title: Internal fluorescence labeling with fluorescent deoxynucleotides in two-label peak-height encoded DNA sequencing by capillary electrophoresis.
abstract: Fluorescently labeled deoxynucleotides were used for internal labeling of DNA sequencing fragments generated in a two-color peak-height encoded protocol. Sequenase and a manganese-containing buffer were used to generate uniform peak heights. Tetramethyl rhodamine - dATP was used in a labeling step, followed by termination with ddATP and ddCTP in a 3:1 ratio. Fluorescein - dATP was used in a second reaction, followed by termination with ddGTP and ddTTP in a 3:1 ratio. The fragments were pooled and separated by capillary gel electrophoresis. The results were compared with peak-height encoded sequencing based on fluorescently labeled primers. The dye-labeled primers produced higher resolution separations for shorter fragments. However, dye-labeled primer fragments suffered from an earlier onset of biased reptation and produced shorter sequencing reads. Fragments from 50 to at least 500 bases in length could be sequenced with the internal labels.
authors.affiliation: Department of Chemistry, University of Alberta, Edmonton, Canada.
authors.forename: H R
authors.initials: HR
authors.lastname: Starke
authors.forename: J Y
authors.initials: JY
authors.lastname: Yan
authors.forename: J Z
authors.initials: JZ
authors.lastname: Zhang
authors.forename: K
authors.initials: K
authors.lastname: Mühlegger
authors.forename: K
authors.initials: K
authors.lastname: Effgen
authors.forename: N J
authors.initials: NJ
authors.lastname: Dovichi
mesh_terms: D002197:Capillary Action; D003838:Deoxyadenine Nucleotides; D003854:Deoxyribonucleotides; D004586:Electrophoresis; D019793:Fluorescein; D005452:Fluoresceins; D005456:Fluorescent Dyes; D007834:Lasers; D012235:Rhodamines; D017422:Sequence Analysis, DNA / Q000379:methods*; D013050:Spectrometry, Fluorescence
chemical_list: D003838:Deoxyadenine Nucleotides; D003854:Deoxyribonucleotides; D005452:Fluoresceins; D005456:Fluorescent Dyes; D012235:Rhodamines; C005358:tetramethylrhodamine; C026600:2'-deoxyadenosine triphosphate; D019793:Fluorescein