Biomedical Engineering Reference
In-Depth Information
Chapter 13
Simple Cloning and DNA Assembly in
Escherichia coli
by Prolonged Overlap Extension PCR
Chun You and Y.-H. Percival Zhang
Abstract
We developed a simple method (Simple Cloning) for subcloning one, two, or three DNA fragments into any
location of a targeted vector without the need for restriction enzyme, ligase, exonuclease, or recombinase.
This cloning technology can be applied to a few common
Escherichia coli
hosts (e.g., BL21(DE3), DH5
α
,
JM109, TOP10). The protocol includes three steps: (a) linear DNA fragments (i.e., the insert DNA and
the vector backbone) with two overlap ends were generated by regular high-fi delity PCR, (b) the DNA
multimers were generated based on these equimolar DNA templates by using prolonged overlap extension
PCR (POE-PCR) without primers added, and (c) the POE-PCR product was transformed to
E
.
coli
strains
directly. Because positive colony effi ciencies are very high, it is not necessary to identify desired clones by
using colony PCR. Simple Cloning provides a new cloning and DNA assembly method with great simplicity
and fl exibility.
Key words
Enzyme-free cloning,
Escherichia coli
, DNA assembly, Prolonged overlap extension PCR,
Simple Cloning, Subcloning
1
Introduction
Escherichia coli
stains have a long history of use in the biotechnology
industry and biological science studies. They are the most pre-
ferred model microorganism for molecular cloning and recombi-
nant protein expression, mainly due to their genetic simplicity, fast
growth rates under laboratory conditions, good transformation
ability for hosting foreign DNA, the availability of their genome
sequence, and laboratory safety [
1
-
3
]. The classic molecular clon-
ing technology in
E
.
coli
is based on the use of restriction enzymes
that digest an inserted DNA fragment and a vector as well as the
use of a DNA ligase that connects two fragments to yield the
desired chimeric plasmid [
4
]. However, limited choices of restric-
tion enzymes, relatively low effi ciencies in digestion and ligation,
and possible self-ligation of the digested vector may result in diffi -
culties in obtaining positive colonies when ligating two fragments,
