Biomedical Engineering Reference
In-Depth Information
DNA overhangs through PCR [
13
,
14
], exonuclease activity [
12
,
17
-
20
], or by excision of uracil [
15
,
16
,
21
-
23
]. These methods
possess a high degree of freedom as they allow for seamless assem-
bly of any DNA stretch and the most well-known commercial
names within this category are In-Fusion
®
(Chapter
15
)
, Gibson
Assembly™ (Chapter
1
)
, and USER™ (Uracil-Specifi c Excision
Reagent). Common for these methods is that they require only a
single-step process of approximately 1 h to assemble the DNA
fragments. Also, they allow for both cloning into plasmids and
joining of PCR fragments, which are features that make these ideal
cloning methods for construction of new vectors; for example,
generating donor plasmids for recombinational systems as
Gateway
®
(Chapter
14
). Although similar possibilities are available
when using methods based on hybridization of ssDNA overhangs,
the methods differ with respect to the way the overhang is gener-
ated and in the recommended length of the overhang. In-Fusion
®
recommends 15 bp sequence overlap and relies on inherent 3
exo-
nuclease function of the vaccinia virus DNA polymerase [
19
],
whereas the Gibson Assembly™ recommends 40 bp overlaps and
includes joint action of a 5
′
exonuclease, a DNA polymerase, and a
DNA ligase [
18
]. In the USER cloning method, the USER primer
used to generate the DNA fragment has uracil incorporated usually
8 nucleotides (nt) from the 5
′
end of the PCR primer. Upon exci-
sion of the uracil, an 8 nt overhang is generated as the remaining
7 nt fragment of the 5
′
end of the USER primer (referred to as
USER tail) is lost from the PCR fragment by diffusion as the few
hydrogen bonds between the USER tail and the PCR fragment
melt at room temperature.. Though coined in the early 1990s [
15
,
16
], the use of uracil excision-based cloning was uncommon
mainly because it was not compatible with high fi delity PCR poly-
merases. This changed when Nour-Eldin and colleagues [
23
]
reported the application of uracil-insensitive proof-reading poly-
merases as well USER cassettes based on restriction enzymes with
8 bp recognition site fl anked by two recognition sites for a nicking
enzyme (Fig.
1
). Uni-directional cloning is obtained by the “direc-
tional bases” placed between the recognition sites of restriction
and nicking enzymes. The overhang created through uracil exci-
sion is the same for a given USER cassette and it is independent of
intrinsic restriction sites in the amplifi ed DNA. The improved
USER technique [
23
] was further developed to include site-
directed mutagenesis [
21
] and fusion of several PCR fragments
into a vector USER cassette through USER fusion [
22
]. Plasmids
carrying USER cassettes have been generated for diverse organ-
isms such as plants [
24
], yeast [
25
], fi lamentous fungi [
26
,
27
],
bacteria [
28
], and mammalian cell lines [
29
]. As an additional
development to USER cloning, the overhangs generated in PCR
products can be used to construct almost any DNA plasmid from
′
