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