Unique Skill ID: KS1233177Z66QMLVD4YS

Hybridization Probe

In molecular biology, a hybridization probe is a fragment of DNA or RNA of variable length which can be radioactively or fluorescently labeled. It can then be used in DNA or RNA samples to detect the presence of nucleotide substances that are complementary to the sequence in the probe. The probe thereby hybridizes to single-stranded nucleic acid whose base sequence allows probe–target base pairing due to complementarity between the probe and target. The labeled probe is first denatured into single stranded DNA (ssDNA) and then hybridized to the target ssDNA or RNA immobilized on a membrane or in situ. To detect hybridization of the probe to its target sequence, the probe is tagged with a molecular marker of either radioactive or fluorescent molecules; commonly used markers are 32P or digoxigenin, which is a non-radioactive, antibody-based marker. DNA sequences or RNA transcripts that have moderate to high sequence similarity to the probe are then detected by visualizing the hybridized probe via autoradiography or other imaging techniques. Normally, either X-ray pictures are taken of the filter, or the filter is placed under UV light. Detection of sequences with moderate or high similarity depends on how stringent the hybridization conditions were applied—high stringency, such as high hybridization temperature and low salt in hybridization buffers, permits only hybridization between nucleic acid sequences that are highly similar, whereas low stringency, such as lower temperature and high salt, allows hybridization when the sequences are less similar. Hybridization probes used in DNA microarrays refer to DNA covalently attached to an inert surface, such as coated glass slides or gene chips, to which a mobile cDNA target is hybridized.

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