title: A 502-Base Free-Solution Electrophoretic DNA Sequencing Method Using End-Attached Wormlike Micelles.
abstract: We demonstrate that the use of wormlike nonionic micelles as drag-tags in end-labeled free-solution electrophoresis ("micelle-ELFSE") provides single-base resolution of Sanger sequencing products up to 502 bases in length, a nearly 2-fold improvement over reported ELFSE separations. "CiEj" running buffers containing 48 mM C12E5, 6 mM C10E5, and 3 M urea (32.5 °C) form wormlike micelles that provide a drag equivalent to an uncharged DNA fragment with a length (α) of 509 bases (effective Rh = 27 nm). Runtime in a 40 cm capillary (30 kV) was 35 min for elution of all products down to the 26-base primer. We also show that smaller Triton X-100 micelles give a read length of 103 bases in a 4 min run, so that a combined analysis of the Sanger products using the two buffers in separate capillaries could be completed in 14 min for the full range of lengths. A van Deemter analysis shows that resolution is limited by diffusion-based peak broadening and wall adsorption. Effects of drag-tag polydispersity are not observed, despite the inherent polydispersity of the wormlike micelles. We ascribe this to a stochastic size-sampling process that occurs as micelle size fluctuates rapidly during the runtime. A theoretical model of the process suggests that fluctuations occur with a time scale less than 10 ms, consistent with the monomer exchange process in nonionic micelles. The CiEj buffer has a low viscosity (2.7 cP) and appears to be semidilute in micelle concentration. The large drag-tag size of the CiEj buffers leads to steric segregation of the DNA and tag for short fragments and attendant mobility shifts.
authors.affiliation: Department of Chemical Engineering and ‡Department of Biomedical Engineering, Carnegie Mellon University , Pittsburgh, Pennsylvania 15213, United States.
authors.forename: Stephen B
authors.initials: SB
authors.lastname: Istivan
authors.affiliation: Department of Chemical Engineering and ‡Department of Biomedical Engineering, Carnegie Mellon University , Pittsburgh, Pennsylvania 15213, United States.
authors.forename: Daniel K
authors.initials: DK
authors.lastname: Bishop
authors.affiliation: Department of Chemical Engineering and ‡Department of Biomedical Engineering, Carnegie Mellon University , Pittsburgh, Pennsylvania 15213, United States.
authors.forename: Angela L
authors.initials: AL
authors.lastname: Jones
authors.affiliation: Department of Chemical Engineering and ‡Department of Biomedical Engineering, Carnegie Mellon University , Pittsburgh, Pennsylvania 15213, United States.
authors.forename: Shane T
authors.initials: ST
authors.lastname: Grosser
authors.affiliation: Department of Chemical Engineering and ‡Department of Biomedical Engineering, Carnegie Mellon University , Pittsburgh, Pennsylvania 15213, United States.
authors.forename: James W
authors.initials: JW
authors.lastname: Schneider
mesh_terms: D002021:Buffers; D004247:DNA / Q000235:genetics; D019075:Electrophoresis, Capillary; D008823:Micelles; D017422:Sequence Analysis, DNA / Q000379:methods*; D012996:Solutions
chemical_list: D002021:Buffers; D008823:Micelles; D012996:Solutions; D004247:DNA