A plasmid-based lacZα gene assay for DNA polymerase fidelity measurement

A significantly improved DNA polymerase fidelity assay, based on a gapped plasmid containing the lacZα reporter gene in a single-stranded region, is described. Nicking at two sites flanking lacZα, and removing the excised strand by thermocycling in the presence of complementary competitor DNA, is used to generate the gap. Simple methods are presented for preparing the single-stranded competitor. The gapped plasmid can be purified, in high amounts and in a very pure state, using benzoylated-naphthoylated DEAE-cellulose, resulting in a low background mutation frequency (~1 × 10(-4)). Two key parameters, the number of detectable sites and the expression frequency, necessary for measuring polymerase error rates have been determined. DNA polymerase fidelity is measured by gap filling in vitro, followed by transformation into Escherichia coli and scoring of blue/white colonies and converting the ratio to error rate. Several DNA polymerases have been used to fully validate this straightforward and highly sensitive system

STOCHASTIC EFFICIENCY ANALYSIS USING MULTIPLE UTILITY FUNCTIONS

Evaluating the risk of a particular decision depends on the risk aversion of the decision maker related to the underlying utility function. The objective of this paper is to use stochastic efficiency with respect to a function (SERF) to compare the ranking of risky alternatives using alternative utility functional forms.Research Methods/ Statistical Methods,

An X-ray absorption spectroscopic study at the mercury LIII edge on phenylmercury(II) oxygen species

The X-ray absorption spectra of the reference and model compounds HgCl2, PhHgCl, PhHgOAc and [(PhHg)2OH][BF4].H2O have been analysed in both the XANES and EXAFS regions, and the technique was extended to determine the structures of (PhHg)2O, PhHgOH, and the basic salts PhHgOH.PhHgNO3 and PhHgOH.(PhHg)2SO4, which were previously structurally uncharacterised. Results indicate that (PhHg)2O is a molecular species with Hg-O-Hg 135°, while PhHgOH contains the [(PhHg)2OH]+ cation and is better formulated as [(PhHg)2OH]OH. The same cation is also featured in the two basic salts. Electrospray mass spectral studies of PhHgOH in aqueous solutions show that [PhHgOH2]+, [(PhHg)2OH]+ and [(PhHg)3O]+ co-exist in solution in a pH-dependent equilibrium

Signal Processing and Propagation for Aeroacoustic Sensor Networking,” Ch

Passive sensing of acoustic sources is attractive in many respects, including the relatively low signal bandwidth of sound waves, the loudness of most sources of interest, and the inherent difficulty of disguising or concealing emitted acoustic signals. The availability of inexpensive, low-power sensing and signal-processing hardware enables application of sophisticated real-time signal processing. Among th

Research Fundamentals: V. The Use of Laboratory Animal Models in Research

Animal research has provided important information about many aspects of the pathophysiology of human disease. Well-performed animal studies can determine the potential benefit of many proposed therapeutic interventions, and experimental results from animal studies have served as the basis for many landmark clinical trials. Many animal research models are described in the research literature, and choosing the appropriate model to answer a research question can be a daunting task. Even more challenging is developing a new model when none of the existing systems are relevant to the proposed question. This article was prepared by members of the SAEM Research Committee to provide an overview of animal modeling. Important considerations in choosing, applying, and developing animal research models are outlined. Practical discussions of potential problems with animal models are also provided

A Computational Model Relating Structure and Reactivity in Enantioselective Oxidations of Secondary Alcohols by (−)-Sparteine−Pd^(II) Complexes

The key interactions responsible for the unique reactivity of (−)-sparteine−PdX_2 complexes (X = chloride, acetate) in the enantioselective oxidation of secondary alcohols have been elucidated using quantum mechanics (B3LYP DFT with the PBF polarizable continuum solvent model). From examining many possible pathways, we find the mechanism involves:  (1) substitution of the alcohol in place of an X-group, (2) deprotonation of the bound alcohol by the deposed anion and free sparteine, (3) β-hydride elimination through a four-coordinate transition state in which the second anion is displaced but tightly associated, (4) replacement of the ketone product with the associated anion. The enantioselectivities observed under base-rich reaction conditions follow directly from calculated energies of diastereomeric β-hydride elimination transition states incorporating (R) and (S) substrates. This relationship reveals an important role of the anion, namely to communicate the steric interaction of the ligand on one side of the PdII square plane and the substrate on the other side. When no anion is included, no enantioselectivity is predicted. Locating these transition states in different solvents shows that higher dielectrics stabilize the charge separation between the anion and metal and draw the anion farther into solution. Thus, the solvent influences the barrier height (rate) and selectivity of the oxidation