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S1 Nuclease
CCP
Scheme 4 Illustration of S1 nuclease mediated digestion of the duplex of long ssDNA target and
the PNA probe, and subsequent FRET between the intact regions bound to the PNA probe
(5
0
-F1-GTA AAT GGT GTT AGG GTT GC-3
0
) was used as the dye-labeled nucleic
acid probe, and energy transfer was examined for the complementary ssDNA
4
(5
0
-
GCA ACC CTA ACA CCA TTT AC-3
0
) and the noncomplementary ssDNA
5
(5
0
-
GAC TCA ATG GCG TTA GAC TG-3
0
). A direct comparison of Fl emission (upon
excitation at 380 nm) with [ssDNA
3
-F1]
10
7
M
is shown in Fig.
2
. Electrostatic attraction between ssDNA-Fl and CCP (1I)does
occur, giving rise to background dye emission in the presence of noncomplementary
ssDNA. Nevertheless, the Fl emission intensity in the presence of ssDNA
4
is threefold
higher than that in the presence of ssDNA
5
. The difference in FRET is attributed to a
closer proximity between Fl and 1I in the presence of ssDNA
4
as compared to that in
the presence of ssDNA
5
. This study indicates that utilization of ssDNA-C*, instead of
PNA-C*, as the probe is less effective for DNA detection. This strategy was also
examined by Leclerc's group using a cationic PT in conjunction with ssDNA labeled
with Alexa Fluor 546, where a detection limit of 3 zMwas achieved [
52
]. In addition,
Wang et al. also used a similar scheme for SNP detection and allele frequency
determination in association with probe extension reaction, where a DNA fragment
as a part of p53 exon8 containing a polymorphic site (Arg282Trp) was effectively
discriminated according to the difference in FRET [
53
].
10
8
Mand[1I]
¼
¼
2.1
5.1
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