Principles of Fluorescence Spectroscopy

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PRINCIPLES OF FLUORESCENCE SPECTROSCOPY 703 ty data for [Ru(dpp(SO 3 Na) 2 ) 3 ]Cl 2 are shown in Figure 20.52. Calculate the oxygen bimolecular Figure 20.52. Frequency-domain intensity decay of [Ru(dpp)(SO 3 Na) 2 ) 3 ]Cl 2 in water, under an atmosphere of argon (o), air (), or oxygen (). quenching constant for this complex. Assume the solubility of oxygen in water is 0.001275 M/atm.
21 During the past 20 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, restriction enzyme fragment analysis, and fluorescence in-situ hybridization (FISH), and to detect polymerase chain reaction products. Molecular beacons can be used to detect messenger RNA within cells. DNA arrays can be used to monitor the expression of thousands of genes using a single microscope slide. Fluorescence was used to sequence the human genome that was reported in 2000. 1–2 Given these advances it is surprising to realize that DNA sequencing by fluorescence was first reported in 1986. It is not practical to describe the many specialized methods used to study DNA in molecular biology, genetics, and medical diagnostics. In this chapter we provide an overview of the dominant users of fluorescence in DNA analysis. 21.1. DNA SEQUENCING DNA sequencing first became practical in 1977. 3–4 The original method involved chemical degradation of the DNA using conditions that were partially selective for one of the four bases. The DNA fragments were then separated by chromatography and the fragments detected using 32 P autoradiography. In the same year a method became available for generating fragments terminating in each of the four bases. 5 This method used termination of enzymatic DNA synthesis using dideoxynucleotides. The fragments were again detected using radioactivity. An overview of the history of DNA sequencing methods can be found in the informative text by Watson et al. 6 The use of radioactive tracers is problematic with regards to cost, safety, and disposal. Also the use of radioactivity was not amenable to the degree of automation needed to sequence long DNA chains, chromosomes or an entire genome. DNA sequencing using fluorescence was first DNA Technology reported in 1986. 7–12 At present essentially all sequencing is done using fluorescence detection. It is unlikely that the human genome would have been sequenced without the use of fluorescence. DNA sequencing is now highly automated 11 and performed in numerous laboratories around the world. 21.1.1. Principle of DNA Sequencing A number of slightly different methods are used for DNA sequencing, but all methods rely on the use of dideoxynucleotide triphosphate (ddNTP) to terminate DNA synthesis. The basic idea of sequencing using ddNTP terminators is shown in Figures 21.1 and 21.2. In DNA the nucleotides are linked in a continuous strand via the 5' and 3' hydroxyl groups of the pentose sugar. DNA is replicated by adding nucleotides to the 3' hydroxyl group. For sequencing a DNA strand with an unknown sequence is replicated using DNA polymerase. Replication is started from a primer location with a known sequence. The most commonly used primer is the M13 sequence, which is 17 nucleotides long. In the example shown in Figure 21.2 a single fluorescent primer is used to initiate the reaction. The sample contains DNA polymerase and the four deoxynucleotide triphosphates. Within a short period of time the DNA polymerase molecules are randomly distributed along the unknown sequence. The strands being synthesized have a sequence complementary to the unknown strand. The reaction mixture is split into four parts, one part for each of the four bases. The DNA polymerase reaction is randomly terminated by adding one of the ddNTPs to each of the four parts of the reaction. The ddNTPs are added along the growing chain. The absence of a 3' hydroxyl group on the ddNTPs prevents further elongation and terminates the reaction. This results in a mixture of oligonucleotides of varying length. The different size oligomers are separated by poly- 705
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Principles of Fluorescence Spectros
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Joseph R. Lakowicz Center for Fluor
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Preface The first edition of Princi
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x GLOSSARY OF ACRONYMS DNS dansyl o
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xii GLOSSARY OF ACRONYMS TRES time-
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xiv GLOSSARY OF MATHEMATICAL TERMS
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xvi CONTENTS 2.9.4. Conversion betw
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xviii CONTENTS 6. Solvent and Envir
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xx CONTENTS 10.4.4. Alignment of Po
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xxii CONTENTS 14.7.1. Simulations o
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xxiv CONTENTS 19.12. New Approaches
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xxvi CONTENTS 25.3. Optical Propert
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2 INTRODUCTION TO FLUORESCENCE Figu
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6 INTRODUCTION TO FLUORESCENCE inst
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10 INTRODUCTION TO FLUORESCENCE Fig
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12 INTRODUCTION TO FLUORESCENCE ns,
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14 INTRODUCTION TO FLUORESCENCE 1.7
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24 INTRODUCTION TO FLUORESCENCE due
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28 INSTRUMENTATION FOR FLUORESCENCE
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3 Fluorescence probes represent the
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PRINCIPLES OF FLUORESCENCE SPECTROS
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98 TIME-DOMAIN LIFETIME MEASUREMENT
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100 TIME-DOMAIN LIFETIME MEASUREMEN
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5 In the preceding chapter we descr
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6 Solvent polarity and the local en
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238 DYNAMICS OF SOLVENT AND SPECTRA
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278 QUENCHING OF FLUORESCENCE 8.1.
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280 QUENCHING OF FLUORESCENCE Figur
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282 QUENCHING OF FLUORESCENCE ly ev
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290 QUENCHING OF FLUORESCENCE relat
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298 QUENCHING OF FLUORESCENCE σ ar
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320 QUENCHING OF FLUORESCENCE 47. R
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322 QUENCHING OF FLUORESCENCE 113.
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328 QUENCHING OF FLUORESCENCE P8.3.
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330 QUENCHING OF FLUORESCENCE P8.11
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332 MECHANISMS AND DYNAMICS OF FLUO
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10 Measurements of fluorescence ani
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11 In the preceding chapter we desc
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12 In the preceding two chapters we
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444 ENERGY TRANSFER Figure 13.1. Fl
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446 ENERGY TRANSFER wavelength is e
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448 ENERGY TRANSFER Table 13.1. Cal
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450 ENERGY TRANSFER Figure 13.6. Ab
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452 ENERGY TRANSFER safe for many p
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454 ENERGY TRANSFER Figure 13.12. P
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456 ENERGY TRANSFER Figure 13.16. E
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458 ENERGY TRANSFER Figure 13.21. R
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460 ENERGY TRANSFER Figure 13.24. R
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462 ENERGY TRANSFER tiple acceptors
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464 ENERGY TRANSFER Figure 13.29. A
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466 ENERGY TRANSFER Figure 13.33. F
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468 ENERGY TRANSFER Table 13.3. Rep
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470 ENERGY TRANSFER 55. Clegg RM. 1
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472 ENERGY TRANSFER Tong AK, Jockus
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474 ENERGY TRANSFER Figure 13.39. O
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14 In the previous chapter we descr
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15 In the previous two chapters on
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16 The biochemical applications of
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578 TIME-RESOLVED PROTEIN FLUORESCE
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608 MULTIPHOTON EXCITATION AND MICR
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19 Fluorescence sensing of chemical
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Page 665 and 666: PRINCIPLES OF FLUORESCENCE SPECTROS
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758 SINGLE-MOLECULE DETECTION Figur
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766 SINGLE-MOLECULE DETECTION small
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770 SINGLE-MOLECULE DETECTION Table
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786 SINGLE-MOLECULE DETECTION face
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788 SINGLE-MOLECULE DETECTION TCSPC
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790 SINGLE-MOLECULE DETECTION 53. H
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792 SINGLE-MOLECULE DETECTION Ke PC
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794 SINGLE-MOLECULE DETECTION Polar
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24 In the previous chapter we descr
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862 RADIATIVE-DECAY ENGINEERING: SU
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Appendix I Corrected Emission Spect
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Appendix II Fluorescence Lifetime S
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890 APPENDIX III P ADDITIONAL READI
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892 APPENDIX III P ADDITIONAL READI
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894 ANSWERS TO PROBLEMS A1.4. A. Th
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896 ANSWERS TO PROBLEMS Energy tran
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898 ANSWERS TO PROBLEMS Figure 4.65
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900 ANSWERS TO PROBLEMS expected to
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904 ANSWERS TO PROBLEMS where f is
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906 ANSWERS TO PROBLEMS [BSA] Obser
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908 ANSWERS TO PROBLEMS emission. T
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910 ANSWERS TO PROBLEMS dx rA A (
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912 ANSWERS TO PROBLEMS B. A covale
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914 ANSWERS TO PROBLEMS The α i va
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920 ANSWERS TO PROBLEMS CHAPTER 24
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Index A Absorption spectroscopy, 12
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