Analytical Chemistry Chemical Cytometry Quantitates Superoxide

of which was constant for all amplicons analyzed in this
experiment, did not show any significant trend, suggesting
approximately constant yields of the PCR products obtained
for any dGTP/dG*TP ratio after the 30 PCR cycles (which was
also confirmed by UV-vis spectrophotometry, not shown).
Dependences of the heights of peak Gox (yielded by the
unmodified 347-bp PCR product) and peak G* ox (yielded by
the same DNA fragment amplified with 100% of dG*TP) on
concentrations of the amplicons followed similar trends (Figure
4A), showing approximately linear regions below 10 µgmL-1and sublinear trends, suggesting saturation of the electrode surface,
at higher DNA concentrations. The lowest detectable DNA
concentrations were around 1 µg mL-1 in both cases. Electrochemical
determination of the modified and unmodified 347bp
amplicons was compared to other commonly used techniques,
based on gel electrophoresis followed by DNA staining
(Figure 4B). The amplicons were separated in native 5% polyacrylamide
gel (1 µL of the reaction mixture, containing about 30
µg mL-1 Figure 2. Electrochemical responses of a PCR-amplified 347-bp
DNA fragment modified with 7-deazapurines. (A) Scheme of the PCR.
Primers at the 5′-ends of both strands of the PCR product (black)
contain only standard nucleobases, regardless of the dNTP composition.
The synthesized stretches contain modified nucleobases depending
on the dNTP mix. (B) Baseline-corrected voltammograms
obtained for DNA fragment resulting from PCR in the presence of
standard dNTP mix (black), for a mix with G* instead of G (red) and
for a mix with A* instead of A (blue). The PCR was conducted in 30
cycles and the products were purified using Qiagen PCR Purification
Kit. Dotted curve corresponds to control PCR mixture (with G*+A)
which was not subjected to thermal cycling.
of the amplicon, and three binary dilutions). After
electrophoresis, the gels were stained with ethidium bromide,
Figure 3. Dependence of the heights of peak G* ox (red), peak G ox
(black) and peak A ox (empty triangles) on the [dG*TP]/[dGTP]+[dG*TP]
ratio in the PCR reaction used for amplification of the 347-bp DNA
fragment. Other conditions as in Figure 2.
SYBR Green I or Stains-All reagent (Figure 4B). Ethidiumstained
bands of the G*-modified PCR products were considerably
weaker than those of the unmodified amplicon (even when only
50% of Gs were substituted by G*, see Figure 4B). Such
observation was in agreement with literature data 26 showing that
ethidium fluorescence is quenched when the dye is intercalated
next to G*. Notably, we observed even stronger quenching effect
of G* on the fluorescence of SYBR Green I (Figure 4B) and SYBR
Gold (not shown) dyes. Thus, results of the fluorescent DNA
staining might be misinterpreted, for the densely G*-modified
DNA, in terms of (strongly) decreasing amount of the PCR
products with increasing G/G* ratio. On the other hand, staining
of the polyacrylamide gel with the Stains-All reagent (Figure 4B)
did not reveal significant differences in the amounts of the
unmodified and G*-substituted amplicons, in agreement with the
electrochemical data.
Despite the approximately same yields of the PCR products
after 30 cycles, we were interested whether we are able to follow
electrochemically differences in the kinetics of the PCR reactions
through analysis of the amplicons after lower number of amplification
cycles. Previous data 15 revealed less efficient DNA amplification
by PCR in the presence of dG*TPs, compared to PCR with
standard dNTPs only. We followed electrochemical signals of the
unmodified and fully G*-substituted PCR products after 5, 10, 20,
and 30 cycles (Figure 5A). For five cycles, peak G ox produced
by the unmodified amplicon was considerably higher than peak
G* ox of the G*-modified PCR product. Even after subtraction
of signal intensity produced by the control PCR mix not
subjected to the thermal cycling (containing initial concentrations
of ODN primers and the primary template), the peak G ox
was at least twice higher than peak G* ox produced by the G*modified
amplicon after the same number of cycles. Large
differences between the signal intensities were also observed
after 10 amplification cycles, while after 20 and 30 cycles both
peaks reached their limiting values. Hence, more cycles were
required to reach the limiting amount of the G*-modified PCR
(26) Latimer, L. J. P.; Lee, J. S. J. Biol. Chem. 1991, 266, 13849–13851.
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