Basic techniques - Principles of Gene Manipulation - page 23

merase substrate and should not interfere with the
amplification primers (Dang & Jayasena 1996).
In order to minimize further the amplification of
spurious products, the strategy of nested primers may
be deployed. Here the products of an initial PCR
amplification are used to seed a second PCR ampli-
fication, in which one or both primers are located
internally with respect to the primers of the first
PCR. Since there is little chance of the spurious prod-
ucts containing sequences capable of hybridizing
with the second primer set, the PCR with these
nested primers selectively amplifies the sought-after
DNA.
As noted above, the Taq DNA polymerase lacks a

Table 2.1 Sources of thermostable DNA polymerases
with proofreading (3′-5′ exonuclease) activity.

DNA polymerase Source

Tma Thermotoga maritima

Deep Vent TM Pyrococcus sp.

Tli Thermococcus litoralis

Pfu Pyrococcus furiosus

Pwo Pyrococcus woesi

• There should be no complementarity between the
two primers. The great majority of primers which
conform with these guidelines can be made to work,
although not all comparable primer sets are equally
effective even under optimized conditions.
In carrying out a PCR it is usual to employ a
hot-start protocol. This entails adding the DNA
polymerase after the heat-denaturation step of the
first cycle, the addition taking place at a temperature
at or above the annealing temperature and just prior
to the annealing step of the first cycle. The hot start
overcomes the problem that would arise if the DNA
polymerase were added to complete the assembly
of the PCR reaction mixture at a relatively low
temperature. At low temperature, below the desired
hybridization temperature for the primer (typically
in the region 45 - 60°C), mismatched primers will
form and may be extended somewhat by the poly-
merase. Once extended, the mismatched primer is
stabilized at the unintended position. Having been
incorporated into the extended DNA during the
first cycle, the primer will hybridize efficiently in
subsequent cycles and hence may cause the ampli-
fication of a spurious product.
Alternatives to the hot-start protocol include the
use of Taq polymerase antibodies, which are inactiv-
ated as the temperature rises (Taylor & Logan 1995),
and AmpliTaq Gold TM , a modified Taq polymerase
that is inactive until heated to 95°C (Birch 1996).
Yet another means of inactivating Taq DNA
polymerase at ambient temperatures is the SELEX
method (systematic evolution of ligands by expo-
nential enrichment). Here the polymerase is
reversibly inactivated by the binding of nanomolar
amounts of a 70-mer, which is itself a poor poly-

3 proofreading exonuclease. This lack appears

to contribute to errors during PCR amplification due
to misincorporation of nucleotides (Eckert & Kunkel
1990). Partly to overcome this problem, other
thermostable DNA polymerases with improved
fidelity have been sought, although the Taq DNA
polymerase remains the most widely used enzyme
for PCR. In certain applications, especially where
amplified DNA is cloned, it is important to check the
nucleotide sequence of the cloned product to reveal
any mutations that may have occurred during the
PCR. The fidelity of the amplification reaction can be
assessed by cloning, sequencing and comparing
several independently amplified molecules.

′ -5 ′

Real-time quantitative PCR

There are many applications of the PCR where it
would be advantageous to be able to quantify the
amount of starting material. Theoretically, there is
a quantitative relationship between the amount of
starting material (target sequence) and the amount
of PCR product at any given cycle. In practice,
replicate reactions yield different amounts of prod-
uct, making quantitation unreliable. Higuchi et al .
(1992, 1993) pioneered the use of ethidium bromide
to quantify PCR products as they accumulate. Ampli-
fication produces increasing amounts of double-
stranded DNA, which binds ethidium bromide,
resulting in an increase in fluorescence. By plotting
the increase in fluorescence versus cycle number it is
possible to analyse the PCR kinetics in real time. This
is much more satisfactory than analysing product
accumulation after a fixed number of cycles.