CPD Course: REAL-TIME PCR

Molecular Biology: Real-time PCR

Molecular Biology:

Real-time PCR

Author: Dr Kgomotso Sibeko-Matjila

Licensed under a Creative Commons Attribution license.

Pre-requisites for the sub-module on Real-time PCR:

1. General theory on Molecular Biology
2. Polymerase chain reaction (PCR)

REAL-TIME PCR DETECTION FORMATS (OR CHEMISTRIES)

Real-time PCR uses fluorescence to monitor the production of amplicons during the amplification reaction. The fluorescent signal is emitted by a fluorescent dye or fluorescent probe included in the PCR reaction mix. FAQ3There are two detection chemistries used with the real-time PCR technology to monitor the accumulation of PCR product during the amplification reaction. These chemistries are based on the type of fluorescent molecule used and can be divided into two broad categories:

• Non-specific detection
• Specific detection

Non-specific detection methods

Non-specific detection methods employ non-specific DNA binding dyes as fluorescent reporters to monitor the real-time PCR reaction. The binding of these dyes to a DNA molecule is independent of that particular DNA sequence. The most commonly used non-specific DNA binding dye is a DNA intercalating agent, SYBRgreen. Although ethidium bromide was the first dye to be used as a DNA-binding fluorophore (Higuchi et al., 1992),SYBR green is the preferred dye because its binding affinity to double-stranded DNA (dsDNA) is more than 100 times higher than that of ethidium bromide and it does not interfere with the amplification process since it binds to the minor groove of the dsDNA (Witter et al., 1997; Morrison et al., 1998). FAQ4This fluorogenic groove-binding dye, binds non-specifically to dsDNA; it does not bind to single-stranded DNA (ssDNA). FAQ5Because SYBR green molecules bind non-specifically to any dsDNA, they will also bind to non-specific products if present in a reaction. Therefore, SYBR green PCR assays need to be properly optimized to avoid amplification of non-specific productsor production of primer dimers, and thus reporting of false positive results. The unbound form of SYBR green exhibits very little fluorescence but emits a strong fluorescent signal once bound to dsDNA (Morrison, 1998). During amplification, the fluorescentsignal increases as the PCR product accumulates with each successive cycle of amplification (Animation 1: the real-time PCR system records the amount of fluorescence emitted allowing the system to monitor the PCR reaction during the amplification process.

Reaction summary (Animation 1)

1. During amplification, the DNA polymerase enzyme amplifies the target sequence, thus producing PCR products.
2. SYBR green I dye then binds to the newly produced double-stranded amplicons resulting in increased fluorescence.
3. As more amplicons are produced, more SYBR green molecules bind to the dsDNA; consequently, the fluorescence intensity increases proportionate to the amount of the PCR product produced.

Specific detection methods

Specific detection methods use fluorogenic-labeled oligonucleotide probes, in addition to primers. FAQ6These probes are designed to bind to a specific sequence on the target DNA, thus increasing the specificity of the PCR. When usingthe specific detection methods, post PCR processing is not necessary because the fluorogenic probes only allow detection of a specific amplification product, consequently eliminating detection of non-specific PCR products.

The following are the two commonly used probe-based real-time PCR chemistries:

1. Hydrolysis probes (or 5’-exonuclease oligonucleotide probes)
2. Hybridization probes(or FRET probes)

Hydrolysis probe chemistry

Hydrolysis probes are dual labeled oligonucleotides of 18-30 bp in size,with a fluorescent reporter dye on the 5’ end and a non-fluorescent quencher dye on the 3’ end. The most commonly used hydrolysis probes are TaqMan®probes (Holland et al., 1991; Heid, 1996). Molecular beacons probes (Tyagi and Kramer, 1996; Tyagi et al., 1998) and Scorpions primers (Whitcombe et al., 1999; Thelwell et al., 2000) also function as hydrolysis probes;for the purpose of these notes, only the TaqMan® probes will be discussed. The TaqMan probe does not release fluorescence when intact because the quencher dye on the probe greatly reduces the fluorescence emitted by the reporter dye. Another essential element of the hydrolysis probe chemistry is the 5’ exonuclease activity of the TaqDNA polymerase which is responsible for the hydrolysis of the probe. FAQ7During annealing, primers and probes hybridize onto the target sequence. At extension, the dsDNA-specific 5’-exonuclease activity of TaqDNA polymerase cleaves the bound probe as the TaqDNA polymerase extends the 3’-end of the primerduring the amplification of the target sequence (Gibson et al., 1996). Consequently, the reporter dye can emit fluorescence because it is separated from the quencher dye (Livak et al., 1995; Heid et al., 1996) (Animation 2: The fluorescent signal that is released due to this process allowsmonitoring of the accumulation of the PCR product. The fluorescence intensity is proportional to the amount of amplicon produced.

Reaction summary (Animation 2)

In the presence of the target sequence, the TaqMan® probe anneals downstream from one of the primer sites.

During amplification the probe is cleaved by the 5’-exonuclease activity of Taq DNA polymerase as the primer is extended,thusdisplacing it from the target strand.

The reporter dye fluorescent signal increases as the activity of the quencher is ended.

The hydrolysis process is repeated in subsequent cycles increasing the reportersignal.

Accumulation of PCR products is detected by monitoring the increase in fluorescence of the reporter dye.

Hybridization probes chemistry

In contrast to hydrolysis probe chemistry whereby a single probe is labeled with two dyes, FAQ8the hybridization probes chemistryuses two short oligonucleotide probes labeled with differentflorescent dyes to measure the transfer of energy between the two fluorophoresattached to the probes (Simon et al, 2004). The two probes aredesigned to hybridize adjacent to each other in a head-to-tail configuration on a nucleotide sequence. The upstream probe contains a fluorophore referred to as a reporter (or donor) dye on the 3’-end and the downstream probe has a non-fluorescent quencher (or acceptor) dye on the 5’-end. When the two fluorophores are inclose proximity, the quencher dye on the one probe greatly reduces the fluorescence emitted by the reporter dye from the second probe using the fluorescence resonance energy transfer (FRET)(Cardullo et al., 1988). FAQ9The FRET phenomenon is distance-dependent, meaning this process will only take place when the two dyes are in close proximity. FRET occurs due to interaction between the electronic excited states of two dye molecules; the excitation is transferred from one dye molecule (the donor) to the other (the acceptor) without emission of a photon. The emission spectrum of one dye should overlap significantly with the excitation spectrum of the other. During FRET, the donor fluorophore is excited by a light source and thus transfers its energy to the acceptor fluorophore when the two are in close proximity. The light source cannot excite the acceptor dye. Once excited by the transfer of energy from the donor fluorophore, the acceptor fluorophore emits light of a longer wavelength, which is detected by the real-time PCR system at a channel specific for that wavelength.The first dye, linked to the donor probe, is a fluorescent dye with an excitation peak of a shorter-wavelength (~ 480-530 nm) while the second, linked to the acceptor probe, can be either a quencher dye or another fluorescent dye which can absorb fluorescent light transferred from the first dye and reemit light at a longer-wavelength,eg.Cyanine dyes Cy3 and Cy5, TET (6-carboxy-4, 7, 2’, 7’-tetrachloro-fluorescein),TAMRA (6-carboxy-N, N, N’, N’-tetramethyrhodamine), ROX (6-carboxyrhodamine) and LightCycler RED-640 and LightCycler RED-705, specifically for use in FRET probes in the Roche LightCycler (Wittwer et al., 1997a, b; Kaltenboeck and Wang, 2005). The 3’-end of the acceptor probe is blocked to preventextension during the amplification process. In contrast to hydrolysis probes, FRET probes are not degraded and fluorescence is reversible, allowing the determination of probe Tm in melt curve analysis (to be discussed in the next section dealing with Analysis).

Reaction summary

1. FAQ10During PCR, the two probes anneal to the target sequence, adjacent to each other.
2. The donor fluorophore is excited by the light source from the real-time PCR system.
3. The activation energy of fluorescein (from the donor probe) is directly transferred to the acceptor dyebyFRET
4. The acceptor fluorophore emits light at a different wavelength.
5. Subsequently the fluorescent signal can be detected and measured.
6. This happens during the annealing phase and first part of the extension phase of the PCR process.
7. After each subsequent PCR cycle more hybridization probes can anneal, resulting in higher fluorescent signals.
8. The fluorescence emitted is proportional to the accumulated PCR product.

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