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

82 Hyndman and Mitsuhashi
3.1. Efficiency
Efficiency can be viewed as the proportion of templates that are used to synthesize
new strands with each round of PCR, assuming the PCR primers are in a high
abundance. A situation in which efficiency is the primary concern is the amplification
of a purified template in which there is no chance of nonspecific PCR amplifications,
so the main issue is that of primer binding to its target.
3.1.1. Melting Temperature
Melting temperature, or Tm, is defined for a given DNA duplex as the temperature
at which half of the strands are hybridized and half of the strands are not hybridized.
The original definition of T m implied that the two complementary strands were in equal
proportions. In cases where the two strands are not in equal proportions, such as a
primer hybridizing to its target in PCR, the definition of Tm must be altered. Because
in a PCR primer concentrations will be orders of magnitude higher than template
concentrations, the interpretation is that the Tm is the temperature at which a primer is
hybridized to half of the template strands. The T m of a given primer/template combination
depends on primer concentration, template concentration, and salt concentration.
Generally, the template concentration is considered to be negligible compared with the
primer concentration, so the formula for calculating Tm is simplified such that it does
not contain a parameter for template concentration.
T m can be expressed as follows:
∆H
T m = ————— –273.15 + 16.6 log [Na + ] (1,2)
∆S + Rln(c)
The primer concentration is c; the ∆H and ∆S refer to the total enthalpy and entropy
of hybridization, respectively; and [Na + ] is the sodium ion concentration but can
refer to the total concentration of most monovalent cations (such as K + ). If there is
Mg 2+ or other divalent cations such as Mn 2+ , the conversion is generally accepted
as follows:
Na + = 4 × [Mg 2+ ] 2 (3)
3.1.2. Calculating T m with the Nearest Neighbor Model
The accurate calculation of T m from a given sequence requires determining the
∆H and ∆S of the hybridization. The most successful method for this is through the
use of the nearest neighbor model (4–7). With the nearest neighbor model, every pair
of adjacent base pairs makes a specific contribution to the overall ∆H and ∆S of the
duplex. The total ∆H and ∆S are calculated by adding all of the component values
plus a value for initiation of duplex formation. A table of ∆H and ∆S nearest neighbor
values is shown in Table 1 (5).
3.1.3. Efficient PCR Primers and T m
The most important issue for designing efficient PCR primers is that they must bind
to the target site efficiently under the conditions of the PCR. This generally means not
only that they bind at the annealing temperature but that if the annealing temperature