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