Principle
The principle of SYBR real-time PCR is a standard PCR reaction carried out in the presence of a dye, SYBR, which fluorescence when intercalated in the DNA helix.
The fluorescence will increase as the amount of the PCR product increases and is quantified after each completed PCR cycle.
The cycle at which the fluorescence exceeds a detection threshold, the Ct (threshold cycle) correlates to the number of target cDNA molecules present in the added cDNA. Therefore, by comparison to a calibration curve, it is possible to quantify in absolute amounts the number of target molecules in added cDNA samples.
Cyclers
There are two very different principles for commercial thermocyclers: block- and air based.
The block based cyclers works by heating and cooling a metal block in which the samples are placed. The heat is usually moved by Peltier elements which are semiconductors which move heat when a current is applied to the cells. If the polarity of the current is reversed, the direction of the heat transport is also reversed.
Heating blocks are relatively slow and changes typically the temperature 1 to a few degree celcius per second. They are usually 96- or even 384-well format.
Heating blocks are difficult to heat uniformly across the plate. The thermogram below shows an example of differences in temperature, especially in the centre of the plate compared with the periphery. It also shows that a hotspot has formed which may happen as the Peltier elements start to fail. This has been investigated in detail by Corbett Life Science. Read more about plate inhomogeneities here.
In air cyclers the temperature changes by means of heated air. The samples are no longer in 96-well plates, but in capillaries which conduct the heat very rapidly. Air cyclers can typically accommodate less samples than plate cyclers, but the time to complete a run is much shorter, due to the rapid heat conduction in the system. The temperature gradient is typically 20 degrees C per second.
1) Capillary
2) Heat coil and fan
3) Optical system reading sample fluorescence
(www.montreal-biotech.com)
