RECOMBINANT DNA TECHNIQUES: CLONING AND MANIPULATION OF DNA - A Guide to Methods in the Biomedical Sciences - page 56

Quantitative real-time PCR
The most sensitive and quantitative approach to PCR is through the
use of “real-time” PCR. The basis for real-time PCR is to utilize a sys-
tem that permits the detection of a PCR product as it accumulates in
the reaction. Real-time PCR can be used experimentally or clinically, for
example to determine the viral load in an HIV-infected individual. The
first real-time PCR used the fluorescent properties of ethidium bromide,
which binds quantitatively to DNA as it is produced (25). Several fully
automated real-time PCR systems have now been developed. These
include a thermal cycler, a light source (often a laser) to induce fluores-
cence, a CCD “charged coupled detector” camera for detection and, of
course, computer software to graphically translate the information col-
lected. Although the different technologies vary somewhat, they have
in common the ability to sample and detect in real-time a fluorescent
product generated during PCR. By comparing this product with the am-
plification of a housekeeping genes (beta-actin is commonly used), true
quantitative comparisons between samples can be accomplished, pro-
viding accurate measurements of the initial quantity of mRNA or DNA
in different samples. A major advantage of quantitative real-time PCR
systems is that the instrumentation provides the readout, and no gel
electrophoresis is required to analyze the PCR product.
One type of quantitative PCR uses the probe system. The
TaqMan probe system is based on FRET (fluorescence resonance en-
ergy transfer) principles, in which the close proximity of two fluorescent
dyes that can transfer energy results in fluorescence quenching (see
Chapter 5 for more information). In the TaqMan probe system, a spe-
cially designed oligonucleotide probe that contains two fluors, the re-
porter and a quencher, hybridizes to the fragment being extended by
PCR. As the PCR product is extended, the exonuclease activity of
the Taq polymerase digests the probe, releasing the reporter from the
quencher, resulting in fluorescence. As the product is amplified, the flu-
orescence increases quantitatively.
A second type of system uses the dye SYBR Green, which is a highly
specific dsDNA binding dye that remains associated with the DNA gener-
ated during the PCR amplification. Standard primers are used to amplify
DNA and the SYBR Green fluorescence is detected and plotted during
the amplification process. At the linear portion of the curve (when plotted
as fluorescence vs the number of rounds of PCR), a comparison
of the number of rounds of PCR required to give a constant amount
of fluorescence can give quantitative comparisons between samples.
This type of real-time PCR is especially useful for ChIP analysis (see
chromatin immunoprecipitation section below).