title: Investigation of the signaling mechanism and verification of the performance of an electrochemical real-time PCR system based on the interaction of methylene blue with DNA.
abstract: The operation of an electrochemical real-time PCR system, based on intercalative binding of methylene blue (MB) with dsDNA, has been demonstrated. PCR was performed on a fabricated electrode-patterned glass chip containing MB while recording the cathodic current peak by measuring the square wave voltammogram (SWV). The current peak signal was found to decrease with an increase in the PCR cycle number. This phenomenon was found to be mainly a consequence of the lower apparent diffusion rate of the MB-DNA complex (D(b) = 6.82 × 10(-6) cm(2) s(-1) with 612 bp dsDNA) as compared to that of free MB (D(f) = 5.06 × 10(-5) cm(2) s(-1)). Utilizing this signal changing mechanism, we successfully demonstrated the feasibility of an electrochemical real-time PCR system by accurately quantifying initial copy numbers of Chlamydia trachomatis DNA templates on a direct electrode chip. A standard calibration plot of the threshold cycle (C(t)) value versus the log of the input template quantity demonstrated reliable linearity and a good PCR efficiency (106%) that is comparable to that of a conventional TaqMan probe-based real time PCR. Finally, the system developed in this effort can be employed as a key technology for the achievement of point-of-care genetic diagnosis based on the electrochemical real-time PCR.
authors.affiliation: Department of Chemical and Biomolecular Engineering, KAIST, Daejeon, 305-701, Republic of Korea.
authors.forename: Byoung Yeon
authors.initials: BY
authors.lastname: Won
authors.forename: Sujeong
authors.initials: S
authors.lastname: Shin
authors.forename: Songyi
authors.initials: S
authors.lastname: Baek
authors.forename: Ye Lim
authors.initials: YL
authors.lastname: Jung
authors.forename: Taihua
authors.initials: T
authors.lastname: Li
authors.forename: Sung Chul
authors.initials: SC
authors.lastname: Shin
authors.forename: Dae-Yeon
authors.initials: DY
authors.lastname: Cho
authors.forename: Sang Bok
authors.initials: SB
authors.lastname: Lee
authors.forename: Hyun Gyu
authors.initials: HG
authors.lastname: Park
mesh_terms: D002692:Chlamydia trachomatis / Q000235:genetics; D004247:DNA / Q000737:chemistry*; D004058:Diffusion; D055664:Electrochemical Techniques / Q000379:methods*; D004566:Electrodes; D018628:Gene Dosage; D008751:Methylene Blue / Q000737:chemistry*; D019095:Point-of-Care Systems; D016133:Polymerase Chain Reaction / Q000379:methods*
chemical_list: D004247:DNA; D008751:Methylene Blue