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)
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
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).
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Molecular Biology: Real-time PCR
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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Molecular Biology: Real-time PCR
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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Molecular Biology: Real-time PCR
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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Molecular Biology: Real-time PCR
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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