In molecular biology, a hybridization probe is a fragment of DNA or RNA of variable length
which is used in DNA or RNA samples to detect the presence of nucleotide sequences 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 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.
Depending on the method, the probe may be synthesized using the phosphoramidite method,
or it can be generated and labeled by PCR amplification or cloning.
In order to increase the in vivo stability of the probe RNA is not used, instead RNA
analogues may be used, in particular morpholino- derivatives.
Molecular DNA- or RNA-based probes are now routinely used in screening gene libraries,
detecting nucleotide sequences with blotting methods, and in other gene technologies, such
as nucleic acid and tissue microarrays.
Examples of probes Scorpion® probes
Molecular Beacon probes TaqMan® probes
LNA® probes Cycling Probe Technology
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.
rRNA probes have enabled scientists to visualize microorganisms, yet to be cultured in laboratory
settings, by retrieval of rRNA sequences directly from the environment.
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.
Achromatium oxaliferum: This huge bacterium 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.
Limitations In some instances, differentiation between
species may be problematic when using 16S rRNA sequences due to similarity.
In such instances, 23S rRNA may be a better alternative.
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 and an undesired/unknown target organism cannot be easily dismissed.
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.
This usually applies when designing group-specific probes.
Probably the greatest practical limitation to this technique is the lack of available
automation.
Use in forensic science In forensic science, hybridization probes
are used, for example, for detection of short tandem repeats regions and in restriction
fragment length polymorphism methods, all of which are widely used as part of DNA profiling
analysis.
References ^ Amann R, Ludwig W. "Ribosomal RNA-targeted
nucleic acid probes for studies in microbial ecology".
FEMS Microbiology Reviews 24: 555–565.
doi:10.1111/j.1574-6976.2000.tb00557.x.
^ Amann, R., Ludwig, W. and Schleifer, K.-H..
"Phylogenetic identification and in situ detection of individual microbial cells without cultivation".
Microbiology Review 59: 143–169.
^ Glöckner, F.O., Babenzien H.D., and Amann R..
"Phylogeny and identification in situ of Nevskia ramosa".
Appl.
Environ.
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^ Glöckner, F.O., Babenzien H.D., and Amann R..
"Phylogeny and diversity of Achromatium oxaliferum".
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Appl.
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doi:10.1016/s0723-2020(99)80025-3.
^ Fox, G.E., Wisotzkey, J.D. and Jurtshuk Jr., P..
"How close is close: 16S rRNA sequence identity may not be sufficient to guarantee species
identity.".
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doi:10.1099/00207713-42-1-166.
^ Olsen, G.J., Lane, D.J., Giovannoni, S.J., Pace, N.R. and Stahl, D.A..
"Microbial ecology and evolution: a ribosomal RNA approach".
Annu. Rev. Microbiol. 40: 337–365.
doi:10.1146/annurev.mi.40.100186.002005.
^ Amann R, Ludwig W. "Ribosomal RNA-targeted nucleic acid probes for studies in microbial
ecology".
FEMS Microbiology Reviews 24: 555–565.
doi:10.1111/j.1574-6976.2000.tb00557.x.