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DNA Amplification

Probe-based qPCR & RT-qPCR

Probe-based quantitative PCR (qPCR) uses real-time fluorescence from 5ʹ-3ʹ exonuclease cleavage of a fluorescently-labeled, target-specific probe to measure DNA amplification at each cycle of a PCR. Because probe-based qPCR is typically more specific than dye-based qPCR, it is often the foundational technology employed in qPCR diagnostic assays. Probe designs vary but the most common type, hydrolysis (e.g., TaqMan®) probes, incorporate a 5’ reporter fluorophore and a 3’ quencher on a short oligonucleotide complementary to the target sequence. Fluorescence resonance energy transfer (FRET) prohibits emission of the fluorophore while the oligo probe is intact. During each PCR cycle, the 5’ flap endonuclease domain of Taq DNA polymerase hydrolyzes the probe as the primer is extended and the target sequence is amplified. This cleavage event separates the reporter fluorophore from the quencher and results in an amplification-dependent increase in fluorescence.  Probe-based qPCR allows multiple targets to be quantified in a single reaction (multiplexing) by using a unique fluorescent dye for each amplicon-specific probe.

Some probe designs do not require the 5’ flap endonuclease activity of Taq such as molecular beacons, Scorpions® and dual-hybridization probes. Molecular beacons form a stem-loop structure in the absence of the target sequence, forcing a 5’ fluorophore in close proximity to a 3’ quencher. Upon binding to the target sequence, the fluorophore and quencher become spatially separated, resulting in the fluorescence of the reporter dye. Scorpions are bifunctional molecules that link a stem-loop structure carrying a reporter fluorophore and quencher to a PCR primer. A portion of the stem-loop sequence is designed to hybridize to the newly synthesized strand, downstream of the primer binding site. Extension of the dual-labeled primer by the polymerase creates a copy of the complementary target sequence. In the subsequent PCR cycle, the stem-loop unfolds and binds intramolecularly to the recently synthesized DNA strand, separating the fluorophore from the quencher, resulting in an increase in fluorescence. A blocker between the stem-loop and primer prohibits any further extension of the target-probe complex. Finally, dual-hybridization probes, or LightCycler® type probes, rely on FRET in order to produce, rather than to quench, a detectable fluorescence signal. Two sequence-specific probes containing compatible FRET dyes bind adjacently to the target sequence, downstream of the PCR primers. Excitation of the donor fluorophore, located on the 3’ end of the first probe, causes emission of the acceptor fluorophore, located on the 5’ end of the second probe. Monitoring the emission wavelength of the acceptor dye results in an increased fluorescence signal as the target sequence is amplified.

 

 

TaqMan® is a registered trademark of Roche Molecular Systems, Inc.
SYBR® is a registered trademark of Molecular Probes, Inc.

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