A novel universal real-time PCR system using the attached universal duplex probes for quantitative analysis of nucleic acids

BACKGROUND: Real-time PCR techniques are being widely used for nucleic acids analysis, but one limitation of current frequently employed real-time PCR is the high cost of the labeled probe for each target molecule. RESULTS: We describe a real-time PCR technique employing attached universal duplex probes (AUDP), which has the advantage of generating fluorescence by probe hydrolysis and strand displacement over current real-time PCR methods. AUDP involves one set of universal duplex probes in which the 5' end of the fluorescent probe (FP) and a complementary quenching probe (QP) lie in close proximity so that fluorescence can be quenched. The PCR primer pair with attached universal template (UT) and the FP are identical to the UT sequence. We have shown that the AUDP technique can be used for detecting multiple target DNA sequences in both simplex and duplex real-time PCR assays for gene expression analysis, genotype identification, and genetically modified organism (GMO) quantification with comparable sensitivity, reproducibility, and repeatability with other real-time PCR methods. CONCLUSION: The results from GMO quantification, gene expression analysis, genotype identification, and GMO quantification using AUDP real-time PCR assays indicate that the AUDP real-time PCR technique has been successfully applied in nucleic acids analysis, and the developed AUDP real-time PCR technique will offer an alternative way for nucleic acid analysis with high efficiency, reliability, and flexibility at low cost.Litao Yang, Wanqi Liang, Lingxi Jiang, Wenquan Li, Wei Cao, Zoe A Wilson, and Dabing Zhan

GMDD: a database of GMO detection methods

<p>Abstract</p> <p>Background</p> <p>Since more than one hundred events of genetically modified organisms (GMOs) have been developed and approved for commercialization in global area, the GMO analysis methods are essential for the enforcement of GMO labelling regulations. Protein and nucleic acid-based detection techniques have been developed and utilized for GMOs identification and quantification. However, the information for harmonization and standardization of GMO analysis methods at global level is needed.</p> <p>Results</p> <p>GMO Detection method Database (GMDD) has collected almost all the previous developed and reported GMOs detection methods, which have been grouped by different strategies (screen-, gene-, construct-, and event-specific), and also provide a user-friendly search service of the detection methods by GMO event name, exogenous gene, or protein information, etc. In this database, users can obtain the sequences of exogenous integration, which will facilitate PCR primers and probes design. Also the information on endogenous genes, certified reference materials, reference molecules, and the validation status of developed methods is included in this database. Furthermore, registered users can also submit new detection methods and sequences to this database, and the newly submitted information will be released soon after being checked.</p> <p>Conclusion</p> <p>GMDD contains comprehensive information of GMO detection methods. The database will make the GMOs analysis much easier.</p

DT-CP: a double-TTPs based contract-signing protocol with lower computational cost

This paper characterizes a contract signing protocol with high efficiency in Internet of Things. Recent studies show that existing contract-signing protocols can achieve abuse-freeness and resist inference attack, but cannot meet the high-efficiency and convenience requirement of the future Internet of things applications. To solve this problem, we propose a novel contract-signing protocol. Our proposed protocol includes two main parts: 1) we use the partial public key of the sender, instead of the zero-knowledge protocol, to verify the intermediate result; 2) we employ two independent Trusted Third Parties (TTPs) to prevent the honest-but-curious TTP. Our analysis shows that our double TTP protocol can not only result in lower computational cost, but also can achieve abuse-freeness with trapdoor commitment scheme. In a word, our proposed scheme performs better than the state of the art in terms of four metrics: encryption time, number of exponentiations, data to be exchanged and exchange steps in one round contract-signing

Tuning the Catalytic Activity of Graphene Nanosheets for Oxygen Reduction Reaction via Size and Thickness Reduction

Currently, the fundamental factors that control the oxygen reduction reaction (ORR) activity of graphene itself, in particular the dependence of the ORR activity on the number of exposed edge sites remain elusive, mainly due to limited synthesis routes of achieving small size graphene. In this work, the synthesis of low oxygen content (< 2.5 +/-0.2 at %), few layer graphene nanosheets with lateral dimensions smaller than a few hundred nm was achieved using a combination of ionic liquid assisted grinding of high purity graphite coupled with sequential centrifugation. We show for the first time, that the graphene nanosheets possessing a plethora of edges exhibited considerably higher electron transfer numbers compared to the thicker graphene nanoplatelets. This enhanced ORR activity was accomplished by successfully exploiting the plethora of edges of the nanosized graphene as well as the efficient electron communication between the active edge sites and the electrode substrate. The graphene nanosheets were characterized by an onset potential of -0.13 V vs. Ag/AgCl and a current density of -3.85 mA/cm2 at -1 V, which represent the best ORR performance ever achieved from an undoped carbon based catalyst. This work demonstrates how low oxygen content nanosized graphene synthesized by a simple route can considerably impact the ORR catalytic activity and hence it is of significance in designing and optimizing advanced metal-free ORR electrocatalysts.Comment: corresponding author: [email protected], ACS Applied Materials and Interfaces 201

Particle Electrophoresis and Magnetophoresis in Microchannels

In current pharmaceutical and biotechnology industries with clinical applications, an increased demand for flow control and cell manipulation on the micrometer scale has emerged. Electrokinetic, magnetic and many other physics fields have been exploited to meet this demand. However, due to the requirement for sophisticated micro-structures and the interference of the increasing significance of many `trivial\u27 physics properties (surface potential, permittivity, etc.) at the smaller scale, most applications encounter poor maneuverability and high operation/fabrication complexity issues. Very few attempts have been made to bypass these requirements while maintaining the same control and efficiency. This thesis research investigates the fundamental behaviors in microfluidic particle transportation. Then, with a thorough comprehension of the governing parameters and key effects, practical applications can be designed and developed to resolve the aforementioned microfluidic technique issues of electrophoresis and magnetophoresis. This thesis consists of two main parts. In the first section, the basic manipulation principle and subsequent applications in particle electrophoresis are discussed. Based on an observed wall-induced particle deflection in a straight microchannel, this thesis developed a method to three-dimensionally focus particle stream to the microchannel center. This application only relied on the particle confinement with respect to the microchannel; no particular external forces had to be exerted since this phenomenon was self-developing along with the traveling in the lengthwise direction. The second half of this work shifted the focus to particle magnetophoresis in a straight microchannel. An analytical model was built that solved the coupled magnetic and flow field, confirmed the experimental observations and enabled predictions for other plausible applications. Following that, this work utilized this negative magnetophoretic deflection to implement a diamagnetic particle focusing in a T-shaped microchannel. Particle ferrofluid flow and axillary sheath flow moved within each half of the microchannel and, the magnetophoretic deflection took effect inside the ferrofluid half where the particles were focused on the interface between the two halves. This arrangement required only one magnet with the help of the sheath flow to restrain the effective magnetophoretic deflection, which tremendously reduced the fabrication complexity and extended the channel-magnet distance to a smaller magnitude, therefore enhanced the throughput. Lastly, the same T-shaped microchannel was proved to perform high efficient particle separation. In addition to the negative magnetophoresis induced deflection for the diamagnetic particle was applied, the `attraction\u27 for the magnetic particle was present at the same time due to the opposite reaction: positive magnetophoresis. Initially mixed diamagnetic and magnetic particle sample were injected into the microchannel and, the opposite responses to the magnetic field formed a continuous separation of these two types at the end of the microchannel. Compared to the batch-mode MACS (magnetic cell sorter), this method undoubtedly made an improvement in both the throughput and operative difficulties