Enzyme-mediated reactions are a useful tool in mutation detection when using a microarray format. Discriminating probes attached to the surface of a DNA chip have to be accessible to target DNA and ...to the enzyme (ligase or polymerase) that catalyses the formation of a new phosphodiester bond. This requires an appropriate chemical platform. Recently, an oligonucleotide hairpin architecture incorporating multiple phosphorothioate moieties along the loop has been proposed as an effective approach to solid-phase minisequencing. We have explored in depth several variables (stem length, number of phosphorothioates, stem–loop architecture versus linear structure) involved in this strategy by using a solid-phase ligation reaction. Microarrays were fabricated either from aminosilyl-modified glass or from aminated polymeric surfaces made of poly-lysine. Both platforms were bromoacetylated and reacted with thiophosphorylated oligonucleotides. The resulting microarrays were tested using either a synthetic template or a PCR-amplified 16S rRNA genomic region as the target sequence. Our results confirm the robustness of the proposed chemistry. We extend its range of application to solid-phase ligation, demonstrating the effectiveness of multiple anchors and suggest that linear oligonucleotides incorporating multiple phosphorothioates are equivalent to their hairpin-structured counterparts.
We present our results in the identification of polymorphic sites within the second exon of the human leukocyte antigen A (HLA-A) region using the DNA microarray technology. Allele specific detection ...was performed by polymerase chain reaction followed by ligase detection reaction (LDR) in combination with a universal array, a powerful method for high throughput DNA sequence analysis. By this approach we confirmed 32 human samples previously characterized by direct DNA sequencing, thus demonstrating the interest of this approach.
In this report we describe two robust procedures for oligonucleotide microarray preparation based on polymeric coatings. The proposed chemical approaches include: 1) a glass functionalisation step ...with appropriate silanes (
γ
-aminopropyltriethoxysilane-APTES or 3-glycid-oxypropyltrimethoxysilane-GOPS), 2) a coating step using polymers (poly-L-Lysine or poly(acrylic acid-co-acrylamide) copolymer) covalently bound to the modified glass and 3) a surface activation step to allow for the attachment of amino-modified oligonucleotides. Results obtained using these chemistries in oligo microarray preparation show: 1) an overall high loading capacity and availability to hybridisation against targets, 2) a good uniformity, 3) resistance to consecutive probing/stripping cycles, 4) stability to thermal cycles, 5) effectiveness in hybridisation-mediated mutation detection procedures and 6) the possibility to perform enzymatic reactions, such as ligation.
A simple, non-destructive procedure is described to determine the quality of DNA arrays before they are used. It consists of a preliminary staining step of the DNA microarray by using SYBR
green II, ...a fluorophore with specific affinity for ssDNA, followed by a laser scan analysis. The surface quality, integrity and homogeneity of each DNA spot of the array can thus be assessed. After this preliminary control, which may avoid further analytical steps that lead to the waste of precious biological samples, a fully reversible staining procedure is performed that produces an array ready for subsequent use.
Detecting foodborne pathogens as quickly and reliably as possible is a matter of public safety. In the last two decades, the field of food microbiology has evolved with increasing speed, also thanks ...to genome sequences available for many of the pathogens involved.
In this chapter, conventional methods, analytical techniques, and recent developments in food pathogen array-based detection, identification, and quantification are described. We report the main applications involving the use of DNA microarrays as detection tools. The chapter, initially, focuses on the most recent developments and experimental results of more traditional array technologies, then new biomolecular techniques, such as integrated platforms for the miniaturized detection of pathogens, are discussed with the description of some interesting approaches and innovative applications. A final paragraph, dedicated to the latest developments and trends in the “omics” field, suggests new opportunities to solve old food safety problems.
In the space environment, instruments onboard of spacecrafts can be affected by displacement damage due to radiation. The differential scattering cross section for screened nucleus--nucleus ...interactions - i.e., including the effects due to screened Coulomb nuclear fields -, nuclear stopping powers and non-ionization energy losses are treated from about 50 keV/nucleon up to relativistic energies.
Energetic (suprathermal) solar particles, accelerated in the interplanetary medium, contribute to the solar wind pressure, in particular during high solar activity periods. We estimated the effect of ...the increase of solar wind pressure due to suprathermal particles on magnetospheric transmissivity of galactic cosmic rays in the case of one recent solar event.
We implemented a website to deal with main effects on Cosmic Ray access to the Earth, i.e. the Solar Modulation and the Geomagnetic Field effect. In helmod.org the end user can easily access a web ...interface to results catalog of the HelMod Monte Carlo Code. This Model uses a Monte Carlo Approach to solves the Parker Transport Equation, obtaining a modulated proton flux for a period (monthly average) between January 1990 and december 2007. geomagsphere.org is instead based on GeoMag Backtracing Code, that solves the Lorentz equation with a Runge-Kutta method of 6th order, and, reversing charge sign and velocity, reconstruct particle trajectories in the Earth Magnetosphere back in time. We use last models of internal (IGRF-11) and external (Tsyganenko 1996 -T96- and 2005 -T05-) field components valid up to 2015. Particles are backtraced to the outer (magnetopause) or inner boundary to separate Primary (allowed trajectory) from Secondary (forbidden) Cosmic Rays. This code has been used both for reproducing known effects as East-West effect and rigidity cutoff calculations. In geomagsphere.org the user can choose the external field model from Tsyganenko (T96 or T05) and obtain for a fixed position and date from 1st Jan. 1968 (T96) and 1st Jan. 1995 (T05) respectively till 31\(^{st}\) Dec 2012, the vertical rigidity cutoff estimation obtained with the backtracing technique with a rigidity step of 0.1 GV. For a more precise calculation (0.01 GV), requiring more CPU time, results are sent to the user by email (mail model)
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