DNA Microarrays, Part A: Array Platforms and Wet-Bench Protocols: 410 (Methods in Enzymology)
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A synchronization circuit may be utilized by the line scanning excitation system to synchronize sampling, for example. The substrate may contact a reaction chamber , encapsulating a buffer solution and making up a real-time PCR microarray reaction system. The refractive index of the substrate may be higher than the buffer solution , for example. The substrate may be glued to the reaction chamber , for example. The fluorescent tag may be imaged in an imaging sensor , such as a cooled CCD camera by imaging lenses An optical filter between the substrate and image lenses may be utilized to block the exciting light and pass the fluorescence.
The element may be a TEC temperature control plate, for example. Variation of any light source intensity may be monitored by detector , such as a photo-electric detector. The following Examples are illustrative of the above invention. One skilled in the art will readily recognize that the techniques and reagents described in the Example suggest many other ways in which the present invention could be practiced.
This example illustrates the fabrication of a microarray reactor for the quantitative analysis of nucleic acids using a polymerase chain reaction PCR process and an evanescent wave detection technique. The reaction chamber is made of a glass cover plate and a thermally conductive polypropylene substrate. The interior surface of the glass cover plate is chemically modified to reduce the adsorption of fluorescent substances and other contaminants. The target nucleic acid probes are tethered to the interior surface of the glass cover plate in a known, two-dimensional pattern.
The glass cover plate is also transparent and suitable for an evanescent wave detection technique. The thermally conductive polypropylene substrate with an interior cavity is fabricated using a molding method. An inlet and an outlet are incorporated into the substrate. The glass cover plate and the polypropylene substrate are assembled and sealed together by a buffer layer to form a reactor.
After the sample is loaded, both the inlet and the outlet are sealed with a rubber plug. To prevent liquid leakage of the reactor with thermal cycling, a buffer layer is used between the substrate and the cover plate. This silicone rubber will also not interfere with the PCR process or exhibit low fluorescence after curing. To prevent damage to the glass cover plate, the polypropylene substrate, and the immobilized target probe the silicon rubber is a room temperature vulcanizing RTV material.
The sample and various analytical reagents and reactants are introduced into the reaction chamber from an external source through inlet port. The outlet port acts as a blowhole when fluid is introduced in through inlet port. After the sample and various analytical reagents and reactants is added, a set of rubber plugs with the proper size, hardness, and chemical resistance are used to seal the inlet port and outlet port of the reaction chamber. When using the reactor for nucleic acid detection, the reactor and the reagent inside is heated and cooled down by a PCR temperature cycling program.
The glass cover plate is suitable for fluorescent detection by evanescent wave. CY5 is excited maximally at nm and emits maximally at nm. The invention has been described with reference to various specific embodiments and techniques. However, it should be understood that many variations and modifications may be made while remaining within the spirit and scope of the invention. The same optical grade was achieved in the final cover plates made of these two kinds of material.
Biofyzikální ústav AV ČR
A beam of excitation light at nm was struck into the cover plate at the side facet with a given incident angle see, e. The moving stage is programmed to move horizontally to ensure the incident light can scan the whole surface of the cover plate with the same incident angle at the interface between the cover plate and the air in the cavity. The scanning process was carried out at a constant speed. The detector was integrated with an optical filter to exclude the excitation light at nm such that only the emission fluorescent light could be captured.
The scanning time was 4 s for each cover plate with the same exposure time in a Coolsnap CCD. The inherent background of the CCD is about 7 RLU, and that the inherent background of quartz and K9 material is below 10 and varying from 20 to 40 respectively including the inherent background of the CCD. Therefore, the inherent: background of the quartz is below 3 most of the quartz slides background is nearly zero , while the inherent background of K9 slides varies from 10 to over Using cover plates that are formed of low fluorescent background difference quartz would allow those cover plates that include obvious background imperfections to be screened out i.
See, e. The signal to noise ratio of quartz and K9 were compared by two DNA oligo probes that are commonly used to detect Staphylococcus aureus , which is a kind of bacterium usually occurring in grapelike clusters and causing boils, septicemia, and other infections. As shown on FIG. The higher signal-to-noise ratio of quartz helps to accurately recognize the initial hybridization signal and the Ct value. One advantage of this process is that the chip quality of inter- or intra-batch cover plates might be controlled by selective examination because those batches with a relatively large signal-to-noise ratio variation could be screened out and discarded.
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Effective date : A reactor for the quantitative analysis of target nucleic acids using an evanescent wave detection technique and a method for the quantitative analysis of target nucleic acids are provided. The reactor includes a substrate with a cavity, a buffer layer arranged over the substrate, a quartz cover plate arranged over the buffer layer, and inlet and outlet ports.
In the drawings: FIG. Examples of such coating agents include polyethylene oxide triblock copolymers, polyethylene glycols PEG having molecular weights ranging from about to about , natural polymers such as bovine serum albumen BSA or any other moieties that provide the desired surface characteristics, particularly those that reduce the sorption of biomolecules such as proteins and nucleic acid A solution containing the sample to be amplified and appropriate buffers and reagents is typically introduced into the reactor via any appropriate methodology.
The Substrate and Cover Plate The materials used to form the substrates and cover plates in the embodiments are selected with regard to physical and chemical characteristics that are desirable for a particular application. In one embodiment, the cover plate is glass. The Buffer Layer In the reactor, a buffer layer is used between the substrate and the cover plate. Fabrication The substrate can be fabricated using any convenient method, including, but not limited to, micromolding and casting techniques, embossing methods, surface micro-machining and bulk-micromachining.
Example 1 This example illustrates the fabrication of a microarray reactor for the quantitative analysis of nucleic acids using a polymerase chain reaction PCR process and an evanescent wave detection technique. What is claimed is: 1. A reactor for the quantitative analysis of target nucleic acids, comprising: a substrate having a first planar opposing surface and a second planar opposing surface, the first planar opposing surface of the substrate having a cavity;. The reactor of claim 1 , wherein the substrate and the buffer layer are each independently comprised of a chemically inert material that is thermally stable and resistant to contamination.
The reactor of claim 1 , wherein the substrate is a glass, a metal, a ceramic, a composite, a polymeric material, or a combination or laminate thereof. The reactor of claim 3 , wherein the polymeric material is a polyimide, polycarbonate, a polyester, a polyamide, a polyether, a polyurethane, a polyfluorocarbon, a polystyrene, a poly acrylonitrile-butadiene-styrene , a polymethyl methacrylate, polyolefin, or a copolymer thereof. The reactor of claim 3 , wherein the substrate is a thermally conductive polypropylene. The reactor of claim 1 , wherein the water-impermeable sealant is a room temperature vulcanizing silicone rubber.
The reactor of claim 1 , further comprising at least one inlet port and at least one outlet port communicating with the reaction chamber through the substrate enabling the passage of fluid from an external source into and through the reaction chamber, and thereby defining a fluid flow path. The reactor of claim 1 , wherein the functional group is a thiocyanate SCN functional group. The reactor of claim 1 , wherein the surface of the cover plate includes unreacted SCN groups that are blocked. Method to increase detection efficiency of real time PCR microarray by quartz material.
CNA en. INDNA en.
DNA microarrays, part a: array platforms and wet-bench protocols
WOA1 en. Imprinting process of hot-melt type curable silicone composition for optical devices. USA en. Block copolymer, method of making the same, diamine precursors of the same, method of making such diamines and end products comprising the block copolymer. Tackified polydiorganosiloxane oligourea segmented copolymers and a process for making same.
Polydiorganosiloxane polyurea segmented copolymers and a process for making same. WOA2 en. Tackified polydiorganosiloxane polyurea segmented copolymers and a process for making same. Method to make fluorescent nucleotide photoproducts for dna sequencing and analysis. USA1 en. Methods and apparatus for analyzing polynucleotide sequences by asynchronous base extension. EPA1 en. Microarray reader based on evanescent wave detection and method of reading a microarray.
USB1 en. Cool Polymers. Dahl, Andreas, et al. Dow Corning.