Color-blind fluorescence detection for four-color DNA sequencing.

TitleColor-blind fluorescence detection for four-color DNA sequencing.
Publication TypeJournal Article
Year of Publication2005
AuthorsLewis, Ernest K., Haaland Wade C., Nguyen Freddy, Heller Daniel A., Allen Matthew J., MacGregor Robert R., C Berger Scott, Willingham Britain, Burns Lori A., Scott Graham B. I., Kittrell Carter, Johnson Bruce R., Curl Robert F., and Metzker Michael L.
JournalProc Natl Acad Sci U S A
Volume102
Issue15
Pagination5346-51
Date Published2005 Apr 12
ISSN0027-8424
KeywordsBase Sequence, Color, Fluorescence, Fluorescent Dyes, Reproducibility of Results, Sensitivity and Specificity, Sequence Analysis, DNA
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.

DOI10.1073/pnas.0501606102
Alternate JournalProc. Natl. Acad. Sci. U.S.A.
PubMed ID15800037
PubMed Central IDPMC556273
Grant ListR21 HG002443 / HG / NHGRI NIH HHS / United States
R41 HG003265 / HG / NHGRI NIH HHS / United States
1R21 HG 002443 / HG / NHGRI NIH HHS / United States
1R41 HG 003265 / HG / NHGRI NIH HHS / United States