Abstract
We have synthesized a set of four energy transfer dyes and demonstrated their use in automated DNA sequencing. The donor dyes are the 5- or 6-carboxy isomers of 4'-aminomethylfluorescein and the acceptor dyes are a novel set of four 4,7-dichloro-substituted rhodamine dyes which have narrower emission spectra than the standard, unsubstituted rhodamines. A rigid amino acid linker, 4-aminomethylbenzoic acid, was used to separate the dyes. The brightness of each dye in an automated sequencing instrument equipped with a dual line argon ion laser (488 and 514 nm excitation) was 2-2.5 times greater than the standard dye-primers with a 2 times reduction in multicomponent noise. The overall improvement in signal-to-noise was 4- to 5-fold. The utility of the new dye set was demonstrated by sequencing of a BAC DNA with an 80 kb insert. Measurement of the extinction coefficients and the relative quantum yields of the dichlororhodamine components of the energy transfer dyes showed their values were reduced by 20-25% compared with the dichlororhodamine dyes alone.
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- Hung S. C., Ju J., Mathies R. A., Glazer A. N. Cyanine dyes with high absorption cross section as donor chromophores in energy transfer primers. Anal Biochem. 1996 Dec 1;243(1):15–27. doi: 10.1006/abio.1996.0477. [DOI] [PubMed] [Google Scholar]
- Ju J., Glazer A. N., Mathies R. A. Cassette labeling for facile construction of energy transfer fluorescent primers. Nucleic Acids Res. 1996 Mar 15;24(6):1144–1148. doi: 10.1093/nar/24.6.1144. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ju J., Kheterpal I., Scherer J. R., Ruan C., Fuller C. W., Glazer A. N., Mathies R. A. Design and synthesis of fluorescence energy transfer dye-labeled primers and their application for DNA sequencing and analysis. Anal Biochem. 1995 Oct 10;231(1):131–140. doi: 10.1006/abio.1995.1512. [DOI] [PubMed] [Google Scholar]
- Ju J., Ruan C., Fuller C. W., Glazer A. N., Mathies R. A. Fluorescence energy transfer dye-labeled primers for DNA sequencing and analysis. Proc Natl Acad Sci U S A. 1995 May 9;92(10):4347–4351. doi: 10.1073/pnas.92.10.4347. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Marra M., Weinstock L. A., Mardis E. R. End sequence determination from large insert clones using energy transfer fluorescent primers. Genome Res. 1996 Nov;6(11):1118–1122. doi: 10.1101/gr.6.11.1118. [DOI] [PubMed] [Google Scholar]
- Metzker M. L., Lu J., Gibbs R. A. Electrophoretically uniform fluorescent dyes for automated DNA sequencing. Science. 1996 Mar 8;271(5254):1420–1422. doi: 10.1126/science.271.5254.1420. [DOI] [PubMed] [Google Scholar]
- Mineno J., Ishino Y., Ohminami T., Kato I. Fluorescent labeling of a DNA sequencing primer. DNA Seq. 1993;4(3):135–141. doi: 10.3109/10425179309015626. [DOI] [PubMed] [Google Scholar]
- Prober J. M., Trainor G. L., Dam R. J., Hobbs F. W., Robertson C. W., Zagursky R. J., Cocuzza A. J., Jensen M. A., Baumeister K. A system for rapid DNA sequencing with fluorescent chain-terminating dideoxynucleotides. Science. 1987 Oct 16;238(4825):336–341. doi: 10.1126/science.2443975. [DOI] [PubMed] [Google Scholar]
- Shipchandler M. T., Fino J. R., Klein L. D., Kirkemo C. L. 4'-[Aminomethyl]fluorescein and its N-alkyl derivatives: useful reagents in immunodiagnostic techniques. Anal Biochem. 1987 Apr;162(1):89–101. doi: 10.1016/0003-2697(87)90013-3. [DOI] [PubMed] [Google Scholar]
- Smith L. M., Sanders J. Z., Kaiser R. J., Hughes P., Dodd C., Connell C. R., Heiner C., Kent S. B., Hood L. E. Fluorescence detection in automated DNA sequence analysis. Nature. 1986 Jun 12;321(6071):674–679. doi: 10.1038/321674a0. [DOI] [PubMed] [Google Scholar]