Download 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, SYBR green. 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 doublestranded 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 products or production of
primer dimers, and thus reporting of false positive results. The unbound form of SYBR green exhibits very
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Molecular Biology: Real-time PCR
little fluorescence but emits a strong fluorescent signal once bound to dsDNA (Morrison, 1998). During
amplification, the fluorescent signal increases as the PCR product accumulates with each successive
cycle
of
amplification
(Animation
1:
http://www.sigmaaldrich.com/life-science/molecularbiology/pcr/learning-center/sybr-green-animation.html). Subsequently, 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 using the 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 Taq DNA polymerase which is responsible for the hydrolysis of the probe. FAQ7During
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Molecular Biology: Real-time PCR
annealing, primers and probes hybridize onto the target sequence. At extension, the dsDNAspecific 5’-exonuclease activity of Taq DNA polymerase cleaves the bound probe as the Taq DNA
polymerase extends the 3’-end of the primer during 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:
http://www.sigmaaldrich.com/life-science/molecular-biology/pcr/learning-center/probed-based-qpcranimation.html). The fluorescent signal that is released due to this process allows monitoring 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, thus displacing 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 reporter signal.
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 chemistry uses two short oligonucleotide probes labeled with different
florescent dyes to measure the transfer of energy between the two fluorophores attached to the
probes (Simon et al, 2004). The two probes are designed 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 nonfluorescent quencher (or acceptor) dye on the 5’-end. When the two fluorophores are in close
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
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Molecular Biology: Real-time PCR
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 prevent
extension 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 dye by FRET
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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