PCR Detection of Microbial Pathogens PCR Detection of Microbial ...

PCR Specificity and Performance 13
Fig. 3. Variation of the efficiency ε of a PCR as a function of [DNA’], the ratio of
amplified product to initial template concentration (85). Point p, where [DNA']1,
corresponds to an efficiency of ε 0.5 (courtesy of Academic Press, Ltd.).
sites) (see Chapter 4), although it will often be necessary to accept compromised
solutions for the sake of versatility and practicability.
Specificity of amplification is mainly determined by the annealing temperature
(T ann) and, to a lesser degree, primer length. The T ann of a PCR assay is
usually set within a few degrees of primer melting temperatures T m. For a given
reaction, the optimal T ann can be calculated using Equation 2 (2, 90):
T ann 0.3 T m-primers 0.7 T m-product 14.9 [Eq. 2]
where the midpoint primer melting temperature is T m-primers 0.5 (T m-primer1
T m-primer 2), and product melting temperature is T m-product 81.5 0.41(% GC)
16.6 log[K ] 675/L (with %G C, molar percentage of guanosine-pluscytosine,
[K], molar potassium ion concentration; L - length of amplicon in
base pairs). The addition of co-solvents, such as dimethyl sulfoxide and
formamide, allows to conduct the reaction at lower T ann and was shown to improve
both yield and specificity in selected cases (90).
If all other conditions are optimal, a rise in T ann can increase yield since
primer-template mismatches are further reduced and co-synthesis of unspecific
products is further suppressed. An example of the effect of T ann on specificity
is shown in Fig. 4.