Biomedical Engineering Reference
In-Depth Information
ligation-based cloning is not well suited for this purpose because
the effi ciency of the method varies considerably and the frequent
occurrence of restriction sites within genes prevents a uniform
strategy. Most successful high-throughput cloning methods are
either based on DNA recombination or on annealing of comple-
mentary single-stranded overhangs [
2
-
5
].
Recombination-based methods rely on phage recombinases to
insert an ORF into a plasmid. These make use of either specifi c
recombination sequences, like Gateway (Chapter
14
)
[
6
], or
sequence overlap between vector and insert, like SLiCE (Chapter
16
)
[
7
]. Methods based on DNA annealing require long single-
stranded overhangs on the insert that are complementary to the
overhangs of the vector to form stable duplexes. Generation of
these overhangs requires the exonuclease activity of DNA poly-
merases, as for LIC [
8
], In-Fusion (Chapter
15
)
[
9
], Quick and
Clean cloning (Chapter
3
) [
10
], SLIC (Chapter
2
) [
11
], and
Gibson assembly (Chapter
1
) [
12
], or alternative approaches, like
EFC [
13
], USER (Chapter
5
) [
14
], and PIPE [
15
]. Next to
recombination- and single-strand annealing-based systems, meth-
ods exclusively relying on assembly PCR, such as RF cloning
(Chapter
6
) [
16
], CPEC (Chapter
8
) [
17
], and TPCR (Chapter
7
)
[
18
] are emerging.
Fragment exchange (FX) cloning was developed as an alterna-
tive to these approaches [
19
]. FX cloning is dedicated to the paral-
lel cloning of multiple ORFs with the fi nal aim of expressing the
corresponding proteins. FX cloning combines several attractive
features that were thus far not unifi ed in one single cloning method.
Like most annealing-based methods, FX cloning is inexpensive and
reduces the extension of the ORF with seams resulting from
cloning-related sequences. The addition of large, up to nine amino
acids long tails at each terminus of the protein, a common feature
of cloning systems like Gateway and LIC [
6
,
8
], is thus prevented.
As a consequence of the minimal seams, only one primer pair per
ORF suffi ces to generate expression constructs for both N- and
C-terminal translational fusions with tags or proteins. Furthermore,
FX cloning allows the subcloning of a sequence-verifi ed ORF to
different expression vectors, similar to Gateway. This feature
assures a correct DNA sequence of the insert in every new expres-
sion construct. In contrast, virtually all other cloning approaches
accept only PCR products as insert and consequently require rese-
quencing. FX cloning requires only minimal handling and is per-
formed in one pot. Transformation effi ciencies are high and only
about tenfold less than for intact plasmids. Finally, the method is
highly robust and thus even accessible to users lacking prior experi-
ence with molecular biology.
FX cloning relies on the use of type IIS restriction enzymes (RE)
[
20
]. These enzymes differ from the commonly used type IIP RE on
two important aspects: (1) type IIS RE have a non-palindromic,
