Editing Hybridization probe (section)

{{short description|Fragment of RNA or DNA able to be chemically labeled}}
{{Refimprove|date=December 2009}}

In [[molecular biology]], a '''hybridization probe''' ('''HP''') is a fragment of [[DNA]] or [[RNA]], usually 15–10000 [[nucleotide]]s long, which can be [[radioactive tracer|radioactively]] or [[Fluorescent tag|fluorescently labeled]]. HPs can be used to detect the presence of  [[nucleotide]] sequences in analyzed RNA or DNA that are [[Complementarity (molecular biology)|complementary]] to the sequence in the probe.<ref>{{Cite web|url=https://www.ndsu.edu/pubweb/~mcclean/plsc731/dna/dna6.htm|title=Nucleic Acid Hybridizations|website=www.ndsu.edu|access-date=2017-05-26}}</ref> The labeled probe is first [[denaturation (biochemistry)|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 blot]]ting) or RNA ([[northern blot]]ting) immobilized on a membrane or ''[[fluorescent in situ hybridization|in situ]]''.

To detect [[Nucleic acid hybridization|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 [[Isotopes of phosphorus|<sup>32</sup>P]] (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 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 microarray]]s refer to DNA covalently attached to an inert surface, such as coated [[Microscope slide|glass slides]] or [[Microarray|gene chips]], to which a mobile [[Complementary DNA|cDNA]] target is hybridized. Depending on the [[nucleic acid methods|method]], the probe may be [[oligonucleotide synthesis|synthesized]] using the [[phosphoramidite]] method, or it can be generated and labeled by [[polymerase chain reaction|PCR]] amplification or [[molecular cloning|cloning]] (both are older methods). In order to increase the ''[[in vivo]]'' stability of the probe RNA is not used. Instead, [[Nucleic acid analogues#Backbone analogues|RNA analogues]] may be used, in particular [[morpholino]]- derivatives. Molecular DNA- or RNA-based probes are routinely used in screening gene libraries, detecting nucleotide sequences with [[Blot (biology)|blotting methods]], and in other gene technologies, such as nucleic acid and tissue [[microarray]]s.