with the plastic membrane slides used by UV laser based microdissection instruments, as these are better suited for collection of larger target areas. In an attempt to optimize laser microdissection slide preparation for tissues of different RNA stability and cryosectioning difficulty, we evaluated the CryoJane system for use with both glass (laser capture microdissection and membrane (laser cutting microdissection slides. We have established a sample preparation protocol for glass and membrane slides including manual coating of membrane slides with CryoJane solutions, cryosectioning, slide staining and dissection procedure, lysis and RNA extraction

Inspecting Targeted Deep Sequencing of Whole Genome Amplified DNA Versus Fresh DNA for Somatic Mutation Detection: A Genetic Study in Myelodysplastic Syndrome Patients.

Science.gov (United States)

Palomo, Laura; Fuster-Tormo, Francisco; Alvira, Daniel; Ademà, Vera; Armengol, María Pilar; Gómez-Marzo, Paula; de Haro, Nuri; Mallo, Mar; Xicoy, Blanca; Zamora, Lurdes; Solé, Francesc

2017-08-01

Whole genome amplification (WGA) has become an invaluable method for preserving limited samples of precious stock material and has been used during the past years as an alternative tool to increase the amount of DNA before library preparation for next-generation sequencing. Myelodysplastic syndromes (MDS) are a group of clonal hematopoietic stem cell disorders characterized by presenting somatic mutations in several myeloid-related genes. In this work, targeted deep sequencing has been performed on four paired fresh DNA and WGA DNA samples from bone marrow of MDS patients, to assess the feasibility of using WGA DNA for detecting somatic mutations. The results of this study highlighted that, in general, the sequencing and alignment statistics of fresh DNA and WGA DNA samples were similar. However, after variant calling and when considering variants detected at all frequencies, there was a high level of discordance between fresh DNA and WGA DNA (overall, a higher number of variants was detected in WGA DNA). After proper filtering, a total of three somatic mutations were detected in the cohort. All somatic mutations detected in fresh DNA were also identified in WGA DNA and validated by whole exome sequencing.

Systematic evaluation and optimization of modification reactions of oligonucleotides with amines and carboxylic acids for the synthesis of DNA-encoded chemical libraries.

Science.gov (United States)

Franzini, Raphael M; Samain, Florent; Abd Elrahman, Maaly; Mikutis, Gediminas; Nauer, Angela; Zimmermann, Mauro; Scheuermann, Jörg; Hall, Jonathan; Neri, Dario

2014-08-20

DNA-encoded chemical libraries are collections of small molecules, attached to DNA fragments serving as identification barcodes, which can be screened against multiple protein targets, thus facilitating the drug discovery process. The preparation of large DNA-encoded chemical libraries crucially depends on the availability of robust synthetic methods, which enable the efficient conjugation to oligonucleotides of structurally diverse building blocks, sharing a common reactive group. Reactions of DNA derivatives with amines and/or carboxylic acids are particularly attractive for the synthesis of encoded libraries, in view of the very large number of building blocks that are commercially available. However, systematic studies on these reactions in the presence of DNA have not been reported so far. We first investigated conditions for the coupling of primary amines to oligonucleotides, using either a nucleophilic attack on chloroacetamide derivatives or a reductive amination on aldehyde-modified DNA. While both methods could be used for the production of secondary amines, the reductive amination approach was generally associated with higher yields and better purity. In a second endeavor, we optimized conditions for the coupling of a diverse set of 501 carboxylic acids to DNA derivatives, carrying primary and secondary amine functions. The coupling efficiency was generally higher for primary amines, compared to secondary amine substituents, but varied considerably depending on the structure of the acids and on the synthetic methods used. Optimal reaction conditions could be found for certain sets of compounds (with conversions >80%), but multiple reaction schemes are needed when assembling large libraries with highly diverse building blocks. The reactions and experimental conditions presented in this article should facilitate the synthesis of future DNA-encoded chemical libraries, while outlining the synthetic challenges that remain to be overcome.

Signal replication in a DNA nanostructure

Science.gov (United States)

Mendoza, Oscar; Houmadi, Said; Aimé, Jean-Pierre; Elezgaray, Juan

2017-01-01

Logic circuits based on DNA strand displacement reaction are the basic building blocks of future nanorobotic systems. The circuits tethered to DNA origami platforms present several advantages over solution-phase versions where couplings are always diffusion-limited. Here we consider a possible implementation of one of the basic operations needed in the design of these circuits, namely, signal replication. We show that with an appropriate preparation of the initial state, signal replication performs in a reproducible way. We also show the existence of side effects concomitant to the high effective concentrations in tethered circuits, such as slow leaky reactions and cross-activation.

Probing DNA-stabilized fluorescent silver nanocluster spectral heterogeneity by time-correlated single photon counting

DEFF Research Database (Denmark)

Carro, Miguel; Paolucci, Valentina; Hooley, Emma Nicole

2016-01-01

DNA-stabilized silver nanoclusters (DNA-AgNCs) are promising fluorophores whose photophysical properties and synthesis procedures have received increased attention in the literature. However, depending on the preparation conditions and the DNA sequence, the DNA-AgNC samples can host a range...... the spectral heterogeneity of other fluorophores, such as luminescent colloidal nanoparticles, and to assess the reproducibility of a synthetic procedure containing an unknown distribution of emissive species....

78 FR 29698 - Availability of an Environmental Assessment for Field Testing a Canine Lymphoma Vaccine, DNA

Science.gov (United States)

2013-05-21

...] Availability of an Environmental Assessment for Field Testing a Canine Lymphoma Vaccine, DNA AGENCY: Animal and... Canine Lymphoma Vaccine, DNA. The environmental assessment, which is based on a risk analysis prepared to... biological product: Requester: Merial, Inc. Product: Canine Lymphoma Vaccine, DNA. Possible Field Test...

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.

Polyphosphate present in DNA preparations from fungal species of Collectotrichum inhibits restriction endonucleases and other enzymes

Science.gov (United States)

Rodriguez, R.J.

1993-01-01

During the development of a procedure for the isolation of total genomic DNA from filamentous fungi (Rodriguez, R. J., and Yoder, 0. C., Exp. Mycol. 15, 232-242, 1991) a cell fraction was isolated which inhibited the digestion of DNA by restriction enzymes. After elimination of DNA, RNA, proteins, and lipids, the active compound was purified by gel filtration to yield a single fraction capable of complete inhibition of restriction enzyme activity. The inhibitor did not absorb uv light above 220 nm, and was resistant to alkali and acid at 25°C and to temperatures as high as 100°C. More extensive analyses demonstrated that the inhibitor was also capable of inhibiting T4 DNA ligase and TaqI DNA polymerase, but not DNase or RNase. Chemical analyses indicated that the inhibitor was devoid of carbohydrates, proteins, lipids, and nucleic acids but rich in phosphorus. A combination of nuclear magnetic resonance, metachromatic shift of toluidine blue, and gel filtration indicated that the inhibitor was a polyphosphate (polyP) containing approximately 60 phosphate molecules. The mechanism of inhibition appeared to involve complexing of polyP to the enzymatic proteins. All species of Colletotrichum analyzed produced polyP equivalent in chain length and concentration. A modification to the original DNA extraction procedure is described which eliminates polyP and reduces the time necessary to obtain DNA of sufficient purity for restriction enzyme digestion and TaqI polymerase amplification.

Parental DNA methylation states are associated with heterosis in epigenetic hybrids

NARCIS (Netherlands)

Lauss, Kathrin; Wardenaar, René; Oka, Rurika; van Hulten, Marieke H A; Guryev, Victor; Keurentjes, Joost J B; Stam, Maike; Johannes, Frank

Despite the importance and wide exploitation of heterosis in commercial crop breeding, the molecular mechanisms behind this phenomenon are not completely understood. Recent studies have implicated changes in DNA methylation and small RNAs in hybrid performance, however, it remains unclear whether

Parental DNA methylation states are associated with heterosis in epigenetic hybrids

NARCIS (Netherlands)

Lauss, K.; Wardenaar, R.; Oka, R.; van Hulten, M.H.A.; Guryev, V.; Keurentjes, J.J.B.; Stam, M.; Johannes, F.

Despite the importance and wide exploitation of heterosis in commercial crop breeding, the molecular mechanisms behind this phenomenon are not completely understood. Recent studies have implicated changes in DNA methylation and small RNAs in hybrid performance; however, it remains unclear whether

Parental DNA methylation states are associated with heterosis in epigenetic hybrids

NARCIS (Netherlands)

Lauss, Kathrin; Wardenaar, R.; Oka, Rurika; Hulten, M.H.A.; Guryev, Victor; Keurentjes, J.J.B.; Stam, Maike; Johannes, Frank

2018-01-01

Despite the importance and wide exploitation of heterosis in commercial crop breeding, the molecular mechanisms behind this phenomenon are not completely understood. Recent studies have implicated changes in DNA methylation and small RNAs in hybrid performance, however, it remains unclear whether

Liquid Chromatographic-Chemometric Techniques for the Simultaneous HPLC Determination of Lansoprazole, Amoxicillin and Clarithromycin in Commercial Preparation.

Science.gov (United States)

Aktas, A Hakan; Saridag, Ayse Mine

2017-09-01

Two multivariate calibration-prediction techniques, principal component regression (PCR) and partial least-squares regression (PLSR) were applied to the chromatographic multicomponent analysis of the drug containing lansoprazole (LAN), clarithromycin (CLA) and amoxicillin (AMO). Optimum chromatographic separation of LAN, CLA and AMO with atorvastatin as the internal standard (IS) was obtained by using Xterra® RP18 column 5 μm 4.6 × 250 mm2, and 25 mM ammonium chloride buffer prepared ammonium chloride, acetonitrile and bidistilled water (45:45:10 v/v) as the mobile phase at flow rate 1.0 mL/min. The high pressure liquid chromatography data sets consisting of the ratios of analyte peak areas to the IS peak area were obtained by using diode array detector detection at five wavelengths (205, 210, 215, 220 and 225 nm). LC-chemometric calibration for LAN, CLA and AMO were separately constructed by using the relationship between the peak-area ratio and training sets for each analyte. A series of synthetic solutions containing different concentrations of LAN, CLA and AMO were used to check the prediction ability of the PCR and PLS. Both of the two-chemometric methods in this study can be satisfactorily used for the quantitative analysis and for dissolutions tests of multicomponent commercial drug. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Bacterial DNA in water and dialysate: detection and significance for patient outcomes.

Science.gov (United States)

Handelman, Garry J; Megdal, Peter A; Handelman, Samuel K

2009-01-01

The fluid used for hemodialysis may contain DNA fragments from bacteria, which could be harmful for patient outcomes. DNA fragments from bacteria, containing the nonmethylated CpG motif, can trigger inflammation through the monocyte and lymphocyte Toll-like receptor 9, and these DNA fragments have been observed in dialysate. The fragments may transfer across the dialyzer into the patient's bloodstream during hemodialysis treatment. During hemodiafiltration, the fragments would be introduced directly into the bloodstream. The DNA fragments may arise from biofilm in the pipes of the water system, from growth of bacteria in the water, or as contaminants in the bicarbonate and salt mixture used for preparation of dialysate. Current filtration methods, such as Diasafe filters, are not able to remove these fragments. It would be prudent to seek to reduce or eliminate these contaminants. However, the cost and effort of decreasing bacterial DNA content may ultimately require substantial facility improvements; we therefore need to fund research studies to determine if modifications to reduce bacterial DNA content are clinically warranted. This research will require methods to accurately determine the species of bacteria that contribute the DNA, since this information will allow the source to be established as biofilm, bicarbonate mixtures, or other problems in the dialysis system such as bacterial growth or leakage during water preparation. In this review, the evidence for bacterial DNA fragments will be examined and suggestions for further studies will be described.

Patterning nanocrystals using DNA

Energy Technology Data Exchange (ETDEWEB)

Williams, Shara Carol [Univ. of California, Berkeley, CA (United States)

2003-01-01

One of the goals of nanotechnology is to enable programmed self-assembly of patterns made of various materials with nanometer-sized control. This dissertation describes the results of experiments templating arrangements of gold and semiconductor nanocrystals using 2'-deoxyribonucleic acid (DNA). Previously, simple DNA-templated linear arrangements of two and three nanocrystals structures have been made.[1] Here, we have sought to assemble larger and more complex nanostructures. Gold-DNA conjugates with 50 to 100 bases self-assembled into planned arrangements using strands of DNA containing complementary base sequences. We used two methods to increase the complexity of the arrangements: using branched synthetic doublers within the DNA covalent backbone to create discrete nanocrystal groupings, and incorporating the nanocrystals into a previously developed DNA lattice structure [2][3] that self-assembles from tiles made of DNA double-crossover molecules to create ordered nanoparticle arrays. In the first project, the introduction of a covalently-branched synthetic doubler reagent into the backbone of DNA strands created a branched DNA ''trimer.'' This DNA trimer templated various structures that contained groupings of three and four gold nanoparticles, giving promising, but inconclusive transmission electron microscopy (TEM) results. Due to the presence of a variety of possible structures in the reaction mixtures, and due to the difficulty of isolating the desired structures, the TEM and gel electrophoresis results for larger structures having four particles, and for structures containing both 5 and 10 nm gold nanoparticles were inconclusive. Better results may come from using optical detection methods, or from improved sample preparation. In the second project, we worked toward making two-dimensional ordered arrays of nanocrystals. We replicated and improved upon previous results for making DNA lattices, increasing the size of the lattices

[Prokaryotic expression of recombinant prochymosin gene and its antiserum preparation].

Science.gov (United States)

Li, Xin-ping; Liu, Huan-huan; Pu, Yan; Zhang, Fu-chun; Li, Yi-jie

2012-07-01

To optimize the prochymosin (pCHY) gene codons and express the gene in Escherichia coli (E.coli), and to prepare its antiserum and detect chymosin protein specifically. According to codon usage bias of E.coli, prochymosin gene sequence was synthesized based on the conserved sequences of prochymosin gene from bovine, lamb and camel, and then cloned into the plasmid pET-30a and pcDNA3-AAT-COMP-C3d3 (pcD-ACC), respectively. pET-30a-pCHY was expressed, as the detected antigen, in E.coli BL21(DE3) after IPTG induction. RT-PCR was used to detect prochymosin mRNA expression in liver from the mice injected pcDNA3-AAT-COMP-pCHY-C3d3(pACCC) by hydrodynamics-based transfection method. To prepare the antiserum of prochymosin, pACCC and GST-pCHY proteins were used to immunize New Zealand rabbits in accordance with DNA prime-protein boost strategy. Antibody levels were tested by ELISA. Western blotting showed the molecular weight of His-pCHY protein was about 55 000, similar to the expected molecular size. ELISA demonstrated that the titer level of prochymosin antiserum was high. Based on the codon optimization, we have obtained high-titer prochymosin antiserum through DNA vaccine vector pcD-ACC combined with DNA prime-protein boost strategy, similar to that by protein vaccine.

Interface Layering Phenomena in Capacitance Detection of DNA with Biochips

Directory of Open Access Journals (Sweden)

Sandro Carrara

2007-02-01

Full Text Available Reliable DNA detection is of great importance for the development of the Lab-on-chip technology. The effort of the most recent projects on this field is to integrate all necessary operations, such as sample preparation (mixing, PCR amplification together with the sensor user for DNA detection. Among the different ways to sense the DNA hybridization, fluorescence based detection has been favored by the market. However, fluorescence based approaches require that the DNA targets are labeled by means of chromophores. As an alternative label-free DNA detection method, capacitance detection was recently proposed by different authors. While this effect has been successfully demonstrated by several groups, the model used for data analysis is far too simple to describe the real behavior of a DNA sensor. The aim of the present paper is to propose a different electrochemical model to describe DNA capacitance detection.

Comparison of nine DNA extraction methods for the diagnosis of bovine tuberculosis by real time PCR

OpenAIRE

Moura, André; Hodon, Mikael Arrais; Soares Filho, Paulo Martins; Issa, Marina de Azevedo; Oliveira, Ana Paula Ferreira de; Fonseca Júnior, Antônio Augusto

2016-01-01

ABSTRACT: Bovine tuberculosis is an infectious disease with a high impact on the cattle industry, particularly in developing countries. PCR is a very sensitive method for detection of infectious agents, but the sensitivity of molecular diagnosis is largely dependent on the efficiency of the DNA extraction methods. The objective of this study was to evaluate DNA extraction methods for direct detection of Mycobacterium bovis in bovine tissue. Nine commercial kits for DNA extraction were evalua...

Preparation of medicines for children - a hierarchy of classification.

Science.gov (United States)

Ernest, Terry B; Craig, Jo; Nunn, Anthony; Salunke, Smita; Tuleu, Catherine; Breitkreutz, Joerg; Alex, Rainer; Hempenstall, John

2012-10-05

There is some confusion about the types of paediatric pharmaceutical preparation (in a regulatory and pharmaceutical development context) that are acceptable for approval by medicines regulators. Some of the confusion relates to terminology which may mean different things to different stakeholders. It may not always be possible to provide authorised, commercially manufactured, age appropriate, ready-to-administer preparations. In terms of assurance of quality and bioavailability there is a continuum from this ideal through intermediate products through authorised compounding and manipulation of commercial dosage forms to ad hoc compounding using only the skills and experience of the individual pharmacist. Additionally, it is widely known that caregivers may manipulate medicines at home, for example by segmenting tablets and by addition to foods or liquids. The first intent of the manufacturer should be to provide for children an age appropriate, ready-to-administer preparation which is commercially manufactured and approved by the competent authorities. However, there will still be a place for providing other age appropriate preparations such as approved products that are 'intermediates' requiring reconstitution before use, or instructions for compounding or manipulation of a dosage form. If compounding or manipulation is likely to be required it is preferable that data are generated by Industry, approved by the competent authorities and provided in the Summary of Product Characteristics (SmPC). It is acknowledged however, that ad hoc compounding or manipulation may also take place in certain circumstances such as logistical difficulties or to satisfy the needs of the child who does not find the authorised product to be 'age appropriate'. This paper explores compounding and manipulation of medicines in relation to approval by medicines regulators and non-approved preparation to fulfil the needs of the individual patient. Definitions are proposed to provide a

Robust Sub-nanomolar Library Preparation for High Throughput Next Generation Sequencing.

Science.gov (United States)

Wu, Wells W; Phue, Je-Nie; Lee, Chun-Ting; Lin, Changyi; Xu, Lai; Wang, Rong; Zhang, Yaqin; Shen, Rong-Fong

2018-05-04

Current library preparation protocols for Illumina HiSeq and MiSeq DNA sequencers require ≥2 nM initial library for subsequent loading of denatured cDNA onto flow cells. Such amounts are not always attainable from samples having a relatively low DNA or RNA input; or those for which a limited number of PCR amplification cycles is preferred (less PCR bias and/or more even coverage). A well-tested sub-nanomolar library preparation protocol for Illumina sequencers has however not been reported. The aim of this study is to provide a much needed working protocol for sub-nanomolar libraries to achieve outcomes as informative as those obtained with the higher library input (≥ 2 nM) recommended by Illumina's protocols. Extensive studies were conducted to validate a robust sub-nanomolar (initial library of 100 pM) protocol using PhiX DNA (as a control), genomic DNA (Bordetella bronchiseptica and microbial mock community B for 16S rRNA gene sequencing), messenger RNA, microRNA, and other small noncoding RNA samples. The utility of our protocol was further explored for PhiX library concentrations as low as 25 pM, which generated only slightly fewer than 50% of the reads achieved under the standard Illumina protocol starting with > 2 nM. A sub-nanomolar library preparation protocol (100 pM) could generate next generation sequencing (NGS) results as robust as the standard Illumina protocol. Following the sub-nanomolar protocol, libraries with initial concentrations as low as 25 pM could also be sequenced to yield satisfactory and reproducible sequencing results.

Controls to validate plasma samples for cell free DNA quantification

DEFF Research Database (Denmark)

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

2015-01-01

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

The nucleotide sequence of human transition protein 1 cDNA

Energy Technology Data Exchange (ETDEWEB)

Luerssen, H; Hoyer-Fender, S; Engel, W [Universitaet Goettingen (West Germany)

1988-08-11

The authors have screened a human testis cDNA library with an oligonucleotide of 81 mer prepared according to a part of the published nucleotide sequence of the rat transition protein TP 1. They have isolated a cDNA clone with the length of 441 bp containing the coding region of 162 bp for human transition protein 1. There is about 84% homology in the coding region of the sequence compared to rat. The human cDNA-clone encodes a polypeptide of 54 amino acids of which 7 are different to that of rat.

Photoluminescence Enhancement of Poly(3-methylthiophene Nanowires upon Length Variable DNA Hybridization

Directory of Open Access Journals (Sweden)

Jingyuan Huang

2018-01-01

Full Text Available The use of low-dimensional inorganic or organic nanomaterials has advantages for DNA and protein recognition due to their sensitivity, accuracy, and physical size matching. In this research, poly(3-methylthiophene (P3MT nanowires (NWs are electrochemically prepared with dopant followed by functionalization with probe DNA (pDNA sequence through electrostatic interaction. Various lengths of pDNA sequences (10-, 20- and 30-mer are conjugated to the P3MT NWs respectively followed with hybridization with their complementary target DNA (tDNA sequences. The nanoscale photoluminescence (PL properties of the P3MT NWs are studied throughout the whole process at solid state. In addition, the correlation between the PL enhancement and the double helix DNA with various lengths is demonstrated.

Fluorescence correlation spectroscopy analysis for accurate determination of proportion of doubly labeled DNA in fluorescent DNA pool for quantitative biochemical assays.

Science.gov (United States)

Hou, Sen; Sun, Lili; Wieczorek, Stefan A; Kalwarczyk, Tomasz; Kaminski, Tomasz S; Holyst, Robert

2014-01-15

Fluorescent double-stranded DNA (dsDNA) molecules labeled at both ends are commonly produced by annealing of complementary single-stranded DNA (ssDNA) molecules, labeled with fluorescent dyes at the same (3' or 5') end. Because the labeling efficiency of ssDNA is smaller than 100%, the resulting dsDNA have two, one or are without a dye. Existing methods are insufficient to measure the percentage of the doubly-labeled dsDNA component in the fluorescent DNA sample and it is even difficult to distinguish the doubly-labeled DNA component from the singly-labeled component. Accurate measurement of the percentage of such doubly labeled dsDNA component is a critical prerequisite for quantitative biochemical measurements, which has puzzled scientists for decades. We established a fluorescence correlation spectroscopy (FCS) system to measure the percentage of doubly labeled dsDNA (PDL) in the total fluorescent dsDNA pool. The method is based on comparative analysis of the given sample and a reference dsDNA sample prepared by adding certain amount of unlabeled ssDNA into the original ssDNA solution. From FCS autocorrelation functions, we obtain the number of fluorescent dsDNA molecules in the focal volume of the confocal microscope and PDL. We also calculate the labeling efficiency of ssDNA. The method requires minimal amount of material. The samples have the concentration of DNA in the nano-molar/L range and the volume of tens of microliters. We verify our method by using restriction enzyme Hind III to cleave the fluorescent dsDNA. The kinetics of the reaction depends strongly on PDL, a critical parameter for quantitative biochemical measurements. Copyright © 2013 Elsevier B.V. All rights reserved.

Polarization Selectivity of Artificial Anisotropic Structures Based on DNA-Like Helices

International Nuclear Information System (INIS)

Semchenko, I. V.; Khakhomov, S. A.; Balmakov, A. P.

2010-01-01

Currently, 2D and 3D structures of different symmetries can be formed from DNA molecules. The electromagnetic properties of this new natural chiral material can be changed by metalizing DNA. Spatial structures of this type can be used in nanotechnology to prepare metamaterials for the far-UV region. It is shown by the example of an octahedron and a cube composed of DNA-like helices that these structures may exhibit polarization selectivity to electromagnetic radiation. In addition, it is suggested that the effect of the polarization selectivity of DNA-like artificial structures may also occur in the soft X-ray region for all living organisms in nature due to the universal DNA form.

Weathering durability of commercial polymeric coatings tested by positron annihilation spectroscopy

International Nuclear Information System (INIS)

Chen, H.; Peng, Q.; Huang, Y.Y.; Zhang, R.; Li, Y.; Zhang, J.; Wu, Y.C.; Richardson, J.R.; Sandreczki, T.C.; Jean, Y.C.

2003-01-01

A series of commercial coatings were prepared according to the industrial specifications and were exposed to Florida natural weathering and controlled UVA irradiation. The Doppler broadening energy spectra (DBES) of positron annihilation were measured as a function of incident positron energy at different periods of weathering. A significant decrease in the S parameter was observed and it was used as an indicator to test coating durability in micro-scale. Application to weathering durability of commercial polymeric coatings under natural weathering and controlled UVA irradiation is investigated by using the S parameter from the DBES

Plasmid fermentation process for DNA immunization applications.

Science.gov (United States)

Carnes, Aaron E; Williams, James A

2014-01-01

Plasmid DNA for immunization applications must be of the highest purity and quality. The ability of downstream purification to efficiently produce a pure final product is directly influenced by the performance of the upstream fermentation process. While several clinical manufacturing facilities already have validated fermentation processes in place to manufacture plasmid DNA for use in humans, a simple and inexpensive laboratory-scale fermentation process can be valuable for in-house production of plasmid DNA for use in animal efficacy studies. This chapter describes a simple fed-batch fermentation process for producing bacterial cell paste enriched with high-quality plasmid DNA. A constant feeding strategy results in a medium cell density culture with continuously increasing plasmid amplification towards the end of the process. Cell banking and seed culture preparation protocols, which can dramatically influence final product yield and quality, are also described. These protocols are suitable for production of research-grade plasmid DNA at the 100 mg-to-1.5 g scale from a typical 10 L laboratory benchtop fermentor.

Ethical, Legal and Social Issues in Japan on the Determination of Blood Relationship via DNA Testing.

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Toya, Waki

2017-01-01

DNA paternity testing has recently become more widely available in Japan. The aim of this paper is to examine the issues surrounding (1) the implementing agency, whether the testing is conducted in a commercial direct-to-consumer (DTC) setting or a judicial non-DTC setting, and (2) the implementation conditions and more specifically the legal capacity of the proband (test subject). Literature research in Japanese and English was conducted. Some countries prohibit commercial DNA testing without the consent of the proband or her or his legally authorized representative. But as in some cases, the results of DTC paternity testing have proven to be unreliable. I propose a complete prohibition of DTC DNA paternity testing in Japan. In many cases of paternity testing, the proband is a minor. This has led to debate about whether proxy consent is sufficient for paternity testing or whether additional safeguards (such as a court order) are required. In cases where commercial DNA testing has been conducted and the test results are produced in court as evidence, the court must judge whether or not to admit these results as evidence. Another important issue is whether or not paternity testing should be legally mandated in certain cases. If we come to the conclusion that DNA test results are the only way to conclusively establish a parent-child relationship, then our society may prioritize even more genetic relatedness over other conceptions of a parent-child relationship. This prioritization could adversely affect families created through assisted reproductive technology (ART), especially in situations where children are not aware of their biological parentage. This paper argues for a complete prohibition of DTC DNA paternity testing in Japan, and highlights that broader ethical and legal deliberation on such genetic services is required.

Stability, protein binding and clearance studies of [99mTc]DTPA. Evaluation of a commercially available dry-kit

DEFF Research Database (Denmark)

Rehling, M

1988-01-01

[99mTc]DTPA has achieved widespread use for the measurement of glomerular filtration rate (GFR) with the single injection plasma clearance technique and for gamma-camera renography. However, the quality of the commercial preparations varies. The purpose of the present investigation was to study...... the quality of a commercial [99mTc]DTPA preparation (C.I.S., France) with reference to stability, protein binding and accuracy of the determined plasma clearance values as a measure of GFR. The stability of the preparations was studied by thin-layer chromatography, the in vitro protein binding by Sephadex.......7% and 1.1%, respectively. The plasma clearance of [99mTc]DTPA was on an average 3.7% higher than that of [51Cr]EDTA in 27 patients. It is concluded that the [99mTc]DTPA preparation is reliable for the measurement of GFR. The preparation is stable for at least six hours at room temperature...

Persistent and heritable structural damage induced in heterochromatic DNA from rat liver by N-nitrosodimethylamine

International Nuclear Information System (INIS)

Ward, E.J.; Stewart, B.W.

1987-01-01

Analysis, by benzoylated DEAE-cellulose chromatography, has been made of structural change in eu- and heterochromatic DNA from rat liver following administration of the carcinogen N-nitrosodimethylamine. Either hepatic DNA was prelabeled with [ 3 H]thymidine administered 2-3 weeks before injection of the carcinogen or the labeled precursor was given during regenerative hyperplasia in rats treated earlier with N-nitrosodimethylamine. Following phenol extraction of either whole liver homogenate or nuclease-fractionated eu- and heterochromatin, carcinogen-modified DNA was examined by stepwise or caffeine gradient elution from benzoylated DEAE-cellulose. In whole DNA, nitrosamine-induced single-stranded character was maximal 4-24 h after treatment, declining rapidly thereafter; gradient elution of these DNA preparations also provided short-term evidence of structural change. Caffeine gradient chromatography suggested short-term nitrosamine-induced structural change in euchromatic DNA, while increased binding of heterochromatic DNA was evident for up to 3 months after carcinogen treatment. Preparations of newly synthesized heterochromatic DNA from animals subjected to hepatectomy up to 2 months after carcinogen treatment provided evidence of heritable structural damage. Carcinogen-induced binding of heterochromatic DNA to benzoylated DEAE-cellulose was indicative of specific structural lesions whose affinity equalled that of single-stranded DNA up to 1.0 kilobase in length. The data suggest that structural lesions in heterochromatin, which may be a consequence of incomplete repair, are preferentially degraded by endogenous nuclease(s)

Differentiating Authentic Adenophorae Radix from Its Adulterants in Commercially-Processed Samples Using Multiplexed ITS Sequence-Based SCAR Markers

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Byeong Cheol Moon

2017-06-01

Full Text Available Determining the precise botanical origin of a traditional herbal medicine is important for basic quality control. In both the Chinese and Korean herbal pharmacopoeia, authentic Adenophorae Radix is defined as the roots of Adenophora stricta and Adenophora triphylla. However, the roots of Codonopsis lanceolata, Codonopsis pilosula, and Glehnia littoralis are frequently distributed as Adenophorae Radix in Korean herbal markets. Unfortunately, correctly identifying dried roots is difficult using conventional methods because the roots of those species are morphologically similar. Therefore, we developed DNA-based markers for the identification of authentic Adenophorae Radix and its common adulterants in commercially-processed samples. To develop a reliable method to discriminate between Adenophorae Radix and its adulterants, we sequenced the nuclear ribosomal DNA internal transcribed spacers (nrDNA-ITS and designed sequence-characterized amplified region (SCAR primers specific to the authentic and adulterant species. Using these primers, we developed SCAR markers for each species and established a multiplex-PCR method that can authenticate the four herbal medicines in a single PCR reaction. Furthermore, we confirmed that commercially-processed herbal medicines, which often have degraded DNA, could be assessed with our method. Therefore, our method is a reliable genetic tool to protect against adulteration and to standardize the quality of Adenophorae Radix.

The impact of different DNA extraction kits and laboratories upon the assessment of human gut microbiota composition by 16S rRNA gene sequencing.

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Kennedy, Nicholas A; Walker, Alan W; Berry, Susan H; Duncan, Sylvia H; Farquarson, Freda M; Louis, Petra; Thomson, John M; Satsangi, Jack; Flint, Harry J; Parkhill, Julian; Lees, Charlie W; Hold, Georgina L

2014-01-01

Determining bacterial community structure in fecal samples through DNA sequencing is an important facet of intestinal health research. The impact of different commercially available DNA extraction kits upon bacterial community structures has received relatively little attention. The aim of this study was to analyze bacterial communities in volunteer and inflammatory bowel disease (IBD) patient fecal samples extracted using widely used DNA extraction kits in established gastrointestinal research laboratories. Fecal samples from two healthy volunteers (H3 and H4) and two relapsing IBD patients (I1 and I2) were investigated. DNA extraction was undertaken using MoBio Powersoil and MP Biomedicals FastDNA SPIN Kit for Soil DNA extraction kits. PCR amplification for pyrosequencing of bacterial 16S rRNA genes was performed in both laboratories on all samples. Hierarchical clustering of sequencing data was done using the Yue and Clayton similarity coefficient. DNA extracted using the FastDNA kit and the MoBio kit gave median DNA concentrations of 475 (interquartile range 228-561) and 22 (IQR 9-36) ng/µL respectively (p<0.0001). Hierarchical clustering of sequence data by Yue and Clayton coefficient revealed four clusters. Samples from individuals H3 and I2 clustered by patient; however, samples from patient I1 extracted with the MoBio kit clustered with samples from patient H4 rather than the other I1 samples. Linear modelling on relative abundance of common bacterial families revealed significant differences between kits; samples extracted with MoBio Powersoil showed significantly increased Bacteroidaceae, Ruminococcaceae and Porphyromonadaceae, and lower Enterobacteriaceae, Lachnospiraceae, Clostridiaceae, and Erysipelotrichaceae (p<0.05). This study demonstrates significant differences in DNA yield and bacterial DNA composition when comparing DNA extracted from the same fecal sample with different extraction kits. This highlights the importance of ensuring that samples

Effect of the use of commercial Saccharomyces strains in a newly established winery in Ronda (Málaga, Spain).

Science.gov (United States)

Clavijo, Almudena; Calderón, Isabel L; Paneque, Patricia

2011-03-01

An ecological study of the yeasts present in a spontaneous and an inoculated fermentation in red wine was carried out in 2005 vintage in a winery located in the Denomination of Origin "Sierras de Málaga" (Málaga, southern of Spain). The winery operated by the first time with the 2003 vintage and since then, has used commercial yeast inocula to start alcoholic fermentation. Yeast isolates were identified by PCR-RFLP analysis of the 5.8S-ITS region from the ribosomal DNA and by mitochondrial DNA RFLP analysis. Except for non-Saccharomyces yeasts found in the fresh must before fermentation, all the isolates were found to be commercial Saccharomyces cerevisiae strains employed by the winery during the successive vintages; thus, no indigenous Saccharomyces yeasts were isolated during fermentation. The same four restriction patterns were found in non inoculated and inoculated vats, although with different frequencies. The use of commercial yeast starter in a new established winery seems to have prevented the development of a resident indigenous Saccharomyces flora.

PCR-Free Enrichment of Mitochondrial DNA from Human Blood and Cell Lines for High Quality Next-Generation DNA Sequencing.

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Meetha P Gould

Full Text Available Recent advances in sequencing technology allow for accurate detection of mitochondrial sequence variants, even those in low abundance at heteroplasmic sites. Considerable sequencing cost savings can be achieved by enriching samples for mitochondrial (relative to nuclear DNA. Reduction in nuclear DNA (nDNA content can also help to avoid false positive variants resulting from nuclear mitochondrial sequences (numts. We isolate intact mitochondrial organelles from both human cell lines and blood components using two separate methods: a magnetic bead binding protocol and differential centrifugation. DNA is extracted and further enriched for mitochondrial DNA (mtDNA by an enzyme digest. Only 1 ng of the purified DNA is necessary for library preparation and next generation sequence (NGS analysis. Enrichment methods are assessed and compared using mtDNA (versus nDNA content as a metric, measured by using real-time quantitative PCR and NGS read analysis. Among the various strategies examined, the optimal is differential centrifugation isolation followed by exonuclease digest. This strategy yields >35% mtDNA reads in blood and cell lines, which corresponds to hundreds-fold enrichment over baseline. The strategy also avoids false variant calls that, as we show, can be induced by the long-range PCR approaches that are the current standard in enrichment procedures. This optimization procedure allows mtDNA enrichment for efficient and accurate massively parallel sequencing, enabling NGS from samples with small amounts of starting material. This will decrease costs by increasing the number of samples that may be multiplexed, ultimately facilitating efforts to better understand mitochondria-related diseases.

Current developments in forensic interpretation of mixed DNA samples (Review)

Science.gov (United States)

HU, NA; CONG, BIN; LI, SHUJIN; MA, CHUNLING; FU, LIHONG; ZHANG, XIAOJING

2014-01-01

A number of recent improvements have provided contemporary forensic investigations with a variety of tools to improve the analysis of mixed DNA samples in criminal investigations, producing notable improvements in the analysis of complex trace samples in cases of sexual assult and homicide. Mixed DNA contains DNA from two or more contributors, compounding DNA analysis by combining DNA from one or more major contributors with small amounts of DNA from potentially numerous minor contributors. These samples are characterized by a high probability of drop-out or drop-in combined with elevated stutter, significantly increasing analysis complexity. At some loci, minor contributor alleles may be completely obscured due to amplification bias or over-amplification, creating the illusion of additional contributors. Thus, estimating the number of contributors and separating contributor genotypes at a given locus is significantly more difficult in mixed DNA samples, requiring the application of specialized protocols that have only recently been widely commercialized and standardized. Over the last decade, the accuracy and repeatability of mixed DNA analyses available to conventional forensic laboratories has greatly advanced in terms of laboratory technology, mathematical models and biostatistical software, generating more accurate, rapid and readily available data for legal proceedings and criminal cases. PMID:24748965

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

Science.gov (United States)

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

2018-06-01

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

The preparation of dental glass-ceramic composites with controlled fraction of leucite crystals

Directory of Open Access Journals (Sweden)

Martina Mrázová