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  • What are the reporter molecules?

    images3Chemical molecules are attached to the probe and that will enable the detection of this after a process of hybridization. There are many types of reporter molecules: radioactive (32P, 35S), affinity (Biotin, Digoxigenin …), enzymatic (phosphatase, peroxidase …) and chemiluminescent (esters of acridine).

    What factors affect the sensitivity and specificity of the hybridization reaction?

    *Concentration of target nucleic acid
    *Complementary target nucleic acid probe
    *Concentration of the probe.
    *Probe Length
    *Specific activity of the reporter molecule.
    *Hybridization conditions (pH, ionic strength, annealing temperature …)
    *Access to the target nucleic acid.
    *Hybridization Format

    Hybridization solution: The probe and target nucleic acid are in a liquid. The hybridization conditions must be in order. The rate of formation of the duplex under these conditions is high. The problem with this type of hybridization is the elimination of unreacted probe, using among other methods nuclease S1-precipitation with trichloroacetic or hybridization protection assay.

    Solid-phase Hybridization:
    The target nucleic acid or the probe are immobilized on filters. These can be of nitrocellulose (heat setting) or nylon (fixing by UV light). Once the attachment is added to the labeled probe will seek its complementary sequence in the target nucleic acid that we identify. The probe does not react, unstable hybrids or non-specific binding are removed by washing. Its sensitivity is lower than the hybridization solution.

    In situ hybridization:
    It allows the detection of genes or gene expression within the context of morphology of the cell or tissue. To achieve this requires that the nucleic acid is liberated from its cellular environment to access the probe, while preserving the morphology for subsequent interpretation and analysis. This is used especially of crosslinking fixatives such as formalin, paraformaldehyde, glutaraldehyde. The use of proteases, facilitating access of the probe to target nucleic acid must be performed carefully to not cause the dismantling of cellular morphology. We recommend the use of oligosondas.

    Now also has managed to fix the nucleic acids plastic microtiter plates.

    One type of solid-phase hybridizations are widely used in molecular biology: the Southern blot (DNA cut with restriction enzymes and hybridized with a probe of DNA) and Northern Blot (RNA and hybridized separately with probes of DNA or RNA). These techniques consist of an initial separation of molecules based on their molecular weight by electrophoresis, capillary transfer these molecules into a filter paper or nylon, fixing, hybridization with the probe of interest and detection.

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