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Figure 1. Hydrolysis probes (e.g., TaqMan® assay).
(Figure omitted. See article PDF.)
Figure 2. Model of a single amplification plot illustrating the nomenclature commonly used in real-time quantitative PCR.
(Figure omitted. See article PDF.)
Figure 3. Double-stranded DNA-intercalating agents/DNA-binding dyes (e.g., SYBR® Green 1).
(Figure omitted. See article PDF.)
Figure 4. Dual hybridization probes.
(Figure omitted. See article PDF.)
Figure 5. Molecular beacons.
(Figure omitted. See article PDF.)
Figure 6. Scorpion probes.
(Figure omitted. See article PDF.)
Even one copy of a specific sequence can be amplified and detected in PCR. The PCR reaction generates copies of a DNA template exponentially. This results in a quantitative relationship between the amount of starting target sequence and amount of PCR product accumulated at any particular cycle. Due to inhibitors of the polymerase reaction found with the template, reagent limitation or accumulation of pyrophosphate molecules, the PCR reaction eventually ceases to generate template at an exponential rate (i.e., the plateau phase) making the end point quantitation of PCR products unreliable. Therefore, duplicate reactions may generate variable amounts of PCR product. Only during the exponential phase of the PCR reaction is it possible to extrapolate back in order to determine the starting quantity of template sequence. The measurement of PCR products as they accumulate (i.e., real-time quantitative PCR) allows quantitation in the exponential phase of the reaction and therefore removes the variability associated with conventional PCR.
Since the first documentation of real-time PCR [1], it has been used for an increasing and diverse number of applications including mRNA expression studies, DNA copy number measurements in genomic or viral DNAs [2-7], allelic discrimination assays [8,9], expression analysis of specific splice variants of genes [10-13] and gene expression in para-ffin-embedded tissues [14,15] and laser captured microdissected cells [13,16-19].
Background & methodology
Real-time quantitative PCR is the reliable detection and measurement of products generated during each cycle of the PCR process, which are directly proportional to the amount of template prior to the start of the PCR pro-cess. Holland and coworkers demonstrated that the thermostable enzyme Thermus aquaticus (i.e., Taq) DNA polymerase had 5´to 3´exonuclease activity. This group also showed that cleavage of a target probe during PCR by the 5´nuclease activity of Taq polymerase can be used to...