Sample records for dna restriction fragment

  1. DNA Restriction

    NSDL National Science Digital Library

    The discovery of enzymes that could cut and paste DNA made genetic engineering possible. Restriction enzymes, found naturally in bacteria, can be used to cut DNA fragment at specific sequences, while another enzyme, DNA ligase, can attach or rejoin DNA fragments with complementary ends. This animation from Cold Spring Harbor Laboratory's Dolan DNA Learning Center presents DNA restriction through a series of illustrations of processes involved.

  2. Estimation of DNA fragment size and generation of DNA restriction endonuclease maps using linear models.

    PubMed

    Holford, T R; Brown, S E; Knudson, D L

    1985-02-01

    A method for the estimation of DNA fragment size and for the generation of DNA restriction endonuclease maps using linear models is discussed, and a computer program which utilizes the SAS (SAS Institute Inc., 1982) statistical package is presented. PMID:2984227

  3. Capillary electrophoresis of DNA restriction fragments: effect of polymer properties.

    PubMed

    Braun, B; Blanch, H W; Prausnitz, J M

    1997-10-01

    The mechanism of DNA separation by dilute polymer solutions in capillary electrophoresis is not well understood. To provide information on the effect of polymer properties on DNA separations, four polymers that differ in size, shape and stiffness were examined. Hydroxyethylcellulose of high molecular weight provides excellent separation of large DNA fragments (2027 bp - 23,130 bp). Polyvinylpyrrolidone separates DNA from 72 bp to 23 kbp; star-poly(ethylene oxide) and linear poly(ethylene oxide) provide separation of fragments to 1353 bp. PMID:9420158

  4. Restriction fragment polymorphisms in the rDNA region among seven species of Alnus and Betula papyrifera

    Microsoft Academic Search

    Jean Bousquet; Eric Girouard; Curtis Strobeck; Bruce P. Dancik; Maurice Lalonde

    1989-01-01

    A study of restriction fragment polymorphisms of ribosomal DNA among seven actinorhizal species (Alnus spp.) and a non-actinorhizal species (Betula papyrifera Marsh.) of the Betulaceae was conducted, using a simple method for the extraction of high molecular weight restrictable nuclear DNA from leaf tissues of perennial angiosperms and nine restriction endonucleases. rDNA restriction fragments were variable within and among the

  5. Methods for producing partially digested restriction DNA fragments and for producing a partially modified PCR product

    DOEpatents

    Wong, Kwong-Kwok (Richland, WA)

    2000-01-01

    The present invention is an improved method of making a partially modified PCR product from a DNA fragment with a polymerase chain reaction (PCR). In a standard PCR process, the DNA fragment is combined with starting deoxynucleoside triphosphates, a primer, a buffer and a DNA polymerase in a PCR mixture. The PCR mixture is then reacted in the PCR producing copies of the DNA fragment. The improvement of the present invention is adding an amount of a modifier at any step prior to completion of the PCR process thereby randomly and partially modifying the copies of the DNA fragment as a partially modified PCR product. The partially modified PCR product may then be digested with an enzyme that cuts the partially modified PCR product at unmodified sites thereby producing an array of DNA restriction fragments.

  6. DNA restriction fragment length polymorphisms and heterozygosity in the human genome

    Microsoft Academic Search

    David N. Cooper; Jörg Schmidtke

    1984-01-01

    A list is presented of published reports of DNA polymorphisms found in the human genome by restriction enzyme analysis. While the list indicates the large number of restriction fragment length polymorphisms (RFLPs) detected to date, the information collated is insufficient to permit an estimate of heterozygosity for the genome as a whole. Data from our laboratory are therefore also presented

  7. Nondetectability of restriction fragments and independence of DNA fragment sizes within and between loci in RFLP typing of DNA.

    PubMed Central

    Chakraborty, R.; Zhong, Y.; Jin, L.; Budowle, B.

    1994-01-01

    We provide experimental evidence showing that, during the restriction-enzyme digestion of DNA samples, some of the HaeIII-digested DNA fragments are small enough to prevent their reliable sizing on a Southern gel. As a result of such nondetectability of DNA fragments, individuals who show a single-band DNA profile at a VNTR locus may not necessarily be true homozygotes. In a population database, when the presence of such nondetectable alleles is ignored, we show that a pseudodependence of alleles within as well as across loci may occur. Using a known statistical method, under the hypothesis of independence of alleles within loci, we derive an efficient estimate of null allele frequency, which may be subsequently used for testing allelic independence within and across loci. The estimates of null allele frequencies, thus derived, are shown to agree with direct experimental data on the frequencies of HaeIII-null alleles. Incorporation of null alleles into the analysis of the forensic VNTR database suggests that the assumptions of allelic independence within and between loci are appropriate. In contrast, a failure to incorporate the occurrence of null alleles would provide a wrong inference regarding the independence of alleles within and between loci. Images Figure 1 Figure 2 PMID:7913584

  8. Differentiation ofCampylobacter jejuniandCampylobacter coli Strains by Using Restriction Endonuclease DNA Profiles and DNA Fragment Polymorphisms

    Microsoft Academic Search

    VICTORIA KOROLIK; LISA MOORTHY; ANDPETER J. COLOE

    The chromosomal DNA fragment patterns from a total of 169 Campylobacter jejuni and Campylobacter coli isolatesfrompoultryandhumanswereanalyzedbyusingDNArestrictionendonucleasesClaIandEcoRV.The DNA restriction patterns produced byClaI andEcoRV consisted of unique DNA fragments of 9 to 9.5 kb and 3.5 kb generated with ClaI and a single unique fragment of 3.0 kb produced by EcoRV. These patterns were obtained with all strains ofC. jejunitested. The DNA

  9. Mapping and ordering of fragments of BK virus DNA produced by restriction endonucleases.

    PubMed Central

    Freund, J; di Mayorca, G; Subramanian, K N

    1979-01-01

    A total of 51 restriction sites were recognized within the BK virus genome by the combination of 10 different restriction endonucleases. These sites were mapped and oriented relative to one another as well as to the five fragments generated by the digestion of BK virus DNA with HindIII and EcoRI. The result was a comprehensive physical map suitable for in-depth characterization of the functions of BK virus at the molecular level. Images PMID:221680

  10. Size-selected genomic libraries: the distribution and size-fractionation of restricted genomic DNA fragments by gel electrophoresis.

    PubMed

    Gondo, Y

    1995-02-01

    By using one-dimensional genome scanning, it is possible to directly identify the restricted genomic DNA fragment that reflects the site of genetic change. The subsequent strategies to obtain the molecular clones of the corresponding restriction fragment are usually as follows: (i) the restriction of a mass quantity of an appropriate genomic DNA, (ii) the size-fractionation of the restricted DNA on a preparative electrophoresis gel in order to enrich the corresponding restriction fragment, (iii) the construction of the size-selected libraries from the fractionated genomic DNA, and (iv) the screening of the library to obtain an objective clone which is identified on the analytical genome scanning gel. A knowledge of the size distribution pattern of restriction fragments of the genomic DNA makes it possible to calculate the heterogeneity or complexity of the restriction fragment in each size-fraction. This manuscript first describes the distribution of the restriction fragments with respect to their length. Some examples of the practical application of this theory to genome scanning is then discussed using presumptive genome scanning gels. The way to calculate such DNA complexities in the prepared size-fractionated samples is also demonstrated. Such information should greatly facilitate the design of experimental strategies for the cloning of a certain size of genomic DNA after digestion with restriction enzyme(s) as is the case with genome scanning. PMID:7774556

  11. Reverse-phase HPLC of DNA restriction fragments and ribooligonucleotides on uncoated Kel-F powder.

    PubMed Central

    Usher, D A

    1979-01-01

    Uncoated Kel-F powder offers some unique features as a support for reverse-phase HPLC of oligonucleotides and DNA restriction fragments. Compounds are eluted from the column by a gradient of acetonitrile (0 tto 18% v/v) in 0.1 M aqueous triethylammonium acetate. In contrast to RPC-5 chromatography, oligonucleotides are not eluted by aqueous salt solutions alone, and the separation of restriction fragments depends only on the chainlength. The packing material is cheap, easy to pack, chemically inert, and does not bleed, so that separations are highly reproducible. The DNA loading capacity for Kel-F is presently inferior to RPC-5, but recovery of microgram amounts of material is typically better than 50%. Images PMID:461189

  12. Acetaldehyde and ethanol are responsible for mitochondrial DNA (mtDNA) restriction fragment length polymorphism (RFLP) in flor yeasts.

    PubMed

    Castrejón, Francisco; Codón, Antonio C; Cubero, Beatriz; Benítez, Tahía

    2002-10-01

    Flor yeasts grow and survive in fino sherry wine where the frequency of respiratory-deficient (petite) mutants is very low. Mitochondria from flor yeasts are highly acetaldehyde- and ethanol-tolerant, and resistant to oxidative stress. However, restriction fragment length polymorphism (RFLP) of mtDNA from flor yeast populations is very high and reflects variability induced by the high concentrations of acetaldehyde and ethanol of sherry wine on mtDNA. mtDNA RFLP increases as the concentration of these compounds also increases, but is followed by a total loss of mtDNA in petite cells. Yeasts with functional mitochondria (grande) are target of continuous variability, so that flor yeast mtDNA can evolve extremely rapidly and may serve as a reservoir of genetic diversity, whereas petite mutants are eventually eliminated because metabolism in sherry wine is oxidative. PMID:12421085

  13. Methods for producing partially digested restriction DNA fragments and for producing a partially modified PCR product

    Microsoft Academic Search

    2000-01-01

    The present invention is an improved method of making a partially modified PCR product from a DNA fragment with a polymerase chain reaction (PCR). In a standard PCR process, the DNA fragment is combined with starting deoxynucleoside triphosphates, a primer, a buffer and a DNA polymerase in a PCR mixture. The PCR mixture is then reacted in the PCR producing

  14. High transmission of paternal plastid DNA in alfalfa plants demonstrated by restriction fragment polymorphic analysis.

    PubMed

    Masoud, S A; Johnson, L B; Sorensen, E L

    1990-01-01

    A high frequency of paternal plastid transmission occurred in progeny from crosses among normal green alfalfa plants. Plastid transmission was analyzed by hybridization of radiolabeled alfalfa plastid DNA (cpDNA) probes to Southern blots of restriction digests of the progeny DNA. Each probe revealed a specific polymorphism differentiating the parental plastid genomes. Of 212 progeny, 34 were heteroplastidic, with their cpDNAs ranging from predominantly paternal to predominantly maternal. Regrowth of shoots from heteroplasmic plants following removal of top growth revealed the persistence of mixed plastids in a given plant. However, different shoots within a green heteroplasmic plant exhibited paternal, maternal, or mixed cpDNAs. Evidence of maternal nuclear genomic influence on the frequency of paternal plastid transmission was observed in some reciprocal crosses. A few tetraploid F1 progeny were obtained from tetraploid (2n=4x=32) Medicago sativa ssp. sativa x diploid (2n=2x=16) M. sativa ssp. falcata crosses, and resulted from unreduced gametes. Here more than the maternal genome alone apparently functioned in controlling plastid transmission. Considering all crosses, only 5 of 212 progeny cpDNAs lacked evidence of a definitive paternal plastid fragment. PMID:24226119

  15. Specific amino acid substitutions in bacterioopsin: replacement of a restriction fragment in the structural gene by synthetic DNA fragments containing altered codons

    SciTech Connect

    Lo, K.M.; Jones, S.S.; Hackett, N.R.; Khorana, H.G.

    1984-04-01

    To study the mechanism of light-dependent proton translocation by bacteriorhodopsin, we have introduced single-codon changes in the gene so as to produce the following specific amino acid substitutions in the protein: Tyr-185 to Phe, Pro-186 to Leu, Trp-189 to Phe, Ser-193 to Ala, and Glu-194 to Gln. The strategy involved replacement of a 62-base-pair restriction fragment by synthetic DNA duplexes containing the modified nucleotide sequences. This required a unique restriction site (Xho I) at Ile-203 which was created by oligonucleotide-directed point mutagenesis. The six DNA duplexes corresponding to the modified native and mutant restriction fragments were all prepared by DNA ligase-catalyzed joining of chemically synthesized deoxyribooligonucleotides. The bacterioopsin expression plasmids reconstructed by using the synthetic DNA fragments were characterized by restriction analysis and DNA sequence determination. An extremely rapid, efficient, and general method for purification of the synthetic oligonucleotides and of DNA fragments was developed. 32 references, 6 figures.

  16. Half-site editing: an in vitro mutagenesis procedure for truncating a DNA fragment and introducing a new restriction site.

    PubMed

    Livak, K J; Whitehorn, E A

    1986-01-01

    Half-site editing is an in vitro mutagenesis procedure designed for use in making precise plasmid constructions. Like many in vitro mutagenesis techniques, this procedure involves hybridization of a mutagenic oligonucleotide primer to single-stranded template DNA followed by polymerization with DNA polymerase I (Klenow). Half-site editing differs from other techniques in two main ways. First, T4 DNA polymerase treatment truncates the target DNA at a point determined by the primer and repairs any mismatches to the sequence specified by the primer. Second, a blunt-end ligation step is included. This ligation exploits the symmetry inherent in most restriction sites to create a desired restriction site at the truncated end of the target DNA fragment. Half-site editing has been used to place ClaI restriction sites at the 3' end of the yeast pyruvate kinase promoter and at two positions at the 5' end of the yeast acetolactate synthase coding sequence. PMID:3006543

  17. Phylogenetic relationships of turfgrasses as revealed by restriction fragment analysis of chloroplast DNA.

    PubMed

    Yaneshita, M; Ohmura, T; Sasakuma, T; Ogihara, Y

    1993-10-01

    Chloroplast DNAs (cpDNAs) were analyzed in order to clarify the phylogenetic relationships among turfgrasses. Physical maps of cpDNAs from Agrostis stolonifera and Zoysia japonica, which are representative species of cool (C3 type) and warm (C4 type) season turfgrasses, respectively, were constructed with four restriction enzymes, i.e., PstI, SalI, SacI, and XhoI. The genome structures of these cpDNAs were found to be similar to each other in terms of genome size and gene orders, showing thereby a similarity to other grass cpDNAs. CpDNAs of 5 species of cool season turfgrasses and 6 species of warm season turfgrasses as well as four species of cereals, distributed among 14 genera of Gramineae, were digested with PstI, XhoI, and BamHI, and their restriction fragment patterns were compared. Their genome sizes were estimated to be 135-140 kbp. Each species showed characteristic RFLP patterns. On the basis of the frequency of commonly shared fragments, a dendrogram showing the phylogenetic relationships among their cpDNAs was constructed. This dendrogram shows that turfgrasses can be divided into three major groups; these correspond to the subfamilies. Cool and warm season turfgrasses are clearly distinguishable from each other, and the latter can be further classified into two subgroups that correspond to Eragrostoideae and Panicoideae. Our classification of turfgrasses and cereals by RFLP analysis of cpDNA agreed in principal with their conventional taxonomy, except for the location of Festuca and Lolium. PMID:24190204

  18. Capillary electrophoretic separation of DNA restriction fragments using dilute polymer solutions

    SciTech Connect

    Braun, B.; Blanch, W.; Prausnitz, J.M.

    1997-02-01

    Because the mechanism of DNA separation in capillary electrophoresis is not well understood, selection of polymers is a {open_quotes}trial-and-error{close_quotes} procedure. We investigated dilute-solution DNA separations by capillary electrophoresis using solutions of four polymers that differ in size, shape and stiffness. Hydroxyethylcellulose of high molecular weight provides excellent separation of large DNA fragments (2027 bp - 23130 bp). Polyvinylpyrrolidone separates DNA from 72 bp to 23 kbp and star-(polyethylene oxide), like linear poly (ethylene oxide), provides separation of fragments up to 1353 bp.

  19. Interspecific rDNA restriction fragment length polymorphism in Globodera species, parasites of Solanaceous plants

    Microsoft Academic Search

    Murielle THIÉRY; Didier MUGNIÉRY

    1996-01-01

    Swnmary - The status of the Globodera species that are parasites of Solanaceous is still unclear due ta low morphological polymorphism. Two 21-mers oligonucleotide primers were used ta amplify the internai Transcribed Spacer (ITS). The amplified product was aproximately 1.2 Kb in the 26 Globodera populations examined. Thirty-one restriction enzymes were tested, of which twelve produced some fragment length polymorphism.

  20. Multi-Niche Crowding in Genetic Algorithms and its Application to the Assembly of DNA Restriction-Fragments

    Microsoft Academic Search

    Walter Cedeño; V. Rao Vemuri; Tom Slezak

    1994-01-01

    The determination of the sequence of all nucleotide base-pairs in a DNA molecule, from restriction-fragment data, is a complex t ask and can be posed as the problem of finding the optima of a multi-modal function. A genetic algorithm that uses multi-niche crowding permits us to do t his. Performance of this algorithm is first t ested using a standard

  1. A phylogenetic analysis of the genus Carica L. (Caricaceae) based on restriction fragment length variation in a cpDNA intergenic spacer region

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The phylogenetic relationships among twelve wild and cultivated species of Carica (Caricaceae) were analyzed using restriction fragment length variation in a 3.2-kb PCR amplified intergenic spacer region of the chloroplast DNA. A total of 138 fragments representing 137 restriction sites accounting f...

  2. A systematic study of HLA class II-beta DNA restriction fragments in insulin-dependent diabetes mellitus.

    PubMed Central

    Cohen-Haguenauer, O; Robbins, E; Massart, C; Busson, M; Deschamps, I; Hors, J; Lalouel, J M; Dausset, J; Cohen, D

    1985-01-01

    DNA restriction fragments of the genes encoding HLA class II-beta antigens were compared in 34 patients with insulin-dependent diabetes mellitus and 34 HLA-DR-matched healthy individuals. Ninety-three fragments, determined by six restriction enzymes (EcoRI, EcoRV, HindIII, BamHI, Pvu II, and Taq I), were analyzed: (i) A DR Taq I 12.7-kilobase-pair fragment might be a marker for the extended haplotype HLA-B8, DR3. (ii) In controls, DR4 haplotypes are associated with two distinct clusters of DQ restriction fragments (DQR4 and DQR5). Almost all (94%) DR4 patients belong to the DQR4 and not to the DQR5 cluster. This suggests that, among HLA-DR4 haplotypes, only DQR4 haplotypes are involved in susceptibility to insulin-dependent diabetes mellitus. (iii) A DR Taq I 14.5-kilobase-pair fragment was found to be strongly associated with DQR4, mainly in DR3/DR4 heterozygous patients (P = 5 X 10(-4). However, these results must be interpreted with caution, taking into account the high number of statistical tests performed. Images PMID:2987920

  3. Flying squirrel-associated Rickettsia prowazekii (epidemic typhus rickettsiae) characterized by a specific DNA fragment produced by restriction endonuclease digestion.

    PubMed Central

    Regnery, R L; Fu, Z Y; Spruill, C L

    1986-01-01

    The DNA from flying squirrel-associated Rickettsia prowazekii was characterized by using a specific DNA fragment produced by digestion with the enzyme BamHI. The DNA fragment was cloned into a plasmid vector and used to readily distinguish between available human- and flying squirrel-associated R. prowazekii DNAs derived from crude cytoplasmic extracts. Images PMID:3009528

  4. A computer simulation analysis of the accuracy of partial genome sequencing and restriction fragment analysis in estimating genetic relationships: an application to papillomavirus DNA sequences

    Microsoft Academic Search

    Baozhen Qiao; Ronald M. Weigel

    2004-01-01

    Background: Determination of genetic relatedness among microorganisms provides information necessary for making inferences regarding phylogeny. However, there is little information available on how well the genetic relationships inferred from different genotyping methods agree with true genetic relationships. In this report, two genotyping methods - restriction fragment analysis (RFA) and partial genome DNA sequencing - were each compared to complete DNA

  5. Chloroplast DNA restriction fragment length polymorphism in Sequoia sempervirens D. Don Endl., Pseudotsuga menziesii (Mirb.) Franco, Calocedrus decurrens (Torr.), and Pinus taeda L

    Microsoft Academic Search

    I. F. Ali; D. B. Neale; K. A. Marshall

    1991-01-01

    The extent and type of chloroplast DNA restriction fragment length polymorphism was determined among individual tree samples of coast redwood, Douglas fir, incense-cedar, and loblolly pine. A total of 107 trees was surveyed for three restriction enzymes (BamHI, EcoRI, HindIII) and six chloroplast DNA probes from petunia (P3, P4, P6, P8, P10, S8). The probes comprise 64% of the petunia

  6. Isolation of a complementary DNA clone for the human complement protein C2 and its use in the identification of a restriction fragment length polymorphism.

    PubMed Central

    Woods, D E; Edge, M D; Colten, H R

    1984-01-01

    Complementary DNA (cDNA) clones corresponding to the major histocompatibility (MHC) class III antigen, complement protein C2, have been isolated from human liver cDNA libraries with the use of a complex mixture of synthetic oligonucleotides (17 mer) that contains 576 different oligonucleotide sequences. The C2 cDNA were used to identify a DNA restriction enzyme fragment length polymorphism that provides a genetic marker within the MHC that was not detectable at the protein level. An extensive search for genomic polymorphisms using a cDNA clone for another MHC class III gene, factor B, failed to reveal any DNA variants. The genomic variants detected with the C2 cDNA probe provide an additional genetic marker for analysis of MHC-linked diseases. Images PMID:6086718

  7. Repair of x-ray-induced DNA double-strand breaks in specific Not I restriction fragments in human fibroblasts: joining of correct and incorrect ends

    NASA Technical Reports Server (NTRS)

    Lobrich, M.; Rydberg, B.; Cooper, P. K.; Chatterjee, A. (Principal Investigator)

    1995-01-01

    An assay that allows measurement of absolute induction frequencies for DNA double-strand breaks (dsbs) in defined regions of the genome and that quantitates rejoining of correct DNA ends has been used to study repair of dsbs in normal human fibroblasts after x-irradiation. The approach involves hybridization of single-copy DNA probes to Not I restriction fragments separated according to size by pulsed-field gel electrophoresis. Induction of dsbs is quantitated from the decrease in the intensity of the hybridizing restriction fragment and an accumulation of a smear below the band. Rejoining of dsbs results in reconstitution of the intact restriction fragment only if correct DNA ends are joined. By comparing results from this technique with results from a conventional electrophoresis assay that detects all rejoining events, it is possible to quantitate the misrejoining frequency. Three Not I fragments on the long arm of chromosome 21 were investigated with regard to dsb induction, yielding an identical induction rate of 5.8 X 10(-3) break per megabase pair per Gy. Correct dsb rejoining was measured for two of these Not I fragments after initial doses of 80 and 160 Gy. The misrejoining frequency was about 25% for both fragments and was independent of dose. This result appears to be representative for the whole genome as shown by analysis of the entire Not I fragment distribution. The correct rejoining events primarily occurred within the first 2 h, while the misrejoining kinetics included a much slower component, with about half of the events occurring between 2 and 24 h. These misrejoining kinetics are similar to those previously reported for production of exchange aberrations in interphase chromosomes.

  8. Restriction-based Multiple-fragment Assembly Strategy to Avoid Random Mutation during Long cDNA Cloning

    PubMed Central

    Wang, Shang; Chen, Wen; Zhang, Kai; Jiao, Peng; Mo, Lihua; Yang, Xiaoxu; Hu, Xiang; Zhang, Jian; Wei, Chenxi; Xiang, Shuanglin

    2015-01-01

    Long fragment cloning is a challenge for its difficulty in accurate amplifying and tendency to get unwanted mutation. Here we discuss Restriction-based Multiple-fragment Assembly Strategy's advantages and limitations. In this strategy, rather than PCR amplifying the entire coding sequence (CDS) at one time, we amplified and sequenced smaller fragments which are shorter than 1.5kb spanning the CDS. After that, the sequence-proved fragments were assembled by digestion-ligation cloning to the target vector. We test its universality in our script programmed in Python. Our data shows that, among the entire human and mouse CDS, at least 70% of long CDS cloning will benefit from this strategy.

  9. DNA sequence polymorphisms in the genus saccharomyces III. Restriction endonuclease fragment patterns of chromosomal regions in brewing and other yeast strains

    Microsoft Academic Search

    Mogens Bohl Pedersen

    1986-01-01

    30 bottom fermenting strains, 13 hybrid lager strains, 11 top fermenting strains. In addition, 24 other yeast species and\\u000a strains have been analysed in this study. Four DNA regions have been monitored by restriction endonuclease fragment pattern\\u000a polymorphisms. The regions analysed are:RDN1 (encoding cytosolic ribosomal RNA molecules) located on chromosome XII in S. cerevisiae,HIS4 (histidine 4) andLEU2 (leucine 2) both

  10. Chloroplast DNA restriction fragment length polymorphism in Sequoia sempervirens D. Don Endl., Pseudotsuga menziesii (Mirb.) Franco, Calocedrus decurrens (Torr.), and Pinus taeda L.

    PubMed

    Ali, I F; Neale, D B; Marshall, K A

    1991-01-01

    The extent and type of chloroplast DNA restriction fragment length polymorphism was determined among individual tree samples of coast redwood, Douglas fir, incense-cedar, and loblolly pine. A total of 107 trees was surveyed for three restriction enzymes (BamHI, EcoRI, HindIII) and six chloroplast DNA probes from petunia (P3, P4, P6, P8, P10, S8). The probes comprise 64% of the petunia chloroplast genome. Polymorphisms were detected in all species but loblolly pine. Coast redwood and incense-cedar had a small number of rare variants, whereas Douglas fir had one highly polymorphic region of insertions/deletions in sequences revealed by the P6 probe from petunia. The mutation hotspot is currently being studied by DNA sequence analysis. PMID:24221163

  11. Interaction of EcoRII restriction and modification enzymes with synthetic DNA fragments. V. Study of single-strand cleavages.

    PubMed Central

    Yolov, A A; Gromova, E S; Kubareva, E A; Potapov, V K; Shabarova, Z A

    1985-01-01

    Concatemer DNA duplexes which contain at the EcoRII restriction endonuclease cleavage sites (formula; see text) phosphodiester, phosphoamide or pyrophosphate internucleotide bonds have been synthesized. It has been shown that this enzyme did not cleave the substrate at phosphoamide bond. EcoRII endonuclease catalyzes single-strand cleavages both in dA- and dT-containing strands of the recognition site if the cleavage of the other strand has been blocked by modification of scissile bond or if the other strand has been cleaved. This enzyme interacts with both strands of the DNA recognition site, each of them being cleaved independently on the cleavage of another one. Nucleotide sequences flanking the EcoRII site on both sides are necessary for effective cleavage of the substrate. Images PMID:3001655

  12. Separation of DNA restriction fragments by high performance capillary electrophoresis with low and zero crosslinked polyacrylamide using continuous and pulsed electric fields.

    PubMed

    Heiger, D N; Cohen, A S; Karger, B L

    1990-09-01

    This paper presents results on the separation of DNA restriction fragments by high performance capillary electrophoresis (HPCE). Capillaries containing polyacrylamide with low amounts of crosslinking agent (i.e. 0.5% C) were first studied. The greater molecular accessibility offered with columns of low crosslinking, relative to higher crosslinked gels (e.g. 5% C), permitted high efficiency separations of double stranded DNA fragments up to 12,000 base pairs in length. Capillaries containing no crosslinking agent, i.e. linear polyacrylamide, were then examined. Ferguson plots (i.e. log mobility vs. %T) were used to assess the size selectivity of linear polyacrylamide capillaries. In another study, it was determined that the relative migration of DNA species was a strong function of applied electric field and molecular size. Lower fields yielded better resolution than higher fields for DNA molecules larger than about 1000 base pairs, albeit at the expense of longer separation time. Based on these results, we have examined pulsed field HPCE and have demonstrated the use of this approach to enhance separation. PMID:1962784

  13. Identification of novel cry-type genes from Bacillus thuringiensis strains on the basis of restriction fragment length polymorphism of the PCR-amplified DNA.

    PubMed Central

    Kuo, W S; Chak, K F

    1996-01-01

    Two pairs of universal oligonucleotide primers were designed to probe the most conserved regions of all known cryI-type gene sequences so that the amplified PCR fragments of the DNA template from Bacillus thuringiensis strains may contain all possible cryI-type gene sequences. The restriction fragment length polymorphism (RFLP) patterns of the PCR-amplified fragments revealed that 14 distinct cry-type genes have been identified from 20 B. thuringiensis strains. Those cry-type genes included cryIA(a), cryIA(a), cryIA(b), cryIA(b), cryIA(c), cryIB, cryIC, cryIC, cryIC(b), cryID, cryIE, cryIF, cryIF, and cryIII (a dagger at the end of a gene designation indicates a novel cry-type gene determined by restriction mapping or DNA sequences). Among them, the sequences of cryIA(a), cryIA(b), cryIB, cryIC, cryIF, and cryIII were found to be different from the corresponding published cry gene sequences. Interestingly, five cry-type genes [cryIA(a)-, cryIB-, cryIC-, cryIC(b)-, and cryIF-type genes] and seven cry-type genes [cryIA(a)-, cryIA(b)-, cryIB-, cryIC-, cryIC(b)-, cryIF-, and cryIII-type genes] have been detected from B. thuringiensis subsp. morrisoni HD-12 and B. thuringiensis subsp. wuhanensis, respectively. Therefore, the PCR-RFLP typing system is a facile method to detect both known and novel cry genes existing in B. thuringiensis strains. PMID:8919799

  14. Identification of novel cry-type genes from Bacillus thuringiensis strains on the basis of restriction fragment length polymorphism of the PCR-amplified DNA.

    PubMed

    Kuo, W S; Chak, K F

    1996-04-01

    Two pairs of universal oligonucleotide primers were designed to probe the most conserved regions of all known cryI-type gene sequences so that the amplified PCR fragments of the DNA template from Bacillus thuringiensis strains may contain all possible cryI-type gene sequences. The restriction fragment length polymorphism (RFLP) patterns of the PCR-amplified fragments revealed that 14 distinct cry-type genes have been identified from 20 B. thuringiensis strains. Those cry-type genes included cryIA(a), cryIA(a), cryIA(b), cryIA(b), cryIA(c), cryIB, cryIC, cryIC, cryIC(b), cryID, cryIE, cryIF, cryIF, and cryIII (a dagger at the end of a gene designation indicates a novel cry-type gene determined by restriction mapping or DNA sequences). Among them, the sequences of cryIA(a), cryIA(b), cryIB, cryIC, cryIF, and cryIII were found to be different from the corresponding published cry gene sequences. Interestingly, five cry-type genes [cryIA(a)-, cryIB-, cryIC-, cryIC(b)-, and cryIF-type genes] and seven cry-type genes [cryIA(a)-, cryIA(b)-, cryIB-, cryIC-, cryIC(b)-, cryIF-, and cryIII-type genes] have been detected from B. thuringiensis subsp. morrisoni HD-12 and B. thuringiensis subsp. wuhanensis, respectively. Therefore, the PCR-RFLP typing system is a facile method to detect both known and novel cry genes existing in B. thuringiensis strains. PMID:8919799

  15. Biochemical profiles, restriction fragment length polymorphism (RFLP), random amplified polymorphic DNA (RAPD) and multilocus variable number tandem repeat analysis (MLVA) for typing Staphylococcus aureus isolated from dairy products.

    PubMed

    Morandi, S; Brasca, M; Lodi, R; Brusetti, L; Andrighetto, C; Lombardi, A

    2010-06-01

    The study concerns 130 Staphylococcus aureus strains isolated from different raw-milk dairy products (122 isolates) and human samples (eight isolates). Four different typing techniques were applied: biochemical profiles (Biolog GP), restriction fragment length polymorphism of coagulase gene (coaRFLP), random amplified polymorphic DNA (RAPD) and multilocus variable number tandem repeat analysis (MLVA). Moreover multiplex-PCR was used to study the distribution of genes encoding staphylococcal enterotoxins. The results of this study reveal marked genomic and phenotypic variability among the tested S. aureus. The considered techniques were all found useful for strain typing, but, based on discriminatory power as the key parameter of the typing system, MLVA and Biolog GP were found to be the most powerful techniques. The methods showed little concordance in terms of discerning the clusters of related strains. PMID:19926103

  16. DNA sequence analysis and restriction fragment length polymorphisms of the P1 gene of Haemophilus influenzae biogroup aegyptius associated with Brazilian purpuric fever.

    PubMed Central

    Reed, R B; Frost, J B; Kort, K; Myers, S D; Lesse, A J

    1996-01-01

    Brazilian purpuric fever (BPF) is a fulminant pediatric disease caused by specific strains of Haemophilus influenzae biogroup aegyptius. A conserved epitope on the P1 protein of strains of H. influenzae biogroup aegyptius is seen on most virulent isolates. The P1 protein from a Brazilian case-clone strain of H. influenzae biogroup aegyptius was analyzed by cloning and sequencing the gene. Three major variable regions are present within the P1 gene of the BPF clone in an architecture similar to that of the previously sequenced P1 genes from H. influenzae. The DNA sequence data of the P1 gene provided information for restriction fragment length polymorphism analyses among strains of H. influenzae biogroup aegyptius. Using PCR for amplification of the P1 gene, we found that AlwI restriction of this gene allowed for a highly accurate segregation of virulent strains of H. influenzae biogroup aegyptius associated with BPF. The strong association of virulent phenotypes with specific AlwI restriction patterns of the P1 gene provides a basis for the convenient and accurate identification of strains of H. influenzae biogroup aegyptius which cause BPF. PMID:8751915

  17. Restriction fragment length polymorphism of 195 bp repeated satellite dna of Trypanosoma cruzi supports the existence of two phylogenetic groups

    Microsoft Academic Search

    Brigitte Bastrenta; Marie France Bosseno; Christian Barnabé; Michel Tibayrenc; Simone Frédérique Brenière

    1999-01-01

    Hinf I) and high molecular weight DNA (Hae III), while group 2 presents a ladder profile for each enzyme, which is a characteristic of tandemly repeated DNA. The two groups, respectively, clustered stocks pertaining to the two principal lineages evidenced by isoenzyme and RAPD markers. The congruence among these three independent genomic markers corroborates the existence of two real phylogenetic

  18. Restriction endonuclease cleavage map of mitochondrial DNA from Aspergillus nidulans.

    PubMed Central

    Stepie?, P P; Bernard, U; Cooke, H J; Küntzel, H

    1978-01-01

    Mitochondrial DNA of the ascomycete fungus Aspergillus nidulans, a circular molecule of 31 500 base pairs, is cleaved by restriction endonucleases Eco R I, Hind II, Hind III and Bgl II into 3, 7, 9 and 5 fragments, respectively. The relative positions of the cleavage sites could be mapped by analysis of fragments obtained by double enzyme digestions of whole DNA and by complete and partial redigestion of isolated restriction fragments. Images PMID:345242

  19. Characterization of primary biogenic aerosol particles in urban, rural, and high-alpine air by DNA sequence and restriction fragment analysis of ribosomal RNA genes

    NASA Astrophysics Data System (ADS)

    Després, V. R.; Nowoisky, J. F.; Klose, M.; Conrad, R.; Andreae, M. O.; Pöschl, U.

    2007-12-01

    This study explores the applicability of DNA analyses for the characterization of primary biogenic aerosol (PBA) particles in the atmosphere. Samples of fine particulate matter (PM2.5) and total suspended particulates (TSP) have been collected on different types of filter materials at urban, rural, and high-alpine locations along an altitude transect in the south of Germany (Munich, Hohenpeissenberg, Mt. Zugspitze). From filter segments loaded with about one milligram of air particulate matter, DNA could be extracted and DNA sequences could be determined for bacteria, fungi, plants and animals. Sequence analyses were used to determine the identity of biological organisms, and terminal restriction fragment length polymorphism analyses (T-RFLP) were applied to estimate diversities and relative abundances of bacteria. Investigations of blank and background samples showed that filter materials have to be decontaminated prior to use, and that the sampling and handling procedures have to be carefully controlled to avoid artifacts in the analyses. Mass fractions of DNA in PM2.5 were found to be around 0.05% in urban, rural, and high-alpine aerosols. The average concentration of DNA determined for urban air was on the order of ~7 ng m-3, indicating that human adults may inhale about one microgram of DNA per day (corresponding to ~108 haploid bacterial genomes or ~105 haploid human genomes, respectively). Most of the bacterial sequences found in PM2.5 were from Proteobacteria (42) and some from Actinobacteria (10) and Firmicutes (1). The fungal sequences were characteristic for Ascomycota (3) and Basidiomycota (1), which are known to actively discharge spores into the atmosphere. The plant sequences could be attributed to green plants (2) and moss spores (2), while animal DNA was found only for one unicellular eukaryote (protist). Over 80% of the 53 bacterial sequences could be matched to one of the 19 T-RF peaks found in the PM2.5 samples, but only 40% of the T-RF peaks did correspond to one of the detected bacterial sequences. The results demonstrate that the T-RFLP analysis covered more of the bacterial diversity than the sequence analysis. Shannon-Weaver indices calculated from both sequence and T-RFLP data indicate that the bacterial diversity in the rural samples was higher than in the urban and alpine samples. Two of the bacterial sequences (Gammaproteobacteria) and five of the T-RF peaks were found at all sampling locations.

  20. Genetic Diversity among Strains of Moraxella catarrhalis: Analysis Using Multiple DNA Probes and a Single-Locus PCR-Restriction Fragment Length Polymorphism Method

    PubMed Central

    Walker, Elaine S.; Preston, Robert A.; Post, J. Christopher; Ehrlich, Garth D.; Kalbfleisch, John H.; Klingman, Karin L.

    1998-01-01

    Moraxella (Branhamella) catarrhalis, a causative agent of otitis media, sinusitis, and exacerbation of bronchitis, has acquired widespread ability to produce ?-lactamase and can be nosocomially transmitted. The typing methods used in epidemiological analyses of M. catarrhalis are not optimal for genetic analyses. Two methods, a multiple-locus Southern blot (SB) method and a single-locus PCR-restriction fragment length polymorphism (RFLP) method, were developed and used to assess genetic diversity and potential clinical and geographic relationships in M. catarrhalis. Nine randomly cloned M. catarrhalis DNA fragments were used as probes of SBs containing DNA from 54 geographically and clinically diverse strains. For comparison, a PCR-RFLP method was developed as a quick, inexpensive, and discriminating alternative. A highly variable 3.7-kb genomic region (M46) was cloned and sequenced, and 3.5 kb of the cloned DNA was targeted for PCR amplification. DNAs from the 54 strains were subjected to PCR-RFLP. SB analysis distinguished all strains that had no apparent epidemiological linkage (40 of 54), and PCR-RFLP distinguished fewer strains (21 of 54). Epidemiologically linked strains appeared genetically identical by both methods. PCR-RFLP was compared to pulsed-field gel electrophoresis (PFGE) for 8 of the 54 strains and 23 additional strains. PCR-RFLP distinguished fewer strains than PFGE typing (16 of 31 versus 20 of 31 strains), but PCR-RFLP was more useful for inferring interstrain relatedness. Separate cluster analyses of multilocus SB and single locus PCR-RFLP data showed high genetic diversity within and across geographic locations and clinical presentations. The resultant dendrograms were not entirely concordant, but both methods often gave similar strain clusters at the terminal branches. High genetic diversity, nonconcordance of cluster analyses from different genetic loci, and shared genotypes among epidemiologically linked strains support a hypothesis of high recombination relative to spread of clones. Single-locus PCR-RFLP may be suitable for short-term epidemiological studies, but the SB data demonstrate that greater strain discrimination may be obtained by sampling variation at multiple genomic sites. PMID:9650948

  1. Genotyping human and bovine isolates of Cryptosporidium parvum by polymerase chain reaction-restriction fragment length polymorphism analysis of a repetitive DNA sequence

    Microsoft Academic Search

    Alain Bonnin; Marie Noelle Fourmaux; Jean François Dubremetz; Richard Gordon Nelson; Philippe Gobet; Géraldine Harly; Marielle Buisson; Dominique Puygauthier-Toubas; Florence Gabriel-Pospisil; Muriel Naciri; Patrick Camerlynck

    1996-01-01

    In order to define transmission routes of cryptosporidiosis and develop markers that distinguish Cryptosporidium parvum isolates, we have identified 2 polymorphic restriction enzyme sites in a C. parvum repetitive DNA sequence. The target sequence was amplified by polymerase chain reaction from 100 to 500 oocysts and the amplified product was subjected to restriction enzyme digestion. Typing of 23 isolates showed

  2. Apolipoprotein E genotyping by capillary electrophoretic analysis of restriction fragments

    Microsoft Academic Search

    Gianluca De Bellis; Giuliana Salani; Silvia Panigone; Ferruccio Betti; Luigia Invernizzi; Massimo Luzzana

    1997-01-01

    We present the genotyping of apolipoprotein (apo) E by means of restriction fragment analysis of amplified genomic DNA by high-performance capillary electro- phoresis and a replaceable non-gel-sieving matrix. This procedure streamlines the genotyping of apo E in large- scale population studies because of the automation and speed of capillary electrophoresis. INDEXING TERMS: genotype determinationAlzheimer diseasehigh-performance capillary electrophoresis

  3. Variability for restriction fragment lengths and phylogenies in lentil

    Microsoft Academic Search

    M. J. Havey; F. J. Muehlbauer

    1989-01-01

    Thirty accessions of domesticated (Lens culinaris ssp. culinaris) and wild (L. culinaris ssp. orientalis, L. culinaris ssp. odemensis, L. nigricans ssp. ervoides and L. nigricans ssp. nigricans) lentil were evaluated for restriction fragment length polymorphisms (RFLPs) using ten relative low-copy-number probes selected from partial genomic and cDNA libraries of lentil. Nei's average gene diversity was used as a measure of

  4. LOCALIZATION IN THE TOMATO GENOME OF DNA RESTRICTION FRAGMENTS CONTAINING SEQUENCES HOMOLOGOUS TO THE RRNA (45S), THE MAJOR CHLOROPHYLL A\\/B BINDING POLYPEPTIDE AND THE RIBULOSE BISPHOSPHATE CARBOXYLASE GENES

    Microsoft Academic Search

    C. E. VALLEJOS; S. D. TANKSLEY; R. BERNATZKY

    DNA restriction fragments containing sequences homologous to the ribosomal RNA (45s), the major chlorophyll a\\/b binding polypeptide (CAB) and the small subunit of ribulose bisphosphate carboxylase (RBCS) genes have been localized and mapped in the tomato nuclear genome by linkage analysis. Ribosomal RNA genes map to a single locus, R45s, which resides in a terminal position on the short arm

  5. Differentiation of Spotted Fever Group Rickettsiae by Sequencing and Analysis of Restriction Fragment Length Polymorphism of PCR Amplified DNA of the Gene Encoding the Protein rOmpA

    Microsoft Academic Search

    VERONIQUE ROUX; PIERRE-EDOUARD FOURNIER; ANDDIDIER RAOULT

    1996-01-01

    Currently, the genotypic identification of the spotted fever group (SFG) rickettsiae is based on restriction fragmentlengthpolymorphismanalysisofPCR-amplifiedgenescodingfortheenzymecitratesynthaseandthe surfaceproteinsrOmpAandrOmpB.Asetofusefulrestrictionendonucleaseswasfoundfollowingcomparison ofRickettsiarickettsiiandR.prowazekiisequences.However,byusingthreePCRamplificationsandfourenzyme digestions with this set, it was impossible to differentiate between all of the known serotypes of the SFG rickettsiae. We amplified by PCR and sequenced using an automated laser fluorescent DNA sequencer a fragment of the gene encoding the protein rOmpA from

  6. Taxonomy and phylogeny of some Eimeria (Apicomplexa: Eimeriidae) species of rodents as determined by polymerase chain reaction\\/restriction-fragment-length polymorphism analysis of 18S rDNA

    Microsoft Academic Search

    J. A. Hnida; D. W. Duszynski

    1999-01-01

    The 18S rDNA genes of 10 Eimeria species from rodents (E. albigulae, E. arizonensis, E. falciformis, E. langebarteli, E. nieschulzi, E. onychomysis, E. papillata, E. reedi, E. separata, E. sevilletensis) were polymerase-chain-reaction (PCR)-amplified, digested with 12 restriction endonucleases, and electophoresed in agarose\\u000a gels. The resulting fragment patterns (riboprints) distinguished all species except E. sevilletensis from E.?falciformis, and E. arizonensis from

  7. Characterization of Bacterial Community Diversity in Cystic Fibrosis Lung Infections by Use of 16S Ribosomal DNA Terminal Restriction Fragment Length Polymorphism Profiling

    Microsoft Academic Search

    G. B. Rogers; M. P. Carroll; D. J. Serisier; P. M. Hockey; G. Jones; K. D. Bruce

    2004-01-01

    Progressive loss of lung function resulting from the inflammatory response to bacterial colonization is the leading cause of mortality in cystic fibrosis (CF) patients. A greater understanding of these bacterial infections is needed to improve lung disease management. As culture-based diagnoses are associated with fundamental drawbacks, we used terminal restriction fragment (T-RF) length polymorphism profiling and 16S rRNA clone data

  8. The linkage mapping of cloned restriction fragment length differences in Caenorabditis elegans

    Microsoft Academic Search

    A. M. Rose; D. L. Baillie; E. P. M. Candido; K. A. Beckenbach; D. Nelson

    1982-01-01

    The genomic DNA of two closely related strains of the nematode, Caenorhabditis elegans, Bristol (N2), and Bergerac (Bo), has different restriction endonuclease sites (Emmons et al. 1979). Since these two strains interbreed, it is possible to regard the restriction fragment length differences (RFLDs) as mutant variants. The N2 and Bo pattern can be segregated and mapped using clasical genetic techniques.

  9. Non-random DNA fragmentation in next-generation sequencing

    NASA Astrophysics Data System (ADS)

    Poptsova, Maria S.; Il'Icheva, Irina A.; Nechipurenko, Dmitry Yu.; Panchenko, Larisa A.; Khodikov, Mingian V.; Oparina, Nina Y.; Polozov, Robert V.; Nechipurenko, Yury D.; Grokhovsky, Sergei L.

    2014-03-01

    Next Generation Sequencing (NGS) technology is based on cutting DNA into small fragments, and their massive parallel sequencing. The multiple overlapping segments termed ``reads'' are assembled into a contiguous sequence. To reduce sequencing errors, every genome region should be sequenced several dozen times. This sequencing approach is based on the assumption that genomic DNA breaks are random and sequence-independent. However, previously we showed that for the sonicated restriction DNA fragments the rates of double-stranded breaks depend on the nucleotide sequence. In this work we analyzed genomic reads from NGS data and discovered that fragmentation methods based on the action of the hydrodynamic forces on DNA, produce similar bias. Consideration of this non-random DNA fragmentation may allow one to unravel what factors and to what extent influence the non-uniform coverage of various genomic regions.

  10. Non-random DNA fragmentation in next-generation sequencing

    PubMed Central

    Poptsova, Maria S.; Il'icheva, Irina A.; Nechipurenko, Dmitry Yu.; Panchenko, Larisa A.; Khodikov, Mingian V.; Oparina, Nina Y.; Polozov, Robert V.; Nechipurenko, Yury D.; Grokhovsky, Sergei L.

    2014-01-01

    Next Generation Sequencing (NGS) technology is based on cutting DNA into small fragments, and their massive parallel sequencing. The multiple overlapping segments termed “reads” are assembled into a contiguous sequence. To reduce sequencing errors, every genome region should be sequenced several dozen times. This sequencing approach is based on the assumption that genomic DNA breaks are random and sequence-independent. However, previously we showed that for the sonicated restriction DNA fragments the rates of double-stranded breaks depend on the nucleotide sequence. In this work we analyzed genomic reads from NGS data and discovered that fragmentation methods based on the action of the hydrodynamic forces on DNA, produce similar bias. Consideration of this non-random DNA fragmentation may allow one to unravel what factors and to what extent influence the non-uniform coverage of various genomic regions. PMID:24681819

  11. Size fractionation of DNA fragments by liquid-liquid chromatography

    PubMed Central

    Müller, Werner; Schuetz, Hans-Jürgen; Guerrier-Takada, Cecilia; Cole, Patricia E.; Potts, Russell

    1979-01-01

    A methods for the fractionation of double-stranded DNA fragments from 150 to 22000 b.p. in size by liquid-liquid chromatography is described. The procedure makes use of the fact that the partitioning of DNA in a polyethylene glycol-dextran system is size dependent and can be altered by alkali metal cations. Cellulose or celite are used as supports for the stationary, dextran-rich phase. Examples show the fractionation of digests of T7 DNA produced by Dpn II and Hind II restriction endonucleases as well as ? DNA digests produced by Hind III and Eco RI restriction endonucleases. Images PMID:160546

  12. Typing of rhizobia by PCR DNA fingerprinting and PCR-restriction fragment length polymorphism analysis of chromosomal and symbiotic gene regions: application to Rhizobium leguminosarum and its different biovars.

    PubMed Central

    Laguerre, G; Mavingui, P; Allard, M R; Charnay, M P; Louvrier, P; Mazurier, S I; Rigottier-Gois, L; Amarger, N

    1996-01-01

    Characterization of 43 strains of Rhizobium leguminosarum biovars viciae, trifolii, and phaseoli was performed by two methodologies based on PCR amplification, i.e., PCR DNA fingerprinting of interrepeat sequences and restriction fragment length polymorphism (RFLP) analysis of PCR -amplified chromosomal and symbiotic gene regions. Groupings generated by PCR DNA fingerprinting with either extragenic palindromic repetitive primers or two different single random primers were correlated with similar levels of resolution. Although less discriminating, PCR-RFLP analysis of intergenic spacer between genes coding for 16S and 23S rRNA (16S and 23S rDNA) yielded intraspecific polymorphisms. The classification of strains was independent of the biovar status and was in agreement with those obtained by PCR DNA fingerprinting. Intrabiovar variation within symbiotic gene regions was detected by PCR-RFLP analysis of nifDK and nodD gene regions, but the strains were grouped according to the biovar. The rDNA intergenic spacer and nif primers were verified to be universal for rhizobial species by testing of various reference strains, whereas the nod primers designed in this study were biovar or species specific for R. leguminosarum and Rhizobium etli. Classifications of R. leguminosarum strains by the PCR-based methods were correlated with those previously obtained by conventional total DNA restriction profile comparisons and RFLP analysis using chromosomal and symbiotic gene probes. Ranges of discriminating powers were also equivalent between the two approaches. However, the PCR-based methods are much less time-consuming and are therefore more convenient. PMID:8787401

  13. Population genetics of the yellow fever mosquito in Trinidad: comparisons of amplified fragment length polymorphism (AFLP) and restriction fragment length polymorphism (RFLP) markers

    Microsoft Academic Search

    G. Yan; J. Romero-Severson; M. Walton; D. D. CHADEEand; D. W. Severson

    1999-01-01

    Recent development of DNA markers provides powerful tools for population genetic analyses. Amplified fragment length polymorphism (AFLP) markers result from a poly- merase chain reaction (PCR)-based DNA fingerprinting technique that can detect multiple restriction fragments in a single polyacrylamide gel, and thus are potentially useful for population genetic studies. Because AFLP markers have to be analysed as dominant loci in

  14. Radiation of human mitochondria DNA types analyzed by restriction endonuclease cleavage patterns

    Microsoft Academic Search

    M. J. Johnson; D. C. Wallace; S. D. Ferris; M. C. Rattazzi; L. L. Cavalli-Sforza

    1983-01-01

    Summary Human mitochondrial DNA (mtDNA) restriction endonuclease fragment patterns were analyzed using total blood cell DNA isolated from 200 individuals representing five different populations. Thirty-two fragment patterns (morphs) were observed with the enzymes Hpa I, Bam HI, Hae II, Msp I and Ava II yielding thirty-five different combinations of fragment patterns (mt DNA types). The major ethnic groups exhibit quantitative

  15. Use of restriction fragment length polymorphisms resolved by pulsed-field gel electrophoresis for subspecies identification of mycobacteria in the Mycobacterium avium complex and for isolation of DNA probes.

    PubMed Central

    Coffin, J W; Condon, C; Compston, C A; Potter, K N; Lamontagne, L R; Shafiq, J; Kunimoto, D Y

    1992-01-01

    Mycobacterial strains from the Mycobacterium avium complex were compared with each other and with Mycobacterium phlei isolates by restriction endonuclease digestion of chromosomal DNA with SspI and analysis by pulsed-field gel electrophoresis. Characteristic profiles were observed for known typed strains, and five groups were identified. Primary bovine isolates identified as Mycobacterium paratuberculosis by classical methods were shown to fall into both the M. paratuberculosis- and M. avium-like groups. M. paratuberculosis 18 was in the latter category. Two Mycobacterium intracellulare strains of different Schaefer serotypes had different digestion profiles. In addition, this system was exploited for the preparation of DNA probes by the isolation, digestion, and subcloning of DNA fragments separated by pulsed-field gel electrophoresis. Probe JC12 hybridized only to M. avium complex strains, but not to M. phlei, showing characteristic hybridization profiles for each of the groups previously identified by pulsed-field gel electrophoresis. The approach taken in the study lends itself to the comparative analysis of members of the M. avium complex and to the isolation and characterization of DNA probes with specificity for these mycobacteria. Images PMID:1352787

  16. Restriction fragment length polymorphisms associated with substance P gene

    SciTech Connect

    de Miguel, C.; Bonner, T.; Detera-Wadleigh, S.

    1987-05-01

    Substance P (SP) is an important neuropepetide detected in a variety of locations in the central nervous system. Variations in SP content or SP receptors in psychiatric disorders have been described. Using SP clones as probes the authors have found three restriction fragment length polymorphisms (RFLPs) in the SP gene. The RFLPs are generated by digestion of genomic DNA with the MspI, and RsaI and NcoI restriction endonucleases. The MspI RFLP is detected by two genomic clones mapping to the 5' end of the gene while the RsaI and NcoI rFLPs are both detected by two genomic clones on the 3' end and also by a full-length cDNA clone of the gene. All three RFLPs are characterized by two alleles. For the MspI RFLP the frequency of both alleles is similar, for the Rsa I and NcoI RFLP one of the alleles is significantly more abundant than the other. These RFLPs are now being used to determine whether any of the alleles correlate with either schizophrenia or affective disorder.

  17. Sizing of DNA fragments by flow cytometry

    SciTech Connect

    Johnson, M.E.; Goodwin, P.M.; Ambrose, W.P.; Martin, J.C.; Marrone, B.L.; Jett, J.H.; Keller, R.A.

    1993-02-01

    Individual, stained DNA fragments were sized using a modified flow cytometer with high sensitivity fluorescence detection. The fluorescent intercalating dye ethidium homodimer was used to stain stoichiometrically lambda phage DNA and a Kpn I digest of lambda DNA. Stained, individual fragments of DNA were passed through a low average power, focused, mode-locked laser beam, and the fluorescence from each fragment was collected and quantified. Time-gated detection was used to discriminate against Raman scattering from the water solvent. The fluorescence burst from each fragment was related directly to its length, thus providing a means to size small quantities of kilobase lengths of DNA quickly. Improvements of several orders of magnitude in analysis time and sample size over current gel electrophoresis techniques were realized. Fragments of 17.1,29.9, and 48.5 thousand base pairs were well resolved, and were sized in 164 seconds. Less than one pg of DNA was required for analysis. We have demonstrated sizing of individual, stained DNA fragments with resolution approaching that of gel electrophoresis for moderately large fragments, but with significant reductions in the analysis time and the amount of sample required. Furthermore, system response is linear with DNA fragment length, in contrast to the logarithmic response in gel electrophoresis. There exists the potential to perform this sizing using relatively simple instrumentation, i.e. a continuous wave laser of low power and current mode detection.

  18. Mapping Flagellar Genes in Chlamydomonas Using Restriction Fragment Length Polymorphisms

    PubMed Central

    Ranum, LPW.; Thompson, M. D.; Schloss, J. A.; Lefebvre, P. A.; Silflow, C. D.

    1988-01-01

    To correlate cloned nuclear DNA sequences with previously characterized mutations in Chlamydomonas and, to gain insight into the organization of its nuclear genome, we have begun to map molecular markers using restriction fragment length polymorphisms (RFLPs). A Chlamydomonas reinhardtii strain (CC-29) containing phenotypic markers on nine of the 19 linkage groups was crossed to the interfertile species Chlamydomonas smithii. DNA from each member of 22 randomly selected tetrads was analyzed for the segregation of RFLPs associated with cloned genes detected by hybridization with radioactive DNA probes. The current set of markers allows the detection of linkage to new molecular markers over approximately 54% of the existing genetic map. This study focused on mapping cloned flagellar genes and genes whose transcripts accumulate after deflagellation. Twelve different molecular clones have been assigned to seven linkage groups. The ?-1 tubulin gene maps to linkage group III and is linked to the genomic sequence homologous to pcf6-100, a cDNA clone whose corresponding transcript accumulates after deflagellation. The ?-2 tubulin gene maps to linkage group IV. The two ?-tubulin genes are linked, with the ?-1 gene being approximately 12 cM more distal from the centromere than the ?-2 gene. A clone corresponding to a 73-kD dynein protein maps to the opposite arm of the same linkage group. The gene corresponding to the cDNA clone pcf6-187, whose mRNA accumulates after deflagellation, maps very close to the tightly linked pf-26 and pf-1 mutations on linkage group V. PMID:2906025

  19. REPK: an analytical web server to select restriction endonucleases for terminal restriction fragment length polymorphism analysis

    Microsoft Academic Search

    Roy Eric Collins; Gabrielle Rocap

    2007-01-01

    Terminal restriction fragment length polymorphism (T-RFLP) analysis is a widespread technique for rapidly fingerprinting microbial communities. Users of T-RFLP frequently overlook the resolving power of well-chosen restriction endonucleases and often fail to report how they chose their enzymes. REPK (Restriction Endonuclease Picker) assists in the rational choice of restriction endonucleases for T-RFLP by finding sets of four restriction endonu- cleases

  20. REMA: A computer-based mapping tool for analysis of restriction sites in multiple DNA sequences

    Microsoft Academic Search

    Jan Szubert; Caroline Reiff; Andrew Thorburn; Brajesh K. Singh

    2007-01-01

    REMA is an interactive web-based program which predicts endonuclease cut sites in DNA sequences. It analyses multiple sequences simultaneously and predicts the number and size of fragments as well as provides restriction maps. The users can select single or paired combinations of all commercially available enzymes. Additionally, REMA permits prediction of multiple sequence terminal fragment sizes and suggests suitable restriction

  1. Restriction fragment length polymorphism analysis of polymerase chain reaction products amplified from mapped loci of rice ( Oryza sativa L.) genomic DNA

    Microsoft Academic Search

    M. N. V. Williams; N. Pande; S. Nair; M. Mohan; J. Bennett

    1991-01-01

    Thirty mapped Indica rice genomic (RG) clones were partially sequenced from each end. From such sequence data, pairs of oligonucleotides were synthesized to act as primers for polymerase chain reaction (PCR) amplification of the corresponding loci in crude total DNA preparations. The PCR products from DNA of Indica varieties were of the sizes expected from the sizes of the corresponding

  2. RESTRICTION FRAGMENT LENGTH POLYMORPHISMS DISTINGUISH ECTOMYCORRHIZAL FUNGI

    EPA Science Inventory

    Basidiomycetous fungi, two saprophytes and three mycorrhizal, were used to assess the specificity of DNA hybridization for distinguishing genera from one another. nterspecific comparisons were done with several isolates of mycorrhizal fungi, Laccaria bicolor and L. laccata, colle...

  3. An integrated restriction fragment length polymorphism--amplified fragment length polymorphism linkage map for cultivated sunflower.

    PubMed

    Gedil, M A; Wye, C; Berry, S; Segers, B; Peleman, J; Jones, R; Leon, A; Slabaugh, M B; Knapp, S J

    2001-04-01

    Restriction fragment length polymorphism (RFLP) maps have been constructed for cultivated sunflower (Helianthus annuus L.) using three independent sets of RFLP probes. The aim of this research was to integrate RFLP markers from two sets with RFLP markers for resistance gene candidate (RGC) and amplified fragment length polymorphism (AFLP) markers. Genomic DNA samples of HA370 and HA372, the parents of the F2 population used to build the map, were screened for AFLPs using 42 primer combinations and RFLPs using 136 cDNA probes (RFLP analyses were performed on DNA digested with EcoRI, HindIII, EcoRV, or DraI). The AFLP primers produced 446 polymorphic and 1101 monomorphic bands between HA370 and HA372. The integrated map was built by genotyping 296 AFLP and 104 RFLP markers on 180 HA370 x HA372 F2 progeny (the AFLP marker assays were performed using 18 primer combinations). The HA370 x HA372 map comprised 17 linkage groups, presumably corresponding to the 17 haploid chromosomes of sunflower, had a mean density of 3.3 cM, and was 1326 cM long. Six RGC RFLP loci were polymorphic and mapped to three linkage groups (LG8, LG13, and LG15). AFLP markers were densely clustered on several linkage groups, and presumably reside in centromeric regions where recombination is reduced and the ratio of genetic to physical distance is low. Strategies for targeting markers to euchromatic DNA need to be tested in sunflower. The HA370 x HA372 map integrated 14 of 17 linkage groups from two independent RFLP maps. Three linkage groups were devoid of RFLP markers from one of the two maps. PMID:11341731

  4. Quantitative Restriction Fragment Length Polymorphism: A Procedure for Quantitation of Diphtheria Toxin Gene CRM197 Allele

    Microsoft Academic Search

    Elena A. Pushnova; Yu Sheng Zhu

    1998-01-01

    Here we present an assay for quantitation of a particular gene allele in DNA mixtures by means of restriction fragment length polymorphism (RFLP) in combination with polymerase chain reaction (PCR). We applied the quantitative RFLP principle for estimation of the relative amount of diphtheria toxin gene CRM197 allele inCorynebacterium diphtheriaeculture DNA samples. The procedure is based on PCR-mediated generation of

  5. Sex in Escherichia coli does not disrupt the clonal structure of the population: evidence from random amplified polymorphic DNA and restriction-fragment-length polymorphism

    Microsoft Academic Search

    Patricia Desjardins; Bertrand Picard; Bernhard Kaltenbiick; Jacques Elion; Erick Denamurl

    1995-01-01

    Analysis of the Escherichia coli population by multilocus enzyme electrophoresis (MLEE) has established its clonal organization, but there is increasing evidence that horizontal DNA transfer occurs in E. coli. We have assessed the genetic structure of the species E. coli and determined the extent to which recombination can affect the clonal structure of bacteria. A panel of 72 E. coli

  6. DNA fragment sizing and sorting by laser-induced fluorescence

    DOEpatents

    Hammond, Mark L. (Angier, NC); Jett, James H. (Los Alamos, NM); Keller, Richard A. (Los Alamos, NM); Marrone, Babetta L. (Los Alamos, NM); Martin, John C. (Los Alamos, NM)

    1996-01-01

    A method is provided for sizing DNA fragments using high speed detection systems, such as flow cytometry to determine unique characteristics of DNA pieces from a sample. In one characterization the DNA piece is fragmented at preselected sites to produce a plurality of DNA fragments. The DNA piece or the resulting DNA fragments are treated with a dye effective to stain stoichiometrically the DNA piece or the DNA fragments. The fluorescence from the dye in the stained fragments is then examined to generate an output functionally related to the number of nucleotides in each one of the DNA fragments. In one embodiment, the intensity of the fluorescence emissions from each fragment is linearly related to the fragment length. The distribution of DNA fragment sizes forms a characterization of the DNA piece for use in forensic and research applications.

  7. DNA POLYMORPHISM DETECTABLE BY RESTRICTION ENDONUCLEASES

    Microsoft Academic Search

    MASATOSHI NE; FUMIO TAJIMA

    1981-01-01

    Data on DNA polymorphisms detected by restriction endonucleases are rapidly accumulating. With the aim of analyzing these data, several different measures of nucleon (DNA segment) diversity within and between popula- tions are proposed, and statistical methods for estimating these quantities are developed. These statistical methods are applicable to both nuclear and non- nuclear DNAs. When evolutionary change of nucleons occurs

  8. DNA Sequence from Cretaceous Period Bone Fragments

    Microsoft Academic Search

    Scott R. Woodward; Nathan J. Weyand; Mark Bunnell

    1994-01-01

    DNA was extracted from 80-million-year-old bone fragments found in strata of the Upper Cretaceous Blackhawk Formation in the roof of an underground coal mine in eastern Utah. This DNA was used as the template in a polymerase chain reaction that amplified and sequenced a portion of the gene encoding mitochondrial cytochrome b. These sequences differ from all other cytochrome b

  9. Restriction fragment length polymorphism species-specific patterns in the identification of white truffles.

    PubMed

    Bertini, L; Potenza, L; Zambonelli, A; Amicucci, A; Stocchi, V

    1998-07-15

    A molecular method for the identification of ectomycorrhizae belonging to five species of white truffle is described. The polymerase chain reaction (PCR) and universal primers were used to amplify internal transcribed spacers and 5.8S rDNA, target sequences present in a high number of copies. The amplified products were digested with restriction enzymes in order to detect interspecific polymorphisms. Species-specific restriction fragment length polymorphism patterns were determined for all five species. The use of PCR in conjunction with restriction enzymes provides a sensitive and efficient tool for use in distinguishing ectomycorrhizal species and monitoring inoculated seedlings or field mycorrhizal populations. PMID:9682488

  10. Restriction analysis of spacers in ribosomal DNA of Drosophila melanogaster.

    PubMed Central

    Long, E O; Dawid, I B

    1979-01-01

    The sequence arrangement of ribosomal DNA (rDNA) spacers in Drosophila melanogaster was analyzed with restriction endonucleases. Spacers, derived from cloned rDNA repeats and from uncloned purified rDNA, are internally repetitive, as demonstrated by the regular 250 base pairs interval between sites recognized by the enzyme Alu I. Length heterogeneity of spacers is due at least in part to varying numbers of repeated sequence elements. All spacers and analyzed, whether derived from X or from Y chromosomal rDNA, have a very similar sequence organization. The distance separating the repeated nontranscribed spacer sequences from the 5' end of the transcribed region is conserved in all ten cloned fragments examined, and is probably less than 150 base pairs, as measured by electron microscopy. Images PMID:114986

  11. Estrogen Receptor Expression in Human Breast Cancer Associated with an Estrogen Receptor Gene Restriction Fragment Length Polymorphism1

    Microsoft Academic Search

    Steven M. Hill; Suzanne A. W. Fuqua; Gary C. Chamness; Geoffrey L. Greene; William L. McGuire

    1989-01-01

    Estrogen receptor (ER) content is a well-known predictor of clinical outcome in human breast cancer. The recent cloning of a human ER complementary DNA has made possible the characterization of the ER gene on a molecular level. We have examined in human breast cancers a single, two-allele restriction fragment length polymorphism using the restriction enzyme Pviill. Initial studies in human

  12. HindIII and Sst I restriction sites mapped on rabbit poxvirus and vaccinia virus DNA.

    PubMed Central

    Wittek, R; Menna, A; Schümperli, D; Stoffel, S; Müller, H K; Wyler, R

    1977-01-01

    The DNAs of two closely related orthopoxviruses, rabbit poxvirus (RPV) and vaccinia virus (VV), were mapped by overlapping-fragment analysis using restriction endonucleases HindIII and Sst I. The exact arrangement of these fragments was accomplished by total digestion of isolated partial restriction products and by end-fragment determination. RPV and VV DNAs showed identical restriction patterns in an internal region comprising approximately 60% of the genome. The size, by electrophoretical analysis of the RPV DNA, was 118 X 10(6) daltons, some 6 X 10(6) daltons less than VV DNA. The two opposite terminal restriction fragments of RPV DNA cross-hybridized to each other. Images PMID:197263

  13. Restriction enzyme analysis of the nuclear 45s ribosomal DNA of six cultivated Alliums ( Alliaceae )

    Microsoft Academic Search

    M. J. Havey

    1992-01-01

    Estimates of the phylogenetic relationships among cultivated and wildAllium species would benefit from identification of objective molecular characters. Restriction fragment length polymorphisms in the nuclear 45s ribosomal DNA (rDNA) were identified among two of five accessions of each of six cultivated Alliums. Restriction enzyme sites forBamHI,DraI,EcoRI,EcoRV,SacI, andXbaI were mapped. Different lengths of the rDNA repeat unit among the cultivated Alliums

  14. DNA fingerprinting of human cell lines using PCR amplification of fragment length polymorphisms

    Microsoft Academic Search

    Rui Yan; Mark Ottenbreit; Bharati Hukku; Michael Mally; Sharong Chou; Joseph Kaplan

    1996-01-01

    Summary  Methods for monitoring cell line identification and authentication include species-specific immunofluorescence, isoenzyme\\u000a phenotyping, chromosome analysis, and DNA fingerprinting. Most previous studies of DNA fingerprinting of cell lines have used\\u000a restriction fragment length polymorphism analysis. In this study, we examined the utility of an alternative and simpler method\\u000a of cell line DNA fingerprinting—polymerase chain reaction (PCR) amplification of fragment length polymorphisms.

  15. Mitochondrial DNA restriction map for the mediterranean fruit fly, Ceratitis capitata

    Microsoft Academic Search

    Bruce A. McPheron; Gail E. Gasparich; Ho-Yeon Han; Gary J. Steck; Walter S. Sheppard

    1994-01-01

    Molecular genetic research on the Mediterranean fruit fly,Ceratitis capitata, will provide tools to permit determination of source populations for new pest infestations. Restriction fragment length polymorphism (RFLP) of mitochondrial DNA provides some interpopulation discrimination. A restriction map, including the informative variableEcoRV andXbaI restriction sites, is constructed for the Mediterranean fruit fly, and several restriction sites are associated with specific gene

  16. Restriction fragment analysis of ‘null’ forms at the Gli-1 loci of bread and durum wheats

    Microsoft Academic Search

    P. A. Sabelli; D. Lafiandra; P. R. Shewry

    1992-01-01

    Wheat accessions lacking some of the ?- and ?-gliadin components encoded by the Gli-1 loci on the short arm of chromosome 1D in bread wheat and chromosome 1A in durum wheat were studied by two-dimensional polyacrylamide gel electrophoresis and restriction fragment analysis. Digested genomic DNAs of ‘normal’ and ‘null’ forms were probed with a cDNA clone related to ?-\\/?-gliadins and

  17. Restriction fragment variation in the nuclear and chloroplast genomes of cultivated and wild Sorghum bicolor

    Microsoft Academic Search

    P. R. Aldrich; J. Doebley

    1992-01-01

    Fifty-six accessions of cultivated and wild sorghum were surveyed for genetic diversity using 50 low-copy-number nuclear DNA sequence probes to detect restriction fragment length polymorphisms (RFLPs). These probes revealed greater genetic diversity in wild sorghum than in cultivated sorghum, including a larger number of alleles per locus and a greater portion of polymorphic loci in wild sorghum. In comparison to

  18. Large-Restriction-Fragment Polymorphism Analysis of Mycobacterium chelonae and Mycobacterium terrae Isolates

    Microsoft Academic Search

    J. Daisy Vanitha; R. Venkatasubramani; K. Dharmalingam; C. N. Paramasivan

    2003-01-01

    Mycobacterium chelonae and Mycobacterium terrae were reported to be frequently present in the environment of the Mycobacterium bovis BCG trial area in south India. Six isolates of M. chelonae and four isolates of M. terrae obtained from different sources in this area were analyzed by pulsed-field gel electrophoresis (PFGE) to examine large-restriction-fragment (LRF) polymorphism using the chromosomal DNA digested with

  19. Bacterial Species Determination from DNA-DNA Hybridization by Using Genome Fragments and DNA Microarrays

    Microsoft Academic Search

    JAE-CHANG CHO; JAMES M. TIEDJE

    2001-01-01

    Whole genomic DNA-DNA hybridization has been a cornerstone of bacterial species determination but is not widely used because it is not easily implemented. We have developed a method based on random genome fragments and DNA microarray technology that overcomes the disadvantages of whole-genome DNA-DNA hybridization. Reference genomes of four fluorescent Pseudomonas species were fragmented, and 60 to 96 genome fragments

  20. Agilent 2100 bioanalyzer for restriction fragment length polymorphism analysis of the Campylobacter jejuni flagellin gene.

    PubMed

    Nachamkin, I; Panaro, N J; Li, M; Ung, H; Yuen, P K; Kricka, L J; Wilding, P

    2001-02-01

    The Agilent 2100 bioanalyzer (Agilent Technologies, Palo Alto, Calif.) utilizes capillary electrophoresis on a microchip device (LabChip 7500; Caliper Technologies, Mountain View, Calif.) that is capable of rapidly sizing small DNA fragments. To determine whether the system could replace conventional restriction fragment length polymorphism (RFLP) typing by agarose gel electrophoresis, we compared the analyzer with conventional flagellin RFLP for typing Campylobacter jejuni. Ninety-seven isolates representing 46 Fla types were initially analyzed. Correct Fla types were detected in 59% of the isolates. The major problem with the system was in resolving samples containing multiple DNA fragments differing from 8 to 20 bp. Overall, the bioanalyzer has the potential to replace conventional RFLP analysis by gel electrophoresis, but improvements in the chip separation are needed. PMID:11158144

  1. Evaluation of PCR Amplification Bias by Terminal Restriction Fragment Length Polymorphism Analysis of Small-Subunit rRNA and mcrA Genes by Using Defined Template Mixtures of Methanogenic Pure Cultures and Soil DNA Extracts

    Microsoft Academic Search

    Tillmann Lueders; Michael W. Friedrich

    2003-01-01

    Terminal restriction fragment length polymorphism (T-RFLP) analysis is a widely used method for profiling microbial community structure in different habitats by targeting small-subunit (SSU) rRNA and also func- tional marker genes. It is not known, however, whether relative gene frequencies of individual community members are adequately represented in post-PCR amplicon frequencies as shown by T-RFLP. In this study, precisely defined

  2. RESTseq – Efficient Benchtop Population Genomics with RESTriction Fragment SEQuencing

    PubMed Central

    Stolle, Eckart; Moritz, Robin F. A.

    2013-01-01

    We present RESTseq, an improved approach for a cost efficient, highly flexible and repeatable enrichment of DNA fragments from digested genomic DNA using Next Generation Sequencing platforms including small scale Personal Genome sequencers. Easy adjustments make it suitable for a wide range of studies requiring SNP detection or SNP genotyping from fine-scale linkage mapping to population genomics and population genetics also in non-model organisms. We demonstrate the validity of our approach by comparing two honeybee and several stingless bee samples. PMID:23691128

  3. Polymerase chain reaction-restriction fragment length polymorphism authentication of raw meats from game birds.

    PubMed

    Rojas, María; González, Isabel; Fajardo, Violeta; Martín, Irene; Hernández, Pablo E; García, Teresa; Martín, Rosario

    2008-01-01

    Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis has been applied to the identification of meats from quail (Coturnix coturnix), pheasant (Phasianus colchicus), red-legged partridge (Alectoris rufa), guinea fowl (Numida meleagris), capercaillie (Tetrao urogallus), Eurasian woodcock (Scolopax rusticola), woodpigeon (Columba palumbus), and song thrush (Turdus philomelos). PCR amplification was performed using a set of primers flanking a conserved region of approximately 720 base pairs (bp) from the mitochondrial 12S rRNA gene. Restriction site analysis based on sequence data from this DNA fragment permitted the selection of AluI and BfaI endonucleases for species identification. The restriction profiles obtained when amplicons were digested with the chosen enzymes allowed the unequivocal identification of all game bird species analyzed. However, the use of the PCR-RFLP technique described is limited to raw meat authentication. It is not suitable for cooked products because thermal treatment strongly accelerates DNA degradation leading to difficulties in amplifying the 720 bp fragment. PMID:19202803

  4. Computerized Analysis of Restriction Fragment Length Polymorphism Patterns: Comparative Evaluation of Two Commercial Software Packages

    PubMed Central

    Gerner-Smidt, P.; Graves, L. M.; Hunter, Susan; Swaminathan, B.

    1998-01-01

    Two computerized restriction fragment length polymorphism pattern analysis systems, the BioImage system and the GelCompar system (Molecular Analyst Fingerprinting Plus in the United States), were compared. The two systems use different approaches to compare patterns from different gels. In GelCompar, a standard reference pattern in one gel is used to normalize subsequent gels containing lanes with the same reference pattern. In BioImage, the molecular sizes of the fragments are calculated from size standards present in each gel. The molecular size estimates obtained with the two systems for 12 restriction fragments of phage ? were between 97 and 101% of their actual sizes, with a standard deviation of less than 1% of the average estimated size for most fragments. At the window sizes used for analysis, the GelCompar system performed somewhat better than BioImage in identifying visually identical patterns generated by electrophoretic separation of HhaI-restricted DNA of Listeria monocytogenes. Both systems require the user to make critical decisions in the analysis. It is very important to visually verify that the systems are finding all bands in each lane and that no artifacts are being detected; both systems allow manual editing. It is also important to verify results obtained in the pattern matching or clustering portions of the analysis. PMID:9574697

  5. Characterization of pearl millet mitochondrial DNA fragments rearranged by reversion from cytoplasmic male sterility to fertility

    Microsoft Academic Search

    R. L. Smith; M. K. U. Chowdhury

    1991-01-01

    Cloned pearl millet [Pennisetum glaucum (L.) R. Br.] mitochondrial (mt) DNA fragments rearranged by spontaneous reversion from cytoplasmic male sterility (cms) to fertility were characterized by restriction mapping, hybridization with maize mt genes, and transcription analyses. The clones characterized were a 4.7-kb fragment found only in the male-sterile cytoplasm and lost upon reversion to fertility, a 10.9-kb fragment found in

  6. Rapid differentiation of vaccine strains and field isolates of infectious laryngotracheitis virus by restriction fragment length polymorphism of PCR products

    Microsoft Academic Search

    Poa-Chun Chang; Yuan-Ling Lee; Jui-Hung Shien; Happy K. Shieh

    1997-01-01

    A procedure was developed for differentiation of vaccine strains and field isolates of infectious laryngotracheitis virus (ILTV) by restriction fragment length polymorphism (RFLP) of DNA fragments amplified from the genome of ILTV by polymerase chain reaction (PCR). RFLP patterns of viral thymidine kinase (TK) gene, glycoprotein C (gC) gene, glycoprotein X (gX) gene and ICP4 gene amplified from different ILT

  7. Sensitive PCR-Restriction Fragment Length Polymorphism Assay for Detection and Genotyping of Giardia duodenalis in Human Feces

    Microsoft Academic Search

    C. F. L. Amar; P. H. Dear; S. Pedraza-Diaz; N. Looker; E. Linnane; J. McLauchlin

    2002-01-01

    An assay that uses heminested PCR-restriction fragment length polymorphism analysis for the detection and genotyping of Giardia duodenalis on the basis of polymorphism in the triose phosphate isomerase (tpi) gene was developed. This assay was evaluated with DNA extracted from purified parasite material, bacterial cultures, whole human feces containing G. duodenalis and other parasites, and their corresponding immunofluorescence- stained fecal

  8. Identification of infectious agents in onychomycoses by PCR-terminal restriction fragment length polymorphism.

    PubMed

    Verrier, Julie; Pronina, Marina; Peter, Corinne; Bontems, Olympia; Fratti, Marina; Salamin, Karine; Schürch, Stéphanie; Gindro, Katia; Wolfender, Jean-Luc; Harshman, Keith; Monod, Michel

    2012-03-01

    A fast and reliable assay for the identification of dermatophyte fungi and nondermatophyte fungi (NDF) in onychomycosis is essential, since NDF are especially difficult to cure using standard treatment. Diagnosis is usually based on both direct microscopic examination of nail scrapings and macroscopic and microscopic identification of the infectious fungus in culture assays. In the last decade, PCR assays have been developed for the direct detection of fungi in nail samples. In this study, we describe a PCR-terminal restriction fragment length polymorphism (TRFLP) assay to directly and routinely identify the infecting fungi in nails. Fungal DNA was easily extracted using a commercial kit after dissolving nail fragments in an Na(2)S solution. Trichophyton spp., as well as 12 NDF, could be unambiguously identified by the specific restriction fragment size of 5'-end-labeled amplified 28S DNA. This assay enables the distinction of different fungal infectious agents and their identification in mixed infections. Infectious agents could be identified in 74% (162/219) of cases in which the culture results were negative. The PCR-TRFLP assay described here is simple and reliable. Furthermore, it has the possibility to be automated and thus routinely applied to the rapid diagnosis of a large number of clinical specimens in dermatology laboratories. PMID:22170903

  9. Large-scale production of palindrome DNA fragments

    SciTech Connect

    Palmer, E.L.; Gewiess, A.; Harp, J.M. [Oak Ridge National Lab., TN (United States)] [and others] [Oak Ridge National Lab., TN (United States); and others

    1995-10-10

    Our structural studies of nucleosomes necessitated the production of over 100 mg of a 146-bp perfect palindrome DNA for use in the reconstitution of perfectly symmetrical nucleosome core particles for detailed X-ray crystallographic analysis. The propagation of palindromic DNA sequences by bacterial culture is hindered by the instability of these sequences during bacterial replication and recombination. While the loss of some palindrome sequences can be elminated by the use of sbcB or sbcC mutants of Escherichia coli, not all palindrome-containing plasmids are faithfully maintained by these strains. The production of large quantities of palindrome DNA can therefore be extremely difficult. After trying several approaches, we were able to develop a reliable procedure for production of large quantities of palindrome DNA that involves production of plasmid containing multiple copies of the repeating unit of the palindrome which are isolated by restriction digestion and ligated in vitro to form the palindrome DNA. The procedure has resulted in the production of over 20 mg of a 146-bp DNA fragment in 2 weeks.

  10. Bacterial natural transformation by highly fragmented and damaged DNA

    E-print Network

    Nielsen, Rasmus

    Bacterial natural transformation by highly fragmented and damaged DNA Søren Overballe-Petersena,1 for review August 14, 2013) DNA molecules are continuously released through decomposition of organic matter and are ubiquitous in most environments. Such DNA becomes fragmented and damaged (often

  11. DNA Extraction Strategies for Amplified Fragment Length Polymorphism Analysis

    Microsoft Academic Search

    Catherine Theisen Comey

    ABSTRACT: A polymerase chain reaction-based DNA typing method, amplified fragment length polymorphism (AMP-FLP) analysis, has shown promise as a means of analyzing forensic biological evidence. A variety of DNA extraction methods,were evaluated,for their suitability for AMP-FLP analysis. Factors that were considered in the evaluation included DNA yield, ability of DNA to be amplified, the presence of DNA fragments other than

  12. Polyethylene glycol derivatives of base and sequence specific DNA ligands: DNA interaction and application for base specific separation of DNA fragments by gel electrophoresis.

    PubMed Central

    Müller, W; Hattesohl, I; Schuetz, H J; Meyer, G

    1981-01-01

    Various base pair specific DNA ligands comprising a phenyl phenazinium dye, a triphenylmethan dye and Hoechst 33258 were covalently bound to polyethylene glycol (PEG) via ester or ether bonds. The DNA interactions of the PEG derivatives formed were shown to exhibit the same base pair specificity as the parent compounds. Since the PEG chains thus bound to the DNA could be expected to increase drastically the frictional coefficient of the DNA, the PEG derivatives were used for base specific DNA separations in agarose and polyacrylamide gel electrophoresis. The procedures, which do not require any special techniques, are described in detail. The resolution observed in agarose gels allows one to separate equally sized DNA fragments differing as little as 1% in base composition at mean travel distances of about 10 cm. Examples of gels showing the base compositional heterogeneity of restriction fragments obtained from lambda DNA, E. coli DNA and calf thymus DNA are given. Images PMID:6259622

  13. Recombination within a Subclass of Restriction Fragment Length Polymorphisms May Help Link Classical and Molecular Genetics

    PubMed Central

    Meagher, R. B.; McLean, M. D.; Arnold, J.

    1988-01-01

    Restriction fragment length polymorphisms (RFLPs) are being used to construct complete linkage maps for many eukaryotic genomes. These RFLP maps can be used to predict the inheritance of important phenotypic loci and will assist in the molecular cloning of linked gene(s) which affect phenotypes of scientific, medical and agronomic importance. However, genetic linkage implies very little about the actual physical distances between loci. An assay is described which uses genetic recombinants to measure physical distance from a DNA probe to linked phenotypic loci. We have defined the subset of all RFLPs which have polymorphic restriction sites at both ends as class II RFLPs. The frequency of class II RFLPs is computed as a function of sequence divergence and total RFLP frequency for highly divergent genomes. Useful frequencies exist between organisms which differ by more than 7% in DNA sequence. Recombination within class II RFLPs will produce fragments of novel sizes which can be assayed by pulsed field electrophoresis to estimate physical distance in kilobase pairs between linked RFLP and phenotypic loci. This proposed assay should have particular applications to crop plants where highly divergent and polymorphic species are often genetically compatible and thus, where class II RFLPs will be most frequent. PMID:2906304

  14. Recognition of Leuconostoc oenos strains by the use of DNA restriction profiles

    Microsoft Academic Search

    M. Lamoureux; H. Prévost; J. F. Cavin; C. Diviès

    1993-01-01

    The chromosome of 41 Leuconostoc oenos strains obtained from collections in different countries was analysed with the aim of differentiating the strains. Pulsed field electrophoresis (TAFE) was used to separate large DNA fragments created by the restriction enzymes NotI, SfiI and ApaI, which specifically recognize guanines or cytosines. The genomic DNA of 11 strains was analysed initially with NotI and

  15. DNA fragment assembly using an ant colony system algorithm

    Microsoft Academic Search

    Prakit Meksangsouy; N. Chaiyaratana

    2003-01-01

    This work presents the use of an ant colony system algorithm in a DNA (deoxyribonucleic acid) fragment assembly. The assembly problem is a combinatorial optimisation problem where the aim of the search is to find the right order and orientation of each fragment in the fragment ordering sequence that leads to the formation of a consensus sequence. In this paper,

  16. Restriction fragment length polymorphism of DQB and DRB class II genes of the ovine major histocompatibility complex.

    PubMed

    Grain, F; Nain, M C; Labonne, M P; Lantier, F; Lechopier, P; Gebuhrer, L; Asso, J; Maddox, J; Betuel, H

    1993-10-01

    The ovine major histocompatibility complex (MhcOvar) class II region was investigated by Southern blot hybridizations using ovine probes specific for the second exons of Ovar-DRB and Ovar-DQB genes. Multiple bands were revealed when genomic DNA was digested with each of five restriction enzymes (BamHI, EcoRI, HindIII, PvuII and TaqI), and successively hybridized with the two radiolabelled ovine probes. Restriction fragment length polymorphisms (RFLPs) were analysed in 89 sheep originating from six inbred families and the inheritance of the fragment patterns was determined. Forty-one fragments were recorded with the DQB probe; 32 were detected with the DRB probe. They constituted 9 DQB and 10 DRB allelic patterns. Twelve DQB-DRB haplotypes were resolved in this study. PMID:7904802

  17. Fiber optic system for rapid analysis of amplified DNA fragments

    Microsoft Academic Search

    J. Matthew M. Mauro; Lynn K. Cao; Joel P. Golden

    1996-01-01

    We have developed a fiber optic sensor for rapid and direct analysis of PCR-amplified DNA fragments with minimal sample processing and real-time data readout. To accomplish this, a novel DNA-recognition system was built onto the surface of fused silica fibers. DNA fragments, labeled with a fluorophore during amplification, are bound to and detected at the fiber surface by means of

  18. Restriction fragment length variations and chromosome mapping of two mouse metallothionein genes, Mt1 and Mt2

    Microsoft Academic Search

    Tomomasa Watanabe; Atsuko Shimizu; Kyoko Ohno; Shigeo Masaki; Kyoji Kondo

    1989-01-01

    Restriction endonuclease fragment length variations (RFLVs) were found through the use of cDNA probes for metallothionein genes 1 (Mt-1) and 2 (Mt-2) in the mouse. RFLVs were detected in restriction patterns generated byBglII andXbaI in theMt-1 gene and byPvuII in theMt-2 gene. All laboratory strains carry theMt-1a andMt-2a alleles. Among strains of wild origin, some Western European subspecies (Mus mus

  19. Restriction fragment length variations and chromosome mapping of two mouse metallothionein genes, Mt1 and Mt2

    Microsoft Academic Search

    Tomomasa Watanabe; Atsuko Shimizu; Kyoko Ohno; Shigeo Masaki; Kyoji Kondo

    1989-01-01

    Restriction endonuclease fragment length variations (RFLVs) were found through the use of cDNA probes for metallothionein\\u000a genes 1 (Mt-1) and 2 (Mt-2) in the mouse. RFLVs were detected in restriction patterns generated byBglII andXbaI in theMt-1 gene and byPvuII in theMt-2 gene. All laboratory strains carry theMt-1\\u000a \\u000a a\\u000a andMt-2\\u000a \\u000a a\\u000a alleles. Among strains of wild origin, some Western European subspecies

  20. Intermediate DNA at low added salt: DNA bubbles slow the diffusion of short DNA fragments

    E-print Network

    Tomislav Vuletic; Sanja Dolanski Babic; Ticijana Ban; Joachim Raedler; Francoise Livolant; Silvia Tomic

    2011-01-05

    We report a study of DNA (150 bp fragments) conformations in very low added salt $DNA concentration range $0.0015\\leq c \\leq 8$~mM (bp). We found an intermediate DNA conformation in the region $0.05 DNA has the diffusion coefficient, $D_p$ reduced below the values for both ssDNA coils and native dsDNA helices of similar polymerization degree $N$. Thus, this DNA population can not be a simple mix of dsDNA and of ssDNA which results from DNA melting. Here, melting occurs due to a reduction in screening concomitant with DNA concentration being reduced, in already very low salt conditions. The intermediate DNA is rationalized through the well known concept of fluctuational openings (DNA bubbles) which we postulate to form in AT-rich portions of the sequence, without the strands coming apart. Within the bubbles, DNA is locally stretched, while the whole molecule remains rod-like due to very low salt environment. Therefore, such intermediate DNA is elongated, in comparison to dsDNA, which accounts for its reduced $D_p$.

  1. DNA fragmentation and sperm head morphometry in cat epididymal spermatozoa.

    PubMed

    Vernocchi, Valentina; Morselli, Maria Giorgia; Lange Consiglio, Anna; Faustini, Massimo; Luvoni, Gaia Cecilia

    2014-10-15

    Sperm DNA fragmentation is an important parameter to assess sperm quality and can be a putative fertility predictor. Because the sperm head consists almost entirely of DNA, subtle differences in sperm head morphometry might be related to DNA status. Several techniques are available to analyze sperm DNA fragmentation, but they are labor-intensive and require expensive instrumentations. Recently, a kit (Sperm-Halomax) based on the sperm chromatin dispersion test and developed for spermatozoa of different species, but not for cat spermatozoa, became commercially available. The first aim of the present study was to verify the suitability of Sperm-Halomax assay, specifically developed for canine semen, for the evaluation of DNA fragmentation of epididymal cat spermatozoa. For this purpose, DNA fragmentation indexes (DFIs) obtained with Sperm-Halomax and terminal deoxynucleotidyl transferase-mediated nick-end labeling (TUNEL) were compared. The second aim was to investigate whether a correlation between DNA status, sperm head morphology, and morphometry assessed by computer-assisted semen analysis exists in cat epididymal spermatozoa. No differences were observed in DFIs obtained with Sperm-Halomax and TUNEL. This result indicates that Sperm-Halomax assay provides a reliable evaluation of DNA fragmentation of epididymal feline spermatozoa. The DFI seems to be independent from all the measured variables of sperm head morphology and morphometry. Thus, the evaluation of the DNA status of spermatozoa could effectively contribute to the completion of the standard analysis of fresh or frozen semen used in assisted reproductive technologies. PMID:25129872

  2. Species-Specific, Nested PCR-Restriction Fragment Length Polymorphism Detection of Single Cryptosporidium parvum Oocysts

    PubMed Central

    Sturbaum, Gregory D.; Reed, Carrie; Hoover, Paul J.; Jost, B. Helen; Marshall, Marilyn M.; Sterling, Charles R.

    2001-01-01

    Concurrent with recent advances seen with Cryptosporidium parvum detection in both treated and untreated water is the need to properly evaluate these advances. A micromanipulation method by which known numbers of C. parvum oocysts, even a single oocyst, can be delivered to a test matrix for detection sensitivity is presented. Using newly developed nested PCR-restriction fragment length polymorphism primers, PCR sensitivity was evaluated with 1, 2, 3, 4, 5, 7, or 10 oocysts. PCR detection rates (50 samples for each number of oocysts) ranged from 38% for single oocysts to 92% for 5 oocysts, while 10 oocysts were needed to achieve 100% detection. The nested PCR conditions amplified products from C. parvum, Cryptosporidium baileyi, and Cryptosporidium serpentis but no other Cryptosporidium sp. or protozoan tested. Restriction enzyme digestion with VspI distinguished between C. parvum genotypes 1 and 2. Restriction enzyme digestion with DraII distinguished C. parvum from C. baileyi and C. serpentis. Use of known numbers of whole oocysts encompasses the difficulty of liberating DNA from the oocyst and eliminates the standard deviation inherent within a dilution series. To our knowledge this is the first report in which singly isolated C. parvum oocysts were used to evaluate PCR sensitivity. This achievement illustrates that PCR amplification of a single oocyst is feasible, yet sensitivity remains an issue, thereby illustrating the difficulty of dealing with low oocyst numbers when working with environmental water samples. PMID:11375178

  3. High-sensitivity stable-isotope probing by a quantitative terminal restriction fragment length polymorphism protocol.

    PubMed

    Andeer, Peter; Strand, Stuart E; Stahl, David A

    2012-01-01

    Stable-isotope probing (SIP) has proved a valuable cultivation-independent tool for linking specific microbial populations to selected functions in various natural and engineered systems. However, application of SIP to microbial populations with relatively minor buoyant density increases, such as populations that utilize compounds as a nitrogen source, results in reduced resolution of labeled populations. We therefore developed a tandem quantitative PCR (qPCR)-TRFLP (terminal restriction fragment length polymorphism) protocol that improves resolution of detection by quantifying specific taxonomic groups in gradient fractions. This method combines well-controlled amplification with TRFLP analysis to quantify relative taxon abundance in amplicon pools of FAM-labeled PCR products, using the intercalating dye EvaGreen to monitor amplification. Method accuracy was evaluated using mixtures of cloned 16S rRNA genes, DNA extracted from low- and high-G+C bacterial isolates (Escherichia coli, Rhodococcus, Variovorax, and Microbacterium), and DNA from soil microcosms amended with known amounts of genomic DNA from bacterial isolates. Improved resolution of minor shifts in buoyant density relative to TRFLP analysis alone was confirmed using well-controlled SIP analyses. PMID:22038597

  4. Genetic Identification of the Main Opportunistic Mucorales by PCR-Restriction Fragment Length Polymorphism

    PubMed Central

    Machouart, M.; Larché, J.; Burton, K.; Collomb, J.; Maurer, P.; Cintrat, A.; Biava, M. F.; Greciano, S.; Kuijpers, A. F. A.; Contet-Audonneau, N.; de Hoog, G. S.; Gérard, A.; Fortier, B.

    2006-01-01

    Mucormycosis is a rare and opportunistic infection caused by fungi belonging to the order Mucorales. Recent reports have demonstrated an increasing incidence of mucormycosis, which is frequently lethal, especially in patients suffering from severe underlying conditions such as immunodeficiency. In addition, even though conventional mycology and histopathology assays allow for the identification of Mucorales, they often fail in offering a species-specific diagnosis. Due to the lack of other laboratory tests, a precise identification of these molds is thus notoriously difficult. In this study we aimed to develop a molecular biology tool to identify the main Mucorales involved in human pathology. A PCR strategy selectively amplifies genomic DNA from molds belonging to the genera Absidia, Mucor, Rhizopus, and Rhizomucor, excluding human DNA and DNA from other filamentous fungi and yeasts. A subsequent digestion step identified the Mucorales at genus and species level. This technique was validated using both fungal cultures and retrospective analyses of clinical samples. By enabling a rapid and precise identification of Mucorales strains in infected patients, this PCR-restriction fragment length polymorphism-based method should help clinicians to decide on the appropriate treatment, consequently decreasing the mortality of mucormycosis. PMID:16517858

  5. Genetic identification of the main opportunistic Mucorales by PCR-restriction fragment length polymorphism.

    PubMed

    Machouart, M; Larché, J; Burton, K; Collomb, J; Maurer, P; Cintrat, A; Biava, M F; Greciano, S; Kuijpers, A F A; Contet-Audonneau, N; de Hoog, G S; Gérard, A; Fortier, B

    2006-03-01

    Mucormycosis is a rare and opportunistic infection caused by fungi belonging to the order Mucorales. Recent reports have demonstrated an increasing incidence of mucormycosis, which is frequently lethal, especially in patients suffering from severe underlying conditions such as immunodeficiency. In addition, even though conventional mycology and histopathology assays allow for the identification of Mucorales, they often fail in offering a species-specific diagnosis. Due to the lack of other laboratory tests, a precise identification of these molds is thus notoriously difficult. In this study we aimed to develop a molecular biology tool to identify the main Mucorales involved in human pathology. A PCR strategy selectively amplifies genomic DNA from molds belonging to the genera Absidia, Mucor, Rhizopus, and Rhizomucor, excluding human DNA and DNA from other filamentous fungi and yeasts. A subsequent digestion step identified the Mucorales at genus and species level. This technique was validated using both fungal cultures and retrospective analyses of clinical samples. By enabling a rapid and precise identification of Mucorales strains in infected patients, this PCR-restriction fragment length polymorphism-based method should help clinicians to decide on the appropriate treatment, consequently decreasing the mortality of mucormycosis. PMID:16517858

  6. Advanced microinstrumentation for rapid DNA sequencing and large DNA fragment separation

    SciTech Connect

    Balch, J.; Davidson, J.; Brewer, L.; Gingrich, J.; Koo, J.; Mariella, R.; Carrano, A.

    1995-01-25

    Our efforts to develop novel technology for a rapid DNA sequencer and large fragment analysis system based upon gel electrophoresis are described. We are using microfabrication technology to build dense arrays of high speed micro electrophoresis lanes that will ultimately increase the sequencing rate of DNA by at least 100 times the rate of current sequencers. We have demonstrated high resolution DNA fragment separation needed for sequencing in polyacrylamide microgels formed in glass microchannels. We have built prototype arrays of microchannels having up to 48 channels. Significant progress has also been made in developing a sensitive fluorescence detection system based upon a confocal microscope design that will enable the diagnostics and detection of DNA fragments in ultrathin microchannel gels. Development of a rapid DNA sequencer and fragment analysis system will have a major impact on future DNA instrumentation used in clinical, molecular and forensic analysis of DNA fragments.

  7. A germline TaqI restriction fragment length polymorphism in the progesterone receptor gene in ovarian carcinoma.

    PubMed Central

    McKenna, N. J.; Kieback, D. G.; Carney, D. N.; Fanning, M.; McLinden, J.; Headon, D. R.

    1995-01-01

    Clinical outcome in ovarian carcinoma is predicted by progesterone receptor status, indicating an endocrine aspect to this disease. Peripheral leucocyte genomic DNAs were obtained from 41 patients with primary ovarian carcinoma and 83 controls from Ireland, as well as from 26 primary ovarian carcinoma patients and 101 controls in Germany. Southern analysis using a human progesterone receptor (hPR) cDNA probe identified a germline TaqI restriction fragment length polymorphism (RFLP) defined by two alleles: T1, represented by a 2.7 kb fragment; and T2, represented by a 1.9 kb fragment and characterised by an additional TaqI restriction site with respect to T1. An over-representation of T2 in ovarian cancer patients compared with controls in the pooled Irish/German population (P < 0.025) was observed. A difference (P < 0.02) in the distribution of the RFLP genotypes between Irish and German control populations was also observed. The allele distributions could not be shown to differ significantly from Hardy-Weinberg distribution in any subgroup. Using hPR cDNA region-specific probes, the extra TaqI restriction site was mapped to intron G of the hPR gene. Images Figure 2 Figure 3 Figure 4 PMID:7880723

  8. SEARCHING FOR AGROBACTERIAL T-DNA FRAGMENTS IN PLANT GENOMES

    Microsoft Academic Search

    SUMMARY Motivation: The aim of this work was to search for the nucleotide sequences in plant genome data banks similar to agrobacterial T-DNA fragments, in order to evaluate the role of naturally associated soilborne agrobacteria in plant evolution. Results: Depending on the variant and length of the T-DNA right-border fragment, we found from 2 to 115 nucleotide sequences within different

  9. Blackboard electrophoresis: An Inexpensive Exercise on the Principles of DNA Restriction Analysis.

    PubMed

    Costa, M J

    2007-09-01

    Undergraduates with little training on molecular biology may find the technical level of the typical introductory restriction laboratory too challenging and have problems with mastering the underlying concepts and processes. Blackboard electrophoresis is an active learning exercise, which focuses student attention on the sequences and principles of DNA restriction. Students convert short strings of letters of A, C, G, and Ts into blackboard electrophoresis profiles analogous to gel electrophoresis of restriction digests. Students work in teams to i) invent short strings of letters representing polynucleotides; ii) identify and count the number of specific restriction sequences in each string; iii) fractionate the strings in restriction fragments and count their size; and iv) predict blackboard electrophoretic patterns in which fragments are represented by sizes. The exercise is inexpensive, since it does not require laboratory equipment or supplies and accommodates a plethora of introductory contexts that can be explored to bring in relevance to students. The exercise has been used with high school and university audiences with very positive outcomes. Blackboard electrophoresis is a valuable complement or alternative to other instructional approaches to teach restriction analysis and DNA diversity. PMID:21591118

  10. Glucocorticosteroids induce DNA fragmentation in human lymphoid leukemia cells.

    PubMed

    Distelhorst, C W

    1988-10-01

    The present study was undertaken to investigate the potential role of glucocorticoid-induced DNA damage in the lysis of human lymphoid leukemia cells by glucocorticoids. Lymphoblasts were isolated from patients with acute lymphoblastic leukemia (ALL) or chronic myelogenous leukemia (CML) in blast crisis and cultured in vitro with or without dexamethasone. DNA was then purified from the cells and analyzed by agarose gel electrophoresis. Only high molecular weight (mol wt) DNA was present in cells cultured without dexamethasone, but a ladder of DNA fragments ranging in size from 180 to 200 base pairs (bp) to greater than 1,500 bp was present in cells cultured with dexamethasone. The DNA fragments were multiples of 180 to 200 bp, suggesting an internucleosomal site of DNA cleavage. The same pattern of DNA fragmentation was detected in normal thymocytes isolated from adrenalectomized rats following in vivo treatment with dexamethasone and in S49 mouse thymoma cells after in vitro incubation with dexamethasone. Dexamethasone-induced DNA fragmentation preceded overt loss of viability in glucocorticoid-sensitive cells but was not detected in two variants of the S49 cell line that are glucocorticoid resistant owing to glucocorticoid receptor defects. The results suggest that glucocorticoids kill human lymphoblastic leukemia cells and both normal and malignant murine thymocytes by a common mechanism that involves glucocorticoid induction of an endonucleolytic activity with cleavage of genomic DNA. PMID:3262387

  11. Sperm DNA fragmentation, recurrent implantation failure and recurrent miscarriage.

    PubMed

    Coughlan, Carol; Clarke, Helen; Cutting, Rachel; Saxton, Jane; Waite, Sarah; Ledger, William; Li, Tinchiu; Pacey, Allan A

    2015-01-01

    Evidence is increasing that the integrity of sperm DNA may also be related to implantation failure and recurrent miscarriage (RM). To investigate this, the sperm DNA fragmentation in partners of 35 women with recurrent implantation failure (RIF) following in vitro fertilization, 16 women diagnosed with RM and seven recent fathers (control) were examined. Sperm were examined pre- and post-density centrifugation by the sperm chromatin dispersion (SCD) test and the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. There were no significant differences in the age of either partner or sperm concentration, motility or morphology between three groups. Moreover, there were no obvious differences in sperm DNA fragmentation measured by either test. However, whilst on average sperm DNA fragmentation in all groups was statistically lower in prepared sperm when measured by the SCD test, this was not seen with the results from the TUNEL assay. These results do not support the hypothesis that sperm DNA fragmentation is an important cause of RIF or RM, or that sperm DNA integrity testing has value in such patients. It also highlights significant differences between test methodologies and sperm preparation methods in interpreting the data from sperm DNA fragmentation tests. PMID:25814156

  12. Agarose gel electrophoresis for the separation of DNA fragments.

    PubMed

    Lee, Pei Yun; Costumbrado, John; Hsu, Chih-Yuan; Kim, Yong Hoon

    2012-01-01

    Agarose gel electrophoresis is the most effective way of separating DNA fragments of varying sizes ranging from 100 bp to 25 kb(1). Agarose is isolated from the seaweed genera Gelidium and Gracilaria, and consists of repeated agarobiose (L- and D-galactose) subunits(2). During gelation, agarose polymers associate non-covalently and form a network of bundles whose pore sizes determine a gel's molecular sieving properties. The use of agarose gel electrophoresis revolutionized the separation of DNA. Prior to the adoption of agarose gels, DNA was primarily separated using sucrose density gradient centrifugation, which only provided an approximation of size. To separate DNA using agarose gel electrophoresis, the DNA is loaded into pre-cast wells in the gel and a current applied. The phosphate backbone of the DNA (and RNA) molecule is negatively charged, therefore when placed in an electric field, DNA fragments will migrate to the positively charged anode. Because DNA has a uniform mass/charge ratio, DNA molecules are separated by size within an agarose gel in a pattern such that the distance traveled is inversely proportional to the log of its molecular weight(3). The leading model for DNA movement through an agarose gel is "biased reptation", whereby the leading edge moves forward and pulls the rest of the molecule along(4). The rate of migration of a DNA molecule through a gel is determined by the following: 1) size of DNA molecule; 2) agarose concentration; 3) DNA conformation(5); 4) voltage applied, 5) presence of ethidium bromide, 6) type of agarose and 7) electrophoresis buffer. After separation, the DNA molecules can be visualized under uv light after staining with an appropriate dye. By following this protocol, students should be able to: Understand the mechanism by which DNA fragments are separated within a gel matrix Understand how conformation of the DNA molecule will determine its mobility through a gel matrix Identify an agarose solution of appropriate concentration for their needs Prepare an agarose gel for electrophoresis of DNA samples Set up the gel electrophoresis apparatus and power supply Select an appropriate voltage for the separation of DNA fragments Understand the mechanism by which ethidium bromide allows for the visualization of DNA bands Determine the sizes of separated DNA fragments. PMID:22546956

  13. Variability in desert bighorn and Rambouillet sheep using restriction fragment length polymorphisms 

    E-print Network

    Lyles, Ingrid Doodeheefver

    1989-01-01

    VARIABILITY IN DESERT BIGHORN AND RAMBOUILLET SHEEP USING RESTRICTION FRAGMENT LENGTH POLYMORPHISMS A Thesis by INGRID DOODEHEEFVER LYLES Approved as to style and content by: Ja es E. Womack (Char of Committee) Joe W. Templeto (Member) ry... E. Hart (Chair of Genetics Faculty) roid J. Price (Member) Kenneth R. Pierce (Head of Department) December 1989 ABSTRACT Variability in Desert Bighorn and Rambouillet Sheep Using Restriction Fragment Length polymorphisms. (December 1989...

  14. Terminal Restriction Fragment Patterns: A Tool for Comparing Microbial Communities and Assessing Community Dynamics

    Microsoft Academic Search

    Dna Polymerase; Christopher L. Kitts

    Terminal Restriction Fragment (TRF) patterns, also known as Terminal Restriction Fragment Length Polymorphisms (T-RFLP), are a recently introduced PCR-based tool for studying microbial community structure and dynamics. Since the first review of TRF methodology (Marsh, 1999. Curr. Op. Microbiol. 2: 323-7), at least 35 new research articles were published that include this powerful tool in some part of their reports.

  15. Neuronal nuclear DNA fragmentation in the aged canine brain: apoptosis or nuclear DNA fragility?

    PubMed

    Borràs, D; Pumarola, M; Ferrer, I

    2000-04-01

    Neuronal DNA fragmentation, as revealed with the method of in situ end-labeling of nuclear DNA fragmentation (TUNEL), has been reported in both the canine and human brains in normal ageing, and in some human age-related neurodegenerative diseases. These results have suggested that apoptosis plays an important role in age-related neuronal loss. It is not clear, however, whether the TUNEL method is highly specific for apoptosis, as DNA fragmentation also occurs in the late stages o necrosis. In this study we have examined 27 dogs aged from 8 to 18 years, to investigate the occurrence of nuclear DNA fragmentation. An autolysis index based on current histological criteria was assigned to each animal to evaluate the effects of autolysis on nuclear DNA integrity. Our results have shown that neuronal nuclear DNA fragmentation is frequent in aged dogs, although it is not accompanied by apoptotic morphology. Yet, a positive relation between TUNEL labelling and the degree of tissue autolysis was observed. In contrast, no TUNEL labelling was detected in young control dogs despite autolysis indices being similar to those in aged dogs. Taken together, these results suggest that neuronal nuclear DNA fragmentation is an age-related phenomenon, not due to apoptosis, whenever other factors render neuronal DNA more susceptible to autolytic fragmentation. We confirm the effect of autolysis in a subpopulation of neurons in the aged canine brain, inducing nuclear DNA fragmentation. PMID:10787039

  16. Rapid detection and characterization from field cases of infectious laryngotracheitis virus by real-time polymerase chain reaction and restriction fragment length polymorphism

    Microsoft Academic Search

    Julie L. Creelan; Viola M. Calvert; David A. Graham; Samuel J. McCullough

    2006-01-01

    A real-time polymerase chain reaction (PCR) assay was developed to specifically amplify infectious laryngotracheitis virus (ILTV) DNA from field samples. The 222-base-pair PCR fragment was amplified using primers located in a conserved region of the infected cell protein 4 gene that was demonstrated in this work to encompass a single nucleotide polymorphism. Subsequent restriction fragment length polymorphism (RFLP) analysis of

  17. Characterization of microbial diversity by determining terminal restriction fragment length polymorphisms of genes encoding 16S rRNA.

    PubMed Central

    Liu, W T; Marsh, T L; Cheng, H; Forney, L J

    1997-01-01

    A quantitative molecular technique was developed for rapid analysis of microbial community diversity in various environments. The technique employed PCR in which one of the two primers used was fluorescently labeled at the 5' end and was used to amplify a selected region of bacterial genes encoding 16S rRNA from total community DNA. The PCR product was digested with restriction enzymes, and the fluorescently labeled terminal restriction fragment was precisely measured by using an automated DNA sequencer. Computer-simulated analysis of terminal restriction fragment length polymorphisms (T-RFLP) for 1,002 eubacterial sequences showed that with proper selection of PCR primers and restriction enzymes, 686 sequences could be PCR amplified and classified into 233 unique terminal restriction fragment lengths or "ribotypes." Using T-RFLP, we were able to distinguish all bacterial strains in a model bacterial community, and the pattern was consistent with the predicted outcome. Analysis of complex bacterial communities with T-RFLP revealed high species diversity in activated sludge, bioreactor sludge, aquifer sand, and termite guts; as many as 72 unique ribotypes were found in these communities, with 36 ribotypes observed in the termite guts. The community T-RFLP patterns were numerically analyzed and hierarchically clustered. The pattern derived from termite guts was found to be distinctly different from the patterns derived from the other three communities. Overall, our results demonstrated that T-RFLP is a powerful tool for assessing the diversity of complex bacterial communities and for rapidly comparing the community structure and diversity of different ecosystems. PMID:9361437

  18. A Computer-Simulated Restriction Fragment Length Polymorphism Analysis of Bacterial Small-Subunit rRNA Genes: Efficacy of Selected Tetrameric Restriction Enzymes for Studies of Microbial Diversity in Nature

    Microsoft Academic Search

    CRAIG L. MOYER; JAMES M. TIEDJE; FRED C. DOBBS; ANDDAVID M. KARL

    An assessment of 10 tetrameric restriction enzymes (TREs) was conducted by using a computer-simulated restriction fragment length polymorphism (RFLP) analysis for over 100 proximally and distally related bacterial small-subunit (SSU) rRNA gene sequences. Screening SSU rDNA clone libraries with TREs has become an effective strategy because of logistic simplicity, commercial availability, and economy. However, the rationale for selecting the type

  19. Molecular typing of Borrelia burgdorferi sensu lato by PCR-restriction fragment length polymorphism analysis.

    PubMed Central

    Liveris, D; Gazumyan, A; Schwartz, I

    1995-01-01

    The etiologic agent of Lyme borreliosis, Borrelia burgdorferi sensu lato, has been isolated from many biologic sources in North America and Eurasia, and isolates have been divided into three distinct genospecies (B. burgdorferi sensu stricto, Borrelia garinii, and Borrelia afzelii). In order to explore the possible association of genospecies with disease manifestation, 60 isolates of B. burgdorferi sensu lato were subjected to 5S rDNA-linked restriction fragment length polymorphism (RFLP) analysis. The results confirmed earlier studies which indicated that virtually all North American isolates are B. burgdorferi sensu stricto, whereas Eurasian strains fall into all three genospecies. Thirty-five isolates were further characterized by PCR amplification of a region of the 16S-23S rDNA spacer and HinfI digestion of the products. This method resulted in the subdivision of B. burgdorferi sensu stricto into two distinct PCR-RFLP types. In contrast, B. garinii isolates all displayed an identical pattern. Additionally, a number of previously unclassified North American isolates (25015, DN127, 19857, 24330) showed distinctively different PCR-RFLP patterns. The application of this method for the typing of uncultured B. burgdorferi directly in biologic samples was demonstrated by analysis of several field-collected Ixodes scapularis tick specimens. The described PCR-RFLP technique should allow for the direct and rapid molecular typing of B. burgdorferi-containing samples and facilitate studies of the relationship between spirochete genotype and clinical disease. PMID:7751362

  20. Identification of blood meal sources of Lutzomyia longipalpis using polymerase chain reaction-restriction fragment length polymorphism analysis of the cytochrome B gene

    PubMed Central

    Soares, Vítor Yamashiro Rocha; da Silva, Jailthon Carlos; da Silva, Kleverton Ribeiro; Cruz, Maria do Socorro Pires e; Santos, Marcos Pérsio Dantas; Ribolla, Paulo Eduardo Martins; Alonso, Diego Peres; Coelho, Luiz Felipe Leomil; Costa, Dorcas Lamounier; Costa, Carlos Henrique Nery

    2014-01-01

    An analysis of the dietary content of haematophagous insects can provide important information about the transmission networks of certain zoonoses. The present study evaluated the potential of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of the mitochondrial cytochrome B (cytb) gene to differentiate between vertebrate species that were identified as possible sources of sandfly meals. The complete cytb gene sequences of 11 vertebrate species available in the National Center for Biotechnology Information database were digested with Aci I, Alu I, Hae III and Rsa I restriction enzymes in silico using Restriction Mapper software. The cytb gene fragment (358 bp) was amplified from tissue samples of vertebrate species and the dietary contents of sandflies and digested with restriction enzymes. Vertebrate species presented a restriction fragment profile that differed from that of other species, with the exception of Canis familiaris and Cerdocyon thous. The 358 bp fragment was identified in 76 sandflies. Of these, 10 were evaluated using the restriction enzymes and the food sources were predicted for four: Homo sapiens (1), Bos taurus (1) and Equus caballus (2). Thus, the PCR-RFLP technique could be a potential method for identifying the food sources of arthropods. However, some points must be clarified regarding the applicability of the method, such as the extent of DNA degradation through intestinal digestion, the potential for multiple sources of blood meals and the need for greater knowledge regarding intraspecific variations in mtDNA. PMID:24821056

  1. Pst I restriction fragment length polymorphism of the human placental alkaline phosphatase gene in normal placentae and tumors

    SciTech Connect

    Tsavaler, L.; Penhallow, R.C.; Kam, W.; Sussman, H.H.

    1987-07-01

    The structure of the human placental alkaline phosphatase gene from normal term placentae was studied by restriction enzyme digestion and Southern blot analysis using a cDNA probe to the gene for the placental enzyme. The DNA digests fall into three distinct patterns based on the presence and intensity of an extra 1.1-kilobase Pst I Band. The extra 1.1-kilobase band is present in 9 of 27 placenta samples, and in 1 of these samples the extra band is present at double intensity. No polymorphism was revealed by digestion with restriction enzymes EcoRI, Sma I, BamHI, or Sac I. The extra Pst I-digestion site may lie in a noncoding region of the gene because no correlation was observed between the restriction fragment length polymorphism and the common placental alkaline phosphatase alleles identified by starch gel electrophoresis. In addition, because placental alkaline phosphatase is frequently re-expressed in neoplasms, the authors examined tissue from ovarian, testicular, and endometrial tumors and from BeWo choriocarcinoma cells in culture. The Pst I-DNA digestion patterns from these cells and tissues were identical to those seen in the normal ovary and term placentae. The consistent reproducible digestion patterns seen in DNA from normal and tumor tissue indicate that a major gene rearrangement is not the basis for the ectopic expression of placental alkaline phosphatase in neoplasia.

  2. Analysis of mer Gene Subclasses within Bacterial Communities in Soils and Sediments Resolved by Fluorescent-PCR-Restriction Fragment Length Polymorphism Profiling

    Microsoft Academic Search

    KENNETH D. BRUCE

    1997-01-01

    Bacterial mer (mercury resistance) gene subclasses in mercury-polluted and pristine natural environments have been profiled by Fluorescent-PCR-restriction fragment length polymorphism (FluRFLP). For FluRFLP, PCR products were amplified from individual mer operons in mercury-resistant bacteria and from DNA isolated directly from bacteria in soil and sediment samples. The primers used to amplify DNA were designed from consensus sequences of the major

  3. Separation of Three Species of Ditylenchus and Some Host Races of D. dipsaci by Restriction Fragment Length Polymorphism

    PubMed Central

    Wendt, Karen R.; Vrain, Thierry C.; Webster, John M.

    1993-01-01

    This study examined the ribosomal cistron of Ditylenchus destructor, D. myceliophagus and seven host races of D. dipsaci from different geographic locations. The three species showed restriction fragment length polymorphisms (RFLPs) in the ribosomal cistron, the 18S rDNA gene, and the ribosomal internal transcribed spacer (ITS). Southern blot analysis with a 7.5-kb ribosomal cistron probe differentiated the five host races of D. dipsaci examined. Polymerase chain reaction (PCR) amplification of the ITS, followed by digestion with some restriction endonucleases (but not others), produced restriction fragments diagnostic of the giant race. Because the PCR product from D. myceliophagus and the host races of D. dipsaci was about 900 base pairs and the ITS size in D. destructor populations was 1,200 base pairs, mixtures of populations could be detected by PCR amplification. ITS fragments differentiated between D. dipsaci and Aphelenchoides rhyntium in mixed populations. This study establishes the feasibility of differentiation of the host races of D. dipsaci by probing Southern blots with the whole ribosomal cistron. PMID:19279809

  4. Restriction fragment length polymorphism of the HLA-DP subregion and correlations to HLA-DP phenotypes

    SciTech Connect

    Hyldig-Nielsen, J.J.; Morling, N.; Oedum, N.; Ryder, L.P.; Platz, P.; Jakobsen, B.; Svejgaard, A.

    1987-03-01

    The restriction fragment length polymorphism (RFLP) of the class II HLA-DP subregion of the major histocompatibility complex (MHC) of humans has been unraveled by Southern blotting using DP/sub ..cap alpha../ and DP/sub ..beta../ probes in a study of 46 unrelated individuals with known HLA-DP types. Contrary to earlier preliminary findings with a limited number of enzymes, the RFLP appears to be quite extensive both with the DP/sub ..beta../ (14 different DNA markers defined by individual fragments or clusters thereof) and the DP/sub ..cap alpha../ (8 markers) probes, especially when enzyme recognizing only four base pairs were used. A few markers were absolutely or strongly associated with individual DP antigens, whereas most were associated with two or more DP antigens as defined by primed lymphocyte typing. Thus, Southern blotting seems feasible for typing for most DP determinants by specific fragments or subtraction between the various more broadly reactive DNA markers, and the RFLP provides further information on the DP subregion in addition to that provided by primed lymphocyte typing. In two recombinant families, the DP/sub ..beta../ and DP/sub ..cap alpha../ DNA markers segregated with DP antigens, whereas the DR/sub ..beta../, DQ/sub ..beta../, DQ/sub ..cap alpha../, and DX/sub ..cap alpha../ markers followed the DR and DQ antigens.

  5. Selective Microbial Genomic DNA Isolation Using Restriction Endonucleases

    PubMed Central

    Barnes, Helen E.; Liu, Guohong; Weston, Christopher Q.; King, Paula; Pham, Long K.; Waltz, Shannon; Helzer, Kimberly T.; Day, Laura; Sphar, Dan; Yamamoto, Robert T.; Forsyth, R. Allyn

    2014-01-01

    To improve the metagenomic analysis of complex microbiomes, we have repurposed restriction endonucleases as methyl specific DNA binding proteins. As an example, we use DpnI immobilized on magnetic beads. The ten minute extraction technique allows specific binding of genomes containing the DpnI Gm6ATC motif common in the genomic DNA of many bacteria including ?-proteobacteria. Using synthetic genome mixtures, we demonstrate 80% recovery of Escherichia coli genomic DNA even when only femtogram quantities are spiked into 10 µg of human DNA background. Binding is very specific with less than 0.5% of human DNA bound. Next Generation Sequencing of input and enriched synthetic mixtures results in over 100-fold enrichment of target genomes relative to human and plant DNA. We also show comparable enrichment when sequencing complex microbiomes such as those from creek water and human saliva. The technique can be broadened to other restriction enzymes allowing for the selective enrichment of trace and unculturable organisms from complex microbiomes and the stratification of organisms according to restriction enzyme enrichment. PMID:25279840

  6. Multiple displacement amplification for complex mixtures of DNA fragments

    PubMed Central

    Shoaib, Muhammad; Baconnais, Sonia; Mechold, Undine; Le Cam, Eric; Lipinski, Marc; Ogryzko, Vasily

    2008-01-01

    Background A fundamental requirement for genomic studies is the availability of genetic material of good quality and quantity. The desired quantity and quality are often hard to obtain when target DNA is composed of complex mixtures of relatively short DNA fragments. Here, we sought to develop a method to representatively amplify such complex mixtures by converting them to long linear and circular concatamers, from minute amounts of starting material, followed by phi29-based multiple displacement amplification. Results We report here proportional amplification of DNA fragments that were first converted into concatamers starting from DNA amounts as low as 1 pg. Religations at low concentration (< 1 ng/?L) preferentially lead to fragment self-circularization, which are then amplified independently, and result in non-uniform amplification. To circumvent this problem, an additional (stuffer) DNA was added during religation (religation concentration > 10 ng/?L), which helped in the formation of long concatamers and hence resulted in uniform amplification. To confirm its usefulness in research, DP1 bound chromatin was isolated through ChIP and presence of DHFR promoter was detected using q-PCR and compared with an irrelevant GAPDH promoter. The results clearly indicated that when ChIP material was religated in presence of stuffer DNA (improved MDA), it allowed to recover the original pattern, while standard MDA and MDA without stuffer DNA failed to do so. Conclusion We believe that this method allows for generation of abundant amounts of good quality genetic material from a complex mixture of short DNA fragments, which can be further used in high throughput genetic analysis. PMID:18793430

  7. Generation of a Restriction Fragment Length Polymorphism Linkage Map for Toxoplasma Gondii

    PubMed Central

    Sibley, L. D.; LeBlanc, A. J.; Pfefferkorn, E. R.; Boothroyd, J. C.

    1992-01-01

    We have constructed a genetic linkage map for the parasitic protozoan, Toxoplasma gondii, using randomly selected low copy number DNA markers that define restriction fragment length polymorphisms (RFLPs). The inheritance patterns of 64 RFLP markers and two phenotypic markers were analyzed among 19 recombinant haploid progeny selected from two parallel genetic crosses between PLK and CEP strains. In these first successful interstrain crosses, these RFLP markers segregated into 11 distinct genetic linkage groups that showed close correlation with physical linkage groups previously defined by molecular karyotype. Separate linkage maps, constructed for each of the 11 chromosomes, indicated recombination frequencies range from approximately 100 to 300 kb per centimorgan. Preliminary linkage assignments were made for the loci regulating sinefungin resistance (snf-1) on chromosome IX and adenine arabinoside (ara-1) on chromosome V by linkage to RFLP markers. Despite random segregation of separate chromosomes, the majority of chromosomes failed to demonstrate internal recombination events and in 3/19 recombinant progeny no intramolecular recombination events were detected. The relatively low rate of intrachromosomal recombination predicts that tight linkage for unknown genes can be established with a relatively small set of markers. This genetic linkage map should prove useful in mapping genes that regulate drug resistance and other biological phenotypes in this important opportunistic pathogen. PMID:1360931

  8. Systematics of Cetaceans Using Restriction Site Mapping of Mitochondrial DNA

    Microsoft Academic Search

    Derek Paul Ohland; Eric H. Harley; Peter B. Best

    1995-01-01

    Phylogenetic analysis of 14 cetacean species, including members from two baleen whale families and three toothed whale families, was undertaken using restriction site mapping of mitochondrial DNA and using cladistic and distance measures to infer phylogenies. The amount of between-taxa sequence divergence inferred from the data was lower than expected from the standard interpretation of the fossil record, but more

  9. Calcium-activated DNA fragmentation kills immature thymocytes

    Microsoft Academic Search

    DAVID J. MCCONKEY; PIA HARTZELL; PIERLUIGI NICOTERA; STEN ORRENIUS

    Glucocorticoid hormones kill immature thymocytes by activating a self-destructive process that involves exten- sive DNA fragmentation. It has been demonstrated that thymocyte suicide is dependent on an early, sustained increase in cytosolic Ca2 concentration, and new protein synthesis, but the biochemical lesion that leads to cell death has not been established. To determine whether endonuclease activation or activation of another

  10. Statistical associations between restriction fragment length polymorphisms and quantitative traits in beef cattle 

    E-print Network

    Rocha, Joao Luis Lopes da Costa

    1990-01-01

    STATISTICAL ASSOCIATIONS BETWEEN RESTRICTION FRAGMENT LENGTH POLYMORPHISMS AND QUANTITATIVE TRAITS IN BEEF CATTLE A Thesis JOAO LUIS LOPES DA COSTA ROCHA Submitted to the Office of Graduate Studies of Texas A&M University in partial... fulfillment of the requirements for the degree of MASTER OF SCIENCE August 1990 Major Subject: Animal Breeding STATISTICAL ASSOCIATIONS BETWEEN RESTRICTION PRAGMENT LENGTH POLYMORPHISMS AND QUANTITATIVE TRAITS IN BEEF CATTLE A Thesis JOAO LUIS LOPES DA...

  11. DNA Oligonucleotide Fragment Ion Rearrangements Upon Collision-Induced Dissociation

    NASA Astrophysics Data System (ADS)

    Harper, Brett; Neumann, Elizabeth K.; Solouki, Touradj

    2015-06-01

    Collision-induced dissociation (CID) of m/z-isolated w type fragment ions and an intact 5' phosphorylated DNA oligonucleotide generated rearranged product ions. Of the 21 studied w ions of various nucleotide sequences, fragment ion sizes, and charge states, 18 (~86%) generated rearranged product ions upon CID in a Synapt G2-S HDMS (Waters Corporation, Manchester, England, UK) ion mobility-mass spectrometer. Mass spectrometry (MS), ion mobility spectrometry (IMS), and theoretical modeling data suggest that purine bases can attack the free 5' phosphate group in w type ions and 5' phosphorylated DNA to generate sequence permuted [phosphopurine]- fragment ions. We propose and discuss a potential mechanism for generation of rearranged [phosphopurine]- and complementary y-B type product ions.

  12. Alternative Okazaki Fragment Ligation Pathway by DNA Ligase III

    PubMed Central

    Arakawa, Hiroshi; Iliakis, George

    2015-01-01

    Higher eukaryotes have three types of DNA ligases: DNA ligase 1 (Lig1), DNA ligase 3 (Lig3) and DNA ligase 4 (Lig4). While Lig1 and Lig4 are present in all eukaryotes from yeast to human, Lig3 appears sporadically in evolution and is uniformly present only in vertebrates. In the classical, textbook view, Lig1 catalyzes Okazaki-fragment ligation at the DNA replication fork and the ligation steps of long-patch base-excision repair (BER), homologous recombination repair (HRR) and nucleotide excision repair (NER). Lig4 is responsible for DNA ligation at DNA double strand breaks (DSBs) by the classical, DNA-PKcs-dependent pathway of non-homologous end joining (C-NHEJ). Lig3 is implicated in a short-patch base excision repair (BER) pathway, in single strand break repair in the nucleus, and in all ligation requirements of the DNA metabolism in mitochondria. In this scenario, Lig1 and Lig4 feature as the major DNA ligases serving the most essential ligation needs of the cell, while Lig3 serves in the cell nucleus only minor repair roles. Notably, recent systematic studies in the chicken B cell line, DT40, involving constitutive and conditional knockouts of all three DNA ligases individually, as well as of combinations thereof, demonstrate that the current view must be revised. Results demonstrate that Lig1 deficient cells proliferate efficiently. Even Lig1/Lig4 double knockout cells show long-term viability and proliferate actively, demonstrating that, at least in DT40, Lig3 can perform all ligation reactions of the cellular DNA metabolism as sole DNA ligase. Indeed, in the absence of Lig1, Lig3 can efficiently support semi-conservative DNA replication via an alternative Okazaki-fragment ligation pathway. In addition, Lig3 can back up NHEJ in the absence of Lig4, and can support NER and HRR in the absence of Lig1. Supporting observations are available in less elaborate genetic models in mouse cells. Collectively, these observations raise Lig3 from a niche-ligase to a universal DNA ligase, which can potentially substitute or backup the repair and replication functions of all other DNA ligases in the cell nucleus. Thus, the old model of functionally dedicated DNA ligases is now replaced by one in which only Lig4 remains dedicated to C-NHEJ, with Lig1 and Lig3 showing an astounding functional flexibility and interchangeability for practically all nuclear ligation functions. The underlying mechanisms of Lig3 versus Lig1 utilization in DNA repair and replication are expected to be partly different and remain to be elucidated. PMID:26110316

  13. Alternative Okazaki Fragment Ligation Pathway by DNA Ligase III.

    PubMed

    Arakawa, Hiroshi; Iliakis, George

    2015-01-01

    Higher eukaryotes have three types of DNA ligases: DNA ligase 1 (Lig1), DNA ligase 3 (Lig3) and DNA ligase 4 (Lig4). While Lig1 and Lig4 are present in all eukaryotes from yeast to human, Lig3 appears sporadically in evolution and is uniformly present only in vertebrates. In the classical, textbook view, Lig1 catalyzes Okazaki-fragment ligation at the DNA replication fork and the ligation steps of long-patch base-excision repair (BER), homologous recombination repair (HRR) and nucleotide excision repair (NER). Lig4 is responsible for DNA ligation at DNA double strand breaks (DSBs) by the classical, DNA-PKcs-dependent pathway of non-homologous end joining (C-NHEJ). Lig3 is implicated in a short-patch base excision repair (BER) pathway, in single strand break repair in the nucleus, and in all ligation requirements of the DNA metabolism in mitochondria. In this scenario, Lig1 and Lig4 feature as the major DNA ligases serving the most essential ligation needs of the cell, while Lig3 serves in the cell nucleus only minor repair roles. Notably, recent systematic studies in the chicken B cell line, DT40, involving constitutive and conditional knockouts of all three DNA ligases individually, as well as of combinations thereof, demonstrate that the current view must be revised. Results demonstrate that Lig1 deficient cells proliferate efficiently. Even Lig1/Lig4 double knockout cells show long-term viability and proliferate actively, demonstrating that, at least in DT40, Lig3 can perform all ligation reactions of the cellular DNA metabolism as sole DNA ligase. Indeed, in the absence of Lig1, Lig3 can efficiently support semi-conservative DNA replication via an alternative Okazaki-fragment ligation pathway. In addition, Lig3 can back up NHEJ in the absence of Lig4, and can support NER and HRR in the absence of Lig1. Supporting observations are available in less elaborate genetic models in mouse cells. Collectively, these observations raise Lig3 from a niche-ligase to a universal DNA ligase, which can potentially substitute or backup the repair and replication functions of all other DNA ligases in the cell nucleus. Thus, the old model of functionally dedicated DNA ligases is now replaced by one in which only Lig4 remains dedicated to C-NHEJ, with Lig1 and Lig3 showing an astounding functional flexibility and interchangeability for practically all nuclear ligation functions. The underlying mechanisms of Lig3 versus Lig1 utilization in DNA repair and replication are expected to be partly different and remain to be elucidated. PMID:26110316

  14. Restricted pollen flow of Dieffenbachia seguine populations in fragmented and continuous tropical forest.

    PubMed

    Cuartas-Hernández, S; Núñez-Farfán, J; Smouse, P E

    2010-08-01

    Habitat fragmentation can change the ecological context of populations, rupturing genetic connectivity among them, changing genetic structure, and increasing the loss of genetic diversity. We analyzed mating system and pollen structure in two population fragments and two continuous forest populations of Dieffenbachia seguine (Araceae), an insect-pollinated understory herb in the tropical rain forest of Los Tuxtlas, México, using nine allozyme loci. Mating system analysis indicated almost complete outcrossing but some inbreeding among the adults. Pollen structure analysis indicated highly restricted pollen flow, both within and among populations. We showed that the effective pollination neighborhood was small in all populations, and slightly (though not significantly) smaller in fragments, partially as a consequence of an increase in density of reproductive individuals in those fragments. Using assignment analysis, we showed that all populations were strongly structured, suggesting that pollen and seed flow across the Los Tuxtlas landscape has been spatially restricted, though sufficient to maintain connectedness. Forest fragmentation at Los Tuxtlas has (so far) had limited impact on pollen dynamics, despite the changing ecological context, with reduced pollinator abundance being partially offset by increased flowering density in fragments. Continued outcrossing and limited pollen immigration, coupled with more extensive seed migration, should maintain genetic connectedness in D. seguine, if fragmentation is not further exacerbated by additional deforestation. PMID:20029453

  15. Genome organization of RNA tumor viruses II. Physical maps of in vitro-synthesized Moloney murine leukemia virus double-stranded DNA by restriction endonucleases.

    PubMed Central

    Verma, I M; McKennett, M A

    1978-01-01

    Physical maps of the genome of Moloney murine leukemia virus (M-MLV) DNA were constructed by using bacterial restriction endonucleases. The in vitro-synthesized M-MLV double-stranded DNA was used as the source of the viral DNA. Restriction endonucleases Sal I and Hind III cleave viral DNA at only one site and, thus, generate two DNA fragments. The two DNA fragments generated by Sal I are Sal IA (molecular weight, 3.5 x 10(6)) and Sal IB (molecular weight, 2.4 x 10(6)) and by Hind III are Hind IIIA (molecular weight, 3.6 x 10(6) and Hind IIIB (molecular weight, 2.3 x 10(6)). Restriction endonuclease Bam I generates four fragments of molecular weights of 2.1 x 10(6) (Bam IA), 2 X 10(6) (Bam IB), 1.25 X 10(6) (Bam IC), and 0.24 x 10(6) (Bam ID), whereas restriction endonuclease Hpa I cleaves the M-MLV double-stranded DNA twice to give three fragments of molecular weights of 4.4 x 10(6) (Hpa IA), 0.84 X 10(6) (Hpa IB), and 0.74 x 10(6) (Hpa IC). Digestion of M-MLV double-stranded DNA with restriction endonuclease Sma I produces four fragments of molecular weights of 3.9 x 10(6) (Sma IA), 1.3 X 10(6) (Sma IB), 0.28 X 10(6) (Sma IC), and 0.21 x 10(6) (Sma ID). A mixture of restriction endonucleases Bgl I and Bgl II (Bgl I + II) cleaves the viral DNA at four sites generating five fragments of approximate molecular weights of 2 x 10(6) (Bgl + IIA), 1.75 X 10(6) (Bgl I + IIB), 1.25 X 10(6) (Bgl I + IIC), 0.40 X 10(6) (Bgl I + IID), and 0.31 x 10(6) (Bgl I + IIE). The order of the fragments in relation to the 5' end and 3' end of the genome was determined either by using fractional-length M-MLV double-stranded DNA for digestion by restriction endonucleases or by redigestion of Sal IA, Sal IB, Hind IIIA, and Hind IIIB fragments with other restriction endonucleases. In addition, a number of other restriction endonucleases that cleave in vitro-synthesized M-MLV double-stranded DNA have also been listed. Images PMID:353301

  16. New Threshold and Confidence Estimates for Terminal Restriction Fragment Length Polymorphism Analysis of Complex Bacterial Communities

    Microsoft Academic Search

    Catherine A. Osborne; Gavin N. Rees; Yaniv Bernstein; Peter H. Janssen

    2006-01-01

    Terminal restriction fragment length polymorphism (T-RFLP) analysis has the potential to be useful for comparisons of complex bacterial communities, especially to detect changes in community structure in re- sponse to different variables. To do this successfully, systematic variations have to be detected above method- associated noise, by standardizing data sets and assigning confidence estimates to relationships detected. We investigated the

  17. Linkages between restriction fragment length, isozyme, and morphological markers in lentil

    Microsoft Academic Search

    M. J. Havey; F. J. Muehlbauer

    1989-01-01

    A genetic linkage map of lentil comprising 333 centimorgans (cM) was constructed from 20 restriction fragment length, 8 isozyme, and 6 morphological markers segregating in a single interspecific cross (Lens culinaris × L. orientalis). Because the genotypes at marker loci were determined for about 66 F2 plants, linkages are only reported for estimates of recombination less than 30 cM. Probes

  18. DNA Fragmentation in mammalian cells exposed to various light ions

    NASA Astrophysics Data System (ADS)

    Belli, M.; Cherubini, R.; Dalla Vecchia, M.; Dini, V.; Esposito, G.; Moschini, G.; Sapora, O.; Signoretti, C.; Simone, G.; Sorrentino, E.; Tabocchini, M. A.

    Elucidation of how effects of densely ionizing radiation at cellular level are linked to DNA damage is fundamental for a better understanding of the mechanisms leading to genomic damage (especially chromosome aberrations) and developing biophysical models to predict space radiation effects. We have investigated the DNA fragmentation patterns induced in Chinese hamster V79 cells by 31 keV/?m protons, 123 keV/?m helium-4 ions and ?-rays in the size range 0.023-5.7 Mbp, using calibrated Pulsed Field Gel Electrophoresis (PFGE). The frequency distributions of fragments induced by the charged particles were shifted towards smaller sizes with respct to that induced by comparable doses of ?-rays. The DSB yields, evaluated from the fragments induced in the size range studied, were higher for protons and helium ions than for ?-rays by a factor of about 1.9 and 1.2, respectively. However, these ratios do not adequately reflect the RBE observed on the same cells for inactivation and mutation induced by these beams. This is a further indication for the lack of correlation between the effects exerted at cellular level and the initial yield of DSB. The dependence on radiation quality of the fragmentation pattern suggests that it may have a role in damage reparability. We have analyzed these patterns with a "random breakage" model generalized in order to consider the initial non-random distribution of the DNA molecules. Our results suggest that a random breakage mechanism can describe with a reasonable approximation the DNA fragmentation induced by ?-rays, while the approximation is not so good for light ions, likely due to the interplay between ion tracks and chromatin organization at the loop level.

  19. Plasmid vectors capable of transferring large DNA fragments to yeast.

    PubMed

    Morris, D W; Noti, J D; Osborne, F A; Szalay, A A

    1981-01-01

    We have constructed several cloning vectors which can be used in vitro packaging and yeast transformation. These plasmids have been designed for the convenient cloning of large segments of DNA and their transfer to yeast. They contain bacterial plasmid DNA sequences for replication and selection in Escherichia coli, yeast 2-microns plasmid DNA sequences or chromosomal replicators and yeast markers necessary for replication and selection in yeast, and the cohesive ends of bacteriophage lambda which allow packaging of recombinant molecules into lambda phage heads. Large fragments (22-38 kb) of Klebsiella pneumoniae and Zea mays DNA were ligated into plasmid vector pBTI-1 to make complete genome libraries. One clone from the K. pneumoniae library was amplified in E. coli and the purified DNA used to transform yeast cells. Transformation of yeast by large DNA fragments occurred at high frequencies. The recombinant plasmid was stably maintained in yeast, provided selective pressure for Leu+ transformants was maintained. The structurally complete recombinant plasmid can be recovered from yeast by transforming E. coli to ampicillin resistance. Fewer than 5% of the recovered plasmids had undergone recombination with endogenous yeast 2-microns plasmid. PMID:6299664

  20. DNA fragment conformations in adducts with Kiteplatin.

    PubMed

    Margiotta, Nicola; Petruzzella, Emanuele; Platts, James A; Mutter, Shaun T; Deeth, Robert J; Ranaldo, Rosa; Papadia, Paride; Marzilli, Patricia A; Marzilli, Luigi G; Hoeschele, James D; Natile, Giovanni

    2015-02-28

    The anticancer activity of cisplatin is triggered by its formation of intrastrand adducts involving adjacent G residues of DNA. To obtain information on the different conformers that can be formed, carrier ligands such as 2,2'-bipiperidine, which provide large steric bulk near the platinum coordination plane and decrease the dynamic motion about the Pt-N7 bonds, were introduced ("retro-modelling" approach). In the present study we investigate the effect of cis-1,4-diaminocyclohexane (cis-1,4-DACH) on the formation, stability, and stereochemistry of (cis-1,4-DACH)Pt(ss-oligo) adducts (ss-oligo = d(GpG) with 3'- and/or 5'-substituents). Interesting features of this ligand, absent in previous retro-modelling studies, include the large bite angle (expected to impede the ease of interconversion between possible conformers), the presence of two protons on each nitrogen (a characteristic associated with antitumor activity), and the absence of chiral centres. The use of cis-1,4-DACH has made it possible to detect different conformers in a system containing a primary diamine carrier ligand associated with anticancer activity and to confirm the previous hypothesis that the coexistence of different conformers established in studies of retro models having relatively bulky ligands is not an artefact resulting from carrier-ligand bulk. Moreover, the data for the (cis-1,4-DACH)Pt(d(GpG)) and (cis-1,4-DACH)Pt(d(GGTTT)) adducts indicate that at a temperature close to the physiological one (40 °C) HH1 and ?HT1 conformers are present in comparable amounts. In contrast, at low temperature (close to 0 °C) the equilibrium shifts dramatically toward the more stable HH1 conformer (for the (cis-1,4-DACH)Pt(d(TGGT)) adduct the HH1 conformer is always dominant, even at high temperature). Notably, (cis-1,4-DACH)PtCl2 (Kiteplatin) has been recently reinvestigated and found to be particularly active against colorectal cancer (including oxaliplatin-resistant phenotypes). PMID:25144401

  1. Hot Fusion: an efficient method to clone multiple DNA fragments as well as inverted repeats without ligase.

    PubMed

    Fu, Changlin; Donovan, William P; Shikapwashya-Hasser, Olga; Ye, Xudong; Cole, Robert H

    2014-01-01

    Molecular cloning is utilized in nearly every facet of biological and medical research. We have developed a method, termed Hot Fusion, to efficiently clone one or multiple DNA fragments into plasmid vectors without the use of ligase. The method is directional, produces seamless junctions and is not dependent on the availability of restriction sites for inserts. Fragments are assembled based on shared homology regions of 17-30 bp at the junctions, which greatly simplifies the construct design. Hot Fusion is carried out in a one-step, single tube reaction at 50 °C for one hour followed by cooling to room temperature. In addition to its utility for multi-fragment assembly Hot Fusion provides a highly efficient method for cloning DNA fragments containing inverted repeats for applications such as RNAi. The overall cloning efficiency is in the order of 90-95%. PMID:25551825

  2. Restriction fragment length polymorphism species-specific patterns in the identification of white truffles

    Microsoft Academic Search

    Luana Bertini; Lucia Potenza; Alessandra Zambonelli; Antonella Amicucci; Vilberto Stocchi

    1998-01-01

    A molecular method for the identification of ectomycorrhizae belonging to five species of white truffle is described. The polymerase chain reaction (PCR) and universal primers were used to amplify internal transcribed spacers and 5.8S rDNA, target sequences present in a high number of copies. The amplified products were digested with restriction enzymes in order to detect interspecific polymorphisms. Species-specific restriction

  3. Sequence specific inhibition of DNA restriction enzyme cleavage by PNA.

    PubMed Central

    Nielsen, P E; Egholm, M; Berg, R H; Buchardt, O

    1993-01-01

    Plasmids containing double-stranded 10-mer PNA (peptide nucleic acid chimera) targets proximally flanked by two restriction enzyme sites were challenged with the complementary PNA or PNAs having one or two mismatches, and the effect on the restriction enzyme cleavage of the flanking sites was assayed. The following PNAs were used: T10-LysNH2, T5CT4-LysNH2 and T2CT2CT4-LysNH2 and the corresponding targets cloned into pUC 19 were flanked by BamH1, Sal1 or Pstl sites, respectively. In all cases it was found that complete inhibition of restriction enzyme cleavage was obtained with the complementary PNA, a significantly reduced effect was seen with a PNA having one mismatch, and no effect was seen with a PNA having two mismatches. These results show that PNA can be used as sequence specific blockers of DNA recognizing proteins. Images PMID:8382793

  4. Controlling DNA fragmentation in MALDI-MS by chemical modification

    SciTech Connect

    Tang, W.; Zhu, L.; Smith, L.M. [Univ. of Wisconsin, Madison, WI (United States)] [Univ. of Wisconsin, Madison, WI (United States)

    1997-02-01

    Fragmentation has proven to be a major factor limiting accessible mass range, sensitivity, and mass resolution in the analysis of DNA by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Previous work has shown that this DNA fragmentation is strongly dependent on both the MALDI matrix and the nucleic acid sequence employed. Fragmentation is initiated by nucleobase protonation, leading to cleavage of the N-glycosidic bond with base loss, followed by cleavage of the phosphodiester backbone. In this study, asymmetric oligonucleotides incorporating cytidine and cytidine analogs such as 5-methyl-2{prime}-deoxycytidine, 5-bromo-2{prime}-deoxycytidine, aracytidine, and 2{prime}-fluorodeoxycytidine nucleosides were used to systematically investigate the influence of the structural changes on the stability of the N-glycosidic bond. Modifications of the deoxyribose sugar ring by replacing the 2{prime}-hydrogen with more electron-withdrawing groups such as the hydroxyl or fluoro group stabilize the N-glycosidic bond to a greater extent than the C5 nucleobase modifications. 2{prime}-Hydroxyl and 2{prime}-fluoro groups respectively are shown to partially or completely block fragmentation at the modified nucleosides. Mixtures of oligonucleotides incorporating such modifications demonstrate remarkably extended accessible mass range, as well as increased sensitivity and mass resolution. 39 refs., 11 figs.

  5. Distribution of the HindIII restriction fragment length polymorphism among patients with systemic lupus erythematosus with different concentrations of CR1

    Microsoft Academic Search

    H Satoh; E Yokota; K Tokiyama; T Kawaguchi; Y Niho

    1991-01-01

    Sixty six patients with systemic lupus erythematosus (SLE) were genotyped using a HindIII restriction fragment length polymorphism identified by CR1.1 cDNA, then were followed up for an average of 50 months to evaluate the stability of their CR1 activities. The gene frequencies for the two alleles which correlate with the numeric expression of CR1 on the erythrocytes were not significantly

  6. SOLVING LARGE DOUBLE DIGESTION PROBLEMS FOR DNA RESTRICTION MAPPING BY USING

    E-print Network

    SOLVING LARGE DOUBLE DIGESTION PROBLEMS FOR DNA RESTRICTION MAPPING BY USING BRANCH;Solving Large Double Digestion Problems for DNA Restriction Mapping by Using Branch-and-Bound Integer.S.A. Abstract. The double digestion problem for DNA restriction mapping has been proved to be NP

  7. Identification of Major Streptococcal Species by rrn-Amplified Ribosomal DNA Restriction Analysis

    PubMed Central

    Schlegel, Laurent; Grimont, Francine; Grimont, Patrick A. D.; Bouvet, Anne

    2003-01-01

    Amplified ribosomal DNA restriction analysis (rrn-ARDRA) is based on PCR amplification and restriction of a fragment of rRNA genes including 16S and 23S genes and the intergenic spacer. rrn-ARDRA was evaluated for the identification of species within the genus Streptococcus. A total of 148 type and reference strains of pyogenic, oral, and group D streptococci were examined in order to construct a database for identification of streptococci. The amplified product was a single band approximately 4,500 bp long. This amplicon was digested separately with three (HhaI, MboII, and Sau3A) restriction endonucleases. Respectively, 27, 26, and 28 major patterns were observed after HhaI, MboII, and Sau3A restrictions. Streptococcal strains belonging to different species had different patterns or different combination of patterns. An identification system based upon a combination of the three restriction patterns in a single database was then proposed. rrn-ARDRA was successfully applied to 11 clinical isolates whose identification to the species level was difficult to obtain by phenotypic analysis. Using a database of well-characterized strains, rrn-ARDRA is a powerful method for the identification of streptococcal isolates. PMID:12574263

  8. Structure and operation of the DNA-translocating type I DNA restriction enzymes

    PubMed Central

    Kennaway, Christopher K.; Taylor, James E.; Song, Chun Feng; Potrzebowski, Wojciech; Nicholson, William; White, John H.; Swiderska, Anna; Obarska-Kosinska, Agnieszka; Callow, Philip; Cooper, Laurie P.; Roberts, Gareth A.; Artero, Jean-Baptiste; Bujnicki, Janusz M.; Trinick, John; Kneale, G. Geoff; Dryden, David T.F.

    2012-01-01

    Type I DNA restriction/modification (RM) enzymes are molecular machines found in the majority of bacterial species. Their early discovery paved the way for the development of genetic engineering. They control (restrict) the influx of foreign DNA via horizontal gene transfer into the bacterium while maintaining sequence-specific methylation (modification) of host DNA. The endonuclease reaction of these enzymes on unmethylated DNA is preceded by bidirectional translocation of thousands of base pairs of DNA toward the enzyme. We present the structures of two type I RM enzymes, EcoKI and EcoR124I, derived using electron microscopy (EM), small-angle scattering (neutron and X-ray), and detailed molecular modeling. DNA binding triggers a large contraction of the open form of the enzyme to a compact form. The path followed by DNA through the complexes is revealed by using a DNA mimic anti-restriction protein. The structures reveal an evolutionary link between type I RM enzymes and type II RM enzymes. PMID:22215814

  9. Characterization of human glucocorticoid receptor complexes formed with DNA fragments containing or lacking glucocorticoid response elements

    SciTech Connect

    Tully, D.B.; Cidlowski, J.A. (Univ. of North Carolina, Chapel Hill (USA))

    1989-03-07

    Sucrose density gradient shift assays were used to study the interactions of human glucocorticoid receptors (GR) with small DNA fragments either containing or lacking glucocorticoid response element (GRE) DNA consensus sequences. When crude cytoplasmic extracts containing ({sup 3}H)triamcinolone acetonide (({sup 3}H)TA) labeled GR were incubated with unlabeled DNA under conditions of DNA excess, a GRE-containing DNA fragment obtained from the 5' long terminal repeat of mouse mammary tumor virus (MMTV LTR) formed a stable 12-16S complex with activated, but not nonactivated, ({sup 3}H)TA receptor. By contrast, if the cytosols were treated with calf thymus DNA-cellulose to deplete non-GR-DNA-binding proteins prior to heat activation, a smaller 7-10S complex was formed with the MMTV LTR DNA fragment. Activated ({sup 3}H)TA receptor from DNA-cellulose pretreated cytosols also interacted with two similarly sized fragments from pBR322 DNA. Stability of the complexes formed between GR and these three DNA fragments was strongly affected by even moderate alterations in either the salt concentration or the pH of the gradient buffer. Under all conditions tested, the complex formed with the MMTV LTR DNA fragment was more stable than the complexes formed with either of the pBR322 DNA fragments. Together these observations indicate that the formation of stable complexes between activated GR and isolated DNA fragments requires the presence of GRE consensus sequences in the DNA.

  10. Restriction fragment length polymorphism typing of infectious bursal disease virus field strains in Turkey.

    PubMed

    Sareyyüpo?lu, B; Akan, M

    2006-12-01

    Infectious bursal disease (IBD), also known as Gumboro disease, is a highly contagious, immunosuppressive disease of immature chickens. It is caused by IBD virus (IBDV) and is responsible for major economic losses in the poultry industry worldwide. In this study, 280 bursa samples from 56 commercially reared chicken flocks in Turkey with clinical symptoms of IBD were examined for IBDVs using the reverse transcription (RT)-polymerase chain reaction (PCR)/restriction fragment length polymorphism (RFLP) assay. The assay was conducted on a 743-bp fragment of the VP2 gene with the restriction enzymes BstNI, MboI, and SspI. The results indicate the existence of field isolates with new molecular patterns different from those previously published that may well be unique and specific to geographical regions. PMID:17274292

  11. Detection of disease-specific restriction fragment length polymorphisms in pemphigus vulgaris linked to the DQwl and DQw3 alleles of the HLA-D region

    SciTech Connect

    Szafer, F.; Brautbar, C.; Tzfoni, E.; Frankel, G.; Sherman, L.; Cohen, I.; Hacham-Zadeh, S.; Aberer, W.; Tappeiner, G.; Holubar, K.; Steinman, L.

    1987-09-01

    Pemphigus vulgaris in Israeli Ashkenazi and non-Ashkenazi Jews and in Austrian non-Jewish patients is strongly associated with the DR4 and DRw6 alleles of the HLA-D region class II genes. Restriction fragment length polymorphism analysis was undertaken with DQ..beta.., DQ..cap alpha.., and DR..beta.. cDNA probes. Hybridization with the DQ..beta.. probe identifies Pvu II, BamHI, and EcoRV fragments that absolutely discriminate pemphigus vulgaris patients from healthy DR-, DQ-, and ethnic-matched controls. In contrast the DQ..cap alpha.. and DR..beta.. probes failed to identify disease-specific restriction fragment length polymorphism fragments. These studies indicate that DQw1 and DQw3 polymorphisms carried by pemphigus vulgaris patients may be directly involved in predisposition to the disease or may be tightly linked to the susceptibility gene itself. To our knowledge, this is the first example of an HLA restriction fragment length polymorphism that is highly associated with susceptibility to autoimmune disease.

  12. Mapping of the Hox-3.1 and Myc-1.2 genes on chromosome 15 of the mouse by restriction fragment length variations

    Microsoft Academic Search

    Tomomasa Watanabe; Kyoko Ohno; Atsuko Shimizu; Yuka Sakai; Masahide Takahashi; Naoki Takahashi

    1990-01-01

    Restriction endonuclease fragment length variations (RFLV) were detected by use of the cDNA probeHox-3.1 for the homeo box-3.1 gene and also thec-myc oncogene probe for exon 2. RFLV ofHox-3.1 were found inHindIII restriction patterns, and RFLV of theMyc-1.2 gene inEcoRV patterns. From the RFLV, theHox-3.1 andMyc-1.2 genes were mapped on chromosome 15. Three-point cross test data showed that the frequency

  13. Analysis of Korean strains of infectious laryngotracheitis virus by nucleotide sequences and restriction fragment length polymorphism

    Microsoft Academic Search

    Myung Guk Han; Sun Joong Kim

    2001-01-01

    Korean field strains of infectious laryngotracheitis virus (ILTV) were analyzed by comparison of nucleotide sequences of thymidine kinase (TK) and glycoprotein G (gG) genes and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) patterns. Main differences among TK gene sequence were found in both amino acid at 252 and mRNA polyadenylation signals. In virulent strains, amino acid 252 of TK gene

  14. Restriction-fragment-length polymorphism and variation in electrophoretic karyotype in Naegleria fowleri from Japan

    Microsoft Academic Search

    Johan F. De Jonckheere; Kenji Yagita; Takuro Endo

    1992-01-01

    Strains ofNaegleria fowleri isolated in Japan from two different places were found to differ in electro-phoretic karyotype and restriction-fragment-length polymorphism (RFLP) both from each other and from strains isolated on other continents. Using the Wagmer parsimony method on the RFLP, we found that the Japanese isolates are most closely related to the Australian isolates and most distinct from the European

  15. Appearance in Europe of Naegleria fowleri displaying the Australian type of restriction-fragment-length polymorphism

    Microsoft Academic Search

    Pierre Pernin; Johan F. De Jonckheere

    1992-01-01

    We report for the first time the isolation in Europe ofNaegleria fowleri showing a type of restriction-fragment-length polymorphism (RFLP) usually found in Australia. The presence of this type as well as the European type fluctuated with time in the cooling waters of the nuclear power station investigated. Two possible explanations for the appearance of the AustralianN. fowleri type in Europe

  16. Detection of Methanotroph Diversity on Roots of Submerged Rice Plants by Molecular Retrieval of pmoA, mmoX, mxaF, and 16S rRNA and Ribosomal DNA, Including pmoA-Based Terminal Restriction Fragment Length Polymorphism Profiling

    Microsoft Academic Search

    HANS-PETER HORZ; MERLIN TCHAWA YIMGA; WERNER LIESACK

    2001-01-01

    The diversity of methanotrophic bacteria associated with roots of submerged rice plants was assessed using cultivation-independent techniques. The research focused mainly on the retrieval of pmoA, which encodes the a subunit of the particulate methane monooxygenase. A novel methanotroph-specific community-profiling method was established using the terminal restriction fragment length polymorphism (T-RFLP) technique. The T-RFLP profiles clearly revealed a more complex

  17. Cavitation Enhancing Nanodroplets Mediate Efficient DNA Fragmentation in a Bench Top Ultrasonic Water Bath

    PubMed Central

    Malc, Ewa P.; Jayakody, Chatura N.; Tsuruta, James K.; Mieczkowski, Piotr A.; Janzen, William P.; Dayton, Paul A.

    2015-01-01

    A perfluorocarbon nanodroplet formulation is shown to be an effective cavitation enhancement agent, enabling rapid and consistent fragmentation of genomic DNA in a standard ultrasonic water bath. This nanodroplet-enhanced method produces genomic DNA libraries and next-generation sequencing results indistinguishable from DNA samples fragmented in dedicated commercial acoustic sonication equipment, and with higher throughput. This technique thus enables widespread access to fast bench-top genomic DNA fragmentation. PMID:26186461

  18. Characterization of Erwinia chrysanthemi by pectinolytic isozyme polymorphism and restriction fragment length polymorphism analysis of PCR-amplified fragments of pel genes.

    PubMed Central

    Nassar, A; Darrasse, A; Lemattre, M; Kotoujansky, A; Dervin, C; Vedel, R; Bertheau, Y

    1996-01-01

    Conserved regions about 420 bp long of the pelADE cluster specific to Erwinia chrysanthemi were amplified by PCR and used to differentiate 78 strains of E. chrysanthemi that were obtained from different hosts and geographical areas. No PCR products were obtained from DNA samples extracted from other pectinolytic and nonpectinolytic species and genera. The pel fragments amplified from the E. chrysanthemi strains studied were compared by performing a restriction fragment length polymorphism (RFLP) analysis. On the basis of similarity coefficients derived from the RFLP analysis, the strains were separated into 16 PCR RFLP patterns grouped in six clusters, These clusters appeared to be correlated with other infraspecific levels of E. chrysanthemi classification, such as pathovar and biovar, and occasionally with geographical origin. Moreover, the clusters correlated well with the polymorphism of pectate lyase and pectin methylesterase isoenzymes. While the pectin methylesterase profiles correlated with host monocot-dicot classification, the pectate lyase polymorphism might reflect the cell wall microdomains of the plants belonging to these classes. PMID:8779560

  19. Epidemic of infectious laryngotracheitis in Italy: characterization of virus isolates by PCR-restriction fragment length polymorphism and sequence analysis.

    PubMed

    Moreno, Ana; Piccirillo, Alessandra; Mondin, Alessandra; Morandini, Emilio; Gavazzi, Luigi; Cordioli, Paolo

    2010-12-01

    Between May 2007 and October 2008, 34 outbreaks of mild to moderate forms of infectious laryngotracheitis (ILT) occurred in commercial broiler flocks in Italy. Affected birds showed watery eyes, conjunctivitis, nasal discharge, reduction of feed and water consumption, and gasping with expectoration of blood-stained mucus. The mortality rate was < 10%. Gross lesions consisted of conjunctivitis, excess of mucus, blood, or presence of diphtheritic membranes in trachea. A real-time PCR assay was performed to confirm the presence of ILT virus (ILTV) DNA in tracheal tissue homogenates. Twenty-three ILTV isolates were propagated on the chorion-allantoic membrane of embryonated chicken eggs showing typical plaques. PCR combined with restriction fragment length polymorphism and gene sequencing of isolates showed a high genetic correlation between field strains and chicken embryo origin vaccines. PMID:21313836

  20. Complementary addressed modification and cleavage of a single stranded DNA fragment with alkylating oligonucleotide derivatives.

    PubMed Central

    Vlassov, V V; Zarytova, V F; Kutiavin, I V; Mamaev, S V; Podyminogin, M A

    1986-01-01

    A single stranded DNA fragment was modified with alkylating derivatives of oligonucleotides complementary to a certain nucleotide sequences in the fragment. The derivatives carried aromatic 2-chloroethylamino groups at their 3'- or 5'-terminal nucleotide residues. Some of the derivatives carried both alkylating group and intercalating phenazine group which stabilized complementary complexes. It was found that these oligonucleotide derivatives modify the DNA fragment in a specific way near the target complementary nucleotide sequences, and the DNA fragment can be cleaved at the alkylated nucleotides positions. Alkylating derivatives carrying phenazine groups were found to be the most efficient in reaction with the DNA fragment. Images PMID:3714471

  1. Molecular typing of Pseudomonas pseudomallei: restriction fragment length polymorphisms of rRNA genes.

    PubMed Central

    Lew, A E; Desmarchelier, P M

    1993-01-01

    The aim of this study was to develop a typing scheme for Pseudomonas pseudomallei by comparison of patterns of restriction fragment length polymorphisms in rRNA genes (ribotyping). BamHI restriction digests of 100 isolates from various animal (34), human (58), and environmental (6) sources, including six reference strains, were hybridized to Escherichia coli 16S and 23S rRNAs. A chemiluminescent labelling and detection system was used to visualize bands. On the basis of patterns, the strains were classified into 22 different groups, with the largest containing 29 isolates. While most of the ribotypes were not exclusive to a particular source, some ribotypes were restricted to a particular geographic area or to either a human or a particular animal species. Application of the typing scheme to isolates of four independent outbreaks among animals showed that certain ribotypes predominated. The study demonstrated ribotyping to be a useful tool in epidemiological investigations of melioidosis. Images PMID:7681436

  2. Does varicocelectomy affect DNA fragmentation in infertile patients?

    PubMed Central

    Telli, Onur; Sarici, Hasmet; Kabar, Mucahit; Ozgur, Berat Cem; Resorlu, Berkan; Bozkurt, Selen

    2015-01-01

    Introduction: The aims of this study were to investigate the effect of varicocelectomy on DNA fragmentation index and semen parameters in infertile patients before and after surgical repair of varicocele. Materials and Methods: In this prospective study, 72 men with at least 1-year history of infertility, varicocele and oligospermia were examined. Varicocele sperm samples were classified as normal or pathological according to the 2010 World Health Organization guidelines. The acridine orange test was used to assess the DNA fragmentation index (DFI) preoperatively and postoperatively. Results: DFI decreased significantly after varicocelectomy from 34.5% to 28.2% (P = 0.024). In addition all sperm parameters such as mean sperm count, sperm concentration, progressive motility and sperm morphology significantly increased from 19.5 × 106 to 30.7 × 106, 5.4 × 106/ml to 14.3 × 106/ml, and 19.9% to 31.2% (P < 0.001) and 2.6% to 3.1% (P = 0.017). The study was limited by the loss to follow-up of some patients and unrecorded pregnancy outcome due to short follow-up. Conclusion: Varicocele causes DNA-damage in spermatozoa. We suggest that varicocelectomy improves sperm parameters and decreases DFI. PMID:25878412

  3. Cloning and expression in Escherichia coli of a DNA fragment from Bacillus sphaericus coding for biocidal activity against mosquito larvae

    Microsoft Academic Search

    Sathyam Ganesan; Haresch Kamdar; Kunthala Jayaraman; Jekisiel Szulmajster

    1983-01-01

    A 3.7 kb DNA fragment from Bacillus sphaericus 1593 was cloned and expressed in E. coli using the plasmid pHV33. The level of larvicidal activity of the hybrid plasmid, pGsp03, against two species of mosquito larvae was comparable to that found with sporulating cell suspensions of B. sphaericus. A limited restriction cleavage map of the cloned insert is given.

  4. Ca2 antagonists inhibit DNA fragmentation and toxic cell death induced by acetaminophen

    Microsoft Academic Search

    SIDHARTHA D. RAY; LISA M KAMENDULIS; MARK W. GURULE; ROBERT D. YORKIN; GEORGE B. CORCORAN

    1993-01-01

    Ca2 accumulates in the nucleus and DNA undergoes enzymatic cleavage into internucleosome- length fragments before acetaminophen and dimethyl- nitrosamine produce hepatic necrosis in vivo and toxic cell death in vitro. However, Ca2-endonudease fragmen- tation of DNA is characteristic of apoptosis, a type of cell death considered biochemically and functionally distinct from toxic cell death. The present studies investigate DNA fragmentation

  5. DNA fragment assembly: an application of graph theory in molecular biology

    E-print Network

    Willems, Wolfgang

    DNA fragment assembly: an application of graph theory in molecular biology Martin Mascher Leibniz Technology Since the central importance of the DNA in storing biological informa- tion had been recognised limitations permit scientists only to obtain contigu- ous DNA fragments whose lengths range from a few dozen

  6. Genetic alterations of hepatocellular carcinoma by random amplified polymorphic DNA analysis and cloning sequencing of tumor differential DNA fragment

    Microsoft Academic Search

    Zhi-Hong Xian; Wen-Ming Cong; Shu-Hui Zhang; Meng-Chao Wu

    2005-01-01

    Abstract Abstract Abstract Abstract Abstract AIM:T o study the genetic alterations and their association with clinicopathological characteristics of hepatocellular carcinoma (HCC), and to find the tumor related DNA fragments. METHODS:DNA isolated from tumors and corresponding

  7. DNA digestion protocol & hints Overview: Although it is pretty standard to digest DNA with restriction enzymes, here

    E-print Network

    Doering, Tamara

    Liu 4/2004 DNA digestion protocol & hints Overview: Although it is pretty standard to digest DNA in molecular biology (3.1.1-3.1.2) Materials: · DNA sample in water or TE buffer · 10x digestion buffer · restriction enzyme · DNA loading buffer · Agarose gel 0.8% (or different depending on expected band sizes

  8. Chloroplast DNA restriction site mapping and the phylogeny of Ranunculus ( Ranunculaceae )

    Microsoft Academic Search

    Jan Thomas Johansson

    1998-01-01

    A chloroplast DNA restriction site map forRanunculus sceleratus (Ranunculaceae) was constructed using 14 restriction endonucleases. The total size of the chloroplast genome is 152.4kb. No inversions were detected relative to the tobacco chloroplast DNA. Cladistic analyses of chloroplast DNA restriction site polymorphism were employed in order to elucidate the phylogeny among 76 species of the genusRanunculus in a wide sense

  9. Phylogenetics of freshwater black basses (Centrarchidae: Micropterus) inferred from restriction endonuclease analysis of mitochondrial DNA.

    PubMed

    Johnson, R L; Magee, J B; Hodge, T A

    2001-12-01

    Geographic isolation and habitat specialization has aided in the evolution and genetic integrity of the micropterid bass species of North America. Members of the genus Micropterus form a close natural unit with little morphologic and meristic variation. Our goals were to measure the genetic characteristics of and distances between six black bass species by using mitochondrial DNA analysis. Mitochondrial DNA restriction fragment length polymorphisms were examined in Guadalupe bass (M. treculi), largemouth bass (M. salmoides), shoal bass (M. cataractae), smallmouth bass (M. dolomieu), spotted bass (M. punctulatus), and Suwannee bass (M. notius), using 15 restriction endonucleases. The bluegill (Lepomis macrochirus) was used as an outgroup. The phylogeny inferred from Dollo parsimony cladistic analysis concurred with published results from allozyme analyses, yet it was inconsistent with published meristic analyses. Genetic distances between species ranged from 0.0659 to 0.2145, with the largemouth and Suwannee basses showing the greatest divergence from the other black basses. The Guadalupe, smallmouth, and spotted basses were most diverged from the bluegill. The black basses diverged over a broad time frame, with estimated black bass speciation occurring during late Miocene-early Pliocene (3.30-10.73 MYA). PMID:11860202

  10. Limits of a rapid identification of common Mediterranean sandflies using polymerase chain reaction-restriction fragment length polymorphism

    PubMed Central

    Bounamous, Azzedine; Lehrter, Véronique; Hadj-Henni, Leila; Delecolle, Jean-Claude; Depaquit, Jérôme

    2014-01-01

    A total of 131 phlebotomine Algerian sandflies have been processed in the present study. They belong to the species Phlebotomus bergeroti, Phlebotomus alexandri, Phlebotomus sergenti, Phlebotomus chabaudi, Phlebotomus riouxi, Phlebotomus perniciosus, Phlebotomus longicuspis, Phlebotomus perfiliewi, Phlebotomus ariasi, Phlebotomus chadlii, Sergentomyia fallax, Sergentomyia minuta, Sergentomyia antennata, Sergentomyia schwetzi, Sergentomyia clydei, Sergentomyia christophersi and Grassomyia dreyfussi. They have been characterised by sequencing of a part of the cytochrome b (cyt b), t RNA serine and NADH1 on the one hand and of the cytochrome C oxidase I of the mitochondrial DNA (mtDNA) on the other hand. Our study highlights two sympatric populations within P. sergenti in the area of its type-locality and new haplotypes of P. perniciosus and P. longicuspis without recording the specimens called lcx previously found in North Africa. We tried to use a polymerase chain reaction-restriction fragment length polymorphism method based on a combined double digestion of each marker. These method is not interesting to identify sandflies all over the Mediterranean Basin. PMID:24936911

  11. Multiple restriction fragment length polymorphisms at the insulin receptor locus: a highly informative marker for linkage analysis.

    PubMed Central

    Elbein, S C; Corsetti, L; Ullrich, A; Permutt, M A

    1986-01-01

    Although resistance to insulin action is a well-studied phenomenon in non-insulin-dependent diabetes and certain genetic syndromes, the role of inherited defects of the insulin receptor in these disorders is unknown. To facilitate the evaluation of that role, restriction fragment length polymorphisms (RFLPs) were identified using various portions of the insulin receptor cDNA to examine digested DNA from American Blacks, Pima Indians, and Caucasians. Five RFLPs were identified in Caucasians. Two of these were detected with a single 1.3-kilobase probe in Rsa I digests with minor allele frequencies of 0.48 and 0.23. An additional RFLP was noted with Bgl II and two more RFLPs with Sac I using a different 1.6-kilobase probe, with minor allele frequencies of 0.17 for Bgl II and 0.12 for both Sac I RFLPs. All RFLPs except for the second Sac I RFLP were present in American Blacks, while only the Rsa I RFLPs were present in Pima Indians. Pairwise analysis showed random association between all sites except for the Bgl II and second Rsa I sites, where the disequilibrium statistic, delta, was -0.70 (different from 0 at P less than 0.001). No association of any RFLP was noted with non-insulin-dependent diabetes in a small population. These studies show that this is a highly informative locus that should be important for mapping of chromosome 19p and for linkage studies. Images PMID:3014538

  12. TeloTool: a new tool for telomere length measurement from terminal restriction fragment analysis with improved probe intensity correction

    PubMed Central

    Göhring, Janett; Fulcher, Nick; Jacak, Jaroslaw; Riha, Karel

    2014-01-01

    Telomeres comprise the protective caps of natural chromosome ends and function in the suppression of DNA damage signaling and cellular senescence. Therefore, techniques used to determine telomere length are important in a number of studies, ranging from those investigating telomeric structure to effects on human disease. Terminal restriction fragment (TRF) analysis has for a long time shown to be one of the most accurate methods for quantification of absolute telomere length and range from a number of species. As this technique centers on standard Southern blotting, telomeric DNA is observed on resulting autoradiograms as a heterogeneous smear. Methods to accurately determine telomere length from telomeric smears have proven problematic, and no reliable technique has been suggested to obtain mean telomere length values. Here, we present TeloTool, a new program allowing thorough statistical analysis of TRF data. Using this new method, a number of methodical biases are removed from previously stated techniques, including assumptions based on probe intensity corrections. This program provides a standardized mean for quick and reliable extraction of quantitative data from TRF autoradiograms; its wide application will allow accurate comparison between datasets generated in different laboratories. PMID:24366880

  13. Limits of a rapid identification of common Mediterranean sandflies using polymerase chain reaction-restriction fragment length polymorphism.

    PubMed

    Bounamous, Azzedine; Lehrter, Véronique; Hadj-Henni, Leila; Delecolle, Jean-Claude; Depaquit, Jérôme

    2014-07-01

    A total of 131 phlebotomine Algerian sandflies have been processed in the present study. They belong to the species Phlebotomus bergeroti, Phlebotomus alexandri, Phlebotomus sergenti, Phlebotomus chabaudi, Phlebotomus riouxi, Phlebotomus perniciosus, Phlebotomus longicuspis, Phlebotomus perfiliewi, Phlebotomus ariasi, Phlebotomus chadlii, Sergentomyia fallax, Sergentomyia minuta, Sergentomyia antennata, Sergentomyia schwetzi, Sergentomyia clydei, Sergentomyia christophersi and Grassomyia dreyfussi. They have been characterised by sequencing of a part of the cytochrome b (cyt b), t RNA serine and NADH1 on the one hand and of the cytochrome C oxidase I of the mitochondrial DNA (mtDNA) on the other hand. Our study highlights two sympatric populations within P. sergenti in the area of its type-locality and new haplotypes of P. perniciosus and P. longicuspis without recording the specimens called lcx previously found in North Africa. We tried to use a polymerase chain reaction-restriction fragment length polymorphism method based on a combined double digestion of each marker. These method is not interesting to identify sandflies all over the Mediterranean Basin. PMID:24936911

  14. A Time-Efficient and User-Friendly Method for Plasmid DNA Restriction Analysis.

    ERIC Educational Resources Information Center

    LaBanca, Frank; Berg, Claire M.

    1998-01-01

    Describes an experiment in which plasmid DNA is digested with restriction enzymes that cleave the plasmid either once or twice. The DNA is stained, loaded on a gel, electrophoresed, and viewed under normal laboratory conditions during electrophoresis. (DDR)

  15. Electric linear dichroism transients of bent DNA fragments.

    PubMed

    Umazano, Juan P; Bertolotto, Jorge A

    2013-03-01

    We study the effect of translational-rotational hydrodynamic coupling on the transient electric linear dichroism of DNA fragments in aqueous solution. As opposed to previous theoretical works, where analytic solutions valid in the limit of low electric field were reported, we present here a numerical approach which allows to obtain numerical results valid independently from the applied electric field strength. Numerical procedures here used are an extension to the transient-state of those developed in a previous work for the study of the problem in the steady-state. The molecular orientational processes induced by an electric field is characterized with statistical arguments solving the Fokker-Planck equation by means of the finite difference method to know the orientational distribution function of molecules. PMID:23485329

  16. Inhibition of Apoptosis-Associated DNA Fragmentation Activity in Nonapoptotic Cells: The Role of DNA Fragmentation Factor45 (DFF45\\/ICAD)

    Microsoft Academic Search

    Steven L. Sabol; Renee Li; Tamara Y. Lee; Rolla Abdul-Khalek

    1998-01-01

    We have investigated the mechanism whereby nuclear DNA fragmentation activity emerging during early apoptosis is inhibited during normal cell life. In a cell-free system, cytosol fractions from diverse nonapoptotic human cell lines (Jurkat T-cell leukemia, HeLa carcinoma, SK-N-MC neuroblastoma, and WI-38 embryonic lung fibroblast) potently neutralized the nuclear DNA fragmentation activity of cytosol from apoptotic anti-Fas treated Jurkat cells. Recombinant

  17. Genome of Hepatitis B Virus: Restriction Enzyme Cleavage and Structure of DNA Extracted from Dane Particles

    Microsoft Academic Search

    Jesse Summers; Anna O'Connell; Irving Millman

    1975-01-01

    DNA extracted from Dane particles has been characterized by gel electrophoresis and restriction enzyme cleavage with endonuclease R\\\\cdot HaeIII (from Hemophilus aegyptius). Dane particle DNA is proposed to be a double-stranded circular DNA approximately 3600 nucleotides in length containing a single-stranded gap of 600-2100 nucleotides. The endogenous DNA polymerase (DNA nucleotidyltransferase; deoxynucleosidetriphosphate:DNA deoxynucleotidyltransferase; EC 2.7.7.7) reaction appears to repair this

  18. Late, but not early, paternal effect on human embryo development is related to sperm DNA fragmentation

    Microsoft Academic Search

    Jan Tesarik; Ermanno Greco; Carmen Mendoza

    BACKGROUND: It is known that repeated failure of assisted reproduction treatment (ART) can be due to a paternal effect. This study was undertaken to analyse the possible relationship between ART failure and sperm DNA fragmentation. METHODS: Zygote morphology and the percentage of spermatozoa with fragmented DNA (assessed by TUNEL) were compared in two groups using donor oocytes for ICSI attempts.

  19. Photochemistry of DNA fragments via semiclassical nonadiabatic dynamics.

    PubMed

    Alexandrova, Anastassia N; Tully, John C; Granucci, Giovanni

    2010-09-23

    Forming upon absorption of a UV photon, excited states of DNA are subject to nonadiabatic evolution, via either internal conversion (IC) back to the ground state or mutagenesis. Nonadiabatic processes following the formation of the first singlet excited states, S1, in 10 different small DNA fragments--4 single 4'H-nucleosides, 2 Watson-Crick base pairs, and 4 nucleotide quartets--have been investigated. Simulations were done via the nonadiabatic direct trajectory surface hopping semiclassical dynamics. The electronic wave function was obtained with configuration interaction, based on the semiempirical AM1 and PM3 Hamiltonians with fractional orbital occupation numbers. The evolution of the electronic wave function was governed by the time-dependent Schrödinger equation with a locally diabatic representation, intrinsically stable near surface crossings. The nuclei evolved on adiabatic potential energy surfaces, as prescribed by classical Newtonian dynamics, with sudden hops between potential energy surfaces to account for nonadiabatic transitions. The "fewest switches" surface hopping algorithm coupled the quantum and classical parts of the system. The dynamics simulations revealed several routes of nonadiabatic relaxation in these systems, which were not reported previously, and also recovered known routes of IC. PMID:20795696

  20. Comparative study of Mycobacterium paratuberculosis strains isolated from Crohn's disease and Johne's disease using restriction fragment length polymorphism and arbitrarily primed polymerase chain reaction.

    PubMed Central

    François, B.; Krishnamoorthy, R.; Elion, J.

    1997-01-01

    To obtain insights into the pathogenic mechanisms involving Mycobacterium paratuberculosis in Crohn's disease (CD) we questioned if the strains of M. paratuberculosis isolated from CD are distinguishable from those involved in Johne's disease (JD), a chronic granulomatous enteritis in cattle. Accordingly we compared human and animal strains at the DNA level, both by the analysis of restriction fragment length polymorphism (RFLP) in and around the insertion sequence IS 900 and by the arbitrarily primed chain reaction (AP-PCR). Results are in favour of a common clonal origin for the 4 strains isolated from CD and for 8 of the 11 strains isolated from cattle and sheep JD. PMID:9207733

  1. Assignment of the {zeta}-crystalin gene (CRYZ) to human chromosome 1p22-p31 and identification of restriction fragment length polymorphisms

    SciTech Connect

    Heinzmann, C.; Kojis, T.L.; Bateman, J.B. [UCLA School of Medicine, Los Angeles, CA (United States)] [and others] [UCLA School of Medicine, Los Angeles, CA (United States); and others

    1994-09-15

    {zeta}-Crystallin is a lens protein that has been associated with autosomal dominant congenital cataracts in guinea pigs and thus is a candidate for human congenital cataracts. The authors have assigned the {zeta}-crystallin gene (CRYZ) to human chromosome 1 using a Southern panel of 17 human-mouse somatic cell hybrids and regionally localized it to 1p22-p31 by fluorescence in situ hybridation. Five restriction fragment length polymorphisms were identified by analyzing the DNA from 10 unrelated, unaffected individuals. The results will permit evaluation of its role in human cataractogenesis. 22 refs., 3 figs., 2 tabs.

  2. Nuclear DNA content variation in Helianthus annus and the detection of repetitive DNA sequences 

    E-print Network

    Michaelson, Martin Joseph

    1986-01-01

    restriction fragments of five H. annuus library clones and the EMBL 3 bacteriophage . . ~ 36 Interpretive drawing of Figure 2, giving sizes of restriction fragments Results of hybridization of biotinylated, whole Ha89 DHA to clone restriction fragments... transferred to a nitrocellulose filter 38 Interpretive drawinp identifying restriction fragments that hybridized the probe 39 Possible restriction maps of clone 7 based on fragment sizes from Figures 3 and 5 INTRODUCTION DNA content varies over 8 orders...

  3. cDNA selection: efficient PCR approach for the selection of cDNAs encoded in large chromosomal DNA fragments.

    PubMed Central

    Parimoo, S; Patanjali, S R; Shukla, H; Chaplin, D D; Weissman, S M

    1991-01-01

    Identification of coding segments in large fragments of genomic DNA is a recurrent problem in genome mapping and positional cloning studies. We have developed a rapid and efficient protocol to achieve this goal, based on hybridization of cDNA fragments to immobilized DNA and recovery of the selected cDNAs by the PCR. The procedure permits rapid cloning of cDNA fragments encoded by large genomic DNA fragments, groups of yeast artificial chromosomes, or cosmids and has the potential to directly enrich cDNAs encoded in chromosome segments. By this approach we have been able to identify several non-major histocompatibility complex class I clones from a yeast artificial chromosome that includes the HLA-A locus. Images PMID:1946377

  4. Natural Transformation of Helicobacter pylori Involves the Integration of Short DNA Fragments Interrupted by Gaps of Variable Size

    PubMed Central

    Lin, Edward A.; Zhang, Xue-Song; Levine, Steven M.; Gill, Steven R.; Falush, Daniel; Blaser, Martin J.

    2009-01-01

    Helicobacter pylori are gram-negative bacteria notable for their high level of genetic diversity and plasticity, features that may play a key role in the organism's ability to colonize the human stomach. Homeologous natural transformation, a key contributor to genomic diversification, has been well-described for H. pylori. To examine the mechanisms involved, we performed restriction analysis and sequencing of recombination products to characterize the length, fragmentation, and position of DNA imported via natural transformation. Our analysis revealed DNA imports of small size (1,300 bp, 95% confidence limits 950–1850 bp) with instances of substantial asymmetry in relation to selectable antibiotic-resistance markers. We also observed clustering of imported DNA endpoints, suggesting a possible role for restriction endonucleases in limiting recombination length. Additionally, we observed gaps in integrated DNA and found evidence suggesting that these gaps are the result of two or more separate strand invasions. Taken together, these observations support a system of highly efficient short-fragment recombination involving multiple recombination events within a single locus. PMID:19282979

  5. Natural transformation of helicobacter pylori involves the integration of short DNA fragments interrupted by gaps of variable size.

    PubMed

    Lin, Edward A; Zhang, Xue-Song; Levine, Steven M; Gill, Steven R; Falush, Daniel; Blaser, Martin J

    2009-03-01

    Helicobacter pylori are gram-negative bacteria notable for their high level of genetic diversity and plasticity, features that may play a key role in the organism's ability to colonize the human stomach. Homeologous natural transformation, a key contributor to genomic diversification, has been well-described for H. pylori. To examine the mechanisms involved, we performed restriction analysis and sequencing of recombination products to characterize the length, fragmentation, and position of DNA imported via natural transformation. Our analysis revealed DNA imports of small size (1,300 bp, 95% confidence limits 950-1850 bp) with instances of substantial asymmetry in relation to selectable antibiotic-resistance markers. We also observed clustering of imported DNA endpoints, suggesting a possible role for restriction endonucleases in limiting recombination length. Additionally, we observed gaps in integrated DNA and found evidence suggesting that these gaps are the result of two or more separate strand invasions. Taken together, these observations support a system of highly efficient short-fragment recombination involving multiple recombination events within a single locus. PMID:19282979

  6. Characterization of the 5-hydroxymethylcytosine-specific DNA restriction endonucleases

    PubMed Central

    Borgaro, Janine G.; Zhu, Zhenyu

    2013-01-01

    In T4 bacteriophage, 5-hydroxymethylcytosine (5hmC) is incorporated into DNA during replication. In response, bacteria may have developed modification-dependent type IV restriction enzymes to defend the cell from T4-like infection. PvuRts1I was the first identified restriction enzyme to exhibit specificity toward hmC over 5-methylcytosine (5mC) and cytosine. By using PvuRts1I as the original member, we identified and characterized a number of homologous proteins. Most enzymes exhibited similar cutting properties to PvuRts1I, creating a double-stranded cleavage on the 3? side of the modified cytosine. In addition, for efficient cutting, the enzymes require two cytosines 21–22-nt apart and on opposite strands where one cytosine must be modified. Interestingly, the specificity determination unveiled a new layer of complexity where the enzymes not only have specificity for 5-?-glucosylated hmC (5?ghmC) but also 5-?-glucosylated hmC (5?ghmC). In some cases, the enzymes are inhibited by 5?ghmC, whereas in others they are inhibited by 5?ghmC. These observations indicate that the position of the sugar ring relative to the base is a determining factor in the substrate specificity of the PvuRts1I homologues. Lastly, we envision that the unique properties of select PvuRts1I homologues will permit their use as an additive or alternative tool to map the hydroxymethylome. PMID:23482393

  7. DNA restriction patterns and DNA-DNA solution hybridization studies of Frankia isolates from Myrica pennsylvanica (bayberry).

    PubMed Central

    Bloom, R A; Mullin, B C; Tate, R L

    1989-01-01

    Sixteen Frankia strains were isolated from Myrica pennsylvanica (bayberry) root nodules collected at diverse sites in New Jersey. Restriction pattern analysis of total genomic DNA was used to group the isolates into gel groups, and the genetic relatedness among the isolates was evaluated by DNA-DNA solution hybridization studies. Restriction pattern analysis provided a distinctive reproducible fingerprint for each isolate. Isolates fell into nine separate groups (strain types). More than one strain type was isolated from most sites. Isolates from two different gel groups were found in 3 of 10 nodules examined. Of the 16 isolates, 10 contained extrachromosomal DNA. Six different extrachromosomal DNA banding patterns were found. Genomically similar isolates carried related, but different, banding patterns. DNA hybridization studies indicated that isolates from a single plant species can be minimally related as determined by total genome homology. Homology ranged from 12 to 99%. Highly divergent strains were isolated from the same plant and found to cohabit the same nodule. Thus, this study demonstrated that Frankia strains which infect the same host plant are not only phenotypically different but also genetically diverse. Images PMID:2802599

  8. Restriction Fragment Length Polymorphism (RFLP) for protein disulfide isomerase (PDI) gene sequences in Triticum and Aegilops species

    Microsoft Academic Search

    M. Ciaffi; L. Dominici; E. Umana; O. A. Tanzarella; E. Porceddu

    2000-01-01

    RFLP variation revealed by protein disulfide isomerase (PDI) coding gene sequences was assessed in 170 accessions belonging\\u000a to 23 species of Triticum and Aegilops. PDI restriction fragments were highly conserved within each species and confirmed that plant PDI is encoded either by single-copy\\u000a sequences or by small gene families. The wheat PDI probe hybridized to single EcoRI or HindIII fragments

  9. Forensic identification of ungulate species using restriction digests of PCR-amplified mitochondrial DNA.

    PubMed

    Murray, B W; McClymont, R A; Strobeck, C

    1995-11-01

    A survey of mitochondrial D-loop variation in 15 species of ungulates was conducted via amplification by the polymerase chain reaction followed by restriction fragment length polymorphism analysis. This survey included moose (Alces alces), caribou (Rangifer tarandus), mule deer (Odocoileus hemionus hemionus), black-tailed deer (O. h. columbianus), white-tailed deer (O. virginianus), waipiti (Cervus elaphus), pronghorn antelope (Antilocapra americana), bighorn sheep (Ovis canadensis), Stone's sheep (O. dalli), domestic sheep (O. aries), moulflon sheep (O. musimon), mountain goat (Oreamnos americanus), domestic goat (Capra hircus), domestic cattle (Bos taurus), and bison (Bison bison). The results of this preliminary survey indicate that there may be sufficient species specific variation in the D-loop region of the mitochondrial genome of the ungulate species examined here, with the exception of deer (Odocoileus) species, to establish the species origin of the mitochondrial haplotypes of this group. The Odocoileus species are known to hybridize and sharing of mtDNA haplotypes was observed. The chelex DNA extraction technique was successfully used on small blood stains. PMID:8522926

  10. GB Virus C/Hepatitis G Virus Groups and Subgroups: Classification by a Restriction Fragment Length Polymorphism Method Based on Phylogenetic Analysis of the 5? Untranslated Region

    PubMed Central

    Quarleri, J. F.; Mathet, V. L.; Feld, M.; Ferrario, D.; della Latta, M. P.; Verdun, R.; Sánchez, D. O.; Oubiña, J. R.

    1999-01-01

    A phylogenetic tree based on 150 5? untranslated region sequences deposited in GenBank database allowed segregation of the sequences into three major groups, including two subgroups, i.e., 1, 2a, 2b, and 3, supported by bootstrap analysis. Restriction site analysis of these sequences predicted that HinfI and either AatII or AciI could be used for genomic typing with 99.4% accuracy. cDNA sequencing and subsequent alignment of 21 Argentine GB virus C/hepatitis G virus strains confirmed restriction fragment length polymorphism patterns theoretically predicted. This method may be useful for a rapid screening of samples when either epidemiological or transmission studies of this agent are carried out. PMID:10203483

  11. Linear mtDNA fragments and unusual mtDNA rearrangements associated with pathological deficiency of MGME1 exonuclease.

    PubMed

    Nicholls, Thomas J; Zsurka, Gábor; Peeva, Viktoriya; Schöler, Susanne; Szczesny, Roman J; Cysewski, Dominik; Reyes, Aurelio; Kornblum, Cornelia; Sciacco, Monica; Moggio, Maurizio; Dziembowski, Andrzej; Kunz, Wolfram S; Minczuk, Michal

    2014-12-01

    MGME1, also known as Ddk1 or C20orf72, is a mitochondrial exonuclease found to be involved in the processing of mitochondrial DNA (mtDNA) during replication. Here, we present detailed insights on the role of MGME1 in mtDNA maintenance. Upon loss of MGME1, elongated 7S DNA species accumulate owing to incomplete processing of 5' ends. Moreover, an 11-kb linear mtDNA fragment spanning the entire major arc of the mitochondrial genome is generated. In contrast to control cells, where linear mtDNA molecules are detectable only after nuclease S1 treatment, the 11-kb fragment persists in MGME1-deficient cells. In parallel, we observed characteristic mtDNA duplications in the absence of MGME1. The fact that the breakpoints of these mtDNA rearrangements do not correspond to either classical deletions or the ends of the linear 11-kb fragment points to a role of MGME1 in processing mtDNA ends, possibly enabling their repair by homologous recombination. In agreement with its functional involvement in mtDNA maintenance, we show that MGME1 interacts with the mitochondrial replicase PolgA, suggesting that it is a constituent of the mitochondrial replisome, to which it provides an additional exonuclease activity. Thus, our results support the viewpoint that MGME1-mediated mtDNA processing is essential for faithful mitochondrial genome replication and might be required for intramolecular recombination of mtDNA. PMID:24986917

  12. Rapid Discrimination among Dermatophytes, Scytalidium spp., and Other Fungi with a PCR-Restriction Fragment Length Polymorphism Ribotyping Method

    PubMed Central

    Machouart-Dubach, Marie; Lacroix, Claire; de Chauvin, Martine Feuilhade; Le Gall, Isabelle; Giudicelli, Catherine; Lorenzo, Frédéric; Derouin, Francis

    2001-01-01

    Dermatomycoses are very common infections caused mainly by dermatophytes. Scytalidiosis is a differential mycological diagnosis, especially in tropical and subtropical areas. Since a culture-based diagnosis takes 2 to 3 weeks, we set up a PCR-restriction fragment length polymorphism (RFLP) method for rapid discrimination of these fungi in clinical samples. The hypervariable V4 domain of the small ribosomal subunit 18S gene was chosen as the target for PCR. The corresponding sequences from 19 fungal species (9 dermatophytes, 2 Scytalidium species, 6 other filamentous fungi, and 2 yeasts) were obtained from databases or were determined in the laboratory. Sequences were aligned to design primers for dermatophyte-specific PCR and to identify digestion sites for RFLP analysis. The reliability of PCR-RFLP for the diagnosis of dermatomycosis was assessed on fungal cultures and on specimens from patients with suspected dermatomycosis. Two sets of primers preferentially amplified fungal DNA from dermatophytes (DH1L and DH1R) or from Scytalidium spp. (DH2L and DH1R) relative to DNA from bacteria, yeasts, some other filamentous fungi, and humans. Digestion of PCR products with EaeI or BamHI discriminated between dermatophytes and Scytalidium species, as shown with cultures of 31 different fungal species. When clinical samples were tested by PCR-RFLP, blindly to mycological findings, the results of the two methods agreed for 74 of 75 samples. Dermatophytes and Scytalidium spp. can thus be readily discriminated by PCR-RFLP within 24 h. This method can be applied to clinical samples and is suited to rapid etiologic diagnosis and treatment selection for patients with dermatomycosis. PMID:11158128

  13. Performance assessment of DNA fragment sizing by high-sensitivity flow cytometry and pulsed-field gel electrophoresis.

    PubMed

    Ferris, Matthew M; Yan, Xiaomei; Habbersett, Robbert C; Shou, Yulin; Lemanski, Cheryl L; Jett, James H; Yoshida, Thomas M; Marrone, Babetta L

    2004-05-01

    The sizing of restriction fragments is the chief analytical technique utilized in the production of DNA fingerprints. Few techniques have been able to compete with pulsed-field gel electrophoresis (PFGE), which is capable of discriminating among bacteria at species and strain levels by resolving restriction fragments. However, an ultrasensitive flow cytometer (FCM) developed in our lab has also demonstrated the ability to discriminate bacteria at species and strain levels. The abilities of FCM warrant a quantitative parallel comparison with PFGE to assess and evaluate the accuracy and precision of DNA fragment sizing by both techniques. Replicate samples of Staphylococcus aureus Mu50 were analyzed along with two clinical S. aureus isolates. The absolute fragment sizing accuracy was determined for PFGE (5% +/- 2%) and FCM (4% +/- 4%), with sequence-predicted Mu50 SmaI fragment sizes used as a reference. Precision was determined by simple arithmetic methods (relative standard deviation for PFGE [RSD(PFGE) ] = 3% +/- 2% and RSD(FCM) = 1.2% +/- 0.8%) as well as by the use of dendrograms derived from Dice coefficient-unweighted pair group method with arithmetic averages (UPGMA) and Pearson-UPGMA analyses. All quantitative measures of PFGE and FCM precision were equivalent, within error. The precision of both methods was not limited by any single sample preparation or analysis step that was tracked in this study. Additionally, we determined that the curve-based clustering of fingerprint data provided a more informative and useful assessment than did traditional band-based methods. PMID:15131156

  14. Use of restriction fragment length polymorphism to identify Candida species, related to onychomycosis

    PubMed Central

    Mohammadi, Rasoul; Badiee, Parisa; Badali, Hamid; Abastabar, Mahdi; Safa, Ahmad Hosseini; Hadipour, Mahboubeh; Yazdani, Hajar; Heshmat, Farnaz

    2015-01-01

    Background: Onychomycosis is one of the most common clinical forms of fungal infections due to both filamentous fungi and yeasts. The genus of Candida is one of the most prominent causes of onychomycosis in all around the world. Although Candida albicans is still the most frequent cause of nail infections, use of broad-spectrum antifungal agents has led to a shift in the etiology of C. albicans to non-albicans species. The aim of the present study is rapid and precise identification of candida species isolated from nail infection by using of PCR-RFLP technique. Materials and Methods: A total of 360 clinical yeast strains were collected from nail infections in Iran. Genomic DNA was extracted using FTA? cards. ITS1-5.8SrDNA-ITS2 region was amplified using universal primers and subsequently products were digested with the restriction enzyme MspI. For identification of newly described species (C. parapsilosis complex), the SADH gene was amplified, followed by digestion with Nla III restriction enzyme. Results: Candida albicans was the most commonly isolated species (41.1%), followed by C. parapsilosis (21.4%), C. tropicalis (12.8%), C. kefyr (9.4%), C. krusei (5.5%), C. orthopsilosis (4.1%), C. glabrata (2.8%), C. guilliermondii (1.4%), C. rugosa (0.8%), and C. lusitaniae (0.5%). Patients in the age groups of 51-60 and 81-90 years had the highest and lowest distribution of positive specimens, respectively. Conclusion: Rapid and precise identification of Candida species from clinical specimens lead to appropriate therapeutic plans. PMID:26015921

  15. Isolation and restriction endonuclease cleavage of Anaplasma marginale DNA in situ in agarose.

    PubMed Central

    Krueger, C M; Buening, G M

    1988-01-01

    Bacterial restriction endonucleases were used to produce DNA cleavage patterns that could be useful as tools to study the relatedness among Anaplasma marginale isolates. Bovine erythrocytes infected with A. marginale were lysed, washed, and embedded in agarose. The embedded erythrocytes and bacterial pathogens were partially digested by sequential infiltration of the agarose with acetone, lysozyme, sodium dodecyl sulfate, and proteinase K. The unfragmented genomic DNA was left supported and protected in a porous matrix. The DNA was digested in situ in agarose under the following conditions: (i) brief treatment with phenol, (ii) brief washing with distilled water, and (iii) adjustment of restriction enzyme digestion mixture to compensate for the volume of the agarose. The cleaved DNA was electrophoresed horizontally to produce a DNA cleavage pattern. Of 19 restriction enzymes screened, 12 produced distinct DNA bands from the genomes of each of the five A. marginale isolates examined. The DNA cleavage pattern produced from each isolate with a given restriction enzyme was reproducible. However, the DNA cleavage patterns produced from different isolates with a given restriction enzyme were not necessarily identical. This procedure could be modified for general bacterial DNA isolation, in situ agarose digestion, and manipulations. Images PMID:2838504

  16. Restriction fragment length polymorphism of polymerase chain reaction products applied to the differentiation of poultry campylobacters for epidemiological investigations

    Microsoft Academic Search

    R. D Ayling; M. J Woodward; S Evans; D. G Newell

    1996-01-01

    A technique for subtyping Campylobacter jejuni isolates has been developed by using the restriction fragment length polymorphism (rflp) of polymerase chain reaction (PCR) products of the flaA and flaB genes. The technique was validated by using strains representing 28 serotypes of C jejuni and it may also be applied to C coli. From these strains 12 distinct RFLP profiles were

  17. Polymorphism of the Human Complement C4 and Steroid 21-Hydroxylase Genes Restriction Fragment Length Polymorphisms Revealing Structural

    E-print Network

    Alper, Chester A.

    Polymorphism of the Human Complement C4 and Steroid 21-Hydroxylase Genes Restriction Fragment associated or closely linked with genetic variants of the fourth component of complement (C4A and C4B from the genes for complement proteins, C2 and factor B (BF) between HLA-B and -DR in the major

  18. Allozyme and Restriction Fragment Length Polymorphism Analyses Confirm Entomophaga maimaiga Responsible for 1989 Epizootics in North American Gypsy Moth Populations

    Microsoft Academic Search

    Ann E. Hajek; Richard A. Humber; Joseph S. Elkinton; Bernie May; Scott R. A. Walsh; Julie C. Silver

    1990-01-01

    In 1989, populations of North American gypsy moth, Lymantria dispar, in seven contiguous northeastern states were severely reduced by a fungal pathogen. Based on morphology, development, and pathology, this organism appeared to be Entomophaga maimaiga. We have now used allozyme and restriction fragment length polymorphism analyses to confirm this identification. Previously, this mycopathogen had been reported only from gypsy moth

  19. DNA Amplification-Restricted Transcription-Translation: Rapid Analysis of Rhesus Rotavirus Neutralization Sites

    Microsoft Academic Search

    Erich R. Mackow; Mark Y. Yamanaka; Minh N. Dang; Harry B. Greenberg

    1990-01-01

    DNA amplification-restricted transcription-translation (DARTT), is based on DNA amplification by the polymerase chain reaction (PCR) and uses PCR to truncate protein-encoding DNA while adding transcriptional and translational initiation signals to the segment. The amplified DNA segments are transcribed into RNA and translated into protein in vitro and the synthesized proteins are used to define functional sites. DARTT was applied to

  20. Helicobacter pylori DprA alleviates restriction barrier for incoming DNA

    PubMed Central

    Dwivedi, Gajendradhar R.; Sharma, Eshita; Rao, Desirazu N.

    2013-01-01

    Helicobacter pylori is a Gram-negative bacterium that colonizes human stomach and causes gastric inflammation. The species is naturally competent and displays remarkable diversity. The presence of a large number of restriction–modification (R–M) systems in this bacterium creates a barrier against natural transformation by foreign DNA. Yet, mechanisms that protect incoming double-stranded DNA (dsDNA) from restriction enzymes are not well understood. A DNA-binding protein, DNA Processing Protein A (DprA) has been shown to facilitate natural transformation of several Gram-positive and Gram-negative bacteria by protecting incoming single-stranded DNA (ssDNA) and promoting RecA loading on it. However, in this study, we report that H. pylori DprA (HpDprA) binds not only ssDNA but also dsDNA thereby conferring protection to both from various exonucleases and Type II restriction enzymes. Here, we observed a stimulatory role of HpDprA in DNA methylation through physical interaction with methyltransferases. Thus, HpDprA displayed dual functional interaction with H. pylori R–M systems by not only inhibiting the restriction enzymes but also stimulating methyltransferases. These results indicate that HpDprA could be one of the factors that modulate the R–M barrier during inter-strain natural transformation in H. pylori. PMID:23355610

  1. Helicobacter pylori DprA alleviates restriction barrier for incoming DNA.

    PubMed

    Dwivedi, Gajendradhar R; Sharma, Eshita; Rao, Desirazu N

    2013-03-01

    Helicobacter pylori is a Gram-negative bacterium that colonizes human stomach and causes gastric inflammation. The species is naturally competent and displays remarkable diversity. The presence of a large number of restriction-modification (R-M) systems in this bacterium creates a barrier against natural transformation by foreign DNA. Yet, mechanisms that protect incoming double-stranded DNA (dsDNA) from restriction enzymes are not well understood. A DNA-binding protein, DNA Processing Protein A (DprA) has been shown to facilitate natural transformation of several Gram-positive and Gram-negative bacteria by protecting incoming single-stranded DNA (ssDNA) and promoting RecA loading on it. However, in this study, we report that H. pylori DprA (HpDprA) binds not only ssDNA but also dsDNA thereby conferring protection to both from various exonucleases and Type II restriction enzymes. Here, we observed a stimulatory role of HpDprA in DNA methylation through physical interaction with methyltransferases. Thus, HpDprA displayed dual functional interaction with H. pylori R-M systems by not only inhibiting the restriction enzymes but also stimulating methyltransferases. These results indicate that HpDprA could be one of the factors that modulate the R-M barrier during inter-strain natural transformation in H. pylori. PMID:23355610

  2. A mini-library of sequenced human DNA fragments: linking bench experiments with informatics

    Microsoft Academic Search

    Raymond Dalgleish; Morag E. Shanks; Karen Monger; Nicola J. Butler

    2012-01-01

    We describe the development of a mini-library of human DNA fragments for use in an enquiry-based learning (EBL) undergraduate practical incorporating ‘wet-lab’ and bioinformatics tasks. In spite of the widespread emergence of the polymerase chain reaction (PCR), the cloning and analysis of DNA fragments in Escherichia coli remains a fundamental skill commonly taught to undergraduate biology students. The individual procedures

  3. Internucleosomal DNA fragmentation during deprived and non-deprived olfactory development

    Microsoft Academic Search

    Joseph Najbauer; Xiao-Xin Yan; Michael Leon

    2002-01-01

    DNA fragmentation is a key marker of neuronal death during development, yet little is known about the size, pattern or quantities of fragments generated during normal and sensory-deprived development. Since there are few neurons dying at any particular time, it has not been possible to obtain sufficient quantities of material to make such a determination. By using a highly sensitive

  4. Internucleosomal DNA fragmentation during deprived and non-deprived olfactory development.

    PubMed

    Najbauer, Joseph; Yan, Xiao-Xin; Leon, Michael

    2002-02-01

    DNA fragmentation is a key marker of neuronal death during development, yet little is known about the size, pattern or quantities of fragments generated during normal and sensory-deprived development. Since there are few neurons dying at any particular time, it has not been possible to obtain sufficient quantities of material to make such a determination. By using a highly sensitive Taq polymerase-based technique, we revealed DNA fragments of 180 base pairs and multiples thereof both in bulbs and cortex of young rats (P4-P31). The bulbs subjected to olfactory deprivation at P1 had higher levels of internucleosomal DNA fragmentation at P16 than the contra-lateral, non-deprived bulbs. Interestingly, the DNA fragmentation induced by olfactory deprivation displayed a characteristic internucleosomal fragmentation pattern, suggesting that the cells induced to die may do so by apoptosis. A significant inverse correlation between DNA fragmentation and the natural variation in normal bulb size was found, suggesting that bulb size may be related to cell death. PMID:11814413

  5. Multiple components in restriction enzyme digests of mammalian (insectivore), avian and reptilian genomic DNA hybridize with murine immunoglobulin VH probes.

    PubMed

    Litman, G W; Berger, L; Jahn, C L

    1982-06-11

    High molecular weight genomic DNAs isolated from an insectivore, Tupaia, and a representative reptilian, Caiman, and avian, Gallus, were digested with restriction endonucleases transferred to nitrocellulose and hybridized with nick-translated probes of murine VH genes. The derivations of the probes designated S107V (1) and mu 107V (2,3) have been described previously. Under conditions of reduced stringency, multiple hybridizing components were observed with Tupaia and Caiman; only mu mu 107V exhibited significant hybridization with the separated fragments of Gallus DNA. The nick-translated S107V probe was digested with Fnu4H1 and subinserts corresponding to the 5' and 3' regions both detected multiple hybridizing components in Tupaia and Caiman DNA. A 5' probe lacking the leader sequence identified the same components as the intact 5' probe, suggesting that VH coding regions distant as the reptilians may possess multiple genetic components which exhibit significant homology with murine immunoglobulin in VH regions. PMID:6285298

  6. Molecular phylogeny of Iva ( Asteraceae, Heliantheae ) based on chloroplast DNA restriction site variation

    Microsoft Academic Search

    B. Miao; B. Turner; T. Mabry

    1995-01-01

    Iva s.str. (comprising ten species) was examined by cpDNA restriction site variation to determine phyletic relationships within the group. The results were compared with relationships proposed from other data. A total of 86 restriction site mutations was detected, 47 of which proved phylogenetically informative. A single most parsimonious tree was obtained using both Wagner and Dollo parsimony. The tree revealed

  7. Intestinal Microbiota is Different in Women with Preterm Birth: Results from Terminal Restriction Fragment Length Polymorphism Analysis

    PubMed Central

    Shiozaki, Arihiro; Yoneda, Satoshi; Yoneda, Noriko; Yonezawa, Rika; Matsubayashi, Takamichi; Seo, Genichiro; Saito, Shigeru

    2014-01-01

    Preterm birth is a leading cause of perinatal morbidity and mortality. Studies using a cultivation method or molecular identification have shown that bacterial vaginosis is one of the risk factors for preterm birth. However, an association between preterm birth and intestinal microbiota has not been reported using molecular techniques, although the vaginal microbiota changes during pregnancy. Our aim here was to clarify the difference in intestinal and vaginal microbiota between women with preterm birth and women without preterm labor. 16S ribosomal ribonucleic acid genes were amplified from fecal and vaginal DNA by polymerase chain reaction. Using terminal restriction fragment length polymorphism (T-RFLP), we compared the levels of operational taxonomic units of both intestinal and vaginal flora among three groups: pregnant women who delivered term babies without preterm labor (non-PTL group) (n?=?20), those who had preterm labor but delivered term babies (PTL group) (n?=?11), and those who had preterm birth (PTB group) (n?=?10). Significantly low levels of Clostridium subcluster XVIII, Clostridium cluster IV, Clostridium subcluster XIVa, and Bacteroides, and a significantly high level of Lactobacillales were observed in the intestinal microbiota in the PTB group compared with those in the non-PTL group. The levels of Clostridium subcluster XVIII and Clostridium subcluster XIVa in the PTB group were significantly lower than those in the PTL group, and these levels in the PTL group were significantly lower than those in non-PTL group. However, there were no significant differences in vaginal microbiota among the three groups. Intestinal microbiota in the PTB group was found to differ from that in the non-PTL group using the T-RFLP method. PMID:25372390

  8. CONSTRUCTION OF CONTIGS OF AEGILOPS TAUSCHII GENOMIC DNA FRAGMENTS CLONED IN BAC AND BIBAC VECTORS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A high-throughput, fully automated, multi-color fluorescent fingerprinting technique for large-insert genomic DNA clones was developed. The technique was used to fingerprint 200,000 genomic DNA fragments of Aegilops tauschii line genetically closely related to the D genome of Chinese Spring wheat. T...

  9. DNA fragmentation and BCL2 expression in infantile spinal muscular atrophy

    Microsoft Academic Search

    D. S. Tews; H. H. Goebel

    1996-01-01

    Chromatin cleavage, a hallmark of apoptosis, was identified by in situ labeling in 55 ± 7% of the muscle fibers in infantile spinal muscular atrophy (ISMA) and, to a lesser extent, in peripheral neuropathy indicating that DNA fragmentation is not specific to ISMA but a common feature in defect innervation. However, as DNA breaks are also known as a temporary

  10. Towards the molecular characterisation of parasitic nematode assemblages: an evaluation of terminal-restriction fragment length polymorphism (T-RFLP) analysis.

    PubMed

    Lott, M J; Hose, G C; Power, M L

    2014-09-01

    Identifying factors which regulate temporal and regional structuring within parasite assemblages requires the development of non-invasive techniques which facilitate both the rapid discrimination of individual parasites and the capacity to monitor entire parasite communities across time and space. To this end, we have developed and evaluated a rapid fluorescence-based method, terminal restriction fragment length polymorphism (T-RFLP) analysis, for the characterisation of parasitic nematode assemblages in macropodid marsupials. The accuracy with which T-RFLP was capable of distinguishing between the constituent taxa of a parasite community was assessed by comparing sequence data from two loci (the ITS+ region of nuclear ribosomal DNA and the mitochondrial CO1) across ?20 species of nematodes (suborder Strongylida). Our results demonstrate that with fluorescent labelling of the forward and reverse terminal restriction fragments (T-RFs) of the ITS+ region, the restriction enzyme Hinf1 was capable of generating species specific T-RFLP profiles. A notable exception was within the genus Cloacina, in which closely related species often shared identical T-RFs. This may be a consequence of the group's comparatively recent evolutionary radiation. While the CO1 displayed higher sequence diversity than the ITS+, the subsequent T-RFLP profiles were taxonomically inconsistent and could not be used to further differentiate species within Cloacina. Additionally, several of the ITS+ derived T-RFLP profiles exhibited unexpected secondary peaks, possibly as a consequence of the restriction enzymes inability to cleave partially single stranded amplicons. These data suggest that the question of T-RFLPs utility in monitoring parasite communities cannot be addressed without considering the ecology and unique evolutionary history of the constituent taxa. PMID:24971699

  11. A homology model of restriction endonuclease SfiI in complex with DNA

    Microsoft Academic Search

    Agnieszka A Chmiel; Janusz M Bujnicki; Krzysztof J Skowronek

    2005-01-01

    BACKGROUND: Restriction enzymes (REases) are commercial reagents commonly used in recombinant DNA technologies. They are attractive models for studying protein-DNA interactions and valuable targets for protein engineering. They are, however, extremely divergent: the amino acid sequence of a typical REase usually shows no detectable similarities to any other proteins, with rare exceptions of other REases that recognize identical or very

  12. Typing and Clustering of Yersinia pseudotuberculosis Isolates by Restriction Fragment Length Polymorphism Analysis Using Insertion Sequences

    PubMed Central

    Voskresenskaya, E.; Savin, C.; Leclercq, A.; Tseneva, G.

    2014-01-01

    Yersinia pseudotuberculosis is an enteropathogen that has an animal reservoir and causes human infections, mostly in temperate and cold countries. Most of the methods previously used to subdivide Y. pseudotuberculosis were performed on small numbers of isolates from a specific geographical area. One aim of this study was to evaluate the typing efficiency of restriction fragment length polymorphism of insertion sequence hybridization patterns (IS-RFLP) compared to other typing methods, such as serotyping, ribotyping, and multilocus sequence typing (MLST), on the same set of 80 strains of Y. pseudotuberculosis of global origin. We found that IS100 was not adequate for IS-RFLP but that both IS285 and IS1541 efficiently subtyped Y. pseudotuberculosis. The discriminatory index (DI) of IS1541-RFLP (0.980) was superior to those of IS285-RFLP (0.939), ribotyping (0.944), MLST (0.861), and serotyping (0.857). The combination of the two IS (2IS-RFLP) further increased the DI to 0.998. Thus, IS-RFLP is a powerful tool for the molecular typing of Y. pseudotuberculosis and has the advantage of exhibiting well-resolved banding patterns that allow for a reliable comparison of strains of worldwide origin. The other aim of this study was to assess the clustering power of IS-RFLP. We found that 2IS-RFLP had a remarkable capacity to group strains with similar genotypic and phenotypic markers, thus identifying robust populations within Y. pseudotuberculosis. Our study thus demonstrates that 2IS- and even IS1541-RFLP alone might be valuable tools for the molecular typing of global isolates of Y. pseudotuberculosis and for the analysis of the population structure of this species. PMID:24671793

  13. Typing and clustering of Yersinia pseudotuberculosis isolates by restriction fragment length polymorphism analysis using insertion sequences.

    PubMed

    Voskresenskaya, E; Savin, C; Leclercq, A; Tseneva, G; Carniel, E

    2014-06-01

    Yersinia pseudotuberculosis is an enteropathogen that has an animal reservoir and causes human infections, mostly in temperate and cold countries. Most of the methods previously used to subdivide Y. pseudotuberculosis were performed on small numbers of isolates from a specific geographical area. One aim of this study was to evaluate the typing efficiency of restriction fragment length polymorphism of insertion sequence hybridization patterns (IS-RFLP) compared to other typing methods, such as serotyping, ribotyping, and multilocus sequence typing (MLST), on the same set of 80 strains of Y. pseudotuberculosis of global origin. We found that IS100 was not adequate for IS-RFLP but that both IS285 and IS1541 efficiently subtyped Y. pseudotuberculosis. The discriminatory index (DI) of IS1541-RFLP (0.980) was superior to those of IS285-RFLP (0.939), ribotyping (0.944), MLST (0.861), and serotyping (0.857). The combination of the two IS (2IS-RFLP) further increased the DI to 0.998. Thus, IS-RFLP is a powerful tool for the molecular typing of Y. pseudotuberculosis and has the advantage of exhibiting well-resolved banding patterns that allow for a reliable comparison of strains of worldwide origin. The other aim of this study was to assess the clustering power of IS-RFLP. We found that 2IS-RFLP had a remarkable capacity to group strains with similar genotypic and phenotypic markers, thus identifying robust populations within Y. pseudotuberculosis. Our study thus demonstrates that 2IS- and even IS1541-RFLP alone might be valuable tools for the molecular typing of global isolates of Y. pseudotuberculosis and for the analysis of the population structure of this species. PMID:24671793

  14. Absence of cinoxacin-induced DNA fragmentation and mutations in the rat granuloma pouch.

    PubMed

    Pino, A; Maura, A; Grillo, P

    1989-01-01

    The mutagenic and DNA-damaging activities of cinoxacin were evaluated in the rat granuloma pouch assay (GPA) in order to assess its genotoxic potential in vivo. High doses of this antimicrobial quinolone, either directly injected into the pouch or administered by gavage, did not induce mutation at the hgprt locus or DNA fragmentation in granuloma cells. Moreover, DNA damage was absent in the liver and kidney of rats given cinoxacin by the oral route. PMID:2917551

  15. Exceptionally High Levels of Restriction Site Polymorphism in DNA near the Maize Adh1 Gene

    PubMed Central

    Johns, Mitrick A.; Strommer, Judith N.; Freeling, Michael

    1983-01-01

    Restriction maps have been prepared for the chromosomal region near seven biochemically and genetically distinct maize alcohol dehydrogenase-1 (Adh1) alleles using a small cDNA probe for Adh1. Five restriction sites spanning about 4 kb in and near the Adh1 transcription unit appear identical in all seven alleles. Outside this conserved region, variation in restriction site position is the rule. Six of the seven alleles are distinguishable, and the alleles appear to fall into four groups. The DNA flanking the 1S-type alleles seems to share no restriction site homology with the DNA near the 1F-type alleles. Several hypotheses are put forward to explain how such high levels of polymorphism could have arisen in a species that has been domesticated for only about 10,000 years. PMID:17246173

  16. Pst I restriction fragment length polymorphism of human placental alkaline phosphatase gene: Mendelian in segregation and localization of mutation site in the gene

    SciTech Connect

    Tsavaler, L.; Penhallow, R.C.; Sussman, H.H. (Stanford Univ., CA (USA))

    1988-10-01

    The pattern of inheritance of a Pst I restriction fragment length polymorphism (RFLP) of the human placental alkaline phosphatase gene was studied in nine nuclear families by Southern blot hybridization analysis of genomic DNA. The dimorphic RFLP is defined by the presence of allelic fragments 1.0 kilobase and 0.8 kilobase long. The results of this study show that the two alleles of the Pst I RFLP of the placental alkaline phosphatase gene segregate as codominant traits according to Mendelian expectations. For a polymorphism to be useful as a genetic marker the probability that an offspring is informative (PIC) must be at least 0.15. The allelic frequency of the 1.0-kilobase allele is 0.21, which correlates to a probability that an offspring is informative of 0.275 and is indicative of a useful polymorphism. By using probes derived from different regions of the placental alkaline phosphatase cDNA, the mutated Pst I site causing the RFLP was located in the penultimate intron 2497 base pairs downstream from the transcriptional initiation site.

  17. Typing Chlamydia trachomatis by Detection of Restriction Fragment Length Polymorphism in the Gene Encoding the Major Outer Membrane Protein

    Microsoft Academic Search

    Eric H. Frost; Sylvie Deslandes; Stéphane Veilleux; Danielle Bourgaux-Ramoisy

    1991-01-01

    A method that avoids culture was devised to determine serovars of Chlamydia trachomatis. Polymerase chain reaction was used first to amplify a part of the chlamydial genome that included the leader sequence and all four variable domains of the major outer membrane protein (MOMP) of the 15 serovars of C. trachomatis. The amplified DNA was then digested simultaneously with restriction

  18. Molecular Scissors: Restriction Enzymes 2009 1 Minority Science Programs School of Biological Sciences University of California, Irvine

    E-print Network

    Rose, Michael R.

    Molecular Scissors: Restriction Enzymes 2009 1 Minority Science Programs a group of enzymes (proteins that aid chemical reactions) in bacteria, which when added to any DNA result fragmented. These "molecular scissors" are called restriction enzymes (RE) How Restriction Enzymes

  19. Identification of opaque-2 genotypes in segregating populations of quality protein maize by analysis of restriction fragment length polymorphisms 

    E-print Network

    Kata, Srinivas Reddy

    1993-01-01

    IDENTIFICATION OF OPAQUE-2 GENOTYPES IN SEGREGATING POPULATIONS OF QUALITY PROTEIN MAIZE BY ANALYSIS OF RESTRICTION FRAGMENT LENGTH POLYMORPHISMS A Thesis by SRINIVAS REDDY KATA Submitted to the Office of Graduate Studies of Texas Adt... LENGTH POLYMORPHISMS A Thesis by SRINIVAS REDDY KATA Submitted to Texas A&M University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Appmved as to style and content by: S. K. Da i (Chair of Genetics Faculty) J...

  20. Rapid Discrimination among Dermatophytes, Scytalidium spp., and Other Fungi with a PCR-Restriction Fragment Length Polymorphism Ribotyping Method

    Microsoft Academic Search

    MARIE MACHOUART-DUBACH; CLAIRE LACROIX; MARTINE FEUILHADE DE CHAUVIN; ISABELLE LE GALL; CATHERINE GIUDICELLI; FREDERIC LORENZO; FRANCIS DEROUIN

    2001-01-01

    Dermatomycoses are very common infections caused mainly by dermatophytes. Scytalidiosis is a differential mycological diagnosis, especially in tropical and subtropical areas. Since a culture-based diagnosis takes 2 to 3 weeks, we set up a PCR-restriction fragment length polymorphism (RFLP) method for rapid discrimination of these fungi in clinical samples. The hypervariable V4 domain of the small ribosomal subunit 18S gene

  1. Routine Use of PCR-Restriction Fragment Length Polymorphism Analysis for Identification of Mycobacteria Growing in Liquid Media

    Microsoft Academic Search

    THERESA B. TAYLOR; CANDY PATTERSON; YVONNE HALE; ANDWILLIAM W. SAFRANEK

    1997-01-01

    A PCR-restriction fragment length polymorphism (PCR-RFLP) procedure capable of rapidly identifying 28 species of clinically encountered mycobacteria was evaluated for use in the routine identification of acid-fast isolates growing in BACTEC 12B and 13A liquid media. PCR-RFLP identified 100 of 103 acid-fast isolates recovered from 610 patient specimens submitted for culture during the study. The three isolates unidentifiable by PCR-RFLP

  2. A Semester-Long Project for Teaching Basic Techniques in Molecular Biology Such as Restriction Fragment Length Polymorphism Analysis to Undergraduate and Graduate Students

    PubMed Central

    DiBartolomeis, Susan M.

    2011-01-01

    Several reports on science education suggest that students at all levels learn better if they are immersed in a project that is long term, yielding results that require analysis and interpretation. I describe a 12-wk laboratory project suitable for upper-level undergraduates and first-year graduate students, in which the students molecularly locate and map a gene from Drosophila melanogaster called dusky and one of dusky's mutant alleles. The mapping strategy uses restriction fragment length polymorphism analysis; hence, students perform most of the basic techniques of molecular biology (DNA isolation, restriction enzyme digestion and mapping, plasmid vector subcloning, agarose and polyacrylamide gel electrophoresis, DNA labeling, and Southern hybridization) toward the single goal of characterizing dusky and the mutant allele dusky73. Students work as individuals, pairs, or in groups of up to four students. Some exercises require multitasking and collaboration between groups. Finally, results from everyone in the class are required for the final analysis. Results of pre- and postquizzes and surveys indicate that student knowledge of appropriate topics and skills increased significantly, students felt more confident in the laboratory, and students found the laboratory project interesting and challenging. Former students report that the lab was useful in their careers. PMID:21364104

  3. Detection of measles virus-induced apoptosis of human monocytic cell line (THP-1) by DNA fragmentation ELISA.

    PubMed

    Ito, M; Yamamoto, T; Watanabe, M; Ihara, T; Kamiya, H; Sakurai, M

    1996-09-01

    Measles virus (wild strain, Toyoshima strain)-induced cell death is characterized by cell shrinkage, chromatin condensation, and nuclear fragmentation in a human monocytic cell line (THP-1). DNA fragmentation of measles virus-infected THP-1 cells was demonstrated by DNA agarose gel electrophoresis as well as by DNA fragmentation ELISA. When measles virus-infected THP-1 cells were cultured on monolayers of fibroblasts or human umbilical vein endothelial cells (HUVEC), the percentage of measles virus antigen-positive THP-1 cells and DNA fragmentation were significantly decreased. Addition of anti-intercellular adhesion molecule (ICAM)-1 (CD54) monoclonal antibody to culture of measles virus-infected THP-1 cells reduced significantly DNA fragmentation induced by measles virus. These findings suggest that inhibition of virus spread by fibroblasts and HUVEC reduces apoptosis, and ICAM-1 (CD54) may participate in the DNA fragmentation pathway. PMID:8880136

  4. Restricted gene flow in fragmented populations of a wind-pollinated tree

    Microsoft Academic Search

    Jim Provan; Gemma E. Beatty; Andrea M. Hunter; Robbie A. McDonald; Emma McLaughlin; S. Jane Preston; Siân Wilson

    2008-01-01

    Fragmentation of natural populations can have negative effects at the genetic level, thus threatening their evolutionary potential.\\u000a Many of the negative genetic impacts of population fragmentation can be ameliorated by gene flow and it has been suggested\\u000a that in wind-pollinated tree species, high or even increased levels of gene flow are a feature of fragmented populations,\\u000a although several studies have

  5. Genetic analysis of autoimmune gld mice. I. Identification of a restriction fragment length polymorphism closely linked to the gld mutation within a conserved linkage group

    PubMed Central

    1988-01-01

    A linkage map of distal mouse chromosome 1 was generated using restriction fragment length polymorphism (RFLP) analysis of DNA prepared from 95 [C3H-gld/gld X Mus spretus)F1 X C3H-gld/gld] backcross mice. The gene order was: (centromere) C4bp, Ren-1,2, Ly-5, [At-3/gld], Apoa-2/Ly-17, Spna-1 (telomere). All mice expressing the phenotype of gld homozygotes were homozygous for the At-3 RFLP characteristic of C3H mice and none of the mice heterozygous for At-3 RFLPs had characteristics of gld homozygotes, demonstrating close linkage between these genes. The identification of an RFLP closely linked to the gld gene provides a starting point for the identification of a genetic defect that results in abnormal T cells and autoimmune disease. PMID:2894402

  6. Estimation of diversity and community structure through restriction fragment length polymorphism distribution analysis of bacterial 16S rRNA genes from a microbial mat at an active, hydrothermal vent system, Loihi Seamount, Hawaii.

    PubMed Central

    Moyer, C L; Dobbs, F C; Karl, D M

    1994-01-01

    PCR was used to amplify (eu)bacterial small-subunit (16S) rRNA genes from total-community genomic DNA. The source of total-community genomic DNA used for this culture-independent analysis was the microbial mats from a deep-sea, hydrothermal vent system, Pele's Vents, located at Loihi Seamount, Hawaii. Oligonucleotides complementary to conserved regions in the 16S rRNA-encoding DNA (rDNA) of bacteria were used to direct the synthesis of PCR products, which were then subcloned by blunt-end ligation into phagemid vector pBluescript II. Restriction fragment length polymorphism patterns, created by using tandem tetrameric restriction endonucleases, revealed the presence of 12 groups of 16S rRNA genes representing discrete operational taxonomic units (OTUs). The rank order abundance of these putative OTUs was measured, and the two most abundant OTUs accounted for 72.9% of all of the 16S rDNA clones. Among the remaining 27.1% of the 16S rDNA clones, none of the 10 OTUs was represented by more than three individual clones. The cumulative OTU distribution for 48 bacterial 16S rDNA clones demonstrated that the majority of taxa represented in the clone library were detected, a result which we assume to be an estimate of the diversity of bacteria in the native hydrothermal vent habitat. 16S rDNA fingerprinting of individual clones belonging to particular OTUs by using an oligonucleotide probe that binds to a universally conserved region of the 16S rDNA fragments was conducted to confirm OTU specificity and 16S rDNA identity. Images PMID:7512808

  7. BIBAC and TAC clones containing potato genomic DNA fragments larger than 100 kb are not stable in Agrobacterium

    Microsoft Academic Search

    J. Song; J. M. Bradeen; S. K. Naess; J. P. Helgeson; J. Jiang

    2003-01-01

    Development of efficient methods to transfer large DNA fragments into plants will greatly facilitate the map-based cloning of genes. The recently developed BIBAC and TAC vectors have shown potential to deliver large DNA fragments into plants via Agrobacterium-mediated transformation. Here we report that BIBAC and TAC clones containing potato genomic DNA fragments larger than 100 kb are not stable in Agrobacterium.

  8. Sperm DNA fragmentation and its role in wildlife conservation.

    PubMed

    Gosálvez, Jaime; Holt, William V; Johnston, Stephen D

    2014-01-01

    Until about 20 years ago, sperm assessment in the laboratory was focused on motility, morphology and acrosomal integrity. Then came the gradual realisation that, because the main objective of a spermatozoon is to deliver an intact genetic payload of DNA to the egg, being able to check DNA quality of spermatozoa would be equally important, if not more so. Research over the last two decades has therefore led to the development of several techniques for reliably detecting DNA strand breaks, and the more recent focus has been directed towards understanding the fertility implications of DNA damage. It is now clear that evolutionary history has played an important role in determining the stability of sperm DNA under stressful conditions, and that the nature of the DNA-protein interactions also influence the extent to which fertility is affected by both technical procedures involved in sperm preservation and the basic biology of the species concerned. Here we present an overview of the principles involved in DNA assessment and also provide some cases studies that illustrate the influences of species diversity. PMID:25091917

  9. DNA methylation protects hematopoietic stem cell multipotency from myeloerythroid restriction

    Microsoft Academic Search

    Ann-Marie Bröske; Lena Vockentanz; Shabnam Kharazi; Matthew R Huska; Elena Mancini; Marina Scheller; Christiane Kuhl; Andreas Enns; Marco Prinz; Rudolf Jaenisch; Claus Nerlov; Achim Leutz; Miguel A Andrade-Navarro; Sten Eirik W Jacobsen; Frank Rosenbauer

    2009-01-01

    DNA methylation is a dynamic epigenetic mark that undergoes extensive changes during differentiation of self-renewing stem cells. However, whether these changes are the cause or consequence of stem cell fate remains unknown. Here, we show that alternative functional programs of hematopoietic stem cells (HSCs) are governed by gradual differences in methylation levels. Constitutive methylation is essential for HSC self-renewal but

  10. Method of preparing an equimolar DNA mixture for one-step DNA assembly of over 50 fragments

    PubMed Central

    Tsuge, Kenji; Sato, Yukari; Kobayashi, Yuka; Gondo, Maiko; Hasebe, Masako; Togashi, Takashi; Tomita, Masaru; Itaya, Mitsuhiro

    2015-01-01

    In the era of synthetic biology, techniques for rapidly constructing a designer long DNA from short DNA fragments are desired. To realize this, we attempted to establish a method for one-step DNA assembly of unprecedentedly large numbers of fragments. The basic technology is the Ordered Gene Assembly in Bacillus subtilis (OGAB) method, which uses the plasmid transformation system of B. subtilis. Since this method doesn’t require circular ligation products but needs tandem repeat ligation products, the degree of deviation in the molar concentration of the material DNAs is the only determinant that affects the efficiency of DNA assembly. The strict standardization of the size of plasmids that clone the DNA block and the measurement of the block in the state of intact plasmid improve the reliability of this step, with the coefficient of variation of the molar concentrations becoming 7%. By coupling this method with the OGAB method, one-step assembly of more than 50 DNA fragments becomes feasible. PMID:25990947

  11. Chloroplast DNA inheritance in Populus

    Microsoft Academic Search

    O. P. Rajora; B. P. Dancik

    1992-01-01

    The inheritance of chloroplast (cp) DNA was examined in F1 hybrid progenies of two Populus deltoides intraspecific controlled crosses and three P. deltoides × P. nigra and two P. deltoides × P. maximowiczii interspecific controlled crosses by restriction fragment analysis. Southern blots of restriction digests of parental and progeny DNAs were hybridized to cloned cpDNA fragments of Petunia hybrida. Sixteen

  12. Electronic and molecular structure of M-DNA fragments

    Microsoft Academic Search

    Yury V. Rubin; Leonid F. Belous; ?natolij ?. Yakuba

    2011-01-01

    To study M-DNA molecular structure (such DNA with transition metal ions placed between the nucleic bases is able to conduct\\u000a the electric current) and its conductivity mechanisms, we carried out ab initio quantum-mechanical calculations of electronic and spatial structures, thermodynamic characteristics of adenine-thymine (??)\\u000a and guanine-cytosine (GC) base pair complexes with Zn2+ and Ni2+. To take into account the influence

  13. Assessment of Microbial Diversity in Four Southwestern United States Soils by 16S rRNA Gene Terminal Restriction Fragment Analysis

    PubMed Central

    Dunbar, John; Ticknor, Lawrence O.; Kuske, Cheryl R.

    2000-01-01

    The ability of terminal restriction fragment (T-RFLP or TRF) profiles of 16S rRNA genes to provide useful information about the relative diversity of complex microbial communities was investigated by comparison with other methods. Four soil communities representing two pinyon rhizosphere and two between-tree (interspace) soil environments were compared by analysis of 16S rRNA gene clone libraries and culture collections (Dunbar et al., Appl. Environ. Microbiol. 65:1662–1669, 1998) and by analysis of 16S rDNA TRF profiles of community DNA. The TRF method was able to differentiate the four communities in a manner consistent with previous comparisons of the communities by analysis of 16S rDNA clone libraries. TRF profiles were not useful for calculating and comparing traditional community richness or evenness values among the four soil environments. Statistics calculated from RsaI, HhaI, HaeIII, and MspI profiles of each community were inconsistent, and the combined data were not significantly different between samples. The detection sensitivity of the method was tested. In standard PCRs, a seeded population comprising 0.1 to 1% of the total community could be detected. The combined results demonstrate that TRF analysis is an excellent method for rapidly comparing the relationships between bacterial communities in environmental samples. However, for highly complex communities, the method appears unable to provide classical measures of relative community diversity. PMID:10877790

  14. Development of procedures for the identification of human papilloma virus DNA fragments in laser plume

    NASA Astrophysics Data System (ADS)

    Woellmer, Wolfgang; Meder, Tom; Jappe, Uta; Gross, Gerd; Riethdorf, Sabine; Riethdorf, Lutz; Kuhler-Obbarius, Christina; Loening, Thomas

    1996-01-01

    For the investigation of laser plume for the existence of HPV DNA fragments, which possibly occur during laser treatment of virus infected tissue, human papillomas and condylomas were treated in vitro with the CO2-laser. For the sampling of the laser plume a new method for the trapping of the material was developed by use of water-soluble gelatine filters. These samples were analyzed with the polymerase chain reaction (PCR) technique, which was optimized in regard of the gelatine filters and the specific primers. Positive PCR results for HPV DNA fragments up to the size of a complete oncogene were obtained and are discussed regarding infectiousity.

  15. Differentiation of mixed biological traces in sexual assaults using DNA fragment analysis

    PubMed Central

    Apostolov, ?leksandar

    2014-01-01

    During the investigation of sexual abuse, it is not rare that mixed genetic material from two or more persons is detected. In such cases, successful profiling can be achieved using DNA fragment analysis, resulting in individual genetic profiles of offenders and their victims. This has led to an increase in the percentage of identified perpetrators of sexual offenses. The classic and modified genetic models used, allowed us to refine and implement appropriate extraction, polymerase chain reaction and electrophoretic procedures with individual assessment and approach to conducting research. Testing mixed biological traces using DNA fragment analysis appears to be the only opportunity for identifying perpetrators in gang rapes. PMID:26019514

  16. Monte Carlo evaluation of DNA fragmentation spectra induced by different radiation qualities.

    PubMed

    Alloni, D; Campa, A; Belli, M; Esposito, G; Mariotti, L; Liotta, M; Friedland, W; Paretzke, H; Ottolenghi, A

    2011-02-01

    The PARTRAC code has been developed constantly in the last several years. It is a Monte Carlo code based on an event-by-event description of the interactions taking place between the ionising radiation and liquid water, and in the present version simulates the transport of photons, electrons, protons, helium and heavier ions. This is combined with an atom-by-atom representation of the biological target, i.e. the DNA target model of a diploid human fibroblast in its interphase (genome of 6 Gigabase pairs). DNA damage is produced by the events of energy depositions, either directly, if they occur in the volume occupied by the sugar-phosphate backbone, or indirectly, if this volume is reached by radiation-induced radicals. This requires the determination of the probabilities of occurrence of DNA damage. Experimental data are essential for this determination. However, after the adjustment of the relevant parameters through the comparison of the simulation data with the DNA fragmentation induced by photon irradiation, the code has been used without further parameter adjustments, and the comparison with the fragmentation induced by charged particle beams has validated the code. In this paper, the results obtained for the DNA fragmentation induced by gamma rays and by charged particle beams of various LET are shown, with a particular attention to the production of very small fragments that are not detected in experiments. PMID:21084331

  17. Synthesis, integration, and restriction and modification of mycoplasma virus L2 DNA

    SciTech Connect

    Dybvig, K.

    1981-01-01

    Mycoplasma virus L2 is an enveloped, nonlytic virus containing double-stranded, superhelical DNA. The L2 virion contains about 7 to 8 major proteins identified by SDS-polyacrylamide gel electrophoresis, but the virion has no discernible capsid structure. It has been suggested that the L2 virion is a DNA-protein condensation surrounded by a lipid-protein membrane. The host for mycoplasma virus L2 is Acholeplasma laidlawii. A. laidlawii has no cell wall and contains a small genome, 1 x 10/sup 9/ daltons, which is two to three times smaller than that of most bacteria. Infection of A. laidlawii by L2 is nonlytic. The studies in this thesis show that L2 DNA synthesis begins at about 1 hour of infection and lasts throughout the infection. Viral DNA synthesis is inhibited by chloramphenicol, streptomycin, and novobiocin. Packaging of L2 DNA into progeny virus is also inhibited by chloramphenicol and novobiocin. It is concluded that protein synthesis and probably DNA gyrase activity are required for L2 DNA synthesis, and for packaging of L2 DNA into progeny virus. DNA-DNA hybridization studies demonstrate that L2 DNA integrates into the host cell during infection, and subsequent to infection the cells are mycoplasma virus L2 lysogens. The viral site of integration has been roughly mapped. L2 virus is restricted and modified by A. laidlawii strains JA1 and K2. The nature of the modification in strain K2 has been elucidated. Two L2 variants containing insertions in the viral DNA were identified in these studies. Restriction endonuclease cleavage maps of these variants have been determined. DNA from L2 and another isolate of L2, MV-Lg-L 172, are compared in these studies. 74 references, 33 figures, 6 tables. (ACR)

  18. Fast multiple gene fragment ligation method based on Type IIs restriction enzyme DraIII

    E-print Network

    Shi, Zhenyu

    2010-10-31

    With the established BioBrick Assembly standards, ligation of different parts has to be accomplished step by step. It can be time-consuming when dealing with multiple fragment ligation. BBF RFC 61 is developed aimed at ...

  19. Sorting Short Fragments of Single-Stranded DNA with an Evolving Electric Double Layer

    PubMed Central

    Wu, Jiamin; Zhao, Shuang-Liang; Gao, Lizeng; Wu, Jianzhong; Gao, Di

    2013-01-01

    We demonstrate a new procedure for separation of single-stranded DNA (ssDNA) fragments that are anchored to the surface of a gold electrode by end hybridization. The new separation procedure takes the advantage of the strong yet evolving non-uniform electric field near the gold surface in contact with a buffer solution gradually being diluted with deionized water. Separation of short ssDNA fragments is demonstrated by monitoring the DNA at the gold surface with in situ fluorescence measurement. The experimental results can be rationalized with a simple theoretical model of electric double layer that relates the strength of the surface pulling force to the ionic concentration of the changing buffer solution. PMID:23356906

  20. Practical identification of human originated Lactobacillus species by amplified ribosomal DNA restriction analysis (ARDRA) for probiotic use.

    PubMed

    Öztürk, Mehmet; Meterelliyöz, Merve

    2015-08-01

    Probiotics are gaining popularity and increasing the importance of their accurate speciation. Lactobacillus species are commonly used as probiotic strains mostly of clinical importance. Present knowledge indicates that at least 14 Lactobacillus species are associated with the human intestinal tract. Currently, researchers are interested in developing efficient techniques for screening and selecting probiotics bacteria, but unfortunately most of these methods are time-consuming, labor-intensive and costly. The aim of this study is to develop reliable, rapid and accurate method to identify 14 references Lactobacillus species that could have been found in the human alimentary tract by 16S ribosomal DNA restriction analysis. In this study, to develop an effective method for the genotype-based identification of the reference Lactobacillus species, 1.5 kb of 16S rRNA nucleotide sequences of 14 Lactobacillus were collected from the Gene Bank aligned, in silico restricted and analyzed in respect to their 16S-rRNA restriction fragment polymorphism. In silico restriction profiles of 16S-rRNA indicated that FspBI, HinfI and DraI restriction enzymes (RE) are convenient for differentiation of 14 Lactobacillus species in human intestinal tract except Lb. casei and Lb. paracasei. The patterns of our experimental findings obtained from 16S PCR-ARDRA completely confirmed our in silico patterns. The present work demonstrated that 16S PCR-ARDRA method with FspBI, HinfI and DraI RE is a rapid, accurate and reliable method for the identification of Lactobacillus species from human alimentary tract, especially during the identification of large numbers of isolates and any laboratory equipped with a thermo cycler for probiotic use. PMID:25860079

  1. Mechanism of DNA Recognition by the Restriction Enzyme EcoRV

    Microsoft Academic Search

    Mai Zahran; Isabella Daidone; Jeremy C. Smith; Petra Imhof

    2010-01-01

    EcoRV, a restriction enzyme in Escherichia coli, destroys invading foreign DNA by cleaving it at the center step of a GATATC sequence. In the EcoRV–cognate DNA crystallographic complex, a sharp kink of 50° has been found at the center base-pair step (TA). Here, we examine the interplay between the intrinsic propensity of the cognate sequence to kink and the induction

  2. Restriction and sequence alterations affect DNA uptake sequence-dependent transformation in Neisseria meningitidis.

    PubMed

    Ambur, Ole Herman; Frye, Stephan A; Nilsen, Mariann; Hovland, Eirik; Tønjum, Tone

    2012-01-01

    Transformation is a complex process that involves several interactions from the binding and uptake of naked DNA to homologous recombination. Some actions affect transformation favourably whereas others act to limit it. Here, meticulous manipulation of a single type of transforming DNA allowed for quantifying the impact of three different mediators of meningococcal transformation: NlaIV restriction, homologous recombination and the DNA Uptake Sequence (DUS). In the wildtype, an inverse relationship between the transformation frequency and the number of NlaIV restriction sites in DNA was observed when the transforming DNA harboured a heterologous region for selection (ermC) but not when the transforming DNA was homologous with only a single nucleotide heterology. The influence of homologous sequence in transforming DNA was further studied using plasmids with a small interruption or larger deletions in the recombinogenic region and these alterations were found to impair transformation frequency. In contrast, a particularly potent positive driver of DNA uptake in Neisseria sp. are short DUS in the transforming DNA. However, the molecular mechanism(s) responsible for DUS specificity remains unknown. Increasing the number of DUS in the transforming DNA was here shown to exert a positive effect on transformation. Furthermore, an influence of variable placement of DUS relative to the homologous region in the donor DNA was documented for the first time. No effect of altering the orientation of DUS was observed. These observations suggest that DUS is important at an early stage in the recognition of DNA, but does not exclude the existence of more than one level of DUS specificity in the sequence of events that constitute transformation. New knowledge on the positive and negative drivers of transformation may in a larger perspective illuminate both the mechanisms and the evolutionary role(s) of one of the most conserved mechanisms in nature: homologous recombination. PMID:22768309

  3. Accurate phylogenetic classification of DNA fragments based onsequence composition

    SciTech Connect

    McHardy, Alice C.; Garcia Martin, Hector; Tsirigos, Aristotelis; Hugenholtz, Philip; Rigoutsos, Isidore

    2006-05-01

    Metagenome studies have retrieved vast amounts of sequenceout of a variety of environments, leading to novel discoveries and greatinsights into the uncultured microbial world. Except for very simplecommunities, diversity makes sequence assembly and analysis a verychallenging problem. To understand the structure a 5 nd function ofmicrobial communities, a taxonomic characterization of the obtainedsequence fragments is highly desirable, yet currently limited mostly tothose sequences that contain phylogenetic marker genes. We show that forclades at the rank of domain down to genus, sequence composition allowsthe very accurate phylogenetic 10 characterization of genomic sequence.We developed a composition-based classifier, PhyloPythia, for de novophylogenetic sequence characterization and have trained it on adata setof 340 genomes. By extensive evaluation experiments we show that themethodis accurate across all taxonomic ranks considered, even forsequences that originate fromnovel organisms and are as short as 1kb.Application to two metagenome datasets 15 obtained from samples ofphosphorus-removing sludge showed that the method allows the accurateclassification at genus level of most sequence fragments from thedominant populations, while at the same time correctly characterizingeven larger parts of the samples at higher taxonomic levels.

  4. Identification of opaque-2 genotypes in segregating populations of quality protein maize by analysis of restriction fragment length polymorphisms

    E-print Network

    Kata, Srinivas Reddy

    1993-01-01

    conversion by eliminating the need for testcrossing and by reducing the population size. To identify the opaque-2 genotypes in segregating populations of Quality Protein Maize, a Restriction Fragment Length Polymorphism (RFLP) marker assay was developed...5855/POP22) X (TX5855)]. 12. BC population [(TX5855/POB67) X (TX5855)] 13. Summary of populations analyzed. 27 30 32 33 35 37 39 41 42 46 CHAPTER I INTRODUCTION Maize (Zea mays L. ) is one of the most important crop species used for food...

  5. Temporal Profile of In Situ DNA Fragmentation After Transient Middle Cerebral Artery Occlusion in the Rat

    Microsoft Academic Search

    Yi Li; Michael Chopp; Ning Jiang; Fayi Yao; Cecylia Zaloga

    1995-01-01

    Summary: We measured the temporal profile and anatomic distribution of cells exhibiting DNA fragmentation at various durations of reperfusion after middle cerebral artery (MCA) occlusion in the rat. Focal cerebral ischemia was induced in male Wistar rats (n = 62) using an intraluminal monofilament blockade of the MCA. After 2 h of MCA occlusion, the animals were killed at different

  6. The influence of cigarette smoking on human sperm quality and DNA fragmentation

    Microsoft Academic Search

    Sandrine Sepaniak; Thierry Forges; Hubert Gerard; Bernard Foliguet; Marie-Christine Bene; Patricia Monnier-Barbarino

    2006-01-01

    The aim of the present study was to evaluate consequences of cigarette smoking on male gametes. In this prospective study, sperm parameters such as sperm density, motility, viability and normal morphology were measured according to the WHO criteria. In addition to these standard parameters, we analysed the degree of DNA fragmentation in spermatozoa using the TUNEL-assay with flow cytometry detection

  7. Construction of long DNA molecules using long PCR-based fusion of several fragments simultaneously

    Microsoft Academic Search

    Nikolai A. Shevchuk; Anton V. Bryksin; Yevgeniya A. Nusinovich; Felipe C. Cabello; Margaret Sutherland; Stephan Ladisch

    2004-01-01

    A procedure for precise assembly of linear DNA constructs as long as 20 kb is proposed. The method, which we call long multiple fusion, has been used to assemble up to four fragments simul- taneously (for a 10.8 kb final product), offering an additional improvement on the combination of long PCR and overlap extension PCR. The method is based on

  8. A WHEAT DNA FRAGMENT EXHIBITS REDUCED POLLEN TRANSMISSION IN TRANSGENIC MAIZE

    Technology Transfer Automated Retrieval System (TEKTRAN)

    An 8.2 kb fragment of wheat genomic DNA containing the Glu1-Dx5 gene has been transferred to maize using biolistic transformation. The Glu1-Dx5 gene encodes the 1Dx5 high molecular weight glutenin subunit, a seed storage protein associated with good bread making properties. The transgenic maize plan...

  9. RESTRICTION FRAGMENT LENGTH POLYMORPHISM ANALYSIS OF PCR-AMPLIFIED NIFH SEQUENCES FROM WETLAND PLANT RHIZOSPHERE COMMUNITIES

    EPA Science Inventory

    We describe a method to assess the community structure of N2-fixing bacteria in the rhizosphere. Total DNA was extracted from Spartina alterniflora and Sesbania macrocarpa root zones by bead-beating and purified by CsCl-EtBr gradient centrifugation. The average DNA yield was 5.5 ...

  10. PCR-Restriction Fragment Length Polymorphism Method for Detection of Cyclospora cayetanensis in Environmental Waters without Microscopic Confirmation

    PubMed Central

    Shields, Joan M.; Olson, Betty H.

    2003-01-01

    We developed an alternative nested-PCR-restriction fragment length polymorphism (RFLP) protocol for the detection of Cyclospora cayetanensis in environmental samples that obviates the need for microscopic examination. The RFLP method, with the restriction enzyme AluI, differentiates the amplified target sequence from C. cayetanensis from those that may cross-react. This new protocol was used to reexamine a subset (121 of 180) of surface water samples. Samples previously positive when the CYCF3E and CYCR4B primers (33) and RFLP with MnlI (20) were used were also PCR positive with the new primers; however, they were RFLP negative. We verified, by sequencing these amplicons, that while two were most likely other Cyclospora species, they were not C. cayetanensis. We can detect as few as one oocyst seeded into an autoclaved pellet flocculated from 10 liters of surface water. This new protocol should be of great use for environmental microbiologists and public health laboratories. PMID:12902255

  11. [Cleavage of DNA fragments induced by UV nanosecond laser excitation at 193 nm].

    PubMed

    Vtiurina, N N; Grokhovski?, S L; Filimonov, I V; Medvedkov, O I; Nechipurenko, D Iu; Vasil'ev, S A; Nechipurenko, Iu D

    2011-01-01

    The cleavage of dsDNA fragments in aqueous solution after irradiation with UV laser pulses at 193 nm has been studied. Samples were investigated using polyacrylamide gel electrophoresis. The intensity of damage of particular phosphodiester bond after hot alkali treatment was shown to depend on the base pair sequence. It was established that the probability of cleavage is twice higher for sites of DNA containing two or more successively running guanine residues. A possible mechanism of damage to the DNA molecule connected with the migration of holes along the helix is discussed. PMID:21786693

  12. Variability of chloroplast DNA in the genus Passiflora L

    Microsoft Academic Search

    Inés Sánchez; Fernando Angel; Mikkel Grum; Miriam C. Duque; Mario Lobo; Joe Tohme; William Roca

    1999-01-01

    Restriction fragment analysis was used to determine the variation of chloroplast DNA (cpDNA) among and within different species\\u000a of Passiflora. Total DNAs of 35 accessions, representing 12 species, were digested with four restriction endonucleases. Southern\\u000a blots of the restriction digests were then probed with eight different cpDNA fragments from Petunia and mung bean. Two hundred\\u000a fragments were scored, and used

  13. A rapid and reliable PCR-restriction fragment length polymorphism (RFLP) marker for the identification of Amaranthus cruentus species

    PubMed Central

    Park, Young-Jun; Nishikawa, Tomotaro; Matsushima, Kenichi; Minami, Mineo; Nemoto, Kazuhiro

    2014-01-01

    A rapid and reliable PCR-restriction fragment length polymorphism (RFLP) marker was developed to identify the Amaranthus cruentus species by comparing sequences of the starch branching enzyme (SBE) locus among the three cultivated grain amaranths. We determined the partial SBE genomic sequence in 72 accessions collected from diverse locations around the world by direct sequence analysis. Then, we aligned the gene sequences and searched for restriction enzyme cleavage sites specific to each species for use in the PCR-RFLP analysis. The result indicated that MseI would recognize the sequence 5?-T/TAA-3? in intron 11 from A. cruentus SBE. A restriction analysis of the amplified 278-bp portion of the SBE gene using the MseI restriction enzyme resulted in species-specific RFLP patterns among A. cruentus, Amaranthus caudatus and Amaranthus hypochondriacus. Two different bands, 174-bp and 104-bp, were generated in A. cruentus, while A. caudatus and A. hypochondriacus remained undigested (278-bp). Thus, we propose that the PCR-RFLP analysis of the amaranth SBE gene provides a sensitive, rapid, simple and useful technique for identifying the A. cruentus species among the cultivated grain amaranths. PMID:25914599

  14. Sequence specific detection of restriction enzymes at DNA-modified carbon nanotube field effect transistors.

    PubMed

    Ordinario, David D; Burke, Anthony M; Phan, Long; Jocson, Jonah-Micah; Wang, Hanfei; Dickson, Mary N; Gorodetsky, Alon A

    2014-09-01

    Protein-DNA interactions play a central role in many cellular processes, and their misregulation has been implicated in a number of human diseases. Thus, there is a pressing need for the development of analytical strategies for interrogating the binding of proteins to DNA. Herein, we report the electrical monitoring of a prototypical DNA-binding protein, the PvuII restriction enzyme, at microfluidic-encapsulated, DNA-modified carbon nanotube field effect transistors. Our integrated platform enables the sensitive, sequence specific detection of PvuII at concentrations as low as 0.5 pM in a volume of 0.025 ?L (corresponding to ~7500 proteins). These figures of merit compare favorably to state of the art values reported for alternative fluorescent and electrical assays. The overall detection strategy represents a step toward the massively parallel electrical monitoring, identification, and quantification of protein-DNA interactions at arrayed nanoscale devices. PMID:25137193

  15. Speciation of organisms within the Mycobacterium avium-Mycobacterium intracellulare-Mycobacterium scrofulaceum (MAIS) complex based on restriction fragment length polymorphisms.

    PubMed

    Picken, R N; Tsang, A Y; Yang, H L

    1988-12-01

    A DNA probe which hybridizes to all pathogenic species of slow-growing mycobacteria has been used to identify restriction-fragment-length-polymorphisms (RFLPs) in Bam Hl digests of chromosomal DNA from members of the Mycobacterium avium-Mycobacterium intracellulare-Mycobacterium scrofulaceum complex. The RFLP patterns so produced were found to fall into distinct categories which were representative of each of the three species. Except for two doubtful isolates, strains of M. avium were found to fall into two related RFLP-types, one of which contained the vast majority of the strains tested. In contrast, M. intracellulare strains were found to be more heterogeneous. For these strains, we found one major RFLP-type and one subsidiary type which appears to be a sub-set of the first. We also found two further RFLP-types which contained serovars 7 and 18 respectively. We conclude from this that M. avium, M. intracellulare and M. scrofulaceum are three distinct species and that serovars belonging to the 'intermediate group' of Meissner and Anz belong to the species M. avium. Utilizing these criteria, we examined a number of isolates from the 'ambiguous' serovar 9 and found that of eight strains tested, six typed as M. avium and two typed as M. intracellulare. PMID:2907775

  16. An interlaboratory comparison of 16S rRNA gene-based terminal restriction fragment length polymorphism and sequencing methods for assessing microbial diversity of seafloor basalts

    PubMed Central

    Orcutt, Beth; Bailey, Brad; Staudigel, Hubert; Tebo, Bradley M; Edwards, Katrina J

    2009-01-01

    We present an interlaboratory comparison between full-length 16S rRNA gene sequence analysis and terminal restriction fragment length polymorphism (TRFLP) for microbial communities hosted on seafloor basaltic lavas, with the goal of evaluating how similarly these two different DNA-based methods used in two independent labs would estimate the microbial diversity of the same basalt samples. Two samples were selected for these analyses based on differences detected in the overall levels of microbial diversity between them. Richness estimators indicate that TRFLP analysis significantly underestimates the richness of the relatively high-diversity seafloor basalt microbial community: at least 50% of species from the high-diversity site are missed by TRFLP. However, both methods reveal similar dominant species from the samples, and they predict similar levels of relative diversity between the two samples. Importantly, these results suggest that DNA-extraction or PCR-related bias between the two laboratories is minimal. We conclude that TRFLP may be useful for relative comparisons of diversity between basalt samples, for identifying dominant species, and for estimating the richness and evenness of low-diversity, skewed populations of seafloor basalt microbial communities, but that TRFLP may miss a majority of species in relatively highly diverse samples. PMID:19508561

  17. Microscopic Varicocelectomy Significantly Decreases the Sperm DNA Fragmentation Index in Patients with Infertility

    PubMed Central

    Kadioglu, Teoman Cem; Aliyev, Emin; Celtik, Murad

    2014-01-01

    Background. Varicocele is associated with high levels of DNA damage in spermatozoa due to oxidative stress and elevated levels of sperm DNA fragmentation, which has been currently proposed to be an essential additional diagnostic test to be recommended for patients with clinical varicocele. The aim of this study was to evaluate the parameters of semen and the DNA fragmentation index (DFI) in patients with varicocele before and after varicocelectomy. Methods. The details of 92 consecutive patients were retrospectively analyzed from January 2010 to December 2012. The sperm samples were evaluated according to the World Health Organization Guidelines. Sperm DNA damage, characterized as DFI, was evaluated by sperm chromatin structure assay using flow cytometry. Results. There was a statistically significant improvement in the semen concentration, the total motile count, the total normal sperm count, and the sperm DNA fragmentation index (DFI; the percentage of sperm with denatured DNA) after varicocelectomy. There was a large decrease in DFI from a preoperative mean of 42.6% to a postoperative mean of 20.5% (P < 0.001). A higher preoperative DFI was associated with a larger decrease in postoperative DFI, and significant negative correlations were observed between the DFI and sperm motility (r = ?0.42, P < 0.01). Conclusion. Our data suggest that varicocelectomy can improve multiple semen parameters and sperm DNA damage in infertile men with varicocele. The patients with preoperative defects in those parameters showed greater improvement postoperatively. Further research in this area is needed to understand the exact mechanisms of DNA damage in infertile men with varicocele. PMID:24712000

  18. Comparative typing of Pseudomonas aeruginosa by random amplification of polymorphic DNA or pulsed-field gel electrophoresis of DNA macrorestriction fragments.

    PubMed Central

    Renders, N; Römling, Y; Verbrugh, H; van Belkum, A

    1996-01-01

    Eighty-seven strains of Pseudomonas aeruginosa were typed by random amplification of polymorphic DNA (RAPD) and pulsed-field gel electrophoresis (PFGE) of macrorestriction fragments. Stains were clustered on the basis of interpretative criteria as presented previously for the PFGE analysis. Clusters of strains were also defined on the basis of epidemiological data and subsequently reanalyzed by RAPD. It was found that in an RAPD assay employing the enterobacterial repetitive intergenic consensus sequence ERIC2 as a primer, single band differences can be ignored; in this case, clonally related strains could be grouped as effectively and reliably as with PFGE. These data could be corroborated by the use of other primer species. However, some primers either showed reduced resolution or, in contrast, identified DNA polymorphisms beyond epidemiologically and PFGE-defined limits. Apparently, different primers define different windows of genetic variation. It is suggested that criteria for interpretation of the ERIC2 PCR fingerprints can be simple and straightforward: when single band differences are ignored, RAPD-determined grouping of P. aeruginosa is congruent with that obtained by PFGE. Consequently, this implies that RAPD can be used with trust as a first screen in epidemiological characterization of P. aeruginosa. The ability to measure the rate of molecular evolution of the P. aeruginosa genome clearly depends on the choice of restriction enzyme or primer when RAPD or PFGE, respectively, is applied for the detection of DNA polymorphisms. PMID:8940470

  19. DNA Detectives

    NSDL National Science Digital Library

    BEGIN:VCARD VERSION:2.1 FN:Suzanne Black N:Black; Suzanne ORG:Inglemoor High School REV:2005-04-09 END:VCARD

    1995-06-30

    Many of the revolutionary changes that have occurred in biology since 1970 can be attributed directly to the ability to manipulate DNA in defined ways. The principal tools for this recombinant DNA technology are enzymes that can "cut and "paste" DNA. Restriction enzymes are the "chemical scissors" of the molecular biologist; these enzymes cut DNA at specific nucleotide sequences. A sample of someone's DNA, incubated with restriction enzymes, is reduced to millions of DNA fragments of varying sizes. A DNA sample from a different person would have a different nucleotide sequence and would thus be enzymatically "chopped up" into a very different collection of fragments. We have been asked to apply DNA fingerprinting to determine which suspect should be charged with a crime perpetrated in our city.

  20. Morphological and polymerase chain reaction-restriction fragment lenght polymorphism characterization of Biomphalaria kuhniana and Biomphalaria amazonica from Colombia.

    PubMed

    Velasquez, Luz E; Caldeira, Roberta L; Estrada, Victoria; Carvalho, Omar S

    2002-10-01

    In Colombia, five Biomphalaria planorbid species are known: B. kuhniana, B. straminea, B. peregrina, B. canonica and B. oligoza(var. B. philippiana). Among them, B. straminea is intermediate host of Schistosoma mansoni and B. peregrina has been found to be experimentally susceptible to this parasite. B. straminea is commonly confused with B. kuhniana and they have been clustered together with B. intermedia in the complex named B. straminea. The difficulties involved in the specific identification, based on morphological data, have motivated the use of new techniques as auxiliary tools in cases of inconclusive morphological identification of such planorbid. In the present study, five Biomphalaria populations from the Colombian Amazon region and from Interandian Valleys were morphologically identified and characterized by polymerase chain reaction-restriction fragment lenght polymorphism directed at the internal transcribed spacer region of the rRNA gene, followed by digestion of the generated fragment with restriction enzymes (DdeI, AluI, RsaI, MvaI and HaeIII). Known profiles of the Brazilian species B. straminea, B. peregrina, B. kuhniana, B. intermedia and B. amazonica, besides B. kuhniana from Colombia, were used for comparison. The five populations under study were morphologically and molecularly identified as B. kuhniana and B. amazonica. PMID:12471427

  1. Modelization of DNA fragmentation induced in human fibroblasts by Fe-56 ions

    NASA Astrophysics Data System (ADS)

    Ballarini, F.; Belli, M.; Campa, A.; Esposito, G.; Friedland, W.; Ottolenghi, A.; Paretzke, H.

    DNA double-strand breaks DSB are widely recognized as cellular critical lesions in the pathways leading from initial energy deposition by radiation to the formation of relevant biological endpoints such as gene mutations chromosome aberrations and cell death Chromatin conformation and radiation track structure are expected to have a strong influence on the spatial modulation of DSB induction at the scale of the nucleosome i e 100 base pairs bp and of the low-level chromatin fiber organization i e 1 kbp At larger scales the DNA fragmentation pattern induced by sparsely ionizing radiation approaches a scenario resulting from a random distribution of DSB However the pattern induced by high-LET irradiation can lead to deviation from randomness also at these scales This feature can have important biological consequences since spatial correlation of DSB is thought to affect their reparability Therefore studies on fragment size distributions induced by radiations of various qualities can help to link the physical characteristics of radiation with the cellular endpoints This is an important issue for understanding the main mechanisms of cell damage induced by HZE particles In this work we have compared the pattern of DNA fragmentation in the range 1-5700 kbp induced in human fibroblasts by gamma -rays with that induced by high-energy Fe-ions which have biological significance for radiation protection issues during long term astronauts travels The study has taken into account the comparison of the experimental fragmentation spectra

  2. Site-dependent cleavage of pBR322 DNA by restriction endonuclease HinfI.

    PubMed Central

    Armstrong, K A; Bauer, W R

    1983-01-01

    Cleavage of pBR322 DNA I by the restriction endonuclease HinfI is preferentially inhibited at specific HinfI cleavage sites. These sites in pBR322 DNA I have been identified and ordered with respect to the frequency with which they are cleaved. The HinfI site most resistant to cleavage in pBR322 DNA I is unique in that runs of G-C base pairs are immediately adjacent on both sites. Two differently permuted linear (DNA III) species were produced by cleavage with two different restriction endonucleases, PstI and AvaI. Only one of these linear molecules, that produced by PstI, exhibits the same preferential cleavage pattern as DNA I. The second linear species, that arising from AvaI digestion, shows pronounced relative inhibition of cleavage at the HinfI sites nearest the ends of the molecule (100 to 120 base pairs away, respectively). This result suggest that proximity to the termini of a linear DNA molecule might also influence preferential cleavage. The possibility of formation of stem-loop structures does not appear to influence preferential cleavage by HinfI. Images PMID:6306575

  3. Comparison of JC and BK Human Papovaviruses with Simian Virus 40: Restriction Endonuclease Digestion and Gel Electrophoresis of Resultant Fragments

    PubMed Central

    Osborn, June E.; Robertson, Sylvia M.; Padgett, Billie L.; Zu Rhein, Gabrielle M.; Walker, Duard L.; Weisblum, Bernard

    1974-01-01

    JC virus was found to have a buoyant density of 1.20 g/cm3 in linear sucrose-D2O and 1.35 g/cm3 in cesium chloride isopycnic gradients. DNA extracted either from JC-infected cultures or from gradient-purified virions occupied a dense position relative to linear DNA in cesium chloride/ethidium bromide gradients, and the circular configuration of the extracted DNA was confirmed by electron microscopy, with a measured molecular weight of 2.93 × 106. DNA from BK virus was similarly prepared and compared to JC and to an SV40 DNA standard by digestion with restriction endonuclease preparations from Haemophilus influenzae, Haemophilus parainfluenzae, and Escherichia coli. Digests were electrophoretically analyzed on gradient polyacrylamide slab gels or agarose gels, and the three viruses were found to have distinctly different cleavage patterns by this form of analysis: JC and BK viruses were almost entirely different from SV40 and significantly different from each other. Thus, JC and BK human papovaviruses appear to be discrete new members of the papovavirus group, rather than SV40 variants. Images PMID:4362864

  4. Modulating mtDNA heteroplasmy by mitochondria-targeted restriction endonucleases in a ‘differential multiple cleavage-site’ model

    PubMed Central

    Bacman, SR; Williams, SL; Hernandez, D; Moraes, CT

    2009-01-01

    The ability to manipulate mitochondrial DNA (mtDNA) heteroplasmy would provide a powerful tool to treat mitochondrial diseases. Recent studies showed that mitochondria-targeted restriction endonucleases can modify mtDNA heteroplasmy in a predictable and efficient manner if it recognizes a single site in the mutant mtDNA. However, the applicability of such model is limited to mutations that create a novel cleavage site, not present in the wild-type mtDNA. We attempted to extend this approach to a ‘differential multiple cleavage site’ model, where an mtDNA mutation creates an extra restriction site to the ones normally present in the wild-type mtDNA. Taking advantage of a heteroplasmic mouse model harboring two haplotypes of mtDNA (NZB/BALB) and using adenovirus as a gene vector, we delivered a mitochondria-targeted Scal restriction endonuclease to different mouse tissues. Scal recognizes five sites in the NZB mtDNA but only three in BALB mtDNA. Our results showed that changes in mtDNA heteroplasmy were obtained by the expression of mitochondria-targeted ScaI in both liver, after intravenous injection, and in skeletal muscle, after intramuscular injection. Although mtDNA depletion was an undesirable side effect, our data suggest that under a regulated expression system, mtDNA depletion could be minimized and restriction endonucleases recognizing multiple sites could have a potential for therapeutic use. PMID:17597792

  5. [Level of DNA fragmentation in human sperm cells in varicocele and prostatitis].

    PubMed

    Osadchuk, L V; Erkovich, A A; Tataru, D A; Markova, E V; Svetlakov, A V

    2014-01-01

    Varicocele and prostatitis are the most common andrological diseases, which may be accompanied by a decrease in the production of sperm cells, the deterioration of their quality and increased risk of infertility. This work was aimed to the evaluation of sperm DNA fragmentation index (DFI) and main indices of sperm fertility (concentration, motility and morphology), and the relationship between these parameters in the men of active reproductive age suffering from prostatitis or varicocele. Assessment of sperm DNA fragmentation was performed by SCSA (sperm chromatin structure assay) using flow cytometry; sperm parameters were evaluated according to WHO recommendations. It was shown that men with prostatitis (n = 9) and varicocele (n = 22) had significantly higher DFI compared with men in the control group (n = 22). Negative influence of these diseases on the concentration and the percentage of motile sperm cells in the ejaculate was revealed. These data suggest that the deterioration in the quality of semen in varicocele and prostatitis may be caused not only by pathospermia, but also, at least partially, by violation of the integrity of the sperm DNA. Evaluation of sperm DNA fragmentation can be recommended for use in laboratory diagnostics for prediction of fertility in infertile men. PMID:25211925

  6. Chromosome loss caused by DNA fragmentation induced in main nuclei and micronuclei of human lymphoblastoid cells treated with colcemid.

    PubMed

    Yamamoto, Mika; Wakata, Akihiro; Aoki, Yoshinobu; Miyamae, Yoichi; Kodama, Seiji

    2014-04-01

    Aneuploidy, a change in the number of chromosomes, plays an essential role in tumorigenesis. Our previous study demonstrated that a loss of a whole chromosome is induced in human lymphocytes by colcemid, a well-known aneugen. Here, to clarify the mechanism for colcemid-induced chromosome loss, we investigated the relationship between chromosome loss and DNA fragmentation in human lymphoblastoid cells treated with colcemid (an aneugen) compared with methyl methanesulfonate (MMS; a clastogen). We analyzed the number of fluorescence in situ hybridization (FISH) signals targeted for a whole chromosome 2 in cytokinesis-blocked binucleated TK6 cells and WTK-1 cells treated with colcemid and MMS, and concurrently detected DNA fragmentation by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Results revealed that DNA fragmentation occurred in 60% of all binucleated TK6 cells harboring colcemid-induced chromosome loss (30% of micronuclei and 30% of main nuclei). DNA fragmentation was observed in colcemid-induced micronuclei containing a whole chromosome but not in MMS-induced micronuclei containing chromosome fragments. In contrast, colcemid-induced nondisjunction had no effect on induction of DNA fragmentation, suggesting that DNA fragmentation was triggered by micronuclei containing a whole chromosome but not by micronuclei containing chromosome fragments or nondisjunction. In addition, the frequency of binucleated cells harboring chromosome loss with DNA fragmentation in micronuclei or main nuclei was higher in wild-type p53 TK6 cells than in mutated-p53 WTK-1 cells treated with colcemid. Taken together, these present and previous results suggest that colcemid-induced chromosome loss is caused by DNA fragmentation, which is triggered by a micronucleus with a whole chromosome and controlled by the p53-dependent pathway. PMID:24582839

  7. Assessment of the degree and the type of restriction fragment length polymorphism in barley ( Hordeum vulgare )

    Microsoft Academic Search

    A. Graner; H. Siedler; A. Jahoor; R. G. Herrmann; G. Wenzel

    1990-01-01

    In order to determine the extent of polymorphism in barley (Hordeum vulgare), DNA from 48 varieties was analyzed with 23 genomic, single-copy probes, distributed across all seven chromosomes. Upon hybridization to wheat-barley addition lines, the probes showed different degrees of homology compared to the wheat genome. Polymorphisms were detected in the barley genome at a frequency of 43% after digestion

  8. Overcoming restriction as a barrier to DNA transformation in Caldicellulosiruptor species results in efficient marker replacement

    PubMed Central

    2013-01-01

    Background Thermophilic microorganisms have special advantages for the conversion of plant biomass to fuels and chemicals. Members of the genus Caldicellulosiruptor are the most thermophilic cellulolytic bacteria known. They have the ability to grow on a variety of non-pretreated biomass substrates at or near ~80°C and hold promise for converting biomass to bioproducts in a single step. As for all such relatively uncharacterized organisms with desirable traits, the ability to genetically manipulate them is a prerequisite for making them useful. Metabolic engineering of pathways for product synthesis is relatively simple compared to engineering the ability to utilize non-pretreated biomass. Results Here we report the construction of a deletion of cbeI (Cbes2438), which encodes a restriction endonuclease that is as a major barrier to DNA transformation of C. bescii. This is the first example of a targeted chromosomal deletion generated by homologous recombination in this genus and the resulting mutant, JWCB018 (?pyrFA ?cbeI), is readily transformed by DNA isolated from E. coli without in vitro methylation. PCR amplification and sequencing suggested that this deletion left the adjacent methyltransferase (Cbes2437) intact. This was confirmed by the fact that DNA isolated from JWCB018 was protected from digestion by CbeI and HaeIII. Plasmid DNA isolated from C. hydrothermalis transformants were readily transformed into C. bescii. Digestion analysis of chromosomal DNA isolated from seven Caldicellulosiruptor species by using nine different restriction endonucleases was also performed to identify the functional restriction-modification activities in this genus. Conclusion Deletion of the cbeI gene removes a substantial barrier to routine DNA transformation and chromosomal modification of C. bescii. This will facilitate the functional analyses of genes as well as metabolic engineering for the production of biofuels and bioproducts from biomass. An analysis of restriction-modification activities in members of this genus suggests a way forward to eliminating restriction as a barrier to DNA transformation and efficient genetic manipulation of this important group of hyperthermophiles. PMID:23714229

  9. Multiple components in restriction enzyme digests of mammalian (insectivore), avian and reptilian genomic DNA hybridize with murine immunoglobulin VH probes.

    PubMed Central

    Litman, G W; Berger, L; Jahn, C L

    1982-01-01

    High molecular weight genomic DNAs isolated from an insectivore, Tupaia, and a representative reptilian, Caiman, and avian, Gallus, were digested with restriction endonucleases transferred to nitrocellulose and hybridized with nick-translated probes of murine VH genes. The derivations of the probes designated S107V (1) and mu 107V (2,3) have been described previously. Under conditions of reduced stringency, multiple hybridizing components were observed with Tupaia and Caiman; only mu mu 107V exhibited significant hybridization with the separated fragments of Gallus DNA. The nick-translated S107V probe was digested with Fnu4H1 and subinserts corresponding to the 5' and 3' regions both detected multiple hybridizing components in Tupaia and Caiman DNA. A 5' probe lacking the leader sequence identified the same components as the intact 5' probe, suggesting that VH coding regions distant as the reptilians may possess multiple genetic components which exhibit significant homology with murine immunoglobulin in VH regions. Images PMID:6285298

  10. DNA fragmentation in human fibroblasts under extremely low frequency electromagnetic field exposure.

    PubMed

    Focke, Frauke; Schuermann, David; Kuster, Niels; Schär, Primo

    2010-01-01

    Extremely low frequency electromagnetic fields (ELF-EMFs) were reported to affect DNA integrity in human cells with evidence based on the Comet assay. These findings were heavily debated for two main reasons; the lack of reproducibility, and the absence of a plausible scientific rationale for how EMFs could damage DNA. Starting out from a replication of the relevant experiments, we performed this study to clarify the existence and explore origin and nature of ELF-EMF induced DNA effects. Our data confirm that intermittent (but not continuous) exposure of human primary fibroblasts to a 50 Hz EMF at a flux density of 1 mT induces a slight but significant increase of DNA fragmentation in the Comet assay, and we provide first evidence for this to be caused by the magnetic rather than the electric field. Moreover, we show that EMF-induced responses in the Comet assay are dependent on cell proliferation, suggesting that processes of DNA replication rather than the DNA itself may be affected. Consistently, the Comet effects correlated with a reduction of actively replicating cells and a concomitant increase of apoptotic cells in exposed cultures, whereas a combined Fpg-Comet test failed to produce evidence for a notable contribution of oxidative DNA base damage. Hence, ELF-EMF induced effects in the Comet assay are reproducible under specific conditions and can be explained by minor disturbances in S-phase processes and occasional triggering of apoptosis rather than by the generation of DNA damage. PMID:19896957

  11. Three dimensional imaging of DNA fragments during electrophoresis using a confocal detector

    SciTech Connect

    Brewer, L.R.; Davidson, C.; Balch, J.; Carrano, A.

    1995-01-30

    We have measured the three dimensional distribution of DNA fragments within an electrophoretic band. The measurements were made using a confocal microscope and a photon counting photomultiplier detector. A DNA sequencing standard was loaded into glass microchannel plates containing polyacrylamide gel. The measurements were made by scanning the plates in three dimensions using a mechanical stage under computer control, while electrophoresis was taking place. We found that the distribution of DNA was the same for all the bands measured in the sequencing ladder with an approximate Gaussian distribution along all three axes. These measurements are important to understand what physical forces shape electrophoretic bands confined by a channel and also to aid in the design of high throughput DNA sequencers.

  12. DNA fragmentation induced by Fe ions in human cells: shielding influence on spatially correlated damage

    NASA Technical Reports Server (NTRS)

    Antonelli, F.; Belli, M.; Campa, A.; Chatterjee, A.; Dini, V.; Esposito, G.; Rydberg, B.; Simone, G.; Tabocchini, M. A.

    2004-01-01

    Outside the magnetic field of the Earth, high energy heavy ions constitute a relevant part of the biologically significant dose to astronauts during the very long travels through space. The typical pattern of energy deposition in the matter by heavy ions on the microscopic scale is believed to produce spatially correlated damage in the DNA which is critical for radiobiological effects. We have investigated the influence of a lucite shielding on the initial production of very small DNA fragments in human fibroblasts irradiated with 1 GeV/u iron (Fe) ions. We also used gamma rays as reference radiation. Our results show: (1) a lower effect per incident ion when the shielding is used; (2) an higher DNA Double Strand Breaks (DSB) induction by Fe ions than by gamma rays in the size range 1-23 kbp; (3) a non-random DNA DSB induction by Fe ions. c2004 COSPAR. Published by Elsevier Ltd. All rights reserved.

  13. DNA fragmentation induced by Fe ions in human cells: shielding influence on spatially correlated damage

    NASA Astrophysics Data System (ADS)

    Antonelli, F.; Belli, M.; Campa, A.; Chatterjee, A.; Dini, V.; Esposito, G.; Rydberg, B.; Simone, G.; Tabocchini, M. A.

    2004-01-01

    Outside the magnetic field of the Earth, high energy heavy ions constitute a relevant part of the biologically significant dose to astronauts during the very long travels through space. The typical pattern of energy deposition in the matter by heavy ions on the microscopic scale is believed to produce spatially correlated damage in the DNA which is critical for radiobiological effects. We have investigated the influence of a lucite shielding on the initial production of very small DNA fragments in human fibroblasts irradiated with 1 GeV/u iron (Fe) ions. We also used ?-rays as reference radiation. Our results show: (1) a lower effect per incident ion when the shielding is used; (2) an higher DNA Double Strand Breaks (DSB) induction by Fe ions than by ?-rays in the size range 1-23 kbp; (3) a non-random DNA DSB induction by Fe ions.

  14. Structure and mutagenesis of the DNA modification-dependent restriction endonuclease AspBHI

    PubMed Central

    Horton, John R.; Nugent, Rebecca L.; Li, Andrew; Mabuchi, Megumu Yamada; Fomenkov, Alexey; Cohen-Karni, Devora; Griggs, Rose M.; Zhang, Xing; Wilson, Geoffrey G.; Zheng, Yu; Xu, Shuang-yong; Cheng, Xiaodong

    2014-01-01

    The modification-dependent restriction endonuclease AspBHI recognizes 5-methylcytosine (5mC) in the double-strand DNA sequence context of (C/T)(C/G)(5mC)N(C/G) (N = any nucleotide) and cleaves the two strands a fixed distance (N12/N16) 3? to the modified cytosine. We determined the crystal structure of the homo-tetrameric AspBHI. Each subunit of the protein comprises two domains: an N-terminal DNA-recognition domain and a C-terminal DNA cleavage domain. The N-terminal domain is structurally similar to the eukaryotic SET and RING-associated (SRA) domain, which is known to bind to a hemi-methylated CpG dinucleotide. The C-terminal domain is structurally similar to classic Type II restriction enzymes and contains the endonuclease catalytic-site motif of DX20EAK. To understand how specific amino acids affect AspBHI recognition preference, we generated a homology model of the AspBHI-DNA complex, and probed the importance of individual amino acids by mutagenesis. Ser41 and Arg42 are predicted to be located in the DNA minor groove 5? to the modified cytosine. Substitution of Ser41 with alanine (S41A) and cysteine (S41C) resulted in mutants with altered cleavage activity. All 19 Arg42 variants resulted in loss of endonuclease activity. PMID:24604015

  15. Chromosomal assignment of human genomic NotI restriction fragments in a two-dimensional electrophoresis profile

    SciTech Connect

    Yoshikawa, Hirohide; Nagai, Hisaki; Matsubara, Kenichi [Osaka Univ. (Japan)] [and others] [Osaka Univ. (Japan); and others

    1996-01-01

    Using DNA from sorted human chromosomes and two-dimensional gel electrophoresis, we assigned 2295 NotI sites, 43% of the total, to specific chromosomes and designated the procedure CA-RLGS (chromosome-assigned restriction landmark genomic scanning). Although the NotI enzyme is sensitive to DNA methylation, our results suggested that the majority of the spots did not seem to be affected by this modification. The NotI sites were distributed at higher levels in chromosomes 17, 19, and 22, suggesting higher gene content in these chromosomes. Most spots were assigned to unique chromosomes, but some spots were found on two or more chromosomes. Quantitative analysis revealed the intensity of the DNA spots on the sex chromosomes to be haploid and that of the chromosome 21 spots in DNA from a male with Down syndrome to be trisomic, although there were exceptions. We report here the first-generation CA-RLGS map of the human genome. 23 refs., 4 figs.

  16. Ejaculate Oxidative Stress Is Related with Sperm DNA Fragmentation and Round Cells

    PubMed Central

    Iommiello, Valeria Maria; Albani, Elena; Di Rosa, Alessandra; Marras, Alessandra; Menduni, Francesca; Morreale, Giovanna; Levi, Shanti Lia; Pisano, Benedetta; Levi-Setti, Paolo Emanuele

    2015-01-01

    Oxidative stress (OS) plays an essential role in male infertility aetiology by affecting sperm quality, function, and also the integrity of sperm DNA. The assessment of oxidative stress in semen may be an important tool to improve the evaluation of sperm reproductive capacity. The purpose of this study was the evaluation of any possible relation between the unbalance of oxidative stress caused by superoxide anion in the ejaculate with the presence of sperm DNA fragmentation and high concentration of round cells. 56 semen samples from males from couples suffering from infertility were evaluated according to World Health Organisation (WHO) 2010 guidelines. Oxidative stress levels from N1 (low) to N4 (high) were assessed in ejaculates using oxiSperm; DFI (sperm DNA fragmentation index) as assessed by the SCSA (Sperm Chromatin Structure Assay) was used for evaluation of sperm chromatin integrity. Our data show that high oxidative stress (N3-N4 levels) correlated positively with a DFI ? 30% (P = 0.0379) and round cells ?1.500.000/mL (P = 0.0084). In conclusion, OS increases sperm DNA damage. Thus evaluation of semen OS extent of sperm DNA damage in infertile man could be useful to develop new therapeutic strategies and improve success of assisted reproduction techniques (ART). PMID:25802519

  17. Ejaculate oxidative stress is related with sperm DNA fragmentation and round cells.

    PubMed

    Iommiello, Valeria Maria; Albani, Elena; Di Rosa, Alessandra; Marras, Alessandra; Menduni, Francesca; Morreale, Giovanna; Levi, Shanti Lia; Pisano, Benedetta; Levi-Setti, Paolo Emanuele

    2015-01-01

    Oxidative stress (OS) plays an essential role in male infertility aetiology by affecting sperm quality, function, and also the integrity of sperm DNA. The assessment of oxidative stress in semen may be an important tool to improve the evaluation of sperm reproductive capacity. The purpose of this study was the evaluation of any possible relation between the unbalance of oxidative stress caused by superoxide anion in the ejaculate with the presence of sperm DNA fragmentation and high concentration of round cells. 56 semen samples from males from couples suffering from infertility were evaluated according to World Health Organisation (WHO) 2010 guidelines. Oxidative stress levels from N1 (low) to N4 (high) were assessed in ejaculates using oxiSperm; DFI (sperm DNA fragmentation index) as assessed by the SCSA (Sperm Chromatin Structure Assay) was used for evaluation of sperm chromatin integrity. Our data show that high oxidative stress (N3-N4 levels) correlated positively with a DFI ? 30% (P = 0.0379) and round cells ?1.500.000/mL (P = 0.0084). In conclusion, OS increases sperm DNA damage. Thus evaluation of semen OS extent of sperm DNA damage in infertile man could be useful to develop new therapeutic strategies and improve success of assisted reproduction techniques (ART). PMID:25802519

  18. Improved Assessment of Denitrifying, N2-Fixing, and Total-Community Bacteria by Terminal Restriction Fragment Length Polymorphism Analysis Using Multiple Restriction Enzymes

    PubMed Central

    Rösch, Christopher; Bothe, Hermann

    2005-01-01

    A database of terminal restriction fragments (tRFs) of the 16S rRNA gene was set up utilizing 13 restriction enzymes and 17,327 GenBank sequences. A computer program, termed TReFID, was developed to allow identification of any of these 17,327 sequences by means of polygons generated from the specific tRFs of each bacterium. The TReFID program complements and exceeds in its data content the Web-based phylogenetic assignment tool recently described by A. D. Kent, D. J. Smith, B. J. Benson, and E. W. Triplett (Appl. Environ. Microb. 69:6768-6766, 2003). The method to identify bacteria is different, as is the region of the 16S rRNA gene employed in the present program. For the present communication the software of the tRF profiles has also been extended to allow screening for genes coding for N2 fixation (nifH) and denitrification (nosZ) in any bacterium or environmental sample. A number of controls were performed to test the reliability of the TReFID program. Furthermore, the TReFID program has been shown to permit the analysis of the bacterial population structure of bacteria by means of their 16S rRNA, nifH, and nosZ gene content in an environmental habitat, as exemplified for a sample from a forest soil. The use of the TReFID program reveals that noncultured denitrifying and dinitrogen-fixing bacteria might play a more dominant role in soils than believed hitherto. PMID:15812035

  19. Identification of the Bacterial Community of Maple Sap by Using Amplified Ribosomal DNA (rDNA) Restriction Analysis and rDNA Sequencing

    Microsoft Academic Search

    L. Lagace; M. Pitre; M. Jacques; D. Roy

    2004-01-01

    The bacterial community of maple sap was characterized by analysis of samples obtained at the taphole of maple trees for the 2001 and 2002 seasons. Among the 190 bacterial isolates, 32 groups were formed according to the similarity of the banding patterns obtained by amplified ribosomal DNA restriction analysis (ARDRA). A subset of representative isolates for each ARDRA group was

  20. Determinants of the Nuclear Localization of the Heterodimeric DNA Fragmentation Factor (Icad\\/Cad)

    Microsoft Academic Search

    Delphine Lechardeur; Luke Drzymala; Manu Sharma; Danuta Zylka; Robert Kinach; Joanna Pacia; Christopher Hicks; Nawaid Usmani; Johanna M. Rommens; Gergely L. Lukacs

    2000-01-01

    Programmed cell death or apoptosis leads to the activation of the caspase-activated DNase (CAD), which degrades chromosomal DNA into nucleosomal fragments. Biochemical studies revealed that CAD forms an inactive heterodimer with the inhibitor of caspase-activated DNase (ICAD), or its alternatively spliced variant, ICAD-S, in the cytoplasm. It was ini- tially proposed that proteolytic cleavage of ICAD by activated caspases causes

  1. Evidence for DNA fragmentation triggered in the self-incompatibility response in pollen of Papaver rhoeas.

    PubMed

    Jordan, N D; Franklin, F C; Franklin-Tong, V E

    2000-08-01

    Studies of the molecular and biochemical basis of self-incompatibility (SI) in Papaver rhoeas have revealed much about the signalling pathways triggered in pollen early in this response. The aim of the current investigation was to begin to study downstream events in order to elucidate some of the later cellular responses involved in the SI response and identification of the mechanisms controlling the irreversible inhibition of pollen tube growth. We have used the FragEL assay to investigate if there is any evidence for DNA fragmentation stimulated in pollen of P. rhoeas in an S-specific manner. Our data clearly demonstrate that S proteins are responsible for triggering this, specifically in incompatible, and not compatible, pollen. DNA fragmentation was first detected in incompatible pollen tubes 4 h after challenge with S proteins, and continued to increase for a further 10 h. This provides the first evidence, to our knowledge, that this phenomenon is associated with the SI response. We also demonstrate that mastoparan, which increases [Ca2+]i, also triggers DNA fragmentation in these pollen tubes, thereby implicating an involvement of Ca2+ signalling in this process. Together, our data represent a significant breakthrough in understanding of the SI response in Papaver pollen. PMID:10972873

  2. Xanthorrhizol induced DNA fragmentation in HepG2 cells involving Bcl-2 family proteins

    SciTech Connect

    Tee, Thiam-Tsui, E-mail: thiamtsu@yahoo.com [School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor (Malaysia)] [School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor (Malaysia); Cheah, Yew-Hoong [School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor (Malaysia) [School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor (Malaysia); Bioassay Unit, Herbal Medicine Research Center, Institute for Medical Research, Jalan Pahang, Kuala Lumpur (Malaysia); Meenakshii, Nallappan [Biology Department, Faculty of Science, Universiti Putra Malaysia, 43400 Serdang, Selangor (Malaysia)] [Biology Department, Faculty of Science, Universiti Putra Malaysia, 43400 Serdang, Selangor (Malaysia); Mohd Sharom, Mohd Yusof; Azimahtol Hawariah, Lope Pihie [School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor (Malaysia)] [School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor (Malaysia)

    2012-04-20

    Highlights: Black-Right-Pointing-Pointer We isolated xanthorrhizol, a sesquiterpenoid compound from Curcuma xanthorrhiza. Black-Right-Pointing-Pointer Xanthorrhizol induced apoptosis in HepG2 cells as observed using SEM. Black-Right-Pointing-Pointer Apoptosis in xanthorrhizol-treated HepG2 cells involved Bcl-2 family proteins. Black-Right-Pointing-Pointer DNA fragmentation was observed in xanthorrhizol-treated HepG2 cells. Black-Right-Pointing-Pointer DNA fragmentation maybe due to cleavage of PARP and DFF45/ICAD proteins. -- Abstract: Xanthorrhizol is a plant-derived pharmacologically active sesquiterpenoid compound isolated from Curcuma xanthorrhiza. Previously, we have reported that xanthorrhizol inhibited the proliferation of HepG2 human hepatoma cells by inducing apoptotic cell death via caspase activation. Here, we attempt to further elucidate the mode of action of xanthorrhizol. Apoptosis in xanthorrhizol-treated HepG2 cells as observed by scanning electron microscopy was accompanied by truncation of BID; reduction of both anti-apoptotic Bcl-2 and Bcl-X{sub L} expression; cleavage of PARP and DFF45/ICAD proteins and DNA fragmentation. Taken together, these results suggest xanthorrhizol as a potent antiproliferative agent on HepG2 cells by inducing apoptosis via Bcl-2 family members. Hence we proposed that xanthorrhizol could be used as an anti-liver cancer drug for future studies.

  3. Characterization of psychrotrophic bacterial communities in modified atmosphere-packed meat with terminal restriction fragment length polymorphism.

    PubMed

    Nieminen, T T; Vihavainen, E; Paloranta, A; Lehto, J; Paulin, L; Auvinen, P; Solismaa, M; Björkroth, K J

    2011-01-01

    Characterization of psychrotrophic lactic acid bacteria (LAB) and Brochothrix thermosphacta communities is needed to understand the microbial ecology of spoilage of modified atmosphere-packed (MAP) meats. To overcome the limitations of the currently used methods for the characterization of psychrotrophic bacterial communities in meat, we developed a culture-independent, 16S rRNA gene-targeted terminal restriction fragment length polymorphism (T-RFLP) method. An identification library consisting of 100 Gram-positive and 30 Gram-negative meat-associated bacterial strains was set up to identify the terminal restriction fragments derived from the communities. The taxonomic resolution level of the T-RFLP method was in between genus and species within the investigated LAB strains and within family and genus within the investigated Gram-negative strains. The established library was applied to identify the members of bacterial communities in MAP minced meat at the end of the shelf life. The T-RFLP results and plate counts on Man-Rogosa-Sharpe, Violet Red Bile Glucose, and Streptomycin sulfate thallium acetate actidione agars indicated that LAB and B. thermosphacta predominated in meat. The bacterial taxa associated with the T-RFLP results were compared to those identified among plate-grown LAB isolates by numerical ribopattern analysis. Both methods agreed that Leuconostoc spp. and Carnobacterium spp. prevailed in the LAB community in minced meat followed by Lactobacillus algidus, Lactococcus spp. and Weissella spp. Colony identification revealed that Leuconostoc gasicomitatum, L. gelidum, Carnobacterium divergens and C. maltaromaticum were the predominant LAB species. The T-RFLP results were shown to correlate with viable counts of Leuconostoc spp. and B. thermosphacta. The T-RFLP method was found to be a useful tool enabling rapid and high-throughput characterization of psychrotrophic bacteria prevailing in MAP meat. PMID:21093087

  4. Cell-free DNA Fragmentation Patterns in Amniotic Fluid Identify Genetic Abnormalities and Changes due to Storage

    PubMed Central

    Peter, Inga; Tighiouart, Hocine; Lapaire, Olav; Johnson, Kirby L.; Bianchi, Diana W.; Terrin, Norma

    2015-01-01

    Circulating cell-free DNA (cfDNA) has become a promising biomarker in prenatal diagnosis. However, despite extensive studies in different body fluids, cfDNA predictive value is uncertain owing to the confounding factors that can affect its levels, such as gestational age, maternal weight, smoking status, and medications. Residual fresh and archived amniotic fluid (AF) supernatants were obtained from gravid women (mean gestational age 17 wk) carrying euploid (N = 36) and aneuploid (N = 29) fetuses, to characterize cfDNA-fragmentation patterns with regard to aneuploidy and storage time (?80°C). AF cfDNA was characterized by the real-time quantitative polymerase chain reaction amplification of glyceraldehyde-3-phosphate dehydrogenase, gel electrophoresis, and pattern recognition of the DNA fragmentation. The distributions of cfDNA fragment lengths were compared using 6 measures that defined the locations and slopes for the first and last peaks, after elimination of the confounding variables. This method allowed for the unique classification of euploid and aneuploid cfDNA samples in AF, which had been matched for storage time. In addition, we showed that archived euploid AF samples gradually lose long cfDNA fragments: this loss accurately distinguishes them from the fresh samples. We present preliminary data using cfDNA-fragmentation patterns, to uniquely distinguish between AF samples of pregnant women with regard to aneuploidy and storage time, independent of gestational age and initial DNA amount. In addition to potential applications in prenatal diagnosis, these data suggest that archived AF samples consist of large amounts of short cfDNA fragments, which are undetectable using standard real-time polymerase chain reaction amplification. PMID:18382362

  5. A Mimicking-of-DNA-Methylation-Patterns Pipeline for Overcoming the Restriction Barrier of Bacteria

    PubMed Central

    Zhang, Guoqiang; Wang, Wenzhao; Deng, Aihua; Sun, Zhaopeng; Zhang, Yun; Liang, Yong; Che, Yongsheng; Wen, Tingyi

    2012-01-01

    Genetic transformation of bacteria harboring multiple Restriction-Modification (R-M) systems is often difficult using conventional methods. Here, we describe a mimicking-of-DNA-methylation-patterns (MoDMP) pipeline to address this problem in three difficult-to-transform bacterial strains. Twenty-four putative DNA methyltransferases (MTases) from these difficult-to-transform strains were cloned and expressed in an Escherichia coli strain lacking all of the known R-M systems and orphan MTases. Thirteen of these MTases exhibited DNA modification activity in Southwestern dot blot or Liquid Chromatography–Mass Spectrometry (LC–MS) assays. The active MTase genes were assembled into three operons using the Saccharomyces cerevisiae DNA assembler and were co-expressed in the E. coli strain lacking known R-M systems and orphan MTases. Thereafter, results from the dot blot and restriction enzyme digestion assays indicated that the DNA methylation patterns of the difficult-to-transform strains are mimicked in these E. coli hosts. The transformation of the Gram-positive Bacillus amyloliquefaciens TA208 and B. cereus ATCC 10987 strains with the shuttle plasmids prepared from MoDMP hosts showed increased efficiencies (up to four orders of magnitude) compared to those using the plasmids prepared from the E. coli strain lacking known R-M systems and orphan MTases or its parental strain. Additionally, the gene coding for uracil phosphoribosyltransferase (upp) was directly inactivated using non-replicative plasmids prepared from the MoDMP host in B. amyloliquefaciens TA208. Moreover, the Gram-negative chemoautotrophic Nitrobacter hamburgensis strain X14 was transformed and expressed Green Fluorescent Protein (GFP). Finally, the sequence specificities of active MTases were identified by restriction enzyme digestion, making the MoDMP system potentially useful for other strains. The effectiveness of the MoDMP pipeline in different bacterial groups suggests a universal potential. This pipeline could facilitate the functional genomics of the strains that are difficult to transform. PMID:23028379

  6. Optimized Quantification of Fragmented, Free Circulating DNA in Human Blood Plasma Using a Calibrated Duplex Real-Time PCR

    Microsoft Academic Search

    Martin Horlitz; Annabelle Lucas; Markus Sprenger-Haussels; Jörg Hoheisel

    2009-01-01

    BackgroundDuplex real-time PCR assays have been widely used to determine amounts and concentrations of free circulating DNA in human blood plasma samples. Circulatory plasma DNA is highly fragmented and hence a PCR-based determination of DNA concentration may be affected by the limited availability of full-length targets in the DNA sample. This leads to inaccuracies when counting PCR target copy numbers

  7. PCR-restriction fragment length polymorphism analysis of the ospC gene for detection of mixed culture and for epidemiological typing of Borrelia burgdorferi sensu stricto.

    PubMed Central

    Masuzawa, T; Komikado, T; Yanagihara, Y

    1997-01-01

    Restriction fragment length polymorphism (RFLP) analysis of the outer surface protein C (ospC) gene amplicon was used for rapid screening for genetic variability within Borrelia burgdorferi sensu stricto species and for detection of multiple borreliae in culture. Primers for the ospC gene amplified a fragment of about 600 bp from Borrelia cultures. After cleavage of the amplified products by MboI and DraI, eight different RFLP types were found among 13 B. burgdorferi sensu stricto strains from various sources and geographical areas, and three RFLP types were found among 10 representative isolates from skin biopsy specimens taken from patients residing on the eastern end of Long Island, New York (B. W. Berger, R. C. Johnson, C. Kodner, and L. Coleman, J. Clin. Microbiol. 30:359-361, 1992). These results suggested that the DNA organization of B. burgdorferi sensu stricto is heterogeneous not only globally but also within a localized geographical area and that the ospC-based typing approach could differentiate the B. burgdorferi sensu stricto. From the results obtained using mixed cultures of two different RFLP types of B. burgdorferi sensu stricto, contamination of at least 0.5% of different types of Borrelia cells in culture could be detected. This method could detect a multiple-B. burgdorferi sensu stricto infection in the bladders of mice experimentally infected with two different RFLP type strains. The present study showed that RFLP analysis of ospC-PCR products is a reliable method for epidemiological typing of B. burgdorferi sensu stricto and could be used for rapid detection of mixed Borrelia culture and multiple B. burgdorferi sensu stricto infections in animals, ticks, and patients. PMID:9008282

  8. Restriction fragment length polymorphisms of chromosome 10S in maize (Zea mays L.) linked to resistance to Southern rust (Puccinia polysora) 

    E-print Network

    Blakey, Cynthia Ann

    1989-01-01

    RESIRICTION FRAGMENT LENGTH POLYMORPHISMS OF CHROMOSOME 10S IN MAIZE (Zea mays I ) LINKED TO RESISTANCE TO SOVFHERN RUSI' (Puccini a polysora) A Thesis CYNTHIA ANN BLAKEY Submitted to the OKce of Graduate Studies of Texas ARM University... in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE December 1989 Major Subject: Genetics RESTRICTION FRAGMENT LENGTH POLYMORPHISMS OF CHROMOSOME 10S IN MAIZE (Zea mays L. ) LINKED TO RESISTANCE TO SOUTHERN RUST (Puccini a...

  9. DNA fragmentation induced by Fe ions in human cell: shielding influence on spatially correlated damage

    NASA Astrophysics Data System (ADS)

    Antonelli, F.; Belli, M.; Chatterjee, A.; Esposito, G.; Rydberg, B.; Simone, G.

    Outside the magnetic field of the Earth, high energy heavy ions (HZE particles) constitute a relevant part of the biologically significant dose to astronauts during the very long travels through the space. For heavy ions the primary ionization sites occur in a correlated manner along the track of the particles and their typical pattern of energy deposition on the microscopic scale is expected to have an important role in their effects on living cells. It has been pointed out that multiple Double Strand Breaks (DSB) can be produced in local proximity by the same particle track, creating a small region of clustered damage. We have investigated the influence of the shielding on the biological effects of heavy ions, studying the initial production of very small DNA fragments in human fibroblasts irradiated with iron ions. Theoretical studies have shown that materials rich in hydrogen, such as polymethylmethacrylate (PMMA), could be more suitable in reducing the radiation risk. This is due mainly to a lower production of secondary neutrons and target fragments in hydrogen-rich materials compared to aluminium, which is the current shield used to protect astronauts. We have measured the size distribution of DNA fragments induced by high-energy Fe ions over a range from 1 kbp to 23 kpb that are produced by DSB occured over distances comparable with the chromatin fiber dimensions. 1 GeV/u Fe ion beams were obtained from the Alternating Gradient Synchrotron at the Brookhaven National Laboratory and irradiations were performed without and with a 190 mm thick PMMA shielding. Plateau phase AG1522 cells were irradiated in agarose plugs with doses up to 600 Gy and DSB induction was determined by DNA fragmentation analysis after Pulsed/Constant Field Gel Electrophoresis. The results until now obtained show that the number of DSB increases linearly either when plotted versus fluence either versus dose. The fragment distribution indicates the occurrence of a spatially correlated damage. When a PMMA shield is used, the dose-rate of the Fe ions beam decreases by a factor around 0.5 and a concomitant marked decrease in the DNA fragments production per unit dose is found.

  10. Functional Coupling of Duplex Translocation to DNA Cleavage in a Type I Restriction Enzyme

    PubMed Central

    Csefalvay, Eva; Lapkouski, Mikalai; Guzanova, Alena; Csefalvay, Ladislav; Baikova, Tatsiana; Bialevich, Vitali; Shamayeva, Katsiaryna; Janscak, Pavel; Kuta Smatanova, Ivana; Panjikar, Santosh; Carey, Jannette; Weiserova, Marie; Ettrich, Rüdiger

    2015-01-01

    Type I restriction-modification enzymes are multifunctional heteromeric complexes with DNA cleavage and ATP-dependent DNA translocation activities located on motor subunit HsdR. Functional coupling of DNA cleavage and translocation is a hallmark of the Type I restriction systems that is consistent with their proposed role in horizontal gene transfer. DNA cleavage occurs at nonspecific sites distant from the cognate recognition sequence, apparently triggered by stalled translocation. The X-ray crystal structure of the complete HsdR subunit from E. coli plasmid R124 suggested that the triggering mechanism involves interdomain contacts mediated by ATP. In the present work, in vivo and in vitro activity assays and crystal structures of three mutants of EcoR124I HsdR designed to probe this mechanism are reported. The results indicate that interdomain engagement via ATP is indeed responsible for signal transmission between the endonuclease and helicase domains of the motor subunit. A previously identified sequence motif that is shared by the RecB nucleases and some Type I endonucleases is implicated in signaling. PMID:26039067

  11. Close correlation between restriction fragment length polymorphism of the L-MYC gene and metastasis of human lung cancer to the lymph nodes and other organs

    SciTech Connect

    Kawashima, Kazuko; Shikama, Hiroshi; Imoto, Kazuhiko; Izawa, Mitsuo; Nishimura, Susumu (National Cancer Center Research Institute, Tokyo (Japan)); Naruke, Tsuguo; Okabayashi, Kenzo (National Cancer Center Hospital, Tokyo (Japan))

    1988-04-01

    Restriction length fragment polymorphism of the L-MYC gene was examined in DNAs from lung cancer tissues and normal tissues of 51 Japanese patients with lung cancer. In individual patients, no difference was seen between the restriction length fragments of the two alleles of L-MYC (6-kilobase (kb)) and 10-kb fragments in EcoRI digests in lung cancer tissues and normal tissues. But a striking correlation was found between the restriction length fragment polymorphism pattern of L-MYC and the extent of metastasis, particularly to the lymph nodes at the time of surgery: Patients with only the L band (10 kb) had few lymph node metastatic lesions, whereas patients with either the S band (6 kb) or the S and L bands almost always had lymph node metastatic lesion. A similar correlation was found between the presence of the S band and metastases to other organs. This correlation was particularly marked in cases of adenocarcinoma. These results indicate a clear genetic influence on metastases and a consequent poor prognosis for certain patients of lung cancer; L-MYC restriction length fragment polymorphism is thus shown to be a useful marker for predicting the metastatic potential of human lung cancer.

  12. Environmental toxicants cause sperm DNA fragmentation as detected by the Sperm Chromatin Structure Assay (SCSA[reg])

    SciTech Connect

    Evenson, Donald P. [HCLD, Department of Chemistry and Biochemistry, South Dakota State University, Brookings, SD 57007 (United States) and SCSA Diagnostics, 807 32nd Avenue, Brookings, SD 57007 (United States)]. E-mail: scsa@brookings.net; Wixon, Regina [SCSA Diagnostics, 807 32nd Avenue, Brookings, SD 57007 (United States)

    2005-09-01

    Studies over the past two decades have clearly shown that reproductive toxicants cause sperm DNA fragmentation. This DNA fragmentation can usually be detected prior to observing alterations of metaphase chromosomes in embryos. Thus, Sperm Chromatin Structure Assay (SCSA)-detected DNA damage is viewed as the molecular precursor to later gross chromosome damage observed under the light microscope. SCSA measurements of animal or human sperm consist of first obtaining a fresh or flash frozen neat semen sample in LN2 or dry ice. Samples are then sent to a SCSA diagnostic laboratory where the samples are thawed, diluted to {approx}1-2 x 106 sperm/ml, treated for 30 s with a pH 1.2 detergent buffer and then stained with acridine orange (AO). The low pH partially denatures DNA at the sites of DNA strand breaks and the AO-ssDNA fluoresces red while the AO-dsDNA fluoresces green. Flow cytometry measurements of 5000 sperm/sample provide statistically robust data on the ratio of red to green sperm, the extent of the DNA fragmentation and the standard deviations of measures. Numerous experiments on rodents treated with reproductive toxicants clearly showed that SCSA measures are highly dose responsive and have a very low CV. Different agents that act on germ cells at various stages of development usually showed sperm DNA fragmentation when that germ cell fraction arrived in the epididymis or ejaculate. Some of these treated samples were capable of successful in vitro fertilization but with frequent embryo failure. A 2-year longitudinal study of men living a valley town with a reported abnormal level of infertility and spontaneous miscarriages and also a seasonal atmospheric smog pollution, showed, for the first time, that SCSA measurements of human sperm DNA fragmentation were detectable and correlated with dosage of air pollution while the classical semen measures were not correlated. Also, young men spraying pesticides without protective gear are at an increased risk for elevated sperm DNA fragmentation. Extensive DNA fragmentation probably cannot be repaired by the egg and the spontaneous abortion rate is {approx}2x higher if a man has more than 30% of sperm showing DNA fragmentation. DNA fragmentation is an excellent marker for exposure to potential reproductive toxicants and a diagnostic/prognostic tool for potential male infertility.

  13. Genetic linkage of two intragenic restriction fragment length polymorphisms with von Willebrand's disease type IIA. Evidence for a defect in the von Willebrand factor gene.

    PubMed Central

    Verweij, C L; Quadt, R; Briët, E; Dubbeldam, K; van Ommen, G B; Pannekoek, H

    1988-01-01

    Restriction fragment length polymorphisms (RFLPs), using the enzymes Bgl II and Xba I in conjunction with human von Willebrand factor (vWF) cDNA probes, have been described previously. In the present study we demonstrate the localization of both genetic markers within the vWF gene. The RFLPs were used to study the segregation of alleles associated with von Willebrand's disease (vWD) type IIA in a comprehensive, affected family. Individuals of this family were tested for their bleeding time and their plasma was analyzed for vWF antigen concentration and vWF ristocetin-cofactor activity. Based on these data, the affected members were diagnosed as vWD type-IIA patients; this conclusion was confirmed by the analysis of the multimeric vWF pattern of some of the patients. It was demonstrated that both RFLPs are completely linked with the vWD type-IIA trait. From this finding, we conclude that the defect that causes the vWD type IIA is most likely due to a mutation in the vWF gene and not to a mutation in a gene involved in posttranslational processing of the vWF protein. Images PMID:2895123

  14. Chloroplast DNA inheritance in the Stellaria longipes complex (Caryophyllaceae)

    Microsoft Academic Search

    D. K. X. Chong; C. C. Chinnappa; F. C. Yeh; S. Chuong

    1994-01-01

    Inheritance of chloroplast DNA (cpDNA) was examined in F1 progenies derived from three crosses and three corresponding reciprocal crosses betweenStellaria porsildii andS. longifolia. Chloroplast DNA restriction fragments were analyzed using methods of nonradioactive digoxigenin-11-dUTP labeling and chemiluminescent detection with Lumi-Phos 530. Distinct interspecific restriction fragment polymorphisms were identified and used to demonstrate the mode of cpDNA inheritance. Mode of cpDNA

  15. A polymerase chain reaction-restriction fragment length polymorphism method for screening ZNF804A gene polymorphism (rs1344706) in patients with schizophrenia: a significant association.

    PubMed

    Sazci, Ali; Ozel, Mavi Deniz; Ergul, Emel; Yildiz, Mustafa

    2012-03-01

    The original ZNF804A rs1344706 risk variant was identified through genome-wide association studies as a risk factor for schizophrenia. Follow-up studies involving meta-analysis have confirmed that rs1344706 is a risk factor for schizophrenia as well as bipolar disorders. We describe here a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method to genotype ZNF804A rs1344706 variant in patients with schizophrenia. We generated a 220 bp fragment through PCR and subsequently cleaved it by the restriction endonuclease BsaBI, creating two fragments of 114 and 106 bp. Upon change in the nucleotide from T to G, the 106 bp fragment is further cleaved by BsaBI, thus creating two fragments of 87 and 19 bp. As a result, when the 220 bp fragment is cleaved by BsaBI restriction endonuclease, the TT genotype yields two fragments of 114 and 106 bp, and TG genotype four fragments of 114, 106, 87, and 19 bp, and the GG genotype three fragments of 114, 87, and 19 bp. Thus, this is a simple, fast, and cost-effective method to genotype the ZNF804A rs1344706 risk variant. Using this method, we were able to replicate an association between ZNF804A rs1344706 variant and schizophrenia in a Turkish population. Stratification analysis of the population according to the gender showed an association that was statistically significant among overall schizophrenia and male schizophrenia and the risk T allele and TT genotype of the ZNF804A gene. PMID:21988329

  16. Geographic distribution and evolution of yellow fever viruses based on direct sequencing of genomic cDNA fragments

    Microsoft Academic Search

    Loic Lepiniec; Lynn Dalgarno; V. T. Q. Huong; T. P. Monath; J.-P. Digoutte; V. Deubel

    1994-01-01

    We have compared the nucleotide sequence of an envelope protein gene fragment encoding amino acids 291 to 406 of 22 yellow fever (YF) virus strains of diverse geographic and host origins isolated over a 63 year time span. The nucleotide fragment of viral RNA was examined by direct sequencing ofa PCR product derived from complementary DNA. Alignment with the proto-

  17. flaB Gene as a Molecular Marker for Distinct Identification of Borrelia Species in Environmental Samples by the PCR-Restriction Fragment Length Polymorphism Method ?

    PubMed Central

    Wodecka, Beata

    2011-01-01

    A new protocol employing nested PCR-restriction fragment length polymorphism (RFLP) based on the flaB gene and two restriction enzymes was worked out. This protocol allows the identification of all Borrelia species transmitted by Ixodes ricinus in Europe, including Borrelia miyamotoi and 3 genetic variants of B. garinii. A dendrogram of flaB sequence similarity was in accordance with RFLP variants. PMID:21841027

  18. Fragmentation of extracellular DNA by long-term exposure to radiation from uranium in aquatic environments.

    PubMed

    Arruda-Neto, J D T; Nieto, L; Righi, H; Cotta, M A; Carrer, H; Rodrigues, T E; Genofre, G C

    2012-08-01

    Persistent harmful scenarios associated with disposal of radioactive waste, high-background radiation areas and severe nuclear accidents are of great concern regarding consequences to both human health and the environment. Of particular concern is the extracellular DNA in aquatic environments contaminated by radiological substances. Strand breaks induced by radiation promote decrease in the transformation efficiency for extracellular DNA. The focus of this study is the quantification of DNA damage following long-term exposure (over one year) to low doses of natural uranium (an alpha particle emitter) to simulate natural conditions, since nothing is known about alpha radiation induced damage to extracellular DNA. A high-resolution Atomic Force Microscope was used to evaluate DNA fragments. Double-stranded plasmid pBS as a model for extracellular DNA was exposed to different amounts of natural uranium. It was demonstrated that low concentrations of U in water (50 to 150 ppm) produce appreciable numbers of double strand breaks, scaling with the square of the average doses. The importance of these findings for environment monitoring of radiological pollution is addressed. PMID:22760469

  19. Multiple determinations of sperm DNA fragmentation show that varicocelectomy is not indicated for infertile patients with subclinical varicocele.

    PubMed

    García-Peiró, Agustín; Ribas-Maynou, Jordi; Oliver-Bonet, María; Navarro, Joaquima; Checa, Miguel A; Nikolaou, Alexandros; Amengual, María J; Abad, Carlos; Benet, Jordi

    2014-01-01

    Varicocele is one of the most common causes of low semen quality, which is reflected in high percentages of sperm cells with fragmented DNA. While varicocelectomy is usually performed to ameliorate a patient's fertility, its impact on sperm DNA integrity in the case of subclinical varicocele is poorly documented. In this study, multiple DNA fragmentation analyses (TUNEL, SCD, and SCSA) were performed on semen samples from sixty infertile patients with varicocele (15 clinical varicoceles, 19 clinical varicoceles after surgical treatment, 16 subclinical varicoceles, and 10 subclinical varicoceles after surgical treatment). TUNEL, SCD, and SCSA assays all showed substantial sperm DNA fragmentation levels that were comparable between subclinical and clinical varicocele patients. Importantly, varicocelectomy did improve sperm quality in patients with clinical varicocele; however, this was not the case in patients with subclinical varicocele. In summary, although infertile patients with clinical and subclinical varicocele have similar sperm DNA quality, varicocelectomy should only be advised for patients with clinical varicocele. PMID:24967335

  20. Multiple Determinations of Sperm DNA Fragmentation Show That Varicocelectomy Is Not Indicated for Infertile Patients with Subclinical Varicocele

    PubMed Central

    García-Peiró, Agustín; Ribas-Maynou, Jordi; Oliver-Bonet, María; Navarro, Joaquima; Checa, Miguel A.; Nikolaou, Alexandros; Amengual, María J.; Abad, Carlos; Benet, Jordi

    2014-01-01

    Varicocele is one of the most common causes of low semen quality, which is reflected in high percentages of sperm cells with fragmented DNA. While varicocelectomy is usually performed to ameliorate a patient's fertility, its impact on sperm DNA integrity in the case of subclinical varicocele is poorly documented. In this study, multiple DNA fragmentation analyses (TUNEL, SCD, and SCSA) were performed on semen samples from sixty infertile patients with varicocele (15 clinical varicoceles, 19 clinical varicoceles after surgical treatment, 16 subclinical varicoceles, and 10 subclinical varicoceles after surgical treatment). TUNEL, SCD, and SCSA assays all showed substantial sperm DNA fragmentation levels that were comparable between subclinical and clinical varicocele patients. Importantly, varicocelectomy did improve sperm quality in patients with clinical varicocele; however, this was not the case in patients with subclinical varicocele. In summary, although infertile patients with clinical and subclinical varicocele have similar sperm DNA quality, varicocelectomy should only be advised for patients with clinical varicocele. PMID:24967335

  1. PCR-Restriction Fragment Length Polymorphism analysis of fljB gene in Salmonella enterica subspecies enterica serovar Typhimurium isolated from avians

    PubMed Central

    Ahmadi, M; Zahraei Salehi, T; Talebi, A; Darvishzadeh, R

    2010-01-01

    Background and Objectives Economic constraint of diseases arising from Salmonella Typhimurium causes the study of this zoonotic organism more important. Most studies on identification and characterization of S. Typhimurium are conducted at DNA level. Flagellin genes (fliC and fljB genes encoding phase-1 and phase-2 flagella, respectively) are useful as a model system for studying genetic differentiation. The objectives of the present study were to identify the polymorphism of fljB among avians in different regions by the PCR-RFLP method. Materials and Methods Fifty-two S. Typhimurium isolates out of 1,870 intestine samples were identified using culture and serotyping as well as multiplex-PCR (broiler (n=13), layer (n=12), duck (n=5), goose (n=5), sparrow (n=8), canary (n=3), pigeon (n=5) and casco parrot (n=1)). Amplification of fljB gene was performed and amplified products subjected to restriction digestion with Hha I enzyme. Results Two RFLP patterns generated DNA fragments between approximately 50 to 800 bps. Pattern A was observed in 33 (63.46%) and pattern B in 19 (36.54%) of isolates. Salmonella Typhimurium recovered from 13 broilers (ten with pattern A and 3 with pattern B) and 8 sparrow (three with pattern A and 5 with pattern B) showed both A and B patterns. Twelve layers, 5 pigeons and 3 canaries showed pattern A and 5 ducks, 5 geese and one casco parrot showed pattern B. None of these patterns was allotted for a special region. Conclusion The results of the present study showed that fljB gene is highly conserved among avians in different geographical regions, suggesting not only the importance of fljB gene in survival of organism in different environmental conditions but also the relation between proteins encoded by fljB gene and serotyping scheme. PMID:22347569

  2. DNA Barcoding for Identification of ‘Candidatus Phytoplasmas’ Using a Fragment of the Elongation Factor Tu Gene

    PubMed Central

    Makarova, Olga; Contaldo, Nicoletta; Paltrinieri, Samanta; Kawube, Geofrey; Bertaccini, Assunta; Nicolaisen, Mogens

    2012-01-01

    Background Phytoplasmas are bacterial phytopathogens responsible for significant losses in agricultural production worldwide. Several molecular markers are available for identification of groups or strains of phytoplasmas. However, they often cannot be used for identification of phytoplasmas from different groups simultaneously or are too long for routine diagnostics. DNA barcoding recently emerged as a convenient tool for species identification. Here, the development of a universal DNA barcode based on the elongation factor Tu (tuf) gene for phytoplasma identification is reported. Methodology/Principal Findings We designed a new set of primers and amplified a 420–444 bp fragment of tuf from all 91 phytoplasmas strains tested (16S rRNA groups -I through -VII, -IX through -XII, -XV, and -XX). Comparison of NJ trees constructed from the tuf barcode and a 1.2 kbp fragment of the 16S ribosomal gene revealed that the tuf tree is highly congruent with the 16S rRNA tree and had higher inter- and intra- group sequence divergence. Mean K2P inter?/intra- group divergences of the tuf barcode did not overlap and had approximately one order of magnitude difference for most groups, suggesting the presence of a DNA barcoding gap. The use of the tuf barcode allowed separation of main ribosomal groups and most of their subgroups. Phytoplasma tuf barcodes were deposited in the NCBI GenBank and Q-bank databases. Conclusions/Significance This study demonstrates that DNA barcoding principles can be applied for identification of phytoplasmas. Our findings suggest that the tuf barcode performs as well or better than a 1.2 kbp fragment of the 16S rRNA gene and thus provides an easy procedure for phytoplasma identification. The obtained sequences were used to create a publicly available reference database that can be used by plant health services and researchers for online phytoplasma identification. PMID:23272216

  3. Chloroplast and Mitochondrial DNA are Paternally Inherited in Sequoia sempervirens D. Don Endl

    Microsoft Academic Search

    David B. Neale; Kimberly A. Marshall; Ronald R. Sederoff

    1989-01-01

    Restriction fragment length polymorphisms in controlled crosses were used to infer the mode of inheritance of chloroplast DNA and mitochondrial DNA in coast redwood (Sequoia sempervirens D. Don Endl.). Chloroplast DNA was paternally inherited, as is true for all other conifers studied thus far. Surprisingly, a restriction fragment length polymorphism detected by a mitochondrial probe was paternally inherited as well.

  4. A baculovirus alkaline nuclease knockout construct produces fragmented DNA and aberrant capsids

    SciTech Connect

    Okano, Kazuhiro [Department of Microbiology, Nash Hall Room 220, Oregon State University, Corvallis, OR 97331-3804 (United States); Vanarsdall, Adam L. [Department of Microbiology, Nash Hall Room 220, Oregon State University, Corvallis, OR 97331-3804 (United States); Rohrmann, George F. [Department of Microbiology, Nash Hall Room 220, Oregon State University, Corvallis, OR 97331-3804 (United States)]. E-mail: rohrmanng@orst.edu

    2007-03-01

    DNA replication of bacmid-derived constructs of the Autographa californica multiple nucleocapsid nucleopolyhedrovirus (AcMNPV) was analyzed by field inversion gel electrophoresis (FIGE) in combination with digestion at a unique Eco81I restriction enzyme site. Three constructs were characterized: a parental bacmid, a bacmid deleted for the alkaline nuclease gene, and a bacmid from which the gp64 gene had been deleted. The latter was employed as a control for comparison with the alkaline nuclease knockout because neither yields infectious virus and their replication is limited to the initially transfected cells. The major difference between DNA replicated by the different constructs was the presence in the alkaline nuclease knockout of high concentrations of relatively small, subgenome length DNA in preparations not treated with Eco81I. Furthermore, upon Eco81I digestion, the alkaline nuclease knockout bacmid also yielded substantially more subgenome size DNA than the other constructs. Electron microscopic examination of cells transfected with the alkaline nuclease knockout indicated that, in addition to a limited number of normal-appearing electron-dense nucleocapsids, numerous aberrant capsid-like structures were observed indicating a defect in nucleocapsid maturation or in a DNA processing step that is necessary for encapsidation. Because of the documented role of the baculovirus alkaline nuclease and its homologs from other viruses in homologous recombination, these data suggest that DNA recombination may play a major role in the production of baculovirus genomes.

  5. DNA fragmentation and nuclear phenotype in tendons exposed to low-intensity infrared laser

    NASA Astrophysics Data System (ADS)

    de Paoli, Flavia; Ramos Cerqueira, Larissa; Martins Ramos, Mayara; Campos, Vera M.; Ferreira-Machado, Samara C.; Geller, Mauro; de Souza da Fonseca, Adenilson

    2015-03-01

    Clinical protocols are recommended in device guidelines outlined for treating many diseases on empirical basis. However, effects of low-intensity infrared lasers at fluences used in clinical protocols on DNA are controversial. Excitation of endogenous chromophores in tissues and free radicals generation could be described as a consequence of laser used. DNA lesions induced by free radicals cause changes in DNA structure, chromatin organization, ploidy degrees and cell death. In this work, we investigated whether low-intensity infrared laser therapy could alter the fibroblasts nuclei characteristics and induce DNA fragmentation. Tendons of Wistar rats were exposed to low-intensity infrared laser (830 nm), at different fluences (1, 5 and 10 J/cm2), in continuous wave (power output of 10mW, power density of 79.6 mW/cm2). Different frequencies were analyzed for the higher fluence (10 J/cm2), at pulsed emission mode (2.5, 250 and 2500 Hz), with the laser source at surface of skin. Geometric, densitometric and textural parameters obtained for Feulgen-stained nuclei by image analysis were used to define nuclear phenotypes. Significant differences were observed on the nuclear phenotype of tendons after exposure to laser, as well as, high cell death percentages was observed for all fluences and frequencies analyzed here, exception 1 J/cm2 fluence. Our results indicate that low-intensity infrared laser can alter geometric, densitometric and textural parameters in tendon fibroblasts nuclei. Laser can also induce DNA fragmentation, chromatin lost and consequently cell death, using fluences, frequencies and emission modes took out from clinical protocols.

  6. Presence of DNA Fragmentation and Lack of Neuroprotective Effect in DFF45 Knockout Mice Subjected to Traumatic Brain Injury

    Microsoft Academic Search

    Alexander G. Yakovlev; Xiao Di; Vilen Movsesyan; Paul G. M. Mullins; Geping Wang; Hamid Boulares; Jianhua Zhang; Ming Xu; Alan I. Faden

    2001-01-01

    Background: Apoptosis plays an important pathophysio- logic role in neuronal cell loss and associated neurologic deficits following traumatic brain injury (TBI). DNA frag- mentation represents one of the characteristic biochemical features of neuronal apoptosis and is observed after ex- perimental TBI. DFF45 and DFF40 are essential for DNA fragmentation in various models of apoptosis. Materials and Methods: We used mice

  7. Differentiation of Helicobacter pylori Strains Directly from Gastric Biopsy Specimens by PCR-Based Restriction Fragment Length Polymorphism Analysis without Culture

    Microsoft Academic Search

    CHUANFU LI; TUANZHU HA; DAVID S. CHI; DONALD A. FERGUSON; CHUANCANG JIANG; JOHN J. LAFFAN; EAPEN THOMAS

    1997-01-01

    Recent studies have shown the usefulness of PCR-based restriction fragment length polymorphism (RFLP) analysis for differentiating Helicobacter pylori strains isolated by culture. For this study, a PCR-based RFLP assay was developed for directly typing H. pylori strains from gastric biopsy specimens. Nineteen gastric biopsy specimens obtained from patients undergoing endoscopy for gastrointestinal complaints were cultured for isolation of H. pylori.

  8. Localization by restriction fragment length polymorphism mapping in potato of a major dominant gene conferring resistance to the potato cyst nematode Globodera rostocbiensis

    Microsoft Academic Search

    Amalia Barone; Enrique Ritter; Undine Schachtschabel; Thomas Debener; Francesco Salamini; Christiane Gebhardt

    1990-01-01

    A major dominant locus conferring resistance against several pathotypes of the root cyst nematode Globodera rostochiensis was mapped on the linkage map of potato using restriction fragment length polymorphism (RFLP) markers. The assessment of resistance versus susceptibility of the plants in the experimental population considered was based on an in vivo (pot) and an in vitro (petri dish) test. By

  9. GENETIC DIVERSITY OF FLAVOBACTERIUM COLUMNARE EXAMINED BY RESTRICTION FRAGMENT LENGTH POLYMORPHISM AND SEQUENCING OF THE 16S RIBOSOMAL RNA GENE AND THE 16S-23S RDNA SPACER

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Genetic variability among strains of Flavobacterium columnare, isolated in the United States, was characterized by restriction fragment length polymorphism (RFLP) and phylogenetic analysis based on the sequence of the 16S rRNA gene. Twenty-seven isolates of F. columnare were differentiated into thr...

  10. The Roles of Family B and D DNA Polymerases in Thermococcus Species 9°N Okazaki Fragment Maturation*

    PubMed Central

    Greenough, Lucia; Kelman, Zvi; Gardner, Andrew F.

    2015-01-01

    During replication, Okazaki fragment maturation is a fundamental process that joins discontinuously synthesized DNA fragments into a contiguous lagging strand. Efficient maturation prevents repeat sequence expansions, small duplications, and generation of double-stranded DNA breaks. To address the components required for the process in Thermococcus, Okazaki fragment maturation was reconstituted in vitro using purified proteins from Thermococcus species 9°N or cell extracts. A dual color fluorescence assay was developed to monitor reaction substrates, intermediates, and products. DNA polymerase D (polD) was proposed to function as the replicative polymerase in Thermococcus replicating both the leading and the lagging strands. It is shown here, however, that it stops before the previous Okazaki fragments, failing to rapidly process them. Instead, Family B DNA polymerase (polB) was observed to rapidly fill the gaps left by polD and displaces the downstream Okazaki fragment to create a flap structure. This flap structure was cleaved by flap endonuclease 1 (Fen1) and the resultant nick was ligated by DNA ligase to form a mature lagging strand. The similarities to both bacterial and eukaryotic systems and evolutionary implications of archaeal Okazaki fragment maturation are discussed. PMID:25814667

  11. Comparative karyology, DNA methylation and restriction pattern analysis of male and female plants of the dioecious alga Chara tomentosa (Charophyceae)

    Microsoft Academic Search

    Agnieszka Kunachowicz; Piotr Luchniak; Maria Olszewska; Tomasz Sakowicz

    2001-01-01

    Comparison of karyology, restriction patterns and DNA methylation levels (as gender separation could be related to some epigenetic mechanism, e.g. DNA methylation) has been performed between male and female individuals of the dioecious species, Chara tomentosa. In contrast to angiosperms, sex determination systems in Charophyceae have not been studied. High-performance liquid chromatography (HPLC) showed that the cytosine methylation level in

  12. Terminal Restriction Fragment Length Polymorphism Analysis of Soil Bacterial Communities under Different Vegetation Types in Subtropical Area

    PubMed Central

    Wu, Zeyan; Lin, Wenxiong; Li, Bailian; Wu, Linkun; Fang, Changxun; Zhang, Zhixing

    2015-01-01

    Soil microbes are active players in energy flow and material exchange of the forest ecosystems, but the research on the relationship between the microbial diversity and the vegetation types is less conducted, especially in the subtropical area of China. In this present study, the rhizosphere soils of evergreen broad-leaf forest (EBF), coniferous forest (CF), subalpine dwarf forest (SDF) and alpine meadow (AM) were chosen as test sites. Terminal-restriction fragment length polymorphisms (T-RFLP) analysis was used to detect the composition and diversity of soil bacterial communities under different vegetation types in the National Natural Reserve of Wuyi Mountains. Our results revealed distinct differences in soil microbial composition under different vegetation types. Total 73 microbes were identified in soil samples of the four vegetation types, and 56, 49, 46 and 36 clones were obtained from the soils of EBF, CF, SDF and AM, respectively, and subsequently sequenced. The Actinobacteria, Fusobacterium, Bacteroidetes and Proteobacteria were the most predominant in all soil samples. The order of Shannon-Wiener index (H) of all soil samples was in the order of EBF>CF>SDF>AM, whereas bacterial species richness as estimated by four restriction enzymes indicated no significant difference. Principal component analysis (PCA) revealed that the soil bacterial communities’ structures of EBF, CF, SDF and AM were clearly separated along the first and second principal components, which explained 62.17% and 31.58% of the total variance, respectively. The soil physical-chemical properties such as total organic carbon (TOC), total nitrogen (TN), total phosphorus (TP) and total potassium (TK) were positively correlated with the diversity of bacterial communities. PMID:26098851

  13. Manning free counterions fraction for a rod-like polyion - short DNA fragments in very low salt

    E-print Network

    Tomislav Vuletic; Sanja Dolanski Babic; Danijel Grgicin; Damir Aumiler; Joachim Raedler; Francoise Livolant; Silvia Tomic

    2011-01-05

    We quantified the Manning free (uncondensed) counterions fraction $\\theta$ for dilute solutions of rod-like polyions - 150bp DNA fragments, in very low salt $salt environment, with the decrease in DNA concentration itself. The extremes of the experimental $\\theta(c)$ range occur towards the highest, above 1 mM and the lowest, below 0.05 mM, DNA concentrations, and correspond to the theoretical $\\theta$ values for dsDNA and ssDNA, respectively. Therefore, we confirmed Manning condensation and conductivity models to be valuable in description of dilute solutions of rod-like polyions.

  14. Assessment of Subtractive Hybridization to Select Species and Subspecies Specific DNA Fragments for the Identification of Xylophilus ampelinus by Polymerase Chain Reaction (PCR)

    Microsoft Academic Search

    Charles Manceau; Marie-Germaine Coutaud; Richard Guyon

    2000-01-01

    Eighteen Bsp143I digested DNA fragments specific to Xylophilus ampelinus were cloned from a library enriched for X. ampelinus obtained after a subtractive hybridization step. It was also possible to clone specific DNA sequences directly after DNA digestion with Bsp143I probably because X. ampelinus is a unique bacterium. Nucleotidic sequences of four cloned specific fragments were determined. They did not share

  15. Validation of a field based chromatin dispersion assay to assess sperm DNA fragmentation in the bottlenose dolphin (Tursiops truncatus).

    PubMed

    Sánchez-Calabuig, M-J; López-Fernández, C; Martínez-Nevado, E; Pérez-Gutiérrez, J F; de la Fuente, J; Johnston, S D; Blyde, D; Harrison, K; Gosálvez, J

    2014-10-01

    Over the last two decades, there have been significant advances in the use of assisted reproductive technology for genetic and reproductive management of captive dolphin populations, including evaluation of sperm DNA quality. This study validated a customized sperm chromatin dispersion test (SCDt) for the bottlenose dolphin (Tursiops truncatus) as a means of assessing sperm DNA damage both in the field and in the laboratory. After performing the SCDt, two different sperm morphotypes were identified: (i) sperm with fragmented DNA showed large haloes of dispersed DNA fragments emerging from a compact sperm nucleoid core and (ii) sperm containing non-fragmented DNA displayed small compact haloes surrounded by a dense core of non-dispersed DNA and protein complex. Estimates of sperm DNA fragmentation by means of SCDt were directly comparable to results obtained following a two-tailed comet assay and showed a significant degree of correlation (r = 0.961; p < 0.001). This investigation also revealed that the SCDt, with minor modifications to the standard protocol, can be successfully conducted in the field using a LED florescence microscopy obtaining a high correlation (r = 0.993; p = 0.01) between the data obtained in the laboratory and in the field. PMID:25130370

  16. DNA fragmentation pattern in human fibroblasts after irradiation with iron ions

    NASA Astrophysics Data System (ADS)

    Campa, Alessandro

    In this work we studied the fragmentation pattern produced by the double stand breaks (DSB) induced in AG1522 primary human fibroblasts by two different iron beams, one of energy 414 MeV/u, and the other of energy 115 MeV/u (with dose-average LET in water equal to 202 keV/µm and 442 keV/µm, respectively). Irradiation with several doses up to 200 Gy was performed at the HIMAC facility of the National Institute of Radiological Sciences, Chiba, Japan. Experimental data, first obtained for fragments belonging to the size ranges 23-1000 kbp and 1000-5700 kbp (Belli et al., 2006), have successively been obtained also for fragments belonging to the size ranges 1-9 kbp and 9-23 kbp; the experimental analysis was performed with pulsed and constant field electrophoresis. The RBE for DSB production was evaluated in two different fragment size ranges (i.e., 23-5700 kbp and 1-5700 kbp), and it was found larger for the wider size range, especially for the beam with the higher LET. The experimental results have been compared to those computed on the basis of the Monte Carlo PARTRAC simulation code, following the line of research started in Campa et al. (2005), and exploiting the recent update of the PARTRAC code to ions heavier than helium (Friedland et al., 2006). Because the agreement has been found satisfactory for both radiation qualities, the spectra outside the experimentally observable fragment size range were also computed in order to evaluate the overall fragmentation pattern. The marked increases of the RBEs for DSB production, obtained when also the very small fragments (< 1 kbp) are included, makes them closer to the RBE values observed for the late cellular effects. This finding is a further indication for the biological significance of the spatial correlation of DSB at short distances. This work was partially supported by ASI (Italian Space Agency, "Mo-Ma/COUNT" project). References M. Belli, A. Campa, V. Dini, G. Esposito, Y. Furusawa, G. Simone, E. Sorrentino and M. A. Tabocchini. DNA fragmentation induced in human fibroblasts by accelerated 56 Fe ions of differing energies. Radiat. Res. 165, 713-720 (2006). A. Campa, F. Ballarini, M. Belli, R. Cherubini, V. Dini, G. Esposito, W. Friedland, S. Gerardi, S. Molinelli, A. Ottolenghi, H. G. Paretzke, G. Simone and M. A. Tabocchini. DNA DSB induced in human cells by charged particles and gamma rays: experimental results and theoretical approaches. Int. J. Radiat. Biol. 81, 841-854 (2005). W. Friedland, P. Jacob, H. G. Paretzke, A. Ottolenghi, F. Ballarini and M. Liotta. Simulation of light ion induced DNA damge patterns. Radiat. Prot. Dosim. 122, 116-120 (2006).

  17. Restriction fragment length polymorphism of flagellin genes of Campylobacter jejuni and/or C. coli isolates from Egypt.

    PubMed Central

    Mohran, Z S; Guerry, P; Lior, H; Murphy, J R; el-Gendy, A M; Mikhail, M M; Oyofo, B A

    1996-01-01

    The conservation of flagellin genes from thermophilic Campylobacter spp. strains isolated in Egypt was evaluated by a restriction fragment length polymorphism (RFLP) assay. The flaA and flaB genes were amplified from 59 independent clinical isolates and digested with EcoRI and PstI, and the resulting patterns were compared with each other and with previously described RFLP groups. The results indicate that the isolates fell into 14 groups for flaA and 11 groups for flaB, 9 of which have been described, and that considerable genetic variability exists among isolates belonging to the same LIO serogroup. In most cases, the flaB gene displayed the same RFLP pattern as that of the flaA gene of the same strain, although some variability was observed. The data suggest that more variability of flagellin genes exists within the LIO serogroups common to Campylobacter field isolates from Egypt than has previously been reported for North American isolates. PMID:8727905

  18. Molecular typing of Borrelia burgdorferi from Lyme disease patients by PCR-restriction fragment length polymorphism analysis.

    PubMed

    Liveris, D; Wormser, G P; Nowakowski, J; Nadelman, R; Bittker, S; Cooper, D; Varde, S; Moy, F H; Forseter, G; Pavia, C S; Schwartz, I

    1996-05-01

    Ninety-three Borrelia burgdorferi isolates obtained from erythema migrans lesions or blood of Lyme disease patients in Westchester County, N.Y., between 1991 and 1994 were characterized by PCR-restriction fragment length polymorphism (PCR-RFLP) analysis of the 16S-23S rRNA gene spacer. All isolates could be classified into three distinct RFLP types. Among the 82 skin biopsy isolates studied, 21 (25.6%) were type 1, 37 (45.1%) were type 2, and 21 (25.6%) were type 3. Three (3.7%) cultures contained a mixture of two isolates with distinct RFLP types. The 11 isolates cultured from blood showed a similar predominance of RFLP type 2 (6 of 11; 54.5%) relative to types 1 (2 of 11; 18.2%) and 3 (3 of 11; 27.3%). For one patient both skin and blood isolates were cultured, and RFLP analysis revealed that these isolates differed from one another. This study demonstrates that there is genotypic heterogeneity in B. burgdorferi strains infecting Lyme disease patients, and this typing approach may allow differentiation of isolates with various degrees of pathogenic potential. PMID:8727927

  19. Restriction fragment length polymorphism mapping of quantitative trait loci for malaria parasite susceptibility in the mosquito Aedes aegypti

    SciTech Connect

    Severson, D.W.; Thathy, V.; Mori, A. [Univ. of Wisconsin, Madison, WI (United States)] [and others

    1995-04-01

    Susceptibility of the mosquito Aedes aegypti to the malarial parasite Plasmodium gallinaceum was investigated as a quantitative trait using restriction fragment length polymorphisms (RFLP). Two F{sub 2} populations of mosquitoes were independently prepared from pairwise matings between a highly susceptible and a refractory strain of A. aegypti. RFLP were tested for association with oocyst development on the mosquito midgut. Two putative quantitative trait loci (QTL) were identified that significantly affect susceptibility. One QTL, pgs [2,LF98], is located on chromosome 2 and accounted for 65 and 49% of the observed phenotypic variance in the two populations, respectively. A second QTL, pgs[3,MalI], is located on chromosome 3 and accounted for 14 and 10% of the observed phenotypic variance in the two populations, respectively. Both QTL exhibit a partial dominance effect on susceptibility, wherein the dominance effect is derived from the refractory parent. No indication of epistasis between these QTL was detected. Evidence suggests that either a tightly linked cluster of independent genes or a single locus affecting susceptibility to various mosquito-borne parasites and pathogens has evolved near the LF98 locus; in addition to P. gallinaceum susceptibility, this general genome region has previously been implicated in susceptibility to the filaria nematode Brugia malayi and the yellow fever virus. 35 refs., 2 figs., 3 tabs.

  20. Restriction fragment length polymorphisms of constant region genes of immunoglobulin lambda chains in Venezuelan patients with systemic lupus erythematosus.

    PubMed

    Blasini, A M; Delgado, M B; Valdivieso, C; Guevara, P; Ramirez, J L; Stekman, I L; Rodriguez, M A; Williams, R C

    1996-08-01

    Previous studies suggest a potential association between human immunoglobulin (Ig) genes and susceptibility to systemic lupus erythematosus (SLE). Ig allotypic determinants seem to confer an increased risk for the disease in various ethnic patient populations. In this study we have examined the pattern of restriction fragment length polymorphisms (RFLP) of constant region lambda (C lambda) light chain genes in a group of 78 Venezuelan patients with SLE and 70 healthy controls. The frequency of the 8-kb allele and the 8/8 genotype was significantly lower in normal Venezuelan controls as compared to healthy British Caucasians (P = 0.0002 and 0.0007 respectively). In turn, Venezuelan controls showed a higher frequency of the 18-kb allele and the 18/18 genotype (P = 0.0002 and 0.0052 respectively). However, there were no statistically significant differences in either parameter between Venezuelan SLE patients and healthy controls. Our study argues against a role for lambda light chain constant region genes in predisposition to SLE. PMID:8869902

  1. Molecular typing of Borrelia burgdorferi from Lyme disease patients by PCR-restriction fragment length polymorphism analysis.

    PubMed Central

    Liveris, D; Wormser, G P; Nowakowski, J; Nadelman, R; Bittker, S; Cooper, D; Varde, S; Moy, F H; Forseter, G; Pavia, C S; Schwartz, I

    1996-01-01

    Ninety-three Borrelia burgdorferi isolates obtained from erythema migrans lesions or blood of Lyme disease patients in Westchester County, N.Y., between 1991 and 1994 were characterized by PCR-restriction fragment length polymorphism (PCR-RFLP) analysis of the 16S-23S rRNA gene spacer. All isolates could be classified into three distinct RFLP types. Among the 82 skin biopsy isolates studied, 21 (25.6%) were type 1, 37 (45.1%) were type 2, and 21 (25.6%) were type 3. Three (3.7%) cultures contained a mixture of two isolates with distinct RFLP types. The 11 isolates cultured from blood showed a similar predominance of RFLP type 2 (6 of 11; 54.5%) relative to types 1 (2 of 11; 18.2%) and 3 (3 of 11; 27.3%). For one patient both skin and blood isolates were cultured, and RFLP analysis revealed that these isolates differed from one another. This study demonstrates that there is genotypic heterogeneity in B. burgdorferi strains infecting Lyme disease patients, and this typing approach may allow differentiation of isolates with various degrees of pathogenic potential. PMID:8727927

  2. Investigation on the Origin of Sperm DNA Fragmentation: Role of Apoptosis, Immaturity and Oxidative Stress

    PubMed Central

    Muratori, Monica; Tamburrino, Lara; Marchiani, Sara; Cambi, Marta; Olivito, Biagio; Azzari, Chiara; Forti, Gianni; Baldi, Elisabetta

    2015-01-01

    Sperm DNA fragmentation (sDF) represents a threat to male fertility, human reproduction and the health of the offspring. The causes of sDF are still unclear, even if apoptosis, oxidative assault and defects in chromatin maturation are hypothesized. Using multicolor flow cytometry and sperm sorting, we challenged the three hypothesized mechanisms by simultaneously evaluating sDF and signs of oxidative damage (8-hydroxy, 2?-deoxyguanosine [8-OHdG] and malondialdehyde [MDA]), apoptosis (caspase activity and cleaved poly[ADP-ribose] polymerase [cPARP]) and sperm immaturity (creatine phosphokinase [CK] and excess of residual histones). Active caspases and c-PARP were concomitant with sDF in a high percentage of spermatozoa (82.6% ± 9.1% and 53.5% ± 16.4%, respectively). Excess of residual histones was significantly higher in DNA-fragmented sperm versus sperm without DNA fragmentation (74.8% ± 17.5% and 37.3% ± 16.6%, respectively, p < 0.005), and largely concomitant with active caspases. Conversely, oxidative damage was scarcely concomitant with sDF in the total sperm population, at variance with live sperm, where 8-OHdG and MDA were clearly associated to sDF. In addition, most live cells with active caspase also showed 8-OHdG, suggesting activation of apoptotic pathways in oxidative-injured live cells. This is the first investigation on the origin of sDF directly evaluating the simultaneous presence of the signs of the hypothesized mechanisms with DNA breaks at the single cell level. The results indicate that the main pathway leading to sperm DNA breaks is a process of apoptosis, likely triggered by an impairment of chromatin maturation in the testis and by oxidative stress during the transit in the male genital tract. These findings are highly relevant for clinical studies on the effects of drugs on sDF and oxidative stress in infertile men and for the development of new therapeutic strategies. PMID:25786204

  3. Structure of 5-hydroxymethylcytosine-specific restriction enzyme, AbaSI, in complex with DNA

    SciTech Connect

    Horton, John R.; Borgaro, Janine G.; Griggs, Rose M.; Quimby, Aine; Guan, Shengxi; Zhang, Xing; Wilson, Geoffrey G.; Zheng, Yu; Zhu, Zhenyu; Cheng, Xiaodong (Emory-MED); (NE Biolabs)

    2014-07-03

    AbaSI, a member of the PvuRts1I-family of modification-dependent restriction endonucleases, cleaves DNA containing 5-hydroxymethylctosine (5hmC) and glucosylated 5hmC (g5hmC), but not DNA containing unmodified cytosine. AbaSI has been used as a tool for mapping the genomic locations of 5hmC, an important epigenetic modification in the DNA of higher organisms. Here we report the crystal structures of AbaSI in the presence and absence of DNA. These structures provide considerable, although incomplete, insight into how this enzyme acts. AbaSI appears to be mainly a homodimer in solution, but interacts with DNA in our structures as a homotetramer. Each AbaSI subunit comprises an N-terminal, Vsr-like, cleavage domain containing a single catalytic site, and a C-terminal, SRA-like, 5hmC-binding domain. Two N-terminal helices mediate most of the homodimer interface. Dimerization brings together the two catalytic sites required for double-strand cleavage, and separates the 5hmC binding-domains by ~ 70 Å, consistent with the known activity of AbaSI which cleaves DNA optimally between symmetrically modified cytosines ~ 22 bp apart. The eukaryotic SET and RING-associated (SRA) domains bind to DNA containing 5-methylcytosine (5mC) in the hemi-methylated CpG sequence. They make contacts in both the major and minor DNA grooves, and flip the modified cytosine out of the helix into a conserved binding pocket. In contrast, the SRA-like domain of AbaSI, which has no sequence specificity, contacts only the minor DNA groove, and in our current structures the 5hmC remains intra-helical. A conserved, binding pocket is nevertheless present in this domain, suitable for accommodating 5hmC and g5hmC. We consider it likely, therefore, that base-flipping is part of the recognition and cleavage mechanism of AbaSI, but that our structures represent an earlier, pre-flipped stage, prior to actual recognition.

  4. Analysis of mitochondrial DNA and its inheritance in Populus

    Microsoft Academic Search

    R. Radetzky

    1990-01-01

    The mitochondrial genome of several poplar clones has been characterized by restriction analysis and hybridization with gene probes from Oenothera. The mitochondrial (mt) DNA of Populus has a complex restriction fragment pattern and its genome size was estimated to be about 450 kilobase pairs (kb). Restriction fragment length polymorphisms (RFLPs) could be detected only between, and not within, species of

  5. Vaccination of mice with DNA encoding a large fragment of botulinum neurotoxin serotype A.

    PubMed

    Clayton, J; Middlebrook, J L

    2000-03-01

    The potential utility of using DNA vaccination to protect mice from the microbial neurotoxin, botulinum toxin type A, was evaluated. A synthetically derived gene encoding a carboxyl-terminal 50 kDa fragment of the toxin was placed in two sites in the DNA inoculation vehicle pCMVint-BL (Vical), one predicted to lead to MHC I processing (pJT-1 construct) and the other to direct MHC II processing (pJT-2 construct). Mice were then inoculated at 3 week intervals with these two constructs and with the vehicle alone and evaluated for protection from botulinum toxin by i.p. challenges with various toxin doses. Protection was observed at about week 10-11 from toxin doses of 25-100 LD(50). Only animals inoculated with pJT-2 exhibited protection. In dose-response experiments, 50 micrograms of DNA was the minimal dose required to elicit a protective response against serotype A, while protection against serotypes B or E was not obtained. With standard ELISA testing, a relationship was observed between the level of protection and the level of ELISA reactive antibody. Our results support the concept that DNA vaccination is a viable methodology to use in cases where protection from toxins is the goal. PMID:10699334

  6. DNA double-strand breaks induced by high-energy neon and iron ions in human fibroblasts. II. Probing individual NotI fragments by hybridization

    SciTech Connect

    Loebrich, M.; Rydberg, B.; Cooper, P.K. [Lawrence Berkeley Lab., CA (United States)

    1994-08-01

    The initial yields of DNA double-strand breaks induced by energetic heavy ions (425 MeV/u neon and 250, 400 and 600 MeV/u iron) in comparison to X rays were measured in normal human diploid fibroblast cells within three small areas of the genome, defined by NotI fragments of 3.2, 2.0 and 1.2 Mbp. The methodology involves NotI restriction endonuclease digestion of DNA from irradiated cells, followed by pulsed-field gel electrophoresis, Southern blotting and hybridization with probes recognizing single-copy sequences within the three NotI fragments. The gradual disappearance of a smear of broken DNA molecules are quantified. Assuming Poisson statistics for the number of double-strand breaks induced per NotI fragment of known size, absolute yields of DNA double-strand breaks were calculated and determined to be linear with dose in all cases, with the neon ion (LET 32 keV/{mu}m) producing 4.4 x 10{sup {minus}3} breaks/Mbp/Gy and all three iron-ion beams (LETs from 190 to 350 keV/{mu}m) producing 2.8 x 10{sup {minus}3} breaks/Mbp/Gy, giving RBE values for production of double-strand breaks of 0.76 for neon and 0.48 for iron in comparison to our previously determined X-ray induction rate of 5.8 x 10{sup {minus}3} breaks/Mbp/Gy. These RBE values are in good agreement with results of measurements over the whole genome as reported in the accompanying paper. The distribution of broken DNA molecules was similar for the various radiations, supporting a random distribution of double-strand breaks induced by the heavy ions over Mbp distances; however, correlated breaks (clusters) over much smaller distances are not ruled out. Reconstitution of the 3.2 Mbp NotI fragment was studied during postirradiation incubation of the cells as a measure of rejoining of correct DNA ends. The proportion of breaks repaired decreased with increasing LET. 41 refs., 6 figs., 1 tab.

  7. Monte Carlo predictions of DNA fragment-size distributions for large sizes after HZE particle irradiation

    NASA Technical Reports Server (NTRS)

    Ponomarev, A. L.; Cucinotta, F. A.; Sachs, R. K.; Brenner, D. J.

    2001-01-01

    DSBs (double-strand breaks) produced by densely ionizing space radiation are not located randomly in the genome: recent data indicate DSB clustering along chromosomes. DSB clustering at large scales, from >100 Mbp down to approximately 2 kbp, is modeled using a Monte-Carlo algorithm. A random-walk model of chromatin is combined with a track model, that predicts the radial distribution of energy from an ion, and the RLC (randomly-located-clusters) formalism, in software called DNAbreak. This model generalizes the random-breakage model, whose broken-stick fragment-size distribution is applicable to low-LET radiation. DSB induction due to track interaction with the DNA volume depends on the radiation quality parameter Q. This dose-independent parameter depends only weakly on LET. Multi-track, high-dose effects depend on the cluster intensity parameter lambda, proportional to fluence as defined by the RLC formalism. After lambda is determined by a numerical experiment, the model reduces to one adjustable parameter Q. The best numerical fits to the experimental data, determining Q, are obtained. The knowledge of lambda and Q allows us to give biophysically based extrapolations of high-dose DNA fragment-size data to low doses or to high LETs.

  8. Patterns of Genomic Integration of Nuclear Chloroplast DNA Fragments in Plant Species

    PubMed Central

    Yoshida, Takanori; Furihata, Hazuka Y.; Kawabe, Akira

    2014-01-01

    The transfer of organelle DNA fragments to the nuclear genome is frequently observed in eukaryotes. These transfers are thought to play an important role in gene and genome evolution of eukaryotes. In plants, such transfers occur from plastid to nuclear [nuclear plastid DNAs (NUPTs)] and mitochondrial to nuclear (nuclear mitochondrial DNAs) genomes. The amount and genomic organization of organelle DNA fragments have been studied in model plant species, such as Arabidopsis thaliana and rice. At present, publicly available genomic data can be used to conduct such studies in non-model plants. In this study, we analysed the amount and genomic organization of NUPTs in 17 plant species for which genome sequences are available. The amount and distribution of NUPTs varied among the species. We also estimated the distribution of NUPTs according to the time of integration (relative age) by conducting sequence similarity analysis between NUPTs and the plastid genome. The age distributions suggested that the present genomic constitutions of NUPTs could be explained by the combination of the rapidly eliminated deleterious parts and few but constantly existing less deleterious parts. PMID:24170805

  9. Binding and cleavage of DNA with the restriction enzyme EcoR1 using time-resolved second harmonic generation.

    PubMed

    Doughty, Benjamin; Kazer, Samuel W; Eisenthal, Kenneth B

    2011-12-13

    The binding of EcoR1 to a 90-bp DNA duplex attached to colloidal microparticles and the subsequent cleavage by the enzyme was observed in real time and label-free with time-resolved second harmonic (SH) spectroscopy. This method provides a unique way to investigate biomolecular interactions based on its sensitivity to changes in structure and electrical charge on formation of a complex and subsequent dynamics. The binding of EcoR1 to the recognition sequence in DNA appears as a rapid increase in the SH signal, which is attributed to the enzyme-induced change in the DNA conformation, going from a rod-like to a bent shape. In the presence of the cofactor Mg(2+), the subsequent decay in the SH signal was monitored in real time as the following processes occurred: cleavage of DNA, dissociation of the enzyme from the DNA, and diffusion of the 74-bp fragment into the bulk solution leaving the 16-bp fragment attached to the microparticle. The observed decay was dependent on the concentration of Mg(2+), which functions as a cofactor and as an electrolyte. With SH spectroscopy the rehybridization dynamics between the rehybridized microparticle bound and free cleaved DNA fragments was observed in real time and label-free following the cleavage of DNA. Collectively, the experiments reported here establish SH spectroscopy as a powerful method to investigate equilibrium and time-dependent biological processes in a noninvasive and label-free way. PMID:22114185

  10. Complementarily addressed modification and cleavage of a single-stranded fragment of DNA with the aid of alkylating derivatives of oligonucleotides

    Microsoft Academic Search

    E. B. Brosalina; V. V. Vlasov; I. V. Kutyavin; S. V. Mamaev; A. G. Pletnev; M. A. Podyminogin

    1986-01-01

    The chemical modification of a 303-nucleotide single-stranded fragment of DNA by alkylating oligonucleotide derivatives bearing 4-(N-methyl-N-(2-chloroethyl)amino)benzyl groups in the 5'-terminal phosphate of the 3'-terminal ribose residue has been investigated. It has been shown that under the conditions of the formation of a complex with the DNA fragment both types of derivatives specifically alkylate nucleotides of the DNA fragments that are

  11. DNA Fingerprinting of Ralstonia paucula by Infrequent-Restriction-Site PCR and Randomly Amplified Polymorphic DNA Analysis

    PubMed Central

    Moissenet, Didier; Vu-Thien, Hoang; Benzerara, Yahia; Arlet, Guillaume

    2003-01-01

    Ralstonia paucula (formerly CDC group IV c-2) is an environmental organism that can cause serious human infections, occasionally clusters of nosocomial infections. In the present work, 26 strains of R. paucula (4 from the American Centers for Disease Control and Prevention collection, 10 from the Belgian Laboratorium voor Microbiologie [LMG] collection, and 12 French clinical isolates) were analyzed with infrequent-restriction-site PCR and randomly amplified polymorphic DNA analysis. Both techniques accurately distinguished between collection strains. Two close patterns obtained for all the French isolates suggested a clonal strain. Two LMG collection strains originating from human sources in the United States also showed patterns close to those of French isolates. PMID:14662974

  12. DNA fingerprinting of Ralstonia paucula by infrequent-restriction-site PCR and randomly amplified polymorphic DNA analysis.

    PubMed

    Moissenet, Didier; Vu-Thien, Hoang; Benzerara, Yahia; Arlet, Guillaume

    2003-12-01

    Ralstonia paucula (formerly CDC group IV c-2) is an environmental organism that can cause serious human infections, occasionally clusters of nosocomial infections. In the present work, 26 strains of R. paucula (4 from the American Centers for Disease Control and Prevention collection, 10 from the Belgian Laboratorium voor Microbiologie [LMG] collection, and 12 French clinical isolates) were analyzed with infrequent-restriction-site PCR and randomly amplified polymorphic DNA analysis. Both techniques accurately distinguished between collection strains. Two close patterns obtained for all the French isolates suggested a clonal strain. Two LMG collection strains originating from human sources in the United States also showed patterns close to those of French isolates. PMID:14662974

  13. Detection of shifts in microbial community structure and diversity in soil caused by copper contamination using amplified ribosomal DNA restriction analysis

    Microsoft Academic Search

    Eric Smit; Paula Leeflang; Karel Wernars

    1997-01-01

    Amplified ribosomal DNA restriction analysis (ARDRA) was used for assessing the effect of copper contamination on the microbial community in soil. ARDRA is a DNA fingerprinting technique based on PCR amplification of 16S ribosomal DNA using primers for conserved regions, followed by restriction enzyme digestions and agarose gel electrophoresis. This results in distinguishable fingerprints for different bacterial species. Microbial community

  14. Host-Mediated Modification ofPvuII Restriction in Mycobacterium tuberculosis

    Microsoft Academic Search

    DICK VANSOOLINGEN; PETRA E. W. DEHAAS; ROBERT M. BLUMENTHAL; KRISTIN KREMER; MARCEL SLUIJTER; JOKE E. M. PIJNENBURG; LEO M. SCHOULS; JELLE E. R. THOLE; MIRJAM W. G. DESSENS-KROON; JAN D. A. VANEMBDEN; ANDPETER W. M. HERMANS

    1996-01-01

    Restriction endonucleasePvuII plays a central role in restriction fragment length polymorphism analysis of MycobacteriumtuberculosiscomplexisolateswithIS6110asageneticmarker.Wehaveinvestigatedthebasisfor an apparent dichotomy in PvuII restriction fragment patterns observed among strains of the M. tuberculosis complex. The chromosomal regions of two modifiedPvuII restriction sites, located upstream of thekatGgene and downstream of an IS1081 insertion sequence, were studied in more detail. An identical 10-bp DNA sequence (CAGCTGGAGC)

  15. Complementarily addressed modification and cleavage of a single-stranded fragment of DNA with the aid of alkylating derivatives of oligonucleotides

    SciTech Connect

    Brosalina, E.B.; Vlasov, V.V.; Kutyavin, I.V.; Mamaev, S.V.; Pletnev, A.G.; Podyminogin, M.A.

    1986-12-01

    The chemical modification of a 303-nucleotide single-stranded fragment of DNA by alkylating oligonucleotide derivatives bearing 4-(N-methyl-N-(2-chloroethyl)amino)benzyl groups in the 5'-terminal phosphate of the 3'-terminal ribose residue has been investigated. It has been shown that under the conditions of the formation of a complex with the DNA fragment both types of derivatives specifically alkylate nucleotides of the DNA fragments that are located directly adjacent to the sections complementary to the oligonucleotides bearing the reactive groups. Alkylation takes place with a high efficiency, and the DNA fragment can be cleaved specifically at the position of the alkylated nucleotides.

  16. A new mtDNA mutation showing accumulation with time and restriction to skeletal muscle.

    PubMed Central

    Weber, K; Wilson, J N; Taylor, L; Brierley, E; Johnson, M A; Turnbull, D M; Bindoff, L A

    1997-01-01

    We have identified a new mutation in mtDNA, involving tRNALeu(CUN) in a patient manifesting an isolated skeletal myopathy. This heteroplasmic A-->G transition at position 12320 affects the T psi C loop at a conserved site and was not found in 120 controls. Analysis of cultured fibroblasts, white blood cells/platelets, and skeletal muscle showed that only skeletal muscle contained the mutation and that only this tissue demonstrated a biochemical defect of respiratory-chain activity. In a series of four muscle-biopsy specimens taken over a 12-year period, there was a gradual increase, from 70% to 90%, in the overall level of mutation, as well as a marked clinical deterioration. Single-fiber PCR confirmed that the proportion of mutant mtDNA was highest in cytochrome c oxidase-negative fibers. This study, which reports a mutation involving tRNALeu(CUN), demonstrates clearly that mtDNA point mutations can accumulate over time and may be restricted in their tissue distribution. Furthermore, clinical deterioration seemed to follow the increase in the level of mutation, although, interestingly, the appearance of fibers deficient in respiratory-chain activity showed a lag period. Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:9012410

  17. Comparison of sieving matrices for on-the-fly fluorescence lifetime detection of dye-labeled DNA fragments

    Microsoft Academic Search

    Lijuan Li; Linda B. McGown

    2001-01-01

    Commercially available, replaceable sieving matrices and their solvent modulated forms were evaluated for use in on-the-fly\\u000a fluorescence lifetime detection of dye-labeled DNA fragments in capillary electrophoresis. The fragments were labeled with\\u000a dyes that can be excited by the 488 nm line of an argon ion laser and have lifetimes in the range of 0.8 ns to 3.8 ns. The\\u000a sieving

  18. Novel apparatus to measure hyperthermal heavy ion damage to DNA: strand breaks, base loss, and fragmentation.

    PubMed

    Sellami, L; Lacombe, S; Hunting, D; Wagner, R J; Huels, M A

    2007-08-01

    We have developed a novel apparatus that allows us to irradiate nonvolatile organic films of high mass (1-100 microg range) spread out over a large surface area (42 cm(2)) with low energy (kT-100 eV) heavy ions and to quantitatively analyze the film substance via standard biochemical techniques afterwards. Here we discuss the details of the apparatus and method and show that it allows us to measure substantial damage to double stranded DNA molecules (plasmids) and its fundamental subunits induced by heavy ions with unprecedented low energies, i.e., 2.5 eV/amu; these energies correspond to track end energies of stopping ions or secondary ions created along primary ion tracks. We find that hyperthermal Ar(+) ions interacting with plasmid DNA will lead to the formation of single and double strand breaks, as well as fragmentation of nucleosides, which also involve chemical modifications and site specific rupture along the N1-C1 glycosidic bond, resulting in base release. In cells, such localized clustered damage will enhance the severity of DNA strand lesions, thus making them harder to repair. PMID:17764359

  19. Identification of individual herbal drugs in tea mixtures using restriction analysis of ITS DNA and real-time PCR.

    PubMed

    Slanc, P; Ravnikar, M; Strukelj, B

    2006-11-01

    We have studied a sedative tea made of Valerianae radix (Valeriana officinalis L.), Lupuli strobuli (Humulus lupulus L.), Melissae folium (Melissa officinalis L.) and Menthae piperitae folium (Mentha piperita L.). In order to identify the constituent drugs a method was established involving amplification of the internal transcribed spacers (ITS) region of nuclear ribosomal DNA on the basis of restriction analysis and real-time PCR. ITS regions of individual drugs were amplified and sequenced. Restriction analysis was performed with selected restriction endonucleases Nae I, PshA I and Xcm I. Real-time PCR was carried out, using primers specifically designed for each individual herbal drug. Real-time PCR proved to be a method for identifying individual herbal drugs in a tea mixture with a single DNA extraction in a single PCR run, since its limit of detection is lower than that for restriction analysis. PMID:17152982

  20. T-BAPS: A Bayesian Statistical Tool for Comparison of Microbial Communities Using Terminal-restriction Fragment Length Polymorphism (T-RFLP) Data

    Microsoft Academic Search

    Jing Tang; Jinglun Tao; Hidetoshi Urakawa; Jukka Corander

    2007-01-01

    The investigation of microbial communities is an essential part of the study of the biosphere. Flexible molecular fingerprinting tools such as terminal-restriction fragment length polymorphism (T-RFLP) analysis are often applied in the studies to enable the characterization of the microbial population. However, such data have so far been primarily analyzed using conventional clustering methods. Here we introduce a Bayesian model-based

  1. Identification of a restriction fragment length polymorphism associated with a deletion that maps in a transcriptionally active open-reading frame, orfX , in Mycobacterium tuberculosis Erdman

    Microsoft Academic Search

    N. Dasgupta; J. Sivaswami Tyagi

    1998-01-01

    A two-component system of Mycobacterium tuberculosis H37Rv, designated as devR-devS , was identified in our laboratory, that encodes a response regulator and a histidine sensor-kinase respectively. Southern analysis of the devR-devS locus in the genomes of M. tuberculosis H37Rv, H37Ra and Erdman indicated the presence of anEco RI restriction fragment length polymorphism (RFLP) in the Erdman strain. Studies employing polymerase

  2. Close association of predominant genotype of herpes simplex virus type 1 with eczema herpeticum analyzed using restriction fragment length polymorphism of polymerase chain reaction

    Microsoft Academic Search

    Masami Yoshida; Kenichi Umene

    2003-01-01

    Herpes simplex virus type 1 (HSV-1) strains belonging to the same genotype can possibly share biological properties and clinical manifestations common to the genotype. We classified previously 66 HSV-1 strains into 35 genotypes (F1–F35) using restriction fragment length polymorphism (RFLP) and F1 and F35 genotypes were revealed to be predominant [Arch. Virol. 13 (1993) 29]. It was found later that

  3. Rapid Detection of K-ras Mutations in Bile by Peptide Nucleic Acid-Mediated PCR Clamping and Melting Curve Analysis: Comparison with Restriction Fragment Length Polymorphism Analysis

    Microsoft Academic Search

    Chiung-Yu Chen; Shu-Chu Shiesh; Sheu-Jen Wu

    Background: Current methods for detection of K-ras gene mutations are time-consuming. We aimed to de- velop a one-step PCR technique using fluorescent hy- bridization probes and competing peptide nucleic acid oligomers to detect K-ras mutations in bile and to compare the efficacy with restriction fragment length polymorphism (RFLP) analysis. Methods: Bile samples were obtained from 116 patients with biliary obstruction,

  4. Cloning of the spoT Gene of “Candidatus Phlomobacter fragariae” and Development of a PCR-Restriction Fragment Length Polymorphism Assay for Detection of the Bacterium in Insects

    PubMed Central

    Foissac, Xavier; Danet, Jean-Luc; Zreik, Leyla; Gandar, Jeanne; Nourrisseau, Jean-Georges; Bové, Joseph-Marie; Garnier, Monique

    2000-01-01

    Marginal chlorosis is a new disease of strawberry in which the uncultured phloem-restricted proteobacterium “Candidatus Phlomobacter fragariae” is involved. In order to identify the insect(s) vector(s) of this bacterium, homopteran insects have been captured. Because a PCR test based on the 16S rRNA gene (rDNA) applied to these insects was unable to discriminate between “P. fragariae” and other insect-associated proteobacteria, isolation of “P. fragariae” genes other than 16S rDNA was undertaken. Using comparative randomly amplified polymorphic DNAs, an amplicon was specifically amplified from “P. fragariae”-infected strawberry plants. It encodes part of a “P. fragariae” open reading frame sharing appreciable homology with the spoT gene from other proteobacteria. A spoT-based PCR test combined with restriction fragment length polymorphisms was developed and was able to distinguish “P. fragariae” from other insect bacteria. None of the many leafhoppers and psyllids captured during several years in and around infected strawberry fields was found to carry “P. fragariae.” Interestingly however, the “P. fragariae” spoT sequence could be easily detected in whiteflies proliferating on “P. fragariae”-infected strawberry plants under confined greenhouse conditions but not on control whiteflies, indicating that these insects can become infected with the bacterium. PMID:10919809

  5. Highly Selective Isolation of Unknown Mutations in Diverse DNA Fragments: Toward New Multiplex Screening in Cancer1

    Microsoft Academic Search

    Subrata Chakrabarti; Brendan D. Price; Sotirios Tetradis; Edward A. Fox; Yuzhi Zhang; Gautam Maulik; Mike Makrigiorgos

    2000-01-01

    Cancer research would greatly benefit from technologies that allow simul- taneous screening of several unknown gene mutations. Lack of such methods currently hampers the large-scale detection of genetic alterations in complex DNA samples. We present a novel mismatch-capture methodology for the highly efficient isolation and amplification of mutation-containing DNA from diverse nucleic acid fragments of unknown sequence. To demonstrate the

  6. Electrochemical Sensors Based on Stationary Electrodes and Immobilized DNA or Its Fragments and the Assessment of Their Analytical Potentials

    Microsoft Academic Search

    S. S. Babkina; E. Palecek; F. Jelen; M. Fojta

    2005-01-01

    Amperometric biosensors were developed on the basis of stationary mercury-film and glassy-carbon electrodes and DNA or its fragments, oligodeoxynucleotides (ODNs), immobilized in a nitrocellulose matrix. Taking into account the high affinity of Cu(II) and Fe(III) ions to denatured DNA ((19.1 ± 0.1) ×105 and (1.4 ± 0.3) × 105 L\\/mol, respectively), a procedure was proposed for the voltammetric determination of

  7. Analysis of the Campylobacter jejuni Genome by SMRT DNA Sequencing Identifies Restriction-Modification Motifs

    PubMed Central

    O’Loughlin, Jason L.; Eucker, Tyson P.; Chavez, Juan D.; Samuelson, Derrick R.; Neal-McKinney, Jason; Gourley, Christopher R.; Bruce, James E.; Konkel, Michael E.

    2015-01-01

    Campylobacter jejuni is a leading bacterial cause of human gastroenteritis. The goal of this study was to analyze the C. jejuni F38011 strain, recovered from an individual with severe enteritis, at a genomic and proteomic level to gain insight into microbial processes. The C. jejuni F38011 genome is comprised of 1,691,939 bp, with a mol.% (G+C) content of 30.5%. PacBio sequencing coupled with REBASE analysis was used to predict C. jejuni F38011 genomic sites and enzymes that may be involved in DNA restriction-modification. A total of five putative methylation motifs were identified as well as the C. jejuni enzymes that could be responsible for the modifications. Peptides corresponding to the deduced amino acid sequence of the C. jejuni enzymes were identified using proteomics. This work sets the stage for studies to dissect the precise functions of the C. jejuni putative restriction-modification enzymes. Taken together, the data generated in this study contributes to our knowledge of the genomic content, methylation profile, and encoding capacity of C. jejuni. PMID:25695747

  8. Xanthorrhizol induced DNA fragmentation in HepG2 cells involving Bcl-2 family proteins.

    PubMed

    Tee, Thiam-Tsui; Cheah, Yew-Hoong; Meenakshii, Nallappan; Mohd Sharom, Mohd Yusof; Azimahtol Hawariah, Lope Pihie

    2012-04-20

    Xanthorrhizol is a plant-derived pharmacologically active sesquiterpenoid compound isolated from Curcuma xanthorrhiza. Previously, we have reported that xanthorrhizol inhibited the proliferation of HepG2 human hepatoma cells by inducing apoptotic cell death via caspase activation. Here, we attempt to further elucidate the mode of action of xanthorrhizol. Apoptosis in xanthorrhizol-treated HepG2 cells as observed by scanning electron microscopy was accompanied by truncation of BID; reduction of both anti-apoptotic Bcl-2 and Bcl-X(L) expression; cleavage of PARP and DFF45/ICAD proteins and DNA fragmentation. Taken together, these results suggest xanthorrhizol as a potent antiproliferative agent on HepG2 cells by inducing apoptosis via Bcl-2 family members. Hence we proposed that xanthorrhizol could be used as an anti-liver cancer drug for future studies. PMID:22465013

  9. Characterization of HIFU ablation using DNA fragmentation labeling as apoptosis stain

    NASA Astrophysics Data System (ADS)

    Anquez, Jeremie; Corréas, Jean-Michel; Pau, Bernard; Lacoste, François; Yon, Sylvain

    2012-11-01

    The goal of this work was to compare modalities to precisely quantify the extent of thermally induced lesions: gross pathology vs. histopathology vs. devascularization. Liver areas of 14 rabbits were targeted with HIFU and RF ablations in an acute study. Contrast enhanced computorized tomography (CE-CT) scan images were acquired two hours after HIFU and RF treatment to obtain the devascularized volumes of the livers. The animals were then euthanized and deep frozen. The livers were sliced and each slice was photographed and stacked yielding a volume of gross pathology. The volume VGP of the HIFU lesions were derived. The area AGP of the lesions were computed on a particular slice. The lesions were segmented as hypo intense (devascularized) regions on CE-CT images and their volumes VC were computed. The ratios VC/VGP were computed for all the HIFU lesions on all the 14 subjects with a mean value of 1.2. Histology was performed on the livers using Hematoxyline Eosine Staining (HES) and DNA Fragmentation labeling (TUNEL® technology) which characterizes apoptosis. Apoptotic regions of area AT were segmented on the images stained by TUNEL®. No necrosis was identified on the HES data. While TUNEL® did not mark the cores of the RF lesions as apoptotic, the periphery of HIFU and RF lesions was always recognized with TUNEL® as apoptotic. The ratio AGP/AT was computed. The mean value was 0.95 and 0.25 for HIFU and RF lesions respectively. These findings show that the devascularized territory seen on CE-CT scan coincide with the coagulated territories seen with gross pathology. Those actually correspond to cells in apoptosis. It is confirmed that HES stain does not show necrosis 2 hours after thermal ablation. TUNEL® technology for DNA fragmentation labeling appears as a useful marker for thermally induced acute lesions in the liver.

  10. A DNA Metabarcoding Study of a Primate Dietary Diversity and Plasticity across Its Entire Fragmented Range

    PubMed Central

    Quéméré, Erwan; Hibert, Fabrice; Miquel, Christian; Lhuillier, Emeline; Rasolondraibe, Emmanuel; Champeau, Julie; Rabarivola, Clément; Nusbaumer, Louis; Chatelain, Cyrille; Gautier, Laurent; Ranirison, Patrick; Crouau-Roy, Brigitte; Taberlet, Pierre; Chikhi, Lounès

    2013-01-01

    In tropical regions, most primary ecosystems have been replaced by mosaic landscapes in which species must cope with a large shift in the distribution of their habitat and associated food resources. Primates are particularly vulnerable to habitat modifications. Most species persist in small fragments surrounded by complex human-mediated matrices whose structure and connectivity may strongly influence their dispersal and feeding behavior. Behavioral plasticity appears to be a crucial parameter governing the ability of organisms to exploit the resources offered by new matrix habitats and thus to persist in fragmented habitats. In this study, we were interested in the dietary plasticity of the golden-crowned sifaka (Propithecus tattersalli), an endangered species of lemur, found only in the Daraina region in north-eastern Madagascar. We used a DNA-based approach combining the barcoding concept and Illumina next-generation sequencing to (i) describe the species diet across its entire range and (ii) evaluate the influence of landscape heterogeneity on diet diversity and composition. Faeces from 96 individuals were sampled across the entire species range and their contents were analyzed using the trnL metabarcoding approach. In parallel, we built a large DNA reference database based on a checklist of the plant species of the Daraina region. Our results suggest that golden-crowned sifakas exhibit remarkable dietary diversity with at least 130 plant species belonging to 80 genera and 49 different families. We highlighted an influence of both habitat type and openness on diet composition suggesting a high flexibility of foraging strategies. Moreover, we observed the presence of numerous cultivated and naturalized plants in the faeces of groups living in forest edge areas. Overall, our findings support our initial expectation that P. tattersalli is able to cope with the current level of alteration of the landscape and confirm our previous results on the distribution and the dispersal ability of this species. PMID:23527060

  11. Selective propagation of functional mtDNA during oogenesis restricts the transmission of a deleterious mitochondrial variant

    PubMed Central

    Hill, Jahda H.; Chen, Zhe; Xu, Hong

    2014-01-01

    Though mitochondrial DNA is prone to mutation and few mtDNA repair mechanisms exist1, crippling mitochondrial mutations are exceedingly rare2. Recent studies demonstrated strong purifying selection in the mouse female germline3,4. However, the mechanisms underlying the positive selection of healthy mitochondria remain to be elucidated. We visualized mtDNA replication during Drosophila oogenesis. We found that mtDNA replication commenced prior to oocyte determination during the late germarium stage, and was dependent on mitochondrial fitness. We isolated a temperature-sensitive lethal mtDNA mutation, mt:CoIT300I, which displayed reduced mtDNA replication in the germarium at the restrictive temperature. Additionally, the frequency of mt:CoIT300I in heteroplasmic flies was decreased both through oogenesis and over multiple generations at the restrictive temperature. Furthermore, we determined that selection against mt:CoIT300I overlaps with the timing of selective replication of mtDNA in the germarium. These findings establish a previously uncharacterized developmental mechanism for selective amplification of healthy mtDNA, which may be evolutionarily conserved to limit transmission of deleterious mutations. PMID:24614072

  12. Structure and cleavage activity of the tetrameric MspJI DNA modification-dependent restriction endonuclease

    PubMed Central

    Horton, John R.; Mabuchi, Megumu Yamada; Cohen-Karni, Devora; Zhang, Xing; Griggs, Rose M.; Samaranayake, Mala; Roberts, Richard J.; Zheng, Yu; Cheng, Xiaodong

    2012-01-01

    The MspJI modification-dependent restriction endonuclease recognizes 5-methylcytosine or 5-hydroxymethylcytosine in the context of CNN(G/A) and cleaves both strands at fixed distances (N12/N16) away from the modified cytosine at the 3?-side. We determined the crystal structure of MspJI of Mycobacterium sp. JLS at 2.05-Å resolution. Each protein monomer harbors two domains: an N-terminal DNA-binding domain and a C-terminal endonuclease. The N-terminal domain is structurally similar to that of the eukaryotic SET and RING-associated domain, which is known to bind to a hemi-methylated CpG dinucleotide. Four protein monomers are found in the crystallographic asymmetric unit. Analytical gel-filtration and ultracentrifugation measurements confirm that the protein exists as a tetramer in solution. Two monomers form a back-to-back dimer mediated by their C-terminal endonuclease domains. Two back-to-back dimers interact to generate a tetramer with two double-stranded DNA cleavage modules. Each cleavage module contains two active sites facing each other, enabling double-strand DNA cuts. Biochemical, mutagenesis and structural characterization suggest three different monomers of the tetramer may be involved respectively in binding the modified cytosine, making the first proximal N12 cleavage in the same strand and then the second distal N16 cleavage in the opposite strand. Both cleavage events require binding of at least a second recognition site either in cis or in trans. PMID:22848107

  13. A population genetics study of Anopheles darlingi (Diptera: Culicidae) from Colombia based on random amplified polymorphic DNA-polymerase chain reaction and amplified fragment lenght polymorphism markers

    Microsoft Academic Search

    Ranulfo González; Richard Wilkerson; Marco Fidel Suárez; Felipe García; Gerardo Gallego; Heiber Cárdenas; Carmen Elisa Posso; Myriam Cristina Duque

    2007-01-01

    The genetic variation and population structure of three populations of Anopheles darlingi from Colombia were studied using random amplified polymorphic markers (RAPDs) and amplified fragment length polymor- phism markers (AFLPs). Six RAPD primers produced 46 polymorphic fragments, while two AFLP primer com- binations produced 197 polymorphic fragments from 71 DNA samples. Both of the evaluated genetic markers showed the presence

  14. Study of mechanisms of some caffeine biological effects via computer simulation of its interactions with DNA fragments

    Microsoft Academic Search

    A. S. Deriabina; T. I. Grokhlina; N. A. Polteva; E. González; V. I. Poltev

    2006-01-01

    To approach atomic level mechanisms of caffeine biological activity, molecular mechanics computations of interactions between caffeine molecule and DNA duplex fragments, containing four nucleotide pairs, have been performed. The calculations reveal a set of energy minima referring to rather compact caffeine arrangement in both major and minor grooves of the helix. The set includes the minima correspondent to the H-bond

  15. Using Restriction Mapping to Teach Basic Skills in the Molecular Biology Lab

    ERIC Educational Resources Information Center

    Walsh, Lauren; Shaker, Elizabeth; De Stasio, Elizabeth A.

    2007-01-01

    Digestion of DNA with restriction enzymes, calculation of volumes and concentrations of reagents for reactions, and the separation of DNA fragments by agarose gel electrophoresis are common molecular biology techniques that are best taught through repetition. The following open-ended, investigative laboratory exercise in plasmid restriction

  16. Comparison of sieving matrices for on-the-fly fluorescence lifetime detection of dye-labeled DNA fragments.

    PubMed

    Li, L; McGown, L B

    2001-02-01

    Commercially available, replaceable sieving matrices and their solvent modulated forms were evaluated for use in on-the-fly fluorescence lifetime detection of dye-labeled DNA fragments in capillary electrophoresis. The fragments were labeled with dyes that can be excited by the 488 nm line of an argon ion laser and have lifetimes in the range of 0.8 ns to 3.8 ns. The sieving matrices and buffer systems included poly(vinylpyrrolidone) (PVP), poly(ethyleneoxide) (PEO), hydroxyethylcellulose (HEC), Tris-borate-EDTA (TBE) and Tris-TAPS-EDTA buffers modified with DMSO and formamide. Selection of the optimal sieving matrix is based on the separation efficiency and the enhancement of lifetime resolution of DNA fragments. Best results for both electrophoretic resolution and lifetime detection were obtained using a poly(ethyleneoxide)/TBE gel buffer in the presence of 10% formamide. PMID:11293703

  17. Denaturing high-performance liquid chromatography using the WAVE DNA fragment analysis system.

    PubMed

    Donohoe, Elizabeth

    2005-01-01

    Denaturing high-performance liquid chromatography (DHPLC) is a chromatographic mutation analysis technique that is based on temperature-dependent separation of DNA containing mismatched base pairs from polymerase chain reaction (PCR)-amplified DNA fragments. The WAVE system, developed for DHPLC analysis, allows for unattended analysis of 96 samples directly from a PCR plate under a number of different conditions. It utilizes a Peltier cooling platform to maintain sample integrity. Sample detection is achieved via UV absorbance at 260 nm, thereby avoiding the cost, safety, variability, or waste disposal issues associated with radioisotopic, enzyme-linked, or fluorescence detection systems. There are four key aspects to successfully detecting mutations on the WAVE: (1) PCR primer design, (2) PCR protocol, (3) separation gradient, and (4) separation temperature. Provided these procedures are carried out correctly, almost 100% detection of single-nucleotide polymorphisms (SNPs) and small deletion/insertion mutations can be achieved. For this reason, DHPLC is a powerful tool for identifying mutations in candidate genes for hypertension. PMID:16028684

  18. Rapid Identification and Typing of Staphylococcus aureus by PCR-Restriction Fragment Length Polymorphism Analysis of the aroA Gene

    Microsoft Academic Search

    JAVIER YUGUEROS MARCOS; ALBERTO CASCON SORIANO; MARIAS ANCHEZ SALAZAR; CARMEN HERNANZ MORAL; SUSANA SUAREZ RAMOS; MARK S. SMELTZER; GERMAN NAHARRO CARRASCO

    1999-01-01

    The Staphylococcus aureus aroA gene, which encodes 5-enolpyruvylshikimate-3-phosphate synthase, was used as a target for the amplification of a 1,153-bp DNA fragment by PCR with a pair of primers of 24 and 19 nucleotides. The PCR products, which were detected by agarose gel electrophoresis, were amplified from all S. aureus strains so far analyzed (reference strains and isolates from cows

  19. Cloning of human heparan sulfate proteoglycan core protein, assignment of the gene (HSPG2) to 1p36.1----p35 and identification of a BamHI restriction fragment length polymorphism.

    PubMed

    Kallunki, P; Eddy, R L; Byers, M G; Kestilä, M; Shows, T B; Tryggvason, K

    1991-10-01

    We have isolated a cDNA coding for the core protein of the large basement membrane heparan sulfate proteoglycan (HSPG) from a human fibrosarcoma cell (HT1080) library. The library was screened with a mouse cDNA probe and one clone obtained, with a 1.5-kb insert, was isolated and sequenced. The sequence contained an open reading frame coding for 507 amino acid residues with a 84% identity to the corresponding mouse sequence. This amino acid sequence contained several cysteine-rich internal repeats similar to those found in component chains of laminin. The HSPG cDNA clone was used to assign the gene (HSPG2) to the p36.1----p35 region of chromosome 1 using both somatic cell hybrid and in situ hybridization. In the study of the polymorphisms of the locus, a BamHI restriction fragment length polymorphism was identified in the gene. This polymorphism displayed bands of 23 and 12 kb with allele frequencies of 76 and 24%, respectively. PMID:1685141

  20. Strains and Clones of Trypanosoma cruzi Can Be Characterized by Pattern of Restriction Endonuclease Products of Kinetoplast DNA Minicircles

    Microsoft Academic Search

    C. Morel; E. Chiari; E. Plessmann Camargo; D. M. Mattei; A. J. Romanha; L. Simpson

    1980-01-01

    A simple method was developed for the characterization of different strains of Trypanosoma cruzi. T. cruzi stocks isolated from vectors or by hemoculture from patients with Chagas disease could be grouped in subpopulations having similar patterns of restriction endonuclease products of kinetoplast DNA minicircles. We designate such subpopulations by the term ``schizodemes.'' Furthermore, it is shown that, from a given

  1. A comparison of synthetic oligodeoxynucleotides, DNA fragments and AAV-1 for targeted episomal and chromosomal gene repair

    PubMed Central

    Leclerc, Xavier; Danos, Olivier; Scherman, Daniel; Kichler, Antoine

    2009-01-01

    Background Current strategies for gene therapy of inherited diseases consist in adding functional copies of the gene that is defective. An attractive alternative to these approaches would be to correct the endogenous mutated gene in the affected individual. This study presents a quantitative comparison of the repair efficiency using different forms of donor nucleic acids, including synthetic DNA oligonucleotides, double stranded DNA fragments with sizes ranging from 200 to 2200 bp and sequences carried by a recombinant adeno-associated virus (rAAV-1). Evaluation of each gene repair strategy was carried out using two different reporter systems, a mutated eGFP gene or a dual construct with a functional eGFP and an inactive luciferase gene, in several different cell systems. Gene targeting events were scored either following transient co-transfection of reporter plasmids and donor DNAs, or in a system where a reporter construct was stably integrated into the chromosome. Results In both episomal and chromosomal assays, DNA fragments were more efficient at gene repair than oligonucleotides or rAAV-1. Furthermore, the gene targeting frequency could be significantly increased by using DNA repair stimulating drugs such as doxorubicin and phleomycin. Conclusion Our results show that it is possible to obtain repair frequencies of 1% of the transfected cell population under optimized transfection protocols when cells were pretreated with phleomycin using rAAV-1 and dsDNA fragments. PMID:19379497

  2. RADOM, an efficient in vivo method for assembling designed DNA fragments up to 10 kb long in Saccharomyces cerevisiae.

    PubMed

    Lin, Qiuhui; Jia, Bin; Mitchell, Leslie A; Luo, Jingchuan; Yang, Kun; Zeller, Karen I; Zhang, Wenqian; Xu, Zhuwei; Stracquadanio, Giovanni; Bader, Joel S; Boeke, Jef D; Yuan, Ying-Jin

    2015-03-20

    We describe rapid assembly of DNA overlapping multifragments (RADOM), an improved assembly method via homologous recombination in Saccharomyces cerevisiae, which combines assembly in yeasto with blue/white screening in Escherichia coli. We show that RADOM can successfully assemble ?3 and ?10 kb DNA fragments that are highly similar to the yeast genome rapidly and accurately. This method was tested in the Build-A-Genome course by undergraduate students, where 125 ?3 kb "minichunks" from the synthetic yeast genome project Sc2.0 were assembled. Here, 122 out of 125 minichunks achieved insertions with correct sizes, and 102 minichunks were sequenced verified. As this method reduces the time-consuming and labor-intensive efforts of yeast assembly by improving the screening efficiency for correct assemblies, it may find routine applications in the construction of DNA fragments, especially in hierarchical assembly projects. PMID:24895839

  3. Elevated dietary intake of Zn-methionate is associated with increased sperm DNA fragmentation in the boar.

    PubMed

    García-Contreras, Adelfa; De Loera, Yasmin; García-Artiga, Carlos; Palomo, Antonio; Guevara, Jesús A; Herrera-Haro, José; López-Fernández, Carmen; Johnston, Steve; Gosálvez, Jaime

    2011-05-01

    Boars fed on ration of 200 ppm Zn methionate showed a significant increase (P < 0.001) in sperm DNA fragmentation when compared to animals fed on non-supplemented and rations containing 150 ppm Zn methionate. There was a positive correlation (R2 = 0.207; P = 0.002) between % sperm DNA fragmentation (SDF) and the concentration of Zn in spermatozoa. Increased Zn in the diet also resulted in a non-proportional increase in Zn concentration in the testis and spermatozoa but not in the epididymis; Zn in sperm accumulated at levels up to 50 times higher than that found in the seminal plasma and 10-13 times that found in the epididymis and testis, respectively. These results show that supplementation of dietary Zn at a concentration of 200 ppm had an adverse effect on boar sperm DNA quality and may be related to the ability of spermatozoa to accumulate Zn during spermiogenesis. PMID:21182932

  4. Specific detection of Mycobacterium paratuberculosis by DNA hybridisation with a fragment of the insertion element IS900.

    PubMed Central

    Moss, M T; Green, E P; Tizard, M L; Malik, Z P; Hermon-Taylor, J

    1991-01-01

    This paper describes the evaluation of a newly developed DNA probe for Mycobacterium paratuberculosis. DNA probe PCR278 is a 278 bp fragment obtained by polymerase chain reaction (PCR) amplification of the 5'-region of IS900, an insertion element contained in the genome of M paratuberculosis. This DNA probe can specifically distinguish M paratuberculosis from a wide range of other organisms, including members of the M avium-M intracellulare complex. When used in conjunction with the PCR amplification technique DNA probe PCR278 could detect as little as 10 fg (equivalent to two genomes) starting material of M paratuberculosis genomic DNA. Use of PCR amplification assays based on IS900, for the detection of M paratuberculosis, and homologous IS elements found in disease isolates of M avium should greatly help our understanding of the role of these organisms in Crohn's disease and other chronic inflammatory disorders. Images Figure 1 Figure 2 Figure 3 PMID:1851124

  5. Phylogenetic relationships of subtribe Ecliptinae (Asteraceae: Heliantheae) based on chloroplast DNA restriction site data.

    PubMed

    Panero, J L; Jansen, R K; Clevinger, J A

    1999-03-01

    Phylogenetic analysis of chloroplast DNA restriction site data for 76 of the 302 genera of Heliantheae sensu lato using 16 restriction endonucleases reveals that subtribe Ecliptinae is polyphyletic and that its genera are distributed in four different lineages. The ecliptinous genera Squamopappus, Podachaenium, Verbesina, and Tetrachyron (of the Neurolaeninae), along with other members of subtribe Neurolaeninae are the basalmost clades of the paleaceous Heliantheae. The mostly temperate species of subtribe Ecliptinae (exemplified by Balsamorhiza, Borrichia, Chrysogonum, Engelmannia, Silphium, Vigethia, and Wyethia) are strongly nested in a clade with the Mesoamerican monotypic genus Rojasianthe as basal. The genera characterized by marcescent ray corollas traditionally classified in subtribe Zinniinae constitute a strongly supported group sister to Acmella, Spilanthes, and Salmea. The largest clade of ecliptinous genera is the most recently derived group within Heliantheae sampled. This large group of mostly Neotropical lowland genera (variously characterized by their winged cypselae, foliaceous phyllaries, and opposite phyllotaxy and exemplified by Perymenium, Wedelia, and Zexmenia) has been and continues to be the most challenging group from a taxonomic standpoint. The study provides new insights as to their relationships that will have a positive impact in future monographic studies of the group. The genera of the Espeletiinae form a monophyletic clade and are sister to members of the Milleriinae and Melampodiinae. This result is consistent with their traditional taxonomic placement with genera such as Smallanthus with which they share a tendency for functionally staminate disc flowers. The phylogenetically enigmatic genus Montanoa is sister to Melampodium. Members of subtribe Galinsoginae are clustered in two main lineages that correspond to the traditional division of the subtribe based on pappus characteristics. There is no support for the monophyly of subtribe Galinsoginae, and the same results indicate some of its genera are paraphyletic. PMID:10077503

  6. Circulating Bacterial-Derived DNA Fragment Level Is a Strong Predictor of Cardiovascular Disease in Peritoneal Dialysis Patients

    PubMed Central

    Szeto, Cheuk-Chun; Kwan, Bonnie Ching-Ha; Chow, Kai-Ming; Kwok, Jeffrey Sung-Shing; Lai, Ka-Bik; Cheng, Phyllis Mei-Shan; Pang, Wing-Fai; Ng, Jack Kit-Chung; Chan, Michael Ho-Ming; Lit, Lydia Choi-Wan; Leung, Chi-Bon; Li, Philip Kam-Tao

    2015-01-01

    Background Circulating bacterial DNA fragment is related to systemic inflammatory state in peritoneal dialysis (PD) patients. We hypothesize that plasma bacterial DNA level predicts cardiovascular events in new PD patients. Methods We measured plasma bacterial DNA level in 191 new PD patients, who were then followed for at least a year for the development of cardiovascular event, hospitalization, and patient survival. Results The average age was 59.3 ± 11.8 years; plasma bacterial DNA level 34.9 ± 1.5 cycles; average follow up 23.2 ± 9.7 months. At 24 months, the event-free survival was 86.1%, 69.8%, 55.4% and 30.8% for plasma bacterial DNA level quartiles I, II, III and IV, respectively (p < 0.0001). After adjusting for confounders, plasma bacterial DNA level, baseline residual renal function and malnutrition-inflammation score were independent predictors of composite cardiovascular end-point; each doubling in plasma bacterial DNA level confers a 26.9% (95% confidence interval, 13.0 – 42.5%) excess in risk. Plasma bacterial DNA also correlated with the number of hospital admission (r = -0.379, p < 0.0001) and duration of hospitalization for cardiovascular reasons (r = -0.386, p < 0.0001). Plasma bacterial DNA level did not correlate with baseline arterial pulse wave velocity (PWV), but with the change in carotid-radial PWV in one year (r = -0.238, p = 0.005). Conclusions Circulating bacterial DNA fragment level is a strong predictor of cardiovascular event, need of hospitalization, as well as the progressive change in arterial stiffness in new PD patients. PMID:26010741

  7. Molecular identification of similar species of the genus Biomphalaria (Mollusca: Planorbidae) determined by a polymerase chain reaction-restriction fragment length polymorphism.

    PubMed

    Caldeira, R L; Vidigal, T H; Paulinelli, S T; Simpson, A J; Carvalho, O S

    1998-01-01

    The freshwater snails Biomphalaria straminea, B. intermedia, B. kuhniana and B. peregrina, are morphologically similar; based on this similarity the first three species were therefore grouped in the complex B. straminea. The morphological identification of these species is based on characters such as vaginal wrinkling, relation between prepuce: penial sheath:deferens vas and number of muscle layers in the penis wall. In this study the polymerase chain reaction restriction fragment length polymorphism technique was used for molecular identification of these molluscs. This technique is based on the amplification of the internal transcribed spacer regions ITS1 e ITS2 of the ribosomal RNA gene and subsequent digestion of these fragments by restriction enzymes. Six enzymes were tested: Dde I, Mn1 I, Hae III, Rsa I, Hpa II e Alu I. The restriction patterns obtained with DdeI presented the best profile for separation of the four species of Biomphalaria. The profiles obtained with all the enzymes were used to estimate the genetic distances among the species through analysis of common banding patterns. PMID:9921355

  8. Chloroplast DNA inheritance and variation in Leucadendron species (Proteaceae) as revealed by PCR-RFLP

    Microsoft Academic Search

    M. Pharmawati; G. Yan; R. Sedgley; P. M. Finnegan

    2004-01-01

    The inheritance of chloroplast DNA (cpDNA) in Leucadendron species was studied by polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) analysis. A total of 100 progeny from five interspecific crosses involving seven parental species were tested, and all progeny exhibited the cpDNA restriction fragment pattern of the female parent, indicating that cpDNA in Leucadendron is maternally inherited. PCR-RFLP was also employed

  9. Crystallization and preliminary crystallographic analysis of the type IIL restriction enzyme MmeI in complex with DNA

    SciTech Connect

    Callahan, Scott J.; Morgan, Richard D.; Jain, Rinku; Townson, Sharon A.; Wilson, Geoffrey G.; Roberts, Richard J.; Aggarwal, Aneel K. (Sinai); (NEB)

    2012-05-29

    Type IIL restriction enzymes have rejuvenated the search for user-specified DNA binding and cutting. By aligning and contrasting the highly comparable amino-acid sequences yet diverse recognition specificities across the family of enzymes, amino acids involved in DNA binding have been identified and mutated to produce alternative binding specificities. To date, the specificity of MmeI (a type IIL restriction enzyme) has successfully been altered at positions 3, 4 and 6 of the asymmetric TCCRAC (where R is a purine) DNA-recognition sequence. To further understand the structural basis of MmeI DNA-binding specificity, the enzyme has been crystallized in complex with its DNA substrate. The crystal belonged to space group P1, with unit-cell parameters a = 61.73, b = 94.96, c = 161.24 {angstrom}, {alpha} = 72.79, {beta} = 89.12, {gamma} = 71.68{sup o}, and diffracted to 2.6 {angstrom} resolution when exposed to synchrotron radiation. The structure promises to reveal the basis of MmeI DNA-binding specificity and will complement efforts to create enzymes with novel specificities.

  10. DNA Fragmentation and DSB correlation Induced in Human Fibroblasts by Accelerated 56Fe Ions of Differing Energies

    NASA Astrophysics Data System (ADS)

    Antonelli, F.; Belli, M.; Campa, A.; Dini, V.; Esposito, G.; Furusawa, Y.; Simone, G.; Sorrentino, E.; Tabocchini, M. A.

    HZE particles from space radiation raise an important protection concern during long-term astronauts travels Although these particles are less abundant than protons they are more effective in damaging biological systems It is thought that this is due to the frequent production of spatially correlated DNA damaged sites particularly double strand breaks DSB since this correlation can strongly affect the repair capability of the cells In this work we have studied the DNA fragmentation induced in human fibroblasts by accelerated 56 Fe ions of four different energies i e 115 MeV u 414 MeV u 1 GeV u and 5 GeV u and by gamma-rays used as reference radiation DNA fragmentation was studied in various size ranges varying from 1 to 5700 kbp using Pulsed or Constant Field Gel Electrophoresis The DSB yields have been derived from fragmentation in the overall range as well as in the two ranges 1-23 and 23-5700 kbp The overall DSB yield slightly increased with the ion energy maily due to the contribution of the 23-5700 kbp fragments while that of small fragments 1-23 kbp was almost constant Accordingly the relative biological effectiveness RBE for DSB induction increased with energy from about 1 3 at 115 MeV u to about 1 8 at about 5 GeV u i e less than the RBE for chromosome aberration and cell inactivation The degree of spatial correlation of DSB was evaluated through the departure from the randomness of the fragment distribution with a simple theoretical tool that we have recently introduced To this aim a parameter R was used

  11. Improved separation of double-stranded DNA fragments by capillary electrophoresis using poly(ethylene oxide) solution containing colloids.

    PubMed

    Huang, Ming-Feng; Huang, Chih-Ching; Chang, Huan-Tsung

    2003-09-01

    The analysis of double-stranded (ds) DNA fragments by capillary electrophoresis (CE) using poly(ethylene oxide) (PEO) solution containing gold nanoparticles (GNPs) is presented, focusing on evaluating size dependence of the GNPs and PEO on resolution and speed. To prevent the interaction of the capillary wall with DNA, the capillary was dynamically coated with polyvinylpyrrolidone. Using different PEO solutions containing GNPs ranging in diameter from 3.5 to 56 nm, we have achieved reproducible, rapid, and high-resolution DNA separations. The results indicate that the sizes of PEO and GNPs as well as the concentration of PEO affect resolution. The separation of DNA ranging in size from 8 to 2176 base pairs (bp) was accomplished in 5 min using 0.2% PEO (8 MDa) containing 56 nm GNPs. We have also demonstrated the separations of the DNA fragments ranging from 5 to 40 kbp using 0.05% PEO (2 MDa) containing 13 nm GNPs or 0.05% PEO (4 MDa) containing 32 nm GNPs. With very low viscosity (< 15 cP), automatic replacement of the sieving matrices is easy, indicating a great potential for high-throughput DNA analysis using capillary array electrophoresis systems. PMID:12973792

  12. Association Mapping of Disease Resistance Traits in Rainbow Trout Using Restriction Site Associated DNA Sequencing

    PubMed Central

    Campbell, Nathan R.; LaPatra, Scott E.; Overturf, Ken; Towner, Richard; Narum, Shawn R.

    2014-01-01

    Recent advances in genotyping-by-sequencing have enabled genome-wide association studies in nonmodel species including those in aquaculture programs. As with other aquaculture species, rainbow trout and steelhead (Oncorhynchus mykiss) are susceptible to disease and outbreaks can lead to significant losses. Fish culturists have therefore been pursuing strategies to prevent losses to common pathogens such as Flavobacterium psychrophilum (the etiological agent for bacterial cold water disease [CWD]) and infectious hematopoietic necrosis virus (IHNV) by adjusting feed formulations, vaccine development, and selective breeding. However, discovery of genetic markers linked to disease resistance offers the potential to use marker-assisted selection to increase resistance and reduce outbreaks. For this study we sampled juvenile fish from 40 families from 2-yr classes that either survived or died after controlled exposure to either CWD or IHNV. Restriction site?associated DNA sequencing produced 4661 polymorphic single-nucleotide polymorphism loci after strict filtering. Genotypes from individual survivors and mortalities were then used to test for association between disease resistance and genotype at each locus using the program TASSEL. After we accounted for kinship and stratification of the samples, tests revealed 12 single-nucleotide polymorphism markers that were highly associated with resistance to CWD and 19 markers associated with resistance to IHNV. These markers are candidates for further investigation and are expected to be useful for marker assisted selection in future broodstock selection for various aquaculture programs. PMID:25354781

  13. Comparison of ribosomal DNA length and restriction site polymorphisms in Gremmeniella and Ascocalyx isolates.

    PubMed Central

    Bernier, L; Hamelin, R C; Ouellette, G B

    1994-01-01

    The small subunit (SSU) and the internal transcribed spacer (ITS) of nuclear ribosomal DNA genes from 27 specimens of the fungal genera Gremmeniella and Ascocalyx were amplified by PCR. Length polymorphisms were observed in the SSU and allowed the differentiation of four groups among the isolates tested: (i) Ascocalyx abietis; (ii) Gremmeniella isolates from Picea spp.; (iii) Gremmeniella isolates from Abies balsamea; and (iv) Gremmeniella isolates from Abies sacchalinensis, Larix spp., and Pinus spp. The amplified ITS was the same length for all Gremmeniella specimens and was 60 bp longer in A. abietis. Phylogenetic analysis of length polymorphisms and of 24 restriction sites in the SSU and ITS showed that Gremmeniella isolates were more related to each other than to the Ascocalyx isolate. Furthermore, seven groups were evident within the genus Gremmeniella. Our results confirm that Gremmeniella and Ascocalyx should be kept as different taxa and suggest that the taxonomy of the former could be revised to consider isolates from Abies balsamea and from Picea spp. to be two different varieties while incorporating Gremmeniella laricina into G. abietina, as a new variety. Images PMID:7912501

  14. Diagnostic value of sperm DNA fragmentation and sperm high-magnification for predicting outcome of assisted reproduction treatment

    PubMed Central

    López, Gemma; Lafuente, Rafael; Checa, Miguel A; Carreras, Ramón; Brassesco, Mario

    2013-01-01

    Over the last years, major improvements in the field of male infertility diagnosis have been achieved. The aim of this study was to determine the diagnostic usefulness of sperm DNA integrity and sperm vacuolisation for predicting outcome in infertile couples undergoing in vitro fertilisation (IVF) and intracytoplasmic sperm injection (ICSI) treatments. A cohort study from 152 infertile couples undergoing sperm DNA fragmentation and high-magnification tests prior to an assisted reproduction treatment was designed. We found that the most predictive cutoff for pregnancy was 25.5% of DNA fragmentation with a negative predictive value of 72.7% (P=0.02). For the degree of vacuolisation, the best predictor of pregnancy was 73.5% of vacuolated sperm grades III+IV with a negative predictive value of 39.4% (P=0.09), which was not statistically significant. In conclusion, sperm DNA fragmentation greater than 25.5% could be associated with higher probability of failure IVF treatment. Regarding the results of the sperm analysis at high magnification, they do not allow us to predict whether or not patients will become pregnant. PMID:23912311

  15. Diagnostic value of sperm DNA fragmentation and sperm high-magnification for predicting outcome of assisted reproduction treatment.

    PubMed

    López, Gemma; Lafuente, Rafael; Checa, Miguel A; Carreras, Ramón; Brassesco, Mario

    2013-11-01

    Over the last years, major improvements in the field of male infertility diagnosis have been achieved. The aim of this study was to determine the diagnostic usefulness of sperm DNA integrity and sperm vacuolisation for predicting outcome in infertile couples undergoing in vitro fertilisation (IVF) and intracytoplasmic sperm injection (ICSI) treatments. A cohort study from 152 infertile couples undergoing sperm DNA fragmentation and high-magnification tests prior to an assisted reproduction treatment was designed. We found that the most predictive cutoff for pregnancy was 25.5% of DNA fragmentation with a negative predictive value of 72.7% (P=0.02). For the degree of vacuolisation, the best predictor of pregnancy was 73.5% of vacuolated sperm grades III+IV with a negative predictive value of 39.4% (P=0.09), which was not statistically significant. In conclusion, sperm DNA fragmentation greater than 25.5% could be associated with higher probability of failure IVF treatment. Regarding the results of the sperm analysis at high magnification, they do not allow us to predict whether or not patients will become pregnant. PMID:23912311

  16. Amplified fragment length polymorphism (AFLP): a review of the procedure and its applications

    Microsoft Academic Search

    M J Blears; S A De Grandis; H Lee; J T Trevors

    1998-01-01

      Amplified fragment length polymorphism (AFLP) is a novel molecular fingerprinting technique that can be applied to DNAs of\\u000a any source or complexity. Total genomic DNA is digested using two restriction enzymes. Double-stranded nucleotide adapters\\u000a are ligated to the DNA fragments to serve as primer binding sites for PCR amplification. Primers complementary to the adapter\\u000a and restriction site sequence, with additional

  17. [Restriction polymorphism of the major noncoding region of mtDNA in the indigenous population of the TUVA republic].

    PubMed

    Golubenko, M V; Puzyrev, V P; Saliukov, V B; Kucher, A N; Sanchat, N O

    1999-08-01

    Mitochondrial DNA sequence variation was examined in three rural populations of the indigenous inhabitants from the Tuva Republic. The frequencies of restriction sites within the D-loop region of mtDNA were determined. The three populations studied demonstrated similar patterns of mtDNA polymorphism. Like other Siberian populations, Tuvinians were characterized by high frequencies of the HaeIII 16517 and AspS9I (Cfr13I) 16516 restriction sites (about 75%). Moreover, in Tuvinians, a relatively low (71 to 81%) frequency of the KpnI 16129 restriction site was observed. The frequency of the mitotype differing from the Cambridge sequence by the HaeIII 16517 and KpnI 16129 sites in Tuvinians was higher than in Mongols and Russians. The features of mtDNA polymorphism point to the similarity between Tuvinians and other Siberian ethnic groups (Sel'kups in particular). This can be explained by the contribution of the Samoyed component, along with the Turkic and Mongoloid ones, to the formation of the Tuvinian ethnic group. PMID:10546115

  18. Caracterización molecular de aislamientos mexicanos del virus de la anemia infecciosa del pollo, a través del polimorfi smo en la longitud de fragmentos de restricción Molecular characterization of Mexican isolates of chicken infectious anemia virus through restriction fragment length polymorphism

    Microsoft Academic Search

    Néstor Ledesma Martínez; Tamas Fehervari; Rogelio Alonso

    Chicken infectious anemia is the main immunosuppressive disease of poultry. It is caused by a Gyrovirus. Thirty positive samples for chicken infectious anemia virus (CIAV) were identifi ed by PCR amplifi cation of a fragment of 675 bp of the amino-terminal end of the VP1 gene protein. Amplifi ed fragments were characterized by restriction fragment length polymorphism, digesting with HaeIII,

  19. Clinical Factors Associated with Sperm DNA Fragmentation in Male Patients with Infertility

    PubMed Central

    Komiya, Akira; Kato, Tomonori; Kawauchi, Yoko; Watanabe, Akihiko; Fuse, Hideki

    2014-01-01

    Objective. The clinical factors associated with sperm DNA fragmentation (SDF) were investigated in male patients with infertility. Materials and Methods. Fifty-four ejaculates from infertile Japanese males were used. Thirty-three and twenty-one were from the patients with varicoceles and idiopathic causes of infertility, respectively. We performed blood tests, including the serum sex hormone levels, and conventional and computer-assisted semen analyses. The sperm nuclear vacuolization (SNV) was evaluated using a high-magnification microscope. The SDF was evaluated using the sperm chromatin dispersion test (SCDt) to determine the SDF index (SDFI). The SDFI was compared with semen parameters and other clinical variables, including lifestyle factors. Results. The SDFI was 41.3 ± 22.2% (mean ± standard deviation) and did not depend on the cause of infertility. Chronic alcohol use increased the SDFI to 49.6 ± 23.3% compared with 33.9 ± 18.0% in nondrinkers. The SDFI was related to adverse conventional semen parameters and sperm motion characteristics and correlated with the serum FSH level. The SNV showed a tendency to increase with the SDFI. The multivariate analysis revealed that the sperm progressive motility and chronic alcohol use were significant predictors of the SDF. Conclusion. The SCDt should be offered to chronic alcohol users and those with decreased sperm progressive motility. PMID:25165747

  20. A nonradioactive restriction enzyme-mediated assay to detect DNA repair by Fe(II)/2-oxoglutarate-dependent dioxygenase.

    PubMed

    Shivange, Gururaj; Kodipelli, Naveena; Anindya, Roy

    2014-11-15

    The Escherichia coli DNA repair enzyme AlkB belongs to the Fe(II)/2-oxoglutarate-dependent dioxygenase family. It removes methyl groups from 1-methyl adenine (1-meA) and 3-methyl cytosine (3-meC) lesions present in single-stranded DNA by oxidative decarboxylation. In the current article, we describe an in vitro assay that permits rapid detection of AlkB activity. To achieve this, we generated methylated oligonucleotide using methyl methanesulfonate and then monitored DNA repair using a methylation-sensitive restriction enzyme and novel agarose gel electrophoresis system capable of resolving small oligonucleotides. Our approach overcomes several drawbacks of NAD(+)-dependent formaldehyde dehydrogenase-coupled assay and radioisotope-based assay for determining AlkB DNA repair activity. PMID:25062850

  1. Community Structure of Denitrifiers, Bacteria, and Archaea along Redox Gradients in Pacific Northwest Marine Sediments by Terminal Restriction Fragment Length Polymorphism Analysis of Amplified Nitrite Reductase (nirS) and 16S rRNA Genes

    PubMed Central

    Braker, Gesche; Ayala-del-Río, Héctor L.; Devol, Allan H.; Fesefeldt, Andreas; Tiedje, James M.

    2001-01-01

    Steep vertical gradients of oxidants (O2 and NO3?) in Puget Sound and Washington continental margin sediments indicate that aerobic respiration and denitrification occur within the top few millimeters to centimeters. To systematically explore the underlying communities of denitrifiers, Bacteria, and Archaea along redox gradients at distant geographic locations, nitrite reductase (nirS) genes and bacterial and archaeal 16S rRNA genes (rDNAs) were PCR amplified and analyzed by terminal restriction fragment length polymorphism (T-RFLP) analysis. The suitablility of T-RFLP analysis for investigating communities of nirS-containing denitrifiers was established by the correspondence of dominant terminal restriction fragments (T-RFs) of nirS to computer-simulated T-RFs of nirS clones. These clones belonged to clusters II, III, and IV from the same cores and were analyzed in a previous study (G. Braker, J. Zhou, L. Wu, A. H. Devol, and J. M. Tiedje, Appl. Environ. Microbiol. 66:2096–2104, 2000). T-RFLP analysis of nirS and bacterial rDNA revealed a high level of functional and phylogenetic diversity, whereas the level of diversity of Archaea was lower. A comparison of T-RFLPs based on the presence or absence of T-RFs and correspondence analysis based on the frequencies and heights of T-RFs allowed us to group sediment samples according to the sampling location and thus clearly distinguish Puget Sound and the Washington margin populations. However, changes in community structure within sediment core sections during the transition from aerobic to anaerobic conditions were minor. Thus, within the top layers of marine sediments, redox gradients seem to result from the differential metabolic activities of populations of similar communities, probably through mixing by marine invertebrates rather than from the development of distinct communities. PMID:11282647

  2. Fibered confocal fluorescence microscopy for imaging apoptotic DNA fragmentation at the single-cell level in vivo

    SciTech Connect

    Al-Gubory, Kais H. [Unite de Biologie du Developpement et de la Reproduction, Departement de Physiologie Animale, Institut National de la Recherche Agronomique, 78352 Jouy-en-Josas Cedex (France)]. E-mail: kais.algubory@jouy.inra.fr

    2005-11-01

    The major characteristic of cell death by apoptosis is the loss of nuclear DNA integrity by endonucleases, resulting in the formation of small DNA fragments. The application of confocal imaging to in vivo monitoring of dynamic cellular events, like apoptosis, within internal organs and tissues has been limited by the accessibility to these sites. Therefore, the aim of the present study was to test the feasibility of fibered confocal fluorescence microscopy (FCFM) to image in situ apoptotic DNA fragmentation in surgically exteriorized sheep corpus luteum in the living animal. Following intra-luteal administration of a fluorescent DNA-staining dye, YO-PRO-1, DNA cleavage within nuclei of apoptotic cells was serially imaged at the single-cell level by FCFM. This imaging technology is sufficiently simple and rapid to allow time series in situ detection and visualization of cells undergoing apoptosis in the intact animal. Combined with endoscope, this approach can be used for minimally invasive detection of fluorescent signals and visualization of cellular events within internal organs and tissues and thereby provides the opportunity to study biological processes in the natural physiological environment of the cell in living animals.

  3. Synthesis of well-defined phosphate-methylated DNA fragments: the application of potassium carbonate in methanol as deprotecting reagent.

    PubMed Central

    Kuijpers, W H; Huskens, J; Koole, L H; van Boeckel, C A

    1990-01-01

    A new deprotection procedure in the synthesis of (partially) phosphate-methylated oligodeoxynucleotides has been developed, involving treatment of fully protected DNA fragments with methanolic potassium carbonate. It is shown that base deprotection can be accomplished in potassium carbonate/methanol without affecting the methyl phosphotriesters. This methodology enables us to synthesize, both in solution and on a solid support, DNA fragments which are phosphate-methylated at defined positions. The solid phase synthesis, however, turns out to be accompanied by considerable demethylation of the phosphotriesters. It is demonstrated that this demethylation does not occur during the deprotection or work-up procedure. Furthermore, it was found that the latter side-reaction is suppressed when the standard capping procedure with acetic anhydride is included. PMID:2402444

  4. Cell-specific DNA fragmentation may be attenuated by a survivin-dependent mechanism after traumatic brain injury in rats

    Microsoft Academic Search

    Erik A. Johnson; Stanislav I. Svetlov; Kevin K. W. Wang; Ronald L. Hayes; Jose A. Pineda

    2005-01-01

    Survivin attenuates apoptosis by inhibiting cleavage of some cell proteins by activated caspase-3. We recently discovered\\u000a strong up-regulation of survivin, primarily in astrocytes and a sub-set of neurons, after traumatic brain injury (TBI) in\\u000a rats. In this study we characterized co-expression of survivin with activated caspase-3 and downstream DNA fragmentation (TUNEL)\\u000a in astrocytes and neurons after TBI. Western blot analysis

  5. Cytochemical identification of programmed cell death in the fusing fetal mouse palate by specific labelling of DNA fragmentation

    Microsoft Academic Search

    Chisato Mori; Noriko Nakamura; Yo Okamoto; Makoto Osawa; Kohei Shiota

    1994-01-01

    The process of palate fusion was examined in 13- and 14-day-old mouse fetuses by using in situ staining for nuclear DNA fragmentation (TUNEL method) and immunofluorescent staining for keratin, with special reference to the disruption of the midline epithelial seam. TUNEL-positive cells were found in the disappearing midline seam and the oral and nasal epithelial triangles at some late stages

  6. A polymer, random walk model for the size-distribution of large DNA fragments after high linear energy transfer radiation

    NASA Technical Reports Server (NTRS)

    Ponomarev, A. L.; Brenner, D.; Hlatky, L. R.; Sachs, R. K.

    2000-01-01

    DNA double-strand breaks (DSBs) produced by densely ionizing radiation are not located randomly in the genome: recent data indicate DSB clustering along chromosomes. Stochastic DSB clustering at large scales, from > 100 Mbp down to < 0.01 Mbp, is modeled using computer simulations and analytic equations. A random-walk, coarse-grained polymer model for chromatin is combined with a simple track structure model in Monte Carlo software called DNAbreak and is applied to data on alpha-particle irradiation of V-79 cells. The chromatin model neglects molecular details but systematically incorporates an increase in average spatial separation between two DNA loci as the number of base-pairs between the loci increases. Fragment-size distributions obtained using DNAbreak match data on large fragments about as well as distributions previously obtained with a less mechanistic approach. Dose-response relations, linear at small doses of high linear energy transfer (LET) radiation, are obtained. They are found to be non-linear when the dose becomes so large that there is a significant probability of overlapping or close juxtaposition, along one chromosome, for different DSB clusters from different tracks. The non-linearity is more evident for large fragments than for small. The DNAbreak results furnish an example of the RLC (randomly located clusters) analytic formalism, which generalizes the broken-stick fragment-size distribution of the random-breakage model that is often applied to low-LET data.

  7. De novo sequencing of sunflower genome for SNP discovery using RAD (Restriction site Associated DNA) approach

    PubMed Central

    2013-01-01

    Background Application of Single Nucleotide Polymorphism (SNP) marker technology as a tool in sunflower breeding programs offers enormous potential to improve sunflower genetics, and facilitate faster release of sunflower hybrids to the market place. Through a National Sunflower Association (NSA) funded initiative, we report on the process of SNP discovery through reductive genome sequencing and local assembly of six diverse sunflower inbred lines that represent oil as well as confection types. Results A combination of Restriction site Associated DNA Sequencing (RAD-Seq) protocols and Illumina paired-end sequencing chemistry generated high quality 89.4 M paired end reads from the six lines which represent 5.3 GB of the sequencing data. Raw reads from the sunflower line, RHA 464 were assembled de novo to serve as a framework reference genome. About 15.2 Mb of sunflower genome distributed over 42,267 contigs were obtained upon assembly of RHA 464 sequencing data, the contig lengths ranged from 200 to 950 bp with an N50 length of 393 bp. SNP calling was performed by aligning sequencing data from the six sunflower lines to the assembled reference RHA 464. On average, 1 SNP was located every 143 bp of the sunflower genome sequence. Based on several filtering criteria, a final set of 16,467 putative sequence variants with characteristics favorable for Illumina Infinium Genotyping Technology (IGT) were mined from the sequence data generated across six diverse sunflower lines. Conclusion Here we report the molecular and computational methodology involved in SNP development for a complex genome like sunflower lacking reference assembly, offering an attractive tool for molecular breeding purposes in sunflower. PMID:23947483

  8. Characterization by restriction fragment length polymorphism and sequence analysis of field and vaccine strains of infectious laryngotracheitis virus involved in severe outbreaks.

    PubMed

    Chacon, Jorge Luis; Mizuma, Matheus Y; Piantino Ferreira, Antonio J

    2010-12-01

    At the end of 2002 and throughout 2003, there was a severe outbreak of infectious laryngotracheitis (ILT) in an intensive production area of commercial hens in the Sao Paulo State of Brazil. ILT virus was isolated from 28 flocks, and 21 isolates were genotyped by polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) using four genes and eight restriction enzymes, and by partial sequencing of the infected cell protein 4 (ICP4) and thymidine kinase (TK) genes. Three groups resulted from the combinations of PCR-RFLP patterns: 19 field isolates formed Group I, and the remaining two isolates together with the chicken embryo origin (CEO) vaccine strains formed Group II. Group III comprised the tissue-culture origin (TCO) vaccine strain by itself. The PCR-RFLP results agreed with the sequencing results of two ICP4 gene fragments. The ICP4 gene sequence analysis showed that the 19 field isolates classified into Group I by RFLP-PCR were identical among themselves, but were different to the TCO and CEO vaccines. The two Group II isolates could not be distinguished from one of the CEO vaccines. The nucleotide and amino acid sequence analyses discriminated between the Brazilian and non-Brazilian isolates, as well as between the TCO and CEO vaccines. Sequence analysis of the TK gene enabled classification of the field isolates (Group I) as virulent and non-vaccine. This work shows that the severe ILT outbreak was caused by a highly virulent, non-vaccine strain. PMID:21154050

  9. Identification of differentially expressed genes by restriction endonuclease-based gene expression fingerprinting.

    PubMed

    Ivanova, N B; Belyavsky, A V

    1995-08-11

    A novel method for identification of differentially expressed genes has been developed. It is based on the consecutive restriction digestions of 3' terminal cDNA fragments to produce a fingerprint of gene expression. cDNA molecules are synthesized using a biotinylated oligo(dT) primer, digested with a frequently cutting restriction endonuclease and the 3'-terminal restriction fragments are isolated using streptavidin microbeads. After amplification by PCR, cDNA fragments are immobilized again on streptavidin beads, radiolabeled and treated sequentially with a set of restriction endonucleases. The products of individual enzymatic reactions from two or more different RNA populations are resolved by polyacrylamide gel electrophoresis and compared to reveal differentially expressed genes. This strategy enabled us to identify and clone the fragments of five genes expressed differentially in murine thymus and spleen. One of the genes was found to encode terminal deoxynucleotidyl transferase; others are apparently previously unknown genes. PMID:7659517

  10. Mitochondrial DNA Restriction Map for the Caribbean Fruit Fly, Anastrepha suspensa , and Occurrence of Mitochondrial DNA Diversity Within Highly Inbred Colonies

    Microsoft Academic Search

    M. Heath; D. Kuhn; R. Schnell; C. Olano

    2002-01-01

    A restriction map has been constructed for Anastrepha suspensa mitochondrial DNA. One HaeIII site was found to be polymorphic among individuals in highly inbred colonies and a feral population. Based on mapping information, the polymorphic site was determined to be in the ATPase 6 gene. Primers TK-J-3804 and C3-N-5460 amplified this region. The amplicon was cut by HaeIII in flies

  11. A Cladistic Analysis of Phenotypic Associations with Haplotypes Inferred from Restriction Endonuclease Mapping and DNA Sequence Data. III. Cladogram Estimation

    PubMed Central

    Templeton, A. R.; Crandall, K. A.; Sing, C. F.

    1992-01-01

    We previously developed a cladistic approach to identify subsets of haplotypes defined by restriction endonuclease mapping or DNA sequencing that are associated with significant phenotypic deviations. Our approach was limited to segments of DNA in which little recombination occurs. In such cases, a cladogram can be constructed from the restriction site or sequence data that represents the evolutionary steps that interrelate the observed haplotypes. The cladogram is used to define a nested statistical design to identify mutational steps associated with significant phenotypic deviations. The central assumption behind this strategy is that any undetected mutation causing a phenotypic effect is embedded within the same evolutionary history that is represented by the cladogram. The power of this approach depends upon the confidence one has in the particular cladogram used to draw inferences. In this paper, we present a strategy for estimating the set of cladograms that are consistent with a particular sample of either restriction site or nucleotide sequence data and that includes the possibility of recombination. We first evaluate the limits of parsimony in constructing cladograms. Once these limits have been determined, we construct the set of parsimonious and nonparsimonious cladograms that is consistent with these limits. Our estimation procedure also identifies haplotypes that are candidates for being products of recombination. If recombination is extensive, our algorithm subdivides the DNA region into two or more subsections, each having little or no internal recombination. We apply this estimation procedure to three data sets to illustrate varying degrees of cladogram ambiguity and recombination. PMID:1385266

  12. CLASSIFICATION OF PHYTOPLASMAS BASED ON COMPUTER-SIMULATED RESTRICTION FRAGMENT LENGTH POLYMORPHISM ANALYSIS OF 16S RRNA GENE SEQUENCES

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Phytoplasmas are cell wall-less pathogenic bacteria that cause numerous plant diseases. Due to the inability to culture phytoplasmas in vitro, it is impossible to differentiate and classify phytoplasmas by the traditional methods that are applied to cultured prokaryotes. To date, restriction fragm...

  13. DEVELOPMENT OF A COMPUTER-SIMULATED 16SRDNA RESTRICTION FRAGMENT LENGTH POLYMORPHISM ANALYSIS SYSTEM FOR CLASSIFICATION OF PHYTOPLASMAS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Phytoplasmas are cell wall-less pathogenic bacteria that cause numerous plant diseases. Due to the inability to culture phytoplasmas in vitro, it is impossible to differentiate and classify phytoplasmas by the traditional methods that are applied to cultured prokaryotes. To date, restriction fragm...

  14. [Interaction of dNTP-binding sites of human DNA polymerase alpha and The Klenow fragment of Escherichia coli DNA polymerase I with nucleotides, pyrophosphate and their analogs].

    PubMed

    Nevinski?, G A; Potapova, I A; Tarusova, N B; Khalabuda, O V; Khomov, V V

    1990-01-01

    AMP and NaF each taken separately were shown to activate DNA polymerization catalyzed by Klenow fragment of DNA polymerase I by means of interaction of AMP or NaF with 3'----5'-exonuclease center of the enzyme. In the presence of NaF which is a selective inhibitor of 3'----5'-exonuclease center, AMP is an inhibitor of polymerization competitive with respect to dATP. Ki values and the pattern of inhibition with respect to dATP were determined for AMP, ADP, ATP, carboxymethylphosphonyl-5'-AMP, Pi, PPi, PPPi, methylenediphosphonic acid and its ethylated esters, phosphonoformic acid, phosphonoacetic acid and its ethylated esters as well as for some bicarbonic acids in the reactions of DNA polymerization catalyzed by Klenow fragment of DNA polymerase I (in the presence of NaF) and DNA polymerase alpha from human placenta in the presence of poly(dT) template and r(pA)10 primer. All nucleotides and their analogs were found to be capable of competing with dATP for the active center of the enzyme. Most of the analogs of PPi and phosphonoacetic acid are inhibitors of Klenow fragment competitive with respect to dATP. Nowever these analogs display a mixed-type inhibition in the case of human DNA polymerase alpha. We postulated a similar mechanism of interaction for dNTP with both DNA-polymerases. It is suggested that each phosphate group of PPi makes equal contribution to the interaction with DNA polymerases and that the distance between the phosphate groups is important for this interaction. beta-phosphate of NTP or dNTP is suggested to make negligible contribution to the efficiency of the formation of enzyme complexes with dNTP. beta-phosphate is likely to be an essential point of PPi interaction with the active center of proteins during the cleavage of the alpha-beta-phosphodiester bond of dNTP in the reaction of DNA polymerization. PMID:2161489

  15. DNA “fingerprints” detect genetic variation in Acer negundo ( Aceraceae )

    Microsoft Academic Search

    Hilde Nybom; Steven H. Rogstad

    1990-01-01

    Genomic DNA samples from 21 box elder plants collected in Missouri (U.S.A.) were digested with restriction enzyme and southern blot hybridized with the M13 minisatellite probe. Each plant was found to have a unique DNA fragment pattern. Moreover, levels of genetic variation estimated from a similarity index appear to be related to sampling distances. However, size of the fragments utilized

  16. Molecular Cloning and Sequence Analysis of Novel Cytochrome P450 cDNA Fragments from Dastarcus helophoroides

    PubMed Central

    Wang, Hai-Dong; Li, Fei-Fei; He, Cai; Cui, Jun; Song, Wang; Li, Meng-Lou

    2014-01-01

    The predatory beetle Dastarcus helophoroides (Fairmaire) (Coleoptera: Bothrideridae) is a natural enemy of many longhorned beetles and is mainly distributed in both China and Japan. To date, no research on D. helophoroides P450 enzymes has been reported. In our study, for the better understanding of P450 enzymes in D. helophoroides, 100 novel cDNA fragments encoding cytochrome P450 were amplified from the total RNA of adult D. helophoroides abdomens using five pairs of degenerate primers designed according to the conserved amino acid sequences of the CYP6 family genes in insects through RT-PCR. The obtained nucleotide sequences were 250 bp, 270 bp, and 420 bp in length depending on different primers. Ninety-six fragments were determined to represent CYP6 genes, mainly from CYP6BK, CYP6BQ, and CYP6BR subfamilies, and four fragments were determined to represent CYP9 genes. Twenty-two fragments, submitted to GenBank, were selected for further homologous analysis, which revealed that some fragments of different sizes might be parts of the same P450 gene. PMID:25373175

  17. Fragmentation of isomeric intrastrand crosslink lesions of DNA in an ion-trap mass spectrometer.

    PubMed

    Cao, Huachuan; Wang, Yinsheng

    2009-04-01

    The collision-induced dissociation pathways of isomeric cytosine-guanine and cytosine-adenine intrastrand crosslink-containing dinucleoside monophosphates were investigated with the stable isotope-labeled compounds to gain insights into the effects of chemical structure on the fragmentation pathways of these DNA modifications. A Dimroth-like rearrangement, which was reported for protonated 2'-deoxycytidine and involved the switching of the exocyclic N4 with the ring N3 nitrogen atom, was also observed for the cytosine component in the protonated ions of C[5-8]G, C[5-2]A, and C[5-8]A, but not C[5-N(2)]G or C[5-N(6)]A. In these two sets of crosslinks, the C5 of cytosine is covalently bonded with its neighboring purine base via a carbon atom on the aromatic ring and an exocyclic nitrogen atom, respectively. On the contrary, the rearrangement could occur for the deprotonated ions of C[5-N(2)]G, C[5-N(6)]A, and unmodified cytosine, but not C[5-8]G, C[5-2]A, or C[5-8]A. In addition, ammonia could be lost more readily from C[5-N(2)]G and C[5-N(6)]A than from C[5-8]G, C[5-2]A, and C[5-8]A. The results from the present study afforded important guidance for the application of mass spectrometry for the structure elucidation of other intrastrand/interstrand crosslink lesions. PMID:19103496

  18. Leaf margin phenotype-specific restriction-site-associated DNA-derived markers for pineapple (Ananas comosus L.)

    PubMed Central

    Urasaki, Naoya; Goeku, Satoko; Kaneshima, Risa; Takamine, Tomonori; Tarora, Kazuhiko; Takeuchi, Makoto; Moromizato, Chie; Yonamine, Kaname; Hosaka, Fumiko; Terakami, Shingo; Matsumura, Hideo; Yamamoto, Toshiya; Shoda, Moriyuki

    2015-01-01

    To explore genome-wide DNA polymorphisms and identify DNA markers for leaf margin phenotypes, a restriction-site-associated DNA sequencing analysis was employed to analyze three bulked DNAs of F1 progeny from a cross between a ‘piping-leaf-type’ cultivar, ‘Yugafu’, and a ‘spiny-tip-leaf-type’ variety, ‘Yonekura’. The parents were both Ananas comosus var. comosus. From the analysis, piping-leaf and spiny-tip-leaf gene-specific restriction-site-associated DNA sequencing tags were obtained and designated as PLSTs and STLSTs, respectively. The five PLSTs and two STSLTs were successfully converted to cleaved amplified polymorphic sequence (CAPS) or simple sequence repeat (SSR) markers using the sequence differences between alleles. Based on the genotyping of the F1 with two SSR and three CAPS markers, the five PLST markers were mapped in the vicinity of the P locus, with the closest marker, PLST1_SSR, being located 1.5 cM from the P locus. The two CAPS markers from STLST1 and STLST3 perfectly assessed the ‘spiny-leaf type’ as homozygotes of the recessive s allele of the S gene. The recombination value between the S locus and STLST loci was 2.4, and STLSTs were located 2.2 cM from the S locus. SSR and CAPS markers are applicable to marker-assisted selection of leaf margin phenotypes in pineapple breeding. PMID:26175625

  19. Caloric restriction decreases mitochondrial free radical generation at complex I and lowers oxidative damage to mitochondrial DNA in the rat heart

    Microsoft Academic Search

    Ricardo Gredilla; Alberto Sanz; Monica Lopez-Torres; Gustavo Barja

    2001-01-01

    The effect of caloric restriction (CR) (40%) on the rates of mitochondrial H 2O2 production and oxygen consumption and oxidative damage to nuclear DNA (nDNA) and mitochondrial DNA (mtDNA) was studied for short-term (6-wk) and long-term (1-year) periods in the heart of young and old rats. Short-term CR did not change any of the parameters measured. However, long-term CR significantly

  20. The effect of sperm DNA fragmentation on the dynamics of the embryonic development in intracytoplasmatic sperm injection.

    PubMed

    Wdowiak, Artur; Bakalczuk, Szymon; Bakalczuk, Grzegorz

    2015-06-01

    The genetic integrity of sperm DNA can contribute to the infertility problems experienced by couples. Sperm DNA fragmentation (SDF) is the most common DNA abnormality in male gametes, and yet its effect on embryo kinetics has not been widely studied. Embryo morphokinetic parameters during the first days of embryo culture after intracytoplasmatic sperm injection (ICSI) are weakly predictive of both embryo development and pregnancy outcome. This study investigated the effect of SDF on embryo morphokinetic parameters following ICSI. The DNA fragmentation index (DFI) in spermatozoa from all males in the study (n=165) was determined before ICSI and the morphokinetic parameters of the embryos (n=165) were monitored via time-lapse recording. We found that a low DFI index in spermatozoa corresponded with embryos that reached the blastocyst stage at a faster rate after ICSI. Overall, lower SDF levels were also found in the group of patients that achieved pregnancy. Thus, higher SDF levels can slow down embryo morphokinetic parameters, and may be predictive of pregnancy outcomes after ICSI. PMID:26051457

  1. Cloning and Identification of lilyturf (Ophiopogon japonicus) RbcL cDNA fragment

    Microsoft Academic Search

    Tianqi Guo; Cong Li; Yingbo Wang; Liebao Han; Wenpan Zhang

    2011-01-01

    This study was aimed to isolate ribulose-1,5- bisphosphate carboxylase\\/oxygenase large subunit (rbcL) from lilyturf (Ophiopogon japonicus). A pair of primers used in amplifying chloroplast fragment from lilyturf was designed according to the corresponding sequences in tobacco, spinach chloroplast genomes. The fragment containing rbcL gene was cloned from lilyturf chloroplast genome. Sequencing analysis indicated that this fragment was 800 base pairs

  2. Huntington disease-linked restriction fragment length polymorphism localized within band p16.1 of chromosome 4 by in situ hybridization.

    PubMed Central

    Magenis, R E; Gusella, J; Weliky, K; Olson, S; Haight, G; Toth-Fejel, S; Sheehy, R

    1986-01-01

    A 5.5-kilobase (kb) single sequence DNA fragment (G8) reveals the DNA polymorphic locus D4S10 on Southern blot analysis. This locus is closely linked to Huntington disease and has been mapped to chromosome 4 short arm using human-mouse somatic cell hybrids, and specifically to chromosome 4 band p16 using DNA from individuals with deletions of chromosome 4 short arm who exhibit Wolf-Hirschhorn syndrome. With in situ hybridization techniques, we have confirmed the location of D4S10 on chromosome 4 and further localized it within band p16 utilizing five patients, four with overlapping chromosome 4 short-arm aberrations. The DNA segment G8 was hybridized to the mataphase chromosomes of the five patients. Two of them have different interstitial deletions of one of the chromosome 4 short arms (TA and BA), two have different chromosome 4 short-arm terminal deletions (RG and DQ), and one has a normal male karyotype. By noting the presence or absence of hybridization to the partially deleted chromosomes with known precise breakpoints, we were able to more accurately localize probe G8 to the distal half of band p16.1 of chromosome 4. Images Fig. 1 Fig. 2 Fig. 3 PMID:2876628

  3. Differentiation between porcine reproductive and respiratory syndrome virus isolates by restriction fragment length polymorphism of their ORFs 6 and 7 genes.

    PubMed Central

    Gagnon, C A; Dea, S

    1998-01-01

    Three distinct antigenic profiles were identified by comparing the reactivities of 15 Canadian field isolates, the attenuated U.S. vaccine (Ingelvac MLV) strain and 2 European reference strains (Lelystad and Weybridge) of the porcine reproductive and respiratory syndrome virus (PRRSV) by indirect immunofluorescence with a set of 4 monoclonal antibodies to the nucleocapsid (N) protein and 2 other to the matrix (M) protein. In the present study, 9 Canadian isolates for which the sequences were determined appeared closely related to 2 U.S. reference strains (ATCC VR-2332 and ATCC VR-2385) with amino acid identities varying between 90 to 98% for the M and N proteins; substitutions in the nucleotide sequences were distributed randomly throughout the ORFs 6 and 7 genes, and most were 3rd base silent mutations. In comparison, more than 30% divergence was demonstrated with the Lelystad virus. Furthermore, differentiation between North American and European isolates, and between field isolates and the MLV strain could be achieved by cutting PCR-amplified products encompassing both ORFs 6 and 7 genes with 4 restriction endonucleases. When taken individually, BsaJI and AluI were the more appropriate restriction enzymes for distinguishing the vaccine strain from field isolates. The results obtained suggest that the restriction fragment length polymorphism of the genomic region covering the ORFs 6 and 7 genes may be a valuable tool to differentiate among PRRSV isolates. Images Figure 1. PMID:9553709

  4. Dynamics of DNA Loop Capture by the SfiI Restriction Endonuclease on Supercoiled and Relaxed DNA

    E-print Network

    Vologodskii, Alexander

    were formed transiently during a burst phase. The burst revealed three sets of doubly cut products was evalu- ated from the burst phases of reactions initiated by adding MgCl2 to SfiI bound to the plasmid for these discrepancies are discussed. q 2004 Elsevier Ltd. All rights reserved. Keywords: DNA looping; DNA

  5. Small variations in crucial steps of TUNEL assay coupled to flow cytometry greatly affect measures of sperm DNA fragmentation.

    PubMed

    Muratori, Monica; Tamburrino, Lara; Tocci, Valentina; Costantino, Antonietta; Marchiani, Sara; Giachini, Claudia; Laface, Ilaria; Krausz, Csilla; Meriggiola, Maria Cristina; Forti, Gianni; Baldi, Elisabetta

    2010-01-01

    Techniques for assessing sperm DNA damage are numerous and various. There are 2 main types of assay: direct and indirect. The former directly detects the amount of sperm DNA damage, whereas the latter reveals the effects of an exogenous insult on sperm chromatin. In addition, even considering the same type of technique, different strategies to reveal or quantify sperm DNA damage, or both, are used. Finally, these techniques, except for sperm chromatin structure assay (SCSA), lack standardized protocols to which all users can adhere to minimize interlaboratory variations. In this study, we investigated the effects of some of the many ways the terminal deoxynucleotidyl transferase-mediated fluorescein-dUTP nick end labeling (TUNEL) assay is performed when measuring sperm DNA fragmentation by flow cytometry. In addition, by using an established procedure, we determined the precision of the technique by calculating intra-assay coefficients of variation (CVs). We found that concentration of the fixative, the time of storage of fixed samples, the fluorochrome used to label DNA breaks, and the method used to analyze flow cytometric data all greatly affect the measures of sperm DNA fragmentation. In particular, we found that treatment with paraformaldehyde produced additional damage in most samples, suggesting that TUNEL also can be considered an indirect assay when performed in semen samples treated with such a fixative reagent. We also showed that 2 different methods used to analyze data yielded results that, albeit correlating, were different and associated differently to semen quality. On the contrary, the TUNEL assay, as measured here, showed low intraassay CVs, resulting in a quite precise technique when performed in established conditions. PMID:19959824

  6. Restriction fragment length polymorphism (RFLP) in exon 2 of the BoLA-DRB3 gene in South American cattle.

    PubMed

    Miretti, M M; Ferro, J A; Lara, M A; Contel, E P

    2001-10-01

    The Bola-DRB3 gene participates in the development of the immune response and is highly polymorphic. For these reasons, it has been a candidate gene in studies of the genetic basis of disease resistance and in population genetic analysis. South American native cattle breeds have been widely replaced by improved exotic breeds leading to a loss of genetic resources. In particular, South American native breeds have high levels of fertility and disease resistance. This work describes genetic variability in the BoLA-DRB3 gene in native (Caracu, Pantaneiro, Argentinean Creole) and exotic (Holstein, Jersey, Nelore, Gir) cattle breeds in Brazil and Argentina. PCR-RFLP alleles were identified by combining the restriction patterns for the BoLA-DRB3.2 locus obtained with RsaI, BstY and HaeIII restriction enzymes. Allelic frequencies and deviations from the Hardy-Weinberg equilibrium were also calculated. Analysis of the 24 BoLA-DRB3 PCR-RFLP alleles identified showed differences in the allele distributions among breeds. PMID:11758727

  7. Application of substrate utilization patterns and terminal restriction fragment length polymorphism analysis to characterize the oral bacterial community of healthy subjects and patients with periodontitis

    PubMed Central

    DING, YI-JIAN; GE, CHAO-RONG; YAO, HUAI-YING

    2015-01-01

    Understanding the association between the bacterial community and oral health status is essential for the diagnosis and therapy of periodontal diseases. The aim of the present study was to apply three methods [conventional culture, substrate utilization using the MicroResp™ system and terminal restriction fragment length polymorphism (T-RFLP)] to investigate the oral bacterial community in saliva from 20 healthy subjects and 20 patients with periodontitis. The three methods all revealed that there was a systematic change in the microbial ecological characteristics associated with oral health status. Compared with the control group, the oral bacterial flora in the patients with chronic periodontitis had a greater culturable population and altered preferred carbon source and TRFLP patterns. TRFLP analysis was found to give more information and exhibit a higher sensitivity than the substrate utilization and conventional culture methods. In conclusion, TRFLP analysis is a potentially rapid method to assess the composition of the oral microbial community and for the diagnosis of chronic periodontitis.

  8. DNA fragmentation in frozen sperm of Equus asinus: Zamorano-Leonés, a breed at risk of extinction.

    PubMed

    Cortés-Gutiérrez, E I; Crespo, F; Gosálvez, A; Dávila-Rodríguez, M I; López-Fernández, C; Gósalvez, J

    2008-05-01

    The dynamics of sperm DNA fragmentation (sDF) and sperm viability were analyzed in frozen-thawed sperm samples of Equus asinus (Zamorano-Leonés), a breed at risk of extinction. Sperm DNA fragmentation was assessed using an adaptation of the sperm chromatin dispersion test developed for stallions in five different frozen samples. Sperm were thawed and incubated at different temperatures (37 degrees C, 25 degrees C, and 4 degrees C) and sDF was assessed at different times and compared. The mean sDF after thawing at the beginning of the experiment was 18.20+/-14.77% and did not differ significantly from the results of a neutral comet assay (22.0+/-19.34%). The tendency in the sDF of all donkeys indicated that sperm DNA is more sensitive to breakage when incubated at 37 degrees C than when incubated at 25 degrees C or 4 degrees C. Interestingly, the tendency was not the same when different animals were compared, and differences in sDF dynamics were established among individuals. sDF correlated negatively with sperm viability in some individuals but not in others. From a conservation perspective, sDF analysis may offer a new way to assess sperm quality in endangered breeds in order to identify and select the best semen samples for artificial reproduction purposes. In particular, we recommend for artificial insemination the use of semen samples with a slow increase in sDF with time after thawing. PMID:18367243

  9. Cloning and Stable Maintenance of 300-Kilobase-Pair Fragments of Human DNA in Escherichia coli Using an F-Factor-Based Vector

    Microsoft Academic Search

    Hiroaki Shizuya; Bruce Birren; Ung-Jin Kim; Valeria Mancino; Tatiana Slepak; Yoshiaki Tachiiri; Melvin Simon

    1992-01-01

    A bacterial cloning system for mapping and analysis of complex genomes has been developed. The BAC system (for bacterial artificial chromosome) is based on Escherichia coli and its single-copy plasmid F factor. It is capable of maintaining human genomic DNA fragments of >300 kilobase pairs. Individual clones of human DNA appear to be maintained with a high degree of structural

  10. CD34+ Cells in the Peripheral Blood Transport Herpes Simplex Virus DNA Fragments to the Skin of Patients with Erythema Multiforme (HAEM)

    Microsoft Academic Search

    Fumitake Ono; Bhuvnesh K. Sharma; Cynthia C. Smith; Joseph W. Burnett; Laure Aurelian

    2005-01-01

    Herpes simplex virus (HSV)-associated erythema multiforme (HAEM) is a recurrent disease characterized by the presence and expression of HSV DNA fragments in lesional skin. Our studies examined the mechanism of viral DNA transport to the skin of HAEM patients. CD34+ cells were isolated from the blood of normal subjects and HSV and HAEM patients during acute lesions and at quiescence.

  11. DNA Fingerprinting of Ralstonia paucula by Infrequent-Restriction-Site PCR and Randomly Amplified Polymorphic DNA Analysis

    Microsoft Academic Search

    Didier Moissenet; Hoang Vu-Thien; Yahia Benzerara

    2003-01-01

    Ralstonia paucula (formerly CDC group IV c-2) is an environmental organism that can cause serious human infections, occasionally clusters of nosocomial infections. In the present work, 26 strains of R. paucula (4 from the American Centers for Disease Control and Prevention collection, 10 from the Belgian Laboratorium voor Microbiologie (LMG) collection, and 12 French clinical isolates) were analyzed with infrequent-restriction-site

  12. Effects of oral antioxidant treatment upon the dynamics of human sperm DNA fragmentation and subpopulations of sperm with highly degraded DNA.

    PubMed

    Abad, C; Amengual, M J; Gosálvez, J; Coward, K; Hannaoui, N; Benet, J; García-Peiró, A; Prats, J

    2013-06-01

    The primary aim of this study was to determine the effect of oral antioxidant treatment (1500 mg of l-Carnitine; 60 mg of vitamin C; 20 mg of coenzyme Q10; 10 mg of vitamin E; 10 mg of zinc; 200 ?g of vitamin B9; 50 ?g of selenium; 1 ?g of vitamin B12) during a time period of 3 months upon the dynamics of sperm DNA fragmentation following varying periods of sperm storage (0 h, 2 h, 6 h, 8 h and 24 h) at 37 °C in a cohort of 20 infertile patients diagnosed with asthenoteratozoospermia. A secondary objective was to use the sperm chromatin dispersion test (SCD) to study antioxidant effects upon a specific subpopulation of highly DNA degraded sperm (DDS). Semen parameters and pregnancy rate (PR) were also determined. Results showed a significant improvement of DNA integrity at all incubation points (P < 0.01). The proportion of DDS was also significantly reduced (P < 0.05). Semen analysis data showed a significant increase in concentration, motility, vitality and morphology parameters. Our results suggest that antioxidant treatment improves sperm quality not only in terms of key seminal parameters and basal DNA damage, but also helps to maintain DNA integrity. Prior administration of antioxidants could therefore promote better outcomes following assisted reproductive techniques. PMID:22943406

  13. Polymerase chain reaction - restriction fragment length polymorphism analysis for the differentiation of Trichinella nativa and Trichinella britovi.

    PubMed

    Mayer-Scholl, A; Broglia, A; Reckinger, S; Nöckler, K

    2014-06-16

    Recently, Trichinella nativa was identified in foxes in Germany and Poland, indicating that the geographical distribution of T. nativa is not restricted to areas north of the isotherm -4°C in January. In the European Union, legislation requires that a regular monitoring of the occurrence of Trichinella spp. in indicator animals such as foxes or raccoon dogs be carried out. The Trichinella isolates must also be identified on a species level. The multiplex PCR recommended by the Community Reference Laboratory for Trichinella allows species identification, yet the differentiation of T. nativa and Trichinella britovi, a widespread Trichinella species in the temperate regions of Europe, is unstable. We therefore describe an easy and reliable method for the differentiation of the two species, which can be utilised to monitor a potential spread of T. nativa in Central Europe. PMID:24572044

  14. Fragmentation of Contaminant and Endogenous DNA in Ancient Samples Determined by Shotgun Sequencing; Prospects for Human Palaeogenomics

    PubMed Central

    Sanchez-Quinto, Federico; Ramirez, Oscar; Calafell, Francesc; Civit, Sergi; Lalueza-Fox, Carles

    2011-01-01

    Background Despite the successful retrieval of genomes from past remains, the prospects for human palaeogenomics remain unclear because of the difficulty of distinguishing contaminant from endogenous DNA sequences. Previous sequence data generated on high-throughput sequencing platforms indicate that fragmentation of ancient DNA sequences is a characteristic trait primarily arising due to depurination processes that create abasic sites leading to DNA breaks. Methodology/Principals Findings To investigate whether this pattern is present in ancient remains from a temperate environment, we have 454-FLX pyrosequenced different samples dated between 5,500 and 49,000 years ago: a bone from an extinct goat (Myotragus balearicus) that was treated with a depurinating agent (bleach), an Iberian lynx bone not subjected to any treatment, a human Neolithic sample from Barcelona (Spain), and a Neandertal sample from the El Sidrón site (Asturias, Spain). The efficiency of retrieval of endogenous sequences is below 1% in all cases. We have used the non-human samples to identify human sequences (0.35 and 1.4%, respectively), that we positively know are contaminants. Conclusions We observed that bleach treatment appears to create a depurination-associated fragmentation pattern in resulting contaminant sequences that is indistinguishable from previously described endogenous sequences. Furthermore, the nucleotide composition pattern observed in 5? and 3? ends of contaminant sequences is much more complex than the flat pattern previously described in some Neandertal contaminants. Although much research on samples with known contaminant histories is needed, our results suggest that endogenous and contaminant sequences cannot be distinguished by the fragmentation pattern alone. PMID:21904610

  15. DNA amplification and restriction endonuclease analysis for differentiation of 12 species and taxa of Nocardia, including recognition of four new taxa within the Nocardia asteroides complex.

    PubMed Central

    Steingrube, V A; Brown, B A; Gibson, J L; Wilson, R W; Brown, J; Blacklock, Z; Jost, K; Locke, S; Ulrich, R F; Wallace, R J

    1995-01-01

    Nineteen reference and 156 clinical strains of the genus Nocardia belonging to 12 taxonomic groups were studied for restriction fragment length polymorphism (RFLP) by using an amplified 439-bp segment of the 65-kDa heat shock protein gene. Of 30 restriction endonucleases, digestion with MspI and then digestion with BsaHI produced RFLP band patterns which separated all 12 groups except N. asteroides type IV from 6 of 12 N. transvalensis isolates and N. carnea from the N. asteroides type VI isolates. Commonly encountered species such as N. nova, N. farcinica, N. brasiliensis sensu stricto, and N. otitidiscaviarum were easily separated. Each taxon resulted in a single RFLP band pattern that included > or = 96% of all biochemically grouped isolates for 9 of 12 taxa with MspI and for 8 of 12 taxa with BsaHI. With the use of both patterns, only 6 of 175 (3.4%) isolates failed to fit the biochemically defined group patterns. These studies provide the first evidence for the separate identities of four antibiogram-defined (but currently unnamed) groups within the N. asteroides complex (types I, II, IV, and VI) and the presence of two subgroups within N. transvalensis. They also provide genotypic evidence for the separate identities of N. nova and N. farcinica. The lack of BstEII recognition sites in amplicons obtained from nocardiae provides a simple and rapid method for the differentiation of nocardiae from mycobacteria. DNA amplification with RFLP analysis is the first rapid method that distinguishes all clinically significant taxa and recognized species within the genus Nocardia. PMID:8586680

  16. Genetic Characterization of Campylobacter Jejuni and C. coli Isolated From Broilers Using flaA PCR-Restriction Fragment Length Polymorphism Method in Shiraz, Southern Iran

    PubMed Central

    Khoshbakht, Rahem; Tabatabaei, Mohammad; Hosseinzadeh, Saeid; Shirzad Aski, Hesamaddin; Seifi, Saeed

    2015-01-01

    Background: Thermophilic campylobacters, particularly Campylobacter jejuni and C. coli are the main agents of human campylobacteriosis. Campylobacter contaminated chicken products is the most important source of foodborne gastroenteritis. Evaluation of genetic diversity among Campylobacter population is critical for understanding the epidemiology of this bacterium and developing effective control strategies against Campylobacter infections and other related disorders. Objectives: The aim of this study was to investigate the polymorphism of thermophilic Campylobacter isolated from broiler fecal samples in Shiraz, southern Iran. Materials and Methods: Ninety Campylobacter isolates were recovered from broiler feces using enrichment process followed by cultivation method. The isolates were species typing on the basis of polymerase chain reaction (PCR) detection of 16SrRNA and multiplex PCR for determining two thermophilic species. To evaluate strain diversity of thermophilic Campylobacter isolates, flaA PCR-Restriction Fragment Length Polymorphism (RFLP) was performed using DdeI restriction enzyme. Results: All 90 Campylobacter isolates confirmed by m-PCR were successfully typed using flaA-PCR-RFLP. Eleven different types were defined according to flaA-typing method and the RFLP patterns were located at three separate clusters in RFLP image analysis dendrogram. Conclusions: Campylobacter jejuni isolates significantly showed more variety than C. coli isolates. A relatively low genetic diversity existed among C. jejuni and C. coli isolated from broilers in Shiraz, southern Iran. In our knowledge, this was the first report of genetic diversity among broiler originated human pathogen thermophilic campylobacters in Shiraz, southern Iran. PMID:26060566

  17. Restriction Endonuclease Cleavage Site and Length Polymorphisms in Mitochondrial DNA of Apis mellifera mellifera and A. m. carnica (Hymenoptera: Apidae)

    E-print Network

    Smith, Deborah; Brown, Wesley M.

    1990-01-01

    variation discussed in Table 2. January 1 9 9 0 SMITH & BROWN: M T D N A POLYMORPHISMS IN A. m. mellifera AND carnica 5 A. m. carnica CM D) m DC o LU CO "O c — a) rt cd H CJ CL -Q £2 O CQ CO X X cQ r U i L o o < rt X) X USA1 CD CO DC o o LU CO... o m i s ) : analysis by mitochondrial DNA and allozyme mark- ers. Genetics 108: 2 3 7 - 2 5 5 . Brown, W. M. 1 9 8 0 . Polymorphisms in mitochondrial DNA of humans as revealed by restriction endonu- clease analysis. Proc. Natl. Acad. Sci. U...

  18. Mitochondrial DNA variation in chinook salmon and chum salmon detected by restriction enzyme analysis of polymerase chain reaction products

    USGS Publications Warehouse

    Cronin, M.; Spearman, R.; Wilmot, R.; Patton, J.; Bickman, J.

    1993-01-01

    We analyze intraspecific mitochondrial DNA variation in chinook salmon from drainages in the Yukon River, the Kenai River, and Oregon and California rivers; and chum salmon from the Yukon River and vancouver Island, and Washington rivers. For each species, three different portions of the mtDNA molecule were amplified seperately using the polymerase chain reaction and then digested with at least 19 restrictions enzymes. Intraspecific sequence divergences between haplotypes were less than 0.01 base subsitution per nucleotide. Nine chum salmon haplotypes were identified. Yukon River chum salmon stocks displayed more haplotypes (8) occurred in all areas. Seven chinook salmon haplotypes were identified. Four haplotypes occurred in the Yukon and Kenai rviers and four occured in the Oregon/California, with only one haplotype shared between the regions. Sample sizes were too small to quantify the degree of stock seperation among drainages, but the patterns of variation that we observed suggest utility of the technique in genetic stock identification.

  19. Detection of Genomic DNA Fragmentation during Apoptosis (DNA Ladder) and the Simultaneous Isolation of RNA from Low Cell Numbers

    Microsoft Academic Search

    Peter T. Daniel; Isrid Sturm; Silke Ritschel; Katrin Friedrich; Bernd Dörken; Peter Bendzko; Timo Hillebrand

    1999-01-01

    In the present paper we describe a rapid and sensitive method for the simultaneous isolation of total RNA and genomic plus low-molecular-weight DNA from apoptotic cells. Using this method, we were able to detect a DNA ladder from as low as 30,000 apoptotic cells in only 45 min including gel electrophoresis. In addition, RNA can be readily obtained from the

  20. Characteristics of cloned repeated DNA sequences in the barley genome

    SciTech Connect

    Anan'ev, E.V.; Bochkanov, S.S.; Ryzhik, M.V.; Sonina, N.V.; Chernyshev, A.I.; Shchipkova, N.I.; Yakovleva, E.Yu.

    1986-12-01

    A partial clone library of barley DNA fragments based on plasmid pBR325 was created. The cloned EcoRI-fragments of chromosomal DNA are from 2 to 14 kbp in length. More than 95% of the barley DNA inserts comprise repeated sequences of different complexity and copy number. Certain of these DNA sequences are from families comprising at least 1% of the barley genome. A significant proportion of the clones hybridize with numerous sets of restriction fragments of genome DNA and they are dispersed throughout the barley chromosomes.

  1. Government restrictions on working with recombinant DNA, Walter GilbertSite: DNA Interactive (www.dnai.org)

    NSDL National Science Digital Library

    2008-10-06

    Interviewee: Walter Gilbert DNAi Location:Manipulation>Production>players Insulin and the moratorium In the late 1970s, there was a moratorium on recombinant DNA work. Gilbert had to go to England's Porton Down facility to try and isolate human insulin.

  2. DNA fragmentation, dATP pool elevation and potentiation of antifolate cytotoxicity in L1210 cells by hypoxanthine.

    PubMed Central

    Kwok, J. B.; Tattersall, M. H.

    1992-01-01

    Exogenous purines (greater than or equal to 10(-5)M) can modulate the cytotoxicity of methotrexate (MTX) in cultured cells, protecting cells at low MTX concentrations (less than or equal to 8 x 10(-8) M) and markedly potentiating its effect at higher concentrations. The ability of hypoxanthine (HX) to modulate the effects of two antifolates-ICI 198583 (an inhibitor of thymidylate synthetase) and piritrexim (PTX, a lipophilic inhibitor of DHFR)-was investigated using cultured mouse leukaemic cells, L1210. HX (10(-4) M) was found to potentiate only the cytotoxicity of DHFR inhibitors (MTS and PTX), increasing cell kill by 20-70 fold to the level achieved by an equivalent concentration (10(-5) M) of ICI 198583 alone. Agarose gel electrophoresis of DNA extracted from cells exposed to antifolates for 24 h demonstrated that the chromatin was cleaved into multimers of 200 base pairs. This pattern of DNA cleavage indicates cell death via apoptosis. The degree of DNA fragmentation was found to be closely linked to cytotoxicity. DNA fragmentation increased from 50% in cells treated with 10(-5) M MTX or PTX to 70% when HX was added with the drugs, a level achieved by 10(-5)M ICI 198583 alone. HX potentiation of cytotoxicity was correlated with a substantial increase in dATP in conjunction with low dTTP pools. The specific potentiation of DHFR inhibitors by HX may be due to their inhibition of purine synthesis with a concurrent rise in PRPP levels. Addition of HX with MTX substantially raised intracellular purine levels via the salvage pathway as indicated by ribonucleotide pool measurements. ICI 198583, on the other hand, stimulated de novo purine synthesis with or without added HX. Treatment with MTX plus HX or ICI 198583 (with or without HX) caused a reduction of dTTP pools to 8% of untreated control and excess dATP accumulation. The subsequent elevation (to 300% of control) of the dATP pool may provide a signal for endonucleolytic fragmentation of DNA and subsequent cell death. Images Figure 3 PMID:1562458

  3. A non-restricting and non-methylating Escherichia coli strain for DNA cloning and high-throughput conjugation to Streptomyces coelicolor.

    PubMed

    Zhou, Hao; Wang, Yemin; Yu, Yanfei; Bai, Tingli; Chen, Li; Liu, Pei; Guo, Hang; Zhu, Chenchen; Tao, Meifeng; Deng, Zixin

    2012-02-01

    Escherichia coli strains are used in secondary metabolism research for DNA cloning and transferring plasmids by intergeneric conjugation. Non-restricting strains are desirable for DNA cloning and non-methylating strains are beneficial for transferring DNA to methyl-restricting hosts, like Streptomyces coelicolor. We have constructed a non-methylating E. coli strain, JTU007, by deleting the DNA methylation genes dcm and dam from the widely used non-restricting cloning host DH10B. JTU007 was tested as donor for the conjugative transfer of a plasmid containing the 39 kb actinorhodin biosynthesis gene cluster to S. lividans and S. coelicolor. The Dcm? Dam? strain JTU007 transferred DNA into S. coelicolor A(3)2 derivatives at high frequency. To demonstrate the usefulness of E. coli JTU007 for gene cloning, we constructed a comprehensive S. toxytricini genomic cosmid library, and transferred it using high-throughput conjugation to the methyl-restricting S. coelicolor. One of the cosmid clones produced a brown pigment, and the clone was revealed to carry a tyrosinase operon. JTU007 is more useful than ET12567 because it does not restrict methylated DNA in primary cloning, and gives higher transformation and cosmid infection frequencies. PMID:22105763

  4. Structural model for the multisubunit Type IC restriction–modification DNA methyltransferase M.EcoR124I in complex with DNA

    PubMed Central

    Obarska, Agnieszka; Blundell, Alex; Feder, Marcin; Vejsadová, Št?pánka; Šišáková, Eva; Weiserová, Marie; Bujnicki, Janusz M.; Firman, Keith

    2006-01-01

    Recent publication of crystal structures for the putative DNA-binding subunits (HsdS) of the functionally uncharacterized Type I restriction–modification (R-M) enzymes MjaXIP and MgeORF438 have provided a convenient structural template for analysis of the more extensively characterized members of this interesting family of multisubunit molecular motors. Here, we present a structural model of the Type IC M.EcoR124I DNA methyltransferase (MTase), comprising the HsdS subunit, two HsdM subunits, the cofactor AdoMet and the substrate DNA molecule. The structure was obtained by docking models of individual subunits generated by fold-recognition and comparative modelling, followed by optimization of inter-subunit contacts by energy minimization. The model of M.EcoR124I has allowed identification of a number of functionally important residues that appear to be involved in DNA-binding. In addition, we have mapped onto the model the location of several new mutations of the hsdS gene of M.EcoR124I that were produced by misincorporation mutagenesis within the central conserved region of hsdS, we have mapped all previously identified DNA-binding mutants of TRD2 and produced a detailed analysis of the location of surface-modifiable lysines. The model structure, together with location of the mutant residues, provides a better background on which to study protein–protein and protein–DNA interactions in Type I R-M systems. PMID:16614449

  5. Original article MtDNA variation among subspecies of Apis cerana

    E-print Network

    Paris-Sud XI, Université de

    until DNA extraction. #12;DNA extraction, digestion and Southern-blot hybridization The total DNA fromOriginal article MtDNA variation among subspecies of Apis cerana using restriction fragment length, Machida, Tokyo, 194; 2 National Institute of Animal Industry, Tsukuba Norindanchi PO Box 5, lbaraki, 305

  6. Cloned plasmid DNA fragments as calibrators for controlling GMOs: different real-time duplex quantitative PCR methods.

    PubMed

    Taverniers, Isabel; Van Bockstaele, Erik; De Loose, Marc

    2004-03-01

    Analytical real-time PCR technology is a powerful tool for implementation of the GMO labeling regulations enforced in the EU. The quality of analytical measurement data obtained by quantitative real-time PCR depends on the correct use of calibrator and reference materials (RMs). For GMO methods of analysis, the choice of appropriate RMs is currently under debate. So far, genomic DNA solutions from certified reference materials (CRMs) are most often used as calibrators for GMO quantification by means of real-time PCR. However, due to some intrinsic features of these CRMs, errors may be expected in the estimations of DNA sequence quantities. In this paper, two new real-time PCR methods are presented for Roundup Ready soybean, in which two types of plasmid DNA fragments are used as calibrators. Single-target plasmids (STPs) diluted in a background of genomic DNA were used in the first method. Multiple-target plasmids (MTPs) containing both sequences in one molecule were used as calibrators for the second method. Both methods simultaneously detect a promoter 35S sequence as GMO-specific target and a lectin gene sequence as endogenous reference target in a duplex PCR. For the estimation of relative GMO percentages both "delta C(T)" and "standard curve" approaches are tested. Delta C(T) methods are based on direct comparison of measured C(T) values of both the GMO-specific target and the endogenous target. Standard curve methods measure absolute amounts of target copies or haploid genome equivalents. A duplex delta C(T) method with STP calibrators performed at least as well as a similar method with genomic DNA calibrators from commercial CRMs. Besides this, high quality results were obtained with a standard curve method using MTP calibrators. This paper demonstrates that plasmid DNA molecules containing either one or multiple target sequences form perfect alternative calibrators for GMO quantification and are especially suitable for duplex PCR reactions. PMID:14689155

  7. Relationship of seminal reactive nitrogen and oxygen species and total antioxidant capacity with sperm DNA fragmentation in infertile couples with normal and abnormal sperm parameters.

    PubMed

    Khosravi, F; Valojerdi, M R; Amanlou, M; Karimian, L; Abolhassani, F

    2012-11-01

    The objective of this study was to investigate the association between the amount of superoxide anion, peroxynitrite as oxidative stress (OS) markers and total antioxidant capacity (TAC) with sperm DNA fragmentation in infertile men with abnormal semen parameters. Semen samples were obtained from 102 infertile couples and divided into groups with normal and abnormal semen parameters according to the World Health Organization (WHO). Peroxynitrite and superoxide anions were detected using spectrofluorometric assays combined with 2,7 dicholorofluorescein (DCF)-DA and 4-chloro-7-nitrobenzo-2-oxa -1, 3-diazole (NBD-CL). Colorimetric assay was used for evaluation of TAC, while DNA fragmentation was studied by using sperm chromatin dispersion test. Superoxide anion, peroxynitrite and DNA fragmentation were significantly higher in infertile couples with abnormal semen parameters as compared to infertile couples with normal semen (P < 0.01). TAC was significantly lower in infertile men with abnormal semen parameters (P < 0.01). There was also a significant positive correlation between OS markers with sperm DNA fragmentation (r = 0.59, P < 0.01 and r = 0.67, P < 0.01, respectively). We have found that imbalance between superoxide anion and peroxynitrite with antioxidant capacity in infertile men with abnormal sperm parameters is associated with higher sperm DNA fragmentation. PMID:23126684

  8. Dietary restriction mitigates age-related accumulation of DNA damage, but not all changes in mouse corneal epithelium.

    PubMed

    Hallam, Dean; Wan, Tengfei; Saretzki, Gabriele

    2015-07-01

    The cornea protects the anterior eye and accounts for two thirds of the eyes refractive capacity. The homeostasis of corneal epithelium is thought to be maintained by putative stem cells residing in the epithelial basal layer. As a tissue constantly exposed to environmental stress, the cornea is hypothesised to accumulate persistent DNA damage events with time in stem cell populations. Recently, telomere associated DNA damage foci (TAFs) have been suggested as a marker for persistent DNA damage which can be used to detect senescent cells during ageing. Dietary restriction (DR) is the only known non-genetic intervention that is able to increase both life and health span among various animal species. The aim of this study was to analyse changes in corneal properties with age and under 16months of DR. We employed immunofluorescence staining for ?H2A.X together with telomere fluorescence in situ hybridisation (immuno-FISH) on mouse corneas from young, old ad libitum (AL) fed as well as dietary restricted (DR) mice. Our data show that the central corneas of old mice had significantly more general and telomere-associated DNA damage compared to young mice while DR treatment was able to reduce the amount of DNA damage significantly. We also found that the thickness of the peripheral region of the cornea, where the putative stem cells may reside, decreased with age regardless of whether the animals underwent DR treatment or not. Number of bullae, which indicates corneal edema, accumulated in old corneas in the central area and DR treatment mitigated the formation of these bullae. Moreover, the protein levels of the stem cell marker TAp63 decreased with age only in the central but not the peripheral region of the cornea. This finding suggests that epithelial progenitors might be better maintained in the peripheral than the central cornea during ageing. Together with the finding that the peripheral corneal showed no increase in DNA damage during age, we speculate that in mice, like humans, the putative stem cells reside in the peripheral cornea. PMID:25937374

  9. Study of a novel sample injection method (floating electrokinetic supercharging) for high-performance microchip electrophoresis of DNA fragments.

    PubMed

    Hirokawa, Takeshi; Takayama, Yoichi; Arai, Akihiro; Xu, Zhongqi

    2008-05-01

    Aiming to achieve high-performance analysis of DNA fragments using microchip electrophoresis, we developed a novel sample injection method, which was given the name of floating electrokinetic supercharging (FEKS). In the method, electrokinetic injection (EKI) and ITP preconcentration of samples was performed in a separation channel, connecting two reservoir ports (P3 and P4) on a cross-geometry microchip. At these two stages, side channels, crossing the separation channel, and their ports (P1 and P2) were electrically floated. After the ITP-stacked zones passed the cross-part, they were eluted for detection by using leading ions from P1 and P2 that enabled electrophoresis mode changing rapidly from ITP to zone electrophoresis (ZE). Possible sample leakage at the cross-part toward P1 and P2 was studied in detail on the basis of computer simulation using a CFD-ACE+ software and real experiments, through which it was validated that the analyte recovery to the separation channel was almost complete. The FEKS method successfully contributed to higher resolution and shorter analysis time of DNA fragments on the cross-microchip owing to more rapid switching from ITP status to ZE separation in comparison with our previous EKS procedure realized on a single-channel microchip. Without any degradation of resolution, the achieved LODs were on average ten times better than using conventional pinched injection. PMID:18393341

  10. Targeted lung expression of interleukin-11 enhances murine tolerance of 100% oxygen and diminishes hyperoxia-induced DNA fragmentation.

    PubMed Central

    Waxman, A B; Einarsson, O; Seres, T; Knickelbein, R G; Warshaw, J B; Johnston, R; Homer, R J; Elias, J A

    1998-01-01

    Acute lung injury is a frequent and treatment-limiting consequence of therapy with hyperoxic gas mixtures. To determine if IL-11 is protective in oxygen toxicity, we compared the effects of 100% O2 on transgenic mice that overexpress IL-11 in the lung and transgene (-) controls. IL-11 markedly enhanced survival in 100% O2 with 100% of transgene (-) animals dying within 72-96 h and > 90% of transgene (+) animals surviving for more than 10 d. This protection was associated with markedly diminished alveolar-capillary protein leak, endothelial and epithelial membrane injury, lipid peroxidation, and pulmonary neutrophil recruitment. Significant differences in copper zinc superoxide dismutase and catalase activities were not noted and the levels of total, reduced and oxidized glutathione were similar in transgene (+) and (-) animals. Glutathione reductase, glutathione peroxidase, and manganese superoxide dismutase activities were slightly higher in transgene (+) as versus (-) mice after 100% O2 exposure, and IL-11 diminished hyperoxia-induced expression of IL-1 and TNF. Hyperoxia also caused cell death with DNA fragmentation in the lungs of transgene (-) animals and IL-11 markedly diminished this cell death response. These studies demonstrate that IL-11 markedly diminishes hyperoxic lung injury. They also demonstrate this protection is associated with small changes in lung antioxidants, diminished hyperoxia-induced IL-1 and TNF production, and markedly suppressed hyperoxia-induced DNA fragmentation. PMID:9576762

  11. Increase in the astaxanthin synthase gene (crtS) dose by in vivo DNA fragment assembly in Xanthophyllomyces dendrorhous

    PubMed Central

    2013-01-01

    Background Xanthophyllomyces dendrorhous is a basidiomycetous yeast that is relevant to biotechnology, as it can synthesize the carotenoid astaxanthin. However, the astaxanthin levels produced by wild-type strains are low. Although different approaches for promoting increased astaxanthin production have been attempted, no commercially competitive results have been obtained thus far. A promising alternative to facilitate the production of carotenoids in this yeast involves the use of genetic modification. However, a major limitation is the few available molecular tools to manipulate X. dendrorhous. Results In this work, the DNA assembler methodology that was previously described in Saccharomyces cerevisiae was successfully applied to assemble DNA fragments in vivo and integrate these fragments into the genome of X. dendrorhous by homologous recombination in only one transformation event. Using this method, the gene encoding astaxanthin synthase (crtS) was overexpressed in X. dendrorhous and a higher level of astaxanthin was produced. Conclusions This methodology could be used to easily and rapidly overexpress individual genes or combinations of genes simultaneously in X. dendrorhous, eliminating numerous steps involved in conventional cloning methods. PMID:24103677

  12. Highly efficient synthesis of DNA-binding polyamides using a convergent fragment-based approach.

    PubMed

    Fallows, Andrew J; Singh, Ishwar; Dondi, Ruggero; Cullis, Paul M; Burley, Glenn A

    2014-09-01

    Two advances in the synthesis of hairpin pyrrole-imidazole polyamides (PAs) are described. First, the application of a convergent synthetic strategy is shown, involving the Boc-based solid phase synthesis of a C-terminal fragment and the solution phase synthesis of the N-terminal fragment. Second a new hybrid resin is developed that allows for the preparation of hairpin PAs lacking a C-terminal ?-alanine tail. Both methods are compatible with a range of coupling reagents and provide a facile, modular route to prepare PA libraries in high yield and crude purity. PMID:25162625

  13. Detection of genomic DNA fragmentation during apoptosis (DNA ladder) and the simultaneous isolation of RNA from low cell numbers.

    PubMed

    Daniel, P T; Sturm, I; Ritschel, S; Friedrich, K; Dörken, B; Bendzko, P; Hillebrand, T

    1999-01-01

    In the present paper we describe a rapid and sensitive method for the simultaneous isolation of total RNA and genomic plus low-molecular-weight DNA from apoptotic cells. Using this method, we were able to detect a DNA ladder from as low as 30,000 apoptotic cells in only 45 min including gel electrophoresis. In addition, RNA can be readily obtained from the same specimen to assess gene expression during apoptosis. This method therefore appears to be advantageous when sensitivity and low amounts of sample material are a limiting factor. PMID:9887219

  14. Strongly structured DNA sequences as targets for genosensing: sensing phase design and coupling to PCR amplification for a highly specific 33-mer gliadin DNA fragment.

    PubMed

    Martín-Fernández, Begoña; Miranda-Ordieres, Arturo J; Lobo-Castañón, María Jesús; Frutos-Cabanillas, Gloria; de-los-Santos-Álvarez, Noemí; López-Ruiz, Beatriz

    2014-10-15

    Electrochemical genosensors are becoming cost-effective miniaturizable alternatives to real-time PCR (RT-PCR) methods for the detection of sequence-specific DNA fragments. We report on the rapid detection of PCR amplicons without the need of purification or strand separation. A challenging target sequence for both PCR amplification and electrochemical detection allowed us to address some difficulties associated to hybridization on electrode surfaces. The target was a highly specific oligonucleotide sequence of wheat encoding the most immunogenic peptide of gliadin that triggers the immune response of celiac disease (CD), the 33-mer. With a sandwich assay format and a rational design of the capture and tagged-signaling probes the problems posed by the strong secondary structure of the target and complementary probes were alleviated. Using a binary self-assembled monolayer and enzymatic amplification, a limit of detection of 0.3 nM was obtained. The genosensor did not respond to other gluten-containing cereals such as rye and barley. Coupling to PCR to analyze wheat flour samples required tailoring both the capture and signaling probes. This is the first time that deleterious steric hindrance from long single-stranded regions adjacent to the electrode surface is reported for relatively short amplicons (less than 200 bp). The importance of the location of the recognition site within the DNA sequence is discussed. Since the selected gene fragment contains several repetitions of short sequences, a careful optimization of the PCR conditions had to be performed to circumvent the amplification of non-specific fragments from wheat flour. PMID:24813914

  15. Mitochondrial DNA polymorphism in Japanese

    Microsoft Academic Search

    Satoshi Horai; Ei Matsunaga

    1986-01-01

    Mitochondrial DNA (mtDNA) from 116 Japanese was analyzed with nine restriction enzymes that recognize a four or five base pair sequence. The sizes of the mtDNA fragments produced by digestion by each enzyme were compared after gel electrophoresis. Double digestion experiments indicated that, in the coding region from URF2 (unidentified reading frame) to tRNAAsn (bp 5274–5691), there is an insertion

  16. The use of the polymerase chain reaction and restriction fragment length polymorphism technique associated with the classical morphology for characterization of Lymnaea columella, L. viatrix, and L. diaphana (Mollusca: Lymnaeidae)

    Microsoft Academic Search

    Omar S Carvalho; Paula CM Cardoso; Pollanah M Lira; Alejandra Rumi; Andrea Roche; Elizabeth Berne; Gertrud Müller; Roberta L Caldeira

    2004-01-01

    The specific identification of Lymnaeid snails is based on a comparison of morphological characters of the shell, radula, renal and reproductive organs. However, the identification is complicated by dissection process, intra and interspecific similarity and variability of morphological characters. In the present study, polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) techniques targeted to the first and second internal

  17. Effect of Ames dwarfism and caloric restriction on spontaneous DNA mutation frequency in different mouse tissues

    Microsoft Academic Search

    Ana Maria Garcia; Rita A. Busuttil; R. Brent Calder; Martijn E. T. Dollé; Vivian Diaz; C. Alex McMahan; Andrzej Bartke; James Nelson; Robert Reddick; Jan Vijg

    2008-01-01

    Genetic instability has been implicated as a causal factor in cancer and aging. Caloric restriction (CR) and suppression of the somatotroph axis significantly increase life span in the mouse and reduce multiple symptoms of aging, including cancer. To test if in vivo spontaneous mutation frequency is reduced by such mechanisms, we crossed long-lived Ames dwarf mice with a C57BL\\/6J line

  18. Alternatively spliced insertions in the paired domain restrict the DNA sequence specificity of Pax6 and Pax8.

    PubMed Central

    Kozmik, Z; Czerny, T; Busslinger, M

    1997-01-01

    Transcription factors of the Pax family bind to their target genes via the paired domain which is known to be composed of two subdomains each recognizing distinct half-sites in adjacent major grooves of the DNA helix. We now demonstrate that the mammalian Pax8 gene gives rise, by alternative mRNA splicing, to a protein isoform containing an extra serine residue in the recognition alpha-helix 3 of the paired domain. This Pax8(S) protein does not interact with bipartite paired domain-binding sites, indicating that inactivation of the N-terminal DNA-binding motif severely restricts the sequence specificity of the paired domain. However, the Pax8(S) protein binds in vitro and in vivo to the 5aCON sequence which was previously identified as a high-affinity binding site for the Pax6(5a) splice variant carrying a 14-amino-acid insertion in the paired domain. The 5aCON sequence is shown to consist of four interdigitated 5' half-sites of the bipartite consensus sequence and is thus bound by four Pax8(S) molecules via the intact C-terminal DNA-binding motif of the paired domain. Together these data suggest that inactivation of the N-terminal region of the paired domain by alternative splicing is used in vivo to selectively target Pax transcription factors to gene regulatory regions containing highly specialized 5aCON-like sequences. PMID:9362493

  19. Gene organization in a central DNA fragment of Oenococcus oeni bacteriophage fOg44 encoding lytic, integrative and non-essential functions

    Microsoft Academic Search

    Ricardo Parreira; Carlos São-José; Anabela Isidro; Susana Domingues; Graça Vieira; Mário A. Santos

    1999-01-01

    The nucleotide sequence of a DNA fragment previously shown to contain the attachment site (attP) of Oenococcus oeni phage fOg44 (Santos et al., 1998. Arch. Virol. 143, 523–536) has been determined. Sequence analysis indicated that this 6226bp EcoRI fragment harbours an integrase gene, in the vicinity of a direct repeat rich region defining attP, as well as genes encoding a

  20. Chromatin Collapse during Caspase-dependent Apoptotic Cell Death Requires DNA Fragmentation Factor, 40-kDa Subunit-/Caspase-activated Deoxyribonuclease-mediated 3?-OH Single-strand DNA Breaks*

    PubMed Central

    Iglesias-Guimarais, Victoria; Gil-Guiñon, Estel; Sánchez-Osuna, María; Casanelles, Elisenda; García-Belinchón, Mercè; Comella, Joan X.; Yuste, Victor J.

    2013-01-01

    Apoptotic nuclear morphology and oligonucleosomal double-strand DNA fragments (also known as DNA ladder) are considered the hallmarks of apoptotic cell death. From a classic point of view, these two processes occur concomitantly. Once activated, DNA fragmentation factor, 40-kDa subunit (DFF40)/caspase-activated DNase (CAD) endonuclease hydrolyzes the DNA into oligonucleosomal-size pieces, facilitating the chromatin package. However, the dogma that the apoptotic nuclear morphology depends on DNA fragmentation has been questioned. Here, we use different cellular models, including MEF CAD?/? cells, to unravel the mechanism by which DFF40/CAD influences chromatin condensation and nuclear collapse during apoptosis. Upon apoptotic insult, SK-N-AS cells display caspase-dependent apoptotic nuclear alterations in the absence of internucleosomal DNA degradation. The overexpression of a wild-type form of DFF40/CAD endonuclease, but not of different catalytic-null mutants, restores the cellular ability to degrade the chromatin into oligonucleosomal-length fragments. We show that apoptotic nuclear collapse requires a 3?-OH endonucleolytic activity even though the internucleosomal DNA degradation is impaired. Moreover, alkaline unwinding electrophoresis and In Situ End-Labeling (ISEL)/In Situ Nick Translation (ISNT) assays reveal that the apoptotic DNA damage observed in the DNA ladder-deficient SK-N-AS cells is characterized by the presence of single-strand nicks/breaks. Apoptotic single-strand breaks can be impaired by DFF40/CAD knockdown, abrogating nuclear collapse and disassembly. In conclusion, the highest order of chromatin compaction observed in the later steps of caspase-dependent apoptosis relies on DFF40/CAD-mediated DNA damage by generating 3?-OH ends in single-strand rather than double-strand DNA nicks/breaks. PMID:23430749

  1. An Interface for a Fragment Assembly Kernel

    Microsoft Academic Search

    Susan Larson; Mudita Jain; Eric Anson; Gene Myers

    ABSTRACT This document,describes the C programming,language interface to our Fragment Assembly Kernel library. Inputs to the Fragment Assembly Kernel are (1) DNA fragment sequences from potentially inaccurate sequencing experiments, and (2) optional constraints on fragment assembly such as known fragment overlaps or relative fragment orientation. Fragment sequence version control is supported. The Fragment Assembly Kernel produces the most probable reconstructions

  2. [Cloning of PCR-copies of Streptomyces coelicolor A3(2) chromosome fragment].

    PubMed

    Luk'ianchuk, V V

    2009-01-01

    Copies of chromosome Streptomyces coelicolor A3(2) fragment (gene coding polyketide syntase of III type) were obtained to use polymerase cycle reaction. Amplified DNA was cloned in shuttle vector pWHM4 on sites for HindIII and EcoRI. The restriction analysis of plasmid DNAs of transformants has demonstrated that molecular size of cloned DNA fragment was 1134 bp. PMID:20458937

  3. A Unified Framework for Mapping Quantitative Trait Loci in Bivalent Tetraploids Using Single-dose Restriction Fragments: A Case Study from Alfalfa

    PubMed Central

    Ma, Chang-Xing; Casella, George; Shen, Zuo-Jun; Osborn, Thomas C.; Wu, Rongling

    2002-01-01

    The development of statistical methodologies for quantitative trait locus (QTL) mapping in polyploids is complicated by complex polysomic inheritance. In this article, we propose a statistical method for mapping QTL in tetraploids undergoing bivalent formation at meiosis by using single-dose restriction fragments. Our method is based on a unified framework, one that uses chromosome bivalent pairing configuration and gametic recombination to discern different mechanisms of gamete formation. Our bivalent polyploid model can not only provide a simultaneous estimation of the linkage and chromosome pairing configuration—a cytological parameter of evolutionary and systematic interest—but also enhances the precision of estimating QTL effects and position by correctly characterizing gene segregation during polyploid meiosis. By using our method and a linkage map constructed in a previous study, we successfully identify several QTL affecting winter hardiness in bivalent tetraploid alfalfa. Moreover, our results reveal significant preferential chromosome pairing at meiosis in an F1 hybrid population, which indicates the importance of reassessing the traditional view of random chromosome segregation in alfalfa. PMID:12466302

  4. Use of polymerase chain reaction: Restriction fragment length polymorphism to detect acaricidal resistance to synthetic pyrethroids in Boophilus microplus ticks of South India

    PubMed Central

    Cattavarayane, Mathivathani; Basith, Abdul; Latha, Bhaskaran Ravi

    2013-01-01

    Background: Boophilus microplus is an important ectoparasite of livestock. Apart from transmitting diseases, heavy tick burden can decrease production and damage hides. The synthetic pyrethroids which are advantageous over other acaricides for treatment of this infestation are now losing their efficacy due to development of resistant strains of ticks. Materials and Methods: Boophilus microplus ticks with a previous history of acaricidal treatment especially synthetic pyrethroids (SP) such as cypermethrin, deltamethrin and flumethrin were randomly collected from different pockets of four Southern States of India namely Tamil Nadu, Kerala, Karnataka, Andhra Pradesh, and Puducherry from cattle. Deoxyribonucleic acid extracted from pooled adult B. microplus tick from each State was subjected to polymerase chain reaction-restriction fragment length polymorphism to detect point mutation in carboxyl esterase gene. Results: A product size of 372 bp was obtained for cattle tick samples collected from all over Southern States of India. Conclusions: B. microplus ticks found in Southern part of India are not resistant to commonly used SP. PMID:23961446

  5. Clustering of Beijing genotype Mycobacterium tuberculosis isolates from the Mekong delta in Vietnam on the basis of variable number of tandem repeat versus restriction fragment length polymorphism typing

    PubMed Central

    2013-01-01

    Background In comparison to restriction fragment length polymorphism (RFLP) typing, variable number of tandem repeat (VNTR) typing is easier to perform, faster and yields results in a simple, numerical format. Therefore, this technique has gained recognition as the new international gold standard in typing of Mycobacterium tuberculosis. However, some reports indicated that VNTR typing may be less suitable for Beijing genotype isolates. We therefore compared the performance of internationally standardized RFLP and 24 loci VNTR typing to discriminate among 100 Beijing genotype isolates from the Southern Vietnam. Methods Hundred Beijing genotype strains defined by spoligotyping were randomly selected and typed by RFLP and VNTR typing. The discriminatory power of VNTR and RFLP typing was compared using the Bionumerics software. Results Among 95 Beijing strains available for analysis, 14 clusters were identified comprising 34 strains and 61 unique profiles in 24 loci VNTR typing ((Hunter Gaston Discrimination Index (HGDI = 0.994)). 13 clusters containing 31 strains and 64 unique patterns in RFLP typing (HGDI = 0.994) were found. Nine RFLP clusters were subdivided by VNTR typing and 12 VNTR clusters were split by RFLP. Five isolates (5%) revealing double alleles or no signal in two or more loci in VNTR typing could not be analyzed. Conclusions Overall, 24 loci VNTR typing and RFLP typing had similar high-level of discrimination among 95 Beijing strains from Southern Vietnam. However, loci VNTR 154, VNTR 2461 and VNTR 3171 had hardly added any value to the level of discrimination. PMID:23375050

  6. Effect of Microbial Inoculants on the Indigenous Actinobacterial Endophyte Population in the Roots of Wheat as Determined by Terminal Restriction Fragment Length Polymorphism

    PubMed Central

    Conn, Vanessa M.; Franco, Christopher M. M.

    2004-01-01

    The effect of single actinobacterial endophyte seed inoculants and a mixed microbial soil inoculant on the indigenous endophytic actinobacterial population in wheat roots was investigated by using the molecular technique terminal restriction fragment length polymorphism (T-RFLP). Wheat was cultivated either from seeds coated with the spores of single pure actinobacterial endophytes of Microbispora sp. strain EN2, Streptomyces sp. strain EN27, and Nocardioides albus EN46 or from untreated seeds sown in soil with and without a commercial mixed microbial soil inoculant. The endophytic actinobacterial population within the roots of 6-week-old wheat plants was assessed by T-RFLP. Colonization of the wheat roots by the inoculated actinobacterial endophytes was detected by T-RFLP, as were 28 to 42 indigenous actinobacterial genera present in the inoculated and uninoculated plants. The presence of the commercial mixed inoculant in the soil reduced the endophytic actinobacterial diversity from 40 genera to 21 genera and reduced the detectable root colonization by approximately half. The results indicate that the addition of a nonadapted microbial inoculum to the soil disrupted the natural actinobacterial endophyte population, reducing diversity and colonization levels. This was in contrast to the addition of a single actinobacterial endophyte to the wheat plant, where the increase in colonization level could be confirmed even though the indigenous endophyte population was not adversely affected. PMID:15528499

  7. dHPLC efficiency for semi-automated cDNA-AFLP analyses and fragment collection in the apple scab-resistance gene model.

    PubMed

    Paris, Roberta; Dondini, Luca; Zannini, Graziano; Bastia, Daniela; Marasco, Elena; Gualdi, Valentina; Rizzi, Valeria; Piffanelli, Pietro; Mantovani, Vilma; Tartarini, Stefano

    2012-05-01

    cDNA-AFLP analysis for transcript profiling has been successfully applied to study many plant biological systems, particularly plant-microbe interactions. However, the separation of cDNA-AFLP fragments by gel electrophoresis is reported to be labor-intensive with only limited potential for automation, and the collection of differential bands for gene identification is even more cumbersome. In this work, we present the use of dHPLC (denaturing high performance liquid chromatography) and automated DNA fragment collection using the WAVE(®) System to analyze and recover cDNA-AFLP fragments. The method is successfully applied to the Malus-Venturia inaequalis interaction, making it possible to collect 66 different transcript-derived fragments for apple genes putatively involved in the defense response activated by the HcrVf2 resistance gene. The results, validated by real time quantitative RT-PCR, were consistent with the plant-pathogen interaction under investigation and this further supports the suitability of dHPLC for cDNA-AFLP transcript profiling. Features and advantages of this new approach are discussed, evincing that it is an almost fully automatable and cost-effective solution for processing large numbers of samples and collecting differential genes involved in other biological processes and non-model plants. PMID:22270558

  8. Phylogenetic relationships of Thlaspi s.l. (subtribe Thlaspidinae, Lepidieae) and allied genera based on chloroplast DNA restriction-site variation

    Microsoft Academic Search

    K. Zunk; K. Mummenhoff; M. Koch; H. Hurka

    1996-01-01

    Chloroplast DNA restriction-site variation was analyzed in 30 accessions representing 20 species from the major lineages in Thlaspi s.l. (previously described as genera by Meyer 1973, 1979) and allied genera from the subtribe Thlaspidinae (Peltaria, Teesdalia, Cochlearia, Ionopsidium, Aethionema). A total of 161 variable restriction sites were detected. Phylogenetic analyses indicated a division of Thlaspi s.l. into three groups consistent

  9. Rapid parallel mutation scanning of gene fragments using a microelectronic protein–DNA chip format

    PubMed Central

    Behrensdorf, Heike A.; Pignot, Marc; Windhab, Norbert; Kappel, Andreas

    2002-01-01

    We have developed a method for the de novo discovery of genetic variations, including single nucleotide polymorphisms and mutations, on microelectronic chip devices. The method combines the features of electronically controlled DNA hybridisation on open-format microarrays, with mutation detection by a fluorescence-labelled mismatch- binding protein. Electronic addressing of DNA strands to distinct test sites of the chip allows parallel analysis of several individuals, as demonstrated for mutations in different exons of the p53 gene. This microelectronic chip-based mutation discovery assay may substitute for time-consuming sequencing studies and will complement existing technologies in genomic research. PMID:12136112

  10. A ribosomal DNA fragment of Listeria monocytogenes and its use as a genus-specific probe in an aqueous-phase hybridization assay.

    PubMed Central

    Emond, E; Fliss, I; Pandian, S

    1993-01-01

    cDNAs were prepared from the total RNA of Listeria monocytogenes ATCC 19118 and used as probes to screen a genomic library of the same strain. Four clones were identified which contained ribosomal DNA fragments. Recombinant DNA from one of them was fractionated and differentially hybridized with the cDNA probes to RNA of L. monocytogenes and Kurthia zopfii. The resulting hybridization pattern revealed an HpaII fragment of 0.8 kb that was specific for the L. monocytogenes strain. The nucleotide sequence of this fragment showed 159 bases of the 3' end of the 16S rRNA gene, 243 bases of the spacer region, and 382 bases of the 5' end of the 23S rRNA gene. In dot blot hybridization assays, the 32P-labeled 784-bp fragment was specific only for Listeria species. Dot blot assays revealed that the 32P-labeled fragment can easily detect > or = 10 pg of total nucleic acids from pure cultures of L. monocytogenes, which corresponds to approximately 300 bacteria. This fragment was also used as a probe in an assay named the heteroduplex nucleic acid (HNA) enzyme-linked immunosorbent assay. In this system, the biotinylated DNA probe is hybridized in the aqueous phase with target RNA molecules and then specific HNAs are captured by HNA-specific antibodies. Captured HNA molecules are revealed with an enzyme conjugate of streptavidin. In a preliminary HNA enzyme-linked immunosorbent assay, the 784-bp fragment maintained its specificity for Listeria spp. and could detect 5 x 10(2) cells in artificially contaminated meat homogenate. Images PMID:8368854

  11. Damage to model DNA fragments from very low-energy (<1 eV) electrons.

    PubMed

    Berdys, Joanna; Anusiewicz, Iwona; Skurski, Piotr; Simons, Jack

    2004-05-26

    Although electrons having enough energy to ionize or electronically excite DNA have long been known to cause strand breaks (i.e., bond cleavages), only recently has it been suggested that even lower-energy electrons (most recently 1 eV and below) can also damage DNA. The findings of the present work suggest that, while DNA bases can attach electrons having kinetic energies in the 1 eV range and subsequently undergo phosphate-sugar O-C sigma bond cleavage, it is highly unlikely (in contrast to recent suggestions) that electrons having kinetic energies near 0 eV can attach to the phosphate unit's P=O bonds. Electron kinetic energies in the 2-3 eV range are required to attach directly to DNA's phosphate group's P=O pi orbital and induce phosphate-sugar O-C sigma bond cleavages if the phosphate groups are rendered neutral (e.g., by nearby counterions). Moreover, significant activation barriers to C-O bond breakage render the rates of both such damage mechanisms (i.e., P=O-attached and base-attached) slow as compared to electron autodetachment and to other damage processes. PMID:15149241

  12. A Fuzzy Classifier to Taxonomically Group DNA Fragments within a Metagenome

    E-print Network

    Nicolescu, Monica

    is influenced by their habitat. Most microorganisms genomes are known from pure cul- tures of organisms isolated natural habitat as part of a community. Research has broadened from studying single species for isolation and lab cultivation of individual species [4]. The whole genome(DNA) or metagenome population can

  13. Intraspecific phylogeographic isolation of Arabian Gulf sailfish Istiophorus platypterus inferred from mitochondrial DNA

    Microsoft Academic Search

    J. P. Hoolihan; J. Premanandh; M.-A. D’Aloia-Palmieri; J. A. H. Benzie

    2004-01-01

    The genetic structure of seven sailfish Istiophorus platypterus populations sampled from three locations inside and four locations outside the Arabian Gulf was determined by restriction fragment length polymorphism analysis of mitochondrial DNA of 147 individuals using eight restriction endonucleases. A total of 39 composite haplotypes derived from 27 presumptive restriction sites demonstrated significant differences in frequency between population groups inside

  14. Extrapolation of the dna fragment-size distribution after high-dose irradiation to predict effects at low doses

    NASA Technical Reports Server (NTRS)

    Ponomarev, A. L.; Cucinotta, F. A.; Sachs, R. K.; Brenner, D. J.; Peterson, L. E.

    2001-01-01

    The patterns of DSBs induced in the genome are different for sparsely and densely ionizing radiations: In the former case, the patterns are well described by a random-breakage model; in the latter, a more sophisticated tool is needed. We used a Monte Carlo algorithm with a random-walk geometry of chromatin, and a track structure defined by the radial distribution of energy deposition from an incident ion, to fit the PFGE data for fragment-size distribution after high-dose irradiation. These fits determined the unknown parameters of the model, enabling the extrapolation of data for high-dose irradiation to the low doses that are relevant for NASA space radiation research. The randomly-located-clusters formalism was used to speed the simulations. It was shown that only one adjustable parameter, Q, the track efficiency parameter, was necessary to predict DNA fragment sizes for wide ranges of doses. This parameter was determined for a variety of radiations and LETs and was used to predict the DSB patterns at the HPRT locus of the human X chromosome after low-dose irradiation. It was found that high-LET radiation would be more likely than low-LET radiation to induce additional DSBs within the HPRT gene if this gene already contained one DSB.

  15. Transgenic DNA integrated into the oat genome is frequently interspersed by host DNA

    PubMed Central

    Pawlowski, Wojciech P.; Somers, David A.

    1998-01-01

    Integration of transgenic DNA into the plant genome was investigated in 13 transgenic oat (Avena sativa L.) lines produced using microprojectile bombardment with one or two cotransformed plasmids. In all transformation events, the transgenic DNA integrated into the plant genome consisted of intact transgene copies that were accompanied by multiple, rearranged, and/or truncated transgene fragments. All fragments of transgenic DNA cosegregated, indicating that they were integrated at single gene loci. Analysis of the structure of the transgenic loci indicated that the transgenic DNA was interspersed by the host genomic DNA. The number of insertions of transgenic DNA within the transgene loci varied from 2 to 12 among the 13 lines. Restriction endonucleases that do not cleave the introduced plasmids produced restriction fragments ranging from 3.6 to about 60 kb in length hybridizing to a probe comprising the introduced plasmids. Although the size of the interspersing host DNA within the transgene locus is unknown, the sizes of the transgene-hybridizing restriction fragments indicated that the entire transgene locus must be at least from 35–280 kb. The observation that all transgenic lines analyzed exhibited genomic interspersion of multiple clustered transgenes suggests a predominating integration mechanism. We propose that transgene integration at multiple clustered DNA replication forks could account for the observed interspersion of transgenic DNA with host genomic DNA within transgenic loci. PMID:9770447

  16. Paternal inheritance of chloroplast DNA and maternal inheritance of mitochondrial DNA in loblolly pine

    Microsoft Academic Search

    D. B. Neale; R. R. Sederoff

    1989-01-01

    The inheritance of organelle DNAs in loblolly pine was studied by using restriction fragment length polymorphisms. Chloroplast DNA from loblolly pine is paternally inherited in pitch pine x loblolly pine hybrids. Mitochondrial DNA is maternally inherited in loblolly pine crosses. The uniparental inheritance of organelle genomes from opposite sexes within the same plant appears to be unique among those higher

  17. Transfer of mitochondrial DNA from the northern red-backed vole ( Clethrionomys rutilus ) to the bank vole ( C. glareolus )

    Microsoft Academic Search

    Håkan Tegelström

    1987-01-01

    Summary Using a silver staining method to detect DNA fragments produced by restriction enzymes, it was possible to compare mitochondrial DNAs (mtDNAs) from 85 individuals of the bank vole (Clethrionomys glareolus) trapped at 25 localities in Fennoscandia. There are two distinctly different mtDNA lineages, one occurring in southern and central Fennoscandia and the other in the northern parts. A fragment

  18. Characterization of mitochondrial DNA in chloramphenicol-resistant interspecific hybrids and a cybrid

    Microsoft Academic Search

    Richard E. Giles; I Iwona Stroynowski; Douglas C. Wallace

    1980-01-01

    We have examined the restriction endonuclease cleavage patterns exhibited by the mitochondrial DNAs (mtDNA) of four chloramphenicolresistant (CAPR)human × mouse hybrids and one CAPRcybrid derived from CAPRHeLa cells and CAPSmouse RAG cells. Restriction fragments of mtDNAs were separated by electrophoresis and transferred by the Southern technique to diazobenzyloxymethyl paper. The covalently bound DNA fragments were hybridized initially with32P-labeled complementary RNA

  19. 16S Ribosomal DNA Terminal Restriction Fragment Pattern Analysis of Bacterial Communities in Feces of Rats Fed Lactobacillus acidophilus NCFM

    Microsoft Academic Search

    CHRISTOPHER W. KAPLAN; JOHANNA C. ASTAIRE; MARY ELLEN SANDERS; BANDARU S. REDDY; CHRISTOPHER L. KITTS

    2001-01-01

    Many efforts to study microbial communities in the gastro- intestinal tract have focused on the large bowel and conse- quently on fecal samples (9, 10, 17, 22, 23, 24). Several methods for following the complex communities in the large bowel have been employed. However, because of the abundance and di- versity of bacteria in feces, this has proved a difficult

  20. Identification ofCandidaSpecies by PCR and Restriction Fragment Length Polymorphism Analysis of Intergenic Spacer Regions of Ribosomal DNA

    Microsoft Academic Search

    DAVID W. WILLIAMS; MELANIE J. WILSON; MICHAEL A. O. LEWIS; JOHN C. POTTS

    1995-01-01

    Candidoses comprise a range of human opportunistic infec- tions which may occur in either acute or chronic forms. Can- dida infections frequently arise on the mucosal surfaces of the mouth or vagina. Chronic hyperplastic candidosis of the oral mucosa is of particular importance since it has been associated with the development of squamous cell carcinoma (3). In ad- dition to

  1. DNA Forensics and Color Pigments

    NSDL National Science Digital Library

    National Science Foundation GK-12 and Research Experience for Teachers (RET) Programs,

    Students perform DNA forensics using food coloring to enhance their understanding of DNA fingerprinting, restriction enzymes, genotyping and DNA gel electrophoresis. They place small drops of different food coloring ("water-based paint") on strips of filter paper and then place one paper strip end in water. As water travels along the paper strips, students observe the pigments that compose the paint decompose into their color components. This is an example of the chromatography concept applied to DNA forensics, with the pigments in the paint that define the color being analogous to DNA fragments of different lengths.

  2. Fragmentation of condensed-phase DNA components by hyperthermal He{sup +} impact

    Microsoft Academic Search

    Deng Zongwu; Marjorie Imhoff; Ilko Bald; Eugen Illenberger; Michael A. Huels

    2006-01-01

    We have observed severe damage to films of DNA components (thymine, D-ribose, 2-deoxy-D-ribose, and thymidine) induced by 10 to 100 eV He{sup +} ions (2.5-25 eV\\/amu). The damage is attributed to the kinetic and potential energies, as well as the chemical reactivity of the He{sup +} projectiles. Hyperthermal He{sup +} ion impact on these films results in the complete destruction

  3. Multi-organ characterization of mitochondrial genomic rearrangements in ad libitum and caloric restricted mice show striking somatic mitochondrial DNA rearrangements with age.

    PubMed Central

    Melov, S; Hinerfeld, D; Esposito, L; Wallace, D C

    1997-01-01

    Mitochondrial DNA (mtDNA) rearrangements have been shown to accumulate with age in the post-mitotic tissues of a variety of animals and have been hypothesized to result in the age-related decline of mitochondrial bioenergetics leading to tissue and organ failure. Caloric restriction in rodents has been shown to extend life span supporting an association between bioenergetics and senescence. In the present study, we use full length mtDNA amplification by long-extension polymerase chain reaction (LX-PCR) to demonstrate that mice accumulate a wide variety of mtDNA rearrangements with age in post mitotic tissues. Similarly, using an alternative PCR strategy, we have found that 2-4 kb minicircles containing the origin of heavy-strand replication accumulate with age in heart but not brain. Analysis of mtDNA structure and conformation by Southern blots of unrestricted DNA resolved by field inversion gel electrophoresis have revealed that the brain mtDNAs of young animals contain the traditional linear, nicked, and supercoiled mtDNAs while old animals accumulate substantial levels of a slower migrating species we designate age-specific mtDNAs. In old caloric restricted animals, a wide variety of rearranged mtDNAs can be detected by LX-PCR in post mitotic tissues, but Southern blots of unrestricted DNA reveals a marked reduction in the levels of the age- specific mtDNA species. These observations confirm that mtDNA mutations accumulate with age in mice and suggest that caloric restriction impedes this progress. PMID:9023106

  4. Direct Biochemical Mapping of Eukaryotic Viral DNA by means of a Linked Transcription-Translation Cell-Free System

    Microsoft Academic Search

    Shmuel Rozenblatt; Richard C. Mulligan; Marian Gorecki; Bryan E. Roberts; Alexander Rich

    1976-01-01

    A method is described for mapping regions of eukaryotic viral DNA coding for specific proteins, utilizing a linked transcription-translation cell-free system primed with DNA fragments generated by restriction endonucleases. Three simian virus 40 (SV40) DNA fragments derived from that region of the DNA expressed late in lytic infection were purified. They were: Hpa I-A (0.76-0.175 map units), Bgl I-EcoRI-B (0.672-0),

  5. A Novel Quantitative Hemolytic Assay Coupled with Restriction Fragment Length Polymorphisms Analysis Enabled Early Diagnosis of Atypical Hemolytic Uremic Syndrome and Identified Unique Predisposing Mutations in Japan

    PubMed Central

    Yoshida, Yoko; Miyata, Toshiyuki; Matsumoto, Masanori; Shirotani-Ikejima, Hiroko; Uchida, Yumiko; Ohyama, Yoshifumi; Kokubo, Tetsuro; Fujimura, Yoshihiro

    2015-01-01

    For thrombotic microangiopathies (TMAs), the diagnosis of atypical hemolytic uremic syndrome (aHUS) is made by ruling out Shiga toxin-producing Escherichia coli (STEC)-associated HUS and ADAMTS13 activity-deficient thrombotic thrombocytopenic purpura (TTP), often using the exclusion criteria for secondary TMAs. Nowadays, assays for ADAMTS13 activity and evaluation for STEC infection can be performed within a few hours. However, a confident diagnosis of aHUS often requires comprehensive gene analysis of the alternative complement activation pathway, which usually takes at least several weeks. However, predisposing genetic abnormalities are only identified in approximately 70% of aHUS. To facilitate the diagnosis of complement-mediated aHUS, we describe a quantitative hemolytic assay using sheep red blood cells (RBCs) and human citrated plasma, spiked with or without a novel inhibitory anti-complement factor H (CFH) monoclonal antibody. Among 45 aHUS patients in Japan, 24% (11/45) had moderate-to-severe (?50%) hemolysis, whereas the remaining 76% (34/45) patients had mild or no hemolysis (<50%). The former group is largely attributed to CFH-related abnormalities, and the latter group has C3-p.I1157T mutations (16/34), which were identified by restriction fragment length polymorphism (RFLP) analysis. Thus, a quantitative hemolytic assay coupled with RFLP analysis enabled the early diagnosis of complement-mediated aHUS in 60% (27/45) of patients in Japan within a week of presentation. We hypothesize that this novel quantitative hemolytic assay would be more useful in a Caucasian population, who may have a higher proportion of CFH mutations than Japanese patients. PMID:25951460

  6. Clonal Complexes of Campylobacter jejuni Identified by Multilocus Sequence Typing Are Reliably Predicted by Restriction Fragment Length Polymorphism Analyses of the flaA Gene?

    PubMed Central

    Djordjevic, Steven P.; Unicomb, Leanne E.; Adamson, Penelope J.; Mickan, Lance; Rios, Rosa

    2007-01-01

    Multilocus sequence typing (MLST) has provided important new insights into the population structure of Campylobacter jejuni and is rapidly becoming the gold standard for typing this species. However, the methodology is comparatively costly and slow to perform for the routine surveillance testing of large numbers of isolates required by public health laboratories. Restriction fragment length polymorphism analysis of the flaA gene (RFLP-flaA) and sequencing of the variable region in the fla locus (SVR-fla) were compared to MLST to determine if a low cost alternative could be found that reliably predicts clonal lineage (as determined by MLST). An isolate of C. jejuni from each of 153 patients from New South Wales, Australia, collected sequentially over a period of 30 months from 1999 to 2001 and comprising 40 sequence types (ST) from 15 clonal complexes (CC) was examined. Of 15 CC, 12 were represented by more than one isolate and a predominant RFLP-flaA type was found for 10 (83%). Of these, seven (70%) correctly predicted the predominant MLST CC with a probability of >0.8. Of 40 STs detected, 19 were reported for the first time, 9 of which were represented by more than one isolate. Eight of these were represented by a single RFLP-flaA type. Only two of eight major SVR-fla types were able to predict CC with a probability of >0.8, indicating that flaA-RFLP is a more reliable predictor of CC than SVR-fla and thus offers an alternative to MLST for use in routine surveillance. PMID:17093018

  7. Distribution Study of Chlamydia trachomatis Serovars among High-Risk Women in China Performed Using PCR-Restriction Fragment Length Polymorphism Genotyping?

    PubMed Central

    Gao, Xing; Chen, Xiang-Sheng; Yin, Yue-Ping; Zhong, Ming-Ying; Shi, Mei-Qin; Wei, Wan-Hui; Chen, Qiang; Peeling, Rosanna W.; Mabey, David

    2007-01-01

    This was one of the first epidemiological studies in China focused on genital Chlamydia trachomatis serotype distribution in high-risk female populations using omp1 gene-based restriction fragment length polymorphism analysis. One thousand seven hundred seventy cervical swab samples from women attending sexually transmitted disease clinics and female sex workers in six cities in China (Shenzhen and Guangzhou in southern China, Nanjing and Shanghai in eastern China, and Nanning and Chengdu in southwestern China) were subjected to serovar genotyping. The proportion of omp1 genes successfully amplified in 240 C. trachomatis plasmid-positive samples was 94.2% (226/240). Serotypes E (n = 63; 27.9%), F (n = 53; 23.5%), G (n = 28; 12.4%), and D (n = 25; 11.1%) were most prevalent. Though there was no significant difference in the geographic distribution of C. trachomatis, serotype E was predominant in the South (32.1%) and East (27.1%), while serotype F was predominant in the Southwest (28.3%). Serotype F infection was associated with young age and single status. Serovar G was associated with lower abdominal pain; 47.5% of asymptomatic patients were infected with serovar E. These results provide information on distribution of genital C. trachomatis serotypes among high-risk women in China and indicate that high-risk women, including those who are asymptomatic, can be infected with multiple serovars of C. trachomatis, revealing exposure to multiple sources of infection. Although the scope for generalizations is limited by our small sample size, our results showing clinical correlations with genotypes are informative. PMID:17301282

  8. Close association of predominant genotype of herpes simplex virus type 1 with eczema herpeticum analyzed using restriction fragment length polymorphism of polymerase chain reaction.

    PubMed

    Yoshida, Masami; Umene, Kenichi

    2003-04-01

    Herpes simplex virus type 1 (HSV-1) strains belonging to the same genotype can possibly share biological properties and clinical manifestations common to the genotype. We classified previously 66 HSV-1 strains into 35 genotypes (F1-F35) using restriction fragment length polymorphism (RFLP) and F1 and F35 genotypes were revealed to be predominant [Arch. Virol. 13 (1993) 29]. It was found later that the F35 genotype seemed to be closely associated with eczema herpeticum [J. Med. Virol. 49 (1996) 329]. In the present study, a convenient method was developed for classification of two predominant genotypes by RFLP of polymerase chain reaction (RFLP-PCR). Using this method, genotypes of 21 strains isolated from eczema herpeticum were analyzed; seven of 21 strains (33.3%) were of F1 and five of 21 (23.8%) were of F35. Genotypes of 19 strains isolated from facial herpes other than eczema herpeticum were as follows; six of 19 (31.6%) strains were of F1 and one of 19 (5.3%) were of F35. Thus, strains belonging to F35 were appear to have been isolated more frequently from eczema herpeticum (5/21) than from facial herpes (1/19). These ratios showed a statistically significant difference. These results support the hypothesis that F35 strains is clearly associated with eczema herpeticum, in agreement with previous study. This is the first report of PCR-based approach for classification of HSV-1 strains into genotypes seeking an association of a genotype with clinical manifestation. PMID:12668262

  9. Reconstructing the history of a fragmented and heavily exploited red deer population using ancient and contemporary DNA

    PubMed Central

    2012-01-01

    Background Red deer (Cervus elaphus) have been an important human resource for millennia, experiencing intensive human influence through habitat alterations, hunting and translocation of animals. In this study we investigate a time series of ancient and contemporary DNA from Norwegian red deer spanning about 7,000 years. Our main aim was to investigate how increasing agricultural land use, hunting pressure and possibly human mediated translocation of animals have affected the genetic diversity on a long-term scale. Results We obtained mtDNA (D-loop) sequences from 73 ancient specimens. These show higher genetic diversity in ancient compared to extant samples, with the highest diversity preceding the onset of agricultural intensification in the Early Iron Age. Using standard diversity indices, Bayesian skyline plot and approximate Bayesian computation, we detected a population reduction which was more prolonged than, but not as severe as, historic documents indicate. There are signs of substantial changes in haplotype frequencies primarily due to loss of haplotypes through genetic drift. There is no indication of human mediated translocations into the Norwegian population. All the Norwegian sequences show a western European origin, from which the Norwegian lineage diverged approximately 15,000 years ago. Conclusions Our results provide direct insight into the effects of increasing habitat fragmentation and human hunting pressure on genetic diversity and structure of red deer populations. They also shed light on the northward post-glacial colonisation process of red deer in Europe and suggest increased precision in inferring past demographic events when including both ancient and contemporary DNA. PMID:23009643

  10. Detection and Identification of Lactobacillus Species in Crops of Broilers of Different Ages by Using PCR-Denaturing Gradient Gel Electrophoresis and Amplified Ribosomal DNA Restriction Analysis

    Microsoft Academic Search

    Luo Guan; Karen E. Hagen; Gerald W. Tannock; Doug R. Korver; Gaylene M. Fasenko; Gwen E. Allison

    2003-01-01

    The microflora of the crop was investigated throughout the broiler production period (0 to 42 days) using PCR combined with denaturing gradient gel electrophoresis (PCR-DGGE) and selective bacteriological culture of lactobacilli followed by amplified ribosomal DNA restriction analysis (ARDRA). The birds were raised under conditions similar to those used in commercial broiler production. Lactobacilli predominated and attained populations of 108

  11. Physical and gene mapping of chloroplast DNA from normal and cytoplasmic male sterile (radish cytoplasm) lines of Brassica napus

    Microsoft Academic Search

    Fernand Vedel; Chantal Mathieu

    1983-01-01

    Using the restriction endonucleases SaII, SmaI, BgII and KpnI, physical maps of chloroplast DNA isolated from normal and cytoplasmic male sterile (radish cytoplasm) lines of B. napus were constructed and compared. In this study, a rapid and simple procedure was developed for the isolation of chloroplast DNA restriction fragments from low gelling temperature agarose gels.

  12. Improved coverage of cDNA-AFLP by sequential digestion of immobilized cDNA

    Microsoft Academic Search

    Arne Weiberg; Dirk Pöhler; Burkhard Morgenstern; Petr Karlovsky

    2008-01-01

    BACKGROUND: cDNA-AFLP is a transcriptomics technique which does not require prior sequence information and can therefore be used as a gene discovery tool. The method is based on selective amplification of cDNA fragments generated by restriction endonucleases, electrophoretic separation of the products and comparison of the band patterns between treated samples and controls. Unequal distribution of restriction sites used to

  13. Development of a PCR-Restriction Fragment Length Polymorphism Assay for the Epidemiological Analysis of Shiga Toxin-Producing Escherichia coli

    PubMed Central

    Shima, Kensuke; Terajima, Jun; Sato, Toshio; Nishimura, Kazuhiko; Tamura, Kazumichi; Watanabe, Haruo; Takeda, Yoshifumi; Yamasaki, Shinji

    2004-01-01

    Six characteristic regions (I to VI) were identified in Shiga toxin 2 (Stx2) phages (T. Sato, T. Shimizu, M. Watarai, M. Kobayashi, S. Kano, T. Hamabata, Y. Takeda, and S. Yamasaki, Gene 309:35-48, 2003). Region V, which is ca. 10 kb in size and is located in the upstream region of the Stx operons, includes the most distinctive region among six Stx phages whose genome sequences have been determined. In this study, we developed a PCR-restriction fragment length polymorphism (RFLP) assay for the epidemiological analysis of Shiga toxin-producing Escherichia coli (STEC) on the basis of the diversity of region V. When region V was amplified by long and accurate-PCR (LA-PCR) with five control E. coli strains carrying six different Stx phages such as E. coli strains C600 (Stx1 phage), C600 (933W phage), C600 (Stx2 phage-I), C600 (Stx2 phage-II), and O157:H7 Sakai strain RIMD0509952 (VT1-Sakai phage and VT2-Sakai phage), an expected size of the band was obtained. Restriction digest of each PCR product with BglI or EcoRV also gave the expected sizes of banding patterns and discriminated the RFLPs of five control strains. When a total of 204 STEC O157 strains were analyzed by LA-PCR, one to three bands whose sizes ranged from 8.2 to 14 kb were obtained. Two STEC O157 strains, however, did not produce any bands. Subsequent restriction digest of the PCR products with BglI or EcoRV differentiated the RFLPs of 202 STEC O157 strains into 24 groups. The RFLP patterns of pulsed-field gel electrophoresis (PFGE) of representative strains of STEC O157 divided into 24 groups were well correlated with those of PCR-RFLP when STEC O157 strains were isolated in the same time period and in the close geographic area. To evaluate the PCR-RFLP assay developed here, ten strains, each isolated from four different outbreaks in different areas in Japan (Tochigi, Hyogo, Aichi, and Fukuoka prefecture), were examined to determine whether the strains in each group showed the same RFLP patterns in the PCR-RFLP assay. In accordance with the results of PFGE except for strains isolated in an area (Fukuoka), which did not produce any amplicon, ten strains in each group demonstrated the same RFLP pattern. Taken together, these data suggest that the PCR-RFLP based on region V is as useful as PFGE but perhaps more simple and rapid than PFGE for the molecular epidemiological analysis of STEC strains during sporadic and common source outbreaks. PMID:15528716

  14. A preliminary trial using multi-target polymerase chain reaction (multiplex PCR) and restriction fragment length polymorphism (PCR-RFLP) on the same feedstuffs to detect tissues of animal origin.

    PubMed

    Colombo, F; Marchisio, E; Trezzi, I E; Peri, V; Pinotti, L; Baldi, A; Soncini, G

    2004-08-01

    A preliminary study using multi-target polymerase chain reaction (multiplex PCR) and restriction fragment length polymorphism (PCR-RFLP) was done on the same feedstuffs to detect animal tissues. The results of the two methods differ somewhat: PCR-RFLP did not detect any signal in any sample, but multiplex PCR detected a signal in one sample. These findings could be a basis for further investigations. PMID:15509020

  15. Analysis of the Endophytic Actinobacterial Population in the Roots of Wheat (Triticum aestivum L.) by Terminal Restriction Fragment Length Polymorphism and Sequencing of 16S rRNA Clones

    Microsoft Academic Search

    Vanessa M. Conn; Christopher M. M. Franco

    2004-01-01

    The endophytic actinobacterial population in the roots of wheat grown in three different soils obtained from the southeast part of South Australia was investigated by terminal restriction fragment length polymorphism (T-RFLP) analysis of the amplified 16S rRNA genes. A new, validated approach was applied to the T-RFLP analysis in order to estimate, to the genus level, the actinobacterial population that

  16. Detection of DNA mutations associated with mitochondrial diseases by Agilent 2100 bioanalyzer

    Microsoft Academic Search

    Ching-You Lu; Dan-Ju Tso; Tom Yang; Yuh-Jyh Jong; Yau-Huei Wei

    2002-01-01

    Background: Molecular analysis of mitochondrial DNA (mtDNA) has provided a final diagnosis for many of the mitochondrial diseases. We evaluated the Agilent 2100 bioanalyzer (Agilent Technologies, Palo Alto, CA) to determine whether the system could replace the conventional restriction fragment length polymorphism (RFLP) analysis by the agarose gel electrophoresis for the detection of the mtDNA mutation. Methods: Three members of

  17. Detection of Polymorphisms of Human DNA by Gel Electrophoresis as Single-Strand Conformation Polymorphisms

    Microsoft Academic Search

    Masato Orita; Hiroyuki Iwahana; Hiroshi Kanazawa; Kenshi Hayashi; Takao Sekiya

    1989-01-01

    We developed mobility shift analysis of single-stranded DNAs on neutral polyacrylamide gel electrophoresis to detect DNA polymorphisms. This method follows digestion of genomic DNA with restriction endonucleases, denaturation in alkaline solution, and electrophoresis on a neutral polyacrylamide gel. After transfer to a nylon membrane, the mobility shift due to a nucleotide substitution of a single-stranded DNA fragment could be detected

  18. Mitochondrial DNA Diversity and Lineage Determination of European Isolates of Fusarium graminearum ( Gibberella zeae )

    Microsoft Academic Search

    M. Láday; Á. Juhász; G. Mulè; A. Moretti; Á. Szécsi; A. Logrieco

    2004-01-01

    Restriction fragment length polymorphisms (RFLPs) were used to assess genetic diversity of mitochondrial DNA (mtDNA) among standard isolates of seven lineages of Fusarium graminearum. The mtDNA patterns within each lineage were very similar (>89%), whereas significant differences were observed between the isolates belonging to different lineages, with the exception of lineages 1 and 4 where strong similarity was found between

  19. Assessing the phylogeographic history of the montane caddisfly Thremma gallicum using mitochondrial and restriction-site-associated DNA (RAD) markers.

    PubMed

    Macher, Jan-Niklas; Rozenberg, Andrey; Pauls, Steffen U; Tollrian, Ralph; Wagner, Rüdiger; Leese, Florian

    2015-02-01

    Repeated Quaternary glaciations have significantly shaped the present distribution and diversity of several European species in aquatic and terrestrial habitats. To study the phylogeography of freshwater invertebrates, patterns of intraspecific variation have been examined primarily using mitochondrial DNA markers that may yield results unrepresentative of the true species history. Here, population genetic parameters were inferred for a montane aquatic caddisfly, Thremma gallicum, by sequencing a 658-bp fragment of the mitochondrial CO1 gene, and 12,514 nuclear RAD loci. T. gallicum has a highly disjunct distribution in southern and central Europe, with known populations in the Cantabrian Mountains, Pyrenees, Massif Central, and Black Forest. Both datasets represented rangewide sampling of T. gallicum. For the CO1 dataset, this included 352 specimens from 26 populations, and for the RAD dataset, 17 specimens from eight populations. We tested 20 competing phylogeographic scenarios using approximate Bayesian computation (ABC) and estimated genetic diversity patterns. Support for phylogeographic scenarios and diversity estimates differed between datasets with the RAD data favouring a southern origin of extant populations and indicating the Cantabrian Mountains and Massif Central populations to represent highly diverse populations as compared with the Pyrenees and Black Forest populations. The CO1 data supported a vicariance scenario (north-south) and yielded inconsistent diversity estimates. Permutation tests suggest that a few hundred polymorphic RAD SNPs are necessary for reliable parameter estimates. Our results highlight the potential of RAD and ABC-based hypothesis testing to complement phylogeographic studies on non-model species. PMID:25691988

  20. Assessing the phylogeographic history of the montane caddisfly Thremma gallicum using mitochondrial and restriction-site-associated DNA (RAD) markers

    PubMed Central

    Macher, Jan-Niklas; Rozenberg, Andrey; Pauls, Steffen U; Tollrian, Ralph; Wagner, Rüdiger; Leese, Florian

    2015-01-01

    Repeated Quaternary glaciations have significantly shaped the present distribution and diversity of several European species in aquatic and terrestrial habitats. To study the phylogeography of freshwater invertebrates, patterns of intraspecific variation have been examined primarily using mitochondrial DNA markers that may yield results unrepresentative of the true species history. Here, population genetic parameters were inferred for a montane aquatic caddisfly, Thremma gallicum, by sequencing a 658-bp fragment of the mitochondrial CO1 gene, and 12,514 nuclear RAD loci. T. gallicum has a highly disjunct distribution in southern and central Europe, with known populations in the Cantabrian Mountains, Pyrenees, Massif Central, and Black Forest. Both datasets represented rangewide sampling of T. gallicum. For the CO1 dataset, this included 352 specimens from 26 populations, and for the RAD dataset, 17 specimens from eight populations. We tested 20 competing phylogeographic scenarios using approximate Bayesian computation (ABC) and estimated genetic diversity patterns. Support for phylogeographic scenarios and diversity estimates differed between datasets with the RAD data favouring a southern origin of extant populations and indicating the Cantabrian Mountains and Massif Central populations to represent highly diverse populations as compared with the Pyrenees and Black Forest populations. The CO1 data supported a vicariance scenario (north–south) and yielded inconsistent diversity estimates. Permutation tests suggest that a few hundred polymorphic RAD SNPs are necessary for reliable parameter estimates. Our results highlight the potential of RAD and ABC-based hypothesis testing to complement phylogeographic studies on non-model species. PMID:25691988

  1. DNA finger printing by oligonucleotide probes specific for simple repeats

    Microsoft Academic Search

    S. Ali; C. R. Müller; J. T. Epplen

    1986-01-01

    Interspersed simple repetitive DNA is a convenient genetic marker for analysis of restriction fragment length polymorphisms (RFLPs) because of the numbers and the frequencies of its alleles. Oligonucleotide probes specific for variations of the GACTA simple repeats have been designed and hybridized to a panel of human DNAs digested with various restriction enzymes. Numerous RFLPs were demonstrated in AluI and

  2. Chloroplast DNA phylogeography of a distylous shrub (Palicourea padifolia, Rubiaceae) reveals past fragmentation and demographic expansion in Mexican cloud forests.

    PubMed

    Gutiérrez-Rodríguez, Carla; Ornelas, Juan Francisco; Rodríguez-Gómez, Flor

    2011-12-01

    Several phylogeographic studies in northern Mesoamerica have examined the influence of Pleistocene glaciations on the genetic structure of temperate tree species with their southern limit by the contact zone between species otherwise characteristic of North or South America, but few have featured plant species that presumably colonized northern Mesoamerica from South America. A phylogeographical study of Palicourea padifolia, a fleshy-fruited, bird dispersed distylous shrub, was conducted to investigate genetic variation at two chloroplast regions (trnS-trnG and rpl32-trnL) across cloud forest areas to determine if such patterns are consistent with the presence of Pleistocene refugia and/or with the historical fragmentation of the Mexican cloud forests. We conducted population and spatial genetic analyses as well as phylogenetic and isolation with migration analyses on 122 individuals from 22 populations comprising the distribution of P. padifolia in Mexico to gain insight of the evolutionary history of these populations. Twenty-six haplotypes were identified after sequencing 1389 bp of chloroplast DNA. These haplotypes showed phylogeographic structure (N(ST) = 0.508, G(ST) = 0.337, N(ST) > G(ST), P < 0.05), including a phylogeographic break at the Isthmus of Tehuantepec, with private haplotypes at either side of the isthmus, and a divergence time of the split in the absence of gene flow dating back c. 309,000-103,000 years ago. The patterns of geographic structure found in this study are consistent with past fragmentation and demographic range expansion, supporting the role of the Isthmus of Tehuantepec as a biogeographical barrier in the dispersal of P. padifolia. Our data suggest that P. padifolia populations were isolated throughout glacial cycles by the Isthmus of Tehuantepec, accumulating genetic differences due to the lack of migration across the isthmus in either direction, but the results of our study are not consistent with the existence of the previously proposed Pleistocene refugia for rain forest plant species in the region. PMID:21930221

  3. The mutT Defect Does Not Elevate Chromosomal Fragmentation in Escherichia coli Because of the Surprisingly Low Levels of MutM/MutY-Recognized DNA Modifications?

    PubMed Central

    Rotman, Ella; Kuzminov, Andrei

    2007-01-01

    Nucleotide pool sanitizing enzymes Dut (dUTPase), RdgB (dITPase), and MutT (8-oxo-dGTPase) of Escherichia coli hydrolyze noncanonical DNA precursors to prevent incorporation of base analogs into DNA. Previous studies reported dramatic AT?CG mutagenesis in mutT mutants, suggesting a considerable density of 8-oxo-G in DNA that should cause frequent excision and chromosomal fragmentation, irreparable in the absence of RecBCD-catalyzed repair and similar to the lethality of dut recBC and rdgB recBC double mutants. In contrast, we found mutT recBC double mutants viable with no signs of chromosomal fragmentation. Overproduction of the MutM and MutY DNA glycosylases, both acting on DNA containing 8-oxo-G, still yields no lethality in mutT recBC double mutants. Plasmid DNA, extracted from mutT mutM double mutant cells and treated with MutM in vitro, shows no increased relaxation, indicating no additional 8-oxo-G modifications. Our ?mutT allele elevates the AT?CG transversion rate 27,000-fold, consistent with published reports. However, the rate of AT?CG transversions in our mutT+ progenitor strain is some two orders of magnitude lower than in previous studies, which lowers the absolute rate of mutagenesis in ?mutT derivatives, translating into less than four 8-oxo-G modifications per genome equivalent, which is too low to cause the expected effects. Introduction of various additional mutations in the ?mutT strain or treatment with oxidative agents failed to increase the mutagenesis even twofold. We conclude that, in contrast to the previous studies, there is not enough 8-oxo-G in the DNA of mutT mutants to cause elevated excision repair that would trigger chromosomal fragmentation. PMID:17616589

  4. Method for identifying mutagenic agents which induce large, multilocus deletions in DNA

    SciTech Connect

    Bradley, W.E.C.; Belouchi, A.; Dewyse, P.

    1993-07-13

    A method of identifying a mutagenic agent is described which includes a large, multilocus deletions in DNA in mammalian cells comprising: (i) exposing a class III heterozygous CHO cell line to a potential mutagenic agent under investigation, and allowing any mutation of the cell line to proceed, said cell line being characterized in that a restriction fragment length variation exists in on mutation it becomes resistant to 2,6-diaminopurine and in that the DNA sequence adjacent to the two alleles of the APRT gene such that the DNA sequence adjacent to one of the two alleles can be digested with the enzyme BclI but the DNA sequence variation adjacent to the other of the two alleles cannot be digested with BclI, (ii) isolating induced mutations of the cell line deficient in APRT function, (iii) isolating DNA from the induced mutants, (iv) digesting the isolated DNA with BclI enzyme to produce digested fragments including a 19 kb fragment and any 2 kb fragment, which fragments hybridize with the labeled probe derived from DNA fragment PDI, (v) separating any digested fragments, (vi) transferring the separated fragments of (v) to a solid support, (vii) hybridizing the supported separated fragments with a labeled probe derived from the clone DNA fragment PD 1, (viii) determining fragments having undergone loss of the 2 kb band identified by the probe, as an identification of parent mutants in which the loss occurred, and (ix) evaluating the mutating ability of the potential mutagenic agent.

  5. Class I and class II restriction pattern polymorphisms associated with independently derived RT1 haplotypes in inbred rats

    Microsoft Academic Search

    Peter J. Wettstein; Susan Faas; David A. Buck

    1985-01-01

    This communication reports the DNA level identification of class I and class II sequences associated with 20 RT1 haplotypes which have been assigned previously to eight RT1 groups. Sixteen to 22 bands in genomic blots hybridized with the mouse pH-2III class I cDNA probe. Only the three RT1khaplotypes associated with identical class I restriction fragment patterns. Differences in restriction bands

  6. An amphioxus RAG1-like DNA fragment encodes a functional central domain of vertebrate core RAG1

    PubMed Central

    Zhang, Yanni; Xu, Ke; Deng, Anqi; Fu, Xing; Xu, Anlong; Liu, Xiaolong

    2014-01-01

    The highly diversified repertoire of antigen receptors in the vertebrate immune system is generated via proteins encoded by the recombination activating genes (RAGs) RAG1 and RAG2 by a process known as variable, diversity, and joining [V(D)J] gene recombination. Based on the study of vertebrate RAG proteins, many hypotheses have been proposed regarding the origin and evolution of RAG. This issue remains unresolved, leaving a significant gap in our understanding of the evolution of adaptive immunity. Here, we show that the amphioxus genome contains an ancient RAG1-like DNA fragment (bfRAG1L) that encodes a virus-related protein that is much shorter than vertebrate RAG1 and harbors a region homologous to the central domain of core RAG1 (cRAG1). bfRAG1L also contains an unexpected retroviral type II nuclease active site motif, DXN(D/E)XK, and is capable of degrading both DNA and RNA. Moreover, bfRAG1L shares important functional properties with the central domain of cRAG1, including interaction with RAG2 and localization to the nucleus. Remarkably, the reconstitution of bfRAG1L into a cRAG1-like protein yielded an enzyme capable of recognizing recombination signal sequences and performing V(D)J recombination in the presence of mouse RAG2. Moreover, this reconstituted cRAG1-like protein could mediate the assembly of antigen receptor genes in RAG1-deficient mice. Together, our results demonstrate that amphioxus bfRAG1L encodes a protein that is functionally equivalent to the central domain of cRAG1 and is well prepared for further evolution to mediate V(D)J recombination. Thus, our findings provide unique insights into the evolutionary origin of RAG1. PMID:24368847

  7. The Opisthorchis felineus paramyosin: cDNA sequence and characterization of its recombinant fragment.

    PubMed

    Shustov, A V; Kotelkin, A T; Sorokin, A V; Ternovoi, V A; Loktev, V B

    2002-08-01

    The cDNA sequence of Opisthorchis felineus paramyosin (PM) was determined and shown to have 66-70% homology with two schistosomes and two cestodes. Phylogenetic analysis revealed an almost equal distance between O. felineus and these distinct clades. Because of its relatively low conservation, the PM gene may be a convenient genetic marker for studying phylogenetic relationships among platyhelminthes.A 25-kDa recombinant polypeptide corresponding to the central part of the full-length PM was produced. In Western blot analysis, murine hyperimmune serum against recombinant PM (recPM) detected 100-kDa polypeptides in the O. felineusegg and somatic antigens. Interactions of recPM with polyclonal anti-parasite antibodies and anti-recPM sera in ELISA with native antigens demonstrated that recPM carries a B cell epitope identical to the O. felineusnative antigen. Our sequence and immunologic data may be helpful in developing new diagnostic tools and candidate vaccines for O. felineus infection. PMID:12122429

  8. DNA variation in the wild plant Arabidopsis thaliana revealed by amplified fragment length polymorphism analysis.

    PubMed Central

    Miyashita, N T; Kawabe, A; Innan, H

    1999-01-01

    To investigate the level and pattern of DNA variation of Arabidopsis thaliana at the entire genome level, AFLP analysis was conducted for 38 ecotypes distributed throughout the world. Ten pairs of selective primers were used to detect a total of 472 bands, of which 374 (79. 2%) were polymorphic. The frequency distribution of polymorphic bands was skewed toward an excess of singleton variation. On the basis of AFLP variation, nucleotide diversity for the entire genome was estimated to be 0.0106, which was within the range reported previously for specific nuclear genes. The frequency distribution of pairwise distance was bimodal because of an ecotype (Fl-3) with a large number of unique bands. Linkage disequilibrium between polymorphic AFLPs was tested. The proportion of significant linkage disequilibria was close to random expectation after neglecting the ecotype Fl-3. This result indicates that the effect of recombination could not be ignored in this selfing species. A neighbor-joining tree was constructed on the basis of the AFLP variation. This tree has a star-like topology and shows no clear association between ecotype and geographic origin, suggesting a recent spread of this plant species and limited migration between its habitats. PMID:10430596

  9. The Orf18 gene product from conjugative transposon Tn916 is an ArdA antirestriction protein that inhibits type I DNA restriction-modification systems.

    PubMed

    Serfiotis-Mitsa, Dimitra; Roberts, Gareth A; Cooper, Laurie P; White, John H; Nutley, Margaret; Cooper, Alan; Blakely, Garry W; Dryden, David T F

    2008-11-28

    Gene orf18, which is situated within the intercellular transposition region of the conjugative transposon Tn916 from the bacterial pathogen Enterococcus faecalis, encodes a putative ArdA (alleviation of restriction of DNA A) protein. Conjugative transposons are generally resistant to DNA restriction upon transfer to a new host. ArdA from Tn916 may be responsible for the apparent immunity of the transposon to DNA restriction and modification (R/M) systems and for ensuring that the transposon has a broad host range. The orf18 gene was engineered for overexpression in Escherichia coli, and the recombinant ArdA protein was purified to homogeneity. The protein appears to exist as a dimer at nanomolar concentrations but can form larger assemblies at micromolar concentrations. R/M assays revealed that ArdA can efficiently inhibit R/M by all four major classes of Type I R/M enzymes both in vivo and in vitro. These R/M systems are present in over 50% of sequenced prokaryotic genomes. Our results suggest that ArdA can overcome the restriction barrier following conjugation and so helps increase the spread of antibiotic resistance genes by horizontal gene transfer. PMID:18838147

  10. EGassembler: online bioinformatics service for large-scale processing, clustering and assembling ESTs and genomic DNA fragments.

    PubMed

    Masoudi-Nejad, Ali; Tonomura, Koichiro; Kawashima, Shuichi; Moriya, Yuki; Suzuki, Masanori; Itoh, Masumi; Kanehisa, Minoru; Endo, Takashi; Goto, Susumu

    2006-07-01

    Expressed sequence tag (EST) sequencing has proven to be an economically feasible alternative for gene discovery in species lacking a draft genome sequence. Ongoing large-scale EST sequencing projects feel the need for bioinformatics tools to facilitate uniform EST handling. This brings about a renewed importance for a universal tool for processing and functional annotation of large sets of ESTs. EGassembler (http://egassembler.hgc.jp/) is a web server, which provides an automated as well as a user-customized analysis tool for cleaning, repeat masking, vector trimming, organelle masking, clustering and assembling of ESTs and genomic fragments. The web server is publicly available and provides the community a unique all-in-one online application web service for large-scale ESTs and genomic DNA clustering and assembling. Running on a Sun Fire 15K supercomputer, a significantly large volume of data can be processed in a short period of time. The results can be used to functionally annotate genes, to facilitate splice alignment analysis, to link the transcripts to genetic and physical maps, design microarray chips, to perform transcriptome analysis and to map to KEGG metabolic pathways. The service provides an excellent bioinformatics tool to research groups in wet-lab as well as an all-in-one-tool for sequence handling to bioinformatics researchers. PMID:16845049

  11. STS map of genes and anonymous DNA fragments on human chromosome 18 using a panel of somatic cell hybrids

    SciTech Connect

    Overhauser, J.; Mewar, R.; Rojas, K.; Kline, A.D. (Thomas Jefferson Univ., Philadelphia, PA (United States)); Lia, K.; Silverman, G.A. (Harvard Medical School, Boston, MA (United States))

    1993-02-01

    Somatic cell hybrids containing different deleted regions of chromosome 18 derived form patients with balanced translocations or terminal deletions were used to create a deletion mapping panel. Twenty-four sequence-tagged sites (STSs) for 17 genes and 7 anonymous polymorphic DNA fragments were identified. These STSs were used to map the 24 loci to 18 defined regions of chromosome 18. Both ERV1, previously mapped to 18q22-q23, and YES1, previously mapped to 18q21.3, were found to map to 18p11.21-pter. Several genes previously mapped to 18q21 were found to be in the order cen-SSAV1-DCC-FECH-GRP-BCL2-PLANH2-tel. The precise mapping of genes to chromosome 18 should help in determining whether these genes may be involved in the etiology of specific chromosomal syndromes associated with chromosome 18. The mapping of the poloymorphic loci will assist in the integration of the physical map with the recombination map of chromosome 18. 43 refs., 2 figs., 1 tab.

  12. A DNA fragment from Xq21 replaces a deleted region containing the entire FVIII gene in a severe hemophilia A patient

    SciTech Connect

    Murru, S.; Casula, L.; Moi, P. [Insituto di Clinica e Biologia dell` Eta Evolutiva, Cagliari (Italy)] [and others] [Insituto di Clinica e Biologia dell` Eta Evolutiva, Cagliari (Italy); and others

    1994-09-15

    In this paper the authors report the molecular characterization of a large deletion that removes the entire Factor VIII gene in a severe hemophilia A patient. Accurate DNA analysis of the breakpoint region revealed that a large DNA fragment replaced the 300-kb one, which was removed by the deletion. Pulsed-field gel electrophoresis analysis revealed that the size of the inserted fragment is about 550 kb. In situ hybridization demonstrated that part of the inserted region normally maps to Xq21 and to the tip of the short arm of the Y chromosome (Yp). In this patient this locus is present both in Xq21 and in Xq28, in addition to the Yp, being thus duplicated in the X chromosome. Sequence analysis of the 3` breakpoint suggested that an illegitimate recombination is probably the cause of this complex rearrangement. 52 refs., 7 figs.

  13. Bovine Parvovirus DNA-binding Proteins: Identification by a Combined DNA Hybridization and Immunodetection Assay

    Microsoft Academic Search

    MURIEL LEDERMAN; BRUCE C. SHULL; ERNEST R. STOUT; ROBERT C. BATES

    1987-01-01

    SUMMARY We have investigated the interaction between bovine parvovirus (BPV) capsid and non-capsid proteins and restriction fragments of the BPV genome by a combined DNA hybridization and immunodetection assay. 3zp-labelled DNA was bound to nitrocellulose membranes bearing lysates of mock-infected and virus-infected cells whose proteins had been separated by SDS-polyacrylamide gel electrophoresis. The position of bound DNA was determined by

  14. Dual-probe electrochemical DNA biosensor based on the "Y" junction structure and restriction endonuclease assisted cyclic enzymatic amplification for detection of double-strand DNA of PML/RAR? related fusion gene.

    PubMed

    Wang, Kun; Lei, Yun; Zhong, Guang-Xian; Zheng, Yan-Jie; Sun, Zhou-Liang; Peng, Hua-Ping; Chen, Wei; Liu, Ai-Lin; Chen, Yuan-Zhong; Lin, Xin-Hua

    2015-09-15

    Taking advantage of "Y" junction structure and restriction endonuclease assisted cyclic enzymatic amplification, a dual-probe electrochemical DNA (DE-DNA) biosensor was designed to detect double-stranded DNA (dsDNA) of acute promyelocytic leukemia (APL) related gene. Two groups of detection probes were designed, and each group was composed of a biotinylated capture probe and an assisted probe. They were separately complementary with two strands of target dsDNA in order to prevent the reannealing of the two separate strands from target dsDNA. First, thiol functionalized capture probes (C1 and C2) were severally assembled onto two different gold electrodes, followed by hybridizing with target dsDNA (S1a-S1b) and assistant probes to form two Y-junction-structure ternary complexes. Subsequently, restriction sites on the ternary complexes were digested by Rsa I, which can release S1a, S1b and biotins from the electrode surfaces. Meanwhile, the released S1a and S1b can further hybridize with the unhybridized corresponding detection probes and then initiate another new hybridization-cleavage-separation cycle. Finally, the current signals were produced by the enzyme-catalyzed reaction of streptavidin-horse reddish peroxidase (streptavidin-HRP). The distinct difference in current signals between different sequences allowed detection of target dsDNA down to a low detection limit of 47fM and presented excellent specificity with discriminating only a single-base mismatched dsDNA sequence. Moreover, this biosensor was also used for assay of polymerase chain reaction (PCR) samples with satisfactory results. According to the results, the power of the DE-DNA biosensor as a promising tool for the detection of APL and other diseases. PMID:25985065

  15. Characterization of a Brucella Species 25-Kilobase DNA Fragment Deleted from Brucella abortus Reveals a Large Gene Cluster Related to the Synthesis of a Polysaccharide

    PubMed Central

    Vizcaíno, Nieves; Cloeckaert, Axel; Zygmunt, Michel S.; Fernández-Lago, Luis

    2001-01-01

    In the present study we completed the nucleotide sequence of a Brucella melitensis 16M DNA fragment deleted from B. abortus that accounts for 25,064 bp and show that the other Brucella spp. contain the entire 25-kb DNA fragment. Two short direct repeats of four nucleotides, detected in the B. melitensis 16M DNA flanking both sides of the fragment deleted from B. abortus, might have been involved in the deletion formation by a strand slippage mechanism during replication. In addition to omp31, coding for an immunogenic protein located in the Brucella outer membrane, 22 hypothetical genes were identified. Most of the proteins that would be encoded by these genes show significant homology with proteins involved in the biosynthesis of polysacharides from other bacteria, suggesting that they might be involved in the synthesis of a Brucella polysaccharide that would be a heteropolymer synthesized by a Wzy-dependent pathway. This polysaccharide would not be synthesized in B. abortus and would be a polysaccharide not identified until present in the genus Brucella, since all of the known polysaccharides are synthesized in all smooth Brucella species. Discovery of a novel polysaccharide not synthesized in B. abortus might be interesting for a better understanding of the pathogenicity and host preference differences observed between the Brucella species. However, the possibility that the genes detected in the DNA fragment deleted in B. abortus no longer lead to the synthesis of a polysaccharide must not be excluded. They might be a remnant of the common ancestor of the alpha-2 subdivision of the class Proteobacteria, with some of its members synthesizing extracellular polysaccharides and, as Brucella spp., living in association with eukaryotic cells. PMID:11598046

  16. Molecular Characterization of a Brucella Species Large DNA Fragment Deleted in Brucella abortus Strains: Evidence for a Locus Involved in the Synthesis of a Polysaccharide

    PubMed Central

    Vizcaíno, Nieves; Cloeckaert, Axel; Zygmunt, Michel S.; Fernández-Lago, Luis

    1999-01-01

    A Brucella melitensis 16M DNA fragment of 17,119 bp, which contains a large region deleted in B. abortus strains and DNA flanking one side of the deletion, has been characterized. In addition to the previously identified omp31 gene, 14 hypothetical genes have been identified in the B. melitensis fragment, most of them showing homology to genes involved in the synthesis of a polysaccharide. Considering that 10 of the 15 genes are missing in B. abortus and that all the polysaccharides described in the Brucella genus (lipopolysaccharide, native hapten, and polysaccharide B) have been detected in all the species, it seems likely that the genes described here might be part of a cluster for the synthesis of a novel Brucella polysaccharide. Several polysaccharides have been identified as important virulence factors, and the discovery of a novel polysaccharide in the brucellae which is probably not synthesized in B. abortus might be interesting for a better understanding of the pathogenicity and host preference differences observed between the Brucella species. However, the possibility that the genes described in this paper no longer encode the synthesis of a polysaccharide cannot be excluded. Brucellae belong to the alpha-2 subdivision of the class Proteobacteria, which includes other microorganisms living in association with eucaryotic cells, some of them synthesizing extracellular polysaccharides involved in the interaction with the host cell. The genes described in this paper might be a remnant of the common ancestor of the alpha-2 subdivision of the class Proteobacteria, and the brucellae might have lost such extracellular polysaccharide during evolution if it was not necessary for survival or for establishment of the infectious process. Nevertheless, further studies are necessary to identify the entire DNA fragment missing in B. abortus strains and to elucidate the mechanism responsible for such deletion, since only 9,948 bp of the deletion was present in the sequenced B. melitensis DNA fragment. PMID:10338472

  17. An RAPD (Random Amplified Polymorphic DNA) Analysis of Genetic Population Structure of Balea biplicata (Gastropoda: Clausiliidae) in Fragmented Floodplain Forests of the Elster\\/Saale Riparian System

    Microsoft Academic Search

    A. Hille; K. Liebal; B. Mosch; H. Pellmann; M. Schlegel

    2003-01-01

    Eight German populations of the land snail Balea biplicata(Mollusca: Clausiliidae) were studied using the randomly amplified polymorphic DNA-polymerase chain reaction and morphometrics (principal component and discriminant analysis) to examine population structure and gene flow patterns in a fragmented landscape mosaic along the Elster\\/Saale riparian system, Germany. A variety of population genetic analyses targeting either more on the geographic scale of

  18. Protective Effects of Grape Seed Proanthocyanidins and Selected Antioxidants against TPA-Induced Hepatic and Brain Lipid Peroxidation and DNA Fragmentation, and Peritoneal Macrophage Activation in Mice

    Microsoft Academic Search

    D Bagchi; A Garg; R. L Krohn; M Bagchi; D. J Bagchi; J Balmoori; S. J Stohs

    1998-01-01

    1.The comparative protective abilities of a grape seed proanthocyanidin extract (GSPE) (25–100 mg\\/kg), vitamin C (100 mg\\/kg), vitamin E succinate (VES) (100 mg\\/kg) and ?-carotene (50 mg\\/kg) on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced lipid peroxidation and DNA fragmentation in the hepatic and brain tissues, as well as production of reactive oxygen species by peritoneal macrophages, were assessed.2.Treatment of mice with GSPE (100 mg\\/kg),

  19. Using restriction mapping to teach basic skills in the molecular biology lab.

    PubMed

    Walsh, Lauren; Shaker, Elizabeth; De Stasio, Elizabeth A

    2007-05-01

    Digestion of DNA with restriction enzymes, calculation of volumes and concentrations of reagents for reactions, and the separation of DNA fragments by agarose gel electrophoresis are common molecular biology techniques that are best taught through repetition. The following open-ended, investigative laboratory exercise in plasmid restriction mapping allows students to gain technical expertise while simultaneously exploring the utility of gel electrophoresis and restriction mapping. Because of its interpretive nature, this project also provides data suitable for a written report, and can thus be used to reinforce lessons on figure presentation and science writing skills. PMID:21591089

  20. Genetic markers in ribosomal DNA for hookworm identification

    Microsoft Academic Search

    Robin B. Gasser; Louise E. Stewart; Richard Speare

    1996-01-01

    Polymerase chain reaction-linked restriction fragment length polymorphism (PCR-RFLP) analysis of ribosomal (r) DNA was conducted on Uncinaria stenocephala, Ancylostoma caninum, A. tubaeforme and A ceylanicum. The rDNA region spanning the first and second internal transcribed spacers (ITS1 and ITS2) plus the 5.8S (ITS+) gene was amplified by PCR from each of the species, digested separately with 9 restriction endonucleases and