Science.gov

Sample records for dna constant-force unzipping

  1. Probing the mechanical unzipping of DNA

    NASA Astrophysics Data System (ADS)

    Voulgarakis, Nikos K.; Bishop, Alan R.; Rasmussen, Kim O.

    2006-03-01

    Recent advances in single-molecule force spectroscopy have made a systematic study of local melting in DNA possible. This provide new insight into important biological processes as replication and transcription. In this work, we present an extensive study of the micromechanical unzipping of DNA in the framework of the Peyrard-Bishop-Dauxois (PBD) model. The force required to separate the doubled strand is derived through analysis of the force-extension curve, while an estimation of the nucleation bubble size of the unzipping process is obtained by the distribution of the rapture force. Our findings are in very good agreement with existing experimental results; for example the force-temperature phase diagram obtained by the PBD model agrees excellently with recent constant-force experimental measurements of the lambda-phage DNA. Fundamental differences between the in vivo and vitro DNA unzipping, as predicted by the PBD model, are also discussed.

  2. Unzipping of DNA with correlated base sequence.

    PubMed

    Allahverdyan, A E; Gevorkian, Zh S; Hu, Chin-Kun; Wu, Ming-Chya

    2004-06-01

    We consider force-induced unzipping transition for a heterogeneous DNA model with a correlated base sequence. Both finite-range and long-range correlated situations are considered. It is shown that finite-range correlations increase stability of DNA with respect to the external unzipping force. Due to long-range correlations the number of unzipped base pairs displays two widely different scenarios depending on the details of the base sequence: either there is no unzipping phase transition at all, or the transition is realized via a sequence of jumps with magnitude comparable to the size of the system. Both scenarios are different from the behavior of the average number of unzipped base pairs (non-self-averaging). The results can be relevant for explaining the biological purpose of correlated structures in DNA.

  3. Role of chain stiffness and end entropy in the unzipping of DNA chain

    NASA Astrophysics Data System (ADS)

    Singh, Amar; Singh, Navin

    2013-06-01

    The force induced unzipping of dsDNA was studied in Constant Force Ensembles (CFE) in presence of salt. The role of end entropy on the force required to unzip the chain has been investigated using Peyrard Bishop Dauxious (PBD) model. The effect of the stiffness and anharmonicity in the stacking interaction of the chain has been investigated. Our results show that the force require to unzip the chain increases with increasing the chain length in case when the other end is free while it decreases in case of when the other end of the chain is restricted. However, as the chain length reaches a critical length, the end effect nullifies and the critical force in both the cases are found to be same.

  4. Statistical Properties of Metastable Intermediates in DNA Unzipping

    NASA Astrophysics Data System (ADS)

    Huguet, J. M.; Forns, N.; Ritort, F.

    2009-12-01

    We unzip DNA molecules using optical tweezers and determine the sizes of the cooperatively unzipping and zipping regions separating consecutive metastable intermediates along the unzipping pathway. Sizes are found to be distributed following a power law, ranging from one base pair up to more than a hundred base pairs. We find that a large fraction of unzipping regions smaller than 10 bp are seldom detected because of the high compliance of the released single stranded DNA. We show how the compliance of a single nucleotide sets a limit value around 0.1N/m for the stiffness of any local force probe aiming to discriminate one base pair at a time in DNA unzipping experiments.

  5. Impacts of magnesium ions on the unzipping of λ-phage DNA

    NASA Astrophysics Data System (ADS)

    Lee, C. H.; Danilowicz, C.; Conroy, R. S.; Coljee, V. W.; Prentiss, M.

    2006-04-01

    We used magnetic tweezers to exert a constant force to separate double stranded λ-phage DNA as a function of temperature and buffer content. The separation was performed at temperatures ranging from 20 to 50 °C in various Mg2+ buffers, including a T4 ligase buffer and a PCR buffer. At 30 °C and pH 7.4 (10 mM Tris), we measured the unzipping force as a function of concentration for Mg2+ concentrations between 0.2 and 50 mM, and determined that the unzipping force is proportional to the logarithm of concentration. For comparison, we performed the analogous experiment as a function of Na+ concentration and found that the unzipping force is also proportional to the log of concentration, but requires a much higher cation concentration to achieve the same unzipping force as in Mg2+ buffer. We also constructed the phase diagram in the force-temperature plane for the unzipping in 10 and 50 mM MgCl2 at pH 7.4 (10 mM Tris). The phase diagram for 10 mM Mg2+ is similar to the one measured previously for phosphate buffer saline (PBS) but the phase diagram for 50 mM Mg2+ deviates significantly from those for 10 mM Mg2+ and PBS at temperatures between 20 and 35 °C.

  6. Probability distribution analysis of force induced unzipping of DNA

    NASA Astrophysics Data System (ADS)

    Kumar, Sanjay; Giri, Debaprasad

    2006-07-01

    We present a semimicroscopic model of dsDNA by incorporating the directional nature of hydrogen bond to describe the force induced unzipping transition. Using exact enumeration technique, we obtain the force-temperature and the force-extension curves and compare our results with the other models of dsDNA. The model proposed by us is rich enough to describe the basic mechanism of dsDNA unzipping and predicts the existence of an "eye phase." We show oscillations in the probability distribution function during unzipping. Effects of stacking energies on the melting profile have also been studied.

  7. Micromechanics of base pair unzipping in the DNA duplex.

    PubMed

    Volkov, Sergey N; Paramonova, Ekaterina V; Yakubovich, Alexander V; Solov'yov, Andrey V

    2012-01-25

    All-atom molecular dynamics (MD) simulations of DNA duplex unzipping in a water environment were performed. The investigated DNA double helix consists of a Drew-Dickerson dodecamer sequence and a hairpin (AAG) attached to the end of the double-helix chain. The considered system is used to examine the process of DNA strand separation under the action of an external force. This process occurs in vivo and now is being intensively investigated in experiments with single molecules. The DNA dodecamer duplex is consequently unzipped pair by pair by means of the steered MD. The unzipping trajectories turn out to be similar for the duplex parts with G·C content and rather distinct for the parts with A·T content. It is shown that during the unzipping each pair experiences two types of motion: relatively quick rotation together with all the duplex and slower motion in the frame of the unzipping fork. In the course of opening, the complementary pair passes through several distinct states: (i) the closed state in the double helix, (ii) the metastable preopened state in the unzipping fork and (iii) the unbound state. The performed simulations show that water molecules participate in the stabilization of the metastable states of the preopened base pairs in the DNA unzipping fork.

  8. Force and kinetic barriers to initiation of DNA unzipping.

    PubMed

    Cocco, Simona; Monasson, Rémi; Marko, John F

    2002-04-01

    A semimicroscopic model of the binding of the two nucleotide strands in a double-stranded DNA is used to describe the effects of applied tension on strand unpairing. We show that the model describes strand separation by elevated temperature, applied torque, and applied force. In particular, we show how the interactions responsible for stabilizing the double helix against thermal denaturation determine the approximately 12 pN force threshold for DNA strand separation. The larger rigidity of the strands when formed into double-stranded DNA, relative to that of isolated strands, gives rise to a potential barrier to unzipping. We show that this barrier results in a approximately 250 pN force barrier opposing the beginning of strand separation. The thermal-fluctuation-assisted "tunnelling" through the barrier is then analyzed using instanton calculations. The resulting kinetics of unzipping initiation is shown to be consistent with solution-phase strand dissociation experiments, and can explain results of two recent unzipping experiments done using atomic-force microscopy.

  9. Probing Nucleosome Remodeling by Unzipping Single DNA Molecules

    NASA Astrophysics Data System (ADS)

    Wang, Michelle

    2006-03-01

    At the core of eukaryotic chromatin is the nucleosome, which consists of 147 bp of DNA wrapped 1.65 turns around an octamer of histone proteins. Even this lowest level of genomic compaction presents a strong barrier to DNA-binding cellular factors that are required for essential processes such as transcription, DNA replication, recombination and repair. Chromatin remodeling enzymes use the energy of ATP hydrolysis to regulate accessibility of the genetic code by altering chromatin structure. While remodeling enzymes have been the subject of extensive research in recent years, their precise mechanism remains unclear. In order to probe the structure of individual nucleosomes and their remodeling, we assembled a histone octamer onto a DNA segment containing a strong nucleosome positioning sequence. As the DNA double helix was unzipped through the nucleosome using a feedback-enhanced optical trap, the presence of the nucleosome was detected as a series of dramatic increases in the tension in the DNA, followed by sudden tension reductions. Analysis of the unzipping force throughout the disruption accurately revealed the spatial location and fine structure of the nucleosome to near base pair precision. Using this approach, we investigate how remodeling enzymes may alter the location and structure of a nucleosome.

  10. DNA Translocation and Unzipping through a Nanopore: Some Geometrical Effects

    PubMed Central

    Muzard, J.; Martinho, M.; Mathé, J.; Bockelmann, U.; Viasnoff, V.

    2010-01-01

    Abstract This article explores the role of some geometrical factors on the electrophoretically driven translocations of macromolecules through nanopores. In the case of asymmetric pores, we show how the entry requirements and the direction of translocation can modify the information content of the blocked ionic current as well as the transduction of the electrophoretic drive into a mechanical force. To address these effects we studied the translocation of single-stranded DNA through an asymmetric α-hemolysin pore. Depending on the direction of the translocation, we measure the capacity of the pore to discriminate between both DNA orientations. By unzipping DNA hairpins from both sides of the pores we show that the presence of single strand or double strand in the pore can be discriminated based on ionic current levels. We also show that the transduction of the electrophoretic drive into a denaturing mechanical force depends on the local geometry of the pore entrance. Eventually we discuss the application of this work to the measurement of energy barriers for DNA unzipping as well as for protein binding and unfolding. PMID:20483325

  11. Resonant optimization in the mechanical unzipping of DNA

    NASA Astrophysics Data System (ADS)

    Bergues-Pupo, Ana Elisa; Falo, Fernando; Fiasconaro, Alessandro

    2014-03-01

    The mechanical separation of the double-stranded DNA in single-molecule experiments is of fundamental importance in the understanding of the replication or transcription processes. Time-dependent forces can influence in different ways the dynamics of this separation. We study here this unzipping of DNA in the framework of the mesoscopic Peyrard-Bishop-Dauxois model under the influence of a periodic driving. Two different protocols, both of them feasible experimentally, have been studied under two modes of pulling: controlled force and controlled position. A strong resonant activation phenomenon has been observed in the magnitudes that characterize the mechanical unzipping such as the mean opening time, the mean opening force, and the mean critical opening force, all of them as a function of the frequency of the driving. This optimal frequency region has been observed for all the cases studied both in a uniform DNA of adenine-thymine nucleotides and in a real DNA sequence. Importantly, a well precise resonant frequency can be determined with the use of one of this protocols.

  12. Double nanohole optical tweezers visualize protein p53 suppressing unzipping of single DNA-hairpins

    PubMed Central

    Kotnala, Abhay; Gordon, Reuven

    2014-01-01

    Here we report on the use of double-nanohole (DNH) optical tweezers as a label-free and free-solution single-molecule probe for protein–DNA interactions. Using this approach, we demonstrate the unzipping of individual 10 base pair DNA-hairpins, and quantify how tumor suppressor p53 protein delays the unzipping. From the Arrhenius behavior, we find the energy barrier to unzipping introduced by p53 to be 2 × 10−20 J, whereas cys135ser mutant p53 does not show suppression of unzipping, which gives clues to its functional inability to suppress tumor growth. This transformative approach to single molecule analysis allows for ultra-sensitive detection and quantification of protein–DNA interactions to revolutionize the fight against genetic diseases. PMID:24940547

  13. Double nanohole optical tweezers visualize protein p53 suppressing unzipping of single DNA-hairpins.

    PubMed

    Kotnala, Abhay; Gordon, Reuven

    2014-06-01

    Here we report on the use of double-nanohole (DNH) optical tweezers as a label-free and free-solution single-molecule probe for protein-DNA interactions. Using this approach, we demonstrate the unzipping of individual 10 base pair DNA-hairpins, and quantify how tumor suppressor p53 protein delays the unzipping. From the Arrhenius behavior, we find the energy barrier to unzipping introduced by p53 to be 2 × 10(-20) J, whereas cys135ser mutant p53 does not show suppression of unzipping, which gives clues to its functional inability to suppress tumor growth. This transformative approach to single molecule analysis allows for ultra-sensitive detection and quantification of protein-DNA interactions to revolutionize the fight against genetic diseases.

  14. DNA unzipping phase diagram calculated via replica theory

    NASA Astrophysics Data System (ADS)

    Roland, C. Brian; Hatch, Kristi Adamson; Prentiss, Mara; Shakhnovich, Eugene I.

    2009-05-01

    We show how single-molecule unzipping experiments can provide strong evidence that the zero-force melting transition of long molecules of natural dsDNA should be classified as a phase transition of the higher-order type (continuous). Toward this end, we study a statistical-mechanics model for the fluctuating structure of a long molecule of dsDNA, and compute the equilibrium phase diagram for the experiment in which the molecule is unzipped under applied force. We consider a perfect-matching dsDNA model, in which the loops are volume-excluding chains with arbitrary loop exponent c . We include stacking interactions, hydrogen bonds, and main-chain entropy. We include sequence heterogeneity at the level of random sequences; in particular, there is no correlation in the base-pairing (bp) energy from one sequence position to the next. We present heuristic arguments to demonstrate that the low-temperature macrostate does not exhibit degenerate ergodicity breaking. We use this claim to understand the results of our replica-theoretic calculation of the equilibrium properties of the system. As a function of temperature, we obtain the minimal force at which the molecule separates completely. This critical-force curve is a line in the temperature-force phase diagram that marks the regions where the molecule exists primarily as a double helix versus the region where the molecule exists as two separate strands. We compare our random-sequence model to magnetic tweezer experiments performed on the 48502 bp genome of bacteriophage λ . We find good agreement with the experimental data, which is restricted to temperatures between 24 and 50°C . At higher temperatures, the critical-force curve of our random-sequence model is very different for that of the homogeneous-sequence version of our model. For both sequence models, the critical force falls to zero at the melting temperature Tc like |T-Tc|α . For the homogeneous-sequence model, α=1/2 almost exactly, while for the random

  15. Stretching short sequences of DNA with constant force axial optical tweezers.

    PubMed

    Raghunathan, Krishnan; Milstein, Joshua N; Meiners, Jens-Christian

    2011-10-13

    the DNA, an active feedback scheme would be needed to apply a constant force and the bandwidth of this will be quite limited, especially at low forces. We circumvent these fundamental problems by directly pulling the DNA away from the coverslip by using a constant force axial optical tweezers. This is achieved by trapping the bead in a linear region of the optical potential, where the optical force is constant-the strength of which can be tuned by adjusting the laser power. Trapping within the linear region also serves as an all optical force-clamp on the DNA that extends for nearly 350 nm in the axial direction. We simultaneously compensate for thermal and mechanical drift by finely adjusting the position of the stage so that a reference microsphere stuck to the coverslip remains at the same position and focus, allowing for a virtually limitless observation period.

  16. Stretching Short Sequences of DNA with Constant Force Axial Optical Tweezers

    PubMed Central

    Raghunathan, Krishnan; Milstein, Joshua N.; Meiners, Jens -Christian

    2011-01-01

    to stretch the DNA, an active feedback scheme would be needed to apply a constant force and the bandwidth of this will be quite limited, especially at low forces. We circumvent these fundamental problems by directly pulling the DNA away from the coverslip by using a constant force axial optical tweezers7,8. This is achieved by trapping the bead in a linear region of the optical potential, where the optical force is constant-the strength of which can be tuned by adjusting the laser power. Trapping within the linear region also serves as an all optical force-clamp on the DNA that extends for nearly 350 nm in the axial direction. We simultaneously compensate for thermal and mechanical drift by finely adjusting the position of the stage so that a reference microsphere stuck to the coverslip remains at the same position and focus, allowing for a virtually limitless observation period. PMID:22025209

  17. Electromechanical unzipping of individual DNA molecules using synthetic sub-2 nm pores.

    PubMed

    McNally, Ben; Wanunu, Meni; Meller, Amit

    2008-10-01

    Nanopores have recently emerged as high-throughput tools for probing and manipulating nucleic acid secondary structure at the single-molecule level. While most studies to date have utilized protein pores embedded in lipid bilayers, solid-state nanopores offer many practical advantages which greatly expand the range of applications in life sciences and biotechnology. Using sub-2 nm solid-state nanopores, we show for the first time that the unzipping kinetics of individual DNA duplexes can be probed by analyzing the dwell-time distributions. We performed high-bandwidth electrical measurements of DNA duplex unzipping as a function of their length, sequence, and temperature. We find that our longer duplexes (>10 bp) follow Arrhenius dependence on temperature, suggesting that unzipping can be approximated as a single-barrier crossing, but the unzipping kinetics of shorter duplexes do not involve a barrier, due to the strong biasing electrical force. Finally, we show that mismatches in the duplex affect unzipping times in a position-sensitive manner. Our results are a crucial step toward sequence variability detection and our single-molecule nanopore sequencing technology, which rely on parallel detection from nanopore arrays.

  18. Conformational Changes Followed by Complete Unzipping of DNA Double Helix by Charge-Tuned Gold Nanoparticles.

    PubMed

    Bera, Subhas C; Sanyal, Kasturi; Senapati, Dulal; Mishra, Padmaja P

    2016-05-12

    The complete unzipping of DNA double helix by small size gold nanoparticles having weakly positive surface charge has been monitored using ensemble and single molecule fluorescence resonance energy transfer (smFRET) techniques. We believe, as the gold nanoparticles have positive charge on the surface, the DNA and nanoparticles were pulled together to form two single strands. The positively charged ligands on the nanoparticles attached to the DNA, and the hydrophobic ligands of the nanoparticles became tangled with each other, pulling the nanoparticles into clusters. At the same time, the nanoparticles pulled the DNA apart. The conformational changes followed by unzipping have been investigated for long DNA (calf thymus DNA) as well as for short DNA (∼40 base pair) using ensemble methods like circular dichroism (CD) spectroscopy, fluorescence intercalation assay, viscometric method, and single molecule FRET imaging. This observation not only reveals a new aspect in the field of nano-bio interface but also provides additional information about DNA dynamics.

  19. A coarse-grained MARTINI-like force field for DNA unzipping in nanopores.

    PubMed

    Stachiewicz, Anna; Molski, Andrzej

    2015-05-15

    In nanopore force spectroscopy (NFS) a charged polymer is threaded through a channel of molecular dimensions. When an electric field is applied across the insulating membrane, the ionic current through the nanopore reports on polymer translocation, unzipping, dissociation, and so forth. We present a new model that can be applied in molecular dynamics simulations of NFS. Although simplified, it does reproduce experimental trends and all-atom simulations. The scaled conductivities in bulk solution are consistent with experimental results for NaCl for a wide range of electrolyte concentrations and temperatures. The dependence of the ionic current through a nanopore on the applied voltage is symmetric and, in the voltage range used in experiments (up to 2 V), linear and in good agreement with experimental data. The thermal stability and geometry of DNA is well represented. The model was applied to simulations of DNA hairpin unzipping in nanopores. The results are in good agreement with all-atom simulations: the scaled translocation times and unzipping sequence are similar. © 2015 Wiley Periodicals, Inc.

  20. Unzipping of Double-stranded DNA in Engineered α-Hemolysin Pores.

    PubMed

    Liu, Aihua; Zhao, Qitao; Krishantha, D M Milan; Guan, Xiyun

    2011-06-12

    Biological protein α-hemolysin nanopore is under intense investigation as a potential platform for rapid and low-cost DNA sequencing. However, due to its narrow constriction, analysis of DNA in the α-hemolysin pore has long time been restricted to single strands. In this paper, we report that by introducing new surface functional groups into the α-hemolysin pore, facilitated unzipping of double-stranded DNA through the channel could be achieved. Since the mean residence time of the DNA events is dependent on the length of the duplex, and also varies with the nucleotide base composition, the modified protein pore approach offers the potential for rapid double-stranded DNA analysis, including sequencing.

  1. Unzipping of Double-stranded DNA in Engineered α-Hemolysin Pores

    PubMed Central

    Liu, Aihua; Zhao, Qitao; Krishantha, D.M. Milan; Guan, Xiyun

    2011-01-01

    Biological protein α-hemolysin nanopore is under intense investigation as a potential platform for rapid and low-cost DNA sequencing. However, due to its narrow constriction, analysis of DNA in the α-hemolysin pore has long time been restricted to single strands. In this paper, we report that by introducing new surface functional groups into the α-hemolysin pore, facilitated unzipping of double-stranded DNA through the channel could be achieved. Since the mean residence time of the DNA events is dependent on the length of the duplex, and also varies with the nucleotide base composition, the modified protein pore approach offers the potential for rapid double-stranded DNA analysis, including sequencing. PMID:21709813

  2. DNA Unzipping and the Unbinding of Directed Polymers in a Random Media

    NASA Astrophysics Data System (ADS)

    Kafri, Yariv; Polkovnikov, Anatoli

    2006-11-01

    We consider the unbinding of a directed polymer in a random media from a wall in d=1+1 dimensions and a simple one-dimensional model for DNA unzipping. Using the replica trick we show that the restricted partition functions of these problems are identical up to an overall normalization factor. Our finding gives an example of a generalization of the stochastic matrix form decomposition to disordered systems, a method which allows us to reduce the dimensionality of the problem. The equivalence between the two problems, for example, allows us to derive the probability distribution for finding the directed polymer a distance z from the wall. We discuss implications of these results for the related Kardar-Parisi-Zhang equation and the asymmetric exclusion process.

  3. DNA unzipping and the unbinding of directed polymers in a random media

    NASA Astrophysics Data System (ADS)

    Kafri, Yariv; Polkovnikov, Anatoli

    2007-03-01

    We consider the unbinding of a directed polymer in a random media from a wall in d=1+1 dimensions and a simple one-dimensional model for DNA unzipping. Using the replica trick we show that the restricted partition functions of these problems are identical up to an overall normalization factor. Our finding gives an example of a generalization of the stochastic matrix form decomposition to disordered systems; a method which effectively allows to reduce the dimensionality of the problem. The equivalence between the two problems, for example, allows us to derive the probability distribution for finding the directed polymer a distance z from the wall. We discuss implications of these results for the related Kardar-Parisi-Zhang equation and the asymmetric exclusion process.

  4. Unzipping DNA by a periodic force: hysteresis loop area and its scaling.

    PubMed

    Kapri, Rajeev

    2014-12-01

    Using Monte Carlo simulations, we study the hysteresis in the unzipping of double-stranded DNA whose ends are subjected to a time-dependent periodic force with frequency (ω) and amplitude (G). For the static force, i.e., ω→0, the DNA is in equilibrium with no hysteresis. On increasing ω, the area of the hysteresis loop initially increases and becomes maximum at frequency ω*(G), which depends on the force amplitude G. If the frequency is increased further, we find that for lower amplitudes the loop area decreases monotonically to zero, but for higher amplitudes it has an oscillatory component. The height of subsequent peaks decreases, and finally the loop area becomes zero at very high frequencies. The number of peaks depends on the length of the DNA. We give a simple analysis to estimate the frequencies at which maxima and minima occur in the loop area. We find that the area of the hysteresis loop scales as 1/ω in the high-frequency regime, whereas it scales as G(α)ω(β) with exponents α=1 and β=5/4 at low frequencies. The values of the exponents α and β are different from the exponents reported earlier based on the hysteresis of small hairpins.

  5. Unraveling siRNA unzipping kinetics with graphene

    NASA Astrophysics Data System (ADS)

    Mogurampelly, Santosh; Panigrahi, Swati; Bhattacharyya, Dhananjay; Sood, A. K.; Maiti, Prabal K.

    2012-08-01

    Using all atom molecular dynamics simulations, we report spontaneous unzipping and strong binding of small interfering RNA (siRNA) on graphene. Our dispersion corrected density functional theory based calculations suggest that nucleosides of RNA have stronger attractive interactions with graphene as compared to DNA residues. These stronger interactions force the double stranded siRNA to spontaneously unzip and bind to the graphene surface. Unzipping always nucleates at one end of the siRNA and propagates to the other end after few base-pairs get unzipped. While both the ends get unzipped, the middle part remains in double stranded form because of torsional constraint. Unzipping probability distributions fitted to single exponential function give unzipping time (τ) of the order of few nanoseconds which decrease exponentially with temperature. From the temperature variation of unzipping time we estimate the energy barrier to unzipping.

  6. Comparison of the measured phase diagrams in the force-temperature plane for the unzipping of two different natural DNA sequences

    NASA Astrophysics Data System (ADS)

    Lee, C. H.; Danilowicz, C.; Coljee, V. W.; Prentiss, M.

    2006-03-01

    In this work, we consider the critical force required to unzip two different naturally occurring sequences of double-stranded DNA (dsDNA) at temperatures ranging from 20 °C to 50 °C, where one of the sequences has a 53% average guanine-cytosine (GC) content and the other has a 40% GC content. We demonstrate that the force required to separate the dsDNA of the 53% GC sequence into single-stranded DNA (ssDNA) is approximately 0.5 pN, or approximately 5% greater than the critical force required to unzip the 40% GC sequence at the same temperature. In the temperature range between 20 and 40 °C the measured critical forces correspond reasonably well to predictions based on a simple theoretical homopolymeric model, but at temperatures above 40 °C the measured critical forces are much smaller than the predicted forces. The correspondence between theory and experiment is not improved by using Monte Carlo simulations that consider the heteropolymeric nature of the sequences.

  7. Unzipping bird feathers.

    PubMed

    Kovalev, Alexander; Filippov, Alexander E; Gorb, Stanislav N

    2014-03-06

    The bird feather vane can be separated into two parts by pulling the barbs apart. The original state can be re-established easily by lightly stroking through the feather. Hooklets responsible for holding vane barbs together are not damaged by multiple zipping and unzipping cycles. Because numerous microhooks keep the integrity of the feather, their properties are of great interest for understanding mechanics of the entire feather structure. This study was undertaken to estimate the separation force of single hooklets and their arrays using force measurement of an unzipping feather vane. The hooklets usually separate in some number synchronously (20 on average) with the highest observed separation force of 1.74 mN (average force 0.27 mN), whereas the single hooklet separation force was 14 μN. A simple numerical model was suggested for a better understanding of zipping and unzipping behaviour in feathers. The model demonstrates features similar to those observed in experiments.

  8. Limitations of Constant-Force-Feedback Experiments

    PubMed Central

    Elms, Phillip J.; Chodera, John D.; Bustamante, Carlos J.; Marqusee, Susan

    2012-01-01

    Single-molecule force spectroscopy has provided important insights into the properties and mechanisms of biological molecules and systems. A common experiment is to measure the force dependence of conformational changes at equilibrium. Here, we demonstrate that the commonly used technique of force feedback has severe limitations when used to evaluate rapid macromolecular conformational transitions. By comparing the force-dependent dynamics of three major classes of macromolecules (DNA, RNA, and protein) using both a constant-force-feedback and a constant-trap-position technique, we demonstrate a problem in force-feedback experiments. The finite response time of the instrument’s force feedback can modify the behavior of the molecule, leading to errors in the reported parameters, such as the rate constants and the distance to the transition state, for the conformational transitions. We elucidate the causes of this problem and provide a simple test to identify and evaluate the magnitude of the effect. We recommend avoiding the use of constant force feedback as a method to study rapid conformational changes in macromolecules. PMID:23062341

  9. Probing molecular pathways for DNA orientational trapping, unzipping and translocation in nanopores by using a tunable overhang sensor

    NASA Astrophysics Data System (ADS)

    Wang, Yong; Tian, Kai; Hunter, Lehr L.; Ritzo, Brandon; Gu, Li-Qun

    2014-09-01

    Nanopores provide a unique single-molecule platform for genetic and epigenetic detection. The target nucleic acids can be accurately analyzed by characterizing their specific electric fingerprints or signatures in the nanopore. Here we report a series of novel nanopore signatures generated by target nucleic acids that are hybridized with a probe. A length-tunable overhang appended to the probe functions as a sensor to specifically modulate the nanopore current profile. The resulting signatures can reveal multiple mechanisms for the orientational trapping, unzipping, escaping and translocation of nucleic acids in the nanopore. This universal approach can be used to program various molecular movement pathways, elucidate their kinetics, and enhance the sensitivity and specificity of the nanopore sensor for nucleic acid detection.Nanopores provide a unique single-molecule platform for genetic and epigenetic detection. The target nucleic acids can be accurately analyzed by characterizing their specific electric fingerprints or signatures in the nanopore. Here we report a series of novel nanopore signatures generated by target nucleic acids that are hybridized with a probe. A length-tunable overhang appended to the probe functions as a sensor to specifically modulate the nanopore current profile. The resulting signatures can reveal multiple mechanisms for the orientational trapping, unzipping, escaping and translocation of nucleic acids in the nanopore. This universal approach can be used to program various molecular movement pathways, elucidate their kinetics, and enhance the sensitivity and specificity of the nanopore sensor for nucleic acid detection. Electronic supplementary information (ESI) available. See DOI: 10.1039/c4nr03195d

  10. Unzipping bird feathers

    PubMed Central

    Kovalev, Alexander; Filippov, Alexander E.; Gorb, Stanislav N.

    2014-01-01

    The bird feather vane can be separated into two parts by pulling the barbs apart. The original state can be re-established easily by lightly stroking through the feather. Hooklets responsible for holding vane barbs together are not damaged by multiple zipping and unzipping cycles. Because numerous microhooks keep the integrity of the feather, their properties are of great interest for understanding mechanics of the entire feather structure. This study was undertaken to estimate the separation force of single hooklets and their arrays using force measurement of an unzipping feather vane. The hooklets usually separate in some number synchronously (20 on average) with the highest observed separation force of 1.74 mN (average force 0.27 mN), whereas the single hooklet separation force was 14 μN. A simple numerical model was suggested for a better understanding of zipping and unzipping behaviour in feathers. The model demonstrates features similar to those observed in experiments. PMID:24352674

  11. Unzipped and loaded: the role of DNA helicases and RFC clamp-loading complexes in sister chromatid cohesion.

    PubMed

    Skibbens, Robert V

    2005-06-20

    It is well known that the products of chromosome replication are paired to ensure that the sisters segregate away from each other during mitosis. A key issue is how cells pair sister chromatids but preclude the catastrophic pairing of nonsister chromatids. The identification of both replication factor C and DNA helicases as critical for sister chromatid pairing has brought new insights into this fundamental process.

  12. Constant-force approach to discontinuous potentials.

    PubMed

    Orea, Pedro; Odriozola, Gerardo

    2013-06-07

    Aiming to approach the thermodynamical properties of hard-core systems by standard molecular dynamics simulation, we propose setting a repulsive constant-force for overlapping particles. That is, the discontinuity of the pair potential is replaced by a linear function with a large negative slope. Hence, the core-core repulsion, usually modeled with a power function of distance, yields a large force as soon as the cores slightly overlap. This leads to a quasi-hardcore behavior. The idea is tested for a triangle potential of short range. The results obtained by replica exchange molecular dynamics for several repulsive forces are contrasted with the ones obtained for the discontinuous potential and by means of replica exchange Monte Carlo. We found remarkable agreements for the vapor-liquid coexistence densities as well as for the surface tension.

  13. A Constant-Force Resistive Exercise Unit

    NASA Technical Reports Server (NTRS)

    Colosky, Paul; Ruttley, Tara

    2010-01-01

    A constant-force resistive exercise unit (CFREU) has been invented for use in both normal gravitational and microgravitational environments. In comparison with a typical conventional exercise machine, this CFREU weighs less and is less bulky: Whereas weight plates and associated bulky supporting structures are used to generate resistive forces in typical conventional exercise machines, they are not used in this CFREU. Instead, resistive forces are generated in this CFREU by relatively compact, lightweight mechanisms based on constant-torque springs wound on drums. Each such mechanism is contained in a module, denoted a resistive pack, that includes a shaft for making a torque connection to a cable drum. During a stroke of resistive exercise, the cable is withdrawn from the cable drum against the torque exerted by the resistance pack. The CFREU includes a housing, within which can be mounted one or more resistive pack(s). The CFREU also includes mechanisms for engaging any combination of (1) one or more resistive pack(s) and (2) one or more spring(s) within each resistive pack to obtain a desired level of resistance.

  14. Star polymers rupture induced by constant forces.

    PubMed

    García, N A; Febbo, M; Vega, D A; Milchev, A

    2014-10-28

    In this work, we study the breakage process of an unknotted three-arm star-shaped polymer when it is pulled from its free ends by a constant force. The star polymer configuration is described through an array of monomers coupled by anharmonic bonds, while the rupture process is tracked in three-dimensional space by means of Langevin Molecular Dynamics simulations. The interaction between monomers is described by a Morse potential, while a Weeks-Chandler-Anderson energetic contribution accounts for the excluded volume interaction. We explore the effect of the molecular architecture on the distributions of rupture times over a broad interval of pulling forces and star configurations. It was found that the rupture time distribution of the individual star arms is strongly affected by the star configuration imposed by the pulling forces and the length of the arms. We also observed that for large pulling forces the rupture time distributions resemble the dominant features observed for linear polymer chains. The model introduced here provides the basic ingredients to describe the effects of tensile forces on stress-induced degradation of branched macromolecules and polymer networks.

  15. Orientation-selective unzipping of carbon nanotubes.

    PubMed

    Zhang, Hongyu; Zhao, Mingwen; He, Tao; Zhang, Xuejuan; Wang, Zhenhai; Xi, Zexiao; Yan, Shishen; Liu, Xiangdong; Xia, Yueyuan; Mei, Liangmo

    2010-11-07

    We carried out first-principles calculations to explore the oxidative longitudinal unzipping of single-walled carbon nanotubes (SWCNTs) of different diameters and chiralities. We found that the initial attack leading to nanotube unzipping prefers to occur in the middle region for armchair tubes, and at the tube ends for zigzag tubes. Once the initial attack has taken place, by overcoming an energy barrier whose value decreases with increasing tube diameter, the subsequent breakage of C-C bonds parallel to the ones broken in the former process is barrierless. The energetically preferred unzipping path is parallel to the tube axis for armchair tubes, resulting in straight zigzag-edged graphene nanoribbons. For zigzag tubes, there are two energetically equivalent unzipping directions corresponding to the opening of two types of C-C bonds tilted towards the tube axis, giving rise to helical unzipping paths. This is disadvantageous for the production of straight graphene ribbons. A local curvature modulation procedure is proposed to efficiently control the location of the initial attack and thus the shape of the produced graphene nanoribbons.

  16. Unzipping mechanism of the double-stranded DNA unwinding by a hexameric helicase: quantitative analysis of the rate of the dsDNA unwinding, processivity and kinetic step-size of the Escherichia coli DnaB helicase using rapid quench-flow method.

    PubMed

    Galletto, Roberto; Jezewska, Maria J; Bujalowski, Wlodzimierz

    2004-10-08

    Kinetics of the double-stranded (ds) DNA unwinding by the Escherichia coli replicative helicase DnaB protein has been examined under single-turnover conditions using the chemical quench-flow technique. The unwinding reaction proceeds through an initial conformational transition followed by the unwinding catalytic steps and the release of the single-stranded (ss) DNA. Analyses of the reaction as a function of the number of base-pairs in the dsDNA reveal that the number of catalytic steps is not strictly proportional to the length of the dsDNA. As the helicase approaches the end of the substrate, the remaining approximately 11 bp of the DNA melts without catalytic participation of the enzyme. The kinetic step-size of the DnaB helicase, i.e. the number of the base-pairs unwound in a single catalytic step is only 1.4(+/- 0.2). The low value of the step-size indicates that the helicase unwinds a single base-pair in a single catalytic step. Thus, the DnaB helicase unzips the dsDNA in a reverse process to the zipping mechanism of the non-enzymatic double helix formation. The protein is a fast helicase that at 25 degrees C unwinds approximately 291 bp/s, much faster than previously thought, and the unwinding rate can be much higher at higher temperatures. However, the ATP-state of the enzyme has an increased dissociation rate, resulting in only a moderate unwinding processivity, P = 0.89(+/- 0.03), little dependent on the temperature. The conformational transition of the DnaB helicase-DNA complex, preceding the unwinding, is an intrinsic transition of the enzyme from the stationary conformation to the ATP-state of the helicase.

  17. Oxygen-driven unzipping of graphitic materials.

    PubMed

    Li, Je-Luen; Kudin, Konstantin N; McAllister, Michael J; Prud'homme, Robert K; Aksay, Ilhan A; Car, Roberto

    2006-05-05

    Optical microscope images of graphite oxide (GO) reveal the occurrence of fault lines resulting from the oxidative processes. The fault lines and cracks of GO are also responsible for their much smaller size compared with the starting graphite materials. We propose an unzipping mechanism to explain the formation of cracks on GO and cutting of carbon nanotubes in an oxidizing acid. GO unzipping is initiated by the strain generated by the cooperative alignment of epoxy groups on a carbon lattice. We employ two small GO platelets to show that through the binding of a new epoxy group or the hopping of a nearby existing epoxy group, the unzipping process can be continued during the oxidative process of graphite. The same epoxy group binding pattern is also likely to be present in an oxidized carbon nanotube and cause its breakup.

  18. Carbon nanoelectronics: unzipping tubes into graphene ribbons.

    PubMed

    Santos, H; Chico, L; Brey, L

    2009-08-21

    We report on the transport properties of novel carbon nanostructures made of partially unzipped carbon nanotubes, which can be regarded as a seamless junction of a tube and a nanoribbon. We find that graphene nanoribbons act at certain energy ranges as perfect valley filters for carbon nanotubes, with the maximum possible conductance. Our results show that a partially unzipped carbon nanotube is a magnetoresistive device, with a very large value of magnetoresistance. We explore the properties of several structures combining nanotubes and graphene nanoribbons, demonstrating that they behave as optimal contacts for each other, and opening a new route for the design of mixed graphene-nanotube devices.

  19. Chemical unzipping of WS2 nanotubes.

    PubMed

    Nethravathi, C; Jeffery, A Anto; Rajamathi, Michael; Kawamoto, Naoyuki; Tenne, Reshef; Golberg, Dmitri; Bando, Yoshio

    2013-08-27

    WS2 nanoribbons have been synthesized by chemical unzipping of WS2 nanotubes. Lithium atoms are intercalated in WS2 nanotubes by a solvothermal reaction with n-butyllithium in hexane. The lithiated WS2 nanotubes are then reacted with various solvents--water, ethanol, and long chain thiols. While the tubes break into pieces when treated with water and ethanol, they unzip through longitudinal cutting along the axes to yield nanoribbons when treated with long chain thiols, 1-octanethiol and 1-dodecanethiol. The slow diffusion of the long chain thiols reduces the aggression of the reaction, leading to controlled opening of the tubes.

  20. Unzipping mechanism of the double-stranded DNA unwinding by a hexameric helicase: the effect of the 3' arm and the stability of the dsDNA on the unwinding activity of the Escherichia coli DnaB helicase.

    PubMed

    Galletto, Roberto; Jezewska, Maria J; Bujalowski, Wlodzimierz

    2004-10-08

    The effect of two structural elements of a replication DNA fork substrate, the length of the 3' arm of the fork and the stability of the double-stranded DNA (dsDNA) part, on the kinetics of the dsDNA unwinding by the Escherichia coli hexameric helicase DnaB protein has been examined under single turnover conditions using the rapid quench-flow technique. The length of the 3' arm of the replication fork, i.e. the number of nucleotides in the arm, is a major structural factor that controls the unwinding rate and processivity of the helicase. The data show the existence of an optimal length of the 3' arm where there is the highest unwinding rate and processivity, indicating that during the unwinding process, the helicase transiently interacts with the 3' arm at a specific distance on the arm with respect to the duplex part of the DNA. Moreover, the area on the enzyme that engages in interactions has also a discrete size. For DNA substrates with the 3' arm containing 14, or less, nucleotide residues, the DnaB helicase becomes a completely distributive enzyme. However, the 3' arm is not a "specific activating cofactor" in the unwinding reaction. Rather, the 3' arm plays a role as a mechanical fulcrum for the enzyme, necessary to provide support for the advancing large helicase molecule on the opposite strand of the DNA. Binding of ATP is necessary to engage the 3' arm with the DnaB helicase, but it does not change the initial distribution of complexes of the enzyme with the DNA fork substrate. Stability of the dsDNA has a significant effect on the unwinding rate and processivity. The unwinding rate constant is a decreasing linear function of the fractional content of GC base-pairs in the dsDNA, indicating that the activation of the unwinding step is proportional to the stability of the nucleic acid.

  1. Gravity-independent constant force resistive exercise unit

    NASA Technical Reports Server (NTRS)

    Colosky, Jr., Paul E. (Inventor); Ruttley, Tara M. (Inventor)

    2004-01-01

    This invention describes a novel gravity-independent exercise unit designed for use in microgravity, or on the ground, as a means by which to counter muscle atrophy and bone degradation due to disuse or underuse. Modular resistive packs comprising constant torque springs provide constant force opposing the withdrawal of an exercise cable from the device. In addition to uses within the space program, the compact resistive packs of the CFREU allow the unit to be small enough for easy use as a home gym for personal use, or as a supplement for rehabilitation programs. Resistive packs may be changed conveniently out of the CFREU according to the desired exercise regimen. Thus, the resistive packs replace the need for expensive, heavy, and bulky traditional weight plates. The CFREU may be employed by hospitals, rehabilitation and physical therapy clinics, and other related professional businesses.

  2. Lieb-Liniger gas in a constant-force potential

    SciTech Connect

    Jukic, D.; Galic, S.; Buljan, H.; Pezer, R.

    2010-08-15

    We use Gaudin's Fermi-Bose mapping operator to calculate exact solutions for the Lieb-Liniger model in a linear (constant-force) potential (the constructed exact stationary solutions are referred to as the Lieb-Liniger-Airy wave functions). The ground-state properties of the gas in the wedgelike trapping potential are calculated in the strongly interacting regime by using Girardeau's Fermi-Bose mapping and the pseudopotential approach in the 1/c approximation (c denotes the strength of the interaction). We point out that quantum dynamics of Lieb-Liniger wave packets in the linear potential can be calculated by employing an N-dimensional Fourier transform as in the case of free expansion.

  3. A gravity-independent constant force resistive exercise unit.

    PubMed

    Ruttley, T M; Colosky, P E; James, S P

    2001-01-01

    This study designed, developed and tested a novel, practical, gravity-independent exercise machine, the Constant Force Resistance Exercise Unit (CFREU). A CFREU prototype was designed and built according to National Aeronautic and Space Administration (NASA) hardware and physiological requirements, and was evaluated for potential exercise countermeasure viability. Life cycle data exhibit lower life than required by NASA guidelines; however, current CFREU re-designs are addressing this issue. Electromyography (EMG) data indicate that the CFREU used on the ground and in microgravity during exercise is capable of providing forces on the muscles that are similar to a standard free-weight machine used in gravity. Given the results of this study, the CFREU has proven to be a viable potential resistive exercise countermeasure to the deconditioning of the musculoskeletal system in microgravity.

  4. Structural destabilization of DNA duplexes containing single-base lesions investigated by nanopore measurements.

    PubMed

    Jin, Qian; Fleming, Aaron M; Ding, Yun; Burrows, Cynthia J; White, Henry S

    2013-11-12

    The influence of DNA duplex structural destabilization introduced by a single base-pair modification was investigated by nanopore measurements. A series of 11 modified base pairs were introduced into the context of an otherwise complementary DNA duplex formed by a 17-mer and a 65-mer such that the overhanging ends comprised poly(dT)23 tails, generating a representative set of duplexes that display a range of unzipping mechanistic behaviors and kinetic stabilities. The guanine oxidation products 8-oxo-7,8-dihydroguanine (OG), guanidinohydantoin (Gh), and spiroiminodihydantoin (Sp) were paired with either cytosine (C), adenine (A), or 2,6-diaminopurine (D) to form modified base pairs. The mechanism and kinetic rate constants of duplex dissociation were determined by threading either the 3' or 5' overhangs into an α-hemolysin (α-HL) channel under an electrical field and measuring the distributions of unzipping times at constant force. In order of decreasing thermodynamic stability (as measured by duplex melting points), the rate of duplex dissociation increases, and the mechanism evolves from a first-order reaction to two sequential first-order reactions. These measurements allow us to rank the kinetic stability of lesion-containing duplexes relative to the canonical G:C base pair in which the OG:C, Gh:C, and Sp:C base pairs are, respectively, 3-200 times less stable. The rate constants also depend on whether unzipping was initiated from the 3' versus 5' side of the duplex. The kinetic stability of these duplexes was interpreted in terms of the structural destabilization introduced by the single base-pair modification. Specifically, a large distortion of the duplex backbone introduced by the presence of the highly oxidized guanine products Sp and Gh leads to a rapid two-step unzipping. The number of hydrogen bonds in the modified base pair plays a lesser role in determining the kinetics of duplex dissociation.

  5. Theoretical aspects of WS₂ nanotube chemical unzipping.

    PubMed

    Kvashnin, D G; Antipina, L Yu; Sorokin, P B; Tenne, R; Golberg, D

    2014-07-21

    Theoretical analysis of experimental data on unzipping multilayered WS₂ nanotubes by consequent intercalation of lithium atoms and 1-octanethiol molecules [C. Nethravathi, et al., ACS Nano, 2013, 7, 7311] is presented. The radial expansion of the tube was described using continuum thin-walled cylinder approximation with parameters evaluated from ab initio calculations. Assuming that the attractive driving force of the 1-octanethiol molecule is its reaction with the intercalated Li ions ab initio calculations of a 1-octanethiol molecule bonding with Li(+) were carried out. In addition, the non-chemical interactions of the 1-octanethiol dipole with an array of positive point charges representing Li(+) were taken into account. Comparing between the energy gain from these interactions and the elastic strain energy of the nanotube allows us to evaluate a value for the tube wall deformation after the implantation of 1-octanethiol molecules. The ab initio molecular dynamics simulation confirmed our estimates and demonstrated that a strained WS₂ nanotube, with a decent concentration of 1-octanethiol molecules, should indeed be unzipped into the WS₂ nanoribbon.

  6. Vacuum-Assisted, Constant-Force Exercise Device

    NASA Technical Reports Server (NTRS)

    Hansen, Christopher P.; Jensen, Scott

    2006-01-01

    The vacuum-assisted, constant-force exercise device (VAC-FED) has been proposed to fill a need for a safe, reliable exercise machine that would provide constant loads that could range from 20 to 250 lb (0.09 to 1.12 kN) with strokes that could range from 6 to 36 in. (0.15 to 0.91 m). The VAC-FED was originally intended to enable astronauts in microgravity to simulate the lifting of free weights, but it could just as well be used on Earth for simulated weight lifting and other constant-force exercises. Because the VAC-FED would utilize atmospheric/vacuum differential pressure instead of weights to generate force, it could weigh considerably less than either a set of free weights or a typical conventional exercise machine based on weights. Also, the use of atmospheric/ vacuum differential pressure to generate force would render the VAC-FED inherently safer, relative to free weights and to conventional exercise machines that utilize springs to generate forces. The overall function of the VAC-FED would be to generate a constant tensile force in an output cable, which would be attached to a bar, handle, or other exercise interface. The primary force generator in the VAC-FED would be a piston in a cylinder. The piston would separate a volume vented to atmosphere at one end of the cylinder from an evacuated volume at the other end of the cylinder (see figure). Hence, neglecting friction at the piston seals, the force generated would be nearly constant equal to the area of the piston multiplied by the atmospheric/vacuum differential pressure. In the vented volume in the cylinder, a direct-force cable would be looped around a pulley on the piston, doubling the stroke and halving the tension. One end of the direct-force cable would be anchored to a cylinder cap; the other end of the direct-force cable would be wrapped around a variable-ratio pulley that would couple tension to the output cable. As its name suggests, the variable-ratio pulley would contain a mechanism that

  7. Zipping and unzipping of cosmic string loops in collision

    SciTech Connect

    Firouzjahi, H.; Karouby, J.; Khosravi, S.; Brandenberger, R.

    2009-10-15

    In this paper the collision of two cosmic string loops is studied. After collision junctions are formed and the loops are entangled. We show that after their formation the junctions start to unzip and the loops disentangle. This analysis provides a theoretical understanding of the unzipping effect observed in numerical simulations of a network of cosmic strings with more than one type of cosmic strings. The unzipping phenomena have important effects in the evolution of cosmic string networks when junctions are formed upon collision, such as in a network of cosmic superstrings.

  8. Oxidative unzipping of stacked nitrogen-doped carbon nanotube cups.

    PubMed

    Dong, Haifeng; Zhao, Yong; Tang, Yifan; Burkert, Seth C; Star, Alexander

    2015-05-27

    We demonstrate a facile synthesis of different nanostructures by oxidative unzipping of stacked nitrogen-doped carbon nanotube cups (NCNCs). Depending on the initial number of stacked-cup segments, this method can yield graphene nanosheets (GNSs) or hybrid nanostructures comprised of graphene nanoribbons partially unzipped from a central nanotube core. Due to the stacked-cup structure of as-synthesized NCNCs, preventing complete exposure of graphitic planes, the unzipping mechanism is hindered, resulting in incomplete unzipping; however, individual, separated NCNCs are completely unzipped, yielding individual nitrogen-doped GNSs. Graphene-based materials have been employed as electrocatalysts for many important chemical reactions, and it has been proposed that increasing the reactive edges results in more efficient electrocatalysis. In this paper, we apply these graphene conjugates as electrocatalysts for the oxygen reduction reaction (ORR) to determine how the increase in reactive edges affects the electrocatalytic activity. This investigation introduces a new method for the improvement of ORR electrocatalysts by using nitrogen dopants more effectively, allowing for enhanced ORR performance with lower overall nitrogen content. Additionally, the GNSs were functionalized with gold nanoparticles (GNPs), resulting in a GNS/GNP hybrid, which shows efficient surface-enhanced Raman scattering and expands the scope of its application in advanced device fabrication and biosensing.

  9. Stretching Submicron Biomolecules with Constant-Force Axial Optical Tweezers

    PubMed Central

    Chen, Yih-Fan; Blab, Gerhard A.; Meiners, Jens-Christian

    2009-01-01

    Optical tweezers have become powerful tools to manipulate biomolecular systems, but are increasingly difficult to use when the size of the molecules is <1 μm. Many important biological structures and processes, however, occur on the submicron length scale. Therefore, we developed and characterized an optical manipulation protocol that makes this length scale accessible by stretching the molecule in the axial direction of the laser beam, thus avoiding limiting artifacts from steric hindrances from the microscope coverslip and other surface effects. The molecule is held under constant mechanical tension by a combination of optical gradient forces and backscattering forces, eliminating the need for electronic feedback. We demonstrate the utility of this method through a measurement of the force-extension relationship of a 1298 bp ds-DNA molecule. PMID:19486692

  10. Using nega'tor springs as a source of constant force

    NASA Astrophysics Data System (ADS)

    Reich, G. R.; Bradshaw, Adam

    2002-03-01

    In recent years there has been interest in having students experiment with nongravitational sources of constant acceleration.We introduce the use of constant force Neg'ator spring motors for this purpose.

  11. Chemical Sharpening, Shortening, and Unzipping of Boron Nitride Nanotubes

    NASA Technical Reports Server (NTRS)

    Liao, Yunlong; Chen, Zhongfang; Connell, John W.; Fay, Catharine C.; Park, Cheol; Kim, Jae-Woo; Lin, Yi

    2014-01-01

    Boron nitride nanotubes (BNNTs), the one-dimensional member of the boron nitride nanostructure family, are generally accepted to be highly inert to oxidative treatments and can only be covalently modifi ed by highly reactive species. Conversely, it is discovered that the BNNTs can be chemically dispersed and their morphology modifi ed by a relatively mild method: simply sonicating the nanotubes in aqueous ammonia solution. The dispersed nanotubes are significantly corroded, with end-caps removed, tips sharpened, and walls thinned. The sonication treatment in aqueous ammonia solution also removes amorphous BN impurities and shortened BNNTs, resembling various oxidative treatments of carbon nanotubes. Importantly, the majority of BNNTs are at least partially longitudinally cut, or "unzipped". Entangled and freestanding BN nanoribbons (BNNRs), resulting from the unzipping, are found to be approximately 5-20 nm in width and up to a few hundred nanometers in length. This is the fi rst chemical method to obtain BNNRs from BNNT unzipping. This method is not derived from known carbon nanotube unzipping strategies, but is unique to BNNTs because the use of aqueous ammonia solutions specifi cally targets the B-N bond network. This study may pave the way for convenient processing of BNNTs, previously thought to be highly inert, toward controlling their dispersion, purity, lengths, and electronic properties.

  12. Molecular dynamics simulations of the morphology transformations in unzipped carbon nanotubes

    NASA Astrophysics Data System (ADS)

    Xu, Jiafang; Zhang, Yingnan; Wang, Tao; Zheng, Xin; Li, Wen; Dong, Zihan; Wang, Wensen

    2016-08-01

    Tuning the assembly of carbon nanomaterials to obtain a kaleidoscope of carbon nanostructures is very important and challenging for the development of nanotechnology. Using molecular dynamics simulations method, we studied the morphology transformations of unzipped CNTs with different unzipping patterns. By modulating the unzipping patterns, the CNTs could self-assemble forming graphene nanoribbons and carbon nanoscrolls. From the energy analyzation, we find that the van der Waals interactions are responsible for the assembly of the unzipped CNTs. This unusual self-assembling method for CNTs could provide clues for further studies on the design of novel nanostructures.

  13. Hydrolytic Unzipping of Boron Nitride Nanotubes in Nitric Acid

    NASA Astrophysics Data System (ADS)

    Kim, Dukeun; Muramatsu, Hiroyuki; Kim, Yoong Ahm

    2017-02-01

    Boron nitride nanoribbons (BNNRs) have very attractive electrical and optical properties due to their unique edge states and width-related properties. Herein, for the first time, BNNRs were produced by a simple reflux of boron nitride nanotubes (BNNTs) in nitric acid containing water, which had led to unzipped sidewalls through hydrolysis. Their high reactivity that originated from edges was verified via a strong interaction with methylene blue.

  14. Hydrolytic Unzipping of Boron Nitride Nanotubes in Nitric Acid.

    PubMed

    Kim, Dukeun; Muramatsu, Hiroyuki; Kim, Yoong Ahm

    2017-12-01

    Boron nitride nanoribbons (BNNRs) have very attractive electrical and optical properties due to their unique edge states and width-related properties. Herein, for the first time, BNNRs were produced by a simple reflux of boron nitride nanotubes (BNNTs) in nitric acid containing water, which had led to unzipped sidewalls through hydrolysis. Their high reactivity that originated from edges was verified via a strong interaction with methylene blue.

  15. Grain-boundary unzipping by oxidation in polycrystalline graphene

    NASA Astrophysics Data System (ADS)

    Alexandre, Simone; Lucio, Aline; Nunes, Ricardo

    2011-03-01

    The need for large-scale production of graphene will inevitably lead to synthesis of the polycrystalline material [1,2]. Understanding the chemical, mechanical, and electronic properties of grain boundaries in graphene polycrystals will be crucial for the development of graphene-based electronics. Oxidation of this material has been suggested to lead to graphene ribbons, by the oxygen-driven unzipping mechanism. A cooperative-strain mechanism, based on the formation of epoxy groups along lines of parallel bonds in the hexagons of graphene's honeycomb lattice, was proposed to explain the unzipping effect in bulk graphene In this work we employ ab initio calculations to study the oxidation of polycrystalline graphene by chemisorption of oxygen at the grain boundaries. Our results indicate that oxygen tends to segregate at the boundaries, and that the unzipping mechanism is also operative along the grain boundaries, despite the lack of the parallel bonds due to the presence of fivefold and sevenfold carbon rings along the boundary core. We acknowledge support from the Brazilian agencies: CNPq, Fapemig, and INCT-Materiais de Carbono.

  16. Observing Stepwise Unzipping of Neuronal Snare Protein with Steered Molecular Dynamics

    NASA Astrophysics Data System (ADS)

    Tekpinar, Mustafa; Zheng, Wenjun

    2013-03-01

    Soluble N-ethylmaleimide-sensitive factor Attachment Protein Receptors (SNARE) play a crucial role in membrane fusion. Neuronal SNAREs are made up of four helices: a snaptobrevin, a syntaxin 1, and two SNAP-25 helices. We applied constant velocity pulling forces to C terminal of snaptobrevin in SNARE complex to understand unzipping mechanism of neuronal SNAREs. We successfully observed unzipping of snaptobrevin from the other three helices in two steps: C terminal unzipping and N terminal unzipping. Our results have good agreement with recent optical tweezer experiments that observe this stepwise unzipping. Additionally, our simulations reveal that these two steps differ from each other. We believe that these different mechanisms can help us to understand SNARE mediated membrane fusion process better.

  17. Two-dimensional transport model of coupled Brownian particles driven by biharmonic forces and constant forces

    NASA Astrophysics Data System (ADS)

    Wu, Weixia; Meng, Tao

    2017-01-01

    A directed transport model of coupled Brownian particles in a two-dimensional potential is established. In this model, the system of Brownian particles is driven by biharmonic forces and constant forces. By numerical simulation, the cooperative transport behaviours of the system are investigated. The results show that the average velocity of the system is significantly independent of the frequencies and intensities of the harmonic forces, the barrier height of the ratchet potential, coupling strength and noise intensity. The average velocity increases monotonically with increasing the intensities of the harmonic forces, and can reach some maximum values when the two frequencies are equal. In addition, the average velocity presents stochastic resonance and generalized resonance for noise intensity, coupling strength and the barrier height of the ratchet potential. Moreover, when a constant force is exerted on the ratchet potential direction, it will promote the directed transport of the system. But, when on the non-ratchet potential direction, the transport will not be affected.

  18. Practical Considerations for Using Constant Force Springs in Space-Based Mechanisms

    NASA Technical Reports Server (NTRS)

    Williams, R. Brett; Fisher, Charles D.; Gallon, John C.

    2013-01-01

    Mechanical springs are a common element in mechanism from all walks of life; cars, watches, appliances, and many others. These springs generally exhibit a linear relationship between force and deflection. In small mechanisms, deflections are small so the variation in spring force between one position and another are generally small and do not influence the design or functionality of the device. However, as the spacecraft industry drives towards larger, deployable satellites, the distances a spring or springs must function over can become considerable so much so that the structural integrity of the device may be impacted. As such, an increasingly common mechanism element is the constant force spring- one that provides a constant force regardless of deflection. These elements are commonly in the conceptual design phase to deal with system-level large deflections, but in the detailed design or integration test phase they can pose significant implementation issues. This article addresses some of the detailed issues in order for these constant force springs to be properly designed into space systems.

  19. Practical Considerations for Using Constant Force Springs in Space-Based Mechanisms

    NASA Technical Reports Server (NTRS)

    Williams, R. Brett; Fisher, Charles D.; Gallon, John C.

    2013-01-01

    Mechanical springs are a common element in mechanism from all walks of life; cars, watches, appliances, and many others. These springs generally exhibit a linear relationship between force and deflection. In small mechanisms, deflections are small so the variation in spring force between one position and another are generally small and do not influence the design or functionality of the device. However, as the spacecraft industry drives towards larger, deployable satellites, the distances a spring or springs must function over can become considerable so much so that the structural integrity of the device may be impacted. As such, an increasingly common mechanism element is the constant force spring- one that provides a constant force regardless of deflection. These elements are commonly in the conceptual design phase to deal with system-level large deflections, but in the detailed design or integration test phase they can pose significant implementation issues. This article addresses some of the detailed issues in order for these constant force springs to be properly designed into space systems.

  20. Theoretical aspects of WS2 nanotube chemical unzipping

    NASA Astrophysics Data System (ADS)

    Kvashnin, D. G.; Antipina, L. Yu.; Sorokin, P. B.; Tenne, R.; Golberg, D.

    2014-06-01

    Theoretical analysis of experimental data on unzipping multilayered WS2 nanotubes by consequent intercalation of lithium atoms and 1-octanethiol molecules [C. Nethravathi, et al., ACS Nano, 2013, 7, 7311] is presented. The radial expansion of the tube was described using continuum thin-walled cylinder approximation with parameters evaluated from ab initio calculations. Assuming that the attractive driving force of the 1-octanethiol molecule is its reaction with the intercalated Li ions ab initio calculations of a 1-octanethiol molecule bonding with Li+ were carried out. In addition, the non-chemical interactions of the 1-octanethiol dipole with an array of positive point charges representing Li+ were taken into account. Comparing between the energy gain from these interactions and the elastic strain energy of the nanotube allows us to evaluate a value for the tube wall deformation after the implantation of 1-octanethiol molecules. The ab initio molecular dynamics simulation confirmed our estimates and demonstrated that a strained WS2 nanotube, with a decent concentration of 1-octanethiol molecules, should indeed be unzipped into the WS2 nanoribbon.

  1. Canine retraction with rare earth magnets: an investigation into the validity of the constant force hypothesis.

    PubMed

    Daskalogiannakis, J; McLachlan, K R

    1996-05-01

    The objective of this study was to test the hypothesis that a prolonged constant force provides more effective tooth movement than an impulsive force of short duration. Six human subjects were selected, the main criterion being a need for extraction of their upper first premolars. Canine retraction on these subjects was executed on one side with the application of a force rapidly declining in magnitude, produced by a vertical loop, and on the other side with the application of a relatively constant force. This type of force was achieved by a similar vertical loop which was constantly activated by three parylene-coated neodymium-iron-boron (Nd2Fe14P) block magnets. The vertical loop on the control side was reactivated 6 weeks after the initial activation. No reactivation was necessary on the experimental side for the duration of the experiment. The rate of tooth movement on the two sides was compared over a period of 3 months, on the basis of maxillary impressions taken at frequent intervals during the course of the study. The canines retracted with a constant force moved statistically significantly more than the control canines (p < 0.05) during the experimental period. The average differences in the mean rates of tooth movement between the two sides were in the order of 2:1 in favor of the experimental side. There were no statistically significant differences in the changes of angulation (tipping) or rotation about the y axis between the two sides. The duration of force application seems to be a critical factor in regulating rate of tooth movement. Conversely, magnitude of the applied force did not appear to be of primary significance.

  2. Dopant-specific unzipping of carbon nanotubes for intact crystalline graphene nanostructures

    PubMed Central

    Lim, Joonwon; Narayan Maiti, Uday; Kim, Na-Young; Narayan, Rekha; Jun Lee, Won; Sung Choi, Dong; Oh, Youngtak; Min Lee, Ju; Yong Lee, Gil; Hun Kang, Seok; Kim, Hyunwoo; Kim, Yong-Hyun; Ouk Kim, Sang

    2016-01-01

    Atomic level engineering of graphene-based materials is in high demand to enable customize structures and properties for different applications. Unzipping of the graphene plane is a potential means to this end, but uncontrollable damage of the two-dimensional crystalline framework during harsh unzipping reaction has remained a key challenge. Here we present heteroatom dopant-specific unzipping of carbon nanotubes as a reliable and controllable route to customized intact crystalline graphene-based nanostructures. Substitutional pyridinic nitrogen dopant sites at carbon nanotubes can selectively initiate the unzipping of graphene side walls at a relatively low electrochemical potential (0.6 V). The resultant nanostructures consisting of unzipped graphene nanoribbons wrapping around carbon nanotube cores maintain the intact two-dimensional crystallinity with well-defined atomic configuration at the unzipped edges. Large surface area and robust electrical connectivity of the synergistic nanostructure demonstrate ultrahigh-power supercapacitor performance, which can serve for AC filtering with the record high rate capability of −85° of phase angle at 120 Hz. PMID:26796993

  3. Dopant-specific unzipping of carbon nanotubes for intact crystalline graphene nanostructures

    NASA Astrophysics Data System (ADS)

    Lim, Joonwon; Narayan Maiti, Uday; Kim, Na-Young; Narayan, Rekha; Jun Lee, Won; Sung Choi, Dong; Oh, Youngtak; Min Lee, Ju; Yong Lee, Gil; Hun Kang, Seok; Kim, Hyunwoo; Kim, Yong-Hyun; Ouk Kim, Sang

    2016-01-01

    Atomic level engineering of graphene-based materials is in high demand to enable customize structures and properties for different applications. Unzipping of the graphene plane is a potential means to this end, but uncontrollable damage of the two-dimensional crystalline framework during harsh unzipping reaction has remained a key challenge. Here we present heteroatom dopant-specific unzipping of carbon nanotubes as a reliable and controllable route to customized intact crystalline graphene-based nanostructures. Substitutional pyridinic nitrogen dopant sites at carbon nanotubes can selectively initiate the unzipping of graphene side walls at a relatively low electrochemical potential (0.6 V). The resultant nanostructures consisting of unzipped graphene nanoribbons wrapping around carbon nanotube cores maintain the intact two-dimensional crystallinity with well-defined atomic configuration at the unzipped edges. Large surface area and robust electrical connectivity of the synergistic nanostructure demonstrate ultrahigh-power supercapacitor performance, which can serve for AC filtering with the record high rate capability of -85° of phase angle at 120 Hz.

  4. A glucose biosensor based on partially unzipped carbon nanotubes.

    PubMed

    Hu, Huifang; Feng, Miao; Zhan, Hongbing

    2015-08-15

    An amperometric glucose biosensor based on direct electron transfer of glucose oxidase (GOD) self-assembled on the surface of partially unzipped carbon nanotubes (PUCNTs) modified glassy carbon electrode (GCE) has been successfully fabricated. PUCNTs were synthesized via a facile chemical oxidative etching CNTs and used as a novel immobilization matrix for GOD. The cyclic voltammetric result of the PUCNT/GOD/GCE showed a pair of well-defined and quasi-reversible redox peaks with a formal potential of -0.470V and a peak to peak separation of 37mV, revealing that the fast direct electron transfer between GOD and the electrode has been achieved. It is notable that the glucose determination has been achieved in mediator-free condition. The developed biosensor displayed satisfactory analytical performance toward glucose including high sensitivity (19.50μA mM(-1)cm(-2)), low apparent Michaelis-Menten (5.09mM), a wide linear range of 0-17mM, and also preventing the interference from ascorbic acid, uric acid and dopamine usually coexisting with glucose in human blood. In addition, the biosensor acquired excellent storage stabilities. This facile, fast, environment-friendly and economical preparation strategy of PUCNT-GOD may provide a new platform for the fabrication of biocompatible glucose biosensors and other types of biosensors.

  5. Unzipping carbon nanotubes into nanoribbons upon oxidation: a first-principles study.

    PubMed

    Li, Feng; Kan, Erjun; Lu, Ruifeng; Xiao, Chuanyun; Deng, Kaiming; Su, Haibin

    2012-02-21

    First-principles calculations are performed to investigate the unzipping mechanism of carbon nanotubes (CNTs) into narrow graphene nanoribbons (GNRs) upon oxidation. By treating possible adsorptive structures, we found that, upon further oxidation, epoxy pairs tear the CNT up with an initial energy barrier of 0.59 eV (armchair) and 0.60 eV (zigzag), and the following steps of unzipping CNT become much easier because of the stress induced by the carbonyl pair. However, for zigzag CNTS, the unzipped edge structures of nanoribbons cannot be controlled, because of the similar stability of different oxidation process, which means zigzag CNTs should be avoided in producing high-quality GNRs. This journal is © The Royal Society of Chemistry 2012

  6. Hand dominance during constant force isometric contractions: evidence of different cortical drive commands.

    PubMed

    Pereira, Rafael; Freire, Ivna Vidal; Cavalcanti, Cláudia Virgínia Galindo; Luz, Carla Patrícia Novais; Neto, Osmar Pinto

    2012-08-01

    The purpose of this study was to investigate force variability and sensoriomotor strategies of dominant and nondominant hands of right and left-handed subjects during a submaximal isometric force production task. Twelve right-handed adults (9 men and 3 women; 23 ± 3 year) and twelve left-handed adults (4 men and 8 women; 24 ± 3 year) performed an isometric constant force contraction sustained at 30 and 50% of maximal force for 10 s. Surface EMG signals were obtained from forearm flexors and extensors. Force signals were analyzed in the time (CV of force) and frequency (0-10 Hz) domain. The neural activation of the involved muscles was investigated from the EMG structure using the cross-wavelet spectra of the interference EMG signals of six different frequency bands of the EMG signals were quantified (5-13, 13-30, 30-60, 60-100, 100-150 and 150-200 Hz). The major findings were: (1) dominant and nondominant hands of right- and left-handed subjects exhibited similar CV of force; (2) the power spectrum of force is influenced by handedness, with greater 1-3 Hz oscillations for left-handed subjects when compared to right-handed subjects; (3) right-handed subjects have greater 30-60 Hz neuromuscular activation when compared to left-handed subjects. Our results indicate that right-handed individuals may rely preferentially in visual feedback to carry out a task with visual and proprioceptive feedback because of the left hemisphere specialization on the visuomotor control.

  7. On the possibility of electrochemical unzipping of multiwalled carbon nanotubes to produce graphene nanoribbons

    SciTech Connect

    Zehtab Yazdi, Alireza; Roberts, Edward P.L.; Sundararaj, Uttandaraman

    2016-08-15

    Highlights: • MWCNTs synthesized and electrochemically oxidized to study the formation of GNR • HRTEM, Raman and XPS confirmed no successful unzipping occurred after oxidation • Electrochemical oxidation very unlikely facilitate formation of intercalated MWCNTs - Abstract: Multiwalled carbon nanotubes (MWCNTs) with different geometrical characteristics and chemical doping have been synthesized and electrochemically oxidized to study the possibility of unzipping, and creating graphene nanoribbon (GNR) nanostructures. Modified glassy carbon electrodes of the MWCNTs have been tested in an aqueous electrolyte via anodic scans in a wide range of potentials, followed by keeping at the maximum potential for different times. The microstructural features, structural defects, and functional groups and their elements have been then studied using high resolution transmission electron microscopy (HRTEM), Raman spectroscopy and X-ray photoelectron spectroscopy (XPS), respectively. All results have confirmed that no successful unzipping occurs in the MWCNTs after electrochemical oxidation, even for the nitrogen-doped MWCNTs (CN{sub x}-MWCNTs) with reactive nitrogen groups and defective bamboo structures. In contrast to the report by Shinde et al. (J. Am. Chem. Soc. 2011, 133, 4168–4171), it has been concluded that the electrochemical oxidation in aqueous electrolytes is very unlikely to facilitate sufficient incorporation of the intercalated molecules among the walls of the MWCNTs. These molecules are, however, responsible for unzipping of MWCNTs.

  8. Natural fire-defense of raw white and brown cotton fibers evidenced by suppressed unzipping depolymerization

    USDA-ARS?s Scientific Manuscript database

    Pre-cleaned (scoured or scoured/bleached), cotton-based materials, whose utilization has greatly been enhanced in support of environmental sustainability, burn rapidly, causing a difficulty in controlling the spread of fire. This high burning rate is primarily associated with the unzipping depolymer...

  9. Sequential electrochemical unzipping of single-walled carbon nanotubes to graphene ribbons revealed by in situ Raman spectroscopy and imaging.

    PubMed

    John, Robin; Shinde, Dhanraj B; Liu, Lili; Ding, Feng; Xu, Zhiping; Vijayan, Cherianath; Pillai, Vijayamohanan K; Pradeep, Thalappil

    2014-01-28

    We report an in situ Raman spectroscopic and microscopic investigation of the electrochemical unzipping of single-walled carbon nanotubes (SWNTs). Observations of the radial breathing modes (RBMs) using Raman spectral mapping reveal that metallic SWNTs are opened up rapidly followed by gradual unzipping of semiconducting SWNTs. Consideration of the resonant Raman scattering theory suggests that two metallic SWNTs with chiralities (10, 4) and (12, 0) get unzipped first at a lower electrode potential (0.36 V) followed by the gradual unzipping of another two metallic tubes, (9, 3) and (10, 1), at a relatively higher potential (1.16 V). The semiconducting SWNTs with chiralities (11, 7) and (12, 5), however, get open up gradually at ±1.66 V. A rapid decrease followed by a subsequent gradual decrease in the metallicity of the SWNT ensemble as revealed from a remarkable variation of the peak width of the G band complies well with the variations of RBM. Cyclic voltammetry also gives direct evidence for unzipping in terms of improved capacitance after oxidation followed by more important removal of oxygen functionalities during the reduction step, as reflected in subtle changes of the morphology confirming the formation of graphene nanoribbons. The density functional-based tight binding calculations show additional dependence of chirality and diameter of nanotubes on the epoxide binding energies, which is in agreement with the Raman spectroscopic results and suggests a possible mechanism of unzipping determined by combined effects of the structural characteristics of SWNTs and applied field.

  10. Intercalation-assisted longitudinal unzipping of carbon nanotubes for green and scalable synthesis of graphene nanoribbons

    PubMed Central

    Li, Yan-Sheng; Liao, Jia-Liang; Wang, Shan-Yu; Chiang, Wei-Hung

    2016-01-01

    We have demonstrated an effective intercalation of multi-walled carbon nanotubes (MWCNTs) for the green and scalable synthesis of graphene nanoribbons (GNRs) using an intercalation-assisted longitudinal unzipping of MWCNTs. The key step is to introduce an intercalation treatment of raw MWCNTs with KNO3 and H2SO4, making it promising to decrease the strong van der Waals attractions in the MWCNTs bundles and between the coaxial graphene walls of CNTs. Systematic micro Raman, X-ray photoelectron spectroscopy (XPS), and X-ray diffraction (XRD) characterizations suggest that potassium, nitrate, and sulfate ions play an important role in the CNT intertube and intratube intercalations during the pretreatment. Detailed scanning electron microscopy (SEM), transmission electron microscopy, XRD, and micro Raman characterizations indicate that the developed methodology possesses the ability to synthesis GNRs effectively with an improved CNT concentration in H2SO4 of 10 mg/ml at 70 °C, which is amenable to industrial-scale production because of the decreased amount of strong acid. Our work provides a scientific understanding how to enhance the GNR formation by accelerating the CNT longitudinal unzipping via suitable molecular intercalation. PMID:26948486

  11. An overlooked riddle of life's origins: energy-dependent nucleic acid unzipping.

    PubMed

    Kovác, Ladislav; Nosek, Jozef; Tomáska, L'ubomír

    2003-01-01

    The imposing progress in understanding contemporary life forms on Earth and in manipulating them has not been matched by a comparable progress in understanding the origins of life. This paper argues that a crucial problem of unzipping of the double helix molecule of nucleic acid during its replication has been underrated, if not plainly overlooked, in the theories of life's origin and evolution. A model is presented of how evolution may have solved the problem in its early phase. Similar to several previous models, the model envisages the existence of a protocell, in which osmotic disbalance is being created by accumulation of synthetic products resulting in expansion and division of the protocell. Novel in the model is the presence in the protocell of a double-stranded nucleic acid, with each of its two strands being affixed by its 3'-terminus to the opposite sides of the membrane of a protocell. In the course of the protocell expansion, osmotic force is utilized to pull the two strands longitudinally in opposite directions, unzipping the helix and partitioning the strands between the two daughter protocells. The model is also being used as a background for arguments of why life need operate in cycles. Many formal models of life's origin and evolution have not taken into account the fact that logical possibility does not equal thermodynamic feasibility. A system of self-replication has to consist of both replicators and replicants.

  12. Intercalation-assisted longitudinal unzipping of carbon nanotubes for green and scalable synthesis of graphene nanoribbons.

    PubMed

    Li, Yan-Sheng; Liao, Jia-Liang; Wang, Shan-Yu; Chiang, Wei-Hung

    2016-03-07

    We have demonstrated an effective intercalation of multi-walled carbon nanotubes (MWCNTs) for the green and scalable synthesis of graphene nanoribbons (GNRs) using an intercalation-assisted longitudinal unzipping of MWCNTs. The key step is to introduce an intercalation treatment of raw MWCNTs with KNO3 and H2SO4, making it promising to decrease the strong van der Waals attractions in the MWCNTs bundles and between the coaxial graphene walls of CNTs. Systematic micro Raman, X-ray photoelectron spectroscopy (XPS), and X-ray diffraction (XRD) characterizations suggest that potassium, nitrate, and sulfate ions play an important role in the CNT intertube and intratube intercalations during the pretreatment. Detailed scanning electron microscopy (SEM), transmission electron microscopy, XRD, and micro Raman characterizations indicate that the developed methodology possesses the ability to synthesis GNRs effectively with an improved CNT concentration in H2SO4 of 10 mg/ml at 70 °C, which is amenable to industrial-scale production because of the decreased amount of strong acid. Our work provides a scientific understanding how to enhance the GNR formation by accelerating the CNT longitudinal unzipping via suitable molecular intercalation.

  13. Intercalation-assisted longitudinal unzipping of carbon nanotubes for green and scalable synthesis of graphene nanoribbons

    NASA Astrophysics Data System (ADS)

    Li, Yan-Sheng; Liao, Jia-Liang; Wang, Shan-Yu; Chiang, Wei-Hung

    2016-03-01

    We have demonstrated an effective intercalation of multi-walled carbon nanotubes (MWCNTs) for the green and scalable synthesis of graphene nanoribbons (GNRs) using an intercalation-assisted longitudinal unzipping of MWCNTs. The key step is to introduce an intercalation treatment of raw MWCNTs with KNO3 and H2SO4, making it promising to decrease the strong van der Waals attractions in the MWCNTs bundles and between the coaxial graphene walls of CNTs. Systematic micro Raman, X-ray photoelectron spectroscopy (XPS), and X-ray diffraction (XRD) characterizations suggest that potassium, nitrate, and sulfate ions play an important role in the CNT intertube and intratube intercalations during the pretreatment. Detailed scanning electron microscopy (SEM), transmission electron microscopy, XRD, and micro Raman characterizations indicate that the developed methodology possesses the ability to synthesis GNRs effectively with an improved CNT concentration in H2SO4 of 10 mg/ml at 70 °C, which is amenable to industrial-scale production because of the decreased amount of strong acid. Our work provides a scientific understanding how to enhance the GNR formation by accelerating the CNT longitudinal unzipping via suitable molecular intercalation.

  14. NiTi Alloy Negator Springs for Long-Stroke Constant-Force Shape Memory Actuators: Modeling, Simulation and Testing

    NASA Astrophysics Data System (ADS)

    Spaggiari, Andrea; Dragoni, Eugenio; Tuissi, Ausonio

    2014-07-01

    This work aims at the experimental characterization and modeling validation of shape memory alloy (SMA) Negator springs. According to the classic engineering books on springs, a Negator spring is a spiral spring made of strip of metal wound on the flat with an inherent curvature such that, in repose, each coil wraps tightly on its inner neighbor. The main feature of a Negator springs is the nearly constant force displacement behavior in the unwinding of the strip. Moreover the stroke is very long, theoretically infinite, as it depends only on the length of the initial strip. A Negator spring made in SMA is built and experimentally tested to demonstrate the feasibility of this actuator. The shape memory Negator spring behavior can be modeled with an analytical procedure, which is in good agreement with the experimental test and can be used for design purposes. In both cases, the material is modeled as elastic in austenitic range, while an exponential continuum law is used to describe the martensitic behavior. The experimental results confirms the applicability of this kind of geometry to the shape memory alloy actuators, and the analytical model is confirmed to be a powerful design tool to dimension and predict the spring behavior both in martensitic and austenitic range.

  15. Dynamical role of phosphorylation on serine/threonine-proline Pin1 substrates from constant force molecular dynamics simulations.

    PubMed

    Velazquez, Hector A; Hamelberg, Donald

    2015-02-21

    Cis-trans isomerization of peptidyl-prolyl bonds of the protein backbone plays an important role in numerous biological processes. Cis-trans isomerization can be the rate-limiting step due its extremely slow dynamics, compared to the millisecond time scale of many processes, and is catalyzed by a widely studied family of peptidyl-prolyl cis-trans isomerase enzymes. Also, mechanical forces along the peptide chain can speed up the rate of isomerization, resulting in "mechanical catalysis," and have been used to study peptidyl-prolyl cis-trans isomerization and other mechanical properties of proteins. Here, we use constant force molecular dynamics simulations to study the dynamical effects of phosphorylation on serine/threonine-proline protein motifs that are involved in the function of many proteins and have been implicated in many aberrant biological processes. We show that the rate of cis-trans isomerization is slowed down by phosphorylation, in excellent agreement with experiments. We use a well-grounded theory to describe the force dependent rate of isomerization. The calculated rates at zero force are also in excellent agreement with experimentally measured rates, providing additional validation of the models and force field parameters. Our results suggest that the slowdown in the rate upon phosphorylation is mainly due to an increase in the friction along the peptidyl-prolyl bond angle during isomerization. Our results provide a microscopic description of the dynamical effects of post-translational phosphorylation on cis-trans isomerization and insights into the properties of proteins under tension.

  16. Hydrogenation, purification, and unzipping of carbon nanotubes by reaction with molecular hydrogen: road to graphane nanoribbons.

    PubMed

    Talyzin, Alexandr V; Luzan, Serhiy; Anoshkin, Ilya V; Nasibulin, Albert G; Jiang, Hua; Kauppinen, Esko I; Mikoushkin, Valery M; Shnitov, Vladimir V; Marchenko, Dmitry E; Noréus, Dag

    2011-06-28

    Reaction of single-walled carbon nanotubes (SWNTs) with hydrogen gas was studied in a temperature interval of 400-550 °C and at hydrogen pressure of 50 bar. Hydrogenation of nanotubes was observed for samples treated at 400-450 °C with about 1/3 of carbon atoms forming covalent C-H bonds, whereas hydrogen treatment at higher temperatures (550 °C) occurs as an etching. Unzipping of some SWNTs into graphene nanoribbons is observed as a result of hydrogenation at 400-550 °C. Annealing in hydrogen gas at elevated conditions for prolonged periods of time (72 h) is demonstrated to result also in nanotube opening, purification of nanotubes from amorphous carbon, and removal of carbon coatings from Fe catalyst particles, which allows their complete elimination by acid treatment.

  17. Unzipped graphene nanoribbons as sensitive O2 sensors: Electron spin resonance probing and dissociation kinetics

    NASA Astrophysics Data System (ADS)

    Rao, S. S.; Stesmans, A.; Keunen, K.; Kosynkin, D. V.; Higginbotham, A.; Tour, J. M.

    2011-02-01

    Electron spin resonance study of unzipped graphene nanoribbons (GNRs) reveals a specific carbon-related signal, denoted GC, at g=2.0032, attributed to GNRs periphery centers. The signal is observed to be readily quenched upon O2 adsorption, which appears to be fully reversible upon room temperature vacuum treatment. Its depassivation behavior, observed from 130 K onward, is well described by first-order kinetics, characterized by the dissociation energy of 0.58±0.04 eV of spread 0.11±0.02 eV. The GC signal is not sensitive to other gases, such as H2, He, N2, and Ar, pointing to a GC-O2 physisorption interaction unique for GNRs, in agreement with theoretical insight. The GC center thus emerges as a highly selective, sensitive, and reversible O2 sensor.

  18. Electron doping effects on the electrical conductivity of zigzag carbon nanotubes and corresponding unzipped armchair graphene nanoribbons

    NASA Astrophysics Data System (ADS)

    Mousavi, Hamze; Jalilvand, Samira; Kurdestany, Jamshid Moradi; Grabowski, Marek

    2017-10-01

    The Kubo formula is used to extract the electrical conductivity (EC) of different diameters of doped zigzag carbon nanotubes and their corresponding unzipped armchair graphene nanoribbons, as a function of temperature and chemical potential, within the tight-binding Hamiltonian model and Green's functions approach. The results reveal more sensitivity to temperature for semiconducting systems in addition to a decrease in EC of all systems with increasing cross-sections.

  19. Mechanical unzipping and rezipping of a single SNARE complex reveals hysteresis as a force-generating mechanism

    PubMed Central

    Min, Duyoung; Kim, Kipom; Hyeon, Changbong; Hoon Cho, Yong; Shin, Yeon-Kyun; Yoon, Tae-Young

    2013-01-01

    Formation of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex provides mechanical thrust for membrane fusion, but its molecular mechanism is still unclear. Here using magnetic tweezers, we observe mechanical responses of a single neuronal SNARE complex under constant pulling force. Single SNARE complexes may be unzipped with 34 pN force. When rezipping is induced by lowering the force to 11 pN, only a partially assembled state results, with the C-terminal half of the SNARE complex remaining disassembled. Reassembly of the C-terminal half occurs only when the force is further lowered below 11 pN. Thus, mechanical hysteresis, characterized by the unzipping and rezipping cycle of a single SNARE complex, produces the partially assembled state. In this metastable state, unzipping toward the N-terminus is suppressed while zippering toward the C-terminus is initiated as a steep function of force. This ensures the directionality of SNARE-complex formation, making the SNARE complex a robust force-generating machine. PMID:23591872

  20. Sharpening the chemical scissors to unzip carbon nanotubes: crystalline graphene nanoribbons.

    PubMed

    Terrones, Mauricio

    2010-04-27

    It has recently been demonstrated that graphene nanoribbons can be mass-produced by unzipping carbon nanotubes. At present, wet chemical routes via acid oxidation appear to be the most effective and scalable. Although it was believed that this route resulted in highly defective nanoribbons with low electrical transport properties, a research group led by James Tour at Rice University has now realized that it is indeed possible to obtain highly crystalline graphene nanoribbons exhibiting high electrical conductivities, which could be used in the fabrication of field effect transistors and other devices. The results indicate that a defect-engineering approach could be used to control the straightness and length of the ribbons using oxidation reactions at relatively high temperatures (e.g., 60 degrees C). It has been shown that defects are critical in tailoring the physicochemical properties of graphene-like nanomaterials such as nanoribbons. However, this is the tip of the iceberg, and more edge chemistry and physics is still needed to develop and to produce real graphene nanoribbon devices for use in the market.

  1. Resonance mode in DNA dynamics

    NASA Astrophysics Data System (ADS)

    Zdravkovic, S.; Sataric, M. V.

    2007-11-01

    In this article we use Peyrard-Bishop-Dauxois model (PBD) to study the nonlinear oscillations of DNA nucleotides of extremely high amplitude (EHA) leading to unzipping of DNA chain in the context of the process of replication. We give arguments that the EHA mode is nothing but the resonance mode (RM). We launched an idea about how molecular mechano-chemical energy transduction can be the origin of the RM. We compared some parameters of the solitonic wave in DNA in resonant and non-resonant regime.

  2. Electrochemical unzipping of multi-walled carbon nanotubes for facile synthesis of high-quality graphene nanoribbons.

    PubMed

    Shinde, Dhanraj B; Debgupta, Joyashish; Kushwaha, Ajay; Aslam, Mohammed; Pillai, Vijayamohanan K

    2011-03-30

    Here we report a remarkable transformation of carbon nanotubes (CNTs) to nanoribbons composed of a few layers of graphene by a two-step electrochemical approach. This consists of the oxidation of CNTs at controlled potential, followed by reduction to form graphene nanoribbons (GNRs) having smooth edges and fewer defects, as evidenced by multiple characterization techniques, including Raman spectroscopy, atomic force microscopy, and transmission electron microscopy. This type of "unzipping" of CNTs (single-walled, multi-walled) in the presence of an interfacial electric field provides unique advantages with respect to the orientation of CNTs, which might make possible the production of GNRs with controlled widths and fewer defects.

  3. Unravelling DNA

    NASA Astrophysics Data System (ADS)

    Conroy, Rs; Danilowicz, C.

    2004-04-01

    The forces involved in the biology of life are carefully balanced between stopping thermal fluctuations ripping our DNA apart and having bonds weak enough to allow enzymes to function. The application of recently developed techniques for measuring piconewton forces and imaging at the nanometre scale on a molecule-by-molecule basis has dramatically increased the impact of single-molecule biophysics. This article describes the most commonly used techniques for imaging and manipulating single biomolecules. Using these techniques, the mechanical properties of DNA can be investigated, for example through measurements of the forces required to stretch and unzip the DNA double helix. These properties determine the ease with which DNA can be folded into the cell nucleus and the size and complexity of the accompanying cellular machinery. Part of this cellular machinery is enzymes, which manipulate, repair and transcribe the DNA helix. Enzymatic function is increasingly being investigated at the single molecule level to give better understanding of the forces and processes involved in the genetic cycle. One of the challenges is to transfer this understanding of single molecules into living systems. Already there have been some notable successes, such as the development of techniques for gene expression through the application of mechanical forces to cells, and the imaging and control of viral infection of a cell. This understanding and control of DNA has also been used to design molecules, which can self-assemble into a range of structures.

  4. Millisecond analysis of double stranded DNA with fluorescent intercalator by micro-thermocontrol-device.

    PubMed

    Arata, Hideyuki F; Gillot, Frederic; Collard, Dominique; Fujita, Hiroyuki

    2009-08-15

    Study of interaction between DNA and intercalator at molecular level is important to understand the mechanisms of DNA replication and repair. A micro-fabricated local heating thermodevice was adapted to perform denaturation experiments of DNA with fluorescent intercalator on millisecond time scale. Response time of complete unzipping of double stranded DNA, 16 microm in length, was measured to be around 5 min by commercial thermocycler. Response time of quenching of double stranded DNA with fluorescent intercalator SYBR Green was measured to be 10 ms. Thus, quenching properties owing to strand unzipping and denaturation at base pair level were distinguished. This method has provided easy access to measure this parameter and may be a powerful methodology in analyzing biomolecules on millisecond time scale.

  5. The Peyrard-Bishop-Dauxois Model of DNA Dynamics

    NASA Astrophysics Data System (ADS)

    Alexandrov, Boian; Bishop, Alan; Usheva, Anny; Rasmussen, Kim

    2008-03-01

    This presentation details aspects of the rapid development of the connection between the dynamics of double strand DNA, and experimental findings that has occurred in the recent years. We will approach this topic by demonstrating the Peyrard-Bishop-Dauxois model's ability to provide useful insight on several experimental observations. Specifically, we will discuss the melting behavior of various DNA sequences, and mechanical unzipping through dynamic force spectroscopy. Focusing on viral transcription initiation we will further show how the connection between DNA dynamics and DNA's biological functionality is becoming increasingly strong. Finally, we will describe a probable connection between DNA dynamics and the ability of repair proteins to recognize UV-radiation damages.

  6. Using DNA looping to measure sequence dependent DNA elasticity

    NASA Astrophysics Data System (ADS)

    Kandinov, Alan; Raghunathan, Krishnan; Meiners, Jens-Christian

    2012-10-01

    We are using tethered particle motion (TPM) microscopy to observe protein-mediated DNA looping in the lactose repressor system in DNA constructs with varying AT / CG content. We use these data to determine the persistence length of the DNA as a function of its sequence content and compare the data to direct micromechanical measurements with constant-force axial optical tweezers. The data from the TPM experiments show a much smaller sequence effect on the persistence length than the optical tweezers experiments.

  7. Oxidative Unzipping and Transformation of High Aspect Ratio Boron Nitride Nanotubes into “White Graphene Oxide” Platelets

    PubMed Central

    Nautiyal, Pranjal; Loganathan, Archana; Agrawal, Richa; Boesl, Benjamin; Wang, Chunlei; Agarwal, Arvind

    2016-01-01

    Morphological and chemical transformations in boron nitride nanotubes under high temperature atmospheric conditions is probed in this study. We report atmospheric oxygen induced cleavage of boron nitride nanotubes at temperatures exceeding 750 °C for the first time. Unzipping is then followed by coalescence of these densely clustered multiple uncurled ribbons to form stacks of 2D sheets. FTIR and EDS analysis suggest these 2D platelets to be Boron Nitride Oxide platelets, with analogous structure to Graphene Oxide, and therefore we term them as “White Graphene Oxide” (WGO). However, not all BNNTs deteriorate even at temperatures as high as 1000 °C. This leads to the formation of a hybrid nanomaterial system comprising of 1D BN nanotubes and 2D BN oxide platelets, potentially having advanced high temperature sensing, radiation shielding, mechanical strengthening, electron emission and thermal management applications due to synergistic improvement of multi-plane transport and mechanical properties. This is the first report on transformation of BNNT bundles to a continuous array of White Graphene Oxide nanoplatelet stacks. PMID:27388704

  8. Cooperative effect of stress and ion displacement on the dynamics of cross-link unzipping and rupture of alginate gels.

    PubMed

    Baumberger, T; Ronsin, O

    2010-06-14

    We study the effect of nonbinding Na(+) ions on the kinetics of rupture of alginate gels cross-linked by Ca(2+). Wetting a crack tip with a saline solution at physiological concentrations is found to be able to induce a quasi-instantaneous, 10-fold velocity jump. This effect is analyzed with a phenomenological model for the rate-dependent fracture energy in physical gels, extended here to account for the role of ions on the rate of cross-link "unzipping". Ionic interaction is found to act cooperatively with mechanical tension, leading to an enhanced rate of rupture. The kinetics turns out to be second order in counterion concentration. The definition of the reference state requires to take into account counterion condensation due to long-range interactions in the polyelectrolyte gel. Surprisingly, the contribution of the Na(+) ions to the free energy of the activated state is essentially entropic, suggesting that the displacement of Ca(2+) is primarily a steric process, electrostatic interactions being reduced to the constraint of charge conservation. This phenomenon may have important consequences on the rate of degradation of alginate based scaffolds for in vivo tissue regeneration.

  9. Oxidative Unzipping and Transformation of High Aspect Ratio Boron Nitride Nanotubes into “White Graphene Oxide” Platelets

    NASA Astrophysics Data System (ADS)

    Nautiyal, Pranjal; Loganathan, Archana; Agrawal, Richa; Boesl, Benjamin; Wang, Chunlei; Agarwal, Arvind

    2016-07-01

    Morphological and chemical transformations in boron nitride nanotubes under high temperature atmospheric conditions is probed in this study. We report atmospheric oxygen induced cleavage of boron nitride nanotubes at temperatures exceeding 750 °C for the first time. Unzipping is then followed by coalescence of these densely clustered multiple uncurled ribbons to form stacks of 2D sheets. FTIR and EDS analysis suggest these 2D platelets to be Boron Nitride Oxide platelets, with analogous structure to Graphene Oxide, and therefore we term them as “White Graphene Oxide” (WGO). However, not all BNNTs deteriorate even at temperatures as high as 1000 °C. This leads to the formation of a hybrid nanomaterial system comprising of 1D BN nanotubes and 2D BN oxide platelets, potentially having advanced high temperature sensing, radiation shielding, mechanical strengthening, electron emission and thermal management applications due to synergistic improvement of multi-plane transport and mechanical properties. This is the first report on transformation of BNNT bundles to a continuous array of White Graphene Oxide nanoplatelet stacks.

  10. Understanding the physics of DNA using nanoscale single-molecule manipulation

    PubMed Central

    Frey, Eric W.; Gooding, Ashton A.; Wijeratne, Sitara; Kiang, Ching-Hwa

    2013-01-01

    Processes for decoding the genetic information in cells, including transcription, replication, recombination and repair, involve the deformation of DNA from its equilibrium structures such as bending, stretching, twisting, and unzipping of the double helix. Single-molecule manipulation techniques have made it possible to control DNA conformation and simultaneously detect the induced changes, revealing a rich variety of mechanically-induced conformational changes and thermodynamic states. These single-molecule techniques helped us to reveal the physics of DNA and the processes involved in the passing on of the genetic code. PMID:23467419

  11. Understanding the physics of DNA using nanoscale single-molecule manipulation.

    PubMed

    Frey, Eric W; Gooding, Ashton A; Wijeratne, Sitara; Kiang, Ching-Hwa

    2012-10-01

    Processes for decoding the genetic information in cells, including transcription, replication, recombination and repair, involve the deformation of DNA from its equilibrium structures such as bending, stretching, twisting, and unzipping of the double helix. Single-molecule manipulation techniques have made it possible to control DNA conformation and simultaneously detect the induced changes, revealing a rich variety of mechanically-induced conformational changes and thermodynamic states. These single-molecule techniques helped us to reveal the physics of DNA and the processes involved in the passing on of the genetic code.

  12. Understanding the physics of DNA using nanoscale single-molecule manipulation

    NASA Astrophysics Data System (ADS)

    Frey, Eric W.; Gooding, Ashton A.; Wijeratne, Sitara; Kiang, Ching-Hwa

    2012-10-01

    Processes for decoding the genetic information in cells, including transcription, replication, recombination and repair, involve the deformation of DNA from its equilibrium structures such as bending, stretching, twisting, and unzipping of the double helix. Single-molecule manipulation techniques have made it possible to control DNA conformation and simultaneously detect the induced changes, revealing a rich variety of mechanically-induced conformational changes and thermodynamic states. These single-molecule techniques helped us to reveal the physics of DNA and the processes involved in the passing on of the genetic code.

  13. DNA.

    ERIC Educational Resources Information Center

    Felsenfeld, Gary

    1985-01-01

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

  14. DNA.

    ERIC Educational Resources Information Center

    Felsenfeld, Gary

    1985-01-01

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

  15. DNA

    ERIC Educational Resources Information Center

    Stent, Gunther S.

    1970-01-01

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

  16. DNA

    ERIC Educational Resources Information Center

    Stent, Gunther S.

    1970-01-01

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

  17. Single molecule high-throughput footprinting of small and large DNA ligands.

    PubMed

    Manosas, Maria; Camunas-Soler, Joan; Croquette, Vincent; Ritort, Felix

    2017-08-21

    Most DNA processes are governed by molecular interactions that take place in a sequence-specific manner. Determining the sequence selectivity of DNA ligands is still a challenge, particularly for small drugs where labeling or sequencing methods do not perform well. Here, we present a fast and accurate method based on parallelized single molecule magnetic tweezers to detect the sequence selectivity and characterize the thermodynamics and kinetics of binding in a single assay. Mechanical manipulation of DNA hairpins with an engineered sequence is used to detect ligand binding as blocking events during DNA unzipping, allowing determination of ligand selectivity both for small drugs and large proteins with nearly base-pair resolution in an unbiased fashion. The assay allows investigation of subtle details such as the effect of flanking sequences or binding cooperativity. Unzipping assays on hairpin substrates with an optimized flat free energy landscape containing all binding motifs allows determination of the ligand mechanical footprint, recognition site, and binding orientation.Mapping the sequence specificity of DNA ligands remains a challenge, particularly for small drugs. Here the authors develop a parallelized single molecule magnetic tweezers approach using engineered DNA hairpins that can detect sequence selectivity, thermodynamics and kinetics of binding for small drugs and large proteins.

  18. Nanomechanical DNA origami 'single-molecule beacons' directly imaged by atomic force microscopy

    PubMed Central

    Kuzuya, Akinori; Sakai, Yusuke; Yamazaki, Takahiro; Xu, Yan; Komiyama, Makoto

    2011-01-01

    DNA origami involves the folding of long single-stranded DNA into designed structures with the aid of short staple strands; such structures may enable the development of useful nanomechanical DNA devices. Here we develop versatile sensing systems for a variety of chemical and biological targets at molecular resolution. We have designed functional nanomechanical DNA origami devices that can be used as 'single-molecule beacons', and function as pinching devices. Using 'DNA origami pliers' and 'DNA origami forceps', which consist of two levers ~170 nm long connected at a fulcrum, various single-molecule inorganic and organic targets ranging from metal ions to proteins can be visually detected using atomic force microscopy by a shape transition of the origami devices. Any detection mechanism suitable for the target of interest, pinching, zipping or unzipping, can be chosen and used orthogonally with differently shaped origami devices in the same mixture using a single platform. PMID:21863016

  19. Threading of Binuclear Ruthenium Complex Through DNA Bases

    NASA Astrophysics Data System (ADS)

    Paramanathan, Thayaparan; Westerlund, Fredrik; McCauley, Micah; Lincoln, Per; Rouzina, Ioulia; Williams, Mark

    2009-03-01

    Due to steric constraints the dumb-bell shaped binuclear ruthenium complex can only intercalate DNA by threading, which requires local melting of the DNA to occur. By mechanically manipulating a single DNA molecule held with optical tweezers, we lower the barrier to threading compared to bulk experiments. Stretching single DNA molecules with different drug concentrations and holding a constant force allows the binding to reach equilibrium. We can obtain the equilibrium fractional ligand binding and length of DNA at saturation. Fitting these results yields quantitative measurements of the binding thermodynamics and kinetics. In addition, we obtain the minimum binding site size, which may be determined by either electrostatic repulsion or steric constraints.

  20. Single-molecule derivation of salt dependent base-pair free energies in DNA

    PubMed Central

    Huguet, Josep M.; Bizarro, Cristiano V.; Forns, Núria; Smith, Steven B.; Bustamante, Carlos; Ritort, Felix

    2010-01-01

    Accurate knowledge of the thermodynamic properties of nucleic acids is crucial to predicting their structure and stability. To date most measurements of base-pair free energies in DNA are obtained in thermal denaturation experiments, which depend on several assumptions. Here we report measurements of the DNA base-pair free energies based on a simplified system, the mechanical unzipping of single DNA molecules. By combining experimental data with a physical model and an optimization algorithm for analysis, we measure the 10 unique nearest-neighbor base-pair free energies with 0.1 kcal mol-1 precision over two orders of magnitude of monovalent salt concentration. We find an improved set of standard energy values compared with Unified Oligonucleotide energies and a unique set of 10 base-pair-specific salt-correction values. The latter are found to be strongest for AA/TT and weakest for CC/GG. Our unique energy values and salt corrections improve predictions of DNA unzipping forces and are fully compatible with melting temperatures for oligos. The method should make it possible to obtain free energies, enthalpies, and entropies in conditions not accessible by bulk methodologies. PMID:20716688

  1. Carbon nanotube proximity influences rice DNA

    NASA Astrophysics Data System (ADS)

    Katti, Dinesh R.; Sharma, Anurag; Pradhan, Shashindra Man; Katti, Kalpana S.

    2015-07-01

    The uptake of carbon nanotubes (CNT) influences the output of plants, potentially through interactions between the DNA and CNTs. However, little is known about the changes in the plant DNA due to CNT proximity. We report changes in rice plant DNA in the proximity of single walled CNT (SWCNT) using molecular dynamics simulations. The DNA experiences breaking and forming of hydrogen bonds due to unzipping of Watson-Crick (WC) nucleobase pairs and wrapping onto SWCNT. The number of hydrogen bonds between water and DNA nucleobases decreases due to the presence of SWCNT. A higher number of guanine-cytosine (Gua-Cyt) WC hydrogen bonds break as compared to adenine-thymine (Ade-Thy), which suggests that Gua and Cyt bases play a dominant role in DNA-SWCNT interactions. We also find that changes to non-WC nucleobase pairs and van der Waals attractive interactions between WC nucleobase pairs and SWCNT cause significant changes in the conformation of the DNA.

  2. A DNA bipedal nanowalker with a piston-like expulsion stroke.

    PubMed

    Yeo, Q Y; Loh, I Y; Tee, S R; Chiang, Y H; Cheng, J; Liu, M H; Wang, Z S

    2017-08-24

    Artificial molecular walkers beyond burn-bridge designs are important for nanotechnology, but their systematic development remains difficult. Herein, we have reported a new rationally designed DNA walker-track system and experimentally verified a previously proposed general expulsion regime for implementing non-burn-bridge nanowalkers. The DNA walker has an optically powered engine motif that reversibly extends and contracts the walker via a quadruplex-duplex conformational change. The walker's extension is an energy-absorbing and force-generating process, which drives the walker's leg dissociation off-track in a piston-like expulsion stroke. The unzipping-shearing asymmetry provides the expulsion stroke a bias, which decides the direction of the walker. Moreover, three candidate walkers of different sizes were fabricated. Fluorescence motility experiments indicated two of them as successful walkers and revealed a distinctive size dependence that was expected for these expulsive walkers, but was not observed in previously reported walkers. This study identifies unique technical requirements for expulsive nanowalkers. The present DNA design is readily adapted for making similar walkers from other molecules since the unzipping-shearing asymmetry is common.

  3. When a DNA triple helix melts: an analogue of the Efimov state

    NASA Astrophysics Data System (ADS)

    Maji, Jaya; Bhattacharjee, Somendra M.; Seno, Flavio; Trovato, Antonio

    2010-08-01

    The base sequences of DNA contain the genetic code, and, to decode it, a double helical DNA has to be unzipped to reveal the bases. Recent studies have shown that a third strand can be used to identify the base sequences, not by opening the double helix but rather by forming a triple helix. It is predicted here that a three-strand DNA exhibits the unusual behaviour of the existence of a three-chain bound state in the absence of any two being bound. Such a state can occur at or above the duplex melting point. This phenomenon is analogous to the Efimov state in three-particle quantum mechanics. A scaling theory is used to justify the Efimov connection. Real space renormalization group (RG) and exact numerical calculations are used to validate the prediction of a biological Efimov effect.

  4. Ab initio bubble-driven denaturation of double-stranded DNA: Self-mechanical theory.

    PubMed

    Kuetche, Victor K

    2016-07-21

    Among the different theoretical models of the open-site-driven DNA-denaturation found in the literature, very few interests are actually paid to the fundamental unzipping process of the double-stranded DNA within the vicinity of its ground state condensate. In this paper, we address an alternative to better understand the process of denaturation of such a macromolecule by investigating the onset of its dynamics around its equilibrium state. We show that from the initiation of the transcription bubble by the promoter to the termination state, the open-states of the strands evolve dynamically while generating some localized waveguide channels with elastic scattering properties. We properly discuss the nonlinear dynamics of these structures within the viewpoint of the self-mechanical theory while inferring to the physical structure of the findings and their potential issues. Copyright © 2016 Elsevier Ltd. All rights reserved.

  5. Modeling the Forced Extension of Nicked DNA

    NASA Astrophysics Data System (ADS)

    Balaeff, Alexander; Craig, Stephen; Beratan, David

    2007-03-01

    The design and study of DNA-based nanodevices has been a topic of considerable interest in the last decade. While the applications of classical continuous DNA structures have been thoroughly studied, nicked DNA structures, i.e., ones that contains breaks (``nicks'') in one or both DNA backbone chains, have received much less attention. Recently, Kersey et al. (JACS, 2004) reported the force spectroscopy of long DNA chains with periodic nicks, self-assembled from short DNA oligomers. We attempt to model the experimental force-extension profiles in a series of steered molecular dynamics simulations. The simulated all-atom model of a basic unit of the long self-assembled chain, a 16bp-long DNA segment with a nick in the middle of one strand, is extended by applying either a constant force or a moving harmonic potential to the DNA ends. The computed force-extension profiles are compared to those for a non-nicked DNA; the dynamics of structural changes in the nicked DNA during the forced extension is discussed. A theoretical framework is established to link the extension and rupture in the simulated basic unit to the corresponding events in the long self-assembled chain.

  6. Mean bubble formation time in DNA denaturation

    NASA Astrophysics Data System (ADS)

    Murthy, K. P. N.; Schütz, G. M.

    2011-12-01

    Using the Poland-Scheraga free energy of the bubble size in a double-stranded DNA we propose a discrete stochastic dynamics for the number of base pairs N of an unzipped bubble. We derive a universal subdiffusive growth TN~A/Γ(b+2)N1+b for the mean formation time (MBFT) TN of a bubble of size N. The amplitude A is determined by the bubble initiation rate and time spent in the denaturated state. We examine critically the significance of these results for experiments. We find: i) Our results provide a new method to determine whether the order of the denaturation transition is discontinuous (b>2) or not. ii) The asymptotic growth law of TN is reached with 10% precision already for small bubbles of sizes >20. However, the amplitude is very sensitive to modeling details for small bubbles. iii) In an equilibrium sample of bubbles up to size N the averaged MBFT grows diffusively, TN*~N2, irrespective of b.

  7. Spring operated accelerator and constant force spring mechanism therefor

    NASA Technical Reports Server (NTRS)

    Shillinger, G. L., Jr. (Inventor)

    1977-01-01

    A spring assembly consisting of an elongate piece of flat spring material formed into a spiral configuration and a free running spool in circumscribing relation to which this spring is disposed was developed. The spring has a distal end that is externally accessible so that when the distal end is drawn along a path, the spring unwinds against a restoring force present in the portion of the spring that resides in a transition region between a relatively straight condition on the path and a fully wound condition on the spool. When the distal end is released, the distal end is accelerated toward the spool by the force existing at the transition region which force is proportional to the cross-sectional area of the spring.

  8. Structural Transitions in Topologically Constrained DNA

    NASA Astrophysics Data System (ADS)

    Leger, J.; Romano, G.; Sarkar, A.; Robert, J.; Bourdieu, L.; Chatenay, D.; Marko, J. F.

    2000-03-01

    We propose a theoretical explanation for results of recent single molecule micromanipulation experiments (Leger et al, PRL 83, 1066, 1999) on double-stranded DNA with fixed linking number. The topological constraint leads to novel structural transitions, including a shift of the usual 60 pN B-form to S-form transition force plateau up to a force of 100 pN when linking is fixed at zero. Our model needs five distinct states to explain the four different observed transitions. The various constant-force plateaus observed for different fixed values of linking correspond to a mixture of different pairs of states, weighted to satisfy the topological constraint. Our model allows us to conclude that sufficiently overtwisted DNA (positive linkage number) undergoes a transition from B-form DNA to a mixture of S-form and P-form DNA at a force plateau near 45 pN, and then to homogeneous P-form DNA at a force plateau near 110 pN. A similar two-step transition occurs for undertwisted DNA, and by analysing the twisting necessary to produce pure S-form DNA we conclude that the S-state has helix repeat of 38 bp. Support from the Whitaker Foundation, the NSF, the ACS-PRF and Research Corporation is gratefully acknowledged.

  9. Dynamics of the Competition Between Nucleosome Unwrapping and DNA Binding Proteins

    NASA Astrophysics Data System (ADS)

    Bundschuh, Ralf

    2015-03-01

    In eukaryotic organisms DNA is tightly wrapped into nucleosomes. This bears the question how this DNA can be accessed in order to be copied, transcribed, or repaired. A process that allows access to the DNA is transient unwrapping of the DNA from the histone proteins. We have developed a quantitative model of this unwrapping process which we calibrate by comparison to nucleosome unzipping experiments by the Wang group. We then apply this model to quantitatively explain the dynamics of transcription factor binding within nucleosomal DNA. In this context, it has been well known that nucleosomes reduce the affinity for transcription factors to binding sites covered by the nucleosome. It has been assumed that this is due to a reduction in on-rate since a transcription factor can only bind when a rare thermal fluctuation of the nucleosome makes the DNA accessible. However, recent experimental data surprisingly shows that the off-rate of transcription factors is also strongly affected in the presence of a nucleosome. The application of our nucleosome unwrapping free energy landscape demonstrates that this increase in off-rate by several orders of magnitude is a consequence of a competition between partial binding events of dimeric transcription factors and the nucleosome. This material is based upon work supported by the National Science Foundation under Grant Nos. 1105458 and 1410172.

  10. Mechanobiology of Short DNA Molecules: A Single Molecule Perspective

    NASA Astrophysics Data System (ADS)

    Raghunathan, Krishnan

    Mechanical properties of DNA are known to play a significant role in several biological processes like wrapping of DNA around histones and looping. Most of these cellular events occur on a DNA length scale of a few hundred basepairs. Single molecule methods have been highly successful in directly investigating heterogeneity in different biomolecular systems and serve as ideal tools to study the mechanical properties of DNA. However, their use in studying DNA of contour lengths less than a kilobase are fraught with experimental difficulties. The research presented in this thesis explores the behavior of short stretches of DNA (≤ 500bp) using existing and novel single molecule methods. We have quantified the variation in persistence lengths between sequences having different elasticity using a constant force axial optical tweezers. Our experiments have also revealed that this difference in persistence lengths manifests itself as a difference in looping lifetimes of lac repressor, in sequences having the aforementioned constructs as the intervening sequence between the operator sites. We have also developed a system to probe DNA dynamics in vivo. We have found that the active processes in the cell have distinct effects on dynamics of DNA and eliminating the active processes causes a 'phase transition' like behavior in the inside the cell. We are currently extending this technique to understand DNA dynamics inside bacterial systems. Our results provide vital insights into mechanical properties of DNA and the effect of athermal fluctuations on DNA dynamics.

  11. Determination of Base Binding Strength and Base Stacking Interaction of DNA Duplex Using Atomic Force Microscope

    PubMed Central

    Zhang, Tian-biao; Zhang, Chang-lin; Dong, Zai-li; Guan, Yi-fu

    2015-01-01

    As one of the most crucial properties of DNA, the structural stability and the mechanical strength are attracting a great attention. Here, we take advantage of high force resolution and high special resolution of Atom Force Microscope and investigate the mechanical force of DNA duplexes. To evaluate the base pair hydrogen bond strength and base stacking force in DNA strands, we designed two modes (unzipping and stretching) for the measurement rupture forces. Employing k-means clustering algorithm, the ruptured force are clustered and the mean values are estimated. We assessed the influence of experimental parameters and performed the force evaluation for DNA duplexes of pure dG/dC and dA/dT base pairs. The base binding strength of single dG/dC and single dA/dT were estimated to be 20.0 ± 0.2 pN and 14.0 ± 0.3 pN, respectively, and the base stacking interaction was estimated to be 2.0 ± 0.1 pN. Our results provide valuable information about the quantitative evaluation of the mechanical properties of the DNA duplexes. PMID:25772017

  12. Determination of base binding strength and base stacking interaction of DNA duplex using atomic force microscope.

    PubMed

    Zhang, Tian-biao; Zhang, Chang-lin; Dong, Zai-li; Guan, Yi-fu

    2015-03-16

    As one of the most crucial properties of DNA, the structural stability and the mechanical strength are attracting a great attention. Here, we take advantage of high force resolution and high special resolution of Atom Force Microscope and investigate the mechanical force of DNA duplexes. To evaluate the base pair hydrogen bond strength and base stacking force in DNA strands, we designed two modes (unzipping and stretching) for the measurement rupture forces. Employing k-means clustering algorithm, the ruptured force are clustered and the mean values are estimated. We assessed the influence of experimental parameters and performed the force evaluation for DNA duplexes of pure dG/dC and dA/dT base pairs. The base binding strength of single dG/dC and single dA/dT were estimated to be 20.0 ± 0.2 pN and 14.0 ± 0.3 pN, respectively, and the base stacking interaction was estimated to be 2.0 ± 0.1 pN. Our results provide valuable information about the quantitative evaluation of the mechanical properties of the DNA duplexes.

  13. Interatomic Coulombic Decay Effects in Theoretical DNA Recombination Systems Involving Protein Interaction Sites

    NASA Astrophysics Data System (ADS)

    Vargas, E. L.; Rivas, D. A.; Duot, A. C.; Hovey, R. T.; Andrianarijaona, V. M.

    2015-03-01

    DNA replication is the basis for all biological reproduction. A strand of DNA will ``unzip'' and bind with a complimentary strand, creating two identical strands. In this study, we are considering how this process is affected by Interatomic Coulombic Decay (ICD), specifically how ICD affects the individual coding proteins' ability to hold together. ICD mainly deals with how the electron returns to its original state after excitation and how this affects its immediate atomic environment, sometimes affecting the connectivity between interaction sites on proteins involved in the DNA coding process. Biological heredity is fundamentally controlled by DNA and its replication therefore it affects every living thing. The small nature of the proteins (within the range of nanometers) makes it a good candidate for research of this scale. Understanding how ICD affects DNA molecules can give us invaluable insight into the human genetic code and the processes behind cell mutations that can lead to cancer. Authors wish to give special thanks to Pacific Union College Student Senate in Angwin, California, for their financial support.

  14. Single-molecule kinetics and footprinting of DNA bis-intercalation: the paradigmatic case of Thiocoraline

    PubMed Central

    Camunas-Soler, Joan; Manosas, Maria; Frutos, Silvia; Tulla-Puche, Judit; Albericio, Fernando; Ritort, Felix

    2015-01-01

    DNA bis-intercalators are widely used in molecular biology with applications ranging from DNA imaging to anticancer pharmacology. Two fundamental aspects of these ligands are the lifetime of the bis-intercalated complexes and their sequence selectivity. Here, we perform single-molecule optical tweezers experiments with the peptide Thiocoraline showing, for the first time, that bis-intercalation is driven by a very slow off-rate that steeply decreases with applied force. This feature reveals the existence of a long-lived (minutes) mono-intercalated intermediate that contributes to the extremely long lifetime of the complex (hours). We further exploit this particularly slow kinetics to determine the thermodynamics of binding and persistence length of bis-intercalated DNA for a given fraction of bound ligand, a measurement inaccessible in previous studies of faster intercalating agents. We also develop a novel single-molecule footprinting technique based on DNA unzipping and determine the preferred binding sites of Thiocoraline with one base-pair resolution. This fast and radiolabelling-free footprinting technique provides direct access to the binding sites of small ligands to nucleic acids without the need of cleavage agents. Overall, our results provide new insights into the binding pathway of bis-intercalators and the reported selectivity might be of relevance for this and other anticancer drugs interfering with DNA replication and transcription in carcinogenic cell lines. PMID:25690887

  15. Transfer-matrix calculations of DNA polymer micromechanics under tension and torque constraints

    NASA Astrophysics Data System (ADS)

    Efremov, Artem K.; Winardhi, Ricksen S.; Yan, Jie

    2016-09-01

    Recent development of single-molecule manipulation technologies has made it possible to exert constant force and torque on individual DNA biopolymers to probe their elastic characteristics and structural stability. It has been previously shown that depending on the nature of applied mechanical constraints, DNA can exist in several forms including B-, L-, and P-DNA. However, there is still a lack of understanding of how structural heterogeneity of DNA, which may naturally arise due to sequence-dependent DNA properties, protein binding, or DNA damage, influences local stability of the above DNA states. To provide a more complete and detailed description of the DNA mechanics, we developed a theoretical framework based on transfer-matrix calculations and demonstrated how it can be used to predict the DNA behavior upon application of a wide range of force and torque constraints. The resulting phase diagram shows DNA structural transitions that are in good agreement with previous experimental and theoretical studies. We further discuss how the constructed formalism can be extended to include local inhomogeneities in the DNA physical properties, thus making it possible to investigate the effect of DNA sequence as well as protein binding on DNA structural stability.

  16. Unzipping of the volcano arc, Japan

    USGS Publications Warehouse

    Stern, R.J.; Smoot, N.C.; Rubin, M.

    1984-01-01

    A working hypothesis for the recent evolution of the southern Volcano Arc, Japan, is presented which calls upon a northward-progressing sundering of the arc in response to a northward-propagating back-arc basin extensional regime. This model appears to explain several localized and recent changes in the tectonic and magrnatic evolution of the Volcano Arc. Most important among these changes is the unusual composition of Iwo Jima volcanic rocks. This contrasts with normal arc tholeiites typical of the rest of the Izu-Volcano-Mariana and other primitive arcs in having alkaline tendencies, high concentrations of light REE and other incompatible elements, and relatively high silica contents. In spite of such fractionated characteristics, these lavas appear to be very early manifestations of a new volcanic and tectonic cycle in the southern Volcano Arc. These alkaline characteristics and indications of strong regional uplift are consistent with the recent development of an early stage of inter-arc basin rifting in the southern Volcano Arc. New bathymetric data are presented in support of this model which indicate: 1. (1) structural elements of the Mariana Trough extend north to the southern Volcano Arc. 2. (2) both the Mariana Trough and frontal arc shoal rapidly northwards as the Volcano Arc is approached. 3. (3) rugged bathymetry associated with the rifted Mariana Trough is replaced just south of Iwo Jima by the development of a huge dome (50-75 km diameter) centered around Iwo Jima. Such uplifted domes are the immediate precursors of rifts in other environments, and it appears that a similar situation may now exist in the southern Volcano Arc. The present distribution of unrifted Volcano Arc to the north and rifted Mariana Arc to the south is interpreted not as a stable tectonic configuration but as representing a tectonic "snapshot" of an arc in the process of being rifted to form a back-arc basin. ?? 1984.

  17. Drug-DNA interactions at single molecule level: A view with optical tweezers

    NASA Astrophysics Data System (ADS)

    Paramanathan, Thayaparan

    Studies of small molecule--DNA interactions are essential for developing new drugs for challenging diseases like cancer and HIV. The main idea behind developing these molecules is to target and inhibit the reproduction of the tumor cells and infected cells. We mechanically manipulate single DNA molecule using optical tweezers to investigate two molecules that have complex and multiple binding modes. Mononuclear ruthenium complexes have been extensively studied as a test for rational drug design. Potential drug candidates should have high affinity to DNA and slow dissociation kinetics. To achieve this, motifs of the ruthenium complexes are altered. Our collaborators designed a dumb-bell shaped binuclear ruthenium complex that can only intercalate DNA by threading through its bases. Studying the binding properties of this complex in bulk studies took hours. By mechanically manipulating a single DNA molecule held with optical tweezers, we lower the barrier to thread and make it fast compared to the bulk experiments. Stretching single DNA molecules with different concentration of drug molecules and holding it at a constant force allows the binding to reach equilibrium. By this we can obtain the equilibrium fractional ligand binding and length of DNA at saturated binding. Fitting these results yields quantitative measurements of the binding thermodynamics and kinetics of this complex process. The second complex discussed in this study is Actinomycin D (ActD), a well studied anti-cancer agent that is used as a prototype for developing new generations of drugs. However, the biophysical basis of its activity is still unclear. Because ActD is known to intercalate double stranded DNA (dsDNA), it was assumed to block replication by stabilizing dsDNA in front of the replication fork. However, recent studies have shown that ActD binds with even higher affinity to imperfect duplexes and some sequences of single stranded DNA (ssDNA). We directly measure the on and off rates by

  18. Invincible DNA tethers: covalent DNA anchoring for enhanced temporal and force stability in magnetic tweezers experiments

    PubMed Central

    Janissen, Richard; Berghuis, Bojk A.; Dulin, David; Wink, Max; van Laar, Theo; Dekker, Nynke H.

    2014-01-01

    Magnetic tweezers are a powerful single-molecule technique that allows real-time quantitative investigation of biomolecular processes under applied force. High pulling forces exceeding tens of picoNewtons may be required, e.g. to probe the force range of proteins that actively transcribe or package the genome. Frequently, however, the application of such forces decreases the sample lifetime, hindering data acquisition. To provide experimentally viable sample lifetimes in the face of high pulling forces, we have designed a novel anchoring strategy for DNA in magnetic tweezers. Our approach, which exploits covalent functionalization based on heterobifunctional poly(ethylene glycol) crosslinkers, allows us to strongly tether DNA while simultaneously suppressing undesirable non-specific adhesion. A complete force and lifetime characterization of these covalently anchored DNA-tethers demonstrates that, compared to more commonly employed anchoring strategies, they withstand 3-fold higher pulling forces (up to 150 pN) and exhibit up to 200-fold higher lifetimes (exceeding 24 h at a constant force of 150 pN). This advance makes it possible to apply the full range of biologically relevant force scales to biomolecular processes, and its straightforward implementation should extend its reach to a multitude of applications in the field of single-molecule force spectroscopy. PMID:25140010

  19. Temperature-induced melting of double-stranded DNA in the absence and presence of covalently bonded antitumour drugs: insight from molecular dynamics simulations.

    PubMed

    Bueren-Calabuig, Juan A; Giraudon, Christophe; Galmarini, Carlos M; Egly, Jean Marc; Gago, Federico

    2011-10-01

    The difference in melting temperature of a double-stranded (ds) DNA molecule in the absence and presence of bound ligands can provide experimental information about the stabilization brought about by ligand binding. By simulating the dynamic behaviour of a duplex of sequence 5'-d(TAATAACGGATTATT)·5'-d(AATAATCCGTTATTA) in 0.1 M NaCl aqueous solution at 400 K, we have characterized in atomic detail its complete thermal denaturation profile in <200 ns. A striking asymmetry was observed on both sides of the central CGG triplet and the strand separation process was shown to be strongly affected by bonding in the minor groove of the prototypical interstrand crosslinker mitomycin C or the monofunctional tetrahydroisoquinolines trabectedin (Yondelis), Zalypsis and PM01183. Progressive helix unzipping was clearly interspersed with some reannealing events, which were most noticeable in the oligonucleotides containing the monoadducts, which maintained an average of 6 bp in the central region at the end of the simulations. These significant differences attest to the demonstrated ability of these drugs to stabilize dsDNA, stall replication and transcription forks, and recruit DNA repair proteins. This stabilization, quantified here in terms of undisrupted base pairs, supports the view that these monoadducts can functionally mimic a DNA interstrand crosslink.

  20. Temperature-induced melting of double-stranded DNA in the absence and presence of covalently bonded antitumour drugs: insight from molecular dynamics simulations

    PubMed Central

    Bueren-Calabuig, Juan A.; Giraudon, Christophe; Galmarini, Carlos M.; Egly, Jean Marc; Gago, Federico

    2011-01-01

    The difference in melting temperature of a double-stranded (ds) DNA molecule in the absence and presence of bound ligands can provide experimental information about the stabilization brought about by ligand binding. By simulating the dynamic behaviour of a duplex of sequence 5′-d(TAATAACGGATTATT)·5′-d(AATAATCCGTTATTA) in 0.1 M NaCl aqueous solution at 400 K, we have characterized in atomic detail its complete thermal denaturation profile in <200 ns. A striking asymmetry was observed on both sides of the central CGG triplet and the strand separation process was shown to be strongly affected by bonding in the minor groove of the prototypical interstrand crosslinker mitomycin C or the monofunctional tetrahydroisoquinolines trabectedin (Yondelis®), Zalypsis® and PM01183®. Progressive helix unzipping was clearly interspersed with some reannealing events, which were most noticeable in the oligonucleotides containing the monoadducts, which maintained an average of 6 bp in the central region at the end of the simulations. These significant differences attest to the demonstrated ability of these drugs to stabilize dsDNA, stall replication and transcription forks, and recruit DNA repair proteins. This stabilization, quantified here in terms of undisrupted base pairs, supports the view that these monoadducts can functionally mimic a DNA interstrand crosslink. PMID:21727089

  1. Opening of DNA chain due to force applied on different locations

    NASA Astrophysics Data System (ADS)

    Singh, Amar; Modi, Tushar; Singh, Navin

    2016-09-01

    We consider a homogeneous DNA molecule and investigate the effect of random force applied on the unzipping profile of the molecule. How the critical force varies as a function of the chain length or number of base pairs is the objective of this study. In general, the ratio of the critical forces that is applied on the middle of the chain to that which is applied on one of the ends is two. Our study shows that this ratio depends on the length of the chain. This means that the force which is applied to a point can be experienced by a section of the chain. Beyond a length, the base pairs have no information about the applied force. In the case when the chain length is shorter than this length, this ratio may vary. Only in the case when the chain length exceeds a critical length, this ratio is found to be two. Based on the de Gennes formulation, we developed a method to calculate these forces at zero temperature. The exact results at zero temperature match numerical calculations.

  2. Temperature and Electrolyte Optimization of the α-Hemolysin Latch Sensing Zone for Detection of Base Modification in Double-Stranded DNA

    PubMed Central

    Johnson, Robert P.; Fleming, Aaron M.; Jin, Qian; Burrows, Cynthia J.; White, Henry S.

    2014-01-01

    The latch region of the wild-type protein pore α-hemolysin (α-HL) constitutes a sensing zone for individual abasic sites (and furan analogs) in double-stranded DNA (dsDNA). The presence of an abasic site or furan within a DNA duplex, electrophoretically captured in the α-HL vestibule and positioned at the latch region, can be detected based on the current blockage prior to duplex unzipping. We investigated variations in blockage current as a function of temperature (12–35°C) and KCl concentration (0.15–1.0 M) to understand the origin of the current signature and to optimize conditions for identifying the base modification. In 1 M KCl solution, substitution of a furan for a cytosine base in the latch region results in an ∼8 kJ mol−1 decrease in the activation energy for ion transport through the protein pore. This corresponds to a readily measured ∼2 pA increase in current at room temperature. Optimal resolution for detecting the presence of a furan in the latch region is achieved at lower KCl concentrations, where the noise in the measured blockage current is significantly lower. The noise associated with the blockage current also depends on the stability of the duplex (as measured from the melting temperature), where a greater noise in the measured blockage current is observed for less stable duplexes. PMID:25140427

  3. Temperature and electrolyte optimization of the α-hemolysin latch sensing zone for detection of base modification in double-stranded DNA.

    PubMed

    Johnson, Robert P; Fleming, Aaron M; Jin, Qian; Burrows, Cynthia J; White, Henry S

    2014-08-19

    The latch region of the wild-type protein pore α-hemolysin (α-HL) constitutes a sensing zone for individual abasic sites (and furan analogs) in double-stranded DNA (dsDNA). The presence of an abasic site or furan within a DNA duplex, electrophoretically captured in the α-HL vestibule and positioned at the latch region, can be detected based on the current blockage prior to duplex unzipping. We investigated variations in blockage current as a function of temperature (12-35°C) and KCl concentration (0.15-1.0 M) to understand the origin of the current signature and to optimize conditions for identifying the base modification. In 1 M KCl solution, substitution of a furan for a cytosine base in the latch region results in an ∼ 8 kJ mol(-1) decrease in the activation energy for ion transport through the protein pore. This corresponds to a readily measured ∼ 2 pA increase in current at room temperature. Optimal resolution for detecting the presence of a furan in the latch region is achieved at lower KCl concentrations, where the noise in the measured blockage current is significantly lower. The noise associated with the blockage current also depends on the stability of the duplex (as measured from the melting temperature), where a greater noise in the measured blockage current is observed for less stable duplexes.

  4. DNA Nanotechnology

    NASA Astrophysics Data System (ADS)

    Taniguchi, Masateru; Kawai, Tomoji

    2002-11-01

    DNA is one candidate of promising molecules for molecular electronic devices, since it has the double helix structure with pi-electron bases for electron transport, the address at 0.4 nm intervals, and the self-assembly. Electrical conductivity and nanostructure of DNA and modified DNA molecules are investigated in order to research the application of DNA in nanoelectronic devices. It has been revealed that DNA is a wide-gap semiconductor in the absence of doping. The conductivity of DNA has been controlled by chemical doping, electric field doping, and photo-doping. It has found that Poly(dG)[middle dot]Poly(dC) has the best conductivity and can function as a conducting nanowire. The pattern of DNA network is controlled by changing the concentration of the DNA solution.

  5. Mitochondrial DNA.

    ERIC Educational Resources Information Center

    Wright, Russell G.; Bottino, Paul J.

    1986-01-01

    Provides background information for teachers on mitochondrial DNA, pointing out that it may have once been a free-living organism. Includes a ready-to-duplicate exercise titled "Using Microchondrial DNA to Measure Evolutionary Distance." (JN)

  6. Mitochondrial DNA.

    ERIC Educational Resources Information Center

    Wright, Russell G.; Bottino, Paul J.

    1986-01-01

    Provides background information for teachers on mitochondrial DNA, pointing out that it may have once been a free-living organism. Includes a ready-to-duplicate exercise titled "Using Microchondrial DNA to Measure Evolutionary Distance." (JN)

  7. Dna Sequencing

    DOEpatents

    Tabor, Stanley; Richardson, Charles C.

    1995-04-25

    A method for sequencing a strand of DNA, including the steps off: providing the strand of DNA; annealing the strand with a primer able to hybridize to the strand to give an annealed mixture; incubating the mixture with four deoxyribonucleoside triphosphates, a DNA polymerase, and at least three deoxyribonucleoside triphosphates in different amounts, under conditions in favoring primer extension to form nucleic acid fragments complementory to the DNA to be sequenced; labelling the nucleic and fragments; separating them and determining the position of the deoxyribonucleoside triphosphates by differences in the intensity of the labels, thereby to determine the DNA sequence.

  8. Quantifying the DNA binding characteristics of ruthenium based threading intercalator Λ Λ -P with optical tweezers

    NASA Astrophysics Data System (ADS)

    Bryden, Nicholas; McCauley, Micah; Westerlund, Fredrik; Lincoln, Per; Rouzina, Ioulia; Williams, Mark; Paramanathan, Thayaparan

    Utilizing optical tweezers, biophysics researchers have been able to study drug-DNA interactions on the single molecule level. Binuclear ruthenium complexes are a particular type of drug molecule that have been found to have potential cancer-fighting qualities, due to their high binding affinity and low dissociation rates. These complexes are threading intercalators, meaning that they must thread their bulky side chains through DNA base pairs to allow the central planar moiety to intercalate between the bases. In this study, we explored the binding properties of the binuclear ruthenium complex, ΛΛ -P (ΛΛ -[µ-bidppz(phen)4Ru2]4+) . A single DNA molecule is held at a constant force and the ΛΛ -P solution introduced to the system in varying concentrations until equilibrium is reached. DNA extension data at various concentrations of ΛΛ -P recorded as a function of time provide the DNA binding kinetics and equilibrium binding affinity. Preliminary data analysis suggests that ΛΛ -P exhibits fast binding kinetics compared to the very similar ΔΔ -P. These complexes have the same chemical structure and only differ in their chirality, which suggests that the left handed (ΛΛ) threading moieties require less DNA structural distortion for threading compared with the right handed (ΔΔ) threading moieties.

  9. DNA Flexibility

    NASA Astrophysics Data System (ADS)

    Widom, Jonathan

    2005-03-01

    Classic experimental and theoretical analyses led to a unified view of DNA as a semiflexible polymer, characterized by a bending persistence length, P, ˜50 nm (˜150 bp). In this view, DNA lengths that are greater than P are, on average, spontaneously gently bent, and require relatively little force to bend significantly, while DNA lengths that are shorter than P are nearly straight, and require great force to bend significantly. Nevertheless, sharply bent DNA plays important roles in biology. We used the method of ligase catalyzed DNA cyclization to investigate the spontaneous looping of short DNAs. Remarkably, DNAs as short as 84 bp spontaneously bend into circles, and 94 bp DNA sequences cyclize up to 10^5 times more easily than predicted from classic theories of DNA bending. In subsequent studies we find that the twistability of sharply looped DNAs exceeds the prediction of classic theories by up to 400-fold. These results can only be understood by greatly enhanced DNA flexibility, not by permanent bends. Our results provide striking support for two new theories of DNA mechanics based on local melted or kinked regions, and they establish DNA as an active participant in the formation and function of looped regulatory complexes in vivo.

  10. DNA Camouflage

    DTIC Science & Technology

    2016-01-08

    1 DNA Camouflage Supplementary Information Bijan Zakeri1,2*, Timothy K. Lu1,2*, Peter A. Carr2,3* 1Department of Electrical Engineering and...ll.mit.edu). Distribution A: Public Release   2 Supplementary Figure 1 DNA camouflage with the 2-state device. (a) In the presence of Cre, DSD-2[α...Supplementary Figure 2 DNA shuffling does not comprise sequencing outside of DSDs. (a) Sequencing of 1 kb downstream of DSD-2[α] produces high quality

  11. DNA Immunization

    PubMed Central

    Wang, Shixia; Lu, Shan

    2013-01-01

    DNA immunization was discovered in early 1990s and its use has been expanded from vaccine studies to a broader range of biomedical research, such as the generation of high quality polyclonal and monoclonal antibodies as research reagents. In this unit, three common DNA immunization methods are described: needle injection, electroporation and gene gun. In addition, several common considerations related to DNA immunization are discussed. PMID:24510291

  12. DNA ligases.

    PubMed

    Tabor, S

    2001-05-01

    DNA ligases catalyze the formation of phosphodiester bonds between juxtaposed 5' phosphate and a 3'-hydroxyl terminus in duplex DNA. This activity can repair single-stranded nicks in duplex DNA and join duplex DNA restriction fragments having either blunt ends or homologous cohesive ends. Two ligases are used for nucleic acid research and their reaction conditions and applications are described in this unit: E. coli ligase and T4 ligase. These enzymes differ in two important properties. One is the source of energy: T4 ligase uses ATP, while E. coli ligase uses NAD. Another important difference is their ability to ligate blunt ends; under normal reaction conditions, only T4 DNA ligase will ligate blunt ends.

  13. Patenting DNA.

    PubMed

    Bobrow, Martin; Thomas, Sandy

    2002-12-01

    The protection of inventions based on human DNA sequences has been achieved mainly through application of the patent system. Over the past decade, there has been continuing debate about whether this use of intellectual property rights is acceptable. Companies and universities have been active during this period in filing thousands of patent applications. Although many have argued that to claim a DNA sequence in a patent is to claim a discovery, patent law allows discoveries that are useful to be claimed as part of an invention. As the technology to isolate DNA sequences has advanced, the criterion for inventiveness, necessary for any invention to be eligible for filing, has become more difficult to justify in the case of claims to DNA sequences. Moreover, the discovery that a gene is associated with a particular disease is, it is argued, to discover a fact about the world and undeserving of the status of an invention. Careful examination of the grounds for allowing the patenting of DNA sequences as research tools suggests such rewards will rarely be justified. The patenting of DNA sequences as chemical intermediates necessary for the manufacture of therapeutic proteins is, however, reasonable given that the information within the sequence is applied to produce a tangible substance which has application as a medicine. Despite the legal, technical and political complexities of applying the flexibilities with the current law, it is argued that much could be achieved in the area of patenting DNA by raising the thresholds for patentability.

  14. [DNA computing].

    PubMed

    Błasiak, Janusz; Krasiński, Tadeusz; Popławski, Tomasz; Sakowski, Sebastian

    2011-01-01

    Biocomputers can be an alternative for traditional "silicon-based" computers, which continuous development may be limited due to further miniaturization (imposed by the Heisenberg Uncertainty Principle) and increasing the amount of information between the central processing unit and the main memory (von Neuman bottleneck). The idea of DNA computing came true for the first time in 1994, when Adleman solved the Hamiltonian Path Problem using short DNA oligomers and DNA ligase. In the early 2000s a series of biocomputer models was presented with a seminal work of Shapiro and his colleguas who presented molecular 2 state finite automaton, in which the restriction enzyme, FokI, constituted hardware and short DNA oligomers were software as well as input/output signals. DNA molecules provided also energy for this machine. DNA computing can be exploited in many applications, from study on the gene expression pattern to diagnosis and therapy of cancer. The idea of DNA computing is still in progress in research both in vitro and in vivo and at least promising results of these research allow to have a hope for a breakthrough in the computer science.

  15. DNA Labeling Using DNA Methyltransferases.

    PubMed

    Tomkuvienė, Miglė; Kriukienė, Edita; Klimašauskas, Saulius

    2016-01-01

    DNA methyltransferases (MTases) uniquely combine the ability to recognize and covalently modify specific target sequences in DNA using the ubiquitous cofactor S-adenosyl-L-methionine (AdoMet). Although DNA methylation plays important roles in biological signaling, the transferred methyl group is a poor reporter and is highly inert to further biocompatible derivatization. To unlock the biotechnological power of these enzymes, two major types of cofactor AdoMet analogs were developed that permit targeted MTase-directed attachment of larger moieties containing functional or reporter groups onto DNA. One such approach (named sequence-specific methyltransferase-induced labeling, SMILing) uses reactive aziridine or N-mustard mimics of the cofactor AdoMet, which render targeted coupling of a whole cofactor molecule to the target DNA. The second approach (methyltransferase-directed transfer of activated groups, mTAG) uses AdoMet analogs with a sulfonium-bound extended side chain replacing the methyl group, which permits MTase-directed covalent transfer of the activated side chain alone. As the enlarged cofactors are not always compatible with the active sites of native MTases, steric engineering of the active site has been employed to optimize their alkyltransferase activity. In addition to the described cofactor analogs, recently discovered atypical reactions of DNA cytosine-5 MTases involving non-cofactor-like compounds can also be exploited for targeted derivatization and labeling of DNA. Altogether, these approaches offer new powerful tools for sequence-specific covalent DNA labeling, which not only pave the way to developing a variety of useful techniques in DNA research, diagnostics, and nanotechnologies but have already proven practical utility for optical DNA mapping and epigenome studies.

  16. Bubbles and mismatches in DNA melting

    NASA Astrophysics Data System (ADS)

    Zeng, Yan

    We obtained the first experimental measurements of the length of the denaturation bubble appearing in the DNA melting transition. This is achieved by working with short oligomers which can form only one bubble per molecule. We used sequences clamped at the ends with GC pairs (strong binding) and possessing AT rich (weaker binding) middle regions in order to have the bubble open in the middle, and sequences with GC pairs at one end and AT pairs at the other end in order to form the bubble at the end. Use a quenching technique to trap the bubble states, we could measure the length of the bubble and the relative weights of the bubble states as a function of temperature. We found that the average bubble size <ℓ> grows for increasing temperature, but reaches a plateau at a length of order B (the length of the AT region). After the plateau, the average bubble length jumps to 1. This jump of the order parameter is a signature of a discontinuous transition, one where the bubble size remains finite up to critical temperature of strand separation. When B increases, the extension of the plateau shrinks. This suggests a continuous transition for a homogenous sequence (e.g. all AT base pairs) in the thermodynamic limit. The presence of the bubble states decreases as B is reduced. By plotting the average statistical weight of the bubble states vs. B, we obtained the first direct measurement of the nucleation size of the bubble. For a bubble flanked by double-stranded regions, the nucleation size is ˜ 3 bases. For bubbles opening at the ends of the molecule there is no nucleation threshold. The measured statistical weights of the bubble states agree with the predictions of the widely used thermodynamic models in the case of unzipping from the ends; however, internal bubble states are not completely described by the model. For the first time we show experimentally that a single mismatch transforms a transition with many intermediates into a nearly two-state transition for

  17. Dancing DNA.

    ERIC Educational Resources Information Center

    Pennisi, Elizabeth

    1991-01-01

    An imaging technique that uses fluorescent dyes and allows scientists to track DNA as it moves through gels or in solution is described. The importance, opportunities, and implications of this technique are discussed. (KR)

  18. DNA Dynamics.

    ERIC Educational Resources Information Center

    Warren, Michael D.

    1997-01-01

    Explains a method to enable students to understand DNA and protein synthesis using model-building and role-playing. Acquaints students with the triplet code and transcription. Includes copies of the charts used in this technique. (DDR)

  19. Dancing DNA.

    ERIC Educational Resources Information Center

    Pennisi, Elizabeth

    1991-01-01

    An imaging technique that uses fluorescent dyes and allows scientists to track DNA as it moves through gels or in solution is described. The importance, opportunities, and implications of this technique are discussed. (KR)

  20. DNA adductomics.

    PubMed

    Balbo, Silvia; Turesky, Robert J; Villalta, Peter W

    2014-03-17

    Systems toxicology is a broad-based approach to describe many of the toxicological features that occur within a living system under stress or subjected to exogenous or endogenous exposures. The ultimate goal is to capture an overview of all exposures and the ensuing biological responses of the body. The term exposome has been employed to refer to the totality of all exposures, and systems toxicology investigates how the exposome influences health effects and consequences of exposures over a lifetime. The tools to advance systems toxicology include high-throughput transcriptomics, proteomics, metabolomics, and adductomics, which is still in its infancy. A well-established methodology for the comprehensive measurement of DNA damage resulting from every day exposures is not fully developed. During the past several decades, the (32)P-postlabeling technique has been employed to screen the damage to DNA induced by multiple classes of genotoxicants; however, more robust, specific, and quantitative methods have been sought to identify and quantify DNA adducts. Although triple quadrupole and ion trap mass spectrometry, particularly when using multistage scanning (LC-MS(n)), have shown promise in the field of DNA adductomics, it is anticipated that high-resolution and accurate-mass LC-MS(n) instrumentation will play a major role in assessing global DNA damage. Targeted adductomics should also benefit greatly from improved triple quadrupole technology. Once the analytical MS methods are fully mature, DNA adductomics along with other -omics tools will contribute greatly to the field of systems toxicology.

  1. Root elongation against a constant force: experiment with a computerized feedback-controlled device

    NASA Technical Reports Server (NTRS)

    Kuzeja, P. S.; Lintilhac, P. M.; Wei, C.

    2001-01-01

    Axial force was applied to the root tip of corn (Zea mays L. cv. Merit) seedlings using a computerized, feedback-controlled mechanical device. The system's feedback capability allowed continuous control of a constant tip load, and the attached displacement transducer provided the time course of root elongation. Loads up to 7.5 g decreased the root elongation rate by 0.13 mm h-1 g-1, but loads 7.5 to 17.5 g decreased the growth rate by only 0.04 mm h-1 g-1. Loads higher than 18 g stopped root elongation completely. Measurement of the cross-sectional areas of the root tips indicated that the 18 g load had applied about 0.98 MPa of axial pressure to the root, thereby exceeding the root's ability to respond with increased turgor pressure. Recorded time-lapse images of loaded roots showed that radial thickening (swelling) occurred behind the root cap, whose cross-sectional area increased with tip load.

  2. Dynamics of equilibrium folding and unfolding transitions of titin immunoglobulin domain under constant forces.

    PubMed

    Chen, Hu; Yuan, Guohua; Winardhi, Ricksen S; Yao, Mingxi; Popa, Ionel; Fernandez, Julio M; Yan, Jie

    2015-03-18

    The mechanical stability of force-bearing proteins is crucial for their functions. However, slow transition rates of complex protein domains have made it challenging to investigate their equilibrium force-dependent structural transitions. Using ultra stable magnetic tweezers, we report the first equilibrium single-molecule force manipulation study of the classic titin I27 immunoglobulin domain. We found that individual I27 in a tandem repeat unfold/fold independently. We obtained the force-dependent free energy difference between unfolded and folded I27 and determined the critical force (∼5.4 pN) at which unfolding and folding have equal probability. We also determined the force-dependent free energy landscape of unfolding/folding transitions based on measurement of the free energy cost of unfolding. In addition to providing insights into the force-dependent structural transitions of titin I27, our results suggest that the conformations of titin immunoglobulin domains can be significantly altered during low force, long duration muscle stretching.

  3. What Is Mitochondrial DNA?

    MedlinePlus

    ... 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 ...

  4. DNA vaccines

    NASA Astrophysics Data System (ADS)

    Gregersen, Jens-Peter

    2001-12-01

    Immunization by genes encoding immunogens, rather than with the immunogen itself, has opened up new possibilities for vaccine research and development and offers chances for new applications and indications for future vaccines. The underlying mechanisms of antigen processing, immune presentation and regulation of immune responses raise high expectations for new and more effective prophylactic or therapeutic vaccines, particularly for vaccines against chronic or persistent infectious diseases and tumors. Our current knowledge and experience of DNA vaccination is summarized and critically reviewed with particular attention to basic immunological mechanisms, the construction of plasmids, screening for protective immunogens to be encoded by these plasmids, modes of application, pharmacokinetics, safety and immunotoxicological aspects. DNA vaccines have the potential to accelerate the research phase of new vaccines and to improve the chances of success, since finding new immunogens with the desired properties is at least technically less demanding than for conventional vaccines. However, on the way to innovative vaccine products, several hurdles have to be overcome. The efficacy of DNA vaccines in humans appears to be much less than indicated by early studies in mice. Open questions remain concerning the persistence and distribution of inoculated plasmid DNA in vivo, its potential to express antigens inappropriately, or the potentially deleterious ability to insert genes into the host cell's genome. Furthermore, the possibility of inducing immunotolerance or autoimmune diseases also needs to be investigated more thoroughly, in order to arrive at a well-founded consensus, which justifies the widespread application of DNA vaccines in a healthy population.

  5. Ancient DNA

    PubMed Central

    Willerslev, Eske; Cooper, Alan

    2004-01-01

    In the past two decades, ancient DNA research has progressed from the retrieval of small fragments of mitochondrial DNA from a few late Holocene specimens, to large-scale studies of ancient populations, phenotypically important nuclear loci, and even whole mitochondrial genome sequences of extinct species. However, the field is still regularly marred by erroneous reports, which underestimate the extent of contamination within laboratories and samples themselves. An improved understanding of these processes and the effects of damage on ancient DNA templates has started to provide a more robust basis for research. Recent methodological advances have included the characterization of Pleistocene mammal populations and discoveries of DNA preserved in ancient sediments. Increasingly, ancient genetic information is providing a unique means to test assumptions used in evolutionary and population genetics studies to reconstruct the past. Initial results have revealed surprisingly complex population histories, and indicate that modern phylogeographic studies may give misleading impressions about even the recent evolutionary past. With the advent and uptake of appropriate methodologies, ancient DNA is now positioned to become a powerful tool in biological research and is also evolving new and unexpected uses, such as in the search for extinct or extant life in the deep biosphere and on other planets. PMID:15875564

  6. DNA codes

    SciTech Connect

    Torney, D. C.

    2001-01-01

    We have begun to characterize a variety of codes, motivated by potential implementation as (quaternary) DNA n-sequences, with letters denoted A, C The first codes we studied are the most reminiscent of conventional group codes. For these codes, Hamming similarity was generalized so that the score for matched letters takes more than one value, depending upon which letters are matched [2]. These codes consist of n-sequences satisfying an upper bound on the similarities, summed over the letter positions, of distinct codewords. We chose similarity 2 for matches of letters A and T and 3 for matches of the letters C and G, providing a rough approximation to double-strand bond energies in DNA. An inherent novelty of DNA codes is 'reverse complementation'. The latter may be defined, as follows, not only for alphabets of size four, but, more generally, for any even-size alphabet. All that is required is a matching of the letters of the alphabet: a partition into pairs. Then, the reverse complement of a codeword is obtained by reversing the order of its letters and replacing each letter by its match. For DNA, the matching is AT/CG because these are the Watson-Crick bonding pairs. Reversal arises because two DNA sequences form a double strand with opposite relative orientations. Thus, as will be described in detail, because in vitro decoding involves the formation of double-stranded DNA from two codewords, it is reasonable to assume - for universal applicability - that the reverse complement of any codeword is also a codeword. In particular, self-reverse complementary codewords are expressly forbidden in reverse-complement codes. Thus, an appropriate distance between all pairs of codewords must, when large, effectively prohibit binding between the respective codewords: to form a double strand. Only reverse-complement pairs of codewords should be able to bind. For most applications, a DNA code is to be bi-partitioned, such that the reverse-complementary pairs are separated

  7. DNA origami nanopores for controlling DNA translocation.

    PubMed

    Hernández-Ainsa, Silvia; Bell, Nicholas A W; Thacker, Vivek V; Göpfrich, Kerstin; Misiunas, Karolis; Fuentes-Perez, Maria Eugenia; Moreno-Herrero, Fernando; Keyser, Ulrich F

    2013-07-23

    We combine DNA origami structures with glass nanocapillaries to reversibly form hybrid DNA origami nanopores. Trapping of the DNA origami onto the nanocapillary is proven by imaging fluorescently labeled DNA origami structures and simultaneous ionic current measurements of the trapping events. We then show two applications highlighting the versatility of these DNA origami nanopores. First, by tuning the pore size we can control the folding of dsDNA molecules ("physical control"). Second, we show that the specific introduction of binding sites in the DNA origami nanopore allows selective detection of ssDNA as a function of the DNA sequence ("chemical control").

  8. DNA Investigations.

    ERIC Educational Resources Information Center

    Mayo, Ellen S.; Bertino, Anthony J.

    1991-01-01

    Presents a simulation activity that allow students to work through the exercise of DNA profiling and to grapple with some analytical and ethical questions involving a couple arranging with a surrogate mother to have a baby. Can be used to teach the principles of restriction enzyme digestion, gel electrophoresis, and probe hybridization. (MDH)

  9. DNA Music.

    ERIC Educational Resources Information Center

    Miner, Carol; della Villa, Paula

    1997-01-01

    Describes an activity in which students reverse-translate proteins from their amino acid sequences back to their DNA sequences then assign musical notes to represent the adenine, guanine, cytosine, and thymine bases. Data is obtained from the National Institutes of Health (NIH) on the Internet. (DDR)

  10. DNA Investigations.

    ERIC Educational Resources Information Center

    Mayo, Ellen S.; Bertino, Anthony J.

    1991-01-01

    Presents a simulation activity that allow students to work through the exercise of DNA profiling and to grapple with some analytical and ethical questions involving a couple arranging with a surrogate mother to have a baby. Can be used to teach the principles of restriction enzyme digestion, gel electrophoresis, and probe hybridization. (MDH)

  11. DNA Music.

    ERIC Educational Resources Information Center

    Miner, Carol; della Villa, Paula

    1997-01-01

    Describes an activity in which students reverse-translate proteins from their amino acid sequences back to their DNA sequences then assign musical notes to represent the adenine, guanine, cytosine, and thymine bases. Data is obtained from the National Institutes of Health (NIH) on the Internet. (DDR)

  12. DNA Methylation

    PubMed Central

    Marinus, M.G.; Løbner-Olesen, A.

    2014-01-01

    The DNA of E. coli contains 19,120 6-methyladenines and 12,045 5-methylcytosines in addition to the four regular bases and these are formed by the postreplicative action of three DNA methyltransferases. The majority of the methylated bases are formed by the Dam and Dcm methyltransferases encoded by the dam (DNA adenine methyltransferase) and dcm (DNA cytosine methyltransferase) genes. Although not essential, Dam methylation is important for strand discrimination during repair of replication errors, controlling the frequency of initiation of chromosome replication at oriC, and regulation of transcription initiation at promoters containing GATC sequences. In contrast, there is no known function for Dcm methylation although Dcm recognition sites constitute sequence motifs for Very Short Patch repair of T/G base mismatches. In certain bacteria (e.g., Vibrio cholerae, Caulobacter crescentus) adenine methylation is essential and in C. crescentus, it is important for temporal gene expression which, in turn, is required for coordinating chromosome initiation, replication and division. In practical terms, Dam and Dcm methylation can inhibit restriction enzyme cleavage; decrease transformation frequency in certain bacteria; decrease the stability of short direct repeats; are necessary for site-directed mutagenesis; and to probe eukaryotic structure and function. PMID:26442938

  13. Prokaryotic DNA ligases unwind superhelical DNA.

    PubMed

    Ivanchenko, M; van Holde, K; Zlatanova, J

    1996-09-13

    We have studied the effect on DNA topology of binding of prokaryotic DNA ligases (T4 and E. coli) to superhelical or nicked circular DNA. Performing topoisomerase I-mediated relaxation in the presence of increasing amounts of T4 ligase led to a shift in the topoisomer distribution to increasingly more negative values. This result suggested that T4 ligase unwound the DNA and was further substantiated by ligation of nicked circular molecules by E. coli DNA ligase in the presence of increasing amounts of T4 ligase. Such an experiment was possible since the two DNA ligases require different cofactors for enzymatic activity. Performing a similar experiment with reverse partners, using E. coli DNA ligase as ligand, and T4 ligase as sealing agent, we observed that the E. coli enzyme also unwound the DNA. Thus, prokaryotic DNA ligases can be added to an ever-growing list of DNA-binding proteins that unwind the DNA upon binding.

  14. Laser induced unzipping: A thermal route to polymer ablation

    NASA Astrophysics Data System (ADS)

    Blanchet, Graciela B.; Fincher, C. R., Jr.

    1994-09-01

    The data presented here show that polytetrafluoroethylene (PTFE) and polymethylmethacrylate (PMMA) can be ablated by the evaporation of solid polymer targets with a pulsed ultraviolet laser. In situ measurements of the plume composition show that the products emitted under laser irradiation are primarily monomer and other species resulting from energetic collisions within the plasma. The similarities between the ablative and pyrolisis mass spectra suggest that ablation of PTFE and PMMA occur through a laser induced pyrolitic decomposition.

  15. Optical DNA

    NASA Astrophysics Data System (ADS)

    Vijaywargi, Deepak; Lewis, Dave; Kirovski, Darko

    A certificate of authenticity (COA) is an inexpensive physical object with a random and unique structure S which is hard to near-exactly replicate. An inexpensive device should be able to scan object’s physical “fingerprint,” a set of features that represents S. In this paper, we explore one set of requirements that optical media such as DVDs should satisfy, to be considered as COAs. As manufacturing of such media produces inevitable errors, we use the locations and count of these errors as a “fingerprint” for each optical disc: its optical DNA. The “fingerprint” is signed using publisher’s private-key and the resulting signature is stored onto the optical medium using a post-production process. Standard DVD players with altered firmware that includes publisher’s public-key, should be able to verify the authenticity of DVDs protected with optical DNA. Our key finding is that for the proposed protocol, only DVDs with exceptional wear-and-tear characteristics would result in an inexpensive and viable anti-counterfeiting technology.

  16. DNA nanostructure meets nanofabrication.

    PubMed

    Zhang, Guomei; Surwade, Sumedh P; Zhou, Feng; Liu, Haitao

    2013-04-07

    Recent advances in DNA nanotechnology have made it possible to construct DNA nanostructures of almost arbitrary shapes with 2-3 nm of precision in their dimensions. These DNA nanostructures are ideal templates for bottom-up nanofabrication. This review highlights the challenges and recent advances in three areas that are directly related to DNA-based nanofabrication: (1) fabrication of large scale DNA nanostructures; (2) pattern transfer from DNA nanostructure to an inorganic substrate; and (3) directed assembly of DNA nanostructures.

  17. DNA ligase I, the replicative DNA ligase.

    PubMed

    Howes, Timothy R L; Tomkinson, Alan E

    2012-01-01

    Multiple DNA ligation events are required to join the Okazaki fragments generated during lagging strand DNA synthesis. In eukaryotes, this is primarily carried out by members of the DNA ligase I family. The C-terminal catalytic region of these enzymes is composed of three domains: a DNA binding domain, an adenylation domain and an OB-fold domain. In the absence of DNA, these domains adopt an extended structure but transition into a compact ring structure when they engage a DNA nick, with each of the domains contacting the DNA. The non-catalytic N-terminal region of eukaryotic DNA ligase I is responsible for the specific participation of these enzymes in DNA replication. This proline-rich unstructured region contains the nuclear localization signal and a PCNA interaction motif that is critical for localization to replication foci and efficient joining of Okazaki fragments. DNA ligase I initially engages the PCNA trimer via this interaction motif which is located at the extreme N-terminus of this flexible region. It is likely that this facilitates an additional interaction between the DNA binding domain and the PCNA ring. The similar size and shape of the rings formed by the PCNA trimer and the DNA ligase I catalytic region when it engages a DNA nick suggest that these proteins interact to form a double-ring structure during the joining of Okazaki fragments. DNA ligase I also interacts with replication factor C, the factor that loads the PCNA trimeric ring onto DNA. This interaction, which is regulated by phosphorylation of the non-catalytic N-terminus of DNA ligase I, also appears to be critical for DNA replication.

  18. DNA modifications: Another stable base in DNA

    NASA Astrophysics Data System (ADS)

    Brazauskas, Pijus; Kriaucionis, Skirmantas

    2014-12-01

    Oxidation of 5-methylcytosine has been proposed to mediate active and passive DNA demethylation. Tracking the history of DNA modifications has now provided the first solid evidence that 5-hydroxymethylcytosine is a stable epigenetic modification.

  19. Sperm DNA oxidative damage and DNA adducts

    PubMed Central

    Jeng, Hueiwang Anna; Pan, Chih-Hong; Chao, Mu-Rong; Lin, Wen-Yi

    2015-01-01

    The objective of this study was to investigate DNA damage and adducts in sperm from coke oven workers who have been exposed to polycyclic aromatic hydrocarbons. A longitudinal study was conducted with repeated measurements during spermatogenesis. Coke-oven workers (n=112) from a coke-oven plant served the PAH-exposed group, while administrators and security personnel (n=67) served the control. Routine semen parameters (concentration, motility, vitality, and morphology) were analyzed simultaneously; the assessment of sperm DNA integrity endpoints included DNA fragmentation, bulky DNA adducts, and 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxo-dGuo). The degree of sperm DNA fragmentation was measured using the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay and sperm chromatin structure assay (SCSA). The PAH-exposed group had a significant increase in bulky DNA adducts and 8-oxo-dGuo compared to the control subjects (Ps = 0.002 and 0.045, respectively). Coke oven workers' percentages of DNA fragmentation and denaturation from the PAH-exposed group were not significantly different from those of the control subjects (Ps = 0.232 and 0.245, respectively). Routine semen parameters and DNA integrity endpoints were not correlated. Concentrations of 8-oxo-dGuo were positively correlated with percentages of DNA fragmentation measured by both TUNEL and SCSA (Ps = 0.045 and 0.034, respectively). However, the concentrations of 8-oxo-dGuo and percentages of DNA fragmentation did not correlate with concentrations of bulky DNA adducts. In summary, coke oven workers with chronic exposure to PAHs experienced decreased sperm DNA integrity. Oxidative stress could contribute to the degree of DNA fragmentation. Bulky DNA adducts may be independent of the formation of DNA fragmentation and oxidative adducts in sperm. Monitoring sperm DNA integrity is recommended as a part of the process of assessing the impact of occupational and environmental toxins on

  20. Synthesis of DNA

    DOEpatents

    Mariella, Jr., Raymond P.

    2008-11-18

    A method of synthesizing a desired double-stranded DNA of a predetermined length and of a predetermined sequence. Preselected sequence segments that will complete the desired double-stranded DNA are determined. Preselected segment sequences of DNA that will be used to complete the desired double-stranded DNA are provided. The preselected segment sequences of DNA are assembled to produce the desired double-stranded DNA.

  1. DNA encoding a DNA repair protein

    DOEpatents

    Petrini, John H.; Morgan, William Francis; Maser, Richard Scott; Carney, James Patrick

    2006-08-15

    An isolated and purified DNA molecule encoding a DNA repair protein, p95, is provided, as is isolated and purified p95. Also provided are methods of detecting p95 and DNA encoding p95. The invention further provides p95 knock-out mice.

  2. DNA systematics. Volume II

    SciTech Connect

    Dutta, S.K.

    1986-01-01

    This book discusses the following topics: PLANTS: PLANT DNA: Contents and Systematics. Repeated DNA Sequences and Polyploidy in Cereal Crops. Homology of Nonrepeated DNA Sequences in Phylogeny of Fungal Species. Chloropast DNA and Phylogenetic Relationships. rDNA: Evolution Over a Billion Years. 23S rRNA-derived Small Ribosomal RNAs: Their Structure and Evolution with Reference to Plant Phylogeny. Molecular Analysis of Plant DNA Genomes: Conserved and Diverged DNA Sequences. A Critical Review of Some Terminologies Used for Additional DNA in Plant Chromosomes and Index.

  3. Molecular DNA switches and DNA chips

    NASA Astrophysics Data System (ADS)

    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.

  4. DNA vaccines: a simple DNA sensing matter?

    PubMed

    Coban, Cevayir; Kobiyama, Kouji; Jounai, Nao; Tozuka, Miyuki; Ishii, Ken J

    2013-10-01

    Since the introduction of DNA vaccines two decades ago, this attractive strategy has been hampered by its low immunogenicity in humans. Studies conducted to improve the immunogenicity of DNA vaccines have shown that understanding the mechanism of action of DNA vaccines might be the key to successfully improving their immunogenicity. Our current understanding is that DNA vaccines induce innate and adaptive immune responses in two ways: (1) encoded protein (or polypeptide) antigen(s) by the DNA plasmid can be expressed in stromal cells (i.e., muscle cells) as well as DCs, where these antigens are processed and presented to naïve CD4 or CD8 T cells either by direct or cross presentation, respectively; and (2) the transfected DNA plasmid itself may bind to an un-identified cytosolic DNA sensor and activate the TBK1-STING pathway and the production of type I interferons (IFNs) which function as an adjuvant. Recent studies investigating double-stranded cytosolic DNA sensor(s) have highlighted new mechanisms in which cytosolic DNA may release secondary metabolites, which are in turn recognized by a novel DNA sensing machinery. Here, we discuss these new metabolites and the possibilities of translating this knowledge into improved immunogenicity for DNA vaccines.

  5. DNA Repair by Reversal of DNA Damage

    PubMed Central

    Yi, Chengqi; He, Chuan

    2013-01-01

    Endogenous and exogenous factors constantly challenge cellular DNA, generating cytotoxic and/or mutagenic DNA adducts. As a result, organisms have evolved different mechanisms to defend against the deleterious effects of DNA damage. Among these diverse repair pathways, direct DNA-repair systems provide cells with simple yet efficient solutions to reverse covalent DNA adducts. In this review, we focus on recent advances in the field of direct DNA repair, namely, photolyase-, alkyltransferase-, and dioxygenase-mediated repair processes. We present specific examples to describe new findings of known enzymes and appealing discoveries of new proteins. At the end of this article, we also briefly discuss the influence of direct DNA repair on other fields of biology and its implication on the discovery of new biology. PMID:23284047

  6. Quantitative DNA fiber mapping

    DOEpatents

    Gray, Joe W.; Weier, Heinz-Ulrich G.

    1998-01-01

    The present invention relates generally to the DNA mapping and sequencing technologies. In particular, the present invention provides enhanced methods and compositions for the physical mapping and positional cloning of genomic DNA. The present invention also provides a useful analytical technique to directly map cloned DNA sequences onto individual stretched DNA molecules.

  7. Poxvirus DNA Replication

    PubMed Central

    Moss, Bernard

    2013-01-01

    Poxviruses are large, enveloped viruses that replicate in the cytoplasm and encode proteins for DNA replication and gene expression. Hairpin ends link the two strands of the linear, double-stranded DNA genome. Viral proteins involved in DNA synthesis include a 117-kDa polymerase, a helicase–primase, a uracil DNA glycosylase, a processivity factor, a single-stranded DNA-binding protein, a protein kinase, and a DNA ligase. A viral FEN1 family protein participates in double-strand break repair. The DNA is replicated as long concatemers that are resolved by a viral Holliday junction endonuclease. PMID:23838441

  8. DNA Damage, DNA Repair, Aging, and Neurodegeneration.

    PubMed

    Maynard, Scott; Fang, Evandro Fei; Scheibye-Knudsen, Morten; Croteau, Deborah L; Bohr, Vilhelm A

    2015-09-18

    Aging in mammals is accompanied by a progressive atrophy of tissues and organs, and stochastic damage accumulation to the macromolecules DNA, RNA, proteins, and lipids. The sequence of the human genome represents our genetic blueprint, and accumulating evidence suggests that loss of genomic maintenance may causally contribute to aging. Distinct evidence for a role of imperfect DNA repair in aging is that several premature aging syndromes have underlying genetic DNA repair defects. Accumulation of DNA damage may be particularly prevalent in the central nervous system owing to the low DNA repair capacity in postmitotic brain tissue. It is generally believed that the cumulative effects of the deleterious changes that occur in aging, mostly after the reproductive phase, contribute to species-specific rates of aging. In addition to nuclear DNA damage contributions to aging, there is also abundant evidence for a causative link between mitochondrial DNA damage and the major phenotypes associated with aging. Understanding the mechanistic basis for the association of DNA damage and DNA repair with aging and age-related diseases, such as neurodegeneration, would give insight into contravening age-related diseases and promoting a healthy life span.

  9. DNA Damage, DNA Repair, Aging, and Neurodegeneration

    PubMed Central

    Maynard, Scott; Fang, Evandro Fei; Scheibye-Knudsen, Morten; Croteau, Deborah L.; Bohr, Vilhelm A.

    2015-01-01

    Aging in mammals is accompanied by a progressive atrophy of tissues and organs, and stochastic damage accumulation to the macromolecules DNA, RNA, proteins, and lipids. The sequence of the human genome represents our genetic blueprint, and accumulating evidence suggests that loss of genomic maintenance may causally contribute to aging. Distinct evidence for a role of imperfect DNA repair in aging is that several premature aging syndromes have underlying genetic DNA repair defects. Accumulation of DNA damage may be particularly prevalent in the central nervous system owing to the low DNA repair capacity in postmitotic brain tissue. It is generally believed that the cumulative effects of the deleterious changes that occur in aging, mostly after the reproductive phase, contribute to species-specific rates of aging. In addition to nuclear DNA damage contributions to aging, there is also abundant evidence for a causative link between mitochondrial DNA damage and the major phenotypes associated with aging. Understanding the mechanistic basis for the association of DNA damage and DNA repair with aging and age-related diseases, such as neurodegeneration, would give insight into contravening age-related diseases and promoting a healthy life span. PMID:26385091

  10. DNA degradation with ozone.

    PubMed

    Cataldo, Franco

    2006-05-30

    DNA was ozonized in solution and the reaction was followed with polarimetry and with iodimetry. Polarimetry was used to determine the molar ratio DNA/O(3) when the DNA optical activity vanishes completely. At a molar ratio DNA/O(3)=2.3 the supramolecular structure of DNA collapses completely. Instead, iodimetry shows that the ozonolysis proceeds until all the nucleobases have been destroyed, an event which occurs at a molar ratio DNA/O(3)=1.1. The ozonolysis of DNA was also followed spectrophotometrically. DNA is reactive with ozone also in the solid state, as fixed bed. Clear indication about its oxidation derives from the FT-IR spectra from polarimetric measurements and from thermal analysis performed by thermogravimetric analysis (TGA), differential thermogravimetric analysis (DTG) and from differential thermal analysis (DTA). Particular remarkable is the fact that RNA has been found much less reactive toward ozone in the solid state than DNA.

  11. Mammalian DNA helicase.

    PubMed Central

    Hübscher, U; Stalder, H P

    1985-01-01

    A forked DNA was constructed to serve as a substrate for DNA helicases. It contains features closely resembling a natural replication fork. The DNA was prepared in large amounts and was used to assay displacement activity during isolation from calf thymus DNA polymerases alpha holoenzyme. One form of DNA polymerase alpha holoenzyme is possibly involved leading strand replication at the replication fork and possesses DNA dependent ATPase activity (Ottiger, H.-P. and Hübscher, U. (1984) Proc. Natl. Acad. Sci. USA 81, 3993-3997). The enzyme can be separated from DNA polymerase alpha by velocity sedimentation in conditions of very low ionic strength and then be purified by chromatography on Sephacryl S-200 and ATP-agarose. At all stages of purification, DNA dependent ATPase and displacement activity profiles were virtually superimposable. The DNA dependent ATPase can displace a hybridized DNA fragment with a short single-stranded tail at its 3'hydroxyl end only in the presence of ATP, and this displacement relies on ATP hydrolysis. Furthermore, homogeneous single-stranded binding proteins from calf thymus as well as from other tissues cannot perform this displacement reaction. By all this token the DNA dependent ATPase appears to be a DNA helicase. It is suggested that this DNA helicase might act in concert with DNA polymerase alpha at the leading strand, possibly pushing the replication fork ahead of the polymerase. Images PMID:3162158

  12. New branched DNA constructs.

    PubMed

    Chandra, Madhavaiah; Keller, Sascha; Gloeckner, Christian; Bornemann, Benjamin; Marx, Andreas

    2007-01-01

    The Watson-Crick base pairing of DNA is an advantageous phenomenon that can be exploited when using DNA as a scaffold for directed self-organization of nanometer-sized objects. Several reports have appeared in the literature that describe the generation of branched DNA (bDNA) with variable numbers of arms that self-assembles into predesigned architectures. These bDNA units are generated by using cleverly designed rigid crossover DNA molecules. Alternatively, bDNA can be generated by using synthetic branch points derived from either nucleoside or non-nucleoside building blocks. Branched DNA has scarcely been explored for use in nanotechnology or from self-assembling perspectives. Herein, we wish to report our results for the synthesis, characterization, and assembling properties of asymmetrical bDNA molecules that are able to generate linear and circular bDNA constructs. Our strategy for the generation of bDNA is based on a branching point that makes use of a novel protecting-group strategy. The bDNA units were generated by means of automated DNA synthesis methods and were used to generate novel objects by employing chemical and biological techniques. The entities generated might be useful building blocks for DNA-based nanobiotechnology.

  13. Amplified DNA Biosensors

    NASA Astrophysics Data System (ADS)

    Willner, Itamar; Shlyahovsky, Bella; Willner, Bilha; Zayats, Maya

    Amplified detection of DNA is a central research topic in modern bioanalytical science. Electronic or optical transduction of DNA recognition events provides readout signals for DNA biosensors. Amplification of the DNA analysis is accomplished by the coupling of nucleic acid-functionalized enzymes or nucleic acid-functionalized nanoparticles (NP) as labels for the DNA duplex formation. This chapter discusses the amplified amperometric analysis of DNA by redox enzymes, the amplified optical sensing of DNA by enzymes or DNAzymes, and the amplified voltammetric, optical, or microgravimetric analysis of DNA using metallic or semiconductor nanoparticles. Further approaches to amplify DNA detection involve the use of micro-carriers of redox compounds as labels for DNA complex formation on electrodes, or the use of micro-objects such as liposomes, that label the resulting DNA complexes on electrodes and alter the interfacial properties of the electrodes. Finally, DNA machines are used for the optical detection of DNA, and the systems are suggested as future analytical procedures that could substitute the polymerase chain reaction (PCR) process.

  14. Study of force-induced melting of dsDNA as a function of length and conformation

    PubMed Central

    Danilowicz, Claudia; Hatch, Kristi; Conover, Alyson; Ducas, Theodore; Gunaratne, Ruwan; Coljee, Vincent; Prentiss, Mara

    2016-01-01

    We measure the constant force required to melt double-stranded DNA (ds) as a function of length for lengths from 12 to 100,000 base pairs, where the force is applied to the 3’3’ or 5’5’ ends of the dsDNA. Molecules with 32 base pairs or less melt before overstretching. For these short molecules, the melting force is independent of the ends to which the force is applied and the shear force as a function of length is well described by de Gennes theory with a de Gennes length of less than 10 bp. Molecules with lengths of 500 base pairs or more overstretch before melting. For these long molecules, the melting force depends on the ends to which the force is applied. The melting force as a function of length increases even when the length exceeds 1000 bp, where the length dependence is inconsistent with de Gennes theory. Finally, we expand de Gennes melting theory to 3’5’ pulling and compare the predictions with experimental results. PMID:21386589

  15. Modeling DNA Replication.

    ERIC Educational Resources Information Center

    Bennett, Joan

    1998-01-01

    Recommends the use of a model of DNA made out of Velcro to help students visualize the steps of DNA replication. Includes a materials list, construction directions, and details of the demonstration using the model parts. (DDR)

  16. Structural Organization of DNA.

    ERIC Educational Resources Information Center

    Banfalvi, Gaspar

    1986-01-01

    Explains the structural organization of DNA by providing information on the primary, secondary, tertiary, and higher organization levels of the molecule. Also includes illustrations and descriptions of sign-inversion and rotating models for supercoiling of DNA. (ML)

  17. DNA tagged microparticles

    DOEpatents

    Farquar, George Roy; Leif, Roald N; Wheeler, Elizabeth

    2015-05-05

    A simulant that includes a carrier and DNA encapsulated in the carrier. Also a method of making a simulant including the steps of providing a carrier and encapsulating DNA in the carrier to produce the simulant.

  18. Modeling DNA Replication.

    ERIC Educational Resources Information Center

    Bennett, Joan

    1998-01-01

    Recommends the use of a model of DNA made out of Velcro to help students visualize the steps of DNA replication. Includes a materials list, construction directions, and details of the demonstration using the model parts. (DDR)

  19. Unusual DNA structures

    SciTech Connect

    Wells, R.D.; Harvey, S.C.

    1988-01-01

    The contents of this book are: Unusual DNS Structures and the Probes Used for Their Detection; The Specificity of Single Strand Specific Endonucleases; Chromatin STructure and DNA Structure at the hsp 26 Locus of Drosophilia; Cruciform Extrusion in Supercoiled DNA-Mechanisms and Contextual Influence; Torsional Stress, Unusual DNA Structures, and Eukaryotic Gene Expression; DNA Sequence and Structure: Bending to Biology. Cruciform Transitions Assayed Using a Psoralen Cross-linking Method: Applications to Measurements of DNA Torisonal Tension; NMR-Distance Geometry Studies of Helical Errors and Sequence Dependent Conformations of DNA in Solution; Hyperreactivity of the B-Z Junctions Probed by Two Aromatic Chemical Carcinogens; Inherently Curved DNA and Its Structural Elements; and DNA Flexibility Under Control: The Juma Algorithm and its Application to BZ Junctions.

  20. Structural Organization of DNA.

    ERIC Educational Resources Information Center

    Banfalvi, Gaspar

    1986-01-01

    Explains the structural organization of DNA by providing information on the primary, secondary, tertiary, and higher organization levels of the molecule. Also includes illustrations and descriptions of sign-inversion and rotating models for supercoiling of DNA. (ML)

  1. DNA Spools under Tension

    NASA Astrophysics Data System (ADS)

    Kulić, I. M.; Schiessel, H.

    2004-06-01

    DNA spools, structures in which DNA is wrapped and helically coiled onto itself or onto a protein core, are ubiquitous in nature. We develop a general theory describing the nonequilibrium behavior of DNA spools under linear tension. Two puzzling and seemingly unrelated recent experimental findings, the sudden quantized unwrapping of nucleosomes and that of DNA toroidal condensates under tension, are theoretically explained and shown to be of the same origin. The study provides new insights into nucleosome and chromatin fiber stability and dynamics.

  2. Nanopores: Flossing with DNA

    NASA Astrophysics Data System (ADS)

    Kasianowicz, John J.

    2004-06-01

    Passing a DNA strand many times back-and-forth through a protein nanopore would enable the interaction between them to be studied more closely. This may now be possible, using a dumbbell-shaped DNA-polymer complex, which may lead to a more reliable analysis of DNA sequences using nanopores.

  3. DNAzymes in DNA Nanomachines and DNA Analysis

    NASA Astrophysics Data System (ADS)

    He, Yu; Tian, Ye; Chen, Yi; Mao, Chengde

    This chapter discusses our efforts in using DNAzymes in DNA nano-machines and DNA analysis systems. 10-23 DNAzymes can cleave specific phos-phodiester bonds in RNA. We use them to construct an autonomous DNA-RNA chimera nanomotor, which constantly extracts chemical energy from RNA substrates and transduces the energy into a mechanical motion: cycles of contraction and extension. The motor's motion can be reversibly turned on and off by a DNA analogue (brake) of the RNA substrate. Addition and removal of the brake stops and restarts, respectively, the motor's motion. Furthermore, when the RNA substrates are preorganized into a one-dimensional track, a DNAzyme can continuously move along the track so long as there are substrates available ahead. Based on a similar mechanism, a novel DNA detection system has been developed. A target DNA activates a DNAzyme to cleave RNA-containing molecular beacons (MB), which generates an enhanced fluorescence signal. A following work integrates two steps of signal amplifications: a rolling-circle amplification (RCA) to synthesize multiple copies of DNAzymes, and the DNAzymes catalyze a chemical reaction to generate a colorimetric signal. This method allows detection of DNA analytes whose concentration is as low as 1 pM.

  4. Three-Dimensional DNA Nanostructures Assembled from DNA Star Motifs.

    PubMed

    Tian, Cheng; Zhang, Chuan

    2017-01-01

    Tile-based DNA self-assembly is a promising method in DNA nanotechnology and has produced a wide range of nanostructures by using a small set of unique DNA strands. DNA star motif, as one of DNA tiles, has been employed to assemble varieties of symmetric one-, two-, three-dimensional (1, 2, 3D) DNA nanostructures. Herein, we describe the design principles, assembly methods, and characterization methods of 3D DNA nanostructures assembled from the DNA star motifs.

  5. The Many Sides of DNA.

    ERIC Educational Resources Information Center

    Flannery, Maura C.

    1997-01-01

    Explores the meaning of DNA. Discusses histories of DNA, literature on DNA, the contributions of Max Delbruck and Barbara McClintock, life, views of control, current research, and the language of DNA. Contains 24 references. (JRH)

  6. The Many Sides of DNA.

    ERIC Educational Resources Information Center

    Flannery, Maura C.

    1997-01-01

    Explores the meaning of DNA. Discusses histories of DNA, literature on DNA, the contributions of Max Delbruck and Barbara McClintock, life, views of control, current research, and the language of DNA. Contains 24 references. (JRH)

  7. Archaeal DNA replication.

    PubMed

    Kelman, Lori M; Kelman, Zvi

    2014-01-01

    DNA replication is essential for all life forms. Although the process is fundamentally conserved in the three domains of life, bioinformatic, biochemical, structural, and genetic studies have demonstrated that the process and the proteins involved in archaeal DNA replication are more similar to those in eukaryal DNA replication than in bacterial DNA replication, but have some archaeal-specific features. The archaeal replication system, however, is not monolithic, and there are some differences in the replication process between different species. In this review, the current knowledge of the mechanisms governing DNA replication in Archaea is summarized. The general features of the replication process as well as some of the differences are discussed.

  8. DNA-Mediated Electrochemistry

    PubMed Central

    Gorodetsky, Alon A.; Buzzeo, Marisa C.

    2009-01-01

    The base pair stack of DNA has been demonstrated as a medium for long range charge transport chemistry both in solution and at DNA-modified surfaces. This chemistry is exquisitely sensitive to structural perturbations in the base pair stack as occur with lesions, single base mismatches, and protein binding. We have exploited this sensitivity for the development of reliable electrochemical assays based on DNA charge transport at self-assembled DNA monolayers. Here we discuss the characteristic features, applications, and advantages of DNA-mediated electrochemistry. PMID:18980370

  9. DNA Sequencing apparatus

    DOEpatents

    Tabor, Stanley; Richardson, Charles C.

    1992-01-01

    An automated DNA sequencing apparatus having a reactor for providing at least two series of DNA products formed from a single primer and a DNA strand, each DNA product of a series differing in molecular weight and having a chain terminating agent at one end; separating means for separating the DNA products to form a series bands, the intensity of substantially all nearby bands in a different series being different, band reading means for determining the position an This invention was made with government support including a grant from the U.S. Public Health Service, contract number AI-06045. The U.S. government has certain rights in the invention.

  10. DNA Functionalization of Nanoparticles.

    PubMed

    Lu, Fang; Gang, Oleg

    2017-01-01

    DNA-nanoparticle conjugates are hybrid nanoscale objects that integrate different types of DNA molecules and inorganic nanoparticles with a typical architecture of a DNA shell around an inorganic core. Such incorporation provides particles with unique properties of DNA, addressability and recognition, but, at the same time, allows exploiting the properties of the particle's inorganic core. Thus, these hybrid nano-objects are advantageous for rational fabrication of functional materials and for biomedical applications. Here, we describe several established DNA functionalization procedures for different types of surface ligands and nanoparticle core materials.

  11. Role of the QBO in Modulating the Influence of the 11 Year Solar Cycle on the Atmosphere Using Constant Forcings

    DTIC Science & Technology

    2010-09-21

    phases is analyzed with special focus on the ozone budget (section 4) and some extratropical signals including a brief description of tropo - spheric...exists in both hemispheres poleward of 50° in the tropo - sphere and lower stratosphere, reaching even into the upper stratosphere in the SH. The solar

  12. DNA structure and function.

    PubMed

    Travers, Andrew; Muskhelishvili, Georgi

    2015-06-01

    The proposal of a double-helical structure for DNA over 60 years ago provided an eminently satisfying explanation for the heritability of genetic information. But why is DNA, and not RNA, now the dominant biological information store? We argue that, in addition to its coding function, the ability of DNA, unlike RNA, to adopt a B-DNA structure confers advantages both for information accessibility and for packaging. The information encoded by DNA is both digital - the precise base specifying, for example, amino acid sequences - and analogue. The latter determines the sequence-dependent physicochemical properties of DNA, for example, its stiffness and susceptibility to strand separation. Most importantly, DNA chirality enables the formation of supercoiling under torsional stress. We review recent evidence suggesting that DNA supercoiling, particularly that generated by DNA translocases, is a major driver of gene regulation and patterns of chromosomal gene organization, and in its guise as a promoter of DNA packaging enables DNA to act as an energy store to facilitate the passage of translocating enzymes such as RNA polymerase.

  13. Supramolecular DNA assembly.

    PubMed

    McLaughlin, Christopher K; Hamblin, Graham D; Sleiman, Hanadi F

    2011-12-01

    The powerful self-assembly features of DNA make it a unique template to finely organize and control matter on the nanometre scale. While DNA alone offers a high degree of fidelity in its self-assembly, a new area of research termed 'supramolecular DNA assembly' has recently emerged. This field combines DNA building blocks with synthetic organic, inorganic and polymeric structures. It thus brings together the toolbox of supramolecular chemistry with the predictable and programmable nature of DNA. The result of this molecular partnership is a variety of hybrid architectures, that expand DNA assembly beyond the boundaries of Watson-Crick base pairing into new structural and functional properties. In this tutorial review we outline this emerging field of study, and describe recent research aiming to synergistically combine the properties inherent to DNA with those of a number of supramolecular scaffolds. This ultimately creates structures with numerous potential applications in materials science, catalysis and medicine.

  14. Drying of DNA droplets.

    PubMed

    Fang, Xiaohua; Li, Bingquan; Petersen, Eric; Seo, Young-Soo; Samuilov, Vladimir A; Chen, Yong; Sokolov, Jonathan C; Shew, Chwen-Yang; Rafailovich, Miriam H

    2006-07-04

    The evaporation kinetics of droplets containing DNA was studied, as a function of DNA concentration. Drops containing very low DNA concentrations dried by maintaining a constant base, whereas those with high concentration dried with a constant contact angle. To understand this phenomenon, the distribution of the DNA inside the droplet was measured using confocal microscopy. The results indicated that the DNA was condensed mostly on the surface of the droplets. In the case of high concentration droplets, it formed a shell, whereas isolated islands were found for droplets of low DNA concentrations. Rheologic results indicate the formation of a hydro gel in the low concentration drops, whereas phase separation between the self-assembled DNA structures and the water phase occurred at higher concentration.

  15. [DNA methylation and epigenetics].

    PubMed

    Vaniushin, B F

    2006-09-01

    In eukaryotic cells, nuclear DNA is subject to enzymatic methylation with the formation of 5-methylcytosine residues, mostly within the CG and CNG sequences. In plants and animals this DNA methylation is species-, tissue-, and organelle-specific. It changes (decreases) with age and is regulated by hormones. On the other hand, genome methylation can control hormonal signal. Replicative and post-replicative DNA methylation types are distinguished. They are mediated by multiple DNA methyltransferases with different site-specificity. Replication is accompanied by the appearance of hemimethylated DNA sites. Pronounced asymmetry of the DNA strand methylation disappears to the end of the cell cycle. A model of methylation-regulated DNA replication is proposed. DNA methylation controls all genetic processes in the cell (replication, transcription, DNA repair, recombination, and gene transposition). It is the mechanism of cell differentiation, gene discrimination and silencing. In animals, suppression of DNA methylation stops development (embryogenesis), switches on apoptosis, and is usually lethal. Disruption of DNA methylation pattern results in the malignant cell transformation and serves as one of the early diagnostic features of carcinogenesis. In malignant cell the pattern of DNA methylation, as well as the set of DNA methyltransferase activities, differs from that in normal cell. In plants inhibition of DNA methylation is accompanied by the induction of seed storage and florescence genes. In eukaryotes one and the same gene can be simultaneously methylated both at cytosine and adenine residues. It can be thus suggested, that the plant cell contains at least two different, and probably, interdependent systems of DNA methylation. The first eukaryotic adenine DNA methyltransferase was isolated from plants. This enzyme methylates DNA with the formation of N6-methyladenine residues in the sequence TGATCA (TGATCA-->TGm6ATCA). Plants possess AdoMet-dependent endonucleases

  16. Forensic DNA analysis.

    PubMed

    McDonald, Jessica; Lehman, Donald C

    2012-01-01

    Before the routine use of DNA profiling, blood typing was an important forensic tool. However, blood typing was not very discriminating. For example, roughly 30% of the United States population has type A-positive blood. Therefore, if A-positive blood were found at a crime scene, it could have come from 30% of the population. DNA profiling has a much better ability for discrimination. Forensic laboratories no longer routinely determine blood type. If blood is found at a crime scene, DNA profiling is performed. From Jeffrey's discovery of DNA fingerprinting to the development of PCR of STRs to the formation of DNA databases, our knowledge of DNA and DNA profiling have expanded greatly. Also, the applications for which we use DNA profiling have increased. DNA profiling is not just used for criminal case work, but it has expanded to encompass paternity testing, disaster victim identification, monitoring bone marrow transplants, detecting fetal cells in a mother's blood, tracing human history, and a multitude of other areas. The future of DNA profiling looks expansive with the development of newer instrumentation and techniques.

  17. Enzymatic initiation of DNA synthesis by yeast DNA polymerases.

    PubMed Central

    Plevani, P; Chang, L M

    1977-01-01

    Partially purified yeast RNA polymerases (RNA nucleotidyltransferases) initiate DNA synthesis by yeast DNA polymerase (DNA nucleotidyltransferase) I and to a lesser extent yeast DNA polymerase II in the replication of single-stranded DNA. The enzymatic initiation of DNA synthesis on phage fd DNA template occurs with dNTPs alone and is further stimulated by the presence of rNTPs in DNA polymerase I reactions. The presence of rNTPs has no effect on the RNA polymerase initiation of the DNA polymerase II reaction. RNA polymerases I and III are more efficient in initiation of DNA synthesis than RNA polymerase II. Analyses of the products of fd DNA replication show noncovalent linkage between the newly synthesized DNA and the template DNA, and covalent linkage between the newly synthesized RNA and DNA. PMID:325562

  18. Functional DNA Nanomaterials

    NASA Astrophysics Data System (ADS)

    Zhao, Zhao

    The discovery of DNA helical structure opened the door of modern molecular biology. Ned Seeman utilized DNA as building block to construct different nanoscale materials, and introduced a new field, know as DNA nanotechnology. After several decades of development, different DNA structures had been created, with different dimension, different morphology and even with complex curvatures. In addition, after construction of enough amounts DNA structure candidates, DNA structure template, with excellent spatial addressability, had been used to direct the assembly of different nanomaterials, including nanoparticles and proteins, to produce different functional nanomaterials. However there are still many challenges to fabricate functional DNA nanostructures. The first difficulty is that the present finite sized template dimension is still very small, usually smaller than 100nm, which will limit the application for large amount of nanomaterials assembly or large sized nanomaterials assembly. Here we tried to solve this problem through developing a new method, superorigami, to construct finite sized DNA structure with much larger dimension, which can be as large as 500nm. The second problem will be explored the ability of DNA structure to assemble inorganic nanomaterials for novel photonic or electronic properties. Here we tried to utilize DNA Origami method to assemble AuNPs with controlled 3D spacial position for possible chiral photonic complex. We also tried to assemble SWNT with discrete length for possible field effect transistor device. In addition, we tried to mimic in vivo compartment with DNA structure to study internalized enzyme behavior. From our results, constructed DNA cage origami can protect encapsulated enzyme from degradation, and internalized enzyme activity can be boosted for up to 10 folds. In summary, DNA structure can serve as an ideal template for construction of functional nanomaterials with lots of possibilities to be explored.

  19. The DNA-polymerase-X family: controllers of DNA quality?

    PubMed

    Ramadan, Kristijan; Shevelev, Igor; Hübscher, Ulrich

    2004-12-01

    Synthesis of the genetic material of the cell is achieved by a large number of DNA polymerases. Besides replicating the genome, they are involved in DNA-repair processes. Recent studies have indicated that certain DNA-polymerase-X-family members can synthesize unusual DNA structures, and we propose that these DNA structures might serve as 'flag wavers' for the induction of DNA-repair and/or DNA-damage-checkpoint pathways.

  20. DNA profiles from fingermarks.

    PubMed

    Templeton, Jennifer E L; Linacre, Adrian

    2014-11-01

    Criminal investigations would be considerably improved if DNA profiles could be routinely generated from single fingermarks. Here we report a direct DNA profiling method that was able to generate interpretable profiles from 71% of 170 fingermarks. The data are based on fingermarks from all 5 digits of 34 individuals. DNA was obtained from the fingermarks using a swab moistened with Triton-X, and the fibers were added directly to one of two commercial DNA profiling kits. All profiles were obtained without increasing the number of amplification cycles; therefore, our method is ideally suited for adoption by the forensic science community. We indicate the use of the technique in a criminal case in which a DNA profile was generated from a fingermark on tape that was wrapped around a drug seizure. Our direct DNA profiling approach is rapid and able to generate profiles from touched items when current forensic practices have little chance of success.

  1. Electrocatalysis in DNA Sensors

    PubMed Central

    Furst, Ariel; Hill, Michael G.; Barton, Jacqueline K.

    2014-01-01

    Electrocatalysis is often thought of solely in the inorganic realm, most often applied to energy conversion in fuel cells. However, the ever-growing field of bioelectrocatalysis has made great strides in advancing technology for both biofuel cells as well as biological detection platforms. Within the context of bioelectrocatalytic detection systems, DNA-based platforms are especially prevalent. One subset of these platforms, the one we have developed, takes advantage of the inherent charge transport properties of DNA. Electrocatalysis coupled with DNA-mediated charge transport has enabled specific and sensitive detection of lesions, mismatches and DNA-binding proteins. Even greater signal amplification from these platforms is now being achieved through the incorporation of a secondary electrode to the platform both for patterning DNA arrays and for detection. Here, we describe the evolution of this new DNA sensor technology. PMID:25435647

  2. Eukaryotic DNA Replication Fork.

    PubMed

    Burgers, Peter M J; Kunkel, Thomas A

    2017-06-20

    This review focuses on the biogenesis and composition of the eukaryotic DNA replication fork, with an emphasis on the enzymes that synthesize DNA and repair discontinuities on the lagging strand of the replication fork. Physical and genetic methodologies aimed at understanding these processes are discussed. The preponderance of evidence supports a model in which DNA polymerase ε (Pol ε) carries out the bulk of leading strand DNA synthesis at an undisturbed replication fork. DNA polymerases α and δ carry out the initiation of Okazaki fragment synthesis and its elongation and maturation, respectively. This review also discusses alternative proposals, including cellular processes during which alternative forks may be utilized, and new biochemical studies with purified proteins that are aimed at reconstituting leading and lagging strand DNA synthesis separately and as an integrated replication fork.

  3. DNA topology and transcription

    PubMed Central

    Kouzine, Fedor; Levens, David; Baranello, Laura

    2014-01-01

    Chromatin is a complex assembly that compacts DNA inside the nucleus while providing the necessary level of accessibility to regulatory factors conscripted by cellular signaling systems. In this superstructure, DNA is the subject of mechanical forces applied by variety of molecular motors. Rather than being a rigid stick, DNA possesses dynamic structural variability that could be harnessed during critical steps of genome functioning. The strong relationship between DNA structure and key genomic processes necessitates the study of physical constrains acting on the double helix. Here we provide insight into the source, dynamics, and biology of DNA topological domains in the eukaryotic cells and summarize their possible involvement in gene transcription. We emphasize recent studies that might inspire and impact future experiments on the involvement of DNA topology in cellular functions. PMID:24755522

  4. DNA supercoiling during transcription.

    PubMed

    Ma, Jie; Wang, Michelle D

    2016-11-01

    The twin-supercoiled-domain model describes how transcription can drive DNA supercoiling, and how DNA supercoiling, in turn plays an important role in regulating gene transcription. In vivo and in vitro experiments have disclosed many details of the complex interactions in this relationship, and recently new insights have been gained with the help of genome-wide DNA supercoiling mapping techniques and single molecule methods. This review summarizes the general mechanisms of the interplay between DNA supercoiling and transcription, considers the biological implications, and focuses on recent important discoveries and technical advances in this field. We highlight the significant impact of DNA supercoiling in transcription, but also more broadly in all processes operating on DNA.

  5. Rifampin and vaccinia DNA.

    PubMed Central

    Esteban, M

    1977-01-01

    The effect of rifampin on the replication of vaccinia DNA was studied in mouse L cells by a cytochemical techinque and by alkaline sucrose sedimentation analysis of newly synthesized viral DNA molecules. By the use of a fluorescent DNA-binding compound (Hoechst 33258), the sequential appearance, size, and location of the viral "factories" in rifampin-treated, virus-infected cells were found to be indistinguishable from those observed in untreated, infected cells. Sedimentation analysis in alkaline scurose gradients of the viral DNA molecules labeled in pulse-chase experiments showed that formation of small fragments, elongation into "intermediate"-sized molecules, and maturation into full-length viral DNA and, finally, into cross-linked viral DNA molecules occurred in the absence or presence of rifampin. The results support the view that the primary effect of the drug is related to assembly or morphogenesis. Images PMID:833950

  6. DNA synthesis security.

    PubMed

    Nouri, Ali; Chyba, Christopher F

    2012-01-01

    It is generally assumed that genetic engineering advances will, inevitably, facilitate the misapplication of biotechnology toward the production of biological weapons. Unexpectedly, however, some of these very advances in the areas of DNA synthesis and sequencing may enable the implementation of automated and nonintrusive safeguards to avert the illicit applications of biotechnology. In the case of DNA synthesis, automated DNA screening tools could be built into DNA synthesizers in order to block the synthesis of hazardous agents. In addition, a comprehensive safety and security regime for dual-use genetic engineering research could include nonintrusive monitoring of DNA sequencing. This is increasingly feasible as laboratories outsource this service to just a few centralized sequencing factories. The adoption of automated, nonintrusive monitoring and surveillance of the DNA synthesis and sequencing pipelines may avert many risks associated with dual-use biotechnology. Here, we describe the historical background and current challenges associated with dual-use biotechnologies and propose strategies to address these challenges.

  7. DNA supercoiling during transcription

    PubMed Central

    Ma, Jie; Wang, Michelle D.

    2017-01-01

    The twin-supercoiled-domain model describes how transcription can drive DNA supercoiling, and how DNA supercoiling, in turn plays an important role in regulating gene transcription. In vivo and in vitro experiments have disclosed many details of the complex interactions in this relationship, and recently new insights have been gained with the help of genome-wide DNA supercoiling mapping techniques and single molecule methods. This review summarizes the general mechanisms of the interplay between DNA supercoiling and transcription, considers the biological implications, and focuses on recent important discoveries and technical advances in this field. We highlight the significant impact of DNA supercoiling in transcription, but also more broadly in all processes operating on DNA.

  8. Detection and quantitation of single nucleotide polymorphisms, DNA sequence variations, DNA mutations, DNA damage and DNA mismatches

    DOEpatents

    McCutchen-Maloney, Sandra L.

    2002-01-01

    DNA mutation binding proteins alone and as chimeric proteins with nucleases are used with solid supports to detect DNA sequence variations, DNA mutations and single nucleotide polymorphisms. The solid supports may be flow cytometry beads, DNA chips, glass slides or DNA dips sticks. DNA molecules are coupled to solid supports to form DNA-support complexes. Labeled DNA is used with unlabeled DNA mutation binding proteins such at TthMutS to detect DNA sequence variations, DNA mutations and single nucleotide length polymorphisms by binding which gives an increase in signal. Unlabeled DNA is utilized with labeled chimeras to detect DNA sequence variations, DNA mutations and single nucleotide length polymorphisms by nuclease activity of the chimera which gives a decrease in signal.

  9. DNA-based machines.

    PubMed

    Wang, Fuan; Willner, Bilha; Willner, Itamar

    2014-01-01

    The base sequence in nucleic acids encodes substantial structural and functional information into the biopolymer. This encoded information provides the basis for the tailoring and assembly of DNA machines. A DNA machine is defined as a molecular device that exhibits the following fundamental features. (1) It performs a fuel-driven mechanical process that mimics macroscopic machines. (2) The mechanical process requires an energy input, "fuel." (3) The mechanical operation is accompanied by an energy consumption process that leads to "waste products." (4) The cyclic operation of the DNA devices, involves the use of "fuel" and "anti-fuel" ingredients. A variety of DNA-based machines are described, including the construction of "tweezers," "walkers," "robots," "cranes," "transporters," "springs," "gears," and interlocked cyclic DNA structures acting as reconfigurable catenanes, rotaxanes, and rotors. Different "fuels", such as nucleic acid strands, pH (H⁺/OH⁻), metal ions, and light, are used to trigger the mechanical functions of the DNA devices. The operation of the devices in solution and on surfaces is described, and a variety of optical, electrical, and photoelectrochemical methods to follow the operations of the DNA machines are presented. We further address the possible applications of DNA machines and the future perspectives of molecular DNA devices. These include the application of DNA machines as functional structures for the construction of logic gates and computing, for the programmed organization of metallic nanoparticle structures and the control of plasmonic properties, and for controlling chemical transformations by DNA machines. We further discuss the future applications of DNA machines for intracellular sensing, controlling intracellular metabolic pathways, and the use of the functional nanostructures for drug delivery and medical applications.

  10. Recombinant DNA. Part F

    SciTech Connect

    Wu, R.

    1987-01-01

    This book contains 36 papers. Some of the titles are: Site-specific cleavage of DNA at 8-, 9-, and 10-bp sequences; The use of DNA methylases to alter the apparent recognition specificities of restriction endonucleases; Random cloning and sequencing by the M13/Dideoxynucleotide chain termination method; Solid-phase methods for sequencing of oligodeoxynucleotides and DNA; and Analysis of modified nucleosides and nucleotide sequence of tRNA.

  11. Forensic DNA testing.

    PubMed

    Butler, John M

    2011-12-01

    Forensic DNA testing has a number of applications, including parentage testing, identifying human remains from natural or man-made disasters or terrorist attacks, and solving crimes. This article provides background information followed by an overview of the process of forensic DNA testing, including sample collection, DNA extraction, PCR amplification, short tandem repeat (STR) allele separation and sizing, typing and profile interpretation, statistical analysis, and quality assurance. The article concludes with discussions of possible problems with the data and other forensic DNA testing techniques.

  12. Multiprotein DNA Looping

    NASA Astrophysics Data System (ADS)

    Vilar, Jose M. G.; Saiz, Leonor

    2006-06-01

    DNA looping plays a fundamental role in a wide variety of biological processes, providing the backbone for long range interactions on DNA. Here we develop the first model for DNA looping by an arbitrarily large number of proteins and solve it analytically in the case of identical binding. We uncover a switchlike transition between looped and unlooped phases and identify the key parameters that control this transition. Our results establish the basis for the quantitative understanding of fundamental cellular processes like DNA recombination, gene silencing, and telomere maintenance.

  13. Phytoplasma plasmid DNA extraction.

    PubMed

    Andersen, Mark T; Liefting, Lia W

    2013-01-01

    Phytoplasma plasmids have generally been detected from DNA extracted from plants and insects using methods designed for the purification of total phytoplasma DNA. Methods include extraction from tissues that are high in phytoplasma titre, such as the phloem of plants, with the use of CsCl-bisbenzimide gradients that exploit the low G+C content of phytoplasma DNA. Many of the methods employed for phytoplasma purification have been described elsewhere in this book. Here we describe in detail two methods that are specifically aimed at isolating plasmid DNA.

  14. Disentangling DNA molecules

    NASA Astrophysics Data System (ADS)

    Vologodskii, Alexander

    2016-09-01

    The widespread circular form of DNA molecules inside cells creates very serious topological problems during replication. Due to the helical structure of the double helix the parental strands of circular DNA form a link of very high order, and yet they have to be unlinked before the cell division. DNA topoisomerases, the enzymes that catalyze passing of one DNA segment through another, solve this problem in principle. However, it is very difficult to remove all entanglements between the replicated DNA molecules due to huge length of DNA comparing to the cell size. One strategy that nature uses to overcome this problem is to create the topoisomerases that can dramatically reduce the fraction of linked circular DNA molecules relative to the corresponding fraction at thermodynamic equilibrium. This striking property of the enzymes means that the enzymes that interact with DNA only locally can access their topology, a global property of circular DNA molecules. This review considers the experimental studies of the phenomenon and analyzes the theoretical models that have been suggested in attempts to explain it. We describe here how various models of enzyme action can be investigated computationally. There is no doubt at the moment that we understand basic principles governing enzyme action. Still, there are essential quantitative discrepancies between the experimental data and the theoretical predictions. We consider how these discrepancies can be overcome.

  15. Disentangling DNA molecules.

    PubMed

    Vologodskii, Alexander

    2016-09-01

    The widespread circular form of DNA molecules inside cells creates very serious topological problems during replication. Due to the helical structure of the double helix the parental strands of circular DNA form a link of very high order, and yet they have to be unlinked before the cell division. DNA topoisomerases, the enzymes that catalyze passing of one DNA segment through another, solve this problem in principle. However, it is very difficult to remove all entanglements between the replicated DNA molecules due to huge length of DNA comparing to the cell size. One strategy that nature uses to overcome this problem is to create the topoisomerases that can dramatically reduce the fraction of linked circular DNA molecules relative to the corresponding fraction at thermodynamic equilibrium. This striking property of the enzymes means that the enzymes that interact with DNA only locally can access their topology, a global property of circular DNA molecules. This review considers the experimental studies of the phenomenon and analyzes the theoretical models that have been suggested in attempts to explain it. We describe here how various models of enzyme action can be investigated computationally. There is no doubt at the moment that we understand basic principles governing enzyme action. Still, there are essential quantitative discrepancies between the experimental data and the theoretical predictions. We consider how these discrepancies can be overcome. Copyright © 2016 Elsevier B.V. All rights reserved.

  16. DNA: An Extensible Molecule

    NASA Astrophysics Data System (ADS)

    Cluzel, Philippe; Lebrun, Anne; Heller, Christoph; Lavery, Richard; Viovy, Jean-Louis; Chatenay, Didier; Caron, Francois

    1996-02-01

    The force-displacement response of a single duplex DNA molecule was measured. The force saturates at a plateau around 70 piconewtons, which ends when the DNA has been stretched about 1.7 times its contour length. This behavior reveals a highly cooperative transition to a state here termed S-DNA. Addition of an intercalator suppresses this transition. Molecular modeling of the process also yields a force plateau and suggests a structure for the extended form. These results may shed light on biological processes involving DNA extension and open the route for mechanical studies on individual molecules in a previously unexplored range.

  17. DNA ELECTROPHORESIS AT SURFACES

    SciTech Connect

    RAFAILOVICH, MIRIAM; SOKOLOV, JONATHAN; GERSAPPE, DILIP

    2003-09-01

    During this year we performed two major projects: I. We developed a detailed theoretical model which complements our experiments on surface DNA electrophoresis. We found that it was possible to enhance the separation of DNA chains by imposing a chemical nanoscale pattern on the surface. This approach utilized the surface interaction effect of the DNA chains with the substrate and is a refinement to our previous method in which DNA chains were separated on homogeneous flat surfaces. By introducing the nano-patterns on the surface, the conformational changes of DNA chains of different lengths can be amplified, which results in the different friction strengths with the substrate surface. Our results also show that, when compared to the DNA electrophoresis performed on homogeneous flat surfaces, nanopatterned surfaces offer a larger window in choosing different surface interactions to achieve separation. II. In collaboration with a large international manufacturer of skin care products we also embarked on a project involving photo toxicity of titanium dioxide nanoparticles, which are a key ingredient in sunscreen and cosmetic lotions. The results clearly implicated the nanoparticles in catalyzing damage to chromosomal DNA. We then used this knowledge to develop a polymer/anti-oxidant coating which prevented the photocatalytic reaction on DNA while still retaining the UV absorptive properties of the nanoparticles. The standard gel electrophoresis was not sufficient in determining the extent of the DNA damage. The conclusions of this study were based predominantly on analysis obtained with the surface electrophoresis method.

  18. Function of transcription factors at DNA lesions in DNA repair.

    PubMed

    Malewicz, Michal; Perlmann, Thomas

    2014-11-15

    Cellular systems for DNA repair ensure prompt removal of DNA lesions that threaten the genomic stability of the cell. Transcription factors (TFs) have long been known to facilitate DNA repair via transcriptional regulation of specific target genes encoding key DNA repair proteins. However, recent findings identified TFs as DNA repair components acting directly at the DNA lesions in a transcription-independent fashion. Together this recent progress is consistent with the hypothesis that TFs have acquired the ability to localize DNA lesions and function by facilitating chromatin remodeling at sites of damaged DNA. Here we review these recent findings and discuss how TFs may function in DNA repair.

  19. Analysis of mtDNA/nDNA Ratio in Mice.

    PubMed

    Quiros, Pedro M; Goyal, Aashima; Jha, Pooja; Auwerx, Johan

    2017-03-02

    Mitochondrial DNA (mtDNA) lacks the protection provided by the nucleosomes in the nuclear DNA and does not have a DNA repair mechanism, making it highly susceptible to damage, which can lead to mtDNA depletion. mtDNA depletion compromises the efficient function of cells and tissues and thus impacts negatively on health. Here, we describe a brief and easy protocol to quantify mtDNA copy number by determining the mtDNA/nDNA ratio. The procedure has been validated using a cohort of young and aged mice. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

  20. Oxidative DNA modifications.

    PubMed

    Poulsen, Henrik E

    2005-07-01

    Oxidative DNA modifications are frequent in mammalian DNA and have been suggested an important mechanism in carcinogenesis, diabetes and ageing. The foundations for this suggestion are: Evidence for the importance of oxidative DNA modifications in cancer development is: high levels of oxidative lesions in cancer tissue; highly conserved and specific DNA repair systems targeting oxidative lesions; high levels of oxidative DNA lesions in oxidative DNA repair knock-out animals; defective repair of oxidative lesions in cancer-prone progeria syndromes; reduced cancer incidence in populations with high dietary antioxidant intake; and increased oxidative stress to DNA in tobacco smokers. Conflicting evidence for a relation between oxidative stress to DNA and cancer is: disagreement about the true levels and occurrence of the oxidative lesions in vivo; failure to identify the localization of oxidative lesions in important genes, e.g. tumor suppressor and oncogenes; lack of evidence that the oxidative lesions induce mutations in vivo; no cancer development in animals knocked-out for specific DNA repair enzymes in spite of high tissue levels of oxidative lesions; and unchanged cancer rates after antioxidant interventions in large clinical controlled and randomized trials. The rate of DNA oxidation has been estimated from urinary excretion of repair products and it is evident that if these lesions were not repaired, a large part of DNA would be oxidized to a degree not compatible with living. The methodologies by which oxidative DNA modifications are measured cover a wide and different range, advantages and disadvantages will be presented. One particular problem is artificial oxidation, and methods to prevent such artifacts will be presented together with results from a large interlaboratory standardization program. The methodology by which the lesions can be measured is complicated and prone to artifacts during DNA isolation, digestion, derivatization and maybe even during

  1. Many Ways to Loop DNA

    PubMed Central

    Griffith, Jack D.

    2013-01-01

    In the 1960s, I developed methods for directly visualizing DNA and DNA-protein complexes using an electron microscope. This made it possible to examine the shape of DNA and to visualize proteins as they fold and loop DNA. Early applications included the first visualization of true nucleosomes and linkers and the demonstration that repeating tracts of adenines can cause a curvature in DNA. The binding of DNA repair proteins, including p53 and BRCA2, has been visualized at three- and four-way junctions in DNA. The trombone model of DNA replication was directly verified, and the looping of DNA at telomeres was discovered. PMID:24005675

  2. Routine DNA testing

    USDA-ARS?s Scientific Manuscript database

    Routine DNA testing. It’s done once you’ve Marker-Assisted Breeding Pipelined promising Qantitative Trait Loci within your own breeding program and thereby established the performance-predictive power of each DNA test for your germplasm under your conditions. By then you are ready to screen your par...

  3. Stool DNA Test

    MedlinePlus

    ... result. A test is considered negative if DNA markers common to colon cancer or precancerous polyps and signs of blood are ... result. A test is considered positive if DNA markers common to colon cancer or precancerous polyps or signs of blood are ...

  4. Metal Sensing by DNA.

    PubMed

    Zhou, Wenhu; Saran, Runjhun; Liu, Juewen

    2017-06-28

    Metal ions are essential to many chemical, biological, and environmental processes. In the past two decades, many DNA-based metal sensors have emerged. While the main biological role of DNA is to store genetic information, its chemical structure is ideal for metal binding via both the phosphate backbone and nucleobases. DNA is highly stable, cost-effective, easy to modify, and amenable to combinatorial selection. Two main classes of functional DNA were developed for metal sensing: aptamers and DNAzymes. While a few metal binding aptamers are known, it is generally quite difficult to isolate such aptamers. On the other hand, DNAzymes are powerful tools for metal sensing since they are selected based on catalytic activity, thus bypassing the need for metal immobilization. In the last five years, a new surge of development has been made on isolating new metal-sensing DNA sequences. To date, many important metals can be selectively detected by DNA often down to the low parts-per-billion level. Herein, each metal ion and the known DNA sequences for its sensing are reviewed. We focus on the fundamental aspect of metal binding, emphasizing the distinct chemical property of each metal. Instead of reviewing each published sensor, a high-level summary of signaling methods is made as a separate section. In principle, each signaling strategy can be applied to many DNA sequences for designing sensors. Finally, a few specific applications are highlighted, and future research opportunities are discussed.

  5. Behavior of supercoiled DNA.

    PubMed Central

    Strick, T R; Allemand, J F; Bensimon, D; Croquette, V

    1998-01-01

    We study DNA supercoiling in a quantitative fashion by micromanipulating single linear DNA molecules with a magnetic field gradient. By anchoring one end of the DNA to multiple sites on a magnetic bead and the other end to multiple sites on a glass surface, we were able to exert torsional control on the DNA. A rotating magnetic field was used to induce rotation of the magnetic bead, and reversibly over- and underwind the molecule. The magnetic field was also used to increase or decrease the stretching force exerted by the magnetic bead on the DNA. The molecule's degree of supercoiling could therefore be quantitatively controlled and monitored, and tethered-particle motion analysis allowed us to measure the stretching force acting on the DNA. Experimental results indicate that this is a very powerful technique for measuring forces at the picoscale. We studied the effect of stretching forces ranging from 0.01 pN to 100 pN on supercoiled DNA (-0.1 < sigma < 0.2) in a variety of ionic conditions. Other effects, such as stretching-relaxing hysteresis and the braiding of two DNA molecules, are discussed. PMID:9545060

  6. Characterization of muntjac DNA

    SciTech Connect

    Davis, R.C.

    1981-05-27

    Sister chromatid exchange (SCE) in muntjac chromosomes is generally proportional to the chromosomal DNA content, but the SCE frequency is reduced in the heterochromatic neck region of the X chromosome. The physical properties of muntjac DNA and the kinetics of repair of UV damage in muntjac heterochromatin and euchromatin were examined and compared with the distribution of sister chromatid exchange.

  7. Curating DNA specimens

    USDA-ARS?s Scientific Manuscript database

    DNA data are used in a variety of ethnobiological disciplines including archaeology, conservation, ecology, medicinal plants and natural products research, taxonomy and systematics, crop evolution and domestication, and genetic diversity. It frequently is convenient to store and share DNA among coop...

  8. DNA-cell conjugates

    DOEpatents

    Hsiao, Shih-Chia; Francis, Matthew B.; Bertozzi, Carolyn; Mathies, Richard; Chandra, Ravi; Douglas, Erik; Twite, Amy; Toriello, Nicholas; Onoe, Hiroaki

    2016-05-03

    The present invention provides conjugates of DNA and cells by linking the DNA to a native functional group on the cell surface. The cells can be without cell walls or can have cell walls. The modified cells can be linked to a substrate surface and used in assay or bioreactors.

  9. Human Mitochondrial DNA Replication

    PubMed Central

    Holt, Ian J.; Reyes, Aurelio

    2012-01-01

    Elucidation of the process of DNA replication in mitochondria is in its infancy. For many years, maintenance of the mitochondrial genome was regarded as greatly simplified compared to the nucleus. Mammalian mitochondria were reported to lack all DNA repair systems, to eschew DNA recombination, and to possess but a single DNA polymerase, polymerase γ. Polγ was said to replicate mitochondrial DNA exclusively via one mechanism, involving only two priming events and a handful of proteins. In this “strand-displacement model,” leading strand DNA synthesis begins at a specific site and advances approximately two-thirds of the way around the molecule before DNA synthesis is initiated on the “lagging” strand. Although the displaced strand was long-held to be coated with protein, RNA has more recently been proposed in its place. Furthermore, mitochondrial DNA molecules with all the features of products of conventional bidirectional replication have been documented, suggesting that the process and regulation of replication in mitochondria is complex, as befits a genome that is a core factor in human health and longevity. PMID:23143808

  10. Recombinant DNA for Teachers.

    ERIC Educational Resources Information Center

    Duvall, James G., III

    1992-01-01

    A science teacher describes his experience at a workshop to learn to teach the Cold Spring Harbor DNA Science Laboratory Protocols. These protocols lead students through processes for taking E. coli cells and transforming them into a new antibiotic resistant strain. The workshop featured discussions of the role of DNA recombinant technology in…

  11. Recombinant DNA for Teachers.

    ERIC Educational Resources Information Center

    Duvall, James G., III

    1992-01-01

    A science teacher describes his experience at a workshop to learn to teach the Cold Spring Harbor DNA Science Laboratory Protocols. These protocols lead students through processes for taking E. coli cells and transforming them into a new antibiotic resistant strain. The workshop featured discussions of the role of DNA recombinant technology in…

  12. Modeling DNA Replication Intermediates

    SciTech Connect

    Broyde, S.; Roy, D.; Shapiro, R.

    1997-06-01

    While there is now available a great deal of information on double stranded DNA from X-ray crystallography, high resolution NMR and computer modeling, very little is known about structures that are representative of the DNA core of replication intermediates. DNA replication occurs at a single strand/double strand junction and bulged out intermediates near the junction can lead to frameshift mutations. The single stranded domains are particularly challenging. Our interest is focused on strategies for modeling the DNA of these types of replication intermediates. Modeling such structures presents special problems in addressing the multiple minimum problem and in treating the electrostatic component of the force field. We are testing a number of search strategies for locating low energy structures of these types and we are also investigating two different distance dependent dielectric functions in the coulombic term of the force field. We are studying both unmodified DNA and DNA damaged by aromatic amines, carcinogens present in the environment in tobacco smoke, barbecued meats and automobile exhaust. The nature of the structure adopted by the carcinogen modified DNA at the replication fork plays a key role in determining whether the carcinogen will cause a mutation during replication that can initiate the carcinogenic process. In the present work results are presented for unmodified DNA.

  13. Multiscale simulation of DNA.

    PubMed

    Dans, Pablo D; Walther, Jürgen; Gómez, Hansel; Orozco, Modesto

    2016-04-01

    DNA is not only among the most important molecules in life, but a meeting point for biology, physics and chemistry, being studied by numerous techniques. Theoretical methods can help in gaining a detailed understanding of DNA structure and function, but their practical use is hampered by the multiscale nature of this molecule. In this regard, the study of DNA covers a broad range of different topics, from sub-Angstrom details of the electronic distributions of nucleobases, to the mechanical properties of millimeter-long chromatin fibers. Some of the biological processes involving DNA occur in femtoseconds, while others require years. In this review, we describe the most recent theoretical methods that have been considered to study DNA, from the electron to the chromosome, enriching our knowledge on this fascinating molecule. Copyright © 2015 Elsevier Ltd. All rights reserved.

  14. DNA replication in thermophiles.

    PubMed

    Majerník, A I; Jenkinson, E R; Chong, J P J

    2004-04-01

    DNA replication enzymes in the thermophilic Archaea have previously attracted attention due to their obvious use in methods such as PCR. The proofreading ability of the Pyrococcus furiosus DNA polymerase has resulted in a commercially successful product (Pfu polymerase). One of the many notable features of the Archaea is the fact that their DNA processing enzymes appear on the whole to be more like those found in eukaryotes than bacteria. These proteins also appear to be simpler versions of those found in eukaryotes. For these reasons, archaeal organisms make potentially interesting model systems to explore the molecular mechanisms of processes such as DNA replication, repair and recombination. Why archaeal DNA-manipulation systems were adopted over bacterial systems by eukaryotic cells remains a most interesting question that we suggest may be linked to thermophily.

  15. Fidelity of DNA polymerases in DNA amplification

    SciTech Connect

    Keohavong, P.; Thilly, W.G. )

    1989-12-01

    Denaturing gradient gel electrophoresis (DGGE) was used to separate and isolate the products of DNA amplification by polymerase chain reaction (PCR). The strategy permitted direct enumeration and identification of point mutations created by T4, modified T7, Klenow fragment of polymerase I, and Thermus aquaticus (Tag) DNA polymerases. Incorrectly synthesized sequences were separated from the wild type by DGGE as mutant/wild-type heteroduplexes and the heteroduplex fraction was used to calculate the average error rate (mutations per base duplication). The error rate induced in the 104-base-pair low-temperature melting domain of exon 3 of the human hypoxanthine/guanine phosphoribosyltransferase (HPRT) gene was {approx} 3.4 {times} 10{sup {minus}5} for modified T7, 1.3 {times} 10{sup {minus}4} for Klenow fragment, and 2.1 {times} 10{sup {minus}4} for Taq polymerases after a 10{sup 6}-fold amplification. The error rate for T4 DNA polymerase was not more than 3 {times} 10{sup {minus}6} error per base duplication. The predominant mutations were sequenced and found to be transitions of G{center dot}C to A{center dot}T for T4 and modified T7 DNA polymerases, and A{center dot}T to G{center dot}C for Taq polymerase. Klenow fragment induced both possible transitions and deletions of 2 and 4 base pairs.

  16. DNA supercoiling and its role in DNA decatenation and unknotting

    PubMed Central

    Witz, Guillaume; Stasiak, Andrzej

    2010-01-01

    Chromosomal and plasmid DNA molecules in bacterial cells are maintained under torsional tension and are therefore supercoiled. With the exception of extreme thermophiles, supercoiling has a negative sign, which means that the torsional tension diminishes the DNA helicity and facilitates strand separation. In consequence, negative supercoiling aids such processes as DNA replication or transcription that require global- or local-strand separation. In extreme thermophiles, DNA is positively supercoiled which protects it from thermal denaturation. While the role of DNA supercoiling connected to the control of DNA stability, is thoroughly researched and subject of many reviews, a less known role of DNA supercoiling emerges and consists of aiding DNA topoisomerases in DNA decatenation and unknotting. Although DNA catenanes are natural intermediates in the process of DNA replication of circular DNA molecules, it is necessary that they become very efficiently decatenated, as otherwise the segregation of freshly replicated DNA molecules would be blocked. DNA knots arise as by-products of topoisomerase-mediated intramolecular passages that are needed to facilitate general DNA metabolism, including DNA replication, transcription or recombination. The formed knots are, however, very harmful for cells if not removed efficiently. Here, we overview the role of DNA supercoiling in DNA unknotting and decatenation. PMID:20026582

  17. Simple & Safe Genomic DNA Isolation.

    ERIC Educational Resources Information Center

    Moss, Robert; Solomon, Sondra

    1991-01-01

    A procedure for purifying DNA using either bacteria or rat liver is presented. Directions for doing a qualitative DNA assay using diphenylamine and a quantitative DNA assay using spectroscopy are included. (KR)

  18. Simple & Safe Genomic DNA Isolation.

    ERIC Educational Resources Information Center

    Moss, Robert; Solomon, Sondra

    1991-01-01

    A procedure for purifying DNA using either bacteria or rat liver is presented. Directions for doing a qualitative DNA assay using diphenylamine and a quantitative DNA assay using spectroscopy are included. (KR)

  19. Studying DNA in the Classroom.

    ERIC Educational Resources Information Center

    Zarins, Silja

    1993-01-01

    Outlines a workshop for teachers that illustrates a method of extracting DNA and provides instructions on how to do some simple work with DNA without sophisticated and expensive equipment. Provides details on viscosity studies and breaking DNA molecules. (DDR)

  20. Studying DNA in the Classroom.

    ERIC Educational Resources Information Center

    Zarins, Silja

    1993-01-01

    Outlines a workshop for teachers that illustrates a method of extracting DNA and provides instructions on how to do some simple work with DNA without sophisticated and expensive equipment. Provides details on viscosity studies and breaking DNA molecules. (DDR)

  1. DNA Based Molecular Scale Nanofabrication

    DTIC Science & Technology

    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...other nanoscale templates, (b) Studied the thermal and chemical stability of DNA origami template. The result shows that the DNA nanostructures can be...potentially used in very harsh chemical environments, (c) Studied the effect of DNA origami template on the growth of self-assembled monolayer (SAM

  2. Quantitive DNA Fiber Mapping

    SciTech Connect

    Lu, Chun-Mei; Wang, Mei; Greulich-Bode, Karin M.; Weier, Jingly F.; Weier, Heinz-Ulli G.

    2008-01-28

    Several hybridization-based methods used to delineate single copy or repeated DNA sequences in larger genomic intervals take advantage of the increased resolution and sensitivity of free chromatin, i.e., chromatin released from interphase cell nuclei. Quantitative DNA fiber mapping (QDFM) differs from the majority of these methods in that it applies FISH to purified, clonal DNA molecules which have been bound with at least one end to a solid substrate. The DNA molecules are then stretched by the action of a receding meniscus at the water-air interface resulting in DNA molecules stretched homogeneously to about 2.3 kb/{micro}m. When non-isotopically, multicolor-labeled probes are hybridized to these stretched DNA fibers, their respective binding sites are visualized in the fluorescence microscope, their relative distance can be measured and converted into kilobase pairs (kb). The QDFM technique has found useful applications ranging from the detection and delineation of deletions or overlap between linked clones to the construction of high-resolution physical maps to studies of stalled DNA replication and transcription.

  3. DNA UVB dosimeters.

    PubMed

    Regan, J D; Yoshida, H

    1995-11-01

    DNA can be used to establish and monitor solar UVB dose. Since the principal molecular site of UVB damage in living organisms is DNA, it is logical to quantitate biologically effective solar UVB in DNA dosimeters. In addition to their particular sensitivity to UVB, DNA dosimeters have the advantage of a 2 pi geometry for collecting diffuse UVB radiation from all vectors, low cost, small size and portability, and no moving parts. Both molecular (cyclobutane pyrimidine dimers) and biological (bacteriophage plaques) dosimeters can be quantitated as endpoints to yield the total dose. DNA dosimeters integrate the absorbed energy of all UVB wavelengths (290-320 nm), are highly sensitive to the differential biological effectiveness of these wavelengths, and also integrate over time in hours, days or weeks of exposure. Our experiments have focused on the demonstration of DNA solar dosimeters in the ocean at various depths, the application of the dosimeters to the terrestrial monitoring of solar UVB under various conditions, and the development of a mini-dosimeter which uses nanograms of DNA and is assayed by polymerase chain reaction.

  4. DNA vaccines against tuberculosis.

    PubMed

    Bruffaerts, Nicolas; Huygen, Kris; Romano, Marta

    2014-12-01

    Tuberculosis (TB) remains a major health problem and novel vaccination regimens are urgently needed. DNA vaccines against TB have been tested in various preclinical models and strategies have been developed to increase their immunogenicity in large animal species. DNA vaccines are able to induce a wide variety of immune responses, including CD8(+) T-cell-mediated cytolytic and IFN-γ responses. DNA vaccination may be valuable in heterologous prime-boost strategies with the currently used bacillus Calmette-Guérin (BCG) vaccine. This approach could broaden the antigenic repertoire of BCG and enhance its weak induction of MHC class I-restricted immune responses. DNA vaccines offer a number of advantages over certain other types of vaccines, such as the induction of robust MHC class I-restricted cytotoxic T lymphocyte (CTL), their generic manufacturing platform and their relatively low manufacturing costs. Because of their strong potential for inducing memory responses, DNA vaccines are particularly suited for priming immune responses. Furthermore, DNA vaccine technology may help antigen discovery by facilitating screening of candidate vaccines. Co-administration of BCG with plasmid DNA coding for immunodominant, subdominant and phase-specific antigens, poorly expressed by BCG, may lead to the development of improved TB vaccines.

  5. DNA Import into Mitochondria.

    PubMed

    Konstantinov, Yu M; Dietrich, A; Weber-Lotfi, F; Ibrahim, N; Klimenko, E S; Tarasenko, V I; Bolotova, T A; Koulintchenko, M V

    2016-10-01

    In recent decades, it has become evident that the condition for normal functioning of mitochondria in higher eukaryotes is the presence of membrane transport systems of macromolecules (proteins and nucleic acids). Natural competence of the mitochondria in plants, animals, and yeasts to actively uptake DNA may be directly related to horizontal gene transfer into these organelles occurring at much higher rate compared to the nuclear and chloroplast genomes. However, in contrast with import of proteins and tRNAs, little is known about the biological role and molecular mechanism underlying import of DNA into eukaryotic mitochondria. In this review, we discuss current state of investigations in this area, particularly specificity of DNA import into mitochondria and its features in plants, animals, and yeasts; a tentative mechanism of DNA import across the mitochondrial outer and inner membranes; experimental data evidencing several existing, but not yet fully understood mechanisms of DNA transfer into mitochondria. Currently available data regarding transport of informational macromolecules (DNA, RNA, and proteins) into the mitochondria do not rule out that the mechanism of protein and tRNA import as well as tRNA and DNA import into the mitochondria may partially overlap.

  6. DNA-PK assay

    DOEpatents

    Anderson, Carl W.; Connelly, Margery A.

    2004-10-12

    The present invention provides a method for detecting DNA-activated protein kinase (DNA-PK) activity in a biological sample. The method includes contacting a biological sample with a detectably-labeled phosphate donor and a synthetic peptide substrate defined by the following features to provide specific recognition and phosphorylation by DNA-PK: (1) a phosphate-accepting amino acid pair which may include serine-glutamine (Ser-Gln) (SQ), threonine-glutamine (Thr-Gln) (TQ), glutamine-serine (Gln-Ser) (QS), or glutamine-threonine (Gln-Thr) (QT); (2) enhancer amino acids which may include glutamic acid or glutamine immediately adjacent at the amino- or carboxyl- side of the amino acid pair and forming an amino acid pair-enhancer unit; (3) a first spacer sequence at the amino terminus of the amino acid pair-enhancer unit; (4) a second spacer sequence at the carboxyl terminus of the amino acid pair-enhancer unit, which spacer sequences may include any combination of amino acids that does not provide a phosphorylation site consensus sequence motif; and, (5) a tag moiety, which may be an amino acid sequence or another chemical entity that permits separating the synthetic peptide from the phosphate donor. A compostion and a kit for the detection of DNA-PK activity are also provided. Methods for detecting DNA, protein phosphatases and substances that alter the activity of DNA-PK are also provided. The present invention also provides a method of monitoring protein kinase and DNA-PK activity in living cells. -A composition and a kit for monitoring protein kinase activity in vitro and a composition and a kit for monitoring DNA-PK activities in living cells are also provided. A method for identifying agents that alter protein kinase activity in vitro and a method for identifying agents that alter DNA-PK activity in living cells are also provided.

  7. Chimeric proteins for detection and quantitation of DNA mutations, DNA sequence variations, DNA damage and DNA mismatches

    DOEpatents

    McCutchen-Maloney, Sandra L.

    2002-01-01

    Chimeric proteins having both DNA mutation binding activity and nuclease activity are synthesized by recombinant technology. The proteins are of the general formula A-L-B and B-L-A where A is a peptide having DNA mutation binding activity, L is a linker and B is a peptide having nuclease activity. The chimeric proteins are useful for detection and identification of DNA sequence variations including DNA mutations (including DNA damage and mismatches) by binding to the DNA mutation and cutting the DNA once the DNA mutation is detected.

  8. Biology of DNA restriction.

    PubMed Central

    Bickle, T A; Krüger, D H

    1993-01-01

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

  9. Making DNA add.

    PubMed

    Guarnieri, F; Fliss, M; Bancroft, C

    1996-07-12

    Recent studies have demonstrated the feasibility of using DNA-based experiments to compute solutions to combinatorial problems. However, a prerequisite for designing a computer useful in a wide range of applications is the ability to perform mathematical calculations. The development of a DNA-based algorithm for addition is presented. The DNA representation of two nonnegative binary numbers is presented in a form permitting a chain of primer extension reactions to carry out the addition operation. To demonstrate the feasibility of this algorithm, a simple example was executed biochemically.

  10. Plasmid DNA manufacturing technology.

    PubMed

    Carnes, Aaron E; Williams, James A

    2007-01-01

    Today, plasmid DNA is becoming increasingly important as the next generation of biotechnology products (gene medicines and DNA vaccines) make their way into clinical trials, and eventually into the pharmaceutical marketplace. This review summarizes recent patents and patent applications relating to plasmid manufacturing, in the context of a comprehensive description of the plasmid manufacturing intellectual property landscape. Strategies for plasmid manufacturers to develop or in-license key plasmid manufacturing technologies are described with the endpoint of efficiently producing kg quantities of plasmid DNA of a quality that meets anticipated European and FDA quality specifications for commercial plasmid products.

  11. Focus: DNA probes

    SciTech Connect

    Not Available

    1986-11-01

    Progress in the development of DNA probes for the identification and quantitation of specific genetic sequences in biological samples is reviewed. Current research efforts in the development of DNA probes for the diagnosis of a wide variety of bacterial, viral, and other infectious diseases, such as herpes simplex and cytomegalovirus, and inherited genetic diseases such as cystic fibrosis and sickle cell anemia are discussed. Progress in development of DNA probe assays for cancer diagnosis, detection of Salmonella food poisoning, tissue typing (detection of histocompatibility antigens), mutagen screening, and animal diseases, among other applications is included.

  12. Development of Phosphorothioate DNA and DNA Thioaptamers.

    PubMed

    Volk, David E; Lokesh, Ganesh L R

    2017-07-13

    Nucleic acid aptamers are short RNA- or DNA-based affinity reagents typically selected from combinatorial libraries to bind to a specific target such as a protein, a small molecule, whole cells or even animals. Aptamers have utility in the development of diagnostic, imaging and therapeutic applications due to their size, physico-chemical nature and ease of synthesis and modification to suit the application. A variety of oligonucleotide modifications have been used to enhance the stability of aptamers from nuclease degradation in vivo. The non-bridging oxygen atoms of the phosphodiester backbones of RNA and DNA aptamers can be substituted with one or two sulfur atoms, resulting in thioaptamers with phosphorothioate or phosphorodithioate linkages, respectively. Such thioaptamers are known to have increased binding affinity towards their target, as well as enhanced resistance to nuclease degradation. In this review, we discuss the development of phosphorothioate chemistry and thioaptamers, with a brief review of selection methods.

  13. DNA-templated nanofabrication.

    PubMed

    Becerril, Héctor A; Woolley, Adam T

    2009-02-01

    Nanofabrication, or the organizational control over matter at the nanometre scale, is an intriguing scientific challenge requiring multidisciplinary tools for its solution. DNA is a biomolecule that can be combined with other nanometre-scale entities through chemical self-assembly to form a broad variety of nanomaterials. In this tutorial review we present the principles that allow DNA to interact with other chemical species, and describe the challenges and potential applications of DNA as a template for making both biological and inorganic features with nanometre resolution. As such, this report should be of interest to chemists, surface and materials scientists, biologists, and nanotechnologists, as well as others who seek to use DNA in nanofabrication.

  14. Retroviral DNA Integration

    PubMed Central

    2016-01-01

    The integration of a DNA copy of the viral RNA genome into host chromatin is the defining step of retroviral replication. This enzymatic process is catalyzed by the virus-encoded integrase protein, which is conserved among retroviruses and LTR-retrotransposons. Retroviral integration proceeds via two integrase activities: 3′-processing of the viral DNA ends, followed by the strand transfer of the processed ends into host cell chromosomal DNA. Herein we review the molecular mechanism of retroviral DNA integration, with an emphasis on reaction chemistries and architectures of the nucleoprotein complexes involved. We additionally discuss the latest advances on anti-integrase drug development for the treatment of AIDS and the utility of integrating retroviral vectors in gene therapy applications. PMID:27198982

  15. FBI's DNA analysis program

    NASA Astrophysics Data System (ADS)

    Brown, John R.

    1994-03-01

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

  16. Making DNA Fingerprints.

    ERIC Educational Resources Information Center

    Nunley, Kathie F.

    1996-01-01

    Presents an activity to simulate electrophoresis using everyday items. Uses adding machine paper to construct a set of DNA fingerprints that can be used to solve crime cases designed by students in any biology class. (JRH)

  17. Multiplex analysis of DNA

    DOEpatents

    Church, George M.; Kieffer-Higgins, Stephen

    1992-01-01

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

  18. Close encounters with DNA

    PubMed Central

    Maffeo, C.; Yoo, J.; Comer, J.; Wells, D. B.; Luan, B.; Aksimentiev, A.

    2014-01-01

    Over the past ten years, the all-atom molecular dynamics method has grown in the scale of both systems and processes amenable to it and in its ability to make quantitative predictions about the behavior of experimental systems. The field of computational DNA research is no exception, witnessing a dramatic increase in the size of systems simulated with atomic resolution, the duration of individual simulations and the realism of the simulation outcomes. In this topical review, we describe the hallmark physical properties of DNA from the perspective of all-atom simulations. We demonstrate the amazing ability of such simulations to reveal the microscopic physical origins of experimentally observed phenomena and we review the frustrating limitations associated with imperfections of present atomic force fields and inadequate sampling. The review is focused on the following four physical properties of DNA: effective electric charge, response to an external mechanical force, interaction with other DNA molecules and behavior in an external electric field. PMID:25238560

  19. Elastic Shapes of DNA

    NASA Astrophysics Data System (ADS)

    Fain, Boris; Rudnick, Joseph

    1997-03-01

    Short segments of DNA assume shapes that minimize their elastic energy. Modeling of the various mechanisms involving the molecule - replication, transcription, packaging, etc. - requires a description of the conformations of DNA under constraints. We develop a formalism that obtains analytic expressions for shape, link, twist and extension of a segment subject to sufficient number of constraints. We apply our technique to two particular cases: a) Stretched twisted linear DNA. This is an interesting test for our formalism, especially in light of recent experiments(Strick T.R., Allemand J.-F., Bensimon A., Croquette V. Science) 271 1835-1837, (1996). The molecule remains extended until a critical twist is reached, at which point it undergoes a plectonemic transition. b) Closed circular DNA. Describing the shapes of such molecules has been an outstanding problem for some time. We obtain a family of curves classified by their deviation in link from the plain circle.

  20. Making DNA Fingerprints.

    ERIC Educational Resources Information Center

    Nunley, Kathie F.

    1996-01-01

    Presents an activity to simulate electrophoresis using everyday items. Uses adding machine paper to construct a set of DNA fingerprints that can be used to solve crime cases designed by students in any biology class. (JRH)

  1. DNA complexes: Durable binders

    NASA Astrophysics Data System (ADS)

    Urbach, Adam R.

    2011-11-01

    A tetra-intercalator compound that threads through a DNA double-helix to form a remarkably stable complex exhibits an unusual combination of sequence specificity and rapid association yet slow dissociation.

  2. DNA reviews: hair.

    PubMed

    Graham, E A M

    2007-06-01

    Human and nonhuman hairs are often recovered during forensic investigation. As with all other biological samples that may be collected, via DNA analysis, hairs have the potential to provide the investigating authority with valuable intelligence pertaining to the identity of offenders, victims and even pets. DNA analysis of hairs is not however a straightforward process. The biochemical make up of hairs provide the DNA analyst with a unique set of challenges that must be overcome before any useful information can be gleaned from the evidence. This short review provides an overview of the location and condition of DNA within hair samples, and discusses the analytical methods that are employed to maximise the information that can be obtained from this sample type.

  3. Automated DNA profile analysis.

    PubMed

    Graham, Eleanor A M

    2005-12-01

    DNA profile analysis is not a simple process. Stringent demands are placed on the accuracy and consistency of forensic evidence so that complex, robust, and reproducible guidelines are necessary to assist the analyst and ensure mistakes are eliminated before a final profile is reported. The guidelines used for forensic DNA profile interpretation are formulated by investigation and statistical evaluation of all aspects of the analytical procedure. All the resulting rules, formulas, and thresholds are perfectly suited to programming of "expert systems"-software programs that imitate the human expert in decision-based processes to formulate a conclusion. Expert systems in forensic DNA analysis will contribute greatly to this field by increasing analytical throughput. The net result of this will be an increase in the human resources available for the research and development of improved methodologies, to ensure that forensic DNA profiling continues to advance at its current impressive rate.

  4. DNA Vaccination in Chickens.

    PubMed

    Gupta, Shishir Kumar; Dey, Sohini; Chellappa, Madhan Mohan

    2016-01-01

    Robust and sustainable development of poultry industry requires prevention of deadly infectious diseases. Vigorous vaccination of the birds is a routine practice; however, the live and inactivated vaccines that are used have inherent disadvantages. New-generation vaccines such as DNA vaccines offer several advantages over conventional vaccines. DNA vaccines, which encode an antigen of interest or multiple antigens in the target host, are stable, easy to produce and administer, do not require cold chain maintenance, and are not affected by the maternal antibodies. In addition, DNA vaccines can also be administered in ovo, and thus, mass vaccination and early induction of immune response can effectively be achieved. In this chapter, we focus on the development of DNA vaccines against important infectious viral as well as parasitic diseases of poultry.

  5. DNA sequencing conference, 2

    SciTech Connect

    Cook-Deegan, R.M.; Venter, J.C.; Gilbert, W.; Mulligan, J.; Mansfield, B.K.

    1991-06-19

    This conference focused on DNA sequencing, genetic linkage mapping, physical mapping, informatics and bioethics. Several were used to study this sequencing and mapping. This article also discusses computer hardware and software aiding in the mapping of genes.

  6. DNA damage and carcinogenesis

    SciTech Connect

    Stelow, R B

    1980-01-01

    Although cancer may arise as a result of many different types of molecular changes, there is little reason to doubt that changes to DNA are one of the more important ones in cancer initiation. Although DNA repair mechanisms seem able to eliminate a very large fraction of deleterious changes to DNA, we not only have little insight into the molecular mechanisms involved in such repair, but have a negligible amount of information to permit us to estimate the shape of dose response relations at low doses. The case of skin cancer is a special one, in that the average population is exposed to sufficient solar uv so that the effects of small increments in uv dose may be estimated. An approximate 85% reduction in DNA repair increases skin cancer incidence 10/sup 4/ fold.

  7. Interaction of DNA and DNA-anti-DNA complexes to fibronectin

    SciTech Connect

    Gupta, R.C.; Simpson, W.A.; Raghow, R.; Hasty, K.

    1986-03-01

    Fibronectin (Fn) is a large multidomain glycoprotein found in the basement membrane, on cell surface and in plasma. The interactions of Fn with DNA may be significant in glomerular deposition of DNA-anti-DNA complexes in patients with systemic lupus erythematosus (SLE). The authors examined the binding of DNA and DNA-anti-DNA complexes to Fn by a solid phase assay in which Fn was coated to microtiter plates and reacted with (/sup 3/H)DNA or DNA complexes with a monoclonal anti-DNA antibody. The optimal interaction of DNA with Fn occurs at <0.1M NaCl suggesting that the binding is charge dependent; the specificity of this binding was shown by competitive inhibition and locking experiments using anti-Fn. The binding was maximum at pH 6.5 and in the absence of Ca/sup 2 +/. The addition of Clq enhanced the binding of DNA and DNA-anti-DNA complexes to Fn, whereas heparan sulfate inhibited such binding. The monomeric or aggregated IgC did not bind Fn but aggregated IgG bound to Fn in the presence of Clq. Furthermore, DNA-anti-DNA complexes in sera from active SLE patients bound Fn which was enhanced in the presence of Clq; DNase abolished this binding indicating that the interaction of these complexes was mediated by DNA. These observations may partially explain the molecular mechanism(s) of the deposition of DNA-anti-DNA complexes in basement membrane.

  8. Patterning nanocrystals using DNA

    SciTech Connect

    Williams, Shara Carol

    2003-01-01

    One of the goals of nanotechnology is to enable programmed self-assembly of patterns made of various materials with nanometer-sized control. This dissertation describes the results of experiments templating arrangements of gold and semiconductor nanocrystals using 2'-deoxyribonucleic acid (DNA). Previously, simple DNA-templated linear arrangements of two and three nanocrystals structures have been made.[1] Here, we have sought to assemble larger and more complex nanostructures. Gold-DNA conjugates with 50 to 100 bases self-assembled into planned arrangements using strands of DNA containing complementary base sequences. We used two methods to increase the complexity of the arrangements: using branched synthetic doublers within the DNA covalent backbone to create discrete nanocrystal groupings, and incorporating the nanocrystals into a previously developed DNA lattice structure [2][3] that self-assembles from tiles made of DNA double-crossover molecules to create ordered nanoparticle arrays. In the first project, the introduction of a covalently-branched synthetic doubler reagent into the backbone of DNA strands created a branched DNA ''trimer.'' This DNA trimer templated various structures that contained groupings of three and four gold nanoparticles, giving promising, but inconclusive transmission electron microscopy (TEM) results. Due to the presence of a variety of possible structures in the reaction mixtures, and due to the difficulty of isolating the desired structures, the TEM and gel electrophoresis results for larger structures having four particles, and for structures containing both 5 and 10 nm gold nanoparticles were inconclusive. Better results may come from using optical detection methods, or from improved sample preparation. In the second project, we worked toward making two-dimensional ordered arrays of nanocrystals. We replicated and improved upon previous results for making DNA lattices, increasing the size of the lattices to a length greater than

  9. Electrochemical DNA Sensors for Detection of DNA Damage

    PubMed Central

    Diculescu, Victor Constantin; Paquim, Ana-Maria Chiorcea; Brett, Ana Maria Oliveira

    2005-01-01

    Electrochemical devices have received particular attention due to their rapid detection and great sensitivity for the evaluation of DNA-hazard compounds interaction mechanisms. Several types of bioanalytical method use nucleic acids probes to detect DNA damage. This article reviews current directions and strategies in the development and applications of electrochemical DNA sensors for the detection of DNA damage.

  10. Blowing DNA bubbles.

    PubMed

    Severin, N; Zhuang, W; Ecker, C; Kalachev, A A; Sokolov, I M; Rabe, J P

    2006-11-01

    We report here experimental observations which indicate that topologically or covalently formed polymer loops embedded in an ultrathin liquid film on a solid substrate can be "blown" into circular "bubbles" during scanning force microscopy (SFM) imaging. In particular, supercoiled vector DNA has been unraveled, moved, stretched, and overstretched to two times its B-form length and then torn apart. We attribute the blowing of the DNA bubbles to the interaction of the tapping SFM tip with the ultrathin liquid film.

  11. Das DNA-Puzzle

    NASA Astrophysics Data System (ADS)

    Kirchner, Stefan

    Im Jahre 1953 wurde von James Watson und Francis Crick erstmalig der strukturelle Aufbau der sogenannten DNA (Desoxyribonukleinsäure) beschrieben, welche das Erbgut jedes Lebewesens enthält. Der wesentliche Teil des Erbguts wird dabei durch eine sehr lange Folge der vier Basen Adenin (A), Cytosin (C), Guanin (G) und Thymin (T) codiert. Seit einigen Jahren ist es möglich, die Folge der vier Basen zu einer gegebenen DNA zu bestimmen. Biologen bezeichnen diesen Vorgang als Sequenzierung.

  12. Programming DNA tube circumferences.

    PubMed

    Yin, Peng; Hariadi, Rizal F; Sahu, Sudheer; Choi, Harry M T; Park, Sung Ha; Labean, Thomas H; Reif, John H

    2008-08-08

    Synthesizing molecular tubes with monodisperse, programmable circumferences is an important goal shared by nanotechnology, materials science, and supermolecular chemistry. We program molecular tube circumferences by specifying the complementarity relationships between modular domains in a 42-base single-stranded DNA motif. Single-step annealing results in the self-assembly of long tubes displaying monodisperse circumferences of 4, 5, 6, 7, 8, 10, or 20 DNA helices.

  13. Issues in DNA Fingerprinting

    DTIC Science & Technology

    1994-07-01

    specialists in forensic science , where one must learn to deal with degraded specimens that are not obtained from well run scientific laboratories. Some of...methods are a most powerful adjunct to forensic science for personal identification and have immense benefit to the public" and later "DNA typing is... forensic science . National Academy Press, Washington, D.C. Devlin, B., Risch, N., and Roeder, K. (1990). No excess of homozygosity at loci used for DNA

  14. DNA methylation and differentiation.

    PubMed Central

    Michalowsky, L A; Jones, P A

    1989-01-01

    The methylation of specific cytosine residues in DNA has been implicated in regulating gene expression and facilitating functional specialization of cellular phenotypes. Generally, the demethylation of certain CpG sites correlates with transcriptional activation of genes. 5-Azacytidine is an inhibitor of DNA methylation and has been widely used as a potent activator of suppressed genetic information. Treatment of cells with 5-azacytidine results in profound phenotypic alterations. The drug-induced hypomethylation of DNA apparently perturbs DNA-protein interactions that may consequently alter transcriptional activity and cell determination. The inhibitory effect of cytosine methylation may be exerted via altered DNA-protein interactions specifically or may be transduced by a change in the conformation of chromatin. Recent studies have demonstrated that cytosine methylation also plays a central role in parental imprinting, which in turn determines the differential expression of maternal and paternal genomes during embryogenesis. In other words, methylation is the mechanism whereby the embryo retains memory of the gametic origin of each component of genetic information. A memory of this type would probably persist during DNA replication and cell division as methylation patterns are stable and heritable. PMID:2466640

  15. Toward larger DNA origami.

    PubMed

    Marchi, Alexandria N; Saaem, Ishtiaq; Vogen, Briana N; Brown, Stanley; LaBean, Thomas H

    2014-10-08

    Structural DNA nanotechnology, and specifically scaffolded DNA origami, is rapidly developing as a versatile method for bottom-up fabrication of novel nanometer-scale materials and devices. However, lengths of conventional single-stranded scaffolds, for example, 7,249-nucleotide circular genomic DNA from the M13mp18 phage, limit the scales of these uniquely addressable structures. Additionally, increasing DNA origami size generates the cost burden of increased staple-strand synthesis. We addressed this 2-fold problem by developing the following methods: (1) production of the largest to-date biologically derived single-stranded scaffold using a λ/M13 hybrid virus to produce a 51 466-nucleotide DNA in a circular, single-stranded form and (2) inexpensive DNA synthesis via an inkjet-printing process on a chip embossed with functionalized micropillars made from cyclic olefin copolymer. We have experimentally demonstrated very efficient assembly of a 51-kilobasepair origami from the λ/M13 hybrid scaffold folded by chip-derived staple strands. In addition, we have demonstrated two-dimensional, asymmetric origami sheets with controlled global curvature such that they land on a substrate in predictable orientations that have been verified by atomic force microscopy.

  16. Variations in brain DNA

    PubMed Central

    Avila, Jesús; Gómez-Ramos, Alberto; Soriano, Eduardo

    2014-01-01

    It is assumed that DNA sequences are conserved in the diverse cell types present in a multicellular organism like the human being. Thus, in order to compare the sequences in the genome of DNA from different individuals, nucleic acid is commonly isolated from a single tissue. In this regard, blood cells are widely used for this purpose because of their availability. Thus blood DNA has been used to study genetic familiar diseases that affect other tissues and organs, such as the liver, heart, and brain. While this approach is valid for the identification of familial diseases in which mutations are present in parental germinal cells and, therefore, in all the cells of a given organism, it is not suitable to identify sporadic diseases in which mutations might occur in specific somatic cells. This review addresses somatic DNA variations in different tissues or cells (mainly in the brain) of single individuals and discusses whether the dogma of DNA invariance between cell types is indeed correct. We will also discuss how single nucleotide somatic variations arise, focusing on the presence of specific DNA mutations in the brain. PMID:25505410

  17. Transcription of mitochondrial DNA.

    PubMed

    Tabak, H F; Grivell, L A; Borst, P

    1983-01-01

    While mitochondrial DNA (mtDNA) is the simplest DNA in nature, coding for rRNAs and tRNAs, results of DNA sequence, and transcript analysis have demonstrated that both the synthesis and processing of mitochondrial RNAs involve remarkably intricate events. At one extreme, genes in animal mtDNAs are tightly packed, both DNA strands are completely transcribed (symmetric transcription), and the appearance of specific mRNAs is entirely dependent on processing at sites signalled by the sequences of the tRNAs, which abut virtually every gene. At the other extreme, gene organization in yeast (Saccharomyces) is anything but compact, with long stretches of AT-rich DNA interspaced between coding sequences and no obvious logic to the order of genes. Transcription is asymmetric and several RNAs are initiated de novo. Nevertheless, extensive RNA processing occurs due largely to the presence of split genes. RNA splicing is complex, is controlled by both mitochondrial and nuclear genes, and in some cases is accompanied by the formation of RNAs that behave as covalently closed circles. The present article reviews current knowledge of mitochondrial transcription and RNA processing in relation to possible mechanisms for the regulation of mitochondrial gene expression.

  18. DNA biosensors that reason.

    PubMed

    Sainz de Murieta, Iñaki; Rodríguez-Patón, Alfonso

    2012-08-01

    Despite the many designs of devices operating with the DNA strand displacement, surprisingly none is explicitly devoted to the implementation of logical deductions. The present article introduces a new model of biosensor device that uses nucleic acid strands to encode simple rules such as "IF DNA_strand(1) is present THEN disease(A)" or "IF DNA_strand(1) AND DNA_strand(2) are present THEN disease(B)". Taking advantage of the strand displacement operation, our model makes these simple rules interact with input signals (either DNA or any type of RNA) to generate an output signal (in the form of nucleotide strands). This output signal represents a diagnosis, which either can be measured using FRET techniques, cascaded as the input of another logical deduction with different rules, or even be a drug that is administered in response to a set of symptoms. The encoding introduces an implicit error cancellation mechanism, which increases the system scalability enabling longer inference cascades with a bounded and controllable signal-noise relation. It also allows the same rule to be used in forward inference or backward inference, providing the option of validly outputting negated propositions (e.g. "diagnosis A excluded"). The models presented in this paper can be used to implement smart logical DNA devices that perform genetic diagnosis in vitro.

  19. Ribonucleotide triggered DNA damage and RNA-DNA damage responses

    PubMed Central

    Wallace, Bret D; Williams, R Scott

    2014-01-01

    Research indicates that the transient contamination of DNA with ribonucleotides exceeds all other known types of DNA damage combined. The consequences of ribose incorporation into DNA, and the identity of protein factors operating in this RNA-DNA realm to protect genomic integrity from RNA-triggered events are emerging. Left unrepaired, the presence of ribonucleotides in genomic DNA impacts cellular proliferation and is associated with chromosome instability, gross chromosomal rearrangements, mutagenesis, and production of previously unrecognized forms of ribonucleotide-triggered DNA damage. Here, we highlight recent findings on the nature and structure of DNA damage arising from ribonucleotides in DNA, and the identification of cellular factors acting in an RNA-DNA damage response (RDDR) to counter RNA-triggered DNA damage. PMID:25692233

  20. Ribonucleotide triggered DNA damage and RNA-DNA damage responses.

    PubMed

    Wallace, Bret D; Williams, R Scott

    2014-01-01

    Research indicates that the transient contamination of DNA with ribonucleotides exceeds all other known types of DNA damage combined. The consequences of ribose incorporation into DNA, and the identity of protein factors operating in this RNA-DNA realm to protect genomic integrity from RNA-triggered events are emerging. Left unrepaired, the presence of ribonucleotides in genomic DNA impacts cellular proliferation and is associated with chromosome instability, gross chromosomal rearrangements, mutagenesis, and production of previously unrecognized forms of ribonucleotide-triggered DNA damage. Here, we highlight recent findings on the nature and structure of DNA damage arising from ribonucleotides in DNA, and the identification of cellular factors acting in an RNA-DNA damage response (RDDR) to counter RNA-triggered DNA damage.

  1. DNA DAMAGE BINDING PROTEIN2 Shapes the DNA Methylation Landscape

    PubMed Central

    Schalk, Catherine; Kramdi, Amira; Ahmed, Ikhlak; Cognat, Valérie; Graindorge, Stéfanie; Bergdoll, Marc; Baumberger, Nicolas; Heintz, Dimitri; Bowler, Chris; Genschik, Pascal; Barneche, Fredy; Molinier, Jean

    2016-01-01

    In eukaryotes, DNA repair pathways help to maintain genome integrity and epigenomic patterns. However, the factors at the nexus of DNA repair and chromatin modification/remodeling remain poorly characterized. Here, we uncover a previously unrecognized interplay between the DNA repair factor DNA DAMAGE BINDING PROTEIN2 (DDB2) and the DNA methylation machinery in Arabidopsis thaliana. Loss-of-function mutation in DDB2 leads to genome-wide DNA methylation alterations. Genetic and biochemical evidence indicate that at many repeat loci, DDB2 influences de novo DNA methylation by interacting with ARGONAUTE4 and by controlling the local abundance of 24-nucleotide short interfering RNAs (siRNAs). We also show that DDB2 regulates active DNA demethylation mediated by REPRESSOR OF SILENCING1 and DEMETER LIKE3. Together, these findings reveal a role for the DNA repair factor DDB2 in shaping the Arabidopsis DNA methylation landscape in the absence of applied genotoxic stress. PMID:27531226

  2. Supramolecular Complexes of DNA

    NASA Astrophysics Data System (ADS)

    Zuber, G.; Scherman, D.

    Deoxyribose nucleic acid or DNA is a linear polymer in the form of a double strand, synthesised by sequential polymerisation of a large number of units chosen from among the nucleic bases called purines (adenosine A and guanosine G) and pyrimidines (cytosine C and thymidine T). DNA contains all the genetic information required for life. It exists in the form of a limited number (a few dozen) of very big molecules, called chromosomes. This genetic information is first of all transcribed. In this process, a restricted fragment of the DNA called a gene is copied in the form of ribonucleic acid, or RNA. This RNA is itself a polymer, but with a single strand in which the sequence of nucleic acids is schematically analogous to the sequence on one of the two strands of the transcribed DNA. Finally, this RNA is translated into a protein, yet another linear polymer. The proteins make up the main part of the active constituents ensuring the survival of the cell. Any loss of information, either by mutation or by deletion of the DNA, will cause an imbalance in the cell's metabolism that may in turn lead to incurable pathologies. Several strategies have been developed to reduce the consequences of such genetic deficiencies or, more generally, to act, by amplifying or suppressing them, on the mechanisms leading from the reading of the genetic information to the production of proteins: Strategies aiming to introduce synthetic DNA or RNA, which selectively block the expression of certain genes, are now being studied by an increasing number of research scientists and pharmacologists. They use antisense oligodeoxyribonucleotides or interfering oligoribonucleotides and they already have clinical applications. This kind of therapy is often called gene pharmacology. Other, more ambitious strategies aim to repair in situ mutated or incomplete DNA within the chromosomes themselves, by introducing short sequences of DNA or RNA which recognise and take the place of mutations. This is the

  3. Novel DNA nanoparticles and networks.

    PubMed

    Seela, Frank; Jawalekar, Anup M; Sirivolu, Venkata R; Rosemeyer, Helmut; He, Yang; Leonard, Peter

    2005-01-01

    Joining the thrombin-binding aptamer 5-d(GGTTGGTGTGGTTGG) and the minihairpin 5-d(GCGAAGC) leads to new DNA nanoparticles, which are different from rod-like helical double-stranded DNA. Covalent interstrand cross-links in DNA duplexes generated by bifunctional alkadiyne chains were used to build-up the DNA networks.

  4. DNA as an Optical Material

    DTIC Science & Technology

    2011-07-01

    of DNA, and a natural DNA source is more appropriate. Currently available sources of natural DNA include fish sperm (e.g., from salmon or herring...harvesting of the fish’s waste milt and roe sacs. (Unlike meat and eggs , these elements are not used for cuisine.) From these, DNA is extracted and

  5. From DNA to transistors

    NASA Astrophysics Data System (ADS)

    Braun, Erez; Keren, Kinneret

    2004-06-01

    The rapid advance in molecular biology and nanotechnology opens up the possibility to explore the interface between biology and electronics at the single-molecule level. We focus on the organization of molecular electronic circuits. Interconnecting an immense number of molecular devices into a functional circuit and constructing a framework for integrated molecular electronics requires new concepts. A promising avenue relies on bottom-up assembly where the information for the circuit connectivity and functionality is embedded in the molecular building blocks. Biology can provide concepts and mechanisms for advancing this approach, but there is no straightforward way to apply them to electronics since biological molecules are essentially electrically insulating. Bridging the chasm between biology and electronics therefore presents great challenges. Circuit organization on the molecular scale is considered and contrasted with the levels of organization presented by the living world. The discussion then focuses on our proposal to harness DNA and molecular biology to construct the scaffold for integrated molecular electronics. DNA metallization is used to convert the DNA scaffold into a conductive one. We present the framework of sequence-specific molecular lithography based on the biological mechanism of homologous genetic recombination and carried out by the bacterial protein RecA. Molecular lithography enables us to use the information encoded in the scaffold DNA molecules for directing the construction of an electronic circuit. We show that it can lead all the way from DNA molecules to working transistors in a test-tube. Carbon nanotubes are incorporated as the active electronic components in the DNA-templated transistors. Our approach can, in principle, be applied to the fabrication of larger-scale electronic circuits. The realization of complex DNA-based circuits will, however, require new concepts and additional biological machinery allowing, for example

  6. Cyclization of short DNA fragments

    NASA Astrophysics Data System (ADS)

    Lam, Pui-Man; Zhen, Yi

    2017-09-01

    From the per unit length free energy for DNA under tension, we have calculated an effective contour length dependent persistence length for short DNA. This effective persistence length results from the enhanced fluctuations in short DNA. It decreases for shorter DNA, making shorter DNA more flexible. The results of the J-factor calculated using this effective persistence length are in good agreement with experimental data.

  7. Forensic DNA Profiling and Database

    PubMed Central

    Panneerchelvam, S.; Norazmi, M.N.

    2003-01-01

    The incredible power of DNA technology as an identification tool had brought a tremendous change in crimnal justice . DNA data base is an information resource for the forensic DNA typing community with details on commonly used short tandem repeat (STR) DNA markers. This article discusses the essential steps in compilation of COmbined DNA Index System (CODIS) on validated polymerase chain amplified STRs and their use in crime detection. PMID:23386793

  8. DNA vaccines: a review.

    PubMed

    Lewis, P J; Babiuk, L A

    1999-01-01

    Therapeutic and prophylactic DNA vaccine clinical trials for a variety of pathogens and cancers are underway (Chattergoon et al., 1997; Taubes, 1997). The speed with which initiation of these trials occurred is no less than astounding; clinical trials for a human immunodeficiency virus (HIV) gp160 DNA-based vaccine were underway within 36 months of the first description of "genetic immunization" (Tang et al., 1992) and within 24 months of publication of the first article describing intramuscular delivery of a DNA vaccine (Ulmer et al., 1993). Despite the relative fervor with which clinical trials have progressed, it can be safely stated that DNA-based vaccines will not be an immunological "silver bullet." In this regard, it was satisfying to see a publication entitled "DNA Vaccines--A Modern Gimmick or a Boon to Vaccinology?" (Manickan et al., 1997b). There is no doubt that this technology is well beyond the phenomenology phase of study. Research niches and models have been established and will allow the truly difficult questions of mechanism and application to target species to be studied. These two aspects of future studies are intricately interwoven and will ultimately determine the necessity for mechanistic understanding and the evolution of target species studies. The basic science of DNA vaccines has yet to be clearly defined and will ultimately determine the success or failure of this technology to find a place in the immunological arsenal against disease. In a commentary on a published study describing DNA vaccine-mediated protection against heterologous challenge with HIV-1 in chimpanzees, Ronald Kennedy (1997) states, "As someone who has been in the trenches of AIDS vaccine research for over a decade and who, together with collaborators, has attempted a number of different vaccine approaches that have not panned out, I have a relatively pessimistic view of new AIDS vaccine approaches." Kennedy then goes on to summarize a DNA-based multigene vaccine

  9. Human DNA polymerase α in binary complex with a DNA:DNA template-primer.

    PubMed

    Coloma, Javier; Johnson, Robert E; Prakash, Louise; Prakash, Satya; Aggarwal, Aneel K

    2016-04-01

    The Polα/primase complex assembles the short RNA-DNA fragments for priming of lagging and leading strand DNA replication in eukaryotes. As such, the Polα polymerase subunit encounters two types of substrates during primer synthesis: an RNA:DNA helix and a DNA:DNA helix. The engagement of the polymerase subunit with the DNA:DNA helix has been suggested as the of basis for primer termination in eukaryotes. However, there is no structural information on how the Polα polymerase subunit actually engages with a DNA:DNA helix during primer synthesis. We present here the first crystal structure of human Polα polymerase subunit in complex with a DNA:DNA helix. Unexpectedly, we find that portion of the DNA:DNA helix in contact with the polymerase is not in a B-form but in a hybrid A-B form. Almost all of the contacts observed previously with an RNA primer are preserved with a DNA primer--with the same set of polymerase residues tracking the sugar-phosphate backbone of the DNA or RNA primer. Thus, rather than loss of specific contacts, the free energy cost of distorting DNA from B- to hybrid A-B form may augur the termination of primer synthesis in eukaryotes.

  10. Facilitated diffusion with DNA coiling.

    PubMed

    Lomholt, Michael A; van den Broek, Bram; Kalisch, Svenja-Marei J; Wuite, Gijs J L; Metzler, Ralf

    2009-05-19

    When DNA-binding proteins search for their specific binding site on a DNA molecule they alternate between linear 1-dimensional diffusion along the DNA molecule, mediated by nonspecific binding, and 3-dimensional volume excursion events between successive dissociation from and rebinding to DNA. If the DNA molecule is kept in a straight configuration, for instance, by optical tweezers, these 3-dimensional excursions may be divided into long volume excursions and short hops along the DNA. These short hops correspond to immediate rebindings after dissociation such that a rebinding event to the DNA occurs at a site that is close to the site of the preceding dissociation. When the DNA molecule is allowed to coil up, immediate rebinding may also lead to so-called intersegmental jumps, i.e., immediate rebindings to a DNA segment that is far away from the unbinding site when measured in the chemical distance along the DNA, but close by in the embedding 3-dimensional space. This effect is made possible by DNA looping. The significance of intersegmental jumps was recently demonstrated in a single DNA optical tweezers setup. Here we present a theoretical approach in which we explicitly take the effect of DNA coiling into account. By including the spatial correlations of the short hops we demonstrate how the facilitated diffusion model can be extended to account for intersegmental jumping at varying DNA densities. It is also shown that our approach provides a quantitative interpretation of the experimentally measured enhancement of the target location by DNA-binding proteins.

  11. Chromatin and DNA Replication

    PubMed Central

    MacAlpine, David M.; Almouzni, Geneviève

    2013-01-01

    The size of a eukaryotic genome presents a unique challenge to the cell: package and organize the DNA to fit within the confines of the nucleus while at the same time ensuring sufficient dynamics to allow access to specific sequences and features such as genes and regulatory elements. This is achieved via the dynamic nucleoprotein organization of eukaryotic DNA into chromatin. The basic unit of chromatin, the nucleosome, comprises a core particle with 147 bp of DNA wrapped 1.7 times around an octamer of histones. The nucleosome is a highly versatile and modular structure, both in its composition, with the existence of various histone variants, and through the addition of a series of posttranslational modifications on the histones. This versatility allows for both short-term regulatory responses to external signaling, as well as the long-term and multigenerational definition of large functional chromosomal domains within the nucleus, such as the centromere. Chromatin organization and its dynamics participate in essentially all DNA-templated processes, including transcription, replication, recombination, and repair. Here we will focus mainly on nucleosomal organization and describe the pathways and mechanisms that contribute to assembly of this organization and the role of chromatin in regulating the DNA replication program. PMID:23751185

  12. Electroeluting DNA fragments.

    PubMed

    Zarzosa-Alvarez, Ana L; Sandoval-Cabrera, Antonio; Torres-Huerta, Ana L; Bermudez-Cruz, Rosa M

    2010-09-05

    Purified DNA fragments are used for different purposes in Molecular Biology and they can be prepared by several procedures. Most of them require a previous electrophoresis of the DNA fragments in order to separate the band of interest. Then, this band is excised out from an agarose or acrylamide gel and purified by using either: binding and elution from glass or silica particles, DEAE-cellulose membranes, "crush and soak method", electroelution or very often expensive commercial purification kits. Thus, selecting a method will depend mostly of what is available in the laboratory. The electroelution procedure allows one to purify very clean DNA to be used in a large number of applications (sequencing, radiolabeling, enzymatic restriction, enzymatic modification, cloning etc). This procedure consists in placing DNA band-containing agarose or acrylamide slices into sample wells of the electroeluter, then applying current will make the DNA fragment to leave the agarose and thus be trapped in a cushion salt to be recovered later by ethanol precipitation.

  13. Yeast DNA plasmids.

    PubMed

    Gunge, N

    1983-01-01

    The study of yeast DNA plasmids has been initiated with the discovery of the 2-micron DNA in Saccharomyces cerevisiae. This multiple copy plasmid, organized into chromatin structure in vivo, probably exists in the nucleus and provides a good system to obtain information on eukaryotic DNA replication. Yeast transformation with the 2-micron DNA or artificially constructed chimeric plasmids had contributed significantly to the study of the molecular biology of yeast and eukaryotes, allowing the isolation and characterization of various genes, ars, centromeres, and telomeres, and also serving as a tool to study the expression of various heterologous genes. Encouraged by these fruitful results, new yeast plasmids have been screened among phylogenetically distant yeasts. The linear DNA plasmids (pGKl1 and pGKl2) from Kluyveromyces lactis are the first case of yeast plasmids associated with biological function (killer phenotype). This plasmid system would be ideal as a model to study the structure and function of eukaryotic linear chromosomes. The extracellular secretion of protein toxin suggests the plasmids to be an excellent candidate for a secretion vector. The importance of yeasts as suitable materials for the study of eukaryotic cell biology would be much enhanced by the advent of new transformation systems with diverse host yeasts of genetically and phylogenetically distinct properties.

  14. Chromatin and DNA replication.

    PubMed

    MacAlpine, David M; Almouzni, Geneviève

    2013-08-01

    The size of a eukaryotic genome presents a unique challenge to the cell: package and organize the DNA to fit within the confines of the nucleus while at the same time ensuring sufficient dynamics to allow access to specific sequences and features such as genes and regulatory elements. This is achieved via the dynamic nucleoprotein organization of eukaryotic DNA into chromatin. The basic unit of chromatin, the nucleosome, comprises a core particle with 147 bp of DNA wrapped 1.7 times around an octamer of histones. The nucleosome is a highly versatile and modular structure, both in its composition, with the existence of various histone variants, and through the addition of a series of posttranslational modifications on the histones. This versatility allows for both short-term regulatory responses to external signaling, as well as the long-term and multigenerational definition of large functional chromosomal domains within the nucleus, such as the centromere. Chromatin organization and its dynamics participate in essentially all DNA-templated processes, including transcription, replication, recombination, and repair. Here we will focus mainly on nucleosomal organization and describe the pathways and mechanisms that contribute to assembly of this organization and the role of chromatin in regulating the DNA replication program.

  15. Programmable Quantitative DNA Nanothermometers.

    PubMed

    Gareau, David; Desrosiers, Arnaud; Vallée-Bélisle, Alexis

    2016-07-13

    Developing molecules, switches, probes or nanomaterials that are able to respond to specific temperature changes should prove of utility for several applications in nanotechnology. Here, we describe bioinspired strategies to design DNA thermoswitches with programmable linear response ranges that can provide either a precise ultrasensitive response over a desired, small temperature interval (±0.05 °C) or an extended linear response over a wide temperature range (e.g., from 25 to 90 °C). Using structural modifications or inexpensive DNA stabilizers, we show that we can tune the transition midpoints of DNA thermometers from 30 to 85 °C. Using multimeric switch architectures, we are able to create ultrasensitive thermometers that display large quantitative fluorescence gains within small temperature variation (e.g., > 700% over 10 °C). Using a combination of thermoswitches of different stabilities or a mix of stabilizers of various strengths, we can create extended thermometers that respond linearly up to 50 °C in temperature range. Here, we demonstrate the reversibility, robustness, and efficiency of these programmable DNA thermometers by monitoring temperature change inside individual wells during polymerase chain reactions. We discuss the potential applications of these programmable DNA thermoswitches in various nanotechnology fields including cell imaging, nanofluidics, nanomedecine, nanoelectronics, nanomaterial, and synthetic biology.

  16. Strandwise translocation of a DNA glycosylase on undamaged DNA

    SciTech Connect

    Qi, Yan; Nam, Kwangho; Spong, Marie C.; Banerjee, Anirban; Sung, Rou-Jia; Zhang, Michael; Karplus, Martin; Verdine, Gregory L.

    2012-05-14

    Base excision repair of genotoxic nucleobase lesions in the genome is critically dependent upon the ability of DNA glycosylases to locate rare sites of damage embedded in a vast excess of undamaged DNA, using only thermal energy to fuel the search process. Considerable interest surrounds the question of how DNA glycosylases translocate efficiently along DNA while maintaining their vigilance for target damaged sites. Here, we report the observation of strandwise translocation of 8-oxoguanine DNA glycosylase, MutM, along undamaged DNA. In these complexes, the protein is observed to translocate by one nucleotide on one strand while remaining untranslocated on the complementary strand. We further report that alterations of single base-pairs or a single amino acid substitution (R112A) can induce strandwise translocation. Molecular dynamics simulations confirm that MutM can translocate along DNA in a strandwise fashion. These observations reveal a previously unobserved mode of movement for a DNA-binding protein along the surface of DNA.

  17. Active DNA unwinding dynamics during processive DNA replication

    PubMed Central

    Morin, José A.; Cao, Francisco J.; Lázaro, José M.; Arias-Gonzalez, J. Ricardo; Valpuesta, José M.; Carrascosa, José L.; Salas, Margarita; Ibarra, Borja

    2012-01-01

    Duplication of double-stranded DNA (dsDNA) requires a fine-tuned coordination between the DNA replication and unwinding reactions. Using optical tweezers, we probed the coupling dynamics between these two activities when they are simultaneously carried out by individual Phi29 DNA polymerase molecules replicating a dsDNA hairpin. We used the wild-type and an unwinding deficient polymerase variant and found that mechanical tension applied on the DNA and the DNA sequence modulate in different ways the replication, unwinding rates, and pause kinetics of each polymerase. However, incorporation of pause kinetics in a model to quantify the unwinding reaction reveals that both polymerases destabilize the fork with the same active mechanism and offers insights into the topological strategies that could be used by the Phi29 DNA polymerase and other DNA replication systems to couple unwinding and replication reactions. PMID:22573817

  18. Active DNA unwinding dynamics during processive DNA replication.

    PubMed

    Morin, José A; Cao, Francisco J; Lázaro, José M; Arias-Gonzalez, J Ricardo; Valpuesta, José M; Carrascosa, José L; Salas, Margarita; Ibarra, Borja

    2012-05-22

    Duplication of double-stranded DNA (dsDNA) requires a fine-tuned coordination between the DNA replication and unwinding reactions. Using optical tweezers, we probed the coupling dynamics between these two activities when they are simultaneously carried out by individual Phi29 DNA polymerase molecules replicating a dsDNA hairpin. We used the wild-type and an unwinding deficient polymerase variant and found that mechanical tension applied on the DNA and the DNA sequence modulate in different ways the replication, unwinding rates, and pause kinetics of each polymerase. However, incorporation of pause kinetics in a model to quantify the unwinding reaction reveals that both polymerases destabilize the fork with the same active mechanism and offers insights into the topological strategies that could be used by the Phi29 DNA polymerase and other DNA replication systems to couple unwinding and replication reactions.

  19. DNA Origami with Double Stranded DNA as a Unified Scaffold

    PubMed Central

    Yang, Yang; Han, Dongran; Nangreave, Jeanette; Liu, Yan; Yan, Hao

    2013-01-01

    Scaffolded DNA origami is a widely used technology for self-assembling precisely structured nanoscale objects that contain a large number of addressable features. Typical scaffolds are long, single strands of DNA (ssDNA) that are folded into distinct shapes through the action of many, short ssDNA staples that are complementary to several different domains of the scaffold. However, sources of long single stranded DNA are scarce, limiting the size and complexity of structures that can be assembled. Here we demonstrated that dsDNA scaffolds can be directly used to fabricate integrated DNA origami structures that incorporate both of the constituent ssDNA molecules. Two basic principles were employed in the design of scaffold folding paths – folding path asymmetry and periodic convergence of the two ssDNA scaffold strands. Asymmetry in the folding path minimizes unwanted complementarity between staples, and incorporating an offset between the folding paths of each ssDNA scaffold strand reduces the number of times that complementary portions of the strands are brought into close proximity with one another, both of which decrease the likelihood of dsDNA scaffold recovery. Meanwhile, the folding paths of the two ssDNA scaffold strands were designed to periodically converge to promote the assembly of a single, unified structure rather than two individual ones. Our results reveal that this basic strategy can be used to reliably assemble integrated DNA nanostructures from dsDNA scaffolds. PMID:22830653

  20. Active DNA demethylation by DNA repair: Facts and uncertainties.

    PubMed

    Schuermann, David; Weber, Alain R; Schär, Primo

    2016-08-01

    Pathways that control and modulate DNA methylation patterning in mammalian cells were poorly understood for a long time, although their importance in establishing and maintaining cell type-specific gene expression was well recognized. The discovery of proteins capable of converting 5-methylcytosine (5mC) to putative substrates for DNA repair introduced a novel and exciting conceptual framework for the investigation and ultimate discovery of molecular mechanisms of DNA demethylation. Against the prevailing notion that DNA methylation is a static epigenetic mark, it turned out to be dynamic and distinct mechanisms appear to have evolved to effect global and locus-specific DNA demethylation. There is compelling evidence that DNA repair, in particular base excision repair, contributes significantly to the turnover of 5mC in cells. By actively demethylating DNA, DNA repair supports the developmental establishment as well as the maintenance of DNA methylation landscapes and gene expression patterns. Yet, while the biochemical pathways are relatively well-established and reviewed, the biological context, function and regulation of DNA repair-mediated active DNA demethylation remains uncertain. In this review, we will thus summarize and critically discuss the evidence that associates active DNA demethylation by DNA repair with specific functional contexts including the DNA methylation erasure in the early embryo, the control of pluripotency and cellular differentiation, the maintenance of cell identity, and the nuclear reprogramming. Copyright © 2016 Elsevier B.V. All rights reserved.

  1. DNA endonuclease activities on psoralen plus ultraviolet light treated DNA

    SciTech Connect

    Lambert, M.W.; Clark, M.

    1986-03-01

    Activities of nuclear DNA endonucleases (Endos) from normal human lymphoblastoid cells on DNA treated with the DNA interstrand cross-linking agents 4,5'8-trimethyl psoralen (TMP) or 8-methoxypsoralen (MOP) plus long-wavelength (320-400 nm) ultraviolet light (UVA) were examined. Chromatin-associated DNA Endos were isolated from both cell lines and subjected to isoelectric focusing (IF). Each IF fraction was assayed for DNA Endo activity. Peaks of activity were pooled and assayed for activity on undamaged PM2 bacteriophage DNA and on PM2 DNA that had been treated with 15 ..mu..g/ml TMP or MOP in the dark and then exposed to UVA light. Unbound psoralen was removed by dialysis and a second dose of UVA light was given in order to increase the number of DNA cross-links. Two Endo activities were found which were active on TMP- and MOP-DNA: a major one, pI 4.6, which is also active on intercalated DNA, and a second, lesser one, pI 7.6, which is active on UVC (254 nm) light irradiated DNA. These results indicate that there are two different DNA Endos which act on both TMP- and MOP-treated DNA and that the major activity recognizes the intercalation of, and/or the cross-link produced by interaction of, psoralen with DNA.

  2. Elongation of primed DNA templates by eukaryotic DNA polymerases.

    PubMed Central

    Ikeda, J E; Longiaru, M; Horwitz, M S; Hurwitz, J

    1980-01-01

    The combined action of DNA polymerase alpha and DNA polymerase beta leads to the synthesis of full-length linear DNA strands with phi X174 DNA templates containing an RNA primer. The reaction can be carried out in two stages. In the first stage, DNA polymerase alpha catalyzes the synthesis of a chain that averaged 230 deoxynucleotides long and was covalently linked to the RNA primer. In the second stage, DNA polymerase beta elongates the DNA strand covalently attached to the RNA primer to full length. With DNA primers, DNA polymerase alpha catalyzes only limited deoxynucleotide addition whereas DNA polymerase beta alone elongates DNA primed templates to full length. DNA polymerase beta can also stimulate the synthesis of adenovirus DNA in vitro in the presence of a cytosol extract from adenovirus-infected cells. In all of these systems, dNMP incorporation catalyzed by DNA polymerase beta was sensitive to N-ethylmaleimide; however, this polymerase activity was resistant to N-ethylmaleimide with poly(rA) x (dT) as the primer template. Images PMID:6160581

  3. dnaA acts before dnaC in the initiation of DNA replication.

    PubMed Central

    Kung, F C; Glaser, D A

    1978-01-01

    We constructed a double mutant of Escherichia coli K-12 carrying dnaA(Ts) and dnaC(Cs) lesions. In this mutant DNA synthesis proeceeds normally at 32 degrees C and initiation is inhibited at both 41 and 20 degrees C. By shifting this culture grown at 32 degrees C to the two restrictive temperatures in different time sequences and assaying protein and DNA synthesis of cells growing at different temperatures, we found that dnaA and dnaC genes work independently with dnaA acting before dnaC. While preparing special strains for this work, we also showed that the order of genes in the neighborhood of dnaA is dnaA-tnaA-phoS-ilv. PMID:627535

  4. DNA replication stress restricts ribosomal DNA copy number

    PubMed Central

    Salim, Devika; Bradford, William D.; Freeland, Amy; Cady, Gillian; Wang, Jianmin

    2017-01-01

    Ribosomal RNAs (rRNAs) in budding yeast are encoded by ~100–200 repeats of a 9.1kb sequence arranged in tandem on chromosome XII, the ribosomal DNA (rDNA) locus. Copy number of rDNA repeat units in eukaryotic cells is maintained far in excess of the requirement for ribosome biogenesis. Despite the importance of the repeats for both ribosomal and non-ribosomal functions, it is currently not known how “normal” copy number is determined or maintained. To identify essential genes involved in the maintenance of rDNA copy number, we developed a droplet digital PCR based assay to measure rDNA copy number in yeast and used it to screen a yeast conditional temperature-sensitive mutant collection of essential genes. Our screen revealed that low rDNA copy number is associated with compromised DNA replication. Further, subculturing yeast under two separate conditions of DNA replication stress selected for a contraction of the rDNA array independent of the replication fork blocking protein, Fob1. Interestingly, cells with a contracted array grew better than their counterparts with normal copy number under conditions of DNA replication stress. Our data indicate that DNA replication stresses select for a smaller rDNA array. We speculate that this liberates scarce replication factors for use by the rest of the genome, which in turn helps cells complete DNA replication and continue to propagate. Interestingly, tumors from mini chromosome maintenance 2 (MCM2)-deficient mice also show a loss of rDNA repeats. Our data suggest that a reduction in rDNA copy number may indicate a history of DNA replication stress, and that rDNA array size could serve as a diagnostic marker for replication stress. Taken together, these data begin to suggest the selective pressures that combine to yield a “normal” rDNA copy number. PMID:28915237

  5. DNA replication stress restricts ribosomal DNA copy number.

    PubMed

    Salim, Devika; Bradford, William D; Freeland, Amy; Cady, Gillian; Wang, Jianmin; Pruitt, Steven C; Gerton, Jennifer L

    2017-09-15

    Ribosomal RNAs (rRNAs) in budding yeast are encoded by ~100-200 repeats of a 9.1kb sequence arranged in tandem on chromosome XII, the ribosomal DNA (rDNA) locus. Copy number of rDNA repeat units in eukaryotic cells is maintained far in excess of the requirement for ribosome biogenesis. Despite the importance of the repeats for both ribosomal and non-ribosomal functions, it is currently not known how "normal" copy number is determined or maintained. To identify essential genes involved in the maintenance of rDNA copy number, we developed a droplet digital PCR based assay to measure rDNA copy number in yeast and used it to screen the yeast conditional temperature-sensitive mutant collection of essential genes. Our screen revealed that low rDNA copy number is associated with compromised DNA replication. Further, subculturing yeast under two separate conditions of DNA replication stress selected for a contraction of the rDNA array independent of the replication fork blocking protein, Fob1. Interestingly, cells with a contracted array grew better than their counterparts with normal copy number under conditions of DNA replication stress. Our data indicate that DNA replication stresses select for a smaller rDNA array. We speculate that this liberates scarce replication factors for use by the rest of the genome, which in turn helps cells complete DNA replication and continue to propagate. Interestingly, tumors from mini chromosome maintenance 2 (MCM2)-deficient mice also show a loss of rDNA repeats. Our data suggest that a reduction in rDNA copy number may indicate a history of DNA replication stress, and that rDNA array size could serve as a diagnostic marker for replication stress. Taken together, these data begin to suggest the selective pressures that combine to yield a "normal" rDNA copy number.

  6. Duplication in DNA Sequences

    NASA Astrophysics Data System (ADS)

    Ito, Masami; Kari, Lila; Kincaid, Zachary; Seki, Shinnosuke

    The duplication and repeat-deletion operations are the basis of a formal language theoretic model of errors that can occur during DNA replication. During DNA replication, subsequences of a strand of DNA may be copied several times (resulting in duplications) or skipped (resulting in repeat-deletions). As formal language operations, iterated duplication and repeat-deletion of words and languages have been well studied in the literature. However, little is known about single-step duplications and repeat-deletions. In this paper, we investigate several properties of these operations, including closure properties of language families in the Chomsky hierarchy and equations involving these operations. We also make progress toward a characterization of regular languages that are generated by duplicating a regular language.

  7. Capillary electrophoresis of DNA.

    PubMed

    Smith, Alan; Nelson, Robert J

    2003-08-01

    Capillary electrophoresis (CE) is an alternative to conventional slab gel electrophoresis for the separation of DNA fragments. CE offers a number of advantages over slab gel separations in terms of speed, resolution, sensitivity, and data handling. Separation times are generally only a few minutes and the DNA is detected either by UV absorption or by fluorescent labeling. The quantity of DNA required for separation is in the nanogram range. Single-base resolution can be obtained on fragments up to several hundred base pairs. In the presence of appropriate standards, fragments can be accurately sized based on relative electrophoretic mobility. A protocol for the analysis of synthetic oligonucleotides in a flowable matrix is described in this unit.

  8. DNA Methylation in Mammals

    PubMed Central

    Li, En; Zhang, Yi

    2014-01-01

    DNA methylation is one of the best characterized epigenetic modifications. In mammals it is involved in various biological processes including the silencing of transposable elements, regulation of gene expression, genomic imprinting, and X-chromosome inactivation. This article describes how DNA methylation serves as a cellular memory system and how it is dynamically regulated through the action of the DNA methyltransferase (DNMT) and ten eleven translocation (TET) enzymes. Its role in the regulation of gene expression, through its interplay with histone modifications, is also described, and its implication in human diseases discussed. The exciting areas of investigation that will likely become the focus of research in the coming years are outlined in the summary. PMID:24789823

  9. Transposon facilitated DNA sequencing

    SciTech Connect

    Berg, D.E.; Berg, C.M.; Huang, H.V.

    1990-01-01

    The purpose of this research is to investigate and develop methods that exploit the power of bacterial transposable elements for large scale DNA sequencing: Our premise is that the use of transposons to put primer binding sites randomly in target DNAs should provide access to all portions of large DNA fragments, without the inefficiencies of methods involving random subcloning and attendant repetitive sequencing, or of sequential synthesis of many oligonucleotide primers that are used to match systematically along a DNA molecule. Two unrelated bacterial transposons, Tn5 and {gamma}{delta}, are being used because they have both proven useful for molecular analyses, and because they differ sufficiently in mechanism and specificity of transposition to merit parallel development.

  10. Tunnelling microscopy of DNA

    NASA Astrophysics Data System (ADS)

    Selci, Stefano; Cricenti, Antonio

    1991-01-01

    Uncoated DNA molecules marked with an activated tris (1-aziridinyl) phosphine oxide (TAPO) solution were deposited on gold substrates and imaged in air with a high resolution Scanning Tunnelling Microscope (STM). The STM operated simultaneously in the constant-current and gap-modulated mode. Highly reproducible STM images have been obtained and interpreted in terms of expected DNA structure. The main periodicity, regularly presented in molecules several hundred Ångstrom long, ranges from 25 Å to 35 Å with an average diameter of 22 Å. Higher resolution images of the minor groove have revealed the phosphate groups along the DNA backbones. Constant-current images of TAPO deposited on gold show a crystalline structure of rows of molecules with a side-by-side spacing of 3 Å.

  11. DNA Vaccination Techniques.

    PubMed

    Fissolo, Nicolás; Montalban, Xavier; Comabella, Manuel

    2016-01-01

    Multiple sclerosis (MS) is the most common inflammatory, demyelinating, and neurodegenerative disorder of the central nervous system (CNS) in humans. Although the etiology of MS remains unknown, several lines of evidence support the notion that autoimmunity against components of the myelin sheath plays a major role in susceptibility to and development of the disease. At present, there are no approved MS therapies aimed specifically toward downregulating antigen-specific autoreactive immune cells. One antigen-specific approach that appears promising for the treatment of MS is DNA vaccination. This technique has demonstrated efficacy in clinical trials while maintaining safety.Here, we describe the generation of DNA vaccines containing immunologically relevant antigens of MS. Moreover, we present a detailed protocol for the prophylactic and therapeutic administration of DNA vaccines via intramuscular injection targeting on the development of experimental autoimmune encephalomyelitis (EAE), an animal model resembling MS.

  12. DNA based molecular motors

    NASA Astrophysics Data System (ADS)

    Michaelis, Jens; Muschielok, Adam; Andrecka, Joanna; Kügel, Wolfgang; Moffitt, Jeffrey R.

    2009-12-01

    Most of the essential cellular processes such as polymerisation reactions, gene expression and regulation are governed by mechanical processes. Controlled mechanical investigations of these processes are therefore required in order to take our understanding of molecular biology to the next level. Single-molecule manipulation and force spectroscopy have over the last 15 years been developed into extremely powerful techniques. Applying these techniques to the investigation of proteins and DNA molecules has led to a mechanistic understanding of protein function on the level of single molecules. As examples for DNA based molecular machines we will describe single-molecule experiments on RNA polymerases as well as on the packaging of DNA into a viral capsid-a process that is driven by one of the most powerful molecular motors.

  13. Principles of DNA architectonics: design of DNA-based nanoobjects

    NASA Astrophysics Data System (ADS)

    Vinogradova, O. A.; Pyshnyi, D. V.

    2012-02-01

    The methods of preparation of monomeric DNA blocks that serve as key building units for the construction of complex DNA objects are described. Examples are given of the formation of DNA blocks based on native and modified oligonucleotide components using hydrogen bonding and nucleic acid-specific types of bonding and also some affinity interactions with RNA, proteins, ligands. The static discrete and periodic two- and three-dimensional DNA objects reported to date are described systematically. Methods used to prove the structures of DNA objects and the prospects for practical application of nanostructures based on DNA and its analogues in biology, medicine and biophysics are considered. The bibliography includes 195 references.

  14. DNA templated magnetic nanoparticles

    NASA Astrophysics Data System (ADS)

    Kinsella, Joseph M.

    Recent discoveries in nanoscience are predicted to potentially revolutionize future technologies in an extensive number of fields. These developments are contingent upon discovering new and often unconventional methods to synthesize and control nanoscale components. Nature provides several examples of working nanotechnology such as the use of programmed self assembly to build and deconstruct complex molecular systems. We have adopted a method to control the one dimensional assembly of magnetic nanoparticles using DNA as a scaffold molecule. With this method we have demonstrated the ability to organize 5 nm particles into chains that stretch up to ˜20 mum in length. One advantage of using DNA compared is the ability of the molecule to interact with other biomolecules. After assembling particles onto DNA we have been able to cleave the molecule into smaller fragments using restriction enzymes. Using ligase enzymes we have re-connected these fragments, coated with either gold or iron oxide, to form long one-dimensional arrangements of the two different types of nanoparticles on a single molecular guide. We have also created a sensitive magnetic field sensor by incorporating magnetic nanoparticle coated DNA strands with microfabricated electrodes. The IV characteristics of the aligned nanoparticles are dependant on the magnitude of an externally applied magnetic field. This transport phenomenon known as tunneling magnetoresistance (TMR) shows room temperature resistance of our devices over 80% for cobalt ferrite coated DNA when a field of 20 kOe is applied. In comparison, studies using two dimensional nanoparticle films of irox oxides xii only exhibit a 35% MR effect. Confinement into one dimension using the DNA guide produces a TMR mechanism which produces significant increases in magnetoresistance. This property can be utilized for applications in magnetic field sensing, data storage, and logic elements.

  15. The DNA methylome

    PubMed Central

    Pelizzola, Mattia; Ecker, Joseph R.

    2010-01-01

    Methylation of cytosines is a pervasive feature of eukaryotic genomes and an important epigenetic layer that is fundamental for cellular differentiation processes and control of transcriptional potential. DNA methylation patterns can be inherited and influenced by the environment, diet and aging, and disrupted in diseases. Complete DNA methylomes for several organisms are now available, helping clarify the evolutionary story of this epigenetic mark and its distribution in key genomic elements. Nonetheless, a complete understanding of its role, the mechanisms responsible for its establishment and maintenance, and its cross talk with other components of cellular machiney remains elusive. PMID:21056564

  16. Towards single molecule DNA sequencing

    NASA Astrophysics Data System (ADS)

    Liu, Hao

    Single molecule DNA Sequencing technology has been a hot research topic in the recent decades because it holds the promise to sequence a human genome in a fast and affordable way, which will eventually make personalized medicine possible. Single molecule differentiation and DNA translocation control are the two main challenges in all single molecule DNA sequencing methods. In this thesis, I will first introduce DNA sequencing technology development and its application, and then explain the performance and limitation of prior art in detail. Following that, I will show a single molecule DNA base differentiation result obtained in recognition tunneling experiments. Furthermore, I will explain the assembly of a nanofluidic platform for single strand DNA translocation, which holds the promised to be integrated into a single molecule DNA sequencing instrument for DNA translocation control. Taken together, my dissertation research demonstrated the potential of using recognition tunneling techniques to serve as a general readout system for single molecule DNA sequencing application.

  17. An autoradiographic demonstration of nuclear DNA replication by DNA polymerase alpha and of mitochondrial DNA synthesis by DNA polymerase gamma.

    PubMed Central

    Geuskens, M; Hardt, N; Pedrali-Noy, G; Spadari, S

    1981-01-01

    The incorporation of thymidine into the DNA of eukaryotic cells is markedly depressed, but not completely inhibited, by aphidicolin, a highly specific inhibitor of DNA polymerase alpha. An electron microscope autoradiographic analysis of the synthesis of nuclear and mitochondrial DNA in vivo in Concanavalin A stimulated rabbit spleen lymphocytes and in Hamster cell cultures, in the absence and in the presence of aphidicolin, revealed that aphidicolin inhibits the nuclear but not the mitochondrial DNA replication. We therefore conclude that DNA polymerase alpha performs the synchronous bidirectional replication of nuclear DNA and that DNA polymerase gamma, the only DNA polymerase present in the mitochondria, performs the "strand displacement" DNA synthesis of these organelles. Images PMID:6262734

  18. DNA banking and DNA databanking by academic and commercial laboratories

    SciTech Connect

    McEwen, J.E. |; Reilly, P.R.

    1994-09-01

    The advent of DNA-based testing is giving rise to DNA banking (the long-term storage of cells, transformed cell lines, or extracted DNA for subsequent retrieval and analysis) and DNA data banking (the indefinite storage of information derived from DNA analysis). Large scale acquisition and storage of DNA and DNA data has important implications for the privacy rights of individuals. A survey of 148 academically based and commercial DNA diagnostic laboratories was conducted to determine: (1) the extent of their DNA banking activities; (2) their policies and experiences regarding access to DNA samples and data; (3) the quality assurance measures they employ; and (4) whether they have written policies and/or depositor`s agreements addressing specific issues. These issues include: (1) who may have access to DNA samples and data; (2) whether scientists may have access to anonymous samples or data for research use; (3) whether they have plans to contact depositors or retest samples if improved tests for a disorder become available; (4) disposition of samples at the end of the contract period if the laboratory ceases operations, if storage fees are unpaid, or after a death or divorce; (5) the consequence of unauthorized release, loss, or accidental destruction of samples; and (6) whether depositors may share in profits from the commercialization of tests or treatments developed in part from studies of stored DNA. The results suggest that many laboratories are banking DNA, that many have already amassed a large number of samples, and that a significant number plan to further develop DNA banking as a laboratory service over the next two years. Few laboratories have developed written policies governing DNA banking, and fewer still have drafted documents that define the rights and obligations of the parties. There may be a need for increased regulation of DNA banking and DNA data banking and for better defined policies with respect to protecting individual privacy.

  19. DNA damage signalling prevents deleterious telomere addition at DNA breaks

    PubMed Central

    Makovets, Svetlana; Blackburn, Elizabeth H.

    2009-01-01

    The response to DNA damage involves regulation of multiple essential processes to maximize the accuracy of DNA damage repair and cell survival 1. Telomerase has the potential to interfere with repair by inappropriately adding telomeres to DNA breaks. It was unknown whether cells modulate telomerase in response to DNA damage, to increase the accuracy of repair. Here we report that telomerase action is regulated as a part of the cellular response to a DNA double-strand break (DSB). Using yeast, we show that the major ATR/Mec1 DNA damage signalling pathway regulates telomerase action at DSBs. Upon DNA damage, MEC1-RAD53-DUN1-dependent phosphorylation of the telomerase inhibitor Pif1 occurs. Utilizing a separation of function PIF1 mutation, we show that this phosphorylation is required for the Pif1-mediated telomerase inhibition that takes place specifically at DNA breaks, but not telomeres. Hence DNA damage signalling down-modulates telomerase action at a DNA break via Pif1 phosphorylation, thus preventing aberrant healing of broken DNA ends by telomerase. These findings uncover a novel regulatory mechanism that coordinates competing DNA end-processing activities and thereby promotes DNA repair accuracy and genome integrity. PMID:19838171

  20. DNA nanomechanical devices for molecular biology and DNA nanotechnology

    NASA Astrophysics Data System (ADS)

    Gu, Hongzhou

    The aim of nanotechnology is to put specific atomic and molecular species where we want them, when we want them there. Achieving such a dynamic and functional control could lead to molecular programming. Structural DNA nanotechnology offers a powerful route to this goal by combining stable branched DNA motifs with cohesive ends to produce objects, programmed nanomechanical devices and fixed or modified patterned lattices. In Chapter II, a two-armed nanorobotic device is built based on a DNA origami substrate. The arms face each other, ready to capture different DNA nanostructures into distinguishable nanopatterns. Combining with a simple error-correction protocol, we are able to achieve this goal in a nearly flawless fashion. In Chapter III, a DNA-based programmable assembly line is developed by combining three PX/JX2 cassettes and a novel DNA walker on a DNA origami substrate. This programmable assembly line can generate eight products by switching the cassettes to PX (ON) or JX2 (OFF) state when the DNA walker passes by. DNA nanomechanical devices hold the promise of controlling structure and performing exquisitely fine measurements on the molecular scale. Several DNA nanomechanical devices based on different biochemistry phenomena have been reported before. In Chapter IV, a scissors-based DNA device is built to measure the work that can be done by a DNA-bending protein (MutS) when it binds to DNA.

  1. Methods of determining DNA conformation

    SciTech Connect

    Mayes, E.L.

    1994-04-01

    While the basic structure of DNA has been known since 1953, there is still a great void in terms of knowing how DNA interacts with its surroundings. Because the structure of biological molecules determines their function, the conformation of DNA is of prime importance in understanding its role in living organisms. We present two methods of determining some components of DNA`s conformation. One uses atomic force microscopy to determine the dimensions of DNA`s base pairs, and the other seeks to computationally determine the structure of DNA in different chemical environments.

  2. DNA Nanotechnology for Cancer Therapy

    PubMed Central

    Kumar, Vinit; Palazzolo, Stefano; Bayda, Samer; Corona, Giuseppe; Toffoli, Giuseppe; Rizzolio, Flavio

    2016-01-01

    DNA nanotechnology is an emerging and exciting field, and represents a forefront frontier for the biomedical field. The specificity of the interactions between complementary base pairs makes DNA an incredible building material for programmable and very versatile two- and three-dimensional nanostructures called DNA origami. Here, we analyze the DNA origami and DNA-based nanostructures as a drug delivery system. Besides their physical-chemical nature, we dissect the critical factors such as stability, loading capability, release and immunocompatibility, which mainly limit in vivo applications. Special attention was dedicated to highlighting the boundaries to be overcome to bring DNA nanostructures closer to the bedside of patients. PMID:27022418

  3. DNA polymerase having modified nucleotide binding site for DNA sequencing

    DOEpatents

    Tabor, Stanley; Richardson, Charles

    1997-01-01

    Modified gene encoding a modified DNA polymerase wherein the modified polymerase incorporates dideoxynucleotides at least 20-fold better compared to the corresponding deoxynucleotides as compared with the corresponding naturally-occurring DNA polymerase.

  4. DNA profiling of trace DNA recovered from bedding.

    PubMed

    Petricevic, Susan F; Bright, Jo-Anne; Cockerton, Sarah L

    2006-05-25

    Trace DNA is often detected on handled items and worn clothing examined in forensic laboratories. In this study, the potential transfer of trace DNA to bedding by normal contact, when an individual sleeps in a bed, is examined. Volunteers slept one night on a new, lower bed sheet in their own bed and one night in a bed foreign to them. Samples from the sheets were collected and analysed by DNA profiling. The results indicate that the DNA profile of an individual can be obtained from bedding after one night of sleeping in a bed. The DNA profile of the owner of the bed could also be detected in the foreign bed experiments. Since mixed DNA profiles can be obtained from trace DNA on bedding, caution should be exercised when drawing conclusions from DNA profiling results obtained from such samples. This transfer may have important repercussions in sexual assault investigations.

  5. DNA polymerase having modified nucleotide binding site for DNA sequencing

    DOEpatents

    Tabor, S.; Richardson, C.

    1997-03-25

    A modified gene encoding a modified DNA polymerase is disclosed. The modified polymerase incorporates dideoxynucleotides at least 20-fold better compared to the corresponding deoxynucleotides as compared with the corresponding naturally-occurring DNA polymerase. 6 figs.

  6. DNA tagged microparticles

    DOEpatents

    Farquar, George R.; Leif, Roald N.; Wheeler, Elizabeth

    2016-03-22

    In one embodiment, a product includes a plurality of particles, each particle including: a carrier that includes a non-toxic material; and at least one DNA barcode coupled to the carrier, where the particles each have a diameter in a range from about 1 nanometer to about 100 microns.

  7. Enzymatic DNA molecules

    NASA Technical Reports Server (NTRS)

    Joyce, Gerald F. (Inventor); Breaker, Ronald R. (Inventor)

    1998-01-01

    The present invention discloses deoxyribonucleic acid enzymes--catalytic or enzymatic DNA molecules--capable of cleaving nucleic acid sequences or molecules, particularly RNA, in a site-specific manner, as well as compositions including same. Methods of making and using the disclosed enzymes and compositions are also disclosed.

  8. Field Deployable DNA analyzer

    SciTech Connect

    Wheeler, E; Christian, A; Marion, J; Sorensen, K; Arroyo, E; Vrankovich, G; Hara, C; Nguyen, C

    2005-02-09

    This report details the feasibility of a field deployable DNA analyzer. Steps for swabbing cells from surfaces and extracting DNA in an automatable way are presented. Since enzymatic amplification reactions are highly sensitive to environmental contamination, sample preparation is a crucial step to make an autonomous deployable instrument. We perform sample clean up and concentration in a flow through packed bed. For small initial samples, whole genome amplification is performed in the packed bed resulting in enough product for subsequent PCR amplification. In addition to DNA, which can be used to identify a subject, protein is also left behind, the analysis of which can be used to determine exposure to certain substances, such as radionuclides. Our preparative step for DNA analysis left behind the protein complement as a waste stream; we determined to learn if the proteins themselves could be analyzed in a fieldable device. We successfully developed a two-step lateral flow assay for protein analysis and demonstrate a proof of principle assay.

  9. DNA Replication Origins

    PubMed Central

    Leonard, Alan C.; Méchali, Marcel

    2013-01-01

    The onset of genomic DNA synthesis requires precise interactions of specialized initiator proteins with DNA at sites where the replication machinery can be loaded. These sites, defined as replication origins, are found at a few unique locations in all of the prokaryotic chromosomes examined so far. However, replication origins are dispersed among tens of thousands of loci in metazoan chromosomes, thereby raising questions regarding the role of specific nucleotide sequences and chromatin environment in origin selection and the mechanisms used by initiators to recognize replication origins. Close examination of bacterial and archaeal replication origins reveals an array of DNA sequence motifs that position individual initiator protein molecules and promote initiator oligomerization on origin DNA. Conversely, the need for specific recognition sequences in eukaryotic replication origins is relaxed. In fact, the primary rule for origin selection appears to be flexibility, a feature that is modulated either by structural elements or by epigenetic mechanisms at least partly linked to the organization of the genome for gene expression. PMID:23838439

  10. Nutrients and DNA Methylation

    USDA-ARS?s Scientific Manuscript database

    Epigenetics is a new mechanism responsible for development, aging, and disease process such as cancer development. One major epigenetic phenomenon is DNA methylation, which attributes to gene expression and integrity. Deepening the knowledge on one-carbon metabolism is very important to understandin...

  11. Psoralen DNA photobiology

    SciTech Connect

    Gasparro, F.P.

    1988-01-01

    This text covers the spectrum of psoralen photobiology from molecular mechanisms to their in vivo impact. HPLC, gel electrophoresis, and ELISA techniques are described. Also described is the most recent information on cellular mechanisms for the repair of psoralen photodamage in DNA, and the use of psoralens as structural probes and the newly ascribed effects of psoralens on membranes and proteins.

  12. Automated DNA Sequencing System

    SciTech Connect

    Armstrong, G.A.; Ekkebus, C.P.; Hauser, L.J.; Kress, R.L.; Mural, R.J.

    1999-04-25

    Oak Ridge National Laboratory (ORNL) is developing a core DNA sequencing facility to support biological research endeavors at ORNL and to conduct basic sequencing automation research. This facility is novel because its development is based on existing standard biology laboratory equipment; thus, the development process is of interest to the many small laboratories trying to use automation to control costs and increase throughput. Before automation, biology Laboratory personnel purified DNA, completed cycle sequencing, and prepared 96-well sample plates with commercially available hardware designed specifically for each step in the process. Following purification and thermal cycling, an automated sequencing machine was used for the sequencing. A technician handled all movement of the 96-well sample plates between machines. To automate the process, ORNL is adding a CRS Robotics A- 465 arm, ABI 377 sequencing machine, automated centrifuge, automated refrigerator, and possibly an automated SpeedVac. The entire system will be integrated with one central controller that will direct each machine and the robot. The goal of this system is to completely automate the sequencing procedure from bacterial cell samples through ready-to-be-sequenced DNA and ultimately to completed sequence. The system will be flexible and will accommodate different chemistries than existing automated sequencing lines. The system will be expanded in the future to include colony picking and/or actual sequencing. This discrete event, DNA sequencing system will demonstrate that smaller sequencing labs can achieve cost-effective the laboratory grow.

  13. Molecular Programming with DNA

    NASA Astrophysics Data System (ADS)

    Winfree, Erik

    2009-05-01

    Information can be stored in molecules and processed by molecular reactions. Molecular information processing is at the heart of all biological systems; might it soon also be at the heart of non-biological synthetic chemical systems? Perhaps yes. One technological approach comes from DNA nanotechnology and DNA computing, where DNA is used as a non-biological informational polymer that can be rationally designed to create a rich class of molecular systems -- for example, DNA molecules that self-assemble precisely, that fold into complex nanoscale objects, that act as mechanical actuators and molecular motors, and that make decisions based on digital and analog logic. I will argue that to fully exploit their design potential, we will need to invent programming languages for specifying the behavior of information-based molecular systems, to create theoretical tools for understanding and analyzing the behavior of molecular programs, to develop compilers that automate the design of molecules with the desired behaviors, and to expand experimental techniques so that the implementation and debugging of complex molecular systems becomes as commonplace and practical as computer programming.

  14. Impact of Alternative DNA Structures on DNA Damage, DNA Repair, and Genetic Instability

    PubMed Central

    Wang, Guliang; Vasquez, Karen M.

    2014-01-01

    Repetitive genomic sequences can adopt a number of alternative DNA structures that differ from the canonical B-form duplex (i.e. non-B DNA). These non-B DNA-forming sequences have been shown to have many important biological functions related to DNA metabolic processes; for example, they may have regulatory roles in DNA transcription and replication. In addition to these regulatory functions, non-B DNA can stimulate genetic instability in the presence or absence of DNA damage, via replication-dependent and/or replication-independent pathways. This review focuses on the interactions of non-B DNA conformations with DNA repair proteins and how these interactions impact genetic instability. PMID:24767258

  15. Fungal DNA barcoding.

    PubMed

    Xu, Jianping

    2016-11-01

    Fungi are ubiquitous in both natural and human-made environments. They play important roles in the health of plants, animals, and humans, and in broad ecosystem functions. Thus, having an efficient species-level identification system could significantly enhance our ability to treat fungal diseases and to monitor the spatial and temporal patterns of fungal distributions and migrations. DNA barcoding is a potent approach for rapid identification of fungal specimens, generating novel species hypothesis, and guiding biodiversity and ecological studies. In this mini-review, I briefly summarize (i) the history of DNA sequence-based fungal identification; (ii) the emergence of the ITS region as the consensus primary fungal barcode; (iii) the use of the ITS barcodes to address a variety of issues on fungal diversity from local to global scales, including generating a large number of species hypothesis; and (iv) the problems with the ITS barcode region and the approaches to overcome these problems. Similar to DNA barcoding research on plants and animals, significant progress has been achieved over the last few years in terms of both the questions being addressed and the foundations being laid for future research endeavors. However, significant challenges remain. I suggest three broad areas of research to enhance the usefulness of fungal DNA barcoding to meet the current and future challenges: (i) develop a common set of primers and technologies that allow the amplification and sequencing of all fungi at both the primary and secondary barcode loci; (ii) compile a centralized reference database that includes all recognized fungal species as well as species hypothesis, and allows regular updates from the research community; and (iii) establish a consensus set of new species recognition criteria based on barcode DNA sequences that can be applied across the fungal kingdom.

  16. DNA-DNA interaction inside bacteriophage modulated by multivalent counterions

    NASA Astrophysics Data System (ADS)

    Nguyen, Toan; Lee, Seil; Le, Tung

    2010-03-01

    The problem of inhibiting viral DNA ejection from bacteriophages by multivalent counterions, especially Mg^+2 counterions, is studied. Experimentally, it is known that MgSO4 salt has a strong and non-monotonic effect on the amount of DNA ejected. There exists an optimal concentration at which the least DNA is ejected from the virus. At lower or higher concentrations, more DNA is ejected from the capsid. We propose that this phenomenon is the result of DNA overcharging by Mg^+2 multivalent counterions. As Mg^+2 concentration increases from zero, DNA net charge changes from negative to positive. The optimal inhibition corresponds to the Mg^+2 concentration where DNA is neutral. At lower/higher concentrations, DNA genome is charged. It prefers to be in solution to lower its electrostatic self-energy, which consequently leads to an increase in DNA ejection. Our theory fits experimental data well. The strength of DNA - DNA short range attraction, mediated by Mg^+2, is found to be - 0.003 kBT per nucleotide base. Results from expanded ensemble Monte-Carlo simulation of hexagonal DNA bundles are discussed and are shown to be in good agreement with theoretical results.

  17. Borrowing Nuclear DNA Helicases to Protect Mitochondrial DNA

    PubMed Central

    Ding, Lin; Liu, Yilun

    2015-01-01

    In normal cells, mitochondria are the primary organelles that generate energy, which is critical for cellular metabolism. Mitochondrial dysfunction, caused by mitochondrial DNA (mtDNA) mutations or an abnormal mtDNA copy number, is linked to a range of human diseases, including Alzheimer’s disease, premature aging‎ and cancer. mtDNA resides in the mitochondrial lumen, and its duplication requires the mtDNA replicative helicase, Twinkle. In addition to Twinkle, many DNA helicases, which are encoded by the nuclear genome and are crucial for nuclear genome integrity, are transported into the mitochondrion to also function in mtDNA replication and repair. To date, these helicases include RecQ-like helicase 4 (RECQ4), petite integration frequency 1 (PIF1), DNA replication helicase/nuclease 2 (DNA2) and suppressor of var1 3-like protein 1 (SUV3). Although the nuclear functions of some of these DNA helicases have been extensively studied, the regulation of their mitochondrial transport and the mechanisms by which they contribute to mtDNA synthesis and maintenance remain largely unknown. In this review, we attempt to summarize recent research progress on the role of mammalian DNA helicases in mitochondrial genome maintenance and the effects on mitochondria-associated diseases. PMID:25984607

  18. The Dynamic Interplay Between DNA Topoisomerases and DNA Topology.

    PubMed

    Seol, Yeonee; Neuman, Keir C

    2016-09-01

    Topological properties of DNA influence its structure and biochemical interactions. Within the cell DNA topology is constantly in flux. Transcription and other essential processes including DNA replication and repair, alter the topology of the genome, while introducing additional complications associated with DNA knotting and catenation. These topological perturbations are counteracted by the action of topoisomerases, a specialized class of highly conserved and essential enzymes that actively regulate the topological state of the genome. This dynamic interplay among DNA topology, DNA processing enzymes, and DNA topoisomerases, is a pervasive factor that influences DNA metabolism in vivo. Building on the extensive structural and biochemical characterization over the past four decades that established the fundamental mechanistic basis of topoisomerase activity, the unique roles played by DNA topology in modulating and influencing the activity of topoisomerases have begun to be explored. In this review we survey established and emerging DNA topology dependent protein-DNA interactions with a focus on in vitro measurements of the dynamic interplay between DNA topology and topoisomerase activity.

  19. Borrowing nuclear DNA helicases to protect mitochondrial DNA.

    PubMed

    Ding, Lin; Liu, Yilun

    2015-05-13

    In normal cells, mitochondria are the primary organelles that generate energy, which is critical for cellular metabolism. Mitochondrial dysfunction, caused by mitochondrial DNA (mtDNA) mutations or an abnormal mtDNA copy number, is linked to a range of human diseases, including Alzheimer's disease, premature aging‎ and cancer. mtDNA resides in the mitochondrial lumen, and its duplication requires the mtDNA replicative helicase, Twinkle. In addition to Twinkle, many DNA helicases, which are encoded by the nuclear genome and are crucial for nuclear genome integrity, are transported into the mitochondrion to also function in mtDNA replication and repair. To date, these helicases include RecQ-like helicase 4 (RECQ4), petite integration frequency 1 (PIF1), DNA replication helicase/nuclease 2 (DNA2) and suppressor of var1 3-like protein 1 (SUV3). Although the nuclear functions of some of these DNA helicases have been extensively studied, the regulation of their mitochondrial transport and the mechanisms by which they contribute to mtDNA synthesis and maintenance remain largely unknown. In this review, we attempt to summarize recent research progress on the role of mammalian DNA helicases in mitochondrial genome maintenance and the effects on mitochondria-associated diseases.

  20. MAMMALIAN DNA IN PCR REAGENTS

    EPA Science Inventory

    Ancient DNA analysis is becoming widespread. These studies use polymerase chain reaction (PCR) to amplify minute quantities of heavily damaged template. Unusual steps are taken to achieve the sensitivity necessary to detect ancient DNA, including high- cycle PCR amplification t...

  1. Structural diversity of supercoiled DNA

    PubMed Central

    Irobalieva, Rossitza N.; Fogg, Jonathan M.; Catanese, Daniel J.; Sutthibutpong, Thana; Chen, Muyuan; Barker, Anna K.; Ludtke, Steven J.; Harris, Sarah A.; Schmid, Michael F.; Chiu, Wah; Zechiedrich, Lynn

    2015-01-01

    By regulating access to the genetic code, DNA supercoiling strongly affects DNA metabolism. Despite its importance, however, much about supercoiled DNA (positively supercoiled DNA, in particular) remains unknown. Here we use electron cryo-tomography together with biochemical analyses to investigate structures of individual purified DNA minicircle topoisomers with defined degrees of supercoiling. Our results reveal that each topoisomer, negative or positive, adopts a unique and surprisingly wide distribution of three-dimensional conformations. Moreover, we uncover striking differences in how the topoisomers handle torsional stress. As negative supercoiling increases, bases are increasingly exposed. Beyond a sharp supercoiling threshold, we also detect exposed bases in positively supercoiled DNA. Molecular dynamics simulations independently confirm the conformational heterogeneity and provide atomistic insight into the flexibility of supercoiled DNA. Our integrated approach reveals the three-dimensional structures of DNA that are essential for its function. PMID:26455586

  2. An Introduction to DNA Fingerprinting.

    ERIC Educational Resources Information Center

    Hepfer, Carol Ely; And Others

    1993-01-01

    Provides background information on DNA fingerprinting, and describes exercises for introducing general biology students at the high school or college level to the methodology and applications of DNA fingerprinting. (PR)

  3. Structural diversity of supercoiled DNA

    NASA Astrophysics Data System (ADS)

    Irobalieva, Rossitza N.; Fogg, Jonathan M.; Catanese, Daniel J.; Sutthibutpong, Thana; Chen, Muyuan; Barker, Anna K.; Ludtke, Steven J.; Harris, Sarah A.; Schmid, Michael F.; Chiu, Wah; Zechiedrich, Lynn

    2015-10-01

    By regulating access to the genetic code, DNA supercoiling strongly affects DNA metabolism. Despite its importance, however, much about supercoiled DNA (positively supercoiled DNA, in particular) remains unknown. Here we use electron cryo-tomography together with biochemical analyses to investigate structures of individual purified DNA minicircle topoisomers with defined degrees of supercoiling. Our results reveal that each topoisomer, negative or positive, adopts a unique and surprisingly wide distribution of three-dimensional conformations. Moreover, we uncover striking differences in how the topoisomers handle torsional stress. As negative supercoiling increases, bases are increasingly exposed. Beyond a sharp supercoiling threshold, we also detect exposed bases in positively supercoiled DNA. Molecular dynamics simulations independently confirm the conformational heterogeneity and provide atomistic insight into the flexibility of supercoiled DNA. Our integrated approach reveals the three-dimensional structures of DNA that are essential for its function.

  4. Multiplexed DNA-modified electrodes.

    PubMed

    Slinker, Jason D; Muren, Natalie B; Gorodetsky, Alon A; Barton, Jacqueline K

    2010-03-03

    We report the use of silicon chips with 16 DNA-modified electrodes (DME chips) utilizing DNA-mediated charge transport for multiplexed detection of DNA and DNA-binding protein targets. Four DNA sequences were simultaneously distinguished on a single DME chip with 4-fold redundancy, including one incorporating a single base mismatch. These chips also enabled investigation of the sequence-specific activity of the restriction enzyme Alu1. DME chips supported dense DNA monolayer formation with high reproducibility, as confirmed by statistical comparison to commercially available rod electrodes. The working electrode areas on the chips were reduced to 10 microm in diameter, revealing microelectrode behavior that is beneficial for high sensitivity and rapid kinetic analysis. These results illustrate how DME chips facilitate sensitive and selective detection of DNA and DNA-binding protein targets in a robust and internally standardized multiplexed format.

  5. Multiplexed DNA-Modified Electrodes

    PubMed Central

    Slinker, Jason D.; Muren, Natalie B.; Gorodetsky, Alon A.; Barton, Jacqueline K.

    2011-01-01

    We report the use of silicon chips with 16 DNA-modified electrodes (DME chips) utilizing DNA-mediated charge transport for multiplexed detection of DNA and DNA-binding protein targets. Four DNA sequences were simultaneously distinguished on a single DME chip with fourfold redundancy, including one incorporating a single base mismatch. These chips also enabled investigation of the sequence-specific activity of the restriction enzyme Alu1. DME chips supported dense DNA monolayer formation with high reproducibility, as confirmed by statistical comparison to commercially available rod electrodes. The working electrode areas on the chips were reduced to 10 µm in diameter, revealing microelectrode behavior that is beneficial for high sensitivity and rapid kinetic analysis. These results illustrate how DME chips facilitate sensitive and selective detection of DNA and DNA-binding protein targets in a robust and internally standardized multiplexed format. PMID:20131780

  6. Conformation-dependent DNA attraction

    NASA Astrophysics Data System (ADS)

    Li, Weifeng; Nordenskiöld, Lars; Zhou, Ruhong; Mu, Yuguang

    2014-05-01

    Understanding how DNA molecules interact with other biomolecules is related to how they utilize their functions and is therefore critical for understanding their structure-function relationships. For a long time, the existence of Z-form DNA (a left-handed double helical version of DNA, instead of the common right-handed B-form) has puzzled the scientists, and the definitive biological significance of Z-DNA has not yet been clarified. In this study, the effects of DNA conformation in DNA-DNA interactions are explored by molecular dynamics simulations. Using umbrella sampling, we find that for both B- and Z-form DNA, surrounding Mg2+ ions always exert themselves to screen the Coulomb repulsion between DNA phosphates, resulting in very weak attractive force. On the contrary, a tight and stable bound state is discovered for Z-DNA in the presence of Mg2+ or Na+, benefiting from their hydrophobic nature. Based on the contact surface and a dewetting process analysis, a two-stage binding process of Z-DNA is outlined: two Z-DNA first attract each other through charge screening and Mg2+ bridges to phosphate groups in the same way as that of B-DNA, after which hydrophobic contacts of the deoxyribose groups are formed via a dewetting effect, resulting in stable attraction between two Z-DNA molecules. The highlighted hydrophobic nature of Z-DNA interaction from the current study may help to understand the biological functions of Z-DNA in gene transcription.Understanding how DNA molecules interact with other biomolecules is related to how they utilize their functions and is therefore critical for understanding their structure-function relationships. For a long time, the existence of Z-form DNA (a left-handed double helical version of DNA, instead of the common right-handed B-form) has puzzled the scientists, and the definitive biological significance of Z-DNA has not yet been clarified. In this study, the effects of DNA conformation in DNA-DNA interactions are explored by

  7. MAMMALIAN DNA IN PCR REAGENTS

    EPA Science Inventory

    Ancient DNA analysis is becoming widespread. These studies use polymerase chain reaction (PCR) to amplify minute quantities of heavily damaged template. Unusual steps are taken to achieve the sensitivity necessary to detect ancient DNA, including high- cycle PCR amplification t...

  8. The Dynamics of DNA Sequencing.

    ERIC Educational Resources Information Center

    Morvillo, Nancy

    1997-01-01

    Describes a paper-and-pencil activity that helps students understand DNA sequencing and expands student understanding of DNA structure, replication, and gel electrophoresis. Appropriate for advanced biology students who are familiar with the Sanger method. (DDR)

  9. An Introduction to DNA Fingerprinting.

    ERIC Educational Resources Information Center

    Hepfer, Carol Ely; And Others

    1993-01-01

    Provides background information on DNA fingerprinting, and describes exercises for introducing general biology students at the high school or college level to the methodology and applications of DNA fingerprinting. (PR)

  10. DNA Methylation within Transcribed Regions

    PubMed Central

    To, Taiko K.; Saze, Hidetoshi; Kakutani, Tetsuji

    2015-01-01

    DNA methylation within transcribed genes is commonly found in diverse animals and plants. Here, we provide an overview of recent advances and the remaining mystery regarding intragenic DNA methylation. PMID:26143255

  11. Complementary DNA libraries: an overview.

    PubMed

    Ying, Shao-Yao

    2004-07-01

    The generation of complete and full-length cDNA libraries for potential functional assays of specific gene sequences is essential for most molecules in biotechnology and biomedical research. The field of cDNA library generation has changed rapidly in the past 10 yr. This review presents an overview of the method available for the basic information of generating cDNA libraries, including the definition of the cDNA library, different kinds of cDNA libraries, difference between methods for cDNA library generation using conventional approaches and a novel strategy, and the quality of cDNA libraries. It is anticipated that the high-quality cDNA libraries so generated would facilitate studies involving genechips and the microarray, differential display, subtractive hybridization, gene cloning, and peptide library generation.

  12. Novel triterpenoids inhibit both DNA polymerase and DNA topoisomerase.

    PubMed Central

    Mizushina, Y; Iida, A; Ohta, K; Sugawara, F; Sakaguchi, K

    2000-01-01

    As described previously, we found that new triterpenoid compounds, designated fomitellic acids A and B, which selectively inhibit the activities of mammalian DNA polymerases alpha and beta [Mizushina, Tanaka, Kitamura, Tamai, Ikeda, Takemura, Sugawara, Arai, Matsukage, Yoshida and Sakaguchi (1998) Biochem. J. 330, 1325-1332; Tanaka, Kitamura, Mizushina, Sugawara and Sakaguchi (1998) J. Nat. Prod. 61, 193-197] and that a known triterpenoid, ursolic acid, is an inhibitor of human DNA topoisomerases I and II (A. Iida, Y. Mizushina and K. Sakaguchi, unpublished work). Here we report that all of these triterpenoids are potent inhibitors of calf DNA polymerase alpha, rat DNA polymerase beta and human DNA topoisomerases I and II, and show moderate inhibitory effects on plant DNA polymerase II and human immunodeficiency virus reverse transcriptase. However, these compounds did not influence the activities of prokaryotic DNA polymerases such as Escherichia coli DNA polymerase I or other DNA metabolic enzymes such as human telomerase, T7 RNA polymerase and bovine deoxyribonuclease I. These triterpenoids were not only mammalian DNA polymerase inhibitors but also inhibitors of DNA topoisomerases I and II even though the enzymic characteristics of DNA polymerases and DNA topoisomerases, including their modes of action, amino acid sequences and three-dimensional structures, differed markedly. These triterpenoids did not bind to DNA, suggesting that they act directly on these enzymes. Because the three-dimensional structures of fomitellic acids were shown by computer simulation to be very similar to that of ursolic acid, the DNA-binding sites of both enzymes, which compete for the inhibitors, might be very similar. Fomitellic acid A and ursolic acid prevented the growth of NUGC cancer cells, with LD(50) values of 38 and 30 microM respectively. PMID:10970789

  13. Strain softening in stretched DNA

    PubMed Central

    Luan, Binquan; Aksimentiev, Aleksei

    2010-01-01

    The microscopic mechanics of DNA stretching was characterized using extensive molecular dynamics simulations. By employing an anisotropic pressure control method, realistic force-extension dependences of effectively infinite DNA molecules were obtained. A coexistence of B- and S-DNA domains was observed during the overstretching transition. The simulations revealed that strain softening may occur in the process of stretching torsionally constrained DNA. The latter observation was qualitatively reconciled with available experimental data using a random-field Ising model. PMID:18851334

  14. Nanostructure-induced DNA condensation

    NASA Astrophysics Data System (ADS)

    Zhou, Ting; Llizo, Axel; Wang, Chen; Xu, Guiying; Yang, Yanlian

    2013-08-01

    The control of the DNA condensation process is essential for compaction of DNA in chromatin, as well as for biological applications such as nonviral gene therapy. This review endeavours to reflect the progress of investigations on DNA condensation effects of nanostructure-based condensing agents (such as nanoparticles, nanotubes, cationic polymer and peptide agents) observed by using atomic force microscopy (AFM) and other techniques. The environmental effects on structural characteristics of nanostructure-induced DNA condensates are also discussed.

  15. DNA ligases as therapeutic targets

    PubMed Central

    Tomkinson, Alan E.; Howes, Timothy R.L.; Wiest, Nathaniel E.

    2013-01-01

    During DNA replication, DNA joining events link Okazaki fragments on the lagging strand. In addition, they are required to repair DNA single- and double-strand breaks and to complete repair events initiated by the excision of mismatched and damaged bases. In human cells, there are three genes encoding DNA ligases. These enzymes are ATP-dependent and contain a conserved catalytic region. Biophysical studies have shown that the catalytic region contains three domains that, in the absence of DNA, are in an extended conformation. When the catalytic region engages a DNA nick, it adopts a compact, ring structure around the DNA nick with each of the three domains contacting the DNA. Protein-protein interactions involving the regions flanking the conserved catalytic regions of human DNA ligases play a major role in directing these enzymes to participate in specific DNA transactions. Among the human LIG genes, the LIG3 gene is unique in that it encodes multiple DNA ligase polypeptides with different N- and C-termini. One of these polypeptides is targeted to mitochondria where it plays an essential role in the maintenance of the mitochondrial genome. In the nucleus, DNA ligases I, III and IV have distinct but overlapping functions in DNA replication and repair. Small molecule inhibitors of human DNA ligases have been identified using structure-based approaches. As expected, these inhibitors are cytotoxic and also potentiate the cytotoxicity of DNA damaging agents. The results of preclinical studies with human cancer cell lines and mouse models of human cancer suggest that DNA ligase inhibitors may have utility as anti-cancer agents. PMID:24224145

  16. Single-stranded DNA library preparation from highly degraded DNA using T4 DNA ligase.

    PubMed

    Gansauge, Marie-Theres; Gerber, Tobias; Glocke, Isabelle; Korlević, Petra; Lippik, Laurin; Nagel, Sarah; Riehl, Lara Maria; Schmidt, Anna; Meyer, Matthias

    2017-01-23

    DNA library preparation for high-throughput sequencing of genomic DNA usually involves ligation of adapters to double-stranded DNA fragments. However, for highly degraded DNA, especially ancient DNA, library preparation has been found to be more efficient if each of the two DNA strands are converted into library molecules separately. We present a new method for single-stranded library preparation, ssDNA2.0, which is based on single-stranded DNA ligation with T4 DNA ligase utilizing a splinter oligonucleotide with a stretch of random bases hybridized to a 3' biotinylated donor oligonucleotide. A thorough evaluation of this ligation scheme shows that single-stranded DNA can be ligated to adapter oligonucleotides in higher concentration than with CircLigase (an RNA ligase that was previously chosen for end-to-end ligation in single-stranded library preparation) and that biases in ligation can be minimized when choosing splinters with 7 or 8 random nucleotides. We show that ssDNA2.0 tolerates higher quantities of input DNA than CircLigase-based library preparation, is less costly and better compatible with automation. We also provide an in-depth comparison of library preparation methods on degraded DNA from various sources. Most strikingly, we find that single-stranded library preparation increases library yields from tissues stored in formalin for many years by several orders of magnitude.

  17. Single-stranded DNA library preparation from highly degraded DNA using T4 DNA ligase

    PubMed Central

    Gerber, Tobias; Glocke, Isabelle; Korlević, Petra; Lippik, Laurin; Nagel, Sarah; Riehl, Lara Maria; Schmidt, Anna

    2017-01-01

    Abstract DNA library preparation for high-throughput sequencing of genomic DNA usually involves ligation of adapters to double-stranded DNA fragments. However, for highly degraded DNA, especially ancient DNA, library preparation has been found to be more efficient if each of the two DNA strands are converted into library molecules separately. We present a new method for single-stranded library preparation, ssDNA2.0, which is based on single-stranded DNA ligation with T4 DNA ligase utilizing a splinter oligonucleotide with a stretch of random bases hybridized to a 3΄ biotinylated donor oligonucleotide. A thorough evaluation of this ligation scheme shows that single-stranded DNA can be ligated to adapter oligonucleotides in higher concentration than with CircLigase (an RNA ligase that was previously chosen for end-to-end ligation in single-stranded library preparation) and that biases in ligation can be minimized when choosing splinters with 7 or 8 random nucleotides. We show that ssDNA2.0 tolerates higher quantities of input DNA than CircLigase-based library preparation, is less costly and better compatible with automation. We also provide an in-depth comparison of library preparation methods on degraded DNA from various sources. Most strikingly, we find that single-stranded library preparation increases library yields from tissues stored in formalin for many years by several orders of magnitude. PMID:28119419

  18. Efficient DNA ligation in DNA-RNA hybrid helices by Chlorella virus DNA ligase.

    PubMed

    Lohman, Gregory J S; Zhang, Yinhua; Zhelkovsky, Alexander M; Cantor, Eric J; Evans, Thomas C

    2014-02-01

    Single-stranded DNA molecules (ssDNA) annealed to an RNA splint are notoriously poor substrates for DNA ligases. Herein we report the unexpectedly efficient ligation of RNA-splinted DNA by Chlorella virus DNA ligase (PBCV-1 DNA ligase). PBCV-1 DNA ligase ligated ssDNA splinted by RNA with kcat ≈ 8 x 10(-3) s(-1) and K(M) < 1 nM at 25 °C under conditions where T4 DNA ligase produced only 5'-adenylylated DNA with a 20-fold lower kcat and a K(M) ≈ 300 nM. The rate of ligation increased with addition of Mn(2+), but was strongly inhibited by concentrations of NaCl >100 mM. Abortive adenylylation was suppressed at low ATP concentrations (<100 µM) and pH >8, leading to increased product yields. The ligation reaction was rapid for a broad range of substrate sequences, but was relatively slower for substrates with a 5'-phosphorylated dC or dG residue on the 3' side of the ligation junction. Nevertheless, PBCV-1 DNA ligase ligated all sequences tested with 10-fold less enzyme and 15-fold shorter incubation times than required when using T4 DNA ligase. Furthermore, this ligase was used in a ligation-based detection assay system to show increased sensitivity over T4 DNA ligase in the specific detection of a target mRNA.

  19. DNA damage and mutation. Types of DNA damage

    NASA Astrophysics Data System (ADS)

    Chakarov, Stoyan; Petkova, Rumena; Russev, George Ch; Zhelev, Nikolai

    2014-02-01

    This review outlines the basic types of DNA damage caused by exogenous and endogenous factors, analyses the possible consequences of each type of damage and discusses the need for different types of DNA repair. The mechanisms by which a minor damaging event to DNA may eventually result in the introduction of heritable mutation/s are reviewed. The major features of the role of DNA damage in ageing and carcinogenesis are outlined and the role of iatrogenic DNA damage in human health and disease (with curative intent as well as a long-term adverse effect of genotoxic therapies) are discussed in detail.

  20. DNA Origami-Graphene Hybrid Nanopore for DNA Detection.

    PubMed

    Barati Farimani, Amir; Dibaeinia, Payam; Aluru, Narayana R

    2017-01-11

    DNA origami nanostructures can be used to functionalize solid-state nanopores for single molecule studies. In this study, we characterized a nanopore in a DNA origami-graphene heterostructure for DNA detection. The DNA origami nanopore is functionalized with a specific nucleotide type at the edge of the pore. Using extensive molecular dynamics (MD) simulations, we computed and analyzed the ionic conductivity of nanopores in heterostructures carpeted with one or two layers of DNA origami on graphene. We demonstrate that a nanopore in DNA origami-graphene gives rise to distinguishable dwell times for the four DNA base types, whereas for a nanopore in bare graphene, the dwell time is almost the same for all types of bases. The specific interactions (hydrogen bonds) between DNA origami and the translocating DNA strand yield different residence times and ionic currents. We also conclude that the speed of DNA translocation decreases due to the friction between the dangling bases at the pore mouth and the sequencing DNA strands.

  1. A survey of DNA diagnostic laboratories regarding DNA banking.

    PubMed

    McEwen, J E; Reilly, P R

    1995-06-01

    This article reports the findings of a survey of 148 academically based and commercial DNA diagnostic labs regarding DNA banking (defined as the storage of individual DNA samples in some form with identifiers for later retrieval). The population surveyed consisted of all laboratories listed with HELIX, a national directory of DNA diagnostic labs that includes a fairly comprehensive listing of clinical service labs as well as a large number of research labs. The survey was concerned primarily with the legal and ethical issues that the long-term storage of DNA may raise. The survey inquired into the respondents' policies and procedures concerning (1) the extent of DNA banking and of interest in developing DNA banking in academia and industry and (2) the degree to which DNA banks had developed written internal policies and/or a written depositor's agreement (a signed document defining the rights and obligations of the person from whom the sample was taken and the bank) designed to anticipate or prevent some of the ethical and legal problems that can arise from the long-term retention of DNA. Our research suggests that (1) the activity of DNA banking is growing, particularly in the academic setting, and (2) most academically based DNA banks lack written internal policies, written depositor's agreements, or other relevant documentation regarding important aspects of this activity.

  2. Efficient Sleeping Beauty DNA Transposition From DNA Minicircles

    PubMed Central

    Sharma, Nynne; Cai, Yujia; Bak, Rasmus O; Jakobsen, Martin R; Schrøder, Lisbeth Dahl; Mikkelsen, Jacob Giehm

    2013-01-01

    DNA transposon-based vectors have emerged as new potential delivery tools in therapeutic gene transfer. Such vectors are now showing promise in hematopoietic stem cells and primary human T cells, and clinical trials with transposon-engineered cells are on the way. However, the use of plasmid DNA as a carrier of the vector raises safety concerns due to the undesirable administration of bacterial sequences. To optimize vectors based on the Sleeping Beauty (SB) DNA transposon for clinical use, we examine here SB transposition from DNA minicircles (MCs) devoid of the bacterial plasmid backbone. Potent DNA transposition, directed by the hyperactive SB100X transposase, is demonstrated from MC donors, and the stable transfection rate is significantly enhanced by expressing the SB100X transposase from MCs. The stable transfection rate is inversely related to the size of circular donor, suggesting that a MC-based SB transposition system benefits primarily from an increased cellular uptake and/or enhanced expression which can be observed with DNA MCs. DNA transposon and transposase MCs are easily produced, are favorable in size, do not carry irrelevant DNA, and are robust substrates for DNA transposition. In accordance, DNA MCs should become a standard source of DNA transposons not only in therapeutic settings but also in the daily use of the SB system. PMID:23443502

  3. A survey of DNA diagnostic laboratories regarding DNA banking.

    PubMed Central

    McEwen, J E; Reilly, P R

    1995-01-01

    This article reports the findings of a survey of 148 academically based and commercial DNA diagnostic labs regarding DNA banking (defined as the storage of individual DNA samples in some form with identifiers for later retrieval). The population surveyed consisted of all laboratories listed with HELIX, a national directory of DNA diagnostic labs that includes a fairly comprehensive listing of clinical service labs as well as a large number of research labs. The survey was concerned primarily with the legal and ethical issues that the long-term storage of DNA may raise. The survey inquired into the respondents' policies and procedures concerning (1) the extent of DNA banking and of interest in developing DNA banking in academia and industry and (2) the degree to which DNA banks had developed written internal policies and/or a written depositor's agreement (a signed document defining the rights and obligations of the person from whom the sample was taken and the bank) designed to anticipate or prevent some of the ethical and legal problems that can arise from the long-term retention of DNA. Our research suggests that (1) the activity of DNA banking is growing, particularly in the academic setting, and (2) most academically based DNA banks lack written internal policies, written depositor's agreements, or other relevant documentation regarding important aspects of this activity. PMID:7762571

  4. DNA methylation reprogramming and DNA repair in the mouse zygote.

    PubMed

    Lepikhov, Konstantin; Wossidlo, Mark; Arand, Julia; Walter, Joern

    2010-01-01

    Here, we summarize current knowledge about epigenetic reprogramming during mammalian preimplantation development, as well as the potential mechanisms driving these processes. We will particularly focus on changes taking place in the zygote, where the paternally derived DNA and chromatin undergo the most striking alterations, such as replacement of protamines by histones, histone modifications and active DNA demethylation. The putative mechanisms of active paternal DNA demethylation have been studied for over a decade, accumulating a lot of circumstantial evidence for enzymatic activities provided by the oocyte, protection of the maternal genome against such activities and possible involvement of DNA repair. We will discuss the various facets of dynamic epigenetic changes related to DNA methylation with an emphasis on the putative involvement of DNA repair in DNA demethylation.

  5. DnaA and ORC: more than DNA replication initiators.

    PubMed

    Scholefield, Graham; Veening, Jan-Willem; Murray, Heath

    2011-03-01

    Mutations in DNA replication initiator genes in both prokaryotes and eukaryotes lead to a pleiotropic array of phenotypes, including defects in chromosome segregation, cytokinesis, cell cycle regulation and gene expression. For years, it was not clear whether these diverse effects were indirect consequences of perturbed DNA replication, or whether they indicated that DNA replication initiator proteins had roles beyond their activity in initiating DNA synthesis. Recent work from a range of organisms has demonstrated that DNA replication initiator proteins play direct roles in many cellular processes, often functioning to coordinate the initiation of DNA replication with essential cell-cycle activities. The aim of this review is to highlight these new findings, focusing on the pathways and mechanisms utilized by DNA replication initiator proteins to carry out a diverse array of cellular functions. Copyright © 2010 Elsevier Ltd. All rights reserved.

  6. Production of random DNA oligomers for scalable DNA computing.

    PubMed

    Wang, Sixue S L; Johnson, John J X; Hughes, Bradley S T; Karabay, Dundar A O; Bader, Karson D W; Austin, Allen; Austin, Alan; Habib, Aisha; Hatef, Husnia; Joshi, Megha; Nguyen, Lawrence; Mills, Allen P

    2009-01-01

    While remarkably complex networks of connected DNA molecules can form from a relatively small number of distinct oligomer strands, a large computational space created by DNA reactions would ultimately require the use of many distinct DNA strands. The automatic synthesis of this many distinct strands is economically prohibitive. We present here a new approach to producing distinct DNA oligomers based on the polymerase chain reaction (PCR) amplification of a few random template sequences. As an example, we designed a DNA template sequence consisting of a 50-mer random DNA segment flanked by two 20-mer invariant primer sequences. Amplification of a dilute sample containing about 30 different template molecules allows us to obtain around 10(11) copies of these molecules and their complements. We demonstrate the use of these amplicons to implement some of the vector operations that will be required in a DNA implementation of an analog neural network.

  7. Single-stranded DNA (ssDNA) production in DNA aptamer generation.

    PubMed

    Marimuthu, Citartan; Tang, Thean-Hock; Tominaga, Junji; Tan, Soo-Choon; Gopinath, Subash C B

    2012-03-21

    The discovery that synthetic short chain nucleic acids are capable of selective binding to biological targets has made them to be widely used as molecular recognition elements. These nucleic acids, called aptamers, are comprised of two types, DNA and RNA aptamers, where the DNA aptamer is preferred over the latter due to its stability, making it widely used in a number of applications. However, the success of the DNA selection process through Systematic Evolution of Ligands by Exponential Enrichment (SELEX) experiments is very much dependent on its most critical step, which is the conversion of the dsDNA to ssDNA. There is a plethora of methods available in generating ssDNA from the corresponding dsDNA. These include asymmetric PCR, biotin-streptavidin separation, lambda exonuclease digestion and size separation on denaturing-urea PAGE. Herein, different methods of ssDNA generation following the PCR amplification step in SELEX are reviewed.

  8. DNA Methylation in Schizophrenia.

    PubMed

    Pries, Lotta-Katrin; Gülöksüz, Sinan; Kenis, Gunter

    2017-01-01

    Schizophrenia is a highly heritable psychiatric condition that displays a complex phenotype. A multitude of genetic susceptibility loci have now been identified, but these fail to explain the high heritability estimates of schizophrenia. In addition, epidemiologically relevant environmental risk factors for schizophrenia may lead to permanent changes in brain function. In conjunction with genetic liability, these environmental risk factors-likely through epigenetic mechanisms-may give rise to schizophrenia, a clinical syndrome characterized by florid psychotic symptoms and moderate to severe cognitive impairment. These pathophysiological features point to the involvement of epigenetic processes. Recently, a wave of studies examining aberrant DNA modifications in schizophrenia was published. This chapter aims to comprehensively review the current findings, from both candidate gene studies and genome-wide approaches, on DNA methylation changes in schizophrenia.

  9. Molecular Motors from DNA

    NASA Astrophysics Data System (ADS)

    Turberfield, Andrew

    2013-03-01

    DNA is a wonderful material for nanoscale construction: its self-assembly can be programmed by making use of its information-carrying capability and its hybridization or hydrolysis can be used as to provide energy for synthetic molecular machinery. With DNA it is possible to design and build three-dimensional scaffolds, to attach molecular components to them with sub-nanometre precision-and then to make them move. I shall describe our work on autonomous, biomimetic molecular motors powered by chemical fuels and the use of synthetic molecular machinery to control covalent chemical synthesis. I shall demonstrate bipedal motors whose operation depends on the coordination of the chemomechanical cycles of two separate catalytic centres and burnt bridges motors that can be programmed to navigate networks of tracks. I shall also discuss the use of kinesin motor proteins to power synthetic devices.

  10. Your DNA, Your Say

    PubMed Central

    Middleton, Anna

    2017-01-01

    Genomic and medical data sharing is pivotal if the promise of genomic medicine is to be fully realised. Social scientists working in the genomics arena ask the public ‘how is the technology working for you?’ Empirical studies on attitudes, values and beliefs are incredibly valuable; they offer a voice from those who are, or will be, directly affected. This is paramount if personalised medicine is to be truly personal. An International attitude study, Your DNA, Your Say, uses film to provide background information and an online survey to gather public views on donating one's own personal DNA and medical data for use by others. In this paper the rationale to the project is introduced together with an overview of the survey and film design. The project has been translated into multiple languages and the results will be used in policy for the Global Alliance for Genomics and Health. PMID:28517993

  11. Fleet DNA (Presentation)

    SciTech Connect

    Walkokwicz, K.; Duran, A.

    2014-06-01

    The Fleet DNA project objectives include capturing and quantifying drive cycle and technology variation for the multitude of medium- and heavy-duty vocations; providing a common data storage warehouse for medium- and heavy-duty vehicle fleet data across DOE activities and laboratories; and integrating existing DOE tools, models, and analyses to provide data-driven decision making capabilities. Fleet DNA advantages include: for Government - providing in-use data for standard drive cycle development, R&D, tech targets, and rule making; for OEMs - real-world usage datasets provide concrete examples of customer use profiles; for fleets - vocational datasets help illustrate how to maximize return on technology investments; for Funding Agencies - ways are revealed to optimize the impact of financial incentive offers; and for researchers -a data source is provided for modeling and simulation.

  12. Facilitated diffusion with DNA coiling

    PubMed Central

    Lomholt, Michael A.; van den Broek, Bram; Kalisch, Svenja-Marei J.; Wuite, Gijs J. L.; Metzler, Ralf

    2009-01-01

    When DNA-binding proteins search for their specific binding site on a DNA molecule they alternate between linear 1-dimensional diffusion along the DNA molecule, mediated by nonspecific binding, and 3-dimensional volume excursion events between successive dissociation from and rebinding to DNA. If the DNA molecule is kept in a straight configuration, for instance, by optical tweezers, these 3-dimensional excursions may be divided into long volume excursions and short hops along the DNA. These short hops correspond to immediate rebindings after dissociation such that a rebinding event to the DNA occurs at a site that is close to the site of the preceding dissociation. When the DNA molecule is allowed to coil up, immediate rebinding may also lead to so-called intersegmental jumps, i.e., immediate rebindings to a DNA segment that is far away from the unbinding site when measured in the chemical distance along the DNA, but close by in the embedding 3-dimensional space. This effect is made possible by DNA looping. The significance of intersegmental jumps was recently demonstrated in a single DNA optical tweezers setup. Here we present a theoretical approach in which we explicitly take the effect of DNA coiling into account. By including the spatial correlations of the short hops we demonstrate how the facilitated diffusion model can be extended to account for intersegmental jumping at varying DNA densities. It is also shown that our approach provides a quantitative interpretation of the experimentally measured enhancement of the target location by DNA-binding proteins. PMID:19420219

  13. Counterintuitive DNA Sequence Dependence in Supercoiling-Induced DNA Melting

    PubMed Central

    Vlijm, Rifka; v.d. Torre, Jaco; Dekker, Cees

    2015-01-01

    The metabolism of DNA in cells relies on the balance between hybridized double-stranded DNA (dsDNA) and local de-hybridized regions of ssDNA that provide access to binding proteins. Traditional melting experiments, in which short pieces of dsDNA are heated up until the point of melting into ssDNA, have determined that AT-rich sequences have a lower binding energy than GC-rich sequences. In cells, however, the double-stranded backbone of DNA is destabilized by negative supercoiling, and not by temperature. To investigate what the effect of GC content is on DNA melting induced by negative supercoiling, we studied DNA molecules with a GC content ranging from 38% to 77%, using single-molecule magnetic tweezer measurements in which the length of a single DNA molecule is measured as a function of applied stretching force and supercoiling density. At low force (<0.5pN), supercoiling results into twisting of the dsDNA backbone and loop formation (plectonemes), without inducing any DNA melting. This process was not influenced by the DNA sequence. When negative supercoiling is introduced at increasing force, local melting of DNA is introduced. We measured for the different DNA molecules a characteristic force Fchar, at which negative supercoiling induces local melting of the dsDNA. Surprisingly, GC-rich sequences melt at lower forces than AT-rich sequences: Fchar = 0.56pN for 77% GC but 0.73pN for 38% GC. An explanation for this counterintuitive effect is provided by the realization that supercoiling densities of a few percent only induce melting of a few percent of the base pairs. As a consequence, denaturation bubbles occur in local AT-rich regions and the sequence-dependent effect arises from an increased DNA bending/torsional energy associated with the plectonemes. This new insight indicates that an increased GC-content adjacent to AT-rich DNA regions will enhance local opening of the double-stranded DNA helix. PMID:26513573

  14. Geant4-DNA simulations using complex DNA geometries generated by the DnaFabric tool

    NASA Astrophysics Data System (ADS)

    Meylan, S.; Vimont, U.; Incerti, S.; Clairand, I.; Villagrasa, C.

    2016-07-01

    Several DNA representations are used to study radio-induced complex DNA damages depending on the approach and the required level of granularity. Among all approaches, the mechanistic one requires the most resolved DNA models that can go down to atomistic DNA descriptions. The complexity of such DNA models make them hard to modify and adapt in order to take into account different biological conditions. The DnaFabric project was started to provide a tool to generate, visualise and modify such complex DNA models. In the current version of DnaFabric, the models can be exported to the Geant4 code to be used as targets in the Monte Carlo simulation. In this work, the project was used to generate two DNA fibre models corresponding to two DNA compaction levels representing the hetero and the euchromatin. The fibres were imported in a Geant4 application where computations were performed to estimate the influence of the DNA compaction on the amount of calculated DNA damage. The relative difference of the DNA damage computed in the two fibres for the same number of projectiles was found to be constant and equal to 1.3 for the considered primary particles (protons from 300 keV to 50 MeV). However, if only the tracks hitting the DNA target are taken into account, then the relative difference is more important for low energies and decreases to reach zero around 10 MeV. The computations were performed with models that contain up to 18,000 DNA nucleotide pairs. Nevertheless, DnaFabric will be extended to manipulate multi-scale models that go from the molecular to the cellular levels.

  15. DNA strand breaks: the DNA template alterations that trigger p53-dependent DNA damage response pathways.

    PubMed Central

    Nelson, W G; Kastan, M B

    1994-01-01

    The tumor suppressor protein p53 serves as a critical regulator of a G1 cell cycle checkpoint and of apoptosis following exposure of cells to DNA-damaging agents. The mechanism by which DNA-damaging agents elevate p53 protein levels to trigger G1/S arrest or cell death remains to be elucidated. In fact, whether damage to the DNA template itself participates in transducing the signal leading to p53 induction has not yet been demonstrated. We exposed human cell lines containing wild-type p53 alleles to several different DNA-damaging agents and found that agents which rapidly induce DNA strand breaks, such as ionizing radiation, bleomycin, and DNA topoisomerase-targeted drugs, rapidly triggered p53 protein elevations. In addition, we determined that camptothecin-stimulated trapping of topoisomerase I-DNA complexes was not sufficient to elevate p53 protein levels; rather, replication-associated DNA strand breaks were required. Furthermore, treatment of cells with the antimetabolite N(phosphonoacetyl)-L-aspartate (PALA) did not cause rapid p53 protein increases but resulted in delayed increases in p53 protein levels temporally correlated with the appearance of DNA strand breaks. Finally, we concluded that DNA strand breaks were sufficient for initiating p53-dependent signal transduction after finding that introduction of nucleases into cells by electroporation stimulated rapid p53 protein elevations. While DNA strand breaks appeared to be capable of triggering p53 induction, DNA lesions other than strand breaks did not. Exposure of normal cells and excision repair-deficient xeroderma pigmentosum cells to low doses of UV light, under conditions in which thymine dimers appear but DNA replication-associated strand breaks were prevented, resulted in p53 induction attributable to DNA strand breaks associated with excision repair. Our data indicate that DNA strand breaks are sufficient and probably necessary for p53 induction in cells with wild-type p53 alleles exposed to DNA

  16. Epigenetics of Ancient DNA

    PubMed Central

    Zhenilo, S. V.; Sokolov, A.S.; Prokhortchouk, E. B.

    2016-01-01

    Initially, the study of DNA isolated from ancient specimens had been based on the analysis of the primary nucleotide sequence. This approach has allowed researchers to study the evolutionary changes that occur in different populations and determine the influence of the environment on genetic selection. However, the improvement of methodological approaches to genome-wide analysis has opened up new possibilities in the search for the epigenetic mechanisms involved in the regulation of gene expression. It was discovered recently that the methylation status of the regulatory elements of the HOXD cluster and MEIS1 gene changed during human evolution. Epigenetic changes in these genes played a key role in the evolution of the limbs of modern humans. Recent works have demonstrated that it is possible to determine the transcriptional activity of genes in ancient DNA samples by combining information on DNA methylation and the DNAaseI hypersensitive sequences located at the transcription start sites of genes. In the nearest future, if a preserved fossils brain is found, it will be possible to identify the evolutionary changes in the higher nervous system associated with epigenetic differences. PMID:27795845

  17. Therapeutic HPV DNA vaccines

    PubMed Central

    Lin, Ken; Roosinovich, Elena; Ma, Barbara; Hung, Chien-Fu

    2010-01-01

    It is now well established that most cervical cancers are causally associated with HPV infection. This realization has led to efforts to control HPV-associated malignancy through prevention or treatment of HPV infection. Currently, commercially available HPV vaccines are not designed to control established HPV infection and associated premalignant and malignant lesions. To treat and eradicate pre-existing HPV infections and associated lesions which remain prevalent in the U.S. and worldwide, effective therapeutic HPV vaccines are needed. DNA vaccination has emerged as a particularly promising form of therapeutic HPV vaccines due to its safety, stability and ability to induce antigen-specific immunity. This review focuses on improving the potency of therapeutic HPV vaccines through modification of dendritic cells (DCs) by [1] increasing the number of antigen-expressing/antigen-loaded DCs, [2] improving HPV antigen expression, processing and presentation in DCs, and [3] enhancing DC and T cell interaction. Continued improvement in therapeutic HPV DNA vaccines may ultimately lead to an effective DNA vaccine for the treatment of HPV-associated malignancies. PMID:20066511

  18. DNA Degradation and Its Defects

    PubMed Central

    Kawane, Kohki; Motani, Kou; Nagata, Shigekazu

    2014-01-01

    DNA is one of the most essential molecules in organisms, containing all the information necessary for organisms to live. It replicates and provides a mechanism for heredity and evolution. Various events cause the degradation of DNA into nucleotides. DNA also has a darker side that has only recently been recognized; DNA that is not properly degraded causes various diseases. In this review, we discuss four deoxyribonucleases that function in the nucleus, cytosol, and lysosomes, and how undigested DNA causes such diseases as cancer, cataract, and autoinflammation. Studies on the biochemical and physiological functions of deoxyribonucleases should continue to increase our understanding of cellular functions and human diseases. PMID:24890510

  19. DNA nanotechnology and fluorescence applications.

    PubMed

    Schlichthaerle, Thomas; Strauss, Maximilian T; Schueder, Florian; Woehrstein, Johannes B; Jungmann, Ralf

    2016-06-01

    Structural DNA nanotechnology allow researchers to use the unique molecular recognition properties of DNA strands to construct nanoscale objects with almost arbitrary complexity in two and three dimensions. Abstracted as molecular breadboards, DNA nanostructures enable nanometer-precise placement of guest molecules such as proteins, fluorophores, or nanoparticles. These assemblies can be used to study biological phenomena with unprecedented control over number, spacing, and molecular identity. Here, we give a general introduction to structural DNA nanotechnology and more specifically discuss applications of DNA nanostructures in the field of fluorescence and plasmonics.

  20. Topoisomerase II minimizes DNA entanglements by proofreading DNA topology after DNA strand passage

    PubMed Central

    Martínez-García, Belén; Fernández, Xavier; Díaz-Ingelmo, Ofelia; Rodríguez-Campos, Antonio; Manichanh, Chaysavanh; Roca, Joaquim

    2014-01-01

    By transporting one DNA double helix (T-segment) through a double-strand break in another (G-segment), topoisomerase II reduces fractions of DNA catenanes, knots and supercoils to below equilibrium values. How DNA segments are selected to simplify the equilibrium DNA topology is enigmatic, and the biological relevance of this activity is unclear. Here we examined the transit of the T-segment across the three gates of topoisomerase II (entry N-gate, DNA-gate and exit C-gate). Our experimental results uncovered that DNA transport probability is determined not only during the capture of a T-segment at the N-gate. When a captured T-segment has crossed the DNA-gate, it can backtrack to the N-gate instead of exiting by the C-gate. When such backtracking is precluded by locking the N-gate or by removing the C-gate, topoisomerase II no longer simplifies equilibrium DNA topology. Therefore, we conclude that the C-gate enables a post-DNA passage proofreading mechanism, which challenges the release of passed T-segments to either complete or cancel DNA transport. This proofreading activity not only clarifies how type-IIA topoisomerases simplify the equilibrium topology of DNA in free solution, but it may explain also why these enzymes are able to solve the topological constraints of intracellular DNA without randomly entangling adjacent chromosomal regions. PMID:24185700

  1. DNA-based soft phases.

    PubMed

    Bellini, Tommaso; Cerbino, Roberto; Zanchetta, Giuliano

    2012-01-01

    This chapter reviews the state-of-the-art in the study of molecular or colloidal systems whose mutual interactions are mediated by DNA molecules. In the last decade, the robust current knowledge of DNA interactions has enabled an impressive growth of self-assembled DNA-based structures that depend crucially on the properties of DNA-DNA interactions. In many cases, structures are built on design by exploiting the programmable selectivity of DNA interactions and the modularity of their strength. The study of DNA-based materials is definitely an emerging field in condensed matter physics, nanotechnology, and material science. This chapter will consider both systems that are entirely constructed by DNA and hybrid systems in which latex or metal colloidal particles are coated by DNA strands. We will confine our discussion to systems in which DNA-mediated interactions promote the formation of "phases," that is structures extending on length scales much larger than the building blocks. Their self-assembly typically involves a large number of interacting particles and often features hierarchical stages of structuring. Because of the possibility of fine-tuning the geometry and strength of the DNA-mediated interactions, these systems are characterized by a wide variety of patterns of self-assembly, ranging from amorphous, to liquid crystalline, to crystalline in one, two, or three dimensions.

  2. Forensic trace DNA: a review

    PubMed Central

    2010-01-01

    DNA analysis is frequently used to acquire information from biological material to aid enquiries associated with criminal offences, disaster victim identification and missing persons investigations. As the relevance and value of DNA profiling to forensic investigations has increased, so too has the desire to generate this information from smaller amounts of DNA. Trace DNA samples may be defined as any sample which falls below recommended thresholds at any stage of the analysis, from sample detection through to profile interpretation, and can not be defined by a precise picogram amount. Here we review aspects associated with the collection, DNA extraction, amplification, profiling and interpretation of trace DNA samples. Contamination and transfer issues are also briefly discussed within the context of trace DNA analysis. Whilst several methodological changes have facilitated profiling from trace samples in recent years it is also clear that many opportunities exist for further improvements. PMID:21122102

  3. Ancient DNA extraction from plants.

    PubMed

    Kistler, Logan

    2012-01-01

    A variety of protocols for DNA extraction from archaeological and paleobotanical plant specimens have been proposed. This is not surprising given the range of taxa and tissue types that may be preserved and the variety of conditions in which that preservation may take place. Commercially available DNA extraction kits can be used to recover ancient plant DNA, but modifications to standard approaches are often necessary to improve yield. In this chapter, I describe two protocols for extracting DNA from small amounts of ancient plant tissue. The CTAB protocol, which I recommend for use with single seeds, utilizes an incubation period in extraction buffer and subsequent chloroform extraction followed by DNA purification and suspension. The PTB protocol, which I recommend for use with gourd rind and similar tissues, utilizes an overnight incubation of pulverized tissue in extraction buffer, removal of the tissue by centrifugation, and DNA extraction from the buffer using commercial plant DNA extraction kits.

  4. DNA Methylation and Cancer Diagnosis

    PubMed Central

    Delpu, Yannick; Cordelier, Pierre; Cho, William C.; Torrisani, Jérôme

    2013-01-01

    DNA methylation is a major epigenetic modification that is strongly involved in the physiological control of genome expression. DNA methylation patterns are largely modified in cancer cells and can therefore be used to distinguish cancer cells from normal tissues. This review describes the main technologies available for the detection and the discovery of aberrantly methylated DNA patterns. It also presents the different sources of biological samples suitable for DNA methylation studies. We discuss the interest and perspectives on the use of DNA methylation measurements for cancer diagnosis through examples of methylated genes commonly documented in the literature. The discussion leads to our consideration for why DNA methylation is not commonly used in clinical practice through an examination of the main requirements that constitute a reliable biomarker. Finally, we describe the main DNA methylation inhibitors currently used in clinical trials and those that exhibit promising results. PMID:23873296

  5. Fragmentation of DNA by sonication.

    PubMed

    Sambrook, Joseph; Russell, David W

    2006-09-01

    INTRODUCTIONDNA fragmentation is often necessary prior to library construction or subcloning for DNA sequencing. This protocol describes a method for DNA fragmentation by sonication. During sonication, DNA samples are subjected to hydrodynamic shearing by exposure to brief periods of sonication. DNA that has been sonicated for excessive periods of time is extremely difficult to clone. Most sonicators will not shear DNA to a size of less than 300-500 bp, and it is tempting to continue sonication until the entire DNA population has been reduced in size. However, the yield of subclones is usually greater if sonication is stopped when the fragments of the target DNA first reach a size of ~700 bp.

  6. Cinnamate-based DNA photolithography

    NASA Astrophysics Data System (ADS)

    Feng, Lang; Romulus, Joy; Li, Minfeng; Sha, Ruojie; Royer, John; Wu, Kun-Ta; Xu, Qin; Seeman, Nadrian C.; Weck, Marcus; Chaikin, Paul

    2013-08-01

    As demonstrated by means of DNA nanoconstructs, as well as DNA functionalization of nanoparticles and micrometre-scale colloids, complex self-assembly processes require components to associate with particular partners in a programmable fashion. In many cases the reversibility of the interactions between complementary DNA sequences is an advantage. However, permanently bonding some or all of the complementary pairs may allow for flexibility in design and construction. Here, we show that the substitution of a cinnamate group for a pair of complementary bases provides an efficient, addressable, ultraviolet light-based method to bond complementary DNA covalently. To show the potential of this approach, we wrote micrometre-scale patterns on a surface using ultraviolet light and demonstrated the reversible attachment of conjugated DNA and DNA-coated colloids. Our strategy enables both functional DNA photolithography and multistep, specific binding in self-assembly processes.

  7. Functionalizing Designer DNA Crystals

    NASA Astrophysics Data System (ADS)

    Chandrasekaran, Arun Richard

    Three-dimensional crystals have been self-assembled from a DNA tensegrity triangle via sticky end interaction. The tensegrity triangle is a rigid DNA motif containing three double helical edges connected pair-wise by three four-arm junctions. The symmetric triangle contains 3 unique strands combined in a 3:3:1 ratio: 3 crossover, 3 helical and 1 central. The length of the sticky end reported previously was two nucleotides (nt) (GA:TC) and the motif with 2-helical turns of DNA per edge diffracted to 4.9 A at beam line NSLS-X25 and to 4 A at beam line ID19 at APS. The purpose of these self-assembled DNA crystals is that they can be used as a framework for hosting external guests for use in crystallographic structure solving or the periodic positioning of molecules for nanoelectronics. This thesis describes strategies to improve the resolution and to incorporate guests into the 3D lattice. The first chapter describes the effect of varying sticky end lengths and the influence of 5'-phosphate addition on crystal formation and resolution. X-ray diffraction data from beam line NSLS-X25 revealed that the crystal resolution for 1-nt (G:C) sticky end was 3.4 A. Motifs with every possible combination of 1-nt and 2-nt sticky-ended phosphorylated strands were crystallized and X-ray data were collected. The position of the 5'-phosphate on either the crossover (strand 1), helical (strand 2), or central strand (3) had an impact on the resolution of the self-assembled crystals with the 1-nt 1P-2-3 system diffracting to 2.62 A at APS and 3.1 A at NSLS-X25. The second chapter describes the sequence-specific recognition of DNA motifs with triplex-forming oligonucleotides (TFOs). This study examined the feasibility of using TFOs to bind to specific locations within a 3-turn DNA tensegrity triangle motif. The TFO 5'-TTCTTTCTTCTCT was used to target the tensegrity motif containing an appropriately embedded oligopurine.oligopyrimidine binding site. As triplex formation involving cytidine

  8. Sequence and Structure Dependent DNA-DNA Interactions

    NASA Astrophysics Data System (ADS)

    Kopchick, Benjamin; Qiu, Xiangyun

    Molecular forces between dsDNA strands are largely dominated by electrostatics and have been extensively studied. Quantitative knowledge has been accumulated on how DNA-DNA interactions are modulated by varied biological constituents such as ions, cationic ligands, and proteins. Despite its central role in biology, the sequence of DNA has not received substantial attention and ``random'' DNA sequences are typically used in biophysical studies. However, ~50% of human genome is composed of non-random-sequence DNAs, particularly repetitive sequences. Furthermore, covalent modifications of DNA such as methylation play key roles in gene functions. Such DNAs with specific sequences or modifications often take on structures other than the canonical B-form. Here we present series of quantitative measurements of the DNA-DNA forces with the osmotic stress method on different DNA sequences, from short repeats to the most frequent sequences in genome, and to modifications such as bromination and methylation. We observe peculiar behaviors that appear to be strongly correlated with the incurred structural changes. We speculate the causalities in terms of the differences in hydration shell and DNA surface structures.

  9. Touch DNA-The prospect of DNA profiles from cables.

    PubMed

    Lim, Sharon; Subhani, Zuhaib; Daniel, Barbara; Frascione, Nunzianda

    2016-05-01

    Metal theft in the railroad industry poses significant challenges to transport investigators. Cable sheaths left behind at crime scenes, if appropriately analysed, could provide valuable evidence in a forensic investigation, but attempts at recovering DNA are not routinely made. Experiments were set up to ascertain the success in DNA recovery from the surface of cable sheaths after deposition of (a) sweat, (b) extracted DNA and (c) fingermarks. Since investigators try to collect fingermarks and often treat the cables with cyanoacrylate fuming (CNA fuming) or wet powder suspensions (WPS) to enhance the marks this study investigated the recovery of DNA from fingermarks pre- and post-enhancement. The double-swab technique and mini-taping were compared as options to recover DNA from the cable sheaths. Results demonstrate that generally, there is no significant difference between using swabs or mini-tapes to recover the DNA from the non-porous cables (p>0.05). It was also illustrated that CNA fuming performed better than WPS in terms of subsequent recovery and profiling of DNA. CNA fuming resulted in an average increase in DNA recovered via swabbing and taping (more than 4× and 8×, respectively), as compared to no treatment, with 50% of the DNA recovered after CNA fuming generating full DNA profiles. Copyright © 2016 The Chartered Society of Forensic Sciences. Published by Elsevier Ireland Ltd. All rights reserved.

  10. DNA-mediated charge transport for DNA repair

    PubMed Central

    Boon, Elizabeth M.; Livingston, Alison L.; Chmiel, Nikolas H.; David, Sheila S.; Barton, Jacqueline K.

    2003-01-01

    MutY, like many DNA base excision repair enzymes, contains a [4Fe4S]2+ cluster of undetermined function. Electrochemical studies of MutY bound to a DNA-modified gold electrode demonstrate that the [4Fe4S] cluster of MutY can be accessed in a DNA-mediated redox reaction. Although not detectable without DNA, the redox potential of DNA-bound MutY is ≈275 mV versus NHE, which is characteristic of HiPiP iron proteins. Binding to DNA is thus associated with a change in [4Fe4S]3+/2+ potential, activating the cluster toward oxidation. Given that DNA charge transport chemistry is exquisitely sensitive to perturbations in base pair structure, such as mismatches, we propose that this redox process of MutY bound to DNA exploits DNA charge transport and provides a DNA signaling mechanism to scan for mismatches and lesions in vivo. PMID:14559969

  11. Multivalent Lipid--DNA Complexes: Distinct DNA Compaction Regimes

    NASA Astrophysics Data System (ADS)

    Evans, Heather M.; Ahmad, A.; Ewert, K.; Safinya, C. R.

    2004-03-01

    Cationic liposomes (CL), while intrinsically advantageous in comparison to viruses, still have limited success for gene therapy and require more study. CL spontaneously self-assemble with DNA via counterion release, forming small particles approximately 200nm in diameter. X-ray diffraction reveals CL-DNA structures that are typically a multilamellar organization of lipids with DNA intercalated between the layers. We explore the structural properties of CL-DNA complexes formed with new multivalent lipids (Ewert et al, J. Med. Chem. 2002; 45:5023) that range from 2+ to 16+. Contrary to a simple prediction for the DNA interaxial spacing d_DNA based on a geometrical space-filling model, these lipids show dramatic DNA compaction, down to d_DNA ˜ 25 ÅVariations in the membrane charge density, σ _M, lead to distinct spacing regimes. We propose that this DNA condensation is controlled by a unique locking mechanism between the DNA double helix and the large, multivalent lipid head groups. Funded by NSF DMR-0203755 and NIH GM-59288.

  12. Herpes Simplex Virus DNA Packaging without Measurable DNA Synthesis

    PubMed Central

    Church, Geoffrey A.; Dasgupta, Anindya; Wilson, Duncan W.

    1998-01-01

    Herpes simplex virus (HSV) type 1 DNA synthesis and packaging occur within the nuclei of infected cells; however, the extent to which the two processes are coupled remains unclear. Correct packaging is thought to be dependent upon DNA debranching or other repair processes, and such events commonly involve new DNA synthesis. Furthermore, the HSV UL15 gene product, essential for packaging, nevertheless localizes to sites of active DNA replication and may link the two events. It has previously been difficult to determine whether packaging requires concomitant DNA synthesis due to the complexity of these processes and of the viral life cycle; however, we have recently described a model system which simplifies the study of HSV assembly. Cells infected with HSV strain tsProt.A accumulate unpackaged capsids at the nonpermissive temperature of 39°C. Following release of the temperature block, these capsids proceed to package viral DNA in a single, synchronous wave. Here we report that, when DNA replication was inhibited prior to release of the temperature block, DNA packaging and later events in viral assembly nevertheless occurred at near-normal levels. We conclude that, under our conditions, HSV DNA packaging does not require detectable levels of DNA synthesis. PMID:9525593

  13. Structural insight into negative DNA supercoiling by DNA gyrase, a bacterial type 2A DNA topoisomerase.

    PubMed

    Papillon, Julie; Ménétret, Jean-François; Batisse, Claire; Hélye, Reynald; Schultz, Patrick; Potier, Noëlle; Lamour, Valérie

    2013-09-01

    Type 2A DNA topoisomerases (Topo2A) remodel DNA topology during replication, transcription and chromosome segregation. These multisubunit enzymes catalyze the transport of a double-stranded DNA through a transient break formed in another duplex. The bacterial DNA gyrase, a target for broad-spectrum antibiotics, is the sole Topo2A enzyme able to introduce negative supercoils. We reveal here for the first time the architecture of the full-length Thermus thermophilus DNA gyrase alone and in a cleavage complex with a 155 bp DNA duplex in the presence of the antibiotic ciprofloxacin, using cryo-electron microscopy. The structural organization of the subunits of the full-length DNA gyrase points to a central role of the ATPase domain acting like a 'crossover trap' that may help to sequester the DNA positive crossover before strand passage. Our structural data unveil how DNA is asymmetrically wrapped around the gyrase-specific C-terminal β-pinwheel domains and guided to introduce negative supercoils through cooperativity between the ATPase and β-pinwheel domains. The overall conformation of the drug-induced DNA binding-cleavage complex also suggests that ciprofloxacin traps a DNA pre-transport conformation.

  14. Structural insight into negative DNA supercoiling by DNA gyrase, a bacterial type 2A DNA topoisomerase

    PubMed Central

    Papillon, Julie; Ménétret, Jean-François; Batisse, Claire; Hélye, Reynald; Schultz, Patrick; Potier, Noëlle; Lamour, Valérie

    2013-01-01

    Type 2A DNA topoisomerases (Topo2A) remodel DNA topology during replication, transcription and chromosome segregation. These multisubunit enzymes catalyze the transport of a double-stranded DNA through a transient break formed in another duplex. The bacterial DNA gyrase, a target for broad-spectrum antibiotics, is the sole Topo2A enzyme able to introduce negative supercoils. We reveal here for the first time the architecture of the full-length Thermus thermophilus DNA gyrase alone and in a cleavage complex with a 155 bp DNA duplex in the presence of the antibiotic ciprofloxacin, using cryo-electron microscopy. The structural organization of the subunits of the full-length DNA gyrase points to a central role of the ATPase domain acting like a ‘crossover trap’ that may help to sequester the DNA positive crossover before strand passage. Our structural data unveil how DNA is asymmetrically wrapped around the gyrase-specific C-terminal β-pinwheel domains and guided to introduce negative supercoils through cooperativity between the ATPase and β-pinwheel domains. The overall conformation of the drug-induced DNA binding–cleavage complex also suggests that ciprofloxacin traps a DNA pre-transport conformation. PMID:23804759

  15. Acetylation of Werner syndrome protein (WRN): relationships with DNA damage, DNA replication and DNA metabolic activities.

    PubMed

    Lozada, Enerlyn; Yi, Jingjie; Luo, Jianyuan; Orren, David K

    2014-08-01

    Loss of Werner syndrome protein function causes Werner syndrome, characterized by increased genomic instability, elevated cancer susceptibility and premature aging. Although WRN is subject to acetylation, phosphorylation and sumoylation, the impact of these modifications on WRN's DNA metabolic function remains unclear. Here, we examined in further depth the relationship between WRN acetylation and its role in DNA metabolism, particularly in response to induced DNA damage. Our results demonstrate that endogenous WRN is acetylated somewhat under unperturbed conditions. However, levels of acetylated WRN significantly increase after treatment with certain DNA damaging agents or the replication inhibitor HU. Use of DNA repair-deficient cells or repair pathway inhibitors further increase levels of acetylated WRN, indicating that induced DNA lesions and their persistence are at least partly responsible for increased acetylation. Notably, acetylation of WRN correlates with inhibition of DNA synthesis, suggesting that replication blockage might underlie this effect. Moreover, WRN acetylation modulates its affinity for and activity on certain DNA structures, in a manner that may enhance its relative specificity for physiological substrates. Our results also show that acetylation and deacetylation of endogenous WRN is a dynamic process, with sirtuins and other histone deacetylases contributing to WRN deacetylation. These findings advance our understanding of the dynamics of WRN acetylation under unperturbed conditions and following DNA damage induction, linking this modification not only to DNA damage persistence but also potentially to replication stalling caused by specific DNA lesions. Our results are consistent with proposed metabolic roles for WRN and genomic instability phenotypes associated with WRN deficiency.

  16. Acetylation of Werner syndrome protein (WRN): relationships with DNA damage, DNA replication and DNA metabolic activities

    PubMed Central

    Lozada, Enerlyn; Yi, Jingjie; Luo, Jianyuan; Orren, David K.

    2014-01-01

    Loss of WRN function causes Werner Syndrome, characterized by increased genomic instability, elevated cancer susceptibility and premature aging. Although WRN is subject to acetylation, phosphorylation and sumoylation, the impact of these modifications on WRN’s DNA metabolic function remains unclear. Here, we examined in further depth the relationship between WRN acetylation and its role in DNA metabolism, particularly in response to induced DNA damage. Our results demonstrate that endogenous WRN is acetylated somewhat under unperturbed conditions. However, levels of acetylated WRN significantly increase after treatment with certain DNA damaging agents or the replication inhibitor hydroxyurea. Use of DNA repair-deficient cells or repair pathway inhibitors further increase levels of acetylated WRN, indicating that induced DNA lesions and their persistence are at least partly responsible for increased acetylation. Notably, acetylation of WRN correlates with inhibition of DNA synthesis, suggesting that replication blockage might underlie this effect. Moreover, WRN acetylation modulates its affinity for and activity on certain DNA structures, in a manner that may enhance its relative specificity for physiological substrates. Our results also show that acetylation and deacetylation of endogenous WRN is a dynamic process, with sirtuins and other histone deacetylases contributing to WRN deacetylation. These findings advance our understanding of the dynamics of WRN acetylation under unperturbed conditions and following DNA damage induction, linking this modification not only to DNA damage persistence but also potentially to replication stalling caused by specific DNA lesions. Our results are consistent with proposed metabolic roles for WRN and genomic instability phenotypes associated with WRN deficiency. PMID:24965941

  17. Chiral DNA packaging in DNA-cationic liposome assemblies.

    PubMed

    Zuidam, N J; Barenholz, Y; Minsky, A

    1999-09-03

    Recent studies have indicated that the structural features of DNA-lipid assemblies, dictated by the lipid composition and cationic lipid-to-DNA ratio, critically affect the efficiency of these complexes in acting as vehicles for cellular delivery of genetic material. Using circular dichroism we find that upon binding DNA, positively-charged liposomes induce a secondary conformational transition of the DNA molecules from the native B form to the C motif. Liposomes composed of positively-charged and neutral 'helper' lipids, found to be particularly effective as transfecting agents, induce - in addition to secondary conformational changes - DNA condensation into a left-handed cholesteric-like phase. A structural model is presented according to which two distinct, yet inter-related modes of DNA packaging coexist within such assemblies. The results underline the notion that subtle changes in the components of a supramolecular assembly may substantially modulate the interplay of interactions which dictate its structure and functional properties.

  18. Ultrasensitive fluorescence detection of DNA

    SciTech Connect

    Mathies, R.A.; Glazer, A.N.

    1992-01-01

    We have shown that a number of polycationic highly fluorescent dyes form complexes with double-stranded DNA (dsDNA) which are stable to electrophoresis and have characterized in detail such dsDNA complexes with TOTO (1,1[prime]-(4,4,7,7-tetramethyl-4,7-diazaundecamethylene)-bis-4-[3-methyl-2,3-dihydro-(benzo-1,3-thiazole)-2-methylidene]-quinolinium tetraiodide) and oxazole yellow dimer (YOYO; an analogue of TOTO with a benzo-1,3-oxazole in place of the benzo-1,3-thiazole). TOTO and YOYO are virtually non-fluorescent in solution, but form highly fluorescent complexes with dsDNA, up to a maximum dye to DNA bp ratio of 1:4, with >1000-fold fluorescence enhancement. We have developed an assay using YOYO for the quantitation of single-stranded and dsDNA in solution applicable over a range of DNA concentrations from 0.5 to 100 ng per ml. The fluorescent dsDNA-dye complexes allow detection of dsDNA on agarose and acrylamide gels with picogram sensitivity. We have applied these complexes in multiplex mapping experiments for accurate sizing and quantitation of restriction fragments. We have shown that in gel shift experiments the stable dsDNA-dye complexes can be used to detect heteroduplex-Muts complexes with a sensitivity comparable to radioisotopic detection.

  19. Topological Behavior of Plasmid DNA

    PubMed Central

    Higgins, N. Patrick; Vologodskii, Alexander V.

    2015-01-01

    The discovery of the B-form structure of DNA by Watson and Crick led to an explosion of research on nucleic acids in the fields of biochemistry, biophysics, and genetics. Powerful techniques were developed to reveal a myriad of different structural conformations that change B-DNA as it is transcribed, replicated, and recombined and as sister chromosomes are moved into new daughter cell compartments during cell division. This article links the original discoveries of superhelical structure and molecular topology to non-B form DNA structure and contemporary biochemical and biophysical techniques. The emphasis is on the power of plasmids for studying DNA structure and function. The conditions that trigger the formation of alternative DNA structures such as left-handed Z-DNA, inter- and intra-molecular triplexes, triple-stranded DNA, and linked catenanes and hemicatenanes are explained. The DNA dynamics and topological issues are detailed for stalled replication forks and for torsional and structural changes on DNA in front of and behind a transcription complex and a replisome. The complex and interconnected roles of topoisomerases and abundant small nucleoid association proteins are explained. And methods are described for comparing in vivo and in vitro reactions to probe and understand the temporal pathways of DNA and chromosome chemistry that occur inside living cells. PMID:26104708

  20. Topological Behavior of Plasmid DNA.

    PubMed

    Higgins, N Patrick; Vologodskii, Alexander V

    2015-04-01

    The discovery of the B-form structure of DNA by Watson and Crick led to an explosion of research on nucleic acids in the fields of biochemistry, biophysics, and genetics. Powerful techniques were developed to reveal a myriad of different structural conformations that change B-DNA as it is transcribed, replicated, and recombined and as sister chromosomes are moved into new daughter cell compartments during cell division. This article links the original discoveries of superhelical structure and molecular topology to non-B form DNA structure and contemporary biochemical and biophysical techniques. The emphasis is on the power of plasmids for studying DNA structure and function. The conditions that trigger the formation of alternative DNA structures such as left-handed Z-DNA, inter- and intra-molecular triplexes, triple-stranded DNA, and linked catenanes and hemicatenanes are explained. The DNA dynamics and topological issues are detailed for stalled replication forks and for torsional and structural changes on DNA in front of and behind a transcription complex and a replisome. The complex and interconnected roles of topoisomerases and abundant small nucleoid association proteins are explained. And methods are described for comparing in vivo and in vitro reactions to probe and understand the temporal pathways of DNA and chromosome chemistry that occur inside living cells.

  1. Mitochondrial DNA maintenance: an appraisal.

    PubMed

    Akhmedov, Alexander T; Marín-García, José

    2015-11-01

    Mitochondria play a crucial role in a variety of cellular processes ranging from energy metabolism, generation of reactive oxygen species (ROS), and Ca(2+) handling to stress responses, cell survival, and death. Malfunction of the organelle may contribute to the pathogenesis of neuromuscular disorders, cancer, premature aging, and cardiovascular diseases, including myocardial ischemia, cardiomyopathy, and heart failure. Mitochondria are unique as they contain their own genome organized into DNA-protein complexes, so-called mitochondrial nucleoids, along with multiprotein machineries, which promote mitochondrial DNA (mtDNA) replication, transcription, and repair. Although the organelle possesses almost all known nuclear DNA repair pathways, including base excision repair, mismatch repair, and recombinational repair, the proximity of mtDNA to the main sites of ROS production and the lack of protective histones may result in increased susceptibility to oxidative stress and other types of mtDNA damage. Defects in the components of these highly organized machineries, which mediate mtDNA maintenance (replication and repair), may result in accumulation of point mutations and/or deletions in mtDNA and decreased mtDNA copy number impairing mitochondrial function. This review will focus on the mechanisms of mtDNA maintenance with emphasis on the proteins implicated in these processes and their functional role in various disease conditions and aging.

  2. DNA reviews: the national DNA database of the United Kingdom.

    PubMed

    Graham, E A M

    2007-12-01

    The national DNA database in United Kingdom has now been operational for over 10 years. This review looks at the history and development of this investigative resource. From the development of commercial DNA profiling kits to the current statistics for matches obtained in relation to criminal investigation in the United Kingdom, before moving onto discussing potential future direction that national DNA databases might take, including international collaboration on a European and global scale.

  3. DNA-based watermarks using the DNA-Crypt algorithm

    PubMed Central

    Heider, Dominik; Barnekow, Angelika

    2007-01-01

    Background The aim of this paper is to demonstrate the application of watermarks based on DNA sequences to identify the unauthorized use of genetically modified organisms (GMOs) protected by patents. Predicted mutations in the genome can be corrected by the DNA-Crypt program leaving the encrypted information intact. Existing DNA cryptographic and steganographic algorithms use synthetic DNA sequences to store binary information however, although these sequences can be used for authentication, they may change the target DNA sequence when introduced into living organisms. Results The DNA-Crypt algorithm and image steganography are based on the same watermark-hiding principle, namely using the least significant base in case of DNA-Crypt and the least significant bit in case of the image steganography. It can be combined with binary encryption algorithms like AES, RSA or Blowfish. DNA-Crypt is able to correct mutations in the target DNA with several mutation correction codes such as the Hamming-code or the WDH-code. Mutations which can occur infrequently may destroy the encrypted information, however an integrated fuzzy controller decides on a set of heuristics based on three input dimensions, and recommends whether or not to use a correction code. These three input dimensions are the length of the sequence, the individual mutation rate and the stability over time, which is represented by the number of generations. In silico experiments using the Ypt7 in Saccharomyces cerevisiae shows that the DNA watermarks produced by DNA-Crypt do not alter the translation of mRNA into protein. Conclusion The program is able to store watermarks in living organisms and can maintain the original information by correcting mutations itself. Pairwise or multiple sequence alignments show that DNA-Crypt produces few mismatches between the sequences similar to all steganographic algorithms. PMID:17535434

  4. DNA-based watermarks using the DNA-Crypt algorithm.

    PubMed

    Heider, Dominik; Barnekow, Angelika

    2007-05-29

    The aim of this paper is to demonstrate the application of watermarks based on DNA sequences to identify the unauthorized use of genetically modified organisms (GMOs) protected by patents. Predicted mutations in the genome can be corrected by the DNA-Crypt program leaving the encrypted information intact. Existing DNA cryptographic and steganographic algorithms use synthetic DNA sequences to store binary information however, although these sequences can be used for authentication, they may change the target DNA sequence when introduced into living organisms. The DNA-Crypt algorithm and image steganography are based on the same watermark-hiding principle, namely using the least significant base in case of DNA-Crypt and the least significant bit in case of the image steganography. It can be combined with binary encryption algorithms like AES, RSA or Blowfish. DNA-Crypt is able to correct mutations in the target DNA with several mutation correction codes such as the Hamming-code or the WDH-code. Mutations which can occur infrequently may destroy the encrypted information, however an integrated fuzzy controller decides on a set of heuristics based on three input dimensions, and recommends whether or not to use a correction code. These three input dimensions are the length of the sequence, the individual mutation rate and the stability over time, which is represented by the number of generations. In silico experiments using the Ypt7 in Saccharomyces cerevisiae shows that the DNA watermarks produced by DNA-Crypt do not alter the translation of mRNA into protein. The program is able to store watermarks in living organisms and can maintain the original information by correcting mutations itself. Pairwise or multiple sequence alignments show that DNA-Crypt produces few mismatches between the sequences similar to all steganographic algorithms.

  5. The DNA Files

    SciTech Connect

    1998-06-09

    The DNA Files is a radio documentary which disseminates genetics information over public radio. The documentaries explore subjects which include the following: How genetics affects society. How human life began and how it evolved. Could new prenatal genetic tests hold the key to disease prevention later in life? Would a national genetic data base sacrifice individual privacy? and Should genes that may lead to the cure for cancer be privately owned? This report serves as a project update for the second quarter of 1998. It includes the spring/summer 1998 newsletter, the winter 1998 newsletter, the program clock, and the latest flyer.

  6. Switchable catalytic DNA catenanes.

    PubMed

    Hu, Lianzhe; Lu, Chun-Hua; Willner, Itamar

    2015-03-11

    Two-ring interlocked DNA catenanes are synthesized and characterized. The supramolecular catenanes show switchable cyclic catalytic properties. In one system, the catenane structure is switched between a hemin/G-quadruplex catalytic structure and a catalytically inactive state. In the second catenane structure the catenane is switched between a catalytically active Mg(2+)-dependent DNAzyme-containing catenane and an inactive catenane state. In the third system, the interlocked catenane structure is switched between two distinct catalytic structures that include the Mg(2+)- and the Zn(2+)-dependent DNAzymes.

  7. Slowing DNA Transport Using Graphene–DNA Interactions

    PubMed Central

    Banerjee, Shouvik; Wilson, James; Shim, Jiwook; Shankla, Manish; Corbin, Elise A.

    2015-01-01

    Slowing down DNA translocation speed in a nanopore is essential to ensuring reliable resolution of individual bases. Thin membrane materials enhance spatial resolution but simultaneously reduce the temporal resolution as the molecules translocate far too quickly. In this study, the effect of exposed graphene layers on the transport dynamics of both single (ssDNA) and double-stranded DNA (dsDNA) through nanopores is examined. Nanopore devices with various combinations of graphene and Al2O3 dielectric layers in stacked membrane structures are fabricated. Slow translocations of ssDNA in nanopores drilled in membranes with layers of graphene are reported. The increased hydrophobic interactions between the ssDNA and the graphene layers could explain this phenomenon. Further confirmation of the hydrophobic origins of these interactions is obtained through reporting significantly faster translocations of dsDNA through these graphene layered membranes. Molecular dynamics simulations confirm the preferential interactions of DNA with the graphene layers as compared to the dielectric layer verifying the experimental findings. Based on our findings, we propose that the integration of multiple stacked graphene layers could slow down DNA enough to enable the identification of nucleobases. PMID:26167144

  8. Applications of mass spectrometry to DNA fingerprinting and DNA sequencing

    SciTech Connect

    Jacobson, K.B.; Buchanan, M.V.; Chen, C.H.; Doktycz, M.J.; McLuckey, S.A.; Arlinghaus, H.F.

    1993-06-01

    DNA fingerprinting and sequencing rely on polyacrylamide gel electrophoresis to determine the sizes of the DNA fragments. Innovative altematives to polyacrylamide gel electrophoresis are under investigation for characterization of such fingerprinting and sequencing. One method uses stable isotopes of tin and other elements to label the DNAwhereas other procedures do not require labels. The detectors in each case are mass spectrometers that detect either the stable isotopes or the DNA fragments themselves. If successful, these methods will speed up the rate of DNA analysis by one or two orders of magnitude.

  9. Applications of mass spectrometry to DNA fingerprinting and DNA sequencing

    SciTech Connect

    Jacobson, K.B.; Buchanan, M.V.; Chen, C.H.; Doktycz, M.J.; McLuckey, S.A. ); Arlinghaus, H.F. )

    1993-01-01

    DNA fingerprinting and sequencing rely on polyacrylamide gel electrophoresis to determine the sizes of the DNA fragments. Innovative altematives to polyacrylamide gel electrophoresis are under investigation for characterization of such fingerprinting and sequencing. One method uses stable isotopes of tin and other elements to label the DNAwhereas other procedures do not require labels. The detectors in each case are mass spectrometers that detect either the stable isotopes or the DNA fragments themselves. If successful, these methods will speed up the rate of DNA analysis by one or two orders of magnitude.

  10. Foldback intercoil DNA and the mechanism of DNA transposition.

    PubMed

    Kim, Byung-Dong

    2014-09-01

    Foldback intercoil (FBI) DNA is formed by the folding back at one point of a non-helical parallel track of double-stranded DNA at as sharp as 180° and the intertwining of two double helixes within each other's major groove to form an intercoil with a diameter of 2.2 nm. FBI DNA has been suggested to mediate intra-molecular homologous recombination of a deletion and inversion. Inter-molecular homologous recombination, known as site-specific insertion, on the other hand, is mediated by the direct perpendicular approach of the FBI DNA tip, as the attP site, onto the target DNA, as the attB site. Transposition of DNA transposons involves the pairing of terminal inverted repeats and 5-7-bp tandem target duplication. FBI DNA configuration effectively explains simple as well as replicative transposition, along with the involvement of an enhancer element. The majority of diverse retrotransposable elements that employ a target site duplication mechanism is also suggested to follow the FBI DNA-mediated perpendicular insertion of the paired intercoil ends by non-homologous end-joining, together with gap filling. A genome-wide perspective of transposable elements in light of FBI DNA is discussed.

  11. Mechanism for priming DNA synthesis by yeast DNA Polymerase α

    PubMed Central

    Perera, Rajika L; Torella, Rubben; Klinge, Sebastian; Kilkenny, Mairi L; Maman, Joseph D; Pellegrini, Luca

    2013-01-01

    The DNA Polymerase α (Pol α)/primase complex initiates DNA synthesis in eukaryotic replication. In the complex, Pol α and primase cooperate in the production of RNA-DNA oligonucleotides that prime synthesis of new DNA. Here we report crystal structures of the catalytic core of yeast Pol α in unliganded form, bound to an RNA primer/DNA template and extending an RNA primer with deoxynucleotides. We combine the structural analysis with biochemical and computational data to demonstrate that Pol α specifically recognizes the A-form RNA/DNA helix and that the ensuing synthesis of B-form DNA terminates primer synthesis. The spontaneous release of the completed RNA-DNA primer by the Pol α/primase complex simplifies current models of primer transfer to leading- and lagging strand polymerases. The proposed mechanism of nucleotide polymerization by Pol α might contribute to genomic stability by limiting the amount of inaccurate DNA to be corrected at the start of each Okazaki fragment. DOI: http://dx.doi.org/10.7554/eLife.00482.001 PMID:23599895

  12. Fluorescently labeled circular DNA molecules for DNA topology and topoisomerases

    PubMed Central

    Gu, Maxwell; Berrido, Andrea; Gonzalez, Walter G.; Miksovska, Jaroslava; Chambers, Jeremy W.; Leng, Fenfei

    2016-01-01

    DNA topology plays essential roles in several fundamental biological processes, such as DNA replication, recombination, and transcription. Typically agarose gel electrophoresis is employed to study DNA topology. Since gel electrophoresis is time-consuming and labor intensive, it is desirable to develop other methods, such as fluorescence-based methods, for such studies. In this paper we report the synthesis of a type of unique fluorescence-labeled DNA molecules that can be used to study DNA topology and topoisomerases by fluorescence resonance energy transfer (FRET). Specifically, we inserted an 82 nt. synthetic DNA oligomer FL905 carrying a 42 nt. AT sequence with fluorescein and dabcyl labels into a gapped DNA molecule to generate relaxed and supercoiled pAB1_FL905. Since the fluorescence intensity of pAB1_FL905 is dependent on its supercoiling status, pAB1_FL905 is a powerful tool to study DNA topology and topoisomerases by FRET. pAB1_FL905 can also be developed into rapid and efficient high-throughput screening assays to identify inhibitors that target various DNA topoisomerases. PMID:27796331

  13. Recent progress in DNA origami technology.

    PubMed

    Endo, Masayuki; Sugiyama, Hiroshi

    2011-06-01

    DNA origami is an emerging technology for designing defined two-dimensional DNA nanostructures. In this review, we focus on and describe several types of DNA origami-related studies, as follows: (1) programmed DNA origami assembly, (2) DNA origami-templated molecular assembly, (3) design and construction of various three-dimensional DNA origami structures, (4) programmed functionalization of DNA origami and combination with top-down nanotechnology, (5) single molecular observation on a designed DNA origami, and (6) DNA nanomachines working on a DNA origami. © 2011 by John Wiley & Sons, Inc.

  14. DNA-based hybrid catalysis.

    PubMed

    Rioz-Martínez, Ana; Roelfes, Gerard

    2015-04-01

    In the past decade, DNA-based hybrid catalysis has merged as a promising novel approach to homogeneous (asymmetric) catalysis. A DNA hybrid catalysts comprises a transition metal complex that is covalently or supramolecularly bound to DNA. The chiral microenvironment and the second coordination sphere interactions provided by the DNA are key to achieve high enantioselectivities and, often, additional rate accelerations in catalysis. Nowadays, current efforts are focused on improved designs, understanding the origin of the enantioselectivity and DNA-induced rate accelerations, expanding the catalytic scope of the concept and further increasing the practicality of the method for applications in synthesis. Herein, the recent developments will be reviewed and the perspectives for the emerging field of DNA-based hybrid catalysis will be discussed. Copyright © 2015 Elsevier Ltd. All rights reserved.

  15. Development of dengue DNA vaccines.

    PubMed

    Danko, Janine R; Beckett, Charmagne G; Porter, Kevin R

    2011-09-23

    Vaccination with plasmid DNA against infectious pathogens including dengue is an active area of investigation. By design, DNA vaccines are able to elicit both antibody responses and cellular immune responses capable of mediating long-term protection. Great technical improvements have been made in dengue DNA vaccine constructs and trials are underway to study these in the clinic. The scope of this review is to highlight the rich history of this vaccine platform and the work in dengue DNA vaccines accomplished by scientists at the Naval Medical Research Center. This work resulted in the only dengue DNA vaccine tested in a clinical trial to date. Additional advancements paving the road ahead in dengue DNA vaccine development are also discussed.

  16. Mismatch repair in heteroduplex DNA.

    PubMed Central

    Wildenberg, J; Meselson, M

    1975-01-01

    DNA with base pair mismatches was prepared by annealing mixtures of genetically marked DNA from bacteriophage lambda. This heteroduplex DNA was used to transfect bacteria under conditions minimizing recombination. Genetic analysis of the progeny phages indicates that: (i) Mismatch repair occurs, usually giving rise to a DNA molecule with one chain with the genotype arising from repair and one parental chain. (ii) The frequency of repair of a given mismatch to wild type depends on the marker, ranging from 3 to 20%. (iii) Excision tracts may extend several hundred nucleotides but are usually shorter than about 2000 nucleotides. (iv) In Rec-mediated bacteriophage crosses, recombination of markers closer than about 10-3 nucleotide pairs frequently occurs by mismatch repair within heteroduplex DNA. (V) The average amount of heteroduplex DNA formed in a Rec-mediated recombination event is a few thousand nucleotide pairs. PMID:1094458

  17. Regulation of DNA replication licensing.

    PubMed

    Niida, Hiroyuki; Kitagawa, Masatoshi

    2012-12-01

    In eukaryotic cells, DNA replication is tightly regulated to occur only once per cell cycle. DNA licensing is a mechanism to guarantee this aim; that is, licensing of replication initiation is permitted during late M phase to G1 phase. The license is canceled by the start of DNA replication. Once DNA replication begins, the license is never given until the next late M phase. The licensing corresponds to the process of assembling components of the pre-replication complex (pre-RC) on the replication origin DNA. This pre-RC is the target of several different regulation systems to prevent rereplication of DNA during a single cell cycle. In this review, the regulation mechanisms mainly in mammals to control assembling components of the pre-RC will be discussed.

  18. Cryptography with DNA binary strands.

    PubMed

    Leier, A; Richter, C; Banzhaf, W; Rauhe, H

    2000-06-01

    Biotechnological methods can be used for cryptography. Here two different cryptographic approaches based on DNA binary strands are shown. The first approach shows how DNA binary strands can be used for steganography, a technique of encryption by information hiding, to provide rapid encryption and decryption. It is shown that DNA steganography based on DNA binary strands is secure under the assumption that an interceptor has the same technological capabilities as sender and receiver of encrypted messages. The second approach shown here is based on steganography and a method of graphical subtraction of binary gel-images. It can be used to constitute a molecular checksum and can be combined with the first approach to support encryption. DNA cryptography might become of practical relevance in the context of labelling organic and inorganic materials with DNA 'barcodes'.

  19. DNA denaturation in ionic solution

    NASA Astrophysics Data System (ADS)

    Maity, Arghya; Singh, Amar; Singh, Navin

    2016-05-01

    Salt or cations, present in solution play an important role in DNA denaturation and folding kinetics of DNA helix. In this work we study the thermal melting of double stranded DNA (dsDNA) molecule using Peyrard Bishop Dauxois (PBD) model. We modify the potential of H-bonding between the bases of the complimentary strands to introduce the salt and solvent effect. We choose different DNA sequences having different contents of GC pairs and calculate the melting temperatures. The melting temperature increases logarithmically with the salt concentration of the solution. The more GC base pairs in the chain enhance the stability of DNA chain at a fix salt concentration. The obtained results are in good accordance with experimental findings.

  20. Labeling nuclear DNA using DAPI.

    PubMed

    Chazotte, Brad

    2011-01-01

    A number of fluorescent stains are available that label DNA and allow easy visualization of the nucleus in interphase cells and chromosomes in mitotic cells, including Hoechst, 4',6-diamidino-2-phenylindole (DAPI), ethidium bromide, propidium iodide, and acridine orange. Although not as bright as the vital Hoechst stains for DNA, DAPI has greater photostability. It is believed that DAPI associates with the minor groove of double-stranded DNA, with a preference for the adenine-thymine clusters. Cells must be permeabilized and/or fixed for DAPI to enter the cell and to bind DNA. Fluorescence increases approximately 20-fold when DAPI is bound to double-stranded DNA. This protocol describes the use of DAPI to label nuclear DNA of cells grown in culture.

  1. Ancient DNA and human history.

    PubMed

    Slatkin, Montgomery; Racimo, Fernando

    2016-06-07

    We review studies of genomic data obtained by sequencing hominin fossils with particular emphasis on the unique information that ancient DNA (aDNA) can provide about the demographic history of humans and our closest relatives. We concentrate on nuclear genomic sequences that have been published in the past few years. In many cases, particularly in the Arctic, the Americas, and Europe, aDNA has revealed historical demographic patterns in a way that could not be resolved by analyzing present-day genomes alone. Ancient DNA from archaic hominins has revealed a rich history of admixture between early modern humans, Neanderthals, and Denisovans, and has allowed us to disentangle complex selective processes. Information from aDNA studies is nowhere near saturation, and we believe that future aDNA sequences will continue to change our understanding of hominin history.

  2. Methods of DNA methylation detection

    NASA Technical Reports Server (NTRS)

    Maki, Wusi Chen (Inventor); Filanoski, Brian John (Inventor); Mishra, Nirankar (Inventor); Rastogi, Shiva (Inventor)

    2010-01-01

    The present invention provides for methods of DNA methylation detection. The present invention provides for methods of generating and detecting specific electronic signals that report the methylation status of targeted DNA molecules in biological samples.Two methods are described, direct and indirect detection of methylated DNA molecules in a nano transistor based device. In the direct detection, methylated target DNA molecules are captured on the sensing surface resulting in changes in the electrical properties of a nano transistor. These changes generate detectable electronic signals. In the indirect detection, antibody-DNA conjugates are used to identify methylated DNA molecules. RNA signal molecules are generated through an in vitro transcription process. These RNA molecules are captured on the sensing surface change the electrical properties of nano transistor thereby generating detectable electronic signals.

  3. DNA vaccine for cancer immunotherapy

    PubMed Central

    Yang, Benjamin; Jeang, Jessica; Yang, Andrew; Wu, T C; Hung, Chien-Fu

    2014-01-01

    DNA vaccination has emerged as an attractive immunotherapeutic approach against cancer due to its simplicity, stability, and safety. Results from numerous clinical trials have demonstrated that DNA vaccines are well tolerated by patients and do not trigger major adverse effects. DNA vaccines are also very cost effective and can be administered repeatedly for long-term protection. Despite all the practical advantages, DNA vaccines face challenges in inducing potent antigen specific cellular immune responses as a result of immune tolerance against endogenous self-antigens in tumors. Strategies to enhance immunogenicity of DNA vaccines against self-antigens have been investigated including encoding of xenogeneic versions of antigens, fusion of antigens to molecules that activate T cells or trigger associative recognition, priming with DNA vectors followed by boosting with viral vector, and utilization of immunomodulatory molecules. This review will focus on discussing strategies that circumvent immune tolerance and provide updates on findings from recent clinical trials. PMID:25625927

  4. Condensed DNA in lipid microcompartments.

    PubMed

    Osfouri, Shahriar; Stano, Pasquale; Luisi, Pier Luigi

    2005-10-27

    DNA was studied in lipid reverse micelles with the aim of investigating the interactions of DNA with lipids in a restricted compartment with minimal water content. Circular dichroic (CD) spectra of DNA at low water content showed the characteristic polymer-salt-induced (psi) spectra of condensed DNA. Dynamic light scattering showed a peak around a radius of 400 nm (corresponding to DNA-containing micelles), and a peak around 2.5 nm (corresponding to "empty" micelles). Fourier Transform-IR (FT-IR) spectroscopy was carried out and analyzed in terms of three distinct states of water inside the micelle water pool, where the local concentration of DNA reached an estimated value of ca. 600 mg/mL, comparable to that found in restricted biological compartments.

  5. DNA vaccine for cancer immunotherapy.

    PubMed

    Yang, Benjamin; Jeang, Jessica; Yang, Andrew; Wu, T C; Hung, Chien-Fu

    2014-01-01

    DNA vaccination has emerged as an attractive immunotherapeutic approach against cancer due to its simplicity, stability, and safety. Results from numerous clinical trials have demonstrated that DNA vaccines are well tolerated by patients and do not trigger major adverse effects. DNA vaccines are also very cost effective and can be administered repeatedly for long-term protection. Despite all the practical advantages, DNA vaccines face challenges in inducing potent antigen specific cellular immune responses as a result of immune tolerance against endogenous self-antigens in tumors. Strategies to enhance immunogenicity of DNA vaccines against self-antigens have been investigated including encoding of xenogeneic versions of antigens, fusion of antigens to molecules that activate T cells or trigger associative recognition, priming with DNA vectors followed by boosting with viral vector, and utilization of immunomodulatory molecules. This review will focus on discussing strategies that circumvent immune tolerance and provide updates on findings from recent clinical trials.

  6. Ancient DNA and human history

    PubMed Central

    Slatkin, Montgomery; Racimo, Fernando

    2016-01-01

    We review studies of genomic data obtained by sequencing hominin fossils with particular emphasis on the unique information that ancient DNA (aDNA) can provide about the demographic history of humans and our closest relatives. We concentrate on nuclear genomic sequences that have been published in the past few years. In many cases, particularly in the Arctic, the Americas, and Europe, aDNA has revealed historical demographic patterns in a way that could not be resolved by analyzing present-day genomes alone. Ancient DNA from archaic hominins has revealed a rich history of admixture between early modern humans, Neanderthals, and Denisovans, and has allowed us to disentangle complex selective processes. Information from aDNA studies is nowhere near saturation, and we believe that future aDNA sequences will continue to change our understanding of hominin history. PMID:27274045

  7. DNA barcoding of billfishes.

    PubMed

    Hanner, Robert; Floyd, Robin; Bernard, Andrea; Collette, Bruce B; Shivji, Mahmood

    2011-10-01

    DNA barcoding is a method promising fast and accurate identification of animal species based on the sequencing of the mitochondrial c oxidase subunit (COI) gene. In this study, we explore the prospects for DNA barcoding in one particular fish group, the billfishes (suborder Xiphioidei--swordfish, marlins, spearfishes, and sailfish). We sequenced the mitochondrial COI gene from 296 individuals from the 10 currently recognized species of billfishes, and combined these data with a further 57 sequences from previously published projects. We also sequenced the rhodopsin gene from a subset of 72 individuals to allow comparison of mitochondrial results against a nuclear marker. Five of the 10 species are readily distinguishable by COI barcodes. Of the rest, the striped marlin (Kajikia audax) and white marlin (K. albida) show highly similar sequences and are not unambiguously distinguishable by barcodes alone, likewise are the three spearfishes Tetrapturus angustirostris, T. belone, and T. pfluegeri. We discuss the taxonomic status of these species groups in light of our and other data, molecular and morphological.

  8. Human DNA ligase III recognizes DNA ends by dynamic switching between two DNA-bound states.

    PubMed

    Cotner-Gohara, Elizabeth; Kim, In-Kwon; Hammel, Michal; Tainer, John A; Tomkinson, Alan E; Ellenberger, Tom

    2010-07-27

    Human DNA ligase III has essential functions in nuclear and mitochondrial DNA replication and repair and contains a PARP-like zinc finger (ZnF) that increases the extent of DNA nick joining and intermolecular DNA ligation, yet the bases for ligase III specificity and structural variation among human ligases are not understood. Here combined crystal structure and small-angle X-ray scattering results reveal dynamic switching between two nick-binding components of ligase III: the ZnF-DNA binding domain (DBD) forms a crescent-shaped surface used for DNA end recognition which switches to a ring formed by the nucleotidyl transferase (NTase) and OB-fold (OBD) domains for catalysis. Structural and mutational analyses indicate that high flexibility and distinct DNA binding domain features in ligase III assist both nick sensing and the transition from nick sensing by the ZnF to nick joining by the catalytic core. The collective results support a "jackknife model" in which the ZnF loads ligase III onto nicked DNA and conformational changes deliver DNA into the active site. This work has implications for the biological specificity of DNA ligases and functions of PARP-like zinc fingers.

  9. Human DNA ligase III bridges two DNA ends to promote specific intermolecular DNA end joining

    PubMed Central

    Kukshal, Vandna; Kim, In-Kwon; Hura, Gregory L.; Tomkinson, Alan E.; Tainer, John A.; Ellenberger, Tom

    2015-01-01

    Mammalian DNA ligase III (LigIII) functions in both nuclear and mitochondrial DNA metabolism. In the nucleus, LigIII has functional redundancy with DNA ligase I whereas LigIII is the only mitochondrial DNA ligase and is essential for the survival of cells dependent upon oxidative respiration. The unique LigIII zinc finger (ZnF) domain is not required for catalytic activity but senses DNA strand breaks and stimulates intermolecular ligation of two DNAs by an unknown mechanism. Consistent with this activity, LigIII acts in an alternative pathway of DNA double strand break repair that buttresses canonical non-homologous end joining (NHEJ) and is manifest in NHEJ-defective cancer cells, but how LigIII acts in joining intermolecular DNA ends versus nick ligation is unclear. To investigate how LigIII efficiently joins two DNAs, we developed a real-time, fluorescence-based assay of DNA bridging suitable for high-throughput screening. On a nicked duplex DNA substrate, the results reveal binding competition between the ZnF and the oligonucleotide/oligosaccharide-binding domain, one of three domains constituting the LigIII catalytic core. In contrast, these domains collaborate and are essential for formation of a DNA-bridging intermediate by adenylated LigIII that positions a pair of blunt-ended duplex DNAs for efficient and specific intermolecular ligation. PMID:26130724

  10. Human DNA Ligase III Recognizes DNA Ends by Dynamic Switching between Two DNA-Bound States

    SciTech Connect

    Cotner-Gohara, Elizabeth; Kim, In-Kwon; Hammel, Michal; Tainer, John A.; Tomkinson, Alan E.; Ellenberger, Tom

    2010-09-13

    Human DNA ligase III has essential functions in nuclear and mitochondrial DNA replication and repair and contains a PARP-like zinc finger (ZnF) that increases the extent of DNA nick joining and intermolecular DNA ligation, yet the bases for ligase III specificity and structural variation among human ligases are not understood. Here combined crystal structure and small-angle X-ray scattering results reveal dynamic switching between two nick-binding components of ligase III: the ZnF-DNA binding domain (DBD) forms a crescent-shaped surface used for DNA end recognition which switches to a ring formed by the nucleotidyl transferase (NTase) and OB-fold (OBD) domains for catalysis. Structural and mutational analyses indicate that high flexibility and distinct DNA binding domain features in ligase III assist both nick sensing and the transition from nick sensing by the ZnF to nick joining by the catalytic core. The collective results support a 'jackknife model' in which the ZnF loads ligase III onto nicked DNA and conformational changes deliver DNA into the active site. This work has implications for the biological specificity of DNA ligases and functions of PARP-like zinc fingers.

  11. Loss of maintenance DNA methylation results in abnormal DNA origin firing during DNA replication

    SciTech Connect

    Haruta, Mayumi; Shimada, Midori; Nishiyama, Atsuya; Johmura, Yoshikazu; Le Tallec, Benoît; Debatisse, Michelle; Nakanishi, Makoto

    2016-01-22

    The mammalian maintenance methyltransferase DNMT1 [DNA (cytosine-5-)-methyltransferase 1] mediates the inheritance of the DNA methylation pattern during replication. Previous studies have shown that depletion of DNMT1 causes a severe growth defect and apoptosis in differentiated cells. However, the detailed mechanisms behind this phenomenon remain poorly understood. Here we show that conditional ablation of Dnmt1 in murine embryonic fibroblasts (MEFs) resulted in an aberrant DNA replication program showing an accumulation of late-S phase replication and causing severely defective growth. Furthermore, we found that the catalytic activity and replication focus targeting sequence of DNMT1 are required for a proper DNA replication program. Taken together, our findings suggest that the maintenance of DNA methylation by DNMT1 plays a critical role in proper regulation of DNA replication in mammalian cells. - Highlights: • DNMT1 depletion results in an abnormal DNA replication program. • Aberrant DNA replication is independent of the DNA damage checkpoint in DNMT1cKO. • DNMT1 catalytic activity and RFT domain are required for proper DNA replication. • DNMT1 catalytic activity and RFT domain are required for cell proliferation.

  12. Human DNA ligase III bridges two DNA ends to promote specific intermolecular DNA end joining.

    PubMed

    Kukshal, Vandna; Kim, In-Kwon; Hura, Gregory L; Tomkinson, Alan E; Tainer, John A; Ellenberger, Tom

    2015-08-18

    Mammalian DNA ligase III (LigIII) functions in both nuclear and mitochondrial DNA metabolism. In the nucleus, LigIII has functional redundancy with DNA ligase I whereas LigIII is the only mitochondrial DNA ligase and is essential for the survival of cells dependent upon oxidative respiration. The unique LigIII zinc finger (ZnF) domain is not required for catalytic activity but senses DNA strand breaks and stimulates intermolecular ligation of two DNAs by an unknown mechanism. Consistent with this activity, LigIII acts in an alternative pathway of DNA double strand break repair that buttresses canonical non-homologous end joining (NHEJ) and is manifest in NHEJ-defective cancer cells, but how LigIII acts in joining intermolecular DNA ends versus nick ligation is unclear. To investigate how LigIII efficiently joins two DNAs, we developed a real-time, fluorescence-based assay of DNA bridging suitable for high-throughput screening. On a nicked duplex DNA substrate, the results reveal binding competition between the ZnF and the oligonucleotide/oligosaccharide-binding domain, one of three domains constituting the LigIII catalytic core. In contrast, these domains collaborate and are essential for formation of a DNA-bridging intermediate by adenylated LigIII that positions a pair of blunt-ended duplex DNAs for efficient and specific intermolecular ligation.

  13. Fluorescence studies with DNA probes: dynamic aspects of DNA structure and DNA-protein interactions

    NASA Astrophysics Data System (ADS)

    Millar, David P.; Carver, Theodore E.

    1994-08-01

    Time-resolved fluorescence measurements of optical probes incorporated at specific sites in DNA provides a new approach to studies of DNA structure and DNA:protein interactions. This approach can be used to study complex multi-state behavior, such as the folding of DNA into alternative higher order structures or the transfer of DNA between multiple binding sites on a protein. In this study, fluorescence anisotropy decay of an internal dansyl probe attached to 17/27-mer oligonucleotides was used to monitor the distribution of DNA 3' termini bound at either the polymerase of 3' to 5' exonuclease sites of the Klenow fragment of DNA polymerase I. Partitioning of the primer terminus between the two active sites of the enzyme resulted in a heterogeneous probe environment, reflected in the associative behavior of the fluorescence anisotropy decay. Analysis of the anisotropy decay with a two state model of solvent-exposed and protein-associated dansyl probes was used to determine the fraction of DNA bound at each site. We examined complexes of Klenow fragment with DNAs containing various base mismatches. Single mismatches at the primer terminus caused a 3-fold increase in the equilibrium partitioning of DNA into the exonuclease site, while two or more consecutive G:G mismatches caused the DNA to bind exclusively at the exonuclease site, with a partitioning constant at least 250- fold greater than that of the corresponding matched DNA sequence. Internal single mismatches located up to four bases from the primer terminus produced larger effects than the same mismatch at the primer terminus. These results provide insight into the recognition mechanisms that enable DNA polymerases to proofread misincorporated bases during DNA replication.

  14. Integrated Sensing Using DNA Nanoarchitectures

    DTIC Science & Technology

    2014-05-20

    Norton. Thiolated Dendrimers as Multi-Point Binding Headgroups for DNA Immobilization on Gold, Langmuir, (10 2011): 0. doi: 10.1021/la202444s...Figure 6, uses dendrimers to provide multipoint adhesion of a single stranded DNA component on a surface. Figure 6 Process for immobilizing... dendrimer (shown as a round species). These dendrimer species are Generation 3 PAMAM dendrimers with ~ 30 thiol groups to bind the dendrimer /DNA construct

  15. DNA Looping, Supercoiling and Tension

    NASA Astrophysics Data System (ADS)

    Finzi, Laura

    2007-11-01

    In complex organisms, activation or repression of gene expression by proteins bound to enhancer or silencer elements located several kilobases away from the promoter is a well recognized phenomenon. However, a mechanistic understanding of any of these multiprotein interactions is still incomplete. Part of the difficulty in characterizing long-range interactions is the complexity of the regulatory systems and also an underestimation of the effect of DNA supercoiling and tension. Supercoiling is expected to promote interactions between DNA sites because it winds the DNA into compact plectonemes in which distant DNA segments more frequently draw close. The idea that DNA is also under various levels of tension is becoming more widely accepted. Forces that stretch the double helix in vivo are the electrostatic repulsion among the negatively charged phosphate groups along the DNA backbone, the action of motor enzymes perhaps acting upon a topologically constrained sequence of DNA or chromosome segregation during cell mitosis following DNA replication. Presently, little is known about the tension acting on DNA in vivo, but characterization of how physiological regulatory processes, such as loop formation, depend on DNA tension in vitro will indicate the stretching force regimes likely to exist in vivo. In this light, the well studied CI protein of bacteriophage l, which was recently found to cause a of 3.8 kbp loop in DNA, is an ideal system in which to characterize long-range gene regulation. The large size of the loop lends itself to single-molecule techniques, which allow characterization of the dynamics of CI-mediated l DNA looping under controlled levels of supercoiling and tension. Such experiments are being used to discover the principles of long-range interactions in l and in more complex systems.

  16. Biosensors for DNA sequence detection

    NASA Technical Reports Server (NTRS)

    Vercoutere, Wenonah; Akeson, Mark

    2002-01-01

    DNA biosensors are being developed as alternatives to conventional DNA microarrays. These devices couple signal transduction directly to sequence recognition. Some of the most sensitive and functional technologies use fibre optics or electrochemical sensors in combination with DNA hybridization. In a shift from sequence recognition by hybridization, two emerging single-molecule techniques read sequence composition using zero-mode waveguides or electrical impedance in nanoscale pores.

  17. Col John Boyds Innovative DNA

    DTIC Science & Technology

    2015-09-01

    September–October 2015 | 99 COMMENTARY Col John Boyd’s Innovative DNA Col Houston R. Cantwell, USAF Surprisingly, few Airmen have heard of Col John...ter traits enabled Boyd’s success? Borrowing from the book The Innovator’s DNA by Jeff Dyer, Hal Gregersen, and Clayton Christensen, this commentary...and Clayton M. Christensen, The Innovator’s DNA : Mastering the Five Skills of Disruptive Innovators (Boston: Harvard Business Press, 2011). 3. Ibid

  18. Exons, Introns, and DNA Thermodynamics

    NASA Astrophysics Data System (ADS)

    Carlon, Enrico; Malki, Mehdi Lejard; Blossey, Ralf

    2005-05-01

    The genes of eukaryotes are characterized by protein coding fragments, the exons, interrupted by introns, i.e., stretches of DNA which do not carry useful information for protein synthesis. We have analyzed the melting behavior of randomly selected human cDNA sequences obtained from genomic DNA by removing all introns. A clear correspondence is observed between exons and melting domains. This finding may provide new insights into the physical mechanisms underlying the evolution of genes.

  19. DNA Damage Induced Neuronal Death

    DTIC Science & Technology

    1999-10-01

    Experiments are proposed to examine the molecular mechanism by which mustard chemical warfare agents induce neuronal cell death . DNA damage is the...proposed underlying mechanism of mustard-induced neuronal cell death . We propose a novel research strategy to test this hypothesis by using mice with...perturbed DNA repair to explore the relationship between mustard-induced DNA damage and neuronal cell death . Initial in vitro studies (Years 1, 2 & 3

  20. Precipitation of DNA with Ethanol.

    PubMed

    Green, Michael R; Sambrook, Joseph

    2016-12-01

    DNA can be precipitated out of solution for the removal of salts and/or for resuspension in an alternative buffer. Either ethanol or isopropanol can be used to achieve this purpose; however, the use of ethanol is generally preferred. Cations, provided as salts, are typically included to neutralize the negative charge of the DNA phosphate backbone. This method describes ethanol precipitation of DNA in microcentrifuge tubes.

  1. Biosensors for DNA sequence detection

    NASA Technical Reports Server (NTRS)

    Vercoutere, Wenonah; Akeson, Mark

    2002-01-01

    DNA biosensors are being developed as alternatives to conventional DNA microarrays. These devices couple signal transduction directly to sequence recognition. Some of the most sensitive and functional technologies use fibre optics or electrochemical sensors in combination with DNA hybridization. In a shift from sequence recognition by hybridization, two emerging single-molecule techniques read sequence composition using zero-mode waveguides or electrical impedance in nanoscale pores.

  2. [Legal implication of DNA profiling].

    PubMed

    Doutremepuich, Christian

    2012-06-01

    In recent years, DNA profiling has been used regularly by the justice system, and has seen a number of improvements, with the need for fewer cells, more efficient DNA extraction and purification, and more rapid genotyping. These methods can now identify an individual more rapidly, from a corpse, blood stain, sperm or epithelial cells, by comparison with familial profiles. In France, DNA profiling can only be ordered by a judge.

  3. Overstretching of a 30 bp DNA duplex studied with steered molecular dynamics simulation: Effects of structural defects on structure and force-extension relation

    NASA Astrophysics Data System (ADS)

    Li, H.; Gisler, T.

    2009-11-01

    Single-molecule experiments on polymeric DNA show that the molecule can be overstretched at nearly constant force by about 70% beyond its relaxed contour length. In this publication we use steered molecular dynamics (MD) simulation to study the effect of structural defects on force-extension curves and structures at high elongation in a 30 base pair duplex pulled by its torsionally unconstrained 5' -5' ends. The defect-free duplex shows a plateau in the force-extension curve at 120pN in which large segments with inclined and paired bases (“S-DNA”) near both ends of the duplex coexist with a central B-type segment separated from the former by small denaturation bubbles. In the presence of a base mismatch or a nick, force-extension curves are very similar to the ones of the defect-free duplex. For the duplex with a base mismatch, S-type segments with highly inclined base pairs are not observed; rather, the overstretched duplex consists of B-type segments separated by denaturation bubbles. The nicked duplex evolves, via a two-step transition, into a two-domain structure characterized by a large S-type segment coexisting with several short S-type segments which are separated by short denaturation bubbles. Our results suggest that in the presence of nicks the force-extension curve of highly elongated duplex DNA might reflect locally highly inhomogeneous stretching. Supplementary material in the form of a PDF file available from the Journal web page at 10.1140/epje/i2009-10524-5 and is accessible for authorised users.

  4. DNA Damage, Homology-Directed Repair, and DNA Methylation

    PubMed Central

    Angrisano, Tiziana; Morano, Annalisa; Lee, Bongyong; Pardo, Alba Di; Messina, Samantha; Iuliano, Rodolfo; Fusco, Alfredo; Santillo, Maria R; Muller, Mark T; Chiariotti, Lorenzo; Gottesman, Max E; Avvedimento, Enrico V

    2007-01-01

    To explore the link between DNA damage and gene silencing, we induced a DNA double-strand break in the genome of Hela or mouse embryonic stem (ES) cells using I-SceI restriction endonuclease. The I-SceI site lies within one copy of two inactivated tandem repeated green fluorescent protein (GFP) genes (DR-GFP). A total of 2%–4% of the cells generated a functional GFP by homology-directed repair (HR) and gene conversion. However, ~50% of these recombinants expressed GFP poorly. Silencing was rapid and associated with HR and DNA methylation of the recombinant gene, since it was prevented in Hela cells by 5-aza-2′-deoxycytidine. ES cells deficient in DNA methyl transferase 1 yielded as many recombinants as wild-type cells, but most of these recombinants expressed GFP robustly. Half of the HR DNA molecules were de novo methylated, principally downstream to the double-strand break, and half were undermethylated relative to the uncut DNA. Methylation of the repaired gene was independent of the methylation status of the converting template. The methylation pattern of recombinant molecules derived from pools of cells carrying DR-GFP at different loci, or from an individual clone carrying DR-GFP at a single locus, was comparable. ClustalW analysis of the sequenced GFP molecules in Hela and ES cells distinguished recombinant and nonrecombinant DNA solely on the basis of their methylation profile and indicated that HR superimposed novel methylation profiles on top of the old patterns. Chromatin immunoprecipitation and RNA analysis revealed that DNA methyl transferase 1 was bound specifically to HR GFP DNA and that methylation of the repaired segment contributed to the silencing of GFP expression. Taken together, our data support a mechanistic link between HR and DNA methylation and suggest that DNA methylation in eukaryotes marks homologous recombined segments. PMID:17616978

  5. Mediators of homologous DNA pairing.

    PubMed

    Zelensky, Alex; Kanaar, Roland; Wyman, Claire

    2014-10-09

    Homologous DNA pairing and strand exchange are at the core of homologous recombination. These reactions are promoted by a DNA-strand-exchange protein assembled into a nucleoprotein filament comprising the DNA-pairing protein, ATP, and single-stranded DNA. The catalytic activity of this molecular machine depends on control of its dynamic instability by accessory factors. Here we discuss proteins known as recombination mediators that facilitate formation and functional activation of the DNA-strand-exchange protein filament. Although the basics of homologous pairing and DNA-strand exchange are highly conserved in evolution, differences in mediator function are required to cope with differences in how single-stranded DNA is packaged by the single-stranded DNA-binding protein in different species, and the biochemical details of how the different DNA-strand-exchange proteins nucleate and extend into a nucleoprotein filament. The set of (potential) mediator proteins has apparently expanded greatly in evolution, raising interesting questions about the need for additional control and coordination of homologous recombination in more complex organisms. Copyright © 2014 Cold Spring Harbor Laboratory Press; all rights reserved.

  6. Mediators of Homologous DNA Pairing

    PubMed Central

    Zelensky, Alex; Kanaar, Roland; Wyman, Claire

    2014-01-01

    Homologous DNA pairing and strand exchange are at the core of homologous recombination. These reactions are promoted by a DNA-strand-exchange protein assembled into a nucleoprotein filament comprising the DNA-pairing protein, ATP, and single-stranded DNA. The catalytic activity of this molecular machine depends on control of its dynamic instability by accessory factors. Here we discuss proteins known as recombination mediators that facilitate formation and functional activation of the DNA-strand-exchange protein filament. Although the basics of homologous pairing and DNA-strand exchange are highly conserved in evolution, differences in mediator function are required to cope with differences in how single-stranded DNA is packaged by the single-stranded DNA-binding protein in different species, and the biochemical details of how the different DNA-strand-exchange proteins nucleate and extend into a nucleoprotein filament. The set of (potential) mediator proteins has apparently expanded greatly in evolution, raising interesting questions about the need for additional control and coordination of homologous recombination in more complex organisms. PMID:25301930

  7. Stabbing simulations and DNA transfer.

    PubMed

    Samie, Lydie; Hicks, Tacha; Castella, Vincent; Taroni, Franco

    2016-05-01

    Technical developments have made it possible to analyze very low amounts of DNA. This has many advantages, but the drawback of this technological progress is that interpretation of the results becomes increasingly complex: the number of mixed DNA profiles increased relatively to single source DNA profiles and stochastic effects in the DNA profile, such as drop-in and drop-out, are more frequently observed. Moreover, the relevance of low template DNA material regarding the activities alleged is not as straightforward as it was a few years ago, when for example large quantities of blood were recovered. The possibility of secondary and tertiary transfer is now becoming an issue. The purpose of this research is twofold: first, to study the transfer of DNA from the handler and secondly, to observe if handlers would transfer DNA from persons closely connected to them. We chose to mimic cases where the offender would attack a person with a knife. As a first approach, we envisaged that the defense would not give an alternative explanation for the origin of the DNA. In our transfer experiments (4 donors, 16 experiments each, 64 traces), 3% of the traces were single DNA profiles. Most of the time, the DNA profile of the person handling the knife was present as the major profile: in 83% of the traces the major contributor profile corresponded to the stabber's DNA profile (in single stains and mixtures). Mixture with no clear major/minor fraction (12%) were observed. 5% of the traces were considered of insufficient quality (more than 3 contributors, presence of a few minor peaks). In that case, we considered that the stabber's DNA was absent. In our experiments, no traces allowed excluding the stabber, however it must be noted that precautions were taken to minimize background DNA as knives were cleaned before the experiments. DNA profiles of the stabber's colleagues were not observed. We hope that this study will allow for a better understanding of the transfer mechanism and

  8. Optical Nonlinearity in DNA Films.

    DTIC Science & Technology

    1988-06-15

    properties of DNA implied by our discovery of counter ion effects on the optical polarizability of DNA ( Weidlich , Lindsay and Rupprechi. Biopolymers 26, 439...spectra. 2a: "The Origin of the A to B Transition in DNA Fibers and Filns" S.M. Lindsay. S.A. Lee. J.W. Powell. T. Weidlich . C. DeMarco, G.D. Lewen...Scattering Study of the Hydration of Li- and Na-DNA films" S.A. Lee. S.M. Lindsay. J.W. Powell. T. Weidlich . N.J. Tao and G.D. Lewen. Biopolymers. 26. 1637

  9. DNA Rearrangements through Spatial Graphs

    NASA Astrophysics Data System (ADS)

    Jonoska, Nataša; Saito, Masahico

    The paper is a short overview of a recent model of homologous DNA recombination events guided by RNA templates that have been observed in certain species of ciliates. This model uses spatial graphs to describe DNA rearrangements and show how gene recombination can be modeled as topological braiding of the DNA. We show that a graph structure, which we refer to as an assembly graph, containing only 1- and 4-valent rigid vertices can provide a physical representation of the DNA at the time of recombination. With this representation, 4-valent vertices correspond to the alignment of the recombination sites, and we model the actual recombination event as smoothing of these vertices.

  10. DNA methylation dynamics in neurogenesis.

    PubMed

    Wang, Zhiqin; Tang, Beisha; He, Yuquan; Jin, Peng

    2016-03-01

    Neurogenesis is not limited to the embryonic stage, but continually proceeds in the adult brain throughout life. Epigenetic mechanisms, including DNA methylation, histone modification and noncoding RNA, play important roles in neurogenesis. For decades, DNA methylation was thought to be a stable modification, except for demethylation in the early embryo. In recent years, DNA methylation has proved to be dynamic during development. In this review, we summarize the latest understanding about DNA methylation dynamics in neurogenesis, including the roles of different methylation forms (5-methylcytosine, 5-hydroxymethylcytosine, 5-formylcytosine and 5-carboxylcytosine), as well as their 'writers', 'readers' and interactions with histone modifications.

  11. DNA attachment to support structures

    DOEpatents

    Balhorn, Rodney L.; Barry, Christopher H.

    2002-01-01

    Microscopic beads or other structures are attached to nucleic acids (DNA) using a terminal transferase. The transferase adds labeled dideoxy nucleotide bases to the ends of linear strands of DNA. The labels, such as the antigens digoxigenin and biotin, bind to the antibody compounds or other appropriate complementary ligands, which are bound to the microscopic beads or other support structures. The method does not require the synthesis of a synthetic oligonucleotide probe. The method can be used to tag or label DNA even when the DNA has an unknown sequence, has blunt ends, or is a very large fragment (e.g., >500 kilobase pairs).

  12. Piezoresistivity in single DNA molecules

    PubMed Central

    Bruot, Christopher; Palma, Julio L.; Xiang, Limin; Mujica, Vladimiro; Ratner, Mark A.; Tao, Nongjian

    2015-01-01

    Piezoresistivity is a fundamental property of materials that has found many device applications. Here we report piezoresistivity in double helical DNA molecules. By studying the dependence of molecular conductance and piezoresistivity of single DNA molecules with different sequences and lengths, and performing molecular orbital calculations, we show that the piezoresistivity of DNA is caused by force-induced changes in the π–π electronic coupling between neighbouring bases, and in the activation energy of hole hopping. We describe the results in terms of thermal activated hopping model together with the ladder-based mechanical model for DNA proposed by de Gennes. PMID:26337293

  13. DNA damage and breast cancer

    PubMed Central

    Davis, Jennifer D; Lin, Shiaw-Yih

    2011-01-01

    Cancer is intimately related to the accumulation of DNA damage, and repair failures (including mutation prone repair and hyperactive repair systems). This article relates current clinical categories for breast cancer and their common DNA damage repair defects. Information is included on the potential for accumulation of DNA damage in the breast tissue of a woman during her lifetime and the role of DNA damage in breast cancer development. We then cover endogenous and exogenous sources of DNA damage, types of DNA damage repair and basic signal transduction pathways for three gene products involved in the DNA damage response system; namely BRCA1, BRIT1 and PARP-1. These genes are often considered tumor suppressors because of their roles in DNA damage response and some are under clinical investigation as likely sources for effective new drugs to treat breast cancers. Finally we discuss some of the problems of DNA damage repair systems in cancer and the conundrum of hyper-active repair systems which can introduce mutations and confer a survival advantage to certain types of cancer cells. PMID:21909479

  14. DNA Methylation-Targeted Drugs.

    PubMed

    Da Costa, Elodie M; McInnes, Gabrielle; Beaudry, Annie; Raynal, Noël J-M

    Targeting DNA hypermethylation, using nucleoside analogs, is an efficient approach to reprogram cancer cell epigenome leading to reduced proliferation, increased differentiation, recognition by the immune system, and ultimately cancer cell death. DNA methyltransferase inhibitors have been approved for the treatment of myelodysplastic syndromes, chronic myelomonocytic leukemia, and acute myelogenous leukemia. To improve clinical efficacy and overcome mechanisms of drug resistance, a second generation of DNA methyltransferase inhibitors has been designed and is currently in clinical trials. Although efficient in monotherapy against hematologic malignancies, the potential of DNA methyltransferase inhibitors to synergize with small molecules targeting chromatin or immunotherapy will provide additional opportunities for their future clinical application against leukemia and solid tumors.

  15. DNA nanotechnology-enabled biosensors.

    PubMed

    Chao, Jie; Zhu, Dan; Zhang, Yinan; Wang, Lianhui; Fan, Chunhai

    2016-02-15

    Biosensors employ biological molecules to recognize the target and utilize output elements which can translate the biorecognition event into electrical, optical or mass-sensitive signals to determine the quantities of the target. DNA-based biosensors, as a sub-field to biosensor, utilize DNA strands with short oligonucleotides as probes for target recognition. Although DNA-based biosensors have offered a promising alternative for fast, simple and cheap detection of target molecules, there still exist key challenges including poor stability and reproducibility that hinder their competition with the current gold standard for DNA assays. By exploiting the self-recognition properties of DNA molecules, researchers have dedicated to make versatile DNA nanostructures in a highly rigid, controllable and functionalized manner, which offers unprecedented opportunities for developing DNA-based biosensors. In this review, we will briefly introduce the recent advances on design and fabrication of static and dynamic DNA nanostructures, and summarize their applications for fabrication and functionalization of DNA-based biosensors. Copyright © 2015 Elsevier B.V. All rights reserved.

  16. DNA/genetic vaccination (minireview).

    PubMed

    Kucerova, L

    1998-01-01

    An important new approach to vaccination is plasmid DNA injection in vivo that can elicit an immune response against protein(s) encoded. Antigen that is expressed from the in vivo transfected cells induces both humoral and cellular immune response. DNA immunization is generally applicable for a wide range of proteins. It can provide an organism with immunity against viruses, bacteria, parasites, and tumors. DNA vaccines can overcome the disadvantages of vaccines presently used as well as provide various new vaccines that are currently not available. This minireview provides an overview of evaluated DNA vaccine candidates against infectious agents and certain cancers.

  17. The human specialized DNA polymerases and non-B DNA: vital relationships to preserve genome integrity.

    PubMed

    Boyer, Anne-Sophie; Grgurevic, Srdana; Cazaux, Christophe; Hoffmann, Jean-Sébastien

    2013-11-29

    In addition to the canonical right-handed double helix, DNA molecule can adopt several other non-B DNA structures. Readily formed in the genome at specific DNA repetitive sequences, these secondary conformations present a distinctive challenge for progression of DNA replication forks. Impeding normal DNA synthesis, cruciforms, hairpins, H DNA, Z DNA and G4 DNA considerably impact the genome stability and in some instances play a causal role in disease development. Along with previously discovered dedicated DNA helicases, the specialized DNA polymerases emerge as major actors performing DNA synthesis through these distorted impediments. In their new role, they are facilitating DNA synthesis on replication stalling sites formed by non-B DNA structures and thereby helping the completion of DNA replication, a process otherwise crucial for preserving genome integrity and concluding normal cell division. This review summarizes the evidence gathered describing the function of specialized DNA polymerases in replicating DNA through non-B DNA structures.

  18. ANIMAL DNA IN PCR REAGENTS PLAGUES ANCIENT DNA RESEARCH

    EPA Science Inventory

    Ancient DNA analysis is becoming widespread. These studies use polymerase chain reaction (PCR) to amplify minute quantities of heavily damaged template. Unusual steps are taken to achieve the sensitivity necessary to detect ancient DNA, including high-cycle PCR amplification targ...

  19. Pea amyloplast DNA is qualitatively similar to pea chloroplast DNA

    NASA Technical Reports Server (NTRS)

    Gaynor, J. J.

    1984-01-01

    Amyloplast DNA (apDNA), when subjected to digestion with restriction endonucleases, yields patterns nearly identical to that of DNA from mature pea chloroplasts (ctDNA). Southern transfers of apDNA and ctDNA, probed with the large subunit (LS) gene of ribulose-1,5-bisphosphate carboxylase (Rubisco), shows hybridization to the expected restriction fragments for both apDNA and ctDNA. However, Northern transfers of total RNA from chloroplasts and amyloplasts, probed again with the LS gene of Rubisco, shows that no detectable LS meggage is found in amyloplasts although LS expression in mature chloroplasts is high. Likewise, two dimensional polyacrylamide gel electrophoresis of etiolated gravisensitive pea tissue shows that both large and small subunits of Rubisco are conspicuously absent; however, in greening tissue these two constitute the major soluble proteins. These findings suggest that although the informational content of these two organelle types is equivalent, gene expression is quite different and is presumably under nuclear control.

  20. Flexible DNA bending in HU-DNA cocrystal structures.

    PubMed

    Swinger, Kerren K; Lemberg, Kathryn M; Zhang, Ying; Rice, Phoebe A

    2003-07-15

    HU and IHF are members of a family of prokaryotic proteins that interact with the DNA minor groove in a sequence-specific (IHF) or non-specific (HU) manner to induce and/or stabilize DNA bending. HU plays architectural roles in replication initiation, transcription regulation and site-specific recombination, and is associated with bacterial nucleoids. Cocrystal structures of Anabaena HU bound to DNA (1P71, 1P78, 1P51) reveal that while underlying proline intercalation and asymmetric charge neutralization mechanisms of DNA bending are similar for IHF and HU, HU stabilizes different DNA bend angles ( approximately 105-140 degrees ). The two bend angles within a single HU complex are not coplanar, and the resulting dihedral angle is consistent with negative supercoiling. Comparison of HU-DNA and IHF-DNA structures suggests that sharper bending is correlated with longer DNA binding sites and smaller dihedral angles. An HU-induced bend may be better modeled as a hinge, not a rigid bend. The ability to induce or stabilize varying bend angles is consistent with HU's role as an architectural cofactor in many different systems that may require differing geometries.

  1. DNA-templated silver nanoclusters for multiplexed fluorescent DNA detection.

    PubMed

    Zhang, Ying; Zhu, Changfeng; Zhang, Lei; Tan, Chaoliang; Yang, Jian; Chen, Bo; Wang, Lianhui; Zhang, Hua

    2015-03-25

    Novel label-free/conjugation-free molecular beacons are designed based on DNA templated-silver nanoclusters for multiplexed DNA detection. The assay is implemented in solution, which makes it easy for the in-situ and real-time analysis. This study demonstrates a new method for multiplexd detection of biological molecules by using fluorescent Ag nanocluster-based molecular beacon probes.

  2. ANIMAL DNA IN PCR REAGENTS PLAGUES ANCIENT DNA RESEARCH

    EPA Science Inventory

    Ancient DNA analysis is becoming widespread. These studies use polymerase chain reaction (PCR) to amplify minute quantities of heavily damaged template. Unusual steps are taken to achieve the sensitivity necessary to detect ancient DNA, including high-cycle PCR amplification targ...

  3. Pea amyloplast DNA is qualitatively similar to pea chloroplast DNA

    NASA Technical Reports Server (NTRS)

    Gaynor, J. J.

    1984-01-01

    Amyloplast DNA (apDNA), when subjected to digestion with restriction endonucleases, yields patterns nearly identical to that of DNA from mature pea chloroplasts (ctDNA). Southern transfers of apDNA and ctDNA, probed with the large subunit (LS) gene of ribulose-1,5-bisphosphate carboxylase (Rubisco), shows hybridization to the expected restriction fragments for both apDNA and ctDNA. However, Northern transfers of total RNA from chloroplasts and amyloplasts, probed again with the LS gene of Rubisco, shows that no detectable LS meggage is found in amyloplasts although LS expression in mature chloroplasts is high. Likewise, two dimensional polyacrylamide gel electrophoresis of etiolated gravisensitive pea tissue shows that both large and small subunits of Rubisco are conspicuously absent; however, in greening tissue these two constitute the major soluble proteins. These findings suggest that although the informational content of these two organelle types is equivalent, gene expression is quite different and is presumably under nuclear control.

  4. Loss of maintenance DNA methylation results in abnormal DNA origin firing during DNA replication.

    PubMed

    Haruta, Mayumi; Shimada, Midori; Nishiyama, Atsuya; Johmura, Yoshikazu; Le Tallec, Benoît; Debatisse, Michelle; Nakanishi, Makoto

    2016-01-22

    The mammalian maintenance methyltransferase DNMT1 [DNA (cytosine-5-)-methyltransferase 1] mediates the inheritance of the DNA methylation pattern during replication. Previous studies have shown that depletion of DNMT1 causes a severe growth defect and apoptosis in differentiated cells. However, the detailed mechanisms behind this phenomenon remain poorly understood. Here we show that conditional ablation of Dnmt1 in murine embryonic fibroblasts (MEFs) resulted in an aberrant DNA replication program showing an accumulation of late-S phase replication and causing severely defective growth. Furthermore, we found that the catalytic activity and replication focus targeting sequence of DNMT1 are required for a proper DNA replication program. Taken together, our findings suggest that the maintenance of DNA methylation by DNMT1 plays a critical role in proper regulation of DNA replication in mammalian cells.

  5. Influence of DNA methylation on positioning and DNA flexibility of nucleosomes with pericentric satellite DNA.

    PubMed

    Osakabe, Akihisa; Adachi, Fumiya; Arimura, Yasuhiro; Maehara, Kazumitsu; Ohkawa, Yasuyuki; Kurumizaka, Hitoshi

    2015-10-01

    DNA methylation occurs on CpG sites and is important to form pericentric heterochromatin domains. The satellite 2 sequence, containing seven CpG sites, is located in the pericentric region of human chromosome 1 and is highly methylated in normal cells. In contrast, the satellite 2 region is reportedly hypomethylated in cancer cells, suggesting that the methylation status may affect the chromatin structure around the pericentric regions in tumours. In this study, we mapped the nucleosome positioning on the satellite 2 sequence in vitro and found that DNA methylation modestly affects the distribution of the nucleosome positioning. The micrococcal nuclease assay revealed that the DNA end flexibility of the nucleosomes changes, depending on the DNA methylation status. However, the structures and thermal stabilities of the nucleosomes are unaffected by DNA methylation. These findings provide new information to understand how DNA methylation functions in regulating pericentric heterochromatin formation and maintenance in normal and malignant cells.

  6. Repetitive DNA in three Gramineae species with low DNA content.

    PubMed

    Deshpande, V G; Ranjekar, P K

    1980-08-01

    The genomes of three Gramineae species, namely finger millet (Eleusine coracana), pearl millet (Pennisetum americanum) and rice (Oryza sativa) are characterized by studying their DNA denaturation-reassociation properties. The reassociation kinetics measurement of the sonicated DNA (500--700 nucleotide pairs) indicate the presence of a heterogeneous, repetitive DNA fraction accounting for 49--54% of the total DNA in all three species. From the cot 1/2 value of the slow reassociating DNA, the genome size is estimated as 3.0 X 10(8) np in finger millet, 7.8 X 10(8) np in pearl millet and 9.0 X 10(8) np in rice. The melting patterns of the total DNAs reveal Tm value of 88.6 degrees C in the case of pearl millet and 85.0 degrees C in the case of finger millet and rice. Total repetitive and cot 1.0 DNA fractions in all the three species are isolated and their melting properties are compared with those of respective sonicated DNAs. In finger millet, the Tm values of cot 25 and cot 1 fractions are lower by 10.8 degrees C and 12.8 degrees C, respectively, than that of sonicated DNA and thus exhibit the presence of a base pair mismatch in the range of 10.8--12.8%. In rice, the Tm values of the fractions cot 50 and cot 1 are slightly lower than that of sonicated DNA and reveal a nucleotide mismatching of only 1.8--3.8%. In the case of pearl millet cot 10 DNA fraction a high-melting DNA component (Tm = 92 degrees C) representing 12% of the total cot 10 DNA and a low-melting component with a Tm of 78 degrees C are present. In cot 1 DNA fraction of pearl millet the proportion of the high-melting component is 35% and it has a Tm or 94.8 degrees C. Optical reassociation studies of cot 1.0 DNA fractions have revealed the presence of two kinetically distinct components, namely minor fast-reassociating and major slow-reassociating, having complexities in the range of 330--390 np and 1.28 X 10(5)--6.0 X 10(5) np, respectively in pearl millet and rice and only one DNA fraction with an

  7. Variola type IB DNA topoisomerase: DNA binding and supercoil unwinding using engineered DNA minicircles.

    PubMed

    Anderson, Breeana G; Stivers, James T

    2014-07-08

    Type IB topoisomerases unwind positive and negative DNA supercoils and play a key role in removing supercoils that would otherwise accumulate at replication and transcription forks. An interesting question is whether topoisomerase activity is regulated by the topological state of the DNA, thereby providing a mechanism for targeting the enzyme to highly supercoiled DNA domains in genomes. The type IB enzyme from variola virus (vTopo) has proven to be useful in addressing mechanistic questions about topoisomerase function because it forms a reversible 3'-phosphotyrosyl adduct with the DNA backbone at a specific target sequence (5'-CCCTT-3') from which DNA unwinding can proceed. We have synthesized supercoiled DNA minicircles (MCs) containing a single vTopo target site that provides highly defined substrates for exploring the effects of supercoil density on DNA binding, strand cleavage and ligation, and unwinding. We observed no topological dependence for binding of vTopo to these supercoiled MC DNAs, indicating that affinity-based targeting to supercoiled DNA regions by vTopo is unlikely. Similarly, the cleavage and religation rates of the MCs were not topologically dependent, but topoisomers with low superhelical densities were found to unwind more slowly than highly supercoiled topoisomers, suggesting that reduced torque at low superhelical densities leads to an increased number of cycles of cleavage and ligation before a successful unwinding event. The K271E charge reversal mutant has an impaired interaction with the rotating DNA segment that leads to an increase in the number of supercoils that were unwound per cleavage event. This result provides evidence that interactions of the enzyme with the rotating DNA segment can restrict the number of supercoils that are unwound. We infer that both superhelical density and transient contacts between vTopo and the rotating DNA determine the efficiency of supercoil unwinding. Such determinants are likely to be important in

  8. Spermatogenesis, DNA damage and DNA repair mechanisms in male infertility.

    PubMed

    Gunes, Sezgin; Al-Sadaan, Maha; Agarwal, Ashok

    2015-09-01

    Spermatogenesis is a complex process of proliferation and differentiation during male germ cell development involving mitosis, meiosis and spermiogenesis. Endogenous and exogenous physical, chemical and biological sources modify the genome of spermatozoa. The genomic integrity and stability of the sperm is protected by DNA repair mechanisms. In the male germline cells, DNA repair mechanisms include nucleotide excision repair, base excision repair, DNA mismatch repair, double strand break repair and post-replication repair. Defects in repair mechanisms cause arrest of spermatogenesis and abnormal recombination, ultimately resulting in male infertility. This review focuses on molecular mechanisms of the DNA repair pathways, DNA repair defects and male infertility. Copyright © 2015 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

  9. Zipping and Unzipping of Adenylate Kinase: Atomistic Insights into the Ensemble of Open ↔ Closed Transitions

    PubMed Central

    Beckstein, Oliver; Denning, Elizabeth J.; Perilla, Juan R.; Woolf, Thomas B.

    2009-01-01

    Adenylate kinase (AdK), a phosphotransferase enzyme, plays an important role in cellular energy homeostasis. It undergoes a large conformational change between an open and a closed state, even in the absence of substrate. We investigate the apo-AdK transition at the atomic level both with free energy calculations and our new dynamic importance sampling (DIMS) molecular dynamics (MD) method. DIMS is shown to sample biologically relevant conformations as verified by comparing an ensemble of hundreds of DIMS transitions to AdK crystal structure intermediates. The simulations reveal in atomic detail how hinge regions partially and intermittently unfold during the transition. Conserved salt bridges are seen to have important structural and dynamic roles; in particular four ionic bonds are identified that open in a sequential, zipper-like fashion and thus dominate the free energy landscape of the transition. Transitions between the closed and open conformations only have to overcome moderate free energy barriers. Unexpectedly, the closed and open state encompass broad free energy basins that contain conformations differing in domain hinge motions by up to 40°. The significance of these extended states is discussed in relation to recent experimental FRET measurements. Taken together, these results demonstrate how a small number of cooperative key interactions can shape the overall dynamics of an enzyme and suggest an “all-or-nothing” mechanism for the opening and closing of AdK. Our efficient DIMS-MD computer simulation approach can provide a detailed picture of a functionally important macromolecular transition and thus help to interpret and suggest experiments to probe the conformational landscape of dynamic proteins such as AdK. PMID:19751742

  10. Unzip the Truth: Results from the Fort Peck Men's Sexual Health Intervention and Evaluation Study

    ERIC Educational Resources Information Center

    Rink, Elizabeth; Ricker, Adriann; FourStar, Kris; Anastario, Michael

    2016-01-01

    American Indian (AI) men experience sexual and reproductive health disparities including sexually transmitted infections, unplanned pregnancy, absent fatherhood, and intimate relationship violence. Using a case-control study within a community-based participatory research framework, we investigated the effectiveness of a sexual and reproductive…

  11. Unzip the Truth: Results from the Fort Peck Men's Sexual Health Intervention and Evaluation Study

    ERIC Educational Resources Information Center

    Rink, Elizabeth; Ricker, Adriann; FourStar, Kris; Anastario, Michael

    2016-01-01

    American Indian (AI) men experience sexual and reproductive health disparities including sexually transmitted infections, unplanned pregnancy, absent fatherhood, and intimate relationship violence. Using a case-control study within a community-based participatory research framework, we investigated the effectiveness of a sexual and reproductive…

  12. Charge‐Induced Unzipping of Isolated Proteins to a Defined Secondary Structure

    PubMed Central

    González Flórez, Ana Isabel; Mucha, Eike; Ahn, Doo‐Sik; Gewinner, Sandy; Schöllkopf, Wieland; Pagel, Kevin

    2016-01-01

    Abstract Here we present a combined experimental and theoretical study on the secondary structure of isolated proteins as a function of charge state. In infrared spectra of the proteins ubiquitin and cytochrome c, amide I (C=O stretch) and amide II (N–H bend) bands can be found at positions that are typical for condensed‐phase proteins. For high charge states a new band appears, substantially red‐shifted from the amide II band observed at lower charge states. The observations are interpreted in terms of Coulomb‐driven transitions in secondary structures from mostly helical to extended C5‐type hydrogen‐bonded structures. Support for this interpretation comes from simple energy considerations as well as from quantum chemical calculations on model peptides. This transition in secondary structure is most likely universal for isolated proteins that occur in mass spectrometric experiments. PMID:26847383

  13. Unzipping the role of chirality in nanoscale self-assembly of tripeptide hydrogels

    NASA Astrophysics Data System (ADS)

    Marchesan, Silvia; Waddington, Lynne; Easton, Christopher D.; Winkler, David A.; Goodall, Liz; Forsythe, John; Hartley, Patrick G.

    2012-10-01

    Change of chirality is a useful tool to manipulate the aqueous self-assembly behaviour of uncapped, hydrophobic tripeptides. In contrast with other short peptides, these tripeptides form hydrogels at a physiological pH without the aid of organic solvents or end-capping groups (e.g. Fmoc). The novel hydrogel forming peptide DLeu-Phe-Phe (DLFF) and its epimer Leu-Phe-Phe (LFF) exemplify dramatic supramolecular effects induced by subtle changes to stereochemistry. Only the d-amino acid-containing peptide instantly forms a hydrogel in aqueous solution following a pH switch, generating long fibres (>100 μm) that entangle into a 3D network. However, unexpected nanostructures are observed for both peptides and they are particularly heterogeneous for LFF. Structural analyses using CD, FT-IR and fluorescent amyloid staining reveal anti-parallel beta-sheets for both peptides. XRD analysis also identifies key distances consistent with beta-sheet formation in both peptides, but suggests additional high molecular order and extended molecular length for DLFF only. Molecular modelling of the two peptides highlights the key interactions responsible for self-assembly; in particular, rapid self-assembly of DLFF is promoted by a phenylalanine zipper, which is not possible because of steric factors for LFF. In conclusion, this study elucidates for the first time the molecular basis for how chirality can dramatically influence supramolecular organisation in very short peptide sequences.Change of chirality is a useful tool to manipulate the aqueous self-assembly behaviour of uncapped, hydrophobic tripeptides. In contrast with other short peptides, these tripeptides form hydrogels at a physiological pH without the aid of organic solvents or end-capping groups (e.g. Fmoc). The novel hydrogel forming peptide DLeu-Phe-Phe (DLFF) and its epimer Leu-Phe-Phe (LFF) exemplify dramatic supramolecular effects induced by subtle changes to stereochemistry. Only the d-amino acid-containing peptide instantly forms a hydrogel in aqueous solution following a pH switch, generating long fibres (>100 μm) that entangle into a 3D network. However, unexpected nanostructures are observed for both peptides and they are particularly heterogeneous for LFF. Structural analyses using CD, FT-IR and fluorescent amyloid staining reveal anti-parallel beta-sheets for both peptides. XRD analysis also identifies key distances consistent with beta-sheet formation in both peptides, but suggests additional high molecular order and extended molecular length for DLFF only. Molecular modelling of the two peptides highlights the key interactions responsible for self-assembly; in particular, rapid self-assembly of DLFF is promoted by a phenylalanine zipper, which is not possible because of steric factors for LFF. In conclusion, this study elucidates for the first time the molecular basis for how chirality can dramatically influence supramolecular organisation in very short peptide sequences. Electronic supplementary information (ESI) available: video showing instant gelation for DLFF (and not LFF), following a pH trigger. Spectroscopic analysis of the two tripeptides. Microscopy images. d spacings from XRD diffraction analysis. Distances obtained for the energetically minimised molecular models. See DOI: 10.1039/c2nr32006a

  14. Authentication of forensic DNA samples.

    PubMed

    Frumkin, Dan; Wasserstrom, Adam; Davidson, Ariane; Grafit, Arnon

    2010-02-01

    Over the past twenty years, DNA analysis has revolutionized forensic science, and has become a dominant tool in law enforcement. Today, DNA evidence is key to the conviction or exoneration of suspects of various types of crime, from theft to rape and murder. However, the disturbing possibility that DNA evidence can be faked has been overlooked. It turns out that standard molecular biology techniques such as PCR, molecular cloning, and recently developed whole genome amplification (WGA), enable anyone with basic equipment and know-how to produce practically unlimited amounts of in vitro synthesized (artificial) DNA with any desired genetic profile. This artificial DNA can then be applied to surfaces of objects or incorporated into genuine human tissues and planted in crime scenes. Here we show that the current forensic procedure fails to distinguish between such samples of blood, saliva, and touched surfaces with artificial DNA, and corresponding samples with in vivo generated (natural) DNA. Furthermore, genotyping of both artificial and natural samples with Profiler Plus((R)) yielded full profiles with no anomalies. In order to effectively deal with this problem, we developed an authentication assay, which distinguishes between natural and artificial DNA based on methylation analysis of a set of genomic loci: in natural DNA, some loci are methylated and others are unmethylated, while in artificial DNA all loci are unmethylated. The assay was tested on natural and artificial samples of blood, saliva, and touched surfaces, with complete success. Adopting an authentication assay for casework samples as part of the forensic procedure is necessary for maintaining the high credibility of DNA evidence in the judiciary system.

  15. RNA-templated DNA repair

    PubMed Central

    Storici, Francesca; Bebenek, Katarzyna; Kunkel, Thomas A.; Gordenin, Dmitry A.; Resnick, Michael A.

    2007-01-01

    RNA can act as a template for DNA synthesis in the reverse transcription of retroviruses and retrotransposons1 and in the elongation of telomeres2. Despite its abundance in the nucleus, there has been no evidence for a direct role of RNA as a template in the repair of any chromosomal DNA lesions, including DNA double-strand breaks (DSBs), which are repaired in most organisms by homologous recombination or by non-homologous end joining3. An indirect role for RNA in DNA repair, following reverse transcription and formation of a complementary DNA, has been observed in the non-homologous joining of DSB ends4,5. In the yeast Saccharomyces cerevisiae, in which homologous recombination is efficient3, RNA was shown to mediate recombination, but only indirectly through a cDNA intermediate6,7 generated by the reverse transcriptase function of Ty retrotransposons in Ty particles in the cytoplasm8. Although pairing between duplex DNA and single-strand (ss)RNA can occur in vitro9,10 and in vivo11, direct homologous exchange of genetic information between RNA and DNA molecules has not been observed. We show here that RNA can serve as a template for DNA synthesis during repair of a chromosomal DSB in yeast. The repair was accomplished with RNA oligonucleotides complementary to the broken ends. This and the observation that even yeast replicative DNA polymerases such as α and δ can copy short RNA template tracts in vitro demonstrate that RNA can transfer genetic information in vivo through direct homologous interaction with chromosomal DNA. PMID:17429354

  16. DNA movies and panspermia.

    PubMed

    Norris, Victor; Grondin, Yohann

    2011-10-20

    There are several ways that our species might try to send a message to another species separated from us by space and/or time. Synthetic biology might be used to write an epitaph to our species, or simply "Kilroy was here", in the genome of a bacterium via the patterns of either (1) the codons to exploit Life's non-equilibrium character or (2) the bases themselves to exploit Life's quasi-equilibrium character. We suggest here how DNA movies might be designed using such patterns. We also suggest that a search for mechanisms to create and preserve such patterns might lead to a better understanding of modern cells. Finally, we argue that the cutting-edge microbiology and synthetic biology needed for the Kilroy project would put origin-of-life studies in the vanguard of research.

  17. DNA Movies and Panspermia

    PubMed Central

    Norris, Victor; Grondin, Yohann

    2011-01-01

    There are several ways that our species might try to send a message to another species separated from us by space and/or time. Synthetic biology might be used to write an epitaph to our species, or simply “Kilroy was here”, in the genome of a bacterium via the patterns of either (1) the codons to exploit Life's non-equilibrium character or (2) the bases themselves to exploit Life's quasi-equilibrium character. We suggest here how DNA movies might be designed using such patterns. We also suggest that a search for mechanisms to create and preserve such patterns might lead to a better understanding of modern cells. Finally, we argue that the cutting-edge microbiology and synthetic biology needed for the Kilroy project would put origin-of-life studies in the vanguard of research. PMID:25382053

  18. Tumorigenic DNA viruses

    SciTech Connect

    Klein, G.

    1989-01-01

    The eighth volume of Advances in Viral Oncology focuses on the three major DNA virus groups with a postulated or proven tumorigenic potential: papillomaviruses, animal hepatitis viruses, and the Epstein-Bar virus. In the opening chapters, the contributors analyze the evidence that papillomaviruses and animal hepatitis viruses are involved in tumorigenesis and describe the mechanisms that trigger virus-host cell interactions. A detailed section on the Epstein-Barr virus (EBV) - comprising more than half the book - examines the transcription and mRNA processing patterns of the virus genome; the mechanisms by which EBV infects lymphoid and epithelial cells; the immunological aspects of the virus; the actions of EBV in hosts with Acquired Immune Deficiency Syndrome; and the involvement of EBV in the etiology of Burkitt's lymphoma.

  19. Chimeric DNA methyltransferases target DNA methylation to specific DNA sequences and repress expression of target genes

    PubMed Central

    Li, Fuyang; Papworth, Monika; Minczuk, Michal; Rohde, Christian; Zhang, Yingying; Ragozin, Sergei; Jeltsch, Albert

    2007-01-01

    Gene silencing by targeted DNA methylation has potential applications in basic research and therapy. To establish targeted methylation in human cell lines, the catalytic domains (CDs) of mouse Dnmt3a and Dnmt3b DNA methyltransferases (MTases) were fused to different DNA binding domains (DBD) of GAL4 and an engineered Cys2His2 zinc finger domain. We demonstrated that (i) Dense DNA methylation can be targeted to specific regions in gene promoters using chimeric DNA MTases. (ii) Site-specific methylation leads to repression of genes controlled by various cellular or viral promoters. (iii) Mutations affecting any of the DBD, MTase or target DNA sequences reduce targeted methylation and gene silencing. (iv) Targeted DNA methylation is effective in repressing Herpes Simplex Virus type 1 (HSV-1) infection in cell culture with the viral titer reduced by at least 18-fold in the presence of an MTase fused to an engineered zinc finger DBD, which binds a single site in the promoter of HSV-1 gene IE175k. In short, we show here that it is possible to direct DNA MTase activity to predetermined sites in DNA, achieve targeted gene silencing in mammalian cell lines and interfere with HSV-1 propagation. PMID:17151075

  20. Humanity and human DNA.

    PubMed

    Mattei, Jean-François

    2012-10-01

    Genetics has marked the second half of the 20th century by addressing such formidable problems as the identification of our genes and their role, their interaction with the environment, and even their therapeutic uses. The identification of genes raises questions about differences between humans and non-humans, as well as about the evolution towards trans-humanism and post-humanism. In practise, however, the main question concerns the limits of prenatal genetic diagnosis, not only on account of the seriousness of the affections involved but also because of the choice to be made between following-up the medical indication and engaging in a systematic public health strategy aimed at eliminating children with certain handicaps. History reminds us that genetic science has already been misused by political forces influenced by the ideas of eugenics, particularly in the Nazi period. We may wonder whether it is reasonable to formulate a judgement on the life of a child yet to be born, merely on the basis of a DNA analysis. My experience as a practising geneticist and my involvement in French politics forces me to stress the dangers of a new eugenics hiding behind a medical mask. As demonstrated by epigenetics, human beings cannot be reduced to their DNA alone. In our society, one of the problems concerns individuals whose lives may be considered by some as simply not worth living. Another problem is the place and the social significance of the handicapped amongst us. Fortunately, recent progresses in gene therapy, biotherapy, and even pharmacology, appear to be opening up promising therapeutic perspectives. We should bear in mind that the chief vocation of medical genetics, which fully belongs to the art of medicine, is to heal and to cure. This is precisely where genetics should concentrate its efforts software.

  1. Method for assaying clustered DNA damages

    DOEpatents

    Sutherland, Betsy M.

    2004-09-07

    Disclosed is a method for detecting and quantifying clustered damages in DNA. In this method, a first aliquot of the DNA to be tested for clustered damages with one or more lesion-specific cleaving reagents under conditions appropriate for cleavage of the DNA to produce single-strand nicks in the DNA at sites of damage lesions. The number average molecular length (Ln) of double stranded DNA is then quantitatively determined for the treated DNA. The number average molecular length (Ln) of double stranded DNA is also quantitatively determined for a second, untreated aliquot of the DNA. The frequency of clustered damages (.PHI..sub.c) in the DNA is then calculated.

  2. DNA: Polymer and molecular code

    NASA Astrophysics Data System (ADS)

    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

  3. Prenatal Cell-Free DNA Screening

    MedlinePlus

    Prenatal cell-free DNA screening Overview By Mayo Clinic Staff Prenatal cell-free DNA (cfDNA) screening, also known as noninvasive prenatal screening, is ... in a developing baby. During prenatal cell-free DNA screening, DNA from the mother and fetus is ...

  4. DNA Extraction Techniques for Use in Education

    ERIC Educational Resources Information Center

    Hearn, R. P.; Arblaster, K. E.

    2010-01-01

    DNA extraction provides a hands-on introduction to DNA and enables students to gain real life experience and practical knowledge of DNA. Students gain a sense of ownership and are more enthusiastic when they use their own DNA. A cost effective, simple protocol for DNA extraction and visualization was devised. Buccal mucosal epithelia provide a…

  5. DNA Extraction Techniques for Use in Education

    ERIC Educational Resources Information Center

    Hearn, R. P.; Arblaster, K. E.

    2010-01-01

    DNA extraction provides a hands-on introduction to DNA and enables students to gain real life experience and practical knowledge of DNA. Students gain a sense of ownership and are more enthusiastic when they use their own DNA. A cost effective, simple protocol for DNA extraction and visualization was devised. Buccal mucosal epithelia provide a…

  6. DNA nanotechnology: a future perspective.

    PubMed

    Zahid, Muniza; Kim, Byeonghoon; Hussain, Rafaqat; Amin, Rashid; Park, Sung Ha

    2013-03-04

    In addition to its genetic function, DNA is one of the most distinct and smart self-assembling nanomaterials. DNA nanotechnology exploits the predictable self-assembly of DNA oligonucleotides to design and assemble innovative and highly discrete nanostructures. Highly ordered DNA motifs are capable of providing an ultra-fine framework for the next generation of nanofabrications. The majority of these applications are based upon the complementarity of DNA base pairing: adenine with thymine, and guanine with cytosine. DNA provides an intelligent route for the creation of nanoarchitectures with programmable and predictable patterns. DNA strands twist along one helix for a number of bases before switching to the other helix by passing through a crossover junction. The association of two crossovers keeps the helices parallel and holds them tightly together, allowing the assembly of bigger structures. Because of the DNA molecule's unique and novel characteristics, it can easily be applied in a vast variety of multidisciplinary research areas like biomedicine, computer science, nano/optoelectronics, and bionanotechnology.

  7. Event extraction for DNA methylation.

    PubMed

    Ohta, Tomoko; Pyysalo, Sampo; Miwa, Makoto; Tsujii, Jun'ichi

    2011-10-06

    We consider the task of automatically extracting DNA methylation events from the biomedical domain literature. DNA methylation is a key mechanism of epigenetic control of gene expression and implicated in many cancers, but there has been little study of automatic information extraction for DNA methylation. We present an annotation scheme for DNA methylation following the representation of the BioNLP shared task on event extraction, select a set of 200 abstracts including a representative sample of all PubMed citations relevant to DNA methylation, and introduce manual annotation for this corpus marking nearly 3000 gene/protein mentions and 1500 DNA methylation and demethylation events. We retrain a state-of-the-art event extraction system on the corpus and find that automatic extraction of DNA methylation events, the methylated genes, and their methylation sites can be performed at 78% precision and 76% recall. Our results demonstrate that reliable extraction methods for DNA methylation events can be created through corpus annotation and straightforward retraining of a general event extraction system. The introduced resources are freely available for use in research from the GENIA project homepage http://www-tsujii.is.s.u-tokyo.ac.jp/GENIA.

  8. Methods of DNA methylation analysis.

    USDA-ARS?s Scientific Manuscript database

    The purpose of this review was to provide guidance for investigators who are new to the field of DNA methylation analysis. Epigenetics is the study of mitotically heritable alterations in gene expression potential that are not mediated by changes in DNA sequence. Recently, it has become clear that n...

  9. DNA repair in mammalian embryos.

    PubMed

    Jaroudi, Souraya; SenGupta, Sioban

    2007-01-01

    Mammalian cells have developed complex mechanisms to identify DNA damage and activate the required response to maintain genome integrity. Those mechanisms include DNA damage detection, DNA repair, cell cycle arrest and apoptosis which operate together to protect the conceptus from DNA damage originating either in parental gametes or in the embryo's somatic cells. DNA repair in the newly fertilized preimplantation embryo is believed to rely entirely on the oocyte's machinery (mRNAs and proteins deposited and stored prior to ovulation). DNA repair genes have been shown to be expressed in the early stages of mammalian development. The survival of the embryo necessitates that the oocyte be sufficiently equipped with maternal stored products and that embryonic gene expression commences at the correct time. A Medline based literature search was performed using the keywords 'DNA repair' and 'embryo development' or 'gametogenesis' (publication dates between 1995 and 2006). Mammalian studies which investigated gene expression were selected. Further articles were acquired from the citations in the articles obtained from the preliminary Medline search. This paper reviews mammalian DNA repair from gametogenesis to preimplantation embryos to late gestational stages.

  10. Forensic applications of DNA profiling.

    PubMed

    Chow, S T; Ng, T L; Chao, T C

    1996-01-01

    Deoxyribonucleic acid (DNA) profiling is a powerful forensic tool to identify and individualise biological evidence recovered at crime scenes. Its discriminating power and evidential value are exemplified by many actual forensic cases. Development of new DNA typing techniques, such as the amplification of fluorescently tagged short tandem repeats, would further enhance this capability in forensic investigation.

  11. Chromatin dynamics during DNA replication

    PubMed Central

    Bar-Ziv, Raz; Voichek, Yoav; Barkai, Naama

    2016-01-01

    Chromatin is composed of DNA and histones, which provide a unified platform for regulating DNA-related processes, mostly through their post-translational modification. During DNA replication, histone arrangement is perturbed, first to allow progression of DNA polymerase and then during repackaging of the replicated DNA. To study how DNA replication influences the pattern of histone modification, we followed the cell-cycle dynamics of 10 histone marks in budding yeast. We find that histones deposited on newly replicated DNA are modified at different rates: While some marks appear immediately upon replication (e.g., H4K16ac, H3K4me1), others increase with transcription-dependent delays (e.g., H3K4me3, H3K36me3). Notably, H3K9ac was deposited as a wave preceding the replication fork by ∼5–6 kb. This replication-guided H3K9ac was fully dependent on the acetyltransferase Rtt109, while expression-guided H3K9ac was deposited by Gcn5. Further, topoisomerase depletion intensified H3K9ac in front of the replication fork and in sites where RNA polymerase II was trapped, suggesting supercoiling stresses trigger H3K9 acetylation. Our results assign complementary roles for DNA replication and gene expression in defining the pattern of histone modification. PMID:27225843

  12. Thermoelectric method for sequencing DNA.

    PubMed

    Nestorova, Gergana G; Guilbeau, Eric J

    2011-05-21

    This study describes a novel, thermoelectric method for DNA sequencing in a microfluidic device. The method measures the heat released when DNA polymerase inserts a deoxyribonucleoside triphosphate into a primed DNA template. The study describes the principle of operation of a laminar flow microfluidic chip with a reaction zone that contains DNA template/primer complex immobilized to the inner surface of the device's lower channel wall. A thin-film thermopile attached to the external surface of the lower channel wall measures the dynamic change in temperature that results when Klenow polymerase inserts a deoxyribonucleoside triphosphate into the DNA template. The intrinsic rejection of common-mode thermal signals by the thermopile in combination with hydrodynamic focused flow allows for the measurement of temperature changes on the order of 10(-4) K without control of ambient temperature. To demonstrate the method, we report the sequencing of a model oligonucleotide containing 12 bases. Results demonstrate that it is feasible to sequence DNA by measuring the heat released during nucleotide incorporation. This thermoelectric method for sequencing DNA may offer a novel new method of DNA sequencing for personalized medicine applications. © The Royal Society of Chemistry 2011

  13. Stereochemical control of DNA biosynthesis

    PubMed Central

    Sosunov, Vasily V.; Santamaria, Fanny; Victorova, Lyubov S.; Gosselin, Gilles; Rayner, Bernard; Krayevsky, Alexander A.

    2000-01-01

    Stereochemical control of DNA biosynthesis was studied using several DNA-synthesizing complexes containing, in each case, a single substitution of a 2′-deoxy-d-nucleotide residue by an enantiomeric l-nucleotide residue in a DNA chain (either in the primer or in the template) as well as 2′-deoxy-l-ribonucleoside 5′-triphosphates (l-dNTPs) as substrates. Three template-dependent DNA polymerases were tested, Escherichia coli DNA polymerase I Klenow fragment, Thermus aquaticus DNA polymerase and avian myeloblastosis virus reverse transcriptase, as well as template-independent calf-thymus terminal deoxynucleotidyl transferase. Very stringent control of stereoselectivity was demonstrated for template-dependent DNA polymerases, whereas terminal deoxynucleotidyl transferase was less selective. DNA polymerase I and reverse transcriptase catalyzed formation of dinucleoside 5′,5′-tetraphosphates when l-dTTP was used as substrate. Comparison between models of template–primer complexes, modified or not by a single l-nucleotide residue, revealed striking differences in their geometry. PMID:10666459

  14. Microscopic Electrohydrodynamics of DNA electrophoresis

    NASA Astrophysics Data System (ADS)

    Aksimentiev, Aleksei; Luan, Binquan

    2008-03-01

    Gel electrophoresis is currently the most successful yet costly method to sequence DNA. Electrophoresis of DNA through solid-state nanopores holds promise for reducing the costs and making personal genomics a reality. The underlying physics of DNA electrophoresis, however, remains controversial. Theoretical models of this process often invoke the notion of the effective charge of a DNA molecule qeff to account for the reduced electric force on DNA in an external field E, i.e. F= qeffE. However, experimental estimates of qeff can differ from each other by as much as ten times. To clarify the physical origin of the reduction of an electric force on DNA in electrophoresis, we investigated this process through extensive all-atom molecular dynamics simulations. Our results demonstrate that the effective screening of the DNA charge arises from the hydrodynamic drag of the electroosmotic flow, not from the counterion condensation. We show that the effective driving force F of an applied electric field E in a nanopore obeys the same law as in a bulk electrolyte: F=ξμE. Here, ξ and μ are, respectively, the friction coefficient and electrophoretic mobility of DNA that depend on the surface properties of a nanopore, such as its roughness. Based on the above law, a method for determining the effective driving force is suggested that does not require a direct force measurement.

  15. DNA/chitosan electrostatic complex.

    PubMed

    Bravo-Anaya, Lourdes Mónica; Soltero, J F Armando; Rinaudo, Marguerite

    2016-07-01

    Up to now, chitosan and DNA have been investigated for gene delivery due to chitosan advantages. It is recognized that chitosan is a biocompatible and biodegradable non-viral vector that does not produce immunological reactions, contrary to viral vectors. Chitosan has also been used and studied for its ability to protect DNA against nuclease degradation and to transfect DNA into several kinds of cells. In this work, high molecular weight DNA is compacted with chitosan. DNA-chitosan complex stoichiometry, net charge, dimensions, conformation and thermal stability are determined and discussed. The influence of external salt and chitosan molecular weight on the stoichiometry is also discussed. The isoelectric point of the complexes was found to be directly related to the protonation degree of chitosan. It is clearly demonstrated that the net charge of DNA-chitosan complex can be expressed in terms of the ratio [NH3(+)]/[P(-)], showing that the electrostatic interactions between DNA and chitosan are the main phenomena taking place in the solution. Compaction of DNA long chain complexed with low molar mass chitosan gives nanoparticles with an average radius around 150nm. Stable nanoparticles are obtained for a partial neutralization of phosphate ionic sites (i.e.: [NH3(+)]/[P(-)] fraction between 0.35 and 0.80).

  16. The enigmatic thymine DNA glycosylase.

    PubMed

    Cortázar, Daniel; Kunz, Christophe; Saito, Yusuke; Steinacher, Roland; Schär, Primo

    2007-04-01

    When it was first isolated from extracts of HeLa cells in Josef Jiricny's laboratory, the thymine DNA glycosylase (TDG) attracted attention because of its ability to remove thymine, i.e. a normal DNA base, from G.T mispairs. This implicated a function of DNA base excision repair in the restoration of G.C base pairs following the deamination of a 5-methylcytosine. TDG turned out to be the founding member of a newly emerging family of mismatch-directed uracil-DNA glycosylases, the MUG proteins, that act on a comparably broad spectrum of base lesion including G.U as the common, most efficiently processed substrate. However, because of its apparent catalytic inefficiency, some have considered TDG a poor DNA repair enzyme without an important biological function. Others have reported 5-meC DNA glycosylase activity to be associated with TDG, thrusting the enzyme into limelight as a possible DNA demethylase. Yet others have found the glycosylase to interact with transcription factors, implicating a function in gene regulation, which appears to be critically important in developmental processes. This article reviews all these developments in view of possible biological functions of this multifaceted DNA glycosylase.

  17. Structural DNA Nanotechnology: An Overview

    PubMed Central

    Seeman, Nadrian C.

    2012-01-01

    Structural DNA Nanotechnology uses unusual DNA motifs to build target shapes and arrangements. These unusual motifs are generated by reciprocal exchange of DNA backbones, leading to branched systems with many strands and multiple helical domains. The motifs may be combined by sticky ended cohesion, involving hydrogen bonding or covalent interactions. Other forms of cohesion involve edge-sharing or paranemic interactions of double helices. A large number of individual species have been developed by this approach, including polyhedral catenanes, such as a cube and a truncated octahedron, a variety of single-stranded knots, and Borromean rings. In addition to these static species, DNA-based nanomechanical devices have been produced that are targeted ultimately to lead to nanorobotics. Many of the key goals of structural DNA nanotechnology entail the use of periodic arrays. A variety of 2D DNA arrays have been produced with tunable features, such as patterns and cavities. DNA molecules have be used successfully in DNA-based computation as molecular representations of Wang tiles, whose self-assembly can be programmed to perform a calculation. The area appears to be at the cusp of a truly exciting explosion of applications, which can be expected to occur by the end of the current decade. PMID:15923682

  18. DNA nanotechnology: a future perspective

    PubMed Central

    2013-01-01

    In addition to its genetic function, DNA is one of the most distinct and smart self-assembling nanomaterials. DNA nanotechnology exploits the predictable self-assembly of DNA oligonucleotides to design and assemble innovative and highly discrete nanostructures. Highly ordered DNA motifs are capable of providing an ultra-fine framework for the next generation of nanofabrications. The majority of these applications are based upon the complementarity of DNA base pairing: adenine with thymine, and guanine with cytosine. DNA provides an intelligent route for the creation of nanoarchitectures with programmable and predictable patterns. DNA strands twist along one helix for a number of bases before switching to the other helix by passing through a crossover junction. The association of two crossovers keeps the helices parallel and holds them tightly together, allowing the assembly of bigger structures. Because of the DNA molecule's unique and novel characteristics, it can easily be applied in a vast variety of multidisciplinary research areas like biomedicine, computer science, nano/optoelectronics, and bionanotechnology. PMID:23497147

  19. Quantitative detection of single DNA molecules on DNA tetrahedron decorated substrates.

    PubMed

    Wang, Zhenguang; Xue, Qingwang; Tian, Wenzhi; Wang, Lei; Jiang, Wei

    2012-10-07

    A single DNA molecule detection method on DNA tetrahedron decorated substrates has been developed. DNA tetrahedra were introduced onto substrates for both preventing nonspecific adsorption and sensitive recognition of single DNA molecules.

  20. Graphene nanodevices for DNA sequencing

    NASA Astrophysics Data System (ADS)

    Heerema, Stephanie J.; Dekker, Cees

    2016-02-01

    Fast, cheap, and reliable DNA sequencing could be one of the most disruptive innovations of this decade, as it will pave the way for personalized medicine. In pursuit of such technology, a variety of nanotechnology-based approaches have been explored and established, including sequencing with nanopores. Owing to its unique structure and properties, graphene provides interesting opportunities for the development of a new sequencing technology. In recent years, a wide range of creative ideas for graphene sequencers have been theoretically proposed and the first experimental demonstrations have begun to appear. Here, we review the different approaches to using graphene nanodevices for DNA sequencing, which involve DNA passing through graphene nanopores, nanogaps, and nanoribbons, and the physisorption of DNA on graphene nanostructures. We discuss the advantages and problems of each of these key techniques, and provide a perspective on the use of graphene in future DNA sequencing technology.