The 5 Nuclease Assay Taqman

TaqMan probes are labeled with two fluorescent dyes that emit at different wavelengths (Figure 4.4). The probe sequence is intended to hybridize specifically in the DNA target region of interest between the two PCR primers (Ong and Irvine 2002). Typically the probe is designed to have a slightly higher annealing temperature compared to the PCR primers so that the probe will be

Figure 4.4

Schematic of TaqMan (5' nuclease) assay.

primer probe ^ Q

Reverse primer

Reverse primer

Reverse primer

Polymerization and Strand Displacement

Probe Cleavage (release of reporter dye)

Fluorescence occurs when reporter dye and quencher dye are no longer in close proximity

Completion of Polymerization hybridized when extension (polymerization) of the primers begins. A minor groove binder is sometimes used near the 3'-end of TaqMan probes to enable the use of shorter sequences that still have high annealing temperatures (Applied Biosystems 2003). The 'reporter' (R) dye is attached at the 5'-end of the probe sequence while the 'quencher' (Q) dye is synthesized on the 3'-end. A popular combination of dyes is FAM or VIC for the reporter dye and TAMRA for the quencher dye (see Chapter 13 for more information on these dyes). When the probe is intact and the reporter dye is in close proximity to the quencher dye, little-to-no fluorescence will result because of suppression of the reporter fluorescence due to an energy transfer between the two dyes.

During polymerization, strand synthesis will begin to displace any TaqMan probes that have hybridized to the target sequence. The Taq DNA polymerase used has a 5'-exonuclease activity and therefore will begin to chew away at any sequences in its path (i.e., those probes that have annealed to the target sequence). When the reporter dye molecule is released from the probe and it is no longer in close proximity to the quencher dye, it can begin to fluoresce (Figure 4.4). Increase in fluorescent signal results if the target sequence is complementary to the TaqMan probe.

Some assays, such as the Quantifiler kit, include an internal PCR control (IPC) that enables verification that the polymerase, the assay, and the detection instrumentation are working correctly. In this case, the IPC is labeled with a VIC (green) reporter dye and hybridizes to a synthetic template added to each reaction. The TaqMan probe for detecting the target region of interest is labeled with a FAM (blue) reporter dye and therefore spectrally resolvable from the green VIC dye. Instruments such as the ABI Prism 7000 Sequence Detection System enable another dye like ROX (red dye) to be placed in each well to adjust for well-to-well differences across a plate through background subtraction.

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