Electroanalytical study of proflavine intercalation in 5-methyl or inosine-containing amplicons |
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Authors: | Despina K Alexiadou Andrea K Ioannou Sofia A Kouidou-Andreou Anastasios N Voulgaropoulos Stella Th Girousi |
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Institution: | (1) Laboratory of Analytical Chemistry, Department of Chemistry, Aristotle University of Thessaloniki, 541 24 Thessaloniki, Greece;(2) Laboratory of Biochemistry, Department of Medicine, Aristotle University of Thessaloniki, 541 24 Thessaloniki, Greece |
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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. |
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Keywords: | Amplicons 5-Methylcytosine Inosine Melting temperature Differential pulse voltammetry Proflavine |
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