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
Schematic of TaqMan (5' nuclease) assay.
primer probe ^ Q
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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This book discusses the futility of curing stammering by common means. It traces various attempts at curing stammering in the past and how wasteful these attempt were, until he discovered a simple program to cure it. The book presents the life of Benjamin Nathaniel Bogue and his struggles with the handicap. Bogue devotes a great deal of text to explain the handicap of stammering, its effects on the body and psychology of the sufferer, and its cure.