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REAL-TIME PCR
Introduction
Over the last 15 years, PCR has become an essential part of most
laboratories involved in biomedical research. PCR amplification turns a few
attograms (
) of the nucleic acid, far too little to be analyzed directly or
used in biochemical reactions, into as much as a microgram of DNA. This is
more than enough for detection, sequencing, or cloning.
In the face of a widely held attitude that “quantitative PCR is an
oxymoron - a contradiction in terms.” years 1991 to 1998 have seen a 10-fold
increase in the number of papers using quantitative PCR methods. The joke is
not without some truth. By nature, an exponential amplification is not ideally
suited to quantification. Small differences in amplification efficiency between
samples can become huge differences in results when they are amplified
through forty doublings. Anyone working with quantitative PCR who forgets
this fact is in danger of making mistakes that are measured in orders of
magnitude.
Why then the continuing increase in the use of quantitative PCR? It
has a sensitivity five orders of magnitude better than the best blotting
procedures and a dynamic range of 10 orders of magnitude. This
unsurpassable sensitivity and range has made the work of turning PCR into a
quantitative tool worthwhile.
Real-time PR is the very best way to do quantitative PCR
A reaction profile can be thought of has having three segments: an
early background phase, an exponential growth phase (or log phase)
and a plateau. The background phase lasts until the fluorescence signal
from the PCR product is greater than the background fluorescence of the
probe system. The exponential growth phase begins when sufficient product
has accumulated to be detected above background, and ends when the
reaction efficiency falls as the reaction enters the plateau.
In real-time PCR fluorescence values are recorded during every
cycle and represent the amount of product amplified to that point in the
amplification reaction. Moreover, these values are recorded separately for
each of the reaction steps: denaturation, annealing and extension. The more
templates present at the beginning of the reaction, the fewer number of
cycles it takes to reach a point in which the fluorescent signal is first
recorded as statistically significant above background , which is the
definition of the (Ct) values. This will increase the throughput, because it is
no longer necessary to analyze dilutions of each sample in order to
obtain accurate results as it is the case with competitive PCR. A number
of options are available for implementing real-time PCR in quantitative
analysis. Homogeneous detection of PCR products can be done using
double- stranded DNA binding dyes; fluorogenic probes, direct labeled
primers and a primer-probe combination termed scorpion.
Advantages
Real time PCR offers numerous advantages over previous attempts at
quantitating PCR. Other methods typically rely on end-point measurements,
when often the reaction has gone beyond the exponential phase because of
limiting reagents. To compensate for such problems, competitive PCR was
devised, which allows for normalization of the end product based on the ratio
between target and competitor. Because this method is cumbersome,
requiring a carefully constructed competitor target for each PCR reaction and
a series of dilutions to ensure that there is a suitable ratio of target to
competitor, it is seldom used successfully. In contrast, with real time PCR, the
dynamic range is much greater than that of competitive PCR (over six orders
of magnitude as compared to one with competitive PCR), post-reaction
processing is eliminated, and the measurements are taken from the
exponential range of the reaction, where component concentrations are not
limiting. And best of all, the entire process is automated.
The theory is straightforward, but a number of technical caveats are
associated with the use of conventional end-point methodologies for
quantitative PCR. In these techniques, PCR results are monitored after a
given number of cycles, by which point factors such as limiting reagent
concentrations and side reactions may have played a significant role in
affecting final product concentration. Quantitative competitive PCR was
developed in response to some of these difficulties. In this approach, the
starting amount of target is calculated based on the ratio of target to
competitor after amplification. However, quantitative competitive PCR is
cumbersome, and it can be associated with a number of drawbacks including
a limited dynamic range and the need to screen multiple dilutions.
The first on the market, the ABI 7700 Sequence Detection System
includes a built-in thermal cycler, a fluorogenic 5' nuclease assay, a laser for
inducing fluorescence, charge-coupled device (CCD) detection, and PCR
application software. The specially designed reaction tube with transparent lid
allows light from a laser, carried on fiber optic cables, to excite the probe and
Applied Biosystems has commercialized the requisite reagents in its
well-known TaqMan® product line still the most widely used real-time reagent.
The fluorogenic probe is complementary to the target sequence, and initially
contains both reporter and quencher moieties. When the probe is not bound to
template DNA, its reporter and quencher dyes are in close proximity, and the
reporter's fluorescent emission is quenched.
Diagram of quantitative RT-PCR (TaqMan) technology
A , in the PCR reaction, an oligonucleotide probe tagged with a 5’
fluorescent reporter and a 3’ quencher is added in addition to the standard
PCR components. The probe is complementary to the target sequence of
interest and anneals during extension. The close proximity of the quencher to
the fluorescent reporter represses fluorescence in the intact probe. As the Taq
polymerase synthesizes the new strand, its 5’ to 3’ nuclease activity cleaves
the probe, separating the quencher and fluorescent reporter. The
fluorescence emitted is proportional to the amount of product accumulated
with each cycle. B , the plot represents a sample analysis from the
experiments shown in the Figure. The plot shows the expression values
obtained for three genes (as indicated) in a matched tumor and normal tissue
sample. The horizontal bold line indicates the fluorescence level used for the
threshold cycle (C t ) determination in this particular example. D Rn is defined
as the cycle-to-cycle change in the reporter fluorescence signal normalized to
a passive reference fluorescence signal (background).
DNA Quantification
Using either the DNA binding dye SYBR Green I or Hybridization
Probes, the progress of amplification is monitored at each cycle. In many
cases, it only takes a few minutes to determine whether or not a sample
contains a certain template molecule.
In the following, the principles of PCR data quantification are explained
using schematic drawings.
In this model experiment, 10
6 , 10
5 and 10
4 template molecules were
amplified. The Y-axis shows the fluorescence whereas the X-axis shows the
number of cycles. As expected, the less template molecules are contained in
a sample the more PCR cycles are required before the amplification signal
Molecular beacons
The ‘‘molecular beacons’’ represent a second approach. These
oligonucleotide probes are chemically modified with a fluorescence donor
(EDANS) at their 5’ end and a non-fluorescent quenching acceptor (DABCYL)
at their 3’ end. In the absence of a perfectly matched target, they assume a
stem-and-loop structure in solution: the loop is a DNA strand complementary
to the target, and the stem is formed by intramolecular base pairing of short
complementary sequences at each end of the loop. This hairpin conformation
positions D and Q in extremely close proximity, much closer than a pair of
fluorophores at opposite ends of a randomly coiled oligonucleotide; this
effectively quenches donor fluorescence. In the presence of a perfectly
matched sequence, the oligonucleotide undergoes a conformational change
that allows the hairpin loop to hybridize to the target, separating D from Q and
resulting in a fluorescence increase (up to 900-fold). The ability of molecular
beacons to form hairpin structures significantly enhances their specificity
compared with standard oligonucleotide probes of the same size, allowing
them to readily distinguish between a perfect match and a single base
mismatch. Recently, molecular beacons with seven additional donors ranging
from blue to red fluorescence emission have been developed, permitting the
simultaneous detection of several targets within the same tube.
Molecular beacon
Molecular Scorpions
Scorpions are bi-functional molecules containing a PCR primer element
covalently linked to a probe element. The molecules also contain a
fluorophore that can interact with a quencher to reduce fluorescence. When
the molecules are used in a PCR reaction the fluorophore and the quencher
are separated which leads to an increase in light output from the reaction
tube.
The benefits of Scorpions derive from the fact that the probe element is
physically coupled to the primer element - this means that the reaction leading
to signal generation is a unimolecular rearrangement. This contrasts to the bi-
molecular collisions required by other technologies such as TaqMan or
Molecular Beacons.
The benefits of a unimolecular rearrangement are significant - as the
reaction is effectively instantaneous it occurs prior to any competing or side
reactions such as target amplicon reannealing or inappropriate target folding.
This leads to stronger signals, more reliable probe design, shorter reaction
times and better discrimination.
The presence of the blocker group is an essential element of the
Scorpions invention. Without such a blocker the Taq DNA polymerase would
be able to read through the Scorpions primer and copy the probe region. This
would generate signal but not in a target specific fashion. Copying the tail in
this way would completely negate the benefits of the Scorpions reaction as
any inappropriate side-reactions, including the formation of primer dimers,
would also generate a signal.
Allelic Discrimination Using the 5´-Nuclease Assay (Mutation detection)
In allelic discrimination assays, the PCR assay includes a specific, fluorescent, dye- labeled probe for each allele. The probes contain different fluorescent reporter dyes (FAM and VIC™) to differentiate the amplification of each allele.
