Color-blind fluorescence detection for four-color DNA sequencing

doi:10.1073/pnas.0501606102

Color-blind fluorescence detection for four-color DNA sequencing

^*Human Genome Sequencing Center, ^†Department of Molecular and Human Genetics, ^§Cell and Molecular Biology Program, Baylor College of Medicine, Houston, TX 77030; and ^¶Department of Chemistry and Rice Quantum Institute, Rice University, Houston, TX 77005

Contributed by Robert F. Curl, February 28, 2005

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.

Footnotes

↵ ‡‡ To whom correspondence should be addressed at: Baylor College of Medicine, One Baylor Plaza, N1409, Houston, TX 77030. E-mail: mmetzker{at}bcm.tmc.edu.
↵ ‡ Present address: LaserGen, Inc., Houston, TX 77054.
↵ ∥ Present address: University of Illinois at Urbana–Champaign, Urbana, IL 61801.
↵ ** Present address: Yale University, New Haven, CT 06520.
↵ †† Present address: Sigma-Aldrich Biotechnology, St. Louis, MO 63106.
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.
Abbreviations: AF, Alexa Fluor; CE, capillary electrophoresis; PME, pulsed multiline excitation; RF, radio frequency.