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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. (www.ambion.com 2003)
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 long-wavelength 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 Frster) 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. (www.probes.com 2003)
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: www.wzw.tum.de.
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.
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 real-time 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. (www.ambion.com 2003)
References:
1. Campbell, Malcolm. PCR movie. <http://www.bio.davidson.edu/misc/movies/pcr2.mov>
Accessed 2/17/03.
2. Campbell, Malcolm. Real-time PCR, molecular beacon method..
<http://www.bio.davidson.edu/courses/genomics/method/realtimepcr.html> Accessed 2/17/03.
3. Colorado State Laboratory homepage. <http://lamar.colostate.edu/~reddy/labtour2.htm> Accessed
2/17/03.
4. Fluorescence Resonance Energy Transfer (FRET).
<http://www.probes.com/handbook/boxes/0422.html> Accessed 2/18/03.
5. History of PCR. <http://usitweb.shef.ac.uk/~mba97cmh/history/history.htm> Accessed 2/15/03.
6. Purves, et al. Life: The Science of Biology. Sixth Edition. Freeman and Company: USA; 2001.
pg. 214-217.
7. "Real-Time" or Kinetic PCR. <http://dna-9.int-med.uiowa.edu/realtime.htm> Accessed 2/17/03.
8. Real-time RT-PCR. <http://www.ambion.com/basics/rtpcr/rtpcr101_page5.html> Accessed
2/17/03.
9. W.M. Keck Center for Comparitive and Functional Genomics.
<http://www.biotech.uiuc.edu/taqman.htm> Accessed 2/17/03.
10. Gene Quantification. <http://www.wzw.tum.de/gene-quantification/relative.html> Accessed
2/16/03.