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. 1997 Nov 15;25(22):4500–4504. doi: 10.1093/nar/25.22.4500

New dye-labeled terminators for improved DNA sequencing patterns.

B B Rosenblum 1, L G Lee 1, S L Spurgeon 1, S H Khan 1, S M Menchen 1, C R Heiner 1, S M Chen 1
PMCID: PMC147091  PMID: 9358158

Abstract

We have used two new dye sets for automated dye-labeled terminator DNA sequencing. One set consists of four, 4,7-dichlororhodamine dyes (d-rhodamines). The second set consists of energy-transfer dyes that use the 5-carboxy-d-rhodamine dyes as acceptor dyes and the 5- or 6-carboxy isomers of 4'-aminomethylfluorescein as the donor dye. Both dye sets utilize a new linker between the dye and the nucleotide, and both provide more even peak heights in terminator sequencing than the dye-terminators consisting of unsubstituted rhodamine dyes. The unsubstituted rhodamine terminators produced electropherograms in which weak G peaks are observed after A peaks and occasionally C peaks. The number of weak G peaks has been reduced or eliminated with the new dye terminators. The general improvement in peak evenness improves accuracy for the automated base-calling software. The improved signal-to-noise ratio of the energy-transfer dye-labeled terminators combined with more even peak heights results in successful sequencing of high molecular weight DNA templates such as bacterial artificial chromosome DNA.

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Selected References

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  1. Bult C. J., White O., Olsen G. J., Zhou L., Fleischmann R. D., Sutton G. G., Blake J. A., FitzGerald L. M., Clayton R. A., Gocayne J. D. Complete genome sequence of the methanogenic archaeon, Methanococcus jannaschii. Science. 1996 Aug 23;273(5278):1058–1073. doi: 10.1126/science.273.5278.1058. [DOI] [PubMed] [Google Scholar]
  2. Fleischmann R. D., Adams M. D., White O., Clayton R. A., Kirkness E. F., Kerlavage A. R., Bult C. J., Tomb J. F., Dougherty B. A., Merrick J. M. Whole-genome random sequencing and assembly of Haemophilus influenzae Rd. Science. 1995 Jul 28;269(5223):496–512. doi: 10.1126/science.7542800. [DOI] [PubMed] [Google Scholar]
  3. Gibbs R. A., Nguyen P. N., Edwards A., Civitello A. B., Caskey C. T. Multiplex DNA deletion detection and exon sequencing of the hypoxanthine phosphoribosyltransferase gene in Lesch-Nyhan families. Genomics. 1990 Jun;7(2):235–244. doi: 10.1016/0888-7543(90)90545-6. [DOI] [PubMed] [Google Scholar]
  4. Hawkins T. L., McKernan K. J., Jacotot L. B., MacKenzie J. B., Richardson P. M., Lander E. S. A magnetic attraction to high-throughput genomics. Science. 1997 Jun 20;276(5320):1887–1889. doi: 10.1126/science.276.5320.1887. [DOI] [PubMed] [Google Scholar]
  5. 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]
  6. Hunkapiller T., Kaiser R. J., Koop B. F., Hood L. Large-scale and automated DNA sequence determination. Science. 1991 Oct 4;254(5028):59–67. doi: 10.1126/science.1925562. [DOI] [PubMed] [Google Scholar]
  7. 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]
  8. 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]
  9. 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]
  10. Lee L. G., Connell C. R., Woo S. L., Cheng R. D., McArdle B. F., Fuller C. W., Halloran N. D., Wilson R. K. DNA sequencing with dye-labeled terminators and T7 DNA polymerase: effect of dyes and dNTPs on incorporation of dye-terminators and probability analysis of termination fragments. Nucleic Acids Res. 1992 May 25;20(10):2471–2483. doi: 10.1093/nar/20.10.2471. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Lee L. G., Spurgeon S. L., Heiner C. R., Benson S. C., Rosenblum B. B., Menchen S. M., Graham R. J., Constantinescu A., Upadhya K. G., Cassel J. M. New energy transfer dyes for DNA sequencing. Nucleic Acids Res. 1997 Jul 15;25(14):2816–2822. doi: 10.1093/nar/25.14.2816. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. 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]
  13. 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]
  14. 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]
  15. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Smith L. M., Fung S., Hunkapiller M. W., Hunkapiller T. J., Hood L. E. The synthesis of oligonucleotides containing an aliphatic amino group at the 5' terminus: synthesis of fluorescent DNA primers for use in DNA sequence analysis. Nucleic Acids Res. 1985 Apr 11;13(7):2399–2412. doi: 10.1093/nar/13.7.2399. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. 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]
  18. Tabor S., Richardson C. C. A single residue in DNA polymerases of the Escherichia coli DNA polymerase I family is critical for distinguishing between deoxy- and dideoxyribonucleotides. Proc Natl Acad Sci U S A. 1995 Jul 3;92(14):6339–6343. doi: 10.1073/pnas.92.14.6339. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Tabor S., Richardson C. C. DNA sequence analysis with a modified bacteriophage T7 DNA polymerase. Effect of pyrophosphorolysis and metal ions. J Biol Chem. 1990 May 15;265(14):8322–8328. [PubMed] [Google Scholar]
  20. Voss H., Schwager C., Wirkner U., Sproat B., Zimmermann J., Rosenthal A., Erfle H., Stegemann J., Ansorge W. Direct genomic fluorescent on-line sequencing and analysis using in vitro amplification of DNA. Nucleic Acids Res. 1989 Apr 11;17(7):2517–2527. doi: 10.1093/nar/17.7.2517. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Wilson R., Ainscough R., Anderson K., Baynes C., Berks M., Bonfield J., Burton J., Connell M., Copsey T., Cooper J. 2.2 Mb of contiguous nucleotide sequence from chromosome III of C. elegans. Nature. 1994 Mar 3;368(6466):32–38. doi: 10.1038/368032a0. [DOI] [PubMed] [Google Scholar]

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