A Riboprobe, abbreviation of RNA probe, is a segment of labelled RNA that can be used to detect a target mRNA or DNA during

in situ hybridization ''In situ'' hybridization (ISH) is a type of hybridization that uses a labeled complementary DNA, RNA or modified nucleic acids strand (i.e., probe) to localize a specific DNA or RNA sequence in a portion or section of tissue (''in situ'') or ...

. RNA probes can be produced by ''

in vitro ''In vitro'' (meaning in glass, or ''in the glass'') studies are performed with microorganisms, cells, or biological molecules outside their normal biological context. Colloquially called "test-tube experiments", these studies in biology and ...

'' transcription of cloned DNA inserted in a suitable

plasmid A plasmid is a small, extrachromosomal DNA molecule within a cell that is physically separated from chromosomal DNA and can replicate independently. They are most commonly found as small circular, double-stranded DNA molecules in bacteria; howev ...

downstream of a viral promoter. Some bacterial

viruses A virus is a submicroscopic infectious agent that replicates only inside the living cells Cell most often refers to: * Cell (biology), the functional basic unit of life Cell may also refer to: Locations * Monastic cell, a small room ...

code for their own

RNA polymerase In molecular biology, RNA polymerase (abbreviated RNAP or RNApol), or more specifically DNA-directed/dependent RNA polymerase (DdRP), is an enzyme that synthesizes RNA from a DNA template. Using the enzyme helicase, RNAP locally opens the ...

s, which are highly specific for the viral promoters. Using these enzymes, labeled NTPs, and inserts inserted in both forward and reverse orientations, both sense and antisense riboprobes can be generated from a cloned

gene In biology, the word gene (from , ; "... Wilhelm Johannsen coined the word gene to describe the Mendelian units of heredity..." meaning ''generation'' or ''birth'' or ''gender'') can have several different meanings. The Mendelian gene is a b ...

. Since

James Watson James Dewey Watson (born April 6, 1928) is an American molecular biologist, geneticist, and zoologist. In 1953, he co-authored with Francis Crick the academic paper proposing the double helix structure of the DNA molecule. Watson, Crick and ...

and

Francis Crick Francis Harry Compton Crick (8 June 1916 – 28 July 2004) was an English molecular biologist, biophysicist, and neuroscientist. He, James Watson, Rosalind Franklin, and Maurice Wilkins played crucial roles in deciphering the helical stru ...

revealed the

double helix A double is a look-alike or doppelgänger; one person or being that resembles another. Double, The Double or Dubble may also refer to: Film and television * Double (filmmaking), someone who substitutes for the credited actor of a character * ...

nature of DNA molecule (Watson & Crick, 1953), the hydrogen bonds between the four bases are well known: adenine always binds to thymine and cytosine always binds to guanine. This binding pattern is the basic principle of modern genetic technologies. Joseph Gall and Mary Lou Pardue published a paper in 1969 demonstrating that radioactive marked ribosomal DNA can be used to detect its complementary DNA sequence in a frog egg, known as the first researchers who use DNA probes to perform in situ hybridization. RNA probes were proved to be able to perform the same function and also used with

. Fluorescent dyed probes replaced radio labeled probes due to the consideration of safety, stability, and ease of detection. Detecting a DNA sequence is similar to "looking for a needle in a haystack, with the needle being the DNA sequence of interest and the haystack being a set of chromosomes". The ability of the DNA helix to disassociate, re-anneal and the remarkable accuracy of base-pairing grants riboprobes the ability to locate its complementary DNA sequence on chromosomes.

Applications

There are two kinds of probes used during in situ hybridization: Riboprobes and DNA oligonucleotides probes. Riboprobes are essential in the study of embryo development in which DNA probes are insufficient. With labelled (fluoresce-dyed, for instance) antisense RNA probes hybridized with developing embryo's mRNA, tracking the expression of genes in different stages of development is possible. RNA probes can be used in detecting either whole embryo's development or just on tissue sections of interest. The ability of riboprobes to bind to transcribed mRNA makes RNA probes important in research on model organisms: Drosophila, zebrafish, chick,

Xenopus ''Xenopus'' () (Gk., ξενος, ''xenos''=strange, πους, ''pous''=foot, commonly known as the clawed frog) is a genus of highly aquatic frogs native to sub-Saharan Africa. Twenty species are currently described within it. The two best-kno ...

and mouse. RNA probes can also be utilized in

immunohistochemistry Immunohistochemistry (IHC) is the most common application of immunostaining. It involves the process of selectively identifying antigens (proteins) in cells of a tissue section by exploiting the principle of antibodies binding specifically to a ...

to identify tissue infection in embryos. Viral mRNA can be targeted by its antisense RNA probes, while infected tissues don't have complementary mRNA that can hybridize with probes; the unique mRNA sequence of each organism makes the detection of expression of certain gene highly effective and accurate.

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 by ...

(FISH)is the most widely used riboprobe technique. A target sequence and a probe are essential in FISH. First, the probe is labeled with either direct or indirect labeling strategy: hapten-modified nucleotides are used in indirect labeling, and fluorophore-modified nucleotides are used in direct labeling. The target DNA and probes are denatured and mixed, allows the re-annealing of DNA sequences. Indirect labeling requires an extra step to produce visualized signals that requires use of enzymatic or immunological system, but provides greater signal amplification than direct labeling. FISH probes can also be used in

karyotype A karyotype is the general appearance of the complete set of metaphase chromosomes in the cells of a species or in an individual organism, mainly including their sizes, numbers, and shapes. Karyotyping is the process by which a karyotype is disce ...

studies. DNA probes can be labeled with various fluorochromes which produce a unique color for each chromosomes. The probes are then hybridized with metaphase chromosomes, producing unique patterns on each chromosomes. This method is useful when people want to study the translocation, deletion and duplication of chromosomes on a larger scale comparing to site-specific FISH.

References

{{Cite book , last= Baynes , first= John W. , author2= Marek H. Dominiczak , title= Medical Biochemistry 2nd. Edition , publisher= Elsevier Mosby , year= 2005 , page
477
, isbn= 978-0-7234-3341-5 , url-access= registration , url= https://archive.org/details/medicalbiochemis0000unse/page/477

External links

YouTube video:in situ hybridization * https://www.youtube.com/watch?v=1iRynlXIJw0 A detailed description of Fluorescence In Situ Hybridization: * http://www.nature.com/scitable/topicpage/fluorescence-in-situ-hybridization-fish-327# Riboprobe In Vitro Transcription Systems Technical Manual: * https://www.promega.com/resources/protocols/technical-manuals/0/riboprobe-in-vitro-transcription-systems-protocol/ Genetic engineering