Biomedical Engineering Reference
In-Depth Information
substitution is suffi cient to remove any type IIS restriction site
(an example is shown in
step 2
in Fig.
3
). For removal of sites
within coding sequences, it is always possible to make a silent
mutation that will not change the protein sequence. However, for
noncoding sequences such as promoters where regulatory sequence
motifs may not necessarily be known, it may be necessary to later
check that the introduced mutation has not affected the genetic
function encoded by the sequence of interest.
3.2 Selection
of Fusion Sites
and Primer Design
Fusion sites are selected at each point where the sequence needs to
be split in several sub-fragments. This includes locations where
restriction sites need to be removed, and any position where sev-
eral variant sequences may need to be recombined. Finally, fusion
sites may also be defi ned at several locations to split a large sequence
into several smaller ones. This may be useful for cloning of large
level 0 modules, since it is sometimes more effi cient to sequence
and screen several small fragments at −1 level than sequencing a
larger fragment at level 0.
Restriction sites are eliminated by amplifi cation with primers
containing a mismatch in the sequence that binds the target
sequence (Figs.
2
and
3
).
n
+ 1 fragments are usually amplifi ed by
PCR to remove n restriction sites (
see
Note 1
). To be able to
reconstitute the entire sequence from the amplifi ed fragments, all
primers have an extension that contains a BpiI restriction site. The
sequence at the cleavage site must correspond exactly to a sequence
from the target sequence (the fusion site) to avoid introducing
unwanted mutations to the sequence of interest. Cleavage of the
amplifi ed fragments using BpiI will release a fragment containing
only target sequence, fl anked on each side by four nucleotide DNA
overhangs derived from the fusion site. The fusion site may be
designed to overlap the mutated site but may also be selected near
the mutated site (as shown in Fig.
3
).
Two fusion sites are also needed at the beginning and the end
of the sequence of interest. These two fusion sites must be compat-
ible with two fusion sites in the destination vector. These sites have
a standard sequence (AATG and GCTT for MoClo level 0 modules
for coding sequences, Fig.
3
), and are therefore not necessarily
found in native sequences. For example, AATG overlaps with the
ATG start codon but an A that is not necessarily native must
be added upstream of the ATG; GCTT is usually not part of the
sequence of interest and is added after the stop codon (fusion sites
of standard sequence are shown in black in Figs.
2
and
3
).
Fusion sites must be carefully selected to all have a different
sequence to avoid assembly of the amplifi ed fragments in the wrong
order. It is important to check that all fusion sites also do not match
the complementary sequence of the other fusion sites, since this
would sometimes lead to ligation of two inappropriate fragments,
one in the inverse orientation. For example, choice of the sequence
