Biomedical Engineering Reference
In-Depth Information
Figure 2.1a-1 The exponential amplification of the gene in PCR. From Vierstraete (1999).
there are two single strands of DNA, called T1 and T2, for
template 1 and template 2, respectively. Once the tem-
perature is cooled to 54 C (signifying the starting of the
annealing stage), we expect that primer 1 (P1) will anneal
with T1 to form a hybrid (H1) and we also expect that P2
and T2 will anneal to form a hybrid (H2). These two re-
actions can be represented by the chemical equations:
Hence, four reactions can occur simultaneously during
the primer annealing stage. These reactions can be
modeled using either thermodynamic (steady state) or
kinetic (transient) equations.
The steady-state thermodynamic model tracks the
total concentration of all products performing active
roles during the annealing stage of PCR, a mass balance is
performed on reactions (2.1a.1) to (2.1a.4). This pro-
cedure results in the following four equations:
P1 þ T14H1
(2.1a.1)
P2 þ T24H2
(2.1a.2)
½ P1 T ¼½ P1 þ½ H1 þ½ D
½ P2 T ¼½ P2 þ½ H2 þ½ D
½ T1 T ¼½ T1 þ½ H1 þ½ HT
½ T2 T ¼½ T2 þ½ H2 þ½ HT
Unfortunately, other reactions can occur during the
annealing stage of PCR. The template strands, T1 and
T2, are complementary and can re-anneal upon contact,
forming a template hybrid (HT) as represented by the
following reaction:
(2.1a.5)
where [X] T is a parameter denoting the total concen-
tration of product X.
To complete the thermodynamic model, we need to
introduce four more parameters into the system
T1 þ T24HT
(2.1a.3)
the
equilibrium constants of each reaction. We define K H1 ,
K H2 ,K HT , and K D to be the equilibrium constants of
(2.1a.1) to (2.1a.4), respectively. Since the equilibrium
d
Finally, depending on primer design, primer-dimers (D)
can form as well:
P1 þ P24D
(2.1a.4)
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