title: Efficient incorporation of positively charged 2', 3'-dideoxynucleoside-5'-triphosphates by DNA polymerases and their application in 'direct-load' DNA sequencing.
abstract: A series of charge-modified, dye-labeled 2', 3'-dideoxynucleoside-5'-triphosphates have been synthesized and evaluated as reagents for dye-terminator DNA sequencing. Unlike the commonly used dye-labeled terminators, these terminators possess a net positive charge and migrate in the opposite direction to dye-labeled Sanger fragments during electrophoresis. Post-sequencing reaction purification is not required to remove unreacted nucleotide or associated breakdown products prior to electrophoresis. Thus, DNA sequencing reaction mixtures can be loaded directly onto a separating medium such as a sequencing gel. The charge-modified nucleotides have also been shown to be more efficiently incorporated by a number of DNA polymerases than regular dye-labeled dideoxynucleotide terminators or indeed normal dideoxynucleoside-5'-triphosphates.
authors.affiliation: Amersham Biosciences, 800 Centennial Avenue, Piscataway, NJ 08855-1327, USA.
authors.forename: Patrick J
authors.initials: PJ
authors.lastname: Finn
authors.forename: Matthew G
authors.initials: MG
authors.lastname: Bull
authors.forename: Haiguang
authors.initials: H
authors.lastname: Xiao
authors.forename: Paula D
authors.initials: PD
authors.lastname: Phillips
authors.forename: John R
authors.initials: JR
authors.lastname: Nelson
authors.forename: Greg
authors.initials: G
authors.lastname: Grossmann
authors.forename: Satyam
authors.initials: S
authors.lastname: Nampalli
authors.forename: Bernard F
authors.initials: BF
authors.lastname: McArdle
authors.forename: J Anthony
authors.initials: JA
authors.lastname: Mamone
authors.forename: Parke K
authors.initials: PK
authors.lastname: Flick
authors.forename: Carl W
authors.initials: CW
authors.lastname: Fuller
authors.forename: Shiv
authors.initials: S
authors.lastname: Kumar
mesh_terms: D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D004396:Coloring Agents / Q000737:chemistry; D004247:DNA / Q000737:chemistry / Q000235:genetics; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D015224:Dideoxynucleosides / Q000737:chemistry*; D008239:Lysine / Q000031:analogs & derivatives* / Q000737:chemistry; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009711:Nucleotides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000379:methods*
chemical_list: D004396:Coloring Agents; D015224:Dideoxynucleosides; D009711:Nucleotides; C003712:trimethyllysine; D004247:DNA; D004259:DNA-Directed DNA Polymerase; D008239:Lysine