Chemistry Reference
In-Depth Information
7.5.2
Enzymatic Ligation of the Fragments Obtained by Site-selective Scission
The present site-selective scission proceeds by an hydrolytic pathway, as is the case in
DNA scission by nucleases. Thus, the scission fragments can be recombined with
various oligonucleotides by using DNA ligase. Figure 7.6 depicts a typical example.
In the presence of the DNA
(L)
-L
0
-P/P-L
0
-DNA
(R)
combination, DNA
(S5)
is first hydrolyzed
by Ce(
IV
)/EDTA (lane 2), and the scission fragments are purified by PAGE. To this re-
action mixture, an oligonucleotide having a monophosphate at the 5
0
-end (DNA
(ligated)
:
arbitrary sequence and length) and DNA
(template22)
template are added, and the mixture
Figure 7.6 Ligation of the scission fragments by T4 DNA ligase in the
presence of various templates. (a) Sequences of the oligonucleotides used.
(b) Lane 1, DNA
(S5)
without treatment (labeled by fluorescein at the 5
0
-end);
lane 2, product of site-selective scission of DNA
(S5)
by Ce(
IV
)/EDTA complex in
the presence of DNA
(L)
-L
0
-P/P-L
0
-DNA
(R)
combination; lane 3, product in lane
2 purified by PAGE (fragments ranging from C1 to T20-G26 were collected);
lane 4, ligation product in the presence of DNA
(template22)
; lane 5, ligation
product in the presence of DNA
(template24)
; lane 6, product obtained by ligation
in the absence of templates.
