Chemistry Reference
In-Depth Information
DNA, and the monophosphate group(s) is placed at the edge of these gaps. Figure 7.4
shows the typical results of polyacrylamide gel electrophoresis (PAGE) for the site-se-
lective scission. In lane 4, DNA
(L)
-L
12
-P and H-DNA
(R)
are combined, and a 5-base gap
is formed from T21 to G25 in the substrate DNA
(S5)
. Thus, the monophosphate group
is placed at the 5
0
-side edge of this 5-base gap. Upon treating this system with Ce(
IV
)/
EDTA complex at pH 7.0 and 37
8
C, the gap-site is selectively hydrolyzed (lane 4). Scis-
sion occurs exactly in the gap-site and ranges from T21 to G25. Site-selective DNA
scission is also successful when one monophosphate group is placed at the 3
0
-side
edge of the gap (DNA
(L)
-H/P-L
12
-DNA
(R)
combination: lane 5). The DNA
(L)
-L
12
-P/P-
L
12
-DNA
(R)
combination, which provides two monophosphate groups to the gap, is
still more effective (lane 6). Without the terminal monophosphate in the oligonucleo-
tide additives, however, DNA scission by Ce(
IV
)/EDTA is far (
>
10-fold) slower (lane 3).
7.5.1.2
Effects of Gap-length on Site-selective DNA Scission
Figure 7.5(a) shows the dependencies of site-selectivity and scission efficiency on gap
length (number of unpaired nucleotides). When a 3-base gap is formed from T21 to
A23inDNA
(S3)
by use of the oligonucleotide additives bearing monophosphate, this
gap-site is selectively hydrolyzed by Ce(
IV
)/EDTA (lane 3). Furthermore, site-selective
hydrolysis of a 2-base gap (lane 2) or a 1-base gap (lane 1) is also successful. The total
Figure 7.5 (a) Site-selective scission of gaps of different lengths by Ce(
IV
)/
EDTA at pH 7.0 and 37
C. Lanes: 1, one-base gap; 2, two-base gap; 3, three-
base gap; 4, five-base gap; M, the markers (authentic oligonucleotides having
3
0
-OH termini). These gaps were formed in DNA
(S1)
-DNA
(S5)
(
32
P-labelled at
the 5
0
-end) using the DNA
(L)
-L
12
-P/P-L
12
-DNA
(R)
combination. (b) Quantitative
analysis of scission efficiencies (solid part is for the formation of 3
0
-OH
terminus and the broken one for the formation of 3
0
-phosphate terminus).
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