An Alternative Hot Start PCR Method Using a Nuclease-Deficient ExoIII from Escherichia coli

Abstract(#br)The Hot Start polymerase chain reaction (Hot Start PCR) is designed to reduce off-target amplification by blocking DNA polymerase extension at room temperature until the desired temperature is reached. In this study, we investigated a new method of Hot Start PCR that uses a modified Escherichia coli Exonuclease III (EcoExoIIIM) by substituting residues in the DNA-binding pocket and catalytic center. The results showed that PCR amplification yield and specificity were significantly promoted by the addition of EcoExoIIIM. We hypothesize that non-specific binding of primers at room temperature is prevented by binding of the primed template by EcoExoIIIM, which is then released from the DNA by heat denaturation before the first PCR cycle. Through this mechanism, PCR would be..

An Alternative Hot Start PCR Method Using a Nuclease-Deficient ExoIII from Escherichia coli.

The Hot Start polymerase chain reaction (Hot Start PCR) is designed to reduce off-target amplification by blocking DNA polymerase extension at room temperature until the desired temperature is reached. In this study, we investigated a new method of Hot Start PCR that uses a modified Escherichia coli Exonuclease III (EcoExoIIIM) by substituting residues in the DNA-binding pocket and catalytic center. The results showed that PCR amplification yield and specificity were significantly promoted by the addition of EcoExoIIIM. We hypothesize that non-specific binding of primers at room temperature is prevented by binding of the primed template by EcoExoIIIM, which is then released from the DNA by heat denaturation before the first PCR cycle. Through this mechanism, PCR would be enhanced by reducing off-target extension at room temperature

Multi-gene-based phylogenetic analysis of oligotrich ciliates with emphasis on two dominant groups: Cyrtostrombidiids and strombidiids (Protozoa, Ciliophora)

publisher: Elsevier articletitle: Multi-gene-based phylogenetic analysis of oligotrich ciliates with emphasis on two dominant groups: Cyrtostrombidiids and strombidiids (Protozoa, Ciliophora) journaltitle: Molecular Phylogenetics and Evolution articlelink: http://dx.doi.org/10.1016/j.ympev.2016.08.019 content_type: article copyright: © 2016 Elsevier Inc. All rights reserved.The file attached is the Accepted/final draft post-refereeing version of the articl

Solution Grown Se/Te Nanowires: Nucleation, Evolution, and The Role of Triganol Te seeds

We have studied the nucleation and growth of Se–Te nanowires (NWs), with different morphologies, grown by a chemical solution process. Through systematic characterization of the Se–Te NW morphology as a function of the Te nanocrystallines (NCs) precursor, the relative ratio between Se and Te, and the growth time, a number of significant insights into Se–Te NW growth by chemical solution processes have been developed. Specifically, we have found that: (i) the growth of Se–Te NWs can be initiated from either long or short triganol Te nanorods, (ii) the frequency of proximal interactions between nanorod tips and the competition between Se and Te at the end of short Te nanorods results in V-shaped structures of Se–Te NWs, the ratio between Se and Te having great effect on the morphology of Se–Te NWs, (iii) by using long Te nanorods as seeds, Se–Te NWs with straight morphology were obtained. Many of these findings on Se–Te NW growth can be further generalized and provide very useful information for the rational synthesis of group VI based semiconductor NW compounds

A Dynamic Energy Budget Model for Kuruma Shrimp Penaeus japonicus: Parameterization and Application in Integrated Marine Pond Aquaculture

Individual growth models can form the basis of population dynamics assessment and ecosystem model construction. In order to provide a basic module for an ecosystem model of an integrated marine aquaculture pond, an individual growth model was constructed for kuruma shrimp (Penaeus japonicus) based on dynamic energy budget (DEB) theory. The model was first parameterized based on a covariation method using the Add-my-Pet (AmP) procedure. The parametric estimation model underestimated the ultimate abdominal length for female shrimp, and the predicted values of other zero-variate parameters were generally consistent with observed values. The relative errors of the predicted and observed values of the univariate data set within three geographical regions showed acceptable goodness of fit. Parameter estimation achieved an overall goodness of fit with a mean relative error of 0.048 and a symmetric mean squared error of 0.066. A DEB model was constructed using the estimated parameters, and the goodness-of-fit indicators (R square, mean bias and absolute and relative root mean square error) showed that the model was able to reproduce the growth of kuruma shrimp in terms of total length and wet weight with high accuracy. The results provide data to support the subsequent development of integrated aquaculture management at the ecosystem level

Binding of Escherichia coli Does Not Protect Tulane Virus from Heat-Inactivation Regardless the Expression of HBGA-Like Molecules

Histo-blood group antigens (HBGAs) are considered as receptors/co-receptors for human norovirus (HuNoV). It has been reported that binding of HuNoV-derived virus-like particles (VLPs) to HBGA-like molecules-expressing bacteria increased the stability of VLPs to heat-denaturation (HD). In this study, we tested for HBGA-like-binding-conveyed protection against HD on viral replication using Tulane virus (TV) and Escherichia coli O86:H2 (O86:H2), with E. coli K-12 (K-12) used as a control. Expression of HBGA type B was confirmed by ELISA in O86:H2 but not in K-12. Binding of TV was confirmed by ELISA in O86:H2 (P/N = 2.23) but not in K-12 (P/N = 1.90). Pre-incubation of TV with free HBGA could completely inhibit its ability to bind to O86:H2 (p = 0.004), while producing no significant change in its ability to bind K-12 (p = 0.635). We utilized a bacterial-capture-RT-qPCR procedure to confirm that both bacterial strains were capable of binding TV, and that O86:H2 exhibited fivefold greater binding capacity than K-12. Pre-incubation of TV with free HBGA would partially inhibit the binding of TV to O86:H2 (p = 0.047). In contrast, not only did pre-incubation of TV with free HBGA not inhibit the binding of TV to K-12, binding was slightly enhanced (p = 0.13). The viral infectivity assay allowed us to conduct a direct evaluation of the ability of HBGA-like-bound bacteria to confer HD protection to TV. Prior to inoculate to LLC-MK2 cells, TV was incubated with each bacterial strain at ratios of 1:0, 1:1 and 100:1, then both partially and fully HD. The viral amplification was quantitated by RT-qPCR 48 h later. The binding of bacteria to TV reduced viral replication in a dose-dependent matter. We found that neither bound O86:H2 nor K-12 conferred protection of TV against partial or full HD conditions. Partial HD reduction of viral replication was not significantly impacted by the binding of either bacterial strain, with infectivity losses of 99.03, 99.42, 96.32, 96.10, and 98.88% for TV w/o bacteria, TV w/O86:H2 (1:1), TV w/O86:H2 (100:1), TV w/K-12 (1:1), and TV w/K-12 (100:1), respectively. Full HD reduction of viral replication was not impacted by the binding of either bacterial strain, as full loss of infectivity was observed in all cases