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Real-Time PCR:
the TaqMan Method
®
Introduction:
The advent of Polymerase Chain Reaction (PCR) by Kary B. Mullis in the mid1980s revolutionized molecular biology as we know it. PCR is a fairly standard
procedure now, and its use is extremely wide-ranging. At its most basic
application, PCR can amplify a small amount of template DNA (or RNA) into
large quantities in a few hours. This is performed by mixing the DNA with
primers on either side of the DNA (forward and reverse), Taq polymerase (of
the species Thermus aquaticus, a thermophile whose polymerase is able to
withstand extremely high temperatures), free nucleotides (dNTPs for DNA,
NTPs for RNA), and buffer. This movie shows PCR in action. The temperature
is then alternated between hot and cold to denature and reanneal the DNA, with
the polymerase adding new complementary strands each time. In addition to the
basic use of PCR, specially designed primers can be made to ligate two
different pieces of DNA together or add a restriction site, in addition to many
other creative uses. Clearly, PCR is a procedure that is an integral addition to
the molecular biologist’s toolbox, and the method has been continually
improved upon over the years. (Purves, et al. 2001)
Fairly recently, a new method of PCR quantification has been invented. This is
called “real-time PCR” because it allows the scientist to actually view the
increase in the amount of DNA as it is amplified. Several different types of
real-time PCR are being marketed to the scientific community at this time, each
with their advantages. This web site will explore one of these types, TaqMan®
real-time PCR, as well as give an overview of the other two types of real-time
PCR, molecular beacon and SYBR® Green.
How TaqMan® works:
TaqMan® utilizes a system that is fairly easy to grasp conceptually. First, we
must take a look at the TaqMan® probe.
Figure 1. The Taqman probe. The red circle represents the quenching dye that disrupts
the observable signal from the reporter dye (green circle) when it is within a short
distance. Image created by Dan Pierce.
The probe consists of two types of fluorophores, which are the fluorescent parts
of reporter proteins (Green Fluorescent Protein (GFP) has an often-used
fluorophore). While the probe is attached or unattached to the template DNA
and before the polymerase acts, the quencher (Q) fluorophore (usually a longwavelength colored dye, such as red) reduces the fluorescence from the
reporter (R) fluorophore (usually a short-wavelength colored dye, such as
green). It does this by the use of Fluorescence (or Förster) Resonance Energy
Transfer (FRET), which is the inhibition of one dye caused by another without
emission of a proton. The reporter dye is found on the 5’ end of the probe and
the quencher at the 3’ end.
Figure 2. The TaqMan® probe binds to the target DNA, and the primer binds as well.
Because the primer is bound, Taq polymerase can now create a complementary strand.
Image created by Dan Pierce.
Once the TaqMan® probe has bound to its specific piece of the template DNA
after denaturation (high temperature) and the reaction cools, the primers anneal
to the DNA. Taq polymerase then adds nucleotides and removes the Taqman®
probe from the template DNA. This separates the quencher from the reporter,
and allows the reporter to give off its emit its energy. This is then quantified
using a computer. The more times the denaturing and annealing takes place, the
more opportunities there are for the Taqman® probe to bind and, in turn, the
more emitted light is detected.
Figure 3. The reporter dye is released from the extending double-stranded DNA created
by the Taq polymerase. Away from the quenching dye, the light emitted from the reporter
dye in an excited state can now be observed. Image created by Dan Pierce.
Quantification:
The specifics in quantification of the light emitted during real-time PCR are
fairly involved and complex. The math involved is above the scope of this
website, but this website explains some specifics not talked about here:
The light emitted from the dye in the excited state is received by a computer
and shown on a graph display, such as this, showing PCR cycles on the X-axis
and a logarithmic indication of intensity on the Y-axis.
Figure 4. A graph printiout of actual data found using the TaqMan® probe. Courtesy
Figure 5. A real-time PCR machine used at Colorado State. Courtesy
Other Images of TaqMan® in Action:
Figure 6. Another three step view of the TaqMan® probe working: before the probe is
met with the Taq polymerase, energy is transferred from a short-wavelength fluorophore
(green) to a long-wavelength fluorophore (red). When the polymerase adds nucleotides to
the template strand, it releases the short-wavelength fluorophore, making it detectable
and the long-wavelength undetectable. Figure courtesy
Figure 7. Another view of TaqMan® in action. The release from the Quencher dye (red
Q) in step 2 eventually causes the Reporter dye (blue R) to be seen in step 4. Figure
courtesty
Other Real-Time PCR Methods:
There are two other types of real-time PCR methods, the molecular beacon
method and the SYBR® Green method. The molecular beacon method utilizes
a reporter probe that is wrapped around into a hairpin. It also has a quencher
dye that must be in close contact to the reporter to work. An important
difference of the molecular beacon method in comparison to the TaqMan®
method is that the probe remains intact throughout the PCR product, and is
rebound to the target at every cycle. Click here to see a web page on the
molecular beacon method of PCR, another type of real-time PCR used in
molecular biology. The SYBR® Green probe was the first to be used in realtime PCR. It binds to double-stranded DNA and emits light when excited.
Unfortunately, it binds to any double-stranded DNA which could result in
inaccurate data, especially compared with the specificity found in the other two
methods.
Applications of real-time polymerase chain reaction
There are numerous applications for real-time polymerase chain reaction in
the laboratory. It is commonly used for both diagnostic and research
applications.
Diagnostically real-time PCR is applied to rapidly detect the presence of
genes involved in infectious diseases, cancer and genetic abnormalities. In
the research setting, real-time PCR is mainly used to provide highly sensitive
quantitative measurements of gene transcription.
The technology may be used in determining how the genetic expression of a
particular gene changes over time, such as in the response of tissue and cell
cultures to an administration of a pharmacological agent, progression of cell
differentiation, or in response to changes in environmental conditions.
Also, the technique is used in Environmental Microbiology, for example to
quantify resistance genes in water samples.