title: Monodisperse, "highly" positively charged protein polymer drag-tags generated in an intein-mediated purification system used in free-solution electrophoretic separations of DNA.
abstract: Free-solution conjugate electrophoresis (FSCE) is a method of DNA sequencing that eliminates the need for viscous polymer solutions by tethering a carefully designed, mobility modifying "drag-tag" to each DNA molecule to achieve size-based separations of DNA. The most successful drag-tags to date are genetically engineered, highly repetitive polypeptides ("protein polymers") that are designed to be large, water-soluble, and completely monodisperse. Positively charged arginines were deliberately introduced at regular intervals into the amino acid sequence to increase the hydrodynamic drag without increasing drag-tag length. Additionally, a one-step purification method that combines affinity chromatography and on-column tag cleavage was devised to achieve the required drag-tag monodispersity. Sequencing with a read length of approximately 180 bases was successfully achieved with a known sequence in free-solution electrophoresis using one of these positively charged drag-tags. This preliminary result is expected to lead to further progress in FSCE sequencing with ~400 bases read length possible when more "highly" positively charged protein polymers of larger size are generated with the intein system.
authors.affiliation: Departments of Bioengineering, Stanford University, Stanford, California 94305, United States.
authors.forename: Xiaoxiao
authors.initials: X
authors.lastname: Wang
authors.forename: Jennifer Coyne
authors.initials: JC
authors.lastname: Albrecht
authors.forename: Jennifer S
authors.initials: JS
authors.lastname: Lin
authors.forename: Annelise E
authors.initials: AE
authors.lastname: Barron
mesh_terms: D004247:DNA / Q000737:chemistry* / Q000235:genetics; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics; D004586:Electrophoresis / Q000379:methods; D047668:Inteins; D017422:Sequence Analysis, DNA / Q000379:methods*
chemical_list: D004268:DNA-Binding Proteins; D004247:DNA