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COMMUNICATION
Journal Name
7.
8.
S. Takahashi, K. T. Kim, P. Podbevsek, J. Plavec, B. H. Kim and N.
homogeneous A sequences and dC rich sequences (see Supporting
Information).
DOI: 10.1039/C8CC09444F
5774.
We also measured the yield of full length products using
quantitation method by ethidium bromide fluorescence emission
(Table S1). Interestingly, we observed decreasing tendency of yield
depends on the increasing the number of dUazoTP (36.78% for C-
T1, 25.0% for C-T2, 16.1% for C-T3). We also observed lower
yield (14.4%) using template G4-1 and G4-2 than homoA1-A3 (23.9
%). Truncated products were detected from C-T2 and C-T3 and
template G4-1 and G4-2, while C-T1 and homoA1-A3 sequence did
not show any truncated product. We suspect that the low yield of
multiple dUazoTP incorporated full length product and G-C rich full
length product using template G4-1and G4-2 (G-C rich full length
product has more probability to form the non-canonical DNA
secondary structure than A-T rich full length product using homo
A1-A3), may be originated from hindrance of non-canonical DNA
secondary structure when the polymerase binds to these modified
DNA. Lower yield of homoA1-A3 (23.9%) sequence relative to the
C-T1 (36.7%) might be from multiple use of Lambda exonuclease (3
times) to remove the template in each step ( homo A1, A2, A3).
From this result, we believe that the secondary structure incorporated
by functional nucleotides and the number of step would be critical
factor to obtain the high yield of full length product using this
method.
In conclusion, we have developed a novel direct enzymatic DNA
labeling method based on step-wise approach using polymerase.
This method employs DNA polymerase to perform the step-by-step
incorporation of functional nucleotides into DNA. We applied
dUazoTP as the quencher and dUpyrTP as the fluorophore in the
step-wise process, and incorporated them, using this direct and site-
specific labeling method, into the CNG repeat sequence, which
forms a hairpin structure, to allow monitoring of the structural
transformations of DNA. The incorporation of multiple units of
dUazoTP and dUpyrTP was possible when using this approach for
direct labeling. Our direct and site-specific method for labeling DNA
with dUazoTP and dUpyrTP resulted in dramatic signal quenching
upon the transformation from single-stranded to hairpin structures,
with the fluorescence recovering after adding the perfect
complementary sequence for the formation of the duplex. This step-
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Acknowledgment
This study was supported by the Basic Science Research Program
through the National Research Foundation of Korea
(2017R1A2B4002398), funded by the Republic of Korea.
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Supplementary data
Electronic Supplementary Information (ESI) available online: Full
experimental details; characterization of compounds; UV absorption
and circular dichroism spectra; UV spectroscopic melting curves for
oligonucleotides.
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