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https://www.readbyqxmd.com/read/28645756/an-aptamer-based-pcr-method-coupled-with-magnetic-immunoseparation-for-sensitive-detection-of-salmonella-typhimurium-in-ground-turkey
#1
Lijun Wang, Ronghui Wang, Hong Wang, Michael Slavik, Hua Wei, Yanbin Li
Aptamers are single-stranded oligonucleotide ligands that can bind to targets with high affinity and specificity. They have been widely studied in the field of diagnostics as alternatives to antibodies due to their favorable features such as easy labeling, temperature tolerance, lower cost and recognition of a wide variety of targets. In this study, an aptamer-based PCR method coupled with magnetic immunoseparation was developed to detect S. Typhimurium from ground turkey. Firstly, biotinylated polyclonal anti-S...
June 20, 2017: Analytical Biochemistry
https://www.readbyqxmd.com/read/28645580/chemostat-studies-of-bacteriophage-m13-infected-escherichia-coli-jm109-for-continuous-ssdna-production
#2
Benjamin Kick, Karl Lorenz Behler, Timm Steffen Severin, Dirk Weuster-Botz
Steady state studies in a chemostat enable the control of microbial growth rate at defined reaction conditions. The effects of bacteriophage M13 infection on maximum growth rate of Escherichia coli JM109 were studied in parallel operated chemostats on a milliliter-scale to analyze the steady state kinetics of phage production. The bacteriophage infection led to a decrease in maximum specific growth rate of 15 % from 0.74 h(-1) to 0.63 h(-1). Under steady state conditions, a constant cell specific ssDNA formation rate of 0...
June 20, 2017: Journal of Biotechnology
https://www.readbyqxmd.com/read/28645379/dna-fiber-analysis-mind-the-gap
#3
Annabel Quinet, Denisse Carvajal-Maldonado, Delphine Lemacon, Alessandro Vindigni
Understanding the mechanisms of replication stress response following genotoxic stress induction is rapidly emerging as a central theme in cell survival and human disease. The DNA fiber assay is one of the most powerful tools to study alterations in replication fork dynamics genome-wide at single-molecule resolution. This approach relies on the ability of many organisms to incorporate thymidine analogs into replicating DNA and is widely used to study how genotoxic agents perturb DNA replication. Here, we review different approaches available to prepare DNA fibers and discuss important limitations of each approach...
2017: Methods in Enzymology
https://www.readbyqxmd.com/read/28641126/dna-requirements-for-interaction-of-the-c-terminal-region-of-ku80-with-the-dna-dependent-protein-kinase-catalytic-subunit-dna-pkcs
#4
Sarvan Kumar Radhakrishnan, Susan P Lees-Miller
Non-homologous end joining (NHEJ) is the major pathway for the repair of ionizing radiation induced DNA double strand breaks (DSBs) in human cells. Critical to NHEJ is the DNA-dependent interaction of the Ku70/80 heterodimer with the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) to form the DNA-PK holoenzyme. However, precisely how Ku recruits DNA-PKcs to DSBs ends to enhance its kinase activity has remained enigmatic, with contradictory findings reported in the literature. Here we address the role of the Ku80 C-terminal region (CTR) in the DNA-dependent interaction of Ku70/80 with DNA-PKcs using purified components and defined DNA structures...
June 9, 2017: DNA Repair
https://www.readbyqxmd.com/read/28636320/dna-loading-and-release-using-custom-tailored-poly-l-lysine-surfaces
#5
Amin Shakiba, Sagar L Patil, Oussama Zenasni, Monika E Schmitt, Preethi H Gunaratne, T Randall Lee
This manuscript describes the generation and study of surfaces modified with custom-crafted poly(L-lysine) (PLL) coatings for use in the loading and delivery of single-stranded DNA (ssDNA). The experimental strategy utilizes bidentate dithiol adsorbates to generate stably-bound azide-terminated self-assembled monolayers (SAMs) on gold possessing an oligo(ethylene glycol) (OEG) spacer. Consequent to the molecular assembly on gold, the azide termini are covalently attached to a maleimide linker moiety via a copper-catalyzed azide-alkyne "click" reaction...
June 21, 2017: ACS Applied Materials & Interfaces
https://www.readbyqxmd.com/read/28634758/a-general-double-library-selex-strategy-for-aptamer-selection-using-unmodified-nonimmobilized-targets
#6
Kyung Hyun Lee, Huaqiang Zeng
Aptamer discovery for unmodified nonimmobilized targets has been constantly presenting itself as a significant challenge to the research community. We demonstrate here a novel double library (DL) SELEX strategy and its usefulness and generality toward discovering both ssDNA- and RNA-based aptamers with nanomolar binding affinities toward unmodified targets of both small (e.g., doxycycline) and large (e.g., VEGF165) sizes. The same selection strategy further allows for concurrent selection of an aptamer pair, recognizing discrete epitopes on the same protein, from the same selection cycles for the sandwich aptamer pair-based biosensor development (e...
June 20, 2017: Analytical and Bioanalytical Chemistry
https://www.readbyqxmd.com/read/28634398/mutations-in-human-aid-differentially-affect-its-ability-to-deaminate-cytidine-and-5-methylcytidine-in-ssdna-substrates-in-vitro
#7
Lucyna Budzko, Paulina Jackowiak, Karol Kamel, Joanna Sarzynska, Janusz M Bujnicki, Marek Figlerowicz
Activation-induced cytidine deaminase (AID) is known for its established role in antibody production. AID induces the diversification of antibodies by deaminating deoxycytidine (C) within immunoglobulin genes. The capacity of AID to deaminate 5-methyldeoxycytidine (5 mC) and/or 5-hydroxymethyldeoxycytidine (5 hmC), and consequently AID involvement in active DNA demethylation, is not fully resolved. For instance, structural determinants of AID activity on different substrates remain to be identified. To better understand the latter issue, we tested how mutations in human AID (hAID) influence its ability to deaminate C, 5 mC, and 5 hmC in vitro...
June 20, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28625157/purification-of-single-stranded-dna-by-co-polymerization-with-acrylamide-and-electrophoresis
#8
Tulsi Ram Damase, Andrew D Ellington, Peter B Allen
Single-stranded DNA (ssDNA) oligonucleotides are useful as aptamers, hybridization probes and for emerging applications in DNA nanotechnology. Current methods to purify ssDNA require both a strand-separation step and a separate size-separation step but may still leave double-stranded DNA (dsDNA) impurities in the sample. Here, we use commercially available acrydite DNA primers to immobilize one strand of a PCR product within a polyacrylamide matrix. Electrophoresis moves the non-crosslinked DNA into the gel where the single-stranded product of desired size can be recovered...
June 1, 2017: BioTechniques
https://www.readbyqxmd.com/read/28623815/colorimetric-sensor-array-for-protein-discrimination-based-on-different-dna-chain-length-dependent-gold-nanoparticles-aggregation
#9
Xiangcong Wei, Yuxian Wang, Yingxin Zhao, Zhengbo Chen
A facile colorimetric sensor array for detection of proteins was demonstrated using DNA as nonspecific receptors. We found that different proteins could trigger the DNA-protected gold nanoparticles (AuNPs) to show different aggregation behaviors in the presence of salt with high concentrations along with various color changes. As a proof-of-concept application, a multi-protein discrimination array was fabricated with two ssDNA strands (15A and 30A bases) as a receptor array. The combinatorial colorimetric response of this sensor array can be analyzed by linear discriminant analysis (LDA) and hierarchical cluster analysis (HCA)...
June 12, 2017: Biosensors & Bioelectronics
https://www.readbyqxmd.com/read/28618091/bacterial-transformation-comfa-is-a-dna-dependent-atpase-that-forms-complexes-with-comfc-and-dpra
#10
Amy Diallo, Hannah R Foster, Katarzyna A Gromek, Thomas N Perry, Annick Dujeancourt, Petya V Krasteva, Francesca Gubellini, Tanya G Falbel, Briana M Burton, Rémi Fronzes
Pneumococcal natural transformation contributes to genomic plasticity, antibiotic resistance development, and vaccine escape. Streptococcus pneumoniae, like many other naturally transformable species, has evolved sophisticated protein machinery for the binding and uptake of DNA. Two proteins encoded by the comF operon, ComFA and ComFC, are involved in transformation but their exact molecular roles remain unknown. In this study, we provide experimental evidence that ComFA binds to single stranded DNA (ssDNA) and has ssDNA-dependent ATPase activity...
June 15, 2017: Molecular Microbiology
https://www.readbyqxmd.com/read/28615444/the-human-mitochondrial-single-strand-dna-binding-protein-displays-distinct-kinetics-and-thermodynamics-of-dna-binding-and-exchange
#11
Yufeng Qian, Kenneth A Johnson
The human mitochondrial SSB (mtSSB) is a homo-tetrameric protein, involved in mtDNA replication and maintenance. Although mtSSB is structurally similar to SSB from Escherichia coli (EcoSSB), it lacks the C-terminal disordered domain but little is known about the biophysics of mtSSB-ssDNA interactions. Here, we characterized the kinetics and thermodynamics of mtSSB binding to ssDNA by equilibrium titrations and stopped-flow kinetic measurements. We show that the mtSSB tetramer can bind to ssDNA in two distinct binding modes: (SSB)30 and (SSB)60, defined by DNA binding site sizes of 30 and 60 nt, respectively...
June 14, 2017: Journal of Biological Chemistry
https://www.readbyqxmd.com/read/28609559/strategies-to-optimize-capsid-protein-expression-and-single-stranded-dna-formation-of-adeno-associated-virus-in-saccharomyces-cerevisiae
#12
Alvaro Galli, Veronica Della Latta, Caterina Bologna, Daniela Pucciarelli, Filippo Cipriani, Ana Backovic, Tiziana Cervelli
AIMS: Adeno-associated virus type 2 (AAV) is a non-pathogenic parvovirus that is a promising tool for gene therapy. We aim to construct plasmids for optimal expression and assembly of capsid proteins, and evaluate Adenovirus (Ad) protein effect on AAV single stranded DNA (ssDNA) formation in Saccharomyces cerevisiae. METHODS AND RESULTS: Yeast expression plasmids have been developed in which the transcription of AAV capsid proteins (VP1,2,3) is driven by the constitutive ADH1 promoter or galactose inducible promoters...
June 13, 2017: Journal of Applied Microbiology
https://www.readbyqxmd.com/read/28607004/rpa-activates-the-xpf-ercc1-endonuclease-to-initiate-processing-of-dna-interstrand-crosslinks
#13
Ummi B Abdullah, Joanna F McGouran, Sanja Brolih, Denis Ptchelkine, Afaf H El-Sagheer, Tom Brown, Peter J McHugh
During replication-coupled DNA interstrand crosslink (ICL) repair, the XPF-ERCC1 endonuclease is required for the incisions that release, or "unhook", ICLs, but the mechanism of ICL unhooking remains largely unknown. Incisions are triggered when the nascent leading strand of a replication fork strikes the ICL Here, we report that while purified XPF-ERCC1 incises simple ICL-containing model replication fork structures, the presence of a nascent leading strand, modelling the effects of replication arrest, inhibits this activity...
June 12, 2017: EMBO Journal
https://www.readbyqxmd.com/read/28606086/analysis-and-prediction-of-single-stranded-and-double-stranded-dna-binding-proteins-based-on-protein-sequences
#14
Wei Wang, Lin Sun, Shiguang Zhang, Hongjun Zhang, Jinling Shi, Tianhe Xu, Keliang Li
BACKGROUND: DNA-binding proteins perform important functions in a great number of biological activities. DNA-binding proteins can interact with ssDNA (single-stranded DNA) or dsDNA (double-stranded DNA), and DNA-binding proteins can be categorized as single-stranded DNA-binding proteins (SSBs) and double-stranded DNA-binding proteins (DSBs). The identification of DNA-binding proteins from amino acid sequences can help to annotate protein functions and understand the binding specificity...
June 12, 2017: BMC Bioinformatics
https://www.readbyqxmd.com/read/28605966/molecular-detection-and-genome-analysis-of-circoviruses-of-european-eel-anguilla-anguilla-and-sichel-pelecus-cultratus
#15
Réka Borzák, Boglárka Sellyei, Csaba Székely, Andor Doszpoly
The prevalence and distribution of piscine circoviruses (CVs) were tested in a routine virus monitoring programme in Lake Balaton, Hungary. A high prevalence of European eel CV (EeCV) was found in the apparently healthy eel population (35.5%). The copy number of the viral DNA in different organs was determined by quantitative real-time PCR. The results suggested that some eel specimens were in active viraemic status despite their asymptomatic condition. Furthermore, a novel, previously undescribed CV was also detected in eel and sichel samples...
June 2017: Acta Veterinaria Hungarica
https://www.readbyqxmd.com/read/28605670/ssb-recruitment-of-exonuclease-i-aborts-template-switching-in-escherichia-coli
#16
Laura T Laranjo, Stephen J Gross, Danna M Zeiger, Susan T Lovett
Misalignment of a nascent strand and the use of an alternative template during DNA replication, a process termed "template-switching", can give rise to frequent mutations and genetic rearrangements. Mutational hotspots are frequently found associated with imperfect inverted repeats ("quasipalindromes" or "QPs") in many organisms, including bacteriophage, bacteria, yeast and mammals. Evidence suggests that QPs mutate by a replication template-switch whereby one copy of the inverted repeat templates synthesis of the other...
June 3, 2017: DNA Repair
https://www.readbyqxmd.com/read/28604877/understanding-and-improving-aggregated-gold-nanoparticle-dsdna-interactions-by-molecular-spectroscopy-and-deconvolution-methods
#17
Jose M Carnerero, Aila Jimenez-Ruiz, Elia M Grueso, Rafael Prado-Gotor
It is well known that single-stranded DNA (ssDNA) is easily able to adsorb on citrate-capped, non-functionalized gold nanoparticles (AuNPs). However, the affinity of double-stranded DNA (dsDNA) for them is much more limited. The present work demonstrates that long dsDNA suffers from a bending conformational change when anionic nanoparticles are present in solution. A striking decrease in the persistence length of the double helix in the absence of salt is observed through dynamic light scattering (DLS), viscometric, and atomic force microscopy (AFM) methods...
June 21, 2017: Physical Chemistry Chemical Physics: PCCP
https://www.readbyqxmd.com/read/28604711/the-anaphase-promoting-complex-impacts-repair-choice-by-protecting-ubiquitin-signalling-at-dna-damage-sites
#18
Kyungsoo Ha, Chengxian Ma, Han Lin, Lichun Tang, Zhusheng Lian, Fang Zhao, Ju-Mei Li, Bei Zhen, Huadong Pei, Suxia Han, Marcos Malumbres, Jianping Jin, Huan Chen, Yongxiang Zhao, Qing Zhu, Pumin Zhang
Double-strand breaks (DSBs) are repaired through two major pathways, homology-directed recombination (HDR) and non-homologous end joining (NHEJ). While HDR can only occur in S/G2, NHEJ can happen in all cell cycle phases (except mitosis). How then is the repair choice made in S/G2 cells? Here we provide evidence demonstrating that APC(Cdh1) plays a critical role in choosing the repair pathways in S/G2 cells. Our results suggest that the default for all DSBs is to recruit 53BP1 and RIF1. BRCA1 is blocked from being recruited to broken ends because its recruitment signal, K63-linked poly-ubiquitin chains on histones, is actively destroyed by the deubiquitinating enzyme USP1...
June 12, 2017: Nature Communications
https://www.readbyqxmd.com/read/28602639/rad52-inverse-strand-exchange-drives-rna-templated-dna-double-strand-break-repair
#19
Olga M Mazina, Havva Keskin, Kritika Hanamshet, Francesca Storici, Alexander V Mazin
RNA can serve as a template for DNA double-strand break repair in yeast cells, and Rad52, a member of the homologous recombination pathway, emerged as an important player in this process. However, the exact mechanism of how Rad52 contributes to RNA-dependent DSB repair remained unknown. Here, we report an unanticipated activity of yeast and human Rad52: inverse strand exchange, in which Rad52 forms a complex with dsDNA and promotes strand exchange with homologous ssRNA or ssDNA. We show that in eukaryotes, inverse strand exchange between homologous dsDNA and RNA is a distinctive activity of Rad52; neither Rad51 recombinase nor the yeast Rad52 paralog Rad59 has this activity...
June 5, 2017: Molecular Cell
https://www.readbyqxmd.com/read/28601788/carbon-nanostructures-as-immobilization-platform-for-dna-a-review-on-current-progress-in-electrochemical-dna-sensors
#20
REVIEW
P Abdul Rasheed, N Sandhyarani
Development of a sensitive, specific and cost-effective DNA detection method is motivated by increasing demand for the early stage diagnosis of genetic diseases. Recent developments in the design and fabrication of efficient sensor platforms based on nanostructures make the highly sensitive sensors which could indicate very low detection limit to the level of few molecules, a realistic possibility. Electrochemical detection methods are widely used in DNA diagnostics as it provide simple, accurate and inexpensive platform for DNA detection...
June 8, 2017: Biosensors & Bioelectronics
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