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 absorpt...

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Published inProceedings of the National Academy of Sciences - PNAS Vol. 102; no. 15; pp. 5346 - 5351
Main Authors Lewis, Ernest K., Haaland, Wade C., Nguyen, Freddy, Heller, Daniel A., Allen, Matthew J., MacGregor, Robert R., Berger, C. Scott, Willingham, Britain, Burns, Lori A., Graham B. I. Scott, Kittrell, Carter, Johnson, Bruce R., Curl, Robert F., Metzker, Michael L.
Format Journal Article
LanguageEnglish
Published United States National Academy of Sciences 12.04.2005
National Acad Sciences
Subjects
Base Sequence
Color
Color blindness
Deoxyribonucleic acid
DNA
Dyes
Electrophoresis
Emission spectra
Fluorescence
Fluorescent Dyes - analysis
Fluorescent Dyes - chemistry
Laser beams
Laser power
Laser spectroscopy
Lasers
Measurement techniques
Physical Sciences
Reproducibility of Results
Sensitivity and Specificity
Sequence Analysis, DNA - instrumentation
Sequence Analysis, DNA - methods
Sequencing
Waveforms
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Summary: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.
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Present address: Yale University, New Haven, CT 06520.
Author contributions: E.K.L., F.N., D.A.H., G.B.I.S., C.K., B.R.J., R.F.C., and M.L.M. designed research; E.K.L., W.C.H., F.N., D.A.H., M.J.A., R.R.M., C.S.B., B.W., L.A.B., and C.K. performed research; W.C.H., C.K., B.R.J., R.F.C., and M.L.M. contributed new reagents/analytic tools; E.K.L., W.C.H., C.K., B.R.J., R.F.C., and M.L.M. analyzed data; and C.K., B.R.J., R.F.C., and M.L.M. wrote the paper.
To whom correspondence should be addressed at: Baylor College of Medicine, One Baylor Plaza, N1409, Houston, TX 77030. E-mail: mmetzker@bcm.tmc.edu.
Contributed by Robert F. Curl, February 28, 2005
Present address: LaserGen, Inc., Houston, TX 77054.
Present address: University of Illinois at Urbana–Champaign, Urbana, IL 61801.
Present address: Sigma-Aldrich Biotechnology, St. Louis, MO 63106.
Abbreviations: AF, Alexa Fluor; CE, capillary electrophoresis; PME, pulsed multiline excitation; RF, radio frequency.
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.0501606102