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Sample records for dna restriction fragment

  1. Separation of DNA restriction fragments using capillary electrophoresis

    SciTech Connect

    Chan, K.C.; Whang, Chenwen; Yeung, E.S. )

    1993-01-01

    Gel-filled and non-gel' capillary electrophoresis (CE) have been applied to the separation of various DNA restriction fragments. 30% HydroLink gel, polymerized inside a 75[mu]m i.d. fused-silica capillary, was used in the gel-filled CE. Primary results show that the HL capillary gel was simple to cast, and its stability was reasonably good under the running conditions. In the non-gel CE experiment, a buffer containing the sieving additive hydroxypropylmethyl cellulose was used to affect the size-dependent separation. The use of GC capillaries eliminates the inconvenience of separately coating the capillary walls for efficient non-gel separation. Finally, the authors demonstrate that it is feasible to detect native DNA fragments using indirect fluorometry in non-gel capillary electrophoresis.

  2. 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.

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

    SciTech Connect

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

    1994-08-01

    The authors 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, they 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, they 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. 47 refs., 2 figs., 4 tabs.

  4. Junction between Z and B conformations in a DNA restriction fragment: evaluation by Raman spectroscopy.

    PubMed Central

    Wartell, R M; Klysik, J; Hillen, W; Wells, R D

    1982-01-01

    Raman vibrational spectra were obtained from two DNA restriction fragments and the DNA polymer (dG-dC)n . (dG-dC)n in 0.01 and 4.5 M NaCl. One fragment contained 95 base pairs (bp) of the Escherichia coli lactose operator-promoter region (95-bp fragment). The other fragment consisted of the 95-bp region flanked by 26 and 32 bp of dC-dG sequences and BamHI ends (157-bp fragment). In 0.01 M NaCl all three DNAs have Raman spectra characteristic of a right-handed B conformation. The high salt spectrum of the 95-bp fragment is also characteristic of a B conformation. However, the spectrum of the 157-bp fragment in 4.5 M NaCl shows major intensity changes from the 0.01 M NaCl spectrum. These changes are also observed in the high salt spectra of (dG-dC)n . (dG-dC)n and are correlated with the presence of a left-handed Z conformation. Comparisons between the high salt Raman spectra of the 157-bp fragment and spectra calculated from (dG-dC)n . (dG-dC)n and the 95-bp fragment indicated that essentially all of the dC-dG regions in the 157-bp fragment are in the Z conformation and a large part (approximately 80%) of the 95-bp region no longer has a B-type backbone vibration. However, this non-B-DNA-like character of the central region is not indicated by base vibrations. PMID:7045865

  5. 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

  6. High interindividual restriction fragment length and copy number of polymorphism of a TVRI family in moderate human DNA repeats

    SciTech Connect

    Rogaev, E.I.; Shapiro, Yu.A.

    1987-06-01

    The authors describe the selection of cloned human DNA sequences, with a copy number not exceeding 1000 copies per diploid genome, and their testing for interindividual restriction fragment lengths and copy number of polymorphism (RFLCP). As a result of the investigation a DNA clone was found (TVRI-6), about 2.8 kilobase-pairs in size, for which an unusually high level of interindividual RFLCP was discovered. The TVRI-6 sequence was obtained from a bank of Pst I restriction fragments of human placental nuclear DNA cloned in pBR 322. The bank was analyzed by hybridization of colonies with phosphorus 32-labelled human nuclear DNA.

  7. 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.

  8. 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...

  9. Identification of Staphylococcus spp. by PCR-Restriction Fragment Length Polymorphism Analysis of dnaJ Gene?

    PubMed Central

    Hauschild, Tomasz; Stepanovi?, Srdjan

    2008-01-01

    A PCR-restriction fragment length polymorphism (RFLP) analysis method that analyzes a part of the dnaJ gene was designed for the rapid and accurate identification of Staphylococcus spp. XapI or Bsp143I digestion of the PCR-generated products rendered distinctive RFLP patterns that allowed 41 reference species and subspecies to be identified with a high degree of specificity. The novel method was validated by the identification of 23 clinical staphylococcal strains, and the results were compared with those obtained by other genotypic identification methods. A 100% concordance of the results was shown. Therefore, PCR-RFLP analysis of the dnaJ gene is proposed as a reliable and reproducible method for the identification of Staphylococcus spp. PMID:18832127

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

    PubMed

    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. PMID:3009528

  11. Separation of fluorescence-labelled terminal restriction fragment DNA on a two-dimensional gel (T-RFs-2D) - an efficient approach for microbial consortium characterization.

    PubMed

    Wang, Shanquan; He, Jianzhong

    2011-09-01

    Fingerprinting techniques provide access to understanding the ecology of uncultured microbial consortia. However, the application of current techniques such as terminal restriction fragment length polymorphism (T-RFLP) and denaturing gradient gel electrophoresis (DGGE) has been hindered due to their limitations in characterizing complex microbial communities. This is due to that different populations possibly share the same terminal restriction fragments (T-RFs) and DNA fragments may co-migrate on DGGE gels. To overcome these limitations, a new approach was developed to separate terminal restriction fragments (T-RFs) of 16S rRNA genes on a two-dimensional gel (T-RFs-2D). T-RFs-2D involves restriction digestion of terminal fluorescence-labelled PCR amplified 16S rRNA gene products and their high-resolution separation via a two-dimensional (2D) gel electrophoresis based on the T-RF fragment size (1(st) D) and its sequence composition on the denaturing gradient gel (2(nd) D). The sequence information of interested T-RFs on 2D gels can be obtained through serial poly(A) tailing reaction, PCR amplification and subsequent DNA sequencing. By employing the T-RFs-2D method, bacteria with MspI digested T-RF size of 436 (±1) bp and 514 (±1) bp were identified to be a Lysobacter sp. and a Dehalococcoides sp. in a polychlorinated biphenyl (PCB) dechlorinating culture. With the high resolution of 2D separation, T-RFs-2D separated 63 DNA fragments in a complex river-sediment microbial community, while traditional DGGE detected only 41 DNA fragments in the same sample. In all, T-RFs-2D has its advantage in obtaining sequence information of interested T-RFs and also in characterization of complex microbial communities. PMID:21824243

  12. 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.

  13. 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. PMID:26078793

  14. [DNA diagnosis of beta-thalassemia. Study of restriction fragment length polymorphisms in families with affected children].

    PubMed

    Solov'ev, G Ia; Luk'ianenko, A V; Tagiev, A F; Dadasheva, T S; Rustamov, R Sh

    1990-10-01

    A kit of DNA-probes directed at the cluster of human beta-globulin genes was used to study the incidence rate of 7 polymorphic restriction sites in beta-thalassemia patients and normal donors in the Azerbaijan SSR. Informative polymorphic sites Hind III were detected in GJ and AJ fetal globin genes, Hinc II in psi beta and Hinc III in 3' area of psi beta gene and Ava II in beta-globine gene differing in the incidence rate in the patients and donors. An analysis of haplotypes with respect to informative sites was made in two Azerbaijan families with an affected child. It has been found that the analysis with respect to one informative site is sufficient for prenatal diagnosis of the status of the following children. PMID:1706673

  15. Stock Structure and Homing Fidelity in Gulf of Mexico Sturgeon (Acipenser Oxyrinchus Desotoi) Based on Restriction Fragment Length Polymorphism and Sequence Analyses of Mitochondrial DNA

    PubMed Central

    Stabile, J.; Waldman, J. R.; Parauka, F.; Wirgin, I.

    1996-01-01

    Efforts have been proposed worldwide to restore sturgeon populations through the use of hatcheries to supplement natural reproduction and to reintroduce sturgeon where they have become extinct. We examined the population structure and inferred the extent of homing in the anadromous Gulf of Mexico (Gulf) sturgeon (Acipenser oxyrinchus desotoi). Restriction fragment length polymorphism and control region sequence analyses of mitochondrial DNA (mtDNA) were used to identify haplotypes of Gulf sturgeon specimens obtained from eight drainages spanning the subspecies' entire distribution from Louisiana to Florida. Significant differences in haplotype frequencies indicated substantial geographic structuring of populations. A minimum of four regional or river-specific populations were identified (from west to east): (1) Pearl River, LA and Pascagoula River, MS, (2) Escambia and Yellow rivers, FL, (3) Choctawhatchee River, FL, and (4) Apalachicola, Ochlockonee, and Suwannee rivers, FL. Estimates of maternally mediated gene flow between any pair of the four regional or river-specific stocks ranged between 0.15 to 1.2. Tandem repeats in the mtDNA control region of Gulf sturgeon were not perfectly conserved. This result, together with an absence of heteroplasmy and length variation in Gulf sturgeon mtDNA, indicates that the molecular mechanisms of mtDNA control region sequence evolution differ among acipenserids. PMID:8889537

  16. Restriction Fragment Length Polymorphisms Detected with Novel DNA Probes Differentiate among Diverse Lineages of Serogroup 4 Listeria monocytogenes and Identify Four Distinct Lineages in Serotype 4b

    PubMed Central

    Tran, Huyen L.; Kathariou, Sophia

    2002-01-01

    Listeria monocytogenes of serotype 4b has been implicated in numerous outbreaks of food-borne listeriosis and in ca. 40% of sporadic cases. Strains of this serotype appear to be relatively homogeneous genetically, and molecular markers specific for distinct serotype 4b lineages have not been frequently identified. Here we show that DNA fragments derived from the putative mannitol permease locus of Listeria monocytogenes had an unexpectedly high potential to differentiate among different strains of serotype 4b when used as probes in Southern blotting of EcoRI-digested genomic DNA, yielding four distinct restriction fragment length polymorphism (RFLP) patterns. Strains of two epidemic-associated lineages, including the major epidemic clone implicated in several outbreaks in Europe and North America, had distinct RFLPs which differed from those of all other serotype 4b strains that we screened but which were encountered among strains of serotypes 1/2b and 3b. In addition, three serogroup 4 lineages were found to have unique RFLPs that were not encountered among any other L. monocytogenes strains. One was an unusual lineage of serotype 4b, and the other two were members of the serotype 4a and 4c group. The observed polymorphisms may reflect evolutionary relationships among lineages of L. monocytogenes and may facilitate detection and population genetic analysis of specific lineages. PMID:11772609

  17. Capillary Electrophoretic Separation of DNA Restriction Fragments in Mixtures of Low-and High-Molecular-Weight

    E-print Network

    Barron, Annelise E.

    , relative to separation media containing solely low-molecular-weight or high-molecular-weight HEC polymers, the mixed polymer solutions provide superior separation over the DNA size range of interest, while providing by 25-fold in comparison to conventional slab-gel electrophoresis. In this work, we report the use

  18. Matrilineage differentiation of the genus Tetragonisca using mitochondrial DNA markers and the polymerase chain reaction-restriction fragment length polymorphism technique.

    PubMed

    Santos, S A; Bronzato, A R; Moreira, B M T; Araujo, K F; Ronqui, L; Mangolin, C A; Toledo, V A A; Ruvolo-Takasusuki, M C C

    2015-01-01

    The Meliponinae are important pollinators of plant species, and one of the most managed species is Tetragonisca angustula. Initially, two subspecies were identified in T. angustula: T. angustula angustula and T. angustula fiebrigi. Subsequently, T. a. fiebrigi was considered a species, based on the coloration of its mesepisternum. The objective of the present study was to obtain genetic markers that could differentiate the two species by amplifying regions of mitochondrial DNA and conducting polymerase chain reaction-restriction fragment length polymorphism analysis. Worker bees were collected in three Brazilian states: Paraná (Maringá, Altônia, and Foz do Iguaçu), São Paulo (Dracena, São Carlos, and Santa Cruz do Rio Pardo), and Rondônia (Ariquemes). Ten pairs of insect heterologous primers were tested and four were used (primer pair 1, ND2 and COI; primer pair 2, COI; primer pair 8, 16S and 12S; and primer pair 9, COII). For the restriction analysis, 13 enzymes were tested: EcoRI, EcoRV, HindIII, HinfI, RsaI, PstI, XbaI, HaeIII, ClaI, XhoI, BglII, PvuII, and ScaI. Markers were obtained (primer pair 8 cleaved with EcoRV and XbaI and primer pair 9 cleaved with HaeIII, RsaI, and XbaI) that enabled matrilineage identification in the nests studied, which confirmed that hybridization could occur between both Tetragonisca species. The beginning of speciation was probably recent, and secondary contact has resulted in crosses between T. angustula females and T. fiebrigi males. Because of this hybridization, it would be appropriate to consider them as two subspecies of T. angustula. PMID:26505434

  19. Analysis of mitochondrial DNA for authentication of meats from chamois (Rupicapra rupicapra), pyrenean ibex (Capra pyrenaica), and mouflon (Ovis ammon) by polymerase chain reaction-restriction fragment length polymorphism.

    PubMed

    Fajardo, Violeta; González, Isabel; López-Calleja, Inés; Martin, Irene; Rojas, Maria; Pavón, Miguel Angel; Hernández, Pablo E; García, Teresa; Martín, Rosario

    2007-01-01

    The prevention of fraudulent labeling of game meat constitutes an important part of food regulatory control and quality assurance systems. A polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis based on mitochondrial deoxyribonucleic acid (DNA) was developed for authentication of meats from chamois (Rupicapra rupicapra), pyrenean ibex (Capra pyrenaica), and mouflon (Ovis ammon). Amplification and restriction site analysis of a DNA fragment about 720 base pairs (bp) from the mitochondrial 12S rRNA gene of all analyzed species permitted the selection of Msel and Apol endonucleases for meat speciation. The 12S rRNA restriction profiles obtained allowed the unequivocal identification of chamois, pyrenean ibex, and mouflon/sheep and their differentiation from meats of domestic species such as cattle, goat, and swine. The highly variable mitochondrial D-loop gene was also targeted to attempt discrimination between mouflon and sheep meats. A D-loop region (700-1000 bp) was amplified and sequenced in all game and domestic species analyzed, and a primer set was designed for the selective amplification of a 370 bp DNA fragment from mouflon and sheep. PCR-RFLP analysis with the selected Maell enzyme generated a single electrophoretic profile characteristic for sheep, whereas 3 different fragment patterns were obtained for mouflon meats. Consequently, the PCR-RFLP technique developed can be routinely applied in inspection programs in order to verify the correct labeling of game species. PMID:17373450

  20. Sizing of single fluorescently stained DNA fragments by scanning microscopy

    PubMed Central

    Laib, Stephan; Rankl, Michael; Ruckstuhl, Thomas; Seeger, Stefan

    2003-01-01

    We describe an approach to determine DNA fragment sizes based on the fluorescence detection of single adsorbed fragments on specifically coated glass cover slips. The brightness of single fragments stained with the DNA bisintercalation dye TOTO-1 is determined by scanning the surface with a confocal microscope. The brightness of adsorbed fragments is found to be proportional to the fragment length. The method needs only minute amount of DNA, beyond inexpensive and easily available surface coatings, like poly-l-lysine, 3-aminoproyltriethoxysilane and polyornithine, are utilizable. We performed DNA-sizing of fragment lengths between 2 and 14 kb. Further, we resolved the size distribution before and after an enzymatic restriction digest. At this a separation of buffers or enzymes was unnecessary. DNA sizes were determined within an uncertainty of 7–14%. The proposed method is straightforward and can be applied to standardized microtiter plates. PMID:14602931

  1. Detection of single lambda DNA fragments by flow cytometry

    SciTech Connect

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

    1993-01-01

    The authors have demonstrated flow cytometric detection and sizing of single pieces of fluorescently stained lambda DNA (48.5 kb) and individual Kpn I restriction fragments of lambda DNA at 17.05 kb and 29.95 kb. DNA fragments were stained stoichiometrically with an intercalating dye such that the fluorescence from each fragment was directly proportional to fragment length. Laser powers range from 10 to 100 mW and transit times through the focused laser beam were several milliseconds. Measurements were made using time-resolved single photon counting of the detected fluorescence emission from individual stained DNA fragments. Samples were analyzed at rates of about 50 fragments per second. The measured fluorescence intensities are linearly correlated with DNA fragment length over the range measured. Detection sensitivity and resolution needed for analysis of small pieces of DNA are discussed and a comparison of single photon counting measurements of DNA fragments to measurements using more conventional flow cytometers is made. Applications of this methodology to DNA sizing and DNA fingerprinting are discussed.

  2. 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.

  3. 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.

    1993-01-01

    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.

  4. 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.

  5. Sequence Assembly Validation by Multiple Restriction Digest Fragment Coverage Analysis

    E-print Network

    Rouchka, Eric

    and restriction enzymes is derived and used to assess the likelihood of detecting rearrangements in genomic sites for restriction enzymes are specific so it is easy to electronically generate a set of predictedSequence Assembly Validation by Multiple Restriction Digest Fragment Coverage Analysis Eric C

  6. 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.

  7. Molecular identification of fecal pollution sources in water supplies by host-specific fecal DNA markers and Terminal Restriction Fragment Length Polymorphism profiles of 16S rRNA gene.

    PubMed

    Jeong, Ju-Yong; Gil, Kyung-Ik; Lee, Kyong-Hee; Ka, Jong-Ok

    2008-12-01

    Specific fecal DNA markers were investigated for major pollution sources, cow, human, and pig, and occurrence of the identified markers was analyzed in river waters using Terminal Restriction Fragment Length Polymorphism (T-RFLP) techniques and sequencing of 16S rDNA of Bacteroides-Prevotella. The unique and specific DNA markers for cow and human were identified as a 222 bp and 60 bp peak in HaeIII T-RFLP profiles, respectively, and the pig-specific marker was not identified but the unique T-RFLP profile of pig could be used as a substitution. Human-specific marker was detected in most of the river waters tested (92.1%) and T-RFLP profiles of river waters were shown to be similar to those of human feces. Cluster analysis of T-RFLP data showed that the fecal sources were multiple (human plus cow and human plus dairy cow) in most of the river waters. The phylogenetic analysis for the clones recovered from the fecal and water samples showed that the clones from cow formed a discreet cluster from those of other sources. The other clones from human, pig, and river water formed two groups all together. The results of this study could be used to identify and control the fecal pollution source in the bodies of water in Korea. PMID:19107387

  8. Restriction endonuclease cleavage map of the DNA of JC virus.

    PubMed Central

    Martin, J D; Frisque, R J; Padgett, B L; Walker, D L

    1979-01-01

    A physical map of the sites cleaved by the following restriction endonucleases was derived for the DNA of JC virus, a human polyomavirus: EcoRI, HpaI, and PstI (one site each); HindII (four sites); and HindIII (three sites). By agarose gel electrophoresis of fragmented DNA, the size of full-length DNA of JC virus was estimated to be 5,125 +/- 105 base pairs (98 +/- 2% of the length of simian virus 40 DNA). Images PMID:221677

  9. Fidelity of Select Restriction Endonucleases in Determining Microbial Diversity by Terminal-Restriction Fragment Length Polymorphism

    PubMed Central

    Engebretson, Jeff J.; Moyer, Craig L.

    2003-01-01

    An evaluation of 18 DNA restriction endonucleases for use in terminal-restriction fragment length polymorphism (T-RFLP) analysis was performed by using richness and density indices in conjunction with computer simulations for 4,603 bacterial small-subunit rRNA gene sequences. T-RFLP analysis has become a commonly used method for screening environmental samples for precursory identification and community comparison studies due to its precision and high-throughput capability. The accuracy of T-RFLP analysis for describing a community has not yet been thoroughly evaluated. In this study, we attempted to classify restriction endonucleases based upon the ability to resolve unique terminal-restriction fragments (T-RFs) or operational taxonomic units (OTUs) from a database of gene sequences. Furthermore, we assessed the predictive accuracy of T-RFLP at fixed values of community richness (n = 1, 5, 10, 50, and 100). Classification of restriction endonuclease fidelity was performed by measuring richness and density for the entire database of T-RFs. Further analysis of T-RFLP accuracy for determining richness was performed by iterative, random sampling from the derived database of T-RFs. It became apparent that two constraints were influential for measuring the fidelity of a given restriction endonuclease: (i) the ability to resolve unique sequence variants and (ii) the number of unique T-RFs that fell within a measurable size range. The latter constraint was found to be more significant for estimating restriction endonuclease fidelity. Of the 18 restriction endonucleases examined, BstUI, DdeI, Sau96I, and MspI had the highest frequency of resolving single populations in model communities. All restriction endonucleases used in this study detected ?70% of the OTUs at richness values greater than 50 OTUs per modeled community. Based on the results of our in silico experiments, the most efficacious uses of T-RFLP for microbial diversity studies are those that address situations where there is low to intermediate species richness (e.g., colonization, early successional stages, biofilm formation). PMID:12902276

  10. 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.

  11. DNA fragmentation by charged particle tracks

    NASA Astrophysics Data System (ADS)

    Stenerlöw, B.; Höglund, E.; Carlsson, J.

    High-LET (linear energy transfer) charged particles induce DNA double-strand breaks (DSB) in a non-random fashion in mammalian cells. The clustering of DSB, probably determined by track structure as well as chromatin conformation, results in an excess of small- and intermediate-sized DNA fragments. DNA fragmentation in normal human fibroblasts (GM5758) was analyzed by pulsed-field gel electrophoresis after irradiation with photons ( 60Co) or 125 keV/?m nitrogen ions. Compared to conventional DSB analysis, i.e. assays only measuring the fraction of DNA smaller than a single threshold, the relative biological effectiveness (RBE) for DSB induction increased with 100%. Further, the size distribution of DNA fragments showed a significant dependence on radiation quality, with an excess of fragments up to 1 Mbp. Irradiation of naked genomic DNA without histone proteins increased the DSB yields 25 and 13 times for photons and nitrogen ions, respectively. The results suggest possible roles of both track structure and chromatin organization in the distribution of DNA double-strand breaks along the chromosome.

  12. 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...

  13. Effects of DNA Extraction Procedures on Bacteroides Profiles in Fecal Samples From Various Animals Determined by Terminal Restriction Fragment Length Polymorphism Analysis

    EPA Science Inventory

    A major assumption in microbial source tracking is that some fecal bacteria are specific to a host animal, and thus provide unique microbial fingerprints that can be used to differentiate hosts. However, the DNA information obtained from a particular sample may be biased dependi...

  14. Factors affecting SFHR gene correction efficiency with single-stranded DNA fragment

    SciTech Connect

    Tsuchiya, Hiroyuki; Harashima, Hideyoshi; Kamiya, Hiroyuki . E-mail: hirokam@pharm.hokudai.ac.jp

    2005-11-04

    A 606-nt single-stranded (ss) DNA fragment, prepared by restriction enzyme digestion of ss phagemid DNA, improves the gene correction efficiency by 12-fold as compared with a PCR fragment, which is the conventional type of fragment used in the small fragment homologous replacement method [H. Tsuchiya, H. Harashima, H. Kamiya, Increased SFHR gene correction efficiency with sense single-stranded DNA, J. Gene Med. 7 (2005) 486-493]. To reveal the characteristic features of this gene correction with the ss DNA fragment, the effects on the gene correction in CHO-K1 cells of the chain length, 5'-phosphate, adenine methylation, and transcription were studied. Moreover, the possibility that the ss DNA fragment is integrated into the target DNA was examined with a radioactively labeled ss DNA fragment. The presence of methylated adenine, but not the 5'-phosphate, enhanced the gene correction efficiency, and the optimal length of the ss DNA fragment ({approx}600 nt) was determined. Transcription of the target gene did not affect the gene correction efficiency. In addition, the target DNA recovered from the transfected CHO-K1 cells was radioactive. The results obtained in this study indicate that length and adenine methylation were important factors affecting the gene correction efficiency, and that the ss DNA fragment was integrated into the double-stranded target DNA.

  15. RNA-Linked DNA Fragments In Vitro*

    PubMed Central

    Sugino, Akio; Okazaki, Reiji

    1973-01-01

    RNA-linked DNA fragments are intermediates in DNA replication in Escherichia coli cells made permeable to nucleoside triphosphates by treatment with toluene. Covalent linkage of a short RNA stretch to the 5? end of the DNA is proved by transfer of 32P from [?-32P]dNTP to ribonucleotides upon digestion with alkali or pancreatic RNase, and by a small decrease in the molecular size upon alkaline hydrolysis. The 32P transfer experiments reveal a unique structure...p(rPy)p(rA)p(rU or rC)p(dC)p... at the RNA-DNA junction. PMID:4567338

  16. DNA fragmentation in mouse organs during endotoxic shock.

    PubMed Central

    Bohlinger, I.; Leist, M.; Gantner, F.; Angermüller, S.; Tiegs, G.; Wendel, A.

    1996-01-01

    The systemic inflammatory response syndrome has still an unpredictable outcome, and patients often die of multiple organ failure despite circulatory stabilization therapy. The still incompletely understood pathophysiological mechanisms include organ damage due to direct toxic actions of cytokines elicited by overactivation of the host response. To study this process of organ failure in experimental septic shock, we injected mice with a lethal dose of endotoxin and examined apoptotic and necrotic tissue damage biochemically, histologically, and ultrastructurally. Endotoxin administration caused oligonucleosomal as well as random DNA fragmentation in liver, lung, kidney, and intestine. In the liver, DNA fragmentation was not restricted to hepatocytes but also occurred in nonparenchymal cells. The DNA fragmentation was mediated by tumor necrosis factor and attenuated by endogenous nitric oxide release. Unlike the situation in D-galactosamine-sensitized mice, in which injection or release of tumor necrosis factor causes massive hepatocyte apoptosis, liver failure due to high doses of endotoxin was characterized by single-cell necrosis, a low incidence of apoptosis, and simultaneous damage to nonparenchymal cells. We conclude that, even though endotoxin causes cytokine-mediated DNA fragmentation in several organs including the liver, hepatocyte apoptosis itself seems to be a minor phenomenon in high-dose endotoxic shock in mice. Images Figure 2 Figure 3 Figure 4 Figure 5 PMID:8863685

  17. Assembling DNA fragments by USER fusion.

    PubMed

    Annaluru, Narayana; Muller, Héloïse; Ramalingam, Sivaprakash; Kandavelou, Karthikeyan; London, Viktoriya; Richardson, Sarah M; Dymond, Jessica S; Cooper, Eric M; Bader, Joel S; Boeke, Jef D; Chandrasegaran, Srinivasan

    2012-01-01

    Recent advances in DNA synthesis technology make it possible to design and synthesize DNA fragments of several kb in size. However, the process of assembling the smaller DNA fragments into a larger DNA segment is still a cumbersome process. In this chapter, we describe the use of the uracil specific excision reaction (USER)-mediated approach for rapid and efficient assembly of multiple DNA fragments both in vitro and in vivo (using Escherichia coli). For USER fusion in vitro assembly, each of the individual building blocks (BBs), 0.75 kb in size (that are to be assembled), was amplified using the appropriate forward and reverse primers containing a single uracil (U) and DNA polymerase. The overlaps between adjoining BBs were 8-13 base pairs. An equimolar of the amplified BBs were mixed together and treated by USER enzymes to generate complementary 3' single-strand overhangs between adjoining BBs, which were then ligated and amplified simultaneously to generate the larger 3-kb segments. The assembled fragments were then cloned into plasmid vectors and sequenced to confirm their identity. For USER fusion in vivo assembly in E. coli, USER treatment of the BBs was performed in the presence of a synthetic plasmid, which had 8-13 base pair overlaps at the 5'-end of the 5' BB and at the 3'-end of the 3' BB in the mixture. The USER treated product was then transformed directly into E. coli to efficiently and correctly reconstitute the recombinant plasmid containing the desired target insert. The latter approach was also used to rapidly assemble three different target genes into a vector to form a new synthetic plasmid construct. PMID:22328427

  18. Effect of ultrasound on the separation of DNA fragments in agarose gel electrophoresis

    SciTech Connect

    Ma, Yinfa; Yeung, E.S. )

    1990-06-01

    Since its first use in 1966 interest in and the applications of electrophoresis of DNA fragments in agarose gel have grown rapidly. Nowadays, agarose gel electrophoresis has become a standard technique with high resolving power for the analysis of DNA structure, for example for the determination of the length of DNA fragments obtained by the action of restriction enzymes. The electrophoretic mobility ({mu}) of DNA fragments is influenced by various parameters-molecular weight, gel concentration, temperature, electric field, and DNA-agarose affinity. A comprehensive study of the influence of these main parameters has been reported. In this paper, the authors investigate a new effect on the electrophoretic mobility of DNA fragments in agarose gels, viz. the influence of ultrasound.

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

    SciTech Connect

    Jett, J.H.; Hammond, M.L.; Keller, R.A.; Marrone, B.L.; Martin, J.C.

    1992-12-31

    A method is provided for obtaining DNA fingerprints using high speed detection systems, such as flow cytometry to determine unique characteristics of DNA pieces from a selected 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 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 directly proportional to the fragment length. Additional dyes can be bound to the DNA piece and DNA fragments to provide information additional to length information. Oligonucleotide specific dyes and/or hybridization probes can be bound to the DNA fragments to provide information on oligonucleotide distribution or probe hybridization to DNA fragments of different sizes.

  20. 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

  1. RESTseq--efficient benchtop population genomics with RESTriction Fragment SEQuencing.

    PubMed

    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

  2. Optical selection and collection of DNA fragments

    DOEpatents

    Roslaniec, Mary C. (Los Alamos, NM); Martin, John C. (Los Alamos, NM); Jett, James H. (Los Alamos, NM); Cram, L. Scott (Los Alamos, NM)

    1998-01-01

    Optical selection and collection of DNA fragments. The present invention includes the optical selection and collection of large (>.mu.g) quantities of clonable, chromosome-specific DNA from a sample of chromosomes. Chromosome selection is based on selective, irreversible photoinactivation of unwanted chromosomal DNA. Although more general procedures may be envisioned, the invention is demonstrated by processing chromosomes in a conventional flow cytometry apparatus, but where no droplets are generated. All chromosomes in the sample are first stained with at least one fluorescent analytic dye and bonded to a photochemically active species which can render chromosomal DNA unclonable if activated. After passing through analyzing light beam(s), unwanted chromosomes are irradiated using light which is absorbed by the photochemically active species, thereby causing photoinactivation. As desired chromosomes pass this photoinactivation point, the inactivating light source is deflected by an optical modulator; hence, desired chromosomes are not photoinactivated and remain clonable. The selection and photoinactivation processes take place on a microsecond timescale. By eliminating droplet formation, chromosome selection rates 50 times greater than those possible with conventional chromosome sorters may be obtained. Thus, usable quantities of clonable DNA from any source thereof may be collected.

  3. Identification of Panulirus homarus puerulus larvae by restriction fragment length polymorphism of mitochondrial cytochrome oxidase I gene.

    PubMed

    Dharani, G; Maitrayee, G A; Karthikayalu, S; Kumar, T S; Anbarasu, M; Vijayakumaran, M

    2009-02-01

    Molecular identification of puerulus larvae of Panulirus homarus of the genus Panulirus from Indian coast was studied by employing Polymerase Chain Reaction, Restriction Fragment Length Polymorphism (PCR-RFLP) analysis of the mitochondrial DNA (mtDNA) Cytochrome Oxidase Gene (COI) by agarose gel electrophoresis and Denaturing Gradient Gel Electrophoresis (DGGE). The size of amplified fragment of COI gene was estimated to be approximately 1300 base pairs (bp). Single fragment amplification was recorded during different stages of the life cycle. The RFLP digestion was carried out using five different restriction enzymes (BsplI, HhaI, RsaI, TaqI and AluI). The RFLP profile of the different endonucleases, varied between 1-5 restriction types. RFLP analysis using endonuclease TaqI enabled identification of P. homarus during different stages of its life history. PMID:19579959

  4. 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

  5. MULTIPLE ENZYME RESTRICTION FRAGMENT LENGTH POLYMORPHISM ANALYSIS FOR HIGH RESOLUTION DISTINCTION OF PSEUDOMONAS (SENSU STRICTO) 16S RRNA GENES

    EPA Science Inventory

    Pseudomonas specific 16S rDNA PCR amplification and multiple enzyme restriction fragment length polymorphism (MERFLP) analysis using a single digestion mixture of Alu I, Hinf I, Rsa I, and Tru 9I distinguished 150 published sequences and reference strains of authentic Pseudomonas...

  6. Identification of Infectious Agents in Onychomycoses by PCR-Terminal Restriction Fragment Length Polymorphism

    PubMed Central

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

    2012-01-01

    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 Na2S 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

  7. 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

  8. A method for selective PCR-amplification of genomic DNA fragments (SAGF method)

    SciTech Connect

    Zheleznaya, L.A.; Menzenyuk, O.Y.; Matvienko, N.N.; Matvienko, N.I.

    1995-09-01

    A method is suggested for dividing into individual sets of the complex mixtures of fragments obtained by DNA cleavage with type IIS and IIN restriction endonucleases producing single-stranded termini with different sequences at the DNA fragment ends. The method is based on the ligation of short double-stranded adapters with single-stranded ends complementary to termini of the selected set of fragments followed by PCR-amplification with the primer representing one of the adapter chains. Using endonucleases BcoKI and Bli736I, recognizing sequences CTCTTC and GGTCTC and producing three- and four nucleotide 5{prime}-termini, respectively, it has been shown that amplification of a set of fragments occurs only upon attachment of the adapters to the DNA fragments with DNA-ligase. Several possible applications of the SAGF method are suggested: obtaining individual bands in DNA fingerprinting; reducing the kinetic complexity of DNA in representative difference analysis (RDA method) of complex genomes; cataloging of DNA fragments; construction of physical genome maps. 13 refs., 3 figs., 2 tabs.

  9. Stacking Interactions in Denaturation of DNA Fragments

    E-print Network

    Zoli, Marco

    2011-01-01

    A mesoscopic model for heterogeneous DNA denaturation is developed in the framework of the path integral formalism. The base pair stretchings are treated as one-dimensional, time dependent paths contributing to the partition function. The size of the paths ensemble, which measures the degree of cooperativity of the system, is computed versus temperature consistently with the model potential physical requirements. It is shown that the ensemble size strongly varies with the molecule backbone stiffness providing a quantitative relation between stacking and features of the melting transition. The latter is an overall smooth crossover which begins from the \\emph{adenine-thymine} rich portions of the fragment. The harmonic stacking coupling shifts, along the $T$-axis, the occurrence of the multistep denaturation but it does not change the character of the crossover. The methods to compute the fractions of open base pairs versus temperature are discussed: by averaging the base pair displacements over the path ensemb...

  10. MINI-REVIEW Advances in the use of terminal restriction fragment length

    E-print Network

    Forney, Larry J.

    enzyme digests greatly facilitate the choice of primers and restriction enzymes for T-RFLP analysisMINI-REVIEW Advances in the use of terminal restriction fragment length polymorphism (T-Verlag 2008 Abstract Terminal restriction fragment length polymor- phism (T-RFLP) analysis is a popular high

  11. Using Terminal Restriction Fragment Length Polymorphism (T-RFLP) Analysis to Assess Microbial Community Structure in Compost Systems

    NASA Astrophysics Data System (ADS)

    Tiquia, Sonia M.

    Terminal restriction fragment length polymorphism (T-RFLP) analysis of PCR-amplified genes is a widely used fingerprinting technique in composting systems. This analysis is based on the restriction endonuclease digestion of fluorescently end-labeled PCR products. The digested product is mixed with a DNA size standard, itself labeled with a distinct fluorescent dye, and the fragments are then separated by capillary or gel electrophoresis using an automated sequencer. Upon analysis, only the terminal end-labeled restriction fragments are detected. An electropherogram is produced, which shows a profile of compost microbial community as a series of peaks of varying height. This technique has also been effectively used in the exploration of complex microbial environments and in the study of bacterial, archaeal, and eukaryal populations in natural habitats.

  12. Reconstructing DNA replication kinetics from small DNA fragments Haiyang Zhang and John Bechhoefer*

    E-print Network

    Bechhoefer, John

    Reconstructing DNA replication kinetics from small DNA fragments Haiyang Zhang and John Bechhoefer 2006; published 5 May 2006 In higher organisms, DNA replicates simultaneously from many origins. Recent in vitro experiments have yielded large amounts of data on the state of replication of DNA fragments. From

  13. Optimizing restriction fragment fingerprinting methods for ordering large genomic libraries

    SciTech Connect

    Branscomb, E.; Slezak, T.; Pae, R.; Carrano, A.V. ); Galas, D.; Waterman, M. )

    1990-01-01

    The authors present a statistical analysis of the problem of ordering large genomic cloned libraries through overlap detection based on restriction fingerprinting. Such ordering projects involve a large investment of effort involving many repetitious experiments. Their primary purpose here is to provide methods of maximizing the efficiency of such efforts. To this end, they adopt a statistical approach that uses the likelihood ratio as a statistic to detect overlap. The main advantages of this approach are that (1) it allows the relatively straightforward incorporation of the observed statistical properties of the data; (2) it permits the efficiency of a particular experimental method for detecting overlap to be quantitatively defined so that alternative experimental designs may be compared and optimized; and (3) it yields a direct estimate of the probability that any two library members overlap. This estimate is a critical tool for the accurate, automatic assembly of overlapping sets of fragments into islands called contigs.' These contigs must subsequently be connected by other methods to provide an ordered set of overlapping fragments covering the entire genome.

  14. Identification of Dermatophytes by Polymerase Chain Reaction-Restriction Fragment Length Polymorphism Analysis of Metalloproteinase-1

    PubMed Central

    Jung, Ho Jung; Kim, Soo Young; Jung, Jae Wook; Park, Hyun Jung; Lee, Yang Won; Choe, Yong Beom

    2014-01-01

    Background Transgenic research on metalloproteinase-1 is an emerging field in the area of plant molecular biology. The new method reported here can similarly be applied in fungal molecular biology to identify different dermatophytes. Our method is more accurate than traditional methods such as molecular analyses. Objective To identify Trichophyton rubrum, T. mentagrophytes var. mentagrophytes, T. tonsurans, T. mentagrophytes var. interdigitale, Microsporum canis and M. gypseum, by using the restriction fragment length polymorphism (RFLP) analysis and polymerase chain reaction (PCR) to detect polymorphisms in the metalloproteinase-1 gene (MEP1). Methods From each fungal strain, we isolated genomic DNA and performed PCR to amplify the region coding for metalloproteinase-1. Primers for the metalloproteinase-1 gene were designed based on the sequence in NCBI GenBank. Subsequently, we purified the amplified PCR product and performed RFLP analysis. After restriction enzyme digestion, BsrDI (NEB, England), the samples were subjected to electrophoresis. Four different patterns of DNA fragments were observed among 6 fungal species. Results The DNA fragments for T. mentagrophytes var. mentagrophytes, T. mentagrophytes var. interdigitale and T. tonsurans showed similar patterns on electrophoresis and were not distinguishable, whereas T. rubrum, M. canis, and M. gypseum showed different patterns. Conclusion To our knowledge, it is the first study to introduce the analysis of the nucleotide sequence of metalloproteinase-1 enzyme to study differentiation in dermatophytes. Based on our results, more accurate differentiation and subtyping of T. rubrum and T. mentagrophytes var. interdigitale might be possible. This might contribute to better understanding of the epidemiology and pathogenesis of dermatophyte. PMID:24966633

  15. Use of Restriction Fragment Length Polymorphisms to Investigate Strain Variation Within Neisseria Meningitidis.

    NASA Astrophysics Data System (ADS)

    Williams, Shelley Diane

    Similarity within bacterial populations is difficult to assess due to the limited number of characters available for evaluation and the heterogeneity of bacterial species. Currently, the preferred method used to evaluate the structure of bacterial populations is multilocus enzyme electrophoresis. However, this method is extremely cumbersome and only offers an indirect measure of genetic similarities. The development of a more direct and less cumbersome method for this purpose is warranted. Restriction fragment length polymorphism analysis was evaluated as a tool for use in the study of bacterial population structures and in the epidemiology and surveillance of infectious disease. A collection of Neisseria meningitidis was available for use in the investigation of this technique. Neisseria meningitidis is the causative agent of epidemic cerebrospinal meningitis and septicemia as well as a variety of other clinical manifestations. Each isolate in the collection was defined in terms of serogroup specificity, clinical history, geographic source, and date of isolation. Forty -six strains were chosen for this study. The DNA from each strain was restricted with Pst1 and EcoR1 and electrophoresed on agarose gels. The DNA was transferred to nylon filters and hybridized with P ^{32} labeled DNA probes. Two randomly generated probes and a gene-specific probe were used to estimate the genetic similarities between and among the strains in the study population. A total of 28 different restriction fragment migration types were detected by the probes used. Data obtained from the RFLP analysis was analysed by cluster analysis and multivariate statistical methods. A total of 7 clones groups were detected. Two of these appear to be major clones that comprise 35% of the population. This analysis demonstrates the lack of structure within Neisseria meningitidis due primarily to a heterogenous population and the lack of geographic segregation. The potential utility of this technique as a tool in epidemiologic surveillance is addressed. Further work is needed in the evaluation of RFLP analysis in the taxonomy bacteria.

  16. Use of restriction fragment length polymorphisms to investigate strain variation within Neisseria meningitidis

    SciTech Connect

    Williams, S.D.

    1989-01-01

    Similarity within bacterial populations is difficult to assess due to the limited number of characters available for evaluation and the heterogeneity of bacterial species. Currently, the preferred method used to evaluate the structure of bacterial populations is multilocus enzyme electrophoresis. However, this method is extremely cumbersome and only offers an indirect measure of genetic similarities. The development of a more direct and less cumbersome method for this purpose is warranted. Restriction fragment length polymorphism analysis was evaluated as a tool for use in the study of bacterial population structures and in the epidemiology and surveillance of infectious disease. A collection of Neisseria meningitidis was available for use in the investigation of this technique. Neisseria meningitidis is the causative agent of epidemic cerebrospinal meningitis and septicemia as well as a variety of other clinical manifestations. Each isolate in the collection was defined in terms of serogroup specificity, clinical history, geographic source, and date of isolation. Forty-six strains were chosen for this study. The DNA from each strain was restricted with Pst1 and EcoR1 and electrophoresed on agarose gels. The DNA was transferred to nylon filters and hybridized with P{sup 32} labeled DNA probes. Two randomly generated probes and a gene-specific probe were used to estimate the genetic similarities between and among the strains in the study population. A total of 28 different restriction fragment migration types were detected by the probes used. Data obtained from the RFLP analysis was analyzed by cluster analysis and multivariate statistical methods. A total of 7 clones groups were detected. Two of these appear to be major clones that comprise 35% of the population.

  17. Stacking Interactions in Denaturation of DNA Fragments

    E-print Network

    Marco Zoli

    2011-06-21

    A mesoscopic model for heterogeneous DNA denaturation is developed in the framework of the path integral formalism. The base pair stretchings are treated as one-dimensional, time dependent paths contributing to the partition function. The size of the paths ensemble, which measures the degree of cooperativity of the system, is computed versus temperature consistently with the model potential physical requirements. It is shown that the ensemble size strongly varies with the molecule backbone stiffness providing a quantitative relation between stacking and features of the melting transition. The latter is an overall smooth crossover which begins from the \\emph{adenine-thymine} rich portions of the fragment. The harmonic stacking coupling shifts, along the $T$-axis, the occurrence of the multistep denaturation but it does not change the character of the crossover. The methods to compute the fractions of open base pairs versus temperature are discussed: by averaging the base pair displacements over the path ensemble we find that such fractions signal the multisteps of the transition in good agreement with the indications provided by the specific heat plots.

  18. DNA studies using atomic force microscopy: capabilities for measurement of short DNA fragments

    PubMed Central

    Pang, Dalong; Thierry, Alain R.; Dritschilo, Anatoly

    2015-01-01

    Short DNA fragments, resulting from ionizing radiation induced DNA double strand breaks (DSBs), or released from cells as a result of physiological processes and circulating in the blood stream, may play important roles in cellular function and potentially in disease diagnosis and early intervention. The size distribution of DNA fragments contribute to knowledge of underlining biological processes. Traditional techniques used in radiation biology for DNA fragment size measurements lack the resolution to quantify short DNA fragments. For the measurement of cell-free circulating DNA (ccfDNA), real time quantitative Polymerase Chain Reaction (q-PCR) provides quantification of DNA fragment sizes, concentration and specific gene mutation. A complementary approach, the imaging-based technique using Atomic Force Microscopy (AFM) provides direct visualization and measurement of individual DNA fragments. In this review, we summarize and discuss the application of AFM-based measurements of DNA fragment sizes. Imaging of broken plasmid DNA, as a result of exposure to ionizing radiation, as well as ccfDNA in clinical specimens offer an innovative approach for studies of short DNA fragments and their biological functions. PMID:25988169

  19. 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 $D_p$.

  20. Identification of cagA tyrosine phosphorylation DNA motifs in Helicobacter pylori isolates from peptic ulcer patients by novel PCR-restriction fragment length polymorphism and real-time fluorescence PCR assays.

    PubMed

    Owen, Robert J; Sharp, Sally I; Chisholm, Stephanie A; Rijpkema, Sjoerd

    2003-07-01

    Cag pathogenicity island-containing Helicobacter pylori (type I) induces signal transduction pathways resulting in tyrosine phosphorylation of proteins adjacent to the site of bacterial adhesion on host gastric epithelial cells. Conventional block PCR-restriction fragment length polymorphism (RFLP) and real-time LightCycler (LC) PCR hybridization assays, validated by direct sequencing, were designed to test for the presence of three nucleotide sequences corresponding to tyrosine phosphorylation motifs (TPMs) A, B, and C in 84 isolates of H. pylori type I from patients in England. Overall, the PCR assays demonstrated that one or more TPMs were present in 62 strains (75%). Motif A was common (71% of strains), whereas motifs B and C were rarer (8% of strains). Strains lacking a TPM were typically vacuolating cytotoxin genotype vacA m2. Motif A was widely distributed in relation to disease severity and was more commonly (but not significantly [P = 0.071]) associated with gastric ulcer than with duodenal ulcer (86 versus 56%). The LC hybridization assay provided a rapid means of detecting all three motifs, but RFLP analysis was more specific for TPM-A. TPMs provide novel additional strain markers for defining cagA variation, including identification of RFLP types within TPM-A. The presence of a particular TPM was not of direct diagnostic value, either singly or in combination, but the higher proportion of TPM-A strains in gastric ulcer patients merits further investigation. PMID:12843050

  1. Phylogenetic analysis of Gossypium L. using restriction fragment length polymorphism of repeated sequences.

    PubMed

    Zhang, Meiping; Rong, Ying; Lee, Mi-Kyung; Zhang, Yang; Stelly, David M; Zhang, Hong-Bin

    2015-10-01

    Cotton is the world's leading textile fiber crop and is also grown as a bioenergy and food crop. Knowledge of the phylogeny of closely related species and the genome origin and evolution of polyploid species is significant for advanced genomics research and breeding. We have reconstructed the phylogeny of the cotton genus, Gossypium L., and deciphered the genome origin and evolution of its five polyploid species by restriction fragment analysis of repeated sequences. Nuclear DNA of 84 accessions representing 35 species and all eight genomes of the genus were analyzed. The phylogenetic tree of the genus was reconstructed using the parsimony method on 1033 polymorphic repeated sequence restriction fragments. The genome origin of its polyploids was determined by calculating the diploid-polyploid restriction fragment correspondence (RFC). The tree is consistent with the morphological classification, genome designation and geographic distribution of the species at subgenus, section and subsection levels. Gossypium lobatum (D7) was unambiguously shown to have the highest RFC with the D-subgenomes of all five polyploids of the genus, while the common ancestor of Gossypium herbaceum (A1) and Gossypium arboreum (A2) likely contributed to the A-subgenomes of the polyploids. These results provide a comprehensive phylogenetic tree of the cotton genus and new insights into the genome origin and evolution of its polyploid species. The results also further demonstrate a simple, rapid and inexpensive method suitable for phylogenetic analysis of closely related species, especially congeneric species, and the inference of genome origin of polyploids that constitute over 70 % of flowering plants. PMID:25877517

  2. Effect of aging and dietary restriction on DNA repair

    SciTech Connect

    Weraarchakul, N.; Strong, R.; Wood, W.G.; Richardson, A.

    1989-03-01

    DNA repair was studied as a function of age in cells isolated from both the liver and the kidney of male Fischer F344 rats. DNA repair was measured by quantifying unscheduled DNA synthesis induced by UV irradiation. Unscheduled DNA synthesis decreased approximately 50% between the ages of 5 and 30 months in both hepatocytes and kidney cells. The age-related decline in unscheduled DNA synthesis in cells isolated from the liver and kidney was compared in rats fed ad libitum and rats fed a calorie-restricted diet; calorie restriction has been shown to increase the survival of rodents. The level of unscheduled DNA synthesis was significantly higher in hepatocytes and kidney cells isolated from the rats fed the restricted diet. Thus, calorie restriction appears to retard the age-related decline in DNA repair.

  3. 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.

  4. Whole CagA gene amplification of Helicobacter pylori and its fingerprinting by restriction fragment length polymorphism.

    PubMed

    Ye, Siying; Ao, Jienan; Peng, Ying; Yue, Haifeng; Liao, Fang; Hu, Guoping; Xu, Yang; Zhang, Zhengmao

    2002-01-01

    To set up a method of amplification for the whole CagA gene of Helicobacter pylori and its fingerprinting by restriction fragment length polymorphism (RFLP), nested PCR was employed in combination with TD-PCR to amplify the gene and EcoRI and Hind III were used to generate the RFLP fingerprinting. Target DNA fragments from 13 of 20 samples were successfully amplified and the relevant RFLP fingerprintings were obtained. It is concluded that the method can be used to amplify the whole CagA gene and CagA gene has apparent diversity of RFLP profile. PMID:12674756

  5. Investigating of yeast species in wine fermentation using terminal restriction fragment length polymorphism method.

    PubMed

    Sun, Yue; Liu, Yanlin

    2014-04-01

    The objective of this study was to examine the potential of terminal restriction fragment length polymorphism (T-RFLP) in monitoring yeast communities during wine fermentation and to reveal new information on yeast community of Chinese enology. Firstly, terminal restriction fragment (TRF) lengths database was constructed using 32 pure yeast species. Ten of these species were firstly documented. The species except for Candida vini, Issatchenkia orientalis/Candida krusei, Saccharomyces bayanus, Saccharomyces pastorianus, Saccharomyces cerevisiae, Saccharomyces kudriarzevii and Zygosaccharomyces bisporus could be distinguished by the T-RFLP targeting 5.8S-ITS rDNA. Moreover, the yeast communities in spontaneous fermentation of Chardonnay and Riesling were identified by T-RFLP and traditional methods, including colony morphology on Wallerstein Nutrient (WLN) medium and 5.8S-ITS-RFLP analysis. The result showed that T-RFLP profiles of the yeast community correlated well with that of the results identified by the traditional methods. The TRFs with the highest intensity and present in all the samples corresponded to Saccharomyces sp. Other species detected by both approaches were Hanseniaspora uvarum, Metschnikowia pulcherrima, Pichia minuta var. minuta, Saccharomycodes ludwigii/Torulaspora delbrueckii and Candida zemplinina. This study revealed that T-RFLP technique is a rapid and useful tool for monitoring the composition of yeast species during wine fermentation. PMID:24290644

  6. Site-specific DNA transesterification catalyzed by a restriction enzyme

    PubMed Central

    Sasnauskas, Giedrius; Connolly, Bernard A.; Halford, Stephen E.; Siksnys, Virginijus

    2007-01-01

    Most restriction endonucleases use Mg2+ to hydrolyze phosphodiester bonds at specific DNA sites. We show here that BfiI, a metal-independent restriction enzyme from the phospholipase D superfamily, catalyzes both DNA hydrolysis and transesterification reactions at its recognition site. In the presence of alcohols such as ethanol or glycerol, it attaches the alcohol covalently to the 5? terminus of the cleaved DNA. Under certain conditions, the terminal 3?-OH of one DNA strand can attack the target phosphodiester bond in the other strand to create a DNA hairpin. Transesterification reactions on DNA with phosphorothioate linkages at the target bond proceed with retention of stereoconfiguration at the phosphorus, indicating, uniquely for a restriction enzyme, a two-step mechanism. We propose that BfiI first makes a covalent enzyme–DNA intermediate, and then it resolves it by a nucleophilic attack of water or an alcohol, to yield hydrolysis or transesterification products, respectively. PMID:17267608

  7. Electronic transport in methylated fragments of DNA

    NASA Astrophysics Data System (ADS)

    de Almeida, M. L.; Oliveira, J. I. N.; Lima Neto, J. X.; Gomes, C. E. M.; Fulco, U. L.; Albuquerque, E. L.; Freire, V. N.; Caetano, E. W. S.; de Moura, F. A. B. F.; Lyra, M. L.

    2015-11-01

    We investigate the electronic transport properties of methylated deoxyribonucleic-acid (DNA) strands, a biological system in which methyl groups are added to DNA (a major epigenetic modification in gene expression), sandwiched between two metallic platinum electrodes. Our theoretical simulations apply an effective Hamiltonian based on a tight-binding model to obtain current-voltage curves related to the non-methylated/methylated DNA strands. The results suggest potential applications in the development of novel biosensors for molecular diagnostics.

  8. Typing of Histoplasma capsulatum by restriction fragment length polymorphisms in a nuclear gene.

    PubMed Central

    Keath, E J; Kobayashi, G S; Medoff, G

    1992-01-01

    We previously described yps-3, a Histoplasma-specific nuclear gene probe useful in the identification of Histoplasma capsulatum. By using restriction fragment length polymorphisms (RFLPs) of DNA detected by the yps-3 gene and mitochondrial DNA, 76 clinical and soil isolates of H. capsulatum were classified. The majority of North American isolates obtained from endemic regions of the midwestern United States were members of the previously characterized class 2, although four clinical isolates from different patients with AIDS from that region were grouped in class 1 with the temperature-sensitive Downs strain. A Florida soil isolate (FLS1) was placed in class 4 on the basis of RFLP with both probes. Two American Type Culture Collection strains (G184B and G186B) from Panama were grouped into class 3 by this analysis. A group of five H. capsulatum isolates obtained from patients with AIDS in New York City were typed into a new class 5 on the basis of yps-3 polymorphisms; those organisms fell into two broad mitochondrial DNA patterns, designated 5b and 5c. Two new isolates from Panama were also members of this broad yps-3 class 5 group, but they exhibited a distinct mitochondrial DNA profile (class 5a). A sixth class was detected in DNA obtained from a patient with AIDS from Panama; that DNA had unique RFLP profiles with respect to both probes. These observations suggest that the Histoplasma-specific yps-3 gene probe is a sensitive tool for typing H. capsulatum in clinical specimens. Additionally, these studies provide molecular support for the hypothesis that AIDS-associated histoplasmosis in nonendemic areas is due to reactivation of a previously acquired infection. Images PMID:1354224

  9. DNA fragments assembly based on nicking enzyme system.

    PubMed

    Wang, Rui-Yan; Shi, Zhen-Yu; Guo, Ying-Ying; Chen, Jin-Chun; Chen, Guo-Qiang

    2013-01-01

    A couple of DNA ligation-independent cloning (LIC) methods have been reported to meet various requirements in metabolic engineering and synthetic biology. The principle of LIC is the assembly of multiple overlapping DNA fragments by single-stranded (ss) DNA overlaps annealing. Here we present a method to generate single-stranded DNA overlaps based on Nicking Endonucleases (NEases) for LIC, the method was termed NE-LIC. Factors related to cloning efficiency were optimized in this study. This NE-LIC allows generating 3'-end or 5'-end ss DNA overlaps of various lengths for fragments assembly. We demonstrated that the 10 bp/15 bp overlaps had the highest DNA fragments assembling efficiency, while 5 bp/10 bp overlaps showed the highest efficiency when T4 DNA ligase was added. Its advantage over Sequence and Ligation Independent Cloning (SLIC) and Uracil-Specific Excision Reagent (USER) was obvious. The mechanism can be applied to many other LIC strategies. Finally, the NEases based LIC (NE-LIC) was successfully applied to assemble a pathway of six gene fragments responsible for synthesizing microbial poly-3-hydroxybutyrate (PHB). PMID:23483947

  10. Heterothallic species of neurospora are distinguishable by restriction analysis of their nuclear rDNA sequences

    SciTech Connect

    Chambers, C.; Dutta, S.K.

    1983-01-01

    Restriction analysis of rDNAs was used to distinguish nuclear rDNA's of three different reference strains of heterothallic species of the genus Neurospora: N. crassa 74A (FGSC number987), N. intermedia P420 (FGSC number2316), and N. sitophila 10B (FGSC number580). Two approaches were adopted: (1) Nuclear DNA's of these three Neurospora species were treated with various restriction enzymes. Against the streaks of nuclear DNAs on the 0.7% agarose gels background bands were visible. These background bands are visible because rDNA sequences of Neurospora species exist in multiple copies within the nuclear DNA's. (2) The second approach was comparison of auto-radiographs of hybrid molecules of Southern blot transfers of restricted nuclear DNAs and /sup 32/P-labelled nick translated rDNA's (referred to as rDNA probe) isolated from N. crassa slime mutant (FGSC number1118), rDNA cloned into pBR322. A summary of restricted fragment sizes as seen in the gels and in autoradiographs of Southern blots of the respective gels is presented.

  11. Increased DNA fragmentation and ultrastructural changes in fibromyalgic muscle fibres

    PubMed Central

    Sprott, H; Salemi, S; Gay, R; Bradley, L; Alarcon, G; Oh, S; Michel, B; Gay, S

    2004-01-01

    Objective: To determine whether there is evidence of increased DNA fragmentation and ultrastructural changes in muscle tissue of patients with fibromyalgia (FM) compared with healthy controls. Methods: Muscle tissues from 10 community residents with FM and 10 age and sex matched healthy controls were examined "blindly" for the presence of DNA fragmentation by two different methods: terminal deoxynucleotidyl transferase (TdT) staining (TUNEL) and the FragEL-Klenow DNA fragmentation detection kit. Ultrastructural analysis of tissue was performed by electron microscopy. Results: DNA fragmentation was detected by both methods in 55.4 (SEM 2.5)% of the nuclei in muscle tissue of patients with FM compared with 16.1 (4.1)% (p<0.001) of the nuclei in healthy controls. Contrary to expectation, no typical features of apoptosis could be detected by electron microscopy. The myofibres and actin filaments were disorganised and lipofuscin bodies were seen; glycogen and lipid accumulation were also found. The number of mitochondria was significantly lower in patients with FM than in controls and seemed to be morphologically altered. Conclusion: The ultrastructural changes described suggest that patients with FM are characterised by abnormalities in muscle tissue that include increased DNA fragmentation and changes in the number and size of mitochondria. These cellular changes are not signs of apoptosis. Persistent focal contractions in muscle may contribute to ultrastructural tissue abnormalities as well as to the induction and/or chronicity of nociceptive transmission from muscle to the central nervous system. PMID:14962957

  12. 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

  13. DNA fragment editing of genomes by CRISPR/Cas9.

    PubMed

    Jinhuan, Li; Jia, Shou; Qiang, Wu

    2015-10-01

    The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease 9 (Cas9) system from bacteria and archaea emerged recently as a new powerful technology of genome editing in virtually any organism. Due to its simplicity and cost effectiveness, a revolutionary change of genetics has occurred. Here, we summarize the recent development of DNA fragment editing methods by CRISPR/Cas9 and describe targeted DNA fragment deletions, inversions, duplications, insertions, and translocations. The efficient method of DNA fragment editing provides a powerful tool for studying gene function, regulatory elements, tissue development, and disease progression. Finally, we discuss the prospects of CRISPR/Cas9 system and the potential applications of other types of CRISPR system. PMID:26496751

  14. Determining a minimum detection threshold in terminal restriction fragment length polymorphism analysis

    E-print Network

    Courtney, Kevin C.; Bainard, Luke D.; Sikes, Benjamin A.; Koch, Alexander M.; Maherali, Hafiz; Klironomos, John N.; Hart, Miranda M.

    2012-01-01

    Terminal restriction fragment length polymorphism (T-RFLP) analysis is a common technique used to characterize soil microbial diversity. The fidelity of this technique in accurately reporting diversity has not been thoroughly evaluated. Here we...

  15. Novel separation and detection methods of DNA fragments in electrophoresis

    SciTech Connect

    Chan, K.C.

    1992-01-01

    A charge-coupled device (CCD) based electrophoresis system was developed. The system allowed non-destructive, sensitive, and on-line detection of native DNA in slab-gel electrophoresis via ultraviolet absorption measurement. The detection limit of double-stranded DNA fragment was 5 ng per band. Since the amount of DNA used in this experiment was typical, the CCD-based system could be readily implemented in molecular biology. Gel-filled and non-gel sieving capillary electrophoresis (CE) was developed for rapid and efficient separation of double-stranded DNA fragments. For the gel-filled CE separation a new gel matrix, the HydroLink gel (HL), was used. The HL capillary gel was easier to cast than the polyacrylamide capillary gel. For the non-gel separation, a GC capillary was used as the separation chamber, and cellulose additive was included in the electrophoresis as the sieving medium. Indirect fluorometry was applied in non-gel and gel electrophoresis for the detection of DNA fragments. This method allowed nondestructive and on-line detection of DNA during electrophoresis. The amount of DNA used with this method was comparable to those obtained with absorption measurement.

  16. Novel separation and detection methods of DNA fragments in electrophoresis

    SciTech Connect

    Chan, King Cheung.

    1993-01-27

    A charge-coupled device (CCD) based electrophoresis system was developed. The system allowed non-destructive, sensitive, and on-line detection of native DNA in slab-gel electrophoresis via ultraviolet absorption measurement. The detection limit of double-stranded DNA fragment was 5 ng per band. Since the amount of DNA used in this experiment was typical, the CCD-based system could be readily implemented in molecular biology. Gel-filled and non-gel sieving capillary electrophoresis was developed for rapid and efficient separation of double-stranded DNA fragments. For the gel-filled CE separation a new gel matrix, the HydroLink gel (HL), was used. The HL capillary gel was easier to cast than the polyacrylamide capillary gel. For the non-gel separation, a GC capillary was used as the separation chamber, and cellulose additive was included in the electrophoresis as the sieving medium. Indirect fluorometry was applied in non-gel and gel electrophoresis for the detection of DNA fragments. This method allowed non-destructive and on-line detection of DNA during electrophoresis. The amount of DNA used with this method was comparable to those obtained with absorption measurement.

  17. FragMatch--a program for the analysis of DNA fragment data.

    PubMed

    Saari, T A; Saari, S K; Campbell, C D; Alexander, I J; Anderson, I C

    2007-03-01

    FragMatch is a user-friendly Java-supported program that automates the identification of taxa present in mixed samples by comparing community DNA fragment data against a database of reference patterns for known species. The program has a user-friendly Windows interface and was primarily designed for the analysis of fragment data derived from terminal restriction fragment length polymorphism analysis of ectomycorrhizal fungal communities, but may be adapted for other applications such as microsatellite analyses. The program uses a simple algorithm to check for the presence of reference fragments within sample files that can be directly imported, and the results appear in a clear summary table that also details the parameters that were used for the analysis. This program is significantly more flexible than earlier programs designed for matching RFLP patterns as it allows default or user-defined parameters to be used in the analysis and has an unlimited database size in terms of both the number of reference species/individuals and the number of diagnostic fragments per database entry. Although the program has been developed with mycorrhizal fungi in mind, it can be used to analyse any DNA fragment data regardless of biological origin. FragMatch, along with a full description and users guide, is freely available to download from the Aberdeen Mycorrhiza Group web page (http://www.aberdeenmycorrhizas.com). PMID:17226045

  18. Chromatin supraorganization, DNA fragmentation, and cell death in snake erythrocytes.

    PubMed

    Miyamoto, Maristela; Vidal, Benedicto C; Mello, Maria Luiza S

    2005-02-01

    In nucleate erythrocytes of several vertebrate groups, the frequency and intensity of DNA fragmentation associated with programmed cell death vary considerably. Although hemoglobin efficiency may be related to erythrocyte life span, and hemoglobin types and erythrocyte life spans are assumed to vary in reptiles, no data on DNA fragmentation and chromatin organization as related to cell death exist for snakes. In the present study, chromatin supraorganization, DNA fragmentation, and cell death were investigated in four snake species (Crotalus durissus terrificus, Bothrops jararaca, Bothrops alternatus, and Bothrops neuwiedii), which differ in their geographical distribution and habitats, by using image analysis of Feulgen hydrolysis kinetics, the TUNEL assay, single-cell gel electrophoresis, and transmission electron microscopy. Relatively few circulating erythrocytes were found to be simultaneously committed to cell death, although there was some variation among the snake species. Conspicuous nuclear and cytoplasmic organelles suggestive of metabolic activity were seen ultrastructurally in most snake erythrocytes. The DNA of the snake erythrocyte chromatin was much more resistant to Feulgen acid hydrolysis (DNA depurination and breakdown) than that of young adult bullfrog erythrocytes, which had a high frequency and intensity of DNA fragmentation. Of the species studied, B. neuwiedii and C. d. terrificus showed the greatest resistance to Feulgen acid hydrolysis and to the DNA fragmentation, revealed by the TUNEL assay. Although B. neuwiedii also showed the lowest frequency of cells with more damaged DNA in the single-cell gel electrophoresis assay, C. d. terrificus had the highest frequency of damaged cells, possibly because of the abundance of alkaline-sensitive DNA sites. The results for DNA fragmentation and cell death in erythrocytes of B. jararaca and B. alternatus generally differed from those for C. d. terrificus and B. neuwiedii and may reflect differences in the biology of these species selected under different geographical habitats. The differences in erythrocyte cell biology reported here may be related to hemoglobin variants selected in the mentioned snake species and that would lead the cells to different resistances to unfavorable environmental conditions. PMID:15746963

  19. RNA-Linked Nascent DNA Fragments in Escherichia coli*

    PubMed Central

    Sugino, Akio; Hirose, Susumu; Okazaki, Reiji

    1972-01-01

    Nucleic acid that is extracted from E. coli labeled by a brief pulse of [3H]dT and depatured by treatment with heat, formamide, or formaldehyde bands in a region with a density higher than that of single-stranded E. coli DNA in a Cs2SO4 equilibrium density gradient. If treated with alkali or RNase, it then exhibits the density of single-stranded DNA. These results suggest the presence of a short strand of RNA covalently linked to the nascent DNA. Evidence for the presence of covalently linked RNA-DNA molecules is also obtained by pulse labeling with [3H]U. Analyses of nascent nucleic acids from cells pulse labeled for various times, and of the molecules with different sizes, support the hypothesis that the short DNA fragments are formed by extension of even shorter RNA chains, which are synthesized on the parental DNA strands and are removed before ligation of the DNA fragments. The synthesis of the RNA segment of the RNA-DNA molecule is much less sensitive to rifampicin than is the synthesis of bulk RNA. Images PMID:4558661

  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. DNA Oligonucleotide Fragment Ion Rearrangements Upon Collision-Induced Dissociation

    NASA Astrophysics Data System (ADS)

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

    2015-08-01

    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.

  2. Cloning of V region fragments from mouse liver DNA and localization of repetitive DNA sequences in the vicinity of immunoglobulin gene segments.

    PubMed Central

    Steinmetz, M; Höchtl, J; Schnell, H; Gebhard, W; Zachau, H G

    1980-01-01

    Two different kappa light chain genes have previously been isolated from one mouse myeloma. The V (variable, abbreviations in ref. 2) gene segments of the two genes were now used to identify their germline counterparts in EcoRI digests of mouse liver DNA. In addition two sets of related V gene segments were found which hybridize with either of the two DNA probes. Five of the V region fragments of one set were cloned in a lambda phage vector and partially characterized by restriction mapping and Southern blot hybridization. Repetitive DNA sequences were found on each of the five fragments as well as on other cloned immunoglobulin gene containing fragments. Cross-hybridization between some but not all of the regions containing repetitive DNA sequences was observed. Images PMID:6253945

  3. Discrimination among individuals using terminal restriction fragment length polymorphism profiling of bacteria derived from forensic evidence.

    PubMed

    Nishi, Eiji; Tashiro, Yukihiro; Sakai, Kenji

    2015-05-01

    DNA typing from forensic evidence is commonly used to identify individuals. However, when the quantity of the forensic evidence is insufficient, successful identification using DNA typing is impossible. Such evidence may also contain DNA from bacteria that occur naturally on the skin. In this study, we aimed to establish a profiling method using terminal restriction fragment length polymorphisms (T-RFLPs) of the amplified bacterial 16S ribosomal RNA (rRNA) gene. First, the extraction and digestion processes were investigated, and the T-RFLP profiling method using the 16S rRNA gene amplicon was optimized. We then used this method to compare the profiles of bacterial flora from the hands of 12 different individuals. We found that the T-RFLP profiles from one person on different days displayed higher similarity than those between individuals. In a principal component analysis (PCA), T-RFLPs from each individual were closely clustered in 11 out of 12 cases. The clusters could be distinguished from each other, even when the samples were collected from different conditions. No major change of the profile was observed after six months except in two cases. When handprints on glass plates were compared, 11 of 12 individuals were assigned to a few clusters including the cluster corresponding to the correct individual. In conclusion, a method for reproducible T-RFLP profiling of bacteria from trace amounts of handprints was established. The profiles were obtained for particular individuals clustered in PCA and were experimentally separable from other individuals in most cases. This technique could provide useful information for narrowing down a suspect in a criminal investigation. PMID:25335807

  4. X chromosome restriction fragment length polymorphisms in five racial groups: rare variant detected with the RC8 (DXS9) probe in the Marathi population, India.

    PubMed

    Wadhwa, R; Papiha, S; Lester, D; Ray, V; Saha, N; Bhattacharya, S

    1989-01-01

    Restriction fragment length polymorphisms were investigated in five racial groups using the X chromosome probes DXS9 and DXS7. The allele frequencies of these polymorphisms showed significant differences and both DNA fragments were found to be highly polymorphic in the populations of south and southeast Asia. In the Marathi population of India, a rare allele B*3 (3 kilobases; kb) and an altered 7-kb fragment instead of the 6.6-kb constant band were found with DXS9. This is the first time that the rare B*3 allele is found in a non-European population. PMID:2575595

  5. Ultra-high-speed DNA fragment separations using microfabricated capillary array electrophoresis chips.

    PubMed Central

    Woolley, A T; Mathies, R A

    1994-01-01

    Capillary electrophoresis arrays have been fabricated on planar glass substrates by photolithographic masking and chemical etching techniques. The photolithographically defined channel patterns were etched in a glass substrate, and then capillaries were formed by thermally bonding the etched substrate to a second glass slide. High-resolution electrophoretic separations of phi X174 Hae III DNA restriction fragments have been performed with these chips using a hydroxyethyl cellulose sieving matrix in the channels. DNA fragments were fluorescently labeled with dye in the running buffer and detected with a laser-excited, confocal fluorescence system. The effects of variations in the electric field, procedures for injection, and sizes of separation and injection channels (ranging from 30 to 120 microns) have been explored. By use of channels with an effective length of only 3.5 cm, separations of phi X174 Hae II DNA fragments from approximately 70 to 1000 bp are complete in only 120 sec. We have also demonstrated high-speed sizing of PCR-amplified HLA-DQ alpha alleles. This work establishes methods for high-speed, high-throughput DNA separations on capillary array electrophoresis chips. Images PMID:7972062

  6. A DNA and restriction enzyme implementation of Turing Ma (Turing machines; Universal Turing machines; recombinant DNA; nonpalindromic endonucleases; class IIS

    E-print Network

    Winfree, Erik

    A DNA and restriction enzyme implementation of Turing Ma­ chines. (Turing machines; Universal://www.ugcs.caltech.edu/~pwkr/oett.html ABSTRACT Bacteria employ restriction enzymes to cut or restrict DNA at or near specific words in a unique way. Many restriction enzymes cut the two strands of double­stranded DNA at different positions

  7. A noninvasive method for distinguishing among canid species: amplification and enzyme restriction of DNA from dung.

    PubMed

    Paxinos, E; McIntosh, C; Ralls, K; Fleischer, R

    1997-05-01

    Endangered San Joaquin kit foxes Vulpes macrotis mutica can be sympatrically distributed with as many as four other canids: red fox, gray fox, coyote and domestic dog. Canid scats are often found during routine fieldwork, but cannot be reliably identified to species. To detect and study the endangered kit fox, we developed mitochondrial DNA markers that can be amplified from small amounts of DNA extracted from scats. We amplified a 412-bp fragment of the mitochondrial cytochrome-b gene from scat samples and digested it with three restriction enzymes. The resulting restriction profiles discriminated among all five canid species and correctly identified 10 'unknown' fox scats to species in blind tests. We have applied our technique to identify canids species for an environmental management study and a conservation study. We envision that our protocol, and similar ones developed for other endangered species will be greatly used for conservation management in the future. PMID:9161016

  8. Detection of Cryptosporidium and Identification to the Species Level by Nested PCR and Restriction Fragment Length Polymorphism

    PubMed Central

    Coupe, Stephane; Sarfati, Claudine; Hamane, Samia; Derouin, Francis

    2005-01-01

    Cryptosporidiosis is an emerging protozoan disease associated with large waterborne outbreaks. Diagnosis relies on microscopic examination of stools, but this method cannot identify the infecting species of Cryptosporidium. We have developed a test based on nested PCR and restriction fragment length polymorphism (RFLP) that offers simple identification of Cryptosporidium hominis, Cryptosporidium parvum, and most other human infective species in stool samples. Purified C. parvum oocysts were used for PCR development. Extracted DNA was amplified by nested PCR targeting a 214-bp fragment of the 18S RNA gene. Enzymatic restriction sites were identified by bioinformatic analysis of all published Cryptosporidium 18S rRNA sequences. Experiments with spiked stool samples gave an estimated PCR detection limit of one oocyst. Specificity was assessed by testing 68 stool samples from patients with microscopically proven cryptosporidiosis and 31 Cryptosporidium-negative stools. Sixty-seven (98.5%) of the 68 stool samples from patients with microscopically proven cryptosporidiosis and 2 of the other stool samples were positive by PCR and could be genotyped. RFLP analysis identified 36 C. hominis, 19 C. parvum, 8 Cryptosporidium meleagridis, and 6 Cryptosporidium felis or Cryptosporidium canis samples. Species determination in 26 PCR-positive cases was in full agreement with DNA sequencing of the 18S rRNA hypervariable region. The excellent sensitivity of PCR, coupled with the accuracy of RFLP for species identification, make this method a suitable tool for routine diagnosis and genotyping of Cryptosporidium in stools. PMID:15750054

  9. Community terminal restriction fragment length polymorphisms reveal insights into the diversity and dynamics of leaf endophytic bacteria

    PubMed Central

    2013-01-01

    Background Plant endophytic bacteria play an important role benefiting plant growth or being pathogenic to plants or organisms that consume those plants. Multiple species of bacteria have been found co-inhabiting plants, both cultivated and wild, with viruses and fungi. For these reasons, a general understanding of plant endophytic microbial communities and their diversity is necessary. A key issue is how the distributions of these bacteria vary with location, with plant species, with individual plants and with plant growing season. Results Five common plant species were collected monthly for four months in the summer of 2010, with replicates from four different sampling sites in the Tallgrass Prairie Preserve in Osage County, Oklahoma, USA. Metagenomic DNA was extracted from ground, washed plant leaf samples, and fragments of the bacterial 16S rDNA genes were amplified for analysis of terminal restriction fragment length polymorphism (T-RFLP). We performed mono-digestion T-RFLP with restriction endonuclease DdeI, to reveal the structures of leaf endophytic bacterial communities, to identify the differences between plant-associated bacterial communities in different plant species or environments, and to explore factors affecting the bacterial distribution. We tested the impacts of three major factors on the leaf endophytic bacterial communities, including host plant species, sampling dates and sampling locations. Conclusions Results indicated that all of the three factors were significantly related (??=?0.05) to the distribution of leaf endophytic bacteria, with host species being the most important, followed by sampling dates and sampling locations. PMID:23286760

  10. Identification of Staphylococcus spp. by PCR-Restriction Fragment Length Polymorphism of gap Gene

    PubMed Central

    Yugueros, Javier; Temprano, Alejandro; Sánchez, María; Luengo, José María; Naharro, Germán

    2001-01-01

    Oligonucleotide primers specific for the Staphylococcus aureus gap gene were previously designed to identify 12 Staphylococcus spp. by PCR. In the present study, AluI digestion of PCR-generated products rendered distinctive restriction fragment length polymorphism patterns that allowed 24 Staphylococcus spp. to be identified with high specificity. PMID:11574593

  11. 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

  12. Congruencein Control-Region Sequenceand Restriction-Site Variation in Mitochondrial DNA of Brook Charr

    E-print Network

    Bernatchez, Louis

    Congruencein Control-Region Sequenceand Restriction- Site Variation in Mitochondrial DNA of Brook mitochondrial DNA (mtDNA) haplotypes detected with restriction-frag- ment-length polymorphisms (RFLPs The analysis of mitochondrial DNA (mtDNA) variation has been widely used to address questions in population

  13. Ion induced fragmentation cross-sections of DNA constituents

    NASA Astrophysics Data System (ADS)

    Rudek, Benedikt; Arndt, Alexander; Bennett, Daniel; Wang, Mingjie; Rabus, Hans

    2015-10-01

    Proton collision with chemical analogs for the base, the sugar and the phosphor residue of the DNA, namely pyrimidine, tetrahydrofuran and trimethyl phosphate, respectively, has been investigated. The impact energies ranged from 300 keV up to 16 MeV. For the first time, relative fragmentation cross-sections for proton impact are reported for tetrahydrofuran and trimethyl phosphate; previously reported cross sections for pyrimidine are extended for energies beyond 2500 keV. Ionization of tetrahydrofuran leads to a ring break in about 80% of all events, trimethyl phosphate predominantly fragments by bond cleavage to one of the three methyl-groups and for pyrimidine the parent ion has the highest abundance. Such comparison supports earlier findings that the sugar is the weak spot for strand breaks.

  14. Phylogenomics of caspase-activated DNA fragmentation factor

    SciTech Connect

    Eckhart, Leopold . E-mail: leopold.eckhart@meduniwien.ac.at; Fischer, Heinz; Tschachler, Erwin

    2007-04-27

    The degradation of nuclear DNA by DNA fragmentation factor (DFF) is a key step in apoptosis of mammalian cells. Using comparative genomics, we have here determined the evolutionary history of the genes encoding the two DFF subunits, DFFA (also known as ICAD) and DFFB (CAD). Orthologs of DFFA and DFFB were identified in Nematostella vectensis, a representative of the primitive metazoan clade cnidarians, and in various vertebrates and insects, but not in representatives of urochordates, echinoderms, and nematodes. The domains mediating the interaction of DFFA and DFFB, a caspase cleavage site in DFFA, and the amino acid residues critical for endonuclease activity of DFFB were conserved in Nematostella. These findings suggest that DFF has been a part of the primordial apoptosis system of the eumetazoan common ancestor and that the ancient cell death machinery has degenerated in several evolutionary lineages, including the one leading to the prototypical apoptosis model, Caenorhabditis elegans.

  15. 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

  16. Taxonomic and ecological discrimination of Fagaceae species based on internal transcribed spacer polymerase chain reaction-restriction fragment length polymorphism.

    PubMed

    Coutinho, João Paulo; Carvalho, Ana; Lima-Brito, José

    2014-01-01

    The internal transcribed spacer (ITS) of ribosomal DNA has been used to confirm taxonomic classifications and define phylogenies in several plant species following sequencing or polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) techniques. In this study, co-dominant ITS PCR-RFLP molecular markers were produced in 30 Fagaceae individuals belonging to the Castanea, Fagus and Quercus genera in order to assess the potential of this technique for taxonomic discrimination and determination of phylogenies. The complete ITS region (ITS1-5.8S rRNA-ITS2) was amplified in most of the Fagaceae individuals as a single fragment of ?700 bp. The ITS amplified products were digested with nine restriction enzymes, but only four (HaeIII, HpaII, TaqI and Sau96I) produced polymorphic/discriminative patterns. The total expected heterozygosity (HE) was 20.31 % and the gene diversity (I), 32.97 %. The ITS polymorphism was higher within the Quercus genus (85.3 %). The ITS PCR-RFLP markers clustered the Fagaceae species according to genus or infrageneric group (in the case of Quercus sp. individuals). Five oaks did not cluster in line with the adopted infrageneric classification, but three of these were grouped according to their actual ecological distributions. The ITS PCR-RFLP markers indicated their potential for phylogenetic studies since all Fagaceae individuals were discriminated according to genus, and most of the oaks were clustered according to infrageneric group or ecological area. PMID:25429047

  17. 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

  18. 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.

  19. Temporal Patterns of Nucleotide Misincorporations and DNA Fragmentation in Ancient DNA

    PubMed Central

    Sawyer, Susanna; Krause, Johannes; Guschanski, Katerina; Savolainen, Vincent; Pääbo, Svante

    2012-01-01

    DNA that survives in museum specimens, bones and other tissues recovered by archaeologists is invariably fragmented and chemically modified. The extent to which such modifications accumulate over time is largely unknown but could potentially be used to differentiate between endogenous old DNA and present-day DNA contaminating specimens and experiments. Here we examine mitochondrial DNA sequences from tissue remains that vary in age between 18 and 60,000 years with respect to three molecular features: fragment length, base composition at strand breaks, and apparent C to T substitutions. We find that fragment length does not decrease consistently over time and that strand breaks occur preferentially before purine residues by what may be at least two different molecular mechanisms that are not yet understood. In contrast, the frequency of apparent C to T substitutions towards the 5?-ends of molecules tends to increase over time. These nucleotide misincorporations are thus a useful tool to distinguish recent from ancient DNA sources in specimens that have not been subjected to unusual or harsh treatments. PMID:22479540

  20. Rapid Identification of Rhizobia by Restriction Fragment Length Polymorphism Analysis of PCR-Amplified 16S rRNA Genes

    PubMed Central

    Laguerre, Gisèle; Allard, Marie-Reine; Revoy, Françoise; Amarger, Noelle

    1994-01-01

    Forty-eight strains representing the eight recognized Rhizobium species, two new Phaseolus bean Rhizobium genomic species, Bradyrhizobium spp., Agrobacterium spp., and unclassified rhizobia from various host plants were examined by restriction fragment length polymorphism (RFLP) analysis of 16S rRNA genes amplified by polymerase chain reaction (PCR). Twenty-one composite genotypes were obtained from the combined data of the RFLP analysis with nine endonucleases. Species assignments were in full agreement with the established taxonomic classification. Estimation from these data of genetic relationships between and within genera and species correlated well with previously published data based on DNA-rRNA hybridizations and sequence analysis of 16S rRNA genes. This PCR-RFLP method provides a rapid tool for the identification of root nodule isolates and the detection of new taxa. Images PMID:16349165

  1. 8 Phylogenetic Relationships Using Restriction Site Variation of the Chloroplast DNA Inverted Repeat

    E-print Network

    Downie, Stephen R.

    genes that contain introns by DNA sequencing. Analyses of restriction site polymorphisms in cpDNA have8 Phylogenetic Relationships Using Restriction Site Variation of the Chloroplast DNA Inverted are largely responsible for variation in size of the molecule. Recent studies of chloroplast DNA (cp

  2. Lactic acid bacterial population dynamics during fermentation and storage of Thai fermented sausage according to restriction fragment length polymorphism analysis.

    PubMed

    Wanangkarn, Amornrat; Liu, Deng-Cheng; Swetwiwathana, Adisorn; Jindaprasert, Aphacha; Phraephaisarn, Chirapiphat; Chumnqoen, Wanwisa; Tan, Fa-Jui

    2014-09-01

    This study applied restriction fragment length polymorphism (RFLP) analysis to identify the lactic acid bacteria (LAB) isolated from "mum" Thai fermented sausages during fermentation and storage. A total of 630 lactic acid bacteria were isolated from the sausages prepared using 2 methods. In Method 1, after stuffing, the sausages were stored at 30 °C for 14 days. In Method 2, after stuffing and storage at 30 °C for 3 days, the sausages were vacuum-packed and stored at 4 °C until Day 28. The sausages were sampled on Days 0, 3, 14, and 28 for analyses. The 16S rDNA was amplified and digested using restriction enzymes. Of the restriction enzymes evaluated, Dde I displayed the highest discrimination capacity. The LAB were classified and 7 species were identified For Methods 1 and 2, during fermentation, the Lactobacillus sakei and Lactobacillus plantarum species were dominant. For Method 2, the proportion of Leuconostoc mesenteroides markedly increased during storage, until L. sakei and Ln. mesenteroides represented the dominant species. The identification of LAB in the sausage samples could facilitate the selection of appropriate microorganisms for candidate starter cultures for future controlled mum production. PMID:25005265

  3. Impacts of degraded DNA on restriction enzyme associated DNA sequencing (RADSeq).

    PubMed

    Graham, Carly F; Glenn, Travis C; McArthur, Andrew G; Boreham, Douglas R; Kieran, Troy; Lance, Stacey; Manzon, Richard G; Martino, Jessica A; Pierson, Todd; Rogers, Sean M; Wilson, Joanna Y; Somers, Christopher M

    2015-11-01

    Degraded DNA from suboptimal field sampling is common in molecular ecology. However, its impact on techniques that use restriction site associated next-generation DNA sequencing (RADSeq, GBS) is unknown. We experimentally examined the effects of in situDNA degradation on data generation for a modified double-digest RADSeq approach (3RAD). We generated libraries using genomic DNA serially extracted from the muscle tissue of 8 individual lake whitefish (Coregonus clupeaformis) following 0-, 12-, 48- and 96-h incubation at room temperature posteuthanasia. This treatment of the tissue resulted in input DNA that ranged in quality from nearly intact to highly sheared. All samples were sequenced as a multiplexed pool on an Illumina MiSeq. Libraries created from low to moderately degraded DNA (12-48 h) performed well. In contrast, the number of RADtags per individual, number of variable sites, and percentage of identical RADtags retained were all dramatically reduced when libraries were made using highly degraded DNA (96-h group). This reduction in performance was largely due to a significant and unexpected loss of raw reads as a result of poor quality scores. Our findings remained consistent after changes in restriction enzymes, modified fold coverage values (2- to 16-fold), and additional read-length trimming. We conclude that starting DNA quality is an important consideration for RADSeq; however, the approach remains robust until genomic DNA is extensively degraded. PMID:25783180

  4. Clusters of DNA damage induced by ionizing radiation: formation of short DNA fragments. II. Experimental detection

    NASA Technical Reports Server (NTRS)

    Rydberg, B.; Chatterjee, A. (Principal Investigator)

    1996-01-01

    The basic 30-nm chromatin fiber in the mammalian cell consists of an unknown (possibly helical) arrangement of nucleosomes, with about 1.2 kb of DNA per 10-nm length of fiber. Track-structure considerations suggest that interactions of single delta rays or high-LET particles with the chromatin fiber might result in the formation of multiple lesions spread over a few kilobases of DNA (see the accompanying paper: W.R. Holley and A. Chatterjee, Radiat. Res. 145, 188-199, 1996). In particular, multiple DNA double-strand breaks and single-strand breaks may form. To test this experimentally, primary human fibroblasts were labeled with [3H]thymidine and exposed at 0 degrees C to X rays or accelerated nitrogen or iron ions in the LET range of 97-440 keV/microns. DNA was isolated inside agarose plugs and subjected to agarose gel electrophoresis under conditions that allowed good separation of 0.1-2 kb size DNA. The bulk of DNA remained in the well or migrated only a small distance into the gel. It was found that DNA fragments in the expected size range were formed linearly with dose with an efficiency that increased with LET. A comparison of the yield of such fragments with the yield of total DNA double-strand breaks suggests that for the high-LET ions a substantial proportion (20-90%) of DNA double-strand breaks are accompanied within 0.1-2 kb by at least one additional DNA double-strand break. It is shown that these results are in good agreement with theoretical calculations based on treating the 30-nm chromatin fiber as the target for ionizing particles. Theoretical considerations also predict that the clusters will contain numerous single-strand breaks and base damages. It is proposed that such clusters be designated "regionally multiply damaged sites." Postirradiation incubation at 37 degrees C resulted in a decline in the number of short DNA fragments, suggesting a repair activity. The biological significance of regionally multiply damaged sites is presently unknown.

  5. Designing and Testing a New DNA Fragment Assembler VEDA-2 Mark K. Goldberg Darren T. Lim

    E-print Network

    Goldberg, Mark

    -generated input formed from the Anthrax DNA. Testing on diverse DNA sequences of lengths of up to 5 million baseDesigning and Testing a New DNA Fragment Assembler VEDA-2 Mark K. Goldberg Darren T. Lim Rensselaer within 5% of the true answer. Keywords: DNA Assembly, Machine Learning, Shotgun sequencing 1 Introduction

  6. The Saccharomyces cerevisiae Dna2 can function as a sole nuclease in the processing of Okazaki fragments in DNA replication

    PubMed Central

    Levikova, Maryna; Cejka, Petr

    2015-01-01

    During DNA replication, synthesis of the lagging strand occurs in stretches termed Okazaki fragments. Before adjacent fragments are ligated, any flaps resulting from the displacement of the 5? DNA end of the Okazaki fragment must be cleaved. Previously, Dna2 was implicated to function upstream of flap endonuclease 1 (Fen1 or Rad27) in the processing of long flaps bound by the replication protein A (RPA). Here we show that Dna2 efficiently cleaves long DNA flaps exactly at or directly adjacent to the base. A fraction of the flaps cleaved by Dna2 can be immediately ligated. When coupled with DNA replication, the flap processing activity of Dna2 leads to a nearly complete Okazaki fragment maturation at sub-nanomolar Dna2 concentrations. Our results indicate that a subsequent nucleolytic activity of Fen1 is not required in most cases. In contrast Dna2 is completely incapable to cleave short flaps. We show that also Dna2, like Fen1, interacts with proliferating cell nuclear antigen (PCNA). We propose a model where Dna2 alone is responsible for cleaving of RPA-bound long flaps, while Fen1 or exonuclease 1 (Exo1) cleave short flaps. Our results argue that Dna2 can function in a separate, rather than in a Fen1-dependent pathway. PMID:26175049

  7. The Saccharomyces cerevisiae Dna2 can function as a sole nuclease in the processing of Okazaki fragments in DNA replication.

    PubMed

    Levikova, Maryna; Cejka, Petr

    2015-09-18

    During DNA replication, synthesis of the lagging strand occurs in stretches termed Okazaki fragments. Before adjacent fragments are ligated, any flaps resulting from the displacement of the 5' DNA end of the Okazaki fragment must be cleaved. Previously, Dna2 was implicated to function upstream of flap endonuclease 1 (Fen1 or Rad27) in the processing of long flaps bound by the replication protein A (RPA). Here we show that Dna2 efficiently cleaves long DNA flaps exactly at or directly adjacent to the base. A fraction of the flaps cleaved by Dna2 can be immediately ligated. When coupled with DNA replication, the flap processing activity of Dna2 leads to a nearly complete Okazaki fragment maturation at sub-nanomolar Dna2 concentrations. Our results indicate that a subsequent nucleolytic activity of Fen1 is not required in most cases. In contrast Dna2 is completely incapable to cleave short flaps. We show that also Dna2, like Fen1, interacts with proliferating cell nuclear antigen (PCNA). We propose a model where Dna2 alone is responsible for cleaving of RPA-bound long flaps, while Fen1 or exonuclease 1 (Exo1) cleave short flaps. Our results argue that Dna2 can function in a separate, rather than in a Fen1-dependent pathway. PMID:26175049

  8. A Mini-Library of Sequenced Human DNA Fragments: Linking Bench Experiments with Informatics

    ERIC Educational Resources Information Center

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

    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…

  9. Identification of Pork Contamination in Meatballs of Indonesia Local Market Using Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) Analysis.

    PubMed

    Erwanto, Yuny; Abidin, Mohammad Zainal; Sugiyono, Eko Yasin Prasetyo Muslim; Rohman, Abdul

    2014-10-01

    This research applied and evaluated a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) using cytochrome b gene to detect pork contamination in meatballs from local markets in Surabaya and Yogyakarta regions, Indonesia. To confirm the effectiveness and specificity of this fragment, thirty nine DNA samples from different meatball shops were isolated and amplified, and then the PCR amplicon was digested by BseDI restriction enzyme to detect the presence of pork in meatballs. BseDI restriction enzyme was able to cleave porcine cytochrome b gene into two fragments (131 bp and 228 bp). Testing the meatballs from the local market showed that nine of twenty meatball shops in Yogyakarta region were detected to have pork contamination, but there was no pork contamination in meatball shops in Surabaya region. In conclusion, specific PCR amplification of cytochrome b gen and cleaved by BseDI restriction enzymes seems to be a powerful technique for the identification of pork presence in meatball because of its simplicity, specificity and sensitivity. Furthermore, pork contamination intended for commercial products of sausage, nugget, steak and meat burger can be checked. The procedure is also much cheaper than other methods based on PCR, immunodiffusion and other techniques that need expensive equipment. PMID:25178301

  10. Identification of Pork Contamination in Meatballs of Indonesia Local Market Using Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) Analysis

    PubMed Central

    Erwanto, Yuny; Abidin, Mohammad Zainal; Sugiyono, Eko Yasin Prasetyo Muslim; Rohman, Abdul

    2014-01-01

    This research applied and evaluated a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) using cytochrome b gene to detect pork contamination in meatballs from local markets in Surabaya and Yogyakarta regions, Indonesia. To confirm the effectiveness and specificity of this fragment, thirty nine DNA samples from different meatball shops were isolated and amplified, and then the PCR amplicon was digested by BseDI restriction enzyme to detect the presence of pork in meatballs. BseDI restriction enzyme was able to cleave porcine cytochrome b gene into two fragments (131 bp and 228 bp). Testing the meatballs from the local market showed that nine of twenty meatball shops in Yogyakarta region were detected to have pork contamination, but there was no pork contamination in meatball shops in Surabaya region. In conclusion, specific PCR amplification of cytochrome b gen and cleaved by BseDI restriction enzymes seems to be a powerful technique for the identification of pork presence in meatball because of its simplicity, specificity and sensitivity. Furthermore, pork contamination intended for commercial products of sausage, nugget, steak and meat burger can be checked. The procedure is also much cheaper than other methods based on PCR, immunodiffusion and other techniques that need expensive equipment. PMID:25178301

  11. Sperm DNA fragmentation is related to sperm morphological staining patterns.

    PubMed

    Sá, Rosália; Cunha, Mariana; Rocha, Eduardo; Barros, Alberto; Sousa, Mário

    2015-10-01

    In this prospective comparative study, sperm DNA fragmentation (sDNAfrag) was compared at each step of a sequential semen preparation, with semen parameters according to their degree of severity. At each step (fractions) of the sequential procedure, sDNAfrag was determined: fresh (Raw), after gradient centrifugation, washing, and swim-up (SU) for 70 infertile men enrolled in intracytoplasmic sperm injection cycles. sDNAfrag significantly (P = 0.04; P < 0.0001) decreased throughout the steps of semen preparation, with centrifugation and washing not increasing it. A negative correlation to sperm motility was observed in Raw and SU fractions, and a higher sDNAfrag was observed in samples with lower semen quality. Our results confirm that the steps of the sequential procedure do not compromise sperm DNA integrity and progressively decreased sDNAfrag regardless of the sperm abnormality and that semen parameters with lower quality present higher sDNAfrag. Four distinct patterns were observed, of which the entire sperm head staining was the pattern most expressed in all studied fractions. Additionally, the sperm head gene-rich region staining pattern was reduced by the procedure. This suggests that pattern quantification might be a useful adjunct when performing sDNAfrag testing for male infertility. PMID:26278809

  12. qPCR-based mitochondrial DNA quantification: Influence of template DNA fragmentation on accuracy

    SciTech Connect

    Jackson, Christopher B.; Gallati, Sabina; Schaller, Andre

    2012-07-06

    Highlights: Black-Right-Pointing-Pointer Serial qPCR accurately determines fragmentation state of any given DNA sample. Black-Right-Pointing-Pointer Serial qPCR demonstrates different preservation of the nuclear and mitochondrial genome. Black-Right-Pointing-Pointer Serial qPCR provides a diagnostic tool to validate the integrity of bioptic material. Black-Right-Pointing-Pointer Serial qPCR excludes degradation-induced erroneous quantification. -- Abstract: Real-time PCR (qPCR) is the method of choice for quantification of mitochondrial DNA (mtDNA) by relative comparison of a nuclear to a mitochondrial locus. Quantitative abnormal mtDNA content is indicative of mitochondrial disorders and mostly confines in a tissue-specific manner. Thus handling of degradation-prone bioptic material is inevitable. We established a serial qPCR assay based on increasing amplicon size to measure degradation status of any DNA sample. Using this approach we can exclude erroneous mtDNA quantification due to degraded samples (e.g. long post-exicision time, autolytic processus, freeze-thaw cycles) and ensure abnormal DNA content measurements (e.g. depletion) in non-degraded patient material. By preparation of degraded DNA under controlled conditions using sonification and DNaseI digestion we show that erroneous quantification is due to the different preservation qualities of the nuclear and the mitochondrial genome. This disparate degradation of the two genomes results in over- or underestimation of mtDNA copy number in degraded samples. Moreover, as analysis of defined archival tissue would allow to precise the molecular pathomechanism of mitochondrial disorders presenting with abnormal mtDNA content, we compared fresh frozen (FF) with formalin-fixed paraffin-embedded (FFPE) skeletal muscle tissue of the same sample. By extrapolation of measured decay constants for nuclear DNA ({lambda}{sub nDNA}) and mtDNA ({lambda}{sub mtDNA}) we present an approach to possibly correct measurements in degraded samples in the future. To our knowledge this is the first time different degradation impact of the two genomes is demonstrated and which evaluates systematically the impact of DNA degradation on quantification of mtDNA copy number.

  13. Evaluating the Assignment of alkB Terminal Restriction Fragments and Sequence Types to Distinct Bacterial Taxa

    PubMed Central

    Giebler, Julia; Wick, Lukas Y.; Schloter, Michael; Harms, Hauke

    2013-01-01

    Sequence and terminal restriction fragment length polymorphism (T-RFLP) analyses revealed multiple alkB gene copies/cell in soil bacterial isolates and an apparently high genetic mobility among various phylogenetic groups. Identifying alkane degraders by alkB terminal restriction fragments (T-RFs) and sequences is strongly biased, as the phylogenetic trees based on 16S rRNA and alkB gene sequences were highly inconsistent. PMID:23455350

  14. Clusters of DNA induced by ionizing radiation: formation of short DNA fragments. I. Theoretical modeling

    NASA Technical Reports Server (NTRS)

    Holley, W. R.; Chatterjee, A.

    1996-01-01

    We have developed a general theoretical model for the interaction of ionizing radiation with chromatin. Chromatin is modeled as a 30-nm-diameter solenoidal fiber comprised of 20 turns of nucleosomes, 6 nucleosomes per turn. Charged-particle tracks are modeled by partitioning the energy deposition between primary track core, resulting from glancing collisions with 100 eV or less per event, and delta rays due to knock-on collisions involving energy transfers >100 eV. A Monte Carlo simulation incorporates damages due to the following molecular mechanisms: (1) ionization of water molecules leading to the formation of OH, H, eaq, etc.; (2) OH attack on sugar molecules leading to strand breaks: (3) OH attack on bases; (4) direct ionization of the sugar molecules leading to strand breaks; (5) direct ionization of the bases. Our calculations predict significant clustering of damage both locally, over regions up to 40 bp and over regions extending to several kilobase pairs. A characteristic feature of the regional damage predicted by our model is the production of short fragments of DNA associated with multiple nearby strand breaks. The shapes of the spectra of DNA fragment lengths depend on the symmetries or approximate symmetries of the chromatin structure. Such fragments have subsequently been detected experimentally and are reported in an accompanying paper (B. Rydberg, Radiat, Res. 145, 200-209, 1996) after exposure to both high- and low-LET radiation. The overall measured yields agree well quantitatively with the theoretical predictions. Our theoretical results predict the existence of a strong peak at about 85 bp, which represents the revolution period about the nucleosome. Other peaks at multiples of about 1,000 bp correspond to the periodicity of the particular solenoid model of chromatin used in these calculations. Theoretical results in combination with experimental data on fragmentation spectra may help determine the consensus or average structure of the chromatin fibers in mammalian DNA.

  15. 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

  16. Restriction Analysis of PCR-Amplified Internal Transcribed Spacers of Ribosomal DNA as a Tool for Species Identification in Different Genera of the Order Glomales

    PubMed Central

    Redecker, D.; Thierfelder, H.; Walker, C.; Werner, D.

    1997-01-01

    A technique combining PCR and restriction fragment length polymorphism analysis was used to generate specific DNA fragment patterns from spore extracts of arbuscular mycorrhizal fungi. With the universal primers ITS1 and ITS4, DNA fragments were amplified from species of Scutellospora and Gigaspora that were approximately 500 bp long. The apparent lengths of the corresponding fragments from Glomus spp. varied between 580 and 600 bp. Within the genus Glomus, the restriction enzymes MboI, HinfI, and TaqI were useful for distinguishing species. Depending on the restriction enzyme used, groups of species with common fragment patterns could be found. Five tropical and subtropical isolates identified as Glomus manihotis and G. clarum could not be distinguished by their restriction patterns, corresponding to the morphological similarity of the spores. The variation of internal transcribed spacer sequences among the Gigaspora species under study was low. Fragment patterns of Scutellospora spp. showed their phylogenetic relationship with Gigaspora and revealed only a slightly higher degree of variation. PMID:16535592

  17. 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

  18. Comparative utility of restriction fragment length polymorphism analysis and gene sequencing to the molecular epidemiological investigation of a viral outbreak.

    PubMed

    Goldberg, T L; Weigel, R M; Hahn, E C; Scherba, G

    2001-06-01

    Restriction fragment length polymorphism (RFLP) analysis and partial-genome DNA sequencing are commonly used to infer genetic relationships among pathogens. This study compares the application of both techniques to the analysis of 16 pseudorabies virus isolates collected during a 1989 outbreak. Genetic distances derived from RFLP and DNA sequence data were not significantly correlated with geographic distances between farms from which isolates were collected. RFLP-based genetic distance was, however, strongly correlated with temporal distance between isolates (days separating time of isolation). Sequence-based genetic distance was significantly correlated with temporal distance only when synonymous changes (nucleotide changes not leading to amino acid changes) were considered separately. Conversely, non-synonymous changes were correlated with the host species of origin of the viral isolate. These results indicate that selectively-neutral genetic changes most accurately reflect historical relationships, but that non-neutral changes most accurately reflect the biological environment of the viral isolate (e.g. host immune system). PMID:11467798

  19. Genome of a mononucleosis Epstein-Barr virus contains DNA fragments previously regarded to be unique to Burkitt's lymphoma isolates.

    PubMed

    Fischer, D K; Miller, G; Gradoville, L; Heston, L; Westrate, M W; Maris, W; Wright, J; Brandsma, J; Summers, W C

    1981-05-01

    We wished to learn whether the genomes of strains of EMB isolated from patients with infectious mononucleosis are consistently distinguishable from those of strains from Burkitt's lymphoma. The genome of a new transforming strains (FF41) of EBV isolated from saliva of a patient with uncomplicated infectious mononucleosis was compared with the DNA of B95-8, the only other available virus from mononucleosis. It had been found previously that B95-8 has a deletion of about 8 Md in the region of the physical map represented by the Eco RI C, Hind III D, and Bam HI I fragments. The W91 and HR-1 isolates for Burkitt's lymphoma are not deleted in this region and it had been proposed that additional information was characteristic of EBV isolates from Burkitt's lymphoma. By means of restriction enzyme analysis, blot hybridization experiments and molecular cloning of FF41 DNA we demonstrate that the deletion found in B95-8 is not present in the new mononucleosis isolate. The FF41 genome contains an extra 8 Md of DNA, represented by Bam HI fragments B', W' and I', which are located in a larger Eco RI C fragment. Thus the genome of this salivary isolate contains DNA that had previously been regarded to be unique to strains from Burkitt's lymphoma. It is therefore unlikely that major insertions or deletions in the EBV genome account for differences in disease manifestation following EBV infection. PMID:6263500

  20. 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)

  1. Effect of site-specific modification on restriction endonucleases and DNA modification methyltransferases.

    PubMed Central

    McClelland, M; Nelson, M; Raschke, E

    1994-01-01

    Restriction endonucleases have site-specific interactions with DNA that can often be inhibited by site-specific DNA methylation and other site-specific DNA modifications. However, such inhibition cannot generally be predicted. The empirically acquired data on these effects are tabulated for over 320 restriction endonucleases. In addition, a table of known site-specific DNA modification methyltransferases and their specificities is presented along with EMBL database accession numbers for cloned genes. PMID:7937074

  2. Diversity analysis of magnetotactic bacteria in Lake Miyun, northern China, by restriction fragment length polymorphism.

    PubMed

    Lin, Wei; Li, Jinhua; Schüler, Dirk; Jogler, Christian; Pan, Yongxin

    2009-08-01

    Magnetotactic bacteria (MTB) synthesize intracellular nano-scale crystals of magnetite or greigite within magnetosomes. MTB are ubiquitous in limnic and marine environments. In order to understand the diversity of MTB better, sediment samples were examined from Lake Miyun near Beijing by restriction fragment length polymorphism (RFLP). First, in silico analysis was used to evaluate the effectiveness of 12 sets of restriction endonucleases for distinguishing MTB sequences retrieved from the GenBank database. It was found that the tested restriction endonucleases had different power in the ability to differentiate the operational taxonomic units (OTUs) of MTB. Specifically, of the 12 sets of enzymes, MspI plus RsaI was found to be the most effective for correctly differentiating the OTUs of selected MTB sequences and it could detect 16 OTUs with appropriate OTUmin and OTUmax values (96.7% and 97.7%, respectively). The MspI plus RsaI RFLP analysis was then utilized to investigate the diversity of MTB in Lake Miyun sediment and it identified 8 OTUs (74.5% of the whole library) as MTB. Among these, 5 were affiliated to Alphaproteobacteria, while the rest belonged to the Nitrospira phylum. Interestingly, OTUs C, D and I displayed 91.8-98.4% similarity to "Magnetobacterium bavaricum". Together, these results demonstrated that the MspI plus RsaI RFLP analysis was useful for studying the diversity and change in community composition of uncultivated MTB from environmental samples. PMID:19168303

  3. Immune response in mice following immunization with DNA encoding fragment C of tetanus toxin.

    PubMed Central

    Anderson, R; Gao, X M; Papakonstantinopoulou, A; Roberts, M; Dougan, G

    1996-01-01

    Tetanus toxin is a potent neurotoxin synthesized by Clostridium tetani. Immunization with fragment C protein, the nontoxic C-terminal domain of tetanus toxin, will protect mice against lethal challenge with tetanus toxin. A synthetic gene encoding fragment C (tetC) had previously been shown to express high levels of fragment C in Saccharomyces cerevisiae. A plasmid, pcDNA3/tetC, which encodes the synthetic tetC gene expressed under the control of the human cytomegalovirus major intermediate-early promoter/enhancer region, was constructed. Expression of fragment C was observed in eukaryotic cells growing in vitro following transfection with pcDNA3/tetC. The immune response induced by intramuscular immunization with pure pcDNA3/tetC DNA was evaluated in a murine model. Anti-fragment C serum immunoglobulin and proliferative responses in splenocytes were observed in BALB/c mice following two immunizations with pcDNA3/tetC. The major immunoglobulin G subclass that recognized fragment C was immunoglobulin G2a, and the stimulated splenocytes secreted high levels of gamma interferon. Immunity to tetanus is dependent on the presence of neutralizing serum antibodies against tetanus toxin. Sufficient anti-fragment C serum immunoglobulins were induced by DNA-mediated immunization to protect mice against lethal challenge with tetanus toxin. PMID:8757849

  4. Effect of cryopreservation on the sperm DNA fragmentation dynamics of the bottlenose dolphin (Tursiops truncatus).

    PubMed

    Sánchez-Calabuig, M J; López-Fernández, C; Johnston, S D; Blyde, D; Cooper, J; Harrison, K; de la Fuente, J; Gosálvez, J

    2015-04-01

    Sperm DNA fragmentation is one of the major causes of infertility; the sperm chromatin dispersion test (SCDt) evaluates this parameter and offers the advantage of species-specific validated protocol and ease of use under field conditions. The main purpose of this study was to evaluate sperm DNA fragmentation dynamics in both fresh and post-thaw bottlenose dolphin sperm using the SCDt following different cryopreservation protocols to gain new information about the post-thaw differential sperm DNA longevity in this species. Fresh and cryopreserved semen samples from five bottlenose dolphins were examined for sperm DNA fragmentation dynamics using the SCDt (Halomax(®)). Sperm DNA fragmentation was assessed immediately at collection and following cryopreservation (T0) and then after 0.5, 1, 4, 8, 24, 48 and 72 h incubation at 37°C. Serially collected ejaculates from four dolphins were frozen using different cryopreservation protocols in a TES-TRIS-fructose buffer (TTF), an egg-yolk-free vegetable lipid LP1 buffer (LP1) and human sperm preservation medium (HSPM). Fresh ejaculated spermatozoa initially showed low levels of DNA fragmentation for up to 48 h. Lower Sperm DNA fragmentation (SDF) was found in the second fresh ejaculate compared to the first when more than one sample was collected on the same day (p < 0.05); this difference was not apparent in any other seminal characteristic. While there was no difference observed in SDF between fresh and frozen-thawed sperm using the different cryopreservation protocols immediately after thawing (T0), frozen-thawed spermatozoa incubated at 37°C showed an increase in the rate of SDF after 24 h. Sperm frozen in the LP1(?) buffer had higher levels (p < 0.05) of DNA fragmentation after 24- and 48-h incubation than those frozen in TTF or HSPM. No correlation was found between any seminal characteristic and DNA fragmentation in either fresh and/or frozen-thawed samples. PMID:25604784

  5. Non-random fragmentation patterns in circulating cell-free DNA reflect epigenetic regulation

    PubMed Central

    2015-01-01

    Background The assessment of cell-free circulating DNA fragments, also known as a "liquid biopsy" of the patient's plasma, is an important source for the discovery and subsequent non-invasive monitoring of cancer and other pathological conditions. Although the nucleosome-guided fragmentation patterns of cell-free DNA (cfDNA) have not yet been studied in detail, non-random representation of cfDNA sequencies may reflect chromatin features in the tissue of origin at gene-regulation level. Results In this study, we investigated the association between epigenetic landscapes of human tissues evident in the patterns of cfDNA in plasma by deep sequencing of human cfDNA samples. We have demonstrated that baseline characteristics of cfDNA fragmentation pattern are in concordance with the ones corresponding to cell lines-derived. To identify the loci differentially represented in cfDNA fragment, we mapped the transcription start sites within the sequenced cfDNA fragments and tested for association of these genomic coordinates with the relative strength and the patterns of gene expressions. Preselected sets of house-keeping and tissue specific genes were used as models for actively expressed and silenced genes. Developed measure of gene regulation was able to differentiate these two sets based on sequencing coverage near gene transcription start site. Conclusion Experimental outcomes suggest that cfDNA retains characteristics previously noted in genome-wide analysis of chromatin structure, in particular, in MNase-seq assays. Thus far the analysis of the DNA fragmentation pattern may aid further developing of cfDNA based biomarkers for a variety of human conditions. PMID:26693644

  6. DNA Fragmentation Simulation Method (FSM) and Fragment Size Matching Improve aCGH Performance of FFPE Tissues

    PubMed Central

    Craig, Justin M.; Vena, Natalie; Ramkissoon, Shakti; Idbaih, Ahmed; Fouse, Shaun D.; Ozek, Memet; Sav, Aydin; Hill, D. Ashley; Margraf, Linda R.; Eberhart, Charles G.; Kieran, Mark W.; Norden, Andrew D.; Wen, Patrick Y.; Loda, Massimo; Santagata, Sandro; Ligon, Keith L.; Ligon, Azra H.

    2012-01-01

    Whole-genome copy number analysis platforms, such as array comparative genomic hybridization (aCGH) and single nucleotide polymorphism (SNP) arrays, are transformative research discovery tools. In cancer, the identification of genomic aberrations with these approaches has generated important diagnostic and prognostic markers, and critical therapeutic targets. While robust for basic research studies, reliable whole-genome copy number analysis has been unsuccessful in routine clinical practice due to a number of technical limitations. Most important, aCGH results have been suboptimal because of the poor integrity of DNA derived from formalin-fixed paraffin-embedded (FFPE) tissues. Using self-hybridizations of a single DNA sample we observed that aCGH performance is significantly improved by accurate DNA size determination and the matching of test and reference DNA samples so that both possess similar fragment sizes. Based on this observation, we developed a novel DNA fragmentation simulation method (FSM) that allows customized tailoring of the fragment sizes of test and reference samples, thereby lowering array failure rates. To validate our methods, we combined FSM with Universal Linkage System (ULS) labeling to study a cohort of 200 tumor samples using Agilent 1 M feature arrays. Results from FFPE samples were equivalent to results from fresh samples and those available through the glioblastoma Cancer Genome Atlas (TCGA). This study demonstrates that rigorous control of DNA fragment size improves aCGH performance. This methodological advance will permit the routine analysis of FFPE tumor samples for clinical trials and in daily clinical practice. PMID:22719973

  7. [The role of duplex ends in the spontaneous interaction of homologous linear DNA fragments].

    PubMed

    Markina, V K; Danilova, O A; Neschastnova, A A; Belitski?, G A; Iakubovskaia, M G

    2002-01-01

    The spontaneous interaction of homologous linear DNA fragments was studied with a model of purified PCR products by agarose gel electrophoresis. To interact, duplexes required not only homology of internal regions, but also complementary ends. Fragments differing in terminal sequences did not interact. The yield of Holliday junctions (HJ), the simplest product of DNA-DNA interaction, depended on dissociation of fragment ends. Compared with genomic fragments, those with low-melting AT ends interacted with each other more efficiently and those with high-melting GC ends, less efficiently. Incubation temperature affected the equilibrium HJ concentration in solution of homologous fragments. A conclusion was made that HJ formation is initiated by nucleation of dissociated duplex ends. PMID:12391850

  8. Fork rotation and DNA precatenation are restricted during DNA replication to prevent chromosomal instability

    PubMed Central

    Schalbetter, Stephanie A.; Mansoubi, Sahar; Chambers, Anna L.; Downs, Jessica A.; Baxter, Jonathan

    2015-01-01

    Faithful genome duplication and inheritance require the complete resolution of all intertwines within the parental DNA duplex. This is achieved by topoisomerase action ahead of the replication fork or by fork rotation and subsequent resolution of the DNA precatenation formed. Although fork rotation predominates at replication termination, in vitro studies have suggested that it also occurs frequently during elongation. However, the factors that influence fork rotation and how rotation and precatenation may influence other replication-associated processes are unknown. Here we analyze the causes and consequences of fork rotation in budding yeast. We find that fork rotation and precatenation preferentially occur in contexts that inhibit topoisomerase action ahead of the fork, including stable protein–DNA fragile sites and termination. However, generally, fork rotation and precatenation are actively inhibited by Timeless/Tof1 and Tipin/Csm3. In the absence of Tof1/Timeless, excessive fork rotation and precatenation cause extensive DNA damage following DNA replication. With Tof1, damage related to precatenation is focused on the fragile protein–DNA sites where fork rotation is induced. We conclude that although fork rotation and precatenation facilitate unwinding in hard-to-replicate contexts, they intrinsically disrupt normal chromosome duplication and are therefore restricted by Timeless/Tipin. PMID:26240319

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

    PubMed

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

    1989-09-01

    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. PMID:2802599

  10. Environmental toxicants cause sperm DNA fragmentation as detected by the Sperm Chromatin Structure Assay (SCSA).

    PubMed

    Evenson, Donald P; Wixon, Regina

    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 approximately 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 approximately 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. PMID:15987647

  11. Sperm nuclear DNA fragmentation and its association with semen quality in Greek men.

    PubMed

    Evgeni, E; Lymberopoulos, G; Touloupidis, S; Asimakopoulos, B

    2015-12-01

    Due to the limitations of conventional semen analysis in predicting a man's fertility potential, sperm DNA fragmentation was recently introduced as a novel marker of sperm quality. This prospective study was undertaken to investigate the associations between conventional seminal parameters and DNA fragmentation in Greek men. A total of 669 subject data were evaluated in two groups, normozoospermic (n = 184) and non-normozoospermic (n = 485), according to the WHO 2010 (WHO Laboratory Manual for the Examination and Processing of Human Semen, 5th edn. World Health Organization), reference limits. For all the subjects, semen volume, sperm concentration, total count, rapid and total progressive motility and morphology were recorded following the WHO 2010 methods and DNA fragmentation was assessed by the sperm chromatin dispersion assay. An inverse correlation was established between DNA fragmentation and all conventional seminal parameters except semen volume in men with seminal profiles below the reference limits, with statistical significance for rapid and total progressive motility. Normozoospermic men exhibited lower levels of DNA fragmentation than their non-normozoospermic counterparts, even though the values were not always below 30%. DNA fragmentation testing and traditional semen analysis should therefore be considered as complementary diagnostic tools in a comprehensive evaluation of male infertility. PMID:25586471

  12. Investigations of Bacterial Inactivation and DNA Fragmentation Induced by Flowing Humid Argon Post-discharge

    NASA Astrophysics Data System (ADS)

    Odic, Emmanuel; Limam, S.; Kirkpatrick, M. J.; Dodet, B.; Salamitou, S.; DuBow, M. S.

    Bio-contaminated surfaces were exposed to an atmospheric pressure flowing post-discharge, i.e. without direct contact of the plasma with the surface. The non-thermal plasma source was a dielectric barrier discharge. Using humid argon as a feed gas, a reduction of six orders of magnitude of survivors could be obtained for Escherichia coli. An investigation of bacterial inactivation mechanisms during the plasma induced treatment was conducted. For this purpose, DNA (plasmid and genomic DNA in aqueous solution) degradation by the plasma process was studied, assuming that the bacterial inactivation is obtained when the bacterial DNA is fragmented. According to the operating conditions (feed gas, reactor geometry and discharge input power), DNA fragmentation was evaluated in correlation with aqueous phase hydrogen peroxide concentration measurements. It appears that hydrogen peroxide is not the only factor responsible for DNA fragmentation and that short-lived species produced by water dissociation are major contributors.

  13. Genetic Diversity among Rhizobium leguminosarum bv. Trifolii Strains Revealed by Allozyme and Restriction Fragment Length Polymorphism Analyses

    PubMed Central

    Demezas, David H.; Reardon, Terry B.; Watson, John M.; Gibson, Alan H.

    1991-01-01

    Allozyme electrophoresis and restriction fragment length polymorphism (RFLP) analyses were used to examine the genetic diversity of a collection of 18 Rhizobium leguminosarum bv. trifolii, 1 R. leguminosarum bv. viciae, and 2 R. meliloti strains. Allozyme analysis at 28 loci revealed 16 electrophoretic types. The mean genetic distance between electrophoretic types of R. leguminosarum and R. meliloti was 0.83. Within R. leguminosarum, the single strain of bv. viciae differed at an average of 0.65 from strains of bv. trifolii, while electrophoretic types of bv. trifolii differed at a range of 0.23 to 0.62. Analysis of RFLPs around two chromosomal DNA probes also delineated 16 unique RFLP patterns and yielded genetic diversity similar to that revealed by the allozyme data. Analysis of RFLPs around three Sym (symbiotic) plasmid-derived probes demonstrated that the Sym plasmids reflect genetic divergence similar to that of their bacterial hosts. The large genetic distances between many strains precluded reliable estimates of their genetic relationships. PMID:16348600

  14. Partial digestion with restriction enzymes of ultraviolet-irradiated human genomic DNA: a method for identifying restriction site polymorphisms

    SciTech Connect

    Nobile, C.; Romeo, G.

    1988-10-01

    A method for partial digestion of total human DNA with restriction enzymes has been developed on the basis of a principle already utilized by P.A. Whittaker and E. Southern for the analysis of phage lambda recombinants. Total human DNA irradiated with uv light of 254 nm is partially digested by restriction enzymes that recognize sequences containing adjacent thymidines because of TT dimer formation. The products resulting from partial digestion of specific genomic regions are detected in Southern blots by genomic-unique DNA probes with high reproducibility. This procedure is rapid and simple to perform because the same conditions of uv irradiation are used for different enzymes and probes. It is shown that restriction site polymorphisms occurring in the genomic regions analyzed are recognized by the allelic partial digest patterns they determine.

  15. Melting profiles may affect detection of residual HPV L1 gene DNA fragments in Gardasil®.

    PubMed

    Lee, Sin Hang

    2014-03-01

    Gardasil® is a quadrivalent human papillomavirus (HPV) protein-based vaccine containing genotype-specific L1 capsid proteins of HPV-16, HPV-18, HPV-6 and HPV-11 in the form of virus-like-particles (VLPs) as the active ingredient. The VLPs are produced by a DNA recombinant technology. It is uncertain if the residual HPV L1 gene DNA fragments in the vaccine products are considered contaminants or excipients of the Gardasil® vaccine. Because naked viral DNA fragments, if present in the vaccine, may bind to the insoluble amorphous aluminum hydroxyphosphate sulfate (AAHS) adjuvant which may help deliver the foreign DNA into macrophages, causing unintended pathophysiologic effects, experiments were undertaken to develop tests for HPV L1 gene DNA fragments in the final products of Gardasil® by polymerase chain reaction (PCR) and direct DNA sequencing. The results showed that while the HPV-11 and HPV-18 L1 gene DNA fragments in Gardasil® were readily amplified by the common GP6/MY11 degenerate consensus primers, the HPV-16 L1 gene DNA may need specially designed non-degenerate PCR primers for amplification at different regions of the L1 gene and different stringency conditions for detection. These variable melting profiles of HPV DNA in the insoluble fraction of the Gardasil® vaccine suggest that the HPV DNA fragments are firmly bound to the aluminum AAHS adjuvant. All methods developed for detecting residual HPV DNA in the vaccine Gardasil® for quality assurance must take into consideration the variable melting profiles of the DNA to avoid false negative results. PMID:24083601

  16. 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...

  17. 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

  18. Chromatin isolated from viable human PBLs contains DNA fragmented to >/=50 kb.

    PubMed

    Szabó, G; Bacsó, Z

    1996-04-01

    Massive chromatin fragmentation (around 50 kb, to several hundred kb) is observed in nuclear lysates of human peripheral blood lymphocytes (PBLs) upon their treatment with nuclease-free protein-denaturants. There is a consistent variation in the fragment size distributions that parallels the proliferative activity of the cells. Predominantly approximately 50 kb fragmentation is exhibited in samples from cells immunsuppressed via CD4 crosslinking, as opposed to the heterogeneous, higher molecular weight DNA of anti-TcR/CD3-, phytohemagglutininor concanavalin A-stimulated cells. Tritiated thymidine incorporated into DNA in the latter cultures can be detected in the approximately 50 kb band. Direct lysis of agarose-embedded, live cells in alkali+detergent also yields fragmented DNA, with a single-strand size of >/=50 kb. These data suggest that (i) the cells yielding fragmented DNA were alive at the time of DNA extraction, (ii) either regularly arranged, preformed nicks or hypersensitive sites may be present at every roughly 50-100 kb in the chromatin of PBLs, (iii) these sites or the fragmentation mechanism acting upon them, appear to be regulated in concert with the transit of cells between the resting and proliferative compartments. PMID:17180088

  19. Characterization of DNA size determination of small fragments by flow cytometry

    SciTech Connect

    Petty, J.T.; Johnson, M.E.; Goodwin, P.M.; Martin, J.C.; Jett, J.H.; Keller, R.A.

    1995-05-15

    DNA fragment lengths were determined using the intensity of fluorescent bursts from single fragments stained with a thiazole orange derivative. The individual stained fragments were introduced into a sheath flow cuvette and passed through a low-power (30 mW), continuous-wave laser beam with transit times in the range 3-5 ms. As little as 50 fg of DNA was analyzed at a rate of 40 fragments/s for times ranging from 1 to 15 min. A detectable lower size limit of 1.5 kilobase pairs (kbp) was demonstrated, and a linear relationship between fluorescence intensity and fragment length was observed. Issues relating to size resolution in the 2-50 kbp range are discussed. 25 refs., 7 figs., 1 tab.

  20. Restriction fragment length polymorphism within the class I gene loci of the equine major histocompatibility complex

    SciTech Connect

    Alexander, A.J.; Bailey, E.; Woodward, J.G.

    1986-03-05

    Fourteen standard bred horses were serotyped as homozygous for 1 of 6 Equine Leukocyte Antigen (ELA) specificities. DNA was purified from peripheral leukocytes and digested with Hind III or Pvu II. Southern blot hybridization analysis was carried out using a /sup 32/P-labeled mouse cDNA probe (PH2IIa) specific for class I MHC genes. Both enzymes generated blots that contained a large number of bands (23 to 30) per horse. Significant polymorphism existed among most fragment sizes, while a dozen highly conserved band sizes suggested the presence of Qa/tla - like genes. Only 2 animals (both W6's) showed identical band patterns. Polymorphism was greatest between horses of different serotypes and was significantly decreased within serotypes. Unique bands were present on both blots for both W1's and W6's and may account for the serologic specificity seen in ELA W1 and W6 horses. This study is consistent with the findings in other higher vertebrates and implies that the MHC of the horse includes a highly polymorphic class I multigene family.

  1. 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

  2. [THE OPTIMAL CONDITIONS OF STORAGE OF SPERMATOZOA FOR ANALYSIS OF DNA FRAGMENTATION].

    PubMed

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

    2015-04-01

    The analysis of fragmentation of DNA of spermatozoons using technique of flow cytometry to evaluate male fertility more and more often begins to be applied in clinical diagnostic. However, development of optimal protocol of storage and preparation of spermatozoons for analysis still is at the stage of experimental elaboration. The studv was carried out to analyse effect of different conditions of preparation of ejaculate for adequate evaluation of index of fragmentation of DNA of spermatozoons using sperm chromatin structure assay technique. The sampling consisted of 20 patients of the Krasnoyarsk center of reproductive medicine. The sperm chromatin structure assay technique was applied to evaluate index of fragmentation of DNA of spermatozoons in fresh native ejaculate and after storage of spermatozoons under different temperature (37, 25 and 4 degrees C) and duration (1-2 and 1-3 days) and conditions of storage (-20 or -70 degrees C) of frozen spermatozoons (as native ejaculate or in TNE-buffer). It is demonstrated that index of fragmentation of DNA of spermatozoons has no significant alterations in ejaculate stored under 4 degrees C during 48 hours. In case of storage of ejaculate under 25 or 37 degrees C index of fragmentation of DNA of spermatozoons significantly increases already after first day of storage. The incubation of ejaculate under 37 degrees C results in increasing of index of fragmentation of DNA of spermatozoons already after first hour. The individual differences are established related to degree of increasing of index of fragmentation of DNA of spermatozoons because of impact of studied temperatures of ejaculate incubation. The storage of spermatozoons under temperature of - 20 and -70 degrees C in native ejaculate or in TNE-buffer has no effect of index of fragmentation of DNA of spermatozoons with measurement during 1-2 hours. Therefore, storage and transportation of native ejaculate under 4 degrees C during 1-2 days or in frozen condition under temperature of -20 degrees C or -70 degrees C can be recommended for adequate evaluation of level of fragmentation of DNA of spermatozoons. PMID:26189292

  3. A New Large-DNA-Fragment Delivery System Based on Integrase Activity from an Integrative and Conjugative Element

    PubMed Central

    van der Meer, Jan Roelof

    2013-01-01

    During the past few decades, numerous plasmid vectors have been developed for cloning, gene expression analysis, and genetic engineering. Cloning procedures typically rely on PCR amplification, DNA fragment restriction digestion, recovery, and ligation, but increasingly, procedures are being developed to assemble large synthetic DNAs. In this study, we developed a new gene delivery system using the integrase activity of an integrative and conjugative element (ICE). The advantage of the integrase-based delivery is that it can stably introduce a large DNA fragment (at least 75 kb) into one or more specific sites (the gene for glycine-accepting tRNA) on a target chromosome. Integrase recombination activity in Escherichia coli is kept low by using a synthetic hybrid promoter, which, however, is unleashed in the final target host, forcing the integration of the construct. Upon integration, the system is again silenced. Two variants with different genetic features were produced, one in the form of a cloning vector in E. coli and the other as a mini-transposable element by which large DNA constructs assembled in E. coli can be tagged with the integrase gene. We confirmed that the system could successfully introduce cosmid and bacterial artificial chromosome (BAC) DNAs from E. coli into the chromosome of Pseudomonas putida in a site-specific manner. The integrase delivery system works in concert with existing vector systems and could thus be a powerful tool for synthetic constructions of new metabolic pathways in a variety of host bacteria. PMID:23686268

  4. Relationship of spermatozoal DNA fragmentation with semen quality in varicocele-positive men.

    PubMed

    Moazzam, A; Sharma, R; Agarwal, A

    2015-10-01

    The aim of the study was to assess the semen quality and levels of spermatozoal nuclear DNA fragmentation in subfertile subjects clinically diagnosed with varicocele, subfertile subjects without varicocele and healthy fertile controls. Semen samples were obtained from 302 subjects. Of them, 115 were healthy fertile controls having normal semen characteristics, 121 subfertile men diagnosed with varicocele, both, clinically and on ultrasonography, while 66 subjects were subfertile with no varicocele. Spermatozoal concentration, percentage motility, morphology and DNA fragmentation were measured. In the study population, deterioration in semen quality-decreased spermatozoal concentration, percentage motility and normal morphology was seen in subfertile subjects, especially with varicocele. Highest spermatozoal DNA fragmentation was observed in varicocele-positive subjects as compared with varicocele-negative subjects and healthy fertile controls. Significant negative correlation was seen between spermatozoal DNA fragmentation and concentration (r = -0.310), motility (r = -0.328) normal morphology, WHO method (r = -0.221) and Tygerberg strict criteria (r = -0.180) in the varicocele-positive subfertile subjects. In conclusion, this study suggests existence of a negative relationship between spermatozoal DNA fragmentation and semen quality in varicocele-positive subfertile subjects. PMID:25346327

  5. Apoptotic DNA fragmentation can be revealed in situ: an ultrastructural approach.

    PubMed

    Burattini, Sabrina; Ferri, Paola; Battistelli, Michela; D'Emilio, Alessandra; Biagiotti, Laura; Sestili, Piero; Rocchi, Marco B L; Falcieri, Elisabetta

    2009-12-01

    A common pattern of apoptotic death is DNA cleavage, initially producing large fragments (50 kbp), followed by the production of nucleosomic/oligonucleosomic fragments. Nevertheless, apoptosis without DNA fragmentation, at least of the nucleosomic type, has been reported. To investigate the spatial relationship between DNA cleavage and chromatin condensation, we applied the TUNEL technique to the ultrastructural analysis of apoptotic cells. A modified method, utilizing a gold-conjugated antidigoxigenin antibody, was carried out on U937 versus Molt-4 cells, both exposed to UVB radiation or staurosporine treatment. Gold particle density in the different domains of apoptotic cells was evaluated by a four-way ANOVA test. Gold labelling was more strongly localised in condensed chromatin than in the diffuse chromatin. U937 cells, which evidenced in vitro oligonucleosomic fragmentation after both UVB and staurosporine treatments, revealed a significantly higher gold particle density, when compared with Molt-4, which did not show, on the other hand, oligonucleosomic cleavage even in the presence of < or = 50 kbp cleavage. Thus, a correlation between DNA fragment sizes and gold particle density appears. TUNEL applied to electron microscopy is an effective approach to study the relationship between apoptotic chromatin condensation and DNA cleavage. Both these events indeed appear in the apoptotic nucleus, but their reciprocal correlation is still greatly unknown. Microsc. Res. Tech. 2009. (c) 2009 Wiley-Liss, Inc. PMID:19484747

  6. Optimizing Restriction Site Placement for Synthetic Genomes

    E-print Network

    Lonardi, Stefano

    in microbiology. · Uses restriction enzymes to cut plasmids for insertion/removal of DNA fragments. · To use a restriction enzyme, the place where it cuts must be unique. · Unique restriction sites regularly distributedOptimizing Restriction Site Placement for Synthetic Genomes Pablo Montes1 Heraldo Memelli1 Charles

  7. Amplified Fragment Length Polymorphism (AFLP) Protocol Introduction taken from

    E-print Network

    Steury, Todd D.

    Amplified Fragment Length Polymorphism (AFLP) Protocol Introduction taken from http://biologi.uio containing high densities of DNA marker loci. The AFLP protocol amplifies restriction fragments obtained and adapter sequence. However, not all restriction fragments are amplified because AFLP primers also contain

  8. Length and restriction site heteroplasmy in the mitochondrial DNA of american shad (alosa sapidissima).

    PubMed

    Bentzen, P; Leggett, W C; Brown, G G

    1988-03-01

    Restriction endonuclease analysis was used to assess mitochondrial DNA (mtDNA) variation in American shad (Alosa sapidissima) collected from 14 rivers ranging from Florida to Quebec. Two types of heteroplasmy were observed, one involving a major length polymorphism and the other a single restriction site. Shad mtDNA occurred in two principal size classes, 18.3 and 19.8 kb. Of 244 shad examined, 30 were heteroplasmic and carried both size classes of mtDNA in varying proportions; the remainder were homoplasmic for the smaller size class of mtDNA. The large mtDNA variant occurred most frequently at the southern end of the range, and except for two individuals from Nova Scotia, was not detected among shad from rivers north of the Delaware. In contrast, ten shad heteroplasmic for a SalI restriction site originated from rivers ranging from South Carolina to Nova Scotia. DNA mapping and hybridization experiments indicated that the length polymorphism is in the D-loop-containing region and consists of a tandemly repeated 1.5-kb DNA sequence occurring in two and three copies, respectively, in the two major size classes of shad mtDNA. Continuous length variation up to approximately 40 bp occurs among copies of the repeat both within and among individuals. Restriction site data support the conclusion that both forms of heteroplasmy in shad mtDNA have originated more than once. PMID:17246419

  9. 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

  10. Fragmentation of DNA in a sub-microliter microfluidic sonication device.

    PubMed

    Tseng, Qingzong; Lomonosov, Alexey M; Furlong, Eileen E M; Merten, Christoph A

    2012-11-21

    Fragmentation of DNA is an essential step for many biological applications including the preparation of next-generation sequencing (NGS) libraries. As sequencing technologies push the limits towards single cell and single molecule resolution, it is of great interest to reduce the scale of this upstream fragmentation step. Here we describe a miniaturized DNA shearing device capable of processing sub-microliter samples based on acoustic shearing within a microfluidic chip. A strong acoustic field was generated by a Langevin-type piezo transducer and coupled into the microfluidic channel via the flexural lamb wave mode. Purified genomic DNA, as well as covalently cross-linked chromatin were sheared into various fragment sizes ranging from ?180 bp to 4 kb. With the use of standard PDMS soft lithography, our approach should facilitate the integration of additional microfluidic modules and ultimately allow miniaturized NGS workflows. PMID:23014736

  11. Comprehensive Study On The Metastable Negative Ion Fragmentation Of Individual Dna Components And Larger Oligonucleotides

    NASA Astrophysics Data System (ADS)

    Ingolfsson, O.; Flosadottir, H. D.; Omarsson, B.; Ilko, B.

    2010-07-01

    Here we present a systematic study on the unimolecular decay pathways of the deprotonated building blocks of DNA and RNA to address the following questions: 1. Are the negative ion fragmentation patterns observed in the metastable decay of individual DNA components still evident when these are combined to larger oligonucleotides? 2. What is the significance of the charge location in determining the fragmentation pathways in the metastable decay process? 3. Are those metastable decay channels relevant in dissociative electron attachment to DNA components? To address these questions we have studied the fragmentation patterns of the deprotonated ribose and ribose 5'-monophosphate, the fragmentation patterns of the individual bases, all nucleosides and all 2'-deoxynucleosides as well as the individual nucleotides and several combinations of hexameric oligonucleotides. Furthermore, to understand the significance of the charge location in determining the fragmentation path in the metastable decay process of these deprotonated ions we have also studied modified uridine and guanosine. These have been modified to block different deprotonation sites and thus to control the initial step in the in the fragmentation process i.e. the site of deprotonation. In addition to our experimental approach we have also simulated the metastable fragmentation of the deprotonated uridine and 2'-deoxyguanosine to clarify the mechanisms and fragmentation patterns observed. Where data is available, the results are compared to dissociative electron attachment to DNA components and discussed in context to the underlying mechanism. Experiments on modified nucleosides where selected deprotonation sites have been blocked are used to verify the predicted reaction paths and imulations on uridine and 2'-deoxyguanosine are compared to the experimental results and used to shed light on the mechanisms involved.

  12. The PML gene is linked to a megabase-scale insertion/deletion restriction fragment length polymorphism

    SciTech Connect

    Goy, A.; Xiao, Y.H.; Passalaris, T.

    1995-03-20

    The PML gene located on chromosome band 15q22 is involved with the RAR{alpha} locus (17q21) in a balanced reciprocal translocation uniquely observed in acute promyelocytic leukemia. Physical mapping studies by pulsed-field gel electrophoresis revealed that the PML gene is flanked by two CpG islands that are separated by a variable distance in normal individuals. Several lines of evidence demonstrate that this is the consequence of a large insertion/deletion polymorphism linked to the PML locus: (1) overlapping fragments obtained with a variety of rare-cutting restriction enzymes demonstrated the same variability in distance between the flanking CpG islands; (2) mapping with restriction enzymes insensitive to CpG methylation confirmed that the findings were not a consequence of variable methylation of CpG dinucleotides; (3) the polymorphism followed a Mendelian inheritance pattern. This polymorphism is localized 3{prime} to the PML locus. There are five common alleles, described on the basis of BssHII fragments, ranging from 220 to 350 kb with increments of approximately 30 kb between alleles. Both heterozygous (61%) and homozygous (391%) patterns were observed in normal individuals. Mega-base-scale insertion/deletion restriction fragment length polymorphisms are very rare and have been described initially in the context of multigene families. Such structures have been also reported as likely regions of genetic instability. High-resolution restriction mapping of this particular structure linked to the PML locus is underway. 47 refs., 4 figs., 1 tab.

  13. Use of Restriction Fragment Length Polymorphism to Rapidly Identify Dermatophyte Species Related to Dermatophytosis

    PubMed Central

    Mohammadi, Rasoul; Abastabar, Mahdi; Mirhendi, Hossein; Badali, Hamid; Shadzi, Shahla; Chadeganipour, Mustafa; Pourfathi, Parinaz; Jalalizand, Niloufar; Haghani, Iman

    2015-01-01

    Background: Dermatophytes are a group of keratinophilic fungi worldwide, which can infect the skin, hair and nails of humans and animals. This genus includes several species that present different features of dermatophytosis. Although, laboratory diagnosis of dermatophytes is based on direct microscopy, biochemical tests and culture, these manners are expensive, time consuming and need skilled staff. Therefore, molecular methods like PCR-RFLP are the beneficial tools for identification, which are rapid and sensitive. Thus, dermatophyte species are able to generate characteristic band patterns on agarose gel electrophoresis using PCR-RFLP technique, which leads to successful identification at the species level within a 5-hour period. Objectives: The purpose of this study was to study inter- and intraspecific genomic variations for identification of clinically important dermatophyte species obtained from clinical specimens in Isfahan, Iran using PCR-RFLP. Materials and Methods: From March 2011 to August 2012, 135 clinical isolates were collected from infected patients at Isfahan, Iran. ITS1-5.8S-ITS2 region of rDNA was amplified using universal fungal primers. Subsequently, amplified products were digested by the MvaI restriction enzyme. Using discriminating band profiles on agarose gel, dermatophyte species were identified. However, DNA sequencing was used for unidentifiable strains. Results: The specimens were obtained from skin scrapings (70.3%), nail (24.4%) and hair (5.1%) clippings. Most patients were between 21 - 30 years and the ratio of male to female was 93/42. Trichophyton interdigitale was the commonest isolate (52.5%) in our findings, followed by Epidermophyton floccosum (24.4%), T. rubrum (16.2%), Microsporum canis (2.2%), T. erinacei (1.4%), T. violaceum (1.4%), T. tonsurans (0.7%) and M. gypseum (0.7%) based on PCR-RFLP. Conclusions: Combination of traditional methods and molecular techniques considerably improves identification of dermatophytes in the species level in clinical laboratories, which can lead to properly antifungal therapy and successful management of infections. However, restriction and specificity and sensitivity should be lowered and increased, respectively, to be useful for a wide variety of clinical applications. PMID:26301058

  14. Terminal strand-switching of E. coli RNA polymerase transcribing a truncated DNA fragment.

    PubMed

    Oostra, B A; Arnberg, A C; Ab, G; Gruber, M

    1981-10-27

    When transcribing a restriction fragment containing the promoters and the first part of the rrnE operon of Escherichia coli, RNA polymerase holoenzyme starts exclusively on the promoters. Besides run-off transcripts, molecules longer than template-size are formed by terminal strand switch. PMID:6269631

  15. Detection and Identification of Decay Fungi in Spruce Wood by Restriction Fragment Length Polymorphism Analysis of Amplified Genes Encoding rRNA†

    PubMed Central

    Jasalavich, Claudia A.; Ostrofsky, Andrea; Jellison, Jody

    2000-01-01

    We have developed a DNA-based assay to reliably detect brown rot and white rot fungi in wood at different stages of decay. DNA, isolated by a series of CTAB (cetyltrimethylammonium bromide) and organic extractions, was amplified by the PCR using published universal primers and basidiomycete-specific primers derived from ribosomal DNA sequences. We surveyed 14 species of wood-decaying basidiomycetes (brown-rot and white-rot fungi), as well as 25 species of wood-inhabiting ascomycetes (pathogens, endophytes, and saprophytes). DNA was isolated from pure cultures of these fungi and also from spruce wood blocks colonized by individual isolates of wood decay basidiomycetes or wood-inhabiting ascomycetes. The primer pair ITS1-F (specific for higher fungi) and ITS4 (universal primer) amplified the internal transcribed spacer region from both ascomycetes and basidiomycetes from both pure culture and wood, as expected. The primer pair ITS1-F (specific for higher fungi) and ITS4-B (specific for basidiomycetes) was shown to reliably detect the presence of wood decay basidiomycetes in both pure culture and wood; ascomycetes were not detected by this primer pair. We detected the presence of decay fungi in wood by PCR before measurable weight loss had occurred to the wood. Basidiomycetes were identified to the species level by restriction fragment length polymorphisms of the internal transcribed spacer region. PMID:11055916

  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. Effect of transition metals on binding of p53 protein to supercoiled DNA and to consensus sequence in DNA fragments.

    PubMed

    Palecek, E; Brázdová, M; Cernocká, H; Vlk, D; Brázda, V; Vojtesek, B

    1999-06-17

    Recently we have shown that wild-type human p53 protein binds preferentially to supercoiled (sc) DNA in vitro in both the presence and absence of the p53 consensus sequence (p53CON). This binding produces a ladder of retarded bands on an agarose gel. Using immunoblotting with the antibody DO-1, we show that the bands obtained correspond to ethidium-stained DNA, suggesting that each band of the ladder contains a DNA-p53 complex. The intensity and the number of these hands are decreased by physiological concentrations of zinc ions. At higher zinc concentrations, binding of p53 to scDNA is completely inhibited. The binding of additional zinc ions to p53 appears much weaker than the binding of the intrinsic zinc ion in the DNA binding site of the core domain. In contrast to previously published data suggesting that 100 microM zinc ions do not influence p53 binding to p53CON in a DNA oligonucleotide, we show that 5-20 microM zinc efficiently inhibits binding of p53 to p53CON in DNA fragments. We also show that relatively low concentrations of dithiothreitol but not of 2-mercaptoethanol decrease the concentration of free zinc ions, thereby preventing their inhibitory effect on binding of p53 to DNA. Nickel and cobalt ions inhibit binding of p53 to scDNA and to its consensus sequence in linear DNA fragments less efficiently than zinc; cobalt ions are least efficient, requiring >100 microM Co2+ for full inhibition of p53 binding. Modulation of binding of p53 to DNA by physiological concentrations of zinc might represent a novel pathway that regulates p53 activity in vivo. PMID:10380883

  18. Clinical value of DNA fragmentation evaluation tests under ART treatments

    PubMed Central

    Tavukçuo?lu, ?lkay ?afak; Al-Azawi, Tahani; Khaki, Amir Afshin; Khaki, Arash; Khalil, Ahmed; Al-Hasani, Safaa

    2012-01-01

    Male reproductive health has been under scrutiny recently. Many studies in the literature have concluded that semen quality is declining and that the incidence of testicular cancers is increasing. The reason for this change has been attributed to damage in sperm chromatin. During in vivo reproduction, the natural selection process ensures that only a spermatozoon with normal genomic material can fertilize an oocyte. However, the assisted reproduction technique (ART) is our selection process, leading to the possibility that abnormal spermatozoa could be used to fertilize an oocyte. We could avoid this by quantifying the amount and type of genomic damage in sperm using well-accepted laboratory methods. The sperm deoxyribonucleic acid (DNA) integrity is important for success of natural or assisted fertilization as well as normal development of the embryo, fetus and child. Intra cytoplasmic sperm injection (ICSI) is bypassing natural sperm selection mechanisms, which increases the risk of transmitting damaged DNA. The significance of required investigations and multiple techniques is that they could evaluate DNA defects in human spermatozoa. The ability of these techniques to accurately estimate sperm DNA damage depends on many technical and biological aspects. The aim of this review is to evaluate the most commonly used methods. PMID:24592055

  19. Mitochondrial and nuclear DNA base excision repair are affected differently by caloric restriction1

    E-print Network

    Stuart, Jeffrey A.

    . Caloric restriction lowers DNA repair activity in brain and kidney but not liver mitochondria Most DNA was assessed by measuring BER activity in mito- chondrial extracts prepared from liver, brain, and kidney and kidney mitochon- dria, CR resulted in 30% reductions of BER activity (t test; P 0.06) compared with PF

  20. A strategy to sequence repetitive DNA based on partial restriction enzyme cleavage

    SciTech Connect

    Abath, F.G.C.; Holder, A.A.

    1995-06-01

    The strategy to sequence repetitive DNA described in this article is based on partial restriction enzyme cleavage. It is an alternative to using nested deletion with exonuclease III or similiar enzymes in which progressively more remote regions of the target DNA are brought into range for sequencing by universal primers. 4 refs., 1 tab.

  1. 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.

  2. 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

  3. Low temperature-induced cell surface membrane vesicle shedding is associated with DNA fragmentation

    SciTech Connect

    Liepins, A.; Younghusband, H.B.

    1985-12-01

    Temperature shift conditions of 0 degree to 22 degrees C or 0 degree to 37 degrees C induce the formation and shedding of membrane vesicles (MV) from P815 tumor cell surfaces. When the MV shedding process takes place at 22 degrees C it occurs without changes in cell surface membrane permeability, whereas at 37 degrees C, changes in permeability to /sup 51/Cr and trypan blue do occur, thus mimicking the lymphocyte-mediated lytic process of tumor cells. The present studies demonstrate that nuclear DNA fragmentation also occurs in both 0 degree to 22 degrees C and 0 degree to 37 degrees C temperature shifts. However, cell surface membrane permeability to DNA fragments occurs only in the latter condition, i.e., 0 degree to 37 degrees C. The microtubule-stabilizing agent deuterium oxide (D/sub 2/O) inhibited the MV shedding process, the changes in membrane permeability, and DNA fragmentation. When P815 cells which had been induced to shed MV by the 0 degree to 22 degrees C temperature shift were labeled with /sup 51/Cr and used as targets for alloimmune lymphocytes, they were found to be as susceptible to T-cell lysis as control P815 cells. This result indicates that the lytic effect of alloimmune T lymphocytes can be exerted at the target cell surface membrane level independently of nuclear DNA fragmentation.

  4. 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...

  5. Allopatric Origin of Sympatric Populations of Lake Whitefish (Coregonus clupeaformis) as Revealed by Mitochondrial-DNA Restriction Analysis

    E-print Network

    Bernatchez, Louis

    recently diverged through sympatric speciation. Thirteen restriction enzymes were used to analyzemt by Mitochondrial-DNA Restriction Analysis Louis Bernatchez; Julian J. Dodson Evolution, Vol. 44, No. 5. (Aug., 1990 WHITEFISH (COREGONUS CLUPEAFORMIS) AS REVEALED BY MITOCHONDRIAL-DNA RESTRICTION ANALYSIS

  6. Non-monotonic density dependence of the diffusion of DNA fragments in low-salt suspensions

    E-print Network

    M. G. McPhie; G. Naegele

    2008-11-26

    The high linear charge density of 20-base-pair oligomers of DNA is shown to lead to a striking non-monotonic dependence of the long-time self-diffusion on the concentration of the DNA in low-salt conditions. This generic non-monotonic behavior results from both the strong coupling between the electrostatic and solvent-mediated hydrodynamic interactions, and from the renormalization of these electrostatic interactions at large separations, and specifically from the dominance of the far-field hydrodynamic interactions caused by the strong repulsion between the DNA fragments.

  7. Chromosomal aneuploidies and DNA fragmentation of human spermatozoa from patients exposed to perfluorinated compounds.

    PubMed

    Governini, L; Guerranti, C; De Leo, V; Boschi, L; Luddi, A; Gori, M; Orvieto, R; Piomboni, P

    2015-11-01

    This study investigated chromosomal aneuploidies and DNA damage in spermatozoa from male patients contaminated by perfluorinated compounds (PFCs) in whole blood and seminal plasma. Sperm aneuploidy and diploidy rate for chromosomes 18, X and Y were evaluated by FISH; sperm DNA fragmentation was assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labelling technique coupled to flow cytometry. Our results indicated that PFC contamination was present in 58% of subjects included in the study. A significant increase in alterations of sperm parameters was observed in PFC-positive subjects compared to PFC-negative subjects. As regards the sperm aneuploidy, both disomy and diploidy rates resulted significantly increased in subjects positive for PFC contamination compared to PFC-negative samples. In addition, sperm DNA fragmentation index resulted significantly increased in PFC-contaminated subjects compared to PFC-non-contaminated subjects, with a significant increased level of dimmer DNA fragmentation index. Our results clearly indicate that PFC contamination may detrimentally affect spermatogenesis, disturbing both meiotic segregation and DNA integrity. We could therefore suggest cautions to reduce or eliminate any contact with these compounds because the long-term effects of PFC accumulation in the body are not predictable. PMID:25382683

  8. Complete mitochondrial genome sequence of a Middle Pleistocene cave bear reconstructed from ultrashort DNA fragments

    PubMed Central

    Dabney, Jesse; Knapp, Michael; Glocke, Isabelle; Gansauge, Marie-Theres; Weihmann, Antje; Nickel, Birgit; Valdiosera, Cristina; García, Nuria; Pääbo, Svante; Arsuaga, Juan-Luis; Meyer, Matthias

    2013-01-01

    Although an inverse relationship is expected in ancient DNA samples between the number of surviving DNA fragments and their length, ancient DNA sequencing libraries are strikingly deficient in molecules shorter than 40 bp. We find that a loss of short molecules can occur during DNA extraction and present an improved silica-based extraction protocol that enables their efficient retrieval. In combination with single-stranded DNA library preparation, this method enabled us to reconstruct the mitochondrial genome sequence from a Middle Pleistocene cave bear (Ursus deningeri) bone excavated at Sima de los Huesos in the Sierra de Atapuerca, Spain. Phylogenetic reconstructions indicate that the U. deningeri sequence forms an early diverging sister lineage to all Western European Late Pleistocene cave bears. Our results prove that authentic ancient DNA can be preserved for hundreds of thousand years outside of permafrost. Moreover, the techniques presented enable the retrieval of phylogenetically informative sequences from samples in which virtually all DNA is diminished to fragments shorter than 50 bp. PMID:24019490

  9. A new PCR system for Agrobacterium tumefaciens detection based on amplification of T-DNA fragment.

    PubMed

    Sachadyn, P; Kur, J

    1997-01-01

    The design of the PCR system presented in this work is based on the knowledge of the molecular character of the crown gall disease. The virulence of Agrobacterium tumefaciens requires the presence of a big (up to 235,000 bp) plasmid Ti (pTi-tumour inducing plasmid). This plasmid carries the so-called T-DNA fragment (T-DNA-transferred DNA), which integrates into cell chromosomes of the infected plants and subsequently changes the plant morphology nad metabolism. In cannot be excluded that after T-DNA integration the presence of Agrobacterium is not necessary for the development of pathological changes. Thus, T-DNA is the only sign that must be present both in virulent bacteria and in infected plants in any stadium of the disease and even before the infection. This is why T-DNA was chosen as the target region for PCR amplification. Primers flanking a 220 bp fragment of one of the conservative regions responsible for Agrobacterium pathogenicity, namely tms2 gene coding for indolacetamide amidohydrolase (the second step of auxin biosynthesis) were designed as the optimal for PCR amplification. The PCR amplification reactions were performed for matrixes isolated from cultures of reference strains giving one predicted product for each sample. First attempts of T-DNA detection in infected soils and plants were performed. We hope that the presented new PCR system for Agrobacterium tumefaciens detection will help to fight the crown gall disease in the nearest future. PMID:9429288

  10. Bioactive beads-mediated transformation of rice with large DNA fragments containing Aegilops tauschii genes.

    PubMed

    Wada, Naoki; Kajiyama, Shin'ichiro; Akiyama, Yukio; Kawakami, Shigeki; No, Daisuke; Uchiyama, Susumu; Otani, Motoyasu; Shimada, Takiko; Nose, Naoko; Suzuki, Go; Mukai, Yasuhiko; Fukui, Kiichi

    2009-05-01

    Transformation with large DNA molecules enables multiple genes to be introduced into plants simultaneously to produce transgenic plants with complex phenotypes. In this study, a large DNA fragment (ca. 100 kb) containing a set of Aegilops tauschii hardness genes was introduced into rice plants using a novel transformation method, called bioactive beads-mediated transformation. Nine transgenic rice plants were obtained and the presence of transgenes in the rice genome was confirmed by PCR and FISH analyses. The results suggested that multiple transgenes were successfully integrated in all transgenic plants. The expression of one of the transgenes, puroindoline b, was confirmed at the mRNA and protein levels in the T(2) generation. Our study clearly demonstrates that the bioactive bead method is capable of producing transgenic rice plants carrying large DNA fragments. This method will facilitate the production of useful transgenic plants by introducing multiple genes simultaneously. PMID:19214515

  11. 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

  12. Entry of bacteriophage T7 DNA into the cell and escape from host restriction

    SciTech Connect

    Moffatt, B.A.; Studier, F.W.

    1988-05-01

    T7 DNA did not become susceptible to degradation by the host restriction enzymes EcoB, EcoK, or EcoP1 until 6 to 7 min after infection (at 30/sup 0/C). During this period, T7 gene 0.3 protein is made and inactivates EcoB and EcoK, allowing wild-type T7, or even a mutant that has recognition sites flanking gene 0.3, to escape restriction by these enzymes. However, T7 failed to escape restriction by EcoP1 even though 0.3 protein was made, evidently because 0.3 protein is unable to inactivate EcoP1. How T7 DNA can be accessible to transcription but not restriction in the first few minutes of infection is not yet understood, but we favor the idea that the entering DNA is initially segregated in a special place. Entry of T7 DNA into the cell is normally coupled to transcription. Tests of degradation of DNAs having their first restriction sites different distances from the end of the DNA indicated that only the first 1000 or so base pairs (2.5%) of the molecule enter the cell without transcription. An exception was the only mutant tested that lacks base pairs 343 to 393 of T7 DNA; most or all of this DNA entered the cell without being transcribed, apparently because it lacks a sequence that normally arrests entry. This block to DNA entry would normally be relieved by the host RNA polymerase transcribing from an appropriately situated promoter, but the block can also be relieved by T7 RNA polymerase, if supplied by the host cell. T7 mutants that lack all three strong early promoters A1, A2, and A3 could grow by using a secondary promoter.

  13. Efficient double fragmentation ChIP-seq provides nucleotide resolution protein-DNA binding profiles.

    PubMed

    Mokry, Michal; Hatzis, Pantelis; de Bruijn, Ewart; Koster, Jan; Versteeg, Rogier; Schuijers, Jurian; van de Wetering, Marc; Guryev, Victor; Clevers, Hans; Cuppen, Edwin

    2010-01-01

    Immunoprecipitated crosslinked protein-DNA fragments typically range in size from several hundred to several thousand base pairs, with a significant part of chromatin being much longer than the optimal length for next-generation sequencing (NGS) procedures. Because these larger fragments may be non-random and represent relevant biology that may otherwise be missed, but also because they represent a significant fraction of the immunoprecipitated material, we designed a double-fragmentation ChIP-seq procedure. After conventional crosslinking and immunoprecipitation, chromatin is de-crosslinked and sheared a second time to concentrate fragments in the optimal size range for NGS. Besides the benefits of increased chromatin yields, the procedure also eliminates a laborious size-selection step. We show that the double-fragmentation ChIP-seq approach allows for the generation of biologically relevant genome-wide protein-DNA binding profiles from sub-nanogram amounts of TCF7L2/TCF4, TBP and H3K4me3 immunoprecipitated material. Although optimized for the AB/SOLiD platform, the same approach may be applied to other platforms. PMID:21152096

  14. Analysis of the promoter-distal region of the tra operon of the F sex factor of Escherichia coli K-12 encoded by EcoRI restriction fragments f17, f19, and f2.

    PubMed Central

    Manning, P A; Kusecek, B; Morelli, G; Fisseau, C; Achtman, M

    1982-01-01

    The promoter-distal region of the tra operon of the F sex factor Escherichia coli K-12 was analyzed, using the chimeric plasmid pRS31, which contains the F EcoRI restriction fragments f17, f19, and f2 cloned into the EcoRI site of pSC101. A series of deletion plasmids of pRS31, extending increasing distances from a site in f17 through f19 and ending in f2, were isolated. These plasmids were examined by heteroduplex analysis with the parent DNA, and a restriction map of this region of DNA was constructed. A series of Tn5 insertion derivatives of pRS31 were also isolated and mapped, using both heteroduplex analysis and restriction mapping. Both the insertion and deletion mutants were tested in minicells for the synthesis of radioactively labeled proteins. This allowed the identification of the individual gene products and mapping of the genes. The result is a saturated physical map of this region of DNA from fragment f17 through to the IS3 insertion sequence near the promoter-distal end of f2. Images PMID:6277873

  15. 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 ...

  16. A restriction enzyme-powered autonomous DNA walking machine: its application for a highly sensitive electrochemiluminescence assay of DNA

    NASA Astrophysics Data System (ADS)

    Chen, Ying; Xiang, Yun; Yuan, Ruo; Chai, Yaqin

    2014-12-01

    The construction of a restriction enzyme (Nt.AlwI)-powered DNA walking machine and its application for highly sensitive detection of DNA are described. DNA nanostructure tracks containing four overhang sequences with electrochemiluminescence (ECL) labels and complementary to the walker (target DNA) are self-assembled on the sensing electrode. The walker hybridizes with the complementary sequences on the tracks and forms specific recognition sites for Nt.AlwI, which cleaves the overhang sequences, releases the ECL labels and enables directional movement of the walker along the tracks. The formation of the nanostructure tracks and the Nt.AlwI-assisted cleavage of the overhang sequences in the presence of the walker are verified by using polyacrylamide gel electrophoresis analysis and cyclic voltammetry. The successive movement of the walker on the nanostructure tracks leads to continuous removal of massive ECL labels from the sensing electrode, which results in a significantly amplified suppression of the ECL emission for highly sensitive detection of sequence-specific DNA down to 0.19 pM. Results show that this DNA walking machine can also offer single-base mismatch discrimination capability. The successful application of the DNA walking machine for sequence-specific DNA detection can thus offer new opportunities for molecular machines in biosensing applications.The construction of a restriction enzyme (Nt.AlwI)-powered DNA walking machine and its application for highly sensitive detection of DNA are described. DNA nanostructure tracks containing four overhang sequences with electrochemiluminescence (ECL) labels and complementary to the walker (target DNA) are self-assembled on the sensing electrode. The walker hybridizes with the complementary sequences on the tracks and forms specific recognition sites for Nt.AlwI, which cleaves the overhang sequences, releases the ECL labels and enables directional movement of the walker along the tracks. The formation of the nanostructure tracks and the Nt.AlwI-assisted cleavage of the overhang sequences in the presence of the walker are verified by using polyacrylamide gel electrophoresis analysis and cyclic voltammetry. The successive movement of the walker on the nanostructure tracks leads to continuous removal of massive ECL labels from the sensing electrode, which results in a significantly amplified suppression of the ECL emission for highly sensitive detection of sequence-specific DNA down to 0.19 pM. Results show that this DNA walking machine can also offer single-base mismatch discrimination capability. The successful application of the DNA walking machine for sequence-specific DNA detection can thus offer new opportunities for molecular machines in biosensing applications. Electronic supplementary information (ESI) available. See DOI: 10.1039/c4nr05387g

  17. Pathological phenotypes and in vivo DNA cleavage by unrestrained activity of a phosphorothioate-based restriction system in Salmonella

    E-print Network

    Cao, Bo

    Prokaryotes protect their genomes from foreign DNA with a diversity of defence mechanisms, including a widespread restriction–modification (R–M) system involving phosphorothioate (PT) modification of the DNA backbone. ...

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

    SciTech Connect

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

    2003-11-19

    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 small gamma, Greek-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 small gamma, Greek-rays in the size range 123 kbp; (3) a non-random DNA DSB induction by Fe ions.

  19. 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.

  20. Distribution of mitochondrial DNA fragments in the nuclear genome of the honeybee.

    PubMed

    Du, W X; Qin, Y C

    2015-01-01

    Nuclear mitochondrial pseudogenes (numts), which originated from mitochondrial DNA (mtDNA) insertions in the nuclear genome, have been detected in many species. The distribution of numts in the honeybee nuclear genome has not yet been fully reported. By referring to the whole honeybee mtDNA sequence and to the recent version of the honeybee nuclear genome, 236 reference sequences were identified by BLAST, with 90 unmapped. The size of the numts ranged from 219 to 3788 bp, and the homologous identity between numts and their corresponding mtDNA fragments varied from 71 to 93%. Furthermore, identified honeybee numts covered nearly all mitochondrial genes and were distributed over all chromosomes. This study provides useful information for further research related to mitochondrial genes and the evolution of the honeybee. PMID:26535652

  1. 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.

  2. Development of a multiplexed polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay to identify common members of the Subgenera Culex (Culex) and Culex (Phenacomyia) in Guatemala.

    PubMed

    Kent, Rebekah J; Deus, Stephen; Williams, Martin; Savage, Harry M

    2010-08-01

    Morphological differentiation of mosquitoes in the subgenera Culex (Culex) and Culex (Phenacomyia) in Guatemala is difficult, with reliable identification ensured only through examination of larval skins from individually reared specimens and associated male genitalia. We developed a multiplexed polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay to identify common Cx. (Cux.) and Cx. (Phc.). Culex (Cux.) chidesteri, Cx. (Cux.) coronator, Cx. (Cux.) interrogator, Cx. (Cux.) quinquefasciatus, Cx. (Cux.) nigripalpus/Cx. (Cux.) thriambus, and Cx. (Phc.) lactator were identified directly with a multiplexed primer cocktail comprising a conserved forward primer and specific reverse primers targeting ribosomal DNA (rDNA). Culex nigripalpus and Cx. thriambus were differentiated by restriction digest of homologous amplicons. The assay was developed and optimized using well-characterized specimens from Guatemala and the United States and field tested with unknown material from Guatemala. This assay will be a valuable tool for mosquito identification in entomological and arbovirus ecology studies in Guatemala. PMID:20682869

  3. HIPK2 restricts SIRT1 activity upon severe DNA damage by a phosphorylation-controlled mechanism.

    PubMed

    Conrad, E; Polonio-Vallon, T; Meister, M; Matt, S; Bitomsky, N; Herbel, C; Liebl, M; Greiner, V; Kriznik, B; Schumacher, S; Krieghoff-Henning, E; Hofmann, T G

    2016-01-01

    Upon severe DNA damage a cellular signalling network initiates a cell death response through activating tumour suppressor p53 in association with promyelocytic leukaemia (PML) nuclear bodies. The deacetylase Sirtuin 1 (SIRT1) suppresses cell death after DNA damage by antagonizing p53 acetylation. To facilitate efficient p53 acetylation, SIRT1 function needs to be restricted. How SIRT1 activity is regulated under these conditions remains largely unclear. Here we provide evidence that SIRT1 activity is limited upon severe DNA damage through phosphorylation by the DNA damage-responsive kinase HIPK2. We found that DNA damage provokes interaction of SIRT1 and HIPK2, which phosphorylates SIRT1 at Serine 682 upon lethal damage. Furthermore, upon DNA damage SIRT1 and HIPK2 colocalize at PML nuclear bodies, and PML depletion abrogates DNA damage-induced SIRT1 Ser682 phosphorylation. We show that Ser682 phosphorylation inhibits SIRT1 activity and impacts on p53 acetylation, apoptotic p53 target gene expression and cell death. Mechanistically, we found that DNA damage-induced SIRT1 Ser682 phosphorylation provokes disruption of the complex between SIRT1 and its activator AROS. Our findings indicate that phosphorylation-dependent restriction of SIRT1 activity by HIPK2 shapes the p53 response. PMID:26113041

  4. Magnetic bead purification of labeled DNA fragments forhigh-throughput capillary electrophoresis sequencing

    SciTech Connect

    Elkin, Christopher; Kapur, Hitesh; Smith, Troy; Humphries, David; Pollard, Martin; Hammon, Nancy; Hawkins, Trevor

    2001-09-15

    We have developed an automated purification method for terminator sequencing products based on a magnetic bead technology. This 384-well protocol generates labeled DNA fragments that are essentially free of contaminates for less than $0.005 per reaction. In comparison to laborious ethanol precipitation protocols, this method increases the phred20 read length by forty bases with various DNA templates such as PCR fragments, Plasmids, Cosmids and RCA products. Our method eliminates centrifugation and is compatible with both the MegaBACE 1000 and ABIPrism 3700 capillary instruments. As of September 2001, this method has produced over 1.6 million samples with 93 percent averaging 620 phred20 bases as part of Joint Genome Institutes Production Process.

  5. 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 ...

  6. DNA shuffling by random fragmentation and reassembly: in vitro recombination for molecular evolution.

    PubMed Central

    Stemmer, W P

    1994-01-01

    Computer simulations of the evolution of linear sequences have demonstrated the importance of recombination of blocks of sequence rather than point mutagenesis alone. Repeated cycles of point mutagenesis, recombination, and selection should allow in vitro molecular evolution of complex sequences, such as proteins. A method for the reassembly of genes from their random DNA fragments, resulting in in vitro recombination is reported. A 1-kb gene, after DNase I digestion and purification of 10- to 50-bp random fragments, was reassembled to its original size and function. Similarly, a 2.7-kb plasmid could be efficiently reassembled. Complete recombination was obtained between two markers separated by 75 bp; each marker was located on a separate gene. Oligonucleotides with 3' and 5' ends that are homologous to the gene can be added to the fragment mixture and incorporated into the reassembled gene. Thus, mixtures of synthetic oligonucleotides and PCR fragments can be mixed into a gene at defined positions based on homology. As an example, a library of chimeras of the human and murine genes for interleukin 1 beta has been prepared. Shuffling can also be used for the in vitro equivalent of some standard genetic manipulations, such as a backcross with parental DNA. The advantages of recombination over existing mutagenesis methods are likely to increase with the numbers of cycles of molecular evolution. Images PMID:7938023

  7. Sperm DNA Fragmentation and Standard Semen Parameters in Algerian Infertile Male Partners

    PubMed Central

    Boushaba, Sarah

    2015-01-01

    Purpose To date, standard semen parameters have been the only parameters investigated in sperm samples of infertile men in Algeria. We investigated, for the first time, semen parameters according to sperm DNA fragmentation (SDF) in these subjects. Materials and Methods SDF was determined by a validated sperm chromatin dispersion test in 26 infertile men. Patients were split into two groups according to the SDF level estimated by the DNA fragmentation index (DFI): the low fragmentation group (LFG; LFG with DFI ?18%) and high fragmentation group (HFG; HFG with DFI >18%). The standard semen parameters were measured in both groups. Results We found that semen concentration and motility were negatively correlated with DFI (r=-0.65, r=-0.45, respectively; p<0.05), while morphology and semen volume were not correlated with it (r=0.24, r=-0.18, respectively; p>0.05). Comparison of the sperm concentration revealed that it was significantly higher in LFG than in HFG (37.57%±13.16% vs. 7.32%±3.59%, respectively; p<0.05), whereas no significant difference was observed regarding sperm motility and morphology. Conclusions Our findings suggest that SDF correlates well with both sperm motility and concentration but not with morphology. Thus, we conclude that SDF evaluation provides additional information regarding sperm quality, and should be used as a complementary test for assessing semen characteristics in infertile males. PMID:25927056

  8. 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

  9. Giardia duodenalis in Damascus, Syria: Identification of Giardia genotypes in a sample of human fecal isolates using polymerase chain reaction and restriction fragment length polymorphism analyzing method.

    PubMed

    Skhal, Dania; Aboualchamat, Ghalia; Al Nahhas, Samar

    2016-02-01

    Giardia duodenalis is a common gastrointestinal parasite that infects humans and many other mammals. It is most prevalent in many developing and industrialized countries. G. duodenalis is considered to be a complex species. While no morphological distinction among different assemblages exist, it can be genetically differentiated into eight major assemblages: A to H. The aim of this study was to determine the genetic heterogeneity of G. duodenalis in human isolates (a study conducted for the first time in Syria). 40 fecal samples were collected from three different hospitals during the hot summer season of 2014. Extraction of genomic DNA from all Giardia positive samples (based on a microscopic examination) was performed using QIAamp DNA Stool Mini Kit. ?-giardin gene was used to differentiate between different Giardia assemblages. The 514bp fragment was amplified using the Polymerase Chain Reaction method, followed by digestion in HaeIII restriction enzyme. Our result showed that genotype A was more frequent than genotype B, 27/40 (67.5%); 4/40 (10%) respectively. A mixed genotype of A+B was only detected in 9 isolates (22.5%). This is the first molecular study performed on G. duodenalis isolates in Syria in order to discriminate among the different genotypes. Further expanded studies using more genes are needed to detect and identify the Giardia parasite at the level of assemblage and sub-assemblage. PMID:26524628

  10. Analysis of the bacterial diversity existing on animal hide and wool: development of a preliminary PCR-restriction fragment length polymorphism fingerprint database for identifying isolates.

    PubMed

    Chen, Yu; Gao, Hongwei; Zhang, Yanming; Deng, Mingjun; Wu, Zhenxing; Zhu, Laihua; Duan, Qing; Xu, Biao; Liang, Chengzhu; Yue, Zhiqin; Xiao, Xizhi

    2012-01-01

    Twenty-one bacterial strains were isolated from imported cattle hide and rabbit wool using two types of media, nutrient broth, and nutrient broth with serum. The bacteria identified were Brevibacillus laterosporus, Leclercia adecarboxylata, Peptococcus niger, Bacillus circulans, Raoultella ornithinolytica, Bacillus subtilis, Bacillus cereus, Bacillus thermobacillus, Bacillus choshinensis, Bacillus sphaericus, Acinetobacter haemolyticus, Sphingomonas paucimobilis, Bacillus thuringiensis, Staphylococcus intermedius, Mycobacteria, Moraxella, Klebsiella pneumoniae, Ralstonia pickettii, Staphylococcus chromogenes, Comamonas testosteroni, and Cupriavidus pauculus. The 16s rDNA gene of each bacterium was amplified using the universal primers 27f and 1492r. The amplicons were digested with AvaI, BamHI, BgII, DraI, EcoRI, EcoRV, HindIII, HinfI, HpaI, PstI, SmaI, TaqII, XbaI, XmaI, AluI, XhoI, and PvuI individually. A specific fingerprint from the PCR-restriction fragment length polymorphism method based on 16s rDNA was obtained for each bacterium. The results showed that the method developed was useful not only for bacterial identification but also for the etiological investigation of pathogens in imported animal hair and wool. PMID:23451394

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

    SciTech Connect

    Tee, Thiam-Tsui; Cheah, Yew-Hoong; Bioassay Unit, Herbal Medicine Research Center, Institute for Medical Research, Jalan Pahang, Kuala Lumpur ; Meenakshii, Nallappan; Mohd Sharom, Mohd Yusof; Azimahtol Hawariah, Lope Pihie

    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.

  12. Gapped DNA and Cyclization of Short DNA Fragments Quan Du,* Maria Vologodskaia,* Heiko Kuhn,y

    E-print Network

    Vologodskii, Alexander

    nicking endonucleases are used to create the 4-nt-long gap. We determined the bending rigidity of the single-stranded region in the gapped DNA. We found that the gap of 4 nt in length makes all torsional orientations of DNA ends equally probable. Our results also show that the gap has isotropic bending rigidity

  13. Modification-dependent restriction endonuclease, MspJI, flips 5-methylcytosine out of the DNA helix

    SciTech Connect

    Horton, J. R.; Wang, H.; Mabuchi, M. Y.; Zhang, X.; Roberts, R. J.; Zheng, Y.; Wilson, G. G.; Cheng, X.

    2014-09-27

    MspJI belongs to a family of restriction enzymes that cleave DNA containing 5-methylcytosine (5mC) or 5-hydroxymethylcytosine (5hmC). MspJI is specific for the sequence 5(h)mC-N-N-G or A and cleaves with some variability 9/13 nucleotides downstream. Earlier, we reported the crystal structure of MspJI without DNA and proposed how it might recognize this sequence and catalyze cleavage. Here we report its co-crystal structure with a 27-base pair oligonucleotide containing 5mC. This structure confirms that MspJI acts as a homotetramer and that the modified cytosine is flipped from the DNA helix into an SRA-like-binding pocket. We expected the structure to reveal two DNA molecules bound specifically to the tetramer and engaged with the enzyme's two DNA-cleavage sites. A coincidence of crystal packing precluded this organization, however. We found that each DNA molecule interacted with two adjacent tetramers, binding one specifically and the other non-specifically. The latter interaction, which prevented cleavage-site engagement, also involved base flipping and might represent the sequence-interrogation phase that precedes specific recognition. MspJI is unusual in that DNA molecules are recognized and cleaved by different subunits. Such interchange of function might explain how other complex multimeric restriction enzymes act.

  14. Large Fragment of DNA Polymerase I from Geobacillus sp. 777: Cloning and Comparison with DNA Polymerases I in Practical Applications.

    PubMed

    Oscorbin, Igor P; Boyarskikh, Ulyana A; Filipenko, Maksim L

    2015-10-01

    A truncated gene of DNA polymerase I from the thermophilic bacteria Geobacillus sp. 777 encoding a large fragment of enzyme (LF Gss pol) was cloned and sequenced. The resulting sequence is 1776-bp long and encodes a 592 aa protein with a predicted molecular mass of 69.8 kDa. Enzyme was overexpressed in E. coli, purified by metal-chelate chromatography, and biochemically characterized. The specific activity of LF Gss pol is 104,000 U/mg (one unit of enzyme was defined as the amount of enzyme that incorporated 10 nmol of dNTP into acid insoluble material in 30 min at 65 °C). The properties of LF Gss pol were compared to commercially available large fragments of DNA polymerase I from G. stearothermophilus (LF Bst pol) and Bacillus smithii (LF Bsm pol). Studied enzymes showed maximum activity at similar pH and concentrations of monovalent/divalent ions, whereas LF Gss pol and LF Bst pol were more thermostable than LF Bsm pol. LF Gss pol is more resistant to enzyme inhibitors (SYBR Green I, heparin, ethanol, urea, blood plasma) in comparison with LF Bst pol and LF Bsm pol. LF Gss pol is also suitable for loop-mediated isothermal amplification and whole genome amplification of human genomic DNA. PMID:26289299

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

    SciTech Connect

    Evenson, Donald P. . E-mail: scsa@brookings.net; Wixon, Regina

    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.

  16. 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

  17. Differentiation of sheeppox and goatpox viruses by polymerase Chain reaction-restriction fragment length polymorphism.

    PubMed

    Venkatesan, Gnanavel; Balamurugan, Vinayagamurthy; Yogisharadhya, Revaniah; Kumar, Amit; Bhanuprakash, Veerakyathappa

    2012-12-01

    In the present study, the partial gene sequences of P32 protein, an immunogenic envelope protein of Capripoxviruses (CaPV), were analyzed to assess the genetic relationship among sheeppox and goatpox virus isolates, and restriction enzyme specific PCR-RFLP was developed to differentiate CaPV strains. A total of six goatpox virus (GTPV) and nine sheeppox virus (SPPV) isolates of Indian origin were included in the sequence analysis of the attachment gene. The sequence analysis revealed a high degree of sequence identity among all the Indian SPPV and GTPV isolates at both nucleotide and amino acid levels. Phylogenetic analysis showed three distinct clusters of SPPV, GTPV and Lumpy skin disease virus (LSDV) isolates. Further, multiple sequence alignment revealed a unique change at G120A in all GTPV isolates resulting in the formation of Dra I restriction site in lieu of EcoR I, which is present in SPPV isolates studied. This change was unique and exploited to develop restriction enzyme specific PCR-RFLP for detection and differentiation of SPPV and GTPV strains. The optimized PCR-RFLP was validated using a total of fourteen (n=14) cell culture isolates and twenty two (n=22) known clinical samples of CaPV. The Restriction Enzyme specific PCR-RFLP to differentiate both species will allow a rapid differential diagnosis during CaPV outbreaks particularly in mixed flocks of sheep and goats and could be an adjunct/supportive tool for complete gene or virus genome sequencing methods. PMID:23271576

  18. Abdominal visceral fat is associated with a BclI restriction fragment length polymorphism at the glucocorticoid receptor gene locus.

    PubMed

    Buemann, B; Vohl, M C; Chagnon, M; Chagnon, Y C; Gagnon, J; Pérusse, L; Dionne, F; Després, J P; Tremblay, A; Nadeau, A; Bouchard, C

    1997-05-01

    Several investigations have suggested that body fat distribution is influenced by nonpathologic variations in the responsiveness to cortisol. Genetic variations in the glucocorticoid receptor (GRL) could therefore potentially have an impact on the level of abdominal fat. A restriction fragment length polymorphism (RFLP) has previously been detected with the BclI restriction enzyme in the GRL gene identifying two alleles with fragment lengths of 4.5 and 2.3 kb. This study investigates whether abdominal fat areas measured by computerized tomography (CT) are associated with this polymorphism in 152 middle-aged men and women. The less frequent 4.5-kb allele was found to be associated with a higher abdominal visceral fat (AVF) area independently of total body fat mass (4.5/4.5 vs. 2.3/2.3 kb genotype; men: 190.7 +/- 30.1 vs. 150.7 +/- 33.3 cm2, p = 0.04; women: 132.7 +/- 37.3 vs. 101.3 +/- 34.5 cm2, p = 0.06). However, the association with AVF was seen only in subjects of the lower tertile of the percent body fat level. In these subjects, the polymorphism was found to account for 41% (p = 0.003) and 35% (p = 0.007), in men and women, respectively, of the total variance in AVF area. The consistent association between the GRL polymorphism detected with BclI and AVF area suggests that this gene or a locus in linkage disequilibrium with the BclI restriction site may contribute to the accumulation of AVF. PMID:9192392

  19. Identification of column edges of DNA fragments by using K-means clustering and mean algorithm on lane histograms of DNA agarose gel electrophoresis images

    NASA Astrophysics Data System (ADS)

    Turan, Muhammed K.; Sehirli, Eftal; Elen, Abdullah; Karas, Ismail R.

    2015-07-01

    Gel electrophoresis (GE) is one of the most used method to separate DNA, RNA, protein molecules according to size, weight and quantity parameters in many areas such as genetics, molecular biology, biochemistry, microbiology. The main way to separate each molecule is to find borders of each molecule fragment. This paper presents a software application that show columns edges of DNA fragments in 3 steps. In the first step the application obtains lane histograms of agarose gel electrophoresis images by doing projection based on x-axis. In the second step, it utilizes k-means clustering algorithm to classify point values of lane histogram such as left side values, right side values and undesired values. In the third step, column edges of DNA fragments is shown by using mean algorithm and mathematical processes to separate DNA fragments from the background in a fully automated way. In addition to this, the application presents locations of DNA fragments and how many DNA fragments exist on images captured by a scientific camera.

  20. 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

  1. Modification-dependent restriction endonuclease, MspJI, flips 5-methylcytosine out of the DNA helix

    DOE PAGESBeta

    Horton, J. R.; Wang, H.; Mabuchi, M. Y.; Zhang, X.; Roberts, R. J.; Zheng, Y.; Wilson, G. G.; Cheng, X.

    2014-09-27

    MspJI belongs to a family of restriction enzymes that cleave DNA containing 5-methylcytosine (5mC) or 5-hydroxymethylcytosine (5hmC). MspJI is specific for the sequence 5(h)mC-N-N-G or A and cleaves with some variability 9/13 nucleotides downstream. Earlier, we reported the crystal structure of MspJI without DNA and proposed how it might recognize this sequence and catalyze cleavage. Here we report its co-crystal structure with a 27-base pair oligonucleotide containing 5mC. This structure confirms that MspJI acts as a homotetramer and that the modified cytosine is flipped from the DNA helix into an SRA-like-binding pocket. We expected the structure to reveal two DNAmore »molecules bound specifically to the tetramer and engaged with the enzyme's two DNA-cleavage sites. A coincidence of crystal packing precluded this organization, however. We found that each DNA molecule interacted with two adjacent tetramers, binding one specifically and the other non-specifically. The latter interaction, which prevented cleavage-site engagement, also involved base flipping and might represent the sequence-interrogation phase that precedes specific recognition. MspJI is unusual in that DNA molecules are recognized and cleaved by different subunits. Such interchange of function might explain how other complex multimeric restriction enzymes act.« less

  2. Restriction enzyme mapping of ribosomal DNA can distinguish between fasciolid (liver fluke) species.

    PubMed

    Blair, D; McManus, D P

    1989-10-01

    Recognition sites for nine different restriction endonucleases were mapped on rDNA genes of fasciolid species. Southern blots of digested DNA from individual worms were probed sequentially with three different probes derived from rDNA of Schistosoma mansoni and known to span between them the entire rDNA repeat unit in that species. Eighteen recognition sites were mapped for Fasciola hepatica, and seventeen for Fasciola gigantica and Fascioloides magna. Each fasciolid species had no more than two unique recognition sites, the remainder being common to one or both of the other two species. No intraspecific variation in restriction sites was noted in F. hepatica (individuals from 11 samples studied; hosts were sheep, cattle and laboratory animals; geographical origins. Australia, New Zealand, Mexico, U.K., Hungary and Spain), or in F. gigantica (two samples; Indonesia and Malaysia). Only one sample of F. magna was available. One specimen of Fasciola sp. from Japan (specific identity regarded in the literature as uncertain) yielded a restriction map identical to that of F. gigantica. Almost all recognition sites occurred in or near the putative rRNA coding regions. The non-transcribed spacer region had few or no cut sites despite the fact that this region is up to about one half of the entire repeat unit in length. Length heterogeneity was noted in the non-transcribed spacer, even within individual worms. PMID:2552311

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

    SciTech Connect

    Okano, Kazuhiro; Vanarsdall, Adam L.; Rohrmann, George F. . 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.

  4. Do Pilea Microphylla Improve Sperm DNA Fragmentation and Sperm Parameters in Varicocelized Rats?

    PubMed

    Heidari, Reza; Alizadeh, Rafieh; Abbasi, Niloufar; Pasbakhsh, Parichehr; Hedayatpour, Azim; Farajpour, Mostafa; Khaleghi, Mohammad Reza; Abbasi, Mehdi; Dehpour, Ahmad Reza

    2015-09-01

    Varicocele is one of the most common causes of primary male infertility. Pilea microphylla (PM) is being used as folk medicine. This study was aimed to investigate the effects of PM in a rat model of varicocele. A total of 30 male Wistar rats were divided into control, sham, varicocele, accessory varicocele and PM-treated groups. After 10 weeks of varicocele induction, sperm parameters and chromatin (Aniline blue, acridine orange and toluidine blue) were evaluated, except for the treated and accessory groups that received 50 mg/kg PM orally daily for 10 weeks and then were sacrificed. Sperm parameters significantly decreased in varicocele groups (P < 0.01).  Moreover, there was a negative correlation between the DNA fragmentation and sperm parameters in varicocelized rats. Administration of PM led to significantly increased sperm parameters and AO staining (P < 0.05). These findings suggest that PM improves sperm parameters and DNA fragmentation in varicocelized rats. PM can reduce the damage to sperm DNA but not chromatin condensation. PMID:26553082

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

    SciTech Connect

    Yoshikawa, Hirohide; Nagai, Hisaki; Matsubara, Kenichi

    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.

  6. 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.

  7. Sequence context effects on 8-methoxypsoralen photobinding to defined DNA fragments

    SciTech Connect

    Sage, E.; Moustacchi, E.

    1987-06-16

    The photoreaction of 8-methoxypsoralen (8-MOP) with DNA fragments of defined sequence was studied. The authors took advantage of the blockage by bulky adducts of the 3'-5'-exonuclease activity associated with the T4 DNA polymerase. The action of the exonuclease is stopped by biadducts as well as by monoadducts. The termination products were analyzed on sequencing gels. A strong sequence specificity was observed in the DNA photobinding of 8-MOP. The exonuclease terminates its digestion near thymine residues, mainly at potentially cross-linkable sites. There is an increasing reactivity of thymine residues in the order T < TT << TTT in a GC environment. For thymine residues in cross-linkable sites, the reactivity follows the order AT << TA approx. TAT << ATA < ATAT < ATATAA. Repeated A-T sequences are hot spots for the photochemical reaction of 8-MOP with DNA. Both monoadducts and interstrand cross-links are formed preferentially in 5'-TpA sites. The results highlight the role of the sequence and consequently of the conformation around a potential site in the photobinding of 8-MOP to DNA.

  8. 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

  9. Calcium ion concentrations and DNA fragmentation in target cell destruction by murine cloned cytotoxic T lymphocytes

    PubMed Central

    1988-01-01

    To investigate the destruction of target cells by murine CTLs, we examined intracellular Ca2+ concentrations ([Ca2+]i) and DNA fragmentation in target cells. Changes in [Ca2+]i were followed by flow cytometry by loading the cells with indo-1, a Ca2+-binding fluorescent dye, and determining the ration of fluorescence intensities at 405 nm (emission maximum for Ca2+-bound dye) over 480 nm (emission maximum for the free dye). Within minutes after interacting with the cytolytic granule fraction that had been isolated from CTLs, [Ca2+]i in target cells was strikingly increased. A pronounced increase in [Ca2+]i was also observed in target cells when they were specifically recognized by intact CTLs. Since ionomycin, a Ca2+ ionophore, caused a similar increase in [Ca2+]i and lysed cells (provided that extracellular Ca2+ was present), it appears that a sustained high level of [Ca2+]i is cytolytic. In contrast with other cells, CTLs, which have been shown to be refractory to granule-mediated lysis and to be poor targets for other CTLs, did not manifest an elevation in [Ca2+]i when they were similarly loaded with indo-1 and treated with isolated granules. The characteristic cleavage of target cell DNA into nucleosome-sized fragments was also induced by isolated granules as well as by valinomycin, a K+ ionophore, but not by ionomycin. The results support the view that lysis of most target cells by cloned CTLs is due primarily to target cell membrane changes that are fundamentally equivalent to the formation of nonspecific ion channels. The resulting large increase in [Ca2+]i is probably responsible for target cell lysis; and changes in intracellular ion concentrations also appear to be responsible for DNA fragmentation, probably by activating endogenous target cell endonucleases. PMID:2450162

  10. Protein-DNA interactions in vitro with 5'-flanking DNA fragments from the chicken vitellogenin gene.

    PubMed

    Bakker, O; Das, A T; Ab, G

    1988-01-01

    The expression of the vitellogenin gene in the liver of oviparous animals is under strict control of estrogen. We have studied the interaction of proteins extracted from nuclei of different estrogen responsive tissues with two fragments (-728 to -470 and -625 to -470) of the upstream region of the chicken vitellogenin gene, using the gel-retardation technique. We found a complex pattern of retarded bands using nuclear extracts from laying hen liver, rooster liver and MCF-7 cells. The patterns observed display differences in the position and intensities of some of the bands, depending on the source of the extract used. The possible significance of these findings will be discussed. PMID:2838691

  11. PCR-restriction fragment length polymorphism for rapid, low-cost identification of isoniazid-resistant Mycobacterium tuberculosis.

    PubMed

    Caws, Maxine; Tho, Dau Quang; Duy, Phan Minh; Lan, Nguyen Thi Ngoc; Hoa, Dai Viet; Torok, Mili Estee; Chau, Tran Thi Hong; Chau, Nguyen Van Vinh; Chinh, Nguyen Tran; Farrar, Jeremy

    2007-06-01

    PCR-restriction fragment length poymorphism (PCR-RFLP) is a simple, robust technique for the rapid identification of isoniazid-resistant Mycobacterium tuberculosis. One hundred consecutive isolates from a Vietnamese tuberculosis hospital were tested by MspA1I PCR-RFLP for the detection of isoniazid-resistant katG_315 mutants. The test had a sensitivity of 80% and a specificity of 100% against conventional phenotypic drug susceptibility testing. The positive and negative predictive values were 1 and 0.86, respectively. None of the discrepant isolates had mutant katG_315 codons by sequencing. The test is cheap (less than $1.50 per test), specific, and suitable for the rapid identification of isoniazid resistance in regions with a high prevalence of katG_315 mutants among isoniazid-resistant M. tuberculosis isolates. PMID:17428939

  12. Use of bifidobacterial specific terminal restriction fragment length polymorphisms to complement next generation sequence profiling of infant gut communities.

    PubMed

    Lewis, Zachery T; Bokulich, Nicholas A; Kalanetra, Karen M; Ruiz-Moyano, Santiago; Underwood, Mark A; Mills, David A

    2013-02-01

    Bifidobacteria are intestinal anaerobes often associated with gut health. Specific bifidobacterial species are particularly common in the gastrointestinal tract of breast-fed infants. Current short read next-generation sequencing approaches to profile fecal microbial ecologies do not discriminate bifidobacteria to the species level. Here we describe a low-cost terminal restriction fragment length polymorphism (TRFLP) procedure to distinguish between the common infant-associated bifidobacterial species. An empirical database of TRF sizes was created from both common reference strains and well-identified isolates from infant feces. Species-specific quantitative PCR validated bifidobacterial-specific TRFLP profiles from infant feces. These results indicate that bifidobacterial-specific TRFLP is a useful method to monitor intestinal bifidobacterial populations from infant fecal samples. When used alongside next generation sequencing methods that detect broader population levels at lower resolution, this high-throughput, low-cost tool can help clarify the role of bifidobacteria in health and disease. PMID:23261904

  13. Differentiation of mixed lactic acid bacteria communities in beverage fermentations using targeted terminal restriction fragment length polymorphism.

    PubMed

    Bokulich, Nicholas A; Mills, David A

    2012-08-01

    Lactic acid bacteria (LAB) are an important group of bacteria in beer and wine fermentations both as beneficial organisms and as spoilage agents. However, sensitive, rapid, culture-independent methods for identification and community analyses of LAB in mixed-culture fermentations are limited. We developed a terminal restriction fragment length polymorphism (TRFLP)-based assay for the detection and identification of lactic acid bacteria and Bacilli during wine, beer, and food fermentations. This technique can sensitively discriminate most species of Lactobacillales, and most genera of Bacillales, in mixed culture, as indicated by both bioinformatic predictions and empirical observations. This method was tested on a range of beer and wine fermentations containing mixed LAB communities, demonstrating the efficacy of this technique for discriminating LAB in mixed culture. PMID:22475950

  14. Tumor cell growth arrest caused by subchromosomal transferable DNA fragments from chromosome 11

    SciTech Connect

    Koi, M.; Johnson, L.A.; Kalikin, L.M.; Feinberg, A.P. ); Little, P.F.R. ); Nakamura, Yusuke )

    1993-04-16

    A fundamental problem in the identification and isolation of tumor suppressor and other growth-inhibiting genes is the loss of power of genetic complementation at the subchromosomal level. A direct genetic strategy was developed to isolate subchromosomal transferable fragments (STFs) from any chromosome, each containing a selectable marker within the human DNA, that could be transferred to any mammalian cells. As a test of the method, several overlapping STFs from 11p15 were shown to cause in vitro growth arrest of rhabdomyosarcoma cells. This activity mapped between the [beta]-globin and insulin genes. 34 refs., 5 figs.

  15. Pathological phenotypes and in vivo DNA cleavage by unrestrained activity of a phosphorothioate-based restriction system in Salmonella

    PubMed Central

    Cao, Bo; Cheng, Qiuxiang; Gu, Chen; Yao, Fen; DeMott, Michael S.; Zheng, Xiaoqing; Deng, Zixin; Dedon, Peter C.; You, Delin

    2015-01-01

    Summary Prokaryotes protect their genomes from foreign DNA with a diversity of defense mechanisms, including a widespread restriction-modification (R-M) system involving phosphorothioate (PT) modification of the DNA backbone. Unlike classical R-M systems, highly partial PT-modification of consensus motifs in bacterial genomes suggests an unusual mechanism of PT-dependent restriction. In Salmonella enterica, PT modification is mediated by four genes dptB-E, while restriction involves additional three genes dptF-H. Here, we performed a series of studies to characterize the PT-dependent restriction, and found that it presented several features distinct with traditional R-M systems. The presence of restriction genes in a PT-deficient mutant was not lethal, but instead resulted in several pathological phenotypes. Subsequent transcriptional profiling revealed the expression of >600 genes was affected by restriction enzymes in cells lacking PT, including induction of bacteriophage, SOS response and DNA repair-related genes. These transcriptional responses are consistent with the observation that restriction enzymes caused extensive DNA cleavage in the absence of PT modifications in vivo. However, over-expression of restriction genes was lethal to the host in spite of the presence PT modifications. These results point to an unusual mechanism of PT-dependent DNA cleavage by restriction enzymes in the face of partial PT modification. PMID:25040300

  16. The AT-Hook motif as a versatile minor groove anchor for promoting DNA binding of transcription factor fragments

    PubMed Central

    Rodríguez, Jéssica; Mosquera, Jesús; Couceiro, Jose R.; Vázquez, M. Eugenio; Mascareñas, José L.

    2015-01-01

    We report the development of chimeric DNA binding peptides comprising a DNA binding fragment of natural transcription factors (the basic region of a bZIP protein or a monomeric zinc finger module) and an AT-Hook peptide motif. The resulting peptide conjugates display high DNA affinity and excellent sequence selectivity. Furthermore, the AT-Hook motif also favors the cell internalization of the conjugates. PMID:26290687

  17. 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.

  18. 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 ); Naruke, Tsuguo; Okabayashi, Kenzo )

    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.

  19. 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

  20. The Evolution of Restricted Recombination and the Accumulation of Repeated DNA Sequences

    PubMed Central

    Charlesworth, Brian; Langley, Charles H.; Stephan, Wolfgang

    1986-01-01

    We suggest hypotheses to account for two major features of chromosomal organization in higher eukaryotes. The first of these is the general restriction of crossing over in the neighborhood of centromeres and telomeres. We propose that this is a consequence of selection for reduced rates of unequal exchange between repeated DNA sequences for which the copy number is subject to stabilizing selection: microtubule binding sites, in the case of centromeres, and the short repeated sequences needed for terminal replication of a linear DNA molecule, in the case of telomeres. An association between proximal crossing over and nondisjunction would also favor the restriction of crossing over near the centromere. The second feature is the association between highly repeated DNA sequences of no obvious functional significance and regions of restricted crossing over. We show that highly repeated sequences are likely to persist longest (over evolutionary time) when crossing over is infrequent. This is because unequal exchange among repeated sequences generates single copy sequences, and a population that becomes fixed for a single copy sequence by drift remains in this state indefinitely (in the absence of gene amplification processes). Increased rates of exchange thus speed up the process of stochastic loss of repeated sequences. PMID:3957013

  1. Improved Genotyping Vaccine and Wild-Type Poliovirus Strains by Restriction Fragment Length Polymorphism Analysis: Clinical Diagnostic Implications

    PubMed Central

    Georgopoulou, Amalia; Markoulatos, Panayotis; Spyrou, Niki; Vamvakopoulos, Nicholas C.

    2000-01-01

    The combination of preventive vaccination and diagnostic typing of viral isolates from patients with clinical poliomyelitis constitutes our main protective shield against polioviruses. The restriction fragment length polymorphism (RFLP) adaptation of the reverse transcriptase (RT)-PCR methodology has advanced diagnostic genotyping of polioviruses, although further improvements are definitely needed. We report here on an improved RFLP procedure for the genotyping of polioviruses. A highly conserved segment within the 5? noncoding region of polioviruses was selected for RT-PCR amplification by the UC53-UG52 primer pair with the hope that it would be most resistant to the inescapable genetic alteration-drift experienced by the other segments of the viral genome. Complete inter- and intratypic genotyping of polioviruses by the present RFLP method was accomplished with a minimum set of four restriction endonucleases (HaeIII, DdeI, NcoI, and AvaI). To compensate for potential genetic drift within the recognition sites of HaeIII, DdeI, or NcoI in atypical clinical samples, the RFLP patterns generated with HpaII and StyI as replacements were analyzed. The specificity of the method was also successfully assessed by RFLP analysis of 55 reference nonpoliovirus enterovirus controls. The concerted implementation of these conditional protocols for diagnostic inter- and intratypic genotyping of polioviruses was evaluated with 21 clinical samples with absolute success. PMID:11101561

  2. Evaluation of antiviral resistant hepatitis B virus subpopulations in patients with chronic hepatitis B by using terminal restriction fragment length polymorphism.

    PubMed

    ?ahin, Ergin

    2015-12-01

    Antiviral therapies with nucleotide analogues (NA) is crucial in the treatment of chronic hepatitis B as it substantially protects patients from the complications of the disease . However in most of the available NA therapies, resistance emerges in the patients' HBV populations. Therefore, detection of antiviral resistance as early as possible by means of genotypically monitoring the patients' HBV pool during NA therapy is critical to manage treatment regime. In this research study we have investigated the sensitivity and specificity of the terminal restriction fragment length polymorphism (T-RFLP) method in detecting HBV subpopulations carrying antiviral resistance mutations. For this aim, differentiation of mutant strains from wild type strains was demonstrated by PCR-RFLP method. With using recombinant plasmids containing mutant and wild type HBV genomes, we constructed artificial HBV genome populations in order to determine the sensitivity of PCR-T-RFLP method in detecting antiviral resistant minor HBV populations. Finally by comparing with the DNA sequencing method, we demonstrated the specificity of T-RFLP method in genotyping HBV populations. As a result we showed that T-RFLP is able to detect HBV subpopulations representing as low as 1 % of the whole viral population. Additionally T-RFLP showed 100 % concordance with the DNA sequencing method in genotyping HBV populations. As a conclusion, considering the other genotyping methods used in evaluating HBV populations, T-RFLP showed high sensitivity and specificity profiles in detecting antiviral resistant HBV subpopulations. Therefore T-RFLP method can be easily employed in genotypic evaluation of patients' HBV populations during the course of antiviral treatment. PMID:26645037

  3. 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

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

    PubMed Central

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

    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 simultaneously (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 Pfu polymerase mix, which has a proofreading activity. We successfully assembled (and confirmed by sequencing) seven different linear constructs ranging from 3 to 20 kb, including two 20 kb products (from fragments of 11, 1.7 and 7.5 kb), two 10.8 kb constructs, and two constructs of 6.1 and 6.2 kb, respectively. Accuracy of the PCR fusion is greater than or equal to one error per 6.6 kb, which is consistent with the expected error rate of the PCR mix. The method is expected to facilitate various kinds of complex genetic engineering projects that require precise in-frame assembly of multiple fragments, such as somatic cell knockout in human cells or creation of whole genomes of viruses for vaccine research. PMID:14739232

  5. Sex determination in highly fragmented human DNA by high-resolution melting (HRM) analysis.

    PubMed

    Álvarez-Sandoval, Brenda A; Manzanilla, Linda R; Montiel, Rafael

    2014-01-01

    Sex identification in ancient human remains is a common problem especially if the skeletons are sub-adult, incomplete or damaged. In this paper we propose a new method to identify sex, based on real-time PCR amplification of small fragments (61 and 64 bp) of the third exon within the amelogenin gene covering a 3-bp deletion on the AMELX-allele, followed by a High Resolution Melting analysis (HRM). HRM is based on the melting curves of amplified fragments. The amelogenin gene is located on both chromosomes X and Y, showing dimorphism in length. This molecular tool is rapid, sensitive and reduces the risk of contamination from exogenous genetic material when used for ancient DNA studies. The accuracy of the new method described here has been corroborated by using control samples of known sex and by contrasting our results with those obtained with other methods. Our method has proven to be useful even in heavily degraded samples, where other previously published methods failed. Stochastic problems such as the random allele drop-out phenomenon are expected to occur in a less severe form, due to the smaller fragment size to be amplified. Thus, their negative effect could be easier to overcome by a proper experimental design. PMID:25098828

  6. Sex Determination in Highly Fragmented Human DNA by High-Resolution Melting (HRM) Analysis

    PubMed Central

    Álvarez-Sandoval, Brenda A.; Manzanilla, Linda R.; Montiel, Rafael

    2014-01-01

    Sex identification in ancient human remains is a common problem especially if the skeletons are sub-adult, incomplete or damaged. In this paper we propose a new method to identify sex, based on real-time PCR amplification of small fragments (61 and 64 bp) of the third exon within the amelogenin gene covering a 3-bp deletion on the AMELX-allele, followed by a High Resolution Melting analysis (HRM). HRM is based on the melting curves of amplified fragments. The amelogenin gene is located on both chromosomes X and Y, showing dimorphism in length. This molecular tool is rapid, sensitive and reduces the risk of contamination from exogenous genetic material when used for ancient DNA studies. The accuracy of the new method described here has been corroborated by using control samples of known sex and by contrasting our results with those obtained with other methods. Our method has proven to be useful even in heavily degraded samples, where other previously published methods failed. Stochastic problems such as the random allele drop-out phenomenon are expected to occur in a less severe form, due to the smaller fragment size to be amplified. Thus, their negative effect could be easier to overcome by a proper experimental design. PMID:25098828

  7. Translocation-coupled DNA cleavage by the Type ISP restriction-modification enzymes.

    PubMed

    Chand, Mahesh K; Nirwan, Neha; Diffin, Fiona M; van Aelst, Kara; Kulkarni, Manasi; Pernstich, Christian; Szczelkun, Mark D; Saikrishnan, Kayarat

    2015-11-01

    Production of endonucleolytic double-strand DNA breaks requires separate strand cleavage events. Although catalytic mechanisms for simple, dimeric endonucleases are known, there are many complex nuclease machines that are poorly understood. Here we studied the single polypeptide Type ISP restriction-modification (RM) enzymes, which cleave random DNA between distant target sites when two enzymes collide after convergent ATP-driven translocation. We report the 2.7-Å resolution X-ray crystal structure of a Type ISP enzyme-DNA complex, revealing that both the helicase-like ATPase and nuclease are located upstream of the direction of translocation, an observation inconsistent with simple nuclease-domain dimerization. Using single-molecule and biochemical techniques, we demonstrate that each ATPase remodels its DNA-protein complex and translocates along DNA without looping it, leading to a collision complex in which the nuclease domains are distal. Sequencing of the products of single cleavage events suggests a previously undescribed endonuclease model, where multiple, stochastic strand-nicking events combine to produce DNA scission. PMID:26389736

  8. Validation of meat inspection results for Taenia saginata cysticercosis by PCR-restriction fragment length polymorphism.

    PubMed

    Geysen, Dirk; Kanobana, Kirezi; Victor, Bjorn; Rodriguez-Hidalgo, Richar; De Borchgrave, Jean; Brandt, Jef; Dorny, Pierre

    2007-01-01

    Bovine cysticercosis is a zoonosis caused by the larval stage (cysticercus) of the human tapeworm Taenia saginata. Infected cattle is an important food safety issue besides an economic concern. Humans get infected by eating raw or undercooked meat containing viable cysticerci. Visual meat inspection of bovines is the only public health measure implemented to control transmission to humans, but it lacks sensitivity and objectivity. It may underestimate the prevalence of the disease by a factor 3 to 10. Furthermore, the success of the method depends on the expertise of the meat inspector as well as on the stage of development of the cysticerci. The focus of this study was to develop and explore the usefulness of a PCR assay as an objective alternative to evaluate the meat inspector's visual inspection results. Hereto, a PCR was developed for the detection of T. saginata DNA in muscle lesions. Based on the laboratory classification of lesions, almost 97% of viable cysts were confirmed by PCR, while for dead cysts, the percentage was approximately 73%. Taken together, these data demonstrate the difficulties of visual meat inspection and their objective interpretation, emphasizing the need to improve current assays to strengthen the control of bovine cysticercosis. PMID:17265888

  9. Exhaustive de novo design of low-molecular-weight fragments against the ATP-binding site of DNA-gyrase.

    PubMed

    Firth-Clark, Stuart; Todorov, Nikolay P; Alberts, Ian L; Williams, Anthony; James, Timothy; Dean, Philip M

    2006-01-01

    We present a de novo design approach to generating small fragments in the DNA-gyrase ATP-binding site using the computational drug design platform SkelGen. We have generated an exhaustive number of structural possibilities, which were subsequently filtered for site complementarity and synthetic tractability. A number of known active fragments are found, but most of the species created are potentially novel and could be valuable for further elaboration and development into lead-like structures. PMID:16711736

  10. Novel apparatus to measure hyperthermal heavy ion damage to DNA: Strand breaks, base loss, and fragmentation

    NASA Astrophysics Data System (ADS)

    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?g range) spread out over a large surface area (42cm2) with low energy (kT-100eV) 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., 2.5eV/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.

  11. Novel apparatus to measure hyperthermal heavy ion damage to DNA: Strand breaks, base loss, and fragmentation

    SciTech Connect

    Sellami, L.; Lacombe, S.; Hunting, D.; Wagner, R. J.; Huels, M. A.

    2007-08-15

    We have developed a novel apparatus that allows us to irradiate nonvolatile organic films of high mass (1-100 {mu}g range) spread out over a large surface area (42 cm{sup 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{sup +} 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.

  12. Microchip-based terminal restriction fragment length polymorphism for on-site analysis of bacterial communities in freshwater.

    PubMed

    Yamaguchi, Nobuyasu; Matsukawa, Syuhei; Shintome, Yoko; Ichijo, Tomoaki; Nasu, Masao

    2013-01-01

    Assessing microbiological quality assurance by monitoring bacteria in various sources of freshwater used for human consumption, recreation, and food preparation is important for a healthy life. Bacterial number and their community structure in freshwater should be determined as quickly as possible, and "real-time" and "on-site" microbiological methods are required. In this study, we examined the protocol for microchip-based terminal restriction fragment length polymorphism (T-RFLP) analysis, which uses microchip electrophoresis for rapid microbial community analysis. The availability of microchip-based T-RFLP was compared with conventional T-RFLP analysis, which uses a capillary electrophoresis system, with freshwater samples (spring water, river water, groundwater, and hydroponics solution). The detection limit of targeted bacteria by on-chip T-RFLP analysis was 1% (10(3) cells/mL). The fragment sizes determined by the two analysis methods were highly correlated (r(2)=0.98). On-chip T-RFLP analysis was completed within 15 min. T-RFLP profiles of nine hydroponics solution samples were analyzed by multidimensional scaling. Considerable changes and stability in bacterial community structure during hydroponic culture were detected by both analyses. These results show that on-chip T-RFLP analysis can monitor changes in bacterial community structure, as well as conventional T-RFLP analysis. The present results indicate that on-chip T-RFLP analysis is an effective tool for rapid and "on-site" bacterial community profiling in freshwater environments, as well as freshwater used for medical and industrial purposes. PMID:23902975

  13. DNA amplification-restricted transcription-translation: rapid analysis of rhesus rotavirus neutralization sites.

    PubMed Central

    Mackow, E R; Yamanaka, M Y; Dang, M N; Greenberg, H B

    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 rhesus rotavirus gene segment 4 cDNA in order to create a series of carboxyl-terminal truncations and new amino termini in the encoded VP4 capsid protein. The truncated VP4 polypeptides were tested for reaction with 11 VP4-specific neutralizing monoclonal antibodies to identify the minimum polypeptides required for antibody recognition. Monoclonal antibodies 2G4, M2, and M7, which neutralize a number of serologically distinct rotaviruses, required amino acids 247-474 of VP4 for binding. DARTT is potentially applicable to the identification of discontinuous epitopes and functional domains on a variety of proteins. Images PMID:2153955

  14. PyroTRF-ID: a novel bioinformatics methodology for the affiliation of terminal-restriction fragments using 16S rRNA gene pyrosequencing data

    PubMed Central

    2012-01-01

    Background In molecular microbial ecology, massive sequencing is gradually replacing classical fingerprinting techniques such as terminal-restriction fragment length polymorphism (T-RFLP) combined with cloning-sequencing for the characterization of microbiomes. Here, a bioinformatics methodology for pyrosequencing-based T-RF identification (PyroTRF-ID) was developed to combine pyrosequencing and T-RFLP approaches for the description of microbial communities. The strength of this methodology relies on the identification of T-RFs by comparison of experimental and digital T-RFLP profiles obtained from the same samples. DNA extracts were subjected to amplification of the 16S rRNA gene pool, T-RFLP with the HaeIII restriction enzyme, 454 tag encoded FLX amplicon pyrosequencing, and PyroTRF-ID analysis. Digital T-RFLP profiles were generated from the denoised full pyrosequencing datasets, and the sequences contributing to each digital T-RF were classified to taxonomic bins using the Greengenes reference database. The method was tested both on bacterial communities found in chloroethene-contaminated groundwater samples and in aerobic granular sludge biofilms originating from wastewater treatment systems. Results PyroTRF-ID was efficient for high-throughput mapping and digital T-RFLP profiling of pyrosequencing datasets. After denoising, a dataset comprising ca. 10?000 reads of 300 to 500 bp was typically processed within ca. 20 minutes on a high-performance computing cluster, running on a Linux-related CentOS 5.5 operating system, enabling parallel processing of multiple samples. Both digital and experimental T-RFLP profiles were aligned with maximum cross-correlation coefficients of 0.71 and 0.92 for high- and low-complexity environments, respectively. On average, 63±18% of all experimental T-RFs (30 to 93 peaks per sample) were affiliated to phylotypes. Conclusions PyroTRF-ID profits from complementary advantages of pyrosequencing and T-RFLP and is particularly adapted for optimizing laboratory and computational efforts to describe microbial communities and their dynamics in any biological system. The high resolution of the microbial community composition is provided by pyrosequencing, which can be performed on a restricted set of selected samples, whereas T-RFLP enables simultaneous fingerprinting of numerous samples at relatively low cost and is especially adapted for routine analysis and follow-up of microbial communities on the long run. PMID:23270314

  15. DNA Methylation Pattern in Overweight Women under an Energy-Restricted Diet Supplemented with Fish Oil

    PubMed Central

    do Amaral, Cátia Lira; Milagro, Fermín I.; Curi, Rui; Martínez, J. Alfredo

    2014-01-01

    Dietary factors modulate gene expression and are able to alter epigenetic signatures in peripheral blood mononuclear cells (PBMC). However, there are limited studies about the effects of omega-3 polyunsaturated fatty acids (n-3 PUFA) on the epigenetic mechanisms that regulate gene expression. This research investigates the effects of n-3-rich fish oil supplementation on DNA methylation profile of several genes whose expression has been reported to be downregulated by n-3 PUFA in PBMC: CD36, FFAR3, CD14, PDK4, and FADS1. Young overweight women were supplemented with fish oil or control in a randomized 8-week intervention trial following a balanced diet with 30% energy restriction. Fatty acid receptor CD36 decreased DNA methylation at CpG +477 due to energy restriction. Hypocaloric diet-induced weight loss also reduced the methylation percentages of CpG sites located in CD14, PDK4, and FADS1. The methylation patterns of these genes were only slightly affected by the fish oil supplementation, being the most relevant to the attenuation of the weight loss-induced decrease in CD36 methylation after adjusting by baseline body weight. These results suggest that the n-3 PUFA-induced changes in the expression of these genes in PBMC are not mediated by DNA methylation, although other epigenetic mechanisms cannot be discarded. PMID:24579084

  16. Rational engineering of type II restriction endonuclease DNA binding and cleavage specificity

    PubMed Central

    Morgan, Richard D.; Luyten, Yvette A.

    2009-01-01

    The type II restriction endonucleases are indispensible tools for molecular biology. Although enzymes recognizing nearly 300 unique sequences are known, the ability to engineer enzymes to recognize any sequence of choice would be valuable. However, previous attempts to engineer new recognition specificity have met limited success. Here we report the rational engineering of multiple new type II specificities. We recently identified a family of MmeI-like type II endonucleases that have highly similar protein sequences but different recognition specificity. We identified the amino-acid positions within these enzymes that determine position specific DNA base recognition at three positions within their recognition sequences through correlations between their aligned amino-acid residues and aligned recognition sequences. We then altered the amino acids at the identified positions to those correlated with recognition of a desired new base to create enzymes that recognize and cut at predictable new DNA sequences. The enzymes so altered have similar levels of endonuclease activity compared to the wild-type enzymes. Using simple and predictable mutagenesis in this family it is now possible to create hundreds of unique new type II restriction endonuclease specificities. The findings suggest a simple mechanism for the evolution of new DNA specificity in Nature. PMID:19567736

  17. Determination of locust bean gum and guar gum by polymerase chain reaction and restriction fragment length polymorphism analysis.

    PubMed

    Meyer, K; Rosa, C; Hischenhuber, C; Meyer, R

    2001-01-01

    A polymerase chain reaction (PCR) was developed to differentiate the thickening agents locust bean gum (LBG) and the cheaper guar gum in finished food products. Universal primers for amplification of the intergenic spacer region between trnL 3' (UAA) exon and trnF (GAA) gene in the chloroplast (cp) genome and subsequent restriction analysis were applied to differentiate guar gum and LBG. The presence of <5% (w/w) guar gum powder added to LBG powder was detectable. Based on data obtained from sequencing this intergenic spacer region, a second PCR method for the specific detection of guar gum DNA was also developed. This assay detected guar gum powder in LBG in amounts as low as 1% (w/w). Both methods successfully detected guar gum and/or LBG in ice cream stabilizers and in foodstuffs, such as dairy products, ice cream, dry seasoning mixes, a finished roasting sauce, and a fruit jelly product, but not in products with highly degraded DNA, such as tomato ketchup and sterilized chocolate cream. Both methods detected guar gum and LBG in ice cream and fresh cheese at levels <0.1%. PMID:11234856

  18. 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

  19. A DNA fragment from the cyanobacterium Synechocystis sp. PCC 6803 mediates gene expression inducible by osmotic stress in E. coli.

    PubMed

    Milkowski, C; Quinones, A; Hagemann, M

    1998-08-01

    Fragments of Synechocystis-DNA driving salt-induced gene expression in E. coli were isolated with translational fusions to a 'lacZ gene. One fragment (fragment 19) showed a NaCl-dependent activation of betaGal expression with the maximum of a ninefold increase in enzyme activity. A similar induction was triggered by the nonionic osmolyte sucrose, indicating an osmotically dependent activation. On the contrary, transcriptional activity of the DNA fragment 19 was only slightly enhanced under salt stress conditions, suggesting a posttranscriptional mechanism of induction. Primer extension assay was performed to identify the transcription initiation site. Upstream regions share weak homology to the "-10" hexamer consensus of E. coli sigma70 promoters. The most thermodynamically stable secondary structure for the nontranslated part of the mRNA indicated that potential translation initiation sites might be blocked, leading to a low basal translation, whereas osmotic stress-induced changes of mRNA structure could be involved to increase translation. In order to analyze the function of fragment 19 in Synechocystis, promoter-probe plasmids were constructed allowing the stable integration of transcriptional and translational reporter gene fusions into the cyanobacterial chromosome. Quantitative assessment of reporter gene expression revealed a weak constitutive promoter activity of fragment 19 in Synechocystis. Sequence analysis showed that fragment 19 comprises 223 bp of the ORF sll0747 of the Synechocystis genome. PMID:9662610

  20. Mechanism of DNA Recognition by the Restriction Enzyme EcoRV

    SciTech Connect

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

    2010-08-01

    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 degrees 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 by the enzyme by performing all-atom molecular dynamics simulations of EcoRV unbound and interacting with three DNA sequences: the cognate sequence, GATATC (TA); the non-cognate sequence, GAATTC (AT); and with the cognate sequence methylated on the first adenine GA(CH(3))TATC (TA-CH(3)). In the unbound EcoRV, the cleft between the two C-terminal subdomains is found to be open. Binding to AT narrows the cleft and forms a partially bound state. However, the intrinsic bending propensity of AT is insufficient to allow tight binding. In contrast, the cognate TA sequence is easier to bend, allowing specific, high-occupancy hydrogen bonds to form in the complex. The absence of cleavage for this methylated sequence is found to arise from the loss of specific hydrogen bonds between the first adenine of the recognition sequence and Asn185. On the basis of the results, we suggest a three-step recognition mechanism. In the first step, EcoRV, in an open conformation, binds to the DNA at a random sequence and slides along it. In the second step, when the two outer base pairs, GAxxTC, are recognized, the R loops of the protein become more ordered, forming strong hydrogen-bonding interactions, resulting in a partially bound EcoRV-DNA complex. In the third step, the flexibility of the center base pair is probed, and in the case of the full cognate sequence the DNA bends, the complex strengthens and the protein and DNA interact more closely, allowing cleavage.

  1. NEAFS CE-DNA Workshop (Butler and Sept 29-30, 2004

    E-print Network

    - Restriction enzymes begin to be used to cut DNA · 1981 - Jorgenson performs CZE · 1984 - Alec Jeffries utilizes restriction fragment digestion of DNA · 1985 - Kary Mullis describes PCR DNA Analysis by RFLP "I polyacrylamide to separate restriction digests · Commercial CE systems (1992) with lasers appear (Beckman P

  2. Calorimetric and Low-Frequency Dielectric Studies of Mesoscopic Ordering in Solutions of Engineered DNA Hairpin Fragments

    NASA Astrophysics Data System (ADS)

    Kashuri, K.; Kashuri, H.; Iannacchione, G. S.

    2012-02-01

    Calorimetry (both AC and MDSC) from 20 to 100 ^oC, as well as low-frequency (0.1 to 100 kHz) isothermal dielectric measurements have been performed on solutions of DNA fragments as a function of concentration. Custom hairpin DNA fragments were obtained with 13-base unit length and samples made in solution at various concentration. Results show a reproducible heat capacity Cp signature on heating and cooling scans. This thermal behavior of a diluted oligonucleotide chain is very different from that seen for mesoscopic ordering of liquid crystals. The AC Cp peak vanishes and new features are revealed as the temperature scan rate is lowered to 0.017 K min-1. The observed real, ?', and imaginary, ?'', permittivity of the suspended DNA show features indicating low-frequency dynamics that in turn suggests large-scale ordering or agglomeration of the DNA hairpin loops.

  3. Polymerase Synthesis and Restriction Enzyme Cleavage of DNA Containing 7-Substituted 7-Deazaguanine Nucleobases.

    PubMed

    Ma?ková, Michaela; Bohá?ová, So?a; Perlíková, Pavla; Poštová Slav?tínská, Lenka; Hocek, Michal

    2015-10-01

    Previous studies of polymerase synthesis of base-modified DNAs and their cleavage by restriction enzymes have mostly related only to 5-substituted pyrimidine and 7-substituted 7-deazaadenine nucleotides. Here we report the synthesis of a series of 7-substituted 7-deazaguanine 2'-deoxyribonucleoside 5'-O-triphosphates (dG(R) TPs), their use as substrates for polymerase synthesis of modified DNA and the influence of the modification on their cleavage by type?II restriction endonucleases (REs). The dG(R) TPs were generally good substrates for polymerases but the PCR products could not be visualised on agarose gels by intercalator staining, due to fluorescence quenching. The presence of 7-substituted 7-deazaguanine residues in recognition sequences of REs in most cases completely blocked the cleavage. PMID:26382079

  4. Comparative assessment of next-generation sequencing, denaturing gradient gel electrophoresis, clonal restriction fragment length polymorphism and cloning-sequencing as methods for characterizing commercial microbial consortia.

    PubMed

    Samarajeewa, A D; Hammad, A; Masson, L; Khan, I U H; Scroggins, R; Beaudette, L A

    2015-01-01

    Characterization of commercial microbial consortia products for human and environmental health risk assessment is a major challenge for regulatory agencies. As a means to develop an approach to assess the potential environmental risk of these products, research was conducted to compare four genomics methods for characterizing bacterial communities; (i) Denaturing Gradient Gel Electrophoresis (DGGE), (ii) Clonal-Restriction Fragment Length Polymorphism (C/RFLP), (iii) partial 16S rDNA amplification, cloning followed by Sanger sequencing (PRACS) and (iv) Next-Generation Sequencing (NGS) based on Ion Torrent technology. A commercially available microbial consortium, marketed as a remediation agent for degrading petroleum hydrocarbon contamination in soil and water, was assessed. The bacterial composition of the commercial microbial product was characterized using the above four methods. PCR amplification of 16S rDNA was performed targeting the variable region V6 for DGGE, C/RFLP and PRACS and V5 for Ion Torrent sequencing. Ion Torrent technology was shown to be a promising tool for initial screening by detecting the majority of bacteria in the consortium that were also detected by DGGE, C/RFLP and PRACS. Additionally, Ion Torrent sequencing detected some of the bacteria that were claimed to be in the product, while three other methods failed to detect these specific bacteria. However, the relative proportions of the microbial composition detected by Ion Torrent were found to be different from DGGE, C/RFLP and PRACS, which gave comparable results across these three methods. The discrepancy of the Ion Torrent results may be due to the short read length generated by this technique and the targeting of different variable regions on the 16S rRNA gene used in this study. Arcobacter spp. a potential pathogenic bacteria was detected in the product by all methods, which was further confirmed using genus and species-specific PCR, RFLP and DNA-based sequence analyses. However, the viability of Arcobacter spp. was not confirmed. This study suggests that a combination of two or more methods may be required to ascertain the microbial constituents of a commercial microbial consortium reliably and for the presence of potentially human pathogenic contaminants. PMID:25479430

  5. Roles for Pbp1 and caloric restriction in genome and lifespan maintenance via suppression of RNA-DNA hybrids.

    PubMed

    Salvi, Jayesh S; Chan, Janet N Y; Szafranski, Kirk; Liu, Tony T; Wu, Jane D; Olsen, Jonathan B; Khanam, Nurussaba; Poon, Betty P K; Emili, Andrew; Mekhail, Karim

    2014-07-28

    Intergenic transcription within repetitive loci such as the ribosomal DNA (rDNA) repeats of yeast commonly triggers aberrant recombination. Major mechanisms suppressing aberrant rDNA recombination rely on chromatin silencing or RNAPII repression at intergenic spacers within the repeats. We find ancient processes operating at rDNA intergenic spacers and other loci to maintain genome stability via repression of RNA-DNA hybrids. The yeast Ataxin-2 protein Pbp1 binds noncoding RNA, suppresses RNA-DNA hybrids, and prevents aberrant rDNA recombination. Repression of RNA-DNA hybrids in Pbp1-deficient cells through RNaseH overexpression, deletion of the G4DNA-stabilizing Stm1, or caloric restriction operating via RNaseH/Pif1 restores rDNA stability. Pbp1 also limits hybrids at non-rDNA G4DNA loci including telomeres. Moreover, cells lacking Pbp1 have a short replicative lifespan that is extended upon hybrid suppression. Thus, we find roles for Pbp1 in genome maintenance and reveal that caloric restriction counteracts Pbp1 deficiencies by engaging RNaseH and Pif1. PMID:25073155

  6. An Improved PCR-Restriction Fragment Length Polymorphism (RFLP) Method for the Identification of cry1-Type Genes

    PubMed Central

    Shu, Changlong; Liu, Dongming; Zhou, Zishan; Cai, Jilin; Peng, Qi; Gao, Jiguo; Song, Fuping

    2013-01-01

    The cry1-type genes of Bacillus thuringiensis represent the largest cry gene family, which contains 50 distinct holotypes. It is becoming more and more difficult to identify cry1-type genes using current methods because of the increasing number of cry1-type genes. In the present study, an improved PCR-restriction fragment length polymorphism (PCR-RFLP) method which can distinguish 41 holotypes of cry1-type genes was developed. This improved method was used to identify cry1-type genes in 20 B. thuringiensis strains that are toxic to lepidoptera. The results showed that the improved method can efficiently identify single and clustered cry1-type genes and can be used to evaluate cry1-type genes in novel strain collections of B. thuringiensis. Among the detected cry1-type genes, we identified four novel genes, cry1Ai, cry1Bb, cry1Ja, and cry1La. The bioassay results from the expressed products of the four novel cry genes showed that Cry1Ai2, Cry1Bb2, and Cry1Ja2 were highly toxic against Plutella xylostella, whereas Cry1La2 exhibited no activity. Moreover, Cry1Ai2 had good lethal activity against Ostrinia furnacalis, Hyphantria cunea, Chilo suppressalis, and Bombyx mori larvae and considerable weight loss activity against Helicoverpa armigera. PMID:23995930

  7. 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.

    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.

  8. 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.

  9. A DNA metabarcoding study of a primate dietary diversity and plasticity across its entire fragmented range.

    PubMed

    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

  10. Single-stranded DNA fragments of insect-specific nuclear polyhedrosis virus act as selective DNA insecticides for gypsy moth control.

    PubMed

    Oberemok, Volodymyr V; Skorokhod, Oleksii A

    2014-07-01

    This paper focuses on the DNA insecticides as a novel preparation against gypsy moth (Lymantria dispar) based on DNA fragments of the anti-apoptotic gene of its nuclear polyhedrosis virus. It was found that the external application of a solution with two single-stranded DNA fragments from BIR and RING domains of LdMNPV (L.dispar multicapsid nuclear polyhedrosis virus) IAP-3 (inhibitor of apoptosis) gene induces a significantly higher mortality of gypsy moth caterpillars in comparison with the application of the control solutions. This effect does not depend on the infection of caterpillars with LdMNPV. The results also show that DNA insecticides based on LdMNPV IAP-3 gene fragments can be selective in action, and at least are not harmful to tobacco hornworm (Manduca sexta) and black cutworm (Agrotis ipsilon). Part of the gypsy moth genome cloned with the fragments of BIR and RING domains of LdMNPV IAP-3 gene as primers, has an overlap with the corresponding part of the LdMNPV IAP-3 gene and L.dispar IAP-1 mRNA for an inhibitor of apoptosis protein with the high cover by query, allows assuming that we cloned a part of gypsy moth anti-apoptosis gene. This finding gives the grounding that proposed here DNA insecticides might act through the blocking of the mechanisms involved in post transcriptional expression of insect anti-apoptosis genes. The results show the insecticidal potential of the viral genome fragments that can be used to create safe and relatively fast-acting DNA insecticides to control the quantity of gypsy moth populations, important task for forestry and agriculture. PMID:25052520

  11. 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

    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.

  12. Variation of Clonal, Mesquite-Associated Rhizobial and Bradyrhizobial Populations from Surface and Deep Soils by Symbiotic Gene Region Restriction Fragment Length Polymorphism and Plasmid Profile Analysis

    PubMed Central

    Thomas, P. M.; Golly, K. F.; Zyskind, J. W.; Virginia, R. A.

    1994-01-01

    Genetic characteristics of 14 Rhizobium and 9 Bradyrhizobium mesquite (Prosopis glandulosa)-nodulating strains isolated from surface (0- to 0.5-m) and deep (4- to 6-m) rooting zones were determined in order to examine the hypothesis that surface- and deep-soil symbiont populations were related but had become genetically distinct during adaptation to contrasting soil conditions. To examine genetic diversity, Southern blots of PstI-digested genomic DNA were sequentially hybridized with the nodDABC region of Rhizobium meliloti, the Klebsiella pneumoniae nifHDK region encoding nitrogenase structural genes, and the chromosome-localized ndvB region of R. meliloti. Plasmid profile and host plant nodulation assays were also made. Isolates from mesquite nodulated beans and cowpeas but not alfalfa, clover, or soybeans. Mesquite was nodulated by diverse species of symbionts (R. meliloti, Rhizobium leguminosarum bv. phaseoli, and Parasponia bradyrhizobia). There were no differences within the groups of mesquite-associated rhizobia or bradyrhizobia in cross-inoculation response. The ndvB hybridization results showed the greatest genetic diversity among rhizobial strains. The pattern of ndvB-hybridizing fragments suggested that surface and deep strains were clonally related, but groups of related strains from each soil depth could be distinguished. Less variation was found with nifHDK and nodDABC probes. Large plasmids (>1,500 kb) were observed in all rhizobia and some bradyrhizobia. Profiles of plasmids of less than 1,000 kb were related to the soil depth and the genus of the symbiont. We suggest that interacting selection pressures for symbiotic competence and free-living survival, coupled with soil conditions that restrict genetic exchange between surface and deep-soil populations, led to the observed patterns of genetic diversity. Images PMID:16349226

  13. Comparison of human gut microbiota in control subjects and patients with colorectal carcinoma in adenoma: Terminal restriction fragment length polymorphism and next-generation sequencing analyses.

    PubMed

    Kasai, Chika; Sugimoto, Kazushi; Moritani, Isao; Tanaka, Junichiro; Oya, Yumi; Inoue, Hidekazu; Tameda, Masahiko; Shiraki, Katsuya; Ito, Masaaki; Takei, Yoshiyuki; Takase, Kojiro

    2016-01-01

    Colorectal cancer (CRC) is the third leading cause of cancer-related deaths in Japan. The etiology of CRC has been linked to numerous factors including genetic mutation, diet, life style, inflammation, and recently, the gut microbiota. However, CRC-associated gut microbiota is still largely unexamined. This study used terminal restriction fragment length polymorphism (T-RFLP) and next-generation sequencing (NGS) to analyze and compare gut microbiota of Japanese control subjects and Japanese patients with carcinoma in adenoma. Stool samples were collected from 49 control subjects, 50 patients with colon adenoma, and 9 patients with colorectal cancer (3/9 with invasive cancer and 6/9 with carcinoma in adenoma) immediately before colonoscopy; DNA was extracted from each stool sample. Based on T-RFLP analysis, 12 subjects (six control and six carcinoma in adenoma subjects) were selected; their samples were used for NGS and species-level analysis. T-RFLP analysis showed no significant differences in bacterial population between control, adenoma and cancer groups. However, NGS revealed that i), control and carcinoma in adenoma subjects had different gut microbiota compositions, ii), one bacterial genus (Slackia) was significantly associated with the control group and four bacterial genera (Actinomyces, Atopobium, Fusobacterium, and Haemophilus) were significantly associated with the carcinoma-in-adenoma group, and iii), several bacterial species were significantly associated with each type (control: Eubacterium coprostanoligens; carcinoma in adenoma: Actinomyces odontolyticus, Bacteroides fragiles, Clostridium nexile, Fusobacterium varium, Haemophilus parainfluenzae, Prevotella stercorea, Streptococcus gordonii, and Veillonella dispar). Gut microbial properties differ between control subjects and carcinoma-in-adenoma patients in this Japanese population, suggesting that gut microbiota is related to CRC prevention and development. PMID:26549775

  14. DNA fragmentation is increased in non-GABAergic neurons in bipolar disorder but not in schizophrenia

    PubMed Central

    Buttner, Ned; Bhattacharyya, Sujoy; Walsh, John; Benes, Francine M.

    2007-01-01

    Apoptosis is thought to contribute to neuronal loss in bipolar disorder and schizophrenia, although empiric evidence in support of this idea has been lacking. In this study, we investigated whether or not apoptosis is associated with GABAergic interneurons in the anterior cingulate cortex in schizophrenia (n = 14) and bipolar disorder (n = 14) when compared to normal controls (n = 14). A double-labeling technique using the Klenow method of in situ end-labeling (ISEL) of single-stranded DNA breaks was combined with an in situ hybridization localization of mRNA for the 67 kiloDalton (kDa) isoform of glutamate decarboxylase (GAD67) and applied to the anterior cingulate cortex of 14 normal controls, 14 schizophrenics, and 14 patients with bipolar disorder matched for age and postmortem interval. An increase in Klenow-positive, GAD67-negative nuclei was observed in layer V/VI of patients with bipolar disorder, but not schizophrenics. Klenow-positive cells that were also positive for GAD67 mRNA did not show differences in either patient group. Conclusions: This is the first demonstration that there is more DNA fragmentation in cells showing no detectable GAD67 mRNA in patients with bipolar disorder than in schizophrenics or controls. These findings suggest that non-GABAergic cells may be selectively vulnerable to oxidative stress in patients with bipolar disorder. PMID:17442540

  15. Transfer of recombinant plasmids containing the gene for DpnII DNA methylase into strains of Streptococcus pneumoniae that produce DpnI or DpnII restriction endonucleases

    SciTech Connect

    Lacks, S.A.; Springhorn, S.S.

    1984-06-01

    Plasmid transfer via the transformation pathway of Streptococcus pneumoniae was weakly restricted by the DpnI or DpnII restriction endonuclease, either of which gave a reduction only to 0.4, compared with phage infection, which was restricted to 10/sup -5/. The greater sensitivity of plasmid transfer compared with chromosomal transformation, which was not at all restricted, can be attributed to partially double-stranded intermediates formed from two complementary donor fragments. However, clustering of potential restriction sites in the plasmids increased the probability of escape from restriction. The recombinant plasmid pMP10, in which the gene for the DpnII DNA methylase was cloned, can be transferred to strains that contain neither restriction enzyme or that contain DpnII as readily as can the vector pMP5. Introduction of pMP10 raised the level of methylase by five times the level normally present in DpnII strains. Transfer of pMP10 to DpnI-containing strains was infrequent, presumably owing to the suicidal methylation of DNA which rendered it susceptible to the host endonuclease. The few clones in which pMP10 was established had lost DpnI. Loss of the plasmid after curing of the cell eliminated the methylase but did not restore DpnI. Although this loss of DpnI could result from spontaneous mutations, its relatively high frequency, 0.1% suggested that the loss was due to a regulatory shift.

  16. Creation of chimeric human/rabbit APOBEC1 with HIV-1 restriction and DNA mutation activities.

    PubMed

    Ikeda, Terumasa; Ong, Eugene Boon Beng; Watanabe, Nobumoto; Sakaguchi, Nobuo; Maeda, Kazuhiko; Koito, Atsushi

    2016-01-01

    APOBEC1 (A1) proteins from lagomorphs and rodents have deaminase-dependent restriction activity against HIV-1, whereas human A1 exerts a negligible effect. To investigate these differences in the restriction of HIV-1 by A1 proteins, a series of chimeric proteins combining rabbit and human A1s was constructed. Homology models of the A1s indicated that their activities derive from functional domains that likely act in tandem through a dimeric interface. The C-terminal region containing the leucine-rich motif and the dimerization domains of rabbit A1 is important for its anti-HIV-1 activity. The A1 chimeras with strong anti-HIV-1 activity were incorporated into virions more efficiently than those without anti-HIV-1 activity, and exhibited potent DNA-mutator activity. Therefore, the C-terminal region of rabbit A1 is involved in both its packaging into the HIV-1 virion and its deamination activity against both viral cDNA and genomic RNA. This study identifies the novel molecular mechanism underlying the target specificity of A1. PMID:26738439

  17. Cloning Should Be Simple: Escherichia coli DH5?-Mediated Assembly of Multiple DNA Fragments with Short End Homologies

    PubMed Central

    Richardson, Ruth E.; Suzuki, Yo

    2015-01-01

    Numerous DNA assembly technologies exist for generating plasmids for biological studies. Many procedures require complex in vitro or in vivo assembly reactions followed by plasmid propagation in recombination-impaired Escherichia coli strains such as DH5?, which are optimal for stable amplification of the DNA materials. Here we show that despite its utility as a cloning strain, DH5? retains sufficient recombinase activity to assemble up to six double-stranded DNA fragments ranging in size from 150 bp to at least 7 kb into plasmids in vivo. This process also requires surprisingly small amounts of DNA, potentially obviating the need for upstream assembly processes associated with most common applications of DNA assembly. We demonstrate the application of this process in cloning of various DNA fragments including synthetic genes, preparation of knockout constructs, and incorporation of guide RNA sequences in constructs for clustered regularly interspaced short palindromic repeats (CRISPR) genome editing. This consolidated process for assembly and amplification in a widely available strain of E. coli may enable productivity gain across disciplines involving recombinant DNA work. PMID:26348330

  18. Divergent dispersion behavior of ssDNA fragments during microchip electrophoresis in pDMA and LPA entangled polymer networks

    PubMed Central

    Fredlake, Christopher P.; Hert, Daniel G.; Niedringhaus, Thomas P.; Lin, Jennifer S.; Barron, Annelise E.

    2015-01-01

    Resolution of DNA fragments separated by electrophoresis in polymer solutions (“matrices”) is determined by both the spacing between peaks and the width of the peaks. Prior research on the development of high-performance separation matrices has been focused primarily on optimizing DNA mobility and matrix selectivity, and gave less attention to peak broadening. Quantitative data are rare for peak broadening in systems in which high electric field strengths are used (> 150 V/cm), which is surprising since capillary and microchip-based systems commonly run at these field strengths. Here, we report results for a study of band broadening behavior for ssDNA fragments on a glass microfluidic chip, for electric field strengths up to 320 V/cm. We compare dispersion coefficients obtained in a poly(N,N-dimethylacrylamide) (pDMA) separation matrix that was developed for chip-based DNA sequencing with a commercially available linear polyacrylamide (LPA) matrix commonly used in capillaries. Much larger DNA dispersion coefficients were measured in the LPA matrix as compared to the pDMA matrix, and the dependences of dispersion coefficient on DNA size and electric field strength were found to differ quite starkly in the two matrices. These observations lead us to propose that DNA migration mechanisms differ substantially in our custom pDMA matrix compared to the commercially available LPA matrix. We discuss the implications of these results in terms of developing optimal matrices for specific separation (microchip or capillary) platforms. PMID:22648809

  19. 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

  20. Detection and mapping of homologous, repeated and amplified DNA sequences by DNA renaturation in agarose gels.

    PubMed Central

    Roninson, I B

    1983-01-01

    A new molecular hybridization approach to the analysis of complex genomes has been developed. Tracer and driver DNAs were digested with the same restriction enzyme(s), and tracer DNA was labeled with 32P using T4 DNA polymerase. Tracer DNA was mixed with an excess amount of driver, and the mixture was electrophoresed in an agarose gel. Following electrophoresis, DNA was alkali-denatured in situ and allowed to reanneal in the gel, so that tracer DNA fragments could hybridize to the driver only when homologous driver DNA sequences were present at the same place in the gel, i.e. within a restriction fragment of the same size. After reannealing, unhybridized single-stranded DNA was digested in situ with S1 nuclease. The hybridized tracer DNA was detected by autoradiography. The general applicability of this technique was demonstrated in the following experiments. The common EcoRI restriction fragments were identified in the genomes of E. coli and four other species of bacteria. Two of these fragments are conserved in all Enterobacteriaceae. In other experiments, repeated EcoRI fragments of eukaryotic DNA were visualized as bands of various intensity after reassociation of a total genomic restriction digest in the gel. The situation of gene amplification was modeled by the addition of varying amounts of lambda phage DNA to eukaryotic DNA prior to restriction enzyme digestion. Restriction fragments of lambda DNA were detectable at a ratio of 15 copies per chicken genome and 30 copies per human genome. This approach was used to detect amplified DNA fragments in methotrexate (MTX)-resistant mouse cells and to identify commonly amplified fragments in two independently derived MTX-resistant lines. Images PMID:6310499

  1. Processive Incorporation of Deoxynucleoside Triphosphate Analogs by Single-Molecule DNA Polymerase I (Klenow Fragment) Nanocircuits.

    PubMed

    Pugliese, Kaitlin M; Gul, O Tolga; Choi, Yongki; Olsen, Tivoli J; Sims, Patrick C; Collins, Philip G; Weiss, Gregory A

    2015-08-01

    DNA polymerases exhibit a surprising tolerance for analogs of deoxyribonucleoside triphosphates (dNTPs), despite the enzymes' highly evolved mechanisms for the specific recognition and discrimination of native dNTPs. Here, individual DNA polymerase I Klenow fragment (KF) molecules were tethered to a single-walled carbon nanotube field-effect transistor (SWCNT-FET) to investigate accommodation of dNTP analogs with single-molecule resolution. Each base incorporation accompanied a change in current with its duration defined by ?closed. Under Vmax conditions, the average time of ?closed was similar for all analog and native dNTPs (0.2 to 0.4 ms), indicating no kinetic impact on this step due to analog structure. Accordingly, the average rates of dNTP analog incorporation were largely determined by durations with no change in current defined by ?open, which includes molecular recognition of the incoming dNTP. All ?-thio-dNTPs were incorporated more slowly, at 40 to 65% of the rate for the corresponding native dNTPs. During polymerization with 6-Cl-2APTP, 2-thio-dTTP, or 2-thio-dCTP, the nanocircuit uncovered an alternative conformation represented by positive current excursions that does not occur with native dNTPs. A model consistent with these results invokes rotations by the enzyme's O-helix; this motion can test the stability of nascent base pairs using nonhydrophilic interactions and is allosterically coupled to charged residues near the site of SWCNT attachment. This model with two opposing O-helix motions differs from the previous report in which all current excursions were solely attributed to global enzyme closure and covalent-bond formation. The results suggest the enzyme applies a dynamic stability-checking mechanism for each nascent base pair. PMID:26147714

  2. Protective roles of Gadd45 and MDM2 in blueberry anthocyanins mediated DNA repair of fragmented and non-fragmented DNA damage in UV-irradiated HepG2 cells.

    PubMed

    Liu, Wei; Lu, Xiangyi; He, Guangyang; Gao, Xiang; Xu, Maonian; Zhang, Jingkai; Li, Meiling; Wang, Lifeng; Li, Zhenjing; Wang, Likui; Luo, Cheng

    2013-01-01

    Growth Arrest and DNA Damage-inducible 45 (Gadd45) and MDM2 proteins, together with p21 and p53, play important roles in cell cycle checkpoints, DNA repair, and genome integrity maintenance. Gadd45 and MDM2 were activated and transcribed instantly by UV irradiation, whereas blueberry anthocyanins (BA) decreased the gene and protein expression levels in HepG2 cells for up to 24 h, and gradually restored the UV-induced fragmented and non-fragmented DNA damage of the nucleus at a time point of 12 h. Nevertheless, UV-irradiated HepG2 cell arrests occurred mainly in the G1 phase, which indicated G1 as a checkpoint. The proteins, p21 and p53, retain cellular integrity, suppressing the oncogenic transformation by interruption of the G1 phase of the cellular cycle, giving time for repairing the damage to DNA, or apoptosis induction if the damage is too severe to be repaired, while MDM2 and Gadd45 concomitantly ensure the presence of p53 and p21. Thus, we conclude that repair, together with Gadd45 and MDM2 genes, were involved in light and dark reaction mechanisms, however, BA could interfere and assist the repair through restoration, although further studies of the complex of the gene cascades triggered and responded to in BA-assisted DNA repair are needed. PMID:24177565

  3. Quantitation of Mitochondrial DNA Deletions Via Restriction Digestion/Long-Range Single-Molecule PCR.

    PubMed

    Kraytsberg, Yevgenya; Guo, Xinhong; Tao, Saisai; Kuznetsov, Alexandra; McLean, Catherine; Ehrlich, Daniel; Feldman, Evan; Dombrovsky, Igor; Yang, Deye; Cloutier, Gregory J; Castaneda-Sceppa, Carmen; Khrapko, Konstantin

    2016-01-01

    Quantification of deletions in mtDNA is a long-standing problem in mutational analysis. We describe here an approach that combines the power of single-molecule PCR of the entire mitochondrial genome with the enrichment of the deletions by restriction digestion. This approach is indispensable if information about wide range of deletion types in a sample is critical, such as in studies concerning distribution of deletion breakpoints (as opposed to approaches where fraction of a single deletion or a limited set of deletions is used as a proxy for total deletion load). Because deletions in a sample are quantified almost exhaustively, the other important application of this approach involves studies where only small amounts of tissue, such as biopsies, are available. PMID:26530673

  4. Crystallization and preliminary diffraction analysis of the HincII restriction endonuclease-DNA complex.

    PubMed

    Horton, N C; Dorner, L F; Schildkraut, I; Perona, J J

    1999-11-01

    Crystals of the 60 kDa dimeric HincII restriction enzyme bound to a 12 base-pair dyad-symmetric duplex DNA carrying the specific 5'-GTCGAC recognition site have been obtained. Crystals grew by hanging-drop vapor diffusion from solutions containing polyethylene glycol 4000 as precipitating agent. The rod-shaped crystals belong to space group I222 (or I2(1)2(1)2(1)), with unit-cell dimensions a = 66.9, b = 176.7, c = 256.0 A. There are most likely to be two dimeric complexes in the asymmetric unit. A complete native data set has been collected from a high-energy synchrotron source to a resolution of 2.5 A at 100 K, with an R(merge) of 4.8%. PMID:10531503

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

    PubMed Central

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

    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 initially proposed that proteolytic cleavage of ICAD by activated caspases causes the dissociation of the ICAD/CAD heterodimer and the translocation of active CAD into the nucleus in apoptotic cells. Here, we show that endogenous and heterologously expressed ICAD and CAD reside predominantly in the nucleus in nonapoptotic cells. Deletional mutagenesis and GFP fusion proteins identified a bipartite nuclear localization signal (NLS) in ICAD and verified the function of the NLS in CAD. The two NLSs have an additive effect on the nuclear targeting of the CAD–ICAD complex, whereas ICAD-S, lacking its NLS, appears to have a modulatory role in the nuclear localization of CAD. Staurosporine-induced apoptosis evoked the proteolysis and disappearance of endogenous and exogenous ICAD from the nuclei of HeLa cells, as monitored by immunoblotting and immunofluorescence microscopy. Similar phenomenon was observed in the caspase-3–deficient MCF7 cells upon expressing procaspase-3 transiently. We conclude that a complex mechanism, involving the recognition of the NLSs of both ICAD and CAD, accounts for the constitutive accumulation of CAD/ICAD in the nucleus, where caspase-3–dependent regulation of CAD activity takes place. PMID:10908575

  6. Development of a real-time PCR method for the detection of fossil 16S rDNA fragments of phototrophic sulfur bacteria

    E-print Network

    Gilli, Adrian

    Development of a real-time PCR method for the detection of fossil 16S rDNA fragments sulfur bacteria populations from sediment samples. We detected fossil 16S rDNA of nine populations's biogeological history. These results provide the first evidence for the presence of 16S rDNA of anoxygenic

  7. Re-evaluating the kinetics of ATP hydrolysis during initiation of DNA sliding by Type III restriction enzymes

    PubMed Central

    Tóth, Júlia; Bollins, Jack; Szczelkun, Mark D.

    2015-01-01

    DNA cleavage by the Type III restriction enzymes requires long-range protein communication between recognition sites facilitated by thermally-driven 1D diffusion. This ‘DNA sliding’ is initiated by hydrolysis of multiple ATPs catalysed by a helicase-like domain. Two distinct ATPase phases were observed using short oligoduplex substrates; the rapid consumption of ?10 ATPs coupled to a protein conformation switch followed by a slower phase, the duration of which was dictated by the rate of dissociation from the recognition site. Here, we show that the second ATPase phase is both variable and only observable when DNA ends are proximal to the recognition site. On DNA with sites more distant from the ends, a single ATPase phase coupled to the conformation switch was observed and subsequent site dissociation required little or no further ATP hydrolysis. The overall DNA dissociation kinetics (encompassing site release, DNA sliding and escape via a DNA end) were not influenced by the second phase. Although the data simplifies the ATP hydrolysis scheme for Type III restriction enzymes, questions remain as to why multiple ATPs are hydrolysed to prepare for DNA sliding. PMID:26538601

  8. 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

  9. A Natural Polymorphism in rDNA Replication Origins Links Origin Activation with Calorie Restriction and Lifespan

    PubMed Central

    Kwan, Elizabeth X.; Foss, Eric J.; Tsuchiyama, Scott; Alvino, Gina M.; Kruglyak, Leonid; Kaeberlein, Matt; Raghuraman, M. K.; Brewer, Bonita J.; Kennedy, Brian K.; Bedalov, Antonio

    2013-01-01

    Aging and longevity are complex traits influenced by genetic and environmental factors. To identify quantitative trait loci (QTLs) that control replicative lifespan, we employed an outbred Saccharomyces cerevisiae model, generated by crossing a vineyard and a laboratory strain. The predominant QTL mapped to the rDNA, with the vineyard rDNA conferring a lifespan increase of 41%. The lifespan extension was independent of Sir2 and Fob1, but depended on a polymorphism in the rDNA origin of replication from the vineyard strain that reduced origin activation relative to the laboratory origin. Strains carrying vineyard rDNA origins have increased capacity for replication initiation at weak plasmid and genomic origins, suggesting that inability to complete genome replication presents a major impediment to replicative lifespan. Calorie restriction, a conserved mediator of lifespan extension that is also independent of Sir2 and Fob1, reduces rDNA origin firing in both laboratory and vineyard rDNA. Our results are consistent with the possibility that calorie restriction, similarly to the vineyard rDNA polymorphism, modulates replicative lifespan through control of rDNA origin activation, which in turn affects genome replication dynamics. PMID:23505383

  10. 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

  11. Fibered confocal fluorescence microscopy for imaging apoptotic DNA fragmentation at the single-cell level in vivo

    SciTech Connect

    Al-Gubory, Kais H. . 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.

  12. Generation of porcine reproductive and respiratory syndrome virus by in vitro assembly of viral genomic cDNA fragments.

    PubMed

    Suhardiman, Maman; Kramyu, Jarin; Narkpuk, Jaraspim; Jongkaewwattana, Anan; Wanasen, Nanchaya

    2015-01-01

    Porcine reproductive and respiratory syndrome virus (PRRSV) is the causative agent for a swine disease affecting the pig industry worldwide. Infection with PRRSV leads to reproductive complications, respiratory illness, and weak immunity to secondary infections. To better control PRRSV infection, novel approaches for generating control measures are critically needed. Here, in vitro Gibson assembly (GA) of viral genomic cDNA fragments was tested for its use as a quick and simple method to recover infectious PRRSV in cell culture. GA involves the activities of T5-exonuclease, Phusion polymerase, and Taq ligase to join overlapping cDNA fragments in an isothermal condition. Four overlapping cDNA fragments covering the entire PRRSV genome and one vector fragment were used to create a plasmid capable of expressing the PRRSV genome. The assembled product was used to transfect a co-culture of 293T and MARC-145 cells. Supernatants from the transfected cells were then passaged onto MARC-145 cells to rescue infectious virus particles. Verification and characterization of the recovered virus confirmed that the GA protocol generated infectious PRRSV that had similar characteristics to the parental virus. This approach was then tested for the generation of a chimeric virus. By replacing one of the four genomic fragments with that of another virus strain, a chimeric virus was successfully recovered via GA. In conclusion, this study describes for the first time the use of GA as a simple, yet powerful tool for generating infectious PRRSV needed for studying PRRSV biology and developing novel vaccines. PMID:25300804

  13. In Vitro Effect of Cell Phone Radiation on Motility, DNA Fragmentation and Clusterin Gene Expression in Human Sperm

    PubMed Central

    Zalata, Adel; El-Samanoudy, Ayman Z; Shaalan, Dalia; El-Baiomy, Youssef; Mostafa, Taymour

    2015-01-01

    Background Use of cellular phones emitting radiofrequency electromagnetic field (RF-EMF) has been increased exponentially and become a part of everyday life. This study aimed to investigate the effects of in vitro RF-EMF exposure emitted from cellular phones on sperm motility index, sperm DNA fragmentation and seminal clusterin (CLU) gene expression. Materials and Methods In this prospective study, a total of 124 semen samples were grouped into the following main categories: i. normozoospermia (N, n=26), ii. asthenozoospermia (A, n=32), iii. asthenoteratozoospermia (AT, n=31) and iv. oligoasthenoteratozoospermia (OAT, n=35). The same semen samples were then divided into two portions non-exposed and exposed samples to cell phone radiation for 1 hour. Before and immediately after exposure, both aliquots were subjected to different assessments for sperm motility, acrosin activity, sperm DNA fragmentation and CLU gene expression. Statistical differences were analyzed using paired t student test for comparisons between two sub-groups where p<0.05 was set as significant. Results There was a significant decrease in sperm motility, sperm linear velocity, sperm linearity index, and sperm acrosin activity, whereas there was a significant increase in sperm DNA fragmentation percent, CLU gene expression and CLU protein levels in the exposed semen samples to RF-EMF compared with non-exposed samples in OAT>AT>A>N groups, respectively (p<0.05). Conclusion Cell phone emissions have a negative impact on exposed sperm motility index, sperm acrosin activity, sperm DNA fragmentation and seminal CLU gene expression, especially in OAT cases. PMID:25918601

  14. 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.

  15. DpnA, a methylase for single-strand DNA in the Dpn II restriction system, and its biological function

    SciTech Connect

    Cerritelli, S.; Springhorn, S.S.; Lacks, S.A. )

    1989-12-01

    The two DNA-adenine methylases encoded by the Dpn II restriction gene cassette were purified, and their activities were compared on various DNA substrates. DpnA was able to methylate single-strand DNA and double-strand DNA, whereas DpnM methylated only double-strand DNA. Although both enzymes act at 5{prime}-GATC-3{prime} in DNA, DpnA can also methylate sequences altered in the guanine position, but at a lower rate. A deletion mutation in the dpnA gene was constructed and transferred to the chromosome. Transmission by way of the transformation pathway of methylated and unmethylated plasmids to dpnA mutant and wild-type recipients was examined. The mutant cells restricted unmethylated donor plasmic establishment much more strongly than did wild-type cells. In the wild type, the single strands of donor plasmid DNA that enter by the transformation pathway are apparently methylated by DpnA prior to conversion of the plasmid to a double-strand form, in which the plasmid would be susceptible to the Dpn II endonuclease. The biological function of DpnA may, therefore, be the enhancement of plasmid transfer to Dpn II-containing strains of Streptococcus pneumoniae.

  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. Is thioredoxin reductase involved in the defense against DNA fragmentation in varicocele?

    PubMed

    Özdemirler Erata, Gül; Küçükgergin, Canan; Aktan, Gülsan; Kadioglu, Ates; Uysal, Müjdat; Koçak-Toker, Necla

    2013-07-01

    We aimed to investigate the role of thioredoxin reductase (TR) and inducible heat shock protein 70 (iHsp70) and their relationship with sperm quality in varicocele (VAR) patients. Semen samples were obtained from 16 subfertile men diagnosed as VAR and 10 fertile men who applied to the Andrology Laboratory of Istanbul Medical Faculty of Istanbul University. The sperm TR and iHsp 70 expression levels were determined using Western blot analysis. The TR activity of the sperm was assayed spectrophometrically. The sperm quality was evaluated both by conventional sperm analysis and by a terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) technique that assayed DNA-fragmented spermatozoa in semen samples. The percentage of TUNEL-positive spermatozoa in the VAR group (16.3%± 5.6%) was higher than that in the fertile group (5.5%± 1.9%). Significant inverse correlations were detected between the percentage of TUNEL-positive cells and both the concentration (r=-0.609; P=0.001) and motility (r=-0.550; P=0.004) of spermatozoa. Both the TR expression and activity were increased significantly in the VAR group (U=22.0; P=0.001 and U=33.5; P=0.012, respectively) as analyzed using the Mann-Whitney U Wilcoxon rank sum W test. Furthermore, significant positive correlations were found between TR expression and activity (r=0.406; P=0.040) and between TR expression and the percentage of TUNEL-positive cells (r=0.665; P=0.001). Sperm iHsp70 expression did not differ between the VAR and fertile groups. In conclusion, increased sperm TR expression might be a defense mechanism against apoptosis in the spermatozoa of men with VAR. PMID:23603921

  18. Software for Automated Analysis of DNA Fingerprinting Gels

    E-print Network

    Saeedi, Parvaneh

    Software for Automated Analysis of DNA Fingerprinting Gels Daniel R. Fuhrmann,1 Martin I software tools for the automated detection of DNA restriction fragments resolved on agarose fingerprinting of genomic DNA, the fingerprinting process (Schein et al. 2003) involves purification of DNA from clones

  19. 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

  20. 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.

    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.

  1. Magnetic activated cell sorting: an effective method for reduction of sperm DNA fragmentation in varicocele men prior to assisted reproductive techniques.

    PubMed

    Degheidy, T; Abdelfattah, H; Seif, A; Albuz, F K; Gazi, S; Abbas, S

    2015-10-01

    Semen parameters of varicocele men have been usually suspected to exhibit higher levels of abnormalities including DNA fragmentation, reactive oxygen species (ROS) and apoptotic markers. Negative correlation between increased level of DNA fragmentation and assisted reproductive techniques (ART) outcome has been studied by several authors. In the current study, we aim to evaluate the possible value of magnetic activated cell sorting (MACs) technology in reduction of DNA fragmentation in infertile varicocele patients prior to ART. Semen samples, collected from 36 varicocele patients, were prepared by density gradient centrifugation (DGC). Every sample was subsequently divided into two aliquots. One aliquot was kept untouched as pre-MACs control while the other aliquot was subjected to MACs technique, for depletion of apoptotic spermatozoa, and serves as post-MACs test. Sperm count, motility and DNA fragmentations were evaluated for both control and test samples. Post-MACs samples showed no deleterious reduction in total sperm motility (80.64 ± 6.97%) compared with control samples (80.97 ± 7.74%) while sperm DNA fragmentations were significantly reduced in post-MACs samples (9.61 ± 5.62%) compared with pre-MACs controls (12.43 ± 6.29%) (P < 0.05). It can be concluded that MACs technique is a simple, noninvasive, technique that can efficiently reduce DNA fragmentation in infertile varicocele patients prior to ART. PMID:25209213

  2. Examination of meat components in commercial dog and cat feed by using polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLPs) technique.

    PubMed

    Wang, Hsien-Chi; Lee, Shu-Hwae; Chang, Tien-Jye; Wong, Min-Liang

    2004-07-01

    It has been shown that certain slow neurological diseases such as bovine spongiform encephalopathy (also known as "mad cow" disease) could be transmitted through contaminated food intake by animals; therefore, the examination of meat components in commercial feeds is important for the control of the disease in public health. The combination of polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLPs) technique was applied to examine the meat components in dog and cat commercial feeds. The partial nucleotide sequence (359 bp) of animal mitochondrial cytochrome b (cytb, CYT) gene was amplified by PCR and then digested with restriction enzyme Alu I or Mbo I. In this work, eight brands of commercial dog and cat feeds available in Taiwan were examined. All brands of dog feeds that were tested contained meat from four different animals (cattle, pig, goat and chicken). In cat feeds, the chicken meat was found in five out of eight brands. PMID:15297759

  3. DNA Methylation Restricts Lineage-specific Functions of Transcription Factor Gata4 during Embryonic Stem Cell Differentiation

    PubMed Central

    Jakt, Lars Martin; Matsuoka, Chisa; Yamagiwa, Akiko; Niwa, Hitoshi; Okano, Masaki

    2013-01-01

    DNA methylation changes dynamically during development and is essential for embryogenesis in mammals. However, how DNA methylation affects developmental gene expression and cell differentiation remains elusive. During embryogenesis, many key transcription factors are used repeatedly, triggering different outcomes depending on the cell type and developmental stage. Here, we report that DNA methylation modulates transcription-factor output in the context of cell differentiation. Using a drug-inducible Gata4 system and a mouse embryonic stem (ES) cell model of mesoderm differentiation, we examined the cellular response to Gata4 in ES and mesoderm cells. The activation of Gata4 in ES cells is known to drive their differentiation to endoderm. We show that the differentiation of wild-type ES cells into mesoderm blocks their Gata4-induced endoderm differentiation, while mesoderm cells derived from ES cells that are deficient in the DNA methyltransferases Dnmt3a and Dnmt3b can retain their response to Gata4, allowing lineage conversion from mesoderm cells to endoderm. Transcriptome analysis of the cells' response to Gata4 over time revealed groups of endoderm and mesoderm developmental genes whose expression was induced by Gata4 only when DNA methylation was lost, suggesting that DNA methylation restricts the ability of these genes to respond to Gata4, rather than controlling their transcription per se. Gata4-binding-site profiles and DNA methylation analyses suggested that DNA methylation modulates the Gata4 response through diverse mechanisms. Our data indicate that epigenetic regulation by DNA methylation functions as a heritable safeguard to prevent transcription factors from activating inappropriate downstream genes, thereby contributing to the restriction of the differentiation potential of somatic cells. PMID:23825962

  4. In vitro assembly of an infectious cDNA clone of infectious bronchitis virus and its application as a gene transfer vector 

    E-print Network

    Youn, Soonjeon

    2005-02-17

    of the restriction enzyme recognition sequence resulting in 3-4 nucleotide overhangs. These nucleotide overhangs are non-palindromic and have unique sequence composition. Each DNA fragment digested with these restriction enzymes can be religated with compatible...

  5. Fusion of GFP to the M.EcoKI DNA methyltransferase produces a new probe of Type I DNA restriction and modification enzymes

    SciTech Connect

    Chen, Kai; Roberts, Gareth A.; Stephanou, Augoustinos S.; Cooper, Laurie P.; White, John H.; Dryden, David T.F.

    2010-07-23

    Research highlights: {yields} Successful fusion of GFP to M.EcoKI DNA methyltransferase. {yields} GFP located at C-terminal of sequence specificity subunit does not later enzyme activity. {yields} FRET confirms structural model of M.EcoKI bound to DNA. -- Abstract: We describe the fusion of enhanced green fluorescent protein to the C-terminus of the HsdS DNA sequence-specificity subunit of the Type I DNA modification methyltransferase M.EcoKI. The fusion expresses well in vivo and assembles with the two HsdM modification subunits. The fusion protein functions as a sequence-specific DNA methyltransferase protecting DNA against digestion by the EcoKI restriction endonuclease. The purified enzyme shows Foerster resonance energy transfer to fluorescently-labelled DNA duplexes containing the target sequence and to fluorescently-labelled ocr protein, a DNA mimic that binds to the M.EcoKI enzyme. Distances determined from the energy transfer experiments corroborate the structural model of M.EcoKI.

  6. Restriction/modification polypeptides, polynucleotides, and methods

    DOEpatents

    Westpheling, Janet; Chung, DaeHwan; Huddleston, Jennifer; Farkas, Joel A

    2015-02-24

    The present invention relates to the discovery of a novel restriction/modification system in Caldicellulosiruptor bescii. The discovered restriction enzyme is a HaeIII-like restriction enzyme that possesses a thermophilic activity profile. The restriction/modification system also includes a methyltransferase, M.CbeI, that methylates at least one cytosine residue in the CbeI recognition sequence to m.sup.4C. Thus, the invention provides, in various aspects, isolated CbeI or M.CbeI polypeptides, or biologically active fragments thereof; isolated polynucleotides that encode the CbeI or M.CbeI polypeptides or biologically active fragments thereof, including expression vectors that include such polynucleotide sequences; methods of digesting DNA using a CbeI polypeptide; methods of treating a DNA molecule using a M.CbeI polypeptide; and methods of transforming a Caldicellulosiruptor cell.

  7. Molecular Mapping of Restriction-Site Associated DNA Markers in Allotetraploid Upland Cotton

    PubMed Central

    Wang, Yangkun; Ning, Zhiyuan; Hu, Yan; Chen, Jiedan; Zhao, Rui; Chen, Hong; Ai, Nijiang; Guo, Wangzhen; Zhang, Tianzhen

    2015-01-01

    Upland cotton (Gossypium hirsutum L., 2n = 52, AADD) is an allotetraploid, therefore the discovery of single nucleotide polymorphism (SNP) markers is difficult. The recent emergence of genome complexity reduction technologies based on the next-generation sequencing (NGS) platform has greatly expedited SNP discovery in crops with highly repetitive and complex genomes. Here we applied restriction-site associated DNA (RAD) sequencing technology for de novo SNP discovery in allotetraploid cotton. We identified 21,109 SNPs between the two parents and used these for genotyping of 161 recombinant inbred lines (RILs). Finally, a high dense linkage map comprising 4,153 loci over 3500-cM was developed based on the previous result. Using this map quantitative trait locus (QTLs) conferring fiber strength and Verticillium Wilt (VW) resistance were mapped to a more accurate region in comparison to the 1576-cM interval determined using the simple sequence repeat (SSR) genetic map. This suggests that the newly constructed map has more power and resolution than the previous SSR map. It will pave the way for the rapid identification of the marker-assisted selection in cotton breeding and cloning of QTL of interest traits. PMID:25894395

  8. Association mapping of disease resistance traits in rainbow trout using restriction site associated DNA sequencing.

    PubMed

    Campbell, Nathan R; LaPatra, Scott E; Overturf, Ken; Towner, Richard; Narum, Shawn R

    2014-12-01

    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

  9. Restriction fragment length polymorphism analysis of rotavirus VP7-encoding gene from humans and animals of Northeast India: a relative study of Indian and global isolates.

    PubMed

    Chakraborty, P; Barman, N N; Sharma, I

    2015-09-01

    A restriction fragment length polymorphism (RFLP) assay was developed to examine the genetic relationship between 67 (29 Indian, 38 global) rotavirus isolates of human, bovine and porcine neonates. The assay involved direct digestion of RT-PCR amplified VP7 cDNAs with three restriction enzymes (VspI, HaeIII, NlaIV) independently. Forty-eight RFLP patterns were identified for all 67 strains, and of these 20 patterns were associated with Indian isolates. A correlation between the restriction patterns and G type was apparent through deduction of enzyme restriction sites from known sequences. Major G serotypes (G1, G2, G6, G8) with a few mixed types could be differentiated where there was a positive assortment of intrinsic serotypes from multiple host origin, and certain single or combined enzyme profiles were highly dominant in the population. Significant genetic variations were established between global and Indian isolates and none of the RFLP patterns were shared between them. These data suggest that the Indian wild-type rotavirus population is distinguishable based on the VP7 gene, and co-circulation of distinct strains in different hosts is foremost, indicating the possible likelihood of inter-species transmission. PMID:25573161

  10. Severe von Willebrand disease due to a defect at the level of von Willebrand factor mRNA expression: Detection by exonic PCR-restriction fragment length polymorphism analysis

    SciTech Connect

    Nichols, W.C.; Lyons, S.E.; Harrison, J.S.; Cody, R.L.; Ginsburg, D. )

    1991-05-01

    von Willebrand disease (vWD), the most common inherited bleeding disorder in humans, results from abnormalities in the plasma clotting protein von Willebrand factor (vWF). Severe (type III) vWD is autosomal recessive in inheritance and is associated with extremely low or undetectable vWF levels. The authors report a method designed to distinguish mRNA expression from the two vWF alleles by PCR analysis of peripheral blood platelet RNA using DNA sequence polymorphisms located within exons of the vWF gene. This approach was applied to a severe-vWD pedigree in which three of eight siblings are affected and the parents and additional siblings are clinically normal. Each parent was shown to carry a vWF allele that is silent at the mRNA level. Family members inheriting both abnormal alleles are affected with severe vWD, whereas individuals with only one abnormal allele are asymptomatic. Given the frequencies of the two exon polymorphisms reported here, this analysis should be applicable to {approx}70% of type I and type III vWD patients. This comparative DNA and RNA PCR-restriction fragment length polymorphism approach may also prove useful in identifying defects at the level of gene expression associated with other genetic disorders.

  11. Is sperm DNA fragmentation a good marker for field AI bull fertility?

    PubMed

    Karoui, S; Díaz, C; González-Marín, C; Amenabar, M E; Serrano, M; Ugarte, E; Gosálvez, J; Roy, R; López-Fernández, C; Carabaño, M J

    2012-08-01

    This paper aimed at investigating the potential use of sperm DNA fragmentation (SDF) to improve the routine screening of infertility of Holstein bulls. Cryopreserved sperm samples from 201 Holstein bulls provided by an AI center were used in the analyses of SDF at 0 (SDF_0) and 6 (SDF_6) h of incubation at 37°C. A refinement of the sperm chromatin dispersion test implemented in the Sperm-Halomax kit was employed to measure SDF. Records on routinely collected semen traits (volume, concentration, mass and individual motility evaluated in the fresh ejaculate, and individual motility in post-thawed semen straws) were provided by the AI center. Artificial insemination bull fertility was obtained from official field recording as successful or failed insemination. The results show that the average SDF was low (around 3.5%) at 0 and 6 h of incubation. A moderate effect of inbreeding depression was found. Estimated heritability for SDF traits were moderately high (0.41 and 0.29 for SDF_0 and SDF_6, respectively) and estimated repeatability of SDF measures in the same animal were high (0.73 and 0.70 for SDF_0 and SDF_6, respectively). An overall estimated service bull value (ESBV) obtained through statistical modeling that allowed for adjustment of systematic environmental effects not specific to a bull and of the female contribution to fertility, and the estimated genetic values (EGV) were obtained from field-recorded AI information. The ESBV and EGV were also obtained for all semen traits. Moderately large and negative Pearson correlation coefficients were observed between SDF traits and male fertility ranging from (-0.43 to -0.50; P <0.001). Results of stepwise regression analyses showed that SDF_6 had the largest partial r(2) (0.15 to 0.26) among all semen characteristics. Overall, the selected semen traits explained 25% and 31% of the observed variability in bull fertility measured as EGV and ESBV, respectively. When looking at the predictive ability of bull fertility categories, the results of discriminant and logistic regression analyses showed that low-fertility bulls (those in the 10th or lower percentile in the fertility distribution) can be accurately identified by using measures of SDF alone or in combination with sperm motility. Values of SDF around 7% to 10% could be used as indicators of low AI success. PMID:22367070

  12. A novel fluorescent biosensor for detection of target DNA fragment from the transgene cauliflower mosaic virus 35S promoter.

    PubMed

    Qiu, Bin; Zhang, Ya-shan; Lin, Yi-bing; Lu, Yu-Jing; Lin, Zhen-yu; Wong, Kwok-Yin; Chen, Guo-nan

    2013-03-15

    In this paper, we reported a convenient fluorescence method for the detection of genetically modified organisms (GMOs). As it is known that the cauliflower mosaic virus (CaMV) 35S promoter is widely used in most transgenic plants (Schnurr and Guerra, 2000), we thus design a simple method based on the detection of a section target DNA (DNA-T) from the transgene CaMV 35S promoter. In this method, the full-length guanine-rich single-strand sequences were split into fragments (Probe 1 and 2) and each part of the fragment possesses two GGG repeats. In the presence of K(+) ion and berberine, if a complementary target DNA of the CaMV 35S promoter was introduced to hybridize with Probe 1 and 2, a G-quadruplex-berberine complex was thus formed and generated a strong fluorescence signal. The generation of fluorescence signal indicates the presence of CaMV 35S promoter. This method is able to identify and quantify Genetically Modified Organisms (GMOs), and it shows wide linear ranges from 5.0×10(-9) to 9.0×10(-7) mol/L with a detection limit of 2.0×10(-9) mol/L. PMID:22959013

  13. 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

  14. Molecular typing of Iranian mycobacteria isolates by polymerase chain reaction-restriction fragment length polymorphism analysis of 360-bp rpoB gene

    PubMed Central

    Hadifar, Shima; Moghim, Sharareh; Fazeli, Hossein; GhasemianSafaei, Hajieh; Havaei, Seyed Asghar; Farid, Fariba; Esfahani, Bahram Nasr

    2015-01-01

    Background: Diagnosis and typing of Mycobacterium genus provides basic tools for investigating the epidemiology and pathogenesis of this group of bacteria. Polymerase chain reaction (PCR)-restriction fragment length polymorphism analysis (PRA) is an accurate method providing diagnosis and typing of species of mycobacteria. The present study is conducted by the purpose of determining restriction fragment profiles of common types of mycobacteria by PRA method of rpoB gene in this geographical region. Materials and Methods: Totally 60 clinical and environmental isolates from February to October, 2013 were collected and subcultured and identified by phenotypic methods. A 360 bp fragment of the rpoB gene amplified by PCR and products were digested by MspI and HaeIII enzymes. Results: In the present study, of all mycobacteria isolates identified by PRA method, 13 isolates (21.66%) were Mycobacterium tuberculosis, 34 isolates (56.66%) were rapidly growing Nontuberculosis Mycobacteria (NTM) that including 26 clinical isolates (43.33%) and 8 environmental isolates (13.33%), 11 isolates (18.33%) were clinical slowly growing NTM. among the clinical NTM isolates, Mycobacterium fortuitum Type I with the frequency of 57.77% was the most prevalent type isolates. Furthermore, an unrecorded of the PRA pattern of Mycobacterium conceptionense (HeaIII: 120/90/80, MspI: 120/105/80) was found. This study demonstrated that the PRA method was high discriminatory power for identification and typing of mycobacteria species and was able to identify 96.6% of all isolates. Conclusion: Based on the result of this study, rpoB gene could be a potentially useful tool for identification and investigation of molecular epidemiology of mycobacterial species. PMID:26380237

  15. 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...

  16. 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...

  17. A combinatorial approach to the restriction of a mouse genome

    PubMed Central

    2013-01-01

    Background A fragmentation of genomic DNA by restriction digestion is a popular step in many applications. Usually attention is paid to the expected average size of the DNA fragments. Another important parameter, randomness of restriction, is regularly implied but rarely verified. This parameter is crucial to the expectation, that either all fragments made by restriction will be suitable for the method of choice, or only a fraction of those will be effectively used by the method. If only a fraction of the fragments are used, we often should know whether the used fragments are representative of the whole genome. With a modern knowledge of mouse, human and many other genomes, frequencies and distributions of restriction sites and sizes of corresponding DNA fragments can be analyzed in silico. In this manuscript, the mouse genome was systematically scanned for frequencies of complementary 4-base long palindromes. Findings and conclusions The study revealed substantial heterogeneity in distribution of those sites genome-wide. Only few palindromes showed close to random pattern of distribution. Overall, the distribution of frequencies for most palindromes is much wider than expected by random occurrence. In practical terms, accessibility of genome upon restriction can be improved by a selective combination of restrictases using a few combinatorial rules. It is recommended to mix at least 3 restrictases, their recognition sequences (palindrome) should be the least similar to each other. Principles of the optimization and optimal combinations of restrictases are provided. PMID:23875927

  18. Cloning and expression of NS3 cDNA fragment of HCV genome of Hebei isolate in E. coli

    PubMed Central

    Zhu, Fen-Lu; Lu, Hao-Ying; Li, Zhuo; Qi, Zhong-Tian

    1998-01-01

    AIM: To obtain greater antigenicity of HCV NS3 protein. METHODS: The HCV NS3 cDNA fragment was amplified by reverse transcription polymerase chain reaction from the sera of the HCV infected patients. The DNA sequence was determined by dideoxy-mediated chain termination method using T7 polymerase. HCV NS3 protein was expressed in E. coli. RESULTS: Sequence analysis indicated that the HCV isolate of this study belongs to HCV-II; SDS-PAGE demonstrated an Mr 23800 and an Mr 22000 recombinant protein band which amount to 14% and 11% of the total bacterial proteins separately. Western blotting and ELISA showed NS3 protein possessed greater antigenicity. CONCLUSION: Recombinant HCV NS3 protein was expressed successfully, which provided the basis for developing HCV diagnostic reagents. PMID:11819265

  19. Restriction endonuclease fingerprinting by SSCP (REF), an efficient method of screening for mutations in long contiguous segments of DNA

    SciTech Connect

    Liu, O.; Sommer, S.S.

    1994-09-01

    Dideoxy fingerprinting is an efficient method of screening for the presence of mutations in short exons ({le}250 bp). Long contiguous segments can be screened by sequential ddF reactions. To screen long contiguous segments in a more rapid manner, REF has been developed. REF will be described in the context of a model system in exon H of the factor IX gene. A 1 kb segment is PCR amplified and digested with each of five groups of restriction endonucleases. The endonucleases are chosen such that, in each group, the average size of the fragments is about 150 bp. After digestion, the products are mixed, 5{prime} end-labeled with T4 polynucleotide kinase, boiled, and electrophoresed under nondenaturing conditions. Each lane screens 1 kb and contains 70 segments (7 fragments per digestion x 5 digestions x 2 strands). The matrices tested were 5.6% polyacrylamide (PA) and 7.5% GeneAmp{sup {trademark}} (GA) at temperatures of either 23{degrees}C (RT) or 8{degrees}C (LT). Point mutations resulted in the gain or loss of a restriction site in 21% of 24 test mutations. In addition, mutations could be detected if any of 5 restriction fragments with the same mutation (producing 10 denatured segments) displayed abnormal mobility (SSCP component). The average sensitivity per segment of the SSCP component for the 24 point mutations ranged from 49% for PA at RT to 68% with GA at LT. REF detected 96% of the mutations with PA at RT and 100% with GA at RT or LT. These latter two conditions detected 100% of a subsequent blinded sample that contained normal controls and 27 different mutations. A blinded analysis is in progress to determine the sensitivity of REF when the segment size is 2 kb.

  20. Reagent kit producing shortened target DNA segments usable in sequencing large DNA segments

    SciTech Connect

    Henikoff, S.; Gelinas, R.E.

    1989-12-26

    This patent describes a reagent kit for producing shortened DNA fragments useful for sequencing a target DNA segment. It comprises: a purified exonuclease reagent capable of unidirectionally digesting nucleotide bases asynchronously from a target DNA segment; and a purified recombinant vector molecule comprising vector DNA, a sequencing primer binding site, and a poly-linker region comprising first, second, and third restriction endonuclease sites. The first restriction site being capable of receiving by ligation the target DNA segment. The second and third restriction sites being located between the first restriction site and the sequencing primer binding site, and the second and third restriction sites being so arranged that cleavage of the polylinker region at the second and third restriction sites will produce a linearized vector molecule having a first terminus adjacent the target DNA segment that is susceptible to digestion by the exonuclease reagent and a second terminus, not susceptible to digestion by the exonuclease reagent adjacent the sequencing primer binding site.

  1. Sperm DNA fragmentation and morphological degeneration in chilled elephant (Elephas maximus and Loxodonta Africana) semen collected by transrectal massage.

    PubMed

    O'Brien, J K; Steinman, K J; Montano, G A; Love, C C; Robeck, T R

    2013-05-01

    Ejaculates from nine Asian and two African elephants were analysed to gain a further understanding of mechanisms underlying variable semen quality after transrectal massage. Semen analysis was performed after collection (0 h; subjective motility parameters only) and after 24 h of chilled storage at 10 °C (24 h; all ejaculate and sperm characteristics). Ejaculates with ?50% total motility (TM) at 24 h, which represented >90% of collection attempts, contained a sperm population with a high degree of DNA damage (64.2 ± 19.2% fragmented DNA) and an elevated incidence of detached heads (43.3 ± 22.5%). In contrast, good quality ejaculates designated as those with >50% TM at 24 h displayed higher (p < 0.05) values of sperm kinetic parameters, DNA integrity and normal morphology. Fertility potential was high for good quality ejaculates from two males (one Asian and one African bull) based on in vitro characteristics after chilled storage for up to 48 h post-collection. Urine contamination of semen, as assessed quantitatively by creatinine concentration, was confirmed as a significant factor in reduced elephant ejaculate quality. However, the identification of considerable DNA damage and morphological degeneration in the majority of ejaculates after only 24 h of chilled storage indicates that sperm ageing could be a primary contributor to inconsistent semen quality in the elephant. PMID:23536498

  2. Fragmentation and plasmid strand breaks in pure and gold-doped DNA irradiated by beams of fast hydrogen atoms

    NASA Astrophysics Data System (ADS)

    Wyer, J. A.; Butterworth, K. T.; Hirst, D. G.; Latimer, C. J.; Montenegro, E. C.; Shah, M. B.; Currell, F. J.

    2009-08-01

    The results of an investigation into the damage caused to dry plasmid DNA after irradiation by fast (keV) hydrogen atoms are presented. Agarose gel electrophoresis was used to assess single and double strand break yields as a function of dose in dry DNA samples deposited on a mica substrate. Damage levels were observed to increase with beam energy. Strand break yields demonstrated a considerable dependence on sample structure and the method of sample preparation. Additionally, the effect of high-Z nanoparticles on damage levels was investigated by irradiating DNA samples containing controlled amounts of gold nanoparticles. In contrast to previous (photonic) studies, no enhancement of strand break yields was observed with the particles showing a slight radioprotective effect. A model of DNA damage as a function of dose has been constructed in terms of the probability for the creation of single and double strand breaks, per unit ion flux. This model provides quantitative conclusions about the effects of both gold nanoparticles and the different buffers used in performing the assays and, in addition, infers the proportion of multiply damaged fragments.

  3. 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

  4. Leaf margin phenotype-specific restriction-site-associated DNA-derived markers for pineapple (Ananas comosus L.).

    PubMed

    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

    2015-06-01

    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

  5. Ionization and fragmentation of DNA, RNA bases induced by proton impact

    NASA Astrophysics Data System (ADS)

    LePadellec, A.; Moretto-Capelle, P.; Richard-Viard, M.; Champeaux, J. P.; Cafarelli, P.

    2008-02-01

    We present recent results obtained in the Toulouse's group that deal with proton to base and nucleoside interactions. We stress the weakness of the sugar part in the nucleoside, i.e. the uridine molecule under scrutiny. Since some parts of the fragmentation spectrum correspond to the fragmentation of a 'pure' uracil molecule, i.e. the RNA base, an 'additivity rule' seems to prevail for the nucleoside, something that still has to be confirmed. Moreover, some results that deal with the secondary electronic emission from uracil are also displayed.

  6. Identification of verotoxin type 2 variant B subunit genes in Escherichia coli by the polymerase chain reaction and restriction fragment length polymorphism analysis.

    PubMed Central

    Tyler, S D; Johnson, W M; Lior, H; Wang, G; Rozee, K R

    1991-01-01

    A set of synthetic oligonucleotide primers was designed for use in a polymerase chain reaction protocol to specifically detect the B subunit genes in vtx2ha and vtx2hb, which code for the production of the VT2 (Shiga-like toxin II) variant cytotoxins VT2v-a and VT2v-b, respectively. An additional set of primers amplified a fragment common to the B subunits of the VT2 and the VT2 variant genes. Subsequent restriction endonuclease digestion of this amplicon permitted prediction of specific VT2 and variant genotypes on the basis of predetermined restriction fragment length polymorphisms. Genotypes of 21 VT2-producing strains of Escherichia coli were determined using this polymerase chain reaction-restriction fragment length polymorphism procedure. Four strains contained B subunit target sequences only for VT2 genes, 9 strains contained sequences only for VT2v-a genes, and 3 strains contained sequences only for VT2v-b. For genes in combination, one strain contained B subunit genes for both VT2 and VT2v-a and two strains contained B subunit genes for VT2 and VT2v-b. Two strains of E. coli O91:H21 contained both VT2v-a and VT2v-b B subunit genes. The VT2 reference strain of E. coli, E32511, was found to contain the targeted sequences from both VT2 and VT2v-a genes, whereas the recombinant E. coli, pEB1, possessed only that of the VT2 gene. The specific activities of extracellular VT2 determined in HeLa cells ranged from 0.3 to 41.7 TCD50 per microgram of protein in strains carrying the VT2 gene target and from 0 to 50.0 TCD50 per microgram of protein in strains carrying only the VT2 variant target (TCD50 is the tissue culture dose by which 50% of the cells were affected), suggesting that phenotypic expression does not correlate with genotype. Images PMID:1679436

  7. 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

  8. 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.

  9. Optimization of STR locus enrichment for STR profiling of fragmented DNA.

    PubMed

    Ham, Seon-Kyu; Kim, Se-Yong; Ahn, Jang-Won; Seo, Bo Young; Woo, Kwang-Man; Choi, Cheol Yong; Lee, Seung-Hwan

    2014-11-01

    DNA degradation is a major obstacle in gaining an accurate profile with standard DNA typing technology. Although alternative genotyping strategies such as mini-STRs and SNPs have proven to be more successful in profiling degraded DNA, these approaches also have limitations. Here, we show that locus enrichment by hybridization of degraded genomic DNA with an STR locus-specific biotinylated oligonucleotide is a powerful approach to overcome problems in STR typing of highly degraded DNA. An experimental investigation of factors affecting the efficiency of this method indicates that the choice of primer and molar ratio of primers to genomic DNA are critical factors in improving enrichment of the STR locus before genotyping with multiplex kits. In addition, we find that indirect capture rather than direct capture with magnetic beads yields better enrichment efficiency for STR locus enrichments. Using these strategies, we demonstrate an improvement in STR typing of DNA from cultured cells damaged by exposure to sunlight or UV. We suggest that this approach could be applied to highly degraded forensic samples alone or in combination with mini-STRs. PMID:25142119

  10. Absence of superoxide dismutase activity causes nuclear DNA fragmentation during the aging process

    SciTech Connect

    Muid, Khandaker Ashfaqul; Karakaya, Hüseyin Çaglar; Koc, Ahmet

    2014-02-07

    Highlights: • Aging process increases ROS accumulation. • Aging process increases DNA damage levels. • Absence of SOD activity does not cause DNA damage in young cells. • Absence of SOD activity accelerate aging and increase oxidative DNA damages during the aging process. - Abstract: Superoxide dismutases (SOD) serve as an important antioxidant defense mechanism in aerobic organisms, and deletion of these genes shortens the replicative life span in the budding yeast Saccharomyces cerevisiae. Even though involvement of superoxide dismutase enzymes in ROS scavenging and the aging process has been studied extensively in different organisms, analyses of DNA damages has not been performed for replicatively old superoxide dismutase deficient cells. In this study, we investigated the roles of SOD1, SOD2 and CCS1 genes in preserving genomic integrity in replicatively old yeast cells using the single cell comet assay. We observed that extend of DNA damage was not significantly different among the young cells of wild type, sod1? and sod2? strains. However, ccs1? mutants showed a 60% higher amount of DNA damage in the young stage compared to that of the wild type cells. The aging process increased the DNA damage rates 3-fold in the wild type and more than 5-fold in sod1?, sod2?, and ccs1? mutant cells. Furthermore, ROS levels of these strains showed a similar pattern to their DNA damage contents. Thus, our results confirm that cells accumulate DNA damages during the aging process and reveal that superoxide dismutase enzymes play a substantial role in preserving the genomic integrity in this process.

  11. 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

  12. Mapping DNA cleavage by the Type ISP restriction-modification enzymes following long-range communication between DNA sites in different orientations.

    PubMed

    van Aelst, Kara; Saikrishnan, Kayarat; Szczelkun, Mark D

    2015-12-01

    The prokaryotic Type ISP restriction-modification enzymes are single-chain proteins comprising an Mrr-family nuclease, a superfamily 2 helicase-like ATPase, a coupler domain, a methyltransferase, and a DNA-recognition domain. Upon recognising an unmodified DNA target site, the helicase-like domain hydrolyzes ATP to cause site release (remodeling activity) and to then drive downstream translocation consuming 1-2 ATP per base pair (motor activity). On an invading foreign DNA, double-strand breaks are introduced at random wherever two translocating enzymes form a so-called collision complex following long-range communication between a pair of target sites in inverted (head-to-head) repeat. Paradoxically, structural models for collision suggest that the nuclease domains are too far apart (>30 bp) to dimerise and produce a double-strand DNA break using just two strand-cleavage events. Here, we examined the organisation of different collision complexes and how these lead to nuclease activation. We mapped DNA cleavage when a translocating enzyme collides with a static enzyme bound to its site. By following communication between sites in both head-to-head and head-to-tail orientations, we could show that motor activity leads to activation of the nuclease domains via distant interactions of the helicase or MTase-TRD. Direct nuclease dimerization is not required. To help explain the observed cleavage patterns, we also used exonuclease footprinting to demonstrate that individual Type ISP domains can swing off the DNA. This study lends further support to a model where DNA breaks are generated by multiple random nicks due to mobility of a collision complex with an overall DNA-binding footprint of ?30 bp. PMID:26507855

  13. Mapping DNA cleavage by the Type ISP restriction-modification enzymes following long-range communication between DNA sites in different orientations

    PubMed Central

    van Aelst, Kara; Saikrishnan, Kayarat; Szczelkun, Mark D.

    2015-01-01

    The prokaryotic Type ISP restriction-modification enzymes are single-chain proteins comprising an Mrr-family nuclease, a superfamily 2 helicase-like ATPase, a coupler domain, a methyltransferase, and a DNA-recognition domain. Upon recognising an unmodified DNA target site, the helicase-like domain hydrolyzes ATP to cause site release (remodeling activity) and to then drive downstream translocation consuming 1–2 ATP per base pair (motor activity). On an invading foreign DNA, double-strand breaks are introduced at random wherever two translocating enzymes form a so-called collision complex following long-range communication between a pair of target sites in inverted (head-to-head) repeat. Paradoxically, structural models for collision suggest that the nuclease domains are too far apart (>30 bp) to dimerise and produce a double-strand DNA break using just two strand-cleavage events. Here, we examined the organisation of different collision complexes and how these lead to nuclease activation. We mapped DNA cleavage when a translocating enzyme collides with a static enzyme bound to its site. By following communication between sites in both head-to-head and head-to-tail orientations, we could show that motor activity leads to activation of the nuclease domains via distant interactions of the helicase or MTase-TRD. Direct nuclease dimerization is not required. To help explain the observed cleavage patterns, we also used exonuclease footprinting to demonstrate that individual Type ISP domains can swing off the DNA. This study lends further support to a model where DNA breaks are generated by multiple random nicks due to mobility of a collision complex with an overall DNA-binding footprint of ?30 bp. PMID:26507855

  14. PCR-restriction fragment length polymorphism identification and host range of single-spore isolates of the flexible Frankia sp. strain UFI 132715.

    PubMed

    Lumini, E; Bosco, M

    1996-08-01

    Twelve single-spore isolates of the flexible Elaeagnus-Frankia strain UFI 132715 fulfilled the third and the fourth of Koch's postulates on both Alnus and Elaeagnus axenic plants. Seminested nifD-nifK PCR-restriction fragment length polymorphisms provided evidence for the genetic uniformity of the single-spore frankiae with the mother strain and its plant reisolates and allowed their molecular identification directly inside Alnus and Elaeagnus nodules. The clonal nature of these single-spore-purified frankiae should allow safe mutagenesis programs, while their flexible phenotype makes them a powerful tool for understanding the molecular interactions between Frankia strains and actinorhizal plants and for identifying Frankia nodulation genes. PMID:8702296

  15. Okazaki Fragment Maturation in Yeast I. DISTRIBUTION OF FUNCTIONS BETWEEN FEN1 AND DNA2*

    E-print Network

    Burgers, Peter M.

    substantially enhanced the rate of maturation and shortened the nick-translation patch (nucleotides ex- cised- tial phenotype (8­10). In addition, DNA2 is required for the proper maintenance of telomeres (11, 12

  16. Assessing terminal restriction fragment length polymorphism suitability for the description of bacterial community structure and dynamics in hydrocarbon-polluted marine environments.

    PubMed

    Denaro, R; D'Auria, G; Di Marco, G; Genovese, M; Troussellier, M; Yakimov, M M; Giuliano, L

    2005-01-01

    The distribution of bacterial communities terminal restriction fragment length polymorphism (T-RFLP) fingerprint patterns was evaluated at three proximal hydrocarbon-contaminated sites located within the harbour of Messina. In order to analyse the short-term variability of the individual terminal restriction fragment (T-RF) patterns, water samples were collected at the three sites on three occasions within 3 months (T(0), T(90) and T(91)). Four sample sizes, from 50 to 1000 ml for each collected sample, were analysed separately (36 total analysed samples) to evaluate the relationship between the sample size and the bacterial diversity estimates. The dominant T-RF groups mostly belonged to signatures of putative hydrocarbon-degrading bacteria, as revealed by the virtual analysis of the obtained bands. In order to test whether significant differences were occurring between the analysed samples, the Kruskal-Wallis non-parametric test was applied to the T-RF data set. Neither significant influence of the sample size nor short spatial variability within the three sampled sites was detected for each sampling time. On the contrary, significant temporal changes in the diversity of the bacterial communities were observed. These results were confirmed by the non-metric multidimensional scales (nMDS) analysis of the whole set of samples, which indicated three main groups corresponding to the three different sampling times. In summary, the T-RFLP technique, although a polymerase chain reaction-based method, proved to be a suitable technique for monitoring polluted marine environments, typically characterized by low diversity and high relative abundances of a few dominant groups. PMID:15643938

  17. Albany DNA Academy Workshop (Butler and McCord) June 13-14, 2005 http://www.cstl.nist.gov/biotech/strbase/NISTpub.htm 1

    E-print Network

    in tubes · 1970 - Restriction enzymes begin to be used to cut DNA · 1981 - Jorgenson performs CZE · 1984 - Alec Jeffries utilizes restriction fragment digestion of DNA · 1985 - Kary Mullis describes PCR sieving polymers in the form of linear polyacrylamide to separate restriction digests · Commercial CE

  18. Present status of radiation interaction with DNA-strand-break cross-section and fragment-size distributions

    NASA Astrophysics Data System (ADS)

    Smirnov, F. A. Castro; Rodriguez, O.; Neto, J. D. T. Arruda

    A cross-section formula for single strand brakes via radiation in the DNA molecule is reviewed and discussed, showing that it describes well-existing data for a wide range of linear energy transfer (LET). Recently published DNA fragment-size distributions (FSDs) data, obtained by irradiation of plasmids with electrons and neutrons, are presented. These results are analyzed and interpreted on the basis of a model developed by the Sao Paulo group, in which by introducing a long-range correlation among the jumps in a random walk, a power-law function is obtained for the probability distribution. The power-law exponent is a function of both the correlation potential and the degree of freedom of the system. It is shown that plasmidial DNA behaves as a nonextensive system, with FSDs described by power laws but not by exponential functions (as in extensive statistics). A pronounced crossover for electron-irradiation data and a change from 1 to 3 in the degree of freedom for neutron irradiation at a high dose were observed. A likely interpretation for these two features is presented and discussed. Perspectives for further developments in this area are presented and addressed.

  19. Methylation profiles of genomic DNA of mouse developmental brain detected by restriction landmark genomic scanning (RLGS) method.

    PubMed Central

    Kawai, J; Hirotsune, S; Hirose, K; Fushiki, S; Watanabe, S; Hayashizaki, Y

    1993-01-01

    Restriction landmark genomic scanning using methylation-sensitive endonucleases (RLGS-M) is a newly developed powerful method for systematic detection of DNA methylation. Using this method, we scanned mouse brain genomic DNAs from various developmental stages to detect the transcriptionally active regions. This approach is based on the assumption that CpG methylation, particularly of CpG islands, might be associated with gene transcriptional regulation. Genomic DNAs were prepared from telencephalons of 9.5-, 13.5- and 16.5-day embryos, 1- and 10-day neonates and adults, followed by subjecting them to RLGS-M and comparing their patterns with each other or with that of the adult liver. We used NotI as a methylation-sensitive restriction enzyme and surveyed the methylation states of 2,600 NotI sites, almost of which should correspond to gene loci. Although almost all RLGS spots (98%) were present constantly at every developmental stages, only a few percent of spots reproducibly appeared and disappeared at different developmental stages of the brain (44 spots, 1.7%) and some were tissue-specific (10 spots, 0.7%). These data suggest that DNA methylation associated with gene transcription is a well-programmed event during the central nervous system (CNS) development. Thus, RLGS-M can offer a means for detecting systematically the genes in which the state of DNA methylation changes during development of the higher organism. Images PMID:8284204

  20. Internucleosomal DNA fragmentation in wild emmer wheat is catalyzed by S1-type endonucleases translocated to the nucleus upon induction of cell death.

    PubMed

    Granot, Gila; Morgenstern, Yaakov; Khan, Asif; Rapp, Yemima Givaty; Pesok, Anat; Nevo, Eviatar; Grafi, Gideon

    2015-03-01

    Leaves of cereal plants display nucleosomal fragmentation of DNA attributed to the action of nucleases induced during program cell death (PCD). Yet, the specific nuclease activity responsible for generating double strand DNA breaks (DSBs) that lead to DNA fragmentation has not been fully described. Here, we characterized a Ca2+/Mg2+-dependent S1-type endonuclease activity in leaves of wild emmer wheat (Triticum dicoccoides Köern.) capable of introducing DSBs as demonstrated by the conversion of supercoiled plasmid DNA into a linear duplex DNA. In-gel nuclease assay revealed a nuclease of about 35 kDa capable of degrading both single stranded DNA and RNA. We further showed that the endonuclease activity can be purified on Concanavalin A and treatment with peptide-N-glycosidase F (PNGase F) did not abolish its activity. Furthermore, ConA-associated endonuclease was capable of generating nucleosomal DNA fragmentation in tobacco nuclei. Since S1-type endonucleases lack canonical nuclear localization signal it was necessary to determine their subcellular localization. To this end, a cDNA encoding for a putative 34 kDa S1-type nuclease, designated TaS1-like (TaS1L) was synthesized based on available sequence data of Triticum aestivum and fused with RFP. Introduction into protoplasts showed that TaS1L-RFP is cytoplasmic 24h post transformation but gradually turn nuclear at 48 h concomitantly with induction of cell death. Our results suggest that DNA fragmentation occurring in leaves of wild emmer wheat may be attributed to S1-type endonuclease(s) that reside in the cytoplasm but translocate to the nucleus upon induction of cell death. PMID:25497371

  1. Development of Fok-I based nested polymerase chain reaction-restriction fragment length polymorphism analysis for detection of hepatitis B virus X region V5M mutation

    PubMed Central

    Kim, Hong; Hong, Seok-Hyun; Lee, Seoung-Ae; Gong, Jeong-Ryeol; Kim, Bum-Joon

    2015-01-01

    AIM: To develop a Fok-I nested polymerase chain reaction (PCR)-restriction fragment length polymorphism analysis (PRA) method for the detection of hepatitis B virus X region (HBx) V5M mutation. METHODS: Nested PCR was applied into DNAs from 198 chronic patients at 2 different stages [121 patients with hepatocellular carcinoma (HCC) and 77 carrier patients]. To identify V5M mutants, digestion of nested PCR amplicons by the restriction enzyme Fok-I (GGA TGN9?) was done. For size comparison, the enzyme-treated products were analyzed by electrophoresis on 2.5% agarose gels, stained with ethidium bromide, and visualized on a UV transilluminator. RESULTS: The assay enabled the identification of 69 patients (sensitivity of 34.8%; 46 HCC patients and 23 carrier patients). Our data also showed that V5M prevalence in HCC patients was significantly higher than in carrier patients (47.8%, 22/46 patients vs 0%, 0/23 patients, P < 0.001), suggesting that HBxAg V5M mutation may play a pivotal role in HCC generation in chronic patients with genotype C infections. CONCLUSION: The Fok-I nested PRA developed in this study is a reliable and cost-effective method to detect HBxAg V5M mutation in chronic patients with genotype C2 infection. PMID:26715821

  2. Restriction endonuclease analysis for the identification of baculovirus pesticides.

    PubMed Central

    Miller, L K; Dawes, K P

    1978-01-01

    Gel electrophoresis of deoxyribonucleic acid (DNA) fragments generated by digesting the DNA genomes of nuclear polyhedrosis viruses (NPV) with restriction endonucleases provides DNA fragment patterns that may be used to identify different viruses of this group. Characteristic fragment patterns were obtained for three NPVs, which are important as biological pesticides (Autographa californica NPV, Orgyia pseudotsugata NPV, and Heliothis zea NPV). The DNA fragment patterns of the A. californica NPV genoms did not change with passage through the alternate insect host, Trichoplusia ni. Heterogeneity in one preparation of O. pseudotsugata NPV was observed. The identification procedure is direct and precise. Applications of this procedure include quality control of commercial preparations of viral pesticides and screening for genetic alterations in the viruses. Images PMID:345968

  3. Introducing Human Population Biology through an Easy Laboratory Exercise on Mitochondrial DNA

    ERIC Educational Resources Information Center

    Pardinas, Antonio F.; Dopico, Eduardo; Roca, Agustin; Garcia-Vazquez, Eva; Lopez, Belen

    2010-01-01

    This article describes an easy and cheap laboratory exercise for students to discover their own mitochondrial haplogroup. Students use buccal swabs to obtain mucosa cells as noninvasive tissue samples, extract DNA, and with a simple polymerase chain reaction-restriction fragment length polymorphism analysis they can obtain DNA fragments of…

  4. 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

  5. The extended version of restriction analysis approach for the examination of the ability of low-molecular-weight compounds to modify DNA in a cell-free system.

    PubMed

    Ko?odziejski, Dominik; Brillowska-D?browska, Anna; Bartoszek, Agnieszka

    2015-01-01

    One of the primary requirements in toxicology is the assessment of ability of chemicals to induce DNA covalent modification. There are several well-established methods used for this purpose such as (32)P-Postlabeling or HPLC-MS. However, all of these approaches have difficult to overcome limitations, which prevents their use in genotoxin screening. Here, we describe the simple protocol exploiting specificity of restriction enzymes for the detection of DNA modification. It uses a specifically designed DNA amplicon, which contains two restriction sites recognized by Tru1I or MspI/HapII endonucleases. Modification of a restriction site abolishes its recognition and thus cleavage by the corresponding enzyme. The inhibition of cleavage indicates the occurrence of DNA modification of the restriction site(s), simultaneously pointing at the kind of base pairs (AT or GC) involved in DNA adduct formation. Previously, the application of this method was demonstrated for two antitumor compounds. Current study shows the extended version, that includes different ways of activation of tested compounds. Moreover, we propose an array of applications being of interest in toxicological research such as monitoring the kinetics of DNA adduct formation, detection of oxidative DNA damage, as well as assessment of the ability of antioxidative phytochemicals to prevent the latter DNA lesions. PMID:25449203

  6. Supplementary Information Intra-Uterine Calorie Restriction Affects Placental DNA Methylation

    E-print Network

    Jacobsen, Steve

    it to a new tube. 0.5ml of 100% isopropanol was added to the tube, mixed well at 55°C with an extraction buffer (750 µl) that contained proteinase K (200 µg bromide-stained DNA products confirmed in comparison with standard DNA size

  7. Recombinant DNA -principles and tools Construct a library -what for, how

    E-print Network

    Linial, Michal

    isolation of DNA fragments from complex mixtures Figure 7-4 7.1 Restriction enzymes cut DNA molecules at specific sequences Figure 7-5a #12;7 7.1 Selected restriction enzymes Site for cutting Length.1 Restriction enzymes cut DNA molecules at specific sequences Figure 7-5b #12;8 15 16 #12;9 7.1 Polylinkers

  8. 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.

  9. Synthesis of PHB by recombinant E. coli harboring an approximately 5 kb genomic DNA fragment from Streptomyces aureofaciens NRRL 2209.

    PubMed

    Ramachander, T V N; Rohini, D; Belhekar, A; Rawal, S K

    2002-12-20

    An approximately 5.0 kb Sau3A I genomic DNA fragment from Streptomyces aureofaciens NRRL 2209 was cloned in a plasmid vector and introduced into Escherichia coli. The recombinant E. coli accumulated polyhydroxyalkanoates (PHAs) as cytoplasmic inclusions. The accumulated PHA was identified as the isotactic homopolymer of PHB with a molecular weight of 2.85x10(5). Purified PHB granules were spherical with an average size of 1.1 microm and of stable configuration. DSC thermogram suggested high crystalline nature of the polymer. Maximum thermal degradation of the biopolymer occurred between 250 and 340 degrees C. Recombinant E. coli cells preferentially utilized glycerol as the carbon source and accumulated 25-28 times more PHB than the native S. aureofaciens. PMID:12559428

  10. Heat-Shocked Monocytes Are Resistant to Staphylococcus aureus-Induced Apoptotic DNA Fragmentation due to Expression of HSP72

    PubMed Central

    Guzik, Krzysztof; Bzowska, Ma?gorzata; Dobrucki, Jerzy; Pryjma, Juliusz

    1999-01-01

    Human peripheral blood monocytes became apoptotic following phagocytosis of Staphylococcus aureus. The consequences of heat stress for monocytes were studied with regard to the effect on S. aureus-induced apoptosis. Exposure of monocytes to 41.5°C for 1 h resulted in HSP72 expression and had no influence on phagocytosis of bacteria; moreover, phagocytosis of S. aureus immediately or shortly after heat shock had no effect on the S. aureus-induced monocyte apoptosis, as evidenced by DNA fragmentation assay. In contrast, cells which recovered from heat shock for 18 to 24 h, although active as phagocytes, were resistant to the S. aureus-induced apoptosis. The observed protective effect was related to the induction of HSP72, since blocking of HSP72 synthesis by an antisense oligomer abolished the protective effect of heat shock on bacterium-induced monocyte apoptosis. PMID:10417194

  11. AB112. Detection of human sperm DNA fragmentation by alkaline comet and neutral comet improved by research center for genetics and reproductive health (CGRH)

    PubMed Central

    Mai, Ma Pham Que; An, Nguyen Thi Thuy; Ha, Nguyen Truong Thai; Tram, Nguyen Bao; Bao, Nguyen Hong Quoc; Tuong, Ho Manh

    2015-01-01

    Objective To optimize the specificity and sensitivity of protocols to detect single-strand and double-strand DNA fragmentation of human sperm by using Neutral Comet and Alkaline Comet assay respectively. In both assays, the same conditions of lysis solution and gel electrophoresis were applied. Materials and methods Thirty samples of semen were collected to assess DNA fragmentation. The inclusion criteria were 18-40-year-old men and the exclusion criteria was azoospermia. Results The sample was prepared on lame at the concentration of 1×106 sperm/mL. The duration of cell lysis was successfully decrease to 30 minutes using the optimized solutions of 1.5 M NaCl and 1 mM DTT. After lysing and removing saline solutions, the gel electrophoresis was run at 20 V/70 mA in 10 minutes. The positive control sample was well-prepared by using 2% H2O2 to detect specificity and sensitivity of lysing and electrophoresis. In assay of Alkaline Comet, the sample was covered in alkaline solution (pH>13), 4 °C in 5 minutes after lysing, then moved into gel electrophoresis. The sample was dyed with SYBR and observed the sperm DNA fragmentation under fluorescent microscope. Four hundred of sperms were randomly counted in every sample. The images were well-captured in terms of detection of fragmented and nonfragmented sperms. Conclusions The optimized protocol allowed to detect the single- and double-strand DNA fragmentation in human sperms by only using the same conditions of lysis solutions and gel electrophoresis; moreover, to reduce the duration of lab performance and the cost. The protocols could be easily applied in andrology labs to provide for useful information together with semen analysis (WHO, 2010). This is the first result in Vietnam to detect DNA fragmentation by using Comet assays.

  12. 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.

  13. A novel whole genome amplification method using type IIS restriction enzymes to create overhangs with random sequences.

    PubMed

    Pan, Xiaoming; Wan, Baihui; Li, Chunchuan; Liu, Yu; Wang, Jing; Mou, Haijin; Liang, Xingguo

    2014-08-20

    Ligation-mediated polymerase chain reaction (LM-PCR) is a whole genome amplification (WGA) method, for which genomic DNA is cleaved into numerous fragments and then all of the fragments are amplified by PCR after attaching a universal end sequence. However, the self-ligation of these fragments could happen and may cause biased amplification and restriction of its application. To decrease the self-ligation probability, here we use type IIS restriction enzymes to digest genomic DNA into fragments with 4-5nt long overhangs with random sequences. After ligation to an adapter with random end sequences to above fragments, PCR is carried out and almost all present DNA sequences are amplified. In this study, whole genome of Vibrio parahaemolyticus was amplified and the amplification efficiency was evaluated by quantitative PCR. The results suggested that our approach could provide sufficient genomic DNA with good quality to meet requirements of various genetic analyses. PMID:24833422

  14. Comparative Genomics of DNA Fragments from Six Antarctic Marine Planktonic Bacteria†

    PubMed Central

    Grzymski, Joseph J.; Carter, Brandon J.; DeLong, Edward F.; Feldman, Robert A.; Ghadiri, Amir; Murray, Alison E.

    2006-01-01

    Six environmental fosmid clones from Antarctic coastal water bacterioplankton were completely sequenced. The genome fragments harbored small-subunit rRNA genes that were between 85 and 91% similar to those of their nearest cultivated relatives. The six fragments span four phyla, including the Gemmatimonadetes, Proteobacteria (? and ?), Bacteroidetes, and high-G+C gram-positive bacteria. Gene-finding and annotation analyses identified 244 total open reading frames. Amino acid comparisons of 123 and 113 Antarctic bacterial amino acid sequences to mesophilic homologs from G+C-specific and SwissProt/UniProt databases, respectively, revealed widespread adaptation to the cold. The most significant changes in these Antarctic bacterial protein sequences included a reduction in salt-bridge-forming residues such as arginine, glutamic acid, and aspartic acid, reduced proline contents, and a reduction in stabilizing hydrophobic clusters. Stretches of disordered amino acids were significantly longer in the Antarctic sequences than in the mesophilic sequences. These characteristics were not specific to any one phylum, COG role category, or G+C content and imply that underlying genotypic and biochemical adaptations to the cold are inherent to life in the permanently subzero Antarctic waters. PMID:16461708

  15. Cotyledon nuclear proteins bind to DNA fragments harboring regulatory elements of phytohemagglutinin genes.

    PubMed Central

    Riggs, C D; Voelker, T A; Chrispeels, M J

    1989-01-01

    The effects of deleting DNA sequences upstream from the phytohemagglutinin-L gene of Phaseolus vulgaris have been examined with respect to the level of gene product produced in the seeds of transgenic tobacco. Our studies indicate that several upstream regions quantitatively modulate expression. Between -1000 and -675, a negative regulatory element reduces expression approximately threefold relative to shorter deletion mutants that do not contain this region. Positive regulatory elements lie between -550 and -125 and, compared with constructs containing only 125 base pairs of upstream sequences (-125), the presence of these two regions can be correlated with a 25-fold and a 200-fold enhancement of phytohemagglutinin-L levels. These experiments were complemented by gel retardation assays, which demonstrated that two of the three regions bind cotyledon nuclear proteins from mid-mature seeds. One of the binding sites maps near a DNA sequence that is highly homologous to protein binding domains located upstream from the soybean seed lectin and Kunitz trypsin inhibitor genes. Competition experiments demonstrated that the upstream regions of a bean beta-phaseolin gene, the soybean seed lectin gene, and an oligonucleotide from the upstream region of the trypsin inhibitor gene can compete differentially for factor binding. We suggest that these legume genes may be regulated in part by evolutionarily conserved protein/DNA interactions. PMID:2535513

  16. Rapid radiation events in the family Ursidae indicated by likelihood phylogenetic estimation from multiple fragments of mtDNA.

    PubMed

    Waits, L P; Sullivan, J; O'Brien, S J; Ward, R H

    1999-10-01

    The bear family (Ursidae) presents a number of phylogenetic ambiguities as the evolutionary relationships of the six youngest members (ursine bears) are largely unresolved. Recent mitochondrial DNA analyses have produced conflicting results with respect to the phylogeny of ursine bears. In an attempt to resolve these issues, we obtained 1916 nucleotides of mitochondrial DNA sequence data from six gene segments for all eight bear species and conducted maximum likelihood and maximum parsimony analyses on all fragments separately and combined. All six single-region gene trees gave different phylogenetic estimates; however, only for control region data was this significantly incongruent with the results from the combined data. The optimal phylogeny for the combined data set suggests that the giant panda is most basal followed by the spectacled bear. The sloth bear is the basal ursine bear, and there is weak support for a sister taxon relationship of the American and Asiatic black bears. The sun bear is sister taxon to the youngest clade containing brown bears and polar bears. Statistical analyses of alternate hypotheses revealed a lack of strong support for many of the relationships. We suggest that the difficulties surrounding the resolution of the evolutionary relationships of the Ursidae are linked to the existence of sequential rapid radiation events in bear evolution. Thus, unresolved branching orders during these time periods may represent an accurate representation of the evolutionary history of bear species. PMID:10508542

  17. Towards the onset of fruit tree growing north of the Alps: ancient DNA from waterlogged apple (Malus sp.) seed fragments.

    PubMed

    Schlumbaum, Angela; van Glabeke, Sabine; Roldan-Ruiz, Isabel

    2012-01-20

    Wild apples (Malus sp.) have been a major food source in the northern Alpine region since prehistory and their use is well understood. The onset of deliberate fruit tree growing in the area is, however, less clear. It is generally assumed that horticulture was practised in Roman times, but it might be even earlier. In the archaeological record seed testa and pericarp remains are particularly frequent at sites with waterlogged preservation such as lakeshore settlements or wells, pits and ditches, but the distinction between wild and domestic plants is not morphologically possible. With waterlogged remains being one main source of information about past fruit cultivation, we have tested the feasibility of analysing ancient DNA from waterlogged preserved bulk samples of testa fragments. We studied apple seeds from three Neolithic and three Roman sites with waterlogged preservation in the Alpine foreland. Chloroplast markers failed in all samples, but nuclear ITS1 (internal transcribed spacer region 1) of the ribosomal DNA was successfully typed in two Roman samples from the site Oedenburg/Biesheim-Kunheim (Haut-Rhin, F). The retrieved ITS1 sequences are identical to each other and are shared with wild Malus sylvestris and Malus sieversii, and with domestic apple cultivars, supporting the potential of using waterlogged remains for identifying the genetic status of apple diachronically. PMID:21501956

  18. The Relationship between Seminal Melatonin with Sperm Parameters, DNA Fragmentation and Nuclear Maturity in Intra-Cytoplasmic Sperm Injection Candidates

    PubMed Central

    Sharbatoghli, Mina; Rezazadeh Valojerdi, Mojtaba; Bahadori, Mohammad Hadi; Salman Yazdi, Reza; Ghaleno, Leila Rashki

    2015-01-01

    Objective Melatonin, the chief secretory product of the pineal gland, regulates dynamic physiological adaptations that occur in seasonally breeding mammals as a response to changes in daylight hours. Because of the presence of melatonin in semen and the mem- brane melatonin receptor in spermatozoa, the impact of melatonin on the regulation of male infertility is still questionable. The aim of this study was to determine the effects of endogenous melatonin on human semen parameters (sperm concentration, motility and normal morphology), DNA fragmentation (DF) and nuclear maturity. Materials and Methods In this clinical prospective study, semen samples from 75 infer- tile men were routinely analyzed and assessed for melatonin and total antioxidant capac- ity (TAC) levels using the enzyme-linked immunosorbent assay (ELISA) and colorimetric assay kits, respectively. DF was examined by the sperm chromatin dispersion (SCD) test. Acidic aniline blue staining was used to detect chromatin defects in the sperm nuclei. Results There was no significant correlation between seminal plasma melatonin and TAC with sperm parameters and nuclear maturity. However, we observed a positive significant correlation between DF and melatonin level (r=0.273, P<0.05). Conclusion Melatonin in seminal plasma is positively correlated with damaged sperm DNA of infertile patients. The mechanism of this phenomenon needs further study. PMID:26464827

  19. Damage to Model DNA Fragments by 0.25-1.0 eV Electrons Attached to a Thymine * Joanna Berdys,, Piotr Skurski,, and Jack Simons*,

    E-print Network

    Simons, Jack

    Damage to Model DNA Fragments by 0.25-1.0 eV Electrons Attached to a Thymine * Orbital Joanna caused by low-energy electrons, we considered electrons that attach either to cytosine's lowest *-orbital or to a PdO *-orbital of a phosphate unit. We examined a range of electron kinetic energies (E) (e

  20. Level of HgCl2-mediated phosphorylation of intracellular proteins determines death of thymic T-lymphocytes with or without DNA fragmentation.

    PubMed

    Akhand, A A; Kato, M; Suzuki, H; Miyata, T; Nakashima, I

    1998-11-01

    Exposure to Hg2+ at a wide range of concentrations (approximately 1-100 microM) more or less caused the death of murine thymic T-lymphocytes, and exposure to 1 microM but not 10 microM (or more) of Hg2- induced DNA fragmentation. Exposure of cells to Hg2+ caused phosphorylation of multiple cellular proteins at the tyrosine residue in a concentration-dependent manner. We found that not only the DNA fragmentation induced by 1 microM Hg2+ but also the cell death bypassing DNA fragmentation caused by 10 microM or more Hg2+ was partly inhibited by protein kinase inhibitors such as staurosporine and herbimycin A. This result suggested the involvement of a protein phosphorylation-linked signal in the mechanism of the Hg2+-mediated cell death with or without DNA fragmentation. Analysis of proteins by both one- and two-dimensional electrophoresis and immunoblot showed that a 52-kDa Shc protein was heavily phosphorylated by an early signal delivered by a high concentration of Hg2+, which also phosphorylated extracellular signal-regulated kinase 1 (ERK1; p44) and ERK2 (p42) of the mitogen-activated protein kinase (MAPK) family in a concentration- and time-dependent manner. The c-Jun amino terminal kinase (p54), which is a distant relative of the MAPK family, was also phosphorylated by the treatment with Hg2+. This eventually formed the signaling cascade that ended with a nuclear target by phosphorylating c-jun at the serine 73. This phosphorylation of c-jun was inhibited by staurosporine. These results suggest that a high level of Hg2+-mediated protein phosphorylation-linked signal induces rapid cell death bypassing DNA fragmentation, whereas a lower level induces cell death accompanying DNA fragmentation. This conclusion in turn implies that DNA fragmentation is not always a prerequisite for the signal transduction-dependent cell death of T-lymphocytes. PMID:9779822

  1. A Chloroplast DNA Phylogeny of the Caryophyllales Based on Structural and Inverted Repeat Restriction Site Variation

    E-print Network

    Downie, Stephen R.

    Restriction Site Variation Stephen R. Downie; Jeffrey D. Palmer Systematic Botany, Vol. 19, No. 2. (Apr. - Jun%3AACDPOT%3E2.0.CO%3B2-0 Systematic Botany is currently published by American Society of Plant Taxonomists

  2. Blackboard Electrophoresis: An Inexpensive Exercise on the Principles of DNA Restriction Analysis

    ERIC Educational Resources Information Center

    Costa, M. J.

    2007-01-01

    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…

  3. Physiological, chemical, morphological, and plant infectivity characteristics of Frankia isolates from Myrica pennsylvanica: correlation to DNA restriction patterns.

    PubMed

    Bloom, R A; Lechevalier, M P; Tate, R L

    1989-09-01

    The filter exclusion method was used to isolate Frankia strains from Myrica pennsylvanica (bayberry) root nodules collected at diverse sites in New Jersey. A total of 16 isolates from five locations were cultured. The isolates were characterized by morphological, chemical, physiological, and plant infectivity criteria and compared with genomic DNA restriction pattern data, which were used to assign the isolates into gel groups (see accompanying paper). The isolates from M. pennsylvanica evaluated in this study were characteristic of Frankia physiological group B strains and were indistinguishable on the basis of whole-cell wall chemistry and diaminopimelic acid isomer analysis. Distinct differences in the spectrum of utilized organic acids and carbohydrates were observed among the isolates and were the only phenotypic criteria by which the isolates could be separated and assigned into separate groups. In general, isolates within a restriction pattern gel group had identical utilization patterns, whereas intragroup isolates had different utilization patterns. Correlation of these phenotypic characteristics with the results of molecular analysis revealed an exclusive carbohydrate and organic acid utilization pattern for each gel group as established by restriction pattern analysis. PMID:2802600

  4. Cytotoxicity of restriction enzyme-induced DNA strand breaks in radiosensitive and radioresistant human tumor cell lines

    SciTech Connect

    Kinashi, Y.; Nagasawa, H.; Little, J.B. )

    1993-09-20

    The purpose was to examine the role of sensitivity to specific types of DNA double strand breaks in human tumor cell response. The X ray-sensitive human squamous carcinoma cell line SCC-61 and the X ray-resistant line SQ-20B were exposed to the restriction enzymes HaeIII, HinfI, PvuII, BamHI by electroporation. Cyotoxicity of these restriction endonucleases was measured by a colony formation assay. Cell killing by each enzyme occurred in a concentration-dependent manner. The radiosensitive cell line was more sensitive to all four restriction enzymes than the radioresistant line, paralleling the response to ionizing radiation. However, the magnitude of the difference was smaller than for radiation. The 5-base sticky ended cutter HinfI and 6-base blunt ended cutter PvuII were much more effective in killing cells from both lines than BamHI, a 6-base sticky ended cutter, whereas the 4-base blunt ended cutter HaeIII was intermediate in its effectiveness. Thus, enzyme sensitivity could not be related to the type of cutter or the distance between cutting sites. 14 refs., 1 fig., 2 tabs.

  5. 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

  6. The Protein Kinase C? Catalytic Fragment Is Critical for Maintenance of the G2/M DNA Damage Checkpoint*

    PubMed Central

    LaGory, Edward L.; Sitailo, Leonid A.; Denning, Mitchell F.

    2010-01-01

    Protein kinase C? (PKC?) is an essential component of the intrinsic apoptotic program. Following DNA damage, such as exposure to UV radiation, PKC? is cleaved in a caspase-dependent manner, generating a constitutively active catalytic fragment (PKC?-cat), which is necessary and sufficient for keratinocyte apoptosis. We found that in addition to inducing apoptosis, expression of PKC?-cat caused a pronounced G2/M cell cycle arrest in both primary human keratinocytes and immortalized HaCaT cells. Consistent with a G2/M arrest, PKC?-cat induced phosphorylation of Cdk1 (Tyr15), a critical event in the G2/M checkpoint. Treatment with the ATM/ATR inhibitor caffeine was unable to prevent PKC?-cat-induced G2/M arrest, suggesting that PKC?-cat is functioning downstream of ATM/ATR in the G2/M checkpoint. To better understand the role of PKC? and PKC?-cat in the cell cycle response to DNA damage, we exposed wild-type and PKC? null mouse embryonic fibroblasts (MEFs) to UV radiation. Wild-type MEFs underwent a pronounced G2/M arrest, Cdk1 phosphorylation, and induction of apoptosis following UV exposure, whereas PKC? null MEFs were resistant to these effects. Expression of PKC?-green fluorescent protein, but not caspase-resistant or kinase-inactive PKC?, was able to restore G2/M checkpoint integrity in PKC? null MEFs. The function of PKC? in the DNA damage-induced G2/M cell cycle checkpoint may be a critical component of its tumor suppressor function. PMID:19917613

  7. T cells detect intracellular DNA but fail to induce type I IFN responses: implications for restriction of HIV replication.

    PubMed

    Berg, Randi K; Rahbek, Stine H; Kofod-Olsen, Emil; Holm, Christian K; Melchjorsen, Jesper; Jensen, David G; Hansen, Anne Louise; Jørgensen, Louise B; Ostergaard, Lars; Tolstrup, Martin; Larsen, Carsten S; Paludan, Søren R; Jakobsen, Martin R; Mogensen, Trine H

    2014-01-01

    HIV infects key cell types of the immune system, most notably macrophages and CD4+ T cells. Whereas macrophages represent an important viral reservoir, activated CD4+ T cells are the most permissive cell types supporting high levels of viral replication. In recent years, it has been appreciated that the innate immune system plays an important role in controlling HIV replication, e.g. via interferon (IFN)-inducible restriction factors. Moreover, innate immune responses are involved in driving chronic immune activation and the pathogenesis of progressive immunodeficiency. Several pattern recognition receptors detecting HIV have been reported, including Toll-like receptor 7 and Retinoic-inducible gene-I, which detects viral RNA. Here we report that human primary T cells fail to induce strong IFN responses, despite the fact that this cell type does express key molecules involved in DNA signaling pathways. We demonstrate that the DNA sensor IFI16 migrates to sites of foreign DNA localization in the cytoplasm and recruits the signaling molecules stimulator of IFN genes and Tank-binding kinase, but this does not result in expression of IFN and IFN-stimulated genes. Importantly, we show that cytosolic DNA fails to affect HIV replication. However, exogenous treatment of activated T cells with type I IFN has the capacity to induce expression of IFN-stimulated genes and suppress HIV replication. Our data suggest the existence of an impaired DNA signaling machinery in T cells, which may prevent this cell type from activating cell-autonomous anti-HIV responses. This phenomenon could contribute to the high permissiveness of CD4+ T cells for HIV-1. PMID:24404168

  8. Major Lineages within Apiaceae Subfamily Apioideae: A Comparison of Chloroplast Restriction Site and DNA Sequence Data

    E-print Network

    Downie, Stephen R.

    and DNA Sequence Data Gregory M. Plunkett; Stephen R. Downie American Journal of Botany, Vol. 86, No. 7%3C1014%3AMLWASA%3E2.0.CO%3B2-L American Journal of Botany is currently published by Botanical Society

  9. of the plasmid DNA prepared by this method is high enough for restriction

    E-print Network

    Bhattacharyya, Madan Kumar

    reaction, nucleotide sequencing and transfection of eukaryotic cells. Moreover, our nov- el procedure. A dye-buoyant-density method for the detection and isolation of closed circular du- plex DNA in HeLa cells. Proc. Natl. Acad. Sci. USA 57:1514. 3.Ward, L.H. and A.W. Jarvis. 1992. Rapid re- moval of cesium

  10. A microfluidic-based electrochemical biochip for label-free diffusion-restricted DNA hybridization analysis

    E-print Network

    Ghodssi, Reza

    online 16 May 2012 Keywords: Electrochemical impedance spectroscopy DNA hybridization biosensor Biochip spectroscopy (EIS) analyses at these length scales. Our highly functional microfluidic biosensor enables-reactivity of 13% following 20 min incubation with the target. This new biosensing approach can be further modeled

  11. New restriction fragment length polymorphisms in the cytochrome oxidase I gene facilitate host strain identification of fall armyworm (Lepidoptera: Noctuidae) populations in the southeastern United States.

    PubMed

    Nagoshi, Rod N; Meagher, Robert L; Adamczyk, John J; Braman, S Kristine; Brandenburg, Rick L; Nuessly, Gregg

    2006-06-01

    Several restriction sites in the cytochrome oxidase I gene of fall armyworm, Spodoptera frugiperda (J.E. Smith), were identified by sequence analysis as potentially being specific to one of the two host strains. Strain specificity was demonstrated for populations in Florida, Texas, Mississippi, Georgia, and North Carolina, with an AciI and SacI site specific to the rice (Oryjza spp.)-strain and a BsmI and HinfI site joining an already characterized MspI site as diagnostic of the corn (Zea mays L.)-strain. All four of these sites can be detected by digestion of a single 568-bp polymerase chain reaction-amplified fragment, but the use of two enzymes in separate digests was found to provide accurate and rapid determination of strain identity. The effectiveness of this method was demonstrated by the analysis of almost 200 adult and larval specimens from the Mississippi delta region. The results indicated that the corn-strain is likely to be the primary strain infesting cotton (Gossypium spp.) and that an unexpected outbreak of fall armyworm on the ornamental tree Paulownia tomentosa (Thunb.) Sieb. & Zucc. ex Steud. was due almost entirely to the rice-strain. PMID:16813297

  12. Response of soybean rhizosphere communities to human hygiene water addition as determined by community level physiological profiling (CLPP) and terminal restriction fragment length polymorphism (TRFLP) analysis

    NASA Technical Reports Server (NTRS)

    Kerkhof, L.; Santoro, M.; Garland, J.

    2000-01-01

    In this report, we describe an experiment conducted at Kennedy Space Center in the biomass production chamber (BPC) using soybean plants for purification and processing of human hygiene water. Specifically, we tested whether it was possible to detect changes in the root-associated bacterial assemblage of the plants and ultimately to identify the specific microorganism(s) which differed when plants were exposed to hygiene water and other hydroponic media. Plants were grown in hydroponics media corresponding to four different treatments: control (Hoagland's solution), artificial gray water (Hoagland's+surfactant), filtered gray water collected from human subjects on site, and unfiltered gray water. Differences in rhizosphere microbial populations in all experimental treatments were observed when compared to the control treatment using both community level physiological profiles (BIOLOG) and molecular fingerprinting of 16S rRNA genes by terminal restriction fragment length polymorphism analysis (TRFLP). Furthermore, screening of a clonal library of 16S rRNA genes by TRFLP yielded nearly full length SSU genes associated with the various treatments. Most 16S rRNA genes were affiliated with the Klebsiella, Pseudomonas, Variovorax, Burkholderia, Bordetella and Isosphaera groups. This molecular approach demonstrated the ability to rapidly detect and identify microorganisms unique to experimental treatments and provides a means to fingerprint microbial communities in the biosystems being developed at NASA for optimizing advanced life support operations.

  13. Mapping the carriage of flaA-restriction fragment length polymorphism Campylobacter genotypes on poultry carcasses through the processing chain and comparison to clinical isolates.

    PubMed

    Duffy, Lesley L; Blackall, Patrick J; Cobbold, Rowland N; Fegan, Narelle

    2015-06-01

    Poultry are considered a major source for campylobacteriosis in humans. A total of 1866 Campylobacter spp. isolates collected through the poultry processing chain were typed using flaA-restriction fragment length polymorphism to measure the impact of processing on the genotypes present. Temporally related human clinical isolates (n = 497) were also typed. Isolates were obtained from whole chicken carcass rinses of chickens collected before scalding, after scalding, before immersion chilling, after immersion chilling and after packaging as well as from individual caecal samples. A total of 32 genotypes comprising at least four isolates each were recognised. Simpson's Index of Diversity (D) was calculated for each sampling site within each flock, for each flock as a whole and for the clinical isolates. From caecal collection to after packaging samples the D value did not change in two flocks, decreased in one flock and increased in the fourth flock. Dominant genotypes occurred in each flock but their constitutive percentages changed through processing. There were 23 overlapping genotypes between clinical and chicken isolates. The diversity of Campylobacter is flock dependant and may alter through processing. This study confirms that poultry are a source of campylobacteriosis in the Australian population although other sources may contribute. PMID:25790999

  14. Use of mitochondrial cytochrome oxidase I polymerase chain reaction-restriction fragment length polymorphism for identifying subclades of Bemisia tabaci Mediterranean group.

    PubMed

    Chu, Dong; Hu, Xiangshun; Gao, Changsheng; Zhao, Huiyan; Nichols, Robert L; Li, Xianchun

    2012-02-01

    The Mediterranean group (commonly known as Q biotype; hereafter MED) of the sweetpotato whitefly, Bemisia tabaci (Gennadius), originated in the Mediterranean region, but it now has been found in at least 10 countries outside the Mediterranean. Collections of B. tabaci from some of these countries exhibit different pest behaviors and pesticide resistance characteristics, yet all may be classified as MED. A phylogenetic analysis of 120 mitochondrial cytochrome oxidase I (mtCOI) sequences (JN966761-JN966880) of MED whiteflies collected in Arizona and of 417 retrieved from the GenBank database resolves the MED into five subclades, designated as Q1-Q5. Only subclades Q1 and Q2 have been detected in the United States. Q1 and the other four subclades (Q2-Q5) differ in the number or position of the AluI recognition sites. Based on the differences in the AluI recognition sites reported here and the previously reported differences in VspI recognition sites, we developed a simple diagnostic technique to identify subclades Q1-Q5 by using mtCOI polymerase chain reaction (PCR)-restriction fragment-length polymorphism (RFLP). A test of a worldwide collection of whiteflies demonstrates that this combination mtCOIPCR-RFLP technique can reliably distinguish not only the MED from the Middle East-Asia Minor 1 group but also the Q1 from any of the other four MED subclades. PMID:22420277

  15. Detection of DNA sequence polymorphisms in human genomic DNA by using denaturing gradient gel blots

    SciTech Connect

    Gray, M.R. )

    1992-02-01

    Denaturing gradient gel electrophoresis can detect sequence differences outside restriction-enzyme recognition sites. DNA sequence polymorphisms can be detected as restriction-fragment melting polymorphisms (RFMPs) in genomic DNA by using blots made from denaturing gradient gels. In contrast to the use of Southern blots to find sequence differences, denaturing gradient gel blots can detect differences almost anywhere, not just at 4-6-bp restriction-enzyme recognition sites. Human genomic DNA was digested with one of several randomly selected 4-bp recognition-site restriction enzymes, electrophoresed in denaturing gradient gels, and transferred to nylon membranes. The blots were hydridized with radioactive probes prepared from the factor VIII, type II collagen, insulin receptor, [beta][sub 2]-adrenergic receptor, and 21-hydroxylase genes; in unrelated individuals, several RFM's were found in fragments from every locus tested. No restriction map or sequence information was used to detect RFMP's.

  16. ClpXP protease targets long-lived DNA translocation states of a helicase-like motor to cause restriction alleviation

    PubMed Central

    Simons, Michelle; Diffin, Fiona M.; Szczelkun, Mark D.

    2014-01-01

    We investigated how Escherichia coli ClpXP targets the helicase-nuclease (HsdR) subunit of the bacterial Type I restriction–modification enzyme EcoKI during restriction alleviation (RA). RA is a temporary reduction in endonuclease activity that occurs when Type I enzymes bind unmodified recognition sites on the host genome. These conditions arise upon acquisition of a new system by a naïve host, upon generation of new sites by genome rearrangement/mutation or during homologous recombination between hemimethylated DNA. Using recombinant DNA and proteins in vitro, we demonstrate that ClpXP targets EcoKI HsdR during dsDNA translocation on circular DNA but not on linear DNA. Protein roadblocks did not activate HsdR proteolysis. We suggest that DNA translocation lifetime, which is elevated on circular DNA relative to linear DNA, is important to RA. To identify the ClpX degradation tag (degron) in HsdR, we used bioinformatics and biochemical assays to design N- and C-terminal mutations that were analysed in vitro and in vivo. None of the mutants produced a phenotype consistent with loss of the degron, suggesting an as-yet-unidentified recognition pathway. We note that an EcoKI nuclease mutant still produces cell death in a clpx? strain, consistent with DNA damage induced by unregulated motor activity. PMID:25260590

  17. Induction of DNA double-strand breaks by restriction enzymes in X-ray-sensitive mutant Chinese hamster ovary cells measured by pulsed-field gel electrophoresis

    SciTech Connect

    Kinashi, Yuko; Nagasawa, Hatsumi; Little, J.B.; Okayasu, Ryuichi; Iliakis, G.E.

    1995-02-01

    This investigation was designed to determine whether the cytotoxic effects of different restriction endonucleases are related to the number and type of DNA double-strand breaks (DSBs) they produce. Chinese hamster ovary (CHO) K1 and xrs-5 cells, a radiosensitive mutant of CHO K1, were exposed to restriction endonucleases HaeIII, HinfI, PvuII and BamHI by electroporation. These enzymes represent both blunt and sticky end cutters with differing recognition sequence lengths. The number of DSBs was measured by pulsed-field gel electrophoresis (PFGE). Two forms of PFGE were employed: asymmetric field-inversion gel electrophoresis (AFIGE) for measuring the kinetics of DNA breaks by enzyme digestion and clamped homogeneous gel electrophoresis (CHEF) for examining the size distributions of damaged DNA. The amount of DNA damage induced by exposure to all four restriction enzymes was significantly greater in xrs-5 compared to CHO K1 cells, consistent with the reported DSB repair deficiency in these cells. Since restriction endonucleases produce DSBs alone as opposed to the various types of DNA damage induced by X rays, these results confirm that the repair defect in this mutant involves the rejoining of DSBs. Although the cutting frequency was directly related to the length of the recognition sequence for four restriction enzymes, there was no simple correlation between the cytotoxic effect and the amount of DNA damage produced by each enzyme in either cell line. This finding suggests that the type or nature of the cutting sequence itself may play a role in restriction enzyme-induced cell killing. 32 refs., 6 figs., 3 tabs.

  18. Conserved Overlapping Gene Arrangement, Restricted Expression, and Biochemical Activities of DNA Polymerase ? (POLN)*

    PubMed Central

    Takata, Kei-ichi; Tomida, Junya; Reh, Shelley; Swanhart, Lisa M.; Takata, Minoru; Hukriede, Neil A.; Wood, Richard D.

    2015-01-01

    DNA polymerase ? (POLN) is one of 16 DNA polymerases encoded in vertebrate genomes. It is important to determine its gene expression patterns, biological roles, and biochemical activities. By quantitative analysis of mRNA expression, we found that POLN from the zebrafish Danio rerio is expressed predominantly in testis. POLN is not detectably expressed in zebrafish embryos or in mouse embryonic stem cells. Consistent with this, injection of POLN-specific morpholino antisense oligonucleotides did not interfere with zebrafish embryonic development. Analysis of transcripts revealed that vertebrate POLN has an unusual gene expression arrangement, sharing a first exon with HAUS3, the gene encoding augmin-like complex subunit 3. HAUS3 is broadly expressed in embryonic and adult tissues, in contrast to POLN. Differential expression of POLN and HAUS3 appears to arise by alternate splicing of transcripts in mammalian cells and zebrafish. When POLN was ectopically overexpressed in human cells, it specifically coimmunoprecipitated with the homologous recombination factors BRCA1 and FANCJ, but not with previously suggested interaction partners (HELQ and members of the Fanconi anemia core complex). Purified zebrafish POLN protein is capable of thymine glycol bypass and strand displacement, with activity dependent on a basic amino acid residue known to stabilize the primer-template. These properties are conserved with the human enzyme. Although the physiological function of pol ? remains to be clarified, this study uncovers distinctive aspects of its expression control and evolutionarily conserved properties of this DNA polymerase. PMID:26269593

  19. 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

  20. Electrospray/ion trap mass spectrometry of single-stranded and duplex fragments of DNA

    SciTech Connect

    Doktycz, M.J.; Habibi-Goudarzi, S.; McLuckey, S.A.

    1994-12-31

    A particularly interesting, and so far unique, feature of electrospray is its ability to form gaseous ions of non-covalently bound species of biological relevance that are known to exist as specific complexes under physiological conditions. The authors are specifically interested in the role quadrupole ion trap mass spectrometry might play in the analysis of ions derived from specific complexes formed in the condensed-phase. The ion trap has already been demonstrated as a mass analyzer for covalently bound biomolecule-ions derived from cesium ion bombardment, matrix assisted laser desorption, and electrospray. A number of useful experiments have been demonstrated with these ions including high mass resolution, multiple stages of mass spectrometry and ion/molecule reactions. In addition to its merits as an analytical mass spectrometer, the relatively small size and low cost of the ion trap are attractive features which can make it more widely accessible to researchers than more expensive forms of mass spectrometry. For these reasons, it is of interest to explore the possibility for injecting and storing non-covalently associated biomolecule-ions in a quadrupole ion trap for the purpose of mass analysis and for determining what other types of information might be obtainable. The authors have recently reported the injection, storage, and mass analysis of the myoglobin ions, which contain a non-covalently bound heme group, in a quadrupole ion trap. The authors have also focused attention on duplex DNA molecules as an extension of ion trap studies of nucleic acid constituents. Studies to date have emphasized the electrospray, interface, and ion trapping conditions required to observe these species.

  1. Biotech SRMs Designed for the Rapidly Evolving Forensic DNA and Human Identity Testing Communities

    E-print Network

    Biotech SRMs Designed for the Rapidly Evolving Forensic DNA and Human Identity Testing Communities, with renewals issued in 1998 and 2003. SRM 2392 Mitochondrial DNA Sequencing Standard (Human) was released of their measurements. SRM 2390 DNA Profiling Standard for Restriction Fragment Length Polymorphism (RFLP) genetic loci

  2. Improved ethanol production from biomass by a rumen metagenomic DNA fragment expressed in Escherichia coli MS04 during fermentation.

    PubMed

    Loaces, Inés; Amarelle, Vanesa; Muñoz-Gutierrez, Iván; Fabiano, Elena; Martinez, Alfredo; Noya, Francisco

    2015-11-01

    With the aim of improving current ethanologenic Escherichia coli strains, we screened a metagenomic library from bovine ruminal fluid for cellulolytic enzymes. We isolated one fosmid, termed Csd4, which was able to confer to E. coli the ability to grow on complex cellulosic material as the sole carbon source such as avicel, carboxymethyl cellulose, filter paper, pretreated sugarcane bagasse, and xylan. Glucanolytic activity obtained from E. coli transformed with Csd4 was maximal at 24 h of incubation and was inhibited when glucose or xylose were present in the media. The 34,406-bp DNA fragment of Csd4 was completely sequenced, and a putative endoglucanase, a xylosidase/arabinosidase, and a laccase gene were identified. Comparison analysis revealed that Csd4 derived from an organism closely related to Prevotella ruminicola, but no homologies were found with any of the genomes already sequenced. Csd4 was introduced into the ethanologenic E. coli MS04 strain and ethanol production from CMC, avicel, sugarcane bagasse, or filter paper was observed. Exogenously expressed ?-glucosidase had a positie effect on cell growth in agreement with the fact that no putative ?-glucosidase was found in Csd4. Ethanol production from sugarcane bagasse was improved threefold by Csd4 after saccharification by commercial Trichoderma reesei cellulases underlining the ability of Csd4 to act as a saccharification enhancer to reduce the enzymatic load and time required for cellulose deconstruction. PMID:26175105

  3. Anethole induces apoptotic cell death accompanied by reactive oxygen species production and DNA fragmentation in Aspergillus fumigatus and Saccharomyces cerevisiae.

    PubMed

    Fujita, Ken-Ichi; Tatsumi, Miki; Ogita, Akira; Kubo, Isao; Tanaka, Toshio

    2014-02-01

    trans-Anethole (anethole), a major component of anise oil, has a broad antimicrobial spectrum, and antimicrobial activity that is weaker than that of other antibiotics on the market. When combined with polygodial, nagilactone E, and n-dodecanol, anethole has been shown to possess significant synergistic antifungal activity against a budding yeast, Saccharomyces cerevisiae, and a human opportunistic pathogenic yeast, Candida albicans. However, the antifungal mechanism of anethole has not been completely determined. We found that anethole stimulated cell death of a human opportunistic pathogenic fungus, Aspergillus fumigatus, in addition to S. cerevisiae. The anethole-induced cell death was accompanied by reactive oxygen species production, metacaspase activation, and DNA fragmentation. Several mutants of S. cerevisiae, in which genes related to the apoptosis-initiating execution signals from mitochondria were deleted, were resistant to anethole. These results suggest that anethole-induced cell death could be explained by oxidative stress-dependent apoptosis via typical mitochondrial death cascades in fungi, including A. fumigatus and S. cerevisiae. PMID:24393541

  4. 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. ); Lia, K.; Silverman, G.A. )

    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.

  5. Ribosomal DNA repeat unit polymorphism in some pulses.

    PubMed

    Ignacimuthu, S

    2000-02-01

    Restriction fragment length polymorphism (RFLP) of nuclear ribosomal RNA genes (rDNA) was carried out in some pulse species such as Lablab purpureus, Dolichos tribolus, Vigna bournii, V. grahmiana, V. unguiculata and V. wightii. Restriction endonuclease digestion and Southern blot analysis with wheat probe pTa 71 revealed a bewildering diversity. The fragment patterns produced by different enzymes such as Dpn II, Hind III, EcoRI and Bam H1 already illustrate considerable polymorphism. PMID:11218841

  6. 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

  7. 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

  8. Characterization of gut microbiota profiles by disease activity in patients with Crohn’s disease using data mining analysis of terminal restriction fragment length polymorphisms

    PubMed Central

    ANDOH, AKIRA; KOBAYASHI, TOSHIO; KUZUOKA, HIROYUKI; TSUJIKAWA, TOMOYUKI; SUZUKI, YASUO; HIRAI, FUMIHITO; MATSUI, TOSHIYUKI; NAKAMURA, SHIRO; MATSUMOTO, TAKAYUKI; FUJIYAMA, YOSHIHIDE

    2014-01-01

    The gut microbiota plays a significant role in the pathogenesis of Crohn’s disease (CD). In this study, we analyzed the disease activity and associated fecal microbiota profiles in 160 CD patients and 121 healthy individuals. Fecal samples from the CD patients were collected during three different clinical phases, the active (n=66), remission-achieved (n=51) and remission-maintained (n=43) phases. Terminal restriction fragment length polymorphism (T-RFLP) and data mining analysis using the Classification and Regression Tree (C&RT) approach were performed. Data mining provided a decision tree that clearly identified the various subject groups (nodes). The majority of the healthy individuals were divided into Node-5 and Node-8. Healthy subjects comprised 99% of Node-5 (91 of 92) and 84% of Node-8 (21 of 25 subjects). Node-3 was characterized by CD (136 of 160 CD subjects) and was divided into Node-6 and Node-7. Node-6 (n=103) was characterized by subjects in the active phase (n=48; 46%) and remission-achieved phase (n=39; 38%) and Node-7 was characterized by the remission-maintained phase (21 of 37 subjects; 57%). Finally, Node-6 was divided into Node-9 and Node-10. Node-9 (n=78) was characterized by subjects in the active phase (n=43; 55%) and Node-10 (n=25) was characterized by subjects in the remission-maintained phase (n=16; 64%). Differences in the gut microbiota associated with disease activity of CD patients were identified. Thus, data mining analysis appears to be an ideal tool for the characterization of the gut microbiota in inflammatory bowel disease. PMID:24748976

  9. Hepatic IGF1 DNA methylation is influenced by gender but not by intrauterine growth restriction in the young lamb.

    PubMed

    Carr, D J; Milne, J S; Aitken, R P; Adam, C L; Wallace, J M

    2015-12-01

    Intrauterine growth restriction (IUGR) and postnatal catch-up growth confer an increased risk of adult-onset disease. Overnourishment of adolescent ewes generates IUGR in ?50% of lambs, which subsequently exhibit increased fractional growth rates. We investigated putative epigenetic changes underlying this early postnatal phenotype by quantifying gene-specific methylation at cytosine:guanine (CpG) dinucleotides. Hepatic DNA/RNA was extracted from IUGR [eight male (M)/nine female (F)] and normal birth weight (12 M/9 F) lambs. Polymerase chain reaction was performed using primers targeting CpG islands in 10 genes: insulin, growth hormone, insulin-like growth factor (IGF)1, IGF2, H19, insulin receptor, growth hormone receptor, IGF receptors 1 and 2, and the glucocorticoid receptor. Using pyrosequencing, methylation status was determined by quantifying cytosine:thymine ratios at 57 CpG sites. Messenger RNA (mRNA) expression of IGF system genes and plasma IGF1/insulin were determined. DNA methylation was independent of IUGR status but sexual dimorphism in IGF1 methylation was evident (MF (both P<0.001). IGF1 mRNA expression correlated negatively with IGF1 methylation (r=-0.507, P=0.002) and positively with plasma IGF1 (r=0.884, P<0.001). Carcass and empty body weights were greater in males (P=0.002-0.014) and this gender difference in early body conformation was mirrored by sexual dimorphism in hepatic IGF1 DNA methylation, mRNA expression and plasma IGF1 concentrations. PMID:26310177

  10. C1q tumor necrosis factor ?-related protein isoform 5 attenuates palmitate-induced DNA fragmentation in myocytes through an AMPK-dependent mechanism

    PubMed Central

    Yang, Won-Mo; Min, Kyung-Ho; Lee, Wan

    2015-01-01

    This article reports the data for the effects of C1q tumor necrosis factor ?-related protein isoform 5 (CTRP5) on the palmitate-induced apoptosis in myocytes. The data obtained from in vitro cultured myocytes shows that the cellular treatment with the globular domain of CTRP5 (gCTRP5) significantly inhibits the palmitate-induced MTT reduction, caspase-3 activation, and DNA fragmentation in a time-dependent manner. The data presented in this article also shows that AraA, an inhibitor of AMPK, almost completely abolished the protective effect of gCTRP5 on the DNA fragmentation induced by palmitate in myocytes. Interpretation of our data and further extensive insights into the protective role of CTRP5 in palmitate-induced apoptosis in myocytes can be found in Yang and Lee (2014) [1]. PMID:26693510

  11. 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.

    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.

  12. Quantification of DNA by Agarose Gel Electrophoresis and Analysis of the Topoisomers of Plasmid and M13 DNA Following Treatment with a Restriction Endonuclease or DNA Topoisomerase I

    ERIC Educational Resources Information Center

    Tweedie, John W.; Stowell, Kathryn M.

    2005-01-01

    A two-session laboratory exercise for advanced undergraduate students in biochemistry and molecular biology is described. The first session introduces students to DNA quantification by ultraviolet absorbance and agarose gel electrophoresis followed by ethidium bromide staining. The second session involves treatment of various topological forms of…

  13. Linear diffusion of the restriction endonuclease EcoRV on DNA is essential for the in vivo function of the enzyme.

    PubMed Central

    Jeltsch, A; Wenz, C; Stahl, F; Pingoud, A

    1996-01-01

    Linear diffusion along DNA is a mechanism of enhancing the association rates of proteins to their specific recognition sites on DNA. It has been demonstrated for several proteins in vitro, but to date in no case in vivo. Here we show that the restriction endonuclease EcoRV slides along the DNA, scanning approximately 1000 bp in one binding event. This process is critically dependent on contacts between amino acid residues of the protein and the backbone of the DNA. The disruption of single hydrogen bonds and, in particular, the alteration of electrostatic interactions between amino acid side chains of the protein and phosphate groups of the DNA interfere with or abolish effective sliding. The efficiency of linear diffusion is dependent on salt concentration, having a maximum at 50 mM NaCl. These results suggest that a nonspecific and mobile binding mode capable of linear diffusion is dependent on a subtle balance of forces governing the interaction of the enzyme and the DNA. A strong correlation between the ability of EcoRV mutants to slide along the DNA in vitro and to protect Escherichia coli cells from phage infection demonstrates that linear diffusion occurs in vivo and is essential for effective phage restriction. Images PMID:8890184

  14. D1/D2 Domain of Large-Subunit Ribosomal DNA for Differentiation of Orpinomyces spp.?

    PubMed Central

    Dagar, Sumit S.; Kumar, Sanjay; Mudgil, Priti; Singh, Rameshwar; Puniya, Anil K.

    2011-01-01

    This study presents the suitability of D1/D2 domain of large-subunit (LSU) ribosomal DNA (rDNA) for differentiation of Orpinomyces joyonii and Orpinomyces intercalaris based on PCR-restriction fragment length polymorphism (RFLP). A variation of G/T in O. intercalaris created an additional restriction site for AluI, which was used as an RFLP marker. The results demonstrate adequate heterogeneity in the LSU rDNA for species-level differentiation. PMID:21784906

  15. 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

  16. Infrared linear dichroism studies of DNA-drug complexes: quantitative determination of the drug-induced restriction of the B-A transition.

    PubMed Central

    Fritzsche, H

    1994-01-01

    The B-A transition of films or fibers of NaDNA occurs at a relative humidity of 75-85%. The fraction of DNA that changed the conformation from B to A form can be determined quantitatively by infrared linear dichroism. DNA-binding drugs can 'freeze' a fraction of DNA in the B form. This fraction of DNA is in the B form and cannot be converted to A-DNA even at a reduced relative humidity of 54%. The 'freezing' potentiality of various drugs can be described by the 'freezing' index, FI, expressed in base pairs per added drug. Drugs with a high value of FI (more than eight base pairs per drug) were observed among both intercalating and groove-binding drugs. High values of FI imply restriction of the conformational flexibility of DNA significantly going beyond the binding site of the drug. This long-range effect of drugs on the conformational flexibility of DNA may be connected with the molecular mechanism of drug action. The freezing index FI is a new quantitative parameter of drug-DNA interaction that should be considered as a valuable tool for drug design. PMID:8139919

  17. DNA DNA DNA (d)DNA DNA DNA

    E-print Network

    Hagiya, Masami

    DNA DNA DNA DNA DNA DNA DNA DNA [ 2008] (d)DNA DNA DNA DNA 2 3 DNA DNA DNA DNA DNA DNA DNA (a) (c) (b) (d) #12;DNA DNA DNA DNA DNA DNA DNA DNA (b) DNA [Tanaka et al.2008] DNA DNA DNA DNA DNA DNA DNA #12;iGEM MIT MIT

  18. Individual and combined effects of ochratoxin A and citrinin on viability and DNA fragmentation in cultured Vero cells and on chromosome aberrations in mice bone marrow cells.

    PubMed

    Bouslimi, Amel; Bouaziz, Chayma; Ayed-Boussema, Imen; Hassen, Wafa; Bacha, Hassen

    2008-09-29

    Ochratoxin A (OTA) and citrinin (CTN) are two common contaminant mycotoxins which can occur jointly in a wide range of food commodities. Both mycotoxins have several toxic effects but share a significant nephrotoxic and carcinogenic potential since OTA and CTN were reported to be responsible for naturally occurring human and animal kidney diseases and tumors. Considering the concomitant production of OTA and CTN, it is very likely that humans and animals are always exposed to the mixture rather than to individual compounds. Therefore, the aim of the present study was to investigate, in vivo and in vitro, whether DNA damage is enhanced by combination of both mycotoxins as compared to their effect separately. To this end, we have assessed their effects individually or combined on cell proliferation and DNA fragmentation in cultured Vero cells and in vivo by monitoring the induction of chromosome aberrations. Our results clearly showed that cultured renal cells respond to OTA and CTN exposure by a moderate and weak inhibition of cell proliferation, respectively. However, when combined, they exert a significant increase in inhibition of cell viability. Similar results were found for the investigated genotoxicity endpoints (DNA fragmentation and chromosome aberrations). Altogether, our study showed that OTA and CTN combination effects are clearly synergistic. The synergistic induction of DNA damage observed with OTA and CTN taken concomitantly could be relevant to explain the molecular basis of the renal diseases and tumorogenesis induced by naturally occurring mycotoxins. PMID:18638518

  19. Inter- and intraspecific resolution of nrDNA TRFLP assessed by computer-simulated restriction analysis of a diverse collection of ectomycorrhizal fungi.

    PubMed

    Edwards, Ivan P; Turco, Ronald F

    2005-02-01

    An assessment of the inter- and intraspecific resolution of 5' nrDNA ITS TRFLP was conducted by computer-simulated restriction analysis of 316 ectomycorrhizal GenBank sequences. Generally, sequences with a similarity of < 90 % could be distinguished with two to three independent enzyme digests, although sequences with similarity > 95 % were likely to remain unresolved. Choice of restriction enzyme strongly influenced resolution, especially when less than three enzymes were used. Although our results showed that 5' nrDNA ITS TRFLP is a powerful tool for distinguishing between species of ectomycorrhizal fungi, closely related species, including species of Rhizopogon, Dermocybe, Hebeloma, and Lactarius produced indistinguishable 5' nrDNA ITS TRFLP--profiles. 5' nrDNA ITS TRFLP may also split a species, and the probability of this occurring reflected intraspecific sequence variation. For groups of closely related species, the use of 3' nrDNA ITS in conjunction with 5' nrDNA ITS produced improved resolution comparable with RFLP analyses. Overall, our results show that nrDNA ITS TRFLP is a valuable addition to the array of molecular tools available to ectomycorrhizal ecologists. However, simple assessments of ectomycorrhizal species diversity based on numbers of unique TRF created with a single restriction enzyme must be viewed with caution, as for each enzyme we examined, identical TRF common to groups of unrelated species were observed. In addition, to maximize the effectiveness of TRFLP in surveys of ectomycorrhizal fungi it will be useful to combine information from at least two independent enzyme digests in order to distinguish and track species in the field. PMID:15839105

  20. SUPPLEMENTARY NOTES DNA Fragmentation

    E-print Network

    Reich, David

    with water or TE before addition. Store MagNA kit in the refrigerator in the dark and vigorously mix before. Separation on a 96-well ring- magnet (e.g. Agencourt SPRIPlate, A32782) is done for different time intervals. The beads are air-dried for about 5- 15min at room temperature on the magnet. Elution is done in low salt

  1. Comparison of Randomly Amplified Polymorphic DNA with Amplified Fragment Length Polymorphism To Assess Genetic Diversity and Genetic Relatedness within Genospecies III of Pseudomonas syringae

    PubMed Central

    Clerc, Agathe; Manceau, Charles; Nesme, Xavier

    1998-01-01

    Recently, DNA pairing analyses showed that Pseudomonas syringae pv. tomato and related pathovars, including P. syringae pv. maculicola, form a genomic species (Pseudomonas tomato) (L. Gardan, H. L. Shafik, and P. A. D. Grimont, p. 445–448, in K. Rudolph, T. J. Burr, J. W. Mansfield, D. Stead, A. Vivian, and J. von Kietzell, ed., Pseudomonas syringae Pathovars and Related Pathogens, 1997). The genetic diversity of 23 strains belonging to this genomic species and 4 outgroup strains was analyzed with randomly amplified polymorphic DNA (RAPD) and amplified fragment length polymorphic (AFLP) techniques. Simple boiling of P. syringae cells was suitable for subsequent DNA amplification to obtain reliable patterns in RAPD and AFLP analyses. In general, the grouping of P. syringae strains by both analysis techniques corresponded well with the classification obtained from an RFLP analysis of ribosomal DNA operons, DNA pairing studies, and an analysis of pathogenicity data. However, two strains of P. syringae pv. maculicola produced distinct DNA patterns compared to the DNA patterns of other P. syringae pv. maculicola strains; these patterns led us to assume that horizontal transfer of DNA could occur between bacterial populations. Both techniques used in this study have high discriminating power because strains of P. syringae pv. tomato and P. syringae pv. maculicola which were indistinguishable by other techniques, including pathogenicity tests on tomato, were separated into two groups by both RAPD and AFLP analyses. In addition, data analysis showed that the AFLP method was more efficient for assessing intrapathovar diversity than RAPD analysis and allowed clear delineation between intraspecific and interspecific genetic distances, suggesting that it could be an alternative to DNA pairing studies. However, it was not possible to distinguish the two races of P. syringae pv. tomato on the basis of an analysis of the data provided by either the AFLP or RAPD technique. PMID:16349533

  2. Panax ginseng extract modulates oxidative stress, DNA fragmentation and up-regulate gene expression in rats sub chronically treated with aflatoxin B1 and fumonisin B 1.

    PubMed

    Hassan, Aziza M; Abdel-Aziem, Sekena H; El-Nekeety, Aziza A; Abdel-Wahhab, Mosaad A

    2015-10-01

    Aflatoxins and fumonisins are important food-borne mycotoxins implicated in human health and have cytotoxic effects. The aims of the current study were to evaluate the protective role of Panax ginseng extract (PGE) against the synergistic effect of subchronic administration of aflatoxin B1 (AFB1) and fumonisin B1 (FB1) on DNA and gene expression in rat. Female Sprague-Dawley rats were divided into eight groups (ten rats/group) and treated for 12 weeks including the control group, the group having received AFB1 (80 µg/kg bw), the group having received FB1 (100 µg/kg bw), the group having received AFB1 plus FB1 and the groups having received PGE (20 mg/kg bw) alone or with AFB1 and/or FB1. At the end of experiment, liver and kidney were collected for the determination of DNA fragmentation, lipid peroxidation (LP), glutathione (GSH) contents and alterations in gene expression. The results indicated that these mycotoxins increased DNA fragmentation, LP and decreased GSH content in liver and kidney and down-regulated gene expression of antioxidants enzymes. The combined treatments with AFB1 and/or FB1 plus PGE suppressed DNA fragmentation only in the liver, normalized LP and increased GSH in the liver and kidney as well as up-regulated the expression of GPx, SOD1 and CAT mRNA. It could be concluded that AFB1 and FB1 have synergistic genotoxic effects. PGE induced protective effects against their oxidative stress and genotoxicity through its antioxidant properties. PMID:24748134

  3. Identification of the coding region for a second Epstein-Barr virus nuclear antigen (EBNA 2) by transfection of cloned DNA fragments.

    PubMed Central

    Mueller-Lantzsch, N; Lenoir, G M; Sauter, M; Takaki, K; Béchet, J M; Kuklik-Roos, C; Wunderlich, D; Bornkamm, G W

    1985-01-01

    Cell lines were established by co-transfection of cloned M-ABA Epstein-Barr virus (EBV) DNA fragments with plasmids conferring resistance to dominant selective markers. A baby hamster kidney cell line carrying the HindIII-I1 fragment exhibits a nuclear antigen of 82 000 daltons, serologically defined as EBV-determined nuclear antigen (EBNA) 1. Furthermore, a Rat-1 cell line transfected with DNA of the clone pM 780-28 containing three large internal repeats (BglII-U) and the adjacent BglII-C fragment expresses a nuclear antigen of 82 000 daltons which can be visualized only by a subset of anti EBNA-positive human sera. Sera recognizing the 82 000-dalton protein of the transfected cell line reacted with a protein of the same size in the non-producer line Raji, designated as EBNA 2. Conversely, sera without reactivity to the 82 000-dalton protein failed to react with EBNA 2 of Raji cells. P3HR-1 and Daudi cells with large deletions in BglII-U and -C are devoid of EBNA 2. The data presented provide evidence that a second EBNA protein is encoded by the region of the EBV genome which is deleted in the non-transforming P3HR-1 strain. Images Fig. 2. Fig. 3. Fig. 4. Fig. 5. PMID:2992944

  4. Electrochemical detection of synthetic DNA and native 16S rRNA fragments on a microarray using a biotinylated intercalator as coupling site for an enzyme label.

    PubMed

    Zimdars, Andreas; Gebala, Magdalena; Hartwich, Gerhard; Neugebauer, Sebastian; Schuhmann, Wolfgang

    2015-10-01

    The direct electrochemical detection of synthetic DNA and native 16S rRNA fragments isolated from Escherichia coli is described. Oligonucleotides are detected via selective post-labeling of double stranded DNA and DNA-RNA duplexes with a biotinylated intercalator that enables high-specific binding of a streptavidin/alkaline phosphatase conjugate. The alkaline phosphatase catalyzes formation of p-aminophenol that is subsequently oxidized at the underlying gold electrode and hence enables the detection of complementary hybridization of the DNA capture strands due to the enzymatic signal amplification. The hybridization assay was performed on microarrays consisting of 32 individually addressable gold microelectrodes. Synthetic DNA strands with sequences representing six different pathogens which are important for the diagnosis of urinary tract infections could be detected at concentrations of 60 nM. Native 16S rRNA isolated from the different pathogens could be detected at a concentration of 30 fM. Optimization of the sensing surface is described and influences on the assay performance are discussed. PMID:26078123

  5. The impact of partial manganese superoxide dismutase (SOD2)-deficiency on mitochondrial oxidant stress, DNA fragmentation and liver injury during acetaminophen hepatotoxicity

    SciTech Connect

    Ramachandran, Anup; Lebofsky, Margitta; Weinman, Steven A.; Jaeschke, Hartmut

    2011-03-15

    Acetaminophen (APAP) hepatotoxicity is the most frequent cause of acute liver failure in many countries. The mechanism of cell death is initiated by formation of a reactive metabolite that binds to mitochondrial proteins and promotes mitochondrial dysfunction and oxidant stress. Manganese superoxide dismutase (SOD2) is a critical defense enzyme located in the mitochondrial matrix. The objective of this investigation was to evaluate the functional consequences of partial SOD2-deficiency (SOD2+/-) on intracellular signaling mechanisms of necrotic cell death after APAP overdose. Treatment of C57Bl/6J wild type animals with 200 mg/kg APAP resulted in liver injury as indicated by elevated plasma alanine aminotransferase activities (2870 {+-} 180 U/L) and centrilobular necrosis at 6 h. In addition, increased tissue glutathione disulfide (GSSG) levels and GSSG-to-GSH ratios, delayed mitochondrial GSH recovery, and increased mitochondrial protein carbonyls and nitrotyrosine protein adducts indicated mitochondrial oxidant stress. In addition, nuclear DNA fragmentation (TUNEL assay) correlated with translocation of Bax to the mitochondria and release of apoptosis-inducing factor (AIF). Furthermore, activation of c-jun-N-terminal kinase (JNK) was documented by the mitochondrial translocation of phospho-JNK. SOD2+/- mice showed 4-fold higher ALT activities and necrosis, an enhancement of all parameters of the mitochondrial oxidant stress, more AIF release and more extensive DNA fragmentation and more prolonged JNK activation. Conclusions: the impaired defense against mitochondrial superoxide formation in SOD2+/- mice prolongs JNK activation after APAP overdose and consequently further enhances the mitochondrial oxidant stress leading to exaggerated mitochondrial dysfunction, release of intermembrane proteins with nuclear DNA fragmentation and more necrosis.

  6. Protective role of probiotic lactic acid bacteria against dietary fumonisin B1-induced toxicity and DNA-fragmentation in sprague-dawley rats.

    PubMed

    Khalil, Ashraf A; Abou-Gabal, Ashgan E; Abdellatef, Amira A; Khalid, Ahmed E

    2015-08-18

    The genus Fusarium, especially F. verticillioides and F. proliferatum, has been found in several agricultural products worldwide, especially in maize. Regardless the occurrence of symptoms, the presence of Fusarium in maize constitutes an imminent risk due to its ability to produce fumonisins, mycotoxins with proven carcinogenic effect on rats, swine, and equines and already classified as possible carcinogens to humans. The toxicity of incremental levels of fumonisin B1 (FB1), that is, 50, 100, and 200 mg FB1/kg diet, and the role of Lactobacillus delbrueckii subsp. lactis DSM 20076 (LL) and Pediococcus acidilactici NNRL B-5627 (PA) supplementation in counteracting the FB1 effects in intoxicated rats were monitored over a period of 4 weeks. Effects on the feed intake and body weight gain were noticed. A significant (p ? 0.05) increase in the level of liver and kidney functions markers and DNA fragmentation was also noticed in rat groups T100 and T200. The lactic acid bacteria (LAB) supplementation could bring back the normal serum biochemical parameters in rats fed on fumonisin B1-contaminated diets (T50 and T100) compared to FB1-treated groups. In rats of high-dosage dietary groups supplemented with LAB (T200-LL and T200-PA), the supplementation reduced the serum activity levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and creatinine by 11.3, 11.9, 32, and 20%, respectively. DNA fragmentations were observed in the rat group treated with 200 mg FB1 after 3 weeks, while fragmentation was noticed in treated groups with 100 and 200 mg FB1 after 4 weeks. No DNA fragmentation was apparent in FB1-treated rats co-administered the LL or PA strain. These results suggest that in male rats consuming diets containing FB1, there is a time- and dose-dependent increase in serum enzyme activities and DNA lesions. Moreover, Lb. delbrueckii subsp. lactis (LL) and P. acidilactici (PA) strains have a protective effect against antigenotoxicity and precancerous lesions. PMID:25036875

  7. The population structure and recent colonization history of Oregon threespine stickleback determined using restriction-site associated DNA-sequencing.

    PubMed

    Catchen, Julian; Bassham, Susan; Wilson, Taylor; Currey, Mark; O'Brien, Conor; Yeates, Quick; Cresko, William A

    2013-06-01

    Understanding how genetic variation is partitioned across genomes within and among populations is a fundamental problem in ecological and evolutionary genetics. To address this problem, we studied the threespine stickleback fish, which has repeatedly undergone parallel phenotypic and genetic differentiation when oceanic fish have invaded freshwater habitats. While significant evolutionary genetic research has been performed using stickleback from geographic regions that have been deglaciated in the last 20 000 years, less research has focused on freshwater populations that predate the last glacial maximum. We performed restriction-site associated DNA-sequencing (RAD-seq) based population genomic analyses on stickleback from across Oregon, which was not glaciated during the last maximum. We sampled stickleback from coastal, Willamette Basin and central Oregon sites, analysed their genetic diversity using RAD-seq, performed structure analyses, reconstructed their phylogeographic history and tested the hypothesis of recent stickleback introduction into central Oregon, where incidence of this species was only recently documented. Our results showed a clear phylogeographic break between coastal and inland populations, with oceanic populations exhibiting the lowest levels of divergence from one another. Willamette Basin and central Oregon populations formed a clade of closely related populations, a finding consistent with a recent introduction of stickleback into central Oregon. Finally, genome-wide analysis of genetic diversity (?) and correlations of alleles within individuals in subpopulations (FIS) supported a role for introgressive hybridization in coastal populations and a recent expansion in central Oregon. Our results exhibit the power of next-generation sequencing genomic approaches such as RAD-seq to identify both historical population structure and recent colonization history. PMID:23718143

  8. Mapping with RAD (restriction-site associated DNA) markers to rapidly identify QTL for stem rust resistance in Lolium perenne.

    PubMed

    Pfender, W F; Saha, M C; Johnson, E A; Slabaugh, M B

    2011-05-01

    A mapping population was created to detect quantitative trait loci (QTL) for resistance to stem rust caused by Puccinia graminis subsp. graminicola in Lolium perenne. A susceptible and a resistant plant were crossed to produce a pseudo-testcross population of 193 F(1) individuals. Markers were produced by the restriction-site associated DNA (RAD) process, which uses massively parallel and multiplexed sequencing of reduced-representation libraries. Additional simple sequence repeat (SSR) and sequence-tagged site (STS) markers were combined with the RAD markers to produce maps for the female (738 cM) and male (721 cM) parents. Stem rust phenotypes (number of pustules per plant) were determined in replicated greenhouse trials by inoculation with a field-collected, genetically heterogeneous population of urediniospores. The F(1) progeny displayed continuous distribution of phenotypes and transgressive segregation. We detected three resistance QTL. The most prominent QTL (qLpPg1) is located near 41 cM on linkage group (LG) 7 with a 2-LOD interval of 8 cM, and accounts for 30-38% of the stem rust phenotypic variance. QTL were detected also on LG1 (qLpPg2) and LG6 (qLpPg3), each accounting for approximately 10% of phenotypic variance. Alleles of loci closely linked to these QTL originated from the resistant parent for qLpPg1 and from both parents for qLpPg2 and qLpPg3. Observed quantitative nature of the resistance may be due to partial-resistance effects against all pathogen genotypes, or qualitative effects completely preventing infection by only some genotypes in the genetically mixed inoculum. RAD markers facilitated rapid construction of new genetic maps in this outcrossing species and will enable development of sequence-based markers linked to stem rust resistance in L. perenne. PMID:21344184

  9. Calorie restriction and resveratrol supplementation prevent age-related DNA and RNA oxidative damage in a non-human primate.

    PubMed

    Marchal, J; Dal-Pan, A; Epelbaum, J; Blanc, S; Mueller, S; Wittig Kieffer, M; Metzger, F; Aujard, F

    2013-09-01

    Oxidative stress is a key factor in the aging process and in the development of age-related diseases. Because nutritional interventions such as caloric restriction (CR) delay the onset of age-related diseases and increase the lifespan of many species, the impact of a moderate CR was tested on male grey mouse lemur (Microcebus murinus), which have a median survival time of 5.7 years in captivity. The effects of CR on these lemurs were compared with a potential mimetic, resveratrol (RSV), a polyphenol naturally found in grapes. We hypothesized that both CR and RSV impact oxidative DNA and RNA damage compared to standard-fed control (CTL) animals. Adult (3-4 years old) male mouse lemurs were assigned to three dietary groups: a CTL group, a CR group receiving 30% fewer calories than the CTL and a RSV group receiving the CTL diet supplemented with RSV (200 mg·day(-1)·kg(-1)). Oxidative stress was estimated after 3, 9, 15 and 21 months of treatment using the measurement of oxidized nucleosides in urine samples by mass spectrometry. The resting metabolic rate, adjusted for changes in body composition, was also measured to assess the potential relationship between oxygen consumption and oxidative damage markers. This study provides evidence for oxidative stress accumulation with age in grey mouse lemur. Dietary interventions resulted in a short-term increase in oxidative stress levels followed by reduced levels with increasing age. Moreover, in this photoperiod-dependent heterotherm primate, seasonal variations in oxidative stress were observed, which was likely due to a season-dependent, cost-benefit trade-off between torpor use and oxidative stress. PMID:23860387

  10. Dna electrophoresis in photopolymerized polyacrylamide gels on a microfluidic device 

    E-print Network

    Lo, Chih-Cheng

    2009-05-15

    and daughter for their patience and love. vii NOMENCLATURE %T Monomer concentration %C Crosslinker concentration bp base pairs BRM Biased reptation model BRF Biased reptation with fluctuations DNA Deoxyribonucleic acid dsDNA Double..., such as DNA sequencing, detection of restriction fragment length polymorphisms (RFLP), and Southern blotting. Crosslinked polyacrylamide and agarose gels are generaly used as the sieving matrices in these sys- tems [47]. A slab gel is cast by loading liquid...

  11. Isolation of Coxiella burnetii by a centrifugation shell-vial assay from ticks collected in Cyprus: detection by nested polymerase chain reaction (PCR) and by PCR-restriction fragment length polymorphism analyses.

    PubMed

    Spyridaki, Ioanna; Psaroulaki, Anna; Loukaides, Fidias; Antoniou, Maria; Hadjichristodolou, Christos; Tselentis, Yannis

    2002-01-01

    Ticks are the principal vectors and reservoirs of Coxiella burnetii. The identification of isolates is necessary for understanding the clinical diversity of Q fever in different geographic areas. This is the first report of isolation of C. burnetii from ticks by the shell-vial assay and by nested polymerase chain reaction (PCR) assay for the detection of this pathogen in ticks. Of 141 ticks collected in Cyprus (Rhipicephalus sanguineus and Hyalloma spp.), 10% were found to be infected with C. burnetii. Three ticks were positive by hemolymph test, and 11 triturated ticks were positive by nested PCR. Three isolates were obtained by the centrifugation shell-vial technique. Analysis by PCR, then restriction fragment length polymorphism showed that the 3 Cyprus isolates had identical restriction profiles to reference strains Nine Mile and Q212. The methods described are useful in studying the epidemiology and ecology of C. burnetii. PMID:12135275

  12. Mitochondrial DNA Fragmentation as a Molecular Tool to Monitor Thermal Processing of Plant-Derived, Low-Acid Foods, and Biomaterials.

    PubMed

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

    2015-08-01

    Cycle threshold (Ct) increase, quantifying plant-derived DNA fragmentation, was evaluated for its utility as a time-temperature integrator. This novel approach to monitoring thermal processing of fresh, plant-based foods represents a paradigm shift. Instead of using quantitative polymerase chain reaction (qPCR) to detect pathogens, identify adulterants, or authenticate ingredients, this rapid technique was used to quantify the fragmentation of an intrinsic plant mitochondrial DNA (mtDNA) gene over time-temperature treatments. Universal primers were developed which amplified a mitochondrial gene common to plants (atp1). These consensus primers produced a robust qPCR signal in 10 vegetables, 6 fruits, 3 types of nuts, and a biofuel precursor. Using sweet potato (Ipomoea batatas) puree as a model low-acid product and simple linear regression, Ct value was highly correlated to time-temperature treatment (R(2) = 0.87); the logarithmic reduction (log CFU/mL) of the spore-forming Clostridium botulinum surrogate, Geobacillus stearothermophilus (R(2) = 0.87); and cumulative F-value (min) in a canned retort process (R(2) = 0.88), all comparisons conducted at 121 °C. D121 and z-values were determined for G. stearothermophilus ATCC 7953 and were 2.71 min and 11.0 °C, respectively. D121 and z-values for a 174-bp universal plant amplicon were 11.3 min and 9.17 °C, respectively, for mtDNA from sweet potato puree. We present these data as proof-of-concept for a molecular tool that can be used as a rapid, presumptive method for monitoring thermal processing in low-acid plant products. PMID:26235411

  13. Identification of Phytophthora citrophthora with Cloned DNA Probes

    PubMed Central

    Goodwin, P. H.; Kirkpatrick, B. C.; Duniway, J. M.

    1990-01-01

    Two different DNA fragments, one of 2.9 kilobases and the other of 5.1 kilobases, were cloned from Phytophthora citrophthora and showed no homology with DNA from plants and other related fungi. These DNA probes hybridized with DNA from 12 different P. citrophthora isolates obtained from a variety of hosts but did not hybridize with DNA from 6 P. citrophthora isolates obtained from cacao. Southern blot analysis revealed that the probes contained repetitive DNA, and restriction fragment length polymorphisms were identified among several P. citrophthora isolates. Of the isolates tested, two major groups were observed whose genetic similarity correlated with geographical distribution. One of the DNA probes was used to detect P. citrophthora growing from infected citrus roots incubated on semiselective medium. P. citrophthora was not detected by a hybridization assay of total DNA extracted directly from infected roots. Images PMID:16348140

  14. A model of EcoRII restriction endonuclease action: the active complex is most likely formed by one protein subunit and one DNA recognition site

    NASA Technical Reports Server (NTRS)

    Karpova, E. A.; Kubareva, E. A.; Shabarova, Z. A.

    1999-01-01

    To elucidate the mechanism of interaction of restriction endonuclease EcoRII with DNA, we studied by native gel electrophoresis the binding of this endonuclease to a set of synthetic DNA-duplexes containing the modified or canonical recognition sequence 5'-d(CCA/TGG)-3'. All binding substrate or substrate analogues tested could be divided into two major groups: (i) duplexes that, at the interaction with endonuclease EcoRII, form two types of stable complexes on native gel in the absence of Mg2+ cofactor; (ii) duplexes that form only one type of complex, observed both in the presence and absence of Mg2+. Unlike the latter, duplexes under the first group can be hydrolyzed by endonuclease. Data obtained suggest that the active complex is most likely formed by one protein subunit and one DNA recognition sequence. A model of EcoRII endonuclease action is presented.

  15. Differentiation of Aujeszky's disease virus strains isolated in Poland using DNA biotinylated probes.

    PubMed

    Kochan, G; Lipowski, A; Fici?ska, J; Szewczyk, B

    1994-01-01

    The aim of this study was to compare 17 different Aujeszky's disease virus (ADV) isolates from clinical outbreaks of AD by using DNA biotinylated probes. All isolates were collected in Poland between 1984 and 1991. The restriction fragment pattern (RFP) analysis done by hybridization to NIA-3 DNA biotinylated probe indicated that all Polish ADV field strains can be classified as type I of Suid herpesvirus 1. Hybridization with BamHI fragment 7 and gI gene biotinylated probes revealed an unusual heterogeneity of BamHI fragment 7 in almost 50% of strains isolated in Poland. The nature of the molecular changes in this fragment will be discussed. PMID:7810432

  16. Evaluation of extraction and purification methods for obtaining PCR-amplifiable DNA from compost

    E-print Network

    Michel Jr., Frederick C.

    Evaluation of extraction and purification methods for obtaining PCR-amplifiable DNA from compost complicate the isolation of PCR- amplifiable DNA from compost and other organic-rich samples. In this study contamination, PCR amplifiability, and microbial community structure assessed by terminal restriction fragment

  17. Validation of a New Test for Schistosoma haematobium Based on Detection of Dra1 DNA Fragments in Urine: Evaluation through Latent Class Analysis

    PubMed Central

    Asaolu, Samuel; Moustaki, Irini; Shiff, Clive

    2012-01-01

    Background Diagnosis of urogenital schistosomiasis in chronically infected adults is challenging but important, especially because long term infection of the bladder and urinary tract can have dire consequences. We evaluated three tests for viable infection: detection of parasite specific DNA Dra1 fragments, haematuria and presence of parasite eggs for sensitivity (Se) and specificity (Sp). Methods Over 400 urine specimens collected from adult volunteers in an endemic area in Western Nigeria were assessed for haematuria then filtered in the field, the filter papers dried and later examined for eggs and DNA. The results were stratified according to sex and age and subjected to Latent Class analysis. Conclusions Presence of Dra1 in males (Se?=?100%; Sp?=?100%) exceeded haematuria (Se?=?87.6%: Sp?=?34.7%) and detection of eggs (Se?=?70.1%; Sp?=?100%). In females presence of Dra1 was Se?=?100%: Sp?=?100%, exceeding haematuria (Se?=?86.7%: Sp?=?77.0%) and eggs (Se?=?70.1%; Sp?=?100%). Dra1 became undetectable 2 weeks after praziquantel treatment. We conclude detection of Dra1 fragment is a definitive test for the presence of Schistosoma haematobium infection. PMID:22235360

  18. Restriction enzyme analysis of American region dengue viruses.

    PubMed

    Vorndam, V; Nogueira, R M; Trent, D W

    1994-01-01

    Restriction fragment heterogeneity of Hae III digestion products of cDNA to virion RNA was used to map the distribution of dengue virus topotypes found in the American region. By comparing the electrophoretic patterns of fragments produced, dengue virus isolates were placed in groups that agreed with those previously determined by oligonucleotide fingerprinting. Dengue-1 and dengue-4 viruses occur throughout the western hemisphere as single genetic types, with most of the isolates sharing at least 70% of their Hae III restriction enzyme fragments. Dengue-2 virus exists as two topotypes in the region with apparently non-overlapping distributions. The Puerto Rico topotype, which has been in the Caribbean for at least 40 years, is genetically diverse, while the Jamaica topotype, first isolated in 1981, is more homogeneous and has expanded its range from the original Caribbean focus to South America. PMID:7516146

  19. Diversity of Staphylococcus Species Strains Based on Partial kat (Catalase) Gene Sequences and Design of a PCR-Restriction Fragment Length Polymorphism Assay for Identification and Differentiation of Coagulase-Positive Species (S. aureus, S. delphini, S. hyicus, S. intermedius, S. pseudintermedius, and S. schleiferi subsp. coagulans)?

    PubMed Central

    Blaiotta, Giuseppe; Fusco, Vincenzina; Ercolini, Danilo; Pepe, Olimpia; Coppola, Salvatore

    2010-01-01

    A set of degenerate PCR primers was designed and used to amplify and sequence about 75% of the catalase (kat) gene from each of 49 staphylococcal strains. In some strains of Staphylococcus xylosus, S. saprophyticus, and S. equorum, two catalase genes, katA and katB, were found. A phylogenetic tree was generated and showed diversities among 66 partial (about 900-bp) staphylococcal kat nucleotide sequences (including 17 sequences found in GenBank) representing 26 different species. The topology of this tree showed a distribution of staphylococcal species similar, but not identical, to those reported previously based on 16S rRNA, hsp60, sodA, rpoB, tuf, and gap genes. The kat gene sequences were less conserved than those of 16S rRNA, rpoB, hsp60, and tuf genes and slightly more conserved than those of the gap gene. Therefore, kat gene sequence analysis may provide an additional marker for inferring phylogenetic relationships of staphylococci. Moreover, the discrete nucleotide polymorphism revealed in this gene could be exploited for rapid, low-cost identification of staphylococcal species through PCR-restriction fragment length polymorphism (RFLP) analysis. In this study, a PCR-RFLP assay performed by using only the TaqI restriction enzyme was successfully developed for rapid unequivocal identification/differentiation, at species and subspecies levels, of coagulase-positive staphylococci (CPS). The assay was validated by testing the DNA from 100 staphylococcal strains, including reference and wild CPS strains isolated from different environments. This reliable, rapid, and low-cost approach (requiring about 6 h from DNA isolation to the achievement of results and <5 Euros for each strain tested) allowed unambiguous identification of all the strains assayed, including the newly described S. delphini and S. pseudintermedius CPS species. PMID:19889901

  20. Diversity of Staphylococcus species strains based on partial kat (catalase) gene sequences and design of a PCR-restriction fragment length polymorphism assay for identification and differentiation of coagulase-positive species (S. aureus, S. delphini, S. hyicus, S. intermedius, S. pseudintermedius, and S. schleiferi subsp. coagulans).

    PubMed

    Blaiotta, Giuseppe; Fusco, Vincenzina; Ercolini, Danilo; Pepe, Olimpia; Coppola, Salvatore

    2010-01-01

    A set of degenerate PCR primers was designed and used to amplify and sequence about 75% of the catalase (kat) gene from each of 49 staphylococcal strains. In some strains of Staphylococcus xylosus, S. saprophyticus, and S. equorum, two catalase genes, katA and katB, were found. A phylogenetic tree was generated and showed diversities among 66 partial (about 900-bp) staphylococcal kat nucleotide sequences (including 17 sequences found in GenBank) representing 26 different species. The topology of this tree showed a distribution of staphylococcal species similar, but not identical, to those reported previously based on 16S rRNA, hsp60, sodA, rpoB, tuf, and gap genes. The kat gene sequences were less conserved than those of 16S rRNA, rpoB, hsp60, and tuf genes and slightly more conserved than those of the gap gene. Therefore, kat gene sequence analysis may provide an additional marker for inferring phylogenetic relationships of staphylococci. Moreover, the discrete nucleotide polymorphism revealed in this gene could be exploited for rapid, low-cost identification of staphylococcal species through PCR-restriction fragment length polymorphism (RFLP) analysis. In this study, a PCR-RFLP assay performed by using only the TaqI restriction enzyme was successfully developed for rapid unequivocal identification/differentiation, at species and subspecies levels, of coagulase-positive staphylococci (CPS). The assay was validated by testing the DNA from 100 staphylococcal strains, including reference and wild CPS strains isolated from different environments. This reliable, rapid, and low-cost approach (requiring about 6 h from DNA isolation to the achievement of results and <5 Euros for each strain tested) allowed unambiguous identification of all the strains assayed, including the newly described S. delphini and S. pseudintermedius CPS species. PMID:19889901

  1. Comparison of serologic typing, sodium dodecyl sulfate-polyacrylamide gel electrophoresis protein analysis, and genetic restriction fragment length polymorphism analysis for identification of rickettsiae: characterization of two new rickettsial strains.

    PubMed Central

    Beati, L; Finidori, J P; Gilot, B; Raoult, D

    1992-01-01

    In 1990, 17 adult Rhipicephalus turanicus ticks were collected in the south of France. Two spotted fever group rickettsiae, Mtu1 and Mtu5, were isolated from the hemolymphs of two of these ticks by the centrifugation shell-vial technique by using HEL cells. These isolates were compared with reference spotted fever group rickettsial serotypes by using three identification methods: microimmunofluorescence serologic typing, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and polymerase chain reaction followed by restriction endonuclease fragment length polymorphism analysis. The results obtained by all these techniques showed that Mtu1 and Mtu5 are each previously undescribed rickettsial serotypes. A comparison of the three methods used to identify the isolates led us to the conclusion that, in large-scale epidemiological studies, the simplest way to identify isolates in ticks is to first use the polymerase chain reaction-restriction fragment length polymorphism analysis directly on triturated ticks as a screening method to detect interesting rickettsiae, and then attempt to isolate rickettsiae from ticks for identification by microimmunofluorescence and SDS-PAGE, both of which are time-consuming and expensive to carry out. Images PMID:1354221

  2. A molecular and cytogenetic analysis of lambda 20p7 fragment DNA from the proximal beta-heterochromatin of Drosophila melanogaster.

    PubMed

    Baiborodin, S I; Baricheva, E M; Bogachev, S S; Borisevich, I V; Strotz, O V; Filippova, M A; Sharakhov, I V; Shilov, A G

    1993-12-01

    A DNA fragment from the Drosophila melanogaster genome, cloned in lambda 20p7, was derived independently from clones lambda 20 and lambda L [Baiborodin et al., Genetika 29 (1993) 403-416; Sharakhov et al., Genetika 29 (1993) 392-402]. In situ hybridization of lambda 20p7 DNA to the chromosomes of D. melanogaster demonstrated preferential hybridization of the fragment to the chromocenter of polytene chromosomes and to pericentric heterochromatin of chromosomes II, IV and X at the metaphase plate. Copy number per haploid genome for lambda 20p7 was estimated as approximately 200. Based on Southern blotting, the major portion of this moderate repeat was localized in the region of a 5.5-kb HindIII digest. In situ hybridization to polytene chromosomes from strain fs(2)B trophocytes revealed that repeats homologous to lambda 20p7 are located in the proximal heterochromatin which undergoes structural reorganization during tissue differentiation. The nucleotide sequence of two segments of the clone lambda 20p7, Dm0.9 and Dm270, was determined. Sequence analysis of the 300-bp Dm0.9 clone revealed that it contains 21-bp and 30-bp d(GT/CA) sequences, a 12-bp AT box, recognition sites for nuclear factors NFI and SpI, and a set of inverted repeats. Clone Dm270 contains an open reading frame (ORF). The deduced amino acid (aa) sequence shares homology with the gag-like gene from type-I (R1) ribosomal DNA insertion and may code for a polypeptide of 10 kDa. The Dm270 sequence was found to contain two direct repeats showing homology to the human CENP-B box. PMID:8262375

  3. Whole genome phylogeny for 21 Drosophila species using predicted 2b-RAD fragments

    PubMed Central

    Stuart, Gary W.

    2013-01-01

    Type IIB restriction endonucleases are site-specific endonucleases that cut both strands of double-stranded DNA upstream and downstream of their recognition sequences. These restriction enzymes have recognition sequences that are generally interrupted and range from 5 to 7 bases long. They produce DNA fragments which are uniformly small, ranging from 21 to 33 base pairs in length (without cohesive ends). The fragments are generated from throughout the entire length of a genomic DNA providing an excellent fractional representation of the genome. In this study we simulated restriction enzyme digestions on 21 sequenced genomes of various Drosophila species using the predicted targets of 16 Type IIB restriction enzymes to effectively produce a large and arbitrary selection of loci from these genomes. The fragments were then used to compare organisms and to calculate the distance between genomes in pair-wise combination by counting the number of shared fragments between the two genomes. Phylogenetic trees were then generated for each enzyme using this distance measure and the consensus was calculated. The consensus tree obtained agrees well with the currently accepted tree for the Drosophila species. We conclude that multi-locus sub-genomic representation combined with next generation sequencing, especially for individuals and species without previous genome characterization, can accelerate studies of comparative genomics and the building of accurate phylogenetic trees. PMID:24432193

  4. 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 R.; Brown, Wesley M.

    1990-01-01

    Cleavage Site and Length Polymorphisms in Mitochondrial DNA of Apis mellifera mellifera and A. m. carnica (Hymenoptera: Apidae) DEBORAH ROAN SMITH12 AND WESLEY M. BROWN2 Museum of Zoology, University of Michigan, Ann Arbor, Michigan 48109 Ann... are compared with previously published maps of mitochondrial DNA from North American bees of European ancestry and Brazilian Africanized bees. A. m. mellifera mitochondrial DNA is characterized by a pattern of cleavage sites unique among the honey bee...

  5. Erratum: A Chloroplast DNA Phylogeny of the Caryophyllales Based on Structural and Inverted Repeat Restriction Site Variation

    E-print Network

    Downie, Stephen R.

    Restriction Site Variation Stephen R. Downie; Jeffrey D. Palmer Systematic Botany, Vol. 19, No. 3. (Jul. - Sep%3AEACDPO%3E2.0.CO%3B2-M Systematic Botany is currently published by American Society of Plant Taxonomists

  6. Base-pair opening and spermine binding--B-DNA features displayed in the crystal structure of a gal operon fragment: implications for protein-DNA recognition.

    PubMed Central

    Tari, L W; Secco, A S

    1995-01-01

    A sequence that is represented frequently in functionally important sites involving protein-DNA interactions is GTG/CAC, suggesting that the trimer may play a role in regulatory processes. The 2.5 A resolution structure of d(CGGTGG)/d(CCACCG), a part of the interior operator (OI, nucleotides +44 to +49) of the gal operon, co-crystallized with spermine, is described herein. The crystal packing arrangement in this structure is unprecedented in a crystal of B-DNA, revealing a close packing of columns of stacked DNA resembling a 5-stranded twisted wire cable. The final structure contains one hexamer duplex, 17 water molecules and 1.5 spermine molecules per crystallographic asymmetric unit. The hexamer exhibits base-pair opening and shearing at T.A resulting in a novel non-Watson-Crick hydrogen-bonding scheme between adenine and thymine in the GTG region. The ability of this sequence to adopt unusual conformations in its GTG region may be a critical factor conferring sequence selectivity on the binding of Gal repressor. In addition, this is the first conclusive example of a crystal structure of spermine with native B-DNA, providing insight into the mechanics of polyamine-DNA binding, as well as possible explanations for the biological action of spermine. Images PMID:7596838

  7. Characterization of mucosa-associated bacterial communities of the mouse intestine by terminal restriction fragment length polymorphism: Utility of sampling strategies

    E-print Network

    Selinger, Brent

    Characterization of mucosa-associated bacterial communities of the mouse intestine by terminal 2009 Keywords: Intestine Mucosa-associated bacteria Single-stranded artifacts T-RFLP Statistical for recovering bacterial community DNA associated with intestinal mucosa of mice (i.e. mechanical agitation

  8. Aging and Calorie Restriction Oppositely Affect Mitochondrial Biogenesis through TFAM Binding at Both Origins of Mitochondrial DNA Replication in Rat Liver

    PubMed Central

    Picca, Anna; Pesce, Vito; Fracasso, Flavio; Joseph, Anna-Maria; Leeuwenburgh, Christiaan; Lezza, Angela M. S.

    2013-01-01

    Aging affects mitochondria in a tissue-specific manner. Calorie restriction (CR) is, so far, the only intervention able to delay or prevent the onset of several age-related changes also in mitochondria. Using livers from middle age (18-month-old), 28-month-old and 32-month-old ad libitum-fed and 28-month-old calorie-restricted rats we found an age-related decrease in mitochondrial DNA (mtDNA) content and mitochondrial transcription factor A (TFAM) amount, fully prevented by CR. We revealed also an age-related decrease, completely prevented by CR, for the proteins PGC-1? NRF-1 and cytochrome c oxidase subunit IV, supporting the efficiency of CR to forestall the age-related decrease in mitochondrial biogenesis. Furthermore, CR counteracted the age-related increase in oxidative damage to proteins, represented by the increased amount of oxidized peroxiredoxins (PRX-SO3) in the ad libitum-fed animals. An unexpected age-related decrease in the mitochondrial proteins peroxiredoxin III (Prx III) and superoxide dismutase 2 (SOD2), usually induced by increased ROS and involved in mitochondrial biogenesis, suggested a prevailing relevance of the age-reduced mitochondrial biogenesis above the induction by ROS in the regulation of expression of these genes with aging. The partial prevention of the decrease in Prx III and SOD2 proteins by CR also supported the preservation of mitochondrial biogenesis in the anti-aging action of CR. To investigate further the age- and CR-related effects on mitochondrial biogenesis we analyzed the in vivo binding of TFAM to specific mtDNA regions and demonstrated a marked increase in the TFAM-bound amounts of mtDNA at both origins of replication with aging, fully prevented by CR. A novel, positive correlation between the paired amounts of TFAM-bound mtDNA at these sub-regions was found in the joined middle age ad libitum-fed and 28-month-old calorie-restricted groups, but not in the 28-month-old ad libitum-fed counterpart suggesting a quite different modulation of TFAM binding at both origins of replication in aging and CR. PMID:24058615

  9. A homicide in the Ukraine: DNA-based identification of a boiled, skeletonized, and varnished human skull, and of bone fragments found in a fireplace.

    PubMed

    Sivolap, Y; Krivda, G; Kozhuhova, N; Chebotar, S; Benecke, M

    2001-12-01

    In an apartment, bone fragments were found in a fireplace. Furthermore, a varnished skull was found elsewhere in the same apartment. The tenant confessed to a murder and stated that the head of a victim, a girl, was boiled for 12 hours. He stated that the soft tissue was then removed and the skull was varnished. Other parts of the body were burned to ashes in an open field. Comparison of loci D19S252, CD4, CYAR04, TII01, F13A01, F13B, and D6S366 from the skull and the bone remains to loci of the mother of a missing girl showed that the skull came from that missing child. Biological maternity was calculated as 99.99%. The bone pieces were DNA typed as male and did not share alleles with the mother in several systems. Therefore, they belonged to a different (human) victim. PMID:11764912

  10. 42 CFR 73.13 - Restricted experiments.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ...) Restricted experiments: (1) Experiments utilizing recombinant DNA that involve the deliberate transfer of a... agriculture. (2) Experiments involving the deliberate formation of recombinant DNA containing genes for...

  11. 42 CFR 73.13 - Restricted experiments.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ...) Restricted experiments: (1) Experiments utilizing recombinant DNA that involve the deliberate transfer of a... agriculture. (2) Experiments involving the deliberate formation of recombinant DNA containing genes for...

  12. 42 CFR 73.13 - Restricted experiments.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ...) Restricted experiments: (1) Experiments utilizing recombinant DNA that involve the deliberate transfer of a... agriculture. (2) Experiments involving the deliberate formation of recombinant DNA containing genes for...

  13. Auxin-induced rapid degradation of inhibitor of caspase-activated DNase (ICAD) induces apoptotic DNA fragmentation, caspase activation, and cell death: a cell suicide module.

    PubMed

    Samejima, Kumiko; Ogawa, Hiromi; Ageichik, Alexander V; Peterson, Kevin L; Kaufmann, Scott H; Kanemaki, Masato T; Earnshaw, William C

    2014-11-01

    Caspase-activated DNase (CAD) is a major apoptotic nuclease, responsible for DNA fragmentation and chromatin condensation during apoptosis. CAD is normally activated in apoptosis as a result of caspase cleavage of its inhibitory chaperone ICAD. Other aspects of CAD regulation are poorly understood. In particular, it has been unclear whether direct CAD activation in non-apoptotic living cells can trigger cell death. Taking advantage of the auxin-inducible degron (AID) system, we have developed a suicide system with which ICAD is rapidly degraded in living cells in response to the plant hormone auxin. Our studies demonstrate that rapid ICAD depletion is sufficient to activate CAD and induce cell death in DT40 and yeast cells. In the vertebrate cells, ectopic CAD activation triggered caspase activation and subsequent hallmarks of caspase-dependent apoptotic changes, including phosphatidylserine exposure and nuclear fragmentation. These observations not only suggest that CAD activation drives apoptosis through a positive feedback loop, but also identify a unique suicide system that can be used for controlling gene-modified organisms. PMID:25248749

  14. MethylRAD: a simple and scalable method for genome-wide DNA methylation profiling using methylation-dependent restriction enzymes

    PubMed Central

    Wang, Shi; Lv, Jia; Zhang, Lingling; Dou, Jinzhuang; Sun, Yan; Li, Xue; Fu, Xiaoteng; Dou, Huaiqian; Mao, Junxia; Hu, Xiaoli; Bao, Zhenmin

    2015-01-01

    Characterization of dynamic DNA methylomes in diverse phylogenetic groups has attracted growing interest for a better understanding of the evolution of DNA methylation as well as its function and biological significance in eukaryotes. Sequencing-based methods are promising in fulfilling this task. However, none of the currently available methods offers the ‘perfect solution’, and they have limitations that prevent their application in the less studied phylogenetic groups. The recently discovered Mrr-like enzymes are appealing for new method development, owing to their ability to collect 32-bp methylated DNA fragments from the whole genome for high-throughput sequencing. Here, we have developed a simple and scalable DNA methylation profiling method (called MethylRAD) using Mrr-like enzymes. MethylRAD allows for de novo (reference-free) methylation analysis, extremely low DNA input (e.g. 1 ng) and adjustment of tag density, all of which are still unattainable for most widely used methylation profiling methods such as RRBS and MeDIP. We performed extensive analyses to validate the power and accuracy of our method in both model (plant Arabidopsis thaliana) and non-model (scallop Patinopecten yessoensis) species. We further demonstrated its great utility in identification of a gene (LPCAT1) that is potentially crucial for carotenoid accumulation in scallop adductor muscle. MethylRAD has several advantages over existing tools and fills a void in the current epigenomic toolkit by providing a universal tool that can be used for diverse research applications, e.g. from model to non-model species, from ordinary to precious samples and from small to large genomes, but at an affordable cost. PMID:26581575

  15. MethylRAD: a simple and scalable method for genome-wide DNA methylation profiling using methylation-dependent restriction enzymes.

    PubMed

    Wang, Shi; Lv, Jia; Zhang, Lingling; Dou, Jinzhuang; Sun, Yan; Li, Xue; Fu, Xiaoteng; Dou, Huaiqian; Mao, Junxia; Hu, Xiaoli; Bao, Zhenmin

    2015-11-01

    Characterization of dynamic DNA methylomes in diverse phylogenetic groups has attracted growing interest for a better understanding of the evolution of DNA methylation as well as its function and biological significance in eukaryotes. Sequencing-based methods are promising in fulfilling this task. However, none of the currently available methods offers the 'perfect solution', and they have limitations that prevent their application in the less studied phylogenetic groups. The recently discovered Mrr-like enzymes are appealing for new method development, owing to their ability to collect 32-bp methylated DNA fragments from the whole genome for high-throughput sequencing. Here, we have developed a simple and scalable DNA methylation profiling method (called MethylRAD) using Mrr-like enzymes. MethylRAD allows for de novo (reference-free) methylation analysis, extremely low DNA input (e.g. 1 ng) and adjustment of tag density, all of which are still unattainable for most widely used methylation profiling methods such as RRBS and MeDIP. We performed extensive analyses to validate the power and accuracy of our method in both model (plant Arabidopsis thaliana) and non-model (scallop Patinopecten yessoensis) species. We further demonstrated its great utility in identification of a gene (LPCAT1) that is potentially crucial for carotenoid accumulation in scallop adductor muscle. MethylRAD has several advantages over existing tools and fills a void in the current epigenomic toolkit by providing a universal tool that can be used for diverse research applications, e.g. from model to non-model species, from ordinary to precious samples and from small to large genomes, but at an affordable cost. PMID:26581575

  16. A Polymorphism in Mitochondrial DNA Associated with IQ?

    ERIC Educational Resources Information Center

    Skuder, Patricia; And Others

    1995-01-01

    Of 100 DNA markers examined in an allelic association study, only 1 showed a replicated association with IQ in samples totaling 107 children. How the gene marked by the particular restriction fragment length polymorphism was tracked and its mitochondrial origin identified is described. (SLD)

  17. A 39 amino acid fragment of the cell cycle regulator p21 is sufficient to bind PCNA and partially inhibit DNA replication in vivo.

    PubMed Central

    Chen, J; Peters, R; Saha, P; Lee, P; Theodoras, A; Pagano, M; Wagner, G; Dutta, A

    1996-01-01

    The cell cycle regulator p21 interacts with and inhibits the DNA replication and repair factor proliferating cell nuclear antigen (PCNA). We have defined a 39 amino acid fragment of p21 which is sufficient to bind PCNA with high affinity (Kd 10-20 nM). This peptide can inhibit DNA replication in vitro and microinjection of a GST fusion protein containing this domain inhibited S phase in vivo. Despite its high affinity for PCNA, the free 39 amino acid peptide does not have a well-defined structure, as judged from circular dichroism and nuclear magnetic resonance measurements, suggesting an induced fit mechanism for the PCNA-p21 interaction. The association of the small peptide with PCNA was thermolabile, suggesting that portions of p21 adjoining the minimal region of contact stabilize the interaction. In addition, a domain containing 67 amino acids from the N-terminus of PCNA was defined as both necessary and sufficient for binding to p21. PMID:8649992

  18. A 39 amino acid fragment of the cell cycle regulator p21 is sufficient to bind PCNA and partially inhibit DNA replication in vivo.

    PubMed

    Chen, J; Peters, R; Saha, P; Lee, P; Theodoras, A; Pagano, M; Wagner, G; Dutta, A

    1996-05-01

    The cell cycle regulator p21 interacts with and inhibits the DNA replication and repair factor proliferating cell nuclear antigen (PCNA). We have defined a 39 amino acid fragment of p21 which is sufficient to bind PCNA with high affinity (Kd 10-20 nM). This peptide can inhibit DNA replication in vitro and microinjection of a GST fusion protein containing this domain inhibited S phase in vivo. Despite its high affinity for PCNA, the free 39 amino acid peptide does not have a well-defined structure, as judged from circular dichroism and nuclear magnetic resonance measurements, suggesting an induced fit mechanism for the PCNA-p21 interaction. The association of the small peptide with PCNA was thermolabile, suggesting that portions of p21 adjoining the minimal region of contact stabilize the interaction. In addition, a domain containing 67 amino acids from the N-terminus of PCNA was defined as both necessary and sufficient for binding to p21. PMID:8649992

  19. Can DNA fragmentation of neat or swim-up spermatozoa be used to predict pregnancy following ICSI of fertile oocyte donors?

    PubMed Central

    Gosálvez, Jaime; Caballero, Pedro; López-Fernández, Carmen; Ortega, Leonor; Guijarro, José Andrés; Fernández, José Luís; Johnston, Stephen D; Nuñez-Calonge, Rocío

    2013-01-01

    This study compared the potential of assessing sperm DNA fragmentation (SDF) from neat semen and the subsequent swim-up (SU) procedure to predict pregnancy when conducting ICSI of fertile donor oocytes. Infertile females (n=81) were transferred embryos resulting from intracytoplasmic sperm injection (ICSI) of their partner's spermatozoa and proven donor oocytes. This model normalized the impact of female factor in putative sperm DNA repair. Semen was blindly assessed for SDF using Halosperm immediately following ejaculation (NS) and after swim-up at the time of ICSI fertilisation. There was a decrease in SDF values of the ejaculated semen sample following the swim-up protocol (P=0.000). Interestingly, pregnancy could be equally predicted from SDF values derived from either neat or swim-up semen samples. Receiver operator curves and the derived Youden's indices determined SDF cutoff values for NS and SU of 24.8% and 17.5%, respectively. Prediction of pregnancy from NS SDF had a sensitivity of 75% and a specificity of 69%, whereas for SU SDF was 78% and 73%, respectively. While increased levels of SDF negatively impact reproductive outcome, we have shown that a reduction in SDF following sperm selection using ICSI with proven donor oocytes is not mandatory for achieving pregnancy. This suggests that a certain level of DNA damage that is not detectable using current technologies could be impacting on the relative success of assisted reproductive technology (ART) procedures. Consequently, we propose a modification of the so called ‘iceberg model' as a possible rationale for understanding the role of SDF in reproductive outcome. PMID:24121979

  20. 2000 Year-old ancient equids: an ancient-DNA lesson from pompeii remains.

    PubMed

    Di Bernardo, Giovanni; Del Gaudio, Stefania; Galderisi, Umberto; Cipollaro, Marilena

    2004-11-15

    Ancient DNA extracted from 2000 year-old equine bones was examined in order to amplify mitochondrial and nuclear DNA fragments. A specific equine satellite-type sequence representing 3.7%-11% of the entire equine genome, proved to be a suitable target to address the question of the presence of aDNA in ancient bones. The PCR strategy designed to investigate this specific target also allowed us to calculate the molecular weight of amplifiable DNA fragments. Sequencing of a 370 bp DNA fragment of mitochondrial control region allowed the comparison of ancient DNA sequences with those of modern horses to assess their genetic relationship. The 16S rRNA mitochondrial gene was also examined to unravel the post-mortem base modification feature and to test the status of Pompeian equids taxon on the basis of a Mae III restriction site polymorphism. PMID:15468050

  1. Pea amyloplast DNA is qualitatively similar to pea chloroplast DNA

    NASA Technical Reports Server (NTRS)

    Gaynor, J. J.

    1984-01-01

    Amyloplast DNA (apDNA), when subjected to digestion with restriction endonucleases, yields patterns nearly identical to that of DNA from mature pea chloroplasts (ctDNA). Southern transfers of apDNA and ctDNA, probed with the large subunit (LS) gene of ribulose-1,5-bisphosphate carboxylase (Rubisco), shows hybridization to the expected restriction fragments for both apDNA and ctDNA. However, Northern transfers of total RNA from chloroplasts and amyloplasts, probed again with the LS gene of Rubisco, shows that no detectable LS meggage is found in amyloplasts although LS expression in mature chloroplasts is high. Likewise, two dimensional polyacrylamide gel electrophoresis of etiolated gravisensitive pea tissue shows that both large and small subunits of Rubisco are conspicuously absent; however, in greening tissue these two constitute the major soluble proteins. These findings suggest that although the informational content of these two organelle types is equivalent, gene expression is quite different and is presumably under nuclear control.

  2. Use of Plasmon Coupling to Reveal the Dynamics of DNA Bending andCleavage by Single EcoRV Restriction Enzymes

    SciTech Connect

    Reinhard, Bjorn; Sheikholeslami, Sassan; Mastroianni, Alexander; Alivisatos, A. Paul; Liphardt, Jan

    2006-09-06

    Pairs of Au nanoparticles have recently been proposed asplasmon rulers based on the dependence of their light scattering on theinterparticle distance. Preliminary work has suggested that plasmonrulers can be used to measure and monitor dynamic distance changes overthe 1 to 100nm length scale in biology. Here, we substantiate thatplasmon rulers can be used to effectively measure dynamical biophysicalprocesses by applying the ruler to a system that has been investigatedextensively using ensemble kinetic measurements: the cleavage of DNA bythe restriction enzyme EcoRV. Temporal resolutions of up to 240 Hz wereobtained, and the end-to-end extension of up to 1000 individual dsDNAenzyme substrates could be monitored in parallel for hours. The singlemolecule cleavage trajectories acquired here agree well with valuesobtained in bulk through other methods, and confirm well-known featuresof the cleavage process, such as the fact that the DNA is bent prior tocleavage. New dynamical information is revealed as well, for instance,the degree of softening of the DNA just prior to cleavage. The unlimitedlife time, high temporal resolution, and high signal/noise make theplasmon ruler an excellent tool for studying macromolecular assembliesand conformational changes at the single molecule level.

  3. New polymorphic mtDNA restriction site in the 12S rRNA gene detected in Tunisian patients with non-syndromic hearing loss

    SciTech Connect

    Mkaouar-Rebai, Emna Tlili, Abdelaziz; Masmoudi, Saber; Charfeddine, Ilhem; Fakhfakh, Faiza

    2008-05-09

    The 12S rRNA gene was shown to be a hot spot for aminoglycoside-induced and non-syndromic hearing loss since several deafness-associated mtDNA mutations were identified in this gene. Among them, we distinguished the A1555G, the C1494T and the T1095C mutations and C-insertion or deletion at position 961. One hundred Tunisian patients with non-syndromic hearing loss and 100 hearing individuals were analysed in this study. A PCR-RFLP analysis with HaeIII restriction enzyme showed the presence of the A1555G mutation in the 12S rRNA gene in only one out of the 100 patients. In addition, PCR-RFLP and radioactive PCR revealed the presence of a new HaeIII polymorphic restriction site in the same gene of 12S rRNA site in 4 patients with non-syndromic hearing loss. UVIDOC-008-XD analyses showed the presence of this new polymorphic restriction site with a variable heteroplasmic rates at position +1517 of the human mitochondrial genome. On the other hand, direct sequencing of the entire mitochondrial 12S rRNA gene in the 100 patients and in 100 hearing individuals revealed the presence of the A750G and A1438G polymorphisms and the absence of the C1494T, T1095C and 961insC mutations in all the tested individuals. Sequencing of the whole mitochondrial genome in the 4 patients showing the new HaeIII polymorphic restriction site revealed only the presence of the A8860G transition in the MT-ATP6 gene and the A4769G polymorphism in the ND2 gene.

  4. Differentiation of mycoplasmalike organisms (MLOs) in European fruit trees by PCR using specific primers derived from the sequence of a chromosomal fragment of the apple proliferation MLO.

    PubMed Central

    Jarausch, W; Saillard, C; Dosba, F; Bové, J M

    1994-01-01

    A 1.8-kb chromosomal DNA fragment of the mycoplasmalike organism (MLO) associated with apple proliferation was sequenced. Three putative open reading frames were observed on this fragment. The protein encoded by open reading frame 2 shows significant homologies with bacterial nitroreductases. From the nucleotide sequence four primer pairs for PCR were chosen to specifically amplify DNA from MLOs associated with European diseases of fruit trees. Primer pairs specific for (i) Malus-affecting MLOs, (ii) Malus- and Prunus-affecting MLOs, and (iii) Malus-, Prunus-, and Pyrus-affecting MLOs were obtained. Restriction enzyme analysis of the amplification products revealed restriction fragment length polymorphisms between Malus-, Prunus, and Pyrus-affecting MLOs as well as between different isolates of the apple proliferation MLO. No amplification with either primer pair could be obtained with DNA from 12 different MLOs experimentally maintained in periwinkle. Images PMID:7916180

  5. Bac clones generated from sheared dna

    SciTech Connect

    Osoegawa, Kazutoyo; Vessere, Gery M.; Shu, Chung Li; Hoskins,Roger A.; Abad, Jose P.; de Pablos, Beatriz; Villasante, Alfredo; deJong, Pieter J.

    2006-08-09

    BAC libraries generated from restriction-digested genomic DNA display representational bias and lack some sequences. To facilitate completion of genome projects, procedures have been developed to create BACs from DNA physically sheared to create fragments extending up to 200kb. The DNA fragments were repaired to create blunt ends and ligated to a new BAC vector. This approach has been tested by generating BAC libraries from Drosophila DNA, with insert lengths of 50 kb to 150 kb. The libraries lack chimeric clone problems as determined by mapping paired BAC-end sequences of one library to the D. melanogaster genome sequence. The utility of ''sheared'' libraries was demonstrated by closure of a previous clone gap and by isolation of clones from telomeric regions, which were notably absent from previous Drosophila BAC libraries.

  6. MITOCHONDRIAL DNA AND ITS1 DIFFERENTIATION IN GEOGRAPHICAL POPULATIONS OF NORTHERN CORN ROOTWORM, DIABROTICA BARBERI (COLEOPTERA: CHRYSOMELIDAE): IDENTIFICATION OF DISTINCT GENETIC POPULATIONS [SEE ABSTRACT FOR ACCESSION NOS.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Genetic variation of mitochondrial DNA (mtDNA) and the nuclear ribosomal spacer, ITS1, in local and dispersed geographical populations of northern corn rootworm, Diabrotica barberi Smith and Lawrence was examined. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was use...

  7. MITOCHONDRIAL DNA AND ITS1 DIFFERENTIATION IN GEOGRAPHICAL POPULATIONS OF NORTHERN CORN ROOTWORM, DIABROTICA BARBERI (COLEOPTERA: CHRYSOMELIDAE): IDENTIFICATION OF DISTINCT GENETIC POPULATIONS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Genetic variation of mitochondrial DNA (mtDNA) and the nuclear ribosomal spacer, ITS1, in local and dispersed geographical populations of northern corn rootworm, Diabrotica barberi Smith and Lawrence was examined. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was use...

  8. Internalization of Staphylococcus aureus in Lymphocytes Induces Oxidative Stress and DNA Fragmentation: Possible Ameliorative Role of Nanoconjugated Vancomycin

    PubMed Central

    Chakraborty, Subhankari Prasad; Kar Mahapatra, Santanu; Sahu, Sumanta Kumar; Das, Sabyasachi; Tripathy, Satyajit; Dash, Sandeep; Pramanik, Panchanan; Roy, Somenath

    2011-01-01

    Staphylococcus aureus is the most frequently isolated pathogen causing bloodstream infections, skin and soft tissue infections and pneumonia. Lymphocyte is an important immune cell. The aim of the present paper was to test the ameliorative role of nanoconjugated vancomycin against Vancomycin-sensitive Staphylococcus aureus (VSSA) and vancomycin-resistant Staphylococcus aureus (VRSA) infection-induced oxidative stress in lymphocytes. VSSA and VRSA infections were developed in Swiss mice by intraperitoneal injection of 5 × 106?CFU/mL bacterial solutions. Nanoconjugated vancomycin was adminstrated to VSSA- and VRSA-infected mice at its effective dose for 10 days. Vancomycin was adminstrated to VSSA- and VRSA-infected mice at a similar dose, respectively, for 10 days. Vancomycin and nanoconjugated vancomycin were adminstrated to normal mice at their effective doses for 10 days. The result of this study reveals that in vivo VSSA and VRSA infection significantly increases the level of lipid peroxidation, protein oxidation, oxidized glutathione level, nitrite generation, nitrite release, and DNA damage and decreases the level of reduced glutathione, antioxidant enzyme status, and glutathione-dependent enzymes as compared to control group, which were increased or decreased significantly near to normal in nanoconjugated vancomycin-treated group. These findings suggest the potential use and beneficial role of nanoconjugated vancomycin against VSSA and VRSA infection-induced oxidative stress in lymphocytes. PMID:21941607

  9. Novel DNA methylation profiles associated with key gene regulation and transcription pathways in blood and placenta of growth-restricted neonates.

    PubMed

    Hillman, Sara L; Finer, Sarah; Smart, Melissa C; Mathews, Chris; Lowe, Robert; Rakyan, Vardhman K; Hitman, Graham A; Williams, David J

    2015-01-01

    Fetal growth is determined by the feto-placental genome interacting with the maternal in utero environment. Failure of this interplay leads to poor placental development and fetal growth restriction (FGR), which is associated with future metabolic disease. We investigated whether whole genome methylation differences existed in umbilical cord blood and placenta, between gestational-matched, FGR, and appropriately grown (AGA) neonates. Using the Infinium HumanMethylation450 BeadChip®, we found that DNA from umbilical cord blood of FGR born at term (n = 19) had 839 differentially methylated positions (DMPs) that reached genome-wide significance compared with AGA (n = 18). Using gestational age as a continuous variable, we identified 76,249 DMPs in cord blood (adj. P < 0.05) of which 121 DMPs were common to the 839 DMPs and were still evident when comparing 12 FGR with 12 AGA [39.9 ± 1.2 vs. 40.0 ± 1.0 weeks (mean ± SD), respectively]. A total of 53 DMPs had a ? methylation difference >10% and 25 genes were co-methylated more than twice within 1000 base pairs. Gene Ontology (GO) analysis of DMPs supported their involvement in gene regulation and transcription pathways related to organ development and metabolic function. A similar profile of DMPs was found across different cell types in the cord blood. At term, no DMPs between FGR and AGA placentae reached genome-wide significance, validated with an external dataset. GO analysis of 284 pre-term, placental DMPs associated with autophagy, oxidative stress and hormonal responses. Growth restricted neonates have distinct DNA methylation profiles in pre-term placenta and in cord blood at birth, which may predispose to future adult disease. PMID:25496377

  10. Down-regulation of a SILENT INFORMATION REGULATOR2-related histone deacetylase gene, OsSRT1, induces DNA fragmentation and cell death in rice.

    PubMed

    Huang, Limin; Sun, Qianwen; Qin, Fujun; Li, Chen; Zhao, Yu; Zhou, Dao-Xiu

    2007-07-01

    The SILENT INFORMATION REGULATOR2 (SIR2) family proteins are NAD(+)-dependent histone deacetylases. Sir2 is involved in chromatin silencing at the mating-type loci, rDNA, and telomeres in yeast and is associated with lifespan extension in yeast, worms, and flies, but also in a broader range of additional functions. In this work, we investigated the role of OsSRT1, one of the two SIR2-related genes found in rice (Oryza sativa). We show that OsSRT1 is a widely expressed nuclear protein with higher levels in rapidly dividing tissues. OsSRT1 RNA interference induced an increase of histone H3K9 (lysine-9 of H3) acetylation and a decrease of H3K9 dimethylation, leading to H(2)O(2) production, DNA fragmentation, cell death, and lesions mimicking plant hypersensitive responses during incompatible interactions with pathogens, whereas overexpression of OsSRT1 enhanced tolerance to oxidative stress. Transcript microarray analysis revealed that the transcription of many transposons and retrotransposons in addition to genes related to hypersensitive response and/or programmed cell death was activated. Chromatin immunoprecipitation assays showed that OsSRT1 down-regulation induced histone H3K9 acetylation on the transposable elements and some of the hypersensitive response-related genes, suggesting that these genes may be among the primary targets of deacetylation regulated by OsSRT1. Our data together suggest that the rice SIR2-like gene is required for safeguard against genome instability and cell damage to ensure plant cell growth, but likely implicates different molecular mechanisms than yeast and animal homologs. PMID:17468215

  11. Random-breakage mapping method applied to human DNA sequences

    NASA Technical Reports Server (NTRS)

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

    1996-01-01

    The random-breakage mapping method [Game et al. (1990) Nucleic Acids Res., 18, 4453-4461] was applied to DNA sequences in human fibroblasts. The methodology involves NotI restriction endonuclease digestion of DNA from irradiated calls, followed by pulsed-field gel electrophoresis, Southern blotting and hybridization with DNA probes recognizing the single copy sequences of interest. The Southern blots show a band for the unbroken restriction fragments and a smear below this band due to radiation induced random breaks. This smear pattern contains two discontinuities in intensity at positions that correspond to the distance of the hybridization site to each end of the restriction fragment. By analyzing the positions of those discontinuities we confirmed the previously mapped position of the probe DXS1327 within a NotI fragment on the X chromosome, thus demonstrating the validity of the technique. We were also able to position the probes D21S1 and D21S15 with respect to the ends of their corresponding NotI fragments on chromosome 21. A third chromosome 21 probe, D21S11, has previously been reported to be close to D21S1, although an uncertainty about a second possible location existed. Since both probes D21S1 and D21S11 hybridized to a single NotI fragment and yielded a similar smear pattern, this uncertainty is removed by the random-breakage mapping method.

  12. Identification of a nuclear-localized nuclease from wheat cells undergoing programmed cell death that is able to trigger DNA fragmentation and apoptotic morphology on nuclei from human cells

    PubMed Central

    Domínguez, Fernando; Cejudo, Francisco J.

    2006-01-01

    PCD (programmed cell death) in plants presents important morphological and biochemical differences compared with apoptosis in animal cells. This raises the question of whether PCD arose independently or from a common ancestor in plants and animals. In the present study we describe a cell-free system, using wheat grain nucellar cells undergoing PCD, to analyse nucleus dismantling, the final stage of PCD. We have identified a Ca2+/Mg2+ nuclease and a serine protease localized to the nucleus of dying nucellar cells. Nuclear extracts from nucellar cells undergoing PCD triggered DNA fragmentation and other apoptotic morphology in nuclei from different plant tissues. Inhibition of the serine protease did not affect DNA laddering. Furthermore, we show that the nuclear extracts from plant cells triggered DNA fragmentation and apoptotic morphology in nuclei from human cells. The inhibition of the nucleolytic activity with Zn2+ or EDTA blocked the morphological changes of the nucleus. Moreover, nuclear extracts from apoptotic human cells triggered DNA fragmentation and apoptotic morphology in nuclei from plant cells. These results show that degradation of the nucleus is morphologically and biochemically similar in plant and animal cells. The implication of this finding on the origin of PCD in plants and animals is discussed. PMID:16613587

  13. Method of preparing and applying single stranded DNA probes to double stranded target DNAs in situ

    DOEpatents

    Gray, Joe W. (Livermore, CA); Pinkel, Daniel (Walnut Creek, CA)

    1991-01-01

    A method is provided for producing single stranded non-self-complementary nucleic acid probes, and for treating target DNA for use therewith. Probe is constructed by treating DNA with a restriction enzyme and an exonuclease to form template/primers for a DNA polymerase. The digested strand is resynthesized in the presence of labeled nucleoside triphosphate precursor. Labeled single stranded fragments are separated from the resynthesized fragments to form the probe. Target DNA is treated with the same restriction enzyme used to construct the probe, and is treated with an exonuclease before application of the probe. The method significantly increases the efficiency and specificity of hybridization mixtures by increasing effective probe concentration by eliminating self-hybridization between both probe and target DNAs, and by reducing the amount of target DNA available for mismatched hybridizations.

  14. Method of preparing and applying single stranded DNA probes to double stranded target DNAs in situ

    DOEpatents

    Gray, J.W.; Pinkel, D.

    1991-07-02

    A method is provided for producing single stranded non-self-complementary nucleic acid probes, and for treating target DNA for use therewith. The probe is constructed by treating DNA with a restriction enzyme and an exonuclease to form template/primers for a DNA polymerase. The digested strand is resynthesized in the presence of labeled nucleoside triphosphate precursor. Labeled single stranded fragments are separated from the resynthesized fragments to form the probe. Target DNA is treated with the same restriction enzyme used to construct the probe, and is treated with an exonuclease before application of the probe. The method significantly increases the efficiency and specificity of hybridization mixtures by increasing effective probe concentration by eliminating self-hybridization between both probe and target DNAs, and by reducing the amount of target DNA available for mismatched hybridizations. No Drawings

  15. Dna Sequencing

    DOEpatents

    Tabor, Stanley (Cambridge, MA); Richardson, Charles C. (Chestnut Hill, MA)

    1995-04-25

    A method for sequencing a strand of DNA, including the steps off: providing the strand of DNA; annealing the strand with a primer able to hybridize to the strand to give an annealed mixture; incubating the mixture with four deoxyribonucleoside triphosphates, a DNA polymerase, and at least three deoxyribonucleoside triphosphates in different amounts, under conditions in favoring primer extension to form nucleic acid fragments complementory to the DNA to be sequenced; labelling the nucleic and fragments; separating them and determining the position of the deoxyribonucleoside triphosphates by differences in the intensity of the labels, thereby to determine the DNA sequence.

  16. Enterocytozoon bieneusi Identification Using Real-Time Polymerase Chain Reaction and Restriction Fragment Length Polymorphism in HIV-Infected Humans from Kinshasa Province of the Democratic Republic of Congo.

    PubMed

    Wumba, Roger; Jean, Menotti; Benjamin, Longo-Mbenza; Madone, Mandina; Fabien, Kintoki; Josué, Zanga; Jean, Sala; Eric, Kendjo; A C, Guillo-Olczyk; Marc, Thellier

    2012-01-01

    Objective. To determine the prevalence and the genotypes of Enterocytozoon bieneusi in stool specimens from HIV patients. Methods. This cross-sectional study was carried out in Kinshasa hospitals between 2009 and 2012. Detection of microsporidia including E. bieneusi and E. intestinalis was performed in 242 HIV-infected patients. Typing was based on DNA polymorphism of the ribosomal DNA ITS region of E. bieneusi. PCRRFLP generated with two restriction enzymes (Nla III and Fnu 4HI) in PCR-amplified ITS products for classifying strains into different lineages. The diagnosis performance of the indirect immune-fluorescence-monoclonal antibody (IFI-AcM) was defined in comparison with real-time PCR as the gold standard. Results. Out of 242 HIV-infected patients, using the real-time PCR, the prevalence of E. bieneusi was 7.9% (n = 19) among the 19 E. bieneusi, one was coinfected with E. intestinalis. In 19 E. bieneusi persons using PCR-RFLP method, 5 type I strains of E. bieneusi (26.3%) and 5 type IV strains of E. bieneusi (26.3%) were identified. The sensitivity of IFI-AcM was poor as estimated 42.1%. Conclusion. Despite different PCR methods, there is possible association between HIVinfection, geographic location (France, Cameroun, Democratic Republic of Congo), and the concurrence of type I and type IV strains. PMID:22811884

  17. Enterocytozoon bieneusi Identification Using Real-Time Polymerase Chain Reaction and Restriction Fragment Length Polymorphism in HIV-Infected Humans from Kinshasa Province of the Democratic Republic of Congo

    PubMed Central

    Wumba, Roger; Jean, Menotti; Benjamin, Longo-Mbenza; Madone, Mandina; Fabien, Kintoki; Josué, Zanga; Jean, Sala; Eric, Kendjo; AC, Guillo-Olczyk; Marc, Thellier

    2012-01-01

    Objective. To determine the prevalence and the genotypes of Enterocytozoon bieneusi in stool specimens from HIV patients. Methods. This cross-sectional study was carried out in Kinshasa hospitals between 2009 and 2012. Detection of microsporidia including E. bieneusi and E. intestinalis was performed in 242 HIV-infected patients. Typing was based on DNA polymorphism of the ribosomal DNA ITS region of E. bieneusi. PCRRFLP generated with two restriction enzymes (Nla III and Fnu 4HI) in PCR-amplified ITS products for classifying strains into different lineages. The diagnosis performance of the indirect immune-fluorescence-monoclonal antibody (IFI-AcM) was defined in comparison with real-time PCR as the gold standard. Results. Out of 242 HIV-infected patients, using the real-time PCR, the prevalence of E. bieneusi was 7.9% (n = 19) among the 19 E. bieneusi, one was coinfected with E. intestinalis. In 19 E. bieneusi persons using PCR-RFLP method, 5 type I strains of E. bieneusi (26.3%) and 5 type IV strains of E. bieneusi (26.3%) were identified. The sensitivity of IFI-AcM was poor as estimated 42.1%. Conclusion. Despite different PCR methods, there is possible association between HIVinfection, geographic location (France, Cameroun, Democratic Republic of Congo), and the concurrence of type I and type IV strains. PMID:22811884

  18. Association Between Chloroplast DNA and Mitochondrial DNA Haplotypes in Prunus spinosa L. (Rosaceae) Populations across Europe

    PubMed Central

    MOHANTY, APARAJITA; MARTÍN, JUAN PEDRO; GONZÁLEZ, LUIS MIGUEL; AGUINAGALDE, ITZIAR

    2003-01-01

    Chloroplast DNA (cpDNA) and mitochondrial DNA (mtDNA) were studied in 24 populations of Prunus spinosa sampled across Europe. The cpDNA and mtDNA fragments were amplified using universal primers and subsequently digested with restriction enzymes to obtain the polymorphisms. Combinations of all the polymorphisms resulted in 33 cpDNA haplotypes and two mtDNA haplotypes. Strict association between the cpDNA haplotypes and the mtDNA haplotypes was detected in most cases, indicating conjoint inheritance of the two genomes. The most frequent and abundant cpDNA haplotype (C20; frequency, 51 %) is always associated with the more frequent and abundant mtDNA haplotype (M1; frequency, 84 %). All but two of the cpDNA haplotypes associated with the less frequent mtDNA haplotype (M2) are private haplotypes. These private haplotypes are phylogenetically related but geographically unrelated. They form a separate cluster on the minimum?length spanning tree. PMID:14534199

  19. Eukaryotic transcriptomics in silico: Optimizing cDNA-AFLP efficiency

    PubMed Central

    2009-01-01

    Background Complementary-DNA based amplified fragment length polymorphism (cDNA-AFLP) is a commonly used tool for assessing the genetic regulation of traits through the correlation of trait expression with cDNA expression profiles. In spite of the frequent application of this method, studies on the optimization of the cDNA-AFLP assay design are rare and have typically been taxonomically restricted. Here, we model cDNA-AFLPs on all 92 eukaryotic species for which cDNA pools are currently available, using all combinations of eight restriction enzymes standard in cDNA-AFLP screens. Results In silco simulations reveal that cDNA pool coverage is largely determined by the choice of individual restriction enzymes and that, through the choice of optimal enzyme combinations, coverage can be increased from <40% to 75% without changing the underlying experimental design. We find evidence of phylogenetic signal in the coverage data, which is largely mediated by organismal GC content. There is nonetheless a high degree of consistency in cDNA pool coverage for particular enzyme combinations, indicating that our recommendations should be applicable to most eukaryotic systems. We also explore the relationship between the average observed fragment number per selective AFLP-PCR reaction and the size of the underlying cDNA pool, and show how AFLP experiments can be used to estimate the number of genes expressed in a target tissue. Conclusion The insights gained from in silico screening of cDNA-AFLPs from a broad sampling of eukaryotes provide a set of guidelines that should help to substantially increase the efficiency of future cDNA-AFLP experiments in eukaryotes. In silico simulations also suggest a novel use of cDNA-AFLP screens to determine the number of transcripts expressed in a target tissue, an application that should be invaluable as next-generation sequencing technologies are adapted for differential display. PMID:19948029

  20. DNA.

    ERIC Educational Resources Information Center

    Felsenfeld, Gary

    1985-01-01

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

  1. Complex of osmium tetroxide with 1,10-phenanthroline binds covalently to double-stranded DNA.

    PubMed

    Palecek, E; Vlk, D; Vojtísková, M; Boublíková, P

    1995-12-01

    Complex of osmium tetroxide with 1,10-phenanthroline (Os,phen) reacts with double-stranded B-DNA in contrast to osmium tetroxide, pyridine and other osmium structural probes which show a strong preference for single-stranded DNA (ssDNA) (Palecek, E. in Abelson, J.N., and Simon, M.I. (eds), Lilley, D.M.J., and Dahlberg, J.E., (volume eds.), Methods in Enzymology, Vol. 212, DNA Structures, part B., Academic Press, 139-155 (1992)). Modification of negatively supercoiled DNA (scDNA) with Os,phen changes the DNA electrophoretic mobility inducing the DNA relaxation at lower degrees of modification followed by formation of positive supercoils at higher modification extents. Electrophoretic mobility of the Os,phen-modified DNA fragments in agarose gel is almost unchanged while a strong retardation of the same fragments is observed in polyacrylamide gels. Os,phen-modified DNA is hypersensitive to nuclease S1. Cleavage of this DNA by restriction enzymes is selectively inhibited showing a preference of Os,phen for TA and AT dinucleotide steps. DNA modification by Os,phen is inhibited by low and moderate concentrations of MgCl2. The covalent binding of Os,phen to double-stranded DNA (dsDNA) is preceded by noncovalent interactions (probably intercalation) inducing DNA structural changes; the shape of the Os,phen-modified DNA molecule appears to be severely deformed. PMID:8825734

  2. Applications of the Restriction Free (RF) cloning procedure for molecular manipulations and protein expression.

    PubMed

    Unger, Tamar; Jacobovitch, Yossi; Dantes, Ada; Bernheim, Reut; Peleg, Yoav

    2010-10-01

    Molecular manipulations, including DNA cloning and mutagenesis are basic tools used on a routine basis in all life-science disciplines. Over the last decade new methodologies have emerged that facilitated and expanded the applications for DNA cloning and mutagenesis. Ligation-Independent Cloning (LIC) techniques were developed and replaced the classical Ligation Dependent Cloning (LDC) platform. Restriction Free (RF) cloning was originally developed for introduction of foreign DNA into a plasmid at any predetermined position. RF cloning is based on PCR amplification of a DNA fragment, which serves as a mega-primer for the linear amplification of the vector and insert. Here we present several novel applications of the Restriction Free (RF) cloning platform for DNA cloning and mutagenesis. The new applications include simultaneous cloning of several DNA fragments into distinct positions within an expression vector, simultaneous multi-component assembly, and parallel cloning of the same PCR product into a series of different vectors. In addition, we have expanded the application of the RF cloning platform for multiple alterations of the target DNA, including simultaneous multiple-site mutagenesis and simultaneous introduction of deletions and insertions at different positions. We further demonstrate the robustness of the new applications for facilitating recombinant protein expression in the Escherichia coli system. PMID:20600952

  3. Sequence-specific cleavage of double-stranded DNA caused by X-ray ionization of the platinum atom in the Pt-bis-netropsin--DNA complex.

    PubMed Central

    Grokhovsky, S L; Zubarev, V E

    1991-01-01

    An analog of the antibiotic netropsin containing two netropsin-like fragments linked covalently via a platinum atom has been synthesized. DNase I and hydroxyl radical footprinting studies have shown that this compound binds at selective sites on a DNA restriction fragment with a known nucleotide sequence. After X-ray irradiation of Pt-bis-netropsin--DNA complexes a platinum-mediated cleavage of DNA is observed at specific DNA sites. This enables one to determine the location of the synthetic ligand on the DNA with a precision of about one nucleotide. The cleavage activity seems to be related to the emission of Auger electrons from the platinum atom that cause rupture of the deoxyribose residues on the two DNA strands near the position of the platinum atom in the complex. Images PMID:1849628

  4. Short communication: Evaluation of the microbiota of kefir samples using metagenetic analysis targeting the 16S and 26S ribosomal DNA fragments.

    PubMed

    Korsak, N; Taminiau, B; Leclercq, M; Nezer, C; Crevecoeur, S; Ferauche, C; Detry, E; Delcenserie, V; Daube, G

    2015-06-01

    Milk kefir is produced by fermenting milk in the presence of kefir grains. This beverage has several benefits for human health. The aim of this experiment was to analyze 5 kefir grains (and their products) using a targeted metagenetic approach. Of the 5 kefir grains analyzed, 1 was purchased in a supermarket, 2 were provided by the Ministry of Agriculture (Namur, Belgium), and 2 were provided by individuals. The metagenetic approach targeted the V1-V3 fragment of the 16S ribosomal (r)DNA for the grains and the resulting beverages at 2 levels of grain incorporation (5 and 10%) to identify the bacterial species population. In contrast, the 26S rDNA pyrosequencing was performed only on kefir grains with the aim of assessing the yeast populations. In parallel, pH measurements were performed on the kefir obtained from the kefir grains using 2 incorporation rates. Regarding the bacterial population, 16S pyrosequencing revealed the presence of 20 main bacterial species, with a dominance of the following: Lactobacillus kefiranofaciens, Lactococcus lactis ssp. cremoris, Gluconobacter frateurii, Lactobacillus kefiri, Acetobacter orientalis, and Acetobacter lovaniensis. An important difference was noticed between the kefir samples: kefir grain purchased from a supermarket (sample E) harbored a much higher proportion of several operational taxonomic units of Lactococcus lactis and Leuconostoc mesenteroides. This sample of grain was macroscopically different from the others in terms of size, apparent cohesion of the grains, structure, and texture, probably associated with a lower level of Lactobacillus kefiranofaciens. The kefir (at an incorporation rate of 5%) produced from this sample of grain was characterized by a lower pH value (4.5) than the others. The other 4 samples of kefir (5%) had pH values above 5. Comparing the kefir grain and the kefir, an increase in the population of Gluconobacter in grain sample B was observed. This was also the case for Acetobacter orientalis in sample D. In relation to 26S pyrosequencing, our study revealed the presence of 3 main yeast species: Naumovozyma spp., Kluyveromyces marxianus, and Kazachastania khefir. For Naumovozyma, further studies are needed to assess the isolation of new species. In conclusion, this study has proved that it is possible to establish the patterns of bacterial and yeast composition of kefir and kefir grain. This was only achieved with the use of high-throughput sequencing techniques. PMID:25828663

  5. Transgenic analysis of a 100-kb human beta-globin cluster-containing DNA fragment propagated as a bacterial artificial chromosome.

    PubMed

    Kaufman, R M; Pham, C T; Ley, T J

    1999-11-01

    To date, the normal transcriptional regulation of the human beta-globin gene cluster has been recapitulated most accurately in transgenic mice that carry large yeast artificial chromosome (YAC) or ligated cosmid constructs. However, these large transgenes still exhibit variegated expression levels, perhaps because they tend to rearrange upon integration, or because the cloning vectors remain attached to the globin inserts. To try to circumvent these potential problems, we investigated the transgenic properties of a 100-kb DNA fragment containing the entire human beta-globin cluster propagated in a bacterial artificial chromosome (BAC). We created 9 independent mouse lines, each carrying 1 to 6 copies of the human beta-globin cluster without the attached BAC vector. Five of the lines carry unrearranged copies of the cluster. Reverse-transcriptase polymerase chain reaction (RT-PCR) analysis of adult F(1) mice showed that 2 lines express human beta globin at levels approximately equivalent to the endogenous mouse beta-major genes. One line expresses no human beta globin, while the remaining 6 lines show intermediate expression levels. Complete gamma-->beta-globin gene switching occurs, but is slightly delayed with respect to the endogenous mouse embryonic-->adult switch. Since these data are similar to what has been obtained using globin YACs or ligated cosmids, we conclude that (1) globin transgenes propagated in BACs are no less likely to rearrange than their cosmid or YAC counterparts, and (2) the retention of YAC vector sequences in a transgene probably has no significant impact on globin expression when using constructs of this size. PMID:10556205

  6. Identification of human papillomavirus type 156, the prototype of a new human gammapapillomavirus species, by a generic and highly sensitive PCR strategy for long DNA fragments.

    PubMed

    Chouhy, Diego; Bolatti, Elisa M; Piccirilli, Gustavo; Sánchez, Adriana; Fernandez Bussy, Ramón; Giri, Adriana A

    2013-03-01

    This study developed a hanging-droplet long PCR, a generic and highly sensitive strategy to facilitate the identification of new human papillomavirus (HPV) genomes. This novel procedure used for the first time the hanging-droplet PCR technique for the amplification of long DNA fragments with generic primers targeting the L1 and E1 regions. It was first applied to the amplification of types belonging to the highly divergent genus Gammapapillovirus (?-PV). The hanging-droplet long PCR was 100-fold more sensitive than a simple long PCR procedure, detecting as few as ten copies of HPV-4. Nineteen skin samples, potentially containing putative HPV types from the ?-PV genus, were also screened. The method identified four ?-PV genomic halves from new and previously described putative types, and made the full characterization of HPV-156 possible. This novel virus meets the criteria for a new species within the ?-PV genus, with nucleotide identities in the L1 ORF ranging from 58.3 to 67.3?% compared with representative types of the current ?-PV species. HPV-156 showed the highest identity to HPV-60 (67.3?%) from species ?-4, and was consistently closely related to it in both late- and early-gene-derived phylogenies. In conclusion, this report provides a versatile and highly sensitive approach that allowed identification of the prototype of a new species within the ?-PV genus. Its application with primers targeting the different genera in which both human and non-human PVs are distributed may facilitate characterization of the missing members of the family Papillomaviridae. PMID:23136368

  7. Problem-Solving Test: Restriction Endonuclease Mapping

    ERIC Educational Resources Information Center

    Szeberenyi, Jozsef

    2011-01-01

    The term "restriction endonuclease mapping" covers a number of related techniques used to identify specific restriction enzyme recognition sites on small DNA molecules. A method for restriction endonuclease mapping of a 1,000-basepair (bp)-long DNA molecule is described in the fictitious experiment of this test. The most important fact needed to…

  8. Restriction endonucleases and methylases

    SciTech Connect

    Chirikjian, J.G.

    1987-01-01

    This volume covers new research advances in molecular biology brought about by the use of restriction endonucleases and methylases. Contributions by authorities in the field focus on research studies that examine both the structure and catalytic properties of this group of DNA enzymes. This volume also serves as a reference for reaction conditions and the general application of restriction endonucleases as reagents for molecular biology.

  9. [Application of DNA polymorphism to forensic medicine].

    PubMed

    Misawa, S

    1994-06-01

    Recent recombinant DNA technology has provided some novel and powerful methods for forensic science application. Human genomic DNA can be analyzed directly for individual identification and paternity testing on the basis of variations in its sequence. The analysis of DNA isolated from forensic biological evidence provides valuable information relating to the identification of the source of the sample. Restriction fragment length polymorphism (RFLP) testing, using a combination of single locus probe (SLP) that vary highly among individuals, produces a DNA fingerprint or profile. The PCR method using STR (microsatellite) and mitochondrial DNA analysis (mtDNA) is suitable for examination of the forensic biological samples (bloodstains, hairs, seminal stains, bones, tooth). For sex identification of bloodstains, bleached skeletons and teeth, Southern blot hybridization with Y-chromosome specific probe (pHY10; 3.4 kb) and PCR amplifying with sex chromosome specific fragments, can be used. Mitochondrial DNA RFLPs and 9-bp deletion type of mtDNA may suggest the characteristics of the human races. For the purpose of resolving the genetic basis of human uniqueness, we reported the isolation of human-specific sequences using the technique of genome subtraction (HS5, 2282 bp). PMID:7914244

  10. Atomic force microscopy of long DNA: imaging in air and under water.

    PubMed Central

    Lyubchenko, Y; Shlyakhtenko, L; Harrington, R; Oden, P; Lindsay, S

    1993-01-01

    We have obtained striking atomic force microscopy images of the intact lambda bacteriophage genome and of several lambda restriction fragments both in air and under water. The DNA is unstained and the images are stable under continuous scanning for up to 30 min. Measured contour lengths of fully imaged restriction fragments and intact lambda DNA are accurate to within a few percent. The key to this development is the use of a process for binding unmodified double-stranded DNA to chemically treated mica surfaces. This procedure leads to strong DNA attachment and yields high-quality images that are stable under repeated scanning, even with the sample submerged in water. This allows normal hydration conditions to be maintained during scanning and in addition leads to a general improvement of image quality. Both the lateral resolution and the contrast increase by a factor of approximately 3 under water. Images Fig. 1 Fig. 2 Fig. 3 PMID:8460119

  11. Restriction Site-Associated DNA Sequencing (RAD-seq) Reveals an Extraordinary Number of Transitions among Gecko Sex-Determining Systems.

    PubMed

    Gamble, Tony; Coryell, Jessi; Ezaz, Tariq; Lynch, Joshua; Scantlebury, Daniel P; Zarkower, David

    2015-05-01

    Sex chromosomes have evolved many times in animals and studying these replicate evolutionary "experiments" can help broaden our understanding of the general forces driving the origin and evolution of sex chromosomes. However this plan of study has been hindered by the inability to identify the sex chromosome systems in the large number of species with cryptic, homomorphic sex chromosomes. Restriction site-associated DNA sequencing (RAD-seq) is a critical enabling technology that can identify the sex chromosome systems in many species where traditional cytogenetic methods have failed. Using newly generated RAD-seq data from 12 gecko species, along with data from the literature, we reinterpret the evolution of sex-determining systems in lizards and snakes and test the hypothesis that sex chromosomes can routinely act as evolutionary traps. We uncovered between 17 and 25 transitions among gecko sex-determining systems. This is approximately one-half to two-thirds of the total number of transitions observed among all lizards and snakes. We find support for the hypothesis that sex chromosome systems can readily become trap-like and show that adding even a small number of species from understudied clades can greatly enhance hypothesis testing in a model-based phylogenetic framework. RAD-seq will undoubtedly prove useful in evaluating other species for male or female heterogamety, particularly the majority of fish, amphibian, and reptile species that lack visibly heteromorphic sex chromosomes, and will significantly accelerate the pace of biological discovery. PMID:25657328

  12. Molecular characterization by amplified ribosomal DNA restriction analysis and antimicrobial potential of endophytic fungi isolated from Luehea divaricata (Malvaceae) against plant pathogenic fungi and pathogenic bacteria.

    PubMed

    Bernardi-Wenzel, J; Garcia, A; Azevedo, J L; Pamphile, J A

    2013-01-01

    Luehea divaricata is an important plant in popular medicine; it is used for its depurative, anti-inflammatory, and other therapeutic activities. We evaluated the antimicrobial activity of endophytic fungi isolated from leaves of L. divaricata against phytopathogens and pathogenic bacteria, and characterized the isolates based on amplified ribosomal DNA restriction analysis (ARDRA). The in vitro antagonistic activity of these endophytes against the phytopathogen Alternaria alternata was assayed by dual culture technique. Based on this evaluation of antimicrobial activity, we extracted secondary metabolites from nine endophytic fungi by partitioning in ethyl acetate and methanol. These were tested against the phytopathogens A. alternata, Colletotrichum sp and Moniliophthora perniciosa, and against the human pathogenic bacteria Escherichia coli and Staphylococcus aureus. Molecular characterization by ARDRA technique was used for phylogenetic analysis, based on comparison with sequences in GenBank. The endophytes had varied effects on A. alternata. One isolate produced an inhibition halo against M. perniciosa and against E. coli. This antibiosis activity indicates a role in the protection of the plant against microbial pathogens in nature, with potential for pharmaceutical and agricultural applications. Based on ARDRA, the 13 isolates were grouped. We found three different haplotypes of Phomopsis sp, showing interspecific variability. It appears that examination of the microbial community associated with medicinal plants of tropical regions has potential as a useful strategy to look for species with biotechnological applications. PMID:24301768

  13. Construction of highly extensive polymorphic DNA libraries by in-gel competitive reassociation procedure

    SciTech Connect

    Inoue, Shinichi; Kiyama, Ryoiti; Oishi, Michio

    1996-02-01

    Differential genomic DNA libraries between two mouse strains and from two human individuals were constructed by means of the in-gel competitive reassociation (IGCR) procedure, a procedure developed for cloning altered anonymous restriction fragments. The libraries were highly enriched fragments, approximately 60 and 40% for the mouse and human libraries, respectively, and, more importantly, maintained most of the original complexities of the RFLP fragments. Therefore, differential genomic DNA libraries constructed by the IGCR procedure, particularly for human genomic DNA, should offer highly extensive sources for polymorphic DNA sequences necessary for a variety of genome analyses, including studies on the origin and mechanism of biological diversity among the same species. 19 refs., 4 figs.

  14. Application of the Restriction-Free (RF) cloning for multicomponents assembly.

    PubMed

    Peleg, Yoav; Unger, Tamar

    2014-01-01

    Molecular manipulations, including DNA cloning and mutagenesis, are currently employed on a routine basis in all life science disciplines. Over the last decade new methodologies have emerged that expanded and facilitated the applications for DNA cloning. The classical Ligation-Dependent Cloning (LDC) is gradually replaced by Ligation-Independent Cloning (LIC) techniques. The Restriction-Free (RF) cloning was originally developed for introduction of a foreign DNA into a plasmid at any desired position. The RF methodology is based on generation of a PCR product, which serves as a set of mega-primers for subsequent incorporation into any desired position within a circular plasmid. We have expanded the applications of the RF methodology for multiple simultaneous alterations of a target DNA and for multicomponents assembly. In the current manuscript we describe a step-by-step protocol for application of the RF methodology for simultaneous multiple DNA fragments assembly in tandem and at distinct positions within an expression vector. PMID:24395358

  15. Functional ?-fragment of ?-galactosidase can be expressed from the mobile group I intron PpLSU3 embedded in yeast pre-ribosomal RNA derived from the chromosomal rDNA locus

    PubMed Central

    Lin, Jue; Vogt, Volker M.

    2000-01-01

    PpLSU3, a mobile group I intron found in the ribosomal RNA genes of Physarum polycephalum, encodes the I-PpoI homing endonuclease. This enzyme represents one of the rare cases in nature where a protein is expressed from an RNA polymerase I transcript. Our previous results showed that the full length intron, but not a further processed species, is the messenger for I-PpoI, implying a role of the untranslated region (UTR) in gene expression. To study the function of the 3?-UTR in expression of the endonuclease and in splicing of the intron, we replaced the I-PpoI gene in PpLSU3 with the gene for the ?-fragment of Escherichia coli ?-galactosidase, and then integrated this chimeric intron into all the chromosomal rDNA repeats of yeast. The resulting cells synthesized functional ?-fragment, as evidenced by a complementation assay analogous to that used in E.coli. The ?-galactosidase activity thus provides an unusual and potentially valuable readout for Pol I transcription from chromosomal rDNA. This is the first example in which a eucaryotic homing endonuclease gene has been successfully replaced by a heterologous gene. Using deletion mutagenesis and a novel randomization approach with the ?-fragment as a reporter, we found that a small segment of the 3?-UTR dramatically influences both splicing and protein expression. PMID:10684939

  16. Bioinorganic Chemistry Special Feature: Gapped DNA is anisotropically bent

    NASA Astrophysics Data System (ADS)

    Guo, Hong; Tullius, Thomas D.

    2003-04-01

    Ionizing radiation damages DNA in several ways, including through formation of a single-nucleoside gap in one DNA strand. We have developed a two-dimensional gel electrophoresis method to investigate the effect of a strand gap on DNA structure. We generate a library of gapped DNA molecules by treating a DNA restriction fragment with the hydroxyl radical, generated by the reaction of Fe(II) EDTA with hydrogen peroxide. The DNA molecule studied contains a fixed bend produced by a set of phased adenine tracts. The A-tract bend serves as a reference bend for investigating the conformational nature of a strand gap. In the first electrophoretic dimension, a bent DNA molecule that has been treated with the hydroxyl radical is electrophoresed on a native gel. Smearing of the band on the native gel indicates that the library of gapped DNA molecules contains a variety of DNA conformations. In the second electrophoretic dimension, gapped DNA molecules having different native gel mobilities are electrophoresed on separate lanes of a denaturing gel to reveal how each strand gap affects the native gel mobility (and thus shape) of the DNA. Our results demonstrate that a single-nucleoside gap in a DNA duplex leads to an anisotropic, directional bend in the DNA helix axis. The implications of our findings for recognition of this lesion by DNA repair proteins are discussed.

  17. Simulation study of natural UV-B radiation on Catla catla and its impact on physiology, oxidative stress, Hsp 70 and DNA fragmentation.

    PubMed

    Singh, Moirangthem Kameshwor; Sharma, Jai Gopal; Chakrabarti, Rina

    2015-08-01

    UV-B radiation is a potential stressor to the aquacultural species. Catla catla, catla larvae (1.08±0.065g) were exposed to different doses of UV-B radiation, 0 (control), 504, 1008, 1512 and 2016mJ/cm(2) at a mean radiant energy of 80?W/cm(2) for 21days. The dose of UV-B radiation was selected on the basis of the field study conducted in Lake Naini, Delhi, India (Latitude: 28°41'26?N and Longitude: 77°12?37?E). Significantly (P<0.05) lower survival, average weight and specific growth rate were found in UV-B irradiated larvae compared to the control one. Food conversion ratio was 1.5-4-fold higher in UV-B treated larvae compared to the control one. The carbonyl protein (CP), thiobarbituric acid reactive substances (TBARS), superoxide dismutase (SOD) levels were significantly (P <0.05) higher in UV-B irradiated larvae compared to the control group. Among the treated larvae, CP and SOD were significantly (P <0.05) higher in larvae exposed at 1512mJ/cm(2) UV-B. A correlation was found between the CP and SOD (R(2)=0.834). Highest TBARS level was found in 2016mJ/cm(2) UV-B exposed catla. Nitric oxide synthase level was significantly (P <0.05) lower in UV-B exposed larvae compared to the control one. A 3-fold increased Hsp 70 level was recorded in UV-B irradiated catla compared to the control larvae. Comet assay analysis indicated that UV-B irradiation enhanced DNA fragmentation. Tail extent moment and the olive tail moment were significantly (P <0.05) higher in 2016mJ/cm(2) UV-B exposed catla compared to others. The tail length was significantly (P <0.05) higher in 1512 and 2016mJ/cm(2) UV-B exposed larvae compared to the other doses. The present study suggests that the catla is a useful species for the biomonitoring of stress in the aquatic environment. PMID:26065818

  18. Detection and identification of phytopathogenic Xanthomonas strains by amplification of DNA sequences related to the hrp genes of Xanthomonas campestris pv. vesicatoria.

    PubMed Central

    Leite, R P; Minsavage, G V; Bonas, U; Stall, R E

    1994-01-01

    Three pairs of oligonucleotide primers specific for different regions of the hrp gene (hypersensitive reaction and pathogenicity) cluster of Xanthomonas campestris pv. vesicatoria were designed and tested for amplification of DNA isolated from a large number of different bacteria. DNA sequences related to the hrp genes were successfully amplified from X. fragariae and from 28 pathovars of X. campestris. No DNA amplification occurred with genomic DNA from phytopathogenic strains of X. campestris pv. secalis, X. campestris pv. translucens, and X. albilineans or from nonpathogenic opportunistic xanthomonads and phytopathogenic strains of the genera Acidovorax, Agrobacterium, Clavibacter, Erwinia, Pseudomonas, and Xylella. The DNA from those bacteria also failed to hybridize to hrp-specific fragments in Southern blot analysis. DNA fragments amplified with a particular primer pair were of identical size from each of the different phytopathogenic xanthomonads. However, restriction analysis of these fragments by using frequently cutting endonucleases revealed variation in the pattern for these hrp-related fragments amplified from the different Xanthomonas strains. The restriction patterns generated for the different fragments allowed distinction of the strains representing a pathovar or species of phytopathogenic xanthomonads. We believe that DNA amplification with hrp-specific oligonucleotide primers is a highly sensitive and specific method that can be applied for detection and identification of phytopathogenic xanthomonads. Images PMID:8017904

  19. [The accumulation of single-stranded breaks does not lead to paired DNA damage--the characteristic of the transcribing fragment of the human ribosomal operon that allows its being detected in biological fluids at the death of different body cells].

    PubMed

    Ve?ko, N N; Spitkovski?, D M

    2000-01-01

    It was shown by blot-hybridization with corresponding DNA probes after electrophoretic separation of control and experimental samples of human genome DNA that accumulation of single-strand breaks in the chains of double-strand fragment of transcribing range of ribosomal gene (TRrDNA) does not result in double-strand breaks. That differs from the other studied DNA sequences (cluster of histon genes, Alu-repetition, telomeric repetition and satellite III). Single-strand breaks and double-strand breaks were induced by endonucleases and by gamma-radiation. In spite of higher chemical modification of TRrDNA by arylazide and dimethylsulfate (because of high content of GC-pairs), under the following fragmentation TRrDNA was found to be more resistant to double-strand breaks than other studied DNA sequences. At the same time in the range of non-transcribing spacer (NTS) of ribosomal gene, the section with higher sensitivity to double-strand breaks was found. Higher resistance of TRrDNA to double breaks makes it possible to identify these fragments in cell material from different tissue after death or in DNA samples after prolonged storage. Resistance of TRrDNA to formation of double-strand breaks can be used for its detection in biological fluids after cell death, including the death initiated by ionizing radiation. PMID:11031484

  20. Supramolecular Complexes of DNA

    NASA Astrophysics Data System (ADS)

    Zuber, G.; Scherman, D.

    Deoxyribose nucleic acid or DNA is a linear polymer in the form of a double strand, synthesised by sequential polymerisation of a large number of units chosen from among the nucleic bases called purines (adenosine A and guanosine G) and pyrimidines (cytosine C and thymidine T). DNA contains all the genetic information required for life. It exists in the form of a limited number (a few dozen) of very big molecules, called chromosomes. This genetic information is first of all transcribed. In this process, a restricted fragment of the DNA called a gene is copied in the form of ribonucleic acid, or RNA. This RNA is itself a polymer, but with a single strand in which the sequence of nucleic acids is schematically analogous to the sequence on one of the two strands of the transcribed DNA. Finally, this RNA is translated into a protein, yet another linear polymer. The proteins make up the main part of the active constituents ensuring the survival of the cell. Any loss of information, either by mutation or by deletion of the DNA, will cause an imbalance in the cell's metabolism that may in turn lead to incurable pathologies. Several strategies have been developed to reduce the consequences of such genetic deficiencies or, more generally, to act, by amplifying or suppressing them, on the mechanisms leading from the reading of the genetic information to the production of proteins: Strategies aiming to introduce synthetic DNA or RNA, which selectively block the expression of certain genes, are now being studied by an increasing number of research scientists and pharmacologists. They use antisense oligodeoxyribonucleotides or interfering oligoribonucleotides and they already have clinical applications. This kind of therapy is often called gene pharmacology. Other, more ambitious strategies aim to repair in situ mutated or incomplete DNA within the chromosomes themselves, by introducing short sequences of DNA or RNA which recognise and take the place of mutations. This is the underlying principle of genetic correction. Yet other strategies aim to reintroduce the deficient DNA fragments into the cells in the form of genes. Indeed, in certain diseases, the only solution is to bring genetic information back into the cells by transferring exogeneous DNA into the cell nucleus. This approach goes by the name of gene therapy.

  1. Sperm DNA oxidative damage and DNA adducts.

    PubMed

    Jeng, Hueiwang Anna; Pan, Chih-Hong; Chao, Mu-Rong; Lin, Wen-Yi

    2015-12-01

    The objective of this study was to investigate DNA damage and adducts in sperm from coke oven workers who have been exposed to polycyclic aromatic hydrocarbons. A longitudinal study was conducted with repeated measurements during spermatogenesis. Coke-oven workers (n=112) from a coke-oven plant served the PAH-exposed group, while administrators and security personnel (n=67) served the control. Routine semen parameters (concentration, motility, vitality, and morphology) were analyzed simultaneously; the assessment of sperm DNA integrity endpoints included DNA fragmentation, bulky DNA adducts, and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dGuo). The degree of sperm DNA fragmentation was measured using the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay and sperm chromatin structure assay (SCSA). The PAH-exposed group had a significant increase in bulky DNA adducts and 8-oxo-dGuo compared to the control subjects (Ps=0.002 and 0.045, respectively). Coke oven workers' percentages of DNA fragmentation and denaturation from the PAH-exposed group were not significantly different from those of the control subjects (Ps=0.232 and 0.245, respectively). Routine semen parameters and DNA integrity endpoints were not correlated. Concentrations of 8-oxo-dGuo were positively correlated with percentages of DNA fragmentation measured by both TUNEL and SCSA (Ps=0.045 and 0.034, respectively). However, the concentrations of 8-oxo-dGuo and percentages of DNA fragmentation did not correlate with concentrations of bulky DNA adducts. In summary, coke oven workers with chronic exposure to PAHs experienced decreased sperm DNA integrity. Oxidative stress could contribute to the degree of DNA fragmentation. Bulky DNA adducts may be independent of the formation of DNA fragmentation and oxidative adducts in sperm. Monitoring sperm DNA integrity is recommended as a part of the process of assessing the impact of occupational and environmental toxins on sperm. PMID:26653986

  2. Isolation and characterization of 25 unique DNA markers for human chromosome 22

    SciTech Connect

    Van Biezen, N.A.; Deprez, R.H.L.; Thijs, A.; Zwarthoff, E.C. ); Heutink, P.; Oostra, B.A.; Kessel, A.H.M.G. van )

    1993-01-01

    Twenty-five single-copy anonymous DNA markers for human chromosome 22 were isolated. These markers were assigned to four different regions on the chromosome. Six markers recognize restriction fragment length polymorphisms. The relative positions of five of these polymorphic markers on the framework map of chromosome 22 were determined by linkage analysis. The sizes of the NotI fragments recognized by 22 markers were determined by pulsed-field gel analysis. The total length of the NotI fragments identified is at least 12 Mb, which represents about 20% of the entire chromosome. 15 refs., 1 fig.

  3. Laser mass spectrometry for DNA fingerprinting for forensic applications

    SciTech Connect

    Chen, C.H.; Tang, K.; Taranenko, N.I.; Allman, S.L.; Chang, L.Y.

    1994-12-31

    The application of DNA fingerprinting has become very broad in forensic analysis, patient identification, diagnostic medicine, and wildlife poaching, since every individual`s DNA structure is identical within all tissues of their body. DNA fingerprinting was initiated by the use of restriction fragment length polymorphisms (RFLP). In 1987, Nakamura et al. found that a variable number of tandem repeats (VNTR) often occurred in the alleles. The probability of different individuals having the same number of tandem repeats in several different alleles is very low. Thus, the identification of VNTR from genomic DNA became a very reliable method for identification of individuals. DNA fingerprinting is a reliable tool for forensic analysis. In DNA fingerprinting, knowledge of the sequence of tandem repeats and restriction endonuclease sites can provide the basis for identification. The major steps for conventional DNA fingerprinting include (1) specimen processing (2) amplification of selected DNA segments by PCR, and (3) gel electrophoresis to do the final DNA analysis. In this work we propose to use laser desorption mass spectrometry for fast DNA fingerprinting. The process and advantages are discussed.

  4. Restrictive cardiomyopathy

    MedlinePLUS

    Cardiomyopathy - restrictive; Infiltrative cardiomyopathy ... In a case of restrictive cardiomyopathy, the heart muscle is normal size or slightly enlarged. Most of the time, it also pumps normally. However, it does not ...

  5. Genetics of the complementary restriction systems DpnI and DpnII revealed by cloning and recombination in Streptococcus pneumoniae

    SciTech Connect

    Lacks, S.A.; Mannarelli, B.M.; Springhorn, S.S.; Greenberg, B.; de la Campa, A.G.

    1986-05-01

    Transformation and cloning of the DpnI and DpnII endonuclease genes has clarified the genetic basis of the two restriction systems. Molecular cloning was carried out in the Gram-positive S. pneumoniae host/vector system. Cloned chromosomal fragments from both DpnI- and DpnII-producing strains were subjected to nucleotide sequence determination and were used as probes for DNA hybridization analysis. It was shown that the restriction enzyme phenotype of S. pneumoniae depended on an intercellular genetic cassette mechanism. In this review some aspects of the evolution of restriction systems in S. pneumoniae and other bacterial will be discussed. 42 refs., 7 figs., 1 tab.

  6. DNA analysis on microfabricated electrophoretic devices with bubble cells.

    PubMed

    Tseng, Wei-Lung; Lin, Yang-Wei; Chen, Ko-Chun; Chang, Huan-Tsung

    2002-08-01

    Microfluidic devices with bubble cells have been fabricated on poly(methyl methacrylate) (PMMA) plates and have been employed for the analysis of DNA using polyethylene oxide (PEO) solutions. First, the separation channel was fabricated using a wire-imprinting method. Then, wires with greater sizes or a razor blade glued in a polycarbonate plate was used to fabricate bubble cells, with sizes of 190-650 microm. The improvements in resolution and sensitivity have been achieved for large DNA (> 603 base pair, bp) using such devices, which depend on the geometry of the bubble cell. The main contributor for optimal resolution is mainly due to DNA migration at lower electric field strengths inside the bubble cell. On the other hand, slight losses of resolution for small DNA fragments have been found mainly due to diffusion, supported by the loss of resolution when separating two small solutes. With a bubble cell of 75 microm (width) x 500 microm (depth), the sensitivity improvement up to 17-fold has been achieved for the 271 bp fragment in the separation of PhiX-174/HaeIII DNA restriction fragments. We have also found that a microfluidic device with a bubble cell of 360 microm x 360 microm is appropriate for DNA analysis. Such a device has been used for separating DNA ranging from 8 to 2176 bp and polymerase chain reaction (PCR) products amplified after 30 cycles, with rapidity and improvements in the sensitivity as well as resolution. PMID:12210206

  7. Mitochondrial DNA variation in screwworm.

    PubMed

    Taylor, D B; Szalanski, A L; Peterson, R D

    1996-04-01

    Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis was used to characterize mitochondrial DNA (mtDNA) variation in screwworms, Cochliomyia hominivorax, and secondary screwworm, C.macellaria, from the Caribbean, North America and South America. Four amplicons, totaling 7.1 kb, were analysed with sixteen restriction enzymes. A total of 133 restriction sites was observed in the two species, 104 in C.hominivorax, of which nineteen were variable, and ninety-five in C.macellaria, none of which was variable. Fourteen mtDNA haplotypes were observed among eighteen C.hominivorax examined. Mean divergence between C.hominivorax haplotypes (d) was 0.0064 substitutions per base-pair and genotypic diversity (G) was 0.97. Mean divergence between C.hominivorax and C.macellaria was 0.0824. Cochliomyia hominivorax haplotypes could be divided into three assemblages representing North America, South America and Jamaica, based on UPGMA clustering with d values. The assemblages did not exhibit complete geographic fidelity. These data were discordant with previously published allozyme data indicating little differentiation between screwworm populations. A scenario invoking historically isolated populations coming into contact with the introduction and movement of European livestock is proposed to explain the observed population structure of screwworm. PMID:8744709

  8. Random Tagging Genotyping by Sequencing (rtGBS), an Unbiased Approach to Locate Restriction Enzyme Sites across the Target Genome

    PubMed Central

    Hilario, Elena; Barron, Lorna; Deng, Cecilia H.; Datson, Paul M.; Davy, Marcus W.; Storey, Roy D.

    2015-01-01

    Genotyping by sequencing (GBS) is a restriction enzyme based targeted approach developed to reduce the genome complexity and discover genetic markers when a priori sequence information is unavailable. Sufficient coverage at each locus is essential to distinguish heterozygous from homozygous sites accurately. The number of GBS samples able to be pooled in one sequencing lane is limited by the number of restriction sites present in the genome and the read depth required at each site per sample for accurate calling of single-nucleotide polymorphisms. Loci bias was observed using a slight modification of the Elshire et al. method: some restriction enzyme sites were represented in higher proportions while others were poorly represented or absent. This bias could be due to the quality of genomic DNA, the endonuclease and ligase reaction efficiency, the distance between restriction sites, the preferential amplification of small library restriction fragments, or bias towards cluster formation of small amplicons during the sequencing process. To overcome these issues, we have developed a GBS method based on randomly tagging genomic DNA (rtGBS). By randomly landing on the genome, we can, with less bias, find restriction sites that are far apart, and undetected by the standard GBS (stdGBS) method. The study comprises two types of biological replicates: six different kiwifruit plants and two independent DNA extractions per plant; and three types of technical replicates: four samples of each DNA extraction, stdGBS vs. rtGBS methods, and two independent library amplifications, each sequenced in separate lanes. A statistically significant unbiased distribution of restriction fragment size by rtGBS showed that this method targeted 49% (39,145) of BamH I sites shared with the reference genome, compared to only 14% (11,513) by stdGBS. PMID:26633193

  9. Quantification of DNA Damage by Real-Time qPCR.

    PubMed

    Wang, Wei; Scheffler, Katja; Esbensen, Ying; Eide, Lars

    2016-01-01

    This chapter describes the use of real-time qPCR to quantify damages in genomic DNA. The method is based on the ability of a lesion in one strand to inhibit restriction enzyme digestion of double-stranded DNA. Subsequent amplification of the complementary strand after restriction cleavage gives a quantitative measure of the damage content in that site (Real-time qPCR Analysis of Damage Frequency; RADF). We compare the RADF assay with the commonly used technique to assess damages by their ability to inhibit amplification of a large PCR fragment relative to a short PCR fragment. The RADF method described here is quick, accurate and allows the detection of nuclear and mitochondrial DNA damage in detailed regions. PMID:26530672

  10. Chameleon Fragmentation

    E-print Network

    Brax, Philippe

    2013-01-01

    A scalar field dark energy candidate could couple to ordinary matter and photons, enabling its detection in laboratory experiments. Here we study the quantum properties of the chameleon field, one such dark energy candidate, in an "afterglow" experiment designed to produce, trap, and detect chameleon particles. In particular, we investigate the possible fragmentation of a beam of chameleon particles into multiple particle states due to the highly non-linear interaction terms in the chameleon Lagrangian. Fragmentation could weaken the constraints of an afterglow experiment by reducing the energy of the regenerated photons, but this energy reduction also provides a unique signature which could be detected by a properly-designed experiment. We show that constraints from the CHASE experiment are essentially unaffected by fragmentation for $\\phi^4$ and $1/\\phi$ potentials, but are weakened for steeper potentials, and we discuss possible future afterglow experiments.

  11. Chameleon fragmentation

    SciTech Connect

    Brax, Philippe

    2014-02-01

    A scalar field dark energy candidate could couple to ordinary matter and photons, enabling its detection in laboratory experiments. Here we study the quantum properties of the chameleon field, one such dark energy candidate, in an ''afterglow'' experiment designed to produce, trap, and detect chameleon particles. In particular, we investigate the possible fragmentation of a beam of chameleon particles into multiple particle states due to the highly non-linear interaction terms in the chameleon Lagrangian. Fragmentation could weaken the constraints of an afterglow experiment by reducing the energy of the regenerated photons, but this energy reduction also provides a unique signature which could be detected by a properly-designed experiment. We show that constraints from the CHASE experiment are essentially unaffected by fragmentation for ?{sup 4} and 1/? potentials, but are weakened for steeper potentials, and we discuss possible future afterglow experiments.

  12. Chameleon Fragmentation

    E-print Network

    Philippe Brax; Amol Upadhye

    2013-12-10

    A scalar field dark energy candidate could couple to ordinary matter and photons, enabling its detection in laboratory experiments. Here we study the quantum properties of the chameleon field, one such dark energy candidate, in an "afterglow" experiment designed to produce, trap, and detect chameleon particles. In particular, we investigate the possible fragmentation of a beam of chameleon particles into multiple particle states due to the highly non-linear interaction terms in the chameleon Lagrangian. Fragmentation could weaken the constraints of an afterglow experiment by reducing the energy of the regenerated photons, but this energy reduction also provides a unique signature which could be detected by a properly-designed experiment. We show that constraints from the CHASE experiment are essentially unaffected by fragmentation for $\\phi^4$ and $1/\\phi$ potentials, but are weakened for steeper potentials, and we discuss possible future afterglow experiments.

  13. Resistance of Spiroplasma citri Lines to the Virus SVTS2 Is Associated with Integration of Viral DNA Sequences into Host Chromosomal and Extrachromosomal DNA

    PubMed Central

    Sha, Y.; Melcher, U.; Davis, R. E.; Fletcher, J.

    1995-01-01

    Spiroplasmavirus SVTS2, isolated from Spiroplasma melliferum TS2, produces plaques when inoculated onto lawns of Spiroplasma citri M200H, a derivative of the type strain Maroc R8A2. S. citri strains MR2 and MR3, originally selected as colonies growing within plaques on a lawn of M200H inoculated with SVTS2, were resistant to SVTS2. Genomic DNA fingerprints and electrophoretic protein profiles of M200H, MR2, and MR3 were similar, but three proteins present in M200H were missing or significantly reduced in both resistant lines. None of these three polypeptides reacted with antiserum against S. citri membrane proteins, indicating that they probably are not surface-located virus receptors. Electroporation with SVTS2 DNA produced 1.5 x 10(sup5) transfectants per (mu)g of DNA in M200H but none in MR2 or MR3, suggesting that resistance may result from inhibition of viral replication. The digestion patterns of the extrachromosomal double-stranded (ds) DNA of these lines were similar. Three TaqI fragments of MR2 extrachromosomal DNA that were not present in M200H extrachromosomal DNA hybridized strongly to an SVTS2 probe, and two of these fragments plus an additional one hybridized with the MR3 extrachromosomal DNA, indicating that a fragment of SVTS2 DNA was present in the extrachromosomal ds DNA of MR2 and MR3 but not of M200H. When the restricted genomes of all three lines were probed with SVTS2 DNA, strong hybridization to two EcoRI fragments of chromosomal MR2 and MR3 DNA but not M200H DNA indicated that SVTS2 DNA had integrated into the genomes of MR2 and MR3 but not of M200H. When MR3 extrachromosomal ds DNA containing a 2.1-kb SVTS2 DNA fragment was transfected into M200H, the transformed spiroplasmas were resistant to SVTS2. These results suggest that SVTS2 DNA fragments, possibly integrated into the chromosomal or extrachromosomal DNA of a previously susceptible spiroplasma, may function as viral incompatibility elements, providing resistance to superinfection by SVTS2. PMID:16535161

  14. Detection and Validation of QTL Affecting Bacterial Cold Water Disease Resistance in Rainbow Trout Using Restriction-Site Associated DNA Sequencing

    PubMed Central

    Gao, Guangtu; Liu, Sixin; Hernandez, Alvaro G.; Rexroad, Caird E.

    2015-01-01

    Bacterial cold water disease (BCWD) causes significant economic loss in salmonid aquaculture. Using microsatellite markers in a genome scan, we previously detected significant and suggestive QTL affecting phenotypic variation in survival following challenge with Flavobacterium psychrophilum, the causative agent of BCWD in rainbow trout. In this study, we performed selective genotyping of SNPs from restriction-site associated DNA (RAD) sequence data from two pedigreed families (2009070 and 2009196) to validate the major QTL from the previous work and to detect new QTL. The use of RAD SNPs in the genome scans increased the number of mapped markers from ~300 to ~5,000 per family. The significant QTL detected in the microsatellites scan on chromosome Omy8 in family 2009070 was validated explaining up to 58% of the phenotypic variance in that family, and in addition, a second QTL was also detected on Omy8. Two novel QTL on Omy11 and 14 were also detected, and the previously suggestive QTL on Omy1, 7 and 25 were also validated in family 2009070. In family 2009196, the microsatellite significant QTL on Omy6 and 12 were validated and a new QTL on Omy8 was detected, but none of the previously detected suggestive QTL were validated. The two Omy8 QTL from family 2009070 and the Omy12 QTL from family 2009196 were found to be co-localized with handling and confinement stress response QTL that our group has previously identified in a separate pedigreed family. With the currently available data we cannot determine if the co-localized QTL are the result of genes with pleiotropic effects or a mere physical proximity on the same chromosome segment. The genetic markers linked to BCWD resistance QTL were used to query the scaffolds of the rainbow trout reference genome assembly and the QTL-positive scaffold sequences were found to include 100 positional candidate genes. Several of the candidate genes located on or near the two Omy8 QTL detected in family 2009070 suggest potential linkages between stress response and the regulation of immune response in rainbow trout. PMID:26376182

  15. Differences in the Ovine HSP90AA1 Gene Expression Rates Caused by Two Linked Polymorphisms at Its Promoter Affect Rams Sperm DNA Fragmentation under Environmental Heat Stress Conditions

    PubMed Central

    González, Carmen; Pérez-Guzmán, M. Dolores; Garde, J. Julián; García-Álvarez, Olga; Maroto-Morales, Alejandro; Calvo, Jorge H.; Serrano, M. Magdalena

    2015-01-01

    Heat shock (HS) is one of the best-studied exogenous cellular stresses. Almost all tissues, cell types, metabolic pathways and biochemical reactions are affected in greater or lesser extent by HS. However, there are some especially thermo sensible cellular types such as the mammalian male germ cells. The present study examined the role of three INDELs in conjunction with the -660G/C polymorphism located at the HSP90AA1 promoter region over the gene expression rate under HS. Specially, the -668insC INDEL, which is very close to the -660G/C transversion, is a good candidate to be implied in the transcriptional regulation of the gene by itself or in a cooperative way with this SNP. Animals carrying the genotype II-668 showed higher transcription rates than those with ID-668 (FC = 3.07) and DD-668 (FC = 3.40) genotypes for samples collected under HS. A linkage between gene expression and sperm DNA fragmentation was also found. When HS conditions were present along or in some stages of the spermatogenesis, alternative genotypes of the -668insC and -660G/C mutations are involved in the effect of HS over sperm DNA fragmentation. Thus, unfavorable genotypes in terms of gene expression induction (ID-668GC-660 and DD-668GG-660) do not produce enough mRNA (stored as messenger ribonucleoprotein particles) and Hsp90? protein to cope with future thermal stress which might occur in posterior stages when transcriptional activity is reduced and cell types and molecular processes are more sensible to heat (spermatocytes in pachytene and spermatids protamination). This would result in the impairment of DNA packaging and the consequent commitment of the events occurring shortly after fertilization and during embryonic development. In the short-term, the assessment of the relationship between sperm DNA fragmentation sensitivity and ram’s fertility will be of interest to a better understanding of the mechanisms of response to HS and its consequences on animal production and reproduction performance. PMID:25671615

  16. 9 CFR 121.13 - Restricted experiments. 10

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... publication, “NIH Guidelines for Research Involving Recombinant DNA Molecules.” This document is available on.... (b) Restricted experiments: (1) Experiments utilizing recombinant DNA that involve the deliberate... medicine, or agriculture. (2) Experiments involving the deliberate formation of recombinant DNA...

  17. 9 CFR 121.13 - Restricted experiments. 10

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... publication, “NIH Guidelines for Research Involving Recombinant DNA Molecules.” This document is available on.... (b) Restricted experiments: (1) Experiments utilizing recombinant DNA that involve the deliberate... medicine, or agriculture. (2) Experiments involving the deliberate formation of recombinant DNA...

  18. 9 CFR 121.13 - Restricted experiments. 10

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... publication, “NIH Guidelines for Research Involving Recombinant DNA Molecules.” This document is available on.... (b) Restricted experiments: (1) Experiments utilizing recombinant DNA that involve the deliberate... medicine, or agriculture. (2) Experiments involving the deliberate formation of recombinant DNA...

  19. DNA Fingerprinting of Pearls to Determine Their Origins

    PubMed Central

    Meyer, Joana B.; Cartier, Laurent E.; Pinto-Figueroa, Eric A.; Krzemnicki, Michael S.; Hänni, Henry A.; McDonald, Bruce A.

    2013-01-01

    We report the first successful extraction of oyster DNA from a pearl and use it to identify the source oyster species for the three major pearl-producing oyster species Pinctada margaritifera, P. maxima and P. radiata. Both mitochondrial and nuclear gene fragments could be PCR-amplified and sequenced. A polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay in the internal transcribed spacer (ITS) region was developed and used to identify 18 pearls of unknown origin. A micro-drilling technique was developed to obtain small amounts of DNA while maintaining the commercial value of the pearls. This DNA fingerprinting method could be used to document the source of historic pearls and will provide more transparency for traders and consumers within the pearl industry. PMID:24130725

  20. Boron Clusters as a Platform for New Materials: Synthesis of Functionalized o-Carborane (C2 B10 H12 ) Derivatives Incorporating DNA Fragments.

    PubMed

    Janczak, Slawomir; Olejniczak, Agnieszka; Balaba?ska, Sandra; Chmielewski, Marcin K; Lupu, Marius; Viñas, Clara; Lesnikowski, Zbigniew J

    2015-10-19

    A synthetic strategy for functionalization of the three vertices of o-carborane and the attachment of the obtained triped to the solid support was developed. Further functionalization of the triped with short DNA sequences by automated DNA synthesis was achieved. The proposed methodology is a first example of boron cluster chemistry on a solid support opening new perspectives in boron cluster functionalization. PMID:26346614

  1. A human systemic lupus erythematosus-related anti-cardiolipin/single-stranded DNA autoantibody is encoded by a somatically mutated variant of the developmentally restricted 51P1 V[sub H] gene

    SciTech Connect

    Van Es, J.H.; Aanstoot, H.; Gmelig-Meyling, F.H.J.; Derksen, R.H.W.M.; Logtenberg, T. )

    1992-09-15

    The authors report the Ig H and L chain V region sequences from the cDNAs encoding a monoclonal human IgG anti-cardiolipin/ssDNA autoantibody (R149) derived from a patient with active SLE. Comparison with the germ-line V-gene repertoire of this patient revealed that R149 likely arose as a consequence of an Ag-driven selection process. The Ag-binding portions of the V regions were characterized by a high number of arginine residues, a property that has been associated with anti-dsDNA autoantibodies from lupus-prone mice and patients with SLE. The V[sub H] gene encoding autoantibody R149 was a somatically mutated variant of the 51P1 gene segment, which is frequently associated with the restricted fetal B cell repertoire, malignant CD5 B cells, and natural antibodies. These data suggest that in SLE patients a common antigenic stimulus may evoke anti-DNA and anti-cardiolipin autoantibodies and provide further evidence that a small set of developmentally restricted V[sub H] genes can give rise to disease-associated autoantibodies through Ag-selected somatic mutations. 42 refs., 5 figs.

  2. Comparison of a semiautomated commercial repetitive-sequence-based PCR method with spoligotyping, 24-locus mycobacterial interspersed repetitive-unit-variable-number tandem-repeat typing, and restriction fragment length polymorphism-based analysis of IS6110 for Mycobacterium tuberculosis typing.

    PubMed

    Brossier, F; Sola, C; Millot, G; Jarlier, V; Veziris, N; Sougakoff, W

    2014-11-01

    Fifty-two multidrug-resistant isolates of Mycobacterium tuberculosis representative of the currently predominant lineages in France were analyzed using repetitive-sequence-based PCR (rep-PCR) DiversiLab (DL), spoligotyping, 24-locus mycobacterial interspersed repetitive-unit-variable-number tandem-repeat typing (MIRU-VNTR), and restriction fragment length polymorphism of IS6110 (IS6110-RFLP). DL, as opposed to MIRU-VNTR and IS6110-RFLP analysis, did not allow discrimination among half of the isolates, an indication of comparatively lower resolving power. PMID:25210067

  3. Induction of chromosome aberrations and mutations by NciI (CC{r_arrow}(C/G)GG), a restrictive enzyme that produces DNA double-strand breaks with 3{prime}-phosphorylated ends

    SciTech Connect

    Winegar, R.A.; Suing, K.R.; Hamer, J.D.; Lutze, L.H.

    1994-12-31

    Restriction enzymes introduced into cells have provided a simplified model of DNA damage produced by ionizing radiation. A drawback of restriction enzymes is that they produce clean, easily ligatable ends (i.e. 5{prime}-P and 3{prime}-OH). Nci I is unique among known restriction enzymes since it produces breaks with 3{prime}-P ends. These ends are nearly impossible to religate in vitro, but characterize a major fraction of DSB induced by ionizing radiation. Therefore, the use of Nci I may provide additional information that cannot be provided by standard restriction enzymes. We have begun studies that analyze the induction of chromosome aberrations and mutations by Nci I in the presence and absence of 3-aminobenzamide (3-AB), which is an inhibitor of poly(ADP-ribosyl)ation. Nci I introduced into CHO cells induced both chromosome- and chromatid-type aberrations. In contrast to other restriction enzymes, Nci I did not induce a high level of interstitial deletions. This may reflect a slower ligational repair of Nci I-induced DSB since it is likely that DSB containing 3{prime}-P ends must be modified prior to ligation, perhaps through excision repair mechanisms. 3-AB caused a marked increase in chromosome aberration induction by Nci I. However, in contrast to findings with other restriction enzymes, most of the increase was in chromatid exchanges rather than interstitial deletions. Nci I also induced a large increase in mutations in the pHAZE shuttle vector, which is maintained in a human lymphoblastoid cell line. These mutations will be sequenced to help determine the mechanisms by which cells deal the 3{prime}-P ends.

  4. Natural interspecies transfer of mitochondrial DNA in amphibians.

    PubMed Central

    Spolsky, C; Uzzell, T

    1984-01-01

    mtDNAs of two Central European water frog species, Rana ridibunda and Rana lessonae, were examined by electrophoresis of restriction enzyme fragments. Two types of mtDNA occur in R. ridibunda. One shares with mtDNA of R. lessonae 25.8% of 132 fragments generated by 19 enzymes, corresponding to a nucleotide sequence divergence of 8.1%; the other has diverged from R. lessonae mtDNA by only 0.3%. This latter type is a variant R. lessonae mtDNA that has been transferred into R. ridibunda; the introgression may have occurred via the hybridogenetic hybrid lineages collectively known as Rana esculenta. Of 37 R. ridibunda from Poland, 59% had the typical R. ridibunda mtDNA; 41% had the modified R. lessonae mtDNA as did a single individual from Switzerland (introduced). A single R. ridibunda from Turkey, outside the present range of R. lessonae, had the typical R. ridibunda mtDNA phenotype. Discordancies between inheritance of mitochondrial and nuclear genomes point up the danger of relying on a single molecular feature in reconstructing phylogeny. In addition, studies of mtDNA provide otherwise inaccessible information on complex evolutionary histories of closely related species. A knowledge of these complexities is important to an understanding of phylogenetic relationships and of the genetic processes that underlie the evolution of clonal taxa. Images PMID:6091109

  5. [Cloning-idependent mapping technology for genomic fidelity, contig linking, C-DNA site analysis, and gene detection]. Final report

    SciTech Connect

    Lerman, L.S.

    1994-12-25

    The project was designed to develop and apply a novel unconventional approach to genome mapping based on physical properties of DNA that are a sensitive function of the base sequence, and so does not depend on the clonability of the sequences to be mapped nor on the presence of particular restriction sites. We have shown that a broad array of DNA fragments are retarded at nearly the same level in denaturing gradient gel electrophoresis (DGGE) if the segment with the lowest thermal stability has the same melting temperature, regardless of the length of the fragment. The retarded pattern remain steady in the gel, changing little with continued field exposure. Mapping proceeds by the analysis of two-dimensional patterns produced by random fragmentation of genomic DNA and denaturing gradient gel electrophoresis. Random fragments are first separated according to length by conventional agarose electrophoresis. The result is a two- dimensional pattern which can be idealized as an array of nearly parallel, mostly separated lines of DNA. The pattern is blotted onto a membrane and probed sequentially with oligos or relevant DNA or RNA fragments. The endpoints on the fragment length scale of each line hybridizing with each probe, the distribution along each line, and the depth in the gradient constitute specific map information.

  6. Matrix-induced fragmentation of P3?-N5? phosphoramidate-containing DNA: high-throughput MALDI-TOF analysis of genomic sequence polymorphisms

    PubMed Central

    Shchepinov, Mikhail S.; Denissenko, Mikhail F.; Smylie, Kevin J.; Wörl, Ralf J.; Leppin, A. Lorieta; Cantor, Charles R.; Rodi, Charles P.

    2001-01-01

    Chemical and enzymatic approaches were used to produce polynucleotide fragments containing acid-labile internucleotide P3?-N5? phosphoramidate bonds, either in a surface-bound form or in solution. The primer extension reaction utilizing 5?-amino-5?-deoxynucleoside 5?-triphosphates generates polynucleotides that can be fragmented into short, easy-to-analyze pieces simply by being premixed with the acidic matrices typically used for MALDI-TOF mass spectrometry of nucleic acids. This leads to detection procedures that are simple, robust and easy to automate. Utilizing this approach, a polymorphic site in the human ADRB3 gene was interrogated. Primer extensions with phosphoramidate analogs of dNTPs allowed for unambiguous discrimination of all possible genotypes. PMID:11557819

  7. Matrix-induced fragmentation of P3'-N5' phosphoramidate-containing DNA: high-throughput MALDI-TOF analysis of genomic sequence polymorphisms.

    PubMed

    Shchepinov, M S; Denissenko, M F; Smylie, K J; Wörl, R J; Leppin, A L; Cantor, C R; Rodi, C P

    2001-09-15

    Chemical and enzymatic approaches were used to produce polynucleotide fragments containing acid-labile internucleotide P3'-N5' phosphoramidate bonds, either in a surface-bound form or in solution. The primer extension reaction utilizing 5'-amino-5'-deoxynucleoside 5'-triphosphates generates polynucleotides that can be fragmented into short, easy-to-analyze pieces simply by being premixed with the acidic matrices typically used for MALDI-TOF mass spectrometry of nucleic acids. This leads to detection procedures that are simple, robust and easy to automate. Utilizing this approach, a polymorphic site in the human ADRB3 gene was interrogated. Primer extensions with phosphoramidate analogs of dNTPs allowed for unambiguous discrimination of all possible genotypes. PMID:11557819

  8. DNA vaccines targeting heavy chain C-terminal fragments of Clostridium botulinum neurotoxin serotypes A, B, and E induce potent humoral and cellular immunity and provide protection from lethal toxin challenge.

    PubMed

    Scott, Veronica L; Villarreal, Daniel O; Hutnick, Natalie A; Walters, Jewell N; Ragwan, Edwin; Bdeir, Khalil; Yan, Jian; Sardesai, Niranjan Y; Finnefrock, Adam C; Casimiro, Danilo R; Weiner, David B

    2015-01-01

    Botulinum neurotoxins (BoNTs) are deadly, toxic proteins produced by the bacterium Clostridium botulinum that can cause significant diseases in humans. The use of the toxic substances as potential bioweapons has raised concerns by the Centers for Disease Control and Prevention and the United States Military. Currently, there is no licensed vaccine to prevent botulinum intoxication. Here we present an immunogenicity study to evaluate the efficacy of novel monovalent vaccines and a trivalent cocktail DNA vaccine targeting the heavy chain C-terminal fragments of Clostridium botulinum neurotoxin serotypes A, B, and E. These synthetic DNA vaccines induced robust humoral and polyfunctional CD4(+) T-cell responses which fully protected animals against lethal challenge after just 2 immunizations. In addition, naïve animals administered immunized sera mixed with the lethal neurotoxin were 100% protected against intoxication. The data demonstrate the protective efficacy induced by a combinative synthetic DNA vaccine approach. This study has importance for the development of vaccines that provide protective immunity against C. botulinum neurotoxins and other toxins. PMID:26158319

  9. Neurospora ribosomal DNA sequences are indistinguishable within cell types but distinguishable among heterothallic species

    SciTech Connect

    Chambers, C.; Dutta, S.K.

    1983-01-01

    High molecular nuclear DNAs were isolated from three developmental cell types of N. crassa: conidia, mycelia and germinated conidia, and from mycelial cells of two other heterothallic species, N. intermedia and N. sitophila. These nuclear DNAs were treated with several restriction enzymes: EcoR1, Bam H1, Hind III, Hinc II, Bgl II, Sma I and Pst 1. All seven restriction enzymes were tested on 0.7% agarose gels. EcoR1, Hind III, Pst 1, and Hinc II showed band differences among the species, but not among the cell types. Southern blot transfers of restricted DNA gels were then hybridized with TSP-labelled pMF2 rDNAs (probe). This later DNA was prepared from N. crassa rDNA cloned into pBR322 plasmid, obtained from Dr. Robert Metzenberg of the University of Wisconsin. Autoradiograms of these hybrids between southern blots and probe DNA revealed similar rDNA band patterns confirming the observations on restriction gels. In the case of EcoR1 restriction analysis there were differences in fragments on 0.7% agarose gel, but after hybridization of southern blots no differences in band patterns were seen in autoradiograms. This raises the question whether the background bands were all of rDNA sequences. These studies are being continued using ITS (internal transcribed spacer) sequences of N. crassa rDNAs cloned in pBR322 plasmid.

  10. Understanding the genetic effects of recent habitat fragmentation in the context of evolutionary history: Phylogeography and landscape genetics of a southern California endemic Jerusalem cricket (Orthoptera: Stenopelmatidae: Stenopelmatus)

    USGS Publications Warehouse

    Vandergast, A.G.; Bohonak, A.J.; Weissman, D.B.; Fisher, R.N.

    2007-01-01

    Habitat loss and fragmentation due to urbanization are the most pervasive threats to biodiversity in southern California. Loss of habitat and fragmentation can lower migration rates and genetic connectivity among remaining populations of native species, reducing genetic variability and increasing extinction risk. However, it may be difficult to separate the effects of recent anthropogenic fragmentation from the genetic signature of prehistoric fragmentation due to previous natural geological and climatic changes. To address these challenges, we examined the phylogenetic and population genetic structure of a flightless insect endemic to cismontane southern California, Stenopelmatus 'mahogani' (Orthoptera: Stenopelmatidae). Analyses of mitochondrial DNA sequence data suggest that diversification across southern California began during the Pleistocene, with most haplotypes currently restricted to a single population. Patterns of genetic divergence correlate with contemporary urbanization, even after correcting for (geographical information system) GIS-based reconstructions of fragmentation during the Pleistocene. Theoretical simulations confirm that contemporary patterns of genetic structure could be produced by recent urban fragmentation using biologically reasonable assumptions about model parameters. Diversity within populations was positively correlated with current fragment size, but not prehistoric fragment size, suggesting that the effects of increased drift following anthropogenic fragmentation are already being seen. Loss of genetic connectivity and diversity can hinder a population's ability to adapt to ecological perturbations commonly associated with urbanization, such as habitat degradation, climatic changes and introduced species. Consequently, our results underscore the importance of preserving and restoring landscape connectivity for long-term persistence of low vagility native species. Journal compilation ?? 2006 Blackwell Publishing Ltd.

  11. Rapid restriction mapping of cosmids by sequence-specific triple-helix-mediated affinity capture

    SciTech Connect

    Ji, Huamin; Francisco, T.; Smith, L.M.; Guilfoyle, R.A.

    1996-01-15

    A simple and rapid strategy for restriction mapping based on sequence-specific triple-helix affinity capture (TAC) was developed. The strategy was applied to the analysis of cosmid clones by the construction of a new cosmid vector, ScosTriplex-II, containing two different triple-helix-forming sequences flanking the cloning site of the original SuperCos-1 cosmid vector. For restriction mapping, the recombinant cosmid DNA is digested with NotI restriction enzyme or with one of four intron-encoded endonucleases for excision of intact inserts followed by controlled partial digestion with a mapping enzyme used in conjunction with the corresponding methyltransferase. The partial digestion products are combined with biotinylated triple-helix-forming oligonucleotides to form a triple-helical complex. The triple-helix complexes are immobilized on streptavidin-coated magnetic beads, washed, and eluted with pH 9 buffer solution. The fragments are separated and directly sized by agarose gel electrophoresis. Bidirectional maps are obtained simultaneously by binding to the two different triple-helix-forming oligonucleotides. No probe labeling, gel drying, blotting to membranes, hybridization, or autoradiography is necessary. Also, TAC conditions that permit gel-free isolation of the terminal restriction fragments from cosmid inserts were found. These advantages afforded by ScosTriplex-II should facilitate the automation of cosmid restriction site fingerprinting needed for large-scale mapping and sequencing projects. 24 refs., 5 figs.

  12. Construction and characterization of human and mouse BAC libraries from sheared DNA

    SciTech Connect

    Shizuya, Hiroaki

    2002-08-23

    We have developed a new way to construct BAC libraries with small inserts using sheared DNA sources. Because of our use of the randomly sheared DNA as DNA sources, some regions of genome may be represented better in our libraries compared to the currently available and more conventional libraries constructed by enzymatic partial digestion. B263 We have developed a new fingerprinting method useful for physical mapping by large insert clones, in particular by BACs. It is based on four-color fluorescent labeling of fragments generated by combination of a type II and a type IIS restriction enzyme.

  13. Genetic variation in chloroplast and nuclear ribosomal DNA in Utah juniper (Juniperus osteosperma, Cupressaceae): evidence for interspecific gene flow.

    PubMed

    Terry, R G; Nowak, R S; Tausch, R J

    2000-02-01

    Geographic patterns of genetic variation in chlorolast (cpDNA) and nuclear ribosomal (nrDNA) DNA were examined to test the hypothesis of hybridization between Juniperus osteosperma and Juniperus occidentalis in the Great Basin of western Nevada. Noncoding DNA from the trnL-trnF intergenic spacer and the trnL intron of the chloroplast genome was sequenced from seven populations of J. osteosperma and four populations of J. occidentalis sampled over a large proportion of their respective ranges. An adenine nucleotide at position 436 in the aligned sequence and within a Tru 9I restriction site was found to be present in individuals of J. osteosperma sampled from western Colorado and central Utah, but absent in sequences of J. osteosperma sampled from central and western Nevada and all sequences of J. occidentalis. Two hundred fourteen individuals from 34 populations of J. osteosperma and J. occidentalis were then screened for cpDNA haplotype by Tru 9I digestion of the trnL-trnF polymerase chain reaction (PCR) product. Two cpDNA haplotypes were evident, each consisting of restriction fragment profiles that differed solely with respect to the presence or absence of the Tru 9I site encompassing the adenine nucleotide at position 436. One of these haplotypes was monomorphic in J. occidentalis and exhibited a decreasing frequency in J. osteosperma with increasing geographic distance from J. occidentalis in west-central Nevada. Geographic patterns in nuclear ribosomal DNA (nrDNA) variation were examined by restriction fragment analysis and, although spatially more restricted, exhibited patterns of clinal variation similar to those observed in cpDNA haplotype. Genetic relationships based on DNA sequences and geographic patterns of genetic variation in chloroplast and nuclear ribosomal DNA are consistent with morphology in suggesting interspecific gene flow between J. occidentalis and J. osteosperma. PMID:10675313

  14. Generating a long DNA fragment of the target ncRNA for quantitative polymerase chain reaction by combining ncRNA-oligos hybridization and oligos ligation.

    PubMed

    Zeng, Zhen; Zhou, Zheng; Wang, Dan; Wang, Zitian; Yang, Huali; Luo, Jianjun; Chen, Run-Sheng

    2016-01-10

    The poor reproducibility of the reverse transcription combined with quantitative polymerase chain reaction (RT-qPCR) results in an unacceptable reliability of publications based on these data. We established a novel method, in which two short complementary DNA oligos were hybridized with target ncRNA molecules and linked by DNA ligase to obtain a long DNA strand (HL-DNA) replacing cDNA for qPCR detection (HL-qPCR). A series of diluted samples prepared from the same total RNA resource were measured by HL-qPCR and RT-qPCR respectively to acquire their relative concentration of RNU4-1, AK026510 and SNORA73B. For every tested sample, the relative concentration of RNU4-1, AK026510 and SNORA73B obtained by HL-qPCR instead of RT-qPCR is closer to its corresponding true value without significant difference, demonstrating that HL-qPCR exhibits higher accuracy compared with RT-qPCR. With three independent repeats, no significant difference was observed among AK026510/RNU4-1 values of four samples diluted from the same RNA resource, by employing HL-qPCR but not RT-qPCR. It strongly suggests that the good reproducibility of HL-qPCR results from the stable efficiency of HL-DNA production regardless of the concentration and individual features of ncRNA. The novel HL-qPCR could be applied for the regular relative ncRNA concentration detection in the future. PMID:26593981

  15. Fragmentation of solids under impulsive stress loading

    SciTech Connect

    Grady, D.E.

    1981-02-10

    An analysis of fragmentation due to dynamic stress loading is presented which provides analytic functions for the distributions in fragment sizes. The analysis is restricted to one-dimensional bodies under uniform tensile loading. Concepts of survival statistics are used to account for spatially random fracture nucleation. Fragment size distribution curves for both brittle and ductile fracture are derived, and the curve for the latter is compared with experimental data. Fragment distribution curves are shown to depend on both material deformation properties and loading conditions.

  16. Development of DNA probes for Candida albicans

    SciTech Connect

    Cheung, L.L.; Hudson, J.B.

    1988-07-01

    An attempt was made to produce DNA probes that could be used as a rapid and efficient means of detecting candidiasis (invasive Candida infection) in immunocompromised patients. Whole DNA from Candida albicans was digested with restriction endonuclease, and the resulting fragments were randomly cloned into a plasmid vector. Several recombinant plasmids were evaluated for cross-hybridization to various other C