Long-Read, High-Resolution Sanger Sequencing by Micelle-Tagging Electrophoresis

We demonstrate a gel-free electrophoretic separation of Sanger sequencing fragments up to 782 bases in length using nonionic wormlike micelles as drag-tags in micelle-tagging electrophoresis (MTE). This is an increase of 280 bases over previous MTE methods and a nearly three-fold improvement over end-labeled free-solution electrophoresis (ELFSE) methods that use covalently attached drag-tags. For MTE, C18 alkane groups are attached to primers prior to their enzymatic extension. This alkane group provides a binding site for wormlike micelles composed of CiEj-type nonionic surfactants in the running buffer. Transient attachment of micelles to the C18 alkane group provides a highly uniform drag, equal to that of an ssDNA fragment 309 bases long. To account for slight mobility differences among the BigDye chain terminators, we developed a two-parameter time-shifting procedure to align the electropherograms for each termination chemistry. The increase in read length for this low-viscosity buffer (2.1 cP) is attributed to the alignment procedure, the large yet uniform drag, and the small degree of adsorption-based band broadening.