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)
TABLE OF CONTENTS
INTRODUCTION........................................................................................................................................... 2
OVERVIEW OF REAL-TIME PCR ............................................................................................................... 2
REAL-TIME PCR vs TRADITIONAL PCR ................................................................................................... 3
REAL-TIME PCR DETECTION FORMATS (OR CHEMISTRIES) ............................................................... 5
Non-specific detection methods ............................................................................................................... 5
Specific detection methods ...................................................................................................................... 6
ANALYSIS .................................................................................................................................................... 8
Qualitative detection: ............................................................................................................................... 8
Quantitative analysis: ............................................................................................................................. 11
ADVANTAGES OF REAL-TIME PCR ........................................................................................................ 14
LIMITATIONS OF REAL-TIME PCR .......................................................................................................... 14
APPLICATIONS OF REAL-TIME PCR ...................................................................................................... 14
FAQ ............................................................................................................................................................. 15
REFERENCES ............................................................................................................................................ 18
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Molecular Biology: Real-time PCR
INTRODUCTION
The polymerase chain reaction (PCR) has become an essential tool for molecular biologists and has
revolutionized the quantitative analysis of DNA and RNA. Consequently, PCR has been accepted as the
gold standard for detecting nucleic acids from a number of origins and it has become an essential tool in
diagnostic and research laboratories. However, existing combinations of PCR and traditional detection
methods, although meant to improve the sensitivity and specificity of PCR-based tests, render these
methods laborious, technically demanding and time-consuming. Furthermore, post-PCR handling steps
increase the risk of the spread of amplicon to the laboratory environment. In contrast to traditional assays,
Higuchi et al. (1992; 1993) pioneered the detection of amplicon which could be visualized as the
amplification progressed and this approach formed the foundation of ‘real-time’ PCR. The development of
real-time PCR technology has improved on the limitations suffered by traditional PCR-based assays
including rapidity, sensitivity, reproducibility and the reduced risk of carry-over contamination (Mackay et
al., 2002). Real-time PCR is currently the most sensitive method to determine the amount of specific DNA
sequences in complex biological samples. Since its introduction, real-time quantitative PCR has
revolutionized the field of molecular diagnostics and the technique is being used in a rapidly expanding
number of applications.
OVERVIEW OF REAL-TIME PCR
FAQ1
Real-time PCR technology allows monitoring of the progress of a PCR reaction in ‘real-time’. This
technology uses a fluorescent signal to monitor the accumulation of PCR products in a PCR reaction. The
fluorescent signal that is detected by the real-time PCR system is produced by a fluorescent dye or
fluorescent probe and it increases as the amplification reaction progresses. Real-time PCR instruments
combine the ability to excite, detect, and record fluorescence using a regular thermocycler making realtime PCR an automated process. Therefore, unlike traditional PCR, real-time PCR technology allows the
detection and analysis of the product of interest in a single closed tube system. The fluorescent dyes or
fluorescent probes used in the PCR mix require an energy source for excitation which is incorporated in
real-time instruments. The fluorophore is excited using this integral light source. Fluorescence is then
measured using a photodetector, such as a camera or photomultiplier tubes. Multiple wavelengths can be
measured at once, which confers the ability to detect multiple targets in a single reaction tube, allowing
multiplex analysis. The instrument measures/records the fluorescence emitted in ‘real-time’. Specific
software is used to collect and elaborate the data in graphic and numeric form for analysis at the
completion of the amplification process.
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Molecular Biology: Real-time PCR
REAL-TIME PCR VS TRADITIONAL PCR
Similar to traditional PCR, a real-time PCR reaction consists of three main steps involved in the
amplification process; these are denaturing, annealing and extension. Traditional PCR methods use
agarose gels for detection of PCR amplification and the detection is performed at the final phase or endpoint of the PCR reaction (Fig. 1). FAQ2In contrast, real-time PCR monitors the progress of a PCR
reaction in real-time and allows detection of the PCR product at the exponential phase of the amplification
process (Fig. 1). Measuring the kinetics of the reaction in the early phases of PCR provides a distinct
advantage
over
traditional
PCR
detection
(http://www.appliedbiosystems.com/absite/us/en/home/applications-technologies/real-time-pcr/real-timepcr-vs-traditional-pcr.html?ICID=EDI-Lrn2).
Fig. 1: Amplification curves produced from replicate samples which have the same starting quantity.
As the PCR reaction progresses, amplification of the target DNA occurs exponentially, that is the
doubling of amplification product (also known as an amplicon) occurs every cycle. This process
occurs at the exponential phase and is promoted by the abundant presence of all the reagents.
At the exponential phase of amplification, reaction efficiency is assumed to be 100%, allowing accurate
quantification of the starting nucleic acid in the PCR reaction. During this phase PCR products double at
every cycle; the reaction is precise since all reagents are still available. Quantification cannot be achieved
with end-point detection because results are collected at the completion of the reaction (at the plateau
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Molecular Biology: Real-time PCR
phase) where amplification has ceased as a result of limitation or exhaustion of reagents. See Table 1
below for a summary of differences between real-time PCR and traditional PCR.
Traditional PCR
Real-time PCR
Non-automated system
Automated fluorometric detection system
End-point detection of PCR product
Real-time PCR product detection
Measures an aliquot of accumulated product
Measures total accumulated product
-
Low sensitivity
Requires post-PCR analysis for product
-
Post-PCR detection analysis is not necessary
detection
–
gel electrophoresis
–
probe hybridization
Prone to contamination
–
–
short turn-around time
–
less contamination
Minimal contamination
–
post-PCR handling
Not suitable for quantitative analysis
High sensitivity
single tube system
Suitable for both qualitative and quantitative
analysis
Not safe
Safe, does not require ethidium bromide or
radioactivity
-
Requires use of carcinogenic
agents, such as ethidium bromide
and radioactivity
Table 1: A summary of differences between real-time PCR and traditional PCR
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Molecular Biology: Real-time 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
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.
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Molecular Biology: Real-time PCR
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
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-qpcr6|Page
Molecular Biology: Real-time PCR
animation.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
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
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Molecular Biology: Real-time PCR
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
ANALYSIS
Real-time PCR amplifies and simultaneously quantifies a targeted DNA or RNA molecule making it
suitable for both qualitative and quantification analysis.
Qualitative detection:
FAQ11Qualitative
detection determines the presence or the absence of target nucleic acid in a biological
material. During amplification, the real-time PCR system monitors the accumulation of the PCR product
using fluorescence. The fluorescent signal increases proportionately to the accumulated PCR product.
When the fluorescent signal reaches detectable levels it is captured by the system and displayed as an
amplification curve. Theoretically, the amplicon concentration is expected to increase exponentially during
the initial phase of the amplification process (Swillens et al., 2008). At the final phase, the amplification
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Molecular Biology: Real-time PCR
curve deviates and bends toward a plateau, as a result of decreasing DNA polymerase activity or
depletion of essential reaction components e.g. primers or fluorescent probe. Consequently, the
amplification curve is generated as a sigmoidal shape plotted from fluorescence data vs cycle (Fig. 2).
Fig. 2: Typical amplification plot. The amplification plot or curve is generated as a sigmoidal shape plotted
from fluorescence data against the cycle number. An amplification curve is only produced when target
nucleic acid has been detected in a sample (blue curve); in the absence of fluorescence, as is the case where
amplification did not occur, a straight line is generated (green line).
An amplification curve is only produced when target nucleic acid has been detected in a sample; in the
absence of fluorescence, as is the case where amplification did not occur, a straight line is generated. An
amplification curve may be observed when there has been contaminating material amplified, resulting in a
false positive result. However,
FAQ12when
using SYBR green and hybridization probes, a melt curve
analysis can be performed on the amplification product to confirm if the product is the desired target
product (Fig. 3).
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Fig. 3: The melting curve is produced when the fluorescence is plotted against temperature and then the
change in fluorescence/change in temperature (-ΔF/ΔT) is plotted against temperature to obtain a clear view
of the melting dynamics as shown by the melting peaks on the figure. Based on the Tm of each amplicon
produced (as determined by the amplicon sequence composition), the melting curve analysis allows
differentiation between specific and non-specific PCR products.
FAQ13Melt
curve analysis measures the dissociation characteristics of double-stranded DNA during
heating. In a SYBR green reaction, the fluorescent signal decreases as a result of the separation of DNA
double strands, ultimately releasing the SYBR green molecules. In a hybridization probes reaction, raising
the temperature causes the probes to melt off the target product resulting in the separation of the donor
and acceptor dye molecules; consequently FRET is reduced and the fluorescence is decreased
(www.roche-applied-science.com/lightcycler/). The temperature at which half the FRET signal is lost is
referred to as the melting temperature (Tm) of the probe. This temperature varies depending on the DNA
sequence, length and GC content. The Tm changes with even a single nucleotide difference. In addition to
differentiating between specific and non-specific products, this characteristic of the melt curve analysis
allows detection of single-nucleotide polymorphisms (SNP), distinction of homozygous and heterozygous
gene alleles by the dissociation patterns produced and discrimination between species of the same
genus.
During melt curve analysis, the fluorescence is measured continuously as the temperature is increased.
The real-time PCR system generates a ‘melting-curve’ by plotting the decreasing fluorescence data
against temperature. The real-time PCR detection systems calculate the first derivatives of the curves,
resulting
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in
curves
with
peaks
at
the
respective
T ms
Molecular Biology: Real-time PCR
(http://www.qiagen.com/resources/info/guidelines_rtpcr/dataanalysis_sybr.aspx) (Fig. 3). Therefore, PCR
products of different sequences have different T ms and products of identical sequences are expected to
have the same Tm. A single nucleotide polymorphism in the target DNA under a hybridization FRET probe
will still generate a signal and the melting curve will display a lower Tm. Therefore, a different Tm may be
an indication of sequence differences under the probes region. When more than three base pair
differences occur under a FRET hybridization probe region, hybridization at typical annealing
temperatures may be prevented and the products are not detected. Primer dimers generate curves with
peaks at a Tm lower than that of the specific PCR product, while non-specific products and smears
produce diverse peaks with different Tms.
Quantitative analysis:
Real-time PCR systems use a fluorescent signal measured during the exponential phase of the
amplification process for quantitative data analysis. The amplification curve generated contains valuable
information that allows the user to determine the concentration, or relative concentration of target DNA or
RNA in unknown samples. To analyze quantitative data, the instrument uses two important parameters:
1.
FAQ14The
Cycle threshold of the sample
Cycle threshold (Ct) is defined as ‘the cycle at which the fluorescence of a sample rises above the
background fluorescence’ (www.roche-applied-science.com/lightcycler); it is the intersection between an
amplification curve and a threshold line (Fig. 4). At this point, a detectable amount of amplicon has been
generated during the early exponential phase of the reaction.
2. The Threshold line
The threshold line is the level of detection at which a reaction reaches a fluorescent signal above
background. The threshold line is set in the exponential phase of the amplification to allow accurate
analysis (Fig. 4) (www.appliedbiosystems.com). Its intersection with the amplification curve determines
the Ct, thus Ct = the number of cycles required to reach the threshold.
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Fig. 4: The graphic representation of PCR data showing several parameters of the real-time reaction
amplification plot (including the threshold cycle, threshold line and baseline). The threshold cycle (Ct) is
the intersection between an amplification curve and a threshold line. It is a relative measure of the
concentration of target in the PCR reaction. The threshold must be set in the linear phase of the
amplification plot. The Ct value increases with a decreasing amount of template.
FAQ15C
t
values in real-time PCR correlate closely with the original quantity of target sequences and are
influenced by the concentration of the target. The Ct value increases with a decreasing amount of target
and vice versa. For absolute quantification, external standards of known concentration are used to
generate a standard curve from which the concentration of an unknown target can be extrapolated
(http://www.qiagen.com/resources/info/guidelines_rtpcr/dataanalysis_sybr.aspx) (Fig. 5). The Cts of the
standards are plotted against the log of the template amount, resulting in a straight line. The Ct values
and the standard curve are then used to calculate the amount of starting template in an unknown sample
(Vaerman et al., 2004; Rutledge and Côté, 2003; He et al., 2002).
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Fig. 5: The amplification plot and standard curve for absolute quantification. External standards of known
concentration are used to generate a standard curve from which the concentration of an unknown target
can be extrapolated. The Cts of the standards are plotted against the log of the template amount, resulting
in a straight line.
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ADVANTAGES OF REAL-TIME PCR

High sensitivity, allowing detection of low copy numbers of pathogen material.

Post-PCR analysis is not necessary.

Closed-tube system significantly reduces the risk of contamination.

Offers low turn-around time which is essential in diagnostics.

The automated technology allows high-throughput.

Allows detection of more than one pathogen through multiplexing.

Collects data in the exponential phase (not at the plateau as in traditional PCR using agarose gel)
allowing more accurate quantitative analysis.

It is safer; does not require use of ethidium bromide or radioactivity.
LIMITATIONS OF REAL-TIME PCR

Setting up requires high technical skill and support.

High equipment cost especially in low-throughput laboratories.

Overlap of emission spectra.

Inability to monitor amplicon size without opening the system.

The incompatibility of some platforms with some fluorogenic chemistries

The relatively restricted multiplex capabilities of current applications.

Risk of false positive results particularly when using DNA binding dyes like SYBR green.
APPLICATIONS OF REAL-TIME PCR
Real-time PCR has been used in various applications, in a number of fields, including:

Molecular diagnostics
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
Single Nucleotide Polymorphisms (SNP) genotyping

Allelic discrimination

Determination of pathogen load

Detection of genetically modified organisms

Quantitation of gene expression

Array verification

Quality control and assay validation

Biosafety and genetic stability testing

Drug therapy efficacy / drug monitoring

DNA damage (microsatellite instability) measurement

Forensics
FAQ
1.
What is real-time PCR?
Real-time PCR technology allows fast real-time monitoring of a PCR reaction. This technology uses
fluorescent detection systems to detect accumulation of amplicon during the amplification reaction.
2.
How is real-time PCR different from traditional PCR?
Traditional PCR methods use agarose gels for detection of PCR amplification and the detection is
performed at the final phase or end-point of the PCR reaction (Fig. 1). FAQ2In contrast, real-time
PCR monitors the progress of a PCR reaction in real-time and allows detection of the PCR product
at the exponential phase of the amplification process
3.
What detection methods do the real-time PCR technology uses?
There 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:
a.
Non-specific detection
b.
Specific detection
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4.
How do SYBR green molecules bind to DNA?
This fluorogenic groove-binding dye, binds non-specifically to dsDNA; it does not bind to singlestranded DNA (ssDNA).
5.
Can SYBR green assays be used for tests that require high specificity?
No, because SYBR green molecules bind non-specifically to any dsDNA, they will also bind to nonspecific products if present in a reaction.
6.
How do fluorogenic–labeled probes improve the specificity of real-time PCR assays?
These probes are designed to bind to a specific sequence on the target DNA, thus increasing the
specificity of the PCR. Thus, post PCR processing is not necessary because the fluorogenic probes
only allow detection of a specific amplification product, consequently eliminating detection of nonspecific PCR products.
7.
How does the hydrolysis probe chemistry works?
During 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.
Consequently, the reporter dye can emit fluorescence because it is separated from the quencher
dye. 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.
8.
What are hybridization probes?
Hybridization probes are two short oligonucleotide probes labeled with different florescent dyes to
measure the transfer of energy between the two fluorophores attached to the probes. 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 non-fluorescent quencher (or acceptor) dye on
the 5’-end.
9.
What is FRET?
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). The 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. 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.
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10. How does the hybridization probe chemistry works?
During PCR, the two probes anneal to the target sequence, adjacent to each other. The donor
fluorophore is excited by the light source from the real-time PCR system. The activation energy of
fluorescein (from the donor probe) is directly transferred to the acceptor dye by FRET. The acceptor
fluorophore emits light at a different wavelength. Subsequently the fluorescent signal can be
detected and measured. This happens during the annealing phase and first part of the extension
phase of the PCR process. After each subsequent PCR cycle more hybridization probes can anneal,
resulting in higher fluorescent signals. The fluorescence emitted is proportional to the accumulated
PCR product
11. How do real-time PCR systems determine the presence or absence of target nucleic acid?
Qualitative detection determines the presence or the absence of target nucleic acid in a biological
material. During amplification, the real-time PCR system monitors the accumulation of the PCR
product using fluorescence. The fluorescent signal increases proportionately to the accumulated
PCR product. When the fluorescent signal reaches detectable levels it is captured by the system and
displayed as an amplification curve. Theoretically, the amplicon concentration is expected to
increase exponentially during the initial phase of the amplification process.
12. How can real-time PCR be used to differentiate between a specific and a non-specific
product?
When using SYBR green and hybridization probes, a melt curve analysis can be performed on the
amplification product to confirm if the product is the desired target product
13. What is melt curve analysis?
Melt curve analysis measures the dissociation characteristics of double-stranded DNA during
heating. In a SYBR green reaction, the fluorescent signal decreases as a result of the separation of
DNA double strands, ultimately releasing the SYBR green molecules. In a hybridization probes
reaction, raising the temperature causes the probes to melt off the target product resulting in the
separation of the donor and acceptor dye molecules; consequently FRET is reduced and the
fluorescence is decreased. The temperature at which half the FRET signal is lost is referred to as the
melting temperature (Tm) of the probe. This temperature varies depending on the DNA sequence,
length and GC content. The Tm changes with even a single nucleotide difference. In addition to
differentiating between specific and non-specific products, this characteristic of the melt curve
analysis allows detection of single-nucleotide polymorphisms (SNP), distinction of homozygous and
heterozygous gene alleles by the dissociation patterns produced and discrimination between species
of the same genus.
14. What is cycle threshold (Ct)?
Cycle threshold (Ct) is defined as ‘the cycle at which the fluorescence of a sample rises above the
background fluorescence’; it is the intersection between an amplification curve and a threshold line.
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At this point, a detectable amount of amplicon has been generated during the early exponential
phase of the reaction.
15. How is Ct used in absolute quantification?
Ct values in real-time PCR correlate closely with the original quantity of target sequences and are
influenced by the concentration of the target. The Ct value increases with a decreasing amount of
target and vice versa. For absolute quantification, external standards of known concentration are
used to generate a standard curve from which the concentration of an unknown target can be
extrapolated. The Cts of the standards are plotted against the log of the template amount, resulting
in a straight line. The Ct values and the standard curve are then used to calculate the amount of
starting template in an unknown sample.
REFERENCES
1.
Cardullo, R.A., Agrawal, S., Flores, C., Zamecnik, P.C., Wolf, D.E., 1988. Detection of nucleic acid
hybridization by nonradiative fluorescence resonance energy transfer. Proc. Natl. Acad. Sci. 85
(23):8790–8794.
2.
Gibson, U.E., Heid, C.A., Williams, P.M., 1996. A novel method for real time quantitative RT‐PCR.
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