Methods in Molecular Biology • 16 Enzymes of Molecular Biology

36 Landgraf and Wolfes

log Primer

incorporation

Efllcienc,

2

1.8

1.6

1.4

1.2

1

0,8

Plateau phase

f I I 1

I I I I

10 20 30 40

Cycles

Fig. 2. Typical PCR. Changes in efficiency and product yield. Graphs were cal-
culated on the basis of: 1 IxM of each primer spanning a stretch of 300 bp, 200 ktM
each dNTP, 2 U enzyme 1 nM (processing 10 templates/cycle, estimated enzyme
degradation 5%/cycle); and 200-ng template (0.05 aMol single copy gene). Vol-
ume of 100 ~L.

this reaction will be close to the theoretical value of 2, which means
that the sequence is doubled in every cycle. After 20-30 cycles are
completed, the product (and therefore the template) is amplified by a
factor of 106. Now the template concentration is in the same order as
the enzyme concentration. The efficiency of the PCR decreases steadily,
and exponential growth is no longer observed (Fig. 2). This phase of
the reaction is called plateau phase (12). Hence, the product yield is
dependent on the enzyme concentration (13) (Fig. 3).