Chemistry Reference
In-Depth Information
Figure 7.12 Agarose gel electrophoresis of the PCR product from
the recombinant DNA. Lane 1, PCR product from the recombinant
DNA; lane 2, PCR product from the mixture without the enzymatic
ligation; M, 200 base pair ladder.
7.6.3
Enzymatic Ligation of the Scission Fragment and Foreign DNA
The scission products were treated with foreign double-stranded DNA in the presence
of T4 DNA ligase (Figure 7.11). For example, the fragment obtained in Figure 7.10(a)
(the one between 2000 and 3000-mer) was combined with DNA
(1)
/DNA
(2)
duplex. The
sticky end of this foreign DNA is complementary with the end of the fragment
(L)
that is
formed when the upper strand of the linearized plasmid DNA is hydrolyzed at the
5
0
-side of A1824 and the lower strand is cleaved at the 5
0
-side of G1837 [see Figure
7.9(b)].
Successful, selective ligation of these two double-stranded DNAs has been substan-
tiated by the following experiments. The ligation product was first amplified by PCR.
Of the two primers used, primer
(1)
is complementary with DNA
(2)
, whereas primer
(2)
is
complementary with G2271-C2292 of the upper strand of the fragment
(L)
. As shown in
lane 1 of Figure 7.12, a PCR product of about 500-mer size was formed. When these
two double-stranded DNAs were successfully ligated and the PCR proceeded with the
ligation product as the template, 492-mer PCR product should be formed. Conversely,
this PCR product should not be formed if the ligation was unsuccessful. Consistently,
no PCR products were formed when a mixture of the longer scission products (includ-
ing the fragment
(L)
) and DNA
(1)
/DNA
(2)
duplex was directly (without treatment with T4
ligase) used for the PCR (lane 2, Figure 7.12). Furthermore, the PCR product obtained
above by using primer
(1)
and primer
(2)
was characterized completely by sequencing
experiments (Figure 7.13).
In the scission of double-stranded DNA by Ce(
IV
)/EDTA in the presence of
pcPNA
(1)
/pcPNA
(2)
, several fragments should be formed. In the enzymatic ligation,
however, the scission fragment (fragment
(L)
here), whose sticky end completely fits
Figure 7.13 Result of sequencing analysis of the recombinant DNA.
The sequence involving DNA
(1)
and the 5
0
-side portion of upper strand
of the fragment
(L)
is shown.
