Principles of Fluorescence Spectroscopy

708 DNA TECHNOLOGY
Figure 21.5. Fluorescent chain-terminating dideoxynucleotides. The letters refer to the DNA base, and the numbers refer to the emission maximum.
Reprinted with permission from Prober JM, Trainor GL, Dam RJ, Hobbs FW, Robertson CW, Zagursky RJ, Cocuzza AJ, Jensen MA, Baumeister K.
1987. A system for rapid DNA sequencing with fluorescent chain-terminating dideoxynucleotides. Science 238:336–343 [9]. Copyright © 1987,
American Association for the Advancement of Science.
Figure 21.6. Absorption (top) and emission spectra (bottom) of the
fluorescent chain terminating dideoxynucleotides in Figure 21.5. The
absorption spectra are of the succinyl fluorescein (SF) dyes, prior to
coupling to the amine reactive ddNTPs. Reprinted with permission
from Prober JM, Trainor GL, Dam RJ, Hobbs FW, Robertson CW,
Zagursky RJ, Cocuzza AJ, Jensen MA, Baumeister K. 1987. A system
for rapid DNA sequencing with fluorescent chain-terminating
dideoxynucleotides. Science 238:336–343 [9]. Copyright © 1987,
American Association for the Advancement of Science.
21.1.4. Example of DNA Sequencing
It is informative to see some actual data from DNA
sequencing, 15–16 which was accomplished using the fluorophores
shown in Figure 21.9. These probes show moderately
distinct emission spectra (Figure 21.10), so that single-lane
sequencing should be possible. However, it is difficult
to excite these four fluorophores using a single excitation
wavelength. 17 Two laser sources were used at 488 and
Figure 21.7. Apparatus for wavelength ratio intensity measurements
from DNA gels. From [9].