During the past 12 years, there have been remarkable advances in the use of fluorescence to study DNA. Fluorescence methods are now used for DNA sequencing, detection of DNA hybridization and restriction enzyme fragments, fluorescence in situ hybridization (FISH), and quantitating polymerase chain reaction products. Because of the rapid introduction of new DNA technology, it is surprising to realize that DNA sequencing by fluorescence was first reported just 12 years ago, in 1986. It is not the purpose of this chapter to describe the many specialized methods used in this extensive area of molecular biology and diagnostics. Instead, we give a brief introduction to each topic, followed by a description of the unique fluorophores and principles used for each application.


Fluorescent Primer Thiazole Orange Pulse Laser Diode Thiazole Orange Pyrene Monomer 
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  1. 1.
    Maxam, A. M., and Gilbert, W., 1977, A new method for sequencing DNA, Proc. Natl. Acad. Sci. U.S.A. 74: 560–564.CrossRefGoogle Scholar
  2. 2.
    Maxam, A. M., and Gilbert, W., 1980, Sequencing end-labeled DNA with base-specific chemical cleavage, Methods Enzymol. 65: 499–560.CrossRefGoogle Scholar
  3. 3.
    Sanger, F., Nicklen, S., and Coulson, A. R., 1977, DNA sequencing with chain-terminating inhibitors, Proc. Natl. Acad. Sei. U.S.A. 74: 5463–5467.CrossRefGoogle Scholar
  4. 4.
    Watson, J. D., Gilman, M., Witkowski, J., and Zoller, M., 1992, Recombinant DNA, 2nd ed., Scientific American Books, New York.Google Scholar
  5. 5.
    Smith, L. M., Sanders, J. Z., Kaiser, R. J., Hughes, P., Dodd, C., Connell, C. R., Heiner, C., Kent. S. B. H., and Hood, L. E., 1986, Fluorescence detection in automated DNA sequence analysis, Nature 321: 674–679.Google Scholar
  6. 6.
    Prober, J. M., Trainor, G. L., Dam, R. J., Hobbs, F. W., Robertson, C. W., Zagursky, R. J., Cocuzza, A. J., Jensen, M. A., and Baumeister, K., 1987, A system for rapid DNA sequencing with fluorescent chain-terminating dideoxynucleotides, Science 238: 336–343.CrossRefGoogle Scholar
  7. 7.
    Ansorge, W., Sproat, B. S., Stegemann, J., and Schwager, C., 1986, A non-radioactive automated method for DNA sequence determination, J. Biochem. Biophys. Methods 13: 315–323.CrossRefGoogle Scholar
  8. 8.
    Flick, P. K., 1995, DNA sequencing by nonisotopic methods, in Nonisotopic Probing, Blotting, and Sequencing, J. J. Kricka (ed.), Academic Press, New York, pp. 475–492.Google Scholar
  9. 9.
    Dhadwal, H. S., Quesada, M. A., and Studier, F. W., 1997, DNA sequencing by multiple capillaries that form a waveguide, Proc. SPIE 2890: 149–162.CrossRefGoogle Scholar
  10. 10.
    Hunkapiller, T., Kaiser, R. J., Koop, B. F., and Hood, L., 1991, Large-scale and automated DNA sequence determination, Science 254: 59–67.CrossRefGoogle Scholar
  11. 11.
    Zimmermann, J., Wiemann, S., Voss, H., Schwager, C., and Ansorge, W., 1994, Improved fluorescent cycle sequencing protocol allows reading nearly 1000 bases, Biotechniques 17 (2): 302–307.Google Scholar
  12. 12.
    Ansorge, W., Sproat, B., Stegermann, J., Schwager, C, and Zenke, M., 1987, Automated DNA sequencing: Ultrasensitive detection of fluorescent bands during electrophoresis, Nucleic Acids Res. 15: 4593–4602.CrossRefGoogle Scholar
  13. 13.
    Brumbaugh, J. A., Middendorf, L. R., Grone, D. L., and Ruth, J. L., 1988, Continuous on-line DNA sequencing using oligodeoxynu-cleotide primers with multiple fluorophores, Proc. Natl. Acad. Sei U.S.A. 85: 5610–5614.CrossRefGoogle Scholar
  14. 14.
    Ju, J., Ruan, C., Fuller, C. W., Glazer, A. N., and Mathies, R. A., 1995, Fluorescence energy transfer dye-labeled primers for DNA sequencing and analysis, Proc. Natl. Acad. Sei, U.S.A. 92: 4347–4351.CrossRefGoogle Scholar
  15. 15.
    Ju, J., Glazer, A. N., and Mathies, R. A., 1996, Energy transfer primers: A new fluorescence labeling paradigm for DNA sequencing and analysis, Nat. Med. 2 (2): 246–249.CrossRefGoogle Scholar
  16. 16.
    Takahashi, S., Murakami, K., Anazawa, T., and Kambara, H., 1994, Multiple sheath-flow gel capillary-array electrophoresis for multicolor fluorescent DNA detection, Anal. Chem. 66: 1021–1026.CrossRefGoogle Scholar
  17. 17.
    Ju, J., Kheterpal, I., Scherer, J. R., Ruan, C., Fuller, C. W., Glazer, A. N., and Mathies, R. A., 1995, Design and synthesis of fluorescence energy transfer dye-labeled primers and their application for DNA sequencing and analysis, Anal. Biochem. 231: 131–140.CrossRefGoogle Scholar
  18. 18.
    Metzker, M. L., Lu, J., and Gibbs, R. A., 1996, Electrophoretically uniform fluorescent dyes for automated DNA sequencing, Science 271: 1420–1422.CrossRefGoogle Scholar
  19. 19.
    Middendorf, L. R., Bruce, J. C., Bruce, R. C., Eckles, R. D., Roemer, S. C., and Sloniker, G. D., 1993, A versatile infrared laser scan-ner/electrophoresis apparatus, Proc. SPIE 1885: 423–434.CrossRefGoogle Scholar
  20. 20.
    Soper, S. A., Flanagan, J. H., Legendre, B. L., Williams, D. C., and Hammer, R. P., 1996, Near-infrared, laser-induced fluorescence detection for DNA sequencing applications, IEEE J. Sei. Top. Quantum Electron. 2 (4): 1–11.Google Scholar
  21. 21.
    Shealy, D. B., Lipowska, M., Lipowski, J., Narayanan, N., Sutter, S., Strekowski, L., and Patonay, G., 1995, Synthesis, chromatographicGoogle Scholar
  22. separation, and characterization of near-infrared labeled DNA oligomers for use in DNA sequencing, Anal. Chem. 67:247–251.Google Scholar
  23. 22.
    Williams, D. C., and Soper, S. A., 1995, Ultrasensitive near-IR fluorescence detection for capillary gel electrophoresis and DNA sequencing applications, Anal. Chem. 67: 3427–3432.CrossRefGoogle Scholar
  24. 23.
    Middendorf, L., Amen, J., Bruce, B., Draney, D., DeGraff, D., Gewecke, J., Grone, D., Humphrey, P., Little, G., Lugade, A., Narayanan, N., Oommen, A., Osterman, H., Peterson, R., Rada, J., Raghavachari, R., and Roemer, S., 1998, Near-infrared fluorescence instrumentation for DNA analysis, S. Daehne et al. (eds.), Near-Infrared Dyes for High Technology Applications, 21–54. © 1998 Kluwer Academic Publishers. Printed in the Netherlands.Google Scholar
  25. 24.
    Middendorf, L. R., Bruce, J. C., Bruce, R. C., Eckles, R. D., Grone, D. L., Roemer, S. C., Sloniker, G. D., Steffens, D. L., Sutter, S. L., Brumbaugh, J. A., and Patonay, G., 1992, Continuous, on-line DNA sequencing using a versatile infrared laser scanner/electrophoresis apparatus, Electrophoresis 13: 487–494.CrossRefGoogle Scholar
  26. 25.
    Middendorf, L., Bruce, R., Brumbaugh, J., Grone, D., Jang, G., Richterich, P., Holtke, H. J., Williams, R. J., and Peralta, J. M., 1995, A two-dimensional infrared fluorescence scanner used for DNA analysis, Proc. SPIE 2388: 44–54.CrossRefGoogle Scholar
  27. 26.
    Han, K.-T., Sauer, M., Schulz, A., Seeger, S., and Wolfram, J., 1993, Time-resolved fluorescence studies of labelled nucleosides, Ber. Bunsenges. Phys. Chem. 97: 1728–1730.CrossRefGoogle Scholar
  28. 27.
    Legendre, B. L., Williams, D. C., Soper, S. A., Erdmann, R., Ort-mann, U., and Enderlein, J., 1996, An all solid-state near-infrared time-correlated single photon counting instrument for dynamic lifetime measurements in DNA sequencing applications, Rev. Sei. Instrum. 67: 3984–3989.CrossRefGoogle Scholar
  29. 28.
    Chang, K., and Force, R. K., 1993, Time-resolved laser-induced fluorescence study on dyes used in DNA sequencing, Appl. Spec-trosc. 47: 24–29.CrossRefGoogle Scholar
  30. 29.
    Sauer, M., Han, K.-T., Ebert, V., Müller, R. Schulz, A., Seeger, S., and Wolfram, J., 1994, Design of multiplex dyes for the detection of different biomolecules, Proc. SPIE 2137: 762–774.Google Scholar
  31. 30.
    Li, L.-C., He, H., Nunnally, B. K., and McGown, L. B., 1997, On-the-fly fluorescence lifetime detection of labeled DNA primers, J. Chromatogr. 695: 85–92.CrossRefGoogle Scholar
  32. 31.
    Li, L.-C., and McGown, L. B., 1996, On-the-fly frequency-domain fluorescence lifetime detection in capillary electrophoresis, Anal. Chem. 68: 2737–2743.CrossRefGoogle Scholar
  33. 32.
    Le Pecq, J.-B., and Paoletti, C., 1967, A fluorescent complex between ethidium bromide and nucleic acids, J. Mol. Biol. 27: 87–106.CrossRefGoogle Scholar
  34. 33.
    Le Pecq, J.-B., Le Bret, M., Barbet, J, and Roques, B., 1975, DNA polyintercalating drugs: DNA binding of diacridine derivatives, Proc. Natl. Acad. Sei. U.S.A. 72: 2915–2919.CrossRefGoogle Scholar
  35. 34.
    Markovits, J., Roques, B. P., and Le Pecq, J.B., 1979, Ethidium dimer. A new reagent for the fluorimetric determination of nucleic acids, Anal. Biochem. 94: 259–264.CrossRefGoogle Scholar
  36. 35.
    Glazer, A. N., Peck, K., and Mathies, R. A., 1990, A stable double-stranded DNA ethidium homodimer complex: Application to pi-cogram fluorescence detection of DNA in agarose gels, Proc. Natl. Acad. Sei. U.S.A. 87: 3851–3855.CrossRefGoogle Scholar
  37. 36.
    Rye, H. S., Yue, S., Wemmer, D. E., Quesada, M. A., Haugland, R. P., Mathies, R. A., and Glazer, A. N., 1992, Stable fluorescent complexes of double-stranded DNA with bis-intercalating asymmetric cyanine dyes: Properties and applications, Nucleic Acids Res. 20: 2803–2812.CrossRefGoogle Scholar
  38. 37.
    Abramo, K. H., Pitner, J. B., and McGown, L. B., 1997, Spectroscopic studies of single-stranded DNA ligands and oxazole yellow dyes, Biospectroscopy 4: 27–35.CrossRefGoogle Scholar
  39. 38.
    Nygren, J., Svanvik, N., and Kubista, M., 1998, The interactions between the fluorescent dye thiazole orange and DNA, Biopolymers 46: 39–51.CrossRefGoogle Scholar
  40. 39.
    Benson, S. C., Mathies, R. A., and Glazer, A. N., 1993, Heterodi-meric DNA-binding dyes designed for energy transfer: Stability and applications of the DNA complexes, Nucleic Acids Res. 21: 5720–5726.CrossRefGoogle Scholar
  41. 40.
    Benson, S. C., Zeng, Z., and Glazer, A. N., 1995, Fluorescence energy-transfer cyanine heterodimers with high affinity for double-stranded DNA, Anal. Biochem. 231: 247–255.CrossRefGoogle Scholar
  42. 41.
    Goodwin, P. M., Johnson, M. E., Martin, J. C., Ambrose, W. P., Marrone, B. L., Jett, J. H., and Keller, R. A., 1993, Rapid sizing of individual fluorescently stained DNA fragments by flow cytometry, Nucleic Acids Res. 21: 803–806.CrossRefGoogle Scholar
  43. 42.
    Petty, J. T., Johnson, M. E., Goodwin, P. M., Martin, J. C., Jett, J. H., and Keller, R. A., 1995, Characterization of DNA size determination of small fragments by flow cytometry, Anal. Chem. 67: 1755–1761.CrossRefGoogle Scholar
  44. 43.
    Huang, Z., Petty, J. T., O’Quinn, B., Longmire, J. L., Brown, N. C., Jett, J. H., and Keller, R. A., 1996, Large DNA fragment sizing by flow cytometry: Application to the characterization of PI artificial chromosome (PAC) clones, Nucleic Acids Res. 24: 4202–4209.CrossRefGoogle Scholar
  45. 44.
    Cardullo, R. A., Agrawal, S., Flores, C., Zamecnik, P. C., and Wolf, D. E., 1988, Detection of nucleic acid hybridization by nonradiative fluorescence resonance energy transfer, Prvc. Natl. Acad. Sci. U.S.A. 85: 8790–8794.CrossRefGoogle Scholar
  46. 45.
    Morrison, L. E., and Stols, L. M., 1993, Sensitive fluorescence-based thermodynamic and kinetic measurements of DNA hybridization in solution, Biochemistry 32: 3095–3104.CrossRefGoogle Scholar
  47. 46.
    Morrison, L. E., 1995, Detection of energy transfer and fluorescence quenching, in Nonisotopic Probing, Blotting, and Sequencing, L. J. Kricka (ed.), Academic Press, New York, pp. 429–471.Google Scholar
  48. 47.
    Asseline, U., Toulme, F., Thuong, N. T., Delarue, M., Montenay- Garestier, T., and Helene, C., 1984, Oligodeoxynucleotides cova-lently linked to intercalating dyes as base sequence-specific ligands. Influence of dye attachment site, EMBO J. 3: 795–800.Google Scholar
  49. 48.
    Asseline, U., Delarue, M., Lancelot, G., Toulme, F., Thuong, N. T., Montenay-Garestier, T., and Helene, C., 1984, Nucleic acid-binding molecules with high affinity and base sequence specificity: Intercalating agents covalently linked to oligodeoxynucleotides, Proc. Natl. Acad. Sci. U.S.A. 81: 3297–3301.CrossRefGoogle Scholar
  50. 49.
    Hélène, C., Montenay-Garestier, T., Saison, T., Takasugi, M., Tolumé, Asseline, U., Lancelot, G., Maurizot, J. C., Tolumé, F., and Thuong, N. T., 1985, Oligodeoxynucleotides covalently linked to intercalating agents: A new class of gene regulatory substances, Biochime 67: 777–783.Google Scholar
  51. 50.
    Morrison, L. E., Haider, T. C., and Stols, L. M., 1989, Solution-phase detection of polynucleotides using interacting fluorescent labels and competitive hybridization, Anal. Biochem. 188: 231–244.CrossRefGoogle Scholar
  52. 51.
    Parkhurst, K. M., and Parkhurst, L. J., 1996, Detection of point mutations in DNA by fluorescence energy transfer, J. Biomed. Opt. 1: 435–441.CrossRefGoogle Scholar
  53. 52.
    Ebata, K., Masuko, M., Ohtani, H., and Kashiwasake-Jibu, M., 1995, Nucleic acid hybridization accompanied with excimer formation from two pyrene-labeled probes, Photochem. Photobiol. 62: 836–839.CrossRefGoogle Scholar
  54. 53.
    Devlin, R., Studholme, R. M., Dandliker, W. B., Fahy, E., Blumeyer, K., and Ghosh, S. S., 1993, Homogeneous detection of nucleic acids by transient state polarized fluorescence, Clin. Chem. 39: 1939–1943.Google Scholar
  55. 54.
    Murakami, A., Nakaura, M., Nakatsuji, Y., Nagahara, S., Tran-Cong, Q., and Makino, K., 1991, Fluorescent-labeled oligonucleotideGoogle Scholar
  56. probes: Detection of hybrid formation in solution by fluorescence polarization spectroscopy, Nucleic Acids Res. 19: 4097–4102.Google Scholar
  57. 55.
    Kumke, M. U., Shu, L., McGown, L. B., Walker, G. T., Pitner, J. B., and Linn, C. P., 1997, Temperature and quenching studies of fluorescence polarization detection of DNA hybridization, Anal. Chem. 69: 500–506.CrossRefGoogle Scholar
  58. 56.
    Livak, K. J., Flood, S. J. A., Marmaro, J., Giusti, W., and Deetz, K., 1995, Oligonucleotides with fluorescent dyes at opposite ends provide a quenched probe system useful for detecting PCR product and nucleic acid hybridization, PCR Methods Appli. 4: 357–362.CrossRefGoogle Scholar
  59. 57.
    Gibson, U. E. M., Heid, C. A., and Williams, P. M., 1996, A novel method for real time quantitative RT-PCR, Genome Res. 6:995-1001.Google Scholar
  60. 58.
    Steffens, D. L., Jang, G. Y., Sutter, S. L., Brumbaugh, J. A., Middendorf, L. R., Muhlegger, K., Mardis, E. R., Weinstock, L. A., and Wilson, R. K., 1995, An infrared fluorescent dATP for labeling DNA, Genome Res. 5: 393–399.CrossRefGoogle Scholar
  61. 59.
    Wittwer, C. T., Herrmann, M. G., Moss, A. A., and Rasmussen, R. P., 1997, Continuous fluorescence monitoring of rapid cycle DNA amplification, BioTechniques 22 (1): 130–138.Google Scholar
  62. 60.
    Pease, A. C., Solas, D., Sullivan, E. J., Cronin, M. T., Holmes, C. P., and Fodor, S. P. A., 1994, Light-generated oligonucleotide arrays for rapid DNA sequence analysis, Proc. Natl. Acad. Sci. U.S.A. 91: 5022–5026.CrossRefGoogle Scholar
  63. 61.
    Lipshutz, R. J., Morris, D., Chee, M., Hubbell, E., Kozal, M. J., Shah, N., Shen, N., Yang, R., and Foder, S. P. A., 1995, Using oligonucleotide probe arrays to access genetic diversity, BioTechniques 19: 442–447.Google Scholar
  64. 62.
    Cronin, M. T., Fucini, R. V., Kim, S. M., Masino, R. S., Wespi, R. M., and Miyada, C. G., 1996, Cystic fibrosis mutation detection by hybridization to light-generated DNA probe arrays, Hum. Mutat. 7: 244–255.CrossRefGoogle Scholar
  65. 63.
    Polak, J. M., and McGee, J. O’D., 1990, In Situ Hybridization, Principles and Practice, Oxford University Press, New York.Google Scholar
  66. 64.
    Wiegant, J., Wiesmeijer, C. C., Hoovers, J. M. N., Schuuring, E., d’Azzo, A., Vrolijk, J., Tanke, H. J., and Raap, A. K., 1993, Multiple and sensitive fluorescence in situ hybridization with rhodamine-, fluorescein-, and coumarin-labeled DNAs, Cytogenet. Cell Genet. 63: 73–76.CrossRefGoogle Scholar
  67. 65.
    Schröck, E., du Manoir, S., Veldman, T., Schoell, B., Wienberg, J., Ferguson-Smith, M. A., Ning, Y., Ledbetter, D. H., Bar-Am, I., Soenksen, D., Garini, Y., and Reid, T., 1996, Multicolor spectral karyotyping of human chromosomes, Science 273: 494–497.CrossRefGoogle Scholar
  68. 66.
    Speicher, M. R., Ballard, S. G., and Ward, D. C., 1996, Karyotyping human chromosomes by combinatorial multi-fluor FISH, Nat. Genet. 12: 368–378.CrossRefGoogle Scholar
  69. 67.
    Nederlof, P. M., van der Flier, S., Wiegant, J., Raap, A. K., Tanke, H. J., Ploem, J. S., and van der Ploeg, M., 1990, Multiple fluorescence in situ hybridization, Cytometry 11: 126–131.CrossRefGoogle Scholar
  70. 68.
    Kallioniemi, A., Kallioniemi, O. P., Sudar, D., Rutovitz, D., Gray, J. W., Waldman, F., and Pinkel, D., 1992, Comparative genomic hybridization for molecular cytogenetic analysis of solid tumors, Science 258: 818–821.CrossRefGoogle Scholar
  71. 69.
    Lewis, R., 1996, Chromosome charting takes a giant step, Photonics Spectra 1996 (June): 48–49.Google Scholar
  72. 70.
    Le Beau, M. M., 1996, One FISH, two FISH, red FISH, blue FISH, Nat. Genet. 12: 341–344.CrossRefGoogle Scholar
  73. 71.
    Speicher, M. R., and Ward, D. C., 1996, The coloring of cytogenetics, Nat. Med. 2: 1046–1048.CrossRefGoogle Scholar
  74. 72.
    Bentz, M., Döhner, H., Cabot, G., and Lichter, P., 1994, Fluorescence in situ hybridization in leukemias: The FISH are spawning, Leuke-mia 8: 1447–1452.Google Scholar
  75. 73.
    Fox, J. L., Hsu, P.-H., Legator, M. S., Morrison, L. E., and Seelig, S. A., 1995, Fluorescence in situ hybridization: Powerful molecular tool for cancer prognosis, Clin. Chem. 41: 1554–1559.Google Scholar
  76. 74.
    Popescu, N. C., and Zimonjic, D. B., 1997, Molecular cytogenetic characterization of cancer cell alterations, Cancer Genet. Cytogenet. 93: 10–21.CrossRefGoogle Scholar
  77. 75.
    Swiger, R. R., and Tucker, J. D., 1996, Fluorescence in situ hybridization, Environ. Mol. Mutagenesis 27: 245–254.CrossRefGoogle Scholar
  78. 76.
    Siadat-Pajouh, M., Periasamy, A., Ayscue, A. H., Moscicki, A. B., Palefsky, J. M., Walton, L., DeMars, L. R., Power, J. D., Herman, B., and Lockett, S. J., 1994, Detection of human papillomavirus type 16/18 DNA in cervicovaginal cells by fluorescence based in situ hybridization and automated image cytometry, Cytometry 15: 245–257.CrossRefGoogle Scholar
  79. 77.
    Pandya, P. P., Cardy, D. L. N„ Jauniaux, E., Campbell, S., and Nicolaides, K. H., 1994, Rapid determination of fetal sex in coelomic and amniotic fluid by fluorescence in situ hybridization, Fetal Di-agn. Ther. 10: 66–70.Google Scholar
  80. 78.
    Matthews, J. A., and Kricka, L. J., 1988, Analytical strategies for the use of DNA probes, Anal. Biochem. 169: 1–25.CrossRefGoogle Scholar
  81. 79.
    Nazarenko, I. A., Bhatnagar, S. K., and Hohman, R. J., 1997, A closed tube format for amplification and detection of DNA based on energy transfer, Nucleic Acids Res. 25: 2516–2521.CrossRefGoogle Scholar
  82. 80.
    Luehrsen, K. R., Marr, L. L., van der Knaap, E., and Cumberledge, S., 1997, Analysis of differential display RT-PCR products using fluorescent primers and GENESCANTM software, BioTechniques 22: 168–174.Google Scholar
  83. 81.
    Alexandre, I., Zammatteo, N., Moris, P., Brancart, F., and Remacle, J., 1997, Comparison of three luminescent assays combined with a sandwich hybridization for the measurement of PCR-amplified human cytomegalovirus DNA, J. Virol. Methods 66: 113–122.CrossRefGoogle Scholar
  84. 82.
    Kostrikis, L. G., tyagi, S., Mhlanga, M. M., Ho, D. D., and Kramer, F. R., 1998, Spectral genotyping of human alleles, Science 279: 1228–1229.CrossRefGoogle Scholar
  85. 83.
    Tyagi,S.,Bratu,D. P., and Kramer, F.R., 1998, Multicolor molecular beacons for allele discrimination, Nat. Biotechnol. 16: 49–52.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 1999

Authors and Affiliations

  • Joseph R. Lakowicz
    • 1
  1. 1.University of Maryland School of MedicineBaltimoreUSA

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