How it Works: Real Time PCR

Brendan Maher

The instrumentation is basic: a thermal cycler for amplification, a light source for excitation of fluorescent probes (see chemistries below), a camera for recording, and a computer to control the instrument and record data. Increasingly sophisticated instruments, such as those capable of multiplex experiments, are becoming affordable in academic labs.

The light source in the Applied Biosystems 7500 (represented here) is a simple halogen lamp shone through one of five different excitation filters over the entire sample. A CCD camera positioned above the sample records fluorescence from behind one of five emission fi lters. Some makes and models use a scanning head that moves over the plate, exciting and reading fluorescence in the wells individually.

Many qPCR instruments including the ABI 7500 use a Peltier element for heating and cooling. Peltier coolers use electron flow between semiconductor couples to heat or cool one side of a plate depending on the direction of current. Other systems use liquid or air fl ow or mechanical transition between blocks of different temperatures to cycle the samples.