ML Estimation of DNA Initial Copy Number in Polymerase Chain Reaction (PCR) Processes

Estimation of DNA copy number in a given biological sample is an extremely important problem in genomics. This problem is especially challenging when the number of the DNA strands is minuscule, which is often the case in applications such as pathogen and genetic mutation detection. A recently developed technique, real-time polymerase chain reaction (PCR), amplifies the number of initial target molecules by replicating them through a series of thermal cycles. Ideally, the number of target molecules doubles at the end of each cycle. However, in practice, due to biochemical noise the efficiency of the PCR reaction, defined as the fraction of target molecules which are successfully copied during a cycle, is always less than 1. In this paper, we formulate the problem of joint maximum-likelihood estimation of the PCR efficiency and the initial DNA copy number. As indicated by simulation studies, the performance of the proposed estimator is superior with respect to competing statistical approaches. Moreover, we compute the Cramer-Rao lower bound on the mean-square estimation error

On joint maximum-likelihood estimation of PCR efficiency and initial amount of target

We consider the problem of estimating unknown parameters of the real-time polymerase chain reaction (RTPCR) from noisy observations. The joint ML estimator of the RT-PCR efficiency and the initial number of DNA target molecules is derived. The mean-square error performance of the estimator is studied via simulations. The simulation results indicate that the proposed estimator significantly outperforms a competing technique

Devices and methods for microarray selection

The present invention relates to a device for the specific selection of target molecules, comprising: (a) at least one reaction zone comprising a microarray, wherein the microarray comprises a substrate, on which one or more species of capture molecules are immobilized, comprising one or more temperature control and/or regulating units for controlling and/or regulating the temperature within the zone; (b) at least one non-reaction zone comprising one or more temperature control and/or regulating units for controlling and/or regulating the temperature within the zone, which is in fluid connection with the reaction zone; and (c) at least one transportation means capable of generating and/or regulating a fluid flow between said reaction zone (a) and said non-reaction zone comprising one or more temperature control and/or regulating units (b). The present invention further relates to a device for the specific selection of target molecules wherein the immobilized capture molecules are organized in the microarray in the form of spots, elongated spots and/or lines. In a further aspect the present invention relates to a method of specifically selecting target molecules, comprising the introducing a medium to such a device, performing interaction reactions in a reaction zone, transporting not interacted or not bound target molecules to a zone allowing reactivation of the target molecules and performing additional interaction reactions with the reactivated target molecules at the reaction zone, as well as the use of such a device for specifically selecting target molecules, e.g. for target enrichment also referred to as microarray based genome selection (MGS) in the literature

Mechanisms of vascular smooth muscle contraction and the basis for pharmacologic treatment of smooth muscle disorders

The smooth muscle cell directly drives the contraction of the vascular wall and hence regulates the size of the blood vessel lumen. We review here the current understanding of the molecular mechanisms by which agonists, therapeutics, and diseases regulate contractility of the vascular smooth muscle cell and we place this within the context of whole body function. We also discuss the implications for personalized medicine and highlight specific potential target molecules that may provide opportunities for the future development of new therapeutics to regulate vascular function.Accepted manuscrip

An Excellent Monitoring System for Surface Ubiquitination-Induced Internalization in Mammals

Background. At present, it is difficult to visualize the internalization of surface receptors induced by ubiquitination that is taken place at the plasma membrane in mammals. This problem makes it difficult to reveal molecular basis for ubiquitinationmediated internalization in mammals. Methodology/Principle Findings. In order to overcome it, we have generated T-REx-c-MIR, a novel mammalian Tet-on B cell line using a constitutively active E3 ubiquitin ligase, c-MIR, and its artificial target molecule. By applying the surface biotinylation method to T-REx-c-MIR, we succeeded to monitor the fate of surface target molecules after initiation of ubiquitination process by doxycycline (Dox)-induced c-MIR expression. Target molecules that preexisted at the plasma membrane before induction of c-MIR expression were oligo-ubiquitinated and degraded by Dox-induced c-MIR expression. Dox-induced c-MIR expression initiated rapid internalization of surface target molecules, and blockage of the internalization induced the accumulation of the surface target molecules that were newly ubiquitinated by c-MIR. Inhibition of the surface ubiquitination by down-regulating ubiquitin conjugating enzyme E2 impaired the internalization of target molecules. Finally, a complex of c-MIR and target molecule was detected at the plasma membrane. Conclusions/ Significances. These results demonstrate that in T-REx-c-MIR, surface target molecule is ubiquitinated at the plasma membrane and followed by being internalized from the plasma membrane. Thus, T-REx-c-MIR is a useful experimental tool t

Fluorescent labeling of plasmid DNA and mRNA : gains and losses of current labeling strategies

Live-cell imaging has provided the life sciences with insights into the cell biology and dynamics. Fluorescent labeling of target molecules proves to be indispensable in this regard. In this Review, we focus on the current fluorescent labeling strategies for nucleic acids, and in particular mRNA (mRNA) and plasmid DNA (pDNA), which are of interest to a broad range of scientific fields. By giving a background of the available techniques and an evaluation of the pros and cons, we try to supply scientists with all the information needed to come to an informed choice of nucleic acid labeling strategy aimed at their particular needs

Screening-Limited Response of NanoBiosensors

Despite tremendous potential of highly sensitive electronic detection of bio-molecules by nanoscale biosensors for genomics and proteomic applications, many aspects of experimentally observed sensor response (S) are unexplained within consistent theoretical frameworks of kinetic response or electrical screening. In this paper, we combine analytic solutions of Poisson-Boltzmann and reaction-diffusion equations to show that the electrical response of nanobiosensor varies logarithmically with the concentration of target molecules, time, the salt concentration, and inversely with the fractal dimension of sensor surface. Our analysis provides a coherent theoretical interpretation of wide variety of puzzling experimental data that have so far defied intuitive explanation.Comment: 7 pages, 2 figure

Label-free biosensors for the detection and quantification of cardiovascular risk markers

This paper presents a biosensor implementation for the detection of protein molecules using specific antibodies. Affinity sensors allow the detection and quantification of target molecules in complex mixtures by affinity-based interactions. Immobilized antibody molecules are the probes that bind to specific protein molecules (targets) in biological fluids. In this study, inter-digitated electrodes in the form of capacitance on glass slide were designed, fabricated and used to measure the changes in the dielectric properties of the inter-digitated capacitances. Our results in this study present that with a careful design of micro-interdigitated capacitors, a wider dynamic range and higher sensitivity can be achieved for the detection and quantification of C-Reeactive Protein

A Revised Design for Microarray Experiments to Account for Experimental Noise and Uncertainty of Probe Response

Background Although microarrays are analysis tools in biomedical research, they are known to yield noisy output that usually requires experimental confirmation. To tackle this problem, many studies have developed rules for optimizing probe design and devised complex statistical tools to analyze the output. However, less emphasis has been placed on systematically identifying the noise component as part of the experimental procedure. One source of noise is the variance in probe binding, which can be assessed by replicating array probes. The second source is poor probe performance, which can be assessed by calibrating the array based on a dilution series of target molecules. Using model experiments for copy number variation and gene expression measurements, we investigate here a revised design for microarray experiments that addresses both of these sources of variance. Results Two custom arrays were used to evaluate the revised design: one based on 25 mer probes from an Affymetrix design and the other based on 60 mer probes from an Agilent design. To assess experimental variance in probe binding, all probes were replicated ten times. To assess probe performance, the probes were calibrated using a dilution series of target molecules and the signal response was fitted to an adsorption model. We found that significant variance of the signal could be controlled by averaging across probes and removing probes that are nonresponsive or poorly responsive in the calibration experiment. Taking this into account, one can obtain a more reliable signal with the added option of obtaining absolute rather than relative measurements. Conclusion The assessment of technical variance within the experiments, combined with the calibration of probes allows to remove poorly responding probes and yields more reliable signals for the remaining ones. Once an array is properly calibrated, absolute quantification of signals becomes straight forward, alleviating the need for normalization and reference hybridizations