Chapter 3

Real-Time PCR – The Basic Principles

Contents

3.1 Traditional PCR Versus Real Time PCR . . . . . . . . . . . . . . . . . . . . 27

3.1.1 PCR Kinetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
3.2 Optimising a Real-Time PCR Reaction . . . . . . . . . . . . . . . . . . . . . 29
3.2.1 Primer Sets and Probe Design . . . . . . . . . . . . . . . . . . . . . . 29
3.2.2 PCR Components and Assay Optimization . . . . . . . . . . . . . . . . 31
3.2.3 Real-Time Fluorescense Reporters . . . . . . . . . . . . . . . . . . . . 32
3.2.4 Melting Curve Dissociation Analysis . . . . . . . . . . . . . . . . . . 35
3.2.5 Probe-Based Chemistry . . . . . . . . . . . . . . . . . . . . . . . . 36
3.2.6 FRET-Based Hybridisation Probes . . . . . . . . . . . . . . . . . . . 38
3.2.7 Scorpion Primers . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
3.2.8 LAMP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43

3.1 Traditional PCR Versus Real Time PCR

Conventional PCR is a powerful technique that allows exponential amplification

of DNA sequences. A PCR reaction needs a pair of primers that are complemen-
tary to the sequence of interest. Primers are extended by the DNA polymerase. The
copies produced after the extension, so called amplicons, are re-amplified with the
same primers leading thus to an exponential amplification of the DNA molecules.
After amplification, gel electrophoresis is used to analyse the amplified PCR prod-
ucts and this makes conventional PCR time consuming; since the reaction must
finish before proceeding with the post-PCR analysis. Real Time PCR overcome this
problem, because of its ability to measure the PCR amplicons at early states of
the reaction as they are accumulate in a “Real Time Detection” mode thus mea-
suring the amount of PCR product where the reaction is still in the exponential
phase (QPCR).

E.A. Pestana et al., Early, Rapid and Sensitive Veterinary Molecular Diagnostics - 27
Real Time PCR Applications, DOI 10.1007/978-90-481-3132-7_3,
Copyright 
C International Atomic Energy Agency 2010
Published by Springer Science+Business Media B.V., Dordrecht 2010. All Rights Reserved.
28 3 Real-Time PCR – The Basic Principles

Real Time PCR allows detection and quantitative measurement of products gen-
erated during each cycle of the PCR process that are directly proportional to the
amount of the template DNA before the start of the PCR process. Such chemistry
requires the use of a method to detect the product formed on each cycle and of a
thermocycler that is adapted to record the results obtained on each amplification
cycle in a Real Time manner.

3.1.1 PCR Kinetics

Differences between conventional PCR and QPCR are much easier to understand
when the kinetics of the PCR reaction are examined. Let us imagine we have
three replicates. As such all samples will begin the PCR cycling process with the
same conditions: the same quantity of all PCR components and the same DNA
concentration.
A conventional PCR reaction usually steps into 3 phases: the exponential, the
non-exponential and plateau or end-point phase (Fig. 3.1).
At the beginning of each PCR reaction, all components are present in a suffi-
ciently high quantity to guarantee good amplification and as the PCR progresses
and fresh components are present, amplification occurs in an exponential manner,
that is the reaction proceed doubling the quantity of initial DNA with every other
cycle. As the cycles progress and reagent components of the reaction start to be
depleted, the reaction will begin to slow down and the PCR product will no longer
be doubled in every cycle, and the non-exponential amplification occurs where sam-
ples begin to diverge in their quantities. After several rounds of amplification, the
PCR reaction will no longer generate template due to the lack of critical components
in the reaction, what it is commonly known as the plateau phase or end-point of the
PCR reaction.

0.3 Exponential phase

Non-exponential
plateau phase
0.2
Fluorescene

0.1 CT value

Threshold line

0 10 20 30 40
Cycle

Fig. 3.1 Amplification plot. Baseline fluorescence is subtracted