Color-blind fluorescence detection for four-color DNA sequencing.
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.
Lewis
Ernest K
EK
Human Genome Sequencing Center, Department of Molecular and Human Genetics, Cell and Molecular Biology Program, Baylor College of Medicine, Houston, TX 77030.
Haaland
Wade C
WC
Nguyen
Freddy
F
Heller
Daniel A
DA
Allen
Matthew J
MJ
MacGregor
Robert R
RR
Berger
C Scott
CS
Willingham
Britain
B
Burns
Lori A
LA
Scott
Graham B I
GB
Kittrell
Carter
C
Johnson
Bruce R
BR
Curl
Robert F
RF
Metzker
Michael L
ML
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*
D005456:Fluorescent Dyes