Appeal No. 95-3606
Application 07/827,691
Innis II restates an earlier analysis of the problem and
his solutions (Innis II, pp. 54-57), including an example of
“PCR Using a 3:1 c dGTP:dGTP Mixture” (Innis II, p. 55).7
However, now Innis II states (Innis II, pp. 56 and 58,
bridging para.):
In our initial publication describing the use of c dGTP7
for structure-independent PCR (McConlogue et al. 1988),
we pointed out that PCRs with c dGTP (or mixtures of7
c dGTP7
and dGTP) appeared to be less efficient on most templates
than were PCRs with dGTP alone. This has now been shown
to be incorrect. We have discovered that PCR products
containing c dGTP simply do not stain efficiently with7
ethidium bromide, presumably because adjacent base
stacking
is diminished in the c dGTP-containing DNA. In fact, PCR7
(including asymmetric PCR) with c dGTP is as efficient7
as it is with dGTP for most templates, and for difficult
templates, is vastly superior to PCR with dGTP alone. . .
.
Indeed, from these results it appears that the only
reason
to use a mixture of c dGTP and dGTP is that incorporation7
of some dGTP is necessary for visualization of the
product
by ethidium staining.
Innis II adds (Innis II, p. 58, last para.):
We (McConlogue et al. 1988) also showed that DNA
amplified in the presence of 100% c dGTP was cleavable by7
Taq I restriction endonuclease (T’CGA). At that time,
we had not tried digesting c dGTP-amplified DNA with7
other restriction enzymes. In contrast, we show here
that incorporation of c dGTP during PCR can interfere7
with subsequent digestion by some enzymes . . . . Under
the conditions of approximately 20-fold overdigestion,
about 95% of the dGTP-containing DNA was cleaved. In
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