Dramatic Increase in the Signal and Sensitivity of Detection via Self-Assembly of Branched DNA

PubMed Central

Kim, Kyung-Tae; Chae, Chi-Bom

2011-01-01

In molecular testing using PCR, the target DNA is amplified via PCR and the sequence of interest is investigated via hybridization with short oligonucleotide capture probes that are either in a solution or immobilized on solid supports such as beads or glass slides. In this report, we report the discovery of assembly of DNA complex(es) between a capture probe and multiple strands of the PCR product. The DNA complex most likely has branched structure. The assembly of branched DNA was facilitated by the product of asymmetric PCR. The amount of branched DNA assembled was increased five fold when the asymmetric PCR product was denatured and hybridized with a capture probe all in the same PCR reaction mixture. The major branched DNA species appeared to contain three reverse strands (the strand complementary to the capture probe) and two forward strands. The DNA was sensitive to S1 nuclease suggesting that it had single-stranded gaps. Branched DNA also appeared to be assembled with the capture probes immobilized on the surface of solid support when the product of asymmetric PCR was hybridized. Assembly of the branched DNA was also increased when hybridization was performed in complete PCR reaction mixture suggesting the requirement of DNA synthesis. Integration of asymmetric PCR, heat denaturation and hybridization in the same PCR reaction mixture with the capture probes immobilized on the surface of solid support achieved dramatic increase in the signal and sensitivity of detection of DNA. Such a system should be advantageously applied for development of automated process for detection of DNA. PMID:21870112

Rapid purification of circular DNA by triplex-mediated affinity capture

DOEpatents

Ji, Huamin; Smith, Lloyd M.

1997-01-01

A single-step capture of a target supercoiled double-stranded DNA molecule is accomplished by forming a local triple-helix among two strands of the supercoiled circular DNA and an oligonucleotide probe. The oligonucleotide is bound to an immobilizing support which facilitates the immobilization and purification of target DNA molecules. Non-target DNA molecules and other contaminating cellular material are easily removed by washing. The triple-helical structure is destabilized by raising the pH, leaving purified target DNA in the supernatant and reusable affinity capture oligonucleotide secured to the immobilizing support.

NF-κB– and AP-1–Mediated DNA Looping Regulates Osteopontin Transcription in Endotoxin-Stimulated Murine Macrophages

PubMed Central

Zhao, Wei; Wang, Lijuan; Zhang, Meng; Wang, Peng; Zhang, Lei; Yuan, Chao; Qi, Jianni; Qiao, Yu; Kuo, Paul C.; Gao, Chengjiang

2013-01-01

Osteopontin (OPN) is expressed by various immune cells and modulates both innate and adaptive immune responses. However, the molecular mechanisms that control opn gene expression, especially at the chromatin level, remain largely unknown. We have previously demonstrated many specific cis- and trans-regulatory elements that determine the extent of endotoxin (LPS)-mediated induction of OPN synthesis in murine macrophages. In the present study, we confirm that NF-κB also plays an important role in the setting of LPS-stimulated OPN expression through binding to a distal regulatory element. Importantly, we demonstrate that LPS stimulates chromosomal loops in the OPN promoter between NF-κB binding site and AP-1 binding site using chromosome conformation capture technology. The crucial role of NF-κB and AP-1 in LPS-stimulated DNA looping was confirmed, as small interfering RNA knock-down of NF-κB p65 and AP-1 c-Jun exhibited decreased levels of DNA looping. Furthermore, we demonstrate that p300 can form a complex with NF-κB and AP-1 and is involved in DNA looping and LPS-induced OPN expression. Therefore, we have identified an essential mechanism to remodel the local chromatin structures and spatial conformations to regulate LPS-induced OPN expression. PMID:21257959

Rapid purification of circular DNA by triplex-mediated affinity capture

DOEpatents

Ji, H.; Smith, L.M.

1997-01-07

A single-step capture of a target supercoiled double-stranded DNA molecule is accomplished by forming a local triple-helix among two strands of the supercoiled circular DNA and an oligonucleotide probe. The oligonucleotide is bound to an immobilizing support which facilitates the immobilization and purification of target DNA molecules. Non-target DNA molecules and other contaminating cellular material are easily removed by washing. The triple-helical structure is destabilized by raising the pH, leaving purified target DNA in the supernatant and reusable affinity capture oligonucleotide secured to the immobilizing support. 3 figs.

Pulling out the 1%: Whole-Genome Capture for the Targeted Enrichment of Ancient DNA Sequencing Libraries

PubMed Central

Carpenter, Meredith L.; Buenrostro, Jason D.; Valdiosera, Cristina; Schroeder, Hannes; Allentoft, Morten E.; Sikora, Martin; Rasmussen, Morten; Gravel, Simon; Guillén, Sonia; Nekhrizov, Georgi; Leshtakov, Krasimir; Dimitrova, Diana; Theodossiev, Nikola; Pettener, Davide; Luiselli, Donata; Sandoval, Karla; Moreno-Estrada, Andrés; Li, Yingrui; Wang, Jun; Gilbert, M. Thomas P.; Willerslev, Eske; Greenleaf, William J.; Bustamante, Carlos D.

2013-01-01

Most ancient specimens contain very low levels of endogenous DNA, precluding the shotgun sequencing of many interesting samples because of cost. Ancient DNA (aDNA) libraries often contain <1% endogenous DNA, with the majority of sequencing capacity taken up by environmental DNA. Here we present a capture-based method for enriching the endogenous component of aDNA sequencing libraries. By using biotinylated RNA baits transcribed from genomic DNA libraries, we are able to capture DNA fragments from across the human genome. We demonstrate this method on libraries created from four Iron Age and Bronze Age human teeth from Bulgaria, as well as bone samples from seven Peruvian mummies and a Bronze Age hair sample from Denmark. Prior to capture, shotgun sequencing of these libraries yielded an average of 1.2% of reads mapping to the human genome (including duplicates). After capture, this fraction increased substantially, with up to 59% of reads mapped to human and enrichment ranging from 6- to 159-fold. Furthermore, we maintained coverage of the majority of regions sequenced in the precapture library. Intersection with the 1000 Genomes Project reference panel yielded an average of 50,723 SNPs (range 3,062–147,243) for the postcapture libraries sequenced with 1 million reads, compared with 13,280 SNPs (range 217–73,266) for the precapture libraries, increasing resolution in population genetic analyses. Our whole-genome capture approach makes it less costly to sequence aDNA from specimens containing very low levels of endogenous DNA, enabling the analysis of larger numbers of samples. PMID:24568772

Gold nanoparticle capture within protein crystal scaffolds.

PubMed

Kowalski, Ann E; Huber, Thaddaus R; Ni, Thomas W; Hartje, Luke F; Appel, Karina L; Yost, Jarad W; Ackerson, Christopher J; Snow, Christopher D

2016-07-07

DNA assemblies have been used to organize inorganic nanoparticles into 3D arrays, with emergent properties arising as a result of nanoparticle spacing and geometry. We report here the use of engineered protein crystals as an alternative approach to biologically mediated assembly of inorganic nanoparticles. The protein crystal's 13 nm diameter pores result in an 80% solvent content and display hexahistidine sequences on their interior. The hexahistidine sequence captures Au25(glutathione)∼17 (nitrilotriacetic acid)∼1 nanoclusters throughout a chemically crosslinked crystal via the coordination of Ni(ii) to both the cluster and the protein. Nanoparticle loading was validated by confocal microscopy and elemental analysis. The nanoparticles may be released from the crystal by exposure to EDTA, which chelates the Ni(ii) and breaks the specific protein/nanoparticle interaction. The integrity of the protein crystals after crosslinking and nanoparticle capture was confirmed by single crystal X-ray crystallography.

Target Capture during Mos1 Transposition*

PubMed Central

Pflieger, Aude; Jaillet, Jerôme; Petit, Agnès; Augé-Gouillou, Corinne; Renault, Sylvaine

2014-01-01

DNA transposition contributes to genomic plasticity. Target capture is a key step in the transposition process, because it contributes to the selection of new insertion sites. Nothing or little is known about how eukaryotic mariner DNA transposons trigger this step. In the case of Mos1, biochemistry and crystallography have deciphered several inverted terminal repeat-transposase complexes that are intermediates during transposition. However, the target capture complex is still unknown. Here, we show that the preintegration complex (i.e., the excised transposon) is the only complex able to capture a target DNA. Mos1 transposase does not support target commitment, which has been proposed to explain Mos1 random genomic integrations within host genomes. We demonstrate that the TA dinucleotide used as the target is crucial both to target recognition and in the chemistry of the strand transfer reaction. Bent DNA molecules are better targets for the capture when the target DNA is nicked two nucleotides apart from the TA. They improve strand transfer when the target DNA contains a mismatch near the TA dinucleotide. PMID:24269942

Target capture during Mos1 transposition.

PubMed

Pflieger, Aude; Jaillet, Jerôme; Petit, Agnès; Augé-Gouillou, Corinne; Renault, Sylvaine

2014-01-03

DNA transposition contributes to genomic plasticity. Target capture is a key step in the transposition process, because it contributes to the selection of new insertion sites. Nothing or little is known about how eukaryotic mariner DNA transposons trigger this step. In the case of Mos1, biochemistry and crystallography have deciphered several inverted terminal repeat-transposase complexes that are intermediates during transposition. However, the target capture complex is still unknown. Here, we show that the preintegration complex (i.e., the excised transposon) is the only complex able to capture a target DNA. Mos1 transposase does not support target commitment, which has been proposed to explain Mos1 random genomic integrations within host genomes. We demonstrate that the TA dinucleotide used as the target is crucial both to target recognition and in the chemistry of the strand transfer reaction. Bent DNA molecules are better targets for the capture when the target DNA is nicked two nucleotides apart from the TA. They improve strand transfer when the target DNA contains a mismatch near the TA dinucleotide.

Mechanisms for RNA capture by ssDNA viruses: grand theft RNA.

PubMed

Stedman, Kenneth

2013-06-01

Viruses contain three common types of packaged genomes; double-stranded DNA (dsDNA), RNA (mostly single and occasionally double stranded) and single-stranded DNA (ssDNA). There are relatively straightforward explanations for the prevalence of viruses with dsDNA and RNA genomes, but the evolutionary basis for the apparent success of ssDNA viruses is less clear. The recent discovery of four ssDNA virus genomes that appear to have been formed by recombination between co-infecting RNA and ssDNA viruses, together with the high mutation rate of ssDNA viruses provide possible explanations. RNA-DNA recombination allows ssDNA viruses to access much broader sequence space than through nucleotide substitution and DNA-DNA recombination alone. Multiple non-exclusive mechanisms, all due to the unique replication of ssDNA viruses, are proposed for this unusual RNA capture. RNA capture provides an explanation for the evolutionary success of the ssDNA viruses and may help elucidate the mystery of integrated RNA viruses in viral and cellular DNA genomes.

Clearing muddied waters: Capture of environmental DNA from turbid waters.

PubMed

Williams, Kelly E; Huyvaert, Kathryn P; Piaggio, Antoinette J

2017-01-01

Understanding the differences in efficiencies of various methods to concentrate, extract, and amplify environmental DNA (eDNA) is vital for best performance of eDNA detection. Aquatic systems vary in characteristics such as turbidity, eDNA concentration, and inhibitor load, thus affecting eDNA capture efficiency. Application of eDNA techniques to the detection of terrestrial invasive or endangered species may require sampling at intermittent water sources that are used for drinking and cooling; these water bodies may often be stagnant and turbid. We present our best practices technique for the detection of wild pig eDNA in water samples, a protocol that will have wide applicability to the detection of elusive vertebrate species. We determined the best practice for eDNA capture in a turbid water system was to concentrate DNA from a 15 mL water sample via centrifugation, purify DNA with the DNeasy mericon Food kit, and remove inhibitors with Zymo Inhibitor Removal Technology columns. Further, we compared the sensitivity of conventional PCR to quantitative PCR and found that quantitative PCR was more sensitive in detecting lower concentrations of eDNA. We show significant differences in efficiencies among methods in each step of eDNA capture, emphasizing the importance of optimizing best practices for the system of interest.

Clearing muddied waters: Capture of environmental DNA from turbid waters

PubMed Central

Huyvaert, Kathryn P.; Piaggio, Antoinette J.

2017-01-01

Understanding the differences in efficiencies of various methods to concentrate, extract, and amplify environmental DNA (eDNA) is vital for best performance of eDNA detection. Aquatic systems vary in characteristics such as turbidity, eDNA concentration, and inhibitor load, thus affecting eDNA capture efficiency. Application of eDNA techniques to the detection of terrestrial invasive or endangered species may require sampling at intermittent water sources that are used for drinking and cooling; these water bodies may often be stagnant and turbid. We present our best practices technique for the detection of wild pig eDNA in water samples, a protocol that will have wide applicability to the detection of elusive vertebrate species. We determined the best practice for eDNA capture in a turbid water system was to concentrate DNA from a 15 mL water sample via centrifugation, purify DNA with the DNeasy mericon Food kit, and remove inhibitors with Zymo Inhibitor Removal Technology columns. Further, we compared the sensitivity of conventional PCR to quantitative PCR and found that quantitative PCR was more sensitive in detecting lower concentrations of eDNA. We show significant differences in efficiencies among methods in each step of eDNA capture, emphasizing the importance of optimizing best practices for the system of interest. PMID:28686659

Comparison of Three Different Sensitive Assays for Hepatitis B Virus DNA in Monitoring of Responses to Antiviral Therapy

PubMed Central

Chan, Henry L. Y.; Leung, Nancy W. Y.; Lau, Tracy C. M.; Wong, May L.; Sung, Joseph J. Y.

2000-01-01

The aim of our study was to compare the performances of two new hepatitis B virus (HBV) DNA assays, a cross-linking assay (NAXCOR) and a hybrid-capture amplification assay (Digene), versus the widely used branched-DNA (bDNA) assay (Chiron) in the monitoring of HBV DNA levels during antiviral treatment. Serial serum samples from 12 chronically HBV infected patients undergoing a phase II trial of an antiviral drug, 2′,3′-dideoxy-5-fluoro-3′-thiacytidine (FTC), were studied. A total of 96 serum samples were tested for HBV DNA using the cross-linking, hybrid-capture amplification, and bDNA assays. In the comparison of the cross-linking and bDNA assays, concordant results were found in 77 (80.3%) samples, no significant difference was found between the median log10 HBV DNA levels (6.66 versus 7.17 meq/ml), and the results of the two assays were closely correlated (r = 0.95). In the comparison of the hybrid-capture amplification and bDNA assays, concordant results were found in 79 (82.3%) samples, no significant difference was found between the median log10 HBV DNA levels (6.98 versus 6.99 meq/ml), and the results of the two assays were closely correlated (r = 0.99). Six (6.3%) samples by the cross-linking assay and 10 (10.4%) samples by the bDNA assay required retesting because of unacceptably high within-run coefficients of variance. No sample required retesting in the hybrid-capture amplification assay according to the internal validation. In conclusion, the cross-linking and hybrid-capture amplification assays were as sensitive as the bDNA assay for HBV DNA detection and can be recommended for monitoring of HBV DNA levels during antiviral treatment. PMID:10970358

Detection of unamplified genomic DNA by a PNA-based microstructured optical fiber (MOF) Bragg-grating optofluidic system.

PubMed

Bertucci, Alessandro; Manicardi, Alex; Candiani, Alessandro; Giannetti, Sara; Cucinotta, Annamaria; Spoto, Giuseppe; Konstantaki, Maria; Pissadakis, Stavros; Selleri, Stefano; Corradini, Roberto

2015-01-15

Microstructured optical fibers containing microchannels and Bragg grating inscribed were internally functionalized with a peptide nucleic acid (PNA) probe specific for a gene tract of the genetically modified Roundup Ready soy. These fibers were used as an optofluidic device for the detection of DNA by measuring the shift in the wavelength of the reflected IR light. Enhancement of optical read-out was obtained using streptavidin coated gold-nanoparticles interacting with the genomic DNA captured in the fiber channels (0%, 0.1%, 1% and 10% RR-Soy), enabling to achieve statistically significant, label-free, and amplification-free detection of target DNA in low concentrations, low percentages, and very low sample volumes. Computer simulations of the fiber optics based on the finite element method (FEM) were consistent with the formation of a layer of organic material with an average thickness of 39 nm for the highest percentage (10% RR soy) analysed. Copyright © 2014 Elsevier B.V. All rights reserved.

Detection of an endangered aquatic heteropteran using environmental DNA in a wetland ecosystem

PubMed Central

Katano, Izumi; Sakata, Yusuke; Souma, Rio; Kosuge, Toshihiro; Nagano, Mariko; Ikeda, Kousuke; Yano, Koki; Tojo, Koji

2017-01-01

The use of environmental DNA (eDNA) has recently been employed to evaluate the distribution of various aquatic macroorganisms. Although this technique has been applied to a broad range of taxa, from vertebrates to invertebrates, its application is limited for aquatic insects such as aquatic heteropterans. Nepa hoffmanni (Heteroptera: Nepidae) is a small (approx. 23 mm) aquatic heteropteran that inhabits wetlands, can be difficult to capture and is endangered in Japan. The molecular tool eDNA was used to evaluate the species distribution of N. hoffmanni in comparison to that determined using hand-capturing methods in two regions of Japan. The eDNA of N. hoffmanni was detected at nearly all sites (10 eDNA-detected sites out of 14 sites), including sites where N. hoffmanni was not captured by hand (five eDNA-detected sites out of six captured sites). Thus, this species-specific eDNA technique can be applied to detect small, sparsely distributed heteropterans in wetland ecosystems. In conclusion, eDNA could be a valuable technique for the detection of aquatic insects inhabiting wetland habitats, and could make a significant contribution to providing distribution data necessary to species conservation. PMID:28791177

Mechanism of How Salt-Gradient-Induced Charges Affect the Translocation of DNA Molecules through a Nanopore

PubMed Central

He, Yuhui; Tsutsui, Makusu; Scheicher, Ralph H.; Fan, Chun; Taniguchi, Masateru; Kawai, Tomoji

2013-01-01

Experiments using nanopores demonstrated that a salt gradient enhances the capture rate of DNA and reduces its translocation speed. These two effects can help to enable electrical DNA sequencing with nanopores. Here, we provide a quantitative theoretical evaluation that shows the positive net charges, which accumulate around the pore entrance due to the salt gradient, are responsible for the two observed effects: they reinforce the electric capture field, resulting in promoted molecule capture rate; and they induce cationic electroosmotic flow through the nanopore, thus significantly retarding the motion of the anionic DNA through the nanopore. Our multiphysical simulation results show that, during the polymer trapping stage, the former effect plays the major role, thus resulting in promoted DNA capture rate, while during the nanopore-penetrating stage the latter effect dominates and consequently reduces the DNA translocation speed significantly. Quantitative agreement with experimental results has been reached by further taking nanopore wall surface charges into account. PMID:23931325

Sequences spanning the leader-repeat junction mediate CRISPR adaptation to phage in Streptococcus thermophilus

PubMed Central

Wei, Yunzhou; Chesne, Megan T.; Terns, Rebecca M.; Terns, Michael P.

2015-01-01

CRISPR-Cas systems are RNA-based immune systems that protect prokaryotes from invaders such as phages and plasmids. In adaptation, the initial phase of the immune response, short foreign DNA fragments are captured and integrated into host CRISPR loci to provide heritable defense against encountered foreign nucleic acids. Each CRISPR contains a ∼100–500 bp leader element that typically includes a transcription promoter, followed by an array of captured ∼35 bp sequences (spacers) sandwiched between copies of an identical ∼35 bp direct repeat sequence. New spacers are added immediately downstream of the leader. Here, we have analyzed adaptation to phage infection in Streptococcus thermophilus at the CRISPR1 locus to identify cis-acting elements essential for the process. We show that the leader and a single repeat of the CRISPR locus are sufficient for adaptation in this system. Moreover, we identified a leader sequence element capable of stimulating adaptation at a dormant repeat. We found that sequences within 10 bp of the site of integration, in both the leader and repeat of the CRISPR, are required for the process. Our results indicate that information at the CRISPR leader-repeat junction is critical for adaptation in this Type II-A system and likely other CRISPR-Cas systems. PMID:25589547

Identification of multi-loci hubs from 4C-seq demonstrates the functional importance of simultaneous interactions.

PubMed

Jiang, Tingting; Raviram, Ramya; Snetkova, Valentina; Rocha, Pedro P; Proudhon, Charlotte; Badri, Sana; Bonneau, Richard; Skok, Jane A; Kluger, Yuval

2016-10-14

Use of low resolution single cell DNA FISH and population based high resolution chromosome conformation capture techniques have highlighted the importance of pairwise chromatin interactions in gene regulation. However, it is unlikely that associations involving regulatory elements act in isolation of other interacting partners that also influence their impact. Indeed, the influence of multi-loci interactions remains something of an enigma as beyond low-resolution DNA FISH we do not have the appropriate tools to analyze these. Here we present a method that uses standard 4C-seq data to identify multi-loci interactions from the same cell. We demonstrate the feasibility of our method using 4C-seq data sets that identify known pairwise and novel tri-loci interactions involving the Tcrb and Igk antigen receptor enhancers. We further show that the three Igk enhancers, MiEκ, 3'Eκ and Edκ, interact simultaneously in this super-enhancer cluster, which add to our previous findings showing that loss of one element decreases interactions between all three elements as well as reducing their transcriptional output. These findings underscore the functional importance of simultaneous interactions and provide new insight into the relationship between enhancer elements. Our method opens the door for studying multi-loci interactions and their impact on gene regulation in other biological settings. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Identification of multi-loci hubs from 4C-seq demonstrates the functional importance of simultaneous interactions

PubMed Central

Jiang, Tingting; Raviram, Ramya; Snetkova, Valentina; Rocha, Pedro P.; Proudhon, Charlotte; Badri, Sana; Bonneau, Richard; Skok, Jane A.; Kluger, Yuval

2016-01-01

Use of low resolution single cell DNA FISH and population based high resolution chromosome conformation capture techniques have highlighted the importance of pairwise chromatin interactions in gene regulation. However, it is unlikely that associations involving regulatory elements act in isolation of other interacting partners that also influence their impact. Indeed, the influence of multi-loci interactions remains something of an enigma as beyond low-resolution DNA FISH we do not have the appropriate tools to analyze these. Here we present a method that uses standard 4C-seq data to identify multi-loci interactions from the same cell. We demonstrate the feasibility of our method using 4C-seq data sets that identify known pairwise and novel tri-loci interactions involving the Tcrb and Igk antigen receptor enhancers. We further show that the three Igk enhancers, MiEκ, 3′Eκ and Edκ, interact simultaneously in this super-enhancer cluster, which add to our previous findings showing that loss of one element decreases interactions between all three elements as well as reducing their transcriptional output. These findings underscore the functional importance of simultaneous interactions and provide new insight into the relationship between enhancer elements. Our method opens the door for studying multi-loci interactions and their impact on gene regulation in other biological settings. PMID:27439714

The identification of cis-regulatory elements: A review from a machine learning perspective.

PubMed

Li, Yifeng; Chen, Chih-Yu; Kaye, Alice M; Wasserman, Wyeth W

2015-12-01

The majority of the human genome consists of non-coding regions that have been called junk DNA. However, recent studies have unveiled that these regions contain cis-regulatory elements, such as promoters, enhancers, silencers, insulators, etc. These regulatory elements can play crucial roles in controlling gene expressions in specific cell types, conditions, and developmental stages. Disruption to these regions could contribute to phenotype changes. Precisely identifying regulatory elements is key to deciphering the mechanisms underlying transcriptional regulation. Cis-regulatory events are complex processes that involve chromatin accessibility, transcription factor binding, DNA methylation, histone modifications, and the interactions between them. The development of next-generation sequencing techniques has allowed us to capture these genomic features in depth. Applied analysis of genome sequences for clinical genetics has increased the urgency for detecting these regions. However, the complexity of cis-regulatory events and the deluge of sequencing data require accurate and efficient computational approaches, in particular, machine learning techniques. In this review, we describe machine learning approaches for predicting transcription factor binding sites, enhancers, and promoters, primarily driven by next-generation sequencing data. Data sources are provided in order to facilitate testing of novel methods. The purpose of this review is to attract computational experts and data scientists to advance this field. Crown Copyright © 2015. Published by Elsevier Ireland Ltd. All rights reserved.

Quantification of Toxoplasma gondii in tissue samples of experimentally infected goats by magnetic capture and real-time PCR.

PubMed

Juránková, Jana; Opsteegh, Marieke; Neumayerová, Helena; Kovařčík, Kamil; Frencová, Anita; Baláž, Vojtěch; Volf, Jiří; Koudela, Břetislav

2013-03-31

Undercooked meat containing tissue cysts is one of the most common sources of Toxoplasma gondii infection in humans. Goats are very susceptible to clinical toxoplasmosis, and especially kids are common food animals, thereby representing a risk for human infection. A sequence-specific magnetic capture method was used for isolation of T. gondii DNA from tissue samples from experimentally infected goat-kids and real-time PCR for the 529 bp repeat element allowed quantification of T. gondii DNA. The contamination level in different types of tissue and in two groups of goats euthanized 30 and 90 dpi was compared. The highest concentration of T. gondii DNA in both groups of goats was found in lung tissue, but only the higher parasite count in lung tissue compared to other organs in group A (euthanized 30 dpi) was statistically significant. T. gondii concentrations were higher in liver and dorsal muscle samples from goats euthanized 90 dpi than in goats euthanized at 30 dpi, while the T. gondii concentration in hearts decreased. This study describes for the first time distribution of T. gondii parasites in post-weaned goat kids. New information about T. gondii predilection sites in goats and about the progression of infection between 30 and 90 dpi was achieved. Copyright © 2012 Elsevier B.V. All rights reserved.

Evaluation by latent class analysis of a magnetic capture based DNA extraction followed by real-time qPCR as a new diagnostic method for detection of Echinococcus multilocularis in definitive hosts.

PubMed

Maas, Miriam; van Roon, Annika; Dam-Deisz, Cecile; Opsteegh, Marieke; Massolo, Alessandro; Deksne, Gunita; Teunis, Peter; van der Giessen, Joke

2016-10-30

A new method, based on a magnetic capture based DNA extraction followed by qPCR, was developed for the detection of the zoonotic parasite Echinococcus multilocularis in definitive hosts. Latent class analysis was used to compare this new method with the currently used phenol-chloroform DNA extraction followed by single tube nested PCR. In total, 60 red foxes and coyotes from three different locations were tested with both molecular methods and the sedimentation and counting technique (SCT) or intestinal scraping technique (IST). Though based on a limited number of samples, it could be established that the magnetic capture based DNA extraction followed by qPCR showed similar sensitivity and specificity as the currently used phenol-chloroform DNA extraction followed by single tube nested PCR. All methods have a high specificity as shown by Bayesian latent class analysis. Both molecular assays have higher sensitivities than the combined SCT and IST, though the uncertainties in sensitivity estimates were wide for all assays tested. The magnetic capture based DNA extraction followed by qPCR has the advantage of not requiring hazardous chemicals like the phenol-chloroform DNA extraction followed by single tube nested PCR. This supports the replacement of the phenol-chloroform DNA extraction followed by single tube nested PCR by the magnetic capture based DNA extraction followed by qPCR for molecular detection of E. multilocularis in definitive hosts. Copyright © 2016 Elsevier B.V. All rights reserved.

SERS detection of indirect viral DNA capture using colloidal gold and methylene blue as a Raman label

USDA-ARS?s Scientific Manuscript database

An indirect capture model assay using colloidal Au nanoparticles is demonstrated for surface enhanced Raman scattering (SERS) spectroscopy detection of DNA. The sequence targeted for capture is derived from the West Nile Virus (WNV) RNA genome and was selected on the basis of exhibiting minimal seco...

Sequence selective capture, release and analysis of DNA using a magnetic microbead-assisted toehold-mediated DNA strand displacement reaction.

PubMed

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

2014-07-21

This paper reports on the modification of magnetic beads with oligonucleotide capture probes with a specially designed pendant toehold (overhang) aimed specifically to capture double-stranded PCR products. After capture, the PCR products were selectively released from the magnetic beads by means of a toehold-mediated strand displacement reaction using short artificial oligonucleotide triggers and analysed using capillary electrophoresis. The approach was successfully shown on two genes widely used in human DNA genotyping, namely human c-fms (macrophage colony-stimulating factor) proto-oncogene for the CSF-1 receptor (CSF1PO) and amelogenin.

Global reorganisation of cis-regulatory units upon lineage commitment of human embryonic stem cells

PubMed Central

Freire-Pritchett, Paula; Schoenfelder, Stefan; Várnai, Csilla; Wingett, Steven W; Cairns, Jonathan; Collier, Amanda J; García-Vílchez, Raquel; Furlan-Magaril, Mayra; Osborne, Cameron S; Fraser, Peter; Rugg-Gunn, Peter J; Spivakov, Mikhail

2017-01-01

Long-range cis-regulatory elements such as enhancers coordinate cell-specific transcriptional programmes by engaging in DNA looping interactions with target promoters. Deciphering the interplay between the promoter connectivity and activity of cis-regulatory elements during lineage commitment is crucial for understanding developmental transcriptional control. Here, we use Promoter Capture Hi-C to generate a high-resolution atlas of chromosomal interactions involving ~22,000 gene promoters in human pluripotent and lineage-committed cells, identifying putative target genes for known and predicted enhancer elements. We reveal extensive dynamics of cis-regulatory contacts upon lineage commitment, including the acquisition and loss of promoter interactions. This spatial rewiring occurs preferentially with predicted changes in the activity of cis-regulatory elements and is associated with changes in target gene expression. Our results provide a global and integrated view of promoter interactome dynamics during lineage commitment of human pluripotent cells. DOI: http://dx.doi.org/10.7554/eLife.21926.001 PMID:28332981

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

PubMed Central

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

2014-01-01

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

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

PubMed

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

2014-01-01

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

Capturing a DNA duplex under near-physiological conditions

NASA Astrophysics Data System (ADS)

Zhang, Huijuan; Xu, Wei; Liu, Xiaogang; Stellacci, Francesco; Thong, John T. L.

2010-10-01

We report in situ trapping of a thiolated DNA duplex with eight base pairs into a polymer-protected gold nanogap device under near-physiological conditions. The double-stranded DNA was captured by electrophoresis and covalently attached to the nanogap electrodes through sulfur-gold bonding interaction. The immobilization of the DNA duplex was confirmed by direct electrical measurements under near-physiological conditions. The conductance of the DNA duplex was estimated to be 0.09 μS. We also demonstrate the control of DNA dehybridization by heating the device to temperatures above the melting point of the DNA.

Recruitment of CRISPR-Cas systems by Tn7-like transposons.

PubMed

Peters, Joseph E; Makarova, Kira S; Shmakov, Sergey; Koonin, Eugene V

2017-08-29

A survey of bacterial and archaeal genomes shows that many Tn7-like transposons contain minimal type I-F CRISPR-Cas systems that consist of fused cas8f and cas5f , cas7f , and cas6f genes and a short CRISPR array. Several small groups of Tn7-like transposons encompass similarly truncated type I-B CRISPR-Cas. This minimal gene complement of the transposon-associated CRISPR-Cas systems implies that they are competent for pre-CRISPR RNA (precrRNA) processing yielding mature crRNAs and target binding but not target cleavage that is required for interference. Phylogenetic analysis demonstrates that evolution of the CRISPR-Cas-containing transposons included a single, ancestral capture of a type I-F locus and two independent instances of type I-B loci capture. We show that the transposon-associated CRISPR arrays contain spacers homologous to plasmid and temperate phage sequences and, in some cases, chromosomal sequences adjacent to the transposon. We hypothesize that the transposon-encoded CRISPR-Cas systems generate displacement (R-loops) in the cognate DNA sites, targeting the transposon to these sites and thus facilitating their spread via plasmids and phages. These findings suggest the existence of RNA-guided transposition and fit the guns-for-hire concept whereby mobile genetic elements capture host defense systems and repurpose them for different stages in the life cycle of the element.

Use of capture-based next-generation sequencing to detect ALK fusion in plasma cell-free DNA of patients with non-small-cell lung cancer.

PubMed

Cui, Shaohua; Zhang, Wei; Xiong, Liwen; Pan, Feng; Niu, Yanjie; Chu, Tianqing; Wang, Huimin; Zhao, Yizhuo; Jiang, Liyan

2017-01-10

Capture-based next-generation sequencing (NGS) is a potentially useful diagnostic method to measure tumor tissue DNA in blood as it can identify concordant mutations between cell-free DNA (cfDNA) and primary tumor DNA in lung cancer patients. In this study, the sensitivity, specificity and accuracy of capture-based NGS for detecting ALK fusion in plasma cfDNA was assessed. 24 patients with tissue ALK-positivity and 15 who did not harbor ALK fusion were enrolled. 13 ALK-positive samples were identified by capture-based NGS among the 24 samples with tissue ALK-positivity. In addition to EML4-ALK, 2 rare fusion types (FAM179A-ALK and COL25A1-ALK) were also identified. The overall sensitivity, specificity and accuracy for all cases were 54.2%, 100% and 71.8%, respectively. For patients without distant metastasis (M0-M1a) and patients with distant metastasis (M1b), the sensitivities were 28.6% and 64.7%, respectively. In the 15 patients who received crizotinib, the estimated median PFS was 9.93 months. Thus, captured-based NGS has acceptable sensitivity and excellent specificity for the detection of ALK fusion in plasma cfDNA, especially for patients with distant metastasis. This non-invasive method is clinically feasible for detecting ALK fusion in patients with advanced-stage NSCLC who cannot undergo traumatic examinations or have insufficient tissue samples for molecular tests.

A High-Throughput Process for the Solid-Phase Purification of Synthetic DNA Sequences

PubMed Central

Grajkowski, Andrzej; Cieślak, Jacek; Beaucage, Serge L.

2017-01-01

An efficient process for the purification of synthetic phosphorothioate and native DNA sequences is presented. The process is based on the use of an aminopropylated silica gel support functionalized with aminooxyalkyl functions to enable capture of DNA sequences through an oximation reaction with the keto function of a linker conjugated to the 5′-terminus of DNA sequences. Deoxyribonucleoside phosphoramidites carrying this linker, as a 5′-hydroxyl protecting group, have been synthesized for incorporation into DNA sequences during the last coupling step of a standard solid-phase synthesis protocol executed on a controlled pore glass (CPG) support. Solid-phase capture of the nucleobase- and phosphate-deprotected DNA sequences released from the CPG support is demonstrated to proceed near quantitatively. Shorter than full-length DNA sequences are first washed away from the capture support; the solid-phase purified DNA sequences are then released from this support upon reaction with tetra-n-butylammonium fluoride in dry dimethylsulfoxide (DMSO) and precipitated in tetrahydrofuran (THF). The purity of solid-phase-purified DNA sequences exceeds 98%. The simulated high-throughput and scalability features of the solid-phase purification process are demonstrated without sacrificing purity of the DNA sequences. PMID:28628204

Multi-scale Modeling of Chromosomal DNA in Living Cells

NASA Astrophysics Data System (ADS)

Spakowitz, Andrew

The organization and dynamics of chromosomal DNA play a pivotal role in a range of biological processes, including gene regulation, homologous recombination, replication, and segregation. Establishing a quantitative theoretical model of DNA organization and dynamics would be valuable in bridging the gap between the molecular-level packaging of DNA and genome-scale chromosomal processes. Our research group utilizes analytical theory and computational modeling to establish a predictive theoretical model of chromosomal organization and dynamics. In this talk, I will discuss our efforts to develop multi-scale polymer models of chromosomal DNA that are both sufficiently detailed to address specific protein-DNA interactions while capturing experimentally relevant time and length scales. I will demonstrate how these modeling efforts are capable of quantitatively capturing aspects of behavior of chromosomal DNA in both prokaryotic and eukaryotic cells. This talk will illustrate that capturing dynamical behavior of chromosomal DNA at various length scales necessitates a range of theoretical treatments that accommodate the critical physical contributions that are relevant to in vivo behavior at these disparate length and time scales. National Science Foundation, Physics of Living Systems Program (PHY-1305516).

Characterization of the repetitive DNA elements in the genome of fish lymphocystis disease viruses.

PubMed

Schnitzler, P; Darai, G

1989-09-01

The complete DNA nucleotide sequence of the repetitive DNA elements in the genome of fish lymphocystis disease virus (FLDV) isolated from two different species (flounder and dab) was determined. The size of these repetitive DNA elements was found to be 1413 bp which corresponds to the DNA sequences of the 5' terminus of the EcoRI DNA fragment B (0.034 to 0.052 m.u.) and to the EcoRI DNA fragment M (0.718 to 0.736 m.u.) of the FLDV genome causing lymphocystis disease in flounder and plaice. The degree of DNA nucleotide homology between both regions was found to be 99%. The repetitive DNA element in the genome of FLDV isolated from other fish species (dab) was identified and is located within the EcoRI DNA fragment B and J of the viral genome. The DNA nucleotide sequence of one duplicate of this repetition (EcoRI DNA fragment J) was determined (1410 bp) and compared to the DNA nucleotide sequences of the repetitive DNA elements of the genome of FLDV isolated from flounder. It was found that the repetitive DNA elements of the genome of FLDV derived from two different fish species are highly conserved and possess a degree of DNA sequence homology of 94%. The DNA sequences of each strand of the individual repetitive element possess one open reading frame.

A novel magneto-DNA duplex probe for bacterial DNA detection based on exonuclease III-aided cycling amplification.

PubMed

Zeng, Yan; Wan, Yi; Zhang, Dun; Qi, Peng

2015-01-01

A novel magneto-DNA duplex probe for bacterial DNA detection based on exonuclease III (Exo-III) aided cycling amplification has been developed. This magneto-DNA duplex probe contains a partly hybrid fluorophore-modified capture probe and a fluorophore-modified signal probe with magnetic microparticle as carrier. In the presence of a perfectly matched target bacterial DNA, blunt 3'-terminus of the capture probe is formed, activating the Exo-III aided cycling amplification. Thus, Exo-III catalyzes the stepwise removal of mononucleotides from this terminus, releasing both fluorophore-modified signal probe, fluorescent dyes of the capture probe and target DNA. The released target DNA then starts a new cycle, while released fluorescent fragments are recovered with magnetic separation for fluorescence signal collection. This system exhibited sensitive detection of bacterial DNA, with a detection limit of 14 pM because of the unique cleavage function of Exo-III, high fluorescence intensity, and separating function of magneto-DNA duplex probes. Besides this sensitivity, this strategy exhibited excellent selectivity with mismatched bacterial DNA targets and other bacterial species targets and good applicability in real seawater samples, hence, this strategy could be potentially used for qualitative and quantitative analysis of bacteria. Copyright © 2014 Elsevier B.V. All rights reserved.

Environmental DNA method for estimating salamander distribution in headwater streams, and a comparison of water sampling methods.

PubMed

Katano, Izumi; Harada, Ken; Doi, Hideyuki; Souma, Rio; Minamoto, Toshifumi

2017-01-01

Environmental DNA (eDNA) has recently been used for detecting the distribution of macroorganisms in various aquatic habitats. In this study, we applied an eDNA method to estimate the distribution of the Japanese clawed salamander, Onychodactylus japonicus, in headwater streams. Additionally, we compared the detection of eDNA and hand-capturing methods used for determining the distribution of O. japonicus. For eDNA detection, we designed a qPCR primer/probe set for O. japonicus using the 12S rRNA region. We detected the eDNA of O. japonicus at all sites (with the exception of one), where we also observed them by hand-capturing. Additionally, we detected eDNA at two sites where we were unable to observe individuals using the hand-capturing method. Moreover, we found that eDNA concentrations and detection rates of the two water sampling areas (stream surface and under stones) were not significantly different, although the eDNA concentration in the water under stones was more varied than that on the surface. We, therefore, conclude that eDNA methods could be used to determine the distribution of macroorganisms inhabiting headwater systems by using samples collected from the surface of the water.

Detection of Helicobacter Pylori Genome with an Optical Biosensor Based on Hybridization of Urease Gene with a Gold Nanoparticles-Labeled Probe

NASA Astrophysics Data System (ADS)

Shahrashoob, M.; Mohsenifar, A.; Tabatabaei, M.; Rahmani-Cherati, T.; Mobaraki, M.; Mota, A.; Shojaei, T. R.

2016-05-01

A novel optics-based nanobiosensor for sensitive determination of the Helicobacter pylori genome using a gold nanoparticles (AuNPs)-labeled probe is reported. Two specific thiol-modified capture and signal probes were designed based on a single-stranded complementary DNA (cDNA) region of the urease gene. The capture probe was immobilized on AuNPs, which were previously immobilized on an APTES-activated glass, and the signal probe was conjugated to different AuNPs as well. The presence of the cDNA in the reaction mixture led to the hybridization of the AuNPs-labeled capture probe and the signal probe with the cDNA, and consequently the optical density of the reaction mixture (AuNPs) was reduced proportionally to the cDNA concentration. The limit of detection was measured at 0.5 nM.

Surface modification of poly(dimethylsiloxane) (PDMS) microchannels with DNA capture-probes for potential use in microfluidic DNA analysis systems

NASA Astrophysics Data System (ADS)

Khodakov, Dmitriy A.; Thredgold, Leigh D.; Lenehan, Claire E.; Andersson, Gunther A.; Kobus, Hilton; Ellis, Amanda V.

2011-12-01

Poly(dimethylsiloxane) (PDMS) is an elastomeric material used for microfluidic devices and is especially suited to medical and forensic applications. This is due to its relatively low cost, ease of fabrication, excellent optical transmission characteristics and its ability to support electroosmotic flow, required during electrophoretic separations. These aspects combined with its large range of surface modification chemistries, make PDMS an attractive substrate in microfluidic devices for, in particular, DNA separation. Here, we report the successful wet chemical surface modification of PDMS microchannels using a simple three step method to produce an isothiocyanate-terminated surface. Initially, PDMS was oxygen plasma treated to produce a silanol-terminated surface, this was then reacted with 3-aminopropyltriethoxysilane with subsequent reaction of the now amine-terminated surface with p-phenylenediisothiocyanate. Water contact angle measurements both before and after modification showed a reduction in hydrophobicity from 101o for native PDMS to 94o for the isothiocyante-terminated PDMS. The isothiocyanate-terminated surface was then coupled with an amineterminated single-stranded DNA (ssDNA) oligonucleotide capture probe via a thiourea linkage. Confirmation of capture probe attachment was observed using fluorescent microscopy after hybridization of the capture probes with fluorescently labeled complimentary ssDNA oligonucleotides.

Genetic Perturbation of the Maize Methylome[W

PubMed Central

Li, Qing; Hermanson, Peter J.; Zaunbrecher, Virginia M.; Song, Jawon; Wendt, Jennifer; Rosenbaum, Heidi; Madzima, Thelma F.; Sloan, Amy E.; Huang, Ji; Burgess, Daniel L.; Richmond, Todd A.; McGinnis, Karen M.; Meeley, Robert B.; Danilevskaya, Olga N.; Vaughn, Matthew W.; Kaeppler, Shawn M.; Jeddeloh, Jeffrey A.

2014-01-01

DNA methylation can play important roles in the regulation of transposable elements and genes. A collection of mutant alleles for 11 maize (Zea mays) genes predicted to play roles in controlling DNA methylation were isolated through forward- or reverse-genetic approaches. Low-coverage whole-genome bisulfite sequencing and high-coverage sequence-capture bisulfite sequencing were applied to mutant lines to determine context- and locus-specific effects of these mutations on DNA methylation profiles. Plants containing mutant alleles for components of the RNA-directed DNA methylation pathway exhibit loss of CHH methylation at many loci as well as CG and CHG methylation at a small number of loci. Plants containing loss-of-function alleles for chromomethylase (CMT) genes exhibit strong genome-wide reductions in CHG methylation and some locus-specific loss of CHH methylation. In an attempt to identify stocks with stronger reductions in DNA methylation levels than provided by single gene mutations, we performed crosses to create double mutants for the maize CMT3 orthologs, Zmet2 and Zmet5, and for the maize DDM1 orthologs, Chr101 and Chr106. While loss-of-function alleles are viable as single gene mutants, the double mutants were not recovered, suggesting that severe perturbations of the maize methylome may have stronger deleterious phenotypic effects than in Arabidopsis thaliana. PMID:25527708

Predictive polymer modeling reveals coupled fluctuations in chromosome conformation and transcription

PubMed Central

Giorgetti, Luca; Galupa, Rafael; Nora, Elphège P.; Piolot, Tristan; Lam, France; Dekker, Job; Tiana, Guido; Heard, Edith

2015-01-01

Summary A new level of chromosome organization, Topologically Associating Domains (TADs), was recently uncovered by chromosome-confirmation-capture (3C) techniques. To explore TAD structure and function, we developed a polymer model that can extract the full repertoire of chromatin conformations within TADs from population-based 3C data. This model predicts actual physical distances and to what extent chromosomal contacts vary between cells. It also identifies interactions within single TADs that stabilize boundaries between TADs and allows us to identify and genetically validate key structural elements within TADs. Combining the model’s predictions with high-resolution DNA FISH and quantitative RNA FISH for TADs within the X-inactivation center (Xic), we dissect the relationship between transcription and spatial proximity to cis-regulatory elements. We demonstrate that contacts between potential regulatory elements occur in the context of fluctuating structures rather than stable loops and propose that such fluctuations may contribute to asymmetric expression in the Xic during X inactivation. PMID:24813616

Sequence capture of ultraconserved elements from bird museum specimens.

PubMed

McCormack, John E; Tsai, Whitney L E; Faircloth, Brant C

2016-09-01

New DNA sequencing technologies are allowing researchers to explore the genomes of the millions of natural history specimens collected prior to the molecular era. Yet, we know little about how well specific next-generation sequencing (NGS) techniques work with the degraded DNA typically extracted from museum specimens. Here, we use one type of NGS approach, sequence capture of ultraconserved elements (UCEs), to collect data from bird museum specimens as old as 120 years. We targeted 5060 UCE loci in 27 western scrub-jays (Aphelocoma californica) representing three evolutionary lineages that could be species, and we collected an average of 3749 UCE loci containing 4460 single nucleotide polymorphisms (SNPs). Despite older specimens producing fewer and shorter loci in general, we collected thousands of markers from even the oldest specimens. More sequencing reads per individual helped to boost the number of UCE loci we recovered from older specimens, but more sequencing was not as successful at increasing the length of loci. We detected contamination in some samples and determined that contamination was more prevalent in older samples that were subject to less sequencing. For the phylogeny generated from concatenated UCE loci, contamination led to incorrect placement of some individuals. In contrast, a species tree constructed from SNPs called within UCE loci correctly placed individuals into three monophyletic groups, perhaps because of the stricter analytical procedures used for SNP calling. This study and other recent studies on the genomics of museum specimens have profound implications for natural history collections, where millions of older specimens should now be considered genomic resources. © 2015 The Authors. Molecular Ecology Resources Published by John Wiley & Sons Ltd.

Methods of DNA methylation detection

NASA Technical Reports Server (NTRS)

Maki, Wusi Chen (Inventor); Filanoski, Brian John (Inventor); Mishra, Nirankar (Inventor); Rastogi, Shiva (Inventor)

2010-01-01

The present invention provides for methods of DNA methylation detection. The present invention provides for methods of generating and detecting specific electronic signals that report the methylation status of targeted DNA molecules in biological samples.Two methods are described, direct and indirect detection of methylated DNA molecules in a nano transistor based device. In the direct detection, methylated target DNA molecules are captured on the sensing surface resulting in changes in the electrical properties of a nano transistor. These changes generate detectable electronic signals. In the indirect detection, antibody-DNA conjugates are used to identify methylated DNA molecules. RNA signal molecules are generated through an in vitro transcription process. These RNA molecules are captured on the sensing surface change the electrical properties of nano transistor thereby generating detectable electronic signals.

Slowing down DNA translocation by a nanofiber meshed layer

NASA Astrophysics Data System (ADS)

Zhao, Yue; Xie, Wanyi; Tian, Enling; Ren, Yiwei; Zhu, Jifeng; Deng, Yunsheng; He, Shixuan; Liang, Liyuan; Wang, Yunjiao; Zhou, Daming; Wang, Deqiang

2018-01-01

Due to the weak interaction between DNA molecules and the inner surface of nanopores, DNA translocation is very fast, just leaving a short current drop without sufficient information to recognise the nucleotide sequence in the strand. In this paper, we propose a nanopore-nanofiber mesh hybridized structure to decelerate DNA translocation speed. Experimental results reveal that due to hydrophobic interaction between the DNA fragments and the nanofibers, the DNA moving speed can be retarded to two orders of magnitude slower. Furthermore, according to theory simulations, the additional fiber layer will reduce the electric field in the channel but elongate the capture region at the pore orifice, which will be helpful for increasing the capture rate and extending the DNA dwelling time in the meanwhile.

Ultrasensitive electrochemical DNA detection based on dual amplification of circular strand-displacement polymerase reaction and hybridization chain reaction.

PubMed

Wang, Cui; Zhou, Hui; Zhu, Wenping; Li, Hongbo; Jiang, Jianhui; Shen, Guoli; Yu, Ruqin

2013-09-15

We developed a novel electrochemical strategy for ultrasensitive DNA detection using a dual amplification strategy based on the circular strand-displacement polymerase reaction (CSDPR) and the hybridization chain reaction (HCR). In this assay, hybridization of hairpin-shaped capture DNA to target DNA resulted in a conformational change of the capture DNA with a concomitant exposure of its stem. The primer was then hybridized with the exposed stem and triggered a polymerization reaction, allowing a cyclic reaction comprising release of target DNA, hybridization of target with remaining capture DNA, polymerization initiated by the primer. Furthermore, the free part of the primer propagated a chain reaction of hybridization events between two DNA hairpin probes with biotin labels, enabling an electrochemical reading using the streptavidin-alkaline phosphatase. The proposed biosensor showed to have very high sensitivity and selectivity with a dynamic response range through 10fM to 1nM, and the detect limit was as low as 8fM. The proposed strategy could have the potential for molecular diagnostics in complex biological systems. Copyright © 2013 Elsevier B.V. All rights reserved.

DNA capture and next-generation sequencing can recover whole mitochondrial genomes from highly degraded samples for human identification

PubMed Central

2013-01-01

Background Mitochondrial DNA (mtDNA) typing can be a useful aid for identifying people from compromised samples when nuclear DNA is too damaged, degraded or below detection thresholds for routine short tandem repeat (STR)-based analysis. Standard mtDNA typing, focused on PCR amplicon sequencing of the control region (HVS I and HVS II), is limited by the resolving power of this short sequence, which misses up to 70% of the variation present in the mtDNA genome. Methods We used in-solution hybridisation-based DNA capture (using DNA capture probes prepared from modern human mtDNA) to recover mtDNA from post-mortem human remains in which the majority of DNA is both highly fragmented (<100 base pairs in length) and chemically damaged. The method ‘immortalises’ the finite quantities of DNA in valuable extracts as DNA libraries, which is followed by the targeted enrichment of endogenous mtDNA sequences and characterisation by next-generation sequencing (NGS). Results We sequenced whole mitochondrial genomes for human identification from samples where standard nuclear STR typing produced only partial profiles or demonstrably failed and/or where standard mtDNA hypervariable region sequences lacked resolving power. Multiple rounds of enrichment can substantially improve coverage and sequencing depth of mtDNA genomes from highly degraded samples. The application of this method has led to the reliable mitochondrial sequencing of human skeletal remains from unidentified World War Two (WWII) casualties approximately 70 years old and from archaeological remains (up to 2,500 years old). Conclusions This approach has potential applications in forensic science, historical human identification cases, archived medical samples, kinship analysis and population studies. In particular the methodology can be applied to any case, involving human or non-human species, where whole mitochondrial genome sequences are required to provide the highest level of maternal lineage discrimination. Multiple rounds of in-solution hybridisation-based DNA capture can retrieve whole mitochondrial genome sequences from even the most challenging samples. PMID:24289217

Densely ionizing radiation affects DNA methylation of selective LINE-1 elements

DOE Office of Scientific and Technical Information (OSTI.GOV)

Prior, Sara; Miousse, Isabelle R.

Long Interspersed Nucleotide Element 1 (LINE-1) retrotransposons are heavily methylated and are the most abundant transposable elements in mammalian genomes. Here, we investigated the differential DNA methylation within the LINE-1 under normal conditions and in response to environmentally relevant doses of sparsely and densely ionizing radiation. We demonstrate that DNA methylation of LINE-1 elements in the lungs of C57BL6 mice is dependent on their evolutionary age, where the elder age of the element is associated with the lower extent of DNA methylation. Exposure to 5-aza-2′-deoxycytidine and methionine-deficient diet affected DNA methylation of selective LINE-1 elements in an age- and promotermore » type-dependent manner. Exposure to densely IR, but not sparsely IR, resulted in DNA hypermethylation of older LINE-1 elements, while the DNA methylation of evolutionary younger elements remained mostly unchanged. We also demonstrate that exposure to densely IR increased mRNA and protein levels of LINE-1 via the loss of the histone H3K9 dimethylation and an increase in the H3K4 trimethylation at the LINE-1 5′-untranslated region, independently of DNA methylation. Our findings suggest that DNA methylation is important for regulation of LINE-1 expression under normal conditions, but histone modifications may dictate the transcriptional activity of LINE-1 in response to exposure to densely IR. - Highlights: • DNA methylation of LINE-1 elements is dependent on their evolutionary age. • Densely ionizing radiation affects DNA methylation of selective LINE-1 elements. • Radiation-induced reactivation of LINE-1 is DNA methylation-independent. • Histone modifications dictate the transcriptional activity of LINE-1.« less

Diversity of abundance patterns of neutron-capture elements in very metal-poor stars

NASA Astrophysics Data System (ADS)

Aoki, Misa; Aoki, Wako; Ishimaru, Yuhri; Wanajo, Shinya

2014-05-01

Observations of Very Metal-Poor stars indicate that there are at least two sites to r-process; "weak r-process" and "main r-process". A question is whether these two are well separated or there exists a variation in the r-process. We present the results of abundance analysis of neutron-capture elements in the two Very Metal-Poor stars HD107752 and HD110184 in the Milky Way halo observed with the Subaru Telescope HDS. The abundance patterns show overabundace at light n-capture elements (e.g. Sr, Y), inferring the element yielding of weak r-process, while heavy neutron-capture elements (e.g. Ba, Eu) are deficient; however, the overabundance of light ones is not as significant as that previously found in stars representing the weak r-process (e.g. HD122563; Honda et al. 2006). Our study show diversity in the abundance patterns from light to heavy neutron-capture elements in VMP stars, suggesting a variation in r-process, which may depend on electron fraction of environment.

Watching the action unfold: New cryo-EM images capture CRISPR’s interaction with target DNA | Center for Cancer Research

Cancer.gov

Using the Nobel-prize winning technique of cryo-EM, researchers led by CCR Senior Investigator Sriram Subramaniam, Ph.D., have captured a series of highly detailed images of a protein complex belonging to the CRISPR system that can be used by bacteria to recognize and destroy foreign DNA. The images reveal the molecule’s form before and after its interaction with DNA and help

Single-molecule imaging of DNA polymerase I (Klenow fragment) activity by atomic force microscopy

NASA Astrophysics Data System (ADS)

Chao, J.; Zhang, P.; Wang, Q.; Wu, N.; Zhang, F.; Hu, J.; Fan, C. H.; Li, B.

2016-03-01

We report a DNA origami-facilitated single-molecule platform that exploits atomic force microscopy to study DNA replication. We imaged several functional activities of the Klenow fragment of E. coli DNA polymerase I (KF) including binding, moving, and dissociation from the template DNA. Upon completion of these actions, a double-stranded DNA molecule was formed. Furthermore, the direction of KF activities was captured and then confirmed by shifting the KF binding sites on the template DNA.We report a DNA origami-facilitated single-molecule platform that exploits atomic force microscopy to study DNA replication. We imaged several functional activities of the Klenow fragment of E. coli DNA polymerase I (KF) including binding, moving, and dissociation from the template DNA. Upon completion of these actions, a double-stranded DNA molecule was formed. Furthermore, the direction of KF activities was captured and then confirmed by shifting the KF binding sites on the template DNA. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr06544e

Telomere healing following DNA polymerase arrest-induced breakages is likely the main mechanism generating chromosome 4p terminal deletions.

PubMed

Hannes, Femke; Van Houdt, Jeroen; Quarrell, Oliver W; Poot, Martin; Hochstenbach, Ron; Fryns, Jean-Pierre; Vermeesch, Joris R

2010-12-01

Constitutional developmental disorders are frequently caused by terminal chromosomal deletions. The mechanisms and/or architectural features that might underlie those chromosome breakages remain largely unexplored. Because telomeres are the vital DNA protein complexes stabilizing linear chromosomes against chromosome degradation, fusion, and incomplete replication, those terminal-deleted chromosomes acquired new telomeres either by telomere healing or by telomere capture. To unravel the mechanisms leading to chromosomal breakage and healing, we sequenced nine chromosome 4p terminal deletion boundaries. A computational analysis of the breakpoint flanking region, including 12 previously published pure terminal breakage sites, was performed in order to identify architectural features that might be involved in this process. All terminal 4p truncations were likely stabilized by telomerase-mediated telomere healing. In the majority of breakpoints multiple genetic elements have a potential to induce secondary structures and an enrichment in replication stalling site motifs were identified. These findings suggest DNA replication stalling-induced chromosome breakage during early development is the first mechanistic step leading toward terminal deletion syndromes. © 2010 Wiley-Liss, Inc.

In and out of the rRNA genes: characterization of Pokey elements in the sequenced Daphnia genome

PubMed Central

2013-01-01

Background Only a few transposable elements are known to exhibit site-specific insertion patterns, including the well-studied R-element retrotransposons that insert into specific sites within the multigene rDNA. The only known rDNA-specific DNA transposon, Pokey (superfamily: piggyBac) is found in the freshwater microcrustacean, Daphnia pulex. Here, we present a genome-wide analysis of Pokey based on the recently completed whole genome sequencing project for D. pulex. Results Phylogenetic analysis of Pokey elements recovered from the genome sequence revealed the presence of four lineages corresponding to two divergent autonomous families and two related lineages of non-autonomous miniature inverted repeat transposable elements (MITEs). The MITEs are also found at the same 28S rRNA gene insertion site as the Pokey elements, and appear to have arisen as deletion derivatives of autonomous elements. Several copies of the full-length Pokey elements may be capable of producing an active transposase. Surprisingly, both families of Pokey possess a series of 200 bp repeats upstream of the transposase that is derived from the rDNA intergenic spacer (IGS). The IGS sequences within the Pokey elements appear to be evolving in concert with the rDNA units. Finally, analysis of the insertion sites of Pokey elements outside of rDNA showed a target preference for sites similar to the specific sequence that is targeted within rDNA. Conclusions Based on the target site preference of Pokey elements and the concerted evolution of a segment of the element with the rDNA unit, we propose an evolutionary path by which the ancestors of Pokey elements have invaded the rDNA niche. We discuss how specificity for the rDNA unit may have evolved and how this specificity has played a role in the long-term survival of these elements in the subgenus Daphnia. PMID:24059783

Long-term exposure of mice to nucleoside analogues disrupts mitochondrial DNA maintenance in cortical neurons.

PubMed

Zhang, Yulin; Song, Fengli; Gao, Ziyun; Ding, Wei; Qiao, Luxin; Yang, Sufang; Chen, Xi; Jin, Ronghua; Chen, Dexi

2014-01-01

Nucleoside analogue reverse transcriptase inhibitor (NRTI), an integral component of highly active antiretroviral therapy (HAART), was widely used to inhibit HIV replication. Long-term exposure to NRTIs can result in mitochondrial toxicity which manifests as lipoatrophy, lactic acidosis, cardiomyopathy and myopathy, as well as polyneuropathy. But the cerebral neurotoxicity of NRTIs is still not well known partly due to the restriction of blood-brain barrier (BBB) and the complex microenvironment of the central nervous system (CNS). In this study, the Balb/c mice were administered 50 mg/kg stavudine (D4T), 100 mg/kg zidovudine (AZT), 50 mg/kg lamivudine (3TC) or 50 mg/kg didanosine (DDI) per day by intraperitoneal injection, five days per week for one or four months, and primary cortical neurons were cultured and exposed to 25 µM D4T, 50 µM AZT, 25 µM 3TC or 25 µM DDI for seven days. Then, single neuron was captured from mouse cerebral cortical tissues by laser capture microdissection. Mitochondrial DNA (mtDNA) levels of the primary cultured cortical neurons, and captured neurons or glial cells, and the tissues of brains and livers and muscles were analyzed by relative quantitative real-time PCR. The data showed that mtDNA did not lose in both NRTIs exposed cultured neurons and one month NRTIs treated mouse brains. In four months NRTIs treated mice, brain mtDNA levels remained unchanged even if the mtDNA levels of liver (except for 3TC) and muscle significantly decreased. However, mtDNA deletion was significantly higher in the captured neurons from mtDNA unchanged brains. These results suggest that long-term exposure to NRTIs can result in mtDNA deletion in mouse cortical neurons.

n-capture elements in the Sculptor dwarf spheroidal galaxy

NASA Astrophysics Data System (ADS)

Skúladóttir, Ása

2018-06-01

Sculptor is a well studied dwarf galaxy in the Local Group, which is dominated by an old stellar population (>10 Gyr) and is therefore an ideal system to study early chemical evolution. With high-resolution VLT/FLAMES spectra, R~20,000, we are able to get accurate abundances of several n-capture elements in ~100 stars, from both the lighter n-capture elements (Y) as well as the heavier ones, both tracers of the s-process (e.g. Ba) and the r-process (e.g. Eu). I will discuss the similarities and differences in the n-capture elements in Sculptor and the Milky Way, as well as other dwarf galaxies.

Room-temperature phosphorescence logic gates developed from nucleic acid functionalized carbon dots and graphene oxide

NASA Astrophysics Data System (ADS)

Gui, Rijun; Jin, Hui; Wang, Zonghua; Zhang, Feifei; Xia, Jianfei; Yang, Min; Bi, Sai; Xia, Yanzhi

2015-04-01

Room-temperature phosphorescence (RTP) logic gates were developed using capture ssDNA (cDNA) modified carbon dots and graphene oxide (GO). The experimental results suggested the feasibility of these developed RTP-based ``OR'', ``INHIBIT'' and ``OR-INHIBIT'' logic gate operations, using Hg2+, target ssDNA (tDNA) and doxorubicin (DOX) as inputs.Room-temperature phosphorescence (RTP) logic gates were developed using capture ssDNA (cDNA) modified carbon dots and graphene oxide (GO). The experimental results suggested the feasibility of these developed RTP-based ``OR'', ``INHIBIT'' and ``OR-INHIBIT'' logic gate operations, using Hg2+, target ssDNA (tDNA) and doxorubicin (DOX) as inputs. Electronic supplementary information (ESI) available: All experimental details, Part S1-3, Fig. S1-6 and Table S1. See DOI: 10.1039/c4nr07620f

DNA-mediated strand displacement facilitates sensitive electronic detection of antibodies in human serums.

PubMed

Dou, Baoting; Yang, Jianmei; Shi, Kai; Yuan, Ruo; Xiang, Yun

2016-09-15

We describe here the development of a sensitive and convenient electronic sensor for the detection of antibodies in human serums. The sensor is constructed by self-assembly formation of a mixed monolayer containing the small molecule epitope conjugated double stranded DNA probes on gold electrode. The target antibody binds the epitope on the dsDNA probe and lowers the melting temperature of the duplex, which facilitates the displacement of the antibody-linked strand of the duplex probe by an invading methylene blue-tagged single stranded DNA (MB-ssDNA) through the strand displacement reaction and leads to the capture of many MB-ssDNA on the sensor surface. Subsequent electrochemical oxidation of the methylene blue labels results in amplified current response for sensitive monitoring of the antibodies. The antibody assay conditions are optimized and the sensor exhibits a linear range between 1.0 and 25.0nM with a detection limit of 0.67nM for the target antibody. The sensor is also selective and can be employed to detect the target antibodies in human serum samples. With the advantages of using small molecule epitope as the antibody recognition element over traditional antigen, the versatile manipulability of the DNA probes and the unique properties of the electrochemical transduction technique, the developed sensor thus hold great potential for simple and sensitive detection of different antibodies and other proteins in real samples. Copyright © 2016 Elsevier B.V. All rights reserved.

Two-Way Gold Nanoparticle Label-Free Sensing of Specific Sequence and Small Molecule Targets Using Switchable Concatemers.

PubMed

Zhu, Longjiao; Shao, Xiangli; Luo, Yunbo; Huang, Kunlung; Xu, Wentao

2017-05-19

A two-way colorimetric biosensor based on unmodified gold nanoparticles (GNPs) and a switchable double-stranded DNA (dsDNA) concatemer have been demonstrated. Two hairpin probes (H1 and H2) were first designed that provided the fuels to assemble the dsDNA concatemers via hybridization chain reaction (HCR). A functional hairpin (FH) was rationally designed to recognize the target sequences. All the hairpins contained a single-stranded DNA (ssDNA) loop and sticky end to prevent GNPs from salt-induced aggregation. In the presence of target sequence, the capture probe blocked in the FH recognizes the target to form a duplex DNA, which causes the release of the initiator probe by FH conformational change. This process then starts the alternate-opening of H1 and H2 through HCR, and dsDNA concatemers grow from the target sequence. As a result, unmodified GNPs undergo salt-induced aggregation because the formed dsDNA concatemers are stiffer and provide less stabilization. A light purple-to-blue color variation was observed in the bulk solution, termed the light-off sensing way. Furthermore, H1 ingeniously inserted an aptamer sequence to generate dsDNA concatemers with multiple small molecule binding sites. In the presence of small molecule targets, concatemers can be disassembled into mixtures with ssDNA sticky ends. A blue-to-purple reverse color variation was observed due to the regeneration of the ssDNA, termed the light-on way. The two-way biosensor can detect both nucleic acids and small molecule targets with one sensing device. This switchable sensing element is label-free, enzyme-free, and sophisticated-instrumentation-free. The detection limits of both targets were below nanomolar.

Probe DNA-Cisplatin Interaction with Solid-State Nanopores

NASA Astrophysics Data System (ADS)

Zhou, Zhi; Hu, Ying; Li, Wei; Xu, Zhi; Wang, Pengye; Bai, Xuedong; Shan, Xinyan; Lu, Xinghua; Nanopore Collaboration

2014-03-01

Understanding the mechanism of DNA-cisplatin interaction is essential for clinical application and novel drug design. As an emerging single-molecule technology, solid-state nanopore has been employed in biomolecule detection and probing DNA-molecule interactions. Herein, we reported a real-time monitoring of DNA-cisplatin interaction by employing solid-state SiN nanopores. The DNA-cisplatin interacting process is clearly classified into three stages by measuring the capture rate of DNA-cisplatin adducts. In the first stage, the negative charged DNA molecules were partially discharged due to the bonding of positive charged cisplatin and forming of mono-adducts. In the second stage, forming of DNA-cisplatin di-adducts with the adjacent bases results in DNA bending and softening. The capture rate increases since the softened bi-adducts experience a lower barrier to thread into the nanopores. In the third stage, complex structures, such as micro-loop, are formed and the DNA-cisplatin adducts are aggregated. The capture rate decreases to zero as the aggregated adduct grows to the size of the pore. The characteristic time of this stage was found to be linear with the diameter of the nanopore and this dynamic process can be described with a second-order reaction model. We are grateful to Laboratory of Microfabrication, Dr. Y. Yao, and Prof. R.C. Yu (Institute of Physics, Chinese Academy of Sciences) for technical assistance.

Capture of unstable protein complex on the streptavidin-coated single-walled carbon nanotubes

NASA Astrophysics Data System (ADS)

Liu, Zunfeng; Voskamp, Patrick; Zhang, Yue; Chu, Fuqiang; Abrahams, Jan Pieter

2013-04-01

Purification of unstable protein complexes is a bottleneck for investigation of their 3D structure and in protein-protein interaction studies. In this paper, we demonstrate that streptavidin-coated single-walled carbon nanotubes (Strep•SWNT) can be used to capture the biotinylated DNA- EcoRI complexes on a 2D surface and in solution using atomic force microscopy and electrophoresis analysis, respectively. The restriction enzyme EcoRI forms unstable complexes with DNA in the absence of Mg2+. Capturing the EcoRI-DNA complexes on the Strep•SWNT succeeded in the absence of Mg2+, demonstrating that the Strep•SWNT can be used for purifying unstable protein complexes.

DNA preservation in skeletal elements from the World Trade Center disaster: recommendations for mass fatality management.

PubMed

Mundorff, Amy Z; Bartelink, Eric J; Mar-Cash, Elaine

2009-07-01

The World Trade Center (WTC) victim identification effort highlights taphonomic influences on the degradation of DNA from victims of mass fatality incidents. This study uses a subset of the WTC-Human Remains Database to evaluate differential preservation of DNA by skeletal element. Recovery location, sex, and victim type (civilian, firefighter, or plane passenger) do not appear to influence DNA preservation. Results indicate that more intact elements, as well as elements encased in soft tissue, produced slightly higher identification rates than more fragmented remains. DNA identification rates by element type conform to previous findings, with higher rates generally found in denser, weight-bearing bones. However, smaller bones including patellae, metatarsals, and foot phalanges yielded rates comparable to both femora and tibiae. These elements can be easily sampled with a disposable scalpel, and thus reduce potential DNA contamination. These findings have implications for DNA sampling guidelines in future mass fatality incidents.

PCR and magnetic bead-mediated target capture for the isolation of short interspersed nucleotide elements in fishes.

PubMed

Liu, Dong; Zhu, Guoli; Tang, Wenqiao; Yang, Jinquan; Guo, Hongyi

2012-01-01

Short interspersed nucleotide elements (SINEs), a type of retrotransposon, are widely distributed in various genomes with multiple copies arranged in different orientations, and cause changes to genes and genomes during evolutionary history. This can provide the basis for determining genome diversity, genetic variation and molecular phylogeny, etc. SINE DNA is transcribed into RNA by polymerase III from an internal promoter, which is composed of two conserved boxes, box A and box B. Here we present an approach to isolate novel SINEs based on these promoter elements. Box A of a SINE is obtained via PCR with only one primer identical to box B (B-PCR). Box B and its downstream sequence are acquired by PCR with one primer corresponding to box A (A-PCR). The SINE clone produced by A-PCR is selected as a template to label a probe with biotin. The full-length SINEs are isolated from the genomic pool through complex capture using the biotinylated probe bound to magnetic particles. Using this approach, a novel SINE family, Cn-SINE, from the genomes of Coilia nasus, was isolated. The members are 180-360 bp long. Sequence homology suggests that Cn-SINEs evolved from a leucine tRNA gene. This is the first report of a tRNA(Leu)-related SINE obtained without the use of a genomic library or inverse PCR. These results provide new insights into the origin of SINEs.

PCR and Magnetic Bead-Mediated Target Capture for the Isolation of Short Interspersed Nucleotide Elements in Fishes

PubMed Central

Liu, Dong; Zhu, Guoli; Tang, Wenqiao; Yang, Jinquan; Guo, Hongyi

2012-01-01

Short interspersed nucleotide elements (SINEs), a type of retrotransposon, are widely distributed in various genomes with multiple copies arranged in different orientations, and cause changes to genes and genomes during evolutionary history. This can provide the basis for determining genome diversity, genetic variation and molecular phylogeny, etc. SINE DNA is transcribed into RNA by polymerase III from an internal promoter, which is composed of two conserved boxes, box A and box B. Here we present an approach to isolate novel SINEs based on these promoter elements. Box A of a SINE is obtained via PCR with only one primer identical to box B (B-PCR). Box B and its downstream sequence are acquired by PCR with one primer corresponding to box A (A-PCR). The SINE clone produced by A-PCR is selected as a template to label a probe with biotin. The full-length SINEs are isolated from the genomic pool through complex capture using the biotinylated probe bound to magnetic particles. Using this approach, a novel SINE family, Cn-SINE, from the genomes of Coilia nasus, was isolated. The members are 180–360 bp long. Sequence homology suggests that Cn-SINEs evolved from a leucine tRNA gene. This is the first report of a tRNALeu-related SINE obtained without the use of a genomic library or inverse PCR. These results provide new insights into the origin of SINEs. PMID:22408437

Highly affine and selective aptamers against cholera toxin as capture elements in magnetic bead-based sandwich ELAA.

PubMed

Frohnmeyer, Esther; Frisch, Farina; Falke, Sven; Betzel, Christian; Fischer, Markus

2018-03-10

Aptamers are single-stranded DNA or RNA oligonucleotides, which have been emerging as recognition elements in disease diagnostics and food control, including the detection of bacterial toxins. In this study, we employed the semi-automated just in time-selection to identify aptamers that bind to cholera toxin (CT) with high affinity and specificity. CT is the main virulence factor of Vibrio cholerae and the causative agent of the eponymous disease. For the selected aptamers, dissociation constants in the low nanomolar range (23-56 nM) were determined by fluorescence-based affinity chromatography and cross-reactivity against related proteins was evaluated by direct enzyme-linked aptamer assay (ELAA). Aptamer CT916 has a dissociation constant of 48.5 ± 0.5 nM and shows negligible binding to Shiga-like toxin 1B, protein A and BSA. This aptamer was chosen to develop a sandwich ELAA for the detection of CT from binding buffer and local tap water. Amine-C6- or biotin-modified CT916 was coupled to magnetic beads to serve as the capture element. Using an anti-CT polyclonal antibody as the reporter, detection limits of 2.1 ng/ml in buffer and 2.4 ng/ml in tap water, with a wide log-linear dynamic range from 1 ng/ml to 1000 ng/ml and 500 ng/ml, respectively, were achieved. Copyright © 2018 Elsevier B.V. All rights reserved.

A DNAzyme-mediated logic gate for programming molecular capture and release on DNA origami.

PubMed

Li, Feiran; Chen, Haorong; Pan, Jing; Cha, Tae-Gon; Medintz, Igor L; Choi, Jong Hyun

2016-06-28

Here we design a DNA origami-based site-specific molecular capture and release platform operated by a DNAzyme-mediated logic gate process. We show the programmability and versatility of this platform with small molecules, proteins, and nanoparticles, which may also be controlled by external light signals.

A universal colorimetry for nucleic acids and aptamer-specific ligands detection based on DNA hybridization amplification.

PubMed

Li, Shuang; Shang, Xinxin; Liu, Jia; Wang, Yujie; Guo, Yingshu; You, Jinmao

2017-07-01

We present a universal amplified-colorimetric for detecting nucleic acid targets or aptamer-specific ligand targets based on gold nanoparticle-DNA (GNP-DNA) hybridization chain reaction (HCR). The universal arrays consisted of capture probe and hairpin DNA-GNP. First, capture probe recognized target specificity and released the initiator sequence. Then dispersed hairpin DNA modified GNPs were cross-linked to form aggregates through HCR events triggered by initiator sequence. As the aggregates accumulate, a significant red-to purple color change can be easily visualized by the naked eye. We used miRNA target sequence (miRNA-203) and aptamer-specific ligand (ATP) as target molecules for this proof-of-concept experiment. Initiator sequence (DNA2) was released from the capture probe (MNP/DNA1/2 conjugates) under the strong competitiveness of miRNA-203. Hairpin DNA (H1 and H2) can be complementary with the help of initiator DNA2 to form GNP-H1/GNP-H2 aggregates. The absorption ratio (A 620 /A 520 ) values of solutions were a sensitive function of miRNA-203 concentration covering from 1.0 × 10 -11  M to 9.0 × 10 -10  M, and as low as 1.0 × 10 -11  M could be detected. At the same time, the color changed from light wine red to purple and then to light blue have occurred in the solution. For ATP, initiator sequence (5'-end of DNA3) was released from the capture probe (DNA3) under the strong combination of aptamer-ATP. The present colorimetric for specific detection of ATP exhibited good sensitivity and 1.0 × 10 -8  M ATP could be detected. The proposed strategy also showed good performances for qualitative analysis and quantitative analysis of intracellular nucleic acids and aptamer-specific ligands. Copyright © 2017 Elsevier Inc. All rights reserved.

Double-probe signal enhancing strategy for toxin aptasensing based on rolling circle amplification.

PubMed

Tong, Ping; Zhao, Wei-Wei; Zhang, Lan; Xu, Jing-Juan; Chen, Hong-Yuan

2012-03-15

On the basis of aptamer-based rolling circle amplification (RCA) and magnetic beads (MBs), a highly sensitive electrochemical method was developed for the determination of Ochratoxin A (OTA). Initially, an amino-modified capture DNA was immobilized onto MBs for the following hybridization with an OTA aptamer and a phosphate labeled padlock DNA. In the presence of OTA, the aptamer would dissociate from the bioconjugate, and the padlock DNA would subsequently hybridize with the capture DNA to form a circular template with the aid of the T4 ligase. Next, capture DNA would act as primer to initiate a linear RCA reaction and hence generate a long tandem repeated sequences by phi29 DNA polymerase and dNTPs. Then, two quantum dots (QDs) labeled DNA probes were tagged on the resulted RCA product to indicate the OTA recognition event by electrochemical readout. This strategy, based on the novel design of OTA-mediated DNA circularization, the combination of RCA and double signal probes introduction, could detect OTA down to the level of 0.2 pg mL(-1) with a dynamic range spanning more than 4 orders of magnitude. The proposed approach is tested to determine OTA in red wines and shows good application potential in real samples. Copyright © 2011 Elsevier B.V. All rights reserved.

DNA-barcode directed capture and electrochemical metabolic analysis of single mammalian cells on a microelectrode array.

PubMed

Douglas, Erik S; Hsiao, Sonny C; Onoe, Hiroaki; Bertozzi, Carolyn R; Francis, Matthew B; Mathies, Richard A

2009-07-21

A microdevice is developed for DNA-barcode directed capture of single cells on an array of pH-sensitive microelectrodes for metabolic analysis. Cells are modified with membrane-bound single-stranded DNA, and specific single-cell capture is directed by the complementary strand bound in the sensor area of the iridium oxide pH microelectrodes within a microfluidic channel. This bifunctional microelectrode array is demonstrated for the pH monitoring and differentiation of primary T cells and Jurkat T lymphoma cells. Single Jurkat cells exhibited an extracellular acidification rate of 11 milli-pH min(-1), while primary T cells exhibited only 2 milli-pH min(-1). This system can be used to capture non-adherent cells specifically and to discriminate between visually similar healthy and cancerous cells in a heterogeneous ensemble based on their altered metabolic properties.

DNA-barcode directed capture and electrochemical metabolic analysis of single mammalian cells on a microelectrode array

PubMed Central

Douglas, Erik S.; Hsiao, Sonny C.; Onoe, Hiroaki; Bertozzi, Carolyn R.; Francis, Matthew B.; Mathies, Richard A.

2010-01-01

A microdevice is developed for DNA-barcode directed capture of single cells on an array of pH-sensitive microelectrodes for metabolic analysis. Cells are modified with membrane-bound single-stranded DNA, and specific single-cell capture is directed by the complementary strand bound in the sensor area of the iridium oxide pH microelectrodes within a microfluidic channel. This bifunctional microelectrode array is demonstrated for the pH monitoring and differentiation of primary T cells and Jurkat T lymphoma cells. Single Jurkat cells exhibited an extracellular acidification rate of 11 milli-pH min−1, while primary T cells exhibited only 2 milli-pH min−1. This system can be used to capture non-adherent cells specifically and to discriminate between visually similar healthy and cancerous cells in a heterogeneous ensemble based on their altered metabolic properties. PMID:19568668

41 CFR 301-71.2 - What are the standard data elements and when must they be captured on a travel accounting system?

Code of Federal Regulations, 2010 CFR

2010-07-01

... data elements and when must they be captured on a travel accounting system? 301-71.2 Section 301-71.2 Public Contracts and Property Management Federal Travel Regulation System TEMPORARY DUTY (TDY) TRAVEL... the standard data elements and when must they be captured on a travel accounting system? The data...

Analytical Devices Based on Direct Synthesis of DNA on Paper.

PubMed

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

2016-01-05

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

Optimizing techniques to capture and extract environmental DNA for detection and quantification of fish.

PubMed

Eichmiller, Jessica J; Miller, Loren M; Sorensen, Peter W

2016-01-01

Few studies have examined capture and extraction methods for environmental DNA (eDNA) to identify techniques optimal for detection and quantification. In this study, precipitation, centrifugation and filtration eDNA capture methods and six commercially available DNA extraction kits were evaluated for their ability to detect and quantify common carp (Cyprinus carpio) mitochondrial DNA using quantitative PCR in a series of laboratory experiments. Filtration methods yielded the most carp eDNA, and a glass fibre (GF) filter performed better than a similar pore size polycarbonate (PC) filter. Smaller pore sized filters had higher regression slopes of biomass to eDNA, indicating that they were potentially more sensitive to changes in biomass. Comparison of DNA extraction kits showed that the MP Biomedicals FastDNA SPIN Kit yielded the most carp eDNA and was the most sensitive for detection purposes, despite minor inhibition. The MoBio PowerSoil DNA Isolation Kit had the lowest coefficient of variation in extraction efficiency between lake and well water and had no detectable inhibition, making it most suitable for comparisons across aquatic environments. Of the methods tested, we recommend using a 1.5 μm GF filter, followed by extraction with the MP Biomedicals FastDNA SPIN Kit for detection. For quantification of eDNA, filtration through a 0.2-0.6 μm pore size PC filter, followed by extraction with MoBio PowerSoil DNA Isolation Kit was optimal. These results are broadly applicable for laboratory studies on carps and potentially other cyprinids. The recommendations can also be used to inform choice of methodology for field studies. © 2015 John Wiley & Sons Ltd.

Densely ionizing radiation affects DNA methylation of selective LINE-1 elements1

PubMed Central

Prior, Sara; Miousse, Isabelle R.; Nzabarushimana, Etienne; Pathak, Rupak; Skinner, Charles; Kutanzi, Kristy R.; Allen, Antiño R.; Raber, Jacob; Tackett, Alan J.; Hauer-Jensen, Martin; Nelson, Gregory A.; Koturbash, Igor

2016-01-01

Long Interspersed Nucleotide Element 1 (LINE-1) retrotransposons are heavily methylated and are the most abundant transposable elements in mammalian genomes. Here, we investigated the differential DNA methylation within the LINE-1 under normal conditions and in response to environmentally relevant doses of sparsely and densely ionizing radiation. We demonstrate that DNA methylation of LINE-1 elements in the lungs of C57BL6 mice is dependent on their evolutionary age, where the elder age of the element is associated with the lower extent of DNA methylation. Exposure to 5-aza-2′-deoxycytidine and methionine-deficient diet affected DNA methylation of selective LINE-1 elements in an age- and promoter type-dependent manner. Exposure to densely IR, but not sparsely IR, resulted in DNA hypermethylation of older LINE-1 elements, while the DNA methylation of evolutionary younger elements remained mostly unchanged. We also demonstrate that exposure to densely IR increased mRNA and protein levels of LINE-1 via the loss of the histone H3K9 dimethylation and an increase in the H3K4 trimethylation at the LINE-1 5′-untranslated region, independently of DNA methylation. Our findings suggest that DNA methylation is important for regulation of LINE-1 expression under normal conditions, but histone modifications may dictate the transcriptional activity of LINE-1 in response to exposure to densely IR. PMID:27419368

DNA Motion Capture Reveals the Mechanical Properties of DNA at the Mesoscale

PubMed Central

Price, Allen C.; Pilkiewicz, Kevin R.; Graham, Thomas G.W.; Song, Dan; Eaves, Joel D.; Loparo, Joseph J.

2015-01-01

Single-molecule studies probing the end-to-end extension of long DNAs have established that the mechanical properties of DNA are well described by a wormlike chain force law, a polymer model where persistence length is the only adjustable parameter. We present a DNA motion-capture technique in which DNA molecules are labeled with fluorescent quantum dots at specific sites along the DNA contour and their positions are imaged. Tracking these positions in time allows us to characterize how segments within a long DNA are extended by flow and how fluctuations within the molecule are correlated. Utilizing a linear response theory of small fluctuations, we extract elastic forces for the different, ∼2-μm-long segments along the DNA backbone. We find that the average force-extension behavior of the segments can be well described by a wormlike chain force law with an anomalously small persistence length. PMID:25992731

DNA purification by triplex-affinity capture and affinity capture electrophoresis

DOEpatents

Cantor, Charles R.; Ito, Takashi; Smith, Cassandra L.

1996-01-01

The invention provides a method for purifying or isolating double stranded DNA intact using triple helix formation. The method includes the steps of complexing an oligonucleotide and double stranded DNA to generate a triple helix and immobilization of the triple helix on a solid phase by means of a molecular recognition system such as avidin/biotin. The purified DNA is then recovered intact by treating the solid phase with a reagent that breaks the bonds between the oligonucleotide and the intact double stranded DNA while not affecting the Watson-Crick base pairs of the double helix. The present invention also provides a method for purifying or isolating double stranded DNA intact by complexing the double stranded DNA with a specific binding partner and recovering the complex during electrophoresis by immobilizing it on a solid phase trap imbedded in an electrophoretic gel.

Portable and sensitive quantitative detection of DNA based on personal glucose meters and isothermal circular strand-displacement polymerization reaction.

PubMed

Xu, Xue-tao; Liang, Kai-yi; Zeng, Jia-ying

2015-02-15

A portable and sensitive quantitative DNA detection method based on personal glucose meters and isothermal circular strand-displacement polymerization reaction was developed. The target DNA triggered target recycling process, which opened capture DNA. The released target then found another capture DNA to trigger another polymerization cycle, which was repeated for many rounds, resulting in the multiplication of the DNA-invertase conjugation on the surface of Streptavidin-MNBs. The DNA-invertase was used to catalyze the hydrolysis of sucrose into glucose for PGM readout. There was a liner relationship between the signal of PGM and the concentration of target DNA in the range of 5.0 to 1000 fM, which is lower than some DNA detection method. In addition, the method exhibited excellent sequence selectivity and there was almost no effect of biological complex to the detection performance, which suggested our method can be successfully applied to DNA detection in real biological samples. Copyright © 2014 Elsevier B.V. All rights reserved.

Fabrication of endothelial progenitor cell capture surface via DNA aptamer modifying dopamine/polyethyleneimine copolymer film

NASA Astrophysics Data System (ADS)

Li, Xin; Deng, Jinchuan; Yuan, Shuheng; Wang, Juan; Luo, Rifang; Chen, Si; Wang, Jin; Huang, Nan

2016-11-01

Endothelial progenitor cells (EPCs) are mainly located in bone marrow and circulate, and play a crucial role in repairmen of injury endothelium. One of the most promising strategies of stents designs were considered to make in-situ endothelialization in vivo via EPC-capture biomolecules on a vascular graft to capture EPCs directly from circulatory blood. In this work, an EPC specific aptamer with a 34 bases single strand DNA sequence was conjugated onto the stent surface via dopamine/polyethyleneimine copolymer film as a platform and linker. The assembled density of DNA aptamer could be regulated by controlling dopamine percentage in this copolymer film. X-ray photoelectron spectroscopy (XPS), water contact angle (WCA) and fluorescence test confirmed the successful immobilization of DNA aptamer. To confirm its biofunctionality and cytocompatibility, the capturing cells ability of the aptamer modified surface and the effects on the growth behavior of human umbilical vein endothelial cells (HUVECs), smooth muscle cells (SMCs) were investigated. The aptamer functionalized sample revealed a good EPC-capture ability, and had a cellular friendly feature for both EPC and EC growth, while not stimulated the hyperplasia of SMCs. And, the co-culture experiment of three types of cells confirmed the specificity capturing of EPCs to aptamer modified surface, rather than ECs and SMCs. These data suggested that this aptamer functionalized surface may have a large potentiality for the application of vascular grafts with targeted endothelialization.

Surface-enhanced Raman scattering detection of DNA derived from the West Nile virus genome using magnetic capture of Raman-active gold nanoparticles

USDA-ARS?s Scientific Manuscript database

A model paramagnetic nanoparticle (MNP) assay is demonstrated for surface-enhanced Raman scattering (SERS) detection of DNA oligonucleotides derived from the West Nile virus (WNV) genome. Detection is based on the capture of WNV target sequences by hybridization with complementary oligonucleotide pr...

NEW NEUTRON-CAPTURE MEASUREMENTS IN 23 OPEN CLUSTERS. I. THE r -PROCESS

DOE Office of Scientific and Technical Information (OSTI.GOV)

Overbeek, Jamie C.; Friel, Eileen D.; Jacobson, Heather R., E-mail: joverbee@indiana.edu

2016-06-20

Neutron-capture elements, those with Z > 35, are the least well understood in terms of nucleosynthesis and formation environments. The rapid neutron-capture, or r -process, elements are formed in the environments and/or remnants of massive stars, while the slow neutron-capture, or s -process, elements are primarily formed in low-mass AGB stars. These elements can provide much information about Galactic star formation and enrichment, but observational data are limited. We have assembled a sample of 68 stars in 23 open clusters that we use to probe abundance trends for six neutron-capture elements (Eu, Gd, Dy, Mo, Pr, and Nd) with clustermore » age and location in the disk of the Galaxy. In order to keep our analysis as homogeneous as possible, we use an automated synthesis fitting program, which also enables us to measure multiple (3–10) lines for each element. We find that the pure r -process elements (Eu, Gd, and Dy) have positive trends with increasing cluster age, while the mixed r - and s -process elements (Mo, Pr, and Nd) have insignificant trends consistent with zero. Pr, Nd, Eu, Gd, and Dy have similar, slight (although mostly statistically significant) gradients of ∼0.04 dex kpc{sup −1}. The mixed elements also appear to have nonlinear relationships with R {sub GC}.« less

A Three-Dimensional Model of the Yeast Genome

NASA Astrophysics Data System (ADS)

Noble, William; Duan, Zhi-Jun; Andronescu, Mirela; Schutz, Kevin; McIlwain, Sean; Kim, Yoo Jung; Lee, Choli; Shendure, Jay; Fields, Stanley; Blau, C. Anthony

Layered on top of information conveyed by DNA sequence and chromatin are higher order structures that encompass portions of chromosomes, entire chromosomes, and even whole genomes. Interphase chromosomes are not positioned randomly within the nucleus, but instead adopt preferred conformations. Disparate DNA elements co-localize into functionally defined aggregates or factories for transcription and DNA replication. In budding yeast, Drosophila and many other eukaryotes, chromosomes adopt a Rabl configuration, with arms extending from centromeres adjacent to the spindle pole body to telomeres that abut the nuclear envelope. Nonetheless, the topologies and spatial relationships of chromosomes remain poorly understood. Here we developed a method to globally capture intra- and inter-chromosomal interactions, and applied it to generate a map at kilobase resolution of the haploid genome of Saccharomyces cerevisiae. The map recapitulates known features of genome organization, thereby validating the method, and identifies new features. Extensive regional and higher order folding of individual chromosomes is observed. Chromosome XII exhibits a striking conformation that implicates the nucleolus as a formidable barrier to interaction between DNA sequences at either end. Inter-chromosomal contacts are anchored by centromeres and include interactions among transfer RNA genes, among origins of early DNA replication and among sites where chromosomal breakpoints occur. Finally, we constructed a three-dimensional model of the yeast genome. Our findings provide a glimpse of the interface between the form and function of a eukaryotic genome.

Mapping Fifteen Trace Elements in Human Seminal Plasma and Sperm DNA.

PubMed

Ali, Sazan; Chaspoul, Florence; Anderson, Loundou; Bergé-Lefranc, David; Achard, Vincent; Perrin, Jeanne; Gallice, Philippe; Guichaoua, Marie

2017-02-01

Studies suggest a relationship between semen quality and the concentration of trace elements in serum or seminal plasma. However, trace elements may be linked to DNA and capable of altering the gene expression patterns. Thus, trace element interactions with DNA may contribute to the mechanisms for a trans-generational reproductive effect. We developed an analytical method to determine the amount of trace elements bound to the sperm DNA, and to estimate their affinity for the sperm DNA by the ratio: R = Log [metal concentration in the sperm DNA/metal concentration in seminal plasma]. We then analyzed the concentrations of 15 trace elements (Al, Cd, Cr, Cu, Hg, Mn, Mo, Ni, Pb, Ti, V, Zn, As, Sb, and Se) in the seminal plasma and the sperm DNA in 64 normal and 30 abnormal semen specimens with Inductively Coupled Plasma/Mass Spectrometry (ICP-MS). This study showed all trace elements were detected in the seminal plasma and only metals were detected in the sperm DNA. There was no correlation between the metals' concentrations in the seminal plasma and the sperm DNA. Al had the highest affinity for DNA followed by Pb and Cd. This strong affinity is consistent with the known mutagenic effects of these metals. The lowest affinity was observed for Zn and Ti. We observed a significant increase of Al linked to the sperm DNA of patients with oligozoospermia and teratozoospermia. Al's reproductive toxicity might be due to Al linked to DNA, by altering spermatogenesis and expression patterns of genes involved in the function of reproduction.

Transcription-induced DNA supercoiling: New roles of intranucleosomal DNA loops in DNA repair and transcription.

PubMed

Gerasimova, N S; Pestov, N A; Kulaeva, O I; Clark, D J; Studitsky, V M

2016-05-26

RNA polymerase II (Pol II) transcription through chromatin is accompanied by formation of small intranucleosomal DNA loops. Pol II captured within a small loop drives accumulation of DNA supercoiling, facilitating further transcription. DNA breaks relieve supercoiling and induce Pol II arrest, allowing detection of DNA damage hidden in chromatin structure.

Revisiting the Zingiberales: using multiplexed exon capture to resolve ancient and recent phylogenetic splits in a charismatic plant lineage

PubMed Central

Iles, William J.D.; Barrett, Craig F.; Smith, Selena Y.; Specht, Chelsea D.

2016-01-01

The Zingiberales are an iconic order of monocotyledonous plants comprising eight families with distinctive and diverse floral morphologies and representing an important ecological element of tropical and subtropical forests. While the eight families are demonstrated to be monophyletic, phylogenetic relationships among these families remain unresolved. Neither combined morphological and molecular studies nor recent attempts to resolve family relationships using sequence data from whole plastomes has resulted in a well-supported, family-level phylogenetic hypothesis of relationships. Here we approach this challenge by leveraging the complete genome of one member of the order, Musa acuminata, together with transcriptome information from each of the other seven families to design a set of nuclear loci that can be enriched from highly divergent taxa with a single array-based capture of indexed genomic DNA. A total of 494 exons from 418 nuclear genes were captured for 53 ingroup taxa. The entire plastid genome was also captured for the same 53 taxa. Of the total genes captured, 308 nuclear and 68 plastid genes were used for phylogenetic estimation. The concatenated plastid and nuclear dataset supports the position of Musaceae as sister to the remaining seven families. Moreover, the combined dataset recovers known intra- and inter-family phylogenetic relationships with generally high bootstrap support. This is a flexible and cost effective method that gives the broader plant biology community a tool for generating phylogenomic scale sequence data in non-model systems at varying evolutionary depths. PMID:26819846

Revisiting the Zingiberales: using multiplexed exon capture to resolve ancient and recent phylogenetic splits in a charismatic plant lineage.

PubMed

Sass, Chodon; Iles, William J D; Barrett, Craig F; Smith, Selena Y; Specht, Chelsea D

2016-01-01

The Zingiberales are an iconic order of monocotyledonous plants comprising eight families with distinctive and diverse floral morphologies and representing an important ecological element of tropical and subtropical forests. While the eight families are demonstrated to be monophyletic, phylogenetic relationships among these families remain unresolved. Neither combined morphological and molecular studies nor recent attempts to resolve family relationships using sequence data from whole plastomes has resulted in a well-supported, family-level phylogenetic hypothesis of relationships. Here we approach this challenge by leveraging the complete genome of one member of the order, Musa acuminata, together with transcriptome information from each of the other seven families to design a set of nuclear loci that can be enriched from highly divergent taxa with a single array-based capture of indexed genomic DNA. A total of 494 exons from 418 nuclear genes were captured for 53 ingroup taxa. The entire plastid genome was also captured for the same 53 taxa. Of the total genes captured, 308 nuclear and 68 plastid genes were used for phylogenetic estimation. The concatenated plastid and nuclear dataset supports the position of Musaceae as sister to the remaining seven families. Moreover, the combined dataset recovers known intra- and inter-family phylogenetic relationships with generally high bootstrap support. This is a flexible and cost effective method that gives the broader plant biology community a tool for generating phylogenomic scale sequence data in non-model systems at varying evolutionary depths.

Immobilization of DNA onto poly(dimethylsiloxane) surfaces and application to a microelectrochemical enzyme-amplified DNA hybridization assay.

PubMed

Liu, Daojun; Perdue, Robbyn K; Sun, Li; Crooks, Richard M

2004-07-06

This paper describes immobilization of DNA onto the interior walls of poly(dimethylsiloxane) (PDMS) microsystems and its application to an enzyme-amplified electrochemical DNA assay. DNA immobilization was carried out by silanization of the PDMS surface with 3-mercaptopropyltrimethoxysilane to yield a thiol-terminated surface. 5'-acrylamide-modified DNA reacts with the pendant thiol groups to yield DNA-modified PDMS. Surface-immobilized DNA oligos serve as capture probes for target DNA. Biotin-labeled target DNA hybridizes to the PDMS-immobilized capture DNA, and subsequent introduction of alkaline phosphatase (AP) conjugated to streptavidin results in attachment of the enzyme to hybridized DNA. Electrochemical detection of DNA hybridization benefits from enzyme amplification. Specifically, AP converts electroinactive p-aminophenyl phosphate to electroactive p-aminophenol, which is detected using an indium tin oxide interdigitated array (IDA) electrode. The IDA electrode eliminates the need for a reference electrode and provides a steady-state current that is related to the concentration of hybridized DNA. At present, the limit of detection of the DNA target is 1 nM in a volume of 20 nL, which corresponds to 20 attomoles of DNA.

Aptamer-based microspheres for highly sensitive protein detection using fluorescently-labeled DNA nanostructures.

PubMed

Han, Daehoon; Hong, Jinkee; Kim, Hyun Cheol; Sung, Jong Hwan; Lee, Jong Bum

2013-11-01

Many highly sensitive protein detection techniques have been developed and have played an important role in the analysis of proteins. Herein, we report a novel technique that can detect proteins sensitively and effectively using aptamer-based DNA nanostructures. Thrombin was used as a target protein and aptamer was used to capture fluorescent dye-labeled DNA nanobarcodes or thrombin on a microsphere. The captured DNA nanobarcodes were replaced by a thrombin and aptamer interaction. The detection ability of this approach was confirmed by flow cytometry with different concentrations of thrombin. Our detection method has great potential for rapid and simple protein detection with a variety of aptamers.

The minimal amount of starting DNA for Agilent’s hybrid capture-based targeted massively parallel sequencing

PubMed Central

Chung, Jongsuk; Son, Dae-Soon; Jeon, Hyo-Jeong; Kim, Kyoung-Mee; Park, Gahee; Ryu, Gyu Ha; Park, Woong-Yang; Park, Donghyun

2016-01-01

Targeted capture massively parallel sequencing is increasingly being used in clinical settings, and as costs continue to decline, use of this technology may become routine in health care. However, a limited amount of tissue has often been a challenge in meeting quality requirements. To offer a practical guideline for the minimum amount of input DNA for targeted sequencing, we optimized and evaluated the performance of targeted sequencing depending on the input DNA amount. First, using various amounts of input DNA, we compared commercially available library construction kits and selected Agilent’s SureSelect-XT and KAPA Biosystems’ Hyper Prep kits as the kits most compatible with targeted deep sequencing using Agilent’s SureSelect custom capture. Then, we optimized the adapter ligation conditions of the Hyper Prep kit to improve library construction efficiency and adapted multiplexed hybrid selection to reduce the cost of sequencing. In this study, we systematically evaluated the performance of the optimized protocol depending on the amount of input DNA, ranging from 6.25 to 200 ng, suggesting the minimal input DNA amounts based on coverage depths required for specific applications. PMID:27220682

Hybridization behavior of mixed DNA/alkylthiol monolayers on gold: characterization by surface plasmon resonance and 32P radiometric assay.

PubMed

Gong, Ping; Lee, Chi-Ying; Gamble, Lara J; Castner, David G; Grainger, David W

2006-05-15

Nucleic acid assay from a complex biological milieu is attractive but currently difficult and far from routine. In this study, DNA hybridization from serum dilutions into mixed DNA/mercaptoundecanol (MCU) adlayers on gold was monitored by surface plasmon resonance (SPR). Immobilized DNA probe and hybridized target densities on these surfaces were quantified using 32P-radiometric assays as a function of MCU diluent exposure. SPR surface capture results correlated with radiometric analysis for hybridization performance, demonstrating a maximum DNA hybridization on DNA/MCU mixed adlayers. The maximum target surface capture produced by MCU addition to the DNA probe layer correlates with structural and conformational data on identical mixed DNA/MCU adlayers on gold derived from XPS, NEXAFS, and fluorescence intensity measurements reported in a related study (Lee, C.-Y.; Gong, P.; Harbers, G. M.; Grainger, D. W.; Castner, D. G.; Gamble, L. J. Anal. Chem. 2006, 78, 3316-3325.). MCU addition into the DNA adlayer on gold also improved surface resistance to both nonspecific DNA and serum protein adsorption. Target DNA hybridization from serum dilutions was monitored with SPR on the optimally mixed DNA/MCU adlayers. Both hybridization kinetics and efficiency were strongly affected by nonspecific protein adsorption from a complex milieu even at a minimal serum concentration (e.g., 1%). No target hybridization was detected in SPR assays from serum concentrations above 30%, indicating nonspecific protein adsorption interference of DNA capture and hybridization from complex milieu. Removal of nonsignal proteins from nucleic acid targets prior to assay represents a significant issue for direct sample-to-assay nucleic acid diagnostics from food, blood, tissue, PCR mixtures, and many other biologically complex sample formats.

DNA purification by triplex-affinity capture and affinity capture electrophoresis

DOEpatents

Cantor, C.R.; Ito, Takashi; Smith, C.L.

1996-01-09

The invention provides a method for purifying or isolating double stranded DNA intact using triple helix formation. The method includes the steps of complexing an oligonucleotide and double stranded DNA to generate a triple helix and immobilization of the triple helix on a solid phase by means of a molecular recognition system such as avidin/biotin. The purified DNA is then recovered intact by treating the solid phase with a reagent that breaks the bonds between the oligonucleotide and the intact double stranded DNA while not affecting the Watson-Crick base pairs of the double helix. The present invention also provides a method for purifying or isolating double stranded DNA intact by complexing the double stranded DNA with a specific binding partner and recovering the complex during electrophoresis by immobilizing it on a solid phase trap imbedded in an electrophoretic gel. 6 figs.

Stellar Abundance Observations and Heavy Element Formation

NASA Astrophysics Data System (ADS)

Cowan, J. J.

2005-05-01

Abundance observations indicate the presence of rapid-neutron capture (i.e., r-process) elements in old Galactic halo and globular cluster stars. These observations provide insight into the nature of the earliest generations of stars in the Galaxy -- the progenitors of the halo stars -- responsible for neutron-capture synthesis of the heavy elements. Abundance comparisons among the r-process-rich halo stars show that the heaviest neutron-capture elements (i.e., Ba and above) are consistent with a scaled solar system r-process abundance distribution, while the lighter neutron-capture elements do not conform to the solar pattern. These comparisons suggest the possibility of two r-process sites in stars. The large star-to-star scatter observed in the abundances of neutron-capture element/iron ratios at low metallicities -- which disappears with increasing metallicity or [Fe/H] -- suggests the formation of these heavy elements (presumably from certain types of supernovae) was rare in the early Galaxy. The stellar abundances also indicate a change from the r-process to the slow neutron capture (i.e., s-) process at higher metallicities in the Galaxy and provide insight into Galactic chemical evolution. Finally, the detection of thorium and uranium in halo and globular cluster stars offers an independent age-dating technique that can put lower limits on the age of the Galaxy, and hence the Universe. This work has been supported in part by NSF grant AST 03-07279 (J.J.C.) and by STScI grants GO-8111, GO-8342 and GO-9359.

Comparisons of non-Gaussian statistical models in DNA methylation analysis.

PubMed

Ma, Zhanyu; Teschendorff, Andrew E; Yu, Hong; Taghia, Jalil; Guo, Jun

2014-06-16

As a key regulatory mechanism of gene expression, DNA methylation patterns are widely altered in many complex genetic diseases, including cancer. DNA methylation is naturally quantified by bounded support data; therefore, it is non-Gaussian distributed. In order to capture such properties, we introduce some non-Gaussian statistical models to perform dimension reduction on DNA methylation data. Afterwards, non-Gaussian statistical model-based unsupervised clustering strategies are applied to cluster the data. Comparisons and analysis of different dimension reduction strategies and unsupervised clustering methods are presented. Experimental results show that the non-Gaussian statistical model-based methods are superior to the conventional Gaussian distribution-based method. They are meaningful tools for DNA methylation analysis. Moreover, among several non-Gaussian methods, the one that captures the bounded nature of DNA methylation data reveals the best clustering performance.

Comparisons of Non-Gaussian Statistical Models in DNA Methylation Analysis

PubMed Central

Ma, Zhanyu; Teschendorff, Andrew E.; Yu, Hong; Taghia, Jalil; Guo, Jun

2014-01-01

As a key regulatory mechanism of gene expression, DNA methylation patterns are widely altered in many complex genetic diseases, including cancer. DNA methylation is naturally quantified by bounded support data; therefore, it is non-Gaussian distributed. In order to capture such properties, we introduce some non-Gaussian statistical models to perform dimension reduction on DNA methylation data. Afterwards, non-Gaussian statistical model-based unsupervised clustering strategies are applied to cluster the data. Comparisons and analysis of different dimension reduction strategies and unsupervised clustering methods are presented. Experimental results show that the non-Gaussian statistical model-based methods are superior to the conventional Gaussian distribution-based method. They are meaningful tools for DNA methylation analysis. Moreover, among several non-Gaussian methods, the one that captures the bounded nature of DNA methylation data reveals the best clustering performance. PMID:24937687

Construction of DNA sandwich electrochemical biosensor with nanoPbS and nanoAu tags on magnetic microbeads.

PubMed

Du, Ping; Li, Hongxia; Cao, Wei

2009-07-15

A novel and sensitive sandwich electrochemical biosensor based on the amplification of magnetic microbeads and Au nanoparticles (NPs) modified with bio bar code and PbS nanoparticles was constructed in the present work. In this method, the magnetic microspheres were coated with 4 layers polyelectrolytes in order to increase carboxyl groups on the surface of the magnetic microbeads, which enhanced the amount of the capture DNA. The amino-functionalized capture DNA on the surface of magnetic microbeads hybridized with one end of target DNA, the other end of which was hybridized with signal DNA probe labelled with Au NPs on the terminus. The Au NPs were modified with bio bar code and the PbS NPs were used as a marker for identifying the target oligoncleotide. The modification of magnetic microbeads could immobilize more amino-group terminal capture DNA, and the bio bar code could increase the amount of Au NPs that combined with the target DNA. The detection of lead ions performed by anodic stripping voltammetry (ASV) technology further improved the sensitivity of the biosensor. As a result, the present DNA biosensor showed good selectivity and sensitivity by the combined amplification. Under the optimum conditions, the linear relationship with the concentration of the target DNA was ranging from 2.0 x 10(-14) M to 1.0 x 10(-12)M and a detection limit as low as 5.0 x 10(-15)M was obtained.

Transcription-induced DNA supercoiling: New roles of intranucleosomal DNA loops in DNA repair and transcription

PubMed Central

Gerasimova, N. S.; Pestov, N. A.; Kulaeva, O. I.; Clark, D. J.; Studitsky, V. M.

2016-01-01

ABSTRACT RNA polymerase II (Pol II) transcription through chromatin is accompanied by formation of small intranucleosomal DNA loops. Pol II captured within a small loop drives accumulation of DNA supercoiling, facilitating further transcription. DNA breaks relieve supercoiling and induce Pol II arrest, allowing detection of DNA damage hidden in chromatin structure. PMID:27115204

Watching the action unfold: New cryo-EM images capture CRISPR’s interaction with target DNA | Center for Cancer Research

Cancer.gov

Using the Nobel-prize winning technique of cryo-EM, researchers led by CCR Senior Investigator Sriram Subramaniam, Ph.D., have captured a series of highly detailed images of a protein complex belonging to the CRISPR system that can be used by bacteria to recognize and destroy foreign DNA. The images reveal the molecule’s form before and after its interaction with DNA and help illuminate both how the complex functions and how it can be blocked. Read more... 

DNA transposon-based gene vehicles - scenes from an evolutionary drive

PubMed Central

2013-01-01

DNA transposons are primitive genetic elements which have colonized living organisms from plants to bacteria and mammals. Through evolution such parasitic elements have shaped their host genomes by replicating and relocating between chromosomal loci in processes catalyzed by the transposase proteins encoded by the elements themselves. DNA transposable elements are constantly adapting to life in the genome, and self-suppressive regulation as well as defensive host mechanisms may assist in buffering ‘cut-and-paste’ DNA mobilization until accumulating mutations will eventually restrict events of transposition. With the reconstructed Sleeping Beauty DNA transposon as a powerful engine, a growing list of transposable elements with activity in human cells have moved into biomedical experimentation and preclinical therapy as versatile vehicles for delivery and genomic insertion of transgenes. In this review, we aim to link the mechanisms that drive transposon evolution with the realities and potential challenges we are facing when adapting DNA transposons for gene transfer. We argue that DNA transposon-derived vectors may carry inherent, and potentially limiting, traits of their mother elements. By understanding in detail the evolutionary journey of transposons, from host colonization to element multiplication and inactivation, we may better exploit the potential of distinct transposable elements. Hence, parallel efforts to investigate and develop distinct, but potent, transposon-based vector systems will benefit the broad applications of gene transfer. Insight and clever optimization have shaped new DNA transposon vectors, which recently debuted in the first DNA transposon-based clinical trial. Learning from an evolutionary drive may help us create gene vehicles that are safer, more efficient, and less prone for suppression and inactivation. PMID:24320156

Intrinsic sequence specificity of the Cas1 integrase directs new spacer acquisition

PubMed Central

Rollie, Clare; Schneider, Stefanie; Brinkmann, Anna Sophie; Bolt, Edward L; White, Malcolm F

2015-01-01

The adaptive prokaryotic immune system CRISPR-Cas provides RNA-mediated protection from invading genetic elements. The fundamental basis of the system is the ability to capture small pieces of foreign DNA for incorporation into the genome at the CRISPR locus, a process known as Adaptation, which is dependent on the Cas1 and Cas2 proteins. We demonstrate that Cas1 catalyses an efficient trans-esterification reaction on branched DNA substrates, which represents the reverse- or disintegration reaction. Cas1 from both Escherichia coli and Sulfolobus solfataricus display sequence specific activity, with a clear preference for the nucleotides flanking the integration site at the leader-repeat 1 boundary of the CRISPR locus. Cas2 is not required for this activity and does not influence the specificity. This suggests that the inherent sequence specificity of Cas1 is a major determinant of the adaptation process. DOI: http://dx.doi.org/10.7554/eLife.08716.001 PMID:26284603

Brain is the predilection site of Toxoplasma gondii in experimentally inoculated pigs as revealed by magnetic capture and real-time PCR.

PubMed

Juránková, Jana; Basso, Walter; Neumayerová, Helena; Baláž, Vojtech; Jánová, Eva; Sidler, Xaver; Deplazes, Peter; Koudela, Břetislav

2014-04-01

Pigs represent an important source of food in many countries, and undercooked pork containing tissue cysts is one of the most common sources of Toxoplasma gondii infection for humans. A magnetic capture method for the isolation of T. gondii DNA and quantitative real-time PCR targeting the 529 bp TOXO repeat element were used to estimate the parasite burden in different tissues of pigs experimentally infected with T. gondii oocysts, and to determine the predilection sites of T. gondii in this host species. The highest concentration of T. gondii DNA was found in brain tissues, equivalent to [median] 553.7 (range 3857.7-121.9) parasites per gram, followed by lungs, heart and dorsal muscles with median values corresponding to 0.3 (range 61.3-0.02); 2.6 (range 7.34-0.37) and 0.6 (range 2.81-0.31) parasites per gram of tissue, respectively. Skeletal muscles from fore and hindlimb, liver and kidney presented very low infection burdens equivalent to [median] ≤0.2 parasites per gram of tissues, and no parasite DNA could be detected in the spleen. This study contributes to understanding the value of different pig tissues as a source of T. gondii infection for humans and shows that the brain, while not being of major importance as human food source, may represent a first-line selection tissue when performing non-serological surveys (e.g. bioassays, histopathological, immunohistochemical or molecular studies) to detect T. gondii infections in pigs. Copyright © 2013 Elsevier Ltd. All rights reserved.

High-throughput assays for DNA gyrase and other topoisomerases

PubMed Central

Maxwell, Anthony; Burton, Nicolas P.; O'Hagan, Natasha

2006-01-01

We have developed high-throughput microtitre plate-based assays for DNA gyrase and other DNA topoisomerases. These assays exploit the fact that negatively supercoiled plasmids form intermolecular triplexes more efficiently than when they are relaxed. Two assays are presented, one using capture of a plasmid containing a single triplex-forming sequence by an oligonucleotide tethered to the surface of a microtitre plate and subsequent detection by staining with a DNA-specific fluorescent dye. The other uses capture of a plasmid containing two triplex-forming sequences by an oligonucleotide tethered to the surface of a microtitre plate and subsequent detection by a second oligonucleotide that is radiolabelled. The assays are shown to be appropriate for assaying DNA supercoiling by Escherichia coli DNA gyrase and DNA relaxation by eukaryotic topoisomerases I and II, and E.coli topoisomerase IV. The assays are readily adaptable to other enzymes that change DNA supercoiling (e.g. restriction enzymes) and are suitable for use in a high-throughput format. PMID:16936317

High-throughput assays for DNA gyrase and other topoisomerases.

PubMed

Maxwell, Anthony; Burton, Nicolas P; O'Hagan, Natasha

2006-01-01

We have developed high-throughput microtitre plate-based assays for DNA gyrase and other DNA topoisomerases. These assays exploit the fact that negatively supercoiled plasmids form intermolecular triplexes more efficiently than when they are relaxed. Two assays are presented, one using capture of a plasmid containing a single triplex-forming sequence by an oligonucleotide tethered to the surface of a microtitre plate and subsequent detection by staining with a DNA-specific fluorescent dye. The other uses capture of a plasmid containing two triplex-forming sequences by an oligonucleotide tethered to the surface of a microtitre plate and subsequent detection by a second oligonucleotide that is radiolabelled. The assays are shown to be appropriate for assaying DNA supercoiling by Escherichia coli DNA gyrase and DNA relaxation by eukaryotic topoisomerases I and II, and E.coli topoisomerase IV. The assays are readily adaptable to other enzymes that change DNA supercoiling (e.g. restriction enzymes) and are suitable for use in a high-throughput format.

DNA motion capture reveals the mechanical properties of DNA at the mesoscale.

PubMed

Price, Allen C; Pilkiewicz, Kevin R; Graham, Thomas G W; Song, Dan; Eaves, Joel D; Loparo, Joseph J

2015-05-19

Single-molecule studies probing the end-to-end extension of long DNAs have established that the mechanical properties of DNA are well described by a wormlike chain force law, a polymer model where persistence length is the only adjustable parameter. We present a DNA motion-capture technique in which DNA molecules are labeled with fluorescent quantum dots at specific sites along the DNA contour and their positions are imaged. Tracking these positions in time allows us to characterize how segments within a long DNA are extended by flow and how fluctuations within the molecule are correlated. Utilizing a linear response theory of small fluctuations, we extract elastic forces for the different, ∼2-μm-long segments along the DNA backbone. We find that the average force-extension behavior of the segments can be well described by a wormlike chain force law with an anomalously small persistence length. Copyright © 2015 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Optimized mtDNA Control Region Primer Extension Capture Analysis for Forensically Relevant Samples and Highly Compromised mtDNA of Different Age and Origin

PubMed Central

Eduardoff, Mayra; Xavier, Catarina; Strobl, Christina; Casas-Vargas, Andrea; Parson, Walther

2017-01-01

The analysis of mitochondrial DNA (mtDNA) has proven useful in forensic genetics and ancient DNA (aDNA) studies, where specimens are often highly compromised and DNA quality and quantity are low. In forensic genetics, the mtDNA control region (CR) is commonly sequenced using established Sanger-type Sequencing (STS) protocols involving fragment sizes down to approximately 150 base pairs (bp). Recent developments include Massively Parallel Sequencing (MPS) of (multiplex) PCR-generated libraries using the same amplicon sizes. Molecular genetic studies on archaeological remains that harbor more degraded aDNA have pioneered alternative approaches to target mtDNA, such as capture hybridization and primer extension capture (PEC) methods followed by MPS. These assays target smaller mtDNA fragment sizes (down to 50 bp or less), and have proven to be substantially more successful in obtaining useful mtDNA sequences from these samples compared to electrophoretic methods. Here, we present the modification and optimization of a PEC method, earlier developed for sequencing the Neanderthal mitochondrial genome, with forensic applications in mind. Our approach was designed for a more sensitive enrichment of the mtDNA CR in a single tube assay and short laboratory turnaround times, thus complying with forensic practices. We characterized the method using sheared, high quantity mtDNA (six samples), and tested challenging forensic samples (n = 2) as well as compromised solid tissue samples (n = 15) up to 8 kyrs of age. The PEC MPS method produced reliable and plausible mtDNA haplotypes that were useful in the forensic context. It yielded plausible data in samples that did not provide results with STS and other MPS techniques. We addressed the issue of contamination by including four generations of negative controls, and discuss the results in the forensic context. We finally offer perspectives for future research to enable the validation and accreditation of the PEC MPS method for final implementation in forensic genetic laboratories. PMID:28934125

Targeted Capture and High-Throughput Sequencing Using Molecular Inversion Probes (MIPs).

PubMed

Cantsilieris, Stuart; Stessman, Holly A; Shendure, Jay; Eichler, Evan E

2017-01-01

Molecular inversion probes (MIPs) in combination with massively parallel DNA sequencing represent a versatile, yet economical tool for targeted sequencing of genomic DNA. Several thousand genomic targets can be selectively captured using long oligonucleotides containing unique targeting arms and universal linkers. The ability to append sequencing adaptors and sample-specific barcodes allows large-scale pooling and subsequent high-throughput sequencing at relatively low cost per sample. Here, we describe a "wet bench" protocol detailing the capture and subsequent sequencing of >2000 genomic targets from 192 samples, representative of a single lane on the Illumina HiSeq 2000 platform.

Target capture enrichment of nuclear SNP markers for massively parallel sequencing of degraded and mixed samples.

PubMed

Bose, Nikhil; Carlberg, Katie; Sensabaugh, George; Erlich, Henry; Calloway, Cassandra

2018-05-01

DNA from biological forensic samples can be highly fragmented and present in limited quantity. When DNA is highly fragmented, conventional PCR based Short Tandem Repeat (STR) analysis may fail as primer binding sites may not be present on a single template molecule. Single Nucleotide Polymorphisms (SNPs) can serve as an alternative type of genetic marker for analysis of degraded samples because the targeted variation is a single base. However, conventional PCR based SNP analysis methods still require intact primer binding sites for target amplification. Recently, probe capture methods for targeted enrichment have shown success in recovering degraded DNA as well as DNA from ancient bone samples using next-generation sequencing (NGS) technologies. The goal of this study was to design and test a probe capture assay targeting forensically relevant nuclear SNP markers for clonal and massively parallel sequencing (MPS) of degraded and limited DNA samples as well as mixtures. A set of 411 polymorphic markers totaling 451 nuclear SNPs (375 SNPs and 36 microhaplotype markers) was selected for the custom probe capture panel. The SNP markers were selected for a broad range of forensic applications including human individual identification, kinship, and lineage analysis as well as for mixture analysis. Performance of the custom SNP probe capture NGS assay was characterized by analyzing read depth and heterozygote allele balance across 15 samples at 25 ng input DNA. Performance thresholds were established based on read depth ≥500X and heterozygote allele balance within ±10% deviation from 50:50, which was observed for 426 out of 451 SNPs. These 426 SNPs were analyzed in size selected samples (at ≤75 bp, ≤100 bp, ≤150 bp, ≤200 bp, and ≤250 bp) as well as mock degraded samples fragmented to an average of 150 bp. Samples selected for ≤75 bp exhibited 99-100% reportable SNPs across varied DNA amounts and as low as 0.5 ng. Mock degraded samples at 1 ng and 10 ng exhibited >90% reportable SNPs. Finally, two-person male-male mixtures were tested at 10 ng in contributor varying ratios. Overall, 85-100% of alleles unique to the minor contributor were observed at all mixture ratios. Results from these studies using the SNP probe capture NGS system demonstrates proof of concept for application to forensically relevant degraded and mixed DNA samples. Copyright © 2018 Elsevier B.V. All rights reserved.

Capture and release of cells using a temperature-responsive surface that immobilizes an antibody through DNA duplex formation.

PubMed

Kimura, Tsuyoshi; Nakamura, Naoko; Umeda, Kanji; Hashimoto, Yoshihide; Kishida, Akio

We synthesized a temperature-responsive surface that immobilized an antibody via DNA duplex formation for selective capture and release of target cells. Polyethylene films were modified by grafting poly(N-isopropylacrylamide-co-acrylic acid) (P(NIPAAm-co-AAc)), which were prepared at various ratios of NIPAAm/AAc. The increased hydrophilicity of P(NIPAAm-co-PAA) film with decreased temperature was confirmed by water contact angle measurement. Single strand DNA (20mer) was chemically immobilized on the surface and then antibody (anti-mouse CD45, mCD45) modified with the complementary single strand DNA was immobilized on the surface through DNA duplex formation. The mCD45 antibody immobilization was confirmed by immunostaining. HeLa cells (mCD45 negative) and mouse bone marrow (BM) cells (mCD45 positive) were adhered on the surfaces at 37 °C. Although HeLa cells were detached by 4 °C incubation, BM cells were still adhered on the surface and then the adhered cells were released by DNase treatment. From these results, it was suggested that cells could be selectively captured and collected by using a film having surface that immobilizes an antibody via DNA duplex formation.

Modular structural elements in the replication origin region of Tetrahymena rDNA.

PubMed Central

Du, C; Sanzgiri, R P; Shaiu, W L; Choi, J K; Hou, Z; Benbow, R M; Dobbs, D L

1995-01-01

Computer analyses of the DNA replication origin region in the amplified rRNA genes of Tetrahymena thermophila identified a potential initiation zone in the 5'NTS [Dobbs, Shaiu and Benbow (1994), Nucleic Acids Res. 22, 2479-2489]. This region consists of a putative DNA unwinding element (DUE) aligned with predicted bent DNA segments, nuclear matrix or scaffold associated region (MAR/SAR) consensus sequences, and other common modular sequence elements previously shown to be clustered in eukaryotic chromosomal origin regions. In this study, two mung bean nuclease-hypersensitive sites in super-coiled plasmid DNA were localized within the major DUE-like element predicted by thermodynamic analyses. Three restriction fragments of the 5'NTS region predicted to contain bent DNA segments exhibited anomalous migration characteristic of bent DNA during electrophoresis on polyacrylamide gels. Restriction fragments containing the 5'NTS region bound Tetrahymena nuclear matrices in an in vitro binding assay, consistent with an association of the replication origin region with the nuclear matrix in vivo. The direct demonstration in a protozoan origin region of elements previously identified in Drosophila, chick and mammalian origin regions suggests that clusters of modular structural elements may be a conserved feature of eukaryotic chromosomal origins of replication. Images PMID:7784181

Comparison of the Second-Generation Digene Hybrid Capture Assay with the Branched-DNA Assay for Measurement of Hepatitis B Virus DNA in Serum

PubMed Central

Ho, Stephen K. N.; Chan, Tak Mao; Cheng, Ignatius K. P.; Lai, Kar Neng

1999-01-01

The optimal hepatitis B virus (HBV) DNA quantitative assay for clinical use remains to be determined. We examined the sensitivity, linearity, and variability of a novel second-generation antibody capture solution hybridization assay, the Digene Hybrid Capture II assay (HCII), and compared it with another widely used solution hybridization assay, the branched-DNA (bDNA) assay (Quantiplex; Chiron Corp.). Our results showed similar and satisfactory assay linearity values, as well as interassay and intra-assay variability values, for both HCII and bDNA assays across different ranges of HBV DNA. Ninety-one percent of 102 serum samples from hepatitis B surface antigen-positive patients showed concordant results with the two assays. The HCII assay was more sensitive than the bDNA assay by 1 dilution, with the lowest reading being 0.9 pg/ml (3.8 pg/ml by bDNA assay). The HBV DNA seropositivity rates for the 102 samples were 58, 67, and 97% by bDNA, HCII, and nested PCR, respectively. While the relationship between results obtained with the bDNA assay and those with the HCII assay was nonlinear, with the bDNA assay yielding values 2.83 ± 0.92-fold higher than those of the HCII assay, especially at high HBV DNA levels, a linear relationship was observed between the two sets of data after logarithmic conversion. The formula for interassay conversion of results was derived as follows: HBV DNA by HCII (picograms per milliliter) = 3.19 × [HBV DNA by bDNA (megaequivalents per milliliter)]0.866. The HCII assay was technically less complex and required a shorter assay time (4 h) than the bDNA assay (24 h). We conclude that the HCII assay compares favorably with the bDNA assay and offers the additional advantages of increased sensitivity and shorter assay time. The increased sensitivity should be particularly useful in monitoring the efficacy of antiviral therapies and detecting the emergence of drug-resistant HBV mutants. PMID:10405385

Comparison of the second-generation digene hybrid capture assay with the branched-DNA assay for measurement of hepatitis B virus DNA in serum.

PubMed

Ho, S K; Chan, T M; Cheng, I K; Lai, K N

1999-08-01

The optimal hepatitis B virus (HBV) DNA quantitative assay for clinical use remains to be determined. We examined the sensitivity, linearity, and variability of a novel second-generation antibody capture solution hybridization assay, the Digene Hybrid Capture II assay (HCII), and compared it with another widely used solution hybridization assay, the branched-DNA (bDNA) assay (Quantiplex; Chiron Corp.). Our results showed similar and satisfactory assay linearity values, as well as interassay and intra-assay variability values, for both HCII and bDNA assays across different ranges of HBV DNA. Ninety-one percent of 102 serum samples from hepatitis B surface antigen-positive patients showed concordant results with the two assays. The HCII assay was more sensitive than the bDNA assay by 1 dilution, with the lowest reading being 0.9 pg/ml (3.8 pg/ml by bDNA assay). The HBV DNA seropositivity rates for the 102 samples were 58, 67, and 97% by bDNA, HCII, and nested PCR, respectively. While the relationship between results obtained with the bDNA assay and those with the HCII assay was nonlinear, with the bDNA assay yielding values 2.83 +/- 0.92-fold higher than those of the HCII assay, especially at high HBV DNA levels, a linear relationship was observed between the two sets of data after logarithmic conversion. The formula for interassay conversion of results was derived as follows: HBV DNA by HCII (picograms per milliliter) = 3.19 x [HBV DNA by bDNA (megaequivalents per milliliter)](0.866). The HCII assay was technically less complex and required a shorter assay time (4 h) than the bDNA assay (24 h). We conclude that the HCII assay compares favorably with the bDNA assay and offers the additional advantages of increased sensitivity and shorter assay time. The increased sensitivity should be particularly useful in monitoring the efficacy of antiviral therapies and detecting the emergence of drug-resistant HBV mutants.

Exploring the read-write genome: mobile DNA and mammalian adaptation.

PubMed

Shapiro, James A

2017-02-01

The read-write genome idea predicts that mobile DNA elements will act in evolution to generate adaptive changes in organismal DNA. This prediction was examined in the context of mammalian adaptations involving regulatory non-coding RNAs, viviparous reproduction, early embryonic and stem cell development, the nervous system, and innate immunity. The evidence shows that mobile elements have played specific and sometimes major roles in mammalian adaptive evolution by generating regulatory sites in the DNA and providing interaction motifs in non-coding RNA. Endogenous retroviruses and retrotransposons have been the predominant mobile elements in mammalian adaptive evolution, with the notable exception of bats, where DNA transposons are the major agents of RW genome inscriptions. A few examples of independent but convergent exaptation of mobile DNA elements for similar regulatory rewiring functions are noted.

A novel multi-functional magnetic Fe-Ti-V spinel catalyst for elemental mercury capture and callback from flue gas.

PubMed

Yang, Shijian; Guo, Yongfu; Yan, Naiqiang; Wu, Daqing; He, Hongping; Xie, Jiangkun; Qu, Zan; Yang, Chen; Jia, Jinping

2010-11-28

A novel magnetic Fe-Ti-V spinel catalyst showed an excellent performance for elemental mercury capture at 100 °C, and the formed HgO can be catalytically decomposed by the catalyst at 300 °C to reclaim elemental mercury and regenerate the catalyst.

A Surrogate Approach to Study the Evolution of Noncoding DNA Elements That Organize Eukaryotic Genomes

PubMed Central

Vermaak, Danielle; Bayes, Joshua J.

2009-01-01

Comparative genomics provides a facile way to address issues of evolutionary constraint acting on different elements of the genome. However, several important DNA elements have not reaped the benefits of this new approach. Some have proved intractable to current day sequencing technology. These include centromeric and heterochromatic DNA, which are essential for chromosome segregation as well as gene regulation, but the highly repetitive nature of the DNA sequences in these regions make them difficult to assemble into longer contigs. Other sequences, like dosage compensation X chromosomal sites, origins of DNA replication, or heterochromatic sequences that encode piwi-associated RNAs, have proved difficult to study because they do not have recognizable DNA features that allow them to be described functionally or computationally. We have employed an alternate approach to the direct study of these DNA elements. By using proteins that specifically bind these noncoding DNAs as surrogates, we can indirectly assay the evolutionary constraints acting on these important DNA elements. We review the impact that such “surrogate strategies” have had on our understanding of the evolutionary constraints shaping centromeres, origins of DNA replication, and dosage compensation X chromosomal sites. These have begun to reveal that in contrast to the view that such structural DNA elements are either highly constrained (under purifying selection) or free to drift (under neutral evolution), some of them may instead be shaped by adaptive evolution and genetic conflicts (these are not mutually exclusive). These insights also help to explain why the same elements (e.g., centromeres and replication origins), which are so complex in some eukaryotic genomes, can be simple and well defined in other where similar conflicts do not exist. PMID:19635763

Construction of hexahedral finite element mesh capturing realistic geometries of a petroleum reserve

DOE PAGES

Park, Byoung Yoon; Roberts, Barry L.; Sobolik, Steven R.

2017-07-27

The three-dimensional finite element mesh capturing realistic geometries of the Bayou Choctaw site has been constructed using the sonar and seismic survey data obtained from the field. The mesh consists of hexahedral elements because the salt constitutive model is coded using hexahedral elements. Various ideas and techniques to construct finite element mesh capturing artificially and naturally formed geometries are provided. The techniques to reduce the number of elements as much as possible to save on computer run time while maintaining the computational accuracy is also introduced. The steps and methodologies could be applied to construct the meshes of Big Hill,more » Bryan Mound, and West Hackberry strategic petroleum reserve sites. The methodology could be applied to the complicated shape masses for various civil and geological structures.« less

Construction of hexahedral finite element mesh capturing realistic geometries of a petroleum reserve

DOE Office of Scientific and Technical Information (OSTI.GOV)

Park, Byoung Yoon; Roberts, Barry L.; Sobolik, Steven R.

The three-dimensional finite element mesh capturing realistic geometries of the Bayou Choctaw site has been constructed using the sonar and seismic survey data obtained from the field. The mesh consists of hexahedral elements because the salt constitutive model is coded using hexahedral elements. Various ideas and techniques to construct finite element mesh capturing artificially and naturally formed geometries are provided. The techniques to reduce the number of elements as much as possible to save on computer run time while maintaining the computational accuracy is also introduced. The steps and methodologies could be applied to construct the meshes of Big Hill,more » Bryan Mound, and West Hackberry strategic petroleum reserve sites. The methodology could be applied to the complicated shape masses for various civil and geological structures.« less

Validation of an entirely in vitro approach for rapid prototyping of DNA regulatory elements for synthetic biology

PubMed Central

Chappell, James; Jensen, Kirsten; Freemont, Paul S.

2013-01-01

A bottleneck in our capacity to rationally and predictably engineer biological systems is the limited number of well-characterized genetic elements from which to build. Current characterization methods are tied to measurements in living systems, the transformation and culturing of which are inherently time-consuming. To address this, we have validated a completely in vitro approach for the characterization of DNA regulatory elements using Escherichia coli extract cell-free systems. Importantly, we demonstrate that characterization in cell-free systems correlates and is reflective of performance in vivo for the most frequently used DNA regulatory elements. Moreover, we devise a rapid and completely in vitro method to generate DNA templates for cell-free systems, bypassing the need for DNA template generation and amplification from living cells. This in vitro approach is significantly quicker than current characterization methods and is amenable to high-throughput techniques, providing a valuable tool for rapidly prototyping libraries of DNA regulatory elements for synthetic biology. PMID:23371936

Evidence for conformational capture mechanism for damage recognition by NER protein XPC/Rad4.

NASA Astrophysics Data System (ADS)

Chakraborty, Sagnik; Steinbach, Peter J.; Paul, Debamita; Min, Jung-Hyun; Ansari, Anjum

Altered flexibility of damaged DNA sites is considered to play an important role in damage recognition by DNA repair proteins. Characterizing lesion-induced DNA dynamics has remained a challenge. We have combined ps-resolved fluorescence lifetime measurements with cytosine analog FRET pair uniquely sensitive to local unwinding/twisting to analyze DNA conformational distributions. This innovative approach maps out with unprecedented sensitivity the alternative conformations accessible to a series of DNA constructs containing 3-base-pair mismatch, suitable model lesions for the DNA repair protein xeroderma pigmentosum C (XPC) complex. XPC initiates eukaryotic nucleotide excision repair by recognizing various DNA lesions primarily through DNA deformability. Structural studies show that Rad4 (yeast ortholog of XPC) unwinds DNA at the lesion site and flips out two nucleotide pairs. Our results elucidate a broad range of conformations accessible to mismatched DNA even in the absence of the protein. Notably, the most severely distorted conformations share remarkable resemblance to the deformed conformation seen in the crystal structure of the Rad4-bound ``recognition'' complex supporting for the first time a possible ``conformational capture'' mechanism for damage recognition by XPC/Rad4. NSF Univ of Illinois-Chicago.

Neutron-capture Nucleosynthesis in the First Stars

NASA Astrophysics Data System (ADS)

Roederer, Ian U.; Preston, George W.; Thompson, Ian B.; Shectman, Stephen A.; Sneden, Christopher

2014-04-01

Recent studies suggest that metal-poor stars enhanced in carbon but containing low levels of neutron-capture elements may have been among the first to incorporate the nucleosynthesis products of the first generation of stars. We have observed 16 stars with enhanced carbon or nitrogen using the MIKE Spectrograph on the Magellan Telescopes at Las Campanas Observatory and the Tull Spectrograph on the Smith Telescope at McDonald Observatory. We present radial velocities, stellar parameters, and detailed abundance patterns for these stars. Strontium, yttrium, zirconium, barium, europium, ytterbium, and other heavy elements are detected. In four stars, these heavy elements appear to have originated in some form of r-process nucleosynthesis. In one star, a partial s-process origin is possible. The origin of the heavy elements in the rest of the sample cannot be determined unambiguously. The presence of elements heavier than the iron group offers further evidence that zero-metallicity rapidly rotating massive stars and pair instability supernovae did not contribute substantial amounts of neutron-capture elements to the regions where the stars in our sample formed. If the carbon- or nitrogen-enhanced metal-poor stars with low levels of neutron-capture elements were enriched by products of zero-metallicity supernovae only, then the presence of these heavy elements indicates that at least one form of neutron-capture reaction operated in some of the first stars. This paper includes data gathered with the 6.5 m Magellan Telescopes located at Las Campanas Observatory, Chile, and The McDonald Observatory of The University of Texas at Austin.

BayMeth: improved DNA methylation quantification for affinity capture sequencing data using a flexible Bayesian approach

PubMed Central

2014-01-01

Affinity capture of DNA methylation combined with high-throughput sequencing strikes a good balance between the high cost of whole genome bisulfite sequencing and the low coverage of methylation arrays. We present BayMeth, an empirical Bayes approach that uses a fully methylated control sample to transform observed read counts into regional methylation levels. In our model, inefficient capture can readily be distinguished from low methylation levels. BayMeth improves on existing methods, allows explicit modeling of copy number variation, and offers computationally efficient analytical mean and variance estimators. BayMeth is available in the Repitools Bioconductor package. PMID:24517713

Destruction of a distal hypoxia response element abolishes trans-activation of the PAG1 gene mediated by HIF-independent chromatin looping

PubMed Central

Schörg, Alexandra; Santambrogio, Sara; Platt, James L.; Schödel, Johannes; Lindenmeyer, Maja T.; Cohen, Clemens D.; Schrödter, Katrin; Mole, David R.; Wenger, Roland H.; Hoogewijs, David

2015-01-01

A crucial step in the cellular adaptation to oxygen deficiency is the binding of hypoxia-inducible factors (HIFs) to hypoxia response elements (HREs) of oxygen-regulated genes. Genome-wide HIF-1α/2α/β DNA-binding studies revealed that the majority of HREs reside distant to the promoter regions, but the function of these distal HREs has only been marginally studied in the genomic context. We used chromatin immunoprecipitation (ChIP), gene editing (TALEN) and chromosome conformation capture (3C) to localize and functionally characterize a 82 kb upstream HRE that solely drives oxygen-regulated expression of the newly identified HIF target gene PAG1. PAG1, a transmembrane adaptor protein involved in Src signalling, was hypoxically induced in various cell lines and mouse tissues. ChIP and reporter gene assays demonstrated that the −82 kb HRE regulates PAG1, but not an equally distant gene further upstream, by direct interaction with HIF. Ablation of the consensus HRE motif abolished the hypoxic induction of PAG1 but not general oxygen signalling. 3C assays revealed that the −82 kb HRE physically associates with the PAG1 promoter region, independent of HIF-DNA interaction. These results demonstrate a constitutive interaction between the −82 kb HRE and the PAG1 promoter, suggesting a physiologically important rapid response to hypoxia. PMID:26007655

Adaptive Shape Functions and Internal Mesh Adaptation for Modelling Progressive Failure in Adhesively Bonded Joints

NASA Technical Reports Server (NTRS)

Stapleton, Scott; Gries, Thomas; Waas, Anthony M.; Pineda, Evan J.

2014-01-01

Enhanced finite elements are elements with an embedded analytical solution that can capture detailed local fields, enabling more efficient, mesh independent finite element analysis. The shape functions are determined based on the analytical model rather than prescribed. This method was applied to adhesively bonded joints to model joint behavior with one element through the thickness. This study demonstrates two methods of maintaining the fidelity of such elements during adhesive non-linearity and cracking without increasing the mesh needed for an accurate solution. The first method uses adaptive shape functions, where the shape functions are recalculated at each load step based on the softening of the adhesive. The second method is internal mesh adaption, where cracking of the adhesive within an element is captured by further discretizing the element internally to represent the partially cracked geometry. By keeping mesh adaptations within an element, a finer mesh can be used during the analysis without affecting the global finite element model mesh. Examples are shown which highlight when each method is most effective in reducing the number of elements needed to capture adhesive nonlinearity and cracking. These methods are validated against analogous finite element models utilizing cohesive zone elements.

Repair of DNA damage caused by cytosine deamination in mitochondrial DNA of forensic case samples.

PubMed

Gorden, Erin M; Sturk-Andreaggi, Kimberly; Marshall, Charla

2018-05-01

DNA sequence damage from cytosine deamination is well documented in degraded samples, such as those from ancient and forensic contexts. This study examined the effect of a DNA repair treatment on mitochondrial DNA (mtDNA) from aged and degraded skeletal samples. DNA extracts from 21 non-probative, degraded skeletal samples (aged 50-70 years) were utilized for the analysis. A portion of each sample extract was subjected to DNA repair using a commercial repair kit, the New England BioLabs' NEBNext FFPE DNA Repair Kit (Ipswich, MA). MtDNA was enriched using PCR and targeted capture in a side-by-side experiment of untreated and repaired DNA. Sequencing was performed using both traditional (Sanger-type; STS) and next-generation sequencing (NGS) methods Although cytosine deamination was evident in the mtDNA sequence data, the observed level of damaged bases varied by sequencing method as well as by enrichment type. The STS PCR amplicon data did not show evidence of cytosine deamination that could be distinguished from background signal in either the untreated or repaired sample set. However, the same PCR amplicons showed 850 C → T/G → A substitutions consistent with cytosine deamination with variant frequencies (VFs) of up to 25% when sequenced using NGS methods The occurrence of base misincorporation due to cytosine deamination was reduced by 98% (to 10) in the NGS amplicon data after repair. The NGS capture data indicated low levels (1-2%) of cytosine deamination in mtDNA fragments that was effectively mitigated by DNA repair. The observed difference in the level of cytosine deamination between the PCR and capture enrichment methods can be attributed to the greater propensity for stochastic effects from the PCR enrichment technique employed (e.g., low template input, increased PCR cycles). Altogether these results indicate that DNA repair may be required when sequencing PCR-amplified DNA from degraded forensic case samples with NGS methods. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

The site-specific ribosomal insertion element type II of Bombyx mori (R2Bm) contains the coding sequence for a reverse transcriptase-like enzyme.

PubMed Central

Burke, W D; Calalang, C C; Eickbush, T H

1987-01-01

Two classes of DNA elements interrupt a fraction of the rRNA repeats of Bombyx mori. We have analyzed by genomic blotting and sequence analysis one class of these elements which we have named R2. These elements occupy approximately 9% of the rDNA units of B. mori and appear to be homologous to the type II rDNA insertions detected in Drosophila melanogaster. Approximately 25 copies of R2 exist within the B. mori genome, of which at least 20 are located at a precise location within otherwise typical rDNA units. Nucleotide sequence analysis has revealed that the 4.2-kilobase-pair R2 element has a single large open reading frame, occupying over 82% of the total length of the element. The central region of this 1,151-amino-acid open reading frame shows homology to the reverse transcriptase enzymes found in retroviruses and certain transposable elements. Amino acid homology of this region is highest to the mobile line 1 elements of mammals, followed by the mitochondrial type II introns of fungi, and the pol gene of retroviruses. Less homology exists with transposable elements of D. melanogaster and Saccharomyces cerevisiae. Two additional regions of sequence homology between L1 and R2 elements were also found outside the reverse transcriptase region. We suggest that the R2 elements are retrotransposons that are site specific in their insertion into the genome. Such mobility would enable these elements to occupy a small fraction of the rDNA units of B. mori despite their continual elimination from the rDNA locus by sequence turnover. Images PMID:2439905

Perceptual organization, visual attention, and objecthood.

PubMed

Kimchi, Ruth; Yeshurun, Yaffa; Spehar, Branka; Pirkner, Yossef

2016-09-01

We have previously demonstrated that the mere organization of some elements in the visual field into an object attracts attention automatically. Here, we explored three different aspects of this automatic attentional capture: (a) Does the attentional capture by an object involve a spatial component? (b) Which Gestalt organization factors suffice for an object to capture attention? (c) Does the strength of organization affect the object's ability to capture attention? Participants viewed multi-elements displays and either identified the color of one element or responded to a Vernier target. On some trials, a subset of the elements grouped by Gestalt factors into an object that was irrelevant to the task and not predictive of the target. An object effect - faster performance for targets within the object than for targets outside the object - was found even when the target appeared after the object offset, and was sensitive to target-object distance, suggesting that the capture of attention by an object is accompanied by a deployment of attention to the object location. Object effects of similar magnitude were found for objects grouped by a combination of factors (collinearity, closure, and symmetry, or closure and symmetry) or by a single factor when it was collinearity, but not symmetry, suggesting that collinearity, or closure combined with symmetry, suffices for automatic capture of attention by an object, but symmetry does not. Finally, the strength of grouping in modal completion, manipulated by varying contrast polarity between and within elements, affected the effectiveness of the attentional capture by the induced object. Copyright © 2015 Elsevier Ltd. All rights reserved.

Multiple data sources improve DNA-based mark-recapture population estimates of grizzly bears.

PubMed

Boulanger, John; Kendall, Katherine C; Stetz, Jeffrey B; Roon, David A; Waits, Lisette P; Paetkau, David

2008-04-01

A fundamental challenge to estimating population size with mark-recapture methods is heterogeneous capture probabilities and subsequent bias of population estimates. Confronting this problem usually requires substantial sampling effort that can be difficult to achieve for some species, such as carnivores. We developed a methodology that uses two data sources to deal with heterogeneity and applied this to DNA mark-recapture data from grizzly bears (Ursus arctos). We improved population estimates by incorporating additional DNA "captures" of grizzly bears obtained by collecting hair from unbaited bear rub trees concurrently with baited, grid-based, hair snag sampling. We consider a Lincoln-Petersen estimator with hair snag captures as the initial session and rub tree captures as the recapture session and develop an estimator in program MARK that treats hair snag and rub tree samples as successive sessions. Using empirical data from a large-scale project in the greater Glacier National Park, Montana, USA, area and simulation modeling we evaluate these methods and compare the results to hair-snag-only estimates. Empirical results indicate that, compared with hair-snag-only data, the joint hair-snag-rub-tree methods produce similar but more precise estimates if capture and recapture rates are reasonably high for both methods. Simulation results suggest that estimators are potentially affected by correlation of capture probabilities between sample types in the presence of heterogeneity. Overall, closed population Huggins-Pledger estimators showed the highest precision and were most robust to sparse data, heterogeneity, and capture probability correlation among sampling types. Results also indicate that these estimators can be used when a segment of the population has zero capture probability for one of the methods. We propose that this general methodology may be useful for other species in which mark-recapture data are available from multiple sources.

Mitochondrial genetic diversity, selection and recombination in a canine transmissible cancer

PubMed Central

Strakova, Andrea; Ní Leathlobhair, Máire; Wang, Guo-Dong; Yin, Ting-Ting; Airikkala-Otter, Ilona; Allen, Janice L; Allum, Karen M; Bansse-Issa, Leontine; Bisson, Jocelyn L; Castillo Domracheva, Artemio; de Castro, Karina F; Corrigan, Anne M; Cran, Hugh R; Crawford, Jane T; Cutter, Stephen M; Delgadillo Keenan, Laura; Donelan, Edward M; Faramade, Ibikunle A; Flores Reynoso, Erika; Fotopoulou, Eleni; Fruean, Skye N; Gallardo-Arrieta, Fanny; Glebova, Olga; Häfelin Manrique, Rodrigo F; Henriques, Joaquim JGP; Ignatenko, Natalia; Koenig, Debbie; Lanza-Perea, Marta; Lobetti, Remo; Lopez Quintana, Adriana M; Losfelt, Thibault; Marino, Gabriele; Martincorena, Inigo; Martínez Castañeda, Simón; Martínez-López, Mayra F; Meyer, Michael; Nakanwagi, Berna; De Nardi, Andrigo B; Neunzig, Winifred; Nixon, Sally J; Onsare, Marsden M; Ortega-Pacheco, Antonio; Peleteiro, Maria C; Pye, Ruth J; Reece, John F; Rojas Gutierrez, Jose; Sadia, Haleema; Schmeling, Sheila K; Shamanova, Olga; Ssuna, Richard K; Steenland-Smit, Audrey E; Svitich, Alla; Thoya Ngoka, Ismail; Vițălaru, Bogdan A; de Vos, Anna P; de Vos, Johan P; Walkinton, Oliver; Wedge, David C; Wehrle-Martinez, Alvaro S; van der Wel, Mirjam G; Widdowson, Sophie AE; Murchison, Elizabeth P

2016-01-01

Canine transmissible venereal tumour (CTVT) is a clonally transmissible cancer that originated approximately 11,000 years ago and affects dogs worldwide. Despite the clonal origin of the CTVT nuclear genome, CTVT mitochondrial genomes (mtDNAs) have been acquired by periodic capture from transient hosts. We sequenced 449 complete mtDNAs from a global population of CTVTs, and show that mtDNA horizontal transfer has occurred at least five times, delineating five tumour clades whose distributions track two millennia of dog global migration. Negative selection has operated to prevent accumulation of deleterious mutations in captured mtDNA, and recombination has caused occasional mtDNA re-assortment. These findings implicate functional mtDNA as a driver of CTVT global metastatic spread, further highlighting the important role of mtDNA in cancer evolution. DOI: http://dx.doi.org/10.7554/eLife.14552.001 PMID:27185408

Concerted formation of macromolecular Suppressor–mutator transposition complexes

PubMed Central

Raina, Ramesh; Schläppi, Michael; Karunanandaa, Balasulojini; Elhofy, Adam; Fedoroff, Nina

1998-01-01

Transposition of the maize Suppressor–mutator (Spm) transposon requires two element-encoded proteins, TnpA and TnpD. Although there are multiple TnpA binding sites near each element end, binding of TnpA to DNA is not cooperative, and the binding affinity is not markedly affected by the number of binding sites per DNA fragment. However, intermolecular complexes form cooperatively between DNA fragments with three or more TnpA binding sites. TnpD, itself not a sequence-specific DNA-binding protein, binds to TnpA and stabilizes the TnpA–DNA complex. The high redundancy of TnpA binding sites at both element ends and the protein–protein interactions between DNA-bound TnpA complexes and between these and TnpD imply a concerted transition of the element from a linear to a protein crosslinked transposition complex within a very narrow protein concentration range. PMID:9671711

Useful DNA polymorphisms are identified by snapback, a midrepetitive element in Tribolium castaneum.

PubMed

Stuart, J J; De Gortari, M J; Hall, P S; Maxwell, M E; Mocelin, G; Brown, S J; Muir, W M

1996-06-01

The red flour bettle, Tribolium castaneum, is both a pest of stored grain products and an important experimental organism. To improve its facility as a genetic model, we are developing DNA fingerprinting methods for this insect. A Tribolium DNA fragment, snapback-1 (SBI), identified among sequences that reassociate before a Cot of 0.03 mol.s/L, was found to produce a banding pattern in restriction endonuclease digested genomic DNA that is characteristic of a midrepetitive element. DNA fingerprints of individual beetles demonstrated that unvarying inherited DNA polymorphism is revealed, and that polymorphism is inherited in a dominant Mendelian fashion. Linkage between bands was minimal. The sequence of SBI was determined, and hybridization experiments indicated that SBI is a fragment of a larger midrepetitive element. Fingerprinting individuals with known inbreeding coefficients indicated that SBI loci have relatively high mutation rates. The possibility that SBI is a fragment of a transposable element is discussed.

Genesis of the heaviest elements in the Milky Way Galaxy.

PubMed

Sneden, Christopher; Cowan, John J

2003-01-03

We review the origin and evolution of the heavy elements, those with atomic numbers greater than 30, in the early history of the Milky Way. There is a large star-to-star bulk scatter in the concentrations of heavy elements with respect to the lighter metals, which suggests an early chemically unmixed and inhomogeneous Galaxy. The relative abundance patterns among the heavy elements are often very different from the solar system mix, revealing the characteristics of the first element donors in the Galaxy. Abundance comparisons among several halo stars show that the heaviest neutron-capture elements (including barium and heavier) are consistent with a scaled solar system rapid neutron-capture abundance distribution, whereas the lighter such elements do not conform to the solar pattern. The stellar abundances indicate an increasing contribution from the slow neutron-capture process (s-process) at higher metallicities in the Galaxy. The detection of thorium in halo and globular cluster stars offers a promising, independent age-dating technique that can put lower limits on the age of the Galaxy.

Highly multiplexed targeted DNA sequencing from single nuclei.

PubMed

Leung, Marco L; Wang, Yong; Kim, Charissa; Gao, Ruli; Jiang, Jerry; Sei, Emi; Navin, Nicholas E

2016-02-01

Single-cell DNA sequencing methods are challenged by poor physical coverage, high technical error rates and low throughput. To address these issues, we developed a single-cell DNA sequencing protocol that combines flow-sorting of single nuclei, time-limited multiple-displacement amplification (MDA), low-input library preparation, DNA barcoding, targeted capture and next-generation sequencing (NGS). This approach represents a major improvement over our previous single nucleus sequencing (SNS) Nature Protocols paper in terms of generating higher-coverage data (>90%), thereby enabling the detection of genome-wide variants in single mammalian cells at base-pair resolution. Furthermore, by pooling 48-96 single-cell libraries together for targeted capture, this approach can be used to sequence many single-cell libraries in parallel in a single reaction. This protocol greatly reduces the cost of single-cell DNA sequencing, and it can be completed in 5-6 d by advanced users. This single-cell DNA sequencing protocol has broad applications for studying rare cells and complex populations in diverse fields of biological research and medicine.

Cell Type-Specific Chromatin Signatures Underline Regulatory DNA Elements in Human Induced Pluripotent Stem Cells and Somatic Cells.

PubMed

Zhao, Ming-Tao; Shao, Ning-Yi; Hu, Shijun; Ma, Ning; Srinivasan, Rajini; Jahanbani, Fereshteh; Lee, Jaecheol; Zhang, Sophia L; Snyder, Michael P; Wu, Joseph C

2017-11-10

Regulatory DNA elements in the human genome play important roles in determining the transcriptional abundance and spatiotemporal gene expression during embryonic heart development and somatic cell reprogramming. It is not well known how chromatin marks in regulatory DNA elements are modulated to establish cell type-specific gene expression in the human heart. We aimed to decipher the cell type-specific epigenetic signatures in regulatory DNA elements and how they modulate heart-specific gene expression. We profiled genome-wide transcriptional activity and a variety of epigenetic marks in the regulatory DNA elements using massive RNA-seq (n=12) and ChIP-seq (chromatin immunoprecipitation combined with high-throughput sequencing; n=84) in human endothelial cells (CD31 + CD144 + ), cardiac progenitor cells (Sca-1 + ), fibroblasts (DDR2 + ), and their respective induced pluripotent stem cells. We uncovered 2 classes of regulatory DNA elements: class I was identified with ubiquitous enhancer (H3K4me1) and promoter (H3K4me3) marks in all cell types, whereas class II was enriched with H3K4me1 and H3K4me3 in a cell type-specific manner. Both class I and class II regulatory elements exhibited stimulatory roles in nearby gene expression in a given cell type. However, class I promoters displayed more dominant regulatory effects on transcriptional abundance regardless of distal enhancers. Transcription factor network analysis indicated that human induced pluripotent stem cells and somatic cells from the heart selected their preferential regulatory elements to maintain cell type-specific gene expression. In addition, we validated the function of these enhancer elements in transgenic mouse embryos and human cells and identified a few enhancers that could possibly regulate the cardiac-specific gene expression. Given that a large number of genetic variants associated with human diseases are located in regulatory DNA elements, our study provides valuable resources for deciphering the epigenetic modulation of regulatory DNA elements that fine-tune spatiotemporal gene expression in human cardiac development and diseases. © 2017 American Heart Association, Inc.

A novel technology for the detection, enrichment, and separation of trace amounts of target DNA based on amino-modified fluorescent magnetic composite nanoparticles.

PubMed

Wang, Guannan; Su, Xingguang

2010-06-01

A novel, highly sensitive technology for the detection, enrichment, and separation of trace amounts of target DNA was developed on the basis of amino-modified fluorescent magnetic composite nanoparticles (AFMN). In this study, the positively charged amino-modified composite nanoparticles conjugate with the negatively charged capture DNA through electrostatic binding. The optimal combination of AFMN and capture DNA was measured by dynamic light scattering (DLS) and UV-vis absorption spectroscopy. The highly sensitive detection of trace amounts of target DNA was achieved through enrichment by means of AFMN. The detection limit for target DNA is 0.4 pM, which could be further improved by using a more powerful magnet. Because of their different melting temperatures, single-base mismatched target DNA could be separated from perfectly complementary target DNA. In addition, the photoluminescence (PL) signals of perfectly complementary target DNA and single-base mismatched DNA as well as the hybridization kinetics of different concentrations of target DNA at different reaction times have also been studied. Most importantly, the detection, enrichment, and separation ability of AFMN was further verified with milk. Simple and satisfactory results were obtained, which show the great potential in the fields of mutation identification and clinical diagnosis.

Sequence-specific sepsis-related DNA capture and fluorescent labeling in monoliths prepared by single-step photopolymerization in microfluidic devices.

PubMed

Knob, Radim; Hanson, Robert L; Tateoka, Olivia B; Wood, Ryan L; Guerrero-Arguero, Israel; Robison, Richard A; Pitt, William G; Woolley, Adam T

2018-05-21

Fast determination of antibiotic resistance is crucial in selecting appropriate treatment for sepsis patients, but current methods based on culture are time consuming. We are developing a microfluidic platform with a monolithic column modified with oligonucleotides designed for sequence-specific capture of target DNA related to the Klebsiella pneumoniae carbapenemase (KPC) gene. We developed a novel single-step monolith fabrication method with an acrydite-modified capture oligonucleotide in the polymerization mixture, enabling fast monolith preparation in a microfluidic channel using UV photopolymerization. These prepared columns had a threefold higher capacity compared to monoliths prepared in a multistep process involving Schiff-base DNA attachment. Conditions for denaturing, capture and fluorescence labeling using hybridization probes were optimized with synthetic 90-mer oligonucleotides. These procedures were applied for extraction of a PCR amplicon from the KPC antibiotic resistance gene in bacterial lysate obtained from a blood sample spiked with E. coli. The results showed similar eluted peak areas for KPC amplicon extracted from either hybridization buffer or bacterial lysate. Selective extraction of the KPC DNA was verified by real time PCR on eluted fractions. These results show great promise for application in an integrated microfluidic diagnostic system that combines upstream blood sample preparation and downstream single-molecule counting detection. Copyright © 2018 Elsevier B.V. All rights reserved.

Synthesis of amino-rich silica-coated magnetic nanoparticles for the efficient capture of DNA for PCR.

PubMed

Bai, Yalong; Cui, Yan; Paoli, George C; Shi, Chunlei; Wang, Dapeng; Zhou, Min; Zhang, Lida; Shi, Xianming

2016-09-01

Magnetic separation has great advantages over traditional bio-separation methods and has become popular in the development of methods for the detection of bacterial pathogens, viruses, and transgenic crops. Functionalization of magnetic nanoparticles is a key factor for efficient capture of the target analytes. In this paper, we report the synthesis of amino-rich silica-coated magnetic nanoparticles using a one-pot method. This type of magnetic nanoparticle has a rough surface and a higher density of amino groups than the nanoparticles prepared by a post-modification method. Furthermore, the results of hydrochloric acid treatment indicated that the magnetic nanoparticles were stably coated. The developed amino-rich silica-coated magnetic nanoparticles were used to directly adsorb DNA. After magnetic separation and blocking, the magnetic nanoparticles and DNA complexes were used directly for the polymerase chain reaction (PCR), without onerous and time-consuming purification and elution steps. The results of real-time quantitative PCR showed that the nanoparticles with higher amino group density resulted in improved DNA capture efficiency. The results suggest that amino-rich silica-coated magnetic nanoparticles are of great potential for efficient bio-separation of DNA prior to detection by PCR. Copyright © 2016. Published by Elsevier B.V.

Comparison of Hybrid Capture 2 Assay with Real-time-PCR for Detection and Quantitation of Hepatitis B Virus DNA.

PubMed

Majid, Farjana; Jahan, Munira; Lutful Moben, Ahmed; Tabassum, Shahina

2014-01-01

Both real-time-polymerase chain reaction (PCR) and hybrid capture 2 (HC2) assay can detect and quantify hepatitis B virus (HBV) DNA. However, real-time-PCR can detect a wide range of HBV DNA, while HC2 assay could not detect lower levels of viremia. The present study was designed to detect and quantify HBV DNA by real-time-PCR and HC2 assay and compare the quantitative data of these two assays. A cross-sectional study was conducted in between July 2010 and June 2011. A total of 66 serologically diagnosed chronic hepatitis B (CHB) patients were selected for the study. Real-time-PCR and HC2 assay was done to detect HBV DNA. Data were analyzed by statistical Package for the social sciences (SPSS). Among 66 serologically diagnosed chronic hepatitis B patients 40 (60.61%) patients had detectable and 26 (39.39%) had undetectable HBV DNA by HC2 assay. Concordant results were obtained for 40 (60.61%) out of these 66 patients by real-time-PCR and HC2 assay with mean viral load of 7.06 ± 1.13 log 10 copies/ml and 6.95 ± 1.08 log 10 copies/ml, respectively. In the remaining 26 patients, HBV DNA was detectable by real-time-PCR in 20 patients (mean HBV DNA level was 3.67 ± 0.72 log 10 copies/ml. However, HBV DNA could not be detectable in six cases by the both assays. The study showed strong correlation (r = 0.915) between real-time-PCR and HC2 assay for the detection and quantification of HBV DNA. HC2 assay may be used as an alternative to real-time-PCR for CHB patients. How to cite this article: Majid F, Jahan M, Moben AL, Tabassum S. Comparison of Hybrid Capture 2 Assay with Real-time-PCR for Detection and Quantitation of Hepatitis B Virus DNA. Euroasian J Hepato-Gastroenterol 2014;4(1):31-35.

Detecting single-abasic residues within a DNA strand immobilized in a biological nanopore using an integrated CMOS sensor.

PubMed

Kim, Jungsuk; Maitra, Raj D; Pedrotti, Ken; Dunbar, William B

2013-02-01

In this paper, we demonstrate the application of a novel current-measuring sensor (CMS) customized for nanopore applications. The low-noise CMS is fabricated in a 0.35μm CMOS process and is implemented in experiments involving DNA captured in an α-hemolysin (α-HL) nanopore. Specifically, the CMS is used to build a current amplitude map as a function of varying positions of a single-abasic residue within a homopolymer cytosine single-stranded DNA (ssDNA) that is captured and held in the pore. Each ssDNA is immobilized using a biotin-streptavidin linkage. Five different DNA templates are measured and compared: one all-cytosine ssDNA, and four with a single-abasic residue substitution that resides in or near the ~1.5nm aperture of the α-HL channel when the strand is immobilized. The CMOS CMS is shown to resolves the ~5Å displacements of the abasic residue within the varying templates. The demonstration represents an advance in application-specific circuitry that is optimized for small-footprint nanopore applications, including genomic sequencing.

New insights on Ba overabundance in open clusters. Evidence for the intermediate neutron-capture process at play?

NASA Astrophysics Data System (ADS)

Mishenina, T.; Pignatari, M.; Carraro, G.; Kovtyukh, V.; Monaco, L.; Korotin, S.; Shereta, E.; Yegorova, I.; Herwig, F.

2015-02-01

Recently, an increasing number of studies were devoted to measure the abundances of neutron-capture elements heavier than iron in stars belonging to Galactic Open Clusters (OCs). OCs span a sizeable range in metallicity (-0.6 ≤ [Fe/H] ≤ +0.4), and they show abundances of light elements similar to disc stars of the same age. A different pattern is observed for heavy elements. A large scatter is observed for Ba, with most OCs showing [Ba/Fe] and [Ba/La] overabundant with respect to the Sun. The origin of this overabundance is not clearly understood. With the goal of providing new observational insights, we determined radial velocities, atmospheric parameters and chemical composition of 27 giant stars members of five OCs: Cr 110, Cr 261, NGC 2477, NGC 2506 and NGC 5822. We used high-resolution spectra obtained with the UVES spectrograph at European Southern Observatory Paranal. We perform a detailed spectroscopic analysis of these stars to measure the abundance of up to 22 elements per star. We study the dependence of element abundance on metallicity and age with unprecedented detail, complementing our analysis with data culled from the literature. We confirm the trend of Ba overabundance in OCs, and show its large dispersion for clusters younger than ˜4 Gyr. Finally, the implications of our results for stellar nucleosynthesis are discussed. We show in this work that the Ba enrichment compared to other neutron-capture elements in OCs cannot be explained by the contributions from the slow neutron-capture process and the rapid neutron-capture process. Instead, we argue that this anomalous signature can be explained by assuming an additional contribution by the intermediate neutron-capture process.

Rat L (long interspersed repeated DNA) elements contain guanine-rich homopurine sequences that induce unpairing of contiguous duplex DNA.

PubMed Central

Usdin, K; Furano, A V

1988-01-01

The L family (long interspersed repeated DNA) of mobile genetic elements is a persistent feature of the mammalian genome. In rats, this family contains approximately equal to 40,000 members and accounts for approximately equal to 10% of the haploid genome. We demonstrate here that the guanine-rich homopurine stretches located at the right end of L-DNA induce oligonucleotide uptake by contiguous duplex DNA. The uptake is dependent on negative supercoiling and the length of the homopurine stretch and occurs even when the L-DNA homopurine stretches are introduced into a different DNA environment. The bound oligomer primes DNA synthesis when DNA polymerase and deoxyribonucleoside triphosphates are added, resulting in a faithful copy of the template to which the oligonucleotide had bound. The implications of this property of the L-DNA guanine-rich homopurine stretches in the amplification, recombination, and dispersal of L elements is discussed. Images PMID:2837766

Crystallization and preliminary X-ray diffraction analysis of the CRISPR-Cas RNA-silencing Cmr complex.

PubMed

Osawa, Takuo; Inanaga, Hideko; Numata, Tomoyuki

2015-06-01

Clustered regularly interspaced short palindromic repeat (CRISPR)-derived RNA (crRNA) and CRISPR-associated (Cas) proteins constitute a prokaryotic adaptive immune system (CRISPR-Cas system) that targets and degrades invading genetic elements. The type III-B CRISPR-Cas Cmr complex, composed of the six Cas proteins (Cmr1-Cmr6) and a crRNA, captures and cleaves RNA complementary to the crRNA guide sequence. Here, a Cmr1-deficient functional Cmr (CmrΔ1) complex composed of Pyrococcus furiosus Cmr2-Cmr3, Archaeoglobus fulgidus Cmr4-Cmr5-Cmr6 and the 39-mer P. furiosus 7.01-crRNA was prepared. The CmrΔ1 complex was cocrystallized with single-stranded DNA (ssDNA) complementary to the crRNA guide by the vapour-diffusion method. The crystals diffracted to 2.1 Å resolution using synchrotron radiation at the Photon Factory. The crystals belonged to the triclinic space group P1, with unit-cell parameters a = 75.5, b = 76.2, c = 139.2 Å, α = 90.3, β = 104.8, γ = 118.6°. The asymmetric unit of the crystals is expected to contain one CmrΔ1-ssDNA complex, with a Matthews coefficient of 2.03 Å(3) Da(-1) and a solvent content of 39.5%.

Simultaneous capture and in situ analysis of circulating tumor cells using multiple hybrid nanoparticles.

PubMed

Lee, Hun Joo; Cho, Hyeon-Yeol; Oh, Jin Ho; Namkoong, Kak; Lee, Jeong Gun; Park, Jong-Myeon; Lee, Soo Suk; Huh, Nam; Choi, Jeong-Woo

2013-09-15

Using hybrid nanoparticles (HNPs), we demonstrate simultaneous capture, in situ protein expression analysis, and cellular phenotype identification of circulating tumor cells (CTCs). Each HNP consists of three parts: (i) antibodies that bind specifically to a known biomarker for CTCs, (ii) a quantum dot that emits fluorescence signals, and (iii) biotinylated DNA that allows capture and release of CTC-HNP complex to an in-house developed capture & recovery chip (CRC). To evaluate our approach, cells representative of different breast cancer subtypes (MCF-7: luminal; SK-BR-3: HER2; and MDA-MB-231: basal-like) were captured onto CRC and expressions of EpCAM, HER2, and EGFR were detected concurrently. The average capture efficiency of CTCs was 87.5% with identification accuracy of 92.4%. Subsequently, by cleaving the DNA portion with restriction enzymes, captured cells were released at efficiencies of 86.1%. Further studies showed that these recovered cells are viable and can proliferate in vitro. Using HNPs, it is possible to count, analyze in situ protein expression, and culture CTCs, all from the same set of cells, enabling a wide range of molecular- and cellular-based studies using CTCs. Copyright © 2013 Elsevier B.V. All rights reserved.

Microfluidic devices with permeable polymer barriers for capture and transport of biomolecules and cells.

PubMed

Lee, Ho Suk; Chu, Wai Keung; Zhang, Kun; Huang, Xiaohua

2013-09-07

We report a method for fabricating permeable polymer microstructure barriers in polydimethylsiloxane (PDMS) microfluidic devices and the use of the devices to capture and transport DNA and cells. The polymer microstructure in a desired location in a fluidic channel is formed in situ by the polymerization of acrylamide and polyethylene diacrylate cross-linker (PEG-DA) monomer in a solution which is trapped in the location using a pair of PDMS valves. The porous polymer microstructure provides a mechanical barrier to convective fluid flow in the channel or between two microfluidic chambers while it still conducts ions or small charged species under an electric field, allowing for the rapid capture and transport of biomolecules and cells by electrophoresis. We have demonstrated the application of the devices for the rapid capture and efficient release of bacteriophage λ genomic DNA, solution exchange and for the transport and capture of HeLa cells. Our devices will enable the multi-step processing of biomolecules and cells or individual cells within a single microfluidic chamber.

Origin of the main r-process elements

NASA Astrophysics Data System (ADS)

Otsuki, K.; Truran, J.; Wiescher, M.; Gorres, J.; Mathews, G.; Frekers, D.; Mengoni, A.; Bartlett, A.; Tostevin, J.

2006-07-01

The r-process is supposed to be a primary process which assembles heavy nuclei from a photo-dissociated nucleon gas. Hence, the reaction flow through light elements can be important as a constraint on the conditions for the r-process. We have studied the impact of di-neutron capture and the neutron-capture of light (Z<10) elements on r-process nucleosynthesis in three different environments: neutrino-driven winds in Type II supernovae; the prompt explosion of low mass supernovae; and neutron star mergers. Although the effect of di-neutron capture is not significant for the neutrino-driven wind model or low-mass supernovae, it becomes significant in the neutron-star merger model. The neutron-capture of light elements, which has been studied extensively for neutrino-driven wind models, also impacts the other two models. We show that it may be possible to identify the astrophysical site for the main r-process if the nuclear physics uncertainties in current r-process calculations could be reduced.

r-process enhanched metal-poor stars

NASA Astrophysics Data System (ADS)

Cowan, John; Sneden, Christopher; Lawler, James E.; Den Hartog, Elizabeth A.

Abundance observations indicate the presence of rapid-neutron capture (i.e., r-process) elements in old Galactic halo and globular cluster stars. These observations provide insight into the nature of the earliest generations of stars in the Galaxy - the progenitors of the halo stars - responsible for neutron-capture synthesis of the heavy elements. The large star-to-star scatter observed in the abundances of neutron-capture element/iron ratios at low metallicities - which diminishes with in- creasing metallicity or [Fe/H] - suggests the formation of these heavy elements (presumably from certain types of supernovae) was rare in the early Galaxy. The stellar abundances also indicate a change from the r-process to the slow neutron capture (i.e., s-) process at higher metallicities in the Galaxy and provide insight into Galactic chemical evolution. Finally, the detection of thorium and uranium in halo and globular cluster stars offers an independent age-dating technique that can put lower limits on the age of the Galaxy, and hence the Universe.

Nucleosynthesis: Stellar and Solar Abundances and Atomic Data

NASA Technical Reports Server (NTRS)

Cowan, John J.; Lawler, James E.; Sneden, Christopher; DenHartog, E. A.; Collier, Jason; Dodge, Homer L.

2006-01-01

Abundance observations indicate the presence of often surprisingly large amounts of neutron capture (i.e., s- and r-process) elements in old Galactic halo and globular cluster stars. These observations provide insight into the nature of the earliest generations of stars in the Galaxy the progenitors of the halo stars responsible for neutron-capture synthesis. Comparisons of abundance trends can be used to understand the chemical evolution of the Galaxy and the nature of heavy element nucleosynthesis. In addition age determinations, based upon long-lived radioactive nuclei abundances, can now be obtained. These stellar abundance determinations depend critically upon atomic data. Improved laboratory transition probabilities have been recently obtained for a number of elements. These new gf values have been used to greatly refine the abundances of neutron-capture elemental abundances in the solar photosphere and in very metal-poor Galactic halo stars. The newly determined stellar abundances are surprisingly consistent with a (relative) Solar System r-process pattern, and are also consistent with abundance predictions expected from such neutron-capture nucleosynthesis.

Multi-Ligand-Binding Flavoprotein Dodecin as a Key Element for Reversible Surface Modification in Nano-biotechnology.

PubMed

Gutiérrez Sánchez, Cristina; Su, Qiang; Schönherr, Holger; Grininger, Martin; Nöll, Gilbert

2015-01-01

In this paper the multiple (re)programming of protein-DNA nanostructures comprising generation, deletion, and reprogramming on the same flavin-DNA-modified surface is introduced. This work is based on a systematic study of the binding affinity of the multi-ligand-binding flavoprotein dodecin on flavin-terminated DNA monolayers by surface plasmon resonance and quartz crystal microbalance with dissipation (QCM-D) measurements, surface plasmon fluorescence spectroscopy (SPFS), and dynamic AFM force spectroscopy. Depending on the flavin surface coverage, a single apododecin is captured by one or more surface-immobilized flavins. The corresponding complex binding and unbinding rate constants kon(QCM) = 7.7 × 10(3) M(-1)·s(-1) and koff(QCM) = 4.5 × 10(-3) s(-1) (Kd(QCM) = 580 nM) were determined by QCM and were found to be in agreement with values for koff determined by SPFS and force spectroscopy. Even though a single apododecin-flavin bond is relatively weak, stable dodecin monolayers were formed on flavin-DNA-modified surfaces at high flavin surface coverage due to multivalent interactions between apododecin bearing six binding pockets and the surface-bound flavin-DNA ligands. If bi- or multivalent flavin ligands are adsorbed on dodecin monolayers, stable sandwich-type surface-DNA-flavin-apododecin-flavin ligand arrays are obtained. Nevertheless, the apododecin flavin complex is easily and quantitatively disassembled by flavin reduction. Binding and release of apododecin are reversible processes, which can be carried out alternatingly several times to release one type of ligand by an external redox trigger and subsequently replace it with a different ligand. Hence the versatile concept of reprogrammable functional biointerfaces with the multi-ligand-binding flavoprotein dodecin is demonstrated.

A rapid and visual aptasensor for Lipopolysaccharides detection based on the bulb-like triplex turn-on switch coupled with HCR-HRP nanostructures.

PubMed

Xu, Wentao; Tian, Jingjing; Shao, Xiangli; Zhu, Longjiao; Huang, Kunlun; Luo, Yunbo

2017-03-15

For previously reported aptasensor, the sensitivity and selectivity of aptamers to targets were often suppressed due to the reporter label of single-stranded molecular beacon or hindrance of the duplex DNA strand displacement. To solve the affinity declining of aptamers showed in traditional way and realize on-site rapid detection of Lipopolysaccharides (LPS), we developed an ingenious structure-switching aptasensor based on the bulb-like triplex turn-on switch (BTTS) as the effective molecular recognition and signal transduction element and streptavidin-horseradish peroxidase modified hybridization chain reaction (HCR-HRP) nanocomposites as the signal amplifier and signal report element. In the presence of LPS, the bulb-like LPS-aptamer (BLA) and LPS formed the LPS/aptamer complex, while the BTTS disassembled and liberated the dissociative bridge probes (BP) to achieve molecular recognition and signal transduction. Immobilized BP, captured by immobilized capture probes (CP), triggered hybridization chain reactions (HCR) to amplify the switching signal, and the HCR products were then modified with streptavidin-horseradish peroxidase (SA-HRP) to form HCR-HRP nanostructures to output colorimetric signals. In less than four hours, the proposed biosensor showed a detection limit of 50pg/mL of LPS quantitatively with the portable spectrophotometer and the observation limit of 20ng/mL semi-quantitatively with the naked eye, opening up new opportunities for LPS detection in future clinical diagnosis, food security and environment monitoring. Copyright © 2016 Elsevier B.V. All rights reserved.

Transformation of a transposon into a derived prolactin promoter with function during human pregnancy

PubMed Central

Emera, Deena; Wagner, Günter P.

2012-01-01

Transposable elements (TEs) are known to provide DNA for host regulatory functions, but the mechanisms underlying the transformation of TEs into cis-regulatory elements are unclear. In humans two TEs—MER20 and MER39—contribute the enhancer/promoter for decidual prolactin (dPRL), which is dramatically induced during pregnancy. We show that evolution of the strong human dPRL promoter was a multistep process that took millions of years. First, MER39 inserted near MER20 in the primate/rodent ancestor, and then there were two phases of activity enhancement in primates. Through the mapping of causal nucleotide substitutions, we demonstrate that strong promoter activity in apes involves epistasis between transcription factor binding sites (TFBSs) ancestral to MER39 and derived sites. We propose a mode of molecular evolution that describes the process by which MER20/MER39 was transformed into a strong promoter, called “epistatic capture.” Epistatic capture is the stabilization of a TFBS that is ancestral but variable in outgroup lineages, and is fixed in the ingroup because of epistatic interactions with derived TFBSs. Finally, we note that evolution of human promoter activity coincides with the emergence of a unique reproductive character in apes, highly invasive placentation. Because prolactin communicates with immune cells during pregnancy, which regulate fetal invasion into maternal tissues, we speculate that ape dPRL promoter activity evolved in response to increased invasiveness of ape fetal tissue. PMID:22733751

A force-based protein biochip

NASA Astrophysics Data System (ADS)

Blank, K.; Mai, T.; Gilbert, I.; Schiffmann, S.; Rankl, J.; Zivin, R.; Tackney, C.; Nicolaus, T.; Spinnler, K.; Oesterhelt, F.; Benoit, M.; Clausen-Schaumann, H.; Gaub, H. E.

2003-09-01

A parallel assay for the quantification of single-molecule binding forces was developed based on differential unbinding force measurements where ligand-receptor interactions are compared with the unzipping forces of DNA hybrids. Using the DNA zippers as molecular force sensors, the efficient discrimination between specific and nonspecific interactions was demonstrated for small molecules binding to specific receptors, as well as for protein-protein interactions on protein arrays. Finally, an antibody sandwich assay with different capture antibodies on one chip surface and with the detection antibodies linked to a congruent surface via the DNA zippers was used to capture and quantify a recombinant hepatitis C antigen from solution. In this case, the DNA zippers enable not only discrimination between specific and nonspecific binding, but also allow for the local application of detection antibodies, thereby eliminating false-positive results caused by cross-reactive antibodies and nonspecific binding.

A signal-on electrochemical aptasensor for ultrasensitive detection of endotoxin using three-way DNA junction-aided enzymatic recycling and graphene nanohybrid for amplification

NASA Astrophysics Data System (ADS)

Bai, Lijuan; Chai, Yaqin; Pu, Xiaoyun; Yuan, Ruo

2014-02-01

Endotoxin, also known as lipopolysaccharide (LPS), is able to induce a strong immune response on its internalization into mammalian cells. To date, aptamer-based biosensors for LPS detection have been rarely reported. This work describes a new signal-on electrochemical aptasensor for the ultrasensitive detection of LPS by combining the three-way DNA hybridization process and nanotechnology-based amplification. With the help of DNA1 (associated with the concentration of target LPS), the capture probe hybridizes with DNA1 and the assistant probe to open its hairpin structure and form a ternary ``Y'' junction structure. The DNA1 can be released from the structure in the presence of nicking endonuclease to initiate the next hybridization process. Then a great deal of cleaved capture probe produced in the cyclic process can bind with DNA2-nanocomposite, which contains the electroactive toluidine blue (Tb) with the amplification materials graphene (Gra) and gold nanoparticles (AuNPs). Thus, an enhanced electrochemical signal can be easily read out. With the cascade signal amplification, this newly designed protocol provides an ultrasensitive electrochemical detection of LPS down to the femtogram level (8.7 fg mL-1) with a linear range of 6 orders of magnitude (from 10 fg mL-1 to 50 ng mL-1). Moreover, the high sensitivity and specificity make this method versatile for the detection of other biomolecules by changing the corresponding sequences of the capture probe and the assistant probe.

Computational Analysis of Enhanced Magnetic Bioseparation in Microfluidic Systems with Flow-Invasive Magnetic Elements

PubMed Central

Khashan, S. A.; Alazzam, A.; Furlani, E. P.

2014-01-01

A microfluidic design is proposed for realizing greatly enhanced separation of magnetically-labeled bioparticles using integrated soft-magnetic elements. The elements are fixed and intersect the carrier fluid (flow-invasive) with their length transverse to the flow. They are magnetized using a bias field to produce a particle capture force. Multiple stair-step elements are used to provide efficient capture throughout the entire flow channel. This is in contrast to conventional systems wherein the elements are integrated into the walls of the channel, which restricts efficient capture to limited regions of the channel due to the short range nature of the magnetic force. This severely limits the channel size and hence throughput. Flow-invasive elements overcome this limitation and enable microfluidic bioseparation systems with superior scalability. This enhanced functionality is quantified for the first time using a computational model that accounts for the dominant mechanisms of particle transport including fully-coupled particle-fluid momentum transfer. PMID:24931437

Immobilization of DNA aptamers via plasma polymerized allylamine film to construct an endothelial progenitor cell-capture surface.

PubMed

Qi, Pengkai; Yan, Wei; Yang, Ying; Li, Yalong; Fan, Yi; Chen, Junying; Yang, Zhilu; Tu, Qiufen; Huang, Nan

2015-02-01

The endothelial progenitor cells (EPCs) capture stent has drawn increasing attentions and become one of the most promising concepts for the next generation vascular stent. In this regard, it is of great significance to immobilize a molecule with the ability to bind EPC for rapid in vivo endothelialization with high specificity. In this work, a facile two-step method aimed at constructing a coating with specific EPC capturing aptamers is reported. The processes involves as the first-step deposition of plasma polymerized allylamine (PPAam) on a substrate to introduce amine groups, followed by the electrostatic adsorption of a 34 bases single strand DNA sequence to the PPAam surface as a second step (PPAam-DNA). Grazing incidence attenuated total reflection Fourier transform infrared spectroscopy (GATR-FTIR) and X-ray photoelectron spectroscopy (XPS) confirmed the successful immobilization of the aptamers. Quartz crystal microbalance with dissipation (QCM-D) real time monitoring result shows that about 175 ng/cm(2) aptamers were conjugated onto the PPAam surface. The interactions between the modified surfaces and human umbilical vein endothelial cells (ECs), smooth muscle cells (SMCs), and murine induced EPCs derived from mesenchymal stem cells (MSCs) were also investigated. It was demonstrated that PPAam-DNA samples could capture more EPCs, and present a cellular friendly surface for the proliferation of both EPCs and ECs but no effect on the hyperplasia of SMCs. Also, the co-culture results of 3 types of cells confirmed that the aptamer could specifically bond EPCs rather than ECs and SMCs, suggesting the competitive adhesion advantage of EPCs to ECs and SMCs. These data demonstrate that the EPC aptamer has large potential for designing an EPC captured stent and other vascular grafts with targeted in situ endothelialization. Copyright © 2014 Elsevier B.V. All rights reserved.

Analysis of warping deformation modes using higher order ANCF beam element

NASA Astrophysics Data System (ADS)

Orzechowski, Grzegorz; Shabana, Ahmed A.

2016-02-01

Most classical beam theories assume that the beam cross section remains a rigid surface under an arbitrary loading condition. However, in the absolute nodal coordinate formulation (ANCF) continuum-based beams, this assumption can be relaxed allowing for capturing deformation modes that couple the cross-section deformation and beam bending, torsion, and/or elongation. The deformation modes captured by ANCF finite elements depend on the interpolating polynomials used. The most widely used spatial ANCF beam element employs linear approximation in the transverse direction, thereby restricting the cross section deformation and leading to locking problems. The objective of this investigation is to examine the behavior of a higher order ANCF beam element that includes quadratic interpolation in the transverse directions. This higher order element allows capturing warping and non-uniform stretching distribution. Furthermore, this higher order element allows for increasing the degree of continuity at the element interface. It is shown in this paper that the higher order ANCF beam element can be used effectively to capture warping and eliminate Poisson locking that characterizes lower order ANCF finite elements. It is also shown that increasing the degree of continuity requires a special attention in order to have acceptable results. Because higher order elements can be more computationally expensive than the lower order elements, the use of reduced integration for evaluating the stress forces and the use of explicit and implicit numerical integrations to solve the nonlinear dynamic equations of motion are investigated in this paper. It is shown that the use of some of these integration methods can be very effective in reducing the CPU time without adversely affecting the solution accuracy.

High-Throughput Analysis of T-DNA Location and Structure Using Sequence Capture.

PubMed

Inagaki, Soichi; Henry, Isabelle M; Lieberman, Meric C; Comai, Luca

2015-01-01

Agrobacterium-mediated transformation of plants with T-DNA is used both to introduce transgenes and for mutagenesis. Conventional approaches used to identify the genomic location and the structure of the inserted T-DNA are laborious and high-throughput methods using next-generation sequencing are being developed to address these problems. Here, we present a cost-effective approach that uses sequence capture targeted to the T-DNA borders to select genomic DNA fragments containing T-DNA-genome junctions, followed by Illumina sequencing to determine the location and junction structure of T-DNA insertions. Multiple probes can be mixed so that transgenic lines transformed with different T-DNA types can be processed simultaneously, using a simple, index-based pooling approach. We also developed a simple bioinformatic tool to find sequence read pairs that span the junction between the genome and T-DNA or any foreign DNA. We analyzed 29 transgenic lines of Arabidopsis thaliana, each containing inserts from 4 different T-DNA vectors. We determined the location of T-DNA insertions in 22 lines, 4 of which carried multiple insertion sites. Additionally, our analysis uncovered a high frequency of unconventional and complex T-DNA insertions, highlighting the needs for high-throughput methods for T-DNA localization and structural characterization. Transgene insertion events have to be fully characterized prior to use as commercial products. Our method greatly facilitates the first step of this characterization of transgenic plants by providing an efficient screen for the selection of promising lines.

CZAEM USER'S GUIDE: MODELING CAPTURE ZONES OF GROUND-WATER WELLS USING ANALYTIC ELEMENTS

EPA Science Inventory

The computer program CZAEM is designed for elementary capture zone analysis, and is based on the analytic element method. CZAEM is applicable to confined and/or unconfined low in shallow aquifers; the Dupuit-Forchheimer assumption is adopted. CZAEM supports the following analyt...

Transcription factor trapping by RNA in gene regulatory elements.

PubMed

Sigova, Alla A; Abraham, Brian J; Ji, Xiong; Molinie, Benoit; Hannett, Nancy M; Guo, Yang Eric; Jangi, Mohini; Giallourakis, Cosmas C; Sharp, Phillip A; Young, Richard A

2015-11-20

Transcription factors (TFs) bind specific sequences in promoter-proximal and -distal DNA elements to regulate gene transcription. RNA is transcribed from both of these DNA elements, and some DNA binding TFs bind RNA. Hence, RNA transcribed from regulatory elements may contribute to stable TF occupancy at these sites. We show that the ubiquitously expressed TF Yin-Yang 1 (YY1) binds to both gene regulatory elements and their associated RNA species across the entire genome. Reduced transcription of regulatory elements diminishes YY1 occupancy, whereas artificial tethering of RNA enhances YY1 occupancy at these elements. We propose that RNA makes a modest but important contribution to the maintenance of certain TFs at gene regulatory elements and suggest that transcription of regulatory elements produces a positive-feedback loop that contributes to the stability of gene expression programs. Copyright © 2015, American Association for the Advancement of Science.

Macacine Herpesvirus 1 in Long-Tailed Macaques, Malaysia, 2009–2011

PubMed Central

Lee, Mei-Ho; Rostal, Melinda K.; Hughes, Tom; Sitam, Frankie; Lee, Chee-Yen; Japning, Jeffrine; Harden, Mallory E.; Griffiths, Anthony; Basir, Misliah; Wolfe, Nathan D.; Daszak, Peter

2015-01-01

Macacine herpesvirus 1 (MaHV1; B virus) naturally infects macaques (Macaca spp.) and can cause fatal encephalitis in humans. In Peninsular Malaysia, wild macaques are abundant, and translocation is used to mitigate human–macaque conflict. Most adult macaques are infected with MaHV1, although the risk for transmission to persons who handle them during capture and translocation is unknown. We investigated MaHV1 shedding among 392 long-tailed macaques (M. fascicularis) after capture and translocation by the Department of Wildlife and National Parks in Peninsular Malaysia, during 2009–2011. For detection of MaHV1 DNA, PCR was performed on urogenital and oropharyngeal swab samples. Overall, 39% of macaques were shedding MaHV1 DNA; rates of DNA detection did not differ between sample types. This study demonstrates that MaHV1 was shed by a substantial proportion of macaques after capture and transport and suggests that persons handling macaques under these circumstances might be at risk for exposure to MaHV1. PMID:26080081

A Method for Selecting Structure-switching Aptamers Applied to a Colorimetric Gold Nanoparticle Assay

PubMed Central

Martin, Jennifer A.; Smith, Joshua E.; Warren, Mercedes; Chávez, Jorge L.; Hagen, Joshua A.; Kelley-Loughnane, Nancy

2015-01-01

Small molecules provide rich targets for biosensing applications due to their physiological implications as biomarkers of various aspects of human health and performance. Nucleic acid aptamers have been increasingly applied as recognition elements on biosensor platforms, but selecting aptamers toward small molecule targets requires special design considerations. This work describes modification and critical steps of a method designed to select structure-switching aptamers to small molecule targets. Binding sequences from a DNA library hybridized to complementary DNA capture probes on magnetic beads are separated from nonbinders via a target-induced change in conformation. This method is advantageous because sequences binding the support matrix (beads) will not be further amplified, and it does not require immobilization of the target molecule. However, the melting temperature of the capture probe and library is kept at or slightly above RT, such that sequences that dehybridize based on thermodynamics will also be present in the supernatant solution. This effectively limits the partitioning efficiency (ability to separate target binding sequences from nonbinders), and therefore many selection rounds will be required to remove background sequences. The reported method differs from previous structure-switching aptamer selections due to implementation of negative selection steps, simplified enrichment monitoring, and extension of the length of the capture probe following selection enrichment to provide enhanced stringency. The selected structure-switching aptamers are advantageous in a gold nanoparticle assay platform that reports the presence of a target molecule by the conformational change of the aptamer. The gold nanoparticle assay was applied because it provides a simple, rapid colorimetric readout that is beneficial in a clinical or deployed environment. Design and optimization considerations are presented for the assay as proof-of-principle work in buffer to provide a foundation for further extension of the work toward small molecule biosensing in physiological fluids. PMID:25870978

Genomic patterns associated with paternal/maternal distribution of transposable elements

NASA Astrophysics Data System (ADS)

Jurka, Jerzy

2003-03-01

Transposable elements (TEs) are specialized DNA or RNA fragments capable of surviving in intragenomic niches. They are commonly, perhaps unjustifiably referred to as "selfish" or "parasitic" elements. TEs can be divided in two major classes: retroelements and DNA transposons. The former include non-LTR retrotransposons and retrovirus-like elements, using reverse transriptase for their reproduction prior to integration into host DNA. The latter depend mostly on host DNA replication, with possible exception of rolling-circle transposons recently discovered by our team. I will review basic information on TEs, with emphasis on human Alu and L1 retroelements discussed in the context of genomic organization. TEs are non-randomly distributed in chromosomal DNA. In particular, human Alu elements tend to prefer GC-rich regions, whereas L1 accumulate in AT-rich regions. Current explanations of this phenomenon focus on the so called "target effects" and post-insertional selection. However, the proposed models appear to be unsatisfactory and alternative explanations invoking "channeling" to different chromosomal regions will be a major focus of my presentation. Transposable elements (TEs) can be expressed and integrated into host DNA in the male or female germlines, or both. Different models of expression and integration imply different proportions of TEs on sex chromosomes and autosomes. The density of recently retroposed human Alu elements is around three times higher on chromosome Y than on chromosome X, and over two times higher than the average density for all human autosomes. This implies Alu activity in paternal germlines. Analogous inter-chromosomal proportions for other repeat families should determine their compatibility with one of the three basic models describing the inheritance of TEs. Published evidence indicates that maternally and paternally imprinted genes roughly correspond to GC-rich and AT-rich DNA. This may explain the observed chromosomal distribution of Alu and L1 elements. Finally, paternal models of inheritance predict rapid accumulation of active TEs on chromosome Y. I will discuss potential implications of this phenomenon for evolution of chromosome Y and transposable elements.

Modern alchemy: Fred Hoyle and element building by neutron capture

NASA Astrophysics Data System (ADS)

Burbidge, E. Margaret

Fred Hoyle's fundamental work on building the chemical elements by nuclear processes in stars at various stages in their lives began with the building of elements around iron in the very dense hot interiors of stars. Later, in the paper by Burbidge, Burbidge, Fowler and Hoyle, we four showed that Hoyle's "equilibrium process" is one of eight processes required to make all of the isotopes of all the elements detected in the Sun and stars. Neutron capture reactions, which Fred had not considered in his epochal 1946 paper, but for which experimental data were just becoming available in 1957, are very important, in addition to the energy-generating reactions involving hydrogen, helium, carbon, nitrogen and oxygen, for building all of the elements. They are now providing clues to the late stages of stellar evolution and the earliest history of our Galaxy. I describe here our earliest observational work on neutron capture processes in evolved stars, some new work on stars showing the results of the neutron capture reactions, and data relating to processes ending in the production of lead, and I discuss where this fits into the history of stars in our own Galaxy.

ELEMENTAL MERCURY CAPTURE BY ACTIVATED CARBON IN A FLOW REACTOR

EPA Science Inventory

The paper gives results of bench-scale experiments in a flow reactor to simulate the entrained-flow capture of elemental mercury (Hgo) using solid sorbents. Adsorption of Hgo by a lignite-based activated carbon (Calgon FGD) was examined at different carbon/mercury (C/Hg) rat...

IN-FLIGHT CAPTURE OF ELEMENTAL MERCURY BY A CHLORINE-IMPREGNATED ACTIVATED CARBON

EPA Science Inventory

The paper discusses the in-flight capture of elemental mercury (Hgo) by a chlorine (C1)-impregnated activated carbon. Efforts to develop sorbents for the control of Hg emissions have demonstrated that C1-impregnation of virgin activated carbons using dilute solutions of hydrogen ...

Effects of sampling conditions on DNA-based estimates of American black bear abundance

USGS Publications Warehouse

Laufenberg, Jared S.; Van Manen, Frank T.; Clark, Joseph D.

2013-01-01

DNA-based capture-mark-recapture techniques are commonly used to estimate American black bear (Ursus americanus) population abundance (N). Although the technique is well established, many questions remain regarding study design. In particular, relationships among N, capture probability of heterogeneity mixtures A and B (pA and pB, respectively, or p, collectively), the proportion of each mixture (π), number of capture occasions (k), and probability of obtaining reliable estimates of N are not fully understood. We investigated these relationships using 1) an empirical dataset of DNA samples for which true N was unknown and 2) simulated datasets with known properties that represented a broader array of sampling conditions. For the empirical data analysis, we used the full closed population with heterogeneity data type in Program MARK to estimate N for a black bear population in Great Smoky Mountains National Park, Tennessee. We systematically reduced the number of those samples used in the analysis to evaluate the effect that changes in capture probabilities may have on parameter estimates. Model-averaged N for females and males were 161 (95% CI = 114–272) and 100 (95% CI = 74–167), respectively (pooled N = 261, 95% CI = 192–419), and the average weekly p was 0.09 for females and 0.12 for males. When we reduced the number of samples of the empirical data, support for heterogeneity models decreased. For the simulation analysis, we generated capture data with individual heterogeneity covering a range of sampling conditions commonly encountered in DNA-based capture-mark-recapture studies and examined the relationships between those conditions and accuracy (i.e., probability of obtaining an estimated N that is within 20% of true N), coverage (i.e., probability that 95% confidence interval includes true N), and precision (i.e., probability of obtaining a coefficient of variation ≤20%) of estimates using logistic regression. The capture probability for the larger of 2 mixture proportions of the population (i.e., pA or pB, depending on the value of π) was most important for predicting accuracy and precision, whereas capture probabilities of both mixture proportions (pA and pB) were important to explain variation in coverage. Based on sampling conditions similar to parameter estimates from the empirical dataset (pA = 0.30, pB = 0.05, N = 250, π = 0.15, and k = 10), predicted accuracy and precision were low (60% and 53%, respectively), whereas coverage was high (94%). Increasing pB, the capture probability for the predominate but most difficult to capture proportion of the population, was most effective to improve accuracy under those conditions. However, manipulation of other parameters may be more effective under different conditions. In general, the probabilities of obtaining accurate and precise estimates were best when p≥ 0.2. Our regression models can be used by managers to evaluate specific sampling scenarios and guide development of sampling frameworks or to assess reliability of DNA-based capture-mark-recapture studies.

Aptamer Selection Express: A Novel Method for Rapid Single-Step Selection and Sensing of Aptamers

PubMed Central

Fan, Maomian; McBurnett, Shelly Roper; Andrews, Carrie J.; Allman, Amity M.; Bruno, John G.; Kiel, Johnathan L.

2008-01-01

Here we describe a new DNA capture element (DCE) sensing system, based on the quenching and dequenching of a double-stranded aptamer. This system shows very good sensitivity and thermal stability. While quenching, dequenching, and separating the DCE systems made from different aptamers (all selected by SELEX), an alternative method to rapidly select aptamers was developed—the Aptamer Selection Express (ASExp). This process has been used to select aptamers against different types of targets (Bacillus anthracis spores, Bacillus thuringiensis spores, MS-2 bacteriophage, ovalbumin, and botulinum neurotoxin). The DCE systems made from botulinum neurotoxin aptamers selected by ASExp have been investigated. The results of this investigation indicate that ASExp can be used to rapidly select aptamers for the DCE sensing system. PMID:19183794

Detection of proteins using a colorimetric bio-barcode assay.

PubMed

Nam, Jwa-Min; Jang, Kyung-Jin; Groves, Jay T

2007-01-01

The colorimetric bio-barcode assay is a red-to-blue color change-based protein detection method with ultrahigh sensitivity. This assay is based on both the bio-barcode amplification method that allows for detecting miniscule amount of targets with attomolar sensitivity and gold nanoparticle-based colorimetric DNA detection method that allows for a simple and straightforward detection of biomolecules of interest (here we detect interleukin-2, an important biomarker (cytokine) for many immunodeficiency-related diseases and cancers). The protocol is composed of the following steps: (i) conjugation of target capture molecules and barcode DNA strands onto silica microparticles, (ii) target capture with probes, (iii) separation and release of barcode DNA strands from the separated probes, (iv) detection of released barcode DNA using DNA-modified gold nanoparticle probes and (v) red-to-blue color change analysis with a graphic software. Actual target detection and quantification steps with premade probes take approximately 3 h (whole protocol including probe preparations takes approximately 3 days).

Destruction of a distal hypoxia response element abolishes trans-activation of the PAG1 gene mediated by HIF-independent chromatin looping.

PubMed

Schörg, Alexandra; Santambrogio, Sara; Platt, James L; Schödel, Johannes; Lindenmeyer, Maja T; Cohen, Clemens D; Schrödter, Katrin; Mole, David R; Wenger, Roland H; Hoogewijs, David

2015-07-13

A crucial step in the cellular adaptation to oxygen deficiency is the binding of hypoxia-inducible factors (HIFs) to hypoxia response elements (HREs) of oxygen-regulated genes. Genome-wide HIF-1α/2α/β DNA-binding studies revealed that the majority of HREs reside distant to the promoter regions, but the function of these distal HREs has only been marginally studied in the genomic context. We used chromatin immunoprecipitation (ChIP), gene editing (TALEN) and chromosome conformation capture (3C) to localize and functionally characterize a 82 kb upstream HRE that solely drives oxygen-regulated expression of the newly identified HIF target gene PAG1. PAG1, a transmembrane adaptor protein involved in Src signalling, was hypoxically induced in various cell lines and mouse tissues. ChIP and reporter gene assays demonstrated that the -82 kb HRE regulates PAG1, but not an equally distant gene further upstream, by direct interaction with HIF. Ablation of the consensus HRE motif abolished the hypoxic induction of PAG1 but not general oxygen signalling. 3C assays revealed that the -82 kb HRE physically associates with the PAG1 promoter region, independent of HIF-DNA interaction. These results demonstrate a constitutive interaction between the -82 kb HRE and the PAG1 promoter, suggesting a physiologically important rapid response to hypoxia. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Abundance patterns of the light neutron-capture elements in very and extremely metal-poor stars

NASA Astrophysics Data System (ADS)

Spite, F.; Spite, M.; Barbuy, B.; Bonifacio, P.; Caffau, E.; François, P.

2018-03-01

Aims: The abundance patterns of the neutron-capture elements in metal-poor stars provide a unique record of the nucleosynthesis products of the earlier massive primitive objects. Methods: We measured new abundances of so-called light neutron-capture of first peak elements using local thermodynamic equilibrium (LTE) 1D analysis; this analysis resulted in a sample of 11 very metal-poor stars, from [Fe/H] = -2.5 to [Fe/H] = -3.4, and one carbon-rich star, CS 22949-037 with [Fe/H] = -4.0. The abundances were compared to those observed in two classical metal-poor stars: the typical r-rich star CS 31082-001 ([Eu/Fe] > +1.0) and the r-poor star HD 122563 ([Eu/Fe] < 0.0), which are known to present a strong enrichment of the first peak neutron-capture elements relative to the second peak. Results: Within the first peak, the abundances are well correlated in analogy to the well-known correlation inside the abundances of the second-peak elements. In contrast, there is no correlation between any first peak element with any second peak element. We show that the scatter of the ratio of the first peak abundance over second peak abundance increases when the mean abundance of the second peak elements decreases from r-rich to r-poor stars. We found two new r-poor stars that are very similar to HD 122563. A third r-poor star, CS 22897-008, is even more extreme; this star shows the most extreme example of first peak elements enrichment to date. On the contrary, another r-poor star (BD-18 5550) has a pattern of first peak elements that is similar to the typical r-rich stars CS 31082-001, however this star has some Mo enrichment. Conclusions: The distribution of the neutron-capture elements in our very metal-poor stars can be understood as the combination of at least two mechanisms: one that enriches the forming stars cloud homogeneously through the main r-process and leads to an element pattern similar to the r-rich stars, such as CS 31082-001; and another that forms mainly lighter, first peak elements. Based on observations collected at the European Organisation for Astronomical Research in the Southern Hemisphere under ESO programme 165.N-0276(A), (PI R.Cayrel).

A Simple Correlation for Neutron Capture Rates from Nuclear Masses

DOE Office of Scientific and Technical Information (OSTI.GOV)

Couture, Aaron Joseph

Recent studies of neutron capture performed at LANL have revealed a previously unrecognized connection between nuclear masses and the average neutron capture cross section. A team of three scientists from Los Alamos (P-27), Yale Univ., and Istanbul Univ. (Turkey) recently discovered this connection and have published their results as a Rapid Communication in Physical Review C. Neutron capture is a reaction in which a free neutron is absorbed by the nucleus, keeping the element unchanged, but changing isotopes. This reaction is typically exothermic. As a result, the reaction can proceed even when many other reaction channels are closed. In anmore » astrophysical environment, this means that neutron capture is the primary mechanism by which all of the elements with atomic number greater than nickel are produced is neutron capture.« less

Single Molecule Visualization of Protein-DNA Complexes: Watching Machines at Work

NASA Astrophysics Data System (ADS)

Kowalczykowski, Stephen

2013-03-01

We can now watch individual proteins acting on single molecules of DNA. Such imaging provides unprecedented interrogation of fundamental biophysical processes. Visualization is achieved through the application of two complementary procedures. In one, single DNA molecules are attached to a polystyrene bead and are then captured by an optical trap. The DNA, a worm-like coil, is extended either by the force of solution flow in a micro-fabricated channel, or by capturing the opposite DNA end in a second optical trap. In the second procedure, DNA is attached by one end to a glass surface. The coiled DNA is elongated either by continuous solution flow or by subsequently tethering the opposite end to the surface. Protein action is visualized by fluorescent reporters: fluorescent dyes that bind double-stranded DNA (dsDNA), fluorescent biosensors for single-stranded DNA (ssDNA), or fluorescently-tagged proteins. Individual molecules are imaged using either epifluorescence microscopy or total internal reflection fluorescence (TIRF) microscopy. Using these approaches, we imaged the search for DNA sequence homology conducted by the RecA-ssDNA filament. The manner by which RecA protein finds a single homologous sequence in the genome had remained undefined for almost 30 years. Single-molecule imaging revealed that the search occurs through a mechanism termed ``intersegmental contact sampling,'' in which the randomly coiled structure of DNA is essential for reiterative sampling of DNA sequence identity: an example of parallel processing. In addition, the assembly of RecA filaments on single molecules of single-stranded DNA was visualized. Filament assembly requires nucleation of a protein dimer on DNA, and subsequent growth occurs via monomer addition. Furthermore, we discovered a class of proteins that catalyzed both nucleation and growth of filaments, revealing how the cell controls assembly of this protein-DNA complex.

DNA damage induced by boron neutron capture therapy is partially repaired by DNA ligase IV.

PubMed

Kondo, Natsuko; Sakurai, Yoshinori; Hirota, Yuki; Tanaka, Hiroki; Watanabe, Tsubasa; Nakagawa, Yosuke; Narabayashi, Masaru; Kinashi, Yuko; Miyatake, Shin-ichi; Hasegawa, Masatoshi; Suzuki, Minoru; Masunaga, Shin-ichiro; Ohnishi, Takeo; Ono, Koji

2016-03-01

Boron neutron capture therapy (BNCT) is a particle radiation therapy that involves the use of a thermal or epithermal neutron beam in combination with a boron ((10)B)-containing compound that specifically accumulates in tumor. (10)B captures neutrons and the resultant fission reaction produces an alpha ((4)He) particle and a recoiled lithium nucleus ((7)Li). These particles have the characteristics of high linear energy transfer (LET) radiation and therefore have marked biological effects. High-LET radiation is a potent inducer of DNA damage, specifically of DNA double-strand breaks (DSBs). The aim of the present study was to clarify the role of DNA ligase IV, a key player in the non-homologous end-joining repair pathway, in the repair of BNCT-induced DSBs. We analyzed the cellular sensitivity of the mouse embryonic fibroblast cell lines Lig4-/- p53-/- and Lig4+/+ p53-/- to irradiation using a thermal neutron beam in the presence or absence of (10)B-para-boronophenylalanine (BPA). The Lig4-/- p53-/- cell line had a higher sensitivity than the Lig4+/+ p53-/-cell line to irradiation with the beam alone or the beam in combination with BPA. In BNCT (with BPA), both cell lines exhibited a reduction of the 50 % survival dose (D 50) by a factor of 1.4 compared with gamma-ray and neutron mixed beam (without BPA). Although it was found that (10)B uptake was higher in the Lig4+/+ p53-/- than in the Lig4-/- p53-/- cell line, the latter showed higher sensitivity than the former, even when compared at an equivalent (10)B concentration. These results indicate that BNCT-induced DNA damage is partially repaired using DNA ligase IV.

Nucleosome core particles containing a poly(dA.dT) sequence element exhibit a locally distorted DNA structure.

PubMed

Bao, Yunhe; White, Cindy L; Luger, Karolin

2006-08-25

Poly(dA.dT) DNA sequence elements are thought to promote transcription by either excluding nucleosomes or by altering their structural or dynamic properties. Here, the stability and structure of a defined nucleosome core particle containing a 16 base-pair poly(dA.dT) element (A16 NCP) was investigated. The A16 NCP requires a significantly higher temperature for histone octamer sliding in vitro compared to comparable nucleosomes that do not contain a poly(dA.dT) element. Fluorescence resonance energy transfer showed that the interactions between the nucleosomal DNA ends and the histone octamer were destabilized in A16 NCP. The crystal structure of A16 NCP was determined to a resolution of 3.2 A. The overall structure was maintained except for local deviations in DNA conformation. These results are consistent with previous in vivo and in vitro observations that poly(dA.dT) elements cause only modest changes in DNA accessibility and modest increases in steady-state transcription levels.

Spectroscopic Analyses of Neutron Capture Elements in Open Clusters

NASA Astrophysics Data System (ADS)

O'Connell, Julia E.

The evolution of elements as a function or age throughout the Milky Way disk provides strong constraints for galaxy evolution models, and on star formation epochs. In an effort to provide such constraints, we conducted an investigation into r- and s-process elemental abundances for a large sample of open clusters as part of an optical follow-up to the SDSS-III/APOGEE-1 near infrared survey. To obtain data for neutron capture abundance analysis, we conducted a long-term observing campaign spanning three years (2013-2016) using the McDonald Observatory Otto Struve 2.1-meter telescope and Sandiford Cass Echelle Spectrograph (SES, R(lambda/Deltalambda) ˜60,000). The SES provides a wavelength range of ˜1400 A, making it uniquely suited to investigate a number of other important chemical abundances as well as the neutron capture elements. For this study, we derive abundances for 18 elements covering four nucleosynthetic families- light, iron-peak, neutron capture and alpha-elements- for ˜30 open clusters within 6 kpc of the Sun with ages ranging from ˜80 Myr to ˜10 Gyr. Both equivalent width (EW) measurements and spectral synthesis methods were employed to derive abundances for all elements. Initial estimates for model stellar atmospheres- effective temperature and surface gravity- were provided by the APOGEE data set, and then re-derived for our optical spectra by removing abundance trends as a function of excitation potential and reduced width log(EW/lambda). With the exception of Ba II and Zr I, abundance analyses for all neutron capture elements were performed by generating synthetic spectra from the new stellar parameters. In order to remove molecular contamination, or blending from nearby atomic features, the synthetic spectra were modeled by a best-fit Gaussian to the observed data. Nd II shows a slight enhancement in all cluster stars, while other neutron capture elements follow solar abundance trends. Ba II shows a large cluster-to-cluster abundance spread, consistent with other open cluster abundance studies. From log(Age) ˜8.5, this large spread as a function of age appears to replicate the findings from an earlier, much debated study by Orazi et al. (2009) which found a linear trend of decreasing barium abundance with increasing age.

Foldback intercoil DNA and the mechanism of DNA transposition.

PubMed

Kim, Byung-Dong

2014-09-01

Foldback intercoil (FBI) DNA is formed by the folding back at one point of a non-helical parallel track of double-stranded DNA at as sharp as 180° and the intertwining of two double helixes within each other's major groove to form an intercoil with a diameter of 2.2 nm. FBI DNA has been suggested to mediate intra-molecular homologous recombination of a deletion and inversion. Inter-molecular homologous recombination, known as site-specific insertion, on the other hand, is mediated by the direct perpendicular approach of the FBI DNA tip, as the attP site, onto the target DNA, as the attB site. Transposition of DNA transposons involves the pairing of terminal inverted repeats and 5-7-bp tandem target duplication. FBI DNA configuration effectively explains simple as well as replicative transposition, along with the involvement of an enhancer element. The majority of diverse retrotransposable elements that employ a target site duplication mechanism is also suggested to follow the FBI DNA-mediated perpendicular insertion of the paired intercoil ends by non-homologous end-joining, together with gap filling. A genome-wide perspective of transposable elements in light of FBI DNA is discussed.

Small RNA-Mediated trans-Nuclear and trans-Element Communications in Tetrahymena DNA Elimination.

PubMed

Noto, Tomoko; Mochizuki, Kazufumi

2018-06-18

Epigenetic inheritance of acquired traits is widespread among eukaryotes, but how and to what extent such information is transgenerationally inherited is still unclear. The patterns of programmed DNA elimination in ciliates are epigenetically and transgenerationally inherited, and it has been proposed that small RNAs, which shuttle between the germline and the soma, regulate this epigenetic inheritance. In this study, we test the existence and role of such small-RNA-mediated communication by epigenetically disturbing the pattern of DNA elimination in Tetrahymena. We show that the pattern of DNA elimination is, indeed, determined by the selective turnover of small RNAs, which is induced by the interaction between germline-derived small RNAs and the somatic genome. In addition, we show that DNA elimination of an element is regulated by small-RNA-mediated communication with other eliminated elements. By contrast, no evidence obtained thus far supports the notion that transfer of epigenetic information from the soma to the germline, if any, regulates DNA elimination. Our results indicate that small-RNA-mediated trans-nuclear and trans-element communication, in addition to unknown information in the germline genome, contributes to determining the pattern of DNA elimination. Copyright © 2018 Elsevier Ltd. All rights reserved.

Satellite DNA and Transposable Elements in Seabuckthorn (Hippophae rhamnoides), a Dioecious Plant with Small Y and Large X Chromosomes

PubMed Central

Puterova, Janka; Razumova, Olga; Martinek, Tomas; Alexandrov, Oleg; Divashuk, Mikhail; Kubat, Zdenek; Hobza, Roman; Karlov, Gennady

2017-01-01

Seabuckthorn (Hippophae rhamnoides) is a dioecious shrub commonly used in the pharmaceutical, cosmetic, and environmental industry as a source of oil, minerals and vitamins. In this study, we analyzed the transposable elements and satellites in its genome. We carried out Illumina DNA sequencing and reconstructed the main repetitive DNA sequences. For data analysis, we developed a new bioinformatics approach for advanced satellite DNA analysis and showed that about 25% of the genome consists of satellite DNA and about 24% is formed of transposable elements, dominated by Ty3/Gypsy and Ty1/Copia LTR retrotransposons. FISH mapping revealed X chromosome-accumulated, Y chromosome-specific or both sex chromosomes-accumulated satellites but most satellites were found on autosomes. Transposable elements were located mostly in the subtelomeres of all chromosomes. The 5S rDNA and 45S rDNA were localized on one autosomal locus each. Although we demonstrated the small size of the Y chromosome of the seabuckthorn and accumulated satellite DNA there, we were unable to estimate the age and extent of the Y chromosome degeneration. Analysis of dioecious relatives such as Shepherdia would shed more light on the evolution of these sex chromosomes. PMID:28057732

Comparison of Hybrid Capture 2 Assay with Real-time-PCR for Detection and Quantitation of Hepatitis B Virus DNA

PubMed Central

Jahan, Munira; Lutful Moben, Ahmed; Tabassum, Shahina

2014-01-01

ABSTRACT Background Both real-time-polymerase chain reaction (PCR) and hybrid capture 2 (HC2) assay can detect and quantify hepatitis B virus (HBV) DNA. However, real-time-PCR can detect a wide range of HBV DNA, while HC2 assay could not detect lower levels of viremia. The present study was designed to detect and quantify HBV DNA by real-time-PCR and HC2 assay and compare the quantitative data of these two assays. Materials and methods A cross-sectional study was conducted in between July 2010 and June 2011. A total of 66 serologically diagnosed chronic hepatitis B (CHB) patients were selected for the study. Real-time-PCR and HC2 assay was done to detect HBV DNA. Data were analyzed by statistical Package for the social sciences (SPSS). Results Among 66 serologically diagnosed chronic hepatitis B patients 40 (60.61%) patients had detectable and 26 (39.39%) had undetectable HBV DNA by HC2 assay. Concordant results were obtained for 40 (60.61%) out of these 66 patients by real-time-PCR and HC2 assay with mean viral load of 7.06 ± 1.13 log10 copies/ml and 6.95 ± 1.08 log10 copies/ml, respectively. In the remaining 26 patients, HBV DNA was detectable by real-time-PCR in 20 patients (mean HBV DNA level was 3.67 ± 0.72 log10 copies/ml. However, HBV DNA could not be detectable in six cases by the both assays. The study showed strong correlation (r = 0.915) between real-time-PCR and HC2 assay for the detection and quantification of HBV DNA. Conclusion HC2 assay may be used as an alternative to real-time-PCR for CHB patients. How to cite this article: Majid F, Jahan M, Moben AL, Tabassum S. Comparison of Hybrid Capture 2 Assay with Real-time-PCR for Detection and Quantitation of Hepatitis B Virus DNA. Euroasian J Hepato-Gastroenterol 2014;4(1):31-35. PMID:29264316

Hybridization chain reaction-based instantaneous derivatization technology for chemiluminescence detection of specific DNA sequences.

PubMed

Wang, Xin; Lau, Choiwan; Kai, Masaaki; Lu, Jianzhong

2013-05-07

We propose here a new amplifying strategy that uses hybridization chain reaction (HCR) to detect specific sequences of DNA, where stable DNA monomers assemble on the magnetic beads only upon exposure to a target DNA. Briefly, in the HCR process, two complementary stable species of hairpins coexist in solution until the introduction of initiator reporter strands triggers a cascade of hybridization events that yield nicked double helices analogous to alternating copolymers. Moreover, a "sandwich-type" detection strategy is employed in our design. Magnetic beads, which are functionalized with capture DNA, are reacted with the target, and sandwiched with the above nicked double helices. Then, chemiluminescence (CL) detection proceeds via an instantaneous derivatization reaction between a specific CL reagent, 3,4,5-trimethoxylphenylglyoxal (TMPG), and the guanine nucleotides within the target DNA, reporter strands and DNA monomers for the generation of light. Our results clearly show that the amplification detection of specific sequences of DNA achieves a better performance (e.g. wide linear response range, low detection limit, and high specificity) as compared to the traditional sandwich type (capture/target/reporter) assays. Upon modification, the approach presented could be extended to detect other types of targets. We believe that this simple technique is promising for improving medical diagnosis and treatment.

Allele frequencies of variants in ultra conserved elements identify selective pressure on transcription factor binding.

PubMed

Silla, Toomas; Kepp, Katrin; Tai, E Shyong; Goh, Liang; Davila, Sonia; Catela Ivkovic, Tina; Calin, George A; Voorhoeve, P Mathijs

2014-01-01

Ultra-conserved genes or elements (UCGs/UCEs) in the human genome are extreme examples of conservation. We characterized natural variations in 2884 UCEs and UCGs in two distinct populations; Singaporean Chinese (n = 280) and Italian (n = 501) by using a pooled sample, targeted capture, sequencing approach. We identify, with high confidence, in these regions the abundance of rare SNVs (MAF<0.5%) of which 75% is not present in dbSNP137. UCEs association studies for complex human traits can use this information to model expected background variation and thus necessary power for association studies. By combining our data with 1000 Genome Project data, we show in three independent datasets that prevalent UCE variants (MAF>5%) are more often found in relatively less-conserved nucleotides within UCEs, compared to rare variants. Moreover, prevalent variants are less likely to overlap transcription factor binding site. Using SNPfold we found no significant influence of RNA secondary structure on UCE conservation. All together, these results suggest UCEs are not under selective pressure as a stretch of DNA but are under differential evolutionary pressure on the single nucleotide level.

Near-Ultraviolet Observations of CS 29497-030: New Constraints on Neutron-Capture Nucleosynthesis Processes

NASA Astrophysics Data System (ADS)

Ivans, Inese I.; Sneden, Christopher; Gallino, Roberto; Cowan, John J.; Preston, George W.

2005-07-01

Employing spectra obtained with the new Keck I HIRES near-UV-sensitive detector, we have performed a comprehensive chemical composition analysis of the binary blue metal-poor star CS 29497-030. Abundances for 29 elements and upper limits for an additional seven have been derived, concentrating on elements largely produced by means of neutron-capture nucleosynthesis. Included in our analysis are the two elements that define the termination point of the slow neutron-capture process, lead and bismuth. We determine an extremely high value of [Pb/Fe]=+3.65+/-0.07 (σ=0.13) from three features, supporting the single-feature result obtained in previous studies. We detect Bi for the first time in a metal-poor star. Our derived Bi/Pb ratio is in accord with those predicted from the most recent FRANEC calculations of the slow neutron-capture process in low-mass asymptotic giant branch (AGB) stars. We find that the neutron-capture elemental abundances of CS 29497-030 are best explained by an AGB model that also includes very significant amounts of pre-enrichment of rapid neutron-capture process material in the protostellar cloud out of which the CS 29497-030 binary system formed. Mass transfer is consistent with the observed [Nb/Zr]~0. Thus, CS 29497-030 is both an r+s and ``extrinsic AGB'' star. Furthermore, we find that the mass of the AGB model can be further constrained by the abundance of the light odd-element Na. The data presented herein were obtained at the W. M. Keck Observatory, which is operated as a scientific partnership among the California Institute of Technology, the University of California, and NASA. The Observatory was made possible by the generous financial support of the W. M. Keck Foundation.

Study of photon emission by electron capture during solar nuclei acceleration. 2: Delimitation of conditions for charge transfert establishment

NASA Technical Reports Server (NTRS)

Perez-Peraza, J.; Alvarez, M.; Gallegos, A.

1985-01-01

The conditions for establishment of charge transfer during acceleration of nuclei up to Fe, for typical conditions of solar flare regions T = 5 x 10 to the 3rd power to 2.5 x 10 to the 8th power degrees K were explored. Results show that such conditions are widely assorted, depending on the acceleration mechanism, the kind of projections and their velocity, the target elements, the source temperature and consequently on the degree of ionization of matter and the local charge state of the accelerated ions. Nevertheless, in spite of that assorted behavior, there are some general tendencies that can be summarized as follows. In atomic H electron capture is systematically established from thermal energies up to high energies, whatever the element and for both acceleration process. For a given element and fixed temperature (T), the probability and energy domain of electron capture and loss with Fermi are higher than with Betatron acceleration. For a given acceleration process the heavier the ion the higher the probability and the wider the energy range for electron capture and loss. For given acceleration mechanism and fixed element the importance and energy domain of capture and loss increase with T: for those reasons, the energy range of charge equilibrium (illustrated with solid lines on the next figs.) is wider with Fermi and increases with temperature and atomic number of projectiles. For the same reasons, electron loss is smaller while the lighter the element, the lower the temperature and the Betatron process, such that there are conditions for which electron loss is not allowed at low energies, but only electron capture is established.

Contribution of Massive Stars to the Production of Neutron Capture Elements

NASA Astrophysics Data System (ADS)

Federman, Steven

2010-09-01

Elements beyond the Fe-peak must be synthesized through neutron-capture processes. With the aim of understanding the contribution of massive stars to the synthesis of neutron-capture elements during the current epoch, we propose an archival survey of interstellar arsenic, cadmium, tin, and lead. Nucleosynthesis via the weak slow process and the rapid process are the routes involving massive stars, while the main slow process arises from the evolution of low-mass stars. Ultraviolet lines for the dominant ions for each element will be used to extract interstellar abundances. The survey involves about forty sight lines, many of which are associated with regions of massive star formation shaped by core-collapse supernovae {SNe II}. The sample will increase the number of published determinations by factors of 2 to 5. HST spectra are the only means for determining the elemental abundances for this set of species in diffuse interstellar clouds. The survey contains directions that are both molecule poor and molecule rich, thereby enabling us to examine the overall level of depletion onto grains as a function of gas density. Complementary laboratory determinations of oscillator strengths will place the interstellar measurements on an absolute scale. The results from the proposed study will be combined with published interstellar abundances for other neutron capture elements and the suite of measurements will be compared to results from stars throughout the history of the Galaxy.

Neutron capture and stellar synthesis of heavy elements.

PubMed

Gibbons, J H; Macklin, R L

1967-05-26

The neutron buildup processes of heavy-element synthesis in stars have left us a number of tantalizing nuclear clues to the early history of solarsystem material. Considerable illumination of our past history has been achieved through studying the correlations between abundance and neutroncapture cross section. Measurement of these cross sections required the development of new techniques for measuring time of flight of pulsed neutron beams. A clear conclusion is that many of our heavy elements were produced inside stars, which can be thought of as giant fast reactors. Extensions of these capture studies have given a clearer picture of additional. violent processes which produced some heavy elements, particularly thorium and uranium. In addition, the correlations have been used for obtaining an independent measure of the time that has elapsed since the solar-system material was synthesized. Finally, data on capture cross section relative to abundance will enable us to determine rather accurately the solar-system abundances of gaseous, volatile, and highly segregated elements.

Two alternative DNA extraction methods to improve the detection of Mycobacterium-tuberculosis-complex members in cattle and red deer tissue samples.

PubMed

Fell, Shari; Bröckl, Stephanie; Büttner, Mathias; Rettinger, Anna; Zimmermann, Pia; Straubinger, Reinhard K

2016-09-15

Bovine tuberculosis (bTB), which is caused by Mycobacterium bovis and M. caprae, is a notifiable animal disease in Germany. Diagnostic procedure is based on a prescribed protocol that is published in the framework of German bTB legislation. In this protocol small sample volumes are used for DNA extraction followed by real-time PCR analyses. As mycobacteria tend to concentrate in granuloma and the infected tissue in early stages of infection does not necessarily show any visible lesions, it is likely that DNA extraction from only small tissue samples (20-40 mg) of a randomly chosen spot from the organ and following PCR testing may result in false negative results. In this study two DNA extraction methods were developed to process larger sample volumes to increase the detection sensitivity of mycobacterial DNA in animal tissue. The first extraction method is based on magnetic capture, in which specific capture oligonucleotides were utilized. These nucleotides are linked to magnetic particles and capture Mycobacterium-tuberculosis-complex (MTC) DNA released from 10 to 15 g of tissue material. In a second approach remaining sediments from the magnetic capture protocol were further processed with a less complex extraction protocol that can be used in daily routine diagnostics. A total number of 100 tissue samples from 34 cattle (n = 74) and 18 red deer (n = 26) were analyzed with the developed protocols and results were compared to the prescribed protocol. All three extraction methods yield reliable results by the real-time PCR analysis. The use of larger sample volume led to a sensitivity increase of DNA detection which was shown by the decrease of Ct-values. Furthermore five samples which were tested negative or questionable by the official extraction protocol were detected positive by real time PCR when the alternative extraction methods were used. By calculating the kappa index, the three extraction protocols resulted in a moderate (0.52; protocol 1 vs 3) to almost perfect agreement (1.00; red deer sample testing with all protocols). Both new methods yielded increased detection rates for MTC DNA detection in large sample volumes and consequently improve the official diagnostic protocol.

Genome-wide colonization of gene regulatory elements by G4 DNA motifs

PubMed Central

Du, Zhuo; Zhao, Yiqiang; Li, Ning

2009-01-01

G-quadruplex (or G4 DNA), a stable four-stranded structure found in guanine-rich regions, is implicated in the transcriptional regulation of genes involved in growth and development. Previous studies on the role of G4 DNA in gene regulation mostly focused on genomic regions proximal to transcription start sites (TSSs). To gain a more comprehensive understanding of the regulatory role of G4 DNA, we examined the landscape of potential G4 DNA (PG4Ms) motifs in the human genome and found that G4 motifs, not restricted to those found in the TSS-proximal regions, are bias toward gene-associated regions. Significantly, analyses of G4 motifs in seven types of well-known gene regulatory elements revealed a constitutive enrichment pattern and the clusters of G4 motifs tend to be colocalized with regulatory elements. Considering our analysis from a genome evolutionary perspective, we found evidence that the occurrence and accumulation of certain progenitors and canonical G4 DNA motifs within regulatory regions were progressively favored by natural selection. Our results suggest that G4 DNA motifs are ‘colonized’ in regulatory regions, supporting a likely genome-wide role of G4 DNA in gene regulation. We hypothesize that G4 DNA is a regulatory apparatus situated in regulatory elements, acting as a molecular switch that can modulate the role of the host functional regions, by transition in DNA structure. PMID:19759215

49 CFR 563.9 - Data capture.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 49 Transportation 6 2010-10-01 2010-10-01 false Data capture. 563.9 Section 563.9 Transportation..., DEPARTMENT OF TRANSPORTATION EVENT DATA RECORDERS § 563.9 Data capture. The EDR must capture and record the data elements for events in accordance with the following conditions and circumstances: (a) In a...

High-throughput analysis of T-DNA location and structure using sequence capture

DOE Office of Scientific and Technical Information (OSTI.GOV)

Inagaki, Soichi; Henry, Isabelle M.; Lieberman, Meric C.

Agrobacterium-mediated transformation of plants with T-DNA is used both to introduce transgenes and for mutagenesis. Conventional approaches used to identify the genomic location and the structure of the inserted T-DNA are laborious and high-throughput methods using next-generation sequencing are being developed to address these problems. Here, we present a cost-effective approach that uses sequence capture targeted to the T-DNA borders to select genomic DNA fragments containing T-DNA—genome junctions, followed by Illumina sequencing to determine the location and junction structure of T-DNA insertions. Multiple probes can be mixed so that transgenic lines transformed with different T-DNA types can be processed simultaneously,more » using a simple, index-based pooling approach. We also developed a simple bioinformatic tool to find sequence read pairs that span the junction between the genome and T-DNA or any foreign DNA. We analyzed 29 transgenic lines of Arabidopsis thaliana, each containing inserts from 4 different T-DNA vectors. We determined the location of T-DNA insertions in 22 lines, 4 of which carried multiple insertion sites. Additionally, our analysis uncovered a high frequency of unconventional and complex T-DNA insertions, highlighting the needs for high-throughput methods for T-DNA localization and structural characterization. Transgene insertion events have to be fully characterized prior to use as commercial products. As a result, our method greatly facilitates the first step of this characterization of transgenic plants by providing an efficient screen for the selection of promising lines.« less

High-throughput analysis of T-DNA location and structure using sequence capture

DOE PAGES

Inagaki, Soichi; Henry, Isabelle M.; Lieberman, Meric C.; ...

2015-10-07

Agrobacterium-mediated transformation of plants with T-DNA is used both to introduce transgenes and for mutagenesis. Conventional approaches used to identify the genomic location and the structure of the inserted T-DNA are laborious and high-throughput methods using next-generation sequencing are being developed to address these problems. Here, we present a cost-effective approach that uses sequence capture targeted to the T-DNA borders to select genomic DNA fragments containing T-DNA—genome junctions, followed by Illumina sequencing to determine the location and junction structure of T-DNA insertions. Multiple probes can be mixed so that transgenic lines transformed with different T-DNA types can be processed simultaneously,more » using a simple, index-based pooling approach. We also developed a simple bioinformatic tool to find sequence read pairs that span the junction between the genome and T-DNA or any foreign DNA. We analyzed 29 transgenic lines of Arabidopsis thaliana, each containing inserts from 4 different T-DNA vectors. We determined the location of T-DNA insertions in 22 lines, 4 of which carried multiple insertion sites. Additionally, our analysis uncovered a high frequency of unconventional and complex T-DNA insertions, highlighting the needs for high-throughput methods for T-DNA localization and structural characterization. Transgene insertion events have to be fully characterized prior to use as commercial products. As a result, our method greatly facilitates the first step of this characterization of transgenic plants by providing an efficient screen for the selection of promising lines.« less

Rapid and sensitive detection of foodborne pathogenic bacteria (Staphylococcus aureus) using an electrochemical DNA genomic biosensor and its application in fresh beef.

PubMed

Abdalhai, Mandour H; Fernandes, António Maximiano; Bashari, Mohand; Ji, Jian; He, Qian; Sun, Xiulan

2014-12-31

Rapid early detection of food contamination is the main key in food safety and quality control. Biosensors are emerging as a vibrant area of research, and the use of DNA biosensor recognition detectors is relatively new. In this study a genomic DNA biosensor system with a fixing and capture probe was modified by a sulfhydryl and amino group, respectively, as complementary with target DNA. After immobilization and hybridization, the following sandwich structure fixing DNA-target DNA-capture DNA-PbS NPs was formed to detect pathogenic bacteria (Staphylococuus aureus EF529607.1) by using GCE modified with (multiwalled carbon nanotubes-chitosan-bismuth) to increase the sensitivity of the electrode. The modification procedure was characterized by cyclic voltammetry and electrochemical impedance spectroscopy. The sandwich structure was dissolved in 1 M nitric acid to become accessible to the electrode, and the PbS NPs was measured in solution by differential pulse voltammetry (DPV). The results showed that the detection limit of the DNA sensor was 3.17 × 10(-14) M S. aureus using PbS NPs, whereas the result for beef samples was 1.23 ng/mL. Thus, according to the experimental results presented, the DNA biosensor exhibited high sensitivity and rapid response, and it will be useful for the food matrix.

NFκB- and AP-1-mediated DNA looping regulates matrix metalloproteinase-9 transcription in TNF-α-treated human leukemia U937 cells.

PubMed

Chen, Ying-Jung; Chang, Long-Sen

2015-10-01

The aim of this study is to explore the spatial association of critical genomic elements in the effect of TNF-α on matrix metalloproteinase-9 (MMP-9) expression in human leukemia U937 cells. TNF-α up-regulated MMP-9 protein expression and mRNA level in U937 cells, and Akt-mediated-NFκB/p65 activation and JNK-mediated c-Jun activation were proven to be involved in TNF-α-induced MMP-9 up-regulation. Promoter luciferase activity assay revealed that NFκB (nt-600) and AP-1 (nt-79) binding sites were crucial for TNF-α-induced transcription of MMP-9 gene. The results of a chromatin immunoprecipitation assay indicated that TNF-α reduced histone deacetylase-1 (HDAC-1) recruitment but increased p300 (a histone acetyltransferase) recruitment to MMP-9 promoter regions surrounding NFκB and AP-1 binding sites. Consistently, TNF-α increased enrichment of the acetylated histone H3 mark on MMP-9 promoter regions. DNA affinity purification assay revealed that p300 and HDAC1 could bind oligonucleotides containing AP-1/c-Jun and NFκB/p65 binding sites. Chromosome conformation capture assay showed that TNF-α stimulated chromosomal loops in the MMP-9 promoter via NFκB/p65 and AP-1/c-Jun. The p300-associated acetyltransferase activity was crucial for p65/c-Jun-mediated DNA looping, and inhibition of HDAC activity increased the level of DNA looping. Reduction in the level of DNA looping eliminated all TNF-α-stimulated MMP-9 up-regulation. Taken together, our data suggest that p65/c-Jun-mediated DNA looping is involved in TNF-α-induced MMP-9 up-regulation and that the recruitment of p300 or HDAC1 to NFκB and AP-1 binding sites modifies the level of DNA looping. Copyright © 2015 Elsevier B.V. All rights reserved.

Regulation of the yeast RAD2 gene: DNA damage-dependent induction correlates with protein binding to regulatory sequences and their deletion influences survival.

PubMed

Siede, W; Friedberg, E C

1992-03-01

In the yeast Saccharomyces cerevisiae the RAD2 gene is absolutely required for damage-specific incision of DNA during nucleotide excision repair and is inducible by DNA-damaging agents. In the present study we correlated sensitivity to killing by DNA-damaging agents with the deletion of previously defined specific promoter elements. Deletion of the element DRE2 increased the UV sensitivity of cells in both the G1/early S and S/G2 phases of the cell cycle as well as in stationary phase. On the other hand, increased UV sensitivity associated with deletion of the sequence-related element DRE1 was restricted to cells irradiated in G1/S. Specific binding of protein(s) to the promoter elements DRE1 and DRE2 was observed under non-inducing conditions using gel retardation assays. Exposure of cells to DNA-damaging agents resulted in increased protein binding that was dependent on de novo protein synthesis.

Connecting micro dynamics and population distributions in system dynamics models

PubMed Central

Rahmandad, Hazhir; Chen, Hsin-Jen; Xue, Hong; Wang, Youfa

2014-01-01

Researchers use system dynamics models to capture the mean behavior of groups of indistinguishable population elements (e.g., people) aggregated in stock variables. Yet, many modeling problems require capturing the heterogeneity across elements with respect to some attribute(s) (e.g., body weight). This paper presents a new method to connect the micro-level dynamics associated with elements in a population with the macro-level population distribution along an attribute of interest without the need to explicitly model every element. We apply the proposed method to model the distribution of Body Mass Index and its changes over time in a sample population of American women obtained from the U.S. National Health and Nutrition Examination Survey. Comparing the results with those obtained from an individual-based model that captures the same phenomena shows that our proposed method delivers accurate results with less computation than the individual-based model. PMID:25620842

Global-Local Finite Element Analysis for Thermo-Mechanical Stresses in Bonded Joints

NASA Technical Reports Server (NTRS)

Shkarayev, S.; Madenci, Erdogan; Camarda, C. J.

1997-01-01

An analysis of adhesively bonded joints using conventional finite elements does not capture the singular behavior of the stress field in regions where two or three dissimilar materials form a junction with or without free edges. However, these regions are characteristic of the bonded joints and are prone to failure initiation. This study presents a method to capture the singular stress field arising from the geometric and material discontinuities in bonded composites. It is achieved by coupling the local (conventional) elements with global (special) elements whose interpolation functions are constructed from the asymptotic solution.

Verification of Orthogrid Finite Element Modeling Techniques

NASA Technical Reports Server (NTRS)

Steeve, B. E.

1996-01-01

The stress analysis of orthogrid structures, specifically with I-beam sections, is regularly performed using finite elements. Various modeling techniques are often used to simplify the modeling process but still adequately capture the actual hardware behavior. The accuracy of such 'Oshort cutso' is sometimes in question. This report compares three modeling techniques to actual test results from a loaded orthogrid panel. The finite element models include a beam, shell, and mixed beam and shell element model. Results show that the shell element model performs the best, but that the simpler beam and beam and shell element models provide reasonable to conservative results for a stress analysis. When deflection and stiffness is critical, it is important to capture the effect of the orthogrid nodes in the model.

Combinatorial events of insertion sequences and ICE in Gram-negative bacteria.

PubMed

Toleman, Mark A; Walsh, Timothy R

2011-09-01

The emergence of antibiotic and antimicrobial resistance in Gram-negative bacteria is incremental and linked to genetic elements that function in a so-called 'one-ended transposition' manner, including ISEcp1, ISCR elements and Tn3-like transposons. The power of these elements lies in their inability to consistently recognize one of their own terminal sequences, while recognizing more genetically distant surrogate sequences. This has the effect of mobilizing the DNA sequence found adjacent to their initial location. In general, resistance in Gram-negatives is closely linked to a few one-off events. These include the capture of the class 1 integron by a Tn5090-like transposon; the formation of the 3' conserved segment (3'-CS); and the fusion of the ISCR1 element to the 3'-CS. The structures formed by these rare events have been massively amplified and disseminated in Gram-negative bacteria, but hitherto, are rarely found in Gram-positives. Such events dominate current resistance gene acquisition and are instrumental in the construction of large resistance gene islands on chromosomes and plasmids. Similar combinatorial events appear to have occurred between conjugative plasmids and phages constructing hybrid elements called integrative and conjugative elements or conjugative transposons. These elements are beginning to be closely linked to some of the more powerful resistance mechanisms such as the extended spectrum β-lactamases, metallo- and AmpC type β-lactamases. Antibiotic resistance in Gram-negative bacteria is dominated by unusual combinatorial mistakes of Insertion sequences and gene fusions which have been selected and amplified by antibiotic pressure enabling the formation of extended resistance islands. © 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

ROLE OF SURFACE FUNCTIONAL GROUPS IN THE CAPTURE OF ELEMENTAL MERCURY AND MERCURIC CHLORIDE BY ACTIVATED CARBONS

EPA Science Inventory

The paper discusses using a laboratory-scale, fixed bed apparatus to study the role of surface functional groups (SFGs) in the capture of mercuric chloride (HgC12) and elemental mercury (Hgo) in nitrogen (N2) prior to flue gas atmosphere studies. The study focused on two activat...

Lecture Capture with Real-Time Rearrangement of Visual Elements: Impact on Student Performance

ERIC Educational Resources Information Center

Yu, P.-T.; Wang, B.-Y.; Su, M.-H.

2015-01-01

The primary goal of this study is to create and test a lecture-capture system that can rearrange visual elements while recording is still taking place, in such a way that student performance can be positively influenced. The system we have devised is capable of integrating and rearranging multimedia sources, including learning content, the…

Capture-SELEX: Selection of DNA Aptamers for Aminoglycoside Antibiotics

PubMed Central

2012-01-01

Small organic molecules are challenging targets for an aptamer selection using the SELEX technology (SELEX—Systematic Evolution of Ligans by EXponential enrichment). Often they are not suitable for immobilization on solid surfaces, which is a common procedure in known aptamer selection methods. The Capture-SELEX procedure allows the selection of DNA aptamers for solute targets. A special SELEX library was constructed with the aim to immobilize this library on magnetic beads or other surfaces. For this purpose a docking sequence was incorporated into the random region of the library enabling hybridization to a complementary oligo fixed on magnetic beads. Oligonucleotides of the library which exhibit high affinity to the target and a secondary structure fitting to the target are released from the beads for binding to the target during the aptamer selection process. The oligonucleotides of these binding complexes were amplified, purified, and immobilized via the docking sequence to the magnetic beads as the starting point of the following selection round. Based on this Capture-SELEX procedure, the successful DNA aptamer selection for the aminoglycoside antibiotic kanamycin A as a small molecule target is described. PMID:23326761

Spontaneous germline excision of Tol1, a DNA-based transposable element naturally occurring in the medaka fish genome.

PubMed

Watanabe, Kohei; Koga, Hajime; Nakamura, Kodai; Fujita, Akiko; Hattori, Akimasa; Matsuda, Masaru; Koga, Akihiko

2014-04-01

DNA-based transposable elements are ubiquitous constituents of eukaryotic genomes. Vertebrates are, however, exceptional in that most of their DNA-based elements appear to be inactivated. The Tol1 element of the medaka fish, Oryzias latipes, is one of the few elements for which copies containing an undamaged gene have been found. Spontaneous transposition of this element in somatic cells has previously been demonstrated, but there is only indirect evidence for its germline transposition. Here, we show direct evidence of spontaneous excision in the germline. Tyrosinase is the key enzyme in melanin biosynthesis. In an albino laboratory strain of medaka fish, which is homozygous for a mutant tyrosinase gene in which a Tol1 copy is inserted, we identified de novo reversion mutations related to melanin pigmentation. The gamete-based reversion rate was as high as 0.4%. The revertant fish carried the tyrosinase gene from which the Tol1 copy had been excised. We previously reported the germline transposition of Tol2, another DNA-based element that is thought to be a recent invader of the medaka fish genome. Tol1 is an ancient resident of the genome. Our results indicate that even an old element can contribute to genetic variation in the host genome as a natural mutator.

Trace elements are associated with urinary 8-hydroxy-2'-deoxyguanosine level: a case study of college students in Guangzhou, China.

PubMed

Lu, Shaoyou; Ren, Lu; Fang, Jianzhang; Ji, Jiajia; Liu, Guihua; Zhang, Jianqing; Zhang, Huimin; Luo, Ruorong; Lin, Kai; Fan, Ruifang

2016-05-01

Many trace heavy elements are carcinogenic and increase the incidence of cancer. However, a comprehensive study of the correlation between multiple trace elements and DNA oxidative damage is still lacking. The aim of this study is to investigate the relationships between the body burden of multiple trace elements and DNA oxidative stress in college students in Guangzhou, China. Seventeen trace elements in urine samples were determined by inductively coupled plasma-mass spectrometry (ICP-MS). Urinary 8-hydroxy-2'-deoxyguanosine (8-OHdG), a biomarker of DNA oxidative stress, was also measured using liquid chromatography tandem mass spectrometer (LC-MS/MS). The concentrations of six essential elements including manganese (Mn), copper (Cu), nickel (Ni), selenium (Se), strontium (Sr), and molybdenum (Mo), and five non-essential elements including arsenic (As), cadmium (Cd), aluminum (Al), stibium (Sb), and thallium (Tl), were found to be significantly correlated with urinary 8-OHdG levels. Moreover, urinary levels of Ni, Se, Mo, As, Sr, and Tl were strongly significantly correlated with 8-OHdG (P < 0.01) concentration. Environmental exposure and dietary intake of these trace elements may play important roles in DNA oxidative damage in the population of Guangzhou, China.

Photoinduced Regeneration of an Aptamer-Based Electrochemical Sensor for Sensitively Detecting Adenosine Triphosphate.

PubMed

Zhang, Xiaoyu; Song, Chunxia; Yang, Ke; Hong, Wenwen; Lu, Ying; Yu, Ping; Mao, Lanqun

2018-04-17

Electrochemical aptasensors generally include three elements, that is, recognition element, signal-transformation element, and regeneration element. In this study, a new adenosine triphosphate (ATP) aptasensor is developed by combining three elements into one DNA oligonucleotide chain. In the DNA oligonucleotide chain, DNA aptamer is used as the recognition element, ferrocene group attached at the 3'-end of the aptamer is used as the signal-transformation element, and azobenzene moiety embedded into the DNA chain is used as the regeneration element. In addition to the similar analytical properties with the traditional ones, the aptasensor developed here is easily regenerated with UV-light irradiation. The current response recorded on the aptasensor increases with increasing the concentration of ATP in the incubation solution and is linear with the logarithm of ATP concentration in the range from 1 nM to 100 μM. The limit of detection is 0.5 nM (S/N = 3). The basal level of ATP in the rat brain cortex microdialysate is determined to be 21.33 ± 4.1 nM ( n = 3). After being challenged with ATP, the aptasensor could be readily regenerated by UV-light irradiation for more than seven cycles. The regeneration of the aptasensor is proposed to be regulated by conversing azobenzene from its trans to cis form under UV irradiation.

Movable Genetic Elements: Detection of Changes in Maize DNA at the Shrunken Locus Due to the Intervention of Ds Elements

DOE R&D Accomplishments Database

Burr, B.; Burr, F.A.

1980-05-28

This report describes our initial attempts at the molecular characterization of a maize controlling element. We have prepared a cDNA probe and used it to detect changes at a locus where Ds elements are found. Evidence of their presence are indicated by changes in the restriction patterns, but there is as yet no information on the physical nature of the controlling elements nor on the kinds of rearrangements they cause.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Ai Cheng; Dai, Ziyu; Chen, Baowei

2008-12-01

We describe a novel electrochemical branched-DNA (bDNA) assay for polymerase chain reaction (PCR)-free detection and quantification of p185 BCR-ABL leukemia fusion transcript in the population of messenger RNA (mRNA) extracted from cell lines. The bDNA amplifier carrying high loading of alkaline phosphatase (ALP) tracers was used to amplify targets signal. The targets were captured on microplate well surfaces through cooperative sandwich hybridization prior to the labeling of bDNA. The activity of captured ALP was monitored by square-wave voltammetric (SWV) analysis of the electroactive enzymatic product in the presence of 1-napthyl-phosphate. The specificity and sensitivity of assay enabled direct detection ofmore » target transcript in as little as 4.6 ng mRNA without PCR amplification. In combination with the use of a well-quantified standard, the electrochemical bDNA assay was capable of direct use for a PCR-free quantitative analysis of target transcript in total mRNA population. The approach thus provides a simple, sensitive, accurate and quantitative tool alternate to the RQ-PCR for early disease diagnosis.« less

Assessment of mitochondrial DNA damage in little brown bats (Myotis lucifugus) collected near a mercury-contaminated river

USGS Publications Warehouse

Karouna-Renier, Natalie K.; White, Carl; Perkins, Christopher R.; Schmerfeld, John J.; Yates, David

2014-01-01

Historical discharges of Hg into the South River near the town of Waynesboro, VA, USA, have resulted in persistently elevated Hg concentrations in sediment, surface water, ground water, soil, and wildlife downstream of the discharge site. In the present study, we examined mercury (Hg) levels in in little brown bats (Myotis lucifugus) from this location and assessed the utility of a non-destructively collected tissue sample (wing punch) for determining mitochondrial DNA (mtDNA) damage in Hg exposed bats. Bats captured 1 and 3 km from the South River, exhibited significantly higher levels of total Hg (THg) in blood and fur than those from the reference location. We compared levels of mtDNA damage using real-time quantitative PCR (qPCR) analysis of two distinct regions of mtDNA. Genotoxicity is among the many known toxic effects of Hg, resulting from direct interactions with DNA or from oxidative damage. Because it lacks many of the protective protein structures and repair mechanisms associated with nuclear DNA, mtDNA is more sensitive to the effects of genotoxic chemicals and therefore may be a useful biomarker in chronically exposed organisms. Significantly higher levels of damage were observed in both regions of mtDNA in bats captured 3 km from the river than in controls. However, levels of mtDNA damage exhibited weak correlations with fur and blood THg levels, suggesting that other factors may play a role in the site-specific differences.

Satellite DNA and Transposable Elements in Seabuckthorn (Hippophae rhamnoides), a Dioecious Plant with Small Y and Large X Chromosomes.

PubMed

Puterova, Janka; Razumova, Olga; Martinek, Tomas; Alexandrov, Oleg; Divashuk, Mikhail; Kubat, Zdenek; Hobza, Roman; Karlov, Gennady; Kejnovsky, Eduard

2017-01-01

Seabuckthorn (Hippophae rhamnoides) is a dioecious shrub commonly used in the pharmaceutical, cosmetic, and environmental industry as a source of oil, minerals and vitamins. In this study, we analyzed the transposable elements and satellites in its genome. We carried out Illumina DNA sequencing and reconstructed the main repetitive DNA sequences. For data analysis, we developed a new bioinformatics approach for advanced satellite DNA analysis and showed that about 25% of the genome consists of satellite DNA and about 24% is formed of transposable elements, dominated by Ty3/Gypsy and Ty1/Copia LTR retrotransposons. FISH mapping revealed X chromosome-accumulated, Y chromosome-specific or both sex chromosomes-accumulated satellites but most satellites were found on autosomes. Transposable elements were located mostly in the subtelomeres of all chromosomes. The 5S rDNA and 45S rDNA were localized on one autosomal locus each. Although we demonstrated the small size of the Y chromosome of the seabuckthorn and accumulated satellite DNA there, we were unable to estimate the age and extent of the Y chromosome degeneration. Analysis of dioecious relatives such as Shepherdia would shed more light on the evolution of these sex chromosomes. © The Author(s) 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

49 CFR 563.9 - Data capture.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 49 Transportation 6 2011-10-01 2011-10-01 false Data capture. 563.9 Section 563.9 Transportation..., DEPARTMENT OF TRANSPORTATION EVENT DATA RECORDERS § 563.9 Data capture. Link to an amendment published at 76 FR 47489, Aug. 5, 2011. The EDR must capture and record the data elements for events in accordance...

Use of Lecture Capture in Higher Education--Lessons from the Trenches

ERIC Educational Resources Information Center

Newton, Genevieve; Tucker, Trent; Dawson, John; Currie, Elliott

2014-01-01

Lecture capture, defined here as the capturing of some or all elements of a live lecture in digital format, is becoming increasingly popular in higher education. Despite this increase in popularity, fewer than 10% of institutes of higher education globally have adopted comprehensive lecture capture systems. So, the majority of instructors wanting…

High-throughput screening of metal-porphyrin-like graphenes for selective capture of carbon dioxide

PubMed Central

Bae, Hyeonhu; Park, Minwoo; Jang, Byungryul; Kang, Yura; Park, Jinwoo; Lee, Hosik; Chung, Haegeun; Chung, ChiHye; Hong, Suklyun; Kwon, Yongkyung; Yakobson, Boris I.; Lee, Hoonkyung

2016-01-01

Nanostructured materials, such as zeolites and metal-organic frameworks, have been considered to capture CO2. However, their application has been limited largely because they exhibit poor selectivity for flue gases and low capture capacity under low pressures. We perform a high-throughput screening for selective CO2 capture from flue gases by using first principles thermodynamics. We find that elements with empty d orbitals selectively attract CO2 from gaseous mixtures under low CO2 pressures (~10−3 bar) at 300 K and release it at ~450 K. CO2 binding to elements involves hybridization of the metal d orbitals with the CO2 π orbitals and CO2-transition metal complexes were observed in experiments. This result allows us to perform high-throughput screening to discover novel promising CO2 capture materials with empty d orbitals (e.g., Sc– or V–porphyrin-like graphene) and predict their capture performance under various conditions. Moreover, these findings provide physical insights into selective CO2 capture and open a new path to explore CO2 capture materials. PMID:26902156

High-throughput screening of metal-porphyrin-like graphenes for selective capture of carbon dioxide.

PubMed

Bae, Hyeonhu; Park, Minwoo; Jang, Byungryul; Kang, Yura; Park, Jinwoo; Lee, Hosik; Chung, Haegeun; Chung, ChiHye; Hong, Suklyun; Kwon, Yongkyung; Yakobson, Boris I; Lee, Hoonkyung

2016-02-23

Nanostructured materials, such as zeolites and metal-organic frameworks, have been considered to capture CO2. However, their application has been limited largely because they exhibit poor selectivity for flue gases and low capture capacity under low pressures. We perform a high-throughput screening for selective CO2 capture from flue gases by using first principles thermodynamics. We find that elements with empty d orbitals selectively attract CO2 from gaseous mixtures under low CO2 pressures (~10(-3) bar) at 300 K and release it at ~450 K. CO2 binding to elements involves hybridization of the metal d orbitals with the CO2 π orbitals and CO2-transition metal complexes were observed in experiments. This result allows us to perform high-throughput screening to discover novel promising CO2 capture materials with empty d orbitals (e.g., Sc- or V-porphyrin-like graphene) and predict their capture performance under various conditions. Moreover, these findings provide physical insights into selective CO2 capture and open a new path to explore CO2 capture materials.

High-throughput screening of metal-porphyrin-like graphenes for selective capture of carbon dioxide

NASA Astrophysics Data System (ADS)

Bae, Hyeonhu; Park, Minwoo; Jang, Byungryul; Kang, Yura; Park, Jinwoo; Lee, Hosik; Chung, Haegeun; Chung, Chihye; Hong, Suklyun; Kwon, Yongkyung; Yakobson, Boris I.; Lee, Hoonkyung

2016-02-01

Nanostructured materials, such as zeolites and metal-organic frameworks, have been considered to capture CO2. However, their application has been limited largely because they exhibit poor selectivity for flue gases and low capture capacity under low pressures. We perform a high-throughput screening for selective CO2 capture from flue gases by using first principles thermodynamics. We find that elements with empty d orbitals selectively attract CO2 from gaseous mixtures under low CO2 pressures (~10-3 bar) at 300 K and release it at ~450 K. CO2 binding to elements involves hybridization of the metal d orbitals with the CO2 π orbitals and CO2-transition metal complexes were observed in experiments. This result allows us to perform high-throughput screening to discover novel promising CO2 capture materials with empty d orbitals (e.g., Sc- or V-porphyrin-like graphene) and predict their capture performance under various conditions. Moreover, these findings provide physical insights into selective CO2 capture and open a new path to explore CO2 capture materials.

Protection of DNA against low-energy electrons by amino acids: a first-principles molecular dynamics study.

PubMed

Gu, Bin; Smyth, Maeve; Kohanoff, Jorge

2014-11-28

Using first-principles molecular dynamics simulations, we have investigated the notion that amino acids can play a protective role when DNA is exposed to excess electrons produced by ionizing radiation. In this study we focus on the interaction of glycine with the DNA nucleobase thymine. We studied thymine-glycine dimers and a condensed phase model consisting of one thymine molecule solvated in amorphous glycine. Our results show that the amino acid acts as a protective agent for the nucleobase in two ways. If the excess electron is initially captured by the thymine, then a proton is transferred in a barrier-less way from a neighboring hydrogen-bonded glycine. This stabilizes the excess electron by reducing the net partial charge on the thymine. In the second mechanism the excess electron is captured by a glycine, which acts as a electron scavenger that prevents electron localization in DNA. Both these mechanisms introduce obstacles to further reactions of the excess electron within a DNA strand, e.g. by raising the free energy barrier associated with strand breaks.

Capturing tumor heterogeneity and clonal evolution in solid cancers using circulating tumor DNA analysis.

PubMed

Perdigones, Nieves; Murtaza, Muhammed

2017-06-01

Circulating tumor DNA analysis has emerged as a potential noninvasive alternative to tissue biopsies for tumor genotyping in patients with metastatic cancer. This is particularly attractive in cases where tissue biopsies are contraindicated or repeat genotyping after progression on treatment is required. However, tissue and plasma analysis results are not always concordant and clinical interpretation of discordant results is not completely understood. Discordant results could arise due to analytical limits of assays used for tumor and plasma DNA analysis or due to low overall contribution of tumor-specific DNA in plasma. Once these factors are ruled out, tissue-plasma concordance and quantitative levels of somatic mutations in plasma can capture tumor heterogeneity. During longitudinal follow-up of patients, this feature can be leveraged to track subclonal evolution and to guide combination or sequential adaptive treatment. Here, we summarize recent results evaluating the opportunities and limitations of circulating tumor DNA analysis in the context of tumor heterogeneity and subclonal evolution in patients with advanced cancers. Copyright © 2017 Elsevier Inc. All rights reserved.

Femtomolar detection of single mismatches by discriminant analysis of DNA hybridization events using gold nanoparticles.

PubMed

Ma, Xingyi; Sim, Sang Jun

2013-03-21

Even though DNA-based nanosensors have been demonstrated for quantitative detection of analytes and diseases, hybridization events have never been numerically investigated for further understanding of DNA mediated interactions. Here, we developed a nanoscale platform with well-designed capture and detection gold nanoprobes to precisely evaluate the hybridization events. The capture gold nanoprobes were mono-laid on glass and the detection probes were fabricated via a novel competitive conjugation method. The two kinds of probes combined in a suitable orientation following the hybridization with the target. We found that hybridization efficiency was markedly dependent on electrostatic interactions between DNA strands, which can be tailored by adjusting the salt concentration of the incubation solution. Due to the much lower stability of the double helix formed by mismatches, the hybridization efficiencies of single mismatched (MMT) and perfectly matched DNA (PMT) were different. Therefore, we obtained an optimized salt concentration that allowed for discrimination of MMT from PMT without stringent control of temperature or pH. The results indicated this to be an ultrasensitive and precise nanosensor for the diagnosis of genetic diseases.

Design and characterization of a nanopore-coupled polymerase for single-molecule DNA sequencing by synthesis on an electrode array

PubMed Central

Stranges, P. Benjamin; Palla, Mirkó; Kalachikov, Sergey; Nivala, Jeff; Dorwart, Michael; Trans, Andrew; Kumar, Shiv; Porel, Mintu; Chien, Minchen; Tao, Chuanjuan; Morozova, Irina; Li, Zengmin; Shi, Shundi; Aberra, Aman; Arnold, Cleoma; Yang, Alexander; Aguirre, Anne; Harada, Eric T.; Korenblum, Daniel; Pollard, James; Bhat, Ashwini; Gremyachinskiy, Dmitriy; Bibillo, Arek; Chen, Roger; Davis, Randy; Russo, James J.; Fuller, Carl W.; Roever, Stefan; Ju, Jingyue; Church, George M.

2016-01-01

Scalable, high-throughput DNA sequencing is a prerequisite for precision medicine and biomedical research. Recently, we presented a nanopore-based sequencing-by-synthesis (Nanopore-SBS) approach, which used a set of nucleotides with polymer tags that allow discrimination of the nucleotides in a biological nanopore. Here, we designed and covalently coupled a DNA polymerase to an α-hemolysin (αHL) heptamer using the SpyCatcher/SpyTag conjugation approach. These porin–polymerase conjugates were inserted into lipid bilayers on a complementary metal oxide semiconductor (CMOS)-based electrode array for high-throughput electrical recording of DNA synthesis. The designed nanopore construct successfully detected the capture of tagged nucleotides complementary to a DNA base on a provided template. We measured over 200 tagged-nucleotide signals for each of the four bases and developed a classification method to uniquely distinguish them from each other and background signals. The probability of falsely identifying a background event as a true capture event was less than 1.2%. In the presence of all four tagged nucleotides, we observed sequential additions in real time during polymerase-catalyzed DNA synthesis. Single-polymerase coupling to a nanopore, in combination with the Nanopore-SBS approach, can provide the foundation for a low-cost, single-molecule, electronic DNA-sequencing platform. PMID:27729524

Determination for Enterobacter cloacae based on a europium ternary complex labeled DNA probe

NASA Astrophysics Data System (ADS)

He, Hui; Niu, Cheng-Gang; Zeng, Guang-Ming; Ruan, Min; Qin, Pin-Zhu; Liu, Jing

2011-11-01

The fast detection and accurate diagnosis of the prevalent pathogenic bacteria is very important for the treatment of disease. Nowadays, fluorescence techniques are important tools for diagnosis. A two-probe tandem DNA hybridization assay was designed for the detection of Enterobacter cloacae based on time-resolved fluorescence. In this work, the authors synthesized a novel europium ternary complex Eu(TTA) 3(5-NH 2-phen) with intense luminescence, high fluorescence quantum yield and long lifetime before. We developed a method based on this europium complex for the specific detection of original extracted DNA from E. cloacae. In the hybridization assay format, the reporter probe was labeled with Eu(TTA) 3(5-NH 2-phen) on the 5'-terminus, and the capture probe capture probe was covalent immobilized on the surface of the glutaraldehyde treated glass slides. The original extracted DNA of samples was directly used without any DNA purification and amplification. The detection was conducted by monitoring the fluorescence intensity from the glass surface after DNA hybridization. The detection limit of the DNA was 5 × 10 -10 mol L -1. The results of the present work proved that this new approach was easy to operate with high sensitivity and specificity. It could be conducted as a powerful tool for the detection of pathogen microorganisms in the environment.

Rapid electrochemical assessment of tumor suppressor gene methylations in raw human serum, and tumor cells and tissues using immuno-magnetic beads and selective DNA hybridization.

PubMed

Povedano, Eloy; Valverde, Alejandro; Ruiz-Valdepeñas Montiel, Víctor; Pedrero, María; Yáñez-Sedeño, Paloma; Barderas, Rodrigo; San Segundo-Acosta, Pablo; Peláez-García, Alberto; Mendiola, Marta; Hardisson, David; Campuzano, Susana; Pingarron, José Manuel

2018-05-09

We report a rapid and sensitive electrochemical strategy for the detection of gene-specific 5-methylcytosine DNA methylation. Magnetic beads (MBs) modified with an antibody specific for 5-methylcytosines (5-mC) are employed for the selective capture of any 5-mC methylated single-stranded (ss)DNA sequence. A flanking region next to the 5-mCs of the captured methylated ssDNA is recognized by selective hybridization with a synthetic biotinylated DNA sequence, further labeled with an HRP streptavidin conjugate. Amperometric transduction at disposable screen-printed carbon electrodes (SPCEs) is employed. The developed biosensor exhibits a dynamic range from 3.9 to 500 pM and a detection limit of 1.2 pM for the methylated synthetic sequence of the tumor suppressor gene O-6-methylguanine-DNA methyltransferase (MGMT) promoter region. The applicability of this strategy is demonstrated through the 45 min-analysis of specific methylation in the MGMT promoter region directly in raw spiked human serum samples and in genomic DNA extracted from U-87 glioblastoma cells and paraffin-embedded brain tumor tissues without any amplification and pretreatment step. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Spatial capture-recapture

USGS Publications Warehouse

Royle, J. Andrew; Chandler, Richard B.; Sollmann, Rahel; Gardner, Beth

2013-01-01

Spatial Capture-Recapture provides a revolutionary extension of traditional capture-recapture methods for studying animal populations using data from live trapping, camera trapping, DNA sampling, acoustic sampling, and related field methods. This book is a conceptual and methodological synthesis of spatial capture-recapture modeling. As a comprehensive how-to manual, this reference contains detailed examples of a wide range of relevant spatial capture-recapture models for inference about population size and spatial and temporal variation in demographic parameters. Practicing field biologists studying animal populations will find this book to be a useful resource, as will graduate students and professionals in ecology, conservation biology, and fisheries and wildlife management.

DETECTION OF DNA DAMAGE USING A FIBEROPTIC BIOSENSOR

EPA Science Inventory

A rapid and sensitive fiber optic biosensor assay for radiation-induced DNA damage is reported. For this assay, a biotin-labeled capture oligonucleotide (38 mer) was immobilized to an avidin-coated quartz fiber. Hybridization of a dye-labeled complementary sequence was observed...

Open-target sparse sensing of biological agents using DNA microarray

PubMed Central

2011-01-01

Background Current biosensors are designed to target and react to specific nucleic acid sequences or structural epitopes. These 'target-specific' platforms require creation of new physical capture reagents when new organisms are targeted. An 'open-target' approach to DNA microarray biosensing is proposed and substantiated using laboratory generated data. The microarray consisted of 12,900 25 bp oligonucleotide capture probes derived from a statistical model trained on randomly selected genomic segments of pathogenic prokaryotic organisms. Open-target detection of organisms was accomplished using a reference library of hybridization patterns for three test organisms whose DNA sequences were not included in the design of the microarray probes. Results A multivariate mathematical model based on the partial least squares regression (PLSR) was developed to detect the presence of three test organisms in mixed samples. When all 12,900 probes were used, the model correctly detected the signature of three test organisms in all mixed samples (mean(R2)) = 0.76, CI = 0.95), with a 6% false positive rate. A sampling algorithm was then developed to sparsely sample the probe space for a minimal number of probes required to capture the hybridization imprints of the test organisms. The PLSR detection model was capable of correctly identifying the presence of the three test organisms in all mixed samples using only 47 probes (mean(R2)) = 0.77, CI = 0.95) with nearly 100% specificity. Conclusions We conceived an 'open-target' approach to biosensing, and hypothesized that a relatively small, non-specifically designed, DNA microarray is capable of identifying the presence of multiple organisms in mixed samples. Coupled with a mathematical model applied to laboratory generated data, and sparse sampling of capture probes, the prototype microarray platform was able to capture the signature of each organism in all mixed samples with high sensitivity and specificity. It was demonstrated that this new approach to biosensing closely follows the principles of sparse sensing. PMID:21801424

Oligonucleotide Based Magnetic Bead Capture of Onchocerca volvulus DNA for PCR Pool Screening of Vector Black Flies

PubMed Central

Gopal, Hemavathi; Hassan, Hassan K.; Rodríguez-Pérez, Mario A.; Toé, Laurent D.; Lustigman, Sara; Unnasch, Thomas R.

2012-01-01

Background Entomological surveys of Simulium vectors are an important component in the criteria used to determine if Onchocerca volvulus transmission has been interrupted and if focal elimination of the parasite has been achieved. However, because infection in the vector population is quite rare in areas where control has succeeded, large numbers of flies need to be examined to certify transmission interruption. Currently, this is accomplished through PCR pool screening of large numbers of flies. The efficiency of this process is limited by the size of the pools that may be screened, which is in turn determined by the constraints imposed by the biochemistry of the assay. The current method of DNA purification from pools of vector black flies relies upon silica adsorption. This method can be applied to screen pools containing a maximum of 50 individuals (from the Latin American vectors) or 100 individuals (from the African vectors). Methodology/Principal Findings We have evaluated an alternative method of DNA purification for pool screening of black flies which relies upon oligonucleotide capture of Onchocerca volvulus genomic DNA from homogenates prepared from pools of Latin American and African vectors. The oligonucleotide capture assay was shown to reliably detect one O. volvulus infective larva in pools containing 200 African or Latin American flies, representing a two-four fold improvement over the conventional assay. The capture assay requires an equivalent amount of technical time to conduct as the conventional assay, resulting in a two-four fold reduction in labor costs per insect assayed and reduces reagent costs to $3.81 per pool of 200 flies, or less than $0.02 per insect assayed. Conclusions/Significance The oligonucleotide capture assay represents a substantial improvement in the procedure used to detect parasite prevalence in the vector population, a major metric employed in the process of certifying the elimination of onchocerciasis. PMID:22724041

Laser capture microdissection: Arcturus(XT) infrared capture and UV cutting methods.

PubMed

Gallagher, Rosa I; Blakely, Steven R; Liotta, Lance A; Espina, Virginia

2012-01-01

Laser capture microdissection (LCM) is a technique that allows the precise procurement of enriched cell populations from a heterogeneous tissue under direct microscopic visualization. LCM can be used to harvest the cells of interest directly or can be used to isolate specific cells by ablating the unwanted cells, resulting in histologically enriched cell populations. The fundamental components of laser microdissection technology are (a) visualization of the cells of interest via microscopy, (b) transfer of laser energy to a thermolabile polymer with either the formation of a polymer-cell composite (capture method) or transfer of laser energy via an ultraviolet laser to photovolatize a region of tissue (cutting method), and (c) removal of cells of interest from the heterogeneous tissue section. Laser energy supplied by LCM instruments can be infrared (810 nm) or ultraviolet (355 nm). Infrared lasers melt thermolabile polymers for cell capture, whereas ultraviolet lasers ablate cells for either removal of unwanted cells or excision of a defined area of cells. LCM technology is applicable to an array of applications including mass spectrometry, DNA genotyping and loss-of-heterozygosity analysis, RNA transcript profiling, cDNA library generation, proteomics discovery, and signal kinase pathway profiling. This chapter describes the unique features of the Arcturus(XT) laser capture microdissection instrument, which incorporates both infrared capture and ultraviolet cutting technology in one instrument, using a proteomic downstream assay as a model.

RNA from the 5' end of the R2 retrotransposon controls R2 protein binding to and cleavage of its DNA target site.

PubMed

Christensen, Shawn M; Ye, Junqiang; Eickbush, Thomas H

2006-11-21

Non-LTR retrotransposons insert into eukaryotic genomes by target-primed reverse transcription (TPRT), a process in which cleaved DNA targets are used to prime reverse transcription of the element's RNA transcript. Many of the steps in the integration pathway of these elements can be characterized in vitro for the R2 element because of the rigid sequence specificity of R2 for both its DNA target and its RNA template. R2 retrotransposition involves identical subunits of the R2 protein bound to different DNA sequences upstream and downstream of the insertion site. The key determinant regulating which DNA-binding conformation the protein adopts was found to be a 320-nt RNA sequence from near the 5' end of the R2 element. In the absence of this 5' RNA the R2 protein binds DNA sequences upstream of the insertion site, cleaves the first DNA strand, and conducts TPRT when RNA containing the 3' untranslated region of the R2 transcript is present. In the presence of the 320-nt 5' RNA, the R2 protein binds DNA sequences downstream of the insertion site. Cleavage of the second DNA strand by the downstream subunit does not appear to occur until after the 5' RNA is removed from this subunit. We postulate that the removal of the 5' RNA normally occurs during reverse transcription, and thus provides a critical temporal link to first- and second-strand DNA cleavage in the R2 retrotransposition reaction.

Multimodal RNA-seq using single-strand, double-strand, and CircLigase-based capture yields a refined and extended description of the C. elegans transcriptome.

PubMed

Lamm, Ayelet T; Stadler, Michael R; Zhang, Huibin; Gent, Jonathan I; Fire, Andrew Z

2011-02-01

We have used a combination of three high-throughput RNA capture and sequencing methods to refine and augment the transcriptome map of a well-studied genetic model, Caenorhabditis elegans. The three methods include a standard (non-directional) library preparation protocol relying on cDNA priming and foldback that has been used in several previous studies for transcriptome characterization in this species, and two directional protocols, one involving direct capture of single-stranded RNA fragments and one involving circular-template PCR (CircLigase). We find that each RNA-seq approach shows specific limitations and biases, with the application of multiple methods providing a more complete map than was obtained from any single method. Of particular note in the analysis were substantial advantages of CircLigase-based and ssRNA-based capture for defining sequences and structures of the precise 5' ends (which were lost using the double-strand cDNA capture method). Of the three methods, ssRNA capture was most effective in defining sequences to the poly(A) junction. Using data sets from a spectrum of C. elegans strains and stages and the UCSC Genome Browser, we provide a series of tools, which facilitate rapid visualization and assignment of gene structures.

Transcription and chromatin determinants of de novo DNA methylation timing in oocytes.

PubMed

Gahurova, Lenka; Tomizawa, Shin-Ichi; Smallwood, Sébastien A; Stewart-Morgan, Kathleen R; Saadeh, Heba; Kim, Jeesun; Andrews, Simon R; Chen, Taiping; Kelsey, Gavin

2017-01-01

Gametogenesis in mammals entails profound re-patterning of the epigenome. In the female germline, DNA methylation is acquired late in oogenesis from an essentially unmethylated baseline and is established largely as a consequence of transcription events. Molecular and functional studies have shown that imprinted genes become methylated at different times during oocyte growth; however, little is known about the kinetics of methylation gain genome wide and the reasons for asynchrony in methylation at imprinted loci. Given the predominant role of transcription, we sought to investigate whether transcription timing is rate limiting for de novo methylation and determines the asynchrony of methylation events. Therefore, we generated genome-wide methylation and transcriptome maps of size-selected, growing oocytes to capture the onset and progression of methylation. We find that most sequence elements, including most classes of transposable elements, acquire methylation at similar rates overall. However, methylation of CpG islands (CGIs) is delayed compared with the genome average and there are reproducible differences amongst CGIs in onset of methylation. Although more highly transcribed genes acquire methylation earlier, the major transitions in the oocyte transcriptome occur well before the de novo methylation phase, indicating that transcription is generally not rate limiting in conferring permissiveness to DNA methylation. Instead, CGI methylation timing negatively correlates with enrichment for histone 3 lysine 4 (H3K4) methylation and dependence on the H3K4 demethylases KDM1A and KDM1B, implicating chromatin remodelling as a major determinant of methylation timing. We also identified differential enrichment of transcription factor binding motifs in CGIs acquiring methylation early or late in oocyte growth. By combining these parameters into multiple regression models, we were able to account for about a fifth of the variation in methylation timing of CGIs. Finally, we show that establishment of non-CpG methylation, which is prevalent in fully grown oocytes, and methylation over non-transcribed regions, are later events in oogenesis. These results do not support a major role for transcriptional transitions in the time of onset of DNA methylation in the oocyte, but suggest a model in which sequences least dependent on chromatin remodelling are the earliest to become permissive for methylation.

Programmable display of DNA-protein chimeras for controlling cell-hydrogel interactions via reversible intermolecular hybridization.

PubMed

Zhang, Zhaoyang; Li, Shihui; Chen, Niancao; Yang, Cheng; Wang, Yong

2013-04-08

Extensive studies have been recently carried out to achieve dynamic control of cell-material interactions primarily through physicochemical stimulation. The purpose of this study was to apply reversible intermolecular hybridization to program cell-hydrogel interactions in physiological conditions based on DNA-antibody chimeras and complementary oligonucleotides. The results showed that DNA oligonucleotides could be captured to and released from the immobilizing DNA-functionalized hydrogels with high specificity via DNA hybridization. Accordingly, DNA-antibody chimeras were captured to the hydrogels, successfully inducing specific cell attachment. The cell attachment to the hydrogels reached the plateau at approximately half an hour after the functionalized hydrogels and the cells were incubated together. The attached cells were rapidly released from the bound hydrogels when triggering complementary oligonucleotides were introduced to the system. However, the capability of the triggering complementary oligonucleotides in releasing cells was affected by the length of intermolecular hybridization. The length needed to be at least more than 20 base pairs in the current experimental setting. Notably, because the procedure of intermolecular hybridization did not involve any harsh condition, the released cells maintained the same viability as that of the cultured cells. The functionalized hydrogels also exhibited the potential to catch and release cells repeatedly. Therefore, this study demonstrates that it is promising to regulate cell-material interactions dynamically through the DNA-programmed display of DNA-protein chimeras.

The long (LINEs) and the short (SINEs) of it: altered methylation as a precursor to toxicity.

PubMed

Carnell, Ammie N; Goodman, Jay I

2003-10-01

Although once thought of as "junk" DNA, the importance of interspersed elements in the genome has become increasingly appreciated in recent years. In a broad sense these are collectively referred to as transposable elements, which encompass both transposons and retrotransposons. The latter include long interspersed nuclear elements (LINEs) and short interspersed nuclear elements (SINEs). Expression of these elements leads to genetic instability. Therefore, it is important that they remain transcriptionally silenced, and DNA methylation plays a key role in this regard. A framework for understanding the possible interplay between altered DNA methylation, an epigenetic change, and mutational events is presented. A case is made as to how retrotransposable elements, specifically LINEs and SINEs, are likely to emerge as key players in furthering our understanding of mechanisms underlying a variety of toxicities, including carcinogenesis but not limited to this endpoint.

Marine ecotoxicity of nitramines, transformation products of amine-based carbon capture technology.

PubMed

Coutris, Claire; Macken, Ailbhe L; Collins, Andrew R; El Yamani, Naouale; Brooks, Steven J

2015-09-15

In the context of reducing CO2 emissions to the atmosphere, chemical absorption with amines is emerging as the most advanced technology for post-combustion CO2 capture from exhaust gases of fossil fuel power plants. Despite amine solvent recycling during the capture process, degradation products are formed and released into the environment, among them aliphatic nitramines, for which the environmental impact is unknown. In this study, we determined the acute and chronic toxicity of two nitramines identified as important transformation products of amine-based carbon capture, dimethylnitramine and ethanolnitramine, using a multi-trophic suite of bioassays. The results were then used to produce the first environmental risk assessment for the marine ecosystem. In addition, the in vivo genotoxicity of nitramines was studied by adapting the comet assay to cells from experimentally exposed fish. Overall, based on the whole organism bioassays, the toxicity of both nitramines was considered to be low. The most sensitive response to both compounds was found in oysters, and dimethylnitramine was consistently more toxic than ethanolnitramine in all bioassays. The Predicted No Effect Concentrations for dimethylnitramine and ethanolnitramine were 0.08 and 0.18 mg/L, respectively. The genotoxicity assessment revealed contrasting results to the whole organism bioassays, with ethanolnitramine found to be more genotoxic than dimethylnitramine by three orders of magnitude. At the lowest ethanolnitramine concentration (1mg/L), 84% DNA damage was observed, whereas 100mg/L dimethylnitramine was required to cause 37% DNA damage. The mechanisms of genotoxicity were also shown to differ between the two compounds, with oxidation of the DNA bases responsible for over 90% of the genotoxicity of dimethylnitramine, whereas DNA strand breaks and alkali-labile sites were responsible for over 90% of the genotoxicity of ethanolnitramine. Fish exposed to >3mg/L ethanolnitramine had virtually no DNA left in their red blood cells. Copyright © 2015 Elsevier B.V. All rights reserved.

DNA-aptamers binding aminoglycoside antibiotics.

PubMed

Nikolaus, Nadia; Strehlitz, Beate

2014-02-21

Aptamers are short, single stranded DNA or RNA oligonucleotides that are able to bind specifically and with high affinity to their non-nucleic acid target molecules. This binding reaction enables their application as biorecognition elements in biosensors and assays. As antibiotic residues pose a problem contributing to the emergence of antibiotic-resistant pathogens and thereby reducing the effectiveness of the drug to fight human infections, we selected aptamers targeted against the aminoglycoside antibiotic kanamycin A with the aim of constructing a robust and functional assay that can be used for water analysis. With this work we show that aptamers that were derived from a Capture-SELEX procedure targeting against kanamycin A also display binding to related aminoglycoside antibiotics. The binding patterns differ among all tested aptamers so that there are highly substance specific aptamers and more group specific aptamers binding to a different variety of aminoglycoside antibiotics. Also the region of the aminoglycoside antibiotics responsible for aptamer binding can be estimated. Affinities of the different aptamers for their target substance, kanamycin A, are measured with different approaches and are in the micromolar range. Finally, the proof of principle of an assay for detection of kanamycin A in a real water sample is given.

Emergence of EGFR G724S mutation in EGFR-mutant lung adenocarcinoma post progression on osimertinib.

PubMed

Oztan, A; Fischer, S; Schrock, A B; Erlich, R L; Lovly, C M; Stephens, P J; Ross, J S; Miller, V; Ali, S M; Ou, S-H I; Raez, L E

2017-09-01

Mutations in the epidermal growth factor receptor (EGFR) are drivers for a subset of lung cancers. Osimertinib is a third-generation tyrosine kinase inhibitor (TKI) recently approved for the treatment of T790M-positive non-small cell lung cancer (NSCLC); however, acquired resistance to osimertinib is evident and resistance mechanisms remain incompletely defined. The EGFR G724S mutation was detected using hybrid-capture based comprehensive genomic profiling (CGP) and a hybrid-capture based circulating tumor DNA (ctDNA) assays in two cases of EGFR-driven lung adenocarcinoma in patients who had progressed on osimertinib treatment. This study demonstrates the importance of both tissue and blood based hybrid-capture based genomic profiling at disease progression to identifying novel resistance mechanisms in the clinic. Copyright © 2017 Elsevier B.V. All rights reserved.

Conserved Elements Vaccine for HIV | NCI Technology Transfer Center | TTC

Cancer.gov

Researchers at the National Cancer Institute (NCI) developed a DNA vaccine using conserved elements of HIV-1 Gag, administered in a prime-boost vaccination protocol. Two of the HIV Gag CE DNA vectors have been tested in a rhesus macaque model. Priming with the Gag CE vaccine and boosting with full length Gag DNA showed increased immune responses when compared to vaccination with Gag alone. Researchers seek licensing and/or co-development research collaborations for development this DNA vaccine.

Extracting DNA from 'jaws': high yield and quality from archived tiger shark (Galeocerdo cuvier) skeletal material.

PubMed

Nielsen, E E; Morgan, J A T; Maher, S L; Edson, J; Gauthier, M; Pepperell, J; Holmes, B J; Bennett, M B; Ovenden, J R

2017-05-01

Archived specimens are highly valuable sources of DNA for retrospective genetic/genomic analysis. However, often limited effort has been made to evaluate and optimize extraction methods, which may be crucial for downstream applications. Here, we assessed and optimized the usefulness of abundant archived skeletal material from sharks as a source of DNA for temporal genomic studies. Six different methods for DNA extraction, encompassing two different commercial kits and three different protocols, were applied to material, so-called bio-swarf, from contemporary and archived jaws and vertebrae of tiger sharks (Galeocerdo cuvier). Protocols were compared for DNA yield and quality using a qPCR approach. For jaw swarf, all methods provided relatively high DNA yield and quality, while large differences in yield between protocols were observed for vertebrae. Similar results were obtained from samples of white shark (Carcharodon carcharias). Application of the optimized methods to 38 museum and private angler trophy specimens dating back to 1912 yielded sufficient DNA for downstream genomic analysis for 68% of the samples. No clear relationships between age of samples, DNA quality and quantity were observed, likely reflecting different preparation and storage methods for the trophies. Trial sequencing of DNA capture genomic libraries using 20 000 baits revealed that a significant proportion of captured sequences were derived from tiger sharks. This study demonstrates that archived shark jaws and vertebrae are potential high-yield sources of DNA for genomic-scale analysis. It also highlights that even for similar tissue types, a careful evaluation of extraction protocols can vastly improve DNA yield. © 2016 John Wiley & Sons Ltd.

Persistence of DNA in carcasses, slime and avian feces may affect interpretation of environmental DNA data

USGS Publications Warehouse

Merkes, Christopher M.; McCalla, S. Grace; Jensen, Nathan R.; Gaikowski, Mark P.; Amberg, Jon J.

2014-01-01

The prevention of non-indigenous aquatic invasive species spreading into new areas is a goal of many resource managers. New techniques have been developed to survey for species that are difficult to capture with conventional gears that involve the detection of their DNA in water samples (eDNA). This technique is currently used to track the invasion of bigheaded carps (silver carp and bighead carp; Hypophthalmichthys molitrix and H. nobilis) in the Chicago Area Waterway System and Upper Mississippi River. In both systems DNA has been detected from silver carp without the capture of a live fish, which has led to some uncertainty about the source of the DNA. The potential contribution to eDNA by vectors and fomites has not been explored. Because barges move from areas with a high abundance of bigheaded carps to areas monitored for the potential presence of silver carp, we used juvenile silver carp to simulate the barge transport of dead bigheaded carp carcasses, slime residue, and predator feces to determine the potential of these sources to supply DNA to uninhabited waters where it could be detected and misinterpreted as indicative of the presence of live bigheaded carp. Our results indicate that all three vectors are feasible sources of detectable eDNA for at least one month after their deposition. This suggests that current monitoring programs must consider alternative vectors of DNA in the environment and consider alternative strategies to minimize the detection of DNA not directly released from live bigheaded carps.

Persistence of DNA in Carcasses, Slime and Avian Feces May Affect Interpretation of Environmental DNA Data

PubMed Central

Merkes, Christopher M.; McCalla, S. Grace; Jensen, Nathan R.; Gaikowski, Mark P.; Amberg, Jon J.

2014-01-01

The prevention of non-indigenous aquatic invasive species spreading into new areas is a goal of many resource managers. New techniques have been developed to survey for species that are difficult to capture with conventional gears that involve the detection of their DNA in water samples (eDNA). This technique is currently used to track the invasion of bigheaded carps (silver carp and bighead carp; Hypophthalmichthys molitrix and H. nobilis) in the Chicago Area Waterway System and Upper Mississippi River. In both systems DNA has been detected from silver carp without the capture of a live fish, which has led to some uncertainty about the source of the DNA. The potential contribution to eDNA by vectors and fomites has not been explored. Because barges move from areas with a high abundance of bigheaded carps to areas monitored for the potential presence of silver carp, we used juvenile silver carp to simulate the barge transport of dead bigheaded carp carcasses, slime residue, and predator feces to determine the potential of these sources to supply DNA to uninhabited waters where it could be detected and misinterpreted as indicative of the presence of live bigheaded carp. Our results indicate that all three vectors are feasible sources of detectable eDNA for at least one month after their deposition. This suggests that current monitoring programs must consider alternative vectors of DNA in the environment and consider alternative strategies to minimize the detection of DNA not directly released from live bigheaded carps. PMID:25402206

Persistence of DNA in carcasses, slime and avian feces may affect interpretation of environmental DNA data.

PubMed

Merkes, Christopher M; McCalla, S Grace; Jensen, Nathan R; Gaikowski, Mark P; Amberg, Jon J

2014-01-01

The prevention of non-indigenous aquatic invasive species spreading into new areas is a goal of many resource managers. New techniques have been developed to survey for species that are difficult to capture with conventional gears that involve the detection of their DNA in water samples (eDNA). This technique is currently used to track the invasion of bigheaded carps (silver carp and bighead carp; Hypophthalmichthys molitrix and H. nobilis) in the Chicago Area Waterway System and Upper Mississippi River. In both systems DNA has been detected from silver carp without the capture of a live fish, which has led to some uncertainty about the source of the DNA. The potential contribution to eDNA by vectors and fomites has not been explored. Because barges move from areas with a high abundance of bigheaded carps to areas monitored for the potential presence of silver carp, we used juvenile silver carp to simulate the barge transport of dead bigheaded carp carcasses, slime residue, and predator feces to determine the potential of these sources to supply DNA to uninhabited waters where it could be detected and misinterpreted as indicative of the presence of live bigheaded carp. Our results indicate that all three vectors are feasible sources of detectable eDNA for at least one month after their deposition. This suggests that current monitoring programs must consider alternative vectors of DNA in the environment and consider alternative strategies to minimize the detection of DNA not directly released from live bigheaded carps.

Interactions between the R2R3-MYB Transcription Factor, AtMYB61, and Target DNA Binding Sites

PubMed Central

Prouse, Michael B.; Campbell, Malcolm M.

2013-01-01

Despite the prominent roles played by R2R3-MYB transcription factors in the regulation of plant gene expression, little is known about the details of how these proteins interact with their DNA targets. For example, while Arabidopsis thaliana R2R3-MYB protein AtMYB61 is known to alter transcript abundance of a specific set of target genes, little is known about the specific DNA sequences to which AtMYB61 binds. To address this gap in knowledge, DNA sequences bound by AtMYB61 were identified using cyclic amplification and selection of targets (CASTing). The DNA targets identified using this approach corresponded to AC elements, sequences enriched in adenosine and cytosine nucleotides. The preferred target sequence that bound with the greatest affinity to AtMYB61 recombinant protein was ACCTAC, the AC-I element. Mutational analyses based on the AC-I element showed that ACC nucleotides in the AC-I element served as the core recognition motif, critical for AtMYB61 binding. Molecular modelling predicted interactions between AtMYB61 amino acid residues and corresponding nucleotides in the DNA targets. The affinity between AtMYB61 and specific target DNA sequences did not correlate with AtMYB61-driven transcriptional activation with each of the target sequences. CASTing-selected motifs were found in the regulatory regions of genes previously shown to be regulated by AtMYB61. Taken together, these findings are consistent with the hypothesis that AtMYB61 regulates transcription from specific cis-acting AC elements in vivo. The results shed light on the specifics of DNA binding by an important family of plant-specific transcriptional regulators. PMID:23741471

Development of DNA Pillar Chip Final Report CRADA No. TSB-2035-01

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ness, K. D.; Long, G. W.

This was a collaborative effort between The Regents of the University of California, Lawrence Livermore National Laboratory (LLNL) and Tetracore, to demonstrate a proof of principal device for the capture and controlled release of DNA moving within a flow stream.

Application of immobilized synthetic anti-lipopolysaccharide peptides for the isolation and detection of bacteria.

PubMed

Sandetskaya, N; Engelmann, B; Brandenburg, K; Kuhlmeier, D

2015-08-01

The molecular detection of microorganisms in liquid samples generally requires their enrichment or isolation. The aim of our study was to evaluate the capture and pre-concentration of bacteria by immobilized particular cationic antimicrobial peptides, called synthetic anti-lipopolysaccharide peptides (SALP). For the proof-of-concept and screening of different SALP, the peptides were covalently immobilized on glass slides, and the binding of bacteria was confirmed by microscopic examination of the slides or their scanning, in case of fluorescent bacterial cells. The most efficient SALP was further tethered to magnetic beads. SALP beads were used for the magnetic capture of Escherichia coli in liquid samples. The efficiency of this strategy was evaluated using polymerase chain reaction (PCR). Covalently immobilized SALP were capable of capturing bacteria in liquid samples. However, PCR was hampered by the unspecific binding of DNA to the positively charged peptide. We developed a method for DNA recovery by the enzymatic digestion of the peptide, which allowed for a successful PCR, though the method had its own adverse impact on the detection and, thus, did not allow for the reliable quantitative analysis of the pathogen enrichment. Immobilized SALP can be used as capture molecules for bacteria in liquid samples and can be recommended for the design of the assays or decontamination of the fluids. For the accurate subsequent detection of bacteria, DNA-independent methods should be used.

[The estimation of possibilities for the application of the laser capture microdissection technology for the molecular-genetic expert analysis (genotyping) of human chromosomal DNA].

PubMed

Ivanov, P L; Leonov, S N; Zemskova, E Iu

2012-01-01

The present study was designed to estimate the possibilities of application of the laser capture microdissection (LCM) technology for the molecular-genetic expert analysis (genotyping) of human chromosomal DNA. The experimental method employed for the purpose was the multiplex multilocus analysis of autosomal DNA polymorphism in the preparations of buccal epitheliocytes obtained by LCM. The key principles of the study were the application of physical methods for contrast enhancement of the micropreparations (such as phase-contrast microscopy and dark-field microscopy) and PCR-compatible cell lysis. Genotyping was carried out with the use of AmpFISTR Minifiler TM PCR Amplification Kits ("Applied Biosynthesis", USA). It was shown that the technique employed in the present study ensures reliable genotyping of human chromosomal DNA in the pooled preparations containing 10-20 dissected diploid cells each. This result fairly well agrees with the calculated sensitivity of the method. A few practical recommendations are offered.

Simple diazonium chemistry to develop specific gene sensing platforms.

PubMed

Revenga-Parra, M; García-Mendiola, T; González-Costas, J; González-Romero, E; Marín, A García; Pau, J L; Pariente, F; Lorenzo, E

2014-02-27

A simple strategy for covalent immobilizing DNA sequences, based on the formation of stable diazonized conducting platforms, is described. The electrochemical reduction of 4-nitrobenzenediazonium salt onto screen-printed carbon electrodes (SPCE) in aqueous media gives rise to terminal grafted amino groups. The presence of primary aromatic amines allows the formation of diazonium cations capable to react with the amines present at the DNA capture probe. As a comparison a second strategy based on the binding of aminated DNA capture probes to the developed diazonized conducting platforms through a crosslinking agent was also employed. The resulting DNA sensing platforms were characterized by cyclic voltammetry, electrochemical impedance spectroscopy and spectroscopic ellipsometry. The hybridization event with the complementary sequence was detected using hexaamineruthenium (III) chloride as electrochemical indicator. Finally, they were applied to the analysis of a 145-bp sequence from the human gene MRP3, reaching a detection limit of 210 pg μL(-1). Copyright © 2014 Elsevier B.V. All rights reserved.

Specific RNP capture with antisense LNA/DNA mixmers

PubMed Central

Rogell, Birgit; Fischer, Bernd; Rettel, Mandy; Krijgsveld, Jeroen; Castello, Alfredo; Hentze, Matthias W.

2017-01-01

RNA-binding proteins (RBPs) play essential roles in RNA biology, responding to cellular and environmental stimuli to regulate gene expression. Important advances have helped to determine the (near) complete repertoires of cellular RBPs. However, identification of RBPs associated with specific transcripts remains a challenge. Here, we describe “specific ribonucleoprotein (RNP) capture,” a versatile method for the determination of the proteins bound to specific transcripts in vitro and in cellular systems. Specific RNP capture uses UV irradiation to covalently stabilize protein–RNA interactions taking place at “zero distance.” Proteins bound to the target RNA are captured by hybridization with antisense locked nucleic acid (LNA)/DNA oligonucleotides covalently coupled to a magnetic resin. After stringent washing, interacting proteins are identified by quantitative mass spectrometry. Applied to in vitro extracts, specific RNP capture identifies the RBPs bound to a reporter mRNA containing the Sex-lethal (Sxl) binding motifs, revealing that the Sxl homolog sister of Sex lethal (Ssx) displays similar binding preferences. This method also revealed the repertoire of RBPs binding to 18S or 28S rRNAs in HeLa cells, including previously unknown rRNA-binding proteins. PMID:28476952

Specific RNP capture with antisense LNA/DNA mixmers.

PubMed

Rogell, Birgit; Fischer, Bernd; Rettel, Mandy; Krijgsveld, Jeroen; Castello, Alfredo; Hentze, Matthias W

2017-08-01

RNA-binding proteins (RBPs) play essential roles in RNA biology, responding to cellular and environmental stimuli to regulate gene expression. Important advances have helped to determine the (near) complete repertoires of cellular RBPs. However, identification of RBPs associated with specific transcripts remains a challenge. Here, we describe "specific ribonucleoprotein (RNP) capture," a versatile method for the determination of the proteins bound to specific transcripts in vitro and in cellular systems. Specific RNP capture uses UV irradiation to covalently stabilize protein-RNA interactions taking place at "zero distance." Proteins bound to the target RNA are captured by hybridization with antisense locked nucleic acid (LNA)/DNA oligonucleotides covalently coupled to a magnetic resin. After stringent washing, interacting proteins are identified by quantitative mass spectrometry. Applied to in vitro extracts, specific RNP capture identifies the RBPs bound to a reporter mRNA containing the Sex-lethal (Sxl) binding motifs, revealing that the Sxl homolog sister of Sex lethal (Ssx) displays similar binding preferences. This method also revealed the repertoire of RBPs binding to 18S or 28S rRNAs in HeLa cells, including previously unknown rRNA-binding proteins. © 2017 Rogell et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

Extended HSR/CARD domain mediates AIRE binding to DNA

DOE Office of Scientific and Technical Information (OSTI.GOV)

Maslovskaja, Julia, E-mail: julia.maslovskaja@ut.ee; Saare, Mario; Liiv, Ingrid

Autoimmune regulator (AIRE) activates the transcription of many genes in an unusual promiscuous and stochastic manner. The mechanism by which AIRE binds to the chromatin and DNA is not fully understood, and the regulatory elements that AIRE target genes possess are not delineated. In the current study, we demonstrate that AIRE activates the expression of transiently transfected luciferase reporters that lack defined promoter regions, as well as intron and poly(A) signal sequences. Our protein-DNA interaction experiments with mutated AIRE reveal that the intact homogeneously staining region/caspase recruitment domain (HSR/CARD) and amino acids R113 and K114 are key elements involved inmore » AIRE binding to DNA. - Highlights: • Promoter and mRNA processing elements are not important for AIRE to activate gene expression from reporter plasmids. • AIRE protein fragment aa 1–138 mediates direct binding to DNA. • Integrity of the HSR/CARD domain is needed for AIRE binding to DNA.« less

Interplay between DNA methylation, histone modification and chromatin remodeling in stem cells and during development.

PubMed

Ikegami, Kohta; Ohgane, Jun; Tanaka, Satoshi; Yagi, Shintaro; Shiota, Kunio

2009-01-01

Genes constitute only a small proportion of the mammalian genome, the majority of which is composed of non-genic repetitive elements including interspersed repeats and satellites. A unique feature of the mammalian genome is that there are numerous tissue-dependent, differentially methylated regions (T-DMRs) in the non-repetitive sequences, which include genes and their regulatory elements. The epigenetic status of T-DMRs varies from that of repetitive elements and constitutes the DNA methylation profile genome-wide. Since the DNA methylation profile is specific to each cell and tissue type, much like a fingerprint, it can be used as a means of identification. The formation of DNA methylation profiles is the basis for cell differentiation and development in mammals. The epigenetic status of each T-DMR is regulated by the interplay between DNA methyltransferases, histone modification enzymes, histone subtypes, non-histone nuclear proteins and non-coding RNAs. In this review, we will discuss how these epigenetic factors cooperate to establish cell- and tissue-specific DNA methylation profiles.

Dwarf galaxies: a lab to investigate the neutron capture elements production

NASA Astrophysics Data System (ADS)

Cescutti, Gabriele

2018-06-01

In this contribution, I focus on the neutron capture elements observed in the spectra of old halo and ultra faint galaxies stars. Adopting a stochastic chemical evolution model and the Galactic halo as a benchmark, I present new constraints on the rate and time scales of r-process events, based on the discovery of the r-process rich stars in the ultra faint galaxy Reticulum 2. I also show that an s-process activated by rotation in massive stars can play an important role in the production of heavy elements.

A Simple Method for Amplifying RNA Targets (SMART)

PubMed Central

McCalla, Stephanie E.; Ong, Carmichael; Sarma, Aartik; Opal, Steven M.; Artenstein, Andrew W.; Tripathi, Anubhav

2012-01-01

We present a novel and simple method for amplifying RNA targets (named by its acronym, SMART), and for detection, using engineered amplification probes that overcome existing limitations of current RNA-based technologies. This system amplifies and detects optimal engineered ssDNA probes that hybridize to target RNA. The amplifiable probe-target RNA complex is captured on magnetic beads using a sequence-specific capture probe and is separated from unbound probe using a novel microfluidic technique. Hybridization sequences are not constrained as they are in conventional target-amplification reactions such as nucleic acid sequence amplification (NASBA). Our engineered ssDNA probe was amplified both off-chip and in a microchip reservoir at the end of the separation microchannel using isothermal NASBA. Optimal solution conditions for ssDNA amplification were investigated. Although KCl and MgCl2 are typically found in NASBA reactions, replacing 70 mmol/L of the 82 mmol/L total chloride ions with acetate resulted in optimal reaction conditions, particularly for low but clinically relevant probe concentrations (≤100 fmol/L). With the optimal probe design and solution conditions, we also successfully removed the initial heating step of NASBA, thus achieving a true isothermal reaction. The SMART assay using a synthetic model influenza DNA target sequence served as a fundamental demonstration of the efficacy of the capture and microfluidic separation system, thus bridging our system to a clinically relevant detection problem. PMID:22691910

LINEs between Species: Evolutionary Dynamics of LINE-1 Retrotransposons across the Eukaryotic Tree of Life

PubMed Central

Ivancevic, Atma M.; Kortschak, R. Daniel; Bertozzi, Terry; Adelson, David L.

2016-01-01

LINE-1 (L1) retrotransposons are dynamic elements. They have the potential to cause great genomic change because of their ability to ‘jump’ around the genome and amplify themselves, resulting in the duplication and rearrangement of regulatory DNA. Active L1, in particular, are often thought of as tightly constrained, homologous and ubiquitous elements with well-characterized domain organization. For the past 30 years, model organisms have been used to define L1s as 6–8 kb sequences containing a 5′-UTR, two open reading frames working harmoniously in cis, and a 3′-UTR with a polyA tail. In this study, we demonstrate the remarkable and overlooked diversity of L1s via a comprehensive phylogenetic analysis of elements from over 500 species from widely divergent branches of the tree of life. The rapid and recent growth of L1 elements in mammalian species is juxtaposed against the diverse lineages found in other metazoans and plants. In fact, some of these previously unexplored mammalian species (e.g. snub-nosed monkey, minke whale) exhibit L1 retrotranspositional ‘hyperactivity’ far surpassing that of human or mouse. In contrast, non-mammalian L1s have become so varied that the current classification system seems to inadequately capture their structural characteristics. Our findings illustrate how both long-term inherited evolutionary patterns and random bursts of activity in individual species can significantly alter genomes, highlighting the importance of L1 dynamics in eukaryotes. PMID:27702814

Whole DNA methylome profiling in mice exposed to secondhand smoke.

PubMed

Tommasi, Stella; Zheng, Albert; Yoon, Jae-In; Li, Arthur Xuejun; Wu, Xiwei; Besaratinia, Ahmad

2012-11-01

Aberration of DNA methylation is a prime epigenetic mechanism of carcinogenesis. Aberrant DNA methylation occurs frequently in lung cancer, with exposure to secondhand smoke (SHS) being an established risk factor. The causal role of SHS in the genesis of lung cancer, however, remains elusive. To investigate whether SHS can cause aberrant DNA methylation in vivo, we have constructed the whole DNA methylome in mice exposed to SHS for a duration of 4 mo, both after the termination of exposure and at ensuing intervals post-exposure (up to 10 mo). Our genome-wide and gene-specific profiling of DNA methylation in the lung of SHS-exposed mice revealed that all groups of SHS-exposed mice and controls share a similar pattern of DNA methylation. Furthermore, the methylation status of major repetitive DNA elements, including long-interspersed nuclear elements (LINE L1), intracisternal A particle long-terminal repeat retrotransposons (IAP-LTR), and short-interspersed nuclear elements (SINE B1), in the lung of all groups of SHS-exposed mice and controls remains comparable. The absence of locus-specific gain of DNA methylation and global loss of DNA methylation in the lung of SHS-exposed mice within a timeframe that precedes neoplastic-lesion formation underscore the challenges of lung cancer biomarker development. Identifying the initiating events that cause aberrant DNA methylation in lung carcinogenesis may help improve future strategies for prevention, early detection and treatment of this highly lethal disease.

FAIRE (Formaldehyde-Assisted Isolation of Regulatory Elements) isolates active regulatory elements from human chromatin

PubMed Central

Giresi, Paul G.; Kim, Jonghwan; McDaniell, Ryan M.; Iyer, Vishwanath R.; Lieb, Jason D.

2007-01-01

DNA segments that actively regulate transcription in vivo are typically characterized by eviction of nucleosomes from chromatin and are experimentally identified by their hypersensitivity to nucleases. Here we demonstrate a simple procedure for the isolation of nucleosome-depleted DNA from human chromatin, termed FAIRE (Formaldehyde-Assisted Isolation of Regulatory Elements). To perform FAIRE, chromatin is crosslinked with formaldehyde in vivo, sheared by sonication, and phenol-chloroform extracted. The DNA recovered in the aqueous phase is fluorescently labeled and hybridized to a DNA microarray. FAIRE performed in human cells strongly enriches DNA coincident with the location of DNaseI hypersensitive sites, transcriptional start sites, and active promoters. Evidence for cell-type–specific patterns of FAIRE enrichment is also presented. FAIRE has utility as a positive selection for genomic regions associated with regulatory activity, including regions traditionally detected by nuclease hypersensitivity assays. PMID:17179217

Electrochemical detection of sequence-specific DNA based on formation of G-quadruplex-hemin through continuous hybridization chain reaction.

PubMed

Sun, Xiaofan; Chen, Haohan; Wang, Shuling; Zhang, Yiping; Tian, Yaping; Zhou, Nandi

2018-08-27

A high-sensitive detection of sequence-specific DNA was established based on the formation of G-quadruplex-hemin complex through continuous hybridization chain reaction (HCR). Taking HIV DNA sequence as an example, a capture probe complementary to part of HIV DNA was firstly self-assembled onto the surface of Au electrode. Then a specially designed assistant probe with both terminals complementary to the target DNA and a G-quadruplex-forming sequence in the center was introduced into the detection solution. In the presence of both the target DNA and the assistant probe, the target DNA can be captured on the electrode surface and then a continuous HCR can be conducted due to the mutual recognition of the target DNA and the assistant probe, leading to the formation of a large number of G-quadruplex on the electrode surface. With the help of hemin, a pronounced electrochemical signal can be observed in differential pulse voltammetry (DPV), due to the formation of G-quadruplex-hemin complex. The peak current is linearly related with the logarithm of the concentration of the target DNA in the range from 10 fM to 10 pM. The electrochemical sensor has high selectivity to clearly discriminate single-base mismatched and three-base mismatched sequences from the original HIV DNA sequence. Moreover, the established DNA sensor was challenged by detection of HIV DNA in human serum samples, which showed the low detection limit of 6.3 fM. Thus it has great application prospect in the field of clinical diagnosis and environmental monitoring. Copyright © 2018 Elsevier B.V. All rights reserved.

Functionalization of quantum rods with oligonucleotides for programmable assembly with DNA origami

NASA Astrophysics Data System (ADS)

Doane, Tennyson L.; Alam, Rabeka; Maye, Mathew M.

2015-02-01

The DNA-mediated self-assembly of CdSe/CdS quantum rods (QRs) onto DNA origami is described. Two QR types with unique optical emission and high polarization were synthesized, and then functionalized with oligonucleotides (ssDNA) using a novel protection-deprotection approach, which harnessed ssDNA's tailorable rigidity and denaturation temperature to increase DNA coverage by reducing non-specific coordination and wrapping. The QR assembly was programmable, and occurred at two different assembly zones that had capture strands in parallel alignment. QRs with different optical properties were assembled, opening up future studies on orientation dependent QR FRET. The QR-origami conjugates could be purified via gel electrophoresis and sucrose gradient ultracentrifugation. Assembly yields, QR stoichiometry and orientation, as well as energy transfer implications were studied in light of QR distances, origami flexibility, and conditions.The DNA-mediated self-assembly of CdSe/CdS quantum rods (QRs) onto DNA origami is described. Two QR types with unique optical emission and high polarization were synthesized, and then functionalized with oligonucleotides (ssDNA) using a novel protection-deprotection approach, which harnessed ssDNA's tailorable rigidity and denaturation temperature to increase DNA coverage by reducing non-specific coordination and wrapping. The QR assembly was programmable, and occurred at two different assembly zones that had capture strands in parallel alignment. QRs with different optical properties were assembled, opening up future studies on orientation dependent QR FRET. The QR-origami conjugates could be purified via gel electrophoresis and sucrose gradient ultracentrifugation. Assembly yields, QR stoichiometry and orientation, as well as energy transfer implications were studied in light of QR distances, origami flexibility, and conditions. Electronic supplementary information (ESI) available: Experimental conditions, DNA origami blueprint and sequences, FRET calculations. Additional Fig. S1-S13. See DOI: 10.1039/c4nr07662a

Ultrasensitive and selective signal-on electrochemical DNA detection via exonuclease III catalysis and hybridization chain reaction amplification.

PubMed

Ren, Wang; Gao, Zhong Feng; Li, Nian Bing; Luo, Hong Qun

2015-01-15

This work reported a novel, ultrasensitive, and selective platform for electrochemical detection of DNA, employing an integration of exonuclease III (Exo-III) assisted target recycling and hybridization chain reaction (HCR) for the dual signal amplification strategy. The hairpin capture probe DNA (C-DNA) with an Exo-III 3' overhang end was self-assembled on a gold electrode. In the presence of target DNA (T-DNA), C-DNA hybridized with the T-DNA to form a duplex region, exposing its 5' complementary sequence (initiator). Exo-III was applied to selectively digest duplex region from its 3-hydroxyl termini until the duplex was fully consumed, leaving the remnant initiator. The intact T-DNA spontaneously dissociated from the structure and then initiated the next hybridization process as a result of catalysis of the Exo-III. HCR event was triggered by the initiator and two hairpin helper signal probes labeled with methylene blue, facilitating the polymerization of oligonucleotides into a long nicked dsDNA molecule. The numerous exposed remnant initiators can trigger more HCR events. Because of integration of dual signal amplification and the specific HCR process reaction, the resultant sensor showed a high sensitivity for the detection of the target DNA in a linear range from 1.0 fM to 1.0 nM, and a detection limit as low as 0.2 fM. The proposed dual signal amplification strategy provides a powerful tool for detecting different sequences of target DNA by changing the sequence of capture probe and signal probes, holding a great potential for early diagnosis in gene-related diseases. Copyright © 2014 Elsevier B.V. All rights reserved.

Microbiological Impact on Carbon Capture and Sequestration: Biotic Processes in Natural CO2 Analogue

EPA Science Inventory

Multiple ground-water based microbial community analyses including membrane lipids assays for phospholipid fatty acid and DNA analysis were performed from hydraulically isolated zones. DGGE results from DNA extracts from vertical profiling of the entire depth of aquifer sampled a...

Mineral inversion for element capture spectroscopy logging based on optimization theory

NASA Astrophysics Data System (ADS)

Zhao, Jianpeng; Chen, Hui; Yin, Lu; Li, Ning

2017-12-01

Understanding the mineralogical composition of a formation is an essential key step in the petrophysical evaluation of petroleum reservoirs. Geochemical logging tools can provide quantitative measurements of a wide range of elements. In this paper, element capture spectroscopy (ECS) was taken as an example and an optimization method was adopted to solve the mineral inversion problem for ECS. This method used the converting relationship between elements and minerals as response equations and took into account the statistical uncertainty of the element measurements and established an optimization function for ECS. Objective function value and reconstructed elemental logs were used to check the robustness and reliability of the inversion method. Finally, the inversion mineral results had a good agreement with x-ray diffraction laboratory data. The accurate conversion of elemental dry weights to mineral dry weights formed the foundation for the subsequent applications based on ECS.

Taking Another Look: Zr and Y abundances in Halo Stars

NASA Astrophysics Data System (ADS)

Burris, Debra L.; Jones, E.

2007-12-01

The elements Zirconium and Yttrium are produced via neutron capture (n-capture). These elements reside in the mass range where there is uncertainty about the production mechanism at early time. The rapid n-capture process (r-process) was believed to be responsible for the production, but no study (Burris et al 2000, Gilroy et al 1988 and others) has been able to successfully use the r-process to reproduce the abundance signature for elements in this mass range for metal-poor halo stars. It has been suggested (Sneden and Cowan 2003) that there may be an undiscovered component to the r-process. New abundance calculations for these elements have been conducted for a sample of metal-poor halo stars. Transition probabilities for Zr II from Malcheva et al (2006) and for YII from Hannaford et al (1982) were utilized in these calculations. This work is supported in part by the AAS Small Grant Program, the Arkansas Space Grant Consortium and the UCA Undergraduate Research Council.

Heavy Element Abundances in Planetary Nebulae from Deep Optical Echelle Spectroscopy

NASA Astrophysics Data System (ADS)

Mashburn, Amanda; Sterling, Nicholas C.; Dinerstein, Harriet L.; Garofali, Kristen; Jensema, Rachael; Turbyfill, Amanda; Wieser, Hannah-Marie N.; Reed, Evan C.; Redfield, Seth

2016-01-01

We present the abundances of neutron(n)-capture elements (atomic number Z > 30) and iron determined from deep optical echelle spectroscopy of 14 Galactic planetary nebulae (PNe). The spectra were obtained with the 2D-coudé spectrograph on the 2.7-m Harlan J. Smith telescope at McDonald Observatory. The abundances of n-capture elements can be enhanced in PNe due to slow n-capture nucleosynthesis in the progenitor asymptotic giant branch (AGB) stars. The high spectral resolution of these data (R = 36,700) allow most n-capture element emission lines to be resolved from other nebular and telluric features. We detect Kr in all of the observed PNe (with multiple ions detected in several objects), while Br, Rb, and Xe were each detected in 4--5 objects. Using the new Kr ionization correction factors (ICFs) of Sterling et al. (2015, ApJS, 218, 25), we find [Kr/O] abundances ranging from 0.05 to 1.1 dex. We utilize approximate ICFs for the other n-capture elements, and find slightly lower enrichments for Br and Rb (-0.1 to 0.7 dex), while Xe is enhanced relative to solar by factors of two to 30. The [Xe/Kr] ratios range from -0.3 to 1.4 dex, indicating a significant range in neutron exposures in PN progenitor stars. Interestingly, the largest [Xe/Kr] ratio is found in the thick-disk PN NGC 6644, which has a lower metallicity than the other observed PNe. We detect iron emission lines in all but one target. Fe can be depleted into dust grains in ionized nebulae, and its abundance thus provides key information regarding dust-to-gas ratios and grain destruction processes. We find that [Fe/O] ranges from -1.3 to -0.7 dex in the observed PNe, a smaller spread of depletion factors than found in recent studies (Delgado-Inglada & Rodriguez 2014, ApJ, 784, 173) though this may be due in part to our smaller sample. These data are part of a larger study of heavy elements in PNe, which will provide more accurate determinations of n-capture element abundances than previous estimates in several PNe, thereby providing key new constraints to models of AGB nucleosynthesis and Galactic chemical evolution. This work was supported by NSF awards AST-0708245 and AST-901432.

Multiplexed evaluation of capture agent binding kinetics using arrays of silicon photonic microring resonators.

PubMed

Byeon, Ji-Yeon; Bailey, Ryan C

2011-09-07

High affinity capture agents recognizing biomolecular targets are essential in the performance of many proteomic detection methods. Herein, we report the application of a label-free silicon photonic biomolecular analysis platform for simultaneously determining kinetic association and dissociation constants for two representative protein capture agents: a thrombin-binding DNA aptamer and an anti-thrombin monoclonal antibody. The scalability and inherent multiplexing capability of the technology make it an attractive platform for simultaneously evaluating the binding characteristics of multiple capture agents recognizing the same target antigen, and thus a tool complementary to emerging high-throughput capture agent generation strategies.

Modeling Analyte Transport and Capture in Porous Bead Sensors

PubMed Central

Chou, Jie; Lennart, Alexis; Wong, Jorge; Ali, Mehnaaz F.; Floriano, Pierre N.; Christodoulides, Nicolaos; Camp, James; McDevitt, John T.

2013-01-01

Porous agarose microbeads, with high surface to volume ratios and high binding densities, are attracting attention as highly sensitive, affordable sensor elements for a variety of high performance bioassays. While such polymer microspheres have been extensively studied and reported on previously and are now moving into real-world clinical practice, very little work has been completed to date to model the convection, diffusion, and binding kinetics of soluble reagents captured within such fibrous networks. Here, we report the development of a three-dimensional computational model and provide the initial evidence for its agreement with experimental outcomes derived from the capture and detection of representative protein and genetic biomolecules in 290μm porous beads. We compare this model to antibody-mediated capture of C-reactive protein and bovine serum albumin, along with hybridization of oligonucleotide sequences to DNA probes. These results suggest that due to the porous interior of the agarose bead, internal analyte transport is both diffusion- and convection-based, and regardless of the nature of analyte, the bead interiors reveal an interesting trickle of convection-driven internal flow. Based on this model, the internal to external flow rate ratio is found to be in the range of 1:3100 to 1:170 for beads with agarose concentration ranging from 0.5% to 8% for the sensor ensembles here studied. Further, both model and experimental evidence suggest that binding kinetics strongly affect analyte distribution of captured reagents within the beads. These findings reveal that high association constants create a steep moving boundary in which unbound analytes are held back at the periphery of the bead sensor. Low association constants create a more shallow moving boundary in which unbound analytes diffuse further into the bead before binding. These models agree with experimental evidence and thus serve as a new tool set for the study of bio-agent transport processes within a new class of medical microdevices. PMID:22250703

Perceptual Grouping Allows for Attention to Cover Noncontiguous Locations and Suppress Capture from Nearby Locations

ERIC Educational Resources Information Center

Kerzel, Dirk; Born, Sabine; Schonhammer, Josef

2012-01-01

A salient stimulus may interrupt visual search because of attentional capture. It has been shown that attentional capture occurs with a wide, but not with a small attentional window. We tested the hypothesis that capture depends more strongly on the shape of the attentional window than on its size. Search elements were arranged in two nested…

Isolation of active regulatory elements from eukaryotic chromatin using FAIRE (Formaldehyde Assisted Isolation of Regulatory Elements)

PubMed Central

Giresi, Paul G.; Lieb, Jason D.

2009-01-01

The binding of sequence-specific regulatory factors and the recruitment of chromatin remodeling activities cause nucleosomes to be evicted from chromatin in eukaryotic cells. Traditionally, these active sites have been identified experimentally through their sensitivity to nucleases. Here we describe the details of a simple procedure for the genome-wide isolation of nucleosome-depleted DNA from human chromatin, termed FAIRE (Formaldehyde Assisted Isolation of Regulatory Elements). We also provide protocols for different methods of detecting FAIRE-enriched DNA, including use of PCR, DNA microarrays, and next-generation sequencing. FAIRE works on all eukaryotic chromatin tested to date. To perform FAIRE, chromatin is crosslinked with formaldehyde, sheared by sonication, and phenol-chloroform extracted. Most genomic DNA is crosslinked to nucleosomes and is sequestered to the interphase, whereas DNA recovered in the aqueous phase corresponds to nucleosome-depleted regions of the genome. The isolated regions are largely coincident with the location of DNaseI hypersensitive sites, transcriptional start sites, enhancers, insulators, and active promoters. Given its speed and simplicity, FAIRE has utility in establishing chromatin profiles of diverse cell types in health and disease, isolating DNA regulatory elements en masse for further characterization, and as a screening assay for the effects of small molecules on chromatin organization. PMID:19303047

The Open Cluster Chemical Abundances and Mapping (OCCAM) Survey: Optical Extension for Neutron Capture Elements

NASA Astrophysics Data System (ADS)

Melendez, Matthew; O'Connell, Julia; Frinchaboy, Peter M.; Donor, John; Cunha, Katia M. L.; Shetrone, Matthew D.; Majewski, Steven R.; Zasowski, Gail; Pinsonneault, Marc H.; Roman-Lopes, Alexandre; Stassun, Keivan G.; APOGEE Team

2017-01-01

The Open Cluster Chemical Abundance & Mapping (OCCAM) survey is a systematic survey of Galactic open clusters using data primarily from the SDSS-III/APOGEE-1 survey. However, neutron capture elements are very limited in the IR region covered by APOGEE. In an effort to fully study detailed Galactic chemical evolution, we are conducting a high resolution (R~60,000) spectroscopic abundance analysis of neutron capture elements for OCCAM clusters in the optical regime to complement the APOGEE results. As part of this effort, we present Ba II, La II, Ce II and Eu II results for a few open clusters without previous abundance measurements using data obtained at McDonald Observatory with the 2.1m Otto Struve telescope and Sandiford Echelle Spectrograph.This work is supported by an NSF AAG grant AST-1311835.

Strategy for Extracting DNA from Clay Soil and Detecting a Specific Target Sequence via Selective Enrichment and Real-Time (Quantitative) PCR Amplification ▿

PubMed Central

Yankson, Kweku K.; Steck, Todd R.

2009-01-01

We present a simple strategy for isolating and accurately enumerating target DNA from high-clay-content soils: desorption with buffers, an optional magnetic capture hybridization step, and quantitation via real-time PCR. With the developed technique, μg quantities of DNA were extracted from mg samples of pure kaolinite and a field clay soil. PMID:19633108

A Comparison Study for DNA Motif Modeling on Protein Binding Microarray.

PubMed

Wong, Ka-Chun; Li, Yue; Peng, Chengbin; Wong, Hau-San

2016-01-01

Transcription factor binding sites (TFBSs) are relatively short (5-15 bp) and degenerate. Identifying them is a computationally challenging task. In particular, protein binding microarray (PBM) is a high-throughput platform that can measure the DNA binding preference of a protein in a comprehensive and unbiased manner; for instance, a typical PBM experiment can measure binding signal intensities of a protein to all possible DNA k-mers (k = 8∼10). Since proteins can often bind to DNA with different binding intensities, one of the major challenges is to build TFBS (also known as DNA motif) models which can fully capture the quantitative binding affinity data. To learn DNA motif models from the non-convex objective function landscape, several optimization methods are compared and applied to the PBM motif model building problem. In particular, representative methods from different optimization paradigms have been chosen for modeling performance comparison on hundreds of PBM datasets. The results suggest that the multimodal optimization methods are very effective for capturing the binding preference information from PBM data. In particular, we observe a general performance improvement if choosing di-nucleotide modeling over mono-nucleotide modeling. In addition, the models learned by the best-performing method are applied to two independent applications: PBM probe rotation testing and ChIP-Seq peak sequence prediction, demonstrating its biological applicability.

Simple and Sensitive Quantification of MicroRNAs via PS@Au Microspheres-Based DNA Probes and DSN-Assisted Signal Amplification Platform.

PubMed

Zhao, Qian; Piao, Jiafang; Peng, Weipan; Wang, Yang; Zhang, Bo; Gong, Xiaoqun; Chang, Jin

2018-01-31

Identifying the microRNA (miRNA) expression level can provide critical information for early diagnosis of cancers or monitoring the cancer therapeutic efficacy. This paper focused on a kind of gold-nanoparticle-coated polystyrene microbeads (PS@Au microspheres)-based DNA probe as miRNA capture and duplex-specific nuclease (DSN) signal amplification platform based on an RGB value readout for detection of miRNAs. In virtue of the outstanding selectivity and simple experimental operation, 5'-fluorochrome-labeled molecular beacons (MBs) were immobilized on PS@Au microspheres via their 3'-thiol, in the wake of the fluorescence quenching by nanoparticle surface energy transfer (NSET). Target miRNAs were captured by the PS@Au microspheres-based DNA probe through DNA/RNA hybridization. DSN enzyme subsequently selectively cleaved the DNA to recycle the target miRNA and release of fluorophores, thereby triggering the signal amplification with more free fluorophores. The RGB value measurement enabled a detection limit of 50 fM, almost 4 orders of magnitude lower than PS@Au microspheres-based DNA probe detection without DSN. Meanwhile, by different encoding of dyes, miRNA-21 and miRNA-10b were simultaneously detected in the same sample. Considering the ability for quantitation, high sensitivity, and convenient merits, the PS@Au microspheres-based DNA probe and DSN signal amplification platform supplied valuable information for early diagnosis of cancers.

First Isolates of Leptospira spp., from Rodents Captured in Angola

PubMed Central

Fortes-Gabriel, Elsa; Carreira, Teresa; Vieira, Maria Luísa

2016-01-01

Rodents play an important role in the transmission of pathogenic Leptospira spp. However, in Angola, neither the natural reservoirs of these spirochetes nor leptospirosis diagnosis has been considered. Regarding this gap, we captured rodents in Luanda and Huambo provinces to identify circulating Leptospira spp. Rodent kidney tissue was cultured and DNA amplified and sequenced. Culture isolates were evaluated for pathogenic status and typing with rabbit antisera; polymerase chain reaction (PCR) and sequencing were also performed. A total of 37 rodents were captured: Rattus rattus (15, 40.5%), Rattus norvegicus (9, 24.3%), and Mus musculus (13, 35.2%). Leptospiral DNA was amplified in eight (21.6%) kidney samples. From the cultures, we obtained four (10.8%) Leptospira isolates belonging to the Icterohaemorrhagiae and Ballum serogroups of Leptospira interrogans and Leptospira borgpetersenii genospecies, respectively. This study provides information about circulating leptospires spread by rats and mice in Angola. PMID:26928840

Primary analysis of repeat elements of the Asian seabass (Lates calcarifer) transcriptome and genome

PubMed Central

Kuznetsova, Inna S.; Thevasagayam, Natascha M.; Sridatta, Prakki S. R.; Komissarov, Aleksey S.; Saju, Jolly M.; Ngoh, Si Y.; Jiang, Junhui; Shen, Xueyan; Orbán, László

2014-01-01

As part of our Asian seabass genome project, we are generating an inventory of repeat elements in the genome and transcriptome. The karyotype showed a diploid number of 2n = 24 chromosomes with a variable number of B-chromosomes. The transcriptome and genome of Asian seabass were searched for repetitive elements with experimental and bioinformatics tools. Six different types of repeats constituting 8–14% of the genome were characterized. Repetitive elements were clustered in the pericentromeric heterochromatin of all chromosomes, but some of them were preferentially accumulated in pretelomeric and pericentromeric regions of several chromosomes pairs and have chromosomes specific arrangement. From the dispersed class of fish-specific non-LTR retrotransposon elements Rex1 and MAUI-like repeats were analyzed. They were wide-spread both in the genome and transcriptome, accumulated on the pericentromeric and peritelomeric areas of all chromosomes. Every analyzed repeat was represented in the Asian seabass transcriptome, some showed differential expression between the gonads. The other group of repeats analyzed belongs to the rRNA multigene family. FISH signal for 5S rDNA was located on a single pair of chromosomes, whereas that for 18S rDNA was found on two pairs. A BAC-derived contig containing rDNA was sequenced and assembled into a scaffold containing incomplete fragments of 18S rDNA. Their assembly and chromosomal position revealed that this part of Asian seabass genome is extremely rich in repeats containing evolutionarily conserved and novel sequences. In summary, transcriptome assemblies and cDNA data are suitable for the identification of repetitive DNA from unknown genomes and for comparative investigation of conserved elements between teleosts and other vertebrates. PMID:25120555

Synthesis of amino-rich silica coated magnetic nanoparticles and their application in the capture of DNA for PCR

USDA-ARS?s Scientific Manuscript database

Magnetic separation has great advantages over traditional bioseparation methods and has become popular in the development of methods for the detection of bacterial pathogens, viruses, and transgenic crops. Functionalization of magnetic nanoparticles is a key factor in allowing efficient capture of t...

Detection of DNA Methylation by Whole-Genome Bisulfite Sequencing.

PubMed

Li, Qing; Hermanson, Peter J; Springer, Nathan M

2018-01-01

DNA methylation plays an important role in the regulation of the expression of transposons and genes. Various methods have been developed to assay DNA methylation levels. Bisulfite sequencing is considered to be the "gold standard" for single-base resolution measurement of DNA methylation levels. Coupled with next-generation sequencing, whole-genome bisulfite sequencing (WGBS) allows DNA methylation to be evaluated at a genome-wide scale. Here, we described a protocol for WGBS in plant species with large genomes. This protocol has been successfully applied to assay genome-wide DNA methylation levels in maize and barley. This protocol has also been successfully coupled with sequence capture technology to assay DNA methylation levels in a targeted set of genomic regions.

Detection of an ALK Fusion in Colorectal Carcinoma by Hybrid Capture-Based Assay of Circulating Tumor DNA.

PubMed

Lai, Andrea Z; Schrock, Alexa B; Erlich, Rachel L; Ross, Jeffrey S; Miller, Vincent A; Yakirevich, Evgeny; Ali, Siraj M; Braiteh, Fadi

2017-07-01

ALK rearrangements have been observed in 0.05%-2.5% of patients with colorectal cancers (CRCs) and are predicted to be oncogenic drivers largely mutually exclusive of KRAS, NRAS, or BRAF alterations. Here we present the case of a patient with metastatic CRC who was treatment naïve at the time of molecular testing. Initial ALK immunohistochemistry (IHC) staining was negative, but parallel genomic profiling of both circulating tumor DNA (ctDNA) and tissue using similar hybrid capture-based assays each identified an identical STRN-ALK fusion. Subsequent ALK IHC staining of the same specimens was positive, suggesting that the initial result was a false negative. This report is the first instance of an ALK fusion in CRC detected using a ctDNA assay. Current guidelines for colorectal cancer (CRC) only recommend genomic assessment of KRAS, NRAS, BRAF, and microsatellite instability (MSI) status. ALK rearrangements are rare in CRC, but patients with activating ALK fusions have responded to targeted therapies ALK rearrangements can be detected by genomic profiling of ctDNA from blood or tissue, and this methodology may be informative in cases where immunohistochemistry (IHC) or other standard testing is negative. © AlphaMed Press 2017.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Park, Byoung Yoon; Roberts, Barry L.

The three-dimensional finite element mesh capturing realistic geometries of Bayou Choctaw site has been constructed using the sonar and seismic survey data obtained from the field. The mesh is consisting of hexahedral elements because the salt constitutive model is coded using hexahedral elements. Various ideas and techniques to construct finite element mesh capturing artificially and naturally formed geometries are provided. The techniques to reduce the number of elements as much as possible to save on computer run time with maintaining the computational accuracy is also introduced. The steps and methodologies could be applied to construct the meshes of Big Hill,more » Bryan Mound, and West Hackberry strategic petroleum reserve sites. The methodology could be applied to the complicated shape masses for not only various civil and geological structures but also biological applications such as artificial limbs.« less

Survival-mediated capture and fusion cross sections for heavy-element synthesis

NASA Astrophysics Data System (ADS)

Yao, L.; Loveland, W.

2018-01-01

The cross section for producing a heavy evaporation residue σEVR in a fusion reaction can be written as a product of three nonseparable factors, i.e., the capture cross section, the fusion probability PCN, and the survival probability Wsur. Each of these factors is dependent on the spin. However, one must remember that the Wsur term is zero or very small for higher spin values, thus effectively limiting the capture and fusion terms. For a series of ˜287 reactions leading to heavy evaporation residues with ZCN≤110 , we point out the implications of this fact for capture cross sections for heavy element formation reactions. From a comparison of calculated and measured evaporation residue cross sections we deduce values of the fusion probability PCN for some of these reactions.

Uncoupling of sgRNAs from their associated barcodes during PCR amplification of combinatorial CRISPR screens

PubMed Central

2018-01-01

Many implementations of pooled screens in mammalian cells rely on linking an element of interest to a barcode, with the latter subsequently quantitated by next generation sequencing. However, substantial uncoupling between these paired elements during lentiviral production has been reported, especially as the distance between elements increases. We detail that PCR amplification is another major source of uncoupling, and becomes more pronounced with increased amounts of DNA template molecules and PCR cycles. To lessen uncoupling in systems that use paired elements for detection, we recommend minimizing the distance between elements, using low and equal template DNA inputs for plasmid and genomic DNA during PCR, and minimizing the number of PCR cycles. We also present a vector design for conducting combinatorial CRISPR screens that enables accurate barcode-based detection with a single short sequencing read and minimal uncoupling. PMID:29799876

A more sensitive, efficient and ISO 17025 validated Magnetic Capture real time PCR method for the detection of archetypal Toxoplasma gondii strains in meat.

PubMed

Gisbert Algaba, Ignacio; Geerts, Manon; Jennes, Malgorzata; Coucke, Wim; Opsteegh, Marieke; Cox, Eric; Dorny, Pierre; Dierick, Katelijne; De Craeye, Stéphane

2017-11-01

Toxoplasma gondii is a globally prevalent, zoonotic parasite of major importance to public health. Various indirect and direct methods can be used for the diagnosis of toxoplasmosis. Whereas serological tests are useful to prove contact with the parasite has occurred, the actual presence of the parasite in the tissues of a seropositive animal is not demonstrated. For this, a bioassay is still the reference method. As an alternative, various PCR methods have been developed, but due to the limited amount of sample that can be tested, combined with a low tissue cyst density, those have proved to be insufficiently sensitive. A major improvement of the sensitivity was achieved with magnetic capture-based DNA extraction. By combining the hybridization of specific, biotinylated probes with the capture of those probes with streptavidin-coated paramagnetic beads, T. gondii DNA can selectively be "fished out" from a large volume of meat lysate. Still, several studies showed an insufficient sensitivity compared with the mouse bioassay. Here we present a method that is more sensitive (99% limit of detection: 65.4 tachyzoites per 100g of meat), economical and reliable (ISO 17025 validated) by adding a non-competitive PCR inhibition control (co-capture of cellular r18S) and making the release of the target DNA from the streptavidin-coated paramagnetic beads UV-dependent. The presented results demonstrate the potential of the modified Magnetic Capture real time PCR as a full alternative to the mouse bioassay for the screening of various types of tissues and meat, with the additional advantage of being quantitative. Copyright © 2017 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.

A Phylogenomic Perspective on the Radiation of Ray-Finned Fishes Based upon Targeted Sequencing of Ultraconserved Elements (UCEs)

PubMed Central

Sorenson, Laurie; Santini, Francesco

2013-01-01

Ray-finned fishes constitute the dominant radiation of vertebrates with over 32,000 species. Although molecular phylogenetics has begun to disentangle major evolutionary relationships within this vast section of the Tree of Life, there is no widely available approach for efficiently collecting phylogenomic data within fishes, leaving much of the enormous potential of massively parallel sequencing technologies for resolving major radiations in ray-finned fishes unrealized. Here, we provide a genomic perspective on longstanding questions regarding the diversification of major groups of ray-finned fishes through targeted enrichment of ultraconserved nuclear DNA elements (UCEs) and their flanking sequence. Our workflow efficiently and economically generates data sets that are orders of magnitude larger than those produced by traditional approaches and is well-suited to working with museum specimens. Analysis of the UCE data set recovers a well-supported phylogeny at both shallow and deep time-scales that supports a monophyletic relationship between Amia and Lepisosteus (Holostei) and reveals elopomorphs and then osteoglossomorphs to be the earliest diverging teleost lineages. Our approach additionally reveals that sequence capture of UCE regions and their flanking sequence offers enormous potential for resolving phylogenetic relationships within ray-finned fishes. PMID:23824177

Establishing gene models from the Pinus pinaster genome using gene capture and BAC sequencing.

PubMed

Seoane-Zonjic, Pedro; Cañas, Rafael A; Bautista, Rocío; Gómez-Maldonado, Josefa; Arrillaga, Isabel; Fernández-Pozo, Noé; Claros, M Gonzalo; Cánovas, Francisco M; Ávila, Concepción

2016-02-27

In the era of DNA throughput sequencing, assembling and understanding gymnosperm mega-genomes remains a challenge. Although drafts of three conifer genomes have recently been published, this number is too low to understand the full complexity of conifer genomes. Using techniques focused on specific genes, gene models can be established that can aid in the assembly of gene-rich regions, and this information can be used to compare genomes and understand functional evolution. In this study, gene capture technology combined with BAC isolation and sequencing was used as an experimental approach to establish de novo gene structures without a reference genome. Probes were designed for 866 maritime pine transcripts to sequence genes captured from genomic DNA. The gene models were constructed using GeneAssembler, a new bioinformatic pipeline, which reconstructed over 82% of the gene structures, and a high proportion (85%) of the captured gene models contained sequences from the promoter regulatory region. In a parallel experiment, the P. pinaster BAC library was screened to isolate clones containing genes whose cDNA sequence were already available. BAC clones containing the asparagine synthetase, sucrose synthase and xyloglucan endotransglycosylase gene sequences were isolated and used in this study. The gene models derived from the gene capture approach were compared with the genomic sequences derived from the BAC clones. This combined approach is a particularly efficient way to capture the genomic structures of gene families with a small number of members. The experimental approach used in this study is a valuable combined technique to study genomic gene structures in species for which a reference genome is unavailable. It can be used to establish exon/intron boundaries in unknown gene structures, to reconstruct incomplete genes and to obtain promoter sequences that can be used for transcriptional studies. A bioinformatics algorithm (GeneAssembler) is also provided as a Ruby gem for this class of analyses.

A multiplexed system for quantitative comparisons of chromatin landscapes

PubMed Central

van Galen, Peter; Viny, Aaron D.; Ram, Oren; Ryan, Russell J.H.; Cotton, Matthew J.; Donohue, Laura; Sievers, Cem; Drier, Yotam; Liau, Brian B.; Gillespie, Shawn M.; Carroll, Kaitlin M.; Cross, Michael B.; Levine, Ross L.; Bernstein, Bradley E.

2015-01-01

Genome-wide profiling of histone modifications can provide systematic insight into the regulatory elements and programs engaged in a given cell type. However, conventional chromatin immunoprecipitation and sequencing (ChIP-seq) does not capture quantitative information on histone modification levels, requires large amounts of starting material, and involves tedious processing of each individual sample. Here we address these limitations with a technology that leverages DNA barcoding to profile chromatin quantitatively and in multiplexed format. We concurrently map relative levels of multiple histone modifications across multiple samples, each comprising as few as a thousand cells. We demonstrate the technology by monitoring dynamic changes following inhibition of P300, EZH2 or KDM5, by linking altered epigenetic landscapes to chromatin regulator mutations, and by mapping active and repressive marks in purified human hematopoietic stem cells. Hence, this technology enables quantitative studies of chromatin state dynamics across rare cell types, genotypes, environmental conditions and drug treatments. PMID:26687680

Complex multi-enhancer contacts captured by Genome Architecture Mapping (GAM)

PubMed Central

Beagrie, Robert A.; Scialdone, Antonio; Schueler, Markus; Kraemer, Dorothee C.A.; Chotalia, Mita; Xie, Sheila Q.; Barbieri, Mariano; de Santiago, Inês; Lavitas, Liron-Mark; Branco, Miguel R.; Fraser, James; Dostie, Josée; Game, Laurence; Dillon, Niall; Edwards, Paul A.W.; Nicodemi, Mario; Pombo, Ana

2017-01-01

Summary The organization of the genome in the nucleus and the interactions of genes with their regulatory elements are key features of transcriptional control and their disruption can cause disease. We developed a novel genome-wide method, Genome Architecture Mapping (GAM), for measuring chromatin contacts, and other features of three-dimensional chromatin topology, based on sequencing DNA from a large collection of thin nuclear sections. We apply GAM to mouse embryonic stem cells and identify an enrichment for specific interactions between active genes and enhancers across very large genomic distances, using a mathematical model ‘SLICE’ (Statistical Inference of Co-segregation). GAM also reveals an abundance of three-way contacts genome-wide, especially between regions that are highly transcribed or contain super-enhancers, highlighting a previously inaccessible complexity in genome architecture and a major role for gene-expression specific contacts in organizing the genome in mammalian nuclei. PMID:28273065

Resistive Exercise Device

NASA Technical Reports Server (NTRS)

Smith, Damon C. (Inventor)

2005-01-01

An exercise device 10 is particularly well suited for use in low gravity environments, and includes a frame 12 with plurality of resistance elements 30,82 supported in parallel on the frame. A load transfer member 20 is moveable relative to the frame for transferring the applied force to the free end of each captured resistance element. Load selection template 14 is removably secured both to the load transfer member, and a plurality of capture mechanisms engage the free end of corresponding resistance elements. The force applying mechanism 53 may be a handle, harness or other user interface for applying a force to move the load transfer member.

Automatic detection of spermatozoa for laser capture microdissection.

PubMed

Vandewoestyne, Mado; Van Hoofstat, David; Van Nieuwerburgh, Filip; Deforce, Dieter

2009-03-01

In sexual assault crimes, differential extraction of spermatozoa from vaginal swab smears is often ineffective, especially when only a few spermatozoa are present in an overwhelming amount of epithelial cells. Laser capture microdissection (LCM) enables the precise separation of spermatozoa and epithelial cells. However, standard sperm-staining techniques are non-specific and rely on sperm morphology for identification. Moreover, manual screening of the microscope slides is time-consuming and labor-intensive. Here, we describe an automated screening method to detect spermatozoa stained with Sperm HY-LITER. Different ratios of spermatozoa and epithelial cells were used to assess the automatic detection method. In addition, real postcoital samples were also screened. Detected spermatozoa were isolated using LCM and DNA analysis was performed. Robust DNA profiles without allelic dropout could be obtained from as little as 30 spermatozoa recovered from postcoital samples, showing that the staining had no significant influence on DNA recovery.

Geographical distribution of genetic polymorphism of the pathogen Histoplasma capsulatum isolated from infected bats, captured in a central zone of Mexico.

PubMed

Taylor, Maria Lucia; Chávez-Tapia, Catalina B; Rojas-Martínez, Alberto; del Rocio Reyes-Montes, Maria; del Valle, Mirian Bobadilla; Zúñiga, Gerardo

2005-09-01

Fourteen Histoplasma capsulatum isolates recovered from infected bats captured in Mexican caves and two human H. capsulatum reference strains were analyzed using random amplification of polymorphic DNA PCR-based and partial DNA sequences of four genes. Cluster analysis of random amplification of polymorphic DNA-patterns revealed differences for two H. capsulatum isolates of one migratory bat Tadarida brasiliensis. Three groups were identified by distance and maximum-parsimony analyses of arf, H-anti, ole, and tub1 H. capsulatum genes. Group I included most isolates from infected bats and one clinical strain from central Mexico; group II included the two isolates from T. brasiliensis; the human G-217B reference strain from USA formed an independent group III. Isolates from group II showed diversity in relation to groups I and III, suggesting a different H. capsulatum population.

Whole DNA methylome profiling in mice exposed to secondhand smoke

PubMed Central

Tommasi, Stella; Zheng, Albert; Yoon, Jae-In; Li, Arthur Xuejun; Wu, Xiwei; Besaratinia, Ahmad

2012-01-01

Aberration of DNA methylation is a prime epigenetic mechanism of carcinogenesis. Aberrant DNA methylation occurs frequently in lung cancer, with exposure to secondhand smoke (SHS) being an established risk factor. The causal role of SHS in the genesis of lung cancer, however, remains elusive. To investigate whether SHS can cause aberrant DNA methylation in vivo, we have constructed the whole DNA methylome in mice exposed to SHS for a duration of 4 mo, both after the termination of exposure and at ensuing intervals post-exposure (up to 10 mo). Our genome-wide and gene-specific profiling of DNA methylation in the lung of SHS-exposed mice revealed that all groups of SHS-exposed mice and controls share a similar pattern of DNA methylation. Furthermore, the methylation status of major repetitive DNA elements, including long-interspersed nuclear elements (LINE L1), intracisternal A particle long-terminal repeat retrotransposons (IAP-LTR), and short-interspersed nuclear elements (SINE B1), in the lung of all groups of SHS-exposed mice and controls remains comparable. The absence of locus-specific gain of DNA methylation and global loss of DNA methylation in the lung of SHS-exposed mice within a timeframe that precedes neoplastic-lesion formation underscore the challenges of lung cancer biomarker development. Identifying the initiating events that cause aberrant DNA methylation in lung carcinogenesis may help improve future strategies for prevention, early detection and treatment of this highly lethal disease. PMID:23051858

Structure and conformational dynamics of scaffolded DNA origami nanoparticles

DTIC Science & Technology

2017-05-08

all-atom molecular dynamics and coarse-grained finite element modeling to DX-based nanoparticles to elucidate their fine-scale and global conforma... finite element (FE) modeling approach CanDo is also routinely used to predict the 3D equilibrium conformation of programmed DNA assemblies based on a...model with both experimental cryo-electron microscopy (cryo-EM) data and all-atom modeling. MATERIALS AND METHODS Lattice-free finite element model

Recovery Based Nanowire Field-Effect Transistor Detection of Pathogenic Avian Influenza DNA

NASA Astrophysics Data System (ADS)

Lin, Chih-Heng; Chu, Chia-Jung; Teng, Kang-Ning; Su, Yi-Jr; Chen, Chii-Dong; Tsai, Li-Chu; Yang, Yuh-Shyong

2012-02-01

Fast and accurate diagnosis is critical in infectious disease surveillance and management. We proposed a DNA recovery system that can easily be adapted to DNA chip or DNA biosensor for fast identification and confirmation of target DNA. This method was based on the re-hybridization of DNA target with a recovery DNA to free the DNA probe. Functionalized silicon nanowire field-effect transistor (SiNW FET) was demonstrated to monitor such specific DNA-DNA interaction using high pathogenic strain virus hemagglutinin 1 (H1) DNA of avian influenza (AI) as target. Specific electric changes were observed in real-time for AI virus DNA sensing and device recovery when nanowire surface of SiNW FET was modified with complementary captured DNA probe. The recovery based SiNW FET biosensor can be further developed for fast identification and further confirmation of a variety of influenza virus strains and other infectious diseases.

Challenges of DNA-based mark-recapture studies of American black bears

USGS Publications Warehouse

Settlage, K.E.; Van Manen, F.T.; Clark, J.D.; King, T.L.

2008-01-01

We explored whether genetic sampling would be feasible to provide a region-wide population estimate for American black bears (Ursus americanus) in the southern Appalachians, USA. Specifically, we determined whether adequate capture probabilities (p >0.20) and population estimates with a low coefficient of variation (CV <20%) could be achieved given typical agency budget and personnel constraints. We extracted DNA from hair collected from baited barbed-wire enclosures sampled over a 10-week period on 2 study areas: a high-density black bear population in a portion of Great Smoky Mountains National Park and a lower density population on National Forest lands in North Carolina, South Carolina, and Georgia. We identified individual bears by their unique genotypes obtained from 9 microsatellite loci. We sampled 129 and 60 different bears in the National Park and National Forest study areas, respectively, and applied closed mark–recapture models to estimate population abundance. Capture probabilities and precision of the population estimates were acceptable only for sampling scenarios for which we pooled weekly sampling periods. We detected capture heterogeneity biases, probably because of inadequate spatial coverage by the hair-trapping grid. The logistical challenges of establishing and checking a sufficiently high density of hair traps make DNA-based estimates of black bears impractical for the southern Appalachian region. Alternatives are to estimate population size for smaller areas, estimate population growth rates or survival using mark–recapture methods, or use independent marking and recapturing techniques to reduce capture heterogeneity.

Adaptation of Hybridization Capture of Chromatin-associated Proteins for Proteomics to Mammalian Cells.

PubMed

Guillen-Ahlers, Hector; Rao, Prahlad K; Perumalla, Danu S; Montoya, Maria J; Jadhav, Avinash Y L; Shortreed, Michael R; Smith, Lloyd M; Olivier, Michael

2018-06-01

The hybridization capture of chromatin-associated proteins for proteomics (HyCCAPP) technology was initially developed to uncover novel DNA-protein interactions in yeast. It allows analysis of a target region of interest without the need for prior knowledge about likely proteins bound to the target region. This, in theory, allows HyCCAPP to be used to analyze any genomic region of interest, and it provides sufficient flexibility to work in different cell systems. This method is not meant to study binding sites of known transcription factors, a task better suited for Chromatin Immunoprecipitation (ChIP) and ChIP-like methods. The strength of HyCCAPP lies in its ability to explore DNA regions for which there is limited or no knowledge about the proteins bound to it. It can also be a convenient method to avoid biases (present in ChIP-like methods) introduced by protein-based chromatin enrichment using antibodies. Potentially, HyCCAPP can be a powerful tool to uncover truly novel DNA-protein interactions. To date, the technology has been predominantly applied to yeast cells or to high copy repeat sequences in mammalian cells. In order to become the powerful tool we envision, HyCCAPP approaches need to be optimized to efficiently capture single-copy loci in mammalian cells. Here, we present our adaptation of the initial yeast HyCCAPP capture protocol to human cell lines, and show that single-copy chromatin regions can be efficiently isolated with this modified protocol.

Evaluation of different strategies for magnetic particle functionalization with DNA aptamers.

PubMed

Pérez-Ruiz, Elena; Lammertyn, Jeroen; Spasic, Dragana

2016-12-25

The optimal bio-functionalization of magnetic particles is essential for developing magnetic particle-based bioassays. Whereas functionalization with antibodies is generally well established, immobilization of DNA probes, such as aptamers, is not yet fully explored. In this work, four different types of commercially available magnetic particles, coated with streptavidin, maleimide or carboxyl groups, were evaluated for their surface coverage with aptamer bioreceptors, efficiency in capturing target protein and non-specific protein adsorption on their surface. A recently developed aptamer against the peanut allergen, Ara h 1 protein, was used as a model system. Conjugation of biotinylated Ara h 1 aptamer to the streptavidin particles led to the highest surface coverage, whereas the coverage of maleimide particles was 25% lower. Carboxylated particles appeared to be inadequate for DNA functionalization. Streptavidin particles also showed the greatest target capturing efficiency, comparable to the one of particles functionalized with anti-Ara h 1 antibody. The performance of streptavidin particles was additionally tested in a sandwich assay with the aptamer as a capture receptor on the particle surface. While the limit of detection obtained was comparable to the same assay system with antibody as capture receptor, it was superior to previously reported values using the same aptamer in similar assay schemes with different detection platforms. These results point to the promising application of the Ara h 1 aptamer-functionalized particles in bioassay development. Copyright © 2016 Elsevier B.V. All rights reserved.

Prospecting for Precious Metals in Ultra-Metal-Poor Stars

NASA Astrophysics Data System (ADS)

French, R. S.

2000-05-01

The chemical compositions of the most metal-poor halo stars are living records of the very early nucleosynthetic history of the Galaxy. Only a few prior generations, if not a single one, of element-donating supernovae could have been responsible for the heavy elements observed in ultra-metal-poor (UMP; [Fe/H] < --2.5) stars. Abundances of the heavy neutron-capture elements (Z > 30) can yield direct information about the supernova progenitors to UMP stars, and abundances of unstable thorium and uranium (Z = 90, 92) can potentially provide age estimates for the Galactic halo. Already, many studies have demonstrated that abundances of rare-earth elements (56 <= Z <= 72) in UMP stars are completely consistent with their production in rapid neutron-capture synthesis (r-process) events, usually believed to occur during supernovae explosions. Therefore, mapping the entire abundance pattern of UMP stars is of significant interest. In particular, abundances of the most massive stable elements (Os -> Pb or 76 <= Z <= 82) could provide crucial information about the so-called ``third r-process peak,'' and are critical to the radioactive-dating technique that uses unstable thorium as a chronometer. Until recently, abundance determinations for these elements have been virtually non-existent, as the strongest relevant transitions lay in the vacuum UV, inaccessible to ground-based observation. The availability of high-resolution space-based spectrometers has opened up new regions of spectral coverage, including precisely the range in wavelength needed to make these sensitive measurements. We have undertaken a study of about 10 metal-poor halo giants to determine the abundances of several of the heaviest neutron-capture elements including platinum, osmium, lead, and gold. Preliminary results indicate that the abundance pattern of heavy neutron-capture elements (56 <= Z <= 82) in UMP stars does mimic a scaled solar system r-process. Thus, the ability to estimate the initial abundances of thorium and uranium is greatly reinforced.

DEPPDB - DNA electrostatic potential properties database. Electrostatic properties of genome DNA elements.

PubMed

Osypov, Alexander A; Krutinin, Gleb G; Krutinina, Eugenia A; Kamzolova, Svetlana G

2012-04-01

Electrostatic properties of genome DNA are important to its interactions with different proteins, in particular, related to transcription. DEPPDB - DNA Electrostatic Potential (and other Physical) Properties Database - provides information on the electrostatic and other physical properties of genome DNA combined with its sequence and annotation of biological and structural properties of genomes and their elements. Genomes are organized on taxonomical basis, supporting comparative and evolutionary studies. Currently, DEPPDB contains all completely sequenced bacterial, viral, mitochondrial, and plastids genomes according to the NCBI RefSeq, and some model eukaryotic genomes. Data for promoters, regulation sites, binding proteins, etc., are incorporated from established DBs and literature. The database is complemented by analytical tools. User sequences calculations are available. Case studies discovered electrostatics complementing DNA bending in E.coli plasmid BNT2 promoter functioning, possibly affecting host-environment metabolic switch. Transcription factors binding sites gravitate to high potential regions, confirming the electrostatics universal importance in protein-DNA interactions beyond the classical promoter-RNA polymerase recognition and regulation. Other genome elements, such as terminators, also show electrostatic peculiarities. Most intriguing are gene starts, exhibiting taxonomic correlations. The necessity of the genome electrostatic properties studies is discussed.

Promoter selection in human mitochondria involves binding of a transcription factor to orientation-independent upstream regulatory elements.

PubMed

Fisher, R P; Topper, J N; Clayton, D A

1987-07-17

Selective transcription of human mitochondrial DNA requires a transcription factor (mtTF) in addition to an essentially nonselective RNA polymerase. Partially purified mtTF is able to sequester promoter-containing DNA in preinitiation complexes in the absence of mitochondrial RNA polymerase, suggesting a DNA-binding mechanism for factor activity. Functional domains, required for positive transcriptional regulation by mtTF, are identified within both major promoters of human mtDNA through transcription of mutant promoter templates in a reconstituted in vitro system. These domains are essentially coextensive with DNA sequences protected from nuclease digestion by mtTF-binding. Comparison of the sequences of the two mtTF-responsive elements reveals significant homology only when one sequence is inverted; the binding sites are in opposite orientations with respect to the predominant direction of transcription. Thus mtTF may function bidirectionally, requiring additional protein-DNA interactions to dictate transcriptional polarity. The mtTF-responsive elements are arrayed as direct repeats, separated by approximately 80 bp within the displacement-loop region of human mitochondrial DNA; this arrangement may reflect duplication of an ancestral bidirectional promoter, giving rise to separate, unidirectional promoters for each strand.

A comprehensive list of cloned human DNA sequences

PubMed Central

Schmidtke, Jörg; Cooper, David N.

1987-01-01

A list of DNA sequences cloned from the human genome is presented. Intended as a guide to clone availability, this list includes published reports of cDNA, genomic and synthetic clones comprising gene and pseudogene sequences, uncharacterised DNA segments and repetitive DNA elements. PMID:3575113

A comprehensive list of cloned human DNA sequences

PubMed Central

Schmidtke, Jörg; Cooper, David N.

1990-01-01

A list of DNA sequences cloned from the human genome is presented. Intended as a guide to clone availability, this list includes published reports of cDNA, genomic and synthetic clones comprising gene and pseudogene sequences, uncharacterised DNA segments and repetitive DNA elements. PMID:2333227

A comprehensive list of cloned human DNA sequences

PubMed Central

Schmidtke, Jörg; Cooper, David N.

1988-01-01

A list of DNA sequences cloned from the human genome is presented. Intended as a guide to clone availability, this list includes published reports of cDNA, genomic and synthetic clones comprising gene and pseudogene sequences, uncharacterised DNA segments and repetitive DNA elements. PMID:3368330

A comprehensive list of cloned human DNA sequences

PubMed Central

Schmidtke, Jörg; Cooper, David N.

1989-01-01

A list of DNA sequences cloned from the human genome is presented. Intended as a guide to clone availability, this list includes published reports of cDNA, genomic and synthetic clones comprising gene and pseudogene sequences, uncharacterised DNA segments and repetitive DNA elements. PMID:2654889

Characterisation of cytoplasmic DNA complementary to non-retroviral RNA viruses in human cells

PubMed Central

Shimizu, Akira; Nakatani, Yoko; Nakamura, Takako; Jinno-Oue, Atsushi; Ishikawa, Osamu; Boeke, Jef D.; Takeuchi, Yasuhiro; Hoshino, Hiroo

2014-01-01

The synthesis and subsequent genomic integration of DNA that is complementary to the genomes of non-retroviral RNA viruses are rarely observed. However, upon infection of various human cell lines and primary fibroblasts with the vesicular stomatitis virus (VSV), we detected DNA complementary to the VSV RNA. The VSV DNA was detected in the cytoplasm as single-stranded DNA fully complementary to the viral mRNA from the poly(A) region to the 7-methyl guanosine cap. The formation of this DNA was cell-dependent. Experimentally, we found that the transduction of cells that do not produce VSV DNA with the long interspersed nuclear element 1 and their infection with VSV could lead to the formation of VSV DNA. Viral DNA complementary to other RNA viruses was also detected in the respective infected human cells. Thus, the genetic information of the non-retroviral RNA virus genome can flow into the DNA of mammalian cells expressing LINE-1-like elements. PMID:24875540

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

PubMed Central

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

2016-01-01

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

Mycobacterium smegmatis strain for detection of Mycobacterium tuberculosis by PCR used as internal control for inhibition of amplification and for quantification of bacteria.

PubMed Central

Kolk, A H; Noordhoek, G T; de Leeuw, O; Kuijper, S; van Embden, J D

1994-01-01

For the detection of Mycobacterium tuberculosis by PCR, the IS6110 sequence was used. A modified target was constructed by insertion of 56 nucleotides in the IS6110 insertion element of Mycobacterium bovis BCG. This modified insertion sequence was integrated into the genome of Mycobacterium smegmatis, a mycobacterium species which does not contain the IS6110 element. When DNA from the modified M. smegmatis 1008 strain was amplified with IS6110-specific primers INS1 and INS2, a band of 301 bp was seen on agarose gel, whereas the PCR product of M. tuberculosis complex DNA was a 245-bp fragment with these primers. The addition of a small number of M. smegmatis 1008 cells to clinical samples before DNA purification enables the detection of problems which may be due to the loss of DNA in the isolation procedure or to the presence of inhibitors. The presence of inhibitors of the amplification reaction can be confirmed by the addition of M. smegmatis 1008 DNA after the DNA isolation procedure. Furthermore, competition between the different target DNAs of M. smegmatis 1008 DNA and M. tuberculosis complex DNA enables the estimation of the number of IS6110 elements in the clinical sample. Images PMID:8051267

VEZF1 Elements Mediate Protection from DNA Methylation

PubMed Central

Strogantsev, Ruslan; Gaszner, Miklos; Hair, Alan; Felsenfeld, Gary; West, Adam G.

2010-01-01

There is growing consensus that genome organization and long-range gene regulation involves partitioning of the genome into domains of distinct epigenetic chromatin states. Chromatin insulator or barrier elements are key components of these processes as they can establish boundaries between chromatin states. The ability of elements such as the paradigm β-globin HS4 insulator to block the range of enhancers or the spread of repressive histone modifications is well established. Here we have addressed the hypothesis that a barrier element in vertebrates should be capable of defending a gene from silencing by DNA methylation. Using an established stable reporter gene system, we find that HS4 acts specifically to protect a gene promoter from de novo DNA methylation. Notably, protection from methylation can occur in the absence of histone acetylation or transcription. There is a division of labor at HS4; the sequences that mediate protection from methylation are separable from those that mediate CTCF-dependent enhancer blocking and USF-dependent histone modification recruitment. The zinc finger protein VEZF1 was purified as the factor that specifically interacts with the methylation protection elements. VEZF1 is a candidate CpG island protection factor as the G-rich sequences bound by VEZF1 are frequently found at CpG island promoters. Indeed, we show that VEZF1 elements are sufficient to mediate demethylation and protection of the APRT CpG island promoter from DNA methylation. We propose that many barrier elements in vertebrates will prevent DNA methylation in addition to blocking the propagation of repressive histone modifications, as either process is sufficient to direct the establishment of an epigenetically stable silent chromatin state. PMID:20062523

AP1 Keeps Chromatin Poised for Action | Center for Cancer Research

Cancer.gov

The human genome harbors gene-encoding DNA, the blueprint for building proteins that regulate cellular function. Embedded across the genome, in non-coding regions, are DNA elements to which regulatory factors bind. The interaction of regulatory factors with DNA at these sites modifies gene expression to modulate cell activity. In cells, DNA exists in a complex with proteins called chromatin that compacts the DNA in the nucleus, strongly restricting access to DNA sequences. As a result, regulatory factors only interact with a small subset of their potential binding elements in a given cell to regulate genes. How factors recognize and select sites in chromatin across the genome is not well understood -- but several discoveries in CCR’s Laboratory of Receptor Biology and Gene Expression (LRBGE) have shed light on the mechanisms that direct factors to DNA.

Performance evaluation of a mitogenome capture and Illumina sequencing protocol using non-probative, case-type skeletal samples: Implications for the use of a positive control in a next-generation sequencing procedure.

PubMed

Marshall, Charla; Sturk-Andreaggi, Kimberly; Daniels-Higginbotham, Jennifer; Oliver, Robert Sean; Barritt-Ross, Suzanne; McMahon, Timothy P

2017-11-01

Next-generation ancient DNA technologies have the potential to assist in the analysis of degraded DNA extracted from forensic specimens. Mitochondrial genome (mitogenome) sequencing, specifically, may be of benefit to samples that fail to yield forensically relevant genetic information using conventional PCR-based techniques. This report summarizes the Armed Forces Medical Examiner System's Armed Forces DNA Identification Laboratory's (AFMES-AFDIL) performance evaluation of a Next-Generation Sequencing protocol for degraded and chemically treated past accounting samples. The procedure involves hybridization capture for targeted enrichment of mitochondrial DNA, massively parallel sequencing using Illumina chemistry, and an automated bioinformatic pipeline for forensic mtDNA profile generation. A total of 22 non-probative samples and associated controls were processed in the present study, spanning a range of DNA quantity and quality. Data were generated from over 100 DNA libraries by ten DNA analysts over the course of five months. The results show that the mitogenome sequencing procedure is reliable and robust, sensitive to low template (one ng control DNA) as well as degraded DNA, and specific to the analysis of the human mitogenome. Haplotypes were overall concordant between NGS replicates and with previously generated Sanger control region data. Due to the inherent risk for contamination when working with low-template, degraded DNA, a contamination assessment was performed. The consumables were shown to be void of human DNA contaminants and suitable for forensic use. Reagent blanks and negative controls were analyzed to determine the background signal of the procedure. This background signal was then used to set analytical and reporting thresholds, which were designated at 4.0X (limit of detection) and 10.0X (limit of quantiation) average coverage across the mitogenome, respectively. Nearly all human samples exceeded the reporting threshold, although coverage was reduced in chemically treated samples resulting in a ∼58% passing rate for these poor-quality samples. A concordance assessment demonstrated the reliability of the NGS data when compared to known Sanger profiles. One case sample was shown to be mixed with a co-processed sample and two reagent blanks indicated the presence of DNA above the analytical threshold. This contamination was attributed to sequencing crosstalk from simultaneously sequenced high-quality samples to include the positive control. Overall this study demonstrated that hybridization capture and Illumina sequencing provide a viable method for mitogenome sequencing of degraded and chemically treated skeletal DNA samples, yet may require alternative measures of quality control. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Automated rendezvous and capture development infrastructure

NASA Technical Reports Server (NTRS)

Bryan, Thomas C.; Roe, Fred; Coker, Cynthia

1992-01-01

The facilities at Marshall Space Flight Center and JSC to be utilized to develop and test an autonomous rendezvous and capture (ARC) system are described. This includes equipment and personnel facility capabilities to devise, develop, qualify, and integrate ARC elements and subsystems into flight programs. Attention is given to the use of a LEO test facility, the current concept and unique system elements of the ARC, and the options available to develop ARC technology.

Rapid and label-free electrochemical DNA biosensor for detecting hepatitis A virus.

PubMed

Manzano, Marisa; Viezzi, Sara; Mazerat, Sandra; Marks, Robert S; Vidic, Jasmina

2018-02-15

Diagnostic systems that can deliver highly specific and sensitive detection of hepatitis A virus (HAV) in food and water are of particular interest in many fields including food safety, biosecurity and control of outbreaks. Our aim was the development of an electrochemical method based on DNA hybridization to detect HAV. A ssDNA probe specific for HAV (capture probe) was designed and tested on DNAs from various viral and bacterial samples using Nested-Reverse Transcription Polymerase Chain Reaction (nRT-PCR). To develop the electrochemical device, a disposable gold electrode was functionalized with the specific capture probe and tested on complementary ssDNA and on HAV cDNA. The DNA hybridization on the electrode was measured through the monitoring of the oxidative peak potential of the indicator tripropylamine by cyclic voltammetry. To prevent non-specific binding the gold surface was treated with 3% BSA before detection. High resolution atomic force microscopy (AFM) confirmed the efficiency of electrode functionalization and on-electrode hybridization. The proposed device showed a limit of detection of 0.65pM for the complementary ssDNA and 6.94fg/µL for viral cDNA. For a comparison, nRT-PCR quantified the target HAV cDNA with a limit of detection of 6.4fg/µL. The DNA-sensor developed can be adapted to a portable format to be adopted as an easy-to- use and low cost method for screening HAV in contaminated food and water. In addition, it can be useful for rapid control of HAV infections as it takes only a few minutes to provide the results. Copyright © 2017. Published by Elsevier B.V.

In situ amplified electrochemical aptasensing for sensitive detection of adenosine triphosphate by coupling target-induced hybridization chain reaction with the assembly of silver nanotags.

PubMed

Zhou, Qian; Lin, Youxiu; Lin, Yuping; Wei, Qiaohua; Chen, Guonan; Tang, Dianping

2016-01-01

Biomolecular immobilization and construction of the sensing platform are usually crucial for the successful development of a high-efficiency detection system. Herein we report on a novel and label-free signal-amplified aptasensing for sensitive electrochemical detection of small molecules (adenosine triphosphate, ATP, used in this case) by coupling with target-induced hybridization chain reaction (HCR) and the assembly of electroactive silver nanotags. The system mainly consisted of two alternating hairpin probes, a partial-pairing trigger-aptamer duplex DNA and a capture probe immobilized on the electrode. Upon target ATP introduction, the analyte attacked the aptamer and released the trigger DNA, which was captured by capture DNA immobilized on the electrode to form a newly partial-pairing double-stranded DNA. Thereafter, the exposed domain at trigger DNA could be utilized as the initator strand to open the hairpin probes in sequence, and propagated a chain reaction of hybridization events between two alternating hairpins to form a long nicked double-helix. The electrochemical signal derived from the assembled silver nanotags on the nicked double-helix. Under optimal conditions, the electrochemical aptasensor could exhibit a high sensitivity and a low detection limit, and allowed the detection of ATP at a concentration as low as 0.03 pM. Our design showed a high selectivity for target ATP against its analogs because of the high-specificity ATP-aptamer reaction, and its applicable for monitoring ATP in the spiking serum samples. Improtantly, the distinct advantages of the developed aptasensor make it hold a great potential for the development of simple and robust sensing strategies for the detection of other small molecules by controlling the apatmer sequence. Copyright © 2015 Elsevier B.V. All rights reserved.

Fluorescence imaging of microbe-containing particles shot from a two-stage Light-gas gun into an aerogel.

PubMed

Kawaguchi, Yuko; Sugino, Tomohiro; Tabata, Makoto; Okudaira, Kyoko; Imai, Eichi; Yano, Hajime; Hasegawa, Sunao; Hashimoto, Hirofumi; Yabuta, Hikaru; Kobayashi, Kensei; Kawai, Hideyuki; Mita, Hajime; Yokobori, Shin-ichi; Yamagishi, Akihiko

2014-02-01

We have proposed an experiment (the Tanpopo mission) to capture microbes on the Japan Experimental Module of the International Space Station. An ultra low-density silica aerogel will be exposed to space for more than 1 year. After retrieving the aerogel, particle tracks and particles found in it will be visualized by fluorescence microscopy after staining it with a DNA-specific fluorescence dye. In preparation for this study, we simulated particle trapping in an aerogel so that methods could be developed to visualize the particles and their tracks. During the Tanpopo mission, particles that have an orbital velocity of ~8 km/s are expected to collide with the aerogel. To simulate these collisions, we shot Deinococcus radiodurans-containing Lucentite particles into the aerogel from a two-stage light-gas gun (acceleration 4.2 km/s). The shapes of the captured particles, and their tracks and entrance holes were recorded with a microscope/camera system for further analysis. The size distribution of the captured particles was smaller than the original distribution, suggesting that the particles had fragmented. We were able to distinguish between microbial DNA and inorganic compounds after staining the aerogel with the DNA-specific fluorescence dye SYBR green I as the fluorescence of the stained DNA and the autofluorescence of the inorganic particles decay at different rates. The developed methods are suitable to determine if microbes exist at the International Space Station altitude.

The twilight zone of cis element alignments.

PubMed

Sebastian, Alvaro; Contreras-Moreira, Bruno

2013-02-01

Sequence alignment of proteins and nucleic acids is a routine task in bioinformatics. Although the comparison of complete peptides, genes or genomes can be undertaken with a great variety of tools, the alignment of short DNA sequences and motifs entails pitfalls that have not been fully addressed yet. Here we confront the structural superposition of transcription factors with the sequence alignment of their recognized cis elements. Our goals are (i) to test TFcompare (http://floresta.eead.csic.es/tfcompare), a structural alignment method for protein-DNA complexes; (ii) to benchmark the pairwise alignment of regulatory elements; (iii) to define the confidence limits and the twilight zone of such alignments and (iv) to evaluate the relevance of these thresholds with elements obtained experimentally. We find that the structure of cis elements and protein-DNA interfaces is significantly more conserved than their sequence and measures how this correlates with alignment errors when only sequence information is considered. Our results confirm that DNA motifs in the form of matrices produce better alignments than individual sequences. Finally, we report that empirical and theoretically derived twilight thresholds are useful for estimating the natural plasticity of regulatory sequences, and hence for filtering out unreliable alignments.

The twilight zone of cis element alignments

PubMed Central

Sebastian, Alvaro; Contreras-Moreira, Bruno

2013-01-01

Sequence alignment of proteins and nucleic acids is a routine task in bioinformatics. Although the comparison of complete peptides, genes or genomes can be undertaken with a great variety of tools, the alignment of short DNA sequences and motifs entails pitfalls that have not been fully addressed yet. Here we confront the structural superposition of transcription factors with the sequence alignment of their recognized cis elements. Our goals are (i) to test TFcompare (http://floresta.eead.csic.es/tfcompare), a structural alignment method for protein–DNA complexes; (ii) to benchmark the pairwise alignment of regulatory elements; (iii) to define the confidence limits and the twilight zone of such alignments and (iv) to evaluate the relevance of these thresholds with elements obtained experimentally. We find that the structure of cis elements and protein–DNA interfaces is significantly more conserved than their sequence and measures how this correlates with alignment errors when only sequence information is considered. Our results confirm that DNA motifs in the form of matrices produce better alignments than individual sequences. Finally, we report that empirical and theoretically derived twilight thresholds are useful for estimating the natural plasticity of regulatory sequences, and hence for filtering out unreliable alignments. PMID:23268451

Evolutionary dynamics of selfish DNA explains the abundance distribution of genomic subsequences

PubMed Central

Sheinman, Michael; Ramisch, Anna; Massip, Florian; Arndt, Peter F.

2016-01-01

Since the sequencing of large genomes, many statistical features of their sequences have been found. One intriguing feature is that certain subsequences are much more abundant than others. In fact, abundances of subsequences of a given length are distributed with a scale-free power-law tail, resembling properties of human texts, such as Zipf’s law. Despite recent efforts, the understanding of this phenomenon is still lacking. Here we find that selfish DNA elements, such as those belonging to the Alu family of repeats, dominate the power-law tail. Interestingly, for the Alu elements the power-law exponent increases with the length of the considered subsequences. Motivated by these observations, we develop a model of selfish DNA expansion. The predictions of this model qualitatively and quantitatively agree with the empirical observations. This allows us to estimate parameters for the process of selfish DNA spreading in a genome during its evolution. The obtained results shed light on how evolution of selfish DNA elements shapes non-trivial statistical properties of genomes. PMID:27488939

BEAMing LAMP: single-molecule capture and on-bead isothermal amplification for digital detection of hepatitis C virus in plasma.

PubMed

Chen, Jiyun; Xu, Xiaomin; Huang, Zhimei; Luo, Yuan; Tang, Lijuan; Jiang, Jian-Hui

2018-01-02

A novel dNAD platform (BEAMing LAMP) by combining emulsion micro-reactors, single-molecule magnetic capture and on-bead loop-mediated isothermal amplification has been developed for DNA detection, which enables absolute and high-precision quantification of a target with a detection limit of 300 copies.

DNA methylation dynamics during early plant life.

PubMed

Bouyer, Daniel; Kramdi, Amira; Kassam, Mohamed; Heese, Maren; Schnittger, Arp; Roudier, François; Colot, Vincent

2017-09-25

Cytosine methylation is crucial for gene regulation and silencing of transposable elements in mammals and plants. While this epigenetic mark is extensively reprogrammed in the germline and early embryos of mammals, the extent to which DNA methylation is reset between generations in plants remains largely unknown. Using Arabidopsis as a model, we uncovered distinct DNA methylation dynamics over transposable element sequences during the early stages of plant development. Specifically, transposable elements and their relics show invariably high methylation at CG sites but increasing methylation at CHG and CHH sites. This non-CG methylation culminates in mature embryos, where it reaches saturation for a large fraction of methylated CHH sites, compared to the typical 10-20% methylation level observed in seedlings or adult plants. Moreover, the increase in CHH methylation during embryogenesis matches the hypomethylated state in the early endosperm. Finally, we show that interfering with the embryo-to-seedling transition results in the persistence of high CHH methylation levels after germination, specifically over sequences that are targeted by the RNA-directed DNA methylation (RdDM) machinery. Our findings indicate the absence of extensive resetting of DNA methylation patterns during early plant life and point instead to an important role of RdDM in reinforcing DNA methylation of transposable element sequences in every cell of the mature embryo. Furthermore, we provide evidence that this elevated RdDM activity is a specific property of embryogenesis.

Modeling Progressive Failure of Bonded Joints Using a Single Joint Finite Element

NASA Technical Reports Server (NTRS)

Stapleton, Scott E.; Waas, Anthony M.; Bednarcyk, Brett A.

2010-01-01

Enhanced finite elements are elements with an embedded analytical solution which can capture detailed local fields, enabling more efficient, mesh-independent finite element analysis. In the present study, an enhanced finite element is applied to generate a general framework capable of modeling an array of joint types. The joint field equations are derived using the principle of minimum potential energy, and the resulting solutions for the displacement fields are used to generate shape functions and a stiffness matrix for a single joint finite element. This single finite element thus captures the detailed stress and strain fields within the bonded joint, but it can function within a broader structural finite element model. The costs associated with a fine mesh of the joint can thus be avoided while still obtaining a detailed solution for the joint. Additionally, the capability to model non-linear adhesive constitutive behavior has been included within the method, and progressive failure of the adhesive can be modeled by using a strain-based failure criteria and re-sizing the joint as the adhesive fails. Results of the model compare favorably with experimental and finite element results.

Moving beyond Watson-Crick models of coarse grained DNA dynamics.

PubMed

Linak, Margaret C; Tourdot, Richard; Dorfman, Kevin D

2011-11-28

DNA produces a wide range of structures in addition to the canonical B-form of double-stranded DNA. Some of these structures are stabilized by Hoogsteen bonds. We developed an experimentally parameterized, coarse-grained model that incorporates such bonds. The model reproduces many of the microscopic features of double-stranded DNA and captures the experimental melting curves for a number of short DNA hairpins, even when the open state forms complicated secondary structures. We demonstrate the utility of the model by simulating the folding of a thrombin aptamer, which contains G-quartets, and strand invasion during triplex formation. Our results highlight the importance of including Hoogsteen bonding in coarse-grained models of DNA.

Giant Reverse Transcriptase-Encoding Transposable Elements at Telomeres.

PubMed

Arkhipova, Irina R; Yushenova, Irina A; Rodriguez, Fernando

2017-09-01

Transposable elements are omnipresent in eukaryotic genomes and have a profound impact on chromosome structure, function and evolution. Their structural and functional diversity is thought to be reasonably well-understood, especially in retroelements, which transpose via an RNA intermediate copied into cDNA by the element-encoded reverse transcriptase, and are characterized by a compact structure. Here, we report a novel type of expandable eukaryotic retroelements, which we call Terminons. These elements can attach to G-rich telomeric repeat overhangs at the chromosome ends, in a process apparently facilitated by complementary C-rich repeats at the 3'-end of the RNA template immediately adjacent to a hammerhead ribozyme motif. Terminon units, which can exceed 40 kb in length, display an unusually complex and diverse structure, and can form very long chains, with host genes often captured between units. As the principal polymerizing component, Terminons contain Athena reverse transcriptases previously described in bdelloid rotifers and belonging to the enigmatic group of Penelope-like elements, but can additionally accumulate multiple cooriented ORFs, including DEDDy 3'-exonucleases, GDSL esterases/lipases, GIY-YIG-like endonucleases, rolling-circle replication initiator (Rep) proteins, and putatively structural ORFs with coiled-coil motifs and transmembrane domains. The extraordinary length and complexity of Terminons and the high degree of interfamily variability in their ORF content challenge the current views on the structural organization of eukaryotic retroelements, and highlight their possible connections with the viral world and the implications for the elevated frequency of gene transfer. © The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

A comparison of radiative capture with decay gamma-ray method in bore hole logging for economic minerals

USGS Publications Warehouse

Senftle, F.E.; Moxham, R.M.; Tanner, A.B.

1972-01-01

The recent availability of borehole logging sondes employing a source of neutrons and a Ge(Li) detector opens up the possibility of analyzing either decay or capture gamma rays. The most efficient method for a given element can be predicted by calculating the decay-to-capture count ratio for the most prominent peaks in the respective spectra. From a practical point of view such a calculation must be slanted toward short irradiation and count times at each station in a borehole. A simplified method of computation is shown, and the decay-to-capture count ratio has been calculated and tabulated for the optimum value in the decay mode irrespective of the irradiation time, and also for a ten minute irradiation time. Based on analysis of a single peak in each spectrum, the results indicate the preferred technique and the best decay or capture peak to observe for those elements of economic interest. ?? 1972.

Coarse-grained simulation of DNA using LAMMPS : An implementation of the oxDNA model and its applications.

PubMed

Henrich, Oliver; Gutiérrez Fosado, Yair Augusto; Curk, Tine; Ouldridge, Thomas E

2018-05-10

During the last decade coarse-grained nucleotide models have emerged that allow us to study DNA and RNA on unprecedented time and length scales. Among them is oxDNA, a coarse-grained, sequence-specific model that captures the hybridisation transition of DNA and many structural properties of single- and double-stranded DNA. oxDNA was previously only available as standalone software, but has now been implemented into the popular LAMMPS molecular dynamics code. This article describes the new implementation and analyses its parallel performance. Practical applications are presented that focus on single-stranded DNA, an area of research which has been so far under-investigated. The LAMMPS implementation of oxDNA lowers the entry barrier for using the oxDNA model significantly, facilitates future code development and interfacing with existing LAMMPS functionality as well as other coarse-grained and atomistic DNA models.

Environmental DNA detection of rare and invasive fish species in two Great Lakes tributaries.

PubMed

Balasingham, Katherine D; Walter, Ryan P; Mandrak, Nicholas E; Heath, Daniel D

2018-01-01

The extraction and characterization of DNA from aquatic environmental samples offers an alternative, noninvasive approach for the detection of rare species. Environmental DNA, coupled with PCR and next-generation sequencing ("metabarcoding"), has proven to be very sensitive for the detection of rare aquatic species. Our study used a custom-designed group-specific primer set and next-generation sequencing for the detection of three species at risk (Eastern Sand Darter, Ammocrypta pellucida; Northern Madtom, Noturus stigmosus; and Silver Shiner, Notropis photogenis), one invasive species (Round Goby, Neogobius melanostomus) and an additional 78 native species from two large Great Lakes tributary rivers in southern Ontario, Canada: the Grand River and the Sydenham River. Of 82 fish species detected in both rivers using capture-based and eDNA methods, our eDNA method detected 86.2% and 72.0% of the fish species in the Grand River and the Sydenham River, respectively, which included our four target species. Our analyses also identified significant positive and negative species co-occurrence patterns between our target species and other identified species. Our results demonstrate that eDNA metabarcoding that targets the fish community as well as individual species of interest provides a better understanding of factors affecting the target species spatial distribution in an ecosystem than possible with only target species data. Additionally, eDNA is easily implemented as an initial survey tool, or alongside capture-based methods, for improved mapping of species distribution patterns. © 2017 John Wiley & Sons Ltd.

Detection of single-nucleotide polymorphisms using an ON-OFF switching of regenerated biosensor based on a locked nucleic acid-integrated and toehold-mediated strand displacement reaction.

PubMed

Gao, Zhong Feng; Ling, Yu; Lu, Lu; Chen, Ning Yu; Luo, Hong Qun; Li, Nian Bing

2014-03-04

Although various strategies have been reported for single-nucleotide polymorphisms (SNPs) detection, development of a time-saving, specific, and regenerated electrochemical sensing platform still remains a realistic goal. In this study, an ON-OFF switching of a regenerated biosensor based on a locked nucleic acid (LNA)-integrated and toehold-mediated strand displacement reaction technique is constructed for detection of SNPs. The LNA-integrated and methylene blue-labeled capture probe with an external toehold is designed to switch on the sensing system. The mutant-type DNA probe completes complementary with the capture probe to trigger the strand displacement reaction, which switches off the sensing system. However, when the single-base mismatched wild-type DNA probe is presented, the strand displacement reaction cannot be achieved; therefore, the sensing system still keeps the ON state. This DNA sensor is stable over five reuses. We further testify that the LNA-integrated sequence has better recognition ability for SNPs detection compared to the DNA-integrated sequence. Moreover, this DNA senor exhibits a remarkable discrimination capability of SNPs among abundant wild-type targets and 6000-fold (m/m) excess of genomic DNA. In addition, it is selective enough in complex and contaminant-ridden samples, such as human urine, soil, saliva, and beer. Overall, these results demonstrate that this reliable DNA sensor is easy to be fabricated, simple to operate, and stable enough to be readily regenerated.

Turnover of R1 (Type I) and R2 (Type Ii) Retrotransposable Elements in the Ribosomal DNA of Drosophila Melanogaster

PubMed Central

Jakubczak, J. L.; Zenni, M. K.; Woodruff, R. C.; Eickbush, T. H.

1992-01-01

R1 and R2 are distantly related non-long terminal repeat retrotransposable elements each of which inserts into a specific site in the 28S rRNA genes of most insects. We have analyzed aspects of R1 and R2 abundance and sequence variation in 27 geographical isolates of Drosophila melanogaster. The fraction of 28S rRNA genes containing these elements varied greatly between strains, 17-67% for R1 elements and 2-28% for R2 elements. The total percentage of the rDNA repeats inserted ranged from 32 to 77%. The fraction of the rDNA repeats that contained both of these elements suggested that R1 and R2 exhibit neither an inhibition of nor preference for insertion into a 28S gene already containing the other type of element. Based on the conservation of restriction sites in the elements of all strains, and sequence analysis of individual elements from three strains, nucleotide divergence is very low for R1 and R2 elements within or between strains (<0.6%). This sequence uniformity is the expected result of the forces of concerted evolution (unequal crossovers and gene conversion) which act on the rRNA genes themselves. Evidence for the role of retrotransposition in the turnover of R1 and R2 was obtained by using naturally occurring 5' length polymorphisms of the elements as markers for independent transposition events. The pattern of these different length 5' truncations of R1 and R2 was found to be diverse and unique to most strains analyzed. Because recombination can only, with time, amplify or eliminate those length variants already present, the diversity found in each strain suggests that retrotransposition has played a critical role in maintaining these elements in the rDNA repeats of D. melanogaster. PMID:1317313

DNA Vaccine for West Nile Virus Infection in Fish Crows (Corvus ossifragus)

DTIC Science & Technology

2003-09-01

SUBJECT TERMS west Nile virus, vaccine , efficacy , crows 16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF ABSTRACT SAR 18. NUMBER OF PAGES 5 19a...A DNA vaccine for West Nile virus (WNV) was evaluat- ed to determine whether its use could protect fish crows (Corvus ossifragus) from fatal WNV...infection. Captured adult crows were given 0.5 mg of the DNA vaccine either orally or by intramuscular (IM) inoculation; control crows were inoculated or

Enumeration and targeted analysis of KRAS, BRAF and PIK3CA mutations in CTCs captured by a label-free platform: Comparison to ctDNA and tissue in metastatic colorectal cancer.

PubMed

Kidess-Sigal, Evelyn; Liu, Haiyan E; Triboulet, Melanie M; Che, James; Ramani, Vishnu C; Visser, Brendan C; Poultsides, George A; Longacre, Teri A; Marziali, Andre; Vysotskaia, Valentina; Wiggin, Matthew; Heirich, Kyra; Hanft, Violet; Keilholz, Ulrich; Tinhofer, Ingeborg; Norton, Jeffrey A; Lee, Mark; Sollier-Christen, Elodie; Jeffrey, Stefanie S

2016-12-20

Treatment of advanced colorectal cancer (CRC) requires multimodal therapeutic approaches and need for monitoring tumor plasticity. Liquid biopsy biomarkers, including CTCs and ctDNA, hold promise for evaluating treatment response in real-time and guiding therapeutic modifications. From 15 patients with advanced CRC undergoing liver metastasectomy with curative intent, we collected 41 blood samples at different time points before and after surgery for CTC isolation and quantification using label-free Vortex technology. For mutational profiling, KRAS, BRAF, and PIK3CA hotspot mutations were analyzed in CTCs and ctDNA from 23 samples, nine matched liver metastases and three primary tumor samples. Mutational patterns were compared. 80% of patient blood samples were positive for CTCs, using a healthy baseline value as threshold (0.4 CTCs/mL), and 81.4% of captured cells were EpCAM+ CTCs. At least one mutation was detected in 78% of our blood samples. Among 23 matched CTC and ctDNA samples, we found a concordance of 78.2% for KRAS, 73.9% for BRAF and 91.3% for PIK3CA mutations. In several cases, CTCs exhibited a mutation that was not detected in ctDNA, and vice versa. Complementary assessment of both CTCs and ctDNA appears advantageous to assess dynamic tumor profiles.

A new family of polymerases related to superfamily A DNA polymerases and T7-like DNA-dependent RNA polymerases.

PubMed

Iyer, Lakshminarayan M; Abhiman, Saraswathi; Aravind, L

2008-10-04

Using sequence profile methods and structural comparisons we characterize a previously unknown family of nucleic acid polymerases in a group of mobile elements from genomes of diverse bacteria, an algal plastid and certain DNA viruses, including the recently reported Sputnik virus. Using contextual information from domain architectures and gene-neighborhoods we present evidence that they are likely to possess both primase and DNA polymerase activity, comparable to the previously reported prim-pol proteins. These newly identified polymerases help in defining the minimal functional core of superfamily A DNA polymerases and related RNA polymerases. Thus, they provide a framework to understand the emergence of both DNA and RNA polymerization activity in this class of enzymes. They also provide evidence that enigmatic DNA viruses, such as Sputnik, might have emerged from mobile elements coding these polymerases.

A new family of polymerases related to superfamily A DNA polymerases and T7-like DNA-dependent RNA polymerases

PubMed Central

Iyer, Lakshminarayan M; Abhiman, Saraswathi; Aravind, L

2008-01-01

Using sequence profile methods and structural comparisons we characterize a previously unknown family of nucleic acid polymerases in a group of mobile elements from genomes of diverse bacteria, an algal plastid and certain DNA viruses, including the recently reported Sputnik virus. Using contextual information from domain architectures and gene-neighborhoods we present evidence that they are likely to possess both primase and DNA polymerase activity, comparable to the previously reported prim-pol proteins. These newly identified polymerases help in defining the minimal functional core of superfamily A DNA polymerases and related RNA polymerases. Thus, they provide a framework to understand the emergence of both DNA and RNA polymerization activity in this class of enzymes. They also provide evidence that enigmatic DNA viruses, such as Sputnik, might have emerged from mobile elements coding these polymerases. This article was reviewed by Eugene Koonin and Mark Ragan. PMID:18834537

Structure, replication efficiency and fragility of yeast ARS elements.

PubMed

Dhar, Manoj K; Sehgal, Shelly; Kaul, Sanjana

2012-05-01

DNA replication in eukaryotes initiates at specific sites known as origins of replication, or replicators. These replication origins occur throughout the genome, though the propensity of their occurrence depends on the type of organism. In eukaryotes, zones of initiation of replication spanning from about 100 to 50,000 base pairs have been reported. The characteristics of eukaryotic replication origins are best understood in the budding yeast Saccharomyces cerevisiae, where some autonomously replicating sequences, or ARS elements, confer origin activity. ARS elements are short DNA sequences of a few hundred base pairs, identified by their efficiency at initiating a replication event when cloned in a plasmid. ARS elements, although structurally diverse, maintain a basic structure composed of three domains, A, B and C. Domain A is comprised of a consensus sequence designated ACS (ARS consensus sequence), while the B domain has the DNA unwinding element and the C domain is important for DNA-protein interactions. Although there are ∼400 ARS elements in the yeast genome, not all of them are active origins of replication. Different groups within the genus Saccharomyces have ARS elements as components of replication origin. The present paper provides a comprehensive review of various aspects of ARSs, starting from their structural conservation to sequence thermodynamics. All significant and conserved functional sequence motifs within different types of ARS elements have been extensively described. Issues like silencing at ARSs, their inherent fragility and factors governing their replication efficiency have also been addressed. Progress in understanding crucial components associated with the replication machinery and timing at these ARS elements is discussed in the section entitled "The replicon revisited". Copyright © 2012 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

A Microwell-Printing Fabrication Strategy for the On-Chip Templated Biosynthesis of Protein Microarrays for Surface Plasmon Resonance Imaging

PubMed Central

Manuel, Gerald; Lupták, Andrej; Corn, Robert M.

2017-01-01

A two-step templated, ribosomal biosynthesis/printing method for the fabrication of protein microarrays for surface plasmon resonance imaging (SPRI) measurements is demonstrated. In the first step, a sixteen component microarray of proteins is created in microwells by cell free on chip protein synthesis; each microwell contains both an in vitro transcription and translation (IVTT) solution and 350 femtomoles of a specific DNA template sequence that together are used to create approximately 40 picomoles of a specific hexahistidine-tagged protein. In the second step, the protein microwell array is used to contact print one or more protein microarrays onto nitrilotriacetic acid (NTA)-functionalized gold thin film SPRI chips for real-time SPRI surface bioaffinity adsorption measurements. Even though each microwell array element only contains approximately 40 picomoles of protein, the concentration is sufficiently high for the efficient bioaffinity adsorption and capture of the approximately 100 femtomoles of hexahistidine-tagged protein required to create each SPRI microarray element. As a first example, the protein biosynthesis process is verified with fluorescence imaging measurements of a microwell array containing His-tagged green fluorescent protein (GFP), yellow fluorescent protein (YFP) and mCherry (RFP), and then the fidelity of SPRI chips printed from this protein microwell array is ascertained by measuring the real-time adsorption of various antibodies specific to these three structurally related proteins. This greatly simplified two-step synthesis/printing fabrication methodology eliminates most of the handling, purification and processing steps normally required in the synthesis of multiple protein probes, and enables the rapid fabrication of SPRI protein microarrays from DNA templates for the study of protein-protein bioaffinity interactions. PMID:28706572

Laser Capture Microdissection for Protein and NanoString RNA analysis

PubMed Central

Golubeva, Yelena; Salcedo, Rosalba; Mueller, Claudius; Liotta, Lance A.; Espina, Virginia

2013-01-01

Laser capture microdissection (LCM) allows the precise procurement of enriched cell populations from a heterogeneous tissue, or live cell culture, under direct microscopic visualization. Histologically enriched cell populations can be procured by harvesting cells of interest directly, or isolating specific cells by ablating unwanted cells. The basic components of laser microdissection technology are a) visualization of cells via light microscopy, b) transfer of laser energy to a thermolabile polymer with either the formation of a polymer-cell composite (capture method) or transfer of laser energy via an ultraviolet laser to photovolatize a region of tissue (cutting method), and c) removal of cells of interest from the heterogeneous tissue section. The capture and cutting methods (instruments) for laser microdissection differ in the manner by which cells of interest are removed from the heterogeneous sample. Laser energy in the capture method is infrared (810nm), while in the cutting mode the laser is ultraviolet (355nm). Infrared lasers melt a thermolabile polymer that adheres to the cells of interest, whereas ultraviolet lasers ablate cells for either removal of unwanted cells or excision of a defined area of cells. LCM technology is applicable to an array of applications including mass spectrometry, DNA genotyping and loss-of-heterozygosity analysis, RNA transcript profiling, cDNA library generation, proteomics discovery, and signal kinase pathway profiling. This chapter describes laser capture microdissection using an ArcturusXT instrument for protein LCM sample analysis, and using a mmi CellCut Plus® instrument for RNA analysis via NanoString technology. PMID:23027006

Phylogenomics of Phrynosomatid Lizards: Conflicting Signals from Sequence Capture versus Restriction Site Associated DNA Sequencing

PubMed Central

Leaché, Adam D.; Chavez, Andreas S.; Jones, Leonard N.; Grummer, Jared A.; Gottscho, Andrew D.; Linkem, Charles W.

2015-01-01

Sequence capture and restriction site associated DNA sequencing (RADseq) are popular methods for obtaining large numbers of loci for phylogenetic analysis. These methods are typically used to collect data at different evolutionary timescales; sequence capture is primarily used for obtaining conserved loci, whereas RADseq is designed for discovering single nucleotide polymorphisms (SNPs) suitable for population genetic or phylogeographic analyses. Phylogenetic questions that span both “recent” and “deep” timescales could benefit from either type of data, but studies that directly compare the two approaches are lacking. We compared phylogenies estimated from sequence capture and double digest RADseq (ddRADseq) data for North American phrynosomatid lizards, a species-rich and diverse group containing nine genera that began diversifying approximately 55 Ma. Sequence capture resulted in 584 loci that provided a consistent and strong phylogeny using concatenation and species tree inference. However, the phylogeny estimated from the ddRADseq data was sensitive to the bioinformatics steps used for determining homology, detecting paralogs, and filtering missing data. The topological conflicts among the SNP trees were not restricted to any particular timescale, but instead were associated with short internal branches. Species tree analysis of the largest SNP assembly, which also included the most missing data, supported a topology that matched the sequence capture tree. This preferred phylogeny provides strong support for the paraphyly of the earless lizard genera Holbrookia and Cophosaurus, suggesting that the earless morphology either evolved twice or evolved once and was subsequently lost in Callisaurus. PMID:25663487

Transcriptional activation of short interspersed elements by DNA-damaging agents.

PubMed

Rudin, C M; Thompson, C B

2001-01-01

Short interspersed elements (SINEs), typified by the human Alu repeat, are RNA polymerase III (pol III)-transcribed sequences that replicate within the genome through an RNA intermediate. Replication of SINEs has been extensive in mammalian evolution: an estimated 5% of the human genome consists of Alu repeats. The mechanisms regulating transcription, reverse transcription, and reinsertion of SINE elements in genomic DNA are poorly understood. Here we report that expression of murine SINE transcripts of both the B1 and B2 classes is strongly upregulated after prolonged exposure to cisplatin, etoposide, or gamma radiation. A similar induction of Alu transcripts in human cells occurs under these conditions. This induction is not due to a general upregulation of pol III activity in either species. Genotoxic treatment of murine cells containing an exogenous human Alu element induced Alu transcription. Concomitant with the increased expression of SINEs, an increase in cellular reverse transcriptase was observed after exposure to these same DNA-damaging agents. These findings suggest that genomic damage may be an important activator of SINEs, and that SINE mobility may contribute to secondary malignancy after exposure to DNA-damaging chemotherapy.

Functional characterization of piggyBat from the bat Myotis lucifugus unveils an active mammalian DNA transposon.

PubMed

Mitra, Rupak; Li, Xianghong; Kapusta, Aurélie; Mayhew, David; Mitra, Robi D; Feschotte, Cédric; Craig, Nancy L

2013-01-02

A revelation of the genomic age has been the contributions of the mobile DNA segments called transposable elements to chromosome structure, function, and evolution in virtually all organisms. Substantial fractions of vertebrate genomes derive from transposable elements, being dominated by retroelements that move via RNA intermediates. Although many of these elements have been inactivated by mutation, several active retroelements remain. Vertebrate genomes also contain substantial quantities and a high diversity of cut-and-paste DNA transposons, but no active representative of this class has been identified in mammals. Here we show that a cut-and-paste element called piggyBat, which has recently invaded the genome of the little brown bat (Myotis lucifugus) and is a member of the piggyBac superfamily, is active in its native form in transposition assays in bat and human cultured cells, as well as in the yeast Saccharomyces cerevisiae. Our study suggests that some DNA transposons are still actively shaping some mammalian genomes and reveals an unprecedented opportunity to study the mechanism, regulation, and genomic impact of cut-and-paste transposition in a natural mammalian host.

Progress in directed energy control of vectors for microbes and other cells

NASA Astrophysics Data System (ADS)

Kiel, Johnathan L.; Parker, Jill E.; Holwitt, Eric A.; Vivekananda, Jeeva; Sloan, Mark A.; Stribling, Lucille J. V.

2004-07-01

Biosynthetic semiconductor, diazoluminomelanin (DALM), is a polymer of tyrosine, luminol, and nitrite. DALM has a very large cross section of absorption for light from ultraviolet to radio frequencies. This polymer can be made efficiently in a genetically engineered E.coli, JM109/pIC2ORNR1.1 (ATCC# 69905). We have been pursuing ways to couple electromagnetic radiation to vectors using this polymer. DNA capture elements (DCEs; formerly aptamers) have made this possible. We incorporated DCEs into the plasmid of this E. coli to direct binding to whatever microbe or cell desired and to produce DALM attached to the plasmid DNA. Using two other vectors pSV2neoNR101 or pSV2neoNR8005 (ATCC # 69617 and 69618, respectively), both propagated in the E. coli host HB101, we have also inserted genes necessary for DALM production into animal and human cell lines (mouse monocytic leukemia: ATCC # CRL- 11771, -11772, -1173, mouse mammary adenocarcinoma: ATCC# CRL-12184, -12185; and human carcinoma of the cervix: ATCC # CRL-12510). The DCE/DALM vectors can be used to tag target cells, detectable by broad-spectrum light absorbance, luminescence, or fluorescence. DCE/DALM can further be activated with light, microwave energy, or by oxidative chemistry to kill the targeted microbes or other cells.

Mycobacterium leprae in six-banded (Euphractus sexcinctus) and nine-banded armadillos (Dasypus novemcinctus) in Northeast Brazil.

PubMed

Frota, Cristiane Cunha; Lima, Luana Nepomuceno Costa; Rocha, Adalgiza da Silva; Suffys, Philip Noel; Rolim, Benedito Neilson; Rodrigues, Laura Cunha; Barreto, Maurício Lima; Kendall, Carl; Kerr, Ligia Regina Sansigolo

2012-12-01

Human beings are the main reservoir of the causative agent of leprosy, Mycobacterium leprae. In the Americas, nine-banded armadillos (Dasypus novemcinctus) also act as a reservoir for the bacillus. In the state of Ceará (CE), which is located in Northeast Brazil and is an endemic area of leprosy, there are several species of armadillos, including D. novemcinctus and Euphractus sexcinctus (six-banded armadillo). Contact between humans and armadillos occur mainly through hunting, cleaning, preparing, cooking and eating. This study identified M. leprae DNA in the two main species of armadillos found in Northeast Brazil. A total of 29 wild armadillos (27 D. novemcinctus and 2 E. sexcinctus) were captured in different environments of CE countryside. Samples from the ear, nose, liver and spleen from each of these animals were tested by a nested M. leprae-specific repetitive element polymerase chain reaction assay. The samples that tested positive were confirmed by DNA sequencing. M. leprae was detected in 21% (6/29) of the animals, including five D. novemcinctus and one E. sexcinctus. This is the first Brazilian study to identify the presence of a biomarker of M. leprae in wild armadillos (D. novemcinctus and E. sexcinctus) in a leprosy hyperendemic area where there is continuous contact between humans and armadillos.

Complex structure of knob DNA on maize chromosome 9. Retrotransposon invasion into heterochromatin.

PubMed Central

Ananiev, E V; Phillips, R L; Rines, H W

1998-01-01

The recovery of maize (Zea mays L.) chromosome addition lines of oat (Avena sativa L.) from oat x maize crosses enables us to analyze the structure and composition of specific regions, such as knobs, of individual maize chromosomes. A DNA hybridization blot panel of eight individual maize chromosome addition lines revealed that 180-bp repeats found in knobs are present in each of these maize chromosomes, but the copy number varies from approximately 100 to 25, 000. Cosmid clones with knob DNA segments were isolated from a genomic library of an oat-maize chromosome 9 addition line with the help of the 180-bp knob-associated repeated DNA sequence used as a probe. Cloned knob DNA segments revealed a complex organization in which blocks of tandemly arranged 180-bp repeating units are interrupted by insertions of other repeated DNA sequences, mostly represented by individual full size copies of retrotransposable elements. There is an obvious preference for the integration of retrotransposable elements into certain sites (hot spots) of the 180-bp repeat. Sequence microheterogeneity including point mutations and duplications was found in copies of 180-bp repeats. The 180-bp repeats within an array all had the same polarity. Restriction maps constructed for 23 cloned knob DNA fragments revealed the positions of polymorphic sites and sites of integration of insertion elements. Discovery of the interspersion of retrotransposable elements among blocks of tandem repeats in maize and some other organisms suggests that this pattern may be basic to heterochromatin organization for eukaryotes. PMID:9691055

Atomic substitution reveals the structural basis for substrate adenine recognition and removal by adenine DNA glycosylase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Seongmin; Verdine, Gregory L.; Harvard)

2010-01-14

Adenine DNA glycosylase catalyzes the glycolytic removal of adenine from the promutagenic A {center_dot} oxoG base pair in DNA. The general features of DNA recognition by an adenine DNA glycosylase, Bacillus stearothermophilus MutY, have previously been revealed via the X-ray structure of a catalytically inactive mutant protein bound to an A:oxoG-containing DNA duplex. Although the structure revealed the substrate adenine to be, as expected, extruded from the DNA helix and inserted into an extrahelical active site pocket on the enzyme, the substrate adenine engaged in no direct contacts with active site residues. This feature was paradoxical, because other glycosylases havemore » been observed to engage their substrates primarily through direct contacts. The lack of direct contacts in the case of MutY suggested that either MutY uses a distinctive logic for substrate recognition or that the X-ray structure had captured a noncatalytically competent state in lesion recognition. To gain further insight into this issue, we crystallized wild-type MutY bound to DNA containing a catalytically inactive analog of 2'-deoxyadenosine in which a single 2'-H atom was replaced by fluorine. The structure of this fluorinated lesion-recognition complex (FLRC) reveals the substrate adenine buried more deeply into the active site pocket than in the prior structure and now engaged in multiple direct hydrogen bonding and hydrophobic interactions. This structure appears to capture the catalytically competent state of adenine DNA glycosylases, and it suggests a catalytic mechanism for this class of enzymes, one in which general acid-catalyzed protonation of the nucleobase promotes glycosidic bond cleavage.« less

Increasing the Analytical Sensitivity by Oligonucleotides Modified with Para- and Ortho-Twisted Intercalating Nucleic Acids – TINA

PubMed Central

Schneider, Uffe V.; Géci, Imrich; Jøhnk, Nina; Mikkelsen, Nikolaj D.; Pedersen, Erik B.; Lisby, Gorm

2011-01-01

The sensitivity and specificity of clinical diagnostic assays using DNA hybridization techniques are limited by the dissociation of double-stranded DNA (dsDNA) antiparallel duplex helices. This situation can be improved by addition of DNA stabilizing molecules such as nucleic acid intercalators. Here, we report the synthesis of a novel ortho-Twisted Intercalating Nucleic Acid (TINA) amidite utilizing the phosphoramidite approach, and examine the stabilizing effect of ortho- and para-TINA molecules in antiparallel DNA duplex formation. In a thermal stability assay, ortho- and para-TINA molecules increased the melting point (Tm) of Watson-Crick based antiparallel DNA duplexes. The increase in Tm was greatest when the intercalators were placed at the 5′ and 3′ termini (preferable) or, if placed internally, for each half or whole helix turn. Terminally positioned TINA molecules improved analytical sensitivity in a DNA hybridization capture assay targeting the Escherichia coli rrs gene. The corresponding sequence from the Pseudomonas aeruginosa rrs gene was used as cross-reactivity control. At 150 mM ionic strength, analytical sensitivity was improved 27-fold by addition of ortho-TINA molecules and 7-fold by addition of para-TINA molecules (versus the unmodified DNA oligonucleotide), with a 4-fold increase retained at 1 M ionic strength. Both intercalators sustained the discrimination of mismatches in the dsDNA (indicated by ΔTm), unless placed directly adjacent to the mismatch – in which case they partly concealed ΔTm (most pronounced for para-TINA molecules). We anticipate that the presented rules for placement of TINA molecules will be broadly applicable in hybridization capture assays and target amplification systems. PMID:21673988

Population-based rare variant detection via pooled exome or custom hybridization capture with or without individual indexing.

PubMed

Ramos, Enrique; Levinson, Benjamin T; Chasnoff, Sara; Hughes, Andrew; Young, Andrew L; Thornton, Katherine; Li, Allie; Vallania, Francesco L M; Province, Michael; Druley, Todd E

2012-12-06

Rare genetic variation in the human population is a major source of pathophysiological variability and has been implicated in a host of complex phenotypes and diseases. Finding disease-related genes harboring disparate functional rare variants requires sequencing of many individuals across many genomic regions and comparing against unaffected cohorts. However, despite persistent declines in sequencing costs, population-based rare variant detection across large genomic target regions remains cost prohibitive for most investigators. In addition, DNA samples are often precious and hybridization methods typically require large amounts of input DNA. Pooled sample DNA sequencing is a cost and time-efficient strategy for surveying populations of individuals for rare variants. We set out to 1) create a scalable, multiplexing method for custom capture with or without individual DNA indexing that was amenable to low amounts of input DNA and 2) expand the functionality of the SPLINTER algorithm for calling substitutions, insertions and deletions across either candidate genes or the entire exome by integrating the variant calling algorithm with the dynamic programming aligner, Novoalign. We report methodology for pooled hybridization capture with pre-enrichment, indexed multiplexing of up to 48 individuals or non-indexed pooled sequencing of up to 92 individuals with as little as 70 ng of DNA per person. Modified solid phase reversible immobilization bead purification strategies enable no sample transfers from sonication in 96-well plates through adapter ligation, resulting in 50% less library preparation reagent consumption. Custom Y-shaped adapters containing novel 7 base pair index sequences with a Hamming distance of ≥2 were directly ligated onto fragmented source DNA eliminating the need for PCR to incorporate indexes, and was followed by a custom blocking strategy using a single oligonucleotide regardless of index sequence. These results were obtained aligning raw reads against the entire genome using Novoalign followed by variant calling of non-indexed pools using SPLINTER or SAMtools for indexed samples. With these pipelines, we find sensitivity and specificity of 99.4% and 99.7% for pooled exome sequencing. Sensitivity, and to a lesser degree specificity, proved to be a function of coverage. For rare variants (≤2% minor allele frequency), we achieved sensitivity and specificity of ≥94.9% and ≥99.99% for custom capture of 2.5 Mb in multiplexed libraries of 22-48 individuals with only ≥5-fold coverage/chromosome, but these parameters improved to ≥98.7 and 100% with 20-fold coverage/chromosome. This highly scalable methodology enables accurate rare variant detection, with or without individual DNA sample indexing, while reducing the amount of required source DNA and total costs through less hybridization reagent consumption, multi-sample sonication in a standard PCR plate, multiplexed pre-enrichment pooling with a single hybridization and lesser sequencing coverage required to obtain high sensitivity.

SINE sequences detect DNA fingerprints in salmonid fishes.

PubMed

Spruell, P; Thorgaard, G H

1996-04-01

DNA probes homologous to two previously described salmonid short interspersed nuclear elements (SINEs) detected DNA fingerprint patterns in 14 species of salmonid fishes. The probes showed more homology to some species than to others and little homology to three nonsalmonid fishes. The DNA fingerprint patterns derived from the SINE probes are individual-specific and inherited in a Mendelian manner. Probes derived from different regions of the same SINE detect only partially overlapping banding patterns, reflecting a more complex SINE structure than has been previously reported. Like the human Alu sequence, the SINEs found in salmonids could provide useful genetic markers and primer sites for PCR-based techniques. These elements may be more desirable for some applications than traditional DNA fingerprinting probes that detect tandemly repeated arrays.

Detection of DNA polymerase λ activity during seed germination and enhancement after salinity stress and dehydration in the plumules of indica rice (Oryza sativa L.

PubMed

Sihi, Sayantani; Bakshi, Sankar; Sengupta, Dibyendu Narayan

2015-02-01

DNA polymerase λ (DNA pol λ) is the only reported X-family DNA polymerases in plants and has been shown to play a significant role in dry quiescent seeds, growth, development and nuclear DNA repair. cDNA for DNA pol λ has been reported in Arabidopsis and japonica rice cultivar and has been characterized from E. coli expressed protein, but very little is known about its activity at protein level in plants. The enzymatic activity of DNA pol λ was studied in dry, imbibed and during different germination stages of indica rice IR-8 (salt sensitive) by in-gel activity assay to determine its physiological role in important stages of growth and development. The upstream sequence was also analyzed using plantCARE database and was found to contain several cis-acting elements, including light responsive elements, dehydration responsive elements, Myb binding sites, etc. Hence, 4-day-old germinating seedlings of IR29, a salt-sensitive, but high yielding indica rice cultivar and Nonabokra, a salt-tolerant, but low yielding cultivar were treated with water (control) or 250 mM NaCl or 20% polyethyleneglycol-6000 for 4 and 8 h. The protein was analyzed by in vitro DNA pol λ activity assay, in-gel activity assay and Western blot analysis. DNA pol λ was not detected in dry seeds, but enhanced after imbibition and detectable from low level to high level during subsequent germination steps. Both salinity and dehydration stress led to the enhancement of the activity and protein level of DNA pol λ, as compared to control tissues. This is the first evidence of the salinity or dehydration stress induced enhancement of DNA pol λ activity in the plumules of rice (Oryza sativa L.) cultivars.

Evolutionary Dynamics of 5S rDNA and Recurrent Association of Transposable Elements in Electric Fish of the Family Gymnotidae (Gymnotiformes): The Case of Gymnotus mamiraua.

PubMed

da Silva, Maelin; Barbosa, Patricia; Artoni, Roberto F; Feldberg, Eliana

2016-01-01

Gymnotidae is a family of electric fish endemic to the Neotropics consisting of 2 genera: Electrophorus and Gymnotus. The genus Gymnotus is widely distributed and is found in all of the major Brazilian river systems. Physical and molecular mapping data for the ribosomal DNA (rDNA) in this genus are still scarce, with its chromosomal location known in only 11 species. As other species of Gymnotus with 2n = 54 chromosomes from the Paraná-Paraguay basin, G. mamiraua was found to have a large number of 5S rDNA sites. Isolation and cloning of the 5S rDNA sequences from G. mamiraua identified a fragment of a transposable element similar to the Tc1/mariner transposon associated with a non-transcribed spacer. Double fluorescence in situ hybridization analysis of this element and the 5S rDNA showed that they were colocalized on several chromosomes, in addition to acting as nonsyntenic markers on others. Our data show the association between these sequences and suggest that the Tc1 retrotransposon may be the agent that drives the spread of these 5S rDNA-like sequences in the G. mamiraua genome. © 2016 S. Karger AG, Basel.

Is junk DNA bunk? A critique of ENCODE.

PubMed

Doolittle, W Ford

2013-04-02

Do data from the Encyclopedia Of DNA Elements (ENCODE) project render the notion of junk DNA obsolete? Here, I review older arguments for junk grounded in the C-value paradox and propose a thought experiment to challenge ENCODE's ontology. Specifically, what would we expect for the number of functional elements (as ENCODE defines them) in genomes much larger than our own genome? If the number were to stay more or less constant, it would seem sensible to consider the rest of the DNA of larger genomes to be junk or, at least, assign it a different sort of role (structural rather than informational). If, however, the number of functional elements were to rise significantly with C-value then, (i) organisms with genomes larger than our genome are more complex phenotypically than we are, (ii) ENCODE's definition of functional element identifies many sites that would not be considered functional or phenotype-determining by standard uses in biology, or (iii) the same phenotypic functions are often determined in a more diffuse fashion in larger-genomed organisms. Good cases can be made for propositions ii and iii. A larger theoretical framework, embracing informational and structural roles for DNA, neutral as well as adaptive causes of complexity, and selection as a multilevel phenomenon, is needed.

Is junk DNA bunk? A critique of ENCODE

PubMed Central

Doolittle, W. Ford

2013-01-01

Do data from the Encyclopedia Of DNA Elements (ENCODE) project render the notion of junk DNA obsolete? Here, I review older arguments for junk grounded in the C-value paradox and propose a thought experiment to challenge ENCODE’s ontology. Specifically, what would we expect for the number of functional elements (as ENCODE defines them) in genomes much larger than our own genome? If the number were to stay more or less constant, it would seem sensible to consider the rest of the DNA of larger genomes to be junk or, at least, assign it a different sort of role (structural rather than informational). If, however, the number of functional elements were to rise significantly with C-value then, (i) organisms with genomes larger than our genome are more complex phenotypically than we are, (ii) ENCODE’s definition of functional element identifies many sites that would not be considered functional or phenotype-determining by standard uses in biology, or (iii) the same phenotypic functions are often determined in a more diffuse fashion in larger-genomed organisms. Good cases can be made for propositions ii and iii. A larger theoretical framework, embracing informational and structural roles for DNA, neutral as well as adaptive causes of complexity, and selection as a multilevel phenomenon, is needed. PMID:23479647

DNA strand displacement reaction for programmable release of biomolecules.

PubMed

Ramezani, Hamid; Jed Harrison, D

2015-05-14

Sample cleanup is a major processing step in many analytical assays. Here, we propose an approach to capture-and-release of analytes based on the DNA strand displacement reaction (SDR) and demonstrate its application to a fluoroimmunoassay on beads for a thyroid cancer biomarker, thyroglobulin. The SDR-based cleanup showed no interference from matrix molecules in serum.

Electrical potential-assisted DNA hybridization. How to mitigate electrostatics for surface DNA hybridization.

PubMed

Tymoczko, Jakub; Schuhmann, Wolfgang; Gebala, Magdalena

2014-12-24

Surface-confined DNA hybridization reactions are sensitive to the number and identity of DNA capture probes and experimental conditions such as the nature and the ionic strength of the electrolyte solution. When the surface probe density is high or the concentration of bulk ions is much lower than the concentration of ions within the DNA layer, hybridization is significantly slowed down or does not proceed at all. However, high-density DNA monolayers are attractive for designing high-sensitivity DNA sensors. Thus, circumventing sluggish DNA hybridization on such interfaces allows a high surface concentration of target DNA and improved signal/noise ratio. We present potential-assisted hybridization as a strategy in which an external voltage is applied to the ssDNA-modified interface during the hybridization process. Results show that a significant enhancement of hybridization can be achieved using this approach.

Identification of endometrial cancer methylation features using combined methylation analysis methods

PubMed Central

Trimarchi, Michael P.; Yan, Pearlly; Groden, Joanna; Bundschuh, Ralf; Goodfellow, Paul J.

2017-01-01

Background DNA methylation is a stable epigenetic mark that is frequently altered in tumors. DNA methylation features are attractive biomarkers for disease states given the stability of DNA methylation in living cells and in biologic specimens typically available for analysis. Widespread accumulation of methylation in regulatory elements in some cancers (specifically the CpG island methylator phenotype, CIMP) can play an important role in tumorigenesis. High resolution assessment of CIMP for the entire genome, however, remains cost prohibitive and requires quantities of DNA not available for many tissue samples of interest. Genome-wide scans of methylation have been undertaken for large numbers of tumors, and higher resolution analyses for a limited number of cancer specimens. Methods for analyzing such large datasets and integrating findings from different studies continue to evolve. An approach for comparison of findings from a genome-wide assessment of the methylated component of tumor DNA and more widely applied methylation scans was developed. Methods Methylomes for 76 primary endometrial cancer and 12 normal endometrial samples were generated using methylated fragment capture and second generation sequencing, MethylCap-seq. Publically available Infinium HumanMethylation 450 data from The Cancer Genome Atlas (TCGA) were compared to MethylCap-seq data. Results Analysis of methylation in promoter CpG islands (CGIs) identified a subset of tumors with a methylator phenotype. We used a two-stage approach to develop a 13-region methylation signature associated with a “hypermethylator state.” High level methylation for the 13-region methylation signatures was associated with mismatch repair deficiency, high mutation rate, and low somatic copy number alteration in the TCGA test set. In addition, the signature devised showed good agreement with previously described methylation clusters devised by TCGA. Conclusion We identified a methylation signature for a “hypermethylator phenotype” in endometrial cancer and developed methods that may prove useful for identifying extreme methylation phenotypes in other cancers. PMID:28278225

Satellite DNA Modulates Gene Expression in the Beetle Tribolium castaneum after Heat Stress

PubMed Central

Feliciello, Isidoro; Akrap, Ivana; Ugarković, Đurđica

2015-01-01

Non-coding repetitive DNAs have been proposed to perform a gene regulatory role, however for tandemly repeated satellite DNA no such role was defined until now. Here we provide the first evidence for a role of satellite DNA in the modulation of gene expression under specific environmental conditions. The major satellite DNA TCAST1 in the beetle Tribolium castaneum is preferentially located within pericentromeric heterochromatin but is also dispersed as single repeats or short arrays in the vicinity of protein-coding genes within euchromatin. Our results show enhanced suppression of activity of TCAST1-associated genes and slower recovery of their activity after long-term heat stress relative to the same genes without associated TCAST1 satellite DNA elements. The level of gene suppression is not influenced by the distance of TCAST1 elements from the associated genes up to 40 kb from the genes’ transcription start sites, but it does depend on the copy number of TCAST1 repeats within an element, being stronger for the higher number of copies. The enhanced gene suppression correlates with the enrichment of the repressive histone marks H3K9me2/3 at dispersed TCAST1 elements and their flanking regions as well as with increased expression of TCAST1 satellite DNA. The results reveal transient, RNAi based heterochromatin formation at dispersed TCAST1 repeats and their proximal regions as a mechanism responsible for enhanced silencing of TCAST1-associated genes. Differences in the pattern of distribution of TCAST1 elements contribute to gene expression diversity among T. castaneum strains after long-term heat stress and might have an impact on adaptation to different environmental conditions. PMID:26275223

Measuring θ13 in the Double Chooz experiment

NASA Astrophysics Data System (ADS)

Crum, Keith

2013-04-01

Double Chooz measures θ13 by searching for the disappearance of reactor electron antineutrinos (νe) interacting via inverse beta decay (IBD) in a liquid scintillator-based detector. The signature of IBD is the coincidence of positron annihilation followed by the capture of a neutron. Although Double Chooz was primarily designed to detect νe by searching for neutron capture on gadolinium, we can also search for neutron capture on hydrogen. We developed separate analyses for neutron capture on hydrogen and gadolinium as the two elements have different capture energies, capture lifetimes, and spatial distributions within our detector.

The elements of design knowledge capture

NASA Technical Reports Server (NTRS)

Freeman, Michael S.

1988-01-01

This paper will present the basic constituents of a design knowledge capture effort. This will include a discussion of the types of knowledge to be captured in such an effort and the difference between design knowledge capture and more traditional knowledge base construction. These differences include both knowledge base structure and knowledge acquisition approach. The motivation for establishing a design knowledge capture effort as an integral part of major NASA programs will be outlined, along with the current NASA position on that subject. Finally the approach taken in design knowledge capture for Space Station will be contrasted with that used in the HSTDEK project.

Single-Stranded DNA Aptamers against Pathogens and Toxins: Identification and Biosensing Applications

PubMed Central

Hong, Ka Lok

2015-01-01

Molecular recognition elements (MREs) can be short sequences of single-stranded DNA, RNA, small peptides, or antibody fragments. They can bind to user-defined targets with high affinity and specificity. There has been an increasing interest in the identification and application of nucleic acid molecular recognition elements, commonly known as aptamers, since they were first described in 1990 by the Gold and Szostak laboratories. A large number of target specific nucleic acids MREs and their applications are currently in the literature. This review first describes the general methodologies used in identifying single-stranded DNA (ssDNA) aptamers. It then summarizes advancements in the identification and biosensing application of ssDNA aptamers specific for bacteria, viruses, their associated molecules, and selected chemical toxins. Lastly, an overview of the basic principles of ssDNA aptamer-based biosensors is discussed. PMID:26199940

Use of spatial capture-recapture modeling and DNA data to estimate densities of elusive animals

USGS Publications Warehouse

Kery, Marc; Gardner, Beth; Stoeckle, Tabea; Weber, Darius; Royle, J. Andrew

2011-01-01

Assessment of abundance, survival, recruitment rates, and density (i.e., population assessment) is especially challenging for elusive species most in need of protection (e.g., rare carnivores). Individual identification methods, such as DNA sampling, provide ways of studying such species efficiently and noninvasively. Additionally, statistical methods that correct for undetected animals and account for locations where animals are captured are available to efficiently estimate density and other demographic parameters. We collected hair samples of European wildcat (Felis silvestris) from cheek-rub lure sticks, extracted DNA from the samples, and identified each animals' genotype. To estimate the density of wildcats, we used Bayesian inference in a spatial capture-recapture model. We used WinBUGS to fit a model that accounted for differences in detection probability among individuals and seasons and between two lure arrays. We detected 21 individual wildcats (including possible hybrids) 47 times. Wildcat density was estimated at 0.29/km2 (SE 0.06), and 95% of the activity of wildcats was estimated to occur within 1.83 km from their home-range center. Lures located systematically were associated with a greater number of detections than lures placed in a cell on the basis of expert opinion. Detection probability of individual cats was greatest in late March. Our model is a generalized linear mixed model; hence, it can be easily extended, for instance, to incorporate trap- and individual-level covariates. We believe that the combined use of noninvasive sampling techniques and spatial capture-recapture models will improve population assessments, especially for rare and elusive animals.

High-throughput identification of antigen-specific TCRs by TCR gene capture.

PubMed

Linnemann, Carsten; Heemskerk, Bianca; Kvistborg, Pia; Kluin, Roelof J C; Bolotin, Dmitriy A; Chen, Xiaojing; Bresser, Kaspar; Nieuwland, Marja; Schotte, Remko; Michels, Samira; Gomez-Eerland, Raquel; Jahn, Lorenz; Hombrink, Pleun; Legrand, Nicolas; Shu, Chengyi Jenny; Mamedov, Ilgar Z; Velds, Arno; Blank, Christian U; Haanen, John B A G; Turchaninova, Maria A; Kerkhoven, Ron M; Spits, Hergen; Hadrup, Sine Reker; Heemskerk, Mirjam H M; Blankenstein, Thomas; Chudakov, Dmitriy M; Bendle, Gavin M; Schumacher, Ton N M

2013-11-01

The transfer of T cell receptor (TCR) genes into patient T cells is a promising approach for the treatment of both viral infections and cancer. Although efficient methods exist to identify antibodies for the treatment of these diseases, comparable strategies to identify TCRs have been lacking. We have developed a high-throughput DNA-based strategy to identify TCR sequences by the capture and sequencing of genomic DNA fragments encoding the TCR genes. We establish the value of this approach by assembling a large library of cancer germline tumor antigen-reactive TCRs. Furthermore, by exploiting the quantitative nature of TCR gene capture, we show the feasibility of identifying antigen-specific TCRs in oligoclonal T cell populations from either human material or TCR-humanized mice. Finally, we demonstrate the ability to identify tumor-reactive TCRs within intratumoral T cell subsets without knowledge of antigen specificities, which may be the first step toward the development of autologous TCR gene therapy to target patient-specific neoantigens in human cancer.

Neutron detector

DOEpatents

Stephan, Andrew C [Knoxville, TN; Jardret,; Vincent, D [Powell, TN

2011-04-05

A neutron detector has a volume of neutron moderating material and a plurality of individual neutron sensing elements dispersed at selected locations throughout the moderator, and particularly arranged so that some of the detecting elements are closer to the surface of the moderator assembly and others are more deeply embedded. The arrangement captures some thermalized neutrons that might otherwise be scattered away from a single, centrally located detector element. Different geometrical arrangements may be used while preserving its fundamental characteristics. Different types of neutron sensing elements may be used, which may operate on any of a number of physical principles to perform the function of sensing a neutron, either by a capture or a scattering reaction, and converting that reaction to a detectable signal. High detection efficiency, an ability to acquire spectral information, and directional sensitivity may be obtained.

Linear combination reading program for capture gamma rays

USGS Publications Warehouse

Tanner, Allan B.

1971-01-01

This program computes a weighting function, Qj, which gives a scalar output value of unity when applied to the spectrum of a desired element and a minimum value (considering statistics) when applied to spectra of materials not containing the desired element. Intermediate values are obtained for materials containing the desired element, in proportion to the amount of the element they contain. The program is written in the BASIC language in a format specific to the Hewlett-Packard 2000A Time-Sharing System, and is an adaptation of an earlier program for linear combination reading for X-ray fluorescence analysis (Tanner and Brinkerhoff, 1971). Following the program is a sample run from a study of the application of the linear combination technique to capture-gamma-ray analysis for calcium (report in preparation).

Comparison of Muon Capture in Light and in Heavy Nuclei

NASA Astrophysics Data System (ADS)

Measday, David F.; Stocki, Trevor J.

2007-10-01

We have recently completed an experimental study at TRIUMF of muon capture in the following elements, N, Al, Si, Ca, Fe, Ni, I, Au, and Bi. We detected the nuclear gamma rays emitted by the product nuclei after muon capture. The energy of the gamma ray identifies the source nuclide, and thus the reaction which has occurred. Our data are of better quality, and more comprehensive than any other data set in the literature. The (μ-,νn) reaction is always dominant. In light nuclei, reactions such as (μ-,νp) and (μ-,νpn) can occur, but not for heavy nuclei. However the reverse is true for reactions such as (μ-,ν3n) and (μ-,ν4n), which are very rare in light nuclei, but easily detected in heavy elements. We shall discuss how such information can be useful in calculations of neutrino-nucleus interactions, and of electron-capture in supernovae.

On-Chip Synthesis of Protein Microarrays from DNA Microarrays Via Coupled In Vitro Transcription and Translation for Surface Plasmon Resonance Imaging Biosensor Applications

PubMed Central

Seefeld, Ting H.; Halpern, Aaron R.; Corn, Robert M.

2012-01-01

Protein microarrays are fabricated from double-stranded DNA (dsDNA) microarrays by a one-step, multiplexed enzymatic synthesis in an on-chip microfluidic format and then employed for antibody biosensing measurements with surface plasmon resonance imaging (SPRI). A microarray of dsDNA elements (denoted as generator elements) that encode either a His-tagged green fluorescent protein (GFP) or a His-tagged luciferase protein is utilized to create multiple copies of messenger RNA (mRNA) in a surface RNA polymerase reaction; the mRNA transcripts are then translated into proteins by cell-free protein synthesis in a microfluidic format. The His-tagged proteins diffuse to adjacent Cu(II)-NTA microarray elements (denoted as detector elements) and are specifically adsorbed. The net result is the on-chip, cell-free synthesis of a protein microarray that can be used immediately for SPRI protein biosensing. The dual element format greatly reduces any interference from the nonspecific adsorption of enzyme or proteins. SPRI measurements for the detection of the antibodies anti-GFP and anti-luciferase were used to verify the formation of the protein microarray. This convenient on-chip protein microarray fabrication method can be implemented for multiplexed SPRI biosensing measurements in both clinical and research applications. PMID:22793370

Surveying DNA Elements within Functional Genes of Heterocyst-Forming Cyanobacteria

PubMed Central

Hilton, Jason A.; Meeks, John C.; Zehr, Jonathan P.

2016-01-01

Some cyanobacteria are capable of differentiating a variety of cell types in response to environmental factors. For instance, in low nitrogen conditions, some cyanobacteria form heterocysts, which are specialized for N2 fixation. Many heterocyst-forming cyanobacteria have DNA elements interrupting key N2 fixation genes, elements that are excised during heterocyst differentiation. While the mechanism for the excision of the element has been well-studied, many questions remain regarding the introduction of the elements into the cyanobacterial lineage and whether they have been retained ever since or have been lost and reintroduced. To examine the evolutionary relationships and possible function of DNA sequences that interrupt genes of heterocyst-forming cyanobacteria, we identified and compared 101 interruption element sequences within genes from 38 heterocyst-forming cyanobacterial genomes. The interruption element lengths ranged from about 1 kb (the minimum able to encode the recombinase responsible for element excision), up to nearly 1 Mb. The recombinase gene sequences served as genetic markers that were common across the interruption elements and were used to track element evolution. Elements were found that interrupted 22 different orthologs, only five of which had been previously observed to be interrupted by an element. Most of the newly identified interrupted orthologs encode proteins that have been shown to have heterocyst-specific activity. However, the presence of interruption elements within genes with no known role in N2 fixation, as well as in three non-heterocyst-forming cyanobacteria, indicates that the processes that trigger the excision of elements may not be limited to heterocyst development or that the elements move randomly within genomes. This comprehensive analysis provides the framework to study the history and behavior of these unique sequences, and offers new insight regarding the frequency and persistence of interruption elements in heterocyst-forming cyanobacteria. PMID:27206019

Surveying DNA Elements within Functional Genes of Heterocyst-Forming Cyanobacteria.

PubMed

Hilton, Jason A; Meeks, John C; Zehr, Jonathan P

2016-01-01

Some cyanobacteria are capable of differentiating a variety of cell types in response to environmental factors. For instance, in low nitrogen conditions, some cyanobacteria form heterocysts, which are specialized for N2 fixation. Many heterocyst-forming cyanobacteria have DNA elements interrupting key N2 fixation genes, elements that are excised during heterocyst differentiation. While the mechanism for the excision of the element has been well-studied, many questions remain regarding the introduction of the elements into the cyanobacterial lineage and whether they have been retained ever since or have been lost and reintroduced. To examine the evolutionary relationships and possible function of DNA sequences that interrupt genes of heterocyst-forming cyanobacteria, we identified and compared 101 interruption element sequences within genes from 38 heterocyst-forming cyanobacterial genomes. The interruption element lengths ranged from about 1 kb (the minimum able to encode the recombinase responsible for element excision), up to nearly 1 Mb. The recombinase gene sequences served as genetic markers that were common across the interruption elements and were used to track element evolution. Elements were found that interrupted 22 different orthologs, only five of which had been previously observed to be interrupted by an element. Most of the newly identified interrupted orthologs encode proteins that have been shown to have heterocyst-specific activity. However, the presence of interruption elements within genes with no known role in N2 fixation, as well as in three non-heterocyst-forming cyanobacteria, indicates that the processes that trigger the excision of elements may not be limited to heterocyst development or that the elements move randomly within genomes. This comprehensive analysis provides the framework to study the history and behavior of these unique sequences, and offers new insight regarding the frequency and persistence of interruption elements in heterocyst-forming cyanobacteria.

WHAEM: PROGRAM DOCUMENTATION FOR THE WELLHEAD ANALYTIC ELEMENT MODEL

EPA Science Inventory

The Wellhead Analytic Element Model (WhAEM) demonstrates a new technique for the definition of time-of-travel capture zones in relatively simple geohydrologic settings. he WhAEM package includes an analytic element model that uses superposition of (many) analytic solutions to gen...

Human Papillomavirus Genotyping After Denaturation of Specimens for Hybrid Capture 2 Testing: Feasibility Study for the HPV Persistence and Progression Cohort†

PubMed Central

LaMere, Brandon J.; Kornegay, Janet; Fetterman, Barbara; Sadorra, Mark; Shieh, Jen; Castle, Philip E.

2009-01-01

Human papillomavirus (HPV) genotyping could be clinically useful, depending on the results of large, prospective studies like the HPV Persistence and Progression cohort. The cohort is based on genotyping and follow-up of Hybrid Capture-positive women at Kaiser Permanente, Northern California. HPV DNA testing by Hybrid Capture 2 requires denaturation with alkali, possibly damaging the DNA for optimal PCR-based genotyping. A feasibility study was conducted on paired aliquots of anonymized specimens from 100 women with low-grade intraepithelial lesion cytology. Test aliquots were left in denaturant for 10 or 18 hours at 4°C and then neutralized; comparison aliquots were not denatured but diluted to match the timing, temperature, concentration and salt conditions of the treated specimens. The masked aliquots were tested using a commercialized PCR-based assay that detects of 37 HPV genotypes. There was no overall effect of treatment on test positivity or number of types. HPV16 was marginally more likely to be detected in untreated versus treated aliquots (P = 0.09) but HPV45 was marginally more likely to be detected in treated than untreated aliquots (P = 0.07), suggesting that these differences represented chance (intra-test variability). It can be concluded that residual Hybrid Capture-positive specimens can be accurately genotyped by PCR after Hybrid Capture 2 processing. PMID:17673302

Multianalyte, dipstick-type, nanoparticle-based DNA biosensor for visual genotyping of single-nucleotide polymorphisms.

PubMed

Litos, Ioannis K; Ioannou, Penelope C; Christopoulos, Theodore K; Traeger-Synodinos, Jan; Kanavakis, Emmanuel

2009-06-15

DNA biosensors involve molecular recognition of the target sequence by hybridization with specific probes and detection by electrochemical, optical or gravimetric transduction. Disposable, dipstick-type biosensors have been developed recently, which enable visual detection of DNA without using instruments. In this context, we report a multianalyte DNA biosensor for visual genotyping of two single-nucleotide polymorphisms (SNPs). As a model, the biosensor was applied to the simultaneous genotyping of two SNPs, entailing the detection of four alleles. A PCR product that flanks both polymorphic sites is subjected to a single primer extension (PEXT) reaction employing four allele-specific primers, each containing a region complementary to an allele and a characteristic segment that enables subsequent capture on a test zone of the biosensor. The primers are extended with dNTPs and biotin-dUTP only if there is perfect complementarity with the interrogated sequence. The PEXT mixture is applied to the biosensor. As the developing buffer migrates along the strip, all the allele-specific primers are captured by immobilized oligonucleotides at the four test zones of the biosensor and detected by antibiotin-functionalized gold nanoparticles. As a result, the test zones are colored red if extension has occurred denoting the presence of the corresponding allele in the original sample. The excess nanoparticles are captured by immobilized biotinylated albumin at the control zone of the sensor forming another red zone that indicates the proper performance of the system. The assay was applied successfully to the genotyping of twenty clinical samples for two common SNPs of MBL2 gene.

New insights into replication origin characteristics in metazoans

PubMed Central

Puy, Aurore; Rialle, Stéphanie; Kaplan, Noam; Segal, Eran

2012-01-01

We recently reported the identification and characterization of DNA replication origins (Oris) in metazoan cell lines. Here, we describe additional bioinformatic analyses showing that the previously identified GC-rich sequence elements form origin G-rich repeated elements (OGREs) that are present in 67% to 90% of the DNA replication origins from Drosophila to human cells, respectively. Our analyses also show that initiation of DNA synthesis takes place precisely at 160 bp (Drosophila) and 280 bp (mouse) from the OGRE. We also found that in most CpG islands, an OGRE is positioned in opposite orientation on each of the two DNA strands and detected two sites of initiation of DNA synthesis upstream or downstream of each OGRE. Conversely, Oris not associated with CpG islands have a single initiation site. OGRE density along chromosomes correlated with previously published replication timing data. Ori sequences centered on the OGRE are also predicted to have high intrinsic nucleosome occupancy. Finally, OGREs predict G-quadruplex structures at Oris that might be structural elements controlling the choice or activation of replication origins. PMID:22373526

The fission yeast CENP-B protein Abp1 prevents pervasive transcription of repetitive DNA elements.

PubMed

Daulny, Anne; Mejía-Ramírez, Eva; Reina, Oscar; Rosado-Lugo, Jesus; Aguilar-Arnal, Lorena; Auer, Herbert; Zaratiegui, Mikel; Azorin, Fernando

2016-10-01

It is well established that eukaryotic genomes are pervasively transcribed producing cryptic unstable transcripts (CUTs). However, the mechanisms regulating pervasive transcription are not well understood. Here, we report that the fission yeast CENP-B homolog Abp1 plays an important role in preventing pervasive transcription. We show that loss of abp1 results in the accumulation of CUTs, which are targeted for degradation by the exosome pathway. These CUTs originate from different types of genomic features, but the highest increase corresponds to Tf2 retrotransposons and rDNA repeats, where they map along the entire elements. In the absence of abp1, increased RNAPII-Ser5P occupancy is observed throughout the Tf2 coding region and, unexpectedly, RNAPII-Ser5P is enriched at rDNA repeats. Loss of abp1 also results in Tf2 derepression and increased nucleolus size. Altogether these results suggest that Abp1 prevents pervasive RNAPII transcription of repetitive DNA elements (i.e., Tf2 and rDNA repeats) from internal cryptic sites. Copyright © 2016 Elsevier B.V. All rights reserved.

The nature of the force-induced conformation transition of dsDNA studied by using single molecule force spectroscopy.

PubMed

Liu, Ningning; Bu, Tianjia; Song, Yu; Zhang, Wei; Li, Jinjing; Zhang, Wenke; Shen, Jiacong; Li, Hongbin

2010-06-15

Single-stranded DNA binding proteins (SSB) interact with single-stranded DNA (ssDNA) specifically. Taking advantage of this character, we have employed Bacillus subtilis SSB protein to investigate the nature of force-induced conformation transition of double-stranded DNA (dsDNA) by using AFM-based single molecule force spectroscopy (SMFS) technique. Our results show that, when a dsDNA is stretched beyond its contour length, the dsDNA is partially melted, producing some ssDNA segments which can be captured by SSB proteins. We have also systematically investigated the effects of stretching length, waiting time, and salt concentration on the conformation transition of dsDNA and SSB-ssDNA interactions, respectively. Furthermore, the effect of proflavine, a DNA intercalator, on the SSB-DNA interactions has been investigated, and the results indicate that the proflavine-saturated dsDNA can be stabilized to the extent that the dsDNA will no longer melt into ssDNA under the mechanical force even up to 150 pN, and no SSB-DNA interactions are detectable.

APPLIED MERCURY CAPTURE

EPA Science Inventory

The first purpose of this project is to complete bench and pilot scale testing of promising mercury sorbents. This work would apply findings from fundamental, mechanistic efforts over the past three years that have developed sorbents which show improved capture of elemental and ...

DNA Charge Transport: From Chemical Principles to the Cell

PubMed Central

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

2016-01-01

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

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

PubMed Central

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

2016-01-01

The apparent dissociation constant (Kd) for specific binding of glucocorticoid receptor (GR) and androgen receptor (AR) to DNA was determined in vivo in Xenopus oocytes. The total nuclear receptor concentration was quantified as specifically retained [3H]-hormone in manually isolated oocyte nuclei. DNA was introduced by nuclear microinjection of single stranded phagemid DNA, chromatin is then formed during second strand synthesis. The fraction of DNA sites occupied by the expressed receptor was determined by dimethylsulphate in vivo footprinting and used for calculation of the receptor-DNA binding affinity. The forkhead transcription factor FoxA1 enhanced the DNA binding by GR with an apparent Kd of ∼1 μM and dramatically stimulated DNA binding by AR with an apparent Kd of ∼0.13 μM at a composite androgen responsive DNA element containing one FoxA1 binding site and one palindromic hormone receptor binding site known to bind one receptor homodimer. FoxA1 exerted a weak constitutive- and strongly cooperative DNA binding together with AR but had a less prominent effect with GR, the difference reflecting the licensing function of FoxA1 at this androgen responsive DNA element. PMID:26657626

Detection of human papillomavirus in pterygium and conjunctival papilloma by hybrid capture II and PCR assays.

PubMed

Takamura, Y; Kubo, E; Tsuzuki, S; Akagi, Y

2008-11-01

To elucidate the putative role of human papillomavirus (HPV) infection in pterygium and conjunctival papilloma. Hybrid capture II (HC-II) and polymerase chain reaction (PCR) assays were performed to detect HPV in pterygium (42 samples obtained from 40 patients) and conjunctival papilloma (8 samples from 6 patients). The amount of HPV DNA was evaluated by measurement of relative light units (RLUs) on a luminometer. All papilloma samples were positive for HPV DNA by PCR and HC-II. The RLU values for specimens of recurrent and re-recurrent papilloma were markedly higher than those for specimens of primary lesions. HPV was detected by PCR in 2 of 42 (4.8%) beta-globin-positive pterygium specimens, whereas HC-II showed that HPV was negative in all pterygium samples. Our results support the hypothesis that HPV DNA is associated with the pathogenesis of conjunctival papilloma, but not pterygium. RLU measurement by HC-II may serve as a marker for evaluating the activity of HPV in conjunctival tumours.

Amplification of a specific repetitive DNA sequence for Trypanosoma rangeli identification and its potential application in epidemiological investigations.

PubMed

Vargas, N; Souto, R P; Carranza, J C; Vallejo, G A; Zingales, B

2000-11-01

Trypanosoma rangeli can infect humans as well as the same domestic and wild animals and triatomine vectors infected by Trypanosoma cruzi in Central and South America. This overlapping distribution complicates the epidemiology of American trypanosomiasis due to the cross-reactivity between T. rangeli and T. cruzi antigens and the presence of conserved DNA sequences in these parasites. We have isolated a T. rangeli-specific DNA repetitive element which is represented in approximately 103 copies per parasite genome and is distributed in several chromosomal bands. The 542-bp nucleotide sequence of this element, named P542, was determined and a PCR assay was standardized for its amplification. The sensitivity of the assay is high, allowing the detection of one tenth of the DNA content of a single parasite. The presence of the P542 element was confirmed in 11 T. rangeli isolates from mammalian hosts and insect vectors originating from several countries in Latin America. Negative amplification was observed with different T. cruzi strains and other trypanosomatids. The potential field application of the P542 PCR assay was investigated in simulated samples containing T. rangeli and/or T. cruzi and intestinal tract and feces of Rhodnius prolixus. Epidemiological studies were conducted in DNA preparations obtained from the digestive tracts of 12 Rhodnius colombiensis insects collected in a sylvatic area in Colombia. Positive amplification of the P542 element was obtained in 9/12 insects. We have also compared in the same samples the diagnostic performance of two PCR assays for the amplification of the variable domain of minicircle kinetoplast DNA (kDNA) and of the large subunit (LSU) of the ribosomal RNA gene of T. cruzi and T. rangeli. Data indicate that the kDNA PCR assay does not allow diagnosis of mixed infections in most insects. On the other hand, the PCR assay of the LSU RNA gene showed lower sensitivity in the detection of T. rangeli than the PCR assay of the P542 element. It is predicted that the use of sensitive detection techniques will indicate that the actual distribution of T. rangeli in America is wider than presumed. Copyright 2000 Academic Press.

Increased methylation of repetitive elements and DNA repair genes is associated with higher DNA oxidation in children in an urbanized, industrial environment.

PubMed

Alvarado-Cruz, Isabel; Sánchez-Guerra, Marco; Hernández-Cadena, Leticia; De Vizcaya-Ruiz, Andrea; Mugica, Violeta; Pelallo-Martínez, Nadia Azenet; Solís-Heredia, María de Jesús; Byun, Hyang-Min; Baccarelli, Andrea; Quintanilla-Vega, Betzabet

2017-01-01

DNA methylation in DNA repair genes participates in the DNA damage regulation. Particulate matter (PM), which has metals and polycyclic aromatic hydrocarbons (PAHs) adsorbed, among others has been linked to adverse health outcomes and may modify DNA methylation. To evaluate PM exposure impact on repetitive elements and gene-specific DNA methylation and DNA damage, we conducted a cross-sectional study in 150 schoolchildren (7-10 years old) from an urbanized, industrial area of the metropolitan area of Mexico City (MAMC), which frequently exhibits PM concentrations above safety standards. Methylation (5mC) of long interspersed nuclear element-1 (LINE1) and DNA repair gene (OGG1, APEX, and PARP1) was assessed by pyrosequencing in peripheral mononuclear cells, DNA damage by comet assay and DNA oxidation by 8-OHdG content. PAH and metal contents in PM 10 (≤10μm aerodynamic diameter) were determined by HPLC-MS and ICP-AES, respectively. Multiple regression analysis between DNA methylation, DNA damage, and PM 10 exposure showed that PM 10 was significantly associated with oxidative DNA damage; a 1% increase in 5mC at all CpG sites in PARP1 promoter was associated with a 35% increase in 8-OHdG, while a 1% increase at 1, 2, and 3 CpG sites resulted in 38, 9, and 56% increments, respectively. An increase of 10pg/m 3 in benzo[b]fluoranthene content of PM 10 was associated with a 6% increase in LINE1 methylation. Acenaphthene, indene [1,2,3-cd] pyrene, and pyrene concentrations correlated with higher dinucleotide methylation in OGG1, APEX and PARP1 genes, respectively. Vanadium concentration correlated with increased methylation at selected APEX and PARP1 CpG sites. DNA repair gene methylation was significantly correlated with DNA damage and with specific PM 10 -associated PAHs and Vanadium. Data suggest that exposure to PM and its components are associated with differences in DNA methylation of repair genes in children, which may contribute to DNA damage. Copyright © 2016 Elsevier B.V. All rights reserved.

Prompt Gamma Activation Analysis (PGAA): Technique of choice for nondestructive bulk analysis of returned comet samples

NASA Technical Reports Server (NTRS)

Lindstrom, David J.; Lindstrom, Richard M.

1989-01-01

Prompt gamma activation analysis (PGAA) is a well-developed analytical technique. The technique involves irradiation of samples in an external neutron beam from a nuclear reactor, with simultaneous counting of gamma rays produced in the sample by neutron capture. Capture of neutrons leads to excited nuclei which decay immediately with the emission of energetic gamma rays to the ground state. PGAA has several advantages over other techniques for the analysis of cometary materials: (1) It is nondestructive; (2) It can be used to determine abundances of a wide variety of elements, including most major and minor elements (Na, Mg, Al, Si, P, K, Ca, Ti, Cr, Mn, Fe, Co, Ni), volatiles (H, C, N, F, Cl, S), and some trace elements (those with high neutron capture cross sections, including B, Cd, Nd, Sm, and Gd); and (3) It is a true bulk analysis technique. Recent developments should improve the technique's sensitivity and accuracy considerably.

Neutron-Capture Elements in Very Metal-Poor Halo Stars

NASA Astrophysics Data System (ADS)

French, R. S.; Sneden, C.; Cowan, J. J.; Lawler, J. E.; Primas, F.; Beers, T. C.; Truran, J. W.

2000-05-01

Abundances of the most massive stable elements (Os -> Pb or 76 <= Z <= 82) in metal-poor stars can provide crucial information about the so-called ``third neutron-capture peak,'' and are critical to the radioactive-dating technique that uses unstable thorium and uranium as chronometers. As the relevant transitions occur in the UV and are inaccessable to ground-based telescopes, we have obtained high resolution (R ~= 30,000) UV spectra of 10 very metal-poor (--3.0 <= [Fe/H] <= --1.4) halo giants using the Space Telescope Imaging Spectrograph (STIS) aboard the Hubble Space Telescope. Using iterative spectrum synthesis techniques, we derive abundances for some of these heavy elements. We compare our abundances to those predicted for very metal-poor stars based on a scaled solar system rapid-process (production in rapid neutron-capture synthesis events, such as occurs during supernovae explosions). This research is supported by NASA STScI grant GO-08342 and NSF grants AST-9618364 to C.S. and AST-9618332 to J.J.C.

The Open Cluster Chemical Abundances and Mapping (OCCAM) Survey: Galactic Neutron Capture Abundance Gradients

NASA Astrophysics Data System (ADS)

O'Connell, Julia; Frinchaboy, Peter M.; Shetrone, Matthew D.; Melendez, Matthew; Cunha, Katia M. L.; Majewski, Steven R.; Zasowski, Gail; APOGEE Team

2017-01-01

The evolution of elements, as a function or age, throughout the Milky Way disk provides a key constraint for galaxy evolution models. In an effort to provide these constraints, we have conducted an investigation into the r- and s- process elemental abundances for a large sample of open clusters as part of an optical follow-up to the SDSS-III/APOGEE-1 survey. Stars were identified as cluster members by the Open Cluster Chemical Abundance & Mapping (OCCAM) survey, which culls member candidates by radial velocity, metallicity, and proper motion from the observed APOGEE sample. To obtain data for neutron capture elements in these clusters, we conducted a long-term observing campaign covering three years (2013-2016) using the McDonald Observatory Otto Struve 2.1-m telescope and Sandiford Cass Echelle Spectrograph (R ~ 60,000). We present Galactic neutron-capture abundance gradients using 30+ clusters, within 6 kpc of the Sun, covering a range of ages from ~80 Myr to ~10 Gyr .

The Open Cluster Chemical Abundances and Mapping (OCCAM) Survey: Galactic Neutron CaptureAbundance Gradients

NASA Astrophysics Data System (ADS)

O'Connell, Julia; Frinchaboy, Peter M.; Shetrone, Matthew D.; Melendez, Matthew; Cunha, Katia; Majewski, Steven R.; Zasowski, Gail; APOGEE Team

2017-06-01

The evolution of elements, as a function or age, throughout the Milky Way disk provides a key constraint for galaxy evolution models. In an effort to provide these constraints, we have conducted an investigation into the r- and s- process elemental abundances for a large sample of open clusters as part of an optical follow-up to the SDSS-III/APOGEE-1 survey. Stars were identified as cluster members by the Open Cluster Chemical Abundance & Mapping (OCCAM) survey, which culls member candidates by radial velocity, metallicity and proper motion from the observed APOGEE sample. To obtain data for neutron capture elements in these clusters, we conducted a long-term observing campaign covering three years (2013-2016) using the McDonald Observatory Otto Struve 2.1-m telescope and Sandiford Cass Echelle Spectrograph (R ~ 60,000). We present Galactic neutron capture abundance gradients using 30+ clusters, within 6 kpc of the Sun, covering a range of ages from ~80 Myr to ~10 Gyr .

Neutron-capture rates for explosive nucleosynthesis: the case of 68Ni(n, γ) 69Ni

DOE PAGES

Spyrou, Artemis; Larsen, Ann-Cecilie; Liddick, Sean N.; ...

2017-02-22

Neutron-capture reactions play an important role in heavy element nucleosynthesis, since they are the driving force for the two processes that create the vast majority of the heavy elements. When a neutron capture occurs on a short-lived nucleus, it is extremely challenging to study the reaction directly and therefore the use of indirect techniques is essential. The present work reports on such an indirect measurement that provides strong constraints on the 68Ni(n,g) 69Ni reaction rate.The commonly used reaction libraries JINA-REACLIB and BRUSLIB are in relatively good agreement with the experimental rate. The impact of the new rate on weak r-processmore » calculations is discussed.« less

The contribution of alu elements to mutagenic DNA double-strand break repair.

PubMed

Morales, Maria E; White, Travis B; Streva, Vincent A; DeFreece, Cecily B; Hedges, Dale J; Deininger, Prescott L

2015-03-01

Alu elements make up the largest family of human mobile elements, numbering 1.1 million copies and comprising 11% of the human genome. As a consequence of evolution and genetic drift, Alu elements of various sequence divergence exist throughout the human genome. Alu/Alu recombination has been shown to cause approximately 0.5% of new human genetic diseases and contribute to extensive genomic structural variation. To begin understanding the molecular mechanisms leading to these rearrangements in mammalian cells, we constructed Alu/Alu recombination reporter cell lines containing Alu elements ranging in sequence divergence from 0%-30% that allow detection of both Alu/Alu recombination and large non-homologous end joining (NHEJ) deletions that range from 1.0 to 1.9 kb in size. Introduction of as little as 0.7% sequence divergence between Alu elements resulted in a significant reduction in recombination, which indicates even small degrees of sequence divergence reduce the efficiency of homology-directed DNA double-strand break (DSB) repair. Further reduction in recombination was observed in a sequence divergence-dependent manner for diverged Alu/Alu recombination constructs with up to 10% sequence divergence. With greater levels of sequence divergence (15%-30%), we observed a significant increase in DSB repair due to a shift from Alu/Alu recombination to variable-length NHEJ which removes sequence between the two Alu elements. This increase in NHEJ deletions depends on the presence of Alu sequence homeology (similar but not identical sequences). Analysis of recombination products revealed that Alu/Alu recombination junctions occur more frequently in the first 100 bp of the Alu element within our reporter assay, just as they do in genomic Alu/Alu recombination events. This is the first extensive study characterizing the influence of Alu element sequence divergence on DNA repair, which will inform predictions regarding the effect of Alu element sequence divergence on both the rate and nature of DNA repair events.

PRESENTED AT TRIANGLE CONSORTIUM OF REPRODUCTIVE BIOLOGY, CHAPEL HILL, NC: GST M1 GENOTYPE INFLUENCES SPERM DNA DAMAGE ASSOCIATED WITH EXPOSURE TO AIR POLLUTION

EPA Science Inventory

Exposure to episodic air pollution in the Czech Republic has been associated with abnormal semen quality and sperm DNA damage (EHP 108:887;2000). A subsequentlongitudinal study evaluated semenfrom 36 men sampled up to 7 times over a period of two years to capture exposures durin...

EFFECT OF DIFFERENT REGIONS OF AMPLIFIED 16S RDNA ON A PERFORMANCE OF A MULTIPLEXED, BEAD-BASED METHOD FOR ANALYSIS OF DNA SEQUENCES IN ENVIRONMENTAL SAMPLES.

EPA Science Inventory

Using a bead-based method for multiplexed analysis of community DNA, the dynamics of aquatic microbial communities can be assessed. Capture probes, specific for a genus or species of bacteria, are attached to the surface of uniquely labeled, microscopic polystyrene beads. Primers...

LINE-1 Elements in Structural Variation and Disease

PubMed Central

Beck, Christine R.; Garcia-Perez, José Luis; Badge, Richard M.; Moran, John V.

2014-01-01

The completion of the human genome reference sequence ushered in a new era for the study and discovery of human transposable elements. It now is undeniable that transposable elements, historically dismissed as junk DNA, have had an instrumental role in sculpting the structure and function of our genomes. In particular, long interspersed element-1 (LINE-1 or L1) and short interspersed elements (SINEs) continue to affect our genome, and their movement can lead to sporadic cases of disease. Here, we briefly review the types of transposable elements present in the human genome and their mechanisms of mobility. We next highlight how advances in DNA sequencing and genomic technologies have enabled the discovery of novel retrotransposons in individual genomes. Finally, we discuss how L1-mediated retrotransposition events impact human genomes. PMID:21801021

SORPTION OF ELEMENTAL MERCURY BY ACTIVATED CARBONS

EPA Science Inventory

The mechanisms and rate of elemental mercury (HgO) capture by activated carbons have been studied using a bench-scale apparatus. Three types of activated carbons, two of which are thermally activated (PC-100 and FGD) and one with elemental sulfur (S) impregnated in it (HGR), were...

The effect of inflammation-related lifestyle exposures and interactions with gene variants on long interspersed nuclear element-1 DNA methylation.

PubMed

Gogna, Priyanka; O'Sullivan, Dylan E; King, Will D

2018-06-11

To examine the relationship between inflammation-related lifestyle factors and long interspersed nuclear element-1 (LINE-1) DNA methylation, and test for interaction by gene variants involved in one-carbon metabolism. The study population consisted of 280 individuals undergoing colonoscopy screening. Multivariable linear regression was employed to examine associations of physical activity, BMI and NSAID use with LINE-1 DNA methylation and interactions with MTR and MTHFR gene variants. The highest quartile of physical activity compared with the lowest was associated with higher LINE-1 DNA methylation (p = 0.005). Long-term NSAID use and a normal BMI were associated with increased LINE-1 DNA methylation among individuals with the variant MTR allele (p = 0.02; p = 0.03). This study provides evidence that inflammation-related exposures may influence LINE-1 DNA methylation.

Comparison of the analytical and clinical performances of Abbott RealTime High Risk HPV, Hybrid Capture 2, and DNA Chip assays in gynecology patients.

PubMed

Park, Seungman; Kang, Youjin; Kim, Dong Geun; Kim, Eui-Chong; Park, Sung Sup; Seong, Moon-Woo

2013-08-01

The detection of high-risk (HR) HPV in cervical cancer screening is important for early diagnosis of cervical cancer or pre-cancerous lesions. We evaluated the analytical and clinical performances of 3 HR HPV assays in Gynecology patients. A total of 991 specimens were included in this study: 787 specimens for use with a Hybrid Capture 2 (HC2) and 204 specimens for a HPV DNA microarray (DNA Chip). All specimens were tested using an Abbott RealTime High Risk HPV assay (Real-time HR), PGMY PCR, and sequence analysis. Clinical sensitivities for severe abnormal cytology (severe than high-grade squamous intraepithelial lesion) were 81.8% for Real-time HR, 77.3% for HC2, and 66.7% for DNA Chip, and clinical sensitivities for severe abnormal histology (cervical intraepithelial neoplasia grade 2+) were 91.7% for HC2, 87.5% for Real-time HR, and 73.3% for DNA Chip. As compared to results of the sequence analysis, HC2, Real-time HR, and DNA Chip showed concordance rates of 94.3% (115/122), 90.0% (117/130), and 61.5% (16/26), respectively. The HC2 assay and Real-time HR assay showed comparable results to each other in both clinical and analytical performances, while the DNA Chip assay showed poor clinical and analytical performances. The Real-time HR assay can be a good alternative option for HR HPV testing with advantages of allowing full automation and simultaneous genotyping of HR types 16 and 18. Copyright © 2013 Elsevier Inc. All rights reserved.

Separation and parallel sequencing of the genomes and transcriptomes of single cells using G&T-seq.

PubMed

Macaulay, Iain C; Teng, Mabel J; Haerty, Wilfried; Kumar, Parveen; Ponting, Chris P; Voet, Thierry

2016-11-01

Parallel sequencing of a single cell's genome and transcriptome provides a powerful tool for dissecting genetic variation and its relationship with gene expression. Here we present a detailed protocol for G&T-seq, a method for separation and parallel sequencing of genomic DNA and full-length polyA(+) mRNA from single cells. We provide step-by-step instructions for the isolation and lysis of single cells; the physical separation of polyA(+) mRNA from genomic DNA using a modified oligo-dT bead capture and the respective whole-transcriptome and whole-genome amplifications; and library preparation and sequence analyses of these amplification products. The method allows the detection of thousands of transcripts in parallel with the genetic variants captured by the DNA-seq data from the same single cell. G&T-seq differs from other currently available methods for parallel DNA and RNA sequencing from single cells, as it involves physical separation of the DNA and RNA and does not require bespoke microfluidics platforms. The process can be implemented manually or through automation. When performed manually, paired genome and transcriptome sequencing libraries from eight single cells can be produced in ∼3 d by researchers experienced in molecular laboratory work. For users with experience in the programming and operation of liquid-handling robots, paired DNA and RNA libraries from 96 single cells can be produced in the same time frame. Sequence analysis and integration of single-cell G&T-seq DNA and RNA data requires a high level of bioinformatics expertise and familiarity with a wide range of informatics tools.

A self-assembled deoxyribonucleic acid concatemer for sensitive detection of single nucleotide polymorphism.

PubMed

Wu, Wei; Chen, Junhua; Fang, Zhiyuan; Ge, Chenchen; Xiang, Zhicheng; Ouyang, Chuanyan; Lie, Puchang; Xiao, Zhuo; Yu, Luxin; Wang, Lin; Zeng, Lingwen

2013-12-04

Polymerase-free and label-free strategies for DNA detection have shown excellent sensitivity and specificity in various biological samples. Herein, we propose a method for single nucleotide polymorphism (SNP) detection by using self-assembled DNA concatemers. Capture probes, bound to magnetic beads, can joint mediator probes by T4 DNA ligase in the presence of target DNA that is complementary to the capture probe and mediator probe. The mediator probes trigger self-assembly of two auxiliary probes on magnetic beads to form DNA concatemers. Separated by a magnetic rack, the double-stranded concatemers on beads can recruit a great amount of SYBR Green I and eventually result in amplified fluorescent signals. In comparison with reported methods for SNP detection, the concatemer-based approach has significant advantages of low background, simplicity, and ultrasensitivity, making it as a convenient platform for clinical applications. As a proof of concept, BRAF(T1799A) oncogene mutation, a SNP involved in diverse human cancers, was used as a model target. The developed approach using a fluorescent intercalator can detect as low as 0.1 fM target BRAF(T1799A) DNA, which is better than those previously published methods for SNP detection. This method is robust and can be used directly to measure the BRAF(T1799A) DNA in complex human serum with excellent recovery (94-103%). It is expected that this assay principle can be directed toward other SNP genes by simply changing the mediator probe and auxiliary probes. Copyright © 2013 Elsevier B.V. All rights reserved.

Sequence of retrovirus provirus resembles that of bacterial transposable elements

NASA Astrophysics Data System (ADS)

Shimotohno, Kunitada; Mizutani, Satoshi; Temin, Howard M.

1980-06-01

The nucleotide sequences of the terminal regions of an infectious integrated retrovirus cloned in the modified λ phage cloning vector Charon 4A have been elucidated. There is a 569-base pair direct repeat at both ends of the viral DNA. The cell-virus junctions at each end consist of a 5-base pair direct repeat of cell DNA next to a 3-base pair inverted repeat of viral DNA. This structure resembles that of a transposable element and is consistent with the protovirus hypothesis that retroviruses evolved from the cell genome.

Molecular Detection of Leishmania DNA in Wild-Caught Phlebotomine Sand Flies (Diptera: Psychodidae) From a Cave in the State of Minas Gerais, Brazil.

PubMed

Carvalho, G M L; Brazil, R P; Rêgo, F D; Ramos, M C N F; Zenóbio, A P L A; Andrade Filho, J D

2017-01-01

Leishmania spp. are distributed throughout the world, and different species are associated with varying degrees of disease severity. In Brazil, Leishmania transmission involves several species of phlebotomine sand flies that are closely associated with different parasites and reservoirs, and thereby giving rise to different transmission cycles. Infection occurs during the bloodmeals of sand flies obtained from a variety of wild and domestic animals, and sometimes from humans. The present study focused on detection of Leishmania DNA in phlebotomine sand flies from a cave in the state of Minas Gerais. Detection of Leishmania in female sand flies was performed with ITS1 PCR-RFLP (internal transcribed spacer 1) using HaeIII enzyme and genetic sequencing for SSUrRNA target. The survey of Leishmania DNA was carried out on 232 pools and the parasite DNA was detected in four: one pool of Lutzomyia cavernicola (Costa Lima, 1932), infected with Le. infantum (ITS1 PCR-RFLP), two pools of Evandromyia sallesi (Galvão & Coutinho, 1939), both infected with Leishmania braziliensis complex (SSUrRNA genetic sequencing analysis), and one pool of Sciopemyia sordellii (Shannon & Del Ponte, 1927), infected with subgenus Leishmania (SSUrRNA genetic sequencing analysis). The present study identified the species for Leishmania DNA detected in four pools of sand flies, all of which were captured inside the cave. These results represent the first molecular detection of Lu cavernicola with Le infantum DNA, Sc sordellii with subgenus Leishmania DNA, and Ev sallesi with Leishmania braziliensis complex DNA. The infection rate in females captured for this study was 0.17%. © The Authors 2016. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Perceptual grouping allows for attention to cover noncontiguous locations and suppress capture from nearby locations.

PubMed

Kerzel, Dirk; Born, Sabine; Schönhammer, Josef

2012-12-01

A salient stimulus may interrupt visual search because of attentional capture. It has been shown that attentional capture occurs with a wide, but not with a small attentional window. We tested the hypothesis that capture depends more strongly on the shape of the attentional window than on its size. Search elements were arranged in two nested rings. The ring containing the search target remained fixed, while a salient color singleton occurred either in the same or in the other ring. We observed that color singletons only disrupted search when shown in the same ring as the search target. It is important to note that, when focusing on the outer array, which presumably required a larger attentional window, singletons on the inner array did not capture attention. In contrast to the original attentional window hypothesis, our results show that attentional capture does not always occur with a large attentional window. Rather, attention can be flexibly allocated to the set of relevant stimulus locations and attentional capture is confined to the attended locations. Further experiments showed that attention was allocated to search elements that were perceptually grouped into "whole" or "Gestalt"-like objects, which prevented attentional capture from nearby locations. However, when attention was allocated to noncontiguous locations that did not form a perceptual Gestalt, nearby locations elicited attentional capture. Perceptual grouping could be based on a combination of color and position, but not on color alone. Thus, the allocation of attention to Gestalt-like objects that were jointly defined by similarity and proximity prevented attentional capture from nearby locations.

Diagnosis of EGFR exon21 L858R point mutation as lung cancer biomarker by electrochemical DNA biosensor based on reduced graphene oxide /functionalized ordered mesoporous carbon/Ni-oxytetracycline metallopolymer nanoparticles modified pencil graphite electrode.

PubMed

Shoja, Yalda; Kermanpur, Ahmad; Karimzadeh, Fathallah

2018-08-15

In this present work we made a novel, fast, selective and sensitive electrochemical genobiosensor to detection of EGFR exon 21 point mutation based on two step electropolymerization of Ni(II)-oxytetracycline conducting metallopolymer nanoparticles (Ni-OTC NPs) on the surface of pencil graphite electrode (PGE) which was modified by reduced graphene oxide/carboxyl functionalized ordered mesoporous carbon (rGO/f-OMC) nanocomposite. ssDNA capture probe with amine groups at the5' end which applied as recognition element was immobilized on the rGO/f-OMC/PGE surface via the strong amide bond. Ni-OTC metallopolymer NPs were electropolymerized to rGO/ssDNA-OMC/PGE surface and then hybridization fallows through the peak current change in differential pulse voltammetry (DPV) using Ni-OTC NPs as a redox label. The biosensor was characterized by field emission scanning electron microscopy (FE-SEM), X-ray diffraction (XRD), FT-IR spectroscopy, energy dispersive X-ray spectroscopy (EDX), cyclic voltammetry and Nitrogen adsorption-desorption analysis. The Ni-OTC current response verified only the complementary sequence indicating a significant reduction current signal in comparison to single point mismatched and non-complementary and sequences. Under optimal conditions, the prepared biosensor showed long-term stability (21 days) with a wide linear range from 0.1 µM to 3 µM with high sensitivity (0.0188 mA/µM) and low detection limit (120 nM). Copyright © 2018 Elsevier B.V. All rights reserved.

Multivalent DNA-binding properties of the HMG-1 proteins.

PubMed Central

Maher, J F; Nathans, D

1996-01-01

HMG-I proteins are DNA-binding proteins thought to affect the formation and function of transcription complexes. Each protein contains three DNA-binding motifs, known as AT-hooks, that bind in the minor groove of AT tracts in DNA. Multiple AT-hooks within a polypeptide chain should contact multiple AT tracts, but the rules governing these interactions have not been defined. In this study, we demonstrate that high-affinity binding uses two or three appropriately spaced AT tracts as a single multivalent binding site. These principles have implications for binding to regulatory elements such as the interferon beta enhancer, TATA boxes, and serum response elements. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:8692884

Control of DEMETER DNA demethylase gene transcription in male and female gamete companion cells in Arabidopsis thaliana.

PubMed

Park, Jin-Sup; Frost, Jennifer M; Park, Kyunghyuk; Ohr, Hyonhwa; Park, Guen Tae; Kim, Seohyun; Eom, Hyunjoo; Lee, Ilha; Brooks, Janie S; Fischer, Robert L; Choi, Yeonhee

2017-02-21

The DEMETER (DME) DNA glycosylase initiates active DNA demethylation via the base-excision repair pathway and is vital for reproduction in Arabidopsis thaliana DME-mediated DNA demethylation is preferentially targeted to small, AT-rich, and nucleosome-depleted euchromatic transposable elements, influencing expression of adjacent genes and leading to imprinting in the endosperm. In the female gametophyte, DME expression and subsequent genome-wide DNA demethylation are confined to the companion cell of the egg, the central cell. Here, we show that, in the male gametophyte, DME expression is limited to the companion cell of sperm, the vegetative cell, and to a narrow window of time: immediately after separation of the companion cell lineage from the germline. We define transcriptional regulatory elements of DME using reporter genes, showing that a small region, which surprisingly lies within the DME gene, controls its expression in male and female companion cells. DME expression from this minimal promoter is sufficient to rescue seed abortion and the aberrant DNA methylome associated with the null dme-2 mutation. Within this minimal promoter, we found short, conserved enhancer sequences necessary for the transcriptional activities of DME and combined predicted binding motifs with published transcription factor binding coordinates to produce a list of candidate upstream pathway members in the genetic circuitry controlling DNA demethylation in gamete companion cells. These data show how DNA demethylation is regulated to facilitate endosperm gene imprinting and potential transgenerational epigenetic regulation, without subjecting the germline to potentially deleterious transposable element demethylation.

Real-time and label-free ring-resonator monitoring of solid-phase recombinase polymerase amplification.

PubMed

Sabaté Del Río, Jonathan; Steylaerts, Tim; Henry, Olivier Y F; Bienstman, Peter; Stakenborg, Tim; Van Roy, Wim; O'Sullivan, Ciara K

2015-11-15

In this work we present the use of a silicon-on-insulator (SOI) chip featuring an array of 64 optical ring resonators used as refractive index sensors for real-time and label-free DNA detection. Single ring functionalisation was achieved using a click reaction after precise nanolitre spotting of specific hexynyl-terminated DNA capture probes to link to an azido-silanised chip surface. To demonstrate detectability using the ring resonators and to optimise conditions for solid-phase amplification, hybridisation between short 25-mer single stranded DNA (ssDNA) fragments and a complementary capture probe immobilised on the surface of the ring resonators was carried out and detected through the shift in the resonant wavelength. Using the optimised conditions demonstrated via the solid-phase hybridisation, a 144-bp double stranded DNA (dsDNA) was then detected directly using recombinase and polymerase proteins through on-chip target amplification and solid-phase elongation of immobilised forward primers on specific rings, at a constant temperature of 37°C and in less than 60min, achieving a limit of detection of 7.8·10(-13)M (6·10(5) copies in 50µL). The use of an automatic liquid handler injection instrument connected to an integrated resealable chip interface (RCI) allowed programmable multiple injection protocols. Air plugs between different solutions were introduced to prevent intermixing and a proportional-integral-derivative (PID) temperature controller minimised temperature based drifts. Published by Elsevier B.V.

Searching for DNA Lesions: Structural Evidence for Lower- and Higher-Affinity DNA Binding Conformations of Human Alkyladenine DNA Glycosylase

PubMed Central

2011-01-01

To efficiently repair DNA, human alkyladenine DNA glycosylase (AAG) must search the million-fold excess of unmodified DNA bases to find a handful of DNA lesions. Such a search can be facilitated by the ability of glycosylases, like AAG, to interact with DNA using two affinities: a lower-affinity interaction in a searching process and a higher-affinity interaction for catalytic repair. Here, we present crystal structures of AAG trapped in two DNA-bound states. The lower-affinity depiction allows us to investigate, for the first time, the conformation of this protein in the absence of a tightly bound DNA adduct. We find that active site residues of AAG involved in binding lesion bases are in a disordered state. Furthermore, two loops that contribute significantly to the positive electrostatic surface of AAG are disordered. Additionally, a higher-affinity state of AAG captured here provides a fortuitous snapshot of how this enzyme interacts with a DNA adduct that resembles a one-base loop. PMID:22148158

Evidence of pervasive biologically functional secondary structures within the genomes of eukaryotic single-stranded DNA viruses.

PubMed

Muhire, Brejnev Muhizi; Golden, Michael; Murrell, Ben; Lefeuvre, Pierre; Lett, Jean-Michel; Gray, Alistair; Poon, Art Y F; Ngandu, Nobubelo Kwanele; Semegni, Yves; Tanov, Emil Pavlov; Monjane, Adérito Luis; Harkins, Gordon William; Varsani, Arvind; Shepherd, Dionne Natalie; Martin, Darren Patrick

2014-02-01

Single-stranded DNA (ssDNA) viruses have genomes that are potentially capable of forming complex secondary structures through Watson-Crick base pairing between their constituent nucleotides. A few of the structural elements formed by such base pairings are, in fact, known to have important functions during the replication of many ssDNA viruses. Unknown, however, are (i) whether numerous additional ssDNA virus genomic structural elements predicted to exist by computational DNA folding methods actually exist and (ii) whether those structures that do exist have any biological relevance. We therefore computationally inferred lists of the most evolutionarily conserved structures within a diverse selection of animal- and plant-infecting ssDNA viruses drawn from the families Circoviridae, Anelloviridae, Parvoviridae, Nanoviridae, and Geminiviridae and analyzed these for evidence of natural selection favoring the maintenance of these structures. While we find evidence that is consistent with purifying selection being stronger at nucleotide sites that are predicted to be base paired than at sites predicted to be unpaired, we also find strong associations between sites that are predicted to pair with one another and site pairs that are apparently coevolving in a complementary fashion. Collectively, these results indicate that natural selection actively preserves much of the pervasive secondary structure that is evident within eukaryote-infecting ssDNA virus genomes and, therefore, that much of this structure is biologically functional. Lastly, we provide examples of various highly conserved but completely uncharacterized structural elements that likely have important functions within some of the ssDNA virus genomes analyzed here.

Evidence of Pervasive Biologically Functional Secondary Structures within the Genomes of Eukaryotic Single-Stranded DNA Viruses

PubMed Central

Muhire, Brejnev Muhizi; Golden, Michael; Murrell, Ben; Lefeuvre, Pierre; Lett, Jean-Michel; Gray, Alistair; Poon, Art Y. F.; Ngandu, Nobubelo Kwanele; Semegni, Yves; Tanov, Emil Pavlov; Monjane, Adérito Luis; Harkins, Gordon William; Varsani, Arvind; Shepherd, Dionne Natalie

2014-01-01

Single-stranded DNA (ssDNA) viruses have genomes that are potentially capable of forming complex secondary structures through Watson-Crick base pairing between their constituent nucleotides. A few of the structural elements formed by such base pairings are, in fact, known to have important functions during the replication of many ssDNA viruses. Unknown, however, are (i) whether numerous additional ssDNA virus genomic structural elements predicted to exist by computational DNA folding methods actually exist and (ii) whether those structures that do exist have any biological relevance. We therefore computationally inferred lists of the most evolutionarily conserved structures within a diverse selection of animal- and plant-infecting ssDNA viruses drawn from the families Circoviridae, Anelloviridae, Parvoviridae, Nanoviridae, and Geminiviridae and analyzed these for evidence of natural selection favoring the maintenance of these structures. While we find evidence that is consistent with purifying selection being stronger at nucleotide sites that are predicted to be base paired than at sites predicted to be unpaired, we also find strong associations between sites that are predicted to pair with one another and site pairs that are apparently coevolving in a complementary fashion. Collectively, these results indicate that natural selection actively preserves much of the pervasive secondary structure that is evident within eukaryote-infecting ssDNA virus genomes and, therefore, that much of this structure is biologically functional. Lastly, we provide examples of various highly conserved but completely uncharacterized structural elements that likely have important functions within some of the ssDNA virus genomes analyzed here. PMID:24284329

Hsmar1 Transposition Is Sensitive to the Topology of the Transposon Donor and the Target

PubMed Central

Claeys Bouuaert, Corentin; Chalmers, Ronald

2013-01-01

Hsmar1 is a member of the Tc1-mariner superfamily of DNA transposons. These elements mobilize within the genome of their host by a cut-and-paste mechanism. We have exploited the in vitro reaction provided by Hsmar1 to investigate the effect of DNA supercoiling on transposon integration. We found that the topology of both the transposon and the target affect integration. Relaxed transposons have an integration defect that can be partially restored in the presence of elevated levels of negatively supercoiled target DNA. Negatively supercoiled DNA is a better target than nicked or positively supercoiled DNA, suggesting that underwinding of the DNA helix promotes target interactions. Like other Tc1-mariner elements, Hsmar1 integrates into 5′-TA dinucleotides. The direct vicinity of the target TA provides little sequence specificity for target interactions. However, transposition within a plasmid substrate was not random and some TA dinucleotides were targeted preferentially. The distribution of intramolecular target sites was not affected by DNA topology. PMID:23341977

Structure of a bacterial RNA polymerase holoenzyme open promoter complex

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bae, Brian; Feklistov, Andrey; Lass-Napiorkowska, Agnieszka

2015-09-08

Initiation of transcription is a primary means for controlling gene expression. In bacteria, the RNA polymerase (RNAP) holoenzyme binds and unwinds promoter DNA, forming the transcription bubble of the open promoter complex (RPo). We have determined crystal structures, refined to 4.14 Å-resolution, of RPo containing Thermus aquaticus RNAP holoenzyme and promoter DNA that includes the full transcription bubble. The structures, combined with biochemical analyses, reveal key features supporting the formation and maintenance of the double-strand/single-strand DNA junction at the upstream edge of the -10 element where bubble formation initiates. The results also reveal RNAP interactions with duplex DNA just upstreammore » of the -10 element and potential protein/DNA interactions that direct the DNA template strand into the RNAP active site. Addition of an RNA primer to yield a 4 base-pair post-translocated RNA:DNA hybrid mimics an initially transcribing complex at the point where steric clash initiates abortive initiation and σA dissociation.« less

Structure of a bacterial RNA polymerase holoenzyme open promoter complex

DOE PAGES

Bae, Brian; Feklistov, Andrey; Lass-Napiorkowska, Agnieszka; ...

2015-09-08

Initiation of transcription is a primary means for controlling gene expression. In bacteria, the RNA polymerase (RNAP) holoenzyme binds and unwinds promoter DNA, forming the transcription bubble of the open promoter complex (RPo). We have determined crystal structures, refined to 4.14 Å-resolution, of RPo containing Thermus aquaticus RNAP holoenzyme and promoter DNA that includes the full transcription bubble. The structures, combined with biochemical analyses, reveal key features supporting the formation and maintenance of the double-strand/single-strand DNA junction at the upstream edge of the -10 element where bubble formation initiates. The results also reveal RNAP interactions with duplex DNA just upstreammore » of the -10 element and potential protein/DNA interactions that direct the DNA template strand into the RNAP active site. Additionally a RNA primer to yield a 4 base-pair post-translocated RNA:DNA hybrid mimics an initially transcribing complex at the point where steric clash initiates abortive initiation and σ A dissociation.« less

LDsplit: screening for cis-regulatory motifs stimulating meiotic recombination hotspots by analysis of DNA sequence polymorphisms.

PubMed

Yang, Peng; Wu, Min; Guo, Jing; Kwoh, Chee Keong; Przytycka, Teresa M; Zheng, Jie

2014-02-17

As a fundamental genomic element, meiotic recombination hotspot plays important roles in life sciences. Thus uncovering its regulatory mechanisms has broad impact on biomedical research. Despite the recent identification of the zinc finger protein PRDM9 and its 13-mer binding motif as major regulators for meiotic recombination hotspots, other regulators remain to be discovered. Existing methods for finding DNA sequence motifs of recombination hotspots often rely on the enrichment of co-localizations between hotspots and short DNA patterns, which ignore the cross-individual variation of recombination rates and sequence polymorphisms in the population. Our objective in this paper is to capture signals encoded in genetic variations for the discovery of recombination-associated DNA motifs. Recently, an algorithm called "LDsplit" has been designed to detect the association between single nucleotide polymorphisms (SNPs) and proximal meiotic recombination hotspots. The association is measured by the difference of population recombination rates at a hotspot between two alleles of a candidate SNP. Here we present an open source software tool of LDsplit, with integrative data visualization for recombination hotspots and their proximal SNPs. Applying LDsplit on SNPs inside an established 7-mer motif bound by PRDM9 we observed that SNP alleles preserving the original motif tend to have higher recombination rates than the opposite alleles that disrupt the motif. Running on SNP windows around hotspots each containing an occurrence of the 7-mer motif, LDsplit is able to guide the established motif finding algorithm of MEME to recover the 7-mer motif. In contrast, without LDsplit the 7-mer motif could not be identified. LDsplit is a software tool for the discovery of cis-regulatory DNA sequence motifs stimulating meiotic recombination hotspots by screening and narrowing down to hotspot associated SNPs. It is the first computational method that utilizes the genetic variation of recombination hotspots among individuals, opening a new avenue for motif finding. Tested on an established motif and simulated datasets, LDsplit shows promise to discover novel DNA motifs for meiotic recombination hotspots.

LDsplit: screening for cis-regulatory motifs stimulating meiotic recombination hotspots by analysis of DNA sequence polymorphisms

PubMed Central

2014-01-01

Background As a fundamental genomic element, meiotic recombination hotspot plays important roles in life sciences. Thus uncovering its regulatory mechanisms has broad impact on biomedical research. Despite the recent identification of the zinc finger protein PRDM9 and its 13-mer binding motif as major regulators for meiotic recombination hotspots, other regulators remain to be discovered. Existing methods for finding DNA sequence motifs of recombination hotspots often rely on the enrichment of co-localizations between hotspots and short DNA patterns, which ignore the cross-individual variation of recombination rates and sequence polymorphisms in the population. Our objective in this paper is to capture signals encoded in genetic variations for the discovery of recombination-associated DNA motifs. Results Recently, an algorithm called “LDsplit” has been designed to detect the association between single nucleotide polymorphisms (SNPs) and proximal meiotic recombination hotspots. The association is measured by the difference of population recombination rates at a hotspot between two alleles of a candidate SNP. Here we present an open source software tool of LDsplit, with integrative data visualization for recombination hotspots and their proximal SNPs. Applying LDsplit on SNPs inside an established 7-mer motif bound by PRDM9 we observed that SNP alleles preserving the original motif tend to have higher recombination rates than the opposite alleles that disrupt the motif. Running on SNP windows around hotspots each containing an occurrence of the 7-mer motif, LDsplit is able to guide the established motif finding algorithm of MEME to recover the 7-mer motif. In contrast, without LDsplit the 7-mer motif could not be identified. Conclusions LDsplit is a software tool for the discovery of cis-regulatory DNA sequence motifs stimulating meiotic recombination hotspots by screening and narrowing down to hotspot associated SNPs. It is the first computational method that utilizes the genetic variation of recombination hotspots among individuals, opening a new avenue for motif finding. Tested on an established motif and simulated datasets, LDsplit shows promise to discover novel DNA motifs for meiotic recombination hotspots. PMID:24533858

ICE Afe 1, an actively excising genetic element from the biomining bacterium Acidithiobacillus ferrooxidans.

PubMed

Bustamante, Paula; Covarrubias, Paulo C; Levicán, Gloria; Katz, Assaf; Tapia, Pablo; Holmes, David; Quatrini, Raquel; Orellana, Omar

2012-01-01

Integrative conjugative elements (ICEs) are self-transferred mobile genetic elements that contribute to horizontal gene transfer. An ICE (ICEAfe1) was identified in the genome of Acidithiobacillus ferrooxidans ATCC 23270. Excision of the element and expression of relevant genes under normal and DNA-damaging growth conditions was analyzed. Bioinformatic tools and DNA amplification methods were used to identify and to assess the excision and expression of genes related to the mobility of the element. Both basal and mitomycin C-inducible excision as well as expression and induction of the genes for integration/excision are demonstrated, suggesting that ICEAfe1 is an actively excising SOS-regulated mobile genetic element. The presence of a complete set of genes encoding self-transfer functions that are induced in response to DNA damage caused by mitomycin C additionally suggests that this element is capable of conjugative transfer to suitable recipient strains. Transfer of ICEAfe1 may provide selective advantages to other acidophiles in this ecological niche through dissemination of gene clusters expressing transfer RNAs, CRISPRs, and exopolysaccharide biosynthesis enzymes, probably by modification of translation efficiency, resistance to bacteriophage infection and biofilm formation, respectively. These data open novel avenues of research on conjugative transformation of biotechnologically relevant microorganisms recalcitrant to genetic manipulation. Copyright © 2013 S. Karger AG, Basel.

Cut-and-Paste Transposons in Fungi with Diverse Lifestyles

PubMed Central

Steczkiewicz, Kamil; Ginalski, Krzysztof

2017-01-01

Abstract Transposable elements (TEs) shape genomes via recombination and transposition, lead to chromosomal rearrangements, create new gene neighborhoods, and alter gene expression. They play key roles in adaptation either to symbiosis in Amanita genus or to pathogenicity in Pyrenophora tritici-repentis. Despite growing evidence of their importance, the abundance and distribution of mobile elements replicating in a “cut-and-paste” fashion is barely described so far. In order to improve our knowledge on this old and ubiquitous class of transposable elements, 1,730 fungal genomes were scanned using both de novo and homology-based approaches. DNA TEs have been identified across the whole data set and display uneven distribution from both DNA TE classification and fungal taxonomy perspectives. DNA TE content correlates with genome size, which confirms that many transposon families proliferate simultaneously. In contrast, it is independent from intron density, average gene distance and GC content. TE count is associated with species’ lifestyle and tends to be elevated in plant symbionts and decreased in animal parasites. Lastly, we found that fungi with both RIP and RNAi systems have more total DNA TE sequences but less elements retaining a functional transposase, what reflects stringent control over transposition. PMID:29228286

Strip biosensor for amplified detection of nerve growth factor-beta based on a molecular translator and catalytic DNA circuit.

PubMed

Liu, Jun; Lai, Ting; Mu, Kejie; Zhou, Zheng

2014-10-07

We have demonstrated a new visual detection approach based on a molecular translator and a catalytic DNA circuit for the detection of nerve growth factor-beta (NGF-β). In this assay, a molecular translator based on the binding-induced DNA strand-displacement reaction was employed to convert the input protein to an output DNA signal. The molecular translator is composed of a target recognition element and a signal output element. Target recognition is achieved by the binding of the anti-NGF-β antibody to the target protein. Polyclonal anti-NGF-β antibody is conjugated to DNA1 and DNA2. The antibody conjugated DNA1 is initially hybridized to DNA3 to form a stable DNA1/DNA3 duplex. In the presence of NGF-β, the binding of the same target protein brings DNA1 and DNA2 into close proximity, resulting in an increase in their local effective concentration. This process triggers the strand-displacement reaction between DNA2 and DNA3 and releases the output DNA3. The released DNA3 is further amplified by a catalytic DNA circuit. The product of the catalytic DNA circuit is detected by a strip biosensor. This proposed assay has high sensitivity and selectivity with a dynamic response ranging from 10 fM to 10 pM, and its detection limit is 10 fM of NGF-β. This work provides a sensitive, enzyme-free, and universal strategy for the detection of other proteins.

Primer-Independent DNA Synthesis by a Family B DNA Polymerase from Self-Replicating Mobile Genetic Elements.

PubMed

Redrejo-Rodríguez, Modesto; Ordóñez, Carlos D; Berjón-Otero, Mónica; Moreno-González, Juan; Aparicio-Maldonado, Cristian; Forterre, Patrick; Salas, Margarita; Krupovic, Mart

2017-11-07

Family B DNA polymerases (PolBs) play a central role during replication of viral and cellular chromosomes. Here, we report the discovery of a third major group of PolBs, which we denote primer-independent PolB (piPolB), that might be a link between the previously known protein-primed and RNA/DNA-primed PolBs. PiPolBs are encoded by highly diverse mobile genetic elements, pipolins, integrated in the genomes of diverse bacteria and also present as circular plasmids in mitochondria. Biochemical characterization showed that piPolB displays efficient DNA polymerization activity that can use undamaged and damaged templates and is endowed with proofreading and strand displacement capacities. Remarkably, the protein is also capable of template-dependent de novo DNA synthesis, i.e., DNA-priming activity, thereby breaking the long-standing dogma that replicative DNA polymerases require a pre-existing primer for DNA synthesis. We suggest that piPolBs are involved in self-replication of pipolins and may also contribute to bacterial DNA damage tolerance. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Rapid Microarray Detection of DNA and Proteins in Microliter Volumes with SPR Imaging Measurements

PubMed Central

Seefeld, Ting Hu; Zhou, Wen-Juan; Corn, Robert M.

2011-01-01

A four chamber microfluidic biochip is fabricated for the rapid detection of multiple proteins and nucleic acids from microliter volume samples with the technique of surface plasmon resonance imaging (SPRI). The 18 mm × 18 mm biochip consists of four 3 μL microfluidic chambers attached to an SF10 glass substrate, each of which contains three individually addressable SPRI gold thin film microarray elements. The twelve element (4 × 3) SPRI microarray consists of gold thin film spots (1 mm2 area; 45 nm thickness) each in individually addressable 0.5 μL volume microchannels. Microarrays of single-stranded DNA and RNA (ssDNA and ssRNA respectively) are fabricated by either chemical and/or enzymatic attachment reactions in these microchannels; the SPRI microarrays are then used to detect femtomole amounts (nanomolar concentrations) of DNA and proteins (single stranded DNA binding protein and thrombin via aptamer-protein bioaffinity interactions). Microarrays of ssRNA microarray elements were also used for the ultrasensitive detection of zeptomole amounts (femtomolar concentrations) of DNA via the technique of RNase H-amplified SPRI. Enzymatic removal of ssRNA from the surface due to the hybridization adsorption of target ssDNA is detected as a reflectivity decrease in the SPR imaging measurements. The observed reflectivity loss was proportional to the log of the target ssDNA concentration with a detection limit of 10 fM or 30 zeptomoles (18,000 molecules). This enzymatic amplified ssDNA detection method is not limited by diffusion of ssDNA to the interface, and thus is extremely fast, requiring only 200 seconds in the microliter volume format. PMID:21488682

Recent Research with the Detector for Advanced Neutron Capture Experiments (dance) at the LOS Alamos Neutron Science Center

NASA Astrophysics Data System (ADS)

Ullmann, J. L.

2014-09-01

The DANCE detector at Los Alamos is a 160 element, nearly 4π BaF2 detector array designed to make measurements of neutron capture on rare or radioactive nuclides. It has also been used to make measurements of gamma-ray multiplicity following capture and gamma-ray output from fission. Several examples of measurements are briefly discussed.

Development of a high-throughput microfluidic integrated microarray for the detection of chimeric bioweapons.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sheppod, Timothy; Satterfield, Brent; Hukari, Kyle W.

2006-10-01

The advancement of DNA cloning has significantly augmented the potential threat of a focused bioweapon assault, such as a terrorist attack. With current DNA cloning techniques, toxin genes from the most dangerous (but environmentally labile) bacterial or viral organism can now be selected and inserted into robust organism to produce an infinite number of deadly chimeric bioweapons. In order to neutralize such a threat, accurate detection of the expressed toxin genes, rather than classification on strain or genealogical decent of these organisms, is critical. The development of a high-throughput microarray approach will enable the detection of unknowns chimeric bioweapons. Themore » development of a high-throughput microarray approach will enable the detection of unknown bioweapons. We have developed a unique microfluidic approach to capture and concentrate these threat genes (mRNA's) upto a 30 fold concentration. These captured oligonucleotides can then be used to synthesize in situ oligonucleotide copies (cDNA probes) of the captured genes. An integrated microfluidic architecture will enable us to control flows of reagents, perform clean-up steps and finally elute nanoliter volumes of synthesized oligonucleotides probes. The integrated approach has enabled a process where chimeric or conventional bioweapons can rapidly be identified based on their toxic function, rather than being restricted to information that may not identify the critical nature of the threat.« less

Improving the prospects of cleavage-based nanopore sequencing engines

NASA Astrophysics Data System (ADS)

Brady, Kyle T.; Reiner, Joseph E.

2015-08-01

Recently proposed methods for DNA sequencing involve the use of cleavage-based enzymes attached to the opening of a nanopore. The idea is that DNA interacting with either an exonuclease or polymerase protein will lead to a small molecule being cleaved near the mouth of the nanopore, and subsequent entry into the pore will yield information about the DNA sequence. The prospects for this approach seem promising, but it has been shown that diffusion related effects impose a limit on the capture probability of molecules by the pore, which limits the efficacy of the technique. Here, we revisit the problem with the goal of optimizing the capture probability via a step decrease in the nucleotide diffusion coefficient between the pore and bulk solutions. It is shown through random walk simulations and a simplified analytical model that decreasing the molecule's diffusion coefficient in the bulk relative to its value in the pore increases the nucleotide capture probability. Specifically, we show that at sufficiently high applied transmembrane potentials (≥100 mV), increasing the potential by a factor f is equivalent to decreasing the diffusion coefficient ratio Dbulk/Dpore by the same factor f. This suggests a promising route toward implementation of cleavage-based sequencing protocols. We also discuss the feasibility of forming a step function in the diffusion coefficient across the pore-bulk interface.

In Vitro Selection of a Single-Stranded DNA Molecular Recognition Element against the Pesticide Fipronil and Sensitive Detection in River Water

PubMed Central

Sooter, Letha J.

2017-01-01

Fipronil is a commonly used insecticide that has been shown to have environmental and human health risks. The current standard methods of detection for fipronil and its metabolites, such as GC-MS, are time consuming and labor intensive. In this study, a variant of systematic evolution of ligands by exponential enrichment (SELEX), was utilized to identify the first single-stranded DNA (ssDNA) molecular recognition element (MRE) that binds to fipronil with high affinity (Kd = 48 ± 8 nM). The selected MRE displayed low cross binding activity on various environmentally relevant, structurally unrelated herbicides and pesticides, in addition to broad-spectrum binding activity on major metabolites of fipronil and a structurally similar pesticide in prepared river samples. Additionally, a proof-of-principle fluorescent detection assay was developed by using the selected ssDNA MRE as a signal-reporting element, with a limit of detection of 105 nM in a prepared river water sample. PMID:29283416

DNA extraction techniques compared for accurate detection of genetically modified organisms (GMOs) in maize food and feed products.

PubMed

Turkec, Aydin; Kazan, Hande; Karacanli, Burçin; Lucas, Stuart J

2015-08-01

In this paper, DNA extraction methods have been evaluated to detect the presence of genetically modified organisms (GMOs) in maize food and feed products commercialised in Turkey. All the extraction methods tested performed well for the majority of maize foods and feed products analysed. However, the highest DNA content was achieved by the Wizard, Genespin or the CTAB method, all of which produced optimal DNA yield and purity for different maize food and feed products. The samples were then screened for the presence of GM elements, along with certified reference materials. Of the food and feed samples, 8 % tested positive for the presence of one GM element (NOS terminator), of which half (4 % of the total) also contained a second element (the Cauliflower Mosaic Virus 35S promoter). The results obtained herein clearly demonstrate the presence of GM maize in the Turkish market, and that the Foodproof GMO Screening Kit provides reliable screening of maize food and feed products.

A dual amplification strategy for DNA detection combining bio-barcode assay and metal-enhanced fluorescence modality.

PubMed

Zhou, Zhenpeng; Li, Tian; Huang, Hongduan; Chen, Yang; Liu, Feng; Huang, Chengzhi; Li, Na

2014-11-11

Silver-enhanced fluorescence was coupled with a bio-barcode assay to facilitate a dual amplification assay to demonstrate a non-enzymatic approach for simple and sensitive detection of DNA. In the assay design, magnetic nanoparticles seeded with silver nanoparticles were modified with the capture DNA, and silver nanoparticles were modified with the binding of ssDNA and the fluorescently labeled barcode dsDNA. Upon introduction of the target DNA, a sandwich structure was formed because of the hybridization reaction. By simple magnetic separation, silver-enhanced fluorescence of barcode DNAs could be readily measured without the need of a further step to liberate barcode DNAs from silver nanoparticles, endowing the method with simplicity and high sensitivity with a detection limit of 1 pM.

Numerical study on determining formation porosity using a boron capture gamma ray technique and MCNP.

PubMed

Liu, Juntao; Zhang, Feng; Wang, Xinguang; Han, Fei; Yuan, Zhelong

2014-12-01

Formation porosity can be determined using the boron capture gamma ray counting ratio with a near to far detector in a pulsed neutron-gamma element logging tool. The thermal neutron distribution, boron capture gamma spectroscopy and porosity response for formations with different water salinity and wellbore diameter characteristics were simulated using the Monte Carlo method. We found that a boron lining improves the signal-to-noise ratio and that the boron capture gamma ray counting ratio has a higher sensitivity for determining porosity than total capture gamma. Copyright © 2014 Elsevier Ltd. All rights reserved.

Tyrosine Recombinase Retrotransposons and Transposons.

PubMed

Poulter, Russell T M; Butler, Margi I

2015-04-01

Retrotransposons carrying tyrosine recombinases (YR) are widespread in eukaryotes. The first described tyrosine recombinase mobile element, DIRS1, is a retroelement from the slime mold Dictyostelium discoideum. The YR elements are bordered by terminal repeats related to their replication via free circular dsDNA intermediates. Site-specific recombination is believed to integrate the circle without creating duplications of the target sites. Recently a large number of YR retrotransposons have been described, including elements from fungi (mucorales and basidiomycetes), plants (green algae) and a wide range of animals including nematodes, insects, sea urchins, fish, amphibia and reptiles. YR retrotransposons can be divided into three major groups: the DIRS elements, PAT-like and the Ngaro elements. The three groups form distinct clades on phylogenetic trees based on alignments of reverse transcriptase/ribonuclease H (RT/RH) and YR sequences, and also having some structural distinctions. A group of eukaryote DNA transposons, cryptons, also carry tyrosine recombinases. These DNA transposons do not encode a reverse transcriptase. They have been detected in several pathogenic fungi and oomycetes. Sequence comparisons suggest that the crypton YRs are related to those of the YR retrotransposons. We suggest that the YR retrotransposons arose from the combination of a crypton-like YR DNA transposon and the RT/RH encoding sequence of a retrotransposon. This acquisition must have occurred at a very early point in the evolution of eukaryotes.

Specific interaction of mutant p53 with regions of matrix attachment region DNA elements (MARs) with a high potential for base-unpairing

PubMed Central

Will, Katrin; Warnecke, Gabriele; Wiesmüller, Lisa; Deppert, Wolfgang

1998-01-01

Mutant, but not wild-type p53 binds with high affinity to a variety of MAR-DNA elements (MARs), suggesting that MAR-binding of mutant p53 relates to the dominant-oncogenic activities proposed for mutant p53. MARs recognized by mutant p53 share AT richness and contain variations of an AATATATTT “DNA-unwinding motif,” which enhances the structural dynamics of chromatin and promotes regional DNA base-unpairing. Mutant p53 specifically interacted with MAR-derived oligonucleotides carrying such unwinding motifs, catalyzing DNA strand separation when this motif was located within a structurally labile sequence environment. Addition of GC-clamps to the respective MAR-oligonucleotides or introducing mutations into the unwinding motif strongly reduced DNA strand separation, but supported the formation of tight complexes between mutant p53 and such oligonucleotides. We conclude that the specific interaction of mutant p53 with regions of MAR-DNA with a high potential for base-unpairing provides the basis for the high-affinity binding of mutant p53 to MAR-DNA. PMID:9811860

Complementary and partially complementary DNA duplexes tethered to a functionalized substrate: a molecular dynamics approach to biosensing.

PubMed

Monti, Susanna; Cacelli, Ivo; Ferretti, Alessandro; Prampolini, Giacomo; Barone, Vincenzo

2011-07-21

Molecular dynamics simulations (90 ns) of different DNA complexes attached to a functionalized substrate in solution were performed in order to clarify the behavior of mismatched DNA sequences captured by a tethered DNA probe (biochip). Examination of the trajectories revealed that the substrate influence and a series of cooperative events, including recognition, reorientation and reorganization of the bases, could induce the formation of stable duplexes having non-canonical arrangements. Major adjustment of the structures was observed when the mutated base was located in the end region of the chain close to the surface. This journal is © the Owner Societies 2011

Observing DNA in live cells.

PubMed

Weiss, Lucien E; Naor, Tal; Shechtman, Yoav

2018-06-19

The structural organization and dynamics of DNA are known to be of paramount importance in countless cellular processes, but capturing these events poses a unique challenge. Fluorescence microscopy is well suited for these live-cell investigations, but requires attaching fluorescent labels to the species under investigation. Over the past several decades, a suite of techniques have been developed for labeling and imaging DNA, each with various advantages and drawbacks. Here, we provide an overview of the labeling and imaging tools currently available for visualizing DNA in live cells, and discuss their suitability for various applications. © 2018 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

A Pilot Study of Noninvasive Prenatal Diagnosis of Alpha- and Beta-Thalassemia with Target Capture Sequencing of Cell-Free Fetal DNA in Maternal Blood.

PubMed

Wang, Wenjuan; Yuan, Yuan; Zheng, Haiqing; Wang, Yaoshen; Zeng, Dan; Yang, Yihua; Yi, Xin; Xia, Yang; Zhu, Chunjiang

2017-07-01

Thalassemia is a dangerous hematolytic genetic disease. In south China, ∼24% Chinese carry alpha-thalassemia or beta-thalassemia gene mutations. Given the fact that the invasive sampling procedures can only be performed by professionals in experienced centers, it may increase the risk of miscarriage or infection. Thus, most people are worried about the invasive operation. As such, a noninvasive and accurate prenatal diagnosis is needed for appropriate genetic counseling for families with high risks. Here we sought to develop capture probes and their companion analysis methods for the noninvasive prenatal detection of deletional and nondeletional thalassemia. Two families diagnosed as carriers of either beta-thalassemia gene or Southeast Asian deletional alpha-thalassemia gene mutation were recruited. The maternal plasma and amniotic fluid were collected for prenatal diagnosis. Probes targeting exons of the genes of interest and the highly heterozygous SNPs within the 1Mb flanking region were designed. The target capture sequencing was performed with plasma DNA from the pregnant woman and genomic DNA from the couples and their children. Then the parental haplotype was constructed by the trios-based strategy. The fetal haplotype was deduced from the parental haplotype with a hidden Markov model-based algorithm. The fetal genotypes were successfully deduced in both families noninvasively. The noninvasively constructed haplotypes of both fetuses were identical to the invasive prenatal diagnosis results with an accuracy rate of 100% in the target region. Our study demonstrates that the effective noninvasive prenatal diagnosis of alpha-thalassemia and beta-thalassemia can be achieved with the targeted capture sequencing and the haplotype-assisted analysis method.

Molecular detection of Leishmania infantum and Leishmania tropica in rodent species from endemic cutaneous leishmaniasis areas in Morocco.

PubMed

Echchakery, Mohamed; Chicharro, Carmen; Boussaa, Samia; Nieto, Javier; Carrillo, Eugenia; Sheila, Ortega; Moreno, Javier; Boumezzough, Ali

2017-10-02

Leishmaniasis remains a major public health problem in African nations, including Morocco, where little is known about the vertebrate reservoirs involved in the causal parasites' transmission cycles. The present study investigates the role of rodent species as potential reservoirs of Leishmania spp. in central Morocco, where both L. tropica and L. infantum have been reported. Rodents were caught from 22 sites in central Morocco, by using Sherman metal traps, and identified morphologically. For each specimen, genomic DNA was extracted from different tissues using the Speed Tools DNA extraction Kit. Then, samples were PCR-analyzed, targeting the SSU rRNA gene to detect Leishmania spp. DNA, followed by amplification of the internal transcribed spacer 1 (ITS1) and its sequencing to identify the species. A total of 197 rodents belonging to ten species were captured and identified: Rattus rattus (40.61%), Mus musculus (25.38%), Apodemus sylvaticus (8.63%), Mus spretus (7.11%), Meriones shawi (5.58%), Rattus norvegicus (4.57%), Meriones libycus (3.05%), Mastomys erythroleucus (2.03%), Gerbillus campestris (2.03%) and Lemniscomys barbarus (1.01%). Molecular analysis revealed the presence of Leishmania species in 18 specimens: six R. rattus (out of 80 captured; 7.5%), 11 M. musculus (out of 50 captured; 22%), and one R. norvegicus (out of 9 captured; 11.11%). To the best of our knowledge, L. infantum and L. tropica were identified in rodent species for the first time in Morocco. These findings suggest that rodent species may be involved in L. infantum and L. tropica transmission cycles in this country but that further studies are needed to confirm their role as reservoirs of Leishmania species in Morocco.

PCR-Free Detection of Genetically Modified Organisms Using Magnetic Capture Technology and Fluorescence Cross-Correlation Spectroscopy

PubMed Central

Zhou, Xiaoming; Xing, Da; Tang, Yonghong; Chen, Wei R.

2009-01-01

The safety of genetically modified organisms (GMOs) has attracted much attention recently. Polymerase chain reaction (PCR) amplification is a common method used in the identification of GMOs. However, a major disadvantage of PCR is the potential amplification of non-target DNA, causing false-positive identification. Thus, there remains a need for a simple, reliable and ultrasensitive method to identify and quantify GMO in crops. This report is to introduce a magnetic bead-based PCR-free method for rapid detection of GMOs using dual-color fluorescence cross-correlation spectroscopy (FCCS). The cauliflower mosaic virus 35S (CaMV35S) promoter commonly used in transgenic products was targeted. CaMV35S target was captured by a biotin-labeled nucleic acid probe and then purified using streptavidin-coated magnetic beads through biotin-streptavidin linkage. The purified target DNA fragment was hybridized with two nucleic acid probes labeled respectively by Rhodamine Green and Cy5 dyes. Finally, FCCS was used to detect and quantify the target DNA fragment through simultaneously detecting the fluorescence emissions from the two dyes. In our study, GMOs in genetically engineered soybeans and tomatoes were detected, using the magnetic bead-based PCR-free FCCS method. A detection limit of 50 pM GMOs target was achieved and PCR-free detection of GMOs from 5 µg genomic DNA with magnetic capture technology was accomplished. Also, the accuracy of GMO determination by the FCCS method is verified by spectrophotometry at 260 nm using PCR amplified target DNA fragment from GM tomato. The new method is rapid and effective as demonstrated in our experiments and can be easily extended to high-throughput and automatic screening format. We believe that the new magnetic bead-assisted FCCS detection technique will be a useful tool for PCR-free GMOs identification and other specific nucleic acids. PMID:19956680

Magnetic Beads-Based Sensor with Tailored Sensitivity for Rapid and Single-Step Amperometric Determination of miRNAs.

PubMed

Vargas, Eva; Torrente-Rodríguez, Rebeca M; Ruiz-Valdepeñas Montiel, Víctor; Povedano, Eloy; Pedrero, María; Montoya, Juan J; Campuzano, Susana; Pingarrón, José M

2017-11-09

This work describes a sensitive amperometric magneto-biosensor for single-step and rapid determination of microRNAs (miRNAs). The developed strategy involves the use of direct hybridization of the target miRNA (miRNA-21) with a specific biotinylated DNA probe immobilized on streptavidin-modified magnetic beads (MBs), and labeling of the resulting heteroduplexes with a specific DNA-RNA antibody and the bacterial protein A (ProtA) conjugated with an horseradish peroxidase (HRP) homopolymer (Poly-HRP40) as an enzymatic label for signal amplification. Amperometric detection is performed upon magnetic capture of the modified MBs onto the working electrode surface of disposable screen-printed carbon electrodes (SPCEs) using the H₂O₂/hydroquinone (HQ) system. The magnitude of the cathodic signal obtained at -0.20 V (vs. the Ag pseudo-reference electrode) demonstrated linear dependence with the concentration of the synthetic target miRNA over the 1.0 to 100 pM range. The method provided a detection limit (LOD) of 10 attomoles (in a 25 μL sample) without any target miRNA amplification in just 30 min (once the DNA capture probe-MBs were prepared). This approach shows improved sensitivity compared with that of biosensors constructed with the same anti-DNA-RNA Ab as capture instead of a detector antibody and further labeling with a Strep-HRP conjugate instead of the Poly-HRP40 homopolymer. The developed strategy involves a single step working protocol, as well as the possibility to tailor the sensitivity by enlarging the length of the DNA/miRNA heteroduplexes using additional probes and/or performing the labelling with ProtA conjugated with homopolymers prepared with different numbers of HRP molecules. The practical usefulness was demonstrated by determination of the endogenous levels of the mature target miRNA in 250 ng raw total RNA (RNA t ) extracted from human mammary epithelial normal (MCF-10A) and cancer (MCF-7) cells and tumor tissues.

A novel metadata management model to capture consent for record linkage in longitudinal research studies.

PubMed

McMahon, Christiana; Denaxas, Spiros

2017-11-06

Informed consent is an important feature of longitudinal research studies as it enables the linking of the baseline participant information with administrative data. The lack of standardized models to capture consent elements can lead to substantial challenges. A structured approach to capturing consent-related metadata can address these. a) Explore the state-of-the-art for recording consent; b) Identify key elements of consent required for record linkage; and c) Create and evaluate a novel metadata management model to capture consent-related metadata. The main methodological components of our work were: a) a systematic literature review and qualitative analysis of consent forms; b) the development and evaluation of a novel metadata model. We qualitatively analyzed 61 manuscripts and 30 consent forms. We extracted data elements related to obtaining consent for linkage. We created a novel metadata management model for consent and evaluated it by comparison with the existing standards and by iteratively applying it to case studies. The developed model can facilitate the standardized recording of consent for linkage in longitudinal research studies and enable the linkage of external participant data. Furthermore, it can provide a structured way of recording consent-related metadata and facilitate the harmonization and streamlining of processes.

Gestalt Perceptual Organization of Visual Stimuli Captures Attention Automatically: Electrophysiological Evidence

PubMed Central

Marini, Francesco; Marzi, Carlo A.

2016-01-01

The visual system leverages organizational regularities of perceptual elements to create meaningful representations of the world. One clear example of such function, which has been formalized in the Gestalt psychology principles, is the perceptual grouping of simple visual elements (e.g., lines and arcs) into unitary objects (e.g., forms and shapes). The present study sought to characterize automatic attentional capture and related cognitive processing of Gestalt-like visual stimuli at the psychophysiological level by using event-related potentials (ERPs). We measured ERPs during a simple visual reaction time task with bilateral presentations of physically matched elements with or without a Gestalt organization. Results showed that Gestalt (vs. non-Gestalt) stimuli are characterized by a larger N2pc together with enhanced ERP amplitudes of non-lateralized components (N1, N2, P3) starting around 150 ms post-stimulus onset. Thus, we conclude that Gestalt stimuli capture attention automatically and entail characteristic psychophysiological signatures at both early and late processing stages. Highlights We studied the neural signatures of the automatic processes of visual attention elicited by Gestalt stimuli. We found that a reliable early correlate of attentional capture turned out to be the N2pc component. Perceptual and cognitive processing of Gestalt stimuli is associated with larger N1, N2, and P3 PMID:27630555

HIGH-RESOLUTION SPECTROSCOPY OF EXTREMELY METAL-POOR STARS IN THE LEAST EVOLVED GALAXIES: BOÖTES II

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ji, Alexander P.; Frebel, Anna; Simon, Joshua D.

2016-01-20

We present high-resolution Magellan/MIKE spectra of the four brightest confirmed red giant stars in the ultra-faint dwarf galaxy Boötes II (Boo II). These stars all inhabit the metal-poor tail of the Boo II metallicity distribution function. The chemical abundance pattern of all detectable elements in these stars is consistent with that of the Galactic halo. However, all four stars have undetectable amounts of neutron-capture elements Sr and Ba, with upper limits comparable to the lowest ever detected in the halo or in other dwarf galaxies. One star exhibits significant radial velocity variations over time, suggesting it to be in a binary system. Itsmore » variable velocity has likely increased past determinations of the Boo II velocity dispersion. Our four stars span a limited metallicity range, but their enhanced α-abundances and low neutron-capture abundances are consistent with the interpretation that Boo II has been enriched by very few generations of stars. The chemical abundance pattern in Boo II confirms the emerging trend that the faintest dwarf galaxies have neutron-capture abundances distinct from the halo, suggesting the dominant source of neutron-capture elements in halo stars may be different than in ultra-faint dwarfs.« less

Space station needs, attributes and architectural options: Architectural options and selection

NASA Technical Reports Server (NTRS)

Nelson, W. G.

1983-01-01

The approach, study results, and recommendations for defining and selecting space station architectural options are described. Space station system architecture is defined as the arrangement of elements (manned and unmanned on-orbit facilities, shuttle vehicles, orbital transfer vehicles, etc.), the number of these elements, their location (orbital inclination and altitude, and their functional performance capability, power, volume, crew, etc.). Architectural options are evaluated based on the degree of mission capture versus cost and required funding rate. Mission capture refers to the number of missions accommodated by the particular architecture.

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

PubMed

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

2016-01-19

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

Plasmid Capture by the Bacillus thuringiensis Conjugative Plasmid pXO16▿

PubMed Central

Timmery, Sophie; Modrie, Pauline; Minet, Olivier; Mahillon, Jacques

2009-01-01

Conjugation, mobilization, and retromobilization are three related mechanisms of horizontal gene transfer in bacteria. They have been extensively studied in gram-negative species, where retromobilization, the capture of DNA from a recipient by a donor cell, was shown to result from two successive steps: the transfer of the conjugative plasmid from the donor to the recipient followed by the retrotransfer of the mobilizable plasmid to the donor. This successive model was established for gram-negative bacteria but was lacking experimental data from the gram-positive counterparts. In the present work, the mobilization and retromobilization abilities of the conjugative plasmid pXO16 from Bacillus thuringiensis subsp. israelensis were studied using the mobilizable plasmids pUB110 and pE194 and the “nonmobilizable” element pC194 lacking the mob and oriT features (all from Staphylococcus aureus). Experimental data suggested a successive model, since different retromobilization frequencies were observed between the small plasmids. More importantly, retromobilization was shown to be delayed by 50 and 150 min for pUB110 and pE194, respectively, compared to pXO16 conjugation. Natural liquid foods (cow milk, soy milk, and rice milk) were used to evaluate the putative ecological impact of these transfers. In cow and soy milk, conjugation, mobilization, and retromobilization were shown to occur at frequencies of 8.0 × 10−1, 1.0 × 10−2, and 1.2 × 10−4 transconjugants per recipient, respectively. These data are comparable to those obtained with LB medium and about 10-fold lower than in the case of rice milk. Taken together, these results emphasize the potential role of plasmid capture played by B. thuringiensis in natural environments. PMID:19181805

Characterization of human glucocorticoid receptor complexes formed with DNA fragments containing or lacking glucocorticoid response elements

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tully, D.B.; Cidlowski, J.A.

1989-03-07

Sucrose density gradient shift assays were used to study the interactions of human glucocorticoid receptors (GR) with small DNA fragments either containing or lacking glucocorticoid response element (GRE) DNA consensus sequences. When crude cytoplasmic extracts containing ({sup 3}H)triamcinolone acetonide (({sup 3}H)TA) labeled GR were incubated with unlabeled DNA under conditions of DNA excess, a GRE-containing DNA fragment obtained from the 5' long terminal repeat of mouse mammary tumor virus (MMTV LTR) formed a stable 12-16S complex with activated, but not nonactivated, ({sup 3}H)TA receptor. By contrast, if the cytosols were treated with calf thymus DNA-cellulose to deplete non-GR-DNA-binding proteins priormore » to heat activation, a smaller 7-10S complex was formed with the MMTV LTR DNA fragment. Activated ({sup 3}H)TA receptor from DNA-cellulose pretreated cytosols also interacted with two similarly sized fragments from pBR322 DNA. Stability of the complexes formed between GR and these three DNA fragments was strongly affected by even moderate alterations in either the salt concentration or the pH of the gradient buffer. Under all conditions tested, the complex formed with the MMTV LTR DNA fragment was more stable than the complexes formed with either of the pBR322 DNA fragments. Together these observations indicate that the formation of stable complexes between activated GR and isolated DNA fragments requires the presence of GRE consensus sequences in the DNA.« less

Integrated in silico and biological validation of the blocking effect of Cot-1 DNA on Microarray-CGH.

PubMed

Kang, Seung-Hui; Park, Chan Hee; Jeung, Hei Cheul; Kim, Ki-Yeol; Rha, Sun Young; Chung, Hyun Cheol

2007-06-01

In array-CGH, various factors may act as variables influencing the result of experiments. Among them, Cot-1 DNA, which has been used as a repetitive sequence-blocking agent, may become an artifact-inducing factor in BAC array-CGH. To identify the effect of Cot-1 DNA on Microarray-CGH experiments, Cot-1 DNA was labeled directly and Microarray-CGH experiments were performed. The results confirmed that probes which hybridized more completely with Cot-1 DNA had a higher sequence similarity to the Alu element. Further, in the sex-mismatched Microarray-CGH experiments, the variation and intensity in the fluorescent signal were reduced in the high intensity probe group in which probes were better hybridized with Cot-1 DNA. Otherwise, those of the low intensity probe group showed no alterations regardless of Cot-1 DNA. These results confirmed by in silico methods that Cot-1 DNA could block repetitive sequences in gDNA and probes. In addition, it was confirmed biologically that the blocking effect of Cot-1 DNA could be presented via its repetitive sequences, especially Alu elements. Thus, in contrast to BAC-array CGH, the use of Cot-1 DNA is advantageous in controlling experimental variation in Microarray-CGH.

Identification of a DNA Segment Exhibiting Rearrangement Modifying Effects upon Transgenic δ-deleting Elements

PubMed Central

Janowski, Karen M.; Ledbetter, Stephanie; Mayo, Matthew S.; Hockett, Richard D.

1997-01-01

Control of the rearrangement and expression of the T cell receptor α and δ chains is critical for determining T cell type. The process of δ deletion is a candidate mechanism for maintaining separation of the α and δ loci. Mice harboring a transgenic reporter δ deletion construct show α/β T cell lineage–specific use of the transgenic elements. A 48-basepair segment of DNA, termed HPS1A, when deleted from this reporter construct, loses tight lineage-specific rearrangement control of transgenic elements, with abundant rearrangements of transgenic δ-deleting elements now in γ/δ T cells. Furthermore, HPS1A augments recombination frequency of extrachromosomal substrates in an in vitro recombination assay. DNA binding proteins recognizing HPS1A have been identified and are restricted to early B and T cells, during the time of active rearrangement of endogenous TCR and immunoglobulin loci. These data are consistent with δ deletion playing an important role in maintaining separate TCR α and δ loci. PMID:9207011

Aptamer based SERS detection of Salmonella typhimurium using DNA-assembled gold nanodimers.

PubMed

Xu, Xumin; Ma, Xiaoyuan; Wang, Haitao; Wang, Zhouping

2018-06-12

The authors describe a surface-enhanced Raman scattering (SERS) based aptasensor for Salmonella typhimurium (S. typhimurium). Gold nanoparticles (AuNPs; 35 nm i.d.) were functionalized with the aptamer (ssDNA 1) and used as the capture probe, while smaller (15 nm) AuNPs were modified with a Cy3-labeled complementary sequence (ssDNA 2) and used as the signalling probe. The asymmetric gold nanodimers (AuNDs) were assemblied with the Raman signal probe and the capture probe via hybridization of the complementary ssDNAs. The gap between two nanoparticles is a "hot spot" in which the Raman reporter Cy3 is localized. It experiences a strong enhancement of the electromagnetic field around the particle. After addition of S. typhimurium, it will be bound by the aptamer which therefore is partially dehybridized from its complementary sequence. Hence, Raman intensity drops. Under the optimal experimental conditions, the SERS signal at 1203 cm -1 increases linearly with the logarithm of the number of colonies in the 10 2 to 10 7  cfu·mL -1 concentration range, and the limit of detection is 35 cfu·mL -1 . The method can be performed within 1 h and was successfully applied to the analysis of spiked milk samples and performed very well and with high specificity. Graphical abstract DNA-assembled asymmetric gold nanodimers (AuNDs) were synthesized and appllied in a SERS-based aptasensor for S. typhimurium. Capture probe was preferentially combined with S. typhimurium and the structure of the AuNDs was destroyed. The "hot spot" vanished partly, this resulting in the decreased Raman intensity of Cy3.

Molecular analysis of population genetic structure and recolonization of rainbow trout following the Cantara spill

USGS Publications Warehouse

Nielsen, J.L.; Heine, Erika L.; Gan, Christina A.; Fountain, Monique C.

2000-01-01

Mitochondrial DNA (mtDNA) sequence and allelic frequency data for 12 microsatellite loci were used to analyze population genetic structure and recolonization by rainbow trout, Oncorhynchus mykiss, following the 1991 Cantara spill on the upper Sacramento River, California. Genetic analyses were performed on 1,016 wild rainbow trout collected between 1993 and 1996 from the mainstem and in 8 tributaries. Wild trout genotypes were compared to genotypes for 79 Mount Shasta Hatchery rainbow trout. No genetic heterogeneity was found 2 years after the spill (1993) between tributary populations and geographically proximate mainstem fish, suggesting recolonization of the upper mainstem directly from adjacent tributaries. Trout collections made in 1996 showed significant year-class genetic variation for mtDNA and microsatellites when compared to fish from the same locations in 1993. Five years after the spill, mainstem populations appeared genetically mixed with no significant allelic frequency differences between mainstem populations and geographically proximate tributary trout. In our 1996 samples, we found no significant genetic differences due to season of capture (summer or fall) or sampling technique used to capture rainbow trout, with the exception of trout collected by electrofishing and hook and line near Prospect Avenue. Haplotype and allelic frequencies in wild rainbow trout populations captured in the upper Sacramento River and its tributaries were found to differ genetically from Mount Shasta Hatchery trout for both years, with the notable exception of trout collected in the lower mainstem river near Shasta Lake, where mtDNA and microsatellite data both suggested upstream colonization by hatchery fish from the reservoir. These data suggest that the chemical spill in the upper Sacramento River produced significant effects over time on the genetic population structure of rainbow trout throughout the entire upper river basin.

Molecular detection of Leishmania (Leishmania) infantum in phlebotomine sandflies from a visceral leishmaniasis endemic area in northwestern of São Paulo State, Brazil.

PubMed

Dos Santos Brighente, Kate Bastos; Cutolo, Andre Antonio; Motoie, Gabriela; da Silva Meira-Strejevitch, Cristina; Pereira-Chioccola, Vera Lucia

2018-05-01

This study identified the natural infection rate of Leishmania (Leishmania) infantum in Lutzomyia longipalpis sandflies collected in a neighborhood around a kennel, in Dracena, northwestern of São Paulo state. This region is highly endemic for visceral leishmaniasis in Brazil. Insects were captured during 2-3 nights monthly for 11 months (January-November 2012) using 10 automatic light traps around a kennel in a transition between periurban and urban neighborhood. Capture aimed the determination of the minimal infection rate (MIR) on the area. A total of 1690 Lu. longipalpis were captured during the studied period. Out of them, 292 (17.25%) were females and were grouped in 165 pools containing 1 to five insects for DNA extraction and PCR analysis. Positive results for L. (L) infantum in conventional PCR and real time PCR were shown in 7.28% (12/165) and 4.85% (8/165) of the analysis respectively. These data confirm that Lu. longipalpis captured in the study area were infected by L. (L.) infantum. The MIR of sandflies during the 11 months of captures was 4.10% for female the total of 292 female sandflies collected. A high DNA concentration of L. (L.) infantum was detected on sandflies especially in kennel, chicken coop and neighboring houses, where higher abundance of hosts for blood source were present. Copyright © 2018 Elsevier B.V. All rights reserved.

Mavericks, a novel class of giant transposable elements widespread in eukaryotes and related to DNA viruses.

PubMed

Pritham, Ellen J; Putliwala, Tasneem; Feschotte, Cédric

2007-04-01

We previously identified a group of atypical mobile elements designated Mavericks from the nematodes Caenorhabditis elegans and C. briggsae and the zebrafish Danio rerio. Here we present the results of comprehensive database searches of the genome sequences available, which reveal that Mavericks are widespread in invertebrates and non-mammalian vertebrates but show a patchy distribution in non-animal species, being present in the fungi Glomus intraradices and Phakopsora pachyrhizi and in several single-celled eukaryotes such as the ciliate Tetrahymena thermophila, the stramenopile Phytophthora infestans and the trichomonad Trichomonas vaginalis, but not detectable in plants. This distribution, together with comparative and phylogenetic analyses of Maverick-encoded proteins, is suggestive of an ancient origin of these elements in eukaryotes followed by lineage-specific losses and/or recurrent episodes of horizontal transmission. In addition, we report that Maverick elements have amplified recently to high copy numbers in T. vaginalis where they now occupy as much as 30% of the genome. Sequence analysis confirms that most Mavericks encode a retroviral-like integrase, but lack other open reading frames typically found in retroelements. Nevertheless, the length and conservation of the target site duplication created upon Maverick insertion (5- or 6-bp) is consistent with a role of the integrase-like protein in the integration of a double-stranded DNA transposition intermediate. Mavericks also display long terminal-inverted repeats but do not contain ORFs similar to proteins encoded by DNA transposons. Instead, Mavericks encode a conserved set of 5 to 9 genes (in addition to the integrase) that are predicted to encode proteins with homology to replication and packaging proteins of some bacteriophages and diverse eukaryotic double-stranded DNA viruses, including a DNA polymerase B homolog and putative capsid proteins. Based on these and other structural similarities, we speculate that Mavericks represent an evolutionary missing link between seemingly disparate invasive DNA elements that include bacteriophages, adenoviruses and eukaryotic linear plasmids.

Approximating the r-Process on Earth with Thermonuclear Explosions. Lessons Learned and Unanswered Questions

DOE Office of Scientific and Technical Information (OSTI.GOV)

Becker, Stephen Allan

2016-01-28

During the astrophysical r-process, multiple neutron captures occur so rapidly on target nuclei that their daughter nuclei generally do not have time to undergo radioactive decay before another neutron is captured. The r-process can be approximately simulated on Earth in certain types of thermonuclear explosions through an analogous process of rapid neutron captures known as the "prompt capture" process. Between 1952 and 1969, 23 nuclear tests were fielded by the US which were involved (at least partially) with the "prompt capture" process. Of these tests, 15 were at least partially successful. Some of these tests were conducted under the Plowsharemore » Peaceful Nuclear Explosion Program as scientific research experiments. It is now known that the USSR conducted similar nuclear tests during 1966 to 1979. The elements einsteinium and fermium were first discovered by this process. The most successful tests achieved 19 successive neutron captures on the initial target nuclei. A review of the US program, target nuclei used, heavy element yields, scientific achievements of the program, and how some of the results have been used by the astrophysical community is given. Finally, some unanswered questions concerning very neutron-rich nuclei that could potentially have been answered with additional nuclear experiments is presented.« less

Complex multi-enhancer contacts captured by genome architecture mapping.

PubMed

Beagrie, Robert A; Scialdone, Antonio; Schueler, Markus; Kraemer, Dorothee C A; Chotalia, Mita; Xie, Sheila Q; Barbieri, Mariano; de Santiago, Inês; Lavitas, Liron-Mark; Branco, Miguel R; Fraser, James; Dostie, Josée; Game, Laurence; Dillon, Niall; Edwards, Paul A W; Nicodemi, Mario; Pombo, Ana

2017-03-23

The organization of the genome in the nucleus and the interactions of genes with their regulatory elements are key features of transcriptional control and their disruption can cause disease. Here we report a genome-wide method, genome architecture mapping (GAM), for measuring chromatin contacts and other features of three-dimensional chromatin topology on the basis of sequencing DNA from a large collection of thin nuclear sections. We apply GAM to mouse embryonic stem cells and identify enrichment for specific interactions between active genes and enhancers across very large genomic distances using a mathematical model termed SLICE (statistical inference of co-segregation). GAM also reveals an abundance of three-way contacts across the genome, especially between regions that are highly transcribed or contain super-enhancers, providing a level of insight into genome architecture that, owing to the technical limitations of current technologies, has previously remained unattainable. Furthermore, GAM highlights a role for gene-expression-specific contacts in organizing the genome in mammalian nuclei.

Personalized Medicine: Ethical Aspects.

PubMed

Sharrer, G Terry

2017-01-01

In our time of genome-based personalized medicine, clinical research and clinical medicine are accelerating at a quick pace. Faster and cheaper DNA sequencing and protein profiling, microfluidic devices for capturing blood biomarkers, nanoparticles for precise drug delivery and enhanced imaging, rapid computational analysis of massive data inputs, and other technological wonders coalesce to create a kind of Moore's Law for medicine. Needs are obvious, knowledge grows, capital becomes available, but these factors are not entirely sufficient to make health more achievable. Personalized medicine also requires social acceptability, not only for accuracy and efficacy but also because medicine is a moral domain. This chapter deals with medical ethics that determine the choices a society makes regarding healthcare; and it has not always been a steady, morally correct course of progress. Indeed, medical ethics has largely derived from socio-scientific calamities in the past. Personalized medicine, with its enhanced capacity to access the individuality of illness, must have a continuously evolving feedback mechanism-the most important element being the physician-patient relationship-which is its ethical footing.

CRISPR Inversion of CTCF Sites Alters Genome Topology and Enhancer/Promoter Function

PubMed Central

Guo, Ya; Xu, Quan; Canzio, Daniele; Shou, Jia; Li, Jinhuan; Gorkin, David U.; Jung, Inkyung; Wu, Haiyang; Zhai, Yanan; Tang, Yuanxiao; Lu, Yichao; Wu, Yonghu; Jia, Zhilian; Li, Wei; Zhang, Michael Q.; Ren, Bing; Krainer, Adrian R.; Maniatis, Tom; Wu, Qiang

2015-01-01

SUMMARY CTCF/cohesin play a central role in insulator function and higher-order chromatin organization of mammalian genomes. Recent studies identified a correlation between the orientation of CTCF-binding sites (CBSs) and chromatin loops. To test the functional significance of this observation, we combined CRISPR/Cas9-based genomic-DNA-fragment editing with chromosome-conformation-capture experiments to show that the location and relative orientations of CBSs determine the specificity of long-range chromatin looping in mammalian genomes, using protocadherin (Pcdh) and β-globin as model genes. Inversion of CBS elements within the Pcdh enhancer reconfigures the topology of chromatin loops between the distal enhancer and target promoters, and alters gene-expression patterns. Thus, although enhancers can function in an orientation-independent manner in reporter assays, in the native chromosome context the orientation of at least some enhancers carrying CBSs can determine both the architecture of topological chromatin domains and enhancer/promoter specificity. The findings reveal how 3D chromosome architecture can be encoded by genome sequence. PMID:26276636

A Runge-Kutta discontinuous finite element method for high speed flows

NASA Technical Reports Server (NTRS)

Bey, Kim S.; Oden, J. T.

1991-01-01

A Runge-Kutta discontinuous finite element method is developed for hyperbolic systems of conservation laws in two space variables. The discontinuous Galerkin spatial approximation to the conservation laws results in a system of ordinary differential equations which are marched in time using Runge-Kutta methods. Numerical results for the two-dimensional Burger's equation show that the method is (p+1)-order accurate in time and space, where p is the degree of the polynomial approximation of the solution within an element and is capable of capturing shocks over a single element without oscillations. Results for this problem also show that the accuracy of the solution in smooth regions is unaffected by the local projection and that the accuracy in smooth regions increases as p increases. Numerical results for the Euler equations show that the method captures shocks without oscillations and with higher resolution than a first-order scheme.

Control of DEMETER DNA demethylase gene transcription in male and female gamete companion cells in Arabidopsis thaliana

PubMed Central

Park, Jin-Sup; Frost, Jennifer M.; Park, Kyunghyuk; Ohr, Hyonhwa; Park, Guen Tae; Kim, Seohyun; Eom, Hyunjoo; Lee, Ilha; Brooks, Janie S.; Fischer, Robert L.; Choi, Yeonhee

2017-01-01

The DEMETER (DME) DNA glycosylase initiates active DNA demethylation via the base-excision repair pathway and is vital for reproduction in Arabidopsis thaliana. DME-mediated DNA demethylation is preferentially targeted to small, AT-rich, and nucleosome-depleted euchromatic transposable elements, influencing expression of adjacent genes and leading to imprinting in the endosperm. In the female gametophyte, DME expression and subsequent genome-wide DNA demethylation are confined to the companion cell of the egg, the central cell. Here, we show that, in the male gametophyte, DME expression is limited to the companion cell of sperm, the vegetative cell, and to a narrow window of time: immediately after separation of the companion cell lineage from the germline. We define transcriptional regulatory elements of DME using reporter genes, showing that a small region, which surprisingly lies within the DME gene, controls its expression in male and female companion cells. DME expression from this minimal promoter is sufficient to rescue seed abortion and the aberrant DNA methylome associated with the null dme-2 mutation. Within this minimal promoter, we found short, conserved enhancer sequences necessary for the transcriptional activities of DME and combined predicted binding motifs with published transcription factor binding coordinates to produce a list of candidate upstream pathway members in the genetic circuitry controlling DNA demethylation in gamete companion cells. These data show how DNA demethylation is regulated to facilitate endosperm gene imprinting and potential transgenerational epigenetic regulation, without subjecting the germline to potentially deleterious transposable element demethylation. PMID:28130550

A methodology to annotate systems biology markup language models with the synthetic biology open language.

PubMed

Roehner, Nicholas; Myers, Chris J

2014-02-21

Recently, we have begun to witness the potential of synthetic biology, noted here in the form of bacteria and yeast that have been genetically engineered to produce biofuels, manufacture drug precursors, and even invade tumor cells. The success of these projects, however, has often failed in translation and application to new projects, a problem exacerbated by a lack of engineering standards that combine descriptions of the structure and function of DNA. To address this need, this paper describes a methodology to connect the systems biology markup language (SBML) to the synthetic biology open language (SBOL), existing standards that describe biochemical models and DNA components, respectively. Our methodology involves first annotating SBML model elements such as species and reactions with SBOL DNA components. A graph is then constructed from the model, with vertices corresponding to elements within the model and edges corresponding to the cause-and-effect relationships between these elements. Lastly, the graph is traversed to assemble the annotating DNA components into a composite DNA component, which is used to annotate the model itself and can be referenced by other composite models and DNA components. In this way, our methodology can be used to build up a hierarchical library of models annotated with DNA components. Such a library is a useful input to any future genetic technology mapping algorithm that would automate the process of composing DNA components to satisfy a behavioral specification. Our methodology for SBML-to-SBOL annotation is implemented in the latest version of our genetic design automation (GDA) software tool, iBioSim.

Determination of the Optimal Chromosomal Location(s) for a DNA Element in Escherichia coli Using a Novel Transposon-mediated Approach.

PubMed

Frimodt-Møller, Jakob; Charbon, Godefroid; Krogfelt, Karen A; Løbner-Olesen, Anders

2017-09-11

The optimal chromosomal position(s) of a given DNA element was/were determined by transposon-mediated random insertion followed by fitness selection. In bacteria, the impact of the genetic context on the function of a genetic element can be difficult to assess. Several mechanisms, including topological effects, transcriptional interference from neighboring genes, and/or replication-associated gene dosage, may affect the function of a given genetic element. Here, we describe a method that permits the random integration of a DNA element into the chromosome of Escherichia coli and select the most favorable locations using a simple growth competition experiment. The method takes advantage of a well-described transposon-based system of random insertion, coupled with a selection of the fittest clone(s) by growth advantage, a procedure that is easily adjustable to experimental needs. The nature of the fittest clone(s) can be determined by whole-genome sequencing on a complex multi-clonal population or by easy gene walking for the rapid identification of selected clones. Here, the non-coding DNA region DARS2, which controls the initiation of chromosome replication in E. coli, was used as an example. The function of DARS2 is known to be affected by replication-associated gene dosage; the closer DARS2 gets to the origin of DNA replication, the more active it becomes. DARS2 was randomly inserted into the chromosome of a DARS2-deleted strain. The resultant clones containing individual insertions were pooled and competed against one another for hundreds of generations. Finally, the fittest clones were characterized and found to contain DARS2 inserted in close proximity to the original DARS2 location.

Novel DNA Motif Binding Activity Observed In Vivo With an Estrogen Receptor α Mutant Mouse

PubMed Central

Li, Leping; Grimm, Sara A.; Winuthayanon, Wipawee; Hamilton, Katherine J.; Pockette, Brianna; Rubel, Cory A.; Pedersen, Lars C.; Fargo, David; Lanz, Rainer B.; DeMayo, Francesco J.; Schütz, Günther; Korach, Kenneth S.

2014-01-01

Estrogen receptor α (ERα) interacts with DNA directly or indirectly via other transcription factors, referred to as “tethering.” Evidence for tethering is based on in vitro studies and a widely used “KIKO” mouse model containing mutations that prevent direct estrogen response element DNA- binding. KIKO mice are infertile, due in part to the inability of estradiol (E2) to induce uterine epithelial proliferation. To elucidate the molecular events that prevent KIKO uterine growth, regulation of the pro-proliferative E2 target gene Klf4 and of Klf15, a progesterone (P4) target gene that opposes the pro-proliferative activity of KLF4, was evaluated. Klf4 induction was impaired in KIKO uteri; however, Klf15 was induced by E2 rather than by P4. Whole uterine chromatin immunoprecipitation-sequencing revealed enrichment of KIKO ERα binding to hormone response elements (HREs) motifs. KIKO binding to HRE motifs was verified using reporter gene and DNA-binding assays. Because the KIKO ERα has HRE DNA-binding activity, we evaluated the “EAAE” ERα, which has more severe DNA-binding domain mutations, and demonstrated a lack of estrogen response element or HRE reporter gene induction or DNA-binding. The EAAE mouse has an ERα null–like phenotype, with impaired uterine growth and transcriptional activity. Our findings demonstrate that the KIKO mouse model, which has been used by numerous investigators, cannot be used to establish biological functions for ERα tethering, because KIKO ERα effectively stimulates transcription using HRE motifs. The EAAE-ERα DNA-binding domain mutant mouse demonstrates that ERα DNA-binding is crucial for biological and transcriptional processes in reproductive tissues and that ERα tethering may not contribute to estrogen responsiveness in vivo. PMID:24713037

Evolution of double-stranded DNA viruses of eukaryotes: from bacteriophages to transposons to giant viruses

PubMed Central

Koonin, Eugene V; Krupovic, Mart; Yutin, Natalya

2015-01-01

Diverse eukaryotes including animals and protists are hosts to a broad variety of viruses with double-stranded (ds) DNA genomes, from the largest known viruses, such as pandoraviruses and mimiviruses, to tiny polyomaviruses. Recent comparative genomic analyses have revealed many evolutionary connections between dsDNA viruses of eukaryotes, bacteriophages, transposable elements, and linear DNA plasmids. These findings provide an evolutionary scenario that derives several major groups of eukaryotic dsDNA viruses, including the proposed order “Megavirales,” adenoviruses, and virophages from a group of large virus-like transposons known as Polintons (Mavericks). The Polintons have been recently shown to encode two capsid proteins, suggesting that these elements lead a dual lifestyle with both a transposon and a viral phase and should perhaps more appropriately be named polintoviruses. Here, we describe the recently identified evolutionary relationships between bacteriophages of the family Tectiviridae, polintoviruses, adenoviruses, virophages, large and giant DNA viruses of eukaryotes of the proposed order “Megavirales,” and linear mitochondrial and cytoplasmic plasmids. We outline an evolutionary scenario under which the polintoviruses were the first group of eukaryotic dsDNA viruses that evolved from bacteriophages and became the ancestors of most large DNA viruses of eukaryotes and a variety of other selfish elements. Distinct lines of origin are detectable only for herpesviruses (from a different bacteriophage root) and polyoma/papillomaviruses (from single-stranded DNA viruses and ultimately from plasmids). Phylogenomic analysis of giant viruses provides compelling evidence of their independent origins from smaller members of the putative order “Megavirales,” refuting the speculations on the evolution of these viruses from an extinct fourth domain of cellular life. PMID:25727355

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

NASA Astrophysics Data System (ADS)

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 recovery of DNA to 45% efficiency. Furthermore, the 225°C-cured PEI-coated microtips recover more DNA than gold-coated microtips when the surface is washed. Heat-cured (225°C) PEI-coated microtips are used for the recovery of human genomic DNA from whole blood. A washing protocol is developed to remove inhibiting particles bound to the PEI-coated microtip surface after DNA extraction. From 1.25 muL of whole blood, an average of 1.83 ng of human genomic DNA is captured, purified, and released using a 225°C-cured PEI-coated microtip in less than 30 minutes. The extracted DNA is profiled by short tandem repeat analysis (STR). For forensic and medical applications, genomic DNA is extracted from dried samples using heat-cured PEI-coated microtips that are integrated into an automated device. DNA extraction from dried samples is critical for forensics. The use of dried samples in the medical field is increasing because dried samples are convenient for storage, biosafety, and contamination. The main challenge is the time required to properly extract DNA in a purified form. Typically, a 1 hour incubation period is required to complete this process. Overnight incubation is sometimes necessary. To address this challenge, a pre-extraction washing step is investigated to remove inhibiting particles from dried blood spots (DBS) before DNA is released from dried form into solution for microtip extraction. The developed protocol is expanded to extract DNA from a variety of dried samples including nasal swabs, buccal swabs, and other forensic samples. In comparison to a commercial kit, the microtip-based extraction reduced the processing time from 1.5 hours to 30 minutes or less with an equivalent concentration of extracted DNA from dried blood spots. The developed assay will benefit genetic studies on newborn screening, forensic investigation, and POC diagnostics.

Gene quantification by the NanoGene assay is resistant to inhibition by humic acids.

PubMed

Kim, Gha-Young; Wang, Xiaofang; Ahn, Hosang; Son, Ahjeong

2011-10-15

NanoGene assay is a magnetic bead and quantum dot nanoparticles based gene quantification assay. It relies on a set of probe and signaling probe DNAs to capture the target DNA via hybridization. We have demonstrated the inhibition resistance of the NanoGene assay using humic acids laden genomic DNA (gDNA). At 1 μg of humic acid per mL, quantitiative PCR (qPCR) was inhibited to 0% of its quantification capability whereas NanoGene assay was able to maintain more than 60% of its quantification capability. To further increase the inhibition resistance of NanoGene assay at high concentration of humic acids, we have identified the specific mechanisms that are responsible for the inhibition. We examined five potential mechanisms with which the humic acids can partially inhibit our NanoGene assay. The mechanisms examined were (1) adsorption of humic acids on the particle surface; (2) particle aggregation induced by humic acids; (3) fluorescence quenching of quantum dots by humic acids during hybridization; (4) humic acids mimicking of target DNA; and (5) nonspecific binding between humic acids and target gDNA. The investigation showed that no adsorption of humic acids onto the particles' surface was observed for the humic acids' concentration. Particle aggregation and fluorescence quenching were also negligible. Humic acids also did not mimic the target gDNA except 1000 μg of humic acids per mL and hence should not contribute to the partial inhibition. Four of the above mechanisms were not related to the inhibition effect of humic acids particularly at the environmentally relevant concentrations (<100 μg/mL). However, a substantial amount of nonspecific binding was observed between the humic acids and target gDNA. This possibly results in lesser amount of target gDNA being captured by the probe and signaling DNA.

Cryptic MCAT enhancer regulation in fibroblasts and smooth muscle cells. Suppression of TEF-1 mediated activation by the single-stranded DNA-binding proteins, Pur alpha, Pur beta, and MSY1.

PubMed

Carlini, Leslie E; Getz, Michael J; Strauch, Arthur R; Kelm, Robert J

2002-03-08

An asymmetric polypurine-polypyrimidine cis-element located in the 5' region of the mouse vascular smooth muscle alpha-actin gene serves as a binding site for multiple proteins with specific affinity for either single- or double-stranded DNA. Here, we test the hypothesis that single-stranded DNA-binding proteins are responsible for preventing a cryptic MCAT enhancer centered within this element from cooperating with a nearby serum response factor-interacting CArG motif to trans-activate the minimal promoter in fibroblasts and smooth muscle cells. DNA binding studies revealed that the core MCAT sequence mediates binding of transcription enhancer factor-1 to the double-stranded polypurine-polypyrimidine element while flanking nucleotides account for interaction of Pur alpha and Pur beta with the purine-rich strand and MSY1 with the complementary pyrimidine-rich strand. Mutations that selectively impaired high affinity single-stranded DNA binding by fibroblast or smooth muscle cell-derived Pur alpha, Pur beta, and MSY1 in vitro, released the cryptic MCAT enhancer from repression in transfected cells. Additional experiments indicated that Pur alpha, Pur beta, and MSY1 also interact specifically, albeit weakly, with double-stranded DNA and with transcription enhancer factor-1. These results are consistent with two plausible models of cryptic MCAT enhancer regulation by Pur alpha, Pur beta, and MSY1 involving either competitive single-stranded DNA binding or masking of MCAT-bound transcription enhancer factor-1.

A User's Guide to the Encyclopedia of DNA Elements (ENCODE)

PubMed Central

2011-01-01

The mission of the Encyclopedia of DNA Elements (ENCODE) Project is to enable the scientific and medical communities to interpret the human genome sequence and apply it to understand human biology and improve health. The ENCODE Consortium is integrating multiple technologies and approaches in a collective effort to discover and define the functional elements encoded in the human genome, including genes, transcripts, and transcriptional regulatory regions, together with their attendant chromatin states and DNA methylation patterns. In the process, standards to ensure high-quality data have been implemented, and novel algorithms have been developed to facilitate analysis. Data and derived results are made available through a freely accessible database. Here we provide an overview of the project and the resources it is generating and illustrate the application of ENCODE data to interpret the human genome. PMID:21526222

In vivo and in vitro characterization of σ70 constitutive promoters by real-time PCR and fluorescent measurements.

PubMed

Chappell, James; Freemont, Paul

2013-01-01

The characterization of DNA regulatory elements such as ribosome binding sites and transcriptional promoters is a fundamental aim of synthetic biology. Characterization of such DNA regulatory elements by monitoring the synthesis of fluorescent proteins is a commonly used technique to resolve the relative or absolute strengths. These measurements can be used in combination with mathematical models and computer simulation to rapidly assess performance of DNA regulatory elements both in isolation and in combination, to assist predictable and efficient engineering of complex novel biological devices and systems. Here we describe the construction and relative characterization of Escherichia coli (E. coli) σ(70) transcriptional promoters by monitoring the synthesis of green fluorescent protein (GFP) both in vivo in E. coli and in vitro in a E. coli cell-free transcription and translation reaction.

Genome-wide DNA methylation patterns in LSH mutant reveals de-repression of repeat elements and redundant epigenetic silencing pathways

PubMed Central

Yu, Weishi; McIntosh, Carl; Lister, Ryan; Zhu, Iris; Han, Yixing; Ren, Jianke; Landsman, David; Lee, Eunice; Briones, Victorino; Terashima, Minoru; Leighty, Robert; Ecker, Joseph R.

2014-01-01

Cytosine methylation is critical in mammalian development and plays a role in diverse biologic processes such as genomic imprinting, X chromosome inactivation, and silencing of repeat elements. Several factors regulate DNA methylation in early embryogenesis, but their precise role in the establishment of DNA methylation at a given site remains unclear. We have generated a comprehensive methylation map in fibroblasts derived from the murine DNA methylation mutant Hells−/− (helicase, lymphoid specific, also known as LSH). It has been previously shown that HELLS can influence de novo methylation of retroviral sequences and endogenous genes. Here, we describe that HELLS controls cytosine methylation in a nuclear compartment that is in part defined by lamin B1 attachment regions. Despite widespread loss of cytosine methylation at regulatory sequences, including promoter regions of protein-coding genes and noncoding RNA genes, overall relative transcript abundance levels in the absence of HELLS are similar to those in wild-type cells. A subset of promoter regions shows increases of the histone modification H3K27me3, suggesting redundancy of epigenetic silencing mechanisms. Furthermore, HELLS modulates CG methylation at all classes of repeat elements and is critical for repression of a subset of repeat elements. Overall, we provide a detailed analysis of gene expression changes in relation to DNA methylation alterations, which contributes to our understanding of the biological role of cytosine methylation. PMID:25170028

Repeated administration of CGP 46381, a gamma-aminobutyric acidB antagonist, and ethosuximide suppresses seizure-associated cyclic adenosine 3'5' monophosphate response element- and activator protein-1 DNA-binding activities in lethargic (lh/lh) mice.

PubMed

Ishige, K; Endo, H; Saito, H; Ito, Y

2001-01-19

To characterize seizure-associated increases in cerebral cortical and thalamic cyclic AMP responsive element (CRE)- and activator protein 1 (AP-1) DNA-binding activities in lethargic (lh/lh) mice, a genetic model of absence seizures, we examined the effects of ethosuximide and CGP 46381 on these DNA-binding activities. Repeated administration (twice a day for 5 days) of ethosuximide (200 mg/kg) or CGP 46381 (60 mg/kg) attenuated both seizure behavior and the increased DNA-binding activities, and was more effective than a single administration of these drugs. These treatments did not affect either normal behavior or basal DNA-binding activities in non-epileptic control (+/+) mice. Gel supershift assays revealed that the increased CRE-binding activity was attributable to activation of the binding activity of CREB, and that the c-Fos-c-Jun complex was a component of the increased AP-1 DNA-binding activity.

Sex-biased gene flow in spectacled eiders (Anatidae): Inferences from molecular markers with contrasting modes of inheritance

USGS Publications Warehouse

Scribner, Kim T.; Petersen, Margaret R.; Fields, Raymond L.; Talbot, Sandra L.; Pearce, John M.; Chesser, Ronald K.

2001-01-01

Genetic markers that differ in mode of inheritance and rate of evolution (a sex-linked Z-specific microsatellite locus, five biparentally inherited microsatellite loci, and maternally inherited mitochondrial [mtDNA] sequences) were used to evaluate the degree of spatial genetic structuring at macro- and microgeographic scales, among breeding regions and local nesting populations within each region, respectively, for a migratory sea duck species, the spectacled eider (Somateria fisheri). Disjunct and declining breeding populations coupled with sex-specific differences in seasonal migratory patterns and life history provide a series of hypotheses regarding rates and directionality of gene flow among breeding populations from the Indigirka River Delta, Russia, and the North Slope and Yukon-Kuskokwim Delta, Alaska. The degree of differentiation in mtDNA haplotype frequency among breeding regions and populations within regions was high (ϕCT = 0.189, P < 0.01; ϕSC = 0.059, P < 0.01, respectively). Eleven of 17 mtDNA haplotypes were restricted to a single breeding region. Genetic differences among regions were considerably lower for nuclear DNA loci (sex-linked: ϕST = 0.001, P > 0.05; biparentally inherited microsatellites: mean θ = 0.001, P > 0.05) than was observed for mtDNA. Using models explicitly designed for uniparental and biparentally inherited genes, estimates of spatial divergence based on nuclear and mtDNA data together with elements of the species' breeding ecology were used to estimate effective population size and degree of male and female gene flow. Differences in the magnitude and spatial patterns of gene correlations for maternally inherited and nuclear genes revealed that females exhibit greater natal philopatry than do males. Estimates of generational female and male rates of gene flow among breeding regions differed markedly (3.67 × 10−4 and 1.28 × 10−2, respectively). Effective population size for mtDNA was estimated to be at least three times lower than that for biparental genes (30,671 and 101,528, respectively). Large disparities in population sizes among breeding areas greatly reduces the proportion of total genetic variance captured by dispersal, which may accelerate rates of inbreeding (i.e., promote higher coancestries) within populations due to nonrandom pairing of males with females from the same breeding population.

Genome-wide inference of transcription factor-DNA binding specificity in cell regeneration using a combination strategy.

PubMed

Wang, Xiaofeng; Zhang, Aiqun; Ren, Weizheng; Chen, Caiyu; Dong, Jiahong

2012-11-01

The cell growth, development, and regeneration of tissue and organ are associated with a large number of gene regulation events, which are mediated in part by transcription factors (TFs) binding to cis-regulatory elements involved in the genome. Predicting the binding affinity and inferring the binding specificity of TF-DNA interactions at the genomic level would be fundamentally helpful for our understanding of the molecular mechanism and biological implication underlying sequence-specific TF-DNA recognition. In this study, we report the development of a combination method to characterize the interaction behavior of a 11-mer oligonucleotide segment and its mutations with the Gcn4p protein, a homodimeric, basic leucine zipper TF, and to predict the binding affinity and specificity of potential Gcn4p binders in the genome-wide scale. In this procedure, a position-mutated energy matrix is created based on molecular modeling analysis of native and mutated Gcn4p-DNA complex structures to describe the position-independent interaction energy profile of Gcn4p with different nucleotide types at each position of the oligonucleotide, and the energy terms extracted from the matrix and their interactives are then correlated with experimentally measured affinities of 19268 distinct oligonucleotides using statistical modeling methodology. Subsequently, the best one of built regression models is successfully applied to screen those of potential high-affinity Gcn4p binders from the complete genome. The findings arising from this study are briefly listed below: (i) The 11 positions of oligonucleotides are highly interactive and non-additive in contribution to Gcn4p-DNA binding affinity; (ii) Indirect conformational effects upon nucleotide mutations as well as associated subtle changes in interfacial atomic contacts, but not the direct nonbonded interactions, are primarily responsible for the sequence-specific recognition; (iii) The intrinsic synergistic effects among the sequence positions of oligonucleotides determine Gcn4p-DNA binding affinity and specificity; (iv) Linear regression models in conjunction with variable selection seem to perform fairly well in capturing the internal dependences hidden in the Gcn4p-DNA system, albeit ignoring nonlinear factors may lead the models to systematically underestimate and overestimate high- and low-affinity samples, respectively. © 2012 John Wiley & Sons A/S.

Flow cytometry sorting of nuclei enables the first global characterization of Paramecium germline DNA and transposable elements.

PubMed

Guérin, Frédéric; Arnaiz, Olivier; Boggetto, Nicole; Denby Wilkes, Cyril; Meyer, Eric; Sperling, Linda; Duharcourt, Sandra

2017-04-26

DNA elimination is developmentally programmed in a wide variety of eukaryotes, including unicellular ciliates, and leads to the generation of distinct germline and somatic genomes. The ciliate Paramecium tetraurelia harbors two types of nuclei with different functions and genome structures. The transcriptionally inactive micronucleus contains the complete germline genome, while the somatic macronucleus contains a reduced genome streamlined for gene expression. During development of the somatic macronucleus, the germline genome undergoes massive and reproducible DNA elimination events. Availability of both the somatic and germline genomes is essential to examine the genome changes that occur during programmed DNA elimination and ultimately decipher the mechanisms underlying the specific removal of germline-limited sequences. We developed a novel experimental approach that uses flow cell imaging and flow cytometry to sort subpopulations of nuclei to high purity. We sorted vegetative micronuclei and macronuclei during development of P. tetraurelia. We validated the method by flow cell imaging and by high throughput DNA sequencing. Our work establishes the proof of principle that developing somatic macronuclei can be sorted from a complex biological sample to high purity based on their size, shape and DNA content. This method enabled us to sequence, for the first time, the germline DNA from pure micronuclei and to identify novel transposable elements. Sequencing the germline DNA confirms that the Pgm domesticated transposase is required for the excision of all ~45,000 Internal Eliminated Sequences. Comparison of the germline DNA and unrearranged DNA obtained from PGM-silenced cells reveals that the latter does not provide a faithful representation of the germline genome. We developed a flow cytometry-based method to purify P. tetraurelia nuclei to high purity and provided quality control with flow cell imaging and high throughput DNA sequencing. We identified 61 germline transposable elements including the first Paramecium retrotransposons. This approach paves the way to sequence the germline genomes of P. aurelia sibling species for future comparative genomic studies.

The GALAH survey: scientific motivation

NASA Astrophysics Data System (ADS)

De Silva, G. M.; Freeman, K. C.; Bland-Hawthorn, J.; Martell, S.; de Boer, E. Wylie; Asplund, M.; Keller, S.; Sharma, S.; Zucker, D. B.; Zwitter, T.; Anguiano, B.; Bacigalupo, C.; Bayliss, D.; Beavis, M. A.; Bergemann, M.; Campbell, S.; Cannon, R.; Carollo, D.; Casagrande, L.; Casey, A. R.; Da Costa, G.; D'Orazi, V.; Dotter, A.; Duong, L.; Heger, A.; Ireland, M. J.; Kafle, P. R.; Kos, J.; Lattanzio, J.; Lewis, G. F.; Lin, J.; Lind, K.; Munari, U.; Nataf, D. M.; O'Toole, S.; Parker, Q.; Reid, W.; Schlesinger, K. J.; Sheinis, A.; Simpson, J. D.; Stello, D.; Ting, Y.-S.; Traven, G.; Watson, F.; Wittenmyer, R.; Yong, D.; Žerjal, M.

2015-05-01

The Galactic Archaeology with HERMES (GALAH) survey is a large high-resolution spectroscopic survey using the newly commissioned High Efficiency and Resolution Multi-Element Spectrograph (HERMES) on the Anglo-Australian Telescope. The HERMES spectrograph provides high-resolution (R ˜ 28 000) spectra in four passbands for 392 stars simultaneously over a 2 deg field of view. The goal of the survey is to unravel the formation and evolutionary history of the Milky Way, using fossil remnants of ancient star formation events which have been disrupted and are now dispersed throughout the Galaxy. Chemical tagging seeks to identify such dispersed remnants solely from their common and unique chemical signatures; these groups are unidentifiable from their spatial, photometric or kinematic properties. To carry out chemical tagging, the GALAH survey will acquire spectra for a million stars down to V ˜ 14. The HERMES spectra of FGK stars contain absorption lines from 29 elements including light proton-capture elements, α-elements, odd-Z elements, iron-peak elements and n-capture elements from the light and heavy s-process and the r-process. This paper describes the motivation and planned execution of the GALAH survey, and presents some results on the first-light performance of HERMES.

Usefulness of circulating free DNA for monitoring epidermal growth factor receptor mutations in advanced non-small cell lung cancer patients: a case report

PubMed Central

Gonzalez-Cao, Maria; Ramirez, Santiago Viteri; Ariza, Nuria Jordana; Balada, Ariadna; Garzón, Mónica; Teixidó, Cristina; Karachaliou, Niki; Morales-Espinosa, Daniela; Molina-Vila, Miguel Ángel; Rosell, Rafael

2016-01-01

Genomic analysis of circulating tumor DNA (ctDNA) released from cancer cells into the bloodstream has been proposed as a useful method to capture dynamic changes during the course of the disease. In particular, the ability to monitor epidermal growth factor receptor (EGFR) mutation status in cell-free circulating DNA (cfDNA) isolated from advanced non-small cell lung cancer (NSCLC) patients EGFR can help to the correct management of the disease and overcome the challenges associated with tumor heterogeneity and insufficient biopsied material to perform key molecular diagnosis. Here, we report a case of long term monitorization of EGFR mutation status in cfDNA from peripheral blood in an NSCLC patient in, with excellent correlation with clinical evolution. PMID:27826535

What triggers differential DNA methylation of genes and TEs: contribution of body methylation?

PubMed

Inagaki, S; Kakutani, T

2012-01-01

Transposable elements (TEs) are epigenetically silenced with extensive DNA methylation. The silent epigenetic marks should, however, be excluded from active genes. By genetic approaches, we study mechanisms to remove the heterochromatin marks from transcribed genes. Based on our observations on control of TE transcription, we propose a possible trigger for the TE-specific accumulation of DNA methylation. A critical difference between TEs and genes could be their responses to the DNA methylation in the internal part of transcribed regions. When their internal region is methylated, genes are still transcribed, but TEs could be silenced, which may reflect the obligatory position of every critical cis-acting element within the TE itself. This initial difference of TEs and genes will be amplified by positive feedback loops to stabilize active or silent states. Thus, the mechanisms to accumulate heterochromatin marks within transcribed regions could provide a trigger to induce differential DNA methylation between genes and TEs.

pH-Driven Reversible Self-Assembly of Micron-Scale DNA Scaffolds.

PubMed

Green, Leopold N; Amodio, Alessia; Subramanian, Hari K K; Ricci, Francesco; Franco, Elisa

2017-12-13

Inspired by cytoskeletal scaffolds that sense and respond dynamically to environmental changes and chemical inputs with a unique capacity for reconfiguration, we propose a strategy that allows the dynamic and reversible control of the growth and breakage of micron-scale synthetic DNA structures upon pH changes. We do so by rationally designing a pH-responsive system composed of synthetic DNA strands that act as pH sensors, regulators, and structural elements. Sensor strands can dynamically respond to pH changes and route regulatory strands to direct the self-assembly of structural elements into tubular structures. This example represents the first demonstration of the reversible assembly and disassembly of micron-scale DNA scaffolds using an external chemical input other than DNA. The capacity to reversibly modulate nanostructure size may promote the development of smart devices for catalysis or drug-delivery applications.

In vitro selection of DNA elements highly responsive to the human T-cell lymphotropic virus type I transcriptional activator, Tax.

PubMed

Paca-Uccaralertkun, S; Zhao, L J; Adya, N; Cross, J V; Cullen, B R; Boros, I M; Giam, C Z

1994-01-01

The human T-cell lymphotropic virus type I (HTLV-I) transactivator, Tax, the ubiquitous transcriptional factor cyclic AMP (cAMP) response element-binding protein (CREB protein), and the 21-bp repeats in the HTLV-I transcriptional enhancer form a ternary nucleoprotein complex (L. J. Zhao and C. Z. Giam, Proc. Natl. Acad. Sci. USA 89:7070-7074, 1992). Using an antibody directed against the COOH-terminal region of Tax along with purified Tax and CREB proteins, we selected DNA elements bound specifically by the Tax-CREB complex in vitro. Two distinct but related groups of sequences containing the cAMP response element (CRE) flanked by long runs of G and C residues in the 5' and 3' regions, respectively, were preferentially recognized by Tax-CREB. In contrast, CREB alone binds only to CRE motifs (GNTGACG[T/C]) without neighboring G- or C-rich sequences. The Tax-CREB-selected sequences bear a striking resemblance to the 5' or 3' two-thirds of the HTLV-I 21-bp repeats and are highly inducible by Tax. Gel electrophoretic mobility shift assays, DNA transfection, and DNase I footprinting analyses indicated that the G- and C-rich sequences flanking the CRE motif are crucial for Tax-CREB-DNA ternary complex assembly and Tax transactivation but are not in direct contact with the Tax-CREB complex. These data show that Tax recruits CREB to form a multiprotein complex that specifically recognizes the viral 21-bp repeats. The expanded DNA binding specificity of Tax-CREB and the obligatory role the ternary Tax-CREB-DNA complex plays in transactivation reveal a novel mechanism for regulating the transcriptional activity of leucine zipper proteins like CREB.

DNA is structured as a linear "jigsaw puzzle" in the genomes of Arabidopsis, rice, and budding yeast.

PubMed

Liu, Yun-Hua; Zhang, Meiping; Wu, Chengcang; Huang, James J; Zhang, Hong-Bin

2014-01-01

Knowledge of how a genome is structured and organized from its constituent elements is crucial to understanding its biology and evolution. Here, we report the genome structuring and organization pattern as revealed by systems analysis of the sequences of three model species, Arabidopsis, rice and yeast, at the whole-genome and chromosome levels. We found that all fundamental function elements (FFE) constituting the genomes, including genes (GEN), DNA transposable elements (DTE), retrotransposable elements (RTE), simple sequence repeats (SSR), and (or) low complexity repeats (LCR), are structured in a nonrandom and correlative manner, thus leading to a hypothesis that the DNA of the species is structured as a linear "jigsaw puzzle". Furthermore, we showed that different FFE differ in their importance in the formation and evolution of the DNA jigsaw puzzle structure between species. DTE and RTE play more important roles than GEN, LCR, and SSR in Arabidopsis, whereas GEN and RTE play more important roles than LCR, SSR, and DTE in rice. The genes having multiple recognized functions play more important roles than those having single functions. These results provide useful knowledge necessary for better understanding genome biology and evolution of the species and for effective molecular breeding of rice.

[Genome-scale sequence data processing and epigenetic analysis of DNA methylation].

PubMed

Wang, Ting-Zhang; Shan, Gao; Xu, Jian-Hong; Xue, Qing-Zhong

2013-06-01

A new approach recently developed for detecting cytosine DNA methylation (mC) and analyzing the genome-scale DNA methylation profiling, is called BS-Seq which is based on bisulfite conversion of genomic DNA combined with next-generation sequencing. The method can not only provide an insight into the difference of genome-scale DNA methylation among different organisms, but also reveal the conservation of DNA methylation in all contexts and nucleotide preference for different genomic regions, including genes, exons, and repetitive DNA sequences. It will be helpful to under-stand the epigenetic impacts of cytosine DNA methylation on the regulation of gene expression and maintaining silence of repetitive sequences, such as transposable elements. In this paper, we introduce the preprocessing steps of DNA methylation data, by which cytosine (C) and guanine (G) in the reference sequence are transferred to thymine (T) and adenine (A), and cytosine in reads is transferred to thymine, respectively. We also comprehensively review the main content of the DNA methylation analysis on the genomic scale: (1) the cytosine methylation under the context of different sequences; (2) the distribution of genomic methylcytosine; (3) DNA methylation context and the preference for the nucleotides; (4) DNA- protein interaction sites of DNA methylation; (5) degree of methylation of cytosine in the different structural elements of genes. DNA methylation analysis technique provides a powerful tool for the epigenome study in human and other species, and genes and environment interaction, and founds the theoretical basis for further development of disease diagnostics and therapeutics in human.

DNA topoisomerase 1α promotes transcriptional silencing of transposable elements through DNA methylation and histone lysine 9 dimethylation in Arabidopsis.

PubMed

Dinh, Thanh Theresa; Gao, Lei; Liu, Xigang; Li, Dongming; Li, Shengben; Zhao, Yuanyuan; O'Leary, Michael; Le, Brandon; Schmitz, Robert J; Manavella, Pablo A; Manavella, Pablo; Li, Shaofang; Weigel, Detlef; Pontes, Olga; Ecker, Joseph R; Chen, Xuemei

2014-07-01

RNA-directed DNA methylation (RdDM) and histone H3 lysine 9 dimethylation (H3K9me2) are related transcriptional silencing mechanisms that target transposable elements (TEs) and repeats to maintain genome stability in plants. RdDM is mediated by small and long noncoding RNAs produced by the plant-specific RNA polymerases Pol IV and Pol V, respectively. Through a chemical genetics screen with a luciferase-based DNA methylation reporter, LUCL, we found that camptothecin, a compound with anti-cancer properties that targets DNA topoisomerase 1α (TOP1α) was able to de-repress LUCL by reducing its DNA methylation and H3K9me2 levels. Further studies with Arabidopsis top1α mutants showed that TOP1α silences endogenous RdDM loci by facilitating the production of Pol V-dependent long non-coding RNAs, AGONAUTE4 recruitment and H3K9me2 deposition at TEs and repeats. This study assigned a new role in epigenetic silencing to an enzyme that affects DNA topology.

Cryo-EM Structures Reveal Mechanism and Inhibition of DNA Targeting by a CRISPR-Cas Surveillance Complex.

PubMed

Guo, Tai Wei; Bartesaghi, Alberto; Yang, Hui; Falconieri, Veronica; Rao, Prashant; Merk, Alan; Eng, Edward T; Raczkowski, Ashleigh M; Fox, Tara; Earl, Lesley A; Patel, Dinshaw J; Subramaniam, Sriram

2017-10-05

Prokaryotic cells possess CRISPR-mediated adaptive immune systems that protect them from foreign genetic elements, such as invading viruses. A central element of this immune system is an RNA-guided surveillance complex capable of targeting non-self DNA or RNA for degradation in a sequence- and site-specific manner analogous to RNA interference. Although the complexes display considerable diversity in their composition and architecture, many basic mechanisms underlying target recognition and cleavage are highly conserved. Using cryoelectron microscopy (cryo-EM), we show that the binding of target double-stranded DNA (dsDNA) to a type I-F CRISPR system yersinia (Csy) surveillance complex leads to large quaternary and tertiary structural changes in the complex that are likely necessary in the pathway leading to target dsDNA degradation by a trans-acting helicase-nuclease. Comparison of the structure of the surveillance complex before and after dsDNA binding, or in complex with three virally encoded anti-CRISPR suppressors that inhibit dsDNA binding, reveals mechanistic details underlying target recognition and inhibition. Published by Elsevier Inc.

A Novel Approach to Assay DNA Methylation in Prostate Cancer

DTIC Science & Technology

2017-12-01

concordant with repression or activation of those nearby genes.15 More recently, a series of chromosome conformation capture (3C)-based approaches ...AWARD NUMBER: W81XWH-13-1-0319 TITLE: A Novel Approach to Assay DNA Methylation in Prostate Cancer PRINCIPAL INVESTIGATOR: Jindan Yu...Department of the Army position, policy or decision unless so designated by other documentation. REPORT DOCUMENTATION PAGE Form Approved OMB No

PRESENTED AT COPENHAGEN WORKSHOP ON ENVIRONMENTAL, REPRODUCTIVE HEALTH AND FERTILITY, COPENHAGEN, DENMARK, 1/15-18/2005: GST M1 GENOTYPE INFLUENCES SPERM DNA DAMAGE ASSOCIATED WITH EXPOSURE TO AIR POLLUTION

EPA Science Inventory

Exposure to episodic air pollution in the Czech Republic has been associated with abnormal semen quality and sperm DNA damage (EHP 108:887;2000). A subsequent longitudinal study evaluated semen from 36 men sampled up to 7 times over a period of two years to capture exposures dur...

Enzymatic cleavage of uracil-containing single-stranded DNA linkers for the efficient release of affinity-selected circulating tumor cells.

PubMed

Nair, Soumya V; Witek, Małgorzata A; Jackson, Joshua M; Lindell, Maria A M; Hunsucker, Sally A; Sapp, Travis; Perry, Caroline E; Hupert, Mateusz L; Bae-Jump, Victoria; Gehrig, Paola A; Wysham, Weiya Z; Armistead, Paul M; Voorhees, Peter; Soper, Steven A

2015-02-21

We report a novel strategy to enzymatically release affinity-selected cells, such as circulating tumor cells (CTCs), from surfaces with high efficiency (∼90%) while maintaining cell viability (>85%). The strategy utilizes single-stranded DNAs that link a capture antibody to the surfaces of a CTC selection device. The DNA linkers contain a uracil residue that can be cleaved.

High-Throughput Sequencing of 16S rRNA Gene Amplicons: Effects of Extraction Procedure, Primer Length and Annealing Temperature

PubMed Central

Sergeant, Martin J.; Constantinidou, Chrystala; Cogan, Tristan; Penn, Charles W.; Pallen, Mark J.

2012-01-01

The analysis of 16S-rDNA sequences to assess the bacterial community composition of a sample is a widely used technique that has increased with the advent of high throughput sequencing. Although considerable effort has been devoted to identifying the most informative region of the 16S gene and the optimal informatics procedures to process the data, little attention has been paid to the PCR step, in particular annealing temperature and primer length. To address this, amplicons derived from 16S-rDNA were generated from chicken caecal content DNA using different annealing temperatures, primers and different DNA extraction procedures. The amplicons were pyrosequenced to determine the optimal protocols for capture of maximum bacterial diversity from a chicken caecal sample. Even at very low annealing temperatures there was little effect on the community structure, although the abundance of some OTUs such as Bifidobacterium increased. Using shorter primers did not reveal any novel OTUs but did change the community profile obtained. Mechanical disruption of the sample by bead beating had a significant effect on the results obtained, as did repeated freezing and thawing. In conclusion, existing primers and standard annealing temperatures captured as much diversity as lower annealing temperatures and shorter primers. PMID:22666455

High-throughput sequencing of 16S rRNA gene amplicons: effects of extraction procedure, primer length and annealing temperature.

PubMed

Sergeant, Martin J; Constantinidou, Chrystala; Cogan, Tristan; Penn, Charles W; Pallen, Mark J

2012-01-01

The analysis of 16S-rDNA sequences to assess the bacterial community composition of a sample is a widely used technique that has increased with the advent of high throughput sequencing. Although considerable effort has been devoted to identifying the most informative region of the 16S gene and the optimal informatics procedures to process the data, little attention has been paid to the PCR step, in particular annealing temperature and primer length. To address this, amplicons derived from 16S-rDNA were generated from chicken caecal content DNA using different annealing temperatures, primers and different DNA extraction procedures. The amplicons were pyrosequenced to determine the optimal protocols for capture of maximum bacterial diversity from a chicken caecal sample. Even at very low annealing temperatures there was little effect on the community structure, although the abundance of some OTUs such as Bifidobacterium increased. Using shorter primers did not reveal any novel OTUs but did change the community profile obtained. Mechanical disruption of the sample by bead beating had a significant effect on the results obtained, as did repeated freezing and thawing. In conclusion, existing primers and standard annealing temperatures captured as much diversity as lower annealing temperatures and shorter primers.

A biologically inspired two-species exclusion model: effects of RNA polymerase motor traffic on simultaneous DNA replication

NASA Astrophysics Data System (ADS)

Ghosh, Soumendu; Mishra, Bhavya; Patra, Shubhadeep; Schadschneider, Andreas; Chowdhury, Debashish

2018-04-01

We introduce a two-species exclusion model to describe the key features of the conflict between the RNA polymerase (RNAP) motor traffic, engaged in the transcription of a segment of DNA, concomitant with the progress of two DNA replication forks on the same DNA segment. One of the species of particles (P) represents RNAP motors while the other (R) represents the replication forks. Motivated by the biological phenomena that this model is intended to capture, a maximum of two R particles only are allowed to enter the lattice from two opposite ends whereas the unrestricted number of P particles constitutes a totally asymmetric simple exclusion process (TASEP) in a segment in the middle of the lattice. The model captures three distinct pathways for resolving the co-directional as well as head-on collision between the P and R particles. Using Monte Carlo simulations and heuristic analytical arguments that combine exact results for the TASEP with mean-field approximations, we predict the possible outcomes of the conflict between the traffic of RNAP motors (P particles engaged in transcription) and the replication forks (R particles). In principle, the model can be adapted to experimental conditions to account for the data quantitatively.

Deep Optical Spectroscopy of Planetary Nebulae: The Search for Neutron-Capture Elements

NASA Astrophysics Data System (ADS)

Sterling, Nicholas C.; Garofali, K.; Dinerstein, H. L.; Hwang, S.; Redfield, S.

2013-01-01

We present deep, high-resolution (R=36,700) optical spectra of five planetary nebulae (PNe), taken with the 2D-coude echelle spectrograph on the 2.7-m Harlan J. Smith Telescope at McDonald Observatory. These observations are part of a larger optical survey of PNe, designed to unambiguously detect emission lines from neutron(n)-capture elements (atomic number Z>30). The abundances of these elements are of particular interest in PNe, since they can be produced by slow n-capture nucleosynthesis (the ``s-process'') during the asymptotic giant branch (AGB) stage of evolution of PN progenitor stars. The first large-scale investigation of n-capture element abundances in PNe (Sterling & Dinerstein 2008, ApJS, 174, 157) surveyed [Kr III] and [Se IV] transitions in the K band spectra of more than 80 PNe. However, the abundances derived from these data relied on ionization corrections that were often large and uncertain due to the detection of only one ion per element. Transitions of other Se and Kr ions, as well as many other trans-iron species, reside at optical wavelengths. High-resolution spectra are essential to unequivocally identify these lines and resolve potential blends with other species. The spectra we present are rich in emission features, with between 125 and 600 distinct lines detected in each PN. Emission from at least one Kr ion is detected in all five objects, and two (Hb 12 and J 900) exhibit emission from multiple Kr ions. We detected multiple Xe ions in J 900, as well as Se, Br, and Rb lines. Hb 12 also exhibits Xe emission, and the first detection of [Se II] in a PN to our knowledge. The spectra display a wealth of other emission lines, including permitted features of second-row elements and forbidden transitions of several iron-peak elements (e.g., Cr, Mn, Fe, Co, Ni, and Cu). Our survey makes it possible to derive more accurate Se and Kr abundances in PNe, and reveals the enrichment of other trans-iron elements. This enables more accurate s-process enrichment factors to be derived for PNe, providing important constraints to models of AGB nucleosynthesis and the chemical evolution of trans-iron nuclides. This research was supported by NSF awards AST-0708425 and AST-901432.

Dead Element Replicating: Degenerate R2 Element Replication and rDNA Genomic Turnover in the Bacillus rossius Stick Insect (Insecta: Phasmida)

PubMed Central

Martoni, Francesco; Eickbush, Danna G.; Scavariello, Claudia; Luchetti, Andrea; Mantovani, Barbara

2015-01-01

R2 is an extensively investigated non-LTR retrotransposon that specifically inserts into the 28S rRNA gene sequences of a wide range of metazoans, disrupting its functionality. During R2 integration, first strand synthesis can be incomplete so that 5’ end deleted copies are occasionally inserted. While active R2 copies repopulate the locus by retrotransposing, the non-functional truncated elements should frequently be eliminated by molecular drive processes leading to the concerted evolution of the rDNA array(s). Although, multiple R2 lineages have been discovered in the genome of many animals, the rDNA of the stick insect Bacillus rossius exhibits a peculiar situation: it harbors both a canonical, functional R2 element (R2Brfun) as well as a full-length but degenerate element (R2Brdeg). An intensive sequencing survey in the present study reveals that all truncated variants in stick insects are present in multiple copies suggesting they were duplicated by unequal recombination. Sequencing results also demonstrate that all R2Brdeg copies are full-length, i. e. they have no associated 5' end deletions, and functional assays indicate they have lost the active ribozyme necessary for R2 RNA maturation. Although it cannot be completely ruled out, it seems unlikely that the degenerate elements replicate via reverse transcription, exploiting the R2Brfun element enzymatic machinery, but rather via genomic amplification of inserted 28S by unequal recombination. That inactive copies (both R2Brdeg or 5'-truncated elements) are not eliminated in a short term in stick insects contrasts with findings for the Drosophila R2, suggesting a widely different management of rDNA loci and a lower efficiency of the molecular drive while achieving the concerted evolution. PMID:25799008

Development and validation of an integrated DNA walking strategy to detect GMO expressing cry genes.

PubMed

Fraiture, Marie-Alice; Vandamme, Julie; Herman, Philippe; Roosens, Nancy H C

2018-06-27

Recently, an integrated DNA walking strategy has been proposed to prove the presence of GMO via the characterisation of sequences of interest, including their transgene flanking regions and the unnatural associations of elements in their transgenic cassettes. To this end, the p35S, tNOS and t35S pCAMBIA elements have been selected as key targets, allowing the coverage of most of GMO, EU authorized or not. In the present study, a bidirectional DNA walking method anchored on the CryAb/c genes is proposed with the aim to cover additional GMO and additional sequences of interest. The performance of the proposed bidirectional DNA walking method anchored on the CryAb/c genes has been evaluated in a first time for its feasibility using several GM events possessing these CryAb/c genes. Afterwards, its sensitivity has been investigated through low concentrations of targets (as low as 20 HGE). In addition, to illustrate its applicability, the entire workflow has been tested on a sample mimicking food/feed matrices analysed in GMO routine analysis. Given the successful assessment of its performance, the present bidirectional DNA walking method anchored on the CryAb/c genes can easily be implemented in GMO routine analysis by the enforcement laboratories and allows completing the entire DNA walking strategy in targeting an additional transgenic element frequently found in GMO.

Ectopic recombination between Ty elements in Saccharomyces cerevisiae is not induced by DNA damage.

PubMed

Parket, A; Kupiec, M

1992-10-01

Mitotic recombination is increased when cells are treated with a variety of physical and chemical agents that cause damage to their DNA. We show here, using Saccharomyces cerevisiae strains that carry marked Ty elements, that recombination between members of this family of retrotransposons is not increased by UV irradiation or by treatment with the radiomimetic drug methyl methanesulfonate. Both ectopic recombination and mutation events were elevated by these agents for non-Ty sequences in the same strain. We discuss possible mechanisms that can prevent the induction of recombination between Ty elements.

DNA transposons have colonized the genome of the giant virus Pandoravirus salinus.

PubMed

Sun, Cheng; Feschotte, Cédric; Wu, Zhiqiang; Mueller, Rachel Lockridge

2015-06-12

Transposable elements are mobile DNA sequences that are widely distributed in prokaryotic and eukaryotic genomes, where they represent a major force in genome evolution. However, transposable elements have rarely been documented in viruses, and their contribution to viral genome evolution remains largely unexplored. Pandoraviruses are recently described DNA viruses with genome sizes that exceed those of some prokaryotes, rivaling parasitic eukaryotes. These large genomes appear to include substantial noncoding intergenic spaces, which provide potential locations for transposable element insertions. However, no mobile genetic elements have yet been reported in pandoravirus genomes. Here, we report a family of miniature inverted-repeat transposable elements (MITEs) in the Pandoravirus salinus genome, representing the first description of a virus populated with a canonical transposable element family that proliferated by transposition within the viral genome. The MITE family, which we name Submariner, includes 30 copies with all the hallmarks of MITEs: short length, terminal inverted repeats, TA target site duplication, and no coding capacity. Submariner elements show signs of transposition and are undetectable in the genome of Pandoravirus dulcis, the closest known relative Pandoravirus salinus. We identified a DNA transposon related to Submariner in the genome of Acanthamoeba castellanii, a species thought to host pandoraviruses, which contains remnants of coding sequence for a Tc1/mariner transposase. These observations suggest that the Submariner MITEs of P. salinus belong to the widespread Tc1/mariner superfamily and may have been mobilized by an amoebozoan host. Ten of the 30 MITEs in the P. salinus genome are located within coding regions of predicted genes, while others are close to genes, suggesting that these transposons may have contributed to viral genetic novelty. Our discovery highlights the remarkable ability of DNA transposons to colonize and shape genomes from all domains of life, as well as giant viruses. Our findings continue to blur the division between viral and cellular genomes, adhering to the emerging view that the content, dynamics, and evolution of the genomes of giant viruses do not substantially differ from those of cellular organisms.

Microsatellite DNA capture from enriched libraries.

PubMed

Gonzalez, Elena G; Zardoya, Rafael

2013-01-01

Microsatellites are DNA sequences of tandem repeats of one to six nucleotides, which are highly polymorphic, and thus the molecular markers of choice in many kinship, population genetic, and conservation studies. There have been significant technical improvements since the early methods for microsatellite isolation were developed, and today the most common procedures take advantage of the hybrid capture methods of enriched-targeted microsatellite DNA. Furthermore, recent advents in sequencing technologies (i.e., next-generation sequencing, NGS) have fostered the mining of microsatellite markers in non-model organisms, affording a cost-effective way of obtaining a large amount of sequence data potentially useful for loci characterization. The rapid improvements of NGS platforms together with the increase in available microsatellite information open new avenues to the understanding of the evolutionary forces that shape genetic structuring in wild populations. Here, we provide detailed methodological procedures for microsatellite isolation based on the screening of GT microsatellite-enriched libraries, either by cloning and Sanger sequencing of positive clones or by direct NGS. Guides for designing new species-specific primers and basic genotyping are also given.

Direct electrical and mechanical characterization of in situ generated DNA between the tips of silicon nanotweezers (SNT).

PubMed

Karsten, Stanislav L; Kumemura, Momoko; Jalabert, Laurent; Lafitte, Nicolas; Kudo, Lili C; Collard, Dominique; Fujita, Hiroyuki

2016-05-24

Previously, we reported the application of micromachined silicon nanotweezers (SNT) integrated with a comb-drive actuator and capacitive sensors for capturing and mechanical characterization of DNA bundles. Here, we demonstrate direct DNA amplification on such a MEMS structure with subsequent electrical and mechanical characterization of a single stranded DNA (ssDNA) bundle generated between the tips of SNT via isothermal rolling circle amplification (RCA) and dielectrophoresis (DEP). An in situ generated ssDNA bundle was visualized and evaluated via electrical conductivity (I-V) and mechanical frequency response measurements. Colloidal gold nanoparticles significantly enhanced (P < 0.01) the electrical properties of thin ssDNA bundles. The proposed technology allows direct in situ synthesis of DNA with a predefined sequence on the tips of a MEMS sensor device, such as SNT, followed by direct DNA electrical and mechanical characterization. In addition, our data provides a "proof-of-principle" for the feasibility of the on-chip label free DNA detection device that can be used for a variety of biomedical applications focused on sequence specific DNA detection.

The site-specific ribosomal DNA insertion element R1Bm belongs to a class of non-long-terminal-repeat retrotransposons.

PubMed Central

Xiong, Y; Eickbush, T H

1988-01-01

Two types of insertion elements, R1 and R2 (previously called type I and type II), are known to interrupt the 28S ribosomal genes of several insect species. In the silkmoth, Bombyx mori, each element occupies approximately 10% of the estimated 240 ribosomal DNA units, while at most only a few copies are located outside the ribosomal DNA units. We present here the complete nucleotide sequence of an R1 insertion from B. mori (R1Bm). This 5.1-kilobase element contains two overlapping open reading frames (ORFs) which together occupy 88% of its length. ORF1 is 461 amino acids in length and exhibits characteristics of retroviral gag genes. ORF2 is 1,051 amino acids in length and contains homology to reverse transcriptase-like enzymes. The analysis of 3' and 5' ends of independent isolates from the ribosomal locus supports the suggestion that R1 is still functioning as a transposable element. The precise location of the element within the genome implies that its transposition must occur with remarkable insertion sequence specificity. Comparison of the deduced amino acid sequences from six retrotransposons, R1 and R2 of B. mori, I factor and F element of Drosophila melanogaster, L1 of Mus domesticus, and Ingi of Trypanosoma brucei, reveals a relatively high level of sequence homology in the reverse transcriptase region. Like R1, these elements lack long terminal repeats. We have therefore named this class of related elements the non-long-terminal-repeat (non-LTR) retrotransposons. Images PMID:2447482

Survival Mediated Heavy Element Capture Cross Sections

NASA Astrophysics Data System (ADS)

Loveland, Walter; Yao, Larry

2017-11-01

Formally, the cross section for producing a heavy evaporation residue, σEVR, in a fusion reaction can be written as where E is the center of mass energy, and T is the probability of the colliding nuclei to overcome the potential barrier in the entrance channel and reach the contact point. PCN is the probability that the projectile-target system will evolve from the contact point to the compound nucleus. Wsur is the probability that the compound nucleus will decay to produce an evaporation residue rather than fissioning. However, one must remember that the Wsur term effectively sets the allowed values of the spin, which in turn, restricts the values of the capture and fusion cross sections. We point out the implications of this fact for capture cross sections for heavy element formation reactions.

Flavonoids and DNA Repair in Prostate Cancer

DTIC Science & Technology

2005-12-01

1-0114 TITLE: Flavonoids and DNA Repair in Prostate Cancer...SUBTITLE Flavonoids and DNA Repair in Prostate Cancer 5a. CONTRACT NUMBER 5b. GRANT NUMBER W81XWH-04-1-0114 5c. PROGRAM ELEMENT NUMBER...test the hypothesis that natural products such as flavonoids are able to stimulate the repair of oxidative DNA damage. For this purpose LNCaP

AP1 Keeps Chromatin Poised for Action | Center for Cancer Research

Cancer.gov

The human genome harbors gene-encoding DNA, the blueprint for building proteins that regulate cellular function. Embedded across the genome, in non-coding regions, are DNA elements to which regulatory factors bind. The interaction of regulatory factors with DNA at these sites modifies gene expression to modulate cell activity. In cells, DNA exists in a complex with proteins

ANALYTIC ELEMENT MODELING FOR SOURCE WATER ASSESSMENTS OF PUBLIC WATER SUPPLY WELLS: CASE STUDIES IN GLACIAL OUTWASH AND BASIN-AND-RANGE

EPA Science Inventory

Over the last 10 years the EPA has invested in analytic elements as a computational method used in public domain software supporting capture zone delineation for source water assessments and wellhead protection. The current release is called WhAEM2000 (wellhead analytic element ...

Stabilization and discontinuity-capturing parameters for space-time flow computations with finite element and isogeometric discretizations

NASA Astrophysics Data System (ADS)

Takizawa, Kenji; Tezduyar, Tayfun E.; Otoguro, Yuto

2018-04-01

Stabilized methods, which have been very common in flow computations for many years, typically involve stabilization parameters, and discontinuity-capturing (DC) parameters if the method is supplemented with a DC term. Various well-performing stabilization and DC parameters have been introduced for stabilized space-time (ST) computational methods in the context of the advection-diffusion equation and the Navier-Stokes equations of incompressible and compressible flows. These parameters were all originally intended for finite element discretization but quite often used also for isogeometric discretization. The stabilization and DC parameters we present here for ST computations are in the context of the advection-diffusion equation and the Navier-Stokes equations of incompressible flows, target isogeometric discretization, and are also applicable to finite element discretization. The parameters are based on a direction-dependent element length expression. The expression is outcome of an easy to understand derivation. The key components of the derivation are mapping the direction vector from the physical ST element to the parent ST element, accounting for the discretization spacing along each of the parametric coordinates, and mapping what we have in the parent element back to the physical element. The test computations we present for pure-advection cases show that the parameters proposed result in good solution profiles.

A paper-based device for double-stranded DNA detection with Zif268

NASA Astrophysics Data System (ADS)

Zhang, Daohong

2017-05-01

Here, a small analytical device was fabricated on both nitrocellulose membrane and filter paper, for the detection of biotinylated double-stranded DNA (dsDNA) from 1 nM. Zif268 was utilized for capturing the target DNA, which was a zinc finger protein that recognized only a dsDNA with specific sequence. Therefore, this detection platform could be utilized for PCR result detection, with the well-designed primers (interpolate both biotin and Zif268 binding sequence). The result of the assay could be recorded by a camera-phone, and analyzed with software. The whole assay finished within 1 hour. Due to the easy fabrication, operation and disposal of this device, this method can be employed in point-of-care detection or on-site monitoring.

A cDNA from a mouse pancreatic beta cell encoding a putative transcription factor of the insulin gene.

PubMed Central

Walker, M D; Park, C W; Rosen, A; Aronheim, A

1990-01-01

Cell specific expression of the insulin gene is achieved through transcriptional mechanisms operating on multiple DNA sequence elements located in the 5' flanking region of the gene. Of particular importance in the rat insulin I gene are two closely similar 9 bp sequences (IEB1 and IEB2): mutation of either of these leads to 5-10 fold reduction in transcriptional activity. We have screened an expression cDNA library derived from mouse pancreatic endocrine beta cells with a radioactive DNA probe containing multiple copies of the IEB1 sequence. A cDNA clone (A1) isolated by this procedure encodes a protein which shows efficient binding to the IEB1 probe, but much weaker binding to either an unrelated DNA probe or to a probe bearing a single base pair insertion within the recognition sequence. DNA sequence analysis indicates a protein belonging to the helix-loop-helix family of DNA-binding proteins. The ability of the protein encoded by clone A1 to recognize a number of wild type and mutant DNA sequences correlates closely with the ability of each sequence element to support transcription in vivo in the context of the insulin 5' flanking DNA. We conclude that the isolated cDNA may encode a transcription factor that participates in control of insulin gene expression. Images PMID:2181401

Application of DNA Aptamers and Quantum Dots to Lateral Flow Test Strips for Detection of Foodborne Pathogens with Improved Sensitivity versus Colloidal Gold

PubMed Central

Bruno, John G.

2014-01-01

Preliminary studies aimed at improving the sensitivity of foodborne pathogen detection via lateral flow (LF) test strips by use of high affinity DNA aptamers for capture and reporter functions when coupled to red-emitting quantum dots (Qdot 655) are reported. A variety of DNA aptamers developed against Escherichia coli, Listeria monocytogenes, and Salmonella enterica were paired in capture and reporter combinations to determine which yielded the strongest detection of their cognate bacteria using a colloidal gold screening system. Several promising sandwich combinations were identified for each of the three bacterial LF strip systems. The best E. coli aptamer-LF system was further studied and yielded a visible limit of detection (LOD) of ~3,000 E. coli 8739 and ~6,000 E. coli O157:H7 in buffer. These LODs were reduced to ~300–600 bacterial cells per test respectively by switching to a Qdot 655 aptamer-LF system. Novel aspects of these assays such as the use of high levels of detergents to avoid quantum dot agglutination and enhance migration in analytical membranes, identification of optimal analytical membrane types, UV-immobilization of capture aptamers, and novel dual biotin/digoxigenin-end labeled aptamer streptavidin-colloidal gold or -Qdot 655 conjugates plus anti-digoxigenin antibody control lines are also discussed. In general, this work provides proof-of-principle for highly sensitive aptamer-Qdot LF strip assays for rapid foodborne pathogen detection. PMID:25437803

Characterization of noncoding regulatory DNA in the human genome.

PubMed

Elkon, Ran; Agami, Reuven

2017-08-08

Genetic variants associated with common diseases are usually located in noncoding parts of the human genome. Delineation of the full repertoire of functional noncoding elements, together with efficient methods for probing their biological roles, is therefore of crucial importance. Over the past decade, DNA accessibility and various epigenetic modifications have been associated with regulatory functions. Mapping these features across the genome has enabled researchers to begin to document the full complement of putative regulatory elements. High-throughput reporter assays to probe the functions of regulatory regions have also been developed but these methods separate putative regulatory elements from the chromosome so that any effects of chromatin context and long-range regulatory interactions are lost. Definitive assignment of function(s) to putative cis-regulatory elements requires perturbation of these elements. Genome-editing technologies are now transforming our ability to perturb regulatory elements across entire genomes. Interpretation of high-throughput genetic screens that incorporate genome editors might enable the construction of an unbiased map of functional noncoding elements in the human genome.

The Human L1 Element Causes DNA Double-Strand Breaks in Breast Cancer

DTIC Science & Technology

2006-08-01

cancer is complex. However, defects in DNA repair genes in the double-strand break repair pathway are cancer predisposing. My lab has characterized...a new potentially important source of double-strand breaks (DSBs) in human cells and are interested in characterizing which DNA repair genes act on...this particular source of DNA damage. Selfish DNA accounts for 45% of the human genome. We have recently demonstrated that one particular selfish

[Molecular-genetic analysis of wheat (T. aestivum L.) genome with introgression of Ae. cylindrica Host genetic elements].

PubMed

Galaev, A V; Sivolap, Iu M

2005-01-01

Wheat-aegilops hybrid plants Triticum aestivum L. (2n = 42) x Aegilops cylindrica Host (2n = 28) were investigated with using microsatellite markers. In two BC1F9 lines some genome modifications connected with losing DNA fragments of initial variety or appearing of Aegilops genome elements were detected. In some investigated hybrids new amplicons lacking in parental plants were found. Substitution of wheat chromosomes for aegilops chromosomes was not revealed. Analysis of microsatellite loci in BC2F5 plants showed stable introgression of aegilops genetic elements into wheat; elimination of some transferred aegilops DNA fragments in the course of backcrossing; decreasing size of introgressive elements after backcrossing. Introgressive lines were classified according to genome changes.

Capturing the radical ion-pair intermediate in DNA guanine oxidation

PubMed Central

Jie, Jialong; Liu, Kunhui; Wu, Lidan; Zhao, Hongmei; Song, Di; Su, Hongmei

2017-01-01

Although the radical ion pair has been frequently invoked as a key intermediate in DNA oxidative damage reactions and photoinduced electron transfer processes, the unambiguous detection and characterization of this species remain formidable and unresolved due to its extremely unstable nature and low concentration. We use the strategy that, at cryogenic temperatures, the transient species could be sufficiently stabilized to be detectable spectroscopically. By coupling the two techniques (the cryogenic stabilization and the time-resolved laser flash photolysis spectroscopy) together, we are able to capture the ion-pair transient G+•⋯Cl− in the chlorine radical–initiated DNA guanine (G) oxidation reaction, and provide direct evidence to ascertain the intricate type of addition/charge separation mechanism underlying guanine oxidation. The unique spectral signature of the radical ion-pair G+•⋯Cl− is identified, revealing a markedly intense absorption feature peaking at 570 nm that is distinctive from G+• alone. Moreover, the ion-pair spectrum is found to be highly sensitive to the protonation equilibria within guanine-cytosine base pair (G:C), which splits into two resolved bands at 480 and 610 nm as the acidic proton transfers along the central hydrogen bond from G+• to C. We thus use this exquisite sensitivity to track the intrabase-pair proton transfer dynamics in the double-stranded DNA oligonucleotides, which is of critical importance for the description of the proton-coupled charge transfer mechanisms in DNA. PMID:28630924

Fully Integrated Microfluidic Device for Direct Sample-to-Answer Genetic Analysis

NASA Astrophysics Data System (ADS)

Liu, Robin H.; Grodzinski, Piotr

Integration of microfluidics technology with DNA microarrays enables building complete sample-to-answer systems that are useful in many applications such as clinic diagnostics. In this chapter, a fully integrated microfluidic device [1] that consists of microfluidic mixers, valves, pumps, channels, chambers, heaters, and a DNA microarray sensor to perform DNA analysis of complex biological sample solutions is present. This device can perform on-chip sample preparation (including magnetic bead-based cell capture, cell preconcentration and purification, and cell lysis) of complex biological sample solutions (such as whole blood), polymerase chain reaction, DNA hybridization, and electrochemical detection. A few novel microfluidic techniques were developed and employed. A micromix-ing technique based on a cavitation microstreaming principle was implemented to enhance target cell capture from whole blood samples using immunomagnetic beads. This technique was also employed to accelerate DNA hybridization reaction. Thermally actuated paraffin-based microvalves were developed to regulate flows. Electrochemical pumps and thermopneumatic pumps were integrated on the chip to provide pumping of liquid solutions. The device is completely self-contained: no external pressure sources, fluid storage, mechanical pumps, or valves are necessary for fluid manipulation, thus eliminating possible sample contamination and simplifying device operation. Pathogenic bacteria detection from ~mL whole blood samples and single-nucleotide polymorphism analysis directly from diluted blood were demonstrated. The device provides a cost-effective solution to direct sample-to-answer genetic analysis, and thus has a potential impact in the fields of point-of-care genetic analysis, environmental testing, and biological warfare agent detection.

Hybrid Methods Reveal Multiple Flexibly Linked DNA Polymerases within the Bacteriophage T7 Replisome

DOE PAGES

Wallen, Jamie R.; Zhang, Hao; Weis, Caroline; ...

2017-01-03

The physical organization of DNA enzymes at a replication fork enables efficient copying of two antiparallel DNA strands, yet dynamic protein interactions within the replication complex complicate replisome structural studies. We employed a combination of crystallographic, native mass spectrometry and small-angle X-ray scattering experiments to capture alternative structures of a model replication system encoded by bacteriophage T7. then, the two molecules of DNA polymerase bind the ring-shaped primase-helicase in a conserved orientation and provide structural insight into how the acidic C-terminal tail of the primase-helicase contacts the DNA polymerase to facilitate loading of the polymerase onto DNA. A third DNA polymerasemore » binds the ring in an offset manner that may enable polymerase exchange during replication. Alternative polymerase binding modes are also detected by small-angle X-ray scattering with DNA substrates present. The collective results unveil complex motions within T7 replisome higher-order structures that are underpinned by multivalent protein-protein interactions with functional implications.« less

Hybrid Methods Reveal Multiple Flexibly Linked DNA Polymerases within the Bacteriophage T7 Replisome

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wallen, Jamie R.; Zhang, Hao; Weis, Caroline

The physical organization of DNA enzymes at a replication fork enables efficient copying of two antiparallel DNA strands, yet dynamic protein interactions within the replication complex complicate replisome structural studies. We employed a combination of crystallographic, native mass spectrometry and small-angle X-ray scattering experiments to capture alternative structures of a model replication system encoded by bacteriophage T7. then, the two molecules of DNA polymerase bind the ring-shaped primase-helicase in a conserved orientation and provide structural insight into how the acidic C-terminal tail of the primase-helicase contacts the DNA polymerase to facilitate loading of the polymerase onto DNA. A third DNA polymerasemore » binds the ring in an offset manner that may enable polymerase exchange during replication. Alternative polymerase binding modes are also detected by small-angle X-ray scattering with DNA substrates present. The collective results unveil complex motions within T7 replisome higher-order structures that are underpinned by multivalent protein-protein interactions with functional implications.« less

Kinetic gating mechanism of DNA damage recognition by Rad4/XPC

NASA Astrophysics Data System (ADS)

Chen, Xuejing; Velmurugu, Yogambigai; Zheng, Guanqun; Park, Beomseok; Shim, Yoonjung; Kim, Youngchang; Liu, Lili; van Houten, Bennett; He, Chuan; Ansari, Anjum; Min, Jung-Hyun

2015-01-01

The xeroderma pigmentosum C (XPC) complex initiates nucleotide excision repair by recognizing DNA lesions before recruiting downstream factors. How XPC detects structurally diverse lesions embedded within normal DNA is unknown. Here we present a crystal structure that captures the yeast XPC orthologue (Rad4) on a single register of undamaged DNA. The structure shows that a disulphide-tethered Rad4 flips out normal nucleotides and adopts a conformation similar to that seen with damaged DNA. Contrary to many DNA repair enzymes that can directly reject non-target sites as structural misfits, our results suggest that Rad4/XPC uses a kinetic gating mechanism whereby lesion selectivity arises from the kinetic competition between DNA opening and the residence time of Rad4/XPC per site. This mechanism is further supported by measurements of Rad4-induced lesion-opening times using temperature-jump perturbation spectroscopy. Kinetic gating may be a general mechanism used by site-specific DNA-binding proteins to minimize time-consuming interrogations of non-target sites.

Context influences on TALE–DNA binding revealed by quantitative profiling

PubMed Central

Rogers, Julia M.; Barrera, Luis A.; Reyon, Deepak; Sander, Jeffry D.; Kellis, Manolis; Joung, J Keith; Bulyk, Martha L.

2015-01-01

Transcription activator-like effector (TALE) proteins recognize DNA using a seemingly simple DNA-binding code, which makes them attractive for use in genome engineering technologies that require precise targeting. Although this code is used successfully to design TALEs to target specific sequences, off-target binding has been observed and is difficult to predict. Here we explore TALE–DNA interactions comprehensively by quantitatively assaying the DNA-binding specificities of 21 representative TALEs to ∼5,000–20,000 unique DNA sequences per protein using custom-designed protein-binding microarrays (PBMs). We find that protein context features exert significant influences on binding. Thus, the canonical recognition code does not fully capture the complexity of TALE–DNA binding. We used the PBM data to develop a computational model, Specificity Inference For TAL-Effector Design (SIFTED), to predict the DNA-binding specificity of any TALE. We provide SIFTED as a publicly available web tool that predicts potential genomic off-target sites for improved TALE design. PMID:26067805

Context influences on TALE-DNA binding revealed by quantitative profiling.

PubMed

Rogers, Julia M; Barrera, Luis A; Reyon, Deepak; Sander, Jeffry D; Kellis, Manolis; Joung, J Keith; Bulyk, Martha L

2015-06-11

Transcription activator-like effector (TALE) proteins recognize DNA using a seemingly simple DNA-binding code, which makes them attractive for use in genome engineering technologies that require precise targeting. Although this code is used successfully to design TALEs to target specific sequences, off-target binding has been observed and is difficult to predict. Here we explore TALE-DNA interactions comprehensively by quantitatively assaying the DNA-binding specificities of 21 representative TALEs to ∼5,000-20,000 unique DNA sequences per protein using custom-designed protein-binding microarrays (PBMs). We find that protein context features exert significant influences on binding. Thus, the canonical recognition code does not fully capture the complexity of TALE-DNA binding. We used the PBM data to develop a computational model, Specificity Inference For TAL-Effector Design (SIFTED), to predict the DNA-binding specificity of any TALE. We provide SIFTED as a publicly available web tool that predicts potential genomic off-target sites for improved TALE design.

Kinetic gating mechanism of DNA damage recognition by Rad4/XPC

DOE PAGES

Chen, Xuejing; Velmurugu, Yogambigai; Zheng, Guanqun; ...

2015-01-06

The xeroderma pigmentosum C (XPC) complex initiates nucleotide excision repair by recognizing DNA lesions before recruiting downstream factors. How XPC detects structurally diverse lesions embedded within normal DNA is unknown. Here we present a crystal structure that captures the yeast XPC orthologue (Rad4) on a single register of undamaged DNA. The structure shows that a disulphide-tethered Rad4 flips out normal nucleotides and adopts a conformation similar to that seen with damaged DNA. Contrary to many DNA repair enzymes that can directly reject non-target sites as structural misfits, our results suggest that Rad4/XPC uses a kinetic gating mechanism whereby lesion selectivitymore » arises from the kinetic competition between DNA opening and the residence time of Rad4/XPC per site. This mechanism is further supported by measurements of Rad4-induced lesion-opening times using temperature-jump perturbation spectroscopy. Lastly, kinetic gating may be a general mechanism used by site-specific DNA-binding proteins to minimize time-consuming interrogations of non-target sites.« less

Altruistic functions for selfish DNA.

PubMed

Faulkner, Geoffrey J; Carninci, Piero

2009-09-15

Mammalian genomes are comprised of 30-50% transposed elements (TEs). The vast majority of these TEs are truncated and mutated fragments of retrotransposons that are no longer capable of transposition. Although initially regarded as important factors in the evolution of gene regulatory networks, TEs are now commonly perceived as neutrally evolving and non-functional genomic elements. In a major development, recent works have strongly contradicted this "selfish DNA" or "junk DNA" dogma by demonstrating that TEs use a host of novel promoters to generate RNA on a massive scale across most eukaryotic cells. This transcription frequently functions to control the expression of protein-coding genes via alternative promoters, cis regulatory non protein-coding RNAs and the formation of double stranded short RNAs. If considered in sum, these findings challenge the designation of TEs as selfish and neutrally evolving genomic elements. Here, we will expand upon these themes and discuss challenges in establishing novel TE functions in vivo.