Biomedical Engineering Reference
In-Depth Information
Fig.
1
A schematic diagram of the proposed CPEC mechanism for cloning a
single insert. The vector and the insert share overlapping regions at the ends.
After denaturation and annealing
(Step 1)
, the hybridized insert and vector
extend using each other as a template until they complete a full circle and reach
their own 5
-ends
(Step 2)
. The fi nal completely assembled plasmid has two
nicks, one on each strand, at the positions marked by an
arrow head
. They can be
used for transformation
(Step 3)
with or without further purifi cation. For library
cloning, the cycle may be repeated in order to increase the yield of complete
plasmids
′
It uses the polymerase extension mechanism [
6
] to join overlapping
DNA fragments into a double-stranded circular form, such as a
plasmid. In a typical CPEC reaction, linear double-stranded
insert(s) and vector are fi rst heat-denatured; the resulting single
strands then anneal with their overlapping ends and extend using
each other as a template to form double-stranded circular plas-
mids. In CPEC, all overlapping regions between insert(s) and the
vector are unique and carefully designed to have very similar and
high melting temperatures (
Tm
), which eliminates vector rean-
nealing and concatenation of inserts and makes CPEC very effi cient
and accurate. The low concentrations of fragments in the reaction
favor plasmid circularization and effectively prevent plasmid con-
catenation. After the CPEC reaction, the perfectly formed double-
stranded circular plasmids, with one nick in each strand, can be
directly transformed into competent host cells (Fig.
1
).
Complex library cloning and multi-way pathway assembly
require high cloning effi ciency and accuracy. Although other
relevant cloning methods only allow the overlapping fragments to
anneal or recombine once, CPEC allows multiple annealing-
extension cycles that not only increase the chance of hybridization
but also permanently join the fragments through polymerase
extension, thereby maximizing the cloning effi ciency. Whereas the
other relevant cloning methods perform the critical annealing/
incubation step under ambient temperature, which tends to cause
