Chemistry Reference
In-Depth Information
Figure 7.9 (a) DNA substrate and pcPNA additives used for site-selective
scission by Ce(
IV
)/EDTA. U and D in pcPNA sequences bear 2-thiouracil and
2,6-diaminopurine residues in place of conventional bases. In the invasion-
complex composed of linearized PBR322, pcPNA
(1)
, and pcPNA
(2)
, the un-
derlined nucleotides remain unpaired. (b) DNA fragments used for enzymatic
ligation. The sticky end of foreign DNA (DNA
(1)
/DNA
(2)
duplex) is comple-
mentary with that of the scission fragment (fragment
(L)
).
site [18]. Furthermore, fragments obtained by the scission are successfully ligated with
foreign DNA to provide recombinant DNA (Section 7.6.3). Of course, the sequences
and lengths of pcPNA additives are freely chosen. Thus, the present method is, in
principle, applicable to site-selective scission of larger DNA substrates, and facilitates
the manipulation of these DNAs for future biotechnology.
7.6.2
Site-selective Hydrolysis of Double-stranded DNA
Both linear and supercoiled double-stranded DNA can be used as substrate for site-
selective scission. Typical methods for the scission of linear double-stranded DNA are
given in Sections 7.6.2.1 and 7.6.2.2. The substrate is obtained by treating supercoiled
PBR322 plasmid DNA by a restriction enzyme EcoRI. Here, the plasmid DNA is cut at
one-site, providing a linear DNA (this DNA is known as form III). Section 7.6.2.3 de-
scribes the method for site-selective hydrolysis of supercoiled DNA. Figure 7.9(a)
