Hybridization probe

Résumé

In molecular biology, a hybridization probe (HP) is a fragment of DNA or RNA of usually 15–10000 nucleotide long which can be radioactively or fluorescently labeled. HP can be used to detect the presence of nucleotide sequences in analyzed RNA or DNA that are complementary to the sequence in the probe. The labeled probe is first denatured (by heating or under alkaline conditions such as exposure to sodium hydroxide) into single stranded DNA (ssDNA) and then hybridized to the target ssDNA (Southern blotting) or RNA (northern blotting) immobilized on a membrane or in situ. To detect hybridization of the probe to its target sequence, the probe is tagged (or "labeled") with a molecular marker of either radioactive or (more recently) fluorescent molecules. Commonly used markers are 32P (a radioactive isotope of phosphorus incorporated into the phosphodiester bond in the probe DNA), digoxigenin, a non-radioactive, antibody-based marker, biotin or fluorescein. DNA sequences or RNA transcr

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https://en.wikipedia.org/wiki/Hybridization_probe

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Detection of mRNA by Whole Mount in situ Hybridization and DNA Extraction for Genotyping of Zebrafish Embryos

Rachna Narayanan, Andrew Charles Oates

In situ hybridization is used to visualize the spatial distribution of gene transcripts in tissues and in embryos, providing important information about disease and development. Current methods involve the use of complementary riboprobes incorporating non-radioactive labels that can be detected by immunohistochemistry and coupled to chromogenic or fluorescent visualization. Although recent fluorescent methods have allowed new capabilities such as single-molecule counting, qualitative chromogenic detection remains important for many applications because of its relative simplicity, low cost and high throughput, and ease of imaging using transmitted light microscopy. A remaining challenge is combining high contrast signals with reliable genotyping after hybridization. Dextran sulfate is commonly added to the hybridization buffer to shorten development times and improve contrast, but this reagent inhibits PCR-based genotyping. This paper describes a modified protocol for in situ hybridization in fixed whole mount zebrafish embryos using digoxigenin (DIG) labeled riboprobes that are detected with alkaline phosphatase conjugated anti-DIG antibodies and nitroblue tetrazolium (NBT)/5-bromo-4-chloro-3-indolyl-phosphate (BCIP) chromogenic substrates. To yield embryos compatible with downstream genotyping after hybridization without sacrificing contrast of the signal, this protocol omits dextran sulfate and utilizes a lower hybridization temperature.

BIO-PROTOCOL2019

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Real-Time Label-Free Direct Electronic Monitoring of Topoisomerase Enzyme Binding Kinetics on Graphene

Kannan Balasubramanian, Klaus Kern

Mono layer graphene field-effect sensors operating in liquid have been widely deployed for detecting a range of analyte species often under equilibrium conditions. Here we report on the real-time detection of the binding kinetics of the essential human enzyme, topoisomerase I interacting with substrate molecules (DNA probes) that are immobilized electrochemically on to monolayer graphene strips. By monitoring the field-effect characteristics of the graphene biosensor in real-time during the enzyme-substrate interactions, we are able to decipher the surface binding constant for the cleavage reaction step of topoisomerase I activity in a label-free manner. Moreover, an appropriate design of the capture probes allows us to distinctly follow the cleavage step of topoisomerase I functioning in real-time down to picomolar concentrations. The presented results are promising for future rapid screening of drugs that are being evaluated for regulating enzyme activity.

Amer Chemical Soc2015

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Microscopy imaging, including fluorescence microscopy and electron microscopy, has a prominent role in life science and medical research. During the past two decades, biological imaging has undergone a revolution by way of the development of new microscopy techniques that allow the visualization of tissues, cells, proteins and macromolecular structures at all levels of resolution, physiological states, chemical composition and dynamic analysis. Thanks to recent advances in optics, digital sensing technologies and labeling probes (i.e., XFP—Colored Fluorescence Protein), we can now visualize subcellular components and organelles at the scale of a few nanometers to several hundreds of nanometers. As a result, fluorescent microscopy has become the workhorse of modern biology. Further technological advances include structured and coherent light sources, faster and more sensitive detectors, smaller and more specific molecular probes and automation processes for image acquisition. Additionally, there is a push towards multimodal imaging in order to gather complementary information such as the concentration of fluorophores at various wavelengths and the refractive index of a given sample (phase imaging).

IEEE2016

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