Taq Polymerase 41
base mismatches. Therefore, new restriction sites can easily be intro-
duced into an amplification product (32). It is also possible to use
primers elongated by a stretch of 40 GC bp (33). Successful amplifi-
cation of sequences derived from known amino acid sequences utiliz-
ing primers with mixed bases at one position has been reported (34, 35).
3.2.3. Template
Normally, very much less than 1 ktg DNA is used for an experiment.
The sensitivity of the method allows a dilution of the sample down to
the theoretical limit of 1 molecule/test, even for routine diagnostics
(36,37). The requirements for DNA purity are rather modest. Intact
cells may be used; they are lysed during the first denaturation step of
the PCR procedure. Some authors recommend a proteinase K digest in the
presence of nonionic detergents in order to render the DNA more acces-
sible (38). However, there is no advantage to employing higher concen-
trations of template; this will make a contamination with inhibitors more
likely. Without any DNA isolation, amplification of bacterial sequences
is feasible directly by incubating 1 × 103 cells at 95°C for 10 min (39). At
higher cell concentrations, the PCR is inhibited. Hematin (hemin) has also
been found to inhibit the PCR at levels as low as 0.8 ~/(38). Therefore,
only nuclear fractions from whole blood samples should be analyzed
by the PCR. The sensitivity of the technique allows the amplification of
degraded DNA from very old, but well-preserved samples (40). Apreced-
ing reverse transcription in the same buffer system permits the direct
amplification of RNA sequences (41,42) (Section 4.2.).
3.2.4. Taq Polymerase
Taq polymerase 1 is commercially available from several manufac-
turers at a concentration of 5 U/ktL. One unit corresponds to 50 fmol
of enzyme (8). Taq polymerase lots from different suppliers may lead
to considerably different quantitative results. One to four units per
100-1aL reaction aliquot are sufficient to amplify a sequence to yield
amounts of DNA detectable by ethidium bromide staining. Larger
enzyme quantities will provoke the amplification of unspecific prod-
ucts (3). If the denaturation temperature for double-stranded DNA is
optimal, enzyme damage can be minimized, and enzyme activity will
be present in the reaction mixture even after 50 or more cycles. The
enzyme dilution should always be made up freshly by pipeting from
stock solution into incubation buffer (Section 3.2.6.).