Biomedical Engineering Reference
In-Depth Information
Fig. 1
In-Fusion™ enzyme action. (
a
) Mechanism of In-Fusion™ enzyme action. The In-Fusion™ enzyme has
a 3
ends of two DNA molecules (1 and 2) that have 15 bp of
homology at the ends. Single-stranded annealing is then promoted giving rise to joined molecules shown
here with small single-stranded gaps. Following transformation into
E. coli
the gaps are repaired and ligated.
(
b
) Design of In-Fusion™ primers. 15 bp of homology are standardly used in In-Fusion™ extensions; however,
the type of restriction endonuclease used determines which vector sequence is used for the homology exten-
sion.
Dark blue boxes
indicate the homology regions on both strands. For blunt digests the 15 bp of homology
is to the cleaved vector DNA. For 5
′
to 5
′
exonuclease activity which degrades the 3
′
′
overhangs the homology should be from the end of the single-stranded
overhang. For 3
overhangs the homology should be to the double-stranded portion of the restricted vector;
that is, the single-stranded region shown in
orange
is removed by exonuclease action and the sequence is
not in the joined vector. If extra amino acids or a stop codon, etc. are required then these may be added to the
3
′
′
ends of all the extensions (indicated in
magenta
)
ends such as a PCR product and a linearized vector can be joined
to make a hybrid molecule ([
4
,
5
]; Fig.
1a
). Mechanistically, when
the double-stranded insert and linearized vector are incubated
with the enzyme in the presence of Mg
2+
and dNTPs, its exonucle-
ase activity degrades from the 3
ends of the molecules, thereby
exposing complementary sequences. These anneal to create a het-
erogeneous population of joined molecules that, at the junctions
where the annealing has taken place, have nicks, small gaps, and/
or short overhangs. The hybrid molecules are relatively resistant
to further exonuclease action and thus accumulate with time [
5
].
′
