1,721,154 research outputs found
Chemical and biochemical strategies for the randomization of protein encoding DNA sequences: library construction methods for directed evolution
No full textDirected molecular evolution and combinatorial methodologies are playing an increasingly important role in the field of protein engineering. The general approach of generating a library of partially randomized genes, expressing the gene library to generate the proteins the library encodes and then screening the proteins for improved or modified characteristics has successfully been applied in the areas of protein-ligand binding, improving protein stability and modifying enzyme selectivity. A wide range of techniques are now available for generating gene libraries with different characteristics. This review will discuss these different methodologies, their accessibility and applicability to non-expert laboratories and the characteristics of the libraries they produce. The aim is to provide an up to date resource to allow groups interested in using directed evolution to identify the most appropriate methods for their purposes and to guide those moving on from initial experiments to more ambitious targets in the selection of library construction techniques. References are provided to original methodology papers and other recent examples from the primary literature that provide details of experimental methods
Thermal equivalence of DNA duplexes for probe design
No full textWe present the theory of thermal equivalence in the framework of the Peyrard–Bishop model and some of its anharmonic variants. The thermal equivalence gives rise to a melting index ? which maps closely the experimental DNA melting temperatures for short DNA sequences. We show that the efficient calculation of the melting index can be used to analyse the parameters of the Peyrard–Bishop model and propose an improved set of Morse potential parameters. With this new set we are able to calculate some of the experimental melting temperatures to ± 1.2 °C. We review some of the concepts of sequencing probe design and show how to use the melting index to explore the possibilities of gene coverage by tuning the model parameters
Probing the microscopic flexibility of DNA from melting temperatures
No full textThe microscopic flexibility of DNA is a key ingredient for understanding its interaction with proteins and drugs but is still poorly understood and technically challenging to measure. Several experimental methods probe very long DNA samples, but these miss local flexibility details. Others mechanically disturb or modify short molecules and therefore do not obtain flexibility properties of unperturbed and pristine DNA. Here, we show that it is possible to extract very detailed flexibility information about unmodified DNA from melting temperatures with statistical physics models. We were able to retrieve, from published melting temperatures, several established flexibility properties such as the presence of highly flexible TATA regions of genomic DNA and support recent findings that DNA is very flexible at short length scales. New information about the nanoscale Na+ concentration dependence of DNA flexibility was determined and we show the key role of ApT and TpA steps when it comes to ion-dependent flexibility and melting temperatures
Introducing structural flexibility into porphyrin–DNA zipper arrays
No full textA more flexible nucleotide building block for the synthesis of new DNA based porphyrin–zipper arrays is described. Changing the rigid acetylene linker between the porphyrin substituent and the 2?-deoxyuridine to a more flexible propargyl amide containing linkage leads in part to an increased duplex stability. The CD spectra reveal different electronic interactions between the porphyrins depending on the type of linker used. Molecular modelling suggests large variation of the relative orientation of the porphyrins within the major groove of the DNA. The porphyrins can be metallated post-synthetically with different metals as shown with zinc, cobalt and copper. The spectroscopic features do not alter drastically upon metallation apart from the CD spectra, and the stability of the metal complex is highly dependent on the nature of the metal. As shown by CD spectroscopy, the zinc porphyrin is rapidly demetallated at high temperatures. Globular structure determination using SAXS indicates that a molecular assembly comprised of a two to four helical bundle dominates in solution at higher concentrations (?50 ?M) which is not observed by spectroscopy at lower concentrations (?1 ?M
Towards a global open scientific notebook infrastructure
No full textThe original World Wide Web vision was of a global and shared information space, and the Web as we now know it is a powerful facilitator of scientific endeavour, as well as being vital for modern commerce, leisure, and public awareness. Recently, calls for scientific research to become more open have gathered momentum: in the UK, the Royal Society has issues a comprehensive report arguing for intelligent openness to become standard. Funding bodies across the world have mandated the sharing of data and other supporting materials generated in the course of publicly funded research. In a press release outlining measure to improve access to scientific information, the European Commission undertook to “develop and support e-infrastructures to host and share scientific information (publications and data) which are interoperable on European and global level”.The infrastructures required comprise more than linked data repositories: there is widespread acceptance of the importance of metadata for describing, classifying, and linking the data. Provenance information is essential: indeed, it has been suggested that the provenance reflects the probability of having quality metadata. However, the utility of any infrastructure will depend heavily on proper curation. Although many researchers regard curation as a burden, capture at source can do much to relieve that perception. Electronic laboratory notebooks (ELNs) are a key enabling technology for scientific data infrastructures, with their built-in provisions for metadata capture, provenance trails, and curation at source. In this paper, we describe LabTrove, a cross-disciplinary ELN, with a range of resources and facilities for assembling a scientific notebook infrastructure. We give examples of international collaborations using LabTrove, and illustrate its application for open notebook science
Replication termination in Escherichia coli: structure and antihelicase activity of the Tus-Ter complex
No full textThe arrest of DNA replication in Escherichia coli is triggered by the encounter of a replisome with a Tus protein-Ter DNA complex. A replication fork can pass through a Tus-Ter complex when traveling in one direction but not the other, and the chromosomal Ter sites are oriented so replication forks can enter, but not exit, the terminus region. The Tus-Ter complex acts by blocking the action of the replicative DnaB helicase, but details of the mechanism are uncertain. One proposed mechanism involves a specific interaction between Tus-Ter and the helicase that prevents further DNA unwinding, while another is that the Tus-Ter complex itself is sufficient to block the helicase in apolar manner, without the need for specific protein -protein interactions. This review integrates three decades of experimental information on the action of the Tus-Ter complex with information available from the Tus-TerA crystal structure. We conclude that while it is possible to explain polar fork arrest by a mechanism involving only the Tus- Ter interaction, there are also strong indications of a role for specific Tus-DnaB interactions. The evidence suggests, therefore, that the termination system is more subtle and complex than may have been assumed. We describe some further experiments and insights that may assist in unraveling the details of this fascinating process
Thermal equivalence of DNA duplexes without calculation of melting temperature
No full textThe common key to nearly all processes involving DNA is the hybridization and melting of the double helix: from transmission of genetic information and RNA transcription, to polymerase chain reaction and DNA microarray analysis, DNA mechanical nanodevices and DNA computing. Selecting DNA sequences with similar melting temperatures is essential for many applications in biotechnology. We show that instead of calculating these temperatures, a single parameter can be derived from a statistical-mechanics model that conveniently represents the thermodynamic equivalence of DNA sequences. This parameter is shown to order experimental melting temperatures correctly, is much more readily obtained than the melting temperature, and is easier to handle than the numerous parameters of empirical regression models
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
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