Analytical Chemistry Chemical Cytometry Quantitates Superoxide

Figure 1. Electrochemical responses of oligonucleotides containing 7-deazapurines incorporated by primer extension (PEX). (A) Scheme of
the experiment. The PEX was performed using a 5′-terminally biotinylated template. After extension of the primer using either standard dNTP
mix, a mixture with dGTP replaced by dG*TP, or a mixture with dATP replaced by dA*TP, the duplex ODN was captured at magnetic beads
covered with streptavidin, the beads were washed and the modified strand released by thermal denaturation, followed by AdTS SWV measurements
at the PGE (shown for temp rnd16 and prim rnd sequences; blue and red letters are used to highlight A and G positions, respectively). (B) SWV
voltammograms of pex rnd16 : standard nucleobases (black); A* instead of A (blue); G* instead of G (red); background electrolyte (dotted); (C)
baseline-corrected curves taken from (B). Inset: PEX products obtained with temp noG and prim noG for dTTP+dATP mix (black) or dTTP+dA*TP
mix (green).
In next experiment, we prepared a 347-bp DNA fragment by
the polymerase chain reaction (PCR) using the pT77 template and
various dNTP mixtures. The PCR products were isolated from
the reaction mixture using Qiagen columns and analyzed by AdTS
SWV as above. During thermal cycling in the PCR, forward and
backward primers (Table 1) are elongated on templates of both
DNA strands, resulting in exponential amplification of the fragment
delimited by the two primers (see Figure 2A). Thus, each
strand of the double-stranded PCR product begins with the primer
at its 5′-terminus, followed by newly synthesized stretch, the
nucleotide composition of which (i.e., presence or absence of the
7-deazapurines) is dictated by the composition of the dNTP mix
(Figure 2A). Accordingly, when using mix of four standard dNTPs
(in Figure 2B denoted as G+A), we observed peak G ox and peak
A ox corresponding to natural purine bases. Replacement of
dGTP with dG*TP resulted in appearance of an intense peak
G* ox (due to G* incorporated into the polymerase-synthesized
strands) and strong decrease of the intensity of peak G ox which,
however, never reached zero. This peak G ox was due to G
residues in the primer stretches of the G*-modified amplicons
(Figure 2A). In addition, certain amount of unconsumed primers
and primary templates remaining in the samples after the
6810 Analytical Chemistry, Vol. 82, No. 16, August 15, 2010
purification step might contribute to the peak G ox intensity, as
indicated by negative PCR control experiment (PCR mixture
not subjected to thermal cycling, dotted curve in Figure 2B).
(It should be however noted that contributions to the peak G ox
from ODN primers in the negative PCR control, as apparent
from Figure 2B, and those from primer stretches in the G*modified
amplicon are not simply additive, because concentration
of residual free primers after the PCR cycling is much lower than
their initial concentration). When dATP was replaced with dA*TP,
we observed increase of the signal close to 1.15 V (peak G ox +
peak A* ox ) and decrease of peak A ox (again, the residual peak
A ox was yielded by A residues in the primer stretches).
Further, we focused our attention on analysis of DNA amplicons
containing G* as an independently detectable marker and
performed PCR experiments using dNTP mixes containing both
dGTP and dG*TP at different ratios (while keeping the total dGTP
+ dG*TP concentration constant). The resulting PCR products
were analyzed electrochemically as above. As shown in Figure 3,
changes in the relative abundances of G and G* in the PCR
product (given by the molar fraction of the respective dNTP) were
reflected in changes of corresponding electrochemical signals
(peak G ox and peak G* ox ). Peak A ox due to adenine, the content