Methods in Molecular Biology • 16 Enzymes of Molecular Biology

Taq Polymerase 37

% primer incorporation

1,2

0,8

0,6

0,4

0,2

0

I1"

I I I I

1 2 3 4

U taq polymerase

Fig. 3. Dependency of primer incorporation on enzyme amounts. O, 22 cycles;
II, 24 cycles; A, 26 cycles.

The initial template concentration hardly influences the product
yield in the plateau phase. Even 1-10 copies of target sequence will be
amplified to give the plateau concentration after 40-50 cycles. Under
the experimental conditions mentioned earlier, incorporation of 10-
100 nM primer can be expected, which corresponds to roughly 1 lag
DNA. Therefore, the amplification has to reach the plateau phase, if
the product is to be analyzed by gel electrophoresis and ethidium
bromide staining.
The PCR is applicable for quantitative determinations only prior to the
onset of the plateau phase. Otherwise, quantitative analysis has to be
performed using competing primer pairs in an experiment (Section 4.7.).
The efficiency, defined as the amplification factor per cycle, has a
theoretical value of 2, in accordance with the doubling of the sequence
in every step. However, in practice, this is not achieved. The efficiency
is dependent on the copy number of the template: The lower the copy
number, down to a certain limit, the better the PCR works. An effi-
ciency close to the theoretical value (> 1.9) can only be expected with
copy numbers of the template in the range of 107-108 (14). Starting