Analytical Chemistry Chemical Cytometry Quantitates Superoxide

Figure 4. (A) Dependence of the intensities of peak G ox (measured
for unmodified 347-bp amplicon, black) and peak G* ox (measured for
the same DNA fragment amplified in the presence of dG*TP instead
of dGTP, red) on DNA concentration. Other conditions as in Figure
2. (B) Staining of the 347-bp amplicon in polyacryalmide gels with
(from top to bottom) ethidium bromide, SYBR Green I and Stains-
All. The PCR reaction contained either four standard dNTPs, G/G*
) 1, or dGTP fully replaced with dG*TP as indicated on the top.
Loading of the PCR products: 1 µL of the undiluted reaction mixture
(about 30 ng of the amplicon), followed by binary dilutions as
indicated.
product, compared to PCR with standard dNTP mix. To support
this conclusion, we performed a real-time PCR experiment
using standard and dG*TP-containing dNTP mixes. Figure 5B
shows increase of the fluorescence of SYBR Green I (which was
used here as fluorescent dye indicating progression of DNA
amplification) as a function of the number of PCR cycles for dNTP
mixes containing 0, 10, 20, 30, 50, and 100% of dG*TP (of
dGTP+dG*TP total). The steepest increase of the signal was
observed for the unmodified amplicon and the amplification rate
decreased with the G* content, showing clear difference even for
10% G*. For 100% of G* the apparent amplification rate was
remarkably depressed. However, it should be taken into consideration
that SYBR Green I fluorescence is quenched by G*
incorporated (see above), and thus the weak signal detected for
the G*-substituted amplicon can have reflected the dense DNA
modification rather than the amount of the PCR product. We
therefore focused on the evaluation of the shapes of the amplification
curves rather than the absolute signal magnitudes. RotorGene-
3000 software enables to compare reactions parameters in the
6812 Analytical Chemistry, Vol. 82, No. 16, August 15, 2010
Figure 5. (A) Effects of the number of PCR cycles on intensities of
peak G ox (measured for unmodified 347-bp amplicon, gray columns)
and peak G* ox (measured for the same DNA fragment amplified in
the presence of dG*TP instead of dGTP, red columns). Other
conditions as in Figure 2. (B) Quantitative (real time) PCR amplification
of the 347-bp DNA fragment: standard dNTP mix (no G*) (1);
10% G* (2); 20% G*(3); 30% G* (4); 50% G (5); G fully replaced with
G* (6); negative “no template” control for standard dNTP mix (7). The
graph shows fluorescence of SYBR Green I complexes with the DNA
amplicons as a function of the number of PCR cycles. Inset, takeoff
graphs derived from the real time PCR data (colors correspond to
the raw data plot). All samples are plotted in duplicate.
comparative quantification mode. The takeoff points for each
reaction (sample) are calculated from the second derivatives of
raw data (Figure 5B, inset). Generally, the takeoff value is not
possible to determine exactly and it is taken as 80% below the
peak of second derivative (i.e., below the point where the
amplification curve is increasing most steeply). In the same mode,
reaction efficiencies of particular reactions are reporting the
amplificability of the template under the given conditions. For
example, amplification factors of 1.72, 1.65, and 1.61 obtained for
reactions with 100% G, G/G* ) 1 and 100% G*, respectively, clearly
show less efficient amplification in the dG*TP-substituted reactions.
Similar effects of G* on the PCR kinetics were obtained
using Taqman hydrolytic probes which release fluorescent indicators
into solution as the PCR progresses, and thus the fluorescence
intensity was not affected by the incorporated G* as it was in the
case of the intercalative dyes (not shown). Hence, results of the
real time PCR experiment were in a qualitative accordance with
the above electrochemical data indicating lower amplification rate
for the reaction with dG*TP.
CONCLUSIONS
7-deazapurines G* and A* enzymatically incorporated into DNA
are electrochemically oxidizable at carbon electrodes, producing