title: A 265-base DNA sequencing read by capillary electrophoresis with no separation matrix.
abstract: Electrophoretic DNA sequencing without a polymer matrix is currently possible only with the use of some kind of "drag-tag" as a mobility modifier. In free-solution conjugate electrophoresis (FSCE), a drag-tag attached to each DNA fragment breaks linear charge-to-friction scaling, enabling size-based separation in aqueous buffer alone. Here we report a 265-base read for free-solution DNA sequencing by capillary electrophoresis using a random-coil protein drag-tag of unprecedented length and purity. We identified certain methods of protein expression and purification that allow the production of highly monodisperse drag-tags as long as 516 amino acids, which are almost charge neutral (+1 to +6) and yet highly water-soluble. Using a four-color LIF detector, 265 bases could be read in 30 min with a 267-amino acid drag-tag, on par with the average read of current next-gen sequencing systems. New types of multichannel systems that allow much higher throughput electrophoretic sequencing should be much more accessible in the absence of a requirement for viscous separation matrix.
authors.affiliation: Department of Chemical Engineering, Stanford University, 318 Campus Drive, W300B James H. Clark Center, Stanford, California 94305-5444, USA.
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: D000595:Amino Acid Sequence; D004247:DNA / Q000737:chemistry; D019075:Electrophoresis, Capillary / Q000379:methods*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA / Q000379:methods*
chemical_list: D011994:Recombinant Proteins; D004247:DNA