Biomedical Engineering Reference
In-Depth Information
7. PCR Plateau
There is a limit to how many product molecules a given PCR can produce.
For a 100-
L PCR, the plateau is about 3-5 pmol
(
7
)
. The plateau effect is
caused by the accumulation of product molecules, which results in a signifi-
cant degree of annealing between complementary product strands rather than
between the primers and templates. Furthermore, the finite amount of enzyme
molecules present will be unable to extend all the primer-template complexes
in the given extension time.
µ
8. Sensitivity
The sensitivity of PCR is related to the number of target molecules, the
complexity of nontarget molecules, and the number of PCR cycles. Generally,
PCR is capable of amplification from a single target molecule
(
8
)
. This single-
molecule capability has allowed the development of single-molecule
genotyping
(
8
,
9
)
and pre-implantation diagnosis
(
10
)
. In these applications, the
single target molecule is essentially bathed in PCR buffer—in other words, in a
low-complexity environment. In situations in which the complexity of the envi-
ronment is high, the reliability of single-molecule PCR decreases and strategies
such as nesting and Hot Start PCR
(
11
,
12
)
are necessary for achieving maxi-
mum sensitivity. The sensitivity of PCR has also allowed it to be used in situa-
tions in which the starting materials have been partially degraded, e.g., with
formalin-fixed, paraffin wax-embedded materials.
9. PCR Fidelity
The fidelity of amplification by PCR is dependent on several factors:
annealing/extension time, annealing temperature, dNTP concentration, MgCl
2
concentration, and the type of DNA polymerase used. In general, the rate of
misincorporation may be reduced by minimizing the annealing/extension time,
maximizing the annealing temperature, and minimizing the dNTP and MgCl
2
concentration
(
13
)
. Eckert and Kunkel reported an error rate per nucleotide
polymerized at 70°C of 10
-5
for base substitution and 10
-6
for frameshift errors
under optimized conditions
(
13
)
. The use of a DNA polymerase with proof-
reading activity reduces the rate of misincorporation. For example, the DNA
polymerase from
Thermococcus litoralis
, which has proofreading activity,
misincorporates at 25% of the rate of the
Taq
polymerase, which lacks such
activity
(
14
)
. Interestingly, the combination of enzymes with and without
proofreading activity has enabled the amplification of extremely long PCR
products.
For most applications, product molecules from individual PCRs are analyzed
as a whole population, and rare misincorporated nucleotides in a small propor-
