Chemistry Reference
In-Depth Information
7.6.2.2
Site-selective Scission
When the invasion complex composed of linearized PBR322, pcPNA
(1)
, and pcPNA
(2)
was treated with Ce(
IV
)/EDTA complex at pH 7.0 and 37
C, two fragments were
formed, as observed in agarose gel electrophoresis [lane 3 in Figure 7.10(a)]. One frag-
ment is between 2000 and 3000-mer, and the other is a little smaller than 2000-mer.
When scission occurred at the target gap-like sites, products of ca. 1830 and 2530-mer
should be formed from the left- and right-hand sides of the substrate DNA, respec-
tively. Apparently, the Ce(
IV
)/EDTA complex hydrolyzed selectively both strands ex-
actly as designed.
For the present site-selective scission of double-stranded DNA, both pcPNA addi-
tives must have flanking portions so that gap-like structures are formed in the double-
stranded DNA. Thus, no site-selective scission was observed when pcPNA
(3)
and
pcPNA
(4)
were combined as additives [Figure 7.10(a), lane 4]. These two pcPNAs
are completely complementary (their duplex is not much formed due to mutual steric
repulsion under the conditions employed), and thus no gap-like structures are pro-
duced in the invasion complex [see structure at the bottom of Figure 7.9(a)]. No scis-
sion occurred in the absence of pcPNA additives, as expected (lane 2).
8
7.6.2.3
Direct Site-selective Scission of Supercoiled Plasmid DNA
In virtually the same way, supercoiled DNA can be hydrolyzed site-selectively by
Ce(
IV
)/EDTA [Figure 7.10(b)]. The invasion complex was obtained by incubating
PBR322 plasmid DNA with pcPNA
(1)
and pcPNA
(2)
, and was then treated with the
Ce(
IV
) complex. In this treatment, the supercoiled DNA was converted into linearized
DNA (form III). When the product was digested by the restriction enzyme EcoRI (to
analyze the scission site), two fragments of expected sizes [the same ones as obtained
in lane 3 of Figure 7.10(a)] were formed [lane 3 in Figure 7.10(b)]. Apparently, the
scission of the supercoiled DNA took place at the same site as that of the linearlized
DNA. The present site-selective scission is applicable to various DNAs.
Figure 7.11 Schematic representation for manipulation of double-stranded DNA
using the present artificial restriction enzyme.
