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TaqMan

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TaqMan

TaqMan probes are hydrolysis probes that are designed to increase the specificity of quantitative PCR. The method was first reported in 1991 by researchers at Cetus Corporation,[1] and the technology was subsequently developed by Roche Molecular Diagnostics for diagnostic assays and by Applied Biosystems for research applications.

The TaqMan probe principle relies on the 5´–3´ exonuclease activity of Taq polymerase to cleave a dual-labeled probe during hybridization to the complementary target sequence and fluorophore-based detection.[2] 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 PacMan (Taq Polymerase + PacMan = TaqMan) as its mechanism is based on the PacMan principle.[3]

Principle

Figure 1: TaqMan probe chemistry mechanism

TaqMan probes consist of a fluorophore covalently attached to the 5’-end of the oligonucleotide probe and a quencher at the 3’-end[4] (Figure 1). Several different fluorophores (e.g. 6-carboxyfluorescein, acronym: FAM, or tetrachlorofluorescein, acronym: TET) and quenchers (e.g. tetramethylrhodamine, acronym: TAMRA, or dihydrocyclopyrroloindole tripeptide minor groove binder, acronym: MGB) are available.[5] The quencher molecule quenches the fluorescence emitted by the fluorophore when excited by the cycler’s light source via FRET (Fluorescence Resonance Energy Transfer).[6] As long as the fluorophore and the quencher are in proximity, quenching inhibits any fluorescence signals (Figure 1).

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.) As the Taq polymerase extends the primer and synthesizes the nascent strand (again, on a single-strand template, but in the direction opposite to that shown in the diagram, i.e. from 3' to 5' of the complementary strand), the 5' to 3' exonuclease 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 close 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:

  • Determine the viral load in clinical specimens (HIV, Hepatitis)
  • Bacterial Identification[7] assays
  • DNA quantification

See also

Notes and references

  1. ^ Holland, P. M.; Abramson, R. D.; Watson, R.; Gelfand, D. H. (1991). "Detection of specific polymerase chain reaction product by utilizing the 5'----3' exonuclease activity of Thermus aquaticus DNA polymerase". Proceedings of the National Academy of Sciences of the United States of America 88 (16): 7276–7280.  
  2. ^ TaqMan Gene Expression - NCBI Projects
  3. ^ The Real-Time TaqMan PCR and Applications in Veterinary Medicine - From PacMan to TaqMan - a computer game revisited
  4. ^ TaqMan Probes: Introduction, functioning and applications
  5. ^ Kutyavin IV, Afonina IA, Mills A, Gorn VV, Lukhtanov EA, Belousov ES, Singer MJ, Walburger DK, Lokhov SG, Gall AA, Dempcy R, Reed MW, Meyer RB, Hedgpeth J (2000). "3′-Minor groove binder-DNA probes increase sequence specificity at PCR extension temperatures". Nucleic Acids Res 28 (2): 655–661.  
  6. ^ Bustin SA (October 2000). "Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays". J. Mol. Endocrinol. 25 (2): 169–93.  
  7. ^ AlleleID - Assay Design for Bacterial Identification

External links

1. TaqMan RT-PCR resources - primer databases, software, protocols.
2. Beacon Designer - Software to design real time PCR primers and probes including SYBR Green primers, TaqMan Probes, Molecular Beacons.

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