WorldWideScience

Sample records for molecular bar-coded dna

  1. DNA Bar-Coding for Phytoplasma Identification

    DEFF Research Database (Denmark)

    Makarova, Olga; Contaldo, Nicoletta; Paltrinieri, Samanta

    2013-01-01

    Phytoplasma identi fi cation has proved dif fi cult due to their inability to be maintained in vitro. DNA barcoding is an identi fi cation method based on comparison of a short DNA sequence with known sequences from a database. A DNA barcoding tool has been developed for phytoplasma identi fi...... cation. While other sequencebased methods may be well adapted to identification of particular strains of phytoplasmas, often they cannot be used for the simultaneous identification of phytoplasmas from different groups. The phytoplasma DNA barcoding protocol in this chapter, based on the tuf and 16Sr...

  2. Bio-bar-code functionalized magnetic nanoparticle label for ultrasensitive flow injection chemiluminescence detection of DNA hybridization.

    Science.gov (United States)

    Bi, Sai; Zhou, Hong; Zhang, Shusheng

    2009-10-07

    A signal amplification strategy based on bio-bar-code functionalized magnetic nanoparticles as labels holds promise to improve the sensitivity and detection limit of the detection of DNA hybridization and single-nucleotide polymorphisms by flow injection chemiluminescence assays.

  3. DNA bar coding and pyrosequencing to analyze adverse events in therapeutic gene transfer.

    Science.gov (United States)

    Wang, Gary P; Garrigue, Alexandrine; Ciuffi, Angela; Ronen, Keshet; Leipzig, Jeremy; Berry, Charles; Lagresle-Peyrou, Chantal; Benjelloun, Fatine; Hacein-Bey-Abina, Salima; Fischer, Alain; Cavazzana-Calvo, Marina; Bushman, Frederic D

    2008-05-01

    Gene transfer has been used to correct inherited immunodeficiencies, but in several patients integration of therapeutic retroviral vectors activated proto-oncogenes and caused leukemia. Here, we describe improved methods for characterizing integration site populations from gene transfer studies using DNA bar coding and pyrosequencing. We characterized 160,232 integration site sequences in 28 tissue samples from eight mice, where Rag1 or Artemis deficiencies were corrected by introducing the missing gene with gamma-retroviral or lentiviral vectors. The integration sites were characterized for their genomic distributions, including proximity to proto-oncogenes. Several mice harbored abnormal lymphoproliferations following therapy--in these cases, comparison of the location and frequency of isolation of integration sites across multiple tissues helped clarify the contribution of specific proviruses to the adverse events. We also took advantage of the large number of pyrosequencing reads to show that recovery of integration sites can be highly biased by the use of restriction enzyme cleavage of genomic DNA, which is a limitation in all widely used methods, but describe improved approaches that take advantage of the power of pyrosequencing to overcome this problem. The methods described here should allow integration site populations from human gene therapy to be deeply characterized with spatial and temporal resolution.

  4. Temporal and spatial trends in prey composition of wahoo Acanthocybium solandri: a diet analysis from the central North Pacific Ocean using visual and DNA bar-coding techniques.

    Science.gov (United States)

    Oyafuso, Z S; Toonen, R J; Franklin, E C

    2016-04-01

    A diet analysis was conducted on 444 wahoo Acanthocybium solandri caught in the central North Pacific Ocean longline fishery and a nearshore troll fishery surrounding the Hawaiian Islands from June to December 2014. In addition to traditional observational methods of stomach contents, a DNA bar-coding approach was integrated into the analysis by sequencing the cytochrome c oxidase subunit 1 (COI) region of the mtDNA genome to taxonomically identify individual prey items that could not be classified visually to species. For nearshore-caught A. solandri, juvenile pre-settlement reef fish species from various families dominated the prey composition during the summer months, followed primarily by Carangidae in autumn months. Gempylidae, Echeneidae and Scombridae were dominant prey taxa from the offshore fishery. Molidae was a common prey family found in stomachs collected north-east of the Hawaiian Archipelago while tetraodontiform reef fishes, known to have extended pelagic stages, were prominent prey items south-west of the Hawaiian Islands. The diet composition of A. solandri was indicative of an adaptive feeder and thus revealed dominant geographic and seasonal abundances of certain taxa from various ecosystems in the marine environment. The addition of molecular bar-coding to the traditional visual method of prey identifications allowed for a more comprehensive range of the prey field of A. solandri to be identified and should be used as a standard component in future diet studies.

  5. Nanogold-based bio-bar codes for label-free immunosensing of proteins coupling with an in situ DNA-based hybridization chain reaction.

    Science.gov (United States)

    Zhou, Jun; Xu, Mingdi; Tang, Dianping; Gao, Zhuangqiang; Tang, Juan; Chen, Guonan

    2012-12-28

    A label-free, non-enzyme immunosensing strategy is designed for ultrasensitive electronic detection of disease-related proteins (carcinoembryonic antigen as a model) by using gold nanoparticle-based bio-bar codes and an in situ amplified DNA-based hybridization chain reaction.

  6. Application of DNA bar codes for screening of industrially important fungi: the haplotype of Trichoderma harzianum sensu stricto indicates superior chitinase formation.

    Science.gov (United States)

    Nagy, Viviana; Seidl, Verena; Szakacs, George; Komoń-Zelazowska, Monika; Kubicek, Christian P; Druzhinina, Irina S

    2007-11-01

    Selection of suitable strains for biotechnological purposes is frequently a random process supported by high-throughput methods. Using chitinase production by Hypocrea lixii/Trichoderma harzianum as a model, we tested whether fungal strains with superior enzyme formation may be diagnosed by DNA bar codes. We analyzed sequences of two phylogenetic marker loci, internal transcribed spacer 1 (ITS1) and ITS2 of the rRNA-encoding gene cluster and the large intron of the elongation factor 1-alpha gene, tef1, from 50 isolates of H. lixii/T. harzianum, which were also tested to determine their ability to produce chitinases in solid-state fermentation (SSF). Statistically supported superior chitinase production was obtained for strains carrying one of the observed ITS1 and ITS2 and tef1 alleles corresponding to an allele of T. harzianum type strain CBS 226.95. A tef1-based DNA bar code tool, TrichoCHIT, for rapid identification of these strains was developed. The geographic origin of the strains was irrelevant for chitinase production. The improved chitinase production by strains containing this haplotype was not due to better growth on N-acetyl-beta-D-glucosamine or glucosamine. Isoenzyme electrophoresis showed that neither the isoenzyme profile of N-acetyl-beta-glucosaminidases or the endochitinases nor the intensity of staining of individual chitinase bands correlated with total chitinase in the culture filtrate. The superior chitinase producers did not exhibit similarly increased cellulase formation. Biolog Phenotype MicroArray analysis identified lack of N-acetyl-beta-D-mannosamine utilization as a specific trait of strains with the chitinase-overproducing haplotype. This observation was used to develop a plate screening assay for rapid microbiological identification of the strains. The data illustrate that desired industrial properties may be an attribute of certain populations within a species, and screening procedures should thus include a balanced mixture of all

  7. Objectivity in Grading: The Promise of Bar Codes

    Science.gov (United States)

    Jae, Haeran; Cowling, John

    2009-01-01

    This article proposes the use of a new technology to assure student anonymity and reduce bias hazards: identifying students by using bar codes. The limited finding suggests that the use of bar codes for assuring student anonymity could potentially cause students to perceive that grades are assigned more fairly and reassure teachers that they are…

  8. Managing purchasing and inventory with bar codes. Part II.

    Science.gov (United States)

    Gandy, J

    1986-04-01

    Automated identification systems (bar coding) have proven their worth in a number of diverse manufacturing and materials handling environments. Whether this technology can be broadly applied with equal effectiveness in the health care setting still remains largely unproven. One thing is certain, however. Before bar code technology can be effectively applied in any setting, the materials manager must understand several basic concepts: How materials flow through his physical plant; How, where and in what amounts they are used; and How and when they are expensed. With this information, it is possible to create a systematized approach to materials cost containment, of which bar coding is one element. In the following article, the author illustrates how purchasing and inventory control can be made more time and labor efficient through the use of bar code technology.

  9. PCR-free quantitative detection of genetically modified organism from raw materials. An electrochemiluminescence-based bio bar code method.

    Science.gov (United States)

    Zhu, Debin; Tang, Yabing; Xing, Da; Chen, Wei R

    2008-05-15

    A bio bar code assay based on oligonucleotide-modified gold nanoparticles (Au-NPs) provides a PCR-free method for quantitative detection of nucleic acid targets. However, the current bio bar code assay requires lengthy experimental procedures including the preparation and release of bar code DNA probes from the target-nanoparticle complex and immobilization and hybridization of the probes for quantification. Herein, we report a novel PCR-free electrochemiluminescence (ECL)-based bio bar code assay for the quantitative detection of genetically modified organism (GMO) from raw materials. It consists of tris-(2,2'-bipyridyl) ruthenium (TBR)-labeled bar code DNA, nucleic acid hybridization using Au-NPs and biotin-labeled probes, and selective capture of the hybridization complex by streptavidin-coated paramagnetic beads. The detection of target DNA is realized by direct measurement of ECL emission of TBR. It can quantitatively detect target nucleic acids with high speed and sensitivity. This method can be used to quantitatively detect GMO fragments from real GMO products.

  10. Bar Coding Platforms for Nucleic Acid and Protein Detection

    Science.gov (United States)

    Müller, Uwe R.

    A variety of novel bar coding systems has been developed as multiplex testing platforms for applications in biological, chemical, and biomedical diagnostics. Instead of identifying a target through capture at a specific locus on an array, target analytes are captured by a bar coded tag, which then uniquely identifies the target, akin to putting a UPC bar code on a product. This requires an appropriate surface functionalization to ensure that the correct target is captured with high efficiency. Moreover the tag, or bar code, has to be readable with minimal error and at high speed, typically by flow analysis. For quantitative assays the target may be labeled separately, or the tag may also serve as the label. A great variety of materials and physicochemical principles has been exploited to generate this plethora of novel bar coding platforms. Their advantages compared to microarray-based assay platforms include in-solution binding kinetics, flexibility in assay design, compatibility with microplate-based assay automation, high sample throughput, and with some assay formats, increased sensitivity.

  11. Innovative application of bar coding technology to breast milk administration.

    Science.gov (United States)

    Fleischman, Ellen K

    2013-01-01

    Hospitalized infants often receive expressed breast milk, either from their mother or from banked milk. Breast milk provides optimal nutrition for infants but because it is a body fluid it carries the risk of disease transmission. Therefore, administering the correct breast milk to hospitalized infants is essential. Bar coding technology, used in hospitals to prevent errors related to medication administration, can be proactively applied to prevent breast milk administration errors. Bar coding systems offer advantages over manual verification processes, including decreasing errors due to human factors and providing for automated entry of feedings in the electronic health record. However, potential barriers to successful implementation must be addressed. These barriers include equipment and training costs, increased time to perform the additional steps with bar coding, and work-arounds.

  12. Denture bar-coding: An innovative technique in forensic dentistry

    Science.gov (United States)

    Dineshshankar, Janardhanam; Venkateshwaran, Rajendran; Vidhya, J.; Anuradha, R.; Mary, Gold Pealin; Pradeep, R.; Senthileagappan, A. R.

    2015-01-01

    Denture markers play an important role in forensic odontology and also in identifying a person. A number of methods are there for identifying dentures from a less expensive technique to a more expensive technique. Out of different denture markers, the bar-coding system is a way of collecting data from the mobile. Even a huge amount of data can be stored in that. It can be easily incorporated during acrylization of the denture and thus could be helpful in identification. This article reviews the strengths of bar-coding and how easily it can be used in the routine procedure. PMID:26538876

  13. Denture bar-coding: An innovative technique in forensic dentistry.

    Science.gov (United States)

    Dineshshankar, Janardhanam; Venkateshwaran, Rajendran; Vidhya, J; Anuradha, R; Mary, Gold Pealin; Pradeep, R; Senthileagappan, A R

    2015-08-01

    Denture markers play an important role in forensic odontology and also in identifying a person. A number of methods are there for identifying dentures from a less expensive technique to a more expensive technique. Out of different denture markers, the bar-coding system is a way of collecting data from the mobile. Even a huge amount of data can be stored in that. It can be easily incorporated during acrylization of the denture and thus could be helpful in identification. This article reviews the strengths of bar-coding and how easily it can be used in the routine procedure.

  14. CERN access card: Introduction of a bar code

    CERN Multimedia

    Relations with the Host States Service

    2004-01-01

    Before the latest version of the implementation measures relating to Operational Circular No. 2 comes into force, we would like to inform you that, in future, CERN access cards may bear a bar code to transcribe the holder's identification number. Relations with the Host States Service http://www.cern.ch/relations/ Tel. 72848

  15. CERN access cards - Introduction of a bar code (Reminder)

    CERN Multimedia

    Relations with the Host States Service

    2004-01-01

    In accordance with the latest revised version of the implementation measures relating to Operational Circular No. 2, CERN access cards may bear a bar code transcribing the holder's identification number (the revised version of this subsidiary document to the aforementioned Circular will be published shortly). Relations with the Host States Service http://www.cern.ch/relations/ relations.secretariat@cern.ch Tel. 72848

  16. Use of bar codes in inpatient drug distribution.

    Science.gov (United States)

    Meyer, G E; Brandell, R; Smith, J E; Milewski, F J; Brucker, P; Coniglio, M

    1991-05-01

    The development and operation of a prototype inpatient drug distribution system that uses bar codes is described, and the impact of bar coding on the cassette-filling and verification process is summarized. A prototype pharmacy dispensing site was created to function in parallel with an existing satellite dispensing site that served 78 general medical-care beds. Supplemental labels encoded with an 11-digit unique product identification number, a 5-digit expiration date, and a 6-character lot number were generated and affixed to all unit dose packages dispensed from the prototype pharmacy site. The unit doses were labeled with Code 49 symbology; each label measured 0.8 x 1.25 inches. Each patient cassette was labeled using Code 39 symbology. A cost-benefit model was developed, and the two dispensing systems were compared with respect to (1) time to fill patient cassettes, (2) time to verify patient cassettes, (3) time to process patient charges and credits, (4) time to correct dispensing errors, (5) accuracy of the cassette-filling process, and (6) accuracy of the cassette verification process. Bar-code dispensing and verification saved 1.52 seconds per dose. Additionally, the cassette verification function was shifted from pharmacists to technicians. Estimated per-dose cost of the bar-code system was 2.73 cents. A measurable improvement in the accuracy of filling patient cassettes was documented. The feasibility of using bar codes in unit dose dispensing was demonstrated, and the prototype system was shown to produce cost efficiencies and patient-care benefits.

  17. Bar code, good for industry and trade--how does it benefit the dentist?

    Science.gov (United States)

    Oehlmann, H

    2001-10-01

    Every dentist who attentively follows the change in product labelling can easily see that the HIBC bar code is on the increase. In fact, according to information from FIDE/VDDI and ADE/BVD, the dental industry and trade are firmly resolved to apply the HIBC bar code to all products used internationally in dental practices. Why? Indeed, at first it looks like extra expense to additionally print a bar code on the packages. Good reasons can only lie in advantages which manufacturers and the trade expect from the HIBC bar code, Indications in dental technician circles are that the HIBC bar code is coming. If there are advantages, what are these, and can the dentist also profit from them? What does HIBC bar code mean and what items of interest does it include? What does bar code cost and does only one code exist? This is explained briefly, concentrating on the benefits bar code can bring for different users.

  18. Applications of Bar Code Technology in the Construction Industry

    Science.gov (United States)

    1991-01-01

    per week. This is an indication that we have much better control of our inventory " ( Ryan 87 ). Producto Machine Company of Bridgeport, Connecticut... marketing and patenting advanced technologies to encourage firms to take on the risks involved. Our country, however, has no such policy. Until it does...assistance in marketing and patenting their achievements. 2) The Department of Defense, who already requires bar codes on all supplies accepted into

  19. Improving radiopharmaceutical supply chain safety by implementing bar code technology.

    Science.gov (United States)

    Matanza, David; Hallouard, François; Rioufol, Catherine; Fessi, Hatem; Fraysse, Marc

    2014-11-01

    The aim of this study was to describe and evaluate an approach for improving radiopharmaceutical supply chain safety by implementing bar code technology. We first evaluated the current situation of our radiopharmaceutical supply chain and, by means of the ALARM protocol, analysed two dispensing errors that occurred in our department. Thereafter, we implemented a bar code system to secure selected key stages of the radiopharmaceutical supply chain. Finally, we evaluated the cost of this implementation, from overtime, to overheads, to additional radiation exposure to workers. An analysis of the events that occurred revealed a lack of identification of prepared or dispensed drugs. Moreover, the evaluation of the current radiopharmaceutical supply chain showed that the dispensation and injection steps needed to be further secured. The bar code system was used to reinforce product identification at three selected key stages: at usable stock entry; at preparation-dispensation; and during administration, allowing to check conformity between the labelling of the delivered product (identity and activity) and the prescription. The extra time needed for all these steps had no impact on the number and successful conduct of examinations. The investment cost was reduced (2600 euros for new material and 30 euros a year for additional supplies) because of pre-existing computing equipment. With regard to the radiation exposure to workers there was an insignificant overexposure for hands with this new organization because of the labelling and scanning processes of radiolabelled preparation vials. Implementation of bar code technology is now an essential part of a global securing approach towards optimum patient management.

  20. 76 FR 49772 - Guidance for Industry: Bar Code Label Requirements-Questions and Answers; Availability

    Science.gov (United States)

    2011-08-11

    ... HUMAN SERVICES Food and Drug Administration Guidance for Industry: Bar Code Label Requirements... Industry: Bar Code Label Requirements--Questions and Answers'' dated August 2011. The guidance announced in... finalizes the draft guidance entitled ``Guidance for Industry: Bar Code Label Requirements--Questions and...

  1. System Design Considerations In Bar-Code Laser Scanning

    Science.gov (United States)

    Barkan, Eric; Swartz, Jerome

    1984-08-01

    The unified transfer function approach to the design of laser barcode scanner signal acquisition hardware is considered. The treatment of seemingly disparate system areas such as the optical train, the scanning spot, the electrical filter circuits, the effects of noise, and printing errors is presented using linear systems theory. Such important issues as determination of depth of modulation, filter specification, tolerancing of optical components, and optimi-zation of system performance in the presence of noise are discussed. The concept of effective spot size to allow for impact of optical system and analog processing circuitry upon depth of modulation is introduced. Considerations are limited primarily to Gaussian spot profiles, but also apply to more general cases. Attention is paid to realistic bar-code symbol models and to implications with respect to printing tolerances.

  2. Nurses' Attitudes Toward the Use of the Bar-coding Medication Administration System

    NARCIS (Netherlands)

    S.D. Marini; A. Hasman; H.A.S. Huijer; H. Dimassi

    2010-01-01

    This study determines nurses' attitudes toward bar-coding medication administration system use. Some of the factors underlying the successful use of bar-coding medication administration systems that are viewed as a connotative indicator of users' attitudes were used to gather data that describe the

  3. Contrast and Comparison Between the Old and New Bar Code for Commodity Management Measures

    Institute of Scientific and Technical Information of China (English)

    Huang Xiaolin; Ma Jing

    2005-01-01

    @@ With the development of socialism market economy, the former Bar Code For Commodity Management Measures (being called Old Measures for short hereafter) issued by the National Bureau of Quality and Technical Supervision can not adapt to the requirement of managing for bar code for commodity.

  4. Accuracy and time requirements of a bar-code inventory system for medical supplies.

    Science.gov (United States)

    Hanson, L B; Weinswig, M H; De Muth, J E

    1988-02-01

    The effects of implementing a bar-code system for issuing medical supplies to nursing units at a university teaching hospital were evaluated. Data on the time required to issue medical supplies to three nursing units at a 480-bed, tertiary-care teaching hospital were collected (1) before the bar-code system was implemented (i.e., when the manual system was in use), (2) one month after implementation, and (3) four months after implementation. At the same times, the accuracy of the central supply perpetual inventory was monitored using 15 selected items. One-way analysis of variance tests were done to determine any significant differences between the bar-code and manual systems. Using the bar-code system took longer than using the manual system because of a significant difference in the time required for order entry into the computer. Multiple-use requirements of the central supply computer system made entering bar-code data a much slower process. There was, however, a significant improvement in the accuracy of the perpetual inventory. Using the bar-code system for issuing medical supplies to the nursing units takes longer than using the manual system. However, the accuracy of the perpetual inventory was significantly improved with the implementation of the bar-code system.

  5. Vision-based fast location of multi-bar code in any direction

    Science.gov (United States)

    Lin, Sheng-Xin; Zhao, Xiao-Fang; Liu, Hua-Zhu

    2017-07-01

    The automatic location of the bar code is a key step in the bar code image recognition system. It is extremely confined that the generalization of the traditional bar code localization algorithms due to the requirements of both direction and quality of bar code, and most of them are only aimed at the single barcode localization. In this paper, we have proposed a novel multi-barcode location algorithm in arbitrary direction based on the accumulation of the linear gray value. First, the line coordinates of the barcode region is determined by the image normalized cross-correlation algorithm. Then the center line of gray value of cumulative distribution is used to analyze the barcode boundary and to determine the number of bar code within the region. Finally, the precise positioning of the barcode region is obtained. The experiments have demonstrated that our proposed method can be used to identify all the bar codes in any area, and automatically locate the bar codes in any direction.

  6. 75 FR 54347 - Draft Guidance for Industry: Bar Code Label Requirements-Questions and Answers (Question 12...

    Science.gov (United States)

    2010-09-07

    ... HUMAN SERVICES Food and Drug Administration Draft Guidance for Industry: Bar Code Label Requirements... document entitled ``Guidance for Industry: Bar Code Label Requirements--Questions and Answers (Question 12... Industry: Bar Code Label Requirements--Questions and Answers (Question 12 Update)'' dated August 2010. FDA...

  7. 19 CFR 142.45 - Use of bar code by entry filer.

    Science.gov (United States)

    2010-04-01

    ... filer and a product description below the bar code. (b) Multiple commodity processing. Multiple... variable allowed. The commodities should be listed on one invoice with C-4 Code labels for each...

  8. Anisotropic Total Variation Regularized L^1-Approximation and Denoising/Deblurring of 2D Bar Codes

    CERN Document Server

    Choksi, Rustum; Oberman, Adam

    2010-01-01

    We consider variations of the Rudin-Osher-Fatemi functional which are particularly well-suited to denoising and deblurring of 2D bar codes. These functionals consist of an anisotropic total variation favoring rectangles and a fidelity term which measure the L^1 distance to the signal, both with and without the presence of a deconvolution operator. Based upon the existence of a certain associated vector field, we find necessary and sufficient conditions for a function to be a minimizer. We apply these results to 2D bar codes to find explicit regimes ---in terms of the fidelity parameter and smallest length scale of the bar codes--- for which a perfect bar code is recoverable via minimization of the functionals. Via a discretization reformulated as a linear program, we perform numerical experiments for all functionals demonstrating their denoising and deblurring capabilities.

  9. Time trend of injection drug errors before and after implementation of bar-code verification system.

    Science.gov (United States)

    Sakushima, Ken; Umeki, Reona; Endoh, Akira; Ito, Yoichi M; Nasuhara, Yasuyuki

    2015-01-01

    Bar-code technology, used for verification of patients and their medication, could prevent medication errors in clinical practice. Retrospective analysis of electronically stored medical error reports was conducted in a university hospital. The number of reported medication errors of injected drugs, including wrong drug administration and administration to the wrong patient, was compared before and after implementation of the bar-code verification system for inpatient care. A total of 2867 error reports associated with injection drugs were extracted. Wrong patient errors decreased significantly after implementation of the bar-code verification system (17.4/year vs. 4.5/year, pcode medication administration is effective for prevention of wrong patient errors. However, ordinary bar-code verification systems are limited in their ability to prevent incorrect drug preparation in hospital wards.

  10. Deblurring, Localization and Geometry Correction of 2D QR Bar Codes Using Richardson Lucy Method

    Directory of Open Access Journals (Sweden)

    Manpreet Kaur

    2014-09-01

    Full Text Available This paper includes the recognition of 2D QR bar codes. This paper describes the deblurring, localization and geometry correction of 2D QR bar codes. The images captured are blurred due motion between the image and the camera. Hence the image containing the QR barcode cannot be read by QR reader. To make the QR barcode readable the images are need to be deblurred. Lucy Richardson method and Weiner Deconvolution Method is used to deblurr and localize the bar code. From both of the methods Lucy Richardson Method is best because this method takes less time for execution than the other method. Simulink Model is used for the Geometry correction of the QR bar code. In future, we would like to investigate the generalization of our algorithm to handle more complicated motion blur.

  11. Molecular DNA switches and DNA chips

    Science.gov (United States)

    Sabanayagam, Chandran R.; Berkey, Cristin; Lavi, Uri; Cantor, Charles R.; Smith, Cassandra L.

    1999-06-01

    We present an assay to detect single-nucleotide polymorphisms on a chip using molecular DNA switches and isothermal rolling- circle amplification. The basic principle behind the switch is an allele-specific oligonucleotide circularization, mediated by DNA ligase. A DNA switch is closed when perfect hybridization between the probe oligonucleotide and target DNA allows ligase to covalently circularize the probe. Mismatches around the ligation site prevent probe circularization, resulting in an open switch. DNA polymerase is then used to preferentially amplify the closed switches, via rolling-circle amplification. The stringency of the molecular switches yields 102 - 103 fold discrimination between matched and mismatched sequences.

  12. Bar coding Biological Diversity: A New Micr ogenomic Identification Approach

    Directory of Open Access Journals (Sweden)

    Bazartseren Boldgiv

    2004-12-01

    Full Text Available Morphology-based taxonomy suffers from its inherent limitations, even though most of biological research depends on reliable identifications of species. A recent microgenomic identification approach, which is now being called the “DNA-barcoding,” presents a promising potential of developing into a real- time, on site tool for identification of organisms, especially animals and of providing an added insight into evolutionary history . For animals, the DNA-barcode seems to have been found in the mitochondrial genome and researchers are in quest of developing similar microgenomic DNA-barcoding systems for other domains of biological diversity . This article discusses the DNA-barcoding technique and considers some of the implications of this approach.

  13. Bar coding Biological Diversity: A New Micr ogenomic Identification Approach

    OpenAIRE

    Bazartseren Boldgiv

    2004-01-01

    Morphology-based taxonomy suffers from its inherent limitations, even though most of biological research depends on reliable identifications of species. A recent microgenomic identification approach, which is now being called the “DNA-barcoding,” presents a promising potential of developing into a real- time, on site tool for identification of organisms, especially animals and of providing an added insight into evolutionary history . For animals, the DNA-barcode seems...

  14. 手机二维码安全研究%Research of Mobile Bar Code's Security

    Institute of Scientific and Technical Information of China (English)

    张焱焱

    2015-01-01

    二维码技术带给人们便利的同时,也给手机安全带来了隐患.通过分析手机二维码技术存在的安全威胁,提出了相应的预防措施.通过这些手段,从一定程度上能够避免由于二维码技术的安全性低所导致的泄密等事件的发生.%The technology of bar code brings up the convenient for people, and the troubles at the same time. The responsible pre-vention methods are given by analyzing the security threats brought by the technology of mobile's bar code. To a certain degree, these methods can avoid leak caused by lower security of bar code technology.

  15. Addressing challenges in bar-code scanning of large-volume infusion bags.

    Science.gov (United States)

    Raman, Kirthana; Heelon, Mark; Kerr, Gary; Higgins, Thomas L

    2011-08-01

    A hospital pharmacy's efforts to identify and address challenges with bedside scanning of bar codes on large-volume parenteral (LVP) infusion bags are described. Bar-code-assisted medication administration (BCMA) has been shown to reduce medication errors and improve patient safety. After the pilot implementation of a BCMA system and point-of-care scanning procedures at a medical center's intensive care unit, it was noted that nurses' attempted bedside scans of certain LVP bags for product identification purposes often were not successful. An investigation and root-cause analysis, including observation of nurses' scanning technique by a multidisciplinary team, determined that the scanning failures stemmed from the placement of two bar-code imprints-one with the product identification code and another, larger imprint with the expiration date and lot number-adjacently on the LVP bags. The nursing staff was educated on a modified scanning technique, which resulted in significantly improved success rates in the scanning of the most commonly used LVP bags. Representatives of the LVP bag manufacturer met with hospital staff to discuss the problem and corrective measures. As part of a subsequent infusion bag redesign, the manufacturer discontinued the use of the bar-code imprint implicated in the scanning failures. Failures in scanning LVP bags were traced to problematic placement of bar-code imprints on the bags. Interdisciplinary collaboration, consultation with the bag manufacturer, and education of the nursing and pharmacy staff resulted in a reduction in scanning failures and the manufacturer's removal of one of the bar codes from its LVP bags.

  16. Solid Warehouse Material Management System Based on ERP and Bar Code Technology

    Institute of Scientific and Technical Information of China (English)

    ZHANG Cheng; WANG Jie; YUAN Bing; WU Chao; HU Qiao-dan

    2004-01-01

    This paper presents a manufacturing material management system based on ERP, which is combined with industrial bar code information collection and material management, and carries out extensive research on the system structure and function model, as well as a detailed application scheme.

  17. Potential use of bar codes to implement automated dispensing quality assurance programs.

    Science.gov (United States)

    Hokanson, J A; Keith, M R; Guernsey, B G; Grudzien, R R; Doutré, W H; Luttman, D J; Trachtenberg, M C

    1985-05-01

    Bar code-based systems have automated many counting, tracking, and sorting functions in health care delivery services. We designed and briefly tested the feasibility of a bar code-based dispensing quality assurance system for a hospital outpatient pharmacy. The use of bar codes to verify the identity of the dispensed product required an extra few seconds processing time for each prescription but did not increase markedly the processing time when compared to a control period. In addition to verifying product identity, the system checked the manufacture's expiration date to reduce the risk of delivering outdated medications to the patient. The on-site test period for this feasibility model was relatively short (one week) and no actual dispensing errors were detected. However, when the system was presented with 100 different prescription forms containing 50 randomly sequenced, precontrived dispensing errors, the system identified all medication errors and outdated products. As shown in other studies, bar code-based systems have the potential to capture information not effectively recorded using manual methods. We suggest they should be considered by pharmacists interested in automating inventory management and work flow functions or establishing automated dispensing quality assurance programs.

  18. The Impact of Bar Code Medication Administration Technology on Reported Medication Errors

    Science.gov (United States)

    Holecek, Andrea

    2011-01-01

    The use of bar-code medication administration technology is on the rise in acute care facilities in the United States. The technology is purported to decrease medication errors that occur at the point of administration. How significantly this technology affects actual rate and severity of error is unknown. This descriptive, longitudinal research…

  19. Challenges implementing bar-coded medication administration in the emergency room in comparison to medical surgical units.

    Science.gov (United States)

    Glover, Nancy

    2013-03-01

    Bar-coded medication administration has been successfully implemented and utilized to decrease medication errors at a number of hospitals in recent years. The purpose of this article was to discuss the varying success in utilization of bar-coded medication administration on medical-surgical units and in the emergency department. Utilization reports were analyzed to better understand the challenges between the units. Many factors negatively impacted utilization in the emergency department, including the inability to use bar-coded medication administration for verbal orders or to document medications distributed by the prescribing providers, unique aspects of emergency department nursing workflow, additional steps to chart when using bar-coded medication administration, and alert fatigue. Hardware problems affected all users. Bar-coded medication administration in its current form is more suitable for use on medical-surgical floors than in the emergency department. New solutions should be developed for bar-coded medication administration in the emergency department, keeping in mind requirements to chart medications when there is no order in the system, document medications distributed by prescribing providers, adapt to unpredictable nursing workflow, minimize steps to chart with bar-coded medication administration, limit alerts to those that are clinically meaningful, and choose reliable hardware with adequate bar-code scanning capability.

  20. A novel photoelectrochemical sensor based on photocathode of PbS quantum dots utilizing catalase mimetics of bio-bar-coded platinum nanoparticles/G-quadruplex/hemin for signal amplification.

    Science.gov (United States)

    Wang, Guang-Li; Liu, Kang-Li; Shu, Jun-Xian; Gu, Tian-Tian; Wu, Xiu-Ming; Dong, Yu-Ming; Li, Zai-Jun

    2015-07-15

    Photocathode based on p-type PbS quantum dots (QDs) combing a novel signal amplification strategy utilizing catalase (CAT) mimetics was designed and utilized for sensitive photoelectrochemical (PEC) detection of DNA. The bio-bar-coded Pt nanoparticles (NPs)/G-quadruplex/hemin exhibited high CAT-like activity following the Michaelis-Menten model for decomposing H2O2 to water and oxygen, whose activity even slightly exceeded that of natural CAT. The bio-bar-code as a catalytic label was conjugated onto the surface of PbS QDs modified electrodes through the formed sandwich-type structure due to DNA hybridization. Oxygen in situ generated by the CAT mimetics of the bio-bar-code of Pt NPs/G-quadruplex/hemin acted as an efficient electron acceptor of illuminated PbS QDs, promoting charge separation and enhancing cathodic photocurrent. Under optimal conditions, the developed PEC biosensor for target DNA exhibited a dynamic range of 0.2pmol/L to 1.0nmol/L with a low detection limit of 0.08pmol/L. The high sensitivity of the method was resulted from the sensitive response of PbS QDs to oxygen and the highly efficient CAT-like catalytic activity of the bio-bar-coded Pt NPs/G-quadruplex/hemin.

  1. Machine-vision-based bar code scanning for long-range applications

    Science.gov (United States)

    Banta, Larry E.; Pertl, Franz A.; Rosenecker, Charles; Rosenberry-Friend, Kimberly A.

    1998-10-01

    Bar code labeling of products has become almost universal in most industries. However, in the steel industry, problems with high temperatures, harsh physical environments and the large sizes of the products and material handling equipment have slowed implementation of bar code based systems in the hot end of the mill. Typical laser-based bar code scanners have maximum scan distances of only 15 feet or so. Longer distance models have been developed which require the use of retro reflective paper labels, but the labels must be very large, are expensive, and cannot stand the heat and physical abuse of the steel mill environment. Furthermore, it is often difficult to accurately point a hand held scanner at targets in bright sunlight or at long distances. An automated product tag reading system based on CCD cameras and computer image processing has been developed by West Virginia University, and demonstrated at the Weirton Steel Corporation. The system performs both the pointing and reading functions. A video camera is mounted on a pan/tilt head, and connected to a personal computer through a frame grabber board. The computer analyzes the images, and can identify product ID tags in a wide-angle scene. It controls the camera to point at each tag and zoom for a closeup picture. The closeups are analyzed and the program need both a barcode and the corresponding alphanumeric code on the tag. This paper describes the camera pointing and bar-code reading functions of the algorithm. A companion paper describes the OCR functions.

  2. Pictorial AR Tag with Hidden Multi-Level Bar-Code and Its Potential Applications

    Directory of Open Access Journals (Sweden)

    Huy Le

    2017-09-01

    Full Text Available For decades, researchers have been trying to create intuitive virtual environments by blending reality and virtual reality, thus enabling general users to interact with the digital domain as easily as with the real world. The result is “augmented reality” (AR. AR seamlessly superimposes virtual objects on to a real environment in three dimensions (3D and in real time. One of the most important parts that helps close the gap between virtuality and reality is the marker used in the AR system. While pictorial marker and bar-code marker are the two most commonly used marker types in the market, they have some disadvantages in visual and processing performance. In this paper, we present a novelty method that combines the bar-code with the original feature of a colour picture (e.g., photos, trading cards, advertisement’s figure. Our method decorates on top of the original pictorial images additional features with a single stereogram image that optically conceals a multi-level (3D bar-code. Thus, it has a larger capability of storing data compared to the general 1D barcode. This new type of marker has the potential of addressing the issues that the current types of marker are facing. It not only keeps the original information of the picture but also contains encoded numeric information. In our limited evaluation, this pictorial bar-code shows a relatively robust performance under various conditions and scaling; thus, it provides a promising AR approach to be used in many applications such as trading card games, educations, and advertisements.

  3. Analysis of the technology acceptance model in examining hospital nurses' behavioral intentions toward the use of bar code medication administration.

    Science.gov (United States)

    Song, Lunar; Park, Byeonghwa; Oh, Kyeung Mi

    2015-04-01

    Serious medication errors continue to exist in hospitals, even though there is technology that could potentially eliminate them such as bar code medication administration. Little is known about the degree to which the culture of patient safety is associated with behavioral intention to use bar code medication administration. Based on the Technology Acceptance Model, this study evaluated the relationships among patient safety culture and perceived usefulness and perceived ease of use, and behavioral intention to use bar code medication administration technology among nurses in hospitals. Cross-sectional surveys with a convenience sample of 163 nurses using bar code medication administration were conducted. Feedback and communication about errors had a positive impact in predicting perceived usefulness (β=.26, Pmodel predicting for behavioral intention, age had a negative impact (β=-.17, Pmodel explained 24% (Ptechnology.

  4. Use of bar code labels on collection tubes for specimen management in the clinical laboratory.

    Science.gov (United States)

    Tilzer, L L; Jones, R W

    1988-12-01

    A new generation in specimen handling has arrived with the introduction of bar code readers on medical laboratory equipment. The incorporation of this technology into laboratory information systems offers a streamlining of specimen workflow never before achievable in a laboratory environment. The use of evacuated collection tubes as the primary sampling container on a random-access chemistry analyzer interfaced to a laboratory information system creates a very simplified sampling/analysis system with tremendous advantages. There are significant labor savings, superior service to clinicians, and reduced chances for clerical error.

  5. DNA: Polymer and molecular code

    Science.gov (United States)

    Shivashankar, G. V.

    1999-10-01

    The thesis work focusses upon two aspects of DNA, the polymer and the molecular code. Our approach was to bring single molecule micromanipulation methods to the study of DNA. It included a home built optical microscope combined with an atomic force microscope and an optical tweezer. This combined approach led to a novel method to graft a single DNA molecule onto a force cantilever using the optical tweezer and local heating. With this method, a force versus extension assay of double stranded DNA was realized. The resolution was about 10 picoN. To improve on this force measurement resolution, a simple light backscattering technique was developed and used to probe the DNA polymer flexibility and its fluctuations. It combined the optical tweezer to trap a DNA tethered bead and the laser backscattering to detect the beads Brownian fluctuations. With this technique the resolution was about 0.1 picoN with a millisecond access time, and the whole entropic part of the DNA force-extension was measured. With this experimental strategy, we measured the polymerization of the protein RecA on an isolated double stranded DNA. We observed the progressive decoration of RecA on the l DNA molecule, which results in the extension of l , due to unwinding of the double helix. The dynamics of polymerization, the resulting change in the DNA entropic elasticity and the role of ATP hydrolysis were the main parts of the study. A simple model for RecA assembly on DNA was proposed. This work presents a first step in the study of genetic recombination. Recently we have started a study of equilibrium binding which utilizes fluorescence polarization methods to probe the polymerization of RecA on single stranded DNA. In addition to the study of material properties of DNA and DNA-RecA, we have developed experiments for which the code of the DNA is central. We studied one aspect of DNA as a molecular code, using different techniques. In particular the programmatic use of template specificity makes

  6. Development of Attendance Database System Using Bar-coded Student Card

    Directory of Open Access Journals (Sweden)

    Abdul Fadlil

    2008-04-01

    Full Text Available The calculation of the level of attendance is very important, because one indicator of a person's credibility can be seen from the level of attendance. For example, at a university, data about the level of attendance of a student in a lecture is very important as one of components in the assessment. The manual presence system is considered less effective. This research presents the draft of presence system using bar codes (barcodes as input data representing the attendance. The presence system is supported by three main components, those are a bar code found on the student card (KTM, a CCD barcode scanner series and a CD-108E computer. Management of attendance list using this system allows for optimization of functions of KTM. The presence system has been tested with several KTM through a variety of distances and positions of the barcode scanner barcode. The test results is obtained at ideal position for reading a barcode when a barcode scanner is at 2 cm from the object with 90 degree. At this position the level of accuracy reach 100%.

  7. Equipment Inventory Management and Transaction Recording Using Bar Coding Scheme via VB6

    Directory of Open Access Journals (Sweden)

    Geoffrey T. Salvador, PECE

    2016-06-01

    Full Text Available The aim of the study is to implement bar coding system developed through the VB6 and Microsoft Access as mechanism for the PUP ECE Laboratory Transaction recording and monitoring. The study was concerned on proper documenting and managing the daily transaction of the ECE Laboratory with the AutoLab System.Results showed that the AutoLab System effectively automated the recording of transactions merging the existing manual method into one recording mechanism. The Automated Laboratory coined as AutoLab merged the ECE Room Utilization Log Book, ECE Borrower’s Slip and the ECE Transaction Log Book into one complete package in terms of transaction recording and equipment inventory monitoring

  8. Thin-layer immunoaffinity chromatography with bar code quantitation of C-reactive protein.

    Science.gov (United States)

    Nilsson, S; Lager, C; Laurell, T; Birnbaum, S

    1995-09-01

    A rapid thin-layer immunoaffinity chromatographic method for quantitation in serum of an acute phase reactant, C-reactive protein (CRP), which can differentiate between viral and bacteria] infections, is described, where material and reagent costs are minimal. The analysis is based on the "sandwich" assay format using monoclonal antibodies directed against two sites of CRP. One of the antibodies is covalently bound to defined zones on a thin-layer immunoaffinity chromatography membrane, while the other antibody is covalently bound to deeply dyed blue latex particles. After incubation (CRP sample and latex particles), the CRP-latex immunocomplex is allowed to migrate along the immunoaffinity chromatography membrane. In the presence of antigen, a sandwich is formed between the CRP-latex immunocomplex and membrane-bound antibodies, which results in the appearance of blue lines on the membrane. Antibody immobilization on the TLC membrane is made with a redesigned piezoelectric-driven ink-jet printer. The time required for the analysis is less than 10 min. Quantitation is achieved either by counting the lines visually, with scanning reflectometry, or with a modified bar code reader. The limit of detection was estimated in the low femtomolar range using the naked eye as detector.

  9. Enhancing transfusion safety with an innovative bar-code-based tracking system.

    Science.gov (United States)

    Askeland, Ryan W; McGrane, Steve P; Reifert, Dan R; Kemp, John D

    2009-01-01

    In an effort to reduce transfusion errors, a novel, comprehensive, computerized wireless bar-code-based tracking system for matching patients, blood samples and blood products was created and deployed at a major academic medical centre. With a grant from the Agency for Healthcare Research and Quality, software was developed to track scans at the times of sample collection, sample arrival in the blood bank, blood product dispensation from the blood bank and blood product administration. The system was deployed in February 2005. The system was well accepted from the outset, and the sample rejection rate due to clerical errors fell from 1.82 to 0.17%; incident reports fell by 83%. At the final blood administration step, the accumulated data as of November 2008 indicated that identification errors were being detected and prevented every 42.4 days and that the scan completion rate was stable at about 99%. Process analysis suggested that these were independent events and, thus, would be expected to coincide (and potentially produce a mis-transfusion) every 4,240 days (11.6 years) on average. We estimate that the system is 10 times safer than the manual system previously employed at our institution and may be 15-20 times safer than most systems employed in the United States.

  10. Pediatric medication administration errors and workflow following implementation of a bar code medication administration system.

    Science.gov (United States)

    Hardmeier, Anna; Tsourounis, Candy; Moore, Mary; Abbott, Wendy E; Guglielmo, B Joseph

    2014-01-01

    Direct observation was used to detect medication errors and Bar Code Medication Administration (BCMA) workarounds on two pediatric units and one neonatal unit at UCSF Benioff Children's Hospital. The study (1) measured the frequency of nursing medication administration-related errors, (2) characterized the types of medication errors, (3) assessed compliance with the institution's six medication administration safety processes, and (4) identified observed workarounds following BCMA implementation. The results of the direct observation were compared to medication administration-related incident reports (IRs) for the same period. The frequency of medication errors was 5% for the three units. Compliance with the process measures was achieved 86% of the time (range 23-100%). Seven medication administration-related IRs were submitted during the same observation period. Three BCMA workarounds were identified; (1) failure to visually confirm patient's identification, (2) failure to compare the medication to the electronic medication administration record at least twice before administration, and (3) charting administration of medication before actual administration. The direct observation methodology identified a low frequency of medication administration errors (MAEs) consistent with post-BCMA implementation. The incident reporting system identified different MAEs than direct observation suggesting that both methods should be used to better characterize the scope of MAEs.

  11. Molecular Combing of DNA: Methods and Applications

    DEFF Research Database (Denmark)

    Nazari, Zeniab Esmail; Gurevich, Leonid

    2013-01-01

    First proposed in 1994, molecular combing of DNA is a technique that allows adsorption and alignment of DNA on the surface with no need for prior modification of the molecule. Since then, many variations of the original method have been devised and used in a wide range of applications from genomic...... of the main methods in molecular combing as well as its major applications in nanotechnology....... studies to nanoelectronics. While molecular combing has been applied in a variety of DNA-related studies, no comprehensive review has been published on different combing methods proposed so far. In this review, the underlying mechanisms of molecular combing of DNA are described followed by discussion...

  12. DNA Based Molecular Scale Nanofabrication

    Science.gov (United States)

    2015-12-04

    water adsorption on DNA origami template and its impact on DNA- mediated chemical reactions. We also extended the concept of DNA- mediated reaction to...addition, we have expanded our efforts to include DNA- mediated HF etching of SiÜ2, DNA- mediated nanoimprinting lithography, DNA-based patterning of self...detailed kinetics study of DNA- mediated chemical reactions. Examples of such reactions include chemical vapor deposition (CVD) of inorganic oxide and HF

  13. A versatile, bar-coded nuclear marker/reporter for live cell fluorescent and multiplexed high content imaging.

    Directory of Open Access Journals (Sweden)

    Irina Krylova

    Full Text Available The screening of large numbers of compounds or siRNAs is a mainstay of both academic and pharmaceutical research. Most screens test those interventions against a single biochemical or cellular output whereas recording multiple complementary outputs may be more biologically relevant. High throughput, multi-channel fluorescence microscopy permits multiple outputs to be quantified in specific cellular subcompartments. However, the number of distinct fluorescent outputs available remains limited. Here, we describe a cellular bar-code technology in which multiple cell-based assays are combined in one well after which each assay is distinguished by fluorescence microscopy. The technology uses the unique fluorescent properties of assay-specific markers comprised of distinct combinations of different 'red' fluorescent proteins sandwiched around a nuclear localization signal. The bar-code markers are excited by a common wavelength of light but distinguished ratiometrically by their differing relative fluorescence in two emission channels. Targeting the bar-code to cell nuclei enables individual cells expressing distinguishable markers to be readily separated by standard image analysis programs. We validated the method by showing that the unique responses of different cell-based assays to specific drugs are retained when three assays are co-plated and separated by the bar-code. Based upon those studies, we discuss a roadmap in which even more assays may be combined in a well. The ability to analyze multiple assays simultaneously will enable screens that better identify, characterize and distinguish hits according to multiple biologically or clinically relevant criteria. These capabilities also enable the re-creation of complex mixtures of cell types that is emerging as a central area of interest in many fields.

  14. Improving transfusion safety: implementation of a comprehensive computerized bar code-based tracking system for detecting and preventing errors.

    Science.gov (United States)

    Askeland, R W; McGrane, S; Levitt, J S; Dane, S K; Greene, D L; Vandeberg, J A; Walker, K; Porcella, A; Herwaldt, L A; Carmen, L T; Kemp, J D

    2008-07-01

    To transfuse blood products safely, health care workers must accurately identify patients, blood samples, and the blood components. A comprehensive bar code-based computerized tracking system was developed and implemented to identify and prevent transfusion errors. A data network, wireless devices, and bar-coded labels were pilot tested before the system was introduced hospitalwide. The system provided a complete audit trail for all transactions. Data from before and after implementation were analyzed. Incident reports decreased from a mean of 41.5 reports per month in the 6 months before the system was implemented to a mean of 7.2 reports per month after implementation. The blood sample rejection rate decreased from 1.82 percent to a mean of 0.17 percent after implementation. Errors detected by the new system were sorted into misscans, skipped steps, wrong steps, and prevented identification errors (PIEs). Misscans and skipped steps were the most common errors in the first 10 months after implementation. During the final transfusion step, PIEs occurred at the rate of about one per month and scans were omitted approximately 1 percent of the time. Therefore, it is estimated that mistransfusions could occur about once every 100 months on average with the new system. The bar code-based computerized tracking system detected and prevented identification and matching errors, thereby reducing the proportion of blood samples rejected and increasing patient safety.

  15. Terahertz molecular resonance of cancer DNA

    Science.gov (United States)

    Cheon, Hwayeong; Yang, Hee-Jin; Lee, Sang-Hun; Kim, Young A.; Son, Joo-Hiuk

    2016-11-01

    Carcinogenesis involves the chemical and structural alteration of biomolecules in cells. Aberrant methylation of DNA is a well-known carcinogenic mechanism and a common chemical modification of DNA. Terahertz waves can directly observe changes in DNA because the characteristic energies lie in the same frequency region. In addition, terahertz energy levels are not high enough to damage DNA by ionization. Here, we present terahertz molecular resonance fingerprints of DNA methylation in cancer DNA. Methylated cytidine, a nucleoside, has terahertz characteristic energies that give rise to the molecular resonance of methylation in DNA. Molecular resonance is monitored in aqueous solutions of genomic DNA from cancer cell lines using a terahertz time-domain spectroscopic technique. Resonance signals can be quantified to identify the types of cancer cells with a certain degree of DNA methylation. These measurements reveal the existence of molecular resonance fingerprints of cancer DNAs in the terahertz region, which can be utilized for the early diagnosis of cancer cells at the molecular level.

  16. Nanomechanical molecular devices made of DNA origami.

    Science.gov (United States)

    Kuzuya, Akinori; Ohya, Yuichi

    2014-06-17

    CONSPECTUS: Eight years have passed since the striking debut of the DNA origami technique ( Rothemund, P. W. K. Nature 2006 , 440 , 297 - 302 ), in which long single-stranded DNA is folded into a designed nanostructure, in either 2D or 3D, with the aid of many short staple strands. The number of proposals for new design principles for DNA origami structures seems to have already reached a peak. It is apparent that DNA origami study is now entering the second phase of creating practical applications. The development of functional nanomechanical molecular devices using the DNA origami technique is one such application attracting significant interest from researchers in the field. Nanomechanical DNA origami devices, which maintain the characteristics of DNA origami structures, have various advantages over conventional DNA nanomachines. Comparatively high assembly yield, relatively large size visible via atomic force microscopy (AFM) or transmission electron microscopy (TEM), and the capability to assemble multiple functional groups with precision using multiple staple strands are some of the advantages of the DNA origami technique for constructing sophisticated molecular devices. This Account describes the recent developments of such nanomechanical DNA origami devices and reviews the emerging target of DNA origami studies. First, simple "dynamic" DNA origami structures with transformation capability, such as DNA origami boxes and a DNA origami hatch with structure control, are briefly summarized. More elaborate nanomechanical DNA origami devices are then reviewed. The first example describes DNA origami pinching devices that can be used as "single-molecule" beacons to detect a variety of biorelated molecules, from metal ions at the size of a few tens of atomic mass number units to relatively gigantic proteins with a molecular mass greater than a hundred kilodaltons, all on a single platform. Clamshell-like DNA nanorobots equipped with logic gates can discriminate

  17. Molecular mechanisms of DNA photodamage

    Energy Technology Data Exchange (ETDEWEB)

    Starrs, S.M

    2000-05-01

    Photodamage in DNA, caused by ultraviolet (UV) light, can occur by direct excitation of the nucleobases or indirectly via the action of photosensitisers. Such, DNA photodamage can be potentially mutagenic or lethal. Among the methods available for detecting UV-induced DNA damage, gel sequencing protocols, utilising synthetic oligodeoxyribonucleotides as targets for UV radiation, allow photolesions to be mapped at nucleotide resolution. This approach has been applied to investigate both DNA damage mechanisms. Following a general overview of DNA photoreactivity, and a description of the main experimental procedures, Chapter 3 identifies the origin of an anomalous mobility shift observed in purine chemical sequence ladders that can confuse the interpretation of DNA cleavage results; measures to abolish this shift are also described. Chapters 4 and 5 examine the alkali-labile DNA damage photosensitised by representative nonsteroidal antiinflammatory drugs (NSAIDs) and the fluoroquinolone antibiotics. Suprofen was the most photoactive NSAID studied, producing different patterns of guanine-specific damage in single-stranded and duplex DNA. Uniform modification of guanine bases, typifying attack by singlet oxygen, was observed in single-stranded oligodeoxyribonucleotides. In duplex molecules, modification was limited to the 5'-G of GG doublets, which is indicative of an electron transfer. The effect of quenchers and photoproduct analysis substantiated these findings. The quinolone, nalidixic acid, behaves similarly. The random base cleavage photosensitised by the fluoroquinolones, has been attributed to free radicals produced during their photodecomposition. Chapter 6 addresses the photoreactivity of purines within unusual DNA structures formed by the repeat sequences (GGA){sub n} and (GA){sub n}, and a minihairpin. There was no definitive evidence for enhanced purine reactivity caused by direct excitation. Finally, Chapter 7 investigates the mutagenic potential of a

  18. Temperature effect on DNA molecular wires

    Science.gov (United States)

    Bui, Christopher Minh

    The demand of technology and information today has further pushed the fabrication process of nanotechnology, yet there are limits and obstacles set by the primary laws of physics. Therefore, researchers are pursuing alternative technologies. Deoxyribonucleic acids (DNA) molecular wire is one advantageous option due to its unique characteristics including self-assembly and naturally small; size. This thesis reports the temperature effect on the electrical properties of a double-stranded ?-DNA molecular wire. The data will help expand the DNA wire application and functionality. Thus, the data supports the charge hopping theory on DNA electrical conductivity. Diverse amount of literatures has demonstrated that DNA experiences a biochemical alteration when exposed under different temperature conditions. This change will also cause a change in the electrical properties. In this research, DNA will hang between two gold covered microelectrodes with a distance of 10 to 12 microns. The microelectrodes are fabricated through negative lithography techniques. Then, the samples were exposed to a numerous range of temperature from 25°C to 180°C and went through varying cycles of heating and cooling. The experimental results revealed that the DNA experienced a hysteresis like behavior where the impedance differed between the heating and cooling phase. The impedance of the DNA molecular wire increased when exposed to higher temperature. Furthermore, the impedance stops increasing after a certain amount of heat cycles before the DNA structure failed. The biology and thermodynamics of the DNA wire was analyzed due to the temperature hysteresis effect. The melting temperature and the bond dissociation temperature were evaluated to determine the cause of the impedance trends. The studies and analysis of the temperature effect provided certain insights towards the charge hopping transport mechanism. The thesis concludes with possible applications relating to the temperature effect of

  19. Scaffolded DNA Origami of a DNA Tetrahedron Molecular Container

    DEFF Research Database (Denmark)

    Ke, Yongang; Sharma, Jaswinder; Liu, Minghui

    2009-01-01

    We describe a strategy of scaffolded DNA origami to design and construct 3D molecular cages of tetrahedron geometry with inside volume closed by triangular faces. Each edge of the triangular face is ∼54 nm in dimension. The estimated total external volume and the internal cavity of the triangular...... pyramid are about 1.8 × 10-23 and 1.5 × 10-23 m3, respectively. Correct formation of the tetrahedron DNA cage was verified by gel electrophoresis, atomic force microscopy, transmission electron microscopy, and dynamic light scattering techniques....

  20. Programmable DNA Nanosystem for Molecular Interrogation

    Science.gov (United States)

    Mathur, Divita; Henderson, Eric R.

    2016-06-01

    We describe a self-assembling DNA-based nanosystem for interrogating molecular interactions. The nanosystem contains a rigid supporting dumbbell-shaped frame, a cylindrical central core, and a mobile ring that is coaxial with the core. Motion of the ring is influenced by several control elements whose force-generating capability is based on the transition of single-stranded DNA to double-stranded DNA. These forces can be directed to act in opposition to adhesive forces between the ring and the frame thereby providing a mechanism for molecular detection and interrogation at the ring-frame interface. As proof of principle we use this system to evaluate base stacking adhesion and demonstrate detection of a soluble nucleic acid viral genome mimic.

  1. Bar Code Technology of Interactive Teaching%条形码技术的互动教学

    Institute of Scientific and Technical Information of China (English)

    林胜青

    2014-01-01

    学习现代科学技术,并结合在生活中实际应用,尽量提倡师生用互动式教学,推动当今的现代化教育,提高教育质量。本文介绍利用互动式教学方法,讲授条形码的制作原理及实际应用。%Learning modern science and technology,and connecting with the practical application in our daily life,try to advocate using interactive teaching between teachers and students,promote the today's modern education,improve the quality of education. This article shows that using interactive teaching methods,teaching of bar code principle and prac-tical application.

  2. High-speed, high-precision thermal printing heads for bar code printers; Kosoku koseisai bar cord printer yo TPH

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    2000-03-01

    Thermal printing heads (TPHs), having the world's first resolution of 24 dots/mm and printing speed of 254 mm/s, have been developed. The high-precision, high-durability TPH is realized, based on the high-precision techniques as one of the company's strong areas, combined with the techniques for high power-resistant film structure and high wear-resistant protective film. At the same time, the structure of high thermal conductivity and thermal efficiency is adopted, to control heat accumulation and realize high-quality images. It is expected to find wide use in various areas, centered by distribution industry, e.g., for bar code label printers, and name plate, postal card and name card printing, with the standardized recording width of A6 size and resolution of 8 to 24 dots/mm. (translated by NEDO)

  3. Study on Bar-coding of Wheat Variety Based on Genetic Diversity of Seed Storage Protein%基于籽粒贮藏蛋白遗传多样性的小麦条形码研究

    Institute of Scientific and Technical Information of China (English)

    康志钰; 王建军

    2012-01-01

    为便于小麦品种管理及保护,针对植物DNA条形码研制存在的问题,以36份品种为材料,分析其HMW-GS和醇溶蛋白组分,并根据谱带的有无,对谱带进行数量化处理,存在的谱带标为1,不存在的谱带标为0,建立谱带二进制代码,再转化为十进制代码,最后通过数据整合,建立了小麦品种身份识别码,并将其转换为条形码,研制出基于籽粒贮藏蛋白遗传多样性的小麦身份识别码制作方法,使原来需要用119位数字表明的品种间差距现在只需37位数字即可表示出来。%To be convenient for the management and protection of wheat varieties, and aimed at the problems on DNA bar-coding of plant, the high molecular weight gluten subunit (HMW-GS) and gli- adin of 36 wheat varieties were investigated and used to establish their codes. By number processing, according to the presence and absence of the bands as presence of band was signed with "1" and ab- sence of band was signed with "0", the binary code system was established and then the binary code system was translated into decimal code system, finally, the identification code system of wheat varie- ty was established through the conformity of data, and translated the identification code for bar-cod- ing. An method for the identification code system of wheat was built based on the genetic diversity of seed storage protein. In this way, the difference between the varieties could be distinguished by 37 digits, instead of 119 digits used in the past.

  4. Design of Bar Code Automatic Printing System Based on LabVIEW%基于LabVIEW的条码自动打印系统设计

    Institute of Scientific and Technical Information of China (English)

    周先飞; 李敏; 杨会伟

    2016-01-01

    汽车行业中各个设备都来自不同的供应商,要实现产品溯源需从条码去区别。针对汽车大灯产品检测系统之后的自动打印条码,实现检测过程的自动化要求。提出了采用单片机制作的数据采集卡和LabVIEW建立的上位机软件,实现对检测完好的产品进行条码的自动打印输出,在上位机中可以对不同型号的产品进行条码的切换,在生产线上具有完全自动化功能,经现场使用,稳定性良好。%In automotive industry ,each equipment comes from different suppliers ,it needs bar codes to distinguish the products. Towards the automatic printing of bar codes after testing of headlight product , the paper realizes the automation of testing process. This paper presents MCU for DAQ and labVIEV for upper computer softwave ,to realize the automatic printing of bar codes tested ,it can change the bar codes in upper computer for differen products. The system has automation function ,and has good stability proved by field use.

  5. Control of DNA hybridization by photoswitchable molecular glue.

    Science.gov (United States)

    Dohno, Chikara; Nakatani, Kazuhiko

    2011-12-01

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

  6. Analysis of run-to-run variation of bar-coded pyrosequencing for evaluating bacterial community shifts and individual taxa dynamics.

    Science.gov (United States)

    Ge, Yuan; Schimel, Joshua P; Holden, Patricia A

    2014-01-01

    Bar-coded pyrosequencing has been increasingly used due to its fine taxonomic resolution and high throughput. Yet, concerns arise regarding the reproducibility of bar-coded pyrosequencing. We evaluated the run-to-run variation of bar-coded pyrosequencing in detecting bacterial community shifts and taxa dynamics. Our results demonstrate that pyrosequencing is reproducible in evaluating community shifts within a run, but not between runs. Also, the reproducibility of pyrosequencing in detecting individual taxa increased as a function of taxa abundance. Based on our findings: (1) for studies with modest sequencing depth, it is doubtful that data from different pyrosequencing runs can be considered comparable; (2) if multiple pyrosequencing runs are needed to increase the sequencing depth, additional sequencing efforts should be applied to all samples, rather than to selected samples; (3) if pyrosequencing is used for estimating bacterial population dynamics, only the abundant taxa should be considered; (4) for less-abundant taxa, the sequencing depth should be increased to ensure an accurate evaluation of taxon variation trends across samples.

  7. Single-molecule studies of DNA by molecular combing

    Institute of Scientific and Technical Information of China (English)

    Liu Yuying; Wang Pengye; Dou Shuoxing

    2007-01-01

    Molecular combing is a powerful method for aligning a large array of DNA molecules onto a surface. It is a process whereby DNA molecules are stretched and aligned on a glass surface by the force via fluid flow. The ability to comb up to several hundred DNAs on a single cover slip allows for a statistically significant number of measurements to be made. These features make molecular combing an attractive tool for genomic studies, such as DNA replication, DNA transcription, DNA-protein interaction and so on. In this review article, we discuss the molecular combing principle, method and its applications.

  8. Envisioning the molecular choreography of DNA base excision repair.

    Science.gov (United States)

    Parikh, S S; Mol, C D; Hosfield, D J; Tainer, J A

    1999-02-01

    Recent breakthroughs integrate individual DNA repair enzyme structures, biochemistry and biology to outline the structural cell biology of the DNA base excision repair pathways that are essential to genome integrity. Thus, we are starting to envision how the actions, movements, steps, partners and timing of DNA repair enzymes, which together define their molecular choreography, are elegantly controlled by both the nature of the DNA damage and the structural chemistry of the participating enzymes and the DNA double helix.

  9. Controlling charge current through a DNA based molecular transistor

    Science.gov (United States)

    Behnia, S.; Fathizadeh, S.; Ziaei, J.

    2017-01-01

    Molecular electronics is complementary to silicon-based electronics and may induce electronic functions which are difficult to obtain with conventional technology. We have considered a DNA based molecular transistor and study its transport properties. The appropriate DNA sequence as a central chain in molecular transistor and the functional interval for applied voltages is obtained. I-V characteristic diagram shows the rectifier behavior as well as the negative differential resistance phenomenon of DNA transistor. We have observed the nearly periodic behavior in the current flowing through DNA. It is reported that there is a critical gate voltage for each applied bias which above it, the electrical current is always positive.

  10. Recent advances in yeast molecular biology: recombinant DNA. [Lead abstract

    Energy Technology Data Exchange (ETDEWEB)

    1982-09-01

    Separate abstracts were prepared for the 25 papers presented at a workshop focusing on chromosomal structure, gene regulation, recombination, DNA repair, and cell type control, that have been obtained by experimental approaches incorporating the new technologies of yeast DNA transformation, molecular cloning, and DNA sequence analysis. (KRM)

  11. Development of a DNA sensor using molecular logic gate

    CERN Document Server

    Bhattacharjee, D; Chakraborty, S; Hussain, Syed Arshad

    2014-01-01

    This communication reports the increase in fluorescence resonance energy transfer (FRET) efficiency between two laser dyes in presence of Deoxyribonucleic acid (DNA). Two types of molecular logic gates have been designed where DNA acts as input signal and fluorescence intensity of different bands are taken as output signal. Use of these logic gates as DNA sensor has been demonstrated

  12. Single DNA molecular manipulation with atomic force microscopy

    Institute of Scientific and Technical Information of China (English)

    L(U) Jun-Hong; WU Shi-Ying; ZHANG Yi; HU Jun; LI Min-Qian

    2004-01-01

    Nanomanipulation of DNA molecules or other biomolecules to form artificial patterns or structures at nanometer scale has potential applications in the construction of molecular devices in future industries. It may also lead to new insights into the interesting properties and behavior of this fantastic nature-selected molecule at the single-molecular level. Here we present a special method based on the combination of macroscopic "molecular combing" and microscopic "molecular cutting" to manipulate DNA molecules and form complex patterns at nanometer scale on solid surfaces. A possible strategy for ordered DNA sequencing based on this nanomanipulation technique has also been proposed.

  13. Streching of (DNA/functional molecules) complex between electrodes towards DNA molecular wire

    Science.gov (United States)

    Kobayashi, Norihisa; Nishizawa, Makoto; Inoue, Shintarou; Nakamura, Kazuki

    2009-08-01

    DNA/functional molecules such as (Ru(bpy)32+ complex, conducting polymer etc.) complex was prepared to study molecular structure and I-V characteristics towards DNA molecular wire. For example, Ru(bpy)32+ was associated with duplex of DNA by not only electrostatic interaction but also intercalation in the aqueous solution. Singlemolecular structure of DNA/Ru(bpy)32+ complex was analyzed with AFM. We found a network structure of DNA/Ru(bpy)32+ complex on the mica substrate, which is similar to native DNA. The height of DNA/Ru(bpy)32+ complex on the mica substrate was ranging from 0.8 to 1.6 nm, which was higher than the naked DNA (0.5-1.0 nm). This indicates that single-molecular DNA/Ru(bpy)32+ complex also connects to each other to form network structure on a mica substrate. In order to stretch DNA complex between electrodes, we employed high frequency and high electric field stretching method proposed by Washizu et al. We stretched and immobilized DNA single molecules between a pair of electrodes and its structures were analyzed with AFM technique. The I-V characteristics of DNA single molecules between electrodes were improved by the association of functional molecules with DNA. The molecular structure and I-V characteristics of DNA complex were discussed.

  14. Electrophoretic High Molecular Weight DNA Purification Enables Optical Mapping

    Science.gov (United States)

    Maydan, Jason; Thomas, Matthew; Tabanfar, Leyla; Mai, Laura; Poon, Hau-Ling; Pe, Joel; Hahn, Kristen; Goji, Noriko; Amoako, Kingsley; Marziali, Andre; Hanson, Dan

    2013-01-01

    Optical mapping generates an ordered restriction map from single, long DNA molecules. By overlapping restriction maps from multiple molecules, a physical map of entire chromosomes and genomes is constructed, greatly facilitating genome assembly in next generation sequencing projects, comparative genomics and strain typing. However, optical mapping relies on a method of preparing high quality DNA >250 kb in length, which can be challenging from some organisms and sample types. Here we demonstrate the ability of Boreal Genomics' Aurora instrument to provide pure, high molecular weight (HMW) DNA 250-1,100 kb in length, ideally suited for optical mapping. The Aurora performs electrophoretic DNA purification within an agarose gel in reusable cartridges, protecting long DNA molecules from shearing forces associated with liquid handling steps common to other purification methods. DNA can be purified directly from intact cells embedded and lysed within an agarose gel, preserving the highest molecular weight DNA possible while achieving exceptional levels of purity. The Aurora delivers DNA in a buffer solution, where DNA can be condensed and protected from shearing during recovery with a pipette. DNA is then returned to its regular coiled state by simple dilution prior to optical mapping. Here we present images showing HMW DNA purification taking place in the Aurora and subsequent images of single DNA molecules on OpGen's Argus® Optical Mapping System. Future work will focus on further optimizing Aurora HMW DNA purification to bias DNA recovery in favor of only the longest molecules in a sample, maximizing the benefits of optical mapping.

  15. DNA Nanostructures-Mediated Molecular Imprinting Lithography.

    Science.gov (United States)

    Tian, Cheng; Kim, Hyojeong; Sun, Wei; Kim, Yunah; Yin, Peng; Liu, Haitao

    2017-01-24

    This paper describes the fabrication of polymer stamps using DNA nanostructure templates. This process creates stamps having diverse nanoscale features with dimensions ranging from several tens of nanometers to micrometers. DNA nanostructures including DNA nanotubes, stretched λ-DNA, two-dimensional (2D) DNA brick crystals with three-dimensional (3D) features, hexagonal DNA 2D arrays, and triangular DNA origami were used as master templates to transfer patterns to poly(methyl methacrylate) and poly(l-lactic acid) with high fidelity. The resulting polymer stamps were used as molds to transfer the pattern to acryloxy perfluoropolyether polymer. This work establishes an approach to using self-assembled DNA templates for applications in soft lithography.

  16. Antibody-controlled actuation of DNA-based molecular circuits

    Science.gov (United States)

    Engelen, Wouter; Meijer, Lenny H. H.; Somers, Bram; de Greef, Tom F. A.; Merkx, Maarten

    2017-02-01

    DNA-based molecular circuits allow autonomous signal processing, but their actuation has relied mostly on RNA/DNA-based inputs, limiting their application in synthetic biology, biomedicine and molecular diagnostics. Here we introduce a generic method to translate the presence of an antibody into a unique DNA strand, enabling the use of antibodies as specific inputs for DNA-based molecular computing. Our approach, antibody-templated strand exchange (ATSE), uses the characteristic bivalent architecture of antibodies to promote DNA-strand exchange reactions both thermodynamically and kinetically. Detailed characterization of the ATSE reaction allowed the establishment of a comprehensive model that describes the kinetics and thermodynamics of ATSE as a function of toehold length, antibody-epitope affinity and concentration. ATSE enables the introduction of complex signal processing in antibody-based diagnostics, as demonstrated here by constructing molecular circuits for multiplex antibody detection, integration of multiple antibody inputs using logic gates and actuation of enzymes and DNAzymes for signal amplification.

  17. Atomistic Molecular Dynamics Simulations of Mitochondrial DNA Polymerase γ

    DEFF Research Database (Denmark)

    Euro, Liliya; Haapanen, Outi; Róg, Tomasz

    2017-01-01

    DNA polymerase γ (Pol γ) is a key component of the mitochondrial DNA replisome and an important cause of neurological diseases. Despite the availability of its crystal structures, the molecular mechanism of DNA replication, the switch between polymerase and exonuclease activities, the site...... of replisomal interactions, and functional effects of patient mutations that do not affect direct catalysis have remained elusive. Here we report the first atomistic classical molecular dynamics simulations of the human Pol γ replicative complex. Our simulation data show that DNA binding triggers remarkable...

  18. 一种改良的镰形棘豆DNA提取方法%A Modified DNA Extraction forLianXingJiDou

    Institute of Scientific and Technical Information of China (English)

    黄聪琳; 郭敏; 王晓琳; 姜华

    2016-01-01

    Objective: To establish a DNA extraction method fromLianXingJiDou(OxytropisfalcateBunge) for DNA bar code analysis. Methods: Leaves ofLianXingJiDouwere used to extract DNA by using a modified DNA method; agarose gel electrophoresis and PCR amplification were used to detect extracted DNA. Results: The DNA extracted by this method could meet basic requirements for PCR molecular identification. The method is simple, time-saving, and low-cost; it is suitable for extraction in large quantities. Conclusion: This method is a proper method of DNA extraction for bar code analysis.%目的:建立适合DNA条形码分析的镰形棘豆DNA提取方法。方法:以镰形棘豆(Oxytropis falcate Bunge)叶片为材料,采用改良的DNA提取方法对其DNA进行提取,并用琼脂糖凝胶电泳和PCR扩增对提取的DNA进行检测。结果:本方法提取的DNA,能够满足以PCR为基础的分子鉴定的基本要求。操作简便,需时短,成本低廉,适于DNA的大批量提取。结论:本方法适合DNA条形码研究的DNA提取。

  19. Molecular dynamics simulations of a single stranded (ss) DNA

    CERN Document Server

    Chatterjee, Subhasish; Thakur, Siddarth; Burin, Alexander

    2012-01-01

    The objective of the present study was to develop an understanding of short single-stranded DNA (ssDNA) to assist the development of new DNA-based biosensors. A ssDNA model containing twelve bases was constructed from the 130-145 codon sequence of the p53 gene. Various thermodynamic macroscopic observables such as temperature, energy distributions, as well as root mean square deviation (RMSD) of the nucleic acid backbone of the ssDNA were studied using molecular dynamics (MD) simulations. The AMBER program was used for building the structural model of the ssDNA, and atomistic MD simulations in three different ensembles were carried out using the NAMD program. The microcanonical (NVE), conical (NVT) and isobaric-isothermal (NPT) ensembles were employed to compare the equilibrium characteristics of ssDNA in aqueous solutions. Our results indicate that the conformational stability of the ssDNA is dependent on the thermodynamic conditions.

  20. Establishing and evaluating bar-code technology in blood sampling system: a model based on human centered human-centered design method.

    Science.gov (United States)

    Chou, Shin-Shang; Yan, Hsiu-Fang; Huang, Hsiu-Ya; Tseng, Kuan-Jui; Kuo, Shu-Chen

    2012-01-01

    This study intended to use a human-centered design study method to develop a bar-code technology in blood sampling process. By using the multilevel analysis to gather the information, the bar-code technology has been constructed to identify the patient's identification, simplify the work process, and prevent medical error rates. A Technology Acceptance Model questionnaire was developed to assess the effectiveness of system and the data of patient's identification and sample errors were collected daily. The average scores of 8 items users' perceived ease of use was 25.21(3.72), 9 items users' perceived usefulness was 28.53(5.00), and 14 items task-technology fit was 52.24(7.09), the rate of patient identification error and samples with order cancelled were down to zero, however, new errors were generated after the new system deployed; which were the position of barcode stickers on the sample tubes. Overall, more than half of nurses (62.5%) were willing to use the new system.

  1. Molecular Mechanisms of DNA Polymerase Clamp Loaders

    Science.gov (United States)

    Kelch, Brian; Makino, Debora; Simonetta, Kyle; O'Donnell, Mike; Kuriyan, John

    Clamp loaders are ATP-driven multiprotein machines that couple ATP hydrolysis to the opening and closing of a circular protein ring around DNA. This ring-shaped clamp slides along DNA, and interacts with numerous proteins involved in DNA replication, DNA repair and cell cycle control. Recently determined structures of clamp loader complexes from prokaryotic and eukaryotic DNA polymerases have revealed exciting new details of how these complex AAA+ machines perform this essential clamp loading function. This review serves as background to John Kuriyan's lecture at the 2010 Erice School, and is not meant as a comprehensive review of the contributions of the many scientists who have advanced this field. These lecture notes are derived from recent reviews and research papers from our groups.

  2. DNA strand exchange catalyzed by molecular crowding in PEG solutions

    KAUST Repository

    Feng, Bobo

    2010-01-01

    DNA strand exchange is catalyzed by molecular crowding and hydrophobic interactions in concentrated aqueous solutions of polyethylene glycol, a discovery of relevance for understanding the function of recombination enzymes and with potential applications to DNA nanotechnology. © 2010 The Royal Society of Chemistry.

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

    Science.gov (United States)

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

    2006-08-01

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

  4. DNA Vaccine Electroporation and Molecular Adjuvants

    Science.gov (United States)

    2016-03-16

    according to the manufacturer’s suggested values (see Note 6). 3. Draw DNA solution into syringe (see Note 7). 4. Anesthetize the animal with the...vaccination is an attractive method for inducing protective immunity to a variety of pathogens, but the low immunogenicity seen in larger animals and...for generating protective immunity against filovirus infection [11]. The demonstration of effective DNA vaccination in small animal models changed the

  5. Molecular threading and tunable molecular recognition on DNA origami nanostructures.

    Science.gov (United States)

    Wu, Na; Czajkowsky, Daniel M; Zhang, Jinjin; Qu, Jianxun; Ye, Ming; Zeng, Dongdong; Zhou, Xingfei; Hu, Jun; Shao, Zhifeng; Li, Bin; Fan, Chunhai

    2013-08-21

    The DNA origami technology holds great promise for the assembly of nanoscopic technological devices and studies of biochemical reactions at the single-molecule level. For these, it is essential to establish well controlled attachment of functional materials to predefined sites on the DNA origami nanostructures for reliable measurements and versatile applications. However, the two-sided nature of the origami scaffold has shown limitations in this regard. We hypothesized that holes of the commonly used two-dimensional DNA origami designs are large enough for the passage of single-stranded (ss)-DNA. Sufficiently long ssDNA initially located on one side of the origami should thus be able to "thread" to the other side through the holes in the origami sheet. By using an origami sheet attached with patterned biotinylated ssDNA spacers and monitoring streptavidin binding with atomic force microscopic (AFM) imaging, we provide unambiguous evidence that the biotin ligands positioned on one side have indeed threaded through to the other side. Our finding reveals a previously overlooked critical design feature that should provide new interpretations to previous experiments and new opportunities for the construction of origami structures with new functional capabilities.

  6. Molecular dynamics simulation of peeling a DNA molecule on substrate

    Institute of Scientific and Technical Information of China (English)

    Xinghua Shi; Yong Kong; Yapu Zhao; Huajian Gao

    2005-01-01

    Molecular dynamics (MD) simulations are performed to study adhesion and peeling of a short fragment of single strand DNA (ssDNA) molecule from a graphite surface. The critical peel-off force is found to depend on both the peeling angle and the elasticity of ssDNA. For the short ssDNA strand under investigation, we show that the simulation results can be explained by a continuum model of an adhesive elastic band on substrate. The analysis suggests that it is often the peak value, rather than the mean value, of adhesion energy which determines the peeling of a nanoscale material.

  7. Single Molecular Demonstration of Modulating Charge Inversion of DNA

    Science.gov (United States)

    Wang, Yanwei; Wang, Ruxia; Cao, Bozhi; Guo, Zilong; Yang, Guangcan

    2016-12-01

    Charge inversion of DNA is a counterintuitive phenomenon in which the effective charge of DNA switches its sign from negative to positive in the presence of multivalent counterions. The underlying microscopic mechanism is still controversial whether it is driven by a specific chemical affinity or electrostatic ion correlation. It is well known that DNA shows no charge inversion in normal aqueous solution of trivalent counterions though they can induce the conformational compaction of DNA. However, in the same trivalent counterion condition, we demonstrate for the first time the occurrence of DNA charge inversion by decreasing the dielectric constant of solution to make the electrophoretic mobility of DNA increase from a negative value to a positive value. In contrast, the charge inversion of DNA induced by quadrivalent counterions can be canceled out by increasing the dielectric constant of solution. The physical modulation of DNA effective charge in two ways unambiguously demonstrates that charge inversion of DNA is a predominantly electrostatic phenomenon driven by the existence of a strongly correlated liquid (SCL) of counterions at the DNA surface. This conclusion is also supported by the measurement of condensing and unraveling forces of DNA condensates by single molecular MT.

  8. Development and application of DNA molecular probes

    Directory of Open Access Journals (Sweden)

    Priya Vizzini

    2017-02-01

    Full Text Available The development of DNA probes started from 1950's for diagnostic purposes and it is still growing. DNA probes are applied in several fields such as food, medical, veterinary, environment and security, with the aim of prevention, diagnosis and treatment. The use of DNA probes permits microorganism identification, including pathogen detection, and their quantification when used in specific systems. Various techniques obtained success by the utilization of specific DNA probes, that allowed the obtainment of rapid and specific results. From PCR, qPCR and blotting techniques that were first used in well equipped laboratories to biosensors such as fiber optic, surface plasmon resonance (SPR, electrochemical, and quartz crystal microbalance (QCM biosensors that use different transduction systems. This review describes i the design and production of primers and probes, and their utilization from the traditional techniques to the new emerging techniques like biosensors used in current applications; ii the possibility to use labelled-free probes and probes labelled with an enzyme/fluorophore, etc.; iii the different sensitivity obtained by using specific systems; and iv the advantage obtained by using biosensors.

  9. Molecular crowding effects on stability of DNA double helix

    Science.gov (United States)

    Singh, Amar; Singh, Navin

    2016-05-01

    Cellular environmental conditions critically affect the structure and stability of double stranded DNA (dsDNA) molecule. It is known that 20-30% of the total volume of the cell is occupied by the molecular crowders. The presence of these crowders, reduces the free space available to the base pairs of a DNA molecule, hence the movement of base pair is restricted. Here, we study the thermal opening of dsDNA molecule using Peyrard Bishop Dauxois (PBD) model. The presence of crowders in the model, that mimic those found in the cell nucleus, is realized through the potential term. Using the equilibrium statistical calculations, we find melting profile and melting probabilities of the chain. The opening of DNA molecule in the presence of these crowders is shown through the density plots. This study reveals that the stability of dsDNA molecule is influenced by entropic as well as enthalpic effects and is more stable in the crowded environment.

  10. Spectroscopic and molecular docking studies on chlorambucil interaction with DNA.

    Science.gov (United States)

    Charak, Sonika; Shandilya, Manish; Tyagi, Gunjan; Mehrotra, Ranjana

    2012-11-01

    Chlorambucil (CMB) is an anticancer drug used for the treatment of variety of cancers. Structural and conformational changes associated with DNA after binding with CMB were explored using spectroscopic techniques to get insight into the mechanism of action of CMB at molecular level. Different molar ratios of CMB-DNA complex were prepared with constant DNA concentration under physiological conditions. FTIR spectroscopy, UV-visible spectroscopy, CD spectroscopy and molecular docking studies were employed to determine the binding site and binding constant of CMB with DNA. The results show CMB binds DNA through nitrogenous bases (thymine, guanine and cytosine). The binding constant was calculated to be 1.3 × 10³ M⁻¹, which suggests weak binding of CMB with DNA double helix. FTIR and CD results show that CMB do not disturb native B-conformation of DNA and it continues to remain in its B conformation even at higher concentrations of CMB. The molecular docking results are in corroboration with our experimental results and provides structural insight into the interaction site. Copyright © 2012 Elsevier B.V. All rights reserved.

  11. Single DNA Condensation Induced by Hexammine Cobalt with Molecular Combing

    Institute of Scientific and Technical Information of China (English)

    Gao-ming Hu; Yu Lin; Shi-yong Ran; Yan-wei Wang; Guang-can Yang

    2012-01-01

    We investigated the interaction between DNA and hexammine cobalt Ⅲ [Co(NH3)6]3+ by a simple molecular combing method and dynamic light scattering.The average extension of λ-DNA-YOYO-1 complex is found to be 20.9 μm,about 30% longer than the contour length of the DNA in TE buffer (10 mmol/L Tris,1 mmol/L EDTA,pH=8.0),due to bis-intercalation of YOYO-1.A multivalent cation,hexammine cobalt,is used for DNA condensation.We find that the length of DNA-[Co(NH3)6]3+ complexes decrease from 20.9 μm to 5.9 μm as the concentration of the [Co(NH3)6]3+ vary from 0 to 3 μmol/L.This observation provides a direct visualization of single DNA condensation induced by hexammine cobalt.The results from the molecular combing studies are supported by dynamic light scattering investigation,where the average hydrodynamic radius of the DNA complex decreases from 203.8 nm to 39.26 nm under the same conditions.It shows that the molecular combing method is feasible for quantitative conformation characterization of single bio-macromolecules.

  12. Metals and DNA: Molecular Left-Handed Complements

    Science.gov (United States)

    Barton, Jacqueline K.

    1986-08-01

    Chiral metal complexes provide unique molecular probes for DNA. Chiral reagents that ``recognize'' different local structures along the DNA strand have been designed by a process in which the asymmetry in shape and size of the complex is matched to that of the DNA helical groove. As a result, the chiral metal complexes provide very sensitive probes for local helical structure, both left- and right-handed. Direct coordination of chiral complexes to the DNA bases adds an element of sequence selectivity to the probe design. With a suitable reactive metal center, reagents that target chemically specific sites along the strand may be developed. One such chiral reagent, which cleaves left-handed DNA sites with photoactivation, has been useful in mapping this distinct conformation and examining its biological role. The conformation-specific molecular cleaver, much like a DNA-binding enzyme, recognizes and reacts at discrete sites along the DNA strand. These site-specific chiral metal complexes provide exciting new tools for probing the local variations in DNA structure and its role in the regulation of gene expression.

  13. Improved Lower Bounds for Constant GC-Content DNA Codes

    CERN Document Server

    Chee, Yeow Meng

    2008-01-01

    The design of large libraries of oligonucleotides having constant GC-content and satisfying Hamming distance constraints between oligonucleotides and their Watson-Crick complements is important in reducing hybridization errors in DNA computing, DNA microarray technologies, and molecular bar coding. Various techniques have been studied for the construction of such oligonucleotide libraries, ranging from algorithmic constructions via stochastic local search to theoretical constructions via coding theory. We introduce a new stochastic local search method which yields improvements up to more than one third of the benchmark lower bounds of Gaborit and King (2005) for n-mer oligonucleotide libraries when n <= 14. We also found several optimal libraries by computing maximum cliques on certain graphs.

  14. Human DNA ligase and DNA polymerase as molecular targets for heavy metals and anticancer drugs

    Energy Technology Data Exchange (ETDEWEB)

    Yang, S.

    1992-01-01

    DNA ligase and DNA polymerase play important roles in DNA replication, repair, and recombination. Frequencies of spontaneous and chemical- and physical-induced mutations are correlated to the fidelity of DNA replication. This dissertation elucidates the mechanisms of the DNA ligation reaction by DNA ligases and demonstrates that human DNA ligase I and DNA polymerase [alpha] are the molecular targets for two metal ions, Zn[sup 2+] and Cd[sup 2+], and an anticancer drug, F-ara-ATP. The formation of the AMP-DNA intermediate and the successive ligation reaction by human DNA ligases were analyzed. Both reactions showed their substrate specificity for ligases I and II, required Mg2+, and were inhibited by ATP. A protein inhibitor from HeLa cells and specific for human DNA ligase I but not ligase II and T4 ligase was discovered. It reversibly inhibited DNA ligation activity but not the AMP-binding activity due to the formation of a reversible ligase I-inhibitor complex. F-ara-ATP inhibited human DNA ligase I activity by competing with ATP for the AMP-binding site of DNA ligase I, forming a ligase I-F-ara-AMP complex, as well as when it was incorporated at 3[prime]-terminus of DNA nick by DNA polymerase [alpha]. All steps of the DNA ligation reaction were inhibited by Zn[sup 2+] and Cd[sup 2+] in a concentration-dependent manner. Both ions did not show the ability to change the fidelity of DNA ligation reaction catalyzed by human DNA ligase I. However, Zn[sup 2+] and Cd[sup 2+] showed their contradictory effects on the fidelity of the reaction by human DNA polymerase [alpha]. Zn[sup 2+] decreased the frequency of misinsertion but less affected that of mispair extension. On the contrary, Cd[sup 2+] increased the frequencies of both misinsertion and mispair extension at very low concentration. The data provided strong evidence in the molecular mechanisms for the mutagenicity of zinc and cadmium, and were comparable with the results previously reported.

  15. Application and Implementation of Bar Code and Two-dimensional Bar Code in Library Personalized Services%条形码/二维码技术在图书馆个性化服务中的应用与实现

    Institute of Scientific and Technical Information of China (English)

    李健; 杨京峰; 张成昱

    2012-01-01

    针对国内图书馆中地理信息的缺失,介绍条形码/二维码在国内外的研究现状,利用Android等技术,设计并实现以图书导航服务为中心的图书馆个性化服务系统,重点介绍该系统的用户需求、设计思路与功能实现。通过对应用效果的评估,认为该系统可以极大地提升图书馆的服务效率和服务质量。%Due to the lack of geographic information in national library,the paper introduces the research of bar code and two-dimensional bar code,using Android and other technology,designs and realizes library personalized services system which is based on book navigation service.It focuses on user needs,design thought and implementation of main functions.According to the assessment of the application,the system can greatly enhance the efficiency and quality of library services.

  16. Molecular Rigidity and Entropy-Enthalpy Compensation in DNA Hybridization

    Science.gov (United States)

    Douglas, Jack; Vargas-Lara, Fernando

    2015-03-01

    Entropy-enthalpy compensation (EEC) is a general and relatively poorly understood pattern in the energetic parameters governing both binding constants and relaxation processes in condensed matter. After defining the basic phenomenology, we focus on how polymer additives, chain confinement, chain length variation affect a well-studied molecular binding process, the hybridization of duplex DNA. Our study is based on a coarse-grained model of DNA that does treat water explicitly. We find that both crowding due to polymer additives and geometrical confinement lead to a change of the effective chain rigidity and that changes in DNA generally lead to a pattern entropy-enthalpy compensation in the DNA association similar to experimental observations. Modulation of the rigidity of binding specifies by constraints associated with chain structure or environmental conditions can greatly influence both the location and cooperativity of molecular binding transition and the relative enthalpy and entropy contributions to the free energy of binding. Entropy-enthalpy compensation arises in numerous synthetic and biological molecular binding processes and we suggest that that changes in molecular rigidity might provide a common explanation of this ubiquitous phenomenon.

  17. Energetics, Thermodynamics, and Molecular Recognition of Piperine with DNA.

    Science.gov (United States)

    Haris, P; Mary, Varughese; Haridas, M; Sudarsanakumar, C

    2015-12-28

    Piperine, the bioactive phytochemical from black pepper (Piper nigrum L.), is a nontoxic natural compound exhibiting many physiological and pharmacological properties. They include antioxidant, anti-inflammatory, antimutagenic, antitumor, antiapoptotic, antigenotoxic, antiarthritic, antifungal, antimicrobial, antidepressant, anti-HBV, and gastro-protective activities. It also enhances the bioavailability of phytochemicals and drugs. The molecular mechanism of action of piperine with DNA has not yet been addressed, while its pharmacological activities have been reported. In this work we report for the first time the interaction of piperine molecule with DNA duplex. We have carried out UV-vis absorption and fluorescence spectroscopy to confirm the binding of piperine with calf thymus DNA (ctDNA). The energetics of interaction of piperine with ctDNA was monitored by isothermal titration calorimetry (ITC). Differential scanning calorimetry (DSC) and melting temperature (Tm) analysis were also performed, confirming a minor groove mode of binding of piperine with ctDNA. The binding free energy ΔG values obtained from molecular dynamics simulation studies agree well with ITC values and reveal a sequence dependent minor groove binding exhibiting a specificity toward AT rich sequences.

  18. Functional self-assembled DNA nanostructures for molecular recognition

    Science.gov (United States)

    Zhang, Xiaojuan; Yadavalli, Vamsi K.

    2012-03-01

    Nucleic acids present a wonderful toolkit of structural motifs for nanoconstruction. Functional DNA nanostructures can enable protein recognition by the use of aptamers attached to a basic core shape formed by DNA self-assembly. Here, we present a facile, programmable strategy for the assembly of discrete aptamer-tagged DNA shapes and nanostructures that can function for molecular recognition and binding in an aqueous environment. These nanostructures, presented here to bind two different protein targets, are easily synthesized in large numbers, and are portable and stable over long periods of time. This construction modality can facilitate on-demand production of libraries of diverse shapes to recognize and bind proteins or catalyze reactions via functional nucleic acid tags.Nucleic acids present a wonderful toolkit of structural motifs for nanoconstruction. Functional DNA nanostructures can enable protein recognition by the use of aptamers attached to a basic core shape formed by DNA self-assembly. Here, we present a facile, programmable strategy for the assembly of discrete aptamer-tagged DNA shapes and nanostructures that can function for molecular recognition and binding in an aqueous environment. These nanostructures, presented here to bind two different protein targets, are easily synthesized in large numbers, and are portable and stable over long periods of time. This construction modality can facilitate on-demand production of libraries of diverse shapes to recognize and bind proteins or catalyze reactions via functional nucleic acid tags. Electronic supplementary information (ESI) available. See DOI: 10.1039/c2nr11711h

  19. Molecular transport through large-diameter DNA nanopores

    Science.gov (United States)

    Krishnan, Swati; Ziegler, Daniela; Arnaut, Vera; Martin, Thomas G.; Kapsner, Korbinian; Henneberg, Katharina; Bausch, Andreas R.; Dietz, Hendrik; Simmel, Friedrich C.

    2016-01-01

    DNA-based nanopores are synthetic biomolecular membrane pores, whose geometry and chemical functionality can be tuned using the tools of DNA nanotechnology, making them promising molecular devices for applications in single-molecule biosensing and synthetic biology. Here we introduce a large DNA membrane channel with an ≈4 nm diameter pore, which has stable electrical properties and spontaneously inserts into flat lipid bilayer membranes. Membrane incorporation is facilitated by a large number of hydrophobic functionalizations or, alternatively, streptavidin linkages between biotinylated channels and lipids. The channel displays an Ohmic conductance of ≈3 nS, consistent with its size, and allows electrically driven translocation of single-stranded and double-stranded DNA analytes. Using confocal microscopy and a dye influx assay, we demonstrate the spontaneous formation of membrane pores in giant unilamellar vesicles. Pores can be created both in an outside-in and an inside-out configuration. PMID:27658960

  20. Molecular transport through large-diameter DNA nanopores

    Science.gov (United States)

    Krishnan, Swati; Ziegler, Daniela; Arnaut, Vera; Martin, Thomas G.; Kapsner, Korbinian; Henneberg, Katharina; Bausch, Andreas R.; Dietz, Hendrik; Simmel, Friedrich C.

    2016-09-01

    DNA-based nanopores are synthetic biomolecular membrane pores, whose geometry and chemical functionality can be tuned using the tools of DNA nanotechnology, making them promising molecular devices for applications in single-molecule biosensing and synthetic biology. Here we introduce a large DNA membrane channel with an ~4 nm diameter pore, which has stable electrical properties and spontaneously inserts into flat lipid bilayer membranes. Membrane incorporation is facilitated by a large number of hydrophobic functionalizations or, alternatively, streptavidin linkages between biotinylated channels and lipids. The channel displays an Ohmic conductance of ~3 nS, consistent with its size, and allows electrically driven translocation of single-stranded and double-stranded DNA analytes. Using confocal microscopy and a dye influx assay, we demonstrate the spontaneous formation of membrane pores in giant unilamellar vesicles. Pores can be created both in an outside-in and an inside-out configuration.

  1. 二维码防伪技术在可变数据印刷中的应用%Application of Anti-counterfeiting Technology Based on Two-dimensional Bar Code in Variable Data Printing

    Institute of Scientific and Technical Information of China (English)

    肖菲菲; 刘真

    2011-01-01

    The principle of two-dimensional bar code anti-counterfeiting technology was analyzed.A plan of applying two-dimensional code anti-counterfeiting technology in variable-data printing was put forward.On the basis of experiments on generation and recognition of two-dimensional bar code,anti-counterfeiting properties of two different two-dimensional bar codes(PDF417 code and QR code) were obtained and their difference was compared.The theoretical analysis and experiment results showed that anti-counterfeiting technology based on two-dimensional bar code can be used in variable data printing,besides,different two-dimensional bar codes have different anti-counterfeiting properties;different two-dimensional bar code should be selected according to specific requirements.%分析了二维码的防伪原理,提出了在可变数据印刷中应用二维码进行防伪的方案,在完成基于可变数据印刷的二维码的生成和识别实验的基础上,设计实验比较得出了不同二维码(PDF417码与QR码)的防伪特性的差异。理论分析及实验结果表明,利用二维码对可变数据印刷进行防伪的方案是可行的,并且不同二维码的防伪特性存在差异,在实际应用中可以根据具体的要求选用不同的二维码。

  2. Single molecular biology: coming of age in DNA replication.

    Science.gov (United States)

    Liu, Xiao-Jing; Lou, Hui-Qiang

    2017-09-20

    DNA replication is an essential process of the living organisms. To achieve precise and reliable replication, DNA polymerases play a central role in DNA synthesis. Previous investigations have shown that the average rates of DNA synthesis on the leading and lagging strands in a replisome must be similar to avoid the formation of significant gaps in the nascent strands. The underlying mechanism has been assumed to be coordination between leading- and lagging-strand polymerases. However, Kowalczykowski's lab members recently performed single molecule techniques in E. coli and showed the real-time behavior of a replisome. The leading- and lagging-strand polymerases function stochastically and independently. Furthermore, when a DNA polymerase is paused, the helicase slows down in a self-regulating fail-safe mechanism, akin to a ''dead-man's switch''. Based on the real-time single-molecular observation, the authors propose that leading- and lagging-strand polymerases synthesize DNA stochastically within a Gaussian distribution. Along with the development and application of single-molecule techniques, we will witness a new age of DNA replication and other biological researches.

  3. Carbon Nanomaterials and DNA: from Molecular Recognition to Applications.

    Science.gov (United States)

    Sun, Hanjun; Ren, Jinsong; Qu, Xiaogang

    2016-03-15

    DNA is polymorphic. Increasing evidence has indicated that many biologically important processes are related to DNA's conformational transition and assembly states. In particular, noncanonical DNA structures, such as the right-handed A-form, the left-handed Z-form, the triplex, the G-quadruplex, the i-motif, and so forth, have been specific targets for the diagnosis and therapy of human diseases. Meanwhile, they have been widely used in the construction of smart DNA nanomaterials and nanoarchitectures. As rising stars in materials science, the family of carbon nanomaterials (CNMs), including two-dimensional graphene, one-dimensional carbon nanotubes (CNTs), and zero-dimensional graphene or carbon quantum dots (GQDs or CQDs), interact with DNA and are able to regulate the conformational transitions of DNA. The interaction of DNA with CNMs not only opens new opportunities for specific molecular recognition, but it also expands the promising applications of CNMs from materials science to biotechnology and biomedicine. In this Account, we focus on our contributions to the field of interactions between CNMs and DNA in which we have explored their promising applications in nanodevices, sensing, materials synthesis, and biomedicine. For one-dimensional CNTs, two-dimensional graphene, and zero-dimensional GQDs and CQDs, the basic principles, binding modes, and applications of the interactions between CNMs and DNA are reviewed. We aim to give prominence to the important status of CNMs in the field of molecular recognition for DNA. First, we summarized our discovery of the interactions between single-walled carbon nanotubes (SWNTs) with duplex, triplex, and human telomeric i-motif DNA and their interesting applications. For example, SWNTs are the first chemical agents that can selectively stabilize human telomeric i-motif DNA and induce its formation under physiological conditions. On the basis of this principle, two types of nanodevices were designed. One was used for

  4. That's nice, but what does IT do? Evaluating the impact of bar coded medication administration by measuring changes in the process of care.

    Science.gov (United States)

    Holden, Richard J; Brown, Roger L; Alper, Samuel J; Scanlon, Matthew C; Patel, Neal R; Karsh, Ben-Tzion

    2011-07-01

    Health information technology (IT) is widely endorsed as a way to improve key health care outcomes, particularly patient safety. Applying a human factors approach, this paper models more explicitly how health IT might improve or worsen outcomes. The human factors model specifies that health IT transforms the work system, which transforms the process of care, which in turn transforms the outcome of care. This study reports on transformations of the medication administration process that resulted from the implementation of one type of IT: bar coded medication administration (BCMA). Registered nurses at two large pediatric hospitals in the US participated in a survey administered before and after one of the hospitals implemented BCMA. Nurses' perceptions of the administration process changed at the hospital that implemented BCMA, whereas perceptions of nurses at the control hospital did not. BCMA appeared to improve the safety of the processes of matching medications to the medication administration record and checking patient identification. The accuracy, usefulness, and consistency of checking patient identification improved as well. In contrast, nurses' perceptions of the usefulness, time efficiency, and ease of the documentation process decreased post-BCMA. Discussion of survey findings is supplemented by observations and interviews at the hospital that implemented BCMA. By considering the way that IT transforms the work system and the work process a practitioner can better predict the kind of outcomes that the IT might produce. More importantly, the practitioner can achieve or prevent outcomes of interest by using design and redesign aimed at controlling work system and process transformations.

  5. PIEZOELECTRIC PROPERTIES OF SINGLE-STRAND DNA MOLECULAR BRUSH BIOLAYERS

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    The paper is devoted to investigations on nanomechanical behaviors of biochips in label-free biodetections. The chip consists of Si-layer, Ti-layer, Au-layer and single-strand DNA (ssDNA) molecular brush biolayer immobilized by self-assembly technology of thiol group. Unlike previous viewpoints, such as force-bending, entropy-bending and curvature electricity effect, etc.,the piezoelectric effect of the biopolymer brush layer is viewed as the main factor that induces nanomechanical bending of biochips, and a classical macroscopic piezoelectric constitutive relation is used to describe the piezoelectric effect. A new laminated cantilever beam model with a piezoelectric biolayer in continuum mechanics, the linearized Poisson-Boltzmann equation in statistical mechanics and the scaling method in polyelectrolyte brush theory are combined to establish a relationship between the nanomechanical deflection of DNA chips and the factors such as nanoscopic structural features of ssDNA molecules, buffer salt concentration, macroscopic mechanical/piezoelectric parameters of DNA chips etc. Curve fitting of experimental data shows that the sign of the piezoelectric constant of the biolayer may control the deflection direction of DNA chips during the packaging process.

  6. DNA-enabled integrated molecular systems for computation and sensing.

    Science.gov (United States)

    LaBoda, Craig; Duschl, Heather; Dwyer, Chris L

    2014-06-17

    CONSPECTUS: Nucleic acids have become powerful building blocks for creating supramolecular nanostructures with a variety of new and interesting behaviors. The predictable and guided folding of DNA, inspired by nature, allows designs to manipulate molecular-scale processes unlike any other material system. Thus, DNA can be co-opted for engineered and purposeful ends. This Account details a small portion of what can be engineered using DNA within the context of computer architectures and systems. Over a decade of work at the intersection of DNA nanotechnology and computer system design has shown several key elements and properties of how to harness the massive parallelism created by DNA self-assembly. This work is presented, naturally, from the bottom-up beginning with early work on strand sequence design for deterministic, finite DNA nanostructure synthesis. The key features of DNA nanostructures are explored, including how the use of small DNA motifs assembled in a hierarchical manner enables full-addressability of the final nanostructure, an important property for building dense and complicated systems. A full computer system also requires devices that are compatible with DNA self-assembly and cooperate at a higher level as circuits patterned over many, many replicated units. Described here is some work in this area investigating nanowire and nanoparticle devices, as well as chromophore-based circuits called resonance energy transfer (RET) logic. The former is an example of a new way to bring traditional silicon transistor technology to the nanoscale, which is increasingly problematic with current fabrication methods. RET logic, on the other hand, introduces a framework for optical computing at the molecular level. This Account also highlights several architectural system studies that demonstrate that even with low-level devices that are inferior to their silicon counterparts and a substrate that harbors abundant defects, self-assembled systems can still

  7. TALENs: Customizable Molecular DNA Scissors for Genome Engineering of Plants

    Institute of Scientific and Technical Information of China (English)

    Kunling Chen; Caixia Gao

    2013-01-01

    Precise genome modification with engineered nucleases is a powerful tool for studying basic biology and applied biotechnology.Transcription activator-like effector nucleases (TALENs),consisting of an engineered specific (TALE) DNA binding domain and a Fok I cleavage domain,are newly developed versatile reagents for genome engineering in different organisms.Because of the simplicity of the DNA recognition code and their modular assembly,TALENs can act as customizable molecular DNA scissors inducing double-strand breaks (DSBs) at given genomic location.Thus,they provide a valuable approach to targeted genome modifications such as mutations,insertions,replacements or chromosome rearrangements.In this article,we review the development of TALENs,and summarize the principles and tools for TALEN-mediated gene targeting in plant cells,as well as current and potential strategies for use in plant research and crop improvement.

  8. Molecular Dynamics Simulations of DNA Translocation through a biological Nanopore

    OpenAIRE

    Barder, Simen Eidsmo

    2012-01-01

    Experimental and simulation studies of nucleic acid transport through nanosized channels, both biological and synthetic, has become a rapidly growing research area over the last decade. While the utilization of the alpha-hemolysin channel as a sequencing device is soon to be realized, other biological nanochannels may hold advantages that are yet unknown. Motivated by this, the first reported molecular dynamics simulations of DNA translocation through a connexon 26 channel were accomplished, ...

  9. DNA as a molecular wire: distance and sequence dependence.

    Science.gov (United States)

    Wohlgamuth, Chris H; McWilliams, Marc A; Slinker, Jason D

    2013-09-17

    Functional nanowires and nanoelectronics are sought for their use in next generation integrated circuits, but several challenges limit the use of most nanoscale devices on large scales. DNA has great potential for use as a molecular wire due to high yield synthesis, near-unity purification, and nanoscale self-organization. Nonetheless, a thorough understanding of ground state DNA charge transport (CT) in electronic configurations under biologically relevant conditions, where the fully base-paired, double-helical structure is preserved, is lacking. Here, we explore the fundamentals of CT through double-stranded DNA monolayers on gold by assessing 17 base pair bridges at discrete points with a redox active probe conjugated to a modified thymine. This assessment is performed under temperature-controlled and biologically relevant conditions with cyclic and square wave voltammetry, and redox peaks are analyzed to assess transfer rate and yield. We demonstrate that the yield of transport is strongly tied to the stability of the duplex, linearly correlating with the melting temperature. Transfer rate is found to be temperature-activated and to follow an inverse distance dependence, consistent with a hopping mechanism of transport. These results establish the governing factors of charge transfer speed and throughput in DNA molecular wires for device configurations, guiding subsequent application for nanoscale electronics.

  10. That’s nice, but what does IT do? Evaluating the impact of bar coded medication administration by measuring changes in the process of care

    Science.gov (United States)

    Holden, Richard J.; Brown, Roger L.; Alper, Samuel J.; Scanlon, Matthew C.; Patel, Neal R.; Karsh, Ben-Tzion

    2011-01-01

    Health information technology (IT) is widely endorsed as a way to improve key health care outcomes, particularly patient safety. Applying a human factors approach, this paper models more explicitly how health IT might improve or worsen outcomes. The human factors model specifies that health IT transforms the work system, which transforms the process of care, which in turn transforms the outcome of care. This study reports on transformations of the medication administration process that resulted from the implementation of one type of IT: bar coded medication administration (BCMA). Registered nurses at two large pediatric hospitals in the US participated in a survey administered before and after one of the hospitals implemented BCMA. Nurses’ perceptions of the administration process changed at the hospital that implemented BCMA, whereas perceptions of nurses at the control hospital did not. BCMA appeared to improve the safety of the processes of matching medications to the medication administration record and checking patient identification. The accuracy, usefulness, and consistency of checking patient identification improved as well. In contrast, nurses’ perceptions of the usefulness, time efficiency, and ease of the documentation process decreased post-BCMA. Discussion of survey findings is supplemented by observations and interviews at the hospital that implemented BCMA. By considering the way that IT transforms the work system and the work process a practitioner can better predict the kind of outcomes that the IT might produce. More importantly, the practitioner can achieve or prevent outcomes of interest by using design and redesign aimed at controlling work system and process transformations. PMID:21686318

  11. The Application of Bar Code Technology in Inspection of Laboratory Information Management System LIS%对在检验实验室信息管理系统LIS中应用条形码技术的探讨

    Institute of Scientific and Technical Information of China (English)

    卢方建

    2011-01-01

    The bar code technology in the clinical laboratory information system can improve laboratory automation,work efficiency, reduce errors and facilitate patient.%将务形码技术应用于检验实验室信息系统,提高实验室的自动化程度、工作效率,减少差错并方便病人。

  12. Application of Wireless Network and Technology of Information Bar Code in the Steel Warehouse Management System%无线网络及信息条码技术在钢材库管理系统的应用

    Institute of Scientific and Technical Information of China (English)

    田宏

    2011-01-01

    本文简要介绍了无线网络及信息条码技术在改造传统钢材库管理中的应用,重点叙述系统的构成及功能。%This paper is a simple statement that the application of wireless network and the technology of information bar code in the steel warehouse management system,the focal point is the constitute and function of the system.

  13. Genotoxicity of formaldehyde: Molecular basis of DNA damage and mutation

    Directory of Open Access Journals (Sweden)

    Masanobu eKawanishi

    2014-09-01

    Full Text Available Formaldehyde is commonly used in the chemical industry and is present in the environment, such as vehicle emissions, some building materials, food and tobacco smoke. It also occurs as a natural product in most organisms, the sources of which include a number of metabolic processes. It causes various acute and chronic adverse effects in humans if they inhale its fumes. Among the chronic effects on human health, we summarize data on genotoxicity and carcinogenicity in this review, and we particularly focus on the molecular mechanisms involved in the formaldehyde mutagenesis. Formaldehyde mainly induces N-hydroxymethyl mono-adducts on guanine, adenine and cytosine, and N-methylene crosslinks between adjacent purines in DNA. These crosslinks are types of DNA damage potentially fatal for cell survival if they are not removed by the nucleotide excision repair pathway. In the previous studies, we showed evidence that formaldehyde causes intra-strand crosslinks between purines in DNA using a unique method (Matsuda et al. Nucleic Acids Res. 26, 1769-1774,1998. Using shuttle vector plasmids, we also showed that formaldehyde as well as acetaldehyde induces tandem base substitutions, mainly at 5’-GG and 5’-GA sequences, which would arise from the intra-strand crosslinks. These mutation features are different from those of other aldehydes such as crotonaldehyde, acrolein, glyoxal and methylglyoxal. These findings provide molecular clues to improve our understanding of the genotoxicity and carcinogenicity of formaldehyde.

  14. 条形码库房管理系统在发电企业的深化应用%Further Application of Bar Code Warehouse Management System on Power Generation Enterprises

    Institute of Scientific and Technical Information of China (English)

    高飞

    2014-01-01

    条形码库房管理系统,改变了原有发电企业传统仓库管理模式,提高了仓库管理的信息化水平。通过条形码系统的实施结合网上采购系统建立了以资产管理信息系统EAM为主线,贯通网上采购、FMIS、条形码系统的物资信息化体系,实现了物资从计划、采购、验收、入库、库存、出库、付款管理的程序化、信息化管理,同时也实现了物资采购的可追溯管理。%The bar code warehouse management system changes the traditional warehouse management mode of original power enterprises; improves the level of warehouse management. Through the implementation of bar code system, and combined with online procurement system, the paper establishes a material information system which set asset management information system EAM as the main line, connect with material information system and FMIS bar code system. Thus it not only achieves the materials sequencing of the planning, procurement, inspection, warehousing, inventory, delivery, payment management and information management, but also realizes the traceability management of material procurement.

  15. Determination of fruit origin by using 26S rDNA fingerprinting of yeast communities by PCR-DGGE: preliminary application to Physalis fruits from Egypt.

    Science.gov (United States)

    El Sheikha, Aly Farag; Condur, Ana; Métayer, Isabelle; Nguyen, Doan Duy Le; Loiseau, Gérard; Montet, Didier

    2009-10-01

    The determination of geographical origin is a demand of the traceability system of import-export food products. One hypothesis for tracing the source of a product is by global analysis of the microbial communities of the food and statistical linkage of this analysis to the geographical origin of the food. For this purpose, a molecular technique employing 26S rDNA profiles generated by PCR-DGGE was used to detect the variation in yeast community structures of three species of Physalis fruit (Physalis ixocarpa Brat, Physalis pubescens L, Physalis pruinosa L) from four Egyptian regions (Qalyoubia, Minufiya, Beheira and Alexandria Governments). When the 26S rDNA profiles were analysed by multivariate analysis, distinct microbial communities were detected. The band profiles of Physalis yeasts from different Governments were specific for each location and could be used as a bar code to discriminate the origin of the fruits. This method is a new traceability tool which provides fruit products with a unique biological bar code and makes it possible to trace back the fruits to their original location.

  16. Intelligent DNA-based molecular diagnostics using linked genetic markers

    Energy Technology Data Exchange (ETDEWEB)

    Pathak, D.K.; Perlin, M.W.; Hoffman, E.P.

    1994-12-31

    This paper describes a knowledge-based system for molecular diagnostics, and its application to fully automated diagnosis of X-linked genetic disorders. Molecular diagnostic information is used in clinical practice for determining genetic risks, such as carrier determination and prenatal diagnosis. Initially, blood samples are obtained from related individuals, and PCR amplification is performed. Linkage-based molecular diagnosis then entails three data analysis steps. First, for every individual, the alleles (i.e., DNA composition) are determined at specified chromosomal locations. Second, the flow of genetic material among the individuals is established. Third, the probability that a given individual is either a carrier of the disease or affected by the disease is determined. The current practice is to perform each of these three steps manually, which is costly, time consuming, labor-intensive, and error-prone. As such, the knowledge-intensive data analysis and interpretation supersede the actual experimentation effort as the major bottleneck in molecular diagnostics. By examining the human problem solving for the task, we have designed and implemented a prototype knowledge-based system capable of fully automating linkage-based molecular diagnostics in X-linked genetic disorders, including Duchenne Muscular Dystrophy (DMD). Our system uses knowledge-based interpretation of gel electrophoresis images to determine individual DNA marker labels, a constraint satisfaction search for consistent genetic flow among individuals, and a blackboard-style problem solver for risk assessment. We describe the system`s successful diagnosis of DMD carrier and affected individuals from raw clinical data.

  17. Molecular modeling and energy refinement of supercoiled DNA.

    Science.gov (United States)

    Hao, M H; Olson, W K

    1989-12-01

    A method is presented for constructing the complete atomic structure of supercoiled DNA starting from a linear description of the double helical pathway. The folding pathway is defined by piecewise B-spline curves and the atoms are initially positioned with respect to the local Frenet trihedra determined by the equations of the curves. The resulting chemical structure is corrected and refined with an energy minimization procedure based on standard potential expressions. The refined molecular structure is then used to study the effects of supercoiling on the local secondary structure of DNA. The minimized structure is found to differ from an isotropic elastic rod model of the double helix, with the base pairs bending in an asymmetric fashion along the supercoiled trajectory. The starting trajectory is chosen so that the refined supercoiled structure is either underwound (10.37 base pairs per turn) or overwound (9.65 base pairs per turn) compared to the standard tenfold B-DNA fiber diffraction model. The underwound supercoil is also lower in energy than the overwound duplex. The variation of base pair sequence in poly(dA).poly(dT).poly(dAT).poly(dTA) and poly(dA5T5).poly(dT5A5) is additionally found to influence the secondary structural features along a given supercoiled pathway. Finally, the detailed features of the refined structures are found to be in agreement with known X-ray crystallographic structures of DNA oligomers.

  18. Pericentric satellite DNA and molecular phylogeny in Acomys (Rodentia).

    Science.gov (United States)

    Kunze, B; Traut, W; Garagna, S; Weichenhan, D; Redi, C A; Winking, H

    1999-01-01

    Satellite DNAs (stDNAs) of four Acomys species (spiny-mice), A. cahirinus, A. cineraceus, A. dimidiatus and A. russatus, belong to closely related sequence families. Monomer sizes range from 338 to 364 bp. Between-species sequence identity was from 81.0% to 97.2%. The molecular phylogeny of the sequences helps to clarify the taxonomy of this 'difficult' group. The A. dimidiatus genome contains about 60000 repeats. According to the restriction patterns, repeats are arranged in tandem. The stDNA maps to the centromeric heterochromatin of most autosomes, both acrocentric and metacentric, but appears to be absent in the centromeric region of Y chromosomes. A well-conserved centromere protein B (CENP-B) box is present in the stDNA of A. russatus while it is degenerated in the other species.

  19. [Molecular dynamics of immune complex of photoadduct-containing DNA with Fab-Anti-DNA antibody fragment].

    Science.gov (United States)

    Akberova, N I; Zhmurov, A A; Nevzorova, T A; Litvinov, R I

    2016-01-01

    Antibodies to DNA play an important role in the pathogenesis of autoimmune diseases. The elucidation of structural mechanisms of both the antigen recognition and the interaction of anti-DNA antibodies with DNA will help to understand the role of DNA-containing immune complexes in various pathologies and can provide a basis for new treatment modalities. Moreover, the DNA-antibody complex is an analog of specific intracellular DNA-protein interactions. In this work, we used in silico molecular dynamic simulations of bimolecular complexes of the dsDNA segment containing the Fab fragment of an anti-DNA antibody to obtain the detailed thermodynamic and structural characteristics of dynamic intermolecular interactions. Using computationally modified crystal structure of the Fab-DNA complex (PDB ID: 3VW3), we studied the equilibrium molecular dynamics of the 64M-5 antibody Fab fragment associated with the dsDNA fragment containing the thymine dimer, the product of DNA photodamage. Amino acid residues that constitute paratopes and the complementary nucleotide epitopes for the Fab-DNA construct were identified. Stacking and electrostatic interactions were found to play the main role in mediating the most specific antibody-dsDNA contacts, while hydrogen bonds were less significant. These findings may shed light on the formation and properties of pathogenic anti-DNA antibodies in autoimmune diseases, such as systemic lupus erythematosus associated with skin photosensitivity and DNA photodamage.

  20. 基于链码跟踪的Data Matrix二维条码快速识别%Quick identification of Data Matrix two-dimensional bar code based on chain code tracking

    Institute of Scientific and Technical Information of China (English)

    徐义钊; 白瑞林; 余震虹; 吉峰

    2012-01-01

    为实现工业现场中Data Matrix二维条码的快速识别,提出一种基于链码跟踪、直线段提取的条码快速识别方法.首先采用Sobel算子提取图像边缘;然后基于链码跟踪方法,跟踪图像的边缘点,记录链码;接着根据快速直线段提取方法,将直线相似度低于阈值的线段剔除,结合线形连接方法合并断裂线段,并使用长度判别剔除不可靠的线段;最后结合Data Matrix二维条码的特征,定位Data Matrix二维条码.实际测试表明,该识别方法能够快速、准确地定位二维条码区域,识别正确率可达99.39%以上,具有实时性强、可靠性好等特点,满足工业现场要求.%A two-dimensional bar code recognition method based on chain code tracking and straight line segment extraction was proposed in order to realize quick identification of the Data Matrix two-dimensional bar code in the industrial field. Firstly, the Sobel operator was used to extract the edge of image. Secondly, the image edge points were tracked and chain codes were recorded based on chain code tracking method. Thirdly, line segment whose straight line similarity was below the threshold was removed with fast straight line segment extraction method. The fracture line was combined with the linear connection method and the unreliable line was excluded by length discrimination. Finally, the Data Matrix bar code was located with the characteristics of the Data Matrix two-dimensional bar code. The application test shows that the two-dimensional bar code area can be quickly and accurately located by the recognition method with the recognition rate of 99. 39%. The proposed method is ready to meet the requirements of industrial field with strong real-time ability and high reliability.

  1. Comparative genomics and molecular dynamics of DNA repeats in eukaryotes.

    Science.gov (United States)

    Richard, Guy-Franck; Kerrest, Alix; Dujon, Bernard

    2008-12-01

    Repeated elements can be widely abundant in eukaryotic genomes, composing more than 50% of the human genome, for example. It is possible to classify repeated sequences into two large families, "tandem repeats" and "dispersed repeats." Each of these two families can be itself divided into subfamilies. Dispersed repeats contain transposons, tRNA genes, and gene paralogues, whereas tandem repeats contain gene tandems, ribosomal DNA repeat arrays, and satellite DNA, itself subdivided into satellites, minisatellites, and microsatellites. Remarkably, the molecular mechanisms that create and propagate dispersed and tandem repeats are specific to each class and usually do not overlap. In the present review, we have chosen in the first section to describe the nature and distribution of dispersed and tandem repeats in eukaryotic genomes in the light of complete (or nearly complete) available genome sequences. In the second part, we focus on the molecular mechanisms responsible for the fast evolution of two specific classes of tandem repeats: minisatellites and microsatellites. Given that a growing number of human neurological disorders involve the expansion of a particular class of microsatellites, called trinucleotide repeats, a large part of the recent experimental work on microsatellites has focused on these particular repeats, and thus we also review the current knowledge in this area. Finally, we propose a unified definition for mini- and microsatellites that takes into account their biological properties and try to point out new directions that should be explored in a near future on our road to understanding the genetics of repeated sequences.

  2. Detection of DNA damage by using hairpin molecular beacon probes and graphene oxide.

    Science.gov (United States)

    Zhou, Jie; Lu, Qian; Tong, Ying; Wei, Wei; Liu, Songqin

    2012-09-15

    A hairpin molecular beacon tagged with carboxyfluorescein in combination with graphene oxide as a quencher reagent was used to detect the DNA damage by chemical reagents. The fluorescence of molecular beacon was quenched sharply by graphene oxide; while in the presence of its complementary DNA the quenching efficiency decreased because their hybridization prevented the strong adsorbability of molecular beacon on graphene oxide. If the complementary DNA was damaged by a chemical reagent and could not form intact duplex structure with molecular beacon, more molecular beacon would adsorb on graphene oxide increasing the quenching efficiency. Thus, damaged DNA could be detected based on different quenching efficiencies afforded by damaged and intact complementary DNA. The damage effects of chlorpyrifos-methyl and three metabolites of styrene such as mandelieaeids, phenylglyoxylieaeids and epoxystyrene on DNA were studied as models. The method for detection of DNA damage was reliable, rapid and simple compared to the biological methods. Copyright © 2012 Elsevier B.V. All rights reserved.

  3. [Detection of DNA human cytomegalovirus of a molecular methods: hybrid capture DNA CMV by immunocompromised].

    Science.gov (United States)

    Mhiri, Leila; Arrouji, Zakia; Slim, Amine; Ben Redjeb, Saida

    2006-10-01

    Human cytomegalovirus (HCMV), a member of the beta-virus herpes family, is a ubiquitous human pathogen. After a primary infection, HCMV establishes life latency. HCMV rarely causes symptomatic disease in an immunocompetent host, however, it is a major cause of infectious morbidity and mortality in immunocompromised individuals and developing fetuses. The HCMV genome consists of 240 kbp of double stranded DNA. Early diagnosis molecular of CMV infection is important. The objective of this study was to develop a molecular methods: Quantitative Hybrid capture for the detection of DNA CMV. We present results for 200 immunocompromised collected from 1999 to 2003 (122 men and 78 women, whom mean age was 35 years). Our results showed that 25% of women and 36% of men were positif for hybrid capture DNA CMV. This simple test (cold probe) provide quantitative and fast results. Also the efficacity of anti-CMV therapy can be followed. More over, in contrary with pp65-antigenemia assay and CMV PCR, this test can be managed on biopsy sample.

  4. A Parallel Biological Optimization Algorithm to Solve the Unbalanced Assignment Problem Based on DNA Molecular Computing.

    Science.gov (United States)

    Wang, Zhaocai; Pu, Jun; Cao, Liling; Tan, Jian

    2015-10-23

    The unbalanced assignment problem (UAP) is to optimally resolve the problem of assigning n jobs to m individuals (m parallel DNA algorithm for solving the unbalanced assignment problem using DNA molecular operations. We reasonably design flexible-length DNA strands representing different jobs and individuals, take appropriate steps, and get the solutions of the UAP in the proper length range and O(mn) time. We extend the application of DNA molecular operations and simultaneity to simplify the complexity of the computation.

  5. Molecular genotyping of Colletotrichum species based on arbitrarily primed PCR, A + T-Rich DNA, and nuclear DNA analyses

    Science.gov (United States)

    Freeman, S.; Pham, M.; Rodriguez, R.J.

    1993-01-01

    Molecular genotyping of Colletotrichum species based on arbitrarily primed PCR, A + T-rich DNA, and nuclear DNA analyses. Experimental Mycology 17, 309-322. Isolates of Colletotrichum were grouped into 10 separate species based on arbitrarily primed PCR (ap-PCR), A + T-rich DNA (AT-DNA) and nuclear DNA banding patterns. In general, the grouping of Colletotrichum isolates by these molecular approaches corresponded to that done by classical taxonomic identification, however, some exceptions were observed. PCR amplification of genomic DNA using four different primers allowed for reliable differentiation between isolates of the 10 species. HaeIII digestion patterns of AT-DNA also distinguished between species of Colletotrichum by generating species-specific band patterns. In addition, hybridization of the repetitive DNA element (GcpR1) to genomic DNA identified a unique set of Pst 1-digested nuclear DNA fragments in each of the 10 species of Colletotrichum tested. Multiple isolates of C. acutatum, C. coccodes, C. fragariae, C. lindemuthianum, C. magna, C. orbiculare, C. graminicola from maize, and C. graminicola from sorghum showed 86-100% intraspecies similarity based on ap-PCR and AT-DNA analyses. Interspecies similarity determined by ap-PCR and AT-DNA analyses varied between 0 and 33%. Three distinct banding patterns were detected in isolates of C. gloeosporioides from strawberry. Similarly, three different banding patterns were observed among isolates of C. musae from diseased banana.

  6. LDFF, the large molecular weight DNA fragmentation factor, is responsible for the large molecular weight DNA degradation during apoptosis in Xenopus egg extracts

    Institute of Scientific and Technical Information of China (English)

    Zhi Gang LU; Chuan Mao ZHANG; Zhong He ZHAI

    2004-01-01

    DNA degradation is a biochemical hallmark in apoptosis. It has been demonstrated in many cell types that there are two stages of DNA fragmentation during the apoptotic execution. In the early stage, chromatin DNA is cut into large molecular weight DNA fragments, although the responsible nuclease(s) has not been recognized. In the late stage, the chromatin DNA is cleaved further into short oligonucleosomal fragments by a well-characterized nuclease in apoptosis,the caspase-activated DNase (CAD/DFF40). In this study, we demonstrate that large molecular weight DNA fragmentation also occurs in Xenopus egg extracts in apoptosis. We show that the large molecular weight DNA fragmentation factor (LDFF) is not the Xenopus CAD homolog XCAD. LDFF is activated by caspase-3. The large molecular weight DNA fragmentation activity of LDFF is Mg2+-dependent and Ca2+-independent, can occur in both acidic and neutral pH conditions and can tolerate 45℃ treatment. These results indicate that LDFF in Xenopus egg extracts might be a new DNase (or DNases) responsible for the large DNA fragmentation.

  7. DNA ligase IV as a new molecular target for temozolomide

    Energy Technology Data Exchange (ETDEWEB)

    Kondo, Natsuko [Department of Biology, School of Medicine, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634-8521 (Japan); Department of Neurosurgery, School of Medicine, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634-8521 (Japan); Takahashi, Akihisa; Mori, Eiichiro; Ohnishi, Ken [Department of Biology, School of Medicine, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634-8521 (Japan); McKinnon, Peter J. [Department of Genetics and Tumor Cell Biology, St. Jude Children' s Research Hospital, Memphis, TN 38105 (United States); Sakaki, Toshisuke; Nakase, Hiroyuki [Department of Neurosurgery, School of Medicine, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634-8521 (Japan); Ohnishi, Takeo, E-mail: tohnishi@naramed-u.ac.jp [Department of Biology, School of Medicine, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634-8521 (Japan)

    2009-10-02

    Temozolomide (TMZ) is a methylating agent used in chemotherapy against glioblastoma. This work was designed to clarify details in repair pathways acting to remove DNA double-strand breaks (DSBs) induced by TMZ. Cultured mouse embryonic fibroblasts were used which were deficient in DSB repair genes such as homologous recombination repair-related genes X-ray repair cross-complementing group 2 (XRCC2)and radiation sensitive mutant54 (Rad54), non-homologous end joining repair-related gene DNAligase IV (Lig4). Cell sensitivity to drug treatments was assessed using colony forming assays. The most effective molecular target which was correlated with TMZ cell sensitivity was Lig4. In addition, it was found that small interference RNAs (siRNA) for Lig4 efficiently enhanced cell lethality induced by TMZ in human glioblastoma A172 cells. These findings suggest that down regulation of Lig4 might provide a useful tool for cell sensitization during TMZ chemotherapy.

  8. Drug-DNA intercalation: from discovery to the molecular mechanism.

    Science.gov (United States)

    Mukherjee, Arnab; Sasikala, Wilbee D

    2013-01-01

    The ability of small molecules to perturb the natural structure and dynamics of nucleic acids is intriguing and has potential applications in cancer therapeutics. Intercalation is a special binding mode where the planar aromatic moiety of a small molecule is inserted between a pair of base pairs, causing structural changes in the DNA and leading to its functional arrest. Enormous progress has been made to understand the nature of the intercalation process since its idealistic conception five decades ago. However, the biological functions were detected even earlier. In this review, we focus mainly on the acridine and anthracycline types of drugs and provide a brief overview of the development in the field through various experimental methods that led to our present understanding of the subject. Subsequently, we discuss the molecular mechanism of the intercalation process, free-energy landscapes, and kinetics that was revealed recently through detailed and rigorous computational studies.

  9. 基于二维码的校园考务信息应用方法%Application procedure of campus examination information based on two-dimensional bar code

    Institute of Scientific and Technical Information of China (English)

    晏燕; 王大程; 王前前; 孙婷婷

    2012-01-01

    In order to reduce the waste of papers used for printing admission tickets for all kinds of examinations and avoid the problem that two-dimensional bar code cannot carry certificate images of high quality, a two-dimensional bar code examination information system was designed and realized based on QR code. In this system a half-on-line and half-off-line method was used to provide major functions such as information gathering of candidates, arrangement of examination information, generation of electronic admission tickets with two dimensional bar code, and identification of examination information. It could not only save the paper resources but also realize triple-sided united authentication among candidates, information of electronic admission tickets, and database of candidate information.%为了减少各类考试中纸质准考证件造成的资源浪费,同时避免二维码无法承载高质量证件图像的难题,设计并实现了基于QR码的二维码考务信息系统.采用半离线半在线的方式提供考生信息采集、考务信息安排、二维码电子准考凭证生成和考务信息识别等主要功能,不但可以节约纸张资源,还可以实现参加考试人员与电子凭证信息和考生信息数据库之间的三方联合认证.

  10. Single and multiple molecular beacon probes for DNA hybridization studies on a silica glass surface

    Science.gov (United States)

    Fang, Xiaohong; Liu, Xiaojing; Tan, Weihong

    1999-05-01

    Surface immobilizable molecular beacons have been developed for DNA hybridization studies on a silica glass plate. Molecular beacons are a new class of oligonucleotide probes that have a loop-and-stem structure with a fluorophore and a quencher attached to the two ends of the stem. They only emit intense fluorescence when hybridize to their target molecules. This provides an excellent selectivity for the detection of DNA molecules. We have designed biotinylated molecular beacons which can be immobilized onto a solid surface. The molecular beacon is synthesized using DABCYL as the quencher and an optical stable dye, tetramethylrhodamine, as the fluorophore. Mass spectrometry is used to confirm the synthesized molecular beacon. The molecular beacons have been immobilized onto a silica surface through biotin-avidin binding. The surface immobilized molecular beacons have been used for the detection of target DNA with subnanomolar analytical sensitivity. have also immobilized two different molecular beacons on a silica surface in spatially resolved microscopic regions. The hybridization study of these two different molecular beacon probes has shown excellent selectivity for their target sequences. The newly designed molecular beacons are intended for DNA molecular interaction studies at an interface and for the development of ultrasensitive DNA sensors for a variety of applications including disease diagnosis, disease mechanism studies, new drug development, and in the investigation of molecular interactions between DNA molecules and other interesting biomolecules.

  11. Prolonged decay of molecular rate estimates for metazoan mitochondrial DNA

    Directory of Open Access Journals (Sweden)

    Martyna Molak

    2015-03-01

    Full Text Available Evolutionary timescales can be estimated from genetic data using the molecular clock, often calibrated by fossil or geological evidence. However, estimates of molecular rates in mitochondrial DNA appear to scale negatively with the age of the clock calibration. Although such a pattern has been observed in a limited range of data sets, it has not been studied on a large scale in metazoans. In addition, there is uncertainty over the temporal extent of the time-dependent pattern in rate estimates. Here we present a meta-analysis of 239 rate estimates from metazoans, representing a range of timescales and taxonomic groups. We found evidence of time-dependent rates in both coding and non-coding mitochondrial markers, in every group of animals that we studied. The negative relationship between the estimated rate and time persisted across a much wider range of calibration times than previously suggested. This indicates that, over long time frames, purifying selection gives way to mutational saturation as the main driver of time-dependent biases in rate estimates. The results of our study stress the importance of accounting for time-dependent biases in estimating mitochondrial rates regardless of the timescale over which they are inferred.

  12. Thermopower of molecular junctions: Tunneling to hopping crossover in DNA

    Science.gov (United States)

    Korol, Roman; Kilgour, Michael; Segal, Dvira

    2016-12-01

    We study the electrical conductance G and the thermopower S of single-molecule junctions and reveal signatures of different transport mechanisms: off-resonant tunneling, on-resonant coherent (ballistic) motion, and multi-step hopping. These mechanisms are identified by studying the behavior of G and S while varying molecular length and temperature. Based on a simple one-dimensional model for molecular junctions, we derive approximate expressions for the thermopower in these different regimes. Analytical results are compared to numerical simulations, performed using a variant of Büttiker's probe technique, the so-called voltage-temperature probe, which allows us to phenomenologically introduce environmentally induced elastic and inelastic electron scattering effects, while applying both voltage and temperature biases across the junction. We further simulate the thermopower of GC-rich DNA sequences with mediating A:T blocks and manifest the tunneling-to-hopping crossover in both the electrical conductance and the thermopower, in accord with measurements by Li et al. [Nat. Commun. 7, 11294 (2016)].

  13. Creating complex molecular topologies by configuring DNA four-way junctions

    Science.gov (United States)

    Liu, Di; Chen, Gang; Akhter, Usman; Cronin, Timothy M.; Weizmann, Yossi

    2016-10-01

    The realization of complex topologies at the molecular level represents a grand challenge in chemistry. This necessitates the manipulation of molecular interactions with high precision. Here we show that single-stranded DNA (ssDNA) knots and links can be created by utilizing the inherent topological properties that pertain to the DNA four-way junction, at which the two helical strands form a node and can be configured conveniently and connected for complex topological construction. Using this strategy, we produced series of ssDNA topoisomers with the same sequences. By finely designing the curvature and torsion, double-stranded DNA knots were accessed by hybridizing and ligating the complementary strands with the knotted ssDNA templates. Furthermore, we demonstrate the use of a constructed ssDNA knot both to probe the topological conversion catalysed by DNA topoisomerase and to study the DNA replication under topological constraint.

  14. Physical mapping and molecular cloning of mung bean yellow mosaic virus DNA.

    Science.gov (United States)

    Morinaga, T; Ikegami, M; Miura, K

    1990-01-01

    Viral single-stranded DNA of mung bean yellow mosaic virus (MYMV) was converted to the double-stranded state in vitro, and physical mapping was carried out. The genome of MYMV was found to consist of two major components (designated as DNA 1 and DNA 2). In addition, some minor components were detected. Molecular cloning of the major components was carried out, using in vitro double-stranded DNA and replicative intermediate DNAs. DNA 1 is about 2.72 and DNA 2 about 2.67 kilobase pairs. No similarities were observed when the two restriction maps of DNA 1 and 2 were compared.

  15. Transverse charge transport through DNA oligomers in large-area molecular junctions

    NARCIS (Netherlands)

    Katsouras, I.; Piliego, C.; Blom, P.W.M.; Leeuw, D.M. de

    2013-01-01

    We investigate the nature of charge transport in deoxyribonucleic acid (DNA) using self-assembled layers of DNA in large-area molecular junctions. A protocol was developed that yields dense monolayers where the DNA molecules are not standing upright, but are lying flat on the substrate. As a result

  16. Hydrodynamic characterization and molecular weight estimation of ultrasonically sheared DNA; Caracterizacion hidrodinamica y estimacion de pesos moleculares de DNA degradado por ultrasonidos

    Energy Technology Data Exchange (ETDEWEB)

    Casal, J. I.; Garces, F.; Garcia-Sacristan, A.

    1981-07-01

    The sedimentation coefficients and intrinsic viscosities of ultrasonically sheared calf thymus DNA have been determined. The molecular weight estimation according to this parameters have been compared with the ones obtained from the electrophoretic migration rates based on the calibration proposed using the known molecular weight restriction fragments of X-ENA. (Author) 35 refs.

  17. 电力仓库管理系统的指纹识别与条形码技术实现%Fingerprint Identification and Bar Code Technology Implementation of Power Warehouse Management System

    Institute of Scientific and Technical Information of China (English)

    欧振国

    2016-01-01

    介绍了利用条形码、指纹识别、数据库以及网络技术的电力仓库管理系统的设计与开发,分析了指纹识别和条形码技术要点,通过C++ Builder可视化编程实现系统功能模块与联网优化,测试结果表明,该系统界面友好、维护简单、操作便捷,提高了仓库管理效率。%Introduction was made to the system design and development of the power warehouse management using bar codes, fingerprint identification, database and network technology. Analysis was made to the main points of fingerprint identification and bar code technology. This paper used c++ Builder visual programming to realize the function module and network optimization. The test results show that the system has a friendly interface with simple maintenance and convenient operation, which improves the efficiency of warehouse management.

  18. Optical Fiber Nanotips Coated with Molecular Beacons for DNA Detection

    Directory of Open Access Journals (Sweden)

    Ambra Giannetti

    2015-04-01

    Full Text Available Optical fiber sensors, thanks to their compactness, fast response and real-time measurements, have a large impact in the fields of life science research, drug discovery and medical diagnostics. In recent years, advances in nanotechnology have resulted in the development of nanotools, capable of entering the single cell, resulting in new nanobiosensors useful for the detection of biomolecules inside living cells. In this paper, we provide an application of a nanotip coupled with molecular beacons (MBs for the detection of DNA. The MBs were characterized by hybridization studies with a complementary target to prove their functionality both free in solution and immobilized onto a solid support. The solid support chosen as substrate for the immobilization of the MBs was a 30 nm tapered tip of an optical fiber, fabricated by chemical etching. With this set-up promising results were obtained and a limit of detection (LOD of 0.57 nM was reached, opening up the possibility of using the proposed nanotip to detect mRNAs inside the cytoplasm of living cells.

  19. Minor Groove Binding between Norfloxacin and DNA Duplexes in Solution: A Molecular Dynamics Study

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    Molecular dynamics were used to investigate the interaction between norfloxacin and DNA duplex. The results showed that norfloxacin was situated in the minor groove of DNA,binding to the TCGA region of d [ATATCGATAT] 2. Specific hydrogen bonds were formed between norfloxacin and guanine base of DNA during the 2 ns MD, which may be the reason for the preferentiality of quinolone antibacterial towards the guanine base of DNA duplex.

  20. More on contamination: the use of asymmetric molecular behavior to identify authentic ancient human DNA

    DEFF Research Database (Denmark)

    Malmström, Helena; Svensson, Emma M; Gilbert, M Thomas P;

    2007-01-01

    the reliability of one of the proposed criteria, that of appropriate molecular behavior. Using real-time polymerase chain reaction (PCR) and pyrosequencing, we have quantified the relative levels of authentic aDNA and contaminant human DNA sequences recovered from archaeological dog and cattle remains. In doing....... Furthermore, we find that there is a substantial increase in the relative proportions of authentic DNA to contaminant DNA as the PCR target fragment size is decreased. We therefore conclude that the degradation pattern in aDNA provides a quantifiable difference between authentic aDNA and modern contamination...

  1. Molecular architecture of the preinitiation complex in adenovirus DNA replication

    OpenAIRE

    Mysiak, Monika Elzbieta

    2004-01-01

    After infection of a host cell, adenovirus (Ad) aims for generation of progeny viruses, and thus it rapidly replicates its genomic DNA. The replication process starts with the assembly of the preinitiation complex (PIC) on the origin DNA. The PIC consists of three viral proteins, DNA polymerase (pol), precursor terminal protein (pTP), DNA binding protein (DBP) and two transcription factors of the host cell, Nuclear Factor I (NFI) and Octamer binding protein (Oct-1). Both transcription factors...

  2. Molecular architecture and function of adenovirus DNA polymerase

    NARCIS (Netherlands)

    Brenkman, A.B. (Arjan Bernard)

    2003-01-01

    Central to this thesis is the role of adenovirus DNA polymerase (Ad pol) in adenovirus DNA replication. Ad pol is a member of the family B DNA polymerases but belongs to a distinct subclass of polymerases that use a protein as primer. As Ad pol catalyses both the initiation and elongation phases and

  3. Molecular mechanics of DNA bricks: in situ structure, mechanical properties and ionic conductivity

    Science.gov (United States)

    Slone, Scott Michael; Li, Chen-Yu; Yoo, Jejoong; Aksimentiev, Aleksei

    2016-05-01

    The DNA bricks method exploits self-assembly of short DNA fragments to produce custom three-dimensional objects with subnanometer precision. In contrast to DNA origami, the DNA brick method permits a variety of different structures to be realized using the same library of DNA strands. As a consequence of their design, however, assembled DNA brick structures have fewer interhelical connections in comparison to equivalent DNA origami structures. Although the overall shape of the DNA brick objects has been characterized and found to conform to the features of the target designs, the microscopic properties of DNA brick objects remain yet to be determined. Here, we use the all-atom molecular dynamics method to directly compare the structure, mechanical properties and ionic conductivity of DNA brick and DNA origami structures different only by internal connectivity of their consistituent DNA strands. In comparison to equivalent DNA origami structures, the DNA brick structures are found to be less rigid and less dense and have a larger cross-section area normal to the DNA helix direction. At the microscopic level, the junction in the DNA brick structures are found to be right-handed, similar to the structure of individual Holliday junctions (HJ) in solution, which contrasts with the left-handed structure of HJ in DNA origami. Subject to external electric field, a DNA brick plate is more leaky to ions than an equivalent DNA origami plate because of its lower density and larger cross-section area. Overall, our results indicate that the structures produced by the DNA brick method are fairly similar in their overall appearance to those created by the DNA origami method but are more compliant when subject to external forces, which likely is a consequence of their single crossover design.

  4. Molecular dynamics simulations demonstrate the regulation of DNA-DNA attraction by H4 histone tail acetylations and mutations.

    Science.gov (United States)

    Korolev, Nikolay; Yu, Hang; Lyubartsev, Alexander P; Nordenskiöld, Lars

    2014-10-01

    The positively charged N-terminal histone tails play a crucial role in chromatin compaction and are important modulators of DNA transcription, recombination, and repair. The detailed mechanism of the interaction of histone tails with DNA remains elusive. To model the unspecific interaction of histone tails with DNA, all-atom molecular dynamics (MD) simulations were carried out for systems of four DNA 22-mers in the presence of 20 or 16 short fragments of the H4 histone tail (variations of the 16-23 a. a. KRHRKVLR sequence, as well as the unmodified fragment a. a.13-20, GGAKRHRK). This setup with high DNA concentration, explicit presence of DNA-DNA contacts, presence of unstructured cationic peptides (histone tails) and K(+) mimics the conditions of eukaryotic chromatin. A detailed account of the DNA interactions with the histone tail fragments, K(+) and water is presented. Furthermore, DNA structure and dynamics and its interplay with the histone tail fragments binding are analysed. The charged side chains of the lysines and arginines play major roles in the tail-mediated DNA-DNA attraction by forming bridges and by coordinating to the phosphate groups and to the electronegative sites in the minor groove. Binding of all species to DNA is dynamic. The structure of the unmodified fully-charged H4 16-23 a.a. fragment KRHRKVLR is dominated by a stretched conformation. The H4 tail a. a. fragment GGAKRHRK as well as the H4 Lys16 acetylated fragment are highly flexible. The present work allows capturing typical features of the histone tail-counterion-DNA structure, interaction and dynamics.

  5. Variety of molecular conformation of plasmid pUC18 DNA and solenoidally supercoiled DNA

    Institute of Scientific and Technical Information of China (English)

    黄熙泰; 王照清; 吴永文; 樊廷玉; 王树荣; 王勖焜

    1996-01-01

    The plasmid pUC18 DNA isolated from Escherichia coli HB101 were analyzed by two-dimensional agarose gel electrophoresis and hybridization. The results show that the DNA sample can be separated into six groups of different structural components. The plectonemically and solenoidally supercoiled pUC18 DNA coexist in it. These two different conformations of supercoiled DNA are interchangeable with the circumstances (ionic strength and type, etc.). The amount of solenoidally supercoiled pUC18 DNA in the samples can be changed by treatment of DNA topoisome rases. Under an electron microscope, the solenoidal supercoiling DNA has a round shape with an average diameter of 45 nm. The facts suggest that solenoidaUy supercoiled DNA be a structural entity independent of histones. The polymorphism of DNA structure may be important to packing of DNA in vivo.

  6. Design and Construction of a One-Dimensional DNA Track for an Artificial Molecular Motor

    Directory of Open Access Journals (Sweden)

    Suzana Kovacic

    2012-01-01

    Full Text Available DNA is a versatile heteropolymer that shows great potential as a building block for a diverse array of nanostructures. We present here a solution to the problem of designing and synthesizing a DNA-based nanostructure that will serve as the track along which an artificial molecular motor processes. This one-dimensional DNA track exhibits periodically repeating elements that provide specific binding sites for the molecular motor. Besides these binding elements, additional sequences are necessary to label specific regions within the DNA track and to facilitate track construction. Designing an ideal DNA track sequence presents a particular challenge because of the many variable elements that greatly expand the number of potential sequences from which the ideal sequence must be chosen. In order to find a suitable DNA sequence, we have adapted a genetic algorithm which is well suited for a large but sparse search space. This algorithm readily identifies long DNA sequences that include all the necessary elements to both facilitate DNA track construction and to present appropriate binding sites for the molecular motor. We have successfully experimentally incorporated the sequence identified by the algorithm into a long DNA track meeting the criteria for observation of the molecular motor's activity.

  7. Spectroscopic and molecular docking studies on the interaction of troxerutin with DNA.

    Science.gov (United States)

    Subastri, A; Ramamurthy, C H; Suyavaran, A; Mareeswaran, R; Lokeswara Rao, P; Harikrishna, M; Suresh Kumar, M; Sujatha, V; Thirunavukkarasu, C

    2015-01-01

    Troxerutin (TXER) is a derivative of naturally occurring bioflavonoid rutin. It possesses different biological activities in rising clinical world. The biological activity possessed by most of the drugs mainly targets on macromolecules. Hence, in the current study we have examined the interaction mechanism of TXER with calf thymus DNA (CT-DNA) by using various spectroscopic methods, isothermal titration calorimetry (ITC) and molecular docking studies. Further, DNA cleavage study was carried out to find the DNA protection activity of TXER. UV-absorption and emission spectroscopy showed low binding constant values via groove binding. Circular dichroism study indicates that TXER does not modify native B-form of DNA, and it retains the native B-conformation. Furthermore, no effective positive potential peak shift was observed in TXER-DNA complex during electrochemical analysis by which it represents an interaction of TXER with DNA through groove binding. Molecular docking study showed thymine guanine based interaction with docking score -7.09 kcal/mol. This result was compared to experimental ITC value. The DNA cleavage study illustrates that TXER does not cause any DNA damage as well as TXER showed DNA protection against hydroxyl radical induced DNA damage. From this study, we conclude that TXER interacts with DNA by fashion of groove binding.

  8. DNA extraction from sea anemone (Cnidaria: Actiniaria tissues for molecular analyses

    Directory of Open Access Journals (Sweden)

    Pinto S.M.

    2000-01-01

    Full Text Available A specific DNA extraction method for sea anemones is described in which extraction of total DNA from eight species of sea anemones and one species of corallimorpharian was achieved by changing the standard extraction protocols. DNA extraction from sea anemone tissue is made more difficult both by the tissue consistency and the presence of symbiotic zooxanthellae. The technique described here is an efficient way to avoid problems of DNA contamination and obtain large amounts of purified and integral DNA which can be used in different kinds of molecular analyses.

  9. 基于条码技术的实验室管理系统的设计与实现%Design and implementation of a laboratory management system based on bar code technique

    Institute of Scientific and Technical Information of China (English)

    王铁流; 秦璐璐

    2009-01-01

    A laboratory management system based on bar code technique was designed. The system mainly includes three important parts: laboratory equipment management, book management, and experiment teaching class management. Combined with internet technology, the system has realized automatic and open laboratory management.%设计了以条码技术为基础的实验室管理系统.该系统主要包括实验室设备管理,图书资料管理和实验教学课堂管理三部分.结合网络技术实现了开放型实验室管理的信息化.

  10. 基于ARM的物品精准定位和二维条码扫描系统设计%Design of Scanning Two-Dimensional Bar Code and Precise Positioning System Based on ARM

    Institute of Scientific and Technical Information of China (English)

    姚立; 刘幺和

    2012-01-01

    GPS positioning and two-dimensional bar code scanning system based on ARM is presented, using the linux operating system and the associated software is provided. System can tin ly locate the positon of the goods on the transporting way and timely read the infor-mation in the two-dimensional code of the goods. Thereby increasing the efficiency of the system.%文章提出了一种以嵌入式ARM为核心,以GPS定位和二维条码扫描为基础的系统,采用Linux操作系统,并给出了相关的软件程序设计.系统可以在物品运输的途中实时的定位,并且还可以实时的了解物品上二维码的信息,从而提高了系统的效率.

  11. Molecular biology. Shielding broken DNA for a quick fix

    DEFF Research Database (Denmark)

    Lukas, Jiri; Lukas, Claudia

    2013-01-01

    A fast-acting DNA repair mechanism involves a protein complex that blocks an alternative process that requires a cell to wait for repair.......A fast-acting DNA repair mechanism involves a protein complex that blocks an alternative process that requires a cell to wait for repair....

  12. Preparation of high molecular weight gDNA and bacterial artificial chromosome (BAC) libraries in plants.

    Science.gov (United States)

    Biradar, Siddanagouda S; Nie, Xiaojun; Feng, Kewei; Weining, Song

    2014-01-01

    Bacterial artificial chromosome (BAC) libraries are extremely valuable large-insert DNA libraries for physical mapping, positional cloning, comparative genomic analysis, complete genome sequencing, and evolutionary studies. Due to their stability and relative simplicity BAC libraries are most preferred over other approaches for cloning large genomic DNA fragments for large-insert libraries. Isolation of intact high molecular weight (HMW) DNA is a critical step underlying the success of large-insert genomic DNA library construction. It requires the isolation of purified nuclei, embedding them into LMP agarose plugs, restriction digestion of the plugs, and quite often size selection using PFGE and electro-elution of insert DNA. The construction of BAC libraries is complex and challenging for most molecular laboratories. To facilitate the construction of BAC libraries, we present a step-by-step protocol for isolation of HMW DNA and construction of plant BAC libraries.

  13. High molecular weight DNA assembly in vivo for synthetic biology applications.

    Science.gov (United States)

    Juhas, Mario; Ajioka, James W

    2017-05-01

    DNA assembly is the key technology of the emerging interdisciplinary field of synthetic biology. While the assembly of smaller DNA fragments is usually performed in vitro, high molecular weight DNA molecules are assembled in vivo via homologous recombination in the host cell. Escherichia coli, Bacillus subtilis and Saccharomyces cerevisiae are the main hosts used for DNA assembly in vivo. Progress in DNA assembly over the last few years has paved the way for the construction of whole genomes. This review provides an update on recent synthetic biology advances with particular emphasis on high molecular weight DNA assembly in vivo in E. coli, B. subtilis and S. cerevisiae. Special attention is paid to the assembly of whole genomes, such as those of the first synthetic cell, synthetic yeast and minimal genomes.

  14. Isolation of high molecular weight DNA suitable for the construction of genomic libraries.

    Science.gov (United States)

    Steven, J; McKechnie, D; Graham, A

    1988-01-01

    Recent advances in molecular biology have made it possible to construct complete gene libraries for any organism that uses DNA as its carrier of genetic information. A gene library should contain a large number of cloned DNA fragments that in total contain the entire donor genome. The construction of a genomic library first requires the isolation of DNA from the donor organism. To be of maximum use in the construction of genomic libraries, DNA isolated from the donor organism should fulfill the following criteria. First, the DNA must represent all sequences in the genome to be cloned. Second, it must be of high molecular weight. Third, no contaminants must taint the DNA so that its use as a substrate for restriction endonucleases and other enzymes used in genetic engineering is uninhibited.

  15. DNA-binding study of anticancer drug cytarabine by spectroscopic and molecular docking techniques.

    Science.gov (United States)

    Shahabadi, Nahid; Falsafi, Monireh; Maghsudi, Maryam

    2017-01-02

    The interaction of anticancer drug cytarabine with calf thymus DNA (CT-DNA) was investigated in vitro under simulated physiological conditions by multispectroscopic techniques and molecular modeling study. The fluorescence spectroscopy and UV absorption spectroscopy indicated drug interacted with CT-DNA in a groove-binding mode, while the binding constant of UV-vis and the number of binding sites were 4.0 ± 0.2 × 10(4) L mol(-1) and 1.39, respectively. The fluorimetric studies showed that the reaction between the drugs with CT-DNA is exothermic. Circular dichroism spectroscopy was employed to measure the conformational change of DNA in the presence of cytarabine. Furthermore, the drug induces detectable changes in its viscosity for DNA interaction. The molecular modeling results illustrated that cytarabine strongly binds to groove of DNA by relative binding energy of docked structure -20.61 KJ mol(-1). This combination of multiple spectroscopic techniques and molecular modeling methods can be widely used in the investigation on the interaction of small molecular pollutants and drugs with biomacromolecules for clarifying the molecular mechanism of toxicity or side effect in vivo.

  16. Simulating Molecular Interactions of Carbon Nanoparticles with a Double-Stranded DNA Fragment

    Directory of Open Access Journals (Sweden)

    Zhuang Wang

    2015-01-01

    Full Text Available Molecular interactions between carbon nanoparticles (CNPs and a double-stranded deoxyribonucleic acid (dsDNA fragment were investigated using molecular dynamics (MD simulations. Six types of CNPs including fullerenes (C60 and C70, (8,0 single-walled carbon nanotube (SWNT, (8,0 double-walled carbon nanotube (DWNT, graphene quantum dot (GQD, and graphene oxide quantum dot (GOQD were studied. Analysis of the best geometry indicates that the dsDNA fragment can bind to CNPs through pi-stacking and T-shape. Moreover, C60, DWNT, and GOQD bind to the dsDNA molecules at the minor groove of the nucleotide, and C70, SWNT, and GQD bind to the dsDNA molecules at the hydrophobic ends. Estimated interaction energy implies that van der Waals force may mainly contribute to the mechanisms for the dsDNA-C60, dsDNA-C70, and dsDNA-SWNT interactions and electrostatic force may contribute considerably to the dsDNA-DWNT, dsDNA-GQD, and dsDNA-GOQD interactions. On the basis of the results from large-scale MD simulations, it was found that the presence of the dsDNA enhances the dispersion of C60, C70, and SWNT in water and has a slight impact on DWNT, GQD, and GOQD.

  17. Temperature gating and competing temperature-dependent effects in DNA molecular wires

    Science.gov (United States)

    Wibowo, Denni; Narenji, Alaleh; Kassegne, Sam

    2017-02-01

    While recent research in electron-transport mechanism on a double strands DNA seems to converge into a consensus, experiments in direct electrical measurements on a long DNA molecules still lead to a conflicting result This study is the continuation of our previous research in electrical characterization of DNA molecular wires, where we furtherly investigate the effects of temperature on the electrical conductivity of DNA molecular wires by measuring its impedance response. We found that at higher temperatures, the expected increase in charge hopping mechanism may account for the decrease in impedance (and hence increase in conductivity) supporting the 'charge hopping mechanism' theory. UV light exposure, on the other hand, causes damage to GC base pairs reducing the path available for hopping mechanism and hence resulting in increased impedance - this again supporting the 'charge hopping mechanism' theory. We also report that λ-DNA molecular wires have differing impedance responses at two temperature regimes: impedance increases between 4 °C - 40 °C and then decreases between 40 °C - melting point (˜110 °C), after which λ-DNA denatures resulting in no current transduction. We submit that the low impedance of λ-DNA molecular wires observed at moderate to high frequencies may have significant implications to the field of DNA-based bionanoelectronics.

  18. Nonlinear microrheology and molecular imaging to map microscale deformations of entangled DNA networks

    Science.gov (United States)

    Wu, Tsai-Chin; Anderson, Rae

    We use active microrheology coupled to single-molecule fluorescence imaging to elucidate the microscale dynamics of entangled DNA. DNA naturally exists in a wide range of lengths and topologies, and is often confined in cell nucleui, forming highly concentrated and entangled biopolymer networks. Thus, DNA is the model polymer for understanding entangled polymer dynamics as well as the crowded environment of cells. These networks display complex viscoelastic properties that are not well understood, especially at the molecular-level and in response to nonlinear perturbations. Specifically, how microscopic stresses and strains propagate through entangled networks, and what molecular deformations lead to the network stress responses are unknown. To answer these important questions, we optically drive a microsphere through entangled DNA, perturbing the system far from equilibrium, while measuring the resistive force the DNA exerts on the bead during and after bead motion. We simultaneously image single fluorescent-labeled DNA molecules throughout the network to directly link the microscale stress response to molecular deformations. We characterize the deformation of the network from the molecular-level to the mesoscale, and map the stress propagation throughout the network. We further study the impact of DNA length (11 - 115 kbp) and topology (linear vs ring DNA) on deformation and propagation dynamics, exploring key nonlinear features such as tube dilation and power-law relaxation.

  19. Suitability of the boiling method of DNA extraction in mosquitoes for routine molecular analyses

    Directory of Open Access Journals (Sweden)

    D. K. Sarma

    2014-09-01

    Full Text Available This communication deals with the experience on suitability of the boiling method of DNA extraction from mosquito tissues. The DNA extracted by this method was found, by and large, stable after 30 months of storage. The method is useful for routine molecular entomological applications.

  20. Understanding the molecular mechanism of formaldehyde-induced DNA-protein crosslink repair

    Science.gov (United States)

    Formaldehyde induces DNA-protein crosslinks (DPCs) in several experimental in vitro and in vivo test systems, as well as in exposed human workers. DPCs are repaired by several DNA repair pathways in different species, but the molecular understanding of DPC repair in human tissues...

  1. Molecular cloning of lupin leghemoglobin cDNA

    DEFF Research Database (Denmark)

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

    1987-01-01

    Poly(A)+ RNA isolated from root nodules of yellow lupin (Lupinus luteus, var. Ventus) has been used as a template for the construction of a cDNA library. The ds cDNA was synthesized and inserted into the Hind III site of plasmid pBR 322 using synthetic Hind III linkers. Clones containing sequences...... its nucleotide sequence was consistent with known amino acid sequence of lupin Lb II. The cloned lupin Lb cDNA hybridized to poly(A)+ RNA from nodules only, which is in accordance with the general concept, that leghemoglobin is expressed exclusively in nodules. Udgivelsesdato: 1987-null...

  2. Molecular cloning of lupin leghemoglobin cDNA

    DEFF Research Database (Denmark)

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

    1987-01-01

    Poly(A)+ RNA isolated from root nodules of yellow lupin (Lupinus luteus, var. Ventus) has been used as a template for the construction of a cDNA library. The ds cDNA was synthesized and inserted into the Hind III site of plasmid pBR 322 using synthetic Hind III linkers. Clones containing sequences...... its nucleotide sequence was consistent with known amino acid sequence of lupin Lb II. The cloned lupin Lb cDNA hybridized to poly(A)+ RNA from nodules only, which is in accordance with the general concept, that leghemoglobin is expressed exclusively in nodules. Udgivelsesdato: 1987-null...

  3. In vitro molecular machine learning algorithm via symmetric internal loops of DNA.

    Science.gov (United States)

    Lee, Ji-Hoon; Lee, Seung Hwan; Baek, Christina; Chun, Hyosun; Ryu, Je-Hwan; Kim, Jin-Woo; Deaton, Russell; Zhang, Byoung-Tak

    2017-08-01

    Programmable biomolecules, such as DNA strands, deoxyribozymes, and restriction enzymes, have been used to solve computational problems, construct large-scale logic circuits, and program simple molecular games. Although studies have shown the potential of molecular computing, the capability of computational learning with DNA molecules, i.e., molecular machine learning, has yet to be experimentally verified. Here, we present a novel molecular learning in vitro model in which symmetric internal loops of double-stranded DNA are exploited to measure the differences between training instances, thus enabling the molecules to learn from small errors. The model was evaluated on a data set of twenty dialogue sentences obtained from the television shows Friends and Prison Break. The wet DNA-computing experiments confirmed that the molecular learning machine was able to generalize the dialogue patterns of each show and successfully identify the show from which the sentences originated. The molecular machine learning model described here opens the way for solving machine learning problems in computer science and biology using in vitro molecular computing with the data encoded in DNA molecules. Copyright © 2017. Published by Elsevier B.V.

  4. Molecular mechanisms of DNA repair inhibition by caffeine

    Energy Technology Data Exchange (ETDEWEB)

    Selby, C.P.; Sancar, A. (Univ. of North Carolina School of Medicine, Chapel Hill (USA))

    1990-05-01

    Caffeine potentiates the mutagenic and lethal effects of genotoxic agents. It is thought that this is due, at least in some organisms, to inhibition of DNA repair. However, direct evidence for inhibition of repair enzymes has been lacking. Using purified Escherichia coli DNA photolyase and (A)BC excinuclease, we show that the drug inhibits photoreactivation and nucleotide excision repair by two different mechanisms. Caffeine inhibits photoreactivation by interfering with the specific binding of photolyase to damaged DNA, and it inhibits nucleotide excision repair by promoting nonspecific binding of the damage-recognition subunit, UvrA, of (A)BC excinuclease. A number of other intercalators, including acriflavin and ethidium bromide, appear to inhibit the excinuclease by a similar mechanism--that is, by trapping the UvrA subunit in nonproductive complexes on undamaged DNA.

  5. Cell and molecular biology of DNA methyltransferase 1.

    Science.gov (United States)

    Mohan, K Naga; Chaillet, J Richard

    2013-01-01

    The DNA cytosine methyltransferase 1 (DNMT1) is a ubiquitous nuclear enzyme that catalyzes the well-established reaction of placing methyl groups on the unmethylated cytosines in methyl-CpG:CpG base pairs in the hemimethylated DNA formed by methylated parent and unmethylated daughter strands. This activity regenerates fully methylated methyl-CpG:methyl-CpG pairs. Despite the straightforward nature of its catalytic activity, detailed biochemical, genetic, and developmental studies revealed intricate details of the central regulatory role of DNMT1 in governing the epigenetic makeup of the nuclear genome. DNMT1 mediates demethylation and also participates in seemingly wide cellular functions unrelated to maintenance DNA methylation. This review brings together mechanistic details of maintenance methylation by DNMT1, its regulation at transcriptional and posttranscriptional levels, and the seemingly unexpected functions of DNMT1 in the context of DNA methylation which is central to epigenetic changes that occur during development and the process of cell differentiation.

  6. Electrophoretic High Molecular Weight DNA Purification Enables Optical Mapping

    OpenAIRE

    Maydan, Jason; THOMAS, Matthew; Tabanfar, Leyla; Mai, Laura; Poon, Hau-Ling; Pe, Joel; HAHN, KRISTEN; Goji, Noriko; Amoako, Kingsley; Marziali, Andre; Hanson, Dan

    2013-01-01

    Optical mapping generates an ordered restriction map from single, long DNA molecules. By overlapping restriction maps from multiple molecules, a physical map of entire chromosomes and genomes is constructed, greatly facilitating genome assembly in next generation sequencing projects, comparative genomics and strain typing. However, optical mapping relies on a method of preparing high quality DNA >250 kb in length, which can be challenging from some organisms and sample types. Here we demonstr...

  7. Kinetic mechanism of DNA translocation by the RSC molecular motor.

    Science.gov (United States)

    Eastlund, Allen; Malik, Shuja Shafi; Fischer, Christopher J

    2013-04-15

    ATP-dependent nucleosome repositioning by chromatin remodeling enzymes requires the translocation of these enzymes along the nucleosomal DNA. Using a fluorescence stopped-flow assay we monitored DNA translocation by a minimal RSC motor and through global analysis of these time courses we have determined that this motor has a macroscopic translocation rate of 2.9 bp/s with a step size of 1.24 bp. From the complementary quantitative analysis of the associated time courses of ATP consumption during DNA translocation we have determined that this motor has an efficiency of 3.0 ATP/bp, which is slightly less that the efficiency observed for several genetically related DNA helicases and which likely results from random pausing by the motor during translocation. Nevertheless, this motor is able to exert enough force during translocation to displace streptavidin from biotinylated DNA. Taken together these results are the necessary first step for quantifying both the role of DNA translocation in nucleosome repositioning by RSC and the efficiency at which RSC couples ATP binding and hydrolysis to nucleosome repositioning.

  8. Expression of exogenous DNA methyltransferases: application in molecular and cell biology.

    Science.gov (United States)

    Dyachenko, O V; Tarlachkov, S V; Marinitch, D V; Shevchuk, T V; Buryanov, Y I

    2014-02-01

    DNA methyltransferases might be used as powerful tools for studies in molecular and cell biology due to their ability to recognize and modify nitrogen bases in specific sequences of the genome. Methylation of the eukaryotic genome using exogenous DNA methyltransferases appears to be a promising approach for studies on chromatin structure. Currently, the development of new methods for targeted methylation of specific genetic loci using DNA methyltransferases fused with DNA-binding proteins is especially interesting. In the present review, expression of exogenous DNA methyltransferase for purposes of in vivo analysis of the functional chromatin structure along with investigation of the functional role of DNA methylation in cell processes are discussed, as well as future prospects for application of DNA methyltransferases in epigenetic therapy and in plant selection.

  9. Molecular Cloning and Analysis of a DNA Repetitive Element from the Mouse Genome

    Science.gov (United States)

    Geisinger, Adriana; Cossio, Gabriela; Wettstein, Rodolfo

    2006-01-01

    We report the development of a 3-week laboratory activity for an undergraduate molecular biology course. This activity introduces students to the practice of basic molecular techniques such as restriction enzyme digestion, agarose gel electrophoresis, cloning, plasmid DNA purification, Southern blotting, and sequencing. Students learn how to carry…

  10. In situ structure and dynamics of DNA origami determined through molecular dynamics simulations.

    Science.gov (United States)

    Yoo, Jejoong; Aksimentiev, Aleksei

    2013-12-10

    The DNA origami method permits folding of long single-stranded DNA into complex 3D structures with subnanometer precision. Transmission electron microscopy, atomic force microscopy, and recently cryo-EM tomography have been used to characterize the properties of such DNA origami objects, however their microscopic structures and dynamics have remained unknown. Here, we report the results of all-atom molecular dynamics simulations that characterized the structural and mechanical properties of DNA origami objects in unprecedented microscopic detail. When simulated in an aqueous environment, the structures of DNA origami objects depart from their idealized targets as a result of steric, electrostatic, and solvent-mediated forces. Whereas the global structural features of such relaxed conformations conform to the target designs, local deformations are abundant and vary in magnitude along the structures. In contrast to their free-solution conformation, the Holliday junctions in the DNA origami structures adopt a left-handed antiparallel conformation. We find the DNA origami structures undergo considerable temporal fluctuations on both local and global scales. Analysis of such structural fluctuations reveals the local mechanical properties of the DNA origami objects. The lattice type of the structures considerably affects global mechanical properties such as bending rigidity. Our study demonstrates the potential of all-atom molecular dynamics simulations to play a considerable role in future development of the DNA origami field by providing accurate, quantitative assessment of local and global structural and mechanical properties of DNA origami objects.

  11. Interaction of anthelmintic drug (thiabendazole with DNA: Spectroscopic and molecular modeling studies

    Directory of Open Access Journals (Sweden)

    Fahimeh Jalali

    2017-05-01

    Full Text Available The interaction between thiabendazole (TBZ and calf-thymus DNA (ct-DNA was studied by experimental and molecular modeling methods. The intrinsic fluorescence of TBZ was quenched in the presence of ct-DNA. In competition experiments, TBZ could displace Hoechst 33258 (a minor groove binder to DNA, whereas it was unable to replace ethidium bromide (an intercalator. Potassium iodide could quench the fluorescence of TBZ, which indicated the nonintercalative mode of binding of TBZ to ct-DNA. UV absorbance of TBZ shows hyperchromic effect on the addition of DNA to the solution with negligible shift in wavelength. Salt effect studies showed the non-electrostatic nature of binding of TBZ to DNA. The viscosity of ct-DNA solution was almost unchanged on addition of TBZ. Circular dichroism (CD spectra of DNA showed small changes in the presence of TBZ which is in agreement with groove binding mode of interaction. Moreover, from molecular modeling methods, a docked structure with minimum energy was obtained in which TBZ was located in minor grooves of ct-DNA.

  12. Effect of gold nanoparticle on stability of the DNA molecule: A study of molecular dynamics simulation.

    Science.gov (United States)

    Izanloo, Cobra

    2017-09-26

    An understanding of the mechanism of DNA interactions with gold nanoparticles is useful in today medicine applications. We have performed a molecular dynamics simulation on a B-DNA duplex (CCTCAGGCCTCC) in the vicinity of a gold nanoparticle with a truncated octahedron structure composed of 201 gold atoms (diameter ∼1.8 nm) to investigate gold nanoparticle (GNP) effects on the stability of DNA. During simulation, the nanoparticle is closed to DNA and phosphate groups direct the particles into the major grooves of the DNA molecule. Because of peeling and untwisting states that are occur at end of DNA, the nucleotide base lies flat on the surface of GNP. The configuration entropy is estimated using the covariance matrix of atom-positional fluctuations for different bases. The results show that when a gold nanoparticle has interaction with DNA, entropy increases. The results of conformational energy and the hydrogen bond numbers for DNA indicated that DNA becomes unstable in the vicinity of a gold nanoparticle. The radial distribution function was calculated for water hydrogen-phosphate oxygen pairs. Almost for all nucleotide, the presence of a nanoparticle around DNA caused water molecules to be released from the DNA duplex and cations were close to the DNA.

  13. Investigation Into the Effects of Nucleotide Content on the Electrical Characteristics of DNA Plasmid Molecular Wires.

    Science.gov (United States)

    Goshi, Noah; Narenji, Alaleh; Bui, Chris; Mokili, John L; Kassegne, Sam

    2016-09-01

    In this study, we investigate the effect of nucleotide content on the conductivity of plasmid length DNA molecular wires covalently bound to high aspect-ratio gold electrodes. The DNA wires were all between [Formula: see text] in length (>6000bp), and contained either 39%, 53%, or 64% GC base-pairs. We compared the current-voltage (I-V) and frequency-impedance characteristics of the DNA wires with varying GC content, and observed statistically significantly higher conductivity in DNA wires containing higher GC content in both AC and DC measurement methods. Additionally, we noted that the conductivity decreased as a function of time for all DNA wires, with the impedance at 100 Hz nearly doubling over a period of seven days. All readings were taken in humidity and temperature controlled environments on DNA wires suspended above an insulative substrate, thus minimizing the effect of experimental and environmental factors as well as potential for nonlinear alternate DNA confirmations. While other groups have studied the effect of GC content on the conductivity of nanoscale DNA molecules (DNA wires at scales that may be required during the fabrication of DNA-based electronics. Furthermore, our results provide further evidence that many of the charge transfer theories developed from experiments using nanoscale DNA molecules may still be applicable for DNA wires at the micro scale.

  14. Investigation of Effects of Nucleotide Content on Electrical Characteristics of DNA Plasmid Molecular Wires.

    Science.gov (United States)

    Goshi, Noah; Narenji, Alaleh; Bui, Chris; Mokili, John L; Kassegne, Sam

    2016-07-28

    In this study, we investigate the effect of nucleotide content on the conductivity of plasmid length DNA molecular wires covalently bound to high aspect-ratio gold electrodes. The DNA wires were all between 2.20-2.35μm in length (>6000bp), and contained either 39%, 53%, or 64% GC base-pairs. We compared the current-voltage (I-V) and frequency-impedance characteristics of the DNA wires with varying GC content, and observed statistically significantly higher conductivity in DNA wires containing higher GC content in both AC and DC measurement methods. Additionally, we noted that the conductivity decreased as a function of time for all DNA wires, with the impedance at 100Hz nearly doubling over a period of seven days. All readings were taken in humidity and temperature controlled environments on DNA wires suspended above an insulative substrate, thus minimizing the effect of experimental and environmental factors as well as potential for nonlinear alternate DNA confirmations. While other groups have studied the effect of GC content on the conductivity of nano-scale DNA molecules (DNA wires at scales that may be required during the fabrication of DNA-based electronics. Furthermore, our results provide further evidence that many of the charge transfer theories developed from experiments using nano-scale DNA molecules may still be applicable for DNA wires at the micro-scale.

  15. Molecular threading: mechanical extraction, stretching and placement of DNA molecules from a liquid-air interface.

    Directory of Open Access Journals (Sweden)

    Andrew C Payne

    Full Text Available We present "molecular threading", a surface independent tip-based method for stretching and depositing single and double-stranded DNA molecules. DNA is stretched into air at a liquid-air interface, and can be subsequently deposited onto a dry substrate isolated from solution. The design of an apparatus used for molecular threading is presented, and fluorescence and electron microscopies are used to characterize the angular distribution, straightness, and reproducibility of stretched DNA deposited in arrays onto elastomeric surfaces and thin membranes. Molecular threading demonstrates high straightness and uniformity over length scales from nanometers to micrometers, and represents an alternative to existing DNA deposition and linearization methods. These results point towards scalable and high-throughput precision manipulation of single-molecule polymers.

  16. Molecular Cloning of the Bovine Liver ADPRT cDNA

    Science.gov (United States)

    1988-12-13

    Sambrook. J. (1982) Molecular Cloning - A Laboratory Manual. Cold Spring Harbor Laboratory, N. Y. Martinez, H (1988) Sequence Analysis Programs. Publ...Sambrook, J. (1982) Molecular Cloning - A Laboratory Manual. Cold Spring Harbor Laboratory, N. Y. 1)..Messing, J. (1983) Methods in Enzymol. 101. 20-78. 1-Actin-S

  17. Rapid methods for the extraction and archiving of molecular grade fungal genomic DNA.

    Science.gov (United States)

    Borman, Andrew M; Palmer, Michael; Johnson, Elizabeth M

    2013-01-01

    The rapid and inexpensive extraction of fungal genomic DNA that is of sufficient quality for molecular approaches is central to the molecular identification, epidemiological analysis, taxonomy, and strain typing of pathogenic fungi. Although many commercially available and in-house extraction procedures do eliminate the majority of contaminants that commonly inhibit molecular approaches, the inherent difficulties in breaking fungal cell walls lead to protocols that are labor intensive and that routinely take several hours to complete. Here we describe several methods that we have developed in our laboratory that allow the extremely rapid and inexpensive preparation of fungal genomic DNA.

  18. DNA extraction methods and multiple sampling to improve molecular diagnosis of Sarcocystis spp. in cattle hearts.

    Science.gov (United States)

    Bräunig, Patrícia; Portella, Luiza Pires; Cezar, Alfredo Skrebsky; Libardoni, Felipe; Sangioni, Luis Antonio; Vogel, Fernanda Silveira Flores; Gonçalves, Paulo Bayard Dias

    2016-10-01

    Molecular detection of Sarcocystis spp. in tissue samples can be useful for experimental and diagnostic purposes. However, the parasite spreads unevenly through tissues, forming tissue cysts, and the cystic wall is an obstacle in DNA extraction protocols. Therefore, adequate sampling and effective disruption of the cysts are essential to improve the accuracy of DNA detection by PCR. The aims of this study were to evaluate the suitability of four protocols for DNA extraction from cysts of Sarcocystis spp. present in bovine myocardium samples or after their harvest in phosphate-buffered saline (PBS) solution as well as determine the effects of single or multiple sampling on the accuracy of molecular diagnosis of sarcocystosis in cattle hearts. Cysts and myocardium samples from nine bovine hearts were randomly distributed to four DNA extraction protocols: kit, kit with modification, DNAzol, and cetyl-trimethyl ammonium bromide (CTAB). Samples were submitted to DNA extraction and PCR as replicates of each heart (simplicate, duplicate, and triplicate), and the probability of a true positive diagnostic was calculated. Among the protocols tested, the kit with modification was determined to be the most suitable for DNA extraction from cysts in PBS solution (92.6 % of DNA detection by PCR); DNAzol resulted in higher DNA detection frequency from bovine myocardium samples (48.1 %). Multiple sampling improved the molecular diagnosis of Sarcocystis spp. infection in cattle hearts, increasing at 22.2 % the rate of true positive diagnostic.

  19. DNA

    Science.gov (United States)

    Stent, Gunther S.

    1970-01-01

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

  20. Interaction of vasicine with calf thymus DNA: Molecular docking, spectroscopic and differential scanning calorimetric insights

    Science.gov (United States)

    R. S., Sai Murali; R. S., Sai Siddhardha; Rajesh Babu, D.; Venketesh, S.; Basavaraju, R.; Nageswara Rao, G.

    2017-06-01

    The present study brings out the interaction between vasicine, an alkaloid and Adhatoda vasica Nees with double stranded DNA. The physico-chemical interaction between small molecules and nucleic acids is a major area of focus in screening drugs against various cancers. Molecular probing in our study using Molecular Operating Environment (MOE) has revealed interaction of vasicine with DNA double helix. Here we report the interaction of vasicine with Calf thymus DNA. We present for the first time the results obtained from UV-visible, fluorescence spectroscopic and differential scanning calorimetric techniques that suggest a moderate to strong electrostatic, hydrophobic and van der Waals interactions mediating the DNA binding properties of vasicine, leading to disruption of DNA secondary structure.

  1. Directed Molecular Evolution of Nitrite Oxido-reductase by DNA-shuffling

    Institute of Scientific and Technical Information of China (English)

    JUN-WEN LI; JIN-LAI ZHENG; XIN-WEI WANG; MIN JIN; FU-HUAN CHAO

    2007-01-01

    Objective To develtop directly molecular evolution of nitrite oxido-reductase using DNA-shuffling technique because nitrobacteria grow extremely slow and are unable to nitrify effectively inorganic nitrogen in wastewater treatment. Methods The norB gene coding the nitrite oxido-reductase in nitrobacteria was cloned and sequenced. Then, directed molecular evolution of nitrite oxido-reductase was developed by DNA-shuffling of 15 norB genes from different nitrobacteria. Results After DNA-shuffling with sexual PCR and staggered extension process PCR, the sequence was different from its parental DNA fragments and the homology ranged from 98% to 99%. The maximum nitrification rate of the modified bacterium of X16 by modified bacterium had the same characteristics of its parental bacteria of E. coli and could grow rapidly in normal cultures.Conclusion DNA-shuffling was successfully used to engineer E. coli, which had norB gene and could degrade inorganic nitrogen effectively.

  2. Synchronized assembly of gold nanoparticles driven by a dynamic DNA-fueled molecular machine.

    Science.gov (United States)

    Song, Tingjie; Liang, Haojun

    2012-07-04

    A strategy for gold nanoparticle (AuNP) assembly driven by a dynamic DNA-fueled molecular machine is revealed here. In this machine, the aggregation of DNA-functionalized AuNPs is regulated by a series of toehold-mediated strand displacement reactions of DNA. The aggregation rate of the AuNPs can be regulated by controlling the amount of oligonucleotide catalyst. The versatility of the dynamic DNA-fueled molecular machine in the construction of two-component "OR" and "AND" logic gates has been demonstrated. This newly established strategy may find broad potential applications in terms of building up an "interface" that allows the combination of the strand displacement-based characteristic of DNA with the distinct assembly properties of inorganic nanoparticles, ultimately leading to the fabrication of a wide range of complex multicomponent devices and architectures.

  3. Extraction of high-molecular-weight genomic DNA for long-read sequencing of single molecules.

    Science.gov (United States)

    Mayjonade, Baptiste; Gouzy, Jérôme; Donnadieu, Cécile; Pouilly, Nicolas; Marande, William; Callot, Caroline; Langlade, Nicolas; Muños, Stéphane

    2016-10-01

    De novo sequencing of complex genomes is one of the main challenges for researchers seeking high-quality reference sequences. Many de novo assemblies are based on short reads, producing fragmented genome sequences. Third-generation sequencing, with read lengths >10 kb, will improve the assembly of complex genomes, but these techniques require high-molecular-weight genomic DNA (gDNA), and gDNA extraction protocols used for obtaining smaller fragments for short-read sequencing are not suitable for this purpose. Methods of preparing gDNA for bacterial artificial chromosome (BAC) libraries could be adapted, but these approaches are time-consuming, and commercial kits for these methods are expensive. Here, we present a protocol for rapid, inexpensive extraction of high-molecular-weight gDNA from bacteria, plants, and animals. Our technique was validated using sunflower leaf samples, producing a mean read length of 12.6 kb and a maximum read length of 80 kb.

  4. Thermoelectric effect and its dependence on molecular length and sequence in single DNA molecules.

    Science.gov (United States)

    Li, Yueqi; Xiang, Limin; Palma, Julio L; Asai, Yoshihiro; Tao, Nongjian

    2016-01-01

    Studying the thermoelectric effect in DNA is important for unravelling charge transport mechanisms and for developing relevant applications of DNA molecules. Here we report a study of the thermoelectric effect in single DNA molecules. By varying the molecular length and sequence, we tune the charge transport in DNA to either a hopping- or tunnelling-dominated regimes. The thermoelectric effect is small and insensitive to the molecular length in the hopping regime. In contrast, the thermoelectric effect is large and sensitive to the length in the tunnelling regime. These findings indicate that one may control the thermoelectric effect in DNA by varying its sequence and length. We describe the experimental results in terms of hopping and tunnelling charge transport models.

  5. Dynamically Arranging Gold Nanoparticles on DNA Origami for Molecular Logic Gates.

    Science.gov (United States)

    Yang, Jing; Song, Zhichao; Liu, Shi; Zhang, Qiang; Zhang, Cheng

    2016-08-31

    In molecular engineering, DNA molecules have been extensively studied owing to their capacity for accurate structural control and complex programmability. Recent studies have shown that the versatility and predictability of DNA origami make it an excellent platform for constructing nanodevices. In this study, we developed a strand-displacing strategy to selectively and dynamically release specific gold nanoparticles (AuNPs) on a rectangular DNA origami. A set of DNA logic gates ("OR", "AND", and "three-input majority gate") were established based on this strategy, in which computing results were identified by disassembly between the AuNPs and DNA origami. The computing results were detected using experimental approaches such as gel electrophoresis and transmission electron microscopy (TEM). This method can be used to assemble more complex nanosystems and may have potential applications for molecular engineering.

  6. Mitochondrial DNA disease—molecular insights and potential routes to a cure

    Energy Technology Data Exchange (ETDEWEB)

    Russell, Oliver; Turnbull, Doug, E-mail: doug.turnbull@newcastle.ac.uk

    2014-07-01

    Mitochondrial DNA diseases are common neurological conditions caused by mutations in the mitochondrial genome or nuclear genes responsible for its maintenance. Current treatments for these disorders are focussed on the management of the symptoms, rather than the correction of biochemical defects caused by the mutation. This review focuses on the molecular effects of mutations, the symptoms they cause and current work focusing on the development of targeted treatments for mitochondrial DNA disease. - Highlights: • We discuss several common disease causing mtDNA mutations. • We highlight recent work linking pathogenicity to deletion size and heteroplasmy. • We discuss recent advances in the development of targeted mtDNA disease treatments.

  7. Specific interactions between DNA and regulatory protein controlled by ligand-binding: Ab initio molecular simulation

    Energy Technology Data Exchange (ETDEWEB)

    Matsushita, Y., E-mail: kurita@cs.tut.ac.jp; Murakawa, T., E-mail: kurita@cs.tut.ac.jp; Shimamura, K., E-mail: kurita@cs.tut.ac.jp; Oishi, M., E-mail: kurita@cs.tut.ac.jp; Ohyama, T., E-mail: kurita@cs.tut.ac.jp; Kurita, N., E-mail: kurita@cs.tut.ac.jp [Department of Computer Science and Engineering, Toyohashi University of Technology, Tempaku-cho, Toyohashi, Aichi, 441-8580 (Japan)

    2015-02-27

    The catabolite activator protein (CAP) is one of the regulatory proteins controlling the transcription mechanism of gene. Biochemical experiments elucidated that the complex of CAP with cyclic AMP (cAMP) is indispensable for controlling the mechanism, while previous molecular simulations for the monomer of CAP+cAMP complex revealed the specific interactions between CAP and cAMP. However, the effect of cAMP-binding to CAP on the specific interactions between CAP and DNA is not elucidated at atomic and electronic levels. We here considered the ternary complex of CAP, cAMP and DNA in solvating water molecules and investigated the specific interactions between them at atomic and electronic levels using ab initio molecular simulations based on classical molecular dynamics and ab initio fragment molecular orbital methods. The results highlight the important amino acid residues of CAP for the interactions between CAP and cAMP and between CAP and DNA.

  8. Molecular interactions of UvrB protein and DNA from Helicobacter pylori: Insight into a molecular modeling approach.

    Science.gov (United States)

    Bavi, Rohit; Kumar, Raj; Rampogu, Shailima; Son, Minky; Park, Chanin; Baek, Ayoung; Kim, Hyong-Ha; Suh, Jung-Keun; Park, Seok Ju; Lee, Keun Woo

    2016-08-01

    Helicobacter pylori (H. pylori) persevere in the human stomach, an environment in which they encounter many DNA-damaging conditions, including gastric acidity. The pathogenicity of H. pylori is enhanced by its well-developed DNA repair mechanism, thought of as 'machinery,' such as nucleotide excision repair (NER). NER involves multi-enzymatic excinuclease proteins (UvrABC endonuclease), which repair damaged DNA in a sequential manner. UvrB is the central component in prokaryotic NER, essential for damage recognition. Therefore, molecular modeling studies of UvrB protein from H. pylori are carried out with homology modeling and molecular dynamics (MD) simulations. The results reveal that the predicted structure is bound to a DNA hairpin with 3-bp stem, an 11-nucleotide loop, and 3-nt 3' overhang. In addition, a mutation of the Y96A variant indicates reduction in the binding affinity for DNA. Free-energy calculations demonstrate the stability of the complex and help identify key residues in various interactions based on residue decomposition analysis. Stability comparative studies between wild type and mutant protein-DNA complexes indicate that the former is relatively more stable than the mutant form. This predicted model could also be useful in designing new inhibitors for UvrB protein, as well as preventing the pathogenesis of H. pylori.

  9. MOLECULAR CLONING OF OVINE cDNA LEPTIN GENE

    Directory of Open Access Journals (Sweden)

    CLAUDIA TEREZIA SOCOL

    2013-12-01

    Full Text Available An efficient bacterial transformation system suitable for cloning the coding sequence of the ovine leptin gene in E. coli DH5α host cells using the pGEMT easy vector it is described in this paper. The necessity of producing leptin is based on the fact that the role of this molecule in the animal and human organism is still unknown, leptin not existing as commercial product on the Romanian market. The results obtained in the bacterial transformation, cloning, recombinant clones selection, control of the insertion experiments and DNA computational analysis represent the first steps in further genetic engineering experiments such as production of DNA libraries, DNA sequencing, protein expression, etc., for a further contribution in elucidating the role of leptin in the animal and human organism.

  10. Towards understanding the molecular basis of bacterial DNA segregation

    DEFF Research Database (Denmark)

    Leonard, Thomas A.; Møller-Jensen, Jakob; Löwe, Jan

    2005-01-01

    Bacteria ensure the fidelity of genetic inheritance by the coordinated control of chromosome segregation and cell division. Here, we review the molecules and mechanisms that govern the correct subcellular positioning and rapid separation of newly replicated chromosomes and plasmids towards the cell...... poles and, significantly, the emergence of mitotic-like machineries capable of segregating plasmid DNA. We further describe surprising similarities between proteins involved in DNA partitioning (ParA/ParB) and control of cell division (MinD/MinE), suggesting a mechanism for intracellular positioning...... common to the two processes. Finally, we discuss the role that the bacterial cytoskeleton plays in DNA partitioning and the missing link between prokaryotes and eukaryotes that is bacterial mechano-chemical motor proteins. Udgivelsesdato: Mar 29...

  11. Use of DNA methylation for cancer detection and molecular classification.

    Science.gov (United States)

    Zhu, Jingde; Yao, Xuebiao

    2007-03-31

    Conjugation of the methyl group at the fifth carbon of cytosines within the palindromic dinucleotide 5'-CpG-3' sequence (DNA methylation) is the best studied epigenetic mechanism, which acts together with other epigenetic entities: histone modification, chromatin remodeling and microRNAs to shape the chromatin structure of DNA according to its functional state. The cancer genome is frequently characterized by hypermethylation of specific genes concurrently with an overall decrease in the level of 5-methyl cytosine, the pathological implication of which to the cancerous state has been well established. While the latest genome-wide technologies have been applied to classify and interpret the epigenetic layer of gene regulation in the physiological and disease states, the epigenetic testing has also been seriously explored in clinical practice for early detection, refining tumor staging and predicting disease recurrence. This critique reviews the latest research findings on the use of DNA methylation in cancer diagnosis, prognosis and staging/classification.

  12. Molecular engineering of chiral colloidal liquid crystals using DNA origami

    Science.gov (United States)

    Siavashpouri, Mahsa; Wachauf, Christian H.; Zakhary, Mark J.; Praetorius, Florian; Dietz, Hendrik; Dogic, Zvonimir

    2017-08-01

    Establishing precise control over the shape and the interactions of the microscopic building blocks is essential for design of macroscopic soft materials with novel structural, optical and mechanical properties. Here, we demonstrate robust assembly of DNA origami filaments into cholesteric liquid crystals, one-dimensional supramolecular twisted ribbons and two-dimensional colloidal membranes. The exquisite control afforded by the DNA origami technology establishes a quantitative relationship between the microscopic filament structure and the macroscopic cholesteric pitch. Furthermore, it also enables robust assembly of one-dimensional twisted ribbons, which behave as effective supramolecular polymers whose structure and elastic properties can be precisely tuned by controlling the geometry of the elemental building blocks. Our results demonstrate the potential synergy between DNA origami technology and colloidal science, in which the former allows for rapid and robust synthesis of complex particles, and the latter can be used to assemble such particles into bulk materials.

  13. Molecular cloning of nif DNA from Azotobacter vinelandii.

    OpenAIRE

    1985-01-01

    Two clones which contained nif DNA were isolated from a clone bank of total EcoRI-digested Azotobacter vinelandii DNA. The clones carrying the recombinant plasmids were identified by use of the 32P-labeled 6.2-kilobase (kb) nif insert from pSA30 (which contains the Klebsiella pneumoniae nifK, nifD, and nifH genes) as a hybridization probe. Hybridization analysis with fragments derived from the nif insert of pSA30 showed that the 2.6-kb insert from one of the plasmids (pLB1) contains nifK wher...

  14. Linearly programmed DNA-based molecular computer operated on magnetic particle surface in test-tube

    Institute of Scientific and Technical Information of China (English)

    ZHAO Jian; ZHANG Zhizhou; SHI Yongyong; Li Xiuxia; HE Lin

    2004-01-01

    The postgenomic era has seen an emergence of new applications of DNA manipulation technologies, including DNA-based molecular computing. Surface DNA computing has already been reported in a number of studies that, however, all employ different mechanisms other than automaton functions. Here we describe a programmable DNA surface-computing device as a Turing machine-like finite automaton. The laboratory automaton is primarily composed of DNA (inputs, output-detectors, transition molecules as software), DNA manipulating enzymes and buffer system that solve artificial computational problems autonomously. When fluoresceins were labeled in the 5′ end of (-) strand of the input molecule, direct observation of all reaction intermediates along the time scale was made so that the dynamic process of DNA computing could be conveniently visualized. The features of this study are: (i) achievement of finite automaton functions by linearly programmed DNA computer operated on magnetic particle surface and (ii) direct detection of all DNA computing intermediates by capillary electrophoresis. Since DNA computing has the massive parallelism and feasibility for automation, this achievement sets a basis for large-scale implications of DNA computing for functional genomics in the near future.

  15. Molecular Behavior of DNA Origami in Higher-Order Self-Assembly

    Energy Technology Data Exchange (ETDEWEB)

    Li, Zhe [Arizona State Univ., Tempe, AZ (United States); Liu, Minghui [Arizona State Univ., Tempe, AZ (United States); Lei, Wang [Arizona State Univ., Tempe, AZ (United States); Shandong Univ., Jinan (China); Nangreave, Jeanette [Arizona State Univ., Tempe, AZ (United States); Yan, Hao [Arizona State Univ., Tempe, AZ (United States); Liu, Yan [Arizona State Univ., Tempe, AZ (United States)

    2010-09-08

    DNA-based self-assembly is a unique method for achieving higher-order molecular architectures made possible by the fact that DNA is a programmable information-coding polymer. In the past decade, two main types of DNA nanostructures have been developed: branch-shaped DNA tiles with small dimensions (commonly up to ~20 nm) and DNA origami tiles with larger dimensions (up to ~100 nm). Here we aimed to determine the important factors involved in the assembly of DNA origami superstructures. We constructed a new series of rectangular-shaped DNA origami tiles in which parallel DNA helices are arranged in a zigzag pattern when viewed along the DNA helical axis, a design conceived in order to relax an intrinsic global twist found in the original planar, rectangular origami tiles. Self-associating zigzag tiles were found to form linear arrays in both diagonal directions, while planar tiles showed significant growth in only one direction. Although the series of zigzag tiles were designed to promote two-dimensional array formation, one-dimensional linear arrays and tubular structures were observed instead. We discovered that the dimensional aspect ratio of the origami unit tiles and intertile connection design play important roles in determining the final products, as revealed by atomic force microscopy imaging. This study provides insight into the formation of higher-order structures from self-assembling DNA origami tiles, revealing their unique behavior in comparison with conventional DNA tiles having smaller dimensions.

  16. 5-Hydroxy-5-methylhydantoin DNA lesion, a molecular trap for DNA glycosylases

    Science.gov (United States)

    Le Bihan, Yann-Vaï; Angeles Izquierdo, Maria; Coste, Franck; Aller, Pierre; Culard, Françoise; Gehrke, Tim H.; Essalhi, Kadija; Carell, Thomas; Castaing, Bertrand

    2011-01-01

    DNA base-damage recognition in the base excision repair (BER) is a process operating on a wide variety of alkylated, oxidized and degraded bases. DNA glycosylases are the key enzymes which initiate the BER pathway by recognizing and excising the base damages guiding the damaged DNA through repair synthesis. We report here biochemical and structural evidence for the irreversible entrapment of DNA glycosylases by 5-hydroxy-5-methylhydantoin, an oxidized thymine lesion. The first crystal structure of a suicide complex between DNA glycosylase and unrepaired DNA has been solved. In this structure, the formamidopyrimidine-(Fapy) DNA glycosylase from Lactococcus lactis (LlFpg/LlMutM) is covalently bound to the hydantoin carbanucleoside-containing DNA. Coupling a structural approach by solving also the crystal structure of the non-covalent complex with site directed mutagenesis, this atypical suicide reaction mechanism was elucidated. It results from the nucleophilic attack of the catalytic N-terminal proline of LlFpg on the C5-carbon of the base moiety of the hydantoin lesion. The biological significance of this finding is discussed. PMID:21486746

  17. DNA Re-EvolutioN: a game for learning molecular genetics and evolution.

    Science.gov (United States)

    Miralles, Laura; Moran, Paloma; Dopico, Eduardo; Garcia-Vazquez, Eva

    2013-01-01

    Evolution is a main concept in biology, but not many students understand how it works. In this article we introduce the game DNA Re-EvolutioN as an active learning tool that uses genetic concepts (DNA structure, transcription and translation, mutations, natural selection, etc.) as playing rules. Students will learn about molecular evolution while playing a game that mixes up theory and entertainment. The game can be easily adapted to different educational levels. The main goal of this play is to arrive at the end of the game with the longest protein. Students play with pawns and dices, a board containing hypothetical events (mutations, selection) that happen to molecules, "Evolution cards" with indications for DNA mutations, prototypes of a DNA and a mRNA chain with colored "nucleotides" (plasticine balls), and small pieces simulating t-RNA with aminoacids that will serve to construct a "protein" based on the DNA chain. Students will understand how changes in DNA affect the final protein product and may be subjected to positive or negative selection, using a didactic tool funnier than classical theory lectures and easier than molecular laboratory experiments: a flexible and feasible game to learn and enjoy molecular evolution at no-cost. The game was tested by majors and non-majors in genetics from 13 different countries and evaluated with pre- and post-tests obtaining very positive results. © 2013 by The International Union of Biochemistry and Molecular Biology.

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

    Energy Technology Data Exchange (ETDEWEB)

    SUTHERLAND, B.M.

    2001-07-26

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

  19. Photoprotection by topical DNA repair enzymes: molecular correlates of clinical studies.

    Science.gov (United States)

    Yarosh, D B; O'Connor, A; Alas, L; Potten, C; Wolf, P

    1999-02-01

    A new approach to photoprotection is to repair DNA damage after UV exposure. This can be accomplished by delivery of a DNA repair enzyme with specificity to UV-induced cyclobutane pyrimidine dimers into skin by means of specially engineered liposomes. Treatment of DNA-repair-deficient xeroderma pigmentosum patients or skin cancer patients with T4N5 liposome lotion containing such DNA repair liposomes increases the removal of DNA damage in the first few hours after treatment. In these studies, a DNA repair effect was observed in some patients treated with heat-inactivated enzyme. Unexpectedly, it was discovered that the heat-inactivated T4 endonuclease V enzyme refolds and recovers enzymatic activity. These studies demonstrate that measurements of molecular changes induced by biological drugs are useful adjuvants to clinical studies.

  20. Plasma DNA integrity index as a potential molecular diagnostic marker for breast cancer.

    Science.gov (United States)

    Kamel, Azza M; Teama, Salwa; Fawzy, Amal; El Deftar, Mervat

    2016-06-01

    Plasma DNA integrity index is increased in various malignancies including breast cancer, the most common cancer in women worldwide; early detection is crucial for successful treatment. Current screening methods fail to detect many cases of breast cancer at an early stage. In this study, we evaluated the level of plasma DNA integrity index in 260 females (95 with breast cancer, 95 with benign breast lesions, and 70 healthy controls) to verify its potential value in discriminating malignant from benign breast lesions. The criteria of the American Joint Committee on Cancer were used for staging of breast cancer patients. DNA integrity index was measured by real-time PCR. DNA integrity index was significantly higher in breast cancer than in benign breast patients and healthy subjects (P = cancer group was 85.3 % at 0.55 DNA integrity index cutoff. In conclusion, the plasma DNA integrity index may be a promising molecular diagnostic marker of malignancy in breast lesions.

  1. DNA Conformational Variations Induced by Stretching 3'5'-Termini Studied by Molecular Dynamics Simulations

    Institute of Scientific and Technical Information of China (English)

    QI Wen-Peng; LEI Xiao-Ling

    2011-01-01

    @@ Investigating the interaction between protein and stretched DNA molecules has become a new way to study the protein DNA interaction.The conformations from different stretching methods give us a further understanding of the interaction between protein and DNA.We study the conformational variations of a 22-mer DNA caused by stretching both 3'-and 5'-termini by molecular dynamics simulations.It requires 250kJ/mol to stretch the DNA molecule by 3'5'-termini for 3.5 run and the force plateau is at 123.8 pN.The stretching 3'5'-termini leads to large values of the angle opening and the dihedral propeller between bases in one base pair, the double helix untwists from 34°to 20°and the successive base pairs rolls to the side of the DNA major groove.The distances between successive base pairs increases from 3.2.(A) to 5.6(A).%Investigating the interaction between protein and stretched DNA molecules has become a new way to study the protein DNA interaction. The conformations from different stretching methods give us a further understanding of the interaction between protein and DNA. We study the conformational variations of a 22-met DNA caused by stretching both 3'- and 5'-termini by molecular dynamics simulations. It requires 250k J/mol to stretch the DNA molecule by 3'5'-termini for 3.5nm and the force plateau is at 123.8pN. The stretching 3'5'-termini leads to large values of the angle opening and the dihedral propeller between bases in one base pair, the double helix untwists from 34° to 20° and the successive base pairs rolls to the side of the DNA major groove. The distances between successive base pairs increases from 3.2 (A) to 5.6 (A).

  2. Characterization of the binding of paylean and DNA by fluorescence, UV spectroscopy and molecular docking techniques.

    Science.gov (United States)

    Zhou, Huifeng; Bi, Shuyun; Wang, Yu; Zhao, Tingting

    2016-06-01

    The interaction of paylean (PL) with calf thymus DNA (ctDNA) was investigated using fluorescence spectroscopy, UV absorption, melting studies, ionic strength, viscosity experiments and molecular docking under simulated physiological conditions. Values for the binding constant Ka between PL and DNA were 5.11 × 10(3) , 2.74 × 10(3) and 1.74 × 10(3)  L mol(-1) at 19, 29 and 39°C respectively. DNA quenched the intrinsic fluorescence of PL via a static quenching procedure as shown from Stern-Volmer plots. The relative viscosity and the melting temperature of DNA were basically unchanged in the presence of PL. The fluorescence intensity of PL-DNA decreased with increasing ionic strength. The value of Ka for PL with double-stranded DNA (dsDNA) was larger than that for PL with single-stranded DNA (ssDNA). All the results revealed that the binding mode was groove binding, and molecular docking further indicated that PL was preferentially bonded to A-T-rich regions of DNA. The values for ΔH, ΔS and ΔG suggested that van der Waals forces or hydrogen bonding might be the main acting forces between PL and DNA. The binding distance was determined to be 3.37 nm based on the theory of Förster energy transference, which indicated that a non-radiation energy transfer process occurred. Copyright © 2015 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.

  3. Understanding DNA Under Oxidative Stress and Sensitization: The Role of Molecular Modeling

    Directory of Open Access Journals (Sweden)

    Antonio eMonari

    2015-07-01

    Full Text Available DNA is constantly exposed to damaging threats coming from oxidative stress, i.e. from the presence of free radicals and reactive oxygen species. Sensitization from exogenous and endogenous compounds that strongly enhance the frequency of light-induced lesions also plays an important role. The experimental determination of DNA lesions, though a difficult subject, is somehow well established and allows to elucidate even extremely rare DNA lesions. In parallel, molecular modeling has become fundamental to clearly understand the fine mechanisms related to DNA defects induction. Indeed, it offers an unprecedented possibility to get access to an atomistic or even electronic resolution. Ab initio molecular dynamics may also describe the time-evolution of the molecular system and its reactivity. Yet the modeling of DNA (photo-reactions does necessitate elaborate multi-scale methodologies to tackle a damage induction reactivity that takes place in a complex environment. The double-stranded DNA environment is first characterized by a very high flexibility, that dynamical effects are to be taken into account, but also a strongly inhomogeneous electrostatic embedding. Additionally, one aims at capturing more subtle effects, such as the sequence selectivity which is of critical important for DNA damage. The structure and dynamics of the DNA/sensitizers complexes, as well as the photo-induced electron- and energy-transfer phenomena taking place upon sensitization, should be carefully modeled. Finally the factors inducing different repair ratios for different lesions should also be rationalized.In this review we will critically analyze the different computational strategies used to model DNA lesions. A clear picture of the complex interplay between reactivity and structural factors will be sketched. The use of proper multi-scale modeling leads to the in-depth comprehension of DNA lesions mechanism and also to the rational design of new chemo-therapeutic agents.

  4. Are glutathione S transferases involved in DNA damage signalling? Interactions with DNA damage and repair revealed from molecular epidemiology studies

    Energy Technology Data Exchange (ETDEWEB)

    Dusinska, Maria, E-mail: Maria.DUSINSKA@nilu.no [CEE-Health Effects Group, NILU - Norwegian Institute for Air Research, Kjeller (Norway); Staruchova, Marta; Horska, Alexandra [Department of Experimental and Applied Genetics, Slovak Medical University, Bratislava (Slovakia); Smolkova, Bozena [Laboratory of Cancer Genetics, Cancer Research Institute of the Slovak Academy of Sciences, Bratislava (Slovakia); Collins, Andrew [Department of Nutrition, Faculty of Medicine, University of Oslo (Norway); Bonassi, Stefano [Unit of Clinical and Molecular Epidemiology, IRCCS San Raffaele Pisana, Rome (Italy); Volkovova, Katarina [Department of Experimental and Applied Genetics, Slovak Medical University, Bratislava (Slovakia)

    2012-08-01

    Glutathione S-transferases (GSTs) are members of a multigene family of isoenzymes that are important in the control of oxidative stress and in phase II metabolism. Acting non-enzymically, GSTs can modulate signalling pathways of cell proliferation, cell differentiation and apoptosis. Using a molecular epidemiology approach, we have investigated a potential involvement of GSTs in DNA damage processing, specifically the modulation of DNA repair in a group of 388 healthy adult volunteers; 239 with at least 5 years of occupational exposure to asbestos, stone wool or glass fibre, and 149 reference subjects. We measured DNA damage in lymphocytes using the comet assay (alkaline single cell gel electrophoresis): strand breaks (SBs) and alkali-labile sites, oxidised pyrimidines with endonuclease III, and oxidised purines with formamidopyrimidine DNA glycosylase. We also measured GST activity in erythrocytes, and the capacity for base excision repair (BER) in a lymphocyte extract. Polymorphisms in genes encoding three GST isoenzymes were determined, namely deletion of GSTM1 and GSTT1 and single nucleotide polymorphism Ile105Val in GSTP1. Consumption of vegetables and wine correlated negatively with DNA damage and modulated BER. GST activity correlated with oxidised bases and with BER capacity, and differed depending on polymorphisms in GSTP1, GSTT1 and GSTM1. A significantly lower BER rate was associated with the homozygous GSTT1 deletion in all asbestos site subjects and in the corresponding reference group. Multifactorial analysis revealed effects of sex and exposure in GSTP1 Ile/Val heterozygotes but not in Ile/Ile homozygotes. These variants affected also SBs levels, mainly by interactions of GSTP1 genotype with exposure, with sex, and with smoking habit; and by an interaction between sex and smoking. Our results show that GST polymorphisms and GST activity can apparently influence DNA stability and repair of oxidised bases, suggesting a potential new role for these

  5. Molecular identification of Paragonimus species by DNA pyrosequencing technology.

    Science.gov (United States)

    Tantrawatpan, Chairat; Intapan, Pewpan M; Janwan, Penchom; Sanpool, Oranuch; Lulitanond, Viraphong; Srichantaratsamee, Chutatip; Anamnart, Witthaya; Maleewong, Wanchai

    2013-06-01

    DNA pyrosequencing for PCR amplicons is an attractive strategy for the identification of microorganisms because of its short time performance for large number of samples. In this study, the primers targeting the fragment of ITS2 region of nuclear ribosomal RNA gene were newly developed for pyrosequencing-based identification of 6 Paragonimus species, Paragonimus bangkokensis, Paragonimus harinasutai, Paragonimus heterotremus, Paragonimus macrorchis, Paragonimus siamensis and Paragonimus westermani. Pyrosequencing determination of 39 nucleotides of partial ITS2 region could discriminate 6 Paragonimus species, and could also detect intra-species genetic variation of P. macrorchis. This DNA pyrosequencing-based identification can be a valuable tool to improve species-level identification of Paragonimus in the endemic areas.

  6. Alignment of Gold Nanoparticle-Decorated DNA Origami Nanotubes: Substrate Prepatterning versus Molecular Combing.

    Science.gov (United States)

    Teschome, Bezu; Facsko, Stefan; Gothelf, Kurt V; Keller, Adrian

    2015-11-24

    DNA origami has become an established technique for designing well-defined nanostructures with any desired shape and for the controlled arrangement of functional nanostructures with few nanometer resolution. These unique features make DNA origami nanostructures promising candidates for use as scaffolds in nanoelectronics and nanophotonics device fabrication. Consequently, a number of studies have shown the precise organization of metallic nanoparticles on various DNA origami shapes. In this work, we fabricated large arrays of aligned DNA origami decorated with a high density of gold nanoparticles (AuNPs). To this end, we first demonstrate the high-yield assembly of high-density AuNP arrangements on DNA origami adsorbed to Si surfaces with few unbound background nanoparticles by carefully controlling the concentrations of MgCl2 and AuNPs in the hybridization buffer and the hybridization time. Then, we evaluate two methods, i.e., hybridization to prealigned DNA origami and molecular combing in a receding meniscus, with respect to their potential to yield large arrays of aligned AuNP-decorated DNA origami nanotubes. Because of the comparatively low MgCl2 concentration required for the efficient immobilization of the AuNPs, the prealigned DNA origami become mobile and displaced from their original positions, thereby decreasing the alignment yield. This increased mobility, on the other hand, makes the adsorbed origami susceptible to molecular combing, and a total alignment yield of 86% is obtained in this way.

  7. Spectroscopic and molecular docking studies on the interaction of the drug olanzapine with calf thymus DNA

    Science.gov (United States)

    Shahabadi, Nahid; Bagheri, Somayeh

    2015-02-01

    The present study investigated the binding interaction between olanzapine and calf thymus DNA (ct-DNA) using emission, absorption, circular dichroism, viscosity measurements and molecular modeling. Thermodynamic parameters (ΔH < 0 and ΔS < 0) indicated that hydrogen bond and van der Waals play main roles in the binding of the drug to ct-DNA. Spectrophotometric studies of the interaction of olanzapine with DNA have shown that it could bind to ct-DNA (Kb = 2 × 103 M-1). The binding constant is comparable to standard groove binding drugs. Competitive fluorimetric studies with Hoechst 33258 have shown that olanzapine exhibits the ability to displace the DNA-bound Hoechst 33258 indicating that binds strongly in minor groove of DNA helix. Furthermore, the drug induces detectable changes in the CD spectrum of ct-DNA as well as changes in its viscosity. All of the experimental results prove that the groove binding must be predominant. The results obtained from experimental data were in good agreement with molecular modeling studies.

  8. Molecular dynamics study of DNA binding by INT-DBD under a polarized force field.

    Science.gov (United States)

    Yao, Xue X; Ji, Chang G; Xie, Dai Q; Zhang, John Z H

    2013-05-15

    The DNA binding domain of transposon Tn916 integrase (INT-DBD) binds to DNA target site by positioning the face of a three-stranded antiparallel β-sheet within the major groove. As the negatively charged DNA directly interacts with the positively charged residues (such as Arg and Lys) of INT-DBD, the electrostatic interaction is expected to play an important role in the dynamical stability of the protein-DNA binding complex. In the current work, the combined use of quantum-based polarized protein-specific charge (PPC) for protein and polarized nucleic acid-specific charge (PNC) for DNA were employed in molecular dynamics simulation to study the interaction dynamics between INT-DBD and DNA. Our study shows that the protein-DNA structure is stabilized by polarization and the calculated protein-DNA binding free energy is in good agreement with the experimental data. Furthermore, our study revealed a positive correlation between the measured binding energy difference in alanine mutation and the occupancy of the corresponding residue's hydrogen bond. This correlation relation directly relates the contribution of a specific residue to protein-DNA binding energy to the strength of the hydrogen bond formed between the specific residue and DNA.

  9. Is there a niche for DNA microarrays in molecular diagnostics?

    Science.gov (United States)

    Jordan, Bertrand R

    2010-10-01

    DNA microarrays, 15 years after their appearance, have achieved presence in a number of medical settings. Several tests have been introduced and have obtained regulatory approval, mostly in the fields of bacterial identification, mutation detection and the global assessment of genome alterations, a particularly successful case being the whole-genome assay of copy-number variations. Gene-expression applications have been less successful because of technical issues (e.g., reproducibility, platform-to-platform consistency and statistical issues in data analysis) and difficulties in demonstrating the clinical utility of expression signatures. In their different applications, DNA arrays have faced competition from PCR-based assays for low and intermediate multiplicity. Now they have a new competitor, new-generation sequencing, that can provide a wealth of direct sequence information, or digital gene-expression data, at a constantly decreasing cost. In this article we evaluate the strengths and weaknesses of the DNA microarray approach to diagnostics, and highlight the fields in which it is most likely to achieve a durable presence.

  10. Ultrahigh molecular recognition specificity of competing DNA oligonucleotide strands in thermal equilibrium

    CERN Document Server

    Schenkelberger, Marc; Mai, Timo; Ott, Albrecht

    2016-01-01

    The specificity of molecular recognition is important to molecular self-organization. A prominent example is the biological cell where, within a highly crowded molecular environment, a myriad of different molecular receptor pairs recognize their binding partner with astonishing accuracy. In thermal equilibrium it is usually admitted that the affinity of recognizer pairs only depends on the nature of the two binding molecules. Accordingly, Boltzmann factors of binding energy differences relate the molecular affinities among different target molecules that compete for the same probe. Here, we consider the molecular recognition of short DNA oligonucleotide single strands. We show that a better matching oligonucleotide strand can prevail against a disproportionally more concentrated competitor that exhibits reduced affinity due to a mismatch. The magnitude of deviation from the simple picture above may reach several orders of magnitude. In our experiments the effective molecular affinity of a given strand remains...

  11. Solvent reorganization energies in A-DNA, B-DNA, and rhodamine 6G-DNA complexes from molecular dynamics simulations with a polarizable force field.

    Science.gov (United States)

    Vladimirov, Egor; Ivanova, Anela; Rösch, Notker

    2009-04-02

    We estimate solvent reorganization energies lambda(s) of electron transfer (ET) in DNA stacks between positively charged guanine (acceptor) and neutral guanine (donor), as well as in rhodamine 6G (R6G)-DNA complexes between R6G (acceptor) and neutral guanine (donor) from molecular dynamics simulations that used a polarizable force field in combination with a polarizable water model. We compare results from the polarizable scheme with those from a common nonpolarizable analogue. We also discuss the influence of charge sets, separate contributions of solute and solvent electronic polarizations, and partial contributions of different molecular groups to changes of lambda(s) due to electronic polarization. Independent of donor-acceptor distances, solvent reorganization energies of ET processes in DNA duplexes from a polarizable force field are about 30% smaller than the corresponding results from a nonpolarizable force field. The effective optical dielectric constant epsilon(infinity) = 1.5, extracted from pertinent scaling factors, is also independent of the donor-acceptor separation over a wide range of distances, from 3.4 to 50.0 A. Reorganization energies calculated with the polarizable force field agree satisfactorily with experimental data for DNA duplexes. Comparison of results for A-DNA and B-DNA forms as well as for the conformational alignment of the dye relative to the duplex in R6G-DNA complexes demonstrates that the conformation of a duplex hardly affects lambda(s). Among these DNA-related systems, the effective parameter epsilon(infinity) is remarkably constant over a broad range of donor-acceptor distances.

  12. Circomics of Cuban geminiviruses reveals the first alpha-satellite DNA in the Caribbean.

    Science.gov (United States)

    Jeske, Holger; Kober, Sigrid; Schäfer, Benjamin; Strohmeier, Stephan

    2014-10-01

    Circomics (circular DNA genomics), the combination of rolling circle amplification (RCA), restriction fragment length polymorphism (RFLP) analysis and pyro-sequencing, has been used recently to identify geminiviruses with high efficiency and low costs. Circular DNAs associated with Cuban geminiviruses were characterised by RCA/RFLP analysis and 454 sequencing of two batches of DNA amplified from selected plant samples as well as individual cloning and Sanger sequencing of DNA components and compared to other geminiviral DNAs by phylogenetic analysis. Cuban geminiviruses that were closely related to each other challenged the circomics approach. Ten geminiviral components and one alpha-satellite DNA were determined and compared to three geminiviral components obtained by conventional cloning. New strains of Sida yellow mottle virus (SiYMoV), tomato yellow distortion leaf virus (ToYDLV), Sida golden mosaic Florida virus (SiGMFV) and Sida golden mosaic Liguanea virus (SiGMLV) are described with host plant species being classified by molecular PCR-based bar coding. A new virus species is named Peristrophe mosaic virus. The first alpha-satellite found in Middle America establishes the New World branch of these elements which are related to nanoviruses and were previously thought to be restricted to the Old World. In conclusion, circomics is efficient for complex infections and closely related viruses to detected unexpected viral DNAs, but may need some scrutinisation by direct sequencing and cloning of individual components for certain cases.

  13. Constructing Bio-molecular Databases on a DNA-based Computer

    CERN Document Server

    Chang, Weng-Long; Ho,; Guo, Minyi

    2007-01-01

    Codd [Codd 1970] wrote the first paper in which the model of a relational database was proposed. Adleman [Adleman 1994] wrote the first paper in which DNA strands in a test tube were used to solve an instance of the Hamiltonian path problem. From [Adleman 1994], it is obviously indicated that for storing information in molecules of DNA allows for an information density of approximately 1 bit per cubic nm (nanometer) and a dramatic improvement over existing storage media such as video tape which store information at a density of approximately 1 bit per 1012 cubic nanometers. This paper demonstrates that biological operations can be applied to construct bio-molecular databases where data records in relational tables are encoded as DNA strands. In order to achieve the goal, DNA algorithms are proposed to perform eight operations of relational algebra (calculus) on bio-molecular relational databases, which include Cartesian product, union, set difference, selection, projection, intersection, join and division. Fu...

  14. [Molecular combing method in the research of DNA replication parameters in isolated organs of Drosophyla melanogaster].

    Science.gov (United States)

    Ivankin, A V; Kolesnikova, T D; Demakov, S A; Andreenkov, O V; Bil'danova, E R; Andreenkova, N G; Zhimulev, I F

    2011-01-01

    Methods of physical DNA mapping and direct visualization of replication and transcription in specific regions of genome play crucial role in the researches of structural and functional organization of eukaryotic genomes. Since DNA strands in the cells are organized into high-fold structure and present as highly compacted chromosomes, the majority of these methods have lower resolution at chromosomal level. One of the approaches to enhance the resolution and mapping accuracy is the method of molecular combing. The method is based on the process of stretching and alignment of DNA molecules that are covalently attached with one of the ends to the cover glass surface. In this article we describe the major methodological steps of molecular combing and their adaptation for researches of DNA replication parameters in polyploidy and diploid tissues of Drosophyla larvae.

  15. A Parallel Biological Optimization Algorithm to Solve the Unbalanced Assignment Problem Based on DNA Molecular Computing

    Directory of Open Access Journals (Sweden)

    Zhaocai Wang

    2015-10-01

    Full Text Available The unbalanced assignment problem (UAP is to optimally resolve the problem of assigning n jobs to m individuals (m < n, such that minimum cost or maximum profit obtained. It is a vitally important Non-deterministic Polynomial (NP complete problem in operation management and applied mathematics, having numerous real life applications. In this paper, we present a new parallel DNA algorithm for solving the unbalanced assignment problem using DNA molecular operations. We reasonably design flexible-length DNA strands representing different jobs and individuals, take appropriate steps, and get the solutions of the UAP in the proper length range and O(mn time. We extend the application of DNA molecular operations and simultaneity to simplify the complexity of the computation.

  16. Molecular characterization and phylogeny of whipworm nematodes inferred from DNA sequences of cox1 mtDNA and 18S rDNA.

    Science.gov (United States)

    Callejón, Rocío; Nadler, Steven; De Rojas, Manuel; Zurita, Antonio; Petrášová, Jana; Cutillas, Cristina

    2013-11-01

    A molecular phylogenetic hypothesis is presented for the genus Trichuris based on sequence data from the mitochondrial cytochrome c oxidase 1 (cox1) and ribosomal 18S genes. The taxa consisted of different described species and several host-associated isolates (undescribed taxa) of Trichuris collected from hosts from Spain. Sequence data from mitochondrial cox1 (partial gene) and nuclear 18S near-complete gene were analyzed by maximum likelihood and Bayesian inference methods, as separate and combined datasets, to evaluate phylogenetic relationships among taxa. Phylogenetic results based on 18S ribosomal DNA (rDNA) were robust for relationships among species; cox1 sequences delimited species and revealed phylogeographic variation, but most relationships among Trichuris species were poorly resolved by mitochondrial sequences. The phylogenetic hypotheses for both genes strongly supported monophyly of Trichuris, and distinct genetic lineages corresponding to described species or nematodes associated with certain hosts were recognized based on cox1 sequences. Phylogenetic reconstructions based on concatenated sequences of the two loci, cox1 (mitochondrial DNA (mtDNA)) and 18S rDNA, were congruent with the overall topology inferred from 18S and previously published results based on internal transcribed spacer sequences. Our results demonstrate that the 18S rDNA and cox1 mtDNA genes provide resolution at different levels, but together resolve relationships among geographic populations and species in the genus Trichuris.

  17. DNA aptamers as molecular probes for colorectal cancer study.

    Directory of Open Access Journals (Sweden)

    Kwame Sefah

    Full Text Available BACKGROUND: Understanding the molecular features of specific tumors can increase our knowledge about the mechanism(s underlying disease development and progression. This is particularly significant for colorectal cancer, which is a heterogeneous complex of diseases developed in a sequential manner through a multistep carcinogenic process. As such, it is likely that tumors with similar characteristics might originate in the same manner and have a similar molecular behavior. Therefore, specific mapping of the molecular features can be potentially useful for both tumor classification and the development of appropriate therapeutic regimens. However, this can only be accomplished by developing high-affinity molecular probes with the ability to recognize specific markers associated with different tumors. Aptamers can most easily meet this challenge based on their target diversity, flexible manipulation and ease of development. METHODOLOGY AND RESULTS: Using a method known as cell-based Systematic Evolution of Ligands by Exponential enrichment (cell-SELEX and colorectal cancer cultured cell lines DLD-1 and HCT 116, we selected a panel of target-specific aptamers. Binding studies by flow cytometry and confocal microscopy showed that these aptamers have high affinity and selectivity. Our data further show that these aptamers neither recognize normal colon cells (cultured and fresh, nor do they recognize most other cancer cell lines tested. CONCLUSION/SIGNIFICANCE: The selected aptamers can identify specific biomarkers associated with colorectal cancers. We believe that these probes could be further developed for early disease detection, as well as prognostic markers, of colorectal cancers.

  18. 丙肝病毒核心抗原荧光定量型生物条形码检测体系的建立与评价%Establishment of fluorescent quantitative bio-bar codes assay (FQ-BCA) for ultrasensitive detection of hepatitis C virus core antigen and its assessment

    Institute of Scientific and Technical Information of China (English)

    张立营; 冯玉奎; 梁冰; 高博; 孙英姿; 苑同业; 孙宇

    2011-01-01

    Objective To establish the highly sensitive assay of hepatitis C virus core antigen (HCVcAg) and to assess its methodology. Methods HCVcAg - and DNA chain-labeled nanoparticle probe (NP) and HCVcAg-labeled magnetic microparticle probe (MMP) with monoclonal antibodies were prepared to form a MMP-HCVcAg-NP sandwich complex. DNA chain was then released by dehybridization. Hepatitis C virus was identified based on the presence of the released DNA chain detected by fluorescent quantitative PCR. Results The fluorescent quantitative bio-bar codes assay (FQ-BCA) of HCVcAg was established with a sensitivity of about 100fg/ml which was 10 times higher than that of conventional ELISA assay. Conclusion This study lays a foundation for establishing the highly sensitive HCV FQ-BCA assay of HCVcAg.%目的 建立丙肝病毒核心抗原(HCVcAg)的荧光定量型生物条形码检测体系,并对检测体系进行方法学评价.方法 制备标记有HCVcAg多抗及特异DNA链的金纳米颗粒探针(NP探针)和标记有HCVcAg单抗的磁性微球探针(MMP探针),形成MMP探针-HCVcAg-NP探针复合物,再利用去杂交将NP探针上标记的DNA链释放出来,通过荧光定量PCR方法鉴定这些释放的DNA链可确定丙肝病毒的存在.结果 建立了HCVcAg的荧光定量型生物条形码检测体系,检测灵敏度可达100fg/ml,是相应的HCVcAg ELISA检测方法的104倍.结论 本研究为发展高灵敏度丙肝病毒的荧光定量型生物条形码检测试剂盒奠定了基础.

  19. Molecular rectifier composed of DNA with high rectification ratio enabled by intercalation.

    Science.gov (United States)

    Guo, Cunlan; Wang, Kun; Zerah-Harush, Elinor; Hamill, Joseph; Wang, Bin; Dubi, Yonatan; Xu, Bingqian

    2016-05-01

    The predictability, diversity and programmability of DNA make it a leading candidate for the design of functional electronic devices that use single molecules, yet its electron transport properties have not been fully elucidated. This is primarily because of a poor understanding of how the structure of DNA determines its electron transport. Here, we demonstrate a DNA-based molecular rectifier constructed by site-specific intercalation of small molecules (coralyne) into a custom-designed 11-base-pair DNA duplex. Measured current-voltage curves of the DNA-coralyne molecular junction show unexpectedly large rectification with a rectification ratio of about 15 at 1.1 V, a counter-intuitive finding considering the seemingly symmetrical molecular structure of the junction. A non-equilibrium Green's function-based model-parameterized by density functional theory calculations-revealed that the coralyne-induced spatial asymmetry in the electron state distribution caused the observed rectification. This inherent asymmetry leads to changes in the coupling of the molecular HOMO-1 level to the electrodes when an external voltage is applied, resulting in an asymmetric change in transmission.

  20. Molecular phylogeny and evolution of Scomber (Teleostei: Scombridae) based on mitochondrial and nuclear DNA sequences

    Institute of Scientific and Technical Information of China (English)

    CHENG Jiao; GAO Tianxiang; MIAO Zhenqing; YANAGIMOTO Takashi

    2011-01-01

    A molecular phylogenetic analysis of the genus Scomber was conducted based on mitochondrial (COI, Cyt b and control region) and nuclear (5S rDNA) DNA sequence data in multigene perspective. A variety of phylogenetic analytic methods were used to clarify the current taxonomic classification and to assess phylogenetic relationships and the evolutionary history of this genus. The present study produced a well-resolved phylogeny that strongly supported the monophyly of Scomber. We confirmed that S. japonicus and S. colias were genetically distinct. Although morphologically and ecologically similar to S. colias, the molecular data showed that S. japonicus has a greater molecular affinity with S. australasicus, which conflicts with the traditional taxonomy. This phyiogenetic pattern was corroborated by the mtDNA data, but incompletely by the nuclear DNA data. Phylogenetic concordance between the mitochondrial and nuclear DNA regions for the basal nodes supports an Atlantic origin for Scomber. The present-day geographic ranges of the species were compared with the resultant molecular phylogeny derived from partition Bayesian analyses of the combined data sets to evaluate possible dispersal routes of the genus. The present-day geographic distribution of Scomber species might be best ascribed to multiple dispersal events. In addition, our results suggest that phylogenies derived from multiple genes and long sequences exhibited improved phylogenetic resolution, from which we conclude that the phylogenetic reconstruction is a reliable representation of the evolutionary history of Scomber.

  1. Feasibility study of molecular memory device based on DNA using methylation to store information

    Science.gov (United States)

    Jiang, Liming; Qiu, Wanzhi; Al-Dirini, Feras; Hossain, Faruque M.; Evans, Robin; Skafidas, Efstratios

    2016-07-01

    DNA, because of its robustness and dense information storage capability, has been proposed as a potential candidate for next-generation storage media. However, encoding information into the DNA sequence requires molecular synthesis technology, which to date is costly and prone to synthesis errors. Reading the DNA strand information is also complex. Ideally, DNA storage will provide methods for modifying stored information. Here, we conduct a feasibility study investigating the use of the DNA 5-methylcytosine (5mC) methylation state as a molecular memory to store information. We propose a new 1-bit memory device and study, based on the density functional theory and non-equilibrium Green's function method, the feasibility of electrically reading the information. Our results show that changes to methylation states lead to changes in the peak of negative differential resistance which can be used to interrogate memory state. Our work demonstrates a new memory concept based on methylation state which can be beneficial in the design of next generation DNA based molecular electronic memory devices.

  2. New Paramecium quadecaurelia strains (P. aurelia spp. complex, Ciliophora) identified by molecular markers (rDNA and mtDNA).

    Science.gov (United States)

    Przyboś, Ewa; Tarcz, Sebastian; Dusi, Eike

    2013-08-01

    Paramecium quadecaurelia is a rare species (previously known only from two locations) belonging to the P. aurelia species complex. In the present paper, fragments of an rDNA gene (ITS1-5.8S-ITS2-5' rDNA) and mtDNA genes (cytochrome oxidase subunit I and cytochrome b regions) were employed to assist in the identification and characterization of three new strains collected from Ecuador and Thailand. Molecular data were confirmed by mating reactions. In rDNA and mtDNA trees constructed for species of the P. aurelia complex, all P. quadecaurelia strains, including the three new strains discussed in this study and two known previously from Australia and Africa, form a monophyletic but differentiated clade. The present study shows that genetic differentiation among the strains of P. quadecaurelia is equal to or even greater than the distances between some other P. aurelia species, e.g., P. primaurelia and P. pentaurelia. Such great intra-specific differentiation may indicate a future splitting of the P. quadecaurelia species into reproductively isolated lines. Copyright © 2012 Elsevier GmbH. All rights reserved.

  3. The pPSU Plasmids for Generating DNA Molecular Weight Markers.

    Science.gov (United States)

    Henrici, Ryan C; Pecen, Turner J; Johnston, James L; Tan, Song

    2017-05-26

    Visualizing nucleic acids by gel electrophoresis is one of the most common techniques in molecular biology, and reference molecular weight markers or ladders are commonly used for size estimation. We have created the pPSU1 & pPSU2 pair of molecular weight marker plasmids which produce both 100 bp and 1 kb DNA ladders when digested with two common restriction enzymes. The 100 bp ladder fragments have been optimized to migrate appropriately on both agarose and native polyacrylamide, unlike many currently available DNA ladders. Sufficient plasmid DNA can be isolated from 100 ml E. coli cultures for the two plasmids to produce 100 bp or 1 kb ladders for 1000 gels. As such, the pPSU1 and pPSU2 plasmids provide reference fragments from 50 to 10000 bp at a fraction of the cost of commercial DNA ladders. The pPSU1 and pPSU2 plasmids are available without licensing restrictions to nonprofit academic users, affording freely available high-quality, low-cost molecular weight standards for molecular biology applications.

  4. Advances in Human Mitochondrial Diseases Molecular Genetic Analysis of Pathogenic mtDNA Mutations.

    Science.gov (United States)

    Davidson, E; King, M P

    1997-01-01

    The mitochondrial diseases are a heterogeneous group of disorders that have been defined by specific morphological alterations in muscle and by deficits of the mitochondrial respiratory chain. The morphological hallmarks of these diseases include ragged-red fibers (an extensive proliferation of mitochondria in muscle fibers) and abnormal paracrystalline inclusions and membrane structures in mitochondria. The identification of pathogenic mutations in mitochondrial DNA (mtDNA) has resulted in a genetic classification of mitochondrial diseases. Investigations are being conducted to understand the molecular basis for the biochemical and morphological alterations of mitochondria associated with mtDNA mutations. © 1997, Elsevier Science Inc. (Trends Cardiovasc Med 1997;7:16-24).

  5. Molecular Dynamics Simulation of Multivalent-Ion Mediated Attraction between DNA Molecules

    Science.gov (United States)

    Dai, Liang; Mu, Yuguang; Nordenskiöld, Lars; van der Maarel, Johan R. C.

    2008-03-01

    All atom molecular dynamics simulations with explicit water were done to study the interaction between two parallel double-stranded DNA molecules in the presence of the multivalent counterions putrescine (2+), spermidine (3+), spermine (4+) and cobalt hexamine (3+). The inter-DNA interaction potential is obtained with the umbrella sampling technique. The attractive force is rationalized in terms of the formation of ion bridges, i.e., multivalent ions which are simultaneously bound to the two opposing DNA molecules. The lifetime of the ion bridges is short on the order of a few nanoseconds.

  6. [From Miescher to molecular DNA technology; a chapter from the medical history of the past century].

    Science.gov (United States)

    Bosman, F T

    1999-10-02

    Although molecular biology is a young discipline, it originated in the second half of the 19th century with the simultaneous discoveries of the laws of heredity by Mendel and of nucleic acid by Miescher. It was not until about 1950 that the structure of DNA was determined and it was proved that DNA governs the hereditary properties. Subsequently, the developments followed in rapid succession with the unravelling of the hereditary code, the elucidation of the mechanism of the translation of DNA into proteins, the discovery of the structure of genes and the finding of the methods for genetic manipulation. These have proved essential for the evolution of modern biotechnology.

  7. Evaluating experimental molecular physics studies of radiation damage in DNA*

    Science.gov (United States)

    Śmiałek, Małgorzata A.

    2016-11-01

    The field of Atomic and Molecular Physics (AMP) is a mature field exploring the spectroscopy, excitation, ionisation of atoms and molecules in all three phases. Understanding of the spectroscopy and collisional dynamics of AMP has been fundamental to the development and application of quantum mechanics and is applied across a broad range of disparate disciplines including atmospheric sciences, astrochemistry, combustion and environmental science, and in central to core technologies such as semiconductor fabrications, nanotechnology and plasma processing. In recent years the molecular physics also started significantly contributing to the area of the radiation damage at molecular level and thus cancer therapy improvement through both experimental and theoretical advances, developing new damage measurement and analysis techniques. It is therefore worth to summarise and highlight the most prominent findings from the AMP community that contribute towards better understanding of the fundamental processes in biologically-relevant systems as well as to comment on the experimental challenges that were met for more complex investigation targets. Contribution to the Topical Issue "Low-Energy Interactions related to Atmospheric and Extreme Conditions", edited by S. Ptasinska, M. Smialek-Telega, A. Milosavljevic, B. Sivaraman.

  8. Molecular Understanding of Efficient DNA Repair Machinery of Photolyase

    Science.gov (United States)

    Tan, Chuang; Liu, Zheyun; Li, Jiang; Guo, Xunmin; Wang, Lijuan; Zhong, Dongping

    2012-06-01

    Photolyases repair the UV-induced pyrimidine dimers in damage DNA with high efficiency, through a cylic light-driven electron transfer radical mechanism. We report here our systematic studies of the repair dynamics in E. coli photolyase with mutation of five active-site residues. The significant loss of repair efficiency by the mutation indicates that those active-site residues play an important role in the DNA repair by photolyase. To understand how the active-site residues modulate the efficiency, we mapped out the entire evolution of each elementary step during the repair in those photolyase mutants with femtosecond resolution. We completely analyzed the electron transfer dynamics using the Sumi-Marcus model. The results suggest that photolyase controls the critical electron transfer and the ring-splitting of pyrimidine dimer through modulation of the redox potentials and reorganization energies, and stabilization of the anionic intermediates, maintaining the dedicated balance of all the reaction steps and achieving the maximum function activity.

  9. Editorial overview: Molecular and genetic bases of disease: the double life of DNA.

    Science.gov (United States)

    McMurray, Cynthia T; Vijg, Jan

    2014-06-01

    This issue of Current Opinions focuses on the dual role of DNA in life and death. In ancient Roman religion and myth, Janus is the god who looks both to the past and to the future. He guides the beginnings of life, its progression from one condition to another, and he foresees distant events. The analogy to DNA could not be stronger. Closely interacting with the environment, our basic genetics provides the origin of life, guides the quality of health with age, predicts disease, and ultimately foresees our end. A shared and deep interest with the origin of life has long prompted our desire to define aging, and, ultimately, to understand whether it can be reversed. In this special issue, the authors collectively review concepts of normative aging, DNA instability, DNA repair, the genetic contribution of age and diet to disease, and how the basic molecular transactions of DNA guide both the transitions to life as well as the transitions to death.

  10. DNA self-assembly-driven positioning of molecular components on nanopatterned surfaces

    Science.gov (United States)

    Szymonik, M.; Davies, A. G.; Wälti, C.

    2016-09-01

    We present a method for the specific, spatially targeted attachment of DNA molecules to lithographically patterned gold surfaces—demonstrated by bridging DNA strands across nanogap electrode structures. An alkanethiol self-assembled monolayer was employed as a molecular resist, which could be selectively removed via electrochemical desorption, allowing the binding of thiolated DNA anchoring oligonucleotides to each electrode. After introducing a bridging DNA molecule with single-stranded ends complementary to the electrode-tethered anchoring oligonucleotides, the positioning of the DNA molecule across the electrode gap, driven by self-assembly, occurred autonomously. This demonstrates control of molecule positioning with resolution limited only by the underlying patterned structure, does not require any alignment, is carried out entirely under biologically compatible conditions, and is scalable.

  11. Experimental genomics: The application of DNA microarrays in cellular and molecular biology studies

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    The genome sequence information in combination with DNA microarrays promises to revolutionize the way of cellular and molecular biological research by allowing complex mixtures of RNA and DNA to interrogated in a parallel and quant itative fashion. DNA microarrays can be used to measure levels of gene expressio n for tens of thousands of gene simultaneously and take advantage of all availab le sequence information for experimental design and data interpretation in pursu it of biological understanding. Recent progress in experimental genomics allows DNA microarrays not simply to provide a catalogue of all the genes and informati on about their function, but to understand how the components work together to comprise functioning cells and organisms. This brief review gives a survey of DNA microarrays technology and its applications in genome and gene function analysis, gene expression studies, biological signal and defense system, cell cyclereg ulation, mechanism of transcriptional regulation, proteomics, and the functional ity of food component.

  12. Binding interaction between sorafenib and calf thymus DNA: Spectroscopic methodology, viscosity measurement and molecular docking

    Science.gov (United States)

    Shi, Jie-Hua; Chen, Jun; Wang, Jing; Zhu, Ying-Yao

    2015-02-01

    The binding interaction of sorafenib with calf thymus DNA (ct-DNA) was studied using UV-vis absorption spectroscopy, fluorescence emission spectroscopy, circular dichroism (CD), viscosity measurement and molecular docking methods. The experimental results revealed that there was obvious binding interaction between sorafenib and ct-DNA. The binding constant (Kb) of sorafenib with ct-DNA was 5.6 × 103 M-1 at 298 K. The enthalpy and entropy changes (ΔH0 and ΔS0) in the binding process of sorafenib with ct-DNA were -27.66 KJ mol-1 and -21.02 J mol-1 K-1, respectively, indicating that the main binding interaction forces were van der Waals force and hydrogen bonding. The docking results suggested that sorafenib preferred to bind on the minor groove of A-T rich DNA and the binding site of sorafenib was 4 base pairs long. The conformation change of sorafenib in the sorafenib-DNA complex was obviously observed and the change was close relation with the structure of DNA, implying that the flexibility of sorafenib molecule played an important role in the formation of the stable sorafenib-ct-DNA complex.

  13. Clinical Utility of Circulating Tumor DNA for Molecular Assessment and Precision Medicine in Pancreatic Cancer.

    Science.gov (United States)

    Takai, Erina; Totoki, Yasushi; Nakamura, Hiromi; Kato, Mamoru; Shibata, Tatsuhiro; Yachida, Shinichi

    2016-01-01

    Pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal malignancies. The genomic landscape of the PDAC genome features four frequently mutated genes (KRAS, CDKN2A, TP53, and SMAD4) and dozens of candidate driver genes altered at low frequency, including potential clinical targets. Circulating cell-free DNA (cfDNA) is a promising resource to detect molecular characteristics of tumors, supporting the concept of "liquid biopsy".We determined the mutational status of KRAS in plasma cfDNA using multiplex droplet digital PCR in 259 patients with PDAC, retrospectively. Furthermore, we constructed a novel modified SureSelect-KAPA-Illumina platform and an original panel of 60 genes. We then performed targeted deep sequencing of cfDNA in 48 patients who had ≥1 % mutant allele frequencies of KRAS in plasma cfDNA.Droplet digital PCR detected KRAS mutations in plasma cfDNA in 63 of 107 (58.9 %) patients with inoperable tumors. Importantly, potentially targetable somatic mutations were identified in 14 of 48 patients (29.2 %) examined by cfDNA sequencing.Our two-step approach with plasma cfDNA, combining droplet digital PCR and targeted deep sequencing, is a feasible clinical approach. Assessment of mutations in plasma cfDNA may provide a new diagnostic tool, assisting decisions for optimal therapeutic strategies for PDAC patients.

  14. Molecular dynamics study on DNA nanotubes as drug delivery vehicle for anticancer drugs.

    Science.gov (United States)

    Liang, Lijun; Shen, Jia-Wei; Wang, Qi

    2017-05-01

    In recent years, self-assembled DNA nanotubes have emerged as a type of nano-biomaterials with great potential for biomedical applications. To develop universal nanocarriers for smart and targeted drug delivery from DNA nanotubes, the understanding of interaction mechanism between DNA nanotubes and drugs is essential. In this study, the interactions between anti-cancer drugs and DNA nanotubes were investigated via molecular dynamics simulation. Our simulation results demonstrated that the DNA nanotubes could serve as a good drug delivery material by absorption of anti-cancer drugs with π-π interactions. At high concentration of anti-cancer drugs, most of the drugs could be absorbed by DNA nanotubes. Therefore, it could greatly decrease the aggregation of anti-cancer drugs in aqueous solution. In addition, the stability of DNA nanotubes could be improved with the absorption of anti-cancer drugs. These findings greatly enhance the understanding of the interaction mechanism of DNA nanotubes and anti-cancer drugs. Our study suggests that DNA nanotubes are promising delivery vehicles by strong absorption of anti-cancer drugs. Copyright © 2017 Elsevier B.V. All rights reserved.

  15. Interaction study of ciprofloxacin with human telomeric DNA by spectroscopy and molecular docking

    Science.gov (United States)

    Li, Huihui; Bu, Xiaoyang; Lu, Jia; Xu, Chongzheng; Wang, Xianlong; Yang, Xiaodi

    2013-04-01

    The interaction of ciprofloxacin (CIP) with human telomeric DNA was studied in vitro using multi-spectroscopy and molecular modeling methods. The hypochromic effect with a red shift in ultraviolet (UV) absorption indicated the occurrence of the interaction between CIP and DNA. The fluorescence quenching of CIP was observed with the addition of DNA and was proved to be the static quenching. The binding constant was found to be 9.62 × 104 L mol-1. Electrospray ionization mass spectrometry (ESI-MS) result further confirmed the formation of 1:1 non-covalent complex between DNA and CIP. Combined with the UV melting results, circular dichroism (CD) results confirmed the existence of groove binding mode, as well as conformational changes of DNA. Molecular docking studies illustrated the visual display of the CIP binding to the GC region in the minor groove of DNA. Specific hydrogen bonds and van der Waals forces were demonstrated as main acting forces between CIP and guanine bases of DNA.

  16. 条码技术与RFID技术在军工物流中的应用前景%Prospects of Bar Code Technology and RFID Technology Application in Military Logistics

    Institute of Scientific and Technical Information of China (English)

    苗卫华; 吴隽

    2011-01-01

    RFID technology and bar code technology has matured in recent years in both military logistics and the application of technology has become the development of China's military logistics information important part.This article describes the basic principles of these two technologies,and the characteristics of both analysis and comparison.This paper presents a combination of two technologies used in military logistics feasibility,and that the combination of both the conditions and prospects.%RFID技术与条码技术近年来已经日趋成熟,在军工物流中应用这两种技术已经成为我国军工物流信息化发展的重要组成部分。作者介绍了这两种技术的基本原理,并对两者的特点进行分析、比较,提出了两种技术结合应用于军工物流的可行性,并指出两者联合应用的条件和前景。

  17. 21st International Conference on DNA Computing and Molecular Programming: 8.1 Biochemistry

    Science.gov (United States)

    2016-04-23

    5454. 4 E. Torelli, M. Marini, S. Palmano, L. Piantanida, C. Polano, A. Scarpellini, M. Lazzarino and G. Firrao, A DNA origami nanorobot controlled...Chemical Engineering, 2012 [2] Douglas et al.A logic-gated nanorobot for targeted transport of molecular payloads Science, 335 (2012), pp. 831–834 [3...logic-gated nanorobot for targeted transport of molecular payloads. Science, 335(6070):831–834, Feb 2012. [2] J.-S. Shin and N. A. Pierce. A synthetic

  18. Recruitment, assembly, and molecular architecture of the SpoIIIE DNA pump revealed by superresolution microscopy.

    Directory of Open Access Journals (Sweden)

    Jean-Bernard Fiche

    Full Text Available ATP-fuelled molecular motors are responsible for rapid and specific transfer of double-stranded DNA during several fundamental processes, such as cell division, sporulation, bacterial conjugation, and viral DNA transport. A dramatic example of intercompartmental DNA transfer occurs during sporulation in Bacillus subtilis, in which two-thirds of a chromosome is transported across a division septum by the SpoIIIE ATPase. Here, we use photo-activated localization microscopy, structured illumination microscopy, and fluorescence fluctuation microscopy to investigate the mechanism of recruitment and assembly of the SpoIIIE pump and the molecular architecture of the DNA translocation complex. We find that SpoIIIE assembles into ∼45 nm complexes that are recruited to nascent sites of septation, and are subsequently escorted by the constriction machinery to the center of sporulation and division septa. SpoIIIE complexes contain 47±20 SpoIIIE molecules, a majority of which are assembled into hexamers. Finally, we show that directional DNA translocation leads to the establishment of a compartment-specific, asymmetric complex that exports DNA. Our data are inconsistent with the notion that SpoIIIE forms paired DNA conducting channels across fused membranes. Rather, our results support a model in which DNA translocation occurs through an aqueous DNA-conducting pore that could be structurally maintained by the divisional machinery, with SpoIIIE acting as a checkpoint preventing membrane fusion until completion of chromosome segregation. Our findings and proposed mechanism, and our unique combination of innovating methodologies, are relevant to the understanding of bacterial cell division, and may illuminate the mechanisms of other complex machineries involved in DNA conjugation and protein transport across membranes.

  19. Recruitment, assembly, and molecular architecture of the SpoIIIE DNA pump revealed by superresolution microscopy.

    Science.gov (United States)

    Fiche, Jean-Bernard; Cattoni, Diego I; Diekmann, Nele; Langerak, Julio Mateos; Clerte, Caroline; Royer, Catherine A; Margeat, Emmanuel; Doan, Thierry; Nöllmann, Marcelo

    2013-01-01

    ATP-fuelled molecular motors are responsible for rapid and specific transfer of double-stranded DNA during several fundamental processes, such as cell division, sporulation, bacterial conjugation, and viral DNA transport. A dramatic example of intercompartmental DNA transfer occurs during sporulation in Bacillus subtilis, in which two-thirds of a chromosome is transported across a division septum by the SpoIIIE ATPase. Here, we use photo-activated localization microscopy, structured illumination microscopy, and fluorescence fluctuation microscopy to investigate the mechanism of recruitment and assembly of the SpoIIIE pump and the molecular architecture of the DNA translocation complex. We find that SpoIIIE assembles into ∼45 nm complexes that are recruited to nascent sites of septation, and are subsequently escorted by the constriction machinery to the center of sporulation and division septa. SpoIIIE complexes contain 47±20 SpoIIIE molecules, a majority of which are assembled into hexamers. Finally, we show that directional DNA translocation leads to the establishment of a compartment-specific, asymmetric complex that exports DNA. Our data are inconsistent with the notion that SpoIIIE forms paired DNA conducting channels across fused membranes. Rather, our results support a model in which DNA translocation occurs through an aqueous DNA-conducting pore that could be structurally maintained by the divisional machinery, with SpoIIIE acting as a checkpoint preventing membrane fusion until completion of chromosome segregation. Our findings and proposed mechanism, and our unique combination of innovating methodologies, are relevant to the understanding of bacterial cell division, and may illuminate the mechanisms of other complex machineries involved in DNA conjugation and protein transport across membranes.

  20. Evaluation of growth conditions and DNA extraction techniques used in the molecular analysis of dermatophytes.

    Science.gov (United States)

    Gnat, S; Nowakiewicz, A; Ziółkowska, G; Trościańczyk, A; Majer-Dziedzic, B; Zięba, P

    2017-05-01

    Recent molecular methods for diagnosis of superficial mycoses have determined the need for a rapid and easy method of extracting DNA. The aim of study was to determine growth conditions and techniques of DNA extraction for Microsporum canis, Trichophyton mentagrophytes and T. verrucosum. Samples were prepared of each of the DNA extraction methods (phenol-chloroform, CTAB and four different kits) for all of the incubation periods (4, 7 and 10 days) of the cultures on the solid and in the liquid medium. The highest DNA concentrations were obtained using the phenol-chloroform method. The concentration of DNA extracted with the CTAB method accounted for 62·21%, for kits it corresponded from 35·53 to 15·41%. The analysis of the DNA weight yield revealed the highest isolation efficiency of the phenol-chloroform method, 1 mg of mycelium yielded 223·8 μg DNA. Lower DNA yield (by 39·32%) was obtained with the CTAB method; in the case of kits by 68·46-85·32%. In most of the techniques, the DNA yield on the solid medium was higher. In summary, the highest DNA yield was noted in the 7-day cultures and extraction with the phenol-chloroform method. Importantly, the type of culture was not relevant for the diagnostic result. Most mycoses are caused by fungi that reside in nature. The severity of the infection depends on the pathogenic attributes, socioeconomic factors and local environmental conditions. Recent diagnosis increasingly relies on not only the clinical features. Molecular identifications have determined the need for a rapid and easy method of extracting DNA. Usually two factors have to be considered: maximize the DNA yield and ensure that the extracted DNA is susceptible to enzymatic reactions. These data suggest that phenol-chloroform methods and a 7-day culture period may be useful for validation and constitute the first step of molecular diagnosis of dermatophytes. © 2017 The Society for Applied Microbiology.

  1. Identification of promising DNA GyrB inhibitors for Tuberculosis using pharmacophore-based virtual screening, molecular docking and molecular dynamics studies.

    Science.gov (United States)

    Islam, Md Ataul; Pillay, Tahir S

    2017-01-21

    In this study, we searched for potential DNA GyrB inhibitors using pharmacophore-based virtual screening followed by molecular docking and molecular dynamics simulation approaches. For this purpose, a set of 248 DNA GyrB inhibitors was collected from the literature and a well-validated pharmacophore model was generated. The best pharmacophore model explained that two each of hydrogen bond acceptors and hydrophobicity regions were critical for inhibition of DNA GyrB. Good statistical results of the pharmacophore model indicated that the model was robust in nature. Virtual screening of molecular databases revealed three molecules as potential antimycobacterial agents. The final screened promising compounds were evaluated in molecular docking and molecular dynamics simulation studies. In the molecular dynamics studies, RMSD and RMSF values undoubtedly explained that the screened compounds formed stable complexes with DNA GyrB. Therefore, it can be concluded that the compounds identified may have potential for the treatment of TB.

  2. Molecular genetics of DNA viruses: recombinant virus technology.

    Science.gov (United States)

    Neuhierl, Bernhard; Delecluse, Henri-Jacques

    2005-01-01

    Recombinant viral genomes cloned onto BAC vectors can be subjected to extensive molecular genetic analysis in the context of E. coli. Thus, the recombinant virus technology exploits the power of prokaryotic genetics to introduce all kinds of mutations into the recombinant genome. All available techniques are based on homologous recombination between a targeting vector carrying the mutated version of the gene of interest and the recombinant virus. After modification, the mutant viral genome is stably introduced into eukaryotic cells permissive for viral lytic replication. In these cells, mutant viral genomes can be packaged into infectious particles to evaluate the effect of these mutations in the context of the complete genome.

  3. Validation of DNA probes for molecular cytogenetics by mapping onto immobilized circular DNA

    Energy Technology Data Exchange (ETDEWEB)

    Greulich-Bode, Karin M.; Wang, Mei; Rhein, Andreas P.; Weier, Jingly F.; Weier, Heinz-Ulli G.

    2008-12-04

    Fluorescence in situ hybridization (FISH) is a sensitive and rapid procedure to detect gene rearrangements in tumor cells using non-isotopically labeled DNA probes. Large insert recombinant DNA clones such as bacterial artificial chromosome (BAC) or P1/PAC clones have established themselves in recent years as preferred starting material for probe preparations due to their low rates of chimerism and ease of use. However, when developing probes for the quantitative analysis of rearrangements involving genomic intervals of less than 100kb, careful probe selection and characterization are of paramount importance. We describe a sensitive approach to quality control probe clones suspected of carrying deletions or for measuring clone overlap with near kilobase resolution. The method takes advantage of the fact that P1/PAC/BAC's can be isolated as circular DNA molecules, stretched out on glass slides and fine-mapped by multicolor hybridization with smaller probe molecules. Two examples demonstrate the application of this technique: mapping of a gene-specific {approx}6kb plasmid onto an unusually small, {approx}55kb circular P1 molecule and the determination of the extent of overlap between P1 molecules homologous to the human NF-{kappa}B2 locus. The relatively simple method presented here does not require specialized equipment and may thus find widespread applications in DNA probe preparation and characterization, the assembly of physical maps for model organisms or in studies on gene rearrangements.

  4. Validation of DNA probes for molecular cytogenetics by mapping onto immobilized circular DNA

    Energy Technology Data Exchange (ETDEWEB)

    Greulich-Bode, Karin; Wang, Mei; Rhein, Andreas; Weier, Jingly; Weier, Heinz-Ulli

    2008-12-16

    Fluorescence in situ hybridization (FISH) is a sensitive and rapid procedure to detect gene rearrangements in tumor cells using non-isotopically labeled DNA probes. Large insert recombinant DNA clones such as bacterial artificial chromosome (BAC) or P1/PAC clones have established themselves in recent years as preferred starting material for probe preparations due to their low rates of chimerism and ease of use. However, when developing probes for the quantitative analysis of rearrangements involving genomic intervals of less than 100kb, careful probe selection and characterization are of paramount importance. We describe a sensitive approach to quality control probe clones suspected of carrying deletions or for measuring clone overlap with near kilobase resolution. The method takes advantage of the fact that P1/PAC/BAC's can be isolated as circular DNA molecules, stretched out on glass slides and fine-mapped by multicolor hybridization with smaller probe molecules. Two examples demonstrate the application of this technique: mapping of a gene-specific {approx}6kb plasmid onto an unusually small, {approx}55kb circular P1 molecule and the determination of the extent of overlap between P1 molecules homologous to the human NF-?B2 locus. The relatively simple method presented here does not require specialized equipment and may thus find widespread applications in DNA probe preparation and characterization, the assembly of physical maps for model organisms or in studies on gene rearrangements.

  5. Validation of DNA probes for molecular cytogenetics by mapping onto immobilized circular DNA

    Directory of Open Access Journals (Sweden)

    Rhein Andreas P

    2008-12-01

    Full Text Available Abstract Background Fluorescence in situ hybridization (FISH is a sensitive and rapid procedure to detect gene rearrangements in tumor cells using non-isotopically labeled DNA probes. Large insert recombinant DNA clones such as bacterial artificial chromosome (BAC or P1/PAC clones have established themselves in recent years as preferred starting material for probe preparations due to their low rates of chimerism and ease of use. However, when developing probes for the quantitative analysis of rearrangements involving genomic intervals of less than 100 kb, careful probe selection and characterization are of paramount importance. Results We describe a sensitive approach to quality control probe clones suspected of carrying deletions or for measuring clone overlap with near kilobase resolution. The method takes advantage of the fact that P1/PAC/BAC's can be isolated as circular DNA molecules, stretched out on glass slides and fine-mapped by multicolor hybridization with smaller probe molecules. Two examples demonstrate the application of this technique: mapping of a gene-specific ~6 kb plasmid onto an unusually small, ~55 kb circular P1 molecule and the determination of the extent of overlap between P1 molecules homologous to the human NF-κB2 locus. Conclusion The relatively simple method presented here does not require specialized equipment and may thus find widespread applications in DNA probe preparation and characterization, the assembly of physical maps for model organisms or in studies on gene rearrangements.

  6. Molecular Basis for DNA Double-Strand Break Annealing and Primer Extension by an NHEJ DNA Polymerase

    Directory of Open Access Journals (Sweden)

    Nigel C. Brissett

    2013-11-01

    Full Text Available Nonhomologous end-joining (NHEJ is one of the major DNA double-strand break (DSB repair pathways. The mechanisms by which breaks are competently brought together and extended during NHEJ is poorly understood. As polymerases extend DNA in a 5′-3′ direction by nucleotide addition to a primer, it is unclear how NHEJ polymerases fill in break termini containing 3′ overhangs that lack a primer strand. Here, we describe, at the molecular level, how prokaryotic NHEJ polymerases configure a primer-template substrate by annealing the 3′ overhanging strands from opposing breaks, forming a gapped intermediate that can be extended in trans. We identify structural elements that facilitate docking of the 3′ ends in the active sites of adjacent polymerases and reveal how the termini act as primers for extension of the annealed break, thus explaining how such DSBs are extended in trans. This study clarifies how polymerases couple break-synapsis to catalysis, providing a molecular mechanism to explain how primer extension is achieved on DNA breaks.

  7. Molecular and cellular functions of the FANCJ DNA helicase defective in cancer and in Fanconi Anemia

    Directory of Open Access Journals (Sweden)

    Robert M. Brosh

    2014-10-01

    Full Text Available The FANCJ DNA helicase is mutated in hereditary breast and ovarian cancer as well as the progressive bone marrow failure disorder Fanconi anemia (FA. FANCJ is linked to cancer suppression and DNA double strand break (DSB repair through its direct interaction with the hereditary breast cancer associated gene product, BRCA1. FANCJ also operates in the FA pathway of interstrand cross-link (ICL repair and contributes to homologous recombination (HR. FANCJ collaborates with a number of DNA metabolizing proteins implicated in DNA damage detection and repair, and plays an important role in cell cycle checkpoint control. In addition to its role in the classical FA pathway, FANCJ is believed to have other functions that are centered on alleviating replication stress. FANCJ resolves G-quadruplex (G4 DNA structures that are known to affect cellular replication and transcription, and potentially plays a role in the preservation and functionality of chromosomal structures such as telomeres. Recent studies suggest that FANCJ helps to maintain chromatin structure and preserve epigenetic stability by facilitating smooth progression of the replication fork when it encounters DNA damage or an alternate DNA structure such as a G4. Ongoing studies suggest a prominent but still not well-understood role of FANCJ in transcriptional regulation, chromosomal structure and function, and DNA damage repair to maintain genomic stability. This review will synthesize our current understanding of the molecular and cellular functions of FANCJ that are critical for chromosomal integrity.

  8. Electrochemical molecular beacon biosensor for sequence-specific recognition of double-stranded DNA.

    Science.gov (United States)

    Miao, Xiangmin; Guo, Xiaoting; Xiao, Zhiyou; Ling, Liansheng

    2014-09-15

    Direct recognition of double-stranded DNA (dsDNA) was crucial to disease diagnosis and gene therapy, because DNA in its natural state is double stranded. Here, a novel sensor for the sequence-specific recognition of dsDNA was developed based on the structure change of ferrocene (Fc) redox probe modified molecular beacon (MB). For constructing such a sensor, gold nanoparticles (AuNPs) were initially electrochemical-deposited onto glass carbon electrode (GCE) surface to immobilize thiolated MB in their folded states with Au-S bond. Hybridization of MB with target dsDNA induced the formation of parallel triplex DNA and opened the stem-loop structure of it, which resulted in the redox probe (Fc) away from the electrode and triggered the decrease of current signals. Under optimal conditions, dsDNA detection could be realized in the range from 350 pM to 25 nM, with a detection limit of 275 pM. Moreover, the proposed method has good sequence-specificity for target dsDNA compared with single base pair mismatch and two base pairs mismatches.

  9. Effect of temperature on DNA double helix: An insight from molecular dynamics simulation

    Indian Academy of Sciences (India)

    Sangeeta Kundu; Sanchita Mukherjee; Dhananjay Bhattacharyya

    2012-07-01

    The three-dimensional structure of DNA contains various sequence-dependent structural information, which control many cellular processes in life, such as replication, transcription, DNA repair, etc. For the above functions, DNA double helices need to unwind or melt locally, which is different from terminal melting, as often seen in molecular dynamics (MD) simulations or even in many DNA crystal structures. We have carried out detailed MD simulations of DNA double helices of regular oligonucleotide fragments as well as in polymeric constructs with water and charge-neutralizing counter-ions at several different temperatures. We wanted to eliminate the end-effect or terminal melting propensity by employing MD simulation of DNA oligonucleotides in such a manner that gives rise to properties of polymeric DNA of infinite length. The polymeric construct is expected to allow us to see local melting at elevated temperatures. Comparative structural analysis of oligonucleotides and its corresponding virtual polymer at various temperatures ranging from 300 K to 400 K is discussed. The general behaviour, such as volume expansion coefficients of both the simulations show high similarity, indicating polymeric construct, does not give many artificial constraints. Local melting of a polymer, even at elevated temperature, may need a high nucleation energy that was not available in the short (7 ns) simulations. We expected to observe such nucleation followed by cooperative melting of the polymers in longer MD runs. Such simulations of different polymeric sequences would facilitate us to predict probable melting origins in a polymeric DNA.

  10. Visualizing DNA domains and sequences by microscopy: a fifty-year history of molecular cytogenetics.

    NARCIS (Netherlands)

    Jong, de J.H.S.G.M.

    2003-01-01

    This short review presents a historical perspective of chromosome research during the last 50 years. It shows how molecular knowledge and technology of DNA entered cytogenetics step by step making it now daily practice in almost every modem chromosome lab. A crucial milestone in these decades has be

  11. A DNAzyme-mediated logic gate for programming molecular capture and release on DNA origami.

    Science.gov (United States)

    Li, Feiran; Chen, Haorong; Pan, Jing; Cha, Tae-Gon; Medintz, Igor L; Choi, Jong Hyun

    2016-06-28

    Here we design a DNA origami-based site-specific molecular capture and release platform operated by a DNAzyme-mediated logic gate process. We show the programmability and versatility of this platform with small molecules, proteins, and nanoparticles, which may also be controlled by external light signals.

  12. Recombinant DNA technology for melanoma immunotherapy: anti-Id DNA vaccines targeting high molecular weight melanoma-associated antigen.

    Science.gov (United States)

    Barucca, A; Capitani, M; Cesca, M; Tomassoni, D; Kazmi, U; Concetti, F; Vincenzetti, L; Concetti, A; Venanzi, F M

    2014-11-01

    Anti-idiotypic MK2-23 monoclonal antibody (anti-Id MK2-23 mAb), which mimics the high molecular weight melanoma-associated antigen (HMW-MAA), has been used to implement active immunotherapy against melanoma. However, due to safety and standardization issues, this approach never entered extensive clinical trials. In the present study, we investigated the usage of DNA vaccines as an alternative to MK2-23 mAb immunization. MK2-23 DNA plasmids coding for single chain (scFv) MK2-23 antibody were constructed via the insertion of variable heavy (V H) and light (V L) chains of MK2-23 into the pVAC-1mcs plasmids. Two alternative MK2-23 plasmids format V H/V L, and V L/V H were assembled. We demonstrate that both polypeptides expressed by scFv plasmids in vitro retained the ability to mimic HMW-MAA antigen, and to elicit specific anti-HMW-MAA humoral and cellular immunoresponses in immunized mice. Notably, MK2-23 scFv DNA vaccines impaired the onset and growth of transplantable B16 melanoma cells not engineered to express HMW-MAA. This pilot study suggests that optimized MK2-23 scFv DNA vaccines could potentially provide a safer and cost-effective alternative to anti-Id antibody immunization, for melanoma immunotherapy.

  13. Size-based molecular diagnostics using plasma DNA for noninvasive prenatal testing.

    Science.gov (United States)

    Yu, Stephanie C Y; Chan, K C Allen; Zheng, Yama W L; Jiang, Peiyong; Liao, Gary J W; Sun, Hao; Akolekar, Ranjit; Leung, Tak Y; Go, Attie T J I; van Vugt, John M G; Minekawa, Ryoko; Oudejans, Cees B M; Nicolaides, Kypros H; Chiu, Rossa W K; Lo, Y M Dennis

    2014-06-10

    Noninvasive prenatal testing using fetal DNA in maternal plasma is an actively researched area. The current generation of tests using massively parallel sequencing is based on counting plasma DNA sequences originating from different genomic regions. In this study, we explored a different approach that is based on the use of DNA fragment size as a diagnostic parameter. This approach is dependent on the fact that circulating fetal DNA molecules are generally shorter than the corresponding maternal DNA molecules. First, we performed plasma DNA size analysis using paired-end massively parallel sequencing and microchip-based capillary electrophoresis. We demonstrated that the fetal DNA fraction in maternal plasma could be deduced from the overall size distribution of maternal plasma DNA. The fetal DNA fraction is a critical parameter affecting the accuracy of noninvasive prenatal testing using maternal plasma DNA. Second, we showed that fetal chromosomal aneuploidy could be detected by observing an aberrant proportion of short fragments from an aneuploid chromosome in the paired-end sequencing data. Using this approach, we detected fetal trisomy 21 and trisomy 18 with 100% sensitivity (T21: 36/36; T18: 27/27) and 100% specificity (non-T21: 88/88; non-T18: 97/97). For trisomy 13, the sensitivity and specificity were 95.2% (20/21) and 99% (102/103), respectively. For monosomy X, the sensitivity and specificity were both 100% (10/10 and 8/8). Thus, this study establishes the principle of size-based molecular diagnostics using plasma DNA. This approach has potential applications beyond noninvasive prenatal testing to areas such as oncology and transplantation monitoring.

  14. [Principles for molecular identification of traditional Chinese materia medica using DNA barcoding].

    Science.gov (United States)

    Chen, Shi-Lin; Yao, Hui; Han, Jian-Ping; Xin, Tian-Yi; Pang, Xiao-Hui; Shi, Lin-Chun; Luo, Kun; Song, Jing-Yuan; Hou, Dian-Yun; Shi, Shang-Mei; Qian, Zhong-Zhi

    2013-01-01

    Since the research of molecular identification of Chinese Materia Medica (CMM) using DNA barcode is rapidly developing and popularizing, the principle of this method is approved to be listed in the Supplement of the Pharmacopoeia of the People's Republic of China. Based on the study on comprehensive samples, the DNA barcoding systems have been established to identify CMM, i.e. ITS2 as a core barcode and psbA-trnH as a complementary locus for identification of planta medica, and COI as a core barcode and ITS2 as a complementary locus for identification of animal medica. This article introduced the principle of molecular identification of CMM using DNA barcoding and its drafting instructions. Furthermore, its application perspective was discussed.

  15. Molecular and immunological characterization of DNA ligase IV deficiency.

    Science.gov (United States)

    Jiang, Jinqiu; Tang, Wenjing; An, Yunfei; Tang, Maozhi; Wu, Junfeng; Qin, Tao; Zhao, Xiaodong

    2016-02-01

    DNA ligase IV (LIG4) deficiency is an extremely rare autosomal recessive primary immunodeficiency disease caused by the LIG4 mutation. To date, fewer than 30 cases of patients have been reported worldwide. No reversion mutations have been previously identified in LIG4. This study enrolled seven Chinese patients with LIG4 deficiency who presented with combined immunodeficiency, microcephaly, and growth retardation. One patient (P1) acquired non-Hodgkin lymphoma. Four patients had impaired T cell proliferation function and skewed T cell receptor diversity. Five novel mutations in LIG4 and a potential hotspot mutation (c.833G>T; p.R278L) in the Chinese population were identified. TA cloning analysis of T cells, NK cells, granulocytes, and oral mucosa cells in P6 revealed wild-type clones and clones that contained both maternally and paternally inherited mutations, indicating possible somatic reversion which need further investigation since no functional or protein assays were possible for all the patients died and no cell lines were available.

  16. DNA detective: a review of molecular approaches to wildlife forensics.

    Science.gov (United States)

    Alacs, E A; Georges, A; FitzSimmons, N N; Robertson, J

    2010-09-01

    Illegal trade of wildlife is growing internationally and is worth more than USD$20 billion per year. DNA technologies are well suited to detect and provide evidence for cases of illicit wildlife trade yet many of the methods have not been verified for forensic applications and the diverse range of methods employed can be confusing for forensic practitioners. In this review, we describe the various genetic techniques used to provide evidence for wildlife cases and thereby exhibit the diversity of forensic questions that can be addressed using currently available genetic technologies. We emphasise that the genetic technologies to provide evidence for wildlife cases are already available, but that the research underpinning their use in forensics is lacking. Finally we advocate and encourage greater collaboration of forensic scientists with conservation geneticists to develop research programs for phylogenetic, phylogeography and population genetics studies to jointly benefit conservation and management of traded species and to provide a scientific basis for the development of forensic methods for the regulation and policing of wildlife trade.

  17. The 5S rDNA in two Abracris grasshoppers (Ommatolampidinae: Acrididae): molecular and chromosomal organization.

    Science.gov (United States)

    Bueno, Danilo; Palacios-Gimenez, Octavio Manuel; Martí, Dardo Andrea; Mariguela, Tatiane Casagrande; Cabral-de-Mello, Diogo Cavalcanti

    2016-08-01

    The 5S ribosomal DNA (rDNA) sequences are subject of dynamic evolution at chromosomal and molecular levels, evolving through concerted and/or birth-and-death fashion. Among grasshoppers, the chromosomal location for this sequence was established for some species, but little molecular information was obtained to infer evolutionary patterns. Here, we integrated data from chromosomal and nucleotide sequence analysis for 5S rDNA in two Abracris species aiming to identify evolutionary dynamics. For both species, two arrays were identified, a larger sequence (named type-I) that consisted of the entire 5S rDNA gene plus NTS (non-transcribed spacer) and a smaller (named type-II) with truncated 5S rDNA gene plus short NTS that was considered a pseudogene. For type-I sequences, the gene corresponding region contained the internal control region and poly-T motif and the NTS presented partial transposable elements. Between the species, nucleotide differences for type-I were noticed, while type-II was identical, suggesting pseudogenization in a common ancestor. At chromosomal point to view, the type-II was placed in one bivalent, while type-I occurred in multiple copies in distinct chromosomes. In Abracris, the evolution of 5S rDNA was apparently influenced by the chromosomal distribution of clusters (single or multiple location), resulting in a mixed mechanism integrating concerted and birth-and-death evolution depending on the unit.

  18. A molecular dynamics simulation of DNA damage induction by ionizing radiation

    CERN Document Server

    Abolfath, Ramin M; Chen, Zhe J; Nath, Ravinder

    2013-01-01

    We present a multi-scale simulation of early stage of DNA damages by the indirect action of hydroxyl ($^\\bullet$OH) free radicals generated by electrons and protons. The computational method comprises of interfacing the Geant4-DNA Monte Carlo with the ReaxFF molecular dynamics software. A clustering method was employed to map the coordinates of $^\\bullet$OH-radicals extracted from the ionization track-structures onto nano-meter simulation voxels filled with DNA and water molecules. The molecular dynamics simulation provides the time evolution and chemical reactions in individual simulation voxels as well as the energy-landscape accounted for the DNA-$^\\bullet$OH chemical reaction that is essential for the first principle enumeration of hydrogen abstractions, chemical bond breaks, and DNA-lesions induced by collection of ions in clusters less than the critical dimension which is approximately 2-3 \\AA. We show that the formation of broken bonds leads to DNA base and backbone damages that collectively propagate ...

  19. Removing external DNA contamination from arthropod predators destined for molecular gut-content analysis.

    Science.gov (United States)

    Greenstone, Matthew H; Weber, Donald C; Coudron, Thomas A; Payton, Mark E; Hu, Jing S

    2012-05-01

    Ecological research requires large samples for statistical validity, typically hundreds or thousands of individuals, which are most efficiently gathered by mass-collecting techniques. For the study of interspecific interactions, molecular gut-content analysis enables detection of arthropod predation with minimal disruption of community interactions. Field experiments have demonstrated that standard mass-collection methods, such as sweep netting, vacuum sampling and foliage beating, sometimes lead to contamination of predators with nontarget DNA, thereby compromising resultant gut-content data. We deliberately contaminated immature Coleomegilla maculata and Podisus maculiventris that had been fed larvae of Leptinotarsa decemlineata by topically applying homogenate of the alternate prey Leptinotarsa juncta. We then attempted to remove contaminating DNA by washing in ethanol or bleach. A 40-min wash with end-over-end rotation in 80% EtOH did not reliably reduce external DNA contamination. Identical treatment with 2.5% commercial bleach removed most externally contaminating DNA without affecting the detectability of the target prey DNA in the gut. Use of this bleaching protocol, perhaps with minor modifications tailored to different predator-prey systems, should reliably eliminate external DNA contamination, thereby alleviating concerns about this possible source of cross-contamination for mass-collected arthropod predators destined for molecular gut-content analysis. Published 2012. This article is a US Government work and is in the public domain in the USA.

  20. Development of a traceable molecular hygiene control method (TMHCM) for human DNA content in foods.

    Science.gov (United States)

    Şakalar, Ergün; Ergün, Şeyma Özçirak; Pala, Çiğdem; Akar, Emine; Ataşoğlu, Cengiz

    2017-06-15

    The aim of this study was to develop a molecular technique to determine the level of human originated DNA contamination in unhygienic food products. In the study, four model foods were prepared under both hygienic (H) and non-hygienic (NH) conditions and the human originated microbial loads of these products were determined. DNA was extracted from the model foods and human buccal samples by GIDAGEN Multi-fast DNA isolation kit. A primer specific region of human mitochondrial D-Loop was designed. The level of human DNA contamination in the model foods was determined by real-time PCR. The sensitivity of the technique developed here was 0.00001ng DNA/PCR. In addition, the applicability of the traceable molecular hygiene control method (TMHCM) was tested in 60 food samples from the market. The results of this study demonstrate that DNA based TMHCM can be used to predict to what extent foods meet the human oriented hygienic conditions. Copyright © 2016. Published by Elsevier Ltd.

  1. 水杉木材DNA提取及条形码分子鉴定%DNA extraction from wood Metasequoia glyptostroboides and molecule barcode identification

    Institute of Scientific and Technical Information of China (English)

    杨星宇; 杨路路; 余志伟; 杨建明

    2011-01-01

    通过对水杉不同年限及不同部位的木材DNA提取及片段扩增实验,结果显示改良后的CTAB(十六烷基三甲基溴化铵)法、SDS (sodium dodecyl sulfate)法及高盐低pH法均可以用于水杉木材DNA的提取,经过纯化后的木材DNA可以进行片段扩增.在提取木材DNA过程中,边材比心材更适合,所提取的DNA数量和质量更有保障;试验显示水杉木材DNA分子大多为23 kbp.运用DNA条形码筛选分析、序列特征分析、遗传距离秩和检验、barcodinggap检验,进行木材DNA分子系列物种鉴定,结果表明序列ITS2、trnL-F比较适合其DNA条形码技术要求,可以作为水杉木材鉴定序列.%Through wood DNA fragments extraction and PCR experiment, in different fixed number of year and in different parts of Metasequoia glyptostroboides , the results showed the method of CTAB (16 alkyl three methyl brominated ammonium), SDS (sodium dodecyl sulfate) and high-salt-low pH after improvement could be used in the Metasequoia glyptostroboides wood's DNA extracted. After purification in wood DNA, the experiment proved it could be PCR. During the extracting, the sapwood was better than heartwood, sapwood DNA s quantity and quality was more reliable. The experiments showed that the Metasequoia glyptostroboides wood's DNA molecules was about 23 kbp. In the use of wood DNA molecular series, we carried the analysis of testing DNA sequence, bar code screening, characteristics of the genetic distance, rank and inspection, barcoding gap inspection, the results indicated that the sequence ITS2, trnL-F was suitable to its DNA bar code technology requirements. It could be used as identification Metasequoia glyptostroboides wood.

  2. Extraction of high molecular weight genomic DNA from soils and sediments.

    Science.gov (United States)

    Lee, Sangwon; Hallam, Steven J

    2009-11-10

    The soil microbiome is a vast and relatively unexplored reservoir of genomic diversity and metabolic innovation that is intimately associated with nutrient and energy flow within terrestrial ecosystems. Cultivation-independent environmental genomic, also known as metagenomic, approaches promise unprecedented access to this genetic information with respect to pathway reconstruction and functional screening for high value therapeutic and biomass conversion processes. However, the soil microbiome still remains a challenge largely due to the difficulty in obtaining high molecular weight of sufficient quality for large insert library production. Here we introduce a protocol for extracting high molecular weight, microbial community genomic DNA from soils and sediments. The quality of isolated genomic DNA is ideal for constructing large insert environmental genomic libraries for downstream sequencing and screening applications. The procedure starts with cell lysis. Cell walls and membranes of microbes are lysed by both mechanical (grinding) and chemical forces (beta-mercaptoethanol). Genomic DNA is then isolated using extraction buffer, chloroform-isoamyl alcohol and isopropyl alcohol. The buffers employed for the lysis and extraction steps include guanidine isothiocyanate and hexadecyltrimethylammonium bromide (CTAB) to preserve the integrity of the high molecular weight genomic DNA. Depending on your downstream application, the isolated genomic DNA can be further purified using cesium chloride (CsCl) gradient ultracentrifugation, which reduces impurities including humic acids. The first procedure, extraction, takes approximately 8 hours, excluding DNA quantification step. The CsCl gradient ultracentrifugation, is a two days process. During the entire procedure, genomic DNA should be treated gently to prevent shearing, avoid severe vortexing, and repetitive harsh pipetting.

  3. Molecular cloaking of H2A.Z on mortal DNA chromosomes during nonrandom segregation.

    Science.gov (United States)

    Huh, Yang Hoon; Sherley, James L

    2011-10-01

    Although nonrandom sister chromatid segregation is a singular property of distributed stem cells (DSCs) that are responsible for renewing and repairing mature vertebrate tissues, both its cellular function and its molecular mechanism remain unknown. This situation persists in part because of the lack of facile methods for detecting and quantifying nonrandom segregating cells and for identifying chromosomes with immortal DNA strands, the cellular molecules that signify nonrandom segregation. During nonrandom segregation, at each mitosis, asymmetrically self-renewing DSCs continuously cosegregate to themselves the set of chromosomes that contain immortal DNA strands, which are the oldest DNA strands. Here, we report the discovery of a molecular asymmetry between segregating sets of immortal chromosomes and opposed mortal chromosomes (i.e., containing the younger set of DNA template strands) that constitutes a new convenient biomarker for detection of cells undergoing nonrandom segregation and direct delineation of chromosomes that bear immortal DNA strands. In both cells engineered with DSC-specific properties and ex vivo-expanded mouse hair follicle stem cells, the histone H2A variant H2A.Z shows specific immunodetection on immortal DNA chromosomes. Cell fixation analyses indicate that H2A.Z is present on mortal chromosomes as well but is cloaked from immunodetection, and the cloaking entity is acid labile. The H2A.Z chromosomal asymmetry produced by molecular cloaking provides a first direct assay for nonrandom segregation and for chromosomes with immortal DNA strands. It also seems likely to manifest an important aspect of the underlying mechanism(s) responsible for nonrandom sister chromatid segregation in DSCs.

  4. Reactive Molecular Dynamics study on the first steps of DNA-damage by free hydroxyl radicals

    CERN Document Server

    Abolfath, Ramin M; Brabec, Thomas

    2011-01-01

    We employ a large scale molecular simulation based on bond-order ReaxFF to simulate the chemical reaction and study the damage to a large fragment of DNA-molecule in the solution by ionizing radiation. We illustrate that the randomly distributed clusters of diatomic OH-radicals that are primary products of megavoltage ionizing radiation in water-based systems are the main source of hydrogen-abstraction as well as formation of carbonyl- and hydroxyl-groups in the sugar-moiety that create holes in the sugar-rings. These holes grow up slowly between DNA-bases and DNA-backbone and the damage collectively propagate to DNA single and double strand break.

  5. Molecular beacon-based enzyme-free strategy for amplified DNA detection.

    Science.gov (United States)

    Huang, Jiahao; Wu, Jueqi; Li, Zhigang

    2016-05-15

    We report an enzyme-free, sensitive strategy for DNA detections through fluorescence amplification. The sensing method employs molecular beacons (MBs) and two single-stranded helper DNA probes. In the presence of a DNA target, it binds and opens an MB. This triggers the hybridizations between the MB and helper probes, and consequently releases the DNA target, which becomes available to react with another MB and enhances the fluorescence emission of the MBs. The detection limit of the proposed strategy is 0.58 pM, which is about 3 orders of magnitude better than the conventional MB-based method. This method is also fast and exhibits good selectivity. It is superior to previous MB-based amplification approaches employing enzymes or nanomaterials.

  6. Taenia hydatigena: isolation of mitochondrial DNA, molecular cloning, and physical mitochondrial genome mapping.

    Science.gov (United States)

    Yap, K W; Thompson, R C; Rood, J I; Pawlowski, I D

    1987-06-01

    Mitochondrial DNA was isolated from Taenia hydatigena, T. crassiceps, and Echinococcus granulosus using a cetyltrimethylammonium bromide precipitation technique. The technique is simple, rapid, reproducible, and does not require extensive high speed ultracentrifugation. The advantage of using mitochondrial DNA from taeniid cestodes for comparative restriction analysis was demonstrated. Mitochondrial DNA of T. hydatigena was isolated as covalently closed circular molecules. These were linearized by single digestion with BamHI and the molecular weight was estimated from the linear form of 17.6 kb. The mitochondrial DNA of T. hydatigena is therefore similar in size and structure to that of many other animal species. The entire mitochondrial genome was cloned into pBR322 in Escherichia coli and a restriction map of the recombinant molecule was constructed. The potential of using the cloned mitochondrial genome as a probe in speciation studies as well as for providing functional information on the role of the cestode mitochondrion is discussed.

  7. DNA Nanostructures as Smart Drug-Delivery Vehicles and Molecular Devices.

    Science.gov (United States)

    Linko, Veikko; Ora, Ari; Kostiainen, Mauri A

    2015-10-01

    DNA molecules can be assembled into custom predesigned shapes via hybridization of sequence-complementary domains. The folded structures have high spatial addressability and a tremendous potential to serve as platforms and active components in a plethora of bionanotechnological applications. DNA is a truly programmable material, and its nanoscale engineering thus opens up numerous attractive possibilities to develop novel methods for therapeutics. The tailored molecular devices could be used in targeting cells and triggering the cellular actions in the biological environment. In this review we focus on the DNA-based assemblies - primarily DNA origami nanostructures - that could perform complex tasks in cells and serve as smart drug-delivery vehicles in, for example, cancer therapy, prodrug medication, and enzyme replacement therapy.

  8. Microbes on building materials - Evaluation of DNA extraction protocols as common basis for molecular analysis

    Energy Technology Data Exchange (ETDEWEB)

    Ettenauer, Joerg D., E-mail: joerg.ettenauer@boku.ac.at [VIBT-BOKU, University of Natural Resources and Life Sciences, Department of Biotechnology, Muthgasse 11, A-1190 Vienna (Austria); Pinar, Guadalupe, E-mail: Guadalupe.Pinar@boku.ac.at [VIBT-BOKU, University of Natural Resources and Life Sciences, Department of Biotechnology, Muthgasse 11, A-1190 Vienna (Austria); Lopandic, Ksenija, E-mail: Ksenija.Lopandic@boku.ac.at [VIBT-BOKU, University of Natural Resources and Life Sciences, Department of Biotechnology, Muthgasse 11, A-1190 Vienna (Austria); Spangl, Bernhard, E-mail: Bernhard.Spangl@boku.ac.at [University of Natural Resources and Life Sciences, Department of Landscape, Spatial and Infrastructure Science, Institute of Applied Statistics and Computing (IASC), Gregor Mendel-Str. 33, A-1180 Vienna (Austria); Ellersdorfer, Guenther, E-mail: Guenther.Ellersdorfer@boku.ac.at [VIBT-BOKU, University of Natural Resources and Life Sciences, Department of Biotechnology, Muthgasse 11, A-1190 Vienna (Austria); Voitl, Christian, E-mail: Christian.Voitl@boku.ac.at [VIBT-BOKU, University of Natural Resources and Life Sciences, Department of Biotechnology, Muthgasse 11, A-1190 Vienna (Austria); Sterflinger, Katja, E-mail: Katja.Sterflinger@boku.ac.at [VIBT-BOKU, University of Natural Resources and Life Sciences, Department of Biotechnology, Muthgasse 11, A-1190 Vienna (Austria)

    2012-11-15

    The study of microbial life in building materials is an emerging topic concerning biodeterioration of materials as well as health risks in houses and at working places. Biodegradation and potential health implications associated with microbial growth in our residues claim for more precise methods for quantification and identification. To date, cultivation experiments are commonly used to gain insight into the microbial diversity. Nowadays, molecular techniques for the identification of microorganisms provide efficient methods that can be applied in this field. The efficiency of DNA extraction is decisive in order to perform a reliable and reproducible quantification of the microorganisms by qPCR or to characterize the structure of the microbial community. In this study we tested thirteen DNA extraction methods and evaluated their efficiency for identifying (1) the quantity of DNA, (2) the quality and purity of DNA and (3) the ability of the DNA to be amplified in a PCR reaction using three universal primer sets for the ITS region of fungi as well as one primer pair targeting the 16S rRNA of bacteria with three typical building materials - common plaster, red brick and gypsum cardboard. DNA concentration measurements showed strong variations among the tested methods and materials. Measurement of the DNA yield showed up to three orders of magnitude variation from the same samples, whereas A260/A280 ratios often prognosticated biases in the PCR amplifications. Visualization of the crude DNA extracts and the comparison of DGGE fingerprints showed additional drawbacks of some methods. The FastDNA Spin kit for soil showed to be the best DNA extraction method and could provide positive results for all tests with the three building materials. Therefore, we suggest this method as a gold standard for quantification of indoor fungi and bacteria in building materials. -- Highlights: Black-Right-Pointing-Pointer Up to thirteen extraction methods were evaluated with three

  9. High-molecular-weight DNA and the sedimentation coefficient: a new perspective based on DNA from T7 bacteriophage and two novel forms of T4 bacteriophage.

    Science.gov (United States)

    Clark, R W; Wever, G H; Wiberg, J S

    1980-01-01

    The DNA molecules from T7 bacteriophage and a recently obtained mutant form of T4D were studied. The DNA of this T4 mutant contains cytosine in place of all of the glucosylated hydroxymethylcytosines normally present in T4. Molecular weights were measured with an electron microscope technique, and sedimentation coefficients were determined in isokinetic sucrose gradients. T7 DNA was found to have an Mr of 26.5 x 10(6). The T4 mutant, which we have termed T4c, produces two distinct phage head and DNA size clases. DNA from the standard heads (T4c DNA) has an Mr of 114.9 x 10(6), and DNA from the petite heads (T4cp DNA) has an Mr of 82.9 x 10(6). This enabled the derivation of an equation of sedimentation coefficient at zero concentration corrected to water at 20 degrees C versus Mr for the molecular weight range of 25 x 10(6) to 115 x 10(6) that is based solely on cytosine-containing DNA standards, thereby avoiding possible anomalies introduced by the glucosylation and hydroxymethylation of cytosine. The theory of Gray et al. provided the best description of the sedimentation coefficient versus Mr relationship, based on the sedimentation coefficients and the molecular weights of the three DNA standards and other evidence.

  10. Dynamic tuning of DNA-nanoparticle superlattices by molecular intercalation of double helix.

    Science.gov (United States)

    Pal, Suchetan; Zhang, Yugang; Kumar, Sanat K; Gang, Oleg

    2015-04-01

    Nanoparticle (NP) assembly using DNA recognition has emerged as a powerful tool for the fabrication of 3D superlattices. In addition to the vast structural diversity, this approach provides an avenue for dynamic 3D NP assembly, which is promising for the modulation of interparticle distances and, hence, for example, for in situ tuning of optical properties. While several approaches have been explored for changing NP separations in the lattices using responsiveness of single-stranded DNA (ss-DNA), far less work has been done for the manipulation of most abundant double-stranded DNA (ds-DNA) motifs. Here, we present a novel strategy for modulation of interparticle distances in DNA linked 3D self-assembled NP lattices by molecular intercalator. We utilize ethidium bromide (EtBr) as a model intercalator to demonstrate selective and isotropic lattice expansion for three superlattice types (bcc, fcc, and AlB2) due to the intercalation of ds-DNA linking NPs. We further show the reversibility of the lattice parameter using n-butanol as a retrieving agent as well as an increased lattice thermal stability by 12-14 °C due to the inclusion of EtBr. The proposed intercalator-based strategy permits the creation of reconfigurable and thermally stable superlattices, which could lead to tunable and functionally responsive materials.

  11. Structural and Molecular Basis for Coordination in a Viral DNA Packaging Motor

    Directory of Open Access Journals (Sweden)

    Huzhang Mao

    2016-03-01

    Full Text Available Ring NTPases are a class of ubiquitous molecular motors involved in basic biological partitioning processes. dsDNA viruses encode ring ATPases that translocate their genomes to near-crystalline densities within pre-assembled viral capsids. Here, X-ray crystallography, cryoEM, and biochemical analyses of the dsDNA packaging motor in bacteriophage phi29 show how individual subunits are arranged in a pentameric ATPase ring and suggest how their activities are coordinated to translocate dsDNA. The resulting pseudo-atomic structure of the motor and accompanying functional analyses show how ATP is bound in the ATPase active site; identify two DNA contacts, including a potential DNA translocating loop; demonstrate that a trans-acting arginine finger is involved in coordinating hydrolysis around the ring; and suggest a functional coupling between the arginine finger and the DNA translocating loop. The ability to visualize the motor in action illuminates how the different motor components interact with each other and with their DNA substrate.

  12. Structural and Molecular Basis for Coordination in a Viral DNA Packaging Motor

    Science.gov (United States)

    Reyes-Aldrete, Emilio; Sherman, Michael B.; Woodson, Michael; Atz, Rockney; Grimes, Shelley; Jardine, Paul J.; Morais, Marc C.

    2016-01-01

    SUMMARY Ring NTPases are a class of ubiquitous molecular motors involved in basic biological partitioning processes. dsDNA viruses encode ring ATPases that translocate their genomes to near-crystalline densities within pre-assembled viral capsids. Here, X-ray crystallography, cryoEM, and biochemical analyses of the dsDNA packaging motor in bacteriophage phi29 show how individual subunits are arranged in a pentameric ATPase ring, and suggest how their activities are coordinated to translocate dsDNA. The resulting pseudo-atomic structure of the motor and accompanying functional analyses show how ATP is bound in the ATPase active site; identify two DNA contacts, including a potential DNA translocating loop; demonstrate that a trans-acting arginine finger is involved in coordinating hydrolysis around the ring; and suggest a functional coupling between the arginine finger and the DNA translocating loop. The ability to visualize the motor in action illuminates how the different motor components interact with each other and with their DNA substrate. PMID:26904950

  13. Influence of molecular weight of DNA on the determination of anti-DNA antibodies in systemic lupus erythematosus (SLE) sera by radioimmunoassay

    Energy Technology Data Exchange (ETDEWEB)

    Geisert, M.; Heicke, B.; Metzmann, E.; Zahn, R.K.

    1975-04-01

    Using a radioimmunoassay (RIA) based on the Farr technique with radioactively labeled /sup 3/H-DNA for quantitative measurements of anti-DNA antibodies in sera of patients with systemic lupus erythematosus (SLE), the influence of molecular weight of DNA (ranging from 0.1 x 10/sup 6/ to 22.0 x 10/sup 6/ daltons) on binding and precipitation in this system has been investigated. Comparing our results with mathematical models it follows that one antibody molecule is fixed on the average to a statistical DNA segment of 2 x 10/sup 6/ to 4 x 10/sup 6/ daltons. Furthermore binding capacity of the DNA was found to be independent of the molecular weight, as demonstrated in a double label experiment using /sup 14/C and /sup 3/H-labeled DNA of different size. However, the amount of radioactivity precipitated was found to depend on the molecular weight of the labeled DNA following a non-linear function. It was calculated that a minimal ratio of fixed antibody molecules per a certain size of DNA was necessary for precipitation. The mathematical treatment of the observed non-linear precipitation dependence will be discussed using various statistical models. The results indicate that the quantitative measurements of anti-DNA antibodies with the Farr technique e.g., for diagnosis and control of SLE in clinical immunology is highly dependent on the molecular weight of the labeled DNA used in the assay system and reliable results are only obtained with DNA of a sufficiently high molecular weight. (auth)

  14. Implications of storing urinary DNA from different populations for molecular analyses.

    Directory of Open Access Journals (Sweden)

    Angela Cannas

    Full Text Available BACKGROUND: Molecular diagnosis using urine is established for many sexually transmitted diseases and is increasingly used to diagnose tumours and other infectious diseases. Storage of urine prior to analysis, whether due to home collection or bio-banking, is increasingly advocated yet no best practice has emerged. Here, we examined the stability of DNA in stored urine in two populations over 28 days. METHODOLOGY: Urine from 40 (20 male healthy volunteers from two populations, Italy and Zambia, was stored at four different temperatures (RT, 4 degrees C, -20 degrees C & -80 degrees C with and without EDTA preservative solution. Urines were extracted at days 0, 1, 3, 7 and 28 after storage. Human DNA content was measured using multi-copy (ALU J and single copy (TLR2 targets by quantitative real-time PCR. Zambian and Italian samples contained comparable DNA quantity at time zero. Generally, two trends were observed during storage; no degradation, or rapid degradation from days 0 to 7 followed by little further degradation to 28 days. The biphasic degradation was always observed in Zambia regardless of storage conditions, but only twice in Italy. CONCLUSION: Site-specific differences in urine composition significantly affect the stability of DNA during storage. Assessing the quality of stored urine for molecular analysis, by using the type of strategy described here, is paramount before these samples are used for molecular prognostic monitoring, genetic analyses and disease diagnosis.

  15. 基于红外条码的盲人扑克游戏辅助仪的设计%Design and implementation of blind poker game auxiliary instrument based on infrared scanning bar code

    Institute of Scientific and Technical Information of China (English)

    景亚霓; 杨海平

    2014-01-01

    The paper designs a blind poker game auxiliary instrument based on bar code technology, embedded system and voice chip technology. The instrument has the characteristics of compact structure , simple and convenient operation and embed popular poker game program, thus users can conveniently select the type of game according to their interest. Its main feature is that it can broadcast information that others played poker and supplies situation of yourself poker in hands by earplug if need. The auxiliary instrument makes the blind to get poke information by "listen" instead of "touch", so that blind and low vision people can play cards as a normal person even if he does not know Braille. In other hand, some problem can be avoided, such as mistake of player distinguishing Braille.%设计了一款基于集条形码技术、嵌入式系统及语音芯片技术于一体、结构紧凑、操作简单方便的盲人扑克游戏辅助仪。该装置设计了常见的扑克游戏程序,使用者可根据兴趣方便地选择游戏种类。其主要特点是利用语音技术能够实时播报其他人的出牌信息并可根据需要耳机播报本人手中牌的信息,让盲人用“听”牌代替了“摸”牌,使盲人以及不懂盲文的低视力人群能像正常人一样打扑克、玩桥牌等,克服了现有低视力群体打牌游戏中常见的问题,如盲文识别错误等。

  16. Preparation of next-generation sequencing libraries using Nextera™ technology: simultaneous DNA fragmentation and adaptor tagging by in vitro transposition.

    Science.gov (United States)

    Caruccio, Nicholas

    2011-01-01

    DNA library preparation is a common entry point and bottleneck for next-generation sequencing. Current methods generally consist of distinct steps that often involve significant sample loss and hands-on time: DNA fragmentation, end-polishing, and adaptor-ligation. In vitro transposition with Nextera™ Transposomes simultaneously fragments and covalently tags the target DNA, thereby combining these three distinct steps into a single reaction. Platform-specific sequencing adaptors can be added, and the sample can be enriched and bar-coded using limited-cycle PCR to prepare di-tagged DNA fragment libraries. Nextera technology offers a streamlined, efficient, and high-throughput method for generating bar-coded libraries compatible with multiple next-generation sequencing platforms.

  17. A molecular dynamics simulation of DNA damage induction by ionizing radiation

    Science.gov (United States)

    Abolfath, Ramin M.; Carlson, David J.; Chen, Zhe J.; Nath, Ravinder

    2013-10-01

    We present a multi-scale simulation of the early stage of DNA damages by the indirect action of hydroxyl (•OH) free radicals generated by electrons and protons. The computational method comprises of interfacing the Geant4-DNA Monte Carlo with ReaxFF molecular dynamics software. A clustering method was employed to map the coordinates of •OH-radicals extracted from the ionization-track-structures onto nano-meter simulation voxels filled with DNA and water molecules. The molecular dynamics simulation provides the time-evolution and chemical reactions in individual simulation voxels as well as the energy-landscape accounted for the DNA-•OH chemical reaction that is essential for the first-principle enumeration of hydrogen abstractions, chemical bond breaks, and DNA-lesions induced by collection of ions in clusters less than the critical dimension which is approximately 2-3 Å. We show that the formation of broken bonds leads to DNA-base and backbone damages that collectively propagate to DNA single and double-strand breaks. For illustration of the methodology, we focused on particles with an initial energy of 1 MeV. Our studies reveal a qualitative difference in DNA damage induced by low energy electrons and protons. Electrons mainly generate small pockets of •OH-radicals, randomly dispersed in the cell volume. In contrast, protons generate larger clusters along a straight-line parallel to the direction of the particle. The ratio of the total DNA double-strand breaks induced by a single proton and electron track is determined to be ≈4 in the linear scaling limit. In summary, we have developed a multi-scale computational model based on first-principles to study the interaction of ionizing radiation with DNA molecules. The main advantage of our hybrid Monte Carlo approach using Geant4-DNA and ReaxFF is the multi-scale simulation of the cascade of both physical and chemical events which result in the formation of biological damage. The tool developed in this

  18. [The application of DNA molecular markers in conservation of the rare and endangered medicinal plants].

    Science.gov (United States)

    Yan, Zhi-Feng; Zhang, Ben-Gang; Zhang, Zhao; Xia, Tian-Rui

    2005-12-01

    In this paper, the advance in DNA molecular markers techniques in recent years was reviewed. The application of DNA markers in conservation of the rare and endangered medicinal plants was explicated, of which included identification of germ-plasm resource, determination of the habitats unite which should be protected in situ, sampling strategies of ex-situ conservation, evaluation of the conservation effects of the rare and endangered medicinal plants, as well as elucidation of their endangered mechanism etc. The information could help drawing up conservation strategies and conservation measures for references.

  19. Molecular verification of the integration of Tripsacum dactyloides DNA into wheat genome through wide hybridization

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    RAPD and RFLP analyses of double haploid lines which derived from hybridization between hexaploid wheat (Triticum aestivum L.2n=42) and eastern gamagrass (Tripsacum dactyloides L.2n=4x=72) are reported.Two of the 340 Operon primers have been screened,which stably amplified Tripsacum dactyloides (male parent) specific bands in the double haploid lines.These results confirm the fact that Tripsacum dactyloides DNA has been integrated into wheat genome by sexual hybridization at molecular level.This idea has been further testified by RFLP analysis.Application and potentials of transferring Tripsacum dactyloides DNA into wheat genome by sexual hybridization in wheat breeding are discussed.

  20. Exploring the Counterion Atmosphere around DNA: What Can Be Learned from Molecular Dynamics Simulations?

    OpenAIRE

    Rueda, Manuel; Cubero, Elena; Laughton, Charles A.; Orozco, Modesto

    2004-01-01

    The counterion distribution around a DNA dodecamer (5′-CGCGAATTCGCG-3′) is analyzed using both standard and novel techniques based on state of the art molecular dynamics simulations. Specifically, we have explored the population of Na+ in the minor groove of DNA duplex, and whether or not a string of Na+ can replace the spine of hydration in the narrow AATT minor groove. The results suggest that the insertion of Na+ in the minor groove is a very rare event, but that when once the ion finds sp...

  1. A reversible DNA-silver nanoclusters-based molecular fluorescence switch and its use for logic gate operation.

    Science.gov (United States)

    Huang, Zhenzhen; Ren, Jinsong; Qu, Xiaogang

    2012-03-01

    Molecule-like silver nanoclusters (AgNCs) with few to tens of atoms are highly sensitive to the sequence and structure of DNA stabilizers. In this paper, a novel pH-triggered reversible molecular fluorescence switch is developed by taking advantage of the DNA-dependent fluorescence pH response of AgNCs. The DNA-AgNCs fluorescence switch simultaneously addresses concerns of simple construction strategy, efficient design and organic-solvent-free operation. Moreover, the excellent photostability and biocompatibility of AgNCs provide great potential for application of the DNA-AgNCs fluorescence switch in the development of functional molecular devices. Specifically, we apply the DNA-AgNCs fluorescence switch combined with the DNA sequence-dependent pH response pattern of AgNCs for construction of molecular logic gates.

  2. HLA DNA sequence variation among human populations: molecular signatures of demographic and selective events.

    Directory of Open Access Journals (Sweden)

    Stéphane Buhler

    Full Text Available Molecular differences between HLA alleles vary up to 57 nucleotides within the peptide binding coding region of human Major Histocompatibility Complex (MHC genes, but it is still unclear whether this variation results from a stochastic process or from selective constraints related to functional differences among HLA molecules. Although HLA alleles are generally treated as equidistant molecular units in population genetic studies, DNA sequence diversity among populations is also crucial to interpret the observed HLA polymorphism. In this study, we used a large dataset of 2,062 DNA sequences defined for the different HLA alleles to analyze nucleotide diversity of seven HLA genes in 23,500 individuals of about 200 populations spread worldwide. We first analyzed the HLA molecular structure and diversity of these populations in relation to geographic variation and we further investigated possible departures from selective neutrality through Tajima's tests and mismatch distributions. All results were compared to those obtained by classical approaches applied to HLA allele frequencies.Our study shows that the global patterns of HLA nucleotide diversity among populations are significantly correlated to geography, although in some specific cases the molecular information reveals unexpected genetic relationships. At all loci except HLA-DPB1, populations have accumulated a high proportion of very divergent alleles, suggesting an advantage of heterozygotes expressing molecularly distant HLA molecules (asymmetric overdominant selection model. However, both different intensities of selection and unequal levels of gene conversion may explain the heterogeneous mismatch distributions observed among the loci. Also, distinctive patterns of sequence divergence observed at the HLA-DPB1 locus suggest current neutrality but old selective pressures on this gene. We conclude that HLA DNA sequences advantageously complement HLA allele frequencies as a source of data used

  3. DNA Barcoding as a Molecular Tool to Track Down Mislabeling and Food Piracy

    Directory of Open Access Journals (Sweden)

    Gianni Barcaccia

    2015-12-01

    Full Text Available DNA barcoding is a molecular technology that allows the identification of any biological species by amplifying, sequencing and querying the information from genic and/or intergenic standardized target regions belonging to the extranuclear genomes. Although these sequences represent a small fraction of the total DNA of a cell, both chloroplast and mitochondrial barcodes chosen for identifying plant and animal species, respectively, have shown sufficient nucleotide diversity to assess the taxonomic identity of the vast majority of organisms used in agriculture. Consequently, cpDNA and mtDNA barcoding protocols are being used more and more in the food industry and food supply chains for food labeling, not only to support food safety but also to uncover food piracy in freshly commercialized and technologically processed products. Since the extranuclear genomes are present in many copies within each cell, this technology is being more easily exploited to recover information even in degraded samples or transformed materials deriving from crop varieties and livestock species. The strong standardization that characterizes protocols used worldwide for DNA barcoding makes this technology particularly suitable for routine analyses required by agencies to safeguard food safety and quality. Here we conduct a critical review of the potentials of DNA barcoding for food labeling along with the main findings in the area of food piracy, with particular reference to agrifood and livestock foodstuffs.

  4. Molecular restructuring of water and lipids upon the interaction of DNA with lipid monolayers.

    Science.gov (United States)

    Campen, R Kramer; Ngo, Thuy T M; Sovago, Maria; Ruysschaert, Jean-Marie; Bonn, Mischa

    2010-06-16

    Understanding the molecular mechanism of DNA/lipid interaction is critical in optimizing the use of lipid cofactors in gene therapy. Here, we address this question by employing label-free vibrational sum frequency (VSF) spectroscopy to study the interaction of DNA with lipid monolayers of the cationic lipids DPTAP(1,2-dipalmitoyl-3-trimethylammonium-propane) and diC14-amidine as well as the zwitterionic lipid DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine) in the presence and absence of calcium. Our approach has the advantage both of allowing us to explicitly probe intermolecular interactions and of providing insight into the structure of water and lipids around DNA at the lipid interface. We find, by examination of the OD stretch of interfacial D(2)O, that water structure differs markedly between systems containing DNA adsorbed to cationic and those that contain DNA adsorbed to zwitterionic lipid monolayers (in the presence or absence of Ca(2+)). The spectral response of interfacial water in the cationic system is consistent with a highly structured, undercoordinated, structural 'type' of water. Further, by investigation of CH stretch modes of the diC14-amidine lipid tails, we demonstrate that the adsorption of DNA to this lipid leads to increased ordering of lipid tails.

  5. Cas9-catalyzed DNA Cleavage Generates Staggered Ends: Evidence from Molecular Dynamics Simulations

    Science.gov (United States)

    Zuo, Zhicheng; Liu, Jin

    2016-11-01

    The CRISPR-associated endonuclease Cas9 from Streptococcus pyogenes (spCas9) along with a single guide RNA (sgRNA) has emerged as a versatile toolbox for genome editing. Despite recent advances in the mechanism studies on spCas9-sgRNA-mediated double-stranded DNA (dsDNA) recognition and cleavage, it is still unclear how the catalytic Mg2+ ions induce the conformation changes toward the catalytic active state. It also remains controversial whether Cas9 generates blunt-ended or staggered-ended breaks with overhangs in the DNA. To investigate these issues, here we performed the first all-atom molecular dynamics simulations of the spCas9-sgRNA-dsDNA system with and without Mg2+ bound. The simulation results showed that binding of two Mg2+ ions at the RuvC domain active site could lead to structurally and energetically favorable coordination ready for the non-target DNA strand cleavage. Importantly, we demonstrated with our simulations that Cas9-catalyzed DNA cleavage produces 1-bp staggered ends rather than generally assumed blunt ends.

  6. The importance of molecular markers and primer design when characterizing biodiversity from environmental DNA.

    Science.gov (United States)

    Freeland, Joanna R

    2017-04-01

    Environmental DNA (eDNA) comprises DNA fragments that have been shed into the environment by organisms, and which can be extracted from environmental samples such as water or soil. Characterization of eDNA can allow researchers to infer the presence or absence of species from a particular site without the need to locate and identify individuals, and therefore may provide an extremely valuable tool for quantifying biodiversity. However, as is often the case with relatively new protocols, methodological challenges remain. A number of earlier reviews have discussed these challenges, but none have provided extensive treatment of the critical decisions surrounding molecular markers and primer development for use in eDNA assays. This review discusses a number of options and approaches that can be used when determining which primers and gene regions are most appropriate for either targeted species detection or metabarcoding macro-organisms from eDNA. The latter represents a new field that is growing rapidly, and which has the potential to revolutionize future assessments of community and ecosystem diversity.

  7. Polyurethane Molecular Stamps for the in situ Synthesis of DNA Microarray

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    Fabrication of polyurethane molecular stamps (PU stamps) based on polypropylene glycol (PPG) and toluene diisocyanate (TDI), using 3, 3(-dichloro-4, 4(-methylenedianiline (MOCA) as the crosslinker, is reported. It was shown from the contact angle measurement that PU stamps surface has good affinity with acetonitrile, guaranteeing the well distribution of DNA monomers on patterned stamps. Laser confocal fluorescence microscopy images of oligonucleotide arrays after hybridization confirmed polyurethane is an excellent material for molecular stamps when transferring polar chemicals and conducting reactions on interfaces by stamping.

  8. Structure and mechanical characterization of DNA i-motif nanowires by molecular dynamics simulation

    CERN Document Server

    Singh, Raghvendra Pratap; Cleri, Fabrizio

    2013-01-01

    We studied the structure and mechanical properties of DNA i-motif nanowires by means of molecular dynamics computer simulations. We built up to 230 nm long nanowires, based on a repeated TC5 sequence from crystallographic data, fully relaxed and equilibrated in water. The unusual stacked C*C+ stacked structure, formed by four ssDNA strands arranged in an intercalated tetramer, is here fully characterized both statically and dynamically. By applying stretching, compression and bending deformation with the steered molecular dynamics and umbrella sampling methods, we extract the apparent Young's and bending moduli of the nanowire, as wel as estimates for the tensile strength and persistence length. According to our results, the i-motif nanowire shares similarities with structural proteins, as far as its tensile stiffness, but is closer to nucleic acids and flexible proteins, as far as its bending rigidity is concerned. Furthermore, thanks to its very thin cross section, the apparent tensile toughness is close to...

  9. Molecular characterization of 18S rDNA partial sequence in Microcosmus (Stolidobranchiata, Pyuridae

    Directory of Open Access Journals (Sweden)

    D. FULGIONE

    2012-12-01

    Full Text Available We present a 18S rDNA based molecular phylogeny of two species of the genus Microcosmus (M. sulcatus and M. claudicans sampled in the Mediterranean, to investigate their phylogenetic position relative to species of the order Stolidobranchiata. The analysis is based on partial sequences (739 bp of the 18S rDNA. Among the 18 variable sites found between the two species, 4 correspond to transitions (ts, 14 to transversions (tv and 4 to deletions/insertions. In the considered Stolidobranchiata, we found 4.3% overall mean number of nucleotide differences and 0.06 (S.E. ±0.01 Kimura 2-parameter distance. The mean number of nucleotide differences between Microcosmus spp. and other Stolidobranchiata species was of 6% and 0.08 (S.E. ±0.01 Kimura 2-parameter distance. A molecular phylogeny obtained by Maximum Parsimony corroborates results of the traditional taxonomy.

  10. Molecular characterization of 18S rDNA partial sequence in Microcosmus (Stolidobranchiata, Pyuridae

    Directory of Open Access Journals (Sweden)

    D. FULGIONE

    2006-12-01

    Full Text Available We present a 18S rDNA based molecular phylogeny of two species of the genus Microcosmus (M. sulcatus and M. claudicans sampled in the Mediterranean, to investigate their phylogenetic position relative to species of the order Stolidobranchiata. The analysis is based on partial sequences (739 bp of the 18S rDNA. Among the 18 variable sites found between the two species, 4 correspond to transitions (ts, 14 to transversions (tv and 4 to deletions/insertions. In the considered Stolidobranchiata, we found 4.3% overall mean number of nucleotide differences and 0.06 (S.E. ±0.01 Kimura 2-parameter distance. The mean number of nucleotide differences between Microcosmus spp. and other Stolidobranchiata species was of 6% and 0.08 (S.E. ±0.01 Kimura 2-parameter distance. A molecular phylogeny obtained by Maximum Parsimony corroborates results of the traditional taxonomy.

  11. Study the effects of metallic ions on the combination of DNA and histones with molecular combing technique

    Institute of Scientific and Technical Information of China (English)

    LIU Yuying; WANG Pengye; DOU Shuoxing; XIE Ping; WANG Weichi; YIN Huawei

    2005-01-01

    The effects of monovalent (Na+, K+) and divalent (Mg2+, Ca2+, Mn2+) ions on the interaction between DNA and histone are studied using the molecular combing technique. λ-DNA molecules and DNA-histone complexes incubated with metal cations (Na+, K+, Mg2+, Ca2+, Mn2+) are stretched on hydrophobic surfaces, and directly observed by fluorescence microscopy. The results indicate that when these cations are added into the DNA solution, the fluorescence intensities of the stained DNA are reduced differently. The monovalent cations (Na+, K+) inhibit binding of histone to DNA. The divalent cations (Mg2+, Ca2+, Mn2+) enhance significantly the binding of histone to DNA and the binding of the DNA-histone complex to the hydrophobic surface. Mn2+ also induces condensation and aggregation of the DNA- histone complex.

  12. [DNA molecular identification of Herba Dendrobii and its adulterant species based on ITS sequence analysis].

    Science.gov (United States)

    Liu, Jing; He, Tao; Chun, Ze

    2009-11-01

    To identify Herba Dendrobii and its adulterant species on molecular level, the rDNA ITS sequences of 17 species of Herba Dendrobii were studied. Genomic DNA of Dendrobium was extracted using the modified cetyltrimethyl ammonium bromide (CTAB) method. The PCR products of the rDNA ITS sequences of Dendrobium (32 materials) were purified and then sequenced. The characteristic of the sequences and the genetic distance were compared between Bulbophyllum odoratissimum and Dendrobium, Dendrobium interspecies and different populations. Phylogenetic trees were constructed using the UPGMA method by the biology softwares including BioEdit, MEGA4.0 etc. The PCR products were purified and then sequenced. It was built up that the database of rDNA ITS sequences of 17 species of Herba Dendrobii (32 materials). The ITS1 was 228-234 bp, the GC content accounting for 45.7%-53.0%. Its variable sites were 167, accounting for 67.34%. The Parsim-Informative positions were 106, accounting for 42.74%. The ITS2 was 241-247 bp, the GC accounting for 44.8% - 55.7%. The variable sites were 165, accounting for 66.27%. The Parsim-Informative positions were 115, accounting for 46.18%. The genetic distance between B. odoratissimum and Dendrobium was 0.295. The average genetic distance was 0.142 between Dendrobium species, and there were 2-156 variable nucleotides. The average genetic distance between different populations was 0.002, and there were 2-156 variable nucleotides. The genetic distance between B. odoratissimum and Dendrobium was greater than that of Denrobium interspecies. Meanwhile, the genetic distance between Denrobium species was also greater than that of different populations (varieties). The molecular phylogeny tree was constructed on the database of rDNA ITS the sequences of 17 species of Herba Dendrobii using the biology softwares. Then 10 materials on molecular level were authenticated. It is concluded that using of the whole sequences database of 17 species of Herba Dendrobii

  13. Dynamic Mechanism of Single-Stranded DNA Encapsulated into Single-Wall Carbon Nanotubes: A Molecular Dynamics Simulation Study

    Science.gov (United States)

    Xing, Yan-Fei; Yang, Chuan-Lu; Mo, Yong-Fang; Wang, Mei-Shan; Ma, Xiao-Guang

    2014-02-01

    Hybrids of single-walled carbon nanotubes (SWCNTs) and biological molecules have been utilized for numerous applications in sensing, imaging, and drug delivery. By molecular dynamics simulation, we investigate the encapsulation of single-strand DNA (ssDNA) containing eight adenine bases with (17,17)-(12,12) SWCNTs. The effects of the diameter and length of SWCNTs on the encapsulation process are explored with the calculated curves of the center-of-mass distance, the van der Waals interaction between the ssDNA and SWCNT, the root-mean-square deviation of the ssDNA, and the radius of gyration of the ssDNA. The free energy of the encapsulated ssDNA for each SWCNT is also obtained via steered molecular dynamics simulation. The most suitable SWCNT for encapsulating the ssDNA is also suggested.

  14. Molecular Detection of Toxoplasmosis Using Specific Primers P30, B1, and rDNA

    Directory of Open Access Journals (Sweden)

    Wisnu Nurcahyo

    2014-04-01

    Full Text Available Study in order to develop molecular techniques using specific primers for the early diagnosis oftoxoplasmosis have been conducted. Detection of Toxoplasma gondii genome was performed usingpolymerase chain reaction (PCR technique. The primers used in this study were rDNA, P30, and B1. ThePCR products were further run using gel electrophoresis (gel 1.5% – 2.0% and the band was documented.Toxoplasma was detected at 500 bp and 600 bp using primer P30 and B1, respectively. Whereas usingprimer rDNA no band was observed. It was assumed that primer rDNA was not sensitive since the targetamplification was 88 bp.

  15. Molecular cloning of GA-suppressed G2 pea genes by cDNA RDA

    Institute of Scientific and Technical Information of China (English)

    朱玉贤; 张翼凤; 李慧英

    1997-01-01

    GA-treated and non-treated G2 pea cDNAs were compared using a newly developed method called cDNA representational difference analysis (cDNA-RDA), and several GA-suppressed mRNAs were found. After cloning of the larger fragments PGAS1-3 ( pea GA-suppressed cDNA 1-3), they were demonstrated to be expressed only in pea tissue not treated with GA3 through Northern analysis. Compared with subtractive hybridization and differ-ential display techniques, this method not only can be easily manipulated but also has a relatively low rate of false posi-tive and is highly repetitive. It is the major progress in molecular cloning techniques.

  16. Molecular cloning of DNA complementary to Drosophila melanogaster alpha-amylase mRNA.

    Science.gov (United States)

    Benkel, B F; Abukashawa, S; Boer, P H; Hickey, D A

    1987-06-01

    Several lambda clones containing cDNAs from Drosophila melanogaster were identified in a lambda cDNA bank using two different approaches: (i) cross-species hybridization using a mouse amylase cDNA probe, and (ii) probing with a differential probe, generated from Drosophila RNA. An amylase cDNA fragment was used, in turn, for the isolation and characterization of amylase genomic clones. The size of the Drosophila amylase mRNA was estimated at 1650 b. This is comparable with the size of the murine amylase messenger that encodes a protein of similar molecular weight. In Drosophila larvae, amylase mRNA can account for as little as 0.01% of the poly(A)+ RNA under conditions of dietary glucose repression or greater than 1% of poly(A)+ RNA under derepressing dietary conditions.

  17. Molecular force spectroscopy with a DNA origami-based nanoscopic force clamp.

    Science.gov (United States)

    Nickels, Philipp C; Wünsch, Bettina; Holzmeister, Phil; Bae, Wooli; Kneer, Luisa M; Grohmann, Dina; Tinnefeld, Philip; Liedl, Tim

    2016-10-21

    Forces in biological systems are typically investigated at the single-molecule level with atomic force microscopy or optical and magnetic tweezers, but these techniques suffer from limited data throughput and their requirement for a physical connection to the macroscopic world. We introduce a self-assembled nanoscopic force clamp built from DNA that operates autonomously and allows massive parallelization. Single-stranded DNA sections of an origami structure acted as entropic springs and exerted controlled tension in the low piconewton range on a molecular system, whose conformational transitions were monitored by single-molecule Förster resonance energy transfer. We used the conformer switching of a Holliday junction as a benchmark and studied the TATA-binding protein-induced bending of a DNA duplex under tension. The observed suppression of bending above 10 piconewtons provides further evidence of mechanosensitivity in gene regulation.

  18. [Analysis of DNA and chromosome damage by the methods of molecular cytogenetics].

    Science.gov (United States)

    Arutiunian, R M; Oganesian, G G

    2011-01-01

    The main hybrid techniques of molecular cytogenetics are described. Methodological aspects of combination of fluorescence in situ hybridization (FISH) with the comet assay and micronuclei (MN) test are discussed along with results of their application to evaluate and locate DNA and chromosome damage in the genome. The experience of the authors with the application of FISH in combination with the comet assay and MN test are reported.

  19. Molecular Processes Studied at a Single-Molecule Level Using DNA Origami Nanostructures and Atomic Force Microscopy

    Directory of Open Access Journals (Sweden)

    Ilko Bald

    2014-09-01

    Full Text Available DNA origami nanostructures allow for the arrangement of different functionalities such as proteins, specific DNA structures, nanoparticles, and various chemical modifications with unprecedented precision. The arranged functional entities can be visualized by atomic force microscopy (AFM which enables the study of molecular processes at a single-molecular level. Examples comprise the investigation of chemical reactions, electron-induced bond breaking, enzymatic binding and cleavage events, and conformational transitions in DNA. In this paper, we provide an overview of the advances achieved in the field of single-molecule investigations by applying atomic force microscopy to functionalized DNA origami substrates.

  20. Allosteric analysis of glucocorticoid receptor-DNA interface induced by cyclic Py-Im polyamide: a molecular dynamics simulation study.

    Directory of Open Access Journals (Sweden)

    Yaru Wang

    Full Text Available BACKGROUND: It has been extensively developed in recent years that cell-permeable small molecules, such as polyamide, can be programmed to disrupt transcription factor-DNA interfaces and can silence aberrant gene expression. For example, cyclic pyrrole-imidazole polyamide that competes with glucocorticoid receptor (GR for binding to glucocorticoid response elements could be expected to affect the DNA dependent binding by interfering with the protein-DNA interface. However, how such small molecules affect the transcription factor-DNA interfaces and gene regulatory pathways through DNA structure distortion is not fully understood so far. METHODOLOGY/PRINCIPAL FINDINGS: In the present work, we have constructed some models, especially the ternary model of polyamides+DNA+GR DNA-binding domain (GRDBD dimer, and carried out molecular dynamics simulations and free energy calculations for them to address how polyamide molecules disrupt the GRDBD and DNA interface when polyamide and protein bind at the same sites on opposite grooves of DNA. CONCLUSIONS/SIGNIFICANCE: We found that the cyclic polyamide binding in minor groove of DNA can induce a large structural perturbation of DNA, i.e. a >4 Å widening of the DNA minor groove and a compression of the major groove by more than 4 Å as compared with the DNA molecule in the GRDBD dimer+DNA complex. Further investigations for the ternary system of polyamides+DNA+GRDBD dimer and the binary system of allosteric DNA+GRDBD dimer revealed that the compression of DNA major groove surface causes GRDBD to move away from the DNA major groove with the initial average distance of ∼4 Å to the final average distance of ∼10 Å during 40 ns simulation course. Therefore, this study straightforward explores how small molecule targeting specific sites in the DNA minor groove disrupts the transcription factor-DNA interface in DNA major groove, and consequently modulates gene expression.

  1. Molecular organization of 5S rDNA in bitterlings (Cyprinidae).

    Science.gov (United States)

    Fujiwara, Mika; Inafuku, Junya; Takeda, Akiko; Watanabe, Akiko; Fujiwara, Atushi; Kohno, Sei-Ichi; Kubota, Souichirou

    2009-04-01

    Molecular organization and nucleotide sequences of the 5S rRNA gene and NTS were investigated in freshwater fish, bitterlings (Acheilognathinae), including 10 species/subspecies of four genera, Acheilognathus, Pseudoperilampus, Rhodeus, and Tanakia, to understand the evolutionary trait of 5S rDNA arrays. Southern hybridization analysis revealed a general trend with tandem repeats of 5S rDNA in all the examined bitterlings. Sequence analysis demonstrated a conserved 120 bp sequence of the 5S rRNA gene and a short NTS of 56-67 bp with two distinct portions, a conserved (5'-flanking portion; at positions -1 to -38) and a variable part (3'-flanking portion), in 6 of 10 species/subspecies examined. The conserved NTS region was most likely an external promoter so far observed in various vertebrates, whereas the variable NTS region could be divided into two types due to its nucleotide polymorphisms. Molecular phylogeny using the 5S rRNA gene and NTS sequences suggested the occurrence of 5S rDNA duplication before speciation and a concerted evolution for the gene and conserved NTS regions, but a birth-and-death process to maintain the variable NTS region. Thus, the 5S rDNA in the examined bitterlings might have evolved under a mixed process of evolution.

  2. Phylogenetic relationships and species delimitation in pinus section trifoliae inferrred from plastid DNA.

    Directory of Open Access Journals (Sweden)

    Sergio Hernández-León

    Full Text Available Recent diversification followed by secondary contact and hybridization may explain complex patterns of intra- and interspecific morphological and genetic variation in the North American hard pines (Pinus section Trifoliae, a group of approximately 49 tree species distributed in North and Central America and the Caribbean islands. We concatenated five plastid DNA markers for an average of 3.9 individuals per putative species and assessed the suitability of the five regions as DNA bar codes for species identification, species delimitation, and phylogenetic reconstruction. The ycf1 gene accounted for the greatest proportion of the alignment (46.9%, the greatest proportion of variable sites (74.9%, and the most unique sequences (75 haplotypes. Phylogenetic analysis recovered clades corresponding to subsections Australes, Contortae, and Ponderosae. Sequences for 23 of the 49 species were monophyletic and sequences for another 9 species were paraphyletic. Morphologically similar species within subsections usually grouped together, but there were exceptions consistent with incomplete lineage sorting or introgression. Bayesian relaxed molecular clock analyses indicated that all three subsections diversified relatively recently during the Miocene. The general mixed Yule-coalescent method gave a mixed model estimate of only 22 or 23 evolutionary entities for the plastid sequences, which corresponds to less than half the 49 species recognized based on morphological species assignments. Including more unique haplotypes per species may result in higher estimates, but low mutation rates, recent diversification, and large effective population sizes may limit the effectiveness of this method to detect evolutionary entities.

  3. Phylogenetic relationships and species delimitation in pinus section trifoliae inferrred from plastid DNA.

    Science.gov (United States)

    Hernández-León, Sergio; Gernandt, David S; Pérez de la Rosa, Jorge A; Jardón-Barbolla, Lev

    2013-01-01

    Recent diversification followed by secondary contact and hybridization may explain complex patterns of intra- and interspecific morphological and genetic variation in the North American hard pines (Pinus section Trifoliae), a group of approximately 49 tree species distributed in North and Central America and the Caribbean islands. We concatenated five plastid DNA markers for an average of 3.9 individuals per putative species and assessed the suitability of the five regions as DNA bar codes for species identification, species delimitation, and phylogenetic reconstruction. The ycf1 gene accounted for the greatest proportion of the alignment (46.9%), the greatest proportion of variable sites (74.9%), and the most unique sequences (75 haplotypes). Phylogenetic analysis recovered clades corresponding to subsections Australes, Contortae, and Ponderosae. Sequences for 23 of the 49 species were monophyletic and sequences for another 9 species were paraphyletic. Morphologically similar species within subsections usually grouped together, but there were exceptions consistent with incomplete lineage sorting or introgression. Bayesian relaxed molecular clock analyses indicated that all three subsections diversified relatively recently during the Miocene. The general mixed Yule-coalescent method gave a mixed model estimate of only 22 or 23 evolutionary entities for the plastid sequences, which corresponds to less than half the 49 species recognized based on morphological species assignments. Including more unique haplotypes per species may result in higher estimates, but low mutation rates, recent diversification, and large effective population sizes may limit the effectiveness of this method to detect evolutionary entities.

  4. Molecular cloning and expression of a novel human cDNA containing CAG repeats.

    Science.gov (United States)

    Takeuchi, T; Chen, B K; Qiu, Y; Sonobe, H; Ohtsuki, Y

    1997-12-19

    A novel human cDNA containing CAG repeats, designated B120, was cloned by PCR amplification. An approximately 300-bp 3' untranslated region in this cDNA was followed by a 3426-bp coding region containing the CAG repeats. A computer search failed to find any significant homology between this cDNA and previously reported genes. The number of CAG trinucleotide repeats appeared to vary from seven to 12 in analyses of genomic DNA from healthy volunteers. An approximately 8-kb band was detected in brain, skeletal muscle and thymus by Northern blot analysis. The deduced amino-acid sequence had a polyglutamine chain encoded by CAG repeats as well as glutamine- and tyrosine-rich repeats, which has also been reported for several RNA binding proteins. We immunized mice with recombinant gene product and established a monoclonal antibody to it. On Western immunoblotting, this antibody detected an approximately 120-kDa protein in human brain tissue. In addition, immunohistochemical staining showed that the cytoplasm of neural cells was stained with this antibody. These findings indicated that B120 is a novel cDNA with a CAG repeat length polymorphism and that its gene product is a cytoplasmic protein with a molecular mass of 120 kDa.

  5. Association of thymine glycol lesioned DNA with repair enzyme endonuclease III-molecular dynamics study

    Energy Technology Data Exchange (ETDEWEB)

    Pinak, Miroslav [Japan Atomic Energy Research Inst., Tokai, Ibaraki (Japan). Tokai Research Establishment

    2001-07-01

    The 2 nanoseconds molecular dynamics (MD) simulation has been performed for the system consisting of repair enzyme and DNA 30-mer with native thymine at position 16 replaced by thymine glycol (TG) solvated in water environment. After 950 picoseconds of MD the enzyme and DNA associated together to form complex that lasted stable up to 2 ns when simulation was terminated. At the contact area of enzyme and DNA there is glutamic acid located as close as 1.6 A to the C3' atom of phosphodiester bond of TG. Initial B-DNA molecule was bent and kinked at the TG during MD. This distortion caused that phosphodiester bond was easier accessible by amino acids of enzyme. The negative value of electrostatic energy (-26 kcal/mol) discriminates TG from nearly neutral native thymine and contributes to the specific recognition of this lesion. Higher number of close water molecules at TG site before formation of complex (compared with other nucleotides) indicates that glycosyl bond of the lesion is easily approached by repair enzyme during scanning of DNA surface and suggests the importance of specific hydration at the lesion during recognition process. (author)

  6. Preparation of high-molecular weight DNA and metagenomic libraries from soils and hot springs.

    Science.gov (United States)

    Reigstad, Laila J; Bartossek, Rita; Schleper, Christa

    2011-01-01

    Metagenomics has become an important tool for the characterization of microorganisms, as it is independent of their enrichment or cultivation in the laboratory. Its application has led to the discovery of metabolisms from widespread, yet uncharacterized organisms such as the ammonia-oxidizing archaea. Different approaches ranging from the generation of short sequence reads by direct use of high-throughput sequencing technologies to the construction and sequencing of large-insert DNA libraries are being employed. For these purposes, DNA of high quality needs to be prepared from an environmental sample, which is a particular challenge for soils and sediments. Here we describe the methods used for the isolation of high-molecular weight (hmw) DNA from soil and hot spring samples, the subsequent production of large-insert metagenomic libraries, and the analysis of the resulting genomic fragments. Detailed step-by-step procedures include (1) how to isolate good-quality hmw DNA from soils and mud; (2) how to prepare the DNA for cloning; (3) how to efficiently establish, grow, pick, replicate, and store the large-insert metagenomic fosmid library; and finally, (4) how to screen the library for genes of interest.

  7. Molecular cytogenetic analysis and genomic organization of major DNA repeats in castor bean (Ricinus communis L.).

    Science.gov (United States)

    Alexandrov, O S; Karlov, G I

    2016-04-01

    This article addresses the bioinformatic, molecular genetic, and cytogenetic study of castor bean (Ricinus communis, 2n = 20), which belongs to the monotypic Ricinus genus within the Euphorbiaceae family. Because castor bean chromosomes are small, karyotypic studies are difficult. However, the use of DNA repeats has yielded new prospects for karyotypic research and genome characterization. In the present study, major DNA repeat sequences were identified, characterized and localized on mitotic metaphase and meiotic pachytene chromosomes. Analyses of the nucleotide composition, curvature models, and FISH localization of the rcsat39 repeat suggest that this repeat plays a key role in building heterochromatic arrays in castor bean. Additionally, the rcsat390 sequences were determined to be chromosome-specific repeats located in the pericentromeric region of mitotic chromosome A (pachytene chromosome 1). The localization of rcsat39, rcsat390, 45S and 5S rDNA genes allowed for the development of cytogenetic landmarks for chromosome identification. General questions linked to heterochromatin formation, DNA repeat distribution, and the evolutionary emergence of the genome are discussed. The article may be of interest to biologists studying small genome organization and short monomer DNA repeats.

  8. Rosalind Franklin and the DNA molecular structure: A case of history of science to learn about the nature of science

    National Research Council Canada - National Science Library

    José Antonio Acevedo-Díaz; Dr. Antonio García-Carmona

    2016-01-01

    The Rosalind Franklin’s case regarding the elucidation of the molecular structure of DNA is presented as an interesting story of the history of science to address a set of questions related to the nature of science (NOS...

  9. Molecular dynamics of DNA quadruplex molecules containing inosine, 6-thioguanine and 6-thiopurine.

    Science.gov (United States)

    Stefl, R; Spacková, N; Berger, I; Koca, J; Sponer, J

    2001-01-01

    The ability of the four-stranded guanine (G)-DNA motif to incorporate nonstandard guanine analogue bases 6-oxopurine (inosine, I), 6-thioguanine (tG), and 6-thiopurine (tI) has been investigated using large-scale molecular dynamics simulations. The simulations suggest that a G-DNA stem can incorporate inosines without any marked effect on its structure and dynamics. The all-inosine quadruplex stem d(IIII)(4) shows identical dynamical properties as d(GGGG)(4) on the nanosecond time scale, with both molecular assemblies being stabilized by monovalent cations residing in the channel of the stem. However, simulations carried out in the absence of these cations show dramatic differences in the behavior of d(GGGG)(4) and d(IIII)(4). Whereas vacant d(GGGG)(4) shows large fluctuations but does not disintegrate, vacant d(IIII)(4) is completely disrupted within the first nanosecond. This is a consequence of the lack of the H-bonds involving the N2 amino group that is not present in inosine. This indicates that formation of the inosine quadruplex could involve entirely different intermediate structures than formation of the guanosine quadruplex, and early association of cations in this process appears to be inevitable. In the simulations, the incorporation of 6-thioguanine and 6-thiopurine sharply destabilizes four-stranded G-DNA structures, in close agreement with experimental data. The main reason is the size of the thiogroup leading to considerable steric conflicts and expelling the cations out of the channel of the quadruplex stem. The G-DNA stem can accommodate a single thioguanine base with minor perturbations. Incorporation of a thioguanine quartet layer is associated with a large destabilization of the G-DNA stem whereas the all-thioguanine quadruplex immediately collapses.

  10. Assessment of the quality of dna extracted by two techniques from Mycobacterium tuberculosis for fast molecular identification and genotyping

    Directory of Open Access Journals (Sweden)

    Marcelo Miyata

    2011-06-01

    Full Text Available We report a comparative study of two DNA extraction techniques, thermolysis and chemical lysis (CTAB, for molecular identification and genotyping of M. tuberculosis. Forty DNA samples were subjected to PCR and the results demonstrated that with thermolysis it is possible to obtain useful data that enables fast identification and genotyping.

  11. Improvement of reliability of molecular DNA computing: solution of inverse problem of Raman spectroscopy using artificial neural networks

    Science.gov (United States)

    Dolenko, T. A.; Burikov, S. A.; Vervald, E. N.; Efitorov, A. O.; Laptinskiy, K. A.; Sarmanova, O. E.; Dolenko, S. A.

    2017-02-01

    Elaboration of methods for the control of biochemical reactions with deoxyribonucleic acid (DNA) strands is necessary for the solution of one of the basic problems in the creation of biocomputers—improvement in the reliability of molecular DNA computing. In this paper, the results of the solution of the four-parameter inverse problem of laser Raman spectroscopy—the determination of the type and concentration of each of the DNA nitrogenous bases in multi-component solutions—are presented.

  12. DNA分子标记在中药鉴定中的应用进展%DNA Molecular Marker in Progress in the Application of Chinese Medicine Identification

    Institute of Scientific and Technical Information of China (English)

    海学忠

    2012-01-01

    DNA molecular markers including molecular hybrid (southern hybridization) as the foundation of DNA molecular markers, PCR-based DNA molecular markers and DNA sequence to the basis of molecular markers and DNA sequencing by technology. This paper summarizes the commonly used DNA molecular markers on the features of DNA molecular markers in recent years Chinese medicine identification in this paper reviewed the application.%DNA分子标记包括以分子杂交(southern杂交)为基础的DNA分子标记技术、以PCR为基础的DNA分子标记技术和以DNA序列为基础的分子标记技术和DNA序列测定技术。本文概述了常用DNA分子标记技术的特点,对近年来DNA分子标记在中药鉴定中的应用进行了综述。

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

    Science.gov (United States)

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

    2015-12-01

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

  14. Molecular Diagnosis of Strongyloides Stercoralis Infection by PCR Detection of Specific DNA in Human Stool Samples

    Directory of Open Access Journals (Sweden)

    Eb Kia

    2011-06-01

    Full Text Available Background: Strongyloidiasis is mostly an asymptomatic infection and diagnosis of latent infec­tions is difficult due to limitations of current parasitological and serological methods. This study was conducted to set up a PCR-based method for molecular diagnosis of Strongyloides stercor­alis infection by detection of copro-DNA in stool samples.Methods: A total of 782 fresh stool samples were collected and examined by agar plate culture. Among those sixteen stool samples, which confirmed to be infected with S. stercoralis were exam­ined as positive control to set up each single and nested PCR, using two primer sets design­ing to amplify partial ribosomal DNA of S. stercoralis genome. Since, single PCR method yielded higher efficacy in detecting positive samples, in the second step, 30 stool samples, which found negative for S. stercoralis by agar plate culture of single stool sample, were examined by sin­gle PCR. Data analysis was performed using McNemar's χ2 test, with consideration of a P-value of <0.05 as indication of significant difference.Results: In amplification of DNA extracted from stool samples, single PCR detected S. stercor­alis DNA target in all 16 positive samples, while nested PCR amplified DNA in only 75% of sam­ples. In the second step, single PCR amplified S. stercoralis extracted DNA in 5 out of 30 sam­ples which were negative by coproculture.Conclusion: Single PCR method amplifying a short (100bp target represented more efficacies for detection of S. stercoralis in faecal examination compared to agar plate culture and nested PCR, which amplified longer target.

  15. In vitro selection of a single-stranded DNA molecular recognition element against atrazine.

    Science.gov (United States)

    Williams, Ryan M; Crihfield, Cassandra L; Gattu, Srikanth; Holland, Lisa A; Sooter, Letha J

    2014-08-18

    Widespread use of the chlorotriazine herbicide, atrazine, has led to serious environmental and human health consequences. Current methods of detecting atrazine contamination are neither rapid nor cost-effective. In this work, atrazine-specific single-stranded DNA (ssDNA) molecular recognition elements (MRE) were isolated. We utilized a stringent Systematic Evolution of Ligands by Exponential Enrichment (SELEX) methodology that placed the greatest emphasis on what the MRE should not bind to. After twelve rounds of SELEX, an atrazine-specific MRE with high affinity was obtained. The equilibrium dissociation constant (Kd) of the ssDNA sequence is 0.62 ± 0.21 nM. It also has significant selectivity for atrazine over atrazine metabolites and other pesticides found in environmentally similar locations and concentrations. Furthermore, we have detected environmentally relevant atrazine concentrations in river water using this MRE. The strong affinity and selectivity of the selected atrazine-specific ssDNA validated the stringent SELEX methodology and identified a MRE that will be useful for rapid atrazine detection in environmental samples.

  16. In Vitro Selection of a Single-Stranded DNA Molecular Recognition Element against Atrazine

    Directory of Open Access Journals (Sweden)

    Ryan M. Williams

    2014-08-01

    Full Text Available Widespread use of the chlorotriazine herbicide, atrazine, has led to serious environmental and human health consequences. Current methods of detecting atrazine contamination are neither rapid nor cost-effective. In this work, atrazine-specific single-stranded DNA (ssDNA molecular recognition elements (MRE were isolated. We utilized a stringent Systematic Evolution of Ligands by Exponential Enrichment (SELEX methodology that placed the greatest emphasis on what the MRE should not bind to. After twelve rounds of SELEX, an atrazine-specific MRE with high affinity was obtained. The equilibrium dissociation constant (Kd of the ssDNA sequence is 0.62 ± 0.21 nM. It also has significant selectivity for atrazine over atrazine metabolites and other pesticides found in environmentally similar locations and concentrations. Furthermore, we have detected environmentally relevant atrazine concentrations in river water using this MRE. The strong affinity and selectivity of the selected atrazine-specific ssDNA validated the stringent SELEX methodology and identified a MRE that will be useful for rapid atrazine detection in environmental samples.

  17. Molecular characterization and evolution of an interspersed repetitive DNA family of oysters.

    Science.gov (United States)

    López-Flores, Inmaculada; Ruiz-Rejón, Carmelo; Cross, Ismael; Rebordinos, Laureana; Robles, Francisca; Navajas-Pérez, Rafael; de la Herrán, Roberto

    2010-12-01

    When genomic DNA from the European flat oyster Ostrea edulis L. was digested by BclI enzyme, a band of about 150 bp was observed in agarose gel. After cloning and sequencing this band and analysing their molecular characteristics and genomic organization by means of Southern blot, in situ hybridisation, and polymerase chain reaction (PCR) protocols, we concluded that this band is an interspersed highly repeated DNA element, which is related in sequence to the flanking regions of (CT)-microsatellite loci of the species O. edulis and Crassostrea gigas. Furthermore, we determined that this element forms part of a longer repetitive unit of 268 bp in length that, at least in some loci, is present in more than one copy. By Southern blot hybridisation and PCR amplifications-using primers designed for conserved regions of the 150-bp BclI clones of O. edulis-we determined that this repetitive DNA family is conserved in five other oyster species (O. stentina, C. angulata, C. gigas, C. ariakensis, and C. sikamea) while it is apparently absent in C. gasar. Finally, based on the analysis of the repetitive units in these oyster species, we discuss the slow degree of concerted evolution in this interspersed repetitive DNA family and its use for phylogenetic analysis.

  18. Use of neuropathological tissue for molecular genetic studies: parameters affecting DNA extraction and polymerase chain reaction.

    Science.gov (United States)

    Kösel, S; Graeber, M B

    1994-01-01

    Nuclear and mitochondrial DNA were extracted from gray matter of human cerebral cortex which had either been formalin-fixed and embedded into paraffin or stored in formalin for up to 26 years. Extraction conditions were optimized for proteinase K digestion, i.e., enzyme concentration, digestion temperature and incubation time. Using the polymerase chain reaction (PCR), DNA was successfully amplified from archival material and sequenced employing a direct nonradioactive cycle sequencing protocol. In general, tissue embedded into paraffin following brief fixation in formalin gave good quantitative results, i.e., up to 1 microgram DNA/mg tissue were extracted. This yield was at least one order of magnitude higher than that obtained with tissue stored in formalin. However, paraffin-embedded neuropathological material was found to contain an as-yet-unidentified PCR inhibitor, and a deleterious effect of long-term fixation in unbuffered low-grade formalin was clearly detectable. Importantly, both paraffin-embedded tissue blocks and human brain that had been stored in formalin for many years yielded DNA sufficient for qualitative analysis. The implications of these findings for the use of neuropathological material in molecular genetic studies are discussed.

  19. The ABCs of molecular dynamics simulations on B-DNA, circa 2012

    Indian Academy of Sciences (India)

    David L Beveridge; Thomas E Cheatham III; Mihaly Mezei

    2012-07-01

    This article provides a retrospective on the ABC initiative in the area of all-atom molecular dynamics (MD) simulations including explicit solvent on all tetranucleotide steps of duplex B-form DNA duplex, ca. 2012. The ABC consortium has completed two phases of simulations, the most current being a set of 50–100 trajectories based on the AMBER ff99 force field together with the parmbsc0 modification. Some general perspectives on the field of MD on DNA and sequence effects on DNA structure are provided, followed by an overview our MD results, including a detailed comparison of the ff99/parmbsc0 results with crystal and NMR structures available for d(CGCGAATTCGCG). Some projects inspired by or related to the ABC initiative and database are also reviewed, including methods for the trajectory analyses, informatics of dealing with the large database of results, compressions of trajectories for efficacy of distribution, DNA solvation by water and ions, parameterization of coarse-grained models with applications and gene finding and genome annotation

  20. Molecular mechanism of immune response induced by foreign plasmid DNA after oral administration in mice

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    AIM: To study immune response induced by foreign plasmid DNA after oral administration in mice.METHODS: Mice were orally administered with 200 μg of plasmid pcDNA3 once and spleen was isolated 4 h and 18 h after administration. Total RNA was extracted from spleen and gene expression profile of BALB/c mice spleen was analyzed by using Affymetrix oligonucleotide GeneChip. Functional cluster analysis was conducted by GenMAPP software.RESULTS: At 4 h and 18 h after oral plasmid pcDNA3 administration, a number of immune-related genes,including cytokine and cytokine receptors, chemokines and chemokine receptor, complement molecule,proteasome, histocompatibility molecule, lymphocyte antigen complex and apoptotic genes, were up-regulated. Moreover, MAPPFinder results also showed that numerous immune response processes were up-regulated. In contrast, the immunoglobulin genes were down-regulated.CONCLUSION: Foreign plasmid DNA can modulate the genes expression related to immune system via the gastrointestinal tract, and further analysis of the related immune process may help understand the molecular mechanisms of immune response induced by foreign plasmid via the gastrointestinal tract.

  1. In Vitro Selection of a Single-Stranded DNA Molecular Recognition Element against Atrazine

    Science.gov (United States)

    Williams, Ryan M.; Crihfield, Cassandra L.; Gattu, Srikanth; Holland, Lisa A.; Sooter, Letha J.

    2014-01-01

    Widespread use of the chlorotriazine herbicide, atrazine, has led to serious environmental and human health consequences. Current methods of detecting atrazine contamination are neither rapid nor cost-effective. In this work, atrazine-specific single-stranded DNA (ssDNA) molecular recognition elements (MRE) were isolated. We utilized a stringent Systematic Evolution of Ligands by Exponential Enrichment (SELEX) methodology that placed the greatest emphasis on what the MRE should not bind to. After twelve rounds of SELEX, an atrazine-specific MRE with high affinity was obtained. The equilibrium dissociation constant (Kd) of the ssDNA sequence is 0.62 ± 0.21 nM. It also has significant selectivity for atrazine over atrazine metabolites and other pesticides found in environmentally similar locations and concentrations. Furthermore, we have detected environmentally relevant atrazine concentrations in river water using this MRE. The strong affinity and selectivity of the selected atrazine-specific ssDNA validated the stringent SELEX methodology and identified a MRE that will be useful for rapid atrazine detection in environmental samples. PMID:25196435

  2. Molecular evidence of apoptotic pathway activation in semen samples with high DNA fragmentation.

    Science.gov (United States)

    Manente, Lucrezia; Pecoraro, Stefano; Picillo, Esther; Gargiulo, Umberto; Gargiulo, Paolo; De Luca, Antonio; Politano, Luisa

    2015-01-01

    Male infertility is diagnosed by semen parameters, such as concentration, motility and morphology; however, these are not sufficient for the prediction of male fertility capacity. In the clinical routine, several other sperm functions have been introduced, including the sperm DNA fragmentation test. The objective of the present study was to evaluate sperm chromatin integrity in semen samples. Sperm chromatin dispersion test (SCD) was used in ejaculates from men divided into five groups: normozoospermic, oligozoospermic, asthenozoospermic, oligoasthenozoospermic and cryptozoospermic. The data obtained showed that the SCD percentage appeared to be significantly associated with oligozoospermia diagnosis. We also evaluated total testosterone, follicle-stimulating hormone (FSH), luteinizing hormone (LH) and inhibin B serum hormonal levels in all samples examined, in order to assess whether DNA fragmentation increase could correlate with abnormal hormonal values. Finally we selected certain samples with an increasing DNA fragmentation and analyzed the molecular activated apoptotic pathways. A significant relationship was found between caspase-3 activation and increased DNA fragmentation. Copyright © 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

  3. Mega primer-mediated molecular cloning strategy for chimaeragenesis and long DNA fragment insertion.

    Science.gov (United States)

    Zhang, Hui; Liu, Chang-Jun; Jiang, Hui; Zhou, Lu; Li, Wen-Ying; Zhu, Ling-Yun; Wu, Lei; Meng, Er; Zhang, Dong-Yi

    2017-04-30

    Molecular cloning methods based on primer and overlap-extension PCR are widely used due to their simplicity, reliability, low cost and high efficiency. In this article, an efficient mega primer-mediated (MP) cloning strategy for chimaeragenesis and long DNA fragment insertion is presented. MP cloning is a seamless, restriction/ligation-independent method that requires only three steps: (i) the first PCR for mega primer generation; (ii) the second PCR for exponential amplification mediated by the mega primers and (iii) DpnI digestion and transformation. Most importantly, for chimaeragenesis, genes can be assembled and constructed into the plasmid vector in a single PCR step. By employing this strategy, we successfully inserted four DNA fragments (approximately 500 bp each) into the same vector simultaneously. In conclusion, the strategy proved to be a simple and efficient tool for seamless cloning.

  4. Small-angle neutron scattering and Molecular Dynamics structural study of gelling DNA nanostars

    CERN Document Server

    Fernandez-Castanon, Javier; Rovigatti, Lorenzo; Zanatta, Marco; Paciaroni, Alessandro; Comez, Lucia; Porcar, Lionel; Jafta, Charl J; Fadda, Giulia C; Bellini, Tommaso; Sciortino, Francesco

    2016-01-01

    DNA oligomers with properly designed sequences self-assemble into well defined constructs. Here, we exploit this methodology to produce bulk quantities of tetravalent DNA nanostars (each one composed by 196 nucleotides) and to explore the structural signatures of their aggregation process. We report small-angle neutron scattering experiments focused on the evaluation of both the form factor and the temperature evolution of the scattered intensity at a nano star concentration where the system forms a tetravalent equilibrium gel. We also perform molecular dynamics simulations of one isolated tetramer to evaluate the form factor theoretically, without resorting to any approximate shape. The numerical form factor is found to be in very good agreement with the experimental one. Simulations predict an essentially temperature independent form factor, offering the possibility to extract the effective structure factor and its evolution during the equilibrium gelation.

  5. A novel reliable method of DNA extraction from olive oil suitable for molecular traceability.

    Science.gov (United States)

    Raieta, Katia; Muccillo, Livio; Colantuoni, Vittorio

    2015-04-01

    Extra virgin olive oil production has a worldwide economic impact. The use of this brand, however, is of great concern to Institutions and private industries because of the increasing number of fraud and adulteration attempts to the market products. Here, we present a novel, reliable and not expensive method for extracting the DNA from commercial virgin and extra virgin olive oils. The DNA is stable overtime and amenable for molecular analyses; in fact, by carrying out simple sequence repeats (SSRs) markers analysis, we characterise the genetic profile of monovarietal olive oils. By comparing the oil-derived pattern with that of the corresponding tree, we can unambiguously identify four cultivars from Samnium, a region of Southern Italy, and distinguish them from reference and more widely used varieties. Through a parentage statistical analysis, we also identify the putative pollinators, establishing an unprecedented and powerful tool for olive oil traceability. Copyright © 2014 Elsevier Ltd. All rights reserved.

  6. Small-angle neutron scattering and molecular dynamics structural study of gelling DNA nanostars

    Science.gov (United States)

    Fernandez-Castanon, J.; Bomboi, F.; Rovigatti, L.; Zanatta, M.; Paciaroni, A.; Comez, L.; Porcar, L.; Jafta, C. J.; Fadda, G. C.; Bellini, T.; Sciortino, F.

    2016-08-01

    DNA oligomers with properly designed sequences self-assemble into well defined constructs. Here, we exploit this methodology to produce bulk quantities of tetravalent DNA nanostars (each one composed of 196 nucleotides) and to explore the structural signatures of their aggregation process. We report small-angle neutron scattering experiments focused on the evaluation of both the form factor and the temperature evolution of the scattered intensity at a nanostar concentration where the system forms a tetravalent equilibrium gel. We also perform molecular dynamics simulations of one isolated tetramer to evaluate the form factor numerically, without resorting to any approximate shape. The numerical form factor is found to be in very good agreement with the experimental one. Simulations predict an essentially temperature-independent form factor, offering the possibility to extract the effective structure factor and its evolution during the equilibrium gelation.

  7. DNA microarray synthesis by using PDMS molecular stamps (Ⅲ)-- Optimization for the reaction conditions

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    Optimization for the technological processes of fabricating oligonucleotide microarray by the molecular stamping method is studied in this note. Three factors that affect the pressing coupling reactions of the nucleosides are focused on: the stability of the chemical activities of the reaction solutions, the contamination of the remain of the reactive nucleotides among the different spots on the chip, and the influence of the capping reaction on the hybridization result. The experiments show that the acetonitrile solution of tetrazole and nucleoside monomer could maintain sufficient reactive activity for more than 10 h. An effective method has been used and proved to eliminate the residual reactive nucleosides on chip with small molecules containing hydroxyl group. Finally, the capping step-- a regular step in the conventional DNA chemical synthesis can be neglected in our on-chip DNA synthetic process, which would not affect its hybridization results.

  8. Bond Orientational Order, Molecular Motion and Free Energy of High Density DNA Mesophases

    CERN Document Server

    Podgornik, R; Gawrisch, K; Rau, D C; Rupprecht, A; Parsegian, V A

    1995-01-01

    By equilibrating condensed DNA arrays against reservoirs of known osmotic stress and examining them with several structural probes, it has been possible to achieve a detailed thermodynamic and structural characterization of the change between two distinct regions on the liquid crystalline phase digram: a higher-density hexagonally packed region with long-range bond orientational order in the plane perpendicular to the average molecular direction; and a lower-density cholesteric region with fluid-like positional order. X-rays scattering on highly ordered DNA arrays at high density and with the helical axis oriented parallel to the incoming beam showed a six-fold azimuthal modulation of the first order diffraction peak that reflects the macroscopic bond-orientational order. Transition to the less-dense cholesteric phase through osmotically controlled swelling shows the loss of this bond orientational order that had been expected from the change in optical birefringence patterns and that is consistent with a rap...

  9. A molecular beacon-based DNA switch for reversible pH sensing in vesicles and live cells.

    Science.gov (United States)

    Narayanaswamy, Nagarjun; Nair, Raji R; Suseela, Y V; Saini, Deepak Kumar; Govindaraju, T

    2016-07-01

    In this Communication, a molecular beacon-based DNA switch (LMB) is developed as an efficient and reversible pH sensing probe. Remarkably, LMB exhibited reversible structural transition between the closed (molecular beacon) and open (A-motif) states very efficiently in synthetic vesicles and live cells without the need for any transfection agents.

  10. Molecular recognition of DNA by ligands: roughness and complexity of the free energy profile.

    Science.gov (United States)

    Zheng, Wenwei; Vargiu, Attilio Vittorio; Vargiu, Attlio Vittorio; Rohrdanz, Mary A; Carloni, Paolo; Clementi, Cecilia

    2013-10-14

    Understanding the molecular mechanism by which probes and chemotherapeutic agents bind to nucleic acids is a fundamental issue in modern drug design. From a computational perspective, valuable insights are gained by the estimation of free energy landscapes as a function of some collective variables (CVs), which are associated with the molecular recognition event. Unfortunately the choice of CVs is highly non-trivial because of DNA's high flexibility and the presence of multiple association-dissociation events at different locations and/or sliding within the grooves. Here we have applied a modified version of Locally-Scaled Diffusion Map (LSDMap), a nonlinear dimensionality reduction technique for decoupling multiple-timescale dynamics in macromolecular systems, to a metadynamics-based free energy landscape calculated using a set of intuitive CVs. We investigated the binding of the organic drug anthramycin to a DNA 14-mer duplex. By performing an extensive set of metadynamics simulations, we observed sliding of anthramycin along the full-length DNA minor groove, as well as several detachments from multiple sites, including the one identified by X-ray crystallography. As in the case of equilibrium processes, the LSDMap analysis is able to extract the most relevant collective motions, which are associated with the slow processes within the system, i.e., ligand diffusion along the minor groove and dissociation from it. Thus, LSDMap in combination with metadynamics (and possibly every equivalent method) emerges as a powerful method to describe the energetics of ligand binding to DNA without resorting to intuitive ad hoc reaction coordinates.

  11. Molecular interactions and residues involved in force generation in the T4 viral DNA packaging motor.

    Science.gov (United States)

    Migliori, Amy D; Smith, Douglas E; Arya, Gaurav

    2014-12-12

    Many viruses utilize molecular motors to package their genomes into preformed capsids. A striking feature of these motors is their ability to generate large forces to drive DNA translocation against entropic, electrostatic, and bending forces resisting DNA confinement. A model based on recently resolved structures of the bacteriophage T4 motor protein gp17 suggests that this motor generates large forces by undergoing a conformational change from an extended to a compact state. This transition is proposed to be driven by electrostatic interactions between complementarily charged residues across the interface between the N- and C-terminal domains of gp17. Here we use atomistic molecular dynamics simulations to investigate in detail the molecular interactions and residues involved in such a compaction transition of gp17. We find that although electrostatic interactions between charged residues contribute significantly to the overall free energy change of compaction, interactions mediated by the uncharged residues are equally if not more important. We identify five charged residues and six uncharged residues at the interface that play a dominant role in the compaction transition and also reveal salt bridging, van der Waals, and solvent hydrogen-bonding interactions mediated by these residues in stabilizing the compact form of gp17. The formation of a salt bridge between Glu309 and Arg494 is found to be particularly crucial, consistent with experiments showing complete abrogation in packaging upon Glu309Lys mutation. The computed contributions of several other residues are also found to correlate well with single-molecule measurements of impairments in DNA translocation activity caused by site-directed mutations.

  12. O6-methylguanine-DNA methyltransferase in equine sarcoids: molecular and epigenetic analysis

    Directory of Open Access Journals (Sweden)

    Altamura Gennaro

    2012-11-01

    Full Text Available Abstract Background Bovine papillomaviruses (BPVs types 1 and 2 are the only known papillomaviruses able to jump the species. In fact, BPVs 1/2 induce neoplasia in their natural bovine host but infection is also associated to neoplastic skin lesions in equids termed sarcoids. The equine sarcoid is considered to be the most common equine cutaneous tumour worldwide for which no effective therapy is available. Very little is known about the molecular mechanisms underlying tumourigenesis, although genes contributing to sarcoid development have been identified. Several studies associate the development of cancer to the loss of function of a number of oncosuppressor genes. In this study the putative role of O6-methylguanine-DNA methyltrasferase (MGMT was investigated for sarcoids. The expression of the oncosuppressor protein was assessed in normal and sarcoid cells and tissues. In addition, the DNA methylation profile was analysed to assess the role of epigenetic mechanism in regulation of MGMT expression. Results A group of 15 equine sarcoids and two primary sarcoid cell lines (fibroblasts were analyzed for the expression of MGMT protein by immunohistochemistry, immunofluorescence and Western blotting techniques. The sarcoid cell line EqSO4b and the tumour samples showed a reduction or absence of MGMT expression. To investigate the causes of deregulated MGMT expression, ten samples were analyzed for the DNA methylation profile of the CpG island associated to the MGMT promoter. The analysis of 73 CpGs encompassing the region of interest showed in 1 out of 10 (10% sarcoids a pronouncedly altered methylation profile when compared to the control epidermal sample. Similarily the EqSO4b cell line showed an altered MGMT methylation pattern in comparison to normal fibroblasts. Conclusion As previously demonstrated for the oncosuppressor gene FHIT, analysis of MGMT expression in sarcoid tissues and a sarcoid-derived fibroblast cell line further suggests that

  13. Improving molecular detection of Candida DNA in whole blood: comparison of seven fungal DNA extraction protocols using real-time PCR.

    Science.gov (United States)

    Metwally, L; Fairley, D J; Coyle, P V; Hay, R J; Hedderwick, S; McCloskey, B; O'Neill, H J; Webb, C H; Elbaz, W; McMullan, R

    2008-03-01

    The limitations of classical diagnostic methods for invasive Candida infections have led to the development of molecular techniques such as real-time PCR to improve diagnosis. However, the detection of low titres of Candida DNA in blood from patients with candidaemia requires the use of extraction methods that efficiently lyse yeast cells and recover small amounts of DNA suitable for amplification. In this study, a Candida-specific real-time PCR assay was used to detect Candida albicans DNA in inoculated whole blood specimens extracted using seven different extraction protocols. The yield and quality of total nucleic acids were estimated using UV absorbance, and specific recovery of C. albicans genomic DNA was estimated quantitatively in comparison with a reference (Qiagen kit/lyticase) method currently in use in our laboratory. The extraction protocols were also compared with respect to sensitivity, cost and time required for completion. The TaqMan PCR assay used to amplify the DNA extracts achieved high levels of specificity, sensitivity and reproducibility. Of the seven extraction protocols evaluated, only the MasterPure yeast DNA extraction reagent kit gave significantly higher total nucleic acid yields than the reference method, although nucleic acid purity was highest using either the reference or YeaStar genomic DNA kit methods. More importantly, the YeaStar method enabled C. albicans DNA to be detected with highest sensitivity over the entire range of copy numbers evaluated, and appears to be an optimal method for extracting Candida DNA from whole blood.

  14. CMOS time-resolved, contact, and multispectral fluorescence imaging for DNA molecular diagnostics.

    Science.gov (United States)

    Guo, Nan; Cheung, Kawai; Wong, Hiu Tong; Ho, Derek

    2014-10-31

    Instrumental limitations such as bulkiness and high cost prevent the fluorescence technique from becoming ubiquitous for point-of-care deoxyribonucleic acid (DNA) detection and other in-field molecular diagnostics applications. The complimentary metal-oxide-semiconductor (CMOS) technology, as benefited from process scaling, provides several advanced capabilities such as high integration density, high-resolution signal processing, and low power consumption, enabling sensitive, integrated, and low-cost fluorescence analytical platforms. In this paper, CMOS time-resolved, contact, and multispectral imaging are reviewed. Recently reported CMOS fluorescence analysis microsystem prototypes are surveyed to highlight the present state of the art.

  15. CMOS Time-Resolved, Contact, and Multispectral Fluorescence Imaging for DNA Molecular Diagnostics

    Directory of Open Access Journals (Sweden)

    Nan Guo

    2014-10-01

    Full Text Available Instrumental limitations such as bulkiness and high cost prevent the fluorescence technique from becoming ubiquitous for point-of-care deoxyribonucleic acid (DNA detection and other in-field molecular diagnostics applications. The complimentary metal-oxide-semiconductor (CMOS technology, as benefited from process scaling, provides several advanced capabilities such as high integration density, high-resolution signal processing, and low power consumption, enabling sensitive, integrated, and low-cost fluorescence analytical platforms. In this paper, CMOS time-resolved, contact, and multispectral imaging are reviewed. Recently reported CMOS fluorescence analysis microsystem prototypes are surveyed to highlight the present state of the art.

  16. Experimental and molecular docking studies on DNA binding interaction of adefovir dipivoxil: Advances toward treatment of hepatitis B virus infections

    Science.gov (United States)

    Shahabadi, Nahid; Falsafi, Monireh

    The toxic interaction of adefovir dipivoxil with calf thymus DNA (CT-DNA) was investigated in vitro under simulated physiological conditions by multi-spectroscopic techniques and molecular modeling study. The fluorescence spectroscopy and UV absorption spectroscopy indicated drug interacted with CT-DNA in a groove binding mode. The binding constant of UV-visible and the number of binding sites were 3.33 ± 0.2 × 104 L mol-1and 0.99, respectively. The fluorimetric studies showed that the reaction between the drug and CT-DNA is exothermic (ΔH = 34.4 kJ mol-1; ΔS = 184.32 J mol-1 K-1). Circular dichroism spectroscopy (CD) was employed to measure the conformational change of CT-DNA in the presence of adefovir dipivoxil, which verified the groove binding mode. Furthermore, the drug induces detectable changes in its viscosity. The molecular modeling results illustrated that adefovir strongly binds to groove of DNA by relative binding energy of docked structure -16.83 kJ mol-1. This combination of multiple spectroscopic techniques and molecular modeling methods can be widely used in the investigation on the toxic interaction of small molecular pollutants and drugs with bio macromolecules, which contributes to clarify the molecular mechanism of toxicity or side effect in vivo.

  17. Synthesis of novel anthraquinones: Molecular structure, molecular chemical reactivity descriptors and interactions with DNA as antibiotic and anti-cancer drugs

    Science.gov (United States)

    Al-Otaibi, Jamelah S.; EL Gogary, Tarek M.

    2017-02-01

    Anthraquinones are well-known anticancer drugs. Anthraquinones anticancer drugs carry out their cytotoxic activities through their interaction with DNA, and inhibition of topoisomerase II activity. Anthraquinones (AQ5 and AQ5H) were synthesized and studied with 1,5-DAAQ by computational and experimental tools. The purpose of this study is to shade more light on mechanism of interaction between anthraquinone DNA affinic agents and different types of DNA. This study will lead to gain of information useful for drug design and development. Molecular structures were optimized using DFT B3LYP/6-31 + G(d). Depending on intramolecular hydrogen bonding interactions four conformers of AQ5 were detected within the range of about 42 kcal/mol. Molecular reactivity of the anthraquinone compounds was explored using global and condensed descriptors (electrophilicity and Fukui functions). NMR and UV-VIS electronic absorption spectra of anthraquinones/DNA were investigated at the physiological pH. The interaction of the anthraquinones (AQ5 and AQ5H) were studied with different DNA namely, calf thymus DNA, (Poly[dA].Poly[dT]) and (Poly[dG].Poly[dC]). UV-VIS electronic absorption spectral data were employed to measure the affinity constants of drug/DNA binding using Scatchard analysis. NMR study confirms qualitatively the drug/DNA interaction in terms of peak shift and broadening.

  18. Therapeutic effect of DNA preparations varying in molecular mass on irradiated rats

    Energy Technology Data Exchange (ETDEWEB)

    Mushkacheva, G.S.; Turdakova, V.A.; Rusina, T.N.; Luzanova, O.V.; Rogacheva, S.A.

    1979-12-01

    Studies were made of the therapeutic effect of rat thymus and spleen DNA as a function of its polymerization in experiments on Wistar rats exposed to radiation in doses of 750, 820, 840 and 875 R. Base preparations with a mean molecular mass of 34.10/sup 6/ dalton, mechanically degraded to 14.5.10/sup 6/, 9.2.10/sup 6/, 5.8.10/sup 6/, dalton and, by means of ultrasound, to 0.9.10/sup 6/ dalton were used. Preparations with average polymerism, with molecular mass of 5.5.10/sup 6/ to 13.10/sup 6/ dalton, had a marked therapeutic effect, increasing survival of irradiated animals by 15 to 25%, as compared to the irradiated control.

  19. Structure-function studies of DNA damage using AB INITIO quantum mechanics and molecular dynamics simulation

    Energy Technology Data Exchange (ETDEWEB)

    Miller, J.; Miaskiewicz, K. [Pacific Northwest Lab., Richland, WA (United States); Osman, R. [Mount Sinai School of Medicine, New York, NY (United States). Dept. of Physiology and Biophysics

    1993-12-01

    Studies of ring-saturated pyrimidine base lesions are used to illustrate an integrated modeling approach that combines quantum-chemical calculations with molecular dynamics simulation. Electronic-structure calculations on the lesions in Isolation reveal strong conformational preferences due to interactions between equatorial substituents to the pyrimidine ring. Large distortions of DNA should result when these interactions force the methyl group of thymine to assume an axial orientation, as is the case for thymine glycol but not for dihydrothymine. Molecular dynamics simulations of the dodecamer d(CGCGAATTCGCG){sub 2} with and without a ring-saturated thymine lesion at position T7 support this conclusion. Implications of these studies for recognition of thymine lesions by endonuclease III are also discussed.

  20. Quantum Mechanics/Molecular Mechanics Free Energy Maps and Nonadiabatic Simulations for a Photochemical Reaction in DNA: Cyclobutane Thymine Dimer.

    Science.gov (United States)

    Mendieta-Moreno, Jesús I; Trabada, Daniel G; Mendieta, Jesús; Lewis, James P; Gómez-Puertas, Paulino; Ortega, José

    2016-11-03

    The absorption of ultraviolet radiation by DNA may result in harmful genetic lesions that affect DNA replication and transcription, ultimately causing mutations, cancer, and/or cell death. We analyze the most abundant photochemical reaction in DNA, the cyclobutane thymine dimer, using hybrid quantum mechanics/molecular mechanics (QM/MM) techniques and QM/MM nonadiabatic molecular dynamics. We find that, due to its double helix structure, DNA presents a free energy barrier between nonreactive and reactive conformations leading to the photolesion. Moreover, our nonadiabatic simulations show that most of the photoexcited reactive conformations return to standard B-DNA conformations after an ultrafast nonradiative decay to the ground state. This work highlights the importance of dynamical effects (free energy, excited-state dynamics) for the study of photochemical reactions in biological systems.

  1. Molecular insights into DNA interference by CRISPR-associated nuclease-helicase Cas3.

    Science.gov (United States)

    Gong, Bei; Shin, Minsang; Sun, Jiali; Jung, Che-Hun; Bolt, Edward L; van der Oost, John; Kim, Jeong-Sun

    2014-11-18

    Mobile genetic elements in bacteria are neutralized by a system based on clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) proteins. Type I CRISPR-Cas systems use a "Cascade" ribonucleoprotein complex to guide RNA specifically to complementary sequence in invader double-stranded DNA (dsDNA), a process called "interference." After target recognition by Cascade, formation of an R-loop triggers recruitment of a Cas3 nuclease-helicase, completing the interference process by destroying the invader dsDNA. To elucidate the molecular mechanism of CRISPR interference, we analyzed crystal structures of Cas3 from the bacterium Thermobaculum terrenum, with and without a bound ATP analog. The structures reveal a histidine-aspartate (HD)-type nuclease domain fused to superfamily-2 (SF2) helicase domains and a distinct C-terminal domain. Binding of ATP analog at the interface of the SF2 helicase RecA-like domains rearranges a motif V with implications for the enzyme mechanism. The HD-nucleolytic site contains two metal ions that are positioned at the end of a proposed nucleic acid-binding tunnel running through the SF2 helicase structure. This structural alignment suggests a mechanism for 3' to 5' nucleolytic processing of the displaced strand of invader DNA that is coordinated with ATP-dependent 3' to 5' translocation of Cas3 along DNA. In agreement with biochemical studies, the presented Cas3 structures reveal important mechanistic details on the neutralization of genetic invaders by type I CRISPR-Cas systems.

  2. Binding of PFOS to serum albumin and DNA: insight into the molecular toxicity of perfluorochemicals

    Directory of Open Access Journals (Sweden)

    Ma Yin-Sheng

    2009-02-01

    Full Text Available Abstract Background Health risk from exposure of perfluorochemicals (PFCs to wildlife and human has been a subject of great interest for understanding their molecular mechanism of toxicity. Although much work has been done, the toxigenicity of PFCs remains largely unknown. In this work, the non-covalent interactions between perfluorooctane sulfonate (PFOS and serum albumin (SA and DNA were investigated under normal physiological conditions, aiming to elucidate the toxigenicity of PFCs. Results In equilibrium dialysis assay, the bindings of PFOS to SA correspond to the Langmuir isothermal model with two-step sequence model. The saturation binding number of PFOS was 45 per molecule of SA and 1 per three base-pairs of DNA, respectively. ITC results showed that all the interactions were spontaneous driven by entropy change. Static quenching of the fluorescence of SA was observed when interacting with PFOS, indicating PFOS bound Trp residue of SA. CD spectra of SA and DNA changed obviously in the presence of PFOS. At normal physiological conditions, 1.2 mmol/l PFOS reduces the binding ratio of Vitamin B2 to SA by more than 30%. Conclusion The ion bond, van der Waals force and hydrophobic interaction contributed to PFOS binding to peptide chain of SA and to the groove bases of DNA duplex. The non-covalent interactions of PFOS with SA and DNA alter their secondary conformations, with the physiological function of SA to transport Vitamin B2 being inhibited consequently. This work provides a useful experimental method for further studying the toxigenicity of PFCs.

  3. Molecular analysis of DNA and construction of genomic libraries of Mycobacterium leprae.

    Science.gov (United States)

    Clark-Curtiss, J E; Jacobs, W R; Docherty, M A; Ritchie, L R; Curtiss, R

    1985-03-01

    Molecular analysis of DNA from Mycobacterium leprae, "Mycobacterium lufu," and Mycobacterium vaccae has demonstrated that the G + C (guanine plus cytosine) contents of the DNAs are 56, 61, and 65%, respectively, and that the genome sizes are 2.2 X 10(9), 3.1 X 10(9), and 3.1 X 10(9) daltons, respectively. Because of the significant differences in both G + C content and genome size among M. leprae, "M. lufu," and M. vaccae DNAs, these species are not related, although hybridization experiments under nonstringent conditions, with two separate cloned M. leprae DNA inserts as probes, indicate that there are some conserved sequences among the DNAs. The G + C content of Dasypus novemcinctus (armadillo, the animal of choice for cultivating M. leprae) DNA was determined to be 36%. Genomic libraries potentially representing more than 99.99% of each genome were prepared by cloning into the cosmid vector, pHC79, in Escherichia coli K-12. A genomic library representing approximately 95% of the genome of M. vaccae was prepared in pBR322. M. leprae DNA was subcloned from the pHC79::M. leprae library into an expression vector, pYA626. This vector is a 3.8-kilobase derivative of pBR322 in which the promoter region of the asd (aspartate semialdehyde dehydrogenase) gene from Streptococcus mutans has been inserted in place of the EcoRI-to-PstI fragment of pBR322. Several (44% of those tested) pYA626::M. leprae recombinants and one pBR322::M. vaccae recombinant synthesized new polypeptides in minicells of E. coli, indicating that mycobacterial DNA can be expressed in E. coli K-12, although expression is probably dependent upon use of nonmycobacterial promoters recognized by the E. coli transcription-translation apparatus.

  4. Design of Engineering Machinery Accessories of the Logistics Information System Based on Bar Code Technology%基于条形码的工程机械配件管理系统设计

    Institute of Scientific and Technical Information of China (English)

    江静岚; 谭桂芹

    2015-01-01

    Engineering machinery consists of thousands of spare parts and has a complicated list of accesso -ries.The characteristic of machinery manufacture is precision and just -in-time, which has strict acquirement in supply and distribution of accessories .Among the current management methods of accessories , many procedures still follow the traditional manual operating methods which are inefficient and easy to make mistakes .In order to es-tablish the standardized enterprise business processes of construction machinery and standardized basic data , and to enhance efficiency , the system, through the combination of the bar code technology and information processing technology , using the Thinkphp framework , which is based on the PHP language , as a system architecture , have designed and implemented a set of engineering machinery spare parts logistics information system based on barcode technology , ensuring the accuracy of the inventory , optimizing the punctuality of the storage management and deliv-ery of the inventory .And for the characteristic of uniqueness of the spare parts ’ code number , the retrospect and dynamic monitoring of every spare part is accomplished .%工程机械设备是由成千上万的配件组成,配件清单十分复杂,而机械制造生产的特点是精确化、准时化,对配件供应和配送要求十分严格。现行的配件管理方法中,许多步骤仍沿用传统的手工作业方式,效率低下,容易出错。为了更好地建立工程机械配件的标准化业务流程、标准化基础资料,提高效率,该系统通过条码技术与信息处理技术相结合,使用以PHP语言为基础的ThinkPHP框架作为系统架构,设计和实现了一套基于条码技术的机械配件物流信息系统,确保配件和产品库存数量的精确性,优化配件和产品在仓储管理和配送管理的准时性,并能利用配件条码编号的唯一性特点,实现每一个工程机械配件的追

  5. 应用UCC/EAN-128编码技术对转基因植物产品进行溯源研究%Tracing of genetically modified crops and their derived products by UCC/EAN-128 bar code

    Institute of Scientific and Technical Information of China (English)

    王醒宇; 杨捷琳; 陈勇; 潘良文; 丁卓平

    2013-01-01

    This paper gathered information from the five sections including the planting origin, products category, harvesting, processing and packaging stages. Based on the national standard and coding rules, the tracing of genetically modified crops by UCC/EAN-128 bar code about five sections was designed and encipher. As the example of soybeans, the five sections were combined to make the integrity the UCC/EAN bar code. The consumer can obtain the information and trace the products through scanning the UCC/EAN-128 bar code, combining with the data received and the data from the computer database.%该文对转基因植物产品从产地、产品、采收、加工、包装等5个环节收集信息,并根据国家标准中规定相应编码规则对这5个环节进行UCC/EAN-128码的设计与编码。最后,以大豆为例,将这5个编码结合,形成一个完整UCC/EAN-128码。消费者通过扫描条形码,获取相关数据,并将获得数据与计算机建立的数据库相结合,进行信息读取,了解转基因植物产品的生产、加工、包装等信息,从而对转基因植物产品进行有效的溯源。

  6. Cleavable DNA-protein hybrid molecular beacon: A novel efficient signal translator for sensitive fluorescence anisotropy bioassay.

    Science.gov (United States)

    Hu, Pan; Yang, Bin

    2016-01-15

    Due to its unique features such as high sensitivity, homogeneous format, and independence on fluorescent intensity, fluorescence anisotropy (FA) assay has become a hotspot of study in oligonucleotide-based bioassays. However, until now most FA probes require carefully customized structure designs, and thus are neither generalizable for different sensing systems nor effective to obtain sufficient signal response. To address this issue, a cleavable DNA-protein hybrid molecular beacon was successfully engineered for signal amplified FA bioassay, via combining the unique stable structure of molecular beacon and the large molecular mass of streptavidin. Compared with single DNA strand probe or conventional molecular beacon, the DNA-protein hybrid molecular beacon exhibited a much higher FA value, which was potential to obtain high signal-background ratio in sensing process. As proof-of-principle, this novel DNA-protein hybrid molecular beacon was further applied for FA bioassay using DNAzyme-Pb(2+) as a model sensing system. This FA assay approach could selectively detect as low as 0.5nM Pb(2+) in buffer solution, and also be successful for real samples analysis with good recovery values. Compatible with most of oligonucleotide probes' designs and enzyme-based signal amplification strategies, the molecular beacon can serve as a novel signal translator to expand the application prospect of FA technology in various bioassays.

  7. Molecular identification of Malaysian Chrysomya megacephala (Fabricius) and Chrysomya rufifacies (Macquart) using life stage specific mitochondrial DNA.

    Science.gov (United States)

    Kavitha, R; Tan, T C; Lee, H L; Nazni, W A; Sofian, A M

    2013-06-01

    DNA identification of blow fly species can be a very useful tool in forensic entomology. One of the potential benefits that mitochondrial DNA (mtDNA) has offered in the field of forensic entomology is species determination. Conventional identification methods have limitations for sibling and closely related species of blow fly and stage and quality of the specimen used. This could be overcome by DNA-based identification methods using mitochondrial DNA which does not demand intact or undamaged specimens. Mitochondrial DNA is usually isolated from whole blow fly and legs. Alternate sources for mitochondrial DNA isolation namely, egg, larva, puparium and empty puparium were explored in this study. The sequence of DNA obtained for each sample for every life cycle stage was 100% identical for a particular species, indicating that the egg, 1st instar, 2nd instar, 3rd instar, pupa, empty puparium and adult from the same species and obtained from same generation will exhibit similar DNA sequences. The present study also highlighted the usefulness of collecting all life cycle stages of blow fly during crime scene investigation with proper preservation and subsequent molecular analysis. Molecular identification provides a strong basis for species identification and will prove an invaluable contribution to forensic entomology as an investigative tool in Malaysia.

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

    Science.gov (United States)

    Guo, Yumei; Yue, Qinyan; Gao, Baoyu

    2011-07-01

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

  9. Biological chemistry as a foundation of DNA genealogy: the emergence of "molecular history".

    Science.gov (United States)

    Klyosov, A A

    2011-05-01

    foundation for "molecular history", in which the principal tool is high-technology analysis of DNA molecules of both our contemporaries and excavated ancient DNA samples, along with their biological kinetics.

  10. New molecular phenotypes in the dst mutants of Arabidopsis revealed by DNA microarray analysis.

    Science.gov (United States)

    Pérez-Amador, M A; Lidder, P; Johnson, M A; Landgraf, J; Wisman, E; Green, P J

    2001-12-01

    In this study, DNA microarray analysis was used to expand our understanding of the dst1 mutant of Arabidopsis. The dst (downstream) mutants were isolated originally as specifically increasing the steady state level and the half-life of DST-containing transcripts. As such, txhey offer a unique opportunity to study rapid sequence-specific mRNA decay pathways in eukaryotes. These mutants show a threefold to fourfold increase in mRNA abundance for two transgenes and an endogenous gene, all containing DST elements, when examined by RNA gel blot analysis; however, they show no visible aberrant phenotype. Here, we use DNA microarrays to identify genes with altered expression levels in dst1 compared with the parental plants. In addition to verifying the increase in the transgene mRNA levels, which were used to isolate these mutants, we were able to identify new genes with altered mRNA abundance in dst1. RNA gel blot analysis confirmed the microarray data for all genes tested and also was used to catalog the first molecular differences in gene expression between the dst1 and dst2 mutants. These differences revealed previously unknown molecular phenotypes for the dst mutants that will be helpful in future analyses. Cluster analysis of genes altered in dst1 revealed new coexpression patterns that prompt new hypotheses regarding the nature of the dst1 mutation and a possible role of the DST-mediated mRNA decay pathway in plants.

  11. Intermolecular hydrogen bonds: From temperature-driven proton transfer in molecular crystals to denaturation of DNA

    Indian Academy of Sciences (India)

    Mark Johnson

    2008-11-01

    We have combined neutron scattering and a range of numerical simulations to study hydrogen bonds in condensed matter. Two examples from a recent thesis will be presented. The first concerns proton transfer with increasing temperature in short inter-molecular hydrogen bonds [1,2]. These bonds have unique physical and chemical properties and are thought to play a fundamental role in processes like enzymatic catalysis. By combining elastic and inelastic neutron scattering results with ab initio, lattice dynamics and molecular dynamics simulations, low frequency lattice modes are identified which modulate the potential energy surface of the hydrogen bond proton and drive proton transfer. The second example concerns base-pair opening in DNA which is the fundamental physical process underlying biological processes like denaturation and transcription. We have used an emprical force field and a large scale, all-atom phonon calculation to gain insight into the base-pair opening modes and the apparent `energy gap' between the accepted frequencies for these modes (∼ 100 cm-1 or ∼ 140 K) and the temperature of the biological processes (room temperature to 100° C) [3]. Inelastic neutron scattering spectra on aligned, highly crystalline DNA samples, produced at the ILL, provide the reference data for evaluating the precision of these simulation results.

  12. Assessing the Value of DNA Barcodes and Other Priority Gene Regions for Molecular Phylogenetics of Lepidoptera

    Science.gov (United States)

    Wilson, John James

    2010-01-01

    Background Despite apparently abundant amounts of observable variation and species diversity, the order Lepidoptera exhibits a morphological homogeneity that has provided only a limited number of taxonomic characters and led to widespread use of nucleotides for inferring relationships. This study aims to characterize and develop methods to quantify the value of priority gene regions designated for Lepidoptera molecular systematics. In particular, I assess how the DNA barcode segment of the mitochondrial COI gene performs across a broad temporal range given its number one position of priority, most sequenced status, and the conflicting opinions on its phylogenetic performance. Methodology/Principal Findings Gene regions commonly sequenced for Lepidoptera phylogenetics were scored using multiple measures across three categories: practicality, which includes universality of primers and sequence quality; phylogenetic utility; and phylogenetic signal. I found that alternative measures within a category often appeared correlated, but high scores in one category did not necessarily translate into high scores in another. The DNA barcode was easier to sequence than other genes, and had high scores for utility but low signal above the genus level. Conclusions/Significance Given limited financial resources and time constraints, careful selection of gene regions for molecular phylogenetics is crucial to avoid wasted effort producing partially informative data. This study introduces an approach to assessing the value of gene regions prior to the initiation of new studies and presents empirical results to help guide future selections. PMID:20479871

  13. Assessing the value of DNA barcodes and other priority gene regions for molecular phylogenetics of Lepidoptera.

    Directory of Open Access Journals (Sweden)

    John James Wilson

    Full Text Available BACKGROUND: Despite apparently abundant amounts of observable variation and species diversity, the order Lepidoptera exhibits a morphological homogeneity that has provided only a limited number of taxonomic characters and led to widespread use of nucleotides for inferring relationships. This study aims to characterize and develop methods to quantify the value of priority gene regions designated for Lepidoptera molecular systematics. In particular, I assess how the DNA barcode segment of the mitochondrial COI gene performs across a broad temporal range given its number one position of priority, most sequenced status, and the conflicting opinions on its phylogenetic performance. METHODOLOGY/PRINCIPAL FINDINGS: Gene regions commonly sequenced for lepidoptera phylogenetics were scored using multiple measures across three categories: practicality, which includes universality of primers and sequence quality; phylogenetic utility; and phylogenetic signal. I found that alternative measures within a category often appeared correlated, but high scores in one category did not necessarily translate into high scores in another. The DNA barcode was easier to sequence than other genes, and had high scores for utility but low signal above the genus level. CONCLUSIONS/SIGNIFICANCE: Given limited financial resources and time constraints, careful selection of gene regions for molecular phylogenetics is crucial to avoid wasted effort producing partially informative data. This study introduces an approach to assessing the value of gene regions prior to the initiation of new studies and presents empirical results to help guide future selections.

  14. Structure and Mechanical Characterization of DNA i-Motif Nanowires by Molecular Dynamics Simulation

    Science.gov (United States)

    Singh, Raghvendra Pratap; Blossey, Ralf; Cleri, Fabrizio

    2013-01-01

    We studied the structure and mechanical properties of DNA i-motif nanowires by means of molecular dynamics computer simulations. We built up to 230 nm-long nanowires, based on a repeated TC5 sequence from crystallographic data, fully relaxed and equilibrated in water. The unusual C⋅C+ stacked structure, formed by four ssDNA strands arranged in an intercalated tetramer, is here fully characterized both statically and dynamically. By applying stretching, compression, and bending deformations with the steered molecular dynamics and umbrella sampling methods, we extract the apparent Young’s and bending moduli of the nanowire, as well as estimates for the tensile strength and persistence length. According to our results, the i-motif nanowire shares similarities with structural proteins, as far as its tensile stiffness, but is closer to nucleic acids and flexible proteins, as far as its bending rigidity is concerned. Furthermore, thanks to its very thin cross section, the apparent tensile toughness is close to that of a metal. Besides their yet to be clarified biological significance, i-motif nanowires may qualify as interesting candidates for nanotechnology templates, due to such outstanding mechanical properties. PMID:24359754

  15. Molecular Characterization of Sudanese and Southern Sudanese Chicken Breeds Using mtDNA D-Loop

    Directory of Open Access Journals (Sweden)

    Charles E. Wani

    2014-01-01

    Full Text Available The objective of this study was to assess the genetic relationships and diversity and to estimate the amount of gene flow among the five chicken populations from Sudan and South Sudan and commercial strain of egg line White Leghorn chickens. The chicken populations were genotyped using mtDNA D-loop as a molecular marker. PCR product of the mtDNA D-loop segment was 600 bp and 14 haplotypes were identified. The neighbor-joining phylogenetic tree indicated that the indigenous Sudanese chickens can be grouped into two clades, IV and IIIa only. Median joining networks analysis showed that haplotype LBB49 has the highest frequency. The hierarchal analysis of molecular variance (AMOVA showed that genetic variation within the population was 88.6% and the differentiation among the population was 11.4%. When the populations was redefined into two geographical zones, rich and poor Savanna, the results were fractioned into three genetic variations: between individuals within population 95.5%, between populations within the group 0.75%, and genetic variation between groups 3.75%. The pair wise Fst showed high genetic difference between Betwil populations and the rest with Fst ranging from 0.1492 to 0.2447. We found that there is large number of gene exchanges within the Sudanese indigenous chicken (Nm=4.622.

  16. Molecular Renormalization Group Coarse-Graining of Polymer Chains: Application to Double-Stranded DNA

    Science.gov (United States)

    Savelyev, Alexey; Papoian, Garegin A.

    2009-01-01

    Coarse-graining of atomistic force fields allows us to investigate complex biological problems, occurring at long timescales and large length scales. In this work, we have developed an accurate coarse-grained model for double-stranded DNA chain, derived systematically from atomistic simulations. Our approach is based on matching correlators obtained from atomistic and coarse-grained simulations, for observables that explicitly enter the coarse-grained Hamiltonian. We show that this requirement leads to equivalency of the corresponding partition functions, resulting in a one-step renormalization. Compared to prior works exploiting similar ideas, the main novelty of this work is the introduction of a highly compact set of Hamiltonian basis functions, based on molecular interaction potentials. We demonstrate that such compactification allows us to reproduce many-body effects, generated by one-step renormalization, at low computational cost. In addition, compact Hamiltonians greatly increase the likelihood of finding unique solutions for the coarse-grained force-field parameter values. By successfully applying our molecular renormalization group coarse-graining technique to double-stranded DNA, we solved, for the first time, a long-standing problem in coarse-graining polymer systems, namely, how to accurately capture the correlations among various polymeric degrees of freedom. Excellent agreement is found among atomistic and coarse-grained distribution functions for various structural observables, including those not included in the Hamiltonian. We also suggest higher-order generalization of this method, which may allow capturing more subtle correlations in biopolymer dynamics. PMID:19450476

  17. Molecular organization of the 5S rDNA gene type II in elasmobranchs.

    Science.gov (United States)

    Castro, Sergio I; Hleap, Jose S; Cárdenas, Heiber; Blouin, Christian

    2016-01-01

    The 5S rDNA gene is a non-coding RNA that can be found in 2 copies (type I and type II) in bony and cartilaginous fish. Previous studies have pointed out that type II gene is a paralog derived from type I. We analyzed the molecular organization of 5S rDNA type II in elasmobranchs. Although the structure of the 5S rDNA is supposed to be highly conserved, our results show that the secondary structure in this group possesses some variability and is different than the consensus secondary structure. One of these differences in Selachii is an internal loop at nucleotides 7 and 112. These mutations observed in the transcribed region suggest an independent origin of the gene among Batoids and Selachii. All promoters were highly conserved with the exception of BoxA, possibly due to its affinity to polymerase III. This latter enzyme recognizes a dT4 sequence as stop signal, however in Rajiformes this signal was doubled in length to dT8. This could be an adaptation toward a higher efficiency in the termination process. Our results suggest that there is no TATA box in elasmobranchs in the NTS region. We also provide some evidence suggesting that the complexity of the microsatellites present in the NTS region play an important role in the 5S rRNA gene since it is significantly correlated with the length of the NTS.

  18. Atomistic details of the molecular recognition of DNA-RNA hybrid duplex by ribonuclease H enzyme

    Indian Academy of Sciences (India)

    Gorle Suresh; U Deva Priyakumar

    2015-10-01

    Bacillus halodurans (ℎ) ribonuclease H (RNase H) belongs to the nucleotidyl-transferase (NT) superfamily and is a prototypical member of a large family of enzymes that use two-metal ion (Mg2+ or Mn2+) catalysis to cleave nucleic acids. Long timescale molecular dynamics simulations have been performed on the ℎRNase H-DNA-RNA hybrid complex and the respective monomers to understand the recognition mechanism, conformational preorganization, active site dynamics and energetics involved in the complex formation. Several structural and energetic analyses were performed and significant structural changes are observed in enzyme and hybrid duplex during complex formation. Hybrid molecule binding to RNase H enzyme leads to conformational changes in the DNA strand. The ability of the DNA strand in the hybrid duplex to sample conformations corresponding to typical A- and B-type nucleic acids and the characteristic minor groove width-seem to be crucial for efficient binding. Sugar moieties in certain positions interacting with the protein structure undergo notable conformational transitions. The water coordination and arrangement around the metal ions in active site region are quite stable, suggesting their important role in enzymatic catalysis. Details of key interactions found at the interface of enzyme-nucleic acid complex that are responsible for its stability are discussed.

  19. Complete nuclear ribosomal DNA sequence amplification and molecular analyses of Bangia (Bangiales, Rhodophyta) from China

    Science.gov (United States)

    Xu, Jiajie; Jiang, Bo; Chai, Sanming; He, Yuan; Zhu, Jianyi; Shen, Zonggen; Shen, Songdong

    2016-09-01

    Filamentous Bangia, which are distributed extensively throughout the world, have simple and similar morphological characteristics. Scientists can classify these organisms using molecular markers in combination with morphology. We successfully sequenced the complete nuclear ribosomal DNA, approximately 13 kb in length, from a marine Bangia population. We further analyzed the small subunit ribosomal DNA gene (nrSSU) and the internal transcribed spacer (ITS) sequence regions along with nine other marine, and two freshwater Bangia samples from China. Pairwise distances of the nrSSU and 5.8S ribosomal DNA gene sequences show the marine samples grouping together with low divergences (00.003; 0-0.006, respectively) from each other, but high divergences (0.123-0.126; 0.198, respectively) from freshwater samples. An exception is the marine sample collected from Weihai, which shows high divergence from both other marine samples (0.063-0.065; 0.129, respectively) and the freshwater samples (0.097; 0.120, respectively). A maximum likelihood phylogenetic tree based on a combined SSU-ITS dataset with maximum likelihood method shows the samples divided into three clades, with the two marine sample clades containing Bangia spp. from North America, Europe, Asia, and Australia; and one freshwater clade, containing Bangia atropurpurea from North America and China.

  20. Molecular Design of Ionization-Induced Proton Switching Element Based on Fluorinated DNA Base Pair.

    Science.gov (United States)

    Tachikawa, Hiroto; Kawabata, Hiroshi

    2016-03-10

    To design theoretically the high-performance proton switching element based on DNA base pair, the effects of fluorine substitution on the rate of proton transfer (PT) in the DNA model base pair have been investigated by means of direct ab initio molecular dynamics (AIMD) method. The 2-aminopyridine dimer, (AP)2, was used as the model of the DNA base pair. One of the hydrogen atoms of the AP molecule in the dimer was substituted by a fluorine (F) atom, and the structures of the dimer, expressed by F-(AP)2, were fully optimized at the MP2/6-311++G(d,p) level. The direct AIMD calculations showed that the proton is transferred within the base pair after the vertical ionization. The rates of PT in F-(AP)2(+) were calculated and compared with that of (AP)2(+) without an F atom. It was found that PT rate is accelerated by the F-substitution. Also, the direction of PT between F-AP and AP molecules can be clearly controlled by the position of F-substitution (AP)2 in the dimer.

  1. Molecular characterization and physical localization of highly repetitive DNA sequences from Brazilian Alstroemeria species.

    Science.gov (United States)

    Kuipers, A G J; Kamstra, S A; de Jeu, M J; Visser, R G F

    2002-01-01

    Highly repetitive DNA sequences were isolated from genomic DNA libraries of Alstroemeria psittacina and A. inodora. Among the repetitive sequences that were isolated, tandem repeats as well as dispersed repeats could be discerned. The tandem repeats belonged to a family of interlinked Sau3A subfragments with sizes varying from 68-127 bp, and constituted a larger HinfI repeat of approximately 400 bp. Southern hybridization showed a similar molecular organization of the tandem repeats in each of the Brazilian Alstroemeria species tested. None of the repeats hybridized with DNA from Chilean Alstroemeria species, which indicates that they are specific for the Brazilian species. In-situ localization studies revealed the tandem repeats to be localized in clusters on the chromosomes of A. inodora and A. psittacina: distal hybridization sites were found on chromosome arms 2PS, 6PL, 7PS, 7PL and 8PL, interstitial sites on chromosome arms 2PL, 3PL, 4PL and 5PL. The applicability of the tandem repeats for cytogenetic analysis of interspecific hybrids and their role in heterochromatin organization are discussed.

  2. A Quick-responsive DNA Nanotechnology Device for Bio-molecular Homeostasis Regulation.

    Science.gov (United States)

    Wu, Songlin; Wang, Pei; Xiao, Chen; Li, Zheng; Yang, Bing; Fu, Jieyang; Chen, Jing; Wan, Neng; Ma, Cong; Li, Maoteng; Yang, Xiangliang; Zhan, Yi

    2016-08-10

    Physiological processes such as metabolism, cell apoptosis and immune responses, must be strictly regulated to maintain their homeostasis and achieve their normal physiological functions. The speed with which bio-molecular homeostatic regulation occurs directly determines the ability of an organism to adapt to conditional changes. To produce a quick-responsive regulatory system that can be easily utilized for various types of homeostasis, a device called nano-fingers that facilitates the regulation of physiological processes was constructed using DNA origami nanotechnology. This nano-fingers device functioned in linked open and closed phases using two types of DNA tweezers, which were covalently coupled with aptamers that captured specific molecules when the tweezer arms were sufficiently close. Via this specific interaction mechanism, certain physiological processes could be simultaneously regulated from two directions by capturing one biofactor and releasing the other to enhance the regulatory capacity of the device. To validate the universal application of this device, regulation of the homeostasis of the blood coagulant thrombin was attempted using the nano-fingers device. It was successfully demonstrated that this nano-fingers device achieved coagulation buffering upon the input of fuel DNA. This nano-device could also be utilized to regulate the homeostasis of other types of bio-molecules.

  3. Susceptibility to DNA damage as a molecular mechanism for non-syndromic cleft lip and palate.

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    Gerson Shigeru Kobayashi

    Full Text Available Non-syndromic cleft lip/palate (NSCL/P is a complex, frequent congenital malformation, determined by the interplay between genetic and environmental factors during embryonic development. Previous findings have appointed an aetiological overlap between NSCL/P and cancer, and alterations in similar biological pathways may underpin both conditions. Here, using a combination of transcriptomic profiling and functional approaches, we report that NSCL/P dental pulp stem cells exhibit dysregulation of a co-expressed gene network mainly associated with DNA double-strand break repair and cell cycle control (p = 2.88×10(-2-5.02×10(-9. This network included important genes for these cellular processes, such as BRCA1, RAD51, and MSH2, which are predicted to be regulated by transcription factor E2F1. Functional assays support these findings, revealing that NSCL/P cells accumulate DNA double-strand breaks upon exposure to H2O2. Furthermore, we show that E2f1, Brca1 and Rad51 are co-expressed in the developing embryonic orofacial primordia, and may act as a molecular hub playing a role in lip and palate morphogenesis. In conclusion, we show for the first time that cellular defences against DNA damage may take part in determining the susceptibility to NSCL/P. These results are in accordance with the hypothesis of aetiological overlap between this malformation and cancer, and suggest a new pathogenic mechanism for the disease.

  4. Molecular sled is an eleven-amino acid vehicle facilitating biochemical interactions via sliding components along DNA.

    Science.gov (United States)

    Mangel, Walter F; McGrath, William J; Xiong, Kan; Graziano, Vito; Blainey, Paul C

    2016-02-02

    Recently, we showed the adenovirus proteinase interacts productively with its protein substrates in vitro and in vivo in nascent virus particles via one-dimensional diffusion along the viral DNA. The mechanism by which this occurs has heretofore been unknown. We show sliding of these proteins along DNA occurs on a new vehicle in molecular biology, a 'molecular sled' named pVIc. This 11-amino acid viral peptide binds to DNA independent of sequence. pVIc slides on DNA, exhibiting the fastest one-dimensional diffusion constant, 26±1.8 × 10(6) (bp)(2) s(-1). pVIc is a 'molecular sled,' because it can slide heterologous cargos along DNA, for example, a streptavidin tetramer. Similar peptides, for example, from the C terminus of β-actin or NLSIII of the p53 protein, slide along DNA. Characteristics of the 'molecular sled' in its milieu (virion, nucleus) have implications for how proteins in the nucleus of cells interact and imply a new form of biochemistry, one-dimensional biochemistry.

  5. Taxonomy-free molecular diatom index for high-throughput eDNA biomonitoring.

    Science.gov (United States)

    Apothéloz-Perret-Gentil, Laure; Cordonier, Arielle; Straub, François; Iseli, Jennifer; Esling, Philippe; Pawlowski, Jan

    2017-03-14

    Current biodiversity assessment and biomonitoring are largely based on the morphological identification of selected bioindicator taxa. Recently, several attempts have been made to use eDNA metabarcoding as an alternative tool. However, until now, most applied metabarcoding studies have been based on the taxonomic assignment of sequences that provides reference to morphospecies ecology. Usually, only a small portion of metabarcoding data can be used due to a limited reference database and a lack of phylogenetic resolution. Here, we investigate the possibility to overcome these limitations using a taxonomy-free approach that allows the computing of a molecular index directly from eDNA data without any reference to morphotaxonomy. As a case study, we use the benthic diatoms index, commonly used for monitoring the biological quality of rivers and streams. We analysed 87 epilithic samples from Swiss rivers, the ecological status of which was established based on the microscopic identification of diatom species. We compared the diatom index derived from eDNA data obtained with or without taxonomic assignment. Our taxonomy-free approach yields promising results by providing a correct assessment for 77% of examined sites. The main advantage of this method is that almost 95% of OTUs could be used for index calculation, compared to 35% in the case of the taxonomic assignment approach. Its main limitations are under-sampling and the need to calibrate the index based on the microscopic assessment of diatoms communities. However, once calibrated, the taxonomy-free molecular index can be easily standardized and applied in routine biomonitoring, as a complementary tool allowing fast and cost-effective assessment of the biological quality of watercourses.

  6. Fluorescent dye labeled DNA size standards for molecular mass detection in visible/infrared range

    Directory of Open Access Journals (Sweden)

    Sreelakshmi Yellamaraju

    2011-01-01

    Full Text Available Abstract Background Targeting Induced Local Lesions in Genomes (TILLING is a high throughput reverse genetics tool which detects mismatches (single point mutations or small indels in large number of individuals of mutagenized populations. Currently, TILLING is intensively used for genomics assisted molecular breeding of several crop plants for desired traits. Most commonly used platform for mutation detection is Li-COR DNA Analyzer, where PCR amplified products treated with single strand mismatch specific nuclease are resolved on denaturing gels. The molecular size of any cut product can be easily estimated by comparing with IR dye labeled markers of known sizes. Similar fluorescent dye labeled size markers are also used for several genotyping experiments. Currently, commercially available size standards are expensive and are restricted up to only 700 bp which renders estimation of products of sizes greater than 700 bases inaccurate. Findings A simple protocol was developed for labeling 5' end of multiple DNA size markers with fluorescent dyes. This method involves cloning a pool of different size markers of DNA in a plasmid vector. PCR amplification of plasmid using IR dye labeled universal primers generates 5' fluorescent labeled products of various sizes. The size of products constituting the ladder can be customized as per the need. The generated size markers can be used without any further purification and were found to be stable up to one year at -20°C. Conclusions A simple method was developed for generating fluorescent dye labeled size standards. This method can be customized to generate different size standards as per experimental needs. The protocol described can also be adapted for developing labeled size standards for detection on platforms other than Li-COR i.e. other than infra red range of the spectrum.

  7. Direct and precise length measurement of single, stretched DNA fragments by dynamic molecular combing and STED nanoscopy.

    Science.gov (United States)

    Kim, Namdoo; Kim, Hyung Jun; Kim, Younggyu; Min, Kyung Suk; Kim, Seong Keun

    2016-09-01

    A combination of DNA stretching method and super-resolution nanoscopy allows an accurate and precise measurement of the length of DNA fragments ranging widely in size from 117 to 23,130 bp. BstEII- and HindIII-treated λDNA fragments were stained with an intercalating dye and then linearly stretched on a coverslip by dynamic molecular combing. The image of individual DNA fragments was obtained by stimulated emission depletion nanoscopy. For DNA fragments longer than ∼1000 bp, the measured lengths of DNA fragments were consistently within ∼0.5 to 1.0 % of the reference values, raising the possibility of this method in a wide range of applications including facile detection for copy number variations and trinucleotide repeat disorder.

  8. Molecular recognition of genomic DNA in a condensate with a model surfactant for potential gene-delivery applications.

    Science.gov (United States)

    Singh, Priya; Choudhury, Susobhan; Chandra, Goutam Kumar; Lemmens, Peter; Pal, Samir Kumar

    2016-04-01

    The functionality of a gene carrying nucleic acid in an artificial gene-delivery system is important for the overall efficiency of the vehicle in vivo. Here, we have studied a well-known artificial gene-delivery system, which is a condensate of calf thymus DNA (CT-DNA) with a model cationic surfactant cetyltrimethylammonium bromide (CTAB) to investigate the molecular recognition of the genomic DNA in the condensate. While dynamic light scattering (DLS) and circular dichroism (CD) reveal structural aspects of the condensate and the constituting DNA respectively, picosecond resolved polarization gated spectroscopy and Förster resonance energy transfer (FRET) reveal molecular recognition of the genomic DNA in the condensate. We have considered ethidium bromide (EB) and crystal violet (CV), which are well known DNA-binding agents through intercalative (specific) and electrostatic (non-specific) interactions, respectively, as model ligands for the molecular recognition studies. A fluorescent cationic surfactant, Nonyl Acridine Orange (NAO) is considered to be a mimic of CTAB in the condensate. The polarization gated fluorescence of NAO at various temperatures has been used to investigate the local microviscosity of the condensate. The excellent spectral overlap of NAO emission and the absorption spectra of both EB and CV allow us to investigate FRET-distances of the ligands with respect to NAO in the condensate at various temperatures and thermal stability of ligand-binding of the genomic DNA. The thermodynamic properties of the molecular recognition have also been explored using Van't Hoff equation. We have also extended our studies to molecular recognition of the genomic DNA in the condensate as dried thin films. This has important implications for its application in bioelectronics.

  9. Molecular phylogeny and barcoding of Caulerpa (Bryopsidales based on the tufA, rbcL, 18S rDNA and ITS rDNA genes.

    Directory of Open Access Journals (Sweden)

    Mudassar Anisoddin Kazi

    Full Text Available The biodiversity assessment of different taxa of the genus Caulerpa is of interest from the context of morphological plasticity, invasive potential of some species and biotechnological and pharmacological applications. The present study investigated the identification and molecular phylogeny of different species of Caulerpa occurring along the Indian coast inferred from tufA, rbcL, 18S rDNA and ITS rDNA nucleotide sequences. Molecular data confirmed the identification of 10 distinct Caulerpa species: C. veravalensis, C. verticillata, C. racemosa, C. microphysa, C. taxifolia, C. sertularioides, C. scalpelliformis, C. serrulata, C. peltata and C. mexicana. All datasets significantly supported the sister relationship between C. veravalensis and C. racemosa var. cylindracea. It was also concluded from the results that the specimen identified previously as C. microphysa and C. lentillifera could not be considered as separate species. The molecular data revealed the presence of multiple lineages for C. racemosa which can be resolved into separate species. All four markers were used to ascertain their utility for DNA barcoding. The tufA gene proved a better marker with monophyletic association as the main criteria for identification at the species level. The results also support the use of 18S rDNA insertion sequences to delineate the Caulerpa species through character-based barcoding. The ITS rDNA (5.8S-ITS2 phylogenetic analysis also served as another supporting tool. Further, more sequences from additional Caulerpa specimens will need to be analysed in order to support the role of these two markers (ITS rDNA and 18S insertion sequence in identification of Caulerpa species. The present study revealed the phylogeny of Caulerpa as complete as possible using the currently available data, which is the first comprehensive report illustrating the molecular phylogeny and barcoding of the genus Caulerpa from Indian waters.

  10. Molecular phylogeny and barcoding of Caulerpa (Bryopsidales) based on the tufA, rbcL, 18S rDNA and ITS rDNA genes.

    Science.gov (United States)

    Kazi, Mudassar Anisoddin; Reddy, C R K; Jha, Bhavanath

    2013-01-01

    The biodiversity assessment of different taxa of the genus Caulerpa is of interest from the context of morphological plasticity, invasive potential of some species and biotechnological and pharmacological applications. The present study investigated the identification and molecular phylogeny of different species of Caulerpa occurring along the Indian coast inferred from tufA, rbcL, 18S rDNA and ITS rDNA nucleotide sequences. Molecular data confirmed the identification of 10 distinct Caulerpa species: C. veravalensis, C. verticillata, C. racemosa, C. microphysa, C. taxifolia, C. sertularioides, C. scalpelliformis, C. serrulata, C. peltata and C. mexicana. All datasets significantly supported the sister relationship between C. veravalensis and C. racemosa var. cylindracea. It was also concluded from the results that the specimen identified previously as C. microphysa and C. lentillifera could not be considered as separate species. The molecular data revealed the presence of multiple lineages for C. racemosa which can be resolved into separate species. All four markers were used to ascertain their utility for DNA barcoding. The tufA gene proved a better marker with monophyletic association as the main criteria for identification at the species level. The results also support the use of 18S rDNA insertion sequences to delineate the Caulerpa species through character-based barcoding. The ITS rDNA (5.8S-ITS2) phylogenetic analysis also served as another supporting tool. Further, more sequences from additional Caulerpa specimens will need to be analysed in order to support the role of these two markers (ITS rDNA and 18S insertion sequence) in identification of Caulerpa species. The present study revealed the phylogeny of Caulerpa as complete as possible using the currently available data, which is the first comprehensive report illustrating the molecular phylogeny and barcoding of the genus Caulerpa from Indian waters.

  11. Comparison of Different DNA-Based Methods for Molecular Typing of Histoplasma capsulatum▿

    Science.gov (United States)

    Muniz, Mauro de Medeiros; Morais e Silva Tavares, Patrícia; Meyer, Wieland; Nosanchuk, Joshua Daniel; Zancope-Oliveira, Rosely Maria

    2010-01-01

    Histoplasma capsulatum is very prevalent in the environment and is one of the most common causes of mycoses in humans and diverse animals in Brazil. Multiple typing methods have been developed to study H. capsulatum epidemiology; however, there is limited information concerning comparisons of results obtained with different methods using the same set of isolates. To explore the diversity of H. capsulatum in Brazil and to determine correlations between the results of three different molecular typing techniques, we examined 51 environmental, animal, and human isolates by M13 PCR fingerprinting, PCR-restriction fragment length polymorphism (RFLP) analysis of the internal transcribed region 1 (ITS1)-5.8S-ITS2 region of the rDNA locus, and DNA sequencing and phylogenetic analysis of parts of four protein-encoding genes, the Arf (ADP ribosylation factor), H-anti (H antigen precursor), Ole (delta-9 fatty acid desaturase), and Tub1 (alpha-tubulin) genes. Each method identified three major genetic clusters, and there was a high level of concordance between the results of the typing techniques. The M13 PCR fingerprinting and PCR-RFLP analyses produced very similar results and separated the H. capsulatum isolates included in this study into three major groups. An additional approach used was comparison of our Brazilian ITS1-5.8S-ITS2 sequences with the sequences deposited previously in NCBI data banks. Our analyses suggest that H. capsulatum can be divided into different molecular types that are dispersed around the world. Our results indicate that the three methods used in this study are reliable and reproducible and that they have similar sensitivities. However, M13 PCR fingerprinting has some advantages over the other two methods as it is faster, cheaper, and more user friendly, which especially increases its utility for molecular typing of Histoplasma in situations where laboratory facilities are relatively limited. PMID:20453140

  12. Direct detection of circulating free DNA extracted from serum samples of breast cancer using locked nucleic acid molecular beacon.

    Science.gov (United States)

    Gui, Zhen; Wang, Quanbo; Li, Jinchang; Zhu, Mingchen; Yu, Lili; Xun, Tang; Yan, Feng; Ju, Huangxian

    2016-07-01

    As an emerging noninvasive blood biomarker, circulating free DNA (cfDNA) can be utilized to assess diagnosis, progression and evaluate prognosis of cancer. However, cfDNAs are not "naked", they can be part of complexes, or are bound to the surface of the cells via proteins, which make the detection more challenging. Here, a simple method for the detection of Ubiquitin-like with PHD and ring finger domains 1 (UHRF1) DNA exacted from serum of breast cancer (BC) has been developed using a novel locked nucleic acid molecular beacon (LNA-MB). In order to enhance the stability and detection efficiency of the probe in biofluids, we design a shared-stem molecular beacon containing a 27-mer loop and a 4-mer stem with DNA/LNA alternating bases. The fluorescence is released in the presence of target. The detection procedure is simple and can be completed within 1h. This method shows a sensitive response to UHRF1 DNA with a dynamic range of 3 orders of magnitude. The limit of detection is 11nM (S/N=3) with excellent selectivity. It can discriminate UHRF1 DNA from three-base mismatched DNA with a high specificity. More importantly, this method can distinguish the expression of serum UHRF1 DNA among 5 breast cancer patients and 5 healthy controls. The mentioned superiority may suggest that this assay can be served as a promising noninvasive detection tool for early BC diagnosis and monitoring.

  13. Molecular Diagnosis of α⁰-Thalassemia Through Urine DNA: A Novel DNA Source to Facilitate Prevention Programs in Remote Geographical Areas.

    Science.gov (United States)

    Suwannakhon, Narutchala; Seeratanachot, Teerapat; Mahingsa, Khwanruedee; Sanguansermsri, Torpong

    2015-01-01

    We assessed whether urinary DNA sediment was a feasible sample type for the molecular diagnosis of α-thalassemia (α-thal) mutations. Urine samples (5-10 mL) were collected from 218 male and female volunteers. The cells were centrifuged, and DNA was isolated according to the protocol of a commercial DNA isolation kit. Detection of the α(0)-thal [Southeast Asian (- -(SEA)) and - -(THAI)] deletions was performed using quantitative real-time polymerase chain reaction (q-PCR), in addition to conventional gap-PCR. The results revealed that DNA extracted from urinary sediment presented an average DNA content of 11.2 ± 5.5 ng/µL, and the 260/280 ratio indicative of DNA purity, was 1.2 ± 0.2. The overall q-PCR threshold cycle was 31.2 ± 2.3. The melting temperature for the - -(SEA) deletion was 87.3 ± 0.1 °C, while that of the wild type sequence was 92.5 ± 0.2 °C. There were 16 (7.3%) α(0)-thal SEA genotypes detected. These results were in agreement with those of the conventional gap-PCR and blood DNA analyses. Thus, DNA from urinary sediment can be efficiently used for the molecular diagnosis of α(0)-thal mutations. This approach allows for rapid diagnosis, is non invasive, and could be useful for preventing Hb Bart's (γ4) hydrops fetalis syndrome.

  14. Deciphering the interactions between chlorambucil and calf thymus DNA: a multi-spectroscopic and molecular docking study.

    Science.gov (United States)

    Rehman, Sayeed Ur; Sarwar, Tarique; Ishqi, Hassan Mubarak; Husain, Mohammed Amir; Hasan, Ziaul; Tabish, Mohammad

    2015-01-15

    Non-covalent interactions of chlorambucil with calf thymus DNA was investigated using multi-spectroscopic techniques and molecular docking study. Binding constant calculated was found to be 1.54×10(4)M(-1) at 290K, significantly lower than various known intercalators. Quenching process was found to be static as evident by biomolecular quenching constant. Thermodynamic parameters revealed the involvement of hydrophobic interactions and hydrogen bonds in the binding. Chlorambucil was found to interact via external binding mode and follow groove binding as it replaces Hoechst (a typical groove binder) from the groove of DNA but does not replace intercalating dyes including ethidium bromide and acridine orange from the DNA helix. These results were further supported by KI quenching experiments, DNA melting studies, CD spectroscopy and molecular docking. Copyright © 2014 Elsevier Inc. All rights reserved.

  15. Study on the interaction of the drug mesalamine with calf thymus DNA using molecular docking and spectroscopic techniques.

    Science.gov (United States)

    Shahabadi, Nahid; Fili, Soraya Moradi; Kheirdoosh, Fahimeh

    2013-11-05

    The interaction of CT-DNA with the drug mesalamine (5-ASA) at physiological pH has been investigated by absorption, emission, circular dichroism (CD), cyclic voltammetry (CV), viscosity studies and molecular modeling. Thermodynamic parameters (ΔH>0 and ΔS<0) indicated that hydrogen bond and van der Waals play main roles in the binding of 5-ASA to CT-DNA. Ethidium bromide (EB) displacement studies revealed that 5-ASA did not have any effect on ethidium bromide (EB) bound DNA which is indicative of groove binding. The results obtained from experimental and molecular modeling showed that 5-ASA is a minor groove binder of DNA and preferentially binds to GC rich regions.

  16. Use of molecular beacons for the rapid analysis of DNA damage induced by exposure to an atmospheric pressure plasma jet

    Energy Technology Data Exchange (ETDEWEB)

    Kurita, Hirofumi, E-mail: kurita@ens.tut.ac.jp, E-mail: mizuno@ens.tut.ac.jp; Miyachika, Saki; Yasuda, Hachiro; Takashima, Kazunori; Mizuno, Akira, E-mail: kurita@ens.tut.ac.jp, E-mail: mizuno@ens.tut.ac.jp [Department of Environmental and Life Sciences, Toyohashi University of Technology, Aichi 441-8580 (Japan)

    2015-12-28

    A rapid method for evaluating the damage caused to DNA molecules upon exposure to plasma is demonstrated. Here, we propose the use of a molecular beacon for rapid detection of DNA strand breaks induced by atmospheric pressure plasma jet (APPJ) irradiation. Scission of the molecular beacon by APPJ irradiation leads to separation of the fluorophore-quencher pair, resulting in an increase in fluorescence that directly correlates with the DNA strand breaks. The results show that the increase in fluorescence intensity is proportional to the exposure time and the rate of fluorescence increase is proportional to the discharge power. This simple and rapid method allows the estimation of DNA damage induced by exposure to a non-thermal plasma.

  17. Use of molecular beacons for the rapid analysis of DNA damage induced by exposure to an atmospheric pressure plasma jet

    Science.gov (United States)

    Kurita, Hirofumi; Miyachika, Saki; Yasuda, Hachiro; Takashima, Kazunori; Mizuno, Akira

    2015-12-01

    A rapid method for evaluating the damage caused to DNA molecules upon exposure to plasma is demonstrated. Here, we propose the use of a molecular beacon for rapid detection of DNA strand breaks induced by atmospheric pressure plasma jet (APPJ) irradiation. Scission of the molecular beacon by APPJ irradiation leads to separation of the fluorophore-quencher pair, resulting in an increase in fluorescence that directly correlates with the DNA strand breaks. The results show that the increase in fluorescence intensity is proportional to the exposure time and the rate of fluorescence increase is proportional to the discharge power. This simple and rapid method allows the estimation of DNA damage induced by exposure to a non-thermal plasma.

  18. Nuclear rDNA-based molecular clock of the evolution of triatominae (Hemiptera: Reduviidae, vectors of Chagas disease

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    Bargues MD

    2000-01-01

    Full Text Available The evolutionary history and times of divergence of triatomine bug lineages are estimated from molecular clocks inferred from nucleotide sequences of the small subunit SSU (18S and the second internal transcribed spacer (ITS-2 of the nuclear ribosomal DNA of these reduviids. The 18S rDNA molecular clock rate in Triatominae, and Prosorrhynchan Hemiptera in general, appears to be of 1.8% per 100 million years (my. The ITS-2 molecular clock rate in Triatominae is estimated to be around 0.4-1% per 1 my, indicating that ITS-2 evolves 23-55 times faster than 18S rDNA. Inferred chronological data about the evolution of Triatominae fit well with current hypotheses on their evolutionary histories, but suggest reconsideration of the current taxonomy of North American species complexes.

  19. Effect of swift-ion irradiation on DNA molecules: A molecular dynamics study using the REAX force field

    Energy Technology Data Exchange (ETDEWEB)

    Bottländer, Dominik [Dept. of Mechanical, Aerospace, and Biomedical Engineering, Knoxville, TN 37996-2210 (United States); Mücksch, Christian [Physics Department and Research Center OPTIMAS, University Kaiserslautern, Erwin-Schrödinger-Straße, D-67663 Kaiserslautern (Germany); Urbassek, Herbert M., E-mail: urbassek@rhrk.uni-kl.de [Physics Department and Research Center OPTIMAS, University Kaiserslautern, Erwin-Schrödinger-Straße, D-67663 Kaiserslautern (Germany)

    2015-12-15

    Modern REAX potentials allow to use molecular dynamics simulation to study bond breaking and reformation in biomolecules. We use this technique to simulate the effects of a swift-ion track on a B-DNA fragment in water. We monitor the number of single- and double-strand breaks as a function of the deposited energy. In addition we compare the results of direct DNA heating with the effect of hydrolysis which we model by heating only the water environment.

  20. Evaluation of the Gibbs Free Energy Changes and Melting Temperatures of DNA/DNA Duplexes Using Hybridization Enthalpy Calculated by Molecular Dynamics Simulation.

    Science.gov (United States)

    Lomzov, Alexander A; Vorobjev, Yury N; Pyshnyi, Dmitrii V

    2015-12-10

    A molecular dynamics simulation approach was applied for the prediction of the thermal stability of oligonucleotide duplexes. It was shown that the enthalpy of the DNA/DNA complex formation could be calculated using this approach. We have studied the influence of various simulation parameters on the secondary structure and the hybridization enthalpy value of Dickerson-Drew dodecamer. The optimal simulation parameters for the most reliable prediction of the enthalpy values were determined. The thermodynamic parameters (enthalpy and entropy changes) of a duplex formation were obtained experimentally for 305 oligonucleotides of various lengths and GC-content. The resulting database was studied with molecular dynamics (MD) simulation using the optimized simulation parameters. Gibbs free energy changes and the melting temperatures were evaluated using the experimental correlation between enthalpy and entropy changes of the duplex formation and the enthalpy values calculated by the MD simulation. The average errors in the predictions of enthalpy, the Gibbs free energy change, and the melting temperature of oligonucleotide complexes were 11%, 10%, and 4.4 °C, respectively. We have shown that the molecular dynamics simulation gives a possibility to calculate the thermal stability of native DNA/DNA complexes a priori with an unexpectedly high accuracy.

  1. Experimental and molecular modeling studies on the DNA-binding of diazacyclam-based acrocyclic copper complex.

    Science.gov (United States)

    Shahabadi, Nahid; Hakimi, Mohammad; Morovati, Teimoor; Falsafi, Monireh; Fili, Soraya Moradi

    2017-02-01

    The interaction of a new macrocyclic copper complex, [CuL(NO3)2] in which L is 1,3,6,10,12,15-hexaaza tricyclo[13.3.1.1(6,10)] eicosane was investigated in vitro under simulated physiological conditions by multi-spectroscopic techniques and molecular modeling study. The fluorescence spectroscopy and UV absorption spectroscopy indicated the complex interacted with ct-DNA in a groove binding mode while the binding constant of UV-vis and the number of binding sites were 1.0±0.2×10(4)Lmol(-1) and 1.01, respectively. The fluorometric studies showed that the reaction between the complex with ct-DNA is exothermic (ΔH=14.85kJmol(-1); ΔS=109.54Jmol(-1)K(-1)). Circular dichroism spectroscopy (CD) was employed to measure the conformational change of DNA in the presence of [CuL(NO3)2] complex. Furthermore, the complex induces detectable changes in the viscosity of DNA. The molecular modeling results illustrated that the complex strongly binds to groove of DNA. Experimental and molecular modeling results showed that Cu(II) complex bound to DNA by a groove binding mode.

  2. Molecular systematics of the Phyllachorales (ascomycota, fungi based on 18S ribosomal DNA sequences

    Directory of Open Access Journals (Sweden)

    Wanderlei-Silva Denise

    2003-01-01

    Full Text Available In order to evaluate the monophyly of the Phyllachorales from a molecular standpoint and elucidate its phylogenetic relationships with other orders, a segment of the 18S rRNA gene from several representatives of the Phyllachorales, including species of Glomerella, Phyllachora, Coccodiella (=Coccostroma, Sphaerodothis, Ophiodothella, as well as Magnaporthe was sequenced. Maximum Parsimony analysis revealed that the Phyllachorales was a polyphyletic assemblage of taxa. None of the other members of the Phyllachorales, which produced either a clypeus or stroma, clustered with Glomerella. Of the taxa examined, was Coccodiella the closest relative of Phyllachora. Magnaporthe was closely related to the Diaporthales. Our 18S rDNA data highly supported Glomerella being accommodated in a separate family.

  3. Exploring Programmable Self-Assembly in Non-DNA based Molecular Computing

    CERN Document Server

    Terrazas, German; Krasnogor, Natalio

    2013-01-01

    Self-assembly is a phenomenon observed in nature at all scales where autonomous entities build complex structures, without external influences nor centralised master plan. Modelling such entities and programming correct interactions among them is crucial for controlling the manufacture of desired complex structures at the molecular and supramolecular scale. This work focuses on a programmability model for non DNA-based molecules and complex behaviour analysis of their self-assembled conformations. In particular, we look into modelling, programming and simulation of porphyrin molecules self-assembly and apply Kolgomorov complexity-based techniques to classify and assess simulation results in terms of information content. The analysis focuses on phase transition, clustering, variability and parameter discovery which as a whole pave the way to the notion of complex systems programmability.

  4. Molecular detection of bacterial pathogens using microparticle enhanced double-stranded DNA probes.

    Science.gov (United States)

    Riahi, Reza; Mach, Kathleen E; Mohan, Ruchika; Liao, Joseph C; Wong, Pak Kin

    2011-08-15

    Rapid, specific, and sensitive detection of bacterial pathogens is essential toward clinical management of infectious diseases. Traditional approaches for pathogen detection, however, often require time-intensive bacterial culture and amplification procedures. Herein, a microparticle enhanced double-stranded DNA probe is demonstrated for rapid species-specific detection of bacterial 16S rRNA. In this molecular assay, the binding of the target sequence to the fluorophore conjugated probe thermodynamically displaces the quencher probe and allows the fluorophore to fluoresce. By incorporation of streptavidin-coated microparticles to localize the biotinylated probes, the sensitivity of the assay can be improved by 3 orders of magnitude. The limit of detection of the assay is as few as eight bacteria without target amplification and is highly specific against other common pathogens. Its applicability toward clinical diagnostics is demonstrated by directly identifying bacterial pathogens in urine samples from patients with urinary tract infections.

  5. PCR amplification of 16S rDNA from lyophilized cell cultures facilitates studies in molecular systematics

    Science.gov (United States)

    Wisotzkey, J. D.; Jurtshuk, P. Jr; Fox, G. E.

    1990-01-01

    The sequence of the major portion of a Bacillus cycloheptanicus strain SCH(T) 16S rRNA gene is reported. This sequence suggests that B. cycloheptanicus is genetically quite distinct from traditional Bacillus strains (e.g., B. subtilis) and may be properly regarded as belonging to a different genus. The sequence was determined from DNA that was produced by direct amplification of ribosomal DNA from a lyophilized cell pellet with straightforward polymerase chain reaction (PCR) procedures. By obviating the need to revive cell cultures from the lyophile pellet, this approach facilitates rapid 16S rDNA sequencing and thereby advances studies in molecular systematics.

  6. Decondensation behavior of DNA chains induced by multivalent cations at high salt concentrations:Molecular dynamics simulations and experiments

    Institute of Scientific and Technical Information of China (English)

    蒋杨伟; 冉诗勇; 何林李; 王向红; 章林溪

    2015-01-01

    Using molecular dynamics simulations and atomic force microscopy (AFM), we study the decondensation process of DNA chains induced by multivalent cations at high salt concentrations in the presence of short cationic chains in solutions. The typical simulation conformations of DNA chains with varying salt concentrations for multivalent cations imply that the concentration of salt cations and the valence of multivalent cations have a strong influence on the process of DNA decondensation. The DNA chains are condensed in the absence of salt or at low salt concentrations, and the compacted conformations of DNA chains become loose when a number of cations and anions are added into the solution. It is explicitly demonstrated that cations can overcompensate the bare charge of the DNA chains and weaken the attraction interactions between the DNA chains and short cationic chains at high salt concentrations. The condensation-decondensation transi-tions of DNA are also experimentally observed in mixing spermidine withλ-phage DNA at different concentrations of NaCl/MgCl2 solutions.

  7. 常见线粒体DNA病的分子遗传学研究进展%Molecular genetics of common mitochondrial DNA disorders

    Institute of Scientific and Technical Information of China (English)

    Lee-Jun C. WONG

    2005-01-01

    SUMMARY Diagnosis of mitochondrial disorders has been difficult due to the clinical and genetic heterogeneity, as well as unique features of mitochondrial genetics. Definitive diagnosis requires the identification of molecular defects in either the mitochondrial or the nuclear genome. We describe the clinical and molecular characteristic of some common mitochondrial syndromes and molecular methodologies available for the detection of mitochondrial DNA mutations. This review provides overview of current molecular diagnosis of mitochondrial DNA disorders that is useful in patient care and genetic counseling.

  8. Molecular phylogeny of black flies (Diptera: Simuliidae) from Thailand, using ITS2 rDNA.

    Science.gov (United States)

    Thanwisai, Aunchalee; Kuvangkadilok, Chaliow; Baimai, Visut

    2006-01-01

    The sequences of the second internal transcribed spacer (ITS2) of ribosomal DNA (rDNA) were determined for 40 black fly species from Thailand, belonging to 4 subgenera of the genus Simulium, namely Gomphostilbia (12 species), Nevermannia (5 species), Montisimulium (1 species), Simulium sensu stricto (21 species), and an unknown subgenus with one species (Simulium baimaii). The length of the ITS2 ranged from 247 to 308 bp. All black fly species had high AT content, ranging from 71 to 83.8%. Intraindividual variation (clonal variation) occurred in 13 species, ranging from 0.3 to 1.1%. Large intrapopulation and interpopulation heterogeneities exist in S. feuerboni from the same and different locations in Doi Inthanon National Park, northern Thailand. Phylogenetic relationships among 40 black fly species were examined using PAUP (version 4.0b10) and MrBAYS (version 3.0B4). The topology of the trees revealed two major monophyletic clades. The subgenus Simulium and Simulium baimaii were placed in the first monophyletic clade, whereas the subgenera Nevermannia + Montisimulium were placed as the sister group to the subgenus Gomphostilbia in the second monophyletic clade. Our results suggest that S. baimaii belongs to the malyschevi-group or variegatum-group in the subgenus Simulium. The molecular phylogeny generally agrees with existing morphology-based phylogenies.

  9. Population genetics and molecular evolution of DNA sequences in transposable elements. I. A simulation framework.

    Science.gov (United States)

    Kijima, T E; Innan, Hideki

    2013-11-01

    A population genetic simulation framework is developed to understand the behavior and molecular evolution of DNA sequences of transposable elements. Our model incorporates random transposition and excision of transposable element (TE) copies, two modes of selection against TEs, and degeneration of transpositional activity by point mutations. We first investigated the relationships between the behavior of the copy number of TEs and these parameters. Our results show that when selection is weak, the genome can maintain a relatively large number of TEs, but most of them are less active. In contrast, with strong selection, the genome can maintain only a limited number of TEs but the proportion of active copies is large. In such a case, there could be substantial fluctuations of the copy number over generations. We also explored how DNA sequences of TEs evolve through the simulations. In general, active copies form clusters around the original sequence, while less active copies have long branches specific to themselves, exhibiting a star-shaped phylogeny. It is demonstrated that the phylogeny of TE sequences could be informative to understand the dynamics of TE evolution.

  10. The crystal structure of sulfamethoxazole, interaction with DNA, DFT calculation, and molecular docking studies

    Science.gov (United States)

    Das, Dipankar; Sahu, Nilima; Roy, Suman; Dutta, Paramita; Mondal, Sudipa; Torres, Elena L.; Sinha, Chittaranjan

    2015-02-01

    Sulfamethoxazole (SMX) [4-amino-N-(5-methyl-1,2-oxazol-3-yl)benzenesulfonamide] is structurally established by single crystal X-ray diffraction measurement. The crystal packing shows H-bonded 2D polymer through N(7)sbnd H(7A)---O(2), N(7)sbnd H(7B)---O(3), N(1)sbnd H(1)---N(2), C(5)sbnd H(5)---O(3)sbnd S(1) and N(7)sbnd (H7A)---O(2)sbnd S(1). Density Functional Theory (DFT) and Time Dependent-DFT (TD-DFT) computations of optimized structure of SMX determine the electronic structure and has explained the electronic spectral transitions. The interaction of SMX with CT-DNA has been studied by absorption spectroscopy and the binding constant (Kb) is 4.37 × 104 M-1. The in silico test of SMX with DHPS from Escherichia coli and Streptococcus pneumoniae helps to understand drug metabolism and accounts the drug-molecule interactions. The molecular docking of SMX-DNA also helps to predict the interaction feature.

  11. Molecular cloning and characterization of a cDNA encoding the Paracoccidioides brasiliensis 135 ribosomal protein.

    Science.gov (United States)

    Jesuino, Rosália S A; Pereira, Maristela; Felipe, M Sueli S; Azevedo, Maristella O; Soares, Célia M A

    2004-06-01

    A 630 bp cDNA encoding an L35 ribosomal protein of Paracoccidioides brasiliensis, designated as Pbl35, was cloned from a yeast expression library. Pbl35 encodes a polypeptide of 125 amino acids, with a predicted molecular mass of 14.5 kDa and a pI of 11.0. The deduced PbL35 shows significant conservation in respect to other described ribosomal L35 proteins from eukaryotes and prokaryotes. Motifs of ribosomal proteins are present in PbL35, including a bipartite nuclear localization signal (NLS) that could be related to the protein addressing to the nucleolus for the ribosomal assembly. The mRNA for PbL35, about 700 nucleotides in length, is expressed at a high level in P. brasiliensis. The PbL35 and the deduced amino acid sequence constitute the first description of a ribosomal protein in P. brasiliensis. The cDNA was deposited in GenBank under accession number AF416509.

  12. The crystal structure of sulfamethoxazole, interaction with DNA, DFT calculation, and molecular docking studies.

    Science.gov (United States)

    Das, Dipankar; Sahu, Nilima; Roy, Suman; Dutta, Paramita; Mondal, Sudipa; Torres, Elena L; Sinha, Chittaranjan

    2015-02-25

    Sulfamethoxazole (SMX) [4-amino-N-(5-methyl-1,2-oxazol-3-yl)benzenesulfonamide] is structurally established by single crystal X-ray diffraction measurement. The crystal packing shows H-bonded 2D polymer through N(7)-H(7A)-O(2), N(7)-H(7B)-O(3), N(1)-H(1)-N(2), C(5)-H(5)-O(3)-S(1) and N(7)-(H7A)-O(2)-S(1). Density Functional Theory (DFT) and Time Dependent-DFT (TD-DFT) computations of optimized structure of SMX determine the electronic structure and has explained the electronic spectral transitions. The interaction of SMX with CT-DNA has been studied by absorption spectroscopy and the binding constant (Kb) is 4.37×10(4)M(-1). The in silico test of SMX with DHPS from Escherichia coli and Streptococcus pneumoniae helps to understand drug metabolism and accounts the drug-molecule interactions. The molecular docking of SMX-DNA also helps to predict the interaction feature.

  13. Detection of endometrial cancer via molecular analysis of DNA collected with vaginal tampons.

    Science.gov (United States)

    Bakkum-Gamez, Jamie N; Wentzensen, Nicolas; Maurer, Matthew J; Hawthorne, Kieran M; Voss, Jesse S; Kroneman, Trynda N; Famuyide, Abimbola O; Clayton, Amy C; Halling, Kevin C; Kerr, Sarah E; Cliby, William A; Dowdy, Sean C; Kipp, Benjamin R; Mariani, Andrea; Oberg, Ann L; Podratz, Karl C; Shridhar, Viji; Sherman, Mark E

    2015-04-01

    We demonstrate the feasibility of detecting EC by combining minimally-invasive specimen collection techniques with sensitive molecular testing. Prior to hysterectomy for EC or benign indications, women collected vaginal pool samples with intravaginal tampons and underwent endometrial brushing. Specimens underwent pyrosequencing for DNA methylation of genes reported to be hypermethylated in gynecologic cancers and recently identified markers discovered by profiling over 200 ECs. Methylation was evaluated individually across CpGs and averaged across genes. Differences between EC and benign endometrium (BE) were assessed using two-sample t-tests and area under the curve (AUC). Thirty-eight ECs and 28 BEs were included. We evaluated 97 CpGs within 12 genes, including previously reported markers (RASSF1, HSP2A, HOXA9, CDH13, HAAO, and GTF2A1) and those identified in discovery work (ASCL2, HTR1B, NPY, HS3ST2, MME, ADCYAP1, and additional CDH13 CpG sites). Mean methylation was higher in tampon specimens from EC v. BE for 9 of 12 genes (ADCYAP1, ASCL2, CDH13, HS3ST2, HTR1B, MME, HAAO, HOXA9, and RASSF1) (all pvaginal pool DNA collected via intravaginal tampon. Identification of additional EC biomarkers and refined collection methods are needed to develop an early detection tool for EC. Copyright © 2015 Elsevier Inc. All rights reserved.

  14. Emerging Molecular and Biological Functions of MBD2, a Reader of DNA Methylation

    Directory of Open Access Journals (Sweden)

    Kathleen H Wood

    2016-05-01

    Full Text Available DNA methylation is an epigenetic mark that is essential for many biological processes and is linked to diseases such as cancer. Methylation is usually associated with transcriptional silencing, but new research has challenged this model. Both transcriptional activation and repression have recently been found to be associated with DNA methylation in a context-specific manner. How DNA methylation patterns are interpreted into different functional output remains poorly understood. One mechanism involves the protein ‘readers’ of methylation, which includes the methyl-CpG binding domain (MBD family of proteins. This review examines the molecular and biological functions of MBD2, which binds to CpG methylation and is an integral part of the nucleosome remodeling and histone deacetylation (NuRD complex. MBD2 has been linked to immune system function and tumorigenesis, yet little is known about its functions in vivo. Recent studies have found the MBD2 protein is ubiquitously expressed, with relatively high levels in the lung, liver and colon. Mbd2 null mice surprisingly show relatively mild phenotypes compared to mice with loss of function of other MBD proteins. This evidence has previously been interpreted as functional redundancy between the MBD proteins. Here we examine and contextualize research that suggests MBD2 has unique properties and functions among the MBD proteins. These functions translate to recently described roles in the development and differentiation of multiple cell lineages, including pluripotent stem cells and various cell types of the immune system, as well as in tumorigenesis. We also consider possible models for the dynamic interactions between MBD2 and NuRD in different tissues in vivo. The functions of MBD2 may have direct therapeutic implications for several areas of human disease, including autoimmune conditions and cancer, in addition to providing insights into the actions of NuRD and chromatin regulation.

  15. Molecular modeling on the recognition of DNA sequence and conformational repair of sheared DNA by novel chiral metal complex D, L-[Co(phen)2hpip]3+

    Institute of Scientific and Technical Information of China (English)

    WU; Yanbo; ZHANG; Cuiping

    2006-01-01

    A study on the recognition of DNA sequence and conformational repair of sheared DNA by Novel Chiral Metal complex D,L-[Co(phen)2hpip]3+ (phen=1,10 phenanthroline, hpip=2-[2-hydroxyphenyl] imidazole [4,5-f][1,10] phenanthroline) is carried out with molecular simulations. The results reveal that two isomers of the complex could both recognize the normal DNA in the minor groove orientation, while recognize the sheared DNA in the major groove orientation and both isomers could convert the conformation of mismatched bases from sheared form to parallel form. Further analysis shows that the steric details of complex's intercalation to base stack determine the results of recognition, which is induced by the steric collision among ancillary ligand phen, bases and DNA backbone, and by the steric crowding occurring in the process of structural expansion of bases and DNA backbone. Detailed analysis reveals that the conformational repair of mismatched bases relates not only to the steric interactions, but also to the π-π stack among normal bases, mismatched bases and hpip ligand.

  16. New insight into the molecular mechanisms of the biological effects of DNA minor groove binders.

    Directory of Open Access Journals (Sweden)

    Xinbo Zhang

    Full Text Available BACKGROUND: Bisbenzimides, or Hoechst 33258 (H258, and its derivative Hoechst 33342 (H342 are archetypal molecules for designing minor groove binders, and widely used as tools for staining DNA and analyzing side population cells. They are supravital DNA minor groove binders with AT selectivity. H342 and H258 share similar biological effects based on the similarity of their chemical structures, but also have their unique biological effects. For example, H342, but not H258, is a potent apoptotic inducer and both H342 and H258 can induce transgene overexpression in in vitro studies. However, the molecular mechanisms by which Hoechst dyes induce apoptosis and enhance transgene overexpression are unclear. METHODOLOGY/PRINCIPAL FINDINGS: To determine the molecular mechanisms underlying different biological effects between H342 and H258, microarray technique coupled with bioinformatics analyses and multiple other techniques has been utilized to detect differential global gene expression profiles, Hoechst dye-specific gene expression signatures, and changes in cell morphology and levels of apoptosis-associated proteins in malignant mesothelioma cells. H342-induced apoptosis occurs in a dose-dependent fashion and is associated with morphological changes, caspase-3 activation, cytochrome c mitochondrial translocation, and cleavage of apoptosis-associated proteins. The antagonistic effect of H258 on H342-induced apoptosis indicates a pharmacokinetic basis for the two dyes' different biological effects. Differential global gene expression profiles induced by H258 and H342 are accompanied by unique gene expression signatures determined by DNA microarray and bioinformatics software, indicating a genetic basis for their different biological effects. CONCLUSIONS/SIGNIFICANCE: A unique gene expression signature associated with H342-induced apoptosis provides a new avenue to predict and classify the therapeutic class of minor groove binders in the drug

  17. In vitro DNA binding, pBR322 plasmid cleavage and molecular modeling study of chiral benzothiazole Schiff-base-valine Cu(II) and Zn(II) complexes to evaluate their enantiomeric biological disposition for molecular target DNA.

    Science.gov (United States)

    Alizadeh, Rahman; Afzal, Mohd; Arjmand, Farukh

    2014-10-15

    Bicyclic heterocyclic compounds viz. benzothiazoles are key components of deoxyribonucleic acid (DNA) molecules and participate directly in the encoding of genetic information. Benzothiazoles, therefore, represent a potent and selective class of antitumor compounds. The design and synthesis of chiral antitumor chemotherapeutic agents of Cu(II) and Zn(II), L- and -D benzothiazole Schiff base-valine complexes 1a &b and 2a &b, respectively were carried out and thoroughly characterized by spectroscopic and analytical techniques. Interaction of 1a and b and 2a and b with CT DNA by employing UV-vis, florescence, circular dichroic methods and cleavage studies of 1a with pBR322 plasmid, molecular docking were done in order to demonstrate their enantiomeric disposition toward the molecular drug target DNA. Interestingly, these studies unambiguously demonstrated the greater potency of L-enantiomer in comparison to D-enantiomer.

  18. In vitro DNA binding, pBR322 plasmid cleavage and molecular modeling study of chiral benzothiazole Schiff-base-valine Cu(II) and Zn(II) complexes to evaluate their enantiomeric biological disposition for molecular target DNA

    Science.gov (United States)

    Alizadeh, Rahman; Afzal, Mohd; Arjmand, Farukh

    2014-10-01

    Bicyclic heterocyclic compounds viz. benzothiazoles are key components of deoxyribonucleic acid (DNA) molecules and participate directly in the encoding of genetic information. Benzothiazoles, therefore, represent a potent and selective class of antitumor compounds. The design and synthesis of chiral antitumor chemotherapeutic agents of Cu(II) and Zn(II), L- and -D benzothiazole Schiff base-valine complexes 1a &b and 2a &b, respectively were carried out and thoroughly characterized by spectroscopic and analytical techniques. Interaction of 1a and b and 2a and b with CT DNA by employing UV-vis, florescence, circular dichroic methods and cleavage studies of 1a with pBR322 plasmid, molecular docking were done in order to demonstrate their enantiomeric disposition toward the molecular drug target DNA. Interestingly, these studies unambiguously demonstrated the greater potency of L-enantiomer in comparison to D-enantiomer.

  19. DNA Duplex-Based Photodynamic Molecular Beacon for Targeted Killing of Retinoblastoma Cell.

    Science.gov (United States)

    Wei, Yanchun; Lu, Cuixia; Chen, Qun; Xing, Da

    2016-11-01

    Retinoblastoma (RB) is the most common primary intraocular malignancy of infancy. An alternative RB treatment protocol is proposed and tested. It is based on a photodynamic therapy (PDT) with a designed molecular beacon that specifically targets the murine double minute x (MDMX) high-expressed RB cells. A MDMX mRNA triggered photodynamic molecular beacon is designed by binding a photosensitizer molecule (pyropheophorbide-a, or PPa) and a black hole quencher-3 (BHQ3) through a complementary oligonucleotide sequence. Cells with and without MDMX high-expression are incubated with the beacon and then irradiated with a laser. The fluorescence and reactive oxygen species are detected in solution to verify the specific activation of PPa by the perfectly matched DNA targets. The cell viabilities are evaluated with CCK-8 and flow cytometry assay. The fluorescence and photo-cytoxicity of PPa is recovered and significantly higher in the MDMX high-expressed Y79 and WERI-Rb1 cells, compared to that with the MDMX low-expressed cells. The synthesized beacon exhibits high PDT efficiency toward MDMX high-expressed RB cells. The data suggest that the designed beacon may provide a potential alternative for RB therapy and secures the ground for future investigation.

  20. Mammalian DNA ligase III: Molecular cloning, chromosomal localization, and expression in spermatocytes undergoing meiotic recombination

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Jingwen; Danehower, S.; Besterman, J.M.; Husain, I. [Glaxo Research Inst., Research Triangle Park, NC (United States)] [and others

    1995-10-01

    Three biochemically distinct DNA ligase activities have been identified in mammalian cell extracts. We have recently purified DNA ligase II and DNA ligase III to near homogeneity from bovine liver and testis tissue, respectively. Amino acid sequencing studies indicated that these enzymes are encoded by the same gene. In the present study, human and murine cDNA clones encoding DNA ligase III were isolated with probes based on the peptide sequences. The human DNA ligase III cDNA encodes a polypeptide of 862 amino acids, whose sequence is more closely related to those of the DNA ligases encoded by poxviruses than to replicative DNA ligases, such as human DNA ligase I. In vitro transcription and translation of the cDNA produced a catalytically active DNA ligase similar in size and substrate specificity to the purified bovine enzyme. The DNA ligase III gene was localized to human chromosome 17, which eliminated this gene as a candidate for the cancer-prone disease Bloom syndrome that is associated with DNA joining abnormalities. DNA ligase III is ubiquitously expressed at low levels, except in the testes, in which the steady-state levels of DNA ligase III mRNA are at least 10-fold higher than those detected in other tissues and cells. Since DNA ligase I mRNA is also present at high levels in the testes, we examined the expression of the DNA ligase genes during spermatogenesis. DNA ligase I mRNA expression correlated with the contribution of proliferating supermatogonia cells to the testes, in agreement with the previously defined role of this enzyme in DNA replications. In contrast, elevated levels of DNA ligase III mRNA were observed in primary supermatocytes undergoing recombination prior to the first meiotic division. Therefore, we suggest that DNA ligase III seals DNA strand breaks that arise during the process of meiotic recombination in germ cells and as a consequence of DNA damage in somatic cells. 62 refs., 7 figs.

  1. Applications of Recombinant DNA Technology in Gastrointestinal Medicine and Hepatology: Basic Paradigms of Molecular Cell Biology. Part A: Eukaryotic Gene Structure and DNA Replication

    Directory of Open Access Journals (Sweden)

    Gary E Wild

    2000-01-01

    Full Text Available Progress in the basic sciences of cell and molecular biology has provided an exciting dimension that has translated into clinically relevant information in every medical subspecialty. Importantly, the application of recombinant DNA technology has played a major role in unravelling the intricacies related to the molecular pathophysiology of disease. This series of review articles constitutes a framework for the integration of the database of new information into the core knowledge base of concepts related to the pathogenesis of gastrointestinal disorders and liver disease. The goal of this series of three articles is to review the basic principles of eukaryotic gene expression. The first article examines the role of DNA in directing the flow of genetic information in eukaryotic cells.

  2. [Lung cancer molecular testing, what role for Next Generation Sequencing and circulating tumor DNA].

    Science.gov (United States)

    Pécuchet, Nicolas; Legras, Antoine; Laurent-Puig, Pierre; Blons, Hélène

    2016-01-01

    Molecular screening has become a standard of care for patients with advanced cancers and impacts on how to treat a patient. Advances in genomic technologies with the development of high throughput sequencing methods will certainly improve the possibilities to access a more accurate molecular diagnosis and to go beyond the identification of validated targets as a large number of genes can be screened for actionable changes. Moreover, accurate high throughput testing may help tumor classification in terms of prognosis and drug sensitivity. Finally, it will be possible to assess tumor heterogeneity and changes in molecular profiles during follow-up using ultra-deep sequencing technologies and circulating tumor DNA characterization. The accumulation of somatic ADN alterations is considered as the main contributing factor in carcinogenesis. The alterations can occur at different levels: mutation, copy number variations or gene translocations resulting in altered expression of the corresponding genes or impaired protein functions. Genes involved are mainly tumor suppressors, oncogenes or ADN repair genes whose modifications in tumors will impinge cell fate and proliferation from tumor initiation to metastasis. The entire genome of various tumor types, have now been sequenced. In lung cancer, the average number of mutations is very high with more than 8.9 mutations/Mb (Network TCGAR, 2014) that is to say more than 10,000 mutations/genome. These alterations need to be classified, indeed, some are true drivers that directly impact proliferation and some are passenger mutations linked to genetic instability. The development of targeted therapies relies on the identification of oncogenic drivers. The identification of genotype-phenotype associations as in the case of EGFR-TKI (Epidermal growth factor receptor-tyrosine kinase inhibitor) and EGFR mutations in lung cancer led to the restriction of drugs to patients for which tumor genotype predicts efficacy. Tumor-molecular

  3. NMR solution structure of the bicoid homeodomain bound to DNA and molecular dynamics simulations of the homeodomain/DNA complex

    Science.gov (United States)

    Baird-Titus, Jamie M.

    The homeodomain is a common DNA recognition motif consisting of three helices and an N-terminal arm that serves as a valuable model for exploring the basis of specific DNA recognition by proteins. Recognition of specific DNA sites, loosely defined by a TAAT core, is dependent on the side-chains of key amino acids in the N-terminal arm and the third "recognition" helix of the homeodomain. While much is known about homeodomain/DNA recognition, key questions concerning the role of individual amino acids and the extent of side-chain, DNA, and water dynamics during recognition remain, often focusing on the dynamic role of position 50 during recognition of the two bases immediately 3' to the 5'-TAAT-3'/3'-ATTA-5' core (ATTANN). The Bicoid homeodomain provides an interesting model system for addressing these and other questions, serving as the only known homeodomain that has a dual role in both transcriptional (DNA-binding) and translational (RNA-binding) control, discriminating between these two functions by a single amino acid, arginine 54. To add to the understanding of both general protein/DNA recognition and to the specific function of the Bicoid transcription factor homeodomain, we have determined the solution structure of the Bicoid homeodomain bound to the consensus duplex B-DNA binding site 5'-TAATCC-3'/3'-ATTAGG-5'. Our structure indicates that the Bicoid homeodomain exhibits variation from other homeodomain structures at the end of helix I, and NMR resonance line broadening of the K50 and R54 side-chains, consistent with side-chain motion and supportive of the adaptive-recognition theory of protein/DNA interactions.

  4. Improving molecular detection of fungal DNA in formalin-fixed paraffin-embedded tissues: comparison of five tissue DNA extraction methods using panfungal PCR.

    Science.gov (United States)

    Muñoz-Cadavid, C; Rudd, S; Zaki, S R; Patel, M; Moser, S A; Brandt, M E; Gómez, B L

    2010-06-01

    DNA extraction from formalin-fixed paraffin-embedded (FFPE) tissues is difficult and requires special protocols in order to extract small amounts of DNA suitable for amplification. Most described methods report an amplification success rate between 60 and 80%; therefore, there is a need to improve molecular detection and identification of fungi in FFPE tissue. Eighty-one archived FFPE tissues with a positive Gomori methenamine silver (GMS) stain were evaluated using five different commercial DNA extraction kits with some modifications. Three different panfungal PCR assays were used to detect fungal DNA, and two housekeeping genes were used to assess the presence of amplifiable DNA and to detect PCR inhibitors. The sensitivities of the five extraction protocols were compared, and the quality of DNA detection (calculated for each kit as the number of housekeeping gene PCR-positive samples divided by the total number of samples) was 60 to 91% among the five protocols. The efficiencies of the three different panfungals used (calculated as the number of panfungal-PCR-positive samples divided by the number of housekeeping gene PCR-positive samples) were 58 to 93%. The panfungal PCR using internal transcribed spacer 3 (ITS3) and ITS4 primers yielded a product in most FFPE tissues. Two of the five DNA extraction kits (from TaKaRa and Qiagen) showed similar and promising results. However, one method (TaKaRa) could extract fungal DNA from 69 of the 74 FFPE tissues from which a housekeeping gene could be amplified and was also cost-effective, with a nonlaborious protocol. Factors such as sensitivity, cost, and labor will help guide the selection of the most appropriate method for the needs of each laboratory.

  5. Specific interactions between lactose repressor protein and DNA affected by ligand binding: ab initio molecular orbital calculations.

    Science.gov (United States)

    Ohyama, Tatsuya; Hayakawa, Masato; Nishikawa, Shin; Kurita, Noriyuki

    2011-06-01

    Transcription mechanisms of gene information from DNA to mRNA are essentially controlled by regulatory proteins such as a lactose repressor (LacR) protein and ligand molecules. Biochemical experiments elucidated that a ligand binding to LacR drastically changes the mechanism controlled by LacR, although the effect of ligand binding has not been clarified at atomic and electronic levels. We here investigated the effect of ligand binding on the specific interactions between LacR and operator DNA by the molecular simulations combined with classical molecular mechanics and ab initio fragment molecular orbital methods. The results indicate that the binding of anti-inducer ligand strengthens the interaction between LacR and DNA, which is consistent with the fact that the binding of anti-inducer enhances the repression of gene transcription by LacR. It was also elucidated that hydrating water molecules existing between LacR and DNA contribute to the specific interactions between LacR and DNA. Copyright © 2011 Wiley Periodicals, Inc.

  6. Comparative molecular dynamics studies of heterozygous open reading frames of DNA polymerase eta (η) in pathogenic yeast Candida albicans

    Science.gov (United States)

    Satpati, Suresh; Manohar, Kodavati; Acharya, Narottam; Dixit, Anshuman

    2017-01-01

    Genomic instability in Candida albicans is believed to play a crucial role in fungal pathogenesis. DNA polymerases contribute significantly to stability of any genome. Although Candida Genome database predicts presence of S. cerevisiae DNA polymerase orthologs; functional and structural characterizations of Candida DNA polymerases are still unexplored. DNA polymerase eta (Polη) is unique as it promotes efficient bypass of cyclobutane pyrimidine dimers. Interestingly, C. albicans is heterozygous in carrying two Polη genes and the nucleotide substitutions were found only in the ORFs. As allelic differences often result in functional differences of the encoded proteins, comparative analyses of structural models and molecular dynamic simulations were performed to characterize these orthologs of DNA Polη. Overall structures of both the ORFs remain conserved except subtle differences in the palm and PAD domains. The complementation analysis showed that both the ORFs equally suppressed UV sensitivity of yeast rad30 deletion strain. Our study has predicted two novel molecular interactions, a highly conserved molecular tetrad of salt bridges and a series of π-π interactions spanning from thumb to PAD. This study suggests these ORFs as the homologues of yeast Polη, and due to its heterogeneity in C. albicans they may play a significant role in pathogenicity.

  7. Fluorescence- and capillary electrophoresis (CE)-based SSR DNA fingerprinting and a molecular identity database for the Louisiana sugarcane industry

    Science.gov (United States)

    A database of Louisiana sugarcane molecular identity has been constructed and is being updated annually using FAM or HEX or NED fluorescence- and capillary electrophoresis (CE)-based microsatellite (SSR) fingerprinting information. The fingerprints are PCR-amplified from leaf DNA samples of current ...

  8. A DNA tetrahedron-based molecular beacon for tumor-related mRNA detection in living cells.

    Science.gov (United States)

    Xie, Nuli; Huang, Jin; Yang, Xiaohai; Yang, Yanjing; Quan, Ke; Wang, He; Ying, Le; Ou, Min; Wang, Kemin

    2016-02-01

    Due to its low cytotoxicity, high resistance to enzymatic degradation, and cellular permeability, a DNA tetrahedron-based molecular beacon (DTMB) is designed for tumor-related TK1 mRNA detection in living cells, where the target sequence can induce the tetrahedron from contraction to extension, resulting in fluorescence restoration.

  9. Synthesis, Spectral Characterization, DNA/ Protein Binding, DNA Cleavage, Cytotoxicity, Antioxidative and Molecular Docking Studies of Cu(II)Complexes Containing Schiff Base-bpy/Phen Ligands.

    Science.gov (United States)

    Anupama, Berelli; Aruna, Airva; Manga, Vijjulatha; Sivan, Sreekanth; Sagar, Madamsetty Vijay; Chandrashekar, Ravula

    2017-05-01

    Ternary Cu(II) complexes [Cu(II)(L)(bpy)Cl] 1, [Cu(II)(L)(Phen)Cl] 2 [L = 2,3-dimethyl-1-phenyl-4(2 hydroxy-5-methyl benzylideneamino)-pyrazol-5-one, bpy = 2,2(') bipyridine, phen =1,10 phenanthroline) were synthesized and characterized by elemental analyses, UV-Visible, FT-IR, ESR, Mass, thermogravimetric and SEM EDAX techniques. The complexes exhibit octahedral geometry. The interaction of the Cu(II) with cailf thymus DNA (CT-DNA) was explored by using absorption and fluorescence spectroscopic methods. The results revealed that the complexes have an affinity constant for DNA in the order of 10(4) M(-1) and mode of interaction is intercalative mode. The DNA cleavage study showed that the complexes cleaved DNA without any external agent. The interaction of Cu(II) complexes with bovine serum albumin (BSA) was also studied using absorption and fluorescence techniques. The cytotoxic activity of the Cu(II) complexes was probed in HeLa (human breast adenocarcinoma cell line), B16F10 (Murine melanoma cell line) and HEPA1-6 celllines, complex 1 has good cytotoxic activity which is comparable with the doxarubicin drug, with IC50 values ranging from 3 to 12.6 μM. A further molecular docking technique was employed to understand the binding of the complexes towards the molecular target DNA. Investigation of the antioxidative properties showed that the metal complexes have significant radical scavenging activity potency against DPPH radical.

  10. Porphyrinic metal-organic framework as electrochemical probe for DNA sensing via triple-helix molecular switch.

    Science.gov (United States)

    Ling, Pinghua; Lei, Jianping; Ju, Huangxian

    2015-09-15

    An electrochemical DNA sensor was developed based on the electrocatalysis of porphyrinic metal-organic framework (MOF) and triple-helix molecular switch for signal transduction. The streptavidin functionalized zirconium-porphyrin MOF (PCN-222@SA) was prepared as signal nanoprobe via covalent method and demonstrated high electrocatalysis for O2 reduction. Due to the large steric effect, the designed nanoprobe was blocked for the interaction with the biotin labeled triple-helix immobilized on the surface of glassy carbon electrode. In the presence of target DNA, the assistant DNA in triple-helix will hybridize with target DNA, resulting in the disassembly of triple-helix molecular. Consequently, the end biotin away from the electrode was ''activated'' for easy access to the signal nanoprobe, PCN-222@SA, on the basis of biotin-streptavidin biorecognition. The introduction of signal nanoprobe to a sensor surface led to a significantly amplified electrocatalytic current towards oxygen reduction. Integrating with DNA recycling amplification of Exonuclease III, the sensitivity of the biosensor was improved significantly with detection limit of 0.29 fM. Moreover, the present method has been successfully applied to detect DNA in complex serum matrix. This porphyrinic MOF-based strategy has promising application in the determination of various analytes for signal transduction and has great potential in bioassays.

  11. Electrical detection of dengue virus (DENV) DNA oligomer using silicon nanowire biosensor with novel molecular gate control.

    Science.gov (United States)

    Nuzaihan M N, M; Hashim, U; Md Arshad, M K; Kasjoo, S R; Rahman, S F A; Ruslinda, A R; Fathil, M F M; Adzhri, R; Shahimin, M M

    2016-09-15

    In this paper, a silicon nanowire biosensor with novel molecular gate control has been demonstrated for Deoxyribonucleic acid (DNA) detection related to dengue virus (DENV). The silicon nanowire was fabricated using the top-down nanolithography approach, through nanostructuring of silicon-on-insulator (SOI) layers achieved by combination of the electron-beam lithography (EBL), plasma dry etching and size reduction processes. The surface of the fabricated silicon nanowire was functionalized by means of a three-step procedure involving surface modification, DNA immobilization and hybridization. This procedure acts as a molecular gate control to establish the electrical detection for 27-mers base targets DENV DNA oligomer. The electrical detection is based on the changes in current, resistance and conductance of the sensor due to accumulation of negative charges added by the immobilized probe DNA and hybridized target DNA. The sensitivity of the silicon nanowire biosensors attained was 45.0µAM(-1), which shows a wide-range detection capability of the sensor with respect to DNA. The limit of detection (LOD) achieved was approximately 2.0fM. The demonstrated results show that the silicon nanowire has excellent properties for detection of DENV with outstanding repeatability and reproducibility performances.

  12. Molecular species identification of Central European ground beetles (Coleoptera: Carabidae using nuclear rDNA expansion segments and DNA barcodes

    Directory of Open Access Journals (Sweden)

    Raupach Michael J

    2010-09-01

    Full Text Available Abstract Background The identification of vast numbers of unknown organisms using DNA sequences becomes more and more important in ecological and biodiversity studies. In this context, a fragment of the mitochondrial cytochrome c oxidase I (COI gene has been proposed as standard DNA barcoding marker for the identification of organisms. Limitations of the COI barcoding approach can arise from its single-locus identification system, the effect of introgression events, incomplete lineage sorting, numts, heteroplasmy and maternal inheritance of intracellular endosymbionts. Consequently, the analysis of a supplementary nuclear marker system could be advantageous. Results We tested the effectiveness of the COI barcoding region and of three nuclear ribosomal expansion segments in discriminating ground beetles of Central Europe, a diverse and well-studied invertebrate taxon. As nuclear markers we determined the 18S rDNA: V4, 18S rDNA: V7 and 28S rDNA: D3 expansion segments for 344 specimens of 75 species. Seventy-three species (97% of the analysed species could be accurately identified using COI, while the combined approach of all three nuclear markers provided resolution among 71 (95% of the studied Carabidae. Conclusion Our results confirm that the analysed nuclear ribosomal expansion segments in combination constitute a valuable and efficient supplement for classical DNA barcoding to avoid potential pitfalls when only mitochondrial data are being used. We also demonstrate the high potential of COI barcodes for the identification of even closely related carabid species.

  13. Effect evaluation of applying two dimensional bar code on quality tracking in the sterilization process of surgical instrument%二维条形码信息管理在手术灭菌器械质量追溯管理中的应用

    Institute of Scientific and Technical Information of China (English)

    梁飞凤; 刘海燕; 李彩婷; 张洁芳

    2013-01-01

      目的探讨应用二维条形码信息管理对手术灭菌器械进行质量追溯的效果。方法将16857件手术灭菌器械分为实验组8356件和对照组8501件,模拟实验组有276件和对照组有288件灭菌器械不合格,实验组使用二维条形码信息管理,对照组采用传统方法对手术灭菌不合格器械质量进行追溯。比较两组器械质量追溯花费时间的差异。结果实验组质量追溯时间短于对照组,两组比较,t=-244.09,t=P<0.001,差异具有统计学意义。结论使用二维条形码信息管理对手术灭菌器械进行质量追溯,不仅能提高工作效率,同时保证手术病人的安全。%Objective To investigate the application of two dimensional bar code on the quality tracking of surgical instrument. Methods Sixty cases of surgical instrument packets without sterilization in sterilization and supply center were selected.The parkets were divided into the observation group and the control group with 30 cases in each group.The observation group was applied two dimensional bar code on the surgical instrument packets.We eraluated the effect of quality tracking according to the using time that started from discorering sterilization items unqualified to tracking to the patient who use this item.The control group was applied traditional methods.Results The quality tracking time of experiment group was significantly shorter than the time of control group. The difference was statistically significant(P<0.001).The staff satisfaction of the observation group was better than that of the control group.The difference was statistically significant(P<0.001).Conclusion Applying two dimensional bar code can improve the work efficiency,the sterilization quality of surgical instrument packet and guarantee the operation safety of patients.

  14. Large-scale molecular dynamics simulation of DNA: implementation and validation of the AMBER98 force field in LAMMPS.

    Science.gov (United States)

    Grindon, Christina; Harris, Sarah; Evans, Tom; Novik, Keir; Coveney, Peter; Laughton, Charles

    2004-07-15

    Molecular modelling played a central role in the discovery of the structure of DNA by Watson and Crick. Today, such modelling is done on computers: the more powerful these computers are, the more detailed and extensive can be the study of the dynamics of such biological macromolecules. To fully harness the power of modern massively parallel computers, however, we need to develop and deploy algorithms which can exploit the structure of such hardware. The Large-scale Atomic/Molecular Massively Parallel Simulator (LAMMPS) is a scalable molecular dynamics code including long-range Coulomb interactions, which has been specifically designed to function efficiently on parallel platforms. Here we describe the implementation of the AMBER98 force field in LAMMPS and its validation for molecular dynamics investigations of DNA structure and flexibility against the benchmark of results obtained with the long-established code AMBER6 (Assisted Model Building with Energy Refinement, version 6). Extended molecular dynamics simulations on the hydrated DNA dodecamer d(CTTTTGCAAAAG)(2), which has previously been the subject of extensive dynamical analysis using AMBER6, show that it is possible to obtain excellent agreement in terms of static, dynamic and thermodynamic parameters between AMBER6 and LAMMPS. In comparison with AMBER6, LAMMPS shows greatly improved scalability in massively parallel environments, opening up the possibility of efficient simulations of order-of-magnitude larger systems and/or for order-of-magnitude greater simulation times.

  15. In-silico screening for DNA-dependent protein kinase (DNA-PK) inhibitors: Combined homology modeling, docking, molecular dynamic study followed by biological investigation.

    Science.gov (United States)

    Tarazi, Hamadeh; Saleh, Ekram; El-Awady, Raafat

    2016-10-01

    DNA-dependent protein kinase (DNA-PK) is a key enzyme in non-homologous DNA end joining (NHEJ) repair pathway. The targeted inhibition of such enzyme would furnish a valuable option for cancer treatment. In this study we report the development of validation of enzyme homology model, and the subsequent use of this model to perform docking-based virtual screening against a database of FDA-approved drugs. The nominated highest ranking hits (Praziquantel and Dutasteride) were subjected to biological investigation. Additionally, molecular dynamic study was carried-out for binding mode exploration. Results of the biological evaluation revealed that both compounds inhibit the DNA-PK enzymatic activity at relatively high concentration levels with an IC50 of 17.3μM for praziquantel and >20μM for dutasteride. Furthermore, both agents enhanced the anti-proliferative effects of doxorubicin and cisplatin on breast cancer (MCF7) and lung cancer (A549) cell lines. This result indicates that these two hits are good candidate as DNA-PK inhibitors and worth further structural modifications to enhance their enzyme inhibitory effects. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  16. Base flip in DNA studied by molecular dynamics simulationsof differently-oxidized forms of methyl-Cytosine.

    Science.gov (United States)

    Helabad, Mahdi Bagherpoor; Kanaan, Natalia; Imhof, Petra

    2014-07-03

    Distortions in the DNA sequence, such as damage or mispairs, are specifically recognized and processed by DNA repair enzymes. Many repair proteins and, in particular, glycosylases flip the target base out of the DNA helix into the enzyme's active site. Our molecular dynamics simulations of DNA with intact and damaged (oxidized) methyl-cytosine show that the probability of being flipped is similar for damaged and intact methyl-cytosine. However, the accessibility of the different 5-methyl groups allows direct discrimination of the oxidized forms. Hydrogen-bonded patterns that vary between methyl-cytosine forms carrying a carbonyl oxygen atom are likely to be detected by the repair enzymes and may thus help target site recognition.

  17. Molecular dynamics simulations of double-stranded DNA in an explicit solvent model with the zero-dipole summation method.

    Directory of Open Access Journals (Sweden)

    Takamasa Arakawa

    Full Text Available Molecular dynamics (MD simulations of a double-stranded DNA with explicit water and small ions were performed with the zero-dipole summation (ZD method, which was recently developed as one of the non-Ewald methods. Double-stranded DNA is highly charged and polar, with phosphate groups in its backbone and their counterions, and thus precise treatment for the long-range electrostatic interactions is always required to maintain the stable and native double-stranded form. A simple truncation method deforms it profoundly. On the contrary, the ZD method, which considers the neutralities of charges and dipoles in a truncated subset, well reproduced the electrostatic energies of the DNA system calculated by the Ewald method. The MD simulations using the ZD method provided a stable DNA system, with similar structures and dynamic properties to those produced by the conventional Particle mesh Ewald method.

  18. Base Flip in DNA Studied by Molecular Dynamics Simulationsof Differently-Oxidized Forms of Methyl-Cytosine

    Directory of Open Access Journals (Sweden)

    Mahdi Bagherpoor Helabad

    2014-07-01

    Full Text Available Distortions in the DNA sequence, such as damage or mispairs, are specifically recognized and processed by DNA repair enzymes. Many repair proteins and, in particular, glycosylases flip the target base out of the DNA helix into the enzyme’s active site. Our molecular dynamics simulations of DNA with intact and damaged (oxidized methyl-cytosine show that the probability of being flipped is similar for damaged and intact methyl-cytosine. However, the accessibility of the different 5-methyl groups allows direct discrimination of the oxidized forms. Hydrogen-bonded patterns that vary between methyl-cytosine forms carrying a carbonyl oxygen atom are likely to be detected by the repair enzymes and may thus help target site recognition.

  19. Optimization of β-glucan synthase gene primers for molecular DNA fingerprinting in Pleurotus pulmonarious

    Science.gov (United States)

    Kadir, Zaiton Abdul; Daud, Fauzi; Mohamad, Azhar; Senafi, Sahidan; Jamaludin, Ferlynda Fazleen

    2015-09-01

    Pleurotus pulmonarius is an edible mushroom in Malaysia and commonly known as Oyster mushroom. The species are important not only for nutritional values but also for pharmaceutical importance related to bioactive compounds in polysaccharides such as β glucan. Hence, β-glucan synthase gene (BGS) pathways which are related to the production of the β-glucan might be useful as marker for molecular DNA fingerprinting in P. pulmonarius. Conserved regions of β-glucan gene were mined from public database and aligned. Consensus from the alignment was used to design the primers by using Primer 3 software. Eight primers were designed and a single primer pair (BGF3: 5' TCTTGGCGAGTTCGAAGAAT 3'; BGR3: 5' TTCCGATCTTGGTCTGGAAG 3') was optimized at Ta (annealing temperature) 57.1°C to produce PCR product ranging from 400-500 bp. Optimum components for PCR reactions were 5.0 µl of 10× PCR buffer, 1.5 µl of 25 mM MgCl2, 1 µl of 10 mM dNTP, 1 µl of β-glucan primers, 0.1 µl of 5 units/ml Taq polymerase and 2 µl DNA template. PCR program was set at 34 PCR cycles by using Bio-Rad T100 Thermal Cycler. Initial denaturation was set at 94°C for 2 min, denaturation at 94°C for 1 minute, primer annealing at 45°C to 60°C (gradient temperature) for 50 seconds, followed by elongation at 72°C for 1 minute and further extension 5 minutes for last cycle PCR prior to end the program cycle. Thus, this information revealed that the primer of β-glucan gene designed could be used as targeted markers in screening population strains of P. pulmonarius.

  20. Molecular Characterization of Indonesian Indigenous Chickens based on Mitochondrial DNA Displacement (D-loop Sequences

    Directory of Open Access Journals (Sweden)

    SRI SULANDARI

    2008-12-01

    Full Text Available The Mitochondrial DNA (mtDNA displacement (D-loop sequences were used to study the genetic diversity and relationship of Indonesian indigenous chickens. A total of 483 individuals belonging to 15 population breeds and 43 individuals belonging to 6 populations of jungle fowl (2 populations of Gallus gallus and 4 populations of Gallus varius were sampled. The hypervariable I (HVI segment of the D-loop was PCR amplified and subsequently sequenced. The sequences of the first 397 nucleotides were used for analysis. Sixty nine haplotypes were identified from 54 polymorphic sites with polymorphism between nucleotides 167 and 397 contributing to 94.5% of the sequence variation. Phylogenetic analysis indicates that Indonesian indigenous chickens can be grouped into five distinct clades (clade I, II, IIIc, IIId, and IV of the previously identified seven clades (clade I, II, IIIa, IIIb, IIIc, IIId, and IV in Asian indigenous chickens. Fifty haplotypes belong to clade II, seven haplotypes are in clade IV, six are in clade IIId, three are in clade I and one haploype is in clade IIIc. There was no breed-specific clade. Analysis of Molecular Variance (AMOVA based on partial D-loop sequences of Indonesian chicken indicates that 67.85% of the total sequence variation between haplotypes was present within the population and 32.15% between populations. One of the haplotypes (represented by PLC4 was shared by all populations, suggesting that these populations may share the same maternal ancestor. These results show a high mitochondrial D-loop diversity and indicate multiple maternal origins for Indonesian indigenous chickens.

  1. Molecular phylogenetic relationship of Eplnephelus based on sequences of mtDNA Cty b

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    The mtDNA Cyt b gene was sequenced partially for Variola louti of Serranidae,Epinephelinae and seven endemic species of groupers-Epinephelus awoara,E.brunneus,E.coioides,E.longispinis,E.sexfasciatus,E.spilotoceps and E.tauvina in China.The seven endemic species and other seven foreign species of groupers--E,aeneus,E.caninus,E.drummondhayi,E,haifensis,E.labriformis,E.marginatus and E.multinotatus from the GenBank were combined and analysed as ingroup,while Variola louti was used as outgroup.We compared the 420 bp sequences of Cyt b among the 15 species and constructed two types of molecular phylogenetic trees with maximum parsimony method (MP)and neighbor-joining method (NJ) respectively.The results were as follows:(1) As to the base composition of mtDNA Cyt b sequence (402 bp) of 14 species of Epinepkelus,the content of (A + T) was 53.6%,higher than that of (G + C) (46.4%).The transition/transversion ratio was 4.78 with no mutation saturation.(2) The duster relationships between E.awoara and E.sexfasciatus,E.coioides and E.tauvina,E.longispinis and E.spilotoceps were consistent with phenotypes in taxonomy.(3) In the phylogenetic tree,the species in the Atlantic Ocean were associated closely with those in the Pacific Ocean,which suggested that the Cyt b sequences of Epinephelus were highly conserved.This may be attributed to the coordinate evolution.(4) In well-bred mating or heredity management,mating Epinephelus of the same branch should be avoided.It is likely to be an effective way to mate the species of the Atlantic Ocean with those of the Pacific Ocean to improve the inheritance species.

  2. Plant molecular biology and biotechnology research in the post-recombinant DNA era.

    Science.gov (United States)

    Tyagi, Akhilesh K; Khurana, Jitendra P

    2003-01-01

    After the beginning of the recombinant DNA era in the mid-1970s, researchers in India started to make use of the new technology to understand the structure of plant genes and regulation of their expression. The outcome started to appear in print in early the 1980s and genes for histones, tubulin, photosynthetic membrane proteins, phototransduction components, organelles and those regulated differentially by developmental and extrinsic signals were sequenced and characterized. Some genes of biotechnological importance like those encoding an interesting seed protein and the enzyme glyoxalase were also isolated. While work on the characterization of genome structure and organization was started quite early, it remained largely focused on the identification of DNA markers and genetic variability. In this context, the work on mustard, rice and wheat is worth mentioning. In the year 2000, India became a member of the international consortium to sequence entire rice genome. Several laboratories have also given attention to regulated expression of plastid and nuclear genes as well as to isolate target-specific promoters or design promoters with improved potential. Simultaneously, transgenic systems for crops like mustard, rice, wheat, cotton, legumes and several vegetables have been established. More recently, genes of agronomic importance like those for insect resistance, abiotic stress tolerance, nutritional improvement and male sterility, isolated in India or abroad, have been utilized for raising transgenics for crop improvement. Some of these transgenics have already shown their potential in containment facility or limited field trials conducted under the stipulated guidelines. Plant molecular biology and biotechnology are thus clearly poised to make an impact on research in basic biology and agriculture in the near future.

  3. Self-consistent treatment of electrostatics in molecular DNA braiding through external forces.

    Science.gov (United States)

    Lee, Dominic J

    2014-06-01

    In this paper we consider a physical system in which two DNA molecules braid about each other. The distance between the two molecular ends, on either side of the braid, is held at a distance much larger than supercoiling radius of the braid. The system is subjected to an external pulling force, and a moment that induces the braiding. In a model, developed for understanding such a system, we assume that each molecule can be divided into a braided and unbraided section. We also suppose that the DNA is nicked so that there is no constraint of the individual linking numbers of the molecules. Included in the model are steric and electrostatic interactions, thermal fluctuations of the braided and unbraided sections of the molecule, as well as the constraint on the braid linking (catenation) number. We compare two approximations used in estimating the free energy of the braided section. One is where the amplitude of undulations of one molecule with respect to the other is determined only by steric interactions. The other is a self-consistent determination of the mean-squared amplitude of these undulations. In this second approximation electrostatics should play an important role in determining this quantity, as suggested by physical arguments. We see that if the electrostatic interaction is sufficiently large there are indeed notable differences between the two approximations. We go on to test the self-consistent approximation-included in the full model-against experimental data for such a system, and we find good agreement. However, there seems to be a slight left-right-handed braid asymmetry in some of the experimental results. We discuss what might be the origin of this small asymmetry.

  4. Early Antiretroviral Therapy Is Associated with Lower HIV DNA Molecular Diversity and Lower Inflammation in Cerebrospinal Fluid but Does Not Prevent the Establishment of Compartmentalized HIV DNA Populations.

    Directory of Open Access Journals (Sweden)

    Michelli F Oliveira

    2017-01-01

    Full Text Available Even when antiretroviral therapy (ART is started early after infection, HIV DNA might persist in the central nervous system (CNS, possibly contributing to inflammation, brain damage and neurocognitive impairment. Paired blood and cerebrospinal fluid (CSF were collected from 16 HIV-infected individuals on suppressive ART: 9 participants started ART 14 months after EDI ("late ART". For each participant, neurocognitive functioning was measured by Global Deficit Score (GDS. HIV DNA levels were measured in peripheral blood mononuclear cells (PBMCs and CSF cell pellets by droplet digital (ddPCR. Soluble markers of inflammation (sCD163, IL-6, MCP-1, TNF-α and neuronal damage (neurofilament light [NFL] were measured in blood and CSF supernatant by immunoassays. HIV-1 partial C2V3 env deep sequencing data (Roche 454 were obtained for 8 paired PBMC and CSF specimens and used for phylogenetic and compartmentalization analysis. Median exposure to ART at the time of sampling was 2.6 years (IQR: 2.2-3.7 and did not differ between groups. We observed that early ART was significantly associated with lower molecular diversity of HIV DNA in CSF (p<0.05, and lower IL-6 levels in CSF (p = 0.02, but no difference for GDS, NFL, or HIV DNA detectability compared to late ART. Compartmentalization of HIV DNA populations between CSF and blood was detected in 6 out of 8 participants with available paired HIV DNA sequences (2 from early and 4 from late ART group. Phylogenetic analysis confirmed the presence of monophyletic HIV DNA populations within the CSF in 7 participants, and the same population was repeatedly sampled over a 5 months period in one participant with longitudinal sampling. Such compartmentalized provirus in the CNS needs to be considered for the design of future eradication strategies and might contribute to the neuropathogenesis of HIV.

  5. Early Antiretroviral Therapy Is Associated with Lower HIV DNA Molecular Diversity and Lower Inflammation in Cerebrospinal Fluid but Does Not Prevent the Establishment of Compartmentalized HIV DNA Populations

    Science.gov (United States)

    Oliveira, Michelli F.; Chaillon, Antoine; Nakazawa, Masato; Vargas, Milenka; Strain, Matthew C.; Morris, Sheldon; Little, Susan J.; Smith, Davey M.; Gianella, Sara

    2017-01-01

    Even when antiretroviral therapy (ART) is started early after infection, HIV DNA might persist in the central nervous system (CNS), possibly contributing to inflammation, brain damage and neurocognitive impairment. Paired blood and cerebrospinal fluid (CSF) were collected from 16 HIV-infected individuals on suppressive ART: 9 participants started ART 14 months after EDI (“late ART”). For each participant, neurocognitive functioning was measured by Global Deficit Score (GDS). HIV DNA levels were measured in peripheral blood mononuclear cells (PBMCs) and CSF cell pellets by droplet digital (dd)PCR. Soluble markers of inflammation (sCD163, IL-6, MCP-1, TNF-α) and neuronal damage (neurofilament light [NFL]) were measured in blood and CSF supernatant by immunoassays. HIV-1 partial C2V3 env deep sequencing data (Roche 454) were obtained for 8 paired PBMC and CSF specimens and used for phylogenetic and compartmentalization analysis. Median exposure to ART at the time of sampling was 2.6 years (IQR: 2.2–3.7) and did not differ between groups. We observed that early ART was significantly associated with lower molecular diversity of HIV DNA in CSF (p<0.05), and lower IL-6 levels in CSF (p = 0.02), but no difference for GDS, NFL, or HIV DNA detectability compared to late ART. Compartmentalization of HIV DNA populations between CSF and blood was detected in 6 out of 8 participants with available paired HIV DNA sequences (2 from early and 4 from late ART group). Phylogenetic analysis confirmed the presence of monophyletic HIV DNA populations within the CSF in 7 participants, and the same population was repeatedly sampled over a 5 months period in one participant with longitudinal sampling. Such compartmentalized provirus in the CNS needs to be considered for the design of future eradication strategies and might contribute to the neuropathogenesis of HIV. PMID:28046096

  6. Molecular phylogenetics of North American phoxinins (Actinopterygii: Cypriniformes: Leuciscidae) based on RAG1 and S7 nuclear DNA sequence data.

    Science.gov (United States)

    Bufalino, Angelo P; Mayden, Richard L

    2010-04-01

    Most molecular phylogenetic hypotheses for North American (NA) phoxinins are based on mitochondrial DNA sequences (mtDNA) and the resulting hypotheses are rather variable, though there is general support for three major lineages of NA phoxinins: western, creek chub-plagopterin (CC-P), and open posterior myodome (OPM) clades. Support for a monophyletic NA phoxinin group has varied among studies. This study utilizes nuclear DNA (nDNA) sequences from the RAG1 (exon 3) and S7 (intron 1) gene regions to evaluate the phylogenetic relationships and monophyly of NA phoxinins. Results from the nDNA analyses provide overall support for the western, CC-P, and OPM clades. The CC-P clade had the best overall resolution and support in the individual and combined analyses of the nDNA data. Resolution of the western clade was fairly good, with most analyses recovering a monophyletic Gila clade. The OPM clade demonstrated the highest degree of topological variability among the analyses. The RAG1 analyses failed to recover a monophyletic NA phoxinin group by resolving the European leuciscins, inclusive Notemigonus crysoleucas, within the NA phoxinin topology. Most analyses recovered a strongly supported shiner clade though, similar to several mtDNA studies; there was a high degree of topological variability among the results.

  7. Molecular Dynamics Study of the Motion of Atomic Components of the DNA Molecule and its Environment.

    Science.gov (United States)

    Perez, Patricio

    It is of great interest for us to come to a better understanding of some biological processes through a study of the behavior of the atomic entities involved in them. One of the most important biological molecules in DNA. In the outer part of its helical structure, phosphate groups are known to be present. The natural environment of this molecule is liquid water. Some cations like Na('+) and Mg('2+) are often found in its surroundings. In this work we show results of applying a polarizable water model (the PE model) to the molecular dynamics simulation of hydrated sodium ion microclusters. We found that the PE model with just two adjustable parameters reproduces the experimental enthalpies of formation of the ion-water microclusters better than a number of other methods. We also found that for the case of six water molecules at O K, they do not form a regular octahedron around the sodium ion as predicted by other models. The predicted change in structure appears to be in agreement with experimental observations. We introduced later a phosphate group and performed a molecular dynamics simulation of its interaction with water and a sodium cation. We use polarizable models for both water and the phosphate group. According to our calculation, a potential fitted to quantum mechanical results produces a strong binding between the sodium and the phosphate group. This binding is such that hydration of the sodium is not clearly observed. Regardless of this we can conclude that the effect of assuming the phosphate group polarizable is not significant.

  8. DNA ligases from rat liver. Purification and partial characterization of two molecular forms

    Energy Technology Data Exchange (ETDEWEB)

    Elder, R.H.; Rossignol, J.M. (Laboratoire de Biologie Moleculaire de la Replication, UPR 272-CNRS, IRSC, Villejuif (France))

    1990-06-26

    The differential ability of mammalian DNA ligases to use oligo(dT).poly(rA) as a substrate has been used to detect, and thereby extensively purify, two immunologically distinct forms of DNA ligase from rat liver. The activity of DNA ligase I, which is unable to use this template, is uniquely increased during liver regeneration, while that of DNA ligase II remains at a low level. Both enzymes require ATP and Mg2+ for activity and form an adenylylated intermediate which is stable and reactive. After SDS-PAGE, such radiolabeled complexes correspond to polypeptides of 130,000 and 80,000 Da for DNA ligase I and to 100,000 Da for DNA ligase II. That these labeled polypeptides do indeed correspond to active polypeptides of two different forms of DNA ligase is shown by the removal of the radiolabeled AMP, only when the intermediate is incubated with an appropriate substrate. In contrast to other eukaryotic DNA ligases, rat liver DNA ligase II has a lower Km for ATP (1.2 X 10(-5) M) than DNA ligase I (6 X 10(-5) M). Also, DNA ligase II can use ATP alpha S as a cofactor in the ligation reaction much more efficiently than DNA ligase I, further discriminating the ATP binding sites of these enzymes. Finally, antibodies raised against the 130,000-Da polypeptide of DNA ligase I specifically recognize this species in an immunoblot and inhibit only the activity of DNA ligase I.

  9. DNA surveillance: web-based molecular identification of whales, dolphins, and porpoises.

    Science.gov (United States)

    Ross, H A; Lento, G M; Dalebout, M L; Goode, M; Ewing, G; McLaren, P; Rodrigo, A G; Lavery, S; Baker, C S

    2003-01-01

    DNA Surveillance is a Web-based application that assists in the identification of the species and population of unknown specimens by aligning user-submitted DNA sequences with a validated and curated data set of reference sequences. Phylogenetic analyses are performed and results are returned in tree and table format summarizing the evolutionary distances between the query and reference sequences. DNA Surveillance is implemented with mitochondrial DNA (mtDNA) control region sequences representing the majority of recognized cetacean species. Extensions of the system to include other gene loci and taxa are planned. The service, including instructions and sample data, is available at http://www.dna-surveillance.auckland.ac.nz.

  10. Label-free molecular beacons-based cascade amplification DNA machine for sensitive detection of telomerase activity.

    Science.gov (United States)

    Li, Kan; Wang, Lei; Xu, Xiaowen; Jiang, Wei

    2017-05-15

    Sensitive detection of telomerase activity is critical to cancer diagnosis, screening of anticancer drugs and evaluation of cancer therapy. Herein, a label-free molecular beacons-based DNA machine was developed for sensitive detection of telomerase activity. The DNA machine consisted of T7 exonuclease (T7 Exo), label-free recognition molecular beacon (RMB) and signal molecular beacon (SMB) with projecting 5'-terminuses, which can protect RMB and SMB from being digested by T7 Exo. Firstly, telomerase elongated telomerase substrate (TS) primer, generating a telomerase elongation production (TEP) with tandem repeats (TTAGGG)n. Next, TEP activated the DNA machine by hybridizing with RMB, unfolding RMB with a recessed 5'-terminus, making RMB deprotection from T7 Exo. Then T7 Exo-assisted cycling cleavage was incurred, releasing intact TEP and numerous DNA fragments (trigger DNA), which got recycling I. Subsequently, trigger DNA specifically opened SMB and was recycled by T7 Exo, liberating multiple G-quadruplex (G4) structures, which got recycling II. Finally, TEP and the liberative G4 structures strongly interacted with N-methyl-mesoporphyrin IX (NMM), yielding a significantly enhanced fluorescence together. In this way, per telomerase-mediated elongation event was efficiently converted into the greatly amplified fluorescence signals. Telomerase activity in crude HeLa cells extracts equivalent to 50 cells/mL was successfully measured with a linear range from 50 cells/mL to 2000 cells/mL. Besides, the strategy was also successfully used to assay the inhibition effect of a telomerase-inhibiting drug, demonstrating the strategy holds the potential to screen telomerase inhibitors.

  11. Molecular cytogenetic mapping of Cucumis sativus and C. melo using highly repetitive DNA sequences.

    Science.gov (United States)

    Koo, Dal-Hoe; Nam, Young-Woo; Choi, Doil; Bang, Jae-Wook; de Jong, Hans; Hur, Yoonkang

    2010-04-01

    Chromosomes often serve as one of the most important molecular aspects of studying the evolution of species. Indeed, most of the crucial mutations that led to differentiation of species during the evolution have occurred at the chromosomal level. Furthermore, the analysis of pachytene chromosomes appears to be an invaluable tool for the study of evolution due to its effectiveness in chromosome identification and precise physical gene mapping. By applying fluorescence in situ hybridization of 45S rDNA and CsCent1 probes to cucumber pachytene chromosomes, here, we demonstrate that cucumber chromosomes 1 and 2 may have evolved from fusions of ancestral karyotype with chromosome number n = 12. This conclusion is further supported by the centromeric sequence similarity between cucumber and melon, which suggests that these sequences evolved from a common ancestor. It may be after or during speciation that these sequences were specifically amplified, after which they diverged and specific sequence variants were homogenized. Additionally, a structural change on the centromeric region of cucumber chromosome 4 was revealed by fiber-FISH using the mitochondrial-related repetitive sequences, BAC-E38 and CsCent1. These showed the former sequences being integrated into the latter in multiple regions. The data presented here are useful resources for comparative genomics and cytogenetics of Cucumis and, in particular, the ongoing genome sequencing project of cucumber.

  12. Molecular phylogenetic analysis of Indonesia Solanaceae based on DNA sequences of internal transcribed spacer region

    Science.gov (United States)

    Hidayat, Topik; Priyandoko, Didik; Islami, Dina Karina; Wardiny, Putri Yunitha

    2016-02-01

    Solanaceae is one of largest family in Angiosperm group with highly diverse in morphological character. In Indonesia, this group of plant is very popular due to its usefulness as food, ornamental and medicinal plants. However, investigation on phylogenetic relationship among the member of this family in Indonesia remains less attention. The purpose of this study was to evaluate the phylogenetics relationship of the family especially distributed in Indonesia. DNA sequences of Internal Transcribed Spacer (ITS) region of 19 species of Solanaceae and three species of outgroup, which belongs to family Convolvulaceae, Apocynaceae, and Plantaginaceae, were isolated, amplified, and sequenced. Phylogenetic tree analysis based on parsimony method was conducted with using data derived from the ITS-1, 5.8S, and ITS-2, separately, and the combination of all. Results indicated that the phylogenetic tree derived from the combined data established better pattern of relationship than separate data. Thus, three major groups were revealed. Group 1 consists of tribe Datureae, Cestreae, and Petunieae, whereas group 2 is member of tribe Physaleae. Group 3 belongs to tribe Solaneae. The use of the ITS region as a molecular markers, in general, support the global Solanaceae relationship that has been previously reported.

  13. Molecular Characterization of Mosquitoes (Diptera: Culicidae) in Northwestern Iran by Using rDNA-ITS2.

    Science.gov (United States)

    Khoshdel-Nezamiha, Farahnaz; Vatandoost, Hassan; Oshaghi, Mohammad Ali; Azari-Hamidian, Shahyad; Mianroodi, Reza Arabi; Dabiri, Farrokh; Bagheri, Masoomeh; Terenius, Olle; Chavshin, Ali Reza

    2016-07-22

    Several mosquito species are vectors of disease; however, to understand their role in disease transmission, accurate species identification is of particular importance. Morphological identification is the main method used, but molecular techniques have emerged as a tool for the identification of closely related species. In this study, mosquitoes from the West Azerbaijan Province in northwestern Iran were characterized on the basis of their rDNA-ITS2 sequences. Nine populations of 6 species of mosquitoes belonging to the genera Anopheles, Culex, Culiseta, and Ochlerotatus were studied. To the best of our knowledge, ITS2 sequences of Culiseta longiareolata and Culex hortensis have been reported for the first time. In addition, ITS2 sequences of Culex theileri and Ochlerotatus caspius have been reported for the first time in Iran. Phylogenetic analysis based on ITS2 showed that subfamilies Anophelinae and Culicinae of the family Culicidae could be differentiated successfully and subgenera Anopheles and Cellia of the genus Anopheles were separated. The analysis showed that the genera Culex, Culiseta, and Ochlerotatus have diverged separately.

  14. Affinity of molecular interactions in the bacteriophage phi29 DNA packaging motor.

    Science.gov (United States)

    Robinson, Mark A; Wood, Jonathan P A; Capaldi, Stephanie A; Baron, Andrew J; Gell, Christopher; Smith, D Alastair; Stonehouse, Nicola J

    2006-01-01

    DNA packaging in the bacteriophage phi29 involves a molecular motor with protein and RNA components, including interactions between the viral connector protein and molecules of pRNA, both of which form multimeric complexes. Data are presented to demonstrate the higher order assembly of pRNA together with the affinity of pRNA:pRNA and pRNA:connector interactions, which are used to propose a model for motor function. In solution, pRNA can form dimeric and trimeric multimers in a magnesium-dependent manner, with dissociation constants for multimerization in the micromolar range. pRNA:connector binding is also facilitated by the presence of magnesium ions, with a nanomolar apparent dissociation constant for the interaction. From studies with a mutant pRNA, it appears that multimerization of pRNA is not essential for connector binding and it is likely that connector protein is involved in the stabilization of higher order RNA multimers. It is proposed that magnesium ions may promote conformational change that facilitate pRNA:connector interactions, essential for motor function.

  15. Affinity of molecular interactions in the bacteriophage φ29 DNA packaging motor

    Science.gov (United States)

    Robinson, Mark A.; Wood, Jonathan P.A.; Capaldi, Stephanie A.; Baron, Andrew J.; Gell, Christopher; Smith, D. Alastair; Stonehouse, Nicola J.

    2006-01-01

    DNA packaging in the bacteriophage φ29 involves a molecular motor with protein and RNA components, including interactions between the viral connector protein and molecules of pRNA, both of which form multimeric complexes. Data are presented to demonstrate the higher order assembly of pRNA together with the affinity of pRNA:pRNA and pRNA:connector interactions, which are used to propose a model for motor function. In solution, pRNA can form dimeric and trimeric multimers in a magnesium-dependent manner, with dissociation constants for multimerization in the micromolar range. pRNA:connector binding is also facilitated by the presence of magnesium ions, with a nanomolar apparent dissociation constant for the interaction. From studies with a mutant pRNA, it appears that multimerization of pRNA is not essential for connector binding and it is likely that connector protein is involved in the stabilization of higher order RNA multimers. It is proposed that magnesium ions may promote conformational change that facilitate pRNA:connector interactions, essential for motor function. PMID:16714447

  16. Construction of a rice immature seeds cDNA library and molecular cloning of oryzacystatin cDNA

    Institute of Scientific and Technical Information of China (English)

    周兆斓; 朱祯; 刘春明; 张海涛; 肖桂芳; 李向辉

    1996-01-01

    Total RNA was extracted from rice immature seeds harvested 2 weeks after flowering; then mRNA was purified. cDNA with NotI and SaiI cohesive ends was synthesized and inserted into λgt22A. After packaged in vitno, the cDNA library was constructed with 1.5×106pfu. A 21-mer oligodeoxynucleotide was synthesized according to the 5’-end conserved coding sequence of oryzacystatin (a thiol proteinase inhibitor) and labeled as a probe. From 2.1 × 104 pfu, 9 positive dones have been isolated, 8 of which contain the entire coding region of oryzacystatin. λOC1 has the longest cDNA insert, which contains an open reading frame of 309 bp coding sequence, 84 bp 5’-end non-coding region and a poly(A) signal AATAAA at the 3’-end followed by 31 Nt of poly(A). The coding sequence is the same compared with oryzacystatin genomic DNA sequence, while there are some obvious differences such as insertion and variation in the non-coding region, especially lots of nonsucoessive insertion in the 3’ region after poly(A) signal.

  17. Molecular cloning and analysis of functional cDNA and genomic clones encoding bovine cellular retinoic acid-binding protein.

    OpenAIRE

    Shubeita, H E; Sambrook, J F; McCormick, A M

    1987-01-01

    A recombinant cDNA clone, pCRABP-HS1, encoding cellular retinoic acid-binding protein was isolated from a bovine adrenal cDNA library. COS-7 cells transfected with pCRABP-HS1 produced a biologically active retinoic acid-binding protein molecule of the expected molecular mass (15.5 kDa). RNA blot hybridization analysis using pCRABP-HS1 as a probe revealed a single 1050-nucleotide mRNA species in bovine adrenal, uterus, and testis, tissues that contain the highest levels of retinoic acid-bindin...

  18. Divergent nuclear 18S rDNA paralogs in a turkey coccidium, Eimeria meleagrimitis, complicate molecular systematics and identification.

    Science.gov (United States)

    El-Sherry, Shiem; Ogedengbe, Mosun E; Hafeez, Mian A; Barta, John R

    2013-07-01

    Multiple 18S rDNA sequences were obtained from two single-oocyst-derived lines of each of Eimeria meleagrimitis and Eimeria adenoeides. After analysing the 15 new 18S rDNA sequences from two lines of E. meleagrimitis and 17 new sequences from two lines of E. adenoeides, there were clear indications that divergent, paralogous 18S rDNA copies existed within the nuclear genome of E. meleagrimitis. In contrast, mitochondrial cytochrome c oxidase subunit I (COI) partial sequences from all lines of a particular Eimeria sp. were identical and, in phylogenetic analyses, COI sequences clustered unambiguously in monophyletic and highly-supported clades specific to individual Eimeria sp. Phylogenetic analysis of the new 18S rDNA sequences from E. meleagrimitis showed that they formed two distinct clades: Type A with four new sequences; and Type B with nine new sequences; both Types A and B sequences were obtained from each of the single-oocyst-derived lines of E. meleagrimitis. Together these rDNA types formed a well-supported E. meleagrimitis clade. Types A and B 18S rDNA sequences from E. meleagrimitis had a mean sequence identity of only 97.4% whereas mean sequence identity within types was 99.1-99.3%. The observed intraspecific sequence divergence among E. meleagrimitis 18S rDNA sequence types was even higher (approximately 2.6%) than the interspecific sequence divergence present between some well-recognized species such as Eimeria tenella and Eimeria necatrix (1.1%). Our observations suggest that, unlike COI sequences, 18S rDNA sequences are not reliable molecular markers to be used alone for species identification with coccidia, although 18S rDNA sequences have clear utility for phylogenetic reconstruction of apicomplexan parasites at the genus and higher taxonomic ranks.

  19. Effects of Spaceflight on Molecular and Cellular Responses to Bleomycin-Induced DNA Damages in Confluent Human Fibroblasts

    Science.gov (United States)

    Lu, Tao; Zhang, Ye; Wong, Michael; Stodieck, Louis; Karouia, Fathi; Wu, Honglu

    2016-01-01

    Spaceflights expose human beings to various risk factors. Among them are microgravity related physiological stresses in immune, cytoskeletal, and cardiovascular systems, and space radiation related elevation of cancer risk. Cosmic radiation consists of energetic protons and other heavier charged particles that induce DNA damages. Effective DNA damage response and repair mechanism is important to maintain genomic integrity and reduce cancer risk. There were studies on effects of spaceflight and microgravity on DNA damage response in cell and animal models, but the published results were mostly conflicting and inconsistent. To investigate effects of spaceflight on molecular and cellular responses to DNA damages, bleomycin, an anti-cancer drug and radiomimetic reagent, was used to induce DNA damages in confluent human fibroblasts flown to the International Space Station (ISS) and on ground. After exposure to 1.0 µg/ml bleomycin for 3 hours, cells were fixed for immunofluorescence assays and for RNA preparation. Extents of DNA damages were quantified by foci and pattern counting of phosphorylated histone protein H2AX (?-H2AX). The cells on the ISS showed modestly increased average foci counts per nucleus while the distribution of patterns was similar to that on the ground. PCR array analysis showed that expressions of several genes, including CDKN1A and PCNA, were significantly changed in response to DNA damages induced by bleomycin in both flight and ground control cells. However, there were no significant differences in the overall expression profile of DNA damage response genes between the flight and ground samples. Analysis of cellular proliferation status with Ki-67 staining showed a slightly higher proliferating population in cells on the ISS than those on ground. Our results suggested that the difference in ?-H2AX focus counts between flight and ground was due to the higher percentage of proliferating cells in space, but spaceflight did not significantly affect

  20. Ultrafast molecular rotor: an efficient sensor for premelting of natural DNA.

    Science.gov (United States)

    Murudkar, Sushant; Mora, Aruna K; Singh, Prabhat K; Nath, Sukhendu

    2012-05-28

    Structural changes in nucleic acids in the premelting region (T sensor to monitor the structural changes in natural DNA at T sensor used in the present study is superior than most commonly used DNA stains.

  1. Dynamic Coupling among Protein Binding, Sliding, and DNA Bending Revealed by Molecular Dynamics.

    Science.gov (United States)

    Tan, Cheng; Terakawa, Tsuyoshi; Takada, Shoji

    2016-07-13

    Protein binding to DNA changes the DNA's structure, and altered DNA structure can, in turn, modulate the dynamics of protein binding. This mutual dependency is poorly understood. Here we investigated dynamic couplings among protein binding to DNA, protein sliding on DNA, and DNA bending by applying a coarse-grained simulation method to the bacterial architectural protein HU and 14 other DNA-binding proteins. First, we verified our method by showing that the simulated HU exhibits a weak preference for A/T-rich regions of DNA and a much higher affinity for gapped and nicked DNA, consistent with biochemical experiments. The high affinity was attributed to a local DNA bend, but not the specific chemical moiety of the gap/nick. The long-time dynamic analysis revealed that HU sliding is associated with the movement of the local DNA bending site. Deciphering single sliding steps, we found the coupling between HU sliding and DNA bending is akin to neither induced-fit nor population-shift; instead they moved concomitantly. This is reminiscent of a cation transfer on DNA and can be viewed as a protein version of polaron-like sliding. Interestingly, on shorter time scales, HU paused when the DNA was highly bent at the bound position and escaped from pauses once the DNA spontaneously returned to a less bent structure. The HU sliding is largely regulated by DNA bending dynamics. With 14 other proteins, we explored the generality and versatility of the dynamic coupling and found that 6 of the 15 assayed proteins exhibit the polaron-like sliding.

  2. DNA as a molecular local thermal probe for the analysis of magnetic hyperthermia.

    Science.gov (United States)

    Dias, Jorge T; Moros, María; Del Pino, Pablo; Rivera, Sara; Grazú, Valeria; de la Fuente, Jesus M

    2013-10-25

    Too hot to handle: The surroundings of magnetic nanoparticles can be heated by applying a magnetic field. Polymer-coated magnetic nanoparticles were functionalized with single-stranded DNA molecules and further hybridized with DNA modified with different fluorophores. By correlating the denaturation profiles of the DNA with the local temperature, temperature gradients for the vicinity of the excited nanoparticles were determined.

  3. Mitochondrial DNA damage: Molecular marker of vulnerable nigral neurons in Parkinson's disease

    NARCIS (Netherlands)

    L.H. Sanders (Laurie); J. McCoy (Jennifer); X. Hu (Xiaoping); P.G. Mastroberardino (Pier); B.C. Dickinson (Bryan); C.J. Chang (Christopher); C.T. Chu (Charleen); B. van Houten (Bennett); J.T. Greenamyre (Timothy)

    2014-01-01

    textabstractDNA damage can cause (and result from) oxidative stress and mitochondrial impairment, both of which are implicated in the pathogenesis of Parkinson's disease (PD). We therefore examined the role of mitochondrial DNA (mtDNA) damage in human postmortem brain tissue and in in vivo and in vi

  4. Comparative Electrostatic Force Microscopy of Tetra- and Intra-Molecular G4-DNA

    CERN Document Server

    Livshits, Gideon I; Borovok, Natalia; Kotlyar, Alexander B; Porath, Danny

    2014-01-01

    Two forms of G4-DNA, with parallel and pairwise anti-parallel strands, are studied using atomic force microscopy. The directionality of the strands affects the molecules' structural properties (different height and length) and their electrical polarizability. Parallel G4-DNA is twice as polarizable as anti-parallel G4-DNA, suggesting it is a better electrical wire for bio-nanoelectronics.

  5. Studying Epigenetic DNA Modifications in Undergraduate Laboratories Using Complementary Bioinformatic and Molecular Approaches

    Science.gov (United States)

    Militello, Kevin T.

    2013-01-01

    Epigenetic inheritance is the inheritance of genetic information that is not based on DNA sequence alone. One type of epigenetic information that has come to the forefront in the last few years is modified DNA bases. The most common modified DNA base in nature is 5-methylcytosine. Herein, we describe a laboratory experiment that combines…

  6. Towards a rapid molecular diagnostic for melioidosis: comparison of DNA extraction methods from clinical specimens

    Science.gov (United States)

    Richardson, Leisha J; Kaestli, Mirjam; Mayo, Mark; Bowers, Jolene R; Tuanyok, Apichai; Schupp, Jim; Engelthaler, David; Wagner, David M; Keim, Paul S; Currie, Bart J

    2011-01-01

    Optimising DNA extraction from clinical samples for Burkholderia pseudomallei Type III secretion system real-time PCR in suspected melioidosis patients confirmed that urine and sputum are useful diagnostic samples. Direct testing on blood remains problematic; testing DNA extracted from plasma was superior to DNA from whole blood or buffy coat. PMID:22108495

  7. Understanding the Molecular Mechanism(s) of Formaldehyde-induced DNA-protein Crosslink Repair

    Science.gov (United States)

    Although formaldehyde has been shown to induce many kinds of DNA damage both in in vitro and in vivo assay systems, initial DNA-protein crosslink (DPC) formation might play a major role in FA-induced mutagenesis and carcinogenesis. Several DNA repair pathways, such as base excisi...

  8. Mitochondrial DNA damage: Molecular marker of vulnerable nigral neurons in Parkinson's disease

    NARCIS (Netherlands)

    L.H. Sanders (Laurie); J. McCoy (Jennifer); X. Hu (Xiaoping); P.G. Mastroberardino (Pier); B.C. Dickinson (Bryan); C.J. Chang (Christopher); C.T. Chu (Charleen); B. van Houten (Bennett); J.T. Greenamyre (Timothy)

    2014-01-01

    textabstractDNA damage can cause (and result from) oxidative stress and mitochondrial impairment, both of which are implicated in the pathogenesis of Parkinson's disease (PD). We therefore examined the role of mitochondrial DNA (mtDNA) damage in human postmortem brain tissue and in in vivo and in

  9. What Is Mitochondrial DNA?

    Science.gov (United States)

    ... DNA What is mitochondrial DNA? What is mitochondrial DNA? Although most DNA is packaged in chromosomes within ... proteins. For more information about mitochondria and mitochondrial DNA: Molecular Expressions, a web site from the Florida ...

  10. The low molecular weight DNA diffusion assay as an indicator of cytotoxicity for the in vitro comet assay.

    Science.gov (United States)

    Speit, Günter; Vesely, Alexandra; Schütz, Petra; Linsenmeyer, Regina; Bausinger, Julia

    2014-07-01

    The low molecular weight DNA diffusion assay (LMW assay) has been recommended as a measure for cytotoxicity for the in vivo comet assay. To better understand the relationship between effects in the LMW assay, DNA migration in the comet assay and effects in established cytotoxicity tests, we performed in vitro experiments with cultured human cell lines (TK6, A549) and comparatively investigated five test substances (methyl methanesulfonate, (±)-benzo[a]pyrene diol epoxide, sodium dodecyl sulphate, menthol and sodium arsenite). We measured DNA migration (tail intensity) in the comet assay and the frequency of 'hedgehogs' (cells with almost all DNA in the tail), DNA diffusion in the LMW assay, cell viability (trypan blue and fluorescein diacetate/ethidium bromide staining) and inhibition of proliferation (relative cell counts). Our in vitro experiments indicate that effects in the LMW assay occur independently from DNA effects in the comet assay and are not related to the occurrence of hedgehogs. Results from the LMW assay are in good agreement with results from viability assays and seem to allow discriminating genotoxic from non-genotoxic substances when appropriate preparation times are considered. Measurements of cytotoxicity by these methods only at an early preparation time after exposure to genotoxic substances may lead to erroneous results.

  11. Ribosomal DNA analysis of tsetse and non-tsetse transmitted Ethiopian Trypanosoma vivax strains in view of improved molecular diagnosis.

    Science.gov (United States)

    Fikru, Regassa; Matetovici, Irina; Rogé, Stijn; Merga, Bekana; Goddeeris, Bruno Maria; Büscher, Philippe; Van Reet, Nick

    2016-04-15

    Animal trypanosomosis caused by Trypanosoma vivax (T. vivax) is a devastating disease causing serious economic losses. Most molecular diagnostics for T. vivax infection target the ribosomal DNA locus (rDNA) but are challenged by the heterogeneity among T. vivax strains. In this study, we investigated the rDNA heterogeneity of Ethiopian T. vivax strains in relation to their presence in tsetse-infested and tsetse-free areas and its effect on molecular diagnosis. We sequenced the rDNA loci of six Ethiopian (three from tsetse-infested and three from tsetse-free areas) and one Nigerian T. vivax strain. We analysed the obtained sequences in silico for primer-mismatches of some commonly used diagnostic PCR assays and for GC content. With these data, we selected some rDNA diagnostic PCR assays for evaluation of their diagnostic accuracy. Furthermore we constructed two phylogenetic networks based on sequences within the smaller subunit (SSU) of 18S and within the 5.8S and internal transcribed spacer 2 (ITS2) to assess the relatedness of Ethiopian T. vivax strains to strains from other African countries and from South America. In silico analysis of the rDNA sequence showed important mismatches of some published diagnostic PCR primers and high GC content of T. vivax rDNA. The evaluation of selected diagnostic PCR assays with specimens from cattle under natural T. vivax challenge showed that this high GC content interferes with the diagnostic accuracy of PCR, especially in cases of mixed infections with T. congolense. Adding betain to the PCR reaction mixture can enhance the amplification of T. vivax rDNA but decreases the sensitivity for T. congolense and Trypanozoon. The networks illustrated that Ethiopian T. vivax strains are considerably heterogeneous and two strains (one from tsetse-infested and one from tsetse-free area) are more related to the West African and South American strains than to the East African strains. The rDNA locus sequence of six Ethiopian T. vivax

  12. Molecular Mechanism of Mot1, a TATA-binding Protein (TBP)-DNA Dissociating Enzyme.

    Science.gov (United States)

    Viswanathan, Ramya; True, Jason D; Auble, David T

    2016-07-22

    The essential Saccharomyces cerevisiae ATPase Mot1 globally regulates transcription by impacting the genomic distribution and activity of the TATA-binding protein (TBP). In vitro, Mot1 forms a ternary complex with TBP and DNA and can use ATP hydrolysis to dissociate the TBP-DNA complex. Prior work suggested an interaction between the ATPase domain and a functionally important segment of DNA flanking the TATA sequence. However, how ATP hydrolysis facilitates removal of TBP from DNA is not well understood, and several models have been proposed. To gain insight into the Mot1 mechanism, we dissected the role of the flanking DNA segment by biochemical analysis of complexes formed using DNAs with short single-stranded gaps. In parallel, we used a DNA tethered cleavage approach to map regions of Mot1 in proximity to the DNA under different conditions. Our results define non-equivalent roles for bases within a broad segment of flanking DNA required for Mot1 action. Moreover, we present biochemical evidence for two distinct conformations of the Mot1 ATPase, the detection of which can be modulated by ATP analogs as well as DNA sequence flanking the TATA sequence. We also show using purified complexes that Mot1 dissociation of a stable, high affinity TBP-DNA interaction is surprisingly inefficient, suggesting how other transcription factors that bind to TBP may compete with Mot1. Taken together, these results suggest that TBP-DNA affinity as well as other aspects of promoter sequence influence Mot1 function in vivo.

  13. Molecular basis for oligomeric-DNA binding and episome maintenance by KSHV LANA.

    Directory of Open Access Journals (Sweden)

    John F Domsic

    Full Text Available LANA is the KSHV-encoded terminal repeat binding protein essential for viral replication and episome maintenance during latency. We have determined the X-ray crystal structure of LANA C-terminal DNA binding domain (LANADBD to reveal its capacity to form a decameric ring with an exterior DNA binding surface. The dimeric core is structurally similar to EBV EBNA1 with an N-terminal arm that regulates DNA binding and is required for replication function. The oligomeric interface between LANA dimers is dispensable for single site DNA binding, but is required for cooperative DNA binding, replication function, and episome maintenance. We also identify a basic patch opposite of the DNA binding surface that is responsible for the interaction with BRD proteins and contributes to episome maintenance function. The structural features of LANADBD suggest a novel mechanism of episome maintenance through DNA-binding induced oligomeric assembly.

  14. An investigation of the structural transitions between different forms of DNA using the Adaptively Biased (ABMD) and Steered Molecular Dynamics Methods

    Science.gov (United States)

    Moradi, Mahmoud; Babin, Volodymyr; Roland, Christopher; Darden, Thomas A.; Sagui, Celeste

    2008-10-01

    Left-handed A-DNA and B-DNA along with right-handed Z-DNA, are believed to be the three main biologically active double-helix structures associated with DNA. The free energy differences associated with the A to B-DNA, and B to Z-DNA transitions in an implicit solvent environment have been investigated using the recently developed Adaptively Biased Molecular Dynamics (ABMD) method, with the RMSD as the collective variable associated with the former transition, and handedness and radius of gyration as the collective variables associated with the latter. The ABMD method belongs to the general category of umbrella sampling methods with a time-dependent potential, and allows for an accurate estimation of the free energy barriers associated with the transitions. The results are compared to those obtained using the Steered Molecular Dynamics method, and ultimately are used in order to gain insight into the microscopics of the DNA transitions.

  15. Molecular cloning and nucleotide sequence of a transforming gene detected by transfection of chicken B-cell lymphoma DNA

    Science.gov (United States)

    Goubin, Gerard; Goldman, Debra S.; Luce, Judith; Neiman, Paul E.; Cooper, Geoffrey M.

    1983-03-01

    A transforming gene detected by transfection of chicken B-cell lymphoma DNA has been isolated by molecular cloning. It is homologous to a conserved family of sequences present in normal chicken and human DNAs but is not related to transforming genes of acutely transforming retroviruses. The nucleotide sequence of the cloned transforming gene suggests that it encodes a protein that is partially homologous to the amino terminus of transferrin and related proteins although only about one tenth the size of transferrin.

  16. Application of steered molecular dynamics (SMD) to study DNA-drug complexes and probing helical propensity of amino acids

    Energy Technology Data Exchange (ETDEWEB)

    Orzechowski, Marek [Faculty of Chemistry, Warsaw University, 1 Pasteura Street, Warsaw, 02-093 (Poland); Cieplak, Piotr [Accelrys Incorporated, 9685 Scranton Road, San Diego, CA 92121 (United States)

    2005-05-11

    We present the preliminary results of two computer experiments involving the application of an external force to molecular systems. In the first experiment we simulated the process of pulling out a simple intercalator, the 9-aminoacridine molecule, from its complex with a short DNA oligonucleotide in aqueous solution. Removing a drug from the DNA is assumed to be an opposite process to the complex formation. The force and energy profiles suggest that formation of the DNA-9-aminoacridine complex is preferred when the acridine approaches the DNA from the minor groove rather than the major groove side. For a given mode of pulling the intercalation process is also shown to be nucleotide sequence dependent. In another computer experiment we performed a series of molecular dynamics simulations for stretching short, containing 15 amino acids, helical polypeptides in aqueous solution using an external force. The purpose of these simulations is to check whether this type of approach is sensitive enough to probe the sequence dependent helical propensity of short polypeptides.

  17. Molecular Mechanism of Dioxin Action: Molecular Cloning of the Ah Receptor Using a DNA Recognition Site Probe

    Science.gov (United States)

    1992-01-13

    DNA with high affinity (Whitlock and Galeazzi , 1984; Henry et al., 1989; Denison and Yao, 1991). Biochemical and genetic studies (Denison et al., 1988a...Pharmacol. Toxicol. 30: 251-277. Whitlock, J. P., Jr. (1987) Pharmacol. Rev. 39: 147-161. Whitlock, J. P., Jr. and Galeazzi , D. R. (1984) J. Biol

  18. Cooperative assembly of Co-Smad4 MH1 with R-Smad1/3 MH1 on DNA: a molecular dynamics simulation study.

    Directory of Open Access Journals (Sweden)

    Guihong Wang

    Full Text Available BACKGROUND: Smads, the homologs of Sma and MAD proteins, play a key role in gene expression regulation in the transforming growth factor-β (TGF-β signaling pathway. Recent experimental studies have revealed that Smad4/R-Smad heterodimers bound on DNA are energetically more favorable than homodimeric R-Smad/R-Smad complexes bound on DNA, which indicates that Smad4 might act as binding vehicle to cooperatively assemble with activated R-Smads on DNA in the nucleus. However, the details of interaction mechanism for cooperative recruitment of Smad4 protein to R-Smad proteins on DNA, and allosteric communication between the Smad4-DNA and R-Smad-DNA interfaces via DNA mediating are not yet clear so far. METHODOLOGY: In the present work, we have constructed a series of Smadn+DNA+Smadn (n = 1, 3, 4 models and carried out molecular dynamics simulations, free energy calculations and DNA dynamics analysis for them to study the interaction properties of Smadn (n = 1, 3, 4 with DNA molecule. RESULTS: The results revealed that the binding of Smad4 protein to DNA molecule facilitates energetically the formation of the heteromeric Smad4+DNA+Smad1/3 complex by increasing the affinity of Smad1/3 with DNA molecule. Further investigations through the residue/base motion correlation and DNA dynamics analyses predicted that the binding of Smad4 protein to DNA molecule in the heteromeric Smad4+DNA+Smad1/3 model induces an allosteric communication from the Smad4-DNA interface to Smad1/Smad3-DNA interface via DNA base-pair helical motions, surface conformation changes and new hydrogen bond formations. The present work theoretically explains the mechanism of cooperative recruitment of Smad4 protein to Smad1/3 protein via DNA-mediated indirect readout mode in the nucleus.

  19. Synthesis, characterization, biological studies (DNA binding, cleavage, antibacterial and topoisomerase I) and molecular docking of copper(II) benzimidazole complexes.

    Science.gov (United States)

    Arjmand, Farukh; Parveen, Shazia; Afzal, Mohd; Shahid, Mohd

    2012-09-03

    To explore the therapeutic potential of copper-based benzimidazole complexes, tetranuclear Cu(II) complex 1 and dinuclear ternary amino acid complexes 2 and 3 {L-trp and L-val, respectively} were synthesized and thoroughly characterized. In vitro DNA binding studies of complexes 1-3 were carried out employing UV-vis titrations, fluorescence, circular dichroic and viscosity measurements which revealed that the complexes 1-3 bind to CT DNA preferably via groove binding. Complex 1 cleaved pBR322 DNA via hydrolytic pathway (validated by T4 DNA ligase assay), accessible to major groove while 2 followed oxidative mechanism, binding to minor groove of DNA double helix; binding events were further validated by molecular docking studies. Additionally, the complexes 1 and 2 exhibit high Topo-I inhibitory activity at different concentrations. The complexes 1-3 were evaluated for antibacterial activity against Escherichia coli and Staphylococcus aureus, and 2 was found to be most effective against Gram-positive bacteria.

  20. The Molecular Mechanisms and the Role of hnRNP K Protein Post- Translational Modification in DNA Damage Repair.

    Science.gov (United States)

    Lu, Jing; Gao, Feng-Hou

    2017-01-01

    DNA damage repair is a kind of cellular self-protection mechanism in which some relevant proteins are activated when DNA damage response occurs in order to maintain the intracellular function stability and structure integrity. Post-translational modifications (PTMs) of proteins can rapidly confer to them more complicated structure and sophisticated function by covalently combining different small molecules with target proteins, which in turn plays an important regulatory role in DNA damage repair. It was reported that heterogeneous nuclear ribonucleoprotein K (hnRNP K) could be involved in DNA damage repair process under the regulation of its many post-translational modifications, including methylation, ubiquitination, sumoylation and phosphorylation. Here, we reviewed molecular mechanisms of hnRNP K protein post-translational modifications and their role in DNA damage repair, which will promote our understanding of how hnRNP K participating in the repair process to maintain the normal operation of biological activities in the cells. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  1. The Molecular Crosstalk between the MET Receptor Tyrosine Kinase and the DNA Damage Response — Biological and Clinical Aspects

    Energy Technology Data Exchange (ETDEWEB)

    Medová, Michaela, E-mail: michaela.medova@dkf.unibe.ch; Aebersold, Daniel M.; Zimmer, Yitzhak, E-mail: michaela.medova@dkf.unibe.ch [Department of Radiation Oncology, Inselspital, Bern University Hospital, and University of Bern, 3010 Bern (Switzerland); Department of Clinical Research, University of Bern, DKF, MEM-E807, Murtenstrasse 35, 3010 Bern (Switzerland)

    2013-12-19

    Radiation therapy remains an imperative treatment modality for numerous malignancies. Enduring significant technical achievements both on the levels of treatment planning and radiation delivery have led to improvements in local control of tumor growth and reduction in healthy tissue toxicity. Nevertheless, resistance mechanisms, which presumably also involve activation of DNA damage response signaling pathways that eventually may account for loco-regional relapse and consequent tumor progression, still remain a critical problem. Accumulating data suggest that signaling via growth factor receptor tyrosine kinases, which are aberrantly expressed in many tumors, may interfere with the cytotoxic impact of ionizing radiation via the direct activation of the DNA damage response, leading eventually to so-called tumor radioresistance. The aim of this review is to overview the current known data that support a molecular crosstalk between the hepatocyte growth factor receptor tyrosine kinase MET and the DNA damage response. Apart of extending well established concepts over MET biology beyond its function as a growth factor receptor, these observations directly relate to the role of its aberrant activity in resistance to DNA damaging agents, such as ionizing radiation, which are routinely used in cancer therapy and advocate tumor sensitization towards DNA damaging agents in combination with MET targeting.

  2. Molecular characterization of a new begomovirus infecting Sida cordifolia and its associated satellite DNA molecules.

    Science.gov (United States)

    Guo, Xiaojian; Zhou, Xueping

    2006-12-01

    Two virus isolates Hn57 and Hn60 were obtained from Sida cordifolia showing mild upward leaf-curling symptoms in Hainan province of China. Comparison of partial sequences of DNA-A like molecule confirmed the existence of a single type of begomovirus. The complete nucleotide sequence of DNA-A of Hn57 was determined to be 2757 nucleotides, with a genomic organization typical of begomoviruses. Complete sequence comparison with other reported begomoviruses revealed that Hn57 DNA-A has the highest sequence identity (71.0%) with that of Tobacco leaf curl Yunnan virus. Consequently, Hn57 was considered to be a new begomovirus species, for which the name Sida leaf curl virus (SiLCV) is proposed. In addition to DNA-A molecule, two additional circular single-stranded satellite DNA molecules corresponding to DNAbeta and DNA1 were found to be associated with SiLCV isolates. Both DNAbeta and DNA1 were approximately half the size of their cognate genomic DNA. Sequence analysis shows that DNAbeta of Hn57 and Hn60 share 93.8% nucleotide sequence identity, and they have the highest sequence identity (58.5%) with DNAbeta associated with Ageratum leaf curl disease (AJ316027). The nucleotide sequence identity between DNA1 of Hn57 and that of Hn60 was 83.8%, they share 58.2-79.3% nucleotide sequence identities in comparison with other previously reported DNAl.

  3. Studies of DNA-binding properties of lafutidine as adjuvant anticancer agent to calf thymus DNA using multi-spectroscopic approaches, NMR relaxation data, molecular docking and dynamical simulation.

    Science.gov (United States)

    Yang, Hongqin; Tang, Peixiao; Tang, Bin; Huang, Yanmei; He, Jiawei; Li, Shanshan; Li, Hui

    2017-06-01

    The interactions between lafutidine (LAF) and calf thymus DNA (ctDNA) have been investigated both experimentally and theoretically. UV-vis absorption studies confirmed that LAF binds to ctDNA through non-covalent interactions. Fluorescence quenching and time-resolved fluorescence spectroscopy studies showed that the binding of LAF with ctDNA occurred through static quenching mechanism, resulting in the formation of a LAF-ctDNA complex. The binding constants (K) of the complex were found to be around 10(3)M(-1) via NMR relaxation rates and fluorescence data, and the calculated thermodynamic parameters indicated that hydrogen bonds and van der Waals forces played major roles in the binding of LAF to ctDNA. The changes in CD spectra indicated that LAF induced a slight perturbation on the base stacking and helicity of B-DNA. A comparative study of the LAF-ctDNA complex with respect to potassium iodide quenching experiments and competition displacement assays with ethidium bromide, acridine orange, and Hoechst 33258 probes suggested that LAF interacted with ctDNA by minor groove mode. Molecular docking analysis further supported the minor groove binding. Molecular dynamics simulation indicated that LAF depart from the C-G region of DNA, but it can steadily bind with the middle part of DNA composed by A-T base pairs. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. Molecular characterization of NAD+-dependent DNA ligase from Wolbachia endosymbiont of lymphatic filarial parasite Brugia malayi.

    Directory of Open Access Journals (Sweden)

    Nidhi Shrivastava

    Full Text Available The lymphatic filarial parasite, Brugia malayi contains Wolbachia endobacteria that are essential for development, viability and fertility of the parasite. Therefore, wolbachial proteins have been currently seen as the potential antifilarial drug targets. NAD(+-dependent DNA ligase is characterized as a promising drug target in several organisms due to its crucial, indispensable role in DNA replication, recombination and DNA repair. We report here the cloning, expression and purification of NAD(+-dependent DNA ligase of Wolbachia endosymbiont of B. malayi (wBm-LigA for its molecular characterization. wBm-LigA has all the domains that are present in nearly all the eubacterial NAD(+-dependent DNA ligases such as N-terminal adenylation domain, OB fold, helix-hairpin-helix (HhH and BRCT domain except zinc-binding tetracysteine domain. The purified recombinant protein (683-amino acid was found to be biochemically active and was present in its native form as revealed by the circular dichroism and fluorescence spectra. The purified recombinant enzyme was able to catalyze intramolecular strand joining on a nicked DNA as well as intermolecular joining of the cohesive ends of BstEII restricted lamda DNA in an in vitro assay. The enzyme was localized in the various life-stages of B. malayi parasites by immunoblotting and high enzyme expression was observed in Wolbachia within B. malayi microfilariae and female adult parasites along the hypodermal chords and in the gravid portion as evident by the confocal microscopy. Ours is the first report on this enzyme of Wolbachia and these findings would assist in validating the antifilarial drug target potential of wBm-LigA in future studies.

  5. Circulating cell-free DNA: an up-coming molecular marker in exercise physiology.

    Science.gov (United States)

    Breitbach, Sarah; Tug, Suzan; Simon, Perikles

    2012-07-01

    The phenomenon of circulating cell-free DNA (cfDNA) concentrations is of importance for many biomedical disciplines including the field of exercise physiology. Increases of cfDNA due to exercise are described to be a potential hallmark for the overtraining syndrome and might be related to, or trigger adaptations of, immune function induced by strenuous exercise. At the same time, exercise provides a practicable model for studying the phenomenon of cfDNA that is described to be of pathophysiological relevance for different topics in clinical medicine like autoimmune diseases and cancer. In this review, we are summarizing the current knowledge of exercise-based acute and chronic alterations in cfDNA levels and their physiological significance. The effects of acute exercise on cfDNA concentrations have been investigated in resistance exercises and in continuous, stepwise and interval endurance exercises of different durations. cfDNA concentrations peaked immediately after acute exercise and showed a rapid return to baseline levels. Typical markers of skeletal muscle damage (creatine kinase, uric acid, C-reactive protein) show delayed kinetics compared with the cfDNA peak response. Exercise parameters such as intensity, duration or average energy expenditure do not explain the extent of increasing cfDNA concentrations after strenuous exercise. This could be due to complex processes inside the human organism during and after physical activity. Therefore, we hypothesize composite effects of different physiological stress parameters that come along with exercise to be responsible for increasing cfDNA concentrations. We suggest that due to acute stress, cfDNA levels increase rapidly by a spontaneous active or passive release mechanism that is not yet known. As a result of the rapid and parallel increase of cfDNA and lactate in an incremental treadmill test leading to exhaustion within 15-20 minutes, it is unlikely that cfDNA is released into the plasma by typical necrosis

  6. Phylogenetic diversity of insecticolous fusaria inferred from multilocus DNA sequence data and their molecular identification via FUSARIUM-ID and Fusarium MLST

    NARCIS (Netherlands)

    O'Donnell, Kerry; Humber, Richard A; Geiser, David M; Kang, Seogchan; Park, Bongsoo; Robert, Vincent A R G; Crous, Pedro W; Johnston, Peter R; Aoki, Takayuki; Rooney, Alejandro P; Rehner, Stephen A

    2011-01-01

    We constructed several multilocus DNA sequence datasets to assess the phylogenetic diversity of insecticolous fusaria, especially focusing on those housed at the Agricultural Research Service Collection of Entomopathogenic Fungi (ARSEF), and to aid molecular identifications of unknowns via the FUSAR

  7. Phylogenetic diversity of insecticolous fusaria inferred from multilocus DNA sequence data and their molecular identification via FUSARIUM-ID and Fusarium MLST

    NARCIS (Netherlands)

    O'Donnell, K.; Humber, R.A.; Geiser, D.M.; Kang, S.; Park, B.; Robert, V.; Crous, P.W.; Johnston, P.R.; Aoki, T.; Rooney, A.P.; Rehner, S.A.

    2012-01-01

    We constructed several multilocus DNA sequence datasets to assess the phylogenetic diversity of insecticolous fusaria, especially focusing on those housed in the Agricultural Research Service Collection of Entomopathogenic Fungi (ARSEF), and to facilitate molecular identifications of unknowns via th

  8. Phylogenetic diversity of insecticolous fusaria inferred from multilocus DNA sequence data and their molecular identification via FUSARIUM-ID and Fusarium MLST

    NARCIS (Netherlands)

    O'Donnell, K.; Humber, R.A.; Geiser, D.M.; Kang, S.; Robert, V.; Park, B.; Crous, P.W.; Johnston, P.; Aoki, T.; Rooney, A.P.; Rehner, S.A.

    2012-01-01

    We constructed several multilocus DNA sequence datasets to assess the phylogenetic diversity of insecticolous fusaria, especially focusing on those housed at the Agricultural Research Service Collection of Entomopathogenic Fungi (ARSEF), and to aid molecular identifications of unknowns via the FUSAR

  9. Phylogenetic diversity of insecticolous fusaria inferred from multilocus DNA sequence data and their molecular identification via FUSARIUM-ID and Fusarium MLST

    Science.gov (United States)

    We constructed several multilocus Deoxyribonucleic acid (DNA) sequence datasets to assess the phylogenetic diversity of insecticolous fusaria, especially focusing on those housed in the Agricultural Research Service Collection of Entomopathogenic Fungi (ARSEF), and to facilitate molecular identifica...

  10. Comparative analysis of four methods to extract DNA from paraffin-embedded tissues: effect on downstream molecular applications

    Directory of Open Access Journals (Sweden)

    Savelkoul Paul HM

    2010-09-01

    Full Text Available Abstract Background A large portion of tissues stored worldwide for diagnostic purposes is formalin-fixed and paraffin-embedded (FFPE. These FFPE-archived tissues are an extremely valuable source for retrospective (genetic studies. These include mutation screening in cancer-critical genes as well as pathogen detection. In this study we evaluated the impact of several widely used DNA extraction methods on the quality of molecular diagnostics on FFPE tissues. Findings We compared 4 DNA extraction methods from 4 identically processed FFPE mammary-, prostate-, colon- and lung tissues with regard to PCR inhibition, real time SNP detection and amplifiable fragment size. The extraction methods, with and without proteinase K pre-treatment, tested were: 1 heat-treatment, 2 QIAamp DNA-blood-mini-kit, 3 EasyMAG NucliSens and 4 Gentra Capture-Column-kit. Amplifiable DNA fragment size was assessed by multiplexed 200-400-600 bp PCR and appeared highly influenced by the extraction method used. Proteinase K pre-treatment was a prerequisite for proper purification of DNA from FFPE. Extractions with QIAamp, EasyMAG and heat-treatment were found suitable for amplification of fragments up to 400 bp from all tissues, 600 bp amplification was marginally successful (best was QIAamp. QIAamp and EasyMAG extracts were found suitable for downstream real time SNP detection. Gentra extraction was unsuitable. Hands-on time was lowest for heat-treatment, followed by EasyMAG. Conclusions We conclude that the extraction method plays an important role with regard to performance in downstream molecular applications.

  11. "DNA Re-EvolutioN": A Game for Learning Molecular Genetics and Evolution

    Science.gov (United States)

    Miralles, Laura; Moran, Paloma; Dopico, Eduardo; Garcia-Vazquez, Eva

    2013-01-01

    Evolution is a main concept in biology, but not many students understand how it works. In this article we introduce the game "DNA Re-EvolutioN" as an active learning tool that uses genetic concepts (DNA structure, transcription and translation, mutations, natural selection, etc.) as playing rules. Students will learn about molecular…

  12. Molecular cloning and expression of Treponema pallidum DNA in Escherichia coli K12.

    NARCIS (Netherlands)

    J.D.A. van Embden; H.J.M. van de Donk; R.V.W. van Eijk (Ron); H.G. v.d. Heide; J.A. de Jong (Jan); M.F. van Olderen; A.D.M.E. Osterhaus (Albert); L.M. Schouls

    1983-01-01

    textabstractA gene bank of Treponema pallidum DNA in Escherichia coli K-12 was constructed by cloning SauI-cleaved T. pallidum DNA into the cosmid pHC79. Sixteen of 800 clones investigated produced one or more antigens that reacted with antibodies from syphilitic patients. According to the separatio

  13. "DNA Re-EvolutioN": A Game for Learning Molecular Genetics and Evolution

    Science.gov (United States)

    Miralles, Laura; Moran, Paloma; Dopico, Eduardo; Garcia-Vazquez, Eva

    2013-01-01

    Evolution is a main concept in biology, but not many students understand how it works. In this article we introduce the game "DNA Re-EvolutioN" as an active learning tool that uses genetic concepts (DNA structure, transcription and translation, mutations, natural selection, etc.) as playing rules. Students will learn about molecular…

  14. Molecular behavior of DNA in a cell-sized compartment coated by lipids

    CERN Document Server

    Hamada, T; Shimobayashi, S F; Ichikawa, M; Takagi, M

    2015-01-01

    The behavior of long DNA molecules in a cell-sized confined space was investigated. We prepared water-in-oil droplets covered by phospholipids, which mimic the inner space of a cell, following the encapsulation of DNA molecules with unfolded coil and folded globule conformations. Microscopic observation revealed that the adsorption of coiled DNA onto the membrane surface depended on the size of the vesicular space. Globular DNA showed a cell-size-dependent unfolding transition after adsorption on the membrane. Furthermore, when DNA interacted with a two-phase membrane surface, DNA selectively adsorbed on the membrane phase, such as an ordered or disordered phase, depending on its conformation. We discuss the mechanism of these trends by considering the free energy of DNA together with a polyamine in the solution. The free energy of our model was consistent with the present experimental data. The cooperative interaction of DNA and polyamines with a membrane surface leads to the size-dependent behavior of molec...

  15. Molecular characterization and physical localization of highly repetitive DNA sequences from Brazilian Alstroemeria species

    NARCIS (Netherlands)

    Kuipers, A.G.J.; Kamstra, S.A.; Jeu, de M.J.; Jacobsen, E.

    2002-01-01

    Highly repetitive DNA sequences were isolated from genomic DNA libraries of Alstroemeria psittacina and A. inodora. Among the repetitive sequences that were isolated, tandem repeats as well as dispersed repeats could be discerned. The tandem repeats belonged to a family of interlinked Sau3A subfragm

  16. Size-based molecular diagnostics using plasma DNA for noninvasive prenatal testing

    NARCIS (Netherlands)

    Yu, S.C.; Chan, K.C.; Zheng, Y.W.; Jiang, P.; Liao, G.J.; Sun, H; Akolekar, R.; Leung, T.Y.; Go, A.T.; Vugt, J.M.G. van; Minekawa, R.; Oudejans, C.B.; Nicolaides, K.H.; Chiu, R.W.; Lo, Y.M.

    2014-01-01

    Noninvasive prenatal testing using fetal DNA in maternal plasma is an actively researched area. The current generation of tests using massively parallel sequencing is based on counting plasma DNA sequences originating from different genomic regions. In this study, we explored a different approach th

  17. Characterization of the DNA of the hamster papovavirus: I. Genom length and molecular cloning.

    Science.gov (United States)

    Vogel, F; Zimmermann, W; Krause, H; Scherneck, S

    1984-01-01

    The complete genome of the hamster papovavirus (HaPV) which was isolated from virions found in multiple skin tumors of the Syrian hamsters was measured by electron microscopy and cloned in Escherichia coli using the certified plasmid vector pBR322. The cloned viral DNA were characterized by digestion of the recombinant DNA with various restriction enzymes followed by comparison of their electrophoretic mobilities in agarose gels with that of similarly digested uncloned DNA and by electron microscopy to determine the genome size of cloned HaPV DNA. The restriction enzyme analysis of the cloned HaPV DNA showed the same cleavage pattern as the corresponding fragments from the uncloned DNA. No major insertions or deletions could be detected by heteroduplex analysis between cloned HaPV DNA and the starting material. The estimated genome size of 5.52 kb for HaPV DNA is approx. 300 bases larger than those determined for other known papovaviruses as SV40 or polyoma.

  18. DNA repair pathways in radiation induced cellular damage: a molecular approach

    NARCIS (Netherlands)

    L.R. van Veelen (Lieneke)

    2005-01-01

    markdownabstract__Abstract__ DNA damage, especially double-strand breaks, can be induced by endogenous or exogenous darnaging agents, such as ionizing radiation. Repair of DNA damage is very important in maintaining genomic stability. Incorrect repair may lead to chromosomal aberrations,

  19. DNA repair pathways in radiation induced cellular damage: a molecular approach

    NARCIS (Netherlands)

    L.R. van Veelen (Lieneke)

    2005-01-01

    markdownabstract__Abstract__ DNA damage, especially double-strand breaks, can be induced by endogenous or exogenous darnaging agents, such as ionizing radiation. Repair of DNA damage is very important in maintaining genomic stability. Incorrect repair may lead to chromosomal aberrations, translocat

  20. Maintenance of Genome Stability and Breast Cancer: Molecular Analysis of DNA Damage-Activated Kinases

    Science.gov (United States)

    2008-03-01

    replication, leading to the generation of abnormally large amounts of RPA-coated single-stranded DNA (12). For this reason, RPA-ssDNA is thought to be...A. Nicolas. 2002. Replication protein A is required for meiotic recombination in Saccharomyces cerevisiae. Genetics 161:535–547. 45. Stauffer, M. E

  1. Synthesis, interactions, molecular structure, biological properties and molecular docking studies on Mn, Co, Zn complexes containing acetylacetone and pyridine ligands with DNA duplex.

    Science.gov (United States)

    Thamilarasan, V; Sengottuvelan, N; Stalin, N; Srinivasan, P; Chakkaravarthi, G

    2016-07-01

    Three metal complexes (1-3) of the type [Mn(acac)2(py)·H2O] (1), [Co(acac)2(py)·H2O] (2) and [Zn(acac)2(py)·H2O] (3), [Where acac=acetylacetone, py=pyridine] were synthesized and characterized by spectral (UV-vis, FT-IR, ESI-mass) analysis. The structure of complex 2 has been determined by single crystal X-ray diffraction studies and the configuration of ligand-coordinated to metal(II) ion was well described as distorted octahedral coordination geometry. The interaction of the complexes with CT-DNA has been explored by absorption, fluorescence, circular dichromism spectroscopy, viscosity measurements and molecular docking studies. The intrinsic binding constant Kb of complexes 1-3 with CT-DNA obtained from UV-vis absorption spectral studies were 2.1×10(4), 2.1×10(5) and 1.98×10(4)M(-1), respectively, which revealed that the complexes could interact with CT-DNA through groove binding. The results indicated that the complexes (1-3) were able to bind to DNA with different binding affinity, in the order: 2>1>3. The interaction of the compounds with bovine serum albumins were also investigated using fluorescence methods and the gel electrophoresis assay demonstrates weak cleavage ability of the pBR322 plasmid DNA in the presence of the metal complexes (1-3) with various activators. Further, the in vitro cytotoxic effect of the complexes were examined on cancerous cell line, with human breast cancer cells MCF-7.

  2. A switchable DNA origami nanochannel for regulating molecular transport at the nanometer scale

    Science.gov (United States)

    Wang, Dianming; Zhang, Yiyang; Wang, Miao; Dong, Yuanchen; Zhou, Chao; Isbell, Mark Antonin; Yang, Zhongqiang; Liu, Huajie; Liu, Dongsheng

    2016-02-01

    A nanochannel with a shutter at one end was built by DNA nanotechnology. Using DNA hybridization the shutter could be opened or closed, influencing the transport of materials through the channel. This process was visualized by an enzyme cascade reaction occurring in the structure.A nanochannel with a shutter at one end was built by DNA nanotechnology. Using DNA hybridization the shutter could be opened or closed, influencing the transport of materials through the channel. This process was visualized by an enzyme cascade reaction occurring in the structure. Electronic supplementary information (ESI) available: Experimental details including methods, materials, ESI figures and DNA sequences. See DOI: 10.1039/c5nr08206d

  3. Molecular cloning and mammalian expression of human beta 2-glycoprotein I cDNA

    DEFF Research Database (Denmark)

    Kristensen, Torsten; Schousboe, Inger; Boel, Espen

    1991-01-01

    Human β2-glycoprotein (β2gpI) cDNA was isolated from a liver cDNA library and sequenced. The cDNA encoded a 19-residue hydrophobic signal peptide followed by the mature β2gpI of 326 amino acid residues. In liver and in the hepatoma cell line HepG2 there are two mRNA species of about 1.4 and 4.3 k......, respectively, hybridizing specifically with the β2gpI cDNA. Upon isoelectric focusing, recombinant β2gpI obtained from expression of β2gpI cDNA in baby hamster kidney cells showed the same pattern of bands as β2gpI isolated from plasma, and at least 5 polypeptides were visible...

  4. Recognition, signaling, and repair of DNA double-strand breaks produced by ionizing radiation in mammalian cells: the molecular choreography.

    Science.gov (United States)

    Thompson, Larry H

    2012-01-01

    The faithful maintenance of chromosome continuity in human cells during DNA replication and repair is critical for preventing the conversion of normal diploid cells to an oncogenic state. The evolution of higher eukaryotic cells endowed them with a large genetic investment in the molecular machinery that ensures chromosome stability. In mammalian and other vertebrate cells, the elimination of double-strand breaks with minimal nucleotide sequence change involves the spatiotemporal orchestration of a seemingly endless number of proteins ranging in their action from the nucleotide level to nucleosome organization and chromosome architecture. DNA DSBs trigger a myriad of post-translational modifications that alter catalytic activities and the specificity of protein interactions: phosphorylation, acetylation, methylation, ubiquitylation, and SUMOylation, followed by the reversal of these changes as repair is completed. "Superfluous" protein recruitment to damage sites, functional redundancy, and alternative pathways ensure that DSB repair is extremely efficient, both quantitatively and qualitatively. This review strives to integrate the information about the molecular mechanisms of DSB repair that has emerged over the last two decades with a focus on DSBs produced by the prototype agent ionizing radiation (IR). The exponential growth of molecular studies, heavily driven by RNA knockdown technology, now reveals an outline of how many key protein players in genome stability and cancer biology perform their interwoven tasks, e.g. ATM, ATR, DNA-PK, Chk1, Chk2, PARP1/2/3, 53BP1, BRCA1, BRCA2, BLM, RAD51, and the MRE11-RAD50-NBS1 complex. Thus, the nature of the intricate coordination of repair processes with cell cycle progression is becoming apparent. This review also links molecular abnormalities to cellular pathology as much a possible and provides a framework of temporal relationships.

  5. Exploration of cell cycle regulation and modulation of the DNA methylation mechanism of pelargonidin: Insights from the molecular modeling approach.

    Science.gov (United States)

    Karthi, Natesan; Karthiga, Arumugasamy; Kalaiyarasu, Thangaraj; Stalin, Antony; Manju, Vaiyapuri; Singh, Sanjeev Kumar; Cyril, Ravi; Lee, Sang-Myeong

    2017-10-01

    Pelargonidin is an anthocyanidin isolated from plant resources. It shows strong cytotoxicity toward various cancer cell lines, even though the carcinogenesis-modulating pathway of pelargonidin is not yet known. One of our previous reports showed that pelargonidin arrests the cell cycle and induces apoptosis in HT29 cells. Flowcytometry and immunoblot analysis confirmed that pelargonidin specifically inhibits the activation of CDK1 and blocks the G2-M transition of the cell cycle. In addition, DNA fragmentation was observed along with induction of cytochrome c release-mediated apoptosis. Hence, the aim of the present study was to investigate the molecular mechanism of pelargonidin's action on cell cycle regulators CDK1, CDK4, and CDK6 as well as the substrate-binding domain of DNMT1 and DNMT3A, which regulate the epigenetic signals related to DNA methylation. The results of docking analysis, binding free energy calculation, and molecular dynamics simulation correlated with the experimental results, and pelargonidin showed a specific interaction with CDK1. In this context, pelargonidin may also inhibit the recognition of DNA and catalytic binding by DNMT1 and DNMT3A. The HOMO-LUMO analysis mapped the functional groups of pelargonidin. Prediction of pharmacological descriptors suggested that pelargonidin can serve as a multitarget inhibitor for cancer treatment. Copyright © 2017 Elsevier Ltd. All rights reserved.

  6. Molecular characteristics of mitochondrial DNA and phylogenetic analysis of the loach (Misgurnus anguillicaudatus) from the Poyang Lake.

    Science.gov (United States)

    Zeng, Liugen; Wang, Junhua; Sheng, Junqing; Gu, Qing; Hong, Yijiang

    2012-06-01

    The goal of our study was to investigate the molecular characteristics of mitochondrial DNA (mtDNA) and phylogenetic construction of the weather loach (Misgurnus anguillicaudatus) in Poyang Lake. The complete mitochondrial genome was 16,634 bp, and the gene order was identical to that of teleost fishes. Compared with the previous reported weather loach in China, there were numerous nucleotide substitutions and length polymorphisms on the structural genes of mitochondrial DNA in the loach from the Poyang Lake. The Phylogenetic tree indicated that the loach had its own molecular characteristics and was somewhat different from those in other regions of China. Fourteen unique haplotypes of the cytochrome b (cyt b) gene were obtained from 300 weather loaches. The Phylogenetic tree based on the cyt b gene showed that the loaches were substructured into two different populations in The Poyang Lake. Results indicated that the loaches in Poyang Lake not only showed the same phylogeny as the loaches in other areas of China, but also generated its own unique phylogenetic relationships.

  7. WRN Exonuclease Structure, Molecular Mechanism, and DNA EndProcessing Role

    Energy Technology Data Exchange (ETDEWEB)

    Perry, J. Jefferson P.; Yannone, Steven M.; Holden, Lauren G.; Hitomi, Chiharu; Asaithamby, Aroumougame; Han, Seungil; Cooper, PriscillaK.; Chen, David J.; Tainer, John A.

    2006-02-15

    WRN is unique among the five human RecQ DNA helicases by having a functional exonuclease domain (WRN-exo) and being defective in the premature aging and cancer-related disorder Werner syndrome. Here, we characterize WRN-exo crystal structures, biochemical activity and participation in DNA end-joining. Metal ion complex structures, active site mutations and activity assays reveal a two-metal-ion mediated nuclease mechanism. The DNA end-binding Ku70/80 complex specifically stimulates WRN-exo activity, and structure-based mutational inactivation of WRN-exo alters DNA end-joining in human cells. We furthermore establish structural and biochemical similarities of WRN-exo to DnaQ family replicative proofreading exonucleases, with WRN-specific adaptations consistent with dsDNA specificity and functionally important conformational changes. These results indicate WRN-exo is a human DnaQ family member and support analogous proof-reading activities that are stimulated by Ku70/80 with implications for WRN functions in age related pathologies and maintenance of genomic integrity.

  8. Utility of Filter Paper for Preserving Insects, Bacteria, and Host Reservoir DNA for Molecular Testing

    Directory of Open Access Journals (Sweden)

    F Karimian

    2011-12-01

    Methods: Total body or haemolymph of individual mosquitoes, sand flies or cockroaches squashed or placed on the paper respectively. Extracted DNA of five different bacteria species as well as blood specimens of human and great gerbil Rhombomys opimus was pipetted directly onto filter paper. The papers were stored in room temperature up to 12 months during 2009 until 2011. At monthly intervals, PCR was conducted using a 1-mm disk from the DNA impregnated filter paper as target DNA. PCR amplification was performed against different target genes of the organisms including the ITS2-rDNA of mosquitoes, mtDNA-COI of the sand flies and cockroaches, 16SrRNA gene of the bacteria, and the mtDNA-CytB of the vertebrates. Results: Successful PCR amplification was observed for all of the specimens regardless of the loci, taxon, or time of storage. The PCR amplification were ranged from 462 to 1500 bp and worked well for the specified target gene/s. Time of storage did not affect the amplification up to one year. Conclusion: The filter paper method is a simple and economical way to store, to preserve, and to distribute DNA samples for PCR analysis.

  9. DNA damage in preserved specimens and tissue samples: a molecular assessment

    Directory of Open Access Journals (Sweden)

    Cantin Elizabeth

    2008-10-01

    Full Text Available Abstract The extraction of genetic information from preserved tissue samples or museum specimens is a fundamental component of many fields of research, including the Barcode of Life initiative, forensic investigations, biological studies using scat sample analysis, and cancer research utilizing formaldehyde-fixed, paraffin-embedded tissue. Efforts to obtain genetic information from these sources are often hampered by an inability to amplify the desired DNA as a consequence of DNA damage. Previous studies have described techniques for improved DNA extraction from such samples or focused on the effect of damaging agents – such as light, oxygen or formaldehyde – on free nucleotides. We present ongoing work to characterize lesions in DNA samples extracted from preserved specimens. The extracted DNA is digested to single nucleosides with a combination of DNase I, Snake Venom Phosphodiesterase, and Antarctic Phosphatase and then analyzed by HPLC-ESI-TOF-MS. We present data for moth specimens that were preserved dried and pinned with no additional preservative and for frog tissue samples that were preserved in either ethanol, or formaldehyde, or fixed in formaldehyde and then preserved in ethanol. These preservation methods represent the most common methods of preserving animal specimens in museum collections. We observe changes in the nucleoside content of these samples over time, especially a loss of deoxyguanosine. We characterize the fragmentation state of the DNA and aim to identify abundant nucleoside lesions. Finally, simple models are introduced to describe the DNA fragmentation based on nicks and double-strand breaks.

  10. Enzymatic treatment of specimens before DNA extraction directly influences molecular detection of infectious agents.

    Directory of Open Access Journals (Sweden)

    Pablo Goldschmidt

    Full Text Available INTRODUCTION: Biological samples, pharmaceuticals or food contain proteins, lipids, polymers, ammoniums and macromolecules that alter the detection of infectious agents by DNA amplification techniques (PCR. Moreover the targeted DNA has to be released from the complex cell walls and the compact nucleoprotein matrixes and cleared from potential inhibitors. The goal of the present work was to assess the efficiency of enzymatic pretreatments on infectious agents to make DNA available for further extraction and amplification. METHODS: Staphylococcus epidermidis, Streptococcus mitis, Propionibacterium acnes, Escherichia coli, Pseudomonas aeruginosa, Candida albicans, Aspergillus niger and Fusarium solani were mixed with an internal control virus and treated with: 1 proteinase K; 2 lyticase and 3 lyticase followed by proteinase K. DNAs was manually extracted using the QIAmp DNA Mini kit or the MagNA Pure Compact automate. DNA extraction yields and the inhibitors were assessed with a phocid Herpesvirus. Bacterial detection was performed using TaqMan real-time PCR and yeasts and filamentous Fungi with HRM (real-time PCR followed by high-resolution melting analysis. RESULTS: Viral DNA was released, extracted and detected using manual and automatic methods without pre enzymatic treatments. Either the manual or the automatic DNA extraction systems did not meet the sensitivity expectations if enzymatic treatments were not performed before: lyticase for Fungi and Proteinase K for Bacteria. The addition of lyticase and proteinase K did not improve results. For Fungi the detection after lyticase was higher than for Proteinase K, for which melting analysis did not allow fungal specification. DISCUSSION: Columns and magnetic beads allowed collecting DNA and separate PCR inhibitors. Detection rates cannot be related to DNA-avidity of beads or to elution but to the lack of proteolysis.

  11. Enzymatic treatment of specimens before DNA extraction directly influences molecular detection of infectious agents.

    Science.gov (United States)

    Goldschmidt, Pablo; Degorge, Sandrine; Merabet, Lilia; Chaumeil, Christine

    2014-01-01

    Biological samples, pharmaceuticals or food contain proteins, lipids, polymers, ammoniums and macromolecules that alter the detection of infectious agents by DNA amplification techniques (PCR). Moreover the targeted DNA has to be released from the complex cell walls and the compact nucleoprotein matrixes and cleared from potential inhibitors. The goal of the present work was to assess the efficiency of enzymatic pretreatments on infectious agents to make DNA available for further extraction and amplification. Staphylococcus epidermidis, Streptococcus mitis, Propionibacterium acnes, Escherichia coli, Pseudomonas aeruginosa, Candida albicans, Aspergillus niger and Fusarium solani were mixed with an internal control virus and treated with: 1) proteinase K; 2) lyticase and 3) lyticase followed by proteinase K. DNAs was manually extracted using the QIAmp DNA Mini kit or the MagNA Pure Compact automate. DNA extraction yields and the inhibitors were assessed with a phocid Herpesvirus. Bacterial detection was performed using TaqMan real-time PCR and yeasts and filamentous Fungi with HRM (real-time PCR followed by high-resolution melting analysis). Viral DNA was released, extracted and detected using manual and automatic methods without pre enzymatic treatments. Either the manual or the automatic DNA extraction systems did not meet the sensitivity expectations if enzymatic treatments were not performed before: lyticase for Fungi and Proteinase K for Bacteria. The addition of lyticase and proteinase K did not improve results. For Fungi the detection after lyticase was higher than for Proteinase K, for which melting analysis did not allow fungal specification. Columns and magnetic beads allowed collecting DNA and separate PCR inhibitors. Detection rates cannot be related to DNA-avidity of beads or to elution but to the lack of proteolysis.

  12. Effects of Spaceflight on Molecular and Cellular Responses to Bleomycin-induced DNA Damages in Confluent Human Fibroblasts

    Science.gov (United States)

    Lu, Tao; Wu, Honglu; Karouia, Fathi; Stodieck, Louis; Zhang, Ye; Wong, Michael

    2016-07-01

    Spaceflights expose human beings to various risk factors. Among them are microgravity related physiological stresses in immune, cytoskeletal, and cardiovascular systems, and space radiation related elevation of cancer risk. Cosmic radiation consists of energetic protons and other heavier charged particles that induce DNA damages. Effective DNA damage response and repair mechanism is important to maintain genomic integrity and reduce cancer risk. There were studies on effects of spaceflight and microgravity on DNA damage response in cell and animal models, but the published results were mostly conflicting and inconsistent. To investigate effects of spaceflight on molecular and cellular responses to DNA damages, bleomycin, an anti-cancer drug and radiomimetic reagent, was used to induce DNA damages in confluent human fibroblasts flown to the International Space Station (ISS) and on ground. After exposure to 1.0 mg/ml bleomycin for 3 hours, cells were fixed for immunofluorescence assays and for RNA preparation. Extents of DNA damages were quantified by focus pattern and focus number counting of phosphorylated histone protein H2AX (γg-H2AX). The cells on the ISS showed modestly increased average focus counts per nucleus while the distribution of patterns was similar to that on the ground. PCR array analysis showed that expressions of several genes, including CDKN1A and PCNA, were significantly changed in response to DNA damages induced by bleomycin in both flight and ground control cells. However, there were no significant differences in the overall expression profiles of DNA damage response genes between the flight and ground samples. Analysis of cellular proliferation status with Ki-67 staining showed a slightly higher proliferating population in cells on the ISS than those on ground. Our results suggested that the difference in γg-H2AX focus counts between flight and ground was due to the higher percentage of proliferating cells in space, but spaceflight did not

  13. Electronic couplings and on-site energies for hole transfer in DNA: Systematic quantum mechanical/molecular dynamic study

    Science.gov (United States)

    Voityuk, Alexander A.

    2008-03-01

    The electron hole transfer (HT) properties of DNA are substantially affected by thermal fluctuations of the π stack structure. Depending on the mutual position of neighboring nucleobases, electronic coupling V may change by several orders of magnitude. In the present paper, we report the results of systematic QM/molecular dynamic (MD) calculations of the electronic couplings and on-site energies for the hole transfer. Based on 15ns MD trajectories for several DNA oligomers, we calculate the average coupling squares ⟨V2⟩ and the energies of basepair triplets XG +Y and XA +Y, where X, Y =G, A, T, and C. For each of the 32 systems, 15 000 conformations separated by 1ps are considered. The three-state generalized Mulliken-Hush method is used to derive electronic couplings for HT between neighboring basepairs. The adiabatic energies and dipole moment matrix elements are computed within the INDO/S method. We compare the rms values of V with the couplings estimated for the idealized B-DNA structure and show that in several important cases the couplings calculated for the idealized B-DNA structure are considerably underestimated. The rms values for intrastrand couplings G-G, A-A, G-A, and A-G are found to be similar, ˜0.07eV, while the interstrand couplings are quite different. The energies of hole states G+ and A+ in the stack depend on the nature of the neighboring pairs. The XG +Y are by 0.5eV more stable than XA +Y. The thermal fluctuations of the DNA structure facilitate the HT process from guanine to adenine. The tabulated couplings and on-site energies can be used as reference parameters in theoretical and computational studies of HT processes in DNA.

  14. Comparative molecular cytogenetic analyses of a major tandemly repeated DNA family and retrotransposon sequences in cultivated jute Corchorus species (Malvaceae).

    Science.gov (United States)

    Begum, Rabeya; Zakrzewski, Falk; Menzel, Gerhard; Weber, Beatrice; Alam, Sheikh Shamimul; Schmidt, Thomas

    2013-07-01

    The cultivated jute species Corchorus olitorius and Corchorus capsularis are important fibre crops. The analysis of repetitive DNA sequences, comprising a major part of plant genomes, has not been carried out in jute but is useful to investigate the long-range organization of chromosomes. The aim of this study was the identification of repetitive DNA sequences to facilitate comparative molecular and cytogenetic studies of two jute cultivars and to develop a fluorescent in situ hybridization (FISH) karyotype for chromosome identification. A plasmid library was generated from C. olitorius and C. capsularis with genomic restriction fragments of 100-500 bp, which was complemented by targeted cloning of satellite DNA by PCR. The diversity of the repetitive DNA families was analysed comparatively. The genomic abundance and chromosomal localization of different repeat classes were investigated by Southern analysis and FISH, respectively. The cytosine methylation of satellite arrays was studied by immunolabelling. Major satellite repeats and retrotransposons have been identified from C. olitorius and C. capsularis. The satellite family CoSat I forms two undermethylated species-specific subfamilies, while the long terminal repeat (LTR) retrotransposons CoRetro I and CoRetro II show similarity to the Metaviridea of plant retroelements. FISH karyotypes were developed by multicolour FISH using these repetitive DNA sequences in combination with 5S and 18S-5·8S-25S rRNA genes which enable the unequivocal chromosome discrimination in both jute species. The analysis of the structure and diversity of the repeated DNA is crucial for genome sequence annotation. The reference karyotypes will be useful for breeding of jute and provide the basis for karyotyping homeologous chromosomes of wild jute species to reveal the genetic and evolutionary relationship between cultivated and wild Corchorus species.

  15. Electronic couplings and on-site energies for hole transfer in DNA: systematic quantum mechanical/molecular dynamic study.

    Science.gov (United States)

    Voityuk, Alexander A

    2008-03-21

    The electron hole transfer (HT) properties of DNA are substantially affected by thermal fluctuations of the pi stack structure. Depending on the mutual position of neighboring nucleobases, electronic coupling V may change by several orders of magnitude. In the present paper, we report the results of systematic QM/molecular dynamic (MD) calculations of the electronic couplings and on-site energies for the hole transfer. Based on 15 ns MD trajectories for several DNA oligomers, we calculate the average coupling squares V(2) and the energies of basepair triplets XG(+)Y and XA(+)Y, where X, Y=G, A, T, and C. For each of the 32 systems, 15,000 conformations separated by 1 ps are considered. The three-state generalized Mulliken-Hush method is used to derive electronic couplings for HT between neighboring basepairs. The adiabatic energies and dipole moment matrix elements are computed within the INDO/S method. We compare the rms values of V with the couplings estimated for the idealized B-DNA structure and show that in several important cases the couplings calculated for the idealized B-DNA structure are considerably underestimated. The rms values for intrastrand couplings G-G, A-A, G-A, and A-G are found to be similar, approximately 0.07 eV, while the interstrand couplings are quite different. The energies of hole states G(+) and A(+) in the stack depend on the nature of the neighboring pairs. The XG(+)Y are by 0.5 eV more stable than XA(+)Y. The thermal fluctuations of the DNA structure facilitate the HT process from guanine to adenine. The tabulated couplings and on-site energies can be used as reference parameters in theoretical and computational studies of HT processes in DNA.

  16. Striking Plasticity of CRISPR-Cas9 and Key Role of Non-target DNA, as Revealed by Molecular Simulations

    Science.gov (United States)

    2016-01-01

    The CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 system recently emerged as a transformative genome-editing technology that is innovating basic bioscience and applied medicine and biotechnology. The endonuclease Cas9 associates with a guide RNA to match and cleave complementary sequences in double stranded DNA, forming an RNA:DNA hybrid and a displaced non-target DNA strand. Although extensive structural studies are ongoing, the conformational dynamics of Cas9 and its interplay with the nucleic acids during association and DNA cleavage are largely unclear. Here, by employing multi-microsecond time scale molecular dynamics, we reveal the conformational plasticity of Cas9 and identify key determinants that allow its large-scale conformational changes during nucleic acid binding and processing. We show how the “closure” of the protein, which accompanies nucleic acid binding, fundamentally relies on highly coupled and specific motions of the protein domains, collectively initiating the prominent conformational changes needed for nucleic acid association. We further reveal a key role of the non-target DNA during the process of activation of the nuclease HNH domain, showing how the nontarget DNA positioning triggers local conformational changes that favor the formation of a catalytically competent Cas9. Finally, a remarkable conformational plasticity is identified as an intrinsic property of the HNH domain, constituting a necessary element that allows for the HNH repositioning. These novel findings constitute a reference for future experimental studies aimed at a full characterization of the dynamic features of the CRISPR-Cas9 system, and—more importantly—call for novel structure engineering efforts that are of fundamental importance for the rational design of new genome-engineering applications. PMID:27800559

  17. Molecular cloning of complementary DNA for human medullasin: an inflammatory serine protease in bone marrow cells.

    Science.gov (United States)

    Okano, K; Aoki, Y; Sakurai, T; Kajitani, M; Kanai, S; Shimazu, T; Shimizu, H; Naruto, M

    1987-07-01

    Medullasin, an inflammatory serine protease in bone marrow cells, modifies the functions of natural killer cells, monocytes, and granulocytes. We have cloned a medullasin cDNA from a human acute promyelocytic cell (ML3) cDNA library using oligonucleotide probes synthesized from the information of N-terminal amino acid sequence of natural medullasin. The cDNA contained a long open reading frame encoding 237 amino acid residues beginning from the second amino acid of natural meduallasin. The deduced amino acid sequence of medullasin shows a typical serine protease structure, with 41% homology with pig elastase 1.

  18. DNA-templated silver nanoclusters based label-free fluorescent molecular beacon for the detection of adenosine deaminase.

    Science.gov (United States)

    Zhang, Kai; Wang, Ke; Xie, Minhao; Zhu, Xue; Xu, Lan; Yang, Runlin; Huang, Biao; Zhu, Xiaoli

    2014-02-15

    A general and reliable fluorescent molecular beacon is proposed in this work utilizing DNA-templated silver nanoclusters (AgNCs). The fluorescent molecular beacon has been employed for sensitive determination of the concentration of adenosine deaminase (ADA) and its inhibition. A well-designed oligonucleotide containing three functional regions (an aptamer region for adenosine assembly, a sequence complementary to the region of the adenosine aptamer, and an inserted six bases cytosine-loop) is adopted as the core element in the strategy. The enzymatic reaction of adenosine catalyzed by ADA plays a key role as well in the regulation of the synthesis of the DNA-templated AgNCs, i.e. the signal indicator. The intensity of the fluorescence signal may thereby determine the concentration of the enzyme and its inhibitor. The detection limit of the ADA can be lowered to 0.05 UL(-1). Also, 100 fM of a known inhibitor erythro-9-(2-hydroxy-3-nonyl) adenine hydrochloride (EHNA) is enough to present distinguishable fluorescence emission. Moreover, since the fluorescent signal indicator is not required to be bound with the oligonucleotide, this fluorescent molecular beacon may integrate the advantages of both the label-free and signal-on strategies.

  19. Molecular cytogenetics of Alstroemeria: identification of parental genomes in interspecific hybrids and characterization of repetitive DNA families in constitutive heterochromatin.

    Science.gov (United States)

    Kuipers, A G; van Os, D P; de Jong, J H; Ramanna, M S

    1997-02-01

    The genus Alstroemeria consists of diploid (2n = 2x = 16) species originating mainly from Chile and Brazil. Most cultivars are triploid or tetraploid interspecific hybrids. C-banding of eight species revealed obvious differentiation of constitutive heterochromatin within the genus. The present study focused on the molecular (cyto)genetic background of this differentiation. Genomic slot-blot analysis demonstrated strong conservation of major parts of the genomes among six species. The chromosomes of A. aurea and A. ligtu, species with pronounced interstitial C-bands, were found to contain large amounts of highly repetitive and species-specific DNA. The variation in size, number and intensity of strongly probed bands of major repetitive DNA families observed in genomic Southern blots of Sau3A, HaeIII, and MseI digests indicated a strong correlation between variation in genomic DNA composition and different C-banding patterns among Alstroemeria species. Genomic in situ hybridization (GISH) revealed a clear distinction between parental chromosomes in the hybrids between Chilean and Brazilian species and also between Chilean species, as long as at least one of the parental species possessed prominent C-banding. Regarding the latter, discriminative hybridization resulted from highly repetitive species specific DNA in the heterochromatic chromosome regions of A. aurea and A. ligtu, and caused GISH banding patterns that coincided with the C-banding patterns.

  20. Molecular characterization of Stictodora tridactyla (Trematoda: Heterophyidae) from Kuwait Bay using rDNA ITS and mtCO1.

    Science.gov (United States)

    Al-Kandari, Wafa Y; Alnaqeeb, Majed A; Isaac, Asha M; Al-Bustan, Suzanne A

    2015-11-01

    Stictodora tridactyla is an intestinal fluke in the family Heterophyidae that parasitizes shorebirds and mammals, including humans. Its metacercarial cyst stage was reported in the Arabian killifish, Aphanius dispar, at Kuwait Bay. In the present study, Cerithidea cingulata was found to serve as the first intermediate host of S. tridactyla. In order to establish the snail-fish link in the life cycle of S. tridactyla, complete sequences of ribosomal DNA internal transcribed spacer region 1 and 2 (rDNA ITS1 and ITS2) and partial sequence of cytochrome oxidase subunit 1 were obtained for metacercarial cysts isolated from the fish A. dispar and rediae isolated from the snail C. cingulata. Sequence alignment demonstrated that these larval stages belong to the same heterophyid species, S. tridactyla. Phylogenetic analysis based on rDNA ITS1, ITS2, and mtCO1 confirmed the position of S. tridactyla within the Heterophyidae and found it to cluster with Haplorchis spp. The present study represents the first molecular study correlating the larval stages of S. tridactyla using rDNA ITS1, ITS2, and mtCO1 and examining the phylogenetic relationships of S. tridactyla with different heterophyid species.