Editing Hybridization probe

{{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.

== Examples of probes ==

*Scorpion® probes
*[[Molecular beacon|Molecular Beacon]] probes
*[[TaqMan]]® probes
*LNA® ([[Locked nucleic acid|Locked Nucleic Acid]]) probes
*[[Cycling probe technology|Cycling Probe Technology]] (CPT)
*[[In situ hybridization|''In situ'' hybridization]]
*Binary (split) probes
*Multicomponent probes

== Uses in microbial ecology ==
Within the field of [[microbial ecology]], oligonucleotide probes are used in order to determine the presence of microbial species, genera, or microorganisms classified on a more broad level, such as [[bacteria]], [[archaea]], and [[eukaryotes]] via [[fluorescence in situ hybridization]] (FISH).<ref>{{cite journal |vauthors=Amann R, Ludwig W | year = 2000 | title = Ribosomal RNA-targeted nucleic acid probes for studies in microbial ecology | journal = FEMS Microbiology Reviews | volume = 24 | issue = 5 | pages = 555–565 | doi=10.1111/j.1574-6976.2000.tb00557.x| pmid = 11077149 | doi-access = free }}</ref> rRNA probes have enabled scientists to visualize microorganisms, yet to be cultured in laboratory settings, by retrieval of rRNA sequences directly from the environment.<ref>{{cite journal | author = Amann, R. |author2=Ludwig, W.  |author3=Schleifer, K.-H. | year = 1995 | title = Phylogenetic identification and in situ detection of individual microbial cells without cultivation | journal = Microbiological Reviews | volume = 59 |issue=1 | pages = 143–169 |doi=10.1128/MMBR.59.1.143-169.1995 |pmid=7535888 |pmc=239358 | doi-access = free }}</ref> Examples of these types of microorganisms include:

*''[[Nevskia ramosa]]'': ''N. ramosa'' is a neuston bacterium that forms typical, dichotomically-branching rosettes on the surface of shallow freshwater habitats.<ref>{{cite journal | author = Glöckner, F.O. |author2=Babenzien H.D.| author3=Amann R. | year = 1998 | title = Phylogeny and identification in situ of ''Nevskia ramosa'' | journal = Appl. Environ. Microbiol. | volume = 64 |issue=5| pages = 1895–1901 |doi=10.1128/AEM.64.5.1895-1901.1998|pmid=9572969|pmc=106248|bibcode=1998ApEnM..64.1895G| doi-access = free }}</ref>
*''[[Achromatium oxaliferum]]'': This huge bacterium (cell length up to >100&nbsp;μm, diameter up to 50&nbsp;μm) contains sulfur globules and massive calcite inclusions and inhabits the upper layers of freshwater sediments. It is visible to the naked eye and has, by its resistance to cultivation, puzzled generations of microbiologists.<ref>{{cite journal | author = Glöckner, F.O. |author2= Babenzien H.D.| author3=Amann R. | year = 1999 | title = Phylogeny and diversity of ''Achromatium oxaliferum'' | journal = Syst. Appl. Microbiol. | volume = 22 |issue= 1| pages = 28–38 | doi=10.1016/s0723-2020(99)80025-3|pmid= 10188276}}</ref>

=== Limitations ===
In some instances, differentiation between species may be problematic when using [[16S ribosomal RNA|16S rRNA]] sequences due to similarity. In such instances, [[23S rRNA]] may be a better alternative.<ref>{{cite journal | author1 = Fox, G.E. |author2= Wisotzkey, J.D. | author3=Jurtshuk Jr., P. | year = 1992 | title = How close is close: 16S rRNA sequence identity may not be sufficient to guarantee species identity. | journal = Int. J. Syst. Bacteriol. | volume = 42 |issue= 1 | pages = 166–170 | doi=10.1099/00207713-42-1-166|pmid= 1371061 | doi-access = free }}</ref> The global standard library of rRNA sequences is constantly becoming larger and continuously being updated, and thus the possibility of a random hybridization event between a specifically-designed probe (based on complete and current data from a range of test organisms) and an undesired/unknown target organism cannot be easily dismissed.<ref>{{cite journal | author = Olsen, G.J. |author2=Lane, D.J. |author3=Giovannoni, S.J. |author4= Pace, N.R.  |author5= Stahl, D.A. | year = 1986 | title = Microbial ecology and evolution: a ribosomal RNA approach | journal = Annu. Rev. Microbiol. | volume = 40 | pages = 337–365 | doi=10.1146/annurev.mi.40.100186.002005|pmid=2430518 }}</ref> On the contrary, it is plausible that there exist microorganisms, yet to be identified, which are phylogenetically members of a probe target group, but have partial or near-perfect target sites, usually applies when designing group-specific probes.

Probably the greatest practical limitation to this technique is the lack of available automation.<ref>{{cite journal |vauthors=Amann R, Ludwig W | year = 2000 | title = Ribosomal RNA-targeted nucleic acid probes for studies in microbial ecology | journal = FEMS Microbiology Reviews | volume = 24 | issue = 5 | pages = 555–565 | doi=10.1111/j.1574-6976.2000.tb00557.x| pmid = 11077149 | doi-access = free }}</ref>

== Use in forensic science ==
In forensic science, hybridization probes are used, for example, for detection of short tandem repeats ([[Microsatellite (genetics)|microsatellite]]) regions<ref>{{cite journal |last1=Tytgat |first1=Olivier |title=STRide probes: Single-labeled short tandem repeat identification probes |journal=Biosensors and Bioelectronics |year=2021 |volume=180 |page=113135 |doi=10.1016/j.bios.2021.113135 |pmid=33690100 |doi-access=free |url=https://biblio.ugent.be/publication/8717666/file/8717669.pdf }}</ref> and in restriction fragment length polymorphism ([[RFLP]]) methods, all of which are widely used as part of [[DNA profiling]] analysis.

==See also==
* [[Molecular probe]]

==References==
{{reflist}}

{{DEFAULTSORT:Hybridization Probe}}
[[Category:Genetics techniques]]
[[Category:Molecular biology]]