Work

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

Year: 2025

Type: article

Abstract: 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.

Source: Electrophoresis

DOI: 10.1002/elps.70047

Authors: Randall Gamble, H. Michael Wenz, Bashar Mullah, James W. Schneider

Institutions: Carnegie Mellon University, Thermo Fisher Scientific (United States)

Language: English

Topic: Microfluidic and Capillary Electrophoresis Applications

Subfield: Biomedical Engineering

Field: Engineering

Domain: Physical Sciences

Open Access status: hybrid

Estimated APC paid: $3,940

Funders: National Science Foundation, Thermo Fisher Scientific