R EAL - TIME PCR S

After the invention of the polymerase chain reaction (PCR) method by Kary Mullis the development of novel chemistries and instrumentation platforms enables the detection of PCR products on a time basis. Traditional PCR is measured at end-point (plateau), while real-time PCR collects data in the exponential growth phase as an indicator of amplicon production during each PCR cycle. An increase in reporter fluorescent signal is directly proportional to the number of amplicons generated. A small amplicon size results in increased amplification efficiency. The real-time progress of the reaction can be viewed in some systems (e.g. Rotor-Gene). Real-time PCR quantitation eliminates post PCR processing of PCR products. This helps to increase throughput and reduce the chances of carryover contamination. In comparison to conventional PCR, real-time PCR also offers a much wider dynamic range. Dynamic range of any assay determines how much target concentration can vary and still be quantified. Available for real-time PCR are five main different chemistries, LightCycler^® HybProbe (Roche Diagnostics, Mannheim, Germany), TaqMan^® (Applied Biosystems, Foster City, CA, U.S.A.), Molecular Beacons, Scorpions^® and SYBR^® Green (Molecular Probes). All of these chemistries allow detection of PCR products via the generation of a fluorescent signal. SYBR Green is a fluorogenic dye that emits a strong fluorescent signal upon binding to double-stranded DNA. LightCycler^® HybProbes, TaqMan probes, Molecular Beacons and Scorpions depend on Förster Resonance Energy Transfer (FRET) to generate the fluorescence signal via the coupling of a fluorogenic dye molecule and a quencher moiety to the same or different oligonucleotide substrates.

9.13.1 SYBR Green

SYBR Green is the most economical choice for real-time PCR. Since the dye binds to double-stranded DNA and upon excitation emits light, there is no need to design a probe for any particular target being analyzed. Thus, as a PCR product accumulates, fluorescence increases.

However, since the dye cannot distinguish between specific and non-specific product accumulated during PCR, an overestimation of the target concentration or a false positive detection may result.

9.13.2 LightCycler^® HybProbe

The LightCycler^® HybProbe format is based on fluorescence resonance energy transfer (FRET). Two sequence-specific oligonucleotide probes are labeled with different dyes (donor and acceptor). During the annealing phase, HybProbe probes hybridize to the target sequences on the amplified DNA fragment in a head-to-tail arrangement, thereby bringing the two dyes close to each other. The energy emitted by the instrument excited donor dye excites the acceptor dye on the second HybProbe probe, which then emits fluorescent light at a different wavelength. This fluorescence is directly proportional to the amount of target DNA generated during PCR. HybProbe probes are displaced during the elongation and denaturation steps.

9.13.3 TaqMan Probes

TaqMan probes depend on the 5'- nuclease activity of the DNA polymerase used for PCR.

TaqMan probes are oligonucleotides that have a fluorescent reporter dye (FAM, TAMRA, TET, ROX) attached to the 5' end and a quencher moiety (e.g. Dabcyl) coupled to the 3' end.

These probes are designed to hybridize to an internal region of a PCR product. In the unhybridized state, the closeness of the reporter and the quench molecules prevents the detection of fluorescent signal from the probe. During PCR, when the polymerase replicates a template on which a TaqMan probe is bound, the 5'- nuclease activity of the polymerase cleaves the probe. This decouples the fluorescent and quenching dyes and FRET no longer occurs. Thus, fluorescence increases in each cycle, proportional to the amount of probe cleavage.

Materials and Methods

Figure 5: TaqMan Probes show an increase in fluorescence with each cycle. The signal depends on hydrolysis after hybridization.

9.13.4 Molecular Beacons

Molecular Beacons use FRET to detect and quantitate the synthesized PCR product via a reporter coupled to the 5' end and a quencher attached to the 3' end of an oligonucleotide.

Molecular Beacons are designed to remain intact during the amplification reaction. They must rebind to the target in every cycle for signal measurement. Molecular Beacons form a stem-loop structure in unbound state. Thus, the close proximity of the reporter and quencher molecules prevents the probe from fluorescing. When a Molecular Beacon hybridizes to a target, the fluorescent dye and quencher are separated, FRET does not occur and the fluorescent dye emits light upon irradiation.

Figure 6: Molecular Beacons are sequence specific probes with self-complementary hairpin configuration. Their ends allow vicinity of reporter and quencher molecule.

9.13.5 Scorpions

With Scorpion probes, sequence-specific priming and PCR product detection is achieved using a single oligonucleotide. The Scorpion probe maintains a stem-loop configuration in the unhybridized state. The fluorophore is attached to the 5' end and is quenched by a moiety coupled to the 3' end. The 3' portion of the stem also contains a sequence that is complementary to the extension product of the primer. This sequence is linked to the 5' end of

specific probe sequence is able to bind to its complement within the extended amplicon thus opening up the hairpin loop. This prevents the fluorescence from being quenched and a signal is observed.

9.13.6 Multiplex PCR

TaqMan probes, Molecular Beacons and Scorpions allow multiple DNA species to be measured in the same reaction (multiplex PCR), since fluorescent dyes with different emission spectra may be attached to the different probes. Multiplex PCR allows for example internal controls to be co-amplified.

9.13.7 Quantitation by standard curves

A standard curve is constructed from RNA, DNA, plasmid dsDNA, in vitro generated ssDNA or any cDNA of known concentration. This curve is then used as a reference standard for extrapolating quantitative information for RNA or DNA targets of unknown concentrations.

The important parameter for quantitation is the cycle threshold (CT). The higher the initial amount of genomic DNA, the sooner accumulated product is detected in the PCR process, and the lower the CT value. Besides being used for quantitation, the CT value can be used for qualitative analysis. The threshold should be placed above any baseline activity and within the exponential increase phase. Some software allows determination of the CT by a mathematical analysis of the growth curve. This provides better run-to-run reproducibility. The calculated CT value is the cycle at which the system begins to detect the increase in the signal associated with an exponential growth of PCR product during the log-linear phase. This phase provides the most useful information about the reaction (certainly more important than the end-point in traditional PCR).

The slope of the log-linear phase is a reflection of the amplification efficiency. The efficiency (E) of the reaction can be calculated by the formula: E =10(-1/slope) – 1

The efficiency of the PCR should be 90 - 100% (– 3.6 > slope > – 3.1). A number of variables can affect the efficiency of the PCR including length of the amplicon, secondary structure and primer quality.

Materials and Methods

9.13.8 Instrumentation

Real-time PCR requires an instrument that consists of a thermal cycler, a computer, optics for fluorescence excitation and emission collection, data acquisition and analysis software. These instruments differ in sample capacity, method of excitation, reaction vessels, data processes and run time. Real-time PCR platforms from three different manufacturers were used:

9.13.8.1 LightCycler® instrument

The LightCycler^® System is offered as two different instruments: the LightCycler® 1.5 Instrument for single dye or duplex assays and the LightCycler® 2.0 Instrument for a wider range of multiplexing (Figure 7).

Figure 7: The LightCycler^® 2.0 (left side) and the LightCycler^® 1.5 (right side).

9.13.8.2 Rotor-Gene™ instrument

The Corbett Research Rotor-Gene™ uses a centrifugal design. After the displacement of the Rotor-Gene™ 2000, the Rotor-Gene™ 3000 is available in two different configurations: the Four Channel model 3000 (also available as artus™ 3000) and the Two Channel model 3000A (Figure 8).

Figure 8: The Rotor-Gene™ 3000 (left side) and the Rotor-Gene™

2000 (right side).

9.13.8.3 ABI PRISM™ instrument

The ABI PRISM™ 7000, 7700 and 7900HT Sequence Detection System were used with main focus on the ABI PRISM™ 7000 SDS (Figure 9). All of the ABI PRISM instruments are Peltier-based thermal cycling systems.

Figure 9: The ABI PRISM™ 7000, 7700 and 7900HT SDS (left to right).