The advent of Polymerase Chain Reaction (PCR) by Kary B. Mullis in the mid-1980s 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. (www.ambion.com 2003)
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 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 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. (www.probes.com 2003)
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. (www.probes.com 2003)
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.
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.
Figure 4. A graph printiout of actual data found using the TaqMan® probe. Courtesy www.biotech.uiuc.edu.
Figure 5. A real-time PCR machine used at Colorado State. Courtesy lamar.colostate.edu.
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 www.probes.com
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 www.ruhr-uni-bochum.de pending.
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 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)
Campbell, Malcolm. PCR movie. <http://www.bio.davidson.edu/misc/movies/pcr2.mov> Accessed 2/17/03.
Campbell, Malcolm. Real-time PCR, molecular beacon method.. <http://www.bio.davidson.edu/courses/genomics/method/realtimepcr.html> Accessed 2/17/03.
Colorado State Laboratory homepage. <http://lamar.colostate.edu/~reddy/labtour2.htm> Accessed 2/17/03.
Fluorescence Resonance Energy Transfer (FRET). <http://www.probes.com/handbook/boxes/0422.html> Accessed 2/18/03.
History of PCR. <http://usitweb.shef.ac.uk/~mba97cmh/history/history.htm> Accessed 2/15/03.
Purves, et al. Life: The Science of Biology. Sixth Edition. Freeman and Company: USA; 2001. pg. 214-217.
"Real-Time" or Kinetic PCR. <http://dna-9.int-med.uiowa.edu/realtime.htm> Accessed 2/17/03.
Real-time RT-PCR. <http://www.ambion.com/basics/rtpcr/rtpcr101_page5.html> Accessed 2/17/03.
W.M. Keck Center for Comparitive and Functional Genomics. <http://www.biotech.uiuc.edu/taqman.htm> Accessed 2/17/03.
Gene Quantification. <http://www.wzw.tum.de/gene-quantification/relative.html> Accessed 2/16/03
مراجعین محترم :
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