Chemistry Reference
In-Depth Information
Figure 7.7 Schematic representation of enzymatic ligation of the scission
fragment and foreign DNA using various templates.
is treated with T4 DNA ligase (lane 4). The 3
0
-side portion of DNA
(template22)
(10 nucleo-
tides) is complementary with G13-T22 of DNA
(S5)
, and the remainder of this template
is complementary with the 5
0
-side of DNA
(ligated)
[Figure 7.6(a)]. Upon treatment with
T4 DNA ligase, a new band appears at the top of the gel. This is the ligation product
of the C1-T22 fragment, formed by cutting DNA
(S5)
between T22 and A23, and the
DNA
(ligated)
(shown schematically on the left-hand side of Figure 7.7). The present
DNA scission occurs at several linkages in DNA
(S5)
, so that there exist several frag-
ments in the reaction mixture. However, only one fragment that completely fits
the template oligonucleotide is picked up and connected with DNA
(ligated)
. Consistently,
the band T22 for the C1-T22 fragment bearing 3
0
-OH terminus becomes weaker in the
enzymatic reaction, while the intensities of other bands hardly change (compare lanes
3 and 4).
These arguments are further supported by the results in lane 5, where the
DNA
(template24)
is used in place of DNA
(template22)
. This template is complementary
with A15-T24 of DNA
(S5)
and with the 5
0
-side of DNA
(ligated)
. A new band of small
mobility also appears after the treatment by DNA ligase, corresponding to the ligation
product between the C1-T24 fragment and DNA
(ligated)
(see right-hand side of Figure
7.7). This product is two bases longer than the one formed in lane 4 and, thus, has
a slightly smaller mobility. The C1-T24 fragment band almost disappears upon enzy-
matic treatment (cf. lanes 3 and 5). In the absence of templates, no ligation products
are formed (lane 6). By designing the template oligonucleotide appropriately, the desired
scission fragment is selectively incorporated to recombinant DNA. Thus, the present
system is promising for future gene manipulation of single-stranded DNA (Figure 7.8).
