Color-blind fluorescence detection for four-color DNA sequencing

Ernest K. Lewis, Wade C. Haaland, Freddy Nguyen, Daniel A. Heller, Matthew J. Allen, Robert R. MacGregor, C. Scott Berger, Britain Willingham, Lori A. Burns, Graham B.I. Scott, Carter Kittrell, Bruce R. Johnson, Robert F. Curl, Michael L. Metzker

Research output: Contribution to journalArticlepeer-review

42 Scopus citations

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.

Original languageEnglish
Pages (from-to)5346-5351
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume102
Issue number15
DOIs
StatePublished - 12 Apr 2005
Externally publishedYes

Keywords

  • Fluorescent detection
  • Instrumentation

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