Principles of Fluorescence Spectroscopy

716 DNA TECHNOLOGY
Figure 21.21. Top: Histogram of photon burst sizes of TOTO-1
stained DNA from a HindIII digest of λ-DNA. Bottom: Correlation of
the photon burst size with DNA fragment length. Excitation was at
514 nm from an argon ion laser, and emission was observed through
a 550-nm interference filter. Modified and reprinted from [80].
Copyright © 1995, American Chemical Society.
One commonly used method is to detect an increase in RET
when complementary donor and acceptor labels hybridize
(upper left). The presence of complementary DNA
sequences can be detected by increased energy transfer
when these sequences are brought into proximity by
hybridization. 82–89 This can occur if the complementary
strands are labeled with donors and acceptors. An example
of this approach was shown in Figure 1.27. Energy transfer
can also occur if the donor- and acceptor-labeled oligonucleotides
bind to adjacent regions of a longer DNA sequence
(Figure 21.22). An example of this approach is
shown below in Figure 21.36. Hybridization can be detected
if a donor intercalates into the double-helical DNA, and
transfers to an acceptor-labeled oligonucleotide. The use of
an intercalating dye has been extended to include a covalently
attached intercalators, whose fluorescence increases
in the presence of double-stranded DNA (d). 90–91 One
example is shown in Figure 21.23, in which the acridine dye
is covalently linked to the 3' phosphate of an oligothymidylate.
Upon binding to a complementary adenine oligonucleotide
the acridine fluorescence increases about twofold.
Hybridization can be competitive where the presence of
increased amounts of target DNA competes with formation
of donor–acceptor pairs. 92 The acceptor can be fluorescent,
or it can be nonfluorescent, in which case the donor appears
to be quenched. A competitive assay (Figure 21.22) was
Figure 21.22. Methods to detect DNA hybridization by energy transfer. D, donor; A, acceptor or quencher; I, intercalating dye.