Electroanalytical study of proflavine intercalation in 5-methyl or inosine-containing amplicons.

Abstract

Amplicons corresponding to the GC-rich p53 exon 5 and its analogues, synthesized by substituting 60% of cytosine by 5-methyl-cytosine, or 60% of guanosine by inosine and GC-poor p53 exon 6 were synthesized and investigated electrochemically, in the presence and absence of proflavine, by differential pulse voltammetry (DPV). Incorporation of base analogues and the thermal stability of the resulting amplicons were tested in the presence of a fluorescent probe (Sybr-Green). Peak current at 1.0 V was lower for methylated than for unmethylated PCR amplicons and was similarly affected by proflavine intercalation. In contrast, considerable peak current differences were observed in the presence of proflavine for unmodified exon 5 v.s. exon 6 or inosine-containing amplicons. Thermal analysis verified the expected shifts in melting temperature (T (m)) due to the base analogue incorporation and GC-content variations. In conclusion, methylated and unmethylated PCR amplicons could be distinguished in model DNA systems using differential pulse voltammetry (DPV) and use of proflavine could serve as an electrochemical probe for identifying different DNA conformations.

Full text

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ORIGINAL PAPER
Electroanalytical study of proflavine intercalation
in 5-methyl or inosine-containing amplicons
Despina K. Alexiadou & Andrea K. Ioannou &
Sofia A. Kouidou-Andreou &
Anastasios N. Voulgaropoulos & Stella Th. Girousi
Received: 22 May 2008 /Revised: 3 July 2008 /Accepted: 4 July 2008 / Published online: 2 August 2008
# Springer-Verlag 2008
Abstract Amplicons corresponding to the GC-rich p53 exon
5 and its analogues, synthesized by substituting 60% of
cytosine by 5-methyl-cytosine, or 60% of guanosine by inosine
and GC-poor p53 exon 6 were synthesized and investigated
electrochemically, in the presence and absence of proflavine,
by differential pulse voltammetry (DPV). Incorporation of
base analogues and the thermal stability of the resulting
amplicons were tested in the presence of a fluorescent probe
(Sybr–Green). Peak current at 1.0 V was lower for
methylated than for unmethylated PCR amplicons and was
similarly affected by proflavine intercalation. In contrast,
considerable peak current differences were observed in the
presence of proflavine for unmodified exon 5 v.s. exon 6 or
inosine-containing amplicons. Thermal analysis verified the
expected shifts in melting temperature (Tm) due to the base
analogue incorporation and GC-content variations. In con-
clusion, methylated and unmethylated PCR amplicons could
be distinguished in model DNA systems using differential
pulse voltammetry (DPV) and use of proflavine could serve as
an electrochemical probe for identifying different DNA
conformations.
Keywords Amplicons.5-Methylcytosine.Inosine.
Meltingtemperature.Differentialpulsevoltammetry.
Proflavine
Introduction
5-Methylcytosine is a modified pyrimidine base, which
frequently substitutes for cytosine, particularly in CpG
sequences[1–4]. Substitution of cytosine by 5-methylcytosine
affects the structural and dynamic properties of the double
helix and leads to helical stabilization [5–7]. Epigenetic
DNA modification via global cytosine methylation is a
dynamic mechanism adopted in differentiation and genetic
reprogramming. Methylation differences and extensive hyper
and hypomethylation loci characterize cancerous versus non-
cancerous DNA[8]. The challenging issue of studying DNA
methylation has been delayed because of its loss in
amplified DNA, for example products of polymerase chain
reaction (PCR), cloning, etc. The relatively recent introduc-
tion of a chemical method for differentiating between
methylated and unmethylated cytosines by PCR has greatly
facilitated the study of this fundamental biological mecha-
nism [9]. However, a direct approach for studying the extent
of methylation based on the physicochemical changes
introduced by cytosine modification would greatly simplify
the evaluation of global DNA methylation.
Inosine (I) is a guanosine derivative formed by hypo-
xanthine and ribose. In comparison with the chemical
structure of guanosine, inosine lacks an amine group but
can still substitute for guanosine in the helix of DNA.
Inosine–cytosine base pairs can form only two hydrogen
bonds instead of the three typical of guanosine-cytosine
base pairs [10]. Inosine has become a favorite tool for
examination of the crucial role of guanine’s exocyclic 2-
amino group in DNA recognition by various drugs [11].
Differential-pulse voltammetry (DPV) is mainly used in
bioelectrochemicalstudies.Theuseofcarbon-pasteelectrodes
(CPE) and transfer differential-pulse voltammetry provides a
signal for guanine’s oxidation [12] and has been used to
Anal Bioanal Chem (2008) 392:533–539
DOI 10.1007/s00216-008-2285-4
D. K. Alexiadou:A. K. Ioannou:A. N. Voulgaropoulos:
S. T. Girousi (*)
Laboratory of Analytical Chemistry, Department of Chemistry,
Aristotle University of Thessaloniki,
541 24 Thessaloniki, Greece
e-mail: girousi@chem.auth.gr
S. A. Kouidou-Andreou
Laboratory of Biochemistry, Department of Medicine,
Aristotle University of Thessaloniki,
541 24 Thessaloniki, Greece

Page 2

investigate DNA damage involving base residues [13].
Lately, genosensors have been developed for detection of
DNA hybridization and DNA modifications [14–16].
Melting-curve analysis of nucleic acids by use of
photometric or fluorimetric methods is also a useful tool
for analyzing nucleic acid biophysical and conformational
properties, which are frequently also studied by gel electro-
phoresis. Real-time PCR technology and fluorescence
techniques have facilitated thermal melting applications
involving the study of DNA biophysical properties, for
example DNA annealing, because fluorescence is greatly
affected by the DNA melting process. Because the melting
temperature of nucleic acids is affected by length, GC
content, and the presence of base mismatches, among other
factors, products can often be distinguished by their melting
characteristics [17–20].
Proflavine is a known intercalator whose electrochemical
behavior has been previously studied [21]. Electrochemical
studies on the interaction of proflavine with calf thymus
DNA have shown that proflavine intercalates within the
DNA helix and induces conformational changes [22–24].
According to a previous study its binding affinity to DNA
depends upon the structure of the double-stranded poly-
nucleotides and their ability to be distorted [25]. In this study
the interaction of proflavine with model DNA systems (PCR
products—amplicons) of different composition (GC content)
(exon 5 and exon 6 of gene p53) containing different base
analogues, for example methylcytosine and inosine, was
studied using amplicon biosensors.
Experimental
Reagents
Natural bases triphosphate monomers (dNTPs) were pur-
chased from Invitrogen (UK). 5-methyldeoxycytosine triphos-
phate (5-methyl-dCTP) and deoxyinosine triphosphate (dITP)
monomers were purchasedfrom Roche(Germany).Proflavine
was purchased from Sigma. Stock solutions of proflavine
(10−1mol L−1) were prepared in doubly-distilled water and
then diluted appropriately, just before use. The supporting
electrolyte for voltammetric experiments was acetate buffer
solution 0.2 mol L−1at pH 5.0+20 mmol L−1NaCl. The
fluorescence dye used for melting curves was Sybr Green I
and was purchased from Molecular Probes (USA). All other
reagents used were of analytical grade.
Clinical sample and DNA isolation
All tissues were frozen in liquid nitrogen immediately after
excision and removal of a specimen for pathological
examination. All specimens used for this study were
obtained from consenting individuals. The tissues were
pulverized in liquid nitrogen and used for isolation of DNA
using organic solvents [26].
Instrumentation
Differential pulse voltammetric measurements were per-
formed using a PalmSens potentiostat controlled by Palm-
SensPC software (IVIUM Technologies, The Netherlands).
The working electrode was a carbon-paste electrode with
3 mm inner and 6 mm outer diameter of the PTFE sleeve. The
reference electrode was Ag/AgCl/3 mol L−1KCl and the
counter-electrode was a platinum wire. A thermocycler was
used for PCR (MJ Research, USA) and real-time PCR (DNA
Engine Opticon 2 System using a CFD-3220 Opticon 2
Detector; MJ Research) was used for melting-curve analysis.
An Eppendorf 5417R centrifuge was used for purification of
the PCR products.
PCR amplification of p53 exon 5 DNA and its analogues
DNA (150 ng) was amplified using 1.25 U Taq DNA
polymerase (recombinant, Invitrogen) in a total volume of
50 μL (1.5 mmol L−1MgCl2, 10 mmol L−1Tris-HCl pH 8.3,
0.5 mmol L−1KCl, 0.2 mmol L−1dNTPs) containing 100
pmol of each primer. p53 ex5 forward: 5′-TTCCTCTTCCTA-
CAGTAC-3′; p53ex5 reverse: 5′-GCCCCAGCTGCTCACC
ATCG-3′. Cycling conditions were: 95°C×5 s; 95°C×45 s;
52°C×45 s; 72°C×1 min, 35 cycles, followed by a step at
72°C for 10 min. The sequence of amplified exon 5 was the
following: TTCCTCTTCCTACAG/TAC-TCC-CCT-GCC-
CTC-AAC-AAG-ATG-TTT-TGC-CAA-CTG-GCC-AAG-
ACC-TGC-CCT-GTG-CAG-CTG-TGG-GTT-GAT-TCC-
ACA-CCC-CGC-CCC-GGC-ACC-CGC-GTC-CGC-GCC-
ATG-GCC-ATC-TAC-AAG-CAG-TCA-CAG-CAC-ATG-
ACG-GAG-GTT-GTG-AGG-CGC-TGC-CCC-CAC-CAT-
CAG-CGC-TGC-TCA-GAT-AGC-GAT-G/GTGAG
CAGCTGGGGC. Amplicons were analyzed by 1% agarose
gel electrophoresis. Amplicons with inosine were synthesized
by substituting dGTP in the dNTPs mixture with dITP (10–
80%). Similarly, amplicons with methylcytosine were pro-
duced by substituting dCTP with 5-methyl-dCTP (10–80%).
PCR amplification of p53 exon 6 DNA
DNA (150 ng) was amplified using 1.25 U Taq DNA
polymerase (recombinant, Invitrogen) in a total volume of
50 μL (1.5 mmol L−1MgCl2, 10 mmol L−1Tris-HCl pH 8.3,
0.5 mmol L−1KCl, 0.2 mmol L−1dNTPs) containing
100 pmol of each primer. p53 ex6 forward: 5′-CACTGA-
TTGCTCTTAGGTCTGGC-3′; p53 ex6 reverse: 5′-AGTTG-
CAAACC AGACCTCAGGCG-3′. Cycling conditions were:
95°C×5 s; 95°C×45 s; 52°C×45 s; 72°C×1 min, 35 cycles,
534D.K. Alexiadou et al.

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followed by a step at 72°C for 10 min. The sequence of
amplified exon 6 was the following: CACTGATTGCTCT
TAG/GT-CTG-GCC-CCT-CCT-CAG-CAT-CTT-ATC-CGA-
GTG-GAA-GGA-AAT-TTG-CGT-GTG-GAG-TAT-TTG-
GAT-GAC-AGA-AAC-ACT-TTT-CGA-CAT-AGT-GTG-
GTG-GTG-CCC-TAT-GAG-CCG-CCT-GAG/GTCT
GGTTTGCAACT.
Melting curves of amplicons
DNA melting curves of the native and modified amplicons
were acquired on the DNA Engine Opticon 2 System by
measuring the fluorescence of Sybr Green I during a linear
temperature transition from 50 to 95°C at 0.1° s−1.
Fluorescence data were converted into melting peaks by
the Opticon Monitor software (Ver. 2.02) to plot the
negative derivative of fluorescence over temperature vs
temperature (−dF/dT vs T). Before melting curve analysis
19 μL amplicon was mixed with 1 μL of a 1:100,000
dilution of Sybr Green I.
DPV measurements
Before DPV measurements, amplicons were purified with cold
ethanolandCH3COONa. Cold absolute ethanol (2.5 volumes)
and 3 mol L−1CH3COONa (0.1 volume) were added to
precipitate the oligonucleotides. The precipitated oligonucleo-
tides were recovered by centrifugation (15 min at 14,000 rpm
and 4°C) and removal of the supernatant. An extra wash with
70% ethanol was included and followed by centrifugation
(5 min at 14,000 rpm and 4°C). After removal of the super-
natant, amplicons were diluted with Tris-HCl (10 mmol L–1,
pH 7) in order to use them in the electroanalytical study.
Adsorptive transfer stripping differential-pulse voltam-
metry was used for the analysis and carbon paste (CPE) as
the working electrode. The carbon paste was prepared in
the usual way by hand-mixing graphite powder and Nujol
oil in the ratio 75:25. The resulting paste was packed tightly
into a Teflon sleeve. Electrical contact was established by
means of a stainless steel screw. The surface was polished
to a smooth finish before use. Prior to the accumulation step
the electrode was pre-treated by applying a potential of
1.7 V for 1 min to the working electrode dipped in the
supporting electrolyte. The electrochemical pre-treatment
produces a more hydrophilic surface state and concomitant
removal of organic layers.
After pretreatment of the CPE, as previously described,
amplicons were immobilized on the activated electrode surface
byadsorptiveaccumulationfor5minat0.5V.Thetransduction
was carried out in blank solution (only supporting electrolyte)
with differential-pulse voltammetry under the following con-
ditions: Ebegin=0.1 V, Eend=1.5 V, Estep=0.005 V, Epulse=
0.025 V, scan rate=0.01 V s−1, and tpulse=0.07 s.
In order to study the intercalation of proflavine, the
electrode with the immobilized amplicon was transferred to
the stirred sample solution (analyte plus supporting elec-
trolyte) for the optimum interaction time. The transduction
was then carried out in blank solution with the same
conditions as mentioned above. Prior to each medium
exchange the electrode was rinsed carefully with water for
5 s. The interactions between the different types of
immobilized amplicon and increasing concentrations of
proflavine in solution were studied.
Results and discussion
Synthesis of modified amplicons
Nucleotide analogues were most efficiently incorporated in
amplicons when 60% of the natural bases were substituted by
the modified bases used (Fig. 1). Eighty percent substitution
of the native bases yielded lower amplification efficiencies
(results not shown). In the substitution of cytosine by 5-
methylcytosine, the amplification efficiency was higher in
the presence of 7% DMSO (Fig. 2). Neither methylcytosine
triphosphate nor inosine triphosphate supported the PCR
reaction, which was strongly inhibited when more than 60%
cytosine or guanosine were substituted by methylcytosine
and inosine, respectively. The degree of incorporation was
evaluated by calculating the melting temperature (Tm) of
these amplicons.
Melting curve analysis of amplicons
Incorporation of the base analogues was verified by thermal
denaturation of the resulting amplicons. The melting
characteristics of amplicons synthesized in the presence of
various concentrations of 5-methylcytosine evaluated by
fluorimetric analysis also showed that in the presence of
60% 5-methylcytosine in the amplification mixture, incor-
poration was maximum and strongly affected the stability
of the PCR product [27] (Fig. 3). In contrast, fluorimetric
results showed that incorporation of inosine was followed
by a decrease of Tm(not shown) related to the decrease of
hydrogen bonds in inosine-containing DNA [11]. Based on
previous data [28, 29] for the Tm values of methylated
synthetic oligonucleotides, the 2.7° of Tmelevation result-
ing from methylcytosine incorporation corresponded to a
substantially less incorporation in the methylated amplicon
used.
The melting curves of amplicons of exon 5, exon 6, and
modified exons 5 (60% 5-methyl-dCTP instead of dCTP or
60% dITP instead of dGTP) were also compared (Fig. 4). As
expected, the amplicon containing 5-methylcytosine was
the most stable (Tm=94°C), while the inosine-containing
Electroanalytical study of proflavine intercalation535

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amplicon was the least stable (Tm=83°C). Furthermore,
exon 6 (Tm=85°C, G:C content 50%) was less stable than
the G:C-rich exon 5 (Tm=91°C, G:C content 61.7%).
Amplicon biosensors
Native double-stranded DNA yielded a positive peak at
1.0 V, which corresponded to oxidation of the guanine
residues [12]. Amplicons also yielded this positive peak
at 1.0 V. The concentration of amplicons increased
gradually and the change in the peak current intensity
at 1.0 V was monitored. The electrode surface assumed
to be saturated only when a plateau was reached. This
concentration, 5×10−3g L−1, was used as the optimum
concentration, for further experiments.
The peak current intensity at 1.0 V was approximately
0.4 μA at the amplicons of exon 5 and exon 6, indicating no
influence due to the GC content. This peak was lower at the
amplicons that contain 5-methylcytosine (0.3 μΑ) than those
containing cytosine, indicating that fewer guanine residues
were exposed to the electrode surface (Table 1). This, most
probably, was a result of more compact oligonucleotide
structure [6] in the case of methylated cytosine compared
with the natural amplicon. The effect of methylcytosine
incorporation in a methylated amplicon was expected to vary
strongly depending on the position of incorporation and the
amplicon sequence. Methylated DNA sequences could form
Fig. 2 PCR amplification of exon-5 by using: 1, natural bases; 2,
natural bases and 7% DMSO; 3, 60% dITP instead of dGTP; 4, 60%
5-methyl-dCTP instead of dCTP; and 5, 60% 5-methyl-dCTP instead
of dCTP and 7% DMSO. L, 100 base-pair DNA ladder
PCR primers for exon 5 a
+

or

+
Taq DNA polymerase

35 cyclesa







a see experimental part
Human genomic DNA
0.2 M (dATP, dTTP, dGTP) +
0.08 M dCTP + 0.12 M 5-methyl-dCTP
0.2 M (dATP, dTTP, dCTP) +
0.08 M dGTP + 0.12 M dITP
exon 5 synthesized in the presence of 60% 5-methyl-dCTP instead of dCTP
or
exon 5 synthesized in the presence of 60% dITP instead of dGTP
Fig. 1 Schematic representation
of the PCR amplification proce-
dure for synthesis of the 5-
methyl and inosine-containing
amplicons
Fig. 3 Melting curves (−dF/dT) of amplicons produced in the
presence of Sybr Green by using: (a) natural bases, (b) 30% 5-
methyl-dCTP instead of dCTP, and (c) 60% of 5-methyl-dCTP instead
of dCTP
536D.K. Alexiadou et al.

Page 5

triple-stranded structures, particularly stable at ambient
temperatures [28, 29]. In the methylated exon 5 amplicon,
synthesized for our experiments, incorporation of methyl-
cytosine was expected to support such structures, which were
probably responsible for the observed alterations of the
electrochemical properties of the corresponding amplicons.
The peak current intensity at inosine-modified amplicons
was only 0.2 μA, because of the substantial number of
guanine molecules substituted by inosine, which is electro-
chemically inactive at 1.0 V. Thus, useful information is
derived by applying differential-pulse voltammetry to differ-
ent amplicons, without using any indicator.
Study of the intercalation of proflavine with amplicons
biosensors
Further investigation of the conformation of amplicons was
performed by using proflavine as a probe. The modified
electrode (amplicons immobilized on its surface) was prepared
aspreviouslydescribed,washed,andsubsequentlyimmersedin
proflavine solutions of concentration ranging from 10−8to
10−6mol L−1(in 0.2 mol L−1sodium acetate, pH 5.0) for
300 s.
The effect of proflavine in all amplicons used in this
work was investigated. The current intensity of the
oxidation peak at 1.0 V was decreased by increasing the
concentration of proflavine (Fig. 5) and two not very well-
separated peaks appeared at 0.8 V and 0.9 V, probably due
to the intercalated PF [22]. Because the peaks of proflavine
were not well-separated, our study focused on guanine’s
oxidation peak at 1.0 V. The decrease of this peak current
intensity was attributed to the stacking of proflavine in the
nucleic acid backbone. Insertion of proflavine caused a
major change in the phosphodiester group orientation [30].
As a result guanine residues were exposed to the electrode
surface to a lesser extent than before the intercalation of
proflavine.
Inordertocomparetheresults,therelativepercentagevalue
of current intensity with reference to the amplicons in the
absence of proflavine was used (Fig. 6). It was obvious that
for concentrations of proflavine below 1.5×10−7mol L−1
there were no distinct differences between amplicons but
there was a significant difference at higher concentrations. It
was also observed that in all amplicons at proflavine
concentrations higher than 5×10−7mol L−1the current
intensity remained almost constant, revealing that no more
proflavine could be intercalated.
The decrease of the peak at 1.0 V by increasing the
concentration of PF was stronger for the inosine-containing
exon 5 than for the other amplicons. This indicated that in
the presence of inosine more proflavine was intercalated
and induced a more pronounced conformational change
compared with the natural amplicons. On the other hand,
the amplicons containing 5-methylcytosine did not favor
the intercalation of proflavine to a large extent, because the
reduction of the oxidation peak current was not so high.
Finally, the decrease of guanine’s peak current was stronger
in exon 6 than in exon 5, indicating that exon 6 favored the
intercalation of proflavine compared with exon 5.
Table 1 Peak-current intensity of the synthesized amplicons at 1.0 V without proflavine, their Tm, and their relative peak-current intensity at
1.0 V when proflavine (C=8×10−7mol L−1) is added
AmpliconI (μΑ)Tm(°C)I (%)a
exon 5 synthesized in the presence of
60% 5-methyl-dCTP instead of dCTP
exon 5
exon 6
exon 5 synthesized in the presence
of 60% dITP instead of dGTP
0.313 (±0.004)93.8 (±0.1) 46.3 (±1.3)
0.395 (±0.004)
0.402 (±0.005)
0.199 (±0.004)
91.1 (±0.1)
84.9 (±0.1)
83.2 (±0.1)
43.8 (±1.0)
30.3 (±1.2)
20.2 (±2.0)
a100% is the peak current intensity at 1.0 V of each amplicon without proflavine
Voltammetric conditions as described in the Experimental section
Fig. 4 Melting curves (−dF/dT) of the following amplicons: (a) exon
5 synthesized in the presence of 60% 5-methyl-dCTP instead of dCTP,
(b) exon 5, (c) exon 6, (d) exon 5 synthesized in the presence of 60%
dITP instead of dGTP
Electroanalytical study of proflavine intercalation537

Page 6

Bytakinginto consideration all the results (Table 1), it was
observed that higher guanosine content and thermal stability
corresponded to lower intercalation. The latter was in
accordance with the intercalation model by which double
helix unwound to form the intercalation site [31]. It could be
assumed that proflavine intercalated preferentially at ampli-
cons that could unwind more easily or had less rigid
structure, for example the inosine-containing amplicons [11].
It could be observed that a difference of 2.7° in Tmof exon
5 and exon 5 containing methylcytosine corresponded to a
difference of 2.5% in current, whereas a difference of 1.7°C
in Tmof exon 6 and exon 5 containing inosine corresponded
0
20
40
60
80
100
010
Cproflavine (10-7 M)
Relative current (%)
a
b
c
d
5
Fig. 6 Relative variation of the peak-current intensity at 1.0 V of
amplicons on increasing the concentration of proflavine. Amplicons of
(a) exon 5 synthesized in the presence of 60% 5-methyl-dCTP instead
of dCTP, (b) exon 5, (c) exon 6, (d) exon 5 synthesized in the presence
of 60% dITP instead of dGTP. Voltammetric conditions as described
in the Experimental section
Fig. 7 Structures of (a) profla-
vine and (b) Sybr Green I
Fig. 5 The peak current inten-
sity at 1.0 V of amplicons (i)
without proflavine and (ii) with
proflavine (C=8×10−7mol
L−1). (a) exon 5 synthesized in
the presence of 60% 5-methyl-
dCTP instead of dCTP, (b) exon
5, (c) exon 6, (d) exon 5
synthesized in the presence of
60% dITP instead of dGTP.
Voltammetric conditions as
described in the Experimental
section
538D.K. Alexiadou et al.

Page 7

to a difference of 10.1% in current, which is much higher
than the former. The reason could be a stereochemical factor
that renders the molecule of PF (Fig. 7a) more labile than the
molecule of Sybr Green I (Fig. 7b), which was used for
evaluation of Tmand yielded significant differences in Tm
between methylated and unmethylated amplicons. Thus,
almost the same number of PF molecules could be
accommodated within both structures of the oligonucleotides
containing either cytosine or 5-methylcytosine despite their
different conformation (the amplicon containing methylcyto-
sine was more rigid than the natural amplicon [6]).
Conclusions
Pulse voltammetry can be used to differentiate between model
DNA systems containing inosine as a guanosine analogue and
depends on the state of methylation of cytosine. Proflavine
intercalation correlates with theguanosine content and thermal
stability of the synthetic DNA systems, being greater for
amplicons of lower guanosine content and lower thermal
stability. A weaker dependence of proflavine intercalation on
amplicon conformation was also observed. Considering the
speed, simplicity of instrumentation, and low cost of this
procedure,theaboveresultsshowelectrochemicalanalysishas
promise for identification of epigenetic modification of DNA.
Acknowledgments
project, implemented within the framework of the “Reinforcement
Programme of Human Research Manpower” (PENED) and co-
financed by National and Community Funds (25% from the Greek
Ministry of Development-General Secretariat of Research and Tech-
nology and 75% from E.U.-European Social Fund).
This paper is part of the 03ED835 research
References
1. Robertson KD, Wolffe AP (2000) Nat Rev Gen 1:11–19
2. Ehrlich M (2002) Oncogene 21:5400–5413
3. Esteller M (2005) Annu Rev Pharmacol Toxicol 45:629–656
4. Watson RE, Goodman JI (2002) Toxicol Sci 67:11–16
5. Mathur P, Xu J, Dedon PC (1997) Biochem (NY) 36:14868–
14873
6. Banyay M, Graslund A (2002) J Mol Biol 324:667–676
7. Hodges-Garcia Y, Hagerman PJ (1995) J Biol Chem 270:197–201
8. Kovalchuk O, Tryndyak VP, Montgomery B, Boyko A, Kutanzi
K, Zemp F, Warbritton AR, Latendresse JR, Kovalchuk I, Beland
FA, Pogribny IP (2007) Cell Cycle 6:2010–2018
9. Herman JG, Graff JR, Myohanen S, Nelkin BD, Baylin SB (1996)
Proc Natl Acad Sci USA 93:9821–9826
10. Chou SH, Chin KH, Chen CW (2001) J Biomol NMR 19:33–48
11. Bailly C, Waring MJ (2001) Meth Enzymol 340:485–502
12. Gherghi IC, Girousi ST, Voulgaropoulos A, Tzimou-Tsitouridou R
(2004) Anal Chim Acta 505:135–144
13. Fojta M (2002) Electroanal 14:1449–1463
14. Ozkan-Ariksoysal D, Tezcanli B, Kosova B, Ozsoz M (2008)
Anal Chem 80:588–596
15. Kara P, Cavdar S, Meric B, Erensoy S, Ozsoz M (2007)
Bioelectrochemistry 71:204–210
16. Eskiocak U, Ozkan-Ariksoysal D, Ozsoz M, Oktem HA (2007)
Anal Chem 79:8807–8811
17. Reed GH, Kent JO, Wittwer CT (2007) Pharmacogen 8:597–608
18. Deligezer U, Esin Akisik E, Dalay N (2007) Clin Chem Lab Med
45:867–873
19. Liu Y, Zhu Q, Zhu N (2007) Immunol Invest 36:507
20. Skow A, Mangold KA, Tajuddin M, Huntington A, Fritz B,
Thomson RB, Kaul KL (2005) J Clin Microbiol 43:2876–2880
21. Girousi ST, Alexiadou DK, Ioannou AK (2008) Microchim Acta
160:435–439
22. Alexiadou DK, Ioannou AK, Girousi ST (2008) Anal Let 41,
1740–1748
23. Aslanoglu M (2006) Anal Sci 22:439–443
24. Vanícková M, Labuda J, Bucková M, Surugiu I, Mecklenburg M,
Danielsson B (2000) Collect Czech Chem Commun 65:1055–
1066
25. Deubel V, Leng M (1974) Biochimie 56:641–648
26. Sambrook J, Fritsch E, Maniatis FT (1989) Molecular Cloning. A
Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press
New York
27. Guldberg P, Worm J, Grønbæk K (2002) Methods 27:121–127
28. Butkus V, Klimasauskas S, Petrauskiene L, Maneliene Z,
Janulaitis A, Minchenkova LE, Schyolkin AK (1987) Nucleic
Acids Res 15:8467–8478
29. Marcourt L, Cordier C, Couesnon T, Dodin G (1999) Eur J
Biochem 265:1032–1042
30. Tang P, Juang CL, Harbison GS (1990) Science 249:70–72
31. Rosu F, Pirotte S, De Pauw E, Gabelica V (2006) Int J Mass
Spectrom 253:156–171
Electroanalytical study of proflavine intercalation539