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.
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)
