Principles of Fluorescence Spectroscopy

710 DNA TECHNOLOGY
Figure 21.11. DNA sequencing using two excitation wavelengths and
the fluorophores shown in Figure 21.10. Revised and reprinted with
permission from [17]. Copyright © 1991, American Chemical
Society.
(R 0 ) using reactive oligonucleotides (Figure 21.12 top) or
DNA-like sugar polymers without the nucleotide bases
(bottom). The lengths of the oligonucleotide linkers were
adjusted to obtain the amount of energy transfer so that the
acceptor intensities are comparable to excitation at 488 nm.
The energy-transfer cassettes displayed nearly equal
intensities when excited at 488 nm (Figure 21.13). The
emission spectra are moderately well separated, which is
easier to see in the normalized emission spectra (Figure
21.14). The bases can be readily identified by measurement
at the four emission wavelengths, which allows DNA
sequencing with capillary electrophoresis using a single
488 nm excitation wavelength. However, Figure 21.14
shows that the emission spectra overlap, and that there is
residual emission from the donors which contributes to the
intensities at shorter wavelengths. Hence, there is still a
need for improved dyes for DNA sequencing. Another set
of energy-transfer primers has been reported based on the
Bodipy fluorophore. 25 These primers are claimed to show
Figure 21.12. Energy-transfer primers used for DNA sequencing.
F10F, F10J, F10T, and F10R are energy-transfer primers in which
the donor and acceptor (see Figure 21.9 for the structures of F, J,
T, and R) have been covalently linked using reactive oligonucleotides.
Excitation is at 488 nm, and the emission wavelengths are
indicated on the right-hand side of the figure. As shown at the bottom
of the figure, donor–acceptor pairs can also be placed on sugar polymers
(—SSS—) prior to the primer sequence. Revised from [15] and
[16].
more distinct emission spectra than shown in Figure 21.13,
but the Bodipy fluorophores are thought to be less photostable.
Given the need for sequencing, there will be continued
development of probes with improved spectral properties.
21.1.6. DNA Sequencing with NIR Probes
The genomes of many organisms are being sequenced,
which must be accomplished as rapidly and inexpensively