Biomedical Engineering Reference
In-Depth Information
utilizes site-specifi c recombination. Alternative recombination-based
technologies such as the In-Fusion™ system (Chapter
15
)
[
2
]
(Clontech, Mountain View, CA) and the Sequence and Ligation-
Independent Cloning (SLIC) (Chapter
2
)
[
3
] rely on homologous
recombination. Other LIC procedures use complementary single-
strand overhangs to combine the vector and the insert [
4
-
6
]. A dif-
ferent LIC approach for DNA cloning is based on whole plasmid
amplifi cation of the insert and the plasmid [
7
-
11
] and designated as
Restriction-Free (RF) cloning [
10
,
11
]. In RF cloning, the gene of
interest is PCR-amplifi ed using two primers, each containing a tar-
get-specifi c sequence and an extension that overlaps the insertion
sites in the destination vector. Following purifi cation, the double-
stranded PCR product is used as a set of megaprimers for the second
reaction. In this step, each of the DNA strands anneals to the desti-
nation vector at a predesigned position and is extended in a linear-
amplifi cation reaction. The two new DNA strands form a
double-stranded nicked plasmid. The parental methylated DNA is
then removed by DpnI treatment and the newly synthesized plas-
mid, containing the DNA insert, is introduced into
Escherichia coli
cells where the nicked DNA is sealed by endogenous enzymatic
activity. Recently, we have expanded the applications of the RF clon-
ing for diverse molecular manipulations including simultaneous
cloning at distinct positions and multicomponent assembly [
10
].
In order to further facilitate and simplify DNA cloning we
developed the Transfer-PCR (TPCR) platform as an attractive and
effi cient alternative to the currently available procedures for recom-
binant DNA cloning [
12
]. Cloning by TPCR combines, in a single
tube, PCR amplifi cation of the gene of interest from a donor vec-
tor, generation of an intermediate PCR product, and its subsequent
integration into the recipient vector (Fig.
1
). As in the previously
described RF cloning, DpnI is used to remove traces of the donor
vector. TPCR allows precise and seamless integration of the DNA
insert into any destination vector, at any position and without any
additional unnecessary sequences. We have developed the TPCR
platform for both DNA cloning and multiple-site-targeted muta-
genesis [
12
]. In the current protocol we describe a detailed proce-
dure for implementation of the TPCR for DNA assembly.
2
Materials
2.1
Bacterial Strains
For DNA cloning and plasmid preparation procedures,
E. coli
DH5
(Agilent Technology, Stratagene division, Santa Clara, CA)
was used. For protein expression
E. coli
BL21(DE3) (Novagen/
EMD Millipore Chemicals, Darmstadt, Germany) was employed.
α
2.2
Vectors
For the experiments described in the methods section the expression
vector pET28-TevH [
13
] was used as a destination vector (
see
Note 1
).
