50 Landgraf and Wolfes
In the plateau phase of the reaction, the processivity of Taq poly-
merase decreases at excess template concentrations. This phenom-
enon is exploited in a very simple DNA sequencing method, where
phosphothioates are incorporated into the newly synthesized DNA.
The product is digested with snake venom phosphodiesterase and
analyzed on a sequencing gel. No product purification is necessary
applying this technique (84).
4.6. PCR and Site-Directed Mutagenesis
High yields of the desired mutations can be produced, when mis-
matched primers are employed in the PCR (85). Deletions, insertions,
and mutations were created in plasmids with the support of mismatched
primers and PCR (86). Furthermore, these primers may generate a new
restriction site, serving as an option to introduce amplified fragments
with the cassette method.
Amplification of the whole plasmid offers the opportunity to intro-
duce mutations in the absence of restriction sites (53); partial deletions
in the plasmid are also feasible (87). In 70% of the amplified plasmids,
3' additions of one base not present in the template were observed.
Prior to cloning, these protruding ends had to be removed by the Klenow
fragment of DNA polymerase to restore the gene sequence (53). The
PCR has proved useful in the precise gene fusion at any chosen loca-
tion (88). Sequencing of mutant clones after mutagenesis is
compulsory, given the relatively high error rate of Taq polymerase
(1.1 × 10 -.4 [6], 2 x 10 -4 [3,9)1). At least 20% of the clones derived
from a 250-bp fragment amplified 106-fold will contain one error in
the sequence (cf Fig. 7).
- Quantitative Interpretation of PCR Results
Standard PCR in itself is not a quantitative method, when the pla-
teau is reached. For a quantitative interpretation of PCR experiments,
low cycle numbers and template concentrations below enzyme con-
centrations are optimal, i.e., conditions where amplification is not
restricted by limiting parameters prevailing in the plateau phase.
Hybridization of 32p-labeled oligodeoxynucleotides revealed a linear
correlation between template DNA and detected signal, provided that
500--4000 copies of DNA as template and 30 amplification cycles
were used (17). In order to increase the incorporation rate, the dNTP
concentration was lowered to 10 JaM (Section 3.2.1.).
- Quantitative Interpretation of PCR Results