Charge transfer through DNA/DNA duplexes and DNA/RNA hybrids: complex theoretical and experimental studies.

Science.gov (United States)

Kratochvílová, Irena; Vala, Martin; Weiter, Martin; Špérová, Miroslava; Schneider, Bohdan; Páv, Ondřej; Šebera, Jakub; Rosenberg, Ivan; Sychrovský, Vladimír

2013-01-01

Oligonucleotides conduct electric charge via various mechanisms and their characterization and understanding is a very important and complicated task. In this work, experimental (temperature dependent steady state fluorescence spectroscopy, time-resolved fluorescence spectroscopy) and theoretical (Density Functional Theory) approaches were combined to study charge transfer processes in short DNA/DNA and RNA/DNA duplexes with virtually equivalent sequences. The experimental results were consistent with the theoretical model - the delocalized nature of HOMO orbitals and holes, base stacking, electronic coupling and conformational flexibility formed the conditions for more effective short distance charge transfer processes in RNA/DNA hybrids. RNA/DNA and DNA/DNA charge transfer properties were strongly connected with temperature affected structural changes of molecular systems - charge transfer could be used as a probe of even tiny changes of molecular structures and settings. © 2013. Published by Elsevier B.V. All rights reserved.

Hybridization change of DNA and nuclear RNA synthesized immediately after ionizing irradiation in spleen cells isolated from 615 mice

International Nuclear Information System (INIS)

Meng Ziqiang

1986-01-01

DNA hybridization with nuclear RNA(nRNA) synthesized immediately after 60 Co Gamma-irradiation in the spleen cells freshly isolated from inbred line 615 mice was investigated, using the technique of Gillespie and Spiegelman. In RNA/DNA hybridization percentage experiment, it was showed that the hybridization of normal DNA with labelled nRNA synthesized in irradiated cells reached the saturation point at a much faster rate than with labelled normal nRNA. The hybridization percentage of nRNA synthesized in irradiated cells was higher than that of normal nRNA during the different reaction time before the saturation point of DNA with nRNA synthesized in irradiated cells, but it was lower than that of normal nRNA after the zone of high repetitive DNA sequences was stimulated, however, the transcription of some base sequences in the zone of low repetitive DNA sequences was seriously inhibited. Measurements of the exhaustion rates of pulse-labelled nRNA were carried out as described by Greene and Flickinger Biochim. In these studies, pulse-labelled nRNA synthesized in unirradiated and irradiated cells were compared by exhausion with DNA at hybridization time of 4 or 24 hours, When the hybridization time was 4 hours, the nRNA synthesized in irradiated cells displayed a faster exhaustion rate than the control nRNA. But if the hybridization time was 24 hours, the exhaustion rate of nRNA synthesized in irradiated cells reduced. These results demostrated that Gamma-irradiation changed the proportion of transcription of some nRNA species and implayed that the sensitivities of the transcription activeties of the different repetitive DNA sequences to Gamma-irradiation were different, and so were the transcription activeties of the different base sequences in the same repetitive DNA sequences

RNA/DNA Hybrid Interactome Identifies DXH9 as a Molecular Player in Transcriptional Termination and R-Loop-Associated DNA Damage.

Science.gov (United States)

Cristini, Agnese; Groh, Matthias; Kristiansen, Maiken S; Gromak, Natalia

2018-05-08

R-loops comprise an RNA/DNA hybrid and displaced single-stranded DNA. They play important biological roles and are implicated in pathology. Even so, proteins recognizing these structures are largely undefined. Using affinity purification with the S9.6 antibody coupled to mass spectrometry, we defined the RNA/DNA hybrid interactome in HeLa cells. This consists of known R-loop-associated factors SRSF1, FACT, and Top1, and yet uncharacterized interactors, including helicases, RNA processing, DNA repair, and chromatin factors. We validate specific examples of these interactors and characterize their involvement in R-loop biology. A top candidate DHX9 helicase promotes R-loop suppression and transcriptional termination. DHX9 interacts with PARP1, and both proteins prevent R-loop-associated DNA damage. DHX9 and other interactome helicases are overexpressed in cancer, linking R-loop-mediated DNA damage and disease. Our RNA/DNA hybrid interactome provides a powerful resource to study R-loop biology in health and disease. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

Fluorescent in situ hybridization of mitochondrial DNA and RNA

Czech Academy of Sciences Publication Activity Database

Alán, Lukáš; Zelenka, Jaroslav; Ježek, Jan; Dlasková, Andrea; Ježek, Petr

2010-01-01

Roč. 57, č. 4 (2010), s. 403-408 ISSN 0001-527X R&D Projects: GA ČR GAP302/10/0346; GA ČR GPP304/10/P204; GA AV ČR KJB500110902 Institutional research plan: CEZ:AV0Z50110509 Keywords : mitochondrial DNA and RNA * nucleoids of mitochondrial DNA * molecular beacon fluorescent hybridization probes Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.234, year: 2010

Atomistic details of the molecular recognition of DNA-RNA hybrid ...

Indian Academy of Sciences (India)

conformations corresponding to typical A- and B-type nucleic acids and the .... protein chains and five base pairs in DNA-RNA hybrid ... employed to treat the long range electrostatic interac- .... The solvent accessible surface areas (SASA) of.

Guanine is indispensable for immunoglobulin switch region RNA-DNA hybrid formation

International Nuclear Information System (INIS)

Mizuta, Ryushin; Mizuta, Midori; Kitamura, Daisuke

2005-01-01

It is suggested that the formation of the switch (S) region RNA-DNA hybrid and the subsequent generation of higher-order chromatin structures including R-loop initiate a class switch recombination of the immunoglobulin gene. The primary factor of this recombination is the S-region derived noncoding RNA. However, the biochemical character of this guanine-rich (G-rich) transcript is poorly understood. The present study was performed to analyze the structure of this G-rich RNA using atomic force microscope (AFM). The in vitro transcribed S-region RNA was spread on a mica plate, air-dried and observed by non-contact mode AFM in air. The G-rich transcripts tend to aggregate on the template DNA and to generate a higher-order RNA-DNA complex. However, the transcripts that incorporated guanine analogues as substitutes for guanine neither aggregated nor generated higher-order structures. Incorporation of guanine analogues in transcribes RNA partially disrupts hydrogen bonds related to guanine, such as Watson-Crick GC-base pair and Hoogsteen bond GG-base pair. Thus, aggregation of S-region RNA and generation of the higher-order RNA-DNA complex are attributed to hydrogen bonds of guanine. (author)

Mechanism and manipulation of DNA:RNA hybrid G-quadruplex formation in transcription of G-rich DNA.

Science.gov (United States)

Zhang, Jia-yu; Zheng, Ke-wei; Xiao, Shan; Hao, Yu-hua; Tan, Zheng

2014-01-29

We recently reported that a DNA:RNA hybrid G-quadruplex (HQ) forms during transcription of DNA that bears two or more tandem guanine tracts (G-tract) on the nontemplate strand. Putative HQ-forming sequences are enriched in the nearby 1000 nt region right downstream of transcription start sites in the nontemplate strand of warm-blooded animals, and HQ regulates transcription under both in vitro and in vivo conditions. Therefore, knowledge of the mechanism of HQ formation is important for understanding the biological function of HQ as well as for manipulating gene expression by targeting HQ. In this work, we studied the mechanism of HQ formation using an in vitro T7 transcription model. We show that RNA synthesis initially produces an R-loop, a DNA:RNA heteroduplex formed by a nascent RNA transcript and the template DNA strand. In the following round of transcription, the RNA in the R-loop is displaced, releasing the RNA in single-stranded form (ssRNA). Then the G-tracts in the RNA can jointly form HQ with those in the nontemplate DNA strand. We demonstrate that the structural cascade R-loop → ssRNA → HQ offers opportunities to intercept HQ formation, which may provide a potential method to manipulate gene expression.

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

Science.gov (United States)

Ares, Manuel

2014-01-01

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

Recognition of RNA by amide modified backbone nucleic acids: molecular dynamics simulations of DNA-RNA hybrids in aqueous solution.

Science.gov (United States)

Nina, Mafalda; Fonné-Pfister, Raymonde; Beaudegnies, Renaud; Chekatt, Habiba; Jung, Pierre M J; Murphy-Kessabi, Fiona; De Mesmaeker, Alain; Wendeborn, Sebastian

2005-04-27

Thermodynamic and structural properties of a chemically modified DNA-RNA hybrid in which a phosphodiester linkage is replaced by a neutral amide-3 linkage (3'-CH(2)-CONH-5') were investigated using UV melting experiments, molecular dynamics simulations in explicit water, and continuum solvent models. van't Hoff analysis of the experimental UV melting curves suggests that the significant increase of the thermodynamic stability of a 15-mer DNA-RNA with seven alternated amide-3 modifications (+11 degrees C) is mainly due to an increased binding enthalpy. To further evaluate the origin in the observed affinities differences, the electrostatic contribution to the binding free energy was calculated by solving the Poisson-Boltzmann equation numerically. The nonelectrostatic contribution was estimated as the product of a hydrophobic surface tension coefficient and the surface area that is buried upon double strand formation. Structures were taken from 10 ns molecular dynamics simulations computed in a consistent fashion using explicit solvent, counterions, and the particle-mesh Ewald procedure. The present preliminary thermodynamic study suggests that the favorable binding free energy of the amide-3 DNA single strand to the complementary RNA is equally driven by electrostatic and nonpolar contributions to the binding compared to their natural analogues. In addition, molecular dynamics simulations in explicit water were performed on an amide-3 DNA single strand and the corresponding natural DNA. Results from the conformations cluster analysis of the simulated amide-3 DNA single strand ensembles suggest that the 25% of the population sampled within 10 ns has a pre-organized conformation where the sugar C3' endo pucker is favored at the 3'-flanking nucleotides. These structural and thermodynamic features contribute to the understanding of the observed increased affinities of the amide-3 DNA-RNA hybrids at the microscopic level.

Inclusion of methoxy groups inverts the thermodynamic stabilities of DNA-RNA hybrid duplexes: A molecular dynamics simulation study.

Science.gov (United States)

Suresh, Gorle; Priyakumar, U Deva

2015-09-01

Modified nucleic acids have found profound applications in nucleic acid based technologies such as antisense and antiviral therapies. Previous studies on chemically modified nucleic acids have suggested that modifications incorporated in furanose sugar especially at 2'-position attribute special properties to nucleic acids when compared to other modifications. 2'-O-methyl modification to deoxyribose sugars of DNA-RNA hybrids is one such modification that increases nucleic acid stability and has become an attractive class of compounds for potential antisense applications. It has been reported that modification of DNA strands with 2'-O-methyl group reverses the thermodynamic stability of DNA-RNA hybrid duplexes. Molecular dynamics simulations have been performed on two hybrid duplexes (DR and RD) which differ from each other and 2'-O-methyl modified counterparts to investigate the effect of 2'-O-methyl modification on their duplex stability. The results obtained suggest that the modification drives the conformations of both the hybrid duplexes towards A-RNA like conformation. The modified hybrid duplexes exhibit significantly contrasting dynamics and hydration patterns compared to respective parent duplexes. In line with the experimental results, the relative binding free energies suggest that the introduced modifications stabilize the less stable DR hybrid, but destabilize the more stable RD duplex. Binding free energy calculations suggest that the increased hydrophobicity is primarily responsible for the reversal of thermodynamic stability of hybrid duplexes. Free energy component analysis further provides insights into the stability of modified duplexes. Copyright © 2015 Elsevier Inc. All rights reserved.

Programmed self-assembly of DNA/RNA for biomedical applications

Science.gov (United States)

Wang, Pengfei

Three self-assembly strategies were utilized for assembly of novel functional DNA/RNA nanostructures. RNA-DNA hybrid origami method was developed to fabricate nano-objects (ribbon, rectangle, and triangle) with precisely controlled geometry. Unlike conventional DNA origami which use long DNA single strand as scaffold, a long RNA single strand was used instead, which was folded by short DNA single strands (staples) into prescribed objects through sequence specific hybridization between RNA and DNA. Single stranded tiles (SST) and RNA-DNA hybrid origami were utilized to fabricate a variety of barcode-like nanostructures with unique patterns by expanding a plain rectangle via introducing spacers (10-bp dsDNA segment) between parallel duplexes. Finally, complex 2D array and 3D polyhedrons with multiple patterns within one structure were assembled from simple DNA motifs. Two demonstrations of biomedical applications of DNA nanotechnology were presented. Firstly, lambda-DNA was used as template to direct the fabrication of multi-component magnetic nanoparticle chains. Nuclear magnetic relaxation (NMR) characterization showed superb magnetic relaxativity of the nanoparticle chains which have large potential to be utilized as MRI contrast agents. Secondly, DNA nanotechnology was introduced into the conformational study of a routinely used catalytic DNAzyme, the RNA-cleaving 10-23 DNAzyme. The relative angle between two flanking duplexes of the catalytic core was determined (94.8°), which shall be able to provide a clue to further understanding of the cleaving mechanism of this DNAzyme from a conformational perspective.

Enzymatic Amplification of DNA/RNA Hybrid Molecular Beacon Signaling in Nucleic Acid Detection

OpenAIRE

Jacroux, Thomas; Rieck, Daniel C.; Cui, Rong; Ouyang, Yexin; Dong, Wen-Ji

2012-01-01

A rapid assay operable under isothermal or non-isothermal conditions is described wherein the sensitivity of a typical molecular beacon (MB) system is improved by utilizing thermostable RNase H to enzymatically cleave an MB comprised of a DNA stem and RNA loop (R/D-MB). Upon hybridization of the R/D-MB to target DNA, there was a modest increase in fluorescence intensity (~5.7x above background) due to an opening of the probe and concomitant reduction in the Förster resonance energy transfer e...

Import of desired nucleic acid sequences using addressing motif of mitochondrial ribosomal 5S-rRNA for fluorescent in vivo hybridization of mitochondrial DNA and RNA.

Science.gov (United States)

Zelenka, Jaroslav; Alán, Lukáš; Jabůrek, Martin; Ježek, Petr

2014-04-01

Based on the matrix-addressing sequence of mitochondrial ribosomal 5S-rRNA (termed MAM), which is naturally imported into mitochondria, we have constructed an import system for in vivo targeting of mitochondrial DNA (mtDNA) or mt-mRNA, in order to provide fluorescence hybridization of the desired sequences. Thus DNA oligonucleotides were constructed, containing the 5'-flanked T7 RNA polymerase promoter. After in vitro transcription and fluorescent labeling with Alexa Fluor(®) 488 or 647 dye, we obtained the fluorescent "L-ND5 probe" containing MAM and exemplar cargo, i.e., annealing sequence to a short portion of ND5 mRNA and to the light-strand mtDNA complementary to the heavy strand nd5 mt gene (5'-end 21 base pair sequence). For mitochondrial in vivo fluorescent hybridization, HepG2 cells were treated with dequalinium micelles, containing the fluorescent probes, bringing the probes proximally to the mitochondrial outer membrane and to the natural import system. A verification of import into the mitochondrial matrix of cultured HepG2 cells was provided by confocal microscopy colocalizations. Transfections using lipofectamine or probes without 5S-rRNA addressing MAM sequence or with MAM only were ineffective. Alternatively, the same DNA oligonucleotides with 5'-CACC overhang (substituting T7 promoter) were transcribed from the tetracycline-inducible pENTRH1/TO vector in human embryonic kidney T-REx®-293 cells, while mitochondrial matrix localization after import of the resulting unlabeled RNA was detected by PCR. The MAM-containing probe was then enriched by three-order of magnitude over the natural ND5 mRNA in the mitochondrial matrix. In conclusion, we present a proof-of-principle for mitochondrial in vivo hybridization and mitochondrial nucleic acid import.

Mycobacterium smegmatis Lhr Is a DNA-dependent ATPase and a 3'-to-5' DNA translocase and helicase that prefers to unwind 3'-tailed RNA:DNA hybrids.

Science.gov (United States)

Ordonez, Heather; Shuman, Stewart

2013-05-17

We are interested in the distinctive roster of helicases of Mycobacterium, a genus of the phylum Actinobacteria that includes the human pathogen Mycobacterium tuberculosis and its avirulent relative Mycobacterium smegmatis. Here, we identify and characterize M. smegmatis Lhr as the exemplar of a novel clade of superfamily II helicases, by virtue of its biochemical specificities and signature domain organization. Lhr is a 1507-amino acid monomeric nucleic acid-dependent ATPase that uses the energy of ATP hydrolysis to drive unidirectional 3'-to-5' translocation along single strand DNA and to unwind duplexes en route. The ATPase is more active in the presence of calcium than magnesium. ATP hydrolysis is triggered by either single strand DNA or single strand RNA, yet the apparent affinity for a DNA activator is 11-fold higher than for an RNA strand of identical size and nucleobase sequence. Lhr is 8-fold better at unwinding an RNA:DNA hybrid than it is at displacing a DNA:DNA duplex of identical nucleobase sequence. The truncated derivative Lhr-(1-856) is an autonomous ATPase, 3'-to-5' translocase, and RNA:DNA helicase. Lhr-(1-856) is 100-fold better RNA:DNA helicase than DNA:DNA helicase. Lhr homologs are found in bacteria representing eight different phyla, being especially prevalent in Actinobacteria (including M. tuberculosis) and Proteobacteria (including Escherichia coli).

Phosphate-methylated DNA aimed at HIV-1 RNA loops and integrated DNA inhibits viral infectivity

NARCIS (Netherlands)

Buck, H. M.; Koole, L. H.; van Genderen, M. H.; Smit, L.; Geelen, J. L.; Jurriaans, S.; Goudsmit, J.

1990-01-01

Phosphate-methylated DNA hybridizes strongly and specifically to natural DNA and RNA. Hybridization to single-stranded and double-stranded DNA leads to site-selective blocking of replication and transcription. Phosphate-methylated DNA was used to interrupt the life cycle of the human

Efficient DNA ligation in DNA–RNA hybrid helices by Chlorella virus DNA ligase

Science.gov (United States)

Lohman, Gregory J. S.; Zhang, Yinhua; Zhelkovsky, Alexander M.; Cantor, Eric J.; Evans, Thomas C.

2014-01-01

Single-stranded DNA molecules (ssDNA) annealed to an RNA splint are notoriously poor substrates for DNA ligases. Herein we report the unexpectedly efficient ligation of RNA-splinted DNA by Chlorella virus DNA ligase (PBCV-1 DNA ligase). PBCV-1 DNA ligase ligated ssDNA splinted by RNA with kcat ≈ 8 x 10−3 s−1 and KM DNA ligase produced only 5′-adenylylated DNA with a 20-fold lower kcat and a KM ≈ 300 nM. The rate of ligation increased with addition of Mn2+, but was strongly inhibited by concentrations of NaCl >100 mM. Abortive adenylylation was suppressed at low ATP concentrations (8, leading to increased product yields. The ligation reaction was rapid for a broad range of substrate sequences, but was relatively slower for substrates with a 5′-phosphorylated dC or dG residue on the 3′ side of the ligation junction. Nevertheless, PBCV-1 DNA ligase ligated all sequences tested with 10-fold less enzyme and 15-fold shorter incubation times than required when using T4 DNA ligase. Furthermore, this ligase was used in a ligation-based detection assay system to show increased sensitivity over T4 DNA ligase in the specific detection of a target mRNA. PMID:24203707

Detection of Hepatitis B virus DNA and Hepatitis δ virus RNA

International Nuclear Information System (INIS)

Smedile, A.; Chiaberge, E.; Brunetto, M.R.; Negro, F.; Baldi, M.; Lavarini, C.; Maran, E.

1987-01-01

The recent availability of DNA probes of the Hepatitis B Virus DNA (HBV-DNA) and of Hepatitis Delta Virus RNA (HDV-RNA) allows the application of nucleic acid hybridization techniques to solve a variety of clinical problems. DNA probes of HBV-DNA and HDV-RNA are labeled by nick translation using 32 P or biotinylated nucleotides and hybridized to filters containing test nucleic acids. Complementary sequences are identified and the degree of blackening of the film at autoradiography or the enzymatic staining of the filter is proportional to the amount of viral nucleic acid hybridized to the probe and present in the sample. These procedures allow rapid examination of multiple specimens and are sensitive and reproducible. Viral nucleic acids can be measured quantitatively and their quantity correlates with the infectivity of sera titered in experimentally infected animals

A novel rat genomic simple repeat DNA with RNA-homology shows triplex (H-DNA)-like structure and tissue-specific RNA expression

International Nuclear Information System (INIS)

Dey, Indranil; Rath, Pramod C.

2005-01-01

Mammalian genome contains a wide variety of repetitive DNA sequences of relatively unknown function. We report a novel 227 bp simple repeat DNA (3.3 DNA) with a d {(GA) 7 A (AG) 7 } dinucleotide mirror repeat from the rat (Rattus norvegicus) genome. 3.3 DNA showed 75-85% homology with several eukaryotic mRNAs due to (GA/CU) n dinucleotide repeats by nBlast search and a dispersed distribution in the rat genome by Southern blot hybridization with [ 32 P]3.3 DNA. The d {(GA) 7 A (AG) 7 } mirror repeat formed a triplex (H-DNA)-like structure in vitro. Two large RNAs of 9.1 and 7.5 kb were detected by [ 32 P]3.3 DNA in rat brain by Northern blot hybridization indicating expression of such simple sequence repeats at RNA level in vivo. Further, several cDNAs were isolated from a rat cDNA library by [ 32 P]3.3 DNA probe. Three such cDNAs showed tissue-specific RNA expression in rat. pRT 4.1 cDNA showed strong expression of a 2.39 kb RNA in brain and spleen, pRT 5.5 cDNA showed strong expression of a 2.8 kb RNA in brain and a 3.9 kb RNA in lungs, and pRT 11.4 cDNA showed weak expression of a 2.4 kb RNA in lungs. Thus, genomic simple sequence repeats containing d (GA/CT) n dinucleotides are transcriptionally expressed and regulated in rat tissues. Such d (GA/CT) n dinucleotide repeats may form structural elements (e.g., triplex) which may be sites for functional regulation of genomic coding sequences as well as RNAs. This may be a general function of such transcriptionally active simple sequence repeats widely dispersed in mammalian genome

Dramatically improved RNA in situ hybridization signals using LNA-modified probes

DEFF Research Database (Denmark)

Thomsen, Rune; Nielsen, Peter Stein; Jensen, Torben Heick

2005-01-01

. This increases the thermal stability of hybrids formed with RNA. The LNA-based probes detect specific RNAs in fixed yeast cells with an efficiency far better than conventional DNA oligonucleotide probes of the same sequence. Using this probe design, we were also able to detect poly(A)+ RNA accumulation within......In situ detection of RNA by hybridization with complementary probes is a powerful technique. Probe design is a critical parameter in successful target detection. We have evaluated the efficiency of fluorescent DNA oligonucleotides modified to contain locked nucleic acid (LNA) residues...

Studies on the effects of persistent RNA priming on DNA replication and genomic stability

OpenAIRE

Stuckey, Ruth

2014-01-01

[EN]: DNA replication and transcription take place on the same DNA template, and the correct interplay between these processes ensures faithful genome duplication. DNA replication must be highly coordinated with other cell cycle events, such as segregation of fully replicated DNA in order to maintain genomic integrity. Transcription generates RNA:DNA hybrids, transient intermediate structures that are degraded by the ribonuclease H (RNaseH) class of enzymes. RNA:DNA hybrids can form R-loops, ...

In situ DNA-RNA hybridization using in vitro 125I-labeled ribosomal RNA of higher plant

International Nuclear Information System (INIS)

Sato, Seiichi; Kikuchi, Tadatoshi; Ishida, M.R.; Tanaka, Ryuso.

1975-01-01

In situ hybridization using 125 I-labeled ribosomal RNA was applied to plant cells. Cytoplasmic 25 s rRNA, which was eluted from acrylamide gels after electrophoretic separation, was labeled in vitro with carrier-free 125 I and hybridized with the interphase nuclei in root tips of Vicia faba. In most of the preparations, the nucleoli were more heavily labeled than the other regions within nuclei, and several types of grain distribution were observed on the nucleoli. From these results, it was confirmed that in situ hybridization using 125 I-labeled rRNA can be used very effectively to detect the annealing sites of different molecular species of rRNA within the nuclei of plant cells, for which it is not as easy to obtain high specific radioactive rRNA in vivo as it is in the case of cultured animal cells. (auth.)

Appendix: a solution hybridization assay to detect radioactive globin messenger RNA nucleotide sequences

Energy Technology Data Exchange (ETDEWEB)

Ross, J

1976-09-15

In view of the sensitivity and specificity of the solution hybridization assay for unlabeled globin mRNA a similar technique has been devised to detect radioactive globin mRNA sequences with unlabeled globin cDNA. Several properties of the hybridization reaction are presented since RNA kinetic experiments reported recently depend on the validity of this assay. Data on hybridization analysis of (/sup 3/H)RNA from mouse fetal liver or erythroleukemia cell cytoplasm are presented. These data indicate that the excess cDNA solution assay for radioactive globin mRNA detection is specific for globin mRNA sequences. It can be performed rapidly and is highly reproducible from experiment. It is at least 500-fold less sensitive than the assay for unlabeled globin mRNA, due to the RNAase backgrounds of 0.05 to 0.15 %. However, this limitation has not affected kinetic experiments with non-dividing fetal liver erythroid cells, which synthesize relatively large quantities of globin mRNA.

Phylogenetic Analysis of Shewanella Strains by DNA Relatedness Derived from Whole Genome Microarray DNA-DNA Hybridization and Comparisons with Other Methods

International Nuclear Information System (INIS)

Wu, Liyou; Yi, T.Y.; Van Nostrand, Joy; Zhou, Jizhong

2010-01-01

Phylogenetic analyses were done for the Shewanella strains isolated from Baltic Sea (38 strains), US DOE Hanford Uranium bioremediation site (Hanford Reach of the Columbia River (HRCR), 11 strains), Pacific Ocean and Hawaiian sediments (8 strains), and strains from other resources (16 strains) with three out group strains, Rhodopseudomonas palustris, Clostridium cellulolyticum, and Thermoanaerobacter ethanolicus X514, using DNA relatedness derived from WCGA-based DNA-DNA hybridizations, sequence similarities of 16S rRNA gene and gyrB gene, and sequence similarities of 6 loci of Shewanella genome selected from a shared gene list of the Shewanella strains with whole genome sequenced based on the average nucleotide identity of them (ANI). The phylogenetic trees based on 16S rRNA and gyrB gene sequences, and DNA relatedness derived from WCGA hybridizations of the tested Shewanella strains share exactly the same sub-clusters with very few exceptions, in which the strains were basically grouped by species. However, the phylogenetic analysis based on DNA relatedness derived from WCGA hybridizations dramatically increased the differentiation resolution at species and strains level within Shewanella genus. When the tree based on DNA relatedness derived from WCGA hybridizations was compared to the tree based on the combined sequences of the selected functional genes (6 loci), we found that the resolutions of both methods are similar, but the clustering of the tree based on DNA relatedness derived from WMGA hybridizations was clearer. These results indicate that WCGA-based DNA-DNA hybridization is an idea alternative of conventional DNA-DNA hybridization methods and it is superior to the phylogenetics methods based on sequence similarities of single genes. Detailed analysis is being performed for the re-classification of the strains examined.

Phylogenetic Analysis of Shewanella Strains by DNA Relatedness Derived from Whole Genome Microarray DNA-DNA Hybridization and Comparison with Other Methods

Energy Technology Data Exchange (ETDEWEB)

Wu, Liyou; Yi, T. Y.; Van Nostrand, Joy; Zhou, Jizhong

2010-05-17

Phylogenetic analyses were done for the Shewanella strains isolated from Baltic Sea (38 strains), US DOE Hanford Uranium bioremediation site [Hanford Reach of the Columbia River (HRCR), 11 strains], Pacific Ocean and Hawaiian sediments (8 strains), and strains from other resources (16 strains) with three out group strains, Rhodopseudomonas palustris, Clostridium cellulolyticum, and Thermoanaerobacter ethanolicus X514, using DNA relatedness derived from WCGA-based DNA-DNA hybridizations, sequence similarities of 16S rRNA gene and gyrB gene, and sequence similarities of 6 loci of Shewanella genome selected from a shared gene list of the Shewanella strains with whole genome sequenced based on the average nucleotide identity of them (ANI). The phylogenetic trees based on 16S rRNA and gyrB gene sequences, and DNA relatedness derived from WCGA hybridizations of the tested Shewanella strains share exactly the same sub-clusters with very few exceptions, in which the strains were basically grouped by species. However, the phylogenetic analysis based on DNA relatedness derived from WCGA hybridizations dramatically increased the differentiation resolution at species and strains level within Shewanella genus. When the tree based on DNA relatedness derived from WCGA hybridizations was compared to the tree based on the combined sequences of the selected functional genes (6 loci), we found that the resolutions of both methods are similar, but the clustering of the tree based on DNA relatedness derived from WMGA hybridizations was clearer. These results indicate that WCGA-based DNA-DNA hybridization is an idea alternative of conventional DNA-DNA hybridization methods and it is superior to the phylogenetics methods based on sequence similarities of single genes. Detailed analysis is being performed for the re-classification of the strains examined.

Detection of hepatitis A virus by hybridization with single-stranded RNA probes

International Nuclear Information System (INIS)

Xi, J.; Estes, M.K.; Metcalf, T.G.

1987-01-01

An improved method of dot-blot hybridization to detect hepatitis A virus (HAV) was developed with single-stranded RNA (ssRNA) probes. Radioactive and nonradioactive ssRNA probes were generated by in vitro transcription of HAV templates inserted into the plasmid pGEM-1. 32 P-labeled ssRNA probes were at least eightfold more sensitive than the 32 P-labeled double-stranded cDNA counterparts, whereas biotin-labeled ssRNA probes showed a sensitivity comparable with that of the 32 P-labeled double-stranded cDNA counterparts. Hybridization of HAV with the ssRNA probes at high stringency revealed specific reactions with a high signal-to-noise ratio. The differential hybridization reactions seen with probes of positive and negative sense (compared with HAV genomic RNA) were used to detect HAV in clinical and field samples. A positive/negative ratio was introduced as an indicator that permitted an semiquantitative expression of a positive HAV reaction. Good agreement of this indicator was observed with normal stool samples and with HAV-seeded samples. By using this system, HAV was detected in estuarine and freshwater samples collected from a sewage-polluted bayou in Houston and a saltwater tributary of Galveston Bay

Cloning the human lysozyme cDNA: Inverted Alu repeat in the mRNA and in situ hybridization for macrophages and Paneth cells

International Nuclear Information System (INIS)

Chung, L.P.; Keshav, S.; Gordon, S.

1988-01-01

Lysozyme is a major secretory product of human and rodent macrophages and a useful marker for myelomonocytic cells. Based on the known human lysozyme amino acid sequence, oligonucleotides were synthesized and used as probes to screen a phorbol 12-myristate 13-acetate-treated U937 cDNA library. A full-length human lysozyme cDNA clone, pHL-2, was obtained and characterized. Sequence analysis shows that human lysozyme, like chicken lysozyme, has in 18-amino-acid-long signal peptide, but unlike the chicken lysozyme cDNA, the human lysozyme cDNA has a >1-kilobase-long 3' nontranslated sequence. Interestingly, within this 3' region, an inverted repeat of the Alu family of repetitive sequences was discovered. In RNA blot analyses, DNA probes prepared from pHL-2 can be used to detect lysozyme mRNA not only from human but also from mouse and rat. Moreover, by in situ hybridization, complementary RNA transcripts have been used as probes to detect lysozyme mRNA in mouse macrophages and Paneth cells. This human lysozyme cDNA clone is therefore likely to be a useful molecular probe for studying macrophage distribution and gene expression

A bio-hybrid DNA rotor-stator nanoengine that moves along predefined tracks.

Science.gov (United States)

Valero, Julián; Pal, Nibedita; Dhakal, Soma; Walter, Nils G; Famulok, Michael

2018-06-01

Biological motors are highly complex protein assemblies that generate linear or rotary motion, powered by chemical energy. Synthetic motors based on DNA nanostructures, bio-hybrid designs or synthetic organic chemistry have been assembled. However, unidirectionally rotating biomimetic wheel motors with rotor-stator units that consume chemical energy are elusive. Here, we report a bio-hybrid nanoengine consisting of a catalytic stator that unidirectionally rotates an interlocked DNA wheel, powered by NTP hydrolysis. The engine consists of an engineered T7 RNA polymerase (T7RNAP-ZIF) attached to a dsDNA nanoring that is catenated to a rigid rotating dsDNA wheel. The wheel motor produces long, repetitive RNA transcripts that remain attached to the engine and are used to guide its movement along predefined ssDNA tracks arranged on a DNA nanotube. The simplicity of the design renders this walking nanoengine adaptable to other biological nanoarchitectures, facilitating the construction of complex bio-hybrid structures that achieve NTP-driven locomotion.

Kinetics of spontaneous displacement of RNA from heteroduplexes by DNA.

OpenAIRE

Landgraf, R; Ramamurthi, K S; Sigman, D S

1996-01-01

We have used R-loop formation and direct hybridization techniques to analyze the kinetics by which RNA is displaced from a heteroduplex by DNA of identical sequence. Using random walk simulations we were able to calculate the step times for a single displacement reaction. For RNA with a GC content of 57-60% the data indicate an RNA exchange probability of 50.06%, which is indicative of a modest destabilization of the heteroduplex compared with a DNA duplex in the presence of magnesium. The av...

HDOCK: a web server for protein–protein and protein–DNA/RNA docking based on a hybrid strategy

Science.gov (United States)

Yan, Yumeng; Zhang, Di; Zhou, Pei; Li, Botong

2017-01-01

Abstract Protein–protein and protein–DNA/RNA interactions play a fundamental role in a variety of biological processes. Determining the complex structures of these interactions is valuable, in which molecular docking has played an important role. To automatically make use of the binding information from the PDB in docking, here we have presented HDOCK, a novel web server of our hybrid docking algorithm of template-based modeling and free docking, in which cases with misleading templates can be rescued by the free docking protocol. The server supports protein–protein and protein–DNA/RNA docking and accepts both sequence and structure inputs for proteins. The docking process is fast and consumes about 10–20 min for a docking run. Tested on the cases with weakly homologous complexes of server. The HDOCK web server is available at http://hdock.phys.hust.edu.cn/. PMID:28521030

Genetic relatedness of orbiviruses by RNA-RNA blot hybridization

International Nuclear Information System (INIS)

Bodkin, D.K.

1985-01-01

RNA-RNA blot hybridization was developed in order to identify type-specific genes among double-stranded (ds) RNA viruses, to assess the genetic relatedness of dsRNA viruses and to classify new strains. Viral dsRNA segments were electrophoresed through 10% polyacrylamide gels, transferred to membranes, and hybridized to [5' 32 P]-pCp labeled genomic RNA from a related strain. Hybridization was performed at 52 0 C, 50% formamide, 5X SSC. Under these conditions heterologous RNA species must share ≥ 74% sequence homology in order to form stable dsRNA hybrids. Cognate genes of nine members of the Palyam serogroup of orbiviruses were identified and their sequence relatedness to the prototype. Palyam virus, was determined. Reciprocal blot hybridizations were performed using radiolabeled genomic RNA of all members of the Palyam serogroup. Unique and variant genes were identified by lack of cross-homology or by weak homology between segments. Since genes 2 and 6 exhibited the highest degree of sequence variability, response to the vertebrate immune system may be a major cause of sequence divergence among members of a single serogroup. Changuinola serogroup isolates were compared by dot-blot hybridization, while Colorado tick fever (CTF) serogroup isolates were compared by the RNA-RNA blot hybridization procedure described for reovirus and Palyam serogroup isolates. Preliminary blot hybridization data were also obtained on the relatedness of members of different Orbivirus serogroups

HDOCK: a web server for protein-protein and protein-DNA/RNA docking based on a hybrid strategy.

Science.gov (United States)

Yan, Yumeng; Zhang, Di; Zhou, Pei; Li, Botong; Huang, Sheng-You

2017-07-03

Protein-protein and protein-DNA/RNA interactions play a fundamental role in a variety of biological processes. Determining the complex structures of these interactions is valuable, in which molecular docking has played an important role. To automatically make use of the binding information from the PDB in docking, here we have presented HDOCK, a novel web server of our hybrid docking algorithm of template-based modeling and free docking, in which cases with misleading templates can be rescued by the free docking protocol. The server supports protein-protein and protein-DNA/RNA docking and accepts both sequence and structure inputs for proteins. The docking process is fast and consumes about 10-20 min for a docking run. Tested on the cases with weakly homologous complexes of server. The HDOCK web server is available at http://hdock.phys.hust.edu.cn/. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Ultra-sensitive DNA assay based on single-molecule detection coupled with fluorescent quantum dot-labeling and its application to determination of messenger RNA

International Nuclear Information System (INIS)

Li Li; Li Xincang; Li Lu; Wang Jinxing; Jin Wenrui

2011-01-01

An ultra-sensitive single-molecule detection (SMD) method for quantification of DNA using total internal reflection fluorescence microscopy (TIRFM) coupled with fluorescent quantum dot (QD)-labeling was developed. In this method, the target DNA (tDNA) was captured by the capture DNA immobilized on the silanized coverslip blocked with ethanolamine and bovine serum albumin. Then, the QD-labeled probe DNA was hybridized to the tDNA. Ten fluorescent images of the QD-labeled sandwich DNA hybrids on the coverslip were taken by a high-sensitive CCD. The tDNA was quantified by counting the bright spots on the images using a calibration curve. The LOD of the method was 1 x 10 -14 mol L -1 . Several key factors, including image acquirement, fluorescence probe, substrate preparation, noise elimination from solutions and glass coverslips, and nonspecific adsorption and binding of solution-phase detection probes were discussed in detail. The method could be applied to quantify messenger RNA (mRNA) in cells. In order to determine mRNA, double-stranded RNA-DNA hybrids consisting of mRNA and corresponding cDNA were synthesized from the cellular mRNA template using reverse transcription in the presence of reverse transcriptase. After removing the mRNA in the double-stranded hybrids using ribonuclease, cDNA was quantified using the SMD-based TIRFM. Osteopontin mRNA in decidual stromal cells was chosen as the model analyte.

Circular RNA (circRNA) was an important bridge in the switch from the RNA world to the DNA world.

Science.gov (United States)

Soslau, Gerald

2018-06-14

osmolarity. Circular RNA, circRNA, is proposed as a critical stable RNA molecule that served as the genetic precursor for the switch to DNA and the replication of circRNA by a rolling circle mechanism gave rise to the RNA complexity required for the genetic functions of the cell. The replicating ribocyte would have required protein synthesis as well as RNA replication and a model for non-coded and primordial coded protein synthesis is proposed. Finally, the switch from the RNA to the DNA world would have involved the synthesis of an RNA:DNA hybrid prior to the formation of dsDNA. If the hybrid was a circular molecule that ultimately yielded a circular dsDNA molecule, it could predict that the primordial DNA cell would evolve into a bacterial cell with a single circular chromosome. One would hope that continued speculation of the origin of life will spur new directions of research that may never fully answer the questions of the past but add to our ability to regulate potentially harmful biological events in the present and in the future. Copyright © 2018 Elsevier Ltd. All rights reserved.

Electrical potential-assisted DNA hybridization. How to mitigate electrostatics for surface DNA hybridization.

Science.gov (United States)

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.

Ultra-sensitive DNA assay based on single-molecule detection coupled with fluorescent quantum dot-labeling and its application to determination of messenger RNA

Energy Technology Data Exchange (ETDEWEB)

Li Li [School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100 (China); Li Xincang [School of Life Sciences, Shandong University, Jinan 250100 (China); Li Lu [School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100 (China); Wang Jinxing [School of Life Sciences, Shandong University, Jinan 250100 (China); Jin Wenrui, E-mail: jwr@sdu.edu.cn [School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100 (China)

2011-01-24

An ultra-sensitive single-molecule detection (SMD) method for quantification of DNA using total internal reflection fluorescence microscopy (TIRFM) coupled with fluorescent quantum dot (QD)-labeling was developed. In this method, the target DNA (tDNA) was captured by the capture DNA immobilized on the silanized coverslip blocked with ethanolamine and bovine serum albumin. Then, the QD-labeled probe DNA was hybridized to the tDNA. Ten fluorescent images of the QD-labeled sandwich DNA hybrids on the coverslip were taken by a high-sensitive CCD. The tDNA was quantified by counting the bright spots on the images using a calibration curve. The LOD of the method was 1 x 10{sup -14} mol L{sup -1}. Several key factors, including image acquirement, fluorescence probe, substrate preparation, noise elimination from solutions and glass coverslips, and nonspecific adsorption and binding of solution-phase detection probes were discussed in detail. The method could be applied to quantify messenger RNA (mRNA) in cells. In order to determine mRNA, double-stranded RNA-DNA hybrids consisting of mRNA and corresponding cDNA were synthesized from the cellular mRNA template using reverse transcription in the presence of reverse transcriptase. After removing the mRNA in the double-stranded hybrids using ribonuclease, cDNA was quantified using the SMD-based TIRFM. Osteopontin mRNA in decidual stromal cells was chosen as the model analyte.

Northern blots: capillary transfer of RNA from agarose gels and filter hybridization using standard stringency conditions.

Science.gov (United States)

Rio, Donald C

2015-03-02

In this protocol, an RNA sample, fractionated by gel electrophoresis, is transferred from the gel onto a membrane by capillary transfer. Short-wave UV light is used to fix the transferred RNA to the membrane. The membrane is then pretreated to block nonspecific probe-binding sites, and hybridization of the immobilized RNA to a (32)P-labeled DNA or RNA probe specific for the mRNA of interest is performed. Finally, the membrane is washed and subjected to autoradiography or phosphorimaging. Because exposure to UV cross-links the RNA to the membrane, the membrane can be stripped and hybridized with other probes. The procedure is suitable for detecting poly(A)(+)-selected mRNA or mRNA in total cellular RNA if the target transcript is relatively abundant. Using DNA or RNA probes labeled to 1 × 10(8)-10 × 10(8) cpm/µg, it should be possible to detect ∼5 pg of a specific RNA. © 2015 Cold Spring Harbor Laboratory Press.

The communication of endogenous biomolecules (RNA, DNA, protein, hormone) via graft union might play key roles in the new traits formation of graft hybrids

International Nuclear Information System (INIS)

Khan, I.A.; Ghazal, L.; Arsalan, M.H.; Siddiqui, M.F.

2018-01-01

Plant graft hybridization is a fact of a sexual hybridization in which heritable changes trigger by grafting, it can produce hereditable variation types desired by breeders and can be used as a new method for germplasm innovation. This paper aimed to discuss new phenotypic variations such as phenotypic diversity and polyploidy formation discovered in recent years, new evidence supporting genetic material exchange between the rootstock and scion, and the timeliness, direction, and genetic stability of trait formation induced by graft hybridization. The unresolved mechanisms of trait variation were also discussed, and the relationship with exchange of chloroplast DNA (cpDNA) genetic information, horizontal gene transfer events of chloroplast and mitochondria, miRNA-mediated post-transcriptional regulation, and pressure-driven trait variations from graft hybridization were performed to clarify the relevant questions. Finally, the future research trend and key questions of graft hybridization were identified. Three clear conclusions for graft hybridization are presented: the new traits of graft hybrids have stable genetic characteristics and can be controlled via selection different genetic plant by grafting, and the products of graft hybrids were safer than those of transgenic plants. (author)

Co-transcriptional formation of DNA:RNA hybrid G-quadruplex and potential function as constitutional cis element for transcription control.

Science.gov (United States)

Zheng, Ke-wei; Xiao, Shan; Liu, Jia-quan; Zhang, Jia-yu; Hao, Yu-hua; Tan, Zheng

2013-05-01

G-quadruplex formation in genomic DNA is considered to regulate transcription. Previous investigations almost exclusively focused on intramolecular G-quadruplexes formed by DNA carrying four or more G-tracts, and structure formation has rarely been studied in physiologically relevant processes. Here, we report an almost entirely neglected, but actually much more prevalent form of G-quadruplexes, DNA:RNA hybrid G-quadruplexes (HQ) that forms in transcription. HQ formation requires as few as two G-tracts instead of four on a non-template DNA strand. Potential HQ sequences (PHQS) are present in >97% of human genes, with an average of 73 PHQSs per gene. HQ modulates transcription under both in vitro and in vivo conditions. Transcriptomal analysis of human tissues implies that maximal gene expression may be limited by the number of PHQS in genes. These features suggest that HQs may play fundamental roles in transcription regulation and other transcription-mediated processes.

Parallel characterization of anaerobic toluene- and ethylbenzene-degrading microbial consortia by PCR-denaturing gradient gel electrophoresis, RNA-DNA membrane hybridization, and DNA microarray technology

Science.gov (United States)

Koizumi, Yoshikazu; Kelly, John J.; Nakagawa, Tatsunori; Urakawa, Hidetoshi; El-Fantroussi, Said; Al-Muzaini, Saleh; Fukui, Manabu; Urushigawa, Yoshikuni; Stahl, David A.

2002-01-01

A mesophilic toluene-degrading consortium (TDC) and an ethylbenzene-degrading consortium (EDC) were established under sulfate-reducing conditions. These consortia were first characterized by denaturing gradient gel electrophoresis (DGGE) fingerprinting of PCR-amplified 16S rRNA gene fragments, followed by sequencing. The sequences of the major bands (T-1 and E-2) belonging to TDC and EDC, respectively, were affiliated with the family Desulfobacteriaceae. Another major band from EDC (E-1) was related to an uncultured non-sulfate-reducing soil bacterium. Oligonucleotide probes specific for the 16S rRNAs of target organisms corresponding to T-1, E-1, and E-2 were designed, and hybridization conditions were optimized for two analytical formats, membrane and DNA microarray hybridization. Both formats were used to characterize the TDC and EDC, and the results of both were consistent with DGGE analysis. In order to assess the utility of the microarray format for analysis of environmental samples, oil-contaminated sediments from the coast of Kuwait were analyzed. The DNA microarray successfully detected bacterial nucleic acids from these samples, but probes targeting specific groups of sulfate-reducing bacteria did not give positive signals. The results of this study demonstrate the limitations and the potential utility of DNA microarrays for microbial community analysis.

Mycobacterium smegmatis HelY Is an RNA-Activated ATPase/dATPase and 3'-to-5' Helicase That Unwinds 3'-Tailed RNA Duplexes and RNA:DNA Hybrids.

Science.gov (United States)

Uson, Maria Loressa; Ordonez, Heather; Shuman, Stewart

2015-10-01

Mycobacteria have a large and distinctive ensemble of DNA helicases that function in DNA replication, repair, and recombination. Little is known about the roster of RNA helicases in mycobacteria or their roles in RNA transactions. The 912-amino-acid Mycobacterium smegmatis HelY (MSMEG_3885) protein is a bacterial homolog of the Mtr4 and Ski2 helicases that regulate RNA 3' processing and turnover by the eukaryal exosome. Here we characterize HelY as an RNA-stimulated ATPase/dATPase and an ATP/dATP-dependent 3'-to-5' helicase. HelY requires a 3' single-strand RNA tail (a loading RNA strand) to displace the complementary strand of a tailed RNA:RNA or RNA:DNA duplex. The findings that HelY ATPase is unresponsive to a DNA polynucleotide cofactor and that HelY is unable to unwind a 3'-tailed duplex in which the loading strand is DNA distinguish HelY from other mycobacterial nucleoside triphosphatases/helicases characterized previously. The biochemical properties of HelY, which resemble those of Mtr4/Ski2, hint at a role for HelY in mycobacterial RNA catabolism. RNA helicases play crucial roles in transcription, RNA processing, and translation by virtue of their ability to alter RNA secondary structure or remodel RNA-protein interactions. In eukarya, the RNA helicases Mtr4 and Ski2 regulate RNA 3' resection by the exosome. Mycobacterium smegmatis HelY, a bacterial homolog of Mtr4/Ski2, is characterized here as a unidirectional helicase, powered by RNA-dependent ATP/dATP hydrolysis, that tracks 3' to 5' along a loading RNA strand to displace the complementary strand of a tailed RNA:RNA or RNA:DNA duplex. The biochemical properties of HelY suggest a role in bacterial RNA transactions. HelY homologs are present in pathogenic mycobacteria (e.g., M. tuberculosis and M. leprae) and are widely prevalent in Actinobacteria and Cyanobacteria but occur sporadically elsewhere in the bacterial domain. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Involvement of DNA topoisomerase I in transcription of human ribosomal RNA genes

International Nuclear Information System (INIS)

Zhang, H.; Wang, J.C.; Liu, L.F.

1988-01-01

Treatment of HeLa cells with a DNA topoisomerase I-specific inhibitor, camptothecin, results in rapid cessation of the synthesis of the 45S rRNA precursor. The inhibition of rRNA synthesis is reversible following drug removal and correlates with the presence of camptothecin-trapped topoisomerase I-DNA abortive complexes, which can be detected as topoisomerase I-linked DNA breaks upon lysis with sodium dodecyl sulfate. These breaks were found to be concentrated within the transcribed region of human rRNA genes. No such sites can be detected in the inactive human rRNA genes in mouse-human hybrid cells, suggesting a preferential association of topoisomerase I with actively transcribed genes. The distribution of RNA polymerase molecules along the transcription unit of human rRNA genes in camptothecin-treated HeLa cells, as assayed by nuclear run-on transcription, shows a graded decrease of the RNA polymerase density toward the 3' end of the transcription unit; the density is minimally affected near the 5' start of the transcription unit. These results suggest that DNA topoisomerase I is normally involved in the elongation step of transcription, especially when the transcripts are long, and that camptothecin interferes with this role

Exciton-controlled fluorescence: application to hybridization-sensitive fluorescent DNA probe.

Science.gov (United States)

Okamoto, Akimitsu; Ikeda, Shuji; Kubota, Takeshi; Yuki, Mizue; Yanagisawa, Hiroyuki

2009-01-01

A hybridization-sensitive fluorescent probe has been designed for nucleic acid detection, using the concept of fluorescence quenching caused by the intramolecular excitonic interaction of fluorescence dyes. We synthesized a doubly thiazole orange-labeled nucleotide showing high fluorescence intensity for a hybrid with the target nucleic acid and effective quenching for the single-stranded state. This exciton-controlled fluorescent probe was applied to living HeLa cells using microinjection to visualize intracellular mRNA localization. Immediately after injection of the probe into the cell, fluorescence was observed from the probe hybridizing with the target RNA. This fluorescence rapidly decreased upon addition of a competitor DNA. Multicoloring of this probe resulted in the simple simultaneous detection of plural target nucleic acid sequences. This probe realized a large, rapid, reversible change in fluorescence intensity in sensitive response to the amount of target nucleic acid, and facilitated spatiotemporal monitoring of the behavior of intracellular RNA.

Comparison of kDNA PCR-hybridization assay with three PCR methods for canines visceral Leishmaniasis diagnosis

Energy Technology Data Exchange (ETDEWEB)

Pilatti, Marcia M.; Andrade, Antero S.R. [Centro de Desenvolvimento da Tecnologia Nuclear (CDTN/CNEN-MG), Belo Horizonte, MG (Brazil)], e-mail: marciapilatti@yahoo.com.br, e-mail: antero@cdtn.br; Ferreira, Sidney A. [Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, MG (Brazil). Dept. de Parasitologia], e-mail: saninoalmeida@gmail.com

2009-07-01

The sensitivity of the kDNA PCR-Hybridization assay, which uses radioactive DNA probes (labeled with {sup 32}P), was compared with three conventional PCR methods used for canine visceral leishmaniasis diagnosis. All PCR methods had two steps: a first amplification followed by hybridization or by a new amplification (nested or semi nested). Two methods (kDNA PCR-Hybridization and kDNA snPCR) used primers addressed to kinetoplast minicircles and the other two methods to the coding (LnPCR) and intergenic noncoding regions (ITS-1 nPCR) of the ribosomal rRNA genes. The comparison was accomplished in two groups of 23 infected dogs using samples collected by the conjunctival swab procedure. In the Group 1 the DNA was extracted from cotton swabs by phenol-chloroform and in Group 2 by boiling. The most efficient PCR methods in the Group 1 were those based on kDNA targets. The kDNA PCR-Hybridization was able to detect parasites in 22/23 dogs (95.6%) and in 40/46 samples (86.9%). The kDNA snPCR was positive for 21/23 dogs (91.3%) and for 40/46 samples (86.9%). The positivities of the kDNA based methods were significantly higher than the positivities verified for the methods based on ribosomal rRNA genes (p<0.05). In the Group 2 the kDNA PCR- Hybridization showed a better performance detecting parasites in 18/23 dogs (78.3%) and in 31/46 samples (67.4%), significantly higher than the other three methods (p<0.05). The higher sensitivity of the minicircle kDNA based assays reported by others was confirmed in this study and kDNA PCR-Hybridization showed the best sensitivity among the assays evaluated. (author)

Comparison of kDNA PCR-hybridization assay with three PCR methods for canines visceral Leishmaniasis diagnosis

International Nuclear Information System (INIS)

Pilatti, Marcia M.; Andrade, Antero S.R.; Ferreira, Sidney A.

2009-01-01

The sensitivity of the kDNA PCR-Hybridization assay, which uses radioactive DNA probes (labeled with 32 P), was compared with three conventional PCR methods used for canine visceral leishmaniasis diagnosis. All PCR methods had two steps: a first amplification followed by hybridization or by a new amplification (nested or semi nested). Two methods (kDNA PCR-Hybridization and kDNA snPCR) used primers addressed to kinetoplast minicircles and the other two methods to the coding (LnPCR) and intergenic noncoding regions (ITS-1 nPCR) of the ribosomal rRNA genes. The comparison was accomplished in two groups of 23 infected dogs using samples collected by the conjunctival swab procedure. In the Group 1 the DNA was extracted from cotton swabs by phenol-chloroform and in Group 2 by boiling. The most efficient PCR methods in the Group 1 were those based on kDNA targets. The kDNA PCR-Hybridization was able to detect parasites in 22/23 dogs (95.6%) and in 40/46 samples (86.9%). The kDNA snPCR was positive for 21/23 dogs (91.3%) and for 40/46 samples (86.9%). The positivities of the kDNA based methods were significantly higher than the positivities verified for the methods based on ribosomal rRNA genes (p<0.05). In the Group 2 the kDNA PCR- Hybridization showed a better performance detecting parasites in 18/23 dogs (78.3%) and in 31/46 samples (67.4%), significantly higher than the other three methods (p<0.05). The higher sensitivity of the minicircle kDNA based assays reported by others was confirmed in this study and kDNA PCR-Hybridization showed the best sensitivity among the assays evaluated. (author)

DNA-DNA hybridization determined in micro-wells using covalent attachment of DNA

DEFF Research Database (Denmark)

Christensen, H.; Angen, Øystein; Mutters, R.

2000-01-01

The present study was aimed at reducing the time and labour used to perform DNA-DNA hybridizations for classification of bacteria at the species level. A micro-well-format DNA hybridization method was developed and validated. DNA extractions were performed by a small-scale method and DNA...... was sheared mechanically into fragments of between 400 and 700 bases. The hybridization conditions were calibrated according to DNA similarities obtained by the spectrophotometric method using strains within the family Pasteurellaceae, Optimal conditions were obtained with 300 ng DNA added per well and bound...... by covalent attachment to NucleoLink. Hybridization was performed with 500 ng DNA, 5% (w/w) of which was labelled with photo-activatable biotin (competitive hybridization) for 2.5 h at 65 degrees C in 2 x SSC followed by stringent washing with 2 x SSC at the same temperature. The criteria for acceptance...

Fluorescence In Situ Hybridization for MicroRNA Detection in Archived Oral Cancer Tissues

Directory of Open Access Journals (Sweden)

Zonggao Shi

2012-01-01

Full Text Available The noncoding RNA designated as microRNA (miRNA is a large group of small single-stranded regulatory RNA and has generated wide-spread interest in human disease studies. To facilitate delineating the role of microRNAs in cancer pathology, we sought to explore the feasibility of detecting microRNA expression in formalin-fixed paraffin-embedded (FFPE tissues. Using FFPE materials, we have compared fluorescent in situ hybridization (FISH procedures to detect miR-146a with (a different synthetic probes: regular custom DNA oligonucleotides versus locked nucleic acid (LNA incorporated DNA oligonucleotides; (b different reporters for the probes: biotin versus digoxigenin (DIG; (c different visualization: traditional versus tyramide signal amplification (TSA system; (d different blocking reagents for endogenous peroxidase. Finally, we performed miR-146a FISH on a commercially available oral cancer tissue microarray, which contains 40 cases of oral squamous cell carcinoma (OSCC and 10 cases of normal epithelia from the human oral cavity. A sample FISH protocol for detecting miR-146a is provided. In summary, we have established reliable in situ hybridization procedures for detecting the expression of microRNA in FFPE oral cancer tissues. This method is an important tool for studies on the involvement of microRNA in oral cancer pathology and may have potential prognostic or diagnostic value.

A rapid and efficient branched DNA hybridization assay to titer lentiviral vectors.

Science.gov (United States)

Nair, Ayyappan; Xie, Jinger; Joshi, Sarasijam; Harden, Paul; Davies, Joan; Hermiston, Terry

2008-11-01

A robust assay to titer lentiviral vectors is imperative to qualifying their use in drug discovery, target validation and clinical applications. In this study, a novel branched DNA based hybridization assay was developed to titer lentiviral vectors by quantifying viral RNA genome copy numbers from viral lysates without having to purify viral RNA, and this approach was compared with other non-functional (p24 protein ELISA and viral RT-qPCR) and a functional method (reporter gene expression) used commonly. The RT-qPCR method requires purification of viral RNA and the accuracy of titration therefore depends on the efficiency of purification; this requirement is ameliorated in the hybridization assay as RNA is measured directly in viral lysates. The present study indicates that the hybridization based titration assay performed on viral lysates was more accurate and has additional advantages of being rapid, robust and not dependent on transduction efficiency in different cell types.

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

Science.gov (United States)

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

2012-04-01

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

Isolation and characterization of human glycophorin A cDNA clones by a synthetic oligonucleotide approach: nucleotide sequence and mRNA structure

International Nuclear Information System (INIS)

Siebert, P.D.; Fukuda, M.

1986-01-01

In an effort to understand the relationships among and the regulation of human glycophorins, the authors have isolated and characterized several glycophorin A-specific cDNA clones obtained from a human erythroleukemic K562 cell cDNA library. This was accomplished by using mixed synthetic oligonucleotides, corresponding to various regions of the known amino acid sequence, to prime the synthesis of the cDNA as well as to screen the cDNA library. They also used synthetic oligonucleotides to sequence the largest of the glycophorin cDNAs. The nucleotide sequence obtained suggests the presence of a potential leader peptide, consistent with the membrane localization of this glycoprotein. Examination of the structure of glycophorin mRNA by blot hybridization revealed the existence of several electrophoretically distinct mRNAs numbering three or four, depending on the size of the glycophorin cDNA used as a hybridization probe. The smaller cDNA hybridized to three mRNAs of approximately 2.8, 1.7, and 1.0 kilobases. In contrast, the larger cDNA hybridized to an additional mRNA of approximately 0.6 kilobases. Further examination of the relationships between these multiple mRNAs by blot hybridization was conducted with the use of exact-sequence oligonucleotide probes constructed from various regions of the cDNA representing portions of the amino acid sequence of glycophorin A with or without known homology with glycophorin B. In total, the results obtained are consistent with the hypothesis that the three larger mRNAs represent glycophorin A gene transcripts and that the smallest (0.6 kilobase) mRNA may be specific for glycophorin B

Simultaneous Detection of CDC Category "A" DNA and RNA Bioterrorism Agents by Use of Multiplex PCR & RT-PCR Enzyme Hybridization Assays

Directory of Open Access Journals (Sweden)

Kelly J. Henrickson

2009-10-01

Full Text Available Assays to simultaneously detect multiple potential agents of bioterrorism are limited. Two multiplex PCR and RT-PCR enzyme hybridization assays (mPCR-EHA, mRT-PCR-EHA were developed to simultaneously detect many of the CDC category “A” bioterrorism agents. The “Bio T” DNA assay was developed to detect: Variola major (VM, Bacillus anthracis (BA, Yersinia pestis (YP, Francisella tularensis (FT and Varicella zoster virus (VZV. The “Bio T” RNA assay (mRT-PCR-EHA was developed to detect: Ebola virus (Ebola, Lassa fever virus (Lassa, Rift Valley fever (RVF, Hantavirus Sin Nombre species (HSN and dengue virus (serotypes 1-4. Sensitivity and specificity of the 2 assays were tested by using genomic DNA, recombinant plasmid positive controls, RNA transcripts controls, surrogate (spiked clinical samples and common respiratory pathogens. The analytical sensitivity (limit of detection (LOD of the DNA asssay for genomic DNA was 1×100~1×102 copies/mL for BA, FT and YP. The LOD for VZV whole organism was 1×10-2 TCID50/mL. The LOD for recombinant controls ranged from 1×102~1×103copies/mL for BA, FT, YP and VM. The RNA assay demonstrated LOD for RNA transcript controls of 1×104~1×106 copies/mL without extraction and 1×105~1×106 copies/mL with extraction for Ebola, RVF, Lassa and HSN. The LOD for dengue whole organisms was ~1×10-4 dilution for dengue 1 and 2, 1×104 LD50/mL and 1×102 LD50/mL for dengue 3 and 4. The LOD without extraction for recombinant plasmid DNA controls was ~1×103 copies/mL (1.5 input copies/reaction for Ebola, RVF, Lassa and HSN. No cross-reactivity of primers and probes used in both assays was detected with common respiratory pathogens or between targeted analytes. Clinical sensitivity was estimated using 264 surrogate clinical samples tested with the BioT DNA assay and 549 samples tested with the BioT RNA assay. The clinical specificity is 99.6% and 99.8% for BioT DNA assay and BioT RNA assay, respectively. The

Cloning of cDNA sequences of a progestin-regulated mRNA from MCF7 human breast cancer cells

Energy Technology Data Exchange (ETDEWEB)

Chalbos, D; Westley, B; Alibert, C; Rochefort, H

1986-01-24

A cDNA clone corresponding to an mRNA regulated by the progestin R5020, has been isolated by differential screening of a cDNA library from the MCF7 breast cancer cell line, which contains estrogen and progesterone receptors. This probe hybridized with a single species of poly A + RNA of 8-kb molecular weight as shown by Northern blot analysis and could also be used to total RNA preparation. This recombinant cone hybridized specifically to an mRNA coding for a 250,000 daltons protein when translated in vitro. This protein was identical to the 250 kDa progestin-regulated protein that the authors previously described as shown by immunoprecipitation with specific rabbit polyclonal antibodies. Dose-response curve and specificity studies show that the accumulation of the Pg8 mRNA and that of the 250-kDa protein was increased by 5 to 30-fold following progestin treatment and that this effect was mediated by the progesterone receptor. Time course of induction indicated that the accumulation of mRNA was rapid and preceded that of the protein. This is the first report on a cloned cDNA probe of progestin-regulated mRNA in human cell lines.

Electrokinetic acceleration of DNA hybridization in microsystems.

Science.gov (United States)

Lei, Kin Fong; Wang, Yun-Hsiang; Chen, Huai-Yi; Sun, Jia-Hong; Cheng, Ji-Yen

2015-06-01

In this work, electrokinetic acceleration of DNA hybridization was investigated by different combinations of frequencies and amplitudes of actuating electric signals. Because the frequencies from low to high can induce different kinds of electrokinetic forces, i.e., electroosmotic to electrothermal forces, this work provides an in-depth investigation of electrokinetic enhanced hybridization. Concentric circular Cr/Au microelectrodes of 350 µm in diameter were fabricated on a glass substrate and probe DNA was immobilized on the electrode surface. Target DNA labeled with fluorescent dyes suspending in solution was then applied to the electrode. Different electrokinetic forces were induced by the application of different electric signals to the circular microelectrodes. Local microfluidic vortexes were generated to increase the collision efficiency between the target DNA suspending in solution and probe DNA immobilized on the electrode surface. DNA hybridization on the electrode surface could be accelerated by the electrokinetic forces. The level of hybridization was represented by the fluorescent signal intensity ratio. Results revealed that such 5-min dynamic hybridization increased 4.5 fold of signal intensity ratio as compared to a 1-h static hybridization. Moreover, dynamic hybridization was found to have better differentiation ability between specific and non-specific target DNA. This study provides a strategy to accelerate DNA hybridization in microsystems. Copyright © 2015 Elsevier B.V. All rights reserved.

Genome-Wide Spectra of Transcription Insertions and Deletions Reveal That Slippage Depends on RNA:DNA Hybrid Complementarity.

Science.gov (United States)

Traverse, Charles C; Ochman, Howard

2017-08-29

Advances in sequencing technologies have enabled direct quantification of genome-wide errors that occur during RNA transcription. These errors occur at rates that are orders of magnitude higher than rates during DNA replication, but due to technical difficulties such measurements have been limited to single-base substitutions and have not yet quantified the scope of transcription insertions and deletions. Previous reporter gene assay findings suggested that transcription indels are produced exclusively by elongation complex slippage at homopolymeric runs, so we enumerated indels across the protein-coding transcriptomes of Escherichia coli and Buchnera aphidicola , which differ widely in their genomic base compositions and incidence of repeat regions. As anticipated from prior assays, transcription insertions prevailed in homopolymeric runs of A and T; however, transcription deletions arose in much more complex sequences and were rarely associated with homopolymeric runs. By reconstructing the relocated positions of the elongation complex as inferred from the sequences inserted or deleted during transcription, we show that continuation of transcription after slippage hinges on the degree of nucleotide complementarity within the RNA:DNA hybrid at the new DNA template location. IMPORTANCE The high level of mistakes generated during transcription can result in the accumulation of malfunctioning and misfolded proteins which can alter global gene regulation and in the expenditure of energy to degrade these nonfunctional proteins. The transcriptome-wide occurrence of base substitutions has been elucidated in bacteria, but information on transcription insertions and deletions-errors that potentially have more dire effects on protein function-is limited to reporter gene constructs. Here, we capture the transcriptome-wide spectrum of insertions and deletions in Escherichia coli and Buchnera aphidicola and show that they occur at rates approaching those of base substitutions

Crystal Structure of a CRISPR RNA-guided Surveillance Complex Bound to a ssDNA Target

Energy Technology Data Exchange (ETDEWEB)

Mulepati, Sabin [Johns Hopkins Univ., Baltimore, MD (United States); Heroux, Annie; Bailey, Scott [Johns Hopkins Univ., Baltimore, MD (United States)

2014-09-19

In prokaryotes, RNA derived from type I and type III CRISPR loci direct large ribonucleoprotein complexes to destroy invading bacteriophage and plasmids. In Escherichia coli, this 405-kilodalton complex is called Cascade. We report the crystal structure of Cascade bound to a single-stranded DNA (ssDNA) target at a resolution of 3.03 angstroms. The structure reveals that the CRISPR RNA and target strands do not form a double helix but instead adopt an underwound ribbon-like structure. This noncanonical structure is facilitated by rotation of every sixth nucleotide out of the RNA-DNA hybrid and is stabilized by the highly interlocked organization of protein subunits. These studies provide insight into both the assembly and the activity of this complex and suggest a mechanism to enforce fidelity of target binding.

Ratiometric FRET-based detection of DNA and micro-RNA in solution

International Nuclear Information System (INIS)

Matveeva, Evgenia G.; Gryczynski, Zygmunt; Stewart, Donald R.; Gryczynski, Ignacy

2009-01-01

A ratiometric method for detecting DNA oligomers in bulk solution based on Foerster resonance energy transfer (FRET) is described. The two fluorescence signals (green and red), originating from Cy3 (donor, green) and Cy5 (acceptor, red) labels, are simultaneously detected from the pre-hybridized Cy3oligomerY:Cy5oligomerX system. The ratio of red to green intensities is sensitive to the presence of the single-stranded complimentary oligomer, which replaces single-stranded Cy3oligomerY in the donor:acceptor complex and perturbs the FRET. The detection scheme is generally applicable to the detection of DNA and RNA, and particularly micro-RNA. The proposed method is applicable to various double-stranded various lengths targets (manipulation of the sample preparation conditions, such as temperature, incubation time, denaturizing agent, may be needed).

DNA:DNA hybridization studies on the pink-pigmented facultative methylotrophs.

Science.gov (United States)

Hood, D W; Dow, C S; Green, P N

1987-03-01

The genomic relatedness among 36 strains of pink-pigmented facultatively methylotrophic bacteria (PPFMs) was estimated by determination of DNA base composition and by DNA:DNA hybridization studies. A reproducible hybridization system was developed for the rapid analysis of multiple DNA samples. Results indicated that the PPFMs comprise four major and several minor homology groups, and that they should remain grouped in a single genus, Methylobacterium.

Control of DNA hybridization by photoswitchable molecular glue.

Science.gov (United States)

Dohno, Chikara; Nakatani, Kazuhiko

2011-12-01

Hybridization of DNA is one of the most intriguing events in molecular recognition and is essential for living matter to inherit life beyond generations. In addition to the function of DNA as genetic material, DNA hybridization is a key to control the function of DNA-based materials in nanoscience. Since the hybridization of two single stranded DNAs is a thermodynamically favorable process, dissociation of the once formed DNA duplex is normally unattainable under isothermal conditions. As the progress of DNA-based nanoscience, methodology to control the DNA hybridization process has become increasingly important. Besides many reports using the chemically modified DNA for the regulation of hybridization, we focused our attention on the use of a small ligand as the molecular glue for the DNA. In 2001, we reported the first designed molecule that strongly and specifically bound to the mismatched base pairs in double stranded DNA. Further studies on the mismatch binding molecules provided us a key discovery of a novel mode of the binding of a mismatch binding ligand that induced the base flipping. With these findings we proposed the concept of molecular glue for DNA for the unidirectional control of DNA hybridization and, eventually photoswitchable molecular glue for DNA, which enabled the bidirectional control of hybridization under photoirradiation. In this tutorial review, we describe in detail how we integrated the mismatch binding ligand into photoswitchable molecular glue for DNA, and the application and perspective in DNA-based nanoscience.

A RNA-DNA Hybrid Aptamer for Nanoparticle-Based Prostate Tumor Targeted Drug Delivery

Directory of Open Access Journals (Sweden)

John C. Leach

2016-03-01

Full Text Available The side effects of radio- and chemo-therapy pose long-term challenges on a cancer patient’s health. It is, therefore, highly desirable to develop more effective therapies that can specifically target carcinoma cells without damaging normal and healthy cells. Tremendous efforts have been made in the past to develop targeted drug delivery systems for solid cancer treatment. In this study, a new aptamer, A10-3-J1, which recognizes the extracellular domain of the prostate specific membrane antigen (PSMA, was designed. A super paramagnetic iron oxide nanoparticle-aptamer-doxorubicin (SPIO-Apt-Dox was fabricated and employed as a targeted drug delivery platform for cancer therapy. This DNA RNA hybridized aptamer antitumor agent was able to enhance the cytotoxicity of targeted cells while minimizing collateral damage to non-targeted cells. This SPIO-Apt-Dox nanoparticle has specificity to PSMA+ prostate cancer cells. Aptamer inhibited nonspecific uptake of membrane-permeable doxorubic to the non-target cells, leading to reduced untargeted cytotoxicity and endocytic uptake while enhancing targeted cytotoxicity and endocytic uptake. The experimental results indicate that the drug delivery platform can yield statistically significant effectiveness being more cytotoxic to the targeted cells as opposed to the non-targeted cells.

In situ hybridization at the electron microscope level: hybrid detection by autoradiography and colloidal gold.

Science.gov (United States)

Hutchison, N J; Langer-Safer, P R; Ward, D C; Hamkalo, B A

1982-11-01

In situ hybridization has become a standard method for localizing DNA or RNA sequences in cytological preparations. We developed two methods to extend this technique to the transmission electron microscope level using mouse satellite DNA hybridization to whole mount metaphase chromosomes as the test system. The first method devised is a direct extension of standard light microscope level using mouse satellite DNA hybridization to whole mount metaphase chromosomes as the test system. The first method devised is a direct extension of standard light microscope in situ hybridization. Radioactively labeled complementary RNA (cRNA) is hybridized to metaphase chromosomes deposited on electron microscope grids and fixed in 70 percent ethanol vapor; hybridixation site are detected by autoradiography. Specific and intense labeling of chromosomal centromeric regions is observed even after relatively short exposure times. Inerphase nuclei present in some of the metaphase chromosome preparations also show defined paatterms of satellite DNA labeling which suggests that satellite-containing regions are associate with each other during interphase. The sensitivity of this method is estimated to at least as good as that at the light microscope level while the resolution is improved at least threefold. The second method, which circumvents the use of autoradiogrphic detection, uses biotin-labeled polynucleotide probes. After hybridization of these probes, either DNA or RNA, to fixed chromosomes on grids, hybrids are detected via reaction is improved at least threefold. The second method, which circumvents the use of autoradiographic detection, uses biotin-labeled polynucleotide probes. After hybridization of these probes, either DNA or RNA, to fixed chromosomes on grids, hybrids are detected via reaction with an antibody against biotin and secondary antibody adsorbed to the surface of over centromeric heterochromatin and along the associated peripheral fibers. Labeling is on average

A plasmonic colorimetric strategy for visual miRNA detection based on hybridization chain reaction

Science.gov (United States)

Miao, Jie; Wang, Jingsheng; Guo, Jinyang; Gao, Huiguang; Han, Kun; Jiang, Chengmin; Miao, Peng

2016-08-01

In this work, a novel colorimetric strategy for miRNA analysis is proposed based on hybridization chain reaction (HCR)-mediated localized surface plasmon resonance (LSPR) variation of silver nanoparticles (AgNPs). miRNA in the sample to be tested is able to release HCR initiator from a solid interface to AgNPs colloid system by toehold exchange-mediated strand displacement, which then triggers the consumption of fuel strands with single-stranded tails for HCR. The final produced long nicked double-stranded DNA loses the ability to protect AgNPs from salt-induced aggregation. The stability variation of the colloid system can then be monitored by recording corresponding UV-vis spectrum and initial miRNA level is thus determined. This sensing system involves only four DNA strands which is quite simple. The practical utility is confirmed to be excellent by employing different biological samples.

Evaluation of the efficacy of the four tests (p16 immunochemistry, PCR, DNA and RNA In situ Hybridization) to evaluate a Human Papillomavirus infection in head and neck cancers: a cohort of 348 French squamous cell carcinomas.

Science.gov (United States)

Augustin, Jérémy; Outh-Gauer, Sophie; Mandavit, Marion; Gasne, Cassandre; Grard, Ophélie; Denize, Thomas; Nervo, Marine; Mirghani, Haïtham; Laccourreye, Ollivier; Bonfils, Pierre; Bruneval, Patrick; Veyer, David; Péré, Hélène; Tartour, Eric; Badoual, Cécile

2018-04-20

It is now established that HPV plays a role in the development of a subset of head and neck squamous cell carcinomas (HNSCCs), notably oropharyngeal squamous cell carcinomas (SCCs). However, it is not clear which test one should use to detect HPV in oropharyngeal (OP) and non-OP SCCs. In this study, using 348 HNSCCs (126 OP SCCs and 222 non-OP SCCs), we evaluated diagnostic performances of different HPV tests in OP and non-OP SCCs: PCR, p16 immunostaining, in situ hybridization targeting DNA (DNA-CISH) and RNA (RNA-CISH), combined p16 + DNA-CISH, and combined p16 + RNA-CISH. HPV DNA (PCR) was detected in 26% of all tumors (44% of OP SCCs and 17% of non-OP SCCs). For OP SCCs, RNA-CISH was the most sensitive standalone test (88%), but p16 + RNA-CISH was even more sensitive (95%). Specificities were the same for RNA-CISH and DNA-CISH (97%) but it was better for p16 + RNA-CISH (100%). For non-OP SCCs, all tests had sensitivities below 50%, and RNA-CISH, DNA-CISH and p16 + DNA-CISH had respectively 100%, 97% and 99% specificities. As a standalone test, RNA-CISH is the most performant assay to detect HPV in OP SCCs, and combined p16 + RNA-CISH test slightly improves its performances. However, RNA-CISH has the advantage of being one single test. Like p16 and DNA-CISH, RNA-CISH performances are poor in non-OP SCCs to detect HPV, and combining tests does not improve performances. Copyright © 2018. Published by Elsevier Inc.

Enzyme-free electrochemical detection of microRNA-21 using immobilized hairpin probes and a target-triggered hybridization chain reaction amplification strategy

International Nuclear Information System (INIS)

Liu, Hongying; Bei, Xiaoqiong; Xia, Qiuting; Fu, Yan; Zhang, Shi; Liu, Maochuan; Fan, Kai; Zhang, Mingzhen; Yang, Yong

2016-01-01

We describe a sensitive enzyme-free bioassay for the determination of microRNA-21. It is based on a combination of target-triggered hybridization chain reaction, tagging with CdTe quantum dots (QDs), and anodic stripping voltammetry. Firstly, a thiolated capture hairpin probe SH-HP1 was immobilized on the surface of a gold electrode. HP1 unfolds in the presence of microRNA-21. If hairpin probe 2 (HP2) is present, a HP1-HP2 complex will be formed which possesses an exposed stem of HP2, and microRNA is released in parallel. The released microRNA-21 triggers a hybridization chain reaction and this leads to form an exposed DNA segment of HP2 and cycle use microRNA-21. With the aid of assistant DNA A1 and A2, the exposed DNA segment of HP2 progressed to a long double strand. The strand is rich in CdTe QDs with the help of QDs-A1. Then, the attached QDs were dissolved with HNO 3 to give dissolved Cd(II) ions. Finally, the corresponding electrochemical current response of Cd(II) is monitored by anodic stripping voltammetry and used to quantify the concentration of microRNA-21. More microRNA-21 participated in this reaction increases the number of CdTe QDs, which results in increased electrochemical current. Thus, an ultrasensitive detection of microRNA-21 is accomplished by anodic stripping voltammetry. This gene assay displays a detection limit as low as 33 aM. It can discriminate between complementary DNA sequence and single-base mismatched DNA, indicating its high specificity. (author)

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

Energy Technology Data Exchange (ETDEWEB)

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

2008-12-01

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

Isolation of Microarray-Grade Total RNA, MicroRNA, and DNA from a Single PAXgene Blood RNA Tube

DEFF Research Database (Denmark)

Kruhøffer, Mogens; Andersen, Lars Dyrskjøt; Voss, Thorsten

2007-01-01

We have developed a procedure for isolation of microRNA and genomic DNA in addition to total RNA from whole blood stabilized in PAXgene Blood RNA tubes. The procedure is based on automatic extraction on a BioRobot MDx and includes isolation of DNA from a fraction of the stabilized blood...... and recovery of small RNA species that are otherwise lost. The procedure presented here is suitable for large-scale experiments and is amenable to further automation. Procured total RNA and DNA was tested using Affymetrix Expression and single-nucleotide polymorphism GeneChips, respectively, and isolated micro......RNA was tested using spotted locked nucleic acid-based microarrays. We conclude that the yield and quality of total RNA, microRNA, and DNA from a single PAXgene blood RNA tube is sufficient for downstream microarray analysis....

Alterations in messenger RNA and small nuclear RNA metabolism resulting from fluorouracil incorporation

International Nuclear Information System (INIS)

Armstrong, R.D.; Cadman, E.C.

1985-01-01

Studies were completed to examine the effect of 5-fluorouracil (FUra) incorporation on messenger RNA (mRNA) and small molecular weight nuclear RNA (SnRNA) metabolism. Studies of mRNA were completed using cDNA-mRNA hybridization methods to specifically examine dihydrofolate reductase (DHFR) mRNA. C 3 -L5178Y murine leukemia cells which are gene-amplified for DHFR, were exposed to FUra for 6, 12 or 24 hr, and the nuclear and cytoplasmic levels of DHFR-mRNA determined by hybridization with 32 P-DHFR-cDNA. FUra produced a dose-dependent increase in nuclear DHFR-mRNA levels, while total cytoplasmic DHFR-mRNA levels appeared to be unchanged. To examine only mRNA synthesized during FUra exposure, cells were also treated concurrently with [ 3 H] cytidine, and the [ 3 H]mRNA-cDNA hybrids measured following S 1 -nuclease treatment. FUra produced a concentration-dependent increase in nascent nuclear DHFR-mRNA levels, and a decrease in nascent cytoplasmic DHFR-mRNAs levels. These results suggest that FUra produces either an inhibition of mRNA processing, or an inhibition of nuclear-cytoplasmic transport. Preliminary experiments to examine ATP-dependent mRNA transport were completed with isolated nuclei from cells treated with FUra for 1 or 24 hr and then pulse-labeled for 1 hr with [ 3 H] cytidine. The results demonstrate a FUra-concentration and time-dependent inhibition of ATP-mediated mRNA efflux

DNA hybridization sensing for cytogenetic analysis

DEFF Research Database (Denmark)

Kwasny, Dorota; Dapra, Johannes; Brøgger, Anna Line

2013-01-01

are rearrangements between two chromosome arms that results in two derivative chromosomes having a mixed DNA sequence. The current detection method is a Fluorescent In situ Hybridization, which requires a use of expensive, fluorescently labeled probes that target the DNA sequences of two chromosomes involved...... in the translocation (Kwasny et al., 2012). We have developed a new double hybridization assay that allows for sorting of the DNA chromosomal fragments into separate compartment, moreover allowing for detection of the translocation. To detect the translocation it is necessary to determine that the two DNA sequences...... forming a derivative chromosome are connected, which is achieved by two subsequent hybridization steps. The first example of the translocation detection was presented on lab-on-a-disc using fluorescently labeled DNA fragments, representing the derivative chromosome (Brøgger et al., 2012). To allow...

Identification of Persistent RNA-DNA Hybrid Structures within the Origin of Replication of Human Cytomegalovirus

OpenAIRE

Prichard, Mark N.; Jairath, Sanju; Penfold, Mark E. T.; Jeor, Stephen St.; Bohlman, Marlene C.; Pari, Gregory S.

1998-01-01

Human cytomegalovirus (HCMV) lytic-phase DNA replication initiates at the cis-acting origin of replication, oriLyt. oriLyt is a structurally complex region containing repeat elements and transcription factor binding sites. We identified two site-specific alkali-labile regions within oriLyt which flank an alkali-resistant DNA segment. These alkali-sensitive regions were the result of the degradation of two RNA species embedded within oriLyt and covalently linked to viral DNA. The virus-associa...

Drosophila interspecific hybrids phenocopy piRNA-pathway mutants.

Directory of Open Access Journals (Sweden)

Erin S Kelleher

Full Text Available The Piwi-interacting RNA (piRNA pathway defends the germline of animals from the deleterious activity of selfish transposable elements (TEs through small-RNA mediated silencing. Adaptation to novel invasive TEs is proposed to occur by incorporating their sequences into the piRNA pool that females produce and deposit into their eggs, which then propagates immunity against specific TEs to future generations. In support of this model, the F1 offspring of crosses between strains of the same Drosophila species sometimes suffer from germline derepression of paternally inherited TE families, caused by a failure of the maternal strain to produce the piRNAs necessary for their regulation. However, many protein components of the Drosophila piRNA pathway exhibit signatures of positive selection, suggesting that they also contribute to the evolution of host genome defense. Here we investigate piRNA pathway function and TE regulation in the F1 hybrids of interspecific crosses between D. melanogaster and D. simulans and compare them with intraspecific control crosses of D. melanogaster. We confirm previous reports showing that intraspecific crosses are characterized by derepression of paternally inherited TE families that are rare or absent from the maternal genome and piRNA pool, consistent with the role of maternally deposited piRNAs in shaping TE silencing. In contrast to the intraspecific cross, we discover that interspecific hybrids are characterized by widespread derepression of both maternally and paternally inherited TE families. Furthermore, the pattern of derepression of TE families in interspecific hybrids cannot be attributed to their paucity or absence from the piRNA pool of the maternal species. Rather, we demonstrate that interspecific hybrids closely resemble piRNA effector-protein mutants in both TE misregulation and aberrant piRNA production. We suggest that TE derepression in interspecific hybrids largely reflects adaptive divergence of piRNA

DNA hybridization sensor based on pentacene thin film transistor.

Science.gov (United States)

Kim, Jung-Min; Jha, Sandeep Kumar; Chand, Rohit; Lee, Dong-Hoon; Kim, Yong-Sang

2011-01-15

A DNA hybridization sensor using pentacene thin film transistors (TFTs) is an excellent candidate for disposable sensor applications due to their low-cost fabrication process and fast detection. We fabricated pentacene TFTs on glass substrate for the sensing of DNA hybridization. The ss-DNA (polyA/polyT) or ds-DNA (polyA/polyT hybrid) were immobilized directly on the surface of the pentacene, producing a dramatic change in the electrical properties of the devices. The electrical characteristics of devices were studied as a function of DNA immobilization, single-stranded vs. double-stranded DNA, DNA length and concentration. The TFT device was further tested for detection of λ-phage genomic DNA using probe hybridization. Based on these results, we propose that a "label-free" detection technique for DNA hybridization is possible through direct measurement of electrical properties of DNA-immobilized pentacene TFTs. Copyright © 2010 Elsevier B.V. All rights reserved.

Interaction of sulforaphane with DNA and RNA.

Directory of Open Access Journals (Sweden)

Farzaneh Abassi Joozdani

Full Text Available Sulforaphane (SFN is an isothiocyanate found in cruciferous vegetables with anti-inflammatory, anti-oxidant and anti-cancer activities. However, the antioxidant and anticancer mechanism of sulforaphane is not well understood. In the present research, we reported binding modes, binding constants and stability of SFN-DNA and -RNA complexes by Fourier transform infrared (FTIR and UV-Visible spectroscopic methods. Spectroscopic evidence showed DNA intercalation with some degree of groove binding. SFN binds minor and major grooves of DNA and backbone phosphate (PO2, while RNA binding is through G, U, A bases with some degree of SFN-phosphate (PO2 interaction. Overall binding constants were estimated to be K(SFN-DNA=3.01 (± 0.035×10(4 M(-1 and K(SFN-RNA= 6.63 (±0.042×10(3 M(-1. At high SFN concentration (SFN/RNA = 1/1, DNA conformation changed from B to A occurred, while RNA remained in A-family structure.

Mechanisms of Surface-Mediated DNA Hybridization

Science.gov (United States)

2015-01-01

Single-molecule total internal reflection fluorescence microscopy was employed in conjunction with resonance energy transfer (RET) to observe the dynamic behavior of donor-labeled ssDNA at the interface between aqueous solution and a solid surface decorated with complementary acceptor-labeled ssDNA. At least 100 000 molecular trajectories were determined for both complementary strands and negative control ssDNA. RET was used to identify trajectory segments corresponding to the hybridized state. The vast majority of molecules from solution adsorbed nonspecifically to the surface, where a brief two-dimensional search was performed with a 7% chance of hybridization. Successful hybridization events occurred with a characteristic search time of ∼0.1 s, and unsuccessful searches resulted in desorption from the surface, ultimately repeating the adsorption and search process. Hybridization was reversible, and two distinct modes of melting (i.e., dehybridization) were observed, corresponding to long-lived (∼15 s) and short-lived (∼1.4 s) hybridized time intervals. A strand that melted back onto the surface could rehybridize after a brief search or desorb from the interface. These mechanistic observations provide guidance for technologies that involve DNA interactions in the near-surface region, suggesting a need to design surfaces that both enhance the complex multidimensional search process and stabilize the hybridized state. PMID:24708278

Isolation of RNA for dot hybridization by heparin-DNase I treatment of whole cell lysate.

Science.gov (United States)

Krawczyk, Z; Wu, C

1987-08-15

We have developed a new procedure for the rapid preparation of undegraded total RNA from cultured cells for specific quantitation by dot blotting analysis. Pelleted cells are resuspended in hypotonic solution containing a ribonuclease inhibitor and heparin and disrupted by freeze-thaw. Heparin is employed as an agent for nuclear lysis, dissociation of chromosomal protein, and release of mRNA from rough endoplasmic reticulum. We eliminate chromosomal DNA by digestion with DNase I and denature the RNA in the lysate with formaldehyde. After centrifugation to remove debris, the supernatant is used directly for dot blotting. All manipulations are performed in the same microfuge tube and recovery of RNA is quantitative. The procedure is especially useful for processing large numbers of samples. We illustrate its versatility by analysis of specific RNAs in Drosophila, rat, and human cell lines. In reconstruction experiments, less than 80 molecules per cell of a small RNA (beta-globin) can be detected under highly stringent hybridization conditions, using only moderately labeled double-stranded plasmid DNA probes and short film exposures.

Increased mRNA expression of a laminin-binding protein in human colon carcinoma: Complete sequence of a full-length cDNA encoding the protein

International Nuclear Information System (INIS)

Yow, Hsiukang; Wong, Jau Min; Chen, Hai Shiene; Lee, C.; Steele, G.D. Jr.; Chen, Lanbo

1988-01-01

Reliable markers to distinguish human colon carcinoma from normal colonic epithelium are needed particularly for poorly differentiated tumors where no useful marker is currently available. To search for markers the authors constructed cDNA libraries from human colon carcinoma cell lines and screened for clones that hybridize to a greater degree with mRNAs of colon carcinomas than with their normal counterparts. Here they report one such cDNA clone that hybridizes with a 1.2-kilobase (kb) mRNA, the level of which is ∼9-fold greater in colon carcinoma than in adjacent normal colonic epithelium. Blot hybridization of total RNA from a variety of human colon carcinoma cell lines shows that the level of this 1.2-kb mRNA in poorly differentiated colon carcinomas is as high as or higher than that in well-differentiated carcinomas. Molecular cloning and complete sequencing of cDNA corresponding to the full-length open reading frame of this 1.2-kb mRNA unexpectedly show it to contain all the partial cDNA sequence encoding 135 amino acid residues previously reported for a human laminin receptor. The deduced amino acid sequence suggests that this putative laminin-binding protein from human colon carcinomas consists of 295 amino acid residues with interesting features. There is an unusual C-terminal 70-amino acid segment, which is trypsin-resistant and highly negatively charged

Hybridization thermodynamics of DNA bound to gold nanoparticles

International Nuclear Information System (INIS)

Lang, Brian

2010-01-01

Isothermal Titration Calorimetry (ITC) was used to study the thermodynamics of hybridization on DNA-functionalized colloidal gold nanoparticles. When compared to the thermodynamics of hybridization of DNA that is free in solution, the differences in the values of the Gibbs free energy of reaction, Δ r G o , the enthalpy, Δ r H o , and entropy, Δ r S o , were small. The change in Δ r G o between the free and bound states was always positive but with statistical significance outside the 95% confidence interval, implying the free DNA is slightly more stable than when in the bound state. Additionally, ITC was also able to reveal information about the binding stoichiometry of the hybridization reactions on the DNA-functionalized gold nanoparticles, and indicates that there is a significant fraction of the DNA on gold nanoparticle surface that is unavailable for DNA hybridization. Furthermore, the fraction of available DNA is dependent on the spacer group on the DNA that is used to span the gold surface from that to the probe DNA.

Modulation of repetitive genes in the parent forms of heterozygous corn hybrids

International Nuclear Information System (INIS)

Gilyazetdinov, S.Ya.; Zimnitskii, A.N.; Yakhin, I.A.; Bikbaeva, E.S.

1987-01-01

The number of copies of the genes of high-molecular-weight rRNA, 5 S r RNA, and certain other families of repetitive sequences of DNA in the genome of different forms of corn is not coordinated but is stably inherited in the same strains. The authors present the results of their investigations into the repetition of the genes of tRNA, 5 S rRNA, histones, and the controlling element Ds of corn for the highly heterozygous hybrid Slava (VIR 44 x VIR 38), the medium-heterozygous hybrid Svetoch (VIR 40 x VIR 43), the low heterozygous hybrid Iskra (VIR 26 x VIR 27), and their parent strains. The relative content of these sequences was studied by the molecular hybridization of DNA immobilized on nitrocellulose filters with [ 125 I]tRNA labeled in vitro, 5 S rRNA, histone DNA of Drosophila, and the Ds-element of corn. The DNA preparations were isolated from the zones of the meristem (1.5-2mm), elongation (4-5mm), differentiation of the roots (3 cm), of 3-4 day seedlings, and from isolated embryos of 4 h and 24 h seedlings. The DNA of the embryos immobilized on the filters was preliminarily incubated with unlabeled high-molecular-weight rRNA in the experiments with tRNA and 5 S rRNA, while when histone DNA and the Ds element of corn were used in the hybridization reaction, it was preliminary incubated with plasmid DNA

An Engineered Kinetic Amplification Mechanism for Single Nucleotide Variant Discrimination by DNA Hybridization Probes.

Science.gov (United States)

Chen, Sherry Xi; Seelig, Georg

2016-04-20

Even a single-nucleotide difference between the sequences of two otherwise identical biological nucleic acids can have dramatic functional consequences. Here, we use model-guided reaction pathway engineering to quantitatively improve the performance of selective hybridization probes in recognizing single nucleotide variants (SNVs). Specifically, we build a detection system that combines discrimination by competition with DNA strand displacement-based catalytic amplification. We show, both mathematically and experimentally, that the single nucleotide selectivity of such a system in binding to single-stranded DNA and RNA is quadratically better than discrimination due to competitive hybridization alone. As an additional benefit the integrated circuit inherits the property of amplification and provides at least 10-fold better sensitivity than standard hybridization probes. Moreover, we demonstrate how the detection mechanism can be tuned such that the detection reaction is agnostic to the position of the SNV within the target sequence. in contrast, prior strand displacement-based probes designed for kinetic discrimination are highly sensitive to position effects. We apply our system to reliably discriminate between different members of the let-7 microRNA family that differ in only a single base position. Our results demonstrate the power of systematic reaction network design to quantitatively improve biotechnology.

Hybrid male sterility is caused by mitochondrial DNA deletion.

Science.gov (United States)

Hayashida, Kenji; Kohno, Shigeru

2009-07-01

Although it is known that the hybrid male mouse is sterile just like any other animal's heterogametic sex, the reason why only the male germ cells are impaired has yet to be discovered. TdT-mediated dUTP nick end labeling assay using a confocal fluorescence microscope and DNA fragmentation assay of hybrid testis indicated destruction of the mitochondrial DNA (mtDNA) rather than the nuclear DNA. Previously we reported that maternal mtDNA inheritance is through selective sperm mtDNA elimination based on the sperm factor and two egg factors, and expression of these three factors was recognized in the hybrid testis. It was thereby assumed that mtDNA destruction caused by the expression of maternal mtDNA inheritance system in male germ cells is implicated in the hybrid male sterility of mice.

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

Directory of Open Access Journals (Sweden)

Jie Zhu

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

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

Science.gov (United States)

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

2013-01-01

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

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

Science.gov (United States)

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

2013-01-01

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

An improved method for RNA isolation and cDNA library construction from immature seeds of Jatropha curcas L

Directory of Open Access Journals (Sweden)

Kaur Jatinder

2010-05-01

Full Text Available Abstract Background RNA quality and quantity is sometimes unsuitable for cDNA library construction, from plant seeds rich in oil, polysaccharides and other secondary metabolites. Seeds of jatropha (Jatropha curcas L. are rich in fatty acids/lipids, storage proteins, polysaccharides, and a number of other secondary metabolites that could either bind and/or co-precipitate with RNA, making it unsuitable for downstream applications. Existing RNA isolation methods and commercial kits often fail to deliver high-quality total RNA from immature jatropha seeds for poly(A+ RNA purification and cDNA synthesis. Findings A protocol has been developed for isolating good quality total RNA from immature jatropha seeds, whereby a combination of the CTAB based RNA extraction method and a silica column of a commercial plant RNA extraction kit is used. The extraction time was reduced from two days to about 3 hours and the RNA was suitable for poly(A+ RNA purification, cDNA synthesis, cDNA library construction, RT-PCR, and Northern hybridization. Based on sequence information from selected clones and amplified PCR product, the cDNA library seems to be a good source of full-length jatropha genes. The method was equally effective for isolating RNA from mustard and rice seeds. Conclusions This is a simple CTAB + silica column method to extract high quality RNA from oil rich immature jatropha seeds that is suitable for several downstream applications. This method takes less time for RNA extraction and is equally effective for other tissues where the quality and quantity of RNA is highly interfered by the presence of fatty acids, polysaccharides and polyphenols.

Bovine viral diarrhea virus: molecular cloning of genomic RNA and its diagnostic application

International Nuclear Information System (INIS)

Brock, K.V.

1987-01-01

Molecular cloning of a field isolate of bovine viral diarrhea virus (BVDV) strain 72 RNA was done in this study. The sensitivity and specificity of cloned cDNA sequences in hybridization assays with various BVDV strains were determined. cDNA was synthesized from polyadenylated BVDV RNA templates with oligo-dT primers, reverse transcriptase, and DNA polymerase I. The newly synthesized double-stranded BVDV cDNA was C-tailed with terminal deoxytransferase and annealed into G-tailed, Pst-1-cut pUC9 plasmid. Escherichia coli was transformed with the recombinant plasmids and a library of approximately 200 BVDV specific cDNA clones varying in length from 0.5 to 2.6 kilobases were isolated. The sensitivity and specificity of hybridization between the labelled cDNA and BVDV target sequences were determined. Cloned BVDV sequences were isolated from pUC9 plasmid DNA and labelled with 32 P by nick translation. The detection limit by dot blot hybridization assay was 20 pg of purified genomic BVDV RNA. cDNA hybridization probes were specific for all strains of BVDV tested, regardless of whether they were noncytopathic and cytopathic, but did not hybridize with heterologous bovine viruses tested. Probes did not hybridize with uninfected cell culture or cellular RNA. Hybridization probes were at least as sensitive as infectivity assays in detecting homologous virus

Nucleic acids in mummified plant seeds: screening of twelve specimens by gel-electrophoresis, molecular hybridization and DNA cloning.

Science.gov (United States)

Rollo, F; La Marca, A; Amici, A

1987-02-01

Twelve seed specimens of varying ages and from different archaeological sites were analyzed for the presence of polymerized DNA and RNA. Amongst the samples tested, one of Vitis vinifera from an archaeological site in Iran (2,000-3,000 B.C.) was found to be completely devoid of nucleic acids. Zea mais seeds of Precolumbial age from Peru (about 800 A.D.) contained depolymerized DNA and RNA. Samples of Vitis vinifera and Rubus sp. from a Lombard archaeological site (800 A.D.) as well as radiocarbon dated seeds from the site of the "Spring Sanctuary" near Metaponto (I-IV century B.C.) were found to contain polymerized DNA and rRNA bands. However the electrophoretic properties of the rRNAs in one case and hybridization experiments performed with cloned seed DNA in the other, clearly demonstrated that the polymerized nucleic acids were not of plant origin.

DNA interrogation by the CRISPR RNA-guided endonuclease Cas9.

Science.gov (United States)

Sternberg, Samuel H; Redding, Sy; Jinek, Martin; Greene, Eric C; Doudna, Jennifer A

2014-03-06

The clustered regularly interspaced short palindromic repeats (CRISPR)-associated enzyme Cas9 is an RNA-guided endonuclease that uses RNA-DNA base-pairing to target foreign DNA in bacteria. Cas9-guide RNA complexes are also effective genome engineering agents in animals and plants. Here we use single-molecule and bulk biochemical experiments to determine how Cas9-RNA interrogates DNA to find specific cleavage sites. We show that both binding and cleavage of DNA by Cas9-RNA require recognition of a short trinucleotide protospacer adjacent motif (PAM). Non-target DNA binding affinity scales with PAM density, and sequences fully complementary to the guide RNA but lacking a nearby PAM are ignored by Cas9-RNA. Competition assays provide evidence that DNA strand separation and RNA-DNA heteroduplex formation initiate at the PAM and proceed directionally towards the distal end of the target sequence. Furthermore, PAM interactions trigger Cas9 catalytic activity. These results reveal how Cas9 uses PAM recognition to quickly identify potential target sites while scanning large DNA molecules, and to regulate scission of double-stranded DNA.

DNA interrogation by the CRISPR RNA-guided endonuclease Cas9

Science.gov (United States)

Sternberg, Samuel H.; Redding, Sy; Jinek, Martin; Greene, Eric C.; Doudna, Jennifer A.

2014-03-01

The clustered regularly interspaced short palindromic repeats (CRISPR)-associated enzyme Cas9 is an RNA-guided endonuclease that uses RNA-DNA base-pairing to target foreign DNA in bacteria. Cas9-guide RNA complexes are also effective genome engineering agents in animals and plants. Here we use single-molecule and bulk biochemical experiments to determine how Cas9-RNA interrogates DNA to find specific cleavage sites. We show that both binding and cleavage of DNA by Cas9-RNA require recognition of a short trinucleotide protospacer adjacent motif (PAM). Non-target DNA binding affinity scales with PAM density, and sequences fully complementary to the guide RNA but lacking a nearby PAM are ignored by Cas9-RNA. Competition assays provide evidence that DNA strand separation and RNA-DNA heteroduplex formation initiate at the PAM and proceed directionally towards the distal end of the target sequence. Furthermore, PAM interactions trigger Cas9 catalytic activity. These results reveal how Cas9 uses PAM recognition to quickly identify potential target sites while scanning large DNA molecules, and to regulate scission of double-stranded DNA.

Cloning and characterization of DNA complementary to the canine distemper virus mRNA encoding matrix, phosphoprotein, and nucleocapsid protein

International Nuclear Information System (INIS)

Rozenblatt, S.; Eizenberg, O.; Englund, G.; Bellini, W.J.

1985-01-01

Double-stranded cDNA synthesized from total polyadenylate-containing mRNA, extracted from monkey kidney cells infected with canine distemper virus (CDV), has been cloned into the PstI site of Escherichia coli plasmid pBR322. Clones containing canine distemper virus DNA were identified by hybridization to a canine distemper virus-specific, 32 P-labeled cDNA. Four specific clones containing different classes of sequences have been identified. The cloned plasmids contain inserts of 800 (clone 44-80), 960 (clone 74-16), 1700 (clone 364), and 950 (clone 40-9) base pairs. The sizes of the mRNA species complementary to these inserts are 1500, 1850, 1850 and 2500 nucleotides, respectively, as determined by the Northern technique. Three of the cloned DNA fragments were further identified as the reverse transcripts of the mRNA coding for the matrix, phosphoprotein, and nucleocapsid protein of CDV

Cloning and characterization of DNA complementary to the canine distemper virus mRNA encoding matrix, phosphoprotein, and nucleocapsid protein

Energy Technology Data Exchange (ETDEWEB)

Rozenblatt, S.; Eizenberg, O.; Englund, G.; Bellini, W.J.

1985-02-01

Double-stranded cDNA synthesized from total polyadenylate-containing mRNA, extracted from monkey kidney cells infected with canine distemper virus (CDV), has been cloned into the PstI site of Escherichia coli plasmid pBR322. Clones containing canine distemper virus DNA were identified by hybridization to a canine distemper virus-specific, /sup 32/P-labeled cDNA. Four specific clones containing different classes of sequences have been identified. The cloned plasmids contain inserts of 800 (clone 44-80), 960 (clone 74-16), 1700 (clone 364), and 950 (clone 40-9) base pairs. The sizes of the mRNA species complementary to these inserts are 1500, 1850, 1850 and 2500 nucleotides, respectively, as determined by the Northern technique. Three of the cloned DNA fragments were further identified as the reverse transcripts of the mRNA coding for the matrix, phosphoprotein, and nucleocapsid protein of CDV.

mRNA related to insulin family in human placenta

International Nuclear Information System (INIS)

Younes, M.A.; D'Agostino, J.B.; Frazier, M.L.; Besch, P.K.

1986-01-01

The authors have previously reported that human term placenta contains mRNA displaying sequence homology to a rat preproinsulin I cDNA clone (p119). When placental poly(A + ) RNA was analyzed for homology to p119 by RNA/DNA blot hybridization, prominent hybridization was observed which was found by densitometric analysis to be three-fold higher than control. To further characterize this insulin-like message, a cDNA library was generated (approx.7000 transformants) using normal term cesarean-sectioned tissue to prepare placental poly(A + ) RNA templates. Five hundred transformants were initially screened by colony hybridization using a 32 P-labeled rat preproinsulin I cDNA as probe. Of the ten initial positives obtained, three were found to be true positives based on Southern hybridization analyses of the recombinant plasmids. Using Taq I digested pBr322 as a size marker, the cDNAs were found to be approximately 300 bp in length. Preliminary DNA sequencing using the Sanger dideoxy chain termination method has revealed that one of these clones displays significant homology to the 5' region of human insulin-like growth factors I and II

mRNA related to insulin family in human placenta

Energy Technology Data Exchange (ETDEWEB)

Younes, M.A.; D' Agostino, J.B.; Frazier, M.L.; Besch, P.K.

1986-03-01

The authors have previously reported that human term placenta contains mRNA displaying sequence homology to a rat preproinsulin I cDNA clone (p119). When placental poly(A/sup +/) RNA was analyzed for homology to p119 by RNA/DNA blot hybridization, prominent hybridization was observed which was found by densitometric analysis to be three-fold higher than control. To further characterize this insulin-like message, a cDNA library was generated (approx.7000 transformants) using normal term cesarean-sectioned tissue to prepare placental poly(A/sup +/) RNA templates. Five hundred transformants were initially screened by colony hybridization using a /sup 32/P-labeled rat preproinsulin I cDNA as probe. Of the ten initial positives obtained, three were found to be true positives based on Southern hybridization analyses of the recombinant plasmids. Using Taq I digested pBr322 as a size marker, the cDNAs were found to be approximately 300 bp in length. Preliminary DNA sequencing using the Sanger dideoxy chain termination method has revealed that one of these clones displays significant homology to the 5' region of human insulin-like growth factors I and II.

Collaborating functions of BLM and DNA topoisomerase I in regulating human rDNA transcription

Energy Technology Data Exchange (ETDEWEB)

Grierson, Patrick M. [Department of Microbiology, Immunology and Medical Genetics, The Ohio State University College of Medicine, Columbus, OH 43210 (United States); Acharya, Samir, E-mail: samir.acharya@osumc.edu [Department of Microbiology, Immunology and Medical Genetics, The Ohio State University College of Medicine, Columbus, OH 43210 (United States); Groden, Joanna [Department of Microbiology, Immunology and Medical Genetics, The Ohio State University College of Medicine, Columbus, OH 43210 (United States)

2013-03-15

Bloom's syndrome (BS) is an inherited disorder caused by loss of function of the recQ-like BLM helicase. It is characterized clinically by severe growth retardation and cancer predisposition. BLM localizes to PML nuclear bodies and to the nucleolus; its deficiency results in increased intra- and inter-chromosomal recombination, including hyper-recombination of rDNA repeats. Our previous work has shown that BLM facilitates RNA polymerase I-mediated rRNA transcription in the nucleolus (Grierson et al., 2012 [18]). This study uses protein co-immunoprecipitation and in vitro transcription/translation (IVTT) to identify a direct interaction of DNA topoisomerase I with the C-terminus of BLM in the nucleolus. In vitro helicase assays demonstrate that DNA topoisomerase I stimulates BLM helicase activity on a nucleolar-relevant RNA:DNA hybrid, but has an insignificant effect on BLM helicase activity on a control DNA:DNA duplex substrate. Reciprocally, BLM enhances the DNA relaxation activity of DNA topoisomerase I on supercoiled DNA substrates. Our study suggests that BLM and DNA topoisomerase I function coordinately to modulate RNA:DNA hybrid formation as well as relaxation of DNA supercoils in the context of nucleolar transcription.

Collaborating functions of BLM and DNA topoisomerase I in regulating human rDNA transcription

International Nuclear Information System (INIS)

Grierson, Patrick M.; Acharya, Samir; Groden, Joanna

2013-01-01

Bloom's syndrome (BS) is an inherited disorder caused by loss of function of the recQ-like BLM helicase. It is characterized clinically by severe growth retardation and cancer predisposition. BLM localizes to PML nuclear bodies and to the nucleolus; its deficiency results in increased intra- and inter-chromosomal recombination, including hyper-recombination of rDNA repeats. Our previous work has shown that BLM facilitates RNA polymerase I-mediated rRNA transcription in the nucleolus (Grierson et al., 2012 [18]). This study uses protein co-immunoprecipitation and in vitro transcription/translation (IVTT) to identify a direct interaction of DNA topoisomerase I with the C-terminus of BLM in the nucleolus. In vitro helicase assays demonstrate that DNA topoisomerase I stimulates BLM helicase activity on a nucleolar-relevant RNA:DNA hybrid, but has an insignificant effect on BLM helicase activity on a control DNA:DNA duplex substrate. Reciprocally, BLM enhances the DNA relaxation activity of DNA topoisomerase I on supercoiled DNA substrates. Our study suggests that BLM and DNA topoisomerase I function coordinately to modulate RNA:DNA hybrid formation as well as relaxation of DNA supercoils in the context of nucleolar transcription

(α,α-dimethyl)glycyl (dmg) PNAs: achiral PNA analogs that form stronger hybrids with cDNA relative to isosequential RNA.

Science.gov (United States)

Gourishankar, Aland; Ganesh, Krishna N

2012-01-01

The design and facile synthesis of sterically constrained new analogs of PNA having gem-dimethyl substitutions on glycine (dmg-PNA-T) is presented. The PNA oligomers [aminoethyl dimethylglycyl (aedmg) and aminopropyl dimethylglycyl (apdmg)] synthesized from the monomers 6 and 12) effected remarkable stabilization of homothyminePNA(2):homoadenine DNA/RNA triplexes and mixed base sequence duplexes with target cDNA or RNA. They show a higher binding to DNA relative to that with isosequential RNA. This may be a structural consequence of the sterically rigid gem-dimethyl group, imposing a pre-organized conformation favorable for complex formation with cDNA. The results complement our previous work that had demonstrated that cyclohexanyl-PNAs favor binding with cRNA compared with cDNA and imply that the biophysical and structural properties of PNAs can be directed by introduction of the right rigidity in PNA backbone devoid of chirality. This approach of tweaking selectivity in binding of PNA constructs by installing gem-dimethyl substitution in PNA backbone can be extended to further fine-tuning by similar substitution in the aminoethyl segment as well either individually or in conjunction with present substitution.

The Spectral Properties and Photostability of DNA, RNA and Oligonucleotides

International Nuclear Information System (INIS)

Kudrya, V.Yu.; Yashchuk, V.M.

2012-01-01

The present work discusses the results of comparative investigations of the optical absorption, luminescence, and photostability of the biomacromolecules (DNA, RNA), as well as synthetic poly- and oligonucleotides. The separate nucleotides in DNA and RNA are examined as almost independent absorbing centers. It is confirmed that the main triplet excitons traps responsible for the DNA phosphorescence emission are AT-complexes in DNA. In contrast to DNA, the main triplet excitons traps in RNA are adenosine bases. These bases are the most photostable against UV-irradiation as compared with all other nucleotides in both DNA and RNA. The fact of the photostability of adenosine bases and the AT-complex provides the existence of the DNA/RNA self-protection mechanisms against a damage caused by UV-irradiation. It is found the deoxyribonucleotides are more photostable than the corresponding ribonucleotides. So, the results presented here show that DNA is more photostable than RNA.

Detection of DNA hybridizations using solid-state nanopores

International Nuclear Information System (INIS)

Balagurusamy, Venkat S K; Weinger, Paul; Sean Ling, Xinsheng

2010-01-01

We report an experimental study of using DNA translocation through solid-state nanopores to detect the sequential arrangement of two double-stranded 12-mer hybridization segments on a single-stranded DNA molecule. The sample DNA is a trimer molecule formed by hybridizing three single-stranded oligonucleotides. A polystyrene bead is attached to the end of the trimer DNA, providing a mechanism in slowing down the translocation and suppressing the thermal diffusion, thereby allowing the detection of short features of DNA by standard patch-clamp electronics. The electrical signature of the translocation of a trimer molecule through a nanopore has been identified successfully in the temporal traces of ionic current. The results reported here represent the first successful attempt in using a solid-state nanopore as an ionic scanning device in resolving individual hybridization segments (or 'probes') on a DNA molecule.

Detection of DNA hybridizations using solid-state nanopores

Energy Technology Data Exchange (ETDEWEB)

Balagurusamy, Venkat S K; Weinger, Paul; Sean Ling, Xinsheng, E-mail: Xinsheng_Ling@brown.edu [Department of Physics, Brown University, Providence, RI 02912 (United States)

2010-08-20

We report an experimental study of using DNA translocation through solid-state nanopores to detect the sequential arrangement of two double-stranded 12-mer hybridization segments on a single-stranded DNA molecule. The sample DNA is a trimer molecule formed by hybridizing three single-stranded oligonucleotides. A polystyrene bead is attached to the end of the trimer DNA, providing a mechanism in slowing down the translocation and suppressing the thermal diffusion, thereby allowing the detection of short features of DNA by standard patch-clamp electronics. The electrical signature of the translocation of a trimer molecule through a nanopore has been identified successfully in the temporal traces of ionic current. The results reported here represent the first successful attempt in using a solid-state nanopore as an ionic scanning device in resolving individual hybridization segments (or 'probes') on a DNA molecule.

Conflict RNA modification, host-parasite co-evolution, and the origins of DNA and DNA-binding proteins1.

Science.gov (United States)

McLaughlin, Paul J; Keegan, Liam P

2014-08-01

Nearly 150 different enzymatically modified forms of the four canonical residues in RNA have been identified. For instance, enzymes of the ADAR (adenosine deaminase acting on RNA) family convert adenosine residues into inosine in cellular dsRNAs. Recent findings show that DNA endonuclease V enzymes have undergone an evolutionary transition from cleaving 3' to deoxyinosine in DNA and ssDNA to cleaving 3' to inosine in dsRNA and ssRNA in humans. Recent work on dsRNA-binding domains of ADARs and other proteins also shows that a degree of sequence specificity is achieved by direct readout in the minor groove. However, the level of sequence specificity observed is much less than that of DNA major groove-binding helix-turn-helix proteins. We suggest that the evolution of DNA-binding proteins following the RNA to DNA genome transition represents the major advantage that DNA genomes have over RNA genomes. We propose that a hypothetical RNA modification, a RRAR (ribose reductase acting on genomic dsRNA) produced the first stretches of DNA in RNA genomes. We discuss why this is the most satisfactory explanation for the origin of DNA. The evolution of this RNA modification and later steps to DNA genomes are likely to have been driven by cellular genome co-evolution with viruses and intragenomic parasites. RNA modifications continue to be involved in host-virus conflicts; in vertebrates, edited cellular dsRNAs with inosine-uracil base pairs appear to be recognized as self RNA and to suppress activation of innate immune sensors that detect viral dsRNA.

Human papillomavirus mRNA and DNA testing in women with atypical squamous cells of undetermined significance

DEFF Research Database (Denmark)

Thomsen, Louise T; Dehlendorff, Christian; Junge, Jette

2016-01-01

In this prospective cohort study, we compared the performance of human papillomavirus (HPV) mRNA and DNA testing of women with atypical squamous cells of undetermined significance (ASC-US) during cervical cancer screening. Using a nationwide Danish pathology register, we identified women aged 30......-65 years with ASC-US during 2005-2011 who were tested for HPV16/18/31/33/45 mRNA using PreTect HPV-Proofer (n = 3,226) or for high-risk HPV (hrHPV) DNA using Hybrid Capture 2 (HC2) (n = 9,405) or Linear Array HPV-Genotyping test (LA) (n = 1,533). Women with ≥1 subsequent examination in the register (n = 13...... those testing HC2 negative (3.2% [95% CI: 2.2-4.2%] versus 0.5% [95% CI: 0.3-0.7%]). Patterns were similar after 18 months and 5 years'; follow-up; for CIN2+ and cancer as outcomes; across all age groups; and when comparing mRNA testing to hrHPV DNA testing using LA. In conclusion, the HPV16...

Structure of the Cpf1 endonuclease R-loop complex after target DNA cleavage

DEFF Research Database (Denmark)

Stella, Stefano; Alcón, Pablo; Montoya, Guillermo

2017-01-01

involved in DNA unwinding to form a CRISPR RNA (crRNA)-DNA hybrid and a displaced DNA strand. The protospacer adjacent motif (PAM) is recognized by the PAM-interacting domain. The loop-lysine helix-loop motif in this domain contains three conserved lysine residues that are inserted in a dentate manner...... and the crRNA-DNA hybrid, avoiding DNA re-annealing. Mutations in key residues reveal a mechanism linking the PAM and DNA nuclease sites. Analysis of the Cpf1 structures proposes a singular working model of RNA-guided DNA cleavage, suggesting new avenues for redesign of Cpf1....

Ddx19 links mRNA nuclear export with progression of transcription and replication and suppresses genomic instability upon DNA damage in proliferating cells.

Science.gov (United States)

Hodroj, Dana; Serhal, Kamar; Maiorano, Domenico

2017-09-03

The DEAD-box Helicase 19 (Ddx19) gene codes for an RNA helicase involved in both mRNA (mRNA) export from the nucleus into the cytoplasm and in mRNA translation. In unperturbed cells, Ddx19 localizes in the cytoplasm and at the cytoplasmic face of the nuclear pore. Here we review recent findings related to an additional Ddx19 function in the nucleus in resolving RNA:DNA hybrids (R-loops) generated during collision between transcription and replication, and upon DNA damage. Activation of a DNA damage response pathway dependent upon the ATR kinase, a major regulator of replication fork progression, stimulates translocation of the Ddx19 protein from the cytoplasm into the nucleus. Only nuclear Ddx19 is competent to resolve R-loops, and down regulation of Ddx19 expression induces DNA double strand breaks only in proliferating cells. Overall these observations put forward Ddx19 as an important novel mediator of the crosstalk between transcription and replication.

Highly selective and sensitive detection of miRNA based on toehold-mediated strand displacement reaction and DNA tetrahedron substrate.

Science.gov (United States)

Li, Wei; Jiang, Wei; Ding, Yongshun; Wang, Lei

2015-09-15

MicroRNAs (miRNAs) play important roles in a variety of biological processes and have been regarded as tumor biomarkers in cancer diagnosis and prognosis. In this work, a single-molecule counting method for miRNA analysis was proposed based on toehold-mediated strand displacement reaction (SDR) and DNA tetrahedron substrate. Firstly, a specially designed DNA tetrahedron was assembled with a hairpin at one of the vertex, which has an overhanging toehold domain. Then, the DNA tetrahedron was immobilized on the epoxy-functional glass slide by epoxy-amine reaction, forming a DNA tetrahedron substrate. Next, the target miRNA perhybridized with the toehold domain and initiated a strand displacement reaction along with the unfolding of the hairpin, realizing the selective recognization of miRNA. Finally, a biotin labeled detection DNA was hybridized with the new emerging single strand and the streptavidin coated QDs were used as fluorescent probes. Fluorescent images were acquired via epi-fluorescence microscopy, the numbers of fluorescence dots were counted one by one for quantification. The detection limit is 5 fM, which displayed an excellent sensitivity. Moreover, the proposed method which can accurately be identified the target miRNA among its family members, demonstrated an admirable selectivity. Furthermore, miRNA analysis in total RNA samples from human lung tissues was performed, suggesting the feasibility of this method for quantitative detection of miRNA in biomedical research and early clinical diagnostics. Copyright © 2015 Elsevier B.V. All rights reserved.

A lncRNA to repair DNA

DEFF Research Database (Denmark)

Lukas, Jiri; Altmeyer, Matthias

2015-01-01

Long non-coding RNAs (lncRNAs) have emerged as regulators of various biological processes, but to which extent lncRNAs play a role in genome integrity maintenance is not well understood. In this issue of EMBO Reports, Sharma et al [1] identify the DNA damage-induced lncRNA DDSR1 as an integral...... player of the DNA damage response (DDR). DDSR1 has both an early role by modulating repair pathway choices, and a later function when it regulates gene expression. Sharma et al [1] thus uncover a dual role for a hitherto uncharacterized lncRNA during the cellular response to DNA damage....

DNA methylation of miRNA coding sequences putatively associated with childhood obesity.

Science.gov (United States)

Mansego, M L; Garcia-Lacarte, M; Milagro, F I; Marti, A; Martinez, J A

2017-02-01

Epigenetic mechanisms may be involved in obesity onset and its consequences. The aim of the present study was to evaluate whether DNA methylation status in microRNA (miRNA) coding regions is associated with childhood obesity. DNA isolated from white blood cells of 24 children (identification sample: 12 obese and 12 non-obese) from the Grupo Navarro de Obesidad Infantil study was hybridized in a 450 K methylation microarray. Several CpGs whose DNA methylation levels were statistically different between obese and non-obese were validated by MassArray® in 95 children (validation sample) from the same study. Microarray analysis identified 16 differentially methylated CpGs between both groups (6 hypermethylated and 10 hypomethylated). DNA methylation levels in miR-1203, miR-412 and miR-216A coding regions significantly correlated with body mass index standard deviation score (BMI-SDS) and explained up to 40% of the variation of BMI-SDS. The network analysis identified 19 well-defined obesity-relevant biological pathways from the KEGG database. MassArray® validation identified three regions located in or near miR-1203, miR-412 and miR-216A coding regions differentially methylated between obese and non-obese children. The current work identified three CpG sites located in coding regions of three miRNAs (miR-1203, miR-412 and miR-216A) that were differentially methylated between obese and non-obese children, suggesting a role of miRNA epigenetic regulation in childhood obesity. © 2016 World Obesity Federation.

Nucleotide sequence of a human tRNA gene heterocluster

International Nuclear Information System (INIS)

Chang, Y.N.; Pirtle, I.L.; Pirtle, R.M.

1986-01-01

Leucine tRNA from bovine liver was used as a hybridization probe to screen a human gene library harbored in Charon-4A of bacteriophage lambda. The human DNA inserts from plaque-pure clones were characterized by restriction endonuclease mapping and Southern hybridization techniques, using both [3'- 32 P]-labeled bovine liver leucine tRNA and total tRNA as hybridization probes. An 8-kb Hind III fragment of one of these γ-clones was subcloned into the Hind III site of pBR322. Subsequent fine restriction mapping and DNA sequence analysis of this plasmid DNA indicated the presence of four tRNA genes within the 8-kb DNA fragment. A leucine tRNA gene with an anticodon of AAG and a proline tRNA gene with an anticodon of AGG are in a 1.6-kb subfragment. A threonine tRNA gene with an anticodon of UGU and an as yet unidentified tRNA gene are located in a 1.1-kb subfragment. These two different subfragments are separated by 2.8 kb. The coding regions of the three sequenced genes contain characteristic internal split promoter sequences and do not have intervening sequences. The 3'-flanking region of these three genes have typical RNA polymerase III termination sites of at least four consecutive T residues

Relationships between 16S-23S rRNA gene internal transcribed spacer DNA and genomic DNA similarities in the taxonomy of phototrophic bacteria

International Nuclear Information System (INIS)

Okamura, K; Hisada, T; Takata, K; Hiraishi, A

2013-01-01

Rapid and accurate identification of microbial species is essential task in microbiology and biotechnology. In prokaryotic systematics, genomic DNA-DNA hybridization is the ultimate tool to determine genetic relationships among bacterial strains at the species level. However, a practical problem in this assay is that the experimental procedure is laborious and time-consuming. In recent years, information on the 16S-23S rRNA gene internal transcribed spacer (ITS) region has been used to classify bacterial strains at the species and intraspecies levels. It is unclear how much information on the ITS region can reflect the genome that contain it. In this study, therefore, we evaluate the quantitative relationship between ITS DNA and entire genomic DNA similarities. For this, we determined ITS sequences of several species of anoxygenic phototrophic bacteria belonging to the order Rhizobiales, and compared with DNA-DNA relatedness among these species. There was a high correlation between the two genetic markers. Based on the regression analysis of this relationship, 70% DNA-DNA relatedness corresponded to 92% ITS sequence similarity. This suggests the usefulness of the ITS sequence similarity as a criterion for determining the genospecies of the phototrophic bacteria. To avoid the effects of polymorphism bias of ITS on similarities, PCR products from all loci of ITS were used directly as genetic probes for comparison. The results of ITS DNA-DNA hybridization coincided well with those of genomic DNA-DNA relatedness. These collective data indicate that the whole ITS DNA-DNA similarity can be used as an alternative to genomic DNA-DNA similarity.

Microcantilver-based DNA hybridization sensors for Salmonella identification

Directory of Open Access Journals (Sweden)

Carlo Ricciardi

2012-02-01

Full Text Available The detection of pathogenic microorganisms in foods remains a challenging since the safety of foodstuffs has to be ensured by the food producing companies. Conventional methods for the detection and identification of bacteria mainly rely on specific microbiological and biochemical identification. Biomolecular methods, are commonly used as a support for traditional techniques, thanks to their high sensitivity, specificity and not excessive costs. However, new methods like biosensors for example, can be an exciting alternative to the more traditional tecniques for the detection of pathogens in food. In this study we report Salmonella enterica serotype Enteritidis DNA detection through a novel class of label-free biosensors: microcantilevers (MCs. In general, MCs can operate as a microbalance and is used to detect the mass of the entities anchored to the cantilever surface using the decrease in the resonant frequency. We use DNA hybridization as model reaction system and for this reason, specific single stranded probe DNA of the pathogen and three different DNA targets (single-stranded complementary DNA, PCR product and serial dilutions of DNA extracted from S. Enteritidis strains were applied. Two protocols were reported in order to allow the probe immobilization on cantilever surface: i MC surface was functionalized with 3-aminopropyltriethoxysilane and glutaraldehyde and an amino-modified DNA probe was used; ii gold-coated sensors and thiolated DNA probes were used in order to generate a covalent bonding (Th-Au. For the first one, measures after hybridization with the PCR product showed related frequency shift 10 times higher than hybridization with complementary probe and detectable signals were obtained at the concentrations of 103 and 106 cfu/mL after hybridization with bacterial DNA. There are currently optimizations of the second protocol, where preliminary results have shown to be more uniform and therefore more precise within each of the

Branched-DNA signal amplification combined with paper chromatography hybridization assay and used in hepatitis B virus DNA detection

International Nuclear Information System (INIS)

Fu, F.Z.; Liu, L.X.; Wang, W.Q.; Sun, S. H.; Liu, L.B.

2002-01-01

Nucleic acids detection method is vital to the clinical pathogen diagnosis. The established method can be classified into target direct amplification and signal amplification format according to the target DNA or RNA being directly amplified or not. Those methods have advantages and disadvantages respectively in the clinical application. In the United States of American, branched-DNA as a strong signal amplifier is broadly used in the quantification of the nucleic acids. To gain satisfied sensitivity, some expensive label molecular and instruments should be adopted. Personnel should be special trained to perform. Hence, those can't be widely carried out in the Third World. To avoid those disadvantages, we used the branched-DNA amplifier in the paper chromatography hybridization assay. Methods: Branched DNA signal amplifier and series of probes complementary to the nucleic acid sequence of hepatitis B virus (HBV) have been synthesized. HBV-DNA or it's capture probe were immobilized on the high flow nitrocellulose strip. Having loaded at one end of the strip in turn, probes or HBV-DNA in the hybridization solution migrate to the opposite end of the strip by capillary forces and hybridizes to the immobilized DNA. The branched-DNA signal amplifier and probe labeled with biotin or 32P were then loaded. Through streptavidin-alkaline phosphatase (SA-AP) conjugate and NBT/BCIP ( the specific chromogenic substrate of AP) or autoradiography, the result can be visualized by color reaction or image production on the X-ray film. Results: The sensitivity of this HBV-DNA detection method used probe labeled with biotin and 32P are 1ng and 10pg. The method using the probe labeled with biotin is simple and rapid (2h) without depending on special instruments, it also avoids the pollution of EtBr which can lead to tumor. And the method using the probe labeled with 32P is simple and sensitive, with the exception of long time autoradiography and the inconvenient isotopic disposal

Ultrasensitive FRET-based DNA sensor using PNA/DNA hybridization.

Science.gov (United States)

Yang, Lan-Hee; Ahn, Dong June; Koo, Eunhae

2016-12-01

In the diagnosis of genetic diseases, rapid and highly sensitive DNA detection is crucial. Therefore, many strategies for detecting target DNA have been developed, including electrical, optical, and mechanical methods. Herein, a highly sensitive FRET based sensor was developed by using PNA (Peptide Nucleic Acid) probe and QD, in which red color QDs are hybridized with capture probes, reporter probes and target DNAs by EDC-NHS coupling. The hybridized probe with target DNA gives off fluorescent signal due to the energy transfer from QD to Cy5 dye in the reporter probe. Compared to the conventional DNA sensor using DNA probes, the DNA sensor using PNA probes shows higher FRET factor and efficiency due to the higher reactivity between PNA and target DNA. In addition, to elicit the effect of the distance between the donor and the acceptor, we have investigated two types of the reporter probes having Cy5 dyes attached at the different positions of the reporter probes. Results show that the shorter the distance between QDs and Cy5s, the stronger the signal intensity. Furthermore, based on the fluorescence microscopy images using microcapillary chips, the FRET signal is enhanced to be up to 276% times stronger than the signal obtained using the cuvette by the fluorescence spectrometer. These results suggest that the PNA probe system conjugated with QDs can be used as ultrasensitive DNA nanosensors. Copyright © 2016. Published by Elsevier B.V.

Molecular cloning of lupin leghemoglobin cDNA

DEFF Research Database (Denmark)

Konieczny, A; Jensen, E O; Marcker, K A

1987-01-01

Poly(A)+ RNA isolated from root nodules of yellow lupin (Lupinus luteus, var. Ventus) has been used as a template for the construction of a cDNA library. The ds cDNA was synthesized and inserted into the Hind III site of plasmid pBR 322 using synthetic Hind III linkers. Clones containing sequences...... specific for nodules were selected by differential colony hybridization using 32P-labeled cDNA synthesized either from nodule poly(A)+ RNA or from poly(A)+ RNA of uninfected root as probes. Among the recombinant plasmids, the cDNA gene for leghemoglobin was identified. The protein structure derived from...... its nucleotide sequence was consistent with known amino acid sequence of lupin Lb II. The cloned lupin Lb cDNA hybridized to poly(A)+ RNA from nodules only, which is in accordance with the general concept, that leghemoglobin is expressed exclusively in nodules. Udgivelsesdato: 1987-null...

Electrochemical DNA Hybridization Sensors Based on Conducting Polymers

Science.gov (United States)

Rahman, Md. Mahbubur; Li, Xiao-Bo; Lopa, Nasrin Siraj; Ahn, Sang Jung; Lee, Jae-Joon

2015-01-01

Conducting polymers (CPs) are a group of polymeric materials that have attracted considerable attention because of their unique electronic, chemical, and biochemical properties. This is reflected in their use in a wide range of potential applications, including light-emitting diodes, anti-static coating, electrochromic materials, solar cells, chemical sensors, biosensors, and drug-release systems. Electrochemical DNA sensors based on CPs can be used in numerous areas related to human health. This review summarizes the recent progress made in the development and use of CP-based electrochemical DNA hybridization sensors. We discuss the distinct properties of CPs with respect to their use in the immobilization of probe DNA on electrode surfaces, and we describe the immobilization techniques used for developing DNA hybridization sensors together with the various transduction methods employed. In the concluding part of this review, we present some of the challenges faced in the use of CP-based DNA hybridization sensors, as well as a future perspective. PMID:25664436

Electrochemical DNA Hybridization Sensors Based on Conducting Polymers

Directory of Open Access Journals (Sweden)

Md. Mahbubur Rahman

2015-02-01

Full Text Available Conducting polymers (CPs are a group of polymeric materials that have attracted considerable attention because of their unique electronic, chemical, and biochemical properties. This is reflected in their use in a wide range of potential applications, including light-emitting diodes, anti-static coating, electrochromic materials, solar cells, chemical sensors, biosensors, and drug-release systems. Electrochemical DNA sensors based on CPs can be used in numerous areas related to human health. This review summarizes the recent progress made in the development and use of CP-based electrochemical DNA hybridization sensors. We discuss the distinct properties of CPs with respect to their use in the immobilization of probe DNA on electrode surfaces, and we describe the immobilization techniques used for developing DNA hybridization sensors together with the various transduction methods employed. In the concluding part of this review, we present some of the challenges faced in the use of CP-based DNA hybridization sensors, as well as a future perspective.

Combined histochemical staining, RNA amplification, regional, and single cell cDNA analysis within the hippocampus.

Science.gov (United States)

Ginsberg, Stephen D; Che, Shaoli

2004-08-01

The use of five histochemical stains (cresyl violet, thionin, hematoxylin & eosin, silver stain, and acridine orange) was evaluated in combination with an expression profiling paradigm that included regional and single cell analyses within the hippocampus of post-mortem human brains and adult mice. Adjacent serial sections of human and mouse hippocampus were labeled by histochemistry or neurofilament immunocytochemistry. These tissue sections were used as starting material for regional and single cell microdissection followed by a newly developed RNA amplification procedure (terminal continuation (TC) RNA amplification) and subsequent hybridization to custom-designed cDNA arrays. Results indicated equivalent levels of global hybridization signal intensity and relative expression levels for individual genes for hippocampi stained by cresyl violet, thionin, and hematoxylin & eosin, and neurofilament immunocytochemistry. Moreover, no significant differences existed between the Nissl stains and neurofilament immunocytochemistry for individual CA1 neurons obtained via laser capture microdissection. In contrast, a marked decrement was observed in adjacent hippocampal sections stained for silver stain and acridine orange, both at the level of the regional dissection and at the CA1 neuron population level. Observations made on the cDNA array platform were validated by real-time qPCR using primers directed against beta-actin and glyceraldehyde-3 phosphate dehydrogenase. Thus, this report demonstrated the utility of using specific Nissl stains, but not stains that bind RNA species directly, in both human and mouse brain tissues at the regional and cellular level for state-of-the-art molecular fingerprinting studies.

Techniques of DNA hybridization detect small numbers of mycobacteria with no cross-hybridization with non-mycobacterial respiratory organisms

International Nuclear Information System (INIS)

Shoemaker, S.A.; Fisher, J.H.; Scoggin, C.H.

1985-01-01

The traditional methods used in identifying mycobacteria, such as acid-fast bacillus stains and culture, are often time-consuming, insensitive, and nonspecific. As part of an ongoing program to improve diagnosis and characterization of mycobacteria, the authors have found that deoxyribonucleic acid (DNA) hybridization techniques using isotopically labeled, single-stranded, total DNA can be used to detect as little as 10(-4) micrograms of Mycobacterium tuberculosis (MTb) DNA. This amount of DNA represents approximately 2 X 10(4) genomes. They have also shown the MTb DNA is sufficiently different from the DNA of non-mycobacterial microorganisms such that cross-hybridization with MTb DNA does not occur under the hybridization conditions employed. The authors speculate that DNA hybridization techniques may allow the rapid, sensitive, and specific identification of mycobacteria

Regulatory mechanisms of RNA function: emerging roles of DNA repair enzymes.

Science.gov (United States)

Jobert, Laure; Nilsen, Hilde

2014-07-01

The acquisition of an appropriate set of chemical modifications is required in order to establish correct structure of RNA molecules, and essential for their function. Modification of RNA bases affects RNA maturation, RNA processing, RNA quality control, and protein translation. Some RNA modifications are directly involved in the regulation of these processes. RNA epigenetics is emerging as a mechanism to achieve dynamic regulation of RNA function. Other modifications may prevent or be a signal for degradation. All types of RNA species are subject to processing or degradation, and numerous cellular mechanisms are involved. Unexpectedly, several studies during the last decade have established a connection between DNA and RNA surveillance mechanisms in eukaryotes. Several proteins that respond to DNA damage, either to process or to signal the presence of damaged DNA, have been shown to participate in RNA quality control, turnover or processing. Some enzymes that repair DNA damage may also process modified RNA substrates. In this review, we give an overview of the DNA repair proteins that function in RNA metabolism. We also discuss the roles of two base excision repair enzymes, SMUG1 and APE1, in RNA quality control.

Phenol emulsion-enhanced DNA-driven subtractive cDNA cloning: isolation of low-abundance monkey cortex-specific mRNAs

International Nuclear Information System (INIS)

Travis, G.H.; Sutcliffe, J.G.

1988-01-01

To isolate cDNA clones of low-abundance mRNAs expressed in monkey cerebral cortex but absent from cerebellum, the authors developed an improved subtractive cDNA cloning procedure that requires only modest quantities of mRNA. Plasmid DNA from a monkey cerebellum cDNA library was hybridized in large excess to radiolabeled monkey cortex cDNA in a phenol emulsion-enhanced reaction. The unhybridized cortex cDNA was isolated by chromatography on hydroxyapatite and used to probe colonies from a monkey cortex cDNA library. Of 60,000 colonies screened, 163 clones were isolated and confirmed by colony hybridization or RNA blotting to represent mRNAs, ranging from 0.001% to 0.1% abundance, specific to or highly enriched in cerebral cortex relative to cerebellum. Clones of one medium-abundance mRNA were recovered almost quantitatively. Two of the lower-abundance mRNAs were expressed at levels reduced by a factor of 10 in Alzheimer disease relative to normal human cortex. One of these was identified as the monkey preprosomatostatin I mRNA

Fluorescent quenching-based quantitative detection of specific DNA/RNA using a BODIPY® FL-labeled probe or primer

Science.gov (United States)

Kurata, Shinya; Kanagawa, Takahiro; Yamada, Kazutaka; Torimura, Masaki; Yokomaku, Toyokazu; Kamagata, Yoichi; Kurane, Ryuichiro

2001-01-01

We have developed a simple method for the quantitative detection of specific DNA or RNA molecules based on the finding that BODIPY® FL fluorescence was quenched by its interaction with a uniquely positioned guanine. This approach makes use of an oligonucleotide probe or primer containing a BODIPY® FL-modified cytosine at its 5′-end. When such a probe was hybridized with a target DNA, its fluorescence was quenched by the guanine in the target, complementary to the modified cytosine, and the quench rate was proportional to the amount of target DNA. This widely applicable technique will be used directly with larger samples or in conjunction with the polymerase chain reaction to quantify small DNA samples. PMID:11239011

RADIA: RNA and DNA integrated analysis for somatic mutation detection.

Directory of Open Access Journals (Sweden)

Amie J Radenbaugh

Full Text Available The detection of somatic single nucleotide variants is a crucial component to the characterization of the cancer genome. Mutation calling algorithms thus far have focused on comparing the normal and tumor genomes from the same individual. In recent years, it has become routine for projects like The Cancer Genome Atlas (TCGA to also sequence the tumor RNA. Here we present RADIA (RNA and DNA Integrated Analysis, a novel computational method combining the patient-matched normal and tumor DNA with the tumor RNA to detect somatic mutations. The inclusion of the RNA increases the power to detect somatic mutations, especially at low DNA allelic frequencies. By integrating an individual's DNA and RNA, we are able to detect mutations that would otherwise be missed by traditional algorithms that examine only the DNA. We demonstrate high sensitivity (84% and very high precision (98% and 99% for RADIA in patient data from endometrial carcinoma and lung adenocarcinoma from TCGA. Mutations with both high DNA and RNA read support have the highest validation rate of over 99%. We also introduce a simulation package that spikes in artificial mutations to patient data, rather than simulating sequencing data from a reference genome. We evaluate sensitivity on the simulation data and demonstrate our ability to rescue back mutations at low DNA allelic frequencies by including the RNA. Finally, we highlight mutations in important cancer genes that were rescued due to the incorporation of the RNA.

Signal-off Electrochemiluminescence Biosensor Based on Phi29 DNA Polymerase Mediated Strand Displacement Amplification for MicroRNA Detection.

Science.gov (United States)

Chen, Anyi; Gui, Guo-Feng; Zhuo, Ying; Chai, Ya-Qin; Xiang, Yun; Yuan, Ruo

2015-06-16

A target induced cycling strand displacement amplification (SDA) mediated by phi29 DNA polymerase (phi29) was first investigated and applied in a signal-off electrochemiluminescence (ECL) biosensor for microRNA (miRNA) detection. Herein, the target miRNA triggered the phi29-mediated SDA which could produce amounts of single-stranded DNA (assistant probe) with accurate and comprehensive nucleotide sequence. Then, the assistant probe hybridized with the capture probe and the ferrocene-labeled probe (Fc-probe) to form a ternary "Y" structure for ECL signal quenching by ferrocene. Therefore, the ECL intensity would decrease with increasing concentration of the target miRNA, and the sensitivity of biosensor would be promoted on account of the efficient signal amplification of the target induced cycling reaction. Besides, a self-enhanced Ru(II) ECL system was designed to obtain a stable and strong initial signal to further improve the sensitivity. The ECL assay for miRNA-21 detection is developed with excellent sensitivity of a concentration variation from 10 aM to 1.0 pM and limit of detection down to 3.3 aM.

Transcription of repetitive DNA in Neurospora crassa

Energy Technology Data Exchange (ETDEWEB)

Dutta, S K; Chaudhuri, R K

1975-01-01

Repeated DNA sequences of Neurospora crassa were isolated and characterized. Approximately 10 to 12 percent of N. crassa DNA sequence were repeated, of which 7.3 percent were found to be transcribed in mid-log phase of mycelial growth as measured by DNA:RNA hybridization. It is suggested that part of repetitive DNA transcripts in N. crassa were mitochondrial and part were nuclear DNA. Most of the nuclear repeated DNAs, however, code for rRNA and tRNA in N. crassa. (auth)

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

Science.gov (United States)

Weihe, Andreas

2014-01-01

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

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

Science.gov (United States)

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

2011-09-12

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

Isolation and characterization of cDNA clones for carrot extensin and a proline-rich 33-kDa protein

International Nuclear Information System (INIS)

Chen, J.; Varner, J.E.

1985-01-01

Extensins are hydroxyproline-rich glycoproteins associated with most dicotyledonous plant cell walls. To isolate cDNA clones encoding extensin, the authors started by isolating poly(A) + RNA from carrot root tissue, and then translating the RNA in vitro, in the presence of tritiated leucine or proline. A 33-kDa peptide was identified in the translation products as a putative extensin precursor. From a cDNA library constructed with poly(A) + RNA from wounded carrots, one cDNA clone (pDC5) was identified that specifically hybridized to poly(A) + RNA encoding this 33-kDa peptide. They isolated three cDNA clones (pDC11, pDC12, and pDC16) from another cDNA library using pCD5 as a probe. DNA sequence data, RNA hybridization analysis, and hybrid released in vitro translation indicate that the cDNA clones pDC11 encodes extensin and that cDNA clones pDC12 and pDC16 encode the 33-kDa peptide, which as yet has an unknown identity and function. The assumption that the 33-kDa peptide was an extensin precursor was invalid. RNA hybridization analysis showed that RNA encoded by both clone types is accumulated upon wounding

Effects of humic substances on fluorometric DNA quantification and DNA hybridization

NARCIS (Netherlands)

Bachoon, DS; Otero, E; Hodson, RE

2001-01-01

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

Alternative splicing of human elastin mRNA indicated by sequence analysis of cloned genomic and complementary DNA

International Nuclear Information System (INIS)

Indik, Z.; Yeh, H.; Ornstein-goldstein, N.; Sheppard, P.; Anderson, N.; Rosenbloom, J.C.; Peltonen, L.; Rosenbloom, J.

1987-01-01

Poly(A) + RNA, isolated from a single 7-mo fetal human aorta, was used to synthesize cDNA by the RNase H method, and the cDNA was inserted into λgt10. Recombinant phage containing elastin sequences were identified by hybridization with cloned, exon-containing fragments of the human elastin gene. Three clones containing inserts of 3.3, 2.7, and 2.3 kilobases were selected for further analysis. Three overlapping clones containing 17.8 kilobases of the human elastin gene were also isolated from genomic libraries. Complete sequence analysis of the six clones demonstrated that: (i) the cDNA encompassed the entire translated portion of the mRNA encoding 786 amino acids, including several unusual hydrophilic amino acid sequences not previously identified in porcine tropoelastin, (ii) exons encoding either hydrophobic or crosslinking domains in the protein alternated in the gene, and (iii) a great abundance of Alu repetitive sequences occurred throughout the introns. The data also indicated substantial alternative splicing of the mRNA. These results suggest the potential for significant variation in the precise molecular structure of the elastic fiber in the human population

Application of DNA-DNA colony hybridization to the detection of catabolic genotypes in environmental samples

International Nuclear Information System (INIS)

Sayler, G.S.; Shields, M.S.; Tedford, E.T.; Breen, A.; Hooper, S.W.; Sirotkin, K.M.; Davis, J.W.

1985-01-01

The application of preexisting DNA hybridization techniques was investigated for potential in determining populations of specific gene sequences in environmental samples. Cross-hybridizations among two degradative plasmids, TOL and NAH, and two cloning vehicles, pLAFR1 and RSF1010, were determined. The detection limits for the TOL plasmid against a nonhomologous plasmid-bearing bacterial background was ascertained. The colony hybridization technique allowed detection of one colony containing TOL plasmid among 10(6) Escherichia coli colonies of nonhomologous DNA. Comparisons between population estimates derived from growth on selective substrates and from hybridizations were examined. Findings indicated that standard sole carbon source enumeration procedures for degradative populations lead to overestimations due to nonspecific growth of other bacteria on the microcontaminant carbon sources present in the media. Population estimates based on the selective growth of a microcosm population on two aromatic substrates (toluene and naphthalene) and estimates derived from DNA-DNA colony hybridizations, using the TOL or NAH plasmid as a probe, corresponded with estimates of substrate mineralization rates and past exposure to environmental contaminants. The applications of such techniques are hoped to eventually allow enumeration of any specific gene sequences in the environment, including both anabolic and catabolic genes. In addition, this procedure should prove useful in monitoring recombinant DNA clones released into environmental situations

Use of molecular hybridization to explore genetic relationships. Progress report, July 1, 1974--March 31, 1975

International Nuclear Information System (INIS)

Atwood, K.C.

1975-01-01

Progress is reported on DNA distribution in pygmy chimpanzees, mountain gorillas, Catarrhine monkeys, baboons, spider monkeys, and marmosets; chromosome mapping of human 5S RNA using 3 H or 125 I labelled RNA; hybridization of 125 I-RNA to chromosomes in mice; mapping of low-multiplicity genes by hybridization in situ; technical improvements in iodination of RNA; formation of RNA aggregates; genetics of DNA and RNA in Drosophila; and x-radioinduced chromosomal aberrations in Drosophila. (U.S.)

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

Science.gov (United States)

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.

DNA-dispersed graphene/NiO hybrid materials for highly sensitive non-enzymatic glucose sensor

International Nuclear Information System (INIS)

Lv Wei; Jin Fengmin; Guo Quangui; Yang Quanhong; Kang Feiyu

2012-01-01

Highlights: ► We investigated the potential of GNS/NiO/DNA hybrid used as a nonenzymatic sensor. ► DNA is a highly efficient disperse agent for GNS/NiO hybrid than ionic surfactants. ► GNS/NiO/DNA hybrid shows fast electron transfer in the electrochemical reaction. ► GNS/NiO/DNA hybrid shows good detection performance towards glucose. - Abstract: We demonstrate graphene nanosheet/NiO hybrids (GNS/NiO) as the active material for high-performance non-enzymatic glucose sensors. Such sensors are fabricated by DNA-dispersed GNS/NiO suspension deposited on glassy carbon electrodes. ss-DNA shows strong dispersing ability for the GNS/NiO hybrid materials resulting in stable water-dispersible GNS/NiO/DNA hybrids with fully separated layers. The GNS/NiO/DNA hybrids show enhanced electron transfer in the electrocatalytic reaction process, and accordingly, such hybrids modified electrodes show good sensing performance towards glucose and are characterized by large detection ranges, short response periods, low detection limit and high sensitivity and stability.

Nicotiana small RNA sequences support a host genome origin of cucumber mosaic virus satellite RNA.

Directory of Open Access Journals (Sweden)

Kiran Zahid

2015-01-01

Full Text Available Satellite RNAs (satRNAs are small noncoding subviral RNA pathogens in plants that depend on helper viruses for replication and spread. Despite many decades of research, the origin of satRNAs remains unknown. In this study we show that a β-glucuronidase (GUS transgene fused with a Cucumber mosaic virus (CMV Y satellite RNA (Y-Sat sequence (35S-GUS:Sat was transcriptionally repressed in N. tabacum in comparison to a 35S-GUS transgene that did not contain the Y-Sat sequence. This repression was not due to DNA methylation at the 35S promoter, but was associated with specific DNA methylation at the Y-Sat sequence. Both northern blot hybridization and small RNA deep sequencing detected 24-nt siRNAs in wild-type Nicotiana plants with sequence homology to Y-Sat, suggesting that the N. tabacum genome contains Y-Sat-like sequences that give rise to 24-nt sRNAs capable of guiding RNA-directed DNA methylation (RdDM to the Y-Sat sequence in the 35S-GUS:Sat transgene. Consistent with this, Southern blot hybridization detected multiple DNA bands in Nicotiana plants that had sequence homology to Y-Sat, suggesting that Y-Sat-like sequences exist in the Nicotiana genome as repetitive DNA, a DNA feature associated with 24-nt sRNAs. Our results point to a host genome origin for CMV satRNAs, and suggest novel approach of using small RNA sequences for finding the origin of other satRNAs.

RNA-DNA Differences Are Generated in Human Cells within Seconds after RNA Exits Polymerase II

Directory of Open Access Journals (Sweden)

Isabel X. Wang

2014-03-01

Full Text Available RNA sequences are expected to be identical to their corresponding DNA sequences. Here, we found all 12 types of RNA-DNA sequence differences (RDDs in nascent RNA. Our results show that RDDs begin to occur in RNA chains ∼55 nt from the RNA polymerase II (Pol II active site. These RDDs occur so soon after transcription that they are incompatible with known deaminase-mediated RNA-editing mechanisms. Moreover, the 55 nt delay in appearance indicates that they do not arise during RNA synthesis by Pol II or as a direct consequence of modified base incorporation. Preliminary data suggest that RDD and R-loop formations may be coupled. These findings identify sequence substitution as an early step in cotranscriptional RNA processing.

RNA-directed DNA methylation: Mechanisms and functions

KAUST Repository

Mahfouz, Magdy M.

2010-01-01

Epigenetic RNA based gene silencing mechanisms play a major role in genome stability and control of gene expression. Transcriptional gene silencing via RNA-directed DNA methylation (RdDM) guides the epigenetic regulation of the genome in response

Radioactive and enzymatic cloned cDNA probes for bovine enteric coronavirus detection by molecular hybridization

International Nuclear Information System (INIS)

Collomb, J.; Finance, C.; Alabouch, S.; Laporte, J.

1992-01-01

Genomic RNA of F15 strain bovine enteric coronavirus (BECV) was cloned in E. coli. Three clones (174, 160, PG 78), selected in the cDNA library, including a large portion of the nucleocapsid (N), matrix (M) and peplomeric (S) protein genes , were used as probes for a slot blot hybridization assay. Two probe labelling techniques were compared, radiolabelling with 32 P and enzymatic labelling through covalent linkage to peroxidase and chemiluminescence detection. The radioactive probe 174 detected as little as 1 to 3 pg of viral RNA, while the less sensitive enzymatic probe could not reveal more than 100 pg of RNA. No significant detection amplification was achieved when a mixture of the three probes was used. Probe 174 allowed specific identification for BECV. No hybridization was noticed either with rotaviruses or even with other antigenically unrelated members of the family Coronaviridae such as transmissible gastroenteritis virus. The test proved valid for detection of BECV in the supernatant of infected HRT-18 cells: genomic RNA could be detected after direct spotting of samples, but prior nucleic acid extraction after proteinase K treatment improved virus detection. BECV diagnosis in faecal samples using enzymatic probe was compared with conventional diagnostic methods. (authors)

Radioactive and enzymatic cloned cDNA probes for bovine enteric coronavirus detection by molecular hybridization

Energy Technology Data Exchange (ETDEWEB)

Collomb, J; Finance, C; Alabouch, S [Lab. de Microbiologie Moleculaire, Faculte des Sciences Pharmaceutiques et Biologiques, Univ. de Nancy I, Nancy (France); Laporte, J [Station de Virologie et d' Immunologie Moleculaires, INRA, Jouy-en-Josas (France)

1992-01-01

Genomic RNA of F15 strain bovine enteric coronavirus (BECV) was cloned in E. coli. Three clones (174, 160, PG 78), selected in the cDNA library, including a large portion of the nucleocapsid (N), matrix (M) and peplomeric (S) protein genes , were used as probes for a slot blot hybridization assay. Two probe labelling techniques were compared, radiolabeled with [sup 32]P and enzymatic labeled through covalent linkage to peroxidase for chemiluminescence detection. The radioactive probe 174 detected as little as 1-3 pg of viral RNA, while the less sensitive enzymatic probe could not reveal more than 100 pg of RNA. No significant detection amplification was achieved when a mixture of the three probes was used. Probe 174 allowed specific identification for BECV. No hybridization was noticed either with rotaviruses or even with other antigenically unrelated members of the family Coronaviridae such as transmissible gastroenteritis virus. The test proved valid for detection of BECV in the supernatant of infected HRT-18 cells: genomic RNA could be detected after direct spotting of samples, but prior nucleic acid extraction after proteinase K treatment improved virus detection. BECV diagnosis in fecal samples using enzymatic probe was compared with conventional diagnostic methods. (authors).

Live-Cell MicroRNA Imaging through MnO2 Nanosheet-Mediated DD-A Hybridization Chain Reaction.

Science.gov (United States)

Ou, Min; Huang, Jin; Yang, Xiaohai; He, Xiaoxiao; Quan, Ke; Yang, Yanjing; Xie, Nuli; Li, Jing; Wang, Kemin

2018-01-18

Innovative techniques to visualize native microRNAs (miRNAs) in live cells can dramatically impact current research on the roles of miRNA in biology and medicine. Here, we report a novel approach for live-cell miRNA imaging using a biodegradable MnO 2 nanosheet-mediated DD-A FRET hybridization chain reaction (HCR). The MnO 2 nanosheets can adsorb DNA hairpin probes and deliver them into live cells. After entering cells, the MnO 2 nanosheets are degraded by cellular GSH. Then, the target miR-21 triggers cascaded assembly of the liberated hairpin probes into long dsDNA polymers, which brings each two FAMs (donor) and one TAMRA (acceptor) into close proximity to generate significantly enhanced DD-A FRET signals, which was discovered and proven by our previous report. We think the developed approach can serve as an excellent intracellular miRNAs detection tool, which promises the potential for biological and disease studies. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

RNA-directed DNA methylation: Mechanisms and functions

KAUST Repository

Mahfouz, Magdy M.

2010-07-01

Epigenetic RNA based gene silencing mechanisms play a major role in genome stability and control of gene expression. Transcriptional gene silencing via RNA-directed DNA methylation (RdDM) guides the epigenetic regulation of the genome in response to disease states, growth, developmental and stress signals. RdDM machinery is composed of proteins that produce and modify 24-nt- long siRNAs, recruit the RdDM complex to genomic targets, methylate DNA and remodel chromatin. The final DNA methylation pattern is determined by either DNA methyltransferase alone or by the combined action of DNA methyltransferases and demethylases. The dynamic interaction between RdDM and demethylases may render the plant epigenome plastic to growth, developmental, and environmental cues. The epigenome plasticity may allow the plant genome to assume many epigenomes and to have the right epigenome at the right time in response to intracellular or extracellular stimuli. This review discusses recent advances in RdDM research and considers future perspectives.

Stereospecificity of oligonucleotide interactions revisited: no evidence for heterochiral hybridization and ribozyme/DNAzyme activity.

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Kai Hoehlig

Full Text Available A major challenge for the application of RNA- or DNA-oligonucleotides in biotechnology and molecular medicine is their susceptibility to abundant nucleases. One intriguing possibility to tackle this problem is the use of mirror-image (l-oligonucleotides. For aptamers, this concept has successfully been applied to even develop therapeutic agents, so-called Spiegelmers. However, for technologies depending on RNA/RNA or RNA/DNA hybridization, like antisense or RNA interference, it has not been possible to use mirror-image oligonucleotides because Watson-Crick base pairing of complementary strands is (thought to be stereospecific. Many scientists consider this a general principle if not a dogma. A recent publication proposing heterochiral Watson-Crick base pairing and sequence-specific hydrolysis of natural RNA by mirror-image ribozymes or DNAzymes (and vice versa prompted us to systematically revisit the stereospecificity of oligonucleotides hybridization and catalytic activity. Using hyperchromicity measurements we demonstrate that hybridization only occurs among homochiral anti-parallel complementary oligonucleotide strands. As expected, achiral PNA hybridizes to RNA and DNA irrespective of their chirality. In functional assays we could not confirm an alleged heterochiral hydrolytic activity of ribozymes or DNAzymes. Our results confirm a strict stereospecificity of oligonucleotide hybridization and clearly argue against the possibility to use mirror-image oligonucleotides for gene silencing or antisense applications.

Label-Free Potentiometry for Detecting DNA Hybridization Using Peptide Nucleic Acid and DNA Probes

Science.gov (United States)

Goda, Tatsuro; Singi, Ankit Balram; Maeda, Yasuhiro; Matsumoto, Akira; Torimura, Masaki; Aoki, Hiroshi; Miyahara, Yuji

2013-01-01

Peptide nucleic acid (PNA) has outstanding affinity over DNA for complementary nucleic acid sequences by forming a PNA-DNA heterodimer upon hybridization via Watson-Crick base-pairing. To verify whether PNA probes on an electrode surface enhance sensitivity for potentiometric DNA detection or not, we conducted a comparative study on the hybridization of PNA and DNA probes on the surface of a 10-channel gold electrodes microarray. Changes in the charge density as a result of hybridization at the solution/electrode interface on the self-assembled monolayer (SAM)-formed microelectrodes were directly transformed into potentiometric signals using a high input impedance electrometer. The charge readout allows label-free, reagent-less, and multi-parallel detection of target oligonucleotides without any optical assistance. The differences in the probe lengths between 15- to 22-mer dramatically influenced on the sensitivity of the PNA and DNA sensors. Molecular type of the capturing probe did not affect the degree of potential shift. Theoretical model for charged rod-like duplex using the Gouy-Chapman equation indicates the dominant effect of electrostatic attractive forces between anionic DNA and underlying electrode at the electrolyte/electrode interface in the potentiometry. PMID:23435052

Label-Free Potentiometry for Detecting DNA Hybridization Using Peptide Nucleic Acid and DNA Probes

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Yuji Miyahara

2013-02-01

Full Text Available Peptide nucleic acid (PNA has outstanding affinity over DNA for complementary nucleic acid sequences by forming a PNA-DNA heterodimer upon hybridization via Watson-Crick base-pairing. To verify whether PNA probes on an electrode surface enhance sensitivity for potentiometric DNA detection or not, we conducted a comparative study on the hybridization of PNA and DNA probes on the surface of a 10-channel gold electrodes microarray. Changes in the charge density as a result of hybridization at the solution/electrode interface on the self-assembled monolayer (SAM-formed microelectrodes were directly transformed into potentiometric signals using a high input impedance electrometer. The charge readout allows label-free, reagent-less, and multi-parallel detection of target oligonucleotides without any optical assistance. The differences in the probe lengths between 15- to 22-mer dramatically influenced on the sensitivity of the PNA and DNA sensors. Molecular type of the capturing probe did not affect the degree of potential shift. Theoretical model for charged rod-like duplex using the Gouy-Chapman equation indicates the dominant effect of electrostatic attractive forces between anionic DNA and underlying electrode at the electrolyte/electrode interface in the potentiometry.

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

Science.gov (United States)

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

2015-10-01

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

An Approach to Detect and Study DNA Double-Strand Break Repair by Transcript RNA Using a Spliced-Antisense RNA Template.

Science.gov (United States)

Keskin, Havva; Storici, Francesca

2018-01-01

A double-strand break (DSB) is one of the most dangerous DNA lesion, and its repair is crucial for genome stability. Homologous recombination is considered the safest way to repair a DNA DSB and requires an identical or nearly identical DNA template, such as a sister chromatid or a homologous chromosome for accurate repair. Can transcript RNA serve as donor template for DSB repair? Here, we describe an approach that we developed to detect and study DNA repair by transcript RNA. Key features of the method are: (i) use of antisense (noncoding) RNA as template for DSB repair by RNA, (ii) use of intron splicing to distinguish the sequence of the RNA template from that of the DNA that generates the RNA template, and (iii) use of a trans and cis system to study how RNA repairs a DSB in homologous but distant DNA or in its own DNA, respectively. This chapter provides details on how to use a spliced-antisense RNA template to detect and study DSB repair by RNA in trans or cis in yeast cells. Our approach for detection of DSB repair by RNA in cells can be applied to cell types other than yeast, such as bacteria, mammalian cells, or other eukaryotic cells. © 2018 Elsevier Inc. All rights reserved.

Nanostructured ZnO-based biosensor: DNA immobilization and hybridization

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Ahmed Mishaal Mohammed

2017-09-01

Full Text Available An electrochemical DNA biosensor was successfully fabricated by using (3-aminopropyl triethoxysilane (APTES with zinc oxide (ZnO nanorods synthesized using microwave-assisted chemical bath deposition method on thermally oxidized SiO2 thin films. The structural quality and morphology of the ZnO nanorods were determined by employing scanning electron microscopy (SEM and X-ray diffraction (XRD, which show a hexagonal wurtzite structure with a preferred orientation along the (101 direction. The surface of the SiO2 thin films was chemically modified with ZnO. Label-free detection DNA immobilization and hybridization were performed using potassium hexacyanoferrate with cyclic voltammetry (CV measurements. The capacitance, permittivity, and conductivity profiles of the fabricated sensor clearly indicate DNA immobilization and hybridization. Results show that the capacitance values of bare, ZnO- modified surface immobilization, and target DNA hybridization were 46×10−12F, 47×10−8F, 27μF, and 17μF, respectively, at 1Hz. The permittivity measurement increased from 3.94×103 to 251×103 and 165×103 at the frequency range of approximately 200 to 1Hz for bare and DNA immobilization and hybridization, respectively. The measured conductivity values for the bare, ZnO, immobilized, and hybridization device were 2.4×10−9, 10×10−8, 1.6×10−7, and 1.3×10−7Scm−1, respectively. Keywords: Zinc oxide, Biosensor, Capacitance, Permittivity, Conductivity

Cutis laxa: reduced elastin gene expression in skin fibroblast cultures as determined by hybridizations with a homologous cDNA and an exon 1-specific oligonucleotide

International Nuclear Information System (INIS)

Olsen, D.R.; Fazio, M.J.; Shamban, A.T.; Rosenbloom, J.; Uitto, J.

1988-01-01

Fibroblast cultures were established from six patients with cutis laxa, and elastin gene expression was analyzed by RNA hybridizations with a 2.5-kilobase human elastin cDNA or an exon 1-specific 35-base oligomer. Northern analyses using either probe detected mRNA transcripts of ∼ 3.5 kilobases, and no qualitative difference between the control and cutis laxa mRNAs was detected. However, quantitation of the elastin mRNA abundance by slot blot hybridizations revealed markedly reduced levels in all cutis laxa cell strains. Assuming equal translational activity of the control and cutix laxa mRNAs, the reduced mRNA levels could result in diminished elastin production, providing an explanation for the paucity of elastic fibers in the skin and other tissues in cutis laxa

Good quality Vitis RNA obtained from an adapted DNA isolation protocol

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Isabel Baiges

2003-03-01

Full Text Available Grapevine is a woody plant, whose high carbohydrate and phenolic compound contents usually interferes with nucleic acid isolation. After we tried several protocols for isolating RNA from the Vitis rootstock Richter- 110 (R-110 with little or no success, we adapted a method reported to be satisfactory for grapevine DNA isolation, to extract RNA. With slight protocol modifications, we succeeded to obtain polysaccharide- and phenolic-free RNA preparations from all vegetative tissues, without excessive sample handling. RNA isolated by the reported method permitted to obtain highly pure mRNA (messenger RNA to construct a cDNA (complementary DNA library and allowed gene transcription analysis by reverse Northern, which guarantees RNA integrity. This method may also be suitable for other plant species with high polysaccharide or phenolic contents.

Mechanism of duplex DNA destabilization by RNA-guided Cas9 nuclease during target interrogation.

Science.gov (United States)

Mekler, Vladimir; Minakhin, Leonid; Severinov, Konstantin

2017-05-23

The prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)-associated 9 (Cas9) endonuclease cleaves double-stranded DNA sequences specified by guide RNA molecules and flanked by a protospacer adjacent motif (PAM) and is widely used for genome editing in various organisms. The RNA-programmed Cas9 locates the target site by scanning genomic DNA. We sought to elucidate the mechanism of initial DNA interrogation steps that precede the pairing of target DNA with guide RNA. Using fluorometric and biochemical assays, we studied Cas9/guide RNA complexes with model DNA substrates that mimicked early intermediates on the pathway to the final Cas9/guide RNA-DNA complex. The results show that Cas9/guide RNA binding to PAM favors separation of a few PAM-proximal protospacer base pairs allowing initial target interrogation by guide RNA. The duplex destabilization is mediated, in part, by Cas9/guide RNA affinity for unpaired segments of nontarget strand DNA close to PAM. Furthermore, our data indicate that the entry of double-stranded DNA beyond a short threshold distance from PAM into the Cas9/single-guide RNA (sgRNA) interior is hindered. We suggest that the interactions unfavorable for duplex DNA binding promote DNA bending in the PAM-proximal region during early steps of Cas9/guide RNA-DNA complex formation, thus additionally destabilizing the protospacer duplex. The mechanism that emerges from our analysis explains how the Cas9/sgRNA complex is able to locate the correct target sequence efficiently while interrogating numerous nontarget sequences associated with correct PAMs.

Nucleic Acid Analogue Induced Transcription of Double Stranded DNA

DEFF Research Database (Denmark)

1998-01-01

RNA is transcribed from a double stranded DNA template by forming a complex by hybridizing to the template at a desired transcription initiation site one or more oligonucleic acid analogues of the PNA type capable of forming a transcription initiation site with the DNA and exposing the complex...... to the action of a DNA dependant RNA polymerase in the presence of nucleoside triphosphates. Equal length transcripts may be obtained by placing a block to transcription downstream from the initiation site or by cutting the template at such a selected location. The initiation site is formed by displacement...... of one strand of the DNA locally by the PNA hybridization....

Mismatch discrimination of lipidated DNA and LNA-probes (LiNAs) in hybridization-controlled liposome assembly

DEFF Research Database (Denmark)

Jakobsen, Ulla; Vogel, Stefan

2016-01-01

Assays for mismatch discrimination and detection of single nucleotide variations by hybridization-controlled assembly of liposomes, which do not require tedious surface chemistry, are versatile for both DNA and RNA targets. We report herein a comprehensive study on different DNA and LNA (locked...... assay in the context of mismatch discrimination and SNP detection are presented. The advantages of membrane-anchored LiNA-probes compared to chemically attached probes on solid nanoparticles (e.g. gold nanoparticles) are described. Key functionalities such as non-covalent attachment of LiNA probes...... without the need for long spacers and the inherent mobility of membrane-anchored probes in lipid-bilayer membranes will be described for several different probe designs....

An RNA Domain Imparts Specificity and Selectivity to a Viral DNA Packaging Motor

Science.gov (United States)

Zhao, Wei; Jardine, Paul J.

2015-01-01

ABSTRACT During assembly, double-stranded DNA viruses, including bacteriophages and herpesviruses, utilize a powerful molecular motor to package their genomic DNA into a preformed viral capsid. An integral component of the packaging motor in the Bacillus subtilis bacteriophage ϕ29 is a viral genome-encoded pentameric ring of RNA (prohead RNA [pRNA]). pRNA is a 174-base transcript comprised of two domains, domains I and II. Early studies initially isolated a 120-base form (domain I only) that retains high biological activity in vitro; hence, no function could be assigned to domain II. Here we define a role for this domain in the packaging process. DNA packaging using restriction digests of ϕ29 DNA showed that motors with the 174-base pRNA supported the correct polarity of DNA packaging, selectively packaging the DNA left end. In contrast, motors containing the 120-base pRNA had compromised specificity, packaging both left- and right-end fragments. The presence of domain II also provides selectivity in competition assays with genomes from related phages. Furthermore, motors with the 174-base pRNA were restrictive, in that they packaged only one DNA fragment into the head, whereas motors with the 120-base pRNA packaged several fragments into the head, indicating multiple initiation events. These results show that domain II imparts specificity and stringency to the motor during the packaging initiation events that precede DNA translocation. Heteromeric rings of pRNA demonstrated that one or two copies of domain II were sufficient to impart this selectivity/stringency. Although ϕ29 differs from other double-stranded DNA phages in having an RNA motor component, the function provided by pRNA is carried on the motor protein components in other phages. IMPORTANCE During virus assembly, genome packaging involves the delivery of newly synthesized viral nucleic acid into a protein shell. In the double-stranded DNA phages and herpesviruses, this is accomplished by a powerful

An RNA Domain Imparts Specificity and Selectivity to a Viral DNA Packaging Motor.

Science.gov (United States)

Zhao, Wei; Jardine, Paul J; Grimes, Shelley

2015-12-01

During assembly, double-stranded DNA viruses, including bacteriophages and herpesviruses, utilize a powerful molecular motor to package their genomic DNA into a preformed viral capsid. An integral component of the packaging motor in the Bacillus subtilis bacteriophage ϕ29 is a viral genome-encoded pentameric ring of RNA (prohead RNA [pRNA]). pRNA is a 174-base transcript comprised of two domains, domains I and II. Early studies initially isolated a 120-base form (domain I only) that retains high biological activity in vitro; hence, no function could be assigned to domain II. Here we define a role for this domain in the packaging process. DNA packaging using restriction digests of ϕ29 DNA showed that motors with the 174-base pRNA supported the correct polarity of DNA packaging, selectively packaging the DNA left end. In contrast, motors containing the 120-base pRNA had compromised specificity, packaging both left- and right-end fragments. The presence of domain II also provides selectivity in competition assays with genomes from related phages. Furthermore, motors with the 174-base pRNA were restrictive, in that they packaged only one DNA fragment into the head, whereas motors with the 120-base pRNA packaged several fragments into the head, indicating multiple initiation events. These results show that domain II imparts specificity and stringency to the motor during the packaging initiation events that precede DNA translocation. Heteromeric rings of pRNA demonstrated that one or two copies of domain II were sufficient to impart this selectivity/stringency. Although ϕ29 differs from other double-stranded DNA phages in having an RNA motor component, the function provided by pRNA is carried on the motor protein components in other phages. During virus assembly, genome packaging involves the delivery of newly synthesized viral nucleic acid into a protein shell. In the double-stranded DNA phages and herpesviruses, this is accomplished by a powerful molecular motor

Physical localization and DNA methylation of 45S rRNA gene loci in Jatropha curcas L.

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Zhiyun Gong

Full Text Available In eukaryotes, 45S rRNA genes are arranged in tandem arrays of repeat units, and not all copies are transcribed during mitosis. DNA methylation is considered to be an epigenetic marker for rDNA activation. Here, we established a clear and accurate karyogram for Jatropha curcas L. The chromosomal formula was found to be 2n=2x=22=12m+10 sm. We found that the 45S rDNA loci were located at the termini of chromosomes 7 and 9 in J. curcas. The distribution of 45S rDNA has no significant difference in J. curcas from different sources. Based on the hybridization signal patterns, there were two forms of rDNA - dispersed and condensed. The dispersed type of signals appeared during interphase and prophase, while the condensed types appeared during different stages of mitosis. DNA methylation analysis showed that when 45S rDNA stronger signals were dispersed and connected to the nucleolus, DNA methylation levels were lower at interphase and prophase. However, when the 45S rDNA loci were condensed, especially during metaphase, they showed different forms of DNA methylation.

Physical Localization and DNA Methylation of 45S rRNA Gene Loci in Jatropha curcas L.

Science.gov (United States)

Gong, Zhiyun; Xue, Chao; Zhang, Mingliang; Guo, Rui; Zhou, Yong; Shi, Guoxin

2013-01-01

In eukaryotes, 45S rRNA genes are arranged in tandem arrays of repeat units, and not all copies are transcribed during mitosis. DNA methylation is considered to be an epigenetic marker for rDNA activation. Here, we established a clear and accurate karyogram for Jatropha curcas L. The chromosomal formula was found to be 2n = 2x = 22 = 12m+10sm. We found that the 45S rDNA loci were located at the termini of chromosomes 7 and 9 in J. curcas. The distribution of 45S rDNA has no significant difference in J. curcas from different sources. Based on the hybridization signal patterns, there were two forms of rDNA - dispersed and condensed. The dispersed type of signals appeared during interphase and prophase, while the condensed types appeared during different stages of mitosis. DNA methylation analysis showed that when 45S rDNA stronger signals were dispersed and connected to the nucleolus, DNA methylation levels were lower at interphase and prophase. However, when the 45S rDNA loci were condensed, especially during metaphase, they showed different forms of DNA methylation. PMID:24386362

Measuring DNA hybridization using fluorescent DNA-stabilized silver clusters to investigate mismatch effects on therapeutic oligonucleotides.

Science.gov (United States)

de Bruin, Donny; Bossert, Nelli; Aartsma-Rus, Annemieke; Bouwmeester, Dirk

2018-04-06

Short nucleic acid oligomers have found a wide range of applications in experimental physics, biology and medicine, and show potential for the treatment of acquired and genetic diseases. These applications rely heavily on the predictability of hybridization through Watson-Crick base pairing to allow positioning on a nanometer scale, as well as binding to the target transcripts, but also off-target binding to transcripts with partial homology. These effects are of particular importance in the development of therapeutic oligonucleotides, where off-target effects caused by the binding of mismatched sequences need to be avoided. We employ a novel method of probing DNA hybridization using optically active DNA-stabilized silver clusters (Ag-DNA) to measure binding efficiencies through a change in fluorescence intensity. In this way we can determine their location-specific sensitivity to individual mismatches in the sequence. The results reveal a strong dependence of the hybridization on the location of the mismatch, whereby mismatches close to the edges and center show a relatively minor impact. In parallel, we propose a simple model for calculating the annealing ratios of mismatched DNA sequences, which supports our experimental results. The primary result shown in this work is a demonstration of a novel technique to measure DNA hybridization using fluorescent Ag-DNA. With this technique, we investigated the effect of mismatches on the hybridization efficiency, and found a significant dependence on the location of individual mismatches. These effects are strongly influenced by the length of the used oligonucleotides. The novel probe method based on fluorescent Ag-DNA functions as a reliable tool in measuring this behavior. As a secondary result, we formulated a simple model that is consistent with the experimental data.

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

Science.gov (United States)

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

2014-02-12

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

Absence of ribosomal DNA amplification in the meroistic (telotrophic) ovary of the large milkweed bug Oncopeltus fasciatus (Dallas) (Hemiptera: Lygaeidae)

Science.gov (United States)

1975-01-01

In the typical meroistic insect ovary, the oocyte nucleus synthesizes little if any RNA. Nurse cells or trophocytes actively synthesize ribosomes which are transported to and accumulated by the oocyte. In the telotrophic ovary a morphological separation exists, the nurse cells being localized at the apical end of each ovariole and communicating with the ooocytes via nutritive cords. In order to determine whether the genes coding for ribosomal RNA (rRNA) are amplified in the telotrophic ovary of the milkweed bug Oncopeltus fasciatus, the percentages of the genome coding for ribosomal RNA in somatic cells, spermatogenic cells, ovarian follicles, and nurse cells were compared. The oocytes and most of the nurse cells of O. fasciatus are uninucleolate. DNA hybridizing with ribosomal RNA is localized in a satellite DNA, the density of which is 1.712 g/cm(-3). The density of main-band DNA is 1.694 g/cm(-3). The ribosomal DNA satellite accounts for approximately 0.2% of the DNA in somatic and gametogenic tissues of both males and females. RNA-DNA hybridization analysis demonstrates that approximately 0.03% of the DNA in somatic tissues, testis, ovarian follicles, and isolated nurse cells hybridizes with ribosomal RNA. The fact that the percentage of DNA hybridizing with rRNA is the same in somatic and in male and female gametogenic tissues indicates that amplification of ribosomal DNA does not occur in nurse cells and that if it occurs in oocytes, it represents less than a 50- fold increase in ribosomal DNA. An increase in total genome DNA accounted by polyploidization appears to provide for increasing the amount of ribosomal DNA in the nurse cells. PMID:1158969

Characterization of Actinomyces with genomic DNA fingerprints and rRNA gene probes.

Science.gov (United States)

Bowden, G; Johnson, J; Schachtele, C

1993-08-01

Cellular DNA from 25 Actinomyces naeslundii and Actinomyces viscosus strains belonging to the 7 taxonomic clusters of Fillery et al. (1978) and several unclustered strains was obtained by enzymatic and N-lauroylsarcosine/guanidine isothiocyanate treatment of whole cells, followed by extraction of the nucleic acid. The DNA samples were digested with restriction endonucleases BamHI or PvuII, and agarose gel electrophoresis was used to obtain DNA fingerprints. The DNA fragments were subjected to Southern blot hybridization with a digoxigenin-labeled cDNA probe transcribed from Escherichia coli 16S and 23S rRNA. The patterns of bands from genomic (DNA fingerprints) and rDNA fingerprints (ribotypes) were used for comparison between the taxonomic cluster strains and strains within clusters. Representative strains from each taxonomic cluster provided different BamHI DNA fingerprints and ribotype patterns with 3 to 9 distinct bands. Some strains within a cluster showed identical ribotype patterns with both endonucleases (A. naeslundii B120 and A. naeslundii B102 from cluster 3), while others showed the same pattern with BamHI but a different pattern with PvuII (A. naeslundii ATCC 12104 and 398A from cluster 5). A viscosus ATCC 15987 (cluster 7) and its parent strain T6 yielded identical fingerprint and ribotype patterns. The genomic diversity revealed by DNA fingerprinting and ribotyping demonstrates that these techniques, which do not require phenotypic expression, are suited for study of the oral ecology of the Actinomyces, and for epidemiological tracking of specific Actinomyces strains associated with caries lesions and sites of periodontal destruction.

In situ hybridization; principles and applications: review article

Directory of Open Access Journals (Sweden)

Zahra Nozhat

2015-06-01

Full Text Available In situ hybridization (ISH is a method that uses labeled complementary single strand DNA or RNA to localize specific DNA or RNA sequences in an intact cell or in a fixed tissue section. The main steps of ISH consist of: probe selection, tissue or sample preparation, pre-hybridization treatment, hybridization and washing, detection and control procedure. Probe selection is one of the important aspects of successful hybridization. ISH sensitivity and specificity can be influenced by: probe construct, efficiency of labeling, percentage of GC, probe length and signal detection systems. Different methods such as nick translation, random priming, end tailing and T4 DNA polymerase replacement are used for probe generation. Both radioactive and non-radioactive labels can be used in order to probe labeling. Nucleic acid maintenance in samples, prevention of morphological changes of samples and probe penetration into tissue section are the main aims of sample preparation step. Then, a small amount of solution containing probe, is added on slides containing tissue sections for hybridization process, then slides are incubated overnight. Next day, washes are carried out to remove the probes which are not bound to target DNA or RNA. Finally, in order to be sure that the observed labeling is specific to the target sequence, using several control procedures is very important. Various techniques based on ISH consist of: Fluorescence in situ hybridization (FISH, chromogenic in situ hybridization (CISH, genomic in situ hybridization (GISH, comparative genomic hybridization (CGH, spectral karyotyping (SKY and multiplex fluorescence in situ hybridization (MFISH. One of the most common techniques of ISH is fluorescence in situ hybridization. FISH can be used to: 1 detect small deletions and duplications that are not visible using microscope analysis, 2 detect how many chromosomes of a certain type are present in each cell and 3 confirm rearrangements that are

Probing of miniPEGγ-PNA-DNA Hybrid Duplex Stability with AFM Force Spectroscopy.

Science.gov (United States)

Dutta, Samrat; Armitage, Bruce A; Lyubchenko, Yuri L

2016-03-15

Peptide nucleic acids (PNA) are synthetic polymers, the neutral peptide backbone of which provides elevated stability to PNA-PNA and PNA-DNA hybrid duplexes. It was demonstrated that incorporation of diethylene glycol (miniPEG) at the γ position of the peptide backbone increased the thermal stability of the hybrid duplexes (Sahu, B. et al. J. Org. Chem. 2011, 76, 5614-5627). Here, we applied atomic force microscopy (AFM) based single molecule force spectroscopy and dynamic force spectroscopy (DFS) to test the strength and stability of the hybrid 10 bp duplex. This hybrid duplex consisted of miniPEGγ-PNA and DNA of the same length (γ(MP)PNA-DNA), which we compared to a DNA duplex with a homologous sequence. AFM force spectroscopy data obtained at the same conditions showed that the γ(MP)PNA-DNA hybrid is more stable than the DNA counterpart, 65 ± 15 pN vs 47 ± 15 pN, respectively. The DFS measurements performed in a range of pulling speeds analyzed in the framework of the Bell-Evans approach yielded a dissociation constant, koff ≈ 0.030 ± 0.01 s⁻¹ for γ(MP)PNA-DNA hybrid duplex vs 0.375 ± 0.18 s⁻¹ for the DNA-DNA duplex suggesting that the hybrid duplex is much more stable. Correlating the high affinity of γ(MP)PNA-DNA to slow dissociation kinetics is consistent with prior bulk characterization by surface plasmon resonance. Given the growing interest in γ(MP)PNA as well as other synthetic DNA analogues, the use of single molecule experiments along with computational analysis of force spectroscopy data will provide direct characterization of various modifications as well as higher order structures such as triplexes and quadruplexes.

High affinity γPNA sandwich hybridization assay for rapid detection of short nucleic acid targets with single mismatch discrimination.

Science.gov (United States)

Goldman, Johnathan M; Zhang, Li Ang; Manna, Arunava; Armitage, Bruce A; Ly, Danith H; Schneider, James W

2013-07-08

Hybridization analysis of short DNA and RNA targets presents many challenges for detection. The commonly employed sandwich hybridization approach cannot be implemented for these short targets due to insufficient probe-target binding strengths for unmodified DNA probes. Here, we present a method capable of rapid and stable sandwich hybridization detection for 22 nucleotide DNA and RNA targets. Stable hybridization is achieved using an n-alkylated, polyethylene glycol γ-carbon modified peptide nucleic acid (γPNA) amphiphile. The γPNA's exceptionally high affinity enables stable hybridization of a second DNA-based probe to the remaining bases of the short target. Upon hybridization of both probes, an electrophoretic mobility shift is measured via interaction of the n-alkane modification on the γPNA with capillary electrophoresis running buffer containing nonionic surfactant micelles. We find that sandwich hybridization of both probes is stable under multiple binding configurations and demonstrate single base mismatch discrimination. The binding strength of both probes is also stabilized via coaxial stacking on adjacent hybridization to targets. We conclude with a discussion on the implementation of the proposed sandwich hybridization assay as a high-throughput microRNA detection method.

ZnO Nanoparticles Protect RNA from Degradation Better than DNA

Directory of Open Access Journals (Sweden)

Jayden McCall

2017-11-01

Full Text Available Gene therapy and RNA delivery require a nanoparticle (NP to stabilize these nucleic acids when administered in vivo. The presence of degradative hydrolytic enzymes within these environments limits the nucleic acids’ pharmacologic activity. This study compared the effects of nanoscale ZnO and MgO in the protection afforded to DNA and RNA from degradation by DNase, serum or tumor homogenate. For double-stranded plasmid DNA degradation by DNase, our results suggest that the presence of MgO NP can protect DNA from DNase digestion at an elevated temperature (65 °C, a biochemical activity not present in ZnO NP-containing samples at any temperature. In this case, intact DNA was remarkably present for MgO NP after ethidium bromide staining and agarose gel electrophoresis where these same stained DNA bands were notably absent for ZnO NP. Anticancer RNA, polyinosinic-polycytidylic acid (poly I:C is now considered an anti-metastatic RNA targeting agent and as such there is great interest in its delivery by NP. For it to function, the NP must protect it from degradation in serum and the tumor environment. Surprisingly, ZnO NP protected the RNA from degradation in either serum-containing media or melanoma tumor homogenate after gel electrophoretic analysis, whereas the band was much more diminished in the presence of MgO. For both MgO and ZnO NP, buffer-dependent rescue from degradation occurred. These data suggest a fundamental difference in the ability of MgO and ZnO NP to stabilize nucleic acids with implications for DNA and RNA delivery and therapy.

Automated DNA electrophoresis, hybridization and detection

International Nuclear Information System (INIS)

Zapolski, E.J.; Gersten, D.M.; Golab, T.J.; Ledley, R.S.

1986-01-01

A fully automated, computer controlled system for nucleic acid hybridization analysis has been devised and constructed. In practice, DNA is digested with restriction endonuclease enzyme(s) and loaded into the system by pipette; 32 P-labelled nucleic acid probe(s) is loaded into the nine hybridization chambers. Instructions for all the steps in the automated process are specified by answering questions that appear on the computer screen at the start of the experiment. Subsequent steps are performed automatically. The system performs horizontal electrophoresis in agarose gel, fixed the fragments to a solid phase matrix, denatures, neutralizes, prehybridizes, hybridizes, washes, dries and detects the radioactivity according to the specifications given by the operator. The results, printed out at the end, give the positions on the matrix to which radioactivity remains hybridized following stringent washing

Preservation of RNA and DNA from mammal samples under field conditions.

Science.gov (United States)

Camacho-Sanchez, Miguel; Burraco, Pablo; Gomez-Mestre, Ivan; Leonard, Jennifer A

2013-07-01

Ecological and conservation genetics require sampling of organisms in the wild. Appropriate preservation of the collected samples, usually by cryostorage, is key to the quality of the genetic data obtained. Nevertheless, cryopreservation in the field to ensure RNA and DNA stability is not always possible. We compared several nucleic acid preservation solutions appropriate for field sampling and tested them on rat (Rattus rattus) blood, ear and tail tip, liver, brain and muscle. We compared the efficacy of a nucleic acid preservation (NAP) buffer for DNA preservation against 95% ethanol and Longmire buffer, and for RNA preservation against RNAlater (Qiagen) and Longmire buffer, under simulated field conditions. For DNA, the NAP buffer was slightly better than cryopreservation or 95% ethanol, but high molecular weight DNA was preserved in all conditions. The NAP buffer preserved RNA as well as RNAlater. Liver yielded the best RNA and DNA quantity and quality; thus, liver should be the tissue preferentially collected from euthanized animals. We also show that DNA persists in nonpreserved muscle tissue for at least 1 week at ambient temperature, although degradation is noticeable in a matter of hours. When cryopreservation is not possible, the NAP buffer is an economical alternative for RNA preservation at ambient temperature for at least 2 months and DNA preservation for at least 10 months. © 2013 John Wiley & Sons Ltd.

Clinical strains of acinetobacter classified by DNA-DNA hybridization

International Nuclear Information System (INIS)

Tjernberg, I.; Ursing, J.

1989-01-01

A collection of Acinetobacter strains consisting of 168 consecutive clinical strains and 30 type and reference strains was studied by DNA-DNA hybridization and a few phenotypic tests. The field strains could be allotted to 13 DNA groups. By means of reference strains ten of these could be identified with groups described by Bouvet and Grimont (1986), while three groups were new; they were given the numbers 13-15. The type strain of A. radioresistens- recently described by Nishimura et al. (1988) - was shown to be a member of DNA group 12, which comprised 31 clinical isolates. Of the 19 strains of A. junii, eight showed hemolytic acitivity on sheep and human blood agar and an additional four strains on human blood agar only. Strains of this species have previously been regarded as non-hemolytic. Reciprocal DNA pairing data for the reference strains of the DNA gropus were treated by UPGMA clustering. The reference strains for A. calcoaceticus, A. baumannii and DNA groups 3 and 13 formed a cluster with about 70% relatedness within the cluster. Other DNA groups joined at levels below 60%. (author)

Clinical strains of acinetobacter classified by DNA-DNA hybridization

Energy Technology Data Exchange (ETDEWEB)

Tjernberg, I; Ursing, J [Department of Medical Microbiology, University of Lund, Malmoe General Hospital, Malmoe (Sweden)

1989-01-01

A collection of Acinetobacter strains consisting of 168 consecutive clinical strains and 30 type and reference strains was studied by DNA-DNA hybridization and a few phenotypic tests. The field strains could be allotted to 13 DNA groups. By means of reference strains ten of these could be identified with groups described by Bouvet and Grimont (1986), while three groups were new; they were given the numbers 13-15. The type strain of A. radioresistens- recently described by Nishimura et al. (1988) - was shown to be a member of DNA group 12, which comprised 31 clinical isolates. Of the 19 strains of A. junii, eight showed hemolytic acitivity on sheep and human blood agar and an additional four strains on human blood agar only. Strains of this species have previously been regarded as non-hemolytic. Reciprocal DNA pairing data for the reference strains of the DNA gropus were treated by UPGMA clustering. The reference strains for A. calcoaceticus, A. baumannii and DNA groups 3 and 13 formed a cluster with about 70% relatedness within the cluster. Other DNA groups joined at levels below 60%. (author).

Microarray-based whole-genome hybridization as a tool for determining procaryotic species relatedness

Energy Technology Data Exchange (ETDEWEB)

Wu, L.; Liu, X.; Fields, M.W.; Thompson, D.K.; Bagwell, C.E.; Tiedje, J. M.; Hazen, T.C.; Zhou, J.

2008-01-15

The definition and delineation of microbial species are of great importance and challenge due to the extent of evolution and diversity. Whole-genome DNA-DNA hybridization is the cornerstone for defining procaryotic species relatedness, but obtaining pairwise DNA-DNA reassociation values for a comprehensive phylogenetic analysis of procaryotes is tedious and time consuming. A previously described microarray format containing whole-genomic DNA (the community genome array or CGA) was rigorously evaluated as a high-throughput alternative to the traditional DNA-DNA reassociation approach for delineating procaryotic species relationships. DNA similarities for multiple bacterial strains obtained with the CGA-based hybridization were comparable to those obtained with various traditional whole-genome hybridization methods (r=0.87, P<0.01). Significant linear relationships were also observed between the CGA-based genome similarities and those derived from small subunit (SSU) rRNA gene sequences (r=0.79, P<0.0001), gyrB sequences (r=0.95, P<0.0001) or REP- and BOX-PCR fingerprinting profiles (r=0.82, P<0.0001). The CGA hybridization-revealed species relationships in several representative genera, including Pseudomonas, Azoarcus and Shewanella, were largely congruent with previous classifications based on various conventional whole-genome DNA-DNA reassociation, SSU rRNA and/or gyrB analyses. These results suggest that CGA-based DNA-DNA hybridization could serve as a powerful, high-throughput format for determining species relatedness among microorganisms.

Oxidatively generated DNA/RNA damage in psychological stress states

DEFF Research Database (Denmark)

Jørgensen, Anders

2013-01-01

age-related somatic disorders. The overall aim of the PhD project was to investigate the relation between psychopathology, psychological stress, stress hormone secretion and oxidatively generated DNA and RNA damage, as measured by the urinary excretion of markers of whole-body DNA/RNA oxidation (8...... between the 24 h urinary cortisol excretion and the excretion of 8-oxodG/8-oxoGuo, determined in the same samples. Collectively, the studies could not confirm an association between psychological stress and oxidative stress on nucleic acids. Systemic oxidatively generated DNA/RNA damage was increased......Both non-pathological psychological stress states and mental disorders are associated with molecular, cellular and epidemiological signs of accelerated aging. Oxidative stress on nucleic acids is a critical component of cellular and organismal aging, and a suggested pathogenic mechanism in several...

An RNA polymerase II-and AGO4-associated protein acts in RNA-directed DNA methylation

KAUST Repository

Gao, Zhihuan

2010-04-21

DNA methylation is an important epigenetic mark in many eukaryotes. In plants, 24-nucleotide small interfering RNAs (siRNAs) bound to the effector protein, Argonaute 4 (AGO4), can direct de novo DNA methylation by the methyltransferase DRM2 (refs 2, 4-6). Here we report a new regulator of RNA-directed DNA methylation (RdDM) in Arabidopsis: RDM1. Loss-of-function mutations in the RDM1 gene impair the accumulation of 24-nucleotide siRNAs, reduce DNA methylation, and release transcriptional gene silencing at RdDM target loci. RDM1 encodes a small protein that seems to bind single-stranded methyl DNA, and associates and co-localizes with RNA polymerase II (Pol II, also known as NRPB), AGO4 and DRM2 in the nucleus. Our results indicate that RDM1 is a component of the RdDM effector complex and may have a role in linking siRNA production with pre-existing or de novo cytosine methylation. Our results also indicate that, although RDM1 and Pol V (also known as NRPE) may function together at some RdDM target sites in the peri-nucleolar siRNA processing centre, Pol II rather than Pol V is associated with the RdDM effector complex at target sites in the nucleoplasm. © 2010 Macmillan Publishers Limited. All rights reserved.

Expression of proto-oncogenes in non-Hodgkin's lymphomas by in situ hybridization with biotinylated DNA probes

International Nuclear Information System (INIS)

Hamatani, Kiyohiro; Yoshida, Kuniko; Abe, Masumi; Shimaoka, Katsutaro; Shiku, Hiroshi; Akiyama, Mitoshi; Kondo, Hisayoshi.

1989-11-01

Expression of six proto-oncogenes (fos, myc, myb, Ki-ras, Ha-ras, and N-ras) in 43 cases of non-Hodgkin's lymphoma was analyzed by means of in situ hybridization. Biotinylated DNA probes of the six oncogenes and those of the immunoglobulin H-chain (IgH) gene and the T cell receptor β-chain (TCRβ) gene were used. The results of in situ hybridization performed under blind conditions by IgH and TCRβ gene probes were compatible with those of typing by cell surface markers. The nuclear protein-related proto-oncogenes, fos myc, and myb, were expressed in about 70 % - 80 % of all cases regardless of phenotypes, histology or histologic grade. On the contrary, genes of the ras family were expressed in fewer cases except for the Ki-ras gene which was more frequently expressed by cases of the T cell immunophenotype with a high malignancy grade. The results of dot hybridization with RNA extracted from some cases were compatible with those of in situ hybridization, further demonstrating the specificity of in situ hybridization. (author)

Isolation and characterization of the human uracil DNA glycosylase gene

International Nuclear Information System (INIS)

Vollberg, T.M.; Siegler, K.M.; Cool, B.L.; Sirover, M.A.

1989-01-01

A series of anti-human placental uracil DNA glycosylase monoclonal antibodies was used to screen a human placental cDNA library in phage λgt11. Twenty-seven immunopositive plaques were detected and purified. One clone containing a 1.2-kilobase (kb) human cDNA insert was chosen for further study by insertion into pUC8. The resultant recombinant plasmid selected by hybridization a human placental mRNA that encoded a 37-kDa polypeptide. This protein was immunoprecipitated specifically by an anti-human placenta uracil DNA glycosylase monoclonal antibody. RNA blot-hybridization (Northern) analysis using placental poly(A) + RNA or total RNA from four different human fibroblast cell strains revealed a single 1.6-kb transcript. Genomic blots using DNA from each cell strain digested with either EcoRI or PstI revealed a complex pattern of cDNA-hydridizing restriction fragments. The genomic analysis for each enzyme was highly similar in all four human cell strains. In contrast, a single band was observed when genomic analysis was performed with the identical DNA digests with an actin gene probe. During cell proliferation there was an increase in the level of glycosylase mRNA that paralleled the increase in uracil DNA glycosylase enzyme activity. The isolation of the human uracil DNA glycosylase gene permits an examination of the structure, organization, and expression of a human DNA repair gene

Influence of thermodynamically unfavorable secondary structures on DNA hybridization kinetics

Science.gov (United States)

Hata, Hiroaki; Kitajima, Tetsuro

2018-01-01

Abstract Nucleic acid secondary structure plays an important role in nucleic acid–nucleic acid recognition/hybridization processes, and is also a vital consideration in DNA nanotechnology. Although the influence of stable secondary structures on hybridization kinetics has been characterized, unstable secondary structures, which show positive ΔG° with self-folding, can also form, and their effects have not been systematically investigated. Such thermodynamically unfavorable secondary structures should not be ignored in DNA hybridization kinetics, especially under isothermal conditions. Here, we report that positive ΔG° secondary structures can change the hybridization rate by two-orders of magnitude, despite the fact that their hybridization obeyed second-order reaction kinetics. The temperature dependence of hybridization rates showed non-Arrhenius behavior; thus, their hybridization is considered to be nucleation limited. We derived a model describing how ΔG° positive secondary structures affect hybridization kinetics in stopped-flow experiments with 47 pairs of oligonucleotides. The calculated hybridization rates, which were based on the model, quantitatively agreed with the experimental rate constant. PMID:29220504

Ultrasensitive electrochemical detection of microRNA-21 combining layered nanostructure of oxidized single-walled carbon nanotubes and nanodiamonds by hybridization chain reaction.

Science.gov (United States)

Liu, Lingzhi; Song, Chao; Zhang, Zhang; Yang, Juan; Zhou, Lili; Zhang, Xing; Xie, Guoming

2015-08-15

Measurement of microRNA (miRNA) levels in body fluids is a crucial tool for the early diagnosis and prognosis of cancers. In this study, we developed an electrochemical assay to detect miRNA-21 by fabricating the electrode with layer-by-layer assembly of oxidized single-walled carbon nanotubes and nanodiamonds. Tetrahedron-structured probes with free-standing probe on the top served as receptors to hybridize with target miRNA directly. The probes were immobilized on the deposited gold nanoparticles through a well-established strong Au-S bond. The electrochemical signal was mainly derived from an ultrasensitive pattern by combining hybridization chain reaction with DNA-functionalized AuNPs, which provided DNAzyme to catalyze H2O2 reduction. Differential pulse voltammetry was applied to record the electrochemical signals, which was increased linearly with the target miRNA-21, and the linear detection range was 10 fM to 1.0 nM. The limit of detection reached 1.95 fM (S/N=3), and the proposed biosensor exhibited good reproducibility and stability, as well as high sensitivity. Hence, this biosensor has a promising potential in clinical application. Copyright © 2015 Elsevier B.V. All rights reserved.

HAMDA: Hybrid Approach for MiRNA-Disease Association prediction.

Science.gov (United States)

Chen, Xing; Niu, Ya-Wei; Wang, Guang-Hui; Yan, Gui-Ying

2017-12-01

For decades, enormous experimental researches have collectively indicated that microRNA (miRNA) could play indispensable roles in many critical biological processes and thus also the pathogenesis of human complex diseases. Whereas the resource and time cost required in traditional biology experiments are expensive, more and more attentions have been paid to the development of effective and feasible computational methods for predicting potential associations between disease and miRNA. In this study, we developed a computational model of Hybrid Approach for MiRNA-Disease Association prediction (HAMDA), which involved the hybrid graph-based recommendation algorithm, to reveal novel miRNA-disease associations by integrating experimentally verified miRNA-disease associations, disease semantic similarity, miRNA functional similarity, and Gaussian interaction profile kernel similarity into a recommendation algorithm. HAMDA took not only network structure and information propagation but also node attribution into consideration, resulting in a satisfactory prediction performance. Specifically, HAMDA obtained AUCs of 0.9035 and 0.8395 in the frameworks of global and local leave-one-out cross validation, respectively. Meanwhile, HAMDA also achieved good performance with AUC of 0.8965 ± 0.0012 in 5-fold cross validation. Additionally, we conducted case studies about three important human cancers for performance evaluation of HAMDA. As a result, 90% (Lymphoma), 86% (Prostate Cancer) and 92% (Kidney Cancer) of top 50 predicted miRNAs were confirmed by recent experiment literature, which showed the reliable prediction ability of HAMDA. Copyright © 2017 Elsevier Inc. All rights reserved.

DNA-Damage Response RNA-Binding Proteins (DDRBPs): Perspectives from a New Class of Proteins and Their RNA Targets.

Science.gov (United States)

Dutertre, Martin; Vagner, Stéphan

2017-10-27

Upon DNA damage, cells trigger an early DNA-damage response (DDR) involving DNA repair and cell cycle checkpoints, and late responses involving gene expression regulation that determine cell fate. Screens for genes involved in the DDR have found many RNA-binding proteins (RBPs), while screens for novel RBPs have identified DDR proteins. An increasing number of RBPs are involved in early and/or late DDR. We propose to call this new class of actors of the DDR, which contain an RNA-binding activity, DNA-damage response RNA-binding proteins (DDRBPs). We then discuss how DDRBPs contribute not only to gene expression regulation in the late DDR but also to early DDR signaling, DNA repair, and chromatin modifications at DNA-damage sites through interactions with both long and short noncoding RNAs. Copyright © 2016 Elsevier Ltd. All rights reserved.

Imaging of RNA in situ hybridization by atomic force microscopy

NARCIS (Netherlands)

Kalle, W.H.J.; Macville, M.V.E.; van de Corput, M.P.C.; de Grooth, B.G.; Tanke, H.J.; Raap, A.K.

In this study we investigated the possibility of imaging internal cellular molecules after cytochemical detection with atomic force microscopy (AFM). To this end, rat 9G and HeLa cells were hybridized with haptenized probes for 28S ribosomal RNA, human elongation factor mRNA and cytomegalovirus

DNA hybridization on membrane-modified carbon electrodes

Czech Academy of Sciences Publication Activity Database

Kouřilová, Alena; Babkina, S. S.; Cahová, Kateřina; Havran, Luděk; Jelen, František; Paleček, Emil; Fojta, Miroslav

2005-01-01

Roč. 38, - (2005), s. 2493-2507 ISSN 0003-2719 R&D Projects: GA MPO(CZ) 1H-PK/42; GA AV ČR(CZ) IAA4004402; GA AV ČR(CZ) IBS5004355 Institutional research plan: CEZ:AV0Z50040507 Keywords : DNA hybridization * electrochemical DNA sensor * nitrocellulose membrane Subject RIV: BO - Biophysics Impact factor: 1.036, year: 2005

DNA/RNA-based formulations for treatment of breast cancer.

Science.gov (United States)

Xie, Zhaolu; Zeng, Xianghui

2017-12-01

To develop a successful formulation for the gene therapy of breast cancer, an effective therapeutic nucleic acid and a proper delivery system are essential. Increased understanding of breast cancer, and developments in biotechnology, material science and nanotechnology have provided a major impetus in the development of effective formulations for the gene therapy of breast cancer. Areas covered: We discuss DNA/RNA-based formulations that can inhibit the growth of breast cancer cells and control the progress of breast cancer. Targets for the gene therapy of breast cancer, DNA/RNA-based therapeutics and delivery systems are summarized. And examples of successful DNA/RNA-based formulations for breast cancer gene therapy are reviewed. Expert opinion: Several challenges remain in developing effective DNA/RNA-based formulations for treatment of breast cancer. Firstly, most of the currently utilized targets are not effective enough as monotherapy for breast cancer. Secondly, the requirements for co-delivery system make the preparation of formulation more complicated. Thirdly, nanoparticles with the modification of tumor-targeting ligands could be more unstable in circulation and normal tissues. Lastly, immune responses against the viral vectors are unfavorable for the gene therapy of breast cancer because of the damage to the host and the impaired therapeutic ability.

Determination of chromium combined with DNA, RNA and protein in chromium-rich brewer's yeast

International Nuclear Information System (INIS)

Ding Wenjun; Qian Qinfang; Hou Xiaolin; Feng Weiyue; Chai Zhifang

2000-01-01

The contents of chromium in the DNA, RNA and protein fractions separated from chromium-rich and normal brewer's yeast were determined with the neutron activation analysis in order to study the combination of Cr with DNA, RNA and protein in chromium-rich brewer's yeast. The results showed that the extracting rats and concentrations of DNA, RNA and protein had no significant difference in two types of yeast, but the chromium contents of DNA, RNA and protein in the chromium-rich yeast were significantly higher than those in the normal. In addition, the content of chromium in DNA was much higher than that in RNA and protein, which indicated that the inorganic chromium compounds entered into the yeast cell, during the yeast cultivation in the culture medium containing chromium were converted into organic chromium compounds combined with DNA, RNA and protein

Presence of albumin mRNA precursors in nuclei of analbuminemic rat liver lacking cytoplasmic albumin mRNA.

OpenAIRE

Esumi, H; Takahashi, Y; Sekiya, T; Sato, S; Nagase, S; Sugimura, T

1982-01-01

Analbuminemic rats, which lack serum albumin, were previously found to have no albumin mRNA in the cytoplasm of the liver. In the present study, the existence of nuclear albumin mRNA precursors in the liver of analbuminemic rats was examined by RNA X cDNA hybridization kinetics. Albumin mRNA precursors were present in the nuclei of analbuminemic rat liver at almost normal levels, despite the absence of albumin mRNA from the cytoplasm. Nuclear RNA of analbuminemic rat liver was subjected to el...

Towards Defined DNA and RNA Delivery Vehicles Using Nucleic Acid Nanotechnology

DEFF Research Database (Denmark)

Okholm, Anders Hauge; Schaffert, David Henning; Kjems, Jørgen

2014-01-01

Both DNA and RNA nanostructures show exceptional programmability, modularity, and self-assembly ability. Using DNA or RNA molecules it is possible to assemble monodisperse particles that are homogeneous in size and shape and with identical positioning of surface modifications. For therapeutic app...

Distribution of 45S rDNA in Modern Rose Cultivars (Rosa hybrida), Rosa rugosa, and Their Interspecific Hybrids Revealed by Fluorescence in situ Hybridization.

Science.gov (United States)

Ding, Xiao-Liu; Xu, Ting-Liang; Wang, Jing; Luo, Le; Yu, Chao; Dong, Gui-Min; Pan, Hui-Tang; Zhang, Qi-Xiang

2016-01-01

To elucidate the evolutionary dynamics of the location and number of rDNA loci in the process of polyploidization in the genus Rosa, we examined 45S rDNA sites in the chromosomes of 6 modern rose cultivars (R. hybrida), 5 R. rugosa cultivars, and 20 hybrid progenies by fluorescence in situ hybridization. Variation in the number of rDNA sites in parents and their interspecific hybrids was detected. As expected, 4 rDNA sites were observed in the genomes of 4 modern rose cultivars, while 3 hybridization sites were observed in the 2 others. Two expected rDNA sites were found in 2 R. rugosa cultivars, while in the other 3 R. rugosa cultivars 4 sites were present. Among the 20 R. hybrida × R. rugosa offspring, 13 carried the expected number of rDNA sites, and 1 had 6 hybridization sites, which exceeded the expected number by far. The other 6 offspring had either 2 or 3 hybridization sites, which was less than expected. Differences in the number of rDNA loci were observed in interspecific offspring, indicating that rDNA loci exhibit instability after distant hybridization events. Abnormal chromosome pairing may be the main factor explaining the variation in rDNA sites during polyploidization. © 2016 S. Karger AG, Basel.

Construction of recombinant DNA clone for bovine viral diarrhea virus

International Nuclear Information System (INIS)

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

1992-01-01

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

Evidence for a Complex Class of Nonadenylated mRNA in Drosophila

Science.gov (United States)

Zimmerman, J. Lynn; Fouts, David L.; Manning, Jerry E.

1980-01-01

The amount, by mass, of poly(A+) mRNA present in the polyribosomes of third-instar larvae of Drosophila melanogaster, and the relative contribution of the poly(A+) mRNA to the sequence complexity of total polysomal RNA, has been determined. Selective removal of poly(A+) mRNA from total polysomal RNA by use of either oligo-dT-cellulose, or poly(U)-sepharose affinity chromatography, revealed that only 0.15% of the mass of the polysomal RNA was present as poly(A+) mRNA. The present study shows that this RNA hybridized at saturation with 3.3% of the single-copy DNA in the Drosophila genome. After correction for asymmetric transcription and reactability of the DNA, 7.4% of the single-copy DNA in the Drosophila genome is represented in larval poly(A+) mRNA. This corresponds to 6.73 x 106 nucleotides of mRNA coding sequences, or approximately 5,384 diverse RNA sequences of average size 1,250 nucleotides. However, total polysomal RNA hybridizes at saturation to 10.9% of the single-copy DNA sequences. After correcting this value for asymmetric transcription and tracer DNA reactability, 24% of the single-copy DNA in Drosophila is represented in total polysomal RNA. This corresponds to 2.18 x 107 nucleotides of RNA coding sequences or 17,440 diverse RNA molecules of size 1,250 nucleotides. This value is 3.2 times greater than that observed for poly(A+) mRNA, and indicates that ≃69% of the polysomal RNA sequence complexity is contributed by nonadenylated RNA. Furthermore, if the number of different structural genes represented in total polysomal RNA is ≃1.7 x 104, then the number of genes expressed in third-instar larvae exceeds the number of chromomeres in Drosophila by about a factor of three. This numerology indicates that the number of chromomeres observed in polytene chromosomes does not reflect the number of structural gene sequences in the Drosophila genome. PMID:6777246

Organophosphonate functionalized silicon nanowires for DNA hybridization studies

Energy Technology Data Exchange (ETDEWEB)

Pedone, Daniel; Cattani Scholz, Anna; Birner, Stefan; Abstreiter, Gerhard [WSI, TU Muenchen (Germany); Dubey, Manish; Schwartz, Jeffrey [Princeton University, NJ (United States); Tornow, Marc [IHT, TU Braunschweig (Germany)

2007-07-01

Semiconductor nanowire field effect devices have great appeal for label-free sensing applications due to their sensitivity to surface potential changes that may originate from charged adsorbates. In addition to requiring high sensitivity, suitable passivation and functionalization of the semiconductor surface is obligatory. We have fabricated both freely suspended and oxide-supported silicon nanowires from Silicon-on-Insulator substrates using standard nanopatterning methods (EBL, RIE) and sacrificial oxide layer etching. Subsequent to nanofabrication, the devices were first coated with an hydroxyalkylphosphonate monolayer and then bound via bifunctional linker groups to single stranded DNA or PNA oligonucleotides, respectively. We investigated DNA hybridization on such functionalized nanowires using a difference resistance setup, where subtracting the reference signal from a second wire could be used to exclude most non-specific effects. A net change in surface potential on the order of a few mV could be detected upon addition of the complementary DNA strand. This surface potential change corresponds to the hybridization of about 10{sup 10}cm{sup -2} probe strands according to our model calculations that takes into account the entire hybrid system in electrolyte solution.

Application of a Novel and Automated Branched DNA in Situ Hybridization Method for the Rapid and Sensitive Localization of mRNA Molecules in Plant Tissues

Directory of Open Access Journals (Sweden)

Andrew J. Bowling

2014-04-01

Full Text Available Premise of the study: A novel branched DNA detection technology, RNAscope in situ hybridization (ISH, originally developed for use on human clinical and animal tissues, was adapted for use in plant tissue in an attempt to overcome some of the limitations associated with traditional ISH assays. Methods and Results: Zea mays leaf tissue was formaldehyde fixed and paraffin embedded (FFPE and then probed with the RNAscope ISH assay for two endogenous genes, phosphoenolpyruvate carboxylase (PEPC and phosphoenolpyruvate carboxykinase (PEPCK. Results from both manual and automated methods showed tissue- and cell-specific mRNA localization patterns expected from these well-studied genes. Conclusions: RNAscope ISH is a sensitive method that generates high-quality, easily interpretable results from FFPE plant tissues. Automation of the RNAscope method on the Ventana Discovery Ultra platform allows significant advantages for repeatability, reduction in variability, and flexibility of workflow processes.

Transcriptional activation of ribosomal RNA genes during compensatory renal hypertrophy

International Nuclear Information System (INIS)

Ouellette, A.J.; Moonka, R.; Zelenetz, A.; Malt, R.A.

1986-01-01

The overall rate of rDNA transcription increases by 50% during the first 24 hours of compensatory renal hypertrophy in the mouse. To study mechanisms of ribosome accumulation after uninephrectomy, transcription rates were measured in isolated kidneys by transcriptional runoff. 32 P-labeled nascent transcripts were hybridized to blots containing linearized, denatured cloned rDNA, and hybridization was quantitated autoradiographically and by direct counting. Overall transcriptional activity of rDNA was increased by 30% above control levels at 6 hrs after nephrectomy and by 50% at 12, 18, and 24 hrs after operation. Hybridizing RNA was insensitive to inhibiby alpha-amanitin, and no hybridization was detected to vector DNA. Thus, accelerated rDNA transcription is one regulatory element in the accretion of ribosomes in renal growth, and the regulatory event is an early event. Mechanisms of activation may include enhanced transcription of active genes or induction of inactive DNA

Tracking fungal community responses to maize plants by DNA- and RNA-based pyrosequencing.

Directory of Open Access Journals (Sweden)

Eiko E Kuramae

Full Text Available We assessed soil fungal diversity and community structure at two sampling times (t1 = 47 days and t2 = 104 days of plant age in pots associated with four maize cultivars, including two genetically modified (GM cultivars by high-throughput pyrosequencing of the 18S rRNA gene using DNA and RNA templates. We detected no significant differences in soil fungal diversity and community structure associated with different plant cultivars. However, DNA-based analyses yielded lower fungal OTU richness as compared to RNA-based analyses. Clear differences in fungal community structure were also observed in relation to sampling time and the nucleic acid pool targeted (DNA versus RNA. The most abundant soil fungi, as recovered by DNA-based methods, did not necessary represent the most "active" fungi (as recovered via RNA. Interestingly, RNA-derived community compositions at t1 were highly similar to DNA-derived communities at t2, based on presence/absence measures of OTUs. We recovered large proportions of fungal sequences belonging to arbuscular mycorrhizal fungi and Basidiomycota, especially at the RNA level, suggesting that these important and potentially beneficial fungi are not affected by the plant cultivars nor by GM traits (Bt toxin production. Our results suggest that even though DNA- and RNA-derived soil fungal communities can be very different at a given time, RNA composition may have a predictive power of fungal community development through time.

RNA Study Using DNA Nanotechnology.

Science.gov (United States)

Tadakuma, Hisashi; Masubuchi, Takeya; Ueda, Takuya

2016-01-01

Transcription is one of the fundamental steps of gene expression, where RNA polymerases (RNAPs) bind to their template genes and make RNAs. In addition to RNAP and the template gene, many molecules such as transcription factors are involved. The interaction and the effect of these factors depend on the geometry. Molecular layout of these factors, RNAP and gene is thus important. DNA nanotechnology is a promising technology that allows controlling of the molecular layout in the range of nanometer to micrometer scale with nanometer resolution; thus, it is expected to expand the RNA study beyond the current limit. Copyright © 2016 Elsevier Inc. All rights reserved.

Mapping of gene transcripts by nuclease protection assays and cDNA primer extension

International Nuclear Information System (INIS)

Calzone, F.J.; Britten, R.J.; Davidson, E.J.

1987-01-01

An important problem often faced in the molecular characterization of genes is the precise mapping of those genomic sequences transcribed into RNA. This requires identification of the genomic site initiating gene transcription, the location of genomic sequences removed from the primary gene transcript during RNA processing, and knowledge of sequences terminating the processed gene transcript. The objective of the protocols described here is the generation of transcription maps utilizing relatively uncharacterized gene fragments. The basic approach is hybridization of a single-stranded DNA probe with cellular RNA, followed by treatment with a single-strand-specific nuclease that does not attack DNA-RNA hybrids, in order to destroy any unreacted probe sequences. Thus the probe sequences included in the hybrid duplexes are protected from nuclease digestion. The sizes of the protected probe fragments determined by gel electrophoresis correspond to the lengths of the hybridized sequence elements

Restriction endonuclease analysis of chloroplast DNA in interspecies somatic Hybrids of Petunia.

Science.gov (United States)

Kumar, A; Cocking, E C; Bovenberg, W A; Kool, A J

1982-12-01

Restriction endonuclease cleavage pattern analysis of chloroplast DNA (cpDNA) of three different interspecific somatic hybrid plants revealed that the cytoplasms of the hybrids contained only cpDNA of P. parodii. The somatic hybrid plants analysed were those between P. parodii (wild type) + P. hybrida (wild type); P. parodii (wild type)+P. inflata (cytoplasmic albino mutant); P. parodii (wild type) + P. parviflora (nuclear albino mutant). The presence of only P. parodii chloroplasts in the somatic hybrid of P. parodii + P. inflata is possibly due to the stringent selection used for somatic hybrid production. However, in the case of the two other somatic hybrids P. parodii + P. hybrida and P. parodii + P. parviflora it was not possible to determine whether the presence of only P. parodii chloroplasts in these somatic hybrid plants was due to the nature of the selection schemes used or simply occurred by chance. The relevance of such somatic hybrid material for the study of genomic-cytoplasmic interaction is discussed, as well as the use of restriction endonuclease fragment patterns for the analysis of taxonomic and evolutionary inter-relationships in the genus Petunia.

Role of the CCA bulge of prohead RNA of bacteriophage ø29 in DNA packaging.

Science.gov (United States)

Zhao, Wei; Morais, Marc C; Anderson, Dwight L; Jardine, Paul J; Grimes, Shelley

2008-11-14

The oligomeric ring of prohead RNA (pRNA) is an essential component of the ATP-driven DNA packaging motor of bacteriophage ø29. The A-helix of pRNA binds the DNA translocating ATPase gp16 (gene product 16) and the CCA bulge in this helix is essential for DNA packaging in vitro. Mutation of the bulge by base substitution or deletion showed that the size of the bulge, rather than its sequence, is primary in DNA packaging activity. Proheads reconstituted with CCA bulge mutant pRNAs bound the packaging ATPase gp16 and the packaging substrate DNA-gp3, although DNA translocation was not detected with several mutants. Prohead/bulge-mutant pRNA complexes with low packaging activity had a higher rate of ATP hydrolysis per base pair of DNA packaged than proheads with wild-type pRNA. Cryoelectron microscopy three-dimensional reconstruction of proheads reconstituted with a CCA deletion pRNA showed that the protruding pRNA spokes of the motor occupy a different position relative to the head when compared to particles with wild-type pRNA. Therefore, the CCA bulge seems to dictate the orientation of the pRNA spokes. The conformational changes observed for this mutant pRNA may affect gp16 conformation and/or subsequent ATPase-DNA interaction and, consequently, explain the decreased packaging activity observed for CCA mutants.

The effects of storage temperature and duration of blood samples on DNA and RNA qualities.

Science.gov (United States)

Huang, Lien-Hung; Lin, Pei-Hsien; Tsai, Kuo-Wang; Wang, Liang-Jen; Huang, Ying-Hsien; Kuo, Ho-Chang; Li, Sung-Chou

2017-01-01

DNA and RNA samples from blood are the common examination target for non-invasive physical tests and/or biomedical studies. Since high-quality DNA and RNA samples guarantee the correctness of these tests and/or studies, we investigated the effects of storage temperature and storage duration of whole blood on DNA and RNA qualities. Subjects were enrolled to donate blood samples which were stored for different durations and at different temperatures, followed by the examinations on RNA quality, qPCR, DNA quality and DNA methylation. For RNA, we observed obvious quality decline with storage duration longer than 24 hours. Storage at low temperature does not keep RNA samples from degradation. And, storing whole blood samples in freezer dramatically damage RNA. For DNA, quality decline was not observed even with storage duration for 15 days. However, DNA methylation significantly altered with storage duration longer than three days. Storage duration within 24 hours is critical for collecting high-quality RNA samples for next-generation sequencing (NGS) assays (RIN≧8). If microarray assays are expected (RIN≧7), storage duration within 32 hours is acceptable. Although DNA is resistant within 15 days when kept in whole blood, DNA quantity dramatically decreases owing to WBC lysis. In addition, duration for more than three days significantly alter DNA methylation status, globally and locally. Our result provides a reference for dealing with blood samples.

Size and Base Composition of RNA in Supercoiled Plasmid DNA

Science.gov (United States)

Williams, Peter H.; Boyer, Herbert W.; Helinski, Donald R.

1973-01-01

The average size and base composition of the covalently integrated RNA segment in supercoiled ColE1 DNA synthesized in Escherichia coli in the presence of chloramphenicol (CM-ColE1 DNA) have been determined by two independent methods. The two approaches yielded similar results, indicating that the RNA segment in CM-ColE1 DNA contains GMP at the 5′ end and comprises on the average 25 to 26 ribonucleotides with a base composition of 10-11 G, 3 A, 5-6 C, and 6-7 U. PMID:4359488

dsRNA silencing of an R2R3-MYB transcription factor affects flower cell shape in a Dendrobium hybrid.

Science.gov (United States)

Lau, Su-Ee; Schwarzacher, Trude; Othman, Rofina Yasmin; Harikrishna, Jennifer Ann

2015-08-11

The R2R3-MYB genes regulate pigmentation and morphogenesis of flowers, including flower and cell shape, and therefore have importance in the development of new varieties of orchids. However, new variety development is limited by the long breeding time required in orchids. In this study, we identified a cDNA, DhMYB1, that is expressed during flower development in a hybrid orchid, Dendrobium hybrida (Dendrobium bobby messina X Dendrobium chao phraya) then used the direct application of dsRNA to observe the effect of gene silencing on flower phenotype and floral epidermal cell shape. Flower bud development in the Dendrobium hybrid was characterised into seven stages and the time of meiosis was determined as between stages 3 to 5 when the bud is approximately half of the mature size. Scanning electron microscopy characterisation of adaxial epidermal cells of the flower perianth, showed that the petals and sepals each are divided into two distinct domains based on cell shape and size, while the labellum comprises seven domains. Thirty-two partial cDNA fragments representing R2R3-MYB gene sequences were isolated from D. hybrida. Phylogenetic analysis revealed that nine of the translated sequences were clustered with MYB sequences that are known to be involved in cell shape development and from these, DhMYB1 was selected for full length cDNA cloning and functional study. Direct application of a 430 bp dsRNA from the 3' region of DhMYB1 to emerging orchid flower buds reduced expression of DhMYB1 RNA compared with untreated control. Scanning electron microscopy of adaxial epidermal cells within domain one of the labellum of flowers treated with DhMYB1 dsRNA showed flattened epidermal cells whilst those of control flowers were conical. DhMYB1 is expressed throughout flower bud development and is involved in the development of the conical cell shape of the epidermal cells of the Dendrobium hybrida flower labellum. The direct application of dsRNA changed the phenotype of

DNA and RNA Quadruplex-Binding Proteins

Czech Academy of Sciences Publication Activity Database

Brázda, Václav; Haroniková, Lucia; Liao, J.C.C.; Fojta, Miroslav

2014-01-01

Roč. 15, č. 10 (2014), s. 17493-17517 E-ISSN 1422-0067 R&D Projects: GA ČR(CZ) GBP206/12/G151 Institutional support: RVO:68081707 Keywords : DNA quadruplex * RNA quadruplex * telomere Subject RIV: BO - Biophysics Impact factor: 2.862, year: 2014

Identification of pathogenic Nocardia species by reverse line blot hybridization targeting the 16S rRNA and 16S-23S rRNA gene spacer regions.

Science.gov (United States)

Xiao, Meng; Kong, Fanrong; Sorrell, Tania C; Cao, Yongyan; Lee, Ok Cha; Liu, Ying; Sintchenko, Vitali; Chen, Sharon C A

2010-02-01

Although 16S rRNA gene sequence analysis is employed most often for the definitive identification of Nocardia species, alternate molecular methods and polymorphisms in other gene targets have also enabled species determinations. We evaluated a combined Nocardia PCR-based reverse line blot (RLB) hybridization assay based on 16S and 16S-23S rRNA gene spacer region polymorphisms to identify 12 American Type Culture Collection and 123 clinical Nocardia isolates representing 14 species; results were compared with results from 16S rRNA gene sequencing. Thirteen 16S rRNA gene-based (two group-specific and 11 species-specific) and five 16S-23S spacer-targeted (two taxon-specific and three species-specific) probes were utilized. 16S rRNA gene-based probes correctly identified 124 of 135 isolates (sensitivity, 92%) but were unable to identify Nocardia paucivorans strains (n = 10 strains) and a Nocardia asteroides isolate with a novel 16S rRNA gene sequence. Nocardia farcinica and Nocardia cyriacigeorgica strains were identified by the sequential use of an N. farcinica-"negative" probe and a combined N. farcinica/N. cyriacigeorgica probe. The assay specificity was high (99%) except for weak cross-reactivity between the Nocardia brasiliensis probe with the Nocardia thailandica DNA product; however, cross-hybridization with closely related nontarget species may occur. The incorporation of 16S-23S rRNA gene spacer-based probes enabled the identification of all N. paucivorans strains. The overall sensitivity using both probe sets was >99%. Both N. farcinica-specific 16S-23S rRNA gene spacer-directed probes were required to identify all N. farcinica stains by using this probe set. The study demonstrates the utility of a combined PCR/RLB assay for the identification of clinically relevant Nocardia species and its potential for studying subtypes of N. farcinica. Where species assignment is ambiguous or not possible, 16S rRNA gene sequencing is recommended.

Sequence-specific RNA Photocleavage by Single-stranded DNA in Presence of Riboflavin

Science.gov (United States)

Zhao, Yongyun; Chen, Gangyi; Yuan, Yi; Li, Na; Dong, Juan; Huang, Xin; Cui, Xin; Tang, Zhuo

2015-10-01

Constant efforts have been made to develop new method to realize sequence-specific RNA degradation, which could cause inhibition of the expression of targeted gene. Herein, by using an unmodified short DNA oligonucleotide for sequence recognition and endogenic small molecue, vitamin B2 (riboflavin) as photosensitizer, we report a simple strategy to realize the sequence-specific photocleavage of targeted RNA. The DNA strand is complimentary to the target sequence to form DNA/RNA duplex containing a G•U wobble in the middle. The cleavage reaction goes through oxidative elimination mechanism at the nucleoside downstream of U of the G•U wobble in duplex to obtain unnatural RNA terminal, and the whole process is under tight control by using light as switch, which means the cleavage could be carried out according to specific spatial and temporal requirements. The biocompatibility of this method makes the DNA strand in combination with riboflavin a promising molecular tool for RNA manipulation.

Detection of DNA hybridization using graphene-coated black phosphorus surface plasmon resonance sensor

Science.gov (United States)

Pal, Sarika; Verma, Alka; Raikwar, S.; Prajapati, Y. K.; Saini, J. P.

2018-05-01

In this paper, graphene-coated black phosphorus at the metal surface for the detection of DNA hybridization event is numerically demonstrated. The strategy consists of placing the sensing medium on top of black phosphorus-graphene-coated SPR which interfaces with phosphate-buffered saline solution carrying single-stranded DNA. Upon hybridization with its complementary DNA, desorption of the nanostructures takes place and thus enables the sensitive detection of the DNA hybridization event. The proposed sensor exhibits a sensitivity (125 ο/RIU), detection accuracy (0.95) and quality factor (13.62 RIU-1) for complementary DNA. In comparison with other reported papers, our suggested sensor provides much better performance. Thus, this label-free DNA detection platform should spur off new interest towards the use of black phosphorus-graphene-coated SPR interfaces.

Amplification of a transcriptionally active DNA sequence in the human brain

International Nuclear Information System (INIS)

Yakovlev, A.G.; Sazonov, A.E.; Spunde, A.Ya.; Gindilis, V.M.

1986-01-01

The authors present their findings of tissue-specific amplification of a DNA fragment actively transcribed in the human brain. This genome fragment was found in the library complement of cDNA of the human brain and evidently belongs to a new class of moderate repetitions of DNA with an unstable copying capacity in the human genome. The authors isolated total cell RNA from various human tissues (brain, placenta), and rat tissues (brain, liver), by the method of hot phenol extraction with guanidine thiocynate. The poly(A + ) RNA fraction was isolated by chromatography. Synthesis of cDNA was done on a matrix of poly(A + ) RNA of human brain. The cDNA obtained was cloned in plasmid pBR322 for the PstI site using (dC/dG) sequences synthesized on the 3' ends of the vector molecule and cDNA respectively. In cloning 75 ng cDNA, the authors obtained approximately 10 5 recombinant. This library was analyzed by the hybridization method on columns with two radioactive ( 32 P) probes: the total cDNA preparation and the total nuclear DNA from the human brain. The number of copies of the cloned DNA fragment in the genome was determined by dot hybridization. Restricting fragments of human and rat DNA genomes homologous to the cloned cDNA were identified on radio-autographs. In each case, 10 micrograms of EcoRI DNA hydrolyzate was fractionated in 1% agarose gel. The probe was also readied with RNA samples fractionated in agarose gel with formaldehyde and transferred to a nitrocellulose filter under weak vacuum. The filter was hybridized with 0.1 micrograms DNA pAG 02, labeled with ( 32 P) to a specific activity of 0.5-1 x 10 9 counts/min x microgram. The autograph was exposed with amplifying screens at -70 0 C for 2 days

Microfluidic Arrayed Lab-On-A-Chip for Electrochemical Capacitive Detection of DNA Hybridization Events.

Science.gov (United States)

Ben-Yoav, Hadar; Dykstra, Peter H; Bentley, William E; Ghodssi, Reza

2017-01-01

A microfluidic electrochemical lab-on-a-chip (LOC) device for DNA hybridization detection has been developed. The device comprises a 3 × 3 array of microelectrodes integrated with a dual layer microfluidic valved manipulation system that provides controlled and automated capabilities for high throughput analysis of microliter volume samples. The surface of the microelectrodes is functionalized with single-stranded DNA (ssDNA) probes which enable specific detection of complementary ssDNA targets. These targets are detected by a capacitive technique which measures dielectric variation at the microelectrode-electrolyte interface due to DNA hybridization events. A quantitative analysis of the hybridization events is carried out based on a sensing modeling that includes detailed analysis of energy storage and dissipation components. By calculating these components during hybridization events the device is able to demonstrate specific and dose response sensing characteristics. The developed microfluidic LOC for DNA hybridization detection offers a technology for real-time and label-free assessment of genetic markers outside of laboratory settings, such as at the point-of-care or in-field environmental monitoring.

Ratiometric FRET-based detection of DNA and micro-RNA on the surface using TIRF detection

International Nuclear Information System (INIS)

Matveeva, Evgenia G.; Gryczynski, Zygmunt; Stewart, Donald R.; Gryczynski, Ignacy

2010-01-01

A new FRET-based method for the ratiometric detection of DNA oligomers on a surface using TIRF detection mode is reported. The dual-labeled system consisting of two hybridized oligomers, Cy3oligoY:Cy5oligoX was immobilized on the surface, and the total internal reflection fluorescence (TIRF) was used to detect emission signals from the surface. Two signals, green and red, which originated from the green donor Cy3 and the red acceptor Cy5, have been simultaneously detected. When the target single-stranded complimentary oligomer was present in the solution, this oligomer replaced the Cy3oligoY in the donor:acceptor complex on the surface and the ratio of red-to-green signal was dramatically changed. This detection scheme is generally applicable to the detection of DNA or RNA on a surface.

The RNA World: Life Before DNA and Protein

Science.gov (United States)

Joyce, Gerald F.

1993-01-01

All of the life that is known, all organisms that exist on Earth today or are known to have existed on Earth in the past, are of the same life form: a life form based on DNA and protein. It does not necessarily have to be that way. Why not have two competing life forms on this planet? Why not have biology as we know it and some other biology that occupies its own distinct niche? Yet that is not how evolution has played out. From microbes living on the surface of antarctic ice to tube worms lying near the deep-sea hydrothermal vents, all known organisms on this planet are of the same biology. Looking at the single known biology on Earth, it is clear that this biology could not have simply sprung forth from the primordial soup. The biological system that is the basis for all known. life is far too complicated to have arisen spontaneously. This brings us to the notion that something else something simpler, must have preceded life based on DNA and protein. One suggestion that has gained considerable acceptance over the past decade is that DNA and protein-based life was preceded by RNA-based life in a period referred to as the 'RNA world'. Even an RNA-based life form would have been fairly complicated - not as complicated as our own DNA- and protein-based life form - but far too complicated, according to prevailing scientific thinking, to have arisen spontaneously from the primordial soup. Thus, it has been argued that something else must have preceded RNA-based life, or even that there was a succession of life forms leading from the primordial soup to RNA-based life. The experimental evidence to support this conjecture is not strong because, after all, the origin of life was a historical event that left no direct physical record. However, based on indirect evidence in both the geological record and the phylogenetic record of evolutionary history on earth, it is possible to reconstruct a rough picture of what life was like before DNA and protein.

Building a Phylogenetic Tree of the Human and Ape Superfamily Using DNA-DNA Hybridization Data

Science.gov (United States)

Maier, Caroline Alexander

2004-01-01

The study describes the process of DNA-DNA hybridization and the history of its use by Sibley and Alquist in simple, straightforward, and interesting language that students easily understand to create their own phylogenetic tree of the hominoid superfamily. They calibrate the DNA clock and use it to estimate the divergence dates of the various…

Design of a Sensitive and Selective Electrochemical Aptasensor for the Determination of the Complementary cDNA of miRNA-145 Based on the Intercalation and Electrochemical Reduction of Doxorubicin.

Science.gov (United States)

Mohamadi, Maryam; Mostafavi, Ali; Torkzadeh-Mahani, Masoud

2017-11-01

The aim of this research was the determination of a microRNA (miRNA) using a DNA electrochemical aptasensor. In this biosensor, the complementary complementary DNA (cDNA) of miRNA-145 (a sense RNA transcript) was the target strand and the cDNA of miRNA-145 was the probe strand. Both cDNAs can be the product of the reverse transcriptase-polymerase chain reaction of miRNA. The proposed aptasensor's function was based on the hybridization of target strands with probes immobilized on the surface of a working electrode and the subsequent intercalation of doxorubicin (DOX) molecules functioning as the electroactive indicators of any double strands that formed. Electrochemical transduction was performed by measuring the cathodic current resulting from the electrochemical reduction of the intercalated molecules at the electrode surface. In the experiment, because many DOX molecules accumulated on each target strand on the electrode surface, amplification was inherently easy, without a need for enzymatic or complicated amplification strategies. The proposed aptasensor also had the excellent ability to regenerate as a result of the melting of the DNA duplex. Moreover, the use of DNA probe strands obviated the challenges of working with an RNA probe, such as sensitivity to RNase enzyme. In addition to the linear relationship between the electrochemical signal and the concentration of the target strands that ranged from 2.0 to 80.0 nM with an LOD of 0.27 nM, the proposed biosensor was clearly capable of distinguishing between complementary (target strand) and noncomplementary sequences. The presented biosensor was successfully applied for the quantification of DNA strands corresponding to miRNA-145 in human serum samples.

Localization of insulin receptor mRNA in rat brain by in situ hybridization

International Nuclear Information System (INIS)

Marks, J.L.; Porte, D. Jr.; Stahl, W.L.; Baskin, D.G.

1990-01-01

Insulin receptor mRNA was demonstrated in rat brain slices by in situ hybridization with three 35 S-oligonucleotide probes and contact film autoradiography. Specificity was confirmed by showing that (a) excess unlabeled probe abolished the signal, (b) an oligonucleotide probe for rat neuropeptide Y mRNA showed a different distribution of hybridization signal, and (c) the distribution of insulin receptor binding was consistent with the distribution of insulin receptor mRNA. Insulin receptor mRNA was most abundant in the granule cell layers of the olfactory bulb, cerebellum and dentate gyrus, in the pyramidal cell body layers of the pyriform cortex and hippocampus, in the choroid plexus and in the arcuate nucleus of the hypothalamus

Surface density dependence of PCR amplicon hybridization on PNA/DNA probe layers

DEFF Research Database (Denmark)

Yao, Danfeng; Kim, Junyoung; Yu, Fang

2005-01-01

at an intermediate sodium concentration (approximately 100 mM). These effects were mainly ascribed to the electrostatic cross talk among the hybridized DNA molecules and the secondary structure of PCR amplicons. For the negatively charged DNA probes, the hybridization reaction was subjected additionally to the DNA....../DNA electrostatic barrier, particularly in lower ionic strength range (e.g., 10 approximately 150 mM Na(+)). The electrostatic cross talk was shown to be largely reduced if the PNA probe layer was sufficiently diluted by following a strategic templated immobilization method. As a consequence, a pseudo...

RNA and DNA Targeting by a Reconstituted Thermus thermophilus Type III-A CRISPR-Cas System.

Directory of Open Access Journals (Sweden)

Tina Y Liu

Full Text Available CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated systems are RNA-guided adaptive immunity pathways used by bacteria and archaea to defend against phages and plasmids. Type III-A systems use a multisubunit interference complex called Csm, containing Cas proteins and a CRISPR RNA (crRNA to target cognate nucleic acids. The Csm complex is intriguing in that it mediates RNA-guided targeting of both RNA and transcriptionally active DNA, but the mechanism is not well understood. Here, we overexpressed the five components of the Thermus thermophilus (T. thermophilus Type III-A Csm complex (TthCsm with a defined crRNA sequence, and purified intact TthCsm complexes from E. coli cells. The complexes were thermophilic, targeting complementary ssRNA more efficiently at 65°C than at 37°C. Sequence-independent, endonucleolytic cleavage of single-stranded DNA (ssDNA by TthCsm was triggered by recognition of a complementary ssRNA, and required a lack of complementarity between the first 8 nucleotides (5' tag of the crRNA and the 3' flanking region of the ssRNA. Mutation of the histidine-aspartate (HD nuclease domain of the TthCsm subunit, Cas10/Csm1, abolished DNA cleavage. Activation of DNA cleavage was dependent on RNA binding but not cleavage. This leads to a model in which binding of an ssRNA target to the Csm complex would stimulate cleavage of exposed ssDNA in the cell, such as could occur when the RNA polymerase unwinds double-stranded DNA (dsDNA during transcription. Our findings establish an amenable, thermostable system for more in-depth investigation of the targeting mechanism using structural biology methods, such as cryo-electron microscopy and x-ray crystallography.

RNA and DNA Targeting by a Reconstituted Thermus thermophilus Type III-A CRISPR-Cas System.

Science.gov (United States)

Liu, Tina Y; Iavarone, Anthony T; Doudna, Jennifer A

2017-01-01

CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated) systems are RNA-guided adaptive immunity pathways used by bacteria and archaea to defend against phages and plasmids. Type III-A systems use a multisubunit interference complex called Csm, containing Cas proteins and a CRISPR RNA (crRNA) to target cognate nucleic acids. The Csm complex is intriguing in that it mediates RNA-guided targeting of both RNA and transcriptionally active DNA, but the mechanism is not well understood. Here, we overexpressed the five components of the Thermus thermophilus (T. thermophilus) Type III-A Csm complex (TthCsm) with a defined crRNA sequence, and purified intact TthCsm complexes from E. coli cells. The complexes were thermophilic, targeting complementary ssRNA more efficiently at 65°C than at 37°C. Sequence-independent, endonucleolytic cleavage of single-stranded DNA (ssDNA) by TthCsm was triggered by recognition of a complementary ssRNA, and required a lack of complementarity between the first 8 nucleotides (5' tag) of the crRNA and the 3' flanking region of the ssRNA. Mutation of the histidine-aspartate (HD) nuclease domain of the TthCsm subunit, Cas10/Csm1, abolished DNA cleavage. Activation of DNA cleavage was dependent on RNA binding but not cleavage. This leads to a model in which binding of an ssRNA target to the Csm complex would stimulate cleavage of exposed ssDNA in the cell, such as could occur when the RNA polymerase unwinds double-stranded DNA (dsDNA) during transcription. Our findings establish an amenable, thermostable system for more in-depth investigation of the targeting mechanism using structural biology methods, such as cryo-electron microscopy and x-ray crystallography.

Colloidal Au-enhanced surface plasmon resonance imaging: application in a DNA hybridization process

International Nuclear Information System (INIS)

Manera, M G; Spadavecchia, J; Taurino, A; Rella, R

2010-01-01

The detection of the DNA hybridization mechanism using monodispersed gold nanoparticles as labels is an interesting alternative to increase the sensitivity of the SPR imaging technique. DNA-modified Au nanoparticles (DNA-Au NPs) containing single-stranded (ss) portions of DNA were prepared by monitoring their monolayer formation by UV–vis spectroscopy. The hybridization process between specific thio-oligonucleotides immobilized on the DNA–Au NPs and the corresponding complementary strands is reported and compared with the traditional hybridization process on properly self-assembled thin gold films deposited on glass substrates. A remarkable signal amplification is observed, following the incorporation of colloidal Au into a SPR biosensing experiment, resulting in an increased SPR response to DNA–DNA interactions. In particular Fusarium thiolated DNA (5'HS poly(T) 15 ATC CCT CAA AAA CTG CCG CT-3) and trichothecenes complementary DNA (5'-AGC GGC AGT TTT TGA GGG AT-3') sequences have been explored due to their possible application to agro-industry for the control of food quality

Nerve growth factor mRNA in brain: localization by in situ hybridization

International Nuclear Information System (INIS)

Rennert, P.D.; Heinrich, G.

1986-01-01

Nerve Growth Factor is a 118 amino acid polypeptide that plays an important role in the differentiation and survival of neurons. The recent discovery that a mRNA that encodes beta Nerve Growth Factor is present in brain suggests that the Nerve Growth Factor gene may not only regulate gene expression of peripheral but also of central neurons. To identify the site(s) of Nerve Growth Factor mRNA production in the brain and to determine which cells express the Nerve Growth Factor gene, the technique of in situ hybridization was employed. A 32P-labeled RNA probe complementary to Nerve Growth Factor mRNA hybridized to cells in the stratum granulosum of the dentate gyrus and the stratum pyramidale of the hippocampus. These observations identify for the first time cellular sites of Nerve Growth Factor gene expression in the central nervous system, and suggest that Nerve Growth Factor mRNA is produced by neurons

Altered minor-groove hydrogen bonds in DNA block transcription elongation by T7 RNA polymerase.

Science.gov (United States)

Tanasova, Marina; Goeldi, Silvan; Meyer, Fabian; Hanawalt, Philip C; Spivak, Graciela; Sturla, Shana J

2015-05-26

DNA transcription depends upon the highly efficient and selective function of RNA polymerases (RNAPs). Modifications in the template DNA can impact the progression of RNA synthesis, and a number of DNA adducts, as well as abasic sites, arrest or stall transcription. Nonetheless, data are needed to understand why certain modifications to the structure of DNA bases stall RNA polymerases while others are efficiently bypassed. In this study, we evaluate the impact that alterations in dNTP/rNTP base-pair geometry have on transcription. T7 RNA polymerase was used to study transcription over modified purines and pyrimidines with altered H-bonding capacities. The results suggest that introducing wobble base-pairs into the DNA:RNA heteroduplex interferes with transcriptional elongation and stalls RNA polymerase. However, transcriptional stalling is not observed if mismatched base-pairs do not H-bond. Together, these studies show that RNAP is able to discriminate mismatches resulting in wobble base-pairs, and suggest that, in cases of modifications with minor steric impact, DNA:RNA heteroduplex geometry could serve as a controlling factor for initiating transcription-coupled DNA repair. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Analysis of experimental mink enteritis virus infection in mink: in situ hybridization, serology, and histopathology

DEFF Research Database (Denmark)

Uttenthal, Åse; Larsen, S; Lund, E

1990-01-01

Strand-specific hybridization probes were used in in situ hybridization studies to localize cells containing mink enteritis virus (MEV) virion DNA or MEV replicative-form DNA and mRNA. Following the experimental MEV infection of 3-month-old unvaccinated mink, a significant increase in serum antib...

Modified method for combined DNA and RNA isolation from peanut and other oil seeds.

Science.gov (United States)

Dang, Phat M; Chen, Charles Y

2013-02-01

Isolation of good quality RNA and DNA from seeds is difficult due to high levels of polysaccharides, polyphenols, and lipids that can degrade or co-precipitate with nucleic acids. Standard RNA extraction methods utilizing guanidinium-phenol-chloroform extraction has not shown to be successful. RNA isolation from plant seeds is a prerequisite for many seed specific gene expression studies and DNA is necessary in marker-assisted selection and other genetic studies. We describe a modified method to isolate both RNA and DNA from the same seed tissue and have been successful with several oil seeds including peanut, soybean, sunflower, canola, and oil radish. An additional LiCl precipitation step was added to isolate both RNA and DNA from the same seed tissues. High quality nucleic acids were observed based on A(260)/A(280) and A(260)/A(230) ratios above 2.0 and distinct bands on gel-electrophoresis. RNA was shown to be suitable for reverse transcriptase polymerase chain reaction based on actin or 60S ribosomal primer amplification and DNA was shown to have a single band on gel-electrophoresis analysis. This result shows that RNA and DNA isolated using this method can be appropriate for molecular studies in peanut and other oil containing seeds.

Nonparametric testing for DNA copy number induced differential mRNA gene expression

NARCIS (Netherlands)

van Wieringen, W.N.; van de Wiel, M.A.

2009-01-01

The central dogma of molecular biology relates DNA with mRNA. Array CGH measures DNA copy number and gene expression microarrays measure the amount of mRNA. Methods that integrate data from these two platforms may uncover meaningful biological relationships that further our understanding of cancer.

Synthesis and structural characterization of piperazino-modified DNA that favours hybridization towards DNA over RNA

DEFF Research Database (Denmark)

Skov, Joan; Bryld, Torsten; Lindegaard, Dorthe

2011-01-01

We report the synthesis of two C4'-modified DNA analogues and characterize their structural impact on dsDNA duplexes. The 4'-C-piperazinomethyl modification stabilizes dsDNA by up to 5°C per incorporation. Extension of the modification with a butanoyl-linked pyrene increases the dsDNA stabilizati...

Effect of uv irradiation on lambda DNA transcription

Energy Technology Data Exchange (ETDEWEB)

Ranade, S S [Cancer Research Inst., Bombay (India)

1977-05-01

The effect of uv irradiation of template DNA has been studied in vitro in the E.coli RNA polymerase system with native and uv treated lambda DNA. Lambda DNA was more susceptible to uv than was calf-thymus DNA, yet a residual activity was observed at a uv dose of 0.5 x 10/sup 4/ erg/mm/sup 2/. From the kinetic analysis of the reaction and the incorporation of lambda /sup 32/P-labelled nucleoside triphosphates, it seems reasonable to conclude that uv irradiation probably did not affect the DNA initiation sites, recognizable by RNA polymerase. The transcription products made with uv irradiated lambda DNA were asymmetrical, and hybridized to the right half (R) and the left half (L) of lambda DNA with the ratio of R/L=4/1, and they showed a lower hybridizability than the transcripts with native lambda DNA. The initiation sites recognizable by RNA polymerase seemed to be the same on both native and uv irradiated lambda DNA, though the transcription of uv treated lambda DNA appeared to terminate with rather short RNA chains.

A populational survey of 45S rDNA polymorphism in the Jefferson salamander Ambystoma jeffersonianum revealed by fluorescence in situ hybridization (FISH

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Jinzhong FU

2009-04-01

Full Text Available The chromosomal localization of 45S ribosomal RNA genes in Ambystoma jeffersonianum was determined by fluorescence in situ hybridization with 18S rDNA fragment as a probe (FISH-rDNA. Our results revealed the presence of rDNA polymorphism among A.jeffersonianum populations in terms of number, location and FISH signal intensity on the chromosomes. Nine rDNA cytotypes were found in ten geographically isolated populations and most of them contained derivative rDNA sites. Our preliminary study provides strong indication of karyotypic diversification of A.jeffersonianum that is demonstrated by intraspecific variation of 45S rDNA cytotypes. rDNA cytotype polymorphism has been described in many other caudate amphibians. We predict that habitat isolation, low dispersal ability and decline of effective population size could facilitate the fixation and accumulation of variable rDNA cytotypes during their chromosome evolution.

A DNA microarray-based methylation-sensitive (MS)-AFLP hybridization method for genetic and epigenetic analyses.

Science.gov (United States)

Yamamoto, F; Yamamoto, M

2004-07-01

We previously developed a PCR-based DNA fingerprinting technique named the Methylation Sensitive (MS)-AFLP method, which permits comparative genome-wide scanning of methylation status with a manageable number of fingerprinting experiments. The technique uses the methylation sensitive restriction enzyme NotI in the context of the existing Amplified Fragment Length Polymorphism (AFLP) method. Here we report the successful conversion of this gel electrophoresis-based DNA fingerprinting technique into a DNA microarray hybridization technique (DNA Microarray MS-AFLP). By performing a total of 30 (15 x 2 reciprocal labeling) DNA Microarray MS-AFLP hybridization experiments on genomic DNA from two breast and three prostate cancer cell lines in all pairwise combinations, and Southern hybridization experiments using more than 100 different probes, we have demonstrated that the DNA Microarray MS-AFLP is a reliable method for genetic and epigenetic analyses. No statistically significant differences were observed in the number of differences between the breast-prostate hybridization experiments and the breast-breast or prostate-prostate comparisons.

Sets of RNA repeated tags and hybridization-sensitive fluorescent probes for distinct images of RNA in a living cell.

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Takeshi Kubota

Full Text Available BACKGROUND: Imaging the behavior of RNA in a living cell is a powerful means for understanding RNA functions and acquiring spatiotemporal information in a single cell. For more distinct RNA imaging in a living cell, a more effective chemical method to fluorescently label RNA is now required. In addition, development of the technology labeling with different colors for different RNA would make it easier to analyze plural RNA strands expressing in a cell. METHODOLOGY/PRINCIPAL FINDINGS: Tag technology for RNA imaging in a living cell has been developed based on the unique chemical functions of exciton-controlled hybridization-sensitive oligonucleotide (ECHO probes. Repetitions of selected 18-nucleotide RNA tags were incorporated into the mRNA 3'-UTR. Pairs with complementary ECHO probes exhibited hybridization-sensitive fluorescence emission for the mRNA expressed in a living cell. The mRNA in a nucleus was detected clearly as fluorescent puncta, and the images of the expression of two mRNAs were obtained independently and simultaneously with two orthogonal tag-probe pairs. CONCLUSIONS/SIGNIFICANCE: A compact and repeated label has been developed for RNA imaging in a living cell, based on the photochemistry of ECHO probes. The pairs of an 18-nt RNA tag and the complementary ECHO probes are highly thermostable, sequence-specifically emissive, and orthogonal to each other. The nucleotide length necessary for one tag sequence is much shorter compared with conventional tag technologies, resulting in easy preparation of the tag sequences with a larger number of repeats for more distinct RNA imaging.

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

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Jahwarhar Izuan Abdul Rashid

2017-11-01

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

Nucleic acid hybridization and radioimmunoassay techniques for studying the interrelationships among the progressive pneumonia viruses of sheep

International Nuclear Information System (INIS)

Weiss, M.J.

1976-01-01

In Section I of this thesis, experiments were performed to determine if three representative ''slow'' viruses of sheep VV, MV and PPV replicate by way of a DNA ''provirus'' in a manner similar to the RNA tumor viruses. The approach used was to determine if unique virus-specific DNA sequences not present in normal cells could be detected in the DNA of infected cell cultures. The results presented demonstrate that infection by VV, MV and PPV results in the synthesis of proviral DNA. Sections II and III examine the similarities among VV, MV and PPV. In Section II, the RNA genomes of these viruses were compared by nucleic acid hybridization. The homology among these viral RNAs was determined from the extensive competition of homologous viral RNA-cDNA hybrids by heterologous RNA and from the thermal stability of homologous and heterologous RNA-cDNA hybrids. The 70S RNAs of visna and maedi virus were indistinguishable but only partially homologous to that of progressive pneumonia virus. Section III describes the purification of the major internal protein component of VV, p27, the development of a radioimmunoassay to study its antigenic relatedness to the corresponding proteins of PPV and MV, and its use in the detection of cross-reacting proteins in progressive pneumonia virus infected sheep lung. The ability to detect unique virus-related DNA sequences and viral antigens in infected sheep tissues makes it now feasible to search for slow virus related DNA sequences and/or antigens in human diseases which bear resemblance to the slow diseases of sheep

Development of species-specific rDNA probes for Giardia by multiple fluorescent in situ hybridization combined with immunocytochemical identification of cyst wall antigens.

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Erlandsen, Stanley L; Jarroll, Edward; Wallis, Peter; van Keulen, Harry

2005-08-01

In this study, we describe the development of fluorescent oligonucleotide probes to variable regions in the small subunit of 16S rRNA in three distinct Giardia species. Sense and antisense probes (17-22 mer) to variable regions 1, 3, and 8 were labeled with digoxygenin or selected fluorochomes (FluorX, Cy3, or Cy5). Optimal results were obtained with fluorochome-labeled oligonucleotides for detection of rRNA in Giardia cysts. Specificity of fluorescent in situ hybridization (FISH) was shown using RNase digestion and high stringency to diminish the hybridization signal, and oligonucleotide probes for rRNA in Giardia lamblia, Giardia muris, and Giardia ardeae were shown to specifically stain rRNA only within cysts or trophozoites of those species. The fluorescent oligonucleotide specific for rRNA in human isolates of Giardia was positive for ten different strains. A method for simultaneous FISH detection of cysts using fluorescent antibody (genotype marker) and two oligonucleotide probes (species marker) permitted visualization of G. lamblia and G. muris cysts in the same preparation. Testing of an environmental water sample revealed the presence of FISH-positive G. lamblia cysts with a specific rDNA probe for rRNA, while negative cysts were presumed to be of animal or bird origin.

The fidelity of reverse transcription differs in reactions primed with RNA versus DNA primers

NARCIS (Netherlands)

Oude Essink, B. B.; Berkhout, B.

1999-01-01

Reverse transcriptase enzymes (RT) convert single-stranded retroviral RNA genomes into double-stranded DNA. The RT enzyme can use both RNA and DNA primers, the former being used exclusively during initiation of minus- and plus-strand synthesis. Initiation of minus-strand DNA synthesis occurs by

The effect of tissue decalcification on mRNA retention within bone for in-situ hybridization studies.

Science.gov (United States)

Walsh, L; Freemont, A J; Hoyland, J A

1993-06-01

Tissue decalcification is a routine part of the preparation of bone tissue for histological studies. Although in-situ hybridization has been employed to localize mRNA of collagenous and non-collagenous bone related proteins in skeletal tissue, little is known regarding the effects of decalcifying agents on mRNA retention within tissue. In this study in-situ hybridization using an oligonucleotide probe (i.e. a poly d(T) probe) to detect total messenger RNA has been employed to investigate the effects of the decalcifying agents nitric acid, formic acid and EDTA on mRNA retention compared to undeacalcified tissue. The results show that formalin fixation and EDTA decalcification preserve substantial amounts of mRNA within the tissue. In particular, this study illustrates that it is possible to perform in-situ hybridization on formalin fixed decalcified paraffin embedded tissue.

Hybrid DNA i-motif: Aminoethylprolyl-PNA (pC5) enhance the stability of DNA (dC5) i-motif structure.

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Gade, Chandrasekhar Reddy; Sharma, Nagendra K

2017-12-15

This report describes the synthesis of C-rich sequence, cytosine pentamer, of aep-PNA and its biophysical studies for the formation of hybrid DNA:aep-PNAi-motif structure with DNA cytosine pentamer (dC 5 ) under acidic pH conditions. Herein, the CD/UV/NMR/ESI-Mass studies strongly support the formation of stable hybrid DNA i-motif structure with aep-PNA even near acidic conditions. Hence aep-PNA C-rich sequence cytosine could be considered as potential DNA i-motif stabilizing agents in vivo conditions. Copyright © 2017 Elsevier Ltd. All rights reserved.

Replication and Transcription of Eukaryotic DNA in Esherichia coli

Science.gov (United States)

Morrow, John F.; Cohen, Stanley N.; Chang, Annie C. Y.; Boyer, Herbert W.; Goodman, Howard M.; Helling, Robert B.

1974-01-01

Fragments of amplified Xenopus laevis DNA, coding for 18S and 28S ribosomal RNA and generated by EcoRI restriction endonuclease, have been linked in vitro to the bacterial plasmid pSC101; and the recombinant molecular species have been introduced into E. coli by transformation. These recombinant plasmids, containing both eukaryotic and prokaryotic DNA, replicate stably in E. coli. RNA isolated from E. coli minicells harboring the plasmids hybridizes to amplified X. laevis rDNA. Images PMID:4600264

The chromosomal constitution of fish hybrid lineage revealed by 5S rDNA FISH.

Science.gov (United States)

Zhang, Chun; Ye, Lihai; Chen, Yiyi; Xiao, Jun; Wu, Yanhong; Tao, Min; Xiao, Yamei; Liu, Shaojun

2015-12-03

The establishment of the bisexual fertile fish hybrid lineage including the allodiploid and allotetraploid hybrids, from interspecific hybridization of red crucian carp (Carassius auratus red var. 2n = 100, 2n = AA) (♀) × common carp (Cyprinus carpio L. 2n = 100, 2n = BB) (♂), provided a good platform to investigate genetic relationship between the parents and their hybrid progenies. The chromosomal inheritance of diploid and allotetraploid hybrid progenies in successive generations, was studied by applying 5S rDNA fluorescence in situ hybridization. Signals of 5S rDNA distinguished the chromosomal constitution of common carp (B-genome) from red crucian carp (A-genome), in which two strong signals were observed on the first submetacentric chromosome, while no major signal was found in common carp. After fish hybridization, one strong signal of 5S rDNA was detected in the same locus on the chromosome of diploid hybrids. As expected, two strong signals were observed in 4nF3 tetraploid hybrids offspring and it is worth mentioning that two strong signals were detected in a separating bivalent of a primary spermatocyte in 4nF3. Furthermore, the mitosis of heterozygous chromosomes was shown normal and stable with blastular tissue histological studies. We revealed that 5S rDNA signal can be applied to discern A-genome from B-genome, and that 5S rDNA bearing chromosomes can be stably passed down in successive generations. Our work provided a significant method in fish breeding and this is important for studies in fish evolutionary biology.

Simulating movement of tRNA through the ribosome during hybrid-state formation.

Science.gov (United States)

Whitford, Paul C; Sanbonmatsu, Karissa Y

2013-09-28

Biomolecular simulations provide a means for exploring the relationship between flexibility, energetics, structure, and function. With the availability of atomic models from X-ray crystallography and cryoelectron microscopy (cryo-EM), and rapid increases in computing capacity, it is now possible to apply molecular dynamics (MD) simulations to large biomolecular machines, and systematically partition the factors that contribute to function. A large biomolecular complex for which atomic models are available is the ribosome. In the cell, the ribosome reads messenger RNA (mRNA) in order to synthesize proteins. During this essential process, the ribosome undergoes a wide range of conformational rearrangements. One of the most poorly understood transitions is translocation: the process by which transfer RNA (tRNA) molecules move between binding sites inside of the ribosome. The first step of translocation is the adoption of a "hybrid" configuration by the tRNAs, which is accompanied by large-scale rotations in the ribosomal subunits. To illuminate the relationship between these rearrangements, we apply MD simulations using a multi-basin structure-based (SMOG) model, together with targeted molecular dynamics protocols. From 120 simulated transitions, we demonstrate the viability of a particular route during P/E hybrid-state formation, where there is asynchronous movement along rotation and tRNA coordinates. These simulations not only suggest an ordering of events, but they highlight atomic interactions that may influence the kinetics of hybrid-state formation. From these simulations, we also identify steric features (H74 and surrounding residues) encountered during the hybrid transition, and observe that flexibility of the single-stranded 3'-CCA tail is essential for it to reach the endpoint. Together, these simulations provide a set of structural and energetic signatures that suggest strategies for modulating the physical-chemical properties of protein synthesis by the

High-resolution NMR studies of chimeric DNA-RNA-DNA duplexes, heteronomous base pairing, and continuous base stacking at junctions

International Nuclear Information System (INIS)

Chou, Shanho; Flynn, P.; Wang, A.; Reid, B.

1991-01-01

Two symmetrical DNA-RNA-DNA duplex chimeras, d(CGCG)r(AAUU)d(CGCG) (designated rAAUU) and d(CGCG)r(UAUA)d(CGCG) (designated rUAUA), and a nonsymmetrical chimeric duplex, d(CGTT)r(AUAA)d(TGCG)/d(CGCA)r(UUAU)d(AACG) (designated rAUAA), as well as their pure DNA analogues, containing dU instead of T, have been synthesized by solid-phase phosphoramidite methods and studied by high-resolution NMR techniques. The 1D imino proton NOE spectra of these d-r-d chimeras indicate normal Watson-Crick hydrogen bonding and base stacking at the junction region. Preliminary qualitative NOESY, COSY, and chemical shift data suggest that the internal RNA segment contains C3'-endo (A-type) sugar conformations except for the first RNA residues (position 5 and 17) following the 3' end of the DNA block, which, unlike the other six ribonucleotides, exhibit detectable H1'-H2' J coupling. The nucleosides of the two flanking DNA segments appear to adopt a fairly normal C2'-endo B-DNA conformation except at the junction with the RNA blocks (residues 4 and 16), where the last DNA residue appears to adopt an intermediate sugar conformation. The data indicate that A-type and B-type conformations can coexist in a single short continuous nucleic acid duplex, but these results differ somewhat from previous theoretical model studies

Interspecific Transmission of Double-Stranded RNA and Hypovirulence from Sclerotinia sclerotiorum to S. minor.

Science.gov (United States)

Melzer, M S; Ikeda, S S; Boland, G J

2002-07-01

ABSTRACT Interspecific transmission of a hypovirulence-associated double-stranded RNA (dsRNA) and hypovirulent phenotype was attempted from hypovirulent isolate Ss275 of Sclerotinia sclerotiorum to five virulent isolates of S. minor. dsRNA and the hypovirulent phenotype were successfully transmitted to one of the five isolates, Sm10. Three putative converted isolates of Sm10 were slow growing and developed atypical colony morphologies characteristic of the hypovirulent phenotype. These isolates were assayed for virulence and produced significantly smaller lesions than isolate Sm10 on detached leaves of Romaine lettuce. One of these putative converted isolates, designated Sm10T, was tested to confirm interspecific transmission of dsRNA. In northern hybridizations, dsRNA isolated from Sm10T hybridized with a digoxigenin-labeled cDNA probe prepared from dsRNA isolated from Ss275. Random amplified polymorphic DNA analysis confirmed that isolate Sm10T was derived from Sm10 and not from Ss275 or a hybrid of the two species. The dsRNA and hypovirulent phenotype were subsequently transmitted intraspecifically from Sm10T to Sm8. To our knowledge, this is the first report of interspecific transmission of dsRNA and an associated hypovirulent phenotype between fungal plant pathogens by hyphal anastomosis.

Direct immobilization and hybridization of DNA on group III nitride semiconductors

Energy Technology Data Exchange (ETDEWEB)

Xu Xiaobin; Jindal, Vibhu; Shahedipour-Sandvik, Fatemeh; Bergkvist, Magnus [College of Nanoscale Science and Engineering, University at Albany (SUNY), 255 Fuller Road, Albany, NY 12203 (United States); Cady, Nathaniel C. [College of Nanoscale Science and Engineering, University at Albany (SUNY), 255 Fuller Road, Albany, NY 12203 (United States)], E-mail: ncady@uamail.albany.edu

2009-03-15

A key concern for group III-nitride high electron mobility transistor (HEMT) biosensors is the anchoring of specific capture molecules onto the gate surface. To this end, a direct immobilization strategy was developed to attach single-stranded DNA (ssDNA) to AlGaN surfaces using simple printing techniques without the need for cross-linking agents or complex surface pre-functionalization procedures. Immobilized DNA molecules were stably attached to the AlGaN surfaces and were able to withstand a range of pH and ionic strength conditions. The biological activity of surface-immobilized probe DNA was also retained, as demonstrated by sequence-specific hybridization experiments. Probe hybridization with target ssDNA could be detected by PicoGreen fluorescent dye labeling with a minimum detection limit of 2 nM. These experiments demonstrate a simple and effective immobilization approach for attaching nucleic acids to AlGaN surfaces which can further be used for the development of HEMT-based DNA biosensors.

Immobilization/hybridization of amino-modified DNA on plasma-polymerized allyl chloride

International Nuclear Information System (INIS)

Zhang Zhihong; Feng Chuanliang

2007-01-01

The present work describes the fabrication and characterization of chloride-derivatized polymer coatings prepared by continuous wave (cw) plasma polymerization as adhesion layers in DNA immobilization/hybridization. The stability of plasma-polymerized allyl chloride (ppAC) in H 2 O was characterized by variation of the thickness of polymer films and its wettability was examined by water contact angle technique. Fourier transform infrared spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS) were used to study polymer matrix properties and oligonucleotide/DNA binding interaction. With the same carrier gas rate and process pressure, plasma polymers deposited at different input powers show various comparable immobilization properties; nevertheless, low input power plasma-polymerized films gives a lower sensitivity toward DNA binding than that from high input power plasma-deposited films. The following DNA immobilization on chloride-functionalized surfaces was found dependence on the macromolecular architecture of the plasma films. The hybridization between probe DNA and total mismatch target DNA shows no non-specific adsorption between target and ppAC

Dendrimer-based biosensor for chemiluminescent detection of DNA hybridization

International Nuclear Information System (INIS)

Liu, P.; Hun, X.; Qing, H.

2011-01-01

We report on a highly sensitive chemiluminescent (CL) biosensor for the sequence-specific detection of DNA using a novel bio barcode DNA probe modified with gold nanoparticles that were covered with a dendrimer. The modified probe is composed of gold nanoparticles, a dendrimer, the CL reagent, and the DNA. The capture probe DNA was immobilized on magnetic beads covered with gold. It first hybridizes with the target DNA and then with one terminal end of the signal DNA on the barcoded DNA probe. CL was generated by adding H 2 O 2 and Co(II) ions as the catalyst. The immobilization of dendrimer onto the gold nanoparticles can significantly enhance sensitivity and gives a detection limit of 6 fmol L -1 of target DNA. (author)

Cloning and sequencing of cDNA encoding human DNA topoisomerase II and localization of the gene to chromosome region 17q21-22

International Nuclear Information System (INIS)

Tsai-Pflugfelder, M.; Liu, L.F.; Liu, A.A.; Tewey, K.M.; Whang-Peng, J.; Knutsen, T.; Huebner, K.; Croce, C.M.; Wang, J.C.

1988-01-01

Two overlapping cDNA clones encoding human DNA topoisomerase II were identified by two independent methods. In one, a human cDNA library in phage λ was screened by hybridization with a mixed oligonucleotide probe encoding a stretch of seven amino acids found in yeast and Drosophila DNA topoisomerase II; in the other, a different human cDNA library in a λgt11 expression vector was screened for the expression of antigenic determinants that are recognized by rabbit antibodies specific to human DNA topoisomerase II. The entire coding sequences of the human DNA topoisomerase II gene were determined from these and several additional clones, identified through the use of the cloned human TOP2 gene sequences as probes. Hybridization between the cloned sequences and mRNA and genomic DNA indicates that the human enzyme is encoded by a single-copy gene. The location of the gene was mapped to chromosome 17q21-22 by in situ hybridization of a cloned fragment to metaphase chromosomes and by hybridization analysis with a panel of mouse-human hybrid cell lines, each retaining a subset of human chromosomes

Protocols for 16S rDNA Array Analyses of Microbial Communities by Sequence-Specific Labeling of DNA Probes

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Knut Rudi

2003-01-01

Full Text Available Analyses of complex microbial communities are becoming increasingly important. Bottlenecks in these analyses, however, are the tools to actually describe the biodiversity. Novel protocols for DNA array-based analyses of microbial communities are presented. In these protocols, the specificity obtained by sequence-specific labeling of DNA probes is combined with the possibility of detecting several different probes simultaneously by DNA array hybridization. The gene encoding 16S ribosomal RNA was chosen as the target in these analyses. This gene contains both universally conserved regions and regions with relatively high variability. The universally conserved regions are used for PCR amplification primers, while the variable regions are used for the specific probes. Protocols are presented for DNA purification, probe construction, probe labeling, and DNA array hybridizations.

NanoRNase from Aeropyrum pernix shows nuclease activity on ssDNA and ssRNA.

Science.gov (United States)

Deng, Yong-Jie; Feng, Lei; Zhou, Huan; Xiao, Xiang; Wang, Feng-Ping; Liu, Xi-Peng

2018-05-01

In cells, degrading DNA and RNA by various nucleases is very important. These processes are strictly controlled and regulated to maintain DNA integrity and to mature or recycle various RNAs. NanoRNase (Nrn) is a 3'-exonuclease that specifically degrades nanoRNAs shorter than 5 nucleotides. Several Nrns have been identified and characterized in bacteria, mainly in Firmicutes. Archaea often grow in extreme environments and might be subjected to more damage to DNA/RNA, so DNA repair and recycling of damaged RNA are very important in archaea. There is no report on the identification and characterization of Nrn in archaea. Aeropyrum pernix encodes three potential Nrns: NrnA (Ape1437), NrnB (Ape0124), and an Nrn-like protein Ape2190. Biochemical characterization showed that only Ape0124 could degrade ssDNA and ssRNA from the 3'-end in the presence of Mn 2+ . Interestingly, unlike bacterial Nrns, Ape0124 prefers ssDNA, including short nanoDNA, and degrades nanoRNA with lower efficiency. The 3'-DNA backbone was found to be required for efficiently hydrolyzing the phosphodiester bonds. In addition, Ape0124 also degrads the 3'-overhang of double-stranded DNA. Interestingly, Ape0124 could hydrolyze pAp into AMP, which is a feature of bacterial NrnA, not NrnB. Our results indicate that Ape0124 is a novel Nrn with a combined substrate profile of bacterial NrnA and NrnB. Copyright © 2018 Elsevier B.V. All rights reserved.

Determination of gene expression patterns using high-throughput RNA in situ hybridizaion to whole-mount Drosophila embryos

Energy Technology Data Exchange (ETDEWEB)

Weiszmann, R.; Hammonds, A.S.; Celniker, S.E.

2009-04-09

We describe a high-throughput protocol for RNA in situ hybridization (ISH) to Drosophila embryos in a 96-well format. cDNA or genomic DNA templates are amplified by PCR and then digoxigenin-labeled ribonucleotides are incorporated into antisense RNA probes by in vitro transcription. The quality of each probe is evaluated before ISH using a RNA probe quantification (dot blot) assay. RNA probes are hybridized to fixed, mixed-staged Drosophila embryos in 96-well plates. The resulting stained embryos can be examined and photographed immediately or stored at 4oC for later analysis. Starting with fixed, staged embryos, the protocol takes 6 d from probe template production through hybridization. Preparation of fixed embryos requires a minimum of 2 weeks to collect embryos representing all stages. The method has been used to determine the expression patterns of over 6,000 genes throughout embryogenesis.

DNA hybridization kinetics: zippering, internal displacement and sequence dependence.

Science.gov (United States)

Ouldridge, Thomas E; Sulc, Petr; Romano, Flavio; Doye, Jonathan P K; Louis, Ard A

2013-10-01

Although the thermodynamics of DNA hybridization is generally well established, the kinetics of this classic transition is less well understood. Providing such understanding has new urgency because DNA nanotechnology often depends critically on binding rates. Here, we explore DNA oligomer hybridization kinetics using a coarse-grained model. Strand association proceeds through a complex set of intermediate states, with successful binding events initiated by a few metastable base-pairing interactions, followed by zippering of the remaining bonds. But despite reasonably strong interstrand interactions, initial contacts frequently dissociate because typical configurations in which they form differ from typical states of similar enthalpy in the double-stranded equilibrium ensemble. Initial contacts must be stabilized by two or three base pairs before full zippering is likely, resulting in negative effective activation enthalpies. Non-Arrhenius behavior arises because the number of base pairs required for nucleation increases with temperature. In addition, we observe two alternative pathways-pseudoknot and inchworm internal displacement-through which misaligned duplexes can rearrange to form duplexes. These pathways accelerate hybridization. Our results explain why experimentally observed association rates of GC-rich oligomers are higher than rates of AT- rich equivalents, and more generally demonstrate how association rates can be modulated by sequence choice.

Molecular modeling of cationic porphyrin-anthraquinone hybrids as DNA topoisomerase IIβ inhibitors.

Science.gov (United States)

Arba, Muhammad; Ruslin; Ihsan, Sunandar; Tri Wahyudi, Setyanto; Tjahjono, Daryono H

2017-12-01

Human DNA Topoisomerase II has been regarded as a promising target in anticancer drug discovery. In the present study, we designed six porphyrin-anthraquinone hybrids bearing pyrazole or pyridine group as meso substituents and evaluated their potentials as DNA Topoisomerase IIβ inhibitor. First, we investigated the binding orientation of porphyrin hybrids into DNA topoisomerase IIβ employing AutoDock 4.2 and then performed 20-ns molecular dynamics simulations to see the dynamic stability of each porphyrin-Topo IIβ complex using Amber 14. We found that the binding of porphyrin hybrids occured through intercalation and groove binding mode in addition interaction with the amino acid residues constituting the active cavity of Topo IIβ. Each porphyrin-Topo IIβ complex was stabilized during 20-ns dynamics simulations. The MM-PBSA free energy calculation shows that the binding affinities of porphyrin hybrids were modified with the number of meso substituent. Interestingly, the affinity of all porphyrin hybrids to Topo IIβ was stronger than that of native ligand (EVP), indicating the potential of the designed porphyrin to be considered in experimental research. Copyright © 2017 Elsevier Ltd. All rights reserved.

New redox-active layer create via epoxy-amine reaction - The base of genosensor for the detection of specific DNA and RNA sequences of avian influenza virus H5N1.

Science.gov (United States)

Malecka, Kamila; Stachyra, Anna; Góra-Sochacka, Anna; Sirko, Agnieszka; Zagórski-Ostoja, Włodzimierz; Dehaen, Wim; Radecka, Hanna; Radecki, Jerzy

2015-03-15

This paper concerns the development of a redox-active monolayer and its application for the construction of an electrochemical genosensor designed for the detection of specific DNA and RNA oligonucleotide sequences related to the avian influenza virus (AIV) type H5N1. This new redox layer was created on a gold electrode surface step by step. Cyclic Voltammetry, Osteryoung Square-Wave Voltammetry and Differential Pulse Voltammetry were used for its characterization. This new redox-active layer was applied for the construction of the DNA biosensor. The NH2-NC3 probe (20-mer) was covalently attached to the gold electrode surface via a "click" reaction between the amine and an epoxide group. The hybridization process was monitored using the Osteryoung Square-Wave Voltammetry. The 20-mer DNA and ca. 280-mer RNA oligonucleotides were used as the targets. The constructed genosensor was capable to determine complementary oligonucleotide sequences with a detection limit in the pM range. It is able to distinguish the different position of the part RNA complementary to the DNA probe. The genosensor was very selective. The 20-mer DNA as well as the 280-mer RNA oligonucleotides without a complementary sequence generated a weak signal. Copyright © 2014 Elsevier B.V. All rights reserved.

RNA polymerase II transcriptional fidelity control and its functional interplay with DNA modifications

Science.gov (United States)

Xu, Liang; Wang, Wei; Chong, Jenny; Shin, Ji Hyun; Xu, Jun; Wang, Dong

2016-01-01

Accurate genetic information transfer is essential for life. As a key enzyme involved in the first step of gene expression, RNA polymerase II (Pol II) must maintain high transcriptional fidelity while it reads along DNA template and synthesizes RNA transcript in a stepwise manner during transcription elongation. DNA lesions or modifications may lead to significant changes in transcriptional fidelity or transcription elongation dynamics. In this review, we will summarize recent progress towards understanding the molecular basis of RNA Pol II transcriptional fidelity control and impacts of DNA lesions and modifications on Pol II transcription elongation. PMID:26392149

Human ribonuclease H1 resolves R-loops and thereby enables progression of the DNA replication fork.

Science.gov (United States)

Parajuli, Shankar; Teasley, Daniel C; Murali, Bhavna; Jackson, Jessica; Vindigni, Alessandro; Stewart, Sheila A

2017-09-15

Faithful DNA replication is essential for genome stability. To ensure accurate replication, numerous complex and redundant replication and repair mechanisms function in tandem with the core replication proteins to ensure DNA replication continues even when replication challenges are present that could impede progression of the replication fork. A unique topological challenge to the replication machinery is posed by RNA-DNA hybrids, commonly referred to as R-loops. Although R-loops play important roles in gene expression and recombination at immunoglobulin sites, their persistence is thought to interfere with DNA replication by slowing or impeding replication fork progression. Therefore, it is of interest to identify DNA-associated enzymes that help resolve replication-impeding R-loops. Here, using DNA fiber analysis, we demonstrate that human ribonuclease H1 (RNH1) plays an important role in replication fork movement in the mammalian nucleus by resolving R-loops. We found that RNH1 depletion results in accumulation of RNA-DNA hybrids, slowing of replication forks, and increased DNA damage. Our data uncovered a role for RNH1 in global DNA replication in the mammalian nucleus. Because accumulation of RNA-DNA hybrids is linked to various human cancers and neurodegenerative disorders, our study raises the possibility that replication fork progression might be impeded, adding to increased genomic instability and contributing to disease. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

16S rRNA Gene Sequence Analysis of Drinking Water Using RNA and DNA Extracts as Targets for Clone Library Development

Science.gov (United States)

The bacterial composition of chlorinated drinking water was analyzed using 16S rRNA gene clone libraries derived from DNA extracts of 12 samples and compared to clone libraries previously generated using RNA extracts from the same samples. Phylogenetic analysis of 761 DNA-based ...

Transcription and DNA Damage: Holding Hands or Crossing Swords?

Science.gov (United States)

D'Alessandro, Giuseppina; d'Adda di Fagagna, Fabrizio

2017-10-27

Transcription has classically been considered a potential threat to genome integrity. Collision between transcription and DNA replication machinery, and retention of DNA:RNA hybrids, may result in genome instability. On the other hand, it has been proposed that active genes repair faster and preferentially via homologous recombination. Moreover, while canonical transcription is inhibited in the proximity of DNA double-strand breaks, a growing body of evidence supports active non-canonical transcription at DNA damage sites. Small non-coding RNAs accumulate at DNA double-strand break sites in mammals and other organisms, and are involved in DNA damage signaling and repair. Furthermore, RNA binding proteins are recruited to DNA damage sites and participate in the DNA damage response. Here, we discuss the impact of transcription on genome stability, the role of RNA binding proteins at DNA damage sites, and the function of small non-coding RNAs generated upon damage in the signaling and repair of DNA lesions. Copyright © 2016 Elsevier Ltd. All rights reserved.

Modeling Hybridization Kinetics of Gene Probes in a DNA Biochip Using FEMLAB

Science.gov (United States)

Munir, Ahsan; Waseem, Hassan; Williams, Maggie R.; Stedtfeld, Robert D.; Gulari, Erdogan; Tiedje, James M.; Hashsham, Syed A.

2017-01-01

Microfluidic DNA biochips capable of detecting specific DNA sequences are useful in medical diagnostics, drug discovery, food safety monitoring and agriculture. They are used as miniaturized platforms for analysis of nucleic acids-based biomarkers. Binding kinetics between immobilized single stranded DNA on the surface and its complementary strand present in the sample are of interest. To achieve optimal sensitivity with minimum sample size and rapid hybridization, ability to predict the kinetics of hybridization based on the thermodynamic characteristics of the probe is crucial. In this study, a computer aided numerical model for the design and optimization of a flow-through biochip was developed using a finite element technique packaged software tool (FEMLAB; package included in COMSOL Multiphysics) to simulate the transport of DNA through a microfluidic chamber to the reaction surface. The model accounts for fluid flow, convection and diffusion in the channel and on the reaction surface. Concentration, association rate constant, dissociation rate constant, recirculation flow rate, and temperature were key parameters affecting the rate of hybridization. The model predicted the kinetic profile and signal intensities of eighteen 20-mer probes targeting vancomycin resistance genes (VRGs). Predicted signal intensities and hybridization kinetics strongly correlated with experimental data in the biochip (R2 = 0.8131). PMID:28555058

Modeling Hybridization Kinetics of Gene Probes in a DNA Biochip Using FEMLAB

Directory of Open Access Journals (Sweden)

Ahsan Munir

2017-05-01

Full Text Available Microfluidic DNA biochips capable of detecting specific DNA sequences are useful in medical diagnostics, drug discovery, food safety monitoring and agriculture. They are used as miniaturized platforms for analysis of nucleic acids-based biomarkers. Binding kinetics between immobilized single stranded DNA on the surface and its complementary strand present in the sample are of interest. To achieve optimal sensitivity with minimum sample size and rapid hybridization, ability to predict the kinetics of hybridization based on the thermodynamic characteristics of the probe is crucial. In this study, a computer aided numerical model for the design and optimization of a flow-through biochip was developed using a finite element technique packaged software tool (FEMLAB; package included in COMSOL Multiphysics to simulate the transport of DNA through a microfluidic chamber to the reaction surface. The model accounts for fluid flow, convection and diffusion in the channel and on the reaction surface. Concentration, association rate constant, dissociation rate constant, recirculation flow rate, and temperature were key parameters affecting the rate of hybridization. The model predicted the kinetic profile and signal intensities of eighteen 20-mer probes targeting vancomycin resistance genes (VRGs. Predicted signal intensities and hybridization kinetics strongly correlated with experimental data in the biochip (R2 = 0.8131.

Fluorescence In situ Hybridization: Cell-Based Genetic Diagnostic and Research Applications.

Science.gov (United States)

Cui, Chenghua; Shu, Wei; Li, Peining

2016-01-01

Fluorescence in situ hybridization (FISH) is a macromolecule recognition technology based on the complementary nature of DNA or DNA/RNA double strands. Selected DNA strands incorporated with fluorophore-coupled nucleotides can be used as probes to hybridize onto the complementary sequences in tested cells and tissues and then visualized through a fluorescence microscope or an imaging system. This technology was initially developed as a physical mapping tool to delineate genes within chromosomes. Its high analytical resolution to a single gene level and high sensitivity and specificity enabled an immediate application for genetic diagnosis of constitutional common aneuploidies, microdeletion/microduplication syndromes, and subtelomeric rearrangements. FISH tests using panels of gene-specific probes for somatic recurrent losses, gains, and translocations have been routinely applied for hematologic and solid tumors and are one of the fastest-growing areas in cancer diagnosis. FISH has also been used to detect infectious microbias and parasites like malaria in human blood cells. Recent advances in FISH technology involve various methods for improving probe labeling efficiency and the use of super resolution imaging systems for direct visualization of intra-nuclear chromosomal organization and profiling of RNA transcription in single cells. Cas9-mediated FISH (CASFISH) allowed in situ labeling of repetitive sequences and single-copy sequences without the disruption of nuclear genomic organization in fixed or living cells. Using oligopaint-FISH and super-resolution imaging enabled in situ visualization of chromosome haplotypes from differentially specified single-nucleotide polymorphism loci. Single molecule RNA FISH (smRNA-FISH) using combinatorial labeling or sequential barcoding by multiple round of hybridization were applied to measure mRNA expression of multiple genes within single cells. Research applications of these single molecule single cells DNA and RNA FISH

Fluorescence In situ Hybridization: Cell-Based Genetic Diagnostic and Research Applications

Directory of Open Access Journals (Sweden)

Chenghua Cui

2016-09-01

Full Text Available Fluorescence in situ hybridization (FISH is a macromolecule recognition technology based on the complementary nature of DNA or DNA/RNA double strands. Selected DNA strands incorporated with fluorophore-coupled nucleotides can be used as probes to hybridize onto the complementary sequences in tested cells and tissues and then visualized through a fluorescence microscope or an imaging system. This technology was initially developed as a physical mapping tool to delineate genes within chromosomes. Its high analytical resolution to a single gene level and high sensitivity and specificity enabled an immediate application for genetic diagnosis of constitutional common aneuploidies, microdeletion/microduplication syndromes and subtelomeric rearrangements. FISH tests using panels of gene-specific probes for somatic recurrent losses, gains and translocations have been routinely applied for hematologic and solid tumors and are one of the fastest-growing areas in cancer diagnosis. FISH has also been used to detect infectious microbials and parasites like malaria in human blood cells. Recent advances in FISH technology involve various methods for improving probe labeling efficiency and the use of super resolution imaging systems for direct visualization of intra-nuclear chromosomal organization and profiling of RNA transcription in single cells. Cas9-mediated FISH (CASFISH allowed in situ labeling of repetitive sequences and single-copy sequences without the disruption of nuclear genomic organization in fixed or living cells. Using oligopaint-FISH and super-resolution imaging enabled in situ visualization of chromosome haplotypes from differentially specified single-nucleotide polymorphism loci. Single molecule RNA FISH (smRNA-FISH using combinatorial labeling or sequential barcoding by multiple round of hybridization were applied to measure mRNA expression of multiple genes within single cells. Research applications of these single molecule single cells

The interplay between polymerase organization and nucleosome occupancy along DNA : How dynamic roadblocks on the DNA induce the formation of RNA polymerase pelotons

NARCIS (Netherlands)

van den Berg, A.A.

2017-01-01

During transcription RNA polymerase (RNAP) moves along a DNA molecule to copy the information on the DNA to an RNA molecule. Many textbook pictures show an RNAP sliding along empty DNA, but in reality it is crowded on the DNA and RNAP competes for space with many proteins such as other RNAP’s and

Determination of chromium combined with DNA, RNA and proteins in chromium-rich brewer's yeast by NAA

International Nuclear Information System (INIS)

Ding, W.J.; Qian, Q.F.; Hou, X.L.; Feng, W.Y.; Chai, Z.F.

2000-01-01

The content of chromium in the DNA, RNA and protein fractions separated from chromium-rich and normal brewer's yeast was determined by neutron activation analysis (NAA). Our results show that the extracted relative amounts and concentrations of DNA, RNA and proteins have no significant difference for two types of yeast, but the chromium content in DNA, RNA and proteins fractions extracted from the chromium-rich yeast are substantially higher than those from the normal. In addition, the concentration of chromium in DNA is much higher than that in RNA and proteins. It is evident that the inorganic chromium compounds can enter the yeast cell during the yeast cultivation in the chromium-containing culture medium and are converted into organic chromium species, which are combined with DNA, RNA and proteins. (author)

Detection of hepatitis C virus RNA using reverse transcription PCR

International Nuclear Information System (INIS)

Yap, S.F.

1998-01-01

Detection of the viral genome (HCV RNA) is by a combination of cDNA synthesis and PCR followed by gel analysis and/or hybridization assay. In principle, cDNA is synthesized using the viral RNA as template and the enzyme, reverse transcriptase. The cDNA is then amplified by PCR and the product detected. Agarose gel electrophoresis provides a rapid and simple detection method; however, it is non-quantitative. The assay protocol described in this paper is adapted from that published by Chan et al. Comments on various aspects of the assay are based on experience with the method in our laboratory

Co-isolation of in vivo 32P-labeled specific transcripts and DNA without phenol extraction of nuclease digestion

International Nuclear Information System (INIS)

Hayes, S.; Hayes, C.; Brand, L.

1981-01-01

A method is described for isolation and quantitation of specific intact transcripts, for which a hybridization probe is available, from 32 P-labeled bacterial cells. The RNA is extracted in the absence of R Nase activity by incorporating an inert, physically removable R Nase inhibitor throughout the spheroplasting, cell lysis, and pronase digestion steps. [/sup 32/P]RNA is separated from [ 32 P]DNA, without recourse to phenol extraction of DNase treatment, on a Cs 2 SO/sub 4-/HCONH 2 step gradient in which the precipitated RNA forms a sharp band. Specific transcripts are purified from [ 32 P]RNA by physical separation of the transcript and hybridization probe using gel-exclusion chromatography. The gentleness of this technique enables the co-isolation of DNA and can facilitate the analysis of covalently joined RNA-DNA replication intermediates

Direct RNA detection without nucleic acid purification and PCR: Combining sandwich hybridization with signal amplification based on branched hybridization chain reaction.

Science.gov (United States)

Xu, Yao; Zheng, Zhi

2016-05-15

We have developed a convenient, robust and low-cost RNA detection system suitable for high-throughput applications. This system uses a highly specific sandwich hybridization to capture target RNA directly onto solid support, followed by on-site signal amplification via 2-dimensional, branched hybridizing chain polymerization through toehold-mediated strand displacement reaction. The assay uses SYBR Green to detect targets at concentrations as low as 1 pM, without involving nucleic acid purification or any enzymatic reaction, using ordinary oligonucleotides without modification or labeling. The system was demonstrated in the detection of malaria RNA in blood and GAPDH gene expression in cell lysate. Copyright © 2015 Elsevier B.V. All rights reserved.

Ultrasensitive Detection of RNA and DNA Viruses Simultaneously Using Duplex UNDP-PCR Assay.

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Yong Huang

Full Text Available Mixed infection of multiple viruses is common in modern intensive pig rearing. However, there are no methods available to detect DNA and RNA viruses in the same reaction system in preclinical level. In this study, we aimed to develop a duplex ultrasensitive nanoparticle DNA probe-based PCR assay (duplex UNDP-PCR that was able to simultaneously detect DNA and RNA viruses in the same reaction system. PCV2 and TGEV are selected as representatives of the two different types of viruses. PCV2 DNA and TGEV RNA were simultaneously released from the serum sample by boiling with lysis buffer, then magnetic beads and gold nanoparticles coated with single and/or duplex specific probes for TGEV and PCV2 were added to form a sandwich-like complex with nucleic acids released from viruses. After magnetic separation, DNA barcodes specific for PCV2 and TGEV were eluted using DTT and characterized by specific PCR assay for specific DNA barcodes subsequently. The duplex UNDP-PCR showed similar sensitivity as that of single UNDP-PCR and was able to detect 20 copies each of PCV2 and TGEV in the serum, showing approximately 250-fold more sensitivity than conventional duplex PCR/RT-PCR assays. No cross-reaction was observed with other viruses. The positive detection rate of single MMPs- and duplex MMPs-based duplex UNDP-PCR was identical, with 29.6% for PCV2, 9.3% for TGEV and 3.7% for PCV2 and TGEV mixed infection. This duplex UNDP-PCR assay could detect TGEV (RNA virus and PCV2 (DNA virus from large-scale serum samples simultaneously without the need for DNA/RNA extraction, purification and reverse transcription of RNA, and showed a significantly increased positive detection rate for PCV2 (29% and TGEV (11.7% preclinical infection than conventional duplex PCR/RT-PCR. Therefore, the established duplex UNDP-PCR is a rapid and economical detection method, exhibiting high sensitivity, specificity and reproducibility.

Ultrasensitive Detection of RNA and DNA Viruses Simultaneously Using Duplex UNDP-PCR Assay

Science.gov (United States)

Wang, Zengguo; Zhang, Xiujuan; Zhao, Xiaomin; Du, Qian; Chang, Lingling; Tong, Dewen

2015-01-01

Mixed infection of multiple viruses is common in modern intensive pig rearing. However, there are no methods available to detect DNA and RNA viruses in the same reaction system in preclinical level. In this study, we aimed to develop a duplex ultrasensitive nanoparticle DNA probe-based PCR assay (duplex UNDP-PCR) that was able to simultaneously detect DNA and RNA viruses in the same reaction system. PCV2 and TGEV are selected as representatives of the two different types of viruses. PCV2 DNA and TGEV RNA were simultaneously released from the serum sample by boiling with lysis buffer, then magnetic beads and gold nanoparticles coated with single and/or duplex specific probes for TGEV and PCV2 were added to form a sandwich-like complex with nucleic acids released from viruses. After magnetic separation, DNA barcodes specific for PCV2 and TGEV were eluted using DTT and characterized by specific PCR assay for specific DNA barcodes subsequently. The duplex UNDP-PCR showed similar sensitivity as that of single UNDP-PCR and was able to detect 20 copies each of PCV2 and TGEV in the serum, showing approximately 250-fold more sensitivity than conventional duplex PCR/RT-PCR assays. No cross-reaction was observed with other viruses. The positive detection rate of single MMPs- and duplex MMPs-based duplex UNDP-PCR was identical, with 29.6% for PCV2, 9.3% for TGEV and 3.7% for PCV2 and TGEV mixed infection. This duplex UNDP-PCR assay could detect TGEV (RNA virus) and PCV2 (DNA virus) from large-scale serum samples simultaneously without the need for DNA/RNA extraction, purification and reverse transcription of RNA, and showed a significantly increased positive detection rate for PCV2 (29%) and TGEV (11.7%) preclinical infection than conventional duplex PCR/RT-PCR. Therefore, the established duplex UNDP-PCR is a rapid and economical detection method, exhibiting high sensitivity, specificity and reproducibility. PMID:26544710

Ultrasensitive Detection of RNA and DNA Viruses Simultaneously Using Duplex UNDP-PCR Assay.

Science.gov (United States)

Huang, Yong; Xing, Na; Wang, Zengguo; Zhang, Xiujuan; Zhao, Xiaomin; Du, Qian; Chang, Lingling; Tong, Dewen

2015-01-01

Mixed infection of multiple viruses is common in modern intensive pig rearing. However, there are no methods available to detect DNA and RNA viruses in the same reaction system in preclinical level. In this study, we aimed to develop a duplex ultrasensitive nanoparticle DNA probe-based PCR assay (duplex UNDP-PCR) that was able to simultaneously detect DNA and RNA viruses in the same reaction system. PCV2 and TGEV are selected as representatives of the two different types of viruses. PCV2 DNA and TGEV RNA were simultaneously released from the serum sample by boiling with lysis buffer, then magnetic beads and gold nanoparticles coated with single and/or duplex specific probes for TGEV and PCV2 were added to form a sandwich-like complex with nucleic acids released from viruses. After magnetic separation, DNA barcodes specific for PCV2 and TGEV were eluted using DTT and characterized by specific PCR assay for specific DNA barcodes subsequently. The duplex UNDP-PCR showed similar sensitivity as that of single UNDP-PCR and was able to detect 20 copies each of PCV2 and TGEV in the serum, showing approximately 250-fold more sensitivity than conventional duplex PCR/RT-PCR assays. No cross-reaction was observed with other viruses. The positive detection rate of single MMPs- and duplex MMPs-based duplex UNDP-PCR was identical, with 29.6% for PCV2, 9.3% for TGEV and 3.7% for PCV2 and TGEV mixed infection. This duplex UNDP-PCR assay could detect TGEV (RNA virus) and PCV2 (DNA virus) from large-scale serum samples simultaneously without the need for DNA/RNA extraction, purification and reverse transcription of RNA, and showed a significantly increased positive detection rate for PCV2 (29%) and TGEV (11.7%) preclinical infection than conventional duplex PCR/RT-PCR. Therefore, the established duplex UNDP-PCR is a rapid and economical detection method, exhibiting high sensitivity, specificity and reproducibility.

An efficient enzyme-powered micromotor device fabricated by cyclic alternate hybridization assembly for DNA detection.

Science.gov (United States)

Fu, Shizhe; Zhang, Xueqing; Xie, Yuzhe; Wu, Jie; Ju, Huangxian

2017-07-06

An efficient enzyme-powered micromotor device was fabricated by assembling multiple layers of catalase on the inner surface of a poly(3,4-ethylenedioxythiophene and sodium 4-styrenesulfonate)/Au microtube (PEDOT-PSS/Au). The catalase assembly was achieved by programmed DNA hybridization, which was performed by immobilizing a designed sandwich DNA structure as the sensing unit on the PEDOT-PSS/Au, and then alternately hybridizing with two assisting DNA to bind the enzyme for efficient motor motion. The micromotor device showed unique features of good reproducibility, stability and motion performance. Under optimal conditions, it showed a speed of 420 μm s -1 in 2% H 2 O 2 and even 51 μm s -1 in 0.25% H 2 O 2 . In the presence of target DNA, the sensing unit hybridized with target DNA to release the multi-layer DNA as well as the multi-catalase, resulting in a decrease of the motion speed. By using the speed as a signal, the micromotor device could detect DNA from 10 nM to 1 μM. The proposed micromotor device along with the cyclic alternate DNA hybridization assembly technique provided a new path to fabricate efficient and versatile micromotors, which would be an exceptional tool for rapid and simple detection of biomolecules.

A novel SERRS sandwich-hybridization assay to detect specific DNA target.

Directory of Open Access Journals (Sweden)

Cécile Feuillie

Full Text Available In this study, we have applied Surface Enhanced Resonance Raman Scattering (SERRS technology to the specific detection of DNA. We present an innovative SERRS sandwich-hybridization assay that allows specific DNA detection without any enzymatic amplification, such as is the case with Polymerase Chain Reaction (PCR. In some substrates, such as ancient or processed remains, enzymatic amplification fails due to DNA alteration (degradation, chemical modification or to the presence of inhibitors. Consequently, the development of a non-enzymatic method, allowing specific DNA detection, could avoid long, expensive and inconclusive amplification trials. Here, we report the proof of concept of a SERRS sandwich-hybridization assay that leads to the detection of a specific chamois DNA. This SERRS assay reveals its potential as a non-enzymatic alternative technology to DNA amplification methods (particularly the PCR method with several applications for species detection. As the amount and type of damage highly depend on the preservation conditions, the present SERRS assay would enlarge the range of samples suitable for DNA analysis and ultimately would provide exciting new opportunities for the investigation of ancient DNA in the fields of evolutionary biology and molecular ecology, and of altered DNA in food frauds detection and forensics.

Structure and assembly of the essential RNA ring component of a viral DNA packaging motor.

Science.gov (United States)

Ding, Fang; Lu, Changrui; Zhao, Wei; Rajashankar, Kanagalaghatta R; Anderson, Dwight L; Jardine, Paul J; Grimes, Shelley; Ke, Ailong

2011-05-03

Prohead RNA (pRNA) is an essential component in the assembly and operation of the powerful bacteriophage 29 DNA packaging motor. The pRNA forms a multimeric ring via intermolecular base-pairing interactions between protomers that serves to guide the assembly of the ring ATPase that drives DNA packaging. Here we report the quaternary structure of this rare multimeric RNA at 3.5 Å resolution, crystallized as tetrameric rings. Strong quaternary interactions and the inherent flexibility helped rationalize how free pRNA is able to adopt multiple oligomerization states in solution. These characteristics also allowed excellent fitting of the crystallographic pRNA protomers into previous prohead/pRNA cryo-EM reconstructions, supporting the presence of a pentameric, but not hexameric, pRNA ring in the context of the DNA packaging motor. The pentameric pRNA ring anchors itself directly to the phage prohead by interacting specifically with the fivefold symmetric capsid structures that surround the head-tail connector portal. From these contacts, five RNA superhelices project from the pRNA ring, where they serve as scaffolds for binding and assembly of the ring ATPase, and possibly mediate communication between motor components. Construction of structure-based designer pRNAs with little sequence similarity to the wild-type pRNA were shown to fully support the packaging of 29 DNA.

Isolation of full-length putative rat lysophospholipase cDNA using improved methods for mRNA isolation and cDNA cloning

International Nuclear Information System (INIS)

Han, J.H.; Stratowa, C.; Rutter, W.J.

1987-01-01

The authors have cloned a full-length putative rat pancreatic lysophospholipase cDNA by an improved mRNA isolation method and cDNA cloning strategy using [ 32 P]-labelled nucleotides. These new methods allow the construction of a cDNA library from the adult rat pancreas in which the majority of recombinant clones contained complete sequences for the corresponding mRNAs. A previously recognized but unidentified long and relatively rare cDNA clone containing the entire sequence from the cap site at the 5' end to the poly(A) tail at the 3' end of the mRNA was isolated by single-step screening of the library. The size, amino acid composition, and the activity of the protein expressed in heterologous cells strongly suggest this mRNA codes for lysophospholipase

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

Directory of Open Access Journals (Sweden)

Vilena V. Ivanova

2014-12-01

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

Identification of DNA viruses by membrane filter hybridization.

OpenAIRE

Stålhandske, P; Pettersson, U

1982-01-01

The use of membrane filter hybridization for the identification of DNA viruses is described. We designed and used a procedure for identification of herpes simplex virus. This method can discriminate between herpes simplex virus types 1 and 2 in a simple way.

Sensitive detection of mercury and copper ions by fluorescent DNA/Ag nanoclusters in guanine-rich DNA hybridization.

Science.gov (United States)

Peng, Jun; Ling, Jian; Zhang, Xiu-Qing; Bai, Hui-Ping; Zheng, Liyan; Cao, Qiu-E; Ding, Zhong-Tao

2015-02-25

In this work, we designed a new fluorescent oligonucleotides-stabilized silver nanoclusters (DNA/AgNCs) probe for sensitive detection of mercury and copper ions. This probe contains two tailored DNA sequence. One is a signal probe contains a cytosine-rich sequence template for AgNCs synthesis and link sequence at both ends. The other is a guanine-rich sequence for signal enhancement and link sequence complementary to the link sequence of the signal probe. After hybridization, the fluorescence of hybridized double-strand DNA/AgNCs is 200-fold enhanced based on the fluorescence enhancement effect of DNA/AgNCs in proximity of guanine-rich DNA sequence. The double-strand DNA/AgNCs probe is brighter and stable than that of single-strand DNA/AgNCs, and more importantly, can be used as novel fluorescent probes for detecting mercury and copper ions. Mercury and copper ions in the range of 6.0-160.0 and 6-240 nM, can be linearly detected with the detection limits of 2.1 and 3.4 nM, respectively. Our results indicated that the analytical parameters of the method for mercury and copper ions detection are much better than which using a single-strand DNA/AgNCs. Copyright © 2014 Elsevier B.V. All rights reserved.

Detection of tmRNA molecules on microarrays at low temperatures using helper oligonucleotides

Directory of Open Access Journals (Sweden)

Palta Priit

2010-04-01

Full Text Available Abstract Background The hybridization of synthetic Streptococcus pneumoniae tmRNA on a detection microarray is slow at 34°C resulting in low signal intensities. Results We demonstrate that adding specific DNA helper oligonucleotides (chaperones to the hybridization buffer increases the signal strength at a given temperature and thus makes the specific detection of Streptococcus pneumoniae tmRNA more sensitive. No loss of specificity was observed at low temperatures compared to hybridization at 46°C. The effect of the chaperones can be explained by disruption of the strong secondary and tertiary structure of the target RNA by the selective hybridization of helper molecules. The amplification of the hybridization signal strength by chaperones is not necessarily local; we observed increased signal intensities in both local and distant regions of the target molecule. Conclusions The sensitivity of the detection of tmRNA at low temperature can be increased by chaperone oligonucleotides. Due to the complexity of RNA secondary and tertiary structures the effect of any individual chaperone is currently not predictable.

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

Science.gov (United States)

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

2006-01-01

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

Quantum Point Contact Single-Nucleotide Conductance for DNA and RNA Sequence Identification.

Science.gov (United States)

Afsari, Sepideh; Korshoj, Lee E; Abel, Gary R; Khan, Sajida; Chatterjee, Anushree; Nagpal, Prashant

2017-11-28

Several nanoscale electronic methods have been proposed for high-throughput single-molecule nucleic acid sequence identification. While many studies display a large ensemble of measurements as "electronic fingerprints" with some promise for distinguishing the DNA and RNA nucleobases (adenine, guanine, cytosine, thymine, and uracil), important metrics such as accuracy and confidence of base calling fall well below the current genomic methods. Issues such as unreliable metal-molecule junction formation, variation of nucleotide conformations, insufficient differences between the molecular orbitals responsible for single-nucleotide conduction, and lack of rigorous base calling algorithms lead to overlapping nanoelectronic measurements and poor nucleotide discrimination, especially at low coverage on single molecules. Here, we demonstrate a technique for reproducible conductance measurements on conformation-constrained single nucleotides and an advanced algorithmic approach for distinguishing the nucleobases. Our quantum point contact single-nucleotide conductance sequencing (QPICS) method uses combed and electrostatically bound single DNA and RNA nucleotides on a self-assembled monolayer of cysteamine molecules. We demonstrate that by varying the applied bias and pH conditions, molecular conductance can be switched ON and OFF, leading to reversible nucleotide perturbation for electronic recognition (NPER). We utilize NPER as a method to achieve >99.7% accuracy for DNA and RNA base calling at low molecular coverage (∼12×) using unbiased single measurements on DNA/RNA nucleotides, which represents a significant advance compared to existing sequencing methods. These results demonstrate the potential for utilizing simple surface modifications and existing biochemical moieties in individual nucleobases for a reliable, direct, single-molecule, nanoelectronic DNA and RNA nucleotide identification method for sequencing.

Thermodynamic framework to assess low abundance DNA mutation detection by hybridization

Science.gov (United States)

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

2017-01-01

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

Counting molecules in cell-free DNA and single cells RNA

OpenAIRE

Karlsson, Kasper

2016-01-01

The field of Molecular Biology got started in earnest with the discovery of the molecular structure of DNA. This lead to a surge of interest into the relationships between DNA, RNA and proteins, and to the development of fundamental tools for manipulating those substances, such as cutting, ligating, amplifying, visualizing and size-selecting DNA. With these tools at hand it was possible to begin sequencing DNA, a process that took a leap forward in 2005 with the advent of Next Generation Sequ...

Inhibition of Hepatitis B virus cccDNA replication by siRNA

International Nuclear Information System (INIS)

Li Guiqiu; Gu Hongxi; Li Di; Xu Weizhen

2007-01-01

The development of an effective therapy for Hepatitis B virus (HBV) infection is still a challenge. Progress in RNA interference (RNAi) has shed slight on developing a new anti-HBV strategy. Here, we present a series of experiments showing a significant reduction in HBV transcripts and replication intermediates in HepG2.2.15 cells by vector-based siRNA targeted nuclear localization signal (NLS) region. More importantly, we showed that siRNA1 markedly inhibited HBV covalently closed circular DNA (cccDNA) replication. Our results indicated that HBV NLS may serve as a novel RNAi target to combat HBV infection, which can enhance anti-HBV efficacy and overcome the drawbacks of current therapies

Isolation and structure of a cDNA encoding the B1 (CD20) cell-surface antigen of human B lymphocytes

International Nuclear Information System (INIS)

Tender, T.F.; Streuli, M.; Schlossman, S.F.; Saito, H.

1988-01-01

The B1 (CD20) molecule is a M/sub r/ 33,000 phosphoprotein on the surface of human B lymphocytes that may serve a central role in the homoral immune response by regulating B-cell proliferation and differentiation. In this report, a cDNA clone that encodes the B1 molecule was isolated and the amino acid sequence of B1 was determined. B-cell-specific cDNA clones were selected from a human tonsillar cDNA library by differential hybridization with labeled cDNA derived from either size-fractionated B-cell mRNA or size-fractionated T-cell mRNA. Of the 261 cDNA clones isolated, 3 cross-hybridizing cDNA clones were chosen as potential candidates for encoding B1 based on their selective hybridization to RNA from B1-positive cell lines. The longest clone, pB1-21, contained a 2.8-kilobase insert with an 891-base-pair open reading frame that encodes a protein of 33 kDa. mRNA synthesized from the pB1-21 cDNA clone in vitro was translated into a protein of the same apparent molecular weight as B1. Limited proteinase digestion of the pB1-21 translation product and B1 generated peptides of the same sizes, indicating that the pB1-21 cDNA encodes the B1 molecule. Gel blot analysis indicated that pB1-21 hybridized with two mRNA species of 2.8 and 3.4 kilobases only in B1-positive cell lines. The amino acid sequence deduced from the pB1-21 nucleotide sequence apparently lacks a signal sequence and contains three extensive hydrophobic regions. The deduced B1 amino acid sequence shows no significant homology with other known patients

Archaeal RNA polymerase arrests transcription at DNA lesions.

Science.gov (United States)

Gehring, Alexandra M; Santangelo, Thomas J

2017-01-01

Transcription elongation is not uniform and transcription is often hindered by protein-bound factors or DNA lesions that limit translocation and impair catalysis. Despite the high degree of sequence and structural homology of the multi-subunit RNA polymerases (RNAP), substantial differences in response to DNA lesions have been reported. Archaea encode only a single RNAP with striking structural conservation with eukaryotic RNAP II (Pol II). Here, we demonstrate that the archaeal RNAP from Thermococcus kodakarensis is sensitive to a variety of DNA lesions that pause and arrest RNAP at or adjacent to the site of DNA damage. DNA damage only halts elongation when present in the template strand, and the damage often results in RNAP arresting such that the lesion would be encapsulated with the transcription elongation complex. The strand-specific halt to archaeal transcription elongation on modified templates is supportive of RNAP recognizing DNA damage and potentially initiating DNA repair through a process akin to the well-described transcription-coupled DNA repair (TCR) pathways in Bacteria and Eukarya.

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

Science.gov (United States)

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.

Transposable Element Misregulation Is Linked to the Divergence between Parental piRNA Pathways in Drosophila Hybrids.

Science.gov (United States)

Romero-Soriano, Valèria; Modolo, Laurent; Lopez-Maestre, Hélène; Mugat, Bruno; Pessia, Eugénie; Chambeyron, Séverine; Vieira, Cristina; Garcia Guerreiro, Maria Pilar

2017-06-01

Interspecific hybridization is a genomic stress condition that leads to the activation of transposable elements (TEs) in both animals and plants. In hybrids between Drosophila buzzatii and Drosophila koepferae, mobilization of at least 28 TEs has been described. However, the molecular mechanisms underlying this TE release remain poorly understood. To give insight on the causes of this TE activation, we performed a TE transcriptomic analysis in ovaries (notorious for playing a major role in TE silencing) of parental species and their F1 and backcrossed (BC) hybrids. We find that 15.2% and 10.6% of the expressed TEs are deregulated in F1 and BC1 ovaries, respectively, with a bias toward overexpression in both cases. Although differences between parental piRNA (Piwi-interacting RNA) populations explain only partially these results, we demonstrate that piRNA pathway proteins have divergent sequences and are differentially expressed between parental species. Thus, a functional divergence of the piRNA pathway between parental species, together with some differences between their piRNA pools, might be at the origin of hybrid instabilities and ultimately cause TE misregulation in ovaries. These analyses were complemented with the study of F1 testes, where TEs tend to be less expressed than in D. buzzatii. This can be explained by an increase in piRNA production, which probably acts as a defence mechanism against TE instability in the male germline. Hence, we describe a differential impact of interspecific hybridization in testes and ovaries, which reveals that TE expression and regulation are sex-biased. © The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Conformationally locked aryl C-nucleosides: synthesis of phosphoramidite monomers and incorporation into single-stranded DNA and LNA (locked nucleic acid)

DEFF Research Database (Denmark)

Babu, B. Ravindra; Prasad, Ashok K.; Trikha, Smriti

2002-01-01

. The phosphoramidite approach was used for automated incorporation of the LNA-type beta-configured C-aryl monomers 17a-17e into short DNA and 2'-OMe-RNA/LNA strands. It is shown that universal hybridization can be obtained with a conformationally restricted monomer as demonstrated most convincingly for the pyrene LNA...... monomer 17d, both in a DNA context and in an RNA-like context. Increased binding affinity of oligonucleotide probes for universal hybridization can be induced by combining the pyrene LNA monomer 17d with affinity-enhancing 2'-OMe-RNA/LNA monomers....

DNA and RNA nanobiotechnologies in medicine diagnosis and treatment of diseases

CERN Document Server

Erdmann, Volker A

2014-01-01

This book covers the design and synthesis of DNA and RNA nanostructures with the aim of using them for drug deliveries, for genetic immunization, for metabolite and nucleic acid detection, gene regulation, and siRNA delivery for cancer treatment.

Nuclear DNA content of the hybrid plant pathogen Phytophthora andina determined by flow cytometry.

Science.gov (United States)

Wang, Jianan; Presser, Jackson W; Goss, Erica M

2016-09-01

Phytophthora andina is a heterothallic plant pathogen of Andean solanaceous hosts and is an interspecific hybrid of P. infestans and an unknown Phytophthora species. The objective of this study was to estimate the nuclear DNA content of isolates in three clonal lineages of P. andina relative to P. infestans Twelve isolates of P. andina and six isolates of P. infestans were measured for nuclear DNA content by propidium iodide-stained flow cytometry. We found that the DNA content of P. andina was similar but slightly smaller, on average, than that of our sample of P. infestans isolates. This is consistent with P. andina being a homoploid hybrid rather than allopolyploid hybrid. Nuclear DNA content was more variable among a smaller sample of P. infestans isolates, including a putative triploid isolate from Mexico, but small differences in nuclear DNA content were also observed among P. andina isolates. Both species appear to be able to tolerate significant variation in genome size. © 2016 by The Mycological Society of America.

Inhibition of Xenograft tumor growth by gold nanoparticle-DNA oligonucleotide conjugates-assisted delivery of BAX mRNA.

Directory of Open Access Journals (Sweden)

Ji-Hyun Yeom

Full Text Available Use of non-biological agents for mRNA delivery into living systems in order to induce heterologous expression of functional proteins may provide more advantages than the use of DNA and/or biological vectors for delivery. However, the low efficiency of mRNA delivery into live animals, using non-biological systems, has hampered the use of mRNA as a therapeutic molecule. Here, we show that gold nanoparticle-DNA oligonucleotide (AuNP-DNA conjugates can serve as universal vehicles for more efficient delivery of mRNA into human cells, as well as into xenograft tumors generated in mice. Injections of BAX mRNA loaded on AuNP-DNA conjugates into xenograft tumors resulted in highly efficient mRNA delivery. The delivered mRNA directed the efficient production of biologically functional BAX protein, a pro-apoptotic factor, consequently inhibiting tumor growth. These results demonstrate that mRNA delivery by AuNP-DNA conjugates can serve as a new platform for the development of safe and efficient gene therapy.

Multilayer DNA Origami Packed on Hexagonal and Hybrid Lattices

DEFF Research Database (Denmark)

Ke, Yonggang; Voigt, Niels Vinther; Shih, William M.

2012-01-01

“Scaffolded DNA origami” has been proven to be a powerful and efficient approach to construct two-dimensional or three-dimensional objects with great complexity. Multilayer DNA origami has been demonstrated with helices packing along either honeycomb-lattice geometry or square-lattice geometry....... Here we report successful folding of multilayer DNA origami with helices arranged on a close-packed hexagonal lattice. This arrangement yields a higher density of helical packing and therefore higher resolution of spatial addressing than has been shown previously. We also demonstrate hybrid multilayer...... DNA origami with honeycomb-lattice, square-lattice, and hexagonal-lattice packing of helices all in one design. The availability of hexagonal close-packing of helices extends our ability to build complex structures using DNA nanotechnology....

Multilayer DNA origami packed on hexagonal and hybrid lattices.

Science.gov (United States)

Ke, Yonggang; Voigt, Niels V; Gothelf, Kurt V; Shih, William M

2012-01-25

"Scaffolded DNA origami" has been proven to be a powerful and efficient approach to construct two-dimensional or three-dimensional objects with great complexity. Multilayer DNA origami has been demonstrated with helices packing along either honeycomb-lattice geometry or square-lattice geometry. Here we report successful folding of multilayer DNA origami with helices arranged on a close-packed hexagonal lattice. This arrangement yields a higher density of helical packing and therefore higher resolution of spatial addressing than has been shown previously. We also demonstrate hybrid multilayer DNA origami with honeycomb-lattice, square-lattice, and hexagonal-lattice packing of helices all in one design. The availability of hexagonal close-packing of helices extends our ability to build complex structures using DNA nanotechnology. © 2011 American Chemical Society

In situ hybridization studies of hepatitis A viral RNA in patients with acute hepatitis A

Energy Technology Data Exchange (ETDEWEB)

Taylor, M; Goldin, R D; Ladva, S [Department of Histopathology, St. Mary' s Hospital Medical School, Imperial College of Science, Technology and Medicine, London (United Kingdom); Scheuer, P J [Department of Histopathology, Royal Free Hospital and School of Medicine, London (United Kingdom); Thomas, H C [Department of Medicine, St. Mary' s Hospital Medical School, Imperial College of Science, Technology and Medicine, London (United Kingdom)

1994-01-01

In situ hybridization with oligonucleotide probes has been used to localise hepatitis A virus RNA genomic sequences in formalin-fixed and routinely processed human liver biopsies from three patients. Using radiolabelled Sulphur-35 antisense probes, viral genomic sequences were found in all three cases, but signal intensity was greatest in cases 1 and 2 with fulminant hepatitis, and was minimal in the third case of resolving hepatitis biopsied 2 months after acute illness. Localisation showed the viral RNA to be present in hepatocytes, sinusoidal cells and inflammatory cells in and around the portal tracts. Both cases showed signal in similar cell types, but the distribution of staining was predominantly periportal in case 1, whereas lobular staining was more apparent in case 2. Hybridization with sense polarity probes failed to detect any evidence of replicative intermediates of antigenomic viral RNA. The presence of hepatitis A RNA in phagocytic cells was confirmed using immunohistochemistryfor a macrophage marker, CD68, combined with in situ hybridization. In all cases the signal was predominantly cytoplasmic, and this was confirmed with the use of tritiated probes. (au).

In situ hybridization studies of hepatitis A viral RNA in patients with acute hepatitis A

International Nuclear Information System (INIS)

Taylor, M.; Goldin, R.D.; Ladva, S.; Scheuer, P.J.; Thomas, H.C.

1994-01-01

In situ hybridization with oligonucleotide probes has been used to localise hepatitis A virus RNA genomic sequences in formalin-fixed and routinely processed human liver biopsies from three patients. Using radiolabelled Sulphur-35 antisense probes, viral genomic sequences were found in all three cases, but signal intensity was greatest in cases 1 and 2 with fulminant hepatitis, and was minimal in the third case of resolving hepatitis biopsied 2 months after acute illness. Localisation showed the viral RNA to be present in hepatocytes, sinusoidal cells and inflammatory cells in and around the portal tracts. Both cases showed signal in similar cell types, but the distribution of staining was predominantly periportal in case 1, whereas lobular staining was more apparent in case 2. Hybridization with sense polarity probes failed to detect any evidence of replicative intermediates of antigenomic viral RNA. The presence of hepatitis A RNA in phagocytic cells was confirmed using immunohistochemistryfor a macrophage marker, CD68, combined with in situ hybridization. In all cases the signal was predominantly cytoplasmic, and this was confirmed with the use of tritiated probes. (au)

Isolation and characterization of 5S rDNA sequences in catfishes genome (Heptapteridae and Pseudopimelodidae): perspectives for rDNA studies in fish by C0t method.

Science.gov (United States)

Gouveia, Juceli Gonzalez; Wolf, Ivan Rodrigo; de Moraes-Manécolo, Vivian Patrícia Oliveira; Bardella, Vanessa Belline; Ferracin, Lara Munique; Giuliano-Caetano, Lucia; da Rosa, Renata; Dias, Ana Lúcia

2016-12-01

Sequences of 5S ribosomal RNA (rRNA) are extensively used in fish cytogenomic studies, once they have a flexible organization at the chromosomal level, showing inter- and intra-specific variation in number and position in karyotypes. Sequences from the genome of Imparfinis schubarti (Heptapteridae) were isolated, aiming to understand the organization of 5S rDNA families in the fish genome. The isolation of 5S rDNA from the genome of I. schubarti was carried out by reassociation kinetics (C 0 t) and PCR amplification. The obtained sequences were cloned for the construction of a micro-library. The obtained clones were sequenced and hybridized in I. schubarti and Microglanis cottoides (Pseudopimelodidae) for chromosome mapping. An analysis of the sequence alignments with other fish groups was accomplished. Both methods were effective when using 5S rDNA for hybridization in I. schubarti genome. However, the C 0 t method enabled the use of a complete 5S rRNA gene, which was also successful in the hybridization of M. cottoides. Nevertheless, this gene was obtained only partially by PCR. The hybridization results and sequence analyses showed that intact 5S regions are more appropriate for the probe operation, due to conserved structure and motifs. This study contributes to a better understanding of the organization of multigene families in catfish's genomes.

Chondroitin sulfate-polyethylenimine copolymer-coated superparamagnetic iron oxide nanoparticles as an efficient magneto-gene carrier for microRNA-encoding plasmid DNA delivery

Science.gov (United States)

Lo, Yu-Lun; Chou, Han-Lin; Liao, Zi-Xian; Huang, Shih-Jer; Ke, Jyun-Han; Liu, Yu-Sheng; Chiu, Chien-Chih; Wang, Li-Fang

2015-04-01

MicroRNA-128 (miR-128) is an attractive therapeutic molecule with powerful glioblastoma regulation properties. However, miR-128 lacks biological stability and leads to poor delivery efficacy in clinical applications. In our previous study, we demonstrated two effective transgene carriers, including polyethylenimine (PEI)-decorated superparamagnetic iron oxide nanoparticles (SPIONs) as well as chemically-conjugated chondroitin sulfate-PEI copolymers (CPs). In this contribution, we report optimized conditions for coating CPs onto the surfaces of SPIONs, forming CPIOs, for magneto-gene delivery systems. The optimized weight ratio of the CPs and SPIONs is 2 : 1, which resulted in the formation of a stable particle as a good transgene carrier. The hydrodynamic diameter of the CPIOs is ~136 nm. The gel electrophoresis results demonstrate that the weight ratio of CPIO/DNA required to completely encapsulate pDNA is >=3. The in vitro tests of CPIO/DNA were done in 293 T, CRL5802, and U87-MG cells in the presence and absence of an external magnetic field. The magnetofection efficiency of CPIO/DNA was measured in the three cell lines with or without fetal bovine serum (FBS). CPIO/DNA exhibited remarkably improved gene expression in the presence of the magnetic field and 10% FBS as compared with a gold non-viral standard, PEI/DNA, and a commercial magnetofection reagent, PolyMag/DNA. In addition, CPIO/DNA showed less cytotoxicity than PEI/DNA and PolyMag/DNA against the three cell lines. The transfection efficiency of the magnetoplex improved significantly with an assisted magnetic field. In miR-128 delivery, a microRNA plate array and fluorescence in situ hybridization were used to demonstrate that CPIO/pMIRNA-128 indeed expresses more miR-128 with the assisted magnetic field than without. In a biodistribution test, CPIO/Cy5-DNA showed higher accumulation at the tumor site where an external magnet is placed nearby.MicroRNA-128 (miR-128) is an attractive therapeutic molecule

Determinazione quantitativa di HCV-RNA: valutazione comparativa dei saggi Abbott Real-Time e Versant bDNA v.3

Directory of Open Access Journals (Sweden)

Aldo Manzin

2007-06-01

Full Text Available Hepatitis C virus (HCV RNA measurement before, during and after antiviral therapy has become an essential tool in the management of interferon-based treatment of HCV-related infections. Conventional Polymerase Chain Reaction (PCR has been largely used to obtain quantitative data, but laborious, time-consuming post-PCR handling steps are required to gain valuable results. Real time (RT PCR now provides advantages over end-point (EP PCR due to its improved rapidity, sensitivity, reproducibility and the reduced risk of carry-over contamination, and has now proven itself to be valuable for the more precise monitoring of viral load kinetics and assessing antiviral response.The Abbott Real-Time HCV-RNA is a recently introduced assay for the automated processing of clinical samples and HCV-RNA quantitation: its basic technology relies on use of fluorescent linear probes (dynamic range using 0.5 ml as input target= 12-108 IU/mL and a hybridization/detection step at low temperature (35°C, which allows target mismatches to be tolerated. To determine the clinical application of the Abbott Real-Time assay and defining its correlation with the Bayer Versant bDNA v.3 assay, 68 consecutive samples from unselected HCV-infected patients were retrospectively analysed with RT and the results obtained using the two tests compared.A good correlation was found between RT-PCR and bDNA: 97% of samples tested had a result within a 0.5 log HCV IU/mL difference (bias=0.15 log, whereas 6 samples negative with bDNA gave positive results with Abbott RT (range, 1.89-3.07 log IU/mL and “in-house” qualitative RT-PCR assays.

RNA Imaging with Multiplexed Error Robust Fluorescence in situ Hybridization

Science.gov (United States)

Moffitt, Jeffrey R.; Zhuang, Xiaowei

2016-01-01

Quantitative measurements of both the copy number and spatial distribution of large fractions of the transcriptome in single-cells could revolutionize our understanding of a variety of cellular and tissue behaviors in both healthy and diseased states. Single-molecule Fluorescence In Situ Hybridization (smFISH)—an approach where individual RNAs are labeled with fluorescent probes and imaged in their native cellular and tissue context—provides both the copy number and spatial context of RNAs but has been limited in the number of RNA species that can be measured simultaneously. Here we describe Multiplexed Error Robust Fluorescence In Situ Hybridization (MERFISH), a massively parallelized form of smFISH that can image and identify hundreds to thousands of different RNA species simultaneously with high accuracy in individual cells in their native spatial context. We provide detailed protocols on all aspects of MERFISH, including probe design, data collection, and data analysis to allow interested laboratories to perform MERFISH measurements themselves. PMID:27241748

Cross-linking of streptomycin to the 16S ribosomal RNA of Escherichia coli

International Nuclear Information System (INIS)

Gravel, M.; Melancon, P.; Barkier-Gingras, L.

1987-01-01

[ 3 H]Dihydrostreptomycin was cross-linked to the 30S ribosomal subunit from Escherichia coli with the bifunctional reagent nitrogen mustard. The cross-linking primarily involved the 16S RNA. To localize the site of cross-linking of streptomycin to the 16S RNA, the authors hybridized RNA labeled with streptomycin to restriction fragments of the 16S RNA gene. Labeled RNA hybridized to DNA fragments corresponding to bases 892-917 and bases 1394-1415. These two segments of the ribosomal RNA must by juxtaposed in the ribosome, since there is a single binding site for streptomycin. This region has been implicated both in the decoding site and in the binding of initiation factor IF-3, indicating its functional importance

Characterization of DNA polymerase. beta. mRNA: cell-cycle growth response in cultured human cells

Energy Technology Data Exchange (ETDEWEB)

Zmudzka, B Z; Fornace, A; Collins, J; Wilson, S H

1988-10-25

DNA polymerase ..beta.. (..beta..-polymerase) is a housekeeping enzyme involved in DNA repair in vertebrate cells. The authors used a cDNA probe to study abundance of ..beta..-polymerase mRNA in cultured human cells. The mRNA level in synchronized HeLa cells, representing different stages of the cell-cycle, varied only slightly. Contact inhibited fibroblasts AG-1522 contained the same level of mRNA as growing cells. The steady-state level of mRNA in fibroblasts is equivalent to 6 molecules per cell. The results indicate that the ..beta..-polymerase transcript is low abundance and is neither cell-cycles nor growth phase responsive.

Genetic determinants of PAM-dependent DNA targeting and pre-crRNA processing in Sulfolobus islandicus

DEFF Research Database (Denmark)

Peng, Wenfang; Li, Huan; Hallstrøm, Søren

2013-01-01

-adjacent motif (PAM)-dependent DNA targeting activity and mature CRISPR RNA (crRNA) production in this organism, mutants deleting individual genes of the type IA system or removing each of other Cas modules were constructed. Characterization of these mutants revealed that Cas7, Cas5, Cas6, Cas3' and Cas3......" are essential for PAM-dependent DNA targeting activity, whereas Csa5, along with all other Cas modules, is dispensable for the targeting in the crenarchaeon. Cas6 is implicated as the only enzyme for pre-crRNA processing and the crRNA maturation is independent of the DNA targeting activity. Importantly, we show...

Transcribed DNA is preferentially located in the perichromatin region of mammalian cell nuclei

Energy Technology Data Exchange (ETDEWEB)

Niedojadlo, Janusz [Centre of Electron Microscopy, University of Lausanne, Bugnon 27, CH-1005 Lausanne (Switzerland); Department of Cell Biology, Institute of General and Molecular Biology, Nicolaus Copernicus University, PL-87-100 Torun (Poland); Perret-Vivancos, Cecile [Centre of Electron Microscopy, University of Lausanne, Bugnon 27, CH-1005 Lausanne (Switzerland); Kalland, Karl-Henning [Centre for Research in Virology, The Gade Institute, University of Bergen, Jonas Liesv. 91, N-5009 Bergen (Norway); Cmarko, Dusan [Centre of Electron Microscopy, University of Lausanne, Bugnon 27, CH-1005 Lausanne (Switzerland); Charles University in Prague, First Faculty of Medicine, Institute of Cellular Biology and Pathology, and Department of Cell Biology, Institute of Physiology, Academy of Sciences of the Czech Republic, v.v.i., Albertov 4, CZ-12801 Prague (Czech Republic); Cremer, Thomas [Department of Biology II, LMU Biozentrum, Grosshaderner Strasse 2, D-82152 Planegg-Martinsried (Germany); Center for Integrated Protein Science Munich (CIPSM), LMU Munich, Munich (Germany); Driel, Roel van, E-mail: r.vandriel@uva.nl [Swammerdam Institute for Life Sciences, University of Amsterdam, P.O. Box 94215, 1090GE Amsterdam (Netherlands); Fakan, Stanislav, E-mail: sfakan@lrz.uni-muenchen.de [Centre of Electron Microscopy, University of Lausanne, Bugnon 27, CH-1005 Lausanne (Switzerland); Department of Biology II, LMU Biozentrum, Grosshaderner Strasse 2, D-82152 Planegg-Martinsried (Germany); Center for Integrated Protein Science Munich (CIPSM), LMU Munich, Munich (Germany)

2011-02-15

The precise localization of transcribed DNA and resulting RNA is an important aspect of the functional architecture of the nucleus. To this end we have developed a novel in situ hybridization approach in combination with immunoelectron microscopy, using sense and anti-sense RNA probes that are derived from total cellular or cytoplasmic poly(A+) RNA. This new technology is much more gentle than classical in situ hybridization using DNA probes and shows excellent preservation of nuclear structure. Carried out on ultrathin sections of fixed and resin-embedded COS-7 cells, it revealed at high resolution the localization of the genes that code for the cellular mRNAs. Quantitative analysis shows that most transcribed DNA is concentrated in the perichromatin region, i.e. the interface between subchromosomal compact chromatin domains and the interchromatin space essentially devoid of DNA. The RNA that is produced is found mainly in the perichromatin region and the interchromatin space. These results imply that in the mammalian nucleus the chromatin fiber is folded so that active genes are predominantly present in the perichromatin region, which is the most prominent site of transcription.

Non-Covalent Fluorescent Labeling of Hairpin DNA Probe Coupled with Hybridization Chain Reaction for Sensitive DNA Detection.

Science.gov (United States)

Song, Luna; Zhang, Yonghua; Li, Junling; Gao, Qiang; Qi, Honglan; Zhang, Chengxiao

2016-04-01

An enzyme-free signal amplification-based assay for DNA detection was developed using fluorescent hairpin DNA probes coupled with hybridization chain reaction (HCR). The hairpin DNAs were designed to contain abasic sites in the stem moiety. Non-covalent labeling of the hairpin DNAs was achieved when a fluorescent ligand was bound to the abasic sites through hydrogen bonding with the orphan cytosine present on the complementary strand, accompanied by quench of ligand fluorescence. As a result, the resultant probes, the complex formed between the hairpin DNA and ligand, showed almost no fluorescence. Upon hybridization with target DNA, the probe underwent a dehybridization of the stem moiety containing an abasic site. The release of ligand from the abasic site to the solution resulted in an effective fluorescent enhancement, which can be used as a signal. Compared with a sensing system without HCR, a 20-fold increase in the sensitivity was achieved using the sensing system with HCR. The fluorescent intensity of the sensing system increased with the increase in target DNA concentration from 0.5 nM to 100 nM. A single mismatched target ss-DNA could be effectively discriminated from complementary target DNA. Genotyping of a G/C single-nucleotide polymorphism of polymerase chain reaction (PCR) products was successfully demonstrated with the sensing system. Therefore, integrating HCR strategy with non-covalent labeling of fluorescent hairpin DNA probes provides a sensitive and cost-effective DNA assay. © The Author(s) 2016.

Sensitive electrochemical monitoring of nucleic acids coupling DNA nanostructures with hybridization chain reaction

International Nuclear Information System (INIS)

Zhuang, Junyang; Fu, Libing; Xu, Mingdi; Yang, Huanghao; Chen, Guonan; Tang, Dianping

2013-01-01

Graphical abstract: -- Highlights: •A new signal-on metallobioassay was developed for detection of nucleic acids. •Target-triggered long-range self-assembled DNA nanostructures are used for amplification of electronic signal. •Hybridization chain reaction is utilized for construction of long-range DNA nanostructures. -- Abstract: Methods based on metal nanotags have been developed for metallobioassay of nucleic acids, but most involve complicated labeling or stripping procedures and are unsuitable for routine use. Herein, we report the proof-of-concept of a novel and label-free metallobioassay for ultrasensitive electronic determination of human immunodeficiency virus (HIV)-related gene fragments at an ultralow concentration based on target-triggered long-range self-assembled DNA nanostructures and DNA-based hybridization chain reaction (HCR). The signal is amplified by silver nanotags on the DNA duplex. The assay mainly consists of capture probe, detection probe, and two different DNA hairpins. In the presence of target DNA, the capture probe immobilized on the sensor sandwiches target DNA with the 3′ end of detection probe. Another exposed part of detection probe at the 5′ end opens two alternating DNA hairpins in turn, and propagates a chain reaction of hybridization events to form a nicked double-helix. Finally, numerous silver nanotags are immobilized onto the long-range DNA nanostructures, each of which produces a strong electronic signal within the applied potentials. Under optimal conditions, the target-triggered long-range DNA nanostructures present good electrochemical behaviors for the detection of HIV DNA at a concentration as low as 0.5 fM. Importantly, the outstanding sensitivity can make this approach a promising scheme for development of next-generation DNA sensors without the need of enzyme labeling or fluorophore labeling

Expression of calmodulin mRNA in rat olfactory neuroepithelium.

Science.gov (United States)

Biffo, S; Goren, T; Khew-Goodall, Y S; Miara, J; Margolis, F L

1991-04-01

A calmodulin (CaM) cDNA was isolated by differential hybridization screening of a lambda gt10 library prepared from rat olfactory mucosa. This cDNA fragment, containing most of the open reading frame of the rat CaMI gene, was subcloned and used to characterize steady-state expression of CaM mRNA in rat olfactory neuroepithelium and bulb. Within the bulb mitral cells are the primary neuronal population expressing CaM mRNA. The major CaM mRNA expressed in the olfactory mucosa is 1.7 kb with smaller contributions from mRNAs of 4.0 and 1.4 kb. CaM mRNA was primarily associated with the olfactory neurons and, despite the cellular complexity of the tissue and the known involvement of CaM in diverse cellular processes, was only minimally evident in sustentacular cells, gland cells or respiratory epithelium. Following bulbectomy CaM mRNA declines in the olfactory neuroepithelium as does olfactory marker protein (OMP) mRNA. In contrast to the latter, CaM mRNA makes a partial recovery by one month after surgery. These results, coupled with those from in situ hybridization, indicate that CaM mRNA is expressed in both mature and immature olfactory neurons. The program regulating CaM gene expression in olfactory neurons is distinct from those controlling expression of B50/GAP43 in immature, or OMP in mature, neurons respectively.

Temporal aspects of DNA and RNA synthesis during human immunodeficiency virus infection: Evidence for differential gene expression

International Nuclear Information System (INIS)

Kim, Sunyoung; Baltimore, D.; Byrn, R.; Groopman, J.

1989-01-01

The kinetics of retroviral DNA and RNA synthesis are parameters vital to understanding viral growth, especially for human immunodeficiency virus (HIV), which encodes several of its own regulatory genes. The authors have established a single-cycle growth condition for HIV in H9 cells, a human CD4 + lymphocyte line. The full-length viral linear DNA is first detectable by 4 h postinfection. During a one-step growth of HIV, amounts of viral DNA gradually increase until 8 to 12 h postinfection and then decrease. The copy number of unintegrated viral DNA is not extraordinarily high even at its peak. Most strikingly, there is a temporal program of RNA accumulation: the earliest RNA is greatly enriched in the 2-kilobase subgenomic mRNA species, while the level of 9.2-kilobase RNA which is both genomic RNA and mRNA remains low until after 24 h of infection. Virus production begins at about 24 h postinfection. Thus, viral DNA synthesis is as rapid as for other retroviruses, but viral RNA synthesis involves temporal alteration in the species that accumulate, presumably as a consequence of viral regulatory genes

The effect of U.V.-irradiation on lambda DNA transcription

International Nuclear Information System (INIS)

Ranade, S.S.

1977-01-01

The effect of U.V.-irradiation of template DNA has been studied in vitro in the E.coli RNA polymerase system with native and U.V.-treated lambda DNA. Lambda DNA was more susceptible to U.V. than was calf-thymus DNA, yet a residual activity was observed at a U.V. dose of 0.5 x 10 4 erg/mm 2 . From the kinetic analysis of the reaction and the incorporation of lambda 32 P-labelled nucleoside triphosphates, it seems reasonable to conclude that U.V.-irradiation probably did not affect the DNA initiation sites, recognizable by RNA polymerase. The transcription products made with U.V.-irradiated lambda DNA were asymmetrical, and hybridized to the right half (R) and the left half (L) of lambda DNA with the ratio of R/L=4/1, and they showed a lower hybridizability than the transcripts with native lambda DNA. The initiation sites recognizable by RNA polymerase seemed to be the same on both native and U.V.-irradiated lambda DNA, though the transcription of U.V.-treated lambda DNA appeared to terminate with rather short RNA chains. (author)

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

Science.gov (United States)

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.

A Novel Method for Rapid Hybridization of DNA to a Solid Support

Science.gov (United States)

Pettersson, Erik; Ahmadian, Afshin; Ståhl, Patrik L.

2013-01-01

Here we present a novel approach entitled Magnetic Forced Hybridization (MFH) that provides the means for efficient and direct hybridization of target nucleic acids to complementary probes immobilized on a glass surface in less than 15 seconds at ambient temperature. In addition, detection is carried out instantly since the beads become visible on the surface. The concept of MFH was tested for quality control of array manufacturing, and was combined with a multiplex competitive hybridization (MUCH) approach for typing of Human Papilloma Virus (HPV). Magnetic Forced Hybridization of bead-DNA constructs to a surface achieves a significant reduction in diagnostic testing time. In addition, readout of results by visual inspection of the unassisted eye eliminates the need for additional expensive instrumentation. The method uses the same set of beads throughout the whole process of manipulating and washing DNA constructs prior to detection, as in the actual detection step itself. PMID:23950946

Intracellular distribution of histone mRNAs in human fibroblasts studied by in situ hybridization

International Nuclear Information System (INIS)

Lawrence, J.B.; Singer, R.H.; Villnave, C.A.; Stein, J.L.; Stein, G.S.

1988-01-01

We have used in situ hybridization to study the intracellular distribution of mRNAs for cell cycle-dependent core and H1 histone proteins in human WI-38 fibroblasts. Because histones are abundant nuclear proteins and histone mRNA expression is tightly coupled to DNA synthesis, it was of interest to determine whether histone mRNAs are localized near the nucleus. Cells were hybridized with tritiated DNA probes specific for either histone H1, histone H4, actin, or poly(A)+ mRNA and were processed for autoradiography. In exponentially growing cultures, the fraction of histone mRNA-positive cells correlated well with the fraction of cells in S phase and was eliminated by hydroxyurea inhibition of DNA synthesis. Within individual cells the label for histone mRNA was widely distributed throughout the cytoplasm and did not appear to be more heavily concentrated near the nucleus. However, histone mRNA appeared to exhibit patchy, nonhomogeneous localization, and a quantitative evaluation confirmed that grain distributions were not as uniform as they were after hybridizations to poly(A)+ mRNA. Actin mRNA in WI-38 cells was also widely distributed throughout the cytoplasm but differed from histone mRNA in that label for actin mRNA was frequently most dense at the outermost region of narrow cell extensions. The localization of actin mRNA was less pronounced but qualitatively very similar to that previously described for chicken embryonic myoblasts and fibroblasts. We conclude that localization of histones in WI-38 cells is not facilitated by localization of histone protein synthesis near the nucleus and that there are subtle but discrete and potentially functional differences in the distributions of histone, actin, and poly(A)+ mRNAs

18S Ribosomal RNA Evaluation as Preanalytical Quality Control for Animal DNA

Directory of Open Access Journals (Sweden)

Cory Ann Leonard

2016-01-01

Full Text Available The 18S ribosomal RNA (rRNA gene is present in all eukaryotic cells. In this study, we evaluated the use of this gene to verify the presence of PCR-amplifiable host (animal DNA as an indicator of sufficient sample quality for quantitative real-time PCR (qPCR analysis. We compared (i samples from various animal species, tissues, and sample types, including swabs; (ii multiple DNA extraction methods; and (iii both fresh and formalin-fixed paraffin-embedded (FFPE samples. Results showed that 18S ribosomal RNA gene amplification was possible from all tissue samples evaluated, including avian, reptile, and FFPE samples and most swab samples. A single swine rectal swab, which showed sufficient DNA quantity and the demonstrated lack of PCR inhibitors, nonetheless was negative by 18S qPCR. Such a sample specifically illustrates the improvement of determination of sample integrity afforded by inclusion of 18S rRNA gene qPCR analysis in addition to spectrophotometric analysis and the use of internal controls for PCR inhibition. Other possible applications for the described 18S rRNA qPCR are preselection of optimal tissue specimens for studies or preliminary screening of archived samples prior to acceptance for biobanking projects.

Anomeric 2'-Deoxycytidines and Silver Ions: Hybrid Base Pairs with Greatly Enhanced Stability and Efficient DNA Mismatch Detection with α-dC.

Science.gov (United States)

Guo, Xiurong; Seela, Frank

2017-09-04

α-d-Nucleosides are rare in nature but can develop fascinating properties when incorporated into DNA. This work reports on the first silver-mediated base pair constructed from two anomeric nucleosides: α-dC and β-dC. The hybrid base pair was integrated into the DNA and DNA/RNA double helix. A 12-mer duplex with α-dC and β-dC pair exhibits a higher thermal stability (T m =43 °C) than that incorporating the β-dC-Ag + -β-dC homo pair (T m =34 °C). Furthermore, α-dC shows excellent mismatch discrimination for DNA single nucleotide polymorphism (SNP). All four SNPs were identified on the basis of large T m value differences measured in the presence of silver ions. High resolution melting was not required. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Circularly polarized luminescence of helically assembled pyrene π-stacks on RNA and DNA duplexes.

Science.gov (United States)

Nakamura, Mitsunobu; Ota, Fuyuki; Takada, Tadao; Akagi, Kazuo; Yamana, Kazushige

2018-05-01

In this report, we describe the circularly polarized luminescence (CPL) of the RNA duplexes having one to four 2'-O-pyrene modified uridines (Upy) and the DNA duplexes having two, four, and six pyrene modified non-nucleosidic linkers (Py). Both the pyrene π-stack arrays formed on the RNA and DNA double helical structures exhibited pyrene excimer fluorescence. In the pyrene-modified RNA systems, the RNA duplex having four Upys gives CPL emission with g lum value of <0.01 at 480 nm. The structure of pyrene stacks on the RNA duplex may be rigidly regulated with increase in the Upy domains, which resulted in the CPL emission. In the DNA systems, the pyrene-modified duplexes containing two and four Pys exhibited CPL emission with g lum values of <0.001 at 505 nm. The pyrene π-stack arrays presented here show CPL emission. However, the g lum values are relatively small when compared with our previous system consisting of the pyrene-zipper arrays on RNA. © 2018 Wiley Periodicals, Inc.

Efficient procedure for transferring specific human genes into Chinese hamster cell mutants: interspecific transfer of the human genes encoding leucyl- and asparaginyl-tRNA synthetases

International Nuclear Information System (INIS)

Cirullo, R.E.; Dana, S.; Wasmuth, J.J.

1983-01-01

A simple and efficient procedure for transferring specific human genes into mutant Chinese hamster ovary cell recipients has been developed that does not rely on using calcium phosphate-precipitated high-molecular-weight DNA. Interspecific cell hybrids between human leukocytes and temperature-sensitive Chinese hamster cell mutants with either a thermolabile leucyl-tRNA synthetase or a thermolabile asparaginyl-tRNA synthetase were used as the starting material in these experiments. These hybrids contain only one or a few human chromosomes and require expression of the appropriate human aminoacyl-tRNA synthetase gene to grow at 39 degrees C. Hybrids were exposed to very high doses of gamma-irradiation to extensively fragment the chromosomes and re-fused immediately to the original temperature-sensitive Chinese hamster mutant, and secondary hybrids were isolated at 39 degrees C. Secondary hybrids, which had retained small fragments of the human genome containing the selected gene, were subjected to another round of irradiation, refusion, and selection at 39 degrees C to reduce the amount of human DNA even further. Using this procedure, Chinese hamster cell lines have been constructed that express the human genes encoding either asparaginyl- or leucyl-tRNA synthetase, yet less than 0.1% of their DNA is derived from the human genome, as quantitated by a sensitive dot-blot nucleic acid hybridization procedure

The use of carrier RNA to enhance DNA extraction from microfluidic-based silica monoliths.

Science.gov (United States)

Shaw, Kirsty J; Thain, Lauren; Docker, Peter T; Dyer, Charlotte E; Greenman, John; Greenway, Gillian M; Haswell, Stephen J

2009-10-12

DNA extraction was carried out on silica-based monoliths within a microfluidic device. Solid-phase DNA extraction methodology was applied in which the DNA binds to silica in the presence of a chaotropic salt, such as guanidine hydrochloride, and is eluted in a low ionic strength solution, such as water. The addition of poly-A carrier RNA to the chaotropic salt solution resulted in a marked increase in the effective amount of DNA that could be recovered (25ng) compared to the absence of RNA (5ng) using the silica-based monolith. These findings confirm that techniques utilising nucleic acid carrier molecules can enhance DNA extraction methodologies in microfluidic applications.

An archaeal CRISPR type III-B system exhibiting distinctive RNA targeting features and mediating dual RNA and DNA interference

DEFF Research Database (Denmark)

Peng, Wenfang; Feng, Mingxia; Feng, Xu

2015-01-01

CRISPR-Cas systems provide a small RNA-based mechanism to defend against invasive genetic elements in archaea and bacteria. To investigate the in vivo mechanism of RNA interference by two type III-B systems (Cmr-α and Cmr-β) in Sulfolobus islandicus, a genetic assay was developed using plasmids...... carrying an artificial mini-CRISPR (AC) locus with a single spacer. After pAC plasmids were introduced into different strains, Northern analyses confirmed that mature crRNAs were produced from the plasmid-borne CRISPR loci, which then guided gene silencing to target gene expression. Spacer mutagenesis....... islandicus Cmr-α mediated transcription-dependent DNA interference, the Cmr-α constitutes the first CRISPR system exhibiting dual targeting of RNA and DNA....

Localized surface plasmon resonance (LSPR) study of DNA hybridization at single nanoparticle transducers

International Nuclear Information System (INIS)

Schneider, T.; Jahr, N.; Jatschka, J.; Csaki, A.; Stranik, O.; Fritzsche, W.

2013-01-01

The effect of DNA–DNA interaction on the localized surface plasmon resonance of single 80 nm gold nanoparticles is studied. Therefore, both the attachment of the capture DNA strands at the particle surface and the sequence-specific DNA binding (hybridization) of analyte DNA to the immobilized capture DNA is subject of investigations. The influence of substrate attachment chemistry, the packing density of DNA as controlled by an assisting layer of smaller molecules, and the distance as increased by a linker on the LSPR efficiency is investigated. The resulting changes in signal can be related to a higher hybridization efficiency of the analyte DNA to the immobilized capture DNA. The subsequent attachment of additional DNA strands to this system is studied, which allows for a multiple step detection of binding and an elucidation of the resulting resonance shifts. The detection limit was determined for the utilized DNA system by incubation with various concentration of analyte DNA. Although the method allows for a marker-free detection, we show that additional markers such as 20 nm gold particle labels increase the signal and thereby the sensitivity significantly. The study of resonance shift for various DNA lengths revealed that the resonance shift per base is stronger for shorter DNA molecules (20 bases) as compared to longer ones (46 bases).

Photolithographic Synthesis of High-Density DNA and RNA Arrays on Flexible, Transparent, and Easily Subdivided Plastic Substrates.

Science.gov (United States)

Holden, Matthew T; Carter, Matthew C D; Wu, Cheng-Hsien; Wolfer, Jamison; Codner, Eric; Sussman, Michael R; Lynn, David M; Smith, Lloyd M

2015-11-17

The photolithographic fabrication of high-density DNA and RNA arrays on flexible and transparent plastic substrates is reported. The substrates are thin sheets of poly(ethylene terephthalate) (PET) coated with cross-linked polymer multilayers that present hydroxyl groups suitable for conventional phosphoramidite-based nucleic acid synthesis. We demonstrate that by modifying array synthesis procedures to accommodate the physical and chemical properties of these materials, it is possible to synthesize plastic-backed oligonucleotide arrays with feature sizes as small as 14 μm × 14 μm and feature densities in excess of 125 000/cm(2), similar to specifications attainable using rigid substrates such as glass or glassy carbon. These plastic-backed arrays are tolerant to a wide range of hybridization temperatures, and improved synthetic procedures are described that enable the fabrication of arrays with sequences up to 50 nucleotides in length. These arrays hybridize with S/N ratios comparable to those fabricated on otherwise identical arrays prepared on glass or glassy carbon. This platform supports the enzymatic synthesis of RNA arrays and proof-of-concept experiments are presented showing that the arrays can be readily subdivided into smaller arrays (or "millichips") using common laboratory-scale laser cutting tools. These results expand the utility of oligonucleotide arrays fabricated on plastic substrates and open the door to new applications for these important bioanalytical tools.

Molecular dynamics simulations of electrostatics and hydration distributions around RNA and DNA motifs

Science.gov (United States)

Marlowe, Ashley E.; Singh, Abhishek; Semichaevsky, Andrey V.; Yingling, Yaroslava G.

2009-03-01

Nucleic acid nanoparticles can self-assembly through the formation of complementary loop-loop interactions or stem-stem interactions. Presence and concentration of ions can significantly affect the self-assembly process and the stability of the nanostructure. In this presentation we use explicit molecular dynamics simulations to examine the variations in cationic distributions and hydration environment around DNA and RNA helices and loop-loop interactions. Our simulations show that the potassium and sodium ionic distributions are different around RNA and DNA motifs which could be indicative of ion mediated relative stability of loop-loop complexes. Moreover in RNA loop-loop motifs ions are consistently present and exchanged through a distinct electronegative channel. We will also show how we used the specific RNA loop-loop motif to design a RNA hexagonal nanoparticle.

Detection of c-myc amplification in formalin-fixed paraffin-embedded tumor tissue by chromogenic in situ hybridization (CISH).

Science.gov (United States)

Todorović-Raković, Nataša

2013-01-01

In situ hybridization (ISH) allows evaluation of genetic abnormalities, such as changes in chromosome number, chromosome translocations or gene amplifications, by hybridization of tagged DNA (or RNA) probes with complementary DNA (or RNA) sequences in interphase nuclei of target tissue. However, chromogenic in situ hybridization (CISH) is also applicable to formalin-fixed, paraffin-embedded (FFPE) tissues, besides metaphase chromosome spreads. CISH is similar to fluorescent in situ hybridization (FISH) regarding pretreatments and hybridization protocols but differs in the way of visualization. Indeed, CISH signal detection is similar to that used in immunohistochemistry, making use of a peroxidase-based chromogenic reaction instead of fluorescent dyes. In particular, tagged DNA probes are indirectly detected using an enzyme-conjugated antibody targeting the tags. The enzymatic reaction of the chromogenic substrate leads to the formation of strong permanent brown signals that can be visualized by bright-field microscopy at 40 × magnification. The advantage of CISH is that it allows the simultaneous observation of gene amplification and tissue morphology and the slides can be stored for a long time.

The chloroplast and mitochondrial DNA type are correlated with the nuclear composition of somatic hybrid calli of Solanum tuberosum and Nicotiana plumbaginifolia.

Science.gov (United States)

Wolters, A M; Koornneef, M; Gilissen, L J

1993-09-01

This paper describes the analysis of chloroplast (cp) DNA and mitochondrial (mt) DNA in 21 somatic hybrid calli of Solanum tuberosum and Nicotiana plumbaginifolia by means of Southern-blot hybridization. Each of these calli contained only one type of cpDNA; 14 had the N. plumbaginifolia (Np) type and seven the S. tuberosum (St) type. N. plumbaginifolia cpDNA was present in hybrids previously shown to contain predominantly N. plumbaginifolia chromosomes whereas hybrids in which S. tuberosum chromosomes predominated possessed cpDNA from potato. We have analyzed the mtDNA of these 21 somatic hybrid calli using four restriction enzyme/probe combinations. Most fusion products had only, or mostly, mtDNA fragments from the parent that predominated in the nucleus. The hybrids containing mtDNA fragments from only one parent (and new fragments) also possessed chloroplasts from the same species. The results suggest the existence of a strong nucleo-cytoplasmic incongruity which affects the genome composition of somatic hybrids between distantly related species.

Design of 240,000 orthogonal 25mer DNA barcode probes.

Science.gov (United States)

Xu, Qikai; Schlabach, Michael R; Hannon, Gregory J; Elledge, Stephen J

2009-02-17

DNA barcodes linked to genetic features greatly facilitate screening these features in pooled formats using microarray hybridization, and new tools are needed to design large sets of barcodes to allow construction of large barcoded mammalian libraries such as shRNA libraries. Here we report a framework for designing large sets of orthogonal barcode probes. We demonstrate the utility of this framework by designing 240,000 barcode probes and testing their performance by hybridization. From the test hybridizations, we also discovered new probe design rules that significantly reduce cross-hybridization after their introduction into the framework of the algorithm. These rules should improve the performance of DNA microarray probe designs for many applications.

RNA-DNA sequence differences spell genetic code ambiguities

DEFF Research Database (Denmark)

Bentin, Thomas; Nielsen, Michael L

2013-01-01

A recent paper in Science by Li et al. 2011(1) reports widespread sequence differences in the human transcriptome between RNAs and their encoding genes termed RNA-DNA differences (RDDs). The findings could add a new layer of complexity to gene expression but the study has been criticized. ...

An efficient algorithm for the stochastic simulation of the hybridization of DNA to microarrays

Directory of Open Access Journals (Sweden)

Laurenzi Ian J

2009-12-01

Full Text Available Abstract Background Although oligonucleotide microarray technology is ubiquitous in genomic research, reproducibility and standardization of expression measurements still concern many researchers. Cross-hybridization between microarray probes and non-target ssDNA has been implicated as a primary factor in sensitivity and selectivity loss. Since hybridization is a chemical process, it may be modeled at a population-level using a combination of material balance equations and thermodynamics. However, the hybridization reaction network may be exceptionally large for commercial arrays, which often possess at least one reporter per transcript. Quantification of the kinetics and equilibrium of exceptionally large chemical systems of this type is numerically infeasible with customary approaches. Results In this paper, we present a robust and computationally efficient algorithm for the simulation of hybridization processes underlying microarray assays. Our method may be utilized to identify the extent to which nucleic acid targets (e.g. cDNA will cross-hybridize with probes, and by extension, characterize probe robustnessusing the information specified by MAGE-TAB. Using this algorithm, we characterize cross-hybridization in a modified commercial microarray assay. Conclusions By integrating stochastic simulation with thermodynamic prediction tools for DNA hybridization, one may robustly and rapidly characterize of the selectivity of a proposed microarray design at the probe and "system" levels. Our code is available at http://www.laurenzi.net.

Cloning of zebrafish activin type IIB receptor (ActRIIB) cDNA and mRNA expression of ActRIIB in embryos and adult tissues.

Science.gov (United States)

Garg, R R; Bally-Cuif, L; Lee, S E; Gong, Z; Ni, X; Hew, C L; Peng, C

1999-07-20

A full-length cDNA encoding for activin type IIB receptor (ActRIIB) was cloned from zebrafish embryos. It encodes a protein with 509 amino acids consisting of a signal peptide, an extracellular ligand binding domain, a single transmembrane region, and an intracellular kinase domain with predicted serine/threonine specificity. The extracellular domain shows 74-91% sequence identity to human, bovine, mouse, rat, chicken, Xenopus and goldfish activin type IIB receptors, while the transmembrane region and the kinase domain show 67-78% and 82-88% identity to these known activin IIB receptors, respectively. In adult zebrafish, ActRIIB mRNA was detected by RT-PCR in the gonads, as well as in non-reproductive tissues, including the brain, heart and muscle. In situ hybridization on ovarian sections further localized ActRIIB mRNA to cytoplasm of oocytes at different stages of development. Using whole-mount in situ hybridization, ActRIIB mRNA was found to be expressed at all stages of embryogenesis examined, including the sphere, shield, tail bud, and 6-7 somite. These results provide the first evidence that ActRIIB mRNA is widely distributed in fish embryonic and adult tissues. Cloning of zebrafish ActRIIB demonstrates that this receptor is highly conserved during vertebrate evolution and provides a basis for further studies on the role of activin in reproduction and development in lower vertebrates.

Multiway study of hybridization in nanoscale semiconductor labeled DNA based on fluorescence resonance energy transfer

DEFF Research Database (Denmark)

Gholami, Somayeh; Kompany Zare, Mohsen

2013-01-01

donor-QD acceptor) upon hybridization with a label free target was monitored by two-dimensional photoluminescence excitation spectroscopy (2D-PLE). Detection of a target oligonucleotide strand, using sandwiched nanoassembly in a separation-free format, was performed with the appearance of a new feature...... and model based analysis of 2D-PLE data was implemented by means of PAR-AFAC and hard trilinear decomposition (HTD), allowing to fit a proper model for FRET-based sandwich DNA hybridization systems. This study is the first successful application of a multiway chemometric technique to consider FRET based DNA...... hybridization in sandwiched nanoassemblies. A multi-equilibria model was properly fitted to the data and confirmed there is a competition between ternary and binary complex formation. Equilibrium constants of DNA hybridization in sandwiched nanoassemblies were estimated for the first time. Equilibrium constants...

Bacillus subtilis DNA polymerases, PolC and DnaE, are required for both leading and lagging strand synthesis in SPP1 origin-dependent DNA replication

Science.gov (United States)

Seco, Elena M.

2017-01-01

Abstract Firmicutes have two distinct replicative DNA polymerases, the PolC leading strand polymerase, and PolC and DnaE synthesizing the lagging strand. We have reconstituted in vitro Bacillus subtilis bacteriophage SPP1 θ-type DNA replication, which initiates unidirectionally at oriL. With this system we show that DnaE is not only restricted to lagging strand synthesis as previously suggested. DnaG primase and DnaE polymerase are required for initiation of DNA replication on both strands. DnaE and DnaG synthesize in concert a hybrid RNA/DNA ‘initiation primer’ on both leading and lagging strands at the SPP1 oriL region, as it does the eukaryotic Pol α complex. DnaE, as a RNA-primed DNA polymerase, extends this initial primer in a reaction modulated by DnaG and one single-strand binding protein (SSB, SsbA or G36P), and hands off the initiation primer to PolC, a DNA-primed DNA polymerase. Then, PolC, stimulated by DnaG and the SSBs, performs the bulk of DNA chain elongation at both leading and lagging strands. Overall, these modulations by the SSBs and DnaG may contribute to the mechanism of polymerase switch at Firmicutes replisomes. PMID:28575448

HIV RNA and proviral HIV DNA can be detected in semen after 6 months of antiretroviral therapy although HIV RNA is undetectable in blood.

Science.gov (United States)

Du, Peiwei; Liu, An; Jiao, Yanmei; Liu, Cuie; Jiang, Taiyi; Zhu, Weijun; Zhu, Yunxia; Wu, Hao; Sun, Lijun

2016-03-01

The risk of sexual transmission of HIV is strongly correlated with amounts of genital HIV RNA. Few studies have reported amounts of HIV RNA and HIV DNA in semen in HIV-infected Chinese patients undergoing antiviral treatment (ART). In this observational study, the amounts of HIV RNA and HIV DNA in semen were assessed after six months of ART in HIV-infected Chinese individuals, when HIV RNA was undetectable in blood . This study included 19 HIV-infected Chinese men undergoing ART for six months. Amounts of HIV in paired semen and blood samples were assessed using real-time PCR. The C2-V5 region of the HIV envelope (env) genes was cloned and sequenced and genotype and co-receptor usage predicted based on the sequence. It was found that HIV RNA was undetectable in the plasma of most patients (17/19), whereas HIV RNA could be detected in the semen of most patients (16/19). HIV DNA could be detected in both semen and blood. Genetic diversity of HIV between the seminal and blood compartments was identified. Thus, amounts of HIV RNA and HIV DNA remain high in semen of HIV-infected Chinese patients after six months of ART treatment, even when HIV RNA was undetectable in blood. © 2016 The Societies and John Wiley & Sons Australia, Ltd.

Influence of DNA treatments on Southern blot hybridization analysis ...

African Journals Online (AJOL)

STORAGESEVER

2008-06-03

Jun 3, 2008 ... DNA samples obtained by a non-phenol/chloroform isolation method, from three races of Fusarium oxysporum f. sp. lycopersici ... Key words: Fusarium oxysporum, DIG-IGS Probe, Southern hybridization. INTRODUCTION .... Detection of Fusarium spp in plants with monoclonal antibody. Ann. Phytopathol.

Hybrid-hybrid matrix structural refinement of a DNA three-way junction from 3D NOESY-NOESY

International Nuclear Information System (INIS)

Thiviyanathan, Varatharasa; Luxon, Bruce A.; Leontis, Neocles B.; Illangasekare, Nishantha; Donne, David G.; Gorenstein, David G.

1999-01-01

Homonuclear 3D NOESY-NOESY has shown great promise for the structural refinement of large biomolecules. A computationally efficient hybrid-hybrid relaxation matrix refinement methodology, using 3D NOESY-NOESY data, was used to refine the structure of a DNA three-way junction having two unpaired bases at the branch point of the junction. The NMR data and the relaxation matrix refinement confirm that the DNA three-way junction exists in a folded conformation with two of the helical stems stacked upon each other. The third unstacked stem extends away from the junction, forming an acute angle (∼60 deg.) with the stacked stems. The two unpaired bases are stacked upon each other and are exposed to the solvent. Helical parameters for the bases in all three strands show slight deviations from typical values expected for right-handed B-form DNA. Inter-nucleotide imino-imino NOEs between the bases at the branch point of the junction show that the junction region is well defined. The helical stems show mobility (± 20 deg.) indicating dynamic processes around the junction region. The unstacked helical stem adjacent to the unpaired bases shows greater mobility compared to the other two stems. The results from this study indicate that the 3D hybrid-hybrid matrix MORASS refinement methodology, by combining the spectral dispersion of 3D NOESY-NOESY and the computational efficiency of 2D refinement programs, provides an accurate and robust means for structure determination of large biomolecules. Our results also indicate that the 3D MORASS method gives higher quality structures compared to the 2D complete relaxation matrix refinement method

Development of Ionic Liquid Modified Disposable Graphite Electrodes for Label-Free Electrochemical Detection of DNA Hybridization Related to Microcystis spp.

Directory of Open Access Journals (Sweden)

Ceren Sengiz

2015-09-01

Full Text Available In this present study, ionic liquid (1-butyl-3-methylimidazolium hexafluorophosphate (IL modified pencil graphite electrode (IL-PGEs was developed for electrochemical monitoring of DNA hybridization related to Microcystis spp. (MYC. The characterization of IL-PGEs was performed using microscopic and electrochemical techniques. DNA hybridization related to MYC was then explored at the surface of IL-PGEs using differential pulse voltammetry (DPV technique. After the experimental parameters were optimized, the sequence-selective DNA hybridization related to MYC was performed in the case of hybridization between MYC probe and its complementary DNA target, noncomplementary (NC or mismatched DNA sequence (MM, or and in the presence of mixture of DNA target: NC (1:1 and DNA target: MM (1:1.

Development of an in situ hybridization assay for the detection of ostreid herpesvirus type 1 mRNAs in the Pacific oyster, Crassostrea gigas.

Science.gov (United States)

Corbeil, Serge; Faury, Nicole; Segarra, Amélie; Renault, Tristan

2015-01-01

An in situ hybridization protocol for detecting mRNAs of ostreid herpesvirus type 1 (OsHV-1) which infects Pacific oysters, Crassostrea gigas, was developed. Three RNA probes were synthesized by cloning three partial OsHV-1 genes into plasmids using three specific primer pairs, and performing a transcription in the presence of digoxigenin dUTP. The RNA probes were able to detect the virus mRNAs in paraffin sections of experimentally infected oysters 26 h post-injection. The in situ hybridization showed that the OsHV-1 mRNAs were mainly present in connective tissues in gills, mantle, adductor muscle, digestive gland and gonads. DNA detection by in situ hybridization using a DNA probe and viral DNA quantitation by real-time PCR were also performed and results were compared with those obtained using RNA probes. Copyright © 2014 Elsevier B.V. All rights reserved.

Compensatory lung growth: protein, DNA and RNA lung contents in undernourished trilobectomized rats Crescimento pulmonar compensatório: conteúdos pulmonares de proteína, DNA e RNA em ratos subnutridos trilobectomizados

Directory of Open Access Journals (Sweden)

Raul Lopes Ruiz Júnior

2005-06-01

Full Text Available PURPOSE: To demonstrate compensatory lung growth (CLG by lung contents of proteins, DNA, and RNA in undernourished young adult rats, submitted to pulmonary trilobectomy. METHODS: We used 137 male Wistar rats, randomly distributed into 9 groups; they were submitted to three treatments (control, thoracotomy, and trilobectomy, and sacrificed at three different times (7, 30, and 90 days. In trilobectomy we removed the right median, accessory, and caudal lobes. We studied lung proteins, DNA, and RNA contents. RESULTS: In the cranial lobe and left lung, protein content was higher in trilobectomized rats however there was insufficient CLG to make up for the loss. The increase of DNA in the cranial lobe and left lung of trilobectomized rats was sufficient to compensate for this loss, resulting in a similar content to controls. RNA content in trilobectomized rats, was higher in the cranial lobe and left lung, more efficient in the cranial lobe, but less than in the other groups. CONCLUSION: CLG occurred in trilobectomized rats, probably with cell hyperplasia and little hypertrophy, due to the large DNA compensation and small RNA compensation. This was markedly different to well-nourished animals, who had pronounced hypertrophy.OBJETIVO: demonstrar se ocorre crescimento pulmonar compensatório (CPC representado pelos conteúdos de proteínas, DNA e RNA no rato adulto jovem, subnutrido, submetido à trilobectomia pulmonar. MÉTODOS: Utilizamos 137 ratos "Wistar", machos, distribuídos por sorteio, em 9 grupos, submetidos a três tratamentos (controle, toracotomia, trilobectomia, sacrificados em três momentos (7, 30 e 90 dias. Na trilobectomia foram extirpados os lobos médio, acessório e caudal direitos. Variáveis estudadas: conteúdos pulmonares de proteínas, DNA e RNA. RESULTADOS: No lobo cranial e pulmão esquerdo o conteúdo protéico foi maior nos trilobectomizados. Ocorreu CPC insuficiente para suprir a perda desta variável, sendo menor nos pulm

Identification and quantification of Bifidobacterium species isolated from food with genus-specific 16S rRNA-targeted probes by colony hybridization and PCR.

Science.gov (United States)

Kaufmann, P; Pfefferkorn, A; Teuber, M; Meile, L

1997-04-01

A Bifidobacterium genus-specific target sequence in the V9 variable region of the 16S rRNA has been elaborated and was used to develop a hybridization probe. The specificity of this probe, named lm3 (5'-CGGGTGCTI*CCCACTTTCATG-3'), was used to identify all known type strains and distinguish them from other bacteria. All of the 30 type strains of Bifidobacterium which are available at the German culture collection Deutsche Sammlung von Mikroorganismen und Zellkulturen, 6 commercially available production strains, and 34 closely related relevant strains (as negative controls) were tested. All tested bifidobacteria showed distinct positive signals by colony hybridization, whereas all negative controls showed no distinct dots except Gardnerella vaginalis DSM4944 and Propionibacterium freudenreichii subsp. shermanii DSM4902, which gave slight signals. Furthermore, we established a method for isolation and identification of bifidobacteria from food by using a PCR assay without prior isolation of DNA but breaking the cells with proteinase K. By this method, all Bifidobacterium strains lead to a DNA product of the expected size. We also established a quick assay to quantitatively measure Bifidobacterium counts in food and feces by dilution plating and colony hybridization. We were able to demonstrate that 2.1 x 10(6) to 2.3 x 10(7) colonies/g of sour milk containing bifidobacteria hybridized with the specific nucleotide probe. With these two methods, genus-specific colony hybridization and genus-specific PCR, it is now possible to readily and accurately detect any bifidobacteria in food and fecal samples and to discriminate between them and members of other genera.

rDNA genetic imbalance and nucleolar chromatin restructuring is induced by distant hybridization between Raphanus sativus and Brassica alboglabra.

Directory of Open Access Journals (Sweden)

Hong Long

Full Text Available The expression of rDNA in hybrids inherited from only one progenitor refers to nucleolar dominance. The molecular basis for choosing which genes to silence remains unclear. We report genetic imbalance induced by distant hybridization correlates with formation of rDNA genes (NORs in the hybrids between Raphanus sativus L. and Brassica alboglabra Bailey. Moreover, increased CCGG methylation of rDNA in F1 hybrids is concomitant with Raphanus-derived rDNA gene silencing and rDNA transcriptional inactivity revealed by nucleolar configuration restriction. Newly formed rDNA gene locus occurred through chromosomal in F1 hybrids via chromosomal imbalance. NORs are gained de novo, lost, and/or transposed in the new genome. Inhibition of methyltransferases leads to changes in nucleolar architecture, implicating a key role of methylation in control of nucleolar dominance and vital nucleolar configuration transition. Our findings suggest that gene imbalance and methylation-related chromatin restructuring is important for rDNA gene silencing that may be crucial for synthesis of specific proteins.

Herpes Simplex Virus 1 DNA Polymerase RNase H Activity Acts in a 3'-to-5' Direction and Is Dependent on the 3'-to-5' Exonuclease Active Site.

Science.gov (United States)

Lawler, Jessica L; Mukherjee, Purba; Coen, Donald M

2018-03-01

The catalytic subunit (Pol) of herpes simplex virus 1 (HSV-1) DNA polymerase has been extensively studied both as a model for other family B DNA polymerases and for its differences from these enzymes as an antiviral target. Among the activities of HSV-1 Pol is an intrinsic RNase H activity that cleaves RNA from RNA-DNA hybrids. There has long been a controversy regarding whether this activity is due to the 3'-to-5' exonuclease of Pol or whether it is a separate activity, possibly acting on 5' RNA termini. To investigate this issue, we compared wild-type HSV-1 Pol and a 3'-to-5' exonuclease-deficient mutant, D368A Pol, for DNA polymerase activity, 3'-to-5' exonuclease activity, and RNase H activity in vitro Additionally, we assessed the RNase H activity using differentially end-labeled templates with 5' or 3' RNA termini. The mutant enzyme was at most modestly impaired for DNA polymerase activity but was drastically impaired for 3'-to-5' exonuclease activity, with no activity detected even at high enzyme-to-DNA substrate ratios. Importantly, the mutant showed no detectable ability to excise RNA with either a 3' or 5' terminus, while the wild-type HSV-1 Pol was able to cleave RNA from the annealed RNA-DNA hairpin template, but only detectably with a 3' RNA terminus in a 3'-to-5' direction and at a rate lower than that of the exonuclease activity. These results suggest that HSV-1 Pol does not have an RNase H separable from its 3'-to-5' exonuclease activity and that this activity prefers DNA degradation over degradation of RNA from RNA-DNA hybrids. IMPORTANCE Herpes simplex virus 1 (HSV-1) is a member of the Herpesviridae family of DNA viruses, several of which cause morbidity and mortality in humans. Although the HSV-1 DNA polymerase has been studied for decades and is a crucial target for antivirals against HSV-1 infection, several of its functions remain to be elucidated. A hypothesis suggesting the existence of a 5'-to-3' RNase H activity intrinsic to this enzyme

High antiviral effect of TiO2·PL–DNA nanocomposites targeted to conservative regions of (−RNA and (+RNA of influenza A virus in cell culture

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Asya S. Levina

2016-08-01

Full Text Available Background: The development of new antiviral drugs based on nucleic acids is under scrutiny. An important problem in this aspect is to find the most vulnerable conservative regions in the viral genome as targets for the action of these agents. Another challenge is the development of an efficient system for their delivery into cells. To solve this problem, we proposed a TiO2·PL–DNA nanocomposite consisting of titanium dioxide nanoparticles and polylysine (PL-containing oligonucleotides.Results: The TiO2·PL–DNA nanocomposites bearing the DNA fragments targeted to different conservative regions of (−RNA and (+RNA of segment 5 of influenza A virus (IAV were studied for their antiviral activity in MDCK cells infected with the H1N1, H5N1, and H3N2 virus subtypes. Within the negative strand of each of the studied strains, the efficiency of DNA fragments increased in the direction of its 3’-end. Thus, the DNA fragment aimed at the 3’-noncoding region of (−RNA was the most efficient and inhibited the reproduction of different IAV subtypes by 3–4 orders of magnitude. Although to a lesser extent, the DNA fragments targeted at the AUG region of (+RNA and the corresponding region of (−RNA were also active. For all studied viral subtypes, the nanocomposites bearing the DNA fragments targeted to (−RNA appeared to be more efficient than those containing fragments aimed at the corresponding (+RNA regions.Conclusion: The proposed TiO2·PL–DNA nanocomposites can be successfully used for highly efficient and site-specific inhibition of influenza A virus of different subtypes. Some patterns of localization of the most vulnerable regions in IAV segment 5 for the action of DNA-based drugs were found. The (−RNA strand of IAV segment 5 appeared to be more sensitive as compared to (+RNA.

Chromosomal organization of the ribosomal RNA genes in the genus Chironomus (Diptera, Chironomidae

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Larisa Gunderina

2015-05-01

Full Text Available Chromosomal localization of ribosomal RNA coding genes has been studied by using FISH (fluorescence in situ hybridization in 21 species from the genus Chironomus Meigen, 1803. Analysis of the data has shown intra- and interspecific variation in number and location of 5.8S rDNA hybridization sites in 17 species from the subgenus Chironomus and 4 species from the subgenus Camptochironomus Kieffer, 1914. In the majority of studied species the location of rDNA sites coincided with the sites where active NORs (nucleolus organizer regions were found. The number of hybridization sites in karyotypes of studied chironomids varied from 1 to 6. More than half of the species possessed only one NOR (12 out of 21. Two rDNA hybridization sites were found in karyotypes of five species, three – in two species, and five and six sites – in one species each. NORs were found in all chromosomal arms of species from the subgenus Chironomus with one of them always located on arm G. On the other hand, no hybridization sites were found on arm G in four studied species from the subgenus Camptochironomus. Two species from the subgenus Chironomus – Ch. balatonicus Devai, Wuelker & Scholl, 1983 and Ch. “annularius” sensu Strenzke, 1959 – showed intraspecific variability in the number of hybridization signals. Possible mechanisms of origin of variability in number and location of rRNA genes in the karyotypes of species from the genus Chironomus are discussed.

Localization of rDNA in small, nucleolus-like structures in human diplotene oocyte nuclei

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Wolgemuth-Jarashow, D.J.; Jagiello, G.M.; Henderson, A.S.

1977-01-01

Small, nucleolus-like structures were demonstrated in the nuclei of human diplotene oocytes. At least some of these bodies were shown to be true micronucleoli by virtue of their ability to bind rRNA during RNA-DNA hybridization in situ.

Analysis of a cDNA clone expressing a human autoimmune antigen: full-length sequence of the U2 small nuclear RNA-associated B antigen

International Nuclear Information System (INIS)

Habets, W.J.; Sillekens, P.T.G.; Hoet, M.H.; Schalken, J.A.; Roebroek, A.J.M.; Leunissen, J.A.M.; Van de Ven, W.J.M.; Van Venrooij, W.J.

1987-01-01

A U2 small nuclear RNA-associated protein, designated B'', was recently identified as the target antigen for autoimmune sera from certain patients with systemic lupus erythematosus and other rheumatic diseases. Such antibodies enabled them to isolate cDNA clone λHB''-1 from a phage λgt11 expression library. This clone appeared to code for the B'' protein as established by in vitro translation of hybrid-selected mRNA. The identity of clone λHB''-1 was further confirmed by partial peptide mapping and analysis of the reactivity of the recombinant antigen with monospecific and monoclonal antibodies. Analysis of the nucleotide sequence of the 1015-base-pair cDNA insert of clone λHB''-1 revealed a large open reading frame of 800 nucleotides containing the coding sequence for a polypeptide of 25,457 daltons. In vitro transcription of the λHB''-1 cDNA insert and subsequent translation resulted in a protein product with the molecular size of the B'' protein. These data demonstrate that clone λHB''-1 contains the complete coding sequence of this antigen. The deduced polypeptide sequence contains three very hydrophilic regions that might constitute RNA binding sites and/or antigenic determinants. These findings might have implications both for the understanding of the pathogenesis of rheumatic diseases as well as for the elucidation of the biological function of autoimmune antigens

Tail-labelling of DNA probes using modified deoxynucleotide triphosphates and terminal deoxynucleotidyl tranferase. Application in electrochemical DNA hybridization and protein-DNA binding assays