title: Color-blind fluorescence detection for four-color DNA sequencing.
abstract: We present an approach called pulsed multiline excitation (PME) for measurements of multicomponent, fluorescence species and demonstrate its application in capillary electrophoresis for DNA sequencing. To fully demonstrate the advantages of PME, a fluorescent dye set has been developed whose absorption maxima span virtually the entire visible spectrum. Unlike emission wavelength-dependent approaches for identifying fluorescent species, the removal of the spectral component in PME confers a number of advantages including higher and normalized signals from all dyes present in the assay, the elimination of spectral cross-talk between dyes, and higher signal collection efficiency. Base-calling is unambiguously determined once dye mobility corrections are made. These advantages translate into significantly enhanced signal quality as illustrated in the primary DNA sequencing data and provide a means for achieving accurate base-calling at lower reagent concentrations.
authors.affiliation: Human Genome Sequencing Center, Department of Molecular and Human Genetics, Cell and Molecular Biology Program, Baylor College of Medicine, Houston, TX 77030.
authors.forename: Ernest K
authors.initials: EK
authors.lastname: Lewis
authors.forename: Wade C
authors.initials: WC
authors.lastname: Haaland
authors.forename: Freddy
authors.initials: F
authors.lastname: Nguyen
authors.forename: Daniel A
authors.initials: DA
authors.lastname: Heller
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: Allen
authors.forename: Robert R
authors.initials: RR
authors.lastname: MacGregor
authors.forename: C Scott
authors.initials: CS
authors.lastname: Berger
authors.forename: Britain
authors.initials: B
authors.lastname: Willingham
authors.forename: Lori A
authors.initials: LA
authors.lastname: Burns
authors.forename: Graham B I
authors.initials: GB
authors.lastname: Scott
authors.forename: Carter
authors.initials: C
authors.lastname: Kittrell
authors.forename: Bruce R
authors.initials: BR
authors.lastname: Johnson
authors.forename: Robert F
authors.initials: RF
authors.lastname: Curl
authors.forename: Michael L
authors.initials: ML
authors.lastname: Metzker
mesh_terms: D001483:Base Sequence; D003116:Color; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000032:analysis* / Q000737:chemistry; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000295:instrumentation* / Q000379:methods*
chemical_list: D005456:Fluorescent Dyes