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الرياض, Saudi Arabia
مسلم، وأناأحوج ما أكون إلى معرفة نفسي

الجمعة، 31 ديسمبر، 2010

TaqMan PCR

INTRODUCTION
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 (Holland, et al. 1991), 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´ nuclease 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). As in other real-time 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 (The Real-Time TaqMan PCR).
Real-time polymerase chain reaction (PCR) methods are relatively recent technological advances that allow routine quantitative analysis of nucleic acid sequences (DNA and RNA). In this report, we provide a brief overview of 5\'-3\' exonuclease-based real-time PCR assays (also known as TaqMan real-time PCR) used in own and in many other laboratories. We discuss the theory, protocols, advantages, drawbacks and utility of these methods in the assessment and quantification of some of the common chromosomal translocations associated with hematopoietic tumors.
The real-time polymerase chain reaction (PCR) assay is a relatively recent technological advance that is quickly becoming widely accepted for routine quantitative analysis of nucleic acid sequences (DNA and RNA). TaqMan Real-time PCR, sometimes referred to as kinetic PCR, or 5' exonuclease-based PCR assay, exploits the 5'-3' exonuclease activity of Taq polymerase first described by Holland and collaborators (Holland, et al. 1991). This assay integrates fluorogenic PCR with a laser-based instrumentation system, the PRISM 7700 Sequence Detector (PE Applied Biosystems, Foster City, CA), to detect and quantitate specific PCR amplicons as the reactions proceed.
TaqMan utilizes a system that is fairly easy to grasp conceptually. First, we must take a look at the TaqMan probe. The probe consists of two types of fluorophores, which are the fluorescent parts of reporter proteins (Green Fluorescent Protein (GFP) has an often-used fluorophore). While the probe is attached or unattached to the template DNA and before the polymerase acts, the quencher (Q) fluorophore (usually a long-wavelength colored dye, such as red) reduces the fluorescence from the reporter (R) fluorophore (usually a short-wavelength colored dye, such as green). It does this by the use of Fluorescence (or Förster) Resonance Energy Transfer (FRET), which is the inhibition of one dye caused by another without emission of a proton. The reporter dye is found on the 5’ end of the probe and the quencher at the 3’ end. Once the TaqMan® probe has bound to its specific piece of the template DNA after denaturation (high temperature) and the reaction cools, the primers anneal to the DNA. Taq polymerase then adds nucleotides and removes the Taqman® probe from the template DNA. This separates the quencher from the reporter, and allows the reporter to give off its emit its energy. This is then quantified using a computer. The more times the denaturing and annealing takes place, the more opportunities there are for the Taqman® probe to bind and, in turn, the more emitted light is detected (www.probes.com 2003).
Figure 1. The Taqman probe. The red circle represents the quenching dye that disrupts the observable signal from the reporter dye (green circle) when it is within a short distance. Image created by Dan Pierce.




Figure 2. The TaqMan® probe binds to the target DNA, and the primer binds as well. Because the primer is bound, Taq polymerase can now create a complementary strand. Image created by Dan Pierce.


Figure 3. The reporter dye is released from the extending double-stranded DNA created by the Taq polymerase. Away from the quenching dye, the light emitted from the reporter dye in an excited state can now be observed. Image created by Dan Pierce.
QUANTIFICATION
The specifics in quantification of the light emitted during real-time PCR are fairly involved and complex. The light emitted from the dye in the excited state is received by a computer and shown on a graph display, such as this, showing PCR cycles on the X-axis and a logarithmic indication of intensity on the Y-axis.
Figure 4. A graph printout of actual data found using the TaqMan probe.

Figure 5. A real-time PCR machine used at Colorado State.
 PRINCIPLE
TaqMan probes consist of a fluorophore covalently attached to the 5’-end of the oligonucleotide probe and a quencher at the 3’-end (Figure 6). Several different fluorophores (e.g. 6-carboxyfluorescein, acronym: FAM, or tetrachlorofluorescin, acronym: TET) and quenchers (e.g. tetramethylrhodamine, acronym: TAMRA, or dihydrocyclopyrroloindole tripeptide minor groove binder, acronym: MGB) are available (Kutyavin, et al. 2000). The quencher molecule quenches the fluorescence emitted by the fluorophore when excited by the cycler’s light source via FRET (Fluorescence Resonance Energy Transfer) (Bustin, 2000).  As long as the fluorophore and the quencher are in proximity, quenching inhibits any fluorescence signals (Figure 6).
TaqMan probes are designed such that they anneal within a DNA region amplified by a specific set of primers. As the Taq polymerase extends the primer and synthesizes the nascent strand, the 5' to 3' exonuclease activity of the 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 real-time PCR thermal cycler is directly proportional to the fluorophore released and the amount of DNA template present in the PCR.

Figure 6: TaqMan probe chemistry mechanism

APPLICATIONS
TaqMan probe-based assays are widely used in real-time PCR:
·   In Gene expression assays.
·   Determine the viral load in clinical specimens.
·   Bacterial Identification (Allele ID - Assay Design for Bacterial Identification) assays.
·   Discriminate alleles.
·   DNA quantification.
·   Verification of microarray results.
REFERENCES
·         Holland, PM; Abramson, RD; Watson, R; Gelfand, DH (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): 7276–80. doi:10.1073/pnas.88.16.7276. PMID 1871133.  edit.
·         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. doi:10.1093/nar/28.2.655. PMID 10606668. 
·         www.probes.com 2003

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