Sample records for dna restriction fragment

  1. Monodisperse DNA restriction fragments

    Microsoft Academic Search

    Karel L. Planken; Gijsberta H. Koenderink; Ramon Roozendaal; Albert P. Philipse

    2005-01-01

    We present a convenient and low-cost method to prepare milligram amounts of completely monodisperse DNA restriction fragments in a physico-chemical laboratory setting to study (in part II) the effect of limited flexibility on the concentration dependent sedimentation velocity. Four fragments of 200, 400, 800, and 1600 bp were designed to span a range of 1–11 persistence lengths. The fragments were

  2. Separation of DNA restriction fragments using capillary electrophoresis

    SciTech Connect

    Chan, K.C.; Whang, Chenwen; Yeung, E.S. (Iowa State Univ., Ames (United States))

    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.

  3. DNA Restriction

    NSDL National Science Digital Library

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

  4. Changes in restricted human cellular DNA fragments containing globin gene sequences in thalassemias and related disorders

    PubMed Central

    Mears, J. Gregory; Ramirez, Francesco; Leibowitz, David; Nakamura, Frank; Bloom, Arthur; Konotey-Ahulu, Felix; Bank, Arthur

    1978-01-01

    Human cellular DNA fragments from cells of normal subjects and patients with thalassemia obtained by restriction enzyme digestion were analyzed for their globin gene content. The fragments were separated on agarose gels, transferred to nitrocellulose filters, hybridized to globin [32P]cDNA, and radioautographed. One to ten picograms of globin gene sequences were detectable. With EcoRI digestion, eight to nine cellular DNA fragments were found to contain globin genes. Three of these contained ?-like gene sequences assayed with ? globin cDNA probe. One ?-like fragment was absent in DNA from a homozygous subject for hemoglobin Lepore. Two of the three ? gene-containing fragments present in normal DNA were absent in DNA from a patient with hereditary persistence of fetal hemoglobin. The same two fragments containing ?-like genes were absent from ?? thalassemic DNA and one new fragment containing ?-like genes was found. Together with results obtained by hybridization of these DNAs in solution, the data are consistent with deletion of specific restriction human DNA fragments in subjects with these disorders and a greater deletion of ?-like gene sequences in subjects with hereditary persistence of fetal hemoglobin than in those with ?? thalassemia. Images PMID:274714

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

    DOEpatents

    Wong, Kwong-Kwok (Richland, WA)

    2000-01-01

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

  6. Effect of magnesium ions and temperature on the sequence-dependent curvature of DNA restriction fragments

    PubMed Central

    Stellwagen, Nancy C.; Lu, Yongjun

    2011-01-01

    Transient electric birefringence has been used to quantitate the curvature of two DNA restriction fragments, a 199-base pair fragment taken from the origin of replication of the M13 bacteriophage and a 207-base pair fragment taken from the VP1 gene in the SV40 minichromosome. Stable curvature in the SV40 and M13 restriction fragments is due a series of closely spaced A-tracts, runs of 4 to 6 contiguous adenine residues located within 40 or 60 base pair “curvature modules” near the center of each fragment. The M13 and SV40 restriction fragments exhibit bends of ~45° in solutions containing monovalent cations and ~58° in solutions containing Mg2+ ions. The curvature is not localized at a single site but is distributed over the various A-tracts in the curvature modules. Thermal denaturation studies indicate that the curvature in the M13 and SV40 restriction fragments remains constant up to 30°C in solutions containing monovalent cations, and up to 40°C in solutions containing Mg2+ ions, before beginning to decrease slowly with increasing temperature. Hence, stable curvature in these DNA restriction fragments exists at the biologically important temperature of 37°C. PMID:21406776

  7. 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. (Univ. of Texas Health Science Center, Houston, TX (United States)); Budowle, B. (FBI Academy, Quantico, VA (United States))

    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.

  8. Comparison of DNA restriction fragment length polymorphisms of Nostoc strains in and from cycads

    Microsoft Academic Search

    Peter Lindblad; Robert Haselkorn; Birgitta Bergman; Sandra A. Nierzwicki-Bauer

    1989-01-01

    DNA was prepared from cyanobacteria freshly isolated from coralloid roots of natural populations of five cycad species: Ceratozamia mexicana mexicana (Mexico), C. mexicana robusta (Mexico), Dioon spinulosum (Mexico), Zamia furfuraceae (Mexico) and Z. skinneri (Costa Rica). Using the Southern blot technique and cloned Anabaena PCC 7120 nifK and glnA genes as probes, restriction fragment length polymorphisms of these cyanobacterial symbionts

  9. Detection of herpes simplex virus type-2 DNA restriction fragments in human cervical carcinoma tissue.

    PubMed Central

    Park, M; Kitchener, H C; Macnab, J C

    1983-01-01

    DNA extracted from eight human cervical carcinomas, one lymph node metastasis and related control tissue was examined for the presence of herpes simplex virus (HSV) DNA sequences. Southern blot transfers of tumour and control DNA were hybridised with radioactively labelled cloned probes representing 70% of the HSV-2 genome. Specific hybridisation to HSV DNA sequences was observed in one of eight carcinoma tissues analysed. Hybridisation of HSV-2 DNA probes to BamHI and XhoI restriction enzyme fragments of tumour cell DNA which co-migrated with authentic HSV-2 viral fragments identified co-linear HSV-2 DNA sequences comprising 3% of the HSV-2 genome, between map coordinates 0.582 and 0.612. The remaining eight tumour and all control tissues analysed, showed no specific hybridisation to any of the probes used at levels of sensitivity which would detect 0.5 copies/cell of HSV-2 DNA restriction fragments of 2 kb or greater. Images Fig. 2. Fig. 3. Fig. 5. PMID:6313349

  10. Correlation between DNA restriction fragment length polymorphisms in Leptospira interrogans serovar pomona type kennewicki and host animal source.

    PubMed Central

    Bolin, C A; Zuerner, R L

    1996-01-01

    Isolates (n = 147) of Leptospira interrogans serovar pomona type kennewicki from cattle, swine, horses, and wildlife were analyzed by DNA restriction endonuclease analysis. Restriction fragment length polymorphisms were identified in DNA digested with HpaII, and the restriction fragment length polymorphisms were correlated with the host animal source of the isolates. These results will be useful in understanding the epidemiology of serovar pomona infections in livestock. PMID:8789028

  11. Chloroplast DNA variation in Populus . I. Intraspecific restriction fragment diversity within Populus deltoides , P. nigra and P. maximowiczii

    Microsoft Academic Search

    O. P. Rajora; B. P. Dancik

    1995-01-01

    We examined intraspecific chloroplast (cp) DNA variation within Populus deltoides, P. nigra, and P. maximowiczii by restriction fragment analysis using 16 restriction endonucleases and six heterologous probes of cloned Petunia cpDNA fragments. All three Populus species showed intraspecific cpDNA variation, which was intra- and inter-varietal in P. deltoides, intervarietal in P. nigra, and origin-specific in P. maximowiczii. Two varieties of

  12. Native fluorescence detection of nucleic acids and DNA restriction fragments in capillary electrophoresis

    SciTech Connect

    Milofsky, R.E.; Yeung, E.S. (Ames Laboratory, IA (United States))

    1993-01-15

    A sensitive laser-induced fluorescence (LIF) detection scheme for native nucleic acids and DNA restriction fragments separated by capillary electrophoresis (CE) has been developed. The 275.4-nm line from an argon ion laser or the 248-nm line from a waveguide KrF laser is used to excite native fluorescence. Detection limits for guanosine and adenosine monophosphate (1.5 [times] 10[sup [minus]8] and 5 [times] 10[sup [minus]8] M, respectively) are up to 3 orders of magnitude lower than UV detection. Sensitivity for native fluorescence of DNA restriction fragments in gel-filled capillaries rivals that of UV absorption. The decrease in performance in gel-filled separations using LIF detection is caused by the high background associated with gel fluorescence, as well as gel quenching of the fluorescence emission. The development of gels exhibiting lower background fluorescence or off-column coupling should lead to significant improvements in sensitivity over UV detection. This novel and practical system enables, for the first time, the sensitive detection of nucleic-acid-containing compounds without the need for fluorescence labeling. 48 refs., 4 figs., 1 tab.

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

    PubMed

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

    1990-01-01

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

  14. A comparison of morphology, pathogenicity and restriction fragment patterns of mitochondrial DNA among isolates of Phytophthora porri Foister

    Microsoft Academic Search

    Arthur W. A. M. De Cock; Afra Neuvel; Günther Bahnweg; Johanna C. J. M. De Cock; Hermann H. Prell

    1992-01-01

    Thirteen strains ofPhytophthora porri from five different hosts were compared with respect to their morphology, cardinal temperatures for growth, pathogenicity to leek and cabbage and restriction fragment patterns of mitochondrial DNA. Morphology of vegetative growth was rather similar in most isolates. Those characters which differed among isolates showed overlapping variability and could not be used to distinguish groups, with the

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

    E-print Network

    Barron, Annelise E.

    Capillary Electrophoretic Separation of DNA Restriction Fragments in Mixtures of Low- and High-Molecular-Weight separation by CE in HEC solutions is strongly influenced by the average HEC molecular weight as well of the effects of a mixture of low- and high-molecular weight HEC polymers, over a range of concentrations

  16. 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.

  17. Intrageneric relationships of maple trees based on the chloroplast DNA restriction fragment length polymorphisms

    Microsoft Academic Search

    Mitsuyasu Hasebe; Toshio Ando; Kunio Iwatsuki

    1998-01-01

    A maple tree genus,Acer is the largest genus in broad-leaved deciduous trees and contains about 200 species. A delimitation of the genus is clear\\u000a but the intrageneric classification was controversial because of homoplasies in morphological characters. In this study, a\\u000a phylogenetic relationship inAcer was inferred based on chloroplast DNA restriction site polymorphisms with 17 restriction endonucleases and previously proposed\\u000a intrageneric

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

    PubMed Central

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

    1985-01-01

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

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

    PubMed Central

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

    1986-01-01

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

  20. Genetic markers in ribosomal DNA for the identification of members of the genus Anisakis (Nematoda: Ascaridoidea) defined by polymerase-chain-reaction-based restriction fragment length polymorphism

    Microsoft Academic Search

    S D'Amelio; K. D Mathiopoulos; C. P Santos; O. N Pugachev; S. C Webb; M Picanço; L Paggi

    2000-01-01

    Polymerase-chain-reaction-based restriction fragment length polymorphism analysis was performed to establish genetic markers in rDNA, for the identification of the three sibling species of the Anisakis simplex complex and morphologically differentiated Anisakis species, i.e. Anisakis physeteris, Anisakis schupakovi, Anisakis typica and Anisakis ziphidarum. Different restriction patterns were found between A. simplex sensu stricto and Anisakis pegreffii with two of the restriction

  1. Restriction Fragment Length Polymorphism Analysis of Ribosomal DNA Intergenic Regions Is Useful for Differentiating Strains of Trichophyton mentagrophytes

    PubMed Central

    Mochizuki, Takashi; Ishizaki, Hiroshi; Barton, Richard C.; Moore, Mary K.; Jackson, Colin J.; Kelly, Steven L.; Evans, E. Glyn V.

    2003-01-01

    Twenty isolates of Tricophyton mentagrophytes var. mentagrophytes and 47 isolates of T. mentagrophytes var. interdigitale, identified by morphological characteristics, were screened by restriction fragment length polymorphism (RFLP) analysis of the PCR-amplified internal transcribed spacer (ITS) region of ribosomal DNA (rDNA). Sixty isolates (14 of 20 T. mentagrophytes var. mentagrophytes isolates and 46 of 47 T. mentagrophytes var. interdigitale isolates) shared an identical ITS RFLP profile and were further investigated by using a probe targeted to the rDNA nontranscribed spacer (NTS) region. Polymorphisms were observed in the NTS regions of both T. mentagrophytes var. mentagrophytes and T. mentagrophytes var. interdigitale isolates. Twenty-three individual RFLP patterns (DNA types P-1 to P-12 and A-1 to A-11) were recognized and divided into two groups depending on the presence (P) or absence (A) of a 2.5-kb band, which correlated to a large extent with the morphological variety. Eleven of 14 T. metagrophytes var. mentagrophytes isolates were A types, and all of the 46 T. mentagrophytes var. interdigitale isolates were P types. A majority of strains (23 of 60 [38.3%]) were characterized by one RFLP pattern (pattern P-1), and eight types (P-1 to P-6, P-8, and P-9) accounted for 75% (45 of 60) of all strains, including all of the T. mentagrophytes var. interdigitale isolates. The remaining 15 types were represented by one only isolate and included all of the T. mentagrophytes var. mentagrophytes isolates. We conclude that RFLP analysis of the rDNA NTS region is a valuable technique for differentiation of T. mentagrophytes strains. Furthermore, by use of this method, there appears to be a greater degree of diversity among T. mentagrophytes var. mentagrophytes isolates than among T. mentagrophytes var. interdigitale isolates. PMID:14532186

  2. Computer-based image analysis of one-dimensional electrophoretic gels used for the separation of DNA restriction fragments.

    PubMed Central

    Gray, A J; Beecher, D E; Olson, M V

    1984-01-01

    A stand-alone, interactive computer system has been developed that automates the analysis of ethidium bromide-stained agarose and acrylamide gels on which DNA restriction fragments have been separated by size. High-resolution digital images of the gels are obtained using a camera that contains a one-dimensional, 2048-pixel photodiode array that is mechanically translated through 2048 discrete steps in a direction perpendicular to the gel lanes. An automatic band-detection algorithm is used to establish the positions of the gel bands. A color-video graphics system, on which both the gel image and a variety of operator-controlled overlays are displayed, allows the operator to visualize and interact with critical stages of the analysis. The principal interactive steps involve defining the regions of the image that are to be analyzed and editing the results of the band-detection process. The system produces a machine-readable output file that contains the positions, intensities, and descriptive classifications of all the bands, as well as documentary information about the experiment. This file is normally further processed on a larger computer to obtain fragment-size assignments. Images PMID:6320097

  3. Trypanosoma evansi: Genetic variability detected using amplified restriction fragment length polymorphism (AFLP) and random amplified polymorphic DNA (RAPD) analysis of Kenyan isolates

    Microsoft Academic Search

    Daniel K. Masiga; Kariuki Ndung’u; Alison Tweedie; Andrew Tait; C. Michael R. Turner

    2006-01-01

    We compared two methods to generate polymorphic markers to investigate the population genetics of Trypanosoma evansi; random amplified polymorphic DNA (RAPD) and amplified restriction fragment length polymorphism (AFLP) analyses. AFLP accessed many more polymorphisms than RAPD. Cluster analysis of the AFLP data showed that 12 T.evansi isolates were very similar (‘type A’) whereas 2 isolates differed substantially (‘type B’). Type

  4. 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.

  5. Biology of DNA restriction.

    PubMed Central

    Bickle, T A; Krüger, D H

    1993-01-01

    Our understanding of the evolution of DNA restriction and modification systems, the control of the expression of the structural genes for the enzymes, and the importance of DNA restriction in the cellular economy has advanced by leaps and bounds in recent years. This review documents these advances for the three major classes of classical restriction and modification systems, describes the discovery of a new class of restriction systems that specifically cut DNA carrying the modification signature of foreign cells, and deals with the mechanisms developed by phages to avoid the restriction systems of their hosts. PMID:8336674

  6. Restriction Fragment Length Polymorphism Linkage Map for Arabidopsis thaliana

    Microsoft Academic Search

    Caren Chang; John L. Bowman; Arthur W. Dejohn; Eric S. Lander; Elliot M. Meyerowitz

    1988-01-01

    We have constructed a restriction fragment length polymorphism linkage map for the nuclear genome of the flowering plant Arabidopsis thaliana. The map, containing 90 randomly distributed molecular markers, is physically very dense; >50% of the genome is within 1.9 centimorgans, or ≈ 270 kilobase pairs, of the mapped DNA fragments. The map was based on the meiotic segregation of markers

  7. Detection of DNA polymorphisms between two inbred mouse strains--limitations of restriction fragment length polymorphisms (RFLPs).

    PubMed

    Knight, A M; Dyson, P J

    1990-12-01

    Type I (insulin-dependent) diabetes in humans is characterized by a T cell mediated destruction of insulin-secreting pancreatic beta cells. This autoimmune response is very similar to that seen in the non-obese diabetic (NOD) mouse strain. Originally bred from the ICR cataract-prone strain, NOD mice spontaneously develop T cell mediated insulitis and type I diabetes by the age of 6 months. Backcross studies with the NOD mouse strain indicate segregation of at least three recessive genes. One of these, Iddm-1, has been shown to be tightly linked to the mouse MHC, H-2 on chromosome 17. Comparative studies with diabetic patients has also shown linkage to human HLA with protective and predisposing haplotypes being present within the population. In this study we have attempted to identify restriction fragment length polymorphisms (RFLPs) between the genomes of the NOD mouse strain and the diabetes-resistant strain C57BL/10. Such polymorphic loci will be used to screen DNAs from backcross animals that are diagnosed diabetic in an attempt to identify probes linked to the non-H2 disease susceptibility genes. PMID:1982336

  8. Restriction Enzymes and DNA Fingerprinting

    NSDL National Science Digital Library

    National Science Foundation GK-12 and Research Experience for Teachers (RET) Programs,

    The discovery of restriction enzymes and their applications in DNA analysis has proven to be essential for biologists and chemists. This lesson focuses on restriction enzymes and their applications to DNA analysis and DNA fingerprinting. Use this lesson and its associated activity in conjunction with biology lessons on DNA analysis and DNA replication.

  9. Restriction fragment length polymorphisms in genetic improvement: methodologies, mapping and costs

    Microsoft Academic Search

    J. S. Beckmann; M. Soller

    1983-01-01

    Recently a new class of genetic polymorphism, restriction fragment length polymorphisms (RFLPs), has been uncovered by the use of restriction endonucleases which cleave DNA molecules at specific sites and cloned DNA probes which detect specific homologous DNA fragments. RFLPs promise to be exceedingly numerous and are expected to have genetic characteristics — lack of dominance, multiple allelic forms and absence

  10. Restriction enzyme-mediated DNA family shuffling.

    PubMed

    Behrendorff, James B Y H; Johnston, Wayne A; Gillam, Elizabeth M J

    2014-01-01

    DNA shuffling is an established recombinatorial method that was originally developed to increase the speed of directed evolution experiments beyond what could be accomplished using error-prone PCR alone. To achieve this, mutated copies of a protein-coding sequence are fragmented with DNase I and the fragments are then reassembled in a PCR without primers. The fragments anneal where there is sufficient sequence identity, resulting in full-length variants of the original gene that have inherited mutations from multiple templates. Subsequent studies demonstrated that directed evolution could be further accelerated by shuffling similar native protein-coding sequences from the same gene family, rather than mutated variants of a single gene. Generally at least 65-75 % global identity between parental sequences is required in DNA family shuffling, with recombination mostly occurring at sites with at least five consecutive nucleotides of local identity. Since DNA shuffling was originally developed, many variations on the method have been published. In particular, the use of restriction enzymes in the fragmentation step allows for greater customization of fragment lengths than DNase I digestion and avoids the risk that parental sequences may be over-digested into unusable very small fragments. Restriction enzyme-mediated fragmentation also reduces the occurrence of undigested parental sequences that would otherwise reduce the number of unique variants in the resulting library. In the current chapter, we provide a brief overview of the alternative methods currently available for DNA shuffling as well as a protocol presented here that improves on several previous implementations of restriction enzyme-mediated DNA family shuffling, in particular with regard to purification of DNA fragments for reassembly. PMID:25055778

  11. Telomeric Terminal Restriction Fragment (TRF).

    PubMed

    Kaushal, S

    1999-01-01

    Telomeres are protein-DNA structures at the ends of all eukaryotic chromosomes that are maintained by a unique ribonucleoprotein known as telomerase. This highly specialized RNA-dependent DNA polymerase provides a critical solution to the end-replication problem by adding TTAGGG repeats to 3' ends of human chromosomes (1-3). Telomere regulation is both cell cycle and developmentally-regulated, and its control is likely to be complex (4,5). There is gradual telomere shortening with age. This chromosomal pruning is presumed to be regulated by a mitotic clock, by which cell divisions are accounted (6,7). After about 100 cell divisions, a cell reaches its senescence "Hayflick" limit and cell division ceases (8). PMID:21380675

  12. An epidemiologically valuable typing method for Neisseria meningitidis by analysis of restriction fragment length polymorphisms

    Microsoft Academic Search

    A. J. Fox; D. M. Jones; S. J. Gray; D. A. Caugant; N. A. SAUNDERSt

    1991-01-01

    Summary. A restriction fragment length polymorphism (RFLP) typing method was developed for Neisseria meningitidis. A cloned EcoRI fragment from a Neisseria meningitidis Group B serotype 15P1.16 sulphonamide-resistant strain was used to probe Southern blots of total chromosomal DNA restriction fragments (enzyme AvaI). A group of 75 apparently unrelated organisms gave rise to 26 different restriction fragment length patterns and two

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

    NASA Astrophysics Data System (ADS)

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

    2007-12-01

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

  14. 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

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

    Microsoft Academic Search

    VERONIQUE ROUX; PIERRE-EDOUARD FOURNIER; ANDDIDIER RAOULT

    1996-01-01

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

  16. Linkage arrangement of restriction fragment length polymorphism loci in Brassica oleracea

    Microsoft Academic Search

    M. K. Slocum; S. S. Figdore; W. C. Kennard; J. Y. Suzuki; T. C. Osborn

    1990-01-01

    A detailed genetic linkage map of Brassica oleracea was constructed based on the segregation of 258 restriction fragment length polymorphism loci in a broccoli × cabbage F2 population. The genetic markers defined nine linkage groups, covering 820 recombination units. A majority of the informative genomic DNA probes hybridized to more than two restriction fragments in the F2 population. “Duplicate” sequences

  17. Genetic Diversity of Mycobacterium africanum Clinical Isolates Based on IS6110Restriction Fragment Length Polymorphism Analysis, Spoligotyping, and Variable Number of Tandem DNA Repeats

    Microsoft Academic Search

    CRISTINA VIANA-NIERO; CRISTINA GUTIERREZ; CHRISTOPHE SOLA; INGRID FILLIOL; FADILA BOULAHBAL; VERONIQUE VINCENT; NALIN RASTOGI

    2001-01-01

    A collection of 105 clinical isolates originally identified as Mycobacterium africanum were characterized using both phenotypic and genotyping methods. The phenotypic methods included routine determination of cultural properties and biochemical tests used to discriminate among the members of the M. tuberculosis complex, whereas genotypic characterization was based on IS6110-restriction fragment length polymorphism (IS6110- RFLP) analysis, IS1081-RFLP analysis, direct repeat-based spacer

  18. Assessment of the degree of restriction fragment length polymorphism in Brassica

    Microsoft Academic Search

    S. S. Figdore; W. C. Kennard; K. M. Song; M. K. Slocum; T. C. Osborn

    1988-01-01

    The feasibility of creating a restriction fragment length polymorphism (RFLP) linkage map in Brassica species was assessed by screening EcoRI-, HindIII-, or EcoRV-digested total genomic DNA from several accessions of B. campestris, B. oleracea, and B. napus using random genomic DNA clones from three Brassica libraries as hybridization probes. Differences in restriction fragment hybridization patterns occurred at frequencies of 95%

  19. Restriction enzyme cutting site distribution regularity for DNA looping technology.

    PubMed

    Shang, Ying; Zhang, Nan; Zhu, Pengyu; Luo, Yunbo; Huang, Kunlun; Tian, Wenying; Xu, Wentao

    2014-01-25

    The restriction enzyme cutting site distribution regularity and looping conditions were studied systematically. We obtained the restriction enzyme cutting site distributions of 13 commonly used restriction enzymes in 5 model organism genomes through two novel self-compiled software programs. All of the average distances between two adjacent restriction sites fell sharply with increasing statistic intervals, and most fragments were 0-499 bp. A shorter DNA fragment resulted in a lower looping rate, which was also directly proportional to the DNA concentration. When the length was more than 500 bp, the concentration did not affect the looping rate. Therefore, the best known fragment length was longer than 500 bp, and did not contain the restriction enzyme cutting sites which would be used for digestion. In order to make the looping efficiencies reach nearly 100%, 4-5 single cohesive end systems were recommended to digest the genome separately. PMID:24211387

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

    E-print Network

    Lyles, Ingrid Doodeheefver

    1989-01-01

    : endonuclease digested genomic DNA restriction fragment length polymorphisms (RFLPs). Nine of the 29 probe/enzyme combinations studied (31. 04) in the desert bighorn sheep revealed restriction site polymorphisms. These 2 captive populations of desert... Species Identification. DNA Extraction. DNA Digestion Southern Blotting Technique Probe Preparation Hybridization Technique RESULTS. 17 17 19 19 20 21 23 Variability Bovine Growth Hormone Bovine Prolactin. Bovine Chymosin Bovine Glucagon...

  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. (Los Alamos National Lab., NM (United States))

    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. DNA Fragmentation in Microorganisms Assessed In Situ?

    PubMed Central

    Fernández, José Luis; Cartelle, Mónica; Muriel, Lourdes; Santiso, Rebeca; Tamayo, María; Goyanes, Vicente; Gosálvez, Jaime; Bou, Germán

    2008-01-01

    Chromosomal DNA fragmentation may be a direct or indirect outcome of cell death. Unlike DNA fragmentation in higher eukaryotic cells, DNA fragmentation in microorganisms is rarely studied. We report an adaptation of a diffusion-based assay, developed as a kit, which allows for simple and rapid discrimination of bacteria with fragmented DNA. Intact cells were embedded in an agarose microgel on a slide, incubated in a lysis buffer to partially remove the cell walls, membranes, and proteins, and then stained with a DNA fluorochrome, SYBR Gold. Identifying cells with fragmented DNA uses peripheral diffusion of DNA fragments. Cells without DNA fragmentation show only limited spreading of DNA fiber loops. These results have been seen in several gram-negative and gram-positive bacteria, as well as in yeasts. Detection of DNA fragmentation was confirmed by fluoroquinolone treatment and by DNA breakage detection-fluorescence in situ hybridization. Proteus mirabilis with spontaneously fragmented DNA during exponential and stationary growth or Escherichia coli with DNA damaged after exposure to hydrogen peroxide or antibiotics, such as ciprofloxacin or ampicillin, was clearly detected. Similarly, fragmented DNA was detected in Saccharomyces cerevisiae after amphotericin B treatment. Our assay may be useful for the simple and rapid evaluation of DNA damage and repair as well as cell death, either spontaneous or induced by exogenous stimuli, including antimicrobial agents or environmental conditions. PMID:18689511

  3. DNA fragmentation in microorganisms assessed in situ.

    PubMed

    Fernández, José Luis; Cartelle, Mónica; Muriel, Lourdes; Santiso, Rebeca; Tamayo, María; Goyanes, Vicente; Gosálvez, Jaime; Bou, Germán

    2008-10-01

    Chromosomal DNA fragmentation may be a direct or indirect outcome of cell death. Unlike DNA fragmentation in higher eukaryotic cells, DNA fragmentation in microorganisms is rarely studied. We report an adaptation of a diffusion-based assay, developed as a kit, which allows for simple and rapid discrimination of bacteria with fragmented DNA. Intact cells were embedded in an agarose microgel on a slide, incubated in a lysis buffer to partially remove the cell walls, membranes, and proteins, and then stained with a DNA fluorochrome, SYBR Gold. Identifying cells with fragmented DNA uses peripheral diffusion of DNA fragments. Cells without DNA fragmentation show only limited spreading of DNA fiber loops. These results have been seen in several gram-negative and gram-positive bacteria, as well as in yeasts. Detection of DNA fragmentation was confirmed by fluoroquinolone treatment and by DNA breakage detection-fluorescence in situ hybridization. Proteus mirabilis with spontaneously fragmented DNA during exponential and stationary growth or Escherichia coli with DNA damaged after exposure to hydrogen peroxide or antibiotics, such as ciprofloxacin or ampicillin, was clearly detected. Similarly, fragmented DNA was detected in Saccharomyces cerevisiae after amphotericin B treatment. Our assay may be useful for the simple and rapid evaluation of DNA damage and repair as well as cell death, either spontaneous or induced by exogenous stimuli, including antimicrobial agents or environmental conditions. PMID:18689511

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed Central

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

    1996-01-01

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

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

    Microsoft Academic Search

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

    1999-01-01

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

  7. 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.

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

    Microsoft Academic Search

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

    1983-01-01

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

  9. Identification of screwworm species by polymerase chain reaction-restriction fragment length polymorphism.

    PubMed

    Taylor, D B; Szalanski, A L; Peterson, R D

    1996-01-01

    Restriction fragment length polymorphisms in polymerase chain reaction amplified fragments (PCR-RFLP) of mitochondrial DNA were used to differentiate species of New World screwworms (Diptera: Calliphoridae). Twenty-seven restriction enzymes were screened on five regions of mtDNA. Eleven restriction fragment length patterns differentiated New World screwworm, Cochliomyia hominivorax (Coquerel), from secondary screwworm, Cochliomyia macellaria (F.). Five restriction fragment length patterns were polymorphic in C.hominivorax while all fragment patterns were fixed in C.macellaria. Diagnostic restriction fragment length patterns were used for species diagnosis, whereas intraspecific variable patterns were used to characterize field samples and laboratory strains. The PCR-RFLP technique is flexible with regard to developmental stage of the sample and method of preservation. We were able to characterize specimens of all life stages from egg to adult including larvae preserved in alcohol and pinned adults. PCR-RFLP is rapid and inexpensive, enabling specimens to be characterized within 24 h for less than $2.50. PMID:8834744

  10. A Stochastic Model of DNA Fragments Rejoining

    PubMed Central

    Li, Yongfeng; Qian, Hong; Wang, Ya; Cucinotta, Francis A.

    2012-01-01

    When cells are exposed to ionizing radiation, DNA damages in the form of single strand breaks (SSBs), double strand breaks (DSBs), base damage or their combinations are frequent events. It is known that the complexity and severity of DNA damage depends on the quality of radiation, and the microscopic dose deposited in small segments of DNA, which is often related to the linear transfer energy (LET) of the radiation. Experimental studies have suggested that under the same dose, high LET radiation induces more small DNA fragments than low-LET radiation, which affects Ku efficiently binding with DNA end and might be a main reason for high-LET radiation induced RBE [1] since DNA DSB is a major cause for radiation-induced cell death. In this work, we proposed a mathematical model of DNA fragments rejoining according to non-homologous end joining (NHEJ) mechanism. By conducting Gillespie's stochastic simulation, we found several factors that impact the efficiency of DNA fragments rejoining. Our results demonstrated that aberrant DNA damage repair can result predominantly from the occurrence of a spatial distribution of DSBs leading to short DNA fragments. Because of the low efficiency that short DNA fragments recruit repair protein and release the protein residue after fragments rejoining, Ku-dependent NHEJ is significantly interfered with short fragments. Overall, our work suggests that inhibiting the Ku-dependent NHEJ may significantly contribute to the increased efficiency for cell death and mutation observed for high LET radiation. PMID:23028515

  11. An analysis of population genetic structure and species history of Drosophila melanogaster and Drosophila simulans using restriction fragment length polymorphisms of mitochondrial DNA

    Microsoft Academic Search

    Lawrence Richard Hale

    1989-01-01

    Animal mitochondrial DNA (mtDNA) has several features that give it great utility in the study of geographic structure of natural populations. Its small size and covalently closed circular conformation make it easy to purify. Strict maternal inheritance and homoplasmy makes the effective copy number of mtDNA as little as 1\\/4 that of nuclear loci; this renders populational complements of mtDNA

  12. 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.

  13. 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.

  14. 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...

  15. Restriction fragment polymorphisms as probes for plant diversity and their development as tools for applied plant breeding

    Microsoft Academic Search

    Tim Helentjaris; Gretchen King; Mary Slocum; Chris Siedenstrang; Sharon Wegman

    1985-01-01

    Maize and tomato cDNA clones have been hybridized in Southern blotting experiments to plant genomic DNA prepared from different lines to detect restriction fragment polymorphisms (RFPs). In maize we have found that a high degree of genetic variability is present, even among domestic inbred lines. Most randomly chosen maize cDNA clones can be used to detect elements of this variability.

  16. THE USE OF RESTRICTION ENDONUCLEASES TO MEASURE MITOCHONDRIAL DNA SEQUENCE RELATEDNESS IN NATURAL

    E-print Network

    Avise, John

    THE USE OF RESTRICTION ENDONUCLEASES TO MEASURE MITOCHONDRIAL DNA SEQUENCE RELATEDNESS IN NATURAL endonucleases to compare mitochon- drial DNA (mtDNA) sequences. We have examined the fragment patterns produced time that there is detectable heterogeneity in mtDNA sequences within and among natural geographic

  17. 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...

  18. Detection of specific sequences among DNA fragments separated by gel electrophoresis

    Microsoft Academic Search

    E. M. Southern

    1975-01-01

    This paper describes a method of transferring fragments of DNA from agarose gels to cellulose nitrate filters. The fragments can then be hybridized to radioactive RNA and hybrids detected by radioautography or fluorography. The method is illustrated by analyses of restriction fragments complementary to ribosomal RNAs from Escherichia coli and Xenopus laevis, and from several mammals.

  19. DNA POLYMORPHISM DETECTABLE BY RESTRICTION ENDONUCLEASES

    Microsoft Academic Search

    MASATOSHI NE; FUMIO TAJIMA

    1981-01-01

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

  20. Restriction Endonuclease Analysis of Mitochondrial DNA from Grande and Genetically Characterized Cytoplasmic Petite Clones of Saccharomyces cerevisiae

    Microsoft Academic Search

    Richard Morimoto; Alfred Lewin; Huey-Juang Hsu; Murray Rabinowitz; Hiroshi Fukuhara

    1975-01-01

    Digestion of grande mitochondrial DNA (mtDNA) by EcoRI restriction endonuclease gives rise to nine fragments with a total molecular weight of 51.8 × 106. HindIII digestion yields six fragments with a similar total molecular weight. Specific restriction fragments can be detected despite the fact that yeast mtDNA consists of a heterogeneous distribution of randomly broken molecules. Digestion patterns of 10

  1. Brassica taxonomy based on nuclear restriction fragment length polymorphisms (RFLPs)

    Microsoft Academic Search

    K. M. Song; T. C. Osborn; P. H. Williams

    1988-01-01

    Restriction fragment length polymorphisms (RFLPs) of nuclear DNAs have been used to explore the origin and evolution of the six cultivated Brassica species. Extensive RFLP variation was found at the species, subspecies and variety levels. Based on RFLP data from Brassica and related genera, a detailed phylogenetic tree was generated using the PAUP microcomputer program, which permits a quantitative analysis

  2. 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.

  3. DNA Sequence from Cretaceous Period Bone Fragments

    Microsoft Academic Search

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

    1994-01-01

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

  4. Reconstructing DNA replication kinetics from small DNA fragments

    Microsoft Academic Search

    Haiyang Zhang; John Bechhoefer

    2006-01-01

    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 measurements of the time dependence of the average size of replicated and nonreplicated domains, one can estimate the rate of initiation of DNA replication origins, as well as the average rate at

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

    E-print Network

    Rouchka, Eric

    Avenue St. Louis, MO 63110-1012, USA Email: ecr@ibc.wustl.edu Abstract DNA sequence analysis depends on the accurate assembly of fragment reads for the determination of a consensus sequence. This report examines assembly of short (400 to 1,000 base pair) sequence reads into contigs that cover extended regions

  6. Isolation of probes detecting restriction fragment length polymorphisms from X chromosome-specific libraries: potential use for diagnosis of Duchenne muscular dystrophy

    Microsoft Academic Search

    M. H. Hofker; M. C. Wapenaar; Nicole Goor; E. Bakker; G.-J. B. Ommen; P. L. Pearson

    1985-01-01

    We have isolated 23 human X chromosome-specific DNA fragments from ? libraries, prepared from flow-sorted X chromosomes. To increase diagnostic potential for X-linked genetic disorders, including Duchenne muscular dystrophy (DMD), the fragments were tested for restriction fragment length polymorphisms (RFLPs) with six restriction enzymes. All fragments were regionally mapped to segments of the X chromosome with a panel of somatic

  7. Quantitative restriction fragment length polymorphism: a procedure for quantitation of diphtheria toxin gene CRM197 allele.

    PubMed

    Pushnova, E A; Zhu, Y S

    1998-06-15

    Here we present an assay for quantitation of a particular gene allele in DNA mixtures by means of restriction fragment length polymorphism (RFLP) in combination with polymerase chain reaction (PCR). We applied the quantitative RFLP principle for estimation of the relative amount of diphtheria toxin gene CRM197 allele in Corynebacterium diphtheriae culture DNA samples. The procedure is based on PCR-mediated generation of an artificial AluI restriction site specifically with the CRM197 DNA template. After AluI digestion of the PCR product and polyacrylamide gel electrophoresis of the restriction fragments, the percentage of CRM197 template in the initial DNA sample was determined by scanning a gel negative. The method was shown to give a linear response when applied to template mixtures containing different amounts of CRM197 reference template. For samples where non-CRM197 DNA was detected by AluI RFLP, we designed a further allele-specific PCR assay to determine whether the non-CRM197 template portion was the wild-type toxin gene allele. PMID:9648648

  8. A linkage map of Brassica rapa (syn. campestris) based on restriction fragment length polymorphism loci

    Microsoft Academic Search

    K. M. Song; J. Y. Suzuki; M. K. Slocum; P. M. Williams; T. C. Osborn

    1991-01-01

    Summary A detailed linkage map ofB. rapa (syn.campestris) was constructed based on segregation of 280 restriction fragment length polymorphism loci, detected by using 188 genomic DNA clones as probes on DNAs from a F2 population of Chinese cabbage ‘MichihilF’בSpring broccoli.’ These genetic markers covered 1,850 centiMorgans (cM) and defined ten linkage groups, which equals the haploid chromosome number of this

  9. Restriction fragment length polymorphism and virulence of Czech Toxoplasma gondii strains.

    PubMed

    Literák, I; Rychlík, I; Svobodová, V; Pospísil, Z

    1998-09-01

    Restriction fragment length polymorphism analysis of chromosomal DNA from 22 strains of Toxoplasma gondii were characterised using SalI and PstI restriction endonucleases and the TGR1E specific repetitive sequence as a probe. Two virulent strains, RH and P-CZ, had previously been isolated from humans, the remaining 20 strains were isolated from animals in the Czech Republic in 1994 and 1995. Among the 20 recently isolated strains, 19 belonged to an avirulent lineage and only one strain from the wild cat Felis silvestris belonged to a virulent lineage. PMID:9770622

  10. 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

    Microsoft Academic Search

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

    2003-01-01

    Cag pathogenicity island-containing Helicobacter pylori (type I) induces signal transduction pathways re- sulting 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

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

    Microsoft Academic Search

    JAE-CHANG CHO; JAMES M. TIEDJE

    2001-01-01

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

  12. Polymerase chain reaction-restriction fragment-length polymorphism method to distinguish Liriomyza huidobrensis from L. Langei (Diptera: Agromyzidae) applied to three recent leafminer invasions.

    PubMed

    Scheffer, S J; Wijesekara, A; Visser, D; Hallett, R H

    2001-10-01

    A molecular method is presented for differentiating the morphologically cryptic leafminers Liriomyza langei Frick and L. huidobrensis (Blanchard). This method requires polymerase chain reaction (PCR) amplification of a 1031-bp region of mitochondrial cytochrome oxidase DNA followed by restriction fragment analysis using the restriction enzymes SpeI and EcoRV. Spel cuts the mitochondrial fragment of L. langei into two fragments, but does not cut the L. huidobrensis fragment. EcoRV cuts the L. huidobrensis fragment into two fragments, but does not cut the L. langei fragment. This PCR-restriction fragment-length polymorphism (RFLP) method is faster and less costly than DNA sequencing,which is currently the only other way to differentiate these two species. We apply the method to samples from recently introduced leafminer populations in Sri Lanka, Canada, and South Africa and find that the invasive leafminer in all three locations is L. huidobrensis. PMID:11681681

  13. Restriction data from chloroplast DNA for phylogenetic reconstruction: Is there only one accurate way of scoring?

    Microsoft Academic Search

    Birgitta Bremer

    1991-01-01

    Information from the same restriction analysis of chloroplast DNA of 33 taxa ofRubiaceae was scored in four different ways, two of which were based on fragments, and two on restriction sites, and they were subsequently analysed with Wagner parsimony. The methods resulted in different phylogenetic trees. The inherent differences between the methods relate to the amount of non-homologous characters and

  14. Restriction fragment length polymorphism and molecular taxonomy in Vitis vinifera L

    Microsoft Academic Search

    J.-C. Bourquin; A. Sonko; L. Otten; B. Walter

    1993-01-01

    Forty-six accessions of grapevine (V. vinifera L.) were compared by restriction fragment length polmorphism (RFLP) analysis, and 111 informative or unique restriction fragments were found that revealed an important level of polymorphism. RFLP patterns were compared in two ways: by calculating electrophoretic similarity degree values further analyzed by principal component analysis and by studying the distribution of rare restriction fragments.

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

    PubMed

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

    2008-01-01

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

  16. Restriction endonuclease analysis of mitochondrial DNA from grande and genetically characterized cytoplasmic petite clones of Saccharomyces cerevisiae.

    PubMed

    Morimoto, R; Lewin, A; Hsu, H J; Rabinowitz, M; Fukuhara, H

    1975-10-01

    Digestion of grande mitochondrial DNA (mtDNA) BY EcoRI restriction endonuclease gives rise to nine fragments with a total molecular weight of 51.8 x 10(6). HindIII digestion yields six fragments with a similar total molecular weight. Specific restriction fragments can be detected despite the fact that yeast mtDNA consists of a heterogeneous distribution of randomly broken molecules. Digestion patterns of 10 genetically characterized petite clones containing various combinations of five antiobiotic resistance markers indicate that the petite mtDNA predominantly represents deletion of the grande genome. The petite mtDNAs contained up to seven EcoRI restriction fragments which comigrate with grande restriction fragments, and at least one fragment that did not correspond to any in the grande. Some strains contained multiple fragments with mobility different from that of grande; these fragments were usually present in less than molar concentrations. The genetic markers were associated with individual sets of restriction fragments. However, several internal inconsistencies prevent the construction of a definitive genetic fragment map. These anomalies, together with the digestion patterns, provide strong evidence that, in addition to single contiguous deletion, other changes such as multiple deletion and heterogeneity of mtDNA populations are present in some of the petite mtDNAs. PMID:1105566

  17. Formation of pseudo-terminal restriction fragments, a PCR-related bias affecting terminal restriction fragment length polymorphism analysis of microbial community structure.

    PubMed

    Egert, Markus; Friedrich, Michael W

    2003-05-01

    Terminal restriction fragment length polymorphism (T-RFLP) analysis of PCR-amplified genes is a widely used fingerprinting technique in molecular microbial ecology. In this study, we show that besides expected terminal restriction fragments (T-RFs), additional secondary T-RFs occur in T-RFLP analysis of amplicons from cloned 16S rRNA genes at high frequency. A total of 50% of 109 bacterial and 78% of 68 archaeal clones from the guts of cetoniid beetle larvae, using MspI and AluI as restriction enzymes, respectively, were affected by the presence of these additional T-RFs. These peaks were called "pseudo-T-RFs" since they can be detected as terminal fluorescently labeled fragments in T-RFLP analysis but do not represent the primary terminal restriction site as indicated by sequence data analysis. Pseudo-T-RFs were also identified in T-RFLP profiles of pure culture and environmental DNA extracts. Digestion of amplicons with the single-strand-specific mung bean nuclease prior to T-RFLP analysis completely eliminated pseudo-T-RFs. This clearly indicates that single-stranded amplicons are the reason for the formation of pseudo-T-RFs, most probably because single-stranded restriction sites cannot be cleaved by restriction enzymes. The strong dependence of pseudo-T-RF formation on the number of cycles used in PCR indicates that (partly) single-stranded amplicons can be formed during amplification of 16S rRNA genes. In a model, we explain how transiently formed secondary structures of single-stranded amplicons may render single-stranded amplicons accessible to restriction enzymes. The occurrence of pseudo-T-RFs has consequences for the interpretation of T-RFLP profiles from environmental samples, since pseudo-T-RFs may lead to an overestimation of microbial diversity. Therefore, it is advisable to establish 16S rRNA gene sequence clone libraries in parallel with T-RFLP analysis from the same sample and to check clones for their in vitro digestion T-RF pattern to facilitate the detection of pseudo-T-RFs. PMID:12732521

  18. Formation of Pseudo-Terminal Restriction Fragments, a PCR-Related Bias Affecting Terminal Restriction Fragment Length Polymorphism Analysis of Microbial Community Structure

    PubMed Central

    Egert, Markus; Friedrich, Michael W.

    2003-01-01

    Terminal restriction fragment length polymorphism (T-RFLP) analysis of PCR-amplified genes is a widely used fingerprinting technique in molecular microbial ecology. In this study, we show that besides expected terminal restriction fragments (T-RFs), additional secondary T-RFs occur in T-RFLP analysis of amplicons from cloned 16S rRNA genes at high frequency. A total of 50% of 109 bacterial and 78% of 68 archaeal clones from the guts of cetoniid beetle larvae, using MspI and AluI as restriction enzymes, respectively, were affected by the presence of these additional T-RFs. These peaks were called “pseudo-T-RFs” since they can be detected as terminal fluorescently labeled fragments in T-RFLP analysis but do not represent the primary terminal restriction site as indicated by sequence data analysis. Pseudo-T-RFs were also identified in T-RFLP profiles of pure culture and environmental DNA extracts. Digestion of amplicons with the single-strand-specific mung bean nuclease prior to T-RFLP analysis completely eliminated pseudo-T-RFs. This clearly indicates that single-stranded amplicons are the reason for the formation of pseudo-T-RFs, most probably because single-stranded restriction sites cannot be cleaved by restriction enzymes. The strong dependence of pseudo-T-RF formation on the number of cycles used in PCR indicates that (partly) single-stranded amplicons can be formed during amplification of 16S rRNA genes. In a model, we explain how transiently formed secondary structures of single-stranded amplicons may render single-stranded amplicons accessible to restriction enzymes. The occurrence of pseudo-T-RFs has consequences for the interpretation of T-RFLP profiles from environmental samples, since pseudo-T-RFs may lead to an overestimation of microbial diversity. Therefore, it is advisable to establish 16S rRNA gene sequence clone libraries in parallel with T-RFLP analysis from the same sample and to check clones for their in vitro digestion T-RF pattern to facilitate the detection of pseudo-T-RFs. PMID:12732521

  19. Specific fragments of phi X174 deoxyribonucleic acid produced by a restriction enzyme from Haemophilus aegyptius, endonuclease Z.

    PubMed

    Middleton, J H; Edgell, M H; Hutchison, C A

    1972-07-01

    A restriction-like enzyme has been purified from Haemophilus aegyptius. This nuclease, endonuclease Z, produces a rapid decrease in the viscosity of native calf thymus and H. influenzae deoxyribonucleic acids (DNA), but does not degrade homologous DNA. The specificity of endonuclease Z is different from that of the similar endonuclease isolated from H. influenzae (endonuclease R). The purified enzyme cleaves the double-stranded replicative form DNA of bacteriophage phiX174 (phiX174 RF DNA) into at least 11 specific limit fragments whose molecular sizes have been estimated by gel electrophoresis. The position of these fragments with respect to the genetic map of phiX174 can be determined by using the genetic assay for small fragments of phiX174 DNA. PMID:4537735

  20. Structural insight into the specificity of the B3 DNA-binding domains provided by the co-crystal structure of the C-terminal fragment of BfiI restriction enzyme

    PubMed Central

    Golovenko, Dmitrij; Manakova, Elena; Zakrys, Linas; Zaremba, Mindaugas; Sasnauskas, Giedrius; Gražulis, Saulius; Siksnys, Virginijus

    2014-01-01

    The B3 DNA-binding domains (DBDs) of plant transcription factors (TF) and DBDs of EcoRII and BfiI restriction endonucleases (EcoRII-N and BfiI-C) share a common structural fold, classified as the DNA-binding pseudobarrel. The B3 DBDs in the plant TFs recognize a diverse set of target sequences. The only available co-crystal structure of the B3-like DBD is that of EcoRII-N (recognition sequence 5?-CCTGG-3?). In order to understand the structural and molecular mechanisms of specificity of B3 DBDs, we have solved the crystal structure of BfiI-C (recognition sequence 5?-ACTGGG-3?) complexed with 12-bp cognate oligoduplex. Structural comparison of BfiI-C–DNA and EcoRII-N–DNA complexes reveals a conserved DNA-binding mode and a conserved pattern of interactions with the phosphodiester backbone. The determinants of the target specificity are located in the loops that emanate from the conserved structural core. The BfiI-C–DNA structure presented here expands a range of templates for modeling of the DNA-bound complexes of the B3 family of plant TFs. PMID:24423868

  1. Restriction analysis of specific DNA sequences of five homothallic species of Neurospora

    SciTech Connect

    Attoh, G.; Dutta, S.K.

    1983-01-01

    Nuclear DNA's from five homothallic species of Neurospora: N. africana, N. dodgei, N. lineolata, N. galapagosensis and N. terricola were isolated and characterized. From these total nuclear DNA's, specific DNA sequences were isolated by CsCl buoyant density gradient equilibrium centrifugation. These selected DNA's were restricted separately with EcoR1, Bam H1, Hind III, XBa 1 and Hinc II. DNA digests were electrophorused in varying agarose gel concentration (0.7%, 0.8% and 1%) and the sizes of fragments generated with these endonucleases were estimated against lambda phage DNA and pMF2 DNA fragments. EcoR1 generated four DNA size variants of about 20.0 kb, 7.5 kb, 5.5 b and 5.23 kb in N. terricola except the 10.5 kb fragment. Instead, N. lineolata showed three distinct DNA size variants of about 15.0 kb, 12.0 kb and 8.2 kb with Bam H1 treatment. Similarly we have noticed unique DNA restriction bands when treated with a few other restriction enzymes like Xba 1 and Hind III. These results suggest distinct rDNA nucleotide sequence differences among homothallic species of Neurospora studied.

  2. 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...

  3. 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.

  4. A Locus with Restriction Fragment-Length Polymorphisms Characteristic of African and European Honey Bee (Hymenoptera: Apidae) Groups of Subspecies

    Microsoft Academic Search

    Alonso Suazo; Myeong-lyeol Lee; H. Glenn Hall

    2002-01-01

    Within an anonymousregion of honey bee DNA (locus227) diges ted with AluI, informative restriction fragment-length polymorphisms (RFLP) were found in Southern blots with a cloned honey bee DNA probe. The probe was subcloned, so that smaller sections of the locus could be analyzed with the polymerase chain reaction (PCR). Further screening of these ampliÞed sections revealed additional useful RFLPs with

  5. An HLA-D region restriction fragment length polymorphism associated with celiac disease

    PubMed Central

    1986-01-01

    This study is the first to describe a molecular marker that distinguishes the celiac disease HLA-D region haplotype from a serologically identical haplotype in unaffected controls. Using a DQ beta chain cDNA probe and the restriction endonuclease Rsa I, we have detected a polymorphic 4.0 kb fragment which, in DQw2 individuals, is associated with a 40-fold increased relative risk of developing celiac disease. This finding should permit the identification of the celiac disease susceptibility gene(s) in the HLA-D region and facilitate a more precise dissection of the molecular and immunogenetic mechanisms involved in the pathogenesis of that disease. PMID:3014038

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

    SciTech Connect

    Branscomb, E.; Slezak, T.; Pae, R.; Carrano, A.V. (Lawrence Livermore National Lab., CA (United States)); Galas, D.; Waterman, M. (Univ. of South California, Los Angeles (United States))

    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.

  7. 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.

  8. 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.

  9. Bacterial natural transformation by highly fragmented and damaged DNA

    E-print Network

    Nielsen, Rasmus

    replication. We show that the adjacent nucleotide variations generated by uptake of short DNA fragments escapeBacterial natural transformation by highly fragmented and damaged DNA Søren Overballe-Petersena,1 for review August 14, 2013) DNA molecules are continuously released through decomposition of organic matter

  10. 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

  11. A restriction-fragment-length difference detected by the anonymous probe DXS199 exhibits non-Mendelian inheritance.

    PubMed Central

    Starr, T; Wood, S

    1988-01-01

    Anonymous DNA probes were isolated from an X chromosome-enriched flow-sorted library. One of these probes, DXS199, identified a restriction-fragment difference that failed to show Mendelian segregation. All normal females were found to have two AvaII fragments of 6.5 kb and 6.0 kb, whereas all normal males had only the 6.5-kb fragment. DNA from a 49,XXXXY male was found to have both 6.0- and 6.5-kb AvaII fragments, in the same 3:1 ratio as seen in the inactive:active number of X chromosomes. This variant, which reflects a structural difference between active and inactive X chromosomes, is likely to be due to a methylation site on the active X chromosome. Images Figure 1 Figure 2 PMID:2893545

  12. 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.

  13. 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.

  14. Formation of Pseudo-Terminal Restriction Fragments, a PCR-Related Bias Affecting Terminal Restriction Fragment Length Polymorphism Analysis of Microbial Community Structure

    Microsoft Academic Search

    Markus Egert; Michael W. Friedrich

    2003-01-01

    Terminal restriction fragment length polymorphism (T-RFLP) analysis of PCR-amplified genes is a widely used fingerprinting technique in molecular microbial ecology. In this study, we show that besides expected terminal restriction fragments (T-RFs), additional secondary T-RFs occur in T-RFLP analysis of amplicons from cloned 16S rRNA genes at high frequency. A total of 50% of 109 bacterial and 78% of 68

  15. Simultaneous splicing of multiple DNA fragments in one PCR reaction

    PubMed Central

    2013-01-01

    Background Rapid and simultaneous splicing of multiple DNA fragments is frequently required in many recombinant DNA projects. However, former overlap extension PCRs, the most common methods for splicing DNA fragments, are not really simultaneous fusing of multiple DNA fragments. Results We performed an optimized method which allowed simultaneous splicing of multiple DNA fragments in one PCR reaction. Shorter outermost primers were prior mixed with other PCR components at the same time. A sequential thermo cycling program was adopted for overlap extension reaction and amplification of spliced DNA. Annealing temperature was relatively higher in the overlap extension reaction stage than in the fused DNA amplification. Finally we successfully harvested target PCR products deriving from fusion of two to seven DNA fragments after 5–10 cycles for overlap extension reaction and then 30 cycles for fused DNA amplification. Conclusions Our method provides more rapid, economical and handy approach to accurately splice multiple DNA fragments. We believe that our simultaneous splicing overlap extension PCR can be used to fuse more than seven DNA fragments as long as the DNA polymerase can match. PMID:24015676

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

    Microsoft Academic Search

    Louis Bernatchez; Julian J. Dodson

    2007-01-01

    Abstract.-In the paper, restriction-fragment length polymorphisms in mitochondria1 DNA (mtDNA) were studied to test the hypothesis that sympatric populations of lake whitefish in the Allegash basin have recently diverged through sympatric speciation. Thirteen restriction enzymes were used to analyze mtDNA of 1 56 specimens representing 1 3 populations from eastern Canada and northern Maine where normal,and dwarf phenotypes,of whitefish exist

  17. Babesia canis: evidence for genetic diversity among isolates revealed by restriction fragment length polymorphism analysis.

    PubMed

    Citard, T; Mähl, P; Boulouis, H J; Chavigny, C; Druilhe, P

    1995-09-01

    The genetic diversity of B. canis was investigated by restriction fragment length polymorphism analysis. For this purpose, we identified a Babesia canis specific DNA probe named pS8. This 1.2 kbp probe can detect as low as 20 pg of B. canis DNA. Results suggest that the pS8 probe is distributed in multiple copies throughout the genome though is probably not itself internally repetitious, i.e. not structured into blocks of tandem units. This probe reveals discrete hybridizing fragments in B. canis enzyme-digested genomic DNA. RFLP patterns obtained with the pS8 probe revealed a large genetic diversity between various isolates and led us to distinguish several clones derived from a single isolate. Results suggest that for a single isolate, the fingerprints obtained reflect those of a few quantitatively dominant clones. This technique can now be routinely applied and provides a convenient tool for the characterization and the identification of B. canis isolates, strains and clones. PMID:8533020

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

    SciTech Connect

    Zheleznaya, L.A.; Menzenyuk, O.Y. [Institute of Theoretical and Experimental Biophysics, Pushchino (Russian Federation); Matvienko, N.N. [Branch of Shemyakin and Ovchinnikov Institute of Bioorganic, Pushchino (Russian Federation); Matvienko, N.I. [Institute of Protein Research, Pushchino (Russian Federation)

    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.

  19. 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 Central

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

    2003-01-01

    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

  20. Autoscreening of Restriction Endonucleases for PCR-Restriction Fragment Length Polymorphism Identification of Fungal Species, with Pleurotus spp. as an Example? †

    PubMed Central

    Yang, Zhi-Hui; Huang, Ji-Xiang; Yao, Yi-Jian

    2007-01-01

    A molecular method based on PCR-restriction fragment length polymorphism (RFLP) analysis of internal transcribed spacer (ITS) ribosomal DNA sequences was designed to rapidly identify fungal species, with members of the genus Pleurotus as an example. Based on the results of phylogenetic analysis of ITS sequences from Pleurotus, a PCR-RFLP endonuclease autoscreening (PRE Auto) program was developed to screen restriction endonucleases for discriminating multiple sequences from different species. The PRE Auto program analyzes the endonuclease recognition sites and calculates the sizes of the fragments in the sequences that are imported into the program in groups according to species recognition. Every restriction endonuclease is scored through the calculation of the average coefficient for the sequence groups and the average coefficient for the sequences within a group, and then virtual electrophoresis maps for the selected restriction enzymes, based on the results of the scoring system, are displayed for the rapid determination of the candidate endonucleases. A total of 85 haplotypes representing 151 ITS sequences were used for the analysis, and 2,992 restriction endonucleases were screened to find the candidates for the identification of species. This method was verified by an experiment with 28 samples representing 12 species of Pleurotus. The results of the digestion by the restriction enzymes showed the same patterns of DNA fragments anticipated by the PRE Auto program, apart from those for four misidentified samples. ITS sequences from 14 samples (of which nine sequences were obtained in this study), including four originally misidentified samples, confirmed the species identities revealed by the PCR-RFLP analysis. The method developed here can be used for the identification of species of other living microorganisms. PMID:17965212

  1. Genetic identification of Mycobacterium bovis BCG by restriction fragment length polymorphism analysis of the direct-repeat region.

    PubMed Central

    Howard, S T; Laszlo, A; Johnson, W M

    1997-01-01

    Restriction fragment length polymorphism (RFLP) analysis was performed on the direct repeat (DR) regions of 14 strains of Mycobacterium bovis BCG. With AluI-digested DNA, BCG Japanese, Russian, and Mexican had differing RFLP patterns but 11 strains, including Pasteur, Glaxo, and Tice, had an identical pattern not detected in over 60 strains of the M. tuberculosis complex. DR analysis can aid in confirming the identification of clinical BCG isolates. PMID:9157163

  2. Distribution of two DNA restriction fragment length polymorphisms (RFLPs) corresponding to Ag(c/g) and Ag(al/d) of the apo B gene in the Orang Asli (aborigines) of West Malaysia

    SciTech Connect

    Candlish, J.K.; Gajra, B; Saha, N. [National Univ. of Singapore, Kuala Lumpur (Malaysia)] [and others

    1994-09-01

    One hundred and ninety five subjects of the Semai group of Orang Asli in peninsular Malaysia were examined for the distribution of Ag(c/g) and Ag(al/d) RFLPs of the apoB gene. Regions of apoB gene corresponding to nt 421 and 1981 representing these two Ags were amplified by polymerase chain reaction using primers of published sequences. Thr{sub 71} to Ile (Ag c/g) was detected as an ApaL I RFLP and Val{sub 591} to Ala (Ag al/d) by Alu I RFLP. DNA fragments were separated by 4% agarose gel electrophoresis and photographed over a UV transilluminator. The frequencies of Ag(d) (absence of ApaL I site) and Ag(d) (presence of Alu I site) were found to be 0.13 and 0.14, respectively, in the Orang Asli compared to frequencies of 0.30 and 0.45 in the Caucasian population. Distribution of the genotypes of these two polymorphisms was at Hardy-Weinberg equiilibrium.

  3. Polymerase chain reaction-restriction fragment length polymorphism analysis: a simple method for species identification in food.

    PubMed

    Meyer, R; Höfelein, C; Lüthy, J; Candrian, U

    1995-01-01

    The polymerase chain reaction (PCR) technique was applied to meat species identification in marinated and heat-treated or fermented products and to the differentiation of closely related species. DNA was isolated from meat samples by using a DNA-binding resin and was subjected to PCR analysis. Primers used were complementary to conserved areas of the vertebrate mitochondrial cytochrome b (cytb) gene and yielded a 359 base-pair (bp) fragment, including a variable 307 bp region. Restriction endonuclease analysis based on sequence data of those fragments was used for differentiation among species. Restriction fragment length polymorphisms (RFLPs) were detected when pig, cattle, wild boar, buffalo, sheep, goat, horse, chicken, and turkey amplicons were cut with AluI, RsaI, TaqI, and HinfI. Analysis of sausages indicates the applicability of this approach to food products containing meat from 3 different species. The PCR-RFLP analytical method detected pork in heated meat mixtures with beef at levels below 1%, and the method was confirmed with porcine- and bovine-specific PCR assays by amplifying fragments of their growth hormone genes. Inter- and intraspecific differences of more than 22 animal species with nearly unknown cytb DNA sequences, including hoofed mammals (ungulates), and poultry were determined with PCR-RFLP typing by using 20 different endonucleases. This typing method allowed the discrimination of game meats, including stag, roe deer, chamois, moose, reindeer, kangaroo, springbok, and other antelopes in marinated and heat-treated products. PMID:8664595

  4. Structure of DNA Polymerase I Klenow Fragment Bound to Duplex DNA

    Microsoft Academic Search

    Lorena S. Beese; Victoria Derbyshire; Thomas A. Steitz

    1993-01-01

    Klenow fragment of Escherichia coli DNA polymerase I, which was cocrystallized with duplex DNA, positioned 11 base pairs of DNA in a groove that lies at right angles to the cleft that contains the polymerase active site and is adjacent to the 3' to 5' exonuclease domain. When the fragment bound DNA, a region previously referred to as the \\

  5. 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

  6. Hyperglycemia Enhances DNA Fragmentation After Transient Cerebral Ischemia

    Microsoft Academic Search

    Ping-An Li; Ingrid Rasquinha; Qing Ping He; Katalin Csiszár; Charles D. Boyd; John P. MacManus

    2001-01-01

    Previous histopathologic results have suggested that one mechanism whereby hyperglycemia (HG) leads to exaggerated ischemic damage involves fragmentation of DNA. DNA fragmentation in normoglycemia (NG) and HG rats subjected to 30 minutes of forebrain ischemia was studied by terminal deoxynucleotidyl transferase mediated DNA nick-labeling (TUNEL) staining, by pulse-field gel electrophoresis (PFGE), and by ligation-mediated polymerase chain reaction (LM-PCR). High molecular

  7. A Mechanism of Gene Amplification Driven by Small DNA Fragments

    PubMed Central

    Mukherjee, Kuntal; Storici, Francesca

    2012-01-01

    DNA amplification is a molecular process that increases the copy number of a chromosomal tract and often causes elevated expression of the amplified gene(s). Although gene amplification is frequently observed in cancer and other degenerative disorders, the molecular mechanisms involved in the process of DNA copy number increase remain largely unknown. We hypothesized that small DNA fragments could be the trigger of DNA amplification events. Following our findings that small fragments of DNA in the form of DNA oligonucleotides can be highly recombinogenic, we have developed a system in the yeast Saccharomyces cerevisiae to capture events of chromosomal DNA amplification initiated by small DNA fragments. Here we demonstrate that small DNAs can amplify a chromosomal region, generating either tandem duplications or acentric extrachromosomal DNA circles. Small fragment-driven DNA amplification (SFDA) occurs with a frequency that increases with the length of homology between the small DNAs and the target chromosomal regions. SFDA events are triggered even by small single-stranded molecules with as little as 20-nt homology with the genomic target. A double-strand break (DSB) external to the chromosomal amplicon region stimulates the amplification event up to a factor of 20 and favors formation of extrachromosomal circles. SFDA is dependent on Rad52 and Rad59, partially dependent on Rad1, Rad10, and Pol32, and independent of Rad51, suggesting a single-strand annealing mechanism. Our results reveal a novel molecular model for gene amplification, in which small DNA fragments drive DNA amplification and define the boundaries of the amplicon region. As DNA fragments are frequently found both inside cells and in the extracellular environment, such as the serum of patients with cancer or other degenerative disorders, we propose that SFDA may be a common mechanism for DNA amplification in cancer cells, as well as a more general cause of DNA copy number variation in nature. PMID:23271978

  8. High-Sensitivity Stable-Isotope Probing by a Quantitative Terminal Restriction Fragment Length Polymorphism Protocol

    PubMed Central

    Andeer, Peter; Stahl, David A.

    2012-01-01

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

  9. 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

  10. Bacterial natural transformation by highly fragmented and damaged DNA

    PubMed Central

    Overballe-Petersen, Søren; Harms, Klaus; Orlando, Ludovic A. A.; Mayar, J. Victor Moreno; Rasmussen, Simon; Dahl, Tais W.; Rosing, Minik T.; Poole, Anthony M.; Sicheritz-Ponten, Thomas; Brunak, Søren; Inselmann, Sabrina; de Vries, Johann; Wackernagel, Wilfried; Pybus, Oliver G.; Nielsen, Rasmus; Johnsen, Pål Jarle; Nielsen, Kaare Magne; Willerslev, Eske

    2013-01-01

    DNA molecules are continuously released through decomposition of organic matter and are ubiquitous in most environments. Such DNA becomes fragmented and damaged (often <100 bp) and may persist in the environment for more than half a million years. Fragmented DNA is recognized as nutrient source for microbes, but not as potential substrate for bacterial evolution. Here, we show that fragmented DNA molecules (?20 bp) that additionally may contain abasic sites, cross-links, or miscoding lesions are acquired by the environmental bacterium Acinetobacter baylyi through natural transformation. With uptake of DNA from a 43,000-y-old woolly mammoth bone, we further demonstrate that such natural transformation events include ancient DNA molecules. We find that the DNA recombination is RecA recombinase independent and is directly linked to DNA replication. We show that the adjacent nucleotide variations generated by uptake of short DNA fragments escape mismatch repair. Moreover, double-nucleotide polymorphisms appear more common among genomes of transformable than nontransformable bacteria. Our findings reveal that short and damaged, including truly ancient, DNA molecules, which are present in large quantities in the environment, can be acquired by bacteria through natural transformation. Our findings open for the possibility that natural genetic exchange can occur with DNA up to several hundreds of thousands years old. PMID:24248361

  11. 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 $D_p$.

  12. DNA sequences of RAPD fragments in artiodactyls.

    PubMed

    Kostia, S; Palo, J; Varvio, S L

    1996-04-01

    A bovine RAPD profile, generated by a 10-mer primer, was analysed by sequencing the major fragments. Three of four different fragments showed homologies to previously characterized mammalian sequences. One was 61-66% identical to LINE sequences and another was 78.5% identical to a human chromosome 2 sequence tagged site. The third fragment was 93.1% identical to the human type 2 inositol 1,4,5-trisphosphate receptor gene. This fragment had counterparts in white-tailed deer and reindeer; fragments of slightly different size in these species showed high sequence similarity and the size differences were due to varying numbers of dinucleotide microsatellite repeats inside the fragment. PMID:8984008

  13. A Sex Chromosomal Restriction-Fragment-Length Marker Linked to Melanoma-Determining Tu Loci in Xiphophorus

    PubMed Central

    Schartl, M.

    1988-01-01

    In Xiphophorus, the causative genetic information for melanoma formation has been assigned by classical genetics to chromosomal loci, which are located on the sex chromosomes. In our attempts to molecularly clone these melanoma-determining loci, named Tu, we have looked for restriction-fragment-length markers (RFLMs) linked to the Tu loci. These RFLMs should be useful in obtaining a physical map of a Tu locus, which will aid in the cloning of the corresponding sequences. DNA samples from various Xiphophorus strains and hybrids including those bearing different Tu wild-type, deletion and translocation chromosomes, were screened for the presence of random RFLMs using homologous or heterologous sequences as hybridization probes. We find an EcoRI restriction fragment which shows limited crosshybridization to the v-erb B gene--but not representing the authentic c-erb B gene of Xiphophorus--to be polymorphic with respect to different sex chromosomes. Linkage analysis revealed that a 5-kb fragment is linked to the Tu-Sd locus on the X chromosome, a 7-kb fragment is linked to the Tu-Sr locus on the Y chromosome, both of Xiphophorus maculatus, and that a 12-kb fragment is linked to the Tu-Li locus on the X chromosome of Xiphophorus variatus. Using different chromosomal mutants this RFLM has been mapped to a frequent deletion/translocation breakpoint of the X chromosome, less than 0.3 cM apart from the Tu locus. PMID:2841190

  14. A sex chromosomal restriction-fragment-length marker linked to melanoma-determining Tu loci in Xiphophorus.

    PubMed

    Schartl, M

    1988-07-01

    In Xiphophorus, the causative genetic information for melanoma formation has been assigned by classical genetics to chromosomal loci, which are located on the sex chromosomes. In our attempts to molecularly clone these melanoma-determining loci, named Tu, we have looked for restriction-fragment-length markers (RFLMs) linked to the Tu loci. These RFLMs should be useful in obtaining a physical map of a Tu locus, which will aid in the cloning of the corresponding sequences. DNA samples from various Xiphophorus strains and hybrids including those bearing different Tu wild-type, deletion and translocation chromosomes, were screened for the presence of random RFLMs using homologous or heterologous sequences as hybridization probes. We find an EcoRI restriction fragment which shows limited crosshybridization to the v-erb B gene--but not representing the authentic c-erb B gene of Xiphophorus--to be polymorphic with respect to different sex chromosomes. Linkage analysis revealed that a 5-kb fragment is linked to the Tu-Sd locus on the X chromosome, a 7-kb fragment is linked to the Tu-Sr locus on the Y chromosome, both of Xiphophorus maculatus, and that a 12-kb fragment is linked to the Tu-Li locus on the X chromosome of Xiphophorus variatus. Using different chromosomal mutants this RFLM has been mapped to a frequent deletion/translocation breakpoint of the X chromosome, less than 0.3 cM apart from the Tu locus. PMID:2841190

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

    Microsoft Academic Search

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

  16. REBASE - enzymes and genes for DNA restriction and modification

    Microsoft Academic Search

    Richard J. Roberts; Tamas Vincze; Janos Posfai; Dana Macelis

    2007-01-01

    REBASE is a comprehensive database of information about restriction enzymes, DNA methyltransferases and related proteins involved in the biological proc- ess of restriction-modification. It contains fully refe- renced information about recognition and cleavage sites, isoschizomers, neoschizomers, commercial availability, methylation sensitivity, crystal and seq- uence data. Experimentally characterized homing endonucleases are also included. All newly seque- nced genomes are analyzed for

  17. Two-step polymerase chain reactions and restriction endonuclease analyses detect and differentiate ompA DNA of Chlamydia spp.

    PubMed

    Kaltenboeck, B; Kousoulas, K G; Storz, J

    1992-05-01

    Specific and sensitive amplification of major outer membrane protein (MOMP) gene (ompA) DNA sequences of Chlamydia species with various MOMP genotypes was achieved by a two-step polymerase chain reaction (PCR). Degenerate, inosine-containing oligonucleotide primers homologous to the 5' and 3' ends of the translated regions of all chlamydial MOMP genes were used in a PCR to amplify a DNA fragment of approximately 1,120 bp. A portion of this DNA fragment was amplified in a second genus-specific reaction that yielded a DNA fragment of approximately 930 bp. A pair of degenerate oligonucleotide primers homologous to internal sequences of the primary DNA fragment was used in this PCR. This method detected three cognate chlamydial genomes in a background of 1 microgram of unrelated DNA. MOMP genes of 13 representative chlamydial MOMP genotypes of the species C. trachomatis, C. pneumoniae, and C. psittaci were amplified. In a secondary PCR, group-specific detection was achieved by the simultaneous use of one genus-specific primer and three primers derived from different fingerprint regions of three major groups of chlamydiae. This multiplex PCR differentiated the groups by the length of the amplified DNA fragments and detected the simultaneous presence of DNA sequences of the Chlamydia spp. with different MOMP genotypes. Further differentiation as ompA restriction fragment length polymorphism types among all chlamydial strains with the various MOMP genotypes analyzed here was achieved by restriction endonuclease analysis of the secondary PCR products. DNA sequences corresponding to the ompA restriction fragment length polymorphism type B577 of C. psittaci were detected in two of seven milk samples from cases of bovine mastitis. PMID:1349899

  18. Two-step polymerase chain reactions and restriction endonuclease analyses detect and differentiate ompA DNA of Chlamydia spp.

    PubMed Central

    Kaltenboeck, B; Kousoulas, K G; Storz, J

    1992-01-01

    Specific and sensitive amplification of major outer membrane protein (MOMP) gene (ompA) DNA sequences of Chlamydia species with various MOMP genotypes was achieved by a two-step polymerase chain reaction (PCR). Degenerate, inosine-containing oligonucleotide primers homologous to the 5' and 3' ends of the translated regions of all chlamydial MOMP genes were used in a PCR to amplify a DNA fragment of approximately 1,120 bp. A portion of this DNA fragment was amplified in a second genus-specific reaction that yielded a DNA fragment of approximately 930 bp. A pair of degenerate oligonucleotide primers homologous to internal sequences of the primary DNA fragment was used in this PCR. This method detected three cognate chlamydial genomes in a background of 1 microgram of unrelated DNA. MOMP genes of 13 representative chlamydial MOMP genotypes of the species C. trachomatis, C. pneumoniae, and C. psittaci were amplified. In a secondary PCR, group-specific detection was achieved by the simultaneous use of one genus-specific primer and three primers derived from different fingerprint regions of three major groups of chlamydiae. This multiplex PCR differentiated the groups by the length of the amplified DNA fragments and detected the simultaneous presence of DNA sequences of the Chlamydia spp. with different MOMP genotypes. Further differentiation as ompA restriction fragment length polymorphism types among all chlamydial strains with the various MOMP genotypes analyzed here was achieved by restriction endonuclease analysis of the secondary PCR products. DNA sequences corresponding to the ompA restriction fragment length polymorphism type B577 of C. psittaci were detected in two of seven milk samples from cases of bovine mastitis. Images PMID:1349899

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

    Microsoft Academic Search

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

    2009-01-01

    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

  20. 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.

  1. Molecular differentiation of Angiostrongylus costaricensis, A. cantonensis, and A. vasorum by polymerase chain reaction-restriction fragment length polymorphism.

    PubMed

    Caldeira, Roberta L; Carvalho, Omar S; Mendonça, Cristiane L; Graeff-Teixeira, Carlos; Silva, Márcia C; Ben, Renata; Maurer, Rafael; Lima, Walter S; Lenzi, Henrique L

    2003-12-01

    Angiostrongylus cantonensis, A. costaricensis, and A. vasorum are etiologic agents of human parasitic diseases. Their identification, at present, is only possible by examining the adult worm after a 40-day period following infection of vertebrate hosts with the third-stage larvae. In order to obtain a diagnostic tool to differentiate larvae and adult worm from the three referred species, polymerase chain reaction-restriction fragment length polymorphism was carried out. The rDNA second internal transcribed spacer (ITS2) and mtDNA cytochrome oxidase I regions were amplified, followed by digestion of fragments with the restriction enzymes RsaI, HapII, AluI, HaeIII, DdeI and ClaI. The enzymes RsaI and ClaI exhibited the most discriminating profiles for the differentiation of the regions COI of mtDNA and ITS2 of rDNA respectively. The methodology using such regions proved to be efficient for the specific differentiation of the three species of Angiostrongylus under study. PMID:15049087

  2. A restriction fragment length polymorphism results in a nonconservative amino acid substitution encoded within the first exon of the human lysyl oxidase gene

    SciTech Connect

    Csiszar, K.; Mariani, T.J.; Gosin, J.S.; Deak, S.B.; Boyd, C.D. [Robert Wood Johnson Medical School, New Brunswick, NJ (United States)] [Robert Wood Johnson Medical School, New Brunswick, NJ (United States)

    1993-05-01

    A cDNA covering most of the coding sequence for human lysyl oxidase was used to screen, by Southern blot analysis, genomic DNA from circulating lymphocytes obtained from unrelated, apparently normal individuals. A heritable restriction fragment length polymorphism (RFLP) within a PstI restriction site was detected in 36% of individuals screened (a total of 72 chromosomes were analyzed). The major allele was represented as a 1.7-kb PstI restriction fragment. The minor allele was detected as 1.4 and 0.3kb restriction fragments. Lambda phage-DNA recombinants were isolated from a human lung fibroblast genomic DNA library using the human lysyl oxidase cDNA clone. DNA sequence analysis of several selected phage recombinants revealed that 83% of the coding sequence of lysyl oxidase was localized in four separate exons. Analysis of the coding sequence within exon 1, the most 5{prime} exon within the lysyl oxidase gene, revealed that the PstI RFLP was due to a G {r_arrow} A transition resulting in a nonconservative arginine to glutamine substitution proximal to a propeptide cleavage domain encoded by exon 1 of the lysyl oxidase gene. 33 refs., 5 figs., 1 tab.

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

    Microsoft Academic Search

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

    2006-01-01

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

  4. Restriction fragment length polymorphism of two HLA-B-associated transcripts genes in five autoimmune diseases.

    PubMed

    Fugger, L; Morling, N; Ryder, L P; Jakobsen, B K; Andersen, V; Oxholm, P; Dalhoff, K; Heilmann, C; Karup Pedersen, F; Friis, J

    1991-01-01

    The restriction fragment length polymorphism of the two human HLA-B-associated transcripts (BATs) genes, BAT1 and BAT2, identifying polymorphic bands of 12, 8, 2.5, and 1.1 kb, and at 3.3, 2.7, 2.3, and 0.9 kb, respectively, was investigated in patients with primary biliary cirrhosis (PBC), systemic lupus erythematosus (SLE), pauciarticular juvenile rheumatoid arthritis (P-JRA), rheumatoid arthritis (RA), and primary Sjögren's syndrome (pSS), and in healthy Danes. The BAT2/RsaI 2.7-kb band fragment was more frequent in PBC, pSS, and SLE than in controls, but the p values did not reach significance when corrected for multiple comparisons. For pSS and SLE, the associations may be secondary to primary associations with HLA-B8 because the BAT2/RsaI 2.3-kb band, which is allelic to the BAT2/RsaI 2.7-kb band, is strongly negatively associated with HLA-B8 and HLA-DR3. The only significance obtained shows that the HLA-B8 frequency is increased in BAT2/RsaI 2.7-kb positive pSS patients as compared to the corresponding controls indicating that the HLA-B8 association may be strongest. No missing or extra DNA fragments were observed in the disease groups when compared with controls indicating that gross deletions or duplications of the BAT1 and BAT2 genes in the patients are unlikely. In conclusions, it cannot be excluded that the BAT2/RsaI 2.7-kb band may contribute to the susceptibility to PBC, pSS, and SLE. PMID:1672123

  5. Rapid identification of filamentous actinomycetes to the genus level using genus-specific 16S rRNA gene restriction fragment patterns

    Microsoft Academic Search

    Andrew E. Cook; Paul R. Meyers

    2003-01-01

    A rapid method for identifying filamentous actinomycete genera was developed based on 16S rRNA gene restriction fragment patterns. The patterns were generated by using specific restriction endonucleases to perform in silico digestions on the 16S rRNA gene sequences of all validly published filamentous actinomycete species. The method was applied to identifying actinomycete isolates from soil. Amplified 16S rDNA of soil

  6. DNA restriction and modification systems in Salmonella

    Microsoft Academic Search

    Charles Colson; Aline Van Pal

    1974-01-01

    Haploid hybrids between Salmonella typhimurium Hfr and Escherichia coli Fexercise two additive types of restriction and modification (SA and SB) on phage ?. System SA had been detected previously in S. typhimurium with phage L. Independent mutants in the SA and SB systems were isolated. P22- and P1-mediated transductions in S. typhimurium and in hybrids established that the genes governing

  7. Cell nucleus and DNA fragmentation are not required for apoptosis

    Microsoft Academic Search

    Klaus Schulze-Osthoff; Herming Walczak; Peter H. Krammer

    1994-01-01

    Apoptosis is the predominant form of cell death and occurs under a variety of physiological and pathological conditions. Cells undergoing apoptotic cell death reveal a characteristic sequence of cytologi- cal alterations including membrane blebbing and nu- clear and cytoplasmic condensation. Activation of an endonuclease which cleaves genomic DNA into inter- nucleosomal DNA fragments is considered to be the hallmark of

  8. Restriction Endonuclease Mapping of the Proviral DNA of the Exogenous RIII Murine Mammary Tumor Virus

    PubMed Central

    Etkind, Polly R.; Szabo, Paul; Sarkar, Nurul H.

    1982-01-01

    Cellular DNA containing integrated murine mammary tumor virus (MuMTV) was isolated from FeI/C6 feline kidney cells and CCL64 mink lung cells infected with milkborne RIII MuMTV. By using restriction enzyme HpaI, intact RIII MuMTV provirus (length, 8.7 kilobases [kb]) was excised from the cellular DNA. Subsequent restriction endonuclease analysis of this HpaI fragment with KpnI, HindIII, EcoRI, BamHI, BglII, PstI, SstI, SalI, and XhoI enabled us to construct a map of the RIII virus genome. A comparison of this map with the maps of the GR and C3H MuMTV's revealed that there are greater sequence differences between the RIII virus and the GR and C3H MuMTV proviruses than there are between the GR and C3H proviruses. The following are features of the restriction map unique to the RIII provirus: the presence of three BamHI and two EcoRI cleavage sites, a HpaI cleavage site in the terminal 3?-5? repeat unit of the provirus, and the absence of an XhoI cleavage site. Another distinguishing feature of the RIII provirus is that the sizes of some of the restriction fragments produced by cleavage of the RIII provirus with PstI are different from the sizes of the fragments obtained by PstI cleavage of the GR and C3H proviruses. Like the GR proviral DNA, the RIII proviral DNA has three SstI (SacI) cleavage sites, whereas the C3H provirus has only two SstI sites. HpaI digestion of MuMTV-infected mink lung cell DNA revealed only one class of provirus (an 8.7-kb fragment); however, we observed several minor classes of RIII proviral DNA in addition to the major class of provirus DNA in infected cat kidney cells. PstI digestion of the HpaI 8.7-kb fragments from both feline and mink cells generated a 3.7-kb DNA fragment identical in size to a PstI-generated fragment that has been found in GR and C3H milkborne virus-infected cells. Although a fragment similar in size to the milkborne 3.7-kb PstI fragment has been found as an endogenous component in many C3H and GR mouse tissues, we did not observe such an endogenous fragment in the RIII mouse strain. Therefore, the 3.7-kb fragment may be useful as a marker for the milkborne RIII MuMTV provirus in RIII mice. Images PMID:6284976

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

    PubMed

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

    2014-06-01

    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

  10. Restriction fragment length polymorphisms in polymerase chain reaction amplified ribosomal DNAs of three Trichogramma (Hymenoptera: Trichogrammatidae) species.

    PubMed

    Sappal, N P; Jeng, R S; Hubbes, M; Liu, F

    1995-06-01

    Restriction fragment length polymorphisms from PCR amplified ribosomal DNAs of three Trichogramma species, T. minutum, T. brassicae, and T. near sibiricum, were studied. Length variation in the internal transcribed spacer (ITS) region was observed. The ITS region of T. brassicae is about 1350 base pairs (bp) in length and those of T. minutum and T. near sibiricum are 1300 bp. These three species also differ in the size of their ITS1 and ITS2 regions. Restriction enzyme digestions of these regions showed unique banding patterns for each species. The amplified 18S region of ribosomal DNA is about 1800 bp in length and showed no length variation between the three species of Trichogramma. Restriction enzyme digestion of this region by BamHI differentiated T. brassicae from the other two species. Restriction site maps of the ITS and 18S regions were constructed for each species. The amplified 28S region is about 1700 bp for these three species. Restriction of this region by RsaI and SacII differentiates these three species. The reported results indicate that these species of Trichogramma can be clearly differentiated from one another by nuclear ribosomal DNA markers. PMID:7557356

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

    SciTech Connect

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

    1987-07-01

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

  12. Brassica taxonomy based on nuclear restriction fragment length polymorphisms (RFLPs)

    Microsoft Academic Search

    K. Song; T. C. Osborn; P. H. Williams

    1990-01-01

    RFLPs were used to study genome evolution and phylogeny in Brassica and related genera. Thirtyeight accessions, including 10 accessions of B. rapa (syn. campestris), 9 cultivated types of B. oleracea, 13 nine-chromosome wild brassicas related to B. oleracea, and 6 other species in Brassica and allied genera, were examined with more then 30 random genomic DNA probes, which identified RFLPs

  13. Restriction fragment length polymorphism caused by a deletion involving Alu sequences within the human. alpha. sub 2 -plasmin inhibitor gene

    SciTech Connect

    Miura, Osamu; Sugahara, Yuichi; Nakamura, Yuichi; Hirosawa, Shinsaku; Aoki, Nobuo (Tokyo Medical and Dental Univ. (Japan))

    1989-06-13

    A restriction fragment length polymorphism within the human {alpha}{sub 2}-plasmin inhibitor gene has been detected by Southern blot hybridization using an {alpha}{sub 2}-plasmin inhibitor cDNA probe. This restriction fragment length polymorphism can be attributed to the presence of two alleles, A and B, that are distributed in Hardy-Weinberg equilibrium with frequencies of 73.5% and 2.65%, respectively, in 66 unrelated Caucasian individuals or with frequencies of 51.0% and 49.0%, respectively, in 50 unrelated Japanese individuals. The minor allele, B, is due to a deletion of about 720 base pairs in intron 8 of the {alpha}{sub 2}-plasmin inhibitor gene. Sequence analysis of the deletion junction in allele B and the corresponding regions of allele A demonstrated the presence of oppositely oriented Alu sequences at the 5{prime} and 3{prime} deletion boundaries. These data suggest that this restriction fragment length polymorphism was caused by intrastrand recombination between Alu sequences.

  14. 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

  15. Typing of Rhizobia by PCR DNA Fingerprinting and PCR-Restriction Fragment Length Polymorphism Analysis of Chromosomal and Symbiotic Gene Regions: Application toRhizobium leguminosarum and Its Different Biovars

    Microsoft Academic Search

    GISELE LAGUERRE; PATRICK MAVINGUI; MARIE-REINE ALLARD; MARIE-PAULE CHARNAY; PHILIPPE LOUVRIER; SYLVIE-ISABELLE MAZURIER; LIONEL RIGOTTIER-GOIS

    1996-01-01

    Characterization of 43 strains of Rhizobium leguminosarum biovars viciae, trifolii, and phaseoli was per- formed by two methodologies based on PCR amplification, i.e., PCR DNA fingerprinting of interrepeat sequencesandrestrictionfragmentlengthpolymorphism(RFLP)analysisofPCR-amplifiedchromosomaland symbiotic gene regions. Groupings generated by PCR DNAfingerprinting with either extragenic palindromic repetitive primers or two different single random primers were correlated with similar levels of resolution. Although less discriminating, PCR-RFLP

  16. Identification of mycobacteria to the species level by automated restriction enzyme fragment length polymorphism analysis

    Microsoft Academic Search

    M. Tötsch; E. Brömmelkamp; A. Stücker; K. Schmid; W. Böcker; B. Dockhorn-Dworniczak; M. Fille; R. Gross; P. Wiesner

    1995-01-01

    An automated method for the restriction fragment length polymorphism (RFLP) analysis for the differentiation of mycobacteria to the species level is described. After polymerase chain reaction (PCR) amplification of a sequence of the gene encoding the 65-kDa surface antigen common to all mycobacteria the product was investigated by RFLP analysis. For accurate determination of fragment sizes the asymmetrically fluorescein-labelled PCR

  17. Fenton fragmentation for faster electrophoretic on chip purification of amplifiable genomic DNA.

    PubMed

    Hakenberg, S; Hügle, M; Meyer, P; Behrmann, O; Dame, G; Urban, G A

    2015-05-15

    With a rapid and simple actuation protocol electrophoretic nucleic acid extraction is easy automatable, requires no moving parts, is easy to miniaturize and furthermore possesses a size dependent cut-off filter adjustable by the pore size of the hydrogel. However electrophoretic nucleic acid extraction from bacteria has so far been applied mainly for short RNA targets. One of the reasons is that electrophoretic processing of unfragmented genomic DNA strands is time-consuming, because of the length. Here DNA fragmentation would accelerate extraction and isolation. We introduce on-chip lysis and non-enzymatic DNA cleavage directly followed by a purifying step for receiving amplifiable DNA fragments from bacteria in less than 25min. In contrast to restriction enzymes the Fenton reaction is known to cleave DNA without nucleotide specificity. The reaction mix contains iron(II) EDTA, sodium ascorbate, hydrogen peroxide and lysozyme. The degree of fragmentation can be adjusted by the concentration of reagents. The results enable electrophoretic extraction methods to unspecifically process long genomic DNA in a short time frame, e.g. for pathogen detection in a lab-on-a-chip format. PMID:24970713

  18. 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.

  19. 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.

  20. Effects of pretreatment on the denaturation and fragmentation of genomic DNA for DNA hybridization.

    PubMed

    Wang, Xiaofang; Son, Ahjeong

    2013-12-01

    DNA hybridization is an important step for a number of bioassays such as fluorescence in situ hybridization, microarrays, as well as the NanoGene assay. Denaturation and fragmentation of genomic DNA are two critical pretreatments for DNA hybridization. However, no thorough and systematic characterization on denaturation and fragmentation has been carried out for the NanoGene assay so far. In this study, we investigated the denaturation and fragmentation of the bacterial gDNA with physical treatments (i.e., heating and sonication) and chemical treatments (i.e., dimethyl sulfoxide). First of all, a simple approach for indicating the denaturation fraction was developed based on the absorbance difference (i.e., hyperchromic effect) between the double-stranded DNA and single-stranded DNA fragments. Then the denaturation capabilities of the treatments to the gDNA were elucidated, followed by the examination of the possible renaturation over time. The fragmentation of the gDNA by each treatment was also investigated. Based on denaturation efficiency, minimum renaturation tendency, and fragmentation, the sonication method was found to be the best among the six methods. We further demonstrated that the sonication method produced the best result among the treatments examined for the DNA hybridization in the NanoGene assay. PMID:24162665

  1. Selective microbial genomic DNA isolation using restriction endonucleases.

    PubMed

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

    2014-01-01

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

  2. Selective Microbial Genomic DNA Isolation Using Restriction Endonucleases

    PubMed Central

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

    2014-01-01

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

  3. Fragmentation dynamics of DNA sequence duplications

    E-print Network

    M. V. Koroteev; J. Miller

    2013-10-29

    Motivated by empirical observations of algebraic duplicated sequence length distributions in a broad range of natural genomes, we analytically formulate and solve a class of simple discrete duplication/substitution models that generate steady-states sharing this property. Continuum equations are derived for arbitrary time-independent duplication length source distribution, a limit that we show can be mapped directly onto certain fragmentation models that have been intensively studied by physicists in recent years. Quantitative agreement with simulation is demonstrated. These models account for the algebraic form and exponent of naturally occuring duplication length distributions without the need for fine-tuning of parameters.

  4. Preparation and characterization of an anti-DNA monoclonal antibody showing size selectivity toward DNA fragments.

    PubMed

    Onishi, Yoshiaki; Kato, Megumi; Hanyu, Yoshiro

    2004-10-01

    Anti-DNA monoclonal antibodies were prepared using an in vitro immunization method. Balb/c mouse splenocytes were immunized with HeLa cell nuclear extract in the presence of N-acetylmuramyl-L-alanyl-D-isoglutamine and fused with P3U1 myeloma cells using PEG 4000. After HAT selection and ELISA using fragmented HeLa genomic DNA, an anti-DNA monoclonal antibody was obtained. The monoclonal antibody D-1-1, whose isotype was IgM, interacted with a variety of double-stranded DNA. The antibody reacted only with DNA fragments longer than 0.8 kbp, and its apparent dissociation constant for a 1.0-kbp DNA fragment was 34 nM. This antibody will be a helpful tool for the detection of DNA structures. PMID:15672610

  5. 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

  6. Clinical significance of telomerase activation and telomeric restriction fragment (TRF) in cervical cancer

    Microsoft Academic Search

    D. K Zhang; H. Y. S Ngan; R. Y. S Cheng; A. N. Y Cheung; S. S Liu; S. W Tsao

    1999-01-01

    Telomerase activation was examined in 50 cases of cervical cancer, 27 normal cervix and five cervical cancer cell lines using the sensitive polymerase chain reaction (PCR)-based TRAP (telomeric repeat amplification protocol) assay. Telomeric restriction fragment (TRF) length of these specimens was measured by Southern hybridisation. Telomerase activation was common in cervical cancers and was detected in 46\\/50 cases (92%). Telomerase

  7. Characterization of avian strains of Pasteurella multocida by restriction endonuclease and amplified fragment length polymorphism

    Microsoft Academic Search

    S. B. Shivachandra; A. A. Kumar; R. Gautam; S. Joseph; M. K. Saxena; P. Chaudhuri; S. K. Srivastava

    2006-01-01

    Avian strains of Pasteurella multocida were typed by employing restriction endonuclease analysis (REA) and single enzyme-amplified fragment length polymorphism (AFLP) to evaluate their applicability for epidemiological studies of fowl cholera outbreaks. A total of 72 strains isolated from different avian species (chicken, duck, turkey, quail and goose) belonging to various geographical regions of India were characterized. REA using two different

  8. Terminal Restriction Fragment Length Polymorphism Data Analysis for Quantitative Comparison of Microbial Communities

    Microsoft Academic Search

    Christopher B. Blackwood; Terry Marsh; Sang-Hoon Kim; Eldor A. Paul

    2003-01-01

    Terminal restriction fragment length polymorphism (T-RFLP) is a culture-independent method of obtaining a genetic fingerprint of the composition of a microbial community. Comparisons of the utility of different methods of (i) including peaks, (ii) computing the difference (or distance) between profiles, and (iii) perform- ing statistical analysis were made by using replicated profiles of eubacterial communities. These samples included soil

  9. A modular assembly cloning technique (aided by the BIOF software tool) for seamless and error-free assembly of long DNA fragments

    PubMed Central

    2012-01-01

    Background Molecular cloning of DNA fragments >5 kbp is still a complex task. When no genomic DNA library is available for the species of interest, and direct PCR amplification of the desired DNA fragment is unsuccessful or results in an incorrect sequence, molecular cloning of a PCR-amplified region of the target sequence and assembly of the cloned parts by restriction and ligation is an option. Assembled components of such DNA fragments can be connected together by ligating the compatible overhangs produced by different restriction endonucleases. However, designing the corresponding cloning scheme can be a complex task that requires a software tool to generate a list of potential connection sites. Findings The BIOF program presented here analyzes DNA fragments for all available restriction enzymes and provides a list of potential sites for ligation of DNA fragments with compatible overhangs. The cloning scheme, which is called modular assembly cloning (MAC), is aided by the BIOF program. MAC was tested on a practical dataset, namely, two non-coding fragments of the translation elongation factor 1 alpha gene from Chinese hamster ovary cells. The individual fragment lengths exceeded 5 kbp, and direct PCR amplification produced no amplicons. However, separation of the target fragments into smaller regions, with downstream assembly of the cloned modules, resulted in both target DNA fragments being obtained with few subsequent steps. Conclusions Implementation of the MAC software tool and the experimental approach adopted here has great potential for simplifying the molecular cloning of long DNA fragments. This approach may be used to generate long artificial DNA fragments such as in vitro spliced cDNAs. PMID:22709633

  10. Dependence on radiation quality of DNA fragmentation spectra

    Microsoft Academic Search

    Alessandro Campa; Andrea Ottolenghi; Daniele Alloni; Francesca Ballarini; Mauro Belli; Giuseppe Esposito; Angelica Facoetti; Werner Friedland; Marco Liotta; Herwig Paretzke

    2008-01-01

    Energy deposition by radiation initially gives rise to cellular critical lesions such as DNA doublestrand breaks (DSB), that later lead to the formation of relevant biological endpoints. Studies on fragment size distributions induced by radiations of various qualities can be of great help in linking the characteristics of radiation to cellular endpoints, providing information for understanding the main mechanisms of

  11. In vitro DNA fragmentation of mitochondrial DNA caused by single-stranded breakage related to macroplasmodial senescence of the true slime mold, Physarum polycephalum.

    PubMed

    Abe, T; Takano, H; Sasaki, N; Mori, K; Kawano, S

    2000-02-01

    We found that mitochondrial DNA (mtDNA) isolated from Physarum polycephalum fragmented itself in weak ionic solutions. The mtDNA was dissolved in STE (saline Tris-EDTA: 150 mM NaCl, 10 mM Tris-HCl, 1 mM EDTA), TE (10 mM Tris-HCl, 1 mM EDTA) and DW, and then electrophoresed in an agarose gel. The intact 86-kbp mtDNA band was seen in STE, but several novel bands appeared in TE and DW. In TE, two discrete bands appeared at 6.7-kbp (alpha-band) and 5.0-kbp (beta-band), whereas at least 17 discrete bands were observed in distilled water (DW). These fragmentation patterns were not stoichiometric, as seen when using restriction endonucleases, but were clearly different from the degradation of DNA caused by a physical shearing force or a contaminating nuclease. In this paper, we characterize this in vitro fragmentation of mtDNA from P. polycephalum. We located 19 fragments, including the alpha and beta fragments, on a mtDNA restriction map, and demonstrated that these cleavage sites were S1 nuclease-sensitive regions, which are single-stranded DNA regions such as nicks and gaps in the mtDNA. The alpha and beta fragments are derived from the region encoding ribosomal RNAs (rRNAs) and the ATP synthase (atpA) gene, while the other 17 fragments are not derived from any specific region, but the cleavage sites are located throughout the mtDNA molecule. In P. polycephalum, it is well known that the growth rate of macroplasmodia decreases with aging. Equal amounts of mtDNA from juvenile and aged macroplasmodia were electrophoresed and the frequency of the beta fragment in each sample was measured. The ratio of the beta band to the total signal including background was estimated to be 3.3-4.0% in juvenile macroplasmodia, whereas it increased to 8.3-28.2% in aged macroplasmodia. This result suggests that the in vitro fragmentation of mtDNA is associated with macroplasmodial senescence. The single-stranded breakage of mtDNA of P. polycephalum may accumulate with age. PMID:10743569

  12. Rapid construction of a Bacterial Artificial Chromosomal (BAC) expression vector using designer DNA fragments.

    PubMed

    Chen, Chao; Zhao, Xinqing; Jin, Yingyu; Zhao, Zongbao Kent; Suh, Joo-Won

    2014-10-17

    Bacterial artificial chromosomal (BAC) vectors are increasingly being used in cloning large DNA fragments containing complex biosynthetic pathways to facilitate heterologous production of microbial metabolites for drug development. To express inserted genes using Streptomyces species as the production hosts, an integration expression cassette is required to be inserted into the BAC vector, which includes genetic elements encoding a phage-specific attachment site, an integrase, an origin of transfer, a selection marker and a promoter. Due to the large sizes of DNA inserted into the BAC vectors, it is normally inefficient and time-consuming to assemble these fragments by routine PCR amplifications and restriction-ligations. Here we present a rapid method to insert fragments to construct BAC-based expression vectors. A DNA fragment of about 130 bp was designed, which contains upstream and downstream homologous sequences of both BAC vector and pIB139 plasmid carrying the whole integration expression cassette. In-Fusion cloning was performed using the designer DNA fragment to modify pIB139, followed by ?-RED-mediated recombination to obtain the BAC-based expression vector. We demonstrated the effectiveness of this method by rapid construction of a BAC-based expression vector with an insert of about 120?kb that contains the entire gene cluster for biosynthesis of immunosuppressant FK506. The empty BAC-based expression vector constructed in this study can be conveniently used for construction of BAC libraries using either microbial pure culture or environmental DNA, and the selected BAC clones can be directly used for heterologous expression. Alternatively, if a BAC library has already been constructed using a commercial BAC vector, the selected BAC vectors can be manipulated using the method described here to get the BAC-based expression vectors with desired gene clusters for heterologous expression. The rapid construction of a BAC-based expression vector facilitates heterologous expression of large gene clusters for drug discovery. PMID:25454071

  13. Sizing Highly Fragmented DNA in Individual Apoptotic Cells Using the Comet Assay and a DNA Crosslinking Agent

    Microsoft Academic Search

    Peggy L. Olive; Judit P. Banáth

    1995-01-01

    TK6 human B lymphoblast cells exposed to ionizing radiation undergo apoptosis in a time and dose-dependent manner. The resulting highly fragmented DNA is easily detected using the comet assay, a sensitive microscopic gel electrophoresis method capable of measuring DNA strand breaks in individual cells. The degree of DNA fragmentation may be indicative of different stages in the fragmentation process, responses

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

    Microsoft Academic Search

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

    1998-01-01

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

  15. Differentiation of Paenibacillus larvae subsp. larvae, the Cause of American Foulbrood of Honeybees, by Using PCR and Restriction Fragment Analysis of Genes Encoding 16S rRNA

    PubMed Central

    Alippi, Adriana M.; López, Ana Claudia; Aguilar, O. Mario

    2002-01-01

    A rapid procedure for the identification of Paenibacillus larvae subsp. larvae, the causal agent of American foulbrood (AFB) disease of honeybees (Apis mellifera L.), based on PCR and restriction fragment analysis of the 16S rRNA genes (rDNA) is described. Eighty-six bacterial strains belonging to 39 species of the genera Paenibacillus, Bacillus, Brevibacillus, and Virgibacillus were characterized. Amplified rDNA was digested with seven restriction endonucleases. The combined data from restriction analysis enabled us to distinguish 35 profiles. Cluster analysis revealed that P. larvae subsp. larvae and Paenibacillus larvae subsp. pulvifaciens formed a group with about 90% similarity; however, the P. larvae subsp. larvae restriction fragment length polymorphism pattern produced by endonuclease HaeIII was found to be unique and distinguishable among other closely related bacteria. This pattern was associated with DNA extracted directly from honeybee brood samples showing positive AFB clinical signs that yielded the restriction profile characteristic of P. larvae subsp. larvae, while no amplification product was obtained from healthy larvae. The method described here is particularly useful because of the short time required to carry it out and because it allows the differentiation of P. larvae subsp. larvae-infected larvae from all other species found in apiarian sources. PMID:12089057

  16. Mutagenicity Assessment of Organophosphates using Polymerase Chain Reaction-Restriction Fragment Length Polymorphism Assay

    PubMed Central

    Bhinder, Preety; Chaudhry, Asha

    2013-01-01

    Objectives: In this study we have evaluated the mutagenicity of organophosphate pesticides acephate, chlorpyrifos, and profenofos using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay with the mosquito Culex quinquefasciatus taken as an experimental model. Materials and Methods: Second instar larvae were treated with LC20 of each pesticide for 24 h and mutations induced in the sequence of mitochondrial COII gene (690bp) were studied from restriction patterns generated with AluI, PacI, and PsiI restriction endonucleases. Results: Variations in the number and size of digested fragments were recorded from treated individuals compared with controls showing that the restriction enzymes created a cut at different locations. In addition, sequences of COII gene from control and treated individuals were also used to confirm the RFLP patterns. From the sequence alignment data, it was found that mutations caused the destruction and generation of restriction sites in the gene sequence of treated individuals. Conclusion: This study indicates that all the three pesticides had potential to induce mutations in the normal sequence of COII gene and also advocates the use of PCR-RFLP assay as an efficient, rapid, and sensitive technique to detect mutagenicity of pesticides. PMID:24403735

  17. Detection of Oxazolidinone-Resistant Enterococcus faecalis and Enterococcus faecium Strains by Real-Time PCR and PCR-Restriction Fragment Length Polymorphism Analysis

    Microsoft Academic Search

    Neil Woodford; Luke Tysall; Cressida Auckland; Mark W. Stockdale; Andrew J. Lawson; Rachel A. Walker; David M. Livermore

    2002-01-01

    A real-time PCR assay identified linezolid-resistant Enterococcus faecalis and Enterococcus faecium isolates with a G2576U rRNA mutation. PCR-restriction fragment length polymorphism analysis of ribosomal DNA amplicons with NheI also detected this mutation. Both assays detected isolates heterozygous at this position. Recognition of isolates with what is presently the most frequent oxazolidinone resistance mutation may aid surveillance and individual case management.

  18. IS1311 and IS1245 Restriction Fragment Length Polymorphism Analyses, Serotypes, and Drug Susceptibilities of Mycobacterium avium Complex Isolates Obtained from a Human Immunodeficiency Virus-Negative Patient

    Microsoft Academic Search

    Lenka Dvorska; Milan Bartos; Oldrich Ostadal; Jarmila Kaustova; Ludmila Matlova; Ivo Pavlik

    2002-01-01

    Six isolates of Mycobacterium avium of genotype dnaJ IS901 IS1311 IS1245 and serotypes 6 (n 1), 6\\/9, (n 2), and 9 (n 3) were obtained within a 5-month period from a human immunodeficiency virus-negative patient treated for tuberculosis. The isolates were identified with PvuII restriction fragment length polymor- phism (RFLP) analysis as a single IS1311 RFLP type and six different

  19. Linkage of the Scrapie-associated Fibril Protein (PrP) Gene and Sinc Using Congenic Mice and Restriction Fragment Length Polymorphism Analysis

    Microsoft Academic Search

    NORA HUNTER; JAMES HOPE; I. McConnell; A. G. Dickinson

    1987-01-01

    SUMMARY Sine, with two alleles p7 and s7, is the major gene determining the incubation period of all strains of scrapie in mice. The major protein (PrP) of scrapie-associated fibrils is encoded by a cellular gene and we have used a cDNA copy of the hamster PrP mRNA to carry out restriction fragment length polymorphism (RFLP) analysis of different inbred

  20. Elimination of Bacterial DNA from Taq DNA Polymerases by Restriction Endonuclease Digestion

    Microsoft Academic Search

    NORA M. CARROLL; PETER ADAMSON; NARCISS OKHRAVI

    1999-01-01

    The incidence of false positives due to the presence of bacterial DNA in Taq DNA polymerase is an obstacle to the use of PCR in the diagnosis of infection. We describe a method that uses a restriction enzyme to destroy the ability of contaminating sequences to act as templates for a nested PCR which uses primers based on the 16S

  1. 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

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

    PubMed Central

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

    2015-01-01

    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

  3. Characterization of Campylobacter jejuni and Campylobacter coli genotypes in poultry flocks by restriction fragment length polymorphism (RFLP) analysis.

    PubMed

    Carreira, Ana Cláudia; Cunha, Mónica V

    2015-01-01

    We describe a simple, rapid, and discriminatory methodology that allows the routine molecular characterization of Campylobacter jejuni and Campylobacter coli isolates. The proposed approach is built on one of the earliest and simplest molecular typing methods ever, consisting on the analysis of the fragments of different lengths generated by digestion of homologous DNA sequences with specific restriction endonucleases, a process known as restriction fragment length polymorphism (RFLP) analysis. The strategy underneath the workflow reported here is meant to explore the polymorphisms of Campylobacter spp. flaA gene (flaA-RFLP) that allows the local investigation of the genetic diversity and distribution of C. coli and C. jejuni isolates from different sources, namely, chickens' caeca. Although not appropriate for global and long-term epidemiological studies as a single approach, flaA-RFLP analysis can be very useful in surveys limited in space and time and, for specific epidemiological settings, an alternative to more modern and resource-demanding techniques. PMID:25399105

  4. Cloning and characterization of a highly reiterated 5.8-kilobase pair nucleolar EcoRI DNA fragment found in Novikoff hepatoma ascites cells.

    PubMed

    Parker, D L; Busch, H; Rothblum, L I

    1981-02-17

    The DNA of Novikoff hepatoma ascites cells was found to contain a 3.6-megadalton EcoRI restriction fragment, referred to as EcoRI fragment A (Parker et al., 1979). C0t analyses demonstrated an enrichment of fragment A sequences in Novikoff hepatoma genome relative to normal rat liver DNA. This fragment was cloned in lambda gtWES to determine its molecular structure and sequence organization. The DNA from a positive clone was labeled by nick translation and hybridized to a Southern blot of EcoRI digested Novikoff DNA. Distinct hybrids formed with the region corresponding to fragment A. The greater degree of hybridization to the nucleolar fraction suggested a nucleolar enrichment of fragment A. Fragment A has a PstI site approximately 300 base pairs from one terminus which was used to generate mono-5'-32P-labeled fragments. The larger PStI subfragment, 5500 base pairs, labeled at a single terminus, was used to evolve a restriction enzyme map. The 300 base pair fragment was partially sequenced, revealing the presence of a repetitive sequence "island", TT(GTCT)8(GAAT)5G-. C0t analysis, utilizing the purified clone as a probe, confirmed the enrichment of fragment A sequences in the tumor relative to the normal rat liver control. PMID:6260140

  5. Differentiation of Porcine Brachyspira Species by a Novel nox PCR-Based Restriction Fragment Length Polymorphism Analysis

    Microsoft Academic Search

    Judith Rohde; Anja Rothkamp; Gerald F. Gerlach

    2002-01-01

    A novel PCR-based restriction fragment length polymorphism analysis of the Brachyspira nox gene was developed. The restriction patterns for Brachyspira hyodysenteriae, B. pilosicoli, B. intermedia, B. murdochii, and B. innocens were highly distinct with two restriction endonucleases only. The assay proved to be user-friendly and robust. The porcine intestinal tract is frequently colonized by dif- ferent Brachyspira species. Brachyspira hyodysenteriae

  6. Cell nucleus and DNA fragmentation are not required for apoptosis

    PubMed Central

    1994-01-01

    Apoptosis is the predominant form of cell death and occurs under a variety of physiological and pathological conditions. Cells undergoing apoptotic cell death reveal a characteristic sequence of cytological alterations including membrane blebbing and nuclear and cytoplasmic condensation. Activation of an endonuclease which cleaves genomic DNA into internucleosomal DNA fragments is considered to be the hallmark of apoptosis. However, no clear evidence exists that DNA degradation plays a primary and causative role in apoptotic cell death. Here we show that cells enucleated with cytochalasin B still undergo apoptosis induced either by treatment with menadione, an oxidant quinone compound, or by triggering APO-1/Fas, a cell surface molecule involved in physiological cell death. Incubation of enucleated cells with the agonistic monoclonal anti-APO-1 antibody revealed the key morphological features of apoptosis. Moreover, in non-enucleated cells inhibitors of endonuclease blocked DNA fragmentation, but not cell death induced by anti-APO-1. These data suggest that DNA degradation and nuclear signaling are not required for induction of apoptotic cell death. PMID:7523418

  7. DNA Flexibility Studied by Covalent Closure of Short Fragments into Circles

    NASA Astrophysics Data System (ADS)

    Shore, David; Langowski, Jorg; Baldwin, Robert L.

    1981-08-01

    The ring closure probability, or j factor, has been measured for DNA restriction fragments of defined sequence bearing EcoRI cohesive ends and ranging in size from 126 to 4361 base pairs (bp). The j factor is defined as the ratio of the equilibrium constants for cyclization and for bimolecular association via the cohesive ends. The end-joining reactions are fast compared to covalent closure of the cohesive ends by T4 DNA ligase. The rate of ligase closure is shown to be proportional to the equilibrium fraction of DNA molecules with joined cohesive ends, both in cyclization and in bimolecular association reactions. The j factor changes by less than 10-fold between 242 and 4361 bp, whereas it decreases by more than 100-fold between 242 and 126 bp as the DNA reaches the size range of the persistence length (150 bp). As regards ring closure, short DNA fragments are surprisingly flexible. These data are in good agreement with predictions by others for the ring closure probability of a wormlike chain.

  8. Variation in Restriction Fragment Length Polymorphisms among Serial Isolates from Patients with Trichophyton rubrum Infection

    PubMed Central

    Gupta, Aditya K.; Kohli, Yatika; Summerbell, Richard C.

    2001-01-01

    Molecular genotyping of strains of Trichophyton rubrum and T. mentagrophytes from patients with onychomycosis of the toes was performed to ascertain whether the fungal genotype changes over the course of time as sequential samples were obtained from patients receiving antifungal therapy and during follow-up. Sixty-six serial strains of T. rubrum and 11 strains of T. mentagrophytes were obtained from 20 patients (16 patients with T. rubrum, 4 with T. mentagrophytes) who were treated with oral antifungal therapy and observed over periods of up to 36 months. These strains were screened for genetic variation by hybridization of EcoRI-digested genomic DNAs with a probe amplified from the small-subunit (18S) ribosomal DNA and adjacent internal transcribed spacer regions. A total of five restriction fragment length polymorphism (RFLP) types were observed among 66 strains of T. rubrum. Two major RFLP types, differentiated by one band shift, represented 68% of the samples. None of the patients had a unique genotype. More than one RFLP type was often observed from a single patient (same nail) over a period of 1, 2, or 3 years, even in cases that did not appear cured at any time. Samples taken from different nails of the same patient had either the same or a different genotype. The genotypic variation did not correspond to any detectable phenotypic variation. Furthermore, no correlation was observed between the efficacy of the treatment administered and the genotype observed. While the DNA region studied distinguished among T. rubrum, T. mentagrophytes, and T. tonsurans, intraspecific RFLP variation was observed for T. rubrum and T. mentagrophytes strains. While independent multiple infection and coinhabitation of multiple strains may explain the presence of different genotypes in a nail, microevolutionary events such as rapid substrain shuffling, as seen in studies of repetitive regions in Candida species, may also produce the same result. The recovery of multiple strains during the course of sequential sampling of uncured patients further suggests that the typing system is not able to distinguish between relapse or reinfection, ongoing infection, and de novo infection. PMID:11526160

  9. Determination of genotypes of hepatitis C virus in Venezuela by restriction fragment length polymorphism.

    PubMed Central

    Pujol, F H; Loureiro, C L; Devesa, M; Blitz, L; Parra, K; Beker, S; Liprandi, F

    1997-01-01

    Hepatitis C virus genotypes in Venezuela were analyzed by restriction fragment length polymorphism in the 5' noncoding region. The absence of BstUI digestion was found to be a useful marker for genotype 2 specimens. From 122 serum samples, 66, 20, and 2.5% were classified as genotypes 1, 2, and 3, respectively; 0.8% were classified as genotype 4; and 10% appeared to be mixed infections. PMID:9196212

  10. Restriction fragment length polymorphism and allozyme linkage map of Cuphea lanceolata

    Microsoft Academic Search

    D. M. Webb; S. J. Knapp; L. A. Tagliani

    1992-01-01

    Cuphea lanceolata Ait. has had a significant role in the domestication of Cuphea and is a useful experimental organism for investigating how medium-chain lipids are synthesized in developing seeds. To expand the genetics of this species, a linkage map of the C. lanceolata genome was constructed using five allozyme and 32 restriction-fragment-length-polymorphism (RFLP) marker loci. These loci were assigned to

  11. Hot Fusion: An Efficient Method to Clone Multiple DNA Fragments as Well as Inverted Repeats without Ligase

    PubMed Central

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

    2014-01-01

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

  12. Rearrangement of DNA fragments: a branch-and-cut algorithm

    Microsoft Academic Search

    C. E. Ferreira; C. Carvalho De Souza; Yoshiko Wakabayashi

    2002-01-01

    We consider a problem called minimum k-contig problem (MkCP), whose specialization to an alphabet with four symbols can be seen as a problem that arises in the process of arranging DNA fragments to reconstruct a molecule. We present a graph theoretical formulation of MkCP and mention some extensions. We show this problem to be NP-hard for every k?1 (for an

  13. 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

  14. DNA Modification Methylase Activity of Escherichia coli Restriction Endonucleases K and P

    Microsoft Academic Search

    Allan Haberman; Janet Heywood; Matthew Meselson

    1972-01-01

    The highly purified restriction endonucleases of E. coli K and coliphage P1 transfer methyl groups from S-adenosylmethionine to adenine residues of unmodified DNA. Incubation of unmodified DNA with endonucleases K or P and S-adenosylmethionine renders the DNA resistant to restriction. The enzymes, therefore, have both restriction endonuclease and modification methylase activities.

  15. DNA-anti-DNA immune complexes. Antibody protection of a discrete DNA fragment from DNase digestion in vitro.

    PubMed Central

    Emlen, W; Ansari, R; Burdick, G

    1984-01-01

    We examined the ability of DNase I to digest DNA that was contained with DNA-anti-DNA immune complexes. IgG isolated from the sera of 20 patients with systemic lupus erythematosus (SLE) and containing antibodies to DNA was incubated with double-stranded DNA to form immune complexes. Excess DNase was added, and digestion of DNA was monitored by the conversion of DNA to TCA soluble products. IgG from 8 of the 20 SLE patients protected DNA from degradation by DNase in direct proportion to the amount of DNA bound to IgG as measured in the Farr binding assay. Using IgG from these sera, we showed that the DNA protected from degradation remained bound to IgG during digestion and was 35-45 base pairs in size. The size of this fragment is the same as that which has been proposed to be the minimal size necessary for monogamous bivalent binding of IgG to DNA. We therefore compared the ability of F(ab')2 and Fab' to protect DNA from DNase digestion and demonstrated that the bivalent F(ab')2 fragments were protective, but that the univalent Fab' fragments were not. These results suggest that some antibodies to DNA that bind to DNA via monogamous bivalent binding can protect a 35-45-base pair DNA fragment from DNase digestion. The implications of this finding are discussed with regard to the in vivo behavior and potential pathogenicity of small DNA-anti-DNA immune complexes. Images PMID:6234327

  16. Diffusional spinning as a probe of DNA fragments conformation

    NASA Astrophysics Data System (ADS)

    Collini, Maddalena; Chirico, Giuseppe; Baldini, Giancarlo

    1996-04-01

    The dependence of the spinning diffusion coefficient of a wormlike chain upon contour length L, persistence length P, and radius R is shown here to follow a ``Lorentzian'' law of width ? vs ??L/R, where ?2?=l0/P is the variance of the bending angles distribution of Monte Carlo simulated chains with bond length l0. This description is equivalent to that of a spinning cylinder of length L and effective radius Reff=R(L,P), with Reff?R. When considering experimental data it is found that fluorescence polarization anisotropy (FPA), a technique very sensitive to spinning, also yields apparent DNA radii depending upon fragment length. In order to derive DNA parameters which are independent of fragment length, we introduce a procedure for fitting FPA data which takes into account thermal distortions and employs the parametric expressions for rigid body rotations, spinning and tumbling, depending only upon L, P, and the actual DNA radius, R. Then the apparent persistence length P can be estimated once a value of R is assumed together with the value of the dynamic persistence length, the latter affecting the internal bending motions of the fragments. Fitting the FPA data is easily accomplished with the value of R=10 Å as suggested by a number of recent measurements.

  17. Small DNA Pieces in C. elegans Are Intermediates of DNA Fragmentation during Apoptosis

    PubMed Central

    Aruscavage, P. Joseph; Hellwig, Sabine; Bass, Brenda L.

    2010-01-01

    While studying small noncoding RNA in C. elegans, we discovered that protocols used for isolation of RNA are contaminated with small DNA pieces. After electrophoresis on a denaturing gel, the DNA fragments appear as a ladder of bands, ?10 nucleotides apart, mimicking the pattern of nuclease digestion of DNA wrapped around a nucleosome. Here we show that the small DNA pieces are products of the DNA fragmentation that occurs during apoptosis, and correspondingly, are absent in mutant strains incapable of apoptosis. In contrast, the small DNA pieces are present in strains defective for the engulfment process of apoptosis, suggesting they are produced in the dying cell prior to engulfment. While the small DNA pieces are also present in a number of strains with mutations in predicted nucleases, they are undetectable in strains containing mutations in nuc-1, which encodes a DNase II endonuclease. We find that the small DNA pieces can be labeled with terminal deoxynucleotidyltransferase only after phosphatase treatment, as expected if they are products of DNase II cleavage, which generates a 3? phosphate. Our studies reveal a previously unknown intermediate in the process of apoptotic DNA fragmentation and thus bring us closer to defining this important pathway. PMID:20585459

  18. Distinguishing Heterodera filipjevi and H. avenae using polymerase chain reaction-restriction fragment length polymorphism and cyst morphology.

    PubMed

    Yan, Guiping; Smiley, Richard W

    2010-03-01

    The cereal cyst nematodes Heterodera filipjevi and H. avenae impede wheat production in the Pacific Northwest (PNW). Accurate identification of cyst nematode species and awareness of high population density in affected fields are essential for designing effective control measures. Morphological methods for differentiating these species are laborious. These species were differentiated using polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) of internal transcribed spacer (ITS)-ribosomal (r)DNA with up to six restriction endonucleases (TaqI, HinfI, PstI, HaeIII, RsaI, and AluI). The method was validated by inspecting underbridge structures of cyst vulval cones. Grid soil sampling of an Oregon field infested by both species revealed that H. filipjevi was present at most of the infested grid sites but mixtures of H. avenae and H. filipjevi also occurred. These procedures also detected and differentiated H. filipjevi and H. avenae in soil samples from nearby fields in Oregon and H. avenae in samples from Idaho and Washington. Intraspecific polymorphism was not observed within H. filipjevi or PNW H. avenae populations based on the ITS-rDNA. However, intraspecific variation was observed between H. avenae populations occurring in the PNW and France. Methods described here will improve detection and identification efficiencies for cereal cyst nematodes in wheat fields. PMID:20128694

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

    PubMed Central

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

    1996-01-01

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

  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. 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

  2. Detection of Irradiated Food: DNA Fragmentation in Grapefruits

    NASA Astrophysics Data System (ADS)

    Delincée, Henry

    1998-06-01

    Employing the simple microgel electrophoresis of single cells - `comet assay' - on grapefruit seeds enabled a rapid identification of irradiated fruits. Fruits were exposed to radiation doses of 0, 0.1, 0.2, 0.3, 0.4 and 0.5 kGy covering the range of potential commercial irradiation for insect disinfestation and quarantine purposes. Seeds were isolated, crushed, and the cells embedded in an agarose layer. After lysis of the cells, they were subjected to microgel electrophoresis for 2.5 minutes, and then stained. Fruits irradiated with 0.2 kGy and higher doses showed typical DNA fragmentation, the DNA fragments stretching or migrating out of the cells forming a tail towards the anode, giving the damaged cells an appearance of a comet. With increasing dose a longer extension of the DNA from the nucleus towards the anode is observed. Undamaged cells will appear as intact nuclei without tails. The DNA comet assay is thus a rapid and inexpensive screening technique to detect irradiated grapefruits. Suspected samples may subsequently be analysed by officially validated methods for detection of irradiated foods.

  3. Secuencia parcial de un fragmento de ADN de patos silvestres homólogo al complejo mayor de histocompatibilidad de Gallus gallus Partial sequence of a DNA fragment from wild ducks homologous to the Gallus gallus major histocompatibility complex

    Microsoft Academic Search

    Sofía González-Guzmán; Elizabeth Loza-Rubio; Virginia León-Régagnonc; Gary García-Espinosa

    In this study, a genomic DNA fragment from domestic duck (Anas domesticus) was amplified by PCR. Such fragment shared 93 % identity to the chicken (Gallus gallus) MHC class II, which correspond to DAB1- (Disabled 1)-like sequence. The DAB1 sequence was also amplified from nine wild duck species of the Anas genus, which after performing a restriction fragment length polymorphism

  4. Structure of DNA polymerase I Klenow fragment bound to duplex DNA.

    PubMed

    Beese, L S; Derbyshire, V; Steitz, T A

    1993-04-16

    Klenow fragment of Escherichia coli DNA polymerase I, which was cocrystallized with duplex DNA, positioned 11 base pairs of DNA in a groove that lies at right angles to the cleft that contains the polymerase active site and is adjacent to the 3' to 5' exonuclease domain. When the fragment bound DNA, a region previously referred to as the "disordered domain" became more ordered and moved along with two helices toward the 3' to 5' exonuclease domain to form the binding groove. A single-stranded, 3' extension of three nucleotides bound to the 3' to 5' exonuclease active site. Although this cocrystal structure appears to be an editing complex, it suggests that the primer strand approaches the catalytic site of the polymerase from the direction of the 3' to 5' exonuclease domain and that the duplex DNA product may bend to enter the cleft that contains the polymerase catalytic site. PMID:8469987

  5. Isolation and characterization of human DNA fragments with nucleotide sequence homologies with the simian virus 40 regulatory region

    SciTech Connect

    Conrad, S.E.; Botchan, M.R.

    1982-08-01

    A recombinant library of human DNA sequences was screened with a segment of simian virus 40 (SV40) DNA that spans the viral origin of replication. One hundred and fifty phage were isolated that hybridized to this probe. Restriction enzyme and hybridization analyses indicated that these sequences were partially homologous to one another. Direct DNA sequencing of two such SV40-hybridizating segments indicated that this was not a highly conserved family of sequences, but rather a set of DNA fragments that contained repetitive regions of high guanine plus cytosine content. These sequences were not members of the previously described Alu family of repeats and hybridized to SV40 DNA more strongly than do Alu family members. The authors tested for the presence of several functional domains on two of these human DNA fragments. One SV40-hybridizing fragment, SVCR29, contained a sequence with enhanced the efficiency of thymidine kinase transformation in human cells by approximately 20-fold. This effect was seen in an orientation-independent manner when the sequence was present at the 3' end of the chicken thymidine kinase gene. They propose that this segment of DNA contains a sequence analogous to the 72-base-pair repeats of SV40. The existence of such an ''activator'' element in cellular DNA raises the possibility that families of these sequences may exist in the mammalian genome.

  6. Does varicocelectomy affect DNA fragmentation in infertile patients?

    PubMed Central

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

    2015-01-01

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

  7. Correlations between two markers of sperm DNA integrity, DNA denaturation and DNA fragmentation, in fertile and infertile men

    Microsoft Academic Search

    Armand Zini; Ryszard Bielecki; Donna Phang; Maria Teresa Zenzes

    2001-01-01

    Objective: To evaluate two different assays of human sperm DNA integrity, DNA denaturation (DD) and DNA fragmentation (DF), and to correlate these with standard semen parameters.Design: Prospective, observational study.Setting: University infertility clinic.Patient(s): Forty consecutive semen samples from 33 nonazoospermic men presenting for infertility evaluation and 7 fertile men presenting for vasectomy.Intervention(s): Assessment of sperm concentration, motility, morphology, DD and DF.Main

  8. Identification of the Genomic Locations of Duplicate Nucleotide Sequences in Maize by Analysis of Restriction Fragment Length Polymorphisms

    Microsoft Academic Search

    Tim Helentjaris; David Weber; Scott Wright

    While preparing a linkage map for maize based upon loci detected through the use of restriction fragment length polymorphisms (RFLPs), it was found that 62 of the 2 17 cloned maize sequences tested (29%) detected more than one fragment on genomic Southern blots. Thus, more than one nucleotide sequence is present within the maize genome which is in part homologous

  9. Phylogenomics of caspase-activated DNA fragmentation factor.

    PubMed

    Eckhart, Leopold; 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. PMID:17343828

  10. Phylogenomics of caspase-activated DNA fragmentation factor

    SciTech Connect

    Eckhart, Leopold [Department of Dermatology, Medical University of Vienna, A-1090 Vienna (Austria)]. E-mail: leopold.eckhart@meduniwien.ac.at; Fischer, Heinz [Department of Dermatology, Medical University of Vienna, A-1090 Vienna (Austria); Tschachler, Erwin [Department of Dermatology, Medical University of Vienna, A-1090 Vienna (Austria)

    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.

  11. A Fuzzy Classifier to Taxonomically Group DNA Fragments within a Metagenome

    E-print Network

    Nicolescu, Monica

    for isolation and lab cultivation of individual species [4]. The whole genome(DNA) or metagenome population canA Fuzzy Classifier to Taxonomically Group DNA Fragments within a Metagenome Sara Nasser, Adrienne classifier is used to group shotgun sequence fragments as small as 500 base pairs according to their DNA

  12. 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

  13. A simple method of detecting K- ras point mutations in stool samples for colorectal cancer screening using one-step polymerase chain reaction\\/restriction fragment length polymorphism analysis

    Microsoft Academic Search

    Takashi Nishikawa; Kentaro Maemura; Ichiro Hirata; Ryouichi Matsuse; Hiroshi Morikawa; Ken Toshina; Mitsuyuki Murano; Keiichi Hashimoto; Yoshihito Nakagawa; Osamu Saitoh; Kazuo Uchida; Kenichi Katsu

    2002-01-01

    Background: We examined a technique for detecting point mutations of K-ras codon 12 in stool samples using one-step polymerase chain reaction\\/restriction fragment length polymorphism (PCR\\/RFLP) analysis, in order to determine whether it could be used to screen for colorectal cancer. Methods: DNA was extracted from 200-mg stool specimens of 5 healthy controls and 31 colorectal cancer patients. A 107-base-pair fragment

  14. Application of qualitative and quantitative real-time PCR, direct sequencing, and terminal restriction fragment length polymorphism analysis for detection and identification of polymicrobial 16S rRNA genes in ascites.

    PubMed

    Krohn, Sandra; Böhm, Stephan; Engelmann, Cornelius; Hartmann, Jan; Brodzinski, Annika; Chatzinotas, Antonis; Zeller, Katharina; Prywerek, Delia; Fetzer, Ingo; Berg, Thomas

    2014-05-01

    Qualitative and quantitative 16S rRNA gene-based real-time PCR and direct sequencing were applied for rapid detection and identification of bacterial DNA (bactDNA) in 356 ascites samples. bactDNA was detected in 35% of samples, with a mean of 3.24 log copies ml(-1). Direct sequencing of PCR products revealed 62% mixed chromatograms predominantly belonging to Gram-positive bacteria. Terminal restriction fragment length polymorphism (T-RFLP) results of a sample subset confirmed sequence data showing polymicrobial DNA contents in 67% of bactDNA-positive ascites samples. PMID:24622095

  15. Dependence on radiation quality of DNA fragmentation spectra

    NASA Astrophysics Data System (ADS)

    Campa, Alessandro; Ottolenghi, Andrea; Alloni, Daniele; Ballarini, Francesca; Belli, Mauro; Esposito, Giuseppe; Facoetti, Angelica; Friedland, Werner; Liotta, Marco; Paretzke, Herwig

    Energy deposition by radiation initially gives rise to cellular critical lesions such as DNA doublestrand breaks (DSB), that later lead to the formation of relevant biological endpoints. Studies on fragment size distributions induced by radiations of various qualities can be of great help in linking the characteristics of radiation to cellular endpoints, providing information for understanding the main mechanisms of cell damage. Here we are concerned with the damage induced by heavy charged particles; this issue is very important in the field of radioprotection of astronauts participating in long term space missions, besides being relevant also in other fields, like hadrontherapy. Galactic Cosmic Rays contain a large component of high-LET particles (HZE), e.g. helium and carbon ions, as well as highcharge particles such as iron ions. These particles are characterized by complex track structures with energy depositions not only along the path of the primary particle, but also at relatively large distance form the path, due to the presence of high energy secondary electrons. In this work we have simulated the irradiation of human fibroblasts with ?-rays, protons, helium, carbon and iron ions at a fixed dose with the biophysical Monte Carlo code PARTRAC,and calculated the induction of DSB. The PARTRAC code includes accurate representation of the chromatin geometry and of the physical and physico-chemical processes associated with the energy deposition by radiation. The results of a first validation of the code have been reported in A. Campa et al. (2005) and D. Alloni et al. (2007a, 2007b). DNA fragment spectra were calculated based on the DSB induction patterns and compared in particular for particles of the same specific energy and for particles of the same LET. Special emphasis has been directed to the calculation of very small fragments (< 1 kbp) that are not detectable by the most common experimental techniques and that can significantly influence the RBE (Relative Biological Effectiveness) of high LET radiation. This work was partially supported by EU ("RISC-RAD" project, Contract no. FI6R-CT 2003- 508842, and "NOTE" project, Contract no. FI6R-036465) and ASI (Italian Space Agency, "Mo-Ma/COUNT" project). References A. Campa, F. Ballarini, M. Belli, R. Cherubini, V. Dini, G. Esposito, W.Friedland, S. Gerardi, S. Molinelli, A. Ottolenghi, H. G. Paretzke, G. Simone and M. A. Tabocchini. DNA DSB induced in human cells by charged particles and gamma rays: experimental results and theoretical approaches. Int. J. Radiat.Biol. 81, 841-854 (2005). D. Alloni, F. Ballarini, M. Belli, A. Campa, G. Esposito, W. Friedland, M.Liotta, A. Ottolenghi and H. G. Paretzke. Modeling of DNA fragmentation induced in human fibroblasts by 56 Fe ions. Adv. Space Res. 40, 1401-1407 (2007a). D. Alloni, F. Antonelli, F. Ballarini, M. Belli, A. Campa, V. Dini, G.Esposito, W. Friedland, M. Liotta, A. Ottolenghi, H. G. Paretzke, G. Simone, E. Sorrentino and M. A. Tabocchini. Small DNA fragments induced in human fibroblasts by 56 Fe ions: experimental data and MC simulations. Proc. "Ion Beams in biology and medicine", Heidelberg, 26-29 September 2007, edited by J. Debus, K. Henrichs, G. Kraft, p. 164 (2207b).

  16. Assessment of fungal diversity using terminal restriction fragment (TRF) pattern analysis: comparison of 18S and ITS ribosomal regions.

    PubMed

    Lord, N S; Kaplan, C W; Shank, P; Kitts, C L; Elrod, S L

    2002-12-01

    Assessment of fungal diversity in environmental samples is currently a challenge. Several recently developed molecular methods offer new avenues for determining the presence and diversity of fungi in complex microbial communities. Terminal restriction fragment (TRF) pattern analysis was tested as a method for assessing the fungal molecular diversity of a terrestrial microbial community. Community DNA was isolated from sand samples taken from a pilot-scale petroleum-contaminated land treatment unit. PCR amplification was carried out using primers, one of which was fluorescently labeled, designed to hybridize to conserved sequences in the fungal ribosomal small subunit (18S) or the internal transcribed spacer ITS1-5.8S-ITS2 (ITS) ribosomal region. Amplicons were then digested separately with HpaII or HaeIII; fluorescently labeled TRFs were detected by capillary gel electrophoresis. ITS region TRF patterns were predicted and observed to generate a greater richness than 18S TRF patterns. Unique TRF patterns were also observed for each community examined. Finally, the ITS region showed a higher degree of specificity in matching observed TRF profiles to those generated from GenBank sequence data for species identification. These data suggest that ITS rDNA TRF pattern analysis has great potential as a rapid and specific method for fungal community analysis and species identification. PMID:19709292

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

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

    2014-02-01

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

  20. 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

  1. RegionSpecific Interrelations between Apoptotic Proteins Expression and DNA Fragmentation in the Neonatal Rat Brain

    Microsoft Academic Search

    Petr N. Menshanov; Anita V. Bannova; Nikolay N. Dygalo

    2006-01-01

    DNA fragmentation, mRNA and protein levels of Bcl-XL, Bax and caspase-3 were determined to characterize interrelations between expression of these apoptotic markers in the neonatal brain regions. High DNA fragmentation intensity in the cortex was in consonance with the lowest Bcl-XL\\/Bax expression ratio, the highest procaspase-3 and active caspase-3 levels. Low and intermediate DNA fragmentation levels in the cerebellum and

  2. Small Fragment Homologous Replacement (SFHR): sequence-specific modification of genomic DNA in eukaryotic cells by small DNA fragments.

    PubMed

    Luchetti, Andrea; Malgieri, Arianna; Sangiuolo, Federica

    2014-01-01

    The sequence-specific correction of a mutated gene (e.g., point mutation) by the Small Fragment Homologous Replacement (SFHR) method is a highly attractive approach for gene therapy. Small DNA fragments (SDFs) were used in SFHR to modify endogenous genomic DNA in both human and murine cells. The advantage of this gene targeting approach is to maintain the physiologic expression pattern of targeted genes without altering the regulatory sequences (e.g., promoter, enhancer), but the application of this technique requires the knowledge of the sequence to be targeted. In our recent study, an optimized SFHR protocol was used to replace the eGFP mutant sequence in SV-40-transformed mouse embryonic fibroblast (MEF-SV40), with the wild-type eGFP sequence. Nevertheless in the past, SFHR has been used to correct several mutant genes, each related to a specific genetic disease (e.g., spinal muscular atrophy, cystic fibrosis, severe combined immune deficiency). Several parameters can be modified to optimize the gene modification efficiency, as described in our recent study. In this chapter we describe the main guidelines that should be followed in SFHR application, in order to increase technique efficiency. PMID:24557898

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

    PubMed

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

    2001-12-01

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

  4. 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

  5. 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)

  6. Screening and characterization of sex-specific DNA fragments in the freshwater fish matrinchã, Brycon amazonicus (Teleostei: Characiformes: Characidae).

    PubMed

    da Silva, Eder Marques; Wong, Marina Sek Lien; Martins, Cesar; Wasko, Adriane Pinto

    2012-10-01

    The matrinchã Brycon amazonicus, a commercially important freshwater fish resource, has no heteromorphic sex chromosomes so far described. In the present study, we performed a screening of sex-associated DNA markers in this species, through the use of a random amplified polymorphic DNA (RAPD) assay and a genomic DNA restriction digestion analysis. DNA digestions evidenced no differences between sexes. Sixty-six random primers were used in pooled and individual DNA samples of males and females, and the analysis of the RAPD fingerprints revealed one female sex-associated band. Cloning and sequencing of this band led to the identification of two distinct DNA segments. While one of the isolated fragments showed a significant identity with a described protein gene (phosphatidylinositol glycan anchor biosynthesis, class W), the other fragment, composed of 535 bp, corresponds to a novel DNA marker. Further experiments were performed with this second DNA fragment in order to verify its sex-specificity. Data on dot blot hybridization, using total DNA of both sexes, confirmed its female-specificity in B. amazonicus. A primer set was designed based on its sequence data and used in PCR with DNA samples of this species, leading to diagnose the animals' sexes with a 100 % overall accuracy through a sequence characterized amplified region approach. No amplification results were found for two other species of the genus--B. orbignyanus and B. lundii. The obtained data can lead to the hypothesis that B. amazonicus may present heteromorphic sex chromosomes that should be in an early phase of differentiation. PMID:22527611

  7. Anatomy of bovine mammillitis DNA. I Restriction endonuclease maps of four populations of molecules that differ in the relative orientation of their long and short components.

    PubMed

    Buchman, T G; Roizman, B

    1978-01-01

    In this paper, we report that the DNA of bovine mammillitis virus (BMV) consists of two covalently linked components that are 71.5 x 10(6) and 15.7 x 10(6) in molecular weight and designated L and S, respectively. We further report that the BMV DNA consists of four equimolar populations differing only in the orientation of the L and S components relative to each other. This conclusion is based on the following: (i) The sum molecular weight of fragments generated by digestion of BMV DNA with Hsu I, Hpa I, Bgl II, or Xba I significantly exceeds the established molecular weight of the intact DNA. (ii) In each digest, the fragments form three groups differing in molar concentration. In reference to the molar concentration of intact DNA, each enzyme digest contained a set of four fragments 0.25 M in concentration, a set of four fragments 0.5 M in concentration, and a variable size set, unique for each enzyme digest, 1.0 M in concentration. (iii) Experiments involving digestion of intact DNA by lambda exonuclease followed by restriction endonuclease digestion established that each of four 0.5 M fragments were positioned at the termini of the BMV DNA. (iv) Complete maps for the fragments generated by each enzyme established that the 0.25 M fragments arise by fusion of the sequences of the terminal fragments when these are juxtaposed as a consequence of the inversion of L and S components. The maps also established the dimensions of the L and S components. We conclude that the structure of BMV DNA is similar to that of HSV DNA previously shown to consist of two unequal size components that invert relative to each other. PMID:202750

  8. Cocrystal Structure of an Editing Complex of Klenow Fragment with DNA

    Microsoft Academic Search

    P. S. Freemont; J. M. Friedman; L. S. Beese; M. R. Sanderson; T. A. Steitz

    1988-01-01

    High-resolution crystal structures of editing complexes of both duplex and single-stranded DNA bound to Escherichia coli DNA polymerase I large fragment (Klenow fragment) show four nucleotides of single-stranded DNA bound to the 3'-5' exonuclease active site and extending toward the polymerase active site. Melting of the duplex DNA by the protein is stabilized by hydophobic interactions between Phe-473, Leu-361, and

  9. 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

  10. Use of multiplex terminal restriction fragment length polymorphism for rapid and simultaneous analysis of different components of the soil microbial community.

    PubMed

    Singh, Brajesh K; Nazaries, Loic; Munro, Stacey; Anderson, Ian C; Campbell, Colin D

    2006-11-01

    A multiplex terminal restriction fragment length polymorphism (M-TRFLP) fingerprinting method was developed and validated for simultaneous analysis of the diversity and community structure of two or more microbial taxa (up to four taxa). The reproducibility and robustness of the method were examined using soil samples collected from different habitats. DNA was PCR amplified separately from soil samples using individual taxon-specific primers for bacteria, archaea, and fungi. The same samples were also subjected to a multiplex PCR with the primers for all three taxa. The terminal restriction fragment length polymorphism profiles generated for the two sets of PCR products were almost identical not only in terms of the presence of peaks but also in terms of the relative peak intensity. The M-TRFLP method was then used to investigate rhizosphere bacterial, fungal, and rhizobial/agrobacterial communities associated with the dwarf shrub Calluna vulgaris growing in either open moorland, a mature pine forest, or a transition zone between these two habitats containing naturally regenerating pine trees. Rhizosphere microbial communities associated with Vaccinium myrtillus collected from the native pine forest were also investigated. In this study, individual PCR products from the three taxa were also pooled before restriction digestion and fragment size analysis. The terminal restriction fragment length polymorphism profiles obtained with PCR products amplified individually and with multiplexed and pooled PCR products were found to be consistent with each other in terms of the number, position, and relative intensity of peaks. The results presented here confirm that M-TRFLP analysis is a highly reproducible and robust molecular tool for simultaneous investigation of multiple taxa, which allows more complete and higher resolution of microbial communities to be obtained more rapidly and economically. PMID:16936053

  11. 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.

  12. Characterization of avian strains of Pasteurella multocida by restriction endonuclease and amplified fragment length polymorphism.

    PubMed

    Shivachandra, S B; Kumar, A A; Gautam, R; Joseph, S; Saxena, M K; Chaudhuri, P; Srivastava, S K

    2006-08-01

    Avian strains of Pasteurella multocida were typed by employing restriction endonuclease analysis (REA) and single enzyme-amplified fragment length polymorphism (AFLP) to evaluate their applicability for epidemiological studies of fowl cholera outbreaks. A total of 72 strains isolated from different avian species (chicken, duck, turkey, quail and goose) belonging to various geographical regions of India were characterized. REA using two different enzymes HhaI and HpaII produced 9 and 18 clusters respectively, whereas Single enzyme-AFLP recognized 32 patterns out of 72 strains typed. The study indicated that REA using HpaII is a simple and resource efficient method, however, further typing with more stringent and rapid method like Single enzyme-AFLP, could drastically enhance investigation in epidemiological studies of fowl cholera outbreaks. PMID:16427104

  13. Caffeine overcomes a restriction point associated with DNA replication, but does not accelerate mitosis

    PubMed Central

    1990-01-01

    Mitotic chromosome condensation is normally dependent on the previous completion of replication. Caffeine spectacularly deranges cell cycle controls after DNA polymerase inhibition or DNA damage; it induces the condensation, in cells that have not completed replication, of fragmented nuclear structures, analogous to the S-phase prematurely condensed chromosomes seen when replicating cells are fused with mitotic cells. Caffeine has been reported to induce S-phase condensation in cells where replication is arrested, by accelerating cell cycle progression as well as by uncoupling it from replication; for, in BHK or CHO hamster cells arrested in early S-phase and given caffeine, condensed chromosomes appear well before the normal time at which mitosis occurs in cells released from arrest. However, we have found that this apparent acceleration depends on the technique of synchrony and cell line employed. In other cells, and in synchronized hamster cells where the cycle has not been subjected to prolonged continual arrest, condensation in replication-arrested cells given caffeine occurs at the same time as normal mitosis in parallel populations where replication is allowed to proceed. This caffeine- induced condensation is therefore "premature" with respect to the chromatin structure of the S-phase nucleus, but not with respect to the timing of the normal cycle. Caffeine in replication-arrested cells thus overcomes the restriction on the formation of mitotic condensing factors that is normally imposed during DNA replication, but does not accelerate the timing of condensation unless cycle controls have previously been disturbed by synchronization procedures. PMID:2161852

  14. 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…

  15. Structural analyses of DNA fragments integrated by illegitimate recombination in Schizosaccharomyces pombe

    Microsoft Academic Search

    Kazuo Tatebayashi; Jun-ichi Kato; Hideo Ikeda

    1994-01-01

    In order to elucidate the mechanisms of illegitimate recombination in eukaryotes, we have studied the structure of DNA fragments integrated by illegitimate recombination into the genome of fission yeast. Nonhomologous recombination was rarely identified when a long region of homology with the chromosomal leu1+ gene was present in the introduced leu1::ura4+ DNA fragment; but a decrease in length of homology

  16. 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.

  17. Differential diagnosis of Perkinsus species by polymerase chain reaction-restriction fragment length polymorphism assay.

    PubMed

    Abollo, Elvira; Casas, Sandra M; Ceschia, Giuseppe; Villalba, Antonio

    2006-12-01

    Perkinsosis is an infection of marine molluscs caused by the protistan parasites of the genus Perkinsus, which has been classified by the OIE as a disease that warrants notification. In the present study, we have applied a molecular genetic approach to develop an optional method for the specific identification of Perkinsus species. A species-specific polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay of the rRNA ITS region was developed to identify and distinguish among Perkinsus species. A taxonomic key was established that allows successful identification of Perkinsus species using a single restriction enzyme (Rsa I) to discriminate P. chesapeaki and P. marinus or by a combination of two endonucleases (Rsa I plus Hinf I) to discriminate P. olseni and P. mediterraneus. In order to validate the RFLP assay, the PCR products were cloned and sequenced, and its phylogenetic affinity was determined. Phylogenetic analysis confirmed the specific identification carried out by RFLPs. Herein is the first report of P. olseni in Manila clams from the NW Adriatic Sea (Italy), which we identified by employing this method. The PCR-RFLP assay herein described may be useful to provide accurate, rapid and inexpensive identification of Perkinsus species, and may aid in ongoing epizooetiological studies and diseases control programmes. PMID:16846717

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

    PubMed Central

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

    2006-01-01

    Terminal restriction fragment length polymorphism (T-RFLP) analysis has the potential to be useful for comparisons of complex bacterial communities, especially to detect changes in community structure in response to different variables. To do this successfully, systematic variations have to be detected above method-associated noise, by standardizing data sets and assigning confidence estimates to relationships detected. We investigated the use of different standardizing methods in T-RFLP analysis of PCR-amplified 16S rRNA genes to elucidate the similarities between the bacterial communities in 17 soil and sediment samples. We developed a robust method for standardizing data sets that appeared to allow detection of similarities between complex bacterial communities. We term this the variable percentage threshold method. We found that making conclusions about the similarities of complex bacterial communities from T-RFLP profiles generated by a single restriction enzyme (RE) may lead to erroneous conclusions. Instead, the use of multiple REs, each individually, to generate multiple data sets allowed us to determine a confidence estimate for groupings of apparently similar communities and at the same time minimized the effects of RE selection. In conjunction with the variable percentage threshold method, this allowed us to make confident conclusions about the similarities of the complex bacterial communities in the 17 different samples. PMID:16461676

  19. Saccharin consumption increases sperm DNA fragmentation and apoptosis in mice

    PubMed Central

    Rahimipour, Marzieh; Talebi, Ali Reza; Anvari, Morteza; Abbasi Sarcheshmeh, Abolghasem; Omidi, Marjan

    2014-01-01

    Background: Saccharin is an artificial non-caloric sweetener that used to sweeten products such as drinks, candies, medicines, and toothpaste, but our bodies cannot metabolize it. Sodium saccharin is considered as an important factor in tumor promotion in male rats but not in humans. Objective: The objective of this study was to investigate the effect of saccharin consumption on sperm parameters and apoptosis in adult mice. Materials and Methods: Totally 14 adult male mice were divided into 2 groups. Group 1 served as control fed on basal diet and group 2 or experimental animals received distilled water containing saccharin (0.2% w/v) for 35 days. After that, the left cauda epididymis of each mouse was cut and placed in Ham’s F10. Swimmed-out spermatozoa were used to analyze count, motility, morphology (Pap-staining) and viability (eosin-Y staining). Sperm DNA integrity, as an indicator of apoptosis, was assessed by SCD (sperm chromatin dispersion) and terminal deoxynucleotidyl transferase (TUNEL) assay. Results: Following saccharin consumption, we had a reduction in sperm motility with respect to control animals (p=0.000). In addition, the sperm count diminished (17.70±1.11 in controls vs. 12.80±2.79 in case group, p=0.003) and the rate of sperm normal morphology decreased from 77.00±6.40 in control animals into 63.85±6.81 in saccharin-treated mice (p=0.001). Also, we saw a statistically significant increase in rates of sperm DNA damage and apoptosis in experimental group when compared to control one (p=0.001, p=0.002 respectively). Conclusion: Saccharin consumption may have negative effects on sperm parameters, and increases the rate of sperm DNA fragmentation and apoptosis in mice. PMID:25031574

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

    SciTech Connect

    Jackson, Christopher B., E-mail: Christopher.jackson@insel.ch [Division of Human Genetics, Departements of Pediatrics and Clinical Research, Inselspital, University of Berne, Freiburgstrasse, CH-3010 Berne (Switzerland); Gallati, Sabina, E-mail: sabina.gallati@insel.ch [Division of Human Genetics, Departements of Pediatrics and Clinical Research, Inselspital, University of Berne, Freiburgstrasse, CH-3010 Berne (Switzerland)] [Division of Human Genetics, Departements of Pediatrics and Clinical Research, Inselspital, University of Berne, Freiburgstrasse, CH-3010 Berne (Switzerland); Schaller, Andre, E-mail: andre.schaller@insel.ch [Division of Human Genetics, Departements of Pediatrics and Clinical Research, Inselspital, University of Berne, Freiburgstrasse, CH-3010 Berne (Switzerland)] [Division of Human Genetics, Departements of Pediatrics and Clinical Research, Inselspital, University of Berne, Freiburgstrasse, CH-3010 Berne (Switzerland)

    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.

  1. Differentiation of slowly growing Mycobacterium species, including Mycobacterium tuberculosis, by gene amplification and restriction fragment length polymorphism analysis.

    PubMed Central

    Plikaytis, B B; Plikaytis, B D; Yakrus, M A; Butler, W R; Woodley, C L; Silcox, V A; Shinnick, T M

    1992-01-01

    A two-step assay combining a gene amplification step and a restriction fragment length polymorphism analysis was developed to differentiate the Mycobacterium species that account for greater than 90% of potentially pathogenic isolates and greater than 86% of all isolates in clinical laboratories in the United States. These species are M. tuberculosis, M. bovis, M. avium, M. intracellulare, M. kansasii, and M. gordonae. With lysates of pure cultures as the template, two oligonucleotide primers that amplified an approximately 1,380-bp portion of the hsp65 gene from all 139 strains of 19 Mycobacterium species tested, but not from the 19 non-Mycobacterium species tested, were identified. Digestion of the amplicons from 126 strains of the six most commonly isolated Mycobacterium species with the restriction enzymes BstNI and XhoI in separate reactions generated restriction fragment patterns that were distinctive for each of these species, except for those of M. tuberculosis and M. bovis, which were not distinguishable. By including size standards in each sample, the restriction fragment profiles could be normalized to a fixed distance and the similarities of patterns could be calculated by using a computer-aided comparison program. The availability of this data base should enable the identification of an unknown Mycobacterium strain to the species level by a comparison of the restriction fragment pattern of the unknown with the data base of known patterns. Images PMID:1352786

  2. Comparison of bacterial communities in New England Sphagnum bogs using terminal restriction fragment length polymorphism (T-RFLP).

    PubMed

    Morales, Sergio E; Mouser, Paula J; Ward, Naomi; Hudman, Stephen P; Gotelli, Nicholas J; Ross, Donald S; Lewis, Thomas A

    2006-07-01

    Wetlands are major sources of carbon dioxide, methane, and other greenhouse gases released during microbial degradation. Despite the fact that decomposition is mainly driven by bacteria and fungi, little is known about the taxonomic diversity of bacterial communities in wetlands, particularly Sphagnum bogs. To explore bacterial community composition, 24 bogs in Vermont and Massachusetts were censused for bacterial diversity at the surface (oxic) and 1 m (anoxic) regions. Bacterial diversity was characterized by a terminal restriction fragment length (T-RFLP) fingerprinting technique and a cloning strategy that targeted the 16S rRNA gene. T-RFLP analysis revealed a high level of diversity, and a canonical correspondence analysis demonstrated marked similarity among bogs, but consistent differences between surface and subsurface assemblages. 16S rDNA sequences derived from one of the sites showed high numbers of clones belonging to the Deltaproteobacteria group. Several other phyla were represented, as well as two Candidate Division-level taxonomic groups. These data suggest that bog microbial communities are complex, possibly stratified, and similar among multiple sites. PMID:16729225

  3. Key morphological features of apoptosis may occur in the absence of internucleosomal DNA fragmentation.

    PubMed Central

    Cohen, G M; Sun, X M; Snowden, R T; Dinsdale, D; Skilleter, D N

    1992-01-01

    Apoptosis, a major form of cell death, is characterized by chromatin condensation, a reduction in cell volume and endonuclease cleavage of DNA into oligonucleosomal length fragments. The detection of these fragments by gel electrophoresis, as a DNA ladder, is currently used as the major biochemical index of apoptosis. Here we report that key morphological changes of apoptosis can be dissociated experimentally from the DNA fragmentation produced by endonuclease activity. Internucleosomal cleavage of DNA is thus likely to be a later event in the apoptotic process. Images Fig. 2. Fig. 3. PMID:1530564

  4. A prediction of the amino acids and structures involved in DNA recognition by type I DNA restriction and modification enzymes

    Microsoft Academic Search

    Shane S. Sturrock; David T. F. Dryden

    1997-01-01

    The S subunits of type I DNA restriction\\/modification enzymes are responsible for recognising the DNA target sequence for the enzyme. They contain two domains of approximately 150 amino acids, each of which is responsible for recognising one half of the bipartite asymmetric target. In the absence of any known tertiary structure for type I enzymes or recog- nisable DNA recognition

  5. 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.

  6. Preparation of covalently linked DNA-RNA hybrids and arabinocytidine containing DNA fragments.

    PubMed Central

    de Vroom, E; Roelen, H C; Saris, C P; Budding, T N; van der Marel, G A; van Boom, J H

    1988-01-01

    It will be demonstrated that 5'-O-DMT-N-acyl-deoxyribonucleosides, 5'-O-Lev-2'-O-MTHP-N-acyl-ribonucleosides and, also, 2'-O-MTHP-N-acyl-ara-cytidine can be coupled, via the hydroxybenzotriazole phosphotriester approach, to afford two types of DNA-RNA hybrids as well as ara-C containing DNA-fragments. The final removal of acid-labile DMT and MTHP groups could be effected by 1 h treatment with 80% acetic acid of the otherwise unprotected DNA-RNA hybrids. The same acidic hydrolysis did not result in complete removal of the 2'-O-MTHP group from the ara-C unit. Complete deblocking was accomplished after an additional 2 h aqueous HC1 (0.01 M; pH 2.00) treatment. PMID:2453027

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

    PubMed Central

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

    1999-01-01

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

  8. Identification of raw and heat-processed meats from game bird species by polymerase chain reaction-restriction fragment length polymorphism of the mitochondrial D-loop region.

    PubMed

    Rojas, M; González, I; Fajardo, V; Martín, I; Hernández, P E; García, T; Martín, R

    2009-03-01

    Polymerase chain reaction-RFLP analysis has been applied to the identification of meats from quail (Coturnix coturnix), pheasant (Phasianus colchicus), red-legged partridge (Alectoris rufa), chukar partridge (Alectoris chukar), guinea fowl (Numida meleagris), capercaillie (Tetrao urogallus), Eurasian woodcock (Scolopax rusticola), and woodpigeon (Columba palumbus). Polymerase chain reaction amplification was carried out using a set of primers flanking a conserved region of approximately 310 bp from the mitochondrial D-loop region. Restriction site analysis based on sequence data from this DNA fragment permitted the selection of HinfI, MboII, and Hpy188III endonucleases for species identification. The restriction profiles obtained when amplicons were digested with the chosen enzymes allowed the unequivocal identification of all game bird species analyzed. Consistent results were obtained with both raw and heat-processed meats. PMID:19211540

  9. Identification of Carnobacterium Species by Restriction Fragment Length Polymorphism of the 16S-23S rRNA Gene Intergenic Spacer Region and Species-Specific PCR

    PubMed Central

    Rachman, Cinta; Kabadjova, Petia; Valcheva, Rosica; Prévost, Hervé; Dousset, Xavier

    2004-01-01

    The genus Carnobacterium is currently divided into the following eight species: Carnobacterium piscicola, C. divergens, C. gallinarum, C. mobile, C. funditum, C. alterfunditum, C. inhibens, and C. viridans. An identification tool for the rapid differentiation of these eight Carnobacterium species was developed, based on the 16S-23S ribosomal DNA (rDNA) intergenic spacer region (ISR). PCR-restriction fragment length polymorphism (PCR-RFLP) analysis of this 16S-23S rDNA ISR was performed in order to obtain restriction profiles for all of the species. Three PCR amplicons, which were designated small ISR (S-ISR), medium ISR (M-ISR), and large ISR (L-ISR), were obtained for all Carnobacterium species. The L-ISR sequence revealed the presence of two tRNA genes, tRNAAla and tRNAIle, which were separated by a spacer region that varied from 24 to 38 bp long. This region was variable among the species, allowing the design of species-specific primers. These primers were tested and proved to be species specific. The identification method based on the 16S-23S rDNA ISR, using PCR-RFLP and specific primers, is very suitable for the rapid low-cost identification and discrimination of all of the Carnobacterium species from other phylogenetically related lactic acid bacteria. PMID:15294774

  10. Identification of Carnobacterium species by restriction fragment length polymorphism of the 16S-23S rRNA gene intergenic spacer region and species-specific PCR.

    PubMed

    Rachman, Cinta; Kabadjova, Petia; Valcheva, Rosica; Prévost, Hervé; Dousset, Xavier

    2004-08-01

    The genus Carnobacterium is currently divided into the following eight species: Carnobacterium piscicola, C. divergens, C. gallinarum, C. mobile, C. funditum, C. alterfunditum, C. inhibens, and C. viridans. An identification tool for the rapid differentiation of these eight Carnobacterium species was developed, based on the 16S-23S ribosomal DNA (rDNA) intergenic spacer region (ISR). PCR-restriction fragment length polymorphism (PCR-RFLP) analysis of this 16S-23S rDNA ISR was performed in order to obtain restriction profiles for all of the species. Three PCR amplicons, which were designated small ISR (S-ISR), medium ISR (M-ISR), and large ISR (L-ISR), were obtained for all Carnobacterium species. The L-ISR sequence revealed the presence of two tRNA genes, tRNA(Ala) and tRNA(Ile), which were separated by a spacer region that varied from 24 to 38 bp long. This region was variable among the species, allowing the design of species-specific primers. These primers were tested and proved to be species specific. The identification method based on the 16S-23S rDNA ISR, using PCR-RFLP and specific primers, is very suitable for the rapid low-cost identification and discrimination of all of the Carnobacterium species from other phylogenetically related lactic acid bacteria. PMID:15294774

  11. DFF, a Heterodimeric Protein That Functions Downstream of Caspase3 to Trigger DNA Fragmentation during Apoptosis

    Microsoft Academic Search

    Xuesong Liu; Hua Zou; Clive Slaughter; Xiaodong Wang

    1997-01-01

    We have identified and purified from HeLa cytosol a protein that induces DNA fragmentation in coincubated nuclei after it is activated by caspase-3. This protein, designated DNA Fragmentation Factor (DFF), is a heterodimer of 40 kDa and 45 kDa subunits. The amino acid sequence of the 45 kDa subunit, determined from its cDNA sequence, reveals it to be a novel

  12. Development of Contamination-Free Restriction Fragment Length Polymorphism Probes for the Obligate Biotroph Peronospora tabacina, an Oomycete Causing Blue Mold of Tobacco.

    PubMed

    Sukno, Serenella A; Taylor, Amy M; Farman, Mark

    2002-11-01

    ABSTRACT Peronospora tabacina is an obligately parasitic oomycete that causes blue mold, a devastating disease of tobacco. Genetic studies of this pathogen have been hampered by the lack of molecular markers. We generated a set of molecular markers for P. tabacina by collecting sporangiospores from infected tobacco leaves, extracting spore DNA, and cloning it in a plasmid vector. The resulting clones were then used to probe DNA from a collection of P. tabacina isolates to survey for polymorphisms. Most probes gave unexpected hybridization patterns with signal intensities that varied significantly from one DNA sample to another or between different DNA preparations of the same isolate. These results indicated that certain DNA preparations contained DNA from a source other than P. tabacina, which in turn suggested that some probes might have been derived from contaminating organisms present in the spore suspensions. Therefore, we characterized the inserts of several recombinant plasmids to determine their origins. Sequence analysis revealed that several of the inserts encoded peptides with similarity to bacterial proteins, suggesting that they were derived from bacterial contaminants. Of the remaining clones, five exhibited similarity to retroelements, one resembled eukaryotic helicase genes, and nine had no similarity to sequences in the databases. These were postulated to be true P. tabacina DNA clones. Verification of the origin of each probe was achieved by filtering a spore suspension, extracting DNA from the retentate and filtrate, and probing Southern blots of these DNA samples. These experiments confirmed the probe origins predicted by sequence analysis, resulting in the generation of 20 different restriction fragment length polymorphism probes that are specific for P. tabacina DNA. These probes should enable identification of reliable genetic markers for population studies of the blue mold organism. PMID:18944249

  13. Identification of Amplified Restriction Fragment Length Polymorphism Markers Linked to Genes Controlling Boar Sperm Viability Following Cryopreservation1

    Microsoft Academic Search

    Lisa M. Thurston; Ken Siggins; Alan J. Mileham; Paul F. Watson; William V. Holt

    This study investigated two hypotheses: 1) that consistent be- tween-boar variation in frozen semen quality exists and is ge- netically determined, and 2) molecular markers linked to genes controlling semen freezability can be identified using amplified restriction fragment length polymorphism (AFLP) technology. Five ejaculates were collected from each of 129 boars. Semen was diluted into a commercial freezing buffer (700

  14. Epidemiologic Import of Tuberculosis Cases Whose Isolates Have Similar but Not Identical IS6110 Restriction Fragment Length Polymorphism Patterns

    Microsoft Academic Search

    M. D. Cave; Z. H. Yang; R. Stefanova; N. Fomukong; K. Ijaz; J. Bates; K. D. Eisenach

    2005-01-01

    Isolates of Mycobacterium tuberculosis from patients with epidemiologic links frequently demonstrate iden- tical IS6110 restriction fragment length polymorphism (RFLP) patterns (i.e., RFLP clustering) because they are infected with the same strain. Uncertainty arises with isolates that differ from one another by a few IS6110 hybridizing bands. During the period from 1 January 1996 to 31 December 1999, isolates from 585

  15. A Genetic Map of Lettuce (Lactuca sativa L.) With Restriction Fragment Length Polymorphism, Isozyme, Disease Resistance and Morphological Markers

    Microsoft Academic Search

    Benoit S. Landry; Rick V. Kesseli; Barry Farrara; Richard W. Michelmore

    1987-01-01

    A detailed linkage map of lettuce was constructed using 53 genetic markers including 4 1 restriction fragment length polymorphism (RFLP) loci, five downy mildew resistance genes, four isozyme loci and three morphological markers. The genetic markers were distributed into nine linkage groups and cover 404 cM which may be 25-30% of the lettuce genome. The majority (31 of 34) of

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

    Microsoft Academic Search

    Stephane Coupe; Claudine Sarfati; Samia Hamane; Francis Derouin

    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 Crypto- sporidium. 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

  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. Phylogenetic relationships and infraspecific variation in Canadian Arctic Poa based on chloroplast DNA restriction site data

    Microsoft Academic Search

    Lynn J. Gillespie; Ruben Boles

    2001-01-01

    Infraspecific variation and phylogenetic relationships of Canadian Arctic species of the genus Poa were stud- ied based on chloroplast DNA (cpDNA) variation. Restriction site analysis of polymerase chain reaction amplified cpDNA was used to reexamine the status of infraspecific taxa, reconstruct phylogenetic relationships, and reexamine previous classification systems and hypotheses of relationships. Infraspecific variation was detected in three species, but

  19. The use of restriction endonucleases to measure mitochondrial DNA sequence relatedness in natural populations

    Microsoft Academic Search

    Robert A. Lansman; Rosemary O. Shade; John F. Shapira; John C. Avise

    1981-01-01

    Summary Restriction endonucleases and agarose gel electrophoresis have been used to demonstrate extensive nucleotide sequence diversity in mitochondrial DNA (mtDNA) within and between conspecific populations of rodents and other mammals. Cleavage of mtDNA samples with a relatively small number of endonucleases provides information concerning the phylogenetic relatedness of individual organisms which cannot now be readily obtained by any other type

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

    E-print Network

    Burgers, Peter M.

    /s). The Dna2 nuclease/helicase alone did not efficiently promote nick translation, nor did it affect nick translation with FEN1. Maturation in the presence of DNA ligase was studied with various downstream primersOkazaki Fragment Maturation in Yeast I. DISTRIBUTION OF FUNCTIONS BETWEEN FEN1 AND DNA2* Received

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

    PubMed Central

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

    2014-01-01

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

  2. Recognition of sequence-directed DNA structure by the Klenow fragment of DNA polymerase I.

    PubMed

    Carver, T E; Millar, D P

    1998-02-17

    Time-resolved fluorescence spectroscopy was used to investigate the influence of sequence-directed DNA structure upon the interaction between the Klenow fragment of DNA polymerase I and a series of defined oligonucleotide primer/templates. 17/27-mer (primer/template) oligonucleotides containing a dansyl fluorophore conjugated to a modified deoxyuridine residue within the primer strand were used as substrates for binding to Klenow fragment. The time-resolved fluorescence anisotropy decay of the dansyl probe was analyzed in terms of two local environments, either solvent-exposed or buried, corresponding to primer/templates positioned with the primer 3' terminus in the polymerase site or the 3'-5' exonuclease site of the enzyme, respectively. Equilibrium constants for partitioning of DNA between the two sites were evaluated from the anisotropy decay data for primer/templates having different (A + T)-rich sequences flanking the primer 3' terminus. Primer/templates with AAAATG/TTTTAC and CGATAT/GCTATA terminal sequences (the nucleotides on the left refer to the last six bases at the 3' end of the primer, and the nucleotides on the right are the corresponding bases in the template) were bound mostly at the polymerase site. The introduction of single mismatches opposite the primer 3' terminus of these DNA substrates increased their partitioning into the 3'-5' exonuclease site, in accord with the results of an earlier study [Carver, T.E., Hochstrasser, R.A., and Millar, D.P. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 10670-10674]. In contrast, a primer/template with the terminal sequence CAATTT/GTTAAA, containing an A-tract element AATTT, exhibited a surprising preference for binding at the 3'-5' exonuclease site, despite the absence of mismatched bases in the DNA substrate. Interruption of the A-tract with a single AG step, to give the terminal sequence CAGTTT/GTCAAA, reversed the effect of the A-tract, causing the DNA to partition in favor of the polymerase site. Moreover, the presence of a single mismatch opposite the primer 3' terminus was also sufficient to reverse the effect of the A-tract, resulting in a distribution of DNA between polymerase and 3'-5' exonuclease sites that was similar to that observed for the other mismatched DNA substrates. Taken together, these results suggest that the A-tract adopts an unusual conformation that is disruptive to binding at the polymerase site. The effect of the A-tract on binding of DNA to the polymerase site is discussed in terms of the unusual helix structural parameters associated with these sequence elements and the difference between the local geometry of the A-tract and the conformation adopted by duplex DNA within the polymerase cleft. The results of this study show that in addition to base mismatches, Klenow fragment can also recognize irregularities in the helix geometry of perfectly base-paired DNA. PMID:9485315

  3. Efficient combination of large DNA in vitro: in gel site specific recombination (IGSSR) of PAC fragments containing alpha satellite DNA and the human HPRT gene locus.

    PubMed Central

    Schindelhauer, D; Cooke, H J

    1997-01-01

    In an attempt to combine a cloned genomic copy of a selectable gene with different cloned centromeric sequences to develop mammalian artificial chromosomes (MAC) we used site specific recombination mediated by purified Cre recombinase acting on the loxP sequence in PAC vector DNA. A new method was required to purify highly concentrated, virtually 100% intact PAC DNA which could be stored for a long period. Here we show the efficient linking of linearized PACs containing alpha satellite DNA from chromosomes X and 17 with sizes of 125 and 140 kb, respectively, to a 95 kb restriction fragment derived from a 175 kb PAC containing the intact human HPRT gene locus. PMID:9153331

  4. A 300 MHz and 600 MHz proton NMR study of a 12 base pair restriction fragment: investigation of structure by relaxation measurements.

    PubMed Central

    Early, T A; Kearns, D R; Hillen, W; Wells, R D

    1980-01-01

    The 1H NMR spectrum of a 12 base pair DNA restriction fragment has been measured at 300 and 600 MHz and resonances from over 70 protons are individually resolved. Relaxation rate measurements have been carried out at 300 MHz and compared with the theoretical predictions obtained using an isotropic rigid rotor model with coordinates derived from a Dreiding model of DNA. The model gives results that are in excellent agreement with experiment for most protons when a 7 nsec rotational correlation time is used, although agreement is improved for certain base protons by using a shorter correlation time for the sugar group, or by increasing the sugar-base interproton distances. A comparison of non-selective and selective spin-lattice relaxation rates for carbon bound protons indicates that there is extensive spin diffusion even in this short DNA fragment. Examination of the spin-spin relaxation rates for the same type of proton on different base pairs reveals little sequence effect on conformation. PMID:6258152

  5. Gene induction by gamma-irradiation leads to DNA fragmentation in lymphocytes

    SciTech Connect

    Sellins, K.S.; Cohen, J.J.

    1987-11-15

    An early event in death of interphase lymphocytes exposed in vivo or in vitro to low doses of gamma-irradiation is the degradation of DNA into nucleosome-sized fragments. Induction of fragmentation required RNA and protein synthesis because actinomycin D and cycloheximide, respectively, are able to inhibit DNA fragmentation in irradiated lymphocytes. Studies adding cycloheximide and actinomycin D at various times postirradiation suggest that once the metabolic process is initiated within an individual cell it proceeds to completion. The reversible RNA synthesis inhibitor, 5,6-dichloro-1-beta-d-ribofuranosylbenzimidazole inhibits DNA fragmentation in irradiated thymocytes. When this drug is removed after 6 hr, irradiated thymocytes proceed to fragment their DNA; this suggests that an inducing signal that is not simply mRNA persists within the irradiated cell for at least 6 hr after irradiation. In contrast to mitogen-activated T and B lymphoblasts, resting T and B cells show significant DNA fragmentation after exposure to 100 to 500 rad. At 2000 rad, all of the splenic subpopulations die rapidly via a different mechanism. By studying the mechanism of DNA fragmentation induced during the interphase death of lymphocytes, we hope to understand better the extreme sensitivity of resting lymphocytes to radiation and what may be the common final pathway of programmed cell death.

  6. Half tetrad analysis in alfalfa using multiple restriction fragment length polymorphism markers S. Tavoletti, E. T. Bingham, B. S. Yandell, F. Veronesi, and T. C. Osborn

    E-print Network

    Yandell, Brian S.

    Half tetrad analysis in alfalfa using multiple restriction fragment length polymorphism markers S Genetics Half tetrad analysis in alfalfa using multiple restriction fragment length polymorphism markers alfalfa clone PG-F9 (2n = 2x = 16) previously selected because of its high frequency of 2n egg production

  7. Cleavage maps for human cytomegalovirus DNA strain AD169 for restriction endonucleases EcoRI, BglII, and HindIII.

    PubMed Central

    Spector, D H; Hock, L; Tamashiro, J C

    1982-01-01

    We have used cloned EcoRI fragments of the human CMV (HCMV) genome, strain AD169, to prepare restriction endonuclease maps of the DNA. Individual 32P-labeled cloned fragments were hybridized to Southern blots of HCMV DNA cleaved to completion with the restriction endonucleases BglII and HindIII and cleaved partially with EcoRI. By determining which EcoRI fragments hybridized to the same band on a Southern blot, we were able to establish linkage groups. This information coupled with the data derived from digestion of the cloned fragments with the enzymes BglII and HindIII (Tamashiro et al., J. Virol. 42:547-557, 1982) provided the basis for the construction of detailed maps for the enzymes EcoRI, BglII, and HindIII. We also identified the EcoRI fragments derived from the termini of this genome and mapped them with respect to the BglII and HindIII terminal fragments. From our mapping data, we conclude that the genome of HCMV is approximately 240 kilobases in length and is divided into long (198 kilobases) and short (42 kilobases) regions. Both regions consist of a unique sequence bounded by inverted repeats (11 to 12 kilobases for the long region and 2 to 3 kilobases for the short region). Furthermore, the long and short regions can invert relative to each other. Images PMID:6283173

  8. 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

  9. Identification of spotted fever group Rickettsia species by polymerase chain reaction-restriction fragment length polymorphism analysis of the sca4 gene.

    PubMed

    Matsumoto, Kotaro; Inokuma, Hisashi

    2009-12-01

    DNA samples from four rickettsial species in Japan--R. japonica Aoki, R. asiatica IO-1, R. helvetica IP-1, and Rickettsia tamurae AT-1--were analyzed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) in which a portion of the sca4 gene was amplified and restriction digested with the MspI enzyme. Each spotted fever group rickettsial species exhibited unique restriction profiles after MspI digestion. To examine the possibility of applying PCR-RFLP analysis to field samples, 36 Ixodes persulcatus ticks were collected from the Tokachi region (Hokkaido, Japan) by the flagging method and subjected to PCR-RFLP analysis. Of the ticks collected, five had identical restriction profiles to that of R. helvetica. Amplification and sequencing of the gltA gene for two of the five positive samples revealed that the sequences were identical to that of the Rickettsia sp. Tokachi-B-IP17m, which exhibits the highest similarity to R. helvetica. These results demonstrate that PCR-RFLP analysis is useful for identifying Rickettsia species in Japan and is applicable to epidemiological studies involving tick samples. PMID:19485769

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

    Microsoft Academic Search

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

    1990-01-01

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

  11. Helicobacter pylori DprA alleviates restriction barrier for incoming DNA

    PubMed Central

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

    2013-01-01

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

  12. Comparison of DNA fragmentation and color thresholding for objective quantitation of apoptotic cells

    NASA Technical Reports Server (NTRS)

    Plymale, D. R.; Ng Tang, D. S.; Fermin, C. D.; Lewis, D. E.; Martin, D. S.; Garry, R. F.

    1995-01-01

    Apoptosis is a process of cell death characterized by distinctive morphological changes and fragmentation of cellular DNA. Using video imaging and color thresholding techniques, we objectively quantitated the number of cultured CD4+ T-lymphoblastoid cells (HUT78 cells, RH9 subclone) displaying morphological signs of apoptosis before and after exposure to gamma-irradiation. The numbers of apoptotic cells measured by objective video imaging techniques were compared to numbers of apoptotic cells measured in the same samples by sensitive apoptotic assays that quantitate DNA fragmentation. DNA fragmentation assays gave consistently higher values compared with the video imaging assays that measured morphological changes associated with apoptosis. These results suggest that substantial DNA fragmentation can precede or occur in the absence of the morphological changes which are associated with apoptosis in gamma-irradiated RH9 cells.

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

    PubMed Central

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

    2014-01-01

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

  14. Oxygen Tension Influences DNA Fragmentation and Cell Death in Glucocorticoid-Treated Thymocytes

    Microsoft Academic Search

    C. Stefanelli; I. Stanic; F. Bonavita; C. Muscari; C. Pignatti; C. Rossoni; C. M. Caldarera

    1995-01-01

    Internucleosomal DNA fragmentation and cell death induced by dexamethasone in rat thymocytes were inhibited when cells were cultured in 95% N2\\/5% CO2 atmosphere, in which oxygen was rapidly reduced to under 0.5%. DNA fragmentation was delayed by a less severe hypoxia in 5% oxygen whilst in cell cultured in high oxygen atmosphere (95% O2) cell death was increased. On the

  15. Fragment-Based Discovery of 6-Azaindazoles As Inhibitors of Bacterial DNA Ligase

    PubMed Central

    2013-01-01

    Herein we describe the application of fragment-based drug design to bacterial DNA ligase. X-ray crystallography was used to guide structure-based optimization of a fragment-screening hit to give novel, nanomolar, AMP-competitive inhibitors. The lead compound 13 showed antibacterial activity across a range of pathogens. Data to demonstrate mode of action was provided using a strain of S. aureus, engineered to overexpress DNA ligase. PMID:24900632

  16. Restriction Enzyme Analysis

    NSDL National Science Digital Library

    Restriction mapping is a cornerstone of modern-day biotechnology. During this three-day exercise, students will digest samples of DNA with three different restriction enzymes. The DNA samples will be electrophoresed and the resulting fragments photographed under and ultraviolet transilluminator. Attached readings and activities will help explain the process of restriction enzyme analysis and how it is used in the DNA fingerprinting procedure. This 17-page pdf contains teacher information for presenting the activity, the complete procedure of the laboratory, and two student activities.

  17. Clinical significance of telomerase activation and telomeric restriction fragment (TRF) in cervical cancer.

    PubMed

    Zhang, D K; Ngan, H Y; Cheng, R Y; Cheung, A N; Liu, S S; Tsao, S W

    1999-01-01

    Telomerase activation was examined in 50 cases of cervical cancer, 27 normal cervix and five cervical cancer cell lines using the sensitive polymerase chain reaction (PCR)-based TRAP (telomeric repeat amplification protocol) assay. Telomeric restriction fragment (TRF) length of these specimens was measured by Southern hybridisation. Telomerase activation was common in cervical cancers and was detected in 46/50 cases (92%). Telomerase activity was weak in normal cervix and was detected only in 2/27 cases (7.4%). Telomerase activity was detected in all stages of cervical cancer suggesting that it is an early event in cancer progression. The clinical significance of telomerase activation was analysed in 47 squamous cell carcinoma of the cervix. High telomerase activity was more frequently detected in advanced diseases (100% in stage III and stage IV cervical cancers combined) compared with early diseases (68.6% in stage I and stage II cancers combined). The difference was statistically significant (P < 0.02). Telomerase activity was not statistically correlated with other clinical parameters examined. This is the first report of telomeric length in human cervical cancer. Both shortening and elongation of TRF length in cervical cancers was observed. Advanced cervical cancers tended to have a wider range of variation of TRF length compared with early disease and normal cervix. There was no obvious relationship between TRF length and the clinical parameters examined including clinical staging, differentiation status of tumour, human papilloma virus (HPV) infection, recurrence rate, tumour size and invasion depth. The clinical significance of TRF length appears to be limited in cervical cancers. Our results indicate that telomerase activity is closely associated with tumour cells and may be useful as a marker for detection of tumour cells in cervical biopsies. PMID:10211104

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

    PubMed Central

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

    2014-01-01

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

  19. Alleviation of Eco K DNA restriction in Escherichia coli and involvement of umuDC activity

    Microsoft Academic Search

    Kevin J. Hiom; Steven G. Sedgwick

    1992-01-01

    The activity of the EcoK DNA restriction system of Escherichia coli reduces both the plating efficiency of unmodified phage ? and the transforming ability of unmodified pBR322 plasmid DNA. However, restriction can be alleviated in wild-type cells, by UV irradiation and expression of the SOS response, so that 103-to 104-fold increases in phage growth and fourfold increases in plasmid transformation

  20. DNA modifying enzymes of Agrobacterium tumefaciens: effect of DNA topoisomerase, restriction endonuclease, and unique DNA endonuclease on plasmid and plant DNA.

    PubMed Central

    LeBon, J M; Kado, C I; Rosenthal, L J; Chirikjian, J G

    1978-01-01

    Extracts from Agrobacterium tumefaciens strain ID135 contain three enzymes that have been characterized and partially purified. The first enzyme, a DNA topoisomerase, appeared to relax only negatively twisted DNA. The second enzyme, Atu I, a type II restriction endonuclease, generated the identical DNA digestion pattern as EcoRII when several DNAs were used. The third enzyme, endonuclease A, showed a preference for superhelical DNAs as substrates. When plasmid pCK135DNA, obtained from the virulent strain IDI135 of A. tumefaciens, or plant DNA was exposed to the three enzymes, changes in DNA patterns were observed due to either conformational changes or digestion of the DNAs. These enzymes may function in vivo in the processing and incorporation of bacterial DNA in plant cells. Images PMID:212732

  1. Definition of restriction, Paul BergSite: DNA Interactive (www.dnai.org)

    NSDL National Science Digital Library

    2008-03-26

    Interviewee: Paul Berg DNAi Location:Manipulation>Techniques>Cutting & Pasting>On the phenomenon of restriction On the phenomenon of restriction Paul Berg speaks about Herbert Boyer's research into the process by which an organism, such as a bacterium, can recognize and destroy foreign DNA.

  2. A new estimate of sequence divergence of mitochondrial DNA using restriction endonuclease mappings

    Microsoft Academic Search

    Norman Kaplan; Charles H. Langley

    1979-01-01

    Summary A new estimate of the sequence divergence of mitochondrial DNA in related species using restriction enzyme maps is constructed. The estimate is derived assuming a simple Posisson-like model for the evolutionary process and is chosen to maximize an expression which is a reasonable approximation to the true likelihood of the restriction map data. Using this estimate, four sets of

  3. Two-dimensional conformation-dependent electrophoresis (2D-CDE) to separate DNA fragments containing unmatched bulge from complex DNA samples

    PubMed Central

    Gunnarsson, Gudmundur H.; Thormar, Hans G.; Gudmundsson, Bjarki; Akesson, Lina; Jonsson, Jon J.

    2004-01-01

    DNA fragments containing mispaired and modified bases, bulges, lesions and specific sequences have altered conformation. Methods for separating complex samples of DNA fragments based on conformation but independent of length have many applications, including (i) separation of mismatched or unmatched DNA fragments from those perfectly matched; (ii) simultaneous, diagnostic, mismatch scanning of multiple fragments; (iii) isolation of damaged DNA fragments from undamaged fragments; and (iv) estimation of reannealing efficiency of complex DNA samples. We developed a two-dimensional conformation-dependent electrophoresis (2D-CDE) method for separating DNA fragments based on length and conformation in the first dimension and only on length in the second dimension. Differences in migration velocity due to conformation were minimized during second dimension electrophoresis by introducing an intercalator. To test the method, we constructed 298 bp DNA fragments containing cytosine bulges ranging from 1 to 5 nt. Bulge-containing DNA fragments had reduced migration velocity in the first dimension due to altered conformation. After 2D-CDE, bulge-containing DNA fragments had migrated in front of an arc comprising heterogeneous fragments with regular conformation. This simple and robust method could be used in both analytical and preparative applications involving complex DNA samples. PMID:14762200

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

    Microsoft Academic Search

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

    1997-01-01

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

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

    E-print Network

    Kata, Srinivas Reddy

    1993-01-01

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

  6. Characterization of Microbial Diversity by Determining Terminal Restriction Fragment Length Polymorphisms of Genes Encoding 16S rRNA

    Microsoft Academic Search

    WEN-TSO LIU; TERENCE L. MARSH; HANS CHENG; LARRY J. FORNEY

    1997-01-01

    A quantitative molecular technique was developed for rapid analysis of microbial community diversity in various environments. The technique employed PCR in which one of the two primers used was fluorescently labeled at the 5* end and was used to amplify a selected region of bacterial genes encoding 16S rRNA from total community DNA. The PCR product was digested with restriction

  7. A simple and reliable PCR-restriction fragment length polymorphism assay to identify Candida albicans and its closely related Candida dubliniensis

    PubMed Central

    Ge, Yi Ping; Wang, Le; Lu, Gui Xia; Shen, Yong Nian; Liu, Wei Da

    2012-01-01

    Candida dubliniensis is an emerging pathogen capable of causing superficial as well as systemic infections. Due to its close similarity to C. albcians, conventional methods based on phenotypic traits are not always reliable in identification of C. dubliniensis. In this study, we developed a PCR-restriction fragment length polymorphism (RFLP) assay to identify and discriminate between the two closely related species. The D1/D2 region of 28S rDNA was amplified by PCR and enzymatically digested by ApaI and BsiEI respectively. PCR products of both species were digested into two fragments by ApaI, but those of other yeast species were undigested. BsiEI cut the PCR products of C. albicans into two fragments but not those of C. dubliniensis. Thus two species were differentiated. We evaluated 10 reference strains representing 10 yeast species, among which C. albicans and C. dubliniensis were successfully identified. A total of 56 phenotypically characterized clinical isolates (42 C. albicans isolates and 14 C. dubliniensis isolates) were also investigated for intra-species variability. All tested isolates produced identical RFLP patterns to their respective reference strains except one initially misidentified isolate. Our method offers a simple, rapid and reliable molecular method for the identification of C. albicans and C. dubliniensis. PMID:24031901

  8. Mitochondrial DNA polymorphism in native Philippine cattle based on restriction endonuclease cleavage patterns

    Microsoft Academic Search

    Tomomasa Watanabe; Joseph S. Masangkay; Shigeharu Wakana; Naruya Saitou; Takeshi Tomita

    1989-01-01

    An analysis of patterns of cleavage of mtDNA by restriction endonucleases was performed for nine individuals from the Philippine\\u000a population of native cattle. MtDNA polymorphisms were detected in the restriction patterns generated by the following six\\u000a enzymes,BamHI,BglII,EcoRV,HindIII,PstI, andScaI. The restriction patterns showing polymorphisms were distributed nonrandomly among the nine individuals examined from the\\u000a Philippine population of native cattle, indicating the

  9. 'Red pigment' from ADE-2 mutants of S. cerevisiae prevents DNA cleavage by restriction endonucleases.

    PubMed

    Meskauskas, A; Ksenzenko, V; Shlyapnikov, M; Kryukov, V; Citavicius, D

    1985-03-25

    Protection of DNA from cleavage by restriction endonucleases EcoRI, HindIII, BamHI, and Bg/II with red pigment, produced by ADE-2 mutants of Saccharomyces cerevisiae is demonstrated. Purification of yeast DNA from pigment can be achieved by chromatography on hydroxyapatite columns. PMID:2984046

  10. 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

  11. Retroviral Restriction Factor APOBEC3G Delays the Initiation of DNA Synthesis by HIV-1 Reverse

    E-print Network

    Levin, Judith G.

    ) by inducing genome mutagenesis through deamination of cytosine to uracil in single-stranded HIV-1 (2)DNA to inactivation of HIV-1 through guanine (G) to adenine (A) hypermutation of the virus genome strand from reverseRetroviral Restriction Factor APOBEC3G Delays the Initiation of DNA Synthesis by HIV-1 Reverse

  12. Isolation and characterization of two repetitive DNA fragments located near the centromere of the mouse X chromosome

    Microsoft Academic Search

    C. M. Disteche; U. Tantravahi; S. Gandy; M. Eisenhard; D. Adler; L. M. Kunkel

    1985-01-01

    Two repetitive DNA fragments located on the mouse X chromosome are described. The fragments were isolated from a lambda phage library enriched in X-chromosomal sequences by flow sorting. Both fragments, which are repeated 20 to 50 times in the genome, were mapped to the mouse X chromosome by Southern blot hybridization to DNA from hybrid cells retaining the mouse X

  13. Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation

    Microsoft Academic Search

    Yael Gavrieli; Yoav Sherman; Shmuel A. Ben-Sasson

    1992-01-01

    Programmed cell death (PCD) plays a key role in developmental biology and in maintenance of the steady state in continuously renewing tissues. Cur- rently, its existence is inferred mainly from gel elec- trophoresis of a pooled DNA extract as PCD was shown to be associated with DNA fragmentation. Based on this observation, we describe here the devel- opment of a

  14. Interaction of fragmented double-stranded DNA with carbon nanotubes in aqueous solution

    NASA Astrophysics Data System (ADS)

    Gladchenko, G. O.; Karachevtsev, M. V.; Leontiev, V. S.; Valeev, V. A.; Glamazda, A. Yu.; Plokhotnichenko, A. M.; Stepanian, S. G.

    Aqueous suspensions of ultrasonically fragmented double-stranded (fds-) DNA and single-walled carbon nanotubes (SWNTs) have been investigated by UV- and IR-absorption, NIR-emission and Raman spectroscopy. According to gel-electrophoresis, the lengths of the polymer fragments were 100-500 base pairs. Analysis of IR and UV data indicates the presence of both double-stranded (ds) and single-stranded (ss)-regions in the fragments. SWNT complex with DNA was revealed by NIR-emission and Raman spectroscopy. It turned out that fds-DNA is less efficient in holding nanotubes in the aqueous solution than ss-DNA. From the UV-data, the character of the helix-coil transition is seen to be like that for fds-DNA off and on nanotube, however, DNA thermostability increased in this latter case. The effective charge density on the DNA sugar-phosphate backbone of the fds-DNA:SWNT hybrid was less than that of DNA alone. Spectroscopic data can be explained by a model in which the formation of hybrids starts due to the interaction between untwisted ss-regions of DNA and the nanotube: the strands wrap on the tube and thus create an 'anchor' for the whole polymer. The ds-part of the polymer is located close to the nanotube.

  15. Babesia canis canis, Babesia canis vogeli, Babesia canis rossi: differentiation of the three subspecies by a restriction fragment length polymorphism analysis on amplified small subunit ribosomal RNA genes.

    PubMed

    Carret, C; Walas, F; Carcy, B; Grande, N; Précigout, E; Moubri, K; Schetters, T P; Gorenflot, A

    1999-01-01

    The parasites Babesia canis and Babesia gibsoni (phylum Apicomplexa) are responsible for canine babesiosis throughout the world. Babesia canis was previously described as a group of three biologically different subspecies, namely B. canis canis, B. canis vogeli, and B. canis rossi. We report partial sequences of small subunit ribosomal RNA gene (ssu-rDNA) of each subspecies amplified in vitro with primers derived from a semi-conserved region of the ssu-rDNA genes in other Babesia species. The polymerase chain reaction combined with a restriction fragment length polymorphism analysis, using HinfI and TaqI restriction enzymes, confirmed the separation of B. canis into three subspecies. These sequences were compared with previously published sequences of other Babesia species. A phylogenetic approach showed that the three subspecies of B. canis belong to the clade of Babesia species sensu stricto where B. canis canis clusters with B. canis rossi whereas B. canis vogeli might form a monophyletic group with the cluster B. divergens and B. odocoilei. Our results show that the three subspecies of B. canis can readily be differentiated at the molecular level and suggest that they might be considered as true species. PMID:10377990

  16. Structure, restriction map and infectivity of the genomic and replicative forms of Aa PV DNA

    Microsoft Academic Search

    Y. Boublik; F.-X. Jousset; M. Bergoin

    1994-01-01

    Summary We have characterized the genomic and replicative form (RF) DNA of theAedes albopictus Parvovirus (AaPV), a virus isolated from a chronically infected C6\\/36 clone ofAedes albopictus cell line [22]. The genome ofAaPV virions is a single-stranded linear DNA molecule approximately 4.2 kb in length, essentially (about 90%) encapsidated as minus strand. A restriction map of the RF DNA isolated

  17. Components of variance in transcriptomics based on electrophoretic separation of cDNA fragments (cDNA-AFLP).

    PubMed

    Weiberg, Arne; Karlovsky, Petr

    2009-07-01

    The sources of variance and errors in transcriptomics based on the electrophoretic separation of amplified cDNA fragments were investigated using cDNA-amplified fragment length polymorphism (AFLP). Transcriptome profiles of the plant-pathogenic fungus Verticillium longisporum were generated by a standard cDNA-AFLP protocol followed by electrophoretic separation of amplified DNA fragments in flatbed polyacrylamide gels with fluorescence detection as well as by capillary electrophoresis (DNA sequencer). The total variance was partitioned into contributions of cDNA synthesis, adapter ligation, preamplification, amplification, and electrophoresis. Parameters of computer-aided peak recognition and matching were investigated and strategies improving matching success based on double passage with different signal intensity thresholds were developed. The overall quality of data was similar for cDNA-AFLP and microarray hybridization. Variance of cDNA-AFLP was independent of signal intensity, whereas microarray data showed higher variance for low-intensity signals. Capillary electrophoresis significantly reduced the number of wrongly matched and unmatched signals as compared with flatbed gels. These results are also likely to apply to related electrophoresis-based transcriptome analysis techniques such as mRNA differential display. PMID:19588459

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

    E-print Network

    Rose, Michael R.

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

  19. Exogenous DNA internalisation by sperm cells is improved by combining lipofection and restriction enzyme mediated integration.

    PubMed

    Churchil, R R; Gupta, J; Singh, A; Sharma, D

    2011-06-01

    1. Three types of exogenous DNA inserts, i.e. complete linearised pVIVO2-GFP/LacZ vector (9620 bp), the LacZ gene (5317 bp) and the GFP gene (2152 bp) were used to transfect chicken spermatozoa through simple incubation of sperm cells with insert. 2. PCR assay, Dot Blot hybridisation and Southern hybridisation showed the successful internalisation of exogenous DNA by chicken sperm cells. 3. Lipofection and Restriction Enzyme Mediated Integration (REMI) were used to improve the rate of internalisation of exogenous DNA by sperm cells. 4. Results from dot blot as well as Southern hybridisation were semi-quantified and improved exogenous DNA uptake by sperm cells through lipofection and REMI. Stronger signals were observed from hybridisation of LacZ as well as GFP specific probe with the DNA from lipofected exogenous DNA transfected sperm DNA in comparison with those transfected with nude exogenous DNA. PMID:21732873

  20. APPLICATION OF POLYMERASE CHAIN REACTION (PCR) AND PCR BASED RESTRICTION FRAGMENT LENGTH POLYMORPHISM FOR DETECTION AND IDENTIFICATION OF DERMATOPHYTES FROM DERMATOLOGICAL SPECIMENS

    PubMed Central

    Bagyalakshmi, R; Senthilvelan, B; Therese, K L; Murugusundram, S; Madhavan, H N

    2008-01-01

    Objective: To develop and optimize polymerase chain reaction-based restriction fragment length polymorphism (PCR-RFLP) targeting 18S rDNA and internal transcribed spacer (ITS) region of fungi for rapid detection and identification of dermatophytes. Materials and Methods: Two PCR-RFLP methods targeting 18S rDNA and ITS regions of fungi were optimized using standard and laboratory isolates of dermatophytes and other fungi. Sixty-eight dermatological clinical specimens (nail clippings (56), material obtained from blisters (8), hair root (2), scraping from scaly plaque of foot (1) and skin scraping (1) collected by the dermatologist were subjected to both the optimized PCR-RFLP and conventional mycological (smear and culture) methods. Results: PCRs targeting 18S rDNA and the ITS region were sensitive to detect 10 picograms and 1 femtogram of T. rubrum DNA, respectively. PCR targeting 18S rDNA was specific for dermatophytes and subsequent RFLP identified them to species level. PCR-RFLP targeting the ITS region differentiated dermatophytes from other fungi with identification to species level. Among the 68 clinical specimens tested, both PCR-RFLP methods revealed the presence of dermatophytes in 27 cases (39.7%), whereas culture revealed the same only in 2 cases (7.40%), increasing the clinical sensitivity by 32.3%. Among 20 smear positive specimens, both PCR-RFLP methods detected dermatophytes in 12 (17.6%). Both the methods detected the presence of dermatophytes in 13 (19.11%) smear and culture negative specimens, increasing the clinical sensitivity by 36.1%. Conclusion: PCR-RFLP methods targeting 18S rDNA and the ITS regions of fungi were specific and highly sensitive for detection and speciation of dermatophytes. PMID:19967012

  1. Viability and DNA fragmentation in differently sorted boar spermatozoa

    Microsoft Academic Search

    M. De Ambrogi; M. Spinaci; G. Galeati; C. Tamanini

    2006-01-01

    Sperm cell defense against DNA damage relies on two factors: the tight packaging of chromatin, based on condensation and substitution of histones with protamines, and the antioxidant agents present in seminal plasma. These defenses are extremely important as mature sperm is unable to repair DNA damage and even if a successful fertilization occurs, embryo undergoes apoptosis at the time of

  2. Mechanism of DNA fragmentation during hypoxia in the cerebral cortex of newborn piglets.

    PubMed

    Chiang, Ming-Chou; Ashraf, Qazi M; Mishra, Om P; Delivoria-Papadopoulos, Maria

    2008-07-01

    We have previously shown that hypoxia results in increased activity of caspase-9, caspase-3 and fragmentation of nuclear DNA in the cerebral cortex of newborn piglets. The present study tested the hypothesis that mechanism of DNA fragmentation during hypoxia in the cerebral cortex of newborn piglets is mediated by caspase-9-dependent caspase-3 activation. Newborn piglets were randomly assigned to normoxic, hypoxic, and hypoxic pretreated with a highly selective caspase-9 inhibitor, Z-LEHD-FMK groups. The data showed that cerebral tissue hypoxia results in increased expression of caspase-activated DNase (CAD) protein in the nucleus and fragmentation of nuclear DNA. A pretreatment with Z-LEHD-FMK attenuated the expression of CAD protein in the nucleus and the fragmentation of nuclear DNA. Based on these results, we conclude that the mechanism by which the nuclear DNA was fragmented is mediated by caspase-9-dependent caspase-3 activation and the consequence of caspase-activated DNase activation in the cerebral cortex of newborn piglets. PMID:18253826

  3. Purification of a 24-kD protease from apoptotic tumor cells that activates DNA fragmentation

    PubMed Central

    1994-01-01

    We report the purification of a protease from tumor cells undergoing apoptosis that is involved in activating DNA fragmentation. Initial studies revealed that two inhibitors of serine proteases, N-1- tosylamide-2-phenylethylchloromethyl ketone and carbobenzoxy-Ala-Ala- borophe (DK120), suppressed tumor necrosis factor or ultraviolet (UV) light-induced DNA fragmentation in the U937 histiocytic lymphoma as well as UV light-induced DNA fragmentation in the BT-20 breast carcinoma, HL-60 myelocytic leukemia, and 3T3 fibroblasts. The protease was purified by affinity chromatography with DK120 as ligand and showed high activity on a synthetic substrate preferred by elastase-like enzymes (Ala-Ala-Pro-Val p-nitroanilide), but was inactive on the trypsin substrate, N-alpha-benzyloxycarbonyl-L-lysine thiobenzyl ester, or the chymotrypsin substrate, Ala-Ala-Pro-Phe p-nitroanilide. The activity of the DK120-binding protease purified from U937 cells undergoing apoptosis was increased approximately 10-fold over that recovered from normal cells. Further purification to homogeneity by heparin-Sepharose affinity chromatography followed by reverse phase high-performance liquid chromatography revealed a single band of 24 kD on a silver-stained sodium dodecyl sulfate gel. In addition to protease activity, the purified enzyme induced DNA fragmentation into multiples of 180 basepairs in isolated U937 nuclei. These findings suggest the 24- kD protease is a novel enzyme that activates DNA fragmentation in U937 cells undergoing apoptosis. PMID:7964487

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

    PubMed

    Moazzam, A; Sharma, R; Agarwal, A

    2014-10-24

    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. 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 ...

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

    PubMed Central

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

    2000-01-01

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

  7. DNA-fragments are transcytosed across CaCo-2 cells by adsorptive endocytosis and vesicular mediated transport.

    PubMed

    Johannessen, Lene E; Spilsberg, Bjørn; Wiik-Nielsen, Christer R; Kristoffersen, Anja B; Holst-Jensen, Arne; Berdal, Knut G

    2013-01-01

    Dietary DNA is degraded into shorter DNA-fragments and single nucleosides in the gastrointestinal tract. Dietary DNA is mainly taken up as single nucleosides and bases, but even dietary DNA-fragments of up to a few hundred bp are able to cross the intestinal barrier and enter the blood stream. The molecular mechanisms behind transport of DNA-fragments across the intestine and the effects of this transport on the organism are currently unknown. Here we investigate the transport of DNA-fragments across the intestinal barrier, focusing on transport mechanisms and rates. The human intestinal epithelial cell line CaCo-2 was used as a model. As DNA material a PCR-fragment of 633 bp was used and quantitative real time PCR was used as detection method. DNA-fragments were found to be transported across polarized CaCo-2 cells in the apical to basolateral direction (AB). After 90 min the difference in directionality AB vs. BA was >10(3) fold. Even undegraded DNA-fragments of 633 bp could be detected in the basolateral receiver compartment at this time point. Transport of DNA-fragments was sensitive to low temperature and inhibition of endosomal acidification. DNA-transport across CaCo-2 cells was not competed out with oligodeoxynucleotides, fucoidan, heparin, heparan sulphate and dextrane sulphate, while linearized plasmid DNA, on the other hand, reduced transcytosis of DNA-fragments by a factor of approximately 2. Our findings therefore suggest that vesicular transport is mediating transcytosis of dietary DNA-fragments across intestinal cells and that DNA binding proteins are involved in this process. If we extrapolate our findings to in vivo conditions it could be hypothesized that this transport mechanism has a function in the immune system. PMID:23409196

  8. DNA Restriction-Modification Systems Mediate Plasmid Maintenance

    Microsoft Academic Search

    SAULIUS KULAKAUSKAS; ARVYDAS LUBYS; DUSKO EHRLICH; Genetique Microbienne; Domaine Vilvert

    1995-01-01

    Two plasmid-carried restriction-modification (R-M) systems, EcoRI (from pMB1 of Escherichia coli) and Bsp6I (from pXH13 ofBacillussp. strain RFL6), enhance plasmid segregational stability inE. coliandBacillus subtilis, respectively. Inactivation of the endonuclease or the presence of the methylase in trans abolish the stabilizing activity of the R-M systems. We propose that R-M systems mediate plasmid segregational stability by postsegregational killing of plasmid-free

  9. Sperm chromatin dispersion test in the assessment of DNA fragmentation and aneuploidy in human spermatozoa.

    PubMed

    Balasuriya, A; Speyer, B; Serhal, P; Doshi, A; Harper, J C

    2011-05-01

    Sperm DNA damage is thought to be increased in men with male factor infertility. Previous studies suggest a correlation between sperm DNA fragmentation and aneuploidy. The sperm chromatin dispersion (SCD) test was modified to produce the Halosperm Kit. The SCD-fluorescent in-situ hybridization (FISH) test allows the simultaneous detection of DNA fragmentation and aneuploidy on the same sperm cell. The objectives of this study were to validate the SCD, SCD-FISH and Halosperm tests for the analysis of sperm DNA fragmentation and compare them to the sperm chromatin structure assay (SCSA). Semen samples from 20 males undergoing IVF/intracytoplasmic sperm injection were processed using FISH, SCD-FISH, SCD and Halosperm, and compared with SCSA results. There was a significant difference between FISH and SCD-FISH results in the detection of aneuploidy (P=0.000) and the level of sperm DNA fragmentation in the samples subjected to SCSA and SCD (P=0.001) or SCSA and SCD-FISH (P=0.001). There was no significant correlation between DNA fragmentation and aneuploidy. If sperm aneuploidy is to be determined, more reliable results will be obtained if FISH is performed rather than SCD-FISH. A lack of validation and unknown clinical significance question the value of DNA fragmentation assays. DNA damage in the male germ line may result in adverse clinical outcomes and the pathophysiology and clinical consequences of sperm DNA damage are being actively researched. Many DNA fragmentation assays such as the Halosperm Kit have been developed recently and are now available at a commercial level. Unfortunately, aimed at vulnerable couples with difficulty conceiving, many of these tests have not been clinically validated. Despite its plausible appeal and fervour of its supporters, the benefits of widespread DNA testing that only achieves the distressing of couples with the knowledge that effectual therapeutic strategies are absent are questionable. Commercially, however, it is no doubt lucrative. Analysis of gametes prior to the initiation of an IVF cycle may improve the quality of embryos transferred. The clinical and scientific community considers it a matter of urgency to translate the basic science behind how a cell prepares for fertilization into routine clinical practice. However, it is equally important, if not more, to allow the science behind such applications to draw level with its practice before its widespread implementation. PMID:21397561

  10. Ribosomal RNA genes in Euglena gracilis mitochondrial DNA: fragmented genes in a seemingly fragmented genome

    Microsoft Academic Search

    David F. Spencer; Michael W. Gray

    2011-01-01

    Because relatively little information is available about mtDNA in the euglenid protozoa, distant relatives of the kinetoplastid\\u000a protozoa, we investigated mitochondrial genome structure and expression in Euglena gracilis. We found that isolated E. gracilis mtDNA comprises a heterodisperse collection of short molecules (modal size ~4 kbp) and that the mitochondrial large subunit\\u000a (LSU) and small subunit (SSU) rRNAs are each split

  11. DNA-Tract Curvature Profile Reconstruction: A Fragment Flipping Algorithm

    Microsoft Academic Search

    Daniele Masotti; Viale Risorgimento

    2002-01-01

    At nanometric level of resolution DNA molecules can be idealized with one dimensional curved line. The curvature value along\\u000a this line is composed by static and dynamic contributions. The first ones constitute the intrinsic curvature, vectorial function\\u000a of the sequence of DNA nucletides, while the second ones, caused by thermal energy, constitute the flexibility. The analysis\\u000a of intrinsic curvature are

  12. DNA Fingerprinting by Infrequent-Restriction-Site Amplification

    Microsoft Academic Search

    GERALD H. MAZUREK; VENKAT REDDY; BARBARA J. MARSTON; WALTER H. HAAS; ANDJACK T. CRAWFORD

    1996-01-01

    Identification of bacterial strains by DNA fingerprinting facilitates epidemiologic studies and improves disease control. For some species of organisms, no typing method is available; for others, typing methods are tedious.WedevelopedamethodofamplifyingDNAsequencesflankinginfrequentrestrictionsitesbyPCRand used the method to produce strain-specific electrophoretic patterns from crude bacterial lysates. This method of fingerprinting is rapid, sensitive, and widely applicable. Identical enzymes, adaptors, primers, and PCR conditions were

  13. Highlights of the DNA cutters: a short history of the restriction enzymes.

    PubMed

    Loenen, Wil A M; Dryden, David T F; Raleigh, Elisabeth A; Wilson, Geoffrey G; Murray, Noreen E

    2014-01-01

    In the early 1950's, 'host-controlled variation in bacterial viruses' was reported as a non-hereditary phenomenon: one cycle of viral growth on certain bacterial hosts affected the ability of progeny virus to grow on other hosts by either restricting or enlarging their host range. Unlike mutation, this change was reversible, and one cycle of growth in the previous host returned the virus to its original form. These simple observations heralded the discovery of the endonuclease and methyltransferase activities of what are now termed Type I, II, III and IV DNA restriction-modification systems. The Type II restriction enzymes (e.g. EcoRI) gave rise to recombinant DNA technology that has transformed molecular biology and medicine. This review traces the discovery of restriction enzymes and their continuing impact on molecular biology and medicine. PMID:24141096

  14. Highlights of the DNA cutters: a short history of the restriction enzymes

    PubMed Central

    Loenen, Wil A. M.; Dryden, David T. F.; Raleigh, Elisabeth A.; Wilson, Geoffrey G.; Murray, Noreen E.

    2014-01-01

    In the early 1950’s, ‘host-controlled variation in bacterial viruses’ was reported as a non-hereditary phenomenon: one cycle of viral growth on certain bacterial hosts affected the ability of progeny virus to grow on other hosts by either restricting or enlarging their host range. Unlike mutation, this change was reversible, and one cycle of growth in the previous host returned the virus to its original form. These simple observations heralded the discovery of the endonuclease and methyltransferase activities of what are now termed Type I, II, III and IV DNA restriction-modification systems. The Type II restriction enzymes (e.g. EcoRI) gave rise to recombinant DNA technology that has transformed molecular biology and medicine. This review traces the discovery of restriction enzymes and their continuing impact on molecular biology and medicine. PMID:24141096

  15. 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.

  16. A Y-chromosomal DNA Fragment Is Conserved in Human and Chimpanzee1

    Microsoft Academic Search

    B. K. A. Rasheed; E. C. Whisenant; R. Fernandez; H. Ostrer

    A human male-specific Y-chromosomal DNA fragment (hYH2D6) has been iso- lated. By deletion-mapping analysis, 2D6 has been localized to the euchromatic portion of the long arm (Yq 11) of the human Y chromosome. Among great apes, this fragment was found to be conserved in male chimpanzee but was lacking in male gorilla and male orangutan. No homologous fmgments were detected

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

    PubMed Central

    Lagacé, L.; Pitre, M.; Jacques, M.; Roy, D.

    2004-01-01

    The bacterial community of maple sap was characterized by analysis of samples obtained at the taphole of maple trees for the 2001 and 2002 seasons. Among the 190 bacterial isolates, 32 groups were formed according to the similarity of the banding patterns obtained by amplified ribosomal DNA restriction analysis (ARDRA). A subset of representative isolates for each ARDRA group was identified by 16S rRNA gene fragment sequencing. Results showed a wide variety of organisms, with 22 different genera encountered. Pseudomonas and Ralstonia, of the ?- and ?-Proteobacteria, respectively, were the most frequently encountered genera. Gram-positive bacteria were also observed, and Staphylococcus, Plantibacter, and Bacillus were the most highly represented genera. The sampling period corresponding to 50% of the cumulative sap flow percentage presented the greatest bacterial diversity according to its Shannon diversity index value (1.1). ?-Proteobacteria were found to be dominant almost from the beginning of the season to the end. These results are providing interesting insights on maple sap microflora that will be useful for further investigation related to microbial contamination and quality of maple products and also for guiding new strategies on taphole contamination control. PMID:15066796

  18. Selective propagation of functional mitochondrial DNA during oogenesis restricts the transmission of a deleterious mitochondrial variant.

    PubMed

    Hill, Jahda H; Chen, Zhe; Xu, Hong

    2014-04-01

    Although mitochondrial DNA (mtDNA) is prone to mutation and few mtDNA repair mechanisms exist, crippling mitochondrial mutations are exceedingly rare. Recent studies have demonstrated strong purifying selection in the mouse female germline. However, the mechanisms underlying positive selection of healthy mitochondria remain to be elucidated. We visualized mtDNA replication during Drosophila melanogaster oogenesis, finding that mtDNA replication commenced before oocyte determination during the late germarium stage and was dependent on mitochondrial fitness. We isolated a temperature-sensitive lethal mtDNA allele, mt:CoI(T300I), which resulted in reduced mtDNA replication in the germarium at the restrictive temperature. Additionally, the frequency of the mt:CoI(T300I) allele in heteroplasmic flies was decreased, both during oogenesis and over multiple generations, at the restrictive temperature. Furthermore, we determined that selection against mt:CoI(T300I) overlaps with the timing of selective replication of mtDNA in the germarium. These findings establish a previously uncharacterized developmental mechanism for the selective amplification of wild-type mtDNA, which may be evolutionarily conserved to limit the transmission of deleterious mutations. PMID:24614072

  19. Variation between observed and true Terminal Restriction Fragment length is dependent on true TRF length and purine content.

    PubMed

    Kaplan, Christopher W; Kitts, Christopher L

    2003-07-01

    Terminal Restriction Fragment (TRF) pattern analysis has become a widely used and informative tool for studying microbial communities. Variation between sequence-determined or true TRF length and observed TRF length (TRF drift) has been previously reported and can significantly affect identification of bacterial species using TRF lengths predicted from sequence databases. In this study TRF drift was determined for 21 bacterial species using an ABI 310 Genetic Analyzer. TRF drift was positively correlated with true TRF length and negatively correlated with TRF purine content. This implies that subtle differences in molecular weight, whether from purine content or dye label, can significantly affect the observed TRF length. PMID:12732430

  20. Allozyme and restriction fragment length polymorphism analyses confirm Entomophaga maimaiga responsible for 1989 epizootics in North American gypsy moth populations.

    PubMed Central

    Hajek, A E; Humber, R A; Elkinton, J S; May, B; Walsh, S R; Silver, J C

    1990-01-01

    In 1989, populations of North American gypsy moth, Lymantria dispar, in seven contiguous northeastern states were severely reduced by a fungal pathogen. Based on morphology, development, and pathology, this organism appeared to be Entomophaga maimaiga. We have now used allozyme and restriction fragment length polymorphism analyses to confirm this identification. Previously, this mycopathogen had been reported only from gypsy moth populations in Japan. During 1989, E. maimaiga occurred only in areas that had been initially defoliated by gypsy moth >10 years ago. E. maimaiga caused 60-88% mortality in late instar larvae on research sites in central Massachusetts. Images PMID:11607100

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

    NASA Astrophysics Data System (ADS)

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

    1996-01-01

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

  2. 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 ...

  3. Polymerase chain reaction-restriction fragment length polymorphism assays to distinguish Liriomyza huidobrensis (Diptera: Agromyzidae) from associated species on lettuce cropping systems in Italy.

    PubMed

    Masetti, Antonio; Luchetti, Andrea; Mantovani, Barbara; Burgio, Giovanni

    2006-08-01

    The pea leafminer, Liriomyza huidobrensis (Blanchard) (Diptera: Agromyzidae), is a serious insect pest infesting open field lettuce plantings in northern Italy. In these cropping systems, it coexists with several other agromyzid species that have negligible economic importance on open field vegetables. The rapid detection of L. huidobrensis is crucial for effective management strategies, but the identification of agromyzids to species can be very difficult at adult as well at immature stages. In this study, a polymerase chain reaction (PCR)-restriction fragment length polymorphism assay is proposed to separate L. huidobrensis from Liriomyza bryoniae (Kaltenbach), Liriomyza trifolii (Burgess), and Chromatomyia horticola (Goureau), which usually occur in the same lettuce plantings. An approximately 1,031-bp region of the mitochondrial genome encompassing the 3' region of cytochrome oxidase I, the whole leucine tRNA, and all of the cytochrome oxidase II was amplified by PCR and digested using the enzymes PvuII and SnaBI separately. Both endonucleases cut the amplicons of L. huidobrensis in two fragments, whereas the original band was not cleaved in the other analyzed species. The presence of Dacnusa spp. DNA does not bias the assay, because the PCR conditions and the primer set here described do not amplify any tract of this endoparasitic wasp genome. PMID:16937681

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

    PubMed

    Tavukçuo?lu, Ilkay ?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

  5. Methylation of single-stranded DNA in vitro introduces new restriction endonuclease cleavage sites

    Microsoft Academic Search

    Bruno Gronenborn; JOACHIM MESSING

    1978-01-01

    RESTRICTION endonucleases recognise specific sequences in DNA, and these endonucleases, especially those which generate cohesive ends, have been widely used to clone DNA1. However, many DNAs lack sequences which are recognised by endonucleases such as EcoRI, HindIII or BamHI. A general method of overcoming this problem has been described recently2. This approach involves the synthesis of oligonucleotides sensitive to a

  6. Macronuclear transformation with specific DNA fragments controls the content of the new macronuclear genome in Paramecium tetraurelia.

    PubMed Central

    You, Y; Aufderheide, K; Morand, J; Rodkey, K; Forney, J

    1991-01-01

    A previously isolated mutant cell line called d48 contains a complete copy of the A surface antigen gene in the micronuclear genome, but the gene is not incorporated into the macronucleus. Previous experiments have shown that a cytoplasmic factor made in the wild-type macronucleus can rescue the mutant. Recently, S. Koizumi and S. Kobayashi (Mol. Cell. Biol. 9:4398-4401, 1989) observed that injection of a plasmid containing the A gene into the d48 macronucleus rescued the cell line after autogamy. It is shown here that an 8.8-kb EcoRI fragment containing only a portion of the A gene coding region is sufficient for the rescue of d48. The inability of other A gene fragments to rescue the mutant shows that this effect is dependent upon specific Paramecium DNA sequences. Rescue results in restoration of the wild-type DNA restriction pattern in the macronucleus. These results are consistent with a model in which the macronuclear A locus normally makes an additional gene product that is required for correct processing of the micronuclear copy of the A gene. Images PMID:1990269

  7. 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.

  8. 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

  9. Electrostatic field of the large fragment of Escherichia coli DNA polymerase I.

    PubMed

    Warwicker, J; Ollis, D; Richards, F M; Steitz, T A

    1985-12-01

    The electrostatic field of the large fragment of Escherichia coli DNA polymerase I (Klenow fragment) has been calculated by the finite difference procedure on a 2 A grid. The potential field is substantially negative at physiological pH (reflecting the net negative charge at this pH). The largest regions of positive potential are in the deep crevice of the C-terminal domain, which is the proposed binding site for the DNA substrate. Within the crevice, the electrostatic potential has a partly helical form. If the DNA is positioned to fulfil stereochemical requirements, then the positive potential generally follows the major groove and (to a lesser extent) the negative potential is in the minor groove. Such an arrangement could stabilize DNA configurations related by screw symmetry. The histidine residues of the Klenow fragment give the positive field of the groove a sensitivity to relatively small pH changes around neutrality. We suggest that the histidine residues could change their ionization states in response to DNA binding, and that this effect could contribute to the protein-DNA binding energy. PMID:3912509

  10. Use of restriction fragment polymorphism analysis of rRNA genes to assign species to unknown clinical isolates of oral viridans streptococci.

    PubMed Central

    Rudney, J D; Larson, C J

    1994-01-01

    This study evaluated restriction fragment length polymorphisms of rRNA genes (ribotyping) for genotypic identification of 53 oral isolates classified as "Streptococcus sanguis" by colony morphology. Isolates were from 8-h buccal plaque on lower first permanent molars of 20 subjects. DNA was digested with AatII and hybridized with digoxygenin-labeled cDNA of Escherichia coli 16S and 23S rRNA. Strains were ribotyped again with AlwNI or PvuII on the basis of the presence or absence of a 2,290-bp AatII band. Band patterns were compared with reference ribotypes for Streptococcus gordonii, Streptococcus sanguis, Streptococcus crista, Streptococcus oralis, Streptococcus mitis, and Streptococcus parasanguis strains. Forty-eight isolates could be assigned to a species (22 S. sanguis, 14 S. oralis, 12 S. gordonii). Multiple species were seen in 14 subjects; multiple strains of the same species occurred in 11 subjects. Our findings suggest that ribotyping can be used for genotypic identification of S. sanguis, S. oralis, and S. gordonii isolates. Images PMID:7512095

  11. 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

  12. Sequence dependent DNA conformations: Raman spectroscopic studies and a model of action of restriction enzymes.

    PubMed

    Nishimura, Y

    1985-01-01

    Raman spectra have been examined to clarify the polymorphic forms of DNA, A, B, and Z forms. From an analysis we found that the guanine ring breathing vibration is sensitive to its local conformation. Examination of nine crystals of guanosine residues in which the local conformations are well established revealed that a guanosine residue with a C3'endo-anti gives a strong line at 666 +/- 2 cm-1, O4'endo-anti at 682 cm-1, C1'exo-anti at 673 cm-1, C2'endo-anti at 677 cm-1 and syn-forms around 625 cm-1. Using this characteristic line, we were able to obtain the local conformations of guanosine moieties in poly(dG-dC). Such a sequence derived variation is suggested to be recognized by sequence specific proteins such as restriction enzymes. We found a correlation between sequence dependent DNA conformation and a mode of action of restriction enzymes. The cutting mode of restriction enzymes is classified into three groups. The classification of whether the products have blunt ends, two-base-long cohesive ends, or four-base-long cohesive ends depends primarily on the substrate, not on the enzyme. It is suggested that sequence dependent DNA conformation causes such a classification by the use of the Calladine-Dickerson analysis. In the recognition of restriction enzymes, the methyl group in a certain sequence is considered to play an important role by changing the local conformation of DNA. PMID:3010660

  13. Characterization of microbial diversity in hypersaline environments by melting profiles and reassociation kinetics in combination with terminal restriction fragment length polymorphism (T-RFLP).

    PubMed

    Øvreås, L; Daae, F L; Torsvik, V; Rodríguez-Valera, F

    2003-10-01

    The diversity of prokaryotes inhabiting solar saltern ponds was determined by thermal melting and reassociation of community DNA. These measurements were compared with fingerprinting techniques such as terminal restriction fragment length polymorphisms (T-RFLP) analysis, denaturant gradient gel electrophoresis (DGGE), and cloning and sequencing approaches. Three ponds with salinities of 22, 32, and 37% (NaCl saturation) were studied. The combination of independent molecular techniques to estimate the total genetic diversity provided a realistic assessment to reveal the microbial diversity in these environments. The changes in the prokaryotic communities at different salinity (22, 32, and 37% salt) were significant and revealed that the total genetic diversity increased from 22% to 32% salinity. At 37% salinity the diversity was reduced again to nearly half that at 22% salinity. Our results revealed that the community "genome" had a DNA complexity that was 7 (in 22% salinity pond), 13 (in 32% salinity pond), and 4 (in 37% salinity pond) times the complexity of an Escherichia coli genome. The base composition profiles showed two abundant populations, which changed in relative amount between the three ponds. They indicated an uneven taxon distribution at 22% and 37% salinity and a more even distribution at 32% salinity. The results indicated a large predominating population at 37% salinity, which might correspond to the abundance of square archaea (SPhT) observed by transmission electron microscopy (TEM) and also indicated by the same T-RFLP fragment as the SPhT. The SPhT phylotype has also been reported to be the most frequently retrieved phylotype from this environment by culture independent techniques. In addition, two different operational taxonomic units (OTU) were detected at 37% salinity based on PCR with bacterial specific primers and T-RFLP. One of these predominant phylotypes is the extreme halophilic bacterium belonging to the bacteroidetes group, Salinibacter ruber. PMID:12904916

  14. Tumor Cell Growth Arrest Caused by Subchromosomal Transferable DNA Fragments from Chromosome 11

    Microsoft Academic Search

    Minoru Koi; Laura A. Johnson; Linda M. Kalikin; Peter F. R. Little; Yusuke Nakamura; Andrew P. Feinberg

    1993-01-01

    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 cell. As

  15. 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...

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

    Microsoft Academic Search

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

    1995-01-01

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

  17. The thermal stability of DNA fragments with tandem mismatches at a d(CXYG).d(CY'X'G) site

    Microsoft Academic Search

    Song-Hua Ke; Roger M. Wartell

    1996-01-01

    Temperature-Gradient Gel Electrophoresis (TGGE) was employed to determine the thermal stabilities of 28 DNA fragments, 373 bp long, with two adjacent mismatched base pairs, and eight DNAs with Watson- Crick base pairs at the same positions. Heteroduplex DNAs containing two adjacent mismatches were formed by melting and reannealing pairs of homo- logous 373 bp DNA fragments differing by two adjac-

  18. A combined molecular and cytogenetic approach to genome evolution in Drosophila using large-fragment DNA cloning

    Microsoft Academic Search

    Elena R. Lozovskaya; Dmitri A. Petrov; Daniel L. Hartl

    1993-01-01

    Methods of genome analysis, including the cloning and manipulation of large fragments of DNA, have opened new strategies for uniting molecular evolutionary genetics with chromosome evolution. We have begun the development of a physical map of the genome of Drosophila virilis based on large DNA fragments cloned in bacteriophage P1. A library of 10,080 P1 clones with average insert sizes

  19. G Protein-Mediated Neuronal DNA Fragmentation Induced by Familial Alzheimer's Disease-Associated Mutants of APP

    Microsoft Academic Search

    Tomoki Yamatsuji; Takashi Matsui; Takashi Okamoto; Katsumi Komatsuzaki; Shizu Takeda; Hiroaki Fukumoto; Takeshi Iwatsubo; Nobuhiro Suzuki; Asano Asami-Odaka; Scott Ireland; T. Bernard Kinane; Ugo Giambarella; Ikuo Nishimoto

    1996-01-01

    Missense mutations in the 695-amino acid form of the amyloid precursor protein (APP695) cosegregate with disease phenotype in families with dominantly inherited Alzheimer's disease. These mutations convert valine at position 642 to isoleucine, phenylalanine, or glycine. Expression of these mutant proteins, but not of normal APP695, was shown to induce nucleosomal DNA fragmentation in neuronal cells. Induction of DNA fragmentation

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

    PubMed Central

    Horton, John R.; Wang, Hua; Mabuchi, Megumu Yamada; Zhang, Xing; Roberts, Richard J.; Zheng, Yu; Wilson, Geoffrey G.; Cheng, Xiaodong

    2014-01-01

    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. PMID:25262349

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

    PubMed

    Horton, John R; Wang, Hua; Mabuchi, Megumu Yamada; Zhang, Xing; Roberts, Richard J; Zheng, Yu; Wilson, Geoffrey G; Cheng, Xiaodong

    2014-10-29

    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. PMID:25262349

  2. Ultrasensitive electrochemical biosensing for DNA using quantum dots combined with restriction endonuclease.

    PubMed

    Zhang, Can; Lou, Jing; Tu, Wenwen; Bao, Jianchun; Dai, Zhihui

    2015-01-21

    A universal and sensitive electrochemical biosensing platform for the detection and identification of DNA using CdSe quantum dots (CdSe QDs) as signal markers was designed. The detection mechanism was based on the specific recognition of MspI endonuclease combined with the signal amplification of gold nanoparticles (AuNPs). MspI endonuclease could recognize its specific sequence in the double-strand DNA (dsDNA) and cleave the dsDNA fragments linked with CdSe QDs from the electrode. The remaining attached CdSe QDs can be easily read out by square-wave voltammetry using an electrodeposited bismuth (Bi) film-modified glass carbon electrode. The concentrations of target DNA could be simultaneously detected by the signal of metal markers. Using mycobacterium tuberculosis (Mtb) DNA as a model, under the optimal conditions, the proposed biosensor could detect Mtb DNA down to 8.7 × 10(-15) M with a linear range of 5 orders of magnitude (from 1.0 × 10(-14) to 1.0 × 10(-9) M) and discriminate mismatched DNA with high selectivity. This strategy presented a universal and convenient biosensing platform for DNA assay, and its satisfactory performances make it a potential candidate for the early diagnosis of gene-related diseases. PMID:25408952

  3. Crystal structure of restriction endonuclease BglI bound to its interrupted DNA recognition sequence.

    PubMed Central

    Newman, M; Lunnen, K; Wilson, G; Greci, J; Schildkraut, I; Phillips, S E

    1998-01-01

    The crystal structure of the type II restriction endonuclease BglI bound to DNA containing its specific recognition sequence has been determined at 2.2 A resolution. This is the first structure of a restriction endonuclease that recognizes and cleaves an interrupted DNA sequence, producing 3' overhanging ends. BglI is a homodimer that binds its specific DNA sequence with the minor groove facing the protein. Parts of the enzyme reach into both the major and minor grooves to contact the edges of the bases within the recognition half-sites. The arrangement of active site residues is strikingly similar to other restriction endonucleases, but the co-ordination of two calcium ions at the active site gives new insight into the catalytic mechanism. Surprisingly, the core of a BglI subunit displays a striking similarity to subunits of EcoRV and PvuII, but the dimer structure is dramatically different. The BglI-DNA complex demonstrates, for the first time, that a conserved subunit fold can dimerize in more than one way, resulting in different DNA cleavage patterns. PMID:9736624

  4. Genetic recombination without using either restriction enzyme or PCR.

    PubMed

    Zhou, Jing-Min; Yamamoto, Yoji; Uehara, Akihiko; Komiyama, Makoto

    2005-01-01

    We recently prepared artificial restriction DNA cutters (ARCUT) for site-selective scission of double-stranded DNA by combining Ce(IV)/EDTA complex with a pair of pseudo-complementary peptide nucleic acids (pcPNAs). Here we report an improved method for genetic recombination using ARCUT. The key point is to treat the scission fragments with nuclease S1 (a single-stranded DNA specific enzyme) and form blunt ends. By this procedure, these scission fragments are efficiently ligated with foreign DNA fragments having blunt ends, providing desired recombinant DNA in high yields. Neither restriction enzyme nor PCR amplification is required. PMID:17150732

  5. 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.

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

    PubMed

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

    2014-09-01

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

  7. Rapid Identification of Nocardia farcinica Clinical Isolates by a PCR Assay Targeting a 314-Base-Pair Species-Specific DNA Fragment

    PubMed Central

    Brown, June M.; Pham, Kim N.; McNeil, Michael M.; Lasker, Brent A.

    2004-01-01

    Nocardia farcinica is the most clinically significant species within the Nocardia asteroides complex. Differentiation of N. farcinica from other members of N. asteroides complex is important because this species characteristically demonstrates resistance to several extended-spectrum antimicrobial agents. Traditional phenotypic characterization of this species is time- and labor-intensive and often leads to misidentification in the clinical microbiology laboratory. We previously observed a 409-bp product for all strains of N. farcinica by using randomly amplified polymorphic DNA analysis with the primer DKU49. In this investigation, the 409-bp fragment was sequenced and then used to design a specific primer pair, Nf1 (16-mer) and Nf2 (16-mer), complementary to the 409-bp fragment. PCR amplification of genomic DNA from 28 N. farcinica isolates with Nf1 and Nf2 generated a single intense 314-bp fragment. The specificity of the assay with these primers was verified, since there were no PCR amplification products observed from heterologous nocardial species (n = 59) or other related bacterial genera (n = 41). Restriction enzyme digestion using CfoI and direct sequencing of the 314-bp fragment further confirmed the specificity of the assay for N. farcinica. This highly sensitive and specific PCR assay provides a rapid (within 1 day of obtaining DNA) method for identification of this medically important emerging pathogen. Rapid diagnosis of N. farcinica infection may allow for earlier initiation of effective therapy, thus improving patient outcome. PMID:15297512

  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. 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

  10. Detection of low-abundance KRAS mutations in colorectal cancer using microfluidic capillary electrophoresis-based restriction fragment length polymorphism method with optimized assay conditions.

    PubMed

    Zhang, Huidan; Song, Jin; Ren, Hui; Xu, Zhangrun; Wang, Xiaonan; Shan, Lianfeng; Fang, Jin

    2013-01-01

    Constitutively active KRAS mutations have been found to be involved in various processes of cancer development, and render tumor cells resistant to EGFR-targeted therapies. Mutation detection methods with higher sensitivity will increase the possibility of choosing the correct individual therapy. Here, we established a highly sensitive and efficient microfluidic capillary electrophoresis-based restriction fragment length polymorphism (µCE-based RFLP) platform for low-abundance KRAS genotyping with the combination of µCE and RFLP techniques. By using our self-built sensitive laser induced fluorescence (LIF) detector and a new DNA intercalating dye YOYO-1, the separation conditions of µCE for ?X174 HaeIII DNA marker were first optimized. Then, a Mav I digested 107-bp KRAS gene fragment was directly introduced into the microfluidic device and analyzed by µCE, in which field amplified sample stacking (FASS) technique was employed to obtain the enrichment of the RFLP digestion products and extremely improved the sensitivity. The accurate analysis of KRAS statuses in HT29, LS174T, CCL187, SW480, Clone A, and CX-1 colorectal cancer (CRC) cell lines by µCE-based RFLP were achieved in 5 min with picoliter-scale sample consumption, and as low as 0.01% of mutant KRAS could be identified from a large excess of wild-type genomic DNA (gDNA). In 98 paraffin-embedded CRC tissues, KRAS codon 12 mutations were discovered in 28 (28.6%), significantly higher than that obtained by direct sequencing (13, 13.3%). Clone sequencing confirmed these results and showed this system could detect at least 0.4% of the mutant KRAS in CRC tissue slides. Compared with direct sequencing, the new finding of the µCE-based RFLP platform was that KRAS mutations in codon 12 were correlated with the patient's age. In conclusion, we established a sensitive, fast, and cost-effective screening method for KRAS mutations, and successfully detected low-abundance KRAS mutations in clinical samples, which will allow provision of more precise individualized cancer therapy. PMID:23355875

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

    PubMed Central

    Rösch, Christopher; Bothe, Hermann

    2005-01-01

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

  13. Sensitive PCR analysis of animal tissue samples for fragments of endogenous and transgenic plant DNA.

    PubMed

    Nemeth, Anne; Wurz, Andreas; Artim, Lori; Charlton, Stacy; Dana, Greg; Glenn, Kevin; Hunst, Penny; Jennings, James; Shilito, Ray; Song, Ping

    2004-10-01

    An optimized DNA extraction protocol for animal tissues coupled with sensitive PCR methods was used to determine whether trace levels of feed-derived DNA fragments, plant and/or transgenic, are detectable in animal tissue samples including dairy milk and samples of muscle (meat) from chickens, swine, and beef steers. Assays were developed to detect DNA fragments of both the high copy number chloroplast-encoded maize rubisco gene (rbcL) and single copy nuclear-encoded transgenic elements (p35S and a MON 810-specific gene fragment). The specificities of the two rbcL PCR assays and two transgenic DNA PCR assays were established by testing against a range of conventional plant species and genetically modified maize crops. The sensitivities of the two rbcL PCR assays (resulting in 173 and 500 bp amplicons) were similar, detecting as little as 0.08 and 0.02 genomic equivalents, respectively. The sensitivities of the p35S and MON 810 PCR assays were approximately 5 and 10 genomic equivalents for 123 bp and 149 bp amplicons, respectively, which were considerably less than the sensitivity of the rbcL assays in terms of plant cell equivalents, but approximately similar when the higher numbers of copies of the chloroplast genome per cell are taken into account. The 173 bp rbcL assay detected the target plant chloroplast DNA fragment in 5%, 15%, and 53% of the muscle samples from beef steers, broiler chickens, and swine, respectively, and in 86% of the milk samples from dairy cows. Reanalysis of new aliquots of 31 of the pork samples that were positive in the 173 bp rbcL PCR showed that 58% of these samples were reproducibly positive in this same PCR assay. The 500 bp rbcL assay detected DNA fragments in 43% of the swine muscle samples and 79% of the milk samples. By comparison, no statistically significant detections of transgenic DNA fragments by the p35S PCR assay occurred with any of these animal tissue samples. PMID:15453677

  14. Comprehensive restriction enzyme lists to update any DNA sequence computer program.

    PubMed

    Raschke, E

    1993-04-01

    Restriction enzyme lists are presented for the practical working geneticist to update any DNA computer program. These lists combine formerly scattered information and contain all presently known restriction enzymes with a unique recognition sequence, a cut site, or methylation (in)sensitivity. The lists are in the shortest possible form to also be functional with small DNA computer programs, and will produce clear restriction maps without any redundancy or loss of information. The lists discern between commercial and noncommercial enzymes, and prototype enzymes and different isoschizomers are cross-referenced. Differences in general methylation sensitivities and (in)sensitivities against Dam and Dcm methylases of Escherichia coli are indicated. Commercial methylases and intron-encoded endonucleases are included. An address list is presented to contact commercial suppliers. The lists are constantly updated and available in electronic form as pure US ASCII files, and in formats for the DNA computer programs DNA-Strider for Apple Macintosh, and DNAsis for IBM personal computers or compatibles via e-mail from the internet address: NETSERV@EMBL-HEIDELBERG.DE by sending only the message HELP RELIBRARY. PMID:8217304

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

    SciTech Connect

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

    1996-01-01

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

  16. DNA vaccination with VP2 gene fragment confers protection against Infectious Bursal Disease Virus in chickens.

    PubMed

    Pradhan, Satya Narayan; Prince, Prabhu Rajaiah; Madhumathi, Jayaprakasam; Arunkumar, Chakkaravarthy; Roy, Parimal; Narayanan, Rangarajan Badri; Antony, Usha

    2014-06-25

    Infectious Bursal Disease Virus (IBDV) causes immunosuppression in young chickens by destruction of antibody producing B cells in the Bursa of Fabricius and poses a potential threat to the poultry industry. We have examined the protective efficacy of a subunit DNA vaccine against IBDV infection in chickens in this study. An immunodominant VP2 gene fragment (VP252-417) was cloned into CMV promoter based DNA vaccine vector pVAX1 and in vitro expression of the DNA encoded antigens was confirmed by transfection of CHO cells with vaccine constructs followed by RT-PCR and western blot analysis using IBDV-antiserum. Two weeks old chickens were immunized intramuscularly with pVAXVP252-417 and the in vivo transcription of the plasmid DNA was confirmed by RT-PCR analysis of DNA injected muscle tissue at different intervals of post immunization. Tissue distribution analysis revealed that the plasmid DNA was extensively distributed in muscle, spleen, kidney, liver, and bursa tissues. Chickens immunized with pVAXVP252-417 developed high titer (1:12,000) of anti-VP252-417 antibodies. Further, chicken splenocytes from pVAXVP252-417 immunized group showed a significantly high proliferation to the whole viral and recombinant antigen (P<0.01) compared to control groups, which implies that pVAXVP252-417 codes for immunogenic fragment which has epitopes capable of eliciting both B and T cell responses. This is evident by the fact that, pVAXVP252-417 immunized chicken conferred 75% protection against virulent IBDV (vIBDV) challenge compared to the control group. Thus, the present study confirms that the immunodominant VP2 fragment can be used as a potential DNA vaccine against IBDV infection in chickens. PMID:24745626

  17. Cocrystal structure of an editing complex of Klenow fragment with DNA.

    PubMed

    Freemont, P S; Friedman, J M; Beese, L S; Sanderson, M R; Steitz, T A

    1988-12-01

    High-resolution crystal structures of editing complexes of both duplex and single-stranded DNA bound to Escherichia coli DNA polymerase I large fragment (Klenow fragment) show four nucleotides of single-stranded DNA bound to the 3'-5' exonuclease active site and extending toward the polymerase active site. Melting of the duplex DNA by the protein is stabilized by hydrophobic interactions between Phe-473, Leu-361, and His-666 and the last three bases at the 3' terminus. Two divalent metal ions interacting with the phosphodiester to be hydrolyzed are proposed to catalyze the exonuclease reaction by a mechanism that may be related to mechanisms of other enzymes that catalyze phospho-group transfer including RNA enzymes. We suggest that the editing active site competes with the polymerase active site some 30 A away for the newly formed 3' terminus. Since a 3' terminal mismatched base pair favors the melting of duplex DNA, its binding and excision at the editing exonuclease site that binds single-stranded DNA is enhanced. PMID:3194400

  18. Sensitive and selective amplification of methylated DNA sequences using helper-dependent chain reaction in combination with a methylation-dependent restriction enzymes

    PubMed Central

    Rand, Keith N.; Young, Graeme P.; Ho, Thu; Molloy, Peter L.

    2013-01-01

    We have developed a novel technique for specific amplification of rare methylated DNA fragments in a high background of unmethylated sequences that avoids the need of bisulphite conversion. The methylation-dependent restriction enzyme GlaI is used to selectively cut methylated DNA. Then targeted fragments are tagged using specially designed ‘helper’ oligonucleotides that are also used to maintain selection in subsequent amplification cycles in a process called ‘helper-dependent chain reaction’. The process uses disabled primers called ‘drivers’ that can only prime on each cycle if the helpers recognize specific sequences within the target amplicon. In this way, selection for the sequence of interest is maintained throughout the amplification, preventing amplification of unwanted sequences. Here we show how the method can be applied to methylated Septin 9, a promising biomarker for early diagnosis of colorectal cancer. The GlaI digestion and subsequent amplification can all be done in a single tube. A detection sensitivity of 0.1% methylated DNA in a background of unmethylated DNA was achieved, which was similar to the well-established Heavy Methyl method that requires bisulphite-treated DNA. PMID:22965136

  19. Human Sperm DNA Fragmentation and its Correlation with Conventional Semen Parameters

    PubMed Central

    Evgeni, Evangelini; Charalabopoulos, Konstantinos; Asimakopoulos, Byron

    2014-01-01

    Background The initial step in the diagnostic investigation of male infertility has been traditionally based on the conventional seminal profile. However, there are significant limitations regarding its ability to determine the underlying mechanisms that cause the disorder. Sperm DNA fragmentation has emerged as a potential causative factor of reproductive failure and its assessment has been suggested as a useful adjunct to the laboratory methodology of male infertility evaluation, especially before the application of assisted reproduction technology (ART). Methods A review of recent bibliography was carried out in PubMed by the use of relevant keywords, in order to evaluate the possible correlation between the conventional seminal parameters and sperm DNA fragmentation assessment as diagnostic tools in male infertility evaluation. Results A comprehensive diagnostic approach of male infertility should be based on a combination of diagnostic attributes, derived from the conventional semen analysis, as well as the investigation of genomic integrity testing. Conclusion Due to its strong correlation with several aspects of ART procedures and further consequences for the offspring, sperm DNA fragmentation is a parameter worth integrating in routine clinical practice. However, additional large scale studies focusing on specific subgroups of infertile men who may benefit from an efficient therapeutic management based on the optimization of sperm DNA integrity are needed. PMID:24696791

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

    PubMed

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

    2014-01-01

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

  1. Polaprezinc, a gastroprotective agent: attenuation of monochloramine-evoked gastric DNA fragmentation.

    PubMed

    Suzuki, H; Mori, M; Seto, K; Nagahashi, S; Kawaguchi, C; Morita, H; Suzuki, M; Miura, S; Yoneta, T; Ishii, H

    1999-01-01

    We previously reported that NH2Cl induced extensive DNA fragmentation in gastric cells. Polaprezinc, a zinc-carnosine chelate compound, is reported to be a potent antioxidant in gastric mucosa. The present study was designed to examine whether polaprezinc could attenuate the NH3Cl-induced DNA damage. Gastric cell lines, MKN45, were exposed to NH2Cl in Ca(2+)-containing Hanks' balanced salt solution. DNA fragmentation was evaluated by photometric enzyme immunoassay for in vitro determination of cytoplasmic mono- and oligonucleosomes. Polaprezinc, L-carnosine, and zinc sulfate (ZnSO4) were added to the cell incubation medium to evaluate the inhibitory effect on the formation of cytoplasmic mono- and oligonucleosomes. Separately, the bleaching level of beta-carotene with the addition of each test solution was evaluated to confirm the inhibitory effect against hypochlorous acid. Polaprezinc or L-carnosine, but not ZnSO4, at a concentration of 0.001 mM, significantly attenuated the increased levels of cytoplasmic mono- and oligonucleosomes evoked by 0.001 mM NH2Cl. Polaprezinc and L-carnosine, but not ZnSO4, also inhibited NH2Cl-induced beta-carotene bleaching in the cell-free system. In conclusion, polaprezinc, especially its subportion L-carnosine, inhibited NH2Cl-evoked gastric epithelial DNA fragmentation, suggesting a role for this agent in preventing the progression of gastric epithelial injury induced by NH2Cl. PMID:10616765

  2. Study of aneuploidy and DNA fragmentation in gametes of patients with severe teratozoospermia.

    PubMed

    Perrin, A; Louanjli, N; Ziane, Y; Louanjli, T; Le Roy, C; Gueganic, N; Amice, V; De Braekeleer, M; Morel, F

    2011-02-01

    This study investigated meiotic segregation in spermatozoa to determine if severe teratozoospermia should prevent the use of intracytoplasmic sperm injection (ICSI) because of the high production of gametes with chromosomal aneuploidies and analysed DNA fragmentation in gametes from the same semen to determine if DNA integrity was worse in patients with severe teratozoospermia. Sperm samples from 12 infertile patients were studied by fluorescence in-situ hybridization for chromosomes X, Y, 13, 18 and 21 and by TdT (terminal deoxynucleotidyl transferase)-mediated dUDP nick-end labelling. Four patients with a majority of macrocephalic forms with multiple flagella had more than 99% spermatozoa with abnormal chromosomal content. The other patients (globozoospermia or other abnormalities concerning sperm heads) had no increased aneuploidy or a slightly significant increase (P<0.05). The rate of DNA fragmentation was significantly higher in infertile patients than in the controls (P<0.001; 14.3% versus 1.20%, respectively) but presented important variability. Therefore, ICSI should not be attempted if men have macrocephalic gametes with multiple flagella but morphology is not always a good predictor of chromosomal content, depending upon the kind of teratozoospermia. Evaluation of the rate of aneuploidy and DNA fragmentation in gametes of patients with severe teratozoospermia is recommended. PMID:21233018

  3. Cloning and characterization of an apoptosis-related DNA fragmentation factor (DFF) from oyster, Crassostrea hongkongensis.

    PubMed

    Xiang, Zhiming; Qu, Fufa; Qi, Lin; Ying, Tong; Li, Jun; Shu, Xiao; Yu, Ziniu

    2014-05-01

    Apoptosis plays an important pathophysiological role in the homeostasis of immune systems. DNA fragmentation factors (DFFs) have been shown to be essential for DNA fragmentation, and the resultant DNA fragments follow a laddering pattern during apoptosis in vertebrates. In invertebrates, the functions of the DFF orthologs are not well characterized; therefore, we cloned and characterized a bivalve DFFA ortholog from the Hong Kong oyster Crassostrea hongkongensis (designated ChDFFA). The full-length cDNA of ChDFFA is 1186 bp in length and encodes a putative protein of 200 amino acids that contains an N-terminal CAD domain and a DFF-C domain at its C-terminus. Real-time RT-PCR results showed that ChDFFA is ubiquitously expressed in several tissues, and its highest expression is in gill. Following a 3- to 48-h challenge by microbial infection, the expression of ChDFFA increased in hemocytes. Using fluorescence microscopy, ChDFFA was localized in nuclei when exogenously expressed in HeLa cells. In addition, over-expression of ChDFFA inhibited the transcriptional activities of p53/p21-Luc reporter genes in HEK293T cells. These results suggest that ChDFFA may be involved in immune response reactions in the Hong Kong oyster C. hongkongensis. PMID:24642253

  4. A framework linkage map of bermudagrass (Cynodon dactylon x transvaalensis) based on single-dose restriction fragments.

    PubMed

    Bethel, C M; Sciara, E B; Estill, J C; Bowers, J E; Hanna, W; Paterson, A H

    2006-02-01

    This study describes the first detailed linkage maps of two bermudagrass species, Cynodon dactylon (T89) and Cynodon transvaalensis (T574), based on single-dose restriction fragments (SDRFs). The mapping population consisted of 113 F1 progeny of a cross between the two parents. Loci were generated using 179 bermudagrass genomic clones and 50 heterologous cDNAs from Pennisetum and rice. The map of T89 is based on 155 SDRFs and 17 double-dose restriction fragments on 35 linkage groups, with an average marker spacing of 15.3 cM. The map of T574 is based on 77 SDRF loci on 18 linkage groups with an average marker spacing of 16.5 cM. About 16 T89 linkage groups were arranged into four complete and eight into four incomplete homologous sets, while 15 T574 linkage groups were arranged into seven complete homologous sets, all on the basis of multi-locus probes and repulsion linkages. Eleven T89 and three T574 linkage groups remain unassigned. In each parent consensus maps were built based on alignments of homologous linkage groups. Four ancestral chromosomes were inferred after aligning T89 and T574 parental consensus maps using multi-locus probes. The inferred ancestral marker orders were used in comparisons to a detailed Sorghum linkage map using 40 common probes, and to the rice genome sequence using 98 significant BLAST hits, to find regions of colinearity. Using these maps we have estimated the recombinational length of the T89 and T574 genomes at 3,012 and 1,569 cM, respectively, which are 61 and 62% covered by our maps. PMID:16395568

  5. Efficient Tracing of Global Isolates of Yersinia pestis by Restriction Fragment Length Polymorphism Analysis Using Three Insertion Sequences as Probes†

    PubMed Central

    Torrea, Gabriela; Chenal-Francisque, Viviane; Leclercq, Alexandre; Carniel, Elisabeth

    2006-01-01

    Yersinia pestis is the etiologic agent of plague, a disease that is transmitted from rodent to rodent and from rodent to humans by fleabites. Multiple copies of three insertion sequences (IS100, IS285, and IS1541) are scattered over the Y. pestis genome. The genomic instability generated by these insertion sequences (IS) creates a polymorphism of the hybridizing restriction fragments (restriction fragment length polymorphism [RFLP]) which can be used to subtype this relatively clonal species. The aim of this work was to evaluate and compare the potential of the three IS-RFLP techniques, individually or in combination, to define clusters of strains according to their focus of origin. The analysis of 61 Y. pestis isolates of worldwide origin indicated that no satisfactory strain clustering was observed with each IS-RFLP used individually. In contrast, the combination of the three IS-RFLP data (3IS-RFLP) resulted in both an efficient strain discrimination (D = 0.999) and a robust clustering of the isolates according to their biovar and geographical origin. This geographical clustering was observed even within the Orientalis group, although these strains had only a short period of time (one century) to diverge from the original clone that spread globally. Therefore, 3IS-RFLP is a technique that may be useful for addressing epidemiological problems and forensic issues. When plague reemerges after several decades of silence in a quiescent focus, it may help in determining whether the disease was reimported or reactivated. It may also be of value to identify the origin of a strain when plague cases appear in a previously plague-free region. Finally, this technique could be useful for the tracing of a Y. pestis isolate that has been used as a biological terrorism threat. PMID:16757602

  6. Role of Magnesium Ions in DNA Recognition by the EcoRV Restriction Endonuclease

    SciTech Connect

    Zahran, Mai [ORNL; Berezniak, Tomasz [University of Heidelberg; Imhof, Petra [University of Heidelberg; Smith, Jeremy C [ORNL

    2011-01-01

    The restriction endonuclease EcoRV binds two magnesium ions. One of these ions, Mg2+A, binds to the phosphate group where the cleavage occurs and is required for catalysis, but the role of the other ion, Mg2+B is debated. Here, multiple independent molecular dynamics simulations suggest that Mg2+B is crucial for achieving a tightly bound protein DNA complex and stabilizing a conformation that allows cleavage. In the absence of Mg2+B in all simulations the protein DNA hydrogen bond network is significantly disrupted and the sharp kink at the central base pair step of the DNA, which is observed in the two-metal complex, is not present. Also, the active site residues rearrange in such a way that the formation of a nucleophile, required for DNA hydrolysis, is unlikely.

  7. In situ ligation simplified: using PCR fragments for detection of double-strand DNA breaks in tissue sections.

    PubMed

    Didenko, Vladimir V

    2011-01-01

    The simplified in situ ligation procedure is described. All reagents for the assay can be easily obtained in any molecular or cell biology laboratory. The technique uses ligation of double-stranded, PCR-derived DNA fragments labeled with digoxigenin or fluorophores for highly selective detection of apoptotic cells in paraffin-embedded tissue sections. Two types of DNA fragments prepared by PCR are employed. The fragment synthesized by Taq polymerase contains single-base 3' overhangs, whereas the Pfu polymerase-made fragment is blunt ended. Both fragments can be used as specific, sensitive and cost-effective DNA damage probes. After ligation to apoptotic nuclei in tissue sections, they indicate the presence of double-strand DNA breaks with single-base 3' overhangs as well as blunt ends. PMID:21057921

  8. IN SITU LIGATION SIMPLIFIED: USING PCR FRAGMENTS FOR DETECTION OF DOUBLE-STRAND DNA BREAKS IN TISSUE SECTIONS

    PubMed Central

    Didenko, Vladimir V.

    2012-01-01

    The simplified in situ ligation procedure is described. All reagents for the assay can be easily obtained in any molecular or cell biology laboratory. The technique uses ligation of double-stranded, PCR-derived DNA fragments labeled with digoxigenin or fluorophores for highly selective detection of apoptotic cells in paraffin-embedded tissue sections. Two types of DNA fragments prepared by PCR are employed. The fragment synthesized by Taq polymerase contains single-base 3' overhangs, whereas the Pfu polymerase-made fragment is blunt ended. Both fragments can be used as specific, sensitive and cost-effective DNA damage probes. After ligation to apoptotic nuclei in tissue sections, they indicate the presence of double-strand DNA breaks with single-base 3' overhangs as well as blunt ends. PMID:21057921

  9. 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

  10. Species Identification of Mycobacteria by PCR-Restriction Fragment Length Polymorphism of the rpoB Gene

    PubMed Central

    Lee, Hyeyoung; Park, Hee-Jung; Cho, Sang-Nae; Bai, Gill-Han; Kim, Sang-Jae

    2000-01-01

    PCR-restriction fragment length polymorphism analysis (PRA) using the novel region of the rpoB gene was developed for rapid and precise identification of mycobacteria to the species level. A total of 50 mycobacterial reference strains and 3 related bacterial strains were used to amplify the 360-bp region of rpoB, and the amplified DNAs were subsequently digested with restriction enzymes such as MspI and HaeIII. The results from this study clearly show that most of the mycobacterial species were easily differentiated at the species level by this PRA method. In addition, species with several subtypes, such as Mycobacterium gordonae, M. kansasii, M. celatum, and M. fortuitum, were also differentiated by this PRA method. Subsequently, an algorithm was constructed based on the results, and a blinded test was carried out with more than 260 clinical isolates that had been identified on the basis of conventional tests. Comparison of these two sets of results clearly indicates that this new PRA method based on the rpoB gene is more simple, more rapid, and more accurate than conventional procedures for differentiating mycobacterial species. PMID:10921960

  11. DNA Detectives

    NSDL National Science Digital Library

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

    1995-06-30

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

  12. Restriction enzyme analysis of cytomegalovirus DNA to study transmission of infection.

    PubMed

    Peckham, C S; Garrett, A J; Chin, K S; Preece, P M; Nelson, D B; Warren, D E

    1986-03-01

    Restriction enzyme analysis of cytomegalovirus deoxyribonucleic acid (DNA) has been used to characterise virus isolates and has provided information on patterns of viral transmission. It was shown that virus isolated from a congenitally infected infant was unlikely to have originated from the 13 congenitally infected children with whom the mother, a nurse, had been in contact. Of nine mother and infant pairs, from whom cytomegalovirus was isolated, seven yielded strains that were indistinguishable for mother and child; one pair showed minor differences and one was clearly distinguishable. Virus isolates from seven children attending a day nursery were typed, and only siblings were excreting similar strains of cytomegalovirus. Further examples of the application of this technique to studies of cytomegalovirus in a family environment are given. It is concluded that characterisation of virus strains by restriction analysis of DNA is a valuable epidemiological tool. PMID:3007581

  13. REBASE--a database for DNA restriction and modification: enzymes, genes and genomes.

    PubMed

    Roberts, Richard J; Vincze, Tamas; Posfai, Janos; Macelis, Dana

    2015-01-01

    REBASE is a comprehensive and fully curated database of information about the components of restriction-modification (RM) systems. It contains fully referenced information about recognition and cleavage sites for both restriction enzymes and methyltransferases as well as commercial availability, methylation sensitivity, crystal and sequence data. All genomes that are completely sequenced are analyzed for RM system components, and with the advent of PacBio sequencing, the recognition sequences of DNA methyltransferases (MTases) are appearing rapidly. Thus, Type I and Type III systems can now be characterized in terms of recognition specificity merely by DNA sequencing. The contents of REBASE may be browsed from the web http://rebase.neb.com and selected compilations can be downloaded by FTP (ftp.neb.com). Monthly updates are also available via email. PMID:25378308

  14. REBASE—a database for DNA restriction and modification: enzymes, genes and genomes

    PubMed Central

    Roberts, Richard J.; Vincze, Tamas; Posfai, Janos; Macelis, Dana

    2015-01-01

    REBASE is a comprehensive and fully curated database of information about the components of restriction-modification (RM) systems. It contains fully referenced information about recognition and cleavage sites for both restriction enzymes and methyltransferases as well as commercial availability, methylation sensitivity, crystal and sequence data. All genomes that are completely sequenced are analyzed for RM system components, and with the advent of PacBio sequencing, the recognition sequences of DNA methyltransferases (MTases) are appearing rapidly. Thus, Type I and Type III systems can now be characterized in terms of recognition specificity merely by DNA sequencing. The contents of REBASE may be browsed from the web http://rebase.neb.com and selected compilations can be downloaded by FTP (ftp.neb.com). Monthly updates are also available via email. PMID:25378308

  15. Cleavage of Phosphorothioated DNA and Methylated DNA by the Type IV Restriction Endonuclease ScoMcrA

    PubMed Central

    Liu, Guang; Ou, Hong-Yu; Wang, Tao; Li, Li; Tan, Huarong; Zhou, Xiufen; Rajakumar, Kumar; Deng, Zixin; He, Xinyi

    2010-01-01

    Many taxonomically diverse prokaryotes enzymatically modify their DNA by replacing a non-bridging oxygen with a sulfur atom at specific sequences. The biological implications of this DNA S-modification (phosphorothioation) were unknown. We observed that simultaneous expression of the dndA-E gene cluster from Streptomyces lividans 66, which is responsible for the DNA S-modification, and the putative Streptomyces coelicolor A(3)2 Type IV methyl-dependent restriction endonuclease ScoA3McrA (Sco4631) leads to cell death in the same host. A His-tagged derivative of ScoA3McrA cleaved S-modified DNA and also Dcm-methylated DNA in vitro near the respective modification sites. Double-strand cleavage occurred 16–28 nucleotides away from the phosphorothioate links. DNase I footprinting demonstrated binding of ScoA3McrA to the Dcm methylation site, but no clear binding could be detected at the S-modified site under cleavage conditions. This is the first report of in vitro endonuclease activity of a McrA homologue and also the first demonstration of an enzyme that specifically cleaves S-modified DNA. PMID:21203499

  16. 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.

  17. Species identification of the Northern shrimp (Pandalus borealis) by polymerase chain reaction-restriction fragment length polymorphism and proteomic analysis.

    PubMed

    Pascoal, Ananias; Ortea, Ignacio; Gallardo, José M; Cañas, Benito; Barros-Velázquez, Jorge; Calo-Mata, Pilar

    2012-02-01

    Genomic and proteomic techniques for species identification of meat and seafood products are being widely used. In this study, a genomic approach was used to differentiate Pandalus borealis (the Northern shrimp), which belongs to the superfamily Pandaloidea, from 30 crustaceans consisting of 19 commercially relevant prawns/shrimps species that belong to the superfamily Penaeoidea, which include the families Penaeidae and Solenoceridae, and 11 other crustacean species, including prawns, shrimps, lobsters, and crabs. For this purpose, a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method was designed based on the amplification of the 16S rRNA/tRNA(Val)/12S rRNA mitochondrial regions using the primers 16S-CruF and 16S-CruR. The 966-bp PCR products were produced and cleaved with the restriction enzymes AluI, TaqI, and HinfI, which provided species-specific restriction patterns. In addition, a proteomic approach, based on matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) and electrospray ionization-ion trap (ESI-IT) mass spectrometry, was used to identify and characterize new P. borealis-specific peptides that could be useful as potential markers of this species in protein-based detection methods. To our knowledge, this is the first time a molecular method has been successfully applied to identify a wide range of prawn and shrimp species, including P. borealis, for either whole individuals or processed products. However, validation of the methods proposed here is required by applying them to a larger sample of individuals from different populations and geographic origins in order to avoid mainly false-negative results. PMID:22080038

  18. Routine use of PCR-restriction fragment length polymorphism analysis for identification of mycobacteria growing in liquid media.

    PubMed Central

    Taylor, T B; Patterson, C; Hale, Y; Safranek, W W

    1997-01-01

    A PCR-restriction fragment length polymorphism (PCR-RFLP) procedure capable of rapidly identifying 28 species of clinically encountered mycobacteria was evaluated for use in the routine identification of acid-fast isolates growing in BACTEC 12B and 13A liquid media. PCR-RFLP identified 100 of 103 acid-fast isolates recovered from 610 patient specimens submitted for culture during the study. The three isolates unidentifiable by PCR-RFLP produced restriction patterns not included in the PCR-RFLP algorithm and could therefore not be assigned to a species. These isolates were characterized by their morphologic and biochemical characteristics. Two of the isolates were identified as M. terrae complex and M. gordonae. The third isolate could not be definitively identified and could only be characterized as a Mycobacterium sp. most closely resembling M. chelonae. PCR-RFLP identifications agreed with the conventional identifications for 96 of the 100 isolates identified by PCR-RFLP. Subsequent identification of the four discordant isolates by gas chromatography analysis supported the PCR-RFLP identification of each isolate. Amplification products were also obtained from isolates of Streptococcus albus and Rhodococcus equi recovered from patient specimens; however, the restriction patterns of these nonmycobacterial species did not resemble the patterns of any mycobacterial species included in the PCR-RFLP algorithm. PCR-RFLP seems to be a reliable procedure for the routine identification of mycobacteria and has the potential for providing identifications of mycobacterial isolates which are more accurate than conventional identification techniques based on morphologic and biochemical characteristics. PMID:8968884

  19. Influence of hydrodynamic coupling on the electric linear dichroism of DNA fragments

    NASA Astrophysics Data System (ADS)

    Umazano, Juan P.; Bertolotto, Jorge A.

    2011-03-01

    In the present work, we study the effect of translational-rotational hydrodynamic coupling on the stationary electric linear dichroism of DNA fragments. The theoretical resolution of the problem has, so far, been dealt with analytic methods valid only in the limit of low electric fields. In this work, we apply numerical methods that allow us to study the problem and also consider electric fields of arbitrary strength. We use the bent rod molecules model to describe DNA fragments with physical properties characterized by their electric charge, electric polarizability tensor, rotational diffusion tensor, and translation-rotation coupling diffusion tensor. The necessary orientational distribution function to calculate electric dichroism is obtained by solving the Fokker-Planck equation through the finite difference method. We analyze the different contributions due to electric polarizability and translational-rotational coupling to the electric dichroism.

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

    PubMed Central

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

    2015-01-01

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

  1. In vitro reconstruction of inflammatory reaction in human semen: effect on sperm DNA fragmentation.

    PubMed

    Fraczek, Monika; Szumala-Kakol, Anna; Dworacki, Grzegorz; Sanocka, Dorota; Kurpisz, Maciej

    2013-11-01

    The study was aimed at evaluating an in vitro induction of DNA damage in three sperm subpopulations exposed to selected inflammatory mediators, such as leukocytes, two combinations of pro-inflammatory cytokines (interleukin [IL]-6 + IL-8 and IL-12 + IL-18) and two bacterial strains (Escherichia coli and Bacteroides ureolyticus). Semen samples from normozoospermic volunteers were differentiated by swim-up (swim-up fraction) and Percoll gradient procedures (90% and 47% Percoll fractions). Leukocytes were isolated from the whole heparinized blood using the density gradient centrifugation technique. DNA fragmentation in sperm fractions was evaluated using flow cytometry with TUNEL labeling and Comet assay. Out of the inflammatory factors tested, bacteria were found to have a greatest toxic effect on sperm DNA, especially in fractions isolated by Percoll gradient, compared with untreated cells (P < 0.05). The results indicate that inflammatory mediators can be a direct cause of DNA fragmentation in ejaculated spermatozoa, which can ultimately lead to limited fertilizing abilities of the germ cells. In contrast to the swim-up technique, the selection of spermatozoa by gradient procedures increases the vulnerability of mature spermatozoa to the harmful effects of infectious agents on DNA integrity. This observation may have some meaning for recommendations concerning laboratory techniques used in assisted reproductive therapy. PMID:24344359

  2. Cutting and pasting DNA, 2D animationSite: DNA Interactive (www.dnai.org)

    NSDL National Science Digital Library

    2008-10-06

    The discovery of enzymes that could cut and paste DNA made genetic engineering possible. Restriction enzymes, found naturally in bacteria, can be used to cut DNA fragments at specific sequences, while another enzyme, DNA ligase, can attach or rejoin DNA fragments with complementary ends.

  3. Restriction-fragment-length polymorphism analysis of small-subunit rRNA genes of Blastocystis hominis isolates from geographically diverse human hosts

    Microsoft Academic Search

    J. Hoevers; P. Holman; K. Logan; M. Hommel; R. Ashford; K. Snowden

    2000-01-01

    Genomic diversity among 14 isolates of Blastocystis hominis from 4 different geographic locations was examined by small-subunit rRNA (ssu rRNA) restriction-fragment-length polymorphisms\\u000a (RFLP) using 5 different restriction endonucleases. On the basis of the observed RFLP patterns among the isolates, a total\\u000a of 12 genotypes were identified, with 7 isolates exhibiting mixed RFLP genotypes. There was no correlation between B. hominis

  4. EcoRV restriction endonuclease: communication between DNA recognition and catalysis.

    PubMed

    Vermote, C L; Vipond, I B; Halford, S E

    1992-07-01

    A genetic system was constructed for the mutagenesis of the EcoRV restriction endonuclease and for the overproduction of mutant proteins. The system was used to make two mutants of EcoRV, with Ala in place of either Asn185 or Asn188. In the crystal structure of the EcoRV-DNA complex, both Asn185 and Asn188 contact the DNA within the EcoRV recognition sequence. But neither mutation affected the ability of the protein to bind to DNA. In the absence of metal ion cofactors, the mutants bound DNA with almost the same affinity as that of the wild-type enzyme. In the presence of Mg2+, both mutants retained the ability to cleave DNA specifically at the EcoRV recognition sequence, but their activities were severely depressed relative to that of the wild-type. In contrast, with Mn2+ as the cofactor, the mutant enzymes cleaved the EcoRV recognition site with activities that were close to that of the wild-type. When bound to DNA at the EcoRV recognition site, the mutant proteins bound Mn2+ ions readily, but they had much lower affinities for Mg2+ ions than the wild-type enzyme. This was the reason for their low activities with Mg2+ as the cofactor. The arrangement of the DNA recognition functions, at one location in the EcoRV restriction enzyme, are therefore responsible for organizing the catalytic functions at a separate location in the protein. PMID:1627552

  5. Molecular Investigation of Quinolone Resistance of Quinolone Resistance-Determining Region in Streptococcus pneumoniae Strains Isolated from Iran Using Polymerase Chain Reaction–Restriction Fragment Length Polymorphism Method

    PubMed Central

    Kargar, Mohammad; Moein Jahromi, Fataneh; Doosti, Abbas; Handali, Somayeh

    2014-01-01

    Objectives The resistance of Streptococcus pneumoniae to the recently available antibiotic treatment has been a growing problem. The aim of the study was to determine the quinolone-resistant strains and detect the presence of mutations in the quinolone resistance-determining regions of the gyrA, parE, and parC genes. Methods In this study, for the first time in Iran, the polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) method was used to investigate the presence of mutations at quinolone resistance-determining regions of topoisomerase IV and DNA gyrase on 82 S. pneumoniae strains, among them 45 clinical samples were from patients and 37 from healthy carriers (control group). Results In clinical samples, 34 (75.56%) strains contained mutations in the parC gene, 31 (68.89%) carried mutations in the gyrA gene, and 14 (31.11%) had parE gene mutations. Antibiotic susceptibility test was performed using the CLSI (Clinical and Laboratory Standards Institute) criteria on three different generations of quinolone family, with nalidixic acid (82.22%) showing the highest resistance and levofloxacin (42.22%) the least resistance. Conclusion Results indicated that there is a significant correlation between quinolone resistance development and mutations in the parE gene as well as in the parC and gyrA genes. PMID:25389509

  6. Molecular variability of house finch Mycoplasma gallisepticum isolates as revealed by sequencing and restriction fragment length polymorphism analysis of the pvpA gene.

    PubMed

    Pillai, S R; Mays, H L; Ley, D H; Luttrell, P; Panangala, V S; Farmer, K L; Roberts, S R

    2003-01-01

    Mycoplasma gallisepticum, a major pathogen of chickens and turkeys, has caused significant declines in house finch (Carpodacus mexicanus) populations in the eastern United States since it was first observed in this species in 1994. There is evidence that M. gallisepticum infection is now endemic among eastern house finches, although disease prevalence has declined, suggesting an evolving host-parasite relationship. Studies based on randomly amplified polymorphic DNA (RAPD) have documented the presence of a single, unique RAPD profile in house finch M. gallisepticum isolates, suggesting a single point source of origin, which agrees with the known epidemiologic observations. In the present study, we evaluated the molecular variability of 55 house finch isolates as well as 11 chicken and turkey isolates including reference strains of M. gallisepticum. Molecular variability was evaluated by polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) analysis and nucleotide sequencing of the pvpA gene, which encodes for the putative cytadhesin protein PvpA. Three different RFLP groups and 16 genotypes were evident from the 55 house finch isolates evaluated. Sequence analysis of pvpA gene PCR products showed that although most house finch M. gallisepticum isolates clustered more closely to each other, others clustered more closely to either turkey or chicken field isolates. These findings suggest that house finch isolates are more polymorphic than previously recognized by RAPD studies. This feature may allow us to learn more about the molecular evolution and epidemiology of this emerging disease host-parasite relationship. PMID:14562892

  7. HIV1 gp120 Produces DNA Fragmentation in the Cerebral Cortex of Rat

    Microsoft Academic Search

    G. Bagetta; M. T. Corasaniti; L. Berliocchi; M. Navarra; A. Finazziagro; G. Nistico

    1995-01-01

    In the present experiments we have used morphological techniques to study the neuropathological profile of the brain of rats after intracerebroventricular (i.c.v.) injection of recombinant HIV-1 gp 120. Using brain cryostat sections (10 ?m) from rats treated with a single, daily dose of gp120 (100 ng\\/rat) given for 7 and 14 consecutive days, in situ DNA fragmentation was revealed in

  8. Structures of the rare-cutting restriction endonuclease NotI reveal a novel metal binding fold involved in DNA recognition

    PubMed Central

    Lambert, Abigail R.; Sussman, Django; Shen, Betty; Maunus, Robert; Nix, Jay; Samuelson, James; Xu, Shuang-yong; Stoddard, Barry L.

    2008-01-01

    The structure of the rare-cutting restriction endonuclease NotI, which recognizes the 8 basepair target 5?-GCGGCCGC-3?, has been solved with and without bound DNA. Because of its specificity (recognizing a site that occurs once per 65kb), NotI is used to generate large genomic fragments and to map DNA methylation status. NotI contains a unique metal-binding fold, found in a variety of putative endonucleases, occupied by an iron atom coordinated within a tetrahedral Cys4 motif. This domain positions nearby structural elements for DNA recognition, and serves a structural role. While recognition of the central six basepairs of the target is accomplished via a saturated hydrogen bond network typical of restriction enzymes, the most peripheral basepairs are engaged in a single direct contact in the major groove, reflecting reduced pressure to recognize those positions. NotI may represent an evolutionary intermediate between mobile endonucleases (which recognize longer target sites) and canonical restriction endonucleases. PMID:18400177

  9. Metal ion dependence of DNA cleavage by SepMI and EhoI restriction endonucleases.

    PubMed

    Belkebir, Abdelkarim; Azeddoug, Houssine

    2013-02-22

    Most of type II restriction endonucleases show an absolute requirement for divalent metal ions as cofactors for DNA cleavage. While Mg(2+) is the natural cofactor other metal ions can substitute it and mediate the catalysis, however Ca(2+) (alone) only supports DNA binding. To investigate the role of Mg(2+) in DNA cleavage by restriction endonucleases, we have studied the Mg(2+) and Mn(2+) concentration dependence of DNA cleavage by SepMI and EhoI. Digestion reactions were carried out at different Mg(2+) and Mn(2+) concentrations at constant ionic strength. These enzymes showed different behavior regarding the ions requirement, SepMI reached near maximal level of activity between 10 and 20mM while no activity was detected in the presence of Mn(2+) and in the presence of Ca(2+) cleavage activity was significantly decreased. However, EhoI was more highly active in the presence of Mn(2+) than in the presence of Mg(2+) and can be activated by Ca(2+). Our results propose the two-metal ion mechanism for EhoI and the one-metal ion mechanism for SepMI restriction endonuclease. The analysis of the kinetic parameters under steady state conditions showed that SepMI had a K(m) value for pTrcHisB DNA of 6.15 nM and a V(max) of 1.79×10(-2)nM min(-1), while EhoI had a K(m) for pUC19 plasmid of 8.66 nM and a V(max) of 2×10(-2)nM min(-1). PMID:23017231

  10. An efficient genetic knockout system based on linear DNA fragment homologous recombination for halophilic archaea.

    PubMed

    Xiaoli, Wang; Chuang, Jiang; Jianhua, Liu; Xipeng, Liu

    2015-04-20

    With the development of functional genomics, gene-knockout is becoming an important tool to elucidate gene functions in vivo. As a good model strain for archaeal genetics, Haloferax volcanii has received more attention. Although several genetic manipulation systems have been developed for some halophilic archaea, it is time-consuming because of the low percentage of positive clones during the second-recombination selection. These classical gene knockout methods are based on DNA recombination between the genomic homologous sequence and the circular suicide plasmid, which carries a pyrE selection marker and two DNA fragments homologous to the upstream and downstream fragments of the target gene. Many wild-type clones are obtained through a reverse recombination between the plasmid and genome in the classic gene knockout method. Therefore, it is necessary to develop an efficient gene knockout system to increase the positive clone percentage. Here we report an improved gene knockout method using a linear DNA cassette consisting of upstream and downstream homologous fragments, and the pyrE marker. Gene deletions were subsequently detected by colony PCR analysis. We determined the efficiency of our knockout method by deleting the xpb2 gene from the H. volcanii genome, with the percentage of positive clones higher than 50%. Our method provides an efficient gene knockout strategy for halophilic archaea. PMID:25881705

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

    SciTech Connect

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

    2012-04-20

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

  12. Phylogenetic Specificity and Reproducibility and New Method for Analysis of Terminal Restriction Fragment Profiles of 16S rRNA Genes from Bacterial Communities

    PubMed Central

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

    2001-01-01

    Terminal restriction fragment (TRF) analysis of 16S rRNA genes is an increasingly popular method for rapid comparison of microbial communities, but analysis of the data is still in a developmental stage. We assessed the phylogenetic resolution and reproducibility of TRF profiles in order to evaluate the limitations of the method, and we developed an essential analysis technique to improve the interpretation of TRF data. The theoretical phylogenetic resolution of TRF profiles was determined based on the specificity of TRFs predicted from 3,908 16S rRNA gene sequences. With sequences from the Proteobacteria or gram-positive division, as much as 73% of the TRFs were phylogenetically specific (representing strains from at most two genera). However, the fraction decreased when sequences from the two divisions were combined. The data show that phylogenetic inference will be most effective if TRF profiles represent only a single bacterial division or smaller group. The analytical precision of the TRF method was assessed by comparing nine replicate profiles of a single soil DNA sample. Despite meticulous care in producing the replicates, numerous small, irreproducible peaks were observed. As many as 85% of the 169 distinct TRFs found among the profiles were irreproducible (i.e., not present in all nine replicates). Substantial variation also occurred in the height of synonymous peaks. To make comparisons of microbial communities more reliable, we developed an analytical procedure that reduces variation and extracts a reproducible subset of data from replicate TRF profiles. The procedure can also be used with other DNA fingerprinting techniques for microbial communities or microbial genomes. PMID:11133445

  13. Metalloporphyrin mediated DNA cleavage by a low concentration of HaeIII restriction enzyme.

    PubMed

    Tabata, M; Nakajima, K; Nyarko, E

    2000-03-01

    The plasmid DNA scission by the restriction enzyme HaeIII was investigated in the presence of tetrakis(1-methylpyridinium-4-yl)porphyrin (H2TMPyP) and its manganese(III), iron(III), nickel(II), cobalt(III) and zinc(II) derivatives. The effect of metalloporphyrins on plasmid DNA cleavage was ascertained by gel electrophoresis. UV-Vis absorption spectroscopy and circular dichroism (CD) spectroscopy. In the absence of the metalloporphyrins, plasmid DNA scission did not occur in the presence of a low concentration of HaeIII (0.2 units microL(-1)) at 37 degrees C after 1 h incubation. However, DNA cleavage occurred in the presence of the metalloporphyrins and HaeIII (0.2 units microL(-1)) at 37 degrees C after 1 h incubation. Gel electrophoresis results indicate the catalytic effect of metalloporphyrins (Mn(III)-, Fe(III)-, Co(III)- and Zn(II)TMPyP) by binding to both DNA and the enzyme through electrostatic interaction, which was confirmed by the change in UV-Vis and CD spectra. A mechanism for the enhanced DNA cleavage is proposed. PMID:10857920

  14. Qualitative analysis of sequence specific binding of flavones to DNA using restriction endonuclease activity assays.

    PubMed

    Duran, Elizabeth; Ramsauer, Victoria P; Ballester, Maria; Torrenegra, Ruben D; Rodriguez, Oscar E; Winkle, Stephen A

    2013-08-01

    Flavones, found in nature as secondary plant metabolites, have shown efficacy as anti-cancer agents. We have examined the binding of two flavones, 5,7-dihydroxy-3,6,8-trimethoxy-2-phenyl-4H-chromen-4-one (5,7-dihydroxy-3,6,8-trimethoxy flavone; FlavA) and 3,5-dihydroxy-6,7,8-trimethoxy-2-phenyl-4H-chromen-4-one (3,5-dihydroxy-6,7,8-trimethoxy flavone; FlavB), to phiX174 RF DNA using restriction enzyme activity assays employing the restriction enzymes Alw44, AvaII, BssHII, DraI, MluI, NarI, NciI, NruI, PstI, and XhoI. These enzymes possess differing target and flanking sequences allowing for observation of sequence specificity analysis. Using restriction enzymes that cleave once with a mixture of supercoiled and relaxed DNA substrates provides for observation of topological effects on binding. FlavA and FlavB show differing sequence specificities in their respective binding to phiX. For example, with relaxed DNA, FlavA shows inhibition of cleavage with DraI (reaction site (5') TTTAAA) but not BssHII ((5') GCGCGC) while FlavB shows the opposite results. Evidence for tolological specificity is also observed, Molecular modeling and conformational analysis of the flavones suggests that the phenyl ring of FlavB is coplanar with the flavonoid ring while the phenyl ring of FlavA is at an angle relative to the flavonoid ring. This may account for aspects of the observed sequence and topological specificities in the effects on restriction enzyme activity. © 2013 Wiley Periodicals, Inc. Biopolymers 99: 530-537, 2013. PMID:23712489

  15. EndoG Links Bnip3-Induced Mitochondrial Damage and Caspase-Independent DNA Fragmentation in Ischemic Cardiomyocytes

    PubMed Central

    Zhang, Jisheng; Ye, Junmei; Altafaj, Albert; Cardona, Maria; Bahi, Núria; Llovera, Marta; Cañas, Xavier; Cook, Stuart A.; Comella, Joan X.; Sanchis, Daniel

    2011-01-01

    Mitochondrial dysfunction, caspase activation and caspase-dependent DNA fragmentation are involved in cell damage in many tissues. However, differentiated cardiomyocytes repress the expression of the canonical apoptotic pathway and their death during ischemia is caspase-independent. The atypical BH3-only protein Bnip3 is involved in the process leading to caspase-independent DNA fragmentation in cardiomyocytes. However, the pathway by which DNA degradation ensues following Bnip3 activation is not resolved. To identify the mechanism involved, we analyzed the interdependence of Bnip3, Nix and EndoG in mitochondrial damage and DNA fragmentation during experimental ischemia in neonatal rat ventricular cardiomyocytes. Our results show that the expression of EndoG and Bnip3 increases in the heart throughout development, while the caspase-dependent machinery is silenced. TUNEL-positive DNA damage, which depends on caspase activity in other cells, is caspase-independent in ischemic cardiomyocytes and ischemia-induced DNA high and low molecular weight fragmentation is blocked by repressing EndoG expression. Ischemia-induced EndoG translocation and DNA degradation are prevented by silencing the expression of Bnip3, but not Nix, or by overexpressing Bcl-xL. These data establish a link between Bnip3 and EndoG-dependent, TUNEL-positive, DNA fragmentation in ischemic cardiomyocytes in the absence of caspases, defining an alternative cell death pathway in postmitotic cells. PMID:21437288

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

    SciTech Connect

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

    2005-09-01

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

  17. Restriction fragment length polymorphisms distinguish Leptospira borgpetersenii serovar hardjo type hardjo-bovis isolates from different geographical locations.

    PubMed Central

    Zuerner, R L; Ellis, W A; Bolin, C A; Montgomery, J M

    1993-01-01

    Genetic variability among Leptospira borgpetersenii serovar hardjo type hardjo-bovis isolates representing several geographical regions was determined by restriction endonuclease analysis. Five previously unidentified EcoRI digestion patterns and one previously unidentified HhaI digestion pattern were seen with the various isolates. The copy number and genomic distribution of an L. borgpetersenii insertion sequence (IS1533) was determined. Hardjo-bovis isolate 033 (the type strain for hardjo-bovis) contained 40 well dispersed copies of IS1533. IS1533 probes were used to compare hardjo-bovis isolates by DNA blot hybridization analysis. Use of these probes showed the presence of additional genetic heterogeneity among hardjo-bovis isolates, which restriction endonuclease analysis did not show. Pulsed-field gel electrophoretic analysis of DNAs from several isolates suggested that some polymorphisms arose by genomic rearrangements. All hardjo-bovis isolates were categorized into 14 distinct groups on the basis of common hybridization and endonuclease digestion patterns. Most of these groups were isolated from distinct geographical regions, suggesting that several different clonal populations of hardjo-bovis exist. Images PMID:7681437

  18. GENETIC DIVERSITY OF FLAVOBACTERIUM COLUMNARE EXAMINED BY RESTRICTION FRAGMENT LENGTH POLYMORPHISM AND SEQUENCING OF THE 16S RIBOSOMAL RNA GENE AND THE 16S-23S RDNA SPACER

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Genetic variability among strains of Flavobacterium columnare, isolated in the United States, was characterized by restriction fragment length polymorphism (RFLP) and phylogenetic analysis based on the sequence of the 16S rRNA gene. Twenty-seven isolates of F. columnare were differentiated into thr...

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

    Microsoft Academic Search

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

    1994-01-01

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

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

    E-print Network

    Bernatchez, Louis

    Allopatric Origin of Sympatric Populations of Lake Whitefish (Coregonus clupeaformis) as Revealed WHITEFISH (COREGONUS CLUPEAFORMIS) AS REVEALED BY MITOCHONDRIAL-DNA RESTRICTION ANALYSIS, sympatric populations of ever, species recognition of sympatric pop- lake whitefish (Coregonus clupeaformis

  1. Versatile biosensing platform for DNA detection based on a DNAzyme and restriction-endonuclease-assisted recycling.

    PubMed

    Yuan, Ling; Tu, Wenwen; Bao, Jianchun; Dai, Zhihui

    2015-01-01

    On the basis of a DNAzyme and a restriction-endonuclease-assisted target recycling strategy using Pd-Au alloy nanocrystals to immobilize probe DNA on an electrode and catalyze the reduction of H2O2 which amplified signal and promoted the detection sensitivity, a versatile biosensing platform for DNA detection was proposed. Using p53 and oral cancer genes as models, hemin/G-quadruplex simultaneously acted as a reduced nicotinamide adenine dinucleotide (NADH) oxidase and a horseradish peroxidase (HRP)-mimicking DNAzyme, and a versatile DNA biosensor was designed for the first time based on the good electrocatalytic activity of Pd-Au alloy nanocrystals. Hemin/G-quadruplex catalyzed the reduction of H2O2, which was generated from NADH in the presence of O2, to produce an electrochemical signal when thionine functioned as the electron mediator. Moreover, the nicking endonuclease N.BstNB I caused the target DNA to cycle for multiple rounds and further amplified the electrochemical response. This versatile DNA biosensor exhibited linear ranges for the detection of p53 and oral cancer genes from 0.1 fmol L(-1) to 0.1 nmol L(-1) and 0.1 fmol L(-1) to 1 nmol L(-1), respectively. The detection limits, established as 3?, were estimated to be 0.03 and 0.06 fmol L(-1) for the p53 and oral cancer genes, respectively. The as-prepared biosensor could discriminate mismatched sequences, indicating a satisfactory selectivity and validating the feasibility of the proposed strategy. More importantly, simply by changing the helper DNA, this versatile DNA biosensor could detect different target DNA species, which could create a new avenue for the potential diagnosis of cancer. PMID:25493424

  2. Pyrrolamide DNA Gyrase Inhibitors: Fragment-Based Nuclear Magnetic Resonance Screening To Identify Antibacterial Agents

    PubMed Central

    Green, Oluyinka; Hales, Neil; Walkup, Grant K.; Bist, Shanta; Singh, Alok; Mullen, George; Bryant, Joanna; Embrey, Kevin; Gao, Ning; Breeze, Alex; Timms, Dave; Andrews, Beth; Uria-Nickelsen, Maria; Demeritt, Julie; Loch, James T.; Hull, Ken; Blodgett, April; Illingworth, Ruth N.; Prince, Bryan; Boriack-Sjodin, P. Ann; Hauck, Sheila; MacPherson, Lawrence J.; Ni, Haihong; Sherer, Brian

    2012-01-01

    DNA gyrase is an essential enzyme in bacteria, and its inhibition results in the disruption of DNA synthesis and, subsequently, cell death. The pyrrolamides are a novel class of antibacterial agents targeting DNA gyrase. These compounds were identified by a fragment-based lead generation (FBLG) approach using nuclear magnetic resonance (NMR) screening to identify low-molecular-weight compounds that bind to the ATP pocket of DNA gyrase. A pyrrole hit with a binding constant of 1 mM formed the basis of the design and synthesis of a focused library of compounds that resulted in the rapid identification of a lead compound that inhibited DNA gyrase with a 50% inhibitory concentration (IC50) of 3 ?M. The potency of the lead compound was further optimized by utilizing iterative X-ray crystallography to yield DNA gyrase inhibitors that also displayed antibacterial activity. Spontaneous mutants were isolated in Staphylococcus aureus by plating on agar plates containing pyrrolamide 4 at the MIC. The resistant variants displayed 4- to 8-fold-increased MIC values relative to the parent strain. DNA sequencing revealed two independent point mutations in the pyrrolamide binding region of the gyrB genes from these variants, supporting the hypothesis that the mode of action of these compounds was inhibition of DNA gyrase. Efficacy of a representative pyrrolamide was demonstrated against Streptococcus pneumoniae in a mouse lung infection model. These data demonstrate that the pyrrolamides are a novel class of DNA gyrase inhibitors with the potential to deliver future antibacterial agents targeting multiple clinical indications. PMID:22183167

  3. Structures of minimal catalytic fragments of topoisomerase V reveals conformational changes relevant for DNA binding.

    PubMed

    Rajan, Rakhi; Taneja, Bhupesh; Mondragón, Alfonso

    2010-07-14

    Topoisomerase V is an archaeal type I topoisomerase that is unique among topoisomerases due to presence of both topoisomerase and DNA repair activities in the same protein. It is organized as an N-terminal topoisomerase domain followed by 24 tandem helix-hairpin-helix (HhH) motifs. Structural studies have shown that the active site is buried by the (HhH) motifs. Here we show that the N-terminal domain can relax DNA in the absence of any HhH motifs and that the HhH motifs are required for stable protein-DNA complex formation. Crystal structures of various topoisomerase V fragments show changes in the relative orientation of the domains mediated by a long bent linker helix, and these movements are essential for the DNA to enter the active site. Phosphate ions bound to the protein near the active site helped model DNA in the topoisomerase domain and show how topoisomerase V may interact with DNA. PMID:20637419

  4. The genome of orf virus: Restriction endonuclease analysis of viral DNA isolated from lesions of orf in sheep

    Microsoft Academic Search

    A. J. Robinson; G. Ellis; T. Balassu

    1982-01-01

    Summary The purification of orf virus directly from scab material from clinical cases of orf in sheep and restriction endonuclease analysis of the viral DNA is described. Between 7×109 and 1.6×1011 virus particles, and 0.7 to 22.8 µg of viral DNA could be recovered from lg of scab material. Considerable heterogeneity was observed between different field isolates when restriction endonuclease

  5. Congruence in Control-Region Sequence and Restriction Site Variation in Mitochondrial DNA of Brook Charr (Salvelinus fontinalis Mitchill)

    Microsoft Academic Search

    Louis Bernatchez; Roy G. Danzmann

    1993-01-01

    We compared the congruence in genetic variation and phylogenetic relationships among mitochondrial DNA (mtDNA) haplotypes detected with restriction-frag- ment-length polymorphisms (RFLPs) and sequence analysis in the brook charr, Sulvelinus fontinalis Mitchill. This was accomplished by analyzing variation both at 172 restriction sites subsampling 903 bp over the entire mitochondrial genome and in the sequence of a 300-bp segment of the

  6. A cladistic analysis of phenotypic associations with haplotypes inferred from restriction endonuclease mapping and DNA sequence data

    Microsoft Academic Search

    Alan R. Templeton; Keith A. Crandall; Charles F. Sing

    1992-01-01

    ABSTRACT We previously developed a cladistic approach to identify subsets of haplotypes defined by restriction endonuclease,mapping,or DNA sequencing that are associated with significant phenotypic deviations. Our approach was limited to segments of DNA in which little recombination occurs. In such cases, a cladogram,can be constructed from the restriction site or , consistent with these limits. Our estimation procedure,also identifies haplotypes

  7. 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

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

    PubMed Central

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

    2014-01-01

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

  9. The Intracellular DNA Sensor IFI16 Gene Acts as Restriction Factor for Human Cytomegalovirus Replication

    PubMed Central

    Gariano, Grazia Rosaria; Dell'Oste, Valentina; Bronzini, Matteo; Gatti, Deborah; Luganini, Anna; De Andrea, Marco; Gribaudo, Giorgio; Gariglio, Marisa; Landolfo, Santo

    2012-01-01

    Human interferon (IFN)-inducible IFI16 protein, an innate immune sensor of intracellular DNA, modulates various cell functions, however, its role in regulating virus growth remains unresolved. Here, we adopt two approaches to investigate whether IFI16 exerts pro- and/or anti-viral actions. First, the IFI16 gene was silenced using specific small interfering RNAs (siRNA) in human embryo lung fibroblasts (HELF) and replication of DNA and RNA viruses evaluated. IFI16-knockdown resulted in enhanced replication of Herpesviruses, in particular, Human Cytomegalovirus (HCMV). Consistent with this, HELF transduction with a dominant negative form of IFI16 lacking the PYRIN domain (PYD) enhanced the replication of HCMV. Second, HCMV replication was compared between HELFs overexpressing either the IFI16 gene or the LacZ gene. IFI16 overexpression decreased both virus yield and viral DNA copy number. Early and late, but not immediate-early, mRNAs and proteins were strongly down-regulated, thus IFI16 may exert its antiviral effect by impairing viral DNA synthesis. Constructs with the luciferase reporter gene driven by deleted or site-specific mutated forms of the HCMV DNA polymerase (UL54) promoter demonstrated that the inverted repeat element 1 (IR-1), located between ?54 and ?43 relative to the transcription start site, is the target of IFI16 suppression. Indeed, electrophoretic mobility shift assays and chromatin immunoprecipitation demonstrated that suppression of the UL54 promoter is mediated by IFI16-induced blocking of Sp1-like factors. Consistent with these results, deletion of the putative Sp1 responsive element from the HCMV UL44 promoter also relieved IFI16 suppression. Together, these data implicate IFI16 as a novel restriction factor against HCMV replication and provide new insight into the physiological functions of the IFN-inducible gene IFI16 as a viral restriction factor. PMID:22291595

  10. Restriction fragment length polymorphism mapping of quantitative trait loci for malaria parasite susceptibility in the mosquito Aedes aegypti

    SciTech Connect

    Severson, D.W.; Thathy, V.; Mori, A. [Univ. of Wisconsin, Madison, WI (United States)] [and others

    1995-04-01

    Susceptibility of the mosquito Aedes aegypti to the malarial parasite Plasmodium gallinaceum was investigated as a quantitative trait using restriction fragment length polymorphisms (RFLP). Two F{sub 2} populations of mosquitoes were independently prepared from pairwise matings between a highly susceptible and a refractory strain of A. aegypti. RFLP were tested for association with oocyst development on the mosquito midgut. Two putative quantitative trait loci (QTL) were identified that significantly affect susceptibility. One QTL, pgs [2,LF98], is located on chromosome 2 and accounted for 65 and 49% of the observed phenotypic variance in the two populations, respectively. A second QTL, pgs[3,MalI], is located on chromosome 3 and accounted for 14 and 10% of the observed phenotypic variance in the two populations, respectively. Both QTL exhibit a partial dominance effect on susceptibility, wherein the dominance effect is derived from the refractory parent. No indication of epistasis between these QTL was detected. Evidence suggests that either a tightly linked cluster of independent genes or a single locus affecting susceptibility to various mosquito-borne parasites and pathogens has evolved near the LF98 locus; in addition to P. gallinaceum susceptibility, this general genome region has previously been implicated in susceptibility to the filaria nematode Brugia malayi and the yellow fever virus. 35 refs., 2 figs., 3 tabs.

  11. Prevalence and distribution of human papillomavirus infection in Korean women as determined by restriction fragment mass polymorphism assay.

    PubMed

    Lee, Eun Hee; Um, Tae Hyun; Chi, Hyun-Sook; Hong, Young-Joon; Cha, Young Joo

    2012-09-01

    The development of a prophylactic vaccine that targets human papillomaviruses (HPV) 6, 11, 16, and 18 to prevent cervical cancer has increased interest in the ethnic and geographical distributions of HPV genotypes. We investigated HPV prevalence and type distribution by restriction fragment mass polymorphism (RFMP) testing a total of 60,775 specimens (aged 18-79 yr, median 44) taken from liquid-based cytology. Overall HPV positive rate of total patients was 34.2%. Among the positive patients, 87.7% was single type infections, and 12.3% was multiple HPV types. HPV-16 was the most prevalent genotype observed in 2,307 (26.0%), followed by type 52 in 2,269 (25.5%), type 58 in 1,090 (12.3%), type 18 in 633 (7.1%), type 56 in 436 (4.9%). The pattern of high risk-HPV positive rate according to age showed U-shape with a peak in HPV prevalence among women less than 30 yr of age, and a second peak among the older females aged 70 to 79 yr. The leading four high-risk HPV genotypes were HPV-16, HPV-52, HPV-58, and HPV-18 in descending order. In conclusion, this study provides the most representative prevalence and type-specific distribution of HPV among Korean women, and demonstrates that the epidemiology of HPV infection is different from that of other regions of the world. PMID:22969258

  12. 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

  13. An improved PCR-restriction fragment length polymorphism (RFLP) method for the identification of cry1-type genes.

    PubMed

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

    2013-11-01

    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

  14. Large DNA fragment sizing by flow cytometry: application to the characterization of P1 artificial chromosome (PAC) clones.

    PubMed Central

    Huang, Z; Petty, J T; O'Quinn, B; Longmire, J L; Brown, N C; Jett, J H; Keller, R A

    1996-01-01

    A flow cytometry-based, ultrasensitive fluorescence detection technique is used to size individual DNA fragments up to 167 kb in length. Application of this technology to the sizing of P1 artificial chromosomes (PACs) in both linear and supercoiled forms is described. It is demonstrated that this method is well suited to characterizing PAC/BAC clones and will be very useful for the analysis of large insert libraries. Fluorescence bursts are recorded as individual, dye stained DNA fragments pass through a low power, focused, continuous laser beam. The magnitudes of the fluorescence bursts are linearly proportional to the lengths of the DNA fragments. The histograms of the burst sizes are generated in <3 min with <1 pg of DNA. Results on linear fragments are consistent with those obtained by pulsed-field gel electrophoresis. In comparison with pulsed-field gel electrophoresis, sizing of large DNA fragments by this approach is more accurate, much faster, requires much less DNA, and is independent of the DNA conformation. PMID:8932373

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

    SciTech Connect

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

    2007-03-01

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

  16. Dynamics of Enzymatic Interactions During Short Flap Human Okazaki Fragment Processing by Two forms of Human DNA Polymerase ?

    PubMed Central

    Lin, Szu Hua Sharon; Wang, Xiaoxiao; Zhang, Sufang; Zhang, Zhongtao; Lee, Ernest Y.C.; Lee, Marietta Y.W.T.

    2013-01-01

    Lagging strand DNA replication requires the concerted actions of DNA polymerase ?, Fen1 and DNA ligase I for the removal of the RNA/DNA primers before ligation of Okazaki fragments. To better understand this process in human cells, we have reconstituted Okazaki fragment processing by the short flap pathway in vitro with purified human proteins and oligonucleotide substrates. We systematically characterized the key events in Okazaki fragment processing: the strand displacement, Pol ?/Fen1 combined reactions for removal of the RNA/DNA primer, and the complete reaction with DNA ligase I. Two forms of human DNA polymerase ? were studied: Pol ?4 and Pol ?3, which represent the heterotetramer and the heterotrimer lacking the p12 subunit, respectively. Pol ?3 exhibits very limited strand displacement activity in contrast to Pol ?4, and stalls on encounter with a 5’-blocking oligonucleotide. Pol ?4 and Pol ?3 exhibit different characteristics in the Pol ?/Fen1 reactions. While Pol ?3 produces predominantly 1 and 2 nt cleavage products irrespective of Fen1 concentrations, Pol ?4 produces cleavage fragments of 1–10 nts at low Fen1 concentrations. Pol ?3 and Pol ?4 exhibit comparable formation of ligated products in the complete system. While both are capable of Okazaki fragment processing in vitro, Pol ?3 exhibits ideal characteristics for a role in Okazaki fragment processing. Pol ?3 readily idles and in combination with Fen1 produces primarily 1 nt cleavage products, so that nick translation predominates in the removal of the blocking strand, avoiding the production of longer flaps that require additional processing. These studies represent the first analysis of the two forms of human Pol ? in Okazaki fragment processing. The findings provide evidence for the novel concept that Pol ?3 has a role in lagging strand synthesis, and that both forms of Pol ? may participate in DNA replication in higher eukaryotic cells. PMID:24035200

  17. PCR and restriction fragment length polymorphism of a pel gene as a tool to identify Erwinia carotovora in relation to potato diseases.

    PubMed Central

    Darrasse, A; Priou, S; Kotoujansky, A; Bertheau, Y

    1994-01-01

    Using a sequenced pectate lyase-encoding gene (pel gene), we developed a PCR test for Erwinia carotovora. A set of primers allowed the amplification of a 434-bp fragment in E. carotovora strains. Among the 89 E. carotovora strains tested, only the Erwinia carotovora subsp. betavasculorum strains were not detected. A restriction fragment length polymorphism (RFLP) study was undertaken on the amplified fragment with seven endonucleases. The Sau3AI digestion pattern specifically identified the Erwinia carotovora subsp. atroseptica strains, and the whole set of data identified the Erwinia carotovora subsp. wasabiae strains. However, Erwinia carotovora subsp. carotovora and Erwinia carotovora subsp. odorifera could not be separated. Phenetic and phylogenic analyses of RFLP results showed E. carotovora subsp. atroseptica as a homogeneous group while E. carotovora subsp. carotovora and E. carotovora subsp. odorifera strains exhibited a genetic diversity that may result from a nonmonophyletic origin. The use of RFLP on amplified fragments in epidemiology and for diagnosis is discussed. Images PMID:7912502

  18. Nuclear DNA fragmentation and morphological alterations in adult rabbit skeletal muscle after short-term immobilization.

    PubMed

    Smith, H K; Maxwell, L; Martyn, J A; Bass, J J

    2000-11-01

    Nuclear DNA fragmentation and ultrastructural changes, indicative of myonuclear apoptosis, were examined in adult skeletal muscle in response to short-term immobilization. Adult rabbits were allocated to 2 days (n=5) or 6 days (n=5) of unilateral casting of the ankle in full plantar flexion or were used as untreated controls (n=2). Atrophy of the soleus muscle was apparent by significant reductions in wet mass of 15% and 26% after 2 days and 6 days of casting (P< or =0.05), respectively. Mean fibre cross-sectional area and myonuclear number per section were also lower (17% and 9.1%, respectively) after 6 days of casting, in comparison with contralateral control muscles (P< or =0.05). Electron-microscopic examination showed condensed chromatin and irregularly shaped myonuclei in muscles immobilized for either 2 days or 6 days. Myofibrillar disruption and abnormalities of the subsarcolemmal mitochondria were also apparent in the absence of inflammation or plasma membrane alterations in cast muscles. Longitudinal and transverse sections showed abundant in situ end-labelling of DNA strand breaks (TUNEL) after 2 days, with less after 6 days, of immobilization. Positive labelling corresponded to myonuclear locations within fibres, yet the number of TUNEL-positive nuclei indicated DNA fragmentation in additional cell types such as capillary endothelial cells or fibroblasts. The data indicate that the immobilization of slow-twitch skeletal muscle in a shortened position rapidly induces morphological alterations consistent with mitochondrial injury and apoptotic myonuclear elimination. PMID:11131134

  19. Association of Self-DNA Mediated TLR9-Related Gene, DNA Methyltransferase, and Cytokeratin Protein Expression Alterations in HT29-Cells to DNA Fragment Length and Methylation Status

    PubMed Central

    F?ri, István; Sipos, Ferenc; Spisák, Sándor; Kiszner, Gerg?; Wichmann, Barnabás; Schöller, Andrea; Tulassay, Zsolt; M?zes, Györgyi; Molnár, Béla

    2013-01-01

    To understand the biologic role of self-DNA bound to Toll-like Receptor 9 (TLR9), we assayed its effect on gene and methyltransferase expressions and cell differentiation in HT29 cells. HT29 cells were incubated separately with type-1 (normally methylated/nonfragmented), type-2 (normally methylated/fragmented), type-3 (hypermethylated/nonfragmented), or type-4 (hypermethylated/fragmented) self-DNAs. Expression levels of TLR9-signaling and proinflammatory cytokine-related genes were assayed by qRT-PCR. Methyltransferase activity and cell differentiation were examined by using DNA methyltransferase (DNMT1, -3A, -3B) and cytokeratin (CK) antibodies. Treatment with type-1 DNA resulted in significant increase in TLR9 expression. Type-2 treatment resulted in the overexpression of TLR9-related signaling molecules (MYD88A, TRAF6) and the IL8 gene. In the case of type-3 treatment, significant overexpression of NFkB, IRAK2, and IL8 as well as downregulation of TRAF6 was detected. Using type-4 DNA, TRAF6 and MYD88A gene expression was upregulated, while MYD88B, IRAK2, IL8, and TNFSF10 were all underexpressed. CK expression was significantly higher only after type-1 DNA treatment. DNMT3A expression could also be induced by type-1 DNA treatment. DNA structure may play a significant role in activation of the TLR9-dependent and even independent proinflammatory pathways. There may be a molecular link between TLR9 signaling and DNMT3A. The mode of self-DNA treatment may influence HT29 cell differentiation. PMID:24459426

  20. Low energy electron induced fragmentation and reactions of DNA and its molecular components

    NASA Astrophysics Data System (ADS)

    Bass, Andrew

    2005-05-01

    Much research has been stimulated by the recognition that ionizing radiation can, in condensed matter, generate large numbers of secondary electrons with energies less than 20 eV [1] and by the experimental demonstration that such electrons may induce both single and double strand breaks in plasmid DNA [2]. Identifying the underlying mechanisms involves several research methodologies, from further experiments with DNA to studies of the electron interaction with the component `sub-units' of DNA in both the gas and condensed phases [3]. In particular, understanding electron-induced strand break damage, the type of damage most difficult for organisms to repair, necessitates study of the sub-units of DNA back-bone, and here Tetrahyrofuran (THF) and its derivatives, provide a useful model for the furyl ring at the centre of the deoxyribose sugar. In this contribution, we review with particular reference to DNA and related molecules, the use of electron spectroscopy and mass spectrometry to study electron-induced fragmentation and reactions in thin molecular solids. We describe a newly completed instrument that combines laser post-ionization with a time-of-flight mass analyzer for highly sensitive ion and neutral detection. Use of the instrument is illustrated with results for THF and derivatives. Anion desorption measurements reveal the role of transient negative ions (TNI) and Dissociative Electron Attachment in significant molecular fragmentation and permit effective cross sections for this electron-induced damage to be obtained. The neutral yield functions also illustrate the importance of TNI, mirroring features seen in recently measured cross sections for electron induced aldehyde production in THF [4]. 1. J. A. Laverne and S. M. Pimblott, Radiat. Res. 141, 208 (1995) 2. B. Boudaiffa, et al, Science 287, 1658 (2000) 3. L. Sanche. Physica Scripta. 68, C108, (2003) 4. S.-P. Breton, et al.,J. Chem. Phys. 121, 11240 (2004)

  1. 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

  2. Sepcific fragmentation of DNA heteroduplex molecules of two bacteriophage lambda mutants with endonuclease Si from Aspergillus oryzae.

    PubMed Central

    Ksënzenko, V N; Tikhomirova, L P; Matvienko, N I

    1976-01-01

    Heteroduplex DNA molecules of two bacteriophage mutants (lambda b2 and lambda i434ct68) were obtained by the method of molecular hybridization. These heteroduplexes possessed two types of loops formed as a result of: a) deletion in one of the DNA strands; and b) substitution of a DNA fragment for nonhomological one. The digestion of heteroduplexes with single-stranded specific nuclease SI from Aspergillus oryzae produced two fragments at 37 degrees C and three ones at 55 degrees C. The separation of fragments and determination of their molecular weight were carried out by means of electrophoresis in agarose. The molecular weights both measured and preliminarily calculated proved to be close. One of the fragments was identificated by its biological activity in CaCl2-dependent infectious system with helperphage. Images PMID:1272803

  3. Colocalization of BAX and BCL2 in small intestine and kidney biopsies with different degrees of DNA fragmentation

    Microsoft Academic Search

    A. P. Aschoff; U. Ott; R. Fünfstück; G. Stein; G. F. Jirikowski

    1999-01-01

    Morphological changes associated with apoptosis are closely correlated with the expression of specific proteins. However, the cause-effect relationships between the expression of these proteins and DNA degradation are barely known. For studying expression of apoptosis-related proteins in relation to different degrees of DNA fragmentation, the small intestine with its spatially organized continuum of proliferation, differentiation and death is a very

  4. Genetic diversity of Dioscorea dumetorum (Kunth) Pax using Amplified Fragment Length Polymorphisms (AFLP) and cpDNA

    Microsoft Academic Search

    Mubo A. Sonibare; Robert Asiedu; Dirk C. Albach

    2010-01-01

    We have utilized Amplified Fragment Length Polymorphisms (AFLP) in conjunction with chloroplast DNA (cpDNA) sequence data to study the genetic diversity in 53 accessions of Dioscorea dumetorum from six countries in West and Central Africa. Our results provide a comparison of the two marker systems with regards to their applicability to differentiate intraspecific genotypes and the grouping of the accessions

  5. 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. (Univ. of Michigan, Ann Arbor (United States)); Little, P.F.R. (Imperial College of Science, Technology, and Medicine, London (United Kingdom)); Nakamura, Yusuke (Cancer Inst., Tokyo (Japan))

    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.

  6. DNA fragmentation pattern in human fibroblasts after irradiation with iron ions

    NASA Astrophysics Data System (ADS)

    Campa, Alessandro

    In this work we studied the fragmentation pattern produced by the double stand breaks (DSB) induced in AG1522 primary human fibroblasts by two different iron beams, one of energy 414 MeV/u, and the other of energy 115 MeV/u (with dose-average LET in water equal to 202 keV/µm and 442 keV/µm, respectively). Irradiation with several doses up to 200 Gy was performed at the HIMAC facility of the National Institute of Radiological Sciences, Chiba, Japan. Experimental data, first obtained for fragments belonging to the size ranges 23-1000 kbp and 1000-5700 kbp (Belli et al., 2006), have successively been obtained also for fragments belonging to the size ranges 1-9 kbp and 9-23 kbp; the experimental analysis was performed with pulsed and constant field electrophoresis. The RBE for DSB production was evaluated in two different fragment size ranges (i.e., 23-5700 kbp and 1-5700 kbp), and it was found larger for the wider size range, especially for the beam with the higher LET. The experimental results have been compared to those computed on the basis of the Monte Carlo PARTRAC simulation code, following the line of research started in Campa et al. (2005), and exploiting the recent update of the PARTRAC code to ions heavier than helium (Friedland et al., 2006). Because the agreement has been found satisfactory for both radiation qualities, the spectra outside the experimentally observable fragment size range were also computed in order to evaluate the overall fragmentation pattern. The marked increases of the RBEs for DSB production, obtained when also the very small fragments (< 1 kbp) are included, makes them closer to the RBE values observed for the late cellular effects. This finding is a further indication for the biological significance of the spatial correlation of DSB at short distances. This work was partially supported by ASI (Italian Space Agency, "Mo-Ma/COUNT" project). References M. Belli, A. Campa, V. Dini, G. Esposito, Y. Furusawa, G. Simone, E. Sorrentino and M. A. Tabocchini. DNA fragmentation induced in human fibroblasts by accelerated 56 Fe ions of differing energies. Radiat. Res. 165, 713-720 (2006). A. Campa, F. Ballarini, M. Belli, R. Cherubini, V. Dini, G. Esposito, W. Friedland, S. Gerardi, S. Molinelli, A. Ottolenghi, H. G. Paretzke, G. Simone and M. A. Tabocchini. DNA DSB induced in human cells by charged particles and gamma rays: experimental results and theoretical approaches. Int. J. Radiat. Biol. 81, 841-854 (2005). W. Friedland, P. Jacob, H. G. Paretzke, A. Ottolenghi, F. Ballarini and M. Liotta. Simulation of light ion induced DNA damge patterns. Radiat. Prot. Dosim. 122, 116-120 (2006).

  7. Structure of 5-hydroxymethylcytosine-specific restriction enzyme, AbaSI, in complex with DNA

    PubMed Central

    Horton, John R.; Borgaro, Janine G.; Griggs, Rose M.; Quimby, Aine; Guan, Shengxi; Zhang, Xing; Wilson, Geoffrey G.; Zheng, Yu; Zhu, Zhenyu; Cheng, Xiaodong

    2014-01-01

    AbaSI, a member of the PvuRts1I-family of modification-dependent restriction endonucleases, cleaves deoxyribonucleic acid (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. PMID:24895434

  8. Variability of chloroplast DNA and nuclear ribosomal DNA in cassava ( Manihot esculenta Crantz) and its wild relatives

    Microsoft Academic Search

    M. A. Fregene; J. Vargas; J. Ikea; F. Angel; J. Tohme; R. A. Asiedu; M. O. Akoroda; W. M. Roca

    1994-01-01

    Chloroplast DNA (cp) and nuclear ribosomal DNA (rDNA) variation was investigated in 45 accessions of cultivated and wild Manihot species. Ten independent mutations, 8 point mutations and 2 length mutations were identified, using eight restriction enzymes and 12 heterologous cpDNA probes from mungbean. Restriction fragment length polymorphism analysis defined nine distinct chloroplast types, three of which were found among the

  9. 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.

  10. The structural basis of differential DNA sequence recognition by restriction–modification controller proteins

    PubMed Central

    Ball, N. J.; McGeehan, J. E.; Streeter, S. D.; Thresh, S.-J.; Kneale, G. G.

    2012-01-01

    Controller (C) proteins regulate the expression of restriction–modification (RM) genes in a wide variety of RM systems. However, the RM system Esp1396I is of particular interest as the C protein regulates both the restriction endonuclease (R) gene and the methyltransferase (M) gene. The mechanism of this finely tuned genetic switch depends on differential binding affinities for the promoters controlling the R and M genes, which in turn depends on differential DNA sequence recognition and the ability to recognize dual symmetries. We report here the crystal structure of the C protein bound to the M promoter, and compare the binding affinities for each operator sequence by surface plasmon resonance. Comparison of the structure of the transcriptional repression complex at the M promoter with that of the transcriptional activation complex at the R promoter shows how subtle changes in protein–DNA interactions, underpinned by small conformational changes in the protein, can explain the molecular basis of differential regulation of gene expression. PMID:22941636

  11. Restricted leucine zipper dimerization and specificity of DNA recognition of the melanocyte master regulator MITF

    PubMed Central

    Pogenberg, Vivian; Ögmundsdóttir, Margrét H; Bergsteinsdóttir, Kristín; Schepsky, Alexander; Phung, Bengt; Deineko, Viktor; Milewski, Morlin; Steingrímsson, Eiríkur; Wilmanns, Matthias

    2012-01-01

    Microphthalmia-associated transcription factor (MITF) is a master regulator of melanocyte development and an important oncogene in melanoma. MITF heterodimeric assembly with related basic helix–loop–helix leucine zipper transcription factors is highly restricted, and its binding profile to cognate DNA sequences is distinct. Here, we determined the crystal structure of MITF in its apo conformation and in the presence of two related DNA response elements, the E-box and M-box. In addition, we investigated mouse and human Mitf mutations to dissect the functional significance of structural features. Owing to an unusual three-residue shift in the leucine zipper register, the MITF homodimer shows a marked kink in one of the two zipper helices to allow an out-of-register assembly. Removal of this insertion relieves restricted heterodimerization by MITF and permits assembly with the transcription factor MAX. Binding of MITF to the M-box motif is mediated by an unusual nonpolar interaction by Ile212, a residue that is mutated in mice and humans with Waardenburg syndrome. As several related transcription factors have low affinity for the M-box sequence, our analysis unravels how these proteins discriminate between similar target sequences. Our data provide a rational basis for targeting MITF in the treatment of important hereditary diseases and cancer. PMID:23207919

  12. MmeI: a minimal Type II restriction-modification system that only modifies one DNA strand for host protection

    PubMed Central

    Morgan, Richard D.; Bhatia, Tanya K.; Lovasco, Lindsay; Davis, Theodore B.

    2008-01-01

    MmeI is an unusual Type II restriction enzyme that is useful for generating long sequence tags. We have cloned the MmeI restriction-modification (R-M) system and found it to consist of a single protein having both endonuclease and DNA methyltransferase activities. The protein comprises an amino-terminal endonuclease domain, a central DNA methyltransferase domain and C-terminal DNA recognition domain. The endonuclease cuts the two DNA strands at one site simultaneously, with enzyme bound at two sites interacting to accomplish scission. Cleavage occurs more rapidly than methyl transfer on unmodified DNA. MmeI modifies only the adenine in the top strand, 5?-TCCRAC-3?. MmeI endonuclease activity is blocked by this top strand adenine methylation and is unaffected by methylation of the adenine in the complementary strand, 5?-GTYGGA-3?. There is no additional DNA modification associated with the MmeI R-M system, as is required for previously characterized Type IIG R-M systems. The MmeI R-M system thus uses modification on only one of the two DNA strands for host protection. The MmeI architecture represents a minimal approach to assembling a restriction-modification system wherein a single DNA recognition domain targets both the endonuclease and DNA methyltransferase activities. PMID:18931376

  13. Bulky DNA adducts in human sperm associated with semen parameters and sperm DNA fragmentation in infertile men: a cross-sectional study

    PubMed Central

    2013-01-01

    Background DNA adducts are widely used marker of DNA damage induced by environmental pollutants. The present study was designed to explore whether sperm polycyclic aromatic hydrocarbon-DNA adducts were associated with sperm DNA integrity and semen quality. Methods A total of 433 Han Chinese men were recruited from an infertility clinic. Immunofluorescence was applied to analyze sperm PAH-DNA adducts. Sperm DNA fragmentation was detected by terminal deoxynucleotidyl transferase (Tdt)-mediated dUTP nick end labelling (TUNEL) assay. Results After adjustment for potential confounders using linear regression, sperm PAH-DNA adducts were negatively associated with sperm concentration, total sperm count, sperm motility, and curvilinear velocity (VCL). In addition, a positive relationship between sperm PAH-DNA adducts and sperm DNA fragmentation was found. Conclusions Our findings suggested an inverse association between sperm PAH-DNA adducts and semen quality, and provided the first epidemiologic evidence of an adverse effect of PAH-DNA adducts on sperm DNA integrity. PMID:24073787

  14. Analysis of rRNA restriction fragment length polymorphisms from Bacteroides spp. and Bacteroides fragilis isolates associated with diarrhea in humans and animals.

    PubMed Central

    Smith, C J; Callihan, D R

    1992-01-01

    The Escherichia coli rRNA operon rrnB was used as a 32P-labeled hybridization probe in Southern blots of genomic DNAs from representative strains of the saccharolytic, gram-negative, obligate anaerobes of the genus Bacteroides. Control experiments with the B. fragilis type strain ATCC 25285 established that nearly identical rRNA fragment patterns were produced when either the E. coli rrnB gene probe or homologous rRNA isolated from B. fragilis was used as the probe. In addition, it was shown that a specific 16S or 23S rrnB gene probe also could be used to produce fragment patterns suitable for analysis. Thirty-one strains from 8 of the 10 recognized Bacteroides species were then examined. The resulting autoradiographs revealed specific fragment patterns for all but one (B. ovatus) of the species tested. Restriction fragment length polymorphisms were observed for many of the strains tested, but these differences did not hinder species classification. The five B. ovatus strains examined did not form a distinct group, and their rRNA fragment patterns displayed a marked heterogeneity. The same approach was applied to a unique set of enterotoxin-producing B. fragilis strains isolated from animals and humans with diarrhea. The results demonstrated that these strains were in fact B. fragilis and that they produce rRNA fragment patterns closely related to those of the type strain ATCC 25285. This set of strains did not appear to form a separate subgroup or genotype within the B. fragilis species, and there were no distinguishable restriction fragment length polymorphisms that could be used to specifically separate enterotoxin-producing strains from nonenterotoxigenic strains. Images PMID:1374078

  15. 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

  16. Monte Carlo predictions of DNA fragment-size distributions for large sizes after HZE particle irradiation

    NASA Technical Reports Server (NTRS)

    Ponomarev, A. L.; Cucinotta, F. A.; Sachs, R. K.; Brenner, D. J.

    2001-01-01

    DSBs (double-strand breaks) produced by densely ionizing space radiation are not located randomly in the genome: recent data indicate DSB clustering along chromosomes. DSB clustering at large scales, from >100 Mbp down to approximately 2 kbp, is modeled using a Monte-Carlo algorithm. A random-walk model of chromatin is combined with a track model, that predicts the radial distribution of energy from an ion, and the RLC (randomly-located-clusters) formalism, in software called DNAbreak. This model generalizes the random-breakage model, whose broken-stick fragment-size distribution is applicable to low-LET radiation. DSB induction due to track interaction with the DNA volume depends on the radiation quality parameter Q. This dose-independent parameter depends only weakly on LET. Multi-track, high-dose effects depend on the cluster intensity parameter lambda, proportional to fluence as defined by the RLC formalism. After lambda is determined by a numerical experiment, the model reduces to one adjustable parameter Q. The best numerical fits to the experimental data, determining Q, are obtained. The knowledge of lambda and Q allows us to give biophysically based extrapolations of high-dose DNA fragment-size data to low doses or to high LETs.

  17. Rates of nuclear DNA evolution in pheasant-like birds: evidence from restriction maps.

    PubMed Central

    Helm-Bychowski, K M; Wilson, A C

    1986-01-01

    To examine the tempo of genomic evolution in birds, we mapped 161 restriction sites in the nuclear DNA of seven species of birds belonging to the pheasant superfamily Phasianoidea. The three regions mapped lie on different chromosomes and bear eight genes, coding for lysozyme c, three "alpha-like" globins, and four "beta-like" globins. Together, the three regions span about 56 kilobases, most of which is presumably noncoding. The maps differed from one another at a minimum of 77 sites and by 9 length mutations. The extent of sequence divergence due to base substitutions was inferred to be similar for all three regions, even though the three coding regions differ by 5-fold from one another in mean rate of evolution at the amino acid level. A tree relating the maps differs in branching order from that implied by the traditional classification of phasianoid birds and is supported by published protein comparisons. Five of the nodes in the tree were associated with fossil evidence and historical biogeographic information, allowing us to estimate the mean rate of DNA divergence to be 0.34-0.40% per million years. This rate is similar to that estimated for the globin gene regions of higher primates, which validates the concept of an evolutionary clock at the DNA level. Our fossil-based calibration of DNA evolution differs by a factor of almost 2 from that proposed by others on the basis of biogeography. In consequence, published estimates of divergence times for birds and primates that are based on a biogeographically calibrated DNA clock may be too long. PMID:3003745

  18. Does quantum entanglement in DNA synchronize the catalytic centers of type II restriction endonucleases?

    E-print Network

    P. Kurian; G. Dunston; J. Lindesay

    2014-03-21

    Several living systems have been examined for their apparent optimization of structure and function for quantum behavior at biological length scales. Orthodox type II endonucleases, the largest class of restriction enzymes, recognize four-to-eight base pair sequences of palindromic DNA, cut both strands symmetrically, and act without an external metabolite such as ATP. While it is known that these enzymes induce strand breaks by attacking phosphodiester bonds, what remains unclear is the mechanism by which cutting occurs in concert at the catalytic centers. Previous studies indicate the primacy of intimate DNA contacts made by the specifically bound enzyme in coordinating the two synchronized cuts. We propose that collective electronic behavior in the DNA helix generates coherent oscillations, quantized through boundary conditions imposed by the endonuclease, that provide the energy required to break two phosphodiester bonds. Such quanta may be preserved in the presence of thermal noise and electromagnetic interference through decoherence shielding, the specific complex's exclusion of water and ions surrounding the helix. Clamping energy imparted by the enzyme decoherence shield is comparable with zero-point modes of the dipole-dipole oscillations in the DNA recognition sequence. The palindromic mirror symmetry of this sequence should conserve parity during the process. Experimental data corroborate that symmetric bond-breaking ceases when the symmetry of the endonuclease complex is violated, or when environmental parameters are perturbed far from biological optima. Persistent correlation between states in DNA sequence across spatial separations of any length--a characteristic signature of quantum entanglement--may be explained by such a physical mechanism.

  19. Single-cell pulsed-field gel electrophoresis to detect the early stage of DNA fragmentation in human sperm nuclei.

    PubMed

    Kaneko, Satoru; Yoshida, Joji; Ishikawa, Hiromichi; Takamatsu, Kiyoshi

    2012-01-01

    Single-cell pulsed-field gel electrophoresis (SCPFGE) with dual electrode pairs was developed to detect the early stage of DNA fragmentation in human sperm. The motile sperm were purified by the commonly used density-gradient centrifugation technique and subsequent swim-up. The sperm were embedded in a thin film of agarose containing bovine trypsin (20 µg/mL) and were then lysed. Prior to SCPFGE, proteolysis of DNA-binding components, such as protamine and the nuclear matrix was essential to separate the long chain fibers from the fibrous and granular fragments derived from a single nucleus. The overall electrophoretic profiles elucidated the course of DNA fragmentation. A few large fibrous fragments were observed at the beginning of the process, however, as the fragmentation advanced, the long chain fibers decreased and shortened, and, conversely, the granular fragments increased until finally almost all the DNA was shredded. Although the ejaculate contained sperm with heterogeneous stages, the purified motile sperm exhibited several dozens of uniformly elongated fibers arising from the tangled DNA at the origin, whereas a part of these fibers gave rise to fibrous fragments beyond the tip of the elongated fibers, and their numbers and sizes varied among the sperm. Conventional intra-cytoplasmic sperm injection (ICSI) usually depends on intra-operative light microscopic observation to select a sperm for injection. The present results revealed that sperm motility could not give full assurance of DNA integrity. SCPFGE is likely to serve an important role in the preoperative differential diagnosis to determine the competence of the sperm population provided for injection. PMID:22848752

  20. 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.

  1. Binding and cleavage of DNA with the restriction enzyme EcoR1 using time-resolved second harmonic generation

    PubMed Central

    Doughty, Benjamin; Kazer, Samuel W.; Eisenthal, Kenneth B.

    2011-01-01

    The binding of EcoR1 to a 90-bp DNA duplex attached to colloidal microparticles and the subsequent cleavage by the enzyme was observed in real time and label-free with time-resolved second harmonic (SH) spectroscopy. This method provides a unique way to investigate biomolecular interactions based on its sensitivity to changes in structure and electrical charge on formation of a complex and subsequent dynamics. The binding of EcoR1 to the recognition sequence in DNA appears as a rapid increase in the SH signal, which is attributed to the enzyme-induced change in the DNA conformation, going from a rod-like to a bent shape. In the presence of the cofactor Mg2+, the subsequent decay in the SH signal was monitored in real time as the following processes occurred: cleavage of DNA, dissociation of the enzyme from the DNA, and diffusion of the 74-bp fragment into the bulk solution leaving the 16-bp fragment attached to the microparticle. The observed decay was dependent on the concentration of Mg2+, which functions as a cofactor and as an electrolyte. With SH spectroscopy the rehybridization dynamics between the rehybridized microparticle bound and free cleaved DNA fragments was observed in real time and label-free following the cleavage of DNA. Collectively, the experiments reported here establish SH spectroscopy as a powerful method to investigate equilibrium and time-dependent biological processes in a noninvasive and label-free way. PMID:22114185

  2. Restriction fragment length polymorphism analysis of major histocompatibility complex genes in the non-obese diabetic mouse strain and its non-diabetic sister strains

    Microsoft Academic Search

    Y. Fujishima; Y. Koide; T. Kaidoh; M. Nishimura; T. O. Yoshida

    1989-01-01

    Summary  It has been suggested that one of the recessive genes controlling diabetes in non-obese diabetic mice is linked to the major histocompatibility complex. We, therefore, performed restriction fragment length polymorphism studies of major histocompatibility complex genes (class I, II, and III) in non-obese diabetic mice in comparison with those of their non-diabetic sister strains, non-obese non-diabetic, cataract, and ILI mice

  3. Distribution Study of Chlamydia trachomatis Serovars among High-Risk Women in China Performed Using PCR-Restriction Fragment Length Polymorphism Genotyping

    Microsoft Academic Search

    Xing Gao; Xiang-Sheng Chen; Yue-Ping Yin; Ming-Ying Zhong; Mei-Qin Shi; Wan-Hui Wei; Qiang Chen; Rosanna W. Peeling; David Mabey

    2007-01-01

    This was one of the first epidemiological studies in China focused on genital Chlamydia trachomatis serotype distribution in high-risk female populations using omp1 gene-based restriction fragment length polymorphism analysis. One thousand seven hundred seventy cervical swab samples from women attending sexually trans- mitted disease clinics and female sex workers in six cities in China (Shenzhen and Guangzhou in southern China,

  4. Comparison of Large Restriction Fragments of Mycobacterium avium Isolates Recovered from AIDS and Non-AIDS Patients with Those of Isolates from Potable Water

    Microsoft Academic Search

    T. ARONSON; A. HOLTZMAN; N. GLOVER; M. BOIAN; S. FROMAN; O. G. W. BERLIN; H. HILL; G. STELMA

    1999-01-01

    We examined potable water in Los Angeles, California, as a possible source of infection in AIDS and non-AIDS patients. Nontuberculous mycobacteria were recovered from 12 (92%) of 13 reservoirs, 45 (82%) of 55 homes, 31 (100%) of 31 commercial buildings, and 15 (100%) of 15 hospitals. Large-restriction-fragment (LRF) pattern analyses were done with AseI. The LRF patterns of Mycobacterium avium

  5. Genetic differentiation of the nucleocapsid protein of Korean isolates of porcine epidemic diarrhoea virus by RT-PCR based restriction fragment length polymorphism analysis

    Microsoft Academic Search

    Changhee Lee; Choi-Kyu Park; Young S. Lyoo; Du Sik Lee

    2008-01-01

    A reverse transcriptase polymerase chain reaction (RT-PCR) based restriction fragment length polymorphism (RFLP) analysis based on the nucleocapsid (N) gene was developed to differentiate between field isolates of porcine epidemic diarrhoea virus (PEDV) and a vaccine strain, J-vac. Thirteen field isolates of PEDV from Korea were distinguishable from the vaccine strain and the prototype PEDV strain CV777 by RFLP using

  6. Rapid Detection of K-ras Mutations in Bile by Peptide Nucleic Acid-Mediated PCR Clamping and Melting Curve Analysis: Comparison with Restriction Fragment Length Polymorphism Analysis

    Microsoft Academic Search

    Chiung-Yu Chen; Shu-Chu Shiesh; Sheu-Jen Wu

    Background: Current methods for detection of K-ras gene mutations are time-consuming. We aimed to de- velop a one-step PCR technique using fluorescent hy- bridization probes and competing peptide nucleic acid oligomers to detect K-ras mutations in bile and to compare the efficacy with restriction fragment length polymorphism (RFLP) analysis. Methods: Bile samples were obtained from 116 patients with biliary obstruction,

  7. Advances in the use of terminal restriction fragment length polymorphism (T-RFLP) analysis of 16S rRNA genes to characterize microbial communities

    Microsoft Academic Search

    Ursel M. E. Schütte; Zaid Abdo; Stephen J. Bent; Conrad Shyu; Christopher J. Williams; Jacob D. Pierson; Larry J. Forney

    2008-01-01

    Terminal restriction fragment length polymorphism (T-RFLP) analysis is a popular high-throughput fingerprinting technique\\u000a used to monitor changes in the structure and composition of microbial communities. This approach is widely used because it\\u000a offers a compromise between the information gained and labor intensity. In this review, we discuss the progress made in T-RFLP\\u000a analysis of 16S rRNA genes and functional genes

  8. Use of 16S rRNA Gene Terminal Restriction Fragment Analysis To Assess the Impact of Solids Retention Time on the Bacterial Diversity of Activated Sludge

    Microsoft Academic Search

    Pascal E. Saikaly; Peter G. Stroot; Daniel B. Oerther

    2005-01-01

    Terminal restriction fragment length polymorphism (T-RFLP) analysis of 16S rRNA genes was used to investigate the reproducibility and stability in the bacterial community structure of laboratory-scale sequenc- ing batch bioreactors (SBR) and to assess the impact of solids retention time (SRT) on bacterial diversity. Two experiments were performed. In each experiment two sets of replicate SBRs were operated for a

  9. Characterization of Microbial Communities Found in the Human Vagina by Analysis of Terminal Restriction Fragment Length Polymorphisms of 16S rRNA Genes

    Microsoft Academic Search

    Marco J. L. Coolen; Eduard Post; Catherine C. Davis; Larry J. Forney

    2005-01-01

    To define and monitor the structure of microbial communities found in the human vagina, a cultivation- independent approach based on analyses of terminal restriction fragment length polymorphisms (T-RFLP) of 16S rRNA genes was developed and validated. Sixteen bacterial strains commonly found in the human vagina were used to construct model communities that were subsequently used to develop efficient means for

  10. Optimization of Terminal-Restriction Fragment Length Polymorphism Analysis for Complex Marine Bacterioplankton Communities and Comparison with Denaturing Gradient Gel Electrophoresis

    Microsoft Academic Search

    MARKUS M. MOESENEDER; JESUS M. ARRIETA; GERARD MUYZER; CHRISTIAN WINTER; GERHARD J. HERNDL

    1999-01-01

    The potential of terminal-restriction fragment length polymorphism (T-RFLP) and the detection of opera- tional taxonomic units (OTUs) by capillary electrophoresis (CE) to characterize marine bacterioplankton communities was compared with that of denaturing gradient gel electrophoresis (DGGE). A protocol has been developed to optimize the separation and detection of OTUs between 20 and 1,632 bp by using CE and laser-induced fluorescence

  11. Identification of Clinical Isolates of Actinomyces Species by Amplified 16S Ribosomal DNA Restriction Analysis

    PubMed Central

    Hall, Val; Talbot, P. R.; Stubbs, S. L.; Duerden, B. I.

    2001-01-01

    Amplified 16S ribosomal DNA (rDNA) restriction analysis (ARDRA), using enzymes HaeIII and HpaII, was applied to 176 fresh and 299 stored clinical isolates of putative Actinomyces spp. referred to the Anaerobe Reference Unit of the Public Health Laboratory Service for confirmation of identity. Results were compared with ARDRA results obtained previously for reference strains and with conventional phenotypic reactions. Identities of some strains were confirmed by analysis of partial 16S rDNA sequences. Of the 475 isolates, 331 (70%) were clearly assigned to recognized Actinomyces species, including 94 isolates assigned to six recently described species. A further 52 isolates in 12 ARDRA profiles were designated as apparently resembling recognized species, and 44 isolates, in 18 novel profiles, were confirmed as members of genera other than Actinomyces. The identities of 48 isolates in nine profiles remain uncertain, and they may represent novel species of Actinomyces. For the majority of species, phenotypic results, published reactions for the species, and ARDRA profiles concurred. However, of 113 stored isolates originally identified as A. meyeri or resembling A. meyeri by phenotypic tests, only 21 were confirmed as A. meyeri by ARDRA; 63 were reassigned as A. turicensis, 7 as other recognized species, and 22 as unidentified actinomycetes. Analyses of incidence and clinical associations of Actinomyces spp. add to the currently sparse knowledge of some recently described species. PMID:11574572

  12. Detection of shifts in microbial community structure and diversity in soil caused by copper contamination using amplified ribosomal DNA restriction analysis

    Microsoft Academic Search

    Eric Smit; Paula Leeflang; Karel Wernars

    1997-01-01

    Amplified ribosomal DNA restriction analysis (ARDRA) was used for assessing the effect of copper contamination on the microbial community in soil. ARDRA is a DNA fingerprinting technique based on PCR amplification of 16S ribosomal DNA using primers for conserved regions, followed by restriction enzyme digestions and agarose gel electrophoresis. This results in distinguishable fingerprints for different bacterial species. Microbial community

  13. Characterization of a Brucella Species 25Kilobase DNA Fragment Deleted from Brucella abortus Reveals a Large Gene Cluster Related to the Synthesis of a Polysaccharide

    Microsoft Academic Search

    NIEVES VIZCAINO; AXEL CLOECKAERT; MICHEL S. ZYGMUNT; LUIS FERNANDEZ-LAGO

    2001-01-01

    In the present study we completed the nucleotide sequence of a Brucella melitensis 16M DNA fragment deleted from B. abortus that accounts for 25,064 bp and show that the other Brucella spp. contain the entire 25-kb DNA fragment. Two short direct repeats of four nucleotides, detected in the B. melitensis 16M DNA flanking both sides of the fragment deleted from

  14. TbPIF5 Is a Trypanosoma brucei Mitochondrial DNA Helicase Involved in Processing of Minicircle Okazaki Fragments

    PubMed Central

    Liu, Beiyu; Wang, Jianyang; Yildirir, Gokben; Englund, Paul T.

    2009-01-01

    Trypanosoma brucei's mitochondrial genome, kinetoplast DNA (kDNA), is a giant network of catenated DNA rings. The network consists of a few thousand 1 kb minicircles and several dozen 23 kb maxicircles. Here we report that TbPIF5, one of T. brucei's six mitochondrial proteins related to Saccharomyces cerevisiae mitochondrial DNA helicase ScPIF1, is involved in minicircle lagging strand synthesis. Like its yeast homolog, TbPIF5 is a 5? to 3? DNA helicase. Together with other enzymes thought to be involved in Okazaki fragment processing, TbPIF5 localizes in vivo to the antipodal sites flanking the kDNA. Minicircles in wild type cells replicate unidirectionally as theta-structures and are unusual in that Okazaki fragments are not joined until after the progeny minicircles have segregated. We now report that overexpression of TbPIF5 causes premature removal of RNA primers and joining of Okazaki fragments on theta structures. Further elongation of the lagging strand is blocked, but the leading strand is completed and the minicircle progeny, one with a truncated H strand (ranging from 0.1 to 1 kb), are segregated. The minicircles with a truncated H strand electrophorese on an agarose gel as a smear. This replication defect is associated with kinetoplast shrinkage and eventual slowing of cell growth. We propose that TbPIF5 unwinds RNA primers after lagging strand synthesis, thus facilitating processing of Okazaki fragments. PMID:19779567

  15. Construction of a YAC library from a Beta vulgaris fragment addition and isolation of a major satellite DNA cluster linked to the beet cyst nematode resistance locus Hs1 (pat-1.).

    PubMed

    Klein-Lankhorst, R M; Salentijn, E M; Dirkse, W G; Arens-de Reuver, M; Stiekema, W J

    1994-10-01

    A YAC library was constructed from the Beta vulgaris fragment addition AN5-203b. This monosomic fragment addition harbors an approximate 12-Mbp fragment of B.patellaris chromosome 1 accomodating the Hs1 (pat-1) conferring resistance to the beet cyst nematode (Heterodera schachtii). The YAC library consists of 20,000 YAC clones having an average size of 140 kb. Screening with organelle-specific probes showed that 12% of the clones contain chloroplast DNA while only 0.2% of the clones hybridizes with a mitochondrial specific probe. On the basis of a sugar beet haploid genome size of 750 Mbp this library represents 3.3 haploid genome equivalents. The addition fragment present in AN5-203b harbors a major satellite DNA cluster that is tightly linked to the Hs1 (pat-1) locus. The cluster is located on a single 250-kb EcoRI restriction fragment and consists of an estimated 700-800 copies of a 159-bp core sequence, most of which are arranged in tandem. Using this core sequence as a probe, we were able to isolate 1 YAC clone from the library that contains the entire 250-kb satellite DNA cluster. PMID:24177891

  16. 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. [Ion Reaction Laboratory, Department of Nuclear Medicine and Radiobiology, Faculty of Medicine and Health Sciences, University of Sherbrooke, Sherbrooke, Quebec J1H 5N4 (Canada)

    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.

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

    PubMed

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

    2007-08-01

    We have developed a novel apparatus that allows us to irradiate nonvolatile organic films of high mass (1-100 microg range) spread out over a large surface area (42 cm(2)) with low energy (kT-100 eV) heavy ions and to quantitatively analyze the film substance via standard biochemical techniques afterwards. Here we discuss the details of the apparatus and method and show that it allows us to measure substantial damage to double stranded DNA molecules (plasmids) and its fundamental subunits induced by heavy ions with unprecedented low energies, i.e., 2.5 eV/amu; these energies correspond to track end energies of stopping ions or secondary ions created along primary ion tracks. We find that hyperthermal Ar(+) ions interacting with plasmid DNA will lead to the formation of single and double strand breaks, as well as fragmentation of nucleosides, which also involve chemical modifications and site specific rupture along the N1-C1 glycosidic bond, resulting in base release. In cells, such localized clustered damage will enhance the severity of DNA strand lesions, thus making them harder to repair. PMID:17764359

  18. The effects of age on DNA fragmentation, chromatin packaging and conventional semen parameters in spermatozoa of oligoasthenoteratozoospermic patients

    Microsoft Academic Search

    Konstantina Plastira; Pavlos Msaouel; Roxani Angelopoulou; Kyriaki Zanioti; Aris Plastiras; Alexios Pothos; Stamatis Bolaris; Nikolaos Paparisteidis; Dimitris Mantas

    2007-01-01

    Purpose  To investigate the effects of male ageing on DNA fragmentation and chromatin packaging in the spermatozoa of oligoasthenoteratozoospermic\\u000a (OAT) patients.\\u000a \\u000a \\u000a \\u000a Methods  Sixty-one OAT patients and 49 men with proven fertility (controls) were included in the present study. DNA fragmentation was\\u000a detected by terminal deoxynucleotidyl transferase-mediated dUTP-nick end labelling (TUNEL) assay, while chromatin packaging\\u000a was assessed by chromomycin A3 (CMA3) staining.\\u000a \\u000a \\u000a \\u000a Results  In

  19. The effects of male aging on semen quality, sperm DNA fragmentation and chromosomal abnormalities in an infertile population

    Microsoft Academic Search

    Sonia Brahem; Meriem Mehdi; Hatem Elghezal; Ali Saad

    2011-01-01

    Purpose  To investigate the effects of male aging on semen quality, DNA fragmentation and chromosomal abnormalities in the spermatozoa\\u000a of infertile patients and fertile men.\\u000a \\u000a \\u000a \\u000a \\u000a Methods  Semen samples of 140 infertile patients (24–76 years) and 50 men with proven fertility (25–65 years) were analyzed according\\u000a to WHO guidelines. DNA fragmentation was detected by TUNEL assay, while aneuploidy was assessed by FISH.\\u000a \\u000a \\u000a \\u000a \\u000a Results  In the patient

  20. Analysis of the Campylobacter jejuni Genome by SMRT DNA Sequencing Identifies Restriction-Modification Motifs

    PubMed Central

    O’Loughlin, Jason L.; Eucker, Tyson P.; Chavez, Juan D.; Samuelson, Derrick R.; Neal-McKinney, Jason; Gourley, Christopher R.; Bruce, James E.; Konkel, Michael E.

    2015-01-01

    Campylobacter jejuni is a leading bacterial cause of human gastroenteritis. The goal of this study was to analyze the C. jejuni F38011 strain, recovered from an individual with severe enteritis, at a genomic and proteomic level to gain insight into microbial processes. The C. jejuni F38011 genome is comprised of 1,691,939 bp, with a mol.% (G+C) content of 30.5%. PacBio sequencing coupled with REBASE analysis was used to predict C. jejuni F38011 genomic sites and enzymes that may be involved in DNA restriction-modification. A total of five putative methylation motifs were identified as well as the C. jejuni enzymes that could be responsible for the modifications. Peptides corresponding to the deduced amino acid sequence of the C. jejuni enzymes were identified using proteomics. This work sets the stage for studies to dissect the precise functions of the C. jejuni putative restriction-modification enzymes. Taken together, the data generated in this study contributes to our knowledge of the genomic content, methylation profile, and encoding capacity of C. jejuni. PMID:25695747

  1. Phylogenomics of Phrynosomatid Lizards: Conflicting Signals from Sequence Capture versus Restriction Site Associated DNA Sequencing.

    PubMed

    Leaché, Adam D; Chavez, Andreas S; Jones, Leonard N; Grummer, Jared A; Gottscho, Andrew D; Linkem, Charles W

    2015-01-01

    Sequence capture and restriction site associated DNA sequencing (RADseq) are popular methods for obtaining large numbers of loci for phylogenetic analysis. These methods are typically used to collect data at different evolutionary timescales; sequence capture is primarily used for obtaining conserved loci, whereas RADseq is designed for discovering single nucleotide polymorphisms (SNPs) suitable for population genetic or phylogeographic analyses. Phylogenetic questions that span both "recent" and "deep" timescales could benefit from either type of data, but studies that directly compare the two approaches are lacking. We compared phylogenies estimated from sequence capture and double digest RADseq (ddRADseq) data for North American phrynosomatid lizards, a species-rich and diverse group containing nine genera that began diversifying approximately 55 Ma. Sequence capture resulted in 584 loci that provided a consistent and strong phylogeny using concatenation and species tree inference. However, the phylogeny estimated from the ddRADseq data was sensitive to the bioinformatics steps used for determining homology, detecting paralogs, and filtering missing data. The topological conflicts among the SNP trees were not restricted to any particular timescale, but instead were associated with short internal branches. Species tree analysis of the largest SNP assembly, which also included the most missing data, supported a topology that matched the sequence capture tree. This preferred phylogeny provides strong support for the paraphyly of the earless lizard genera Holbrookia and Cophosaurus, suggesting that the earless morphology either evolved twice or evolved once and was subsequently lost in Callisaurus. PMID:25663487

  2. Increased circulating cell-free DNA levels and mtDNA fragments in interventional cardiologists occupationally exposed to low levels of ionizing radiation.

    PubMed

    Borghini, Andrea; Mercuri, Antonella; Turchi, Stefano; Chiesa, Maria Rosa; Piccaluga, Emanuela; Andreassi, Maria Grazia

    2015-04-01

    Circulating cell-free DNA (ccf-DNA) and mtDNA (ccf-mtDNA) have often been used as indicators of cell death and tissue damage in acute and chronic disorders, but little is known about changes in ccf-DNA and ccf-mtDNA concentrations following radiation exposure. The aim of the study was to investigate the impact of chronic low-dose radiation exposure on serum ccf-DNA levels and ccf-mtDNA fragments (mtDNA-79 and mtDNA-230) of interventional cardiologists working in high-volume cardiac catheterization laboratory to assess their possible role as useful radiation biomarkers. We enrolled 50 interventional cardiologists (26 males; age?=?48.4?±?10 years) and 50 age- and gender-matched unexposed controls (27 males; age?=?47.6?±?8.3 years). Quant-iT™ dsDNA High-Sensitivity assay was used to measure circulating ccf-DNA isolated from serum samples. Quantitative analysis of mtDNA fragments was performed by real-time PCR. No significant relationships were found between ccf-DNA and ccf-mtDNA, and age, gender, smoking, or other clinical parameters. Ccf-DNA levels (44.2?±?31.1 vs. 30.6?±?19.2 ng/ml, P?=?0.013), ccf-mtDNA-79 (2.6?±?2.1 vs. 1.1?±?0.8, P < 0.01), and ccf-mtDNA-230 copies (2.0?±?1.8 vs. 1.04?±?0.9, P?=?0.02) were significantly higher in interventional cardiologists compared with the non-exposed group. In a subset (n?=?15) of interventional cardiologists with a reliable reconstruction of cumulative professional exposure (59.7?±?48.4 mSv; range: 1.4-182 mS), ccf-DNA (53.2?±?41.3 vs. 36.4?±?22.9 and 32.2?±?20.5, P?=?0.08), mtDNA-79 (2.4?±?2.1 vs. 2.03?±?1.7 and 1.09?±?0.82, P?=?0.05), and mtDNA-230 (2.0?±?2.2 vs. 1.5?±?1.4 and 1.04?±?0.9, P?=?0.09) tended to be significantly increased in high-exposure subjects compared with both low-exposure interventional cardiologists and controls. Our results provide evidence for a possible role of circulating DNA as a relevant biomarker of cellular damage induced by exposure to chronic low-dose radiation. Environ. Mol. Mutagen. 56:293-300, 2015. © 2014 Wiley Periodicals, Inc. PMID:25327629

  3. 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.

  4. Targeting individual subunits of the FokI restriction endonuclease to specific DNA strands

    PubMed Central

    Sanders, Kelly L.; Catto, Lucy E.; Bellamy, Stuart R. W.; Halford, Stephen E.

    2009-01-01

    Many restriction endonucleases are dimers that act symmetrically at palindromic DNA sequences, with each active site cutting one strand. In contrast, FokI acts asymmetrically at a non-palindromic sequence, cutting ‘top’ and ‘bottom’ strands 9 and 13 nucleotides downstream of the site. FokI is a monomeric protein with one active site and a single monomer covers the entire recognition sequence. To cut both strands, the monomer at the site recruits a second monomer from solution, but it is not yet known which DNA strand is cut by the monomer bound to the site and which by the recruited monomer. In this work, mutants of FokI were used to show that the monomer bound to the site made the distal cut in the bottom strand, whilst the recruited monomer made in parallel the proximal cut in the top strand. Procedures were also established to direct FokI activity, either preferentially to the bottom strand or exclusively to the top strand. The latter extends the range of enzymes for nicking specified strands at specific sequences, and may facilitate further applications of FokI in gene targeting. PMID:19223323

  5. Mechanisms of bypass by replicative DNA polymerases through the Escherichia coli restriction endonuclease site NarI (GGCGCC)

    Microsoft Academic Search

    Joshua P Gill

    2005-01-01

    N-acetyl-2-amino fluorene (AAF) and 2-aminofluorene (AF) are chemical carcinogens that react with guanines at the C8 position in DNA to form a structure that interferes with DNA replication. In bacteria, the NarI restriction enzyme recognition sequence (G 1G2CG3CC) is a very strong mutational hotspot when an AAF adduct is positioned at G3 of this sequence, causing predominantly a -2 frameshift

  6. Mitochondrial DNA from a spider mite: isolation, restriction map and partial sequence of the cytochrome oxidase subunit I gene

    Microsoft Academic Search

    Didier Fournier; Jean Marc Bride; Maria Navajas

    1994-01-01

    Mitochondrial (mt) DNA of the phytophagous miteTetranychus urticae was purified and a restriction map was constructed. The 12.5 kb long genome is the shortest animal mtDNA known. A 564 bp clone comprising part of the gene for cytochrome oxidase subunit I was sequenced. As has been found in insects, the mitochondrial sequences of mites are extremely A+T rich (75% on

  7. 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.

  8. A DNA Metabarcoding Study of a Primate Dietary Diversity and Plasticity across Its Entire Fragmented Range

    PubMed Central

    Quéméré, Erwan; Hibert, Fabrice; Miquel, Christian; Lhuillier, Emeline; Rasolondraibe, Emmanuel; Champeau, Julie; Rabarivola, Clément; Nusbaumer, Louis; Chatelain, Cyrille; Gautier, Laurent; Ranirison, Patrick; Crouau-Roy, Brigitte; Taberlet, Pierre; Chikhi, Lounès

    2013-01-01

    In tropical regions, most primary ecosystems have been replaced by mosaic landscapes in which species must cope with a large shift in the distribution of their habitat and associated food resources. Primates are particularly vulnerable to habitat modifications. Most species persist in small fragments surrounded by complex human-mediated matrices whose structure and connectivity may strongly influence their dispersal and feeding behavior. Behavioral plasticity appears to be a crucial parameter governing the ability of organisms to exploit the resources offered by new matrix habitats and thus to persist in fragmented habitats. In this study, we were interested in the dietary plasticity of the golden-crowned sifaka (Propithecus tattersalli), an endangered species of lemur, found only in the Daraina region in north-eastern Madagascar. We used a DNA-based approach combining the barcoding concept and Illumina next-generation sequencing to (i) describe the species diet across its entire range and (ii) evaluate the influence of landscape heterogeneity on diet diversity and composition. Faeces from 96 individuals were sampled across the entire species range and their contents were analyzed using the trnL metabarcoding approach. In parallel, we built a large DNA reference database based on a checklist of the plant species of the Daraina region. Our results suggest that golden-crowned sifakas exhibit remarkable dietary diversity with at least 130 plant species belonging to 80 genera and 49 different families. We highlighted an influence of both habitat type and openness on diet composition suggesting a high flexibility of foraging strategies. Moreover, we observed the presence of numerous cultivated and naturalized plants in the faeces of groups living in forest edge areas. Overall, our findings support our initial expectation that P. tattersalli is able to cope with the current level of alteration of the landscape and confirm our previous results on the distribution and the dispersal ability of this species. PMID:23527060

  9. Comparative analysis of genetic variability among Borrelia burgdorferi isolates from Europe and the United States by restriction enzyme analysis, gene restriction fragment length polymorphism, and pulsed-field gel electrophoresis.

    PubMed Central

    Zingg, B C; Anderson, J F; Johnson, R C; LeFebvre, R B

    1993-01-01

    The genomes of 62 North American and European Borrelia burgdorferi isolates were examined by restriction endonuclease analysis (REA), gene probe restriction fragment length polymorphism, and pulsed-field gel electrophoresis (PFGE). Hybridization of restriction fragments with the immunologically relevant 83-kDa antigen gene revealed polymorphisms and divided the isolates into three major groups. Group I included all but two of the American isolates and some of the European isolates. One of two Californian isolates (DN 127) and one Ixodes dammini isolate from New York (strain 25015), previously described as atypical, were distinct from the isolates in the three groups. Plasmid profile analysis and REA, the method with the highest level of discrimination, revealed extensive heterogeneity among isolates of the same major group. Our study demonstrates the usefulness of the 83-kDa antigen gene probe for dividing the isolates into major genogroups, whereas REA and plasmid profile analysis allow for a distinction of individual strains within these groups. Images PMID:7905881

  10. Assessment of four DNA fragments (COI, 16S rDNA, ITS2, 12S rDNA) for species identification of the Ixodida (Acari: Ixodida)

    PubMed Central

    2014-01-01

    Background The 5’ region of cytochrome oxidase I (COI) is the standard marker for DNA barcoding. However, COI has proved to be of limited use in identifying some species, and for some taxa, the coding sequence is not efficiently amplified by PCR. These deficiencies lead to uncertainty as to whether COI is the most suitable barcoding fragment for species identification of ticks. Methods In this study, we directly compared the relative effectiveness of COI, 16S ribosomal DNA (rDNA), nuclear ribosomal internal transcribed spacer 2 (ITS2) and 12S rDNA for tick species identification. A total of 307 sequences from 84 specimens representing eight tick species were acquired by PCR. Besides the 1,834 published sequences of 189 tick species from GenBank and the Barcode of Life Database, 430 unpublished sequences representing 59 tick species were also successfully screened by Bayesian analyses. Thereafter, the performance of the four DNA markers to identify tick species was evaluated by identification success rates given by these markers using nearest neighbour (NN), BLASTn, liberal tree-based or liberal tree-based (+threshold) methods. Results Genetic divergence analyses showed that the intra-specific divergence of each marker was much lower than the inter-specific divergence. Our results indicated that the rates of correct sequence identification for all four markers (COI, 16S rDNA, ITS2, 12S rDNA) were very high (> 96%) when using the NN methodology. We also found that COI was not significantly better than the other markers in terms of its rate of correct sequence identification. Overall, BLASTn and NN methods produced higher rates of correct species identification than that produced by the liberal tree-based methods (+threshold or otherwise). Conclusions As the standard DNA barcode, COI should be the first choice for tick species identification, while 16S rDNA, ITS2, and 12S rDNA could be used when COI does not produce reliable results. Besides, NN and BLASTn are efficient methods for species identification of ticks. PMID:24589289

  11. Structure and cleavage activity of the tetrameric MspJI DNA modification-dependent restriction endonuclease

    PubMed Central

    Horton, John R.; Mabuchi, Megumu Yamada; Cohen-Karni, Devora; Zhang, Xing; Griggs, Rose M.; Samaranayake, Mala; Roberts, Richard J.; Zheng, Yu; Cheng, Xiaodong

    2012-01-01

    The MspJI modification-dependent restriction endonuclease recognizes 5-methylcytosine or 5-hydroxymethylcytosine in the context of CNN(G/A) and cleaves both strands at fixed distances (N12/N16) away from the modified cytosine at the 3?-side. We determined the crystal structure of MspJI of Mycobacterium sp. JLS at 2.05-Å resolution. Each protein monomer harbors two domains: an N-terminal DNA-binding domain and a C-terminal endonuclease. The N-terminal domain is structurally similar to that of the eukaryotic SET and RING-associated domain, which is known to bind to a hemi-methylated CpG dinucleotide. Four protein monomers are found in the crystallographic asymmetric unit. Analytical gel-filtration and ultracentrifugation measurements confirm that the protein exists as a tetramer in solution. Two monomers form a back-to-back dimer mediated by their C-terminal endonuclease domains. Two back-to-back dimers interact to generate a tetramer with two double-stranded DNA cleavage modules. Each cleavage module contains two active sites facing each other, enabling double-strand DNA cuts. Biochemical, mutagenesis and structural characterization suggest three different monomers of the tetramer may be involved respectively in binding the modified cytosine, making the first proximal N12 cleavage in the same strand and then the second distal N16 cleavage in the opposite strand. Both cleavage events require binding of at least a second recognition site either in cis or in trans. PMID:22848107

  12. Structure and cleavage activity of the tetrameric MspJI DNA modification-dependent restriction endonuclease.

    PubMed

    Horton, John R; Mabuchi, Megumu Yamada; Cohen-Karni, Devora; Zhang, Xing; Griggs, Rose M; Samaranayake, Mala; Roberts, Richard J; Zheng, Yu; Cheng, Xiaodong

    2012-10-01

    The MspJI modification-dependent restriction endonuclease recognizes 5-methylcytosine or 5-hydroxymethylcytosine in the context of CNN(G/A) and cleaves both strands at fixed distances (N(12)/N(16)) away from the modified cytosine at the 3'-side. We determined the crystal structure of MspJI of Mycobacterium sp. JLS at 2.05-Å resolution. Each protein monomer harbors two domains: an N-terminal DNA-binding domain and a C-terminal endonuclease. The N-terminal domain is structurally similar to that of the eukaryotic SET and RING-associated domain, which is known to bind to a hemi-methylated CpG dinucleotide. Four protein monomers are found in the crystallographic asymmetric unit. Analytical gel-filtration and ultracentrifugation measurements confirm that the protein exists as a tetramer in solution. Two monomers form a back-to-back dimer mediated by their C-terminal endonuclease domains. Two back-to-back dimers interact to generate a tetramer with two double-stranded DNA cleavage modules. Each cleavage module contains two active sites facing each other, enabling double-strand DNA cuts. Biochemical, mutagenesis and structural characterization suggest three different monomers of the tetramer may be involved respectively in binding the modified cytosine, making the first proximal N(12) cleavage in the same strand and then the second distal N(16) cleavage in the opposite strand. Both cleavage events require binding of at least a second recognition site either in cis or in trans. PMID:22848107

  13. 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

  14. Restriction enzyme analysis of American region dengue viruses

    Microsoft Academic Search

    V. Vorndam; R. M. R. Nogueira; D. W. Trent

    1994-01-01

    Summary 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

  15. Cloning and expression of small cDNA fragment encoding strong antiviral peptide from Celosia cristata in Escherichia coli.

    PubMed

    Gholizadeh, A; Kohnehrouz, B Baghban; Santha, I M; Lodha, M L; Kapoor, H C

    2005-09-01

    A small cDNA fragment containing a ribosome-inactivating site was isolated from the leaf cDNA population of Celosia cristata by polymerase chain reaction (PCR). PCR was conducted linearly using a degenerate primer designed from the partially conserved peptide of ribosome-inactivating/antiviral proteins. Sequence analysis showed that it is 150 bp in length. The cDNA fragment was then cloned in a bacterial expression vector and expressed in Escherichia coli as a ~57 kD fused protein, and its presence was further confirmed by Western blot analysis. The recombinant protein was purified by affinity chromatography. The purified product showed strong antiviral activity towards tobacco mosaic virus on host plant leaves, Nicotiana glutinosa, indicating the presence of a putative antiviral determinant in the isolated cDNA product. It is speculated that antiviral site is at, or is separate but very close to, the ribosome-inactivating site. We nominate this short cDNA fragment reported here as a good candidate to investigate further the location of the antiviral determinants. The isolated cDNA sequence was submitted to EMBL databases under accession number of AJ535714. PMID:16266271

  16. 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

  17. Capillary array scanner for time-resolved detection and identification of fluorescently labelled DNA fragments.

    PubMed

    Neumann, M; Herten, D P; Dietrich, A; Wolfrum, J; Sauer, M

    2000-02-25

    The first capillary array scanner for time-resolved fluorescence detection in parallel capillary electrophoresis based on semiconductor technology is described. The system consists essentially of a confocal fluorescence microscope and a x,y-microscope scanning stage. Fluorescence of the labelled probe molecules was excited using a short-pulse diode laser emitting at 640 nm with a repetition rate of 50 MHz. Using a single filter system the fluorescence decays of different labels were detected by an avalanche photodiode in combination with a PC plug-in card for time-correlated single-photon counting (TCSPC). The time-resolved fluorescence signals were analyzed and identified by a maximum likelihood estimator (MLE). The x,y-microscope scanning stage allows for discontinuous, bidirectional scanning of up to 16 capillaries in an array, resulting in longer fluorescence collection times per capillary compared to scanners working in a continuous mode. Synchronization of the alignment and measurement process were developed to allow for data acquisition without overhead. Detection limits in the subzeptomol range for different dye molecules separated in parallel capillaries have been achieved. In addition, we report on parallel time-resolved detection and separation of more than 400 bases of single base extension DNA fragments in capillary array electrophoresis. Using only semiconductor technology the presented technique represents a low-cost alternative for high throughput DNA sequencing in parallel capillaries. PMID:10735310

  18. The metabolic enhancer piracetam attenuates mitochondrion-specific endonuclease G translocation and oxidative DNA fragmentation.

    PubMed

    Gupta, Sonam; Verma, Dinesh Kumar; Biswas, Joyshree; Rama Raju, K Siva; Joshi, Neeraj; Wahajuddin; Singh, Sarika

    2014-08-01

    This study was performed to investigate the involvement of mitochondrion-specific endonuclease G in piracetam (P)-induced protective mechanisms. Studies have shown the antiapoptotic effects of piracetam but the mechanism of action of piracetam is still an enigma. To assess the involvement of endonuclease G in piracetam-induced protective effects, astrocyte glial cells were treated with lipopolysaccharide (LPS) and piracetam. LPS treatment caused significantly decreased viability, mitochondrial activity, oxidative stress, chromatin condensation, and DNA fragmentation, which were attenuated by piracetam cotreatment. Cotreatment of astrocytes with piracetam showed its significantly time-dependent absorption as observed with high-performance liquid chromatography. Astrocytes treated with piracetam alone showed enhanced mitochondrial membrane potential (MMP) in comparison to control astrocytes. However, in LPS-treated cells no significant alteration in MMP was observed in comparison to control cells. Protein and mRNA levels of the terminal executor of the caspase-mediated pathway, caspase-3, were not altered significantly in LPS or LPS + piracetam-treated astrocytes, whereas endonuclease G was significantly translocated to the nucleus in LPS-treated astrocytes. Piracetam cotreatment attenuated the LPS-induced endonuclease G translocation. In conclusion this study indicates that LPS treatment of astrocytes caused decreased viability, oxidative stress, mitochondrial dysfunction, chromatin condensation, DNA damage, and translocation of endonuclease G to the nucleus, which was inhibited by piracetam cotreatment, confirming that the mitochondrion-specific endonuclease G is one of the factors involved in piracetam-induced protective mechanisms. PMID:24882422

  19. Amplified fragment length polymorphism versus random amplified polymorphic DNA markers: clonal diversity in Saxifraga cernua.

    PubMed

    Kjølner, S; Såstad, S M; Taberlet, P; Brochmann, C

    2004-01-01

    Random amplified polymorphic DNA (RAPD) markers are sensitive to changes in reaction conditions and may express polymorphisms of nongenetic origin. Taxa with variable chromosome numbers are particularly challenging cases, as differences in DNA content may also influence marker reproducibility. We addressed these problems by comparing RAPD and amplified fragment length polymorphism (AFLP) analyses of clonal identity and relationships in a chromosomally variable arctic plant, the polyploid Saxifraga cernua, which has been thought to be monoclonal over large geographical distances. Fifty-seven plants from four Greenland populations were analysed using a conservative scoring approach. In total, 26 AFLP and 32 RAPD multilocus phenotypes (putative clones) were identified, of which 21 were identical and each of the remaining five AFLP clones was split into two to three very similar RAPD clones. This minor difference can be explained by sampling error and stochastic variation. The pattern observed in Greenland corroborates our previous results from Svalbard, suggesting that rare sexual events in S. cernua are sufficient to maintain high levels of clonal diversity even at small spatial scales. We conclude that although AFLP analysis is superior in terms of efficiency, RAPDs may still be used as reliable markers in small low-tech laboratories. PMID:14653790

  20. DNA Fragmentation Follows Delayed Neuronal Death in CA1 Neurons Exposed to Transient Global Ischemia in the Rat

    Microsoft Academic Search

    Carol K. Petito; Jorge Torres-Munoz; Brenda Roberts; John-Paul Olarte; Thaddeus S. Nowak; William A. Pulsinelli

    1997-01-01

    Apoptosis is an active, gene-directed process of cell death in which early fragmentation of nuclear DNA precedes morphological changes in the nucleus and, later, in the cytoplasm. In ischemia, biochemical studies have detected oligonucleosomes of apoptosis whereas sequential morphological studies show changes consistent with necrosis rather than apoptosis. To resolve this apparent discrepancy, we subjected rats to 10 minutes of

  1. Does the marine biotoxin okadaic acid cause DNA fragmentation in the blue mussel and the pacific oyster?

    PubMed

    McCarthy, Moira; O'Halloran, John; O'Brien, Nora M; van Pelt, Frank F N A M

    2014-10-01

    Two bivalve species of global economic importance: the blue mussel, Mytilus edulis and the pacific oyster, Crassostrea gigas were exposed in vivo, to the diarrhoetic shellfish toxin okadaic acid (OA), and impacts on DNA fragmentation were measured. Shellfish were exposed using two different regimes, the first was a single (24 h) exposure of 2.5 nM OA (?0.1 ?g/shellfish) and algal feed at the beginning of the trial (T0), after which shellfish were only fed algae. The second was daily exposure of shellfish to two different concentrations of OA mixed with the algal feed over 7 days; 1.2 nM OA (?0.05 ?g OA/shellfish/day) and 50 nM OA (?2 ?g OA/shellfish/day). Haemolymph and hepatopancreas cells were extracted following 1, 3 and 7 days exposure. Cell viability was measured using the trypan blue exclusion assay and remained above 85% for both cell types. DNA fragmentation was examined using the single-cell gel electrophoresis (comet) assay. A significant increase in DNA fragmentation was observed in the two cell types from both species relative to the controls. This increase was greater in the pacific oyster at the higher toxin concentration. However, there was no difference in the proportion of damage measured between the two cell types, and a classic dose response was not observed, increasing toxin concentration did not correspond to increased DNA fragmentation. PMID:25440785

  2. Effect of Sperm DNA Fragmentation on Clinical Outcome of Frozen-Thawed Embryo Transfer and on Blastocyst Formation

    PubMed Central

    Ni, Wuhua; Xiao, Shiquan; Qiu, Xiufang; Jin, Jianyuan; Pan, Chengshuang; Li, Yan; Fei, Qianjin; Yang, Xu; Zhang, Liya; Huang, Xuefeng

    2014-01-01

    During the last decades, many studies have shown the possible influence of sperm DNA fragmentation on assisted reproductive technique outcomes. However, little is known about the impact of sperm DNA fragmentation on the clinical outcome of frozen-thawed embryo transfer (FET) from cycles of conventional in vitro fertilization (IVF) and intra-cytoplasmic sperm injection (ICSI). In the present study, the relationship between sperm DNA fragmentation (SDF) and FET clinical outcomes in IVF and ICSI cycles was analyzed. A total of 1082 FET cycles with cleavage stage embryos (C-FET) (855 from IVF and 227 from ICSI) and 653 frozen-thawed blastocyst transfer cycles (B-FET) (525 from IVF and 128 from ICSI) were included. There was no significant change in clinical pregnancy, biochemical pregnancy and miscarriage rates in the group with a SDF >30% compared with the group with a SDF ?30% in IVF and ICSI cycles with C-FET or B-FET. Also, there was no significant impact on the FET clinic outcome in IVF and ICSI when different values of SDF (such as 10%, 20%, 25%, 35%, and 40%) were taken as proposed threshold levels. However, the blastulation rates were significantly higher in the SDF ?30% group in ICSI cycle. Taken together, our data show that sperm DNA fragmentation measured by Sperm Chromatin Dispersion (SCD) test is not associated with clinical outcome of FET in IVF and ICSI. Nonetheless, SDF is related to the blastocyst formation in ICSI cycles. PMID:24733108

  3. Effect of sperm DNA fragmentation on clinical outcome of frozen-thawed embryo transfer and on blastocyst formation.

    PubMed

    Ni, Wuhua; Xiao, Shiquan; Qiu, Xiufang; Jin, Jianyuan; Pan, Chengshuang; Li, Yan; Fei, Qianjin; Yang, Xu; Zhang, Liya; Huang, Xuefeng

    2014-01-01

    During the last decades, many studies have shown the possible influence of sperm DNA fragmentation on assisted reproductive technique outcomes. However, little is known about the impact of sperm DNA fragmentation on the clinical outcome of frozen-thawed embryo transfer (FET) from cycles of conventional in vitro fertilization (IVF) and intra-cytoplasmic sperm injection (ICSI). In the present study, the relationship between sperm DNA fragmentation (SDF) and FET clinical outcomes in IVF and ICSI cycles was analyzed. A total of 1082 FET cycles with cleavage stage embryos (C-FET) (855 from IVF and 227 from ICSI) and 653 frozen-thawed blastocyst transfer cycles (B-FET) (525 from IVF and 128 from ICSI) were included. There was no significant change in clinical pregnancy, biochemical pregnancy and miscarriage rates in the group with a SDF >30% compared with the group with a SDF ?30% in IVF and ICSI cycles with C-FET or B-FET. Also, there was no significant impact on the FET clinic outcome in IVF and ICSI when different values of SDF (such as 10%, 20%, 25%, 35%, and 40%) were taken as proposed threshold levels. However, the blastulation rates were significantly higher in the SDF ?30% group in ICSI cycle. Taken together, our data show that sperm DNA fragmentation measured by Sperm Chromatin Dispersion (SCD) test is not associated with clinical outcome of FET in IVF and ICSI. Nonetheless, SDF is related to the blastocyst formation in ICSI cycles. PMID:24733108

  4. Use of cloned DNA fragments for event-specific quantification of genetically modified organisms in pure and mixed food products

    Microsoft Academic Search

    Isabel Taverniers; Pieter Windels; Erik Van Bockstaele; Marc De Loose

    2001-01-01

    An event-specific PCR method for detection and quantification of genetically modified Roundup Ready soybean (RRS) is described in this article. The complete DNA sequence at both the right and left integration sites of this genetically modified organism has recently been determined. Based on these sequence data, transformation event-specific primer pairs were developed. These primers amplify a fragment of the unique

  5. Close genetic similarity of Atlantic and Pacific skipjack tuna ( Katsuwonus pelamis ) demonstrated with restriction endonuclease analysis of mitochondrial DNA

    Microsoft Academic Search

    J. E. Graves; S. D. Ferris; A. E. Dizon

    1984-01-01

    Restriction endonuclease analysis of mitochondrial DNA indicated a surprisingly high degree of genetic similarity between skipjack tuna (Katsuwonus pelamis) from the Atlantic and Pacific Oceans. The present results (1983) support the findings of previous morphological and electrophoretic studies. Evidently, since the uplift of the Panamá land bridge about 3.1 million years ago, there has been continued genetic contact between Atlantic

  6. 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

  7. Transcriptional and translational mapping of a 6.6-kilobase-pair DNA fragment containing the junction of the terminal repetition and unique sequence at the left end of the vaccinia virus genome.

    PubMed Central

    Wittek, R; Cooper, J A; Moss, B

    1981-01-01

    The penultimate EcoRI fragments from the left and right ends of the vaccinia virus genomes were cloned in phage lambda. Heteroduplex analysis and comparison of restriction fragments indicated that the inverted terminal repetition extended 780 base pairs (bp) beyond the EcoRI site or about 9,800 bp from each end of the genome. Detailed physical, transcriptional, and translational maps of the 6,600-bp left penultimate EcoRI fragment were prepared so as to extend previous maps of the 9,000-bp terminal EcoRI fragment. Polypeptides with molecular weights of 6,000 (6K polypeptide), 13,000, 19,000, 21,000, and 60,000 were synthesized in a reticulocyte cell-free system programmed with immediate early RNA (made in the presence of cycloheximide) or early RNA (made in the presence of cytosine arabinoside) and selected by hybridization to immobilized recombinant DNA. A 22K polypeptide was detected as a translation product of late RNA that hybridized to this DNA fragment. A variety of biochemical procedures were used to size and map the mRNA's. Of the five messages that hybridized to this 6,600-bp EcoRI fragment, only the one for the 21K polypeptide was encoded within the inverted terminal repetition and hybridized to the rightward-reading DNA strand. (Three additional early polypeptides were encoded within the first 9,000 bp of the inverted terminal repetition.) The remaining early polypeptides were encoded within the unique portion of the penultimate EcoRI fragment and were transcribed from the leftward-reading strand. Additional high-molecular-weight early RNAs of unknown function were also detected; however, there was no evidence indicating that mature mRNA's were spliced. Images PMID:6270347

  8. 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. (Brookhaven National Lab., Upton, NY (USA))

    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.

  9. Cloning and characterization of a DNA region encoding a stress-sensitive restriction system from Corynebacterium glutamicum ATCC 13032 and analysis of its role in intergeneric conjugation with Escherichia coli.

    PubMed Central

    Schäfer, A; Schwarzer, A; Kalinowski, J; Pühler, A

    1994-01-01

    RP4-mediated transfer of mobilizable plasmids in intergeneric conjugation of Escherichia coli donors with Corynebacterium glutamicum ATCC 13032 is severely affected by a restriction system in the recipient that can be inactivated by a variety of exogenous stress factors. In this study a rapid test procedure based on intergeneric conjugal plasmid transfer that permitted the distinction between restriction-negative and restriction-positive C. glutamicum clones was developed. By using this procedure, clones of the restriction-deficient mutant strain C. glutamicum RM3 harboring a plasmid library of the wild-type chromosome were checked for their restriction properties. A complemented clone with a restriction-positive phenotype was isolated and found to contain a plasmid with a 7-kb insertion originating from the wild-type chromosome. This plasmid, termed pRES806, is able to complement the restriction-deficient phenotype of different C. glutamicum mutants. Sequence analysis revealed the presence of two open reading frames (orf1 and orf2) on the complementing DNA fragment. The region comprising orf1 and orf2 displayed a strikingly low G+C content and was present exclusively in C. glutamicum strains. Gene disruption experiments with the wild type proved that orf1 is essential for complementation, but inactivation of orf2 also resulted in a small but significant increase in fertility. These results were confirmed by infection assays with the bacteriophage CL31 from Corynebacterium lilium ATCC 15990. Images PMID:7961503

  10. Terminal restriction fragment patterns (TRFPs), a rapid, PCR-based method for the comparison of complex bacterial communities

    Microsoft Academic Search

    Brian G Clement; Lucia E Kehl; Kristin L DeBord; Christopher L Kitts

    1998-01-01

    Microbial populations in complex environmental samples are difficult to characterize; current techniques are incomplete and time consuming. We investigated a polymerase chain reaction (PCR)-based method for rapidly comparing bacterial communities independent of culture or cloning. Community 16S rRNA genes were amplified and fluorescently labeled by PCR. The labeled products were digested by a restriction enzyme and the labeled, terminal restriction

  11. Large amplification of a 35-kb DNA fragment carrying two penicillin biosynthetic genes in high penicillin producing strains of Penicillium chrysogenum

    Microsoft Academic Search

    Jose L. Barredo; Bruno Díez; Emilio Alvarez; Juan F. Martín

    1989-01-01

    The isopenicillin N synthase (pcbC) and acyl-CoA:6-APA acyltransferase (penDE) genes of Penicillium chrysogenum were located in a 19.5-kb DNA fragment that had been previously cloned in phage vector EMBL3. This 19.5-kb DNA fragment was mapped with several endonucleases, and the (pcbC) and penDE genes were located by hybridization with probes corresponding to internal fragments of each gene. A low penicillin

  12. Caspase 3 inhibition attenuates hydrogen peroxide-induced DNA fragmentation but not cell death in neuronal PC12 cells.

    PubMed

    Jiang, D; Jha, N; Boonplueang, R; Andersen, J K

    2001-03-01

    Exposure of neurons to H(2)O(2) results in both necrosis and apoptosis. Caspases play a pivotal role in apoptosis, but exactly how they are involved in H(2)O(2)-mediated cell death is unknown. We examined H(2)O(2)-induced toxicity in neuronal PC12 cells and the effects of inducible overexpression of the H(2)O(2)-scavenging enzyme catalase on this process. H(2)O(2) caused cell death in a time- and concentration-dependent manner. Cell death induced by H(2)O(2) was found to be mediated in part through an apoptotic pathway as H(2)O(2)-treated cells exhibited cell shrinkage, nuclear condensation and marked DNA fragmentation. H(2)O(2) also triggered activation of caspase 3. Genetic up-regulation of catalase not only significantly reduced cell death but also suppressed caspase 3 activity and DNA fragmentation. While the caspase 3 inhibitor DEVD inhibited both caspase 3 activity and DNA fragmentation induced by H(2)O(2) it did not prevent cell death. Treatment with the general caspase inhibitor ZVAD, however, resulted in complete attenuation of H(2)O(2)-mediated cellular toxicity. These results suggest that DNA fragmentation induced by H(2)O(2) is attributable to caspase 3 activation and that H(2)O(2) may be critical for signaling leading to apoptosis. However, unlike inducibly increased catalase expression and general caspase inhibition both of which protect cells from cytotoxicity, caspase 3 inhibition alone did not improve cell survival suggesting that prevention of DNA fragmentation is insufficient to prevent H(2)O(2)-mediated cell death. PMID:11259492

  13. DNA of a circular minichromosome linearized by restriction enzymes or other reagents is resistant to further cleavage: an influence of chromatin topology on the accessibility of DNA

    PubMed Central

    Kumala, S?awomir; Hadj-Sahraoui, Yasmina; Rzeszowska-Wolny, Joanna; Hancock, Ronald

    2012-01-01

    The accessibility of DNA in chromatin is an essential factor in regulating its activities. We studied the accessibility of the DNA in a ?170?kb circular minichromosome to DNA-cleaving reagents using pulsed-field gel electrophoresis and fibre-fluorescence in situ hybridization on combed DNA molecules. Only one of several potential sites in the minichromosome DNA was accessible to restriction enzymes in permeabilized cells, and in growing cells only a single site at an essentially random position was cut by poisoned topoisomerase II, neocarzinostatin and ?-radiation, which have multiple potential cleavage sites; further sites were then inaccessible in the linearized minichromosomes. Sequential exposure to combinations of these reagents also resulted in cleavage at only a single site. Minichromosome DNA containing single-strand breaks created by a nicking endonuclease to relax any unconstrained superhelicity was also cut at only a single position by a restriction enzyme. Further sites became accessible after ?95% of histones H2A, H2B and H1, and most non-histone proteins were extracted. These observations suggest that a global rearrangement of the three-dimensional packing and interactions of nucleosomes occurs when a circular minichromosome is linearized and results in its DNA becoming inaccessible to probes. PMID:22848103

  14. 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

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

    E-print Network

    Blakey, Cynthia Ann

    1989-01-01

    (Drosophilia melanogaster) (NAOOSHI and GELBART, 1987), tomato (BURR et al. 1986; VALLEJOS et al. 1986; WELLER et al. 1988), lettuce (Lactuca sativa L. ) (LANURY, 1987), and maize (Zea mays L. ) (HELENTJARis et al. 1986a; BURR et al. 1988; HELENTJARIs et al... Committee: Dr. James D. Smith F, seedling populations of the maize inbred crosses B73 x B37R, Mo17 x B37R, and Tx5855 x B37R were examined for linkage of restriction fragment length polymorphisms (RFLPs) to the Rpp9 allele from the B37R parent, which...

  16. RADOM, an Efficient In Vivo Method for Assembling Designed DNA Fragments up to 10 kb Long in Saccharomyces cerevisiae.

    PubMed

    Lin, Qiuhui; Jia, Bin; Mitchell, Leslie A; Luo, Jingchuan; Yang, Kun; Zeller, Karen I; Zhang, Wenqian; Xu, Zhuwei; Stracquadanio, Giovanni; Bader, Joel S; Boeke, Jef D; Yuan, Ying-Jin

    2015-03-20

    We describe rapid assembly of DNA overlapping multifragments (RADOM), an improved assembly method via homologous recombination in Saccharomyces cerevisiae, which combines assembly in yeasto with blue/white screening in Escherichia coli. We show that RADOM can successfully assemble ?3 and ?10 kb DNA fragments that are highly similar to the yeast genome rapidly and accurately. This method was tested in the Build-A-Genome course by undergraduate students, where 125 ?3 kb "minichunks" from the synthetic yeast genome project Sc2.0 were assembled. Here, 122 out of 125 minichunks achieved insertions with correct sizes, and 102 minichunks were sequenced verified. As this method reduces the time-consuming and labor-intensive efforts of yeast assembly by improving the screening efficiency for correct assemblies, it may find routine applications in the construction of DNA fragments, especially in hierarchical assembly projects. PMID:24895839

  17. Polymerase Chain Reaction and Restriction Fragment Length Polymorphism Mediated Detection and Speciation of Candida spp Causing Intraocular Infection

    Microsoft Academic Search

    Narciss Okbravi; Peter Adamson; Rebecca Mant; Melville M. Matheson; Gillian Midgley; Hamish M. A. Towler; Susan Lightman

    RESULTS. The sensitivity of the amplification reaction after two rounds of PCR was 10 fg genomic C. albicans DNA or one copy of the gene. No amplification product was obtained when DNA from C. guilliermondii, Aspergillus fumigatus, Fusarium solani, human leukocytes, or 10 species of bacteria was used as a template. Experiments with spiked normal vitreous demonstrated equal sensitivity as

  18. Identification of shrimp species in raw and processed food products by means of a polymerase chain reaction-restriction fragment length polymorphism method targeted to cytochrome b mitochondrial sequences.

    PubMed

    Pascoal, Ananías; Barros-Velázquez, Jorge; Cepeda, Alberto; Gallardo, José M; Calo-Mata, Pilar

    2008-08-01

    A novel PCR-RFLP method has been developed for the identification of six commercially relevant penaeid shrimp species in raw and processed food products. The method can be completed within 8 h. To implement the method, PCR amplification with the crustF/crustR primers, targeted to the amplification of a ca. 181 bp region of the cytochrome b (cytb) mitochondrial gene in penaeid shrimps, was coupled to restriction analysis with CviJI, DdeI and NlaIV. The method was also applied successfully to the identification of shrimp species in complex processed foods, including this type of shellfish as an added-value food ingredient. The small size of this molecular target facilitates amplification from fresh, frozen, or precooked samples, where DNA fragmentation may be relevant and fragment size critical. We also report the first cytb mitochondrial sequences described to date for the species Farfantepenaeus notialis, Parapenaeus longirostris and Pleoticus muelleri, and these nearly triplicate current knowledge of reference nucleotide sequences in this mitochondrial region for this group of species. The cytb mitochondrial gene may also be considered as a molecular marker for identification and phylogenetic purposes in penaeid shrimp species. PMID:18604873

  19. Association Mapping of Disease Resistance Traits in Rainbow Trout Using Restriction Site Associated DNA Sequencing

    PubMed Central

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

    2014-01-01

    Recent advances in genotyping-by-sequencing have enabled genome-wide association studies in nonmodel species including those in aquaculture programs. As with other aquaculture species, rainbow trout and steelhead (Oncorhynchus mykiss) are susceptible to disease and outbreaks can lead to significant losses. Fish culturists have therefore been pursuing strategies to prevent losses to common pathogens such as Flavobacterium psychrophilum (the etiological agent for bacterial cold water disease [CWD]) and infectious hematopoietic necrosis virus (IHNV) by adjusting feed formulations, vaccine development, and selective breeding. However, discovery of genetic markers linked to disease resistance offers the potential to use marker-assisted selection to increase resistance and reduce outbreaks. For this study we sampled juvenile fish from 40 families from 2-yr classes that either survived or died after controlled exposure to either CWD or IHNV. Restriction site?associated DNA sequencing produced 4661 polymorphic single-nucleotide polymorphism loci after strict filtering. Genotypes from individual survivors and mortalities were then used to test for association between disease resistance and genotype at each locus using the program TASSEL. After we accounted for kinship and stratification of the samples, tests revealed 12 single-nucleotide polymorphism markers that were highly associated with resistance to CWD and 19 markers associated with resistance to IHNV. These markers are candidates for further investigation and are expected to be useful for marker assisted selection in future broodstock selection for various aquaculture programs. PMID:25354781

  20. Molecular mapping of restriction-site associated DNA markers in allotetraploid upland cotton.

    PubMed

    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

  1. Crystallization and preliminary crystallographic analysis of the type IIL restriction enzyme MmeI in complex with DNA

    SciTech Connect

    Callahan, Scott J.; Morgan, Richard D.; Jain, Rinku; Townson, Sharon A.; Wilson, Geoffrey G.; Roberts, Richard J.; Aggarwal, Aneel K. (Sinai); (NEB)

    2012-05-29

    Type IIL restriction enzymes have rejuvenated the search for user-specified DNA binding and cutting. By aligning and contrasting the highly comparable amino-acid sequences yet diverse recognition specificities across the family of enzymes, amino acids involved in DNA binding have been identified and mutated to produce alternative binding specificities. To date, the specificity of MmeI (a type IIL restriction enzyme) has successfully been altered at positions 3, 4 and 6 of the asymmetric TCCRAC (where R is a purine) DNA-recognition sequence. To further understand the structural basis of MmeI DNA-binding specificity, the enzyme has been crystallized in complex with its DNA substrate. The crystal belonged to space group P1, with unit-cell parameters a = 61.73, b = 94.96, c = 161.24 {angstrom}, {alpha} = 72.79, {beta} = 89.12, {gamma} = 71.68{sup o}, and diffracted to 2.6 {angstrom} resolution when exposed to synchrotron radiation. The structure promises to reveal the basis of MmeI DNA-binding specificity and will complement efforts to create enzymes with novel specificities.

  2. DNA Fragmentation and DSB correlation Induced in Human Fibroblasts by Accelerated 56Fe Ions of Differing Energies

    NASA Astrophysics Data System (ADS)

    Antonelli, F.; Belli, M.; Campa, A.; Dini, V.; Esposito, G.; Furusawa, Y.; Simone, G.; Sorrentino, E.; Tabocchini, M. A.

    HZE particles from space radiation raise an important protection concern during long-term astronauts travels Although these particles are less abundant than protons they are more effective in damaging biological systems It is thought that this is due to the frequent production of spatially correlated DNA damaged sites particularly double strand breaks DSB since this correlation can strongly affect the repair capability of the cells In this work we have studied the DNA fragmentation induced in human fibroblasts by accelerated 56 Fe ions of four different energies i e 115 MeV u 414 MeV u 1 GeV u and 5 GeV u and by gamma-rays used as reference radiation DNA fragmentation was studied in various size ranges varying from 1 to 5700 kbp using Pulsed or Constant Field Gel Electrophoresis The DSB yields have been derived from fragmentation in the overall range as well as in the two ranges 1-23 and 23-5700 kbp The overall DSB yield slightly increased with the ion energy maily due to the contribution of the 23-5700 kbp fragments while that of small fragments 1-23 kbp was almost constant Accordingly the relative biological effectiveness RBE for DSB induction increased with energy from about 1 3 at 115 MeV u to about 1 8 at about 5 GeV u i e less than the RBE for chromosome aberration and cell inactivation The degree of spatial correlation of DSB was evaluated through the departure from the randomness of the fragment distribution with a simple theoretical tool that we have recently introduced To this aim a parameter R was used

  3. 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

  4. Diagnostic value of sperm DNA fragmentation and sperm high-magnification for predicting outcome of assisted reproduction treatment

    PubMed Central

    López, Gemma; Lafuente, Rafael; Checa, Miguel A; Carreras, Ramón; Brassesco, Mario

    2013-01-01

    Over the last years, major improvements in the field of male infertility diagnosis have been achieved. The aim of this study was to determine the diagnostic usefulness of sperm DNA integrity and sperm vacuolisation for predicting outcome in infertile couples undergoing in vitro fertilisation (IVF) and intracytoplasmic sperm injection (ICSI) treatments. A cohort study from 152 infertile couples undergoing sperm DNA fragmentation and high-magnification tests prior to an assisted reproduction treatment was designed. We found that the most predictive cutoff for pregnancy was 25.5% of DNA fragmentation with a negative predictive value of 72.7% (P=0.02). For the degree of vacuolisation, the best predictor of pregnancy was 73.5% of vacuolated sperm grades III+IV with a negative predictive value of 39.4% (P=0.09), which was not statistically significant. In conclusion, sperm DNA fragmentation greater than 25.5% could be associated with higher probability of failure IVF treatment. Regarding the results of the sperm analysis at high magnification, they do not allow us to predict whether or not patients will become pregnant. PMID:23912311

  5. Lactobacillus species identification by amplified ribosomal 16S-23S rRNA restriction fragment length polymorphism analysis.

    PubMed

    Sandes, S H C; Alvin, L B; Silva, B C; Zanirati, D F; Jung, L R C; Nicoli, J R; Neumann, E; Nunes, A C

    2014-12-01

    Lactic acid bacteria strains are commonly used for animal and human consumption due to their probiotic properties. One of the major genera used is Lactobacillus, a highly diverse genus comprised of several closely related species. The selection of new strains for probiotic use, especially strains of Lactobacillus, is the focus of several research groups. Accurate identification to species level is fundamental for research on new strains, as well as for safety assessment and quality assurance. The 16S-23S internal transcribed spacer (ITS-1) is a deeply homologous region among prokaryotes that is commonly used for identification to the species level because it is able to acquire and accumulate mutations without compromising general bacterial metabolism. In the present study, 16S-23S ITS regions of 45 Lactobacillus species (48 strains) were amplified and subjected to independent enzymatic digestions, using 12 restriction enzymes that recognise six-base sequences. Twenty-nine species showed unique restriction patterns, and could therefore be precisely identified solely by this assay (64%). This approach proved to be reproducible, allowing us to establish simplified restriction patterns for each evaluated species. The restriction patterns of each species were similar among homologous strains, and to a large extent reflected phylogenetic relationships based on 16S rRNA sequences, demonstrating the promising nature of this region for evolutionary studies. PMID:24902955

  6. 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...

  7. 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...

  8. Restriction Enzyme Digestion: How does it work? Why is it useful?

    NSDL National Science Digital Library

    2012-12-27

    In this activity related to plant biotechnology, learners use restriction enzymes to cut up DNA from a virus called Bacteriophage ?, a process known as restriction digestion. After overnight digestion, the reaction is stopped by addition of a loading buffer. The DNA fragments are separated by electrophoresis, a process that involves application of an electric field to cause the DNA fragments to migrate into an agarose gel. The gel is then stained with a methylene blue stain to visualize the DNA bands and may be photographed. This lab will help learners understand what a DNA restriction enzyme is and how it works, how to use a micropipette, how to separate DNA using electrophoresis, and how to use a restriction digestion map to identify a sample DNA. This lesson guide includes background information, safety precautions and notes, and questions with answers for learners. For safety reasons, adult supervision is recommended. Modifications for younger learners are included in a related PDF (see related resources).

  9. Amplified fragment length polymorphism (AFLP): a review of the procedure and its applications

    Microsoft Academic Search

    M J Blears; S A De Grandis; H Lee; J T Trevors

    1998-01-01

      Amplified fragment length polymorphism (AFLP) is a novel molecular fingerprinting technique that can be applied to DNAs of\\u000a any source or complexity. Total genomic DNA is digested using two restriction enzymes. Double-stranded nucleotide adapters\\u000a are ligated to the DNA fragments to serve as primer binding sites for PCR amplification. Primers complementary to the adapter\\u000a and restriction site sequence, with additional

  10. Identification of healed terminal DNA fragments in linear minichromosomes of Schizosaccharomyces pombe.

    PubMed Central

    Matsumoto, T; Fukui, K; Niwa, O; Sugawara, N; Szostak, J W; Yanagida, M

    1987-01-01

    The minichromosome Ch16 of the fission yeast Schizosaccharomyces pombe is derived from the centromeric region of chromosome III. We show that Ch16 and a shorter derivative, Ch12, made by gamma-ray cleavage, are linear molecules of 530 and 280 kilobases, respectively. Each minichromosome has two novel telomeres, as shown by genomic Southern hybridization with an S. pombe telomere probe. Comparison by hybridization of the minichromosomes and their chromosomal counterparts showed no signs of gross rearrangement. Cosmid clones covering the ends of the long arms of Ch16 and Ch12 were isolated, and subcloned fragments that contained the breakage sites were identified. They are apparently unique in the genome. By hybridization and Bal 31 digestion, the ends appear to consist of the broken-end sequences directly associated with short stretches (about 300 base pairs) of new DNA that hybridizes to a cloned S. pombe telomere. They do not contain the telomere-adjacent repeated sequences that are present in the normal chromosomes. The sizes of the short telomeric stretches are roughly the same as those of the normal chromosomes. Our results show that broken chromosomal ends in S. pombe can be healed by the de novo addition of the short telomeric repeats. The formation of Ch16 must have required two breakage-healing events, whereas a single cleavage-healing event in the long arm of Ch16 yielded Ch12. Images PMID:2830493

  11. Clinical Factors Associated with Sperm DNA Fragmentation in Male Patients with Infertility

    PubMed Central

    Komiya, Akira; Kato, Tomonori; Kawauchi, Yoko; Watanabe, Akihiko; Fuse, Hideki

    2014-01-01

    Objective. The clinical factors associated with sperm DNA fragmentation (SDF) were investigated in male patients with infertility. Materials and Methods. Fifty-four ejaculates from infertile Japanese males were used. Thirty-three and twenty-one were from the patients with varicoceles and idiopathic causes of infertility, respectively. We performed blood tests, including the serum sex hormone levels, and conventional and computer-assisted semen analyses. The sperm nuclear vacuolization (SNV) was evaluated using a high-magnification microscope. The SDF was evaluated using the sperm chromatin dispersion test (SCDt) to determine the SDF index (SDFI). The SDFI was compared with semen parameters and other clinical variables, including lifestyle factors. Results. The SDFI was 41.3 ± 22.2% (mean ± standard deviation) and did not depend on the cause of infertility. Chronic alcohol use increased the SDFI to 49.6 ± 23.3% compared with 33.9 ± 18.0% in nondrinkers. The SDFI was related to adverse conventional semen parameters and sperm motion characteristics and correlated with the serum FSH level. The SNV showed a tendency to increase with the SDFI. The multivariate analysis revealed that the sperm progressive motility and chronic alcohol use were significant predictors of the SDF. Conclusion. The SCDt should be offered to chronic alcohol users and those with decreased sperm progressive motility. PMID:25165747

  12. 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. (Univ. of Michigan, Ann Arbor (United States))

    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.

  13. The helicase-like domains of type III restriction enzymes trigger long-range diffusion along DNA.

    PubMed

    Schwarz, Friedrich W; Tóth, Júlia; van Aelst, Kara; Cui, Guanshen; Clausing, Sylvia; Szczelkun, Mark D; Seidel, Ralf

    2013-04-19

    Helicases are ubiquitous adenosine triphosphatases (ATPases) with widespread roles in genome metabolism. Here, we report a previously undescribed functionality for ATPases with helicase-like domains; namely, that ATP hydrolysis can trigger ATP-independent long-range protein diffusion on DNA in one dimension (1D). Specifically, using single-molecule fluorescence microscopy we show that the Type III restriction enzyme EcoP15I uses its ATPase to switch into a distinct structural state that diffuses on DNA over long distances and long times. The switching occurs only upon binding to the target site and requires hydrolysis of ~30 ATPs. We define the mechanism for these enzymes and show how ATPase activity is involved in DNA target site verification and 1D signaling, roles that are common in DNA metabolism: for example, in nucleotide excision and mismatch repair. PMID:23599494

  14. 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. [School of Chemistry, University of Edinburgh, The King's Buildings, Edinburgh, EH9 3JJ (United Kingdom)] [School of Chemistry, University of Edinburgh, The King's Buildings, Edinburgh, EH9 3JJ (United Kingdom); Dryden, David T.F., E-mail: david.dryden@ed.ac.uk [School of Chemistry, University of Edinburgh, The King's Buildings, Edinburgh, EH9 3JJ (United Kingdom)

    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.

  15. DNA Fragments in the Blood Plasma of Cancer Patients: Quantitations and Evidence for Their Origin from Apoptotic and Necrotic Cells1

    Microsoft Academic Search

    Sabine Jahr; Hannes Hentze; Sabine Englisch; Dieter Hardt; Frank O. Fackelmayer; Rolf-Dieter Hesch; Rolf Knippers

    2001-01-01

    Increased levels of DNA fragments have frequently been found in the blood plasma of cancer patients. Published data suggest that only a fraction of the DNA in blood plasma is derived from cancer cells. How- ever, it is not known how much of the circulating DNA is from cancer or from noncancer cells. By quantitative methylation-specific PCR of the promoter

  16. Differing Effects of T4 DNA Ligase in the Modulation of the Damage Induced in Mammalian Cells by either X-Rays or Restriction Endonucleases

    Microsoft Academic Search

    Trinidad Ortiz; Joaquín Piñero

    2007-01-01

    Background: Of the different lesions induced by X-rays, DNA double-strand breaks (DSBs) are considered the main cause of chromosomal aberrations and cell death. Restriction endonucleases (REs) induce only DNA DSBs and have frequently been used to mimic the effects of ionizing radiation in the study of DNA damage and repair. Methods: The present work makes use of clonogenic and cytogenetic

  17. De novo sequencing of sunflower genome for SNP discovery using RAD (Restriction site Associated DNA) approach

    PubMed Central

    2013-01-01

    Background Application of Single Nucleotide Polymorphism (SNP) marker technology as a tool in sunflower breeding programs offers enormous potential to improve sunflower genetics, and facilitate faster release of sunflower hybrids to the market place. Through a National Sunflower Association (NSA) funded initiative, we report on the process of SNP discovery through reductive genome sequencing and local assembly of six diverse sunflower inbred lines that represent oil as well as confection types. Results A combination of Restriction site Associated DNA Sequencing (RAD-Seq) protocols and Illumina paired-end sequencing chemistry generated high quality 89.4 M paired end reads from the six lines which represent 5.3 GB of the sequencing data. Raw reads from the sunflower line, RHA 464 were assembled de novo to serve as a framework reference genome. About 15.2 Mb of sunflower genome distributed over 42,267 contigs were obtained upon assembly of RHA 464 sequencing data, the contig lengths ranged from 200 to 950 bp with an N50 length of 393 bp. SNP calling was performed by aligning sequencing data from the six sunflower lines to the assembled reference RHA 464. On average, 1 SNP was located every 143 bp of the sunflower genome sequence. Based on several filtering criteria, a final set of 16,467 putative sequence variants with characteristics favorable for Illumina Infinium Genotyping Technology (IGT) were mined from the sequence data generated across six diverse sunflower lines. Conclusion Here we report the molecular and computational methodology involved in SNP development for a complex genome like sunflower lacking reference assembly, offering an attractive tool for molecular breeding purposes in sunflower. PMID:23947483

  18. [Effect of N-stearoylethanolamine on the DNA fragmentation intensity in tumour and extratumoral tissues of the human adrenal cortex].

    PubMed

    Levchuk, N I; Pushkar'ov, V M; Kovzun, O I; Mykosha, O S; Hula, N M; Tron'ko, M D

    2012-01-01

    The effect of different concentrations of N-stearoylethanolamine (NSE 18:0) on fragmentation of DNA in the tumoural and extratumour tissues of the adrenal glands in vitro was studied. In this work the following types of tissue were investigated: extratumoural tissue from patients with hormonally active tumours, benign tumour tissue (hormonally active and hormonally inactive), tissue of malignant tumours and hyperplasic tissue of the adrenal glands (Itsenko-Cushing disease). It has been established that the NSE increases the intensity of DNA fragmentation only in the tissue of hormonally inactive tumours. Benign hormonally active tumours, malignant tumours and hyperplastic tissue of the adrenal glands were resistant to the NSE. The possible mechanisms of resistance to the drug are discussed. PMID:22946300

  19. 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

  20. 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. [Mayo Clinic/Foundation, Rochester, MN (United States)

    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.