Hybridization probe
Encyclopedia
In molecular biology
, a hybridization probe is a fragment of DNA
or RNA
of variable length (usually 100-1000 bases long), which is used in DNA or RNA samples to detect the presence of nucleotide
sequences (the DNA target) that are complementary
to the sequence in the probe. The probe thereby hybridizes to single-stranded nucleic acid (DNA or RNA) whose base sequence allows probe-target base pair
ing due to complementarity between the probe and target. The labeled probe is first 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 in situ
.
To detect hybridization of the probe to its target sequence, the probe is tagged (or labelled) with a molecular marker of either radioactive or (more recently) fluorescent molecules; commonly used markers are 32P
(a radioactive isotope
of phosphorus
incorporated into the phosphodiester bond in the probe DNA) or Digoxigenin
, which is 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 microarray
s refer to DNA covalently attached to an inert surface, such as coated glass slides or gene chips, and to which a mobile cDNA target is hybridized.
Depending on the method
the probe may be synthesized
using phosphoramidite
method or generated and labeled by PCR
amplification or cloning
(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
. 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 like microarray
s.
Molecular Beacon
probes
TaqMan
® probes
LNA® (Locked Nucleic Acid
)probes
Cycling Probe Technology
(CPT)
Molecular biology
Molecular biology is the branch of biology that deals with the molecular basis of biological activity. This field overlaps with other areas of biology and chemistry, particularly genetics and biochemistry...
, a hybridization probe is a fragment of DNA
DNA
Deoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...
or RNA
RNA
Ribonucleic acid , or RNA, is one of the three major macromolecules that are essential for all known forms of life....
of variable length (usually 100-1000 bases long), which is used in DNA or RNA samples to detect the presence of nucleotide
Nucleotide
Nucleotides are molecules that, when joined together, make up the structural units of RNA and DNA. In addition, nucleotides participate in cellular signaling , and are incorporated into important cofactors of enzymatic reactions...
sequences (the DNA target) that are complementary
Complementarity (molecular biology)
In molecular biology, complementarity is a property of double-stranded nucleic acids such as DNA, as well as DNA:RNA duplexes. Each strand is complementary to the other in that the base pairs between them are non-covalently connected via two or three hydrogen bonds...
to the sequence in the probe. The probe thereby hybridizes to single-stranded nucleic acid (DNA or RNA) whose base sequence allows probe-target base pair
Base pair
In molecular biology and genetics, the linking between two nitrogenous bases on opposite complementary DNA or certain types of RNA strands that are connected via hydrogen bonds is called a base pair...
ing due to complementarity between the probe and target. The labeled probe is first denatured
Denaturation (biochemistry)
Denaturation is a process in which proteins or nucleic acids lose their tertiary structure and secondary structure by application of some external stress or compound, such as a strong acid or base, a concentrated inorganic salt, an organic solvent , or heat...
(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
Southern blot
A Southern blot is a method routinely 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 detection by probe hybridization. The method is named...
ting) or RNA (northern blot
Northern blot
The northern blot is a technique used in molecular biology research to study gene expression by detection of RNA in a sample. With northern blotting it is possible to observe cellular control over structure and function by determining the particular gene expression levels during differentiation,...
ting) immobilized on a membrane or in situ
Fluorescent in situ hybridization
FISH is a cytogenetic technique developed by biomedical researchers in the early 1980s that is used to detect and localize the presence or absence of specific DNA sequences on chromosomes. FISH uses fluorescent probes that bind to only those parts of the chromosome with which they show a high...
.
To detect hybridization of the probe to its target sequence, the probe is tagged (or labelled) with a molecular marker of either radioactive or (more recently) fluorescent molecules; commonly used markers are 32P
Isotopes of phosphorus
Although phosphorus has 24 isotopes from 24P to 47P, only one of these isotopes is stable 31P; as such, it is considered a monoisotopic element. The longest-lived radioactive isotopes are 33P with a half-life of 25.34 days and 32P with a half-life of 14.263 days. All other have half-lives under...
(a radioactive isotope
Isotope
Isotopes are variants of atoms of a particular chemical element, which have differing numbers of neutrons. Atoms of a particular element by definition must contain the same number of protons but may have a distinct number of neutrons which differs from atom to atom, without changing the designation...
of phosphorus
Phosphorus
Phosphorus is the chemical element that has the symbol P and atomic number 15. A multivalent nonmetal of the nitrogen group, phosphorus as a mineral is almost always present in its maximally oxidized state, as inorganic phosphate rocks...
incorporated into the phosphodiester bond in the probe DNA) or Digoxigenin
Digoxigenin
Digoxigenin is a steroid found exclusively in the flowers and leaves of the plants Digitalis purpurea, Digitalis orientalis and Digitalis lanata , where it is attached to sugars, to form the glycosides Digoxigenin (DIG) is a steroid found exclusively in the flowers and leaves of the plants...
, which is non-radioactive antibody
Antibody
An antibody, also known as an immunoglobulin, is a large Y-shaped protein used by the immune system to identify and neutralize foreign objects such as bacteria and viruses. The antibody recognizes a unique part of the foreign target, termed an antigen...
-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
Nucleic acid
Nucleic acids are biological molecules essential for life, and include DNA and RNA . Together with proteins, nucleic acids make up the most important macromolecules; each is found in abundance in all living things, where they function in encoding, transmitting and expressing genetic information...
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
Microarray
A microarray is a multiplex lab-on-a-chip. It is a 2D array on a solid substrate that assays large amounts of biological material using high-throughput screening methods.Types of microarrays include:...
s refer to DNA covalently attached to an inert surface, such as coated glass slides or gene chips, and to which a mobile cDNA target is hybridized.
Depending on the method
Nucleic acid methods
Nucleic acid methods are the techniques used to study nucleic acids .Purification*Phenol-chloroform extraction*minicolumn purification*RNA extractionQuantification*Abundance in weight: spectroscopic quantification...
the probe may be synthesized
Oligonucleotide synthesis
Oligonucleotide synthesis is the chemical synthesis of relatively short fragments of nucleic acids with defined chemical structure . The technique is extremely useful in current laboratory practice because it provides a rapid and inexpensive access to custom-made oligonucleotides of the desired...
using phosphoramidite
Phosphoramidite
Nucleoside phosphoramidites are derivatives of natural or synthetic nucleosides. They are used to synthesize oligonucleotides, relatively short fragments of nucleic acid and their analogs. Nucleoside phosphoramidites were first introduced in 1981 by Beaucage and Caruthers...
method or generated and labeled by PCR
Polymerase chain reaction
The polymerase chain reaction is a scientific technique in molecular biology to amplify a single or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence....
amplification or cloning
Molecular cloning
Molecular cloning refers to a set of experimental methods in molecular biology that are used to assemble recombinant DNA molecules and to direct their replication within host organisms...
(older methods). In order to increase the in vivo
In vivo
In vivo is experimentation using a whole, living organism as opposed to a partial or dead organism, or an in vitro controlled environment. Animal testing and clinical trials are two forms of in vivo research...
stability of the probe RNA is not used, instead RNA analogues may be used, in particular morpholino
Morpholino
In molecular biology, a Morpholino is a molecule in a particular structural family that is used to modify gene expression. Morpholino oligomers are an antisense technology used to block access of other molecules to specific sequences within nucleic acid...
. 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 like microarray
Microarray
A microarray is a multiplex lab-on-a-chip. It is a 2D array on a solid substrate that assays large amounts of biological material using high-throughput screening methods.Types of microarrays include:...
s.
Examples of probes
Scorpion® probesMolecular Beacon
Molecular beacon
Molecular beacons are oligonucleotide hybridization probes that can report the presence of specific nucleic acids in homogenous solutions. The terms more often used is molecular beacon probes. Molecular beacons are hairpin shaped molecules with an internally quenched fluorophore whose fluorescence...
probes
TaqMan
TaqMan
TaqMan probes are hydrolysis probes that are designed to increase the specificity of real-time PCR assays. The method was first reported in 1991 by researchers at Cetus Corporation, and the technology was subsequently developed by Roche Molecular Diagnostics for diagnostic assays and by Applied...
® probes
LNA® (Locked Nucleic Acid
Locked nucleic acid
A locked nucleic acid , often referred to as inaccessible RNA, is a modified RNA nucleotide. The ribose moiety of an LNA nucleotide is modified with an extra bridge connecting the 2' oxygen and 4' carbon. The bridge "locks" the ribose in the 3'-endo conformation, which is often found in the A-form...
)probes
Cycling Probe Technology
Cycling probe technology
Cycling probe technology is a molecular biological technique for detecting specific DNA sequences. CPT operates under isothermal conditions. In some applications, CPT offers an alternative to PCR...
(CPT)