Capillary gel electrophoresis with sinusoidal voltammetric detection: a strategy to allow four-"color" DNA sequencing.

Abstract

A novel detection strategy for DNA sequencing applications that utilizes a frequency-based electrochemical method is reported. Sinusoidal voltammetry is used to selectively identify four unique redox molecules that are covalently attached to the 5'-end of a 20-base sequencing primer. The tags used in this work are ferrocene derivatives with different substituents attached to the ferrocene ring, where the electron-donating or -withdrawing character of the substituent alters the half-wave potential of the modified ferrocene. Therefore, each tag has a unique SV frequency spectrum that can be easily identified in the frequency domain. In this work, the discrimination of one tag versus all others is accomplished through a "phase-nulling" technique. The signal for each tag is selectively eliminated while the other three responses remain virtually unchanged. This analysis scheme allows for the selective identification of each tagged oligonucleotide eluting in sieving polymer capillary gel electrophoresis with a separation efficiency of 2 x 10(6) theoretical plates per meter. This separation efficiency is sufficient to perform "low-resolution" DNA sequencing; the conditions used in this work have not yet been optimized for high-resolution sequencing applications.