John M. S. Bartlett.pdf - Bio-Nica.info

PCR Primer Design 83
Table 1
Nearest Neighbor Thermodynamic Values
for DNA Base Pairs
Base Pair ∆H ∆S ∆G
aa/tt 1–8.4 –23.6 –1.21
at/ta 1– 6.5 –18.8 – 0.73
aa/at 1– 6.3 –18.5 – 0.61
ca/gt 1–7.4 –19.3 –1.38
gt/ca 1–8.6 –231. –1.43
ct/ga 1– 6.1 –16.1 –1.16
ga/ct 1–7.7 –20.3 –1.46
gc/cg –11.1 –28.4 –2.28
gg/cc 1– 6.7 –15.6 –1.77
is so low that the thermostable DNA polymerase is not active, the primer must be
bound at the temperature at which the polymerase becomes active in order to begin
extension.
3.1.4. Typical Three-Step PCR
As an illustration, let us examine a typical PCR cycle, where there is a dissociation
step of 30 s at 95°C, an annealing step of 1 min at 37°C, and an extension step of 3
min at 72°C. If the T m of the primer, in the conditions of the reaction, is 65°C, then
the following will happen. As the temperature decreases from 95°C to 37°C, at some
point the primer will hybridize to the template. At this temperature, however, the
thermostable DNA polymerase may be almost totally inactive. As the temperature rises
towards 72°C, at some point the polymerase becomes active and will start to extend the
primer along the template. As the temperature rises above 65°C, some of the primers
will dissociate if they haven’t been extended. Those that have extended sufficiently,
however will form a more stable duplex as a result of their added base pairs from the
extension, and they will not dissociate before reaching 72°C. During the extension at
72°C, the primers still hybridized will be fully extended to generate new strands.
Let’s now imagine that with this scenario the T m of the primer is 75°C. In this
case, as the temperature rises from annealing to extension, even primers that have
not extended will not dissociate. In this scenario, there will be almost total extension
of every possible target strand. Therefore, this PCR would have a high degree of
efficiency.
3.1.5. Two-Step PCR
PCR is sometimes performed in two steps without a discrete annealing step. In this
case, the annealing takes place at the same temperature as the extension. This requires
that the primers will hybridize to some degree at the extension temperature. If the
extension temperature is 72°C, then a primer with a T m of 72°C would be an efficient
primer. Because the definition of T m is the point at which half of the templates are in
a duplex, a short time after reaching 72°C, half of the templates will be bound by a
primer. Shortly after that, a large percentage of those templates will be extended by