TaqMan probes are hydrolysis probes that are designed to increase the specificity of quantitative PCR. The method was first reported in 1991 by researcher

Kary Mullis Kary Banks Mullis (December 28, 1944August 7, 2019) was an American biochemist. In recognition of his role in the invention of the polymerase chain reaction (PCR) technique, he shared the 1993 Nobel Prize in Chemistry with Michael Smith and w ...

at Cetus Corporation, and the technology was subsequently developed by

Hoffmann-La Roche F. Hoffmann-La Roche AG, commonly known as Roche, is a Swiss multinational healthcare company that operates worldwide under two divisions: Pharmaceuticals and Diagnostics. Its holding company, Roche Holding AG, has shares listed on the SIX ...

for diagnostic assays and by Applied Biosystems (now part of Thermo Fisher Scientific) for research applications. The TaqMan probe principle relies on the 5´–3´

exonuclease Exonucleases are enzymes that work by cleaving nucleotides one at a time from the end (exo) of a polynucleotide chain. A hydrolyzing reaction that breaks phosphodiester bonds at either the 3′ or the 5′ end occurs. Its close relative is the ...

activity of ''Taq'' polymerase to cleave a dual-labeled probe during hybridization to the complementary target sequence and fluorophore-based detection.TaqMan Gene Expression - NCBI Projects
/ref> As in other quantitative PCR methods, the resulting fluorescence signal permits quantitative measurements of the accumulation of the product during the exponential stages of the PCR; however, the TaqMan probe significantly increases the specificity of the detection. TaqMan probes were named after the videogame

Pac-Man originally called ''Puck Man'' in Japan, is a 1980 maze action video game developed and released by Namco for arcades. In North America, the game was released by Midway Manufacturing as part of its licensing agreement with Namco America. Th ...

(''Taq'' Polymerase + PacMan = TaqMan) as its mechanism is based on the Pac-Man principle.The Real-Time TaqMan PCR and Applications in Veterinary Medicine - From PacMan to TaqMan - a computer game revisited

Principle

TaqMan probes consist of a fluorophore covalently attached to the 5’-end of the oligonucleotide probe and a quencher at the 3’-end.TaqMan Probes: Introduction, functioning and applications
/ref> Several different fluorophores (e.g.

6-carboxyfluorescein 6-Carboxyfluorescein (6-FAM) is a fluorescent dye with an absorption wavelength of 495 nm and an emission wavelength of 517 nm. A carboxyfluorescein molecule is a fluorescein molecule with a carboxyl group added. They are commonly used a ...

, acronym: ''FAM'', or tetrachlorofluorescein, acronym: TET) and '' quenchers'' (e.g. tetramethyl rhodamine, acronym: TAMRA) are available. The quencher molecule quenches the fluorescence emitted by the fluorophore when excited by the cycler’s light source via Förster resonance energy transfer (FRET). As long as the fluorophore and the quencher are in proximity, quenching inhibits any fluorescence signals. TaqMan probes are designed such that they anneal within a DNA region amplified by a specific set of primers. (Unlike the diagram, the probe binds to single stranded DNA.) TaqMan probes can be conjugated to a minor groove binder (MGB) moiety, dihydrocyclopyrroloindole tripeptide (DPI₃), in order to increase its binding affinity to the target sequence; MGB-conjugated probes have a higher melting temperature (T_m) due to increased stabilization of van der Waals forces. As the ''Taq'' polymerase extends the

primer Primer may refer to: Arts, entertainment, and media Films * ''Primer'' (film), a 2004 feature film written and directed by Shane Carruth * ''Primer'' (video), a documentary about the funk band Living Colour Literature * Primer (textbook), a t ...

and synthesizes the nascent strand (from the single-stranded template), the 5' to 3'

activity of the ''Taq'' polymerase degrades the probe that has annealed to the template. Degradation of the probe releases the fluorophore from it and breaks the proximity to the quencher, thus relieving the quenching effect and allowing fluorescence of the fluorophore. Hence, fluorescence detected in the quantitative PCR thermal cycler is directly proportional to the fluorophore released and the amount of DNA template present in the PCR.

Applications

TaqMan probe-based assays are widely used in quantitative PCR in research and

medical laboratories A medical laboratory or clinical laboratory is a laboratory where tests are conducted out on clinical specimens to obtain information about the health of a patient to aid in diagnosis, treatment, and prevention of disease. Clinical Medical la ...

: *

Gene expression Gene expression is the process by which information from a gene is used in the synthesis of a functional gene product that enables it to produce end products, protein or non-coding RNA, and ultimately affect a phenotype, as the final effect. The ...

assays * Pharmacogenomics * Human Leukocyte Antigen (HLA) genotyping *Determination of viral load in clinical specimens (

HIV The human immunodeficiency viruses (HIV) are two species of '' Lentivirus'' (a subgroup of retrovirus) that infect humans. Over time, they cause acquired immunodeficiency syndrome (AIDS), a condition in which progressive failure of the immu ...

, Hepatitis) *Bacterial IdentificationAlleleID - Assay Design for Bacterial Identification
/ref> assays * DNA quantification * SNP genotyping *Verification of

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 silicon t ...

results

Notes and references

External links

1
TaqMan RT-PCR resources
primer databases, software, protocols
2

- Software to design real time PCR primers and probes including SYBR Green primers, TaqMan Probes, Molecular Beacons. {{DEFAULTSORT:Taqman Chemical reactions Gene expression Polymerase chain reaction

Principle

Applications

See also

Notes and references

External links