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molecular biology Molecular biology is the branch of biology that seeks to understand the molecular basis of biological activity in and between cells, including biomolecular synthesis, modification, mechanisms, and interactions. The study of chemical and physi ...
, a hybridization probe (HP) is a fragment of DNA or
RNA Ribonucleic acid (RNA) is a polymeric molecule essential in various biological roles in coding, decoding, regulation and expression of genes. RNA and deoxyribonucleic acid ( DNA) are nucleic acids. Along with lipids, proteins, and carbohyd ...
of usually 15–10000 nucleotide long which can be radioactively or fluorescently labeled. HP can be used to detect the presence of
nucleotide Nucleotides are organic molecules consisting of a nucleoside and a phosphate. They serve as monomeric units of the nucleic acid polymers – deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), both of which are essential biomolecules with ...
sequences in analyzed RNA or DNA that are
complementary A complement is something that completes something else. Complement may refer specifically to: The arts * Complement (music), an interval that, when added to another, spans an octave ** Aggregate complementation, the separation of pitch-class ...
to the sequence in the probe. The labeled probe is first denatured (by heating or under
alkaline In chemistry, an alkali (; from ar, القلوي, al-qaly, lit=ashes of the saltwort) is a basic, ionic salt of an alkali metal or an alkaline earth metal. An alkali can also be defined as a base that dissolves in water. A solution of ...
conditions such as exposure to
sodium hydroxide Sodium hydroxide, also known as lye and caustic soda, is an inorganic compound with the formula NaOH. It is a white solid ionic compound consisting of sodium cations and hydroxide anions . Sodium hydroxide is a highly caustic base and alka ...
) into single stranded DNA (ssDNA) and then hybridized to the target ssDNA (
Southern blot A Southern blot is a method used in molecular biology for detection of a specific DNA sequence in DNA samples. Southern blotting combines transfer of electrophoresis-separated DNA fragments to a filter membrane and subsequent fragment detecti ...
ting) or RNA (
northern blot The northern blot, or RNA blot,Gilbert, S. F. (2000) Developmental Biology, 6th Ed. Sunderland MA, Sinauer Associates. is a technique used in molecular biology research to study gene expression by detection of RNA (or isolated mRNA) in a sample.Ke ...
ting) immobilized on a membrane or
in situ ''In situ'' (; often not italicized in English) is a Latin phrase that translates literally to "on site" or "in position." It can mean "locally", "on site", "on the premises", or "in place" to describe where an event takes place and is used in ...
. To detect hybridization of the probe to its target sequence, the probe is tagged (or "labeled") with a
molecular marker A molecular marker is a molecule, sampled from some source, that gives information about its source. For example, DNA is a molecular marker that gives information about the organism from which it was taken. For another example, some proteins can be ...
of either radioactive or (more recently) fluorescent molecules. Commonly used markers are 32P (a
radioactive Radioactive decay (also known as nuclear decay, radioactivity, radioactive disintegration, or nuclear disintegration) is the process by which an unstable atomic nucleus loses energy by radiation. A material containing unstable nuclei is consid ...
isotope Isotopes are two or more types of atoms that have the same atomic number (number of protons in their nuclei) and position in the periodic table (and hence belong to the same chemical element), and that differ in nucleon numbers ( mass numbers ...
of
phosphorus Phosphorus is a chemical element with the symbol P and atomic number 15. Elemental phosphorus exists in two major forms, white phosphorus and red phosphorus, but because it is highly reactive, phosphorus is never found as a free element on Ea ...
incorporated into the
phosphodiester In chemistry, a phosphodiester bond occurs when exactly two of the hydroxyl groups () in phosphoric acid react with hydroxyl groups on other molecules to form two ester bonds. The "bond" involves this linkage . Discussion of phosphodiesters is d ...
bond in the probe DNA),
digoxigenin Digoxigenin (DIG) is a steroid found exclusively in the flowers and leaves of the plants ''Digitalis purpurea'', '' Digitalis orientalis'' and ''Digitalis lanata'' (foxgloves), where it is attached to sugars, to form the glycosides (e.g. Lanato ...
, a non-radioactive,
antibody An antibody (Ab), also known as an immunoglobulin (Ig), is a large, Y-shaped protein used by the immune system to identify and neutralize foreign objects such as pathogenic bacteria and viruses. The antibody recognizes a unique molecule of the ...
-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 An autoradiograph is an image on an X-ray film or nuclear emulsion produced by the pattern of decay emissions (e.g., beta particles or gamma rays) from a distribution of a radioactive substance. Alternatively, the autoradiograph is also availab ...
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 Nucleic acids are biopolymers, macromolecules, essential to all known forms of life. They are composed of nucleotides, which are the monomers made of three components: a 5-carbon sugar, a phosphate group and a nitrogenous base. The two main clas ...
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 A DNA microarray (also commonly known as DNA chip or biochip) is a collection of microscopic DNA spots attached to a solid surface. Scientists use DNA microarrays to measure the expression levels of large numbers of genes simultaneously or to g ...
s refer to DNA covalently attached to an inert surface, such as coated glass slides or gene chips, to which a mobile
cDNA In genetics, complementary DNA (cDNA) is DNA synthesized from a single-stranded RNA (e.g., messenger RNA (mRNA) or microRNA (miRNA)) template in a reaction catalyzed by the enzyme reverse transcriptase. cDNA is often used to express a spe ...
target is hybridized. Depending on the
method Method ( grc, μέθοδος, methodos) literally means a pursuit of knowledge, investigation, mode of prosecuting such inquiry, or system. In recent centuries it more often means a prescribed process for completing a task. It may refer to: *Scien ...
, the probe may be synthesized using the
phosphoramidite A phosphoramidite (RO)2PNR2 is a monoamide of a phosphite diester. The key feature of phosphoramidites is their markedly high reactivity towards nucleophiles catalyzed by weak acids ''e.c''., triethylammonium chloride or 1''H''-tetrazole. In these ...
method, or it can be generated and labeled by
PCR PCR or pcr may refer to: Science * Phosphocreatine, a phosphorylated creatine molecule * Principal component regression, a statistical technique Medicine * Polymerase chain reaction ** COVID-19 testing, often performed using the polymerase chain r ...
amplification or
cloning Cloning is the process of producing individual organisms with identical or virtually identical DNA, either by natural or artificial means. In nature, some organisms produce clones through asexual reproduction. In the field of biotechnology, c ...
(both are older methods). In order to increase the '' in vivo'' stability of the probe RNA is not used. Instead, RNA analogues may be used, in particular
morpholino A Morpholino, also known as a Morpholino oligomer and as a phosphorodiamidate Morpholino oligomer (PMO), is a type of oligomer molecule (colloquially, an oligo) used in molecular biology to modify gene expression. Its molecular structure contains ...
- derivatives. Molecular DNA- or RNA-based probes are routinely used in screening gene libraries, detecting nucleotide sequences with blotting methods, and in other gene technologies, such as nucleic acid and tissue
microarray A microarray is a multiplex lab-on-a-chip. Its purpose is to simultaneously detect the expression of thousands of genes from a sample (e.g. from a tissue). It is a two-dimensional array on a solid substrate—usually a glass slide or silico ...
s.


Examples of probes

*Scorpion® probes *
Molecular Beacon Molecular beacons, or molecular beacon probes, are oligonucleotide hybridization probes that can report the presence of specific nucleic acids in homogenous solutions. Molecular beacons are hairpin-shaped molecules with an internally quenched fluor ...
probes * TaqMan® probes *LNA® (
Locked Nucleic Acid A locked nucleic acid (LNA), also known as bridged nucleic acid (BNA), and often referred to as inaccessible RNA, is a modified RNA nucleotide in which the ribose moiety is modified with an extra bridge connecting the 2' oxygen and 4' carbon. The ...
) probes * Cycling Probe Technology (CPT)


Uses in microbial ecology

Within the field of
microbial ecology Microbial ecology (or environmental microbiology) is the ecology of microorganisms: their relationship with one another and with their environment. It concerns the three major domains of life— Eukaryota, Archaea, and Bacteria—as well as ...
, 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 Eukaryotes () are organisms whose cells have a nucleus. All animals, plants, fungi, and many unicellular organisms, are Eukaryotes. They belong to the group of organisms Eukaryota or Eukarya, which is one of the three domains of life. Bact ...
via
fluorescence in situ hybridization Fluorescence ''in situ'' hybridization (FISH) is a molecular cytogenetic technique that uses fluorescent probes that bind to only particular parts of a nucleic acid sequence with a high degree of sequence complementarity. It was developed b ...
(FISH). 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 (cell length up to >100 µm, diameter up to 50 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.


Limitations

In some instances, differentiation between species may be problematic when using
16S rRNA 16S rRNA may refer to: * 16S ribosomal RNA 16 S ribosomal RNA (or 16 S rRNA) is the RNA component of the 30S subunit of a prokaryotic ribosome ( SSU rRNA). It binds to the Shine-Dalgarno sequence and provides most of the SSU structure. The g ...
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 (based on complete and current data from a range of test organisms) 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, 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 (
microsatellite A microsatellite is a tract of repetitive DNA in which certain DNA motifs (ranging in length from one to six or more base pairs) are repeated, typically 5–50 times. Microsatellites occur at thousands of locations within an organism's genome. ...
) regions and in restriction fragment length polymorphism ( RFLP) methods, all of which are widely used as part of
DNA profiling DNA profiling (also called DNA fingerprinting) is the process of determining an individual's DNA characteristics. DNA analysis intended to identify a species, rather than an individual, is called DNA barcoding. DNA profiling is a forensic tec ...
analysis.


See also

*
Molecular probe A molecular probe is a group of atoms or molecules used in molecular biology or chemistry to study the properties of other molecules or structures. If some measurable property of the molecular probe used changes when it interacts with the analyte ...


References

{{DEFAULTSORT:Hybridization Probe Genetics techniques Molecular biology