Analytical Chemistry Chemical Cytometry Quantitates Superoxide
Analytical Chemistry Chemical Cytometry Quantitates Superoxide
Analytical Chemistry Chemical Cytometry Quantitates Superoxide
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of which was constant for all amplicons analyzed in this<br />
experiment, did not show any significant trend, suggesting<br />
approximately constant yields of the PCR products obtained<br />
for any dGTP/dG*TP ratio after the 30 PCR cycles (which was<br />
also confirmed by UV-vis spectrophotometry, not shown).<br />
Dependences of the heights of peak Gox (yielded by the<br />
unmodified 347-bp PCR product) and peak G* ox (yielded by<br />
the same DNA fragment amplified with 100% of dG*TP) on<br />
concentrations of the amplicons followed similar trends (Figure<br />
4A), showing approximately linear regions below 10 µgmL-1and sublinear trends, suggesting saturation of the electrode surface,<br />
at higher DNA concentrations. The lowest detectable DNA<br />
concentrations were around 1 µg mL-1 in both cases. Electrochemical<br />
determination of the modified and unmodified 347bp<br />
amplicons was compared to other commonly used techniques,<br />
based on gel electrophoresis followed by DNA staining<br />
(Figure 4B). The amplicons were separated in native 5% polyacrylamide<br />
gel (1 µL of the reaction mixture, containing about 30<br />
µg mL-1 Figure 2. Electrochemical responses of a PCR-amplified 347-bp<br />
DNA fragment modified with 7-deazapurines. (A) Scheme of the PCR.<br />
Primers at the 5′-ends of both strands of the PCR product (black)<br />
contain only standard nucleobases, regardless of the dNTP composition.<br />
The synthesized stretches contain modified nucleobases depending<br />
on the dNTP mix. (B) Baseline-corrected voltammograms<br />
obtained for DNA fragment resulting from PCR in the presence of<br />
standard dNTP mix (black), for a mix with G* instead of G (red) and<br />
for a mix with A* instead of A (blue). The PCR was conducted in 30<br />
cycles and the products were purified using Qiagen PCR Purification<br />
Kit. Dotted curve corresponds to control PCR mixture (with G*+A)<br />
which was not subjected to thermal cycling.<br />
of the amplicon, and three binary dilutions). After<br />
electrophoresis, the gels were stained with ethidium bromide,<br />
Figure 3. Dependence of the heights of peak G* ox (red), peak G ox<br />
(black) and peak A ox (empty triangles) on the [dG*TP]/[dGTP]+[dG*TP]<br />
ratio in the PCR reaction used for amplification of the 347-bp DNA<br />
fragment. Other conditions as in Figure 2.<br />
SYBR Green I or Stains-All reagent (Figure 4B). Ethidiumstained<br />
bands of the G*-modified PCR products were considerably<br />
weaker than those of the unmodified amplicon (even when only<br />
50% of Gs were substituted by G*, see Figure 4B). Such<br />
observation was in agreement with literature data 26 showing that<br />
ethidium fluorescence is quenched when the dye is intercalated<br />
next to G*. Notably, we observed even stronger quenching effect<br />
of G* on the fluorescence of SYBR Green I (Figure 4B) and SYBR<br />
Gold (not shown) dyes. Thus, results of the fluorescent DNA<br />
staining might be misinterpreted, for the densely G*-modified<br />
DNA, in terms of (strongly) decreasing amount of the PCR<br />
products with increasing G/G* ratio. On the other hand, staining<br />
of the polyacrylamide gel with the Stains-All reagent (Figure 4B)<br />
did not reveal significant differences in the amounts of the<br />
unmodified and G*-substituted amplicons, in agreement with the<br />
electrochemical data.<br />
Despite the approximately same yields of the PCR products<br />
after 30 cycles, we were interested whether we are able to follow<br />
electrochemically differences in the kinetics of the PCR reactions<br />
through analysis of the amplicons after lower number of amplification<br />
cycles. Previous data 15 revealed less efficient DNA amplification<br />
by PCR in the presence of dG*TPs, compared to PCR with<br />
standard dNTPs only. We followed electrochemical signals of the<br />
unmodified and fully G*-substituted PCR products after 5, 10, 20,<br />
and 30 cycles (Figure 5A). For five cycles, peak G ox produced<br />
by the unmodified amplicon was considerably higher than peak<br />
G* ox of the G*-modified PCR product. Even after subtraction<br />
of signal intensity produced by the control PCR mix not<br />
subjected to the thermal cycling (containing initial concentrations<br />
of ODN primers and the primary template), the peak G ox<br />
was at least twice higher than peak G* ox produced by the G*modified<br />
amplicon after the same number of cycles. Large<br />
differences between the signal intensities were also observed<br />
after 10 amplification cycles, while after 20 and 30 cycles both<br />
peaks reached their limiting values. Hence, more cycles were<br />
required to reach the limiting amount of the G*-modified PCR<br />
(26) Latimer, L. J. P.; Lee, J. S. J. Biol. Chem. 1991, 266, 13849–13851.<br />
<strong>Analytical</strong> <strong>Chemistry</strong>, Vol. 82, No. 16, August 15, 2010<br />
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