Science.gov

Sample records for acid dna analysis

  1. Genomewide expression analysis in amino acid-producing bacteria using DNA microarrays.

    PubMed

    Polen, Tino; Wendisch, Volker F

    2004-01-01

    DNA microarray technology has become an important research tool for biotechnology and microbiology. It is now possible to characterize genetic diversity and gene expression in a genomewide manner. DNA microarrays have been applied extensively to study the biology of many bacteria including Escherichia coli, but only recently have they been developed for the Gram-positive Corynebacterium glutamicum. Both bacteria are widely used for biotechnological amino acid production. In this article, in addition to the design and generation of microarrays as well as their use in hybridization experiments and subsequent data analysis, we describe recent applications of DNA microarray technology regarding amino acid production in C. glutamicum and E. coli. We also discuss the impact of functional genomics studies on fundamental as well as applied aspects of amino acid production with C. glutamicum and E. coli. PMID:15304751

  2. DNA nanotechnology for nucleic acid analysis: DX motif-based sensor.

    PubMed

    Kolpashchikov, Dmitry M; Gerasimova, Yulia V; Khan, Mohammad S

    2011-11-25

    A light on the tiles: A sensor that fluoresces in the presence of specific nucleic acids was designed and characterized. The sensor uses a molecular beacon probe and three adaptor strands to form a five-stranded assembly, a DX-tile, with a specific analyte. This sensor is a highly selective and affordable tool for the real-time analysis of DNA and RNA.

  3. Technical approaches for efficient high-precision nucleic acid analysis using DNA microarrays

    NASA Astrophysics Data System (ADS)

    Pinkel, Daniel; Hamilton, Gregory; Brown, Nils; Segraves, Richard; Huey, Bing; Snijders, Anoine; Blackwood, Stephanie; Hindle, Kate; Law, Sindy; Gray, Joe W.; Jain, Ajay; Hanson, John; Nordmeyer, Robert; Albertson, Donna

    2002-06-01

    Microarray measurements offer the potential to compare the abundances of numerous nucleic acid sequences in parallel. Using linker-adapter PCR products from mapped BAC clones we have made arrays that permit scanning the human genome for single copy gains and losses of DNA sequence, which requires reliable detection of 50 percent changes. The DNA is printed at high concentration on amino-silane or chromium coated surface using a custom-built capillary pin printing system. Spots are printed on 130 micrometers centers or closer to minimize the size of the arrays. Hybridization occurs in a dextran sulfate/formamide buffer at 37 degrees C, using slow rocking to mix the reaction. The entire array is imaged in a single CCD frame using a custom built system that employs mercury arc illumination. Up to four fluorochromes can be imaged from a single array with adequate spectral separation. Typically we use DAPI to stain the DNA in the array spots to facilitate automatic image segmentation during analysis, and fluorescein, Cy3, and Cy5 or their spectral equivalents, for labeling specimen nucleic acids. Array spots are segmented and quantitative fluorescence intensities and intensity ratios are automatically calculated in < 1 minute per approximately 8000 element array using the custom software UCSF SPOT.

  4. Development of an efficient fungal DNA extraction method to be used in random amplified polymorphic DNA-PCR analysis to differentiate cyclopiazonic acid mold producers.

    PubMed

    Sánchez, Beatriz; Rodríguez, Mar; Casado, Eva M; Martín, Alberto; Córdoba, Juan J

    2008-12-01

    A variety of previously established mechanical and chemical treatments to achieve fungal cell lysis combined with a semiautomatic system operated by a vacuum pump were tested to obtain DNA extract to be directly used in randomly amplified polymorphic DNA (RAPD)-PCR to differentiate cyclopiazonic acid-producing and -nonproducing mold strains. A DNA extraction method that includes digestion with proteinase K and lyticase prior to using a mortar and pestle grinding and a semiautomatic vacuum system yielded DNA of high quality in all the fungal strains and species tested, at concentrations ranging from 17 to 89 ng/microl in 150 microl of the final DNA extract. Two microliters of DNA extracted with this method was directly used for RAPD-PCR using primer (GACA)4. Reproducible RAPD fingerprints showing high differences between producer and nonproducer strains were observed. These differences in the RAPD patterns did not differentiate all the strains tested in clusters by cyclopiazonic acid production but may be very useful to distinguish cyclopiazonic acid producer strains from nonproducer strains by a simple RAPD analysis. Thus, the DNA extracts obtained could be used directly without previous purification and quantification for RAPD analysis to differentiate cyclopiazonic acid producer from nonproducer mold strains. This combined analysis could be adaptable to other toxigenic fungal species to enable differentiation of toxigenic and non-toxigenic molds, a procedure of great interest in food safety. PMID:19244904

  5. Development of an efficient fungal DNA extraction method to be used in random amplified polymorphic DNA-PCR analysis to differentiate cyclopiazonic acid mold producers.

    PubMed

    Sánchez, Beatriz; Rodríguez, Mar; Casado, Eva M; Martín, Alberto; Córdoba, Juan J

    2008-12-01

    A variety of previously established mechanical and chemical treatments to achieve fungal cell lysis combined with a semiautomatic system operated by a vacuum pump were tested to obtain DNA extract to be directly used in randomly amplified polymorphic DNA (RAPD)-PCR to differentiate cyclopiazonic acid-producing and -nonproducing mold strains. A DNA extraction method that includes digestion with proteinase K and lyticase prior to using a mortar and pestle grinding and a semiautomatic vacuum system yielded DNA of high quality in all the fungal strains and species tested, at concentrations ranging from 17 to 89 ng/microl in 150 microl of the final DNA extract. Two microliters of DNA extracted with this method was directly used for RAPD-PCR using primer (GACA)4. Reproducible RAPD fingerprints showing high differences between producer and nonproducer strains were observed. These differences in the RAPD patterns did not differentiate all the strains tested in clusters by cyclopiazonic acid production but may be very useful to distinguish cyclopiazonic acid producer strains from nonproducer strains by a simple RAPD analysis. Thus, the DNA extracts obtained could be used directly without previous purification and quantification for RAPD analysis to differentiate cyclopiazonic acid producer from nonproducer mold strains. This combined analysis could be adaptable to other toxigenic fungal species to enable differentiation of toxigenic and non-toxigenic molds, a procedure of great interest in food safety.

  6. Determination of DNA adducts by combining acid-catalyzed hydrolysis and chromatographic analysis of the carcinogen-modified nucleobases.

    PubMed

    Leung, Elvis M K; Deng, Kailin; Wong, Tin-Yan; Chan, Wan

    2016-01-01

    The commonly used method of analyzing carcinogen-induced DNA adducts involves the hydrolysis of carcinogen-modified DNA samples by using a mixture of enzymes, followed by (32)P-postlabeling or liquid chromatography (LC)-based analyses of carcinogen-modified mononucleotides/nucleosides. In the present study, we report the development and application of a new approach to DNA adduct analysis by combining the H(+)/heat-catalyzed release of carcinogen-modified nucleobases and the use of LC-based methods to analyze DNA adducts. Results showed that heating the carcinogen-modified DNA samples at 70 °C for an extended period of 4 to 6 h in the presence of 0.05% HCl can efficiently induce DNA depurination, releasing the intact carcinogen-modified nucleobases for LC analyses. After optimizing the hydrolysis conditions, DNA samples with C8- and N (2) -modified 2'-deoxyguanosine, as well as N (6) -modified 2'-deoxyadenosine, were synthesized by reacting DNA with 1-nitropyrene, acetaldehyde, and aristolochic acids, respectively. These samples were then hydrolyzed, and the released nucleobase adducts were analyzed using LC-based analytical methods. Analysis results demonstrated a dose-dependent release of target DNA adducts from carcinogen-modified DNA samples, indicating that the developed H(+)/heat-catalyzed hydrolysis method was quantitative. Comparative studies with enzymatic digestion method on carcinogen-modified DNA samples revealed that the two hydrolysis methods did not yield systematically different results.

  7. In silico comparative analysis of DNA and amino acid sequences for prion protein gene.

    PubMed

    Kim, Y; Lee, J; Lee, C

    2008-01-01

    Genetic variability might contribute to species specificity of prion diseases in various organisms. In this study, structures of the prion protein gene (PRNP) and its amino acids were compared among species of which sequence data were available. Comparisons of PRNP DNA sequences among 12 species including human, chimpanzee, monkey, bovine, ovine, dog, mouse, rat, wallaby, opossum, chicken and zebrafish allowed us to identify candidate regulatory regions in intron 1 and 3'-untranslated region (UTR) in addition to the coding region. Highly conserved putative binding sites for transcription factors, such as heat shock factor 2 (HSF2) and myocite enhancer factor 2 (MEF2), were discovered in the intron 1. In 3'-UTR, the functional sequence (ATTAAA) for nucleus-specific polyadenylation was found in all the analysed species. The functional sequence (TTTTTAT) for maturation-specific polyadenylation was identically observed only in ovine, and one or two nucleotide mismatches in the other species. A comparison of the amino acid sequences in 53 species revealed a large sequence identity. Especially the octapeptide repeat region was observed in all the species but frog and zebrafish. Functional changes and susceptibility to prion diseases with various isoforms of prion protein could be caused by numeric variability and conformational changes discovered in the repeat sequences.

  8. Phylogenetic Diversity of Lactic Acid Bacteria Associated with Paddy Rice Silage as Determined by 16S Ribosomal DNA Analysis

    PubMed Central

    Ennahar, Saïd; Cai, Yimin; Fujita, Yasuhito

    2003-01-01

    A total of 161 low-G+C-content gram-positive bacteria isolated from whole-crop paddy rice silage were classified and subjected to phenotypic and genetic analyses. Based on morphological and biochemical characters, these presumptive lactic acid bacterium (LAB) isolates were divided into 10 groups that included members of the genera Enterococcus, Lactobacillus, Lactococcus, Leuconostoc, Pediococcus, and Weissella. Analysis of the 16S ribosomal DNA (rDNA) was used to confirm the presence of the predominant groups indicated by phenotypic analysis and to determine the phylogenetic affiliation of representative strains. The virtually complete 16S rRNA gene was PCR amplified and sequenced. The sequences from the various LAB isolates showed high degrees of similarity to those of the GenBank reference strains (between 98.7 and 99.8%). Phylogenetic trees based on the 16S rDNA sequence displayed high consistency, with nodes supported by high bootstrap values. With the exception of one species, the genetic data was in agreement with the phenotypic identification. The prevalent LAB, predominantly homofermentative (66%), consisted of Lactobacillus plantarum (24%), Lactococcus lactis (22%), Leuconostoc pseudomesenteroides (20%), Pediococcus acidilactici (11%), Lactobacillus brevis (11%), Enterococcus faecalis (7%), Weissella kimchii (3%), and Pediococcus pentosaceus (2%). The present study, the first to fully document rice-associated LAB, showed a very diverse community of LAB with a relatively high number of species involved in the fermentation process of paddy rice silage. The comprehensive 16S rDNA-based approach to describing LAB community structure was valuable in revealing the large diversity of bacteria inhabiting paddy rice silage and enabling the future design of appropriate inoculants aimed at improving its fermentation quality. PMID:12514026

  9. [Classification of amino acids based on a comparative analysis of contacts in DNA-protein complexes and specific DNA-protein interactions].

    PubMed

    Anashkina, A A; Kuznetsov, E N; Batianovskiĭ, A V; Gnuchev, N V; Tumanian, V G; Esipova, N G

    2013-01-01

    The classification of amino acid residues based on the events of contact formation between distinct amino acid and selected nucleotides was constructed. Thus, the most integral properties, that characterize interactions in organization of DNA-protein complexes, were used. We applied the Voronoi-Delaunay tessellation to draw statistics of contacts and area of contacts for the set included 1937 DNA-protein complexes. Similarities of amino acid residues have been searched for based on the comparison of corresponded rows and matrixes of contacts and areas of contacts. Nine measures of distance were used for estimation of rows similarity degree. The procedure of clustering amino acids in groups included three hierarchical and two nonhierarchical methods. A total tree was built using nine techniques of estimating distance with three hierarchical clustering methods. It was shown that clustering centers in the main groups are always constant while other relationships between objects vary. Clustering of binary associations was found for the most amino acids. Major classes of up to six amino acids correspond to the certain local structures of the polypeptide chain in the context of amino acid composition. These data should be taken into account when designing DNA-protein ligands. PMID:25486755

  10. [Classification of amino acids based on a comparative analysis of contacts in DNA-protein complexes and specific DNA-protein interactions].

    PubMed

    2013-01-01

    The classification of amino acid residues based on the events of contact formation between distinct amino acid and selected nucleotides was constructed. Thus, the most integral properties, that characterize interactions in organization of DNA-protein complexes, were used. We applied the Voronoi-Delaunay tessellation to draw statistics of contacts and area of contacts for the set included 1937 DNA-protein complexes. Similarities of amino acid residues have been searched for based on the comparison of corresponded rows and matrixes of contacts and areas of contacts. Nine measures of distance were used for estimation of rows similarity degree. The procedure of clustering amino acids in groups included three hierarchical and two nonhierarchical methods. A total tree was built using nine techniques of estimating distance with three hierarchical clustering methods. It was shown that clustering centers in the main groups are always constant while other relationships between objects vary. Clustering of binary associations was found for the most amino acids. Major classes of up to six amino acids correspond to the certain local structures of the polypeptide chain in the context of amino acid composition. These data should be taken into account when designing DNA-protein ligands. PMID:25508886

  11. Molecular cloning and sequence analysis of complementary DNA encoding rat mammary gland medium-chain S-acyl fatty acid synthetase thio ester hydrolase

    SciTech Connect

    Safford, R.; de Silva, J.; Lucas, C.; Windust, J.H.C.; Shedden, J.; James, C.M.; Sidebottom, C.M.; Slabas, A.R.; Tombs, M.P.; Hughes, S.G.

    1987-03-10

    Poly(A) + RNA from pregnant rat mammary glands was size-fractionated by sucrose gradient centrifugation, and fractions enriched in medium-chain S-acyl fatty acid synthetase thio ester hydrolase (MCH) were identified by in vitro translation and immunoprecipitation. A cDNA library was constructed, in pBR322, from enriched poly(A) + RNA and screened with two oligonucleotide probes deduced from rat MCH amino acid sequence data. Cross-hybridizing clones were isolated and found to contain cDNA inserts ranging from approx. 1100 to 1550 base pairs (bp). A 1550-bp cDNA insert, from clone 43H09, was confirmed to encode MCH by hybrid-select translation/immunoprecipitation studies and by comparison of the amino acid sequence deduced from the DNA sequence of the clone to the amino acid sequence of the MCH peptides. Northern blot analysis revealed the size of the MCH mRNA to be 1500 nucleotides, and it is therefore concluded that the 1550-bp insert (including G x C tails) of clone 43H09 represents a full- or near-full-length copy of the MCH gene. The rat MCH sequence is the first reported sequence of a thioesterase from a mammalian source, but comparison of the deduced amino acid sequences of MCH and the recently published mallard duck medium-chain S-acyl fatty acid synthetase thioesterase reveals significant homology. In particular, a seven amino acid sequence containing the proposed active serine of the duck thioesterase is found to be perfectly conserved in rat MCH.

  12. Folic acid binds DNA and RNA at different locations.

    PubMed

    Bourassa, P; Tajmir-Riahi, H A

    2015-03-01

    We located multiple binding sites for folic acid on DNA and tRNA at physiological conditions, using FTIR, CD, fluorescence spectroscopic methods and molecular modeling. Structural analysis revealed that folic acid binds DNA and tRNA at multiple sites via hydrophilic, hydrophobic and H-bonding contacts with overall binding constants of Kfolic acid-DNA=1.1 (±0.3)×10(4) M(-1) and Kfolic acid-tRNA=6.4 (±0.5)×10(3) M(-1). Molecular modeling showed the participation of several nucleobases in folic acid complexes with DNA and tRNA, stabilized by H-bonding network. Two types of complexes were located for folic acid-tRNA adducts, one at the major groove and the other with TΨC loop, while acid binding occurs at major and minor grooves of DNA duplex. Folic acid complexation induced more alterations of DNA structure than tRNA.

  13. Image analysis for DNA sequencing

    NASA Astrophysics Data System (ADS)

    Palaniappan, Kannappan; Huang, Thomas S.

    1991-07-01

    There is a great deal of interest in automating the process of DNA (deoxyribonucleic acid) sequencing to support the analysis of genomic DNA such as the Human and Mouse Genome projects. In one class of gel-based sequencing protocols autoradiograph images are generated in the final step and usually require manual interpretation to reconstruct the DNA sequence represented by the image. The need to handle a large volume of sequence information necessitates automation of the manual autoradiograph reading step through image analysis in order to reduce the length of time required to obtain sequence data and reduce transcription errors. Various adaptive image enhancement, segmentation and alignment methods were applied to autoradiograph images. The methods are adaptive to the local characteristics of the image such as noise, background signal, or presence of edges. Once the two-dimensional data is converted to a set of aligned one-dimensional profiles waveform analysis is used to determine the location of each band which represents one nucleotide in the sequence. Different classification strategies including a rule-based approach are investigated to map the profile signals, augmented with the original two-dimensional image data as necessary, to textual DNA sequence information.

  14. Genotyping of velvet antlers for identification of country of origin using mitochondrial DNA and fluorescence melting curve analysis with locked nucleic acid probes.

    PubMed

    Ahn, Jeong Jin; Kim, Youngjoo; Hong, Ji Young; Kim, Gi Won; Hwang, Seung Yong

    2016-07-01

    Velvet antlers are used medicinally in Asia and possess various therapeutic effects. Prices are set according to the country of origin, which is unidentifiable to the naked eye, and therefore counterfeiting is prevalent. Additionally, antlers of the Canadian elk, which can generate chronic wasting disease, are prevalently smuggled and distributed in the market. Thus, a method for identifying the country of origin of velvet antlers was developed, using polymorphisms in mitochondrial DNA, fluorescence melting curve analysis and analysis of locked nucleic acids (LNA). This combined method is capable of identifying five genotypes of velvet antlers in a single experiment using two probes. It also has advantages in multiplexing, simplicity and efficiency in genotyping, when compared to real-time PCR or microarrays. The developed method can be used to improve identification rates in the velvet antler market and, by extension, research based on polymorphisms in DNA sequences.

  15. Femtosecond spectroscopic study of carminic acid DNA interactions

    NASA Astrophysics Data System (ADS)

    Comanici, Radu; Gabel, Bianca; Gustavsson, Thomas; Markovitsi, Dimitra; Cornaggia, Christian; Pommeret, Stanislas; Rusu, Catalin; Kryschi, Carola

    2006-06-01

    Photo-excited carminic acid and carminic acid-DNA complexes in a buffer solution at pH 7 have been examined using a variety of spectroscopy techniques, that are in particular, the femtosecond resolved fluorescence upconversion and transient absorption spectroscopy. The observation of dual fluorescence emission, one peaks at 470 nm and the other at 570 nm, indicates to an excited-state (S 1) intramolecular proton transfer (ESIPT). A detailed analysis of the transient absorption measurements of an aqueous carminic-acid solution at pH 7 yielded four lifetimes for the excited-state (S 1): 8, 15, 33 and 46 ps. On the other hand, only two lifetimes, 34 and 47 ps, were observed by fluorescence upconversion spectroscopy because of the detection limitation to the long wavelength edge of the carminic-acid spectrum. The four S 1 lifetimes were ascribed to the coexistence of respectively two tautomer (normal and tautomer) forms of carminic acid, in the non-dissociated state (CAH) and in the deprotonated state (CA -). The fluorescence upconversion measurements of carminic acid-DNA complexes exhibited a prolongation of the fluorescence lifetimes. This effect was accepted as evidence for the formation of intercalation complexes between the carminic acid and the DNA. The intercalative binding of the carminic acid to DNA was confirmed by the fluorescence titration experiments resulting to a binding constant of 2 × 10 5 M -1 that is typical for anthracycline-DNA complexes.

  16. Optimizing Phytoplasma DNA purification for genome analysis.

    PubMed

    Tran-Nguyen, L T T; Gibb, K S

    2007-04-01

    Genome analysis of uncultivable plant pathogenic phytoplasmas is hindered by the difficulty in obtaining sufficient quantities of phytoplasma enriched DNA. We investigated a combination of conventional enrichment techniques such as cesium chloride (CsCl) buoyant gradient centrifugation, and new methods such as rolling circle amplification (RCA), suppression subtractive hybridization (SSH), and mirror orientation selection (MOS) to obtain DNA with a high phytoplasma:host ratio as the major first step in genome analysis of Candidatus Phytoplasma australiense. The phytoplasma:host ratio was calculated for five different plasmid libraries. Based on sequence data, 90% of clones from CsCl DNA enrichment contained chromosomal phytoplasma DNA, compared to 60% from RCA CsCl DNA and 20% from SSH subtracted libraries. Based on an analysis of representative libraries, none contained plant DNA. A high percentage of clones (80-100%) from SSH libraries contained extrachromosomal DNA (eDNA), and we speculate that eDNA in the original DNA preparation was amplified in subsequent SSH manipulations. Despite the availability of new techniques for nucleic acid amplification, we found that conventional CsCl gradient centrifugation was the best enrichment method for obtaining chromosomal phytoplasma DNA with low host DNA content.

  17. Analysis of several methods for the extraction of high quality DNA from acetic acid bacteria in wine and vinegar for characterization by PCR-based methods.

    PubMed

    Jara, C; Mateo, E; Guillamón, J M; Torija, M J; Mas, A

    2008-12-10

    Acetic acid bacteria (AAB) are fastidious microorganisms with poor recovery in culture. Culture-independent methods are currently under examination. Good DNA extraction is a strict requirement of these methods. We compared five methods for extracting the DNA of AAB directly from wine and vinegar samples. Four matrices (white wine, red wine, superficial vinegar and submerged vinegar) contaminated with two AAB strains belonging to Acetobacter pasteurianus and Gluconacetobacter hansenii were assayed. To improve the yield and quality of the extracted DNA, a sample treatment (washing with polyvinyl pyrrolidone or NaCl) was also tested. DNA quality was measured by amplification of the 16S rRNA gene with conventional PCR. DNA recovery rate was assessed by real-time PCR. DNA amplification was always successful with the Wizard method though DNA recovery was poor. A CTAB-based method and NucleoSpin protocol extracted the highest DNA recoveries from wine and vinegar samples. Both of these methods require treatment to recover suitable DNA for amplification with maximum recovery. Both may therefore be good solutions for DNA extraction in wine and vinegar samples. DNA extraction of Ga hansenii was more effective than that of A. pasteurianus. The fastest and cheapest method we evaluated (the Thermal shock protocol) produced the worst results both for DNA amplification and DNA recovery.

  18. Molecular cloning and analysis of a pea cDNA that is expressed in darkness and very rapidly induced by gibberellic acid.

    PubMed

    Li, H Y; Guo, Z F; Zhu, Y X

    1998-09-01

    Using cDNA representational difference analysis (cDNA RDA), we isolated a cDNA named GDA-1 from a cDNA library constructed with mRNA from short-day (SD) grown G2 pea apical tissue. The amino acid sequence deduced from GDA-1 shares partial identity with the B2 protein which is expressed during embryogenesis of carrot cells. Northern analysis showed that GDA-1 mRNA is abundant in SD-grown G2 pea apical buds. In long-day (LD) conditions, there was almost no detectable GDA-1 mRNA. When LD-grown G2 peas were kept in continuous darkness for 24 h, the GDA-1 mRNA content reached a level equivalent to about 50% of that in the SD samples. On the other hand, when SD-grown peas were transferred into the light for 24 h, the amount of hybridizable GDA-1 mRNA dropped to the same as that of LD-grown plants. GDA-1 expression was found to be independent of flower initiation time. GA3 application in vitro resulted in rapid accumulation of GDA-1 mRNA in LD-grown G2 pea apical buds, which is compatible with its delaying effect on apical senescence. Time-course experiments revealed that GDA-1 is induced within 15 min of GA3 application. Exogenous GA3 did not influence the expression of GDA-1 in SD-grown G2 peas. Since both photoperiod and GA induce the expression of GDA-1, we speculate that they may activate similar signal transduction pathways in G2 peas. Our work also shows that photoperiod may change the efficiency of gibberellin perception by plants. PMID:9790595

  19. Complexing of amino acids to DNA by chromate in intact cells.

    PubMed Central

    Voitkun, V; Zhitkovich, A; Costa, M

    1994-01-01

    Using o-pthaldialdehyde (OPT) fluorescence, the amino acids associated with DNA were studied following exposure of intact Chinese hamster ovary cells to chromate. Rigorous extraction with EDTA, acid, or base was required to release the amino acids cross-linked to the DNA isolated from control or chromate-treated cells by standard procedures (i.e., proteinase K, phenol, etc.). Amino acids resisting extraction from DNA were not studied since analysis was limited to those that could be released by these procedures. There was a chromate dose-dependent increase in amino acids complexed with the DNA that could be released by EDTA, acid, and base, and these amino acids were separated by HPLC and identified. Substantial increases in cysteine, glutamine, glutamic acid, histidine, threonine, and tyrosine were found as a function of increasing concentrations of chromate. There was also a time-dependent increase in complexing of these amino acids to the DNA by chromate. The amino acids found complexed to DNA in intact cells by chromate were thought to originate from reactions of free amino acids or small peptides with the DNA rather than being proteolytic products derived from larger proteins that were cross-linked to the DNA. This was supported by a number of experiments: a) free amino acids or bovine serum albumin (BSA) were cross-linked by chromium to DNA in vitro and the DNA was isolated by standard procedures.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7843108

  20. Identification of PCR-amplified genetically modified organisms (GMOs) DNA by peptide nucleic acid (PNA) probes in anion-exchange chromatographic analysis.

    PubMed

    Rossi, Stefano; Lesignoli, Francesca; Germini, Andrea; Faccini, Andrea; Sforza, Stefano; Corradini, Roberto; Marchelli, Rosangela

    2007-04-01

    PCR products obtained by selective amplification of transgenic DNA derived from food samples containing Roundup Ready soybean or Bt-176 maize have been analyzed by anion-exchange HPLC. Peptide nucleic acids (PNAs), oligonucleotide analogues known to bind to complementary single-stranded DNA with high affinity and specificity, have been used as specific probes in order to assess the identity of the peaks observed. Two different protocols were adopted in order to obtain single-stranded DNA: amplification with an excess of one primer or digestion of one DNA strand. The single-stranded DNA was mixed with the PNA probe, and the presence of a specific sequence was revealed through detection of the corresponding PNA:DNA peak with significantly different retention time. Advantages and limits of this approach are discussed. The method was tested with reference materials and subsequently applied to commercial samples.

  1. DNA analysis in human disease.

    PubMed Central

    Wright, A F

    1986-01-01

    The analysis of human DNA using recombinant DNA technology is fast becoming an integral part of the diagnosis, assessment, and prevention of inherited and somatic genetic disease. The rationale underlying these methods of analysis is discussed, and the nature and extent of mutational change in heritable disorders and neoplastic development is outlined. PMID:3543064

  2. A Column Experiment To Determine Black Shale Degradation And Colonization By Means of δ13C and 14C Analysis Of Phospholipid Fatty Acids And DNA Extraction

    NASA Astrophysics Data System (ADS)

    Seifert, A.; Gleixner, G.

    2008-12-01

    We investigated the degradation of black shale organic matter by microbial communities. We inoculated two columns respectively, with the fungi Schizophyllum commune, the gram-positive bacterium Pseudomonas putida and the gram-negative bacteria Streptomyces griseus and Streptomyces chartreusis. These microorganisms are known to degrade a wide variety of organic macromolecules. Additionally, we had two sets of control columns. To one set the same nutrient solution was added as to the inoculated columns and to the other set only sterile deionised water was supplied. All columns contained 1.5 kg of freshly crushed not autoclaved black shale material with a particle size of 0.63-2 mm. The columns were incubated at 28° C and 60% humidity in the dark. The aim was to investigate, which microorganisms live on black shales and if these microorganisms are able to degrade ancient organic matter. We used compound specific stable isotope measurement techniques and compound specific 14C-dating methods. After 183 days PLFAs were extracted from the columns to investigate the microbial community, furthermore we extracted on one hand total-DNA of column material and on the other hand DNA from pure cultures isolates which grew on Kinks-agar B, Starch-casein-nitrate-agar (SCN) and on complete-yeast-medium-agar (CYM). According to the PLFA analysis bacteria dominated in the columns, whereas in pure cultures more fungi were isolated. A principal component analysis revealed differences between the columns in accordance with the inoculation, but it seems that the inoculated microorganisms were replaced by the natural population. For AMS measurements palmitic acid (C 16:0) was re-isolated from total-PLFA-extract with a preparative fraction collector (PFC). Preliminary results of the study revealed that microorganisms are able to degrade black shale material and that PLFA analysis are useful methods to be combined with analysis of stable isotope and 14C measurements to study microbial

  3. Multiplex analysis of DNA

    DOEpatents

    Church, George M.; Kieffer-Higgins, Stephen

    1992-01-01

    This invention features vectors and a method for sequencing DNA. The method includes the steps of: a) ligating the DNA into a vector comprising a tag sequence, the tag sequence includes at least 15 bases, wherein the tag sequence will not hybridize to the DNA under stringent hybridization conditions and is unique in the vector, to form a hybrid vector, b) treating the hybrid vector in a plurality of vessels to produce fragments comprising the tag sequence, wherein the fragments differ in length and terminate at a fixed known base or bases, wherein the fixed known base or bases differs in each vessel, c) separating the fragments from each vessel according to their size, d) hybridizing the fragments with an oligonucleotide able to hybridize specifically with the tag sequence, and e) detecting the pattern of hybridization of the tag sequence, wherein the pattern reflects the nucleotide sequence of the DNA.

  4. FBI's DNA analysis program

    NASA Astrophysics Data System (ADS)

    Brown, John R.

    1994-03-01

    Forensic DNA profiling technology is a significant law enforcement tool due to its superior discriminating power. Applying the principles of population genetics to the DNA profile obtained in violent crime investigations results in low frequency of occurrence estimates for the DNA profile. These estimates often range from a frequency of occurrence of 1 in 50 unrelated individuals to 1 in a million unrelated individuals or even smaller. It is this power to discriminate among individuals in the population that has propelled forensic DNA technology to the forefront of forensic testing in violent crime cases. Not only is the technology extremely powerful in including or excluding a criminal suspect as the perpetrator, but it also gives rise to the potential of identifying criminal suspects in cases where the investigators of unknown suspect cases have exhausted all other available leads.

  5. Amino Acid Racemization and the Preservation of Ancient DNA

    NASA Technical Reports Server (NTRS)

    Poinar, Hendrik N.; Hoss, Matthias

    1996-01-01

    The extent of racemization of aspartic acid, alanine, and leucine provides criteria for assessing whether ancient tissue samples contain endogenous DNA. In samples in which the D/L ratio of aspartic acid exceeds 0.08, ancient DNA sequences could not be retrieved. Paleontological finds from which DNA sequences purportedly millions of years old have been reported show extensive racemization, and the amino acids present are mainly contaminates. An exception is the amino acids in some insects preserved in amber.

  6. Amino acid analysis

    NASA Technical Reports Server (NTRS)

    Winitz, M.; Graff, J. (Inventor)

    1974-01-01

    The process and apparatus for qualitative and quantitative analysis of the amino acid content of a biological sample are presented. The sample is deposited on a cation exchange resin and then is washed with suitable solvents. The amino acids and various cations and organic material with a basic function remain on the resin. The resin is eluted with an acid eluant, and the eluate containing the amino acids is transferred to a reaction vessel where the eluant is removed. Final analysis of the purified acylated amino acid esters is accomplished by gas-liquid chromatographic techniques.

  7. DNA microarray analysis reveals a role for lysophosphatidic acid in the regulation of anti-inflammatory genes in MC3T3-E1 cells

    SciTech Connect

    Waters, Katrina M.; Tan, Ruimin; Genetos, Damian C.; Verma, Seema; Yellowley, Clare E.; Karin, Norm J.

    2007-11-01

    DNA microarray analysis revealed that treatment of bone cells with a lipid growth factor led to extensive changes in gene expression. Particular relevance to fracture healing and inflammation was revealed.

  8. Microfabricated integrated DNA analysis systems

    SciTech Connect

    Woolley, A.T.; Mathies, R.A.; Northrup, M.A.

    1996-12-31

    Microfabrication has the potential to revolutionize chemical analysis, from reactions to separations to molecular biotechnology. Microfabricated devices allow high speed separations, automated sample handling, and the study of reactions in the pl to {mu}l volume range. Our research has focused on microfabricated integrated DNA analysis systems (MIDAS). As a first step, we have demonstrated high-speed DNA restriction fragment sizing, and DNA sequencing on microfabricated capillary electrophoresis (CE) chips. We have recently coupled microfabricated PCR reactors and CE chips to make integrated DNA analysis devices. With these devices, rapid PCR can be performed and the reaction products immediately analyzed on the CE chip, eliminating the need for manual transfer of the amplified sample. PCR amplifications have been done in less than 16 minutes, followed by CE analysis in under 100 seconds. These PCR-CE chips represent an important step towards completely integrated sample manipulation on microfabricated devices.

  9. Analysis of DNA strand breaks induced in rodent liver in vivo, hepatocytes in primary culture, and a human cell line by chlorinated acetic acids and chlorinated acetaldehydes

    SciTech Connect

    Chang, L.W.; Daniel, F.B. ); DeAngelo, A.B. )

    1992-01-01

    An alkaline unwinding assay was used to quantitate the induction of DNA strand breaks (DNA SB) in the livers of rats and mice treated in vivo, in rodent hepatocytes in primary culture, and in CCRF-CEM cells, a human lymphoblastic leukemia cell line, following treatment with tri-(TCA), di-(CA), and mono-(MCA) chloroacetic acid and their corresponding aldehydes, tri-(chloralhydrate, CH), di(DCAA) and mono-(CAA) chloroacetaldehyde. None of the chloracetic acids induced DNA SB in the livers of rats at 4 hr following a single administration of 1-10 mmole/kg. TCA (10 mmole/kg) and DCA (5 and 10 mmole/kg) did produce a small amount of strand breakage in mice (7% at 4hr) but not at 1 hr. N-nitrosodiethylamine (DENA), an established alkylating agent and a rodent hepatocarcinogen, produced DNA SB in the livers of both species. TCA, DCA, and MCA also failed to induce DNA strand breaks in splenocytes and epithelial cells derived from the stomach and duodenum of mice treated in vivo. None of the three chloroacetaldehydes induced DNA SB in either mouse or rat liver. These studies provide further evidence that the chloroacetic acids lack genotoxic activity not only in rodent liver, a tissue in that they induce tumors, but in a variety of other rodent tissues and cultured cell types. Two of the chloroacetaldehydes, DCAA and CAA, are direct acting DNA damaging agents in CCRF-CEM cells, but not in liver or splenocytes in vivo or in cultured hepatocytes. CH showed no activity in any system investigated. 58 refs., 6 figs., 2 tabs.

  10. Genetic and epigenetic transgenerational implications related to omega-3 fatty acids. Part I: maternal FADS2 genotype and DNA methylation correlate with polyunsaturated fatty acid status in toddlers: an exploratory analysis.

    PubMed

    Lupu, Daniel S; Cheatham, Carol L; Corbin, Karen D; Niculescu, Mihai D

    2015-11-01

    Polyunsaturated fatty acid metabolism in toddlers is regulated by a complex network of interacting factors. The contribution of maternal genetic and epigenetic makeup to this milieu is not well understood. In a cohort of mothers and toddlers 16 months of age (n = 65 mother-child pairs), we investigated the association between maternal genetic and epigenetic fatty acid desaturase 2 (FADS2) profiles and toddlers' n-6 and n-3 fatty acid metabolism. FADS2 rs174575 variation and DNA methylation status were interrogated in mothers and toddlers, as well as food intake and plasma fatty acid concentrations in toddlers. A multivariate fit model indicated that maternal rs174575 genotype, combined with DNA methylation, can predict α-linolenic acid plasma concentration in all toddlers and arachidonic acid concentrations in boys. Arachidonic acid intake was predictive for its plasma concentration in girls, whereas intake of 3 major n-3 species (eicosapentaenoic, docosapentaenoic, and docosahexaenoic acids) were predictive for their plasma concentrations in boys. FADS2 genotype and DNA methylation in toddlers were not related to plasma concentrations or food intakes, except for CpG8 methylation. Maternal FADS2 methylation was a predictor for the boys' α-linolenic acid intakes. This exploratory study suggests that maternal FADS2 genetic and epigenetic status could be related to toddlers' polyunsaturated fatty acid metabolism.

  11. Nitrous acid induced damage in T7 DNA and phage

    SciTech Connect

    Scearce, L.M.; Masker, W.E.

    1986-05-01

    The response of bacteriophage T7 to nitrous acid damage was investigated. The T7 system allows in vitro mimicry of most aspects of in vivo DNA metabolism. Nitrous acid is of special interest since it has been previously shown to induce deletions and point mutations as well as novel adducts in DNA. T7 phage was exposed to 56 mM nitrous acid at pH 4.6 in vivo, causing a time dependent 98% decrease in survival for each 10 min duration of exposure to nitrous acid. These studies were extended to include examination of pure T7 DNA exposed in vitro to nitrous acid conditions identical to those used in the in vivo survival studies. The treated DNA was dialyzed to remove the nitrous acid and the DNA was encapsulated into empty phage heads. These in vitro packaged phage showed a survival curve analogous to the in vivo system. There was no change in survival when either in vitro or in vivo exposed phage were grown on wild type E. coli or on E. coli strains deficient in DNA repair due to mutations in DNA polymerase I, exonuclease III or a uvrA mutation. Survival was not increased when nitrous acid treated T7 were grown on E. coli induced for SOS repair. In vitro replication of nitrous acid treated DNA showed a time dependent decrease in the total amount of DNA synthesized.

  12. The interaction of amino acids, peptides, and proteins with DNA.

    PubMed

    Solovyev, Andrey Y; Tarnovskaya, Svetlana I; Chernova, Irina A; Shataeva, Larisa K; Skorik, Yury A

    2015-01-01

    Amino acids that carry charges on their side groups can bind to double stranded DNA (dsDNA) and change the strength of the double helix. Measurement of the DNA melting temperature (Tm) confirmed that acidic amino acids (Glu, Asp) weaken the H-bonds between DNA strands, whereas basic amino acids (Arg, Lys) strengthen the interaction between the strands. A rank correlation exists between the amino acid isoelectric points and the observed changes in Tm. A similar dependence of the hyperchromic effect on the isoelectric point of a protein (pepsin, insulin, cortexin, and protamine) was observed for DNA-protein complexes at room temperature. Short peptides (KE, AEDG, and KEDP) containing a mixture of acidic and basic amino acid residues also affect Tm and the stability of the double helix. A model for binding Glu and Lys to dsDNA was explored by a docking simulation. The model shows that Glu, in an untwisted shape, binds to dsDNA in its major groove and disrupts three H-bonds between the strands, thereby destabilizing the double helix. Lys, in an untwisted shape, binds to the external side of the dsDNA and forms two bonds with O atoms of neighboring phosphodiester groups, thereby strengthening the DNA helix.

  13. Human acidic ribosomal phosphoproteins P0, P1, and P2: Analysis of cDNA clones, in vitro synthesis, and assembly

    SciTech Connect

    Rich, B.E.; Steitz, J.A.

    1987-11-01

    cDNA clones encoding three antigenically related human ribosomal phosophoproteins (P-proteins) P0, P1, and P2 were isolated and sequenced. P1 and P2 are analogous to Escherichia coli ribosomal protein L7/L12, and P0 is likely to be an analog of L10. The three proteins have a nearly identical carboxy-terminal 17-amino-acid sequence (KEESEESD(D/E)DMGFGLFD-COOH) that is the basis of their immunological cross-reactivity. The identifies of the P1 and P2 cDNAs were confirmed by the strong similarities of their encoded amino acid sequences to published primary structures of the homologous rat, brine shrimp, and Saccharomyces cerevisiae proteins. The P0 cDNA was initially identified by translation of hybrid-selected mRNA and immunoprecipitation of the products. To demonstrate that the coding sequences are full length, the P0, P1, and P2 cDNAs were transcribed in vitro by bacteriophage T7 RNA polymerase and the resulting mRNAs were translated in vitro. The synthetic P0, P1, and P2 proteins were serologically and electrophoretically identical to P-proteins extracted from HeLa cells. These synthetic P-proteins were incorporated into 60S but not 40S ribosomes and also assembled into a complex similar to that described for E. coli L7/L12 and L10.

  14. 3DNA: a versatile, integrated software system for the analysis, rebuilding, and visualization of three-dimensional nucleic-acid structures

    PubMed Central

    Lu, Xiang-Jun; Olson, Wilma K.

    2010-01-01

    We present a set of protocols showing how to use the 3DNA suite of programs to analyze, rebuild, and visualize three-dimensional nucleic-acid structures. The software determines a wide range of conformational parameters, including the identities and rigid-body parameters of interacting bases and base-pair steps, the nucleotides comprising helical fragments, the area of overlap of stacked bases, etc. The reconstruction of three-dimensional structure takes advantage of rigorously defined rigid-body parameters, producing rectangular block representations of the nucleic-acid bases and base pairs and all-atom models with approximate sugar-phosphate backbones. The visualization components create vector-based drawings and scenes that can be rendered as raster-graphics images, allowing for easy generation of publication-quality figures. The utility programs use geometric variables to control the view and scale of an object, for comparison of related structures. The commands run in seconds even for large structures. The software and related information are available at http://3dna.rutgers.edu/. PMID:18600227

  15. Antioxidant and DNA damage protection potentials of selected phenolic acids.

    PubMed

    Sevgi, Kemal; Tepe, Bektas; Sarikurkcu, Cengiz

    2015-03-01

    In this study, ten different phenolic acids (caffeic, chlorogenic, cinnamic, ferulic, gallic, p-hydroxybenzoic, protocatechuic, rosmarinic, syringic, and vanillic acids) were evaluated for their antioxidant and DNA damage protection potentials. Antioxidant activity was evaluated by using four different test systems named as β-carotene bleaching, DPPH free radical scavenging, reducing power and chelating effect. In all test systems, rosmarinic acid showed the maximum activity potential, while protocatechuic acid was determined as the weakest antioxidant in β-carotene bleaching, DPPH free radical scavenging, and chelating effect assays. Phenolic acids were also screened for their protective effects on pBR322 plasmid DNA against the mutagenic and toxic effects of UV and H2O2. Ferulic acid was found as the most active phytochemical among the others. Even at the lowest concentration value (0.002 mg/ml), ferulic acid protected all of the bands in the presence of H2O2 and UV. It is followed by caffeic, rosmarinic, and vanillic acids. On the other hand, cinnamic acid (at 0.002 mg/ml), gallic acid (at 0.002 mg/ml), p-hydroxybenzoic acid (at 0.002 and 0.004 mg/ml), and protocatechuic acid (at 0.002 and 0.004 mg/ml) could not protect plasmid DNA. PMID:25542528

  16. Analysis of Bile Acids

    NASA Astrophysics Data System (ADS)

    Sjövall, Jan; Griffiths, William J.; Setchell, Kenneth D. R.; Mano, Nariyasu; Goto, Junichi

    Bile acids constitute a large family of steroids in vertebrates, normally formed from cholesterol and carrying a carboxyl group in a side-chain of variable length. Bile alcohols, also formed from cholesterol, have similar structures as bile acids, except for the absence of a carboxyl group in the steroid skeleton. The conversion of cholesterol to bile acids and/or bile alcohols is of major importance for maintenance of cholesterol homeostasis, both from quantitative and regulatory points of view (Chiang, 2004; Kalaany and Mangelsdorf, 2006; Moore, Kato, Xie, et al., 2006; Scotti, Gilardi, Godio, et al., 2007). Appropriately conjugated bile acids and bile alcohols (also referred to as bile salts) are secreted in bile and serve vital functions in the absorption of lipids and lipid-soluble compounds (Hofmann, 2007). Reliable analytical methods are required for studies of the functions and pathophysiological importance of the variety of bile acids and bile alcohols present in living organisms. When combined with genetic and proteomic studies, analysis of these small molecules (in today's terminology: metabolomics, steroidomics, sterolomics, cholanoidomics, etc.) will lead to a deeper understanding of the integrated metabolic processes in lipid metabolism.

  17. Immunological responses of turbot (Psetta maxima) to nodavirus infection or polyriboinosinic polyribocytidylic acid (pIC) stimulation, using expressed sequence tags (ESTs) analysis and cDNA microarrays.

    PubMed

    Park, Kyoung C; Osborne, Jane A; Montes, Ariana; Dios, Sonia; Nerland, Audun H; Novoa, Beatriz; Figueras, Antonio; Brown, Laura L; Johnson, Stewart C

    2009-01-01

    To investigate the immunological responses of turbot to nodavirus infection or pIC stimulation, we constructed cDNA libraries from liver, kidney and gill tissues of nodavirus-infected fish and examined the differential gene expression within turbot kidney in response to nodavirus infection or pIC stimulation using a turbot cDNA microarray. Turbot were experimentally infected with nodavirus and samples of each tissue were collected at selected time points post-infection. Using equal amount of total RNA at each sampling time, we made three tissue-specific cDNA libraries. After sequencing 3230 clones we obtained 3173 (98.2%) high quality sequences from our liver, kidney and gill libraries. Of these 2568 (80.9%) were identified as known genes and 605 (19.1%) as unknown genes. A total of 768 unique genes were identified. The two largest groups resulting from the classification of ESTs according to function were the cell/organism defense genes (71 uni-genes) and apoptosis-related process (23 uni-genes). Using these clones, a 1920 element cDNA microarray was constructed and used to investigate the differential gene expression within turbot in response to experimental nodavirus infection or pIC stimulation. Kidney tissue was collected at selected times post-infection (HPI) or stimulation (HPS), and total RNA was isolated for microarray analysis. Of the 1920 genes studied on the microarray, we identified a total of 121 differentially expressed genes in the kidney: 94 genes from nodavirus-infected animals and 79 genes from those stimulated with pIC. Within the nodavirus-infected fish we observed the highest number of differentially expressed genes at 24 HPI. Our results indicate that certain genes in turbot have important roles in immune responses to nodavirus infection and dsRNA stimulation.

  18. Statistical analysis of molecular nanotemplate driven DNA adsorption on graphite.

    PubMed

    Dubrovin, E V; Speller, S; Yaminsky, I V

    2014-12-30

    In this work, we have studied the conformation of DNA molecules aligned on the nanotemplates of octadecylamine, stearyl alcohol, and stearic acid on highly oriented pyrolytic graphite (HOPG). For this purpose, fluctuations of contours of adsorbed biopolymers obtained from atomic force microscopy (AFM) images were analyzed using the wormlike chain model. Moreover, the conformations of adsorbed biopolymer molecules were characterized by the analysis of the scaling exponent ν, which relates the mean squared end-to-end distance and contour length of the polymer. During adsorption on octadecylamine and stearyl alcohol nanotemplates, DNA forms straight segments, which order along crystallographic axes of graphite. In this case, the conformation of DNA molecules can be described using two different length scales. On a large length scale (at contour lengths l > 200-400 nm), aligned DNA molecules have either 2D compact globule or partially relaxed 2D conformation, whereas on a short length scale (at l ≤ 200-400 nm) their conformation is close to that of rigid rods. The latter type of conformation can be also assigned to DNA adsorbed on a stearic acid nanotemplate. The different conformation of DNA molecules observed on the studied monolayers is connected with the different DNA-nanotemplate interactions associated with the nature of the functional group of the alkane derivative in the nanotemplate (amine, alcohol, or acid). The persistence length of λ-DNA adsorbed on octadecylamine nanotemplates is 31 ± 2 nm indicating the loss of DNA rigidity in comparison with its native state. Similar values of the persistence length (34 ± 2 nm) obtained for 24-times shorter DNA molecules adsorbed on an octadecylamine nanotemplate demonstrate that this rigidity change does not depend on biopolymer length. Possible reasons for the reduction of DNA persistence length are discussed in view of the internal DNA structure and DNA-surface interaction.

  19. Gibberellic Acid enhancement of DNA turnover in barley aleurone cells.

    PubMed

    Taiz, L; Starks, J E

    1977-08-01

    When imbibed, deembryonated halfseeds from barley (Hordeum vulgare L., var. Himalaya) are incubated in buffer, the DNA content of the aleurone layer increases 25 to 40% over a 24-hour period. In contrast, the DNA of isolated aleurone layers declines by 20% over the same time period. Gibberellic acid (GA) causes a reduction in DNA levels in both halfseed aleurone layers and isolated aleurone layers. GA also increases the specific radioactivity of [(3)H]thymidine-labeled halfseed aleurone layer DNA during the first 12 hours of treatment. Pulse-chase studies demonstrated that the newly synthesized DNA is metabolically labile.The buoyant density on CsCl density gradients of hormone-treated aleurone DNA is identical with that of DNA extracted from whole seedlings. After density-labeling halfseed DNA with 5-bromodeoxyuridine, a bimodal absorption profile is obtained in neutral CsCl. The light band (1.70 g/ml) corresponds to unsubstituted DNA, while the heavy band (1.725-1.74 g/ml) corresponds to a hybrid density-labeled species. GA increases the relative amount of the heavy (hybrid) peak in halfseed aleurone layer DNA, further suggesting that the hormone enhances semiconservative replication in halfseeds.DNA methylation was also demonstrated. Over 60% of the radioactivity from [(3)H-Me]methionine is incorporated into 5-methylcytosine. GA has no effect on the percentage distribution of label among the bases.It was concluded that GA enhances the rate of DNA degradation and DNA synthesis (turnover) in halfseeds, but primarily DNA degradation in isolated aleurone layers. Incorporation by isolated aleurone layers is due to DNA repair. Semiconservative replication apparently plays no physiological role in the hormone response, since both isolated aleurone layers and gamma-irradiated halfseeds respond normally. The hypothesis was advanced that endoreduplication and DNA degradation are means by which the seed stores and mobilizes deoxyribonucleotides for the embryo during

  20. Nucleic Acid Engineering: RNA Following the Trail of DNA.

    PubMed

    Kim, Hyejin; Park, Yongkuk; Kim, Jieun; Jeong, Jaepil; Han, Sangwoo; Lee, Jae Sung; Lee, Jong Bum

    2016-02-01

    The self-assembly feature of the naturally occurring biopolymer, DNA, has fascinated researchers in the fields of materials science and bioengineering. With the improved understanding of the chemical and structural nature of DNA, DNA-based constructs have been designed and fabricated from two-dimensional arbitrary shapes to reconfigurable three-dimensional nanodevices. Although DNA has been used successfully as a building block in a finely organized and controlled manner, its applications need to be explored. Hence, with the myriad of biological functions, RNA has recently attracted considerable attention to further the application of nucleic acid-based structures. This Review categorizes different approaches of engineering nucleic acid-based structures and introduces the concepts, principles, and applications of each technique, focusing on how DNA engineering is applied as a guide to RNA engineering.

  1. Sequence-specific DNA damage induced by ultraviolet A-irradiated folic acid via its photolysis product.

    PubMed

    Hirakawa, Kazutaka; Suzuki, Hiroyuki; Oikawa, Shinji; Kawanishi, Shosuke

    2003-02-15

    DNA damage mediated by photosensitizers participates in solar carcinogenesis. Fluorescence measurement and high-performance liquid chromatography analysis demonstrated that photoirradiated folic acid, one of the photosensitizers in cells, generates pterine-6-carboxylic acid (PCA). Experiments using 32P-labeled DNA fragments obtained from a human gene showed that ultraviolet A-irradiated folic acid or PCA caused DNA cleavage specifically at consecutive G residues in double-stranded DNA after Escherichia coli formamidopyrimidine-DNA glycosylase or piperidine treatment. The amount of 8-oxo-7,8-dihydro-2(')-deoxyguanosine formed through this DNA photoreaction in double-stranded DNA exceeded that in single-stranded DNA. Kinetic studies suggested that DNA damage is caused mainly by photoexcited PCA generated from folic acid rather than by folic acid itself. In conclusion, photoirradiated folic acid generates PCA, which induces DNA photooxidation specifically at consecutive G residues through electron transfer. Excess intake of folic acid supplements may increase a risk of skin cancer by solar ultraviolet light. PMID:12573286

  2. Evaluation of DNA/RNAshells for room temperature nucleic acids storage.

    PubMed

    Liu, Xiaopan; Li, Qiyuan; Wang, Xian; Zhou, Xiaolin; He, Xuheng; Liao, Qiuyan; Zhu, Fengqin; Cheng, Le; Zhang, Yong

    2015-02-01

    Traditional nucleic acids preservation methods rely on maintaining samples in cold environments, which are costly to operate and time sensitive. Recent work validated that using room temperature for the storage of nucleic acids is possible if the samples are completely protected from water and oxygen. Here, we conducted accelerated aging and real-time degradation studies to evaluate the new technology DNAshell and RNAshell, which preserves DNA and RNA at room temperature, including the DNA and RNA yield, purity, and integrity. DNA and RNA solutions are dried in the presence of stabilizers in stainless steel minicapsules, then redissolved after different time points of heating and storing at room temperature. Results show that DNAshell and RNAshell ensure the safe storage of nucleic acids at room temperature for long periods of time, and that the quality of these nucleic acids is suitable for common downstream analysis.

  3. Intracavity DNA melting analysis with optofluidic lasers.

    PubMed

    Lee, Wonsuk; Fan, Xudong

    2012-11-01

    DNA melting analysis holds great promise for simple and fast DNA sequence discrimination. However, conventional fluorescence-based methods suffer from a small differential signal and demanding melting curve analysis, both of which make it difficult to distinguish the target DNA from the mismatched one. Herein, we propose and demonstrate a highly specific intracavity DNA melting analysis scheme utilizing an optofluidic laser. The laser optically amplifies the small yet intrinsic thermal dynamic difference between the target and the single-base-mismatched DNA, resulting in a differential signal that is orders of magnitude greater than with fluorescence-based methods. In particular, the existence of a phase transition between the stimulated laser emission and fluorescence (i.e., spontaneous emission) enables accurate determination of the DNA transition temperature difference. Furthermore, the high differential signal in the intracavity detection allows for scanning of the laser excitation at a fixed temperature to distinguish two DNA sequences, which provides another means for rapid DNA analysis. In this paper, we first theoretically investigate DNA melting analysis using an optofluidic laser and then experimentally explore this scheme with a high-quality optofluidic ring resonator. Distinction of two DNA sequences of up to 100 bases long is demonstrated. The intracavity detection developed here will lead to novel optofluidic devices that enable rapid and simple analysis of DNAs with very long sequences.

  4. Laccaic Acid A Is a Direct, DNA-competitive Inhibitor of DNA Methyltransferase 1*

    PubMed Central

    Fagan, Rebecca L.; Cryderman, Diane E.; Kopelovich, Levy; Wallrath, Lori L.; Brenner, Charles

    2013-01-01

    Methylation of cytosines in CpG dinucleotides is the predominant epigenetic mark on vertebrate DNA. DNA methylation is associated with transcriptional repression. The pattern of DNA methylation changes during development and with disease. Human DNA methyltransferase 1 (Dnmt1), a 1616-amino acid multidomain enzyme, is essential for maintenance of DNA methylation in proliferating cells and is considered an important cancer drug target. Using a fluorogenic, endonuclease-coupled DNA methylation assay with an activated form of Dnmt1 engineered to lack the replication foci targeting sequence domain, we discovered that laccaic acid A (LCA), a highly substituted anthraquinone natural product, is a direct inhibitor with a 310 nm Ki. LCA is competitive with the DNA substrate in in vitro methylation assays and alters the expression of methylated genes in MCF-7 breast cancer cells synergistically with 5-aza-2′-deoxycytidine. LCA represents a novel class of Dnmt-targeted molecular probes, with biochemical properties that allow it to distinguish between non DNA-bound and DNA-bound Dnmt1. PMID:23839987

  5. Investigation of perfluorooctanoic acid induced DNA damage using electrogenerated chemiluminescence associated with charge transfer in DNA.

    PubMed

    Lu, Liping; Guo, Linqing; Li, Meng; Kang, Tianfang; Cheng, Shuiyuan; Miao, Wujian

    2016-10-01

    An electrogenerated chemiluminescence (ECL)-DNA sensor was designed and fabricated for the investigation of DNA damage by a potential environmental pollutant, perfluorooctanoic acid (PFOA). The ECL-DNA sensor consisted of a Au electrode that had a self-assembled monolayer of 15 base-pair double-stranded (ds) DNA oligonucleotides with covalently attached semiconductor CdSe quantum dots (QDs) at the distal end of the DNA. Characterization of the ECL-DNA sensor was conducted with X-ray photoelectron spectroscopy (XPS), electrochemical impedance spectroscopy (EIS), ECL, and cyclic voltammetry before and after the exposure of the sensor to PFOA. Consistent data revealed that the dsDNA on Au was severely damaged upon the incubation of the electrode in PFOA, causing significant increase in charge (or electron) transfer (CT) resistance within DNA strands. Consequently, the cathodic coreactant ECL responses of the Au/dsDNA-QDs electrode in the presence of K2S2O8 were markedly decreased. The strong interaction between DNA and PFOA via the hydrophobic interaction, especially the formation of F···H hydrogen bonds by insertion of the difluoro-methylene group of PFOA into the DNA base pairs, was believed to be responsible for the dissociation or loosening of dsDNA structure, which inhibited the CT through DNA. A linear relationship between the ECL signal of the sensor and the logarithmical concentration of PFOA displayed a dynamic range of 1.00 × 10(-14)-1.00 × 10(-4) M, with a limit of detection of 1.00 × 10(-15) M at a signal-to-noise ratio of 3. Graphical Abstract Illustration of ECL detection of PFOA on a Au/dsDNA-QDs ECL-DNA sensor.

  6. Investigation of perfluorooctanoic acid induced DNA damage using electrogenerated chemiluminescence associated with charge transfer in DNA.

    PubMed

    Lu, Liping; Guo, Linqing; Li, Meng; Kang, Tianfang; Cheng, Shuiyuan; Miao, Wujian

    2016-10-01

    An electrogenerated chemiluminescence (ECL)-DNA sensor was designed and fabricated for the investigation of DNA damage by a potential environmental pollutant, perfluorooctanoic acid (PFOA). The ECL-DNA sensor consisted of a Au electrode that had a self-assembled monolayer of 15 base-pair double-stranded (ds) DNA oligonucleotides with covalently attached semiconductor CdSe quantum dots (QDs) at the distal end of the DNA. Characterization of the ECL-DNA sensor was conducted with X-ray photoelectron spectroscopy (XPS), electrochemical impedance spectroscopy (EIS), ECL, and cyclic voltammetry before and after the exposure of the sensor to PFOA. Consistent data revealed that the dsDNA on Au was severely damaged upon the incubation of the electrode in PFOA, causing significant increase in charge (or electron) transfer (CT) resistance within DNA strands. Consequently, the cathodic coreactant ECL responses of the Au/dsDNA-QDs electrode in the presence of K2S2O8 were markedly decreased. The strong interaction between DNA and PFOA via the hydrophobic interaction, especially the formation of F···H hydrogen bonds by insertion of the difluoro-methylene group of PFOA into the DNA base pairs, was believed to be responsible for the dissociation or loosening of dsDNA structure, which inhibited the CT through DNA. A linear relationship between the ECL signal of the sensor and the logarithmical concentration of PFOA displayed a dynamic range of 1.00 × 10(-14)-1.00 × 10(-4) M, with a limit of detection of 1.00 × 10(-15) M at a signal-to-noise ratio of 3. Graphical Abstract Illustration of ECL detection of PFOA on a Au/dsDNA-QDs ECL-DNA sensor. PMID:27108285

  7. DNA analysis of digested tomato seeds in stomach contents.

    PubMed

    Lee, Cheng-Lung; Coyle, Heather Miller; Carita, Eric; Ladd, Carll; Yang, Nicholas C S; Palmbach, Timothy M; Hsu, Ian C; Lee, Henry C

    2006-06-01

    Examination of stomach contents is one of the important steps in medical legal autopsy. Vegetative materials such as stems, roots, and seeds in stomach contents can be valuable evidence for providing investigative leads in death investigation. Currently, the identification of plant materials relies on microscopic and morphologic examination. We have found that many seeds are often protected from acid degradation during stomach digestion by their tough exterior seed coat. Tomato seeds were selected as a model system to assess DNA analysis and plant variety marker identification. The DNA-amplified fragment length polymorphism method was performed to determine if the DNA obtained from single seeds could be used for PCR analysis. From the amplified fragment length polymorphism results, some candidate markers for individualizing seeds from morphologically distinct tomatoes were identified. These data on DNA analysis of tomato seeds indicate amplified fragment length polymorphism is a viable procedure for the individualization of seeds from stomach contents in forensic investigations.

  8. Human liver apolipoprotein B-100 cDNA: complete nucleic acid and derived amino acid sequence.

    PubMed Central

    Law, S W; Grant, S M; Higuchi, K; Hospattankar, A; Lackner, K; Lee, N; Brewer, H B

    1986-01-01

    Human apolipoprotein B-100 (apoB-100), the ligand on low density lipoproteins that interacts with the low density lipoprotein receptor and initiates receptor-mediated endocytosis and low density lipoprotein catabolism, has been cloned, and the complete nucleic acid and derived amino acid sequences have been determined. ApoB-100 cDNAs were isolated from normal human liver cDNA libraries utilizing immunoscreening as well as filter hybridization with radiolabeled apoB-100 oligodeoxynucleotides. The apoB-100 mRNA is 14.1 kilobases long encoding a mature apoB-100 protein of 4536 amino acids with a calculated amino acid molecular weight of 512,723. ApoB-100 contains 20 potential glycosylation sites, and 12 of a total of 25 cysteine residues are located in the amino-terminal region of the apolipoprotein providing a potential globular structure of the amino terminus of the protein. ApoB-100 contains relatively few regions of amphipathic helices, but compared to other human apolipoproteins it is enriched in beta-structure. The delineation of the entire human apoB-100 sequence will now permit a detailed analysis of the conformation of the protein, the low density lipoprotein receptor binding domain(s), and the structural relationship between apoB-100 and apoB-48 and will provide the basis for the study of genetic defects in apoB-100 in patients with dyslipoproteinemias. PMID:3464946

  9. DNA Methylation Screening and Analysis

    PubMed Central

    Sant, Karilyn E.; Nahar, Muna S.; Dolinoy, Dana C.

    2013-01-01

    DNA methylation is an epigenetic form of gene regulation that is universally important throughout the life course, especially during in utero and postnatal development. DNA methylation aids in cell cycle regulation and cellular differentiation processes. Previous studies have demonstrated that DNA methylation profiles may be altered by diet and the environment, and that these profiles are especially vulnerable during development. Thus, it is important to understand the role of DNA methylation in developmental governance and subsequent disease progression. A variety of molecular methods exist to assay for global, gene-specific, and epigenome-wide methylation. Here we describe these methods and discuss their relative strengths and limitations. PMID:22669678

  10. Laser desorption mass spectrometry for fast DNA analysis

    SciTech Connect

    Chen, C.H.; Ch`ang, L.Y.; Taranenko, N.I.; Allman, S.L.; Tang, K.; Matteson, K.J.

    1995-09-01

    During the past few years, major effort has been directed toward developing mass spectrometry to measure biopolymers because of the great potential benefit to biomedical research. Hellenkamp and his co-workers were the first to report that large polypeptide molecules can be ionized and detected without significant fragmentation when a greater number of nicotinic acid molecules are used as a matrix. This method is now well known as matrix-assisted laser desorption/ionization (MALDI). Since then, various groups have reported measurements of very large proteins by MALDI. Reliable protein analysis by MALDI is more or less well established. However, the application of MALDI to nucleic acids analysis has been found to be much more difficult. Most research on the measurement of nucleic acid by MALDI were stimulated by the Human Genome Project. Up to now, the only method for reliable routine analysis of nucleic acid is gel electrophoresis. Different sizes of nucleic acids can be separated in gel medium when a high electric field is applied to the gel. However, the time needed to separate different sizes of DNA segments usually takes from several minutes to several hours. If MALDI can be successfully used for nucleic acids analysis, the analysis time can be reduced to less than I millisecond. In addition, no tagging with radioactive materials or chemical dyes is needed. In this work, we will review recent progress related to MALDI for DNA analysis.

  11. Analysis of environmental transcriptomes by DNA microarrays.

    PubMed

    Parro, Víctor; Moreno-Paz, Mercedes; González-Toril, Elena

    2007-02-01

    In this work we investigated the correlations between global gene expression patterns and environmental parameters in natural ecosystems. We studied the preferential gene expression of the iron oxidizer bacterium Leptospirillum ferrooxidans to adapt its physiology to changes in the physicochemical parameters in its natural medium. Transcriptome analysis by DNA microarrays can proportionate an instant picture about the preferential gene expression between two different environmental samples. However, this type of analysis is very difficult and complex in natural ecosystems, mainly because of the broad biodiversity and multiple environmental parameters that may affect gene expression. The necessity of high-quality RNA preparations as well as complicated data analysis are also technological limitations. The low prokaryotic diversity of the extremely acidic and iron-rich waters of the Tinto River (Spain) ecosystem, where L. ferrooxidans is abundant, allows the opportunity to achieve global gene expression studies and to associate gene function with environmental parameters. We applied a total RNA amplification protocol validated previously for the amplification of the environmental transcriptome (meta-transcriptome). The meta-transcriptome of two sites from the Tinto River mainly differing in the salt and oxygen contents were amplified and analysed by a L. ferrooxidans DNA microarray. The results showed a clear preferential induction of genes involved in certain physicochemical parameters like: high salinity (ectAB, otsAB), low oxygen concentration (cydAB), iron uptake (fecA-exbBD-tonB), oxidative stress (carotenoid synthesis, oxyR, recG), potassium (kdpBAC) or phosphate concentrations (pstSCAB), etc. We conclude that specific gene expression patterns can be useful indicators for the physiological conditions in a defined ecosystem. Also, the upregulation of certain genes and operons reveals information about the environmental conditions (nutrient limitations, stresses

  12. Analysis of Organic Acids.

    ERIC Educational Resources Information Center

    Griswold, John R.; Rauner, Richard A.

    1990-01-01

    Presented are the procedures and a discussion of the results for an experiment in which students select unknown carboxylic acids, determine their melting points, and investigate their solubility behavior in water and ethanol. A table of selected carboxylic acids is included. (CW)

  13. Label-free potentiometry for detecting DNA hybridization using peptide nucleic acid and DNA probes.

    PubMed

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

    2013-02-07

    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.

  14. DNA Tetrominoes: The Construction of DNA Nanostructures Using Self-Organised Heterogeneous Deoxyribonucleic Acids Shapes

    PubMed Central

    Ong, Hui San; Rahim, Mohd Syafiq; Firdaus-Raih, Mohd; Ramlan, Effirul Ikhwan

    2015-01-01

    The unique programmability of nucleic acids offers alternative in constructing excitable and functional nanostructures. This work introduces an autonomous protocol to construct DNA Tetris shapes (L-Shape, B-Shape, T-Shape and I-Shape) using modular DNA blocks. The protocol exploits the rich number of sequence combinations available from the nucleic acid alphabets, thus allowing for diversity to be applied in designing various DNA nanostructures. Instead of a deterministic set of sequences corresponding to a particular design, the protocol promotes a large pool of DNA shapes that can assemble to conform to any desired structures. By utilising evolutionary programming in the design stage, DNA blocks are subjected to processes such as sequence insertion, deletion and base shifting in order to enrich the diversity of the resulting shapes based on a set of cascading filters. The optimisation algorithm allows mutation to be exerted indefinitely on the candidate sequences until these sequences complied with all the four fitness criteria. Generated candidates from the protocol are in agreement with the filter cascades and thermodynamic simulation. Further validation using gel electrophoresis indicated the formation of the designed shapes. Thus, supporting the plausibility of constructing DNA nanostructures in a more hierarchical, modular, and interchangeable manner. PMID:26258940

  15. Associations between whole peripheral blood fatty acids and DNA methylation in humans.

    PubMed

    de la Rocha, Carmen; Pérez-Mojica, J Eduardo; León, Silvia Zenteno-De; Cervantes-Paz, Braulio; Tristán-Flores, Fabiola E; Rodríguez-Ríos, Dalia; Molina-Torres, Jorge; Ramírez-Chávez, Enrique; Alvarado-Caudillo, Yolanda; Carmona, F Javier; Esteller, Manel; Hernández-Rivas, Rosaura; Wrobel, Katarzyna; Wrobel, Kazimierz; Zaina, Silvio; Lund, Gertrud

    2016-01-01

    Fatty acids (FA) modify DNA methylation in vitro, but limited information is available on whether corresponding associations exist in vivo and reflect any short-term effect of the diet. Associations between global DNA methylation and FAs were sought in blood from lactating infants (LI; n = 49) and adult males (AMM; n = 12) equally distributed across the three conventional BMI classes. AMM provided multiple samples at 2-hour intervals during 8 hours after either a single Western diet-representative meal (post-prandial samples) or no meal (fasting samples). Lipid/glucose profile, HDAC4 promoter and PDK4 5'UTR methylation were determined in AMM. Multiple regression analysis revealed that global (in LI) and both global and PDK4-specific DNA methylation (in AMM) were positively associated with eicosapentaenoic and arachidonic acid. HDAC4 methylation was inversely associated with arachidonic acid post-prandially in AMM. Global DNA methylation did not show any defined within-day pattern that would suggest a short-term response to the diet. Nonetheless, global DNA methylation was higher in normal weight subjects both post-prandially and in fasting and coincided with higher polyunsaturated relative to monounsaturated and saturated FAs. We show for the first time strong associations of DNA methylation with specific FAs in two human cohorts of distinct age, diet and postnatal development stage. PMID:27181711

  16. Associations between whole peripheral blood fatty acids and DNA methylation in humans

    PubMed Central

    de la Rocha, Carmen; Pérez-Mojica, J. Eduardo; León, Silvia Zenteno-De; Cervantes-Paz, Braulio; Tristán-Flores, Fabiola E.; Rodríguez-Ríos, Dalia; Molina-Torres, Jorge; Ramírez-Chávez, Enrique; Alvarado-Caudillo, Yolanda; Carmona, F. Javier; Esteller, Manel; Hernández-Rivas, Rosaura; Wrobel, Katarzyna; Wrobel, Kazimierz; Zaina, Silvio; Lund, Gertrud

    2016-01-01

    Fatty acids (FA) modify DNA methylation in vitro, but limited information is available on whether corresponding associations exist in vivo and reflect any short-term effect of the diet. Associations between global DNA methylation and FAs were sought in blood from lactating infants (LI; n = 49) and adult males (AMM; n = 12) equally distributed across the three conventional BMI classes. AMM provided multiple samples at 2-hour intervals during 8 hours after either a single Western diet-representative meal (post-prandial samples) or no meal (fasting samples). Lipid/glucose profile, HDAC4 promoter and PDK4 5’UTR methylation were determined in AMM. Multiple regression analysis revealed that global (in LI) and both global and PDK4-specific DNA methylation (in AMM) were positively associated with eicosapentaenoic and arachidonic acid. HDAC4 methylation was inversely associated with arachidonic acid post-prandially in AMM. Global DNA methylation did not show any defined within-day pattern that would suggest a short-term response to the diet. Nonetheless, global DNA methylation was higher in normal weight subjects both post-prandially and in fasting and coincided with higher polyunsaturated relative to monounsaturated and saturated FAs. We show for the first time strong associations of DNA methylation with specific FAs in two human cohorts of distinct age, diet and postnatal development stage. PMID:27181711

  17. Oxidative DNA damage induced by aminoacetone, an amino acid metabolite.

    PubMed

    Hiraku, Y; Sugimoto, J; Yamaguchi, T; Kawanishi, S

    1999-05-01

    We investigated DNA damage induced by aminoacetone, a metabolite of threonine and glycine. Pulsed-field gel electrophoresis revealed that aminoacetone caused cellular DNA cleavage. Aminoacetone increased the amount of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) in human cultured cells in a dose-dependent manner. The formation of 8-oxodG in calf thymus DNA increased due to aminoacetone only in the presence of Cu(II). DNA ladder formation was observed at higher concentrations of aminoacetone than those causing DNA cleavage. Flow cytometry showed that aminoacetone enhanced the generation of hydrogen peroxide (H2O2) in cultured cells. Aminoacetone caused damage to 32P-5'-end-labeled DNA fragments, obtained from the human c-Ha-ras-1 and p53 genes, at cytosine and thymine residues in the presence of Cu(II). Catalase and bathocuproine inhibited DNA damage, suggesting that H2O2 and Cu(I) were involved. Analysis of the products generated from aminoacetone revealed that aminoacetone underwent Cu(II)-mediated autoxidation in two different pathways: the major pathway in which methylglyoxal and NH+4 are generated and the minor pathway in which 2,5-dimethylpyrazine is formed through condensation of two molecules of aminoacetone. These findings suggest that H2O2 generated by the autoxidation of aminoacetone reacts with Cu(I) to form reactive species capable of causing oxidative DNA damage.

  18. High-quality DNA from fingernails for genetic analysis.

    PubMed

    Preuner, Sandra; Danzer, Martin; Pröll, Johannes; Pötschger, Ulrike; Lawitschka, Anita; Gabriel, Christian; Lion, Thomas

    2014-07-01

    The availability of high-quality germline DNA is an important prerequisite for a variety of genetic analyses. We have shown previously that fingernail clippings provide an optimal source of autologous, constitutional DNA for PCR-based applications. However, most existing protocols for nucleic acid purification from nails do not provide sufficiently high yields of pure and intact DNA for more demanding downstream analyses such as next generation sequencing (NGS). We have extensively tested and systematically modified a number of different protocols for DNA purification from nail material to optimize the yield and quality. The integrity of DNA was determined by PCR amplification of short (<300 bp), mid-range (>400 bp), and long-range (>2 kb) sequences using different target genes. Among the methods tested, the Prepfiler Forensic DNA Extraction kit was identified as the most appropriate approach to isolation of high-quality DNA from nail clippings. A standardized input of 20 mg nail material (1 to 10 pieces of fingernail clippings) yielded a mean of 1 μg DNA (range, 0.5 to 2.3 μg). Subsequent PCR-analysis revealed efficient amplifiability of short and mid-range targets in 93% and 90%, and long-range fragments in 60% of the samples tested. The adequacy for next generation sequencing applications was demonstrated by successful high-resolution HLA-typing in ten transplant recipients. Hence, the protocol presented facilitates the exploitation of fingernail material even for demanding genomic analyses both in research and diagnostics.

  19. Is amino acid racemization a useful tool for screening for ancient DNA in bone?

    PubMed Central

    Collins, Matthew J.; Penkman, Kirsty E. H.; Rohland, Nadin; Shapiro, Beth; Dobberstein, Reimer C.; Ritz-Timme, Stefanie; Hofreiter, Michael

    2009-01-01

    Many rare and valuable ancient specimens now carry the scars of ancient DNA research, as questions of population genetics and phylogeography require larger sample sets. This fuels the demand for reliable techniques to screen for DNA preservation prior to destructive sampling. Only one such technique has been widely adopted: the extent of aspartic acid racemization (AAR). The kinetics of AAR are believed to be similar to the rate of DNA depurination and therefore a good measure of the likelihood of DNA survival. Moreover, AAR analysis is only minimally destructive. We report the first comprehensive test of AAR using 91 bone and teeth samples from temperate and high-latitude sites that were analysed for DNA. While the AAR range of all specimens was low (0.02–0.17), no correlation was found between the extent of AAR and DNA amplification success. Additional heating experiments and surveys of the literature indicated that d/l Asx is low in bones until almost all the collagen is lost. This is because aspartic acid is retained in the bone within the constrained environment of the collagen triple helix, where it cannot racemize for steric reasons. Only if the helix denatures to soluble gelatin can Asx racemize readily, but this soluble gelatine is readily lost in most burial environments. We conclude that Asx d/l is not a useful screening technique for ancient DNA from bone. PMID:19493899

  20. Flexibility of nucleic acids: From DNA to RNA

    NASA Astrophysics Data System (ADS)

    Lei, Bao; Xi, Zhang; Lei, Jin; Zhi-Jie, Tan

    2016-01-01

    The structural flexibility of nucleic acids plays a key role in many fundamental life processes, such as gene replication and expression, DNA-protein recognition, and gene regulation. To obtain a thorough understanding of nucleic acid flexibility, extensive studies have been performed using various experimental methods and theoretical models. In this review, we will introduce the progress that has been made in understanding the flexibility of nucleic acids including DNAs and RNAs, and will emphasize the experimental findings and the effects of salt, temperature, and sequence. Finally, we will discuss the major unanswered questions in understanding the flexibility of nucleic acids. Project supported by the National Basic Research Program of China (Grant No. 2011CB933600), the National Natural Science Foundation of China (Grant Nos. 11175132, 11575128, and 11374234), and the Program for New Century Excellent Talents, China (Grant No. NCET 08-0408).

  1. Associations between Serum Perfluoroalkyl Acids and LINE-1 DNA Methylation

    PubMed Central

    Watkins, Deborah J.; Wellenius, Gregory A.; Butler, Rondi A.; Bartell, Scott M.; Fletcher, Tony; Kelsey, Karl T.

    2014-01-01

    Perfluoroalkyl acids (PFAAs) are persistent, synthetic compounds that are used in a number of consumer products. Perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS) have been associated with cardiovascular risk factors, and changes in gene expression and DNA methylation in animals and cellular systems. However, whether PFAA exposure is associated with LINE-1 DNA methylation, a potential marker of cardiovascular risk, in humans remains unknown. We sought to evaluate the cross-sectional associations between serum PFAAs and LINE-1 DNA methylation in a population highly exposed to PFOA. We measured serum PFAAs twice four to five years apart in 685 adult participants (47% male, mean age ± SD=42 ± 11 years). We measured percent LINE-1 DNA methylation in peripheral blood leukocytes at the second time point (follow-up), and estimated absolute differences in LINE-1 methylation associated with an interquartile (IQR) shift in mean PFAA serum levels. IQR increases in mean serum PFOA, PFOS, perfluorononanoic acid (PFNA), and perfluorohexane sulfonate (PFHxS) were associated with differences of −0.04 (p=0.16), 0.20 (p=0.001), 0.06 (p=0.19), and 0.02 (p=0.57), respectively, in % LINE-1 methylation at follow-up after adjustment for potential confounders. We observed a monotonic increase in LINE-1 DNA methylation across tertiles of PFOS and PFNA (ptrend=0.02 for both associations), but not across tertiles of PFOA or PFHxS (ptrend=0.71 and 0.44, respectively). In summary, serum PFOS was associated with LINE-1 methylation, while serum PFOA, PFHxS, and PFNA were not. Additional research is needed to more precisely determine whether these compounds are epigenetically active. PMID:24263140

  2. Analysis of Ancient DNA in Microbial Ecology.

    PubMed

    Gorgé, Olivier; Bennett, E Andrew; Massilani, Diyendo; Daligault, Julien; Pruvost, Melanie; Geigl, Eva-Maria; Grange, Thierry

    2016-01-01

    The development of next-generation sequencing has led to a breakthrough in the analysis of ancient genomes, and the subsequent genomic analyses of the skeletal remains of ancient humans have revolutionized the knowledge of the evolution of our species, including the discovery of a new hominin, and demonstrated admixtures with more distantly related archaic populations such as Neandertals and Denisovans. Moreover, it has also yielded novel insights into the evolution of ancient pathogens. The analysis of ancient microbial genomes allows the study of their recent evolution, presently over the last several millennia. These spectacular results have been attained despite the degradation of DNA after the death of the host, which results in very short DNA molecules that become increasingly damaged, only low quantities of which remain. The low quantity of ancient DNA molecules renders their analysis difficult and prone to contamination with modern DNA molecules, in particular via contamination from the reagents used in DNA purification and downstream analysis steps. Finally, the rare ancient molecules are diluted in environmental DNA originating from the soil microorganisms that colonize bones and teeth. Thus, ancient skeletal remains can share DNA profiles with environmental samples and identifying ancient microbial genomes among the more recent, presently poorly characterized, environmental microbiome is particularly challenging. Here, we describe the methods developed and/or in use in our laboratory to produce reliable and reproducible paleogenomic results from ancient skeletal remains that can be used to identify the presence of ancient microbiota. PMID:26791510

  3. Analysis of Ancient DNA in Microbial Ecology.

    PubMed

    Gorgé, Olivier; Bennett, E Andrew; Massilani, Diyendo; Daligault, Julien; Pruvost, Melanie; Geigl, Eva-Maria; Grange, Thierry

    2016-01-01

    The development of next-generation sequencing has led to a breakthrough in the analysis of ancient genomes, and the subsequent genomic analyses of the skeletal remains of ancient humans have revolutionized the knowledge of the evolution of our species, including the discovery of a new hominin, and demonstrated admixtures with more distantly related archaic populations such as Neandertals and Denisovans. Moreover, it has also yielded novel insights into the evolution of ancient pathogens. The analysis of ancient microbial genomes allows the study of their recent evolution, presently over the last several millennia. These spectacular results have been attained despite the degradation of DNA after the death of the host, which results in very short DNA molecules that become increasingly damaged, only low quantities of which remain. The low quantity of ancient DNA molecules renders their analysis difficult and prone to contamination with modern DNA molecules, in particular via contamination from the reagents used in DNA purification and downstream analysis steps. Finally, the rare ancient molecules are diluted in environmental DNA originating from the soil microorganisms that colonize bones and teeth. Thus, ancient skeletal remains can share DNA profiles with environmental samples and identifying ancient microbial genomes among the more recent, presently poorly characterized, environmental microbiome is particularly challenging. Here, we describe the methods developed and/or in use in our laboratory to produce reliable and reproducible paleogenomic results from ancient skeletal remains that can be used to identify the presence of ancient microbiota.

  4. Nucleic Acid-Peptide Complex Phase Controlled by DNA Hybridization

    NASA Astrophysics Data System (ADS)

    Vieregg, Jeffrey; Lueckheide, Michael; Leon, Lorraine; Marciel, Amanda; Tirrell, Matthew

    When polyanions and polycations are mixed, counterion release drives formation of polymer-rich complexes that can either be solid (precipitates) or liquid (coacervates) depending on the properties of the polyelectrolytes. These complexes are important in many fields, from encapsulation of industrial polymers to membrane-free segregation of biomolecules such as nucleic acids and proteins. Condensation of long double-stranded DNA has been studied for several decades, but comparatively little attention has been paid to the polyelectrolyte behavior of oligonucleotides. We report here studies of DNA oligonucleotides (10 - 88 nt) complexed with polylysine (10 - 100 aa). Unexpectedly, we find that the phase of the resulting complexes is controlled by the hybridization state of the nucleic acid, with double-stranded DNA forming precipitates and single-stranded DNA forming coacervates. Stability increases with polyelectrolyte length and decreases with solution salt concentration, with complexes of the longer double-stranded polymers undergoing precipitate/coacervate/soluble transitions as ionic strength is increased. Mixing coacervates formed by complementary single-stranded oligonucleotides results in precipitate formation, raising the possibility of stimulus-responsive material design.

  5. An integrated workflow for DNA methylation analysis.

    PubMed

    Li, Pingchuan; Demirci, Feray; Mahalingam, Gayathri; Demirci, Caghan; Nakano, Mayumi; Meyers, Blake C

    2013-05-20

    The analysis of cytosine methylation provides a new way to assess and describe epigenetic regulation at a whole-genome level in many eukaryotes. DNA methylation has a demonstrated role in the genome stability and protection, regulation of gene expression and many other aspects of genome function and maintenance. BS-seq is a relatively unbiased method for profiling the DNA methylation, with a resolution capable of measuring methylation at individual cytosines. Here we describe, as an example, a workflow to handle DNA methylation analysis, from BS-seq library preparation to the data visualization. We describe some applications for the analysis and interpretation of these data. Our laboratory provides public access to plant DNA methylation data via visualization tools available at our "Next-Gen Sequence" websites (http://mpss.udel.edu), along with small RNA, RNA-seq and other data types. PMID:23706300

  6. Improved algorithm for analysis of DNA sequences using multiresolution transformation.

    PubMed

    Inbamalar, T M; Sivakumar, R

    2015-01-01

    Bioinformatics and genomic signal processing use computational techniques to solve various biological problems. They aim to study the information allied with genetic materials such as the deoxyribonucleic acid (DNA), the ribonucleic acid (RNA), and the proteins. Fast and precise identification of the protein coding regions in DNA sequence is one of the most important tasks in analysis. Existing digital signal processing (DSP) methods provide less accurate and computationally complex solution with greater background noise. Hence, improvements in accuracy, computational complexity, and reduction in background noise are essential in identification of the protein coding regions in the DNA sequences. In this paper, a new DSP based method is introduced to detect the protein coding regions in DNA sequences. Here, the DNA sequences are converted into numeric sequences using electron ion interaction potential (EIIP) representation. Then discrete wavelet transformation is taken. Absolute value of the energy is found followed by proper threshold. The test is conducted using the data bases available in the National Centre for Biotechnology Information (NCBI) site. The comparative analysis is done and it ensures the efficiency of the proposed system.

  7. Improved algorithm for analysis of DNA sequences using multiresolution transformation.

    PubMed

    Inbamalar, T M; Sivakumar, R

    2015-01-01

    Bioinformatics and genomic signal processing use computational techniques to solve various biological problems. They aim to study the information allied with genetic materials such as the deoxyribonucleic acid (DNA), the ribonucleic acid (RNA), and the proteins. Fast and precise identification of the protein coding regions in DNA sequence is one of the most important tasks in analysis. Existing digital signal processing (DSP) methods provide less accurate and computationally complex solution with greater background noise. Hence, improvements in accuracy, computational complexity, and reduction in background noise are essential in identification of the protein coding regions in the DNA sequences. In this paper, a new DSP based method is introduced to detect the protein coding regions in DNA sequences. Here, the DNA sequences are converted into numeric sequences using electron ion interaction potential (EIIP) representation. Then discrete wavelet transformation is taken. Absolute value of the energy is found followed by proper threshold. The test is conducted using the data bases available in the National Centre for Biotechnology Information (NCBI) site. The comparative analysis is done and it ensures the efficiency of the proposed system. PMID:26000337

  8. Biological characterization of liver fatty acid binding gene from miniature pig liver cDNA library.

    PubMed

    Gao, Y H; Wang, K F; Zhang, S; Fan, Y N; Guan, W J; Ma, Y H

    2015-01-01

    Liver fatty acid binding proteins (L-FABP) are a family of small, highly conserved, cytoplasmic proteins that bind to long-chain fatty acids and other hydrophobic ligands. In this study, a full-length enriched cDNA library was successfully constructed from Wuzhishan miniature pig, and then the L-FABP gene was cloned from this cDNA library and an expression vector (pEGFP-N3-L-FABP) was constructed in vitro. This vector was transfected into hepatocytes to test its function. The results of western blotting analysis demonstrated that the L-FABP gene from our full-length enriched cDNA library regulated downstream genes, including the peroxisome proliferator-activated receptor family in hepatocytes. This study provides a theoretical basis and experimental evidence for the application of L-FABP for the treatment of liver injury. PMID:26345909

  9. A Concentrated Hydrochloric Acid-based Method for Complete Recovery of DNA from Bone.

    PubMed

    Huynen, Leon; Lambert, David M

    2015-11-01

    The successful extraction of DNA from historical or ancient animal bone is important for the analysis of discriminating genetic markers. Methods used currently rely on the digestion of bone with EDTA and proteinase K, followed by purification with phenol/chloroform and silica bed binding. We have developed a simple concentrated hydrochloric acid-based method that precludes the use of phenol/chloroform purification and can lead to a several-fold increase in DNA yield when compared to other commonly used methods. Concentrated hydrochloric acid was shown to dissolve most of the undigested bone and allowed the efficient recovery of DNA fragments <100 bases in length. This method should prove useful for the recovery of DNAs from highly degraded animal bone, such as that found in historical or ancient samples.

  10. Biological characterization of liver fatty acid binding gene from miniature pig liver cDNA library.

    PubMed

    Gao, Y H; Wang, K F; Zhang, S; Fan, Y N; Guan, W J; Ma, Y H

    2015-01-01

    Liver fatty acid binding proteins (L-FABP) are a family of small, highly conserved, cytoplasmic proteins that bind to long-chain fatty acids and other hydrophobic ligands. In this study, a full-length enriched cDNA library was successfully constructed from Wuzhishan miniature pig, and then the L-FABP gene was cloned from this cDNA library and an expression vector (pEGFP-N3-L-FABP) was constructed in vitro. This vector was transfected into hepatocytes to test its function. The results of western blotting analysis demonstrated that the L-FABP gene from our full-length enriched cDNA library regulated downstream genes, including the peroxisome proliferator-activated receptor family in hepatocytes. This study provides a theoretical basis and experimental evidence for the application of L-FABP for the treatment of liver injury.

  11. Characterization of DNA Binding and Retinoic Acid Binding Properties of Retinoic Acid Receptor

    NASA Astrophysics Data System (ADS)

    Yang, Na; Schule, Roland; Mangelsdorf, David J.; Evans, Ronald M.

    1991-05-01

    High-level expression of the full-length human retinoic acid receptor (RAR) α and the DNA binding domain of the RAR in Escherichia coli was achieved by using a T7 RNA polymerase-directed expression system. After induction, full-length RAR protein was produced at an estimated level of 20% of the total bacterial proteins. Both intact RAR molecules and the DNA binding domain bind to the cognate DNA response element with high specificity in the absence of retinoic acid. However, this binding is enhanced to a great extent upon the addition of eukaryotic cell extracts. The factor responsible for this enhancement is heat-sensitive and forms a complex with RAR that binds to DNA and exhibits a distinct migration pattern in the gel-mobility-shift assay. The interaction site of the factor with RAR is localized in the 70-amino acid DNA binding region of RAR. The hormone binding ability of the RARα protein was assayed by a charcoal absorption assay and the RAR protein was found to bind to retinoic acid with a K_d of 2.1 x 10-10 M.

  12. Pyrosequencing Analysis for Breast Cancer DNA Methylome.

    PubMed

    Kuscu, Cem; Kuscu, Canan

    2016-01-01

    Unraveling DNA methylation profile of tumor is important for the diagnosis and treatment of cancer patients. Because of the heterogeneity of clinical samples, it is very difficult to get methylation profile of only tumor cells. Laser capture Microdissection (LCM) is giving us a chance to isolate the DNA only from the tumor cells without any stroma cell's DNA contamination. Once we capture the breast tumor cells, we can isolate the genomic DNA which is followed by the bisulfite treatment in which unmethylated cytosines of the CG pairs are converted into uracil; however, methylated cytosine does not go into any chemical change during this reaction. Next, bisulfite treated DNA is used in the regular PCR reaction to get a single band PCR amplicon which will be used as a template for the pyrosequencing. Pyrosequencing is a powerful method to make a quantitative methylation analysis for each specific CG pair.

  13. Mitochondrial DNA analysis: polymorphisms and pathogenicity

    PubMed Central

    Chinnery, P.; Howell, N.; Andrews, R.; Turnbull, D.

    1999-01-01

    The investigation of mtDNA disease can be relatively straightforward if a person has a recognisable phenotype and if it is possible to identify a known pathogenic mtDNA mutation. The difficulties arise when no known mtDNA defect can be found, or when the clinical abnormalities are complex and not easily matched to those of the more common mitochondrial disorders. We will describe here the difficulties that can be encountered during the identification of pathogenic mtDNA mutations and the approaches that can be used to confirm, or eliminate, a likely pathogenic role, in either single gene diseases or in multifactorial disorders.


Keywords: mitochondrial DNA; phylogenetic analysis; Leber's hereditary optic neuropathy; Alzheimer's disease PMID:10424809

  14. Analysis of Chiral Carboxylic Acids in Meteorites

    NASA Technical Reports Server (NTRS)

    Burton, A. S.; Elsila, J. E.; Hein, J. E.; Aponte, J. C.; Parker, E. T.; Glavin, D. P.; Dworkin, J. P.

    2015-01-01

    Homochirality of amino acids in proteins and sugars in DNA and RNA is a critical feature of life on Earth. In the absence of a chiral driving force, however, reactions leading to the synthesis of amino acids and sugars result in racemic mixtures. It is currently unknown whether homochirality was necessary for the origins of life or if it was a product of early life. The observation of enantiomeric excesses of certain amino acids of extraterrestrial origins in meteorites provides evidence to support the hypothesis that there was a mechanism for the preferential synthesis or destruction of a particular amino acid enantiomer [e.g., 1-3]. The cause of the observed chiral excesses is un-clear, although at least in the case of the amino acid isovaline, the degree of aqueous alteration that occurred on the meteorite parent body is correlated to the isovaline L-enantiomeric excess [3, 4]. This suggests that chiral symmetry is broken and/or amplified within the meteorite parent bodies. Besides amino acids, there have been only a few reports of other meteoritic compounds found in enantiomeric excess: sugars and sugar acids [5, 6] and the hydroxy acid lactic acid [7]. Determining whether or not additional types of molecules in meteorites are also present in enantiomeric excesses of extraterrestrial information will provide insights into mechanisms for breaking chiral symmetry. Though the previous measurements (e.g., enantiomeric composition of lactic acid [7], and chiral carboxylic acids [8]) were made by gas chromatography-mass spectrometry, the potential for increased sensitivity of liquid chromatography-mass spectrometry (LC-MS) analyses is important because for many meteorite samples, only small sample masses are available for study. Furthermore, at least in the case of amino acids, many of the largest amino acid enantiomeric excesses were observed in samples that contained lower abundances (tens of ppb) of a given amino acid enantiomer. In the present work, we describe

  15. Interaction of photosensitive surfactant with DNA and poly acrylic acid

    SciTech Connect

    Zakrevskyy, Yuriy Paasche, Jens; Lomadze, Nino; Santer, Svetlana; Cywinski, Piotr; Cywinska, Magdalena; Reich, Oliver; Löhmannsröben, Hans-Gerd

    2014-01-28

    In this paper, we investigate interactions and phase transitions in polyelectrolyte-surfactant complexes formed between a cationic azobenzene-containing surfactant and two types of polyelectrolytes: natural (DNA) or synthetic (PAA: poly acrylic acid). The construction of a phase diagram allowed distancing between four major phases: extended coil conformation, colloidally stable compacted globules, colloidal instability range, and surfactant-stabilized compact state. Investigation on the complexes’ properties in different phases and under irradiation with UV light provides information about the role of the surfactant's hydrophobic trans isomers both in the formation and destruction of DNA and PAA globules as well as in their colloidal stabilization. The trans isomer shows much stronger affinity to the polyelectrolytes than the hydrophilic cis counterpart. There is no need for complete compensation of the polyelectrolyte charges to reach the complete compaction. On contrary to the findings previously reported in the literature, we demonstrate – for the first time – complete polyelectrolyte compaction which occurs already at 20% of DNA (and at 50% of PAA) charge compensation. The trans isomer plays the main role in the compaction. The aggregation between azobenzene units in the photosensitive surfactant is a driving force of this process. The decompaction can be realized during UV light irradiation and is strongly influenced by the interplay between surfactant-surfactant and surfactant-DNA interactions in the compacted globules.

  16. Interaction of photosensitive surfactant with DNA and poly acrylic acid.

    PubMed

    Zakrevskyy, Yuriy; Cywinski, Piotr; Cywinska, Magdalena; Paasche, Jens; Lomadze, Nino; Reich, Oliver; Löhmannsröben, Hans-Gerd; Santer, Svetlana

    2014-01-28

    In this paper, we investigate interactions and phase transitions in polyelectrolyte-surfactant complexes formed between a cationic azobenzene-containing surfactant and two types of polyelectrolytes: natural (DNA) or synthetic (PAA: poly acrylic acid). The construction of a phase diagram allowed distancing between four major phases: extended coil conformation, colloidally stable compacted globules, colloidal instability range, and surfactant-stabilized compact state. Investigation on the complexes' properties in different phases and under irradiation with UV light provides information about the role of the surfactant's hydrophobic trans isomers both in the formation and destruction of DNA and PAA globules as well as in their colloidal stabilization. The trans isomer shows much stronger affinity to the polyelectrolytes than the hydrophilic cis counterpart. There is no need for complete compensation of the polyelectrolyte charges to reach the complete compaction. On contrary to the findings previously reported in the literature, we demonstrate - for the first time - complete polyelectrolyte compaction which occurs already at 20% of DNA (and at 50% of PAA) charge compensation. The trans isomer plays the main role in the compaction. The aggregation between azobenzene units in the photosensitive surfactant is a driving force of this process. The decompaction can be realized during UV light irradiation and is strongly influenced by the interplay between surfactant-surfactant and surfactant-DNA interactions in the compacted globules. PMID:25669583

  17. The cDNA-derived amino acid sequence of hemoglobin II from Lucina pectinata.

    PubMed

    Torres-Mercado, Elineth; Renta, Jessicca Y; Rodríguez, Yolanda; López-Garriga, Juan; Cadilla, Carmen L

    2003-11-01

    Hemoglobin II from the clam Lucina pectinata is an oxygen-reactive protein with a unique structural organization in the heme pocket involving residues Gln65 (E7), Tyr30 (B10), Phe44 (CD1), and Phe69 (E11). We employed the reverse transcriptase-polymerase chain reaction (RT-PCR) and methods to synthesize various cDNA(HbII). An initial 300-bp cDNA clone was amplified from total RNA by RT-PCR using degenerate oligonucleotides. Gene-specific primers derived from the HbII-partial cDNA sequence were used to obtain the 5' and 3' ends of the cDNA by RACE. The length of the HbII cDNA, estimated from overlapping clones, was approximately 2114 bases. Northern blot analysis revealed that the mRNA size of HbII agrees with the estimated size using cDNA data. The coding region of the full-length HbII cDNA codes for 151 amino acids. The calculated molecular weight of HbII, including the heme group and acetylated N-terminal residue, is 17,654.07 Da.

  18. Fractal analysis of DNA sequence data

    SciTech Connect

    Berthelsen, C.L.

    1993-01-01

    DNA sequence databases are growing at an almost exponential rate. New analysis methods are needed to extract knowledge about the organization of nucleotides from this vast amount of data. Fractal analysis is a new scientific paradigm that has been used successfully in many domains including the biological and physical sciences. Biological growth is a nonlinear dynamic process and some have suggested that to consider fractal geometry as a biological design principle may be most productive. This research is an exploratory study of the application of fractal analysis to DNA sequence data. A simple random fractal, the random walk, is used to represent DNA sequences. The fractal dimension of these walks is then estimated using the [open quote]sandbox method[close quote]. Analysis of 164 human DNA sequences compared to three types of control sequences (random, base-content matched, and dimer-content matched) reveals that long-range correlations are present in DNA that are not explained by base or dimer frequencies. The study also revealed that the fractal dimension of coding sequences was significantly lower than sequences that were primarily noncoding, indicating the presence of longer-range correlations in functional sequences. The multifractal spectrum is used to analyze fractals that are heterogeneous and have a different fractal dimension for subsets with different scalings. The multifractal spectrum of the random walks of twelve mitochondrial genome sequences was estimated. Eight vertebrate mtDNA sequences had uniformly lower spectra values than did four invertebrate mtDNA sequences. Thus, vertebrate mitochondria show significantly longer-range correlations than to invertebrate mitochondria. The higher multifractal spectra values for invertebrate mitochondria suggest a more random organization of the sequences. This research also includes considerable theoretical work on the effects of finite size, embedding dimension, and scaling ranges.

  19. Fractal Analysis of DNA Sequence Data

    NASA Astrophysics Data System (ADS)

    Berthelsen, Cheryl Lynn

    DNA sequence databases are growing at an almost exponential rate. New analysis methods are needed to extract knowledge about the organization of nucleotides from this vast amount of data. Fractal analysis is a new scientific paradigm that has been used successfully in many domains including the biological and physical sciences. Biological growth is a nonlinear dynamic process and some have suggested that to consider fractal geometry as a biological design principle may be most productive. This research is an exploratory study of the application of fractal analysis to DNA sequence data. A simple random fractal, the random walk, is used to represent DNA sequences. The fractal dimension of these walks is then estimated using the "sandbox method." Analysis of 164 human DNA sequences compared to three types of control sequences (random, base -content matched, and dimer-content matched) reveals that long-range correlations are present in DNA that are not explained by base or dimer frequencies. The study also revealed that the fractal dimension of coding sequences was significantly lower than sequences that were primarily noncoding, indicating the presence of longer-range correlations in functional sequences. The multifractal spectrum is used to analyze fractals that are heterogeneous and have a different fractal dimension for subsets with different scalings. The multifractal spectrum of the random walks of twelve mitochondrial genome sequences was estimated. Eight vertebrate mtDNA sequences had uniformly lower spectra values than did four invertebrate mtDNA sequences. Thus, vertebrate mitochondria show significantly longer-range correlations than do invertebrate mitochondria. The higher multifractal spectra values for invertebrate mitochondria suggest a more random organization of the sequences. This research also includes considerable theoretical work on the effects of finite size, embedding dimension, and scaling ranges.

  20. Non-intercalative, deoxyribose binding of boric acid to calf thymus DNA.

    PubMed

    Ozdemir, Ayse; Gursaclı, Refiye Tekiner; Tekinay, Turgay

    2014-05-01

    The present study characterizes the effects of the boric acid binding on calf thymus DNA (ct-DNA) by spectroscopic and calorimetric methods. UV-Vis absorbance spectroscopy, circular dichroism (CD) spectroscopy, transmission electron microscopy (TEM), isothermal titration calorimetry (ITC), and Fourier transform infrared (FT-IR) spectroscopy were employed to characterize binding properties. Changes in the secondary structure of ct-DNA were determined by CD spectroscopy. Sizes and morphologies of boric acid-DNA complexes were determined by transmission electron microscopy (TEM). The kinetics of boric acid binding to calf thymus DNA (ct-DNA) was investigated by isothermal titration calorimetry (ITC). ITC results revealed that boric acid exhibits a moderate affinity to ct-DNA with a binding constant (K a) of 9.54 × 10(4) M(-1). FT-IR results revealed that boric acid binds to the deoxyribose sugar of DNA without disrupting the B-conformation at tested concentrations.

  1. A DNA origami nanorobot controlled by nucleic acid hybridization.

    PubMed

    Torelli, Emanuela; Marini, Monica; Palmano, Sabrina; Piantanida, Luca; Polano, Cesare; Scarpellini, Alice; Lazzarino, Marco; Firrao, Giuseppe

    2014-07-23

    A prototype for a DNA origami nanorobot is designed, produced, and tested. The cylindrical nanorobot (diameter of 14 nm and length of 48 nm) with a switchable flap, is able to respond to an external stimulus and reacts by a physical switch from a disarmed to an armed configuration able to deliver a cellular compatible message. In the tested design the robot weapon is a nucleic acid fully contained in the inner of the tube and linked to a single point of the internal face of the flap. Upon actuation the nanorobot moves the flap extracting the nucleic acid that assembles into a hemin/G-quadruplex horseradish peroxidase mimicking DNAzyme catalyzing a colorimetric reaction or chemiluminescence generation. The actuation switch is triggered by an external nucleic acid (target) that interacts with a complementary nucleic acid that is beard externally by the nanorobot (probe). Hybridization of probe and target produces a localized structural change that results in flap opening. The flap movement is studied on a two-dimensional prototype origami using Förster resonance energy transfer and is shown to be triggered by a variety of targets, including natural RNAs. The nanorobot has potential for in vivo biosensing and intelligent delivery of biological activators.

  2. Evaluation of DNA typing as a positive identification method for soft and hard tissues immersed in strong acids.

    PubMed

    Robino, C; Pazzi, M; Di Vella, G; Martinelli, D; Mazzola, L; Ricci, U; Testi, R; Vincenti, M

    2015-11-01

    Identification of human remains can be hindered by several factors (e.g., traumatic mutilation, carbonization or decomposition). Moreover, in some criminal cases, offenders may purposely adopt various expedients to thwart the victim's identification, including the dissolution of body tissues by the use of corrosive reagents, as repeatedly reported in the past for Mafia-related murders. By means of an animal model, namely porcine samples, we evaluated standard DNA typing as a method for identifying soft (muscle) and hard (bone and teeth) tissues immersed in strong acids (hydrochloric, nitric and sulfuric acid) or in mixtures of acids (aqua regia). Samples were tested at different time intervals, ranging between 2 and 6h (soft tissues) and 2-28 days (hard tissues). It was shown that, in every type of acid, complete degradation of the DNA extracted from soft tissues preceded tissue dissolution and could be observed within 4h of immersion. Conversely, high molecular weight DNA amenable to STR analysis could be isolated from hard tissues as long as cortical bone fragments were still present (28 days for sulfuric acid, 7 days for nitric acid, 2 days for hydrochloric acid and aqua regia), or the integrity of the dental pulp chamber was preserved (7 days, in sulfuric acid only). The results indicate that DNA profiling of acid-treated body parts (in particular, cortical bone) is still feasible at advanced stages of corrosion, even when the morphological methods used in forensic anthropology and odontology can no longer be applied for identification purposes.

  3. DNA.

    ERIC Educational Resources Information Center

    Felsenfeld, Gary

    1985-01-01

    Structural form, bonding scheme, and chromatin structure of and gene-modification experiments with deoxyribonucleic acid (DNA) are described. Indicates that DNA's double helix is variable and also flexible as it interacts with regulatory and other molecules to transfer hereditary messages. (DH)

  4. Flow cytometric DNA analysis of corneal epithelium.

    PubMed

    Burns, E R; Roberson, M C; Brown, M F; Shock, J P; Pipkin, J L; Hinson, W G; Anson, J F

    1990-03-01

    We have modified an existing technique in order to perform DNA analysis by flow cytometry (FCM) of corneal epithelium from the mouse, rat, chicken, rabbit, and human. This protocol permitted an investigation of human corneal scrapings from several categories: normal, aphakic bullous keratopathy (ABK), keratoconus (KC), Fuch's dystrophy, edema, epithelial dysplasia, and lipid degeneration. No abnormal characteristic cell-kinetic profile was detected when averaged DNA histograms were compared statistically between the normal and either ABK, KC, edema, or Fuch's dystrophy groups. Abnormal DNA histograms were recorded for cell samples that were taken 1) from three individuals who had epithelial dysplasia and 2) from one individual diagnosed with lipid degeneration. The former condition was characterized by histograms that had a subpopulation of cells with an aneuploid amount of DNA or had higher than normal percentages of cells in the S and G2 + M phases of the cell cycle. Corneal cells from the patient who had lipid degeneration had an abnormally high percentage of cells in the G2 + M phases of the cell cycle. The availability of accurate DNA flow cytometric analysis of corneal epithelium allows further studies on this issue from both experimental and clinical situations.

  5. DNA-seq analysis of Garcinia mangostana

    PubMed Central

    Abu Bakar, Syuhaidah; Sampathrajan, Sureshkumar; Loke, Kok-Keong; Goh, Hoe-Han; Mohd Noor, Normah

    2015-01-01

    Mangosteen (Garcinia mangostana Linn.) is a tropical tree mainly found in South East Asia and considered as “the queen of fruits”. The asexually produced fruit is dark purple or reddish in color, with white flesh which is slightly acidic with sweet flavor and a pleasant aroma. The purple pericarp tissue is rich in xanthones which are useful for medical purposes. We performed the first genome sequencing of this commercially important fruit tree to study its genome composition and attempted draft genome assembly. Raw reads of the DNA sequencing project have been deposited to SRA database with the accession number SRX1426419. PMID:26981362

  6. DNA-seq analysis of Garcinia mangostana.

    PubMed

    Abu Bakar, Syuhaidah; Sampathrajan, Sureshkumar; Loke, Kok-Keong; Goh, Hoe-Han; Mohd Noor, Normah

    2016-03-01

    Mangosteen (Garcinia mangostana Linn.) is a tropical tree mainly found in South East Asia and considered as "the queen of fruits". The asexually produced fruit is dark purple or reddish in color, with white flesh which is slightly acidic with sweet flavor and a pleasant aroma. The purple pericarp tissue is rich in xanthones which are useful for medical purposes. We performed the first genome sequencing of this commercially important fruit tree to study its genome composition and attempted draft genome assembly. Raw reads of the DNA sequencing project have been deposited to SRA database with the accession number SRX1426419.

  7. Inhibition of N-nitrosamine carcinogenesis and aflatoxin DNA damage by ellagic acid

    SciTech Connect

    Mandal-Chaudhuri, S.

    1988-01-01

    The effect of ellagic acid (EA), on the tumorigenicity of N-nitrosobenzylmethylamine (NBMA) in the rat esophagus was investigated. Groups of 30 male F-344 rats were fed a semipurified diet containing EA for 27 weeks. N-nitrosobenzylmethylamine was administered subcutaneously, once a week for 18 weeks. Ellagic acid produced a significant inhibition in the average number of esophageal tumors at both 20 weeks and 27 weeks. To investigate the mechanism(s) of this inhibition, EA was tested for its effect on the metabolism, DNA-binding and DNA-adduct formation of NBMA in cultured explants of rat esophagus. Explants were incubated in medium containing EA at concentrations of 10, 50, and 100 {mu}M for 16 hours, followed by the addition of 1{mu}M ({sup 3}H)NBMA and EA for 12 hours. Explant DNA was isolated by phenol extraction and hydroxylapatite chromatography, and benzaldehyde formation was determined by h.p.l.c. analysis of the culture medium. Finally, EA was examined for its ability to inhibit DNA damage induced by aflatoxin B{sub 1} (AFB{sub 1}) in cultured explants of rat trachea and esophagus, and human tracheobronchus.

  8. Ancient DNA analysis of dental calculus.

    PubMed

    Weyrich, Laura S; Dobney, Keith; Cooper, Alan

    2015-02-01

    Dental calculus (calcified tartar or plaque) is today widespread on modern human teeth around the world. A combination of soft starchy foods, changing acidity of the oral environment, genetic pre-disposition, and the absence of dental hygiene all lead to the build-up of microorganisms and food debris on the tooth crown, which eventually calcifies through a complex process of mineralisation. Millions of oral microbes are trapped and preserved within this mineralised matrix, including pathogens associated with the oral cavity and airways, masticated food debris, and other types of extraneous particles that enter the mouth. As a result, archaeologists and anthropologists are increasingly using ancient human dental calculus to explore broad aspects of past human diet and health. Most recently, high-throughput DNA sequencing of ancient dental calculus has provided valuable insights into the evolution of the oral microbiome and shed new light on the impacts of some of the major biocultural transitions on human health throughout history and prehistory. Here, we provide a brief historical overview of archaeological dental calculus research, and discuss the current approaches to ancient DNA sampling and sequencing. Novel applications of ancient DNA from dental calculus are discussed, highlighting the considerable scope of this new research field for evolutionary biology and modern medicine.

  9. DNA adsorption to and elution from silica surfaces: influence of amino acid buffers.

    PubMed

    Vandeventer, Peter E; Mejia, Jorge; Nadim, Ali; Johal, Malkiat S; Niemz, Angelika

    2013-09-19

    Solid phase extraction and purification of DNA from complex samples typically requires chaotropic salts that can inhibit downstream polymerase amplification if carried into the elution buffer. Amino acid buffers may serve as a more compatible alternative for modulating the interaction between DNA and silica surfaces. We characterized DNA binding to silica surfaces, facilitated by representative amino acid buffers, and the subsequent elution of DNA from the silica surfaces. Through bulk depletion experiments, we found that more DNA adsorbs to silica particles out of positively compared to negatively charged amino acid buffers. Additionally, the type of the silica surface greatly influences the amount of DNA adsorbed and the final elution yield. Quartz crystal microbalance experiments with dissipation monitoring (QCM-D) revealed multiphasic DNA adsorption out of stronger adsorbing conditions such as arginine, glycine, and glutamine, with DNA more rigidly bound during the early stages of the adsorption process. The DNA film adsorbed out of glutamate was more flexible and uniform throughout the adsorption process. QCM-D characterization of DNA elution from the silica surface indicates an uptake in water mass during the initial stage of DNA elution for the stronger adsorbing conditions, which suggests that for these conditions the DNA film is partly dehydrated during the prior adsorption process. Overall, several positively charged and polar neutral amino acid buffers show promise as an alternative to methods based on chaotropic salts for solid phase DNA extraction.

  10. Protective Effect of Folic Acid on Oxidative DNA Damage

    PubMed Central

    Guo, Xiaojuan; Cui, Huan; Zhang, Haiyang; Guan, Xiaoju; Zhang, Zheng; Jia, Chaonan; Wu, Jia; Yang, Hui; Qiu, Wenting; Zhang, Chuanwu; Yang, Zuopeng; Chen, Zhu; Mao, Guangyun

    2015-01-01

    Abstract Although previous reports have linked DNA damage with both transmissions across generations as well as our own survival, it is unknown how to reverse the lesion. Based on the data from a Randomized, Double-blind, Placebo Controlled Clinical Trial, this study aimed to assess the efficacy of folic acid supplementation (FAS) on DNA oxidative damage reversal. In this randomized clinical trial (RCT), a total of 450 participants were enrolled and randomly assigned to 3 groups to receive folic acid (FA) 0.4 mg/day (low-FA), 0.8 mg/day (high-FA), or placebo (control) for 8 weeks. The urinary 8-hydroxy-2’-deoxyguanosine (8-OHdG) and creatinine (Cr) concentration at pre- and post-FAS were measured with modified enzyme-linked immunosorbent assay (ELISA) and high-performance liquid chromatography (HPLC), respectively. A multivariate general linear model was applied to assess the individual effects of FAS and the joint effects between FAS and hypercholesterolemia on oxidative DNA damage improvement. This clinical trial was registered with ClinicalTrials.gov, number NCT02235948. Of the 438 subjects that received FA fortification or placebo, the median (first quartile, third quartile) of urinary 8-OHdG/Cr for placebo, low-FA, and high-FA groups were 58.19 (43.90, 82.26), 53.51 (38.97, 72.74), 54.73 (39.58, 76.63) ng/mg at baseline and 57.77 (44.35, 81.33), 51.73 (38.20, 71.30), and 50.65 (37.64, 76.17) ng/mg at the 56th day, respectively. A significant decrease of urinary 8-OHdG was observed after 56 days FA fortification (P < 0.001). Compared with the placebo, after adjusting for some potential confounding factors, including the baseline urinary 8-OHdG/Cr, the urinary 8-OHdG/Cr concentration significantly decreased after 56 days FAS [β (95% confidence interval) = −0.88 (−1.62, −0.14) and P = 0.020 for low-FA; and β (95% confidence interval) = −2.68 (−3.42, −1.94) and P < 0.001 for high-FA] in a dose-response fashion (Ptrend

  11. Phylogenetic analysis of ancient DNA using BEAST.

    PubMed

    Ho, Simon Y W

    2012-01-01

    Under exceptional circumstances, it is possible to obtain DNA sequences from samples that are up to hundreds of thousands of years old. These data provide an opportunity to look directly at past genetic diversity, to trace the evolutionary process through time, and to infer demographic and phylogeographic trends. Ancient DNA (aDNA) data sets have some degree of intrinsic temporal structure because the sequences have been obtained from samples of different ages. When analyzing these data sets, it is usually necessary to take the sampling times into account. A number of phylogenetic methods have been designed with this purpose in mind. Here I describe the steps involved in Bayesian phylogenetic analysis of aDNA data. I outline a procedure that can be used to co-estimate the genealogical relationships, mutation rate, evolutionary timescale, and demographic history of the study species in a single analytical framework. A number of modifications to the methodology can be made in order to deal with complicating factors such as postmortem damage, sequences from undated samples, and data sets with low information content.

  12. Phylogenetic analysis of ancient DNA using BEAST.

    PubMed

    Ho, Simon Y W

    2012-01-01

    Under exceptional circumstances, it is possible to obtain DNA sequences from samples that are up to hundreds of thousands of years old. These data provide an opportunity to look directly at past genetic diversity, to trace the evolutionary process through time, and to infer demographic and phylogeographic trends. Ancient DNA (aDNA) data sets have some degree of intrinsic temporal structure because the sequences have been obtained from samples of different ages. When analyzing these data sets, it is usually necessary to take the sampling times into account. A number of phylogenetic methods have been designed with this purpose in mind. Here I describe the steps involved in Bayesian phylogenetic analysis of aDNA data. I outline a procedure that can be used to co-estimate the genealogical relationships, mutation rate, evolutionary timescale, and demographic history of the study species in a single analytical framework. A number of modifications to the methodology can be made in order to deal with complicating factors such as postmortem damage, sequences from undated samples, and data sets with low information content. PMID:22237538

  13. Ligand-activated PPARα-dependent DNA demethylation regulates the fatty acid β-oxidation genes in the postnatal liver.

    PubMed

    Ehara, Tatsuya; Kamei, Yasutomi; Yuan, Xunmei; Takahashi, Mayumi; Kanai, Sayaka; Tamura, Erina; Tsujimoto, Kazutaka; Tamiya, Takashi; Nakagawa, Yoshimi; Shimano, Hitoshi; Takai-Igarashi, Takako; Hatada, Izuho; Suganami, Takayoshi; Hashimoto, Koshi; Ogawa, Yoshihiro

    2015-03-01

    The metabolic function of the liver changes sequentially during early life in mammals to adapt to the marked changes in nutritional environment. Accordingly, hepatic fatty acid β-oxidation is activated after birth to produce energy from breast milk lipids. However, how it is induced during the neonatal period is poorly understood. Here we show DNA demethylation and increased mRNA expression of the fatty acid β-oxidation genes in the postnatal mouse liver. The DNA demethylation does not occur in the fetal mouse liver under the physiologic condition, suggesting that it is specific to the neonatal period. Analysis of mice deficient in the nuclear receptor peroxisome proliferator-activated receptor α (PPARα) and maternal administration of a PPARα ligand during the gestation and lactation periods reveal that the DNA demethylation is PPARα dependent. We also find that DNA methylation of the fatty acid β-oxidation genes are reduced in the adult human liver relative to the fetal liver. This study represents the first demonstration that the ligand-activated PPARα-dependent DNA demethylation regulates the hepatic fatty acid β-oxidation genes during the neonatal period, thereby highlighting the role of a lipid-sensing nuclear receptor in the gene- and life-stage-specific DNA demethylation of a particular metabolic pathway.

  14. [Amplification, cloning and sequence analysis of spider dragline silk cDNA].

    PubMed

    Zhang, Li-Shu; Ma, He-Wen; Lu, Yi-Ming; Zhang, Yu-Jing

    2002-09-01

    Spider dragline silk is synthesized in special gland named major ampulate (MA) gland. The MA glands were dissected from the abdomen of the spiders Nephila clavata and the total RNA was extracted by the TRIZOL. The cDNA of dragline silk was amplificated by RT-PCR (reverse transcription polymerase chain reaction), multiplex PCR and cloned. PCR identification, restriction analysis and DNA sequence analysis were carried out to verify the recombinant plasmids. The codon usage frequencies of the cloned cDNA were added up, and the predicted amino acid sequence was compared with Spidroin2 of Nephila clavipes. Predicted secondary structure of the predicted amino-acid sequence was analysized by DNAStar software. All results showed that the cloned cDNA we got (GenBank Accession No. AF441245) was the very fragment of spider dragline silk Spidroin2 cDNA.

  15. PLASMID DNA DAMAGE CAUSED BY METHYLATED ARSENICALS, ASCORBIC ACID AND HUMAN LIVER FERRITIN

    EPA Science Inventory

    PLASMID DNA DAMAGE CAOUSED BY METHYLATED ARSENICALS, ASCORBIC ACID AND HUMAN LIVER FERRITIN

    ABSTRACT

    Both dimethylarsinic acid (DMA(V)) and dimethylarsinous acid (DMA(III)) release iron from human liver ferritin (HLF) with or without the presence of ascorbic acid. ...

  16. Applications of cellular fatty acid analysis.

    PubMed Central

    Welch, D F

    1991-01-01

    More than ever, new technology is having an impact on the tools of clinical microbiologists. The analysis of cellular fatty acids by gas-liquid chromatography (GLC) has become markedly more practical with the advent of the fused-silica capillary column, computer-controlled chromatography and data analysis, simplified sample preparation, and a commercially available GLC system dedicated to microbiological applications. Experience with applications in diagnostic microbiology ranges from substantial success in work with mycobacteria, legionellae, and nonfermentative gram-negative bacilli to minimal involvement with fungi and other nonbacterial agents. GLC is a good alternative to other means for the identification of mycobacteria or legionellae because it is rapid, specific, and independent of other specialized testing, e.g., DNA hybridization. Nonfermenters show features in their cellular fatty acid content that are useful in identifying species and, in some cases, subspecies. Less frequently encountered nonfermenters, including those belonging to unclassified groups, can ideally be characterized by GLC. Information is just beginning to materialize on the usefulness of cellular fatty acids for the identification of gram-positive bacteria and anaerobes, despite the traditional role of GLC in detecting metabolic products as an aid to identification of anaerobes. When species identification of coagulase-negative staphylococci is called for, GLC may offer an alternative to biochemical testing. Methods for direct analysis of clinical material have been developed, but in practical and economic terms they are not yet ready for use in the clinical laboratory. Direct analysis holds promise for detecting markers of infection due to an uncultivable agent or in clinical specimens that presently require cultures and prolonged incubation to yield an etiologic agent. PMID:1747860

  17. Analysis of single nucleic acid molecules with protein nanopores

    PubMed Central

    Maglia, Giovanni; Heron, Andrew J.; Stoddart, David; Japrung, Deanpen; Bayley, Hagan

    2011-01-01

    We describe the methods used in our laboratory for the analysis of single nucleic acid molecules with protein nanopores. The technical section is preceded by a review of the variety of experiments that can be done with protein nanopores. The end goal of much of this work is single-molecule DNA sequencing, although sequencing is not discussed explicitly here. The technical section covers the equipment required for nucleic acid analysis, the preparation and storage of the necessary materials, and aspects of signal processing and data analysis. PMID:20627172

  18. Microfabricated structures for integrated DNA analysis.

    PubMed Central

    Burns, M A; Mastrangelo, C H; Sammarco, T S; Man, F P; Webster, J R; Johnsons, B N; Foerster, B; Jones, D; Fields, Y; Kaiser, A R; Burke, D T

    1996-01-01

    Photolithographic micromachining of silicon is a candidate technology for the construction of high-throughput DNA analysis devices. However, the development of complex silicon microfabricated systems has been hindered in part by the lack of a simple, versatile pumping method for integrating individual components. Here we describe a surface-tension-based pump able to move discrete nanoliter drops through enclosed channels using only local heating. This thermocapillary pump can accurately mix, measure, and divide drops by simple electronic control. In addition, we have constructed thermal-cycling chambers, gel electrophoresis channels, and radiolabeled DNA detectors that are compatible with the fabrication of thermocapillary pump channels. Since all of the components are made by conventional photolithographic techniques, they can be assembled into more complex integrated systems. The combination of pump and components into self-contained miniaturized devices may provide significant improvements in DNA analysis speed, portability, and cost. The potential of microfabricated systems lies in the low unit cost of silicon-based construction and in the efficient sample handling afforded by component integration. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:8643614

  19. Capture Hybridization Analysis of DNA Targets.

    PubMed

    Sexton, Alec N; Machyna, Martin; Simon, Matthew D

    2016-01-01

    There are numerous recent cases where chromatin modifying complexes associate with long noncoding RNA (lncRNA), stoking interest in lncRNA genomic localization and associated proteins. Capture Hybridization Analysis of RNA Targets (CHART) uses complementary oligonucleotides to purify an RNA with its associated genomic DNA or proteins from formaldehyde cross-linked chromatin. Deep sequencing of the purified DNA fragments gives a comprehensive profile of the potential lncRNA biological targets in vivo. The combined identification of the genomic localization of RNA and its protein partners can directly inform hypotheses about RNA function, including recruitment of chromatin modifying complexes. Here, we provide a detailed protocol on how to design antisense capture oligos and perform CHART in tissue culture cells. PMID:27659977

  20. Analysis of Electric Properties of DNA Nucleotides

    NASA Astrophysics Data System (ADS)

    Zikic, R.; Zhang, X.-G.; Krstic, P. S.; Wells, J. C.; Fuentes-Cabrera, M.

    2006-05-01

    Calculation of the quantum tunnelling conductance through the DNA nucleotides between gold nanoelectrodes and analysis of the corresponding molecular spectra reveals that the tunneling conductance at low electric bias can be separated into two simple and approximately independent factors. The first is an exponential factor due to the potential barrier between the molecule and the electrode. The second factor is different for each molecule, but follows a universal form that can be expressed in terms of the bending angle of the DNA base relative to the sugar-phosphate group. This factor is also oscillatory indicating interference and resonance effects inside the molecule. Distinguishable conductances of Adenine (A), Cytosine (C), Guanine (G), and Thymine (T) are correlated to their differences in geometric dimensions.

  1. Inhibition of DNA methylation by caffeic acid and chlorogenic acid, two common catechol-containing coffee polyphenols.

    PubMed

    Lee, Won Jun; Zhu, Bao Ting

    2006-02-01

    We studied the modulating effects of caffeic acid and chlorogenic acid (two common coffee polyphenols) on the in vitro methylation of synthetic DNA substrates and also on the methylation status of the promoter region of a representative gene in two human cancer cells lines. Under conditions that were suitable for the in vitro enzymatic methylation of DNA and dietary catechols, we found that the presence of caffeic acid or chlorogenic acid inhibited in a concentration-dependent manner the DNA methylation catalyzed by prokaryotic M.SssI DNA methyltransferase (DNMT) and human DNMT1. The IC50 values of caffeic acid and chlorogenic acid were 3.0 and 0.75 microM, respectively, for the inhibition of M.SssI DNMT-mediated DNA methylation, and were 2.3 and 0.9 microM, respectively, for the inhibition of human DNMT1-mediated DNA methylation. The maximal in vitro inhibition of DNA methylation was approximately 80% when the highest concentration (20 microM) of caffeic acid or chlorogenic acid was tested. Kinetic analyses showed that DNA methylation catalyzed by M.SssI DNMT or human DNMT1 followed the Michaelis-Menten curve patterns. The presence of caffeic acid or chlorogenic acid inhibited DNA methylation predominantly through a non-competitive mechanism, and this inhibition was largely due to the increased formation of S-adenosyl-L-homocysteine (SAH, a potent inhibitor of DNA methylation), resulting from the catechol-O-methyltransferase (COMT)-mediated O-methylation of these dietary catechols. Using cultured MCF-7 and MAD-MB-231 human breast cancer cells, we also demonstrated that treatment of these cells with caffeic acid or chlorogenic acid partially inhibited the methylation of the promoter region of the RARbeta gene. The findings of our present study provide a general mechanistic basis for the notion that a variety of dietary catechols can function as inhibitors of DNA methylation through increased formation of SAH during the COMT-mediated O-methylation of these dietary

  2. [Comparative analysis of 2 methods of DNA extraction].

    PubMed

    Sánchez García, I; Martín Seisdedos, C; Pérez Grande, R; Corral de la Calle, J; Rodríguez Arias, C; González-Sarmiento, R

    1990-10-01

    The study of the organization of immunoglobulin and T cell receptor genes in leukaemias and lymphomas depends on our ability to obtain high molecular weight DNA. We have compared two different methods of DNA extraction: one being enzymatic, using the enzyme proteinase K, and the other chemical, using urea as a substance purifying DNA. Since the amount and purity of the DNA obtained are similar with each of the two methods of DNA extraction, both are valid to make such a genetic analysis.

  3. Molecular beacons: a novel DNA probe for nucleic acid and protein studies.

    PubMed

    Tan, W; Fang, X; Li, J; Liu, X

    2000-04-01

    A new concept has been introduced for molecular beacon DNA molecules. Molecular beacons are a new class of oligonucleotides that can report the presence of specific nucleic acids in both homogeneous solutions and at the liquid-solid interface. They emit an intense fluorescent signal only when hybridized to their target DNA or RNA molecules. Biotinylated molecular beacons have been designed and used for the development of ultrasensitive DNA sensors and for DNA molecular interaction studies at a solid-liquid interface. Molecular beacons have also been used to study protein-DNA interactions. They have provided a variety of exciting opportunities in DNA/RNA/protein studies.

  4. Food Fish Identification from DNA Extraction through Sequence Analysis

    ERIC Educational Resources Information Center

    Hallen-Adams, Heather E.

    2015-01-01

    This experiment exposed 3rd and 4th y undergraduates and graduate students taking a course in advanced food analysis to DNA extraction, polymerase chain reaction (PCR), and DNA sequence analysis. Students provided their own fish sample, purchased from local grocery stores, and the class as a whole extracted DNA, which was then subjected to PCR,…

  5. Chimeric RNA-DNA molecular beacons for quantification of nucleic acids, single nucleotide polymophisms, and nucleic acid damage.

    PubMed

    El-Yazbi, Amira F; Loppnow, Glen R

    2013-05-01

    Single nucleotide polymorphisms (SNPs) are the main cause for variations in the human genome. DNA lesions, such as cyclobutane pyrimidine dimers (CPDs), [6-4] pyrimidine-pyrimidinones, dewar pyrimidinones, and photohydrates, can subsequently lead to mutagenesis, carcinogenesis, and cell death. Much effort has focused on methods for detecting DNA, SNPs, or damaged nucleic acids. However, almost all of the proposed methods consist of multistep procedures, are limited to specific types of damage, some of these methods require expensive instruments, and some suffer from a high level of interferences. In this paper, we present a novel, simple, mix-and-read assay for the detection of nucleic acids that is general for all types of SNPs and nucleic acid damage. This method uses a chimeric RNA-DNA molecular beacon (chMB). The calibration curve of the chMB for detecting single base mismatch and ultraviolet (UV)-induced DNA damage shows good linearity (R(2) = 0.981 and 0.996, respectively) and limits of detection of 10.4 ± 2.2 and 8.64 ± 1.2 nM, respectively. The chimeric RNA-DNA MB proves to be a more sensitive and selective tool for the quantification of nucleic acids, DNA mismatches, and UV-induced DNA damage than DNA MBs.

  6. Highly sensitive fluorescence quantitative detection of specific DNA sequences with molecular beacons and nucleic acid dye SYBR Green I.

    PubMed

    Xiang, Dongshan; Zhai, Kun; Xiang, Wenjun; Wang, Lianzhi

    2014-11-01

    A highly sensitive fluorescence method of quantitative detection for specific DNA sequence is developed based on molecular beacon (MB) and nucleic acid dye SYBR Green I by synchronous fluorescence analysis. It is demonstrated by an oligonucleotide sequence of wild-type HBV (target DNA) as a model system. In this strategy, the fluorophore of MB is designed to be 6-carboxyfluorescein group (FAM), and the maximum excitation wavelength and maximum emission wavelength are both very close to that of SYBR Green I. In the presence of targets DNA, the MBs hybridize with the targets DNA and form double-strand DNA (dsDNA), the fluorophore FAM is separated from the quencher BHQ-1, thus the fluorophore emit fluorescence. At the same time, SYBR Green I binds to dsDNA, the fluorescence intensity of SYBR Green I is significantly enhanced. When targets DNA are detected by synchronous fluorescence analysis, the fluorescence peaks of FAM and SYBR Green I overlap completely, so the fluorescence signal of system will be significantly enhanced. Thus, highly sensitive fluorescence quantitative detection for DNA can be realized. Under the optimum conditions, the total fluorescence intensity of FAM and SYBR Green I exhibits good linear dependence on concentration of targets DNA in the range from 2×10(-11) to 2.5×10(-9)M. The detection limit of target DNA is estimated to be 9×10(-12)M (3σ). Compared with previously reported methods of detection DNA with MB, the proposed method can significantly enhance the detection sensitivity.

  7. DNA analysis of epithelial cell suspensions

    SciTech Connect

    Wilson, J.S.; Johnson, N.F.; Holland, L.M.

    1985-01-01

    Cell suspensions of skin were obtained by animals exposed by skin painting of several crude oils. DNA analysis of these cell suspensions labeled with mithramycin provide determination of percentages of cells in the G/sub 1/, S and G/sub 2/M phases of the cell cycle. Data acquired showed differences from control animals occurring as early as 7 days after treatment and persisting through 21 days afterwards. There was histological evidence of erythema and hyperplasia in shale oil-exposed skins. Flow cytometric analysis of DNA content in shale-oil-exposed skin cells showed an increased percentage of cycling cells plus evidence of aneuploidy. Similar data from simply abraded skin showed increased percentages of cycling cells, but no aneuploidy. The shale-oil-exposed group, when compared to a standard petroleum-exposed group, had significantly increased percentages of cycling cells. This early indication of differing response to different complex mixtures was also seen in long-term skin exposures to these compounds. Similar analytical techniques were applied to tracheal cell suspensions from ozone-exposed rats. 12 refs., 4 figs., 4 tabs. (DT)

  8. Method for nucleic acid hybridization using single-stranded DNA binding protein

    DOEpatents

    Tabor, Stanley; Richardson, Charles C.

    1996-01-01

    Method of nucleic acid hybridization for detecting the presence of a specific nucleic acid sequence in a population of different nucleic acid sequences using a nucleic acid probe. The nucleic acid probe hybridizes with the specific nucleic acid sequence but not with other nucleic acid sequences in the population. The method includes contacting a sample (potentially including the nucleic acid sequence) with the nucleic acid probe under hybridizing conditions in the presence of a single-stranded DNA binding protein provided in an amount which stimulates renaturation of a dilute solution (i.e., one in which the t.sub.1/2 of renaturation is longer than 3 weeks) of single-stranded DNA greater than 500 fold (i.e., to a t.sub.1/2 less than 60 min, preferably less than 5 min, and most preferably about 1 min.) in the absence of nucleotide triphosphates.

  9. GENETIC AND MOLECULAR ANALYSIS OF DNA DAMAGE REPAIR AND TOLERANCE PATHWAYS.

    SciTech Connect

    SUTHERLAND, B.M.

    2001-07-26

    Radiation can damage cellular components, including DNA. Organisms have developed a panoply of means of dealing with DNA damage. Some repair paths have rather narrow substrate specificity (e.g. photolyases), which act on specific pyrimidine photoproducts in a specific type (e.g., DNA) and conformation (double-stranded B conformation) of nucleic acid. Others, for example, nucleotide excision repair, deal with larger classes of damages, in this case bulky adducts in DNA. A detailed discussion of DNA repair mechanisms is beyond the scope of this article, but one can be found in the excellent book of Friedberg et al. [1] for further detail. However, some DNA damages and paths for repair of those damages important for photobiology will be outlined below as a basis for the specific examples of genetic and molecular analysis that will be presented below.

  10. Carcinoma of the anal canal and flow cytometric DNA analysis.

    PubMed Central

    Scott, N. A.; Beart, R. W.; Weiland, L. H.; Cha, S. S.; Lieber, M. M.

    1989-01-01

    Using flow cytometric DNA analysis of paraffin embedded tissue, DNA histograms were successfully obtained from the anal cancers of 117 patients. DNA diploid patterns were given by 82 cancers (70%) and DNA non-diploid patterns by 35 cancers (30%): 15 DNA aneuploid, 20 DNA tetraploid. Well differentiated squamous cell cancers were mainly DNA diploid, while a larger proportion of poorly differentiated and small cell cancers were DNA non-diploid. The large majority of stage A cancers were DNA diploid. A greater proportion of tumours that had invaded through the anal sphincter or had lymph node metastases or distant spread were DNA non-diploid. Prognosis was slightly poorer for patients with DNA non-diploid cancers when compared to patients with DNA diploid tumours (P = 0.08) and significantly poorer for individuals with DNA aneuploid anal cancers (P = 0.037). However, in a multivariate analysis model, the DNA ploidy pattern of an anal cancer was not of independent prognostic significance alongside tumour histology and tumour stage. PMID:2803916

  11. Nucleic acid chemistry in the organic phase: from functionalized oligonucleotides to DNA side chain polymers.

    PubMed

    Liu, Kai; Zheng, Lifei; Liu, Qing; de Vries, Jan Willem; Gerasimov, Jennifer Y; Herrmann, Andreas

    2014-10-01

    DNA-incorporating hydrophobic moieties can be synthesized by either solid-phase or solution-phase coupling. On a solid support the DNA is protected, and hydrophobic units are usually attached employing phosphoramidite chemistry involving a DNA synthesizer. On the other hand, solution coupling in aqueous medium results in low yields due to the solvent incompatibility of DNA and hydrophobic compounds. Hence, the development of a general coupling method for producing amphiphilic DNA conjugates with high yield in solution remains a major challenge. Here, we report an organic-phase coupling strategy for nucleic acid modification and polymerization by introducing a hydrophobic DNA-surfactant complex as a reactive scaffold. A remarkable range of amphiphile-DNA structures (DNA-pyrene, DNA-triphenylphosphine, DNA-hydrocarbon, and DNA block copolymers) and a series of new brush-type DNA side-chain homopolymers with high DNA grafting density are produced efficiently. We believe that this method is an important breakthrough in developing a generalized approach to synthesizing functional DNA molecules for self-assembly and related technological applications.

  12. Knowledge on DNA Success Rates to Optimize the DNA Analysis Process: From Crime Scene to Laboratory.

    PubMed

    Mapes, Anna A; Kloosterman, Ate D; van Marion, Vincent; de Poot, Christianne J

    2016-07-01

    DNA analysis has become an essential intelligence tool in the criminal justice system for the identification of possible offenders. However, it appears that about half of the processed DNA samples contains too little DNA for analysis. This study looks at DNA success rates within 28 different categories of trace exhibits and relates the DNA concentration to the characteristics of the DNA profile. Data from 2260 analyzed crime samples show that cigarettes, bloodstains, and headwear have relatively high success rates. Cartridge cases, crowbars, and tie-wraps are on the other end of the spectrum. These objective data can assist forensics in their selection process.The DNA success probability shows a positive relation with the DNA concentration. This finding enables the laboratory to set an evidence-based threshold value in the DNA analysis process. For instance, 958 DNA extracts had a concentration value of 6 pg/μL or less. Only 46 of the 958 low-level extracts provided meaningful DNA profiling data. PMID:27364287

  13. Site-Selective Binding of Nanoparticles to Double-Stranded DNA via Peptide Nucleic Acid "Invasion"

    SciTech Connect

    Stadler, A.L.; van der Lelie, D.; Sun, D.; Maye, M. M.; Gang, O.

    2011-04-01

    We demonstrate a novel method for by-design placement of nano-objects along double-stranded (ds) DNA. A molecular intercalator, designed as a peptide nucleic acid (PNA)-DNA chimera, is able to invade dsDNA at the PNA-side due to the hybridization specificity between PNA and one of the duplex strands. At the same time, the single-stranded (ss) DNA tail of the chimera, allows for anchoring of nano-objects that have been functionalized with complementary ssDNA. The developed method is applied for interparticle attachment and for the fabrication of particle clusters using a dsDNA template. This method significantly broadens the molecular toolbox for constructing nanoscale systems by including the most conventional not yet utilized DNA motif, double helix DNA.

  14. DETECTION OF DNA DAMAGE USING MELTING ANALYSIS TECHNIQUES

    EPA Science Inventory

    A rapid and simple fluorescence screening assay for UV radiation-, chemical-, and enzyme-induced DNA damage is reported. This assay is based on a melting/annealing analysis technique and has been used with both calf thymus DNA and plasmid DNA (puc 19 plasmid from E. coli). DN...

  15. Fractal landscape analysis of DNA walks

    NASA Astrophysics Data System (ADS)

    Peng, C.-K.; Buldyrev, S. V.; Goldberger, A. L.; Havlin, S.; Sciortino, F.; Simons, M.; Stanley, H. E.

    1992-12-01

    By mapping nucleotide sequences onto a “DNA walk”, we uncovered remarkably long-range power law correlations [Nature 356 (1992) 168] that simply a new scale invariant property of DNA. We found such long-range correlations in intron-containing genes and in non-transcribed regulatory DNA sequences, but not in cDNA sequences or intron-less genes. In this paper, we present more explicit evidences to support our findings.

  16. Fractal landscape analysis of DNA walks

    NASA Technical Reports Server (NTRS)

    Peng, C. K.; Buldyrev, S. V.; Goldberger, A. L.; Havlin, S.; Sciortino, F.; Simons, M.; Stanley, H. E.

    1992-01-01

    By mapping nucleotide sequences onto a "DNA walk", we uncovered remarkably long-range power law correlations [Nature 356 (1992) 168] that imply a new scale invariant property of DNA. We found such long-range correlations in intron-containing genes and in non-transcribed regulatory DNA sequences, but not in cDNA sequences or intron-less genes. In this paper, we present more explicit evidences to support our findings.

  17. Comprehensive analysis of hydrogen bonds in regulatory protein DNA-complexes: in search of common principles.

    PubMed

    Mandel-Gutfreund, Y; Schueler, O; Margalit, H

    1995-10-20

    A systematic analysis of hydrogen bonds between regulatory proteins and their DNA targets is presented, based on 28 crystallographically solved complexes. All possible hydrogen bonds were screened and classified into different types: those that involve the amino acid side-chains and DNA base edges and those that involve the backbone atoms of the molecules. For each interaction type, all bonds were characterized and a statistical analysis was performed to reveal significant amino acid-base interdependence. The interactions between the amino acid side-chains and DNA backbone constitute about half of the interactions, but did not show any amino acid-base correlation. Interactions via the protein backbone were also observed, predominantly with the DNA backbone. As expected, the most significant pairing preference was demonstrated for interactions between the amino acid side-chains and the DNA base edges. The statistically significant relationships could mostly be explained by the chemical nature of the participants. However, correlations that could not be trivially predicted from the hydrogen bonding potential of the residues were also identified, like the preference of lysine for guanine over adenine, or the preference of glutamic acid for cystosine over adenine. While Lys x G interactions were very frequent and spread over various families, the Glu x C interactions were found mainly in the basic helix-loop-helix family. Further examination of the side-chain-base edge contacts at the atomic level revealed a trend of the amino acids to contact the DNA by their donor atoms, preferably at position W2 in the major groove. In most cases it seems that the interactions are not guided simply by the presence of a required atom in a specific position in the groove, but that the identity of the base possessing this atom is crucial. This may have important implications in molecular design experiments.

  18. A High Phosphorus Diet Affects Lipid Metabolism in Rat Liver: A DNA Microarray Analysis.

    PubMed

    Chun, Sunwoo; Bamba, Takeshi; Suyama, Tatsuya; Ishijima, Tomoko; Fukusaki, Eiichiro; Abe, Keiko; Nakai, Yuji

    2016-01-01

    A high phosphorus (HP) diet causes disorders of renal function, bone metabolism, and vascular function. We previously demonstrated that DNA microarray analysis is an appropriate method to comprehensively evaluate the effects of a HP diet on kidney dysfunction such as calcification, fibrillization, and inflammation. We reported that type IIb sodium-dependent phosphate transporter is significantly up-regulated in this context. In the present study, we performed DNA microarray analysis to investigate the effects of a HP diet on the liver, which plays a pivotal role in energy metabolism. DNA microarray analysis was performed with total RNA isolated from the livers of rats fed a control diet (containing 0.3% phosphorus) or a HP diet (containing 1.2% phosphorus). Gene Ontology analysis of differentially expressed genes (DEGs) revealed that the HP diet induced down-regulation of genes involved in hepatic amino acid catabolism and lipogenesis, while genes related to fatty acid β-oxidation process were up-regulated. Although genes related to fatty acid biosynthesis were down-regulated in HP diet-fed rats, genes important for the elongation and desaturation reactions of omega-3 and -6 fatty acids were up-regulated. Concentrations of hepatic arachidonic acid and eicosapentaenoic acid were increased in HP diet-fed rats. These essential fatty acids activate peroxisome proliferator-activated receptor alpha (PPARα), a transcription factor for fatty acid β-oxidation. Evaluation of the upstream regulators of DEGs using Ingenuity Pathway Analysis indicated that PPARα was activated in the livers of HP diet-fed rats. Furthermore, the serum concentration of fibroblast growth factor 21, a hormone secreted from the liver that promotes fatty acid utilization in adipose tissue as a PPARα target gene, was higher (p = 0.054) in HP diet-fed rats than in control diet-fed rats. These data suggest that a HP diet enhances energy expenditure through the utilization of free fatty acids

  19. A High Phosphorus Diet Affects Lipid Metabolism in Rat Liver: A DNA Microarray Analysis

    PubMed Central

    Chun, Sunwoo; Bamba, Takeshi; Suyama, Tatsuya; Ishijima, Tomoko; Fukusaki, Eiichiro; Abe, Keiko; Nakai, Yuji

    2016-01-01

    A high phosphorus (HP) diet causes disorders of renal function, bone metabolism, and vascular function. We previously demonstrated that DNA microarray analysis is an appropriate method to comprehensively evaluate the effects of a HP diet on kidney dysfunction such as calcification, fibrillization, and inflammation. We reported that type IIb sodium-dependent phosphate transporter is significantly up-regulated in this context. In the present study, we performed DNA microarray analysis to investigate the effects of a HP diet on the liver, which plays a pivotal role in energy metabolism. DNA microarray analysis was performed with total RNA isolated from the livers of rats fed a control diet (containing 0.3% phosphorus) or a HP diet (containing 1.2% phosphorus). Gene Ontology analysis of differentially expressed genes (DEGs) revealed that the HP diet induced down-regulation of genes involved in hepatic amino acid catabolism and lipogenesis, while genes related to fatty acid β-oxidation process were up-regulated. Although genes related to fatty acid biosynthesis were down-regulated in HP diet-fed rats, genes important for the elongation and desaturation reactions of omega-3 and -6 fatty acids were up-regulated. Concentrations of hepatic arachidonic acid and eicosapentaenoic acid were increased in HP diet-fed rats. These essential fatty acids activate peroxisome proliferator-activated receptor alpha (PPARα), a transcription factor for fatty acid β-oxidation. Evaluation of the upstream regulators of DEGs using Ingenuity Pathway Analysis indicated that PPARα was activated in the livers of HP diet-fed rats. Furthermore, the serum concentration of fibroblast growth factor 21, a hormone secreted from the liver that promotes fatty acid utilization in adipose tissue as a PPARα target gene, was higher (p = 0.054) in HP diet-fed rats than in control diet-fed rats. These data suggest that a HP diet enhances energy expenditure through the utilization of free fatty acids

  20. Homodinuclear lanthanide complexes of phenylthiopropionic acid: synthesis, characterization, cytotoxicity, DNA cleavage, and antimicrobial activity.

    PubMed

    Shiju, C; Arish, D; Kumaresan, S

    2013-03-15

    Lanthanide complexes of La(III), Pr(III), Nd(III), Sm(III), and Ho(III) with phenylthiopropionic acid were synthesized and characterized by elemental analysis, mass, IR, electronic spectra, molar conductance, TGA, and powder XRD. The results show that the lanthanide complexes are homodinuclear in nature. The two lanthanide ions are bridged by eight oxygen atoms from four carboxylate groups. Thermal decomposition profiles are consistent with the proposed formulations. Powder XRD studies show that all the complexes are amorphous in nature. Antimicrobial studies indicate that these complexes exhibit more activity than the ligand itself. The DNA cleavage activity of the ligand and its complexes were assayed on Escherichia coli DNA using gel electrophoresis in the presence of H(2)O(2). The result shows that the Pr(III) and Nd(III) complexes have completely cleaved the DNA. The anticancer activities of the complexes have also been studied towards human cervical cancer cell line (HeLa) and colon cancer cells (HCT116) and it was found that the La(III) and Nd(III) complexes are more active than the corresponding Pr(III), Sm(III), Ho(III) complexes, and the free ligand on both the cancer cells.

  1. Homodinuclear lanthanide complexes of phenylthiopropionic acid: Synthesis, characterization, cytotoxicity, DNA cleavage, and antimicrobial activity

    NASA Astrophysics Data System (ADS)

    Shiju, C.; Arish, D.; Kumaresan, S.

    2013-03-01

    Lanthanide complexes of La(III), Pr(III), Nd(III), Sm(III), and Ho(III) with phenylthiopropionic acid were synthesized and characterized by elemental analysis, mass, IR, electronic spectra, molar conductance, TGA, and powder XRD. The results show that the lanthanide complexes are homodinuclear in nature. The two lanthanide ions are bridged by eight oxygen atoms from four carboxylate groups. Thermal decomposition profiles are consistent with the proposed formulations. Powder XRD studies show that all the complexes are amorphous in nature. Antimicrobial studies indicate that these complexes exhibit more activity than the ligand itself. The DNA cleavage activity of the ligand and its complexes were assayed on Escherichia coli DNA using gel electrophoresis in the presence of H2O2. The result shows that the Pr(III) and Nd(III) complexes have completely cleaved the DNA. The anticancer activities of the complexes have also been studied towards human cervical cancer cell line (HeLa) and colon cancer cells (HCT116) and it was found that the La(III) and Nd(III) complexes are more active than the corresponding Pr(III), Sm(III), Ho(III) complexes, and the free ligand on both the cancer cells.

  2. Peptide nucleic acid-fluorescence in situ hybridization (PNA-FISH) assay for specific detection of Mycobacterium immunogenum and DNA-FISH assay for analysis of pseudomonads in metalworking fluids and sputum.

    PubMed

    Selvaraju, Suresh B; Kapoor, Renuka; Yadav, Jagjit S

    2008-01-01

    Specific and rapid detection and quantification of mycobacteria in contaminated metalworking fluid (MWF) are problematic due to complexity of the matrix and heavy background co-occurring microflora. Furthermore, cross-reactivity among neighboring species of Mycobacterium makes species differentiation difficult for this genus. Here, we report for the first time a species-specific peptide nucleic acid-fluorescence in situ hybridization (PNA-FISH) method for Mycobacterium immunogenum, a non-tuberculous Mycobacterium species prevalent in MWF and implicated in occupational lung disease hypersensitivity pneumonitis and pseudo-outbreaks. A novel species-specific 14-bp PNA probe was designed for M. immunogenum based on its 16S rRNA gene sequence and was validated for specificity, by testing against a panel of other phylogenetically closely related rapidly growing mycobacteria and representative species of gram-positive, gram-negative, and acid fast organisms. In addition, a DNA-FISH protocol was optimized for co-detection of Pseudomonas, the most predominantly co-occurring genus in contaminated MWF. Reliable quantification for both the test organisms was achieved at or above a cell density of 10(3)cellsml(-1), a recognized minimum limit for microscopic quantification. The mycobacterial PNA-FISH assay was successfully adapted to human sputum demonstrating its potential for clinical diagnostic applications in addition to industrial MWF monitoring, to assess MWF-associated exposures and pseudo-outbreaks.

  3. The future of forensic DNA analysis.

    PubMed

    Butler, John M

    2015-08-01

    The author's thoughts and opinions on where the field of forensic DNA testing is headed for the next decade are provided in the context of where the field has come over the past 30 years. Similar to the Olympic motto of 'faster, higher, stronger', forensic DNA protocols can be expected to become more rapid and sensitive and provide stronger investigative potential. New short tandem repeat (STR) loci have expanded the core set of genetic markers used for human identification in Europe and the USA. Rapid DNA testing is on the verge of enabling new applications. Next-generation sequencing has the potential to provide greater depth of coverage for information on STR alleles. Familial DNA searching has expanded capabilities of DNA databases in parts of the world where it is allowed. Challenges and opportunities that will impact the future of forensic DNA are explored including the need for education and training to improve interpretation of complex DNA profiles.

  4. The future of forensic DNA analysis

    PubMed Central

    Butler, John M.

    2015-01-01

    The author's thoughts and opinions on where the field of forensic DNA testing is headed for the next decade are provided in the context of where the field has come over the past 30 years. Similar to the Olympic motto of ‘faster, higher, stronger’, forensic DNA protocols can be expected to become more rapid and sensitive and provide stronger investigative potential. New short tandem repeat (STR) loci have expanded the core set of genetic markers used for human identification in Europe and the USA. Rapid DNA testing is on the verge of enabling new applications. Next-generation sequencing has the potential to provide greater depth of coverage for information on STR alleles. Familial DNA searching has expanded capabilities of DNA databases in parts of the world where it is allowed. Challenges and opportunities that will impact the future of forensic DNA are explored including the need for education and training to improve interpretation of complex DNA profiles. PMID:26101278

  5. [DNA extraction methods of compost for molecular ecology analysis].

    PubMed

    Yang, Zhao-Hui; Xiao, Yong; Zeng, Guang-Ming; Liu, Yun-Guo; Deng, Jiu-Hua

    2006-08-01

    Molecular ecology provides new techniques for studying compost microbes, and the DNA extraction is the basis of molecular techniques. Because of the contamination of humic acids, it turns to be more difficult for compost microbial DNA extraction. Three different approaches, named as lysozyme lysis, ultrasonic lysis and proteinase K lysis with CTAB, were used to extract the total DNA from compost. The detection performed on a nucleic acids and protein analyzer showed that all the three approaches produced high DNA yields. The agarose gel electrophoresis showed that the DNA fragments extracted from compost had a length of about 23 kb. A eubacterial 16S rRNA gene targeted primer pair (27F and 1 495R) was used for PCR amplification, and all the samples got almost the full length 16S rDNA sequence (about 1.5 kb). After digested by restriction endonucleases (Hae Ill and Alu I), the restriction map showed relatively identical microbial diversity in the DNA, which was extracted by the three different approaches. All the compost microbial DNA extracted by the three different approaches could be used for molecular ecological study, and researchers should choose the right approach for extracting microbial DNA from compost based on the facts.

  6. DNA interaction with octahedral and square planar Ni(II) complexes of aspartic-acid Schiff-bases

    NASA Astrophysics Data System (ADS)

    Sallam, S. A.; Orabi, A. S.; Abbas, A. M.

    2011-12-01

    Ni(II) complexes of (S,E)-2-(2-OHbenzilydene)aspartic acid; (S,E)-2-(2,3-diOHbenzilydene)aspartic acid-; (S,E)-2-(2,4-diOH-benzilydene)aspartic acid; (S,E)-2-(2,5-diOHbenzilydene)aspartic acid and (S,E)-2-((2-OHnaphthalene-1-yl)methylene)aspartic acid Schiff-bases have been synthesized by template method in ethanol or ammonia media. They were characterized by elemental analyses, conductivity measurements, magnetic moment, UV, IR and 1H nmr spectra as well as thermal analysis (TG, DTG, DTA). The Schiff-bases are dibasic tridentate or tetradentate donors and the complexes have square planar and octahedral structures. The complexes decompose in two or three steps where kinetic and thermodynamic parameters of the decomposition steps were computed. The interactions of the formed complexes with FM-DNA were monitored by UV and fluorescence spectroscopy.

  7. Analysis of Translesion DNA Synthesis by the Mitochondrial DNA Polymerase γ.

    PubMed

    Copeland, William C; Kasiviswanathan, Rajesh; Longley, Matthew J

    2016-01-01

    Mitochondrial DNA is replicated by the nuclear-encoded DNA polymerase γ (pol γ) which is composed of a single 140 kDa catalytic subunit and a dimeric 55 kDa accessory subunit. Mitochondrial DNA is vulnerable to various forms of damage, including several types of oxidative lesions, UV-induced photoproducts, chemical adducts from environmental sources, as well as alkylation and inter-strand cross-links from chemotherapy agents. Although many of these lesions block DNA replication, pol γ can bypass some lesions by nucleotide incorporation opposite a template lesion and further extension of the DNA primer past the lesion. This process of translesion synthesis (TLS) by pol γ can occur in either an error-free or an error-prone manner. Assessment of TLS requires extensive analysis of oligonucleotide substrates and replication products by denaturing polyacrylamide sequencing gels. This chapter presents protocols for the analysis of translesion DNA synthesis.

  8. Analysis of Translesion DNA Synthesis by the Mitochondrial DNA Polymerase γ

    PubMed Central

    Copeland, William C.; Kasiviswanathan, Rajesh; Longley, Matthew J.

    2016-01-01

    Summary Mitochondrial DNA is replicated by the nuclear encoded DNA polymerase γ (pol γ) which is composed of a single 140 kDa catalytic subunit and a dimeric 55 kDa accessory subunit. Mitochondrial DNA is vulnerable to various forms of damage, including several types of oxidative lesions, UV-induced photoproducts, chemical adducts from environmental sources, as well as alkylation and inter-strand crosslinks from chemotherapy agents. Although many of these lesions block DNA replication, Pol γ can bypass some lesions by nucleotide incorporation opposite a template lesion and further extension of the DNA primer past the lesion. This process of translesion synthesis (TLS) by Pol γ can occur in either an error-free or an error-prone manner. Assessment of TLS requires extensive analysis of oligonucleotide substrates and replication products by denaturing polyacrylamide sequencing gels. This chapter presents protocols for the analysis of translesion DNA synthesis. PMID:26530671

  9. Improved reproducibility in genome-wide DNA methylation analysis for PAXgene-fixed samples compared with restored formalin-fixed and paraffin-embedded DNA.

    PubMed

    Andersen, Gitte Brinch; Hager, Henrik; Hansen, Lise Lotte; Tost, Jörg

    2015-01-01

    Formalin fixation has been the standard method for conservation of clinical specimens for decades. However, a major drawback is the high degradation of nucleic acids, which complicates its use in genome-wide analyses. Unbiased identification of biomarkers, however, requires genome-wide studies, precluding the use of the valuable archives of specimens with long-term follow-up data. Therefore, restoration protocols for DNA from formalin-fixed and paraffin-embedded (FFPE) samples have been developed, although they are cost-intensive and time-consuming. An alternative to FFPE and snap-freezing is the PAXgene Tissue System, developed for simultaneous preservation of morphology, proteins, and nucleic acids. In the current study, we compared the performance of DNA from either PAXgene or formalin-fixed tissues to snap-frozen material for genome-wide DNA methylation analysis using the Illumina 450K BeadChip. Quantitative DNA methylation analysis demonstrated that the methylation profile in PAXgene-fixed tissues showed, in comparison with restored FFPE samples, a higher concordance with the profile detected in frozen samples. We demonstrate, for the first time, that DNA from PAXgene conserved tissue performs better compared with restored FFPE DNA in genome-wide DNA methylation analysis. In addition, DNA from PAXgene tissue can be directly used on the array without prior restoration, rendering the analytical process significantly more time- and cost-effective.

  10. Energy transfer analysis of Fos-Jun dimerization and DNA binding.

    PubMed Central

    Patel, L R; Curran, T; Kerppola, T K

    1994-01-01

    The protooncogenes fos and jun encode proteins that bind to DNA as dimeric complexes and regulate gene expression. Protein dimerization is mediated by a leucine zipper and results in juxtaposition of regions of each protein rich in basic amino acids that comprise a bimolecular DNA binding domain. We have developed an approach based on resonance energy transfer for the quantitative analysis of dimerization and DNA binding by Fos and Jun in solution. Fos-(118-211) and Jun-(225-334) polypeptides were labeled with either 5-iodoacetamidofluorescein or rhodamine X iodoacetamide on unique cysteine residues located in their DNA binding domains. Formation of heterodimeric complexes between the labeled proteins allowed resonance energy transfer between the donor fluorescein and the acceptor rhodamine fluorophores. DNA binding induced a conformational transition that increased the efficiency of resonance energy transfer. This increase was consistent with a 3-A reduction in the distance between the fluorophores. Using this assay, we determined the affinity of the Fos-Jun interaction and examined the kinetics of dimerization and DNA binding as well as the rate of subunit exchange. Dimerization and DNA binding by Fos and Jun were rapid, with half-times of < 10 s. In the absence of DNA, Fos and Jun subunits exchanged rapidly, with a half-time of < 10 s. In contrast, in the presence of DNA, the complex was extremely stable. Thus, leucine zipper-containing transcription factors may exchange subunits readily when free in solution, but not when bound to DNA. Images PMID:8041795

  11. DNA Diagnostics: Nanotechnology-enhanced Electrochemical Detection of Nucleic Acids

    PubMed Central

    Wei, Fang; Lillehoj, Peter B.; Ho, Chih-Ming

    2010-01-01

    The detection of mismatched base pairs in DNA plays a crucial role in the diagnosis of genetic-related diseases and conditions, especially for early stage treatment. Among the various biosensors that have been employed for DNA detection, electrochemical sensors show great promise since they are capable of precise DNA recognition and efficient signal transduction. Advancements in micro- and nanotechnologies, specifically fabrication techniques and new nanomaterials, have enabled for the development of highly sensitive, highly specific sensors making them attractive for the detection of small sequence variations. Furthermore, the integration of sensors with sample preparation and fluidic processes enables for rapid, multiplexed DNA detection for point-of-care (POC) clinical diagnostics. PMID:20075759

  12. Docosahexaenoic Acid Induces Oxidative DNA Damage and Apoptosis, and Enhances the Chemosensitivity of Cancer Cells.

    PubMed

    Song, Eun Ah; Kim, Hyeyoung

    2016-01-01

    The human diet contains low amounts of ω-3 polyunsaturated fatty acids (PUFAs) and high amounts of ω-6 PUFAs, which has been reported to contribute to the incidence of cancer. Epidemiological studies have shown that a high consumption of fish oil or ω-3 PUFAs reduced the risk of colon, pancreatic, and endometrial cancers. The ω-3 PUFA, docosahexaenoic acid (DHA), shows anticancer activity by inducing apoptosis of some human cancer cells without toxicity against normal cells. DHA induces oxidative stress and oxidative DNA adduct formation by depleting intracellular glutathione (GSH) and decreasing the mitochondrial function of cancer cells. Oxidative DNA damage and DNA strand breaks activate DNA damage responses to repair the damaged DNA. However, excessive DNA damage beyond the capacity of the DNA repair processes may initiate apoptotic signaling pathways and cell cycle arrest in cancer cells. DHA shows a variable inhibitory effect on cancer cell growth depending on the cells' molecular properties and degree of malignancy. It has been shown to affect DNA repair processes including DNA-dependent protein kinases and mismatch repair in cancer cells. Moreover, DHA enhanced the efficacy of anticancer drugs by increasing drug uptake and suppressing survival pathways in cancer cells. In this review, DHA-induced oxidative DNA damage, apoptotic signaling, and enhancement of chemosensitivity in cancer cells will be discussed based on recent studies. PMID:27527148

  13. Docosahexaenoic Acid Induces Oxidative DNA Damage and Apoptosis, and Enhances the Chemosensitivity of Cancer Cells

    PubMed Central

    Song, Eun Ah; Kim, Hyeyoung

    2016-01-01

    The human diet contains low amounts of ω-3 polyunsaturated fatty acids (PUFAs) and high amounts of ω-6 PUFAs, which has been reported to contribute to the incidence of cancer. Epidemiological studies have shown that a high consumption of fish oil or ω-3 PUFAs reduced the risk of colon, pancreatic, and endometrial cancers. The ω-3 PUFA, docosahexaenoic acid (DHA), shows anticancer activity by inducing apoptosis of some human cancer cells without toxicity against normal cells. DHA induces oxidative stress and oxidative DNA adduct formation by depleting intracellular glutathione (GSH) and decreasing the mitochondrial function of cancer cells. Oxidative DNA damage and DNA strand breaks activate DNA damage responses to repair the damaged DNA. However, excessive DNA damage beyond the capacity of the DNA repair processes may initiate apoptotic signaling pathways and cell cycle arrest in cancer cells. DHA shows a variable inhibitory effect on cancer cell growth depending on the cells’ molecular properties and degree of malignancy. It has been shown to affect DNA repair processes including DNA-dependent protein kinases and mismatch repair in cancer cells. Moreover, DHA enhanced the efficacy of anticancer drugs by increasing drug uptake and suppressing survival pathways in cancer cells. In this review, DHA-induced oxidative DNA damage, apoptotic signaling, and enhancement of chemosensitivity in cancer cells will be discussed based on recent studies. PMID:27527148

  14. Synthesis of nucleoside and nucleotide conjugates of bile acids, and polymerase construction of bile acid-functionalized DNA.

    PubMed

    Ikonen, Satu; Macícková-Cahová, Hana; Pohl, Radek; Sanda, Miloslav; Hocek, Michal

    2010-03-01

    Aqueous Sonogashira cross-coupling reactions of 5-iodopyrimidine or 7-iodo-7-deazaadenine nucleosides with bile acid-derived terminal acetylenes linked via an ester or amide tether gave the corresponding bile acid-nucleoside conjugates. Analogous reactions of halogenated nucleoside triphosphates gave directly bile acid-modified dNTPs. Enzymatic incorporation of these modified nucleotides to DNA was successfully performed using Phusion polymerase for primer extension. One of the dNTPs (dCTP bearing cholic acid) was also efficient for PCR amplification. PMID:20165813

  15. Sequencing and Analysis of Neanderthal Genomic DNA

    PubMed Central

    Noonan, James P.; Coop, Graham; Kudaravalli, Sridhar; Smith, Doug; Krause, Johannes; Alessi, Joe; Chen, Feng; Platt, Darren; Pääbo, Svante; Pritchard, Jonathan K.; Rubin, Edward M.

    2008-01-01

    Our knowledge of Neanderthals is based on a limited number of remains and artifacts from which we must make inferences about their biology, behavior, and relationship to ourselves. Here, we describe the characterization of these extinct hominids from a new perspective, based on the development of a Neanderthal metagenomic library and its high-throughput sequencing and analysis. Several lines of evidence indicate that the 65,250 base pairs of hominid sequence so far identified in the library are of Neanderthal origin, the strongest being the ascertainment of sequence identities between Neanderthal and chimpanzee at sites where the human genomic sequence is different. These results enabled us to calculate the human-Neanderthal divergence time based on multiple randomly distributed autosomal loci. Our analyses suggest that on average the Neanderthal genomic sequence we obtained and the reference human genome sequence share a most recent common ancestor ~706,000 years ago, and that the human and Neanderthal ancestral populations split ~370,000 years ago, before the emergence of anatomically modern humans. Our finding that the Neanderthal and human genomes are at least 99.5% identical led us to develop and successfully implement a targeted method for recovering specific ancient DNA sequences from metagenomic libraries. This initial analysis of the Neanderthal genome advances our understanding of the evolutionary relationship of Homo sapiens and Homo neanderthalensis and signifies the dawn of Neanderthal genomics. PMID:17110569

  16. Sequence and transcription analysis of the human cytomegalovirus DNA polymerase gene

    SciTech Connect

    Kouzarides, T.; Bankier, A.T.; Satchwell, S.C.; Weston, K.; Tomlinson, P.; Barrell, B.G.

    1987-01-01

    DNA sequence analysis has revealed that the gene coding for the human cytomegalovirus (HCMV) DNA polymerase is present within the long unique region of the virus genome. Identification is based on extensive amino acid homology between the predicted HCMV open reading frame HFLF2 and the DNA polymerase of herpes simplex virus type 1. The authors present here a 5280 base-pair DNA sequence containing the HCMV pol gene, along with the analysis of transcripts encoded within this region. Since HCMV pol also shows homology to the predicted Epstein-Barr virus pol, they were able to analyze the extent of homology between the DNA polymerases of three distantly related herpes viruses, HCMV, Epstein-Barr virus, and herpes simplex virus. The comparison shows that these DNA polymerases exhibit considerable amino acid homology and highlights a number of highly conserved regions; two such regions show homology to sequences within the adenovirus type 2 DNA polymerase. The HCMV pol gene is flanked by open reading frames with homology to those of other herpes viruses; upstream, there is a reading frame homologous to the glycoprotein B gene of herpes simplex virus type I and Epstein-Barr virus, and downstream there is a reading frame homologous to BFLF2 of Epstein-Barr virus.

  17. Evaluation of a facile method of template DNA preparation for PCR-based detection and typing of lactic acid bacteria.

    PubMed

    Singh, Atul Kumar; Ramesh, Aiyagari

    2009-08-01

    The objective of our investigation was to develop a convenient and reliable method of generating template DNA for routine PCR-based detection and typing of lactic acid bacteria (LAB). Template DNA extracted from Lactobacillus, Lactococcus, Pediococcus and Leuconostoc using a combination of urea, SDS and NaOH yielded amplicons of expected size in PCR with genus-specific primers. Apart from LAB, the proposed method could also be adopted to generate PCR-compatible template DNA from a number of Gram-positive and Gram-negative bacterial strains. DNA template prepared by the proposed method from various standard strains of Lactobacillus sp. also generated discriminating fingerprints with BOXA1R primer in rep-PCR. A significant finding of the investigation was that a comparable banding profile of LAB strains was obtained in rep-PCR using template DNA prepared by urea-SDS-NaOH method and a commercially available DNA isolation kit. This was further evidenced by high dice coefficient values obtained in the range of 81.8-96.7 when cluster analysis was performed by UPGAMA method. The application potential of this DNA extraction method for PCR-based direct detection of LAB in fermented food samples such as dahi, idli batter and salt-fermented cucumber was validated by detecting specific amplicons of LAB genera in the fermented samples. The applicability of the proposed template DNA extraction method was further substantiated when 29 bacteriocinogenic LAB strains (Bac+) previously detected in salt-fermented cucumber by PCR [Singh, A.K., Ramesh, A., 2008. Succession of dominant and antagonistic lactic acid bacteria in fermented cucumber: Insights from a PCR-based approach. Food. Microbiol. 25, 278-287] generated differentiating fingerprints in BOX element based rep-PCR and formed clusters with reference LAB strains. PMID:19465247

  18. Superior structure stability and selectivity of hairpin nucleic acid probes with an L-DNA stem.

    PubMed

    Kim, Youngmi; Yang, Chaoyong James; Tan, Weihong

    2007-01-01

    Hairpin nucleic acid probes have been highly useful in many areas, especially for intracellular and in vitro nucleic acid detection. The success of these probes can be attributed to the ease with which their conformational change upon target binding can be coupled to a variety of signal transduction mechanisms. However, false-positive signals arise from the opening of the hairpin due mainly to thermal fluctuations and stem invasions. Stem invasions occur when the stem interacts with its complementary sequence and are especially problematic in complex biological samples. To address the problem of stem invasions in hairpin probes, we have created a modified molecular beacon that incorporates unnatural enantiomeric l-DNA in the stem and natural d-DNA or 2'-O-Me-modified RNA in the loop. l-DNA has the same physical characteristics as d-DNA except that l-DNA cannot form stable duplexes with d-DNA. Here we show that incorporating l-DNA into the stem region of a molecular beacon reduces intra- and intermolecular stem invasions, increases the melting temperature, improves selectivity to its target, and leads to enhanced bio-stability. Our results suggest that l-DNA is useful for designing functional nucleic acid probes especially for biological applications.

  19. DNA Cloning of Plasmodium falciparum Circumsporozoite Gene: Amino Acid Sequence of Repetitive Epitope

    NASA Astrophysics Data System (ADS)

    Enea, Vincenzo; Ellis, Joan; Zavala, Fidel; Arnot, David E.; Asavanich, Achara; Masuda, Aoi; Quakyi, Isabella; Nussenzweig, Ruth S.

    1984-08-01

    A clone of complementary DNA encoding the circumsporozoite (CS) protein of the human malaria parasite Plasmodium falciparum has been isolated by screening an Escherichia coli complementary DNA library with a monoclonal antibody to the CS protein. The DNA sequence of the complementary DNA insert encodes a four-amino acid sequence: proline-asparagine-alanine-asparagine, tandemly repeated 23 times. The CS β -lactamase fusion protein specifically binds monoclonal antibodies to the CS protein and inhibits the binding of these antibodies to native Plasmodium falciparum CS protein. These findings provide a basis for the development of a vaccine against Plasmodium falciparum malaria.

  20. Polymerase/DNA interactions and enzymatic activity: multi-parameter analysis with electro-switchable biosurfaces

    NASA Astrophysics Data System (ADS)

    Langer, Andreas; Schräml, Michael; Strasser, Ralf; Daub, Herwin; Myers, Thomas; Heindl, Dieter; Rant, Ulrich

    2015-07-01

    The engineering of high-performance enzymes for future sequencing and PCR technologies as well as the development of many anticancer drugs requires a detailed analysis of DNA/RNA synthesis processes. However, due to the complex molecular interplay involved, real-time methodologies have not been available to obtain comprehensive information on both binding parameters and enzymatic activities. Here we introduce a chip-based method to investigate polymerases and their interactions with nucleic acids, which employs an electrical actuation of DNA templates on microelectrodes. Two measurement modes track both the dynamics of the induced switching process and the DNA extension simultaneously to quantitate binding kinetics, dissociation constants and thermodynamic energies. The high sensitivity of the method reveals previously unidentified tight binding states for Taq and Pol I (KF) DNA polymerases. Furthermore, the incorporation of label-free nucleotides can be followed in real-time and changes in the DNA polymerase conformation (finger closing) during enzymatic activity are observable.

  1. Polymerase/DNA interactions and enzymatic activity: multi-parameter analysis with electro-switchable biosurfaces

    PubMed Central

    Langer, Andreas; Schräml, Michael; Strasser, Ralf; Daub, Herwin; Myers, Thomas; Heindl, Dieter; Rant, Ulrich

    2015-01-01

    The engineering of high-performance enzymes for future sequencing and PCR technologies as well as the development of many anticancer drugs requires a detailed analysis of DNA/RNA synthesis processes. However, due to the complex molecular interplay involved, real-time methodologies have not been available to obtain comprehensive information on both binding parameters and enzymatic activities. Here we introduce a chip-based method to investigate polymerases and their interactions with nucleic acids, which employs an electrical actuation of DNA templates on microelectrodes. Two measurement modes track both the dynamics of the induced switching process and the DNA extension simultaneously to quantitate binding kinetics, dissociation constants and thermodynamic energies. The high sensitivity of the method reveals previously unidentified tight binding states for Taq and Pol I (KF) DNA polymerases. Furthermore, the incorporation of label-free nucleotides can be followed in real-time and changes in the DNA polymerase conformation (finger closing) during enzymatic activity are observable. PMID:26174478

  2. Kinetic Analysis of an Efficient DNA-Dependent TNA Polymerase

    PubMed Central

    Horhota, Allen; Zou, Keyong; Ichida, Justin K.; Yu, Biao; McLaughlin, Larry W.; Szostak, Jack W.

    2005-01-01

    α-l-Threofuranosyl nucleoside triphosphates (tNTPs) are tetrafuranose nucleoside derivatives and potential progenitors of present-day β-d-2‘-deoxyribofuranosyl nucleoside triphosphates (dNTPs). Therminator DNA polymerase, a variant of the 9°N DNA polymerase, is an efficient DNA-directed threosyl nucleic acid (TNA) polymerase. Here we report a detailed kinetic comparison of Therminator-catalyzed TNA and DNA syntheses. We examined the rate of single-nucleotide incorporation for all four tNTPs and dNTPs from a DNA primer−template complex and carried out parallel experiments with a chimeric DNA−TNA primer−DNA template containing five TNA residues at the primer 3‘-terminus. Remarkably, no drop in the rate of TNA incorporation was observed in comparing the DNA−TNA primer to the all-DNA primer, suggesting that few primer-enzyme contacts are lost with a TNA primer. Moreover, comparison of the catalytic efficiency of TNA synthesis relative to DNA synthesis at the downstream positions reveals a difference of no greater than 5-fold in favor of the natural DNA substrate. This disparity becomes negligible when the TNA synthesis reaction mixture is supplemented with 1.25 mM MnCl2. These results indicate that Therminator DNA polymerase can recognize both a TNA primer and tNTP substrates and is an effective catalyst of TNA polymerization despite changes in the geometry of the reactants. PMID:15898792

  3. Biodegradable DNA-Brush Block Copolymer Spherical Nucleic Acids Enable Transfection Agent-Free Intracellular Gene Regulation.

    PubMed

    Zhang, Chuan; Hao, Liangliang; Calabrese, Colin M; Zhou, Yu; Choi, Chung Hang J; Xing, Hang; Mirkin, Chad A

    2015-10-28

    By grafting multiple DNA strands onto one terminus of a polyester chain, a DNA-brush block copolymer that can assemble into micelle structure is constructed. These micelle spherical nucleic acids have a density of nucleic acids that is substantively higher than linear DNA block copolymer structures, which makes them effective cellular transfection and intracellular gene regulation agents.

  4. Amino acid racemization in amber-entombed insects: implications for DNA preservation

    NASA Technical Reports Server (NTRS)

    Bada, J. L.; Wang, X. S.; Poinar, H. N.; Paabo, S.; Poinar, G. O.

    1994-01-01

    DNA depurination and amino acid racemization take place at similar rates in aqueous solution at neutral pH. This relationship suggests that amino acid racemization may be useful in accessing the extent of DNA chain breakage in ancient biological remains. To test this suggestion, we have investigated the amino acids in insects entombed in fossilized tree resins ranging in age from <100 years to 130 million years. The amino acids present in 40 to 130 million year old amber-entombed insects resemble those in a modern fly and are probably the most ancient, unaltered amino acids found so far on Earth. In comparison to other geochemical environments on the surface of the Earth, the amino acid racemization rate in amber insect inclusions is retarded by a factor of >10(4). These results suggest that in amber insect inclusions DNA depurination rates would also likely be retarded in comparison to aqueous solution measurements, and thus DNA fragments containing many hundreds of base pairs should be preserved. This conclusion is consistent with the reported successful retrieval of DNA sequences from amber-entombed organisms.

  5. End invasion of peptide nucleic acids (PNAs) with mixed-base composition into linear DNA duplexes.

    PubMed

    Smolina, Irina V; Demidov, Vadim V; Soldatenkov, Viatcheslav A; Chasovskikh, Sergey G; Frank-Kamenetskii, Maxim D

    2005-01-01

    Peptide nucleic acid (PNA) is a synthetic DNA mimic with valuable properties and a rapidly growing scope of applications. With the exception of recently introduced pseudocomplementary PNAs, binding of common PNA oligomers to target sites located inside linear double-stranded DNAs (dsDNAs) is essentially restricted to homopurine-homopyrimidine sequence motifs, which significantly hampers some of the PNA applications. Here, we suggest an approach to bypass this limitation of common PNAs. We demonstrate that PNA with mixed composition of ordinary nucleobases is capable of sequence-specific targeting of complementary dsDNA sites if they are located at the very termini of DNA duplex. We then show that such targeting makes it possible to perform capturing of designated dsDNA fragments via the DNA-bound biotinylated PNA as well as to signal the presence of a specific dsDNA sequence, in the case a PNA beacon is employed. We also examine the PNA-DNA conjugate and prove that it can initiate the primer-extension reaction starting from the duplex DNA termini when a DNA polymerase with the strand-displacement ability is used. We thus conclude that recognition of duplex DNA by mixed-base PNAs via the end invasion has a promising potential for site-specific and sequence-unrestricted DNA manipulation and detection.

  6. Evaluation of extraction and purification methods for obtaining PCR-amplifiable DNA from compost for microbial community analysis.

    PubMed

    LaMontagne, M G; Michel, F C; Holden, P A; Reddy, C A

    2002-05-01

    Analysis of microbial community structure in complex environmental samples using nucleic acid techniques requires efficient unbiased DNA extraction procedures; however, humic acids and other contaminants complicate the isolation of PCR-amplifiable DNA from compost and other organic-rich samples. In this study, combinations of DNA extraction and purification methods were compared based on DNA yield, humic acid contamination, PCR amplifiability, and microbial community structure assessed by terminal restriction fragment length polymorphisms (TRFLP) of amplified 16S rRNA genes. DNA yield and humic acid contamination, determined by A230, varied significantly between extraction methods. Humic acid contamination of DNA obtained from compost decreased with increasing salt concentration in the lysis buffer. DNA purified by gel permeation chromatography (Sepharose 4B columns) gave satisfactory PCR amplification with universal eubacterial 16S rRNA gene primers only when A260/A280 ratios exceeded 1.5. DNA purified with affinity chromatography (hydroxyapatite columns), and showing A260/A280 ratios as high as 1.8, did not show consistently satisfactory PCR amplification using the same 16S rRNA primers. Almost all DNA samples purified by agarose gel electrophoresis showed satisfactory PCR amplification. Principal components analysis (PCA) of TRFLP patterns differentiated compost types based on the presence/absence of peaks and on the height of the peaks, but differences in TRFLP patterns were not appreciable between extraction methods that yielded relatively pure DNA. High levels of humic acid contamination in extracted DNA resulted in TRFLP patterns that were not consistent and introduced a bias towards lower estimates of diversity.

  7. Synthesis, physicochemical studies, embryos toxicity and DNA interaction of some new Iron(II) Schiff base amino acid complexes

    NASA Astrophysics Data System (ADS)

    Abdel-Rahman, Laila H.; El-Khatib, Rafat M.; Nassr, Lobna A. E.; Abu-Dief, Ahmed M.

    2013-05-01

    New Fe(II) Schiff base amino acid complexes derived from the condensation of o-hydroxynaphthaldehyde with L-alanine, L-phenylalanine, L-aspartic acid, L-histidine and L-arginine were synthesized and characterized by elemental analysis, IR, electronic spectra, and conductance measurements. The stoichiometry and the stability constants of the complexes were determined spectrophotometrically. The investigated Schiff bases exhibited tridentate coordination mode with the general formulae [Fe(HL)2]·nH2O for all amino acids except L-histidine. But in case of L-histidine, the ligand acts as tetradentate ([FeL(H2O)2]·2H2O), where HL = mono anion and L = dianion of the ligand. The structure of the prepared complexes is suggested to be octahedral. The prepared complexes were tested for their toxicity on chick embryos and found to be safe until a concentration of 100 μg/egg with full embryos formation. The interaction between CT-DNA and the investigated complexes were followed by spectrophotometry and viscosity measurements. It was found that, the prepared complexes bind to DNA via classical intercalative mode and showed a different DNA cleavage activity with the sequence: nhi > nari > nali > nasi > nphali. The thermodynamic Profile of the binding of nphali complex and CT-DNA was constructed by analyzing the experimental data of absorption titration and UV melting studies with the McGhee equation, van't Hoff's equation, and the Gibbs-Helmholtz equation.

  8. Statistical analysis of structural determinants for protein-DNA-binding specificity.

    PubMed

    Corona, Rosario I; Guo, Jun-Tao

    2016-08-01

    DNA-binding proteins play critical roles in biological processes including gene expression, DNA packaging and DNA repair. They bind to DNA target sequences with different degrees of binding specificity, ranging from highly specific (HS) to nonspecific (NS). Alterations of DNA-binding specificity, due to either genetic variation or somatic mutations, can lead to various diseases. In this study, a comparative analysis of protein-DNA complex structures was carried out to investigate the structural features that contribute to binding specificity. Protein-DNA complexes were grouped into three general classes based on degrees of binding specificity: HS, multispecific (MS), and NS. Our results show a clear trend of structural features among the three classes, including amino acid binding propensities, simple and complex hydrogen bonds, major/minor groove and base contacts, and DNA shape. We found that aspartate is enriched in HS DNA binding proteins and predominately binds to a cytosine through a single hydrogen bond or two consecutive cytosines through bidentate hydrogen bonds. Aromatic residues, histidine and tyrosine, are highly enriched in the HS and MS groups and may contribute to specific binding through different mechanisms. To further investigate the role of protein flexibility in specific protein-DNA recognition, we analyzed the conformational changes between the bound and unbound states of DNA-binding proteins and structural variations. The results indicate that HS and MS DNA-binding domains have larger conformational changes upon DNA-binding and larger degree of flexibility in both bound and unbound states. Proteins 2016; 84:1147-1161. © 2016 Wiley Periodicals, Inc.

  9. Uracil misincorporation into DNA and folic acid supplementation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    BACKGROUND: Folate deficiency decreases thymidylate synthesis from deoxyuridylate, which results in an imbalance of deoxyribonucleotide that may lead to excessive uracil misincorporation (UrMis) into DNA during replication and repair. OBJECTIVE: We evaluated the relation between UrMis in different ...

  10. Flow cytometric fluorescence lifetime analysis of DNA binding fluorochromes

    SciTech Connect

    Crissman, Harry A.; Cui, H. H.; Steinkamp, J. A.

    2002-01-01

    Most flow cytometry (FCM) applications monitor fluorescence intensity to quantitate the various cellular parameters; however, the fluorescence emission also contains information relative to the fluorescence lifetime. Recent developments in FCM (Pinsky et al., 1993; Steinkamp & Crissman, 1993; Steinkamp et al., 1993), provide for the measurement of fluorescence lifetime which is also commonly referred to as fluorescence decay, or the time interval in which a fluorochrome remains in the excited state. Many unbound fluorochromes have characteristic lifetime values that are determined by their molecular structure; however, when the probe becomes bound, the lifetime value is influenced by a number of factors that affect the probe interaction with a target molecule. Monitoring the changes in the lifetime of the probe yields information relating to the molecular conformation, the functional state or activity of the molecular target. In addition, the lifetime values can be used as signatures to resolve the emissions of multiple fluorochrome labels with overlapping emission spectra that cannot be resolved by conventional FCM methodology. Such strategies can increase the number of fluorochrome combinations used in a flow cytometer with a single excitation source. Our studies demonstrate various applications of lifetime measurements for the analysis of the binding of different fluorochromes to DNA in single cells. Data presented in this session will show the utility of lifetime measurements for monitoring changes in chromatin structure associated with cell cycle progression, cellular differentiation, or DNA damage, such as induced during apoptosis. Several studies show that dyes with specificity for nucleic acids display different lifetime values when bound to DNA or to dsRNA. The Phase Sensitive Flow Cytometer is a multiparameter instrument, capable of performing lifetime measurements in conjunction with all the conventional FCM measurements. Future modifications of this

  11. Single molecule DNA interaction kinetics of retroviral nucleic acid chaperone proteins

    NASA Astrophysics Data System (ADS)

    Williams, Mark

    2010-03-01

    Retroviral nucleocapsid (NC) proteins are essential for several viral replication processes including specific genomic RNA packaging and reverse transcription. The nucleic acid chaperone activity of NC facilitates the latter process. In this study, we use single molecule biophysical methods to quantify the DNA interactions of wild type and mutant human immunodeficiency virus type 1 (HIV-1) NC and Gag and human T-cell leukemia virus type 1 (HTLV-1) NC. We find that the nucleic acid interaction properties of these proteins differ significantly, with HIV-1 NC showing rapid protein binding kinetics, significant duplex destabilization, and strong DNA aggregation, all properties that are critical components of nucleic acid chaperone activity. In contrast, HTLV-1 NC exhibits significant destabilization activity but extremely slow DNA interaction kinetics and poor aggregating capability, which explains why HTLV-1 NC is a poor nucleic acid chaperone. To understand these results, we developed a new single molecule method for quantifying protein dissociation kinetics, and applied this method to probe the DNA interactions of wild type and mutant HIV-1 and HTLV-1 NC. We find that mutations to aromatic and charged residues strongly alter the proteins' nucleic acid interaction kinetics. Finally, in contrast to HIV-1 NC, HIV-1 Gag, the nucleic acid packaging protein that contains NC as a domain, exhibits relatively slow binding kinetics, which may negatively impact its ability to act as a nucleic acid chaperone.

  12. Switchable reconfiguration of nucleic acid nanostructures by stimuli-responsive DNA machines.

    PubMed

    Liu, Xiaoqing; Lu, Chun-Hua; Willner, Itamar

    2014-06-17

    CONSPECTUS: The base sequence in DNA dictates structural and reactivity features of the biopolymer. These properties are implemented to use DNA as a unique material for developing the area of DNA nanotechnology. The design of DNA machines represents a rapidly developing research field in the area of DNA nanotechnology. The present Account discusses the switchable reconfiguration of nucleic acid nanostructures by stimuli-responsive DNA machines, and it highlights potential applications and future perspectives of the area. Programmed switchable DNA machines driven by various fuels and antifuels, such as pH, Hg(2+) ions/cysteine, or nucleic acid strands/antistrands, are described. These include the assembly of DNA tweezers, walkers, a rotor, a pendulum, and more. Using a pH-oscillatory system, the oscillatory mechanical operation of a DNA pendulum is presented. Specifically, the synthesis and "mechanical" properties of interlocked DNA rings are described. This is exemplified with the preparation of interlocked DNA catenanes and a DNA rotaxane. The dynamic fuel-driven reconfiguration of the catenane/rotaxane structures is followed by fluorescence spectroscopy. The use of DNA machines as functional scaffolds to reconfigurate Au nanoparticle assemblies and to switch the fluorescence features within fluorophore/Au nanoparticle conjugates between quenching and surface-enhanced fluorescence states are addressed. Specifically, the fluorescence features of the different DNA machines are characterized as a function of the spatial separation between the fluorophore and Au nanoparticles. The experimental results are supported by theoretical calculations. The future development of reconfigurable stimuli-responsive DNA machines involves fundamental challenges, such as the synthesis of molecular devices exhibiting enhanced complexities, the introduction of new fuels and antifuels, and the integration of new payloads being reconfigured by the molecular devices, such as enzymes or

  13. Affinity purification of DNA and RNA from environmental samples with peptide nucleic acid clamps.

    PubMed

    Chandler, D P; Stults, J R; Cebula, S; Schuck, B L; Weaver, D W; Anderson, K K; Egholm, M; Brockman, F J

    2000-08-01

    Bispeptide nucleic acids (bis-PNAs; PNA clamps), PNA oligomers, and DNA oligonucleotides were evaluated as affinity purification reagents for subfemtomolar 16S ribosomal DNA (rDNA) and rRNA targets in soil, sediment, and industrial air filter nucleic acid extracts. Under low-salt hybridization conditions (10 mM NaPO(4), 5 mM disodium EDTA, and 0.025% sodium dodecyl sulfate [SDS]) a PNA clamp recovered significantly more target DNA than either PNA or DNA oligomers. The efficacy of PNA clamps and oligomers was generally enhanced in the presence of excess nontarget DNA and in a low-salt extraction-hybridization buffer. Under high-salt conditions (200 mM NaPO(4), 100 mM disodium EDTA, and 0.5% SDS), however, capture efficiencies with the DNA oligomer were significantly greater than with the PNA clamp and PNA oligomer. Recovery and detection efficiencies for target DNA concentrations of > or =100 pg were generally >20% but depended upon the specific probe, solution background, and salt condition. The DNA probe had a lower absolute detection limit of 100 fg of target (830 zM [1 zM = 10(-21) M]) in high-salt buffer. In the absence of exogenous DNA (e.g., soil background), neither the bis-PNA nor the PNA oligomer achieved the same absolute detection limit even under a more favorable low-salt hybridization condition. In the presence of a soil background, however, both PNA probes provided more sensitive absolute purification and detection (830 zM) than the DNA oligomer. In varied environmental samples, the rank order for capture probe performance in high-salt buffer was DNA > PNA > clamp. Recovery of 16S rRNA from environmental samples mirrored quantitative results for DNA target recovery, with the DNA oligomer generating more positive results than either the bis-PNA or PNA oligomer, but PNA probes provided a greater incidence of detection from environmental samples that also contained a higher concentration of nontarget DNA and RNA. Significant interactions between probe

  14. DNA

    ERIC Educational Resources Information Center

    Stent, Gunther S.

    1970-01-01

    This history for molecular genetics and its explanation of DNA begins with an analysis of the Golden Jubilee essay papers, 1955. The paper ends stating that the higher nervous system is the one major frontier of biological inquiry which still offers some romance of research. (Author/VW)

  15. DNA fingerprinting of lactic acid bacteria in sauerkraut fermentations

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Previous studies using traditional biochemical methods to study the ecology of commercial sauerkraut fermentations revealed that four lactic acid bacteria species, Leuconostoc mesenteroides, Lactobacillus plantarum, Pediococcus pentosaceus, and Lactobacillus brevis were the primary microorganisms in...

  16. A beta-D-allopyranoside-grafted Ru(II) complex: synthesis and acid-base and DNA-binding properties.

    PubMed

    Ma, Yan-Zi; Yin, Hong-Ju; Wang, Ke-Zhi

    2009-08-01

    A new ruthenium(II) complex grafted with beta-d-allopyranoside, Ru(bpy)(2)(Happip)(ClO(4))(2) (where bpy = 2,2'-bipyridine; Happip = 2-(4-(beta-d-allopyranoside)phenyl)imidazo[4,5-f][1,10]phenanthroline), has been synthesized and characterized by elemental analysis, (1)H NMR spectroscopy, and mass spectrometry. The acid-base properties of the complex have been studied by UV-visible and luminescence spectrophotometric pH titrations, and ground- and excited-state ionization constants have been derived. The Ru(II) complex functions as a DNA intercalator as revealed by UV-visible and emission titrations, salt effects, steady-state emission quenching by [Fe(CN)(6)](4-), DNA competitive binding with ethidium bromide, DNA melting experiment, and viscosity measurements.

  17. The nitrosated bile acid DNA lesion O6-carboxymethylguanine is a substrate for the human DNA repair protein O6-methylguanine-DNA methyltransferase

    PubMed Central

    Senthong, Pattama; Millington, Christopher L.; Wilkinson, Oliver J.; Marriott, Andrew S.; Watson, Amanda J.; Reamtong, Onrapak; Eyers, Claire E.; Williams, David M.; Margison, Geoffrey P.; Povey, Andrew C.

    2013-01-01

    The consumption of red meat is a risk factor in human colorectal cancer (CRC). One hypothesis is that red meat facilitates the nitrosation of bile acid conjugates and amino acids, which rapidly convert to DNA-damaging carcinogens. Indeed, the toxic and mutagenic DNA adduct O6-carboxymethylguanine (O6-CMG) is frequently present in human DNA, increases in abundance in people with high levels of dietary red meat and may therefore be a causative factor in CRC. Previous reports suggested that O6-CMG is not a substrate for the human version of the DNA damage reversal protein O6-methylguanine-DNA methyltransferase (MGMT), which protects against the genotoxic effects of other O6-alkylguanine lesions by removing alkyl groups from the O6-position. We now show that synthetic oligodeoxyribonucleotides containing the known MGMT substrate O6-methylguanine (O6-MeG) or O6-CMG effectively inactivate MGMT in vitro (IC50 0.93 and 1.8 nM, respectively). Inactivation involves the removal of the O6-alkyl group and its transfer to the active-site cysteine residue of MGMT. O6-CMG is therefore an MGMT substrate, and hence MGMT is likely to be a protective factor in CRC under conditions where O6-CMG is a potential causative agent. PMID:23335782

  18. Possible role of mtDNA depletion and respiratory chain defects in aristolochic acid I-induced acute nephrotoxicity

    SciTech Connect

    Jiang, Zhenzhou Bao, Qingli Sun, Lixin Huang, Xin Wang, Tao Zhang, Shuang Li, Han Zhang, Luyong

    2013-01-15

    This report describes an investigation of the pathological mechanism of acute renal failure caused by toxic tubular necrosis after treatment with aristolochic acid I (AAI) in Sprague–Dawley (SD) rats. The rats were gavaged with AAI at 0, 5, 20, or 80 mg/kg/day for 7 days. The pathologic examination of the kidneys showed severe acute tubular degenerative changes primarily affecting the proximal tubules. Supporting these results, we detected significantly increased concentrations of blood urea nitrogen (BUN) and creatinine (Cr) in the rats treated with AAI, indicating damage to the kidneys. Ultrastructural examination showed that proximal tubular mitochondria were extremely enlarged and dysmorphic with loss and disorientation of their cristae. Mitochondrial function analysis revealed that the two indicators for mitochondrial energy metabolism, the respiratory control ratio (RCR) and ATP content, were reduced in a dose-dependent manner after AAI treatment. The RCR in the presence of substrates for complex I was reduced more significantly than in the presence of substrates for complex II. In additional experiments, the activity of respiratory complex I, which is partly encoded by mitochondrial DNA (mtDNA), was more significantly impaired than that of respiratory complex II, which is completely encoded by nuclear DNA (nDNA). A real-time PCR assay revealed a marked reduction of mtDNA in the kidneys treated with AAI. Taken together, these results suggested that mtDNA depletion and respiratory chain defects play critical roles in the pathogenesis of kidney injury induced by AAI, and that the same processes might contribute to aristolochic acid-induced nephrotoxicity in humans. -- Highlights: ► AAI-induced acute renal failure in rats and the proximal tubule was the target. ► Tubular mitochondria were morphologically aberrant in ultrastructural examination. ► AAI impair mitochondrial bioenergetic function and mtDNA replication.

  19. Calcium-activated gene transfection from DNA/poly(amic acid-co-imide) complexes.

    PubMed

    Wu, Szu-Yuan; Chang, Li-Ting; Peng, Sydeny; Tsai, Hsieh-Chih

    2015-01-01

    In this study, we synthesized a water-soluble poly(amic acid-co-imide) (PA-I) from ethylenediaminetetraacetic dianhydride (EDTA) and 2,2'-(ethylenedioxy)bis(ethylamine) that possesses comparable transfection efficiency to that of polyethylenimine (PEI), when prepared in combination with divalent calcium cations. The polycondensation of monomers afforded poly(amic acid) (PA) precursors, and subsequent thermal imidization resulted in the formation of PA-I. At a polymer/DNA ratio (indicated by the molar ratio of nitrogen in the polymer to phosphate in DNA) of 40, complete retardation of the DNA band was observed by gel electrophoresis, indicating the strong association of DNA with PA-I. A zeta potential of -22 mV was recorded for the PA-I polymer solution, and no apparent cytotoxicity was observed at concentrations up to 500 μg·mL(-1). In the presence of divalent Ca(2+), the transfection efficiency of PA-I was higher than that of PA, due to the formation of a copolymer/Ca(2+)/DNA polyplex and the reduction in negative charge due to thermal cyclization. Interestingly, a synergistic effect of Ca(2+) and the synthesized copolymer on DNA transfection was observed. The use of Ca(2+) or copolymer alone resulted in unsatisfactory delivery, whereas the formation of three-component polyplexes synergistically increased DNA transfection. Our findings demonstrated that a PA-I/Ca(2+)/DNA polyplex could serve as a promising candidate for gene delivery.

  20. Canine amino acid transport system Xc(-): cDNA sequence, distribution and cystine transport activity in lens epithelial cells.

    PubMed

    Maruo, Takuya; Kanemaki, Nobuyuki; Onda, Ken; Sato, Reiichiro; Ichihara, Nobuteru; Ochiai, Hideharu

    2014-04-01

    The cystine transport activity of a lens epithelial cell line originated from a canine mature cataract was investigated. The distinct cystine transport activity was observed, which was inhibited to 28% by extracellular 1 mM glutamate. The cDNA sequences of canine cysteine/glutamate exchanger (xCT) and 4F2hc were determined. The predicted amino acid sequences were 527 and 533 amino acid polypeptides, respectively. The amino acid sequences of canine xCT and 4F2hc showed high similarities (>80%) to those of humans. The expression of xCT in lens epithelial cell line was confirmed by western blot analysis. RT-PCR analysis revealed high level expression only in the brain, and it was below the detectable level in other tissues.

  1. DNA sequence analysis by MALDI mass spectrometry.

    PubMed Central

    Kirpekar, F; Nordhoff, E; Larsen, L K; Kristiansen, K; Roepstorff, P; Hillenkamp, F

    1998-01-01

    Conventional DNA sequencing is based on gel electrophoretic separation of the sequencing products. Gel casting and electrophoresis are the time limiting steps, and the gel separation is occasionally imperfect due to aberrant mobility of certain fragments, leading to erroneous sequence determination. Furthermore, illegitimately terminated products frequently cannot be distinguished from correctly terminated ones, a phenomenon that also obscures data interpretation. In the present work the use of MALDI mass spectrometry for sequencing of DNA amplified from clinical samples is implemented. The unambiguous and fast identification of deletions and substitutions in DNA amplified from heterozygous carriers realistically suggest MALDI mass spectrometry as a future alternative to conventional sequencing procedures for high throughput screening for mutations. Unique features of the method are demonstrated by sequencing a DNA fragment that could not be sequenced conventionally because of gel electrophoretic band compression and the presence of multiple non-specific termination products. Taking advantage of the accurate mass information provided by MALDI mass spectrometry, the sequence was deduced, and the nature of the non-specific termination could be determined. The method described here increases the fidelity in DNA sequencing, is fast, compatible with standard DNA sequencing procedures, and amenable to automation. PMID:9592136

  2. Role of amino acid insertions on intermolecular forces between arginine peptide condensed DNA helices: implications for protamine-DNA packaging in sperm.

    PubMed

    DeRouchey, Jason E; Rau, Donald C

    2011-12-01

    In spermatogenesis, chromatin histones are replaced by arginine-rich protamines to densely compact DNA in sperm heads. Tight packaging is considered necessary to protect the DNA from damage. To better understand the nature of the forces condensing protamine-DNA assemblies and their dependence on amino acid content, the effect of neutral and negatively charged amino acids on DNA-DNA intermolecular forces was studied using model peptides containing six arginines. We have previously observed that the neutral amino acids in salmon protamine decrease the net attraction between protamine-DNA helices compared with the equivalent homo-arginine peptide. Using osmotic stress coupled with x-ray scattering, we have investigated the component attractive and repulsive forces that determine the net attraction and equilibrium interhelical distance as a function of the chemistry, position, and number of the amino acid inserted. Neutral amino acids inserted into hexa-arginine increase the short range repulsion while only slightly affecting longer range attraction. The amino acid content alone of salmon protamine is enough to rationalize the forces that package DNA in sperm heads. Inserting a negatively charged amino acid into hexa-arginine dramatically weakens the net attraction. Both of these observations have biological implications for protamine-DNA packaging in sperm heads.

  3. Principal component analysis of phenolic acid spectra

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Phenolic acids are common plant metabolites that exhibit bioactive properties and have applications in functional food and animal feed formulations. The ultraviolet (UV) and infrared (IR) spectra of four closely related phenolic acid structures were evaluated by principal component analysis (PCA) to...

  4. Amino acid isotopic analysis in agricultural systems

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A relatively new approach to stable isotopic analysis—referred to as compound-specific isotopic analysis (CSIA)—has emerged, centering on the measurement of 15N:14N ratios in amino acids (glutamic acid and phenylalanine). CSIA has recently been used to generate trophic position estimates among anima...

  5. Switchable mechanical DNA ``arms'' operating on nucleic acid scaffolds associated with electrodes or semiconductor quantum dots

    NASA Astrophysics Data System (ADS)

    Pelossof, Gilad; Tel-Vered, Ran; Liu, Xiaoqing; Willner, Itamar

    2013-09-01

    Functional footholds linked to DNA scaffolds associated with surfaces provide nano-engineered assemblies acting as switching devices. By the assembly of a β-cyclodextrin receptor on one foothold, and a ferrocene-modified nucleic acid on a second foothold, the switchable and reversible, fuel-driven activation of ``molecular arms'' proceeds, transduced by electrochemical or optical signals.Functional footholds linked to DNA scaffolds associated with surfaces provide nano-engineered assemblies acting as switching devices. By the assembly of a β-cyclodextrin receptor on one foothold, and a ferrocene-modified nucleic acid on a second foothold, the switchable and reversible, fuel-driven activation of ``molecular arms'' proceeds, transduced by electrochemical or optical signals. Electronic supplementary information (ESI) available: Experimental procedures, time-dependent deactivation of a DNA ``arm'' using a DNA anti-fuel, and control experiments, excluding β-cyclodextrin from the systems. See DOI: 10.1039/c3nr02653a

  6. Boric Acid in Kjeldahl Analysis

    ERIC Educational Resources Information Center

    Cruz, Gregorio

    2013-01-01

    The use of boric acid in the Kjeldahl determination of nitrogen is a variant of the original method widely applied in many laboratories all over the world. Its use is recommended by control organizations such as ISO, IDF, and EPA because it yields reliable and accurate results. However, the chemical principles the method is based on are not…

  7. Pyroglutamic acid stimulates DNA synthesis in rat primary hepatocytes through the mitogen-activated protein kinase pathway.

    PubMed

    Inoue, Shinjiro; Okita, Yoichi; de Toledo, Andreia; Miyazaki, Hiroyuki; Hirano, Eiichi; Morinaga, Tetsuo

    2015-01-01

    We purified pyroglutamic acid from human placental extract and identified it as a potent stimulator of rat primary hepatocyte DNA synthesis. Pyroglutamic acid dose-dependently stimulated DNA synthesis, and this effect was inhibited by PD98059, a dual specificity mitogen-activated protein kinase kinase 1 (MAP2K1) inhibitor. Therefore, pyroglutamic acid stimulated DNA synthesis in rat primary hepatocytes via MAPK signaling.

  8. Beyond DNA origami: A look on the bright future of nucleic acid nanotechnology

    PubMed Central

    Michelotti, Nicole; Johnson-Buck, Alexander; Manzo, Anthony J.

    2012-01-01

    Nucleic acid nanotechnology exploits the programmable molecular recognition properties of natural and synthetic nucleic acids to assemble structures with nanometer-scale precision. In 2006, DNA origami transformed the field by providing a versatile platform for self-assembly of arbitrary shapes from one long DNA strand held in place by hundreds of short, site-specific (spatially addressable) DNA ”staples”. This revolutionary approach has led to the creation of a multitude of 2D and 3D scaffolds that form the basis for functional nanodevices. Not limited to nucleic acids, these nanodevices can incorporate other structural and functional materials, such as proteins and nanoparticles, making them broadly useful for current and future applications in emerging fields such as nanomedicine, nanoelectronics, and alternative energy. PMID:22131292

  9. Automated DNA extraction of single dog hairs without roots for mitochondrial DNA analysis.

    PubMed

    Bekaert, Bram; Larmuseau, Maarten H D; Vanhove, Maarten P M; Opdekamp, Anouschka; Decorte, Ronny

    2012-03-01

    Dogs are intensely integrated in human social life and their shed hairs can play a major role in forensic investigations. The overall aim of this study was to validate a semi-automated extraction method for mitochondrial DNA analysis of telogenic dog hairs. Extracted DNA was amplified with a 95% success rate from 43 samples using two new experimental designs in which the mitochondrial control region was amplified as a single large (± 1260 bp) amplicon or as two individual amplicons (HV1 and HV2; ± 650 and 350 bp) with tailed-primers. The results prove that the extraction of dog hair mitochondrial DNA can easily be automated to provide sufficient DNA yield for the amplification of a forensically useful long mitochondrial DNA fragment or alternatively two short fragments with minimal loss of sequence in case of degraded samples.

  10. Microscopic and spectroscopic analysis of chitosan-DNA conjugates.

    PubMed

    Agudelo, D; Kreplak, L; Tajmir-Riahi, H A

    2016-02-10

    Conjugations of DNA with chitosans 15 kD (ch-15), 100 kD (ch-100) and 200 kD (ch-200) were investigated in aqueous solution at pH 5.5-6.5. Multiple spectroscopic methods and atomic force microscopy (AFM) were used to locate the chitosan binding sites and the effect of polymer conjugation on DNA compaction and particle formation. Structural analysis showed that chitosan-DNA conjugation is mainly via electrostatic interactions through polymer cationic charged NH2 and negatively charged backbone phosphate groups. As polymer size increases major DNA compaction and particle formation occurs. At high chitosan concentration major DNA structural changes observed indicating a partial B to A-DNA conformational transition.

  11. Microfluidic Devices for Forensic DNA Analysis: A Review.

    PubMed

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

    2016-01-01

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

  12. Microfluidic Devices for Forensic DNA Analysis: A Review

    PubMed Central

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

    2016-01-01

    Microfluidic devices may offer various advantages for forensic DNA analysis, such as reduced risk of contamination, shorter analysis time and direct application at the crime scene. Microfluidic chip technology has already proven to be functional and effective within medical applications, such as for point-of-care use. In the forensic field, one may expect microfluidic technology to become particularly relevant for the analysis of biological traces containing human DNA. This would require a number of consecutive steps, including sample work up, DNA amplification and detection, as well as secure storage of the sample. This article provides an extensive overview of microfluidic devices for cell lysis, DNA extraction and purification, DNA amplification and detection and analysis techniques for DNA. Topics to be discussed are polymerase chain reaction (PCR) on-chip, digital PCR (dPCR), isothermal amplification on-chip, chip materials, integrated devices and commercially available techniques. A critical overview of the opportunities and challenges of the use of chips is discussed, and developments made in forensic DNA analysis over the past 10–20 years with microfluidic systems are described. Areas in which further research is needed are indicated in a future outlook. PMID:27527231

  13. Microfluidic Devices for Forensic DNA Analysis: A Review.

    PubMed

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

    2016-01-01

    Microfluidic devices may offer various advantages for forensic DNA analysis, such as reduced risk of contamination, shorter analysis time and direct application at the crime scene. Microfluidic chip technology has already proven to be functional and effective within medical applications, such as for point-of-care use. In the forensic field, one may expect microfluidic technology to become particularly relevant for the analysis of biological traces containing human DNA. This would require a number of consecutive steps, including sample work up, DNA amplification and detection, as well as secure storage of the sample. This article provides an extensive overview of microfluidic devices for cell lysis, DNA extraction and purification, DNA amplification and detection and analysis techniques for DNA. Topics to be discussed are polymerase chain reaction (PCR) on-chip, digital PCR (dPCR), isothermal amplification on-chip, chip materials, integrated devices and commercially available techniques. A critical overview of the opportunities and challenges of the use of chips is discussed, and developments made in forensic DNA analysis over the past 10-20 years with microfluidic systems are described. Areas in which further research is needed are indicated in a future outlook. PMID:27527231

  14. Binding of retinoic acid receptor heterodimers to DNA. A role for histones NH2 termini.

    PubMed

    Lefebvre, P; Mouchon, A; Lefebvre, B; Formstecher, P

    1998-05-15

    The retinoic acid signaling pathway is controlled essentially through two types of nuclear receptors, RARs and RXRs. Ligand dependent activation or repression of retinoid-regulated genes is dependent on the binding of retinoic acid receptor (RAR)/9-cis-retinoic acid receptor (RXR) heterodimers to retinoic acid response element (RARE). Although unliganded RXR/RAR heterodimers bind constitutively to DNA in vitro, a clear in vivo ligand-dependent occupancy of the RARE present in the RARbeta2 gene promoter has been reported (Dey, A., Minucci, S., and Ozato, K. (1994) Mol. Cell. Biol. 14, 8191-8201). Nucleosomes are viewed as general repressors of the transcriptional machinery, in part by preventing the access of transcription factors to DNA. The ability of hRXRalpha/hRARalpha heterodimers to bind to a nucleosomal template in vitro has therefore been examined. The assembly of a fragment from the RARbeta2 gene promoter, which contains a canonical DR5 RARE, into a nucleosome core prevented hRXRalpha/hRARalpha binding to this DNA, in conditions where a strong interaction is observed with a linear DNA template. However, histone tails removal by limited proteolysis and histone hyperacetylation yielded nucleosomal RAREs able to bind to hRXRalpha/hRARalpha heterodimers. These data establish therefore the role of histones NH2 termini as a major impediment to retinoid receptors access to DNA, and identify histone hyperacetylation as a potential physiological regulator of retinoid-induced transcription.

  15. Extraction of DNA from paraffin blocks for Southern blot analysis.

    PubMed

    Mies, C; Houldsworth, J; Chaganti, R S

    1991-02-01

    Tissues stored as paraffin blocks are a potential source of DNA for retrospective clinicogenetic analysis. To assess the feasibility of Southern blot analysis, DNA extracted from paraffin blocks was compared with DNA obtained from fresh-frozen controls of the same tissues. Sections 50-100 microns thick cut from paraffin blocks of 11 normal tissues, 18 lymphoid lesions, and 9 gastric carcinoma samples were deparaffinized and incubated at 45 degrees C for 48 to 72 h in a sodium dodecyl sulfate (SDS)/proteinase K solution. Following organic extraction, alcohol precipitation, restriction endonuclease digestion, and gel electrophoresis, DNA was transferred to nylon membranes. 32P-labelled DNA probes for the immunoglobulin heavy-chain locus and T-cell receptor beta-chain gene were hybridized to the normal tissue and lymphoid samples; the gastric cancers were probed for the HER-2/neu protooncogene. Intact DNA was obtained from the majority of formalin-fixed samples, yielding results qualitatively similar to those from fresh tissues. Degradation is the most significant problem in analyzing DNA extracted from paraffin blocks and compromises accurate quantitation. DNA analysis using paraffin-embedded tissue has potential clinical and research applications and may be a particularly useful way to study gene abnormalities in unusual tumors infrequently available as fresh specimens.

  16. Real-time DNA microarray analysis

    PubMed Central

    Hassibi, Arjang; Vikalo, Haris; Riechmann, José Luis; Hassibi, Babak

    2009-01-01

    We present a quantification method for affinity-based DNA microarrays which is based on the real-time measurements of hybridization kinetics. This method, i.e. real-time DNA microarrays, enhances the detection dynamic range of conventional systems by being impervious to probe saturation in the capturing spots, washing artifacts, microarray spot-to-spot variations, and other signal amplitude-affecting non-idealities. We demonstrate in both theory and practice that the time-constant of target capturing in microarrays, similar to all affinity-based biosensors, is inversely proportional to the concentration of the target analyte, which we subsequently use as the fundamental parameter to estimate the concentration of the analytes. Furthermore, to empirically validate the capabilities of this method in practical applications, we present a FRET-based assay which enables the real-time detection in gene expression DNA microarrays. PMID:19723688

  17. Real-time DNA microarray analysis.

    PubMed

    Hassibi, Arjang; Vikalo, Haris; Riechmann, José Luis; Hassibi, Babak

    2009-11-01

    We present a quantification method for affinity-based DNA microarrays which is based on the real-time measurements of hybridization kinetics. This method, i.e. real-time DNA microarrays, enhances the detection dynamic range of conventional systems by being impervious to probe saturation in the capturing spots, washing artifacts, microarray spot-to-spot variations, and other signal amplitude-affecting non-idealities. We demonstrate in both theory and practice that the time-constant of target capturing in microarrays, similar to all affinity-based biosensors, is inversely proportional to the concentration of the target analyte, which we subsequently use as the fundamental parameter to estimate the concentration of the analytes. Furthermore, to empirically validate the capabilities of this method in practical applications, we present a FRET-based assay which enables the real-time detection in gene expression DNA microarrays. PMID:19723688

  18. Complete cDNA and derived amino acid sequence of human factor V.

    PubMed Central

    Jenny, R J; Pittman, D D; Toole, J J; Kriz, R W; Aldape, R A; Hewick, R M; Kaufman, R J; Mann, K G

    1987-01-01

    cDNA clones encoding human factor V have been isolated from an oligo(dT)-primed human fetal liver cDNA library prepared with vector Charon 21A. The cDNA sequence of factor V from three overlapping clones includes a 6672-base-pair (bp) coding region, a 90-bp 5' untranslated region, and a 163-bp 3' untranslated region within which is a poly(A) tail. The deduced amino acid sequence consists of 2224 amino acids inclusive of a 28-amino acid leader peptide. Direct comparison with human factor VIII reveals considerable homology between proteins in amino acid sequence and domain structure: a triplicated A domain and duplicated C domain show approximately equal to 40% identity with the corresponding domains in factor VIII. As in factor VIII, the A domains of factor V share approximately 40% amino acid-sequence homology with the three highly conserved domains in ceruloplasmin. The B domain of factor V contains 35 tandem and approximately 9 additional semiconserved repeats of nine amino acids of the form Asp-Leu-Ser-Gln-Thr-Thr/Asn-Leu-Ser-Pro and 2 additional semiconserved repeats of 17 amino acids. Factor V contains 37 potential N-linked glycosylation sites, 25 of which are in the B domain, and a total of 19 cysteine residues. Images PMID:3110773

  19. Complete cDNA and derived amino acid sequence of human factor V

    SciTech Connect

    Jenny, R.J.; Pittman, D.D.; Toole, J.J.; Kriz, R.W.; Aldape, R.A.; Hewick, R.M.; Kaufman, R.J.; Mann, K.G.

    1987-07-01

    cDNA clones encoding human factor V have been isolated from an oligo(dT)-primed human fetal liver cDNA library prepared with vector Charon 21A. The cDNA sequence of factor V from three overlapping clones includes a 6672-base-pair (bp) coding region, a 90-bp 5' untranslated region, and a 163-bp 3' untranslated region within which is a poly(A)tail. The deduced amino acid sequence consists of 2224 amino acids inclusive of a 28-amino acid leader peptide. Direct comparison with human factor VIII reveals considerable homology between proteins in amino acid sequence and domain structure: a triplicated A domain and duplicated C domain show approx. 40% identity with the corresponding domains in factor VIII. As in factor VIII, the A domains of factor V share approx. 40% amino acid-sequence homology with the three highly conserved domains in ceruloplasmin. The B domain of factor V contains 35 tandem and approx. 9 additional semiconserved repeats of nine amino acids of the form Asp-Leu-Ser-Gln-Thr-Thr/Asn-Leu-Ser-Pro and 2 additional semiconserved repeats of 17 amino acids. Factor V contains 37 potential N-linked glycosylation sites, 25 of which are in the B domain, and a total of 19 cysteine residues.

  20. Genetic analysis of yeast RPA1 reveals its multiple functions in DNA metabolism.

    PubMed Central

    Umezu, K; Sugawara, N; Chen, C; Haber, J E; Kolodner, R D

    1998-01-01

    Replication protein A (RPA) is a single-stranded DNA-binding protein identified as an essential factor for SV40 DNA replication in vitro. To understand the in vivo functions of RPA, we mutagenized the Saccharomyces cerevisiae RFA1 gene and identified 19 ultraviolet light (UV) irradiation- and methyl methane sulfonate (MMS)-sensitive mutants and 5 temperature-sensitive mutants. The UV- and MMS-sensitive mutants showed up to 10(4) to 10(5) times increased sensitivity to these agents. Some of the UV- and MMS-sensitive mutants were killed by an HO-induced double-strand break at MAT. Physical analysis of recombination in one UV- and MMS-sensitive rfa1 mutant demonstrated that it was defective for mating type switching and single-strand annealing recombination. Two temperature-sensitive mutants were characterized in detail, and at the restrictive temperature were found to have an arrest phenotype and DNA content indicative of incomplete DNA replication. DNA sequence analysis indicated that most of the mutations altered amino acids that were conserved between yeast, human, and Xenopus RPA1. Taken together, we conclude that RPA1 has multiple roles in vivo and functions in DNA replication, repair, and recombination, like the single-stranded DNA-binding proteins of bacteria and phages. PMID:9539419

  1. Genetic analysis of yeast RPA1 reveals its multiple functions in DNA metabolism.

    PubMed

    Umezu, K; Sugawara, N; Chen, C; Haber, J E; Kolodner, R D

    1998-03-01

    Replication protein A (RPA) is a single-stranded DNA-binding protein identified as an essential factor for SV40 DNA replication in vitro. To understand the in vivo functions of RPA, we mutagenized the Saccharomyces cerevisiae RFA1 gene and identified 19 ultraviolet light (UV) irradiation- and methyl methane sulfonate (MMS)-sensitive mutants and 5 temperature-sensitive mutants. The UV- and MMS-sensitive mutants showed up to 10(4) to 10(5) times increased sensitivity to these agents. Some of the UV- and MMS-sensitive mutants were killed by an HO-induced double-strand break at MAT. Physical analysis of recombination in one UV- and MMS-sensitive rfa1 mutant demonstrated that it was defective for mating type switching and single-strand annealing recombination. Two temperature-sensitive mutants were characterized in detail, and at the restrictive temperature were found to have an arrest phenotype and DNA content indicative of incomplete DNA replication. DNA sequence analysis indicated that most of the mutations altered amino acids that were conserved between yeast, human, and Xenopus RPA1. Taken together, we conclude that RPA1 has multiple roles in vivo and functions in DNA replication, repair, and recombination, like the single-stranded DNA-binding proteins of bacteria and phages.

  2. DNA methylation in Folbp1 knockout mice supplemented with folic acid during gestation.

    PubMed

    Finnell, Richard H; Spiegelstein, Ofer; Wlodarczyk, Bogdan; Triplett, Aleata; Pogribny, Igor P; Melnyk, Stepan; James, Jill S

    2002-08-01

    Periconceptional folic acid supplementation has been shown to prevent up to 70% of neural tube and other birth defects in humans; however, the mechanism is still unknown. In this study, we tested whether defective intracellular folate transport, as achieved by inactivation of the murine folate-binding protein 1 (Folbp1), affects global DNA methylation in the liver and brain from gestational day (GD) 15 embryos. Complete Folbp1 inactivation is embryolethal but can be reversed by maternal folinic acid (FA) supplementation, and thus we also tested the effect of FA supplementation on DNA methylation in Folbp1 fetuses. Overall, the extent of global DNA methylation seems to be similar across all genotypes in unsupplemented control Folbp1 mice; however, explicit conclusions regarding Folbp1(-/-) fetuses were not possible because only a single living unsupplemented fetus was viable at GD 15. FA supplementation induced global DNA hypomethylation across all genotypes. FA-induced hypomethylation is most likely due to its ability to inhibit the enzyme glycine hydroxymethyltransferase, thereby inhibiting the homocysteine remethylation cycle necessary to regenerate S-adenosylmethionine, the methyl donor for DNA methyltransferases. Our hypothesis was that due to defective folate transport in Folbp1(-/-) embryos and fetuses, DNA would be hypomethylated, thereby altering the temporal expression of critical genes necessary for normal embryonic development. However, these results suggest that an extended examination of changes in DNA methylation prior to GD 15 is required to unequivocally prove or disprove the hypothesis. PMID:12163711

  3. Laser Desorption Mass Spectrometry for DNA Sequencing and Analysis

    NASA Astrophysics Data System (ADS)

    Chen, C. H. Winston; Taranenko, N. I.; Golovlev, V. V.; Isola, N. R.; Allman, S. L.

    1998-03-01

    Rapid DNA sequencing and/or analysis is critically important for biomedical research. In the past, gel electrophoresis has been the primary tool to achieve DNA analysis and sequencing. However, gel electrophoresis is a time-consuming and labor-extensive process. Recently, we have developed and used laser desorption mass spectrometry (LDMS) to achieve sequencing of ss-DNA longer than 100 nucleotides. With LDMS, we succeeded in sequencing DNA in seconds instead of hours or days required by gel electrophoresis. In addition to sequencing, we also applied LDMS for the detection of DNA probes for hybridization LDMS was also used to detect short tandem repeats for forensic applications. Clinical applications for disease diagnosis such as cystic fibrosis caused by base deletion and point mutation have also been demonstrated. Experimental details will be presented in the meeting. abstract.

  4. DNA analysis for ornithine transcarbamylase deficiency.

    PubMed

    Rozen, R; Fox, J E; Hack, A M; Fenton, W A; Horwich, A L; Rosenberg, L E

    1986-01-01

    We have utilized the Southern blotting technique to analyse genomic DNA from males with ornithine transcarbamylase (OTC) deficiency and their families. Using a nearly full-length human cDNA probe, we have identified 3 patients with deletions at this locus and have characterized 4 different restriction fragment length polymorphisms that can be used as linkage markers for the OTC mutation. These polymorphisms occur at sufficiently high frequencies so as to enable us to distinguish the two X-chromosomes in approximately 80% of OTC carriers. As a direct consequence of these findings, prenatal diagnosis and carrier assessment can be offered to a large fraction of families at risk for OTC deficiency.

  5. Analysis of amino acid constituents of gallstones

    PubMed Central

    Chen, Ying; Wang, Lian-Lian; Xiao, Yu-Xia; Ni, Jing-Hua; Yu, Yan

    1997-01-01

    AIM: To seek drugs that will efficaciously dissolve bilirubin, glycoprotein and black stones and that will represent improved lithotriptic agents to resolve cholesterol stones, and to study the amino acid constituents of gallstones. METHODS: According to characteristics determined by infrared spectroscopy and to the contents of bilirubin determined by semi-quantitative chemical analysis, 30 of 148 cases of gallstones were selected and divided into 5 groups. Amino acids of the 30 cases were detected by high-speed chromatography. RESULTS: The quantity of amino acids was highest in black stones (226.9 mg/g) and lowest in pure cholesterol stones (1.4 mg/g). In the 5 groups of gallstones, the quantity of amino acids followed the hierarchy of black stone > mixed bilirubin stone and glucoprotein stone > mixed cholesterol stone > pure cholesterol stone. The proportions were: 95.95:29.02 and 28.05:5.78:1. Aliphatic amino acids accounted for approximately 50% of the total amino acids in the gallstones, with glycine accounting for 15.3% of the total amount of the 17 kinds of amino acids. CONCLUSION: For mixed stones, the higher level of bilirubin, the higher content of amino acids. Acidic amino acids were relatively higher in bilirubin stones than in cholesterol stones. PMID:27053886

  6. DNA Microarray Analysis of Estrogen-Responsive Genes.

    PubMed

    Eyster, Kathleen M

    2016-01-01

    DNA microarray is a powerful, non-biased discovery technology that allows the analysis of the expression of thousands of genes at a time. The technology can be used for the identification of differential gene expression, genetic mutations associated with diseases, DNA methylation, single-nucleotide polymorphisms, and microRNA expression, to name a few. This chapter describes microarray technology for the analysis of differential gene expression in response to estrogen treatment.

  7. DNA Methylation Profiling at Single-Base Resolution Reveals Gestational Folic Acid Supplementation Influences the Epigenome of Mouse Offspring Cerebellum

    PubMed Central

    Barua, Subit; Kuizon, Salomon; Brown, W. Ted; Junaid, Mohammed A.

    2016-01-01

    It is becoming increasingly more evident that lifestyle, environmental factors, and maternal nutrition during gestation can influence the epigenome of the developing fetus and thus modulate the physiological outcome. Variations in the intake of maternal nutrients affecting one-carbon metabolism may influence brain development and exert long-term effects on the health of the progeny. In this study, we investigated whether supplementation with high maternal folic acid during gestation alters DNA methylation and gene expression in the cerebellum of mouse offspring. We used reduced representation bisulfite sequencing to analyze the DNA methylation profile at the single-base resolution level. The genome-wide DNA methylation analysis revealed that supplementation with higher maternal folic acid resulted in distinct methylation patterns (P < 0.05) of CpG and non-CpG sites in the cerebellum of offspring. Such variations of methylation and gene expression in the cerebellum of offspring were highly sex-specific, including several genes of the neuronal pathways. These findings demonstrate that alterations in the level of maternal folic acid during gestation can influence methylation and gene expression in the cerebellum of offspring. Such changes in the offspring epigenome may alter neurodevelopment and influence the functional outcome of neurologic and psychiatric diseases. PMID:27199632

  8. DNA Methylation Profiling at Single-Base Resolution Reveals Gestational Folic Acid Supplementation Influences the Epigenome of Mouse Offspring Cerebellum.

    PubMed

    Barua, Subit; Kuizon, Salomon; Brown, W Ted; Junaid, Mohammed A

    2016-01-01

    It is becoming increasingly more evident that lifestyle, environmental factors, and maternal nutrition during gestation can influence the epigenome of the developing fetus and thus modulate the physiological outcome. Variations in the intake of maternal nutrients affecting one-carbon metabolism may influence brain development and exert long-term effects on the health of the progeny. In this study, we investigated whether supplementation with high maternal folic acid during gestation alters DNA methylation and gene expression in the cerebellum of mouse offspring. We used reduced representation bisulfite sequencing to analyze the DNA methylation profile at the single-base resolution level. The genome-wide DNA methylation analysis revealed that supplementation with higher maternal folic acid resulted in distinct methylation patterns (P < 0.05) of CpG and non-CpG sites in the cerebellum of offspring. Such variations of methylation and gene expression in the cerebellum of offspring were highly sex-specific, including several genes of the neuronal pathways. These findings demonstrate that alterations in the level of maternal folic acid during gestation can influence methylation and gene expression in the cerebellum of offspring. Such changes in the offspring epigenome may alter neurodevelopment and influence the functional outcome of neurologic and psychiatric diseases. PMID:27199632

  9. DNA adducts of aristolochic acid II: total synthesis and site-specific mutagenesis studies in mammalian cells

    PubMed Central

    Attaluri, Sivaprasad; Bonala, Radha R.; Yang, In-Young; Lukin, Mark A.; Wen, Yujing; Grollman, Arthur P.; Moriya, Masaaki; Iden, Charles R.; Johnson, Francis

    2010-01-01

    Aristolochic acids I and II (AA-I, AA-II) are found in all Aristolochia species. Ingestion of these acids either in the form of herbal remedies or as contaminated wheat flour causes a dose-dependent chronic kidney failure characterized by renal tubulointerstitial fibrosis. In ∼50% of these cases, the condition is accompanied by an upper urinary tract malignancy. The disease is now termed aristolochic acid nephropathy (AAN). AA-I is largely responsible for the nephrotoxicity while both AA-I and AA-II are genotoxic. DNA adducts derived from AA-I and AA-II have been isolated from renal tissues of patients suffering from AAN. We describe the total synthesis, de novo, of the dA and dG adducts derived from AA-II, their incorporation site-specifically into DNA oligomers and the splicing of these modified oligomers into a plasmid construct followed by transfection into mouse embryonic fibroblasts. Analysis of the plasmid progeny revealed that both adducts blocked replication but were still partly processed by DNA polymerase(s). Although the majority of coding events involved insertion of correct nucleotides, substantial misincorporation of bases also was noted. The dA adduct is significantly more mutagenic than the dG adduct; both adducts give rise, almost exclusively, to misincorporation of dA, which leads to AL-II-dA→T and AL-II-dG→T transversions. PMID:19854934

  10. PCR-based analysis of mitochondrial DNA copy number, mitochondrial DNA damage, and nuclear DNA damage

    PubMed Central

    Gonzalez-Hunt, Claudia P.; Rooney, John P.; Ryde, Ian T.; Anbalagan, Charumathi; Joglekar, Rashmi

    2016-01-01

    Because of the role DNA damage and depletion play in human disease, it is important to develop and improve tools to assess these endpoints. This unit describes PCR-based methods to measure nuclear and mitochondrial DNA damage and copy number. Long amplicon quantitative polymerase chain reaction (LA-QPCR) is used to detect DNA damage by measuring the number of polymerase-inhibiting lesions present based on the amount of PCR amplification; real-time PCR (RT-PCR) is used to calculate genome content. In this unit we provide step-by-step instructions to perform these assays in Homo sapiens, Mus musculus, Rattus norvegicus, Caenorhabditis elegans, Drosophila melanogaster, Danio rerio, Oryzias latipes, Fundulus grandis, and Fundulus heteroclitus, and discuss the advantages and disadvantages of these assays. PMID:26828332

  11. Chemical repair of base lesions, AP-sites, and strand breaks on plasmid DNA in dilute aqueous solution by ascorbic acid

    SciTech Connect

    Hata, Kuniki; Urushibara, Ayumi; Yamashita, Shinichi; Shikazono, Naoya; Yokoya, Akinari; Katsumura, Yosuke

    2013-05-03

    Highlights: •We report a novel mechanism of radiation protection of DNA by chemical activity of ascorbic acid. •The “chemical repair” of DNA damage was revealed using biochemical assay and chemical kinetics analysis. •We found that ascorbic acid significantly repairs precursors of nucleobase lesions and abasic sites. •However, ascorbic acid seldom repairs precursors of DNA-strand breaks. -- Abstract: We quantified the damage yields produced in plasmid DNA by γ-irradiation in the presence of low concentrations (10–100 μM) of ascorbic acid, which is a major antioxidant in living systems, to clarify whether it chemically repairs radiation damage in DNA. The yield of DNA single strand breaks induced by irradiation was analyzed with agarose gel electrophoresis as conformational changes in closed circular plasmids. Base lesions and abasic sites were also observed as additional conformational changes by treating irradiated samples with glycosylase proteins. By comparing the suppression efficiencies to the induction of each DNA lesion, in addition to scavenging of the OH radicals derived from water radiolysis, it was found that ascorbic acid promotes the chemical repair of precursors of AP-sites and base lesions more effectively than those of single strand breaks. We estimated the efficiency of the chemical repair of each lesion using a kinetic model. Approximately 50–60% of base lesions and AP-sites were repaired by 10 μM ascorbic acid, although strand breaks were largely unrepaired by ascorbic acid at low concentrations. The methods in this study will provide a route to understanding the mechanistic aspects of antioxidant activity in living systems.

  12. Bioinformatic analysis of the protein/DNA interface

    PubMed Central

    Schneider, Bohdan; Černý, Jiří; Svozil, Daniel; Čech, Petr; Gelly, Jean-Christophe; de Brevern, Alexandre G.

    2014-01-01

    To investigate the principles driving recognition between proteins and DNA, we analyzed more than thousand crystal structures of protein/DNA complexes. We classified protein and DNA conformations by structural alphabets, protein blocks [de Brevern, Etchebest and Hazout (2000) (Bayesian probabilistic approach for predicting backbone structures in terms of protein blocks. Prots. Struct. Funct. Genet., 41:271–287)] and dinucleotide conformers [Svozil, Kalina, Omelka and Schneider (2008) (DNA conformations and their sequence preferences. Nucleic Acids Res., 36:3690–3706)], respectively. Assembling the mutually interacting protein blocks and dinucleotide conformers into ‘interaction matrices’ revealed their correlations and conformer preferences at the interface relative to their occurrence outside the interface. The analyzed data demonstrated important differences between complexes of various types of proteins such as transcription factors and nucleases, distinct interaction patterns for the DNA minor groove relative to the major groove and phosphate and importance of water-mediated contacts. Water molecules mediate proportionally the largest number of contacts in the minor groove and form the largest proportion of contacts in complexes of transcription factors. The generally known induction of A-DNA forms by complexation was more accurately attributed to A-like and intermediate A/B conformers rare in naked DNA molecules. PMID:24335080

  13. Bioinformatics analysis of circulating cell-free DNA sequencing data.

    PubMed

    Chan, Landon L; Jiang, Peiyong

    2015-10-01

    The discovery of cell-free DNA molecules in plasma has opened up numerous opportunities in noninvasive diagnosis. Cell-free DNA molecules have become increasingly recognized as promising biomarkers for detection and management of many diseases. The advent of next generation sequencing has provided unprecedented opportunities to scrutinize the characteristics of cell-free DNA molecules in plasma in a genome-wide fashion and at single-base resolution. Consequently, clinical applications of circulating cell-free DNA analysis have not only revolutionized noninvasive prenatal diagnosis but also facilitated cancer detection and monitoring toward an era of blood-based personalized medicine. With the remarkably increasing throughput and lowering cost of next generation sequencing, bioinformatics analysis becomes increasingly demanding to understand the large amount of data generated by these sequencing platforms. In this Review, we highlight the major bioinformatics algorithms involved in the analysis of cell-free DNA sequencing data. Firstly, we briefly describe the biological properties of these molecules and provide an overview of the general bioinformatics approach for the analysis of cell-free DNA. Then, we discuss the specific upstream bioinformatics considerations concerning the analysis of sequencing data of circulating cell-free DNA, followed by further detailed elaboration on each key clinical situation in noninvasive prenatal diagnosis and cancer management where downstream bioinformatics analysis is heavily involved. We also discuss bioinformatics analysis as well as clinical applications of the newly developed massively parallel bisulfite sequencing of cell-free DNA. Finally, we offer our perspectives on the future development of bioinformatics in noninvasive diagnosis.

  14. An osmium-DNA interstrand complex: application to facile DNA methylation analysis.

    PubMed

    Tanaka, Kazuo; Tainaka, Kazuki; Umemoto, Tadashi; Nomura, Akiko; Okamoto, Akimitsu

    2007-11-21

    Nucleic acids often acquire new functions by forming a variety of complexes with metal ions. Osmium, in an oxidized state, also reacts with C5-methylated pyrimidines. However, control of the sequence specificity of osmium complexation with DNA is still immature, and the value of the resulting complexes is unknown. We have designed a bipyridine-attached adenine derivative for sequence-specific osmium complexation. Sequence-specific osmium complexation was achieved by hybridization of a short DNA molecule containing this functional nucleotide to a target DNA sequence and resulted in the formation of a cross-linked structure. The interstrand cross-link clearly distinguished methylated cytosines from unmethylated cytosines and was used to quantify the degree of methylation at a specific cytosine in the genome.

  15. Detection of methylglyoxal as a degradation product of DNA and nucleic acid components treated with strong acid.

    PubMed

    Chaplen, F W; Fahl, W E; Cameron, D C

    1996-05-01

    The 1,2-diaminobenzene derivation assay for methylglyoxal in biological systems involves the use of perchloric acid, both as a deproteinizing agent and to prevent the spontaneous formation of methylglyoxal from glycolytic pathway intermediates. However, while using a modification of the standard literature assay to measure methylglyoxal in Chinese hamster ovary cells, we found that oxidation of nucleic acids and related compounds by perchloric or trichloroacetic acid results in the formation of methylglyoxal. Compounds containing 2-deoxyribose gave higher levels of methylglyoxal than those containing ribose; purine nucleotides and deoxynucleotides gave more methylglyoxal than did the pyrimidines. Nucleic acids were the most susceptible to degradation, with 12-fold more methylglyoxal being formed from DNA than RNA. Oxidation of nucleic acids increased with higher temperatures and with decreasing nucleic acid fragment size. Another product of nucleic acid oxidation was 2,3-butanedione, the 1,2-diaminobenzene derivative of which is sometimes used as an internal standard during methylglyoxal measurement. Unless accounted for during the assay procedure, the generation of methylglyoxal and 2,3-butanedione due to the oxidation of nucleic acids may lead to substantial errors in the determination of methylglyoxal concentrations in biological systems.

  16. Cloning and sequence analysis of Hemonchus contortus HC58cDNA.

    PubMed

    Muleke, Charles I; Ruofeng, Yan; Lixin, Xu; Xinwen, Bo; Xiangrui, Li

    2007-06-01

    The complete coding sequence of Hemonchus contortus HC58cDNA was generated by rapid amplification of cDNA ends and polymerase chain reaction using primers based on the 5' and 3' ends of the parasite mRNA, accession no. AF305964. The HC58cDNA gene was 851 bp long, with open reading frame of 717 bp, precursors to 239 amino acids coding for approximately 27 kDa protein. Analysis of amino acid sequence revealed conserved residues of cysteine, histidine, asparagine, occluding loop pattern, hemoglobinase motif and glutamine of the oxyanion hole characteristic of cathepsin B like proteases (CBL). Comparison of the predicted amino acid sequences showed the protein shared 33.5-58.7% identity to cathepsin B homologues in the papain clan CA family (family C1). Phylogenetic analysis revealed close evolutionary proximity of the protein sequence to counterpart sequences in the CBL, suggesting that HC58cDNA was a member of the papain family.

  17. Immobilization-free electrochemical DNA detection with anthraquinone-labeled pyrrolidinyl peptide nucleic acid probe.

    PubMed

    Kongpeth, Jutatip; Jampasa, Sakda; Chaumpluk, Piyasak; Chailapakul, Orawon; Vilaivan, Tirayut

    2016-01-01

    Electrochemical detection provides a simple, rapid, sensitive and inexpensive method for DNA detection. In traditional electrochemical DNA biosensors, the probe is immobilized onto the electrode. Hybridization with the DNA target causes a change in electrochemical signal, either from the intrinsic signal of the probe/target or through a label or a redox indicator. The major drawback of this approach is the requirement for probe immobilization in a controlled fashion. In this research, we take the advantage of different electrostatic properties between PNA and DNA to develop an immobilization-free approach for highly sequence-specific electrochemical DNA sensing on a screen-printed carbon electrode (SPCE) using a square-wave voltammetric (SWV) technique. Anthraquinone-labeled pyrrolidinyl peptide nucleic acid (AQ-PNA) was employed as a probe together with an SPCE that was modified with a positively-charged polymer (poly quaternized-(dimethylamino-ethyl)methacrylate, PQDMAEMA). The electrostatic attraction between the negatively-charged PNA-DNA duplex and the positively-charged modified SPCE attributes to the higher signal of PNA-DNA duplex than that of the electrostatically neutral PNA probe, resulting in a signal change. The calibration curve of this proposed method exhibited a linear range between 0.35 and 50 nM of DNA target with a limit of detection of 0.13 nM (3SD(blank)/Slope). The sub-nanomolar detection limit together with a small sample volume required (20 μL) allowed detection of <10 fmol (<1 ng) of DNA. With the high specificity of the pyrrolidinyl PNA probe used, excellent discrimination between complementary and various single-mismatched DNA targets was obtained. An application of this new platform for a sensitive and specific detection of isothermally-amplified shrimp's white spot syndrome virus (WSSV) DNA was successfully demonstrated. PMID:26695270

  18. Immobilization-free electrochemical DNA detection with anthraquinone-labeled pyrrolidinyl peptide nucleic acid probe.

    PubMed

    Kongpeth, Jutatip; Jampasa, Sakda; Chaumpluk, Piyasak; Chailapakul, Orawon; Vilaivan, Tirayut

    2016-01-01

    Electrochemical detection provides a simple, rapid, sensitive and inexpensive method for DNA detection. In traditional electrochemical DNA biosensors, the probe is immobilized onto the electrode. Hybridization with the DNA target causes a change in electrochemical signal, either from the intrinsic signal of the probe/target or through a label or a redox indicator. The major drawback of this approach is the requirement for probe immobilization in a controlled fashion. In this research, we take the advantage of different electrostatic properties between PNA and DNA to develop an immobilization-free approach for highly sequence-specific electrochemical DNA sensing on a screen-printed carbon electrode (SPCE) using a square-wave voltammetric (SWV) technique. Anthraquinone-labeled pyrrolidinyl peptide nucleic acid (AQ-PNA) was employed as a probe together with an SPCE that was modified with a positively-charged polymer (poly quaternized-(dimethylamino-ethyl)methacrylate, PQDMAEMA). The electrostatic attraction between the negatively-charged PNA-DNA duplex and the positively-charged modified SPCE attributes to the higher signal of PNA-DNA duplex than that of the electrostatically neutral PNA probe, resulting in a signal change. The calibration curve of this proposed method exhibited a linear range between 0.35 and 50 nM of DNA target with a limit of detection of 0.13 nM (3SD(blank)/Slope). The sub-nanomolar detection limit together with a small sample volume required (20 μL) allowed detection of <10 fmol (<1 ng) of DNA. With the high specificity of the pyrrolidinyl PNA probe used, excellent discrimination between complementary and various single-mismatched DNA targets was obtained. An application of this new platform for a sensitive and specific detection of isothermally-amplified shrimp's white spot syndrome virus (WSSV) DNA was successfully demonstrated.

  19. DNA Methylation Analysis: Choosing the Right Method

    PubMed Central

    Kurdyukov, Sergey; Bullock, Martyn

    2016-01-01

    In the burgeoning field of epigenetics, there are several methods available to determine the methylation status of DNA samples. However, choosing the method that is best suited to answering a particular biological question still proves to be a difficult task. This review aims to provide biologists, particularly those new to the field of epigenetics, with a simple algorithm to help guide them in the selection of the most appropriate assay to meet their research needs. First of all, we have separated all methods into two categories: those that are used for: (1) the discovery of unknown epigenetic changes; and (2) the assessment of DNA methylation within particular regulatory regions/genes of interest. The techniques are then scrutinized and ranked according to their robustness, high throughput capabilities and cost. This review includes the majority of methods available to date, but with a particular focus on commercially available kits or other simple and straightforward solutions that have proven to be useful. PMID:26751487

  20. [Diagnosis of Lepore hemoglobinopathy with DNA analysis].

    PubMed

    Lavinha, J; Sampaio, V; da Mata, E

    1992-07-01

    We illustrate the application of recombinant DNA methods (namely Southern blotting) for the genotype diagnosis of haemoglobin Lepore Boston: the use of the restriction endonucleases PstI and XbaI along with a beta globin gene specific probe make it possible to detect a deletion of approximately 7kb which typically characterizes the delta beta Boston gene. The impact of using such methods in the prenatal diagnosis of major haemoglobinopathies is discussed.

  1. DNA immobilization on a polypyrrole nanofiber modified electrode and its interaction with salicylic acid/aspirin.

    PubMed

    Yousef Elahi, M; Bathaie, S Z; Kazemi, S H; Mousavi, M F

    2011-04-15

    A double-stranded calf thymus DNA (dsDNA) was physisorbed onto a polypyrrole (PPy) nanofiber film that had been electrochemically deposited onto a Pt electrode. The surface morphology of the polymeric film was characterized using scanning electron microscopy (SEM). The electrochemical characteristics of the PPy film and the DNA deposited onto the PPy modified electrode were investigated by cyclic voltammetry (CV), differential pulse voltammetry (DPV), and electrochemical impedance spectroscopy (EIS). Then the interaction of DNA with salicylic acid (SA) and acetylsalicylic acid (ASA), or aspirin, was studied on the electrode surface with DPV. An increase in the DPV current was observed due to the oxidation of guanine, which decreased with the increasing concentrations of the ligands. The interactions of SA and ASA with the DNA follow the saturation isotherm behavior. The binding constants of these interactions were 1.15×10(4)M for SA and 7.46×10(5)M for ASA. The numbers of binding sites of SA and ASA on DNA were approximately 0.8 and 0.6, respectively. The linear dynamic ranges of the sensors were 0.1-2μM (r(2)=0.996) and 0.05-1mM (r(2)=0.996) with limits of detection of 8.62×10(-1) and 5.24×10(-6)μM for SA and ASA, respectively.

  2. PLASMID DNA DAMAGE CAUSED BY METHYLATED ARSENICALS, ASCORBIC ACID AND HUMAN LIVER FERRITIN

    EPA Science Inventory

    Plasmid DNA damage caused by methylated arsenicals, ascorbic acid and human liver ferritin.

    Arsenic causes cancer in human skin, urinary bladder, lung, liver and kidney and is a significant world-wide public health problem. Although the metabolism of inorganic arsenic is ...

  3. DNA tetrahedron and star trigon nanostructures for target recycling detection of nucleic acid.

    PubMed

    Li, Yueran; Chen, Xifeng; Wang, Bidou; Liu, Guangxing; Tang, Yuguo; Miao, Peng

    2016-06-01

    Human immunodeficiency virus (HIV) is a retrovirus which attacks the human body's immune system and further leads to acquired immunodeficiency syndrome (AIDS). Nucleic acid detection is of great importance in the medical diagnosis of such diseases. Herein, we develop a simple and enzyme-free electrochemical method for the target recycling detection of nuclei acid. DNA tetrahedron and star trigon nanostructures are designed and constructed on the electrode interface for target capture and signal enrichment. This strategy is convenient and sensitive, with a limit of detection as low as 1 fM, and can also successfully distinguish single-base mismatched DNA. Therefore, the proposed method has a promising potential application for HIV DNA detection. PMID:27170090

  4. DNA tetrahedron and star trigon nanostructures for target recycling detection of nucleic acid.

    PubMed

    Li, Yueran; Chen, Xifeng; Wang, Bidou; Liu, Guangxing; Tang, Yuguo; Miao, Peng

    2016-06-01

    Human immunodeficiency virus (HIV) is a retrovirus which attacks the human body's immune system and further leads to acquired immunodeficiency syndrome (AIDS). Nucleic acid detection is of great importance in the medical diagnosis of such diseases. Herein, we develop a simple and enzyme-free electrochemical method for the target recycling detection of nuclei acid. DNA tetrahedron and star trigon nanostructures are designed and constructed on the electrode interface for target capture and signal enrichment. This strategy is convenient and sensitive, with a limit of detection as low as 1 fM, and can also successfully distinguish single-base mismatched DNA. Therefore, the proposed method has a promising potential application for HIV DNA detection.

  5. A universal molecular translator for non-nucleic acid targets that enables dynamic DNA assemblies and logic operations.

    PubMed

    Tang, Wei; Hu, Shichao; Wang, Huaming; Zhao, Yan; Li, Na; Liu, Feng

    2014-11-28

    A universal molecular translator based on the target-triggered DNA strand displacement was developed, which was able to convert various kinds of non-nucleic acid targets into a unique output DNA. This translation strategy was successfully applied in directing dynamic DNA assemblies and in realizing three-input logic gate operations. PMID:25295484

  6. NMR analysis of base-pair opening kinetics in DNA.

    PubMed

    Szulik, Marta W; Voehler, Markus; Stone, Michael P

    2014-12-12

    Base pairing in nucleic acids plays a crucial role in their structure and function. Differences in the base-pair opening and closing kinetics of individual double-stranded DNA sequences or between chemically modified base pairs provide insight into the recognition of these base pairs by DNA processing enzymes. This unit describes how to quantify the kinetics for localized base pairs by observing changes in the imino proton signals by nuclear magnetic resonance spectroscopy. The determination of all relevant parameters using state-of-the art techniques and NMR instrumentation, including cryoprobes, is discussed.

  7. Intelligent DNA machine for the ultrasensitive colorimetric detection of nucleic acids.

    PubMed

    Xu, Jianguo; Qian, Jun; Li, Hongling; Wu, Zai-Sheng; Shen, Weiyu; Jia, Lee

    2016-01-15

    As DNA is employed to serve as a smart building block, an increasing interest has been devoted to the development of different DNA-based machines for the specific purpose, for example, the exploration of inter- or intramolecular interaction. In the current contribution, we developed an intelligent DNA machine and its operation can be designed to execute the ultrasensitive colorimetric detection of target nucleic acids. The DNA machine consists of a hairpin probe (HP) and an assistant template (AT). Using p53 gene as the target model to trigger the molecular machine operation, cyclic nucleic acid strand displacement polymerization (CNDP) was specifically induced, leading to the DNAzyme mediated catalytic reaction for signal readout. Specifically, with the help of polymerase and nickase, one target molecule was able to drive DNA nano-mechanical devices one-by-one through the hybridization/polymerization displacement cycles, and every initiated machine continued to operate, causing the dramatic accumulation of G-quadruplex-contained products. The G-quadruplex structure after binding to hemin could act as a horseradish peroxidase (HRP)-mimicking DNAzyme and catalyzed the oxidation of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) by H2O2. As a result, an enhanced color change could be detected because of the generation of oxidation product ABTS•(+). In this way, the DNA machine has no any signal loss and enables the quantitative measurement of p53 DNA with a detection limit of 10fM, indicating great promise for unique application in biomedical research and early clinical diagnosis.

  8. Efficient in vivo gene transfection by stable DNA/PEI complexes coated by hyaluronic acid.

    PubMed

    Ito, Tomoko; Iida-Tanaka, Naoko; Koyama, Yoshiyuki

    2008-05-01

    Plasmid DNA was mixed with polyethyleneimine (PEI) and hyaluronic acid (HA) to afford ternary complexes with negative surface charge regardless of the mixing order. They showed reduced non-specific interactions with blood components. When DNA and PEI were mixed at a high concentration such as that used in in vivo experiments, they soon aggregated, and large particles were formed. On the other hand, pre-addition of HA to DNA prior to PEI effectively diminished the aggregation, and 10% (in volume) of the complexes remained as small particles with a diameter below 80 nm. Those negatively charged small ternary complexes induced a much stronger extra-gene expression in tumor than binary DNA/PEI complex after intratumoral or intravenous injection into the mice bearing B16 cells. PMID:18446606

  9. DNA detection using water-soluble conjugated polymers and peptide nucleic acid probes

    NASA Astrophysics Data System (ADS)

    Gaylord, Brent S.; Heeger, Alan J.; Bazan, Guillermo C.

    2002-08-01

    The light-harvesting properties of cationic conjugated polymers are used to sensitize the emission of a dye on a specific peptide nucleic acid (PNA) sequence for the purpose of homogeneous, "real-time" DNA detection. Signal transduction is controlled by hybridization of the neutral PNA probe and the negative DNA target. Electrostatic interactions bring the hybrid complex and cationic polymer within distances required for Förster energy transfer. Conjugated polymer excitation provides fluorescein emission >25 times higher than that obtained by exciting the dye, allowing detection of target DNA at concentrations of 10 pM with a standard fluorometer. A simple and highly sensitive assay with optical amplification that uses the improved hybridization behavior of PNA/DNA complexes is thus demonstrated.

  10. Analysis-Driven Lossy Compression of DNA Microarray Images.

    PubMed

    Hernández-Cabronero, Miguel; Blanes, Ian; Pinho, Armando J; Marcellin, Michael W; Serra-Sagristà, Joan

    2016-02-01

    DNA microarrays are one of the fastest-growing new technologies in the field of genetic research, and DNA microarray images continue to grow in number and size. Since analysis techniques are under active and ongoing development, storage, transmission and sharing of DNA microarray images need be addressed, with compression playing a significant role. However, existing lossless coding algorithms yield only limited compression performance (compression ratios below 2:1), whereas lossy coding methods may introduce unacceptable distortions in the analysis process. This work introduces a novel Relative Quantizer (RQ), which employs non-uniform quantization intervals designed for improved compression while bounding the impact on the DNA microarray analysis. This quantizer constrains the maximum relative error introduced into quantized imagery, devoting higher precision to pixels critical to the analysis process. For suitable parameter choices, the resulting variations in the DNA microarray analysis are less than half of those inherent to the experimental variability. Experimental results reveal that appropriate analysis can still be performed for average compression ratios exceeding 4.5:1.

  11. Detection of Adult Green Sturgeon Using Environmental DNA Analysis

    PubMed Central

    Bergman, Paul S.; Schumer, Gregg; Blankenship, Scott; Campbell, Elizabeth

    2016-01-01

    Environmental DNA (eDNA) is an emerging sampling method that has been used successfully for detection of rare aquatic species. The Identification of sampling tools that are less stressful for target organisms has become increasingly important for rare and endangered species. A decline in abundance of the Southern Distinct Population Segment (DPS) of North American Green Sturgeon located in California’s Central Valley has led to its listing as Threatened under the Federal Endangered Species Act in 2006. While visual surveys of spawning Green Sturgeon in the Central Valley are effective at monitoring fish densities in concentrated pool habitats, results do not scale well to the watershed level, providing limited spatial and temporal context. Unlike most traditional survey methods, environmental DNA analysis provides a relatively quick, inexpensive tool that could efficiently monitor the presence and distribution of aquatic species. We positively identified Green Sturgeon DNA at two locations of known presence in the Sacramento River, proving that eDNA can be effective for monitoring the presence of adult sturgeon. While further study is needed to understand uncertainties of the sampling method, our study represents the first documented detection of Green Sturgeon eDNA, indicating that eDNA analysis could provide a new tool for monitoring Green Sturgeon distribution in the Central Valley, complimenting traditional on-going survey methods. PMID:27096433

  12. Detection of Adult Green Sturgeon Using Environmental DNA Analysis.

    PubMed

    Bergman, Paul S; Schumer, Gregg; Blankenship, Scott; Campbell, Elizabeth

    2016-01-01

    Environmental DNA (eDNA) is an emerging sampling method that has been used successfully for detection of rare aquatic species. The Identification of sampling tools that are less stressful for target organisms has become increasingly important for rare and endangered species. A decline in abundance of the Southern Distinct Population Segment (DPS) of North American Green Sturgeon located in California's Central Valley has led to its listing as Threatened under the Federal Endangered Species Act in 2006. While visual surveys of spawning Green Sturgeon in the Central Valley are effective at monitoring fish densities in concentrated pool habitats, results do not scale well to the watershed level, providing limited spatial and temporal context. Unlike most traditional survey methods, environmental DNA analysis provides a relatively quick, inexpensive tool that could efficiently monitor the presence and distribution of aquatic species. We positively identified Green Sturgeon DNA at two locations of known presence in the Sacramento River, proving that eDNA can be effective for monitoring the presence of adult sturgeon. While further study is needed to understand uncertainties of the sampling method, our study represents the first documented detection of Green Sturgeon eDNA, indicating that eDNA analysis could provide a new tool for monitoring Green Sturgeon distribution in the Central Valley, complimenting traditional on-going survey methods. PMID:27096433

  13. Detection of Adult Green Sturgeon Using Environmental DNA Analysis.

    PubMed

    Bergman, Paul S; Schumer, Gregg; Blankenship, Scott; Campbell, Elizabeth

    2016-01-01

    Environmental DNA (eDNA) is an emerging sampling method that has been used successfully for detection of rare aquatic species. The Identification of sampling tools that are less stressful for target organisms has become increasingly important for rare and endangered species. A decline in abundance of the Southern Distinct Population Segment (DPS) of North American Green Sturgeon located in California's Central Valley has led to its listing as Threatened under the Federal Endangered Species Act in 2006. While visual surveys of spawning Green Sturgeon in the Central Valley are effective at monitoring fish densities in concentrated pool habitats, results do not scale well to the watershed level, providing limited spatial and temporal context. Unlike most traditional survey methods, environmental DNA analysis provides a relatively quick, inexpensive tool that could efficiently monitor the presence and distribution of aquatic species. We positively identified Green Sturgeon DNA at two locations of known presence in the Sacramento River, proving that eDNA can be effective for monitoring the presence of adult sturgeon. While further study is needed to understand uncertainties of the sampling method, our study represents the first documented detection of Green Sturgeon eDNA, indicating that eDNA analysis could provide a new tool for monitoring Green Sturgeon distribution in the Central Valley, complimenting traditional on-going survey methods.

  14. cDNA-derived amino acid sequences of myoglobins from nine species of whales and dolphins.

    PubMed

    Iwanami, Kentaro; Mita, Hajime; Yamamoto, Yasuhiko; Fujise, Yoshihiro; Yamada, Tadasu; Suzuki, Tomohiko

    2006-10-01

    We determined the myoglobin (Mb) cDNA sequences of nine cetaceans, of which six are the first reports of Mb sequences: sei whale (Balaenoptera borealis), Bryde's whale (Balaenoptera edeni), pygmy sperm whale (Kogia breviceps), Stejneger's beaked whale (Mesoplodon stejnegeri), Longman's beaked whale (Indopacetus pacificus), and melon-headed whale (Peponocephala electra), and three confirm the previously determined chemical amino acid sequences: sperm whale (Physeter macrocephalus), common minke whale (Balaenoptera acutorostrata) and pantropical spotted dolphin (Stenella attenuata). We found two types of Mb in the skeletal muscle of pantropical spotted dolphin: Mb I with the same amino acid sequence as that deposited in the protein database, and Mb II, which differs at two amino acid residues compared with Mb I. Using an alignment of the amino acid or cDNA sequences of cetacean Mb, we constructed a phylogenetic tree by the NJ method. Clustering of cetacean Mb amino acid and cDNA sequences essentially follows the classical taxonomy of cetaceans, suggesting that Mb sequence data is valid for classification of cetaceans at least to the family level. PMID:16962803

  15. Calcium-activated gene transfection from DNA/poly(amic acid-co-imide) complexes

    PubMed Central

    Wu, Szu-Yuan; Chang, Li-Ting; Peng, Sydeny; Tsai, Hsieh-Chih

    2015-01-01

    In this study, we synthesized a water-soluble poly(amic acid-co-imide) (PA-I) from ethylenediaminetetraacetic dianhydride (EDTA) and 2,2′-(ethylenedioxy)bis(ethylamine) that possesses comparable transfection efficiency to that of polyethylenimine (PEI), when prepared in combination with divalent calcium cations. The polycondensation of monomers afforded poly(amic acid) (PA) precursors, and subsequent thermal imidization resulted in the formation of PA-I. At a polymer/DNA ratio (indicated by the molar ratio of nitrogen in the polymer to phosphate in DNA) of 40, complete retardation of the DNA band was observed by gel electrophoresis, indicating the strong association of DNA with PA-I. A zeta potential of −22 mV was recorded for the PA-I polymer solution, and no apparent cytotoxicity was observed at concentrations up to 500 μg·mL−1. In the presence of divalent Ca2+, the transfection efficiency of PA-I was higher than that of PA, due to the formation of a copolymer/Ca2+/DNA polyplex and the reduction in negative charge due to thermal cyclization. Interestingly, a synergistic effect of Ca2+ and the synthesized copolymer on DNA transfection was observed. The use of Ca2+ or copolymer alone resulted in unsatisfactory delivery, whereas the formation of three-component polyplexes synergistically increased DNA transfection. Our findings demonstrated that a PA-I/Ca2+/DNA polyplex could serve as a promising candidate for gene delivery. PMID:25767385

  16. Strand displacement and duplex invasion into double-stranded DNA by pyrrolidinyl peptide nucleic acids.

    PubMed

    Bohländer, Peggy R; Vilaivan, Tirayut; Wagenknecht, Hans-Achim

    2015-09-21

    The so-called acpcPNA system bears a peptide backbone consisting of 4'-substituted proline units with (2'R,4'R) configuration in an alternating combination with (2S)-amino-cyclopentane-(1S)-carboxylic acids. acpcPNA forms exceptionally stable hybrids with complementary DNA. We demonstrate herein (i) strand displacements by single-stranded DNA from acpcPNA-DNA hybrids, and by acpcPNA strands from DNA duplexes, and (ii) strand invasions by acpcPNA into double-stranded DNA. These processes were studied in vitro using synthetic oligonucleotides and by means of our concept of wavelength-shifting fluorescent nucleic acid probes, including fluorescence lifetime measurements that allow quantifying energy transfer efficiencies. The strand displacements of preannealed 14mer acpcPNA-7mer DNA hybrids consecutively by 10mer and 14mer DNA strands occur with rather slow kinetics but yield high fluorescence color ratios (blue : yellow or blue : red), fluorescence intensity enhancements, and energy transfer efficiencies. Furthermore, 14mer acpcPNA strands are able to invade into 30mer double-stranded DNA, remarkably with quantitative efficiency in all studied cases. These processes can also be quantified by means of fluorescence. This remarkable behavior corroborates the extraordinary versatile properties of acpcPNA. In contrast to conventional PNA systems which require 3 or more equivalents PNA, only 1.5 equivalents acpcPNA are sufficient to get efficient double duplex invasion. Invasions also take place even in the presence of 250 mM NaCl which represents an ionic strength nearly twice as high as the physiological ion concentration. These remarkable results corroborate the extraordinary properties of acpcPNA, and thus acpcPNA represents an eligible tool for biological analytics and antigene applications.

  17. Inactivation of Mycobacterium tuberculosis for DNA Typing Analysis

    PubMed Central

    Bemer-Melchior, P.; Drugeon, H. B.

    1999-01-01

    DNA fingerprinting analysis of Mycobacterium tuberculosis is used for epidemiological studies and the control of laboratory cross-contamination. Because standardized procedures are not entirely safe for mycobacteriology laboratory staff, the paper proposes a new technique for the processing of specimens. The technique ensures the inactivation of M. tuberculosis before DNA extraction without the loss of DNA integrity. The control of inactivated cultures should be rigorous and should involve the use of two different culture media incubated for at least 4 months. PMID:10364613

  18. Analysis of genomic DNA with the UCSC genome browser.

    PubMed

    Pevsner, Jonathan

    2009-01-01

    Genomic DNA is being sequenced and annotated at a rapid rate, with terabases of DNA currently deposited in GenBank and other repositories. Genome browsers provide an essential collection of resources to visualize and analyze chromosomal DNA. The University of California, Santa Cruz (UCSC) Genome Browser provides annotations from the level of single nucleotides to whole chromosomes for four dozen metazoan and other species. The Genome Browser may be used to address a wide range of problems in bioinformatics (e.g., sequence analysis), comparative genomics, and evolution.

  19. Collection of Samples for DNA Analysis.

    PubMed

    van Oorschot, Roland A H; Verdon, Timothy J; Ballantyne, Kaye N

    2016-01-01

    Effective sampling of biological material is critical to the ability to acquire DNA profiles of probative value. The main methods of collection are swabbing, tapelifting, or direct excision. This chapter describes the key aspects to consider when applying these methods, in addition to suggested procedures for swabbing and tapelifting. Important issues to be considered, such as exhibit triaging, pre-examination preparation, contamination risk reduction, sample localization, sample identification, and sample prioritization as well as aspects of record keeping, packaging, and storage, are also raised. PMID:27259727

  20. A Mathematical Approach to the Analysis of Multiplex DNA Profiles

    PubMed Central

    Goor, Robert M.; Neall, Lisa Forman; Hoffman, Douglas; Sherry, Stephen T.

    2010-01-01

    Multiplex DNA profiles are used extensively for biomedical and forensic purposes. However, while DNA profile data generation is automated, human analysis of those data is not, and the need for speed combined with accuracy demands a computer-automated approach to sample interpretation and quality assessment. In this paper, we describe an integrated mathematical approach to modeling the data and extracting the relevant information, while rejecting noise and sample artifacts. We conclude with examples showing the effectiveness of our algorithms. PMID:21103945

  1. Laser Desorption Mass Spectrometry for High Throughput DNA Analysis and Its Applications

    SciTech Connect

    Allman, S.L.; Chen, C.H.; Golovlev, V.V.; Isola, N.R.; Matteson, K.J.; Potter, N.T.; Taranenko, N.I.

    1999-01-23

    Laser desorption mass spectrometry (LDMS) has been developed for DNA sequencing, disease diagnosis, and DNA Fingerprinting for forensic applications. With LDMS, the speed of DNA analysis can be much faster than conventional gel electrophoresis. No dye or radioactive tagging to DNA segments for detection is needed. LDMS is emerging as a new alternative technology for DNA analysis.

  2. Laser desorption mass spectrometry for high-throughput DNA analysis and its applications

    NASA Astrophysics Data System (ADS)

    Chen, C. H. Winston; Golovlev, Valeri V.; Taranenko, N. I.; Allman, S. L.; Isola, Narayana R.; Potter, N. T.; Matteson, K. J.; Chang, Linus Y.

    1999-05-01

    Laser desorption mass spectrometry (LDMS) has been developed for DNA sequencing, disease diagnosis, and DNA fingerprinting for forensic applications. With LDMS, the speed of DNA analysis can be much faster than conventional gel electrophoresis. No dye or radioactive tagging to DNA segments for detection is needed. LDMS is emerging as a new alternative technology for DNA analysis.

  3. Hydration of nucleic acid fragments: comparison of theory and experiment for high-resolution crystal structures of RNA, DNA, and DNA-drug complexes.

    PubMed

    Hummer, G; García, A E; Soumpasis, D M

    1995-05-01

    A computationally efficient method to describe the organization of water around solvated biomolecules is presented. It is based on a statistical mechanical expression for the water-density distribution in terms of particle correlation functions. The method is applied to analyze the hydration of small nucleic acid molecules in the crystal environment, for which high-resolution x-ray crystal structures have been reported. Results for RNA [r(ApU).r(ApU)] and DNA [d(CpG).d(CpG) in Z form and with parallel strand orientation] and for DNA-drug complexes [d(CpG).d(CpG) with the drug proflavine intercalated] are described. A detailed comparison of theoretical and experimental data shows positional agreement for the experimentally observed water sites. The presented method can be used for refinement of the water structure in x-ray crystallography, hydration analysis of nuclear magnetic resonance structures, and theoretical modeling of biological macromolecules such as molecular docking studies. The speed of the computations allows hydration analyses of molecules of almost arbitrary size (tRNA, protein-nucleic acid complexes, etc.) in the crystal environment and in aqueous solution.

  4. [Determination of body fluid based on analysis of nucleic acids].

    PubMed

    Korabečná, Marie

    2015-01-01

    Recent methodological approaches of molecular genetics allow isolation of nucleic acids (DNA and RNA) from negligible forensic samples. Analysis of these molecules may be used not only for individual identification based on DNA profiling but also for the detection of origin of the body fluid which (alone or in mixture with other body fluids) forms the examined biological trace. Such an examination can contribute to the evaluation of procedural, technical and tactical value of the trace. Molecular genetic approaches discussed in the review offer new possibilities in comparison with traditional spectrum of chemical, immunological and spectroscopic tests especially with regard to the interpretation of mixtures of biological fluids and to the confirmatory character of the tests. Approaches based on reverse transcription of tissue specific mRNA and their subsequent polymerase chain reaction (PCR) and fragmentation analysis are applicable on samples containing minimal amounts of biological material. Methods for body fluid discrimination based on examination of microRNA in samples provided so far confusing results therefore further development in this field is needed. The examination of tissue specific methylation of nucleotides in selected gene sequences seems to represent a promising enrichment of the methodological spectrum. The detection of DNA sequences of tissue related bacteria has been established and it provides satisfactory results mainly in combination with above mentioned methodological approaches. PMID:26419517

  5. Presentation of large DNA molecules for analysis as nanoconfined dumbbells

    PubMed Central

    Kounovsky-Shafer, Kristy L.; Hernández-Ortiz, Juan P.; Jo, Kyubong; Odijk, Theo; de Pablo, Juan J.; Schwartz, David C.

    2014-01-01

    The analysis of very large DNA molecules intrinsically supports long-range, phased sequence information, but requires new approaches for their effective presentation as part of any genome analysis platform. Using a multi-pronged approach that marshaled molecular confinement, ionic environment, and DNA elastic properties–but tressed by molecular simulations–we have developed an efficient and scalable approach for presentation of large DNA molecules within nanoscale slits. Our approach relies on the formation of DNA dumbbells, where large segments of the molecules remain outside the nanoslits used to confine them. The low ionic environment, synergizing other features of our approach, enables DNA molecules to adopt a fully stretched conformation, comparable to the contour length, thereby facilitating analysis by optical microscopy. Accordingly, a molecular model is proposed to describe the conformation and dynamics of the DNA molecules within the nanoslits; a Langevin description of the polymer dynamics is adopted in which hydrodynamic effects are included through a Green’s function formalism. Our simulations reveal that a delicate balance between electrostatic and hydrodynamic interactions is responsible for the observed molecular conformations. We demonstrate and further confirm that the “Odijk regime” does indeed start when the confinement dimensions size are of the same order of magnitude as the persistence length of the molecule. We also summarize current theories concerning dumbbell dynamics. PMID:24683272

  6. Cloning and sequence analysis of a cDNA encoding rat preprocholecystokinin.

    PubMed Central

    Deschenes, R J; Lorenz, L J; Haun, R S; Roos, B A; Collier, K J; Dixon, J E

    1984-01-01

    Poly(A) RNA was isolated from a rat medullary thyroid carcinoma that exhibited high levels of immunoreactive cholecystokinin (CCK). Double-stranded cDNA was synthesized from the poly(A) RNA and inserted into the Pst I site of pBR322. Bacterial colonies containing CCK cDNA were identified using the hybridization probe d(T-C-C-A-T-C-C-A-N-C-C-C-A-T-G-T-A-G-T-C). The sequence of the probe was deduced from the known amino acid sequence of porcine CCK-8, Asp-Tyr-Met-Gly-Trp-Met-Asp-Phe-NH2. The nucleotide sequence of the cDNA complementary to the mRNA of rat preprocholecystokinin was determined. The cDNA contains 33 nucleotides in the 5'-noncoding region, 199 nucleotides in the 3'-noncoding region, and 345 nucleotides coding for a precursor to CCK, which is 115 amino acids (Mr, 12,826). Examination of the rat CCK gene revealed a suggested transcriptional control sequence analogous to the "TATA" sequence located 33 nucleotides upstream from a proposed transcriptional start site. The amino acid sequence of CCK-39 is flanked by both amino-terminal and carboxyl-terminal extensions. Analysis of CCK mRNA showed that it is approximately equal to 750 nucleotides long. CCK mRNA of the rat brain and intestine appeared to be identical in size to the CCK mRNA of the carcinoma. Images PMID:6199787

  7. Analysis of Oligonucleotide DNA Binding and Sedimentation Properties of Montmorillonite Clay Using Ultraviolet Light Spectroscopy

    PubMed Central

    Beall, Gary W.; Sowersby, Drew S.; Roberts, Rachel D.; Robson, Michael H.; Lewis, L. Kevin

    2009-01-01

    Smectite clays such as montmorillonite form complexes with a variety of biomolecules, including the nucleic acids DNA and RNA. Most previous studies of DNA adsorption onto clay have relied upon spectrophotometric analysis after separation of free nucleic acids from bound complexes by centrifugation. In the current work we demonstrate that such studies produce a consistent error due to (a) incomplete sedimentation of montmorillonite and (b) strong absorbance of the remaining clay at 260 nm. Clay sedimentation efficiency was strongly dependent upon cation concentration (Na+ or Mg2+) and on the level of dispersion of the original suspension. An improved clay:DNA adsorption assay was developed and utilized to assess the impact of metal counterions on binding of single-stranded DNA to montmorillonite. X-ray diffraction demonstrated, for the first time, formation of intercalated structures consistent with orientation of the DNA strands parallel to the clay surface. Observed gallery spacings were found to closely match values calculated utilizing atomistic modeling techniques. PMID:19061334

  8. DNA damage and oxidative stress induced by acetylsalicylic acid in Daphnia magna.

    PubMed

    Gómez-Oliván, Leobardo Manuel; Galar-Martínez, Marcela; Islas-Flores, Hariz; García-Medina, Sandra; SanJuan-Reyes, Nely

    2014-08-01

    Acetylsalicylic acid is a nonsteroidal anti-inflammatory widely used due to its low cost and high effectiveness. This compound has been found in water bodies worldwide and is toxic to aquatic organisms; nevertheless its capacity to induce oxidative stress in bioindicators like Daphnia magna remains unknown. This study aimed to evaluate toxicity in D. magna induced by acetylsalicylic acid in water, using oxidative stress and DNA damage biomarkers. An acute toxicity test was conducted in order to determine the median lethal concentration (48-h LC50) and the concentrations to be used in the subsequent subacute toxicity test in which the following biomarkers were evaluated: lipid peroxidation, oxidized protein content, activity of the antioxidant enzymes superoxide dismutase, catalase, and glutathione peroxidase, and level of DNA damage. Lipid peroxidation level and oxidized protein content were significantly increased (p<0.05), and antioxidant enzymes significantly altered with respect to controls; while the DNA damage were significantly increased (p<0.05) too. In conclusion, acetylsalicylic acid induces oxidative stress and DNA damage in D. magna.

  9. Comparison of commercial systems for extraction of nucleic acids from DNA/RNA respiratory pathogens.

    PubMed

    Yang, Genyan; Erdman, Dean E; Kodani, Maja; Kools, John; Bowen, Michael D; Fields, Barry S

    2011-01-01

    This study compared six automated nucleic acid extraction systems and one manual kit for their ability to recover nucleic acids from human nasal wash specimens spiked with five respiratory pathogens, representing Gram-positive bacteria (Streptococcus pyogenes), Gram-negative bacteria (Legionella pneumophila), DNA viruses (adenovirus), segmented RNA viruses (human influenza virus A), and non-segmented RNA viruses (respiratory syncytial virus). The robots and kit evaluated represent major commercially available methods that are capable of simultaneous extraction of DNA and RNA from respiratory specimens, and included platforms based on magnetic-bead technology (KingFisher mL, Biorobot EZ1, easyMAG, KingFisher Flex, and MagNA Pure Compact) or glass fiber filter technology (Biorobot MDX and the manual kit Allprep). All methods yielded extracts free of cross-contamination and RT-PCR inhibition. All automated systems recovered L. pneumophila and adenovirus DNA equivalently. However, the MagNA Pure protocol demonstrated more than 4-fold higher DNA recovery from the S. pyogenes than other methods. The KingFisher mL and easyMAG protocols provided 1- to 3-log wider linearity and extracted 3- to 4-fold more RNA from the human influenza virus and respiratory syncytial virus. These findings suggest that systems differed in nucleic acid recovery, reproducibility, and linearity in a pathogen specific manner.

  10. Mitochondrial DNA Fragmentation to Monitor Processing Parameters in High Acid, Plant-Derived Foods.

    PubMed

    Caldwell, Jane M; Pérez-Díaz, Ilenys M; Harris, Keith; Hassan, Hosni M; Simunovic, Josip; Sandeep, K P

    2015-12-01

    Mitochondrial DNA (mtDNA) fragmentation was assessed in acidified foods. Using quantitative polymerase chain reaction, Ct values measured from fresh, fermented, pasteurized, and stored cucumber mtDNA were determined to be significantly different (P > 0.05) based on processing and shelf-life. This indicated that the combination of lower temperature thermal processes (hot-fill at 75 °C for 15 min) and acidified conditions (pH = 3.8) was sufficient to cause mtDNA fragmentation. In studies modeling high acid juices, pasteurization (96 °C, 0 to 24 min) of tomato serum produced Ct values which had high correlation to time-temperature treatment. Primers producing longer amplicons (approximately 1 kb) targeting the same mitochondrial gene gave greater sensitivity in correlating time-temperature treatments to Ct values. Lab-scale pasteurization studies using Ct values derived from the longer amplicon differentiated between heat treatments of tomato serum (95 °C for <2 min). MtDNA fragmentation was shown to be a potential new tool to characterize low temperature (<100 °C) high acid processes (pH < 4.6), nonthermal processes such as vegetable fermentation and holding times of acidified, plant-derived products. PMID:26556214

  11. Mitochondrial DNA Fragmentation to Monitor Processing Parameters in High Acid, Plant-Derived Foods.

    PubMed

    Caldwell, Jane M; Pérez-Díaz, Ilenys M; Harris, Keith; Hassan, Hosni M; Simunovic, Josip; Sandeep, K P

    2015-12-01

    Mitochondrial DNA (mtDNA) fragmentation was assessed in acidified foods. Using quantitative polymerase chain reaction, Ct values measured from fresh, fermented, pasteurized, and stored cucumber mtDNA were determined to be significantly different (P > 0.05) based on processing and shelf-life. This indicated that the combination of lower temperature thermal processes (hot-fill at 75 °C for 15 min) and acidified conditions (pH = 3.8) was sufficient to cause mtDNA fragmentation. In studies modeling high acid juices, pasteurization (96 °C, 0 to 24 min) of tomato serum produced Ct values which had high correlation to time-temperature treatment. Primers producing longer amplicons (approximately 1 kb) targeting the same mitochondrial gene gave greater sensitivity in correlating time-temperature treatments to Ct values. Lab-scale pasteurization studies using Ct values derived from the longer amplicon differentiated between heat treatments of tomato serum (95 °C for <2 min). MtDNA fragmentation was shown to be a potential new tool to characterize low temperature (<100 °C) high acid processes (pH < 4.6), nonthermal processes such as vegetable fermentation and holding times of acidified, plant-derived products.

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

    PubMed Central

    2011-01-01

    Background High throughput sequencing (HTS) technologies have revolutionized the field of genomics by drastically reducing the cost of sequencing, making it feasible for individual labs to sequence or resequence plant genomes. Obtaining high quality, high molecular weight DNA from plants poses significant challenges due to the high copy number of chloroplast and mitochondrial DNA, as well as high levels of phenolic compounds and polysaccharides. Multiple methods have been used to isolate DNA from plants; the CTAB method is commonly used to isolate total cellular DNA from plants that contain nuclear DNA, as well as chloroplast and mitochondrial DNA. Alternatively, DNA can be isolated from nuclei to minimize chloroplast and mitochondrial DNA contamination. Results We describe optimized protocols for isolation of nuclear DNA from eight different plant species encompassing both monocot and eudicot species. These protocols use nuclei isolation to minimize chloroplast and mitochondrial DNA contamination. We also developed a protocol to determine the number of chloroplast and mitochondrial DNA copies relative to the nuclear DNA using quantitative real time PCR (qPCR). We compared DNA isolated from nuclei to total cellular DNA isolated with the CTAB method. As expected, DNA isolated from nuclei consistently yielded nuclear DNA with fewer chloroplast and mitochondrial DNA copies, as compared to the total cellular DNA prepared with the CTAB method. This protocol will allow for analysis of the quality and quantity of nuclear DNA before starting a plant whole genome sequencing or resequencing experiment. Conclusions Extracting high quality, high molecular weight nuclear DNA in plants has the potential to be a bottleneck in the era of whole genome sequencing and resequencing. The methods that are described here provide a framework for researchers to extract and quantify nuclear DNA in multiple types of plants. PMID:21599914

  13. Vibrational analysis of α-cyanohydroxycinnamic acid

    NASA Astrophysics Data System (ADS)

    Mojica, Elmer-Rico E.; Vedad, Jayson; Desamero, Ruel Z. B.

    2015-08-01

    In the present study, a comparative Raman vibrational analysis of alpha-cyano-4-hydroxycinnamic acid (4CHCA) and its derivative, alpha-cyano-3-hydroxycinnamic acid (3CHCA), was performed. The Raman spectra of the 4CHCA and 3CHCA in solid form were obtained and analyzed to determine differences between the two structurally similar derivatives. For comparison, the CHCA derivatives cyanocinnamic acid (CCA) and coumaric acid (CA) were also studied. The plausible vibrational assignments were made and matched with those obtained theoretically using density functional theory (DFT) based method employing a 6-31 g basis set. The computational wavenumbers obtained were in good agreement with the observed experimental results. This was the first reported Raman study of CCA, 3CHCA and 4CHCA.

  14. Mining the bitter melon (momordica charantia l.) seed transcriptome by 454 analysis of non-normalized and normalized cDNA populations for conjugated fatty acid metabolism-related genes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Seeds of Momordica charantia (bitter melon) produce high levels of eleostearic acid, an unusual conjugated fatty acid with industrial value. Deep sequencing of non-normalized and normalized cDNAs from developing bitter melon seeds was conducted to uncover key genes required for biotechnological tran...

  15. Quantitative analysis of genomic DNA degradation in whole blood under various storage conditions for molecular diagnostic testing.

    PubMed

    Permenter, Jessalyn; Ishwar, Arjun; Rounsavall, Angie; Smith, Maddie; Faske, Jennifer; Sailey, Charles J; Alfaro, Maria P

    2015-12-01

    Proper storage of whole blood is crucial for isolating nucleic acids from leukocytes and to ensure adequate performance of downstream assays in the molecular diagnostic laboratory. Short-term and long-term storage recommendations are lacking for successful isolation of genomic DNA (gDNA). Container type (EDTA or heparin), temperature (4 °C and room temperature) and time (1-130 days) were assessed as criterion for sample acceptance policies. The percentage of integrated area (%Ti) between 150 and 10,000 bp from the 2200 TapeStation electropherogram was calculated to measure gDNA degradation. Refrigerated EDTA samples yielded gDNA with low %Ti (high quality). Heparinized samples stored at room temperature yielded gDNA of worst quality. Downstream analysis demonstrated that the quality of the gDNA correlated with the quality of the data; samples with high %Ti generated significantly lower levels of high molecular weight amplicons. Recommendations from these analyses include storing blood samples intended for nucleic acid isolation in EDTA tubes at 4 °C for long term storage (>10 days). gDNA should be extracted within 3 days when blood is stored at room temperature regardless of the container. Finally, refrigerated heparinized samples should not be stored longer than 9 days if expecting high quality gDNA isolates. Laboratories should consider many factors, in addition to the results obtained herein, to update their policies for sample acceptance for gDNA extraction intended for molecular genetic testing.

  16. One-stop genomic DNA extraction by salicylic acid-coated magnetic nanoparticles.

    PubMed

    Zhou, Zhongwu; Kadam, Ulhas S; Irudayaraj, Joseph

    2013-11-15

    Salicylic acid-coated magnetic nanoparticles were prepared via a modified one-step synthesis and used for a one-stop extraction of genomic DNA from mammalian cells. The synthesized magnetic particles were used for magnetic separation of cells from the media by nonspecific binding of the particles as well as extraction of genomic DNA from the lysate. The quantity and quality were confirmed by agarose gel electrophoresis and polymerase chain reaction. The entire process of extraction and isolation can be completed within 30 min. Compared with traditional methods based on centrifugation and filtration, the established method is fast, simple, reliable, and environmentally friendly.

  17. Sequence selective double strand DNA cleavage by peptide nucleic acid (PNA) targeting using nuclease S1.

    PubMed Central

    Demidov, V; Frank-Kamenetskii, M D; Egholm, M; Buchardt, O; Nielsen, P E

    1993-01-01

    A novel method for sequence specific double strand DNA cleavage using PNA (peptide nucleic acid) targeting is described. Nuclease S1 digestion of double stranded DNA gives rise to double strand cleavage at an occupied PNA strand displacement binding site, and under optimized conditions complete cleavage can be obtained. The efficiency of this cleavage is more than 10 fold enhanced when a tandem PNA site is targeted, and additionally enhanced if this site is in trans rather than in cis orientation. Thus in effect, the PNA targeting makes the single strand specific nuclease S1 behave like a pseudo restriction endonuclease. Images PMID:8502550

  18. Immobilization of DNA via oligonucleotides containing an aldehyde or carboxylic acid group at the 5' terminus.

    PubMed Central

    Kremsky, J N; Wooters, J L; Dougherty, J P; Meyers, R E; Collins, M; Brown, E L

    1987-01-01

    A general method for the immobilization of DNA through its 5'-end has been developed. A synthetic oligonucleotide, modified at its 5'-end with an aldehyde or carboxylic acid, was attached to latex microspheres containing hydrazide residues. Using T4 polynucleotide ligase and an oligonucleotide splint, a single stranded 98mer was efficiently joined to the immobilized synthetic fragment. After impregnation of the latex microspheres with the fluorescent dye, Nile Red and attachment of an aldehyde 16mer, 5 X 10(5) bead-DNA conjugates could be detected with a conventional fluorimeter. Images PMID:3562241

  19. Acetylsalicylic acid, aging and coronary artery disease are associated with ABCA1 DNA methylation in men

    PubMed Central

    2014-01-01

    Background Previous studies have suggested that DNA methylation contributes to coronary artery disease (CAD) risk variability. DNA hypermethylation at the ATP-binding cassette transporter A1 (ABCA1) gene, an important modulator of high-density lipoprotein cholesterol and reverse cholesterol transport, has been previously associated with plasma lipid levels, aging and CAD, but the association with CAD has yet to be replicated. Results ABCA1 DNA methylation levels were measured in leucocytes of 88 men using bis-pyrosequencing. We first showed that DNA methylation at the ABCA1 gene promoter locus is associated with aging and CAD occurrence in men (P < 0.05). The latter association is stronger among older men with CAD (≥61 years old; n = 19), who showed at least 4.7% higher ABCA1 DNA methylation levels as compared to younger men with CAD (<61 years old; n = 19) or men without CAD (n = 50; P < 0.001). Higher ABCA1 DNA methylation levels in older men were also associated with higher total cholesterol (r = 0.34, P = 0.03), low-density lipoprotein cholesterol (r = 0.32, P = 0.04) and triglyceride levels (r = 0.26, P = 0.09). Furthermore, we showed that acetylsalicylic acid therapy is associated with 3.6% lower ABCA1 DNA methylation levels (P = 0.006), independent of aging and CAD status of patients. Conclusions This study provides new evidence that the ABCA1 epigenetic profile is associated with CAD and aging, and highlights that epigenetic modifications might be a significant molecular mechanism involved in the pathophysiological processes associated with CAD. Acetylsalicylic acid treatment for CAD prevention might involve epigenetic mechanisms. PMID:25093045

  20. Flow-cytometry-based DNA hybidization and polymorphism analysis

    NASA Astrophysics Data System (ADS)

    Cai, Hong; Kommander, Kristina; White, P. S.; Nolan, John P.

    1998-05-01

    Functional analysis of the human genome, including the quantification of differential gene expression and the identification of polymorphic sites and disease genes, is an important element of the Human Genome Project. Current methods of analysis are mainly gel-based assays that are not well- suited to rapid genome-scale analyses. To analyze DNA sequence on a large scale, robust and high throughput assays are needed. We are developing a suite of microsphere-based approaches employing fluorescence detection to screen and analyze genomic sequence. Our approaches include competitive DNA hybridization to measure DNA or RNA targets in unknown samples, and oligo ligation or extension assays to analyze single-nucleotide polymorphisms. Apart from the advantages of sensitivity, simplicity, and low sample consumption, these flow cytometric approaches have the potential for high throughput multiplexed analysis using multicolored microspheres and automated sample handling.

  1. Signal amplification architecture for electrochemical aptasensor based on network-like thiocyanuric acid/gold nanoparticle/ssDNA.

    PubMed

    Chen, Zhengbo; Li, Lidong; Tian, Yu; Mu, Xiaojiao; Guo, Lin

    2012-01-01

    In this work, we described signal amplification architecture for electronic aptamer-based sensor (E-AB), which is applicable to a wide range of aptamers. Herein, we only take lysozyme as the representative sensing target. The amplification method was based on the network of thiocyanuric acid (TCA)/gold nanoparticles (AuNPs) modified with ssDNA. The binding event can be detected by a decrease in the integrated charge of the surface-bound [Ru(NH(3))(6)](3+) which electrostatically absorbed onto the negatively charged phosphate backbones of DNA. In the presence of target molecules, a large amount of TCA/AuNP/ssDNA network associated with [Ru(NH(3))(6)](3+) would be removed from the electrode surface, leading to a significant decrease of redox current. Cyclic voltammetry (CV) signals of [Ru(NH(3))(6)](3+) provides quantitative measures of the concentrations of lysozyme, with a linear calibration ranging from 5 pM to 1 nM and a detection limit is 0.1 pM. The detection limit of the proposed sensor is one order of magnitude and three orders of magnitude more sensitive than the detection limits in the absence of TCA (5 pM) and in the absence of TCA/AuNP/ssDNA network (0.5 nM). This amplification method is promising for broad potential application in clinic assay and various protein analysis.

  2. NDI and DAN DNA: Nucleic Acid-Directed Assembly of NDI and DAN

    PubMed Central

    2015-01-01

    Two novel DNA base surrogate phosphoramidites 1 and 2, based upon relatively electron-rich 1,5-dialkoxynaphthalene (DAN) and relatively electron-deficient 1,4,5,8-naphthalenetetracarboxylic diimide (NDI), respectively, were designed, synthesized, and incorporated into DNA oligonucleotide strands. The DAN and NDI artificial DNA bases were inserted within a three-base-pair region within the interior of a 12-mer oligonucleotide duplex in various sequential arrangements and investigated with CD spectroscopy and UV melting curve analysis. The CD spectra of the modified duplexes indicated B-form DNA topology. Melting curve analyses revealed trends in DNA duplex stability that correlate with the known association of DAN and NDI moieties in aqueous solution as well as the known favorable interactions between NDI and natural DNA base pairs. This demonstrates that DNA duplex stability and specificity can be driven by the electrostatic complementarity between DAN and NDI. In the most favorable case, an NDI–DAN–NDI arrangement in the middle of the DNA duplex was found to be approximately as stabilizing as three A–T base pairs. PMID:24502543

  3. Optimal Arrangement of Four Short DNA Strands for Delivery of Immunostimulatory Nucleic Acids to Immune Cells.

    PubMed

    Ohtsuki, Shozo; Matsuzaki, Noriyuki; Mohri, Kohta; Endo, Masayuki; Emura, Tomoko; Hidaka, Kumi; Sugiyama, Hiroshi; Takahashi, Yuki; Ishiyama, Kenichi; Kadowaki, Norimitsu; Takakura, Yoshinobu; Nishikawa, Makiya

    2015-10-01

    Nanosized DNA assemblies are useful for delivering immunostimulatory cytosine-phosphate-guanine (CpG) DNA to immune cells, but little is known about the optimal structure for such delivery. In this study, we designed three different DNA nanostructures using four 55-mer oligodeoxynucleotides (ODNs), that is, tetrapod-like structured DNA (tetrapodna), tetrahedral DNA (tetrahedron), and tetragonal DNA (tetragon), and compared their potencies. Electrophoresis showed that tetrapodna was obtained with high yield and purity, whereas tetrahedron formed multimers at high ODN concentrations. Atomic force microscopy revealed that all preparations were properly constructed under optimal conditions. The thermal stability of tetrapodna was higher than those of the others. Dynamic light scattering analysis showed that all of the assemblies were about 8 nm in diameter. Upon addition to mouse macrophage-like RAW264.7 cells, tetrahedron was most efficiently taken up by the cells. Then, a CpG DNA, a ligand for toll-like receptor 9, was linked to these DNA nanostructures and added to RAW264.7 cells. CpG tetrahedron induced the largest amount of tumor necrosis factor-α, followed by CpG tetrapodna. Similar results were obtained using human peripheral blood mononuclear cells. Taken together, these results indicate that tetrapodna is the best assembly with the highest yield and high immunostimulatory activity, and tetrahedron can be another useful assembly for cellular delivery if its preparation yield is improved.

  4. Symmetric curvature descriptors for label-free analysis of DNA

    NASA Astrophysics Data System (ADS)

    Buzio, Renato; Repetto, Luca; Giacopelli, Francesca; Ravazzolo, Roberto; Valbusa, Ugo

    2014-09-01

    High-resolution microscopy techniques such as electron microscopy, scanning tunnelling microscopy and atomic force microscopy represent well-established, powerful tools for the structural characterization of adsorbed DNA molecules at the nanoscale. Notably, the analysis of DNA contours allows mapping intrinsic curvature and flexibility along the molecular backbone. This is particularly suited to address the impact of the base-pairs sequence on the local conformation of the strands and plays a pivotal role for investigations relating the inherent DNA shape and flexibility to other functional properties. Here, we introduce novel chain descriptors aimed to characterize the local intrinsic curvature and flexibility of adsorbed DNA molecules with unknown orientation. They consist of stochastic functions that couple the curvatures of two nanosized segments, symmetrically placed on the DNA contour. We show that the fine mapping of the ensemble-averaged functions along the molecular backbone generates characteristic patterns of variation that highlight all pairs of tracts with large intrinsic curvature or enhanced flexibility. We demonstrate the practical applicability of the method for DNA chains imaged by atomic force microscopy. Our approach paves the way for the label-free comparative analysis of duplexes, aimed to detect nanoscale conformational changes of physical or biological relevance in large sample numbers.

  5. Direct DNA Analysis with Paper-Based Ion Concentration Polarization.

    PubMed

    Gong, Max M; Nosrati, Reza; San Gabriel, Maria C; Zini, Armand; Sinton, David

    2015-11-01

    DNA analysis is essential for diagnosis and monitoring of many diseases. Conventional DNA testing is generally limited to the laboratory. Increasing access to relevant technologies can improve patient care and outcomes in both developed and developing regions. Here, we demonstrate direct DNA analysis in paper-based devices, uniquely enabled by ion concentration polarization at the interface of patterned nanoporous membranes in paper (paper-based ICP). Hepatitis B virus DNA targets in human serum are simultaneously preconcentrated, separated, and detected in a single 10 min operation. A limit of detection of 150 copies/mL is achieved without prior viral load amplification, sufficient for early diagnosis of hepatitis B. We clinically assess the DNA integrity of sperm cells in raw human semen samples. The percent DNA fragmentation results from the paper-based ICP devices strongly correlate (R(2) = 0.98) with the sperm chromatin structure assay. In all cases, agreement was 100% with respect to the clinical decision. Paper-based ICP can provide inexpensive and accessible advanced molecular diagnostics.

  6. Direct DNA Analysis with Paper-Based Ion Concentration Polarization.

    PubMed

    Gong, Max M; Nosrati, Reza; San Gabriel, Maria C; Zini, Armand; Sinton, David

    2015-11-01

    DNA analysis is essential for diagnosis and monitoring of many diseases. Conventional DNA testing is generally limited to the laboratory. Increasing access to relevant technologies can improve patient care and outcomes in both developed and developing regions. Here, we demonstrate direct DNA analysis in paper-based devices, uniquely enabled by ion concentration polarization at the interface of patterned nanoporous membranes in paper (paper-based ICP). Hepatitis B virus DNA targets in human serum are simultaneously preconcentrated, separated, and detected in a single 10 min operation. A limit of detection of 150 copies/mL is achieved without prior viral load amplification, sufficient for early diagnosis of hepatitis B. We clinically assess the DNA integrity of sperm cells in raw human semen samples. The percent DNA fragmentation results from the paper-based ICP devices strongly correlate (R(2) = 0.98) with the sperm chromatin structure assay. In all cases, agreement was 100% with respect to the clinical decision. Paper-based ICP can provide inexpensive and accessible advanced molecular diagnostics. PMID:26447553

  7. ³²P-postlabeling analysis of DNA adducts.

    PubMed

    Phillips, David H; Arlt, Volker M

    2014-01-01

    (32)P-Postlabeling analysis is an ultra-sensitive method for the detection of DNA adducts, such as those formed directly by the covalent binding of carcinogens and mutagens to bases in DNA and other DNA lesions resulting from modification of bases by endogenous or exogenous agents (e.g., oxidative damage). The procedure involves four main steps: enzymatic digestion of the DNA sample; enrichment of the adducts; radiolabeling of the adducts by T4 kinase-catalyzed transference of (32)P-orthophosphate from [γ-(32)P]ATP; chromatographic separation of labeled adducts; and detection and quantification by means of their radioactive decay. Using 10 μg of DNA or less, it is capable of detecting adduct levels as low as 1 adduct in 10(9)-10(10) normal nucleotides. It is applicable to a wide range of investigations, including monitoring human exposure to environmental or occupational carcinogens, determining whether a chemical has genotoxic properties, analysis of the genotoxicity of complex mixtures, elucidation of the pathways of activation of carcinogens, and monitoring DNA repair. PMID:24623224

  8. Cheminformatics methods for novel nanopore analysis of HIV DNA termini

    PubMed Central

    Winters-Hilt, Stephen; Landry, Matthew; Akeson, Mark; Tanase, Maria; Amin, Iftekhar; Coombs, Amy; Morales, Eric; Millet, John; Baribault, Carl; Sendamangalam, Srikanth

    2006-01-01

    Background Channel current feature extraction methods, using Hidden Markov Models (HMMs) have been designed for tracking individual-molecule conformational changes. This information is derived from observation of changes in ionic channel current blockade "signal" upon that molecule's interaction with (and occlusion of) a single nanometer-scale channel in a "nanopore detector". In effect, a nanopore detector transduces single molecule events into channel current blockades. HMM analysis tools described are used to help systematically explore DNA dinucleotide flexibility, with particular focus on HIV's highly conserved (and highly flexible/reactive) viral DNA termini. One of the most critical stages in HIV's attack is the binding between viral DNA and the retroviral integrase, which is influenced by the dynamic-coupling induced high flexibility of a CA/TG dinucleotide positioned precisely two base-pairs from the blunt terminus of the duplex viral DNA. This suggests the study of a family of such CA/TG dinucleotide molecules via nanopore measurement and cheminformatics analysis. Results HMMs are used for level identification on the current blockades, HMM/EM with boosted variance emissions are used for level projection pre-processing, and time-domain FSAs are used to parse the level-projected waveform for kinetic information. The observed state kinetics of the DNA hairpins containing the CA/TG dinucleotide provides clear evidence for HIV's selection of a peculiarly flexible/interactive DNA terminus. PMID:17118144

  9. RNA:DNA Ratio and Other Nucleic Acid Derived Indices in Marine Ecology

    PubMed Central

    Chícharo, Maria Alexandra; Chícharo, Luis

    2008-01-01

    Some of most used indicators in marine ecology are nucleic acid-derived indices. They can be divided by target levels in three groups: 1) at the organism level as ecophysiologic indicators, indicators such as RNA:DNA ratios, DNA:dry weight and RNA:protein, 2) at the population level, indicators such as growth rate, starvation incidence or fisheries impact indicators, and 3) at the community level, indicators such as trophic interactions, exergy indices and prey identification. The nucleic acids derived indices, especially RNA:DNA ratio, have been applied with success as indicators of nutritional condition, well been and growth in marine organisms. They are also useful as indicators of natural or anthropogenic impacts in marine population and communities, such as upwelling or dredge fisheries, respectively. They can help in understanding important issues of marine ecology such as trophic interactions in marine environment, fish and invertebrate recruitment failure and biodiversity changes, without laborious work of counting, measuring and identification of small marine organisms. Besides the objective of integrate nucleic acid derived indices across levels of organization, the paper will also include a general characterization of most used nucleic acid derived indices in marine ecology and also advantages and limitations of them. We can conclude that using indicators, such RNA:DNA ratios and other nucleic acids derived indices concomitantly with organism and ecosystems measures of responses to climate change (distribution, abundance, activity, metabolic rate, survival) will allow for the development of more rigorous and realistic predictions of the effects of anthropogenic climate change on marine systems. PMID:19325815

  10. Binding-Induced DNA Nanomachines Triggered by Proteins and Nucleic Acids.

    PubMed

    Zhang, Hongquan; Lai, Maode; Zuehlke, Albert; Peng, Hanyong; Li, Xing-Fang; Le, X Chris

    2015-11-23

    We introduce the concept and operation of a binding-induced DNA nanomachine that can be activated by proteins and nucleic acids. This new type of nanomachine harnesses specific target binding to trigger assembly of separate DNA components that are otherwise unable to spontaneously assemble. Three-dimensional DNA tracks of high density are constructed on gold nanoparticles functionalized with hundreds of single-stranded oligonucleotides and tens of an affinity ligand. A DNA swing arm, free in solution, is linked to a second affinity ligand. Binding of a target molecule to the two ligands brings the swing arm to AuNP and initiates autonomous, stepwise movement of the swing arm around the AuNP surface. The movement of the swing arm, powered by enzymatic cleavage of conjugated oligonucleotides, cleaves hundreds of oligonucleotides in response to a single binding event. We demonstrate three nanomachines that are specifically activated by streptavidin, platelet-derived growth factor, and the Smallpox gene. Substituting the ligands enables the nanomachine to respond to other molecules. The new nanomachines have several unique and advantageous features over DNA nanomachines that rely on DNA self-assembly.

  11. Structural analysis of hyperperiodic DNA from Caenorhabditis elegans.

    PubMed

    Moreno-Herrero, Fernando; Seidel, Ralf; Johnson, Steven M; Fire, Andrew; Dekker, Nynke H

    2006-01-01

    Several bioinformatics studies have identified an unexpected but remarkably prevalent approximately 10 bp periodicity of AA/TT dinucleotides (hyperperiodicity) in certain regions of the Caenorhabditis elegans genome. Although the relevant C.elegans DNA segments share certain sequence characteristics with bent DNAs from other sources (e.g. trypanosome mitochondria), the nematode sequences exhibit a much more extensive and defined hyperperiodicity. Given the presence of hyperperiodic structures in a number of critical C.elegans genes, the physical characteristics of hyperperiodic DNA are of considerable interest. In this work, we demonstrate that several hyperperiodic DNA segments from C.elegans exhibit structural anomalies using high-resolution atomic force microscopy (AFM) and gel electrophoresis. Our quantitative analysis of AFM images reveals that hyperperiodic DNA adopts a significantly smaller mean square end-to-end distance, hence a more compact coil structure, compared with non-periodic DNA of similar length. While molecules remain capable of adopting both bent and straight (rod-like) configurations, indicating that their flexibility is still retained, examination of the local curvatures along the DNA contour length reveals that the decreased mean square end-to-end distance can be attributed to the presence of long-scale intrinsic bending in hyperperiodic DNA. Such bending is not detected in non-periodic DNA. Similar studies of shorter, nucleosome-length DNAs that survived micrococcal nuclease digestion show that sequence hyperperiodicity in short segments can likewise induce strong intrinsic bending. It appears, therefore, that regions of the C.elegans genome display a significant correlation between DNA sequence and unusual mechanical properties. PMID:16738142

  12. NOMAD: a versatile strategy for in vitro DNA manipulation applied to promoter analysis and vector design.

    PubMed Central

    Rebatchouk, D; Daraselia, N; Narita, J O

    1996-01-01

    Molecular analysis of complex modular structures, such as promoter regions or multi-domain proteins, often requires the creation of families of experimental DNA constructs having altered composition, order, or spacing of individual modules. Generally, creation of every individual construct of such a family uses a specific combination of restriction sites. However, convenient sites are not always available and the alternatives, such as chemical resynthesis of the experimental constructs or engineering of different restriction sites onto the ends of DNA fragments, are costly and time consuming. A general cloning strategy (nucleic acid ordered assembly with directionality, NOMAD; WWW resource locator http:@Lmb1.bios.uic.edu/NOMAD/NOMAD.htm l) is proposed that overcomes these limitations. Use of NOMAD ensures that the production of experimental constructs is no longer the rate-limiting step in applications that require combinatorial rearrangement of DNA fragments. NOMAD manipulates DNA fragments in the form of "modules" having a standardized cohesive end structure. Specially designed "assembly vectors" allow for sequential and directional insertion of any number of modules in an arbitrary predetermined order, using the ability of type IIS restriction enzymes to cut DNA outside of their recognition sequences. Studies of regulatory regions in DNA, such as promoters, replication origins, and RNA processing signals, construction of chimeric proteins, and creation of new cloning vehicles, are among the applications that will benefit from using NOMAD. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:8855278

  13. Persistence of DNA damage following exposure of human bladder cells to chronic monomethylarsonous acid

    SciTech Connect

    Wnek, S.M.; Medeiros, M.K.; Eblin, K.E.; Gandolfi, A.J.

    2009-12-01

    Malignant transformation was demonstrated in UROtsa cells following 52-weeks of exposure to 50 nM monomethylarsonous acid (MMA{sup III}); the result was the malignantly transformed cell line, URO-MSC. URO-MSC cells were used to study the induction of DNA damage and the alteration of DNA repair enzymes in both the presence of MMA{sup III} [URO-MSC(+)] and after subsequent removal of MMA{sup III} [URO-MSC(-)] following chronic, low-level exposure. In the presence of MMA{sup III}, URO-MSC(+) cells demonstrated a sustained increase in DNA damage following 12-weeks of exposure; in particular, a significant increase in DNA single-strand breaks at 12-weeks of exposure consistently elevated through 52 weeks. The persistence of DNA damage in URO-MSC cells was assessed after a 2-week removal of MMA{sup III}. URO-MSC(-) cells demonstrated a decrease in DNA damage compared to URO-MSC(+); however, DNA damage in URO-MSC(-) remained significantly elevated when compared to untreated UROtsa and increased in a time-dependent manner. Reactive oxygen species (ROS) were demonstrated to be a critical component in the generation of DNA damage determined through the incubation of ROS scavengers with URO-MSC cells. Poly (ADP-ribose) polymerase (PARP) is a key repair enzyme in DNA single-strand break repair. URO-MSC(+) resulted in a slight increase in PARP activity after 36-weeks of MMA{sup III} exposure, suggesting the presence of MMA{sup III} is inhibiting the increase in PARP activity. In support, PARP activity in URO-MSC(-) increased significantly, coinciding with a subsequent decrease in DNA damage demonstrated in URO-MSC(-) compared to URO-MSC(+). These data demonstrate that chronic, low-level exposure of UROtsa cells to 50 nM MMA{sup III} results in: the induction of DNA damage that remains elevated upon removal of MMA{sup III}; increased levels of ROS that play a role in MMA{sup III} induced-DNA damage; and decreased PARP activity in the presence of MMA{sup III}.

  14. 28 CFR 28.13 - Analysis and indexing of DNA samples.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 28 Judicial Administration 1 2013-07-01 2013-07-01 false Analysis and indexing of DNA samples. 28.13 Section 28.13 Judicial Administration DEPARTMENT OF JUSTICE DNA IDENTIFICATION SYSTEM DNA Sample Collection, Analysis, and Indexing § 28.13 Analysis and indexing of DNA samples. (a) The Federal Bureau...

  15. 28 CFR 28.13 - Analysis and indexing of DNA samples.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 28 Judicial Administration 1 2010-07-01 2010-07-01 false Analysis and indexing of DNA samples. 28.13 Section 28.13 Judicial Administration DEPARTMENT OF JUSTICE DNA IDENTIFICATION SYSTEM DNA Sample Collection, Analysis, and Indexing § 28.13 Analysis and indexing of DNA samples. (a) The Federal Bureau...

  16. 28 CFR 28.13 - Analysis and indexing of DNA samples.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 28 Judicial Administration 1 2011-07-01 2011-07-01 false Analysis and indexing of DNA samples. 28.13 Section 28.13 Judicial Administration DEPARTMENT OF JUSTICE DNA IDENTIFICATION SYSTEM DNA Sample Collection, Analysis, and Indexing § 28.13 Analysis and indexing of DNA samples. (a) The Federal Bureau...

  17. 28 CFR 28.13 - Analysis and indexing of DNA samples.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 28 Judicial Administration 1 2014-07-01 2014-07-01 false Analysis and indexing of DNA samples. 28.13 Section 28.13 Judicial Administration DEPARTMENT OF JUSTICE DNA IDENTIFICATION SYSTEM DNA Sample Collection, Analysis, and Indexing § 28.13 Analysis and indexing of DNA samples. (a) The Federal Bureau...

  18. 28 CFR 28.13 - Analysis and indexing of DNA samples.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 28 Judicial Administration 1 2012-07-01 2012-07-01 false Analysis and indexing of DNA samples. 28.13 Section 28.13 Judicial Administration DEPARTMENT OF JUSTICE DNA IDENTIFICATION SYSTEM DNA Sample Collection, Analysis, and Indexing § 28.13 Analysis and indexing of DNA samples. (a) The Federal Bureau...

  19. Effect of nucleic acid binding dyes on DNA extraction, amplification, and STR typing.

    PubMed

    Haines, Alicia M; Tobe, Shanan S; Kobus, Hilton J; Linacre, Adrian

    2015-10-01

    We report on the effects of six dyes used in the detection of DNA on the process of DNA extraction, amplification, and detection of STR loci. While dyes can be used to detect the presence of DNA, their use is restricted if they adversely affect subsequent DNA typing processes. Diamond™ Nucleic Acid Dye, GelGreen™, GelRed™, RedSafe™, SYBR(®) Green I, and EvaGreen™ were evaluated in this study. The percentage of dye removed during the extraction process was determined to be: 70.3% for SYBR(®) Green I; 99.6% for RedSafe™; 99.4% for EvaGreen™; 52.7% for Diamond™ Dye; 50.6% for GelRed™, and; could not be determined for GelGreen™. It was then assumed that the amount of dye in the fluorescent quantification assay had no effect on the DNA signal. The presence of all six dyes was then reviewed for their effect on DNA extraction. The t-test showed no significant difference between the dyes and the control. These extracts were then STR profiled and all dyes and control produced full DNA profiles. STR loci in the presence of GelGreen(TM) at 1X concentration showed increased amplification products in comparison to the control samples. Full STR profiles were detected in the presence of EvaGreen™ (1X), although with reduced amplification products. RedSafe™ (1X), Diamond™ Dye (1X), and SYBR(®) Green I (1X) all exhibited varying degrees of locus drop-out with GelRed™ generating no loci at all. We provide recommendations for the best dye to visualize the presence of DNA profile as a biological stain and its subsequent amplification and detection. PMID:26202628

  20. Sequencing and Analysis of Neanderthal Genomic DNA

    SciTech Connect

    Noonan, James P.; Coop, Graham; Kudaravalli, Sridhar; Smith,Doug; Krause, Johannes; Alessi, Joe; Chen, Feng; Platt, Darren; Paabo,Svante; Pritchard, Jonathan K.; Rubin, Edward M.

    2006-06-13

    Recovery and analysis of multiple Neanderthal autosomalsequences using a metagenomic approach reveals that modern humans andNeanderthals split ~;400,000 years ago, without significant evidence ofsubsequent admixture.

  1. Virtual DNA analysis as a platform for interlaboratory data exchange of HLA DNA typing results.

    PubMed

    Helmberg, W; Zahn, R; Keller, E; Weinmair, B; Lanzer, G; Albert, E

    1999-10-01

    In 1998, the German DNA Exchange offered the possibility to report typing data as virtual DNA. Participating labs have been equipped with software based on the principle of Virtual DNA Analysis (VDA). This approach allows the combination of sequence-specific oligonucleotide (SSO), sequence-specific primer (SSP) and sequence-based typing (SBT) results. The use of all types of test kits has been allowed without any limitations, as long as basic sequence information on SSOs or SSPs was available, at least the sequence and the position of the detected motif on the sample DNA. Typing raw data of the actual SSO-SSP and, if performed, SBT information was collected. Participating labs received 20 DNA samples to type. Fourteen labs returned data on 1,250 single-locus testings. Reported data consisted of 317 SBT data, 452 SSO kits and 1,795 SSP kits with 43,312 single SSO/ SSP reactivities. One hundred and twenty-six different typing kits (unique laboratory-specific kits, commercial kits from 7 companies) have been used. In 30 (2.4%) single-locus testings, at least one single SSO/SSP reactivity has been false-positive or -negative, thus not leading to a valid result on primary evaluation. Eight of these 30 cases were due to the presence of a new DRB1*14 allele in sample no. 2. Thirty-five tests (2.8%) showed wrong allele assignments. This first attempt to collect raw typing data instead of typing interpretation on a larger scale shows the advantages of Virtual DNA Analysis like interlaboratory data exchange without loss of information, transparency of typing interpretation and reinterpretation of typing data with an updated allele database. The VDA format is a useful tool for workshops and bone marrow donor registries. PMID:10551421

  2. Virtual DNA analysis as a platform for interlaboratory data exchange of HLA DNA typing results.

    PubMed

    Helmberg, W; Zahn, R; Keller, E; Weinmair, B; Lanzer, G; Albert, E

    1999-10-01

    In 1998, the German DNA Exchange offered the possibility to report typing data as virtual DNA. Participating labs have been equipped with software based on the principle of Virtual DNA Analysis (VDA). This approach allows the combination of sequence-specific oligonucleotide (SSO), sequence-specific primer (SSP) and sequence-based typing (SBT) results. The use of all types of test kits has been allowed without any limitations, as long as basic sequence information on SSOs or SSPs was available, at least the sequence and the position of the detected motif on the sample DNA. Typing raw data of the actual SSO-SSP and, if performed, SBT information was collected. Participating labs received 20 DNA samples to type. Fourteen labs returned data on 1,250 single-locus testings. Reported data consisted of 317 SBT data, 452 SSO kits and 1,795 SSP kits with 43,312 single SSO/ SSP reactivities. One hundred and twenty-six different typing kits (unique laboratory-specific kits, commercial kits from 7 companies) have been used. In 30 (2.4%) single-locus testings, at least one single SSO/SSP reactivity has been false-positive or -negative, thus not leading to a valid result on primary evaluation. Eight of these 30 cases were due to the presence of a new DRB1*14 allele in sample no. 2. Thirty-five tests (2.8%) showed wrong allele assignments. This first attempt to collect raw typing data instead of typing interpretation on a larger scale shows the advantages of Virtual DNA Analysis like interlaboratory data exchange without loss of information, transparency of typing interpretation and reinterpretation of typing data with an updated allele database. The VDA format is a useful tool for workshops and bone marrow donor registries.

  3. Amino acid and cDNA sequences of lysozyme from Hyalophora cecropia

    PubMed Central

    Engström, Å.; Xanthopoulos, K. G.; Boman, H. G.; Bennich, H.

    1985-01-01

    The amino acid and cDNA sequences of lysozyme from the giant silk moth Hyalophora cecropia have been determined. This enzyme is one of several immune proteins produced by the diapausing pupae after injection of bacteria. Cecropia lysozyme is composed of 120 amino acids, has a mol. wt. of 13.8 kd and shows great similarity with vertebrate lysozymes of the chicken type. The amino acid residues responsible for the catalytic activity and for the binding of substrate are essentially conserved. Three allelic variants of the Cecropia enzyme are identified. A comparison of the chicken and the Cecropia lysozymes shows that there is a 40% identity at both the amino acid and the nucleotide level. Some evolutionary aspects of the sequence data are discussed. PMID:16453632

  4. An alternate method for extracting DNA from environmentally challenged teeth for improved DNA analysis.

    PubMed

    Hughes-Stamm, Sheree; Warnke, Frauke; van Daal, Angela

    2016-01-01

    A grinding-free method to extract DNA from teeth via a direct minimal-invasive retrograde approach to the pulp cavity and dentine was compared to a standard grinding/pulverisation method. This alternate method uses endodontic dental files to access the root canals and pulp cavity for tissue and dentine harvest via the apical end of the roots and avoids mechanical damage to the crown and root morphology. In contrast, other methods require pulverisation of the whole root or tooth, transection or destruction of the occlusal surface to gain access to the DNA in the root canals and pulp chamber. This study compared two methods for preparing dentine powder from the roots of environmentally challenged teeth for forensic DNA analysis. We found that although the filing method was more laborious, and produced less dentine powder, the amount of amplifiable DNA per milligram of powder was substantially higher with the filing method compared to grinding the entire root. In addition, the number of short tandem repeat (STR) alleles detected and the peak height ratios of the STR profiles were notably higher. Although several other methods of extracting DNA-rich tissue from the pulp chamber of teeth have previously been reported, the method presented in this study is minimally invasive, thereby allowing the preservation of tooth and crown morphology. PMID:26832373

  5. DNA Sequence and Expression Variation of Hop (Humulus lupulus) Valerophenone Synthase (VPS), a Key Gene in Bitter Acid Biosynthesis

    PubMed Central

    Castro, Consuelo B.; Whittock, Lucy D.; Whittock, Simon P.; Leggett, Grey; Koutoulis, Anthony

    2008-01-01

    Background The hop plant (Humulus lupulus) is a source of many secondary metabolites, with bitter acids essential in the beer brewing industry and others having potential applications for human health. This study investigated variation in DNA sequence and gene expression of valerophenone synthase (VPS), a key gene in the bitter acid biosynthesis pathway of hop. Methods Sequence variation was studied in 12 varieties, and expression was analysed in four of the 12 varieties in a series across the development of the hop cone. Results Nine single nucleotide polymorphisms (SNPs) were detected in VPS, seven of which were synonymous. The two non-synonymous polymorphisms did not appear to be related to typical bitter acid profiles of the varieties studied. However, real-time quantitative reverse-transcription polymerase chain reaction (qRT-PCR) analysis of VPS expression during hop cone development showed a clear link with the bitter acid content. The highest levels of VPS expression were observed in two triploid varieties, ‘Symphony’ and ‘Ember’, which typically have high bitter acid levels. Conclusions In all hop varieties studied, VPS expression was lowest in the leaves and an increase in expression was consistently observed during the early stages of cone development. PMID:18519445

  6. Gibberellic-acid-induced cell elongation in pea epicotyls: Effect on polyploidy and DNA content.

    PubMed

    Boeken, G; Van Oostveldt, P

    1977-01-01

    In gibberellic-acid(GA3)-treated epicotyls of dwarf peas (Pisum sativum L.) grown in the light, DNA (per cell and per epicotyl) is followed. Histofluorometric DNA determinations show that GA3-promoted cell elongation is not accompanied by increased endomitosis, but chemical estimations show an increased DNA content per epicotyl. This difference must therefore be the result of increased mitotic activity in the GA3-treated tissue. Epicotyls of seedlings grown with or without cotyledons under continuous light with GA3 are tetraploid, as are those of ecotylized embryos grown in darkness. These epicotyls reach no more than half the length of octaploid epicotyls of seedlings grown in darkness. This result provides evidence for a relationship between polyploidy and final possible cell length. PMID:24419898

  7. Hyaluronic acid and fibrin hydrogels with concentrated DNA/PEI polyplexes for local gene delivery.

    PubMed

    Lei, Yuguo; Rahim, Maha; Ng, Quinn; Segura, Tatiana

    2011-08-10

    Local delivery of DNA through a hydrogel scaffold would increase the applicability of gene therapy in tissue regeneration and cancer therapy. However, the delivery of DNA/cationic polymer nanoparticles (polyplexes) using hydrogels is challenging due to the aggregation and inactivation of polyplexes during their incorporation into hydrogel scaffolds. We developed a novel process (termed caged nanoparticle encapsulation or CnE) to load concentrated and unaggregated non-viral gene delivery nanoparticles into various hydrogels. Previously, we showed that PEG hydrogels loaded with DNA/PEI polyplexes through this process were able to deliver genes both in vitro and in vivo. In this study, we found that hyaluronic acid and fibrin hydrogels with concentrated and unaggregated polyplexes loaded through CnE were able to deliver genes in vivo as well, demonstrating the universality of the process.

  8. Mapping of replication initiation sites in human ribosomal DNA by nascent-strand abundance analysis.

    PubMed Central

    Yoon, Y; Sanchez, J A; Brun, C; Huberman, J A

    1995-01-01

    New techniques for mapping mammalian DNA replication origins are needed. We have modified the existing nascent-strand size analysis technique (L. Vassilev and E.M. Johnson, Nucleic Acids Res. 17:7693-7705, 1989) to provide an independent means of studying replication initiation sites. We call the new method nascent-strand abundance analysis. We confirmed the validity of this method with replicating simian virus 40 DNA as a model. We then applied nascent-strand abundance and nascent-strand size analyses to mapping of initiation sites in human (HeLa) ribosomal DNA (rDNA), a region previously examined exclusively by two-dimensional gel electrophoresis methods (R.D. Little, T.H.K. Platt, and C.L. Schildkraut, Mol. Cell. Biol. 13:6600-6613, 1993). Our results partly confirm those obtained by two-dimensional gel electrophoresis techniques. Both studies suggest that replication initiates at relatively high frequency a few kilobase pairs upstream of the transcribed region and that many additional low-frequency initiation sites are distributed through most of the remainder of the ribosomal DNA repeat unit. PMID:7739533

  9. High-resolution melt analysis of DNA methylation to discriminate semen in biological stains.

    PubMed

    Antunes, Joana; Silva, Deborah S B S; Balamurugan, Kuppareddi; Duncan, George; Alho, Clarice S; McCord, Bruce

    2016-02-01

    The goal of this study was to develop a method for the detection of semen in biological stains using high-resolution melt (HRM) analysis and DNA methylation. To perform this task, we used an epigenetic locus that targets a tissue-specific differentially methylated region for semen. This specific locus, ZC3H12D, contains methylated CpG sites that are hypomethylated in semen and hypermethylated in blood and saliva. Using this procedure, DNA from forensic stains can be isolated, processed using bisulfite-modified polymerase chain reaction (PCR), and detected by real-time PCR with HRM capability. The method described in this article is robust; we were able to obtain results from samples with as little as 1 ng of genomic DNA. Samples inhibited by humic acid still produced reliable results. Furthermore, the procedure is specific and will not amplify non-bisulfite-modified DNA. Because this process can be performed using real-time PCR and is quantitative, it fits nicely within the workflow of current forensic DNA laboratories. As a result, it should prove to be a useful technique for processing trace evidence samples for serological analysis.

  10. Optoelectronic studies on heterocyclic bases of deoxyribonucleic acid for DNA photonics.

    PubMed

    El-Diasty, Fouad; Abdel-Wahab, Fathy

    2015-10-01

    The optoelectronics study of large molecules, particularly π-stacking molecules, such as DNA is really an extremely difficult task. We perform first electronic structure calculations on the heterocyclic bases of 2'-deoxyribonucleic acid based on Lorentz-Fresnel dispersion theory. In the UV-VIS range of spectrum, many of the optoelectronic parameters for DNA four bases namely adenine, guanine, cytosine and thymine are calculated and discussed. The results demonstrate that adenine has the highest hyperpolarizability, whereas thymine has the lowest hyperpolarizability. Cytosine has the lower average oscillator energy and the higher lattice energy. Thymine infers the most stable nucleic base with the lower phonon energy. Thymine also has the highest average oscillator energy and the lower lattice energy. Moreover, the four nucleic acid bases have large band gap energies less than 5 eV with a semiconducting behavior. Guanine shows the smallest band gap and the highest Fermi level energy, whereas adenine elucidates the highest band gap energy.

  11. DNA methylation landscape of fat deposits and fatty acid composition in obese and lean pigs

    PubMed Central

    Zhang, Shunhua; Shen, Linyuan; Xia, Yudong; Yang, Qiong; Li, Xuewei; Tang, Guoqing; Jiang, Yanzhi; Wang, Jinyong; Li, Mingzhou; Zhu, Li

    2016-01-01

    Obese and lean type pig breeds exhibit differences in their fat deposits and fatty acid composition. Here, we compared the effect of genome-wide DNA methylation on fatty acid metabolism between Landrace pigs (LP, leaner) and Rongchang pigs (RP, fatty). We found that LP backfat (LBF) had a higher polyunsaturated fatty acid content but a lower adipocyte volume than RP backfat (RBF). LBF exhibited higher global DNA methylation levels at the genome level than RBF. A total of 483 differentially methylated regions (DMRs) were located in promoter regions, mainly affecting olfactory and sensory activity and lipid metabolism. In LBF, the promoters of genes related to ATPase activity had significantly stronger methylation. This fact may suggest lower energy metabolism levels, which may result in less efficient lipid synthesis in LBF. Furthermore, we identified a DMR in the miR-4335 and miR-378 promoters and validated their methylation status by bisulfite sequencing PCR. The hypermethylation of the promoters of miR-4335 and miR-378 in LBF and the resulting silencing of the target genes may result in LBF’s low content in saturated fatty acids and fat deposition capacity. This study provides a solid basis for exploring the epigenetic mechanisms affecting fat deposition and fatty acid composition. PMID:27721392

  12. Multiplexed analysis of genes using nucleic acid-stabilized silver-nanocluster quantum dots.

    PubMed

    Enkin, Natalie; Wang, Fuan; Sharon, Etery; Albada, H Bauke; Willner, Itamar

    2014-11-25

    Luminescent nucleic acid-stabilized Ag nanoclusters (Ag NCs) are applied for the optical detection of DNA and for the multiplexed analysis of genes. Two different sensing modules including Ag NCs as luminescence labels are described. One sensing module involves the assembly of a three-component sensing module composed of a nucleic acid-stabilized Ag NC and a quencher-modified nucleic acid hybridized with a nucleic acid scaffold that is complementary to the target DNA. The luminescence of the Ag NCs is quenched in the sensing module nanostructure. The strand displacement of the scaffold by the target DNA separates the nucleic acid-functionalized Ag NCs, leading to the turned-on luminescence of the NCs and to the optical readout of the sensing process. By implementing two different-sized Ag NC-modified sensing modules, the parallel multiplexed analysis of two genes (the Werner Syndrome gene and the HIV, human immunodeficiency, gene), using 615 and 560 nm luminescent Ag NCs, is demonstrated. The second sensing module includes the nucleic acid functionalized Ag NCs and the quencher-modified nucleic acid hybridized with a hairpin DNA scaffold. The luminescence of the Ag NCs is quenched in the sensing module. Opening of the hairpin by the target DNA triggers the luminescence of the Ag NCs, due to the spatial separation of the Ag NCs/quencher units. The system is applied for the optical detection of the BRAC1 gene. In addition, by implementing two-sized Ag NCs, the multiplexed analysis of two genes by the hairpin sensing module approach is demonstrated.

  13. Polyanionic Carboxyethyl Peptide Nucleic Acids (ce-PNAs): Synthesis and DNA Binding

    PubMed Central

    Kirillova, Yuliya; Boyarskaya, Nataliya; Dezhenkov, Andrey; Tankevich, Mariya; Prokhorov, Ivan; Varizhuk, Anna; Eremin, Sergei; Esipov, Dmitry; Smirnov, Igor; Pozmogova, Galina

    2015-01-01

    New polyanionic modifications of polyamide nucleic acid mimics were obtained. Thymine decamers were synthesized from respective chiral α- and γ-monomers, and their enantiomeric purity was assessed. Here, we present the decamer synthesis, purification and characterization by MALDI-TOF mass spectrometry and an investigation of the hybridization properties of the decamers. We show that the modified γ-S-carboxyethyl-T10 PNA forms a stable triplex with polyadenine DNA. PMID:26469337

  14. Information transfer from DNA to peptide nucleic acids by template-directed syntheses

    NASA Technical Reports Server (NTRS)

    Schmidt, J. G.; Christensen, L.; Nielsen, P. E.; Orgel, L. E.; Bada, J. L. (Principal Investigator)

    1997-01-01

    Peptide nucleic acids (PNAs) are analogs of nucleic acids in which the ribose-phosphate backbone is replaced by a backbone held together by amide bonds. PNAs are interesting as models of alternative genetic systems because they form potentially informational base paired helical structures. Oligocytidylates have been shown to act as templates for formation of longer oligomers of G from PNA G2 dimers. In this paper we show that information can be transferred from DNA to PNA. DNA C4T2C4 is an efficient template for synthesis of PNA G4A2G4 using G2 and A2 units as substrates. The corresponding synthesis of PNA G4C2G4 on DNA C4G2C4 is less efficient. Incorporation of PNA T2 into PNA products on DNA C4A2C4 is the least efficient of the three reactions. These results, obtained using PNA dimers as substrates, parallel those obtained using monomeric activated nucleotides.

  15. Preamplification Procedure for the Analysis of Ancient DNA Samples

    PubMed Central

    Del Gaudio, Stefania; Cirillo, Alessandra; Di Bernardo, Giovanni; Galderisi, Umberto; Thanassoulas, Theodoros; Pitsios, Theodoros; Cipollaro, Marilena

    2013-01-01

    In ancient DNA studies the low amount of endogenous DNA represents a limiting factor that often hampers the result achievement. In this study we extracted the DNA from nine human skeletal remains of different ages found in the Byzantine cemetery of Abdera Halkidiki and in the medieval cemetery of St. Spiridion in Rhodes (Greece). Real-time quantitative polymerase chain reaction (qPCR) was used to detect in the extracts the presence of PCR inhibitors and to estimate the DNA content. As mitochondrial DNA was detected in all samples, amplification of nuclear targets, as amelogenin and the polymorphism M470V of the transmembrane conductance regulator gene, yielded positive results in one case only. In an effort to improve amplification success, we applied, for the first time in ancient DNA, a preamplification strategy based on TaqMan PreAmp Master Mix. A comparison between results obtained from nonpreamplified and preamplified samples is reported. Our data, even if preliminary, show that the TaqMan PreAmp procedure may improve the sensitivity of qPCR analysis. PMID:24187523

  16. Diagnosis of Lung Cancer by Fractal Analysis of Damaged DNA

    PubMed Central

    Namazi, Hamidreza; Kiminezhadmalaie, Mona

    2015-01-01

    Cancer starts when cells in a part of the body start to grow out of control. In fact cells become cancer cells because of DNA damage. A DNA walk of a genome represents how the frequency of each nucleotide of a pairing nucleotide couple changes locally. In this research in order to study the cancer genes, DNA walk plots of genomes of patients with lung cancer were generated using a program written in MATLAB language. The data so obtained was checked for fractal property by computing the fractal dimension using a program written in MATLAB. Also, the correlation of damaged DNA was studied using the Hurst exponent measure. We have found that the damaged DNA sequences are exhibiting higher degree of fractality and less correlation compared with normal DNA sequences. So we confirmed this method can be used for early detection of lung cancer. The method introduced in this research not only is useful for diagnosis of lung cancer but also can be applied for detection and growth analysis of different types of cancers. PMID:26539245

  17. Nonenzymatic synthesis of RNA and DNA oligomers on hexitol nucleic acid templates: the importance of the A structure

    NASA Technical Reports Server (NTRS)

    Kozlov, I. A.; Politis, P. K.; Van Aerschot, A.; Busson, R.; Herdewijn, P.; Orgel, L. E.; Bada, J. L. (Principal Investigator); Dolan, M. (Principal Investigator)

    1999-01-01

    Hexitol nucleic acid (HNA) is an analogue of DNA containing the standard nucleoside bases, but with a phosphorylated 1,5-anhydrohexitol backbone. HNA oligomers form duplexes having the nucleic acid A structure with complementary DNA or RNA oligomers. The HNA decacytidylate oligomer is an efficient template for the oligomerization of the 5'-phosphoroimidazolides of guanosine or deoxyguanosine. Comparison of the oligomerization efficiencies on HNA, RNA, and DNA decacytidylate templates under various conditions suggests strongly that only nucleic acid double helices with the A structure support efficient template-directed synthesis when 5'-phosphoroimidazolides of nucleosides are used as substrates.

  18. DNA-osmium complexes: recent developments in the operative chemical analysis of DNA epigenetic modifications.

    PubMed

    Okamoto, Akimitsu

    2014-09-01

    The development of a reaction for the detection of one epigenetic modification in a long DNA strand is a chemically and biologically challenging research subject. Herein, we report and discuss the formation of 5-methylcytosine-osmium complexes that are used as the basis for a bisulfite-free chemical assay for DNA methylation analysis. Osmium in the oxidized state reacts with C5-methylated pyrimidines in the presence of a bipyridine ligand to give a stable ternary complex. On the basis of this reaction, an adenine derivative with a tethered bipyridine moiety has been designed for sequence-specific osmium complex formation. Osmium complexation is then achieved by hybridization of a short DNA molecule containing this functional nucleotide to a target DNA sequence and results in the formation of a cross-linked structure. This novel concept of methylation-specific reaction, based on a straightforward chemical process, expands the range of methods available for the analysis of epigenetic modifications. Advantages of the described method include amplification-insensitive detection, 5-hydroxymethylcytosine complexation, and visualization through methylation-specific in situ hybridization.

  19. Effects of altered maternal folic acid, vitamin B12 and docosahexaenoic acid on placental global DNA methylation patterns in Wistar rats.

    PubMed

    Kulkarni, Asmita; Dangat, Kamini; Kale, Anvita; Sable, Pratiksha; Chavan-Gautam, Preeti; Joshi, Sadhana

    2011-01-01

    Potential adverse effects of excess maternal folic acid supplementation on a vegetarian population deficient in vitamin B(12) are poorly understood. We have previously shown in a rat model that maternal folic acid supplementation at marginal protein levels reduces brain omega-3 fatty acid levels in the adult offspring. We have also reported that reduced docosahexaenoic acid (DHA) levels may result in diversion of methyl groups towards DNA in the one carbon metabolic pathway ultimately resulting in DNA methylation. This study was designed to examine the effect of normal and excess folic acid in the absence and presence of vitamin B(12) deficiency on global methylation patterns in the placenta. Further, the effect of maternal omega 3 fatty acid supplementation on the above vitamin B(12) deficient diets was also examined. Our results suggest maternal folic acid supplementation in the absence of vitamin B(12) lowers plasma and placental DHA levels (p<0.05) and reduces global DNA methylation levels (p<0.05). When this group was supplemented with omega 3 fatty acids there was an increase in placental DHA levels and subsequently DNA methylation levels revert back to the levels of the control group. Our results suggest for the first time that DHA plays an important role in one carbon metabolism thereby influencing global DNA methylation in the placenta. PMID:21423696

  20. Differential DNA Methylation Analysis without a Reference Genome.

    PubMed

    Klughammer, Johanna; Datlinger, Paul; Printz, Dieter; Sheffield, Nathan C; Farlik, Matthias; Hadler, Johanna; Fritsch, Gerhard; Bock, Christoph

    2015-12-22

    Genome-wide DNA methylation mapping uncovers epigenetic changes associated with animal development, environmental adaptation, and species evolution. To address the lack of high-throughput methods for DNA methylation analysis in non-model organisms, we developed an integrated approach for studying DNA methylation differences independent of a reference genome. Experimentally, our method relies on an optimized 96-well protocol for reduced representation bisulfite sequencing (RRBS), which we have validated in nine species (human, mouse, rat, cow, dog, chicken, carp, sea bass, and zebrafish). Bioinformatically, we developed the RefFreeDMA software to deduce ad hoc genomes directly from RRBS reads and to pinpoint differentially methylated regions between samples or groups of individuals (http://RefFreeDMA.computational-epigenetics.org). The identified regions are interpreted using motif enrichment analysis and/or cross-mapping to annotated genomes. We validated our method by reference-free analysis of cell-type-specific DNA methylation in the blood of human, cow, and carp. In summary, we present a cost-effective method for epigenome analysis in ecology and evolution, which enables epigenome-wide association studies in natural populations and species without a reference genome.

  1. Differential DNA Methylation Analysis without a Reference Genome.

    PubMed

    Klughammer, Johanna; Datlinger, Paul; Printz, Dieter; Sheffield, Nathan C; Farlik, Matthias; Hadler, Johanna; Fritsch, Gerhard; Bock, Christoph

    2015-12-22

    Genome-wide DNA methylation mapping uncovers epigenetic changes associated with animal development, environmental adaptation, and species evolution. To address the lack of high-throughput methods for DNA methylation analysis in non-model organisms, we developed an integrated approach for studying DNA methylation differences independent of a reference genome. Experimentally, our method relies on an optimized 96-well protocol for reduced representation bisulfite sequencing (RRBS), which we have validated in nine species (human, mouse, rat, cow, dog, chicken, carp, sea bass, and zebrafish). Bioinformatically, we developed the RefFreeDMA software to deduce ad hoc genomes directly from RRBS reads and to pinpoint differentially methylated regions between samples or groups of individuals (http://RefFreeDMA.computational-epigenetics.org). The identified regions are interpreted using motif enrichment analysis and/or cross-mapping to annotated genomes. We validated our method by reference-free analysis of cell-type-specific DNA methylation in the blood of human, cow, and carp. In summary, we present a cost-effective method for epigenome analysis in ecology and evolution, which enables epigenome-wide association studies in natural populations and species without a reference genome. PMID:26673328

  2. Differential DNA Methylation Analysis without a Reference Genome

    PubMed Central

    Klughammer, Johanna; Datlinger, Paul; Printz, Dieter; Sheffield, Nathan C.; Farlik, Matthias; Hadler, Johanna; Fritsch, Gerhard; Bock, Christoph

    2015-01-01

    Summary Genome-wide DNA methylation mapping uncovers epigenetic changes associated with animal development, environmental adaptation, and species evolution. To address the lack of high-throughput methods for DNA methylation analysis in non-model organisms, we developed an integrated approach for studying DNA methylation differences independent of a reference genome. Experimentally, our method relies on an optimized 96-well protocol for reduced representation bisulfite sequencing (RRBS), which we have validated in nine species (human, mouse, rat, cow, dog, chicken, carp, sea bass, and zebrafish). Bioinformatically, we developed the RefFreeDMA software to deduce ad hoc genomes directly from RRBS reads and to pinpoint differentially methylated regions between samples or groups of individuals (http://RefFreeDMA.computational-epigenetics.org). The identified regions are interpreted using motif enrichment analysis and/or cross-mapping to annotated genomes. We validated our method by reference-free analysis of cell-type-specific DNA methylation in the blood of human, cow, and carp. In summary, we present a cost-effective method for epigenome analysis in ecology and evolution, which enables epigenome-wide association studies in natural populations and species without a reference genome. PMID:26673328

  3. Educational Software for the Analysis of DNA and Protein Sequences.

    ERIC Educational Resources Information Center

    Maloy, Stanley; Olson, Sue

    1989-01-01

    Describes the development of the microcomputer-based educational software, DNAzoom, which was designed to introduce undergraduates in molecular biology to computer analysis of DNA protein sequences. Highlights include graphical presentation of data, the functional use of color, a menu-oriented interface, and students' evaluations of the software.…

  4. Restriction fragment length polymorphism DNA analysis by the FBI Laboratory protocol using a simple, convenient hardware system.

    PubMed

    Lewis, M E; Kouri, R E; Latorra, D; Berka, K M; Lee, H C; Gaensslen, R E

    1990-09-01

    Restriction fragment length polymorphism analysis of human deoxyribonucleic acid (DNA) using two probes, pYNH24 and CMM101, was performed on the BIOS Timeframe system following the Federal Bureau of Investigation (FBI) Laboratory protocol and some variations of it. Comparable results were obtained by the different methods used.

  5. Proteomic analysis of DNA-protein cross-linking by antitumor nitrogen mustards.

    PubMed

    Loeber, Rachel L; Michaelson-Richie, Erin D; Codreanu, Simona G; Liebler, Daniel C; Campbell, Colin R; Tretyakova, Natalia Y

    2009-06-01

    Nitrogen mustards are antitumor agents used clinically for the treatment of a variety of neoplastic conditions. The biological activity of these compounds is typically attributed to their ability to induce DNA-DNA cross-links. However, nitrogen mustards are able to produce a variety of other lesions, including DNA-protein cross-links (DPCs). DPCs induced by nitrogen mustards are not well-characterized because of their structural complexity and the insufficient specificity and sensitivity of previously available experimental methodologies. In the present work, affinity capture methodology in combination with mass spectrometry-based proteomics was employed to identify mammalian proteins that form covalent cross-links to DNA in the presence of a simple nitrogen mustard, mechlorethamine. Following incubation of 5'-biotinylated DNA duplexes with nuclear protein extracts, DPCs were isolated by affinity capture on streptavidin beads, and the cross-linked proteins were identified by high-performance liquid chromatography-electrospray tandem mass spectrometry of tryptic peptides. Mechlorethamine treatment resulted in the formation of DPCs with nuclear proteins involved in chromatin regulation, DNA replication and repair, cell cycle control, transcriptional regulation, and cell architecture. Western blot analysis was employed to confirm protein identification and to quantify the extent of drug-mediated cross-linking. Mass spectrometry of amino acid-nucleobase conjugates found in total proteolytic digests revealed that mechlorethamine-induced DPCs are formed via alkylation of the N7 position of guanine in duplex DNA and cysteine thiols within the proteins to give N-[2-[S-cysteinyl]ethyl]-N-[2-(guan-7-yl)ethyl]methylamine lesions. The results described herein suggest that cellular exposure to nitrogen mustards leads to cross-linking of a large spectrum of nuclear proteins to chromosomal DNA, potentially contributing to the cytotoxic and mutagenic effects of these drugs.

  6. Filtration Isolation of Nucleic Acids: A Simple and Rapid DNA Extraction Method.

    PubMed

    McFall, Sally M; Neto, Mário F; Reed, Jennifer L; Wagner, Robin L

    2016-01-01

    FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad to extract cellular DNA from whole blood in less than 2 min. The blood specimen is treated with detergent, mixed briefly and applied by pipet to the separation membrane. The lysate wicks into the blotting pad due to capillary action, capturing the genomic DNA on the surface of the separation membrane. The extracted DNA is retained on the membrane during a simple wash step wherein PCR inhibitors are wicked into the absorbent blotting pad. The membrane containing the entrapped DNA is then added to the PCR reaction without further purification. This simple method does not require laboratory equipment and can be easily implemented with inexpensive laboratory supplies. Here we describe a protocol for highly sensitive detection and quantitation of HIV-1 proviral DNA from 100 µl whole blood as a model for early infant diagnosis of HIV that could readily be adapted to other genetic targets. PMID:27583575

  7. DNA delivery from hyaluronic acid-collagen hydrogels via a substrate-mediated approach

    PubMed Central

    Segura, Tatiana; Chung, Peter H.; Shea, Lonnie D.

    2008-01-01

    Efficient and controlled gene delivery from biodegradable materials can be employed to stimulate cellular processes that lead to tissue regeneration. In this report, a substrate-mediated approach was developed to deliver DNA from hyaluronic acid-collagen hydrogels. The hydrogels were formed by crosslinking HA with poly(ethylene glycol) diglycidyl ether. Poly(ethylene imine)(PEI)/DNA complexes were immobilized to the substrate using either biotin/neutravidin or non-specific adsorption. Complexes were formed in the presence or absence of salt to regulate complex size, and resulted in complexes with z-average diameters of 1221.7±152.3 and 139.4±1.3 nm, respectively. During 48-h incubation in PBS or hyaluronidase, DNA was released slowly from the hydrogel substrate (<30% of immobilized DNA), which was enhanced by incubation with conditioned media (≈50% of immobilized DNA). Transgene expression mediated by immobilized, large diameter complexes was 3 to 7-fold greater than for small diameter complexes. However, the percentage of cells expressing the transgene was greater for small diameter complexes (48.7%) than for large diameter complexes (22.3%). Spatially controlled gene transfer was achieved by topographically patterning the hydrogel to pattern cell adhesion. Biomaterial-based gene delivery can be applicable to numerous tissue engineering applications, or as a tool to examine tissue formation. PMID:15522759

  8. Plant flavone apigenin binds to nucleic acid bases and reduces oxidative DNA damage in prostate epithelial cells.

    PubMed

    Sharma, Haripaul; Kanwal, Rajnee; Bhaskaran, Natarajan; Gupta, Sanjay

    2014-01-01

    Oxidative stress has been linked to prostate carcinogenesis as human prostate tissue is vulnerable to oxidative DNA damage. Apigenin, a dietary plant flavone, possesses anti-proliferative and anticancer effects; however, its antioxidant properties have not been fully elucidated. We investigated sub-cellular distribution of apigenin, it's binding to DNA and protective effects against H2O2-induced DNA damage using transformed human prostate epithelial RWPE-1 cells and prostate cancer LNCaP, PC-3 and DU145 cells. Exposure of cells to apigenin exhibited higher accumulation in RWPE-1 and LNCaP cells, compared to PC-3 and DU145 cells. The kinetics of apigenin uptake in LNCaP cells was estimated with a Km value of 5 µmole/L and Vmax of 190 pmoles/million cells/h. Sub-cellular fractionation demonstrated that nuclear matrix retains the highest concentration of apigenin (45.3%), followed by cytosol (23.9%), nuclear membranes (17.9%) and microsomes (12.9%), respectively. Spectroscopic analysis of apigenin with calf-thymus DNA exhibited intercalation as the dominant binding mode to DNA duplex. Apigenin exposure resulted in significant genoprotective effects in H2O2-stressed RWPE-1 cells by reduction in reactive oxygen species levels. In addition, apigenin exposure suppressed the formation of 8-hydroxy-2' deoxyguanosine and protected exposed cells from apoptosis. Our studies demonstrate that apigenin is readily taken up by normal prostatic epithelial cells and prostate cancer cells, and is incorporated into their nuclei, where its intercalation with nucleic acid bases may account for its antioxidant and chemopreventive activities. PMID:24614817

  9. Deep image analysis of entangled ring-shaped DNA

    NASA Astrophysics Data System (ADS)

    Kim, Hyeongju; Jee, Ah-Young; Granick, Steve

    Ring-shaped DNA entangled in aqueous actin networks and observed by super-resolution microscopy (STED; stimulated emission depletion) offers rich data for comparison with unresolved questions of polymer physics. Using home-written software, we calculated not only the center of mass (CoM) and CoM trajectories of hundreds of molecules, but also analyzed conformation dynamics with statistical analysis including wavelet transformation and a correlation matrix approach. The analysis reveals some surprising aspects unanticipated by classical theories.

  10. Molecular-beacon-based tricomponent probe for SNP analysis in folded nucleic acids.

    PubMed

    Nguyen, Camha; Grimes, Jeffrey; Gerasimova, Yulia V; Kolpashchikov, Dmitry M

    2011-11-11

    Hybridization probes are often inefficient in the analysis of single-stranded DNA or RNA that are folded in stable secondary structures. A molecular beacon (MB) probe is a short DNA hairpin with a fluorophore and a quencher attached to opposite sides of the oligonucleotide. The probe is widely used in real-time analysis of specific DNA and RNA sequences. This study demonstrates how a conventional MB probe can be used for the analysis of nucleic acids that form very stable (T(m) > 80 °C) hairpin structures. Here we demonstrate that the MB probe is not efficient in direct analysis of secondary structure-folded analytes, whereas a MB-based tricomponent probe is suitable for these purposes. The tricomponent probe takes advantage of two oligonucleotide adaptor strands f and m. Each adaptor strand contains a fragment complementary to the analyte and a fragment complementary to a MB probe. In the presence of a specific analyte, the two adaptor strands hybridize to the analyte and the MB probe, thus forming a quadripartite complex. DNA strand f binds to the analyte with high affinity and unwinds its secondary structure. Strand m forms a stable complex only with the fully complementary analyte. The MB probe fluorescently reports the formation of the quadripartite associate. It was demonstrated that the DNA analytes folded in hairpin structures with stems containing 5, 6, 7, 8, 9, 11, or 13 base pairs can be detected in real time with the limit of detection (LOD) lying in the nanomolar range. The stability of the stem region in the DNA analyte did not affect the LOD. Analytes containing single base substitutions in the stem or in the loop positions were discriminated from the fully complementary DNA at room temperature. The tricomponent probe promises to simplify nucleic acid analysis at ambient temperatures in such applications as in vivo RNA monitoring, detection of pathogens, and single nucleotide polymorphism (SNP) genotyping by DNA microarrays.

  11. Molecular analysis of Neanderthal DNA from the northern Caucasus.

    PubMed

    Ovchinnikov, I V; Götherström, A; Romanova, G P; Kharitonov, V M; Lidén, K; Goodwin, W

    2000-03-30

    The expansion of premodern humans into western and eastern Europe approximately 40,000 years before the present led to the eventual replacement of the Neanderthals by modern humans approximately 28,000 years ago. Here we report the second mitochondrial DNA (mtDNA) analysis of a Neanderthal, and the first such analysis on clearly dated Neanderthal remains. The specimen is from one of the eastern-most Neanderthal populations, recovered from Mezmaiskaya Cave in the northern Caucasus. Radiocarbon dating estimated the specimen to be approximately 29,000 years old and therefore from one of the latest living Neanderthals. The sequence shows 3.48% divergence from the Feldhofer Neanderthal. Phylogenetic analysis places the two Neanderthals from the Caucasus and western Germany together in a clade that is distinct from modern humans, suggesting that their mtDNA types have not contributed to the modern human mtDNA pool. Comparison with modern populations provides no evidence for the multiregional hypothesis of modern human evolution.

  12. Air-sampled Filter Analysis for Endotoxins and DNA Content.

    PubMed

    Lang-Yona, Naama; Mazar, Yinon; Pardo, Michal; Rudich, Yinon

    2016-01-01

    Outdoor aerosol research commonly uses particulate matter sampled on filters. This procedure enables various characterizations of the collected particles to be performed in parallel. The purpose of the method presented here is to obtain a highly accurate and reliable analysis of the endotoxin and DNA content of bio-aerosols extracted from filters. The extraction of high molecular weight organic molecules, such as lipopolysaccharides, from sampled filters involves shaking the sample in a pyrogen-free water-based medium. The subsequent analysis is based on an enzymatic reaction that can be detected using a turbidimetric measurement. As a result of the high organic content on the sampled filters, the extraction of DNA from the samples is performed using a commercial DNA extraction kit that was originally designed for soils and modified to improve the DNA yield. The detection and quantification of specific microbial species using quantitative polymerase chain reaction (q-PCR) analysis are described and compared with other available methods. PMID:27023725

  13. Selection and Characterization of Single Stranded DNA Aptamers for the Hormone Abscisic Acid

    PubMed Central

    Gonzalez, Victor M.; Millo, Enrico; Sturla, Laura; Vigliarolo, Tiziana; Bagnasco, Luca; Guida, Lucrezia; D'Arrigo, Cristina; De Flora, Antonio; Salis, Annalisa; Martin, Elena M.; Bellotti, Marta; Zocchi, Elena

    2013-01-01

    The hormone abscisic acid (ABA) is a small molecule involved in pivotal physiological functions in higher plants. Recently, ABA has been also identified as an endogenous hormone in mammals, regulating different cell functions including inflammatory processes, stem cell expansion, insulin release, and glucose uptake. Aptamers are short, single-stranded (ss) oligonucleotidesable to recognize target molecules with high affinity. The small size of the ABA molecule represented a challenge for aptamer development and the aim of this study was to develop specific anti-ABA DNA aptamers. Biotinylated abscisic acid (bio-ABA) was immobilized on streptavidin-coated magnetic beads. DNA aptamers against bio-ABA were selected with 7 iterative rounds of the systematic evolution of ligands by exponential enrichment method (SELEX), each round comprising incubation of the ABA-binding beads with the ssDNA sequences, DNA elution, electrophoresis, and polymerase chain reaction (PCR) amplification. The PCR product was cloned and sequenced. The binding affinity of several clones was determined using bio-ABA immobilized on streptavidin-coated plates. Aptamer 2 and aptamer 9 showed the highest binding affinity, with dissociation constants values of 0.98±0.14 μM and 0.80±0.07 μM, respectively. Aptamers 2 and 9 were also able to bind free, unmodified ABA and to discriminate between different ABA enantiomers and isomers. Our findings indicate that ssDNA aptamers can selectively bind ABA and could be used for the development of ABA quantitation assays. PMID:23971905

  14. Synthesis, spectroscopic characterization and in vitro antimicrobial, anticancer and antileishmanial activities as well interaction with Salmon sperm DNA of newly synthesized carboxylic acid derivative, 4-(4-methoxy-2-nitrophenylamino)-4-oxobutanoic acid.

    PubMed

    Sirajuddin, Muhammad; Ali, Saqib; McKee, Vickie; Ullah, Hameed

    2015-03-01

    This paper stresses on the synthesis, characterization of novel carboxylic acid derivative and its application in pharmaceutics. Carboxylic acid derivatives have a growing importance in medicine, particularly in oncology. A novel carboxylic acid, 4-(4-methoxy-2-nitrophenylamino)-4-oxobutanoic acid, was synthesized and characterized by elemental analysis, FT-IR, NMR ((1)H, and (13)C), mass spectrometry and single crystal X-ray structural analysis. The structure of the title compound, C11H12N2O6, shows the molecules dimerised by short intramolecular OH⋯O hydrogen bonds. The compound was screened for in vitro antimicrobial, anticancer, and antileishmanial activities as well as interaction with SS-DNA. The compound was also checked for in vitro anticancer activity against BHK-21, H-157 and HCEC cell lines, and showed significant anticancer activity. The compound was almost non-toxic towards human corneal epithelial cells (HCEC) and did not show more than 7.4% antiproliferative activity when used at the 2.0μg/mL end concentration. It was also tested for antileishmanial activity against the promastigote form of leishmania major and obtained attractive result. DNA interaction study exposes that the binding mode of the compound with SS-DNA is an intercalative as it results in hypochromism along with minor red shift. A new and efficient strategy to identify pharmacophores sites in carboxylic acid derivative for antibacterial/antifungal activity using Petra, Osiris and Molinspiration (POM) analyses was also carried out.

  15. Synthesis, spectroscopic characterization and in vitro antimicrobial, anticancer and antileishmanial activities as well interaction with Salmon sperm DNA of newly synthesized carboxylic acid derivative, 4-(4-methoxy-2-nitrophenylamino)-4-oxobutanoic acid

    NASA Astrophysics Data System (ADS)

    Sirajuddin, Muhammad; Ali, Saqib; McKee, Vickie; Ullah, Hameed

    2015-03-01

    This paper stresses on the synthesis, characterization of novel carboxylic acid derivative and its application in pharmaceutics. Carboxylic acid derivatives have a growing importance in medicine, particularly in oncology. A novel carboxylic acid, 4-(4-methoxy-2-nitrophenylamino)-4-oxobutanoic acid, was synthesized and characterized by elemental analysis, FT-IR, NMR (1H, and 13C), mass spectrometry and single crystal X-ray structural analysis. The structure of the title compound, C11H12N2O6, shows the molecules dimerised by short intramolecular Osbnd H⋯O hydrogen bonds. The compound was screened for in vitro antimicrobial, anticancer, and antileishmanial activities as well as interaction with SS-DNA. The compound was also checked for in vitro anticancer activity against BHK-21, H-157 and HCEC cell lines, and showed significant anticancer activity. The compound was almost non-toxic towards human corneal epithelial cells (HCEC) and did not show more than 7.4% antiproliferative activity when used at the 2.0 μg/mL end concentration. It was also tested for antileishmanial activity against the promastigote form of leishmania major and obtained attractive result. DNA interaction study exposes that the binding mode of the compound with SS-DNA is an intercalative as it results in hypochromism along with minor red shift. A new and efficient strategy to identify pharmacophores sites in carboxylic acid derivative for antibacterial/antifungal activity using Petra, Osiris and Molinspiration (POM) analyses was also carried out.

  16. Cytogenetic analysis from DNA by comparative genomic hybridization.

    PubMed

    Tachdjian, G; Aboura, A; Lapierre, J M; Viguié, F

    2000-01-01

    Comparative genomic hybridization (CGH) is a modified in situ hybridization technique which allows detection and mapping of DNA sequence copy differences between two genomes in a single experiment. In CGH analysis, two differentially labelled genomic DNA (study and reference) are co-hybridized to normal metaphase spreads. Chromosomal locations of copy number changes in the DNA segments of the study genome are revealed by a variable fluorescence intensity ratio along each target chromosome. Since its development, CGH has been applied mostly as a research tool in the field of cancer cytogenetics to identify genetic changes in many previously unknown regions. CGH may also have a role in clinical cytogenetics for detection and identification of unbalanced chromosomal abnormalities.

  17. Boswellic acid induces epigenetic alterations by modulating DNA methylation in colorectal cancer cells

    PubMed Central

    Shen, Yan; Takahashi, Masanobu; Byun, Hyang-Min; Link, Alexander; Sharma, Nupur; Balaguer, Francesc; Leung, Hon-Chiu; Boland, C. Richard; Goel, Ajay

    2012-01-01

    Accumulating evidence suggests that chemopreventive effects of some dietary polyphenols may in part be mediated by their ability to influence epigenetic mechanisms in cancer cells. Boswellic acids, derived from the plant Boswellia serrata, have long been used for the treatment of various inflammatory diseases due to their potent anti-inflammatory activities. Recent preclinical studies have also suggested that this compound has anti-cancer potential against various malignancies. However, the precise molecular mechanisms underlying their anti-cancer effects remain elusive. Herein, we report that boswellic acids modulate DNA methylation status of several tumor suppressor genes in colorectal cancer (CRC) cells. We treated RKO, SW48 and SW480 CRC cell lines with the active principle present in boswellic acids, acetyl-keto-β-boswellic acid (AKBA). Using genome-wide DNA methylation and gene expression microarray analyses, we discovered that AKBA induced a modest genome-wide demethylation that permitted simultaneous re-activation of the corresponding tumor suppressor genes. The quantitative methylation-specific PCR and RT-PCR validated the gene demethylation and re-expression in several putative tumor suppressor genes including SAMD14 and SMPD3. Furthermore, AKBA inhibited DNMT activity in CRC cells. Taken together, these results lend further support to the growing notion that anti-cancer effect of boswellic acids may in part be due to its ability to demethylate and reactivate methylation-silenced tumor suppressor genes. These results suggest that not only boswellic acid might be a promising epigenetic modulator in the chemoprevention and treatment of CRC, but also provide a rationale for future investigations on the usefulness of such botanicals for epigenetic therapy in other human malignancies. PMID:22415137

  18. Categorical spectral analysis of periodicity in nucleosomal DNA

    PubMed Central

    Jin, Hu; Rube, H. Tomas; Song, Jun S.

    2016-01-01

    DNA helical twist imposes geometric constraints on the location of histone–DNA interaction sites along nucleosomal DNA. Certain 10.5-bp periodic nucleotides in phase with these geometric constraints have been suggested to facilitate nucleosome positioning. However, the extent of nucleotide periodicity in nucleosomal DNA and its significance in directing nucleosome positioning still remain unclear. We clarify these issues by applying categorical spectral analysis to high-resolution nucleosome maps in two yeast species. We find that only a small fraction of nucleosomal sequences contain significant 10.5-bp periodicity. We further develop a spectral decomposition method to show that the previously observed periodicity in aligned nucleosomal sequences mainly results from proper phasing among nucleosomal sequences, and not from a preponderant occurrence of periodicity within individual sequences. Importantly, we show that this phasing may arise from the histones’ proclivity for putting preferred nucleotides at some of the evenly spaced histone–DNA contact points with respect to the dyad axis. We demonstrate that 10.5-bp periodicity, when present, significantly facilitates rotational, but not translational, nucleosome positioning. Finally, although periodicity only moderately affects nucleosome occupancy genome wide, reduced periodicity is an evolutionarily conserved signature of nucleosome-depleted regions around transcription start/termination sites. PMID:26893354

  19. Image Analysis of DNA Fiber and Nucleus in Plants.

    PubMed

    Ohmido, Nobuko; Wako, Toshiyuki; Kato, Seiji; Fukui, Kiichi

    2016-01-01

    Advances in cytology have led to the application of a wide range of visualization methods in plant genome studies. Image analysis methods are indispensable tools where morphology, density, and color play important roles in the biological systems. Visualization and image analysis methods are useful techniques in the analyses of the detailed structure and function of extended DNA fibers (EDFs) and interphase nuclei. The EDF is the highest in the spatial resolving power to reveal genome structure and it can be used for physical mapping, especially for closely located genes and tandemly repeated sequences. One the other hand, analyzing nuclear DNA and proteins would reveal nuclear structure and functions. In this chapter, we describe the image analysis protocol for quantitatively analyzing different types of plant genome, EDFs and interphase nuclei. PMID:27557694

  20. Involvement of aberrant DNA methylation on reduced expression of lysophosphatidic acid receptor-1 gene in rat tumor cell lines

    SciTech Connect

    Tsujiuchi, Toshifumi . E-mail: ttujiuch@life.kindai.ac.jp; Shimizu, Kyoko; Onishi, Mariko; Sugata, Eriko; Fujii, Hiromasa; Mori, Toshio; Honoki, Kanya; Fukushima, Nobuyuki

    2006-10-27

    Lysophosphatidic acid (LPA) is a bioactive phospholipid that stimulates cell proliferation, migration, and protects cells from apoptosis. It interacts with specific G protein-coupled transmembrane receptors. Recently, it has been reported that alterations of LPA receptor expression might be important in the malignant transformation of tumor cells. Therefore, to assess an involvement of DNA methylation in reduced expression of the LPA receptor-1 (lpa1) gene, we investigated the expression of the lpa1 gene and its DNA methylation patterns in rat tumor cell lines. Both rat brain-derived neuroblastoma B103 and liver-derived hepatoma RH7777 cells used in this study indicated no expression of lpa1. For the analysis of methylation status, bisulfite sequencing was performed with B103 and RH7777 cells, comparing with other lpa1 expressed cells and normal tissues of brain and liver. The lpa1 expressed cells and tissues were all unmethylated in this region of lpa1. In contrast, both B103 and RH7777 cells were highly methylated, correlating with reduced expression of the lpa1. Treatment with 5-aza 2'-deoxycytidine induced expression of lpa1 gene in B103 and RH7777 cells after 24 h. In RH7777 cells treated with 5-aza 2'-deoxycytidine, stress fiber formation was also observed in response to LPA in RH7777 cells, but not in untreated RH7777 cells. These results suggest that aberrant DNA methylation of the lpa1 gene may be involved in its reduced expression in rat tumor cells.

  1. Genomic localization, sequence analysis, and transcription of the putative human cytomegalovirus DNA polymerase gene

    SciTech Connect

    Heilbronn, T.; Jahn, G.; Buerkle, A.; Freese, U.K.; Fleckenstein, B.; Zur Hausen, H.

    1987-01-01

    The human cytomegalovirus (HCMV)-induced DNA polymerase has been well characterized biochemically and functionally, but its genomic location has not yet been assigned. To identify the coding sequence, cross-hybridization with the herpes simplex virus type 1 (HSV-1) polymerase gene was used, as suggested by the close similarity of the herpes group virus-induced DNA polymerases to the HCMV DNA polymerase. A cosmid and plasmid library of the entire HCMV genome was screened with the BamHI Q fragment of HSF-1 at different stringency conditions. One PstI-HincII restriction fragment of 850 base pairs mapping within the EcoRI M fragment of HCMV cross-hybridized at T/sub m/ - 25/degrees/C. Sequence analysis revealed one open reading frame spanning the entire sequence. The amino acid sequence showed a highly conserved domain of 133 amino acids shared with the HSV and putative Esptein-Barr virus polymerase sequences. This domain maps within the C-terminal part of the HSV polymerase gene, which has been suggested to contain part of the catalytic center of the enzyme. Transcription analysis revealed one 5.4-kilobase early transcript in the sense orientation with respect to the open reading frame identified. This transcript appears to code for the 140-kilodalton HCMV polymerase protein.

  2. Molecular docking study investigating the possible mode of binding of C.I. Acid Red 73 with DNA.

    PubMed

    Guo, Yumei; Yue, Qinyan; Gao, Baoyu

    2011-07-01

    C.I. Acid Red 73 is a reactive azo dye with a variable potential carcinogenicity. The mechanism mediating interactions that occur between the dye and DNA have not been completely understood thus far. In this study, molecular docking techniques were applied to describe the most probable mode of DNA binding as well as the sequence selectivity of the C.I. Acid Red 73 dye. These docking experiments revealed that the dye is capable of interacting with the minor groove of the DNA on the basis of its curved shape, which fits well with the topology of double-stranded DNA. In addition, the dye can bind selectively to the minor groove of the DNA by applying CGT sequence selectivity. Further, the minor groove can be recognized although DNA targets present intercalation gaps. However, intercalative binding can also occur when the DNA target possesses an appropriate intercalation gap. Compared with the other eight DNA sequences that were studied, the DNA dodecamer d(CGCGATATCGCG)(2) (PDB ID: 1DNE) presents a very favorable target for the binding of C.I. Acid Red 73 to the minor groove, with the lowest binding free energy -9.19 kcal/mol. Results reported from this study are expected to provide useful information for research involving further simulations of molecular dynamics and toxicology investigations of the dye.

  3. The cytotoxicity, DNA crosslinking ability and DNA sequence selectivity of the aniline mustards melphalan, chlorambucil and 4-[bis(2-chloroethyl)amino] benzoic acid.

    PubMed

    Sunters, A; Springer, C J; Bagshawe, K D; Souhami, R L; Hartley, J A

    1992-07-01

    Three aniline derivatives melphalan (L-PAM), chlorambucil (CHL) and 4-[bis(2-chloroethyl)amino] benzoic acid (BAM) have been compared on the basis of their in vitro cytotoxicities, DNA interstrand crosslinking ability and DNA sequence selectivity. Cytotoxicity was assessed in the human colonic adenocarcinoma LS174T and leukaemic K562 cell lines using the sulpho-rhodamine B and tetrazolium dye reduction assays. The order of cytotoxicities was L-PAM greater than CHL greater than BAM in both cell lines with K562 being less sensitive than LS174T. This was different from the order CHL greater than L-PAM greater than BAM which would be predicted from simple chemical reactivity or rate of hydrolysis, parameters which have been used previously as indicators of biological potency for aromatic nitrogen mustards. DNA interstrand crosslinking in cells as determined by alkaline elution showed a correlation with IC50 values. The ranking order of activity was further predicted by the ability of the agents to produce interstrand crosslinks in isolated DNA. The extent of guanine N-7 alkylation, assessed using a modified DNA sequencing technique, mirrored cytotoxicity and crosslinking ability, but at equivalent levels of alkylation there was no significant difference in DNA sequence selectivity. These data demonstrates that simple chemical reactivity or hydrolysis rate is not a good indicator of DNA reactivity or cytotoxicity for a number of aniline mustards, whereas DNA interstrand crosslinking ability either measured directly in cells or in isolated DNA, gives a good indication of biological activity.

  4. Analysis of Peptides and Conjugates by Amino Acid Analysis.

    PubMed

    Højrup, Peter

    2015-01-01

    Amino acid analysis is a highly accurate method for characterization of the composition of synthetic peptides. Together with mass spectrometry, it gives a reliable control of peptide quality and quantity before conjugation and immunization. Peptides are hydrolyzed, preferably in gas phase, with 6 M HCl at 110 °C for 20-24 h and the resulting amino acids analyzed by ion-exchange chromatography with post-column ninhydrin derivatization. Depending on the hydrolysis conditions, tryptophan is destroyed, and cysteine also, unless derivatized, and the amides, glutamine and asparagine, are deamidated to glutamic acid and aspartic acid, respectively. Three different ways of calculating results are suggested, and taking the above limitations into account, a quantitation better than 5% can usually be obtained. PMID:26424264

  5. Detection of orthopoxvirus DNA by real-time PCR and identification of variola virus DNA by melting analysis.

    PubMed

    Nitsche, Andreas; Ellerbrok, Heinz; Pauli, Georg

    2004-03-01

    Although variola virus was eradicated by the World Health Organization vaccination program in the 1970s, the diagnosis of smallpox infection has attracted great interest in the context of a possible deliberate release of variola virus in bioterrorist attacks. Obviously, fast and reliable diagnostic tools are required to detect variola virus and to distinguish it from orthopoxviruses that have identical morphological characteristics, including vaccinia virus. The advent of real-time PCR for the clinical diagnosis of viral infections has facilitated the detection of minute amounts of viral nucleic acids in a fast, safe, and precise manner, including the option to quantify and to genotype the target reliably. In this study a complete set of four hybridization probe-based real-time PCR assays for the specific detection of orthopoxvirus DNA is presented. Melting analysis following PCR enables the identification of variola virus by the PCR product's characteristic melting temperature, permitting the discrimination of variola virus from other orthopoxviruses. In addition, an assay for the specific amplification of variola virus DNA is presented. All assays can be performed simultaneously in the same cycler, and results of a PCR run are obtained in less than 1 h. The application of more than one assay for the same organism significantly contributes to the diagnostic reliability, reducing the risk of false-negative results due to unknown sequence variations. In conclusion, the assays presented will improve the speed and reliability of orthopoxvirus diagnostics and variola virus identification.

  6. Genome-wide analysis of DNA methylation in hepatoblastoma tissues

    PubMed Central

    Cui, Ximao; Liu, Baihui; Zheng, Shan; Dong, Kuiran; Dong, Rui

    2016-01-01

    DNA methylation has a crucial role in cancer biology. In the present study, a genome-wide analysis of DNA methylation in hepatoblastoma (HB) tissues was performed to verify differential methylation levels between HB and normal tissues. As alpha-fetoprotein (AFP) has a critical role in HB, AFP methylation levels were also detected using pyrosequencing. Normal and HB liver tissue samples (frozen tissue) were obtained from patients with HB. Genome-wide analysis of DNA methylation in these tissues was performed using an Infinium HumanMethylation450 BeadChip, and the results were confirmed with reverse transcription-quantitative polymerase chain reaction. The Infinium HumanMethylation450 BeadChip demonstrated distinctively less methylation in HB tissues than in non-tumor tissues. In addition, methylation enrichment was observed in positions near the transcription start site of AFP, which exhibited lower methylation levels in HB tissues than in non-tumor liver tissues. Lastly, a significant negative correlation was observed between AFP messenger RNA expression and DNA methylation percentage, using linear Pearson's R correlation coefficients. The present results demonstrate differential methylation levels between HB and normal tissues, and imply that aberrant methylation of AFP in HB could reflect HB development. Expansion of these findings could provide useful insight into HB biology. PMID:27446465

  7. The xeroderma pigmentosum pathway: decision tree analysis of DNA quality.

    PubMed

    Naegeli, Hanspeter; Sugasawa, Kaoru

    2011-07-15

    The nucleotide excision repair (NER) system is a fundamental cellular stress response that uses only a handful of DNA binding factors, mutated in the cancer-prone syndrome xeroderma pigmentosum (XP), to detect an astounding diversity of bulky base lesions, including those induced by ultraviolet light, electrophilic chemicals, oxygen radicals and further genetic insults. Several of these XP proteins are characterized by a mediocre preference for damaged substrates over the native double helix but, intriguingly, none of them recognizes injured bases with sufficient selectivity to account for the very high precision of bulky lesion excision. Instead, substrate versatility as well as damage specificity and strand selectivity are achieved by a multistage quality control strategy whereby different subunits of the XP pathway, in succession, interrogate the DNA double helix for a distinct abnormality in its structural or dynamic parameters. Through this step-by-step filtering procedure, the XP proteins operate like a systematic decision making tool, generally known as decision tree analysis, to sort out rare damaged bases embedded in a vast excess of native DNA. The present review is focused on the mechanisms by which multiple XP subunits of the NER pathway contribute to the proposed decision tree analysis of DNA quality in eukaryotic cells. PMID:21684221

  8. Unsaturated fatty acids bind Myc-Max transcription factor and inhibit Myc-Max-DNA complex formation.

    PubMed

    Chung, Sunah; Park, Seyeon; Yang, Chul Hak

    2002-12-15

    Oncoprotein Myc, hetero-dimerized with Max through a b/HLH/Zip region, is a transcription factor that governs important cellular processes such as cell cycle entry, proliferation and differentiation. We found that linoleic acid, isolated from Pollen Typhae, and other unsaturated fatty acids have strong inhibitory effects on the binding of Myc-Max heterodimer to an E-box DNA site (CA(C/T)GTG). The interaction of a fatty acid with a protein dimer, not with DNA, is assumed to block the entire Myc-Max-DNA complex formation. Unsaturated fatty acids also showed cytotoxicity against a SNU16 human stomach cancer cell line and conjugated linoleic acid suppressed mRNA expression of several myc-target genes; ornithine decarboxylase, p53, cdc25a in the SNU16 cells.

  9. Spatiotemporal analysis of DNA repair using charged particle radiation.

    PubMed

    Tobias, F; Durante, M; Taucher-Scholz, G; Jakob, B

    2010-01-01

    Approaches to visualise the dynamics of the DNA lesion processing substantially contributes to the understanding of the hierarchical organisation of the DNA damage response pathways. Charged particle irradiation has recently emerged as a tool to generate discrete sites of subnuclear damage by its means of extremely localised dose deposition at low energies, thus facilitating the spatiotemporal analysis of repair events. In addition, they are of high interest for risk estimations of human space exploration (e.g. mars mission) in the high energy regime (HZE). In this short review we will give examples for the application of charged particle irradiation to study spatiotemporal aspects of DNA damage recognition and repair in the context of recent achievements in this field. Beamline microscopy allows determining the exact kinetics of repair-related proteins after irradiation with different charged particles that induce different lesion densities. The classification into fast recruited proteins like DNA-PK or XRCC1 or slower recruited ones like 53BP1 or MDC1 helps to establish the hierarchical organisation of damage recognition and subsequent repair events. Additionally, motional analysis of DNA lesions induced by traversing particles proved information about the mobility of DSBs. Increased mobility or the absence of large scale motion has direct consequences on the formation of chromosomal translocations and, thus, on mechanisms of cancer formation. Charged particle microbeams offer the interesting perspective of precise nuclear or subnuclear targeting with a defined number of ions, avoiding the Poisson distribution of traversals inherent to broad beam experiments. With the help of the microbeam, geometrical patterns of traversing ions can be applied facilitating the analysis of spatial organisation of repair. PMID:19944777

  10. Adansonian Analysis and Deoxyribonucleic Acid Base Composition of Serratia marcescens

    PubMed Central

    Colwell, R. R.; Mandel, M.

    1965-01-01

    Colwell, R. R. (Georgetown University, Washington, D.C.), and M. Mandel. Adansonian analysis and deoxyribonucleic acid base composition of Serratia marcescens. J. Bacteriol. 89:454–461. 1965.—A total of 33 strains of Serratia marcescens were subjected to Adansonian analysis for which more than 200 coded features for each of the organisms were included. In addition, the base composition [expressed as moles per cent guanine + cytosine (G + C)] of the deoxyribonucleic acid (DNA) prepared from each of the strains was determined. Except for four strains which were intermediate between Serratia and the Hafnia and Aerobacter group C of Edwards and Ewing, the S. marcescens species group proved to be extremely homogeneous, and the different strains showed high affinities for each other (mean similarity, ¯S = 77%). The G + C ratio of the DNA from the Serratia strains ranged from 56.2 to 58.4% G + C. Many species names have been listed for the genus, but only a single clustering of the strains was obtained at the species level, for which the species name S. marcescens was retained. S. kiliensis, S. indica, S. plymuthica, and S. marinorubra could not be distinguished from S. marcescens; it was concluded, therefore, that there is only a single species in the genus. The variety designation kiliensis does not appear to be valid, since no subspecies clustering of strains with negative Voges-Proskauer reactions could be detected. The characteristics of the species are listed, and a description of S. marcescens is presented. PMID:14255714

  11. DNA/polyethyleneimine/hyaluronic acid small complex particles and tumor suppression in mice.

    PubMed

    Ito, Tomoko; Yoshihara, Chieko; Hamada, Katsuyuki; Koyama, Yoshiyuki

    2010-04-01

    The highest barriers for non-viral vectors to an efficient in vivo gene transfection would be (1) non-specific interaction with biological molecules, and (2) large size of the DNA complex particles. Protective coating of the DNA/polyethyleneimine (PEI) complexes by hyaluronic acid (HA) effectively diminished the adverse interactions with biological molecules. Here we found HA also protected the DNA/PEI complexes against aggregation and inactivation through lyophilization-and-rehydration procedures. It allows us to prepare the concentrated very small DNA complex particles (<70 nm) suspension by preparing the complexes at highly diluted conditions, followed by lyophilized-and-rehydrated to a small volume. In vivo gene expression efficiency of the small complex was examined with mice subcutaneously inoculated with B16 melanoma cells. These formulations showed high reporter-gene expression level in tumor after intravenous injection into tumor-bearing mice. Small complex was then made of the plasmid encoding GM-CSF gene, and injected into the mice bearing subcutaneous solid B16 tumor. After intravenous injection, it induced apparent tumor growth suppression in 50% of the mice. Notably, significant therapeutic effect was detected in the mice that received intratumoral injection, and 75% of the mice were completely cured with disappearance of tumor. PMID:20047759

  12. Hybrid polymeric hydrogels via peptide nucleic acid (PNA)/DNA complexation.

    PubMed

    Chu, Te-Wei; Feng, Jiayue; Yang, Jiyuan; Kopeček, Jindřich

    2015-12-28

    This work presents a new concept in hybrid hydrogel design. Synthetic water-soluble N-(2-hydroxypropyl)methacrylamide (HPMA) polymers grafted with multiple peptide nucleic acids (PNAs) are crosslinked upon addition of the linker DNA. The self-assembly is mediated by the PNA-DNA complexation, which results in the formation of hydrophilic polymer networks. We show that the hydrogels can be produced through two different types of complexations. Type I hydrogel is formed via the PNA/DNA double-helix hybridization. Type II hydrogel utilizes a unique "P-form" oligonucleotide triple-helix that comprises two PNA sequences and one DNA. Microrheology studies confirm the respective gelation processes and disclose a higher critical gelation concentration for the type I gel when compared to the type II design. Scanning electron microscopy reveals the interconnected microporous structure of both types of hydrogels. Type I double-helix hydrogel exhibits larger pore sizes than type II triple-helix gel. The latter apparently contains denser structure and displays greater elasticity as well. The designed hybrid hydrogels have potential as novel biomaterials for pharmaceutical and biomedical applications.

  13. Antioxidant, DNA protective efficacy and HPLC analysis of Annona muricata (soursop) extracts.

    PubMed

    George, V Cijo; Kumar, D R Naveen; Suresh, P K; Kumar, R Ashok

    2015-04-01

    Annona muricata is a naturally occurring edible plant with wide array of therapeutic potentials. In India, it has a long history of traditional use in treating various ailments. The present investigation was carried out to characterize the phytochemicals present in the methanolic and aqueous leaf extracts of A. muricata, followed by validation of its radical scavenging and DNA protection activities. The extracts were also analyzed for its total phenolic contents and subjected to HPLC analysis to determine its active metabolites. The radical scavenging activities were premeditated by various complementary assays (DRSA, FRAP and HRSA). Further, its DNA protection efficacy against H2O2 induced toxicity was evaluated using pBR322 plasmid DNA. The results revealed that the extracts were highly rich in various phytochemicals including luteolin, homoorientin, tangeretin, quercetin, daidzein, epicatechin gallate, emodin and coumaric acid. Both the extracts showed significant (p < 0.05) radical scavenging activities, while methanolic extract demonstrated improved protection against H2O2-induced DNA damage when compared to aqueous extract. A strong positive correlation was observed for the estimated total phenolic contents and radical scavenging potentials of the extracts. Further HPLC analysis of the phyto-constituents of the extracts provides a sound scientific basis for compound isolation.

  14. Bioinformatic tools for DNA/protein sequence analysis, functional assignment of genes and protein classification.

    PubMed

    Rehm, B H

    2001-12-01

    The development of efficient DNA sequencing methods has led to the achievement of the DNA sequence of entire genomes from (to date) 55 prokaryotes, 5 eukaryotic organisms and 10 eukaryotic chromosomes. Thus, an enormous amount of DNA sequence data is available and even more will be forthcoming in the near future. Analysis of this overwhelming amount of data requires bioinformatic tools in order to identify genes that encode functional proteins or RNA. This is an important task, considering that even in the well-studied Escherichia coli more than 30% of the identified open reading frames are hypothetical genes. Future challenges of genome sequence analysis will include the understanding of gene regulation and metabolic pathway reconstruction including DNA chip technology, which holds tremendous potential for biomedicine and the biotechnological production of valuable compounds. The overwhelming volume of information often confuses scientists. This review intends to provide a guide to choosing the most efficient way to analyze a new sequence or to collect information on a gene or protein of interest by applying current publicly available databases and Web services. Recently developed tools that allow functional assignment of genes, mainly based on sequence similarity of the deduced amino acid sequence, using the currently available and increasing biological databases will be discussed.

  15. Determination of the dissociation constants for recombinant c-Myc, Max, and DNA complexes: The inhibitory effect of linoleic acid on the DNA-binding step

    SciTech Connect

    Jung, Kyung Chae; Rhee, Ho Sung; Park, Chi Hoon; Yang, Chul-Hak . E-mail: chulyang@plaza.snu.ac.kr

    2005-08-19

    c-Myc, the protein product of protooncogene c-myc, functions in cell proliferation, differentiation, and neoplastic disease. In this study, recombinant c-Myc and Max proteins, encompassing DNA binding (basic region) and dimerization (helix-loop-helix/leucine zipper) domain of human origin, were expressed in bacteria as Myc87 and Max85. Myc87 was purified under denatured conditions and was renatured again. The dissociation constant for the protein dimers and for dimer/DNA complexes were not detectable by isothermal titration calorimetry because of the low degree of solubility of Myc87 and Max85. Therefore, we set up equations which were used to determine the dissociation constants from the proportion of protein-DNA complexes. The dimer dissociation constants in TBS were 5.90({+-}0.54) x 10{sup -7} M for Max85/Max85 homodimer, 6.85({+-}0.25) x 10{sup -3} M for Myc87/Myc87 homodimer, and 2.55({+-}0.29) x 10{sup -8} M for Myc87/Max85 heterodimer, and the DNA-binding dissociation constants in TBS were 1.33({+-}0.21) x 10{sup -9} M for Max85/Max85/DNA, 2.27({+-}0.08) x 10{sup -12} M for Myc87/Myc87/DNA, and 4.43({+-}0.37) x 10{sup -10} M for Myc87/Max85/DNA. In addition, we revealed that linoleic acid which is known as an inhibitor for the formation of Max/Max/DNA complex reduced the affinity of Max homodimer for DNA. This result indicates that linoleic acid may bind to the DNA-binding region of Max homodimer.

  16. Random amplified polymorphic DNA analysis of genetically modified organisms.

    PubMed

    Yoke-Kqueen, Cheah; Radu, Son

    2006-12-15

    Randomly amplified polymorphic DNA (RAPD) was used to analyzed 78 samples comprises of certified reference materials (soya and maize powder), raw seeds (soybean and maize), processed food and animal feed. Combination assay of two arbitrary primers in the RAPD analysis enable to distinguish genetically modified organism (GMO) reference materials from the samples tested. Dendrogram analysis revealed 13 clusters at 45% similarity from the RAPD. RAPD analysis showed that the maize and soybean samples were clustered differently besides the GMO and non-GMO products.

  17. Random amplified polymorphic DNA analysis of genetically modified organisms.

    PubMed

    Yoke-Kqueen, Cheah; Radu, Son

    2006-12-15

    Randomly amplified polymorphic DNA (RAPD) was used to analyzed 78 samples comprises of certified reference materials (soya and maize powder), raw seeds (soybean and maize), processed food and animal feed. Combination assay of two arbitrary primers in the RAPD analysis enable to distinguish genetically modified organism (GMO) reference materials from the samples tested. Dendrogram analysis revealed 13 clusters at 45% similarity from the RAPD. RAPD analysis showed that the maize and soybean samples were clustered differently besides the GMO and non-GMO products. PMID:16860900

  18. Dualities in the analysis of phage DNA packaging motors

    PubMed Central

    Serwer, Philip; Jiang, Wen

    2012-01-01

    The DNA packaging motors of double-stranded DNA phages are models for analysis of all multi-molecular motors and for analysis of several fundamental aspects of biology, including early evolution, relationship of in vivo to in vitro biochemistry and targets for anti-virals. Work on phage DNA packaging motors both has produced and is producing dualities in the interpretation of data obtained by use of both traditional techniques and the more recently developed procedures of single-molecule analysis. The dualities include (1) reductive vs. accretive evolution, (2) rotation vs. stasis of sub-assemblies of the motor, (3) thermal ratcheting vs. power stroking in generating force, (4) complete motor vs. spark plug role for the packaging ATPase, (5) use of previously isolated vs. new intermediates for analysis of the intermediate states of the motor and (6) a motor with one cycle vs. a motor with two cycles. We provide background for these dualities, some of which are under-emphasized in the literature. We suggest directions for future research. PMID:23532204

  19. Nucleic Acid Aptamers for Living Cell Analysis

    NASA Astrophysics Data System (ADS)

    Xiong, Xiangling; Lv, Yifan; Chen, Tao; Zhang, Xiaobing; Wang, Kemin; Tan, Weihong

    2014-06-01

    Cells as the building blocks of life determine the basic functions and properties of a living organism. Understanding the structure and components of a cell aids in the elucidation of its biological functions. Moreover, knowledge of the similarities and differences between diseased and healthy cells is essential to understanding pathological mechanisms, identifying diagnostic markers, and designing therapeutic molecules. However, monitoring the structures and activities of a living cell remains a challenging task in bioanalytical and life science research. To meet the requirements of this task, aptamers, as “chemical antibodies,” have become increasingly powerful tools for cellular analysis. This article reviews recent advances in the development of nucleic acid aptamers in the areas of cell membrane analysis, cell detection and isolation, real-time monitoring of cell secretion, and intracellular delivery and analysis with living cell models. Limitations of aptamers and possible solutions are also discussed.

  20. Analysis of DNA methylation and gene expression in radiation-resistant head and neck tumors.

    PubMed

    Chen, Xiaofei; Liu, Liang; Mims, Jade; Punska, Elizabeth C; Williams, Kristin E; Zhao, Weiling; Arcaro, Kathleen F; Tsang, Allen W; Zhou, Xiaobo; Furdui, Cristina M

    2015-01-01

    Resistance to radiation therapy constitutes a significant challenge in the treatment of head and neck squamous cell cancer (HNSCC). Alteration in DNA methylation is thought to play a role in this resistance. Here, we analyzed DNA methylation changes in a matched model of radiation resistance for HNSCC using the Illumina HumanMethylation450 BeadChip. Our results show that compared to radiation-sensitive cells (SCC-61), radiation-resistant cells (rSCC-61) had a significant increase in DNA methylation. After combining these results with microarray gene expression data, we identified 84 differentially methylated and expressed genes between these 2 cell lines. Ingenuity Pathway Analysis revealed ILK signaling, glucocorticoid receptor signaling, fatty acid α-oxidation, and cell cycle regulation as top canonical pathways associated with radiation resistance. Validation studies focused on CCND2, a protein involved in cell cycle regulation, which was identified as hypermethylated in the promoter region and downregulated in rSCC-61 relative to SCC-61 cells. Treatment of rSCC-61 and SCC-61 with the DNA hypomethylating agent 5-aza-2'deoxycitidine increased CCND2 levels only in rSCC-61 cells, while treatment with the control reagent cytosine arabinoside did not influence the expression of this gene. Further analysis of HNSCC data from The Cancer Genome Atlas found increased methylation in radiation-resistant tumors, consistent with the cell culture data. Our findings point to global DNA methylation status as a biomarker of radiation resistance in HNSCC, and suggest a need for targeted manipulation of DNA methylation to increase radiation response in HNSCC. PMID:25961636

  1. Analysis of DNA methylation and gene expression in radiation-resistant head and neck tumors

    PubMed Central

    Chen, Xiaofei; Liu, Liang; Mims, Jade; Punska, Elizabeth C; Williams, Kristin E; Zhao, Weiling; Arcaro, Kathleen F; Tsang, Allen W; Zhou, Xiaobo; Furdui, Cristina M

    2015-01-01

    Resistance to radiation therapy constitutes a significant challenge in the treatment of head and neck squamous cell cancer (HNSCC). Alteration in DNA methylation is thought to play a role in this resistance. Here, we analyzed DNA methylation changes in a matched model of radiation resistance for HNSCC using the Illumina HumanMethylation450 BeadChip. Our results show that compared to radiation-sensitive cells (SCC-61), radiation-resistant cells (rSCC-61) had a significant increase in DNA methylation. After combining these results with microarray gene expression data, we identified 84 differentially methylated and expressed genes between these 2 cell lines. Ingenuity Pathway Analysis revealed ILK signaling, glucocorticoid receptor signaling, fatty acid α-oxidation, and cell cycle regulation as top canonical pathways associated with radiation resistance. Validation studies focused on CCND2, a protein involved in cell cycle regulation, which was identified as hypermethylated in the promoter region and downregulated in rSCC-61 relative to SCC-61 cells. Treatment of rSCC-61 and SCC-61 with the DNA hypomethylating agent 5-aza-2'deoxycitidine increased CCND2 levels only in rSCC-61 cells, while treatment with the control reagent cytosine arabinoside did not influence the expression of this gene. Further analysis of HNSCC data from The Cancer Genome Atlas found increased methylation in radiation-resistant tumors, consistent with the cell culture data. Our findings point to global DNA methylation status as a biomarker of radiation resistance in HNSCC, and suggest a need for targeted manipulation of DNA methylation to increase radiation response in HNSCC. PMID:25961636

  2. Characterization of a Single-Stranded DNA-Binding-Like Protein from Nanoarchaeum equitans—A Nucleic Acid Binding Protein with Broad Substrate Specificity

    PubMed Central

    Olszewski, Marcin; Balsewicz, Jan; Nowak, Marta; Maciejewska, Natalia; Cyranka-Czaja, Anna; Zalewska-Piątek, Beata; Piątek, Rafał; Kur, Józef

    2015-01-01

    Background SSB (single-stranded DNA-binding) proteins play an essential role in all living cells and viruses, as they are involved in processes connected with ssDNA metabolism. There has recently been an increasing interest in SSBs, since they can be applied in molecular biology techniques and analytical methods. Nanoarchaeum equitans, the only known representative of Archaea phylum Nanoarchaeota, is a hyperthermophilic, nanosized, obligatory parasite/symbiont of Ignicoccus hospitalis. Results This paper reports on the ssb-like gene cloning, gene expression and characterization of a novel nucleic acid binding protein from Nanoarchaeum equitans archaeon (NeqSSB-like protein). This protein consists of 243 amino acid residues and one OB fold per monomer. It is biologically active as a monomer like as SSBs from some viruses. The NeqSSB-like protein displays a low sequence similarity to the Escherichia coli SSB, namely 10% identity and 29% similarity, and is the most similar to the Sulfolobus solfataricus SSB (14% identity and 32% similarity). The NeqSSB-like protein binds to ssDNA, although it can also bind mRNA and, surprisingly, various dsDNA forms, with no structure-dependent preferences as evidenced by gel mobility shift assays. The size of the ssDNA binding site, which was estimated using fluorescence spectroscopy, is 7±1 nt. No salt-dependent binding mode transition was observed. NeqSSB-like protein probably utilizes a different model for ssDNA binding than the SSB proteins studied so far. This protein is highly thermostable; the half-life of the ssDNA binding activity is 5 min at 100°C and melting temperature (Tm) is 100.2°C as shown by differential scanning calorimetry (DSC) analysis. Conclusion NeqSSB-like protein is a novel highly thermostable protein which possesses a unique broad substrate specificity and is able to bind all types of nucleic acids. PMID:25973760

  3. Quantification of false positive reduction in nucleic acid purification on hemorrhagic fever DNA.

    SciTech Connect

    James, Conrad D.; Pohl, Kenneth Roy; Derzon, Mark Steven; McClain, Jaime; Achyuthan, Komandoor

    2006-11-01

    Columbia University has developed a sensitive highly multiplexed system for genetic identification of nucleic acid targets. The primary obstacle to implementing this technology is the high rate of false positives due to high levels of unbound reporters that remain within the system after hybridization. The ability to distinguish between free reporters and reporters bound to targets limits the use of this technology. We previously demonstrated a new electrokinetic method for binary separation of kb pair long DNA molecules and oligonucleotides. The purpose of this project 99864 is to take these previous demonstrations and further develop the technique and hardware for field use. Specifically, our objective was to implement separation in a heterogeneous sample (containing target DNA and background oligo), to perform the separation in a flow-based device, and to develop all of the components necessary for field testing a breadboard prototype system.

  4. MicroRNA and DNA methylation alterations mediating retinoic acid induced neuroblastoma cell differentiation.

    PubMed

    Stallings, Raymond L; Foley, Niamh H; Bray, Isabella M; Das, Sudipto; Buckley, Patrick G

    2011-10-01

    Many neuroblastoma cell lines can be induced to differentiate into a mature neuronal cell type with retinoic acid and other compounds, providing an important model system for elucidating signalling pathways involved in this highly complex process. Recently, it has become apparent that miRNAs, which act as regulators of gene expression at a post-transcriptional level, are differentially expressed in differentiating cells and play important roles governing many aspects of this process. This includes the down-regulation of DNA methyltransferases that cause the de-methylation and transcriptional activation of numerous protein coding gene sequences. The purpose of this article is to review involvement of miRNAs and DNA methylation alterations in the process of neuroblastoma cell differentiation. A thorough understanding of miRNA and genetic pathways regulating neuroblastoma cell differentiation potentially could lead to targeted therapies for this disease.

  5. Quantitative DNA Methylation Analysis of Candidate Genes in Cervical Cancer

    PubMed Central

    Siegel, Erin M.; Riggs, Bridget M.; Delmas, Amber L.; Koch, Abby; Hakam, Ardeshir; Brown, Kevin D.

    2015-01-01

    Aberrant DNA methylation has been observed in cervical cancer; however, most studies have used non-quantitative approaches to measure DNA methylation. The objective of this study was to quantify methylation within a select panel of genes previously identified as targets for epigenetic silencing in cervical cancer and to identify genes with elevated methylation that can distinguish cancer from normal cervical tissues. We identified 49 women with invasive squamous cell cancer of the cervix and 22 women with normal cytology specimens. Bisulfite-modified genomic DNA was amplified and quantitative pyrosequencing completed for 10 genes (APC, CCNA, CDH1, CDH13, WIF1, TIMP3, DAPK1, RARB, FHIT, and SLIT2). A Methylation Index was calculated as the mean percent methylation across all CpG sites analyzed per gene (~4-9 CpG site) per sequence. A binary cut-point was defined at >15% methylation. Sensitivity, specificity and area under ROC curve (AUC) of methylation in individual genes or a panel was examined. The median methylation index was significantly higher in cases compared to controls in 8 genes, whereas there was no difference in median methylation for 2 genes. Compared to HPV and age, the combination of DNA methylation level of DAPK1, SLIT2, WIF1 and RARB with HPV and age significantly improved the AUC from 0.79 to 0.99 (95% CI: 0.97–1.00, p-value = 0.003). Pyrosequencing analysis confirmed that several genes are common targets for aberrant methylation in cervical cancer and DNA methylation level of four genes appears to increase specificity to identify cancer compared to HPV detection alone. Alterations in DNA methylation of specific genes in cervical cancers, such as DAPK1, RARB, WIF1, and SLIT2, may also occur early in cervical carcinogenesis and should be evaluated. PMID:25826459

  6. Quantitative DNA methylation analysis of candidate genes in cervical cancer.

    PubMed

    Siegel, Erin M; Riggs, Bridget M; Delmas, Amber L; Koch, Abby; Hakam, Ardeshir; Brown, Kevin D

    2015-01-01

    Aberrant DNA methylation has been observed in cervical cancer; however, most studies have used non-quantitative approaches to measure DNA methylation. The objective of this study was to quantify methylation within a select panel of genes previously identified as targets for epigenetic silencing in cervical cancer and to identify genes with elevated methylation that can distinguish cancer from normal cervical tissues. We identified 49 women with invasive squamous cell cancer of the cervix and 22 women with normal cytology specimens. Bisulfite-modified genomic DNA was amplified and quantitative pyrosequencing completed for 10 genes (APC, CCNA, CDH1, CDH13, WIF1, TIMP3, DAPK1, RARB, FHIT, and SLIT2). A Methylation Index was calculated as the mean percent methylation across all CpG sites analyzed per gene (~4-9 CpG site) per sequence. A binary cut-point was defined at >15% methylation. Sensitivity, specificity and area under ROC curve (AUC) of methylation in individual genes or a panel was examined. The median methylation index was significantly higher in cases compared to controls in 8 genes, whereas there was no difference in median methylation for 2 genes. Compared to HPV and age, the combination of DNA methylation level of DAPK1, SLIT2, WIF1 and RARB with HPV and age significantly improved the AUC from 0.79 to 0.99 (95% CI: 0.97-1.00, p-value = 0.003). Pyrosequencing analysis confirmed that several genes are common targets for aberrant methylation in cervical cancer and DNA methylation level of four genes appears to increase specificity to identify cancer compared to HPV detection alone. Alterations in DNA methylation of specific genes in cervical cancers, such as DAPK1, RARB, WIF1, and SLIT2, may also occur early in cervical carcinogenesis and should be evaluated.

  7. [DNA fingerprinting analysis of silkworm embryo cell lines].

    PubMed

    Pan, Min Hui; Feng, Zhen Yue; Tian, Zhi Qiang; Liu, Min; Lu, Cheng

    2006-12-01

    DNA extraction and the polymerase chain reaction (PCR) were used on DNA genomes study of cell lines of Bombyx mori. DNA polymorphic marker analysis was conducted and DNA fingerprint of cell lines of Bombyx mori. was carried out using ISSR and RAPD. Primers that can reliably find polymorphic bands were screened out. 26 ISSR primers were selected from them any available, and 797 polymorphic bands were abtained through PCR amplification in 9 samples, including 3 embryo cell lines of Bombyx mori (BmE-SWU1, BmE-SWU2, BmE-SWU3), 5 passage cell lines (BmE, BmN, Sf9, Sf21, Hi5) and the embryos from which BmE-SWU1 originated. The ration of polymorphic bands was 89.9%. 43 RAPD primers were selected out through PCR amplification, and 1205 polymorphic bands were obtained in 9 samples. The ration of polymorphic bands was 76.6%. There were many DNA polymorphic bands differences in the cell lines of Bombyx mori. The special DNA markers of the 3 embryo cell lines were found respectively. The similarity index Nei and genetic distance of the 9 samples were calculated and the phylogeny tree of 9 samples was constructed by UPGMA. Results showed that 2 groups were divided,one group including the 3 embryo cell lines and the embryo of XQ has close relative. Another group constructed by five insect cell lines came from different species, their genetic distance was closer than the 3 embryo cell lines.

  8. Quantitative analysis of TALE-DNA interactions suggests polarity effects.

    PubMed

    Meckler, Joshua F; Bhakta, Mital S; Kim, Moon-Soo; Ovadia, Robert; Habrian, Chris H; Zykovich, Artem; Yu, Abigail; Lockwood, Sarah H; Morbitzer, Robert; Elsäesser, Janett; Lahaye, Thomas; Segal, David J; Baldwin, Enoch P

    2013-04-01

    Transcription activator-like effectors (TALEs) have revolutionized the field of genome engineering. We present here a systematic assessment of TALE DNA recognition, using quantitative electrophoretic mobility shift assays and reporter gene activation assays. Within TALE proteins, tandem 34-amino acid repeats recognize one base pair each and direct sequence-specific DNA binding through repeat variable di-residues (RVDs). We found that RVD choice can affect affinity by four orders of magnitude, with the relative RVD contribution in the order NG > HD ≈ NN > NI > NK. The NN repeat preferred the base G over A, whereas the NK repeat bound G with 10(3)-fold lower affinity. We compared AvrBs3, a naturally occurring TALE that recognizes its target using some atypical RVD-base combinations, with a designed TALE that precisely matches 'standard' RVDs with the target bases. This comparison revealed unexpected differences in sensitivity to substitutions of the invariant 5'-T. Another surprising observation was that base mismatches at the 5' end of the target site had more disruptive effects on affinity than those at the 3' end, particularly in designed TALEs. These results provide evidence that TALE-DNA recognition exhibits a hitherto un-described polarity effect, in which the N-terminal repeats contribute more to affinity than C-terminal ones.

  9. Quantitative analysis of TALE–DNA interactions suggests polarity effects

    PubMed Central

    Meckler, Joshua F.; Bhakta, Mital S.; Kim, Moon-Soo; Ovadia, Robert; Habrian, Chris H.; Zykovich, Artem; Yu, Abigail; Lockwood, Sarah H.; Morbitzer, Robert; Elsäesser, Janett; Lahaye, Thomas; Segal, David J.; Baldwin, Enoch P.

    2013-01-01

    Transcription activator-like effectors (TALEs) have revolutionized the field of genome engineering. We present here a systematic assessment of TALE DNA recognition, using quantitative electrophoretic mobility shift assays and reporter gene activation assays. Within TALE proteins, tandem 34-amino acid repeats recognize one base pair each and direct sequence-specific DNA binding through repeat variable di-residues (RVDs). We found that RVD choice can affect affinity by four orders of magnitude, with the relative RVD contribution in the order NG > HD ∼ NN ≫ NI > NK. The NN repeat preferred the base G over A, whereas the NK repeat bound G with 103-fold lower affinity. We compared AvrBs3, a naturally occurring TALE that recognizes its target using some atypical RVD-base combinations, with a designed TALE that precisely matches ‘standard’ RVDs with the target bases. This comparison revealed unexpected differences in sensitivity to substitutions of the invariant 5′-T. Another surprising observation was that base mismatches at the 5′ end of the target site had more disruptive effects on affinity than those at the 3′ end, particularly in designed TALEs. These results provide evidence that TALE–DNA recognition exhibits a hitherto un-described polarity effect, in which the N-terminal repeats contribute more to affinity than C-terminal ones. PMID:23408851

  10. A general DNA analysis program for the Hewlett-Packard Model 86/87 microcomputer.

    PubMed

    Harley, E H

    1986-01-10

    A program is described to perform general DNA sequence analysis on the Hewlett-Packard Model 86/87 microcomputer operating on 128 K of RAM. The following analytical procedures can be performed: 1. display of the sequence, in whole or part, or its complement; 2. search for specified sequences e.g. restriction sites, and in the case of the latter give fragment sizes; 3. perform a comprehensive search for all known restriction enzyme sites; 4. map sites graphically; 5. perform editing functions; 6. base frequency analysis; 7. search for repeated sequences; 8. search for open reading frames or translate into the amino acid sequence and analyse for basic and acidic amino acids, hydrophobicity, and codon usage. Two sequences, or parts thereof, can be merged in various orientations to mimic recombination strategies, or can be compared for homologies. The program is written in HP BASIC and is designed principally as a tool for the laboratory investigator manipulating a defined set of vectors and recombinant DNA constructs.

  11. Circulating nucleic acids damage DNA of healthy cells by integrating into their genomes.

    PubMed

    Mittra, Indraneel; Khare, Naveen Kumar; Raghuram, Gorantla Venkata; Chaubal, Rohan; Khambatti, Fatema; Gupta, Deepika; Gaikwad, Ashwini; Prasannan, Preeti; Singh, Akshita; Iyer, Aishwarya; Singh, Ankita; Upadhyay, Pawan; Nair, Naveen Kumar; Mishra, Pradyumna Kumar; Dutt, Amit

    2015-03-01

    Whether nucleic acids that circulate in blood have any patho-physiological functions in the host have not been explored.We report here that far from being inert molecules, circulating nucleic acids have significant biological activities of their own that are deleterious to healthy cells of the body. Fragmented DNA and chromatin (DNAfs and Cfs) isolated from blood of cancer patients and healthy volunteers are readily taken up by a variety of cells in culture to be localized in their nuclei within a few minutes. The intra-nuclear DNAfs and Cfs associate themselves with host cell chromosomes to evoke a cellular DNA-damage-repair-response (DDR) followed by their incorporation into the host cell genomes. Whole genome sequencing detected the presence of tens of thousands of human sequence reads in the recipient mouse cells. Genomic incorporation of DNAfs and Cfs leads to dsDNA breaks and activation of apoptotic pathways in the treated cells. When injected intravenously into Balb/C mice, DNAfs and Cfs undergo genomic integration into cells of their vital organs resulting in activation of DDR and apoptotic proteins in the recipient cells. Cfs have significantly greater activity than DNAfs with respect to all parameters examined, while both DNAfs and Cfs isolated from cancer patients are more active than those from normal volunteers. All the above pathological actions of DNAfs and Cfs described above can be abrogated by concurrent treatment with DNase I and/or anti-histone antibody complexed nanoparticles both in vitro and in vivo. Taken together, our results suggest that circulating DNAfs and Cfs are physiological, continuously arising, endogenous DNA damaging agents with implications to ageing and a multitude of human pathologies including initiation of cancer.

  12. Clinical application of forensic DNA analysis: a literature review.

    PubMed

    Manasatienkij, Chairat; Ra-ngabpai, Chatchawin

    2012-10-01

    Polymorphic short tandem repeat (STR) analysis after polymerase chain reaction amplification is presently the most important analytical method of forensic DNA laboratory. Beside the forensic-purpose application, STR analysis is also necessary for chimerism testing after bone marrow or allogeneic stem cell transplantation. To increase success rate of the treatment and avoid adverse effect of the transplantation procedure, serial chimerism study has to be closely monitored. Many analytical techniques have been therefore developed to improve the accuracy and sensitivity. STR-PCR is currently the most commonly used analytical method for chimerism testing. Sensitivity of the method is about 3% of the minor DNA in a specimen. The method consumes a short period of time and is convenient due to availability of the commercial kits. High discriminative ability and well-validated interpretation procedure are the attractive characteristics of the method.

  13. High-Resolution DNA Melting Analysis in Plant Research.

    PubMed

    Simko, Ivan

    2016-06-01

    Genetic and genomic studies provide valuable insight into the inheritance, structure, organization, and function of genes. The knowledge gained from the analysis of plant genes is beneficial to all aspects of plant research, including crop improvement. New methods and tools are continually being developed to facilitate rapid and accurate mapping, sequencing, and analyzing of genes. Here, I review the recent progress in the application of high-resolution melting (HRM) analysis of DNA, a method that allows detecting polymorphism in double-stranded DNA by comparing profiles of melting curves. Use of HRM has expanded considerably in the past few years as the method was successfully applied for high-throughput genotyping, mapping genes, testing food products and seeds, and other areas of plant research. PMID:26827247

  14. GENSTYLE: exploration and analysis of DNA sequences with genomic signature.

    PubMed

    Fertil, Bernard; Massin, Matthieu; Lespinats, Sylvain; Devic, Caroline; Dumee, Philippe; Giron, Alain

    2005-07-01

    GENSTYLE (http://Genstyle.imed.jussieu.fr) is a workspace designed for the characterization and classification of nucleotide sequences. Based on the genomic signature paradigm, GENSTYLE focuses on oligonucleotide frequencies in DNA sequences. Users can select sequences of interest in the GENSTYLE companion database, where the whole set of GenBank sequences is grouped per species, or upload their own sequences to work with. Tools for the exploration and analysis of signatures allow (i) identification of the origin of DNA segments (detection of rare species or species for which technical problems prevent fast characterization, such as micro-organisms with slow growth), (ii) analysis of the homogeneity of a genome and isolation of areas with novel functionality (horizontal transfers for example)--and (iii) molecular phylogeny and taxonomy.

  15. HLA class II genes: typing by DNA analysis.

    PubMed

    Bidwell, J L; Bidwell, E A; Bradley, B A

    1990-04-01

    A detailed understanding of the structure and function of the human major histocompatibility complex (MHC) has ensued from studies by molecular biologist during the last decade. Virtually all of the HLA genes have now been cloned, and the nucleotide sequences of their different allelic forms have been determined. Typing for these HLA alleles is a fundamental prerequisite for tissue matching in allogeneic organ transplantation. Until very recently, typing procedures have been dominated by serological and cellular methods. The availability of cloned DNA from HLA genes has now permitted the technique of restriction fragment length polymorphism (RFLP) analysis to be applied, with remarkable success and advantage, to phenotyping of both HLA Class I and Class II determinants. For the HLA Class II genes DR and DQ, a simple two-stage RFLP analysis permits the accurate identification of all specificities defined by serology, and of many which are defined by cellular typing. At the present time, however, RFLP typing of HLA Class I genes is not as practicable or as informative as that for HLA Class II genes. The present clinical applications of HLA-DR and DQ RFLP typing are predominantly in phenotyping of living donors, including selection of HLA-matched volunteer bone marrow donors, in allograft survival studies, and in studies of HLA Class II-associated diseases. However, the time taken to perform RFLP analysis precludes its use for the typing of cadaveric kidney donors. Nucleotide sequence data for the alleles of HLA Class II genes have now permitted the development of allele-specific oligonucleotide (ASO) typing, a second category of DNA analysis. This has been greatly facilitated by the ability to amplify specific HLA Class II DNA 'target' sequences using the polymerase chain reaction (PCR) technique. The accuracy of DNA typing techniques should ensure that this methodology will eventually replace conventional HLA phenotyping.

  16. Amino acid sequence homology between Piv, an essential protein in site-specific DNA inversion in Moraxella lacunata, and transposases of an unusual family of insertion elements.

    PubMed Central

    Lenich, A G; Glasgow, A C

    1994-01-01

    Deletion analysis of the subcloned DNA inversion region of Moraxella lacunata indicates that Piv is the only M. lacunata-encoded factor required for site-specific inversion of the tfpQ/tfpI pilin segment. The predicted amino acid sequence of Piv shows significant homology solely with the transposases/integrases of a family of insertion sequence elements, suggesting that Piv is a novel site-specific recombinase. Images PMID:8021196

  17. Voltammetric behavior of uric acid on carbon paste electrode modified with salmon sperm dsDNA and its application as label-free electrochemical sensor.

    PubMed

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

    2014-04-15

    A simple and sensitive label-free electrochemical DNA biosensor was proposed for the rapid determination of uric acid (UA) using a carbon nano tube paste electrode (CNTPE) modified with salmon sperm dsDNA. At first, the interaction between UA and the DNA was studied using differential pulse voltammetry (DPV). The addition of the DNA to UA solution resulted in a decrease in the peak current of UA and at the same time, a positive shift in the peak potential indicating an intercalative interaction. Then, the voltammetric response of a DNA-immobilized CNTPE was investigated for the determination of UA. The immobilization of the DNA was carried out using acid-functionalized carbon nanotubes and studied using Fe(CN)6(3-)/Fe(CN)6(4-) redox indicator. Compared with unmodified CNTPE, the oxidation signal of UA showed a significant increase at the DNA-coated electrode, and shifted to more positive potentials attributed to the pre-concentration of UA at the electrode surface due to interaction with the surface-confined DNA layer. This interaction was used for the fabrication of a simple and sensitive biosensor for determining UA. After the optimization of operational parameters, a linear dependence of the peak current on the UA concentration was observed in the range of 7.0×10(-7) to 1.1×10(-4) mol L(-1), with the detection and quantification limits of 1.8×10(-7) and 5.8×10(-7) mol L(-1), respectively. The proposed biosensor was successfully applied to validate its capability for the analysis of UA in human serum and urine samples.

  18. APE1-mediated DNA damage repair provides survival advantage for esophageal adenocarcinoma cells in response to acidic bile salts.

    PubMed

    Hong, Jun; Chen, Zheng; Peng, Dunfa; Zaika, Alexander; Revetta, Frank; Washington, M Kay; Belkhiri, Abbes; El-Rifai, Wael

    2016-03-29

    Chronic Gastroesophageal Reflux Disease (GERD) is the main risk factor for the development of Barrett's esophagus (BE) and its progression to esophageal adenocarcinoma (EAC). Accordingly, EAC cells are subjected to high levels of oxidative stress and subsequent DNA damage. In this study, we investigated the expression and role of Apurinic/apyrimidinic endonuclease 1 (APE1) protein in promoting cancer cell survival by counteracting the lethal effects of acidic bile salts (ABS)-induced DNA damage. Immunohistochemistry analysis of human tissue samples demonstrated overexpression of APE1 in more than half of EACs (70 of 130), as compared to normal esophagus and non-dysplastic BE samples (P < 0.01). To mimic in vivo conditions, we treated in vitro cell models with a cocktail of ABS. The knockdown of endogenous APE1 in EAC FLO-1 cells significantly increased oxidative DNA damage (P < 0.01) and DNA single- and double-strand breaks (P < 0.01), whereas overexpression of APE1 in EAC OE33 cells reversed these effects. Annexin V/PI staining indicated that the APE1 expression in OE33 cells protects against ABS-induced apoptosis. In contrast, knockdown of endogenous APE1 in FLO-1 cells increased apoptosis under the same conditions. Mechanistic investigations indicated that the pro-survival function of APE1 was associated with the regulation of stress response c-Jun N-terminal protein kinase (JNK) and p38 kinases. Pharmacological inhibition of APE1 base excision repair (BER) function decreased cell survival and enhanced activation of JNK and p38 kinases by ABS. Our findings suggest that constitutive overexpression of APE1 in EAC may be an adaptive pro-survival mechanism that protects against the genotoxic lethal effects of bile reflux episodes. PMID:26934647

  19. Self-assembling DNA hydrogel-based delivery of immunoinhibitory nucleic acids to immune cells.

    PubMed

    Nishida, Yu; Ohtsuki, Shozo; Araie, Yuki; Umeki, Yuka; Endo, Masayuki; Emura, Tomoko; Hidaka, Kumi; Sugiyama, Hiroshi; Takahashi, Yuki; Takakura, Yoshinobu; Nishikawa, Makiya

    2016-01-01

    Immunoinhibitory oligodeoxynucleotides (INH-ODNs) are promising inhibitors of Toll-like receptor 9 (TLR9) activation. To efficiently deliver INH-ODNs to TLR9-positive cells, we designed a Takumi-shaped DNA (Takumi) consisting of two partially complementary ODNs as the main component of a DNA hydrogel. Polyacrylamide gel electrophoresis showed that Takumi-containing INH-ODNs (iTakumi) and iTakumi-based DNA hydrogel (iTakumiGel) were successfully generated. Their activity was examined in murine macrophage-like RAW264.7 cells and DC2.4 dendritic cells by measuring tumor necrosis factor-α and interleukin-6 release after the addition of a TLR9 ligand (CpG ODN). Cytokine release was efficiently inhibited by the iTakumiGel. Flow cytometry analysis and confocal microscopy showed that cellular uptake of INH-ODN was greatly increased by the iTakumiGel. These results indicate that a Takumi-based DNA hydrogel is useful for the delivery of INH-ODNs to immune cells to inhibit TLR9-mediated hyperinduction of proinflammatory cytokines. From the Clinical Editor: Toll-like receptor 9 activation has been reported to be associated with many autoimmune diseases. DNA inhibition using oligodeoxynucleotides is one of the potential treatments. In this article, the authors described hydrogel-based platform for the delivery of the inhibitory oligodeoxynucleotides for enhanced efficacy. The positive findings could indicate a way for the future.

  20. An Unprecedented Nucleic Acid Capture Mechanism for Excision of DNA Damage

    PubMed Central

    Rubinson, Emily H.; Gowda, A.S. Prakasha; Spratt, Thomas E.; Gold, Barry; Eichman, Brandt F.

    2014-01-01

    DNA glycosylases that remove alkylated and deaminated purine nucleobases are essential DNA repair enzymes that protect the genome, and at the same time confound cancer alkylation therapy, by excising cytotoxic N3-methyladenine bases formed by DNA targeting anticancer compounds. The basis for glycosylase specificity toward N3- and N7-alkylpurines is believed to result from intrinsic instability of the modified bases and not from direct enzyme functional group chemistry. Here, we present crystal structures of the recently discovered Bacillus cereus AlkD glycosylase in complex with DNAs containing alkylated, mismatched, and abasic nucleotides. Unlike other glycosylases, AlkD captures the extrahelical lesion in a solvent-exposed orientation, providing the first illustration for how hydrolysis of N3- and N7-alkylated bases may be facilitated by increased lifetime out of the DNA helix. The structures and supporting biochemical analysis of base flipping and catalysis reveal how AlkD’s HEAT-repeats distort the DNA backbone to detect non-Watson-Crick base pairs without duplex intercalation. PMID:20927102

  1. An unprecedented nucleic acid capture mechanism for excision of DNA damage

    SciTech Connect

    Rubinson, Emily H.; Prakasha Gowda, A.S.; Spratt, Thomas E.; Gold, Barry; Eichmanbrand, Brandt F.

    2010-11-18

    DNA glycosylases that remove alkylated and deaminated purine nucleobases are essential DNA repair enzymes that protect the genome, and at the same time confound cancer alkylation therapy, by excising cytotoxic N3-methyladenine bases formed by DNA-targeting anticancer compounds. The basis for glycosylase specificity towards N3- and N7-alkylpurines is believed to result from intrinsic instability of the modified bases and not from direct enzyme functional group chemistry. Here we present crystal structures of the recently discovered Bacillus cereus AlkD glycosylase in complex with DNAs containing alkylated, mismatched and abasic nucleotides. Unlike other glycosylases, AlkD captures the extrahelical lesion in a solvent-exposed orientation, providing an illustration for how hydrolysis of N3- and N7-alkylated bases may be facilitated by increased lifetime out of the DNA helix. The structures and supporting biochemical analysis of base flipping and catalysis reveal how the HEAT repeats of AlkD distort the DNA backbone to detect non-Watson-Crick base pairs without duplex intercalation.

  2. DNA Three Way Junction Core Decorated with Amino Acids-Like Residues-Synthesis and Characterization.

    PubMed

    Addamiano, Claudia; Gerland, Béatrice; Payrastre, Corinne; Escudier, Jean-Marc

    2016-01-01

    Construction and physico-chemical behavior of DNA three way junction (3WJ) functionalized by protein-like residues (imidazole, alcohol and carboxylic acid) at unpaired positions at the core is described. One 5'-C(S)-propargyl-thymidine nucleotide was specifically incorporated on each strand to react through a post synthetic CuACC reaction with either protected imidazolyl-, hydroxyl- or carboxyl-azide. Structural impacts of 5'-C(S)-functionalization were investigated to evaluate how 3WJ flexibility/stability is affected. PMID:27563857

  3. Effect of alpha-ketoglutarate and oxaloacetate on brain mitochondrial DNA damage and seizures induced by kainic acid in mice.

    PubMed

    Yamamoto, Hiro-aki; Mohanan, Parayanthala V

    2003-07-20

    The effects of alpha-ketoglutarate and oxaloacetate on brain mitochondrial DNA (mtDNA) damage and seizures induced by kainic acid were examined both in vivo and in vitro. An intraperitoneal (ip) injection of kainic acid (45 mg/kg) produced broad-spectrum limbic and severe sustained seizures in all of the treated mice. The seizures were abolished when alpha-ketoglutarate (2 g/kg) or oxaloacetate (1 g/kg) was injected intraperitoneally in the animals 1 min before kainic acid administration. In addition, the administration of kainic acid caused damage to mtDNA in brain frontal and middle cortex of mice. These effects were completely abolished by the ip preinjection of alpha-ketoglutarate (2 g/kg) or oxaloacetate (1 g/kg). In vitro exposure of kainic acid (0.25, 0.5 or 1.0 mM) to brain homogenate inflicted damage to mtDNA in a concentration-dependent manner. The damage of mtDNA induced by 1.0 mM kainic acid was attenuated by the co-treatment with alpha-ketoglutarate (2.5 or 5.0 mM) or oxaloacetate (0.75 or 1.0 mM). Furthermore, in vivo and in vitro exposure of kainic acid elicited an increase in lipid peroxidation. However, the increased lipid peroxidation was completely inhibited by cotreatment of alpha-ketoglutarate or oxaloacetate. These results suggest that alpha-keto acids such as alpha-ketoglutarate and oxaloacetate play a role in the inhibition of seizures and subsequent mtDNA damage induced by the excitotoxic/neurotoxic agent, kainic acid.

  4. Impact of boric acid exposure at different concentrations on testicular DNA and male rats fertility.

    PubMed

    El-Dakdoky, Mai H; Abd El-Wahab, Hanan M F

    2013-06-01

    The aim of this study was to investigate the consequences of exposure to three levels of boric acid (BA) on male rats reproduction, fertility and progeny outcome, with emphasis on testicular DNA level and quality. Adult male rats (12 weeks old) were treated orally with 125, 250 and 500 mg/kg bwt/d of BA for 60 d. The results indicated that BA administration at 125 mg/kg bwt had no adverse effects on fertility, sperm characteristics or prenatal development of the impregnated females. However, at dose 250 mg, BA treatment significantly increased serum nitric oxide, testosterone, estradiol levels and testicular boron and calcium levels and also significantly reduced serum arginase activity, sperm quality and testicular DNA content with minor DNA fragmentation. The impact of BA exposure at dose 250 mg on male rats fertility was translated into increases in pre-implantation loss with a resulting decrease in the number of live fetuses/litter. In addition to the significant alteration of biochemical measurements, observed at dose 250 mg, administration of BA at 500 mg caused testicular atrophy, severe damage of spermatogenesis, spermiation failure and significant reduction of Mg and Zn testicular levels. None of the male rats, treated with 500 mg/kg bwt, could impregnate untreated females, suggesting the occurrence of definitive loss of fertility. In conclusion, BA impaired fertility, in a dose-dependant manner, by targeting the highly proliferative cells, the germ cells, through decreasing DNA synthetic rate rather than the induction of DNA damage. PMID:23301826

  5. Impact of boric acid exposure at different concentrations on testicular DNA and male rats fertility.

    PubMed

    El-Dakdoky, Mai H; Abd El-Wahab, Hanan M F

    2013-06-01

    The aim of this study was to investigate the consequences of exposure to three levels of boric acid (BA) on male rats reproduction, fertility and progeny outcome, with emphasis on testicular DNA level and quality. Adult male rats (12 weeks old) were treated orally with 125, 250 and 500 mg/kg bwt/d of BA for 60 d. The results indicated that BA administration at 125 mg/kg bwt had no adverse effects on fertility, sperm characteristics or prenatal development of the impregnated females. However, at dose 250 mg, BA treatment significantly increased serum nitric oxide, testosterone, estradiol levels and testicular boron and calcium levels and also significantly reduced serum arginase activity, sperm quality and testicular DNA content with minor DNA fragmentation. The impact of BA exposure at dose 250 mg on male rats fertility was translated into increases in pre-implantation loss with a resulting decrease in the number of live fetuses/litter. In addition to the significant alteration of biochemical measurements, observed at dose 250 mg, administration of BA at 500 mg caused testicular atrophy, severe damage of spermatogenesis, spermiation failure and significant reduction of Mg and Zn testicular levels. None of the male rats, treated with 500 mg/kg bwt, could impregnate untreated females, suggesting the occurrence of definitive loss of fertility. In conclusion, BA impaired fertility, in a dose-dependant manner, by targeting the highly proliferative cells, the germ cells, through decreasing DNA synthetic rate rather than the induction of DNA damage.

  6. Recent advances in microchip electrophoresis for amino acid analysis.

    PubMed

    Ou, Gaozhi; Feng, Xiaojun; Du, Wei; Liu, Xin; Liu, Bi-Feng

    2013-10-01

    With the maturation of microfluidic technologies, microchip electrophoresis has been widely employed for amino acid analysis owing to its advantages of low sample consumption, reduced analysis time, high throughput, and potential for integration and automation. In this article, we review the recent progress in amino acid analysis using microchip electrophoresis during the period from 2007 to 2012. Innovations in microchip materials, surface modification, sample introduction, microchip electrophoresis, and detection methods are documented, as well as nascent applications of amino acid analysis in single-cell analysis, microdialysis sampling, food analysis, and extraterrestrial exploration. Without doubt, more applications of microchip electrophoresis in amino acid analysis may be expected soon.

  7. Recent advances in microchip electrophoresis for amino acid analysis.

    PubMed

    Ou, Gaozhi; Feng, Xiaojun; Du, Wei; Liu, Xin; Liu, Bi-Feng

    2013-10-01

    With the maturation of microfluidic technologies, microchip electrophoresis has been widely employed for amino acid analysis owing to its advantages of low sample consumption, reduced analysis time, high throughput, and potential for integration and automation. In this article, we review the recent progress in amino acid analysis using microchip electrophoresis during the period from 2007 to 2012. Innovations in microchip materials, surface modification, sample introduction, microchip electrophoresis, and detection methods are documented, as well as nascent applications of amino acid analysis in single-cell analysis, microdialysis sampling, food analysis, and extraterrestrial exploration. Without doubt, more applications of microchip electrophoresis in amino acid analysis may be expected soon. PMID:23436170

  8. Characterization of a novel inhibitor of human DNA polymerases: 3,4,5-tri-O-galloylquinic acid.

    PubMed

    Parker, W B; Nishizawa, M; Fisher, M H; Ye, N; Lee, K H; Cheng, Y C

    1989-11-01

    Various galloyl derivatives of quinic acid were found to be inhibitors of human DNA polymerases. Among them, 3,4,5-tri-O-galloylquinic acid (TGQA) was the most potent inhibitor of DNA polymerase alpha. Under identical conditions, this compound was 60-fold more potent than aphidicolin as an inhibitor of DNA polymerase alpha. The inhibition of DNA polymerase alpha by this compound was not competitive with either the template or any of the deoxynucleoside triphosphates with a Ki of 0.28 microM. Under similar reaction conditions, DNA polymerases beta and gamma were much less sensitive to the effects of these compounds and, in contrast to the effect seen with DNA polymerase alpha, the inhibition of DNA polymerases beta and gamma by TGQA was competitive with respect to the template with Ki values of 44.4 and 7.5 microM respectively. The potency of these compounds against DNA polymerase gamma varied according to the assay conditions used. The inhibition of DNA polymerase gamma by TGQA could be increased substantially by using MnCl2 in place of MgCl2 and by including 50 mM potassium phosphate, pH 7.5, in the assay mixture. DNA polymerase beta was also more sensitive to TGQA when measured with MnCl2. However, potassium phosphate had little, if any, effect on the inhibition by TGQA of either DNA polymerase alpha or beta. DNA polymerase alpha was less sensitive to TGQA when assayed with MnCl2. TGQA was not a potent inhibitor of human KB cell growth in culture, which could be due to its degradation or poor uptake. Nevertheless, this compound could serve as a model for developing antitumor drugs targeted at DNA polymerases.

  9. Body fluid identification by integrated analysis of DNA methylation and body fluid-specific microbial DNA.

    PubMed

    Choi, Ajin; Shin, Kyoung-Jin; Yang, Woo Ick; Lee, Hwan Young

    2014-01-01

    Identification of body fluids found at crime scenes provides important information that can support a link between sample donors and actual criminal acts. Previous studies have reported that DNA methylation analysis at several tissue-specific differentially methylated regions (tDMRs) enables successful identification of semen, and the detection of certain bacterial DNA can allow for identification of saliva and vaginal fluid. In the present study, a method for detecting bacterial DNA was integrated into a previously reported multiplex methylation-sensitive restriction enzyme-polymerase chain reaction. The developed multiplex PCR was modified by the addition of a new semen-specific marker and by including amplicons for the 16S ribosomal RNA gene of saliva- and vaginal fluid-specific bacteria to improve the efficacy to detect a specific type of body fluid. Using the developed multiplex system, semen was distinguishable by unmethylation at the USP49, DACT1, and PFN3 tDMRs and by hypermethylation at L81528, and saliva could be identified by detection of saliva-specific bacteria, Veillonella atypica and/or Streptococcus salivarius. Additionally, vaginal fluid and menstrual blood were differentiated from other body fluids by hypomethylation at the PFN3 tDMR and the presence of vaginal fluid-specific bacteria, Lactobacillus crispatus and/or Lactobacillus gasseri. Because the developed multiplex system uses the same biological source of DNA for individual identification profiling and simultaneously analyses various types of body fluid in one PCR reaction, this method will facilitate more efficient body fluid identification in forensic casework.

  10. Molecular cloning of a cDNA encoding a novel fatty acid-binding protein from rat skin.

    PubMed

    Watanabe, R; Fujii, H; Odani, S; Sakakibara, J; Yamamoto, A; Ito, M; Ono, T

    1994-04-15

    A novel skin-type fatty acid-binding protein, termed cutaneous(C)-FABP, has been purified from rat skin and a cDNA clone for this protein has been identified. The purified protein had the ability to bind long chain fatty acids like other rat FABPs. The deduced amino acid sequence of the cDNA clone comprises residues yielding a molecular mass for the polypeptide of 15.1 kDa and exhibits around 50% identity to myelin P2 protein, adipocyte FABP and heart FABP. Our results propose that C-FABP is a new member of the FABP family.

  11. Functional analysis of abscisic acid 8'-hydroxylase.

    PubMed

    Endo, Akira; Kimura, Mitsuhiro; Kawakami, Naoto; Nambara, Eiji

    2011-01-01

    Abscisic acid (ABA) plays an important role in the control of seed dormancy and germination. Identification of hormone metabolism genes from a particular plant species of interest is an essential step in hormone research. The function of these gene products is validated by biochemical analysis using heterologous expression systems, such as E. coli and yeast. ABA 8'-hydroxylase is a subfamily of P450 monooxygenases and is encoded by CYP707A genes. CYP707A catalyzes the committed step in the major ABA catabolic pathway. In this chapter, we describe the methods for RNA extraction from seeds, cloning the CYP707A cDNAs, protein expression in yeast, and biochemical analysis of their gene products.

  12. Electrochemical Interrogation of Kinetically-Controlled Dendritic DNA/PNA Assembly for Immobilization-Free and Enzyme-Free Nucleic Acids Sensing.

    PubMed

    Xuan, Feng; Fan, Tsz Wing; Hsing, I-Ming

    2015-05-26

    We present an immobilization-free and enzyme-free electrochemical nucleic acid sensing strategy, which uses kinetically controlled dendritic assembly of DNA and peptide nucleic acid (PNA). In the presence of a target sequence, ferrocene-labeled PNA probes (Fc-PNAs) and specially designed DNA strands are autonomously assembled into dendritic nanostructures through a cascade of toehold-mediated strand displacement reactions. The consumption of freely diffusible Fc-PNAs (neutrally charged), due to incorporation to DNA/PNA dendrimer, results in a significant electrochemical signal reduction of Fc on a negatively charged electrode from which the hyperbranched and negatively charged dendrimer of DNA/PNA would be electrostatically repelled. The cascade-like assembly process and large electrostatic affinity difference between Fc-PNAs and DNA/PNA dendrimer toward the sensing electrode offer a detection limit down to 100 fM and an inherently high specificity for detecting single nucleotide polymorphisms. The target-triggered mechanism was examined by PAGE analysis, and morphologies of the assembled dendrimers were verified by AFM imaging. PMID:25872652

  13. Development of a nucleic Acid extraction procedure for simultaneous recovery of DNA and RNA from diverse microbes in water.

    PubMed

    Hill, Vincent R; Narayanan, Jothikumar; Gallen, Rachel R; Ferdinand, Karen L; Cromeans, Theresa; Vinjé, Jan

    2015-01-01

    Drinking and environmental water samples contain a diverse array of constituents that can interfere with molecular testing techniques, especially when large volumes of water are concentrated to the small volumes needed for effective molecular analysis. In this study, a suite of enteric viruses, bacteria, and protozoan parasites were seeded into concentrated source water and finished drinking water samples, in order to investigate the relative performance of nucleic acid extraction techniques for molecular testing. Real-time PCR and reverse transcription-PCR crossing threshold (CT) values were used as the metrics for evaluating relative performance. Experimental results were used to develop a guanidinium isothiocyanate-based lysis buffer (UNEX buffer) that enabled effective simultaneous extraction and recovery of DNA and RNA from the suite of study microbes. Procedures for bead beating, nucleic acid purification, and PCR facilitation were also developed and integrated in the protocol. The final lysis buffer and sample preparation procedure was found to be effective for a panel of drinking water and source water concentrates when compared to commercial nucleic acid extraction kits. The UNEX buffer-based extraction protocol enabled PCR detection of six study microbes, in 100 L finished water samples from four drinking water treatment facilities, within three CT values (i.e., within 90% difference) of the reagent-grade water control. The results from this study indicate that this newly formulated lysis buffer and sample preparation procedure can be useful for standardized molecular testing of drinking and environmental waters.

  14. Contributions of the TEL-patch amino acid cluster on TPP1 to telomeric DNA synthesis by human telomerase.

    PubMed

    Dalby, Andrew B; Hofr, Ctirad; Cech, Thomas R

    2015-03-27

    Telomere maintenance is a highly coordinated process, and its misregulation is linked to cancer and telomere-shortening syndromes. Recent studies have shown that the TEL-patch--a cluster of amino acids on the surface of the shelterin component TPP1--is necessary for the recruitment of telomerase to the telomere in human cells. However, there has been only basic biochemical analysis of the role of TPP1 in the telomerase recruitment process. Here we develop an in vitro assay to quantitatively measure the contribution of the TEL-patch to telomerase recruitment--binding and extension of the first telomeric repeat. We also demonstrate that the TEL-patch contributes to the translocation step of the telomerase reaction. Finally, our quantitative observations indicate that the TEL-patch stabilizes the association between telomerase and telomeric DNA substrates, providing a molecular explanation for its contributions to telomerase recruitment and action.

  15. Complete VAX/VMS DNA/protein sequence analysis system

    SciTech Connect

    Smith, D.W.

    1987-05-01

    A complete yet flexible system of programs and database libraries for analysis of DNA, RNA and protein sequences is implemented for VAX/VMS computers. Types of analysis include 1) construction and analysis of chimeric sequences (cloning in the VAX), 2) multiple analysis of one or more single sequences, 3) search and comparison studies using sequence libraries, and 4) direct input and analysis of experimental data. Published groups of programs, including the Staden, Los Alamos, Zuker, Pearson, and PHYLIP programs, are used. GenBank and EMBL DNA libraries and PIR and Doolittle NEWAT protein libraries are available, with associated programs. The system is tutorial, with online documentation for relevent VAX software, the programs, and the databases. The complete documentation is flexibly maintained on reserve via computer printout placed in 3-ring binders. Command files are used extensively; porting of the entire system to another VAX/VMS system requires modification of a single command. Users of the system are members of a VAX group, with automatic implementation of the system upon login. The present system occupies about 140,000 blocks, and is easily expanded, or contracted, as desired. The UCSD system is used extensively for both teaching and research purposes. Use of microcomputers emulating Tektronix 4014 graphics terminals permits saving of graphics output to disk for subsequent modification to generate high quality publishable figures.

  16. Single molecule analysis of Trypanosoma brucei DNA replication dynamics.

    PubMed

    Calderano, Simone Guedes; Drosopoulos, William C; Quaresma, Marina Mônaco; Marques, Catarina A; Kosiyatrakul, Settapong; McCulloch, Richard; Schildkraut, Carl L; Elias, Maria Carolina

    2015-03-11

    Eukaryotic genome duplication relies on origins of replication, distributed over multiple chromosomes, to initiate DNA replication. A recent genome-wide analysis of Trypanosoma brucei, the etiological agent of sleeping sickness, localized its replication origins to the boundaries of multigenic transcription units. To better understand genomic replication in this organism, we examined replication by single molecule analysis of replicated DNA. We determined the average speed of replication forks of procyclic and bloodstream form cells and we found that T. brucei DNA replication rate is similar to rates seen in other eukaryotes. We also analyzed the replication dynamics of a central region of chromosome 1 in procyclic forms. We present evidence for replication terminating within the central part of the chromosome and thus emanating from both sides, suggesting a previously unmapped origin toward the 5' extremity of chromosome 1. Also, termination is not at a fixed location in chromosome 1, but is rather variable. Importantly, we found a replication origin located near an ORC1/CDC6 binding site that is detected after replicative stress induced by hydroxyurea treatment, suggesting it may be a dormant origin activated in response to replicative stress. Collectively, our findings support the existence of more replication origins in T. brucei than previously appreciated. PMID:25690894

  17. Single molecule analysis of Trypanosoma brucei DNA replication dynamics

    PubMed Central

    Calderano, Simone Guedes; Drosopoulos, William C.; Quaresma, Marina Mônaco; Marques, Catarina A.; Kosiyatrakul, Settapong; McCulloch, Richard; Schildkraut, Carl L.; Elias, Maria Carolina

    2015-01-01

    Eukaryotic genome duplication relies on origins of replication, distributed over multiple chromosomes, to initiate DNA replication. A recent genome-wide analysis of Trypanosoma brucei, the etiological agent of sleeping sickness, localized its replication origins to the boundaries of multigenic transcription units. To better understand genomic replication in this organism, we examined replication by single molecule analysis of replicated DNA. We determined the average speed of replication forks of procyclic and bloodstream form cells and we found that T. brucei DNA replication rate is similar to rates seen in other eukaryotes. We also analyzed the replication dynamics of a central region of chromosome 1 in procyclic forms. We present evidence for replication terminating within the central part of the chromosome and thus emanating from both sides, suggesting a previously unmapped origin toward the 5′ extremity of chromosome 1. Also, termination is not at a fixed location in chromosome 1, but is rather variable. Importantly, we found a replication origin located near an ORC1/CDC6 binding site that is detected after replicative stress induced by hydroxyurea treatment, suggesting it may be a dormant origin activated in response to replicative stress. Collectively, our findings support the existence of more replication origins in T. brucei than previously appreciated. PMID:25690894

  18. MitoAge: a database for comparative analysis of mitochondrial DNA, with a special focus on animal longevity

    PubMed Central

    Toren, Dmitri; Barzilay, Thomer; Tacutu, Robi; Lehmann, Gilad; Muradian, Khachik K.; Fraifeld, Vadim E.

    2016-01-01

    Mitochondria are the only organelles in the animal cells that have their own genome. Due to a key role in energy production, generation of damaging factors (ROS, heat), and apoptosis, mitochondria and mtDNA in particular have long been considered one of the major players in the mechanisms of aging, longevity and age-related diseases. The rapidly increasing number of species with fully sequenced mtDNA, together with accumulated data on longevity records, provides a new fascinating basis for comparative analysis of the links between mtDNA features and animal longevity. To facilitate such analyses and to support the scientific community in carrying these out, we developed the MitoAge database containing calculated mtDNA compositional features of the entire mitochondrial genome, mtDNA coding (tRNA, rRNA, protein-coding genes) and non-coding (D-loop) regions, and codon usage/amino acids frequency for each protein-coding gene. MitoAge includes 922 species with fully sequenced mtDNA and maximum lifespan records. The database is available through the MitoAge website (www.mitoage.org or www.mitoage.info), which provides the necessary tools for searching, browsing, comparing and downloading the data sets of interest for selected taxonomic groups across the Kingdom Animalia. The MitoAge website assists in statistical analysis of different features of the mtDNA and their correlative links to longevity. PMID:26590258

  19. Theoretical study of the adsorption of DNA bases on the acidic external surface of montmorillonite.

    PubMed

    Mignon, Pierre; Sodupe, Mariona

    2012-01-14

    In the present study, DFT periodic plane wave calculations, at the PBE-D level of theory, were carried out to investigate the interaction of DNA nucleobases with acidic montmorillonite. The surface model was considered in its octahedral (Osub) and tetrahedral (Tsub) substituted forms, known to have different acidic properties. The adsorption of adenine, guanine and cytosine was considered in both orthogonal and coplanar orientations with the surface, interacting with the proton via a given heteroatom. In almost all considered cases, adsorption involved the spontaneous proton transfer to the nucleobase, with a more pronounced character in the Osub structures. The binding energy is about 10 kcal mol(-1) larger for Osub than for Tsub complexes mainly due to the larger acidity in Osub surfaces and due to the better stabilization by H-bond contacts between the negatively charged surface and the protonated base. The binding energy of coplanar orientations of the base is observed to be as large as the orthogonal ones due to a balance between electrostatic and dispersion contributions. Finally the binding of guanine and adenine on the acidic surface amounts to 50 kcal mol(-1) while that of cytosine rises to 44 kcal mol(-1).

  20. Using Willie's Acid-Base Box for Blood Gas Analysis

    ERIC Educational Resources Information Center

    Dietz, John R.

    2011-01-01

    In this article, the author describes a method developed by Dr. William T. Lipscomb for teaching blood gas analysis of acid-base status and provides three examples using Willie's acid-base box. Willie's acid-base box is constructed using three of the parameters of standard arterial blood gas analysis: (1) pH; (2) bicarbonate; and (3) CO[subscript…

  1. Porous hyaluronic acid hydrogels for localized nonviral DNA delivery in a diabetic wound healing model.

    PubMed

    Tokatlian, Talar; Cam, Cynthia; Segura, Tatiana

    2015-05-01

    The treatment of impaired wounds requires the use of biomaterials that can provide mechanical and biological queues to the surrounding environment to promote angiogenesis, granulation tissue formation, and wound closure. Porous hydrogels show promotion of angiogenesis, even in the absence of proangiogenic factors. It is hypothesized that the added delivery of nonviral DNA encoding for proangiogenic growth factors can further enhance this effect. Here, 100 and 60 μm porous and nonporous (n-pore) hyaluronic acid-MMP hydrogels with encapsulated reporter (pGFPluc) or proangiogenic (pVEGF) plasmids are used to investigate scaffold-mediated gene delivery for local gene therapy in a diabetic wound healing mouse model. Porous hydrogels allow for significantly faster wound closure compared with n-pore hydrogels, which do not degrade and essentially provide a mechanical barrier to closure. Interestingly, the delivery of pDNA/PEI polyplexes positively promotes granulation tissue formation even when the DNA does not encode for an angiogenic protein. And although transfected cells are present throughout the granulation tissue surrounding, all hydrogels at 2 weeks, pVEGF delivery does not further enhance the angiogenic response. Despite this, the presence of transfected cells shows promise for the use of polyplex-loaded porous hydrogels for local gene delivery in the treatment of diabetic wounds.

  2. DNA binding mode of novel tetradentate amino acid based 2-hydroxybenzylidene-4-aminoantipyrine complexes

    NASA Astrophysics Data System (ADS)

    Raman, N.; Sobha, S.; Selvaganapathy, M.; Mahalakshmi, R.

    2012-10-01

    Few transition metal complexes of tetradentate N2O2 donor Schiff base ligands containing 2-hydroxybenzylidene-4-aminoantipyrine and amino acids (alanine/valine) abbreviated to KHL1/KHL2 have been synthesized. All the metal complexes have been fully characterized with the help of elemental analyses, molecular weights, molar conductance values, magnetic moments and spectroscopic data. The Schiff bases KHL1/KHL2 are found to act as tetradentate ligands using N2O2 donor set of atoms leading to a square-planar geometry for the complexes around the metal ions. The binding behaviors of the complexes to calf thymus DNA have been investigated by absorption spectra, viscosity measurements and cyclic voltammetry. The DNA binding constants reveal that all these complexes interact with DNA through minor groove binding mode. The studies on mechanism of photocleavage reveal that singlet oxygen (1O2) and superoxide anion radical (O2rad -) may play an important role in the photocleavage. The Schiff bases and their metal complexes have been screened for their in vitro antibacterial activities against Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Staphylococcus epidermidis, Klebsiella pneumoniae and antifungal activities against Aspergillus niger, Fusarium solani, Culvularia lunata, Rhizoctonia bataicola and Candida albicans by MIC method.

  3. DNA binding mode of novel tetradentate amino acid based 2-hydroxybenzylidene-4-aminoantipyrine complexes.

    PubMed

    Raman, N; Sobha, S; Selvaganapathy, M; Mahalakshmi, R

    2012-10-01

    Few transition metal complexes of tetradentate N(2)O(2) donor Schiff base ligands containing 2-hydroxybenzylidene-4-aminoantipyrine and amino acids (alanine/valine) abbreviated to KHL(1)/KHL(2) have been synthesized. All the metal complexes have been fully characterized with the help of elemental analyses, molecular weights, molar conductance values, magnetic moments and spectroscopic data. The Schiff bases KHL(1)/KHL(2) are found to act as tetradentate ligands using N(2)O(2) donor set of atoms leading to a square-planar geometry for the complexes around the metal ions. The binding behaviors of the complexes to calf thymus DNA have been investigated by absorption spectra, viscosity measurements and cyclic voltammetry. The DNA binding constants reveal that all these complexes interact with DNA through minor groove binding mode. The studies on mechanism of photocleavage reveal that singlet oxygen ((1)O(2)) and superoxide anion radical (O(2)(-)) may play an important role in the photocleavage. The Schiff bases and their metal complexes have been screened for their in vitro antibacterial activities against Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Staphylococcus epidermidis, Klebsiella pneumoniae and antifungal activities against Aspergillus niger, Fusarium solani, Culvularia lunata, Rhizoctonia bataicola and Candida albicans by MIC method. PMID:22885083

  4. Enzymatic amplification of DNA/RNA hybrid molecular beacon signaling in nucleic acid detection.

    PubMed

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

    2013-01-15

    A rapid assay operable under isothermal or nonisothermal conditions is described, where the sensitivity of a typical molecular beacon (MB) system is improved by using thermostable RNase H to enzymatically cleave an MB composed of a DNA stem and an RNA loop (R/D-MB). On hybridization of the R/D-MB to target DNA, there was a modest increase in fluorescence intensity (~5.7× above background) due to an opening of the probe and a concomitant reduction in the Förster resonance energy transfer efficiency. The addition of thermostable RNase H resulted in the cleavage of the RNA loop, which eliminated energy transfer. The cleavage step also released bound target DNA, enabling it to bind to another R/D-MB probe and rendering the approach a cyclic amplification scheme. Full processing of R/D-MBs maximized the fluorescence signal to the fullest extent possible (12.9× above background), resulting in an approximately 2- to 2.8-fold increase in the signal-to-noise ratio observed isothermally at 50 °C following the addition of RNase H. The probe was also used to monitor real-time polymerase chain reactions by measuring enhancement of donor fluorescence on R/D-MB binding to amplified pUC19 template dilutions. Hence, the R/D-MB-RNase H scheme can be applied to a broad range of nucleic acid amplification methods.

  5. Entropy Beacon: A Hairpin-Free DNA Amplification Strategy for Efficient Detection of Nucleic Acids.

    PubMed

    Lv, Yifan; Cui, Liang; Peng, Ruizi; Zhao, Zilong; Qiu, Liping; Chen, Huapei; Jin, Cheng; Zhang, Xiao-Bing; Tan, Weihong

    2015-12-01

    Here, we propose an efficient strategy for enzyme- and hairpin-free nucleic acid detection called an entropy beacon (abbreviated as Ebeacon). Different from previously reported DNA hybridization/displacement-based strategies, Ebeacon is driven forward by increases in the entropy of the system, instead of free energy released from new base-pair formation. Ebeacon shows high sensitivity, with a detection limit of 5 pM target DNA in buffer and 50 pM in cellular homogenate. Ebeacon also benefits from the hairpin-free amplification strategy and zero-background, excellent thermostability from 20 °C to 50 °C, as well as good resistance to complex environments. In particular, based on the huge difference between the breathing rate of a single base pair and two adjacent base pairs, Ebeacon also shows high selectivity toward base mutations, such as substitution, insertion, and deletion and, therefore, is an efficient nucleic acid detection method, comparable to most reported enzyme-free strategies.

  6. In vitro study of DNA damage induced by acid orange 52 and its biodegradation derivatives.

    PubMed

    Ben Mansour, Hedi; Barillier, Daniel; Corroler, David; Ghedira, Kamel; Chekir-Ghedira, Leila; Mosrati, Ridha

    2009-03-01

    Mutagenicity of acid orange 52 (AO52) and its degradation products by Pseudomonas putida mt-2 was evaluated with the use of Salmonella Typhimurium TA102 and TA104 with and without the metabolic activation system (S9). No mutagenicity was observed in the absence of S9 and in the presence of S9 for biodegradation under shaking conditions, but it increased significantly in the presence of S9 after biodegradation under static conditions. In addition, the ability of tested compounds to induce DNA damage in vitro was evaluated with the DNA strand scission assay. The toxicity generated by the pure azo dye and the corresponding azoreduction products (4-aminobenzenesulfonic acid and N,N'-dimethyl-p-phenylenediamine) were compared. We suggest that the mutagenicity mechanism of these molecules occurs through free radical generation processes. In this study, we demonstrate that P. putida mt-2 incubated under aerobic conditions undergoes catabolism that enables it to degrade AO52 completely and, especially, to detoxify the dye mixtures.

  7. Arginine as a general acid catalyst in serine recombinase-mediated DNA cleavage.

    PubMed

    Keenholtz, Ross A; Mouw, Kent W; Boocock, Martin R; Li, Nan-Sheng; Piccirilli, Joseph A; Rice, Phoebe A

    2013-10-01

    Members of the serine family of site-specific DNA recombinases use an unusual constellation of amino acids to catalyze the formation and resolution of a covalent protein-DNA intermediate. A recent high resolution structure of the catalytic domain of Sin, a particularly well characterized family member, provided a detailed view of the catalytic site. To determine how the enzyme might protonate and stabilize the 3'O leaving group in the strand cleavage reaction, we examined how replacing this oxygen with a sulfur affected the cleavage rate by WT and mutant enzymes. To facilitate direct comparison of the cleavage rates, key experiments used suicide substrates that prevented religation after cleavage. The catalytic defect associated with mutation of one of six highly conserved arginine residues, Arg-69 in Sin, was partially rescued by a 3' phosphorothiolate substrate. We conclude that Arg-69 has an important role in stabilizing the 3'O leaving group and is the prime candidate for the general acid that protonates the 3'O, in good agreement with the position it occupies in the high resolution structure of the active site of Sin.

  8. DNA microarray data and contextual analysis of correlation graphs

    PubMed Central

    Rougemont, Jacques; Hingamp, Pascal

    2003-01-01

    Background DNA microarrays are used to produce large sets of expression measurements from which specific biological information is sought. Their analysis requires efficient and reliable algorithms for dimensional reduction, classification and annotation. Results We study networks of co-expressed genes obtained from DNA microarray experiments. The mathematical concept of curvature on graphs is used to group genes or samples into clusters to which relevant gene or sample annotations are automatically assigned. Application to publicly available yeast and human lymphoma data demonstrates the reliability of the method in spite of its simplicity, especially with respect to the small number of parameters involved. Conclusions We provide a method for automatically determining relevant gene clusters among the many genes monitored with microarrays. The automatic annotations and the graphical interface improve the readability of the data. A C++ implementation, called Trixy, is available from . PMID:12720549

  9. Analysis of DNA methylation in pancreatic cancer: an update.

    PubMed

    Pilarsky, Christian; Grützmann, Robert

    2015-01-01

    Pancreatic ductal adenocarcinoma (PDAC) is an aggressive tumor and the fourth common cause of cancer death in the Western world. The lack of effective therapeutic strategies is due to the late diagnosis of this disease. Methylation markers could improve early detection and help in the surveillance of PDAC after treatment. Analysis of hypermethylation in the tumor tissue might help to identify new therapeutic strategies and aid in the understanding of the pathophysiological changes occurring in pancreatic cancer. There are several methods for the detection of methylated events, but methylation-specific PCR (MSP-PCR) is the method of choice if a small number of genes will be tested in a larger set of patients samples. After isolation of the DNA by standard procedure, the DNA is then modified using sodium bisulfide. PMID:25421660

  10. Cladistic analysis of iridoviruses based on protein and DNA sequences.

    PubMed

    Wang, J W; Deng, R Q; Wang, X Z; Huang, Y S; Xing, K; Feng, J H; He, J G; Long, Q X

    2003-11-01

    Cladograms of iridoviruses were inferred from bootstrap analysis of molecular data sets comprising all published protein and DNA sequences of the major capsid protein, ATPase and DNA polymerase genes of members of the Iridoviridae family Iridovirus. All data sets yielded cladograms supporting the separation of the Iridovirus, Ranavirus and Lymphocystivirus genera, and the cladogram based on data derived from major capsid proteins further divided both the Iridovirus and Ranavirus genera into two groups. Tests of alternative hypotheses of topological constraints were also performed to further investigate relationships between infectious spleen and kidney necrosis virus (ISKNV), an unclassified fish iridovirus for which the complete genome sequence data is available, and other iridoviruses. Cladograms inferred and results of Shimodaira-Hasegawa tests indicated that ISKNV is more closely related to the Ranavirus genus than it is to the other genera of the family.

  11. Role of retinoic acid in the modulation of benzo(a)pyrene-DNA adducts in human hepatoma cells: Implications for cancer prevention

    SciTech Connect

    Zhou Guodong; Richardson, Molly; Fazili, Inayat S.; Wang, Jianbo; Donnelly, Kirby C.; Wang Fen; Amendt, Brad; Moorthy, Bhagavatula

    2010-12-15

    Carcinogen-DNA adducts could lead to mutations in critical genes, eventually resulting in cancer. Many studies have shown that retinoic acid (RA) plays an important role in inducing cell apoptosis. Here we have tested the hypothesis that levels of carcinogen-DNA adducts can be diminished by DNA repair and/or by eliminating damaged cells through apoptosis. Our results showed that the levels of total DNA adducts in HepG2 cells treated with benzo(a)pyrene (BP, 2 {mu}M) + RA (1 {mu}M) were significantly reduced compared to those treated with BP only (P = 0.038). In order to understand the mechanism of attenuation of DNA adducts, further experiments were performed. Cells were treated with BP (4 {mu}M) for 24 h to initiate DNA adduct formation, following which the medium containing BP was removed, and fresh medium containing 1 {mu}M RA was added. The cells were harvested 24 h after RA treatment. Interestingly, the levels of total DNA adducts were lower in the BP/RA group (390 {+-} 34) than those in the BP/DMSO group (544 {+-} 33), P = 0.032. Analysis of cell apoptosis showed an increase in BP + RA group, compared to BP or RA only groups. Our results also indicated that attenuation of BP-DNA adducts by RA was not primarily due to its effects on CYP1A1 expression. In conclusion, our results suggest a mechanistic link between cellular apoptosis and DNA adduct formation, phenomena that play important roles in BP-mediated carcinogenesis. Furthermore, these results help understand the mechanisms of carcinogenesis, especially in relation to the chemopreventive properties of nutritional apoptosis inducers.

  12. Simultaneous Non-invasive Analysis of DNA Condensation and Stability by Two-step QD-FRET

    PubMed Central

    Chen, Hunter H.; Ho, Yi-Ping; Jiang, Xuan; Mao, Hai-Quan; Wang, Tza-Huei; Leong, Kam W.

    2009-01-01

    Summary Nanoscale vectors comprised of cationic polymers that condense DNA to form nanocomplexes are promising options for gene transfer. The rational design of more efficient nonviral gene carriers will be possible only with better mechanistic understanding of the critical rate-limiting steps, such as nanocomplex unpacking to release DNA and degradation by nucleases. We present a two-step quantum dot fluorescence resonance energy transfer (two-step QD-FRET) approach to simultaneously and non-invasively analyze DNA condensation and stability. Plasmid DNA, double-labeled with QD (525 nm emission) and nucleic acid dyes, were complexed with Cy5-labeled cationic gene carriers. The QD donor drives energy transfer stepwise through the intermediate nucleic acid dye to the final acceptor Cy5. At least three distinct states of DNA condensation and integrity were distinguished in single particle manner and within cells by quantitative ratiometric analysis of energy transfer efficiencies. This novel two-step QD-FRET method allows for more detailed assessment of the onset of DNA release and degradation simultaneously. PMID:20161048

  13. Antibacterial activity of lichen secondary metabolite usnic acid is primarily caused by inhibition of RNA and DNA synthesis.

    PubMed

    Maciąg-Dorszyńska, Monika; Węgrzyn, Grzegorz; Guzow-Krzemińska, Beata

    2014-04-01

    Usnic acid, a compound produced by various lichen species, has been demonstrated previously to inhibit growth of different bacteria and fungi; however, mechanism of its antimicrobial activity remained unknown. In this report, we demonstrate that usnic acid causes rapid and strong inhibition of RNA and DNA synthesis in Gram-positive bacteria, represented by Bacillus subtilis and Staphylococcus aureus, while it does not inhibit production of macromolecules (DNA, RNA, and proteins) in Escherichia coli, which is resistant to even high doses of this compound. However, we also observed slight inhibition of RNA synthesis in a Gram-negative bacterium, Vibrio harveyi. Inhibition of protein synthesis in B. subtilis and S. aureus was delayed, which suggest indirect action (possibly through impairment of transcription) of usnic acid on translation. Interestingly, DNA synthesis was halted rapidly in B. subtilis and S. aureus, suggesting interference of usnic acid with elongation of DNA replication. We propose that inhibition of RNA synthesis may be a general mechanism of antibacterial action of usnic acid, with additional direct mechanisms, such as impairment of DNA replication in B. subtilis and S. aureus.

  14. Seed response to strigolactone is controlled by abscisic acid-independent DNA methylation in the obligate root parasitic plant, Phelipanche ramosa L. Pomel.

    PubMed

    Lechat, Marc-Marie; Brun, Guillaume; Montiel, Grégory; Véronési, Christophe; Simier, Philippe; Thoiron, Séverine; Pouvreau, Jean-Bernard; Delavault, Philippe

    2015-06-01

    Seed dormancy release of the obligate root parasitic plant, Phelipanche ramosa, requires a minimum 4-day conditioning period followed by stimulation by host-derived germination stimulants, such as strigolactones. Germination is then mediated by germination stimulant-dependent activation of PrCYP707A1, an abscisic acid catabolic gene. The molecular mechanisms occurring during the conditioning period that silence PrCYP707A1 expression and regulate germination stimulant response are almost unknown. Here, global DNA methylation quantification associated with pharmacological approaches and cytosine methylation analysis of the PrCYP707A1 promoter were used to investigate the modulation and possible role of DNA methylation during the conditioning period and in the PrCYP707A1 response to GR24, a synthetic strigolactone analogue. Active global DNA demethylation occurs during the conditioning period and is required for PrCYP707A1 activation by GR24 and for subsequent seed germination. Treatment with 5-azacytidine, a DNA-hypomethylating molecule, reduces the length of the conditioning period. Conversely, hydroxyurea, a hypermethylating agent, inhibits PrCYP707A1 expression and seed germination. Methylated DNA immunoprecipitation followed by PCR experiments and bisulfite sequencing revealed that DNA demethylation particularly impacts a 78-nucleotide sequence in the PrCYP707A1 promoter. The results here demonstrate that the DNA methylation status during the conditioning period plays a crucial role independently of abscisic acid in the regulation of P. ramosa seed germination by controlling the strigolactone-dependent expression of PrCYP707A1.

  15. Seed response to strigolactone is controlled by abscisic acid-independent DNA methylation in the obligate root parasitic plant, Phelipanche ramosa L. Pomel

    PubMed Central

    Lechat, Marc-Marie; Brun, Guillaume; Montiel, Grégory; Véronési, Christophe; Simier, Philippe; Thoiron, Séverine; Pouvreau, Jean-Bernard; Delavault, Philippe

    2015-01-01

    Seed dormancy release of the obligate root parasitic plant, Phelipanche ramosa, requires a minimum 4-day conditioning period followed by stimulation by host-derived germination stimulants, such as strigolactones. Germination is then mediated by germination stimulant-dependent activation of PrCYP707A1, an abscisic acid catabolic gene. The molecular mechanisms occurring during the conditioning period that silence PrCYP707A1 expression and regulate germination stimulant response are almost unknown. Here, global DNA methylation quantification associated with pharmacological approaches and cytosine methylation analysis of the PrCYP707A1 promoter were used to investigate the modulation and possible role of DNA methylation during the conditioning period and in the PrCYP707A1 response to GR24, a synthetic strigolactone analogue. Active global DNA demethylation occurs during the conditioning period and is required for PrCYP707A1 activation by GR24 and for subsequent seed germination. Treatment with 5-azacytidine, a DNA-hypomethylating molecule, reduces the length of the conditioning period. Conversely, hydroxyurea, a hypermethylating agent, inhibits PrCYP707A1 expression and seed germination. Methylated DNA immunoprecipitation followed by PCR experiments and bisulfite sequencing revealed that DNA demethylation particularly impacts a 78-nucleotide sequence in the PrCYP707A1 promoter. The results here demonstrate that the DNA methylation status during the conditioning period plays a crucial role independently of abscisic acid in the regulation of P. ramosa seed germination by controlling the strigolactone-dependent expression of PrCYP707A1. PMID:25821070

  16. Effective DNA binding and cleaving tendencies of malonic acid coupled transition metal complexes

    NASA Astrophysics Data System (ADS)

    Pravin, Narayanaperumal; Utthra, Ponnukalai Ponya; Kumaravel, Ganesan; Raman, Natarajan

    2016-11-01

    Eight transition metal complexes were designed to achieve maximum biological efficacy. They were characterized by elemental analysis and various other spectroscopic techniques. The monomeric complexes were found to espouse octahedral geometry and non-electrolytic nature. The DNA interaction propensity of the complexes with calf thymus DNA (CT-DNA), studied at physiological pH by spectrophotometric, spectrofluorometric, cyclic voltammetry, and viscometric techniques revealed intercalation as the possible binding mode. Fascinatingly, the complexes were found to exhibit greater binding strength than that of the free ligands. A strong hypochromism and a slight red shift were exhibited by complex 5 among the other complexes. The intrinsic binding constant values of all the complexes compared to cisplatin reveal that they are excellent metallonucleases than that of cisplatin. The complexes were also shown to reveal displacement of the ethidium bromide, a strong intercalator using fluorescence titrations. Gel electrophoresis was used to divulge the competence of the complexes in cleaving the supercoiled pBR322 plasmid DNA. From the results, it is concluded that the complexes, especially 5, are excellent chemical nucleases in the presence of H2O2. Furthermore, the in vitro antimicrobial screening of the complexes exposes that these complexes are excellent antimicrobial agents. Overall the effect of coligands is evident from the results of all the investigations.

  17. Folic acid, polymorphism of methyl-group metabolism genes, and DNA methylation in relation to GI carcinogenesis.

    PubMed

    Fang, Jing Yuan; Xiao, Shu Dong

    2003-01-01

    DNA methylation is the main epigenetic modification after replication in humans. DNA (cytosine-5)-methyltransferase (DNMT) catalyzes the transfer of a methyl group from S-adenosyl-L-methionine (SAM) to C5 of cytosine within CpG dinucleotide sequences in the genomic DNA of higher eukaryotes. There is considerable evidence that aberrant DNA methylation plays an integral role in carcinogenesis. Folic acid or folate is crucial for normal DNA synthesis and can regulate DNA methylation, and through this, it affects cellular SAM levels. Folate deficiency results in DNA hypomethylation. Epidemiological studies have indicated that folic acid protects against gastrointestinal (GI) cancers. Methylene-tetrahydrofolate reductase (MTHFR) and methionine synthase (MS) are the enzymes involved in folate metabolism and are thought to influence DNA methylation. MTHFR is highly polymorphic, and the variant genotypes result in decreased MTHFR enzyme activity and lower plasma folate level. Two common MTHFR polymorphisms, 677CT (or 677TT) and A1298C, and an MS polymorphism, A-->G at 2756, have been identified. Most studies support an inverse association between folate status and the rate of colorectal adenomas and carcinomas. During human GI carcinogenesis, MTHFR is highly polymorphic, and the variant genotypes result in decreased MTHFR enzyme activity and lower plasma folate level, as well as aberrant methylation.

  18. A Novel Method for Alignment-free DNA Sequence Similarity Analysis Based on the Characterization of Complex Networks

    PubMed Central

    Zhou, Jie; Zhong, Pianyu; Zhang, Tinghui

    2016-01-01

    Determination of sequence similarity is one of the major steps in computational phylogenetic studies. One of the major tasks of computational biologists is to develop novel mathematical descriptors for similarity analysis. DNA clustering is an important technology that automatically identifies inherent relationships among large-scale DNA sequences. The comparison between the DNA sequences of different species helps determine phylogenetic relationships among species. Alignment-free approaches have continuously gained interest in various sequence analysis applications such as phylogenetic inference and metagenomic classification/clustering, particularly for large-scale sequence datasets. Here, we construct a novel and simple mathematical descriptor based on the characterization of cis sequence complex DNA networks. This new approach is based on a code of three cis nucleotides in a gene that could code for an amino acid. In particular, for each DNA sequence, we will set up a cis sequence complex network that will be used to develop a characterization vector for the analysis of mitochondrial DNA sequence phylogenetic relationships among nine species. The resulting phylogenetic relationships among the nine species were determined to be in agreement with the actual situation. PMID:27746676

  19. Analysis of proteins responsive to acetic acid in Acetobacter: molecular mechanisms conferring acetic acid resistance in acetic acid bacteria.

    PubMed

    Nakano, Shigeru; Fukaya, Masahiro

    2008-06-30

    Acetic acid bacteria are used for industrial vinegar production because of their remarkable ability to oxidize ethanol and high resistance to acetic acid. Although several molecular machineries responsible for acetic acid resistance in acetic acid bacteria have been reported, the entire mechanism that confers acetic acid resistance has not been completely understood. One of the promising methods to elucidate the entire mechanism is global analysis of proteins responsive to acetic acid by two-dimensional gel electrophoresis. Recently, two proteins whose production was greatly enhanced by acetic acid in Acetobacter aceti were identified to be aconitase and a putative ABC-transporter, respectively; furthermore, overexpression or disruption of the genes encoding these proteins affected acetic acid resistance in A. aceti, indicating that these proteins are involved in acetic acid resistance. Overexpression of each gene increased acetic acid resistance in Acetobacter, which resulted in an improvement in the productivity of acetic acid fermentation. Taken together, the results of the proteomic analysis and those of previous studies indicate that acetic acid resistance in acetic acid bacteria is conferred by several mechanisms. These findings also provide a clue to breed a strain having high resistance to acetic acid for vinegar fermentation.

  20. Cloning and sequence analysis of cDNA encoding urotensin I precursor.

    PubMed Central

    Ishida, I; Ichikawa, T; Deguchi, T

    1986-01-01

    The primary structure of the precursor of urotensin I, a neuropeptide hormone from the caudal neurosecretory system of the carp Cyprinus carpio, has been determined by analyzing the nucleotide sequence of cloned DNA complementary to the mRNA encoding it. The precursor consists of 145 amino acid residues and the carboxyl terminus represents the 41-amino acid sequence of urotensin I, preceded by Lys-Arg and followed by Gly-Lys. Sequence homology as well as similar organization of the precursors of urotensin I and mammalian corticotropin-releasing factors suggest that they are evolutionarily related. RNA transfer blot analysis indicates that mRNA encoding the precursor of urotensin I is present only in the spinal cord and not in the brain, intestine, liver, or kidney of the carp. Images PMID:3484550

  1. Comparison of Rapid Methods for Analysis of Bacterial Fatty Acids

    PubMed Central

    Moss, C. Wayne; Lambert, M. A.; Merwin, W. H.

    1974-01-01

    When rapid gas-liquid chromatography methods for determination of bacterial fatty acids were compared, results showed that saponification was required for total fatty acid analysis. Transesterification with boron-trihalide reagents (BF3-CH3OH, BCl3-CH3OH) caused extensive degradation of cyclopropane acids and was less effective than saponification in releasing cellular hydroxy fatty acids. Digestion of cells with tetramethylammonium hydroxide was unsatisfactory because of extraneous gas-liquid chromatography peaks and because of lower recovery of branched-chain and hydroxy fatty acids. A simple, rapid saponification procedure which can be used for total cellular fatty acid analysis of freshly grown cells is described. PMID:4844271

  2. Target-induced self-assembly of DNA nanomachine on magnetic particle for multi-amplified biosensing of nucleic acid, protein, and cancer cell.

    PubMed

    Bi, Sai; Cui, Yangyang; Dong, Ying; Zhang, Ningbo

    2014-03-15

    A biosensing system is established for the multi-amplified detection of DNA or specific substrates of aptamers under isothermal conditions, which combines nicked rolling circle amplification (N-RCA) and beacon assisted amplification (BAA) with sensitive colorimetric technique by using DNAzymes as reporter units. According to the configuration, the analysis of DNA is accomplished by recognizing the target to capture nucleic acid-functionalized magnetic particles, followed by the self-assembly of the other two nucleic acids into multicomponent DNA supramolecular structure on magnetic particles. After magnetic separation, the circularization with ligase and the fragmentation with polymerase activate N-RCA and BAA in the presence of polymerase, dNTPs, and the nicking endonuclease, successively producing horseradish peroxidase (HRP)-mimicking DNAzymes that act as colorimetric reporter to catalyze the oxidation of ABTS(2-) by H2O2 in the presence of hemin. Under the optimized conditions, we obtain a wide dynamic range for DNA analysis over 6 orders of magnitude from 1.0 × 10(-14) to 1.0 × 10(-9)M with a low limit of detection of 6.8 × 10(-15)M. In the absence of a target, neither self-assembly of nucleic acids nor amplification process can be initiated, indicating an excellent selectivity of the proposed strategy. Similarly, an analogous system is activated by cancer cells or lysozyme through cooperative self-assembly of nucleic acids on magnetic particles in the presence of respective substrates of aptamers to synthesize HRP-mimicking DNAzymes that give the readout signal for the recognition events, achieving LODs of 81 Ramos cells and 7.2 × 10(-15)M lysozyme, respectively.

  3. Evaluating the effects of galbanic acid on hydrogen peroxide-induced oxidative DNA damage in human lymphocytes

    PubMed Central

    Shirani, Kobra; Behravan, Javad; Mosaffa, Fatemeh; Iranshahi, Mehrdad; Mehmankhah, Babak; Razavi-Azarkhiavi, Kamal; Karimi, Gholamreza

    2014-01-01

    Objective: Ferula szowitsiana has been widely used for medicinal purposes around the world. The anti-oxidant effect of F. szowitsiana had been proved. The current study aims to determine the protective effects of galbanic acid, a sesquiterpene coumarin from F. szowitsiana, against hydrogen peroxide (H2O2) - induced oxidative DNA damage in human lymphocytes. Materials and Methods: Human lymphocytes were incubated with H2O2 (0, 25, 50, 100, and 200 µM), galbanic acid (200 and 400 µM) and a combination of galbanic acid (200 and 400 µM) and H2O2 (25 µM) at 4 C for 30 minutes. Solvents of galbanic acid without H2O2 were used as negative controls. Results: The findings of this study demonstrated that H2O2 exposure leads to a significant concentration-dependent increase in DNA damage. Galbanic acid did not cause DNA damage compared with the control cells. Data showed that galbanic acid does not have a protective effect against H2O2-induced oxidative DNA damage in human lymphocytes. Conclusion: According to the results, it is concluded that the capability of F. szowitsiana in reducing reactive oxygen species and the anti-inflammatory property of its methanolic extract may be due to its other ingredients. PMID:25386396

  4. Proteogenomic analysis reveals unanticipated adaptations of colorectal tumor cells to deficiencies in DNA mismatch repair

    PubMed Central

    Halvey, Patrick J.; Wang, Xiaojing; Wang, Jing; Bhat, Ajaz A.; Dhawan, Punita; Li, Ming; Zhang, Bing; Liebler, Daniel C.; Slebos, Robbert J.C.

    2014-01-01

    Summary A growing body of genomic data on human cancers poses the critical question of how genomic variations translate to cancer phenotypes. We employed standardized shotgun proteomics and targeted protein quantitation platforms to analyze a panel of 10 colon cancer cell lines differing by mutations in DNA mismatch repair (MMR) genes. In addition, we performed transcriptome sequencing (RNA-seq) to enable detection of protein sequence variants from the proteomic data. Biological replicate cultures yielded highly consistent proteomic inventories with a cumulative total of 6,513 protein groups with a protein FDR of 3.17% across all cell lines. Networks of co-expressed proteins with differential expression based on MMR status revealed impact on protein folding, turnover and transport, on cellular metabolism and on DNA and RNA synthesis and repair. Analysis of variant amino acid sequences suggested higher stability of proteins affected by naturally occurring germline polymorphisms than of proteins affected by somatic protein sequence changes. The data provide evidence for multi-system adaptation to MMR deficiency with a stress response that targets misfolded proteins for degradation through the ubiquitin-dependent proteasome pathway. Enrichment analysis suggested epithelial-to-mesenchymal transition (EMT) in RKO cells, as evidenced by increased mobility and invasion properties compared to SW480. The observed proteomic profiles demonstrate previously unknown consequences of altered DNA repair and provide an expanded basis for mechanistic interpretation of MMR phenotypes. PMID:24247723

  5. Bile acids in combination with low pH induce oxidative stress and oxidative DNA damage: relevance to the pathogenesis of Barrett's oesophagus

    PubMed Central

    Dvorak, Katerina; Payne, Claire M; Chavarria, Melissa; Ramsey, Lois; Dvorakova, Barbora; Bernstein, Harris; Holubec, Hana; Sampliner, Richard E; Guy, Naihsuan; Condon, Amanda; Bernstein, Carol; Green, Sylvan B; Prasad, Anil; Garewal, Harinder S

    2007-01-01

    Background Barrett's oesophagus is a premalignant condition associated with an increased risk for the development of oesophageal adenocarcinoma (ADCA). Previous studies indicated that oxidative damage contributes to the development of ADCA. Objective To test the hypothesis that bile acids and gastric acid, two components of refluxate, can induce oxidative stress and oxidative DNA damage. Methods Oxidative stress was evaluated by staining Barrett's oesophagus tissues with different degrees of dysplasia with 8‐hydroxy‐deoxyguanosine (8‐OH‐dG) antibody. The levels of 8‐OH‐dG were also evaluated ex vivo in Barrett's oesophagus tissues incubated for 10 min with control medium and medium acidified to pH 4 and supplemented with 0.5 mM bile acid cocktail. Furthermore, three oesophageal cell lines (Seg‐1 cells, Barrett's oesophagus cells and HET‐1A cells) were exposed to control media, media containing 0.1 mM bile acid cocktail, media acidified to pH 4, and media at pH 4 supplemented with 0.1 mM bile acid cocktail, and evaluated for induction of reactive oxygen species (ROS). Results Immunohistochemical analysis showed that 8‐OH‐dG is formed mainly in the epithelial cells in dysplastic Barrett's oesophagus. Importantly, incubation of Barrett's oesophagus tissues with the combination of bile acid cocktail and acid leads to increased formation of 8‐OH‐dG. An increase in ROS in oesophageal cells was detected after exposure to pH 4 and bile acid cocktail. Conclusions Oxidative stress and oxidative DNA damage can be induced in oesophageal tissues and cells by short exposures to bile acids and low pH. These alterations may underlie the development of Barrett's oesophagus and tumour progression. PMID:17145738

  6. Channel Size Conversion of Phi29 DNA-Packaging Nanomotor for Discrimination of Single- and Double-Stranded Nucleic Acids

    PubMed Central

    Geng, Jia; Wang, Shaoying; Fang, Huaming; Guo, Peixuan

    2013-01-01

    Nanopores have been utilized to detect the conformation and dynamics of polymers, including DNA and RNA. Biological pores are extremely reproducible at the atomic level with uniform channel sizes. The channel of the bacterial virus phi29 DNA packaging motor is a natural conduit for the transportation of double-stranded DNA (dsDNA), and has the largest diameter among the well-studied biological channels. The larger channel facilitates translocation of dsDNA, and offers more space for further channel modification and conjugation. Interestingly, the relatively large wild type channel, which translocates dsDNA, cannot detect single-stranded nucleic acids (ssDNA or ssRNA) under the current experimental conditions. Herein, we reengineered this motor channel by removing the internal loop segment of the channel. The modification resulted in two classes of channels. One class was the same size as the wild type channel, while the other class had a cross-sectional area about 60% of the wild type. This smaller channel was able to detect the real-time translocation of single stranded nucleic acids at single-molecule level. While the wild type connector exhibited a one-way traffic property with respect to dsDNA translocation, the loop deleted connector was able to translocate ssDNA and ssRNA with equal competencies from both termini. This finding of size alterations in reengineered motor channels expands the potential application of the phi29 DNA packaging motor in nanomedicine, nanobiotechnology, and high-throughput single pore DNA sequencing. PMID:23488809

  7. Flow cytometry reliability analysis and variations in sugarcane DNA content.

    PubMed

    Oliveira, A C L; Pasqual, M; Bruzi, A T; Pio, L A S; Mendonça, P M S; Soares, J D R

    2015-01-01

    The aim of this study was to evaluate the reliability of flow cytometry analysis and the use of this technique to differentiate species and varieties of sugarcane (Saccharum spp) according to their relative DNA content. We analyzed 16 varieties and three species belonging to this genus. To determine a reliable protocol, we evaluated three extraction buffers (LB01, Marie, and Tris·MgCl2), the presence and absence of RNase, six doses of propidium iodide (10, 15, 20, 25, and 30 μg), four periods of exposure to propidium iodide (0, 5, 10, and 20 min), and seven external reference standards (peas, beans, corn, radish, rye, soybean, and tomato) with reference to the coefficient of variation and the DNA content. For statistical analyses, we used the programs Sisvar(®) and Xlstat(®). We recommend using the Marie extraction buffer and at least 15 μg propidium iodide. The samples should not be analyzed immediately after the addition of propidium iodide. The use of RNase is optional, and tomato should be used as an external reference standard. The results show that sugarcane has a variable genome size (8.42 to 12.12 pg/2C) and the individuals analyzed could be separated into four groups according to their DNA content with relative equality in the genome sizes of the commercial varieties.

  8. DNA analysis in the case of Kaspar Hauser.

    PubMed

    Weichhold, G M; Bark, J E; Korte, W; Eisenmenger, W; Sullivan, K M

    1998-01-01

    In 1828 a mysterious young man appeared in Nürnberg, Germany, who was barely able to speak or walk but could write down his name, Kaspar Hauser. He quickly became the centre of social interest but also the victim of intrigue. His appearance, his origin and assassination in 1833 were, and still are, the source of much debate. The most widely accepted theory postulates that Kaspar Hauser was the son of Grand Duke Carl von Baden and his wife Stephanie de Beauharnais, an adopted daughter of Napoleon Bonaparte. To check this theory, DNA analysis was performed on the clothes most likely worn by Kaspar Hauser when he was stabbed on December 14th, 1833. A suitable bloodstain from the underpants was divided and analysed independently by the Institute of Legal Medicine, University of Munich (ILM) and the Forensic Science Service Laboratory, Birmingham (FSS). Mitochondrial DNA (mtDNA) was sequenced from the bloodstain and from blood samples obtained from two living maternal relatives of Stephanie de Beauharnais. The sequence from the bloodstained clothing differed from the sequence found in both reference blood samples at seven confirmed positions. This proves that the bloodstain does not originate from a son of Stephanie de Beauharnais. Thus, it is becoming clear that Kaspar Hauser was not the Prince of Baden.

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

    SciTech Connect

    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.

  10. Vibrational spectroscopy and principal component analysis for conformational study of virus nucleic acids

    NASA Astrophysics Data System (ADS)

    Dovbeshko, G. I.; Repnytska, O. P.; Pererva, T.; Miruta, A.; Kosenkov, D.

    2004-07-01

    Conformation analysis of mutated DNA-bacteriophages (PLys-23, P23-2, P47- the numbers have been assigned by T. Pererva) induced by MS2 virus incorporated in Ecoli AB 259 Hfr 3000 has been done. Surface enhanced infrared absorption (SEIRA) spectroscopy and principal component analysis has been applied for solving this problem. The nucleic acids isolated from the mutated phages had a form of double stranded DNA with different modifications. The nucleic acid from phage P47 was undergone the structural rearrangement in the most degree. The shape and position ofthe fine structure of the Phosphate asymmetrical band at 1071cm-1 as well as the stretching OH vibration at 3370-3390 cm-1 has indicated to the appearance ofadditional OH-groups. The Z-form feature has been found in the base vibration region (1694 cm-1) and the sugar region (932 cm-1). A supposition about modification of structure of DNA by Z-fragments for P47 phage has been proposed. The P23-2 and PLys-23 phages have showed the numerous minor structural changes also. On the basis of SEIRA spectra we have determined the characteristic parameters of the marker bands of nucleic acid used for construction of principal components. Contribution of different spectral parameters of nucleic acids to principal components has been estimated.

  11. Crystal structures of the DNA-binding domain of Escherichia coli proline utilization A flavoprotein and analysis of the role of Lys9 in DNA recognition

    PubMed Central

    Larson, John D.; Jenkins, Jermaine L.; Schuermann, Jonathan P.; Zhou, Yuzhen; Becker, Donald F.; Tanner, John J.

    2006-01-01

    PutA (proline utilization A) from Escherichia coli is a 1320-amino-acid residue protein that is both a bifunctional proline catabolic enzyme and an autogenous transcriptional repressor. Here, we report the first crystal structure of a PutA DNA-binding domain along with functional analysis of a mutant PutA defective in DNA binding. Crystals were grown using a polypeptide corresponding to residues 1–52 of E. coli PutA (PutA52). The 2.1 Å resolution structure of PutA52 mutant Lys9Met was determined using Se-Met MAD phasing, and the structure of native PutA52 was solved at 1.9 Å resolution using molecular replacement. Residues 3–46 form a ribbon–helix–helix (RHH) substructure, thus establishing PutA as the largest protein to contain an RHH domain. The PutA RHH domain forms the intertwined dimer with tightly packed hydrophobic core that is characteristic of the RHH family. The structures were used to examine the three-dimensional context of residues conserved in PutA RHH domains. Homology modeling suggests that Lys9 and Thr5 contact DNA bases through the major groove, while Arg15, Thr28, and His30 may interact with the phosphate backbone. Lys9 is shown to be essential for specific recognition of put control DNA using gel shift analysis of the Lys9Met mutant of full-length PutA. Lys9 is disordered in the PutA52 structure, which implies an induced-fit binding mechanism in which the side chain of Lys9 becomes ordered through interaction with DNA. These results provide new insights into the structural basis of DNA recognition by PutA and reveal three-dimensional structural details of the PutA dimer interface. PMID:17001030

  12. A Δ-9 Fatty Acid Desaturase Gene in the Microalga Myrmecia incisa Reisigl: Cloning and Functional Analysis.

    PubMed

    Xue, Wen-Bin; Liu, Fan; Sun, Zheng; Zhou, Zhi-Gang

    2016-01-01

    The green alga Myrmecia incisa is one of the richest natural sources of arachidonic acid (ArA). To better understand the regulation of ArA biosynthesis in M. incisa, a novel gene putatively encoding the Δ9 fatty acid desaturase (FAD) was cloned and characterized for the first time. Rapid-amplification of cDNA ends (RACE) was employed to yield a full length cDNA designated as MiΔ9FAD, which is 2442 bp long in sequence. Comparing cDNA open reading frame (ORF) sequence to genomic sequence indicated that there are 8 introns interrupting the coding region. The deduced MiΔ9FAD protein is composed of 432 amino acids. It is soluble and localized in the chloroplast, as evidenced by the absence of transmembrane domains as well as the presence of a 61-amino acid chloroplast transit peptide. Multiple sequence alignment of amino acids revealed two conserved histidine-rich motifs, typical for Δ9 acyl-acyl carrier protein (ACP) desaturases. To determine the function of MiΔ9FAD, the gene was heterologously expressed in a Saccharomyces cerevisiae mutant strain with impaired desaturase activity. Results of GC-MS analysis indicated that MiΔ9FAD was able to restore the synthesis of monounsaturated fatty acids, generating palmitoleic acid and oleic acid through the addition of a double bond in the Δ9 position of palmitic acid and stearic acid, respectively. PMID:27438826

  13. A Δ-9 Fatty Acid Desaturase Gene in the Microalga Myrmecia incisa Reisigl: Cloning and Functional Analysis

    PubMed Central

    Xue, Wen-Bin; Liu, Fan; Sun, Zheng; Zhou, Zhi-Gang

    2016-01-01

    The green alga Myrmecia incisa is one of the richest natural sources of arachidonic acid (ArA). To better understand the regulation of ArA biosynthesis in M. incisa, a novel gene putatively encoding the Δ9 fatty acid desaturase (FAD) was cloned and characterized for the first time. Rapid-amplification of cDNA ends (RACE) was employed to yield a full length cDNA designated as MiΔ9FAD, which is 2442 bp long in sequence. Comparing cDNA open reading frame (ORF) sequence to genomic sequence indicated that there are 8 introns interrupting the coding region. The deduced MiΔ9FAD protein is composed of 432 amino acids. It is soluble and localized in the chloroplast, as evidenced by the absence of transmembrane domains as well as the presence of a 61-amino acid chloroplast transit peptide. Multiple sequence alignment of amino acids revealed two conserved histidine-rich motifs, typical for Δ9 acyl-acyl carrier protein (ACP) desaturases. To determine the function of MiΔ9FAD, the gene was heterologously expressed in a Saccharomyces cerevisiae mutant strain with impaired desaturase activity. Results of GC-MS analysis indicated that MiΔ9FAD was able to restore the synthesis of monounsaturated fatty acids, generating palmitoleic acid and oleic acid through the addition of a double bond in the Δ9 position of palmitic acid and stearic acid, respectively. PMID:27438826

  14. Artificial intelligence approach in analysis of DNA sequences.

    PubMed

    Brézillon, P J; Zaraté, P; Saci, F

    1993-01-01

    We present an approach for designing a knowledge-based system, called Sequence Acquisition In Context (SAIC), that will be able to cooperate with a biologist in the analysis of DNA sequences. The main task of the system is the acquisition of the expert knowledge that the biologist uses for solving ambiguities from gel autoradiograms, with the aim of re-using it later for solving similar ambiguities. The various types of expert knowledge constitute what we call the contextual knowledge of the sequence analysis. Contextual knowledge deals with the unavoidable problems that are common in the study of the living material (eg noise on data, difficulties of observations). Indeed, the analysis of DNA sequences from autoradiograms belongs to an emerging and promising area of investigation, namely reasoning with images. The SAIC project is developed in a theoretical framework that is shared with other applications. Not all tasks have the same importance in each application. We use this observation for designing an intelligent assistant system with three applications. In the SAIC project, we focus on knowledge acquisition, human-computer interaction and explanation. The project will benefit research in the two other applications. We also discuss our SAIC project in the context of large international projects that aim to re-use and share knowledge in a repository.

  15. FUNGAL-SPECIFIC PCR PRIMERS DEVELOPED FOR ANALYSIS OF THE ITS REGION OF ENVIRONMENTAL DNA EXTRACTS

    EPA Science Inventory

    Background The Internal Transcribed Spacer (ITS) regions of fungal ribosomal DNA (rDNA) are highly variable sequences of great importance in distinguishing fungal species by PCR analysis. Previously published PCR primers available for amplifying these sequences from environmenta...

  16. Electron microscopic analysis of partially replicated bacteriophage T7 DNA.

    PubMed Central

    Burck, K B; Scraba, D G; Miller, R C

    1979-01-01

    Partially replicated bacteriophage T7 DNA was isolated from Escherichia coli infected with UV-irradiated T7 bacteriophage and was analyzed by electron microscopy. The analysis determined the distribution of eye forms and forks in the partially replicated molecules. Eye forms and forks in unit length molecules were aligned with respect to the left end of the T7 genome, and segments were scored for replication in each molecule. The resulting histogram showed that only the left 25 to 30% of the molecules was replicated. Several different origins of DNA replication were used to initiate replication in the UV-irradiated experiments in which 32P-labeled progeny DNA from UV-irradiated phage was annealed with ordered restriction fragments of T7 DNA (K. B. Burck and R. C. Miller, Jr., Proc. Natl. Acad. Sci. U.S.A. 75:6144--6148, 1978). Both analyses support partial-replica hypotheses (N. A. Barricelli and A. H. Doermann, Virology 13:460--476, 1961; Doermann et al., J. Cell. comp. Physiol. 45[Suppl.]:51--74, 1955) as an explanation for the distribution of marker rescue frequencies during cross-reactivation; i.e., replication proceeds in a bidirectional manner from an origin to a site of UV damage, and those regions of the genome which replicate most efficiently are rescued most efficiently by a coinfecting phage. In addition, photoreactivation studies support the hypothesis that thymine dimers are the major UV damage blocking cross-reactivation in the right end of the T7 genome. Images PMID:291738

  17. Analysis of DNA Methylation in Various Swine Tissues

    PubMed Central

    Niu, Weiping; Yang, Runjun; Zhang, Yonghong; Qiu, Zhengyan; Sun, Boxing; Zhao, Zhihui

    2011-01-01

    DNA methylation is known to play an important role in regulating gene expression during biological development and tissue differentiation in eukaryotes. In this study, we used the fluorescence-labeled methylation-sensitive amplified polymorphism (F-MSAP) method to assess the extent and pattern of cytosine methylation in muscle, heart, liver, spleen, lung, kidney and stomach from the swine strain Laiwu, and we also examined specific methylation patterns in the seven tissues. In total, 96,371 fragments, each representing a recognition site cleaved by either or both EcoRI + HpaII and EcoRI + MspI, the HpaII and MspI are isoschizomeric enzymes, were amplified using 16 pairs of selective primers. A total of 50,094 sites were found to be methylated at cytosines in seven tissues. The incidence of DNA methylation was approximately 53.99% in muscle, 51.24% in the heart, 50.18% in the liver, 53.31% in the spleen, 51.97% in the lung, 51.15% in the kidney and 53.39% in the stomach, as revealed by the incidence of differential digestion. Additionally, differences in DNA methylation levels imply that such variations may be related to specific gene expression during tissue differentiation, growth and development. Three types of bands were generated in the F-MSAP profile, the total numbers of these three types of bands in the seven tissues were 46,277, 24,801 and 25,293, respectively. In addition, different methylation patterns were observed in seven tissues from pig, and almost all of the methylation patterns detected by F-MSAP could be confirmed by Southern analysis using the isolated amplified fragments as probes. The results clearly demonstrated that the F-MSAP technique can be adapted for use in large-scale DNA methylation detection in the pig genome. PMID:21283691

  18. Comparative DNA Sequence Analysis of Wheat and Rice Genomes

    PubMed Central

    Sorrells, Mark E.; La Rota, Mauricio; Bermudez-Kandianis, Catherine E.; Greene, Robert A.; Kantety, Ramesh; Munkvold, Jesse D.; Miftahudin; Mahmoud, Ahmed; Ma, Xuefeng; Gustafson, Perry J.; Qi, Lili L.; Echalier, Benjamin; Gill, Bikram S.; Matthews, David E.; Lazo, Gerard R.; Chao, Shiaoman; Anderson, Olin D.; Edwards, Hugh; Linkiewicz, Anna M.; Dubcovsky, Jorge; Akhunov, Eduard D.; Dvorak, Jan; Zhang, Deshui; Nguyen, Henry T.; Peng, Junhua; Lapitan, Nora L.V.; Gonzalez-Hernandez, Jose L.; Anderson, James A.; Hossain, Khwaja; Kalavacharla, Venu; Kianian, Shahryar F.; Choi, Dong-Woog; Close, Timothy J.; Dilbirligi, Muharrem; Gill, Kulvinder S.; Steber, Camille; Walker-Simmons, Mary K.; McGuire, Patrick E.; Qualset, Calvin O.

    2003-01-01

    The use of DNA sequence-based comparative genomics for evolutionary studies and for transferring information from model species to crop species has revolutionized molecular genetics and crop improvement strategies. This study compared 4485 expressed sequence tags (ESTs) that were physically mapped in wheat chromosome bins, to the public rice genome sequence data from 2251 ordered BAC/PAC clones using BLAST. A rice genome view of homologous wheat genome locations based on comparative sequence analysis revealed numerous chromosomal rearrangements that will significantly complicate the use of rice as a model for cross-species transfer of information in nonconserved regions. PMID:12902377

  19. Alkylation of nucleic acids by DNA-targeted 4-anilinoquinolinium aniline mustards: kinetic studies.

    PubMed

    O'Connor, C J; Denny, W A; Fan, J Y

    1991-01-01

    The rate of constant for hydrolysis of a series of 4-substituted aniline mustards Ar-X-pC6H4-N(CH2CH2Cl)2, where Ar is 4-anilinoquinolinium and X = O, CH2, CONH and CO, have been measured in water and 0.02 M imidazole buffer at 37 degrees C and in 50% aqueous acetone at 66 degrees C. The equilibrium binding constants of the compounds and their hydrolysis products to nucleic acids of differing base composition have been determined at varying ionic strengths, and the results are consistent with the compounds binding as expected in the DNA minor groove. The alkylating reactivity of the mustards towards these nucleic acids has been measured in water at 37 degrees C and in 0.01 M HEPES buffer over a range of temperatures from 25 degrees C to 60 degrees C. Evaluation of the thermodynamic parameters for these kinetic and equilibrium studies suggests that the interaction with nucleic acids is via an internal SN2 mechanism involving an aziridinium ion.

  20. Effect of tannic acid on benzo[a]pyrene-DNA adduct formation in mouse epidermis: comparison with synthetic gallic acid esters.

    PubMed

    Baer-Dubowska, W; Gnojkowski, J; Fenrych, W

    1997-01-01

    Tannic acid, a naturally occurring plant phenol, was shown to inhibit the mutagenicity and/or tumorigenicity of several polycyclic aromatic hydrocarbons in mouse skin. In this study the effect of topical application of tannic acid on epidermal aryl hydrocarbon hydroxylase, glutathione S-transferase, and binding of benzo[a]pyrene (B[a]P) to epidermal DNA was compared with the activity of synthetic gallic acid esters. Single topical application of 8 mumol octyl and dodecyl gallate had no effect on the induction of aryl hydrocarbon hydroxylase, whereas propyl gallate and tannic acid increased the enzyme activity by nearly 200%. Application of the phenolics one hour before 0.2 mumol of B[a]P enhanced the enzyme activity, but the observed differences were not significant in comparison with a B[a]P-treated group of mice. Application of dodecyl and octyl gallates to mouse skin resulted in three- and twofold increases, respectively, in the activity of glutathione S-transferase. Combined treatment with dodecyl gallate and B[a]P also resulted in significant enhancement of this enzyme activity. Application of the same dose of tannic acid to mouse skin one hour before the application of 0.2 or 1 mumol of B[a]P afforded 60% inhibition of covalent benzo[a]pyrene-diol-epoxide binding to epidermal DNA. Gallic acid esters with the exception of dodecyl gallate were less effective inhibitors of benzo[a]pyrene-diol-epoxide binding, especially when the higher dose of B[a]P was used. These results indicate that the antitumorigenic activity of tannic acid involves the interaction of the ultimate carcinogen with DNA rather than an altered metabolism. The linkage between gallic acid and glucose in natural plant phenols is also more effective at inhibiting B[a]P binding to epidermal DNA than the linkage with the alkyl group in synthetic gallates.

  1. DNA curtains: novel tools for imaging protein-nucleic acid interactions at the single-molecule level.

    PubMed

    Collins, Bridget E; Ye, Ling F; Duzdevich, Daniel; Greene, Eric C

    2014-01-01

    Interactions between proteins and nucleic acids are at the molecular foundations of most key biological processes, including DNA replication, genome maintenance, the regulation of gene expression, and chromosome segregation. A complete understanding of these types of biological processes requires tackling questions with a range of different techniques, such as genetics, cell biology, molecular biology, biochemistry, and structural biology. Here, we describe a novel experimental approach called "DNA curtains" that can be used to complement and extend these more traditional techniques by providing real-time information about protein-nucleic acid interactions at the level of single molecules. We describe general features of the DNA curtain technology and its application to the study of protein-nucleic acid interactions in vitro. We also discuss some future developments that will help address crucial challenges to the field of single-molecule biology.

  2. Acute acidic exposure induces p53-mediated oxidative stress and DNA damage in tilapia (Oreochromis niloticus) blood cells.

    PubMed

    Mai, Wei-jun; Yan, Jun-lun; Wang, Lei; Zheng, Ying; Xin, Yu; Wang, Wei-na

    2010-11-01

    Acid rain and inputs of acidic effluent can result in increased acidity in aquatic ecosystems, where it is known to have a significant impact and possibly, to cause the decline of some populations of aquatic organisms. In previous studies, intracellular acid-induced oxidative stress has been shown to cause DNA damage, and cooperatively activate the expression of the p53 gene. The acute effects of acidic environments on shrimp and fish have been widely studied. However, the molecular mechanism of acid-induced injury remains largely unknown. In this study, we examined the cellular responses of tilapia to acidic exposure-induced oxidative stress and antioxidant enzyme gene expression. Furthermore, we determined how acute acid stress activates the ATM-p53 signal pathway. We measured the upregulation of reactive oxygen species (ROS) production, the intracellular Ca(2)(+) concentration ([Ca(2)(+)](i)), the tail DNA values, the malondialdehyde (MDA) level in the blood cells and the percentage of dead and damaged blood cells. Our results suggest that oxidative stress and DNA damage occurred in tilapia in conditions where the pH was 5.3. Apoptosis was detected by Hoechst staining, which was mainly associated with changes in cell viability. The parameters that we measured were related to acid-induced DNA damage, and all parameters changed in the blood cells through time. The effects of acute acid exposure (pH 5.3) on the expression of ATM, p53, p21, Bax, manganese superoxide dismutase (MnSOD), glutathione peroxidase (GPx) were investigated in tilapia blood cells. The results showed that acute acid stress induced upregulation of ATM, p53 and p21, associated with increasing of DNA damage and apoptosis in blood cells. Additionally, the expression of Bax was slightly increased. Moreover, consensus p53-binding sequences were identified in tilapia MnSOD and GPx gene promoter regions and increased levels of ROS in the blood cells coincided with increased mRNA expression of p53, Mn

  3. Protective effect of borage seed oil and gamma linolenic acid on DNA: in vivo and in vitro studies.

    PubMed

    Tasset-Cuevas, Inmaculada; Fernández-Bedmar, Zahira; Lozano-Baena, María Dolores; Campos-Sánchez, Juan; de Haro-Bailón, Antonio; Muñoz-Serrano, Andrés; Alonso-Moraga, Angeles

    2013-01-01

    Borage (Borago officinalis L.) seed oil has been used as a treatment for various degenerative diseases. Many useful properties of this oil are attributed to its high gamma linolenic acid content (GLA, 18:3 ω-6). The purpose of this study was to demonstrate the safety and suitability of the use of borage seed oil, along with one of its active components, GLA, with respect to DNA integrity, and to establish possible in vivo toxic and in vitro cytotoxic effects. In order to measure these properties, five types of assays were carried out: toxicity, genotoxicity, antigenotoxicity, cytotoxicity (using the promyelocytic leukaemia HL60 cell line), and life span (in vivo analysis using the Drosophila model). Results showed that i) Borage seed oil is not toxic to D. melanogaster at physiological concentrations below 125 µl/ml and the studies on GLA indicated non-toxicity at the lowest concentration analyzed ii) Borage seed oil and GLA are DNA safe (non-genotoxic) and antimutagenic compared to hydrogen peroxide, thereby confirming its antioxidant capacity; iii) Borage seed oil and GLA exhibited cytotoxic activity in low doses (IC50 of 1 µl/ml and 0.087 mM, respectively) iv) Low doses of borage seed oil (0.19%) increased the health span of D. melanogaster; and v) GLA significantly decreased the life span of D. melanogaster.Based on the antimutagenic and cytotoxic effects along with the ability to increase the health span, we propose supplementation with borage seed oil rather than GLA, because it protects DNA by modulating oxidative genetic damage in D. melanogaster, increases the health span and exerts cytotoxic activity towards promyelocytic HL60 cells. PMID:23460824

  4. Protective Effect of Borage Seed Oil and Gamma Linolenic Acid on DNA: In Vivo and In Vitro Studies

    PubMed Central

    Tasset-Cuevas, Inmaculada; Fernández-Bedmar, Zahira; Lozano-Baena, María Dolores; Campos-Sánchez, Juan; de Haro-Bailón, Antonio; Muñoz-Serrano, Andrés; Alonso-Moraga, Ángeles

    2013-01-01

    Borage (Borago officinalis L.) seed oil has been used as a treatment for various degenerative diseases. Many useful properties of this oil are attributed to its high gamma linolenic acid content (GLA, 18:3 ω-6). The purpose of this study was to demonstrate the safety and suitability of the use of borage seed oil, along with one of its active components, GLA, with respect to DNA integrity, and to establish possible in vivo toxic and in vitro cytotoxic effects. In order to measure these properties, five types of assays were carried out: toxicity, genotoxicity, antigenotoxicity, cytotoxicity (using the promyelocytic leukaemia HL60 cell line), and life span (in vivo analysis using the Drosophila model). Results showed that i) Borage seed oil is not toxic to D. melanogaster at physiological concentrations below 125 µl/ml and the studies on GLA indicated non-toxicity at the lowest concentration analyzed ii) Borage seed oil and GLA are DNA safe (non-genotoxic) and antimutagenic compared to hydrogen peroxide, thereby confirming its antioxidant capacity; iii) Borage seed oil and GLA exhibited cytotoxic activity in low doses (IC50 of 1 µl/ml and 0.087 mM, respectively) iv) Low doses of borage seed oil (0.19%) increased the health span of D. melanogaster; and v) GLA significantly decreased the life span of D. melanogaster. Based on the antimutagenic and cytotoxic effects along with the ability to increase the health span, we propose supplementation with borage seed oil rather than GLA, because it protects DNA by modulating oxidative genetic damage in D. melanogaster, increases the health span and exerts cytotoxic activity towards promyelocytic HL60 cells. PMID:23460824

  5. Protective effect of borage seed oil and gamma linolenic acid on DNA: in vivo and in vitro studies.

    PubMed

    Tasset-Cuevas, Inmaculada; Fernández-Bedmar, Zahira; Lozano-Baena, María Dolores; Campos-Sánchez, Juan; de Haro-Bailón, Antonio; Muñoz-Serrano, Andrés; Alonso-Moraga, Angeles

    2013-01-01

    Borage (Borago officinalis L.) seed oil has been used as a treatment for various degenerative diseases. Many useful properties of this oil are attributed to its high gamma linolenic acid content (GLA, 18:3 ω-6). The purpose of this study was to demonstrate the safety and suitability of the use of borage seed oil, along with one of its active components, GLA, with respect to DNA integrity, and to establish possible in vivo toxic and in vitro cytotoxic effects. In order to measure these properties, five types of assays were carried out: toxicity, genotoxicity, antigenotoxicity, cytotoxicity (using the promyelocytic leukaemia HL60 cell line), and life span (in vivo analysis using the Drosophila model). Results showed that i) Borage seed oil is not toxic to D. melanogaster at physiological concentrations below 125 µl/ml and the studies on GLA indicated non-toxicity at the lowest concentration analyzed ii) Borage seed oil and GLA are DNA safe (non-genotoxic) and antimutagenic compared to hydrogen peroxide, thereby confirming its antioxidant capacity; iii) Borage seed oil and GLA exhibited cytotoxic activity in low doses (IC50 of 1 µl/ml and 0.087 mM, respectively) iv) Low doses of borage seed oil (0.19%) increased the health span of D. melanogaster; and v) GLA significantly decreased the life span of D. melanogaster.Based on the antimutagenic and cytotoxic effects along with the ability to increase the health span, we propose supplementation with borage seed oil rather than GLA, because it protects DNA by modulating oxidative genetic damage in D. melanogaster, increases the health span and exerts cytotoxic activity towards promyelocytic HL60 cells.

  6. Cytoprotection against Cr(6+)-induced DNA damage by alpha-lipoic acid: implications in reducing occupational cancer risk.

    PubMed

    Kumar, Sushil; Budhwar, Roli; Nigam, Akanksha; Priya, Shivam

    2009-11-01

    Alpha-lipoic acid (LA), the metabolic antioxidant, was evaluated for its potential to protect against Cr(6+)-induced DNA damage. Potassium dichromate was administered to Swiss albino mice orally ad libitum at the doses of 5, 10 or 25 mg/kg body weight in drinking water to set DNA damage in cells, which was characterized in mouse peripheral blood mononuclear cells and bone marrow cells using single-cell gel electrophoresis and analyses of generated comets for Tail moment, Tail DNA and Tail length. DNA damage was dose dependent. Cytoprotection by LA was remarkable. LA (5, 10 and 25 mg/kg body weight intraperitoneally) in pre-, co- and post-toxicant administration schedule abrogated DNA damage substantially in both cell types. Protection by LA was also dose dependent. LA annulled DNA damage by Cr(6+) in plasmid relaxation assay. A negligible DNA damage resulted during interaction of Cr(6+) and LA. Compared to ascorbate, LA emerged as a better antioxidant and least DNA damaging. In conclusion, our study advocated an experimental therapeutic research potential in LA against Cr(6+)-induced DNA damage for reduction of occupational cancer risk in humans. PMID:19710206

  7. Enzyme-free translation of DNA into sequence-defined synthetic polymers structurally unrelated to nucleic acids

    NASA Astrophysics Data System (ADS)

    Niu, Jia; Hili, Ryan; Liu, David R.

    2013-04-01

    The translation of DNA sequences into corresponding biopolymers enables the production, function and evolution of the macromolecules of life. In contrast, methods to generate sequence-defined synthetic polymers with similar levels of control have remained elusive. Here, we report the development of a DNA-templated translation system that enables the enzyme-free translation of DNA templates into sequence-defined synthetic polymers that have no necessary structural relationship with nucleic acids. We demonstrate the efficiency, sequence-specificity and generality of this translation system by oligomerizing building blocks including polyethylene glycol, α-(D)-peptides, and β-peptides in a DNA-programmed manner. Sequence-defined synthetic polymers with molecular weights of 26 kDa containing 16 consecutively coupled building blocks and 90 densely functionalized β-amino acid residues were translated from DNA templates using this strategy. We integrated the DNA-templated translation system developed here into a complete cycle of translation, coding sequence replication, template regeneration and re-translation suitable for the iterated in vitro selection of functional sequence-defined synthetic polymers unrelated in structure to nucleic acids.

  8. An Investigation into the Association between DNA Damage and Dietary Fatty Acid in Men with Prostate Cancer

    PubMed Central

    Bishop, Karen S.; Erdrich, Sharon; Karunasinghe, Nishi; Han, Dug Yeo; Zhu, Shuotun; Jesuthasan, Amalini; Ferguson, Lynnette R.

    2015-01-01

    Prostate cancer is a growing problem in New Zealand and worldwide, as populations adopt a Western style dietary pattern. In particular, dietary fat is believed to be associated with oxidative stress, which in turn may be associated with cancer risk and development. In addition, DNA damage is associated with the risk of various cancers, and is regarded as an ideal biomarker for the assessment of the influence of foods on cancer. In the study presented here, 20 men with prostate cancer adhered to a modified Mediterranean style diet for three months. Dietary records, blood fatty acid levels, prostate specific antigen, C-reactive protein and DNA damage were assessed pre- and post-intervention. DNA damage was inversely correlated with dietary adherence (p = 0.013) and whole blood monounsaturated fatty acids (p = 0.009) and oleic acid (p = 0.020). DNA damage was positively correlated with the intake of dairy products (p = 0.043), red meat (p = 0.007) and whole blood omega-6 polyunsaturated fatty acids (p = 0.015). Both the source and type of dietary fat changed significantly over the course of the dietary intervention. Levels of DNA damage were correlated with various dietary fat sources and types of dietary fat. PMID:25580814

  9. Inhibition of non-templated nucleotide addition by DNA polymerases in primer extension using twisted intercalating nucleic acid modified templates.

    PubMed

    Güixens-Gallardo, Pedro; Hocek, Michal; Perlíková, Pavla

    2016-01-15

    A simple and elegant method for inhibition of non-templated nucleotide addition by DNA polymerases and for following DNA 3'-heterogeneity in enzymatic DNA synthesis by primer extension (PEX) is described. When template bearing ortho-twisted intercalating nucleic acid (ortho-TINA) at the 5'-end is used, non-templated nucleotide addition is reduced in both the A- and B-family DNA polymerases (KOD XL, KOD (exo-), Bst 2.0, Therminator, Deep Vent (exo-) and Taq). Formation of a single oligonucleotide product was observed with ortho-TINA modified template and KOD XL, KOD (exo-), Bst 2.0, Deep Vent (exo-) and Taq DNA polymerases. This approach can be applied to the synthesis of both unmodified and base-modified oligonucleotides. PMID:26707394

  10. In vitro study of damaging effects of 2,4-dichlorophenoxyacetic acid on DNA structure by spectroscopic and voltammetric techniques.

    PubMed

    Ahmadi, Farhad; Bakhshandeh, Fatemeh

    2009-10-01

    2,4-Dichlorophenoxyacetic acid (2,4-D) is a genotoxic organochlorinated herbicide, and its interaction with DNA was studied by UV/Vis, fluorescence, circular dichroism viscosity measurements, and alternative current voltammetry techniques. Using these analyses, the binding constant of 2,4-D to DNA has been calculated by two different techniques. The binding constant of 2,4-D to DNA calculated by fluorescence and circular dichroism spectra was found to be 3.5 x 10(3) M(-1) and 5.02 x 10(3) M(-1), respectively. Analyses of fluorescence spectra, viscosity measurements, and alternative current voltammetry interactions indicated that 2,4-D is a groove binder of DNA. Ethidium bromide displacement studies revealed that 2,4-D does not have any effect on ethidium bromide-bound DNA, which is indicative of groove binding.

  11. GENETIC ANALYSIS OF ABSCISIC ACID BIOSYNTHESIS

    SciTech Connect

    MCCARTY D R

    2012-01-10

    The carotenoid cleavage dioxygenases (CCD) catalyze synthesis of a variety of apo-carotenoid secondary metabolites in plants, animals and bacteria. In plants, the reaction catalyzed by the 11, 12, 9-cis-epoxy carotenoid dioxygenase (NCED) is the first committed and key regulated step in synthesis of the plant hormone, abscisic acid (ABA). ABA is a key regulator of plant stress responses and has critical functions in normal root and seed development. The molecular mechanisms responsible for developmental control of ABA synthesis in plant tissues are poorly understood. Five of the nine CCD genes present in the Arabidopsis genome encode NCED's involved in control of ABA synthesis in the plant. This project is focused on functional analysis of these five AtNCED genes as a key to understanding developmental regulation of ABA synthesis and dissecting the role of ABA in plant development. For this purpose, the project developed a comprehensive set of gene knockouts in the AtNCED genes that facilitate genetic dissection of ABA synthesis. These mutants were used in combination with key molecular tools to address the following specific objectives: (1) the role of ABA synthesis in root development; (2) developmental control of ABA synthesis in seeds; (3) analysis of ATNCED over-expressers; (4) preliminary crystallography of the maize VP14 protein.

  12. An analysis of issues concerning acid rain

    SciTech Connect

    Not Available

    1984-01-01

    GAO examines the implications of current scientific knowledge for policy decisions on acid rain and offers a series of observations on the following issues involved in the debate: to what extent has it been scientifically demonstrated that acid rain is resulting in damage to the environment. What are the causes of acid rain and where is it most prevalent. What alternatives exist for controlling acid rain and what are their economic effects.

  13. Affordable Hands-On DNA Sequencing and Genotyping: An Exercise for Teaching DNA Analysis to Undergraduates

    ERIC Educational Resources Information Center

    Shah, Kushani; Thomas, Shelby; Stein, Arnold

    2013-01-01

    In this report, we describe a 5-week laboratory exercise for undergraduate biology and biochemistry students in which students learn to sequence DNA and to genotype their DNA for selected single nucleotide polymorphisms (SNPs). Students use miniaturized DNA sequencing gels that require approximately 8 min to run. The students perform G, A, T, C…

  14. Combined treatment with vitamin B12b and ascorbic acid causes in vitro DNA degradation in tumor cells.

    PubMed

    Medvedev, A I; Akatov, V S; Kreshchenko, N D; Solov'eva, M E; Leshchenko, V V; Lezhnev, E I; Yakubovskaya, R I

    2001-04-01

    Incubation of Ehrlich ascites carcinoma and HEp-2 human epidermoid laryngeal carcinoma cells with hydroxycobalamin (vitamin B12b) and ascorbic acid induced generation and accumulation of double-stranded DNA fragments (23,000 b.p. and longer) in cells. The same vitamins alone in the same concentrations produced no such effects. DNA degradation in HEp-2 cells caused by long-term (4 h) incubation with 5-25 microM hydroxycobalamin and ascorbic acid (1:10-1:40 molar ratio) at 37 degrees C was comparable with that induced by gamma-irradiation in a dose of 150 Gy at 4 degrees C.

  15. Platelet hexosaminidase a enzyme assay effectively detects carriers missed by targeted DNA mutation analysis.

    PubMed

    Nakagawa, Sachiko; Zhan, Jie; Sun, Wei; Ferreira, Jose Carlos; Keiles, Steven; Hambuch, Tina; Kammesheidt, Anja; Mark, Brian L; Schneider, Adele; Gross, Susan; Schreiber-Agus, Nicole

    2012-01-01

    Biochemical testing of hexosaminidase A (HexA) enzyme activity has been available for decades and has the ability to detect almost all Tay-Sachs disease (TSD) carriers, irrespective of ethnic background. This is increasingly important, as the gene pool of those who identify as Ashkenazi Jewish is diversifying. Here we describe the analysis of a cohort of 4,325 individuals arising from large carrier screening programs and tested by the serum and/or platelet HexA enzyme assays and by targeted DNA mutation analysis. Our results continue to support the platelet assay as a highly effective method for TSD carrier screening, with a low inconclusive rate and the ability to detect possible disease-causing mutation carriers that would have been missed by targeted DNA mutation analysis. Sequence analysis performed on one such platelet assay carrier, who had one non-Ashkenazi Jewish parent, identified the amino acid change Thr259Ala (A775G). Based on crystallographic modeling, this change is predicted to be deleterious, as threonine 259 is positioned proximal to the HexA alpha subunit active site and helps to stabilize key residues therein. Accordingly, if individuals are screened for TSD in broad-based programs by targeted molecular testing alone, they must be made aware that there is a more sensitive and inexpensive test available that can identify additional carriers. Alternatively, the enzyme assays can be offered as a first tier test, especially when screening individuals of mixed or non-Jewish ancestry. PMID:23430931

  16. Complementary DNA cloning of the pear 1-aminocyclopropane-1-carboxylic acid oxidase gene and agrobacterium-mediated anti-sense genetic transformation.

    PubMed

    Qi, Jing; Dong, Zhen; Zhang, Yu-Xing

    2015-12-01

    The aim of the present study was to genetically modify plantlets of the Chinese yali pear to reduce their expression of ripening-associated 1-aminocyclopropane-1-carboxylic acid oxidase (ACO) and therefore increase the shelf-life of the fruit. Primers were designed with selectivity for the conserved regions of published ACO gene sequences, and yali complementary DNA (cDNA) cloning was performed by reverse transcription quantitative polymerase chain reaction (PCR). The obtained cDNA fragment contained 831 base pairs, encoding 276 amino acid residues, and shared no less than 94% nucleotide sequence identity with other published ACO genes. The cDNA fragment was inversely inserted into a pBI121 expression vector, between the cauliflower mosaic virus 35S promoter and the nopaline synthase terminator, in order to construct the anti‑sense expression vector of the ACO gene; it was transfected into cultured yali plants using Agrobacterium LBA4404. Four independent transgenic lines of pear plantlets were obtained and validated by PCR analysis. A Southern blot assay revealed that there were three transgenic lines containing a single copy of exogenous gene and one line with double copies. The present study provided germplasm resources for the cultivation of novel storage varieties of pears, therefore providing a reference for further applications of anti‑sense RNA technology in the genetic improvement of pears and other fruit.

  17. Effect of pollution on DNA damage and essential fatty acid profile in Cirrhinus mrigala from River Chenab

    NASA Astrophysics Data System (ADS)

    Hussain, Bilal; Sultana, Tayyaba; Sultana, Salma; Al-Ghanim, K. A.; Mahboob, Shahid

    2016-05-01

    The objective of this study was to evaluate the effect of anthropogenic pollution on DNA damage and the fatty acid profile of the bottom dweller fish (Cirrhinus mrigala), collected from the River Chenab, in order to assess the effect of the toxicants on the quality of the fish meat. The levels of Cd, Hg, Cu, Mn, Zn, Pb, Cr and Sn and of phenols from this river were significantly higher than the permissible limits set by the USEPA. Comet assays showed DNA damage in Cirrhinus mrigala collected from three different sampling sites in the polluted area of the river. Significant differences were observed for DNA damage through comet assay in fish collected from polluted compared to control sites. No significant differences were observed for DNA damage between farmed and fish collected from upstream. The micronucleus assay showed similar trends. Fish from the highly polluted sites showed less number of fatty acids and more saturated fatty acids in their meat compared to fish from less polluted areas. Several fatty acids were missing in fish with higher levels of DNA in comet tail and micronucleus induction. Long-chain polyunsaturated fatty acids, eicosapentaenoic acid (20:5n-3) was found missing in the fish from polluted environment while it was found in considerable amount in farmed fish 7.8±0.4%. Docosahexaenoic acid (22:6n-3) also showed significant differences as 0.1±0.0 and 7.0±0.1% respectively, in wild polluted and farmed fishes.

  18. Analysis of genetic diversity in earthworms using DNA markers.

    PubMed

    Sharma, Anshul; Sonah, Humira; Deshmukh, Rupesh K; Gupta, Navneet K; Singh, Nagendra K; Sharma, Tilak R

    2011-01-01

    Earthworms are one of the most important and beneficial macrofauna, and are used extensively in organic farming. Earthworms mediate soil biological regulation systems, and produce biogenic structures. They help to maintain soil structure, water infiltration, and regulate the availability of nutrients assimilated by plants. The objectives of this study were to perform morphological and molecular characterizations of 24 earthworm individuals collected from geographically diverse locations to assess the level of genetic variation. For molecular analysis, the effectiveness of RAPD, ISSR, and Universal rice primers (URPs) markers was investigated to identify polymorphism among 24 isolates of earthworms. A total of 62 molecular markers were used for amplification of genomic DNA of earthworms. Of these, 10 RAPD, 10 ISSR, and 10 URPs markers were used for characterization, which showed 95.7%, 96.7% and 98.3% polymorphism, respectively. The dendrogram, generated from the DNA markers by the unweighted pair group method using arithmetic averages, grouped all the isolates into two main clusters. All Eisenia fetida isolates were clustered in group A, whereas group B included three isolates belonging to Eudrilus eugeniae. Molecular markers allowed a rapid assessment of genetic variation among these closely related isolates of earthworms. These results suggest that molecular markers are a good choice for diversity analysis of earthworm individuals. PMID:21186943

  19. Probing DNA Stiffness through Optical Fluctuation Analysis of Plasmon Rulers.

    PubMed

    Chen, Tianhong; Hong, Yan; Reinhard, Björn M

    2015-08-12

    The distance-dependent plasmon coupling between biopolymer tethered gold or silver nanoparticles forms the foundation for the so-called plasmon rulers. While conventional plasmon ruler applications focus on the detection of singular events in the far-field spectrum, we perform in this Letter a ratiometric analysis of the continuous spectral fluctuations arising from thermal interparticle separation variations in plasmon rulers confined to fluid lipid membranes. We characterized plasmon rulers with different DNA tethers and demonstrate the ability to detect and quantify differences in the plasmon ruler potential and tether stiffness. The influence of the nature of the tether (single-stranded versus double-stranded DNA) and the length of the tether is analyzed. The characterization of the continuous variation of the interparticle separation in individual plasmon rulers through optical fluctuation analysis provides additional information about the conformational flexibility of the tether molecule(s) located in the confinement of the deeply subdiffraction limit interparticle gap and enhances the versatility of plasmon rulers as a tool in Biophysics and Nanotechnology.

  20. Trophic spectra under the lens of amino acid isotopic analysis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Recent advances in compound specific isotopic ratio analysis (CSIRA) have allowed researchers to measure trophic fractionation of 15N in specific amino acids, namely glutamic acid and phenylalanine. These amino acids have proven useful in food web studies because of the wide and consistent disparity...

  1. DNA methylation patterns are associated with n-3 fatty acid intake in Yup'ik people.

    PubMed

    Aslibekyan, Stella; Wiener, Howard W; Havel, Peter J; Stanhope, Kimber L; O'Brien, Diane M; Hopkins, Scarlett E; Absher, Devin M; Tiwari, Hemant K; Boyer, Bert B

    2014-04-01

    A large body of evidence links a high dietary intake of n-3 (ω-3) polyunsaturated fatty acids (PUFAs) with improved cardiometabolic outcomes. Recent studies suggested that the biologic processes underlying the observed associations may involve epigenetic changes, specifically DNA methylation. To evaluate changes in methylation associated with n-3 PUFA intake, we conducted an epigenome-wide methylation association study of long-chain n-3 PUFA intake and tested associations between the diabetes- and cardiovascular disease-related traits. We assessed DNA methylation at ∼470,000 cytosine-phosphate-guanine (CpG) sites in a cross-sectional study of 185 Yup'ik Alaska Native individuals representing the top and bottom deciles of PUFA intake. Linear regression models were used to test for the associations of interest, adjusting for age, sex, and community group. We identified 27 differentially methylated CpG sites at biologically relevant regions that reached epigenome-wide significance (P < 1 × 10⁻⁷). Specifically, regions on chromosomes 3 (helicase-like transcription factor), 10 (actin α 2 smooth muscle/Fas cell surface death receptor), and 16 (protease serine 36/C16 open reading frame 67) each harbored 2 significant correlates of n-3 PUFA intake. In conclusion, we present promising evidence of association between several biologically relevant epigenetic markers and long-term intake of marine-derived n-3 PUFAs. PMID:24477300

  2. In depth analysis of the quenching of three fluorene-phenylene-based cationic conjugated polyelectrolytes by DNA and DNA bases.

    PubMed

    Davies, Matthew L; Douglas, Peter; Burrows, Hugh D; Martincigh, Bice; Miguel, Maria da Graça; Scherf, Ullrich; Mallavia, Ricardo; Douglas, Alastair

    2014-01-16

    The interaction of three cationic poly {9,9-bis[N,N-(trimethylammonium)hexyl]fluorene-co-1,4-phenylene} polymers with average chain lengths of ∼6, 12, and 100 repeat units (PFP-NR36(I),12(Br),100(Br)) with both double and single stranded, short and long, DNA and DNA bases have been studied by steady state and time-resolved fluorescence techniques. Fluorescence of PFP-NR3 polymers is quenched with high efficiency by DNA (both double and single stranded) and DNA bases. The resulting quenching plots are sigmoidal and are not accurately described by using a Stern-Volmer quenching mechanism. Here, the quenching mechanism is well modeled in terms of an equilibrium in which a PFP-NR3/DNA aggregate complex is formed which brings polymer chains into close enough proximity to allow interchain excitation energy migration and quenching at aggregate or DNA base traps. Such an analysis gives equilibrium constants of 8.4 × 10(6) (±1.2 × 10(6)) M(-1) for short-dsDNA and 8.6 × 10(6) (±1.7 × 10(6)) M(-1) for short-ssDNA with PFP-NR36(I).

  3. mtDNA-Server: next-generation sequencing data analysis of human mitochondrial DNA in the cloud

    PubMed Central

    Weissensteiner, Hansi; Forer, Lukas; Fuchsberger, Christian; Schöpf, Bernd; Kloss-Brandstätter, Anita; Specht, Günther; Kronenberg, Florian; Schönherr, Sebastian

    2016-01-01

    Next generation sequencing (NGS) allows investigating mitochondrial DNA (mtDNA) characteristics such as heteroplasmy (i.e. intra-individual sequence variation) to a higher level of detail. While several pipelines for analyzing heteroplasmies exist, issues in usability, accuracy of results and interpreting final data limit their usage. Here we present mtDNA-Server, a scalable web server for the analysis of mtDNA studies of any size with a special focus on usability as well as reliable identification and quantification of heteroplasmic variants. The mtDNA-Server workflow includes parallel read alignment, heteroplasmy detection, artefact or contamination identification, variant annotation as well as several quality control metrics, often neglected in current mtDNA NGS studies. All computational steps are parallelized with Hadoop MapReduce and executed graphically with Cloudgene. We validated the underlying heteroplasmy and contamination detection model by generating four artificial sample mix-ups on two different NGS devices. Our evaluation data shows that mtDNA-Server detects heteroplasmies and artificial recombinations down to the 1% level with perfect specificity and outperforms existing approaches regarding sensitivity. mtDNA-Server is currently able to analyze the 1000G Phase 3 data (n = 2,504) in less than 5 h and is freely accessible at https://mtdna-server.uibk.ac.at. PMID:27084948

  4. mtDNA-Server: next-generation sequencing data analysis of human mitochondrial DNA in the cloud.

    PubMed

    Weissensteiner, Hansi; Forer, Lukas; Fuchsberger, Christian; Schöpf, Bernd; Kloss-Brandstätter, Anita; Specht, Günther; Kronenberg, Florian; Schönherr, Sebastian

    2016-07-01

    Next generation sequencing (NGS) allows investigating mitochondrial DNA (mtDNA) characteristics such as heteroplasmy (i.e. intra-individual sequence variation) to a higher level of detail. While several pipelines for analyzing heteroplasmies exist, issues in usability, accuracy of results and interpreting final data limit their usage. Here we present mtDNA-Server, a scalable web server for the analysis of mtDNA studies of any size with a special focus on usability as well as reliable identification and quantification of heteroplasmic variants. The mtDNA-Server workflow includes parallel read alignment, heteroplasmy detection, artefact or contamination identification, variant annotation as well as several quality control metrics, often neglected in current mtDNA NGS studies. All computational steps are parallelized with Hadoop MapReduce and executed graphically with Cloudgene. We validated the underlying heteroplasmy and contamination detection model by generating four artificial sample mix-ups on two different NGS devices. Our evaluation data shows that mtDNA-Server detects heteroplasmies and artificial recombinations down to the 1% level with perfect specificity and outperforms existing approaches regarding sensitivity. mtDNA-Server is currently able to analyze the 1000G Phase 3 data (n = 2,504) in less than 5 h and is freely accessible at https://mtdna-server.uibk.ac.at. PMID:27084948

  5. [Infrared fluorescent markers for microarray DNA analysis on biological microchip].

    PubMed

    Spitsyn, M A; Shershov, V E; Kuznetsova, V E; Barsky, V E; Egorov, E E; Emelyanova, M A; Kreindlin, E Ya; Lysov, Yu P; Guseinov, T O; Fesenko, D E; Lapa, S A; Surzhikov, S A; Abramov, I S; Nasedkina, T V; Zasedatelev, A S; Chudinov, A V

    2015-01-01

    To expand the informational capabilities of molecular genetic research, on the biological microchips, new indotricarbocyanine dyes that fluoresce in the near infrared (IR) spectral region have been synthesized. The developed IR dyes were studied using a biochip-based test system for detection of mutations in the BRCA1/BRCA2 and CHECK2 genes associated with breast cancer. The fluorescent label was introduced to the analyzed DNA during PCR using primers labeled with the synthesized IR dyes. An analyzer that allows recording and processing of images of fluorescent microarrays in the IR spectral region was designed and manufactured. It has been shown that the use of the synthesized dyes enables to conduct analysis in the IR region and improve the reliability of medical diagnostic tests due to low fluorescence intensity of sample components as well as of a biochip substrate and the reagents used for analysis. PMID:26510593

  6. [Infrared fluorescent markers for microarray DNA analysis on biological microchip].

    PubMed

    Spitsyn, M A; Shershov, V E; Kuznetsova, V E; Barsky, V E; Egorov, E E; Emelyanova, M A; Kreindlin, E Ya; Lysov, Yu P; Guseinov, T O; Fesenko, D E; Lapa, S A; Surzhikov, S A; Abramov, I S; Nasedkina, T V; Zasedatelev, A S; Chudinov, A V

    2015-01-01

    To expand the informational capabilities of molecular genetic research, on the biological microchips, new indotricarbocyanine dyes that fluoresce in the near infrared (IR) spectral region have been synthesized. The developed IR dyes were studied using a biochip-based test system for detection of mutations in the BRCA1/BRCA2 and CHECK2 genes associated with breast cancer. The fluorescent label was introduced to the analyzed DNA during PCR using primers labeled with the synthesized IR dyes. An analyzer that allows recording and processing of images of fluorescent microarrays in the IR spectral region was designed and manufactured. It has been shown that the use of the synthesized dyes enables to conduct analysis in the IR region and improve the reliability of medical diagnostic tests due to low fluorescence intensity of sample components as well as of a biochip substrate and the reagents used for analysis.

  7. Analysis of alcohol-induced DNA damage in Escherichia coli by visualizing single genomic DNA molecules.

    PubMed

    Kang, Yujin; Lee, Jinyong; Kim, Jisoo; Oh, Yeeun; Kim, Dogeun; Lee, Jungyun; Lim, Sangyong; Jo, Kyubong

    2016-07-21

    Consumption of alcohol injures DNA, and such damage is considered to be a primary cause for the development of cancer and many other diseases essentially due to reactive oxygen species generated from alcohol. To sensitively detect alcohol-induced DNA lesions in a biological system, we introduced a novel analytical platform for visualization of single genomic DNA molecules using E. coli. By fluorescently labelling the DNA lesions, our approach demonstrated, with the highest sensitivity, that we could count the number of DNA lesions induced by alcohol metabolism in a single bacterial cell. Moreover, our results showed a linear relationship between ethanol concentration and the number of DNA lesions: 0.88 lesions per 1% ethanol. Using this approach, we quantitatively analysed the DNA damage induced by exposure to alcoholic beverages such as beer (5% ethanol), rice wine (13%), soju (20%), and whisky (40%). PMID:27186604

  8. Role of Accessory DNA Polymerases in DNA Replication in Escherichia coli: Analysis of the dnaX36 Mutator Mutant▿

    PubMed Central

    Gawel, Damian; Pham, Phuong T.; Fijalkowska, Iwona J.; Jonczyk, Piotr; Schaaper, Roel M.

    2008-01-01

    The dnaX36(TS) mutant of Escherichia coli confers a distinct mutator phenotype characterized by enhancement of transversion base substitutions and certain (−1) frameshift mutations. Here, we have further investigated the possible mechanism(s) underlying this mutator effect, focusing in particular on the role of the various E. coli DNA polymerases. The dnaX gene encodes the τ subunit of DNA polymerase III (Pol III) holoenzyme, the enzyme responsible for replication of the bacterial chromosome. The dnaX36 defect resides in the C-terminal domain V of τ, essential for interaction of τ with the α (polymerase) subunit, suggesting that the mutator phenotype is caused by an impaired or altered α-τ interaction. We previously proposed that the mutator activity results from aberrant processing of terminal mismatches created by Pol III insertion errors. The present results, including lack of interaction of dnaX36 with mutM, mutY, and recA defects, support our assumption that dnaX36-mediated mutations originate as errors of replication rather than DNA damage-related events. Second, an important role is described for DNA Pol II and Pol IV in preventing and producing, respectively, the mutations. In the system used, a high fraction of the mutations is dependent on the action of Pol IV in a (dinB) gene dosage-dependent manner. However, an even larger but opposing role is deduced for Pol II, revealing Pol II to be a major editor of Pol III mediated replication errors. Overall, the results provide insight into the interplay of the various DNA polymerases, and of τ subunit, in securing a high fidelity of replication. PMID:18156258

  9. A convenient and adaptable microcomputer environment for DNA and protein sequence manipulation and analysis.

    PubMed Central

    Pustell, J; Kafatos, F C

    1986-01-01

    We describe the further development of a widely used package of DNA and protein sequence analysis programs for microcomputers (1,2,3). The package now provides a screen oriented user interface, and an enhanced working environment with powerful formatting, disk access, and memory management tools. The new GenBank floppy disk database is supported transparently to the user and a similar version of the NBRF protein database is provided. The programs can use sequence file annotation to automatically annotate printouts and translate or extract specified regions from sequences by name. The sequence comparison programs can now perform a 5000 X 5000 bp analysis in 12 minutes on an IBM PC. A program to locate potential protein coding regions in nucleic acids, a digitizer interface, and other additions are also described. PMID:3753784

  10. Using quantitative acid-base analysis in the ICU.

    PubMed

    Lloyd, P; Freebairn, R

    2006-03-01

    The quantitative acid-base 'Strong Ion' calculator is a practical application of quantitative acid-base chemistry, as developed by Peter Stewart and Peter Constable. It quantifies the three independent factors that control acidity, calculates the concentration and charge of unmeasured ions, produces a report based on these calculations and displays a Gamblegram depicting measured ionic species. Used together with the medical history, quantitative acid-base analysis has advantages over traditional approaches.

  11. Novel zinc-based fixative for high quality DNA, RNA and protein analysis

    PubMed Central

    Lykidis, Dimitrios; Van Noorden, Susan; Armstrong, Alan; Spencer-Dene, Bradley; Li, Jie; Zhuang, Zhengping; Stamp, Gordon W. H.

    2007-01-01

    We have developed a reliable, cost-effective and non-toxic fixative to meet the needs of contemporary molecular pathobiology research, particularly in respect of RNA and DNA integrity. The effects of 25 different fixative recipes on the fixed quality of tissues from C57BL/6 mice were investigated. Results from IHC, PCR, RT–PCR, RNA Agilent Bioanalyser and Real-Time PCR showed that a novel zinc-based fixative (Z7) containing zinc trifluoroacetate, zinc chloride and calcium acetate was significantly better than the standard zinc-based fixative (Z2) and neutral buffered formalin (NBF) for DNA, RNA and protein preservation. DNA sequences up to 2.4 kb in length and RNA fragments up to 361 bp in length were successfully amplified from Z7 fixed tissues, as demonstrated by PCR, RT–PCR and Real-Time PCR. Total protein analysis was achieved using 2-D gel electrophoresis. In addition, nucleic acids and proteins were very stable over a 6–14-month period. This improved, non-toxic and economical tissue fixative could be applied for routine use in pathology laboratories to permit subsequent genomic/proteomic studies. PMID:17576663

  12. Molecular mechanisms of McArdle's disease (muscle glycogen phosphorylase deficiency). RNA and DNA analysis.

    PubMed Central

    Gautron, S; Daegelen, D; Mennecier, F; Dubocq, D; Kahn, A; Dreyfus, J C

    1987-01-01

    Lack of muscle glycogen phosphorylase activity leads to McArdle's disease, a rare metabolic myopathy. To investigate its molecular basis at the nucleic acid level, we isolated muscle phosphorylase cDNA clones from a human cDNA library in Escherichia coli plasmid pBR 322. Subcloning of one insertion of M13 bacteriophage permitted its definite identification by sequencing. Northern blot experiments revealed one specific messenger RNA of 3.4 kilobases found uniquely in tissues expressing muscle phosphorylase. We show that McArdle's disease exhibits a molecular heterogeneity at the messenger RNA level. In eight unrelated cases of McArdle's disease in which no inactive proteins had been detected, we assayed muscle biopsies for phosphorylase mRNA by Northern blotting. In five cases, no muscle phosphorylase mRNA could be detected, while in three other cases, normal length mRNA was present in lower amounts. Moreover, Southern blot analysis of DNA isolated from white blood cells in four McArdle patients revealed no major deletion or rearrangements of the phosphorylase gene as compared with controls. Images PMID:3466902

  13. Direct detection of circulating free DNA extracted from serum samples of breast cancer using locked nucleic acid molecular beacon.

    PubMed

    Gui, Zhen; Wang, Quanbo; Li, Jinchang; Zhu, Mingchen; Yu, Lili; Xun, Tang; Yan, Feng; Ju, Huangxian

    2016-07-01

    As an emerging noninvasive blood biomarker, circulating free DNA (cfDNA) can be utilized to assess diagnosis, progression and evaluate prognosis of cancer. However, cfDNAs are not "naked", they can be part of complexes, or are bound to the surface of the cells via proteins, which make the detection more challenging. Here, a simple method for the detection of Ubiquitin-like with PHD and ring finger domains 1 (UHRF1) DNA exacted from serum of breast cancer (BC) has been developed using a novel locked nucleic acid molecular beacon (LNA-MB). In order to enhance the stability and detection efficiency of the probe in biofluids, we design a shared-stem molecular beacon containing a 27-mer loop and a 4-mer stem with DNA/LNA alternating bases. The fluorescence is released in the presence of target. The detection procedure is simple and can be completed within 1h. This method shows a sensitive response to UHRF1 DNA with a dynamic range of 3 orders of magnitude. The limit of detection is 11nM (S/N=3) with excellent selectivity. It can discriminate UHRF1 DNA from three-base mismatched DNA with a high specificity. More importantly, this method can distinguish the expression of serum UHRF1 DNA among 5 breast cancer patients and 5 healthy controls. The mentioned superiority may suggest that this assay can be served as a promising noninvasive detection tool for early BC diagnosis and monitoring. PMID:27154709

  14. Direct detection of circulating free DNA extracted from serum samples of breast cancer using locked nucleic acid molecular beacon.

    PubMed

    Gui, Zhen; Wang, Quanbo; Li, Jinchang; Zhu, Mingchen; Yu, Lili; Xun, Tang; Yan, Feng; Ju, Huangxian

    2016-07-01

    As an emerging noninvasive blood biomarker, circulating free DNA (cfDNA) can be utilized to assess diagnosis, progression and evaluate prognosis of cancer. However, cfDNAs are not "naked", they can be part of complexes, or are bound to the surface of the cells via proteins, which make the detection more challenging. Here, a simple method for the detection of Ubiquitin-like with PHD and ring finger domains 1 (UHRF1) DNA exacted from serum of breast cancer (BC) has been developed using a novel locked nucleic acid molecular beacon (LNA-MB). In order to enhance the stability and detection efficiency of the probe in biofluids, we design a shared-stem molecular beacon containing a 27-mer loop and a 4-mer stem with DNA/LNA alternating bases. The fluorescence is released in the presence of target. The detection procedure is simple and can be completed within 1h. This method shows a sensitive response to UHRF1 DNA with a dynamic range of 3 orders of magnitude. The limit of detection is 11nM (S/N=3) with excellent selectivity. It can discriminate UHRF1 DNA from three-base mismatched DNA with a high specificity. More importantly, this method can distinguish the expression of serum UHRF1 DNA among 5 breast cancer patients and 5 healthy controls. The mentioned superiority may suggest that this assay can be served as a promising noninvasive detection tool for early BC diagnosis and monitoring.

  15. Analysis of Replicating Yeast Chromosomes by DNA Combing.

    PubMed

    Gallo, David; Wang, Gang; Yip, Christopher M; Brown, Grant W

    2016-02-01

    Molecular combing of DNA fibers is a powerful technique to monitor origin usage and DNA replication fork progression in the budding yeast Saccharomyces cerevisiae. In contrast to traditional flow cytometry, microarray, or sequencing techniques, which provide population-level data, DNA combing provides DNA replication profiles of individual molecules. DNA combing uses yeast strains that express human thymidine kinase, which facilitates the incorporation of thymidine analogs into nascent DNA. First, DNA is isolated and stretched uniformly onto silanized glass coverslips. Following immunodetection with antibodies that recognize the thymidine analog and the DNA, the DNA fibers are imaged using a fluorescence microscope. Finally, the lengths of newly replicated DNA tracks are measured and converted to base pairs, allowing calculations of the speed of the replication fork and of interorigin distances. DNA combing can be applied to monitor replication defects caused by gene mutations or by chemical agents that induce replication stress. Here, we present a methodology for studying replicating yeast chromosomes by molecular DNA combing. We begin with procedures for the preparation of silanized coverslips and for assembly of a DNA combing machine (DCM) and conclude by presenting a detailed protocol for molecular DNA combing in yeast.

  16. A Magnetic Nanoparticle Based Nucleic Acid Isolation and Purification Instrument for DNA Extraction of Escherichia Coli O157: H7.

    PubMed

    Chen, Yahui; Lin, Jianhan; Jiang, Qin; Chen, Qi; Zhang, Shengjun; Li, Li

    2016-03-01

    The objective of this study was to evaluate the performance of a nucleic acid isolation and purification instrument using Escherichia coli O157:H7 as the model. The instrument was developed with magnetic nanoparticles for efficiently capturing nucleic acids and an intelligent mechanical unit for automatically performing the whole nucleic acid extraction process. A commercial DNA extraction kit from Huier Nano Company was used as reference. Nucleic acids in 1 ml of E. coli O157: H7 at a concentration of 5 x 10(8) CFU/mL were extracted by using this instrument and the kit in parallel and then detected by an ultraviolet spectrophotometer to obtain A260 values and A260/A280 values for the determination of the extracted DNA's quantity and purity, respectively. The A260 values for the instrument and the kit were 0.78 and 0.61, respectively, and the A260/A280 values were 1.98 and 1.93. The coefficient of variations of these parallel tests ranged from 10.5% to 16.7%. The results indicated that this nucleic acid isolation and purification instrument could extract a comparable level of nucleic acid within 50 min compared to the commercial DNA extraction kit.

  17. Analysis of issues concerning acid rain

    SciTech Connect

    Bowsher, C.A.

    1984-12-11

    Although science has largely determined that man-made emissions cause acid rain, there is uncertainty concerning the extent and timing of its anticipated effects. Thus, at the present time scientific information alone does not lead unequivocally to a conclusion on whether it is appropriate to begin control actions now or to await better understanding. Given this uncertainty, decisionmakers must weigh the risks of further, potentially avoidable environmental damage against the risks of economic impacts from acid rain control actions which may ultimately prove to be unwarranted. GAO examines the implications of current scientific knowledge for policy decisions on acid rain and offers a series of observations on the following issues involved in the debate: To what extent has it been scientifically demonstrated that acid rain is resulting in damage to the environment. What are the causes of acid rain and where is it most prevalent. What alternatives exist for controlling acid rain and what are their economic effects. 5 figures, 20 tables.

  18. Targeted DNA methylation analysis by next-generation sequencing.

    PubMed

    Masser, Dustin R; Stanford, David R; Freeman, Willard M

    2015-02-24

    The role of epigenetic processes in the control of gene expression has been known for a number of years. DNA methylation at cytosine residues is of particular interest for epigenetic studies as it has been demonstrated to be both a long lasting and a dynamic regulator of gene expression. Efforts to examine epigenetic changes in health and disease have been hindered by the lack of high-throughput, quantitatively accurate methods. With the advent and popularization of next-generation sequencing (NGS) technologies, these tools are now being applied to epigenomics in addition to existing genomic and transcriptomic methodologies. For epigenetic investigations of cytosine methylation where regions of interest, such as specific gene promoters or CpG islands, have been identified and there is a need to examine significant numbers of samples with high quantitative accuracy, we have developed a method called Bisulfite Amplicon Sequencing (BSAS). This method combines bisulfite conversion with targeted amplification of regions of interest, transposome-mediated library construction and benchtop NGS. BSAS offers a rapid and efficient method for analysis of up to 10 kb of targeted regions in up to 96 samples at a time that can be performed by most research groups with basic molecular biology skills. The results provide absolute quantitation of cytosine methylation with base specificity. BSAS can be applied to any genomic region from any DNA source. This method is useful for hypothesis testing studies of target regions of interest as well as confirmation of regions identified in genome-wide methylation analyses such as whole genome bisulfite sequencing, reduced representation bisulfite sequencing, and methylated DNA immunoprecipitation sequencing.

  19. DNA Methylation Perturbations in Genes Involved in Polyunsaturated Fatty Acid Biosynthesis Associated with Depression and Suicide Risk

    PubMed Central

    Haghighi, Fatemeh; Galfalvy, Hanga; Chen, Sean; Huang, Yung-yu; Cooper, Thomas B.; Burke, Ainsley K.; Oquendo, Maria A.; Mann, J. John; Sublette, M. Elizabeth

    2015-01-01

    Polyunsaturated fatty acid (PUFA) status has been associated with neuropsychiatric disorders, including depression and risk of suicide. Long-chain PUFAs (LC-PUFAs) are obtained in the diet or produced by sequential desaturation and elongation of shorter-chain precursor fatty acids linoleic acid (LA, 18:2n-6) and α-linolenic acid (ALA, 18:3n-3). We compared DNA methylation patterns in genes involved in LC-PUFA biosynthesis in major depressive disorder (MDD) with (n = 22) and without (n = 39) history of suicide attempt, and age- and sex-matched healthy volunteers (n = 59). Plasma levels of selected PUFAs along the LC-PUFA biosynthesis pathway were determined by transesterification and gas chromatography. CpG methylation levels for the main human LC-PUFA biosynthetic genes, fatty acid desaturases 1 (Fads1) and 2 (Fads2), and elongation of very long-chain fatty acids protein 5 (Elovl5), were assayed by bisulfite pyrosequencing. Associations between PUFA levels and diagnosis or suicide attempt status did not survive correction for multiple testing. However, MDD diagnosis and suicide attempts were significantly associated with DNA methylation in Elovl5 gene regulatory regions. Also the relative roles of PUFA levels and DNA methylation with respect to diagnostic and suicide attempt status were determined by least absolute shrinkage and selection operator logistic regression analyses. We found that PUFA associations with suicide attempt status were explained by effects of Elovl5 DNA methylation within the regulatory regions. The observed link between plasma PUFA levels, DNA methylation, and suicide risk may have implications for modulation of disease-associated epigenetic marks by nutritional intervention. PMID:25972837

  20. Effect of human polymorphonuclear and mononuclear leukocytes on chromosomal and plasmid DNA of Escherichia coli. Role of acid DNase

    SciTech Connect

    Rozenberg-Arska, M.; van Strijp, J.A.; Hoekstra, W.P.; Verhoef, J.

    1984-05-01

    Phagocytosis and killing by polymorphonuclear and mononuclear leukocytes are important host resistance factors against invading microorganisms. Evidence showing that killing is rapidly followed by degradation of bacterial components is limited. Therefore, we studied the fate of Escherichia coli DNA following phagocytosis of E. coli by polymorphonuclear and mononuclear leukocytes. (/sup 3/H)Thymidine-labeled, unencapsulated E. coli PC2166 and E. coli 048K1 were incubated in serum, washed, and added to leukocytes. Uptake and killing of the bacteria and degradation of DNA were measured. Although phagocytosis and killing by mononuclear leukocytes was less efficient than that by polymorphonuclear leukocytes, only mononuclear leukocytes were able to degrade E. coli PC2166 DNA. Within 2 h, 60% of the radioactivity added to mononuclear leukocytes was released into the supernate, of which 40% was acid soluble. DNA of E. coli 048K1 was not degraded. To further analyze the capacity of mononuclear leukocytes to degrade E. coli DNA, chromosomal and plasmid DNA was isolated from ingested bacteria and subjected to agarose gel-electrophoresis. Only chromosomal DNA was degraded after phagocytosis. Plasmid DNA of E. coli carrying a gene coding for ampicillin resistance remained intact for a 2-h period after ingestion, and was still able to transform recipient E. coli cells after this period. Although we observed no DNA degradation during phagocytosis by polymorphonuclear leukocytes, lysates of both polymorphonuclear and mononuclear leukocytes contained acid-DNase activity with a pH optimum of 4.9. However, the DNase activity of mononuclear leukocytes was 20 times higher than that of polymorphonuclear leukocytes. No difference was observed between DNase activity from polymorphonuclear and mononuclear leukocytes from a chronic granulomatous disease patient with DNase activity from control polymorphonuclear and mononuclear leukocytes.

  1. A kinetic analysis of strand breaks on large DNA induced by cigarette smoke extract

    NASA Astrophysics Data System (ADS)

    Kurita, Hirofumi; Takata, Tatsuya; Yasuda, Hachiro; Takashima, Kazunori; Mizuno, Akira

    2010-06-01

    We report a kinetic analysis of strand breakages on large DNA molecules induced by cigarette smoke extract (CSE), an extract of soluble cigarette smoke components. Previously, this DNA damage was analyzed by agarose gel electrophoresis, whereas we used fluorescence to kinetically analyze damage to individual DNA molecules. CSE caused a marked change in length of DNA molecules. The rate of CSE-induced double-strand breakage on large random-coiled DNA molecules was determined using a simple theoretical model, allowing the facile estimation of the rate of double-strand breaks on large DNA molecules.

  2. Isolation of acetylated bile acids from the sponge Siphonochalina fortis and DNA damage evaluation by the comet assay.

    PubMed

    Patiño Cano, Laura P; Bartolotta, Susana A; Casanova, Natalia A; Siless, Gastón E; Portmann, Erika; Schejter, Laura; Palermo, Jorge A; Carballo, Marta A

    2013-10-01

    From the organic extracts of the sponge Siphonochalina fortis, collected at Bahía Bustamante, Chubut, Argentina, three major compounds were isolated and identified as deoxycholic acid 3, 12-diacetate (1), cholic acid 3, 7, 12-triacetate (2) and cholic acid, 3, 7, 12-triacetate. (3). This is the first report of acetylated bile acids in sponges and the first isolation of compound 3 as a natural product. The potential induction of DNA lesions by the isolated compounds was investigated using the comet assay in lymphocytes of human peripheral blood as in vitro model. The results showed that the administration of the bile acid derivatives would not induce DNA damages, indicating that acetylated bile acids are nontoxic metabolites at the tested concentrations. Since the free bile acids were not detected, it is unlikely that the acetylated compounds may be part of the sponge cells detoxification mechanisms. These results may suggest a possible role of acetylated bile acids as a chemical defense mechanism, product of a symbiotic relationship with microorganisms, which would explain their seasonal and geographical variation, and their influence on the previously observed genotoxicity of the organic extract of S. fortis.

  3. FRETmatrix: a general methodology for the simulation and analysis of FRET in nucleic acids

    PubMed Central

    Preus, Søren; Kilså, Kristine; Miannay, Francois-Alexandre; Albinsson, Bo; Wilhelmsson, L. Marcus

    2013-01-01

    Förster resonance energy transfer (FRET) is a technique commonly used to unravel the structure and conformational changes of biomolecules being vital for all living organisms. Typically, FRET is performed using dyes attached externally to nucleic acids through a linker that complicates quantitative interpretation of experiments because of dye diffusion and reorientation. Here, we report a versatile, general methodology for the simulation and analysis of FRET in nucleic acids, and demonstrate its particular power for modelling FRET between probes possessing limited diffusional and rotational freedom, such as our recently developed nucleobase analogue FRET pairs (base–base FRET). These probes are positioned inside the DNA/RNA structures as a replacement for one of the natural bases, thus, providing unique control of their position and orientation and the advantage of reporting from inside sites of interest. In demonstration studies, not requiring molecular dynamics modelling, we obtain previously inaccessible insight into the orientation and nanosecond dynamics of the bases inside double-stranded DNA, and we reconstruct high resolution 3D structures of kinked DNA. The reported methodology is accompanied by a freely available software package, FRETmatrix, for the design and analysis of FRET in nucleic acid containing systems. PMID:22977181

  4. Multilayered Polyelectrolyte Assemblies as Platforms for the Delivery of DNA and Other Nucleic Acid-Based Therapeutics

    PubMed Central

    Jewell, Christopher M.; Lynn, David M.

    2008-01-01

    Materials that provide spatial and temporal control over the delivery of DNA and other nucleic acid-based agents from surfaces play important roles in the development of localized gene-based therapies. This review focuses on a relatively new approach to the immobilization and release of DNA from surfaces: methods based on the layer-by-layer assembly of thin multilayered films (or polyelectrolyte multilayers, PEMs). Layer-by-layer methods provide convenient, nanometer-scale control over the incorporation of DNA, RNA, and oligonucleotide constructs into thin polyelectrolyte films. Provided that these assemblies can be designed in ways that permit controlled film disassembly under physiological conditions, this approach can contribute new methods for spatial and/or temporal control over the delivery of nucleic acid-based therapeutics in vitro and in vivo. We describe applications of layer-by-layer assembly to the fabrication of DNA-containing films that can be used to provide control over the release of plasmid DNA from the surfaces of macroscopic objects and promote surface-mediated cell transfection. We also highlight the application of these methods to the coating of colloidal substrates and the fabrication of hollow micrometer-scale capsules that can be used to encapsulate and control the release or delivery of DNA and oligonucleotides. Current challenges, gaps in knowledge, and new opportunities for the development of these methods in the general area of gene delivery are discussed. PMID:18395291

  5. Rapid DNA hybridization analysis using a PDMS microfluidic sensor and a molecular beacon.

    PubMed

    Kim, Sungyong; Chen, Lingxin; Lee, Sangyeop; Seong, Gi Hun; Choo, Jaebum; Lee, Eun Kyu; Oh, Chil-Hwan; Lee, Sanghoon

    2007-04-01

    A rapid DNA analysis has been developed based on a fluorescence intensity change of a molecular beacon in a PDMS microfluidic channel. Recently, we reported a new analytical method of DNA hybridization involving a PDMS microfluidic sensor using fluorescence energy transfer (FRET). However, there are some limitations in its application to real DNA samples because the target DNA must be labelled with a suitable fluorescent dye. To resolve this problem, we have developed a new DNA microfluidic sensor using a molecular beacon. By monitoring the change in the restored fluorescence intensity along the channel length, it is possible to rapidly detect any hybridization of the molecular beacon to the target DNA. In this case, the target DNA does not need to be labelled. Our experimental results demonstrate that this microfluidic sensor using a molecular beacon is a promising diagnostic tool for rapid DNA hybridization analysis.

  6. Conformational dynamics of abasic DNA upon interactions with AP endonuclease 1 revealed by stopped-flow fluorescence analysis.

    PubMed

    Kanazhevskaya, Lyubov Yu; Koval, Vladimir V; Vorobjev, Yury N; Fedorova, Olga S

    2012-02-14

    Apurinic/apyrimidinic (AP) sites are abundant DNA lesions arising from exposure to UV light, ionizing radiation, alkylating agents, and oxygen radicals. In human cells, AP endonuclease 1 (APE1) recognizes this mutagenic lesion and initiates its repair via a specific incision of the phosphodiester backbone 5' to the AP site. We have investigated a detailed mechanism of APE1 functioning using fluorescently labeled DNA substrates. A fluorescent adenine analogue, 2-aminopurine, was introduced into DNA substrates adjacent to the abasic site to serve as an on-site reporter of conformational transitions in DNA during the catalytic cycle. Application of a pre-steady-state stopped-flow technique allows us to observe changes in the fluorescence intensity corresponding to different stages of the process in real time. We also detected an intrinsic Trp fluorescence of the enzyme during interactions with 2-aPu-containing substrates. Our data have revealed a conformational flexibility of the abasic DNA being processed by APE1. Quantitative analysis of fluorescent traces has yielded a minimal kinetic scheme and appropriate rate constants consisting of four steps. The results obtained from stopped-flow data have shown a substantial influence of the 2-aPu base location on completion of certain reaction steps. Using detailed molecular dynamics simulations of the DNA substrates, we have attributed structural distortions of AP-DNA to realization of specific binding, effective locking, and incision of the damaged DNA. The findings allowed us to accurately discern the step that corresponds to insertion of specific APE1 amino acid residues into the abasic DNA void in the course of stabilization of the precatalytic complex. PMID:22243137

  7. Conformational dynamics of abasic DNA upon interactions with AP endonuclease 1 revealed by stopped-flow fluorescence analysis.

    PubMed

    Kanazhevskaya, Lyubov Yu; Koval, Vladimir V; Vorobjev, Yury N; Fedorova, Olga S

    2012-02-14

    Apurinic/apyrimidinic (AP) sites are abundant DNA lesions arising from exposure to UV light, ionizing radiation, alkylating agents, and oxygen radicals. In human cells, AP endonuclease 1 (APE1) recognizes this mutagenic lesion and initiates its repair via a specific incision of the phosphodiester backbone 5' to the AP site. We have investigated a detailed mechanism of APE1 functioning using fluorescently labeled DNA substrates. A fluorescent adenine analogue, 2-aminopurine, was introduced into DNA substrates adjacent to the abasic site to serve as an on-site reporter of conformational transitions in DNA during the catalytic cycle. Application of a pre-steady-state stopped-flow technique allows us to observe changes in the fluorescence intensity corresponding to different stages of the process in real time. We also detected an intrinsic Trp fluorescence of the enzyme during interactions with 2-aPu-containing substrates. Our data have revealed a conformational flexibility of the abasic DNA being processed by APE1. Quantitative analysis of fluorescent traces has yielded a minimal kinetic scheme and appropriate rate constants consisting of four steps. The results obtained from stopped-flow data have shown a substantial influence of the 2-aPu base location on completion of certain reaction steps. Using detailed molecular dynamics simulations of the DNA substrates, we have attributed structural distortions of AP-DNA to realization of specific binding, effective locking, and incision of the damaged DNA. The findings allowed us to accurately discern the step that corresponds to insertion of specific APE1 amino acid residues into the abasic DNA void in the course of stabilization of the precatalytic complex.

  8. Homo-DNA templated chemistry and its application to nucleic acid sensing.

    PubMed

    Stoop, Matthias; Leumann, Christian J

    2011-07-14

    We have investigated the homo-DNA templated Staudinger reduction of the profluorophore rhodamine azide and have applied this reaction to the detection of natural DNA with a hybrid homo-DNA/DNA molecular beacon. In this system the sensing and the reporting unit are bioorthogonal to each other which facilitates sequence design and increases fidelity.

  9. The Effects of Topically Applied Glycolic Acid and Salicylic Acid on Ultraviolet Radiation-Induced Erythema, DNA Damage and Sunburn Cell Formation in Human Skin

    PubMed Central

    Kornhauser, Andrija; Wei, Rong-Rong; Yamaguchi, Yuji; Coelho, Sergio G.; Kaidbey, Kays; Barton, Curtis; Takahashi, Kaoruko; Beer, Janusz Z.; Miller, Sharon A.; Hearing, Vincent J.

    2009-01-01

    Background α-Hydroxy acids (αHA) are reported to reduce signs of aging in the skin and are widely used cosmetic ingredients. Several studies suggest that αHA can increase the sensitivity of skin to ultraviolet radiation. More recently, β-hydroxy acids (βHA), or combinations of αHA and βHA have also been incorporated into antiaging skin care products. Concerns have also arisen about increased sensitivity to ultraviolet radiation following use of skin care products containing β-HA. Objective To determine whether topical treatment with glycolic acid, a representative αHA, or with salicylic acid, a βHA, modifies the short-term effects of solar simulated radiation (SSR) in human skin. Methods Fourteen subjects participated in this study. Three of the four test sites on the mid-back of each subject were treated daily Monday - Friday, for a total of 3.5 weeks, with glycolic acid (10%), salicylic acid (2%), or vehicle (control). The fourth site received no treatment. After the last treatment, each site was exposed to SSR, and shave biopsies from all 4 sites were obtained. The endpoints evaluated in this study were erythema (assessed visually and instrumentally), DNA damage and sunburn cell formation. Results Treatment with glycolic acid resulted in increased sensitivity of human skin to SSR, measured as an increase in erythema, DNA damage and sunburn cell formation. Salicylic acid did not produce significant changes in any of these biomarkers. Conclusions Short-term topical application of glycolic acid in a cosmetic formulation increased the sensitivity of human skin to SSR, while a comparable treatment with salicylic acid did not. PMID:19411163

  10. Localization and DNA sequence analysis of the C gene of bacteriophage Mu, the positive regulator of Mu late transcription.

    PubMed Central

    Margolin, W; Howe, M M

    1986-01-01

    The C gene of bacteriophage Mu, required for transcription of the phage late genes, was localized by construction and analysis of a series of deleted derivatives of pKN50, a plasmid containing a 9.4 kb Mu DNA fragment which complements Mu C amber mutant phages for growth. One such deleted derivative, pWM10, containing only 0.5 kb of Mu DNA, complements C amber phages and transactivates the mom gene, one of the Mu late genes dependent on C for activation. The DNA sequence of the 0.5 kb fragment predicts a single long open reading frame coding for a 140 amino acid protein. Sequence analysis of DNA containing a C amber mutation located the base change to the second codon of this reading frame. Generation of a frameshift mutation by filling in a BglII site spanning codon 114 of this reading frame resulted in the loss of C complementation and transactivation activity. These results indicate that this open reading frame encodes the Mu C gene product. Comparison of the predicted amino acid sequence of the C protein with those of other transcriptional regulatory proteins revealed some similarity to a region highly conserved among bacterial sigma factors. Images PMID:3014438

  11. Synthesis, structural characterization and DNA interaction of zinc complex from 2,6-diacetylpyridine dihydrazone and {4-[(2E)-2-(hydroxyimino)acetyl]phenoxy} acetic acid.

    PubMed

    Gup, Ramazan; Gökçe, Cansu; Dilek, Nefise

    2015-03-01

    A new water soluble zinc complex has been prepared and structurally characterized. The Zn(II) complex was synthesized by the reaction of 2,6-diacetylpyridine dihydrazone (dph) with {4-[(2E)-2-(hydroxyimino)acetyl]phenoxy} acetic acid (H₂L) in the presence of zinc(II) acetate. Single crystal X-ray diffraction study revealed that the zinc ion is situated in distorted trigonal-bipyramidal environment where the equatorial position is occupied by the nitrogen atom of pyridine ring and the oxygen atoms of acetate groups of two oxime ligands (H₂L) whereas the axial positions of the zinc complex are occupied by the imine nitrogen atoms of dph ligand. Characterization of the complex with FTIR, (1)H and (13)C NMR, UV-vis and elemental analysis also confirmed the proposed structure. Interaction of the Zn(II) complex with calf-thymus DNA (CT-DNA) was investigated through UV-vis spectroscopy and viscosity measurements. The results suggest that the complex preferably bind to DNA through the groove binding mode. The zinc complex cleaves plasmid pBR 322 DNA in the presence and absence of an oxidative agent (H₂O₂), possibly through a hydrolytic pathway which is also supported by DNA cleave experiments in the presence of different radical scavengers. The nuclease activity of the zinc complex significantly depends on concentration of the complex and incubation time both in the presence and absence of H₂O₂. DNA cleave activity is inhibited in the presence of methyl green indicating that the zinc complex seems to bind the major groove of DNA.

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-08-01

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

  14. Hypoxically inducible barley lactate dehydrogenase: cDNA cloning and molecular analysis

    SciTech Connect

    Hondred, D. ); Hanson, A.D. Univ. de Montreal, Quebec )

    1990-09-01

    In the roots of barley and other cereals, hypoxia induces a set of five isozymes of L-lactate dehydrogenase (LDH; (S)-lactate:NADH oxidoreductase, EC 1.1.1.27). Biochemical and genetic data indicate that the five LDH isozymes are tetramers that arise from random association of the products of two Ldh loci. To investigate this system, cDNA clones of LDH were isolated from a {lambda}gt11 cDNA library derived from hypoxically treated barley roots. The library was screened with antiserum raised against barley LDH purified {approx}3,000-fold by an improved three-step procedure. Immunopositive clones were rescreened with a cDNA probe synthesized by the polymerase chain reaction using primers modeled from the amino acid sequences of two tryptic LDH peptides. Two types of LDH clones were found. Nucleotide sequence analysis of one representative insert of each type (respectively, 1,305 and 1,166 base pairs) revealed open reading framed encoding 10 peptide fragments of LDH. The 1,305-base-pair insert included the entire coding region of a 356-residue LDH monomer. The nucleotide sequences of the two LDH cDNAs were 92% identical in the coding region, but highly divergent in the 3{prime} noncoding region, and thus probably correspond to the two postulated Ldh loci. The deduced amino acid sequences of the two barley LDHs were 96% identical to each other and very similar to those from vertebrate and bacterial LDHs. RNA blot hybridization showed a single mRNA band of 1.5 kilobases whose level rose about 8-fold in roots during hypoxic induction, as did the level of translatable LDH message.

  15. Breaking the dogma: PCB-derived semiquinone free radicals do not form covalent adducts with DNA, GSH, and amino acids.

    PubMed

    Wangpradit, Orarat; Rahaman, Asif; Mariappan, S V Santhana; Buettner, Garry R; Robertson, Larry W; Luthe, Gregor

    2016-02-01

    Covalent bond formations of free radical metabolites with biomolecules like DNA and proteins are thought to constitute a major mechanism of toxicity and carcinogenesis. Glutathione (GSH) is generally accepted as a radical scavenger protecting the cell. In the present study, we investigated a semiquinone radical (SQ(●-)) metabolite of the semivolatile 4-chlorobiphenyl, using electron paramagnetic resonance spectroscopy, and oxygen consumption. Proton nuclear magnetic resonance ((1)H NMR) and liquid chromatography-mass spectrometry (LC-MS) were also employed to elucidate the radical interaction with DNA, amino acids, and GSH. We found that DNA and oligonucleotides stabilized SQ(●-) by electron delocalization in the π-stacking system, resulting in persistent radical intercalated, rather than forming a covalent bond with SQ(●-). This finding was strongly supported by the semiempirical calculation of the semioccupied molecular orbital and the linear combination of the atomic orbitals, indicating 9.8 kcal mol(-1) energy gain. The insertion of SQ(●-) into the DNA strand may result in DNA strand breaks and interruption of DNA replication process or even activate radical mediated secondary reactions. The presence of amino acids resulted in a decrease of the electron paramagnetic resonance (EPR) signal of SQ(●-) and correlated with their isoelectric points. The pH shifts the equilibrium of the dianions of hydroquinone and influenced indirectly the formation of SQ(●-). Similar findings were observed with GSH and Cys. GSH and Cys functioned as indirect radical scavengers; their activities depend on their chemical equilibria with the corresponding quinones, and their further reaction via Michael addition. The generally accepted role of GSH as radical scavenger in biological systems should be reconsidered based upon these findings, questioning the generally accepted view of radical interaction of semiquinones with biologically active compounds, like DNA, amino acids

  16. Ion-exchange chromatographic analysis of peroxynitric acid.

    PubMed

    Nakashima, Yoichi; Ikawa, Satoshi; Tani, Atsushi; Kitano, Katsuhisa

    2016-01-29

    Ion-exchange chromatographic analysis of peroxynitric acid (O2NOOH) was performed by combining an acidic eluate with an UV-vis detector and immersing the separation column in an ice-water bath. The decomposition behavior of peroxynitric acid in the solution was also studied using this system. The fraction for the peroxynitric acid peak was collected. Ion-exchange chromatographic analysis of this fraction, after standing at room temperature for 24h, showed that the decomposition products were mainly nitrate ions with a very small amount of nitrous acid. The peroxynitric acid peak area correlated perfectly with the total amount of decomposition products. The ion-exchange chromatographic isolation allowed us to evaluate the molar extinction coefficient of peroxynitric acid precisely in a wider wavelength range than previous reports. The value decreases monotonically from 1729±26M(-1)cm(-1) at 200nm to 12.0±0.5M(-1)cm(-1) at 290nm.

  17. Characterization and directed evolution of a methyl-binding domain protein for high-sensitivity DNA methylation analysis.

    PubMed

    Heimer, Brandon W; Tam, Brooke E; Sikes, Hadley D

    2015-12-01

    Methyl-binding domain (MBD) family proteins specifically bind double-stranded, methylated DNA which makes them useful for DNA methylation analysis. We displayed three of the core members MBD1, MBD2 and MBD4 on the surface of Saccharomyces cerevisiae cells. Using the yeast display platform, we determined the equilibrium dissociation constant of human MBD2 (hMBD2) to be 5.9 ± 1.3 nM for binding to singly methylated DNA. The measured affinity for DNA with two methylated sites varied with the distance between the sites. We further used the yeast display platform to evolve the hMBD2 protein for improved binding affinity. Affecting five amino acid substitutions doubled the affinity of the wild-type protein to 3.1 ± 1.0 nM. The most prevalent of these mutations, K161R, occurs away from the DNA-binding site and bridges the N- and C-termini of the protein by forming a new hydrogen bond. The F208Y and L170R mutations added new non-covalent interactions with the bound DNA strand. We finally concatenated the high-affinity MBD variant and expressed it in Escherichia coli as a green fluorescent protein fusion. Concatenating the protein from 1× to 3× improved binding 6-fold for an interfacial binding application. PMID:26384511

  18. Nonlinear analysis of correlations in Alu repeat sequences in DNA

    NASA Astrophysics Data System (ADS)

    Xiao, Yi; Huang, Yanzhao; Li, Mingfeng; Xu, Ruizhen; Xiao, Saifeng

    2003-12-01

    We report on a nonlinear analysis of deterministic structures in Alu repeats, one of the richest repetitive DNA sequences in the human genome. Alu repeats contain the recognition sites for the restriction endonuclease AluI, which is what gives them their name. Using the nonlinear prediction method developed in chaos theory, we find that all Alu repeats have novel deterministic structures and show strong nonlinear correlations that are absent from exon and intron sequences. Furthermore, the deterministic structures of Alus of younger subfamilies show panlike shapes. As young Alus can be seen as mutation free copies from the “master genes,” it may be suggested that the deterministic structures of the older subfamilies are results of an evolution from a “panlike” structure to a more diffuse correlation pattern due to mutation.

  19. Phylogenetic and epidemiological analysis of Neisseria meningitidis using DNA probes.

    PubMed Central

    Ni, H.; Knight, A. I.; Cartwright, K. A.; McFadden, J. J.

    1992-01-01

    The genetic relationships between various serotypes and serogroups of meningococcal strains were investigated by restriction fragment-length polymorphism (RFLP) analysis using a number of random DNA probes and a probe containing a truncated copy of the meningococcal insertion sequence IS1106. The data were used to estimate genetic distance between all pairs of strains and to construct phylogenetic trees for meningococcal strains. B15:P1.16R strains isolated from cases of systemic meningococcal disease in two health districts with a high incidence of disease were clonal in contrast to similar strains from cases occurring in other parts of the UK. Strains from these areas, which contain a similar genomic deletion, were found to be derived from two distinct lineages within the B15:P1.16R phylogenetic group. RFLP data demonstrated that present serological typing systems for the meningococcus do not necessarily reflect true genetic relationships. Images Fig. 1 PMID:1356820

  20. Applying machine learning techniques to DNA sequence analysis

    SciTech Connect

    Shavlik, J.W.

    1992-01-01

    We are developing a machine learning system that modifies existing knowledge about specific types of biological sequences. It does this by considering sample members and nonmembers of the sequence motif being learned. Using this information (which we call a domain theory''), our learning algorithm produces a more accurate representation of the knowledge needed to categorize future sequences. Specifically, the KBANN algorithm maps inference rules, such as consensus sequences, into a neural (connectionist) network. Neural network training techniques then use the training examples of refine these inference rules. We have been applying this approach to several problems in DNA sequence analysis and have also been extending the capabilities of our learning system along several dimensions.

  1. Freshwater foraminiferans revealed by analysis of environmental DNA samples.

    PubMed

    Holzmann, Maria; Habura, Andrea; Giles, Hannah; Bowser, Samuel S; Pawlowski, Jan

    2003-01-01

    Sediment-dwelling protists are among the most abundant meiobenthic organisms, ubiquitous in all types of aquatic ecosystems. Yet, because their isolation and identification are difficult, their diversity remains largely unknown. In the present work, we applied molecular methods to examine the diversity of freshwater Foraminifera, a group of granuloreticulosan protists largely neglected until now. By using specific PCR primers, we detected the presence of Foraminifera in all sediment samples examined. Phylogenetic analysis of amplified SSU rDNA sequences revealed two distinct groups of freshwater foraminiferans. All obtained sequences branched within monothalamous (single-chambered), marine Foraminifera, suggesting a repeated colonization of freshwater environments. The results of our study challenge the traditional view of Foraminifera as essentially marine organisms, and provide a conceptual framework for charting the molecular diversity of freshwater granuloreticulosan protists.

  2. Cytometric analysis of shape and DNA content in mammalian sperm

    SciTech Connect

    Gledhill, B.L.

    1983-10-10

    Male germ cells respond dramatically to a variety of insults and are important reproductive dosimeters. Semen analyses are very useful in studies on the effects of drugs, chemicals, and environmental hazards on testicular function, male fertility and heritable germinal mutations. Sperm were analyzed by flow cytometry and slit-scan flow analysis for injury following the exposure of testes to mutagens. The utility of flow cytometry in genotoxin screening and monitoring of occupational exposure was evaluated. The technique proved valuable in separation of X- and Y-chromosome bearing sperm and the potential applicability of this technique in artificial insemination and a solution, of accurately assessing the DNA content of sperm were evaluated-with reference to determination of X- and Y-chromosome bearing sperm.

  3. A mushroom-derived amino acid, ergothioneine, is a potential inhibitor of inflammation-related DNA halogenation.

    PubMed

    Asahi, Takashi; Wu, Xiaohong; Shimoda, Hiroshi; Hisaka, Shinsuke; Harada, Etsuko; Kanno, Tomomi; Nakamura, Yoshimasa; Kato, Yoji; Osawa, Toshihiko

    2016-01-01

    Myeloperoxidase (MPO)-generated halogenating molecules, such as hypochlorous acid and hypobromous acid (HOBr), in inflammatory regions are postulated to contribute to disease progression. In this study, we showed that ergothioneine (EGT), derived from an edible mushroom, inhibited MPO activity as well as the formation of 8-bromo-2'-deoxyguanosine in vitro. The HOBr scavenging effect of EGT is higher than those of ascorbic acid and glutathione. We initially observed that the administration of Coprinus comatus, an edible mushroom containing a high amount of EGT, inhibited the UV-B-induced inflammatory responses and DNA halogenation, suggesting that EGT is a promising anti-inflammatory agent from mushrooms.

  4. Pre-Steady-State Kinetic Analysis of Single-Nucleotide Incorporation by DNA Polymerases.

    PubMed

    Su, Yan; Peter Guengerich, F

    2016-06-01

    Pre-steady-state kinetic analysis is a powerful and widely used method to obtain multiple kinetic parameters. This protocol provides a step-by-step procedure for pre-steady-state kinetic analysis of single-nucleotide incorporation by a DNA polymerase. It describes the experimental details of DNA substrate annealing, reaction mixture preparation, handling of the RQF-3 rapid quench-flow instrument, denaturing polyacrylamide DNA gel preparation, electrophoresis, quantitation, and data analysis. The core and unique part of this protocol is the rationale for preparation of the reaction mixture (the ratio of the polymerase to the DNA substrate) and methods for conducting pre-steady-state assays on an RQF-3 rapid quench-flow instrument, as well as data interpretation after analysis. In addition, the methods for the DNA substrate annealing and DNA polyacrylamide gel preparation, electrophoresis, quantitation and analysis are suitable for use in other studies. © 2016 by John Wiley & Sons, Inc.

  5. Analysis of beta-carotene hydroxylase gene cDNA isolated from the American oil-palm (Elaeis oleifera) mesocarp tissue cDNA library

    PubMed Central

    Bhore, Subhash J; Kassim, Amelia; Loh, Chye Ying; Shah, Farida H

    2010-01-01

    It is well known that the nutritional quality of the American oil-palm (Elaeis oleifera) mesocarp oil is superior to that of African oil-palm (Elaeis guineensis Jacq. Tenera) mesocarp oil. Therefore, it is of important to identify the genetic features for its superior value. This could be achieved through the genome sequencing of the oil-palm. However, the genome sequence is not available in the public domain due to commercial secrecy. Hence, we constructed a cDNA library and generated expressed sequence tags (3,205) from the mesocarp tissue of the American oil-palm. We continued to annotate each of these cDNAs after submitting to GenBank/DDBJ/EMBL. A rough analysis turned our attention to the beta-carotene hydroxylase (Chyb) enzyme encoding cDNA. Then, we completed the full sequencing of cDNA clone for its both strands using M13 forward and reverse primers. The full nucleotide and protein sequence was further analyzed and annotated using various Bioinformatics tools. The analysis results showed the presence of fatty acid hydroxylase superfamily domain in the protein sequence. The multiple sequence alignment of selected Chyb amino acid sequences from other plant species and algal members with E. oleifera Chyb using ClustalW and its phylogenetic analysis suggest that Chyb from monocotyledonous plant species, Lilium hubrid, Crocus sativus and Zea mays are the most evolutionary related with E. oleifera Chyb. This study reports the annotation of E. oleifera Chyb. Abbreviations ESTs - expressed sequence tags, EoChyb - Elaeis oleifera beta-carotene hydroxylase, MC - main cluster PMID:21364789

  6. Simultaneous analysis of micro-RNA and DNA for determining the body fluid origin of DNA profiles.

    PubMed

    van der Meer, Donny; Uchimoto, Mari L; Williams, Graham

    2013-07-01

    Micro-RNAs (miRNAs) can be specifically expressed in forensically relevant body fluids such as blood or saliva. The aim of the study was to develop a simultaneous extraction and analysis protocol that allows for the acquisition of a DNA profile and the identity of the body fluid using a single process. DNA and micro-RNA were extracted from blood and saliva before undergoing a cDNA synthesis step by using stem-loop reverse transcription PCR. The resulting extracts containing DNA and cDNA synthesized from body fluid-specific miRNA markers then underwent standard STR analysis using a modified ABI AmpFℓSTR(®) NGM SElect™ kit. In all samples, a full DNA profile was obtained along with additional peaks corresponding to the miRNA marker targeted. In all cases, blood samples profiled exhibited a peak indicating the presence of the blood-specific miRNA marker and the saliva sample profiled exhibited a peak indicating the presence of the saliva-specific miRNA marker.

  7. Computational investigation of locked nucleic acid (LNA) nucleotides in the active sites of DNA polymerases by molecular docking simulations.

    PubMed

    Poongavanam, Vasanthanathan; Madala, Praveen K; Højland, Torben; Veedu, Rakesh N

    2014-01-01

    Aptamers constitute a potential class of therapeutic molecules typically selected from a large pool of oligonucleotides against a specific target. With a scope of developing unique shorter aptamers with very high biostability and affinity, locked nucleic acid (LNA) nucleotides have been investigated as a substrate for various polymerases. Various reports showed that some thermophilic B-family DNA polymerases, particularly KOD and Phusion DNA polymerases, accepted LNA-nucleoside 5'-triphosphates as substrates. In this study, we investigated the docking of LNA nucleotides in the active sites of RB69 and KOD DNA polymerases by molecular docking simulations. The study revealed that the incoming LNA-TTP is bound in the active site of the RB69 and KOD DNA polymerases in a manner similar to that seen in the case of dTTP, and with LNA structure, there is no other option than the locked C3'-endo conformation which in fact helps better orienting within the active site. PMID:25036012

  8. Crystallization of bFGF-DNA Aptamer Complexes Using a Sparse Matrix Designed for Protein-Nucleic Acid Complexes

    NASA Technical Reports Server (NTRS)

    Cannone, Jaime J.; Barnes, Cindy L.; Achari, Aniruddha; Kundrot, Craig E.; Whitaker, Ann F. (Technical Monitor)

    2001-01-01

    The Sparse Matrix approach for obtaining lead crystallization conditions has proven to be very fruitful for the crystallization of proteins and nucleic acids. Here we report a Sparse Matrix developed specifically for the crystallization of protein-DNA complexes. This method is rapid and economical, typically requiring 2.5 mg of complex to test 48 conditions. The method was originally developed to crystallize basic fibroblast growth factor (bFGF) complexed with DNA sequences identified through in vitro selection, or SELEX, methods. Two DNA aptamers that bind with approximately nanomolar affinity and inhibit the angiogenic properties of bFGF were selected for co-crystallization. The Sparse Matrix produced lead crystallization conditions for both bFGF-DNA complexes.

  9. Fluorescence determination of DNA with 1-pyrenebutyric acid nanoparticles coated with β-cyclodextrin as a fluorescence probe

    NASA Astrophysics Data System (ADS)

    Wang, Lun; Bian, Guirong; Wang, Leyu; Dong, Ling; Chen, Hongqi; Xia, Tingting

    2005-04-01

    A novel ultrasonication method has been successfully developed for the preparation of 1-pyrenebutyric acid (PBAC)/β-cyclodextrin(β-CD) complex nanoparticles. The as-prepared nanoparticles are characterized by transmission electron microscopy (TEM), fluorescence excitation and emission spectroscopy. Complex nanoparticles prepared with ultrasonication are smaller and better dispersed than single PBAC nanoparticles. At pH 3.0, the relative fluorescence intensity of complex nanoparticles of PBAC/β-CD can be quenched by the concentration of DNA. Based on this, a novel fluorimetric method has been developed for rapid determination of DNA. In comparison with single organic fluorophores, these nanoparticle probes are better water-solubility, more stable and do not suffer from blinking. Under optimum conditions, the calibration graphs are linear over the range 0.2-15 μg mL -1 for calf thymus DNA (ct-DNA) and 0.3-12 μg mL -1 for fish sperm DNA (fs-DNA). The corresponding detection limit is 0.01 μg mL -1 for ct-DNA and 0.02 μg mL -1 for fs-DNA. The relative standard deviation of seven replicate measurements is 1.2% for 2.0 μg mL -1 ct-DNA and 1.4% for 2.0 μg mL -1 fs-DNA, respectively. The method is simple and sensitive. The recovery and relative standard deviation are very satisfactory. A mechanism proposed to explain the process also has been studied.

  10. Gas chromatographic analysis of total fatty acids in cider.

    PubMed

    Blanco-Gomis, D; Alonso, J J; Cabrales, I M; Abrodo, P A

    2001-03-01

    This paper reports the composition of total fatty acids in an apple beverage, cider. Fatty acids are present in the free or esterified form and contribute to both the flavor and foam properties of cider. Fatty acids were separated and identified as methyl esters by GC-MS, and 12 of these were subsequently determined by GC-FID. The major fatty acids found in cider were caproic, caprylic, capric, and palmitic acid, the saturated acids predominating over the unsaturated ones. The proposed method was applied to 59 ciders from three consecutive harvests (1996, 1997, and 1998), which were made by 19 cider-makers from the region of Asturias (Spain). Linear discriminant analysis of fatty acids in these samples allowed selection of palmitoleic, pentadecanoic, linoleic, myristic, and linolenic acid as the most predictive variables to differentiate ciders made from apples grown in the Asturias region (1997 harvest) and ciders made from apples grown outside this region (1996 and 1998 harvests). PMID:11312846

  11. Products of ozonized arachidonic acid potentiate the formation of DNA single strand breaks in cultured human lung cells

    SciTech Connect

    Kozumbo, W.J.; Hanley, N.M.; Agarwal, S.

    1996-12-31

    In this study we examined the potential for environmental levels of ozone (O{sub 3}) to degrade arachidonic acid (AA), a polyunsaturated fatty acid abundantly present in the lung, into products that can produce DNA single strand breaks (ssb) in cultured human lung cells. Human lung fibroblasts were incubated with 60 {mu}M AA that had been previously exposed to an degraded by 0.4 ppm O{sub 3} (1 hr). Incubation of the cells with O{sub 3}-exposed AA (but not with vehicle alone) for 1 hr at 4{degrees}C and 37{degrees}C produced 555 and 245 rad-equivalents of DNA ssb, respectively, as determined by the DNA alkaline elution technique. These breaks were completely eliminated when the ozonized AA solution was incubated with catalase prior to cell treatment, indicating that H{sub 2}O{sub 2} was solely responsible for damaging DNA. Superoxide dismutase, bovine serum albumin, or heat-inactivated catalase showed little, if any, inhibitory activity. The H{sub 2}O{sub 2} content for only about 40% of the observed breaks. Potentiation of the H{sub 2}O{sub 2}-induced DNA ssb persisted after removal of the carbonyl substances by chromatographic procedures, suggesting that the non-carbonyl component of ozonized AA was the responsible component for inducing augmentation of the observed increases in DNA ssb. Ozonized AA also induced DNA ssb in cultures of the human bronchial epithelial cell line BEAS-2B. Again, these breaks were shown to exceed levels that could be attributed to the presence of H{sub 2}O{sub 2} alone. These results indicate that products of ozonized AA can interact to potentiate DNA ssb in human lung cells. 42 refs., 6 figs., 3 tabs.

  12. Target-catalyzed autonomous assembly of dendrimer-like DNA nanostructures for enzyme-free and signal amplified colorimetric nucleic acids detection.

    PubMed

    He, Hongfei; Dai, Jianyuan; Duan, Zhijuan; Meng, Yan; Zhou, Cuisong; Long, Yuyin; Zheng, Baozhan; Du, Juan; Guo, Yong; Xiao, Dan

    2016-12-15

    Self-assembly of DNA nanostructures is of great importance in nanomedicine, nanotechnology and biosensing. Herein, a novel target-catalyzed autonomous assembly pathway for the formation of dendrimer-like DNA nanostructures that only employing target DNA and three hairpin DNA probes was proposed. We use the sticky-ended Y shape DNA (Y-DNA) as the assembly monomer and it was synthesized by the catalyzed hairpin assembly (CHA) instead of the DNA strand annealing method. The formed Y-DNA was equipped with three ssDNA sticky ends and two of them were predesigned to be complementary to the third one, then the dendrimer-like DNA nanostructures can be obtained via an autonomous assembly among these sticky-ended Y-DNAs. The resulting nanostructure has been successfully applied to develop an enzyme-free and signal amplified gold nanoparticle (AuNP)-based colorimetric nucleic acids assay. PMID:27498325

  13. A new antifungal peptide from the seeds of Phytolacca americana: characterization, amino acid sequence and cDNA cloning.

    PubMed

    Shao, F; Hu, Z; Xiong, Y M; Huang, Q Z; WangCG; Zhu, R H; Wang, D C

    1999-03-19

    An antifungal peptide from seeds of Phytolacca americana, designated PAFP-s, has been isolated. The peptide is highly basic and consists of 38 residues with three disulfide bridges. Its molecular mass of 3929.0 was determined by mass spectrometry. The complete amino acid sequence was obtained from automated Edman degradation, and cDNA cloning was successfully performed by 3'-RACE. The deduced amino acid sequence of a partial cDNA corresponded to the amino acid sequence from chemical sequencing. PAFP-s exhibited a broad spectrum of antifungal activity, and its activities differed among various fungi. PAFP-s displayed no inhibitory activity towards Escherichia coli. PAFP-s shows significant sequence similarities and the same cysteine motif with Mj-AMPs, antimicrobial peptides from seeds of Mirabilis jalapa belonging to the knottin-type antimicrobial peptide.

  14. Vertically integrated analysis of human DNA. Final technical report

    SciTech Connect

    Olson, M.

    1997-10-01

    This project has been oriented toward improving the vertical integration of the sequential steps associated with the large-scale analysis of human DNA. The central focus has been on an approach to the preparation of {open_quotes}sequence-ready{close_quotes} maps, which is referred to as multiple-complete-digest (MCD) mapping, primarily directed at cosmid clones. MCD mapping relies on simple experimental steps, supported by advanced image-analysis and map-assembly software, to produce extremely accurate restriction-site and clone-overlap maps. We believe that MCD mapping is one of the few high-resolution mapping systems that has the potential for high-level automation. Successful automation of this process would be a landmark event in genome analysis. Once other higher organisms, paving the way for cost-effective sequencing of these genomes. Critically, MCD mapping has the potential to provide built-in quality control for sequencing accuracy and to make possible a highly integrated end product even if there are large numbers of discontinuities in the actual sequence.

  15. Non-Viral DNA Delivery from Porous Hyaluronic Acid Hydrogels in Mice

    PubMed Central

    Tokatlian, Talar; Cam, Cynthia; Segura, Tatiana

    2013-01-01

    The lack of vascularization within tissue-engineered constructs remains the primary cause of construct failure following implantation. Porous constructs have been successful in allowing for vessel infiltration without requiring extensive matrix degradation. We hypothesized that the rate and maturity of infiltrating vessels could be enhanced by complementing the open pore structure with the added delivery of DNA encoding for angiogenic growth factors. Both 100 and 60 μm porous and non-porous hyaluronic acid hydrogels loaded with pro-angiogenic (pVEGF) or reporter (pGFPluc) plasmid nanoparticles were used to study the effects of pore size and DNA delivery on angiogenesis in a mouse subcutaneous implant model. GFP-expressing transfected cells were found inside all control hydrogels over the course of the study, although transfection levels peaked by week 3 for 100 and 60 μm porous hydrogels. Transfection in non-porous hydrogels continued to increase over time corresponding with continued surface degradation. pVEGF transfection levels were not high enough to enhance angiogenesis by increasing vessel density, maturity, or size, although by 6 weeks for all pore size hydrogels more hydrogel implants were positive for vascularization when pVEGF polyplexes were incorporated compared to control hydrogels. Pore size was found to be the dominant factor in determining the angiogenic response with 60 μm porous hydrogels having more vessels/area present than 100 μm porous hydrogels at the initial onset of angiogenesis at 3 weeks. The results of this study show promise for the use of polyplex loaded porous hydrogels to transfect infiltrating cells in vivo and guide tissue regeneration and repair. PMID:24210142

  16. Development of an optical biosensor based on surface-enhanced Raman scattering for DNA analysis

    NASA Astrophysics Data System (ADS)

    Yigit, Tugce; Akdogan, Ebru; Karagoz, Isık. Didem; Kahraman, Mehmet

    2016-03-01

    Rapid, accurate and sensitive DNA analysis is critically important for the diagnostic of genetic diseases. The most common method preferred in practice is fluorescence based microarrays to analyze the DNA. However, there exist some disadvantages related to the above-mentioned method such as the overlapping of the fluorescence emission wavelengths that can diminish in the performance of multiplexing, needed to obtain fluorescence spectra from each dye and photo degradation. In this study, a novel SERS based DNA analysis approach, which is Raman active dye-free and independent of SERS substrate properties, is developed. First, the single strand DNA probe is attached to the SERS substrate and half of the complimentary DNA is attached to gold nanoparticles, as well. We hypothesize that in the presence of target DNA, the complimentary DNA coupled colloids will bind to the SERS substrate surface via hybridization of single strand target DNA. To test this hypothesis, we used UV/Vis spectroscopy, atomic for microscopy (AFM) and dynamic light scattering (DLS). DNA analysis is demonstrated by a peak shift of the certain peak of the small molecules attached to the SERS substrate surface instead of SERS spectrum obtained in the presence of target DNA from the Raman reporter molecules. The degree of peak shifting will be used for the quantification of the target DNA in the sample. Plasmonic properties of SERS substrates and reproducibility issues will not be considerable due to the use of peak shifting instead of peak intensity for the qualitative analysis.

  17. DNA-LCEB: a high-capacity and mutation-resistant DNA data-hiding approach by employing encryption, error correcting codes, and hybrid twofold and fourfold codon-based strategy for synonymous substitution in amino acids.

    PubMed

    Hafeez, Ibbad; Khan, Asifullah; Qadir, Abdul

    2014-11-01

    Data-hiding in deoxyribonucleic acid (DNA) sequences can be used to develop an organic memory and to track parent genes in an offspring as well as in genetically modified organism. However, the main concerns regarding data-hiding in DNA sequences are the survival of organism and successful extraction of watermark from DNA. This implies that the organism should live and reproduce without any functional disorder even in the presence of the embedded data. Consequently, performing synonymous substitution in amino acids for watermarking becomes a primary option. In this regard, a hybrid watermark embedding strategy that employs synonymous substitution in both twofold and fourfold codons of amino acids is proposed. This work thus presents a high-capacity and mutation-resistant watermarking technique, DNA-LCEB, for hiding secret information in DNA of living organisms. By employing the different types of synonymous codons of amino acids, the data storage capacity has been significantly increased. It is further observed that the proposed DNA-LCEB employing a combination of synonymous substitution, lossless compression, encryption, and Bose-Chaudary-Hocquenghem coding is secure and performs better in terms of both capacity and robustness compared to existing DNA data-hiding schemes. The proposed DNA-LCEB is tested against different mutations, including silent, miss-sense, and non-sense mutations, and provides substantial improvement in terms of mutation detection/correction rate and bits per nucleotide. A web application for DNA-LCEB is available at http://111.68.99.218/DNA-LCEB.

  18. Extraterrestrial material analysis: loss of amino acids during liquid-phase acid hydrolysis

    NASA Astrophysics Data System (ADS)

    Buch, Arnaud; Brault, Amaury; Szopa, Cyril; Freissinet, Caroline

    2015-04-01

    Searching for building blocks of life in extraterrestrial material is a way to learn more about how life could have appeared on Earth. With this aim, liquid-phase acid hydrolysis has been used, since at least 1970 , in order to extract amino acids and other organic molecules from extraterrestrial materials (e.g. meteorites, lunar fines) or Earth analogues (e.g. Atacama desert soil). This procedure involves drastic conditions such as heating samples in 6N HCl for 24 h, either under inert atmosphere/vacuum, or air. Analysis of the hydrolyzed part of the sample should give its total (free plus bound) amino acid content. The present work deals with the influence of the 6N HCl hydrolysis on amino acid degradation. Our experiments have been performed on a standard solution of 17 amino acids. After liquid-phase acid hydrolysis (6N HCl) under argon atmosphere (24 h at 100°C), the liquid phase was evaporated and the dry residue was derivatized with N-Methyl-N-(t-butyldimethylsilyl)trifluoroacetamide (MTBSTFA) and dimethylformamide (DMF), followed by gas chromatography-mass spectrometry analysis. After comparison with derivatized amino acids from the standard solution, a significant reduction of the chromatographic peak areas was observed for most of the amino acids after liquid-phase acid hydrolysis. Furthermore, the same loss pattern was observed when the amino acids were exposed to cold 6N HCl for a short amount of time. The least affected amino acid, i.e. glycine, was found to be 73,93% percent less abundant compared to the non-hydrolyzed standard, while the most affected, i.e. histidine, was not found in the chromatograms after hydrolysis. Our experiments thereby indicate that liquid-phase acid hydrolysis, even under inert atmosphere, leads to a partial or total loss of all of the 17 amino acids present in the standard solution, and that a quick cold contact with 6N HCl is sufficient to lead to a loss of amino acids. Therefore, in the literature, the reported increase

  19. Analysis of bacterial diversity in acidic pond water and compost after treatment of artificial acid mine drainage for metal removal.

    PubMed

    Morales, Teresita A; Dopson, Mark; Athar, Rana; Herbert, Roger B

    2005-06-01

    The microbial population of a sludge amended leaf compost material utilized for treatment of artificial acid mine drainage was studied by culture-independent molecular methods. Iron-rich and sulfurous wastewater (artificial acid mine drainage) was circulated through a column bioreactor for 16 months. After 12 months the column was inoculated with a mixed culture from an acidic pond receiving acid mine drainage from a tailings impoundment at a decommissioned site in Kristineberg, North Sweden. Hydrogen sulfide odor and the formation of black precipitates indicated that sulfate-reduction occurred in the column. 16S rDNA gene analysis by denaturing gradient gel electrophoresis, cloning, and sequencing as well as fluorescent in situ hybridization confirmed the presence of microorganisms closely related to sulfate-reducing bacteria and microorganisms from the genera Pseudoxanthmonas, Dechlorosoma, Desulfovibrio, Agrobacterium, Methylocapsa, Rhodococcus, Sulfobacillus, and some unidentified bacteria. Sulfate-reducing bacteria were found in the column bioreactor 2 weeks after inoculation, but not thereafter. This suggests they were in low abundance, even though sulfate remediation rates were significant. Instead, the population contained species similar to those previously found to utilize humic substances released from the compost material. PMID:15818559

  20. Structure and DNA Hybridization Properties of Mixed Nucleic Acid/Maleimide-Ethylene Glycol Monolayers

    SciTech Connect

    Lee,C.; Nguyen, P.; Grainger, D.; Gamble, L.; Castner, D.

    2007-01-01

    The surface structure and DNA hybridization performance of thiolated single-strand DNA (HS-ssDNA) covalently attached to a maleimide-ethylene glycol disulfide (MEG) monolayer on gold have been investigated. Monolayer immobilization chemistry and surface coverage of reactive ssDNA probes were studied by X-ray photoelectron spectroscopy and time-of-flight secondary ion mass spectrometry. Orientation of the ssDNA probes was determined by near-edge X-ray absorption fine structure (NEXAFS). Target DNA hybridization on the DNA-MEG probe surfaces was measured by surface plasmon resonance (SPR) to demonstrate the utility of these probe surfaces for detection of DNA targets from both purified target DNA samples and complex biological mixtures such as blood serum. Data from complementary techniques showed that immobilized ssDNA density is strongly dependent on the spotted bulk DNA concentration and buffer ionic strength. Variation of the immobilized ssDNA density had a profound influence on the DNA probe orientation at the surface and subsequent target hybridization efficiency. With increasing surface probe density, NEXAFS polarization dependence results (followed by monitoring the N 1s {yields} {pi}* transition) indicate that the immobilized ssDNA molecules reorient toward a more upright position on the MEG monolayer. SPR assays of DNA targets from buffer and serum showed that DNA hybridization efficiency increased with decreasing surface probe density. However, target detection in serum was better on the 'high-density' probe surface than on the 'high-efficiency' probe surface. The amounts of target detected for both ssDNA surfaces were several orders of magnitude poorer in serum than in purified DNA samples due to nonspecific serum protein adsorption onto the sensing surface.

  1. Folic Acid Inhibits Amyloid β-Peptide Production through Modulating DNA Methyltransferase Activity in N2a-APP Cells.

    PubMed

    Li, Wen; Jiang, Mingyue; Zhao, Shijing; Liu, Huan; Zhang, Xumei; Wilson, John X; Huang, Guowei

    2015-10-20

    Alzheimer's disease (AD) is a common neurodegenerative disease resulting in progressive dementia, and is a principal cause of dementia among older adults. Folate acts through one-carbon metabolism to support the methylation of multiple substrates. We hypothesized that folic acid supplementation modulates DNA methyltransferase (DNMT) activity and may alter amyloid β-peptide (Aβ) production in AD. Mouse Neuro-2a cells expressing human APP695 were incubated with folic acid (2.8-40 μmol/L), and with or without zebularine (the DNMT inhibitor). DNMT activity, cell viability, Aβ and DNMTs expression were then examined. The results showed that folic acid stimulated DNMT gene and protein expression, and DNMT activity. Furthermore, folic acid decreased Aβ protein production, whereas inhibition of DNMT activity by zebularine increased Aβ production. The results indicate that folic acid induces methylation potential-dependent DNMT enzymes, thereby attenuating Aβ production.

  2. Sequence analysis of the cDNA for the human casein kinase I {delta} (CSNK1D) gene and its chromosomal localization

    SciTech Connect

    Kusuda, Jun; Hidari, Nobuko; Hirai, Momoki; Hashimoto, Katsuyuki

    1996-02-15

    This article reports on the genetic mapping of a cDNA clone encoding human casein kinase I (CK1) using fluorescence in situ hybridization and polymerase chain reaction analysis of human-rodent hybrid cell panels. When compared to the amino acid sequence in the kinase domain of the rat, this cDNA seems to be a human homologue of the CK1 {delta} isoform. Sequence similarity to the kinase domains and function in DNA repair in Saccharomyces cerevisiae and Saccharomyces pombe are discussed. 14 refs., 2 figs.

  3. Tissue extraction of DNA and RNA and analysis by the polymerase chain reaction.

    PubMed

    Jackson, D P; Lewis, F A; Taylor, G R; Boylston, A W; Quirke, P

    1990-06-01

    Several DNA extraction techniques were quantitatively and qualitatively compared using both fresh and paraffin wax embedded tissue and their suitability investigated for providing DNA and RNA for the polymerase chain reaction (PCR). A one hour incubation with proteinase K was the most efficient DNA extraction procedure for fresh tissue. For paraffin wax embedded tissue a five day incubation with proteinase K was required to produce good yields of DNA. Incubation with sodium dodecyl sulphate produced very poor yields, while boiling produced 20% as much DNA as long enzyme digestion. DNA extracted by these methods was suitable for the PCR amplification of a single copy gene. Proteinase K digestion also produced considerable amounts of RNA which has previously been shown to be suitable for PCR analysis. A delay before fixation had no effect on the amount of DNA obtained while fixation in Carnoy's reagent results in a much better preservation of DNA than formalin fixation, allowing greater yields to be extracted.

  4. Effect of food processing on plant DNA degradation and PCR-based GMO analysis: a review.

    PubMed

    Gryson, Nicolas

    2010-03-01

    The applicability of a DNA-based method for GMO detection and quantification depends on the quality and quantity of the DNA. Important food-processing conditions, for example temperature and pH, may lead to degradation of the DNA, rendering PCR analysis impossible or GMO quantification unreliable. This review discusses the effect of several food processes on DNA degradation and subsequent GMO detection and quantification. The data show that, although many of these processes do indeed lead to the fragmentation of DNA, amplification of the DNA may still be possible. Length and composition of the amplicon may, however, affect the result, as also may the method of extraction used. Also, many techniques are used to describe the behaviour of DNA in food processing, which occasionally makes it difficult to compare research results. Further research should be aimed at defining ingredients in terms of their DNA quality and PCR amplification ability, and elaboration of matrix-specific certified reference materials. PMID:20012944

  5. Effect of food processing on plant DNA degradation and PCR-based GMO analysis: a review.

    PubMed

    Gryson, Nicolas

    2010-03-01

    The applicability of a DNA-based method for GMO detection and quantification depends on the quality and quantity of the DNA. Important food-processing conditions, for example temperature and pH, may lead to degradation of the DNA, rendering PCR analysis impossible or GMO quantification unreliable. This review discusses the effect of several food processes on DNA degradation and subsequent GMO detection and quantification. The data show that, although many of these processes do indeed lead to the fragmentation of DNA, amplification of the DNA may still be possible. Length and composition of the amplicon may, however, affect the result, as also may the method of extraction used. Also, many techniques are used to describe the behaviour of DNA in food processing, which occasionally makes it difficult to compare research results. Further research should be aimed at defining ingredients in terms of their DNA quality and PCR amplification ability, and elaboration of matrix-specific certified reference materials.

  6. Affordable hands-on DNA sequencing and genotyping: an exercise for teaching DNA analysis to undergraduates.

    PubMed

    Shah, Kushani; Thomas, Shelby; Stein, Arnold

    2013-01-01

    In this report, we describe a 5-week laboratory exercise for undergraduate biology and biochemistry students in which students learn to sequence DNA and to genotype their DNA for selected single nucleotide polymorphisms (SNPs). Students use miniaturized DNA sequencing gels that require approximately 8 min to run. The students perform G, A, T, C Sanger sequencing reactions. They prepare and run the gels, perform Southern blots (which require only 10 min), and detect sequencing ladders using a colorimetric detection system. Students enlarge their sequencing ladders from digital images of their small nylon membranes, and read the sequence manually. They compare their reads with the actual DNA sequence using BLAST2. After mastering the DNA sequencing system, students prepare their own DNA from a cheek swab, polymerase chain reaction-amplify a region of their DNA that encompasses a SNP of interest, and perform sequencing to determine their genotype at the SNP position. A family pedigree can also be constructed. The SNP chosen by the instructor was rs17822931, which is in the ABCC11 gene and is the determinant of human earwax type. Genotypes at the rs178229931 site vary in different ethnic populations.

  7. Primers and a specific DNA probe for detecting lactic acid bacteria producing 3-hydroxypropionaldehyde from glycerol in spoiled ciders.

    PubMed

    Claisse, O; Lonvaud-Funel, A

    2001-06-01

    Of the 40 strains isolated from several spoiled ciders where glycerol was degraded, 36 were identified as Lactobacillus collinoides, three were Lactobacillus hilgardii, and one was Lactobacillus mali. However, only 30 L. collinoides and two L. hilgardii could degrade glycerol. The glycerol dehydratase activity was shown. The main product of the transformation was 1.3 propanediol. Two DNA primers GD1 and GD2 were chosen in the region encoding one of the subunits of glycerol dehydratase of Citrobacter freundii, Klebsiella pneumoniae, Klebsiella oxytoca, Salmonella Typhimurium, and Clostridium pasteurianum. A 279-bp amplicon in polymerase chain reaction amplification was obtained with the genomic L. collinoides IOEB 9527 DNA as template. The amino acid sequence deduced from the amplicon DNA sequence showed a very high similarity and identity with the gene of gram-negative and C. pasteurianum species. After labeling, the amplicon was used as DNA probe in dot-blot hybridization with the genomic DNA of all the tested strains. Only strains that could degrade glycerol hybridized. Moreover, polymerase chain reactions using GDI and GD2 revealed only glycerol dehydratase genes of positive L. collinoides and L. hilgardii strains. The primers and the amplicon proved to be suitable and reliable tools to detect the lactic acid bacteria involved in the deterioration of cider. PMID:11403134

  8. Effect of DNA interaction involving antioxidative 4-aminoantipyrine incorporating mixed ligand complexes having alpha-amino acid as co-ligand

    NASA Astrophysics Data System (ADS)

    Raman, Natarajan; Sakthivel, Arunagiri; Selvaganapathy, Muthusamy; Mitu, Liviu

    2014-02-01

    Few new mixed ligand transition metal complexes of the stoichiometry [ML(A)2], where M = Co(II), Ni(II), Cu(II) and Zn(II), L = FFAP (furfurylidene-4-aminoantipyrine) and A = amino acid (glycine/alanine/valine), have been designed, synthesized and characterized. The molar conductivity of the complexes in DMF at 10-3 M concentration shows that they are non-electrolytes. The interaction of these complexes with CT-DNA indicates that the valine mixed ligand complexes are having higher binding constant than alanine and glycine mixed ligand complexes. This analysis reveals that binding constant depends on the size of the alkyl group present in the amino acid. The binding constants of valine mixed ligand complexes are in the order of 104 to 105 M-1 revealing that the complexes interact with DNA through moderate intercalation mode. The metal complexes exhibit effective cleavage of pUC19 DNA but it is not preceded via radical cleavage an