title: Use of non-cross-linked polyacrylamide for four-color DNA sequencing by capillary electrophoresis separation of fragments up to 640 bases in length in two hours.
abstract: Four-color DNA cycle sequencing was performed on an M13mp18 template using dye-labeled primers. Sequencing fragments were separated by capillary electrophoresis at 60 degrees C and at an electric field of 150 V/cm. The sieving medium was 5%T, non-cross-linked polyacrylamide in 7 M urea. The use of high temperature for the separation reduces formation of secondary structures in the sequencing fragments, generating a sequence that is free of compressions without the use of strongly denaturing gels. The use of high temperatures also increases the separation rate compared with room-temperature operation. Fragments up to 640 bases are separated in less than 2 h.
authors.affiliation: Department of Chemistry, University of Alberta, Edmonton, Canada.
authors.forename: J
authors.initials: J
authors.lastname: Zhang
authors.forename: Y
authors.initials: Y
authors.lastname: Fang
authors.forename: J Y
authors.initials: JY
authors.lastname: Hou
authors.forename: H J
authors.initials: HJ
authors.lastname: Ren
authors.forename: R
authors.initials: R
authors.lastname: Jiang
authors.forename: P
authors.initials: P
authors.lastname: Roos
authors.forename: N J
authors.initials: NJ
authors.lastname: Dovichi
mesh_terms: D001483:Base Sequence; D004247:DNA / Q000737:chemistry*; D017931:DNA Primers / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D012015:Reference Standards; D017422:Sequence Analysis, DNA / Q000379:methods*
chemical_list: D017931:DNA Primers; D004247:DNA